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Validated stability-indicating HPLC-DAD method for determination of the recently approved hepatitis C antiviral agent daclatasvir
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ORIGINAL ARTICLE
Validated stability-indicating HPLC-DAD method for determination of the recently approved hepatitis C antiviral agent daclatasvir Méthode de détermination stabilité-indicative par CLHP-barrette de diodes de l’agent antiviral de l’hépatite C récemment approuvé, le daclatasvir M.M. Baker a, D.S. El-Kafrawy b, M.S. Mahrous b, T.S. Belal c,∗ a
Methodology Department, Pharco Pharmaceuticals Company, Alexandria, Egypt Pharmaceutical Chemistry Department, Faculty of Pharmacy, University of Alexandria, Elmessalah, 21521 Alexandria, Egypt c Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, University of Alexandria, Elmessalah, 21521 Alexandria, Egypt b
Received 27 October 2016; accepted 16 December 2016
KEYWORDS HPLC; Diode array detection; Daclatasvir; HCV; Stability-indicating assay; Forced degradation
∗
Summary A comprehensive stability indicating HPLC with diode array detection method was developed for the determination of the recently approved antiviral drug daclatasvir dihydrochloride (DCV) which is used for the treatment of chronic Hepatitis C Virus (HCV) genotype 3 infection. Effective chromatographic separation was achieved using Waters C8 column (4.6 × 250 mm, 5 m particle size) with isocratic elution of the mobile phase composed of mixed phosphate buffer pH 2.5 and acetonitrile in the ratio of 75:25 (by volume). The mobile phase was pumped at a flow rate of 1.2 mL/min, and quantification of DCV was based on measuring its peak areas at 306 nm. DCV eluted at retention time 5.4 min. Analytical performance of the proposed HPLC procedure was thoroughly validated with respect to system suitability, linearity, range, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity range was 0.6—60 g/mL with correlation coefficient > 0.99999. The drug was subjected to forced degradation conditions of neutral, acidic and alkaline hydrolysis, oxidation and thermal degradation. The proposed method proved to be stability-indicating by resolution
Corresponding author. E-mail address: [email protected] (T.S. Belal).
http://dx.doi.org/10.1016/j.pharma.2016.12.005 0003-4509/© 2017 Acad´ emie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Baker MM, et al. Validated stability-indicating HPLC-DAD method for determination of the recently approved hepatitis C antiviral agent daclatasvir. Ann Pharm Fr (2017), http://dx.doi.org/10.1016/j.pharma.2016.12.005
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M.M. Baker et al. of the drug from its forced-degradation products. The validated HPLC method was successfully applied to analysis of the cited drug in its tablets. © 2017 Acad´ emie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.
MOTS CLÉS CLHP ; Détection barette de diode ; Daclatasvir ; HCV ; Methode indicatrice de stabilité ; Dégradation forcée
Résumé Une méthode de détermination par CLHP à barrette de diode du médicament antiviral récemment approuvé, le daclatasvir dichlorhydrate (DCV) utilisé pour le traitement de l’infection par le génotype 3 du virus de l’hépatite C (HCV), est proposée. Une séparation chromatographique efficace a été obtenue en utilisant une colonne Waters C8 (4,6 × 250 mm, taille de particule 5 m) avec élution isocratique avec une phase mobile composée de tampon phosphate à pH 2,5 et d’acétonitrile dans un rapport de 75:25 (en volume). La phase mobile a été pompée à un débit de 1,2 mL/min et la quantification du DCV était basée sur la détection à 306 nm. Le DCV élue à un temps de rétention de 5,4 min. La performance analytique de la méthode CLHP a été validée de fac ¸on approfondie en ce qui concerne la pertinence du système, la linéarité, le range, la précision, la spécificité, la robustesse, les limites de détection et de quantification. La plage de linéarité était de 0,6—60 g/mL avec un coefficient de corrélation > 0,99999. Le médicament a été soumis à des conditions de dégradation forcée d’hydrolyse neutre, acide et alcaline, d’oxydation et de dégradation thermique. La méthode proposée s’est avérée être une indicatrice de stabilité par la résolution du médicament et de ses produits de dégradation forcée. La méthode CLHP validée a été appliquée avec succès à l’analyse du médicament dans des comprimés. emie Nationale de Pharmacie. Publi´ e par Elsevier Masson SAS. Tous droits © 2017 Acad´ r´ eserv´ es.
Introduction Hepatitis C is a chronic infection associated with considerable morbidity and mortality. In recent years, there has been a shift in treatment methods with the discovery and approval of agents that target specific proteins vital for hepatitis C virus (HCV) replication. Daclatasvir (DCV) (Fig. 1) is an inhibitor of HCV nonstructural protein NS5A. DCV is an oral, direct-acting antiviral with potent activity that has been recently approved in many countries worldwide. In vitro data show that DCV exerts a very potent antiviral effect against several HCV genotypes. Clinical trials proved that oral regimen comprising DCV plus sofosbuvir with or without ribavirin is an important option for use in patients with chronic HCV genotype 1, 3 or 4 infection, including patients with advanced liver disease, post-transplant recurrence and HIV-1 co-infection [1—4]. Few methods of analysis for DCV can be found in the scientific literature. Determination of DCV in human plasma has been carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS) [5,6] and UPLC-MS/MS [7]. Recently, a chiral HPLC method has been described for separation of DCV enantiomers [8]. On the other hand, a RP-HPLC with UV detection method has been proposed for assay of DCV tablet dosage form and for dissolution study [9]. To the best of our knowledge, no comprehensive forced degradation and stability-indicating report could be found for the estimation of DCV. The objective of this work is to develop a simple, rapid, selective and reliable HPLC with diode array detection method for the quantitative analysis of DCV in pure and in
tablets dosage form. The method was thoroughly validated and tested for its specificity and stability-indicating properties by resolution of DCV from its forced hydrolytic, oxidative and dry heat degradation products.
Experimental Instrumentation The HPLC system with diode array detector (DAD) consisted of Waters 2695 Alliance (quaternary pump, vacuum degasser heater, diode array and multiple wavelength detector Waters 2996) connected to a computer loaded with MILLENNIUM32 Login Version 4.00 Software. An automated injector model SM7 with loop capacity 100 L was used. The column used was Waters C8 (4.6 × 250 mm, 5 m particle size). Filtration of solutions prior injection to the column was done using cellulose nitrate membrane filters (0.45 m pore size) (Sartorius Stedim Biotech GmbH, Goettingen, Germany).
Materials and chemicals Daclatasvir dihydrochloride was kindly donated by Pharco Pharmaceuticals Co., Alexandria, Egypt. HPLC-grade acetonitrile (Carbon Group Ringaskiddy, Cork, Ireland), HPLCgrade methanol (Lab-scan, Gliwice, Poland), reagent-grade potassium dihydrogen phosphate, dipotassium hydrogen phosphate and phosphoric acid (Scharlau Chemie S.A., Sentmenat, Spain), analytical grade of hydrochloric acid (BDH
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Figure 1. Chemical structure of DCV. Structure chimique du DCV.
