Investigation of endogenous blood lipids components

Drug Testing and Analysis

Research article Received: 20 July 2012

Revised: 31 August 2012

Accepted: 31 August 2012

Published online in Wiley Online Library: 10 October 2012

(www.drugtestinganalysis.com) DOI 10.1002/dta.1421

Investigation of endogenous blood lipids components that contribute to matrix effects in dried blood spot samples by liquid chromatography-tandem mass spectrometry Omnia A. Ismaiel,a* Rand G. Jenkinsb and H. Thomas Karnesc Dried blood spot (DBS) sampling coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a rapidly developing approach in the field of biopharmaceutical analysis. DBS sampling enables analysis of small sample volumes with high sensitivity and selectivity while providing a convenient easy to store and ship format. Lipid components that may be extracted during biological sample processing may result in matrix ionization effects and can significantly affect the precision and accuracy of the results. Glycerophosphocholines (GPChos), cholesterols and triacylglycerols (TAG) are the main lipid components that contribute to matrix effects in LC-MS/MS. Various organic solvents such as methanol, acetonitrile, methyl tertiary butyl ether, ethyl ether, dichloromethane and n-hexane were investigated for elution of these lipid components from DBS samples. Methanol extracts demonstrated the highest levels of GPChos whereas ethyl ether and n-hexane extracts contained less than 1.0 % of the GPChos levels in the methanol extracts. Ethyl ether extracts contained the highest levels of cholesterols and TAG in comparison to other investigated organic solvents. Acetonitrile is recommended as an elution solvent due to low lipid recoveries. Matrix effects resulted from different extracted lipid components should be studied and assessed carefully in DBS samples. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: matrix effects; dried blood spot samples

Introduction

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Matrix ionization effects are variable from one biofluid source to another,[1] and can significantly affect the accuracy, precision and reproducibility of liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.[2,3] Phospholipids are well known as the major cause of matrix effects in LC-MS/MS analysis. It was found that other endogenous lipid components such as cholesterols (C) and triacylglycerols (TAG) may also result in ion suppression.[4] Dried blood spot (DBS) technique is an advanced method for human blood sampling. It has many advantages including simplicity of sample collection, less invasive sampling, the ability of the patient to collect samples at home, small sample volumes are required, and inexpensive ambient temperature shipment with long term storage stability.[5,6] DBS coupled to LC-MS/MS is a promising technique that enables analysis of micro quantities of blood samples with good sensitivity and selectivity.[6] DBS-LC-MS and DBS-LC-MS/MS techniques have been used for quantitative determination of dexamethasone, dextromethorphane, 25OH vitamin D3 and 25OH vitamin D2 in biological samples.[7–9] A DBS-LC-MS/MS method has been developed and validated for the simultaneous quantification of the most commonly used protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs).[10] DBS is also a promising technique for bioavailability and pharmacokinetic (PK) studies; the PK profiles for naproxen in plasma and in DBS samples have been compared using LC-MS/MS. A strong correlation between PK data from both methods has been obtained.[11]

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The DBS on-card derivatization concept has been evaluated to simplify sample handling and allow accurate quantification of unstable compounds in biological matrices. In-house pretreated cards with the derivatizing agent have been used for derivatization and analysis of thiorphan, which can be easily oxidized in biological matrices and requires complex handling procedures at the clinical site for stabilization.[12] The pre-cut dried blood spot (PCDBS) approach has been presented as an alternative to the traditional DBS technique to overcome the factors affecting precision and accuracy, such as blood viscosity, analyte nature, spotting technique, spotting conditions, and primarily the hematocrit impact.[13] Fully automated DBS systems are available recently for LC-MS/MS analysis to allow automated card handling and online sample extraction. Organic solvents such as methanol (MeOH), acetonitrile (ACN), and methyl tertiary butyl ether (MTBE) have been used to elute analytes from the DBS. A mixture of water and organic solvent can also be used for analyte extraction.[14] These solvents may

* Correspondence to: Omnia A. Ismaiel, Zagazig University Faculty of Pharmacy Department of Analytical Chemistry, Zagazig, Egypt, 44519. E–mail: oismaiel@ yahoo.com a Zagazig University Faculty of Pharmacy, Department of Analytical Chemistry, Egypt b PPD, Richmond, Virginia, USA c Virginia Commonwealth University, School of Pharmacy, Department of Pharmaceutics, USA

