Development and validation of a HPLC method for the determination of voriconazole in pharmaceutical formulation using an experimental design

Talanta 71 (2007) 1424–1429

Short communication

Development and validation of a HPLC method for the determination of voriconazole in pharmaceutical formulation using an experimental design G. Srinubabu a,∗ , Ch. A.I. Raju a , N. Sarath b , P. Kiran Kumar c , J.V.L.N. Seshagiri Rao b a

b

Center for Biotechnology, College of Engineering, Andhra University, Visakhapatnam 530003, India Department of Pharmaceutical Sciences, College of Engineering, Andhra University, Visakhapatnam 530003, India c Department of Pharmaceutical Analysis, Rolands Institute of Pharmaceutical Sciences, Bhermpur, Orissa, India Received 8 February 2006; received in revised form 28 April 2006; accepted 28 April 2006 Available online 10 January 2007

Abstract A rapid and sensitive RP–HPLC method with UV detection (260 nm) for routine analysis of voriconazole in a pharmaceutical formulation (Vfend® ) was developed. Chromatography was performed with mobile phase containing a mixture of acetonitrile and water (50:50, v/v) with flow rate was of 1.0 ml min−1 . Quantitation was accomplished with internal standard method. The procedure was validated for linearity (correlation coefficient = 0.9999), accuracy, robustness and intermediate precision. Experimental design was used for validation of robustness and intermediate precision. To test robustness, three factors were considered. Percentage of acetonitrile in mobile phase, flow rate and pH ; an increase in the flow rate results in a decrease of the drug found concentration, while the percentage of organic modifier and pH have no important effect on the response. For intermediate precision measure the variables considered were: analyst, equipment and number of days. The R.S.D. value (0.45%, n = 24) indicated a good precision of the analytical method. The proposed method was simple, highly sensitive, precise and accurate and retention time less than 4 min indicating that the method is useful for routine quality control. © 2007 Published by Elsevier B.V. Keywords: Voriconazole; HPLC; Validation; Robustness testing; Experimental design

1. Introduction Voriconazole is designated chemically as (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol1-yl)-2-butanol with an empirical formula of C16 H14 F3 N5 O and a molecular weight of 349.3 (Fig. 1). This antifungal agent is a derivative of fluconazole, having one triazole moiety replaced by a fluoropyrimidine ring and a methyl group added to the propanol backbone [1]. This change in structure results in a potent and wide-spectrum activity against various mold species including Aspergillus [2]. In common with other azole antifungal agents, such as fluconazole and itraconazole, its primary mode of action is by inhibition of the fungal cytochrome P450-dependent 14␣-sterol demethylase, an essential enzyme in ergosterol biosynthesis [3]. Voriconazole shows a greater

∗ Corresponding author. Tel.: +91 891 2844933/91 891 2535316; fax: +91 891 2755324.

0039-9140/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.talanta.2006.04.042

selectivity for the fungal enzyme than for the corresponding rat liver enzyme as compared ketoconazole and itraconazole [4]. Voriconazole is moderately lipophilic (log D7.4 = 1.8) and a single diastereomer with R- and S-stereochemistry by virtue of two chiral centers (2R, 3S) as shown in Fig. 1. Human pharmacokinetic data for voriconazole have been published by Purkins et al. [5]. A few methods were reported for the determination of voriconazole in human serum [6–8], and in aqueous humor [9], reports regarding the determination of impurities [10] and separation of stereoisomers [11] also appear in literature. So far, no systematic HPLC method has been reported for determination of voriconazole in pharmaceutical injections. This paper reports a rapid and sensitive HPLC determination method with UV detection, useful for routine quality control of voriconazole in pharmaceutical formulations. The method was validated by parameters such as linearity, accuracy, precision and robustness. Experimental design was used for validation to evaluate the robustness and intermediate precision.

