Determination of Calcipotriene in Calcipotriene Cream 0.05% w/w by RP-HPLC Method Development and Validation
Mayanka Singh*, Manoj Charde, Rajesh Shukla and Rita M. Charde
NRI Institute of Pharmacy, Bhopal
*Corresponding Author E-mail: mayanka.s@rediffmail.com
ABSTRACT:
A simple, specific, sensitive, precise stability-indicating high-performance liquid chromatography method for determination of Calcipotriene was developed and validated. A Zorbax 300 SB-C18 column (250*4.6 mm, 3.5 μ) in isocratic mode, with mobile phase consisting of a mixture of solution methanol: water (70:30) was used. The quantitation performed at flow rate of 1.0 mL/min at 264 nm and run time was 7.5 min. The analytical method was validated as per ICH guideline for linearity, accuracy, precision, specificity, limit of detection, limit of quantification, and stability and method can be extended to the analysis of Calcipotriene in semis olid formulations. The relative standard deviation values for precision was less than 2%, and % recovery was greater than 98% for Calcipotriene. The drug is photo and thermal sensitive, so the analysis should be done in moderate conditions only.
KEYWORDS: Calcipotriene, RP-HPLC, Validation, Specificity
INTRODUCTION:
In HPLC, separation occurs due to partitioning between a stationary phase contained in a column and a liquid phase, which is pumped under pressure through this column. Each of the components will have a certain affinity for the stationary phase and a certain affinity for the mobile phase. Provided there is sufficient difference between the analytes in their relative affinities for the two phases, then in HPLC system they will separate. Method development of most samples is often done with RP columns1-12.
Calcipotriene is belonging to the chemical class of vitamin D derivatives and chemically, it is (5Z, 7E, 22E, 24S)-24-Cyclopropyl 9,10-secochola-5,7,10(19),22 tetraene-1a,3b,24-triol. with molecular formula C27H40O3 and CAS number 112828-00-9 (1); Calcipotriene is official in BP 200713-16. The precise mechanism of calcipotriol in remitting psoriasis is not well-understood. However, it has been shown to have comparable affinity with calcitriol (Vit D) for the Vitamin D receptor, while being less than 1% as active as the calcitriol in regulating calcium metabolism.
The Vitamin D receptor (VDR) belongs to the steroid/thyroid receptor superfamily, and is found on the cells of many different tissues including the thyroid, bone, kidney, and T cells of the immune system. T cells are known to play a role in psoriasis, and it is thought that the binding of calcipotriol to the VDR modulates the T cells gene transcription of cell differentiation and proliferation related genes. Its metabolism is still not so clear17.
Literature review for Calcipotriene analysis revealed very few analytical methods. However, there is no method reported for quantification of Calcipotriene forms in the literature18-25.
Stress testing carried out to elucidate the inherent stability characteristic of the active substances and forms an important part of the API and drug product development. It suggests that degradation products that are formed under a variety of conditions should be identified and degradation pathways be established. The purpose of stress testing is to provide evidence on how the quality of drug substance varies with time under the effect of varieties of environmental factors such as acid, alkali, temperature, humidity, light and presence of oxygen. An ideal stability-indicating method is one that quantifies the drug and also resolves its degradation products26. The aim of present work is to develop a simple, specific, sensitive, accurate and stability indicating HPLC analytical procedure for the analysis of Calcipotriene in the presence of its degradation products and related impurities as per ICH guideline.
EXPERIMENTAL:
Reagent and Materials:
Calcipotriene working standard and sample cream was provided by ZYG Pharma Pvt. Ltd., Pithampur (Label claim: 0.005%; Calcipotriene cream; and manufacturer: ZYG Pharma Pvt. Ltd., Pithampur). Methanol, and water (HPLC grade), were used. Instrument used was Agilent Technologies 1200 series27.
Methods:
Preparation of Mobile Phase:
Mobile phase was prepared by mixing 750.0 mL of methanol with 250.0 mL of water. (75:25 v/v).
