A New Simultaneous Determination of Rosuvastatin Calcium and its Lactone Impurity by Reverse Phase HPLC method

Prabhu Venkatesh Moodbidri*, Varadaraji Dhayanithi, Ganesh Belavadi Manjunathashastry, Hari Narayan Pati , Pardhasaradhi Vasireddy

Advinus Therapeutics Ltd., 21 and 22, Phase II, Peenya Industrial Area, Bengaluru-560058, Karnataka, India.

*Corresponding Author E-mail: vprabhu74@yahoo.com; venkatesh.prabhu@advinus.com

ABSTRACT:

A reliable, sensitive and stability –indicating reverse phase HPLC method was developed for the determination of Rosuvastatin calcium and its lactone impurity in drug substance and pharmaceutical dosage form. Rosuvastatin calcium was fourth highest selling drug in the United States, accounting approximately $5.2 billion in the year of 2013, which is used for the treatment of hypercholesterolemia. The resolution between Rosuvastatin and lactone impurity was good with resolution factors more than 10.0. The chromatographic separation was achieved on a sunfire column C18 (250 x 4.6 mm, 5 µm) with mobile phase containing a gradient mixture of solvent-A (10 mM ammonium acetate) and solvent-B (acetonitrile: methanol (50:50 v/v)). The eluted compounds were monitored at 242 nm and the total run time was 15 minutes. Degradation behavior of the Rosuvastatin calcium was studied under various degradation stress conditions. The Limit of detection and limit of quantitation of lactone impurity was found to be 0.01µg/mL and 0.04µg/mL, respectively, for 10µL injection volume. The sample solution and mobile phase were stable for at least 48 hours. The proposed accurate method can be useful for quantification of Rosuvastatin calcium and its lactone impurity in the bulk drug substance and also dosage form.

KEY WORDS: Rosuvastatin calcium, HPLC, Lactone impurity, Assay, Method validation.

INTRODUCTION:

Rosuvastatin calcium is a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is designated as (3R, 5S)-7-[4-(4-fluorophenyl)-2-(N-methylmethanesulfonamido)-6-(propan-2-yl) pyrimidin-5-yl]-3,5-dihydroxyhept-6-enoic acid (Fig. 1). Rosuvastatin calcium is a synthetic lipid-lowering agent. Nowadays impurities separations are playing more important role for the analysis of active pharmaceutical ingredient in the field of pharmaceutical industries. It is not uncommon for active pharmaceutical ingredient to be active while other small percentage impurity is toxic in biological systems. HMG-CoA reductase inhibitors reduce the production of mevalomic acid from HMG-CoA, resulting in a reduction in hepatic cholesterol synthesis.^1-3

Received on 26.11.2015 Accepted on 14.12.2015

Asian J. Pharm. Res. 5(4): October- December, 2015; Page 175-182

DOI: 10.5958/2231-5691.2015.00027.1

The Rosuvastatin calcium is more potent than other statins such as atorvastatin, simvastatin and is 8 fold more potent than the hydrophilic comparator, pravastatin.^4-6

According to our knowledge, there is no HPLC method for the simultaneous determination of Rosuvastatin calcium and its lactone impurity at the 0.04µg/mL level available in the literature of Rosuvastatin calcium. The reported analytical methods are three stability-indicating HPLC methods,^7-9 their mass detection methods for the determination of Rosuvastatin calcium in plasma and biological fluids,^10-12 a stability indicating related substance method by UPLC¹³, a stability indicating assay method for determination of Rosuvastatin calcium in the presence of its degradation products using high performance liquid chromatography,¹⁴ a stability-indicating RP-UPLC method for Rosuvastatin calcium and its related substance¹⁵ and three UV spectrometric methods.^13-17

In these above references assay method run time is around 35 minutes and its applicable only for Rosuvastatin calcium and some of the references used UPLC, which is not available in assay of the pharmaceutical companies to quantify. But none of the methods either individually or combined are capable of simultaneous quantification of Rosuvastatin calcium and its lactone impurity at the level of 0.04 µg/mL.

The aim of this work is to optimize the HPLC analysis condition in terms of mobile phase composition in order to separate, identify and quantify the Rosuvasatin calcium and its lactone impurity. The developed RP-HPLC method is precise and accurate for the quantitative determination. Thereafter, the developed method was successfully validated according to international conference on harmonization (ICH) guidelines¹⁸to show the stability-indicating capability of the method. This method was successfully used to determination of Rosuvastatin calcium and its lactone impurity in drug substance and tablet formulation.