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From this solution, a working standard solution of 100 ␮g ml−1 of strength was prepared from this dilution of 5, 10, 20, 30, 40, and 50 ␮g ml−1 were made in 10 ml volumetric flasks with the mobile phase here in every standard solution contains 5 ␮g ml−1 of famciclovir internal standard. To carry out the assay aliquots of voriconazole solution equal to 10,20,30,40 and 50 ␮g ml−1 were accurately withdrawn. 2.4. Calibration procedure Fig. 1. Structure of voriconazole.

2. Experimental 2.1. Apparatus Two different HPLC systems were used for the study. The corresponding specifications were provided below. HPLC system 1: the HPLC 1 apparatus was a Waters chromatographic system equipped with an injection valve (Rheodyne 033381); Waters 2487 UV dual ␭ absorbance detector was used. A reversed-phase C18 column (25 cm × 4.6 mm i.d., particle size 5 ␮m). Peak area integration was performed using Breeze software. HPLC system 2: The HPLC 2 apparatus was a Shimadzu chromatographic system with two LC-10AT VP pumps, variable wavelength programmable UV–vis detector SPD-10A, VP CTO, -10 AS VP column oven (Shimadzu) A reversedphase C18 column (25 cm × 4.6 mm i.d., particle size 5 ␮m; YMC, IMC, Wilmington, NC, 28403, U.S.A.) and the HPLC system was monitored by software “Class-VP series version 5.03 (Shimadzu)”. A model SL-164 UV–vis spectrophotometer (Elico Ltd., India) was employed for spectrophotometric study. The experimental design and statistical analysis of the data were performed, by statistica [15] data analysis software system, with using central composite design (CCD) and 16 runs for robustness study. 2.2. Reagents HPLC grade acetonitrile, potassium dihydrogenphosphate (A.R. grade) and orthophosphoric acid (A.R. grade) was obtained from Quiligens (Mumbai, India). Pure sample of drug and internal standard were obtained from Sun pharmaceuticals Ltd., India. Ultra pure water was obtained using a Milli-Q® UFPlus apparatus (Millipore) was used to prepare all solutions for the method. The determination of voriconazole in commercial formulation was carried out (Vfend® ) 250 mg vials, which are to be constituted with water before use.

The calibration curve was plotted with five concentrations of the standard solution 5–50 ␮g/ml solutions and chromatography was repeated thrice for each dilution. The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method. Before injecting solutions, the column was equilibrated for at least 30 min. with the mobile phase flowing through the system. Quantitation was accomplished using an internal standard method. Five determinations were carried out for each solution. Peak area ratios were recorded for all the solutions. The correlation graph was constructed by plotting the peak area ratios obtained at the optimum wavelength of detection versus the injected amounts. 2.5. Chromatographic conditions The mobile phase was a mixture of acetonitrile and water (50:50, v/v) and flow rate was 1.0 mL min−1 . The UV detector wavelength was set at 256 nm and the temperature was set at 23 ± 1 ◦ C. 3. Results and discussion The applied chromatographic conditions permitted a good separation of voriconazole 10 ␮g ml−1 and the internal standard famciclovir 5 ␮g ml−1 (Fig. 2), no drug decomposition was observed during the analysis. The LC method was validated for the parameters reported below. 3.1. System suitability The chromatographic separation, as explained above was carried out with HPLC 1 to evaluate the chromatographic param-

2.3. Preparation of the standard solution An accurately weighed sample (100 mg) of voriconazole reference standard was transferred to a 100 ml volumetric flask and dissolved in triple distilled water to make a solution (1 mg ml−1 ).

Fig. 2. Chromatogram of a solution containing voriconazole at the described chromatographic conditions.