Preparation of Standard Solution:
About 10.0 mg of Calcipotriene reference standard was weighed out accurately, and transferred into a 200.0 mL volumetric flask. 100.0 mL of methanol was added into it, sonicated for 5 minutes or until dissolved, if necessary. Volume was make up with methanol, and mixed well (Stock Std). This solution contained about 0.05 mg/mL of Calcipotriene.
Further 2.0 mL of stock standard was diluted to 100.0 mL volumetric flask with sample solvent, and mixed well. A portion was filtered through the sample filtration kit into auto sampler vials. Discard minimum 2.0 mL of filtrate prior to collecting for analysis (Std). This solution contained about 1.0 µg/mL of Calcipotriene.
Preparation of Sample preparation:
1.0 g of the Calcipotriene Cream was weighed out accurately into a 50.0 mL volumetric flask. 45.0 mL of sample solvent was added and vortex to disperse the cream. Sonicated for 30 minutes at room temperature. Diluted to volume with diluting solvent. Mixed well by shaking and vortexing. Using a disposable pipette, 10.0 mL of the top solution was discarded and placed the sample at 2–8°C for 2 hrs to settle the solution. The supernatant clear solution was filtered through 0.2 µm nylon filtered discarding the first few mL. This solution contained about 1.0 µg/ml of Calcipotriene.
Chromatographic condition:
Chromatographic separation was achieved at 25°C on a reverse phase Zorbax SB-300 column28 using mobile phase consisting of methanol and water in the ratio of 70:30 (v/v). The flow rate was kept at 1.0 mL/min and detection was carried out at 264 nm. The sample was injected using 50 μl fixed loop, and the total run time was 7.5 min.
System suitability parameters:
Depending on the theoretical plate count, and other parameters as pressure etc system suitability parameters were studied.
Calibration curve:
The linearity of the response for Calcipotriene assay method was determined by preparing and injecting standard solutions with concentrations of 0.8-1.4 PPM Calcipotriene (Figure 2).
Analysis of the marketed formulation:
The developed method was applied to the analysis of Calcipotriene from marketed product named as Calcipotriene Cream (Label claim 0.005% w/w)29. The contents of marketed dosage form were found to be in the range of 98-102% with % RSD less than 2% which indicate suitability for routine analysis of Calcipotriene in dosage form.
% ASSAY = Aspl/Astd x mg Std x f /200 x 2/100 x 50/ g Spl x 100/LC
Where,
f = correction factor for potency
Expressed as % of Calcipotriene reference standard x 10-2 = 98.6 x 10-2 = 0.986
LC = Label claim of Calcipotriene in Calcipotriene cream 0.005% = 0.05 mg/g
Recovery Studies:
Recovery studies were performed by standard deviation method to validate the accuracy of developed method to different concentrations of preanalyzed sample solution of cream, varying concentration of standard drug was added and then its recovery was analyzed.
Preparation of Reference Standard: Calcipotriene (10 ppm): 50.0 mg of Calcipotriene reference standard was dissolved in 50.0 mL of methanol. 1.0 mL of above solution was taken and diluted to 100.0 mL with sample solvent (stock std solution)
Preparation of solution: About 1.0 g of placebo was weighed accurately in a stoppered test tube and added in it 2.5 mL Calcipotriene stock std solution and heated on a water bath and cooled to room temperature and the volume was make up to 50.0 mL with sample solvent, mixed and filtered(50 %).
About 1.0 g of placebo was weighed accurately in a stoppered test tube and added in it 5.0 mL Calcipotriene Stock std solution and heated on a water bath and cooled to room temperature and the volume was make up to 50.0 mL with sample solvent and mixed and filtered(100 %).
About 1.0 g of placebo was weighed accurately in a stoppered test tube and added in it 7.5 mL Calcipotriene Stock std solution and heated on a water bath and cooled to room temperature and the volume was make up to 50.0 mL with sample solvent and mixed and filtered (150%).
