Published:Journal of Chromatographic Science,
ISSN 0021-9665 Volume
47, Number 5, May/June 2009, pp. 365-372
Effect of Mobile Phase pH and Organic Content
on LC–MS Analysis of Nucleoside and Nucleotide HIV Reverse
Transcriptase Inhibitors
Zsuzsanna Kuklenyik1, Amy Martin2,
Chou-Pong Pau2, J. Gerardo Garcia-Lerma2,
Walid Heneine2, James L. Pirkle1,
and John R. Barr1, 1Division of Laboratory Sciences, National Center for Environmental
Health, Centers for Disease Control and Prevention, Atlanta,
Georgia; 2Division of HIV/AIDS Prevention, Centers for Disease
Control and Prevention, Atlanta, Georgia
The HIV-1 reverse transcriptase inhibitors tenofovir
(TFV), emtricitabine (FTC), and lamivudine (3TC) are widely used
in the treatment of HIV-1-infected persons and are now being
considered as chemoprophylactic drugs for the prevention of sexual
HIV transmission. Assays that measure these drugs after either
oral or topical application are critical to the understanding
of the pharmacokinetic profiles of the drugs and allow a rational
design of chemoprophylaxis modalities for evaluation in macaque
models and human trials. We developed a high-performance liquid
chromatography–tandem mass spectrometry (HPLC–MS–MS)
method for sensitive measurement of FTC, 3TC, and TFV in plasma
from macaques. To achieve detection limits of 10 pg on column,
the plasma analytes were measured using acidic mobile phase and
positive electrospray ionization MS–MS detection. However,
this caused various chromatographic peak distortions, which were
minimized by using mobile phase additives that induced ion-pairing
interactions. Chromatographic peak tailing was minimized by adjusting
the organic mobile phase concentration while considering the
simultaneous effect of organic content on buffer and analyte
pKa+. Injection solution interferences were corrected
by chromatographic peak focusing using column switching. The
final method provides simultaneous measurement of all three analytes
with a wide linear range of 1–3000 ng/mL using 0.1 mL plasma
(10 pg on column) and coefficients of variation from 5% to 15%
in the high ng/mL concentration range and from 16% to 20% in
the low ng/mL concentration range.
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