Development and Validation of a new improved RP-HPLC Method for estimation of Rosuvastatin calcium in Pharmaceutical dosage form

 

Kallol Jana*, Beduin Mahanti

School of Pharmacy, Techno India University, West Bengal, EM 4, Sector –V, Salt Lake,

Kolkata-700091, West Bengal, India.

 

ABSTRACT:

 

 

INTRODUCTION:

Rosuvastatin calcium is the calcium salt from of Rosuvastatin, a statin with antilipidemic activity. Rosuvastatin selectively and competitively binds to and inhibits hepatic hydroxymethyl-glutaryl coenzyme A (HMG-CoA) Reductase, the enzyme which catalyzed the conversion of HMG-CoA to mevalonate, a precursor of cholesterol. This leads to decrease in hepatic cholesterol level and increase in uptake of LDL (Low density lipoprotein) cholesterol^1-3. Rosuvastatin calcium is chemically bis[(E)-7[4-(4-flurophenyl)-6-isopropyl-2-[methyl(methyl-sulphonyl) amino] pyrimidin-5-yl](3R,5S)-3,5dihydroxyhept-6-enoicacid]calcium salt (Fig-1).

 

Fig-1: Rosuvastatin calcium

 

Rosuvastatin calcium is available in tablet form containing 5mg, 10mg, 20mg and 40mg. In this work a simple, first, economic and isocratic RP-HPLC method was developed to estimate tablet formulation. The most of the HPLC method developed for the Rosuvastatin calcium use buffer, THF, ortho phosphoric acid and triethylamine in the mobile phase preparation^4-14 Table-1. An acute use buffer and acid decrease the column life, increase cost of estimation. The proposed research work was developed, validated and optimized according to International Conference on Harmonization (ICH) guideline¹⁵.

Table-1:Comparison of published methods with developed method.

ROSUVASTATIN CALCIUM
Sl. No.	Mobile phase	Column	Wave length	Injection volume	Flow rate	Retention time
1	KH2PO4 (Potassium dihydrogen phosphate): Methanol = ( 30:70)	RP-18 150 X 3.0 mm ( 5 micron )	234 nm	20 µL	1.0 ml /min	5.969
2	0.02 M phosphate buffer pH 6.8: Acetonitrile = ( 60: 40 )	C-18, 100 x 4.6 mm, (3 micron)	242 nm	20 µL	0.6 ml /min	3.424
3.	Acetonitrile :THF: Water ( pH 3.0) = (68:12:20)	C-18, 250 x 4.6 mm, (5 micron), 50°C	251 nm	10 µL	0.5 ml /min	5.4
4.	KH2PO4 (Potassium dihydrogen phosphate):Acetonitrile = ( 50:50) pH 3.0 by phosphoric acid	C-18, 100 x 4.6 mm, (5 micron)	243 nm	20 µL	0.5 ml /min	3.33
5.	Methanol : 0.02 M ammonium di-hydrgen phosphate buffer ( 75:25) pH 5.5 by phosphoric acid	C-18, 250 x 4.6 mm, (5 micron)	272 nm	20 µL	1.0 ml /min	4.18
6.	Acetonitrile:water pH 3.5 by phosphoric acid =( 40:60)	C-8, 150 x 4.6 mm, (5 micron)	242 nm	20 µL	1.5 ml /min	5.2
7.	Acetonitrile : water ( 75: 25)	C-18, 250 x 4.6 mm, (5 micron)	252 nm	20 µL	0.6 ml /min	8.0
8.	Buffer pH 4.8 (0.78 % w/v sod. Dihydrogen orthophosphate : acetonitrile =( 50:50)	C-18, 250 x 4.6 mm, (5 micron)	241 nm	20 µL	1.0 ml /min	4.72
9.	Buffer pH4.5 ( 0.05 M sod.dihydrogen phosphate) : Acetonitrile = ( 50:50 )	C-8, 250 x 4.6 mm, (5 micron)	245 nm	10 µL	1.2 ml /min	3.684
10.	Water pH 2.6: acetonitrile = ( 30:70)	C-18, 150 x 4.6 mm, (5 micron)	220 nm	20 µL	1.0 ml /min	1.89
11.	Phosphate buffer pH 2.8 : Acetonitrile = ( 65:35) pH 3.8 by triethylamin	C-18, 250 x 4.6 mm, (5 micron)	252 nm	20 µL	1.0 ml /min	2.147
As per IP 2018	Ammonium acetate buffer : Acetonitrile: THF = (585:360: 50)	C-18, 250 x 4.6 mm, (5 micron)	248 nm	20 µL	1.5 ml /min	5.600
Developed method	methanol : acetonitrile : water (40:40:20,v/v).	Thermo scientific C8 column, 250 x 4.6 mm, particle size 5 µm	248 nm	5 µL	1.0 ml /min	3.427

