Elsevier

Journal of Chromatography B

Volume 879, Issue 25, 1 September 2011, Pages 2554-2560
Journal of Chromatography B

Development of a novel LC–MS/MS method for the determination of letosteine in human plasma and its application on pharmacokinetic studies

https://doi.org/10.1016/j.jchromb.2011.07.011Get rights and content

Abstract

Letosteine has been found to be effective in treating patients with chronic bronchopneumopathies in clinical practice. To provide robust support for its pharmacokinetic and clinical studies, a rapid and sensitive method based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) was developed and validated for the analysis of letosteine in plasma samples. After protein precipitation, the plasma samples were separated on a reversed-phase C18 column in less than 1.5 min. The LC–MS/MS system was performed in the positive ion multiple-reaction-monitoring (MRM) mode to produce intensive product ions of m/z 280.1  160.0 for letosteine and m/z 248.1  121.1 for the internal standard, tinidazole. The method was found to have excellent linearity (r  0.9974), precision (RSD  5.83%), extraction recovery (71.8–73.0%) and stability (RE, −8.45% to 9.03%) over a concentration range of 0.1140–152.0 μg L−1. Compared to the previous published radioactive method, LC–MS/MS method showed many advantages including shorter analysis time, simpler preparation procedure, increased sensitivity as well as lower safety risks. In addition, this method was successfully applied to study the pharmacokinetics of letosteine following a single and multiple dose oral administration in Chinese healthy volunteers.

Introduction

Letosteine, a cyclic cysteine derivative as mucolytic agent, has proved to be highly effective in the treatment of upper respiratory tract infections and chronic bronchopneumopathies, such as chronic bronchitis, pulmonary emphysema and asthma [1], [2], [3], [4], [5], [6]. Letosteine may also have a therapeutic effect in preventing oxidative tissue damage induced by the respiratory burst; this can be attributed to its antioxidant activity by scavenging the reactive oxygen species [7]. What's more, letosteine was reported to be effective in children suffering from acute bronchitis [8].

However, no analytical methods using LC–MS/MS for determination of letosteine in biological samples have been published up to now, except for traditional radioactive approaches using letosteine labeled either with 14C or 35S [9], [10]. Unfortunately, the methods of radioactivity measurements have many inherent drawbacks, including expensive radioactive sources and measuring devices [11], complicated radioactive labeling techniques [12], long exposure time for autoradiography, especially the ethical and safety issues associated with the use and disposal of radioactive materials [13], which highly limit their applications in handling of large numbers of samples from pharmacokinetic researches or clinical use.

To provide adequate support for the pre-clinical and clinical studies of letosteine, a sensitive and reliable bioanalytical method is highly required for the determination of letosteine in human plasma. So, in this research work, an LC–MS/MS method with high speed and satisfactory sensitivity was developed. A lower limit of quantification (LLOQ) of 0.1140 μg L−1 was attained for letosteine in human plasma with retention time of 1.1 min. What's more, the present LC–MS/MS method was successfully applied to study the pharmacokinetics of letosteine in Chinese healthy volunteers after single (25, 50, and 100 mg) and multiple (50 mg, three times daily) dose administration.

Section snippets

Reagents and materials

The letosteine tablets containing 25 mg of letosteine per tablet (batch No. 0812002) and the reference standard of letosteine (batch No. 0905001, 99%, Fig. 1a) were both supplied by Xi’ an BoHua Pharmaceutical Co. Ltd. (Shanxi, China). Tinidazole (99.5% purity, Fig. 1b), used as an internal standard (IS), was supplied by Hubei Institute for Food and Dug Control (Hubei, China). Acetonitrile and methanol for HPLC use were from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid and ammonium

Method development

Tandem MS spectrometric parameters were first optimized in order to obtain the most specific and sensitive detection. To achieve the optimal sensitivity, direct infusion of standard solution was carried out to optimize the electrospray ion source parameters. The full scan mass spectra were recorded, and the protonated ion was selected as the precursor ion since it provided high sensitivity and a better, more stable fragmentation compared to the deprotonated ion. The precursor ions of letosteine

Conclusions

In this paper, a rapid and sensitive LC–MS/MS method for the determination of letosteine in human plasma is described for the first time. The method consists of simple sample preparation process by protein precipitation, chromatographic separation and tandem MS detection. No interfering peaks were observed at the elution times of letosteine and IS. Adequate precision, accuracy, stability and suitability of the proposed method were demonstrated over the concentration range of 0.1140–152.0 μg L−1 (r

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