Development and validation a new stability indicating RP-UFLC method for the quantification of Voriconazole
Kalyani Lingamaneni*, Mukthinuthalapati Mathrusri Annapurna
Department of Pharmaceutical Analysis and Quality Assurance
*Corresponding Author E-mail: kalyani.lingamaneni@gmail.com
ABSTRACT:
Voriconazole is anti-fungal agent. Voriconazole is used for treating invasive candidiasis, invasive aspergillosis and emerging fungal infections. A new stability indicating RP-UFLC method has been proposed for the quantification of Voriconazole in pharmaceutical formulations and the method was validated as per ICH guidelines. Mobile phase consisting of a mixture of Acetonitrile: 0.01% Acetic acid (50: 50, v/v) (pH 5.0) with a flow rate 1 mL/min and UV detection at 240 nm was used for the assay of Voriconazole. The drug was exposed to different stress conditions such as alkaline, acidic, thermal and oxidation degradation. Linearity was observed over the concentration range 0.5-50 μg/mL with linear regression equation y = 23554x- 774.8 (r2 = 0.9999). The LOQ and LOD were found to be 0.3987 μg/mL was found to be 0.1313 μg/mL. The present method can be useful for the quantification of pharmaceutical formulations such as tablets and injections and also for the bioanalytical studies.
KEYWORDS: Voriconazole, RP-UFLC, Validation, Stability indicating, ICH guidelines.
INTRODUCTION:
Figure 1: Chemical structure of Voriconazole
MATERIALS AND METHODS:
Shimadzu Model HPLC system (Shimadzu Co., Kyoto, Japan) equipped with PDA detector and C8 Luna column (250 mm × 4.60 mm i. d. 5µm particle size) was used or the chromatographic study. The system was maintained at 25 ºC. A mixture of Acetonitrile: 0.01% Acetic acid (50: 50, v/v) (pH 5.0) was used as mobile phase with a flow rate 1 mL/min (UV detection at 240 nm) for the present study. The mobile phase was sonicated and filtered through 0.22µm membrane filter prior to use.
Voriconazole was obtained as gift sample from Glenmark Pharmaceuticals Ltd. Voriconazole is available as tablets with brand names Voraze (Sun Pharma), Vorizol (Natco Pharma), Vonaz (United Biotech Pvt. Ltd.), Voritek (Cipla Limited) and Vosicaz (Glenmark Pharmaceutical Ltd) (Labelled claim 200 mg) and as I.V. injection with brand name Vfend (Pfizer).
Preparation of Voriconazole drug solution
Voriconazole (25 mg) was weighed accurately and dissolved in a 25 mL volumetric flask, sonicated and made up to volume with HPLC grade acetonitrile (MERCK) (1000 μg/mL) and further dilutions were made with mobile phase, sonicated and filtered through 0.22 μm membrane filter prior to injection.
Method validation
Linearity
Dilute solutions (0.5-50 μg/mL) of Voriconazole were prepared from the stock solution with mobile phase Acetonitrile: 0.01% Acetic acid (50: 50, v/v) (pH 5.0) and 20 μL of each were injected in to the UFLC system. The mean peak area (n=3) of Voriconazole was calculated from the chromatograms obtained and a calibration curve was drawn by taking the concentration of drug solutions on the x-axis and the corresponding mean peak area values on the y-axis.
Precision, Accuracy and Robustness
Intraday and inter-day precision were studied using three different concentrations (10, 20 and 30 μg/mL) of Voriconazole on the same day and on three consecutive days respectively and the % RSD was calculated. The accuracy of the assay method was evaluated in triplicate at three concentration levels (50, 100 and 150 %), and the percentage recoveries were calculated. Standard addition and recovery experiments were conducted and the percentage recovery was calculated. The robustness of the method was assessed by exposing the drug solution to different analytical conditions purposely changing from the original optimized conditions.
Stress degradation studies
Forced degradation studies were performed in the applied stress conditions. Voriconazole was exposed to different stress conditions such as acidic, basic, oxidation and thermal treatment.
Acidic degradation was performed by treating the drug solution with 1mL of 0.1N HCl, heated at 80 ºC for about 30 minutes on a water bath. The stressed sample is then cooled neutralized with 1mL 0.1N sodium hydroxide solution and the solution was made up to volume to the required concentration with the mobile phase. 20 μl of the solution was injected in to the UFLC system.
Alkaline degradation, was performed by treating the drug solution with 1mL 0.01 N NaOH heated at 80 ºC for about 30 minutes on a water bath. The solution is then cooled and neutralized with 1mL 0.01N hydrochloric acid and diluted with mobile phase. 20 µl of the solution was injected in to the UFLC system.