 

AIMS AND OBJECTIVES:

The main aim and objective of this research work was to develop cost effective, time effective, rapid, reliable, precise and simple isocratic RP-HPLC chromatographic analytical method for determine the content uniformity of tablet formulation and assay of Rosuvastatin calcium in accordance with ICH guidelines. Determination of content of uniformity is now a day’s an important test included in USP 32¹⁶.

 

EXPERIMENTAL:

Material and Reagents:

Methanol, acetonitrile and water were procured from Rankem and Rosuvastatin were received as gift sample from MSN Laboratories Pvt. Ltd. Telangana, India. 0.2µm PTFE syringe filter and also 0.2µm Nylon filter were procured from Millipore.

 

Equipment:

The chromatographic separation was performed for development and validation of this assay method of model Agilent Technology 1260 Infinity II having UV detector, Quaternary pump and multisampler with 100 µl loop volume. Open Lab software was applied for data collecting and processing.

 

HPLC condition:

Chromatographic analysis was performed on Thermo scientific C₈ column, 250 x 4.6mm, particle size 5µm column. A mixture of methanol, acetonitrile and water (40:40:20,v/v) was used as a mobile phase. The mobile phase was pumped at 1ml/min and eluents were monitored by using UV detector at 248nm. The maintained column temperature 27°C and injection volumes of sample, standard were 5µl. The mobile phase was filtered through 0.2µm filter prior to use.

 

Preparation of standard solution:

20mg of Rosuvastatin calcium was weight accurately and transfer into a 100ml volumetric flask. Add 70ml of mobile phase, dissolve and volume made up to 100ml with the same solvent. Then it is filtered through 0.2µm PTFE syringe filter. The chromatogram of standard solution was shown in Fig.2.

 

Fig-2 Chromatogram of Rosuvastatin Calcium

 

Preparation of sample solution:

Commercial tablets of three different brands were taken and their average weight was determined. Crushed the 20 tablets of each brand separately to fine powder. Weight accurately 10mg equivalent of powder of three different brands separately three different 50ml volumetric flask and dissolve in mobile phase with shaking for 5-10 minutes and then sonicate. Then it is filtered through 0.2µm PTFE syringe filter. For content uniformity one tablet was transfer in to a 25ml volumetric flask use the same procedure as in assay.

 

Analysis of commercial formulations:

The proposed method was developed to determine of Rosuvastatin calcium in RosuRite-5 (MSN Laboratories private Limited) (Sample A), Rosuvas 5 (Sun pharma laboratories) (Sample B) and Rozucor 5 (Torrent Pharma) (Sample C).

 

RESULTS:

The amount of Rosuvastatin calcium in each brand was calculated by comparing the peak area of standard solution and sample. The assay % of the drugs and content uniformity were calculated and tabulated in Table-2 and Table-5.

 

Table-2: The Assay % of Three Different Brands.

Different Brands	Percentage Label claim (%w/w)
Sample-A (MSN Laboratories private Limited)	100.16
Sample-B (Sun pharma laboratories)	98.84
Sample-C (Torrent Pharma)	99.38

 

HPLC METHOD VALIDATION AND OPTIMIZATION:

For development of mobile phase different composition and ratio was tried including methanol: water (60:40), Acetonitrile: water (40:60), flow rate ( 0.7ml, 1.0ml, 1.2ml and 1.5ml/min) but peak shape was not so good. Scanning wavelengths (200 - 400nm) was also tried. Preliminary development involved trying C18, C8 reversed phase columns. The best development was done by Thermo scientific C₈ column, 250 x 4.6mm, particle size 5µm with flow rate of 1.0ml/min. Detection was monitor at 248nm. The mobile phase consisted of methanol : acetonitrile : water (40:40:20, v/v). This selected developing method allows sharp peak with good retention time and without tailing.

