Simple and fast LC-MS/MS method for quantification of terazosin in human plasma and application to bioequivalence study

Highlights

• A simple and sensitive LCMSMS method for quantification of terazosin in human plasma.

• Simple protein precipitation in sample preparation.

• Impact of analytical column on response intensity and peak symmetrical.

• Effect of CPD and EDTA plasma on sensitivity.

• Application of validated method in bioequivalence study.

Abstract

A simple, fast and sensitive LC-MS/MS method was developed to quantify terazosin in human plasma. The mobile phase consisted of acetonitrile-0.1% (v/v) formic acid (70:30, v/v). Prazosin was used as internal standard (IS). As deproteinization agent, acetonitrile produced a clean sample. A higher response intensity with more symmetrical peak was obtained using Agilent Poroshell 120 EC-C18 – Fast LC column (100 × 2.1mmID, 2.7 μm) compared with Kinetex XB-C18 (100 × 2.1 mm, 2.6 µm) column. The response of terazosin and IS were approximately two times in citrate phosphate dextrose (CPD) plasma compared with dipotassium ethylenediaminetetraacetic acid (K₂EDTA) plasma. Plasma calibration curve was linear from 1.0 to 100.0 ng/mL, with coefficient of determination r² ≥ 0.99. The within-run and between-run precision values (CV, %) were <5.2% and <7.8%, while accuracy values were 102.8–112.7% and 103.4–112.2%. The extended run accuracy was 98.6–102.8% and precision (CV, %) 4.3–10.4%. The recovery of analyte was >98% and IS >94%. Terazosin in plasma kept at benchtop was stable for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h, for 7 freeze-thaw cycles and in freezer for 140 days. Terazosin and IS stock standard solutions were stable for 140 days at room temperature and in the chiller. The high throughput method was successfully utilized to measure 935 samples in a bioequivalence study of terazosin.

Introduction

Terazosin is an α1-adrenoceptor antagonist is used to treat the symptoms of hypertension and benign prostatic hyperplasia (BPH) [1], [2], [3], [4]. It is a selective, quinazoline-derived post-synaptic alpha-1 antagonist [5], [6]. Its hypotensive action is due to the relaxation of peripheral blood vessels by the competitive antagonism of post-synaptic alpha-adrenoceptors. [7].

Several terazosin analytical methods in human plasma [8], [9], [10], [11], [12], [13], [14], [15], [16], rabbit plasma [17] and pharmaceutical products [14], [16], [18] have been published. The limitations with the use of high performance liquid chromatography (HPLC) methods were insufficient sensitivity, utilization of large plasma sample volume, huge injection volume or long test run time [11], [12], [13]. The disadvantages of liquid-liquid extraction (LLE) methods [8], [11], [12], [15], [19] used for quantification of terazosin in biological samples were labour-intensive, utilization of large volumes of organic solvents and the recycling of used organic solvents is costly while the limitations with the use of solid phase extraction (SPE) methods [13] were time consuming, cumbersome and the SPE cartridges are expensive. These methods may not be suitable for the analysis of large number of samples. The fluorescence spectrofluorimetry using protein precipitation technique [16], [18], [20], [21] was not chromatography method and not feasible to be used in pharmacokinetic and bioequivalence study. Zavitsanos and Alebic-Kolbah [10] reported LC-MS method with LLOQ of 0.0625 ng/mL but using liquid-liquid extraction and injection volume of 20 μL. Therefore, there is a need to develop a fast, simple, high throughput and specific method for determination of terazosin in human plasma for pharmacokinetic and bioequivalence study using simple protein precipitation method.

In the current study, we aim to develop a fast, simple, sensitive and high throughput ultra-fast HPLC tandem mass spectrometry (UHPLC-MS/MS) method for the quantification of terazosin in human plasma using simple protein precipitation method. The effect of different anticoagulants on sensitivity of terazosin was studied. The method was validated in accordance with international guidelines and utilized in a bioequivalence study of 2 mg terazosin in Malaysian healthy volunteers.