EXPERIMENTAL:

Chemicals and Reagents:

Rosuvastatin calcium and lactone impurity were obtained from the Spiralifesciences, Hyderabad, India. Rosuvastatin calcium is available as tablets with the brand name Zyrova with a lable claim of 10 mg of the drug, manufactured by Zydus Cadila, Ahmedabad, India were procured from local pharmacy. HPLC grade, acetonitrile and methanol were purchased from Merck, Mumbai, India. The Sunfire C18 (250 x 4.6mm, 5µm) column was procured from Waters, India. The AR grade ammonium acetate was purchased from S.D.Fine-Chem Limited, Mumbai, India.

Chromatographic condition:

Chromatographic method was carried out by using Waters instrument equipped with column oven, photo diode array detector, Alliances 2695 series low pressure quaternary gradient pump equipped with auto sampler has been used for the analysis of samples and the data was processed using a software program, Empower. The chromatographic conditions were optimized using a C18 stationary phase, Sunfire column (250 mm x 4.6 mm, 5µm). The mobile phase A was 10-mM ammonium acetate, while mobile phase B was combination of acetonitrile and methanol (50:50 v/v). The mixture was pumped at a flow rate of 1.0 mL/minute.

The initial constant composition 50% of mobile phase A and 50 % of mobile phase B was applied from 0 to 4 minutes. From 4 to 6 minutes, the mobile phase composition was changed to 80 % of mobile phase B and 20% mobile phase A. From 6 to 11 minutes constant 80 % mobile phase B and 20 % mobile phase A was used. And from 11 to 12 minutes, the mobile phase composition was changed to 50 % of mobile phase B and 50 % mobile phase A. Finally, from 12 to 15 minutes the mobile phase composition was kept constant to 50 % of mobile phase B and 50 % mobile phase A. The temperature of the column was maintained at 25°C and the eluent was monitored at a wavelength of 242 nm (Fig. 2) which was selected based on the UV spectrum of Rosuvastatin and lactone impurity. The water: methanol (20:80 v/v) was used as diluent. The injection volume was 10µL.

Standard preparation:

Stock solutions of Rosuvastatin calcium (1mg/mL) and lactone impurity (1mg/mL) were prepared separately by dissolving appropriate amount of the substances in diluent. The analyte concentration of Rosuvastatin calcium was fixed as 1000 µg/mL. The 0.15% w/w level of lactone impurity solution was spiked with calculated amount of Rosuvastatin calcium stock solution. The system suitability solution containing 1mg/mL of Rosuvastatin calcium and 0.15% w/w of lactone impurity was prepared (Fig. 4).

Sample preparation:

For the preparation of drug substance, 50 mg of powder was accurately transferred into a 50 mL standard volumetric flask. Approximately 40 mL of diluent was added to the volumetric flask, which was then sonicated in an ultrasonic bath for 5 minutes. The resulting solution was then diluted up to the mark with diluent and mixed well. For the preparation of drug product, an equivalent of 50 mg of tablets of powder was accurately transferred into a 50 mL standard volumetric flask.

Approximately 40 mL of diluent was added to the volumetric flask, which was then sonicated in an ultrasonic bath for 5 minutes. The resulting solution was then diluted up to the mark with diluent and mixed well. This solution was filtered through a 0.45-µm nylon syringe filter.

Forced degradation:

Forced degradation studies were carried out at an initial concentration of 1000 µg/mL of Rosuvastatin calcium to provide an indication of the stability-indicating property and specificity of the proposed method. For this method, it is necessary to ensure that no degradation product, which may form under various stress conditions, interferes with the Rosuvastatin and lactone impurity peaks. The intentional degradation was attempted for the following stress conditions (Table 1). Peak purity was carried out for the Rosuvastatin and lactone impurity by using PDA (photo diode array) detector in all stressed samples to confirm there is no co-elution.

Validation of method:

The method was validated for the following parameters as per ICH guideline Q2 (R1). The specificity of the method is performed by injecting Rosuvastatin calcium and lactone impurity individually. Specificity of the method was evaluated to ensure there was no interference from placebo components (prepared in solution) and from the forced degradation samples. The specificity was determined by using peak purity and resolution. The system suitability of the method was performed by using known concentration of Rosuvastatin calcium and lactone impurity. The system suitability is confirmed by using resolution between Rosuvastatin and lactone impurity, tailing factor and theoretical plates of Rosuvastatin calcium and lactone impurity (Table 2).

The limit of detection (LoD) and limit of quantitation (LoQ) for lactone impurity of Rosuvastatin was achieved by injecting series of dilute solutions and by using signal to noise ratio method ICH Q2 (R1). The precision at LoQ of the developed method for lactone impurity of Rosuvastatin was checked by analyzing six injections of lactone impurity of Rosuvastatin prepared at LoQ level and calculated the percentage relative standard deviation.

Method reproducibility was determined by measuring repeatability and intermediate precision of retention time and peak area of each of Rosuvastatin and lactone impurity. The repeatability of the method was determined by analyzing six replicate injections containing Rosuvastatin (1000 µg/mL) spiked with lactone impurity of Rosuvastatin (0.15% w/w, 1.5µg/mL). The intermediate precision was determined by analyzing the spiked samples in six replicates (n=6) on a different day, different HPLC system by a different analyst and with a different column (different lot number).

