INTRODUCTION:

Rosuvastatin is used for the treatment of hyperlipidemia1 and for preventing cardiovascular diseases2. Chemically, Rosuvastatin (RVS) is (3R, 5S, 6E) 7-{4-(4-fluorophenyl)-6-(1-methylethyl)-2-methyl (methyl sulfonyl) amino] pyrimidin-5-yl}-3, 5 dihydroxy hept-6-enoic acid and is an inhibitor of 3-hydroxy-3- methyl glutaryl coenzyme (HMG-CoA) reductase3. Rosuvastatin (Figure 1) is a white amorphous powder sparingly soluble in water, methanol and has a molecular formula C22H28FN3O6S with molecular weight 481.5 grams/mole.

Figure 1: Structure of Rosuvastatin

Literature survey for the analytical methods for the estimation of Rosuvastatin includes HPTLC4-5, capillary electrophoresis6, spectrophotometry7-13, LC-MS14-19 and LC-MS/MS20 (Human plasma), UPLC21-22, HPLC23-32. In the present study the authors have proposed a new stability indicating liquid chromatographic method (RP-UFLC) for the assay of Rosuvastatin tablets and the method was validated as per ICH guidelines.

MATERIALS AND METHODS:

Rosuvastatin API was obtained from Alembic Pharmaceuticals, India. Acetonitrile (HPLC grade), sodium hydroxide (NaOH) and hydrochloric acid (HCl) and Hydrogen peroxide (H₂O₂) were obtained from Merck (India). Rosuvastatin is available with brand names ARVAST (Intas Pharmaceuticals Ltd), ROSAVE (Alembic Pharmaceuticals), ROSULIP (Cipla Ltd) etc as tablets with label claim: 5 mg, 10 mg and 20 mg and all chemicals are of AR grade.

Shimadzu Model CBM-20A/20 Alite HPLC system, equipped with Phenomenex C18 column (250mm × 4.6mm i.d., 5 µm particle size) was used for the Chromatographic separation. Mobile phase consisting of a mixture of sodium acetate buffer (pH 4.0) and acetonitrile (30:70, %v/v) (Isocratic mode) with flow rate was 1.2 mL/min was chosen for the present study. The overall run time was 10 min and the injection volume was 20 µl.

Preparation of Rosuvastatin drug solution:

Rosuvastatin stock solution (1000 μg/mL) was prepared by weighing accurately 25 mg of Rosuvastatin in a 25 mL volumetric flask and made up to volume with acetonitrile. Working standard solutions were prepared from the stock solution and diluted with mobile phase (Sodium acetate buffer (pH 4.0): Acetonitrile (28:72, %v/v). All the solutions were filtered through 0.45 μm membrane filter prior to injection.

Method validation³³

Linearity, Limit of quantification (LOQ) and limit of detection (LOD):

The method was validated for linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, robustness and selectivity. A series of Rosuvastatin drug solutions 0.5-200 μg/mL were prepared from the drug stock solution on dilution with mobile phase and 20 µL of each solution was injected (n=3) in to the UFLC system. The peak area of the chromatograms was recorded and a calibration curve was drawn by plotting the concentration of the drug solutions on the x-axis and the corresponding mean peak area on the y-axis.

Precision, Accuracy and Robustness

The intra-day and inter-day precision studies were evaluated by carrying out independent assays of Rosuvastatin at three concentration levels (10, 20 and 50 µg/mL) (n=3) on the same day and also on different days (Day 1, Day 2 and Day 3) respectively and the %RSD of the peak areas obtained was calculated.

The accuracy of the assay method was evaluated in triplicate by standard addition method (80, 100 and 120%) i.e. by the addition of API drug solution to the pre-analyzed formulation and thereby the percentage recovery was calculated. The robustness of the analytical method denotes the ability of the method to remain unaffected by the small and deliberate variations in the method optimized conditions.

