INTRODUCTION:

Pravastatin sodium (PVS) is hexahydro-6-hydroxy-2-methyl-8-(2-methylbutyryloxy)-1-naphthyl-3,5-dihydroxy heptanoate, is one of a class of lipid-lowering compounds, the HMG-CoA reductase inhibitors, which reduce cholesterol biosynthesis. These compounds are used for the treatment of hypercholesterolemia. Pravastatin is characterized as one of the best, due to the hydroxyl group attached to its decalin ring, which results in a greater hydrophilicity than other HMG-CoA reductase inhibitors^1-3.

Literature survey reveals that PVS is official in British Pharmacopœia⁴. Several analytical methods are available for the determination of the latter compound in pharmaceutical dosage forms, these include spectrophotometric method is based on the reduction of ferric to ferrous ions followed by complication reaction with 2,2́-bipyridyl or 1,10-phenanthroline to produce orange red chromogen⁵, high performance liquid chromatography (HPLC) with ultraviolet detector^6-10,

capillary electrophoresis (CE)^11,12, high performance liquid chromatography and capillary electrophoresis¹³, high performance thin layer chromatography (HPTLC)¹⁴ and electro-analytical methods by square-wave adsorptive-stripping voltammetry (SWAdSV)¹⁵, differential puls polarography¹⁶. PVS has been determined by high performance liquid chromatography (HPLC) with laser-induced fluorescence detector¹⁷ and ultraviolet detector^18-22, liquid chromatography/tandem mass spectrometry (LC/MS)^23-25, liquid chromatography/atmospheric-pressure chemical ionization mass spectrometry (LC/APCIMS)²⁶, and gas chromatographic employing chemical ionization mass spectrometry (GC/CIMS)^27-29in biological samples.

Bromocresol purple (BCP)which is a sulphonphthalein dye commonly used as indicator and spectrophotometric reagent. Pravastatin sodium has Chemical formula: C₂₃H₃₅O₇Na and Bromocresol purple has Chemical formula: C₂₁H₁₆Br₂O₅S.

MATERIALS AND METHODS:

Apparatus:

A Jasco V-630 UV–VIS spectrophotometer (Japan) with 1 cm quartz cells. Ultrasonic bath Daihan (China), and stirrer Velp Scientifica (Europe), Sartorius balance, sensitivity 10^-5g.

Chemical regents:

Pravastatin sodium (PVS(C₂₃H₃₅O₇Na, 446.52g/mol from (India),its purity 101.5%. Methanol from Merck (Germany). Bromocresol purple from Merck (Germany). Acetonitrile from fine-chem limited (Mumbai).

Standard Preparation:

Pravastatin sodium stock solutions:

Stock solution 1×10^-2 M of Pravastatin sodium (M_W= 446.52g/mol) was prepared by dissolving 44.652mg of raw material in volumetric flask 10mL and complete the volume with Methanol, then 1mL of the solution was taken to volumetric flask 10mL and diluted with Acetonitrile to give concentration 1×10^-3 M equivalent to 446.52𝜇g/mL. The working standard solutions of Pravastatin sodium were prepared by appropriate dilutions among (50 - 600)𝜇Lof 446.52𝜇g/mL solution in volumetric flask 10mL and added to each one of BCP 5 × 10^-3 M equals to five times of Pravastatin sodium concentration then completed to volume with Acetonitrile to give concentrations between (2.23 – 26.79)𝜇g/mL of Pravastatin sodium.

Reagent stock solution:

Bromocresol purple 5 × 10^-3M was prepared by dissolving 135.055mg of Bromocresol purple (M_W = 540.22g/mol) in volumetric flask 50mL and completing to volume with Acetonitrile.

Calibration Curve:

To construct the calibration curve, five standard solutions for each concentration were prepared and the absorbance was measured of each solution five times.

