Both Artemisinin And Dexamethasone Biology Essay

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An improved RP- HPLC method with PDA detection has been developed and validated for the simultaneous estimation of artemisinin and dexamethasone in ocular nanodispersion formulations containing artemisinin and dexamethasone, and it has been successfully adopted to check the ex vivo corneal transport. The chromatographic separation was achieved using phenomenex C- 18 (250 X 4.6 mm, 5 µm) analytical column and the mobile phase consisting of acetonitrile and methanol: water (4: 6), (45:55, v/v) at a flow rate of 1.0 mLmin-1 and injection volume of 200 µl. The column temperature was maintained at 50°C. The UV detection was carried out at 219 nm using photodiode array detector. The retention time of dexamethasone and artemisinin were found to be 4.5 and 7.9 min. Artemisinin and dexamethasone calibration curves were linear with correlation coefficient of 0.9617 and 0.9954 at a dynamic concentration ranging from 250 ngmL-1 to 1250 ngmL-1. Recovery was between the ranges of 98.9% - 116.8% for both artemisinin and dexamethasone. The developed method is very sensitive as both peaks were well separated with a short analysis time of 10 minutes and does not require any preliminary treatment of sample.

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Keywords: Artemisinin, Dexamethasone, RP- HPLC, Quantification, Nanodispersions etc.

1. Introduction

Artemisinin is an effective schistocidal anti-malarial drug, isolated from Artemisia annua belongs to the family Compositae (Chun et al., 2002). Structurally it possesses a sesquiterpene lactone containing an endoperoxide bridge (Fig. 1) which is essential for its anti-malarial activity against Plasmodium falciparum (Napawan et al., 2007). The capacity of larger iron uptake of cancerous cells in comparison with normal cells makes artemisinin also as a selective cytotoxic agent (Steve et al., 2009). The anti- angiogenic effect of artemisinin and dihydro artemisinin have been tested in human cervical cancer, uterus chorion cancer, embryo transversal cancer and ovarian cancer cell lines (Huan et al., 2003). The derivatives of artemisinin such as artesunate, artemether, arteether and dihydroartemisinin (Steven et al., 2002) are able to inhibit the growth of fibrosacroma, breast, cervical, leukemia and ovarian cancer cell lines (Yong et al., 2010), and they are eliminated quickly from the system.

Dexamethasone acetate (Fig. 2), (9-fluoro-11 ß-17, 21- trioxy-16 α-methylpregna-1, 4-diene-3, 20- dione 21-acetate monohydrate) is an epimeric synthetic glucocorticoidal drug having high binding affinity with GC receptor (Cato et al., 1996) and possesses anti- inflammatory, anti allergic immunosuppresor (Maria et al., 2009) and antipyretic activities (Li et al., 2010). Dexamethasone used in the treatment of ocular diseases like allergic conjunctivitis, herpes zoster keratitis, corneal injury, age related macular degeneration, proliferative vitreoretinopathy and diabetic macular edema (Ravinder et al., 2010) due to its anti- angiogenic, anti edematous, anti apoptotic and anti proliferative properties. Dexamethasone was reported to have maximum residual limits of 2 µg/kg in liver, 0.75 µg/kg in muscle and kidney and 0.3 µg/kg in milk (Li et al., 2010). Combinations of artemisinin and dexamethasone nanodispersions were developed for the treatment of ocular diseases. Corneal permeation of drugs is essential to provide sufficient drug concentration for therapeutic activity. Therefore, specific and sensitive method is very essential for the identification and quantification of dexamethasone and artemisinin during the ex- vivo corneal transport study and in the anterior segment of eye such as cornea and aqueous humour.

Literature survey revealed that in the past 25 years many analytical techniques had been reported for individual estimation of artemisinin, dexamethasone and in combination with other drugs. Ultra- performance liquid chromatography- tandem mass spectrometry (UPLC- MS/MS) had been developed to detect artemisinin in rat serum (Lie et al., 2008), lengthy derivatization procedures were reported for artemisinin detection using HPLC and UV but these methods lack the sensitivity (Iqbual et al., 2010). Previously, gas chromatography- mass spectrometry (GC- MS) and liquid chromatography- mass spectrometry have been developed and reported for the estimation of dexamethasone and betamethasone combinations in biological matrixes (Li et al., 2010). The different methods for the estimation of dexamethasone in biological samples such as HPLC (Shibata et al., 1998), gas chromatography (Shibasaki et al., 2008) and radioimmunoassay (Tunn et al., 1998) have been described. However, no report has been found for simultaneous determination of artemisinin and dexamethasone in pharmaceutical preparations up to be best of our knowledge. In this manuscript, we describe a reproducible and selective isocratic reverse phase- HPLC method for the simultaneous determination of artemisinin and dexamethasone in nanodispersions and in the samples of ex- vivo corneal transport study.

2. Materials and methods

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2.1. Chemicals

Artemisinin was obtained from Herbochem, Hyderabad, India; Dexamethasone was obtained from Star Drugs and Research lab Ltd, Hosur, India. Methanol (HPLC grade) was purchased from SD Fine Chemicals Ltd, Mumbai. Acetonitrile (HPLC grade) was purchased from Merck, Mumbai. All other chemicals used were of analytical grade.