Oxidation degradation was performed by treating the drug solution with 1ml of 30% v/v H2O2 heated at 80 ºC for about 30 minutes on a water bath. The solution is then cooled and diluted with mobile phase.20 μl of the solution was injected in to the UFLC system.
Thermal degradation was performed by heating the drug solution at 80 ºC for about 30 minutes on a water bath. The solution is then cooled and diluted with mobile phase. 20 μl of the solution was injected in to the UFLC system.
Assay of Voriconazole tablets
20 Tablets of Voriconazole (Label claim: 200 mg) were procured and powdered. Powder equivalent to 25 mg Voriconazole was extracted using acetonitrile and then with the mobile phase in a 25 ml volumetric flask. The solution was sonicated for half an hour and filtered through membrane filter and 20 µL of this solution was injected in to the UFLC system. The peak area observed was noted at its retention time from the resultant chromatogram and the mean peak area was calculated (n=3).
RESULTS AND DISCUSSION:
A new stability indicating RP-UFLC method was developed and validated for the estimation of Voriconazole in tablets. A review of analytical methods so far published for the quantification of Voriconazole was given in Table 1.
Table 1: Literature survey
|
Mobile phase or Reagent / Detection (nm) |
Column |
Linearity (µg/mL) |
Remarks |
Ref |
|
Spectrophotometric methods |
|
|||
|
Water / 252 |
- |
5-80 |
- |
5 |
|
0.1N HCl / 256 |
- |
10-70 |
- |
6 |
|
Water / 255 |
- |
5-35 |
- |
7 |
|
0.1N HCl / 256 |
- |
10-60 |
- |
8 |
|
Methanol / 256 |
- |
5-30 |
- |
9 |
|
Sodium Hydroxide Phosphate buffer pH 2.0 Phosphate buffer pH 4.0 / 256 Phosphate buffer pH 6.8 Phosphate buffer pH 7.0 |
- |
5-60 |
- |
10 |
|
HPTLC Methods |
|
|||
|
Toluene: Methanol: triethylamine (6: 4: 0.1) / 254 |
Silica gel 60 F254 |
50–400 ng/band |
Human plasma Cephalexin (Internal standard) |
11 |
|
Acetonitrile: Water (60:40) / 257 |
Silica gel 60RP-18F-254S |
200–1200 ng/band |
Cream |
12 |
|
LC-MS Method |
|
|||
|
Acetonitrile: 0.1% Formic acid in 10 mM Ammonium acetate (50:50) |
Poroshell 120 EC‐C18 |
0.05-10 |
Human plasma (Protein precipitation) Fluconazole (Internal standard) |
13 |
|
Liquid chromatographic methods |
|
|||
|
50 mM Ammonium phosphate dibasic buffer (48:52) (pH adjusted to 6.0 with dilute ortho phosphoric acid): Acetonitrile / 250 |
Agilent Zorbax SB-C18 |
10-100 |
HPLC Related substances (4 impurities) |
14 |
|
Water: Acetonitrile: Methanol (50:25:25) / 256 |
Hypersil BDS C18 |
20-400 |
HPLC Rt is more (12.986 min) |
15 |
|
Acetonitrile: Water: Acetic acid (55:45:0.25) (pH 4.0) / 256 |
Diamonsil C18 |
0.1-50 |
HPLC Rat & Beagle dog plasma |
16 |
|
Acetonitrile: Water (70:30) / 255 |
Symmetry C18 |
0.2-15 |
HPLC Plasma (Protein precipitation) |
17 |
|
Acetonitrile: Water (60:40) / 256 |
enable C18G |
10-50 |
HPLC Not stability indicating method |
18 |
|
0.05M Ammonium acetate: Acetonitrile: Methanol (40:20:40) / 256 (Voriconazole) / 310 |
Microsorb MV- C18 |
0.1-10 |
HPLC Plasma Serum Clonazepam (Internal standard) |
19 |
|
Methanol: Water (45:55) |
Hypersil ODS C18 |
|
HPLC Chiral Separation & Analysis of enantiomers |
20 |
|
Acetonitrile: Water (40:60) / 256 |
C18 Hypersil BDS |
5-25 |
HPLC Rt is more (8.0 min) |
21 |
|
Acetonitrile: Water (50:50) / 256 (pH 3.0) |
RP C18 |
5-50 |
HPLC Experimental design Famciclovir (Internal standard) |
22 |
|
Acetonitrile: 0.01% Acetic acid (50: 50) (pH 5.0) / 240 |
C8 (2) 100A (Luna) |
0.5-50 |
UFLC Stability indicating Rt is less (6.45 min) |
Present method |
Mobile phase consisting of a mixture of Acetonitrile: 0.01% Acetic acid (50: 50, v/v) (pH 5.0) with a flow rate 1 mL/min and UV detection at 240 nm was used for the assay of Voriconazole using C8 Luna column (250 mm × 4.60 mm i.d. 5µm particle size). A symmetrical and sharp peak was observed at 6.45 ± 0.05 min. The chromatograms obtained were shown in Figure 2.