% of Calcipotriene
% Recovery = ------------------------------------ x 100
% of Calcipotriene added
Validation of the method:
Validation of the developed method was done according to ICH Q2B guideline 199630-35.
Accuracy:
The accuracy of the method was determined by adding working standard into the solution. The recovery studies were performed by standard addition method, at 50%, 100%, 150% level. The resulting solutions were assayed in triplicate and the results obtained were compared with expected results and expressed as percentage.
Precision:
The precision of analytical method is determined by assaying a sufficient number of aliquots of homogenous sample to be able to calculate statistically valid estimate of % RSD (Relative Standard deviation). Precision of a standard sample were carried out using three replicates of three different solutions (0.8, 1, 1.2 PPM).
Specificity:
Preparation of Reference Standard: 50.0 mg of Calcipotriene reference standard was dissolved in 50.0 mL of methanol. 1.0 mL of above solution was taken and diluted to 100.0 mL with sample solvent. (stock std solution). 5.0 mL of stock std solution was taken and diluted to 50.0 mL with sample solvent. (1ppm)
Sample preparation: A quantity of the cream containing 0.05 mg of Calcipotriene was transfered (about 1.0 g) in a stoppered test tube, heated on a water bath and cooled to room temperature and the volume was make up 50.0 mL with sample solvent and mixed.
Linearity:
The linearity of detector response was studied for Calcipotriene by HPLC at the concentration of 80%, 90%, 100%, 110%, 120% and 140% by one analyst on the same day.
Preparation of reference standard: 50.0 mg of Calcipotriene reference standard was dissolved in 50.0 mL of methanol. 1.0 mL of above solution was taken and diluted to 100.0 mL with sample solvent. (stock std solution)
Limit of Detection and Limit of Quantitation:
Preparation of reference standard (10 ppm): 50.0 mg of Calcipotriene reference standard was dissolved in 50.0 mL of methanol. 1.0 mL of above solution was taken and diluted to 100.0 mL sample solvent. (stock std solution)
The determination of LOD and LOQ is done as per the formula below:
Height of signal
S/N Ratio = ----------------------- x 100
Height of noise
RESULTS AND DISCUSSION:
The chromatographic conditions were optimized with a view to develop a stability-indicating assay method for Calcipotriene in semisolid dosage forms. Different mobile phases were tried as under chromatographic conditions. The column Zorbax SB-300 15 cm x 4.6 mm, gave good peak shape with response at affordable retention time with peak purity of Calcipotriene in presence its degradation products. The isocratic profile, methanol and water in mobile phase varied from 30-70% (v/v), was also altered to give the best separation of the peaks and Calcipotriene peak amongst degradation products peaks were found resolved. The final chromatographic system comprising reverse-phase C8 column (250 × 4.6 mm, 3.5μ) with a mobile phase consisting of a mixture of solution methanol and water in a ratio of 70:30 at a flow rate 1.0 mL/min was found optimum. Detection was performed at 264 nm (Table 1). Typical UV-Visible chromatograms are shown in Figure 1.
Table 1 Optimized chromatographic condition of Calcipotriene for the proposed method
Variable |
Condition |
|
Column |
||
Dimension |
Zorbax SB-300 15 cm x 4.6 mm |
|
Particle Size |
3.5 m |
|
Bonded Phase |
Octadecylsilane (C18) |
|
Mobile Phase |
||
Methanol |
750 ml |
|
Water |
250 ml |
|
Chromatographic conditions |
||
Column Temperature |
25° C |
|
Flow rate |
1.0 ml/min |
|
Sample cooler Temperature |
20° C |
|
Injection volume |
50 ml |
|
Detection wavelength |
264 nm |
|
Total run time |
10 min |
|
Retention time |
7.5 min |
|
Std-5 |
7.592 |
125.335 |
Mean |
125.100 |
|
S.D. |
0.998 |
|
% R.S.D. |
0.89 |
Figure 1 Chromatogram of standard solution
Figure 2 Linearity curve for Calcipotriene by the proposed method
As per USP, system suitability tests were carried out at before performing each of validation parameters for Calcipotriene by the proposed HPLC method summarized in Table 4. “RP-HPLC estimation of Calcipotriene in the dosage form” suggested only estimation of Calcipotriene in the dosage form with methanol: water-70:30, as a mobile phase; difference in ratio for establishing stability indicating method of Calcipotriene. The Calibration of the standard solution was done (Table 2).
