INTRODUCTION:

Fluvastatin (FVT) chemically Sodium (3 R*5S*6E)-7-[3-(4-Fluoro-phenyl)-1-(propan-2-yl)-1H-indol-2-yl]-3,5-dihydroxy-6 heptanoate and pharmacologically Fluvastatin sodium - A 3 hydroxy 3 methyl glutaryl coenzyme (HMG-10A) reductase inhibitor is a lipid regulating drug with the actions on plasma lipids similar to those of simvastatin.

It is used to reduce total cholesterol, LDL cholesterol, apolipoprotein B and triglycerides and to increase HDL-cholesterol in the treatment of hyperlipidaemias^1-4.

For estimation of FVT methods such as Spectrophotometric method^5-9, voltammetry¹⁰, Green method¹¹, GC-MS/MS¹², UPLC¹³, Stability indicating HPLC¹⁴, HPLC^15,16, Bio HPLC¹⁷havebeen reported for estimation of FVT alone or in combination with other drugs.

The drug is official in recently published British Pharmacopoeia¹⁷ and Indian Pharmacopoeia¹⁸. Chemical structure of drug is shown in (Fig No 1).

Fig No 1: Chemical structure of Drug molecule

Product with predefined quality will be assured by applying Quality by design concept in the development of pharmaceutical process. QBD concepts are mentioned in ICH guidelines Q8 (R1) (Pharmaceutical development), Q9 (Quality risk management), and Q10 (Pharmaceutical quality system)^19-21. ICH guidelines Q8 (R2) defines QBD as a “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management”²². QBD approach in analytical method summarizes a complete understanding of how the analytical technique attributes and operating conditions affect the analytical performance^{23, 24}. Factors to study in analytical quality by design (AQbD) approach may include the type of analytical technique chosen; reagents used and instrument parameters^{25, 26}.

There are similar advantages of applying QbD principles to analytical methods as to manufacturing processes and product²⁷. A QbD approach can be beneficial in the development of suitable, robust, low cost and eco-friendly (eco-friendly solvent, chemicals) method which is applicable at any stage of the lifecycle of the product. Also some regulatory guidelines have mentioned flexibility of changing analytical method without revalidation if the AQbD approach has been implemented during analytical method development. The first stage of AQbD approach is to fix an analytical target profile (ATP) for the method. ATP defines the goal of the analytical method development process and it is the sign of method performance^{28, 29}. For analytical method validation ICH Q2 (R1) has given various method performance characteristics. Thus a QbD based UV spectrophotometric was developed, QbD approach was implemented with the study of the effect of method input variables on spectral shape, intensity of absorbance, and absorbance maxima λ_max and critical parameters were selected for the proposed method and method was validated as per ICH guidelines Q2 (R1).

MATERIALS AND METHODS:

Instrumentation:

Analysis was performed with a Shimadzu Double beam UV - Visible spectrophotometer (Shimadzu, Kyoto, Japan) with spectral bandwidth of 2 nm and wavelength accuracy of ± 1 nm with 10 mm matched Quartz cells was used. Electronic balance Afcoset balance (The Bombay Burmah Trading corpo Ltd) with accuracy ±0.1 mg Model No. ER 200A was used for weighing and for degassing the solutions Digital Ultrasonic cleaner 1.8 Ltr (Labman scientific Instruments Chennai) was used.

Reagents and Chemicals:

Pharmaceutically pure samples of FVT from Swapnroop Drugs and Pharmaceuticals, Aurangabad Maharashtra was procured as a gift-samples and the commercial formulation Lescol XL containing Fluvastatin 80 mg was procured from local market.

AQbD approach application in method development:

Many literatures have given efforts to elaborate QbD and its significance in the pharmaceutical development^30-32. (Fig No 2) broadly explains the advantages on implementation of QbD. As a AQbD approach the influence of input variable parameters on spectrophotometric analytical method performance was studied shown in diagram (Fig No 3).