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eters (capacity factor (K ), asymmetry of the peaks, tailing factor and resolution between two consecutive peaks). In Fig. 2 representative chromatogram was shown, which corresponds to the chromatographic separation of these substances. The capacity factor (K ) of the first peak was 3.6 and second was 5.1, the resolution factor was 1.6, results obtained for asymmetry of the peak and tailing factor parameters were the following 0.781 and 0.645, respectively, for IS famciclovir, 0.424 and 0.852 for voriconazole, respectively. It was concluded that the developed method is the optimum according to the studied parameters. The capacity factor obtained is within the accepted values, above 2 for the first peak and less than 10 for the second peak. The tailing factor to be controlled was within the limits established by these guidelines. Lastly, good resolution was obtained between two consecutive peaks in the developed method. Therefore, this method can be applied to its intended purpose with no problems, its suitability being proved. 3.2. Stability of the solution Results obtained in the study of the solution (both reference and sample solution) where it can be noticed that solutions were stable for 48 h, as during this time the results does not decrease below the minimum percentage (96%). 3.3. Linearity Voriconazole and internal standard were chromatographed using the mobile phase. The linearity of peak area responses versus concentrations was studied from 5 to 50 ␮g ml−1 for voriconazole. A linear response was observed over the examined concentration range. The results are tabulated Table 1. 3.4. Accuracy and repeatability Accuracy was studied using three different solutions, containing 10, 20 and 30 ␮g ml−1 of voriconazole. Recovery data are reported in Table 2. The obtained values were within the range of 99.45 and 100.50%, mean R.S.D.% was 0.53%, satisfying the acceptance criteria for the study. The system repeatability was calculated from five replicate injections of voriconazole at the analytical concentration of about 10 ␮g ml−1 ; the R.S.D.% found was 0.79. Table 1 Results of the data analysis for the quantitative determination of voriconazole by the proposed method Statical parameter

HPLC

Concentration range (␮g/ml) Regression equation Correlation coefficient (r) Stand error on estimation (Se ) Standard deviation on slope (Sb ) Standard deviation on intercept (Sa ) Limit of detection (LOD) (␮g/ml) Limit of quantification (LOQ) (␮g/ml)

5–50 y = 0.31949x − 0.08903 0.9999 0.09693 0.00235 0.05383 0.55 2

Table 2 Accuracy for voriconazole Concentration (␮g ml−1 )

Recovery (%)a

R.S.D. (%)

10 20 30

99.45 100.27 99.82

0.25 0.53 0.82

99.84

0.52

Mean a

Mean of five determinations.

3.5. Robustness As defined by the ICH, the robustness of an analytical procedure refers to its capability to remain unaffected by small and deliberate variations in method parameters [12,13]. In order to study the simultaneous variation of the factors on the considered responses, a multivariate approach using design of experiments is recommended in robustness testing. A response surface method was carried out to obtain more information and to investigate the behavior of the response around the nominal values of the factors. Response surface methodology (RSM) has the following advantages: (a) to allow a complete study where all interaction effects are estimated; and (b) to give an accurate description of an experimental region around a center of interest with validity of interpolation [14,16,17]. Generally the large numbers of experiments required by standard designs applied in RSM discourage their use in the validation procedure. However, if an analytical method is fast and requires the testing of a few factors (three or less), a good choice for robustness testing may be the central composite design (CCD), widely employed because of its high efficiency with respect to the number of runs required. A CCD in k factors requires 2k factorial runs, 2k axial experiments, symmetrically spaced at ±α along each variable axis, and at least one center point [18]. Two to five center repetitions are generally carried out in order to know the experimental error variance and to test the predictive validity of the model [19]. In order to study the variables at no more than three levels (−1, 0, +1), the design used in robustness testing of voriconazole was a central composite design (CCD) with α = ±1 [16,20]. Three factors were considered: percentage v/v of acetonitrile (x1 ); flow rate ml min−1 (x2 ) and pH (x3 ). The experimental domain of the selected variables is reported in Table 3. The ranges examined were small deviations from the method settings and the corresponding responses in the peak area ratio considered (Y) were observed. A three-factor CCD requires 16 experiments, including two replicates of the center point. The experimental plan and the corresponding responses are reported in Table 4. All experiments were performed in randomized order to minimize the effects of uncontrolled factors that may introduce a bias on the response. A classical second-degree model with a cubic Table 3 Chromatographic conditions and range investigated during robustness testing Variable

Optimized value

Range investigated

Mobile phase (ACN/buffer) Flow rate (ml min−1 ) pH

50:50 1.0 3.0

45/55–55/45 0.8–1.2 2.5–3.5

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Table 4 Experimental plan for robustness testing and obtained responses No. exp.

