New Validated Analytical Method for Determination of Simvastatin in Tablet Formulations by RP–HPLC

A B S T R A C T

RP–HPLC method has been developed and validated for the determination of simvastatin (SVS) in pure form and in tablets. Fluvastatin sodium was used as internal standard. The determination was performed on Nucleodur column C8 (250×4.6 mm i.d., 5 µm particle size); the mobile phase consisted of a mixture of phosphate buffer solution (KH₂PO₄ 0.05 M, pH 4.83) and methanol (20:80, v/v), pumped at a flow rate 1.0 mL min⁻¹. Analyte was monitored by UV detection at 230 nm. The mean retention times for fluvastatin and simvastatin were about 4.40 and 9.00 min, respectively. The method was proved linear in the range of 3.5–550.0 µg mL^-1 and exhibited good correlation coefficient (r>0.9998) and excellent mean recovery (100.71–102.18%). Very good limit of detection of 0.63 µg mL^-1 was found for SVS. The method was validated statistically and by recovery studies for linearity, precision, repeatability, and reproducibility. This method was successfully applied to the determination of simvastatin content in four marketed brands from Syria. A good agreement between this method with the pharmacopoeial method for the determination of simvastatin in some real samples demonstrates that the proposed method is suitable to quantify simvastatin in pharmaceutical formulations.

Keywords

Simvastatin, high–performance liquid chromatography, tablets

Introduction

Simvastatin, 2,2'-dimethylbutanoic acid (1S,3R,7S,8S,8aR-(1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-[(2R,4R–(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl]ethyl]-1-naphthalenyl ester is used to reduce the amount of fatty substances such as low-density lipoprotein (LDL) cholesterol and triglycerides in the blood and to increase the amount of high-density lipoprotein (HDL) cholesterol in the blood. Simvastatin is also used to decrease the risk of heart attacks, strokes, and death, and to decrease the need for surgery to improve blood flow in people who have medical conditions that put them at high risk of developing heart and blood vessel problems [1]. The USP describes HPLC method for the estimation of simvastatin [2]. Spectrophotometric, colorimetric, modified glassy carbon electrode, RP-HPLC methods and various voltammetric techniques are reported for determination of simvastatin in pharmaceutical products [3-9]. Simvastatin with other drugs are determined by HPTLC, HPLC with UV detection and spectrophotometry in combined dosage forms [10-32]. Spectrophotometry and liquid chromatography–UV detection are used for determination of simvastatin either alone or in combination with other drugs in biological samples [32-35].

This manuscript deals the development and validation of RP-HPLC method serve as a reliable and rapid method for the determination of simvastatin in pharmaceutical preparations. In the proposed method, the separation and elution of simvastatin and internal standard were during 9 min run time. The precision of the described method has been checked regarding F-test using a pharmacopeia method as reference. The developed method can be serves as an alternative to the methods described in pharmacopeias.

Experimental

I Instrumentation

The L–2000 HPLC system composed of L-2130 binary pump, degasser, L-2350 column oven, autosampler and photodiode array (PDA) detector containing a quartz flow cell (all from Hitachi, Japan). Chromatograms were analysed and integrated automatically using the EzChrom Elite Hitachi Software. Metrohm compact titrator, Sartorius analytical balance, WTW pH meter and Dihan sonicator were used for all preparations.

II Chromatographic Conditions

Nucleodur C8 column (250×4.6 mm, 5 µm particle size, Macherey–Nagel Germany) was used to achieve the separation. The mobile phase was a mixture of a KH₂PO₄ solution (0.05 M–pH 4.83) and methanol (20:80, v/v), filtered through a nylon 0.45 µm membrane filter and degassed by ultrasonic agitation before use. The mobile phase was prepared weekly and was delivered at a flow rate of 1.0 mL min-1. The injection volume was 10 µL with ambient column oven temperature. Isocratic elution of all analytes was monitored at 230 nm.

III Chemicals and Materials

Analytical reagent grade KH₂PO₄ and HPLC grade methanol and water were purchased from Merck (Darmstadt, Germany). Simvastatin (SVS) was supplied by Cipal Ltd, India, and its purity was found to be 100.3%. Fluvastatin sodium (FVS) was used as the internal standard, and obtained from Zhejiang Materials Industry Chemical Group Ltd, China. The chemical structure of simvastatin and fluvastatin sodium is shown in (Figure 1). Tablets were purchased from Syrian market, containing simvastatin 5, 10, 20 and 40 mg per tablet.