Linearity
Voriconazole obeys Beer-Lambert’s law and follows linearity over the concentration range 0.5–50 μg/mL (Table 3) (% RSD 0.19-1.65) and the linear regression equation was found to be y = 23554x- 774.8 (r2 = 0.9999) (Figure 3). The LOQ and LOD were found to be 0.3987 μg/mL was found to be 0.1313 μg/mL.
Table 2: Linearity of Voriconazole
|
Conc.(µg/mL) |
*Mean peak area |
% RSD |
|
0.05 |
1173 |
1.42 |
|
1 |
23513 |
0.62 |
|
5 |
117377 |
0.92 |
|
10 |
235839 |
0.87 |
|
15 |
343778 |
1.65 |
|
20 |
471654 |
0.54 |
|
25 |
589550 |
1.34 |
|
30 |
707503 |
1.52 |
|
40 |
943657 |
0.63 |
|
50 |
1175195 |
0.19 |
*mean of three replicates
|
|
|
|
A |
B |
|
|
|
|
C |
|
|
Figure 2: Representative chromatograms of Vorconazole A) Placebo B) Vorconazole standard (API) and C) Vosicaz tablet (Labelled claim: 200 mg) |
|
Figure 3: Calibration curve of Voriconazole
Precision, Accuracy and Robustness
Intraday and inter-day precision were studied at three different concentration levels of Voriconazole on the same day and on three consecutive days respectively and the % RSD was found to be 0.73-1.42 (Intraday) (Table 4) and 0.52-1.54 (Inter day) (Table 5) respectively (<2.0) demonstrating that the method is precise. The accuracy of the method was proved by the standard addition method and the % RSD values were 0.36-1.15 (<2.0) with a recovery of 99.72-99.93 % (Table 6). The robustness of the assay method was established by introducing small changes in the chromatographic conditions which include detection wavelength (242 and 238 nm), percentage of organic phase i.e. Acetonitrile in the mobile phase (48 and 52%) and flow rate (± 0.1 ml/min). Robustness of the method was studied using 10 μg/mL of Voriconazole (Table 7) and the % RSD was found to be 0.43-1.23 (<2.0).
Table 4: Intraday precision study of Voriconazole
|
Conc. (µg/mL) |
*Mean peak area |
Statistical Analysis |
|
*Mean ± SD (% RSD) |
||
|
10 |
235883 |
235857.67 ± 3372.76 (1.42) |
|
10 |
235839 |
|
|
10 |
235851 |
|
|
20 |
471734 |
471732 ± 3962.55 (0.84) |
|
20 |
471671 |
|
|
20 |
471793 |
|
|
30 |
707498 |
707521.67 ± 5164.91 (0.73) |
|
30 |
707504 |
|
|
30 |
707563 |
*mean of three replicates
Table 5: Interday precision study of Voriconazole
|
Conc. (µg/mL) |
*Mean peak area |
*Mean ± SD (% RSD) |
||
|
Day 1 |
Day 2 |
Day 3 |
||
|
10 |
235839 |
235756 |
235799 |
235798 ± 3631.29 (1.54) |
|
20 |
471634 |
471539 |
471698 |
471623.67 ± 4055.96 (0.86) |
|
30 |
707513 |
707629 |
707436 |
707526 ± 3679.13 (0.52) |
*mean of three replicates
Table 6: Accuracy study of Voriconazole
|
Spiked conc.((µg/mL) |
Formulation ( µg/mL) |
Total Conc. ( µg/mL) |
*Mean Conc. (µg/mL) ± SD (%RSD) |
% Recovery |
|
5 (50%) |
10 10 10 |
15 15 15 |
15.01 ± 0.163 (1.09) |
99.93 |
|
10 (100%) |
10 10 10 |
20 20 20 |
19.97 ± 0.231 (1.15) |
99.85 |
|
15 (150%) |
10 10 10 |
25 25 25 |
24.93 ± 0.090 (0.36) |
99.72
|
*mean of three replicates
Table7: Robustness study of Voriconazole
|
Parameter |
Condition |
*Mean peak area |
*Mean peak area ± SD (% RSD) |
|
Flow rate (± 0.1 ml/min) |
1.1 |
235712 |
235710 ± 2899.23 (1.23) |
|
1.0 |
235839 |
||
|
0.9 |
235579 |
||
|
Detection wavelength (± 2 nm) |
242 |
235847 |
252524.33 ± 1085.86 (0.43) |
|
240 |
235839 |
||
|
238 |
235887 |
||
|
Mobile phase composition Acetonitrile: 0.01% Acetic acid (50: 50, v/v) (pH 5.0) (± 2 %, v/v) |
48:52 |
235698 |
235772.67 ± 1626.83 (0.69) |
|
50:50 |
235839 |
||
|
50:48 |
235781 |
*Mean of three replicates
Assay of Voriconazole tablets
Two different brands of Voriconazole tablets consisting of 200 mg API were procured from the pharmacy store and the method developed was applied. It was found that the amount of Voriconazole found in tablets was 98.93-99.49 (Table 8) and there is no interference of excipients.
