Development and Validation of Stability Indicating RP ...

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International Journal of Pharmaceutical Sciences and Nanotechnology

Volume 3 • Issue 2 • July-September 2010

International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 • Issue 2 • July - September 2010

Research Paper Development and Validation of Stability Indicating RP-HPLC Method for Estimation of Voriconazole in Bulk Drug V.V. Wamorkar*, C.S Ramaa1, S.Y. Manjunath , V. Malla Reddy Srikrupa Institute of Pharmaceutical Sciences, Vill: Velkatta, Mdl: Kondapak: RD: Siddipet, Dist: Medak, AP 502277 India. 1

Bharti Vidyapeeth’s College of Pharmacy, Sector-8, Belapur CBD, Navi Mumbai. India.

ABSTRACT: RP-HPLC method has been developed and validated for the determination of voricaonazole in bulk drug.

The developed method is found to be specific, reproducible, and stability indicating. The Hypersil, C18 (250 X 4.6 mm) 5μ column was used and mobile phase consisting of water:acetonitrile to achieve good resolution and retention of the analyte and its impurities. The detector linearity was established from concentrations ranging from 5-100 μg/ml. The method was tested at different levels of specificity and accuracy as per requirements given in ICH guidelines. The molecule was exposed to the stress conditions such as acid, base, oxidation, heat and light as per the recommendations of ICH guidelines. The method was proved to be robust with respect to changes in flow rate, mobile phase composition and allied columns. The proposed method is found to be sensitive, precise, rapid, reproducible, and offers good column life.

KEY WORDS: Voriconazole; HPLC method; Validation; Stability indicating; ICH guidelines

Introduction

It is important to recognize and be aware of the potential for instability in both manufactured and an extemporaneous product. Therefore, it’s important to specify storage conditions and shelf life, to ensure effective stock control and pay attention to the packaging used in dispensing. (J.Winfield 1998).The ICH guidelines have been incorporated as law in the EU, Japan and in US, but in reality, besides these other countries are also using them. The ICH guideline Q1A on Stability Testing of New Drug Substances and Products emphasis that the testing of those features which are susceptible to change during storage and are likely to influence quality, safety and/or efficacy must be done by validating stability indicating testing methods. The ICH guideline Q3B entitled ‘Impurities in New Drug Products’ emphasizes on providing documented evidence that the analytical procedures are validated and suitable for the detection and quantitation of degradation products. (ICH Q3B 1996).

In recent years, stability testing of pharmaceutical products has advanced tremendously from a somewhat haphazard exercise that showed dramatic variation in quality, both within and between various jurisdictions to an operation based on sound scientific principles that show a significant degree of commonality. It is necessary to conduct stability studies to predict, evaluate, and ensure drug product safety. The legal requirements of stability are aimed at ensuring that the product remains within the specifications established to ensure its identity, strength, quality, and purity. Stability is interpreted as the length of time under specific conditions and storage that a product will remain within the predefined limits for all of its important characterization. It is important to remember that stability is fundamental for product characterization, including safety and efficacy. Operating a dynamic, compliant stability program requires more than just knowing and adhering to various regulatory requirements. It also requires management of stability samples, the environmental chambers, and all of the associated documentation. In short, it is important to manage both the operating elements and the regulatory compliances issued to ensure a successful inspection by any regulatory agency.

Any degradation product observed in stability studies conducted at the recommended storage condition should be identified when present at a level greater than (>) the identification thresholds (Table 1). When identification of a degradation product is not feasible, a summary of the laboratory studies demonstrating the unsuccessful efforts to identify it should be included in the degradation products present at a level of not more than (1g