Solvent selection:

Research article³³was focused on effect of solvent on the retention time of fluvastatin and pH effect, drug interaction was precisely discussed in the research article^{34, 35}. FVT is freely soluble in 0.1 N NaOH, methanol, soluble in water and poorly soluble in 0.1 N HCl. Although the solubility of the procured drug was studied in methanol, 0.1 N HCl and 0.1 N NaOH, 0.05 N HCl and 0.05 N NaOH separately; and each solution with known conc of analyte were scanned in UV range of 220 nm to 400 nm. The recorded spectra in respective solvent are shown in (Fig No 4, 5 and 6). It was found that suitable solvent is 0.1 N NaOH with respect to more absorbance of FVT in this solvent, low cost, robust and precise in producing result.

Fig No 2: Significance of QbD approach in pharmaceutical development

Fig No 3: Diagram showing the relationship between input variable parameters and the spectrophotometric method performance characteristics

Preparation of stock solutions and standard solutions:

10 mg of pure drug FVT was accurately weighed; and transferred into separate 50 ml volumetric flask. Dissolved into 0.1 N NaOH and volume was made to 50 ml with solvent. Subsequent standard solution of drug with conc 10 μg/ml was prepared by diluting aliquot 0.5 ml of stock solution to 10 ml capacity volumetric flask.

Selection of wavelength and conc range:

From UV spectra it was found that FVT has measurable absorbance at 302.4 nm. From the nature of spectra of FVT working conc range 5 to 40 mcg/ml (μg/ml) was selected in solvent 0.1 N NaOH. Also drug solution was prepared simulated to marketed formulation. Above discussed observations was guided to select critical parameters listed in Table No 1 and by using these; method was validated as per ICH guidelines and by analysing marketed preparations.

Table No 1: Selected critical parameter for UV-VIS analytical method of FVT

Parameter	Selected variables For FVT
Wavelength range	220-400
Wavelength	302.4 nm
Solvent	0.1 N NaOH
Scan speed	Fast
Sampling interval	0.2 nm

Experimental Method for estimation:

Two simple methods calibration curve method and single point absorbance method were applied for estimation of the formulation/dosage form. Calibration curve method employed preparation of six standard solutions from stock solution in the working conc range and measurement of absorbance was recorded at selected wavelength; followed by plot of calibration curve absorbance against conc. Best linear relationship between conc and absorbance was ascertained after three replicates of calibration curve prepared from different stock solutions. The regression line equation Y = mX + c where m is the slope and c is the intercept was used for the calculation of sample concentration. Also instruments quantitation mode was utilised to know the conc of sample/formulation solution.

Single point absorption method:

Standard solution and sample solution were prepared and conc of sample solution was calculated by applying formula

Csample = A sample × (C std / A std)

Where

A _std = absorbance of FVT standard solution at 302.4 nm

C _std = Conc of FVT standard solution

A _sample = absorbance of sample solution

C _sample = Conc of sample solution

Validation of the Method:

To attain analytical target profile of the method, selected critical parameters should meet the performance characteristics of the analytical method. In order to implement AQbD approach an ICH guideline Q2 R1 was applied to study methods performance with critical parameters. The method was validated as per ICH guidelines.

System suitability:

System suitability is studied to demonstrate the suitability of the developed procedure under consideration for the analytical method. Six replicates of working standard solutions with conc 10 mcg/ml of FVT were prepared separately and absorbance was recorded; SD and % RSD of the response was calculated. Stability of the solution was also studied by bench top stability at laboratory temp.

Linearity:

The linearity of an analytical method is its ability to obtain response i.e. absorbance which is directly proportional to the conc of analyte. series of working standard solutions were prepared in conc. range of 5 to 40 mcg/ml (μg/ml) and scanned in 220 to 400 nm range in spectrum mode of the spectrophotometer, absorbance of the standard solutions were recorded at 302.4 nm for FVT in spectrum order. Microsoft office excel software tool was used to obtain the standard regression curve and its analysis as slope, intercept, and correlation coefficient.

Assay of formulation:

Assay was carried out by proposed methods and method was validated by statistical parameters.

