Simultaneous determination of Aspirin and Rosuvastatin Calcium in capsules by using RP-HPLC coupled with photo diode array detection

A simple, sensitive, specific, and cost effective method for simultaneous determination of Aspirin and Rosuvastatin calcium was developed and validated in single dosage formulation. The sample solution of ASP and RSTC was prepared using methanol as a solvent. Separation of ASP and RSTC was achieved with a mobile phase consisting of 20 mM KH 2 PO 4 : Methanol (30:70 v/v) at a flow rate of 1.0 ml/min. Separations were performed on Merck hibar 250-4.6 RP18 (5 µm) column (150 mm X 3.0 mm), using a Shimadzu Prominence HPLC system equipped with a Shimadzu SPD-20A detector, Rhenodyne 7725i injector with 20 μL loop, LC-20 AD pump, CBM-20 Alite controller and LC Solution software. Retention times of ASP and RSTC were 3.747 and 5.969 minutes respectively. Absolute recovery of ASP and RSTC was 100.3 and 100.03 % respectively. The lower limit of quantification (LLOQ) of ASP and RSTC was 0.3097 and 0.1063 ppm and lower limit of detection (LLOD) of ASP and RSTC was 0.01535 and 0.01358 ppm respectively. Linearity was established for the range of concentrations 15.00-90.0 μg/ml and 2.0-12.0 μg/ml for ASP and RSTC respectively with the coefficient of determination ( R 2 ) of 0.994 and 0.999 for both the compounds. The inter- and intra-day precision in the measurement of ASP quality control (QC) sample 75 μg/ml, were in the range 0.1-0.2 % relative standard deviation (R.S.D.) and 0.2-0.3 % R.S.D., respectively. The inter- and intra-day precision in the measurement of RST quality control (QC) sample 10 μg/ml, were in the range 0.1-0.2 % R.S.D., and 0.0-0.3 % R.S.D., respectively. The developed method would be applicable for routine quality control of ASP And RSTC in bulk as well as in pharmaceutical formulations.

However the comprehensive literature study revealed that none of the pharmacopoeias or any journals includes these drugs in combination for the simultaneous quantification of ASP and RSTC. With this regards, the present investigation was carried out to develop a reverse phase high performance liquid chromatography (RP-HPLC) procedure which will serve a reliable, accurate, sensitive and fast method for the simultaneous determination of ASP and RSTC.

1. Chemicals and Reagents
The reference standards of ASP and RSTC were gifted from Cadila Pharmaceuticals (Ahmedabad, India). Capsule (Unistar (10+75), Unichem Laboratories Ltd. India) containing ASP and RSTC of 75 mg and 10 mg respectively were purchased from local market. Water, methanol and ortho-phosphoric acid of HPLC grade were procured from Merck, India and Rankem, Ltd. India. The other chemicals used in experiments were of HPLC grade and purchased from local market.

Instrumentation and Chromatographic Conditions
Analytical RP-HPLC separations were performed on Merck hibar 250-4.6 RP18 (5 µm) column (150 mm X 3.0 mm), using a Shimadzu Prominence HPLC system equipped with a Shimadzu SPD-20A detector, Rhenodyne 7725i injector with 20 μL loop, LC-20 AD pump, CBM-20 Alite controller and LC Solution software. Eluents A (20 mM KH 2 PO 4 ) and B (Methanol) in the ratio of 30:70 were used as the mobile phases and flow rate was set at 1 mL/min.

3. Preparation of stock and standard solutions
Primary stock solutions of ASP and RSTC for preparation of standard samples were prepared from separate weighing. The primary stock solutions of the analyte were prepared in methanol (1.0 mg/ml) and stored at -20 °C, which were found to be stable for one month. Appropriate dilutions were made in methanol for ASP and RSTC to produce working stock solutions (WSS) of 15, 30, 45, 60, 75, 90 μg/ml and 2, 4, 6, 8, 10, 12 μ/ml, respectively, on the day of analysis and these stocks were used to prepare calibration curve (CC).

4. Sample preparation
Twenty capsules were weighed each containing 75 mg of Aspirin entric coated tablet and 10 mg of Rosuvastatin calcium granules, gelatin shell were removed and drug content was powered using mortar and pestle. An amount of pharmaceutical products powder

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ILCPA Volume 33 equivalent to 75 mg of ASP and 10 mg of RSTC were accurately weighed and transferred into a 100 ml volumetric flask and dilute with 50 ml of methanol. Solution was subjected to sonication for 15 minutes for complete extraction of drug and the solution was mark up with the methanol to get final concentration of 750 μg/ml of ASP and 100 μg/ml of RSTC respectively. Sample stock solution (SSS) was filtered through 0.45 µm PVDF filter paper (0.45 µm) before further dilution.

5. Validation
Validation studies were performed using the optimized assay conditions following the principles of validation described in the ICH guideline [25]. Key analytical parameters, including, specificity, accuracy, precision, linearity, limit of detection (LOD) and limit of quantification (LOQ) were evaluated.

5. 1. Selectivity/selectivity
The specificity/selectivity of the assay method was investigated by verifying the absolute separation and resolution of all the desired peaks of the analytes in mobile phase, and in mixture of excipients and standard. The interference of excipients with drug was measured by recording the retention time and % recovery.

5. Linearity and Range
For linearity study, six solutions at different concentrations (15,30,45, 60, 75, 90 µg/ml of Aspirin and 2, 4, 6, 8, 10, 12 µg/ml of Rosuvastatin calcium) were prepared using six different aliquots of WSS, and the obtained data were used for the linearity calibration plot. Lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for the assay were also calculated using equation 1 and 2 and also determined experimentally by visual evaluation (Table 6).