 

Method validation:

The chromatographic conditions were validated by system suitability, accuracy, method and system precision, linearity, recovery, solution stability, limit of detection (LOD), limit of quantification (LOQ), robustness and ruggedness studies in accordance with ICH guideline^17-19.

 

System suitability:

System suitability is use to verify reproducibility of the chromatographic system. The parameters like number of theoretical plates, tailing factor, Retention time were investigated by injecting standard solutions of drug in six replicates. The results are given in Table-3. From the results it was observed that system suitability parameters fall within ±2% relative standard deviation (RSD) during performance of the method. Hear asymmetric factor is less than 2 and peak is sharp and have clear base line separation.

 

Table-3:Analytical Parameters.

Parameters	Rosuvastatin calcium
Linear dynamic range	140-260 µg/ml
R2 value	0.999
Retention time	3.427
Theoretical plates	8915
Tailing Factor	1.0195
LOD (µg/mL)	3.26
LOQ (µg/mL)	9.88
RSD%	0.09

 

Accuracy:

The accuracy of the method was determined by recovery experiments. Recovery study was carried out by applying the method to drug content present in tablet from which known amount of standard added at three concentration levels (80,100 and 120%) and percentage of recovery in each case was calculated. The technique involves addition of standard drug solution to pre analyzed sample solution. The sample solutions were injected and the chromatograms were recorded. These data are shown in Table-4.

 

Precision:

The intraday precision studies were studies by six replicate measurements at 200µg/ml. Inter-day precision of Content uniformity was determined for same analytical samples of concentration 200µg/ml of drug concentration. The result is shown in Table-5 and Table-6 respectively. The % RSD was found less than 2 for both intraday and inter-day precision. Method precision was performed by preparing six different samples from the same sample pool. Each solution was injected under the same condition and mean value of peak area response of each solution was taken. The inter-day precision of Content uniformity of sample was measured on three different days.

 

Table-4:Comparison of recovery study of Three Different Brand

% Target	Sample-A (%Recovery)	Sample-B (%Recovery)	Sample-C (%Recovery)	Mean
80%	106.17	105.96	106.40	106.58
	105.92	106.60	106.22
	105.86	106.37	107.13
100%	99.42	99.60	100.00	100.18
	100.11	100.03	100.33
	99.69	100.03	100.20
120%	102.67	102.64	102.78	102.81
	102.78	102.68	102.79
	102.74	102.79	102.87

 

Table-5:Comparison of Intraday Precision.

Sl. No.	Percentage Label claim (%w/w) Sample-A	Percentage Label claim (%w/w) Sample-B	Percentage Label claim (%w/w) Sample-C
Set-1	100.16	98.84	99.38
Set-2	99.06	98.88	99.48
Set-3	99.14	99.21	100.21
Set-4	99.36	99.68	100.48
Set-5	100.52	98.76	99.68
Set-6	99.94	99.94	99.56
RSD%	0.5985	0.4941	0.4439

 

Table-6: Inter-day content uniformity of Three Different Brands

Content uniformity  Day-1	Percentage Label claim (%w/w) Sample-A	Percentage Label claim (%w/w) Sample-B	Percentage Label claim (%w/w) Sample-C
Sample-1	101.74	101.81	101.79
Sample-2	102.37	101.88	101.37
Sample-3	101.51	102.32	101.91
Sample-4	101.64	102.32	101.86
Sample-5	101.42	102.27	101.87
Sample-6	101.86	101.88	101.66
Sample-7	101.40	102.56	101.47
Sample-8	101.62	102.53	101.59
Sample-9	102.23	102.10	102.10
Sample-10	102.67	101.28	101.88

 

Content uniformity  Day-2	Percentage Label claim (%w/w) Sample-A	Percentage Label claim (%w/w) Sample-B	Percentage Label claim (%w/w) Sample-C
Sample-1	100.30	100.17	99.79
Sample-2	105.09	100.24	99.24
Sample-3	99.20	100.10	100.75
Sample-4	99.92	100.10	98.73
Sample-5	99.35	99.22	98.80
Sample-6	99.64	100.47	99.05
Sample-7	99.39	99.67	99.03
Sample-8	99.96	99.43	100.53
Sample-9	100.91	99.22	99.73
Sample-10	102.53	98.95	99.10

