Ultrasensitive Liquid
Chromatography–Tandem
Mass Spectrometric Methodologies for Quantification of Five HIV‑1
Integrase Inhibitors in Plasma for a Microdose Clinical Trial
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Abstract
HIV-1 integrase strand transfer inhibitors are an important
class
of compounds targeted for the treatment of HIV-1 infection. Microdosing
has emerged as an attractive tool to assist in drug candidate screening
for clinical development, but necessitates extremely sensitive bioanalytical
assays, typically in the pg/mL concentration range. Currently, accelerator
mass spectrometry is the predominant tool for microdosing support,
which requires a specialized facility and synthesis of radiolabeled
compounds. There have been few studies attempted to comprehensively
assess a liquid chromatography–tandem mass spectrometry (LC–MS/MS)
approach in the context of microdosing applications. Herein, we describe
the development of automated LC–MS/MS methods to quantify five
integrase inhibitors in plasma with the limits of quantification at
1 pg/mL for raltegravir and 2 pg/mL for four proprietary compounds.
The assays involved double extractions followed by UPLC coupled with
negative ion electrospray MS/MS analysis. All methods were fully validated
to the rigor of regulated bioanalysis requirements, with intraday
precision between 1.20 and 14.1% and accuracy between 93.8 and 107%
at the standard curve concentration range. These methods were successfully
applied to a human microdose study and demonstrated to be accurate,
reproducible, and cost-effective. Results of the study indicate that
raltegravir displayed linear pharmacokinetics between a microdose
and a pharmacologically active dose