We show results from experiments designed to determine the relative importance of gas phase processes and solution phase processes into ionization suppression observed in biological sample extracts. The data indicate that gas phase reactions leading to the loss of net charge on the analyte is not likely to be the most important process involved in ionization suppression. The results point to changes in the droplet solution properties caused by the presence of nonvolatile solutes as the main cause of ionization suppression in electrospray ionization of biological extracts.

A post-column infusion system was developed in order to analyze suppression of electrospray ionization (ESI) tandem mass spectrometry response in the presence of endogenous plasma interferences. By enabling direct detection of these interfering components, this experimental system was used to analyze the ability of several common extraction procedures to remove endogenous plasma components that cause changes in the ESI response of model drug substances. Methyl-t-butyl ether (MTBE) liquid-liquid, Oasis and Empore solid-phase, and acetonitrile (ACN) protein precipitation sample preparation methods were tested using the post-column infusion system. In all cases, ACN protein precipitation samples showed the greatest amount of ESI response suppression while liquid-liquid extracts demonstrated the least. In addition, the three test compounds, phenacetin, caffeine, and a representative Merck compound, demonstrated that ESI response suppression is compound dependent. Suppression was greatest with caffeine, the most polar analyte, and the smallest for the Merck compound, the least polar analyte. Copyright 1999 John Wiley & Sons, Ltd.

The present study describes a new analytical approach for the detection and characterization of GSH-trapped reactive metabolites using multiple reaction monitoring (MRM) as the survey scan to trigger the acquisition of enhanced product ion (EPI) spectra on a triple quadrupole linear ion mass spectrometer. The MRM scan step was carried out following up to 114 MRM transitions from the protonated molecules of potential GSH adducts to their product ions derived from a neutral loss of 129 or 307 Da. MRM transition protocols were constructed on the basis of common bioactivation reactions predicted to occur in human liver microsomes (HLM). The effectiveness and reliability of the approach were evaluated using acetaminophen, diclofenac, and carbamazepine as model compounds. The total ion chromatograms of the MRM for the HLM incubations with these compounds and GSH clearly displayed a number of GSH adducts, including acetaminophen-GSH adducts and carbamazepine-GSH adducts that were not previously observed in HLM incubations. In addition, clomipramine and mefenamic acid that have the frame structures susceptible to P450-mediated bioactivation were investigated. As a result, the MRM-EPI analysis revealed multiple GSH adducts of clomipramine and mefenamic acid in HLM incubations possibly mediated by epoxide and/or quinone imine intermediates. Compared with the neutral loss (NL) and precursor ion (PI) scanning analysis, the MRM-based approach provided superior sensitivity and selectivity for GSH adducts. It also enabled the sensitive acquisition of EPI spectra with rich fragmentation in the same LC/MS run, which were useful for the rapid structure elucidation of GSH adducts and the elimination of false positives. The MRM-EPI experiment can be employed for high throughput screening of reactive metabolites and should be especially applicable to compounds of the same chemotype. Also, it can be applied in conjunction with the PI or NL scan as a comprehensive method for the analysis of reactive metabolites in a drug discovery setting.

A novel analytical method has been developed for direct quantification of intracellular nucleoside triphosphates (NTPs). Lysates of human peripheral blood mononuclear cells (PBMCs) were extracted by protein precipitation, and the filtered extracts were analyzed by weak anion exchange liquid chromatography (WAX-LC) coupled to detection by mass spectrometry (MS). Compared with ion pairing (IP)-LC/MS/MS, the only MS-compatible direct detection method for NTPs currently available, the new method completely avoids the usage of ion-pairing reagents and has a shorter analytical time of only 2 min. The method was validated and is being used to determine the amount of the triphosphate metabolite of D-D4FC (DPC817), an investigational HIV nucleoside reverse transcriptase inhibitor (NRTI), in human PBMC samples from clinical studies. By using a PE Sciex API 4000 triple quadrupole instrument operating in positive ion MRM mode, the method was able to achieve a lower limit of quantitation (LLOQ) of 5 fmol/10(6) cells in samples containing 3 x 10(6) lysed cells (6 fmol on-column). With minor adaptation, the method described here may be suitable for analyzing other NTPs. This paper also provides a discussion of the unique retention characteristics of WAX-LC, the principles of which may prove to be valuable for designing other forms of directly coupled ion-exchange (IX)-LC/MS methods suited for high sensitivity quantitative analysis.