 

Content uniformity  Day-3	Percentage Label claim (%w/w) Sample-A	Percentage Label claim (%w/w) Sample-B	Percentage Label claim (%w/w) Sample-C
Sample-1	99.80	99.38	99.02
Sample-2	99.53	100.46	99.16
Sample-3	102.79	99.31	99.12
Sample-4	103.72	99.31	99.47
Sample-5	101.82	99.51	99.33
Sample-6	106.41	99.63	99.32
Sample-7	102.23	99.63	99.52
Sample-8	100.63	99.27	99.02
Sample-9	100.18	99.00	99.43
Sample-10	99.90	99.23	98.86

 

Linearity:

The peak areas of Rosuvastatin calcium were linear with concentration range of 140-260 µg/ml in three replicates. The slope and intercept value for calibration curve Y= 37027 X+ 12490. R²=0.999, Y= 37040 X+ 12200. R²=0.999 and Y= 37115 X+ 75788. R²=0.999 respectively. The excellent correlation exists between peak area and concentration of the drugs within the concentration range. The data was analyzed by “linear regression least squares fit” and the result is shown in Fig-3.

 

Linearity:

 

Solution stability:

Solution stability of samples solution during analysis was analyzed over a period of 12 hr at room temperature. The results indicated that peak area of Rosuvastatin calcium did not show much variation. There was no significant degradation with the indicated period. The % of RSD is less than 2.0. Hence it was concluded that both the solution were stable for 12 hr at room temperature. The results are displayed in Table-7.

 

Fig-3 Calibration Curve of Rosuvastatin Calcium

 

 

Table-7:Stability of the solution.
Time interval:	Area of sample A	Area of sample B	Area of sample C	Acceptance criteria
2 hours	37372120	37403221	37428569
4 hours	37409707	37608623	37312783
6 hours	37679193	37219919	37401772
10 hours	36916788	36933584	36789965	% of RSD is ≤2.0
12 hours	36770162	37043206	36545773
Mean	37229594	37241710.6	37095772
SD	375439.3994	271813.939	402337.06
% of RSD	1.01	0.73	1.08

 

Ruggedness (Intermediate precision):

Intermediate precision expresses within laboratories variation: different days, different analysts and different equipments. The good results are presented in Table-8.

 

Table-8:Comparison of Intermediate Precision of Three Different Brands.

Sl. No.	Percentage Label claim (%w/w) Sample-A	Percentage Label claim (%w/w) Sample-B	Percentage Label claim (%w/w) Sample-C
Set-1	100.64	100.85	100.58
Set-2	100.81	100.19	99.18
Set-3	100.33	100.69	100.49
Set-4	99.89	100.43	99.16
Set-5	100.67	99.04	98.69
Set-6	100.16	99.39	99.72

 

Robustness:

The robustness of the proposed method was estimated in analytical development stage where the effect of different factor on the method was studies to obtain the optimum parameters for assay. Robustness of the method was studied by deliberately varying parameters like acetonitrile composition (±5%), methanol composition (±5%), column temperature (±2°C), flow rate (±0.1 ml/min) and wavelength was set at 246 and 250 (±2 nm). The results are given in Table-9. indicate that the robustness of the proposed method.

 

Table-9:Change of Acetonitrile concentration (±5%), methanol concentration (±5%), column temperature (±2°C), flow rate (±0.1 ml/min) and wavelength (±2 nm).

Change of mobile phase composition methanol : acetonitrile : water (42:38:20,v/v) Percentage Label claim (%w/w)		Change of mobile phase composition methanol : acetonitrile : water (38:42:20,v/v) Percentage Label claim (%w/w)
Sample-A	99.09	Sample-A	100.88
Sample-B	99.26	Sample-B	98.78
Sample-C	100.52	Sample-C	100.56

 

Change of Wavelength Wavelength = 246, Percentage Label claim (%w/w)		Change of Wavelength Wavelength = 250, Percentage Label claim (%w/w)
Wavelength = 246	101.16	Sample-A	99.45
Sample-B	101.31	Sample-B	100.46
Sample-C	99.66	Sample-C	98.54
Change of column Temperature Column Temperature = 25°C, Percentage Label claim (%w/w)		Change of column Temperature Column Temperature = 29°C, Percentage Label claim (%w/w)
Wavelength = 246	100.2	Sample-A	98.89
Sample-B	99.32	Sample-B	98.93
Sample-C	99.8	Sample-C	100.63

