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Development of a Selective LC Method for the Determination of Pravastatin Sodium

2006, 64, 157–162

¨ nal&, O. Sagirli A. O Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul University, 34116, Istanbul Turkey; E-Mail: [email protected]

Received: 13 March 2006 / Revised: 18 May 2006 / Accepted: 22 May 2006 Online publication: 20 July 2006

Abstract A novel, simple and rapid stability-indicating high-performance liquid chromatographic (HPLC) method for pravastatin sodium (PRA) was successfully developed and validated for the assay of in tablets. Chromatographic separation was achieved isocratically on a C18 column (150 mm · 4.6 mm) utilizing a mobile phase of methanol-phosphate buffer (pH 7; 0.02 M) (57:43, v/v) at a flow rate of 1.0 mL min)1 with UV detection at 238 nm. A linear response (r = 0.9999) was observed in the range of 1–5 lg mL)1. The method showed good recoveries (100.50%) and the relative standard deviation of intra and inter-day were 1.40%. The method can be used for both quality control assay of pravastatin in tablets and for stability studies as the method separates pravastatin from its degradation products and tablet excipients.

Keywords Column liquid chromatography Validation Quality control Pravastatin

Introduction Pravastatin sodium (PRA), a mono-sodium salt of 1S-(1a(bS,dS,2a6a,8b(R),8a (a)))-l,2,6,7,8,8a-hexahydro b d, 6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy) -1-naphthaleneheptanoic acid (Fig. 1), is an anti-hypercholesterolemic agent having an inhibitory activity against 3-hydroxy-3methylglutaryl coenzyme A (HMG CoA) reductase, the rate-determining enzyme in the cholesterol synthesis. A number of HMG-CoA reductase inhibitors have been introduced into clinical therapy since the introduction of the first substance, lovastatin. These compounds are used for the Original DOI: 10.1365/s10337-006-0843-5 0009-5893/06/08

treatment of hypercholesterolemia. Many pharmacokinetic studies have been performed and different HMG-CoA reductase inhibitors have been compared. 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]. Pravastatin undergoes extensive firstpass extraction in liver. The main metabolite of pravastatin, a 3a-hydroxy isomeric compound has approximately one-tenth to one-fortieth of the HMGCoA reductase inhibitory activity of pravastatin [4], i.e. the enzyme inhibi-

tion is mainly attributable to the parent drug. Several methods including high-performance liquid chromatography with UV detection [5–7], liquid chromatography/tandem mass spectrometry (LC/ MS/MS) [8–10] or gas chromatographyMS [11] have been reported for determination of pravastatin in plasma. Ertu¨rk et al. [12] have recently reviewed the analytical methods for determination of HMG-CoA inhibitors in biological fluids. Only one capillary electrophoretic method [13] has been described for its determination in tablets. As to our best knowledge, no HPLC method has been described for the determination of this drug. Hence, the aim of this study is to develop a selective and sensitive HPLCUV method for the determination of pravastatin sodium in drug substance and formulated products (tablets) suitable for routine quality control analysis and stability tests.

Experimental Chemicals Pravastatin sodium (PRA) and its tablets (10 mg per tablet) were from BristolMyers Squibb (Istanbul, Turkey). All solvents and reagents were of analytical or HPLC grade. HPLC-grade water was prepared by using AquaMAX-ultra water purification system from Young Lin Inst. (Korea).

Chromatographia 2006, 64, August (No. 3/4) 2006 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH

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Limit of Detection and Limit of Quantitation

Fig. 1. Structure of Pravastatin sodium

Instrumentation and Analytical Conditions The analyses were performed on a Thermo Separation Products Liquid Chromatograph (TX, USA) which consisted of a P4000 solvent delivery system equipped with a Rheodyne injection valve with a 20 lL loop, a UV3000 detector set at 238 nm and an SN4000 automation system software. Separations were carried out at room temperature on a Alltima C18 column (1504.6 mm I.D, 5 lm; Alltech Associates, Deerfield, IL, USA), with a guard column (43 mm I.D, Phenomenex, Texas, USA) packed with the same material. The mobile phase consists of methanol: phosphate buffer (pH 7; 0.02 M) (57:43, v=v) at a flow rate of 1.0 mL min)1. Before use, the mobile phase was degassed by an ultrasonic bath and filtered by a Millipore vacuum filter system equipped with a 0.45 lm HV filter. Peak identity was confirmed by comparison of the UV spectra obtained from the scanning dedector.

