Quantitative LC Analysis of Cyclosporine A in Ocular Samples

Chromatographia (2011) 73:817–821 DOI 10.1007/s10337-011-1963-0 LIMITED SHORT COMMUNICATION Quantitative LC Analysis of Cyclosporine A in Ocular Samples Bizhan Malaekeh-Nikouei • Toka Banaee • Javad Aghamohammadian • Navid Mosallaei Seyed Ahmad Mohajeri • Received: 14 November 2010 / Revised: 17 January 2011 / Accepted: 4 February 2011 / Published online: 25 February 2011 Ó Springer-Verlag 2011 Abstract An isocratic reversed-phase HPLC method with ultraviolet detection at 205 nm has been developed for analysis of cyclosporine A (CyA) in rabbit ocular samples. Neither internal standard nor extraction was needed for sample preparation. Acetonitrile (ACN; 1 mL) was added to 250 lL aqueous and vitreous samples to precipitate proteins. The supernatant was dried and the residue was reconstituted in mobile phase and injected for HPLC analysis. Chromatography was performed on an octadecyl silane-A (ODSA) C18 (4.6 9 250 mm, 5 lm) column. The column temperature was fixed at 70 °C and the mobile phase was ACN 65%, methanol 20% and water 15% at a flow rate of 1.5 mL min-1. The calibration curve for CyA in rabbit ocular samples was linear over the concentration range 0.2 and 10 lg mL-1 with a correlation coefficient of 0.9992. Intra-day and inter-day precision were 4.61–7.83% and 5.27–10.70%, respectively. Intra-day and inter-day accuracy were 89.2–108% and 83.4–111%, respectively. The limits of detection (LOD) and quantification (LOQ) were 5.7 and 38 ng mL-1, respectively. The method was successfully used for analysis of CyA in real aqueous and vitreous humor samples from New Zealand albino rabbits. B. Malaekeh-Nikouei  N. Mosallaei Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran T. Banaee Khatam-al-Anbia Eye Hospital and Eye Research Center, Mashhad University of Medical Sciences, Mashhad, Iran J. Aghamohammadian  S. A. Mohajeri (&) Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran e-mail: [email protected] The method is therefore suitable for analysis of CyA in ocular samples. Keywords Column liquid chromatography  Ocular samples  Cyclosporine A Introduction Cyclosporine A (CyA) is a vital immunosuppressive drug widely used to prevent rejection of transplanted organs and for the treatment of autoimmune diseases [1–3]. It has also been used for new applications, for example reversal of multi drug resistance, herpes virus infection, rheumatoid arthritis, type I diabetes, and as a potent anti-human immunodeficiency virus 1 (HIV-1) agent [4–6]. The drug has some serious side effects, including chronic nephrotoxicity, increased blood pressure, dyslipidemia, and renal hypoperfusion [7, 8]. The therapeutic window of CyA is narrow and the consequences of therapeutic and toxic levels are graft rejection on the one hand and nephrotoxicity on the other [9, 10]. Because of poor correlation between dose administered and clinical response and large inter-subject variability in the bioavailability and metabolism of this drug, therapeutic monitoring of CyA is of critical importance [9, 11]. Systemically administered CyA penetrates the eye only if an inflammation such as severe uveitis occurs [12]. Because of the adverse effects and low ocular delivery of the drug after systemic administration, many efforts have been made to prepare an ophthalmic CyA delivery system which improves the efficacy of drug in different ocular diseases [13–15]. These studies include the use of drug-delivery systems such as nanoparticles [13] and nanocapsules [16]. Therefore, analysis and quantification of CyA in ophthalmic fluids is very important for 123 818 evaluation of ocular bioavailability from drug-delivery systems. Many papers have been published on the analysis of CyA in biological fluids [17–20]. Analytical methods such as HPLC [17, 21, 22], liquid chromatography–mass spectrometry [8], capillary electrophoresis [18], and immunoassay [19, 23] have been reported for this purpose. Sample preparation and extraction is usually needed in analytical methods [17, 21]. The objective of this study was to develop a simple and reliable liquid chromatographic method for quantitative analysis of CyA in aqueous and vitreous humor samples with high linearity. The method was validated by determination of the concentration of the drug in rabbit ocular samples. Experimental Materials CyA was purchased from LC Laboratories (Woburn, MA, USA). Acetonitrile (ACN) and methanol were of HPLCgrade and obtained from Duksan (Ansan, South Korea). Preparation of Stock and Standard Solutions Stock solution (1 mg mL-1) of CyA was prepared by dissolving 10 mg CyA in 10 mL methanol. Standard solutions (0.2, 0.5, 1, 2.5, 5 and 10 lg mL-1) were prepared from the stock solution by dilution with mobile phase and a calibration curve was plotted over this concentration range. Calibration Standard solutions (2–100 lg mL-1) in water, used for spiking calibration samples, were also prepared from stock solution. For calibration, 25 lL standard solutions were added to 225 lL vitreous humor to obtain 0.2–10 lg mL-1 calibration standards. The ocular samples were frozen and stored at -20 °C. Quantification of the CyA was performed by plotting nominal CyA concentrations against the corresponding area under UV absorption peak for spiked samples. Sample Preparation ACN (1 mL ) was added to 250 lL ocular sample in a 1.5 mL microtube to precipitate proteins. After centrifugation at 11,337g, 0.5 mL supernatant was dried under a stream of nitrogen and reconstituted in 100 lL mobile phase, and 20 lL of this solution was injected for HPLC analysis. 