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Investigation of matrix effects in dried blood spot samples by LC-MS/MS

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analytes from interference peaks and additional sample clean-up steps have been found to be required.[24] A novel on-line desorption DBS approach has been developed for the direct LC-MS analysis of m-whole blood samples; experimental conditions were optimized to ensure selective desorption of target analytes compared to matrix interferences. Organic solvent desorption resulted in a precipitation of the proteins such as haemoglobin into the paper fibres and prevented their elution into the desorption flow.[25] Endogenous matrix components that may be extracted from DBS samples and contribute to matrix effects in DBS-LC-MS/MS analysis did not investigated before; the aim of this study is to investigate the ability of various organic solvents to extract GPChos (e.g. PC), Cholesterols and TAGs as the main endogenous matrix components that may result in matrix effects in DBS samples, as a general guide for the expected matrix effects in DBS samples

Experimental Materials 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (PC), 1-palmitoyl2-hydroxy-sn-glycerol-3-phosphocholine (LPC), were purchased from Avanti Polar lipids, Inc. (Alabaster, AL, USA). cholesterol (C), cholesteryl oleate (CE), 1,3-dipalmitoyl-2-oleoylglycerol (TAG), formic acid (FA), ammonium formate, Harris UNI-CORE ™ 3.0 mm were purchased from Sigma-Aldrich, (St Louis, MO, USA ). Acetonitrile (ACN), methanol (MeOH) and isopropyl alcohol (IPA), methyl tertiary butyl ether (MTBE), ethyl ether, dichloromethane (DCM), n-hexane (HEX) were from Burdicks& Jackson (B&J) (Muskegon, MI, USA). Human whole blood with K2EDTA was from BioChemed services (Winchester, VA, USA). Whatman FTA DMPK-A cards were purchased from WhatmanW (Piscataway, NJ, USA). Apparatus The high performance liquid chromatography (HPLC) system consisted of a Shimadzu, System Controller, SCL-10A VP, Pump, LC 10AD VP, Agilent 1100 Series HPLC system (Agilent Technologies, Palo Alto, CA, USA), Solvent Degasser, DGU14A, and autosampler CTC PAL (Zwingen, Switzerland). The mass spectrometer was a Micromass Quattro API Micro, Waters Corp., with Masslynx version 4.0 and 4.1 data acquisition software installed on an HP computer (Waters Corp, Milford, MA, USA), that was operated in the electrospray ionization (ESI) positive multiple reaction monitoring (MRM) mode. Gradient method for the analysis of endogenous components The optimized method for monitoring endogenous lipid components, selection of columns, mobile phases and the switching valve diagram have been described previously.[4] The optimized method was found to be suitable for different sample preparation extracts (e.g. protein precipitation, liquid-liquid extraction and solid phase extraction). A Luna Silica 50  2.0 mm, 5mm particle size, analytical column, Phenomenex (Torrance, CA, USA) along with a Gemini C18 4.0 x 2.0 mm guard column, Phenomenex (Torrance, CA, USA) were used. Mobile phase A was (20:80) methanol (MeOH): H2O, v/v with 10 mM ammonium formate and 0.3% formic acid. Mobile phase B was (5:25:70) H2O: acetonitrile (ACN): MeOH, v/v/v with 10 mM ammonium formate and 0.3% formic acid, and mobile phase C