The robustness of the method was evaluated by incorporating small changes in the chromatographic conditions such as mobile phase composition (± 2%; 26:74 v/v and 30:70 v/v), detection wavelength (± 2 nm; 252 and 256 nm), pH (± 0.1 unit; 3.9 and 4.1) and flow rate (± 0.1 mL; 1.1 and 1.3 mL/min).

Assay of Rosuvastatin marketed formulations (Tablets)

Twenty tablets from two different brands were bought from the local pharmacy medical store, weighed, crushed in to fine powder and then powder equivalent to 25 mg Rosuvastatin from each brand was accurately weighed and dissolved in mobile phase in different 25 mL volumetric flasks. The contents of the flask were sonicated for 30 min and filtered through 0.45μm membrane.

Forced degradation studies³⁴:

Stability indicating studies were performed to define the specificity of the method. Rosuvastatin drug solution was exposed to different stress conditions such as acidic hydrolysis, alkaline hydrolysis, oxidation, thermal degradation and photolysis as per ICH guidelines.

Acidic hydrolysis:

Acidic hydrolysis was performed by treating the Rosuvastatin drug solution with 0.1 N HCl at 70ºC in a thermostat for 1 hour, cooled and then neutralized with 0.1N NaOH followed by dilution with the mobile phase as per the requirement. The resultant solution was filtered through membrane filter and 20 µl was injected (n=3) in to the UFLC system and the area under the curve or the peak area was noted.

Alkaline hydrolysis:

Alkaline hydrolysis was performed by treating the Rosuvastatin drug solution with 0.1 N NaOH at 70ºC in a thermostat for 1 hour, cooled and then neutralized with 0.1N HCl followed by dilution with the mobile phase as per the requirement. The resultant solution was filtered through membrane filter and 20 µl was injected (n=3) in to the UFLC system and the area under the curve or the peak area was noted.

Oxidation degradation

Oxidation degradation study was performed by treating the Rosuvastatin drug solution with 0.1 ml 30% H₂O₂ at 70ºC in a thermostat for 1 hour, cooled and diluted with mobile phase as per the requirement. The resultant solution was filtered through membrane filter and 20 µl was injected (n=3) in to the UFLC system and the area under the curve or the peak area was noted.

Photolytic degradation:

Photolytic degradation was performed by exposing the Rosuvastatin drug powder (solid state) to UV radiation (365 nm) in a UV chamber for 24 hours and then Rosuvastatin drug solution was prepared using this powder (1 mg/mL) and diluted with mobile phase. The resultant solution was filtered through membrane filter and 20 µl was injected (n=3) in to the UFLC system and the area under the curve or the peak area was noted.

RESULTS AND DISCUSSION:

In the literature no stability-indicating RP-UFLC method was found for the determination of Rosuvastatin in pharmaceutical formulations. A thorough comparative study of the previously published methods with the present proposed method was summarised in Table 1. The chromatographic method was developed using a mixture of sodium acetate buffer and acetonitrile and C18 column with UV detection at 254 nm.

Table 1: Literature survey

Method /Reagent	λ (nm)	Linearity (mg/mL)	Comments	Ref.
0.1% Tri fluoro acetic acid: Methanol (Gradient mode)	240	0.075-11.84	UPLC Related substances	21
0.1% Tri fluoro acetic acid: Methanol (50:50)	240	25-75	UPLC Related impurities	22
Acetonitrile: 0.05 m formic acid (45:55)	240	0.02-10	HPLC (UV detector) (Rat Plasma)	23
Acetonitrile: Water (40:60) (pH 3.5)	242	0.5-80	HPLC	24
Acetonitrile: Methanol: 0.05 m Sodium dihydrogen phosphate buffer (20: 40:40) (pH 2.0)	242	0.05–70	HPLC Stability indicating	25
Acetonitrile: Potassium dihydrogen ortho phosphate (50:50) (pH 3.0)	243	5-30	HPLC	26
Buffer (pH 4.5): Acetonitrile: methanol (45:25:35)	248	25-75	HPLC	27
Acetonitrile: (0.02 M) Potassium dihydrogen ortho phosphate (35:65) (pH 3.0 ± 0.1)	242	0.8-60	HPLC	28
0.1M Formic acid: Methanol (25:75) (Internal standard: Fluvastatin)	280	3.0-1602	HPLC	29
Acetonitrile: 0.5% formic acid (50:50) And HPTLC (6: 3.5: 0.5: 0.2) (Ethyl acetate: toluene: Acetonitrile: Formic acid)	248 243	5-300 and 0.318-3.816 /spot	HPLC UV detector Stability indicating	30
Acetonitrile: phosphate buffer (pH 2.6) (70:30)	241	2-20	HPLC	31
Methanol: Water (pH 3.0) (60:40)	238	6.0-22	HPLC Stability indicating	32
Sodium acetate: Acetonitrile (28: 72)	254	0.5-250	UFLC Stability indicating (PDA detector)	Present work

