Atlantis® Columns Applications Notebook - Waters

A P P L I C AT I O N S N O T E B O O K

www.waters.com

Introduction Most chromatographers have experienced problems retaining and separating polar compounds using conventional reversed-phase (RP) chromatography. These difficult-to-analyze compounds either pass through the column unretained or, if retained at all, co-elute at the beginning of the chromatogram. Although today’s sensitive and selective mass spectrometers (MS) may help identify these early co-eluting compounds, MS ion suppression often occurs if these analytes are not sufficiently separated from the solvent front. Waters Atlantis™ columns are designed for these types of challenging separations. Atlantis™ columns are available in two fully LC/MS compatible chemistries: dC18 and HILIC Silica.

Atlantis™dC18 Columns – The Ideal Reversed-Phase Column Waters spent two years designing a new and unique stationary phase material that would offer the perfect balance of RP retention for polar and non-polar compounds. Atlantis™ dC18 columns exhibit superior retention of polar compounds as compared to conventional RP columns while not exhibiting excessive retention of hydrophobic compounds. Stationary phase physical attributes such as endcapping, silica pore size, bonded phase ligand density and ligand type were all studied and optimized in order to create a column that exhibits superior peak shape, low pH stability, resistance to dewetting (hydrophobic collapse) and enhanced polar compound retention.

Atlantis™ HILIC Silica Columns – Hydrophilic Interaction Chromatography for Very Polar Basic Compounds Another result of the Atlantis™ dC18 columns stationary phase creation project was the realization that no single RP material can retain all polar, water-soluble compounds. Another mode of retention and separation needed to be considered for very polar compounds such as actives, metabolites, and peptides. Hydrophilic Interaction Chromatography (HILIC) is a variation of normal-phase chromatography that uses RP solvents. HILIC is also referred to as “aqueous normal phase” or “reverse reversed-phase” since elution is in the order of increasing hydrophilicity and the organic portion of the mobile phase (typically acetonitrile) is the weak solvent and the aqueous portion is the strong eluting solvent. Retention Mechanisms in Hydrophilic Interaction Chromatography (HILIC)

Atlantis™ dC18 Columns—an Intelligent Design

Designing the Ideal Column Endcapping

Ligand Type

Peak Shape Stability Dewetting Retention

Pore Size

Ligand Ligand Density Density

Optimizing key stationary phase attributes results in the optimal combination of peak shape, low pH stability, resistance to dewetting and polar compound retention.

One result of this stationary phase creation project was a thorough understanding of the stationary phase’s role in polar compound retention. Only by studying and creating such a stationary phase can all the desirable characteristics of an ideal RP column be combined. Atlantis™ dC18 columns are compatible with aqueous mobile phases, provide enhanced low pH stability, and are suitable for separating polar compounds as well as standard RP applications. Atlantis™ dC18 columns are available in a wide range of column configurations ranging from nanoscale to preparative including Intelligent Speed (IS™) columns for fast, high-throughput analysis and Optimum Bed Density (OBD™) isolation and purification columns which provide analytical column performance in preparative column dimensions.

The combination of partitioning and weak cation-exchange results in retention of polar bases with Atlantis™ HILIC Silica columns.

The reason for developing Atlantis™ HILIC Silica columns was to offer a simple, rugged stationary phase which provided improved LC/ESI-MS response, direct SPE solvent compatibility and complementary selectivity to RP. This complementary selectivity is important to AR&D and drug metabolism scientists since the impurity or metabolite is often more polar and present at much lower concentrations than the parent compound. With Atlantis™ HILIC Silica columns, these very polar compounds elute later than the higher concentration hydrophobic parent compound, thus minimizing the MS ion suppression that can occur at the beginning of the chromatogram.

For More Information For more information about the Atlantis™ family of columns please visit the Waters website at www.waters.com.

Atlantis™ Columns Brochure Literature Code 720000793EN

©2004 Waters Corporation.Waters and Atlantis are trademarks of Waters Corporation.

1

Click anywhere. Type desired page number. Click OK. Table of Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Compound Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 List of Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Troubleshooting and Problem Solving Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-88 Care and Use Instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89-94 Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95-96 Compound Index 2’-Deoxyguanosine.........................56, 58, 59, 60 2,4,5-Trihydroxybutyrophenone ...........................44 2,6-Di-tert-butyl-4-hydroxymethylphenol ..................44 2-Acetamidophenol ...........................................16 2-Acetylfuran ..............................................63, 64 2-Amino-7chloro-5-oxo-5H-[1]benzopyrano [2,3-b]pyridine-3-carbonitrile..................68, 69, 70, ......................................................... 71, 72, 73 2-Bromofluorene ................68, 69, 70, 71, 72, 73 2-’Deoxyadenosine............................................56 2’-Deoxycytidine................................................56 3,4-Dihydroxybenzylamine (DHBA) ......................32 3,4-Dihydroxyphenylacetic acid (DOPAC) ......30, 31 3,4-Dihydroxyphenylalanine (DOPA) ....................31 3-Aminofluoranthene ......................68, 69, 70, 71, ................................................................72, 73 3-Methoxy-4-hydroxyphenylglycol (MHPG) ............31 4-Hydroxy-3-methoxymandelic acid (VMA)............31 4-Hydroxy-3-methoxyphenylacetic acid (HVA) .................................................30, 31 4-Hydroxybenzoic acid......................................19 5-Fluorocytosine .......................22, 52, 53, 54, 55 5-Fluorouracil .................................52, 53, 54, 55 5-Hydroxy-3-indoleacetic acid (5-HIAA) ................30 6α-Methyl-17α-hydroxyprogesterone.......68, 69, 70, ..........................................................71, 72, 73 8-Hydroxy-2’-Deoxyguanosine ................58, 59, 60 8-Bromoguanosine...............................68, 69, 70, ..........................................................71, 72, 73 A6677............................................................62 Acenaphthene...................................................67 Acephate.....................................................7, 74 Acetaminophen ..........................................16, 34 Acetanilide.............................16, 63, 64, 67, 68, .............................................69, 70, 71, 72, 73 Acetic acid.......................................................57 Acetophenone ............................................63, 64 Acetylcholine.......................................................8 Acetylsalicylic acid ............................................16 Acrylamide.........................................................9 Acyclovir .............................................10, 11, 42 Adenine.....................................................22, 54 Adenosine........................................................56 Albuterol..............................................23, 24, 25 Allantoin...........................................................12 Amitrole ...........................................................43 Amoxicillin..................................................14, 15 Angiotensin I.....................................................62 Angiotensin II ....................................................62 Arbutin.............................................................18 Asulam ......................................................21, 43 Atenolol .....................................................26, 27 Bamethan............................................23, 24, 25 Benzocaine ......................................................17

2

Benzophenone ...........................................63, 64 Bradykinin ........................................................62 Butacaine.........................................................17 Butylated hydroxyanisole ....................................44 Butylated hydroxytoluene ....................................44 Butyrophenone............................................63, 64 Caffeine.....................16, 28, 39, 77, 86, 87, 88 Catechin ..........................................................28 Catechin gallate................................................28 Chlorpheniramine........................................34, 39 Chlorsulfuron.....................................................43 Chlorthalidone ..................................................38 Choline..............................................................8 Choline-d9 (I.S.) ..................................................8 Cinoxacin .........................46, 47, 48, 49, 50, 51 Ciprofloxacin ....................................................40 Clonidine .........................................................33 Corticosterone ............................................78, 79 Cortisone ...................................................78, 79 Cytidine ...........................................................56 Cytochrome C ..................................................35 Cytosine........................................52, 53, 54, 55 Dextromethorphan .......................................34, 36 DHBA..............................................................32 Diazepam ........................................................37 Dibutyl phthalate................................................66 Dimethyl phthalate .............................................66 Dioctyl phthalate................................................66 Diphenhydramine ..............................................34 Dopamine .....................................29, 30, 31, 32 Enrofloxacin......................................................40 Epicatechin.......................................................28 Epicatechin gallate ............................................28 Epigallocatechin................................................28 Epigallocatechin gallate .....................................28 Epinephrine ...................................29, 30, 31, 32 Ergocalciferol....................................................86 Estradiol.....................................................78, 79 Estrone ......................................................78, 79 Fenoprofen.................................................75, 76 Flurbiprofen ................................................75, 76 Folic acid...................................................86, 87 Fumaric acid.....................................................57 Furosemide.......................................................38 Gallic Acid ................................................19, 28 Gallocatechin gallate.........................................28 Gentisic acid ....................................................19 Ginsenoside Rb2 ..............................................41 Ginsenoside Rc.................................................41 Ginsenoside Rd.................................................41 Ginsenoside Rg1 ..............................................41 Guaiacol glyceryl ether ......................................34 Guanine ....................................................42, 54 Guanosine .................................................22, 56

Guanosine-5’-monophosphate .............................22 Heptanophenone........................................63, 64 Hexanophenone .........................................63, 64 Homocysteine ...................................................84 Hydrochlorothiazide...........................................38 Hydrocortisone..............................67, 68, 69, 70, ..........................................................71, 72, 73 Hydroxylated Diazepam Metabolite.....................37 Ibuprofen ...................................................75, 76 Imazethapyr......................................................43 Insulin B Chain..................................................62 Isophthalic acid.................................................65 Ketoprofen .................................................75, 76 L-ascorbic acid............................................86, 87 L2275.............................................................62 Labetolol ..........................................................26 Lactic acid........................................................57 Lauryl Gallate ...................................................44 Lidocaine .........................................................17 Maleic acid ...............................................39, 57 Malic acid .......................................................57 Melittin.............................................................62 Methamidophos............................................7, 74 Methionine .......................................................84 Metoprolol .................................................26, 27 Morphine .........................................................45 Morphine 3-ß-D-Glucuronide ...............................45 N-Dealkylated Dextromethorphan Metabolite........................................................36 Nalidixic acid ...................46, 47, 48, 49, 50, 51 Naphtho(2,3-a)pyrene..........................68, 69, 70, ..........................................................71, 72, 73 Neomycin ........................................................13 Niacin .............................................................86 Nicotinic acid ............................................86, 87 Norepinephrine..............................29, 30, 31, 32 Octanophenone .........................................63, 64 Octyl Gallate....................................................44 Oxalic acid ......................................................57 Oxolinic acid ....................46, 47, 48, 49, 50, 51 Oxprenolol .......................................................26 Paclitaxel..........................................................61 Perylene ...........................68, 69, 70, 71, 72, 73 Phenacetin........................................................16 Phenol .............................................................77 Phenylalanine....................................................20 Phenylephrine....................................................39 Phenylpropanolamine.........................................39 Phthalic acid.....................................................65 Picloram...........................................................43 Pindolol............................................................26 Prednisolone...............................................78, 79 Procaine...........................................................17 Progesterone...............................................78, 79

Propiophenone ...........................................63, 64 Propranolol.................................................26, 27 Propyl Gallate...................................................44 Protocatechuic acid ...........................................19 Pseudophendrine...............................................34 Pyridoxal....................................................86, 87 Pyridoxine............................................86, 87, 88 Pyruvic acid......................................................57 Renin Substrate..................................................62 Retinol..............................................................86 Riboflavin ...................................................86, 87 Serotonin (5-HT) ..........................................30, 31 Streptomycin .....................................................13 Substance P......................................................62 Succinic acid....................................................57 Sulfadiazine.........................................80, 81, 82 Sulfadimethoxine.........................................80, 81 Sulfadimidine..............................................80, 81 Sulfadmethazine................................................82 Sulfamerazine ......................................80, 81, 82 Sulfamethazine ...........................................82, 83 Sulfamethoxazole ...........................80, 81, 82, 83 Sulfanilamide ....................................................83 Sulfathiazole ..................................80, 81, 82, 83 Sulfisoxazole ..............................................80, 81 Syringic acid ....................................................19 Tartaric acid......................................................57 Terephthalic acid ...............................................65 Tert-butylhydroquinone ........................................44 Tetracaine.........................................................17 Theobromine.....................................................77 Theophylline.........................................63, 64, 77 Thiamine....................................................86, 87 Thiourea...........................................................22 Thymidine.........................................................56 Thymine.....................................................22, 54 Tocopherol .......................................................86 Triamcinolone....................68, 69, 70, 71, 72, 73 Tryptophan .......................................................20 Tyrosine............................................................20 Uracil...............................52, 53, 54, 55, 72, 73 Uridine.............................................................56 Valerophenone ...........................................63, 64 Vancomycin......................................................85 Vanillic Acid......................................................19 Vitamin A .........................................................86 Vitamin B1 .................................................86, 87 Vitamin B11...............................................86, 87 Vitamin B2 .................................................86, 87 Vitamin C...................................................86, 87 Vitamin D2 .......................................................86 Vitamin E..........................................................86


Click anywhere. Type desired page number. Click OK. List of Applications Acephate in River Water................................................................7

Fluoroquinolone Antibiotics in Beef Kidney......................................40

Acetylcholine and Choline .............................................................8

Ginsenosides .............................................................................41

Acrylamide ..................................................................................9

Guanine and Acyclovir: HILIC vs. Reversed-Phase ...........................42

Acyclovir – Isocratic ....................................................................10

Herbicides by LC/MS/MS .........................................................43

Acyclovir – Gradient ...................................................................11

Lipid Soluble Antioxidants ............................................................44

Allantoin....................................................................................12

Morphine and Morphine-3-β-D-Glucuronide....................................45

Aminoglycoside Antibiotics...........................................................13

Nalidixic Acid Antibiotics.............................46, 47, 48, 49, 50, 51

Amoxicillin – Isocratic ..................................................................14

Nucleic Acid Bases .................................................52, 53, 54, 55

Amoxicillin – Gradient.................................................................15

Nucleosides...............................................................................56

Analgesics.................................................................................16

Organic Acids ...........................................................................57

Anesthetics.................................................................................17

Oxidative Stress Markers – ACN, pH 3.0 ........................58, 59, 60

Arbutin ......................................................................................18

Paclitaxel...................................................................................61

Aromatic Acids...........................................................................19

Peptides ....................................................................................62

Aromatic Amino Acids.................................................................20

Phenones – 2.1 x 20 mm IS™ Column ....................................63, 64

Asulam in River Water.................................................................21

Phthalic Acids ............................................................................65

Atlantis™ dC18100% Aqueous QC Batch Test .................................22

Phthalic Esters.............................................................................66

Bamethan and Albuterol .................................................23, 24, 25

Polar & Non-Polar Compounds ..............67, 68, 69, 70, 71, 72, 73

ß-Blockers............................................................................26, 27

Polar Pesticides...........................................................................74

Catechins..................................................................................28

Profens................................................................................75, 76

Catecholamines ......................................................29, 30, 31, 32

Purine Alkaloids..........................................................................77

Clonidine ..................................................................................33

Steroids ..............................................................................78, 79

Cold Medicine Products ..............................................................34

Sulfonamides ..........................................................80, 81, 82, 83

Cytochrome C Tryptic Digest........................................................35

Thiols ........................................................................................84

Dextromethorphan and N-Dealkylated Metabolite ...........................36

Vancomycin ...............................................................................85

Diazepam and Hydroxylated Metabolite .......................................37

Water and Fat Soluble Vitamins....................................................86

Diuretics ....................................................................................38

Water Soluble Vitamins .........................................................87, 88

Drugs for Rhinitis .........................................................................39


3

Problem Solving and Troubleshooting Using Atlantis™ dC18 Columns Problem Little or no retention of polar compounds

Impact

Solution and Benefit

l

l

l

Re-run samples using separate methods for polar compounds Increased method development time and labor

l

l

Method requires 100% aqueous mobile phase for desired separation

Sudden loss of analyte retention observed when using highly aqueous mobile phase

l

l

l l l

Short column lifetime in acidic mobile phases

l l l

Retaining polar compounds on a conventional C column results in increased or infinite retention

l

18

of non-polar compounds

l

l

Severe peak tailing for polar bases is observed

l

l

Column bleed is observed on MS

l l

Loss of retention is observed

Run organic modifier through column to rewet and regenerate column Increased labor and solvent costs Decreased throughput Reproducibility issues High cost due to frequent column replacement Increased instrument downtime Retention time reproducibility issues

Multiple columns are required to separate analytes with a wide range of polarities Increased method development time, labor and column costs Decreased throughput Method fails system suitability guidelines for peak tailing Increased method development time Frequent cleaning of MS source Incorrect or inconsistent results

l

l

l l

l

l

l

l l

l

l

l

l

Column to column reproducibility is inconsistent (e.g., selectivity, retention, etc.)

l

l

4

Increased labor costs due to individual column QC testing Revalidate/redevelop method with each new batch of columns

l

l

Polar compounds are retained longer with Atlantis™ dC18 columns One Atlantis™ dC18 column and method can be used for polar and non-polar compounds Decreased labor costs Atlantis™ dC18 packing material is tested with highly polar analytes in 100% aqueous conditions, thereby ensuring its utility in aqueous conditions Atlantis™ dC18 columns do not lose retention in 100% aqueous mobile phases Less time spent rewetting columns resulting in lower labor costs Increased throughput

The proprietary difunctional bonding chemistry of Atlantis™ dC18 columns results in low pH stability and longer column lifetime Decreased costs associated with column replacement and instrument maintenance One Atlantis™ dC18 column and method can be used for polar and non-polar compounds Easier and faster method development Increased throughput

Atlantis™ dC18 columns are optimally endcapped and provide excellent peak shapes using MS compatible mobile phases Easier and faster method development Atlantis™ dC18 columns do not exhibit MS detectable column bleed Decreased instrument downtime and maintenance costs The stringent Atlantis™ dC18 packing material QC batch test separates highly polar analytes in 100% aqueous mobile phase conditions Decreased method revalidation and development time


Problem Solving and Troubleshooting Using Atlantis™ HILIC Silica Columns Problem Polar metabolites or contaminants not retained by reversed-phase HPLC

Impact

Solution and Benefit

l

l

l l

Severe peak tailing for polar bases is observed on reversed-phase column

l

l

Evaporation and reconstitution step in sample preparation is too time consuming

l

l

Re-run samples using alternate, non-MS compatible chromatographic techniques Metabolites or contaminants are not detected Increased method development time Method fails system suitability guidelines for peak tailing Increased method development time Increased labor costs and higher cost per analysis Decreased sample throughput

l

l

l

l

l l

Evaporation and reconstitution step in sample preparation results in poor analyte recoveries

l

l

l

Insufficient MS sensitivity due to highly aqueous reversed-phase mobile phases

l

l

Poor polar stationary phase column lifetime (e.g., amino, diol, etc.)

l l

Unstable or mobile analytes are lost and/or not detected Evaporated sample does not completely reconstitute Method fails recovery and limits of detection requirements Samples need to be concentrated and re-analyzed Low concentration metabolites or contaminants not detected Frequent column replacement Retention time reproducibility issues

l

l l

l

l

l

l

Polar bonded phase bleed is observed on MS, UV and/or ELSD

l l l

Noisy baselines resulting in poor sensitivity Incorrect false positive peaks Frequent system cleaning

l

l l

Column to column reproducibility is inconsistent (e.g., selectivity, retention, etc.)

l

l

Difficult separation requires complementary chromatographic selectivity

l l

Increased labor costs due to individual column QC testing Revalidate/redevelop method with each new batch of columns Multiple dedicated LC systems required Separation techniques must be developed independently

l

l

l l

l


Atlantis™ HILIC Silica columns retain polar metabolites that cannot be retained by reversed-phase HPLC Faster and easier method development

Atlantis™ HILIC Silica columns provide superior peak shapes for polar bases Faster and easier method development Evaporation and reconstitution step is not necessary with Atlantis™ HILIC Silica columns since the mobile phases used are compatible with sample preparation organic solvents Lower analysis costs Increased throughput Evaporation and reconstitution step is not necessary with Atlantis™ HILIC Silica columns since the mobile phases used are compatible with sample preparation organic solvents Greater analyte recoveries Lower limits of detection

Highly volatile mobile phases used with Atlantis™ HILIC Silica columns provide increased ESI-MS sensitivity Lower limits of detection

Atlantis™ HILIC Silica columns do not have an unstable polar bonded phase Decreased costs associated with frequent column replacement Atlantis™ HILIC Silica columns do not exhibit detectable column bleed Increased sensitivity Decreased instrument downtime and maintenance costs Atlantis™ HILIC Silica columns are tested under actual HILIC conditions Decreased method revalidation and development time

Atlantis™ HILIC Silica columns use reversed-phase solvents Single LC system running reversed-phase and HILIC separations can be easily automated Greater flexibility, lower instrumentation costs, faster method development

5

Retaining Polar Compounds START HERE 1

base

Is the polar compound an acid, neutral or base?

neutral

2

2

Can it be retained by reversed-phase?