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extract various endogenous lipid components and result in matrix ionization effects in LC-MS/MS analysis. The composition of endogenous lipid components in human plasma and erythrocytes and the expected matrix effects resulting from various lipid components have been described previously.[4] Postcolumn infusion experiments have been conducted by injecting standard solutions of different lipid components and infusing chlorpheniramine or loratadine as test compounds.[4] A concentration normalized suppression factor (%CNSF) has been created to compare the relative potential ion suppression effects of these lipids components based on the approximate lipid concentrations in human plasma and erythrocytes.[4] Glycerophosphocholines (GPChos) are well known as the primary cause of matrix effects in LC-MS/MS. The %CNSF results showed that other lipid components which present at relatively high concentrations in biological matrices such as cholesterol, cholesterol esters, and triacylglycerols can also result in significant ion suppression effects.[4] Human whole blood is composed of approximately 54% plasma and 46% circulating cells (mainly erythrocytes).[15] C, CE, phospholipids, and TAG constitute 45, 18, 17, and 7 % of the total lipids in human whole blood, respectively.[16] Endogenous lipid concentrations differ from one individual to another and the extracted lipid components are based on many factors such as organic solvents used for analyte elution and pH. Matrix effects are also compound-dependent; the chemical nature of the analyte strongly affects the likelihood of either ion suppression or enhancement. The chromatographic elution profile of endogenous lipid components and target analytes are keys factor in the matrix effects extent. Phospholipids are separated on normal phase chromatography according to the polarity of the head group; each class will be eluted as one peak. However, reversed phase chromatography separates phospholipids according to the lipophilicity of the fatty acid side chain; GPChos will be eluted as several peaks.[17] Retention of phospholipids on hydrophilic interaction chromatography (HILIC) has been investigated.[18] It was found that phospholipids retention significantly vary under different HILIC conditions and changing pH is a potential factor to chromatographically resolve the analyte peaks from phospholipids.[18] Late eluting peaks may be eluted in subsequent injections and result in inconsistent matrix ionization effects. Monitoring phospholipids during method development and sometimes during sample analysis was found to be a successful approach to minimize matrix effects and avoid co-elution with phospholipid peaks.[19] Other approaches such as the appropriate selection of sample preparation technique, comparison of different ionization mode (ESI vs APCI) and adjusting of the chromatographic conditions have been applied to minimize matrix effects.[20,21] DBS card-induced interferences in LC-MS have been investigated. It was found that one of the DBS card constituents (sodium dodecyl sulfate) was responsible for the interferences due to ionpair formation with compounds containing basic amine groups, which resulted in retention time shifting, peak shape distortion, and MS signal intensity suppression.[22] Non-acidic mobile phases were found to overcome these interferences.[22] The presence of red and/or white blood cells in the whole blood may interfere with some assays. The components of the ruptured cells were found to be an instrumental problem in the measurement of some analytes using DBS.[23] Hemolysis effect has been found to have a significant impact on LC-MS/MS quantitation of some analytes in human plasma spiked with hemolyzed whole blood. Method modifications such as adjusting chromatographic conditions to resolve target

Drug Testing and Analysis

Drug Testing and Analysis

O. A. Ismaiel, R. G. Jenkins and H. T. Karnes

was (5:55:40) H2O: MeOH: isopropyl alcohol (IPA), v/v/v with 10 mM ammonium formate and 0.3% formic acid. The total run time was 10 min. A Phenomenx Gemini C18 4.0 mm  2.0 mm guard column with a loading mobile phase consisting of (20:80) MeOH: H2O, v/v with 10 mM ammonium formate and 0.3% formic acid at a 0.5 ml/min flow rate for 1 min was used to trap the target lipids and flush salts and other highly polar non-retained compounds into waste. The loading pump ran an isocratic 100% mobile phase A, the elution pumps ran a step gradient at 0.5 ml/min flow rate as follows: (0–2.9) min 100% B, (3.0–4.9) min 100% C, (5.0–8.9) min 50/50 B/C at 0.75 ml/min flow rate and (9.0–10) min 100% B at 0.5 ml/min flow rate. A switching valve was used to direct the guard column effluent either to the waste (0–1) min during the loading step or to transfer lipids to the analytical column (1–9) min then return back to the initial position (9–10) min.

Mass spectrometry In-source multiple reaction monitoring (IS-MRM)[26] using a high cone voltage (90 volts) and low collision energy (7 eV) was used for detection of all GPChos using the m/z 184/184 mass transition; other phospholipids such as lysoglycerophosphocholines (LGPChos) and sphingomyelines (SMs) can also be monitored using the same mass transition.[26] LGPChos (e.g. lysophosphatidylcholine) can be also monitored separately using the m/z 104/104 mass transition.[26] The characteristic ion fragment m/z 369 [M + H-H2O]+ for cholesterol was used without further fragmentation to monitor cholesterols and cholesterol esters using m/z 369/369 mass transition.[4] 1,3-dipalmitoyl-2-oleoylglycerol has been used as a marker for TAGs, it formed [M + NH4]+ at m/z 850.73 and the MS/MS of the ammonium adduct showed an intense ion at m/z 577.83 [OP]+. The MS/MS system parameters were: capillary (4.00 kV), extractor (2.00 volts), source temperature (120  C), desolvation temperature (350  C), desolvation gas flows (600 L/h and 100 L/h). Collision gas (Argon), collision energy, mass transitions and MRM parameters were as shown in Table 1.

