Development and validation of a fast and sensitive bioanalytical method for the quantitative determination of glucocorticoids—Quantitative measurement of dexamethasone in rabbit ocular matrices by liquid chromatography tandem mass spectrometry

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Abstract

A sensitive, selective, accurate and robust LC–MS/MS method was developed and validated for the quantitative determination of glucocorticoids in rabbit ocular tissues. Samples were processed by a simple liquid–liquid extraction procedure. Chromatographic separation was performed on Phenomenex reversed phase C18 gemini column (50 mm × 4.6 mm i.d.,) with an isocratic mobile phase composed of 30% of acetonitrile in water containing 0.1% of formic acid, at a flow rate 0.2 mL/min. Dexamethasone (DEX), prednisolone (PD) and hydrocortisone (HD) were detected with proton adducts at m/z 393.20  355.30, 361.30  147.20 and 363.20  121.0 in multiple reaction monitoring (MRM) positive mode respectively. Finally, 50 μL of 0.1% novel DEX mixed micellar formulation was topically administered to a rabbit eye and concentrations were measured. The method was validated over a linear concentration range of 2.7–617.6 ng/mL. Lower limit of quantitation (LLOQ) of DEX and PD was measured in the concentration range of 2.7 and 11.0 ng/mL respectively. The resulting method demonstrated intra and inter-day precision within 13.3% and 11.1% and accuracy within 19.3% and 12.5% for DEX and PD, respectively. Both analytes were found to be stable throughout freeze–thaw cycles and during bench top and postoperative stability studies (r2 > 0.999). DEX concentrations in various ocular tissue samples i.e., aqueous humor, cornea, iris ciliary body, sclera and retina choroid were found to be 344.0, 1050.07, 529.6, 103.9 and 48.5 ng/mg protein respectively. Absorption of DEX after topical administration from a novel aqueous mixed micellar formulation achieved therapeutic concentration levels in posterior segment of the rabbit eye.

Introduction

Glucocorticoids (GC) belong to a class of steroid hormones high binding affinities with GC receptor [1]. Such binding trigger analogous effects, which are either mediated slowly via nuclear receptors or rapidly via non-genomically, mediated membrane-associated receptors [2]. GC receptors are found inside the cells of most vertebrate tissues [3]. Cortisol or hydrocortisone (HD) is a naturally occurring GC in humans, which regulates vital metabolic, immunologic, cardiovascular and homeostatic functions. GC exhibit anti-inflammatory properties and inhibit inflammatory response in all stages [4], [5], [19]. GC derivatives are synthetic compounds with pharmacologic properties similar to cortisol and include dexamethasone (DEX), prednisolone (PD), triamcinolone and betamethasone [6], [7]. These compounds are widely recommended in replacement therapy for glucocorticoid deficient patients. Owing to the potential anti-angiogenic, anti-edematous, anti-apoptotic and anti-proliferative effects GC based drugs gained wide use in the treatment of both anterior and posterior ocular segment diseases such as allergic conjunctivitis, herpes zoster keratitis, corneal injury, age-related macular degeneration, proliferative vitreoretinopathy and diabetic macular edema [8].

Drug delivery to retina, choroid and vitreous is virtually important for controlling posterior segment disorders. An ideal drug delivery system should be capable of delivering therapeutic drug concentrations to this target tissues with minimal or no side effect [9]. Currently corticosteroids are administered through local (eye-drop suspensions, ointments, implants and intravitreal injections) and systemic routes (oral and parenteral). Though intravitreal injection directly delivers the drug to vitreous bathing neural retina, it is associated with inherent potential side effects such as retinal detachment, increased intraocular pressure, hemorrhage, endophthalmitis, cataract which limit long term therapy [10]. Topical administration of drugs in the form of eye drops is considered to be the most patient compliant treatment. However, only a small fraction of eye dose reach the posterior segment after topical administration [3].

Quantification of steroids following topical administration in various ocular tissues especially retina requires a rapid, sensitive and robust bioanalytical method. Analysis of GC molecules is somewhat distribute, because of its lipophilic nature. Moreover, these lipophilic compounds are extensively distributed in tissue space resulting in very low biological matrix concentrations. Various analytical methods have been reported in literature to estimate GC concentrations in biological samples. These include high performance liquid chromatography (HPLC) [11], [12] gas chromatography [13], [14], [19], radioimmunoassay (RIA) [15] and capillary separations coupled with various detectors [16], [17]. HPLC technique utilizing ultra-violet (UV) detection suffers from disadvantages such longer retention time, tedious extraction procedure and inadequate limit of quantitation (LOQ) [18]. Though fluorescence and gas chromatography are highly sensitive they require derivatization steps which are time consuming [12]. RIA usually suffers from cross-reactivity and hence resulting in lack of adequate selectivity for GC [15], [20].

Recently, LC–MS/MS has been applied extensively for the quantitative estimation of drugs in various biological matrices such as plasma, serum and urine due to its sensitivity, selectivity and reproducibility. To the best of our knowledge, no validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been reported in literature for the quantitative estimation of corticosteroids in ocular matrices and vitreous humor samples.

In particular, tandem triple-quadrupole mass spectrometry in the multiple reaction monitoring mode provides uncompromised sensitivity and selectivity [21]. The aim of the present study is to develop and validate a selective, rapid, rugged and reproducible LC–MS/MS assay for the quantitative determination of corticosteroids in ocular tissue matrices. This method is successfully validated and applied to the quantitative determination of DEX in rabbit ocular tissues following topical administration in a novel mixed micellar eye drop formulation. DEX, PD and HD structures are shown in Fig. 1.

Section snippets

Materials

DEX, PD and HD were purchased from Sigma Chemical Co (St. Louis, MO). HPLC grade methanol, acetonitrile, diethyl ether, dichloromethane, isopropyl alcohol and analytical grade formic acid, perchloric acid and potassium chloride were procured from Fisher Scientific (New Brunswick, NJ). Ultrapure water from MilliQ-system (Millipore, Molshecin France) was used through the study. All chemicals were of HPLC grade and used as received without further purification.

Preparation of stock and standard solutions

DEX, PD and HD stock solutions were

Liquid-chromatographic operating conditions

The goal of this work was to develop and validate a simple, rapid and sensitive assay method for quantitative determination of glucocorticoids by extraction of ocular tissues. Chromatographic conditions, especially the composition and acidic nature of the mobile phase, were optimized to achieve best resolution. It also increased the signal to mix ratio and to minimized run times. Total run time of the method was 7.0 min at 200 μL/min flow rate and injection volume was 25 μL. Under these conditions

Conclusion

A sensitive and selective LC–MS/MS method for the determination of DEX and PD in rabbit ocular matrix was developed and validated. Total run time for the method was 7 min. Ion suppression was not a significant occurrence for both analytes and internal standard. Protein precipitation followed by liquid–liquid extraction was a useful method development process to avoid matrix ionization effects. Quantitative measurements of samples resulted in consistent values with high accuracy. The peak area

Acknowledgements

This research was supported by the National Institutes of Health grants R01 EY 09171-12 and R01 EY 10659-10.

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