The linearity test was carried out by preparing seven calibration solutions of lactone impurity and Rosuvastatin covering from 0.04 µg/mL (LoQ) to 3.0 µg/mL (0.04, 0.2, 0.6, 1.0, 1.5, 2.0, 3.0µg/mL) and 500 µg/mL to 1500 µg/mL (500, 600, 700, 900, 1000, 1200, 1500 µg/mL), respectively, in diluent. The regression curve was obtained by plotting average peak area versus concentration. The accuracy of the method was determined by analyzing samples with known concentrations of lactone impurity and Rosuvastatin calcium.

Table 1. Forced degradation conditions

Type of degradation	Condition
Base degradation	1 N Sodium hydroxide solution was left under stirring for 48 hours at room temperature.
Acid degradation	1 N Hydrochloric acid solution was left under stirring for 48 hours at room temperature.
Oxidative degradation	10 % Hydrogen peroxide solution was left under stirring for 48 hours at room temperature.
Thermal degradation	Hot air oven maintained at 80°C for 3 days.
Photo degradation	1.2 million lux hours and 200 watt hours/square meter.

Table 2. Results of specificity and system suitability

Name	Purity Angle	Purity Threshold	Peak Purity	USP Resolution	USP Tailing	USP Plate Count
Rosuvastatin	0.3265	0.6254	Pass	-	1.43	6806
Lactone impurity	0.2541	0.5896	Pass	10.9	1.14	90211

Fig. 6. LoD chromatogram of lactone impurity

The accuracy was calculated in terms of recovery (%). This accuracy test was carried out in triplicate at each concentration covering from 0.04 µg/mL (LoQ) to 3.0 µg/mL (0.04, 0.6, 1.5, 3.0µg/mL) in diluent for lactone impurity and 500 µg/mL to 1500 µg/mL (500, 700, 1000, 1500 µg/mL) in diluent for Rosuvastatin calcium. The solution stability of Rosuvastatin calcium and lactone impurity of Rosuvastatin was studied by keeping the solution in tightly stoppered volumetric flask at room temperature on a laboratory bench for 48 hours. The area of lactone impurity and Rosuvastatin calcium was checked at every 4 hours interval during the storage period.

RESULTS AND DISCUSSION:

Optimization of chromatographic conditions:

To develop suitable HPLC method for the separation of lactone impurity from Rosuvastatin, different stationary phases and mobile phases were tried. Inertsil C18, Thermo C18 and different combinations of mobile phases consisting acetonitrile and methanol were used. Noticeable separation could be achieved in Inertsil C18 column but the peak shape of Rosuvastatin and lactone impurity was not good (Fig. 3). Hence the efforts were continued to select the best stationary phase and mobile phase combinations that would give optimum resolution and selectivity for the lactone impurity from Rosuvastatin. This lead to an excellent separation on Sunfire column (250 mm x 4.6 mm, 5µm) using mobile phase consisting of 10 mM ammonium acetate, acetonitrile and methanol combination. The results of resolution between the lactone impurity and Rosuvastatin are summarized (Table 2). Based on the data obtained from the method development and optimization activities, Sunfire column (250 mm x 4.6 mm, 5µm) with mobile phase A (10 mM ammonium acetate), and mobile phase B (combination of acetonitrile and methanol (50:50 v/v)) was selected from the method development. The flow rate of final method was 1.0 mL/minute with injection volume 10 µL. The column temperature was 25°C and the detection wavelength was 242 nm. Under these conditions, the lactone impurity from Rosuvastatin was well separated. In the optimized method, the typical retention time of Rosuvastatin and lactone impurity were 6.2 and 9.8 minutes, respectively (Fig. 4). Excellent base line separation was obtained with the total run time of 15 minutes.

Validation results of the method:

The optimized reverse phase HPLC method of the final method was evaluated for its specificity, precision, LoD, LoQ, linearity, accuracy and solution stability.

Table 3. Forced degradation results

Type of degradation	Peak purity at Rosuvast-atin retention time	Observation
Base degradation	Pass	No significant degradation
Acid degradation	Pass	Small degradation
Oxidative degradation	Pass	No significant degradation
Thermal degradation	Pass	No significant degradation
Photo degradation	Pass	No significant degradation

Table 4. Precision study results of Rosuvastatin and lactone impurity

Study	% RSD of Rosuvastatin	% RSD of Lactone impurity
Repeatability (Standard injections)	0.95	1.56
Method precision (n=6)	0.05	1.68
Intermediate precision (n=6)	0.07	1.74

The specificity of the method was determined through photo diode array detector by using peak purity (Table 2). Forced degradation studies were performed to demonstrate the selectivity and stability-indicating capacity of the proposed reverse phase HPLC method. It is clearly evident that there is no interference at the retention time of Rosuvatstain and lactone impurity from the blank, other excipients and forced degradation samples (Table 3).

