Liquid chromatography–mass spectrometry/mass spectrometry method development for drug metabolism studies: Examining lipid matrix ionization effects in plasma
Introduction
Offering very high selectivity and sensitivity, liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) is the preferred method for drug metabolism studies. However, matrix effects may lead to significant analytical errors [1]. The Guidance for Industry on Bioanalytical Method Validation [2] states that “In the case of LC–MS and LC–MS/MS based procedures, matrix effects should be investigated to ensure that precision, selectivity, and sensitivity will not be compromised.” The specific methods to evaluate matrix effects are not specified; the approaches to expose matrix effects are left to the discretion of the investigator. In this regard, many researchers have described methods to probe and/or control matrix effects [1], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15].
Phospholipids are extremely abundant in biological membranes [16] and are formed from glycerol (phosphoglycerides) or sphingosine (sphingomyelins). Phosphoglycerides are composed of glycerol, one or two fatty acid ester chains, and a phosphorylated alcohol; whereas sphingomyelin is composed of sphingosine, an amide linked fatty acid, and a phosphatidyl choline. The glycerophosphocholines (GPCho's) constitute the major phospholipids in plasma [17], [18] and are known to cause significant LC–MS/MS matrix ionization effects [1], [6], [7], [8] in the positive ion electrospray mode (+ESI). The glycerine group in these GPCho's can be either 1-mono (2-lyso) or 1,2-disubstituted (diradyl). The 1,2-disubstituted GPCho (phosphatidylcholine) is commonly referred to as lecithin.
We have developed a new approach [19], which we refer to as “in-source multiple reaction monitoring” (IS-MRM) for monitoring GPCho's. Our method simultaneously monitors all GPCho's using only one channel in an MRM LC–MS/MS experiment. A similar approach can be employed in selected ion recording mode utilizing a single quadrupole mass spectrometer. Thus, this approach is conveniently employed to develop LC–MS/MS or LC–MS methods for the analysis of drugs and/or drug metabolites in protein-precipitated plasma samples that avoid deleterious matrix ionization effects from GPCho's. However, there are a variety of other exogenous and endogenous chemical species in plasma samples that yield matrix ionization effects and require additional strategies for evaluation [1].
Section snippets
Standards and reagents
Tamoxifen (>99%) and itraconazole (≥98%) standards were obtained from Apin Chemicals, Ltd. (Abingdon, Oxon, UK). Diphenhydramine hydrochloride (≥98%) and 4-hydroxytamoxifen (98% Z-isomer, remainder primarily E-isomer) standards were acquired from Sigma–Aldrich (St. Louis, MO, USA). N-desmethyltamoxifen (>98%) was obtained from Toronto Research Chemicals Inc. (North York, Ont., Canada). Itraconazole-d3 (97.8%) and hydroxyitraconazole (98.6%) were acquired from SynFine Research Inc. (Richmond
Monitoring choline-containing phospholipids via IS-MRM
As summarized in Fig. 1, there is a wide variety of GPCho's found in plasma (mouse, rat, rabbit, dog, monkey, and human). The lipid alkyl, acyl, and 1-alkenyl ester group chain lengths may vary from 16–22 carbons and contain from 0 to 6 sites of unsaturation [17], [25]. Consequently, monitoring all ion transitions for these individual components in an MRM experiment is not practical. The large number of required transitions would decrease the method's sensitivity.
We have developed a method [19]
Conclusions
A wide variety of strategies are needed to evaluate LC–MS/MS matrix ionization effects resulting from endogenous and exogenous chemical species in plasma [1]. We found our IS-MRM approach to be a useful new tool for probing the matrix ionization effects of endogenous GPCho's. Depending on the positive ion electrospray and chromatographic conditions, these GPCho's can either suppress or enhance ionization. Our approach was utilized to develop reliable LC–MS/MS methods for the analysis of several
Acknowledgements
We gratefully acknowledge John W. Dolan for valuable discussions, William W. Christie for literature references on lipids, and Stephen K. Haynes for sample preparation.
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