Can it be retained by reversed-phase?

Yes

Atlantis™ dC18 or XTerra®

No

Sufficient Mass Spec sensitivity?

Yes

Atlantis™ dC18

No

Yes

3

acid

Alternate separation technique such as ion exchange, ion-pair, etc.

No Atlantis™ HILIC Silica

NOTE: Atlantis™ dC18 = High aqueous, Low pH

To learn more about polar compound retention, contact your local Waters representative or visit us online at www.waters.com/atlantis

6

Atlantis™ HILIC Silica = High organic, Low pH XTerra® = High aqueous, High pH

©2004 Waters Corporation.Waters and Atlantis and XTerra are trademarks of Waters Corporation.

Acephate in River Water Oasis® HLB Extraction Method

LC Conditions: Column: Part Number: Mobile Phase: Flow Rate: Injection Volume: Temperature: Instrument:

Atlantis dC18, 2.1 x 100 mm, 3 µm 186001295 15% MeOH in H2O 0.2 mL/min 20 µL 25 ˚C Alliance® HT 2795

MS Conditions: Instrument: Ion Source: Source Temp: Desolvation Temp: Cone Gas: Desolvation Gas: Collision Gas:

Quattro micro Electrospray positive 150 ˚C 450 ˚C 50 L/hr 500 L/hr Argon

™

Oasis® HLB Extraction Cartridge, 6 cc/200 mg Prepare Sample: 5 g ammonium sulfate/20 mL sample Condition/Equilibrate: 1 mL DCM1/1mL mthanol/1 mL water Load: 20 mL sample2

™

Wash: 1 mL water Elute: 2 mL 10:90 methanol/DCM Evaporate and Reconstitute: 250 µL water 1

Compound methamidophos acephate

MRM 142>112 184>143

Cone(V) 27 20

2

Coll.Energy(eV) 15 10

200ng/L spiked river water

Acephate H 3 CS

O

O

P

C N H

H CO 3

methylene chloride 25 mL maximum sample size

100

MRM 184 >143

acephate

CH 3

%

- contact Acephate and systematic organophosphorous insecticide - highly polar, water soluble

0 100

MRM 142 >112

methamidophos %

Acephate/Methamidophos Daughter Ion Spectra

Time

0

1

2

3

4

5

6

200ng/L spiked river water - same injection as MRM data

Acephate/Methamidophos Recovery Data 113

100

125

External standard calculation (n=5) acephate

%

143

112

Methamidophos 184

0

100

110

120

130

150

160

170

180

m/z 190

142

125

112

140

methamidophos

143

%

Spike Level (ng/L) 50 200 400 800

Acephate Recovery1 50% 62% 66% 60%

RSD 25% 9.8% 12% 6.8%

Spike Level (ng/L) 50 200 400 800

Recovery1

RSD

92% 91% 90% 105%

12% 5.4% 15% 12%

110 0

110

120

130

140

150

160

170

180

m/z 190

1 Calculated against standard prepared in matrix. Matrix suppression was approximately 40%. Without Oasis® HLB cleanup, matrix supression was approximately 80%.

©2004 Waters Corporation. Waters, Oasis, Alliance, Quattro micro and Atlantis are trademarks of Waters Corporation.

7

Acetylcholine and Choline LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Sample Diluent: Temperature: Instrument: MS Conditions: Ionization Mode: Capillary (kV): Cone (V): Extractor: RF Lens: Source Temp (°C): Desolvation Temp (°C): Cone Gas Flow (L/Hr): Desolvation Gas Flow (L/Hr): SIR m/z:

Atlantis™ HILIC Silica 2.1 x 50 mm, 3 µm 186002011 H2O ACN 200 mM NH4COOH, pH 3.0 0.3 mL/min 9% A; 86% B; 5% C 20 µL 60:40 IPA:ACN with 0.2% HCOOH Ambient Alliance® HT 2795 with Waters ZQ™

H3C N

+

H C 3

CH3

CH3

N

O

H C 3

H 3C O

Acetylcholine D 3C

D3C

3

OH

CD 3

Choline-d9 (I.S.)

3.0x105

50 700 146.2 103.9 113.1

CH

Choline

+ N

ES+ 1.0 15 (ACh); 30 (Ch, Ch-d9) 3V 0.3 V 150 350

OH

+

(ACh) (Ch) (Ch-d9)

Acetylcholine 100 fg/µL

2.0x105

1.0x105 1.4x106

Intensity

Choline 1,000 fg/µL

2.0x105

1.0x107 Choline-d9 (I.S.) 5,000 fg/µL

5.0x106

2.00

4.00

6.00

Minutes

8

©2004 Waters Corporation. Waters, Alliance, ZQ and Atlantis are trademarks of Waters Corporation.

Acrylamide LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Isocratic Mobile Phase Composition: Flow Rate: Injection Volume: Temperature: Instrument:

99.5% A; 0.5% B 0.2 mL/min 20 µL 26° C Alliance® 2695, Quattro Premier™

MS Conditions: Ionization Mode: Capillary (kV): Cone (V): Extractor: RF Lens: Source Temperature (°C):

ES+ 3.5 20 5V 0.0 V 120

MS Conditions: The mass detector was calibrated between m/z 19 to m/z 2000 with unit resolution.

Atlantis™ dC18 2.1 x 150 mm, 3 µm 186001299 0.1% CH3COOH MeOH

Desolvation Temp (°C): Desolvation Gas Flow (L/hr): Cone Gas Flow (L/hr): Gas Cell Pressure:

MS/MS Analysis Function (MRM of 4 mass pairs) Transition Dwell Time Collision Energy 71.7 > 43.9 0.4 11.0 71.7 > 54.8 0.4 10.0 74.7 > 44.9 0.4 11.0 74.7 > 57.8 0.4 10.0

Delay 0.01 0.01 0.01 0.01

The 74.7>57.8 and 71.7>44.9 for for the 13C3-Acrylamide Internal Standard at 50 ppb.

450 800 50 1.1 x 10-2 mbar

4.2 x 10 3

Acrylamide

2 pg on column (20 mL of 0.1 ppb)

S/N = 10.8 71.7 > 54.8 71.7 > 43.9

O Smooth (Mn,1x2)

CH2

H2 N

1.00

2.00

3.00

Acrylamide Acrylamide

4.00

2.4 x 10 3

Acrylamide

S/N = 10.5

3.5 x 10

4

50 pg on column (20 µL of 2.5 ppb)

2.50

3.00

3.50

4.00

71.7 > 54.8 71.7 > 43.9

Smooth (Mn,1x2)

1.00

2.00

3.00

4.00

Minutes

©2004 Waters Corporation. Waters, Alliance, Quattro Premier and Atlantis are trademarks of Waters Corporation.

9

Acyclovir - Isocratic LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Sample Concentration: Temperature: Detection: Instrument:

Atlantis™ dC18 4.6 x 50 mm, 3 µm 186001329 H2O ACN 100 mM HCOONH4, pH 3.0 1.0 mL/min 87% A; 3% B; 10% C 5 µL 10 µg/mL 30° C UV @ 254 nm Alliance® HT 2795 with 2996 PDA

Acyclovir

USP Tailing = 1.34

V0 = 1.0

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

6.50

7.00

7.50

8.00

8.50

9.00

9.50

10.00

Minutes

10

©2004 Waters Corporation. Waters, Alliance and Atlantis are trademarks of Waters Corporation.

Acyclovir - Gradient LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

Injection Volume: Sample Concentration: Temperature: Detection: Instrument:

Atlantis™ dC18 4.6 x 50 mm, 3 µm 186001329 H2O ACN 100 mM HCOONH4, pH 3.0 1.0 mL/min Time Profile (min) %A %B %C 0.0 90 0 10 5.0 85 5 10 5 µL 10 µg/mL 30° C UV @ 254 nm Alliance® HT 2795 with 2996 PDA

Acyclovir

USP Tailing = 1.00

V0 = 1.0

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

6.50

7.00

7.50

8.00

8.50

9.00

9.50

10.00

Minutes


11

Allantoin LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Injection Solvent: Sample Concentration: Temperature: Detection: Instrument:

Atlantis™ HILIC Silica 4.6 x 50 mm, 3 µm 186002027 100 mM NH4COOH, pH 3.0 ACN 1.0 mL/min

H N

NH

H2N

O O

5% A; 95% B 5 µL 75:25 ACN:H2O 250 µg/mL 30° C UV @ 205 nm Alliance® 2695 with 2996 PDA

N H

O

Allantoin

V0 = 1.15

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes

12


Aminoglycoside Antibiotics LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

NH2

Atlantis™ dC18 4.6 x 20 mm IS™, 3 µm 186002062 H2O ACN 0.50% Hexafluorobutyric acid in H2O 3.0 mL/min Time Profile (min) %A %B %C 0.0 75 5 20 3.0 20 60 20 10 µL 0.1 mg/mL 35° C Evaporative Light Scattering Alliance® 2695 with 2420 ELSD


H3C

NH

HN O

O

OH

O HO O

HO

O

NH

HO

HO

OH H2N

NH OH

NH

CH3

Streptomycin

NH2 O

O H2N HO

NH2

HO

OH

Compounds: 1. Streptomycin 2. Neomycin

NH2 OH

Neomycin

2

1

V0

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Minutes

©2004 Waters Corporation. Waters, Alliance, IS and Atlantis are trademarks of Waters Corporation.

13

Amoxicillin—Isocratic LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow rate: Isocratic Mobile Phase Composition: Injection Volume: Sample Concentration: Temperature: Detection: Instrument:

Atlantis™ dC18 4.6 x 50 mm, 3 µm 186001329 H2O ACN 100 mM HCOONH4, pH 3.0 1.0 mL/min 87% A; 3% B; 10% C 10 µL 40 µg/mL 30° C UV @ 254 nm Alliance® HT 2795 with 2996 PDA Amoxicillin

V0 = 1.0

0.00

1.00

USP Tailing = 1.19

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

14


Amoxicillin—Gradient LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:



Amoxicillin

USP Tailing = 1.01

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes


15

Analgesics LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

O

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O ACN 1% HCOOH, pH 2.3 1.0 mL/min Time Profile (min) %A %B %C 0.0 75 15 10 10.0 30 60 10 10 µL 30° C UV @ 260 nm Alliance® 2695 with 2996 PDA

Injection Volume: Temperature: Detection: Instrument:

O

OH

O

HN

O

O

Acetylsalicylic acid

Acetaminophen

Phenacetin

HO

O H N

N

N

HN O

O

O

N

N

2-Acetamidophenol Acetanilide

Compounds: 1. Acetaminophen 2. Caffeine 3. 2-Acetamidophenol 4. Acetanilide 5. Acetylsalicyic acid 6. Phenacetin

USP Tailing 1.00 0.99 0.98 1.00 0.96 0.96

Caffeine

Sample Concentrations (µg/mL) 16 18 55 22 55 18

1

6 2

4

3

V0 = 1.83

0.00

1.00

5

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

16


Anesthetics LC Conditions: Column: Part Number: Mobile Phase A:

Atlantis™ dC18 4.6 x 150 mm, 5 µm

O

186001344 H2O

Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

N

MeOH 100 mM HCOONH4, pH 3.75 1.4 mL/min Time Profile (min) %A %B %C 0.0 65 25 10 5.0 40 50 10

O H2N O

NH2

Benzocaine

10.0 40 50 10 20 µL 30° C UV @ 254 nm Alliance® 2695 with 2996 PDA


O

Butacaine

H2N

NH

N

O N

O

O

Lidocaine

Procaine

Compounds: 1. Procaine 2. Lidocaine 3. Butacaine 4. Benzocaine 5. Tetracaine

Sample Concentrations (µg/mL) 7 9 59 59 59

USP Tailing 1.07 1.04 1.04 0.98 1.20

N O HN O

Tetracaine

4

3

1

5 2

V0 = 1.37

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes


17

Arbutin LC Conditions: Column: Part Number: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Sample Concentration: Temperature: Detection: Instrument:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 2.0 mL/min 10 mM HCOONH4, pH 3.75 5 µL 175 µg/mL 30° C UV @ 280 nm Alliance® 2695 with 2996 PDA

Arbutin

USP Tailing = 0.96

V0 = 0.98

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

18


Aromatic Acids LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

OH

O

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O ACN 100 mM HCOONH4, pH 3.0 2.0 mL/min Time Profile (min) %A %B %C 0.0 90 0 10 10.0 65 25 10 10 µL 30° C UV @ 285 nm Alliance® 2695 with 2996 PDA


O

OH

HO OH

OH O

HO

OH

4-Hydroxybenzoic Acid

O

Gallic Acid

OH

Vanillic Acid O OH

HO

OH

HO OH

OH

O

Gentisic Acid

Protocatechuic Acid O HO

OH

O O

Compounds: USP Tailing 1. Gallic acid 0.87 2. Protocatechuic acid 1.03 3. Gentisic acid 1.02 4. 4-Hydroxybenzoic acid 1.10 5. Vanillic acid 1.08 6. Syringic acid 1.09

Syringic acid


2 5 6 1

V0 = 0.98

4

3

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes


19

Aromatic Amino Acids LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:


Atlantis™ dC18 4.6 x 50 mm, 3 µm 186001329 H2O MeOH 100 mM HCOONH4, pH 3.0 1.0 mL/min Time Profile (min) %A %B %C 0.0 90 0 10 10.0 10 80 10 10 µL 30° C UV @ 254 nm Alliance® 2695 with 2996 PDA

Compounds: 1. Tyrosine 2. Phenylalanine 3. Tryptophan

USP Tailing 1.18 1.13 1.11

O

O

OH

OH

NH2

NH2

HO

Phenylalanine

Tyrosine O

OH HN

NH2

Tryptophan

Sample Concentrations (µg/mL) 150 200 30

3

1

V0 = 1.18

0.00

1.00

2

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

20


Asulam in River Water LC Conditions: Column: Part number: Mobile Phase A: Mobile Phase B: Gradient:

Oasis® MCX Extraction Method Oasis MCX Extraction Cartridge, 6 cc/150 mg (LP) Part Number: 186000255

Atlantis d C18, 2.1 x 100 mm, 3 µm 186001295 15 mM HCOONH4, pH 4.5 ACN Time Profile (min) %A %B 0.0 90 10 19.0 10 90 0.2 mL/min 20 µL Alliance® 2695 Separations Module ™

Flow rate: Injection volume: Instrument:

®

Prepare Sample: acidify to pH 2.7 Condition: 5 mL methanol/1 mL water Load: 250 mL sample (5 mL/min) Wash: 2 mL 5% MeOH/water

MS Conditions: Instrument: Ion Source: Mode: Source Temp: Desolvation Temp: Cone Gas: Desolvation Gas: Collision Gas:

Quattro Premier™ Electrospray positive and negative Multiple Reaction Monitoring 150 ˚C 450 ˚C 50 L/Hr 500 L/hr Argon

Elute: 2.5 mL MeOH/MTBE/NH4OH(22:75:3) Evaporate and Reconstitute: 250 µL 20% acetonitrile

O

MRM (ESI+) 231>156

Cone(V) 30

Coll. Energy (eV) 10

MRM (ESI+) 229>197

Cone(V) 25

Coll. Energy (eV) 15

H2 N

S O

NH COOCH3

Asulam

250 ng/L Spiked River Water

ESIAsulam Recovery 1 (n=4) 50 ng/L 81% (14%RSD) 250 ng/L 78% (7.6%RSD) 1000 ng/L 71% (12%RSD)

ESI+

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

time

Minutes

©2004 Waters Corporation. Waters, Oasis, Alliance, Quattro Premier and Atlantis are trademarks of Waters Corporation.

