Simultaneous quantification of carteolol and dorzolamide in rabbit aqueous humor and ciliary body by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

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Abstract

A rapid, sensitive and selective method for the simultaneous quantification of carteolol and dorzolamide in rabbit aqueous humor (AH) and ciliary body (CB) has been developed and validated using reversed phase-high performance liquid chromatography (RP-HPLC) with isocratic elution coupled with atmospheric pressure chemical ionization mass spectrometry/mass spectrometry (APCI-MS/MS). The analytes and nadolol (used as internal standard, IS) were purified from AH by protein precipitation. The sample preparation from CB was based on a two steps extraction procedure at different pH, utilizing a liquid–liquid extraction with a mixture of ethyl acetate, toluene and isopropanol 50:40:10 (v/v) at pH 8, followed by a second extraction with ethyl acetate at pH 11. The combined organic extracts were then back extracted into 0.1% aqueous trifluoroacetic acid (TFA). The accuracy and precision values, calculated from three different sets of quality control samples analyzed in sestuplicate on three different days, were within the generally accepted criteria for analytical methods (<15%). The assay proved to be accurate and precise when applied to the in vivo study of carteolol and dorzolamide in rabbit AH and CB after single administration of an eye drops containing both drugs.

Introduction

β-Blockers or β-adrenoceptor antagonists are a class of therapeutically important drugs widely used for the management of angina pectoris, cardiac arrhythmias, hypertension and myocardial infarction. They are also used in the treatment of glaucoma and ocular hypertension [1], [2] due to their property of lowering the intraocular pressure (IOP) by decreasing the production of aqueous humor (AH) at the ciliary process [3], [4]. Carteolol (Fig. 1a) is a β-blocking agent with an intrinsic sympathomimetic action. All β-blockers, such as carteolol, with intrinsic sympathomimetic activity, are drugs that exert a partial agonism at the adrenergic receptor while simultaneously block natural endogenous agonist from binding to the receptor. This activity has proved effective in a real reduction of some side effects [5]. Oral carbonic anhydrase inhibitors (CAIs) have been used to lower IOP for the past 40 years. However, the use of these agents have been limited due to associated side effects that include general malaise, fatigue, depression, loss of appetite, gastrointestinal disturbances, weight loss, paresthesias and renal calculi [6], [7]. Dorzolamide (Fig. 1b) is a potent and selective inhibitor of human carbonic anhydrase II (CAII), an enzyme that is present in the ciliary epithelium where it plays an important role in the formation of AH [8], [9], [10]. The inhibition of CAII by dorzolamide, that presumably takes place by slowing the formation of bicarbonate ions with subsequent reduction in sodium and fluid transport, decreases AH production and lowers IOP in patients with glaucoma or ocular hypertension and who are insufficiently responsive to β-blockers.

Modern fixed combinations pair β-blockers with either prostaglandin analogs [11] or carbonic anhydrase inhibitors [12]. Potential benefits of these combinations include fewer drops per day, fewer bottles of medication (and potentially fewer copayments), reduction in exposure to preservatives, and elimination of the washout effect. For these reasons, a fixed combination for ophthalmic use containing carteolol and dorzolamide at various concentrations has been formulated. Consequently, it is high desirable to have an analytical assay for the simultaneous extraction and determination of carteolol and dorzolamide in the ocular matrices, so as to reduce the time of analysis, with a considerable economic saving, especially when the number of samples to be analyzed is very large.

In the last years, mass spectrometry (ESI or APCI) coupled with RP-HPLC has become an alternative and powerful technique for quantitative biological analyses. Chromatographic procedures have been reported for the quantification of several β-blockers [13] and dorzolamide [14] in human plasma based on HPLC/APCI-MS. In this paper we describe a rapid, sensitive and selective RP-HPLC/APCI-MS/MS method for the simultaneous determination of carteolol and dorzolamide in rabbit AH and ciliary body (CB) and its application to an in vivo study.

Section snippets

Chemicals and reagents

Carteolol hydrochloride (purity > 99%) was purchased from Otsuka Pharmaceutical (Tokyo, Japan), dorzolamide hydrochloride (purity > 98%) from Neuland Laboratories Limited (Andhra Pradesh, India) and nadolol (purity 99.8%) from Sigma (Milan, Italy). Formic acid (FA), acetic acid (AA) and trifluoroacetic acid (TFA) were obtained from Fluka (Milan, Italy). Sodium hydroxide, mono- and dibasic sodium phosphate, ethyl acetate, toluene, isopropanol and methanol (HPLC grade) were purchased from Merck

Extraction procedures

Sample preparation is a basilar step for determination of analytes in biological samples. Protein precipitation using methanol was found appropriate for the quantification of carteolol and dorzolamide in rabbit AH. In fact, this ocular fluid contains smaller amounts of proteins compared to other samples. Therefore, satisfying results were obtained adopting a simpler preparation procedure. On the contrary, the quantitative extraction of the two analytes together with the IS from CB required the

Conclusions

A sensitive, selective and rapid method was developed for the simultaneous quantification of carteolol and dorzolamide in rabbit AH and CB by HPLC–MS/MS. Because no gradient elution and no chromatographic separation are necessary, the need of “fine tuning” of the HPLC is eliminated. After the extraction procedures, the recovery of two analytes is greater than 98% and 93% in AH and CB, respectively. The results obtained for selectivity, accuracy and precision, short-term and long-term stability

Acknowledgements

This work was supported by a grant from MIUR. The authors wish to thank Dr. Eileen Collazo for revising the English language used in the paper.

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