Novel direct detection method for quantitative determination of intracellular nucleoside triphosphates using weak anion exchange liquid chromatography/tandem mass spectrometry

Abstract

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.

Pharmacokinetics of Lamivudine and Lamivudine-Triphosphate after Administration of 300 Milligrams and 150 Milligrams Once Daily to Healthy Volunteers: Results of the ENCORE 2 Study

“…The elimination half-life did not differ between the two doses. Bioequivalence in plasma 3TC pharmacokinetic parameters, following 300-mg and 150-mg QD regimens, was not demonstrated as the GMR (90% CI) were 0.57 (0.55 to 0.60) for AUC 24 , 0.63 (0.59 to 0.67) for C 24 , and 0.56 (0.53 to 0.60) for C max .…”

“…The GM (90% CI) intracellular 3TC-TP AUC 24 (pmol.h/10 6 cells), C 24 , and C max (pmol/10 6 cells) for the 300-mg dose were 59.5 (51.8 to 68.3), 1.49 (1.19 to 1.86), and 4.10 (3.59 to 4.69), respectively. For the 150-mg dose, they were 44.0 (38.0 to 51.0), 1.23 (1.00 to 1.52), and 2.95 (2.47 to 3.51), respectively.…”

“…For the 150-mg dose, they were 44.0 (38.0 to 51.0), 1.23 (1.00 to 1.52), and 2.95 (2.47 to 3.51), respectively. Bioequivalence in intracellular 3TC-TP pharmacokinetic parameters, following 300-mg and 150-mg QD regimens, was not demonstrated since the GMRs (90% CI) were 0.73 (0.64 to 0.83) for AUC 24 , 0.82 (0.68 to 0.99) for C 24 , and 0.70 (0.61 to 0.82) for C max .…”

Gradient chromatofocusing-mass spectrometry: A new technique in protein analysis

“…A few reports have been published using volatile displacer ions/acids in the mobile phase, such as ammonium acetate/acetic acid or ammonium formate/ formic acid, for ion-exchange chromatography techniques directly interfaced to an electrospray mass spectrometer in the analysis of drugs [15,16], chlormequat [17], nucleoside triphosphates [18], and peptides [19]. However, these reports have not led to widespread use of ion-exchange chromatography interfaced to MS because the volatile ions have relatively weak displacing power [20,21].…”

HILIC LC‐MS for the determination of 2′‐C‐methyl‐cytidine‐triphosphate in rat liver

“…Due to the strong ionic nature of nucleotides, RP LC with classical elution with ACN and phosphate buffer gradients leads to a significantly faster elution disabling a successful separation analysis. In contrast, a number of methods based on ion-pairing [10 -16], ion exchange [17,18] and column-switching [19] LC have been described. Even though a number of these chromatographic methods have been coupled to mass spectrometric detection, it is well known that the nature of salts that are used in the mobile phases as ion exchange and/or ion-pairing reagents are not suitable for MS detection.…”