A New High-Throughput LC-MS/MS Assay for Therapeutic Level Monitoring of Valproic Acid in Human Plasma

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. Keywords

HPLC-UV and GC-MS Methods for Determination of Chlorambucil and Valproic Acid in Plasma for Further Exploring a New Combined Therapy of Chronic Lymphocytic Leukemia

“…One previously described HPLC method, based on the extraction of VPA with hexane from acidified plasma, evaporation and derivatization with 2-bromo-2´-acetonaphtone, offered the LLOQ of 0.05 µg/mL using 160 µL of plasma [ 14 ]. When LC-MS/MS methods were applied, the LLOQ of 0.2–5 µg/mL using 200 µL of plasma were reported [ 20 , 21 , 23 ]. In one of these methods, MS/MS detection required the extraction of VPA from acidified plasma with methylene chloride, then two step derivatization with 2-chloro-1-methylpyridinium iodide and 4-dimethylaminobenzylamine dihydrochloride, shaking for 1 h and using evaporation [ 23 ].…”

“…In turn, the centrifugal ultrafiltration followed by a direct GC method offered the linearity range from 0.56 µg/mL [ 24 ]. Because the mean concentration of VPA in plasma of patients with epilepsy is usually between 40 and 120 μg/mL [ 14 , 20 , 21 ], these LLOQ are optimal for the determination of VPA in patients with epilepsy after typical dosing. However, effective VPA dosing in different types of cancer is yet to be discovered.…”

“…Finally, we could conclude that the elaborated GC-MS method with simple derivatization ensured the quantitative determination of VPA, with desired sensitivity, precision and accuracy. Consequently, it could be a good alternative to previously described LC-MS methods [ 20 , 21 , 23 , 24 ] when used for monitoring VPA levels in biological samples.…”

“…It is a well known anticonvulsant drug which nowadays is widely examined for new therapeutic indications [ 13 ]. Analytical methods, such as HPLC [ 14 , 15 , 16 , 17 , 18 ], LC-MS/MS [ 19 , 20 , 21 , 22 , 23 ], gas chromatography (GC) [ 24 , 25 , 26 ] and gas chromatography–mass spectrometry (GC-MS) [ 27 ] were reported for the determination of VPA in biological matrices. Most of the previously reported HPLC and GC methods required complex sample pretreatment or detection, e.g., multistep derivatization for UV detection [ 14 , 17 ], multistep derivatization for fluorimetric detection [ 15 , 16 ], or complex extraction [ 18 , 24 , 25 , 26 , 27 ].…”

Plantaginaceae Juss.

“…Other methodology is available employing varying versions of LC -MS technology (Beyer et al 2007;Vlase et al 2009). In addition, a fl uorescence polarization immunoassay ( FPIA) method has been adapted to allow the determination of cardenolide levels in animal tissues and plants as well as in the plasma (Schultz et al 2005).…”