The 8th Mass Spectrometry CVG Annual Symposium - Full Day Event!!!
Presentation No. 3 - Quantitation of several antiretroviral drugs in human plasma by LC tandem MS
David W. Blank; Brian J. Gilfix; Marcos DiFalco; Line Roy; Bernard F. Gibbs
McGill University, Montreal , Canada
Novel Aspect: Identification and quantitation of several HIV antiretroviral drugs in body fluids
Introduction: The human immunodeficiency virus (HIV) is responsible for causing acquired immuno-deficiency syndrome (AIDS), a devastating and potentially fatal disease. Infection can be treated as a chronic illness with antiretroviral drugs (ARVs). Therapeutic drug monitoring is the standard procedure to optimize the effectiveness of antiretroviral drugs used to treat infected patients with. Several ARVs are commonly prescribed simultaneously in order to control the virus and their concentration levels in the blood stream is critical Ideally, the analytical method should be able to monitor any combination of commonly used ARV drugs simultaneously in body fluids. The method should possess excellent specificities and precision with short analysis times. An LC tandem MS method was developed and implemented to monitor several ARVs.
Methods: Calibration curves (0.1 - 10.0 ug/mL) were prepared in heparinized plasma. Samples were heated for 30 min. at 56°C to deactivate the virus. One hundred µL of plasma were precipitated with 400 µL of acetonitrile containing 1 µg of cimetidine (IS). A similar procedure was used for the preparation of quality control samples at 0.4, 4.0, 8.0 µg/mL. QC samples were prepared with a separate weighing of reference standards. Tubes were capped, vortexed 30 s and centrifuged at 14,000 g for 10 min. Supernatants were injected onto a C18 column in Agilent 1100LC connected to an ABI/Sciex QSTAR MS.. Data acquisition time was 2.0 min with a 2.5 min injection cycle time.
Results: Eighteen approved ARVs were monitored in several patients. Results from these analyses were crucial in allowing physicians to adjust dose regimens appropriately. Sample preparation was relatively straightforward and analysis time required was 3 min for simultaneous detection and quantitation of all analytes. Calibration standards, QC samples and study samples were injected in a randomized fashion to ensure that the analytical system was performing properly Calibration curves were analysed by linear regression with a 1/concentration weighting. A correlation coefficient (R2) of > 0.69 was established for quantitation of each analyte. Within run and between run precision (CV) was approximately 4 %. The method was linear for each analyte over an excellent dynamic range (four orders of magnitude. Precision (CV) for the various analytes with this method varied from 2.0 to .6 % for within run data while CVs for between run data varied from 2.1 to 5.8 %. The robustness of the system was tested with several analyses were performed in triplicate. As opposed to a previous method developed in this laboratory, this study utilized a modern instrument with better sensitivity and response time. The number of drugs quantitated was increased from 12 to 18 (50%) and the analysis tine was simultaneously decreased from 5 to 2.5 min.
