UHPLC-HIGH RESOLUTION-MS vs UPLC-MS/MS ...

UHPLC-HIGH RESOLUTION-MS vs UPLC-MS/MS FOR THE DETERMINATION OF SEDATIVES IN KIDNEY Nuria León a, b, Carmen Igualada a, b , Francisco Moragues a, b and Vicent Yusà a, b, c. a Veterinary

drug residues unit - Public Health Laboratory of Valencia. Avda. Cataluña nº 21.46020 Valencia. Spain. Safety Research Area - Foundation for the Promotion of Health and Biomedical Research in the Valencian Region, FISABIO – Public Health. Avda. Cataluña nº 21. 46020 Valencia. Spain. c Analytical Chemistry Department, Universidad de Valencia, Edifici Jeroni Muñoz, 50, Dr. Moliner, 46100 Burjassot, Valencia, Spain. b Food

e-mail: [email protected], [email protected], [email protected] and [email protected]

INTRODUCTION Sedatives are widely used in food producing animals for the prevention of stress caused during animal transportation from the farm to the slaughterhouse. According to the Council's Regulation 37/2010 the use of chlorpromazine has been banned and maximum residue levels (MRLs) have been established for carazolol, azaperone and azaperol for kidney and other matrices. A rapid extraction procedure was developed allowing the determination of nine sedatives (acepromazine, propionylpromazine, chlorpromazine, carazolol, azaperone, azaperol, haloperidol, xylazine and atenolol) in kidney tissue. While triple quadrupole MS remains the workhorse for the analysis of veterinary medicines, LC coupled with high-resolution MS (LC-HRMS) is making headway in this field. Despite the multiple advantages of LCHRMS such as reduction of matrix effects, number of molecules in one run, confidence in identifications and post-acquisition data mining, this instrumentation has not been broadly adopted in routine laboratories. The methods were validated in accordance with the Commission Decision 2002/657/EC. The relevant validation parameters for both analytical tools (LC-HRMS and LC-MS/MS)such as precision, accuracy, linearity, specificity, decision limits (CCα) and detection capabilities (CCβ), were investigated. The suitability of LC-HRMS method for quantitative and confirmatory purposes was evaluated for each substance on the basis of the 2002/657/EC.

EXPERIMENTAL

RESULTS 5 ml Acetonitrile extract

8 ml H2O+ 10 ml Acetonitrile + extraction salts

MS/MS settings settings and and monitored ions MS/MS

LC-MS/MS Extracted ion chromatogram of a porcine kidney at MRL level 1.82 3591

Collision Energy (eV)

Product ions (quantification ion in bold)

0

Adquisition Mode

1.25

EXTRACTION

1.50

1.75

2.00

3.00

3.25

3.50

3.75

4.00

4.25

4.50

4.75

5.00

5.25

5.50

5.75

1.50

1.75

2.00

2.25

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

4.25

4.50

4.75

5.00

5.25

5.50

5.75

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

4.50

4.75

5.00

5.25

5.50

5.75

4.75

5.00

5.25

5.50

5.75

5.25

5.50

5.75

5.50

5.75

5.50

5.75

5.50

5.75

1.79 2008

2.50

2.75

ATENOLOL

%

100

evaporate to dryness under stream of N2

ATENOLOL-D7

25

274

20

145

ESI +

ATENOLOL

25

267

30

145

ESI +

30

133

0 1.25

3.28 11686

100

0 1.25

XYLAZINE

30

221

20

90

20

164

15

330

25

121

30

149

%

AZAPEROL

0 1.25

AZAPEROL

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

328

20

165

30

123

15

116

20

222

4.25

4.50

4.25

4.50

%

AZAPERONE

0 1.25

25

4.00

3.86 83406

ESI + 100

AZAPERONE

4.25

3.38 79183

ESI + 100

1 ml H2O:MeOH (80:20)

XYLAZINE

%

1 g KIDNEY SAMPLE

ATENOLOL-D7

%

100

Cone Precursor ion Voltage (V)

Compound name

4.00

ESI +

4.34 5404

Chromatographic conditions

15

299

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

Column: BEH C18 (50 x 2.1 mm, 1.7 µ)

Column: Hypersil Gold (100 x 2.1 mm, 1.9 µ)

Flow rate: 400 µL/min

Flow rate: 400 µL/min

Injection volume: 10 µL Time (min)

0

3

20

PROPIONYLPROMAZINE

327

20

341

5.5

6

6.5*

Time (min)