0.05 % Identification Thresholds

< 1mg

1.0 % or 5 µg TDI, Whichever is lower

1mg-10mg

0.5 % or 20 µg TDI, Whichever is lower

>10mg- 2g

0.2% or 2mg TDI, Whichever is lower

>2g

0.1% Qualification Thresholds

< 10 mg

1.0 % or 50 µg TDI, Whichever is lower

10mg-100mg

0.5 % or 200 µg TDI, Whichever is lower

>100 mg- 2 g

0.2% or 3mg TDI, Whichever is lower

>2 g

0.15%

Voriconazole (Figure 1) is a newly approved antifungal agent belonging to azoles class. Azoles, in general work primarily through inhibition of cytochrome P-450-14-α-demethylase, thereby inhibiting the steps in the synthesis pathway for fungal membrane production and growth. Voriconazole interferes with both 14-αdemethylase and 2,4-methylenedihydrolanosterol demethylase, which might explain its increased activity against certain molds (Wellington M, et al, 2001, Patterson BE, et al 1995). Literature revels that there is no report on HPLC method of analysis for this new drug. Therefore, it was considered worthwhile to develop a robust method while subjecting the drug to various stress conditions.

extractions were of Analytical Grade (Apchem Ashonuj Pvt. Ltd.). The HPLC grade chemicals used were Methanol, Acetonitrile, and procured from SD-Fine CHEM Ltd Mumbai. Hydrogen peroxide 6% (Research lab, Mumbai.), and 50% (Qualigens Fine chemicals, Mumbai.) were used for oxidation reactions. All the aqueous reagents were prepared using carbon dioxide free distilled water and whereas the sample solutions in carbon dioxide free double distilled water for HPLC purpose.

Apparatus U.V.-Visible double beam spectrophotometer Shimadzu (1601), along with two matched cuvettes was used. Stock solutions of the samples were prepared in AR grade methanol and used for analysis.The HPLC system consisted of a pump (model Jasco-2080 Plus with intelligent Pump) with manual Rheodyne injector facility operate at 20 μl capacity per injection. The column used was Hypersil, C18 (250 X 4.6 mm) and allied columns of Thermo and Symmetry C18 (250 X 4.6 mm) for validation studies. The detector consisted of UV/VIS (Jasco U.V 2080-Plus) operated at 255 nm. The data were acquired and processed by the use of Borwin software version 2.0.

Chromatographic Conditions

Fig. 1 Structure of voriconazole.

Materials and Methods Pharmaceutical grade voriconazole was supplied as gift sample by Alkem Laboratories Ltd, Mumbai, India and used without further purification. The solvents used for

Different mobile phases were tested in order to find the suitability for separation of the drug and its degradation products. In view of solubility of the drug in methanol and also in acetonitrile, initially the mobile phase containing atleast one of these two was tried. The experimentation was started with 1:1 composition of methanol and water and tried at different levels of combination containing these solvents. The optimal composition of mobile phase was determined as acetonitrile: water (50:50 v/v). The mobile

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International Journal of Pharmaceutical Sciences and Nanotechnology

phase was filtered through 0.45 μm nylon filter, then sonicated for atleast 20 min. The injection volume was 20 μl and mobile phase flow rate was maintained at 1 ml/min.

Validation of RP-HPLC method for voriconazole in bulk drug

Standard Stock Solution

The standard solutions were prepared by dilution of stock solution with methanol in concentration range 5-70 µg/ml, injected in triplicate and chromatogram were taken under conditions mentioned above. The peak area was plotted against corresponding concentrations to obtain the calibration graph. (Figure 2)

Accurately weighed about 100 mg of voriconazole was taken in 100 ml volumetric flask and dissolved in methanol (HPLC grade) and volume was made upto 100 ml (1000 µg/ml solution A). 0.2 ml of solution A was pipetted out in 10 ml volumetric flask and volume was made upto 10 ml (20 µg/ml solution B) with mobile phase containing acetonitrile: water (1:1 v/v). 20 µl of solution B was injected in HPLC system. Corresponding peak area was measured and respective concentration was calculated from the calibration curve. A system suitability test was performed for six replicate standard injections.

Calibration curve (Linearity and Range)

Precision Inter and intra day precision of method was determined by analyzing standard drug in triplicate at three different levels/day for consecutive six days and results were expressed as % RSD. (25 µg/ml was taken as 100%). (Table 2)

Name

RT

Asymmetry

Capacity

Plates

Standard

5.828

1.154

581.833

11707.27

Fig. 2 HPLC Chromatogram of voriconazole (Std. Drug). Table 2 Intra and Inter-day Precision. Intra-Day Precision: (n=6) Parameter Average value SD % RSD Injection repeatability

75 % 18.55 0.3278 1.7 1.538

100 % 25.04 0.4500 1.796 1.2106

125 % 31.196 0.3592 1.151 1.6039

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Accuracy

Dry heat conditions

Recovery studies were carried out by using bulk drug sample at three levels of 75, 100 and 125% (Standard Addition Method). At each level six determinations were performed and the results obtained were compared with expected results. (25 µg/ml was taken as 100%) (Table 3)

The drug sample was kept in dry-heat oven at 80ºC for 48 hours and analyzed. The chromatogram showed neither any decrease in areas of drug sample nor any additional peaks.