Method I: Estimation of formulations by calibration curve method:

Tablets were weighed, powdered and tablet powder equivalent to 10 mg FVT was weighed and transferred into 50 ml volumetric flask. Dissolved into 0.1 N NaOH and volume was made with solvent. Solution was filtered through whatman filter paper No 40 and aliquots of solution were diluted to obtain tablet solution. Solution was scanned in the range of 220 to 400 nm to obtain absorbance of tablet solution at 302.4 nm in spectrum order. Obtained absorbance was utilised to estimate unknown conc of formulation; and results are statistically validated to obtain % of nominal conc, standard deviation and % of RSD.

Method II: Estimation of formulations by single point absorbance method:

Standard solutions of conc 10 mcg/ml and 20 mcg/ml were prepared separately scanned in 220 – 400 nm range; and absorbance at 302.4 nm was recorded. Also above prepared tablet Solution was scanned in 220 to 400 nm range and absorbance was recorded. Equation was applied to determine conc of sample solution; and obtained results are statistically validated to obtain % of nominal conc, standard deviation and % of RSD.

Accuracy and Precision:

The accuracy of an analytical method expresses the closeness of an agreement between test result and true result. Accuracy study was performed by recovery study i.e. standard addition method; diluted standard solution of FVT was prepared and standard solutions added in 50,100 and 150% proportionate to the tablet solution. Three replicates at each of these three levels were prepared and measured and % of conc, SD and RSD of replicates were calculated.

The precision study was carried out by performing assay of tablet six times; also the reproducibility in result was studied by interday and intraday precision.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ of FVT by the proposed method were determined using calibration graph method and calculated as 3.3σ/s and 10 σ/s for LOD and LOQ respectively. σ is the standard deviation of calibration curve and s is the slope of regression line.

Robustness and Ruggedness:

It is measure of capacity of analytical procedure to remain unaffected by small but deliberate variations in method parameter.

RESULTS AND DISCUSSION:

Method development comprises numerous steps of which solvent selection, method for measurement selection are significant one. Uses of eco-friendly solvents have got remarkable weightage due to low cost, readily available and environmentally sound. Drugs underlying analysis must have appreciable solubility in the selected solvent. Chemical structure of the drug and physico-chemical properties available in the literature guides about use of appropriate solvent in the method. Solubility of FVT was studied in each solvent and spectra of drug shown in Fig No 4, 5 and 6. In solvent 0.1 N NaOH drug FVT was shown maximum and consistent absorbance as compare to other solvent.

Fig No 4: UV-VIS spectra of Fluvastatin 20 mcg/ml in 0.1 N HCl

Fig No 5: UV-VIS spectra of Fluvastatin 40 mcg/ml in water

Fig No 6: UV-VIS spectra of Fluvastatin 20 mcg/ml in 0.1 N NaOH

System Suitability:

The absorbances of six replicates of standard solutions (10 mcg / ml) are reported in Table No 2. The SD and % RSD was found for FVT and meets the system suitability requirements indicates method was suitable for analysis.

Table No 2: System suitability study of FVT

Sr No	Conc in mcg/ml	Absorbance of FVT
1	10 mcg/ml	0.221
2	10 mcg/ml	0.217
3	10 mcg/ml	0.219
4	10 mcg/ml	0.219
5	10 mcg/ml	0.227
6	10 mcg/ml	0.222
	SD RSD	0.004560 2.0787

Linearity:

The overlay spectra obtained in linearity study was shown in Fig No 8 and the calibration curve of drug found to be linear in the conc range of 5-40 μg/ml as shown in Fig No 8. The regression equation of line and its parameters slope, r²value and intercept are tabulated in Table No 3, which proved the linear relationship between conc and obtained response.

