CLEANING VALIDATION OF A SIMPLE AND RAPID REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE SIMULTANEOUS ESTIMATION OF ASPIRIN AND ROSUVASTATIN

Objective: This study describes a new, simple, precise, accurate, and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) cleaning validation method for simultaneous estimation of rosuvastatin and aspirin. Methods: The proposed RP-HPLC method was carried out on AGILENT-ZORBAX RP-Inertsil column (250 mm × 4.6 mm, 5 μm) in an isocratic mode utilizing potassium dihydrogen phosphate buffer (pH 2.5 with OPA):acetonitrile (50:50,v/v) as mobile phase, at a flow rate of 1.5 ml/min. Detection was carried out at 243 nm using UV detector. Results: The method was found specific as there was no swab interference. The Beer–Lambert’s law was obeyed in the concentration range of 0.5–20 μg/ml for both rosuvastatin and aspirin. The mean percentage recoveries at 100% level were 89.4% for rosuvastatin and 82.1% for aspirin. The limit of detection and limit of quantification for rosuvastatin and aspirin were 0.03 μg/ml and 0.1 μg/ml, respectively. The method was found to be robust and precise with percentage RSD <2.0%. Conclusion: A simple, novel, and economical RP-HPLC method for cleaning validation has been developed for the simultaneous estimation of rosuvastatin and aspirin. The method was validated as per ICH guidelines for specificity, linearity, accuracy, precision, and robustness. The developed method can be used as a sensitive analytical tool for ensuring the effectiveness of the cleaning procedure adopted.


INTRODUCTION
Rosuvastatin (Fig. 1) is a hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor. It acts in the liver. Chemically, rosuvastatin is (3R,5S,6E)-7-[4-(4-fluorophenyl)-2-(N-methylmethanesulfonamido)-6-(propan-2-yl)pyrimidin-5-yl]-3,5-dihydroxyhept-6-enoic acid. It is a statin with antilipidemic and potential antineoplastic activities. It selectively and competitively binds to and inhibits hepatic hydroxymethyl-glutaryl coenzyme A (HMG-CoA) reductase, the enzyme which catalyzes the conversion of HMG-CoA to mevalonate, which is a precursor of cholesterol. This leads to a decrease in hepatic cholesterol levels and increase in uptake of LDL cholesterol [1,2]. Aspirin (Fig. 2) is an anticoagulant agent. Chemically, it is 2-acetobenzoic acid. It blocks the production of prostaglandins by inhibiting cyclooxygenase (prostaglandin H synthase), with greater selectivity toward the COX-1 isoform. The antithrombotic effect is due to the inhibition of COX-1 in platelets that block thromboxane production and platelet aggregation. It is chemopreventive against colorectal and other solid tumors [1,2]. Equipment contamination may come from any of the materials that have been in contact with the equipment surfaces. It is critical to avoid carryover of the trace amounts of either active or other materials from one batch to another to avoid cross-contamination of the subsequent product. Hence, equipment used in pharmaceutical manufacturing must be cleaned meticulously and the cleaning procedure used must be validated [3,4].
Cleaning validation is required in the pharmaceutical field to avoid potential, clinically significant synergistic interactions between pharmacologically active chemicals. The objective of the determination of the residue of drugs during cleaning validation is to verify the effectiveness of the cleaning procedure for the removal of product residues, degradation products, preservatives, excipients, and/or cleaning agents as well as the control of potential microbial contaminants. In addition, it is needed to ensure that there is no risk associated with cross-contamination of active ingredients [20,21].
Hence, it was thought worthwhile to develop a cleaning validation HPLC method for the simultaneous determination of rosuvastatin calcium and aspirin and validate it as per ICH guidelines.

METHODS
The active pharmaceutical ingredients rosuvastatin and aspirin were supplied as gift samples by Vergo Pharma Research Laboratories, Verna, Goa. Water used for analysis was Milli Q water. Other chemicals used were of analytical/HPLC grade.
The HPLC system used was Agilent-Zorbax RP with a UV detector. Processing was done using Openlab software. The column used was Inertsil (250 mm × 4.6 mm, 5 µm). The mobile phase consisted of pH 2.5 phosphate buffer:acetonitrile (50:50, v/v). The flow rate was 1.5 ml/min. Injection volume was 25 µl and ultraviolet detection wavelength was set at 243 nm.

Preparation of orthophosphoric acid (OPA)
The OPA was prepared by dissolving 1 g of OPA in 1000 ml of water. It was stirred well by adjusting the pH with KOH solution to get pH 2.5 using digital pH meter.

Preparation of KH 2 PO 4 buffer pH 2.5
Preparation of the buffer was carried out by dissolving 1.36 g of KH 2 PO 4 in 1000 ml of water. It was stirred well and the pH was adjusted to 2.5 using OPA and filtered.

Preparation of mobile phase
The mobile phase was prepared by adding 500 ml of ACN and 500 ml of previously prepared KH 2 PO 4 buffer pH 2.5 in a 1000 ml flask. Further, it was sonicated for 10 min.