Laboratory Suppliers, Poole, England), sodium hydroxide (El-Nasr Pharmaceutical Chemicals Co., Egypt), 50% hydrogen peroxide (Chemajet Chemical Co., Egypt) and high purity distilled water were used. Pharmaceutical formu® lation assayed in the study was Daclanork tablets (Mash for Pharmaceutical and Cosmetics Industries-Mash Premiere, Badr City, Egypt) labeled to contain 65.92 mg daclatasvir dihydrochloride equivalent to 60 mg daclatasvir per tablet.
General procedure Chromatographic conditions The column used was Waters C8 (4.6 × 250 mm, 5 m particle size). A mobile phase system consisting of mixed phosphate buffer pH 2.5 and acetonitrile in the ratio of 75:25 (by volume) was used. The mixed phosphate buffer was prepared by dissolving 2.5 g potassium dihydrogen phosphate and 2.5 g dipotassium hydrogen phosphate into 800 mL distilled water, solution was adjusted to pH 2.5 using 0.1 M phosphoric acid solution then it was completed to 1000 mL with distilled water. The separation was achieved with isocratic elution. The flow rate was 1.2 mL/min. The injection volume was 25 L. The eluant was monitored by DAD from 190 to 400 nm, and chromatograms were extracted at 306 nm. All determinations were performed at ambient temperature.
Preparation of standard solutions and construction of calibration graph DCV stock solution (600 g/mL) was prepared in HPLC-grade methanol. The working standard solutions were prepared by dilution of aliquots of the stock solution with the mobile phase to reach the concentration range 0.6—60 g/mL. Triplicate injections were made for each concentration and chromatographed under the previously described chromatographic conditions. Peak areas were plotted against the corresponding concentrations to construct the calibration graph, and the linear regression equation was calculated.
Assay of tablets dosage form ®
Ten Daklanork tablets were finely powdered. An accurate weight of the powdered tablets equivalent to 60 mg DCV was extracted into 50 mL methanol with the aid of sonication
for 10 min then filtered into a 100 mL-volumetric flask. The residue was washed with 2 × 20 mL portions of methanol, washings were added to the filtrate and finally the solution was completed to volume with methanol. Aliquots of the prepared tablet solutions were diluted with the mobile phase to obtain final concentrations within the specified range. The prepared sample solution was chromatographed using the previously described conditions. Recovered concentrations were calculated from the corresponding external standard (simultaneously prepared standard DCV solution). Standard addition technique was applied by spiking sample solution with portion of DCV stock standard solution to obtain total concentration within the previously specified range then treated as previously described. Recovered concentration was calculated by comparing the drug response in tablet solution with the increment response obtained after addition of the standard.
Preparation of forced-degradation solutions Forced degradation studies were carried out on DCV standard according to the following conditions.
Neutral (water) degradation DCV solution was treated with 2 mL of distilled water. Solution was placed in a water bath at 90 ◦ C for 1 h, then they were diluted to volume with the mobile phase to obtain a final concentration of 15 g/mL.
Acidic and basic degradation DCV solutions were treated with 2 mL of 1 M hydrochloric acid solution or 1 M sodium hydroxide solution. Acidic degradation solutions were placed in a water bath at 90 ◦ C for 1 h, while basic degradation solutions were placed in a water bath at 90 ◦ C for 30 min. After the specified time intervals, all solutions were neutralized by adjusting the pH to 7.0 and then diluted to volume with the mobile phase to obtain a final concentration of 15 g/mL.
Oxidative degradation DCV solution was treated with 2 mL of H2 O2 5% solution. Solution was placed in a water bath at 90 ◦ C for 1 h. After the specified time interval, the solution was diluted to
Please cite this article in press as: Baker MM, et al. Validated stability-indicating HPLC-DAD method for determination of the recently approved hepatitis C antiviral agent daclatasvir. Ann Pharm Fr (2017), http://dx.doi.org/10.1016/j.pharma.2016.12.005
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volume with the mobile phase to obtain a final concentration of 15 g/mL.
Dry heat degradation An amount of DCV powder (100 mg) was kept in an oven at 120 ◦ C for 12 h. After the specified time, the powder was dissolved in methanol, and aliquots of the methanolic stock were diluted to volume with the mobile phase to obtain a final concentration of 15 g/mL. After the previous treatments, all forced degradation solutions were filtered with a 0.45 m membrane filter prior to injection to the column.
Results and discussion Optimization of chromatographic conditions An isocratic liquid chromatographic method coupled with diode array detection was developed to provide a suitable and reliable procedure for the routine quality control analysis of DCV. The developed method was carefully designed and optimized to separate the investigated drug from its forced degradation products. The most important aspect in LC method development is the achievement of sufficient resolution of the target drug from all other compounds present in the sample with acceptable peak symmetry in a reasonable analysis time. To achieve this goal, several experiments were carried out to optimize both the stationary and mobile phases. For optimization of the stationary phase, several reversed phase columns such as Thermo-C18 (4.6 × 250 mm, 5 m particle size), Waters-C8 (4.6 × 250 mm, 5 m particle size) and Venusil XBP CN (4.6 × 250 mm, 5 m particle size) were tested. The best clear separation between all the eluting peaks (DCV and its degradation products), better system suitability parameters, symmetric DCV peak and relatively short retention time was attained using the
Waters-C8 (4.6 × 250 mm); hence, it became the column of choice for this study. Several mobile phases were evaluated using various proportions of different aqueous phases and organic modifiers. The best mobile phase combination was mixed phosphate buffer pH 2.5 and acetonitrile in the ratio of 75:25 (by volume). Methanol was tried as an organic modifier and mixed phosphate buffer was substituted by other aqueous phases such as water. In these trials, DCV suffered from delayed elution and/or peak asymmetry. It is generally preferable to use a mobile phase pH that is at least 1—2 units away from the pKa values of the analyte. DCV pKa values are 6.09 and 11.15 [10]. Consequently, mobile phase pH 2.5 was considered ideal for analysis of DCV. Besides, changing the buffer pH to 5.0 or 7.5 caused longer retention times for DCV. Flow rate was kept constant at 1.2 mL/min and all determinations were performed at ambient temperature. The DAD enhances the power of HPLC and is an elegant option for assessing method specificity by monitoring the recorded spectra of the eluted peaks especially during running forced degradation samples. DCV exhibits considerable absorption all over the range 210—350 nm with a maximum at 306 nm (Fig. 2). Accordingly, all chromatograms in this study were recorded at 306 nm. Quantification was based on peak area measurement. The previously described chromatographic conditions showed well-defined symmetric DCV peak at about 5.42 ± 0.04 min. Fig. 3 shows a typical chromatogram for DCV using the optimized conditions. Retention factor (k ) is 4.42, tailing factor is 1.15 and column performance (apparent efficiency) expressed by the number of theoretical plates (N) is 6957.