IV Standard Solutions

Standard stock solutions of SVS (1.0 mg mL^-1) and FVS (1.0 mg mL^-1) were prepared by direct weighing of standard substance with subsequent dissolution in methanol. From these stock solutions, the working standard solutions were prepared by further diluting of stock solution using methanol. These solutions were stored in the dark at 2–8 °C and were found to be stable for two days.

V Assay Procedure for Dosage Forms

Twenty tablets containing SVS were weighed and finely powdered. Five accurately weighed quantities of the powder equivalent to 40 mg of SVS were transferred into 100 mL separated volumetric flasks. A 80 mL of methanol was then added to each flask and the mixture was sonicated for 10 min. Then, the volume of each mixture was adjusted to 100 mL with methanol. The sample solutions were filtered, and a suitable concentration was prepared in 10 mL volumetric flasks containing 1 mL of the internal standard FVS. Finally, 10 µL of each diluted sample was injected into the column. Peak area ratios of SVS to that of FVS were then measured for the determination. SVS concentrations in the samples were then calculated using peak data and standard curve.

VI Method Validation

The method was validated in accordance with the International Conference on Harmonization (ICH) guidelines [36]. The following validation characteristics were addressed:

i Linearity

A series working standard solutions of SVS (3.5–550.0 µg mL^-1) were prepared by diluting the stock standard solution with the methanol. In each sample 1 mL of FVS was added (100 µg mL^-1 in the final volume). Standard solutions were found to be stable during the analysis time. To construct the calibration curve five replicates (10 µL) of each standard solution were injected immediately after preparation into the column and the peak area of the chromatograms was measured. The calibration graph was constructed for the determination of SVS. Graph of the mean peak area ratio of SVS to that of FVS (R_SVS/FVS) versus the corresponding concentration of SVS (C_SVS) is described by regression equation, R_SVS/FVS=mC_SVS+b (where m is the slope, b is the intercept and C is the concentration of the cited drug in µg/mL) obtained by least-squares method [37].

ii Sensitivity

The sensitivity of the method was determined on the limit of detection (LOD) and limit of quantification (LOQ). LOD) and LOQ of the SVS assay were determined experimentally by calibration curve method. LOD was expressed as the concentration of drug that generated a response to three times of the signal-to-noise (S/N) ratio, and LOQ was 10 times of the S/N ratio, thus, a calculation by using the following equations [38]:

$$ \mathrm{LOD}=\frac{\mathrm{3.3}\times S D\ of\ \ y-intercept}{Slope\ of\ calibration\ curve} $$

$$ \mathrm{LOQ}=\frac{10\times S D\ of\ \ y-intercept}{Slope\ of\ calibration\ curve} $$

iii Precision

The intra-day precision was determined by measuring SVS samples of 3.5, 15, 55, 210 and 550 µg mL^-1 and of FVS at the concentration used in the assay (100 µg mL^-1), from bulk or formulations, injected five times on the same day. The percent relative standard deviation (RSD) of the predicted concentrations from the regression equation was taken as precision (Table 3).

iv Accuracy

To determine the accuracy, assay the same different levels of drug concentration mentioned above were analysed by the proposed method. The percentage relative error and mean percentage recovery of SVS were calculated. Absolute recoveries at each concentration were measured by comparing the response of pretreated standards with the response of standards which had not been subjected to sample pretreatment.

v Specificity

The specificity of the method was established through the study of resolution factor of drug peaks from nearest resolving peak and also among all other peaks. Peak purity of SVS was assessed to evaluate the specificity of the method.

vi System Suitability

The system suitability test was performed to confirm that the LC system to be used was suitable for intended application. A standard solution containing 100 µg mL^-1 of SVS in the presence of 100 µg mL^-1 of internal standard were injected six times. The parameters peak area, resolution (Rs), capacity factor (k'), number of theoretical plates (N), tailing factor (peak symmetry, T) and % RSD were determined.

vii Robustness

Robustness of the proposed method was determined by estimating the effect of a small variation of certain parameters like the percentage of methanol in the mobile phase, the concentration of the aqueous component in the mobile phase and the flow rate. When the effect altering one set of conditions was tested, the other conditions were held constant at optimum values.