Preparation of the standard stock solution of aspirin and rosuvastatin (10 ppm)
About 20 mg of aspirin and rosuvastatin each were weighed accurately and transferred into two separate 200 ml volumetric flask. About 30 ml of diluent was added and sonicated for 5 min and diluted up to the mark with 170 ml diluent and mixed well. Further, 10 ml of each solution was pipetted into two separate 100 ml volumetric flask and 30 ml of diluent was added and sonicated for 5 min and diluted up to the mark with 60 ml diluent and mixed well.

Preparation of mixed standard solution (1 ppm)
About 1 ml from each standard stock solution of rosuvastatin and aspirin were taken and transferred to 10 ml volumetric flask and the volume was made up with diluent.

Method validation
The method validation was performed according to ICH guidelines [22,23].

System suitability
To evaluate system suitability, the mixed drug standard solution was injected 6 times in the HPLC system. The system suitability was then established by calculating the percentage RSD, resolution, tailing factor, and the number of theoretical plates.

Specificity
Swabs used to evaluate the specificity (swab interference) for the proposed method were pretreated by transferring swab sticks in a clean test tube containing 10 ml of diluent and sonicated for 5 min and then drained. The whole procedure was repeated in water and then back in diluent. Two pretreated swabs were then taken and put in two separate test tubes containing 10 ml of diluent and sonicated for about 5 min with intermittent shaking. For the study of specificity, the two prepared swab solutions were injected into the HPLC system.

Linearity
To evaluate the linearity of the proposed method, a 10 ppm solution of rosuvastatin and aspirin was prepared by taking 10 ml of aspirin and 10 ml of rosuvastatin (100 ppm stock solution) and diluting to 100 ml using diluent. The concentration range selected was 0.1-20 ppm. The solutions for linearity study were injected in the HPLC system and the linearity range was determined by plotting calibration curves for both the drugs. The linear regression equation and correlation coefficient (r 2 ) were calculated.

Accuracy
To evaluate the accuracy of the method, recovery study was performed at 100% level concentration of the drugs. A 10 ppm solution of the drugs was prepared by taking 10 ml each of 100 ppm stock solution of rosuvastatin and aspirin and diluting to 100 ml using diluent. For recovery at 100% level, 1 ml of rosuvastatin and aspirin 10 ppm solution was dispersed over 4" × 4" stainless steel plate and dried using an electric fan. After drying, the plate was swabbed and the swabbed samples were collected in a test tube containing 10 ml of diluent, mixed, and sonicated for about 5 min with intermittent shaking to result in approximate 1 ppm solution. The procedure was performed in triplicate to result in three samples which were then injected as per test method and mean percentage recovery for both the drugs was calculated.

Precision
The evaluation of precision was done by injecting six replicates of rosuvastatin and aspirin mixed standard solution at limit of quantification (LOQ) level. The peak areas were recorded and the relative standard deviation for rosuvastatin and aspirin was calculated.

Limit of detection (LOD) and LOQ
The evaluation of the LOD and LOQ of rosuvastatin and aspirin was done based on "signal-to-noise ratio" method. The concentration which gave a signal-to-noise ratio of about 3 for LOD and about 10 for LOQ was derived. The LOD and LOQ tests were carried out using 0.1 ppm solution.

Robustness
The robustness for the developed method was demonstrated by bringing about small deliberate changes in organic phase composition, flow rate, pH of buffer in mobile phase, and column temperature and calculating its impact on system suitability parameters.

DISCUSSION
Preliminary experiments were carried out to achieve the best chromatographic conditions for the simultaneous determination of both the drugs. With the optimized chromatographic conditions, the HPLC instrument was subjected to system suitability. A representative chromatogram is depicted in Fig. 3. The system suitability parameters as summarized in Table 1, complied with the acceptance criteria. Hence, the system was found suitable for the analysis.
A representative chromatogram for swab interference is shown in Fig. 4. The chromatogram showed no interference at the retention time of the drugs. From the data tabulated in Table 2, none of the swabs showed interference. Hence, the method was found to be specific and the swabs were suitable for use.
The calibration curves (Figs. 5 and 6) showed a good correlation between peak areas and concentration of the drugs within the concentration range specified. The correlation coefficient (r 2 ) values for both the drugs were >0.999. The linearity data are summarized in Table 3. The linearity range was thus established as 0.1-20 µg/ml for both the drugs.
The mean % recovery at 100% level as shown in Table 4 was in the range of 70.0-110.0%. Hence, the method was found to be accurate for both the drugs.
The results of precision study as depicted in Table 5 showed % RSD value for peak areas of both drugs <2.0%. Hence, the method was found to be precise at the LOQ level.
The calculated LOD and LOQ values are shown in Table 6, which proved that the method was sensitive for both the drugs.
For robustness study, it was observed that there were no marked changes in any of the tested method parameters, which demonstrated that the developed method was robust. The results of robustness study are summarized in Table 7.

CONCLUSION
A simple, novel, and economical reversed-phase high-performance liquid chromatography method for cleaning validation has been developed for the simultaneous estimation of rosuvastatin and aspirin. The method was validated as per ICH guidelines for specificity, linearity, accuracy, precision, and robustness. The developed method is thus a sensitive analytical tool for ensuring the effectiveness of the cleaning procedure adopted.

ACKNOWLEDGMENT
The authors are grateful to Vergo Pharma Research Laboratories, Verna, Goa, for the gift sample of the active pharmaceutical ingredients.