Elsevier

Journal of Chromatography B

Volumes 881–882, 15 January 2012, Pages 34-41
Journal of Chromatography B

Development and validation of a sensitive solid-phase-extraction (SPE) method using high-performance liquid chromatography/tandem mass spectrometry (LC–MS/MS) for determination of risedronate concentrations in human plasma

https://doi.org/10.1016/j.jchromb.2011.11.031Get rights and content

Abstract

Risedronate is a commonly prescribed bisphosphonate for the treatment of bone disorders. Due to its high polarity and low oral bioavailability, low concentrations of risedronate are expected in human plasma and therefore a sensitive assay is required to serve in pharmacokinetic studies. Here, we describe the development and validation of an LC–MS/MS assay for the measurement of risedronate concentrations in human plasma. Risedronate and the internal standard, risedronate-d4, were derivatized on an anion exchange solid-phase extraction cartridge. Trimethylsilyl-diazomethane which is a thermally stable and relatively non-toxic derivatization agent was used to methylate the risedronate phosphonic acid groups and decrease analyte polarity. Following extraction, the analytes were separated on a Phenomenex Gemini C18 column (150 mm × 2.0 mm, 5 μm), using a gradient of ammonium acetate 10 mM and acetonitrile with a flow rate of 300 μL/min. The assay calibration range was 0.2–25 ng/mL. The calibration curve of risedronate standards spiked in six individual plasma samples was linear (r2 = 0.9998). Accuracy (percent deviation from nominal) and precision (percent coefficient of variation) at concentrations 0.5, 5 and 20 ng/mL, and at the lower limit of quantification (LLOQ) of 0.2 ng/mL were excellent at <6%. Mean recovery was 54% for risedronate and 51% for the internal standard. Risedronate was stable in human plasma samples for at least 5 h at room temperature, 101 days frozen at −80 °C, 72 h in an autosampler at 10 °C, and for three freeze/thaw cycles. The validated assay method successfully quantified the concentrations of risedronate in plasma samples from informed consenting healthy volunteers administered a single 35 mg risedronate tablet.

Introduction

Risedronate (RDR) is a member of the bisphosphonate class of drugs that are widely used for the treatment of bone disorders such as osteoporosis, bone metastasis, and Paget's disease [1]. Osteoporosis affects almost 200 million women worldwide and 75 million people in Europe, USA and Japan. During the year 2000, it was estimated that there were nine million new osteoporotic fractures [2]. Bisphosphonates have been prescribed by clinicians since the 1970s and they are first-line therapy for patients with osteoporosis [3]. Alendronate and RDR are the most popular nitrogen-containing bisphosphonates in this therapeutic class [4].

Bioanalysis of bisphosphonates in complex matrices such as human plasma is challenging due to their high polarity (Fig. 1) (structurally similar to endogenous phosphorylated compounds), metal chelating properties and low bioavailability [5]. Previously reported methods have generally utilized a combination of multiple extraction steps, derivatization to less polar or more volatile compounds, together with ion pair chromatography [6] and capillary electrophoresis [7] using HPLC [8], GC [9], LC–MS, or ELISA [9] to improve the efficacy of the assays [4]. LC–MS/MS is preferable for measuring bisphosphonate concentrations in plasma samples due to higher sensitivity and selectivity, as well as shorter analytical run times. An LLOQ of less than 1 ng/mL is required for the quantification of RDR in plasma and urine samples. Solid phase extraction (SPE) has been used for sample clean-up, ion-pairing and derivatization of various bisphosphonates of interest. Due to their high polarity, liquid–liquid extraction is unsuitable for extraction of bisphosphonates from plasma; however, it may have a role for removal of excess derivatization agents [4].

The concept of derivatization of bisphosphonates whilst retained on an SPE cartridge followed by elution and quantitation of derivatized analytes using LC–MS/MS was first proposed by Zhu et al. [10]. Here, we report the development and validation of an improved LC–MS/MS method using SPE derivatization of RDR with a more efficient and less toxic derivatization agent, trimethylsilyldiazomethane (TMS-diazomethane). This method is both sensitive and robust for the quantification of RDR in human plasma samples.

Section snippets

Chemicals

RDR sodium and RDR-d4 were purchased from Toronto Research Chemicals, (North York, Ontario, Canada). TMS-diazomethane and bovine serum albumin were bought from Sigma–Aldrich (Sydney, NSW, Australia). Sodium bicarbonate, sodium carbonate, ammonium acetate, formic acid were purchased from Lomb Scientific (Sydney, NSW, Australia). Methanol and acetonitrile (HPLC grade) were bought from Lab-Scan (Brisbane, QLD, Australia). Human blank plasma was purchased from BioCore Pty. Ltd. (Sydney, NSW,

Mass spectrometry and liquid chromatography

Using the method described herein, RDR and RDR-d4 were separated chromatographically with a 6.5 min overall run time. Retention times for RDR and RDR-d4 were 3.39–3.77 min and 3.35–3.72 min, respectively with the respective times being precise within-batches. Chromatograms of RDR and internal standard after extraction from a QC (5 ng/mL), plasma blank, at LLOQ, and extracted from a healthy volunteer plasma have been presented in Fig. 3, Fig. 4, Fig. 5. It was confirmed that drug-free plasma from

Discussion

Measurement of low concentrations of bisphosphonates in biological matrices has been a challenge for analytical chemists. Various tedious, time-consuming, and not practical methods have been used by previous investigators including an enzyme-linked immunosorbent assay (ELISA) [11], ion-pair chromatography [5], co-precipitation of RDR with calcium in combination with a clean-up step such as protein precipitation [5], or ion-pair solid phase extraction [12]. Co-precipitation with calcium has also

Conclusions

Compared with previously published methods for the quantification of bisphosphonates in biological samples [4], the present SPE method using, on-cartridge derivatization with a relatively safe agent and LC–MS/MS, is simple, sensitive and robust. These features coupled with a short run time at 5-min, facilitates its application to high throughput measurement of RDR concentrations in human plasma samples collected from large cohorts of patients/volunteers in clinical studies. This derivatization

Acknowledgements

This work was supported financially by an R&D contract with Q-Pharm Pty Ltd.

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