Sample preparation:

Twenty tablets from Pravastatin-ELSaad 20 (Syrian product) was weighed and finely powdered and an accurate weight equivalent to 20mg (PVS) was weighed accurately, dissolved in volumetric flask 10ml of Methanol, then 1mL of the solution was taken to volumetric flask 10mL and diluted to volume with Acetonitrile. 0.3mL of the last solution was taken to volumetric flask 10mL and added 0.3mL of Bromocresol purple 5 × 10^-3M, then diluted to volume with Acetonitrile, equivalent theoretically to 6.099𝜇g/mL for (PVS).

RESULTS AND DISCUSSION:

Pravastatin sodium forms with Bromocresol purple at 25 ±5ºC yellow ion-pair complex. The stability of the complex was 4 hours. The result solution was scanned in the range of wavelengths 300-550nm against a blank of BCP prepared in Acetonitrile and then measured the absorbance at maximum wavelength 398nm. We studied all the parameters of the colored result solutions to obtain the optimal conditions.

Stability of stock solution:

Time effect on standard stock solution of Pravastatin sodium in Acetonitrile was studied in three different concentrations 1×10^-5, 2×10^-5 and 3×10^-5M. We did not notice any significant changes during the absorption measurement within one month.

Effect of reagent concentration:

To study the effect of reagent concentration on the colored complex solution, we made a series of 10mL of separated volumetric flasks, by adding 0.4mL of Pravastatin sodium 40 ×10^-6M equivalent to 40µM and added between (0.02 – 0.44mL) of (BCP) 1 × 10^-2 M,equivalent to (20 - 440µM) after completing the volume to 10mL by Acetonitrile. The absorbance at 398 nm for every added (BCP) reagent was measured against the blank of Acetonitrile. It was found that the completed colored complex formation in the best condition was 200 µM of (BCP) equivalent to 0.2 mL of (BCP) which equal to five times of Pravastatin sodium concentration, as it is shown in Fig. 1.

Fig. 1: Effect of reagent concentration.

Pravastatin sodium concentration 40 µM.

Correlation ratio by molecular ratio

We have prepared a series of complex solutions PVS-BCP in the medium of the Acetonitrile. The concentration of the reagent changes within the ratio (5.0 × 10^-6 - 1.1 × 10^-4) M while the concentration of Pravastatin sodium was constant in each solution and equal to 5 × 10^-5 M. We measured the absorbance values of these solutions at the wavelength of the maximum absorbance 398nm according to the used reagent percentage (using Acetonitrile as a blank). The absorption changes of the molecular ratio of the reagent to the Pravastatin sodium permitted us to measure correlation ratio. We obtained the curve A = f([BCP]/[PVS]) shown in Fig. 2 where the correlation ratio is (1:1).

Fig. 2: Correlation molecular ratio (1:1).

Correlation ratios by continuous variation:

We have prepared a series of complex solutions PVS-BCP in the medium of the Acetonitrile. The concentration of the reagent and the concentration of Pravastatin sodium changes in solutions between (0.1 – 1) 10^-4M where the sum of both concentrations remains constant and equal to 1 × 10^-4 M.

We measured the absorbance values of these solutions at the wavelength of the maximum absorbance 398 nm according to the used reagent percentage of the formed complex in terms of molecular fraction of Pravastatin sodium. We obtained the curve A = f ([BCP]/{[BCP] + [PVS]}) shown in fig. 3:where the correlation ratio is (1:1).

Fig. 3: Correlation ratio by continuous variation (1:1).

Calculation of formation constant for the (PVS:BCP) complex:

The conditional stability constant (𝐾𝑓) of the ion-pair complexes were calculated from molecular ratio and the continuous variation curves. Data using the following equation^30-32:

Where 𝐴𝑚 and 𝐴are the observed maximum absorbance and the absorbance value when all the Pravastatin sodium is completely associated with Bromocresol purple, respectively. 𝐶𝑀 is the mole concentration of Pravastatin sodium at the maximum absorbance and 𝑛 is the stoichiometry which dye ion associates with Pravastatin sodium. The log 𝐾𝑓 values for PVS-BCP ion-pair association at correlation ratio (1:1) by molecular ratio and continuous variation were 7.96 and 8.05 respectively, where the average log 𝐾𝑓 is 8.01.