Table 3 Data indicating Accuracy of RP-HPLC method for voriconazole in bulk drug. Added amount (µg/ml ) (n=6)

Amount found ±S.D.

% Recovery

18

17.69±0.3278

98.33

25

25.04±0.450

100.16

32

31.96 ±0.3592

99.8272

Forced Degradation

Photo-stability studies The photo-degradation was performed exposing the drug material to 1.2 million lux hours and 200 watt hours/M2. (Bakshi 2002) Accelerated stability studies Voriconazole was kept under different stability conditions and examined at a regular interval of one month. No degradation was found under accelerated stability conditions at 45ºC and 55º C and 75 % RH.

Forced degradation studies were performed under different stress conditions on drug.

Results and Discussions

Acid hydrolysis

Optimization of Chromatographic conditions

The drug (10 mg) was added to 10 ml of 0.1 N HCl. This solution was heated at 80ºC for 1-8 hours. It was found that drug degraded rapidly in 0.1 N HCl and after 8 hours complete degradation was observed. The chromatograms of acid samples showed two additional peaks at Rt 3.210 and 3.843 min after 4 hours at 80ºC temperature.

Several columns were used for optimizing the chromatographic condition. The parameters being focused were improvisation of retention time, separation of degradation products and column life. The Symmetry C18, Thermo C18 and Hypersil BDS C18 columns provided good peak shapes and no peak splitting was observed with any impurity.

Alkali hydrolysis 10 mg of drug was weighed accurately and added in 10 ml of 0.1 N NaOH and heated at 80ºC for 1-8 hours. In alkaline conditions drug was found to decompose rapidly than that of acidic conditions and degradation was completed in 4 hours. Therefore, degradation studies were carried out at 40ºC for 2 hours in 10 ml of 0.01 N NaOH. Two additional peaks other than drug were observed at 3.8 and 4.2 min.

Linearity and range For linearity, all solutions were prepared in mobile phase. All solutions showed linear responses with concentrations level ranging from 5-100μg/ml. The correlation coefficient value was found to be 0.999. The relative response factor (RRF) was determined by slope method. (Figure 3)

Neutral conditions

2500000

The drug (10 mg) was weighed accurately, added in 10 ml of purified water and heated at 60 ºC for 6 hours. In neutral conditions, mild degradation occurred as two new peaks other than that of drug appeared at 3.90 and 4.2 min.

2000000

y = 29554x - 3023.6 R2 = 0.9996

1500000

Series1

1000000

Linear (Series1)

500000

Oxidative conditions In order to find oxidative degradation, different strengths of hydrogen peroxide were tried. Voriconazole (10 mg) was added in 10 ml of 3, 6, 10, 30 % hydrogen peroxide individually and kept at room temperature for 8 hours. No degradation products were observed.

0 0

20

40

60

80

Fig. 3 Calibration Curve indicating linearity of RPHPLC response for drug concentrations.

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Precision

Specificity

The method was found to be precise after six replications for the quantification of voriconazole and its degradation products and %RSD was found to be less than 2.0% (Table 2).

Forced degradation of drug was carried out as per the ICH guidelines (ICH Q2B) by subjecting to acidic, alkaline, oxidative, thermal and photolytic conditions. The acidic, alkaline, neutral and oxidative stress studies were carried out by refluxing drug for 8 hours with 10ml 0.1N HCl (Figure 4), 0.1N NaOH (Figure 5), water (Figure 6) and 30% and 50% hydrogen peroxide (Figure 7), respectively. Under thermal and photo stability conditions, drug was found to be stable.

Accuracy The recovery of drug was determined by spiking drug at three different levels ranging from 75-125 % of the label claim. The recovery range was found to be between 98108% with RSD between 0.35% and 0.45% (Table 3).

