INTRODUCTION:

Simvastatin (Fig-1), a methylated analogue of lovastatin, is -(+)-{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]-naphthyl-2,2-dimethyl butanoate. It acts by inhibiting HMG CoA reductase and is used for the treatment of hypercholesterolemia. After oral administration, this prodrug is converted into ß-hydroxy acid of Simvastatin, which is a potent inhibitor of HMG CoA reductase, a key enzyme required for the synthesis of cholesterol in liver.

Fig 1: Structure of Simvastatin

Niacin (Figure -2) chemically designated as Pyridine-3-carboxylic acid used in treating hyperlipedimic condition and has found to effective for increasing serum HDL levels. It has also been demonstrated that this drug lowers the occurrence of coronary heart disease..

Fig 2: Structure of Niacin

The literature review suggested that there were methods available for the estimation of Niacin in pharmaceutical formulations or in bio fluids either alone or in combination with other drugs. The methods include determination of niacin by LC-MS, HPLC, flow injection and spectrofluorimetric analysis [3-13] and Simvastatin [1 ,2] individually with the help of instruments like UV-Spectrophotometer, HPLC and for simultaneous determination there were methods found on UV-Spectrophotometer [14-16] and a very few on HPLC [17, 18]. An attempt was made to develop a method which is precise, simple, robust and most economic method so far for their determination.

MATERIALS AND METHODS:

All the reagents used in the experiment were HPLC grade solvents. After many trails it was observed that a mixture of phosphate buffer, methanol and acetonitrile (45:20:35) at pH 3.5 suits best for the elution. Pure drugs of Simvastatin and Niacin were obtained from Chandra labs, Hyderabad and the tablets were obtained from local pharmacy. The specifications of the instruments and all the conditions maintained were shown in Table-1.

Table 1: Optimized chromatographic conditions

Parameters	Method
Column	Inertsil ODS, RP-18, 250×4.6mm ID, 5µm
Mobile phase	Phosphate buffer: Methanol: Acetonitrile (45:20:35)
Flow rate (ml/min)	1.0
Pump	LC-20 AT Vp Series
Detector	PDA Detector
Operating temperature	20-25 ºC
Selected wave length	220 nm.
Diluent	mobile phase
Injection volume	20 µl
Run time (min)	6
Retention time	2.970 min for SIMVASTATIN 4.747 min for NIACIN

Preparation of Mobile Phase:

A mixture of 45 volumes of Phosphate buffer pH 2.5, 20 volumes of Methanol and 35 volumes of Acetonitrile. The mobile phase was sonicated for 10min to remove gases.

Preparation of Phosphate buffer:

1.36 gm of potassium di hydrogen phosphate (KH₂PO₄) was weighed and dissolved in 100ml of water and volume was made up to 1000ml with water. Adjust the pH to 2.5 using ortho phosphoric acid. The buffer was filtered through 0.45µ filters to remove all fine particles and gases.

Preparation of standard stock solution:

Standard stock solutions of Niacin and Simvastatin (μ/ml) were prepared by dissolving 125 mg of Niacin and 5 mg of Simvastatin in 100 ml of mobile phase. After that filtered the solution using 0.45-micron syringe filter and Sonicated for 5 min. and dilute 100ml with mobile phase.

Calibration of standards:

Based on the label claim of dosage form to be analyzed, different volumes of stock solutions of each drug were transferred accurately to 10ml volumetric flask and diluted to mark to give a series of concentrations of solution equal to 75, 100, 125, 150 and 175 µg/ml of Niacin and 3,4,5,6and 7 µg/ml of simvastatin. These solutions were injected into the optimized condition. The calibration line was plotted with mean peak area on Y-axis and concentration of drugs on X-axis.