Results and discussion Post-column infusion experiments have shown that phosphatidylcholines (PC), TAG, C and cholesterol esters (CE) at equal concentration resulted in approximately 55%, 35%, 45%, and 40% ion suppression for chlorpheniramine, respectively, and resulted in approximately 85%, 70%, 60%, and 70% ion suppression for loratadine, respectively.[4] Glycerophosphocholines (GPChos) Polar lipids, such as glycerophosphocholines are soluble in polar solvents such as methanol and insoluble in non-polar solvents such as n-hexane. PCs with short side chain fatty acids are soluble in methanol and insoluble in ether. However, PCs with long side chain fatty are soluble in both methanol and ether.[27] MeOH extracts showed the highest levels of GPChos in comparison to the other investigated organic solvents likely due to the solubility of PCs with both short and long side chain fatty acids in methanol. IPA and ACN, extracts contained approximately, 16.0 and 2.5% of the GPChos levels that were found in MeOH extracts, respectively. Ether, n-hexane and DCM showed less than 1.0 % of PCs levels in the MeOH extract (Figure 1). LGPChos showed the same extractability trend. Combined responses of cholesterol (C) and cholesterol esters (CE) Non-polar lipids such as TAGs and CE are soluble in non polar solvents such as n-hexane, and slightly polar solvents such as diethyl ether, but insoluble in methanol. Their solubility in alcohol increases either by decreasing the fatty acid side chain length or by increasing the alcohol side chain length.[27] Cholesterol solubility in acetonitrile and methanol (at 26  C) was found to be 0.161 and 0.648 g/100 g of solvent, respectively.[28] The final extracts of DBS samples resulting from the studied organic solvents showed that ether extracts contained the highest

Sample preparation 30 mL aliquots of blank human whole blood with K2EDTA were spotted in triplicate onto Whatman FTA DMPK-A cards. After drying the cards for at least 4 h, disks were removed from the cards using a punch (3 mm) and extracted by vortex mixing for 15 min using 250 ml of MeOH, ACN, IPA, DCM, MTBE, ether or HEX, in a 96-well plate. The eluate was evaporated to dryness under a nitrogen stream at approximately 40  C. The residue was reconstituted with 250 ml of (75:25) MeOH: ACN (v/v), vortex mixed for 5 min, and 25 ml of the resulting solution was injected into the LC-MS/MS

Figure 1. Absolute area responses of GPChos (m/z 184/184) in final extracts of DBS samples using 30 mL aliquots of blank human whole blood with K2EDTA and 250 ml of (1) MeOH, (2) ACN, (3) IPA, (4) DCM, (5) MTBE, (6) Ether and (7) HEX as elution solvents.

Table 1. Multiple reaction monitoring (MRM) parameters Compound

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Glycerophosphocholines (GPChos) 1,3-Dipalmitoyl,2oleoyl-glycerol [PPO] (TAG) Cholesterol (C) Cholesteryl oleate (CE)

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MS/MS2 (m/z)

Cone (Volts)

CE (eV)

184/184 [H2PO4-(CH2)2-N(CH3)3]+ 850.73/577.83 [M + NH4]+ /[OP]+ 369.2/369.2 [M + H-H2O]+ 369.2/369.2 [M + H-Oliec acid]+

90 40 40 40

7 25 10 10

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Investigation of matrix effects in dried blood spot samples by LC-MS/MS

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concentrations of C and CE compared to the other investigated extraction solvents. MeOH extracts showed 88% of the C and CE levels that were found in ether extract which may be due to the solubility of cholesterol and CE (with short chain fatty acid) in MeOH. Other organic solvents – MTBE, n-hexane, IPA, ACN and DCM extracts – contained approximately 80, 70, 38, 26, and 13% of C and CE levels in ether extracts, respectively (Figure 2).