Acetonitrile (%)

Flow rate (ml min−1 )

pH

Peak area ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

45 55 45 55 45 55 45 55 45 55 50 50 50 50 50 50

0.8 0.8 1.2 1.2 0.8 0.8 1.2 1.2 0.8 1.2 1.0 1.0 1.0 1.0 1.0 1.0

2.5 2.5 2.5 2.5 3.5 3.5 3.5 3.5 3 3 3 3 2.5 3.5 3 3

2.15 2.18 1.95 1.84 2.13 2.14 1.86 1.92 1.91 2.00 1.95 2.11 2.14 2.16 2.16 2.15

Fig. 3. Representative graphic to show the influence of variables studied in the response of voriconazole.

Fig. 4. Three-dimensional plot of the response surface for Y (found drug peak area ratio). (a) Variation of the response Y as a function of x1 (% acetonitrile) and x2 (flow rate); fixed factor: x3 (pH ) = 3.0. (b) Variation of the response Y as a function of x1 (% acetonitrile) and x3 (pH ) fixed factor: x2 (flow rate) = 1.0 ml min−1 . (c) Variation of the response Y as a function of x2 (flow rate) and x3 (pH ); fixed factor: x1 (% acetonitrile) = 50% v/v.

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Table 5 ANOVA results

Table 6 Experimental plan for intermediate precision testing and obtained responses

Parameter

SS

MS

F

P

No. exp.

Analyst

Instrument

Day

Peak area ratio

ACN (%) Flow rate (ml/min) pH

0.007 0.095 0.002

0.007 0.095 0.003

0.692 8.813 0.023

0.432 0.020 0.883

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Analyst 1 Analyst 1 Analyst 1 Analyst 2 Analyst 2 Analyst 2 Analyst 1 Analyst 1 Analyst 1 Analyst 2 Analyst 2 Analyst 2 Analyst 1 Analyst 1 Analyst 1 Analyst 2 Analyst 2 Analyst 2 Analyst 1 Analyst 1 Analyst 1 Analyst 2 Analyst 2 Analyst 2

HPLC 1 HPLC 1 HPLC 1 HPLC 1 HPLC 1 HPLC 1 HPLC 2 HPLC 2 HPLC 2 HPLC 2 HPLC 2 HPLC 2 HPLC 1 HPLC 1 HPLC 1 HPLC 1 HPLC 1 HPLC 1 HPLC 2 HPLC 2 HPLC 2 HPLC 2 HPLC 2 HPLC 2

Day 1 Day 1 Day 1 Day 1 Day 1 Day 1 Day 1 Day 1 Day 1 Day 1 Day 1 Day 1 Day 2 Day 2 Day 2 Day 2 Day 2 Day 2 Day 2 Day 2 Day 2 Day 2 Day 2 Day 2

2.085 2.087 2.086 2.086 2.087 2.085 2.12 2.15 2.13 2.14 2.23 2.18 2.08 2.082 2.079 2.079 2.085 2.14 2.15 2.11 2.09 2.16 2.11 2.10