Stability-indicating aspects Forced degradation experiments were carried out on standard DCV to produce the possible relevant degradation products and test their chromatographic behavior using the developed method. Hydrolytic, using neutral (water), strong acidic (1 M HCl) and strong basic (1 M NaOH) media,
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Figure 3. Representative HPLC chromatogram of DCV (15 g/mL). Chromatogramme représentatif du DCV (15 g/mL).
oxidative (5% H2 O2 ) and dry heat degradation experiments were conducted, and the resulting chromatograms were compared with those obtained from a standard untreated solution of the drug. No strong signs of degradation of DCV were observed after heating for 1 h at 90 ◦ C in neutral conditions. DCV peak appeared at its specific retention time with area almost identical (98.4%) to that of a standard of the same concentration and the chromatogram did not show any extra peaks (Fig. 4a). Likewise, in case of degradation under acidic conditions after heating with 1 M HCl solution at 90 ◦ C for 1 h, no significant change in the peak area of the parent compound was detected. DCV peak appeared at its specific retention time with area almost identical (98.6%) to that of a standard of the same concentration with only a tiny degradation peak at 3.05 (Fig. 4b). Excellent resolution (Rs = 12.6) was observed between DCV and such degradation peak. The situation was quite different in case of degradation under basic conditions. About 18% decrease in DCV peak area as well as the appearance of a major degradation peak at 3.05 min in addition to several minor peaks at 1.97, 2.37, 4.20, 4.52, 4.76, 7.45 and 9.55 min were clearly noticed after heating with 1 M NaOH solution at 90 ◦ C for 30 min (Fig. 4c). Resolution was calculated between DCV and both the preceding and succeeding degradation peaks
at 4.76 and 7.45 min, respectively. Resolution was found not less than 3.3 which implied an adequate baseline separation between DCV and the nearest degradation peaks. Oxidative degradation with 5% H2 O2 at 90 ◦ C for 1 h caused about 7.5% reduction in the peak area of DCV as well as the appearance of a minor degradation product peak at 4.75 min was noticed (Fig. 4d). Again resolution value between DCV and the degradation peak was 2.9 which implied a sufficient separation between both peaks. It is noteworthy to mention that the extra peak eluting at about 2.25 min is due to the reagent (hydrogen peroxide) which usually elutes with the solvent peak (Fig. 4d). Finally, DCV was found stable under thermal (dry heat) degradation conditions. Its peak appeared at its exact retention time with area comparable to that of standard of the same concentration, and the chromatogram did not show any extra peaks (Fig. 4e). In all these forced degradation experiments, DCV was successfully separated from the degradation products as confirmed by the resolution values calculated for each chromatogram (Rs > 1.5). Results of the forced degradation of DCV in different conditions are summarized in Table 1. DCV Peak purity was checked in all the forced degradation chromatograms by using the diode array detector. Purity angle is a measure of spectral homogeneity. The obtained purity angles were
Table 1 Summary of the forced-degradation results of DCV. Récapitulatif des essais de dégradation forcée. Degradation type
Degradation conditions
% remaining of DCV
Retention times of the degradation peaks (min)
Neutral (water) Acidic (1 M HCl) Basic (1 M NaOH)
90 ◦ C for 1 h 90 ◦ C for 1 h 90 ◦ C for 30 min
98.4 98.6 81.6
Oxidation (5% H2 O2 ) Thermal (dry heat)
90 ◦ C for 1 h 120 ◦ C for 12 h
92.4 99.6
— 3.05 1.97, 2.37, 3.05, 4.20 4.52, 4.76, 5.44, 9.55 4.75 —
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Figure 4. HPLC chromatograms of DCV (15 g/mL) after exposure to neutral (a), acid (b), base (c), oxidative (d) and thermal dry heat degradation (e). Chromatogrammes of DCV (15 g/mL) après exposition en milieu neutre (a), acide (b), basique (c), oxydatif (d) et à la chaleur sèche (e).
within the purity threshold limits which confirm that DCV peaks are homogenous and pure in all the analyzed samples subjected to forced degradation conditions.
Validation of the proposed method The proposed HPLC method was validated as per the International Conference on Harmonization (ICH) guidelines on validation of analytical procedures [11].
Linearity and concentration range The linearity of the proposed HPLC procedure was evaluated by analyzing a series of different concentrations for the analyte (n = 9). The linear regression equation was generated by least squares treatment of the calibration data. Under the optimized conditions described above, the measured peak areas were found perfectly proportional to concentrations
of DCV. Table 2 presents the analytical parameters including linear regression equation, concentration range, correlation coefficient, standard deviations of the intercept (Sa ), slope (Sb ) and standard deviations of residuals (Sy/x ). Regression analysis shows good linearity as indicated from the correlation coefficient values (> 0.99999). In addition, deviation around the slope can be further evaluated by calculating the RSD % of the slope (Sb %) which was found to be less than 0.2%. Linearity can be further guaranteed by the analysis of variance (ANOVA) test. The most important statistic in this test is the F-value which is the ratio of the mean of squares due to regression divided by the mean of squares due to residuals. High F value reveals an increase in the mean of squares due to regression and a decrease in the mean of squares due to residuals. The greater the mean of squares due to regression, the steeper is the regression line. The smaller the mean of squares due to residuals, the less is the scatter of experimental points around the regression line.
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Table 2 Analytical parameters for the determination of DCV using the proposed HPLC-DAD method. Paramètres analytiques pour la détermination du DCV par la méthode CLHP-barrette de diode.
the developed method for estimation of the analyte in its bulk form (Table 3).
Parameter
Result
Concentration range (g/mL) Intercept (a) Sa Slope (b) Sb RSD % of the slope Correlation coefficient (r) Sy/x F Significance F LOD (g/mL) LOQ (g/mL)
0.6—60 3540 3262 81395 119 0.15 0.99999 7277 471715 3.66 × 10−18 0.12 0.40
Specificity is defined as the ability to access unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products and matrix components [11]. Method specificity was already demonstrated by the successful resolution of the intact drug from its forced degradation products (Fig. 4). DAD played an essential role to confirm the purity of DCV peaks in all forced degradation chromatograms.
Consequently, regression lines with high F values (low significance F) are much better than those with lower ones. Good regression lines show high values for both r and F statistical parameters (Table 2).
Limits of detection and quantification The limit of detection (LOD) is defined as the concentration of the analyte which has a signal-to-noise ratio of 3:1. For the limit of quantification (LOQ), the ratio considered is 10:1. The LOD and LOQ values of DCV were calculated using the signal-to-noise ratio method and are given in Table 2. Both LOD and LOQ values confirm the sensitivity of the proposed HPLC method.