21

Atlantis™ dC18 100% Aqueous QC Batch Test LC Conditions: Column: Part Number: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Temperature: Detection: Instrument:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 1.2 mL/min

H N

HN

N

F

10 mM HCOONH4, pH 3.0 7 µL Ambient UV @ 254 nm Alliance® 2690 with 2996 PDA

NH2

O

USP Tailing 1.11 1.09 1.13 1.11 1.03

Adenine

5-Fluorocytosine


N

N

NH2

Thiourea

Guanosine-5’-monophosphate

Compounds: 1. Thiourea 2. 5-Fluorocytosine 3. Adenine 4. Guanosine-5’-monophosphate 5. Thymine

N

H N

O NH

H3C O

Thymine

1

2 4 5

3

V0 = 1.20

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

13.00

14.00

15.00

Minutes

22


Bamethan and Albuterol—LC/MS LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

Injection Volume: Sample Diluent: Sample Concentration: Temperature: Instrument:

MS Conditions: Ionization Mode: Capillary (kV): Cone (V): Extractor: RF Lens: Source Temperature (°C): Desolvation Temperature (°C): Cone Gas Flow (L/Hr): Desolvation Gas Flow (L/Hr): SIR m/z:

Atlantis™ HILIC Silica 2.1 x 50 mm, 3 µm 186002011 H2O ACN 200 mM NH4COOH, pH 3.0 0.2 mL/min Time Profile (min) %A %B %C 0.0 0 95 5 5.0 45 50 5 10 µL 75:25 ACN:MeOH with 0.2% HCOOH 100 ng/mL Albuterol, 50 ng/mL Bamethan Ambient Alliance® HT 2795, Waters ZQ™

H3C

ES+ 3.5 20 3V 0.3 V 100 250 50 450 209.9 (Bamethan) 239.9 (Albuterol)

OH

NH

HO C3H

HO

NH

CH3 CH3

OH

OH

Bamethan

Albuterol

Bamethan 2.0x10

6

intens

1.5x10 6

1.0x10 6

5.0x10 5

0.0

Albuterol

500000

intens

400000 300000 200000 100000

0 0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes


23

Bamethan and Albuterol—LC/UV HILIC vs. Reversed-Phase HILIC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Isocratic Mobile Phase Conditions: Injection Volume: Injection Solvent: Sample Concentration: Temperature: Detection: Instrument:

Reversed Phase Conditions: Column: Atlantis™ dC18 4.6 x 50 mm, 3 µm Part Number: 186001329 Mobile Phase A: H2O Mobile Phase B: ACN Mobile Phase C: 100 mM NH4COOH, pH 3.0 Flow Rate: 1.4 mL/min Isocratic Mobile Phase Conditions: 82% A; 8% B: 10%C Injection Volume: 2 µL Injection Solvent: 75:25 H2O:ACN Sample Concentration: 125 µg/mL Temperature: 30° C Detection: UV @ 280 nm Instrument: Alliance® 2695 with 2996 PDA

Atlantis™ HILIC Silica 4.6 x 50 mm, 3 µm 186002027 100 mM NH4COOH, pH 3.0 ACN 2.0 mL/min 10% A; 90% B 2 µL 75:25 ACN:MeOH 125 µg/mL 30° C UV @ 280 nm Alliance® 2695 with 2996 PDA

H3C

2

1

OH

NH

HO C3H

HO

HILIC

NH

1. Bamethan

0.00

0.50

CH3

OH

OH

V0 = 0.5

CH3

2. Albuterol

Atlantis™ HILIC Silica

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

4.00

4.50

5.00

2

ReversedPhase

1

V0 = 0.65

0.00

0.50

1.00

1.50

2.00

Atlantis™ dC18

2.50

3.00

3.50

Minutes

24


Bamethan and Albuterol in Plasma—LC/MS LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

Injection Volume: Sample Concentration: Temperature: Instrument:


Atlantis™ HILIC Silica 2.1 x 50 mm, 3 µm 186002011 H2O ACN 200 mM NH4COOH, pH 3.0 0.2 mL/min Time Profile (min) %A %B %C 0.0 0 95 5 5.0 45 50 5 10 µL 50 pg/µL Albuterol 10 pg/µL Bamethan Ambient Alliance® HT 2795 with Waters ZQ™

H3C

ES+ 3.5 20 3V 0.3 V 100 250 50 450 209.9, 239.9 OH

NH

HO C3H

HO

NH

CH3 CH3

OH

OH

1. Bamethan

2. Albuterol

Oasis® HLB Extraction Method Oasis® HLB µElution Plate 186001828BA Condition/Equilibrate:* 200 µL methanol/200 µL water Load: 75 µL spiked plasma sample 75 µL internal standard with 2% ammonium hydroxide Wash: 200 µL 5% methanol in water 1

Elute: 75 µL 40% acetonitrile/60% isopropanol with 2% formic acid

2

100

Inject eluent directly onto column: NO Evaporation and Reconstitution % SIR of 2 Channels ES+ 239.8 209.9 8.97e4

0 0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes


25

β-Blockers LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O ACN 100 mM HCOONH4, pH 3.0 2.0 mL/min Time Profile (min) %A %B %C 0.0 85 5 10 3.0 65 25 10 10.0 65 25 10 10 µL 30° C UV @ 280 nm Alliance® 2695 with 2996 PDA


NH2

O N H

O

O OH

Atenolol

O

NH

OH

Oxprenolol O HO

N H

N H

O OH

Metoprolol

O

Propranolol

OH NH2

Compounds: 1. Atenolol 2. Pindolol 3. Metoprolol 4. Oxprenolol 5. Labetolol 6. Propranolol


USP Tailing 1.02 1.06 1.13 1.32 1.31 1.41

O N H

O

NH

HO

OH

HN

Pindolol

Labetolol

2

1

3 4

V0 = 0.98

6 5

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

26


β-Blockers— Analytical to Preparative Scale Up LC Conditions: Mobile Phase A: Mobile Phase B: Sample Concentration: Temperature: Instrument: Detection:

0.1% HCOOH in H2O ACN/1% HCOOH (90/10) 5, 25, and 25 mg/mL in DMSO/MeOH (50/50) Ambient AutoPurification™ System UV @ 280 nm

NH2

O N H

O OH

Atenolol HO N H

Compounds: 1. Atenolol (5 mg/mL) 2. Metoprolol (25 mg/mL) 3. Propranolol (25 mg/mL)

O O

N H

O OH

Metoprolol Propranolol

Column:


Part Number: Total Mass Load:

186001340 1,650 µg

Flow Rate: Gradient:

3

V0 = 1.76

2

Injection Volume:

1.0 mL/min Time Profile (min) %A 0.0 95 1.0 95 2.0 80 4.0 50 6.0 50 30 µL

%B 5 5 20 50 50

1

Column: Part Number: Total Mass Load:

Atlantis™ dC18 Prep OBD™ 30 x 100 mm, 5 µm 186001375 70,125 µg


3

V0 = 1.73 2

Injection Volume:


%B 5 5 20 50 50

1

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

Minutes

©2004 Waters Corporation. Waters, OBD, AutoPurification and Atlantis are trademarks of Waters Corporation.

27

Catechins LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O MeOH 1% HCOOH, pH 2.3 1.0 mL/min Time Profile (min) %A %B %C 0.0 70 20 10 10.0 60 30 10 20.0 30 60 10 10 µL 30° C UV @ 280 nm Alliance® 2695 with 2996 PDA


Compounds: USP Tailing 1. Gallic Acid 1.05 2. Epigallocatechin 0.94 3. Catechin 1.00 4. Caffeine 0.97 5. Epigallocatechin gallate 1.08 6. Epicatechin 0.98 7. Gallocatechin gallate 0.98 8. Epicatechin gallate 0.98 9. Catechin gallate 0.99

O

OH HO

N

N

OH O

N

O

N

OH

Gallic acid

Caffeine Catechin

Epicatechin Epigallocatechin gallate

Epigallocatechin

Sample Concentrations (µg/mL) 11 43 22 11 22 43 33 22 33

Catechin gallate

Epicatechin gallate

Gallocatechin gallate

9 1

3

7 8 4

V0 = 1.83

5

6

2

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

Minutes

28


Catecholamines—HILIC LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

Injection Volume: Injection Solvent: Analyte Concentrations:

Detection: Temperature: Instrument:

Atlantis™ HILIC Silica 4.6 x 50 mm, 5 µm 186002028 H2O ACN 200 mM NH4COOH, pH 3.0 2.0 mL/min Time Profile (min) %A %B %C 0.0 0 95 5 5.0 25 70 5 5 µL 75:25 ACN:MeOH with 0.2% HCOOH 42 µg/mL Norepinephrine 17 µg/mL Epinephrine

OH

NH2 HO

HO OH HO

H N

CH3

HO

Norepinephrine

Epinephrine

NH2

HO HO

Dopamine

83 µg/mL Dopamine UV @ 280 nm Ambient Alliance® 2695 with 2996 PDA

Norepinephrine

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

4.50

5.00

4.50

5.00

Epinephrine

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Dopamine

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Minutes


29

Catecholamines—Isocratic LC/UV LC Conditions: Columns: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Temperature: Detection: Instrument:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O ACN 100 mM CH3COONH4, pH 5.0 1.0 mL/min

H N HO

NH2

HO HO

5-HT

Dopamine

88% A; 2% B; 10% C 10 µL 30° C UV @ 280 nm Alliance® 2695 with 2996 PDA

NH2 OH

OH

H N

HO

O

HO

HO

N H

Epinephrine

5-HIAA OH HO

Compounds: 1. Norepinephrine (NE) 2. Epinephrine (E) 3. Dopamine (DA) 4. 3,4-Dihydroxyphenylacetic acid (DOPAC) 5. Serotonin (5-HT) 6. 5-Hydroxy-3-indoleacetic acid (5-HIAA) 7. 4-Hydroxy-3-methoxyphenylacetic acid (HVA)

USP Tailing 1.21 1.20 1.21 1.00 1.10 0.97 0.97

Sample Concentrations (µg/mL) 23 9 23 23 27 45 23

NH2

HO

Norepinephrine

HO O

HO

OH

DOPAC

OH

2 1

O HO

3

HVA

O

4

H3C

6

V0 = 1.83

5

7

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

26.00

28.00

30.00

Minutes

30


Catecholamines—Gradient LC/UV LC Conditions: Column: Part Number: Mobile Phase A:

H N

186001344 H2O

Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

ACN 100 mM HCOONH4, pH 3.0 1.0 mL/min Time Profile (min) %A %B 0.0 90 0

OH

HO HO

Compounds: 1. Norepinephrine (NE) 2. Epinephrine (E) 3. 3,4-Dihydroxyphenylalanine (DOPA) 4. Dopamine (DA) 5. 3-Methoxy-4-hydroxyphenylglycol (MHPG) 6. 4-Hydroxy-3-methoxymandelic acid (VMA) 7. Serotonin (5-HT) 8. 3,4-Dihydroxyphenylacetic acid (DOPAC) 9. 5-Hydroxy-3-indoleacetic acid (5-HIAA) 10. 4-Hydroxy-3-methoxyphenylacetic acid (HVA)

USP Tailing 1.16 1.17 1.15 1.04 1.16 1.17 1.10 1.09 1.11 1.12

NH2

HO O

HO HO

Sample Concentrations (µg/mL) 25 20 HO 20 25 25 HO 25 30 25 25 25

OH

HO

DOPAC

Dopamine OH

O

H N OH HO O

Epinephrine

OH

OH

O

OH

OH

NH2

HO

Norepinephrine

MHPG 9

HO

DOPA

N H

2

V0 = 1.83

7

8 10

6

1 3

2.00

HO

HO

NH2

0.00

HVA

O

O

5-HIAA

NH2

HO

OH

HO

VMA O

5-HT

%C 10

5.0 90 0 10 15.0 65 25 10 20.0 65 25 10 10 µL 30° C UV @ 280 nm Alliance® 2695 with 2996 PDA


O

HO


4.00

5

4

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

Minutes


31

Catecholamines—LC/ECD LC Conditions: Column: Part Number: Mobile Phase:

Flow Rate: Injection Volume: Sample Concentration: Temperature: Detection: Instrument:

Atlantis™ dC18 2.1 x 150 mm, 3 µm 186001299 3% MeOH 97% Buffer: 10.5 g/L Citric Acid 20 mg/L EDTA 20 mg/L Octanesulfonic acid 135 mg/L NaCl pH 2.9 with NaOH 0.25 mL/min 10 µL 2.0 ng/mL 40° C Electrochemical, +600 mV (Glassy Carbon vs. ISSAC) Alliance® 2695 with 2465 ECD

OH

HO

NH2

HO

H N

HO

HO

Dopamine

Epinephrine

OH HO

HO

NH2

NH 2 HO

HO

3,4-Dihydroxybenzylamine

Norepinephrine

Compounds: 1. Norepinephrine (NE) 2. Epinephrine (E) 3. 3,4-Dihydroxybenzylamine (DHBA, Internal Standard) 4. Dopamine (DA)

1.20

1

2

1.10 1.00

3

4

nA

0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

6.50

7.00

7.50

8.00

8.50

9.00

9.50

Minutes

32


Clonidine LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Isocratic Mobile Phase Conditions: Injection Volume: Sample Diluent: Sample Concentration: Temperature: Instrument:

Atlantis™ HILIC Silica 2.1 x 50 mm, 3 µm 186002011 200 mM NH4COOH, pH 3.0 ACN 0.3 mL/min

Cl H N

5% A; 95% B 5 µL 75:25 ACN:MeOH 10 ng/mL Ambient Alliance® HT 2795 with Waters ZQ™

MS Conditions: Ionization Mode: Capillary (kV):

ES+ 2.0

Cone (V): Extractor: RF Lens: Source Temperature (oC): Desolvation Temperature (oC):

40 3V 0.3 V 150 350

Cone Gas Flow (L/Hr): Desolvation Gas Flow (L/Hr): SIR m/z:

50 700 230.2

N

N H

Cl

Clonidine

1.0x10 7

Intensity

8.0x10 6

6.0x10 6

4.0x10 6

2.0x10 6

0.0 0.00

1.00

2.00

3.00

4.00

5.00

Minutes


33

Cold Medicine Products O

LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 0.1% TFA ACN 1.0 mL/min Time Profile (min) %A %B 0.0 77 23 1.0 77 23 3.0 52 48 10.0 52 48 10 µL 30° C UV @ 265 nm Alliance® 2695 with 2996 PDA


HN

Cl

N

N

OH

Chlorpheniramine

Acetaminophen O

N O

H N

Diphenhydramine Dextromethorphan Compounds: USP Tailing 1. Acetaminophen 1.04 2. Pseudoephedrine 1.11 3. Chlorpheniramine 1.04 4. Guaiacol glyceryl ether 1.01 5. Dextromethorphan 1.00 6. Diphenhydramine 1.08


NH

OH

Pseudophendrine

O

3

O

OH

1 4

OH

5

6

Guaiacol glyceryl ether

V0 = 1.83 2

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

34


Cytochrome C Tryptic Digest— HILIC vs. Reversed-Phase HILIC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:


100

Reversed-Phase Conditions: Column: Atlantis™ dC18 4.6 x 50 mm, 5 µm Part Number: 186001331 Mobile Phase A: 0.1% HCOOH in H2O Mobile Phase B: 0.1% HCOOH in ACN Flow Rate: 1.0 mL/min Gradient: Time Profile (min) %A %B 0.0 100 0 45.0 60 40 Injection Volume: 10 µL Sample Concentration: 1 µg/µL Temperature: 40° C Instrument: Alliance® 2690 with Waters ZQ™

Atlantis™ HILIC Silica 4.6 x 50 mm, 5 µm 186002028 90:10 ACN:NH4CH3COOH, pH 4.5 6.5 mM NH4CH3COOH, pH 4.5 1.0 mL/min Time Profile (min) %A %B 0.0 90 10 45.0 60 40 10 µL 1 µg/µL 40° C Alliance® 2690 with Waters ZQ™

204 (T2) 261 (T6,11) 361 (T18) 434 (T21)

ReversedPhase

Peak Annotation M/Z(Fragment ID)

Atlantis™ dC18

779 (T15) %

678(T14) 634 (T4)

729 (T10)

964 (T19)

1005(T12)

585 (T8) 0

634 (T4) 100

HILIC 779 (T15) %

678 (T14) 434 (T21)

Atlantis™ HILIC Silica 585 (T8)

261 (T6,11) 204 (T2)

729 (T10) 964 (T19) 1005(T12) 361 (T18) 0 0

45 Minutes


35

Dextromethorphan and N-Dealkylated Metabolite LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

Injection Volume: Temperature: Instrument: Sample:

Atlantis™ dC18 2.1 x 150 mm, 5 µm 186001301 0.1% HCOOH in H2O 0.1% HCOOH in ACN 0.3 mL/min Time Profile (min) %A %B 0.0 90 10 1.5 90 10 7.0 15 85 8.0 15 85 8.1 90 10 13.0 90 10 10 µL 30° C Alliance® HT 2795, Q-Tof micro™ ES+ Dextromethorphan rat microsomal incubation at 0.5 mM

CH3

CH3

O

O

H

H N

CH3

1. Dextromethorphan

NH

2. N-Dealkylated Dextromethorphan Metabolite

1

2

Minutes

36

©2004 Waters Corporation. Waters, Q-Tof micro, Alliance and Atlantis are trademarks of Waters Corporation.

