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Application of a novel ultra-low elution volume 96-well solid-phase extraction method to the LC/MS/MS determination of simvastatin and simvastatin acid in human plasma

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Abstract

A novel extraction method has been utilized in the LC/MS/MS determination of simvastatin and simvastatin acid in human plasma. In this method, 300 μl of plasma sample was loaded onto a Waters Oasis 96-well HLB μElution plate, the stationary phase was washed using 2 × 400 μl of 5% methanol in water, and the analytes were eluted using 35 μl of 95/5 acetonitrile/H2O twice. The sample extracts were diluted with 40 μl of methyl ammonium acetate (1 mM, pH 4.5). Chromatography was performed on a Phenomenex Synergi Max-RP column (2.0 mm × 50 mm, 4 μm). A PE Sciex API 3000 tandem mass spectrometer interfaced with a turbo ionspray source was used for mass detection. Compared to solid-phase extraction, liquid–liquid extraction and solid-supported liquid–liquid extraction methods that were developed and previously used in our laboratory, this method reduced the labor cost and was less time consuming in sample preparation, due to the fact that post-extraction solvent evaporation and reconstitution steps were avoided using this μElution solid-phase extraction plate. The method has been proved to be fast, reliable and reproducible.

Introduction

In recent years, LC/MS/MS has been widely used for the quantitative determination of drugs and their metabolites in biological matrices. With the large amount of samples generated from clinical studies, sample preparation has become a major bottleneck during high-throughput analysis. The recent introduction of the Waters Oasis μElution solid-phase extraction (SPE) plate provides a step forward for sample preparation technology. This technology allows for the rapid isolation of analytes from complex matrices using an ultra-low elution volume thus eliminating the need for post-extraction solvent evaporation and reconstitution steps which can be costly in terms of time.

Simvastatin (Fig. 1) is a highly effective agent for the treatment of hypercholesterolemia [1]. Following oral administration, simvastatin, an inactive lactone, is hydrolyzed in vivo rapidly to its corresponding β-hydroxy acid, simvastatin acid (SVA) (Fig. 1). The latter is a potent inhibitor of HMG-CoA reductase. LC/MS/MS methods for the quantitative determination of SV and SVA in human plasma have been previously reported using various extraction procedures [2], [3], [4], [5], including solid-phase extraction [2], liquid–liquid extraction [3] and solid-supported liquid–liquid extraction [4] used in our laboratory and an on-line extraction [5] by Jemel et al. The on-line extraction provided a simple and labor-saving sample clean-up, however, its relatively high lower limit of quantitation (LLOQ) of 0.5 ng ml−1 and higher interconversion between SV and SVA (≤1.0%) do not meet our requirement. Among the off-line extraction methods, the solid-supported liquid–liquid extraction on Chem Elut cartridges has been extensively used: it was sensitive (LLOQ was 0.05 ng ml−1), reproducible, and showed excellent extraction efficiency with no or negligible interconversion between SV and SVA. However, the automated version of the solid-supported liquid–liquid extraction [6], [7] was not very straightforward. The solvent evaporation step was very tedious due to the fact that a large volume of elution solvent (methyl t-butyl ether, MTBE) is required to achieve optimum recovery while the capacity (loading volume) of 96-well plate is limited. This resulted in the necessity of multiple elution and evaporation steps.

In this work, we report the development and validation of a new method using the Oasis HLB μElution SPE for the quantitative LC/MS/MS determination of SV and SVA in human plasma, which requires no solvent evaporation and reconstitution and greatly reduces sample preparation time and improves assay efficiency.

Section snippets

Materials, chemicals and reagents

Standard compounds of SV and SVA were synthesized by Merck Research Laboratories. Stable isotope labeled SV and SVA, 13CD3-SV and 13CD3-SVA, were synthesized by Drug Metabolism of Merck Research Laboratories and were used as the internal standards for SV and SVA, respectively. The isotopic purities for both internal standards were >98.5%.

The μElution plates packed with Oasis HLB SPE sorbent were obtained from Waters (Milford, MA, USA). Ammonium acetate (HPLC grade), methylamine (40% solution in

Optimization of sample preparation conditions

Sample transfer, internal standard and buffer additions were performed on the Packard Multiprobe II liquid handling system and the Tomtec Qudra-96 station. Sample mixture was then loaded onto the Waters Oasis HLB μElution plate for extraction. The novel 96-well HLB μElution SPE plate consists of a 2 mg high-capacity SPE sorbent and a focusing tip which allows loading of up to 750 μl of plasma and the use of ultra-low elution volume [8], [11]. The sorbent incorporates both a lipophilic

Conclusion

A novel μElution SPE technique has been successfully applied to LC/MS/MS determination of SV and SVA in human plasma. In this method, SV and SVA were successfully extracted on an Oasis μElution SPE plate. The analytes and internal standards were separated on a Phenomenex Synergi Max-RP column and detected on a PE Sciex API 3000 tandem mass spectrometer. The method had an LLOQ of 0.05 ng ml−1 with a linear calibration range of 0.05–50 ng ml−1 using 0.3 ml of sample. The extraction efficiency was 66%

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