A simple reversed phase high-performance liquid chromatography (RP-HPLC) method for determination of curcumin in aqueous humor of rabbit

Abstract

This article describes a simple and rapid method for determination of curcumin (diferuloylmethane) in aqueous humor of rabbit using high-performance liquid chromatography (HPLC). Analysis was performed using a C-18 column (250 × 4.6 mm, 5 μ luna) by isocratic elution with a mobile phase containing 25 mM potassium dihydrogen orthophosphate (pH 3.5): Acetonitrile (40:60) and detection at 424 nm using a photodiode array (PDA) detector for curcumin. The regression data for curcumin showed a good linear relationship with r²> 0.998 over the concentration range of 0.1-10 μg ml⁻¹. Relative standard deviations (RSD) for the intraday and interday coefficient of variations for the assay were less than 5.0 and 8.5, respectively. The recovery of the method was between 79.8-83.6%. The quantification limit of the method for curcumin was 0.01 μg ml⁻¹. This method has good accuracy, precision, and quantitation limit. It is also concluded that the method is useful for measuring very low curcumin concentrations in aqueous humor.

Turmeric has been used historically as a component of Indian Ayurvedic medicine.[1] It has properties like anti-inflammatory, antimicrobial, antioxidant, antiarthritis, antifungal, and anticancer.[2,3] Many preclinical studies suggest that curcumin may be useful for the prevention and treatment of several diseases.[4] Currently, curcumin is being examined in phase II clinical trials for cancer treatment[5,6] and many pharmacological activities e.g. antioxidant, anti-inflammatory, antimicrobial, and hepatoprotective have been attributed to curcumin,[5] and thus, its quality control is a matter of interest. Turmeric has curcumin: Principle curcuminoid. Curcumin has two form viz., Enol and Keto forms.[7,8] In vitro, animal studies have suggested a wide range of potential therapeutic and preventive effects associated with curcumin.[9] Curcumin having molecular weight of 368.38 is slightly soluble in water and moderately soluble in ethanol (10 mg ml⁻¹) and also stable but may be light sensitive. Its pharmacokinetics in animal system is scarcely available in literature. Analysis of curcumin is essential to study disposition kinetics of the drug in aqueous humor during diseased condition.

However, no methods were reported for determination of curcumin in aqueous humor of rabbits. The purpose of the present study was to evaluate low concentrations curcumin in aqueous humor of New Zealand white rabbit by an isocratic high-performance liquid chromatography (HPLC) method with photo diode array (PDA) detector. The developed method was validated as per the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines.[10]

All the solvents were of analytical grade purchased from Rankem (India) and SRL laboratories (India). Water purified by milli Q Elix (USA) was used. The analytical grade standard curcumin was purchased from Sigma Aldrich (USA).

Ten adult New Zealand white rabbits weighing 1.0-1.5 kg of either sex of about 1 year of age were used for this study. This study was approved by the Institutional Animal Ethics Committee, Faculty of Veterinary and Animal sciences, West Bengal University of Animal and Fishery Sciences, Kolkata, India.

Samples were collected according to established method of previously published article.[11] The animals were anesthetized with 6 mg kg⁻¹ of xylazine hydrochloride (Xylaxine^®; Indian Immunologicals Ltd., Hyderabad, India) and 30 mg kg⁻¹ of ketamine hydrochloride (Ketamine 50^®; Themis, Haridwar, Uttarakhand, India). A retrobulbar injection of 4% lignocaine hydrochloride was made into the extraconal space for local anesthesia and also to prevent eye movements. The pupil was dilated with topical tropicamide eye drops (Tropicacyl^®, 1% w/v; Sunways, Pvt. Ltd., Mumbai, India) and phenyephrine eye drops (Ocurest^®0.12%; Centaur Pharmaceuticals Pvt. Ltd., Mumbai, India). The ocular surface was disinfected with 5% povidone-iodine, and the lids were retracted with a lid speculum.

A fresh aqueous was drawn from anterior chamber of all 10 rabbits with a 26-gauge needle and collected in a test tube. A stock solution containing 100 μg ml⁻¹ of curcumin was prepared in mobile phase. This stock solution was stored in dark brown color container which is light resistant. The aliquot of curcumin 0.1 to 10 μg ml⁻¹ was prepared in mobile phase and analyzed to check the linearity of response. A good linearity was observed for the standard curve [Figure 1].

Figure 1.

Linear standard curve of curcumin in aqueous humor of rabbit

For the assessment of method precision, reproducibility of the results obtained for different concentrations of curcumin in aqueous humor was determined at five different days and five times in one day. The results of reproducibility study are shown in [Table 1] as coefficient of variations. Five different concentrations of curcumin (0.1, 1.0, 2.0, 5.0, and 10.0 μg ml⁻¹) in fresh aqueous humor of rabbits from stock solution (100 μg ml⁻¹) were made and subjected to deproteinization using HPLC grade acetonitrile and kept in deep freeze for 15 minutes, then vortex mixing followed by centrifugation and supernatant was taken and filtered through 0.2 μm membrane filter before injection to HPLC column. The sample and standard chromatograms have been shown in [Figures 2 and 3]. The HPLC system includes a Shimadzu LC-20AT solvent delivery system, a SPD-M10A VP photo diode array detector and a 7725i rheodyne injector with 20-μl loop volume. The class VP 6.01 data system software was utilized for integration. Separation was achieved using a phenomenex C-18 column (250 × 4.6 mm, 5 μ ID, USA). The solvent system consisted of 25 mM potassium dihydrogen orthophosphate (pH adjusted to 3.5 using orthophosphoric acid): Acetonitrile (40:60 V/V) was pumped isocratically at a flow rate of 1 ml minute⁻¹. The detection was carried out using a diode array detector.

Table 1.

Assessment of reproducibility, recovery of the method for quantitative determination of curcumin (n=5 replicate)

Figure 2.

Typical chromatogram of standard curcumin (analytical grade, 10 ppm)

Figure 3.

Typical chromatogram of curcumin after work-up in sample (analytical grade, 10 ppm)

The limit of detection and limit of quantification of the developed method were determined by injecting progressively low concentration of solutions using the Reverse Phase HPLC (RP-HPLC) method. The limit of detection is the smallest concentration of the analyte that gives a measurable response. The limit of detection for this method was 0.01 μg ml⁻¹. The limit of quantification is the smallest concentration of the analyte which gives a response that can be accurately quantified. The limit of quantitation of curcumin in aqueous humor was 0.1 μg ml⁻¹.

Various methods of HPLC for the determination of curcumin in biological fluids have been described so far which differ in chromatography type (reverse phase or ion-pair chromatography) or detection system (UV or fluorescence).[12,13,14,15] RP-HPLC with ultraviolet detection has been recommended.[16] But most of those methods are tedious and expensive because they used more material and have many stages of experiments. In this article, a simple and rapid method for the determination of curcumin in aqueous humor has been described that has equal precision and accuracy to other similar methods.[16] A full chromatography including sample preparation time, it may need 30 minutes for the whole of each analysis, which is comparatively a short time. The short duration of assay time is of quite importance in routine monitoring of the drug in aqueous humor to predict. On the other hand, this method has a satisfactory quantitation limit that makes it ideal for pharmacokinetic studies and therapeutic drug monitoring of curcumin after administration of different doses of this drug in diseased condition of aqueous humor.