Diazepam and Hydroxylated Metabolite LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

Injection Volume: Temperature: Instrument: Sample:

CH3

Atlantis™ dC18 2.1 x 150 mm, 5 µm 186001301 0.1 % HCOOH in H2O 0.1% HCOOH in ACN 0.3 mL/min Time Profile (min) %A %B 0.0 90 10 1.5 90 10 7.0 15 85 8.0 15 85 8.1 90 10 13.0 90 10 10 µL 30° C Alliance® HT 2795, Q-Tof micro™ ES+ Diazepam rat microsomal incubation at 1 mM

CH3

O

N

O

N OH

N

Cl

1. Diazepam

Cl

N

2. Hydroxylated Diazepam Metabolite

1

2

Minutes


37

Diuretics LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:



Chlorthalidone

Furosemide

Compounds: 1. Hydrochlorothiazide 2. Chlorthalidone 3. Furosemide

USP Tailing 0.94 0.99 0.99


Hydrochlorothiazide

3 2 1

V0 = 1.37

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

38


Drugs for Rhinitis LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

O

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O ACN 1% HCOOH, pH 2.3 1.4 mL/min Time Profile (min) %A %B %C 0.0 90 0 10 10.0 50 40 10 10 µL 30° C UV @ 254 nm Alliance® 2695 with 2996 PDA


Cl

N

N

O

N

N

N

N

Caffeine

Chlorpheniramine

Maleic acid

NH2

OH

Phenylpropanolamine

Compounds: 1. Phenylephrine 2. Maleic acid 3. Phenylpropanolamine 4. Caffeine 5. Chlorpheniramine

USP Tailing 1.10 1.17 1.26 1.05 1.26


Phenylephrine

4

5

2 1

V0 = 1.37 3

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes


39

Fluoroquinolone Antibiotics in Beef Kidney (LC/MS/MS) LC Conditions: Column: Part number: Mobile Phase A: Mobile Phase B: Flow rate: Gradient:

Oasis® MAX Extraction Method Oasis MAX Extraction Cartridge, 6 cc/150 mg, 30 µm Part Number: 186000369

Atlantis dC18, 4.6 x 150 mm, 5 µm 1860001344 0.2% NFPA* in H2O MeOH 0.8 mL/min Time Profile (min) %A %B 0.0 60 40 10.0 20 80 50 µL 30 ˚C Alliance® 2695 ™

Injection volume: Temperature: Instrument:

®

Prepare Sample: Extract 2 g of kidney with 30 mL of 50 mM phosphate buffer (pH 7.4); centrifuge at 10000 rpm for 10 min; take 5 mL allquot for SPE Condition: 1 mL methanol, 1 mL 5 N NaOH, 1 mL water N COOH N

HN

*NFPA- nonafluoropentanoic acid (NFPA) - C4F9COOH

Load: 5 mL of prepared sample

O F

Wash 1: 1 mL 5% ammonia in water

Ciprofloxacin

MS Conditions: Instrument: Ion Source: Mode: Corona: Source Temp: Desolvation Temp: Cone Gas (N2): Desolvation Gas (N2): Collision Gas:

Quattro micro™ APCI+ Multiple Reaction Monitoring 0.8 V 150 ˚C 625 ˚C 175 L/Hr 250 L/Hr Argon

H3C

N N

Wash 2: 1 mL methanol

N

F

COOH

Elute: 3 mL methanol (4% formic acid)

Enrofloxacin

9.32

flumequine

262.2 > 244.1

Compound

10µg/kg % Recovery (± % RSD)

50µg/kg % Recovery (± % RSD)

flumequin enoxacin norfloxacin

74 (± 9.1) 63 (± 5.7) 64 (± 8.1)

70 (± 17) 65 (± 13) 65 (± 9.9)

sarafloxacin ofloxacin enrofloxacin

68 (± 9.5) 72 (± 7.0) 73 (± 5.3)

71 (± 9.8) 80 (± 8.6) 76 (± 8.9)

danofloxacin lomefloxacin ciprofloxacin

64 (± 8.8) 76 (± 6.9) 70 (± 8.7)

68 (± 8.2) 76 (± 7.9) 62 (± 6.1)

7.67

enoxacin

321.2 > 303.1 7.87

norfloxacin

320.2 > 302.1 8.46

sarafloxacin

386.2 > 368.2 External standard calculation Results calculated against standards in matrix (n=5)

7.65

ofloxacin

362.2 > 344.1 8.06

enrofloxacin

360.2 > 342.1 8.02

danofloxacin

358.2 > 340.1 8.12

lomefloxacin

352.2 > 334.1 7.98

ciprofloxacin 5

40

332.2 > 314.1 6

7

8

9

10

11

12

Compound Energy(eV)

MW

(MRM)

flumequine enoxacin norfloxacin sarafloxacin ofloxacin enrofloxacin danofloxacin lomefloxacin ciprofloxacin

261 320 319 385 361 359 357 351 331

262 321 320 386 362 360 358 352 332

> > > > > > > > >

244 303 302 368 344 342 340 334 214

Cone(V)

Coll.

50 50 50 50 50 50 50 50 50

20 20 23 25 20 20 25 20 20

time

©2004 Waters Corporation. Waters, Oasis, Alliance, Quattro micro and Atlantis are trademarks of Waters Corporation.

Ginsenosides LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

Injection Volume: Sample Concentrations: Temperature: Detection: Instrument:

OH

OH OH Atlantis™ dC18 4.6 x 150 mm, 5 µm HO 186001344 H2O O HO ACN 1.0 mL/min O H3C CH3 Time Profile HO CH3 (min) %A %B CH3 0.0 75 25 CH3 4.0 60 40 10.0 60 40 OH O 10 µL O 63 µg/mL each

O HO

O

H3C CH3

OH

H3C

CH3

1. Ginsenoside Rg1

1.01

3. Ginsenoside Rb2 4. Ginsenoside Rd

1.13 0.99

OH

O O

CH3

H3C

HO

CH3

OH

CH3

OH

H3C

HO

O

CH3

H

H

OH

OH

CH3

OH H CH3

HO

1.02

2. Ginsenoside Rc

OH OH

Ginsenoside Rc

CH3

HO

OH

O

CH3CH3

OH

USP Tailing

O

H3C

O

Ginsenoside Rg1

Compounds:

OH

OH

O

HO HO

OH

OH

OH

OH

30° C UV @ 203 nm Alliance® 2695 with 2996 PDA

HO

O

OH

O

O

O

H

CH3

OH

Ginsenoside Rb2

O H H3C O H CH3 OH OH

O OH OH

CH3

H3C O

OH

OH

O

HO OH

CH3 HO

O

O OH

OH

CH3

OH

O

1

O OH

OH

2

CH3

OH H C 3

CH3

OH

Ginsenoside Rd 4

3

V0 = 1.83

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes


41

Guanine and Acyclovir— HILIC vs. Reversed-Phase HILIC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Injection Solvent: Sample Concentration: Temperature: Detection: Instrument:

Reversed-Phase Conditions: Column: Atlantis™ dC18 4.6 x 50 mm, 3 µm Part Number: 186001329 Mobile Phase A: 0.2% HCOOH in H2O Mobile Phase B: 0.2% HCOOH in ACN Flow Rate: 1.4 mL/min Gradient: Time Profile (min) %A %B %C 0.0 100 0 0 5.0 95 5 0 Injection Volume: 1 µL Sample Diluent: 0.02N NaOH Sample Concentration: 83 µg/mL Temperature: 30° C Detection: UV @ 205 nm Instrument: Alliance® 2695 with 2996 PDA

Atlantis™ HILIC Silica 4.6 x 50 mm, 3 µm 186002027 0.2% HCOOH in H2O 0.2% HCOOH in ACN 2.0 mL/min 10% A; 90% B 1 µL 60:40 ACN:0.02N NaOH 83 µg/mL 30° C UV @ 254 nm Alliance® 2695 with 2996 PDA

O N

HN

H2N

O

N

N

HN

OH

N

H N 2

N

N H

O

1. Acyclovir

2. Guanine

1 2

V0 = 0.5

HILIC


2 1

V0 = 0.65

0.00

0.50

ReversedPhase 1.00

1.50

Atlantis™ dC18

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes

42


Herbicides, (Five Polar Herbicides, 200 ng/mL) by LC/MS/MS LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow rate: Gradient:

Injection Volume: Instrument:

C2H5

Atlantis™ dC18, 2.1 x 100 mm, 3 µm 186001295 15mM HCOONH4, pH 4.5 ACN 200 µL/min Time Profile (min) %A %B 0.0 90 10 19.0 10 90 20 µL Alliance® 2690

COOH

N

H2 N

CH3

S

Imazethapyr (acid)

NH COOCH3

O

NH O

Asulam (base)

CH3

OCH 3 O

O S

Cl

N

NH

N

OCH 3

O

Chlorsulfuron (neutral)

Cl NH 2

HC

Picloram (acid)

Quattro Ultima™ Electrospray (ESI- and ESI+) Multiple Reaction Monitoring

N

C NH

COOH Cl

Cl

MS Conditions: Mass spectrometer Instrument:

O

H N CH3

HN

N NH2 N H

Amitrole (base)

200 ng/L Spiked River Water Sample 4 3

ESI-

Compounds 1. Amitrole (80%) 2. Picloram (75%) 3. Asulam (60%) 4. Imazethapyr (75%) 5. Chlorsulfuron (90%)

2

2.7

Compound

4.2

3.7

Picloram 3 5

ESI+

MW

Mode

(mrm)

cone (V)

Coll. Energy(ev)

241.5

ESI-

239-195

20

10

Imazethapyr

289

ESI-

288--244

25

15

Asulam

230

ESI-

229-197

30

10

Asulam

230

ESI+

231-156

30

10

Chlorsulfron

357

ESI+

358-141

20

17

Amitrole

84

ESI+

85-57

20

15

1

2.5

5.0

3.7 Minutes

©2004 Waters Corporation. Waters, Quattro Ultima and Atlantis are trademarks of Waters Corporation.

43

Lipid Soluble Antioxidants LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Mobile Phase D: Flow Rate: Gradient:

Injection Volume: Temperature:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O ACN 1% HCOOH, pH 2.3 MeOH 2.0 mL/min Time Profile (min) %A %B %C 0.0 55 17.5 10 4.0 35 27.5 10 6.0 4 45 10 15.0 4 45 10 10 µL 30° C

Detection: Instrument:

UV @ 280 nm Alliance® 2695 with 2996 PDA

Compounds: 1. Propyl gallate (PG) 2. 2,4,5-Trihydroxybutyrophenone (THBP) 3. Tert-butylhydroquinone (TBHQ) 4. Butylated hydroxyanisole (BHA) 5. 2,6-Di-tert-butyl-4-hydroxymethylphenol 6. Octyl gallate (OG) 7. Butylated hydroxytoluene (BHT) 8. Lauryl gallate (LG)

USP Tailing 1.08 1.09 1.01 1.02 1.06 1.11 0.95 1.16

Propyl gallate 2,4,5-Trihydroxybutyrophenone %D 17.5 27.5 4 4 Butylated hydroxyanisole

Tert-butylhydroquinone

Sample Concentrations (µg/mL) 10 10 19 38 38 19 38 23

Lauryl gallate

2,6-Di-tert-butyl-4-hydroxymethylphenol

6

Octyl gallate

4

8

2 1

V0 = 0.98

5 7

Butylated hydroxytoluene

3

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

13.00

14.00

15.00

Minutes

44


Morphine and Morphine-3--β-Glucuronide— HILIC vs. Reversed-Phase HILIC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

Injection Volume: Injection Solvent: Sample Concentration: Temperature: Detection: Instrument:

Reversed-Phase Conditions: Column: Atlantis™ dC18 4.6 x 50 mm, 3 µm Part Number: 186001329 Mobile Phase A: H2O Mobile Phase B: ACN Mobile Phase C: 100 mM NH4COOH, pH 3.0 Flow Rate: 1.4 mL/min Isocratic Mobile Phase Conditions: 88% A: 2% B: 10% C Injection Volume: 5 µL Injection Solvent 75:25 H2O:MeOH Sample Concentration: 125 µg/mL Temperature: 30° C Detection: UV @ 280 nm Instrument: Alliance® 2695 with 2996 PDA

Atlantis™ HILIC Silica 4.6 x 50 mm, 3 µm 186002027 H2O ACN 100 mM NH4COOH, pH 3.0 2.0 mL/min Time Profile (min) %A %B %C 0.0 0 90 10 5.0 40 50 10 5 µL 75:25 ACN:MeOH 125 µg/mL 30° C UV @ 280 nm Alliance® 2695 with 2996 PDA

COOH OO

HO

1 2

HO

O

HILIC

H

OH OH

NCH3

O

HO

H

NCH3

HO

Morphine

Morphine 3-ß-D-Glucuronide

V0 = 0.5


2

ReversedPhase 1

V0 = 0.65

Atlantis™ dC18

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes


45

Nalidixic Acid Antibiotics by LC/MS 1.0 Minute Gradient LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:


MS Conditions: Waters ZQ™ ES+ Capillary (kV) Cone (V) Extractor RF Lens Source Temp (˚C) Desolvation Temp (˚C) Cone Gas Flow (L/Hr) Desolvation Gas FLow(L/Hr) LM Resolution HM Resolution Ion Energy Multiplier (V)

Atlantis™ dC18, 2.1 x 20 mm IS™, 3 µm 186002058 H2O MeOH 1% HCOOH in H2O 0.4 mL/min Time Profile (min) %A %B %C 0.0 50 40 10 1.0 30 60 10 2 µL 10 µg/mL 30˚ C Alliance® HT 2795 with Waters ZQ™

The top figure is the total ion current, followed by the extracted ion signals for each of the three analytes. 0.88

*The “extra” peak in the cinoxacin panel is the isotope from oxolinic acid.

3.5 5.0 3.0 0.1 150 400 50 500 15 15 0.5 650

1: Scan ES+ TIC 1.15e9

0.65

Compounds 1. Cinoxacin 2. Oxolinic Acid 3. Nalidixic Acid


0.59

0 0.59 0.65

*

MW 262.2 261.2 232.2

1: Scan ES+ 263 4.05e7

Cinoxacin

Cinoxacin

0.88

O

1: Scan ES+ 263 4.05e7

O

O

O

0 O

1: Scan ES+ 262 1: Scan1.19e8 ES+

Oxolinic acid

262 1.19e8

Oxolinic acid

O

N

0.65

N

N HO

HO

O

O

Oxolinic acid

Cinoxacin

0 0.89

1: Scan ES+ 233 1.28e8

Nalidixic Nalidixic acid acid 0.00

0.20

0.40

1: Scan ES+ 233 1.28e8

0.60

0.80

N

N

OH

O

O

Nalidixic acid

1.00

Minutes

46

©2004 Waters Corporation. Waters, IS, Alliance, ZQ and Atlantis are trademarks of Waters Corporation.



MS Conditions: Waters ZQ™ ES+ Capillary (kV) Cone (V) Extractor RF Lens Source Temp (˚C) Desolvation Temp (˚C) Cone Gas Flow (L/Hr) Desolvation Gas FLow(L/Hr) LM Resolution HM Resolution Ion Energy Multiplier (V)

Atlantis™ dC18, 2.1 x 20 mm IS™, 3 µm 186002058 H2O MeOH 1% HCOOH in H2O 0.4 mL/min Time Profile (min) %A %B %C 0.0 60 30 10 2.0 40 50 10 2 µL 10 µg/mL 30˚ C Alliance® HT 2795 with Waters ZQ™

The top figure is the total ion current, followed by the extracted ion signals for each of the three analytes.

1: Scan ES+ 1.62

TIC 2.44e9

*The “extra” peak in the cinoxacin panel is the isotope from oxolinic acid.


1.15 0.98

0.98

3.5 5.0 3.0 0.1 150 400 50 500 15 15 0.5 650


1: Scan ES+ 263 8.37e7

1.15*

MW 262.2 261.2 232.2

Cinoxacin 1: Scan ES+ 263 8.37e7

Cinoxacin

1: Scan ES+ 262 2.31e8

1.15

O

O

O

O

O

N

N

N HO

HO

O

O

Oxolinic acid

1: Scan ES+ 262 2.31e8

Oxolinic acid

1: Scan ES+ 233 2.74e8

1.62

Oxolinic acid

Cinoxacin

N

N

OH

Nalidixic acid 1: Scan ES+ 233 2.74e8

Nalidixic acid 0.00

0.25

0.50

O

0.75

1.00

1.25

1.50

1.75

O

O

Nalidixic acid

2.00

Minutes


47



MS Conditions: Waters ZQ™ ES+ Capillary (kV) Cone (V) Extractor RF Lens Source Temp (˚C) Desolvation Temp (˚C) Cone Gas Flow (L/Hr) Desolvation Gas FLow (L/Hr) LM Resolution HM Resolution Ion Energy Multiplier (V)

Atlantis™ dC18, 2.1 x 20 mm IS™, 3 µm 186002058 H2O MeOH 1% HCOOH in H2O 0.4 mL/min Time Profile (min) %A %B %C 0.0 60 30 10 3.0 40 50 10 2 µL 10 µg/mL 30˚C Alliance® HT 2795 with Waters ZQ™

3.5 5.0 3.0 0.1 150 400 50 500 15 15 0.5 650

The top figure is the total ion current, followed by the extracted ion signals for each of the three analytes. *The “extra” peak in the cinoxacin panel is the isotope from oxolinic acid.

1.09

1.95

1: Scan ES+ TIC 4.72e9 1: Scan ES+ TIC 4.72e9

1.33

0 1.10

Cinoxacin Cinoxacin

1.33

*

Compounds 1. Cinoxacin

MW 262.2

2. Oxolinic Acid

261.2

3. Nalidixic Acid

232.2

1: Scan ES+ 263 2.08e8

1: Scan ES+ 263 2.08e8

O

O

O

O

0 1.33

Oxolinic acid

1: Scan ES+ 262 3.11e8

O

N

N

N HO

HO

O

O

1: Scan ES+ 262 3.11e8

Oxolinic acid

O

Oxolinic acid

Cinoxacin

0

1: Scan ES+ 233 4.87e8 1: Scan ES+ 233 4.87e8

Nalidixic acid

Nalidixic acid 0 0.00

N

N

OH

O

O

Nalidixic acid 0.50

1.00

1.50

2.00

2.50

3.00

Minutes

48




MS Conditions: Waters ZQ™ ES+ Capillary (kV) Cone (V) Extractor RF Lens Source Temp (˚C) Desolvation Temp (˚C) Cone Gas Flow (L/Hr) Desolvation Gas FLow (L/Hr) LM Resolution HM Resolution Ion Energy Multiplier (V)

Atlantis™ dC18, 2.1 x 20 mm IS™, 3 µm 186002058 H2O MeOH 1% HCOOH in H2O 0.2 mL/min Time Profile (min) %A %B %C 0.0 60 30 10 5.0 40 50 10 5 µL 10 µg/mL 30˚C Alliance® HT 2795 with Waters ZQ™

3.5 5.0 3.0 0.1 150 400 50 500 15 15 0.5 650

The top figure is the total ion current, followed by the extracted ion signals for each of the three analytes. *The “extra” peak in the cinoxacin panel is the isotope from oxolinic acid.