Precision and accuracy The within-day (intra-day) precision and accuracy for the proposed method were studied at three concentration levels for DCV using three replicate determinations for each concentration within the same day. Similarly, the between day (inter-day) precision and accuracy were tested by analyzing the same three concentrations using three replicate determinations repeated on three days. Recoveries were calculated using the corresponding regression equation and they were satisfactory. The percentage relative standard deviation (RSD %) and percentage relative error (Er %) did not exceed 0.9% proving the high precision and accuracy of
Specificity
Robustness The robustness of an analytical procedure is a measure of its capability to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage [11]. Robustness was examined by making small changes in acetonitrile content in the mobile phase (± 1%), pH of mixed phosphate buffer solution (± 0.3), flow rate (± 0.05 mL/min) or working wavelength (± 2 nm) and recording the chromatograms of DCV standard solution. These variations did not have significant effects on the measured response (peak area) or retention time of DCV. RSD% for the measured peak areas using these variations did not exceed 3.85%. Table 4 shows the effects of the studied variations on the peak area and retention time of DCV.
Stability of solutions The stability of DCV working solutions as well as sample solutions in the mobile phase was examined, and no chromatographic changes were observed within 12 h at room temperature. Also, the stock solutions prepared in HPLCgrade methanol were stable for at least 2 days when stored under refrigeration at 4 ◦ C. Retention times and peak areas of the drug remained unchanged and no significant degradation was observed during these periods.
Assay of tablets dosage forms The developed method was applied for the assay of the drug ® in its commercial pharmaceutical formulation (Daklanork tablets). The active ingredient eluted at its specific retention time, and no interfering peaks were observed from any of the excipients of the assayed tablets. The diode array detection enables peak purity verification where no signs of
Table 3 Precision and accuracy for the determination of DCV in bulk form using the proposed method. Précision and justesse pour la détermination du DCV principe actif. Nominal value
Within-day
(g/mL)
Found ± SDa (g/mL)
RSD(%)
Er (%)
Found ± SDa (g/mL)
RSD (%)
Er (%)
3 15 45
3.01 ± 0.02 14.93 ± 0.02 45.15 ± 0.05
0.67 0.13 0.11
0.33 −0.47 0.33
3.01 ± 0.03 14.92 ± 0.03 45.20 ± 0.07
1.00 0.20 0.16
0.33 −0.53 0.44
a
Between-day
Mean ± standard deviation for three determinations.
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M.M. Baker et al. Table 4 Robustness evaluation for the determination of DCV (15 g/mL) using the proposed HPLC-DAD method. Évaluation de la robustesse pour la détermination du DCV (15 g/mL) par la méthode proposée par CLHP-barrette de diode. Parameter
Peak area ± SD
RSD %
Retention time ± SD
Acetonitrile ratio in mobile phase ± 1% pH of mixed phosphate buffer solution ± 0.3 Flow rate ± 0.05 mL/min Wavelength ± 2 nm
1270888 ± 8640
0.68
5.328 ± 0.81
1283387 ± 19949
1.55
5.409 ± 0.73
1264263 ± 48722
3.85
5.324 ± 0.17
1251087 ± 6753
0.54
5.419 ± 0.04
Figure 5.
®
Purity plot of DCV in its commercial dosage form (Daklanork tablets). ®
Tracé de pureté du DCV dans sa forme commercialisée (Daklanork comprimés).
Table 5 Application of the proposed HPLC-DAD method ® to the analysis of DCV in its dosage form (Daklanork tablets). Application de la méthode proposée par CLHP-barrette de diode pour l’analyse du DCV dans sa forme ® d’administration (Daklanork comprimés).
%Recovery ± SDa RSD% a
External standard
Standard addition
99.63 ± 0.31 0.31
99.22 ± 0.49 0.50
Mean ± standard deviation for five determinations.
co-elution from any of the excipients were detected (Fig. 5). Recoveries were calculated using both external standard and standard addition methods. Assay results revealed satisfactory accuracy and precision as indicated from % recovery, SD and RSD% values (Table 5). It is evident from these results that the proposed method is applicable to the assay of DCV in tablets with minimum sample preparation and satisfactory level of accuracy and precision.
Conclusion This study described a simple, specific and reliable HPLCDAD procedure for the assay of DCV in tablets dosage form.
To our present knowledge, no attempts have been made yet for the forced degradation or the stability indicating assay of DCV in pure and in its dosage form by any analytical methodology, furthermore; only very few methods were reported for the determination of DCV in human plasma. A significant advantage in the study is the separation of DCV from the degradation peaks obtained by various forced degradation experiments in relatively short run time. Reliability was guaranteed by testing various validation parameters of the method and the successful application to commercial tablet dosage form. The method can thus be used for routine analysis, quality control, and for checking quality during stability studies of pharmaceutical preparations containing the drug.
Disclosure of interest The authors declare that they have no competing interest.
References [1] Gentile I, Borgia F, Coppola N, Buonomo AR, Castaldo G, Borgia G, et al. The first of a new class of drugs targeted against hepatitis C virus NS5A. Curr Med Chem 2014;21(12):1391—404. [2] McConachie SM, Wilhelm SM, Kale-Pradhan PB. New directacting antivirals in hepatitis C therapy: a review of sofosbuvir, ledipasvir, daclatasvir, simeprevir, paritaprevir, ombitasvir and dasabuvir. Expert Rev Clin Pharmacol 2016;9(2):287—302.
Please cite this article in press as: Baker MM, et al. Validated stability-indicating HPLC-DAD method for determination of the recently approved hepatitis C antiviral agent daclatasvir. Ann Pharm Fr (2017), http://dx.doi.org/10.1016/j.pharma.2016.12.005
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Stability indicating HPLC-DAD determination of daclatasvir [3] McCormack PL, Lyseng-Williamson KA. Daclatasvir in hepatitis C virus infection: a guide to its use in the EU. Drugs Ther Perspect 2016;32(2):42—9. [4] Keating GM. Daclatasvir: a review in chronic hepatitis C. Drugs 2016;76(14):1381—91. [5] Jiang H, Zeng J, Kandoussi H, Liu Y, Wang X, Bifano M, et al. A sensitive and accurate liquid chromatography-tandem mass spectrometry method for quantitative determination of the novel hepatitis C NS5A inhibitor BMS-790052 (daclastasvir) in human plasma and urine. J Chromatogr A 2012;1245:117—21. [6] Jiang H, Kandoussi H, Zeng J, Wang J, Demers R, Eley T, et al. Multiplexed LC-MS/MS method for the simultaneous quantitation of three novel hepatitis C antivirals, daclatasvir, asunaprevir, and beclabuvir in human plasma. J Pharm Biomed Anal 2015;107:409—18. [7] Rezk MR, Bendas ER, Basalious EB, Karim IA. Development and validation of sensitive and rapid UPLC-MS/MS method for
9
[8]
[9]
[10] [11]
quantitative determination of daclatasvir in human plasma: application to a bioequivalence study. J Pharm Biomed Anal 2016;128:61—6. Srinivasu G, Nagesh Kumar K, Thirupathi C, Lakshmi Narayana C, Parameswara Murthy C. Development and validation of the chiral HPLC method for daclatasvir in gradient elution mode on amylose-based immobilized chiral stationary phase. Chromatographia 2016;79(21):1457—67. Ashok Chakravarthy V, Sailaja BBV. Method development and validation of assay and dissolution methods for the estimation of daclatasvir in tablet dosage forms by reverse phase HPLC. Eur J Pharm Med Res 2016;3(7):356—64. http://www.drugbank.ca/ (last time accessed: October 1st , 2016). ICH. Validation of analytical procedures: text and methodology, Q2(R1). International Conference on Harmonisation; 2005.