Assay:

Preparation of Standard Sample solution:

Standard stock solutions of Simvastatin and Niacin (microgram/ml) were prepared by dissolving 5 mg of Simvastatin and 125 mg of Niacin dissolved in sufficient mobile phase. After that filtered the solution using 0.45-micron syringe filter and Sonicated for 5min and dilute to 100 ml with mobile phase. Further dilutions are prepared in 5 replicates of 5 μg/ml of Simvastatin and 125 μg/ml of Niacin was made by adding 1 ml of stock solution to 10 ml of mobile phase.

Preparation of Tablet Sample solution:

20 tablets (each tablet contains 5 mg of Simvastatin and 125 mg of Niacin) were weighed and taken into a mortar and crushed to fine powder and uniformly mixed. Tablet stock solutions of Simvastatin and Niacin (μg/ml) were prepared by dissolving weight equivalent to 5 mg of Simvastatin and 125 mg of Niacin and dissolved in sufficient mobile phase. After that filtered the solution using 0.45-micron syringe filter and Sonicated for 5 min and dilute to 100ml with mobile phase. Further dilutions are prepared in 5 replicates of 5 μg/ml of Simvastatin and 125 μg/ml of Niacin was made by adding 1 ml of stock solution to 10 ml of mobile phase. The resulting solution was analyzed under optimized condition. The Results were shown in the Table 2.

Table 2: Assay data for Simvastatin and Niacin

Parameter	NIACIN		SIMVASTATIN
Parameter	Standard Area	Sample Area	Standard Area	Sample Area
Injection-1	3116.234	3120.396	164.234	180.009
Injection-2	3046.600	3078.485	167.520	177.820
Injection-3	3094.364	3316.605	161.55	170.251
Injection-4	3316.605	3119.161	170.251	122.16
Injection-5	3109.694	3108.647	159.005	169.38
Average Area	3136.699	3148.659	164.512	163.924
Tablet average weight	300.5 mg		300.5 mg
Standard weight	5 mg		125 mg
Sample weight	300.5 mg		300.5 mg
Label amount	5 mg		125 mg
Std. purity	99.2		99.3
Amount found in mg	4.94 mg		124.60 mg
Assay(%purity)	98.85 %		99.68 %

Method Validation:

The developed method was validated as per ICH guidelines. All the solutions were prepared according to the procedures given under preparation of standard and sample solutions.

RESULTS AND DISCUSSION:

An attempt was made to develop a RP-HPLC method which is accurate, precise, economic and robust for the determination of Simvastatin and Niacin in combined dosage form. Chromatographic separation was achieved on a Inertsil ODS C18 (250×4.6mm ID,5µm) column and the chromatographic conditions were optimized by changing the composition of mobile phase, pH, buffers and their concentration during many trails run on the instrument. Finally a mixture of 45 parts of phosphate buffer, 20 parts of Methanol and 35 parts of acetonitrile at pH 3.5 with detection of 220nm was found suitable for best separation of two components. Different concentrations of standard solutions were injected to predict the linearity range for both drugs. Niacin was found linear between the concentrations 75 to 175 µg/ml and 3 to 7µg/ml for Simvastatin. Their correlation coefficients were found from the linear graph as 0.9946 for Simvastatin and 0.9906 for Niacin. The retention times were found as 2.970 and 4.747 for Simvastatin and Niacin respectively.

The assay (Table -3) was made for the combination tablets by preparing the solutions of concentrations from tablet powder which falls between the linear ranges of standard solutions. The accuracy (Table-4) of the method was checked by performing recovery studies. The recovery was determined at three levels, they are 80, 100 and 120% of the selected concentrations and performing three replicates at each recovery level. The precision (Table-5) of the method was determined from one lot of combined dosage forms. To determine the robustness (Table-6) of the developed method, experimental conditions were purposefully altered and the assay was performed. LOD and LOQ also determined for the developed method, results were showed in table-7. The system suitability parameters were eluted and results were showed in table-8.