Method’s validation:

The validity and suitability of the proposed method was assessed by linearity (evaluated by regression equation), limit of detection (LOD), limit of quantification (LOQ), accuracy (reported as percent %), precision (reported as RSD %), robustness, and Sandellʹs sensitivity.

Linearity:

We studied the linearity of Pravastatin sodium concentrations at the optimal conditions where we made a series of 10mL of separated volumetric flasks, each one contains 1mL of BCP 5 × 10^-3 M, and variable concentrations of PVS stock solution 1 × 10^-3 M, and completed to 10mL with Acetonitrile. Finally, we measured the absorbance at 398nm for each concentration against the blank of BCP in Acetonitrile. Fig.4 presents the PVS spectra. The range of linearity was obeyed to Beer’s law in concentration (2.23 - 26.79) μg/mL and the linearity curve is presented in Fig. 5.

Fig. 4: spectra of (PVS-BCP):

C₁: 4.46 𝜇g/mL, C₂: 8.93𝜇g/mL,

C₃: 13.39𝜇g/mL,C₄: 17.86𝜇g/mL,

C₅: 22.32𝜇g/mL, C6: 26.79𝜇g/mL.

Fig. 5: Calibration curve for (PVS-BCP):

C₁: 4.46 𝜇g/mL, C₂: 8.93 𝜇g/mL,

C₃: 13.39𝜇g/mL, C₄: 17.86 𝜇g/mL,

C₅: 22.32 𝜇g/mL, C₆: 26.79 𝜇g/mL.

n = 5 for each concentration.

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

In spite of the measurement LOD and LOQ, five concentrations were analyzed in five replicates.

LOD and LOQ for Pravastatin sodium were calculated by using the following equations:

;

Where SD, is the standard deviation of y intercepts of regression lines and m is the slope of the calibration curve. The limit of detection (LOD) and limit of quantification (LOQ) were to be 0.158 and 0.479 𝜇g/mL respectively.

Accuracy

To determine the precision and accuracy of the proposed method, five replicates determinations were carried out on three different concentrations of standards (PVS). The validation results are presented in table 1.

Table1: Precision and accuracy for determination of Pravastatin sodium.

Raw material	Theoretical concentration (μg/mL)	Observed concentration (μg/mL)	SD µg/mL	Precision RSD %)	Accuracy (%)	LC = ± [t.SD/(n)½] µg/mL
Pravastatin sodium	8.93	9.05	0.1038	1.147	101.34	9.05 ± 0.129
	13.39	13.33	0.1333	1.000	99.55	13.33 ± 0.165
	17.86	17.96	0.1503	0.837	100.56	17.96 ± 0.187

: mean of five replicated determinations, Accuracy (%) = (observed concentration/theoretical concentration)_˟ 100,

Precision (RSD %) = (standard deviation/mean concentration)_˟100.

LC: Limit of confidence at 95 %; t = 2.78.

Table 2: Intra-day and inter-day precision for determination of Pravastatin sodium.

Intra-day
Found concentration μg/mL						Concentration 𝜇g/mL	Sample
Precision RSD%	* Time III	Precision RSD %	* Time II	Precision RSD%	* Time I	Concentration 𝜇g/mL	Sample
2.51	4.31	3.68	4.44	2.45	4.41	4.46	Pravastatin sodium
1.01	8.99	0.51	8.94	1.38	8.91	8.93
1.62	13.54	1.09	13.44	0.89	13.34	13.39
Inter-day
Found Concentration μg/mL						concentration 𝜇g/mL	Sample
Precision RSD%	*Day III	Precision RSD %	*Day II	Precision RSD%	* Day I	concentration 𝜇g/mL	Sample
1.54	4.40	0.99	4.40	3.68	4.44	4.46	Pravastatin sodium
0.77	8.94	0.58	8.96	0.51	8.94	8.93
0.76	13.50	1.13	13.38	1.09	13.44	13.39

*n = 5.

Precision:

In order to demonstrate the precision of the proposed method, intra-day and inter-day variability studies were performed at three different concentrations (4.46, 8.93, and 13.39) 𝜇g/mL for Pravastatin sodium at the same day and also at three different days. Method efficiency was tested in terms of RSD% for both intra-day and inter-day precisions.