Name

RT

Plates

Selectivity

Asymmetry

Capacity

D-I

3.210

11633.805

0.000

1.26

320.000

D-II

3.843

11980.228

1.198

1.205

383.333

Fig. 4 HPLC chromatogram representing acid degradation of drug.

V.V. Wamorkar et al. : Development and Validation of Stability Indicating RP-HPLC Method for…

Name

RT

Plates

Selectivity

Asymmetry

Capacity

D-I

3.820

11118.055

0.000

1.210

381.000

D-II

4.218

12640.097

1.105

1.238

420.833

Voriconazole

5.66

12854.179

1.343

1.205

565.333

Fig. 5 HPLC Chromatogram representing alkaline degradation of drug.

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Volume 3 • Issue 2 • July-September 2010

RT

Plates

Selectivity

Asymmetry

Capacity

D-I

3.900

11397.851

0.000

1.114

389.000

D-II

4.298

13048.391

1.102

1.114

428.833

Voriconazole

5.933

12781.074

1.381

1.051

529.333

Fig. 6 HPLC Chromatogram representing neutral degradation of drug.

Name

RT

Plates

Selectivity

Asymmetry

Capacity

H2O2

2.673

8584.445

0.000

1.234

266.333

Voriconazole

5.540

13006.244

2.0076

1.262

553.000

Fig. 7 HPLC chromatogram representing oxidative degradation of drug. Robustness

Conclusion

The robustness was investigated by varying the conditions w. r. t. change in flow rate (± 0.1 ml/min), mobile phase composition (± 2 ml) and using two allied columns other than regularly used column. The method was found to be robust with respect to flow rate, compositions and column with out any changes in system suitability parameters such as tailing, resolution and theoretical plate.

The chemistry of column employed in present method allows working with aqueous mobile phase showing excellent chromatographic features with respect to voriconazole and related substances. The method also provides selective quantification of voriconazole and impurities without interference from blank, thereby affirming stability- indicating nature of method. The proposed method is highly sensitive, reproducible, specific and rapid. The method was completely validated showing satisfactory data for all the method validation parameters tested. The developed method was robust in the separation

Voriconazole was exposed to different humidity conditions as per ICH guidelines and found to be stable for 6 months.

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and quantification of voriconazole and related substances. This method can be used for the routine analysis of production samples. The information presented herein could be very useful for quality monitoring of bulk samples and as well employed to check the quality during stability studies.

ICH guidelines Q3A and Q3B ICH Harmonized Tripartite guideline: Impurities in new drug substance and drug products 2004.

References

International Conference on Harmonization Steering Committee. Q1A - Stability Testing of New Drug Substance and Products, 1999.

Bakshi M, Singh S. The ICH guidelines in practice: stress degradation studies on Ornidazole and development of validated stability indicating assay. J. of Pharm. and Biomed. Ana. 26 (2001) pp 891-897. Hitchcock5 C, et al.UK-109,496. A novel, wide spectrum triazole derivative for treatment of fungal infection: antifungal activity and selectivity in vitro (abstract F76). Presented at 35th Intersciences Conferences on Antimicrobial Agents and Chemotherapy, Toronto, Canada, sept 28-Oct 1, 1995. ICH guidelines Q2B Validation of Analytical Procedures: Methodology International Conference on Harmonisation of Technical requirements for registration of Pharmaceuticals for Human use, Geneva, Swizterland, 1996.

ICH stability testing of new drug substance and product in: proceeding of the international conference on Harmonization, IFPMA. Geneva. 2000

Patterson BE, Coates PE.1995.UK-109, 496, A novel wide spectrum triazole derivative for treatment of fungal infection; pharmacokinetics in man ,abstr.F78,p-126.In human program and abstract of the 35th Intersciences Conferences on Antimicrobial Agents and Chemotherapy. American society for microbiology, Washington, D.C. Wellington M, Gigliotti F. Update on antifungal agents .Pediatric Infections Diseases J, 2001; 20; pp 993-996. Winfield J., R.M.E.Richards, “Storage and stability of medicine”, Pharmaceutical Practice, 2nd edition, pp 83, 1998.