Determination of Rizatriptan in Human Plasma by Liquid Chromatography Stable Isotope Dilution Electrospray MS–MS for Application in Bioequivalence Study

Abstract

A simple, sensitive, selective, rapid, rugged, reproducible and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used for quantitative estimation of rizatriptan (RZ) in human plasma using rizatriptan-d 6 (RZD6) as internal standard (IS). Chromatographic separation was performed on Ascentis Express RP Amide C18, 50 9 4.6 mm, 2.7 lm column with isocratic mobile phase composed of 10 mM ammonium formate:acetonitrile (20:80 v/v) at flow rate of 0.5 mL min -1 . RZ and RZD6 were detected with proton adducts at m/z (amu) 270.2 ? 201.2 and 276.1 ? 207.1, respectively, in multiple reaction monitoring (MRM) positive mode. Liquid-liquid extraction was used and validated over a linear concentration range of 0.1-100.0 ng mL -1 with correlation coefficient r 2 C 0.9981. The limit of quantification (LOQ) and limit of detection (LOD) were found to be 0.1 ng mL -1 and 12.5 fg, respectively. Intra-and inter-day precision were within 1.7-3.1% and 2.8-3.7%, and accuracy within 96.0-101.7% and 99.7-101.4% for RZ. Drug was found to be stable throughout three freeze-thaw cycles. The method was successfully employed for analysis of plasma samples following oral administration of RZ (10 mg) in 25 healthy Indian male human volunteers under fasting conditions.

Application of a Reliable LC-MS/MS Method for Determination of Rizatriptan in Healthy Subject Samples: Demonstration of Assay Reproducibility by Incurred Sample Reanalysis

“…Several methods are reported for determination of RIZ in different biological matrices, either alone [9][10][11][12][13][14] or in combination with other triptans [15,16]. RIZ has been determined in dog plasma [9], urine [10], human serum [16], and human plasma [10][11][12][13][14][15] mainly by LC-MS except [11]. Two highly sensitive methods (0.05 ng/mL) have been described by Chen et al [12] and Guo et al [13], employing 100 µL and 500 µL human plasma, respectively, for processing.…”

“…The linear calibration curves were established from 1 to 100 ng/mL and the mechanism of formation of collision-induced dissociation products for all the triptans was proposed. Recently, Mogili et al [14] have developed a sensitive (0.1 ng/mL) and rapid (3.0 min) isotope dilution LC-MS/MS method for quantitative analysis of RIZ in human plasma.…”

“…There was no effect of IS on analyte recovery, sensitivity, or ion suppression. For sample clean-up previous reports have used liquidliquid extraction (LLE) under alkaline conditions with high recovery [11][12][13][14]. Since RIZ and IS are weakly basic compounds, the presence of relatively stronger base during LLE improves the extraction recovery [12].…”

“…Since RIZ and IS are weakly basic compounds, the presence of relatively stronger base during LLE improves the extraction recovery [12]. Use of different basic compounds like sodium hydroxide and sodium carbonate for extraction of RIZ in solvents like methyl tert-butyl ether [11,14] and ethyl acetate [12,13] has been reported. In the present work, diethyl ether was used as the extraction solvents in presence of 4.5 M ammonium hydroxide to obtain high consistent and quantitative recovery (>78%) at all QC levels using 300 µL plasma volume for processing.…”

“…Based on the response it was possible to lower the LLOQ by fourfold; however, it was not required based on subject sample analysis results. Mogili et al [14] have achieved an LLOQ of 0.1 ng/mL with S/N above 12, while the LOD was 0.0125 ng/mL at S/N > 3.…”