Liquid chromatography–mass spectrometry/mass spectrometry method development for drug metabolism studies: Examining lipid matrix ionization effects in plasma

Abstract

Glycerophosphocholines (GPCho's) are known to cause liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) matrix ionization effects during the analysis of biological samples (i.e. blood, plasma). We have developed a convenient new method, which we refer to as “in-source multiple reaction monitoring” (IS-MRM), for detecting GPCho's during LC–MS/MS method development. The approach uses high energy in-source collisionally induced dissociation (CID) to yield trimethylammonium-ethyl phosphate ions (m/z 184), which are formed from mono- and disubstituted GPCho's. The resulting ion is selected by the first quadrupole (Q1), passed through the collision cell (Q2) in the presence of collision gas at low energy to minimize fragmentation, and m/z 184 selected by the third quadrupole. This approach can be combined with standard multiple reaction monitoring (MRM) transitions with little compromise in sensitivity during method development and sample analysis. Hence, this approach was used to probe ionization matrix effects in plasma samples. The resulting information was employed to develop LC–MS/MS analyses for drugs and their metabolites with cycle times less than 5 min.

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].