Fig No 7: UV-VIS overlay spectra of Fluvastatin in 0.1 N NaOH

Fig No 8: Calibration curve of FVT in 0.1 N NaOH solvent

Table No 3: Parameters of regression equation obtained in Microsoft excel

Parameters	FVT
Detection wavelength	302.4
Beer’s law limit (μg/ml)	5 – 40 mcg/ml
Correlation coefficient (r²)	0.9986
Regression equation (y = mx + c)	Y = 0.02761X+0.0145

Fig No 9: UV-VIS spectra of Fluvastatin Tablet solution 8 mcg/ml

Table No 4: Results of assay of formulation by proposed method

Formulation

Drug

Label Claim (mg/Tablet)

Amount found/mg; n=6

Drug

Content %

Std

Deviation

% RSD

Lescol XL

By I method

FVT

80 mg

81.8952

102.442

0.30815

2.0052

Lescol XL

By II method

FVT

80 mg

82.5008

103.126

0.23265

1.5039

Table No 5: Results of accuracy and precision

S. No.	Parameter	Level of study	Data Title	Obtd Data	S.D.	RSD
1	Precision study of FVT	Intraday Precision	Mean of Abs n= 6	0.602 (Abs of 25 mcg/ml)	0.3753	0.6259
1	Precision study of FVT	Interday precision	Mean of Abs n= 6	0.595 ( Abs of 25 mcg/ml)	0.3907	0.6559
2	Accuracy study of FVT	50%	% Purity found	102.25% 112.45 % 108.18 %	4.2461	3.9454
		100%		101.77 % 100.88 % 106.22 %	5.4723	2.3391
		150%		101.75 % 108 .18 % 108.13 %	3.0189	2.8478

Assay:

The assay was carried out by both the methods. The spectra of formulation were shown in Fig No 9. The assay of formulation was carried out by proposed method and calculated % of nominal conc and RSD was found within acceptable limits are summarized in Table No 4. The results indicated applicability of the method for estimation of Formulation.

Accuracy and Precision:

The results of accuracy are summarised in Table No 5, the obtained results were within acceptable limit; and methods accuracy was justified by calculating % drug content. The precision study was carried out by performing assay of solutions; further the reproducibility in result was studied by interday and intraday precision. The values obtained SD and % RSD was shown methods precision and are summarised in Table No 5.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ of AMD and TEL by the proposed method were shown in Table No 6. The standard deviation of the calibration curve was obtained in Microsoft office excel word.

Robustness and Ruggedness:

Robustness was studied and capacity of analytical procedure to measure analyte was remain unaffected by small but deliberate variations in method parameter like variation in the wavelength ± 1 nm, variation in the solvent strength by ± 0.01 N. The analytical method was found rugged during development; similarity the result was produced by performing the analysis by different analyst given in Table No 6.

Table No.6: Results of LOD and LOQ, robustness

Parameters		FVT
LOD mcg/ml		1.08902
LOQ mcg/ml		3.7278
Robustness	± 1 nm	0.4416 (conc 20 mcg/ml)
	0.011 N NaOH 0.09 N NaOH	0.439 (conc 20 mcg/ml) 0.448 (conc 20 mcg/ml)
Ruggedness	Analyst 1	SD ± 0.007797
	Analyst 2	SD ± 0.0085264

CONCLUSION:

The method was developed with eco-friendly and readily available aqueous 0.1 N NaOH solvent. Fluvastatin was estimated from the formulation by both the method and satisfactory results were obtained. The calibration graph method was given reproducible results; however obtained results of both the methods were within acceptable limits given in the pharmacopoeia. The validated method is economical, precise, accurate, robust and reproducible hence can be routinely used for estimation of fluvastatin from the dosage form.

CONFLICT OF INTEREST:

All Authors declared that there is no conflict of interest.

ACKNOWLEDGEMENT:

Authors are thankful to Swapnroop Drugs and Pharmaceuticals, Aurangabad for providing pure drug FVT as a gift sample. Authors are thankful to Principal Dr. R. B. Jadhav, SVPM’S college of Pharmacy Malegaon Dist. Pune for providing facilities for research.

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Received on 03.12.2021 Modified on 05.02.2022

Asian J. Pharm. Ana. 2022; 12(2):87-93.

DOI: 10.52711/2231-5675.2022.00016