Results and Discussion

I Optimization of Chromatographic Conditions

FVS was used as internal standard, to improve the analytical performance and thus control undetermined changes in active pharmaceutical ingredient concentration and instrument response fluctuations, and also to reduce the problem of the many-fold dilution required in the classical batch procedures. The effect of the composition of the mobile phase on the retention time of SVS and the internal standard, FVS, was investigated. Drug peak was eluted fast with the solvent front when many solvents and water was used. When methanol and water were used as mobile phase drug eluted late and had broadened. Results of the effect of methanol percentage in the mobile phase are presented in (Figure 2A). An increase in the percentage of methanol decreases the retention of SVS and FVS. Increasing methanol percentage to more than 85% FVS peak is eluted with the solvent front, while at methanol percentage lower than 75% the elution of SVS peak is seriously delayed. The optimum methanol percentage was found to be 80%.

The effect of pH in the chromatographic elution of the compounds was also investigated by change the concentration values of the aqueous component of the mobile phase from 0.01 to 0.5 M. A concentration value of 0.05 M KH₂PO₄ (pH 4.83) was chosen for the optimum separation of the compounds, as at this concentration, a satisfactory separation and peak asymmetry for the drug was obtained with mobile phase consisting of methanol: 0.05 M KH₂PO₄ pH 4.83 (80:20, v/v), pumped at a flow rate 1.0 mL min-1 (Figure 2B) at 25 oC. Nucleodur C8 column (250×4.6 mm, 5 µm particle size) gave the most suitable resolution between FVS and SVS peaks (>4) according to the pharmacopeial requirement while the other columns (Supelcosil C8, Hichrom 5 C8, Nucleodur C18, ODS Hypersil C18) cause the peaks of the FVS and SVS either to be overlapped or to have unsuitable resolution (<4).

The use of isocratic elution was proven to be short retention time for the SVS peak and helped in the separation of FVS and SVS. Figure 3 shows a typical chromatogram obtained by the proposed RP-HPLC method, demonstrating the resolution of the symmetrical peaks corresponding to FVS and SVS with a flow rate of 1.0 mL min^-1. The retention time of FSV and SVS was about 4.407 and 9.047 min, respectively. The retention time observed allows a fast determination of the drug, which is suitable for QC laboratories. Quantitation was achieved with UV detection at 230 nm based on peak area.

II Method Validation

i System Suitability

The system suitability requirements for 100 µg mL^-1 of SVS in the presence of 100 µg mL^-1 of internal standard was a %RSD for peak area less than 1.30, a peak tailing factor less than 1.1 and R_s greater than 14 between adjacent peaks for all analytes. This method met these requirements. The results are shown in (Table 1).

Table 1: System suitability parameters.

Parameters	FVS	SVS	Preferable levels
Capacity factor (k')	4.47	8.05	2–10
Selectivity (α)	–	1.8	1.0–2.0
Resolution (Rs)	–	14.75	> 1.5
Number of theoretical plates (N)	32753	37516	> 2500
Tailing factor (T)	1.21	1.04	< 1.5
% RSD for six injections	1.26	1.28

ii Linearity

Under the optimal conditions for HPLC, the calibration curve obtained was linear over the concentration range of 3.5–550.0 µg mL^-1. Correlation coefficients (r) of the regression equations were greater than 0.999 in all cases. Characteristic parameters for regression equations and (r) were given in (Table 2). The linearity of the calibration graph was validated by the high value of (r) of the regression.

Table 2: Calibration data for the estimation of SVS by HPLC.

Parameters	Simvastatin
Retention time (min)	9.04
Optimum concentration range (µg mL-1)	3.5–550.0
Regression equation	RSVS/FVS=0.0078CSVS+0.0643
Correlation coefficient (r)	0.9999
Standard deviation of slope	7.07×10-5
Standard deviation of intercept	0.00167
LOQ (µg mL-1)	2.11
LOD (µg mL-1)	0.63

iii Specificity

The specificity of the chromatographic method was determined to ensure separation of SVS and FVS as illustrated in (Figure 3) where complete separation was noticed. The HPLC chromatogram recorded for the analyte in tablets (Figure 4) showed almost no peaks within a retention time range of 10 min. The Figures show that SVS are clearly separated, and the peak of analyte was pure and the excipients in the formulation did not interfere the analyte. Thus, the HPLC method presented in this study is selective for SVS.