2.2. Instrumentation

Analysis was performed on a chromatographic system of Shimadzu prominence consisting of LC 20 AD liquid pump equipped with manual 200 µl sample injection loop. Chromatographic separation was achieved on phenomenex C18 (250 - 4.6 mm, 5 µm) analytical column. Data acquisition was made with LC solution v.1.24 Spinchrome-1 soft ware.

2.3 Standard preparation

Standard stock solution of artemisinin (1 mgmL-1) and dexamethasone (1 mgmL-1) were prepared by transferring 10 mg of artemisinin and 10 mg of dexamethasone into a 10 mL volumetric flask containing 4 mL ethanol. It was then sonicated for 15 minutes. The solution was diluted up to volume with ethanol. From these further dilutions were made using pH 7.4 buffer to produce solutions containing artemisinin (10 µgmL-1) and dexamethasone (10 µgmL-1).

2.4 HPLC assay of Artemisnin and Dexamethasone in Nanodispersions

Nanodispersion containing artemisinin and dexamethasone was accurately weighed and dissolved in 3 mL of ethanol sonicated for 15 mins made up to 10 mL using pH 7.4 buffer, filtered through 0.22 µm membrane filter and analyzed by HPLC.

2.5 Method Development (Optimization of the chromatographic conditions)

The goal of this study was to develop a single isocratic reverse phase HPLC method for the simultaneous determination of artemisinin and dexamethasone. During optimizing the method two organic solvents (methanol, acetonitrile) were tested. The chromatographic conditions were also studied by extensive preliminary experiments using different mobile phase combinations and ratios of mobile phase like acetonitrile: 0.2 % formic acid (68: 32 % v/v), acetonitrile: water (70: 30 % v/v) and methanol: water (68: 32 % v/v) but split peaks were obtained for dexamethasone and the response was poor for artemisinin. After a series of screening experiments, it was concluded that the mobile phase composition of acetonitrile and methanol: water (4: 6), (45:55, v/v) gave better peak shape for both drugs. Aqueous methanol generally gives better peak shape than aqueous acetonitrile (Lapkin et al., 2009), but artemisinin is highly soluble in acetonitrile in comparison with methanol, hence we attempted to use a combination of water, methanol and acetonitrile as mobile phase. Increasing column temperature decreased retention times and sharpened the peaks and little improvement in separation of artemisinin has been observed. Hence for the simultaneous quantitative determinations (to achieve good reproducibility and to improve peak sharpening due to signal to noise ratio) the column temperature of 50-C was maintained.

The chromatographic separation was achieved on a Shimadzu Prominence consisting of LC 20 AD liquid pump, Phenomenex C- 18 column (250 X 4.6 mm, 5 µm), using acetonitrile and methanol : water (4: 6), (45:55, v/v) as mobile phase at an injection volume of 200 µl and column temperature of 50°C. The absorption maximum were found to be 219 nm for artemisinin was and 242 nm for dexamethasone. The absorption maximum wavelength of artemisinin (219 nm) was fixed as detection wavelength and at this point the peak response was maxima for both artemisinin and dexamethasone and the peak intensity of dexamethasone is also not changed. The selected chromatographic conditions provided optimum resolution of artemisinin and dexamethasone.

2.6 Method validation

The chromatographic conditions were validated by evaluating linearity, recovery, method and system precision, robustness, intra and inter- day variabilities. Preliminary experiments were performed with the aim to select best and optimum conditions.

2.6.1 Linearity and Recovery

Linearity was evaluated by five- point standard curves for artemisinin and dexamethasone in two levels with concentration range of 5- 25 µgmL-1 and 250- 1250 ngmL-1. In order to ascertain the quality and applicability of method the recovery analysis was performed at three levels like low, middle and high concentrations (80%, 100% and 120%) by standard addition method containing the known amount of each drug. These mixtures were determined by the proposed method in triplicates.

2.6.2. Method and system precision

Precision of an analytical method expresses the closeness of agreement and to check the variability of results between a series of measurements obtained from repeated analysis of same homogenous sample under prescribed identical experimental conditions. Method precision was determined by injecting the standard solution of the analytes five times (Siddiqui et al., 2009, Dhoka et al., 2010). The system precision (injection repeatability) is a measure of the method variability that can be expected for a given analyst performing the analysis for five repeated analysis of the same working solution.

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2.6.3. Intra- inter day and analyst variability

The intra-day (repeatability), inter-day (intermediate precision) variability and analyst to analyst variations were determined using known concentration solutions. These experiments were repeated on second day to evaluate a day- day variability (Siddiqui et al., 2009, Dhoka et al., 2010).

2.6.4. Robustness

The capacity to remain unaffected by small changes has been studied by robustness. The robustness of the method was checked by creating deliberate variations in the chromatographic conditions (Dhoka et al., 2010) like flow rate and wavelength. The altered conditions studied were flow rate of (1.0 ± 0.1 mLminute-1) and wavelength of (219 ± 3).