Standard Solutions and Linearity A stock solution of pravastatin sodium (PRA) (1 mg mL)1, calculated as free base) was prepared in methanol and diluted further with the mobile phase to obtain standard solutions of 50 lg mL)1. By appropriate dilution of the PRA standard solution with the mobile phase, five working solutions ranging between 1–5 lg mL)1 were prepared. The solutions (20 lL) were injected and chromatographed (n = 4) according to the chromatographic conditions previously given. For PRA quantitation, the chromatographic signals were evaluated on the basis of peak area.

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Detection limit was determined by reducing the concentration of a standard solution until the pravastatin peak response was approximately three times greater than the noise. The lowest concentrations assayed where the signal/ noise ratio was at least 10:1, this concentration was regarded as LOQ.

Precision and Accuracy The intra-day and inter-day precision were determined by analyzing the samples of PRA at concentrations of 1, 3 and 5 lg mL)1. Determinations were performed with five replicates on the same day as well as on five different days. A recovery study was performed by adding known ammounts of pravastatin (1, 2 and 3 lg mL)1 ) to sample solution. The actual and measured concentrations were then compared. Sample solution of 2 lg mL)1 was prepared, as described in sample preparation. The mixtures were then analyzed by the proposed method. The experiments were conducted five times.

Robustness Robustness was assessed by testing the susceptibility of measurements to deliberate variation of the analytical conditions. Thus, different columns were used and pH, mobile phase composition, and flow rate were modified.

Specificity Forced degradation or stress testing is undertaken to demonstrate specificity when developing stability-indicating methods, particularly when little information is available about potential degradation products. The stress conditions were as follows: Hydrolysis

Individually, 5 mg PRA (calculated as base) were dissolved in 5 mL methanol in a 10 mL volumetric flask and boiled for 1 h at 80 oC after adding: (a) 5 mL water for neutral hydrolysis, (b) 5 mL 1 N HCl for acid hydrolysis (c) 5 mL 1 N NaOH for basic hydrolysis.

Chemical Oxidation

5 mg PRA (calculated as base) were dissolved in 5 mL methanol in a 10 mL volumetric flask and 100 lL 30% H2O2 solution (v=v) were added and mixed. The solution was boiled for 1 h at 80 oC. Photochemical Degradation

PRA solution was added into a transparent container. The photochemical stability of the PRA was studied by exposing the methanolic stock solution to UV light (k = 366 nm) for 10 h. Thermal Stress

Bulk drug was subjected to dry heat at 105 C for 5 h. Each of the stressed solutions was diluted with the mobile phase to obtain a theoretical concentration of 2 lg mL)1 for PRA (for photochemical stability and thermal stress, 5 lg mL)1 PRA solutions were used). Each solution was analyzed in triplicate. Preparation of Sample Solution

Twenty tablets were individually weighed to get the average weight of the tablets. A sample of the powdered tablets, claimed to contain 100 mg of PRA was transferred to 100 mL volumetric flask. About 75 mL of methanol was added and then extraction was performed mechanically for 20 min and sonicated for 20 more min. The volume was brought to 100 mL with methanol. The content was centrifuged for 10 min at 3000 g, and then a 1 mL aliquot of the supernatant was diluted to 100 mL with the mobile phase. 1 mL of this solution was transferred into a 5 mL calibrated flask and diluted to the volume with the mobile phase. A 20 lL of its aliquot was injected and chromatographed (n = 5).

Results and Discussion Development of the HPLC Method In order to separate PRA, and degradation products produced under stressed conditions, aqueous buffer-methanol phases were used and adjusted to obtain a rapid and simple assay method with a reasonable run time, suitable retention

Chromatographia 2006, 64, August (No. 3/4)

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time and the sharpness of the peak. Satisfactory resolution was obtained using the mobile phase system of methanol:phosphate buffer (pH 7; 0.02 M) (57:43, v=v) with a flow rate of 1 mL min)1. As PRA showed maximum absorption at 238 nm (Fig. 2B), the detector was set at 238 nm. Under the experimental conditions, the chromatogram of PRA (Fig. 2A) showed a single peak of the drug around 5 min. Chromatograms of stressed reaction solutions were given in Fig. 3. They indicate that the developed method was successful to separate the drug and its chromophoric degradation products.