Table 2 Calibration of the standard for the proposed method
Std Injection |
RT |
AUC |
Std-1 |
7.597 |
125.534 |
Std-2 |
7.591 |
125.628 |
Std-3 |
7.593 |
125.946 |
Std-4 |
7.589 |
123.165 |
Table 3: Summary of validation parameters for the proposed method
VALIDATION PARAMETERS |
CALCIPOTRIENE |
Linearity (R) |
0.9997 |
Accuracy by recovery study (% found) |
100.23 |
Precision (% RSD) |
|
RT |
AUC |
0.03 |
0.57 |
Specificity |
Implies as per the RT |
The developed method was applied to the analysis of Calcipotriene in marketed as Calcipotriene cream (Label claim 0.005% strength, ZYG Pharma Pvt. Ltd.). The results of analysis are given in Table 5. The contents of marketed tablet dosage form were found to be in the range of 98.79±.89% with RSD less than 2% which indicate suitability for routine analysis of Calcipotriene.
Table 4 System suitability parameters for Calcipotriene by the proposed method
· Pressure at start: 179 bar, Pressure at end: 181 bar |
· Left temp. at start: 25°C, Left temp. at end: 24.9°C |
· Right temp. at start: 25°C, Right temp. at end: 25°C |
· USP Tailing: 1.524 |
· Symmetry: 0.722 |
· Theoretical plates: Tangent method: 5766 |
Half width method: 5872 |
5 sigma method: 3182 |
Statistical: 3274 |
· Height: 7.56 |
· Width at half height: 0.233 |
5 sigma: 0.673 |
Tangent: 0.400 |
· Amount: 8.0904 ng/ul |
* mean of replicate determinations.
Recovery of Calcipotriene in the range of 99.68-100.23% shows method may be used for routine analysis of Calcipotriene. The percent recovery indicates the accuracy of the developed method. Precision showed RSD of 0.580. This shows method is precise as relative standard deviation is below 2.0%. Validation parameters for analysis of Calcipotriene are presented in Table 3. The linear regression data for the calibration curves indicate that the response is linear over the concentration range studied with correlation coefficient, r2 value as 0.9999. The value of slope was 1.24. The limit of detection was found to be 0.005ppm and limit of quantitation was found to be 0.02ppm.
Table 5: Analysis of marketed formulations
Std Injection |
RT |
AUC |
% Assay |
Std-1 |
7.589 |
125.877 |
97.81 |
Std-2 |
7.586 |
128.408 |
99.77 |
Average |
98.79 |
Specificity was carried out to identify the retention time of the drug and the obtained degradation products was identified, which were formed due to isomerization. The identified product was pre-calcipotriol, since the relative retention time was found to be 0.9.
Accuracy for the developed method was studied and percentage recovery was found to be within the limit of 98 – 102% and % RSD was found to be not more than 2.
CONCLUSION:
The developed HPLC method is specific, accurate and stability indicating. Statistical analysis proves that method is precise, and selective for analysis of Calcipotriene. The developed method is suitable for the quality control analysis of Calcipotriene.
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Received on 02.12.2010 Modified on 23.01.2011
Accepted on 25.02.2011 © RJPT All right reserved
Research J. Pharm. and Tech. 4(8): August 2011; Page 1219-1223