2.6.5 Ex- vivo corneal transport study

Ex vivo corneal drug permeation was studied using side by side diffusion cell consist of donor and receptor compartments.

The goat eyes were obtained from slaughter house and rinsed with phosphate buffer saline (PBS); the vitreous humor of the enucleated eye was aspirated using 1 ml tuberculin syringe, the sclera was cutted to open the eye, lens and iris- ciliary body was separated from the cornea using foreceps and the cornea was carefully excised leaving some adhered scleral portion, which was utilized to hold the tissue in place between the half cells during experiments.

The excised cornea was rinsed with PBS and placed vertically on the donor half cell with endothelial surface facing the reservoir solution. The donor half cell was placed vertically over the receptor half cell and clamped, temperature was maintained at 34°C throughout the experiment using circulating water bath (Majumdar et al., 2003). The contents in both chambers were stirred continuously using magnetic stir bars. The set up was made such that the volume of the receiver chamber was kept at higher than the donor chamber so that the hydrostatic pressure difference maintains the natural curvature of the cornea. Aliquots of 200 µl of sample were withdrawn from the receiver chamber at selected time points up to 2 hrs through the sample port using micro syringe and placed into eppendorf tubes and analyzed using RP- HPLC (Shaul et al., 1997).

3. Results and Discussion

Previously published works focused on analyzing either artemisinin or dexamethasone individually. Iqbal et al., 2010 reported a HPLC method where artemisinin was derivatized using 0.2 % sodium hydroxide and refluxed at 50°C analyzed using ODS column. But in this method, we reported combination of artemisinin and dexamethasone estimation using a simple technique without derivatization, temperature maintained at 50°C using column oven. Hyung et al., 1995 determined dexamethasone by HPLC on C18 column using methanol- water (50: 50, v/v) as mobile phase.

The major disadvantage of HPLC method for the estimation of artemisinin is very low UV absorbance at the selected wavelength. Scientists attempted for pre/post column derivatization to hydrolyze artemisinin into more UV active compounds to be analyzed by common HPLC/UV instruments. The accurate estimation of artemisinin quantity was not resolved even by changing the detectors of LC systems (Lapkin et al., 2009). Nevertheless, in the case of simultaneous determination of dexamethasone and artemisinin for derivatization of artemisinin to highly UV active compounds, this might alter the absorption character of dexamethasone. Further, the effective derivatization of analytes obtained from corneal permeation studies might be difficult. Hence, in the present study, we developed HPLC method for the simultaneous determination of dexamethasone and artemisinin without derivatizing the artemisinin and the UV detection has been fixed at the absorption wavelength of artemisinin.

The linearity curves obtained for dexamethasone, and artemisinin were linear over a wide range of studied concentrations at two levels (5- 25 µgmL-1 and 250- 1250 ngmL-1). The R2 value of artemisinin and dexamethasone at microgram levels were 0.9929 and 0.9883, at nanogram levels were 0.9617 and 0.9954 respectively. Typically, the regression equation for the calibration curve was found to be y = 3.4479x + 203.83 for artemisinin and y = 260.12x + 81311 for dexamethasone at nanogram levels. The established standard calibration curves reflected good linearity of the drugs at two levels. The tailing factor was found to be less than 1.2 for dexamethasone and less than 1.0 for artemisinin and the resolution between two peaks was found to be 5.1. The validation parameters studied were shown in Table. 1.

The developed simultaneous method for the determination of dexamethasone and artemisinin was validated as per ICH guidelines Q2 (R1). The mean percentage recoveries obtained for artemisinin and dexamethasone were between 98.9% and 116.8%. The % RSD of peak area for recovery was found to be less than 5.0 %. In peak purity, analysis purity angle was less than the purity threshold for both artemisinin and dexamethasone, which indicates that the peak of analytes was pure and excipients in formulation did not interfere with the analytes. The relative standard deviation values (% RSD) were calculated from the ratios of the standard deviation to the mean being expressed in percentage (Hammam et al., 2012). The % RSD of peak area for system and method precisions, inter intra day and analyst - analyst variability was found to be less than 2.0 %. The system precision of the method is also illustrated in Fig.3. The peak area and retention time remains almost unchanged. The system suitability results (Table. 2) depicts there was no co eluted peaks and chromatographic interference peaks. The degree of reproducibility of results (% RSD < 2.0 %) obtained as a result of small deliberate variations in flow rate and detection wavelength has proven that the method is robust (Table.3). The mean drug permeation across the cornea following a single dose of dexamethasone and artemisinin containing nanodispersions is illustrated in Fig. 4, the results represent that 1763 ng of dexamethasone and 5566 ng of artemisinin has been permeated at 120 mins.

4. Conclusion

The developed and validated HPLC method outlined is very obvious, affordable, low cost, rapid and easy to perform with good repeatability. The robustness study data indicates the reliability of the method during normal usage. It can be adopted for the simultaneous determination of artemisinin and dexamethasone in the developed nanodispersions and other formulations and in ocular matrixes after proper separation because of good separation and resolution of the chromatographic peaks.