experimental domain was postulated. Experimental results were computed by statistica [15]. The coefficients of the second-order polynomial model were estimated by the least squares regression. The equation model for Y (found peak area ratio) was as follows: Y = 2.0950000 + 0.02791x1 − 0.12875x2 − 0.09958x3 −0.0050x12 + 0.05502x22 − 0.011250x32 +0.01875x1 x2 + 0.00750x1 x3 + 0.00625x2 x3 The factor flow rate (x2 ) was significant for the regression model assumed. The model was validated by the analysis of variance (ANOVA). The statistical analysis showed (Table 5) that the model represents the phenomenon quite well and the variation of the response was correctly related to the variation of the factors. Fig. 3 shows the influence of each of the variables studied in the voriconazole as a response where none of them exceeds the limit except the flow rate as shown in the paerto graph. The interpretation of the results has to start from the analysis of the whole model equation rather than from the analysis of the single coefficients. It is important for the response surface study, to consider also the factors whose coefficients are statistically nonsignificant. For this reason the analysis of the response surface plot is necessary. As shown in Fig. 4(a)–(c), the analysis produces three-dimensional graphs by plotting the response model against two of the factors, while the third is held constant at a specified level, usually the proposed optimum. Fig. 3a shows a graphical representation of the isoresponse surface for variation of percentage of ACN (x1 ) and flow rate (x2 ), while the pH (x3 ) is maintained constant at its optimum of 3.0. An increase in the flow rate results in a decrease of the observed peak area ratio (Y), while the percentage of organic modifier had no important effect on the response. Analogous interpretation may be derived by examining Fig. 4c that plots the factors flow rate (x2 ) versus pH (x3 ). In Fig. 4b, where the factor flow rate is maintained constant, the method can be considered robust for the studied experimental response. In conclusion, by examining the ANOVA results and analysis of response surface confirms that Y is not robust for factor x2 , thus a precautionary statement should be included in the analytical procedure for this factor.

The intermediate precision is obtained when the assay is performed by multiple analysts, using multiple instruments, on multiple days, in one laboratory [12]. In order to study these effects simultaneously, a multivariate approach was used. The considered variables included analysts (1 and 2), equipment (HPLC 1 and 2) and days (1 and 2). The considered

3.6. Intermediate precision The intermediate precision is a measure of precision between repeatability and reproducibility and it should be established according to the circumstances under which the procedure is intended to be used [21]. The analyst should establish the effects of random events on the precision of the analytical procedure.

Fig. 5. UV spectrum of voriconazole 10 ␮g ml−1 concentration.

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response was the found drug peak area ratio. A linear model (y = b0 + b1 x1 + b2 x2 + b3 x3 ) was postulated and a 23 full factorial design was employed to estimate the model coefficients. Each experiment was repeated three times in order to evaluate the experimental error variance. The analyses were carried out in a randomized order according to the experimental plan reported in Table 6. The concentration of voriconazole was about 10 ␮g ml−1 . No considered factor was found significant for the regression model assumed. The R.S.D. found (0.45%, n = 24) was acceptable, indicating a good precision of the analytical procedure. The proposed method was compared to the UV spectrophotometry (Fig. 5) to verify the results obtained from HPLC. A calibration equation was obtained in the concentration range of 10–50 ␮g ml−1 and at the wavelength of 256 nm by using water as a blank. The relation between absorbance (A) and concentration of voriconazole (C) was [A = 1854C − 0.0267; r = 0.9998]. The injection vial analysis results were found to be 9.6 ± 0.2 (mean ± S.D.; n = 6) by UV spectrophotometry [22]. High reproducibility and insignificant differences between the two methods were obtained at the 95% probability level for t- and F-test of significance of 1.75 < 2.57 and 1.11 < 5.05, respectively. 4. Conclusion The proposed high-performance liquid chromatographic method has been evaluated over the linearity, precision, accuracy, and specificity and proved to be convenient and effective for the quality control of voriconazole in pharmaceutical dosage forms. The measured signal was shown to be precise, accurate, and linear over the concentration range tested (5.0–50.0 ␮g ml−1 ) with a correlation coefficient better than 0.9999. The proposed method was further compared with an UV procedure proposed by us and proved to be more accurate and precise. Moreover, the lower solvent consumption along with the short analytical run time of 4.0 min leads to a cost effective and environmentally friendly chromatographic procedure. Thus, the proposed methodology is rapid, selective, requires a simple sample preparation procedure, and represents a good procedure of voriconazole in vials. Acknowledgements The authors thank to M/s Sun pharmaceuticals Ltd., India for providing pure drugs to develop the method, Dr. K. Rama

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