iv Limit of Detection and Limit of Quantification

The limit of detection (LOD) and limit of quantification (LOQ) were determined experimentally. LOD was found to be 0.63 µg mL−1, and LOQ was 2.11 µg mL−1 for SVS showed a good sensitivity of the proposed method.

v Accuracy and Precision

The precision and accuracy of the method were evaluated by analysis of five samples for the drug. The proposed method was successfully applied for the analysis of the drug by intra-day (analysis of standard solutions of SVS in replicates of six in the same day), and the recovery experiments were carried out by spiking the already analysed samples with five different concentrations of standard SVS. The percent recoveries obtained were from 100.71 to 102.18%. The standard deviation, relative standard deviation and relative error % of different amounts tested were determined from the calibration curve. Acceptable repeatability of the results within one day was observed. The accuracy of the method is indicated by the excellent recovery, and the precision is supported by the low relative standard deviation, as recorded in (Table 3).

Table 3: Accuracy and precision of the determination of simvastatin by HPLC.

Nominal concentration (mg mL-1)	Intra–day (n=6)
	Mean±SD (mg mL-1)	RSD (%)	Recovery (%)	Relative error (%)
3.50	3.55±0.09	2.53	101.43	1.43
15.00	15.23±0.28	1.84	101.53	1.53
55.00	55.39±0.77	1.39	100.71	0.71
210.00	214.57±2.76	1.29	102.18	2.18
550.00	558.28±5.97	1.07	101.51	1.51

vi Robustness

The robustness of the method was studied by using five replicates at SVS concentration level of 100 µg mL^-1. The obtained results are shown in (Table 4). The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters has proven that the method is robust.

Table 4: Results of robustness study of SVS.

Parameter	SVS
	Assay %	RSD %	tr	T
Change in methanol percentage
75%	100.92	1.29	14.853	1.05
80%	100.57	1.20	9.047	1.04
85%	99.89	1.33	6.0	1.03
Change in concentration of KH2PO4
0.025 M	101.04	1.31	9.133	1.05
0.050 M	100.51	1.24	9.047	1.03
0.075 M	100.21	1.29	9.093	1.04
Change in flow rate
0.9 mL/min	99.91	1.35	10.02	1.04
1.0 mL/min	100.54	1.22	9.047	1.02
1.1 mL/min	100.87	1.20	8.240	1.03

III Application of the Assay

The validity of the proposed method for the determination of simvastatin was assessed by measuring drug concentration of pharmaceutical dosage forms (Figure 4). The results obtained with the proposed method were compared with the pharmacopeial method and are shown in (Table 5) [2]. Mean values were obtained with a Student's t- and F-tests at 95% confidence limits for four degrees of freedom. The results showed comparable accuracy (t-test) and precision (F-test), since the calculated values of t- and F-tests were less than the theoretical data. The proposed method is simple, rapid, accurate, highly sensitive and suitable for the routine quality control without interference from the excipients and additives.

Table 5: Determination of SVS in tablets by the proposed and official methods.

Product a	Pharmaceutical company (country of origin)	%Recoveryb ±SD
		Proposed method	Official method [2]
Adacor 20mg	Adamco Pharma (Syria)	101.30±1.50 t =1.94 F = 3.79	100.87±0.77 t =1.96
Simvacor 40mg	Alfares (Syria)	101.84±2.07 t =1.99 F = 3.08	101.00±1.18 t =1.47
Zocorine 10mg	Asia (Syria)	101.67±1.62 t =2.92 F = 1.26	101.93±1.44 t =2.31
Zukol 5mg	K.C. pharma (Syria)	100.75±0.81 t =2.05 F = 1.60	99.46±0.73 t =1.28

^a The dose is 5, 10, 20 and 40 mg expressed as simvastatin for all products.

^bFive independent analyses. At 95% confidence level t-value is 2.776 and F-value is 6.26.

Conclusion

The proposed HPLC method was successfully applied to the determination of simvastatin in bulk powder and tablet dosage form. The suggested method was found to be simple, accurate, precise and robust. Moreover, the method is fast with respect to analysis time as compared to sophisticated chromatographic techniques. The method provided excellent specificity and linearity with LOQ of 2.11 µg mL⁻¹ and LOD of 0.63 µg mL⁻¹. The sample recoveries from all formulations were in good agreement with their respective label claims, which suggested non-interference of formulations excipients in the estimation. Hence this method can be conveniently used for routine quality control analysis of simvastatin in its pharmaceutical formulation.