Table-3: Assay data for Simvastatin and Niacin

Drug	Amount found (mg)	Labelled amount (mg)	% Assay
Simvastatin	124.60	125	99.68
Niacin	4.94	5	98.8

Table-4: Accuracy studies of Simvastatin and Niacin

Recovery Level	Simvastatin			Niacin
Recovery Level	Amount taken (µg/ml)	Amount recovered (µg/ml)	% Recovery	Amount taken (µg/ml)	Amount recovered (µg/ml)	% Recovery
80%	5	4.91	98.26	125	122.66	99.5
	5	4.91	98.26	125	122.66	99.0
	5	4.91	98.26	125	122.66	99.5
100%	6	5.96	99.38	150	147.65	99.2
	6	5.96	99.38	150	147.65	99.6
	6	5.96	99.38	150	147.65	98.8
120%	7	6.88	98.30	175	178.28	99.3
	7	6.88	98.30	175	178.28	99.6
	7	6.88	98.30	175	29.8	99.3
Mean % recovery: 98.64				Mean % recovery: 99.47

Table-5: Precision studies of Simvastatin and Niacin

S. No	Simvastatin		Niacin
S. No	Rt	Area	Rt	Area
1.	3.053	3189.674	4.907	175.7
2.	3.063	3164.022	4.877	174.027
3.	3.047	3144.053	4.837	176.034
4.	3.027	3109.693	4.810	179.005
5.	2.99	3135.882	4.770	173.804
6	3.003	3146.222	4.770	178.211
Mean	3.0305	3148.258	4.829	176.130
SD	0.0291	26.930	0.056	2.127
%RSD	0.96	0.86	1.16	1.21

Table-6: Robustness data for Simvastatin and Niacin

Parameter

Niacin

Simvastatin

Rt (min)

Tailing factor

Rt (min)

Tailing factor

Flow Rate

0.8 ml/min

1.0 ml/min

1.2 ml/min

3.693

3.053

2.503

1.590

1.559

1.414

5.857
4.907

3.980

1.311

1.524

1.588

Wavelength

218nm

220nm

222nm

2.983

3.053

2.970

1.515

1.559

1.441

4.723

4.907

4.703

1.525

1.524

1.368

Table-7: LOD and LOQ for Simvastatin and Niacin

Parameter	Measured Value (µg/mL)
Parameter	Simvastatin	Niacin
Limit of detection (LOD)	6.53	0.18
Limit of quantification (LOQ)	19.79	0.53

Table-8: System Suitability parameters of Simvastatin and Niacin

Parameters	Niacin	Simvastatin
Retention time (min)	4.747	2.970
Peak Area	3146.222	178.211
Theoretical plates	2931	4362
Tailing factor	1.5	1.42
Resolution	-	7.008

All the validated parameters were checked by applying statistical formulas such as standard and relative standard deviation. The results were found to fall within the prescribed limits.

CONCLUSION:

The fixed dose combination tablet of Simvastatin and Niacin was subjected to simultaneous estimation by reverse phase HPLC method. The proposed HPLC method was validated by evaluation of the validation parameters. The relative standard deviation of slope, correlation coefficient, within and between day repeatability, resolution and tailing factors for this techniques were obtained. Assay parameters used in this study reduced tailing for all peaks and improved the resolution. Highly reliable and cost efficient HPLC method was developed for the quantitative estimation of Simvastatin and Niacin in combined tablet dosage form. The results obtained were reproducible and reliable. The validity and precision of the methods were evident from the statistical and analytical parameters obtained. From the forgoing it is concluded that the method developed is accurate, simple, rapid, specific and precise hence suitable for application in routine analysis of pharmaceutical preparations.

ACKNOWLEDGEMENT:

The authors are thankful to the management of Procadence Institute of Pharmaceutical Sciences and Chandra Labs, Hyderabad for providing samples of Simvastatin and Niacin for the research work.

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Received on 19.01.2016 Modified on 27.01.2016

Asian J. Research Chem. 9(2): Feb., 2016; Page 62-66

DOI: 10.5958/0974-4150.2016.00011.0