The LoQ and LoD concentrations were estimated to be 0.04µg/mL and 0.01µg/mL for lactone impurity of Rosuvastatin. The LoQ and LoD were calculated by using signal to noise ratio method. The method precision for lactone impurity of Rosuvastatin at LoQ was less than 5% RSD. Therefore, this method had adequate sensitivity for the detection and estimation of lactone impurity of Rosuvastatin calcium. The LoQ and LoD chromatograms are shown in Figures 5 and 6.

Table 5. Linearity results of lactone impurity

Concentration in µg/mL	Average Peak area (n=6)	% RSD
0.04	1272	1.78
0.20	4807	1.01
0.60	13086	1.43
1.00	19305	1.81
1.50	26283	1.25
2.01	36752	1.03
3.01	51865	0.98

Table 6. Linearity results of Rosuvastatin

Concentration in µg/mL	Average Peak area (n=6)	% RSD
502.0	10126625	0.93
602.4	11969358	0.86
702.8	13940712	0.94
803.2	16498400	0.54
1004.0	20118275	0.56
1204.8	24242878	0.62
1506.0	29956714	0.72

Linearity of lactone impurity and Rosuvastatin was evaluated over seven levels from 0.04µg/mL to 3.01µg/mL and 502 µg/mL to 1506 µg/mL (502, 602.4, 702.8, 803.2, 1004, 1204.8, 1506 µg/mL), respectively (Table 5 and 6), with the linear regression equation y = mx + c, where ‘x’ is the concentration in µg/mL and ‘y’ is the corresponding peak area in mV/s. The correlation coefficient value is more than 0.997 and 0.999 for lactone impurity and Rosuvastatin, respectively, linear graph was shown in Figure 7 and 8. The accuracy (as percent recovery) was determined by standard addition experiment. The recovery experiments were conducted for lactone impurity of Rosuvastatin and Rosuvastatin in triplicate at 0.04, 1.0, 1.50 and 3.01µg/mL, and 502, 803.2, 1004.0 and 1204.8 µg/mL, respectively. The recovery of lacton impurity and Rosuvastatin was calculated by back calculated concentration at each level in each preparation and the recovery was within 98.4 and 100.8; and 99.6 and 100.2, respectively (Tables 7 and 8).

Table 7. Accuracy results of lactone impurity

Entry	Spiked amount (µg)	% RSD (n=3)	Recovery (%)
1	0.04	1.96	98.4
2	1.00	1.49	100.8
3	1.50	1.15	99.6
4	3.01	1.32	100.2

Table 8. Accuracy results of Rosuvastatin

Entry	Spiked amount (µg)	% RSD (n=3)	Recovery (%)
1	502.0	0.91	100.1
2	803.2	1.03	99.9
3	1004.0	0.83	100.2
4	1204.8	0.69	99.6

The stability of the solution in this method was tested over 48 hours. No significant change in lactone impurity and Rosuvastatin content was observed in Rosuvastatin calcium sample during solution stability study. The calculated RSD % for lactone impurity and Rosuvastatin for replicate analysis was 1.54% and 0.62%. No unknown peak was observed in the above solution stability study indicating that the solution of lactone impurity of Rosuvastatin in the presence of Rosuvastatin calcium solution was stable for at least 48 hours. The repeatability and intermediate precision were expressed as relative standard deviation (RSD). For this study, solution of Rosuvastatin calcium (1000 µg/mL) spiked with lactone impurity of Rosuvastatin 0.15 % w/w (1.5µg/mL) was analyzed in six replicates to establish repeatability. The RSD % values were calculated for retention time and peak area of Rosuvasatain and lactone impurity of Rosuvastatin.

In the intermediate precision, results shown that % RSD values are in the same order of magnitude as those obtained for repeatability studies (Table 4). All the obtained validation results indicated that the method is precise, accurate, specific and linear in the tested range.

CONCLUSION:

A simple, specific, linear, accurate, precise and sensitive reverse phase HPLC method was successfully developed and validated for simultaneous quantification of lactone impurity and Rosuvastatin by using ammonium acetate, acetonitrile and methanol, which was capable of separating the Rosuvastatin calcium and its lactone impurity. The developed and validated method can be used for determining lactone impurity and Rosuvastatin in Rosuvastatin calcium bulk drug and dosages form. This developed method can be also applied to find the identification of unknown impurities by LC-MS in Rosuvastatin calcium.

ACKNOWLEDGMENT:

The authors would like to thank the management of Advinus Therapeutics Limited, Bengaluru, India, for providing necessary facilities to carry out this research work.

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