1: Scan ES+ 1: Scan ES+ TIC TIC 2.94e9 2.94e9

3.54 2.44

2.05

2.44 2.05

*


MW 262.2

2. Oxolinic Acid

261.2

3. Nalidixic Acid

232.2

1: Scan 1: Scan ES+ES+ 263263 1.32e8 1.32e8

Cinoxacin

O

O

O

O

Cinoxacin O

2.44

1: Scan ES+ES+ 1: Scan 262262 4.07e8 4.07e8

Oxolinic acid

O

N

N

N HO

HO

O

O

Oxolinic acid

Cinoxacin Oxolinic acid 1: Scan 1: Scan ES+ES+ 233233 2.91e8 2.91e8

3.54

Nalidixic acid

N

N

OH

O

Nalidixic acid 0.00

0.50

1.00

O

Nalidixic acid 1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes


49

Nalidixic Acid Antibiotics —LC/MS LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:


Atlantis™ dC18 2.1 x 30 mm, 3 µm 186001287 H2O MeOH 1% HCOOH in H2O 0.8 mL/min with 0.2 mL/min to MS Time Profile (min) %A %B %C 0.0 75 15 10 5.0 40 50 10 10 µL 20 µg/mL 30° C UV @ 254 nm and MS ESI+ Alliance® 2795 with 2996 PDA and Waters ZQ™

Waters ZQ Settings: Source: ESI+ Capillary (kV) Cone (V) Extractor RF Lens Source Temp Desolvation Temp Cone gas Flow (L/Hr) Desolvation Gas Flow (L/Hr) LM Resolution HM Resolution Ion Energy Multiplier (V)

3

100

Scan ES+ TIC 5.35e9

3.5 50 3.0 0.1 150 400 50 500 15 15 0.5 650


MW 262.2

2. Oxolinic Acid

261.2

3. Nalidixic Acid

232.2

O

%

1

O

O

O

2

O

O

N

N

N HO

HO

0

O

O

100

Oxolinic acid

Cinoxacin

Diode Array 254nm 5.22e4

%

N

N

OH

O

O

Nalidixic acid

-16 0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes

50


Nalidixic Acid Antibiotics —Isocratic LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Mobile Phase D: Flow Rate: Isocratic MobilePhase Composition: Injection Volume: Sample Concentration: Temperature: Detection: Instrument:

Atlantis™ dC18, 4.6 x 20 mm IS™, 3 µm, 186002062 H2O MeOH ACN 1% HCOOH in H2O 4 mL/min 55% A, 30% B, 5% C, 10% D 5 µL 5 µg/mL 30˚ C UV @ 254 nm Alliance® 2965 with 2996 PDA


O

MW 262.2 261.2 232.2

O

O

O

O

O

N

N

N HO

HO

O

O

Oxolinic acid

Cinoxacin 2

V0 = 0.09 1

3

N

N

OH

O

O

Nalidixic acid

0.00

0.50

1.00

1.50

2.00

Minutes Minutes

©2004 Waters Corporation. Waters, IS, Alliance and Atlantis are trademarks of Waters Corporation.

51

Nucleic Acid Bases—Gradient Effect of Polar Solvent on Retention in HILIC LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:


H N

Atlantis™ HILIC Silica, 4.6 x 50 mm, 3 µm 186002027 H2O, MeOH, or IPA ACN 200 mM NH4COOH, pH 3.0 1.0 mL/min Time Profile (min) %A %B %C 0.0 5 90 5 5.0 45 50 5 2 µL 75:25 ACN:MeOH 25 µg/mL 30° C UV @ 260 nm Alliance® 2695 with 2996 PDA

H N

O N

O N

F

NH2

NH2 Cytosine

5-Fluorocytosine

O

H N

O

F

HN

NH

O

O Uracil

N H

5-Fluorouracil

Compounds 1. 5-Fluorouracil 2. Uracil 3. 5-Fluorocytosine 4. Cytosine

2 4

Initial 90 ACN: 5 H2O: 5 buffer

1

Final 50 ACN: 45 H2O: 5 buffer

3

Replace H2O with MeOH 2 1

Initial 90 ACN: 5 MeOH: 5 buffer Final 50 ACN: 45 MeOH: 5 buffer

4 3

Replace MeOH with IPA 2 1

Initial 90 ACN: 5 IPA: 5 buffer Final 50 ACN: 45 IPA: 5 buffer 4

3

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes

52


Nucleic Acid Bases— HILIC vs. Reversed-Phase HILIC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:


Reversed-Phase Conditions: Column: Atlantis™ dC18 4.6 x 50 mm, 3 µm Part Number: 1860001329 Mobile Phase: 10 mM CH3COONH4 pH 5.0 Flow Rate: 1.4 mL/min Isocratic Mobile Phase Conditions: 100% A Injection Volume: 2 µL Injection Solvent: H2O Sample Concentration: 25 µg/mL Temperature: 30° C Detection: UV @ 254 nm Instrument: Alliance® 2695 with 2996 PDA

Atlantis™ HILIC Silica 4.6 x 50 mm, 3 µm 186002027 MeOH ACN 200 mM CH3COONH4, pH 5.0 2.0 mL/min Time Profile (min) %A %B %C 0.0 3 95 2 5.0 40 50 10 2 µL 75:25 ACN:MeOH 25 µg/mL 30° C UV @ 254 nm Alliance® 2695 with 2996 PDA

H N

H N

O N

O N

F

NH2

NH2 Cytosine

5-Fluorocytosine


O

H N

O NH

O

O Uracil

4

2

F

HN N H

5-Fluorouracil

3

1

HILIC

V0 = 0.5


4 2

Reversed-Phase

3 1

V0 = 0.65

0.20

0.40

0.60

Atlantis™ dC18 0.80

1.00

1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

Minutes


53

Nucleic Acid Bases —Gradient LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

H N

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O ACN 100 mM CH3COONH4, pH 5.0 1.0 mL/min Time Profile (min) %A %B %C 0.0 90 0 10 10.0 84 6 10 10 µL 30° C UV @ 254 nm Alliance® 2695 with 2996 PDA


H N

O N

N

F

NH2

NH2 Cytosine

5-Fluorocytosine

O

H N

O

F

HN

NH

O

O Uracil

N H

5-Fluorouracil

H N

O H N USP Tailing 1.11

Sample Concentrations (µg/mL) 82

2. 5-Fluorocytosine 3. Uracil 4. 5-Fluorouracil

1.08 1.05 1.18

82 4 41

5. Guanine 6. Thymine

1.16 1.18

10 82

7. Adenine

1.25

164

Compounds: 1. Cytosine

NH

N

O

Guanine

NH2

O NH

H3C

NH2

N

HN

O

Thymine

N N

N Adenine

7 4

1 5

2

6

3

V0 = 1.83

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

54


Nucleic Acid Bases —Isocratic LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Injection Solvent: Sample Concentration: Temperature: Detection: Instrument:

H N

Atlantis™ HILIC Silica, 4.6 x 50 mm, 3 µm 186002027 100 mM NH4COOH ACN 1.0 mL/min

H N

O N

N

F

NH2

NH2 Cytosine

12% A; 88% B 10 µL 75:25 ACN:MeOH 25 µg/mL 30° C UV @ 254 nm Alliance® 2695 with 2996 PDA

O

5-Fluorocytosine

O

H N

O

F

HN

NH

O

O Uracil

N H

5-Fluorouracil


2

3

1

0.00

0.50

1.00

1.50

4

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes


55

Nucleosides LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:


Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O ACN 1% HCOOH, pH 2.3 1.4 mL/min Time Profile (min) %A %B %C 0.0 90 0 10 10.0 82 8 10 10 µL 19 µg/mL 30° C UV @ 260 nm Alliance® 2695 with 2996 PDA

2’-Deoxycytidine Cytidine

2’-Deoxyadenosine

Uridine

2’-Deoxyguanosine

Compounds:

USP Tailing

1. Cytidine

1.05

2. 2’-Deoxycytidine

1.02

3. Uridine 4. Adenosine

1.08 1.07

5. 2’-Deoxyadenosine

1.08

6. Guanosine 7. 2’-Deoxyguanosine

1.09 1.10 1.10

8. Thymidine

Adenosine

Thymidine Guanosine

5 4

3

1

7 6 8 2

V0 = 1.37

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

56


Organic Acids LC Conditions: Column: Part Number: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Temperature: Detection: Instrument:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 0.5 mL/min 20 mM NaH2PO4, pH 2.7 10 µL 30° C UV @ 210 nm Alliance® 2695 with 2996 PDA

Oxalic acid

Malic acid

O

OH Acetic acid Pyruvic acid

H

Succinic acid

Compounds: 1. Oxalic acid 2. Pyruvic acid 3. Tartaric acid 4. Malic acid 5. Lactic acid 6. Acetic acid 7. Maleic acid 8. Fumaric acid 9. Succinic acid

Sample Concentrations (µg/mL) 25 30 15 61 243 243 3 3 158

USP Tailing 1.35 1.12 1.13 1.19 1.24 1.30 1.24 1.23 1.26

Lactic acid Tartaric acid

Maleic acid

Fumaric acid

3

1 7 8

V0 = 3.51 5 4

6

2

0.00

2.00

4.00

6.00

9

8.00

10.00

12.00

14.00

16.00

18.00

20.00

Minutes


57

Oxidative Stress Markers —5% ACN, pH 3.0 LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Temperature: Detection: Instrument:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O ACN 100 mM HCOONH4, pH 3.0 1.4 mL/min

2’-Deoxyguanosine

85% A; 5% B; 10% C 10.0 µL 30° C UV @ 280 nm Alliance® 2695 with 2996 PDA

Compounds: 1. 2’-Deoxyguanosine 2. 8-Hydroxy-2’-deoxyguanosine

USP Tailing 1.04 0.99

Sample Concentrations (µg/mL) 75 75

8- Hydroxy-2’-Deoxyguanosine

1

2

V0 = 1.37

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

58


Oxidative Stress Markers —5% MeOH, pH 5.0 LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Temperature: Detection: Instrument:

Atlantis™ dC18 4.6 x 100 mm, 3 µm 186001337 H2O MeOH 100 mM CH3COONH4, pH 5.0 1.0 mL/min

2’-Deoxyguanosine


Compounds: 1. 2’-Deoxyguanosine 2. 8-Hydroxy-2’-deoxyguanosine




1

2

V0 = 1.83

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

Minutes


59

Oxidative Stress Markers —10% MeOH, pH 5.0 LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Temperature: Detection: Instrument:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O MeOH 100 mM CH3COONH4, pH 5.0 1.0 mL/min

2’-Deoxyguanosine


Compounds: 1. 2’-Deoxyguanosine

USP Tailing 1.15


1.11

75

2. 8-Hydroxy-2’-deoxyguanosine


1

2

V0 = 1.83

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

60


Paclitaxel LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Sample Concentration: Temperature: Detection: Instrument:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O ACN 100 mM HCOONH4, pH 3.0 1.4 mL/min 30% A; 60% B; 10% C 2 µL 0.25 µg/mL 30° C UV @ 238 nm Alliance® 2695 with 2996 PDA

Paclitaxel

V0 = 1.38 USP Tailing = 1.01

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes


61

Peptides LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

Atlantis™ dC18 4.6 x 50 mm, 5 µm 186001331 0.02% TFA in H2O 0.016% TFA in ACN 0.75 mL/min Time Profile (min) %A %B 0.0 100 0 25.0 50 50 20 µL 40° C 18 µg UV @ 220 nm Alliance® 2690 with 2996 PDA

Injection Volume: Temperature: Total Mass Load: Detection: Instrument:

Compounds: 1. L2275 2. A6677 3. Bradykinin 4. Angiotensin II 5. Angiotensin I 6. Substance P 7. Renin Substrate 8. Insulin B Chain 9. Melittin

7

4

5

6

8 9

3

V0 = 0.79

1 2

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

Minutes

62


Phenones— 2.1 x 20 mm IS™ Column LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

O

Atlantis™ dC18 2.1 x 20 mm IS™, 3 µm 186002058 0.1% HCOOH in Water 0.1% HCOOH in ACN 0.6 mL/min Time Profile (min) %A %B 0.0 100 0 4.0 0 100 5 µL 20 µg/mL 30° C UV @ 254 nm Alliance® 2795 with 996 PDA


O

H N

H3C N

N

N

O

O

O

CH3

Theophylline

2-Acetylfuran

O

CH3

Acetophenone

Propiophenone

Butyrophenone

O

O

O

Heptanophenone

O

Benzophenone

6. Butyrophenone 7. Benzophenone 8. Valerophenone

NH

Acetanilide

O

O

Octanophenone Compounds: 1. Theophylline 2. 2-Acetylfuran 3. Acetanilide 4. Acetophenone 5. Propiophenone

H3C

O

Hexanophenone

Valerophenone

9. Hexanophenone 10. Heptanophenone 11. Octanophenone

2 7

V0 = 0.17

4

1

6 8

11

9

5

10

3

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Minutes


63

Phenones— 4.6 x 20 mm IS™ Column Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

O

Atlantis™ dC18 4.6 x 20 mm IS™, 3 µm 186002062 0.1% HCOOH in Water 0.1% HCOOH in ACN 3.0 mL/min Time Profile (min) %A %B 0.0 100 0 4.0 0 100 10 µL 20 µg/mL 30° C UV @ 254 nm Alliance® 2795 with 996 PDA


O

H N

H3C N

N

N

O

O

O

CH3

Theophylline

Acetanilide

O

O

O

CH3

Acetophenone

Propiophenone

Butyrophenone

O

O

O

Heptanophenone

O

Benzophenone

6. Butyrophenone 7. Benzophenone 8. Valerophenone

NH

2-Acetylfuran

Octanophenone Compounds: 1. Theophylline 2. 2-Acetylfuran 3. Acetanilide 4. Acetophenone 5. Propiophenone

H3C

O

Hexanophenone

Valerophenone

9. Hexanophenone 10. Heptanophenone 11. Octanophenone

7 2

4

6

11

8

V0 = 0.15

1

9

5

10

3

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Minutes

64


Phthalic Acids Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O MeOH 100 mM HCOONH4, pH 3.75 1.4 mL/min Time Profile (min) %A %B %C 0.0 55 35 10 5.0 30 60 10 10 µL 30° C UV @ 280 nm Alliance® 2695 with 2996 PDA


Isophthalic acid Phthalic acid

Terephthalic acid

Compounds: 1. Phthalic acid 2. Terephthalic acid 3. Isophthalic acid

USP Tailing 1.10 1.03 1.00


1 2

3

V0 = 1.37

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes


65

Phthalic Esters Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

Injection Volume: Sample Concentrations: Temperature: Detection: Instrument:

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O ACN 1.0 mL/min Time Profile (min) %A %B 0.0 10 90 4.0 0 100 10 0 100 10.0 µL 200 µg/mL each 30° C UV @ 285 nm Alliance® 2695 with 2996 PDA

Dibutyl phthalate

Dimethyl phthalate

Compounds:

USP Tailing

1. Dimethyl phthalate

1.06

2. Dibutyl phthalate

1.04

3. Dioctyl phthalate

0.99 Dioctyl phthalate

1

2

V0 = 1.83 3

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

66


Polar and Non-Polar Compounds— Analytical to Preparative Scale Up Conditions: Mobile Phase A: Mobile Phase B: Sample Concentration: Temperature: Instrument: Detection:

OH

0.1% HCOOH in H2O ACN/1% HCOOH (90/10) 10 mg/mL each in DMSO Ambient AutoPurification™ System UV @ 260 nm

O

H3C

O

NH

OH

HO

Acetanilide

H

H

H

O

Hydrocortisone

Compounds: 1. Acetanilide 2. Hydrocortisone 3. Acenaphthene

Acenaphthene

Atlantis™ dC18 4.6 x 100 mm, 5 µm 186001340 1 2 2,700 µg



3

V0 = 1.76 Injection Volume:

Atlantis™ dC18 Prep OBD™ 19 x 100 mm, 5 µm


186002116 48,000 µg


1 2

3

V0 = 1.74 Injection Volume:

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

1.0 mL/min Time Profile (min) %A 0.0 95 1.0 95 14.0 0 20.0 0 90 µL

17.06 mL/min Profile Time (min) %A 95 0.0 95 3.02 16.02 0 22.02 0 1600 µL

%B 5 5 100 100

%B 5 5 100 100

22.00

Minutes


67

Polar and Non-Polar Compound Test Mix— 2.1 x 20 mm IS™ Column Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

Atlantis™ dC18 2.1 x 20 mm IS™, 3 µm 186002058 0.1% HCOOH in H2O 0.1% HCOOH in ACN 0.6 mL/min Time Profile (min) %A %B 0.0 100 0 4.0 0 100 5 µL 20 µg/mL 30° C UV @ 254 nm Alliance® HT 2795 with 996 PDA


2-Bromofluorene 8-Bromoguanosine O CI

CN

N

O

NH2

2-Amino-7chloro-5-oxo-5H-[1]benzopyrano[2,3-b]pyridine-3-carbonitrile Triamcinolone

Compounds:

USP Tailing

1. 8-bromoguanosine 2. Acetanilide

1.15 1.01

3. Triamcinolone 4. Hydrocortisone 5. 2-Amino-7-chloro-5-oxo-5H[1]benzopyrano [2,3-b]pyridine-3-carbonitrile 6. 6α-Methyl-17α-hydroxyprogesterone 7. 3-Aminofluoranthene 8. 2-Bromofluorene 9. Perylene 10. Naphtho(2,3-a)pyrene

0.99 1.01 6α-Methyl-17α-hydroxyprogesterone Perylene

3-Aminofluoranthene

1.02 0.96 1.01 1.03 1.01 1.03

Acetanilide

Naphtho(2,3-a)pyrene

4

Hydrocortisone 9 2

V0 = 0.25

8 5

1

6 7

3

10

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Minutes

68


Polar and Non-Polar Compound Test Mix— 4.6 x 20 mm IS™ Column Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:




CN

N

O

NH2


Compounds:

USP Tailing

1. 2. 3. 4. 5.

