Determination of stavudine in human serum by on‐line solid‐phase extraction coupled to high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry: application to a bioequivalence study

Abstract

A method based on solid-phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the determination of stavudine in human serum, using didanosine as internal standard. The acquisition was performed in multiple reaction monitoring (MRM) mode. The method was linear over the studied range (10-2000 ng/mL), with r(2) > 0.99, and the run time was 4 min. The intra- and inter-assay precisions (%) were in the ranges 0.1-13.6 and 2.6-9.9, respectively, and the intra- and inter-assay accuracies were >92%. The absolute recoveries were approximately 100% (10 ng/mL), 98% (30 ng/mL), 105% (750 ng/mL) and 105% (1500 ng/mL). The limits of detection and quantitation were 4 and 10 ng/mL, respectively. The analytical method was applied to a bioequivalence study, in which 24 healthy adult volunteers (12 men) received single oral doses (40 mg) of reference and two test stavudine formulations, in an open, three-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration), AUC(0-10) and AUC(0-inf) (areas under the serum concentration vs. time curve from time zero to 10 h and to infinity, respectively), were in the range 80-125%, which supports the conclusion that the two test formulations are bioequivalent to the reference formulation with respect to the rate and extent of stavudine absorption.

Quantitative determination of salidroside in rat plasma by on-line solid-phase extraction integrated with high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

“…In order to obtain samples which are clean enough to enter the mass spectrometer, various sample preparation methods have already been applied [12][13][14][15][16]. As compared to liquid-liquid extraction, protein precipitation, and off-line SPE, on-line SPE offers advantage in term of being easier to handle and more time-efficient [17][18][19][20][21][22][23]. To extend the life of SPE extraction column, a simple protein precipitation with acetonitrile was applied prior to injection.…”

Liquid chromatographic–tandem mass spectrometric assay for the simultaneous determination of didanosine and stavudine in human plasma, bronchoalveolar lavage fluid, alveolar cells, peripheral blood mononuclear cells, seminal plasma, cerebrospinal fluid and tonsil tissue

“…Their high polarities prevent them from being extracted by organic solvents. Two sample preparation methods have been reported for ddI and d4T in human plasma [3][4][5][6][7][8][9][10][11][12]. One is ultrafiltration; the other is solid phase extraction (SPE).…”

“…Most of them utilize HPLC with UV detection. Recently, high-pressure liquid chromatographic-tandem mass spectrometric (LC/MS/MS) methods to determine ddI or d4T concentrations in human plasma have been reported [9][10][11][12]. However, no assay methods have been reported to detect ddI and d4T levels in other human matrices.…”

Determination of bromazepam in human plasma by high‐performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study

“…Some workers have reported the use of on-line solidphase extraction (SPE) coupled with LC/MS/MS applied to bioequivalence studies, 13,14 as this procedure combines the universality, sensitivity and specificity of MS/MS with the high recovery and easy automation characteristics of SPE as sample treatment prior to HPLC. Nevertheless, the cost of the SPE instrument and the respective cartridges limits its use.…”