HPLC method development and optimization:

A mobile phase consisting of water: acetonitrile (50:50, %v/v) was used as mobile phase initially (Flow rate 0.8 mL/min) where the drug peak was eluted with retention time more than 10 mins and therefore the mobile phase composition was modified as 45:55, 40:60, 35:65 and 30: 70 the same flow rate where the retention time was shifted to a lower value but slight tailing was observed. Then the flow rate was slowly modified and with mobile phase ratio (sodium acetate buffer (pH 4.0): acetonitrile) 28:72, v/v a sharp peak was eluted keeping flow rate at 1.2 ml/min.

Method validation:

Linearity:

Rosuvastatin was obeying Beer-Lambert’s law over the concentration range 0.5-200 μg/mL (Table 2) with the linear regression equation, y = 32548x - 16862 (r² = 0.9999) (Figure 2). The LOQ was found to be 0.04291 μg/mL and the LOD was found to be 0.01502 μg/mL. The % RSD range was 0.257-0.628. The chromatogram so obtained for Rosuvastatin with the above optimised chromatographic conditions was shown in Figure 3.

Table 2: Linearity of Rosuvastatin

Conc. (mg/mL)	*Mean peak area ± SD	RSD (%)
0.5	18569 ± 53.85	0.29
1	34269 ± 119.94	0.35
5	174698 ± 541.56	0.31
10	348963 ± 1605.22	0.46
20	672369 ± 1680.92	0.25
50	1689267 ± 4729.94	0.28
100	3275986 ± 107035.12	0.52
150	4918691 ± 49186.90	0.57
200	6497698 ± 14294.93	0.22

*Mean of three replicates

Figure 2: Calibration curve of Rosuvastatin


Placebo	Rosuvastatin (100 μg/mL) (API)

Rosuvastatin tablet (100 μg/mL) (Brand I)(Label claim: 20 mg)	Rosuvastatin tablet (100 μg/mL) (Brand II)(Label claim: 20 mg)
Figure 3: Representative chromatograms of Rosuvastatin

Table 3: Precision and accuracy study of Rosuvastatin

Conc. (mg/mL)	Intra-day precision	Inter-day precision
Conc. (mg/mL)	*Mean peak area ± SD (%RSD)	*Mean peak area ± SD (% RSD)
10	349156 ± 1256.96 (0.36)	351003 ± 3194.12 (0.91)
20	673681 ± 3301.03 (0.49)	679268 ± 6928.53 (1.02)
50	1696584 ± 10688.47 (0.63)	1689654 ± 20106.88 (1.19)
Accuracy
Conc. (mg/mL)	*Mean peak area ± SD (%RSD)	Drug found (µg/mL)	% Recovery
18	603195 ± 3154.81 (0.52)	18.01	100.08
20	664156 ± 4853.92 (0.61)	19.89	99.44
22	731821 ± 5341.11 (0.72)	21.97	99.85

*Mean of three replicates

Table 4: Robustness study of Rosuvastatin (20 mg/mL)