 

Change of flow rate Flow = 0.9 ml/min, Percentage Label claim (%w/w)		Change of flow rate Flow = 1.1 ml/min, Percentage Label claim (%w/w)
Wavelength = 246	98.35	Sample-A	100.01
Sample-B	100.14	Sample-B	99.54
Sample-C	100.4	Sample-C	99.73

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

These approaches were based on the standard deviation of the response and slope. A specific calibration curve was studied using concentration range of 140-260 µg/ml. The linear graph was plotted and correlation coefficient was determined. The residual standard deviation of a regression line or the standard deviation of y-intercept of regression line may be used as the standard deviation. LOD = 3.3σ/slop and LOQ =10σ/slop, where σ = standard deviation of the response. Table-6.

 

Table-6:Limit of detection (LOD) and limit of quantification (LOQ)

Sl.No.	Slope S	y-intercepts
Set-1	37040	12200
Set-2	37027	12490
Set-3	37115	75788
SD	47.50087718	36629.12
Mean	37060.66667	33492.667

 

 

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

                                                      3.3σ

Limit of detection (LOD) =--------------------  = 3.26

                                                         S

 

                                                      10σ

Limit of detection (LOD) =--------------------  = 9.88

                                                          S

 

DISCUSSION:

In this work an analytical RP-HPLC method for assay and determination of content uniformity of Rosuvastatin calcium in tablet formulation was develop and validated. The simple, easy to use, reproduce and selective chromatographic conditions were designed. For example, Thermo scientific C₈ column, 250 x 4.6mm, particle size 5µm with flow rate of 1.0ml/min and detection was monitor at 248nm. The mobile phase consisted of methanol: acetonitrile: water (40:40:20,v/v). The drug substance was easily dissolved from pharmaceutical dosage form by using mobile phase. The tablet disperses in mobile phase and standard, sample solutions were found to be stable in mobile phase. After development of the analytical method, it was validated in accordance with ICH and USP guideline.

 

To determine linearity, calibration graph was obtained by plotting Rosuvastatin calcium concentration against peak area. Linearity was in the concentration range 140-260µg/ml. The regration equation was Y= 37027 X+ 12490, Y= 37040 X+ 12200 and Y= 37115 X+ 75788, where X is concentration in µg/ml and Y is the peak area in absorbance units; the correlation coefficient was 0.999.

 

 

For the determination of content uniformity RSD on the same day ( intra-day) 0.4266% for Sample A, 0.3829% for Sample B, 0.2197% for Sample C and different days (inter-day) 1.68% for Sample A, 1.2632% for Sample B and 1.2312% for Sample C. The mean values of method precision (repeatability) were 99.70% for Sample A, 99.22% for Sample B and 99.80% for Sample C, RSD 0.5985% for Sample A, 0.4941% for Sample B and 0.4439% for Sample C respectively.

 

This HPLC method for assay and determination of content uniformity of Rosuvastatin calcium in tablet formulation was successfully developed and validated. The method was shown to specific, linear, precise, accurate and robust.

 

CONCLUSION:

The developed method represents a fast, cost effective, simple analytical procedure for the estimation of Rosuvastatin Calcium in pharmaceutical dosage forms. The sample preparation and the mobile phase preparation are simple, the analysis time is very short and elution is isocratic. The proposed method has an advantage of low solvent consumption and the large number of sample can be analyzed with excellent precision and accuracy. The method is accurate, precise, rapid and selective for estimation of Rosuvastatin Calcium in tablet dosage form. Hence this method can be applied for routine analysis of Rosuvastatin Calcium in formulation.

 

ACKNOWLEDGMENT:

All authors are grateful to School of Pharmacy, Techno India University, West Bengal for providing research facilities to carry out this work successfully and MSN Laboratories Pvt.Ltd. Telangana, India for providing gift sample of Rosuvastatin Calcium. I am also grateful to my friends and scholars for their kind help from time to time at each and every step of this work.

 

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Accepted on 13.02.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(6): 2886-2892.