Method Validation The calibration curve was prepared by plotting the peak area of PRA against drug concentration and it was linear in the range of 1–5 lg mL)1. Peak area and concentration was subjected to least square linear regression analysis to calculate the calibration equation and correlation coefficients. The regression equation was found as A = 101682C – 1981 (r = 0.9999, n = 4) (A = aC þ b , where A is the peak area of PRA, a is the slope, b is the intercept and C is the concentration of the measured solution in lg mL)1). The results show that there is an excellent correlation between the peak area and the concentrations of PRA in the range tested. The LOD was found to be 0.1 lg mL)1when the signalto-noise ratios 3:1. Under the developed HPLC conditions, the limit of quantitation was determined to be 0.35 lg mL)1 with a R.S.D. % of less than 1.33%. The intra-day (n=5) and inter-day (n=5, at five different day) reproducibilities expressed as relative standard deviation (RSD) were found to be 0.80–1.26% and 1.08–1.40% respectively, indicating good precision (Table 1). To examine the accuracy of the method, recovery studies were carried out by standard addition method. The percent recovery of the added standard to the assay samples was calculated from: Recovery % = [(Ct Cu Þ=Ca ] 100 where Ct is the total concentration of the analyte found; Cu is the concentration of the analyte present in the formulation; and Ca is the concentration of the pure analyte added to the formulation. The results are shown in Table 2. The average

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Fig. 2. (A) A typical chromatogram of 5 lg mL)1 PRA standard, 20 lL injection, (B) UV spectrum of PRA standard in mobile phase

Table 1. Intra-day and inter-day precision and accuracy of PRA Actual concentration (lg mL)1)

1 3 5

Intra-day (n=5)

Inter-day*(n=5)

Found concentration (lg mL)1)

RSD (%)

Found concentration (lg mL)1)

RSD (%)

1.008 3.009 5.021

1.26 1.12 0.80

0.996 2.994 4.993

1.40 1.52 1.08

* Results of five different days

Table 2. Results of recovery studies by standard addition method

a b c

Amount of drug in tablet (lg)a

Amount of pure drug added (lg)

Total found (mg)b (Mean± S.D.c )

RSD(%)

Recovery of pure drug added (%)

2 2 2

1 2 3

3.01 ± 0.05 4.02 ± 0.02 4.99 ± 0.05

1.65 0.55 1.09

101.00 101.00 99.67

Pravachol tablet (10 mg) Five independent analyses Standard deviation


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percent recoveries obtained as 99.53– 100.50% indicate good accuracy of the method. The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small variations in method conditions. With respect to the composition of HPLC mobile phase, no significant influence in peak area was found when changing the mobile phase composition to 60:40 (methanol:buffer solution) and also 55:45 and flow rates at 1 ± 0.05 mL min1 . The effect of pH was studied by varying ±0.2 pH units (at 7.2 and 6.8 buffer pH). Two analytical columns, one Luna C18 column and the other Shim-Pack CLS-ODS were used to evaluate for the robustness of the method. The assay results indicated that different columns do not lead to significantly different results. Low RSD values were indicative of the robustness of the method (Table 3).

Specificity Forced degradation or stress testing is undertaken to demonstrate specificity when developing stability-indicating methods, particularly when little information is available about potential degradation products. Forced degradation studies may help facilitate pharmaceutical development as well in areas such as formulation development, manufacturing, and packaging, in which knowledge of chemical behavior can be used to improve a drug product [14]. The International Conference on Harmonization (ICH) guideline entitled ‘‘Stability Testing of New Drug Substances and Products’’ requires the stress testing to be carried out to elucidate the inherent stability characteristics of the active substances [15]. The hydrolytic and photolytic stabilities are also required. An ideal stability-indicating method is one that quantifies the drug per se and also resolves its degradation products. The following degradation behaviour of drug was observed during the abovementioned HPLC studies:

Neutral ðwaterÞ Condition Fig. 3. Chromatograms corresponding to PRA solution subjected to (A) neutral hydrolysis (B) alkaline hydrolysis, (C) chemical oxidation, (D) acid hydrolysis, (E) thermal stress and (F) exposure to sunlight

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Upon heating the drug in water at 80 C for 1 h, 10% fall in the original drug peak


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Table 3. Results from robustness experiments Condition

Value

RSD (%) (n=5)

Recovery (%)

Flow rate (mL min)1)

0.95 1.05 55 60 6.8 7.2 Lunaa Shim-Packb

1.33 1.77 1.72 1.65 0.95 1.06 1.39 1.84 1.12

100.86 100.60 101.27 100.94 100.79 101.67 100.60 100.72 100.33

Mobile phase composition (% methanol) pH of buffer used in mobile phase C18 column Fig. 4. Effect of UV light (k=366 nm) on the peak area of PRA during 10 h exposure