Table 8: Assay of Voriconazole tablets
|
Brand |
Label claim (mg) |
Observed amount (mg) |
% Recovery* |
Manufacturer (India) |
|
I |
200 |
198.97 |
99.49 |
Glenmark Pharmaceutical Ltd |
|
II |
200 |
197.86 |
98.93 |
Cipla Limited |
*Mean of three replicates
Stress degradation studies
Voriconazole (10 µg/mL) was eluted as a sharp peak at 3.022 min. During the acidic degradation the drug was eluted at 6.480 min and in alkaline degradation Voriconazole was eluted at 4.436 min. During the oxidation along with the drug peak was eluted at 6.504 min and in thermal degradation the drug was eluted at 6.502 min. Voriconazole was very sensitive towards alkaline degradation conditions. Initially the degradation was performed by using 1 ml of 0.1N NaOH but the total amount of drug has undergone degradation and therefore the alkaline degradation was continued by using 0.05, 0.01 N NaOH but even then total drug was decomposed and two degradants were eluted at 4.436 and 1.656 min. The fluoro phenyl moiety may be responsible for the complete degradation of the drug due to the alkali effect. In all the degradation studies the degradants were well separated without interfering with the main drug peak indicating that the method is selective and specific. The system suitability parameters were within the acceptable criteria i.e. the tailing factor was less than 1.5 and the theoretical plates were more than 2000 (Table 9). The individual chromatograms obtained during the stress degradation studies were shown in Figure 4.
|
|
|
|
(A) Drug standard |
(B) Acidic degradation |
|
|
|
|
(C) Alkaline degradation |
(D) Oxidative degradation |
|
|
|
|
(E)Thermal degradation |
|
|
Figure 4: Typical chromatograms of Voriconazole A) Drug standard (10 µg/mL) B) Acidic degradation C) Alkaline degradation D) Oxidative degradation E) Thermal degradation |
|
Table 9: Stress degradation studies of Voriconazole
|
Stress condition Medium / Temp. / Time |
Rt (min) |
% Recovery |
%Drug degradation |
Theoretical plates |
Tailing factor |
|
Standard drug |
6.471 |
100 |
- |
9856 |
1.196 |
|
Acidic degradation 0.1N HCl/ 80°C/ 30 min |
6.480 |
84.63 |
84.63 |
10033 |
1.404 |
|
Alkaline degradation 0.01N NaOH/ 80°C/ 30 min |
4.436 1.656 |
- |
100.0 |
7846 |
1.032 |
|
Oxidation 30% H2O2/ 80°C/ 30 min |
6.504 1.950 |
79.69 |
20.30 |
10298 |
1.411 |
|
Thermal degradation 80°C/ 30 min |
6.502 |
71.72 |
28.27 |
10572 |
1.224 |
CONCLUSION:
The RP-UFLC techniques were validated as per ICH guidelines and found to be simple, precise, accurate and robust for the quantification of Voriconazole tablets. There is no interference of excipients and the degradants were well separated without interfering with the drug peak. The method is specific and the system suitability parameters are within the acceptable criteria. The method can be successfully applied or the determination of Voriconazole in pharmaceutical formulations.
ACKNOWLEDGEMENT:
The authors are grateful to Glenmark Pharmaceutical Ltd (India) for supplying gift samples of Voriconazole. The authors have no conflict of interest.
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Received on 21.10.2020 Modified on 27.11.2020
Accepted on 30.12.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(1):420-426.
DOI: 10.5958/0974-360X.2021.00076.7