8-bromoguanosine Acetanilide Triamcinolone Hydrocortisone 2-Amino-7-chloro-5-oxo-5H[1]benzopyrano [2,3-b]pyridine-3-carbonitrile 6. 6α-Methyl-17α-hydroxyprogesterone 7. 3-Aminofluoranthene 8. 2-Bromofluorene 9. Perylene 10. Naphtho(2,3-a)pyrene

1.15 1.01 0.99 1.01

6α-Methyl-17α-hydroxyprogesterone Perylene

3-Aminofluoranthene

1.02 0.96 1.01 1.03 1.01 1.03

Acetanilide


4

Hydrocortisone

V0 = 0.17 1

2

7

3

0.50

1.00

9

8

6

5

1.50

10

2.00

2.50

3.00

3.50

4.00

Minutes


69

Polar and Non-Polar Compound Test Mix— 2.1 x 30 mm Column Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

Atlantis™ dC18 2.1 x 30 mm, 3 µm 186001287 0.1% HCOOH in H2O 0.1% HCOOH in ACN 0.8 mL/min Time Profile (min) %A %B 0.0 100 0 0.2 100 0 3.2 0 100 4.0 0 100 2 µL Ambient UV @ 254 nm 10 pts/sec Alliance® 2795 with 2996 PDA

Injection Volume: Temperature: Detection: Sampling Rate: Instrument:


CN

N

O

3-Aminofluoranthene

NH2


Acetanilide 6α-Methyl-17α-hydroxyprogesterone Compounds: 1. 8-bromoguanosine 2. Acetanilide



0.99 1.01

31 12

1.02 0.96 1.01

12 25 31

1.03 1.01 1.03

16 12 12

3. Triamcinolone 4. Hydrocortisone 5. 2-Amino-7-chloro-5-oxo-5H[1]benzopyrano [2,3-b]pyridine-3-carbonitrile 6. 6α-Methyl-17α-hydroxyprogesterone 7. 3-Aminofluoranthene 8. 2-Bromofluorene 9. Perylene 10. Naphtho(2,3-a)pyrene

Perylene


7

Hydrocortisone

2

0.40

10

1

V0 = 0.20

0.20

9

8

6

3

0.60

0.80

1.00

1.20

4 5

1.40

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

3.20

3.40

3.60

3.80

4.00

Minutes

70


Polar and Non-Polar Compound Test Mix— 4.6 x 150 mm Column Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:


Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 0.1% HCOOH in H2O 0.1% HCOOH in ACN 2.0 mL/min Time Profile (min) %A %B 8-Bromoguanosine 0.0 100 0 0.5 100 0 10.0 0 100 15.0 0 100 10 µL Ambient UV @ 254 nm Triamcinolone Alliance® 2695 with 2996 PDA

2-Bromofluorene

O CI

CN

O

N

3-Aminofluoranthene

NH2

2-Amino-7chloro-5-oxo-5H-[1]benzopyrano[2,3-b]pyridine-3-carbonitrile

Acetanilide 6α-Methyl-17α-hydroxyprogesterone Compounds: 1. 8-bromoguanosine 2. Acetanilide 3. Triamcinolone 4. Hydrocortisone 5. 2-Amino-7-chloro-5-oxo-5H[1]benzopyrano [2,3-b]pyridine-3-carbonitrile 6. 6α-Methyl-17α-hydroxyprogesterone 7. 3-Aminofluoranthene 8. 2-Bromofluorene 9. Perylene 10. Naphtho(2,3-a)pyrene



0.99 1.01

31 12

1.02 0.96 1.01

12 25 31

1.03 1.01 1.03

16 12 12

Perylene


7

Hydrocortisone

9 2

V0 = 0.98

8

1 3

10

6

4 5

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

13.00

14.00

15.00

Minutes


71

Polar and Non-Polar Compound Test Mix— (with Uracil), 4.6 x 150 mm Column Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

O

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 0.1% HCOOH in H2O 0.1% HCOOH in ACN 2.0 mL/min Time Profile (min) %A %B 0.0 100 0 0.5 100 0 10.0 0 100 15.0 0 100 10 µL Ambient UV @ 254 nm Alliance® 2695 with 2996 PDA


HN O

2-Bromofluorene

N H Uracil

O CI

CN

O

N

3-Aminofluoranthene

NH2


Acetanilide 6α-Methyl-17α-hydroxyprogesterone Compounds: 1. Uracil 2. Acetanilide 3. Triamcinolone 4. Hydrocortisone 5. 2-Amino-7-chloro-5-oxo-5H[1]benzopyrano [2,3-b]pyridine-3-carbonitrile 6. 6α-Methyl-17α-hydroxyprogesterone 7. 3-Aminofluoranthene 8. 2-Bromofluorene 9. Perylene 10. Naphtho(2,3-a)pyrene

USP Tailing 1.15


1.01 0.99 1.01

19 31 12

1.02 0.96 1.01

12 25 31

1.03 1.01 1.03

16 12 12

Perylene


7

Hydrocortisone 9

2

1

3

V0 = 0.98

0.00

1.00

8

10

6 4

2.00

3.00

4.00

5.00

5

6.00

7.00

8.00

9.00

10.00

11.00

12.00

13.00

14.00

15.00

Minutes

72


Polar and Non-Polar Compound Test Mix— (with Uracil) 4.6 x 150 mm Column Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

O

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O ACN 100 mM HCOONH4, pH 3.0 2.0 mL/min Time Profile (min) %A %B %C 0.0 80 10 10 10.0 0 95 5 15.0 0 95 5 5 µL 30° C UV @ 254 nm Alliance® 2695 with 2996 PDA


HN O

2-Bromofluorene

N H Uracil

O CI

CN

O

N

3-Aminofluoranthene

NH2


Acetanilide 6α-Methyl-17α-hydroxyprogesterone Compounds: USP Tailing 1. Uracil 1.15 2. Acetanilide 1.01 3. Triamcinolone 0.99 4. Hydrocortisone 1.01 5. 2-Amino-7-chloro-5-oxo-5H[1]benzopyrano 1.02 [2,3-b]pyridine-3-carbonitrile 6. 6α-Methyl-17α-hydroxyprogesterone 0.96 7. 3-Aminofluoranthene 8. 2-Bromofluorene 9. Perylene 10. Naphtho(2,3-a)pyrene

Perylene

Sample Concentrations (µg/mL) 31 19 31 12

Naphtho(2,3-a)pyrene 12

1.01 1.03

25 31 16

1.01 1.03

22 22

7

Hydrocortisone

8

9

2 3

4

1

0.00

1.00

10

6

V0 = 0.98

2.00

3.00

4.00

5

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

13.00

14.00

15.00

Minutes


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Polar Pesticides LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Isocratic Mobile Phase Composition: Injection Volume: Sample Concentration: Temperature: Detection: Instrument:

Atlantis™ dC18 2.1 x 100 mm, 3 µm 186001295 H2O ACN 0.17 mL/min 95% A; 5% B 20 µL 10 ppb 25˚ C MS(ESI+) Alliance® 2795 with Waters ZQ™

MS Conditions: ESI+ Capillary (kV) Extractor RF Lens Source Temp Desolvation Temp

3.1 3.0 0.1 150 350

Desolvation Gas Flow (L/Hr) LM Resolution HM Resolution Ion Energy Multipler (V)

500 12.5 12.5 1.0 650

Methamidophos

Acephate

SIR of 5 Masses mass (m/z) 112a 113b 142a 143b 184b

cone (V) 40 40 20 25 15

ion for methamidophos ion for acephate

a b

2

Compounds: 1. Methamidophos 2. Acephate

1

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

Minutes

74


Profens—Gradient 4.6 x 20 mm IS™ Column LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:


CH3

O


O O

O

OH

OH

CH3

Ketoprofen

O

Fenoprofen

CH3

OH

O CH3

CH3

OH H3C

Ibuprofen

F

Flurbiprofen

Compounds: 1. Ketoprofen 2. Fenoprofen 3. Flurbiprofen 4. Ibuprofen

2

1 3

V0 = 0.14

0.20

0.40

0.60

0.80

1.00

1.20

1.40

4

1.60

1.80

2.00

Minutes


75

Profens—Isocratic 4.6 x 20 mm IS™ Column LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Isocratic Mobile Phase Conditions: Injection Volume: Sample Concentration: Temperature: Detection: Instrument:

CH3

O

Atlantis™ dC18 4.6 x 20 mm IS™, 3 µm 186002062 0.1% HCOOH in H2O 0.1% HCOOH in ACN 3.0 mL/min

O O

O

OH

OH

CH3

Ketoprofen

65% A, 35% B 10 µL 20 µg/mL 30° C UV @ 220 nm Alliance® 2695 with 2996 PDA

O

Fenoprofen

CH3

OH

O CH3

CH3

OH H3C

Ibuprofen

F

Flurbiprofen

Compounds: 1. Ketoprofen 2. Fenoprofen 3. Flurbiprofen 4. Ibuprofen

2

1

V0 = 0.14

0.50

3

1.00

1.50

2.00

4

2.50

3.00

3.50

4.00

Minutes

76


Purine Alkaloids LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:



Theobromine Theophylline

O N

N

O

Compounds: 1. Theobromine 2. Theophylline 3. Caffeine 4. Phenol

USP Tailing 1.00 1.00 0.98 0.97

Sample Concentrations (µg/mL) 42 50 42 42

N

N

Phenol

Caffeine

2

1

3

V0 = 0.98 4

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes


77

Steroids— 2.1 x 20 mm IS™ Column LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:


Atlantis™ dC18 2.1 x 20 mm IS™, 3 µm 186002058 H2O ACN 0.6 mL/min Time Profile (min) %A %B 0.0 80 20 4.0 36 64 5 µL 20 µg/mL 30° C UV @ 220 nm Alliance® 2795 with 2996 PDA

O

O OH

CH3

HO CH3

OH

O

H

H

CH3

CH3

H

H

H

O

H

O

Prednisolone

Cortisone

O OH

CH3

CH3 H

H

CH3

H

H

H

HO

O

Corticosterone Compounds: 1. Prednisolone

Estradiol

CH3

H3C

O

O CH3

2. Cortisone H

3. Corticosterone 4. Estradiol 5. Estrone

CH3

H

H

6. Progesterone

H

O

Estrone

0.50

H H

HO

V0 = 0.32

OH

HO

H

2

OH OH

Progesterone

3

1

5 4

1.00

1.50

6

2.00

2.50

3.00

3.50

4.00

Minutes

78


Steroids— 4.6 x 20 mm IS™ Column LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:


Atlantis™ dC18 4.6 x 20 mm IS™, 3 µm 186002062 H2O ACN 3.0 mL/min Time Profile (min) %A %B 0.0 80 20 4.0 36 64 10 µL 20 µg/mL 30° C UV @ 220 nm Alliance® HT 2795 with 2996 PDA

O

O OH

CH3

HO CH3

OH

O

H

H

CH3

CH3

H

H

H

O

H

O

Prednisolone

Cortisone

O OH

CH3

CH3 H

H

CH3

H

H

H

HO

O

Corticosterone Compounds: 1. Prednisolone

Estradiol

CH3

H3C

O

O CH3

2. Cortisone H

3. Corticosterone 4. Estradiol 5. Estrone

H

CH3

H

6. Progesterone

H

O

Estrone

3

H H

HO

V0 = 0.17

OH

HO

H

2

OH OH

Progesterone

5

1

4 6

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Minutes


79

Sulfonamides— 2.1 x 20 mm IS™ Column LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:


O

Atlantis™ dC18 2.1 x 20 mm IS™, 3 µm 186002058 0.1% HCOOH in H2O 0.1% HCOOH in MeOH 0.6 mL/min Time Profile (min) %A %B 0.0 100 0 4.0 50 50 10 µL 20 µg/mL 30° C UV @ 270nm Alliance® HT 2795 with 2996 PDA

NH N

H2N

NH2

S

S

N

NH

O

Sulfadiazine

N

Sulfathiazole

CH3

H3C

NH2

NH2

N

O O NH

S H3C

O

N

H3C

O O

O S

N

O

Sulfadimidine

NH2 N

NH

N

Sulfamethoxazole

H3C

O

S

N

Compounds: 1. Sulfadiazine 2. Sulfathiazole 3. Sulfamerazine 4. Sulfadimidine 5. Sulfamethoxazole 6. Sulfisoxazole 7. Sulfadimethoxine

S

O

O

NH

H3C

NH

O

S

NH2

O

Sulfamerazine

Sulfisoxazole

H3C

O NH2

N H3C

N

O S

O

NH

O

Sulfadimethoxine

1

5 2

6

3

7

4

V0 = 0.19

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Minutes

80


Sulfonamides — 4.6 x 20 mm IS™ Column LC Conditions: Column:

O

Atlantis™ dC18 4.6 x 20 mm IS™, 3 µm

Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

Injection Volume:

186002062 0.1% HCOOH in H2O 0.1% HCOOH in MeOH 3.0 mL /min Time Profile (min) %A %B 0.0 100 0 4.0 50 50 10 µL

Sample Concentration: Temperature: Detection: Instrument:

20 µg/mL 30° C UV @ 270 nm Alliance® HT 2795 with 2996 PDA

NH N

H2N

NH2

S

S

N

NH

O

Sulfadiazine

N

Sulfathiazole

CH3

H3C

NH2

NH2

N

O O

O

S

N

NH

S H3C

O

N

N

H3C

O O

O S

H3C

O

Sulfadimidine

NH2 N

NH

N

Sulfamethoxazole

Compounds: 1. Sulfadiazine 2. Sulfathiazole 3. Sulfamerazine 4. Sulfadimidine 5. Sulfamethoxazole 6. Sulfisoxazole 7. Sulfadimethoxine

S

O

O

NH

H3C

NH

O

S

NH2

O

Sulfamerazine

Sulfisoxazole

H3C

O NH2

N H3C

N

O S

O

NH

O

Sulfadimethoxine

1

2

5 3

7

6 4

V0 = 0.17

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Minutes


81

Sulfonamides LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

O

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 H2O MeOH 1% HCOOH, pH 2.4 2.0 mL/min Time Profile (min) %A %B %C 0.0 75 15 10 10.0 50 40 10 5 µL 30° C UV @ 270 nm Alliance® 2695 with 2996 PDA


NH N

H2N

NH2

S

S

N

Sulfadiazine

Sulfathiazole

H3C

H2 N

NH2

N

O N

N

NH

O

O

S

O

O

HN S

NH

S

N O

O

O

Sulfamethoxazole

Sulfadmethazine

NH2

Compounds: 1. Sulfadiazine 2. Sulfathiazole 3. Sulfamerazine 4. Sulfamethazine 5. Sulfamethoxazole

USP Tailing 1.01 1.02 1.00 0.99 0.99


1

N

S N

H3C

NH

O

Sulfamerazine

2

5

3

4

V0 = 0.98

0.00

O

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes

82


Sulfonamides— Analytical to Preparative Scale Up LC Conditions: Mobile Phase A: Mobile Phase B: Sample Concentration: Temperature: Instrument:

0.1% HCOOH in H2O ACN/1% HCOOH (90/10) 1.0 mg/mL each in DMSO Ambient AutoPurification™ System

H2N

O H2 N

S

S O

NH

O

N

Sulfathiazole

Sulfanilamide

Compounds: 1. Sulfanilamide 2. Sulfathiazole 3. Sulfamethazine 4. Sulfamethoxazole

O H2N

S

H3C

H N

N

NH2

CH 3 O

O

O

N

N

The flattened peak profiles on analytical chromatogram reflects the

Atlantis™ dC18 4.6 x 100 mm, 5 µm 186001340

Total Mass Load:

280 µg

Detection:

UV @ 300 nm 1


3

2

4

3

2

4

1

V0 = 1.76

Injection Volume:

3 mm, 5 µm Atlantis™ dC18 Prep OBD™ 19 x 100 2 186002116 3 4,800 µg 4 2 1UV @ 280 nm


4

1

V0 = 1.74 Injection Volume: 1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

O

Sulfamethoxazole

Sulfamethazine

Column: Part Number:

S NH

CH 3

saturation of PDA detector, not column overload.