0

8

8.5

10

20

86

20

58

5.29 24565

%

ACEPROMAZINE

100

5.25

%

58

ESI + 0 1.25

ESI +

1.50

1.75

2.00

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

2.50

2.75

3.00

3.25

3.50

3.75

4.00

4.25

4.50

4.75

5.00

5.25

5.44 14568

PROPIONYLPROMAZINE

100 0

1.25

ESI +

2.25

4.25

4.50

4.75

5.00

5.25

5.50

5.75

5.51 4127

CHLORPROMAZINE

100

CHLORPROMAZINE-D6

30

325

20

92

ESI +

CHLORPROMAZINE

30

319

25

86

ESI +

25

58

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

4.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

4.25

4.50

4.75

5.00

5.25

5.50

5.75

5.50 4704

%

100 0

1.25

HCD OFF (50-500 m/z); positive mode

10.5*

1st Diagnostic/ Quan ion Compound name

75

75

20

20

100

%A

100

50

50

5

5

100

%B

0

25

25

80

80

0

%B

0

50

50

95

95

0

* Equilibration time: 5min

Adduct

Accurate mass (m/z)

4.50

4.75

5.00

5.25

5.50

5.75

HCD ON event (25eV) (50-500 m/z); positive mode

2nd Diagnostic ion

3rd Diagnostic ion

Accurate Chemical Formula mass (m/z)

Chemical Formula

1st Fragment

Accurate mass (m/z)

Chemical Formula

2nd Fragment

3rd Fragment

Accurate mass Accurate Chemical Chemical Formula (m/z) mass (m/z) Formula

C14D7H15N2O3

[M+H]+

274.2143

C1313CD7H16N2O3+

275.2176

C14H22N2O3

[M+H]+

267.1703

C1313CH23N2O3+

268.1737

C10H9O+

145.0648

C11H12NO2+

190.0863

XYLAZINE

C12H16N2S

[M+H]+

221.1107

C1113CH17N2S+

222.1141

C4H8NS+

102.0372

C8H12N+

122.0964

AZAPEROL

C19H24FN3O

[M+H]+

330.1976

C1813CH25FN3O+

331.2010

C10H10F+

149.0761

C7H9N2+

121.0760

AZAPERONE

C19H22FN3O

[M+H]+

328.1820

C1813CH23FN3O+

329.1853

CARAZOLOL

C18H22N2O2

[M+H]+

299.1754

C1713CH23N2O2+

300.1788

C21H23ClFNO2

[M+H]+

376.1474

C21H2437ClFNO2+

378.1445 C2013CH24ClFNO2+

HALOPERIDOL

MS conditions

Elemental composition (M)

ATENOLOL

ATENOLOL-D7

377.1508

C10H10FO+

165.0710

C7H4FO+

123.0241

C15H12NO+

222.0913

C12H10N+

168.0808

C10H10FO+

165.0710

C7H4FO+

123.0241

C19H22N2OS

[M+H]+

327.1526

C1813CH23N2OS+

328.1559

C5H12N+

86.0964

C3H8N+

58.0641

PROPIONYLPROMAZINE C20H24N2OS

[M+H]+

341.1682

C1913CH25N2OS+

342.1716

C5H12N+

86.0964

C3H8N+

58.0641

CHLORPROMAZINE-D6 C17D6H13ClN2S

[M+H]+

325.1407

C17D6H1437ClN2S+

327.1377

C5D6H6N+

92.1341

[M+H]+

319.1030

C17H2037ClN2S+

321.1001 C1613CH19ClN2S+

C5H12N+

86.0964

C3H8N+

58.0641

ACEPROMAZINE

CHLORPROMAZINE

C17H19ClN2S

319.0986

4th Fragment

Accurate Accurate Chemical Formula mass (m/z) mass (m/z)

C9H9O+

133.0648

C7H9N2+

121.0760

C11H14NO3+

208.0968

UHPLC-HRMS system: Orbitrap ExactiveTM/ HESI II

MS Settings:

MS Settings: Automatic gain control : 106(Balanced) Resolving power (FWHM): 50.000 (High) Positive mode mass range (m/z): In one run: Full scan:50-500 HCD on (25eV): 50-500 Spray voltage: 3.5 kV(+) Sheath gas flow rate: 50 Auxiliary gas flow rate: 10 Skimmer voltage: 30 V Heater temperature: 425 ºC Capillary temperature: 125 ºC Tube lens voltage: 120 V Mass accuracy