5. 3. Precision
As per the Complementary Guideline on Methodology (dated 6 November 1996 incorporated in November 2005) of ICH. Method precision was performed both in expressions of repeatability (injection and analysis) and intermediate precision (intra-day and inter-days reproducibility).

5. 4. Repeatability
To determine the repeatability of assay method sample with 75 µg/ml of ASP and 10 µg/ml of RSTC were injected 6 times into HPLC system and repeatability of the retention time and peak area was determined and expressed as mean and % RSD calculated from the data obtained.

5. 5. Intermediate precision
Intermediate precision (intra-day and inter-days reproducibility), were performed at three different concentration levels (45, 60, 75, µg/ml of Aspirin and 6, 8, 10, µg/ml of International Letters of Chemistry, Physics and Astronomy Vol. 33 Rosuvastatin calcium) were analyzed three times a day in triplicate injections over three consecutive days and expressed as mean ±SD and % RSD.

5. 6. Accuracy
The study were performed in triplicates using a mixture of pure drug spiked with its formulation i. e. ASP (750 µg/ml) & RSTC (100 µg/ml) solution with three different concentrations of standards at 80 %, 100 % and 120 % (60, 75 and 90 µg/ml for ASP and 8, 10 and 12 µg/ml for RSTC respectively). Accuracy was determined in terms of percent recovery.

5 .7. Robustness
The robustness of the developed assay method was studied by evaluating the manipulating small deliberate variations in procedure variables like column temperature (±1 °C), flow rate (±5 %) and pH of the mobile phase (±0.2 units).

1. Sample preparation
Numerous organic solvents were tried to prepare the stock solution of ASP and RSTC. Both ASP and RSTC were having the great solubility in methanol, thus selected as a solvent for this experiment. The corresponding working solutions of ASP and RSTC were prepared by diluting their stock solutions with methanol.

2. Method development and optimization
Feasibility of different solvent system such as water-methanol, buffer-methanol, wateracetonitrile mixture in different strength, pH (2)(3)(4)(5)(6)(7)(8), and flow rate (0.8-1.2 ml/min) were experimented. Desired separations were achieved using 20 mM phosphate buffer -methanol in the ratio of 30:70 v/v (pH adjusted to 3 with ortho phosphoric acid) at a flow rate of 1 ml/min. While optimization of the ratio of different solvents in mobile phase, pH was fixed to 3.0 with ortho phosphoric acid, at a flow rate of 1 ml/min, the mobile phase composition found better resolution and separation in buffer (20 mM KH 2 PO 4 ) -methanol (30:70 v/v). The pKa values of both RSTC and ASP were of 4.6 and 3.49 respectively. Resolution and retention of drug component depends upon the pH of the mobile phase, pH range from 2 to 8 were studied to optimize the mobile phase. Buffer : methanol ratio (30:70 v/v) and flow rate (1 ml/min.) were kept constant, pH 3 were found suitable for the current experimentation.

Validation of the analytical method
The linear response of ASP and RSTC was determined by analyzing six independent levels of the calibration curve in the range of 15-90 µg/ml for ASP and 2-12 µg/ml for RSTC. The linearity equations and standard errors for the calibration curves of both ASP and RSTC are presented in Table 1. Average percent recoveries for ASP and RSTC were of 100.3 % and 100.03 %, respectively, while % RSD values for both ASP and RSTC were 0.607 and 0.485, which was less than 1 % and indicates accuracy of the reported method. Method specificity was verified using standard solutions of each drug alone, with excipients, and solvents shows that the resulting peaks on chromatograms at retention times of 3.747 and 5.969 min coincided with ASP and RSTC respectively with no interference by other substances.

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ILCPA Volume 33 The average plate numbers over the concentration range were 2269.153 and 2071.9 for ASP and RSTC respectively. The chromatograms of blank and the mobile phase do not show any interference at the retention time of Aspirin and Rosuvastatin calcium as it can be seen from the respective chromatograms (Figure 2, 3).
System precision experiment was performed by preparing the standard solution of ASP (75 µg/ml) and RSTC (10 µg/ml) for six times and analyzed as per the ICH guideline (Table  2). Method precision experiment was performed by preparing the test solution of ASP (75 µg/ml) and RSTC (10 µg/ml) for six times from different capsule units and analyzed ( Table  2). As per the ICH guideline, it expresses within laboratory variations as on different days analysis or equipment within the laboratory. The Intra-day precision was determined for standard solution of ASP (75 µg/ml) and RSTC (10 µg/ml) for different hours in same day, (like 0, 2, 4, 6, 8 and 10 Hours) and results were found 0.1 % and 0.2 % respectively, which were within the limit prescribed by ICH ( Table 3). The Inter-day precision was determined for standard solution of ASP (75 µg/ml) and RSTC (10 µg/ml) for different days, (like 1, 2, 3, 4, 5 and 6 Days) ( Table 3). The accuracy of the method was determined by recovery studies and the percentage recovery was calculated, overall mean percentage recovery was found 99.966 % and 99.347 % for ASP and RSTC respectively (Table 4). Minor deliberate changes in different experimental parameters such as flow rate (±5 %), pH (±0.2 units) and mobile phase ratio did not significantly affect area under the curve and retention time of both ASP and RSTC indicating that the proposed method is robust ( Table 5). The LOD for ASP and RSTC standard solution were found to be 0.0445 µg/ml and 0.0046 µg/ml respectively, while LOQ were found to be 0.134697 µg/ml and 0.01386 µg/ml respectively (Table 6).