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ORIGINAL ARTICLE
Validated stability-indicating HPLC-DAD method for determination of the recently approved hepatitis C antiviral agent daclatasvir Méthode de détermination stabilité-indicative par CLHP-barrette de diodes de l’agent antiviral de l’hépatite C récemment approuvé, le daclatasvir M.M. Baker a, D.S. El-Kafrawy b, M.S. Mahrous b, T.S. Belal c,∗ a
Methodology Department, Pharco Pharmaceuticals Company, Alexandria, Egypt Pharmaceutical Chemistry Department, Faculty of Pharmacy, University of Alexandria, Elmessalah, 21521 Alexandria, Egypt c Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, University of Alexandria, Elmessalah, 21521 Alexandria, Egypt b
Received 27 October 2016; accepted 16 December 2016
KEYWORDS HPLC; Diode array detection; Daclatasvir; HCV; Stability-indicating assay; Forced degradation
∗
Summary A comprehensive stability indicating HPLC with diode array detection method was developed for the determination of the recently approved antiviral drug daclatasvir dihydrochloride (DCV) which is used for the treatment of chronic Hepatitis C Virus (HCV) genotype 3 infection. Effective chromatographic separation was achieved using Waters C8 column (4.6 × 250 mm, 5 m particle size) with isocratic elution of the mobile phase composed of mixed phosphate buffer pH 2.5 and acetonitrile in the ratio of 75:25 (by volume). The mobile phase was pumped at a flow rate of 1.2 mL/min, and quantification of DCV was based on measuring its peak areas at 306 nm. DCV eluted at retention time 5.4 min. Analytical performance of the proposed HPLC procedure was thoroughly validated with respect to system suitability, linearity, range, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity range was 0.6—60 g/mL with correlation coefficient > 0.99999. The drug was subjected to forced degradation conditions of neutral, acidic and alkaline hydrolysis, oxidation and thermal degradation. The proposed method proved to be stability-indicating by resolution
Corresponding author. E-mail address: [email protected] (T.S. Belal).
http://dx.doi.org/10.1016/j.pharma.2016.12.005 0003-4509/© 2017 Acad´ emie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.
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M.M. Baker et al. of the drug from its forced-degradation products. The validated HPLC method was successfully applied to analysis of the cited drug in its tablets. © 2017 Acad´ emie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.
MOTS CLÉS CLHP ; Détection barette de diode ; Daclatasvir ; HCV ; Methode indicatrice de stabilité ; Dégradation forcée
Résumé Une méthode de détermination par CLHP à barrette de diode du médicament antiviral récemment approuvé, le daclatasvir dichlorhydrate (DCV) utilisé pour le traitement de l’infection par le génotype 3 du virus de l’hépatite C (HCV), est proposée. Une séparation chromatographique efficace a été obtenue en utilisant une colonne Waters C8 (4,6 × 250 mm, taille de particule 5 m) avec élution isocratique avec une phase mobile composée de tampon phosphate à pH 2,5 et d’acétonitrile dans un rapport de 75:25 (en volume). La phase mobile a été pompée à un débit de 1,2 mL/min et la quantification du DCV était basée sur la détection à 306 nm. Le DCV élue à un temps de rétention de 5,4 min. La performance analytique de la méthode CLHP a été validée de fac ¸on approfondie en ce qui concerne la pertinence du système, la linéarité, le range, la précision, la spécificité, la robustesse, les limites de détection et de quantification. La plage de linéarité était de 0,6—60 g/mL avec un coefficient de corrélation > 0,99999. Le médicament a été soumis à des conditions de dégradation forcée d’hydrolyse neutre, acide et alcaline, d’oxydation et de dégradation thermique. La méthode proposée s’est avérée être une indicatrice de stabilité par la résolution du médicament et de ses produits de dégradation forcée. La méthode CLHP validée a été appliquée avec succès à l’analyse du médicament dans des comprimés. emie Nationale de Pharmacie. Publi´ e par Elsevier Masson SAS. Tous droits © 2017 Acad´ r´ eserv´ es.
Introduction Hepatitis C is a chronic infection associated with considerable morbidity and mortality. In recent years, there has been a shift in treatment methods with the discovery and approval of agents that target specific proteins vital for hepatitis C virus (HCV) replication. Daclatasvir (DCV) (Fig. 1) is an inhibitor of HCV nonstructural protein NS5A. DCV is an oral, direct-acting antiviral with potent activity that has been recently approved in many countries worldwide. In vitro data show that DCV exerts a very potent antiviral effect against several HCV genotypes. Clinical trials proved that oral regimen comprising DCV plus sofosbuvir with or without ribavirin is an important option for use in patients with chronic HCV genotype 1, 3 or 4 infection, including patients with advanced liver disease, post-transplant recurrence and HIV-1 co-infection [1—4]. Few methods of analysis for DCV can be found in the scientific literature. Determination of DCV in human plasma has been carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS) [5,6] and UPLC-MS/MS [7]. Recently, a chiral HPLC method has been described for separation of DCV enantiomers [8]. On the other hand, a RP-HPLC with UV detection method has been proposed for assay of DCV tablet dosage form and for dissolution study [9]. To the best of our knowledge, no comprehensive forced degradation and stability-indicating report could be found for the estimation of DCV. The objective of this work is to develop a simple, rapid, selective and reliable HPLC with diode array detection method for the quantitative analysis of DCV in pure and in
tablets dosage form. The method was thoroughly validated and tested for its specificity and stability-indicating properties by resolution of DCV from its forced hydrolytic, oxidative and dry heat degradation products.
Experimental Instrumentation The HPLC system with diode array detector (DAD) consisted of Waters 2695 Alliance (quaternary pump, vacuum degasser heater, diode array and multiple wavelength detector Waters 2996) connected to a computer loaded with MILLENNIUM32 Login Version 4.00 Software. An automated injector model SM7 with loop capacity 100 L was used. The column used was Waters C8 (4.6 × 250 mm, 5 m particle size). Filtration of solutions prior injection to the column was done using cellulose nitrate membrane filters (0.45 m pore size) (Sartorius Stedim Biotech GmbH, Goettingen, Germany).
Materials and chemicals Daclatasvir dihydrochloride was kindly donated by Pharco Pharmaceuticals Co., Alexandria, Egypt. HPLC-grade acetonitrile (Carbon Group Ringaskiddy, Cork, Ireland), HPLCgrade methanol (Lab-scan, Gliwice, Poland), reagent-grade potassium dihydrogen phosphate, dipotassium hydrogen phosphate and phosphoric acid (Scharlau Chemie S.A., Sentmenat, Spain), analytical grade of hydrochloric acid (BDH
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Figure 1. Chemical structure of DCV. Structure chimique du DCV.