Column: Part Number: Total Mass Load: Detection:

S

O

NH2

9.00

10.00

11.00

12.00

13.00

14.00

9.00

10.00

11.00

12.00

13.00

14.00

1.0 mL/min Time

Profile

(min)

%A

%B

0.0

85

15

1.0

85

15

2.0

70

30

12.0 14.0 70 µL

30 30

70 70


%B 15 15 30 70 70

Minutes


83

Thiols LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow Rate: Gradient:

Injection Volume: Sample Diluent: Sample Concentration: Temperature: Instrument:


Atlantis™ HILIC Silica 2.1 x 50 mm, 3 µm 186002011 H2O ACN 200 mM NH4COOH, pH 3.0 0.3 mL/min Time Profile (min) %A %B %C 0.0 0 95 5 5.0 45 50 5 5.0 µL 75:25 ACN:MeOH with 0.2% HCOOH 50 µg/mL Ambient Alliance® HT 2795 with Waters ZQ™

ES+ 3.0 20 3V 0.3 V 150 350 50 700 136.1 150.2

(Homocysteine) (Methionine)

OH

1.0x10 7

SH O

Intensity

1.5x10 7

NH 2 1.0x10 7

Homocysteine

5.0x10 6

0.0 0.00

1.00

2.00

3.00

4.00

5.00

6x10 7

OH 4x10 7

S

Intensity

H3C

O NH2

2x10 7

Methionine

0.0 0.00

1.00

2.00

3.00

4.00

5.00

Minutes

84


Vancomycin LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Mobile Phase C: Flow rate: Gradient:



Vancomycin

USP Tailing = 1.03

V0 = 0.51

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Minutes


85

Water and Fat Soluble Vitamins Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

OH

Atlantis™ dC18 4.6 x 150 mm, 5 µm 186001344 0.1% TFA in H2O 0.1% TFA in ACN 1.4 mL/min Time Profile (min) %A %B 0.0 100 0 5.0 97 3 6.0 85 15 10.0 80 20 12.0 0 100 25.0 0 100 10 µL H3C 82:4:14 H2O:EtOH:MeOH 30° C H3C UV @ 268 nm Alliance® 2695 with 2996 PDA

Injection Volume: Injection Solvent*: Temperature: Detection: Instrument:

HO

O C C H C H H2

O

HC HC

OH

HO

L-ascorbic acid (Vitamin C)

N

Sample Concentration (µg/mL)* 19.61 9.81 H3C 19.61 HO 39.21 39.21 23.51 9.81 3.91

9. Retinol (vitamin A) 10. Tocopherol (vitamin E) 11. Ergocalciferol (vitamin D2)

19.62 39.23 9.83

CH

C H2

C H

C H2

CH3

H2 C

Nicotinic acid (Niacin)

HO

HC

CH2

C H OH HO H C

HO C H2

C H2C H OH

H C

N

C H

N

O

N

HC

CH3

H3C

OH

HO

N

H2C

O

Pyridoxal

N N CH3

Caffeine

C H2

OH

OH

NH

N O

H N

H2N

N

CH

N

CH2 HN HC

CH3 H CH3 H2 CH3 H CH3 2 2 O C C C C C C C C H C C H C C H CH3 CHH2 2 H H2 H2 H2 H2 2 C H2

C H

CH

CH3 N CH N

Pyridoxine

O

CH3

OH

O H3C O

Folic acid (Vitamin B11) H C C H

CH

O

O H2 H N H C C OH C H2 O

OH

Tocopherol (Vitamin E)

H3C

CH3

H2C C H2

Sample Preparation Solvents (1) H2O

H C

CH3 C H

CH3

C H

H C

CH3 C H

H2 C

C H

OH

Retinol (Vitamin A)

CH3 H C H CH3 H3C H C C H C C H 2 H3C H H CH3 C CH2 H2C CH2

H2C HC

MeOH/EtOH (50/50) MeOH

H2C

CH

H

Ergocalciferol (Vitamin D2)

CH2

C CH2 HO H C H2

(*) – Analytes were dissolved in the solvent listed above. Aliquots were then combined in a single vial to yield the concentrations and final injection solvent shown.

7

4

+

N

Thiamine (Vitamin B1)

N

H2C

(2) (3)

N

O

Riboflavin (Vitamin B2) Compounds: 1. L-ascorbic acid (vitamin C) 2. Nicotinic acid (niacin) 3. Thiamine (vitamin B1) 4. Pyridoxal 5. Pyridoxine 6. Folic acid (vitamin B11) 7. Caffeine 8. Riboflavin (vitamin B2)

NH2 H C S

N

H3C

OH

H C

5

V0 = 1.37

3

1 2

8 6

11

9 10

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

Minutes

86


Water Soluble Vitamins LC Conditions: Column: Part Number: Mobile Phase A: Mobile Phase B: Flow Rate: Gradient:

OH

OH


H3C

C H

N

O

1. L-ascorbic acid (vitamin C)

1.12

23.8

2. Nicotinic acid (niacin)

1.27

11.9

3. Thiamine (vitamin B1) 4. Pyridoxal

1.20 1.13

23.8 47.6

5. Pyridoxine

1.04

47.6

6. Folic acid (vitamin B11) 7. Caffeine 8. Riboflavin (vitamin B2)

1.10 1.10 1.14

42.9 11.9 6.0

CH3

C H2

OH

O H3C

N N CH3

O N

H3C

CH

H2C

CH3 N CH N

Caffeine

C H2

HO

OH

OH

Pyridoxine

N

USP Tailing

C H2

C H

H2 C

Thiamine (Vitamin B1)

H N

H2N

Riboflavin (Vitamin B2)

Compounds:

+

N

NH

N

Sample Concentrations (µg/mL)

NH2 H C S

N

HC

3 Atlantis™ dC18 4.6 x 150 mm, 5 µm H O HO C C O 186001344 C H C O HC H H2 0.1% TFA CH HC N OH HO ACN 1.4 mL/min L-ascorbic acid Nicotinic acid Time Profile (Vitamin C) (Niacin) (min) %A %B 0.0 100 0 4.0 97 3 N HO 6.0 85 15 CH3 HC CH2 15.0 80 20 C H OH HO C OH HO H C 10 µL H2 HC 30° C C O H2C H OH H UV @ 260 nm H C C N N O 3 Pyridoxal Alliance® 2695 with 2996 PDA

N

CH

N O

CH2 HN HC

Folic acid (Vitamin B11) H C

CH

C H

O

O H2 H N H C C OH C H2 O

OH

3

2 1 7 8

V0 = 1.37

4 5

0.00

1.00

2.00

3.00

4.00

5.00

6.00

6

7.00

8.00

9.00

10.00

11.00

12.00

13.00

14.00

15.00

Minutes


87

Water Soluble Vitamins— Analytical to Preparative Scale Up LC Conditions: Mobile Phase A: Mobile Phase B: Sample Concentration: Temperature: Instrument: Detection:

100

100


H3C

Compounds: 1. Pyridoxine (5 mg/mL) 2. Caffeine (25 mg/mL))

0.1% HCOOH in H2O ACN/1% HCOOH (90/10) 5, 25 mg/mL in water Ambient AutoPurification™ System UV @ 290 nm

N

O

CH

HO H2C

C H2

OH

H3C O

OH

Pyridoxine

Atlantis™ dC18 4.6 x 100 mm, 5 µm 186001340 3,000 µg

2


2

1

100 %

2 1

%

1

Injection Volume:

N N CH3

CH3 N CH N

Caffeine


%B 0 0 20 40

% 0 100 0

0 100 100 %

2

Column:

Atlantis™ dC18 Prep OBD™ 19 x 100 mm, 5 µm

Part Number: Total Mass Load:

186002116 50,000 µg


2

1

2 1

%

1

% 1

Injection Volume:

100 1

17.06 mL/min Time Profile (min) %A %B 0.0 3.02 8.02 18.02 1700 µL

100 100 80 60

0 0 20 40

2

100 1

2 1

100 % %


Atlantis™ dC18 Prep OBD™ 30 x 100 mm, 5 µm 186001375 1 126,000 µg


2

1

% 1 2.00

4.00

6.00

8.00

10.00

12.00

2.00

4.00

6.00

8.00

10.00

12.00

2.00

4.00

6.00

8.00

10.00

12.00

1

Injection Volume:


%B 0 0 20 40

1

Minutes

88


Atlantis™ Column Care and Use Instructions PLEASE READ CAREFULLY BEFORE USING ATLANTIS™ COLUMNS.

Cartridge Connection

Thank you for choosing a Waters Atlantis column. The manufacture of Atlantis dC18 and Atlantis™ HILIC Silica columns begins with ultrapure reagents to control the chemical composition and purity of the final product. Atlantis™ columns are manufactured in a cGMP, ISO 9002 certified plant with each step being conducted within narrow tolerances. Every column is individually tested and Certificates of Batch Analysis and a Performance Chromatogram are provided with each column. ™

™

Waters recommends the use of Waters Sentry™ guard columns to maximize column lifetime and protect the column from contaminants. Connecting the Column or Cartridge to the HPLC

Handle the cartridge with care. Do not drop or hit the cartridge on a hard surface as it may disturb the bed and affect its performance. Refer to Figure 1 for an exploded view of an Atlantis™ cartridge column with a Sentry™ guard column. 1. Unscrew end connectors from the old cartridge. Leave them connected to the inlet and outlet lines of the instrument. 2. Attach new cartridge column between connectors so that the direction of the flow arrow on the label is pointing in the direction of mobile phase flow (toward detector). 3. Fingertighten all fittings.

Column Connection

It is important to realize that extra column peak broadening can destroy a successful separation. The choice of appropriate column connectors and system tubing is discussed in detail below.

Handle the column with care. Do not drop or hit the column on a hard surface as it may disturb the bed and affect its performance.

Column Connectors and System Tubing Considerations

1. Correct connection of 1/16 inch outer diameter stainless steel tubing leading to and from the column is essential for high-quality chromatographic results. 2. When using standard stainless steel compression screw fittings, it is important to ensure proper fit of the 1/16 inch outer diameter stainless steel tubing. When tightening or loosening the compression screw, place a 5/16 inch wrench on the compression screw and a 3/8 inch wrench on the hex head of the column endfitting. Note: If one of the wrenches is placed on the column flat during this process, the endfitting will be loosened and leak.

Due to the absence of an industry standard, various column manufacturers have employed different types of chromatographic column connectors. The chromatographic performance of the separation can be negatively affected if the style of the column endfittings does not match the existing tubing ferrule setting. This section explains the differences between Waters style and Parker style ferrules and endfittings (Figure 2). Each endfitting style varies in the required length of the tubing protruding from the ferrule. The Atlantis™ column is equipped with Waters style endfittings, which require a 0.130 inch ferrule. If a non-Waters style column is presently being used, it is critical that ferrule depth be reset for optimal performance prior to installing an Atlantis™ column.

3. If a leak occurs between the stainless steel compression screw fitting and the column endfitting, a new compression screw fitting, tubing and ferrule must be assembled.

The presence of a void in the flow stream reduces column performance. This can occur if a Parker ferrule is connected to a Waters style endfitting (Figure 4).

4. An arrow on the column identification label indicates correct direction of solvent flow.

Note: A void appears if tubing with a Parker ferrule is connected to a Waters style column.

Figure 1: Installation of Atlantis™ Cartridge Column with Sentry™ Guard Column Spacer

0.25 mm (0.009 inch) Tubing

Sentry™ Guard Column

Figure 2: Waters and Parker Ferrule Types

Waters Ferrule Setting

C-Clip

Atlantis™ Cartridge Column

End Connector

Figure 3: Proper Tubing/Column Connection Tubing touches the bottom of the column endfitting, with no void between them.

Parker Ferrule Setting


89

Atlantis™ Column Care and Use Instructions Figure 4: Parker Ferrule in a Waters Style Endfitting

Table 1. Waters Part Numbers for SLIPFREE® Connectors SLIPFREE® Type and Tubing Length

To fix this problem: Cut the end of the tubing with the ferrule, place a new ferrule on the tubing and make a new connection. Before tightening the screw, make sure that the tubing bottoms out in the endfitting of the column. Conversely, if tubing with a Waters ferrule is connected to a column with Parker style endfitting, the end of the tubing will bottom out before the ferrule reaches its proper sealing position. This will leave a gap and create a leak (Figure 5).

0.005”

Single 6 cm Single 10 cm Single 20 cm Double 6 cm Double 10 cm Double 20 cm

Tubing Internal Diameter 0.010” 0.020”

PSL 618000 PSL 618002 PSL 618004 PSL 618001 PSL 618003 PSL 618005



Band Spreading Minimization Internal tubing diameter influences system band spreading and peak shape. Larger tubing diameters cause excessive peak broadening and lower sensitivity (Figure 7). Figure 7: Effect of Connecting Tubing on System

Note: The connection leaks if a Waters ferrule is connected to a column with a Parker style endfitting. 0.005 inches

Figure 5: Waters Ferrule in a Parker Style Endfitting

0.020 inches 0.040 inches

Diluted/Distorted Sample Band

There are two ways to fix the problem: 1. Tighten the screw a bit more. The ferrule moves forward, and reaches the sealing surface. Do not overtighten since this may break the screw. 2. Cut the tubing, replace the ferrule and make a new connection. Alternatively, replace the conventional compression screw fitting with an all-in-one PEEK fitting (Waters part number PSL613315) that allows resetting of the ferrule depth. Another approach is to use a Thermo Hypersil Keystone, Inc. SLIPFREE® connector to ensure the correct fit. The fingertight SLIPFREE® connectors automatically adjust to fit all compression screw type fittings without the use of tools (Figure 6). Figure 6: Single and Double SLIPFREE® Connectors

Measuring System Bandspread Volume This test should be performed on an HPLC system with a single wavelength UV detector (not a Photodiode Array (PDA)). 1. Disconnect column from system and replace with a zero dead volume union. 2. Set flow rate to 1 mL/min. 3. Dilute a test mix in mobile phase to give a detector sensitivity 0.5 - 1.0 AUFS (system start up test mix can be used which contains uracil, ethyl and propyl parabens; Waters part number WAT034544). 4. Inject 2 to 5 µL of this solution. 5. Using 5 sigma method measure the peak width at 4.4% of peak height: Band Spread (µL) = Peak Width (min) x Flow Rate (µL/min) = 0.1 min x 1000 µL/min = 100 µL

90


Atlantis™ Column Care and Use Instructions Figure 8: Determination of System Bandspread Volume Using 5-Sigma Method

System optimization, especially in a system that contains a flow splitter, can have dramatic effects on sensitivity and resolution. Optimization includes using correct-depth ferrules and minimizing tubing diameter and lengths. System optimization results in a doubling of sensitivity and resolution of the metabolite in an LC/MS/MS system (Figure 10). Figure 10: Non-Optimized vs. Optimized LC/MS/MS System

7.00

In a typical HPLC system, the Bandspread Volume should be 100 µL ± 30 µL.

7.50

8.00

Non-optimized LC/MS/MS System

7.00

7.50

In a microbore (≤2.1mm i.d.) system, the Bandspread Volume should be no greater than 20 to 40 µL.

System Modification Recommendations

Measuring Gradient Delay Volume

1. Use a microbore detector flowcell with ≤2.1 mm i.d. columns.

1. Replace the column with a zero dead volume union.

8.00

Optimized System

Note: Detector sensitivity is reduced with the shorter flowcell pathlength in order to achieve lower bandspread volume.

2. Prepare eluent A (pure solvent, such as methanol) and eluent B (solvent plus sample, such as 5.6 mg/mL propylparaben in methanol).

2. Minimize injector sample loop volume.

3. Equilibrate the system with eluent A until a stable baseline is achieved.

3. Use 0.009 inch (0.25 mm) tubing between pump and injector.

4. Switch to 100% eluent B.

4. Use 0.009 inch (0.25 mm) tubing for rest of connections in standard systems and 0.005 inch (0.12 mm) tubing for narrowbore (≤2.1 mm i.d.) systems.

5. Record the half height of the step and determine dwell volume (Figure 9). Figure 9: Determination of Dwell Volume

6. Detector time constants should be shortened to less than 0.2 seconds.

1.0

Waters Small Particle Size (3 µm) Columns – Fast Chromatography

0.8

0.6 Height

5. Use perfect (pre-cut) connections (with a variable depth inlet if using columns from different suppliers).

Dwell Volume

Waters columns with 3 µm particles provide faster and more efficient separations without sacrificing column lifetime. This section describes five parameters to consider when performing separations with columns containing 3 µm particles.

0.4

0.2

0.0 Volume (µL)

The dwell volume should be less than 1 mL. If the dwell volume is greater than 1 mL, see System Modification Recommendations section on how to reduce system volume. Use of Smaller i.d. Columns A 3.0 mm i.d. narrow-bore column usually requires no system modifications. A 2.1 mm i.d. microbore column, however, requires modifications to the HPLC system to eliminate excessive system bandspread volume. Without proper system modifications, excessive system bandspread volume causes peak broadening and has a large impact on peak width as peak volume decreases. Impact of Bandspread Volume on 2.1 mm i.d. Column Performance System with 70 µL bandspread: 10,000 plates System with 130 µL bandspread: 8,000 plates (same column)

Note: Columns that contain 3 µm particles have smaller outlet frits to retain packing material. These columns should not be backflushed. 1. Flow Rate — Compared to columns with 5 µm particles, columns with 3 µm particles have higher optimum flow rates and are used when high efficiency and short analysis times are required. These higher flow rates, however, lead to increased backpressure. Note: Use a flow rate that is practical for your system. 2. Backpressure — Backpressures for columns with 3 µm particles are higher than for 5 µm columns with the same dimensions. Waters suggests using a shorter column to compensate for increased backpressure and to obtain a shorter analysis time. 3. Temperature — Use a higher temperature to reduce backpressure caused by smaller particle sizes. The recommended temperature range for Atlantis™ columns is 20°C to 45°C. See Column Usage section for a discussion of elevated temperature use with Atlantis™ columns. 4. Sampling Rate — Use a sampling rate of about 10 points per second. 5. Detector Time Constant — Use a time constant of 0.1 seconds for fast analyses.

Note: Flow splitters after the column will introduce additional bandspreading.


91

Atlantis™ Column Care and Use Instructions Column Performance Validation

Column Installation Procedure

Each Atlantis column comes with Certificates of Batch Analysis and a Performance Test Chromatogram. The Certificates of Analysis are specific to each batch of packing material and includes the gel lot batch number, analysis of unbonded particles, analysis of bonded particles (Atlantis™ dC18), and chromatographic results and conditions. The Performance Test Chromatogram is specific to each individual column and contains information such as gel lot batch number, column serial number, USP plate count, USP tailing factor, capacity factor and chromatographic results and conditions.

Note: The flow rates given in the procedure below are for a typical 4.6 mm i.d. column. Scale the flow rate up or down accordingly based upon the column i.d., length, particle size and backpressure of the Atlantis™ column being installed. See Scaling Up/Down section for calculating flow rates when changing column i.d. and/or length.

These data should be stored for future reference.

3. When the mobile phase is flowing freely from the column outlet, attach the column to the detector. This prevents entry of air into the detector and provides more rapid baseline equilibration.

™

Initial Column Efficiency Determination 1. Perform an efficiency test on the column before using it. Waters recommends using a suitable solute mixture, as found in the “Performance Test Chromatogram”, to analyze the column upon receipt. 2. Determine the number of theoretical plates (N) and use for periodic comparison. 3. Repeat the test periodically to track column performance over time. Slight variations may be obtained on two different HPLC systems due to the quality of the connections, operating environment, system electronics, reagent quality, column condition and operator technique. Column Equilibration Atlantis™ columns are packed and shipped in 100% acetonitrile. It is important to ensure solvent compatibility before changing to a new solvent. Atlantis dC18 - Equilibrate with a minimum of 10 column volumes of the mobile phase to be used (refer to Table 2 for a listing of standard column volumes). ™

1. Purge the pumping system and connect the inlet end of the column to the injector outlet. 2. Set the pump flow to 0.1 mL/min. and increase to 1 mL/min over 5 minutes.