123 B. Malaekeh-Nikouei et al. Precision and Accuracy Assay precision was determined by analysis of ocular samples spiked with known concentrations of CyA. Drug concentrations were determined in batches of four on the same day and on different days. Intra and inter-day assay precision was determined by calculating relative standard deviation (RSD, %) of the results obtained. Relative accuracy was determined by use of the equation: Accuracy ð%Þ ¼ Observedconcentration  100 Nominal concentration Chromatographic Conditions High-performance liquid chromatography of CyA was performed with a Young Lin (Anyang, South Korea) Acme 9000 system, consisting of an SP930D solvent-delivery module, an SDV50A solvent-mixing vacuum degasser, a CTS30 column oven, a UV730 dual-wavelength UV–visible detector, and an ODSA C18 (4.6 9 250 mm, 5 lm) column. Data were analyzed by use of Autochro-3000 software supplied by Young Lin. The injection volume was 20 lL, the flow rate was 1.5 mL min-1, and the column temperature was fixed at 70 °C. The UV detector was set to 205 nm. An isocratic method was used for chromatographic analysis of CyA. The composition of the mobile phase was: ACN 65%, methanol 20%, and water 15%. Application of Analytical Method for Ocular Samples To test the applicability of the assay method for analysis of CyA in ocular samples, New Zealand albino rabbits were chosen. Nanoliposomes containing CyA were prepared according to our previous study [24]. The nanoliposomal formulation applied topically to the rabbit ocular surface every day. After 1 month, rabbits were killed by intravenous pentobarbital overdose. Both eyes were immediately enucleated, the corneas were cut off, the lenses were removed, the posterior capsule of the lens and anterior hyaloid face was severed, and the whole of the vitreous was aspirated. The vitreous samples were kept at -80 °C for quantitative analysis. Before analysis, the eye samples were defrosted overnight at 4 °C. The animal protocol was approved by the local ethics committee. Results and Discussion Several reports have been published on HPLC analysis of CyA in blood or other biological fluids [8, 9, 12, 14, 21]. Most of these have focused on HPLC analysis of CyA in Analysis of Cyclosporine in Ocular Samples blood samples rather than ocular fluids. In previous works, extraction was usually performed and an internal standard was necessary to increase the precision and accuracy of the method [9, 25]. Many of the methods used in these studies were expensive and complicated. Sometimes the lack of availability of an internal standard was a problem. In this study we developed an HPLC method for analysis of CyA in ocular samples. We found that HPLC analysis could be performed with good accuracy and precision without adding an internal standard or use of an extraction procedure. Aqueous and vitreous humor are not as complicated as blood. For example, the protein content of aqueous humor is both quantitatively and qualitatively different from that of plasma. The protein content of aqueous humor and vitreous humor is a factor of 200 and 10, respectively, less than that of plasma [26, 27]. This means that ocular humors are dilute aqueous solutions, which could be a reason for not adding an internal standard or using an extraction procedure. Thus, simple protein precipitation was sufficient before HPLC. Sometimes low sample volume is a problem. Most reported methods for analysis of CyA required 1–2 mL sample [21]. In this study the volume of some ocular samples was no more than 250 lL. Thus, we calibrated the method for a sample volume as low as 250 lL. ACN (1 mL) was used for protein precipitation. After centrifugation, 500 lL supernatant was dried and re-dissolved in 100 lL mobile phase before injection for HPLC analysis. This concentrating step was performed to improve the quantification and detection limits of the method. As described in other studies, relatively high temperature (70 °C) affected the results [9]. When the temperature is reduced the CyA peaks become wider and the resolution of the peaks decreases. Thus, we fixed the column temperature at 70 °C. Under these conditions no interfering peaks were observed in the chromatogram (Fig. 1). Method Validation 819 Fig. 1 Chromatograms obtained from a blank aqueous humor sample (a) and from an aqueous humor sample spiked with a standard solution of CyA (b) concentration that produces a signal-to-noise ratio of 3, was 5.7 ng mL-1 and the limit of quantification (LOQ), defined as the concentration that produces a signal-to noise ratio of 20, was 38 ng mL-1. In a study by Khoschsorur et al. [21], the LOD and LOQ for analysis of CyA in whole blood were 15 and 31 ng mL-1, respectively. In another study, the LOQ obtained for determination of CyA in rat plasma was 50 ng mL-1 [8]. Recovery of CyA in this calibrated analytical method was between 77 and 101%. Linearity Precision and Accuracy A calibration curve for standard solutions (y = 40.705x - 0.5018, R2 = 0.999) was plotted over the range 0.2–10 lg mL-1. A calibration curve for assay of CyA in rabbit ocular samples (y = 35.243x ? 