Laboratory Suppliers, Poole, England), sodium hydroxide (El-Nasr Pharmaceutical Chemicals Co., Egypt), 50% hydrogen peroxide (Chemajet Chemical Co., Egypt) and high purity distilled water were used. Pharmaceutical formu® lation assayed in the study was Daclanork tablets (Mash for Pharmaceutical and Cosmetics Industries-Mash Premiere, Badr City, Egypt) labeled to contain 65.92 mg daclatasvir dihydrochloride equivalent to 60 mg daclatasvir per tablet.
General procedure Chromatographic conditions The column used was Waters C8 (4.6 × 250 mm, 5 m particle size). A mobile phase system consisting of mixed phosphate buffer pH 2.5 and acetonitrile in the ratio of 75:25 (by volume) was used. The mixed phosphate buffer was prepared by dissolving 2.5 g potassium dihydrogen phosphate and 2.5 g dipotassium hydrogen phosphate into 800 mL distilled water, solution was adjusted to pH 2.5 using 0.1 M phosphoric acid solution then it was completed to 1000 mL with distilled water. The separation was achieved with isocratic elution. The flow rate was 1.2 mL/min. The injection volume was 25 L. The eluant was monitored by DAD from 190 to 400 nm, and chromatograms were extracted at 306 nm. All determinations were performed at ambient temperature.
Preparation of standard solutions and construction of calibration graph DCV stock solution (600 g/mL) was prepared in HPLC-grade methanol. The working standard solutions were prepared by dilution of aliquots of the stock solution with the mobile phase to reach the concentration range 0.6—60 g/mL. Triplicate injections were made for each concentration and chromatographed under the previously described chromatographic conditions. Peak areas were plotted against the corresponding concentrations to construct the calibration graph, and the linear regression equation was calculated.
Assay of tablets dosage form ®
Ten Daklanork tablets were finely powdered. An accurate weight of the powdered tablets equivalent to 60 mg DCV was extracted into 50 mL methanol with the aid of sonication
for 10 min then filtered into a 100 mL-volumetric flask. The residue was washed with 2 × 20 mL portions of methanol, washings were added to the filtrate and finally the solution was completed to volume with methanol. Aliquots of the prepared tablet solutions were diluted with the mobile phase to obtain final concentrations within the specified range. The prepared sample solution was chromatographed using the previously described conditions. Recovered concentrations were calculated from the corresponding external standard (simultaneously prepared standard DCV solution). Standard addition technique was applied by spiking sample solution with portion of DCV stock standard solution to obtain total concentration within the previously specified range then treated as previously described. Recovered concentration was calculated by comparing the drug response in tablet solution with the increment response obtained after addition of the standard.
Preparation of forced-degradation solutions Forced degradation studies were carried out on DCV standard according to the following conditions.
Neutral (water) degradation DCV solution was treated with 2 mL of distilled water. Solution was placed in a water bath at 90 ◦ C for 1 h, then they were diluted to volume with the mobile phase to obtain a final concentration of 15 g/mL.
Acidic and basic degradation DCV solutions were treated with 2 mL of 1 M hydrochloric acid solution or 1 M sodium hydroxide solution. Acidic degradation solutions were placed in a water bath at 90 ◦ C for 1 h, while basic degradation solutions were placed in a water bath at 90 ◦ C for 30 min. After the specified time intervals, all solutions were neutralized by adjusting the pH to 7.0 and then diluted to volume with the mobile phase to obtain a final concentration of 15 g/mL.
Oxidative degradation DCV solution was treated with 2 mL of H2 O2 5% solution. Solution was placed in a water bath at 90 ◦ C for 1 h. After the specified time interval, the solution was diluted to
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volume with the mobile phase to obtain a final concentration of 15 g/mL.
Dry heat degradation An amount of DCV powder (100 mg) was kept in an oven at 120 ◦ C for 12 h. After the specified time, the powder was dissolved in methanol, and aliquots of the methanolic stock were diluted to volume with the mobile phase to obtain a final concentration of 15 g/mL. After the previous treatments, all forced degradation solutions were filtered with a 0.45 m membrane filter prior to injection to the column.
Results and discussion Optimization of chromatographic conditions An isocratic liquid chromatographic method coupled with diode array detection was developed to provide a suitable and reliable procedure for the routine quality control analysis of DCV. The developed method was carefully designed and optimized to separate the investigated drug from its forced degradation products. The most important aspect in LC method development is the achievement of sufficient resolution of the target drug from all other compounds present in the sample with acceptable peak symmetry in a reasonable analysis time. To achieve this goal, several experiments were carried out to optimize both the stationary and mobile phases. For optimization of the stationary phase, several reversed phase columns such as Thermo-C18 (4.6 × 250 mm, 5 m particle size), Waters-C8 (4.6 × 250 mm, 5 m particle size) and Venusil XBP CN (4.6 × 250 mm, 5 m particle size) were tested. The best clear separation between all the eluting peaks (DCV and its degradation products), better system suitability parameters, symmetric DCV peak and relatively short retention time was attained using the
Waters-C8 (4.6 × 250 mm); hence, it became the column of choice for this study. Several mobile phases were evaluated using various proportions of different aqueous phases and organic modifiers. The best mobile phase combination was mixed phosphate buffer pH 2.5 and acetonitrile in the ratio of 75:25 (by volume). Methanol was tried as an organic modifier and mixed phosphate buffer was substituted by other aqueous phases such as water. In these trials, DCV suffered from delayed elution and/or peak asymmetry. It is generally preferable to use a mobile phase pH that is at least 1—2 units away from the pKa values of the analyte. DCV pKa values are 6.09 and 11.15 [10]. Consequently, mobile phase pH 2.5 was considered ideal for analysis of DCV. Besides, changing the buffer pH to 5.0 or 7.5 caused longer retention times for DCV. Flow rate was kept constant at 1.2 mL/min and all determinations were performed at ambient temperature. The DAD enhances the power of HPLC and is an elegant option for assessing method specificity by monitoring the recorded spectra of the eluted peaks especially during running forced degradation samples. DCV exhibits considerable absorption all over the range 210—350 nm with a maximum at 306 nm (Fig. 2). Accordingly, all chromatograms in this study were recorded at 306 nm. Quantification was based on peak area measurement. The previously described chromatographic conditions showed well-defined symmetric DCV peak at about 5.42 ± 0.04 min. Fig. 3 shows a typical chromatogram for DCV using the optimized conditions. Retention factor (k ) is 4.42, tailing factor is 1.15 and column performance (apparent efficiency) expressed by the number of theoretical plates (N) is 6957.
