Abstract
A new high-throughput liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay for the quantification of valproic acid in human plasma was developed and validated. The separation was performed on a Zorbax SB-C18 column under isocratic conditions using a 48:52 (v/v) mixture of acetonitrile and 0.1% (v/v) acetic acid in water at 45 °C with a flow rate of 0.8 mL/min. The detection of valproic acid was performed in SIM mode (m/z 143.1). The human plasma samples (0.2 mL) were deproteinized with methanol and aliquots of 2 μL from supernatants obtained after centrifugation were directly injected into the chromatographic system. The method shows a good linearity (r > 0.9972), precision (CV > 7.8 %) and accuracy (bias > 5.7 %) over the range of 5-200 μg/mL plasma. Lower limit of quantification (LLOQ) was 5 μg/mL and the recovery was between 98-106 %. The method is not expensive, it needs a minimum time for plasma sample preparation and has a run-time of 2.4 min for instrument analysis (retention time of valproic acid was 1.8 min). The developed and validated high-throughput method is very simple, rapid and efficient, with wide applications in clinical level monitoring, pharmacokinetics and bioequivalence studies.
Highlights
Valproic acid (VA; 2-n-propylpentanoic acid – Fig. 1) is an anticonvulsant drug, commonly used in the treatment of many forms of epilepsy and many types of seizures, affecting both children and adults
The aim of this work was to develop and validate a new simple and efficiently high throughput LC-MS/MS assay for quantification of VA in human plasma in clinical level monitoring
We propose a very simple and rapid pretreatment of plasma samples including only precipitation of proteins (PP) by methanol and direct injection into chromatographic system from supernatant obtained after centrifugation
Summary
Valproic acid (VA; 2-n-propylpentanoic acid – Fig. 1) is an anticonvulsant drug, commonly used in the treatment of many forms of epilepsy and many types of seizures, affecting both children and adults. Periodic plasma level monitoring is very important both for successful therapy and for evaluating potential drug interactions and adverse effects of VA [1, 2]. Being an acid, it shows peaks with considerable tails on the GC chromatograms. Because VA is strongly bound to proteins, most methods involve primarily precipitation of proteins, followed by extraction of VA in organic solvents and derivatization. VA is isolated by liquid-liquid extraction (LLE) [8, 9, 11] or solid-phase extraction (SPE) [12,13,14] from acidic medium Both extraction and derivatization are time-consuming steps, increase the cost of assay and can affect the recovery. The aim of this work was to develop and validate a new simple and efficiently high throughput LC-MS/MS assay for quantification of VA in human plasma in clinical level monitoring
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