Method conditionsc a b c

Luna C18 column (250 4.6 mm I.D, 5 lm; Phenomenex USA) Shim-Pack CLS-ODS column (250 4.0 mm I.D, 5 lm; Shimadzu, Kyoto, Japan) Flow rate= 1.0 mL min)1, methanol-phosphate buffer (pH 7; 0.02 M) (57:43, v=v)

Table 4. Degradation trial for PRA

Fig. 5. The UV spectrum of the chromatographic peak corresponding to the parent drug after subjected to neutral, alkaline hydrolysis, chemical oxidation, thermal stress and exposure to UV light (k=366 nm)

b

Degradation in Alkali

When the PRA solution was exposed to basic hydrolysis the chromatographic peak corresponding to the parent drug diminishes about 90% and showed an additional peak at Rt values of 0.99 and 1.82 (Fig. 3B). The peak of degraded products was well resolved from the drug peak.

Oxidative Conditions

When the PRA solution was exposed to chemical oxidation with H2 O2 , the chromatographic peak corresponding to the parent drug diminishes about 30% and a new signal can be observed, which do not interfere with the parent drug peak (Fig. 3C).

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Time (h)

Recovery (%)

Rt values of degradation products

Acid, 1 N HCl, (80 oC) Base, 1 N NaOH, (80 oC) Neutral hydrolysis, (80 oC) H2 O2 , 30%, (80 oC) Dry heat (100 oC) UV light (k = 366 nm )

1 1 1 1 5 10

Not detected 10.52 87.50 68.25 100.21 58.70

1.53, 1.71 0.99, 1.82 1.53, 3.20 1.54 Not detected 1.60

Table 5. Analysis of PRA in tablets

a

area was observed. Two additional peaks were observed in the chromatograms. The peaks of degraded products were well resolved from the drug peak (Fig. 3A).

Condition

Formulation

Label Claim mg/per tablet)

Amount found (mg) ± S.Db (n=5)

Recovery (%)

RSD (%)

Pravachola

10

10.05 ± 0.09

100.50

1.81

Marketed by Bristol-Myers Squibb Standard deviation

Acidic Condition

On heating the drug solution in 1 M HCl at 80 C for 1 h, the peak corresponding to the parent drug substantially disappeared (Fig. 3D) and two new signals can be observed in the chromatogram. These peaks did not interfere with the signal corresponding to the parent drug, which has a retention time of 5 min.

Thermal Stress

When PRA powder standard drug was subjected to dry heat at 105 C for 5 h, no change in the original chromatogram was observed, i.e. diminish in the original signal or the appearance of new ones (Fig. 3E). Photolytic Conditions

When the PRA solution was irridated with UV light (k=366 nm) during 10 h,


50% of the drug was decomposed and a new small peak appeared in the chromatogram. As can be seen from Fig. 3F, resolution of PRA from its photodegradation product could be achieved. Effect of UV light (k=366 nm) on the peak area of PRA during 10 h is shown in Fig. 4. The peak of the parent drug except acidic condition was checked by its UV spectrum (Fig. 5). In conclusion, forced degradation studies under described conditions showed that PRA solution remained stable under thermal stress but degraded partially with neutral hydrolysis, exposure to UV light (k=366 nm) and chemical oxidation, and totally with acid hydrolysis. Recovery data of the degradation tests for PRA are given in Table 4. The proposed method can be used as a stability-indicating one because the peak of the parent drug, PRA, is not interfered by any other signal in the chromatogram.

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Assay Procedure for Tablets

The proposed method was successfully applied to the analysis of marketed product (Pravachol tablet) and the results obtained are given in Table 5. A placebo formulation (formulation without analyte) was analyzed, concluding from these experiments that excipients included in the drug formulation (povidone K30, lactose monohydrate, croscarmellose stearate, magnesium stearate, cellulose microcrystalline, magnesium oxide lourd, fer oxide jaune) do not interfere with the selectivity of the method.

analysis for determination of pravastatin sodium, without any interference from the excipients and in the presence of its acidic, alkaline, oxidative and photolytic degradation products. Hence, this method is suitable for routine analysis and stability tests of pravastatin.

References

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The proposed liquid chromatographic method provides a simple, accurate, reproducible assay for quantitative

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Conclusion

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