4. When the mobile phase is changed, gradually increase the flow rate of the new mobile phase from 0.0 mL/min to 1.0 mL/min in 0.1 mL/min increments. 5. Once a steady backpressure and baseline have been achieved, the column is ready to be used (or equilibrated). Note: If mobile phase additives are present in low concentrations (e.g., ion-pairing reagents), 100 to 200 column volumes may be required for complete equilibration. In addition, mobile phases that contain formate (e.g., ammonium formate, formic acid, etc.) may also require slightly longer initial column equilibration times. Please see additional equilibration information for Atlantis™ HILIC Silica columns in the HILIC Getting Started section. Column Usage To ensure the continued high performance of Atlantis™ columns and cartridges, follow these guidelines: Guard columns

Atlantis™ HILIC Silica – Upon receipt, equilibrate with 50 column volumes of 50:50 acetonitrile:water with 10 mM final buffer concentration (refer to Table 2 for a listing of standard column volumes). Prior to the first injection, equilibrate with 20 column volumes of initial mobile phase conditions. See HILIC Getting Started section for additional information.

Use a Waters Sentry™ guard cartridge of matching chemistry and particle size between the injector and main column. It is important to use a high-performance matching guard column to protect the main column while not compromising or changing analytical resolution.

Table 2. Standard column volumes in mL (multiply by 10 for flush solvent volumes)

1. Sample impurities often contribute to column contamination. Use Waters Oasis® or Sep-Pak® solid-phase extraction cartridges/columns of the appropriate chemistry to clean up the sample before analysis.

Column Volume (mL) Column Length

1.0

2.1

30

50

15 mm

–

0.1

–

–

–

–

–

–

–

–

20 mm

–

0.1

0.1

–

0.3

–

–

–

–

–

30 mm

–

0.1

0.2

–

0.5

–

2.4

8

–

–

50 mm

0.1

0.2

0.3

–

0.8

2.4

4

14

35

98

100 mm

0.1

0.4

0.7

1.2

1.7

5

8

28

70

–

150 mm

0.1

0.5

1.0

1.8

2.5

7

12

42

106

294

250 mm

–

0.9

1.8

–

4

–

20

70

176

490

300 mm

–

–

–

–

–

14

24

85

212

589

92

Column internal diameter (mm) 3.0 3.9 4.6 7.8 10 19

Sample Preparation

2. For reversed-phase separations (Atlantis™ dC18) prepare the sample in mobile phase or a solvent that is weaker (less organic modifier) than the mobile phase. For Hydrophilic Interaction Chromatography (HILIC) separations (Atlantis™ HILIC Silica), the samples must be prepared in 100% organic solvents (e.g., acetonitrile). See HILIC Getting Started section for additional information. 3. If the sample is not dissolved in the mobile phase, ensure that the sample, solvent and mobile phases are miscible in order to avoid sample and/or buffer precipitation. 4. Filter sample through a 0.2 µm membrane to remove particulates. If the sample is dissolved in a solvent that contains an organic modifier (e.g., acetonitrile, methanol, etc.) ensure that the membrane material does not dissolve in the solvent. Contact the membrane manufacturer with solvent compatibility questions.


Atlantis™ Column Care and Use Instructions pH Range

Scaling Up/Down Isocratic Methods

Atlantis dC18 - The pH range for Atlantis dC18 columns is 2 to 7. ™

The following formulas will allow scale up or scale down, while maintaining the same linear velocity (retention time), and provide new sample loading values:

™

Atlantis HILIC Silica - The pH range for Atlantis HILIC Silica columns is 1 to 6. ™

™

Column lifetime will vary depending upon the temperature, type and concentration of buffer used. A listing of recommended and non-recommended buffers is given in Table 3. Please use this as a guideline when developing methods.

If column i.d. and length are altered:

Note: Operating at the upper end of the pH range in combination with elevated temperatures will lead to shorter column lifetime.

Where:

Table 3: Buffer recommendations for using Atlantis™ columns from pH 1 to 7 Additive or Buffer

Buffer range (±1 pH unit)

pKa

Volatility

Used for Mass Spec?

TFA

0.3

Volatile

Yes

Ion pair additive, can suppress MS signal. Used in the 0.01-0.1% range.

Formic Acid

3.75

Volatile

Yes

Maximum buffering obtained when used with Ammonium Formate salt. Used in 0.1-1.0% range.

Acetic Acid

4.76

Volatile

Yes

Maximum buffering obtained when used with Ammonium Acetate salt. Used in 0.1-1.0% range.

Formate (NH4COOH)

3.75

2.75 – 4.75

Volatile

Yes

Used in the 1-10mM range. Note: sodium or potassium salts are not volatile.

Acetate (NH4CH2COOH)

4.76

3.76 – 5.76

Volatile

Yes

Used in the 1-10mM range. Note: sodium or potassium salts are not volatile.

Phosphate 1

2.15

1.15 – 3.15

Non-volatile

No

Traditional low pH buffer, good UV transparency. Not recommended for HILIC

Phosphate 2

7.2

6.20 – 8.20

Non-volatile

No

Above pH 7, reduce temperature/concentration and use guard column to maximize lifetime. Not recommended for HILIC

F2 = F1(r2/r1)2

or

Load2 = Load1(r2/r1)2(L2/L1)

or

Inj vol1 = Inj vol2 (r2/r1)2 (L2/L1)

r = Radius of the column, in mm F = Flow rate, in mL/min L = Length of column, in mm 1 = Original, or reference column

Comments

2 = New column HILIC Getting Started Equilibration of Atlantis™ HILIC Silica Columns 1. Upon receipt, equilibrate in 50% acetonitrile/50% aqueous buffer (10 mM final buffer concentration) for 50 column volumes. 2. Prior to first injection, equilibrate with 20 column volumes of initial mobile phase conditions. 3. When running gradients, equilibrate with 10 column volumes between injections.

* Phosphate salt buffers are not recommeded for HILIC (phosphoric acid is OK).

Failure to appropriately equilibrate the column could result in drifting retention times.

Solvents To maintain maximum column performance, use high quality chromatography grade solvents. Filter all aqueous buffers prior to use. Pall Gelman Laboratory Acrodisc® filters are recommended. Solvents containing suspended particulate materials will generally clog the outside surface of the inlet distribution frit of the column. This will result in higher operating pressure and poorer performance. Degas all solvents thoroughly before use to prevent bubble formation in the pump and detector. The use of an on-line degassing unit is also recommended. This is especially important when running low pressure gradients since bubble formation can occur as a result of aqueous and organic solvent mixing during the gradient. Pressure Atlantis™ columns can tolerate pressures of up to 6,000 psi (400 bar or 40 Mpa) although pressures greater than 4,000 – 5,000 psi should be avoided in order to maximize column and system lifetimes. Temperature

HILIC Mobile Phase Considerations 1. Always maintain at least 5% polar solvent in the mobile phase or gradient (e.g., 5% water, 5% methanol or 3% methanol/2% aqueous buffer, etc.). This ensures that the Atlantis™ HILIC Silica particle is always hydrated. 2. Maintain at least 40% organic solvent (e.g., acetonitrile) in your mobile phase or gradient. 3. Avoid phosphate salt buffers to avoid precipitation in HILIC mobile phases (phosphoric acid is OK). 4. Buffers such as ammonium formate or ammonium acetate will produce more reproducible results than additives such as formic acid or acetic acid. If an additive (e.g., formic acid) must be used instead of a buffer, use 0.2% (v:v) instead of 0.1%. 5. For best peak shape, maintain a buffer concentration of 10 mM in your mobile phase/gradient at all times. Injection Solvents for HILIC

Temperatures between 20˚C – 45˚C are recommended for operating Waters Atlantis columns in order to enhance selectivity, lower solvent viscosity and increase mass transfer rates. However, any temperature rise above ambient will have a negative effect on lifetime which will vary depending on the pH and buffer conditions used. ™

1. If possible, injection solvents should be 100% organic solvent. Water must be eliminated or minimized. Choose weak HILIC solvents such as acetonitrile, isopropanol, methanol, etc. 2. A generic injection solvent is 75:25 acetonitrile:methanol. This is a good compromise between analyte solubility and peak shape. 3. Avoid water and dimethylsulfoxide (DMSO) as injection solvents. These solvents will produce very poor peak shapes.


93

Atlantis™ Column Care and Use Instructions 4. Exchange water or DMSO with acetonitrile by using reversed-phase solid phase extraction. If this is not possible, dilute the water or DMSO with organic solvent. Additional HILIC Recommendations 1. For initial scouting conditions, run a gradient from 95% acetonitrile to 50% acetonitrile. If no retention occurs, run isocratically with 95:3:2 acetonitrile:methanol:aqueous buffer. 2. Alternate polar solvents such as methanol, acetone or isopropanol can also be used in place of water in the mobile phase to increase retention. 3. Be sure that your needle wash solvent/purge solvent contains the same high organic solvent concentration as your mobile phase, else peak shapes will suffer. Column Cleaning, Regenerating and Storage

Storage Atlantis™ dC18 – For periods longer than four days, store the column in 100% acetonitrile. Do not store columns in buffered eluents. If the mobile phase contained a buffer salt, flush the column with 10 column volumes of HPLC grade water (see Table 2 for common column volumes) and replace with 100% acetonitrile for storage. Failure to perform this intermediate step could result in precipitation of the buffer salt in the column when 100% acetonitrile is introduced. Atlantis™ HILIC Silica – For periods longer than four days, store the column in 95:5 acetonitrile: water. Do not store columns in buffered eluents. If the mobile phase contained a buffer salt, flush the column with 10 column volumes of 95:5 acetonitrile:water (see Table 2 for common column volumes) prior to storage.

Cleaning and Regeneration

Completely seal column to avoid evaporation and drying out of the bed.

A sudden increase in pressure or shift in retention or resolution may indicate contamination of the column.

Note: If a column has been run with a formate-containing mobile phase (e.g., ammonium formate, formic acid, etc.) and is flushed to remove the buffer, slightly longer equilibration times may be required after the column is re-installed and run again with a formate-containing mobile phase.

Atlantis™ dC18 – Flush with a neat organic solvent to remove the non-polar contaminant(s). If this flushing procedure does not solve the problem, purge the column with a sequence of progressively more non-polar solvents. For example, switch from water to tetrahydrofuran to methylene chloride. Return to the standard mobile phase conditions by reversing the sequence. Atlantis™ HILIC Silica – Flush with 50:50 acetonitrile:water to remove the polar contaminant(s). If this flushing procedure does not solve the problem, purge the column with 5:95 acetonitrile:water.

Troubleshooting Changes in retention time, resolution, or backpressure are often due to column contamination. See the Column Cleaning, Regeneration and Storage section of this Care and Use Manual. Information on column troubleshooting problems may be found in HPLC Columns Theory, Technology and Practice, U.D. Neue, (Wiley-VCH, 1997) or the Waters HPLC Troubleshooting Guide (Literature Code 720000181EN).

Guard columns require replacement at regular intervals as determined by sample contamination. When system backpressure increases above a set pressure limit, it is usually an indication that the guard column should be replaced. A sudden appearance of split peaks is also indicative of a need to replace the guard column.

94


Ordering Information— Atlantis™ dC18 Columns Atlantis™ dC18 Analytical Columns Hardware Type

Dimensions

NanoEase NanoEase™ NanoEase™ NanoEase™ NanoEase™ NanoEase™ NanoEase™ NanoEase™ NanoEase™ Column Column Column Column Column Column Column Guard Guard Direct Connect Direct Connect Guard Guard IS™ Column IS™ Column Column Column Column Column Column Column Column Column IS™ Column IS™ Column

0.075 x 50 mm 0.075 x 100 mm 0.075 x 150 mm 0.100 x 50 mm 0.100 x 100 mm 0.100 x 150 mm 0.150 x 50 mm 0.150 x 100 mm 0.150 x 150 mm 0.320 x 50 mm 0.320 x 100 mm 0.320 x 150 mm 1.0 x 50 mm 1.0 x 50 mm 1.0 x 150 mm 1.0 x 150 mm 2.1 x 10 mm 2.1 x 10 mm 2.1 x 15 mm 2.1 x 15 mm 2.1 x 20 mm 2.1 x 20 mm 2.1 x 20 mm 2.1 x 20 mm 2.1 x 30 mm 2.1 x 30 mm 2.1 x 50 mm 2.1 x 50 mm 2.1 x 100 mm 2.1 x 100 mm 2.1 x 150 mm 2.1 x 150 mm 3.0 x 20 mm 3.0 x 20 mm

™

Atlantis™ dC18 Analytical Columns Particle Size

Part No.

3 µm 3 µm 3 µm 3 µm 3 µm 3 µm 3 µm 3 µm 3 µm 3 µm 3 µm 3 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm

186002194 186002195 186002197 186002207 186002208 186002209 186002466 186002467 186002468 186002304 186002305 186002306 186001279 186001281 186001283 186001285 1860013771 1860013791 186002064 186002065 1860013812 1860013832 186002058 186002059 186001287 186001289 186001291 186001293 186001295 186001297 186001299 186001301 186002060 186002061

Hardware Type

Dimensions

Particle Size

Part No.

Column Column Column Column Column Column Column Guard Guard Cartridge Column Cartridge Column Column Column Column Column Guard Guard IS™ Column IS™ Column Column Column Column Column Column Column Column Column Column Column Column

3.0 x 50 mm 3.0 x 50 mm 3.0 x 100 mm 3.0 x 100 mm 3.0 x 150 mm 3.0 x 150 mm 3.0 x 250 mm 3.9 x 20 mm 3.9 x 20 mm 3.9 x 50 mm 3.9 x 50 mm 3.9 x 100 mm 3.9 x 100 mm 3.9 x 150 mm 3.9 x 150 mm 4.6 x 20 mm 4.6 x 20 mm 4.6 x 20 mm 4.6 x 20 mm 4.6 x 30 mm 4.6 x 30 mm 4.6 x 50 mm 4.6 x 50 mm 4.6 x 75 mm 4.6 x 75 mm 4.6 x 100 mm 4.6 x 100 mm 4.6 x 150 mm 4.6 x 150 mm 4.6 x 250 mm

3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 5 µm

186001389 186001391 186001303 186001305 186001307 186001309 186001311 1860013133 1860013153 1860013854 1860013874 186001393 186001395 186001317 186001319 1860013213 1860013233 186002062 186002063 186001325 186001327 186001329 186001331 186001333 186001335 186001337 186001340 186001342 186001344 186001346

1

Requires Sentry Guard Holder

WAT097958

2


186000262

3


WAT046910

4

Requires Cartridge End Fittings

WAT037525

Part No.

5

Requires Cartridge Guard Holder

289000779

1860023005 1860024525 186002298 186002299 186002453 186002454 1860013616 1860013636 186001365 186001367 186001369 186001371 186001373 186001375 186002417 186002418

6


186000709

Atlantis™ dC18 Preparative Columns Hardware Type Guard Guard Column Column Column Column Guard Guard Column Column Column Column Column Column Column Column

Dimensions 10 x 10 mm 10 x 10 mm 10 x 50 mm 10 x 100 mm 10 x 150 mm 10 x 250 mm 19 x 10 mm 19 x 10 mm 19 x 50 mm 19 x 100 mm 19 x 150 mm 19 x 250 mm 30 x 50 mm 30 x 100 mm 30 x 150 mm 30 x 250 mm

Particle Size 5 µm 10 µm 5 µm 5 µm 10 µm 10 µm 5 µm 10 µm 5 µm 5 µm 10 µm 10 µm 5 µm 5 µm 10 µm 10 µm


Atlantis™ dC18 Method Validation Kits Dimensions

Particle Size

4.6 x 150 mm 4.6 x 150 mm 4.6 x 250 mm

3 µm 5 µm 5 µm

Part No. 186002312 186002311 186002313

95

Ordering Information— Atlantis™ HILIC Silica Columns Atlantis™ HILIC Silica Analytical Columns

Atlantis™ HILIC Silica Method Validation Kits

Hardware Type

Dimensions

Particle Size

Part No.

Dimensions

Particle Size

Column Column Guard Guard Direct Connect Direct Connect Column Column Column Column Column Column Column Column Column Column Column Column Guard Guard Guard Guard Column Column Column Column Column Column Column Column Column

1.0 x 50 mm 1.0 x 50 mm 2.1 x 10 mm 2.1 x 10 mm 2.1 x 15 mm 2.1 x 15 mm 2.1 x 30 mm 2.1 x 30 mm 2.1 x 50 mm 2.1 x 50 mm 2.1 x 100 mm 2.1 x 100 mm 2.1 x 150 mm 2.1 x 150 mm 3.0 x 50 mm 3.0 x 50 mm 3.0 x 100 mm 3.0 x 100 mm 3.9 x 20 mm 3.9 x 20 mm 4.6 x 20 mm 4.6 x 20 mm 4.6 x 30 mm 4.6 x 30 mm 4.6 x 50 mm 4.6 x 50 mm 4.6 x 100 mm 4.6 x 100 mm 4.6 x 150 mm 4.6 x 150 mm 4.6 x 250 mm

3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 5 µm

186002003 186002004 1860020051 1860020061 186002007 186002008 186002009 186002010 186002011 186002012 186002013 186002014 186002015 186002016 186002017 186002018 186002019 186002020 1860020213 1860020223 1860020233 1860020243 186002025 186002026 186002027 186002028 186002029 186002030 186002031 186002032 186002033

4.6 x 150 mm 4.6 x 150 mm 4.6 x 250 mm

3 µm 5 µm 5 µm

96

1


WAT097958

2


186000262

3


WAT046910

4

Requires Cartridge End Fittings

WAT037525

5


289000779

6


186000709

Part No. 186002315 186002314 186002316


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The quality management system of Waters' manufacturing facilities in Taunton, Massachusetts and Wexford, Ireland complies with the International Standard ISO 9002 Quality Management and Quality Assurance Standards. Waters' quality management system is periodically audited by the registering body to ensure compliance.

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Atlantis® Columns Applications Notebook - Waters

Atlantis® Columns Applications Notebook - Waters

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