Triacylglycerols (TAG) Ether extracts showed the highest levels of the extracted TAG whereas MeOH extracts demonstrated very low levels of TAG which may be due to the solubility of TAG as a non-polar lipid in a slightly polar solvent (ether) and limited solubility in MeOH. Other organic solvents – MTBE, n-hexane, IPA, ACN and DCM extracts – contained approximately 89, 77, 43, 21, and 18% of TAG levels that were found in ether extracts, respectively (Figure 3). The results showed that different lipid components will be extracted to a different extent during analyte elution from DBS samples using different organic solvents. Figures 4 and 5 show the various levels of extracted lipid components using ACN and n-hexane, respectively. Although the sample volume used for DBS was very small, high levels of lipids were observed in the final extracts as shown in Figure 4 and 5. The presence of these lipids has the potential to impact sensitivity and selectivity of DBS-LC-MS/MS results. The organic solvent used for analyte elution should be selected carefully not only in terms of analyte recovery but also in terms of lipid extractability. Lipid components should be monitored during method development and matrix effects should be assessed for DBS samples.

Figure 4. LC/MS/MS analysis of the final extract of a DBS sample from a 30 mL aliquot of blank human whole blood with K2EDTA and using 250 mL of ACN as an elution solvent (A) GPChos (B) C&CE (C) TAG.

ACN is recommended as an elution solvent over MeOH due to low recoveries of GPChos, C, CE, and TAG. Matrix effects should be investigated during method development. The previous results and the proposed mass transitions may help scientist to

Figure 2. Absolute area responses of TAG (m/z 850.73/577.83 ) in final extracts of DBS samples using 30 mL aliquots of blank human whole blood with K2EDTA and 250 ml of (1) MeOH, (2) ACN, (3) IPA, (4) DCM, (5) MTBE, (6) Ether and (7) HEX, as elution solvents.

Drug Test. Analysis 2013, 5, 710–715

Figure 5. LC/MS/MS analysis of the final extract of a DBS sample from a 30 mL aliquot of blank human whole blood with K2EDTA and using 250 mL of hexane as an elution solvent (A) GPChos (B) C&CE (C) TAG.

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Figure 3. Combined area responses of C and CE (m/z 369.2/369.2) in final extracts of DBS samples using 30 mL aliquots of blank human whole blood with K2EDTA and 250 ml of (1) MeOH, (2) ACN, (3) IPA, (4) DCM, (5) MTBE, (6) Ether and (7) HEX, as elution solvents.

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O. A. Ismaiel, R. G. Jenkins and H. T. Karnes

monitor the extracted lipid components in the final extracts of DBS samples and expect the extracted lipid components based on the organic solvent used for analyte elution. Polar solvents such as MeOH would be expected to extract more phospholipids. GPChos (e.g. phosphatidylcholine) are characterized by the polar head group (trimethyl-ammonium ethyl phosphate)[26] that may act as an active site for charge transfer interactions, analytes with either electron donor site or proton acceptor site may react with the positively charged trimethyl-ammonium site or the hydroxyl group, respectively, which may lead to ionization matrix effects in case of chromatographic co-elution with phospholipids. Phospholipid elution profiles are different from normal phase (NP) to reversed phase (RP) and late eluting peaks may result in unanticipated matrix effects in later injections. Non-polar solvents such as n-hexane and slightly polar solvents such as ether will extract more cholesterols and TAGs. As shown previously, presence of these lipids at high concentrations can also result in ion suppression.[4] These effects may be due to charge transfer interactions with (hydroxyl and ester groups) or due to change the efficiency of droplets formation and droplets evaporation in ESI mode. Selection of the appropriate elution solvent, monitoring extracted lipids during method development and adjustment of the chromatographic conditions to avoid co-elution can be a successful approach to avoid matrix ionization effects in DBS samples.

Conclusions The DBS technique coupled to LC-MS/MS is a very promising bioanalytical tool; however, the presence of lipids in the final extracts may significantly affect sensitivity, selectivity, and reproducibility of the results. Different lipid components may be extracted and resulted in matrix ionization effects. ACN is recommended as an elution solvent over MeOH due to the low recoveries for lipid components. Matrix effects in DBS samples should be investigated during method development. GPChos, cholesterols and TAGs should be monitored in the final extracts to ensure absence of co-elution with target analyte and to minimize matrix effects.

Acknowledgements The authors would like to acknowledge the valuable help given by Jordan Honrine and Michael P. Waldron, PPD, Richmond, Virginia, USA.

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