2.6793, R2 = 0.9992) was plotted over the same range. This range of concentrations was within the acceptable limit for analysis of real ocular samples. Linearity was tested using aqueous and vitreous humor spiked with standard solutions containing known concentrations of the drug. The correlation coefficient obtained for the calibration curve demonstrated the linearity of the results from in this analysis. The limit of detection (LOD), defined as the Table 1 shows the precision and accuracy of the assay for ocular samples. Intra-day and inter-day precision were determined as the relative standard deviation (RSD%) of the results obtained. Intra-day precision for CyA concentrations of 0.2, 0.5, and 1 lg mL-1 ranged from 4.61 to 7.83%, and inter-day precision for these concentrations was between 5.27 and 10.70%. In other studies, intra-day and inter-day precision ranged between 0.75 and 21.87% [9, 17, 21]. Accuracy, calculated to reflect the difference between nominal and observed concentrations, was in the range 83.4–111%, which is acceptable. 123 820 B. Malaekeh-Nikouei et al. Table 1 Intra-day and inter-day precision and accuracy of determination of CyA in rabbit ocular samples Nominal concentration (lg mL-1) Intra-day Inter-day Observed mean concentration (lg mL-1) SD RSD (%) 0.2 0.194 0.01 5.47 0.5 0.54 0.024 4.61 1 0.892 0.069 7.83 Stability of the Analyte CyA was stable in solutions at room temperature for at least 12 h; it also remained intact at -20 °C for up to 4 weeks. With regard to the run-time stability of processed samples, no significant loss of CyA was observed at room temperature. Thus, no degradation was observed during the day of an experiment. This information suggests repeated analysis of samples within 12 h can be performed with confidence. Ruggedness The ruggedness of the method was evaluated as the repeatability of results obtained by analysis of the same sample under a variety conditions, for example different experimenters or instruments. No significant difference was observed between results, thus proving the method was rugged. Robustness of the Method To study the robustness of the method, deliberate slight variations were made in method conditions, for example changes of flow rate, ACN content of the mobile phase, and column temperature. These slight variations had no significant effect on the area and shape of the peak. The results indicated that the resolution of the peak is more sensitive to changes in column temperature than to changes in the other conditions. Accuracy (%) 97.19 108 89.25 Observed mean concentration (lg mL-1) SD RSD (%) Accuracy (%) 0.222 0.02 9.03 111.09 0.517 0.027 5.27 103.53 0.834 0.089 10.7 83.4 Application of the Analytical Method to Ocular Samples The optimized and calibrated method was used for analysis of CyA in aqueous and vitreous humor samples from New Zealand albino rabbits. After topical administration of a nanoliposomal formulation of CyA to the rabbit ocular surface for 1 month the concentrations of the drug in ocular samples were determined by HPLC. Fourteen aqueous and 14 vitreous samples were tested in this study. The concentration of CyA in aqueous humor was significantly higher than that in vitreous humor (P = 0.01 \ 0.05). The average CyA concentration in aqueous humor was 0.412 ± 0.157 lg mL-1 whereas that in vitreous humor was 0.291 ± 0.093 lg mL-1. Conclusion In this study a simple, fast, and reliable HPLC method was developed for analysis CyA in ocular samples. CyA was not extracted from ocular samples and no internal standard was needed. Because of the low volume of some ocular samples, the assay was calibrated for a volume as low as 250 lL. The intra-day and inter-day precision and accuracy results were within acceptable limits. Concentrations of CyA as low as 0.2 lg mL-1 in ocular samples were measured. The calibrated HPLC method was used for analysis of drug concentration in real rabbit ocular samples. Specificity To verify the specificity of the method, blank ocular samples were analyzed to evaluate the absence of endogenous compounds at the retention time of CyA. Ocular samples spiked with betamethasone, dexamethasone, prednisolone acetate, and dorzolamide, which could be administered topically, simultaneously, with CyA were also analyzed. The results indicated that no interference was observed at retention time of CyA. 123 References 1. 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