Conflicts of Interest

None.

Article Info

Article Type

Research Article

Publication history

Received: Wed 10, Jun 2020
Accepted: Wed 08, Jul 2020
Published: Wed 29, Jul 2020

Copyright

© 2023 Safwan Ashour. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Hosting by Science Repository.
DOI: 10.31487/j.DDA.2010.01.02

Author Info

Safwan Ashour Soulafa Omar

Corresponding Author

Safwan Ashour
Department of Chemistry, Faculty of Science, University of Aleppo, Aleppo, Syria

Figures & Tables

Table 1: System suitability parameters.

Parameters	FVS	SVS	Preferable levels
Capacity factor (k')	4.47	8.05	2–10
Selectivity (α)	–	1.8	1.0–2.0
Resolution (Rs)	–	14.75	> 1.5
Number of theoretical plates (N)	32753	37516	> 2500
Tailing factor (T)	1.21	1.04	< 1.5
% RSD for six injections	1.26	1.28

Table 2: Calibration data for the estimation of SVS by HPLC.

Parameters	Simvastatin
Retention time (min)	9.04
Optimum concentration range (µg mL-1)	3.5–550.0
Regression equation	RSVS/FVS=0.0078CSVS+0.0643
Correlation coefficient (r)	0.9999
Standard deviation of slope	7.07×10-5
Standard deviation of intercept	0.00167
LOQ (µg mL-1)	2.11
LOD (µg mL-1)	0.63

Table 3: Accuracy and precision of the determination of simvastatin by HPLC.

Nominal concentration (mg mL-1)	Intra–day (n=6)
	Mean±SD (mg mL-1)	RSD (%)	Recovery (%)	Relative error (%)
3.50	3.55±0.09	2.53	101.43	1.43
15.00	15.23±0.28	1.84	101.53	1.53
55.00	55.39±0.77	1.39	100.71	0.71
210.00	214.57±2.76	1.29	102.18	2.18
550.00	558.28±5.97	1.07	101.51	1.51

Table 4: Results of robustness study of SVS.

Parameter	SVS
	Assay %	RSD %	tr	T
Change in methanol percentage
75%	100.92	1.29	14.853	1.05
80%	100.57	1.20	9.047	1.04
85%	99.89	1.33	6.0	1.03
Change in concentration of KH2PO4
0.025 M	101.04	1.31	9.133	1.05
0.050 M	100.51	1.24	9.047	1.03
0.075 M	100.21	1.29	9.093	1.04
Change in flow rate
0.9 mL/min	99.91	1.35	10.02	1.04
1.0 mL/min	100.54	1.22	9.047	1.02
1.1 mL/min	100.87	1.20	8.240	1.03

Table 5: Determination of SVS in tablets by the proposed and official methods.

Product a	Pharmaceutical company (country of origin)	%Recoveryb ±SD
		Proposed method	Official method [2]
Adacor 20mg	Adamco Pharma (Syria)	101.30±1.50 t =1.94 F = 3.79	100.87±0.77 t =1.96
Simvacor 40mg	Alfares (Syria)	101.84±2.07 t =1.99 F = 3.08	101.00±1.18 t =1.47
Zocorine 10mg	Asia (Syria)	101.67±1.62 t =2.92 F = 1.26	101.93±1.44 t =2.31
Zukol 5mg	K.C. pharma (Syria)	100.75±0.81 t =2.05 F = 1.60	99.46±0.73 t =1.28

^a The dose is 5, 10, 20 and 40 mg expressed as simvastatin for all products.

^bFive independent analyses. At 95% confidence level t-value is 2.776 and F-value is 6.26.

Figure 1: The chemical structure of A) simvastatin (C₂₅H₃₈O₅=418.56 g/mole) and B) fluvastatin sodium (C₂₄H₂₅FNO₄.Na=433.45 g/mole).

Figure 2: Variation of the retention time of FVS and SVS as a function of A) methanol percentage in the mobile phase and B) flow rate of the mobile phase.

Figure 3: A typical chromatogram of a mixture of FVS (100 μg mL^-1) and SVS (100 μg mL^-1) at retention times 4.407 and 9.047 min, respectively, under optimal conditions.

Figure 4: A typical chromatogram of a mixture of FVS (100 μg mL^-1) and SVS (210 μg mL^-1) in the mobile phase, prepared from Adacor 20 mg tablets, under optimal conditions.

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