Simple and rapid docetaxel assay in plasma by protein precipitation and high-performance liquid chromatography–tandem mass spectrometry

https://doi.org/10.1016/j.jchromb.2004.01.021Get rights and content

Abstract

A simple, rapid and low cost sample preparation method was developed for quantification of docetaxel in mouse plasma by high-performance liquid chromatography/tandem mass spectrometry with paclitaxel as the internal standard. A small volume of plasma (40 μl) and one-step protein precipitation using methanol and acetonitrile (1:1 (v/v)) were used for sample preparation. The calibration curve for docetaxel in mouse plasma was linear over the range 25–2500 nM. The detection limit was 8 nM. The lower limit of quantitation is 25 nM. The intra- and inter-day precisions (CV) of analysis were 9.5 and 9.7% for the low quality control (LQC), 5.5 and 4.9% for the medium quality control (MQC) and 3.9 and 6.3% for the high quality control (HQC), respectively. The accuracy was 102.5% for LQC, 97.9% for MQC and 108.8% for HQC. This assay has now been applied to evaluation of mouse pharmacogenetics and other clinical pharmacology applications.

Introduction

Docetaxel is a semi-synthetic analogue of paclitaxel, prepared from a non-cytotoxic precursor extract from the needles of the European yew tree (Taxus baccata L.) [1]. It is an inhibitor of microtubule depolymerization [2] and has a broad antitumour activity against various solid tumors including breast, non-small cell lung, head and neck and ovarian carcinomas [3], [4]. Large scale pharmacokinetic analysis has identified a relationship between docetaxel plasma pharmacokinetics and both toxicity and antitumor activity [5]. However, limited volumes of blood samples are often used not only for pharmacokinetic studies, but also for pharmacogenomic investigations. This is especially true in the preclinical setting where only semi-micro (<0.1 ml) sample volumes are available for research in small animal models. Therefore, sensitive and selective assays are required to evaluate the pharmacokinetics of docetaxel in this setting. Current methods for the determination of docetaxel are high-performance liquid chromatography (HPLC) with UV [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], HPLC/MS [16] and HPLC/MS/MS [17], [18], [19]. These assays employ manual solid-phase extraction (SPE), liquid–liquid extraction, or a combination of liquid and solid extraction. The solid-phase extraction often involves multi-step purification and nitrogen evaporation, and 0.5–1 ml plasma is usually needed. The liquid–liquid extraction often needs to use high-purity organic solvents, is hard to handle and nitrogen evaporation is needed. It is often also expensive and time-consuming. So far there are only a few papers published [15], [17] in which a small volume of plasma (at least 50 μl) can be used and both use liquid–liquid extraction for sample preparation.

This paper describes HPLC/MS/MS method for the determination of docetaxel in mouse plasma with a micro-sample volume using one-step protein precipitation. The sample preparation is very simple, fast and low cost. This HPLC/MS/MS method is very specific compared with commonly used HPLC/UV methods and no interfering plasma peaks were found for the docetaxel assay.

Section snippets

Chemical and reagents

Docetaxel (purity 99%) and paclitaxel (purity 99%) were obtained from Hande Tech Development Co. (Houston, TX, USA). Methanol and acetonitrile (HPLC grade) were from Fisher Scientific. (Houston, TX, USA). Whatman nylon membrane filters, 47 mm (Maidstone, UK), 0.2 μm filter was used for filtering the organic solvents. Millipore 0.22 μm GP Express plus membrane (Bedford, MA, USA) was used for filtering water-based solution for HPLC mobile phase. Minimum 95% formic acid was from Sigma (St. Louis, MO,

Selectivity and specificity

Docetaxel and paclitaxel formed predominantly protonated molecules ([M+H]+) in the mobile phase containing formic acid using the electrospray ion source. The most sensitive daughter ions for docetaxel and paclitaxel were found at m/z 527.05 and 285.9, respectively. The MS/MS parameters were optimized to maximize the response for the docetaxel parent/daughter ion combination of m/z 808.2>527.05 and for the paclitaxel of 854.2>285.9 in the positive ion mode.

The formic acid modifier in mobile

Conclusions

In conclusion, a simple and rapid assay for the quantitative determination of docetaxed in mouse plasma has been described. The method is accurate, precise, sensitive and selective. Plasma matrix components do not interfere with the analysis. As sample preparation consists of a simple, rapid one-step protein precipitation, it avoids the use of the complicated and expensive manual solid phase extraction techniques and time-consuming liquid–liquid solvent extraction with evaporation that were

Acknowledgements

This study was supported by the Siteman Cancer Center Pharmacology Core (P30 CA091842).

References (19)

  • J.C. Vergniol et al.

    J. Chromatogr.

    (1992)
  • H. Rosing et al.

    J. Chromatogr. B

    (1997)
  • M.R. Rouini et al.

    J. Pharm. Biomed. Anal.

    (1998)
  • M.B. Garg et al.

    J. Chromatogr. B

    (2000)
  • A. Sparreboom et al.

    J. Chromatogr. B

    (1998)
  • W.J. Loos et al.

    J. Chrmatogr. B

    (1997)
  • N. Martin et al.

    J. Chromatogr. B

    (1998)
  • R.A. Parise et al.

    J. Chromatogr. B

    (2003)
  • S.D. Baker et al.

    Anal. Biochem.

    (2004)
There are more references available in the full text version of this article.

Cited by (0)

View full text