Stability-indicating aspects Forced degradation experiments were carried out on standard DCV to produce the possible relevant degradation products and test their chromatographic behavior using the developed method. Hydrolytic, using neutral (water), strong acidic (1 M HCl) and strong basic (1 M NaOH) media,
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oxidative (5% H2 O2 ) and dry heat degradation experiments were conducted, and the resulting chromatograms were compared with those obtained from a standard untreated solution of the drug. No strong signs of degradation of DCV were observed after heating for 1 h at 90 ◦ C in neutral conditions. DCV peak appeared at its specific retention time with area almost identical (98.4%) to that of a standard of the same concentration and the chromatogram did not show any extra peaks (Fig. 4a). Likewise, in case of degradation under acidic conditions after heating with 1 M HCl solution at 90 ◦ C for 1 h, no significant change in the peak area of the parent compound was detected. DCV peak appeared at its specific retention time with area almost identical (98.6%) to that of a standard of the same concentration with only a tiny degradation peak at 3.05 (Fig. 4b). Excellent resolution (Rs = 12.6) was observed between DCV and such degradation peak. The situation was quite different in case of degradation under basic conditions. About 18% decrease in DCV peak area as well as the appearance of a major degradation peak at 3.05 min in addition to several minor peaks at 1.97, 2.37, 4.20, 4.52, 4.76, 7.45 and 9.55 min were clearly noticed after heating with 1 M NaOH solution at 90 ◦ C for 30 min (Fig. 4c). Resolution was calculated between DCV and both the preceding and succeeding degradation peaks
at 4.76 and 7.45 min, respectively. Resolution was found not less than 3.3 which implied an adequate baseline separation between DCV and the nearest degradation peaks. Oxidative degradation with 5% H2 O2 at 90 ◦ C for 1 h caused about 7.5% reduction in the peak area of DCV as well as the appearance of a minor degradation product peak at 4.75 min was noticed (Fig. 4d). Again resolution value between DCV and the degradation peak was 2.9 which implied a sufficient separation between both peaks. It is noteworthy to mention that the extra peak eluting at about 2.25 min is due to the reagent (hydrogen peroxide) which usually elutes with the solvent peak (Fig. 4d). Finally, DCV was found stable under thermal (dry heat) degradation conditions. Its peak appeared at its exact retention time with area comparable to that of standard of the same concentration, and the chromatogram did not show any extra peaks (Fig. 4e). In all these forced degradation experiments, DCV was successfully separated from the degradation products as confirmed by the resolution values calculated for each chromatogram (Rs > 1.5). Results of the forced degradation of DCV in different conditions are summarized in Table 1. DCV Peak purity was checked in all the forced degradation chromatograms by using the diode array detector. Purity angle is a measure of spectral homogeneity. The obtained purity angles were
Table 1 Summary of the forced-degradation results of DCV. Récapitulatif des essais de dégradation forcée. Degradation type
Degradation conditions
% remaining of DCV
Retention times of the degradation peaks (min)
Neutral (water) Acidic (1 M HCl) Basic (1 M NaOH)
90 ◦ C for 1 h 90 ◦ C for 1 h 90 ◦ C for 30 min
98.4 98.6 81.6
Oxidation (5% H2 O2 ) Thermal (dry heat)
90 ◦ C for 1 h 120 ◦ C for 12 h
92.4 99.6
— 3.05 1.97, 2.37, 3.05, 4.20 4.52, 4.76, 5.44, 9.55 4.75 —
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Figure 4. HPLC chromatograms of DCV (15 g/mL) after exposure to neutral (a), acid (b), base (c), oxidative (d) and thermal dry heat degradation (e). Chromatogrammes of DCV (15 g/mL) après exposition en milieu neutre (a), acide (b), basique (c), oxydatif (d) et à la chaleur sèche (e).
within the purity threshold limits which confirm that DCV peaks are homogenous and pure in all the analyzed samples subjected to forced degradation conditions.
Validation of the proposed method The proposed HPLC method was validated as per the International Conference on Harmonization (ICH) guidelines on validation of analytical procedures [11].
Linearity and concentration range The linearity of the proposed HPLC procedure was evaluated by analyzing a series of different concentrations for the analyte (n = 9). The linear regression equation was generated by least squares treatment of the calibration data. Under the optimized conditions described above, the measured peak areas were found perfectly proportional to concentrations
of DCV. Table 2 presents the analytical parameters including linear regression equation, concentration range, correlation coefficient, standard deviations of the intercept (Sa ), slope (Sb ) and standard deviations of residuals (Sy/x ). Regression analysis shows good linearity as indicated from the correlation coefficient values (> 0.99999). In addition, deviation around the slope can be further evaluated by calculating the RSD % of the slope (Sb %) which was found to be less than 0.2%. Linearity can be further guaranteed by the analysis of variance (ANOVA) test. The most important statistic in this test is the F-value which is the ratio of the mean of squares due to regression divided by the mean of squares due to residuals. High F value reveals an increase in the mean of squares due to regression and a decrease in the mean of squares due to residuals. The greater the mean of squares due to regression, the steeper is the regression line. The smaller the mean of squares due to residuals, the less is the scatter of experimental points around the regression line.
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Table 2 Analytical parameters for the determination of DCV using the proposed HPLC-DAD method. Paramètres analytiques pour la détermination du DCV par la méthode CLHP-barrette de diode.
the developed method for estimation of the analyte in its bulk form (Table 3).
Parameter
Result
Concentration range (g/mL) Intercept (a) Sa Slope (b) Sb RSD % of the slope Correlation coefficient (r) Sy/x F Significance F LOD (g/mL) LOQ (g/mL)
0.6—60 3540 3262 81395 119 0.15 0.99999 7277 471715 3.66 × 10−18 0.12 0.40
Specificity is defined as the ability to access unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products and matrix components [11]. Method specificity was already demonstrated by the successful resolution of the intact drug from its forced degradation products (Fig. 4). DAD played an essential role to confirm the purity of DCV peaks in all forced degradation chromatograms.
Consequently, regression lines with high F values (low significance F) are much better than those with lower ones. Good regression lines show high values for both r and F statistical parameters (Table 2).
Limits of detection and quantification The limit of detection (LOD) is defined as the concentration of the analyte which has a signal-to-noise ratio of 3:1. For the limit of quantification (LOQ), the ratio considered is 10:1. The LOD and LOQ values of DCV were calculated using the signal-to-noise ratio method and are given in Table 2. Both LOD and LOQ values confirm the sensitivity of the proposed HPLC method.
Precision and accuracy The within-day (intra-day) precision and accuracy for the proposed method were studied at three concentration levels for DCV using three replicate determinations for each concentration within the same day. Similarly, the between day (inter-day) precision and accuracy were tested by analyzing the same three concentrations using three replicate determinations repeated on three days. Recoveries were calculated using the corresponding regression equation and they were satisfactory. The percentage relative standard deviation (RSD %) and percentage relative error (Er %) did not exceed 0.9% proving the high precision and accuracy of
Specificity
Robustness The robustness of an analytical procedure is a measure of its capability to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage [11]. Robustness was examined by making small changes in acetonitrile content in the mobile phase (± 1%), pH of mixed phosphate buffer solution (± 0.3), flow rate (± 0.05 mL/min) or working wavelength (± 2 nm) and recording the chromatograms of DCV standard solution. These variations did not have significant effects on the measured response (peak area) or retention time of DCV. RSD% for the measured peak areas using these variations did not exceed 3.85%. Table 4 shows the effects of the studied variations on the peak area and retention time of DCV.