The precision was ascertained by carrying out five replicates of standard Pravastatin sodium under study and the mean was calculated. The results are showed in Table 2. The RSD % results were not more than 3.68 during the determination in one day or three days, where the method is considered very precise.

Robustness:

The robustness of an analytical procedure is a measure of its capacity to maintain unaffected results by a very small variation of some parameters and provides an indication of its reliability during normal usage. The studied variables parameters were slit, scan, speed and the wavelength which performed at concentration (8.93 𝜇g/mL) for Pravastatin sodium Table 3.

Table 3: Robustness test.

Initial conditions

Measured deviation

µg/mL

RSD

Percent (%)

Step size

0.5 nm

0.2 nm

1 nm

8.98

8.96

0.087

0.059

0.97

0.66

100.56

100.34

Scan speed medium

Fast

Slow

8.97

8.96

0.075

0.104

0.84

1.16

100.45

100.34

Wavelength

398 nm

2 nm+

2 nm-

8.93

8.94

0.098

0.100

1.10

1.12

100.00

100.11

*n=5.

Sensitivity Sandell’s and molar absorptivity ε:

Sensitivity of the proposed method for Pravastatin sodium was determined by calculating Sandell’s sensitivity (SS), it was to be SS = 0.0298 µg/cm².The mean molar absorptivity ε was found equal to 29967 L/mol.cm.

RECOVERY:

The recovery was studied by three addition standards of Pravastatin sodium for Pravastatin- ELSaad 20 product. Table 4 presents the recoveries results.

APPLICATION:

The developed method was applied for quantitative determination and identification of Pravastatin sodium in Syrian pharmaceutical (Pravastatin-ELSaad 20) product. The samples were prepared as described in the section of samples preparation and analyzed. Quantitative analysis was done by using calibration curve.

In general, the concentrations of the detected Pravastatin sodium compounds in one product were within the allowed limits under USP legislation³³) the tablet must contain not less than 90.00% and not more than 110.00 % of labeled amount(. So the obtained results are conformed to USP legislation³³.

The relative standard deviations RSD% of the quantitative results were in the range of (2.50 - 3.75)%. Table 5 presents the determination results of Pravastatin sodium in (Pravastatin-ELSaad 20) product, for three different batches.

Table 5: Results of Pravastatin sodium in (Pravastatin-ELSaad 20) tablets.

No. of batches	Pravastatin-ELSaad 20 mg/tab.
No. of batches	Concentration mg/tab	SD mg/tab	RSD %	Per %
1	19.95	0.748	3.75	99.75
2	19.70	0.492	2.50	98.50
3	20.19	0.712	3.53	100.95
Range of concentration mg/tab.	19.70 – 20.19
Range RSD%	2.50–3.75
Range Per %	98.50 - 100.95

Mean for five replicates.

CONCLUSION:

We developed a new method which is suitable for the identification and quantification of Pravastatin sodium in raw material and tablets formulation. A good percentage of recovery shows that the method can be successfully used in routine analyses. The proposed method is simple, sensitive, rapid, specific, a little cost and could be applied for quality control of Pravastatin sodium. The levels of Pravastatin sodium compounds were within the permissible limits set by the USP legislation³³.

ACKNOWLEDGEMENT:

The Ministry of High Education in Syria financially and technically supported this work through department of Chemistry, Faculty of Science, University of Aleppo, Syria.

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Received on 16.01.2020 Modified on 21.03.2020

Research J. Pharm. and Tech. 2021; 14(2):741-746.

DOI: 10.5958/0974-360X.2021.00129.3

Recovery Average %	RSD%	SD µg/mL	Recovery %	Total Found µg/mL	Added µg/mL	Sample µg/mL	Pharmaceutical dosage	Product
99.51	1.81	0.195	99.37	10.77	4.80	6.00	Pravastatin sodium 20 mg/tab.	PravastatinELSaad 20
	2.66	0.318	99.16	11.95	6.00
	0.96	0.127	100.00	13.20	7.20