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Abstract INTRODUCTION:
Protease inhibitors (PIs) are a class of anti-viral drugs that prevent viral replication by selectively binding to viral proteases and inhibiting their function. The development of PI-based therapies has been of enormous benefit to people infected with HIV. Unfortunately, the effectiveness of protease inhibitors can fade over time. Mutations during viral replication can result in viruses that produce new, different proteases that are not targeted by current PI therapies. The best way to avoid this drug resistance is to reduce or stop HIV replication. With less HIV replication, there is less of a chance of a new strain that is resistant to antiHIV drugs. To keep HIV levels as low as possible, PIs are typically taken in combination with at least two other anti-HIV drugs. Such combination therapies are referred to as highly active antiretroviral therapy (HAART). Lopinavir and ritonavir are two protease inhibitors that are often used as part of a fixed-dose combination, and serve as the model compounds in this study.
OBJECTIVE:
To develop a method utilizing Acoustic Ejection Mass Spectrometry (AEMS), as implemented in the Echo® MS System with a SCIEX Triple Quad™ 6500+ LCMS/MS system, which offers clear benefits for quantification of lopinavir and ritonavir in human plasma. Requiring minimal sample preparation and no chromatographic separation, it provides high sample throughput without sacrificing robustness or reproducibility.
METHODS:
Lopinavir and ritonavir were spiked into human plasma samples in the range of 0.5 ng/mL to 250 ng/mL each. Samples were processed using a liquid-liquid extraction method and 0.4 mL of supernatant liquid was collected and dried under a nitrogen stream. Samples were reconstituted in 100 µL of 25% v/v methanol in water and transferred to a 384-well plate for analysis by AEMS. Methanol with 0.1% v/v formic acid was used as carrier solvent at a flow rate of 425 µL/min in the AEMS with 50 nL sample volumes. MS/MS detection was performed using the SCIEX Triple Quad 6500+ LC-MS/MS.
RESULTS:
Calibration curves, along with quality control samples, all analyzed in six replicates, demonstrated the high reproducibility of AEMS when combined with liquid-liquid extraction. Excellent %CVs were achieved across all concentration levels with no interference in blank human plasma samples. Even at the extremely short analysis time of 3 seconds per sample, the method yielded LLOQs of 0.5 ng/mL for both lopinavir and ritonavir. The assay accuracy was 85.31–112.34% for ritonavir and 85.51–113.52% for lopinavir. The calibration curve covered approximately 3 orders of magnitude (0.5–250 ng/mL) for both analytes and displayed linearity with regression coefficients (r2) of 0.9934 for ritonavir and 0.9946 for lopinavir using a weighting of 1/x2.
CONCLUSION:
The Echo® MS System produced very sensitive, accurate and reproducible results for the simultaneous quantitative analysis of lopinavir and ritonavir in human plasma. Having a very short analysis time (3 sec/sample) enabled rapid generation of quantitative data for high numbers of samples, and with the assay showing great reproducibility even without using labeled internal standards. Future use of labeled internal standards is recommended to further improve these results. |
= Discovery stage. (24.37%, 2023)
= Translation stage. (39.50%, 2023)
= Clinically available. (36.13%, 2023)