Parameter	Condition	*Mean peak area	*Mean peak area ± SD (% RSD)	*(Drug recovery) Assay (%)**
Flow rate (± 0.1 mL min^-1)	1.1	662695	667378.67 ± 7875.06 (1.18)	(19.99) 99.93
	1.2	672315
	1.3	667126
Detection wavelength (± 2 nm)	252	672651	671898.5 ± 1948.50 (0.29)	(20.13) 100.07
	254	671256
	256	672541
Mobile phase composition (Sodium acetate buffer: Acetonitrile) (± 2 % v/v)	28:72	672638	673829.33 ± 3975.59 (0.59)	(20.18) 100.90
	30:70	675698
	32:68	673152
pH (± 0.1 unit)	3.9	663984	670628.33 ± 6907.47 (1.03)	(20.09) 100.43
	4.0	672685
	4.1	675216

*Mean of three replicates

Precision, Accuracy and Robustness:

The % RSD value for the intra-day precision was 0.36-0.63 and that of inter-day precision study was 0.91-1.19 respectively which was less than 2% indicating that the method is precise. The % RSD value for the accuracy study was 0.52-0.72 which was less than 2% indicating that the method is accurate and the percentage recovery was found to be 99.44-100.08% (Table 3). The % RSD value for the robustness study was found to be 0.29-1.18 which was less than 2% indicating that the method is robust (Table 4).

Assay of Rosuvastatin marketed formulations (Tablets):

The marketed formulations of two different brands were analysed using the present proposed method. The % recovery was found to be 99.05- 99.70 (Table 5) and there is no interference of excipients. The resultant chromatograms obtained during the assay of Rosuvastatin were shown in Figure 3C and Figure 3D.

Table 5: Analysis of Rosuvastatin commercial formulation (Tablets)

S. No.	Formulation	Labelled claim (mg)	*Amount found (mg)	*Recovery (%)
1	Brand I	20	19.94	99.70
2	Brand I	20	19.81	99.05

*Mean of three replicates

Forced degradation studies

The specificity of the method can be determined using stress degradation studies.

The stability indicating capability of the proposed method was recognised from the separation of Rosuvastatin drug peak from that of the degradants peaks. Forced degradation studies of Rosuvastatin were performed using 100 μg/mL.

Table 6: Forced degradation studies of Rosuvastatin

Stress Conditions	*Mean peak area	*Drug recovered (%)	*Drug decomposed (%)	Theoretical plates	Tailing factor
Standard Drug	3277956	100	-	5991	1.385
Acidic hydrolysis	3197365	97.54	2.46	5933	1.412
Alkaline hydrolysis	3189487	97.30	2.70	5834	1.364
Oxidative degradation	2872103	87.62	12.38	5799	1.319
Thermal degradation	3269521	99.74	0.26	5982	1.321
Photolytic degradation	2324632	70.92	29.08	5968	1.404

*Mean of three replicates

Rosuvastatin has shown 2.46 % degradation in acidic conditions and 2.70% during the alkaline degradation. Rosuvastatin has shown less than 1% degradation in thermal degradation and 12.38% degradation during oxidation and less than 30% (29.08) degradation was observed during photolysis. Rosuvastatin is found to be more sensitive towards photolysis. The chromatograms obtained during the forced degradation study was shown in Figure 4. The system suitability parameters such as theoretical plates, tailing factor etc were within the acceptable criteria (Table 6).

CONCLUSION:

The proposed new stability-indicating RP-UFLC method has been validated as per ICH guidelines and this method can be applied for the quantification of Rosuvastain in pharmaceutical dosage forms. The proposed method was found to be precise, accurate and robust

ACKNOWLEDGEMENT:

The authors are grateful to Alembic Pharmaceuticals (India) for providing the gift samples of Rosuvastatin (API).

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Received on 21.10.2020 Modified on 12.12.2020

Research J. Pharm. and Tech 2021; 14(3):1673-1679.

DOI: 10.5958/0974-360X.2021.00297.3


Acidic hydrolysis	Alkaline hydrolysis

Oxidative degradation	Thermal degradation

Photolytic degradation
Figure 4: Typical chromatograms of Rosuvastatin during forced degradation studies