Determination of tamsulosin in human aqueous humor and serum by liquid chromatography–electrospray ionization tandem mass spectrometry

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Abstract

A simple, sensitive and selective LC–MS/MS method was developed for the determination of tamsulosin in human aqueous humor and serum to study the recently reported eye-related adverse effects of this α1-blocker drug. Aqueous humor samples were analyzed by direct injection, after addition of the internal standard, labetalol. Liquid–liquid extraction with ethyl acetate was used for serum sample preparation. The chromatographic separation was performed on a reversed phase column by gradient elution with acetonitrile −0.1% formic acid at a flow-rate of 0.2 ml/min. Detection and quantification of the analytes were carried out with a linear ion trap mass spectrometer, using positive electrospray ionization (ESI) and multiple reaction monitoring (MRM). The limit of quantification was 0.1 ng/ml for both aqueous humor and serum samples and linearity was obtained over the concentration ranges of 0.1–4.7 ng/ml and 0.1–19.3 ng/ml for aqueous humor and serum samples, respectively. Acceptable accuracy and precision were obtained for concentrations within the standard curve ranges. The method has been used for the determination of tamsulosin in aqueous humor and serum samples from patients that were on tamsulosin medication and underwent cataract surgery.

Introduction

Tamsulosin (−)-(R)-5-[2-[[2-(O-ethoxyphenoxy)ethyl]amino]propyl]-2-methoxybenzensulfon-amide (Fig. 1) is an α1A1D subtype selective α1-adrenoceptor antagonist (α1-blocker) that is the most frequently prescribed medication for the treatment of lower urinary tract symptoms suggestive of benign prostatic hyperplasia (BPH) [1], [2]. The α1-adrenoceptors (α1A, α1B, α1D) take part in the modulation of blood pressure and thus the α1-blockers were originally developed as antihypertensive agents. Non-selective α1-receptor blockage has also additional effects, including relaxation of the smooth muscles of bladder neck and prostatic urethra, which are found to relieve the symptoms associated with BPH. Consequently, the first drugs for the treatment of the BPH were non-subtype-selective α1-blockers. Unfortunately these drugs were commonly associated with cardiovascular adverse effects. These side effects were later avoided by selectively blocking the α1-adrenoceptor subtypes α1A and α1D with uroselective α1-blockers like tamsulosin, resulting in the relaxation of the smooth muscles in lower urinary tract with minimal blood pressure-related adverse effects [3].

However, despite the uroselectivity of tamsulosin, recent findings suggest that there is a strong association between systemic use of tamsulosin and iris hypotony [4] or intraoperative floppy iris syndrome (IFIS) [5]. This phenomenon is described to be an inadequate pupil dilation and a sluggish iris during eye operation that compromise cataract surgery and increase the risk for complications. Although the pharmacologic mechanism is not known, it is hypothesized that in addition to blocking the α1A-receptors in the prostate, tamsulosin selectively blocks the iris dilator muscle in which the same receptor subtype dominates [4], [5]. After the finding of this new syndrome, it has been suggested that temporarily withholding tamsulosin perioperatively might be a solution to avoid the IFIS [6], [7]. However, this has not always been effective and it seems that receptor binding of tamsulosin might continue for some time beyond the disappearance of a plasma level of tamsulosin [8], [9].

In order to study this phenomenon and the proposed ophthalmic penetration of tamsulosin, a method was needed to estimate tamsulosin levels in the aqueous humor of patients that were on tamsulosin medication and underwent cataract surgery. Also the serum concentration of tamsulosin at the time of the eye operation was needed to compare the state of medication to the aqueous humor concentrations and to the clinical observations. A single instrumental method that would enable the analysis of both aqueous humor and serum samples in one analytical sequence was preferred over two separate methods. However, to the author's knowledge, no methods have been reported for the measurement of tamsulosin in aqueous humor or serum. Moreover, bioanalytical methods available for the quantification of tamsulosin in body fluids are few, being mostly developed for plasma concentration measurements relating to pharmacokinetic studies [10], [11], [12], [13], [14], [15]. Some papers also describe methods for plasma dialysate, urine or tissue analysis [9], [16]. These methods were considered unsuitable for the analysis of small volumes of aqueous humor samples and also, with few exceptions, the internal standards used in these methods are not commercially available.

In this study, a simple and sensitive method for the analysis of tamsulosin in aqueous humor and serum samples was developed. The method enables both types of samples to be analyzed within the same analytical sequence using identical instrument conditions and an easily available internal standard. The method is based on a HPLC with gradient elution and MS/MS detection by multiple reaction monitoring with highly selective and sensitive linear ion trap mass spectrometer. The method was validated in terms of selectivity, linearity, accuracy, precision and stability for both serum and aqueous humor sample analysis and successfully applied to study the IFIS and the effect of withholding tamsulosin before cataract surgery of patients on tamsulosin medication. A detailed report of these studies is under preparation.

Section snippets

Chemicals

Tamsulosin hydrochloride was obtained from Fermion Oy (Espoo, Finland). Labetalol hydrochloride (internal standard, IS) was purchased from Sigma (St. Louis, MO, USA), HPLC-grade acetonitrile was from Rathburn (Walkerburn, UK). Formic acid and sodium bicarbonate were from Riedel-de Haen (Seelze, Germany) and ethyl acetate from LabScan (Dublin, Ireland). Water was purified using a Milli-Q Gradient system (Millipore, Milford, MA, USA). All the reagents used were of analytical grade.

Chromatographic equipment and conditions

The HPLC system

Chromatography

Concentration of tamsulosin in the aqueous humor samples was presumed to be minimal because of the very high plasma protein binding of tamsulosin [9]. In addition, the small volume of aqueous humor recovered during the eye operation made sample concentration by means of evaporation unfeasible. For these reasons, the focus of the chromatographic method development was to find suitable instrument conditions to allow direct injection of large volumes of aqueous humor without causing excessive peak

Conclusions

A simple and sensitive method using liquid chromatography with electrospray ionization ion trap mass spectrometry (LC–ESI-MS/MS) was developed for the determination of tamsulosin in human serum and aqueous humor. The method enables the analysis of both serum and aqueous humor samples within the same analytical sequence using easily available internal standard, offers high selectivity and sensitivity and enables direct injection of aqueous humor samples. The method is considered adequate for its

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