Stability of solutions The stability of DCV working solutions as well as sample solutions in the mobile phase was examined, and no chromatographic changes were observed within 12 h at room temperature. Also, the stock solutions prepared in HPLCgrade methanol were stable for at least 2 days when stored under refrigeration at 4 ◦ C. Retention times and peak areas of the drug remained unchanged and no significant degradation was observed during these periods.
Assay of tablets dosage forms The developed method was applied for the assay of the drug ® in its commercial pharmaceutical formulation (Daklanork tablets). The active ingredient eluted at its specific retention time, and no interfering peaks were observed from any of the excipients of the assayed tablets. The diode array detection enables peak purity verification where no signs of
Table 3 Precision and accuracy for the determination of DCV in bulk form using the proposed method. Précision and justesse pour la détermination du DCV principe actif. Nominal value
Within-day
(g/mL)
Found ± SDa (g/mL)
RSD(%)
Er (%)
Found ± SDa (g/mL)
RSD (%)
Er (%)
3 15 45
3.01 ± 0.02 14.93 ± 0.02 45.15 ± 0.05
0.67 0.13 0.11
0.33 −0.47 0.33
3.01 ± 0.03 14.92 ± 0.03 45.20 ± 0.07
1.00 0.20 0.16
0.33 −0.53 0.44
a
Between-day
Mean ± standard deviation for three determinations.
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M.M. Baker et al. Table 4 Robustness evaluation for the determination of DCV (15 g/mL) using the proposed HPLC-DAD method. Évaluation de la robustesse pour la détermination du DCV (15 g/mL) par la méthode proposée par CLHP-barrette de diode. Parameter
Peak area ± SD
RSD %
Retention time ± SD
Acetonitrile ratio in mobile phase ± 1% pH of mixed phosphate buffer solution ± 0.3 Flow rate ± 0.05 mL/min Wavelength ± 2 nm
1270888 ± 8640
0.68
5.328 ± 0.81
1283387 ± 19949
1.55
5.409 ± 0.73
1264263 ± 48722
3.85
5.324 ± 0.17
1251087 ± 6753
0.54
5.419 ± 0.04
Figure 5.
®
Purity plot of DCV in its commercial dosage form (Daklanork tablets). ®
Tracé de pureté du DCV dans sa forme commercialisée (Daklanork comprimés).
Table 5 Application of the proposed HPLC-DAD method ® to the analysis of DCV in its dosage form (Daklanork tablets). Application de la méthode proposée par CLHP-barrette de diode pour l’analyse du DCV dans sa forme ® d’administration (Daklanork comprimés).
%Recovery ± SDa RSD% a
External standard
Standard addition
99.63 ± 0.31 0.31
99.22 ± 0.49 0.50
Mean ± standard deviation for five determinations.
co-elution from any of the excipients were detected (Fig. 5). Recoveries were calculated using both external standard and standard addition methods. Assay results revealed satisfactory accuracy and precision as indicated from % recovery, SD and RSD% values (Table 5). It is evident from these results that the proposed method is applicable to the assay of DCV in tablets with minimum sample preparation and satisfactory level of accuracy and precision.
Conclusion This study described a simple, specific and reliable HPLCDAD procedure for the assay of DCV in tablets dosage form.
To our present knowledge, no attempts have been made yet for the forced degradation or the stability indicating assay of DCV in pure and in its dosage form by any analytical methodology, furthermore; only very few methods were reported for the determination of DCV in human plasma. A significant advantage in the study is the separation of DCV from the degradation peaks obtained by various forced degradation experiments in relatively short run time. Reliability was guaranteed by testing various validation parameters of the method and the successful application to commercial tablet dosage form. The method can thus be used for routine analysis, quality control, and for checking quality during stability studies of pharmaceutical preparations containing the drug.
Disclosure of interest The authors declare that they have no competing interest.
References [1] Gentile I, Borgia F, Coppola N, Buonomo AR, Castaldo G, Borgia G, et al. The first of a new class of drugs targeted against hepatitis C virus NS5A. Curr Med Chem 2014;21(12):1391—404. [2] McConachie SM, Wilhelm SM, Kale-Pradhan PB. New directacting antivirals in hepatitis C therapy: a review of sofosbuvir, ledipasvir, daclatasvir, simeprevir, paritaprevir, ombitasvir and dasabuvir. Expert Rev Clin Pharmacol 2016;9(2):287—302.
Please cite this article in press as: Baker MM, et al. Validated stability-indicating HPLC-DAD method for determination of the recently approved hepatitis C antiviral agent daclatasvir. Ann Pharm Fr (2017), http://dx.doi.org/10.1016/j.pharma.2016.12.005
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ARTICLE IN PRESS
Stability indicating HPLC-DAD determination of daclatasvir [3] McCormack PL, Lyseng-Williamson KA. Daclatasvir in hepatitis C virus infection: a guide to its use in the EU. Drugs Ther Perspect 2016;32(2):42—9. [4] Keating GM. Daclatasvir: a review in chronic hepatitis C. Drugs 2016;76(14):1381—91. [5] Jiang H, Zeng J, Kandoussi H, Liu Y, Wang X, Bifano M, et al. A sensitive and accurate liquid chromatography-tandem mass spectrometry method for quantitative determination of the novel hepatitis C NS5A inhibitor BMS-790052 (daclastasvir) in human plasma and urine. J Chromatogr A 2012;1245:117—21. [6] Jiang H, Kandoussi H, Zeng J, Wang J, Demers R, Eley T, et al. Multiplexed LC-MS/MS method for the simultaneous quantitation of three novel hepatitis C antivirals, daclatasvir, asunaprevir, and beclabuvir in human plasma. J Pharm Biomed Anal 2015;107:409—18. [7] Rezk MR, Bendas ER, Basalious EB, Karim IA. Development and validation of sensitive and rapid UPLC-MS/MS method for
9
[8]
[9]
[10] [11]
quantitative determination of daclatasvir in human plasma: application to a bioequivalence study. J Pharm Biomed Anal 2016;128:61—6. Srinivasu G, Nagesh Kumar K, Thirupathi C, Lakshmi Narayana C, Parameswara Murthy C. Development and validation of the chiral HPLC method for daclatasvir in gradient elution mode on amylose-based immobilized chiral stationary phase. Chromatographia 2016;79(21):1457—67. Ashok Chakravarthy V, Sailaja BBV. Method development and validation of assay and dissolution methods for the estimation of daclatasvir in tablet dosage forms by reverse phase HPLC. Eur J Pharm Med Res 2016;3(7):356—64. http://www.drugbank.ca/ (last time accessed: October 1st , 2016). ICH. Validation of analytical procedures: text and methodology, Q2(R1). International Conference on Harmonisation; 2005.
Please cite this article in press as: Baker MM, et al. Validated stability-indicating HPLC-DAD method for determination of the recently approved hepatitis C antiviral agent daclatasvir. Ann Pharm Fr (2017), http://dx.doi.org/10.1016/j.pharma.2016.12.005