Detection and Quantification of Some Antiepileptic Drugs in Human Plasma using Hydrophilic Interaction Liquid Chromatography- Triple Quadrupole Mass Spectrometry

MastersA fast, sensitive and selective methods for the detection and quantification of Carbamazepine (CBZ) , Vigabatrin (VGB), Phenytoin (PHT) and Lamotrigine (LTG) in plasma are described using high-performance liquid chromatographic separation with Triple Quadrupole Mass Spectrometry (QQQ). Samples were purified using liquid–liquid extraction and separated on a Phenomenex Luna HILIC 3 μ (2× 150 mm) column using 5 μL as volume of injection. The first method determined the CBZ and VGB in plasma using a mobile phase consisting (75:25, v/v) acetonitril/ ammonium formate (5 mM, pH 3.5) at a flow-rate of 0.4 ml/min. Detection was performed by a triple quadrupole model G6410A mass spectrometer in the multiple reaction monitoring mode (MRM) using electro spray ionisation (ESI), monitoring the transition of the protonated molecular ion for CBZ at m/z 237 and VGB at m/z 130 to the predominant ions of m/z 194 and 71, respectively. The mean recovery was 81% for CBZ and 100% for VGB. The calibration curves were linear (r=0.991) over the concentration ranges 250–8000 ng/ml for CBZ and 15-960 ng/ml (r=0.999) for VGB. The limits of detection (LODs) for CBZ and VGB in human plasma were 5.43 ng/ml and 3 ng/mL, respectively. The limits of quantification (LOQs) for both analytes in human plasma were 18.2 ng/ml and 10 ng/ml, respectively. Both intra-and inter-day coefficient of variation (CV % or RSD %) values ranged from 1.79–5.89% at three QC levels (750, 4000 and 6400 ng/ml.) for carbamazepine and 1.145-7.03% at three QC levels (i.e., 45, 480 and 768 ng/ml.) for vigabatrin. The second method determined PHT and LTG in plasma using a mobile phase consisting (97.5:2.5, v/v) acetonitril/ ammonium formate (5 mM, pH 3.5) at a flow-rate of 0.2 ml/min. Detection was performed by a triple quadrupole model G6410A mass spectrometer in the MRM mode for PHT and single ion monitoring mode (SIM) for LTG , monitoring the transition of the deprotonated molecular ion for PHT at m/z 251 to the predominant ions of m/z 102 and protonated molecular ion for LTG at m/z 256. The mean recovery was 90% for PHT and 80% for LTG. The calibration curves were linear over the concentration range 250–1885 ng/mL for PHT (r=0.997) and LTG (r=0.998). The LODs for PHT and LTG in human plasma were 5.17 ng/ml and 0.13 ng/ml, respectively. The LOQs for both analytes in human plasma were 17.24 ng/ml and 0.46 ng/ml, respectively. Both intra- and inter-day CV values ranged from 1.66–8 % at three QC levels (250, 500 and 1250 ng/ml) for PHT and 0.71-3.7 % at three QC levels (250, 500 and 1250 ng/ml) for LTG