INTRODUCTION:
Calcipotriol (1S, 3R, 5Z, 7E, 22E, 24S)-24-Cyclopropyl-9, 10 secochola-5,7,10 (19), 22-tetraene-1,3,24-triol is a non-steroidal antipsoriatic agent derived from vitamin D. Calcipotriol (CP) is a non steroidal, synthetic vitamin D3 analogue, chemically known as (1S, 3R, 5Z, 7E, 22E, 24S)-24-Cyclopropyl-9, 10 secochola-5,7,10 (19), 22-tetraene-1,3,24-triol [Figure 1].
Figure 1: Chemical structure of calcipotriol
It is frequently used alone or in combination for topical treatment of autoimmune skin disease like psoriasis1,2. Calcipotriol are chiefly possessing antiproliferative property and they quickly block the keratinocyte differentiation, which are major culprit for psoriasis3.
D-vitamin receptor expressed by keratinocytes is presesnt in the lower epidermis (the target site for calcipotriol) where it controls the over proliferation and differentiation of keratinocytes during psoriasis.4,5 Furthermore, calcipotriol have been reported to reduce the expression of psoriasin and koebnerisin which are the key proteins involved in the enhancement proinflammatory cytokines in keratinocytes which consequently leads to amplification of inflammatory reaction 6,7.
In literatures, a handful of methods have been described for determination of calcipotriol in topical dosage form8, simultaneous estimation of calcipotriene and betamethasone dipropionate9.However, there exist a great need to develop and validate a new-fangled precise technique for routine analysis of drug which could be applied for topical dosage forms like ointment.
The projected method has been developed and validated with an objective to provide a method which is simple in operation, economic, and competent to examine bulk material. The method has been validated according to guidelines of ICH Q2 (R1). Classic validation parameters such as precision, accuracy, repeatability, linearity, range, limit of detection, limit of quantification (LOQ) and robustness were examined.
MATERIAL AND METHODS:
Materials
Calcipotriol anhydrous was obtained from Manus Aktteva Biopharma Llp. Ahmedabad, India. Calcipotriol ointment (Pasitrex Ointment 0.005% w/w) was procured from local market. Methanol, and water (HPLC grade) were purchased from Sigma Aldrich (India). Instrument used was RP-HPLC (LC-10AT-vp, Shimadzu).
Instrument
HPLC system consists of a Shimadzu LC-10AT-vp Solvent delivery system (pump), SPD – 10Avp –UV visible detector, Rheodyne injector with 20μL loop volume and a Phenomenex Luna C18 column (250mm X 4.6 mm in diameter) with an average particle size of 5μ.
Chromatographic conditions
The optimized chromatographic conditions that have been employed in the ultimate development of RP-HPLC method has been shown in the Table 1.
Table 1: Chromatographic conditions
|
Stationary Phase |
Phenomenex Luna C18 column (250mm X 4.6 mm in diameter) with average particle size of 5μ |
|
Mobile Phase |
Methanol: water 80:20 (v/v) |
|
Wave length |
264nm |
|
Run Time |
8.2 min |
|
Flow rate |
1mL/min |
|
Injection volume |
20µL |
|
Mode of operation |
Gradient elution |
Selection Preparation of Mobile Phase
On the basis of solubility and stability of CP, a variety of mobile phase compositions were tried in order to obtain a fine resolution with sharp peaks. The standard solution was run with different composition of mobile phases. Finally, methanol: water 80:20 (v/v) was opted with detection wavelength 264 nm, as it resulted into jagged peak with fine symmetry within limits.
Preparation of Standard stock solution
Accurately weighed quantity of CP (10mg) was transferred into a volumetric flask (100 mL), a small amount of methanol was added to it, shaken well to dissolve the drug and the final volume was made up to the mark using methanol to get a solution containing 100 μg/mL of CP.
Method validation
The optimized Chromatographic method was entirely validated according to ICH guidelines Q2 (R1) for the validation of analytical methods10. Validation parameters were linearity, precision, repeatability, accuracy, LOD, LOQ and robustness as described below.
Linearity
The linearity of an analytical method is its potential to obtain a test result, which possess a mathematical correlation with the concentration of sample under examination11. Different volumes of standard stock solution were transferred into 10 mL volumetric flasks separately and diluted with mobile phase to yield 5.0–30.0 µg/mL concentration range. The prepared dilutions were serially injected, peak area was measured for every dilution and graph was constructed for peak areas versus the concentration.
Precision
The precision of the method was determined in terms of method precision (repeatability) and intermediate precision (interday) 12.
Method precision (repeatability)
Method precision (repeatability) is the outcome of the method operating over a short time period under the similar setting (inter-day precision)13. Five replicates of standard solution of CP (10.0 μg/mL) were examined. The mean area and % relative standard deviation (RSD) for all the injections were calculated.
Intermediate precision
Intermediate precision of the method was measured by repeating the whole process on the subsequent day for 5 replicates of standard solution of CP (10.0μg/mL). The mean area and % relative standard deviation (RSD) for all the injections were calculated.
Accuracy as recovery
The accuracy of the developed method was examined by recovery from drug solution. Recovery study was performed thrice for dissimilar concentrations of CP equivalent to 50%, 100%, and 150% of the standard amount. Samples were introduced into the HPLC column and analysis was made as per the developed procedure. The average % recovery of CP was calculated14.
Limit of detection and limit of quantification
Limit of detection (LOD) and Limit of quantification (LOQ) for the assay were determined employing the following equations-
where σ = the standard deviation of the response, S = the slope of the calibration curve of analyte 15,16.
Robustness
The robustness of an analytical method is degree of its capability to stay unaltered by minute, but purposeful changes in method parameters17. It offers a hint of its reliability during regular usage. The factors opted for this study was flow rate (mL/min) and mobile phase (percentage methanol).
Analysis of Marketed Formulation
About 1 gm of the ointment (0.005%w/w) was weighed and transferred to a conical flask. To the flask about 20 mL methanol was added. The contents were mixed properly and heated between 50-55 ºC until the ointment base got melted. It was set to cool at room temperature and then filtered. The same process was repeated two times using 10 mL of methanol for extraction of remained content of ointment. The extracts so obtained were combined together in a volumetric flask and the volume was adjusted to 100 mL with methanol so as to obtain ultimate concentration of 5μg/mL of CP. The solution was then sonicated for 10min and introduced into column as per the optimized chromatographic conditions and peak areas were documented. The quantifications were done by putting these figures to the straight line equation of calibration curve.
RESULTS AND DISCUSSION:
The chromatogram of CP obtained by developed method has been presented in Figure 2. CP was eluted at retention time of 8.2 min.
Figure 2: Representative chromatogram of calcipotriol using a developed analytical method
Linearity
The Linearity data showing peak area and % RSD has been shown in Table 2.
Table 2: Linearity data for CP with methanol–water 80:20 (%, v/v) as mobile phase
|
Concentration (µg/ml) |
Mean Peak area ± SD (n = 3) |
% RSD |
|
5 |
53527±363.037 |
0.68 |
|
10 |
117323±1810.299 |
1.54 |
|
25 |
179486±322.856 |
0.18 |
|
20 |
237166±4499.24 |
1.90 |
|
25 |
304638.0±370.405 |
0.12 |
|
30 |
367483.0 ±2208.755 |
0.060 |
Where, SD= Standard deviation; % RSD= Percent relative standard deviation
The developed method was observed to be linear within in the experimental range of concentration. The regression coefficient was observed 0.9993 which demonstrates its appropriateness for analysis. The linearity curve of CP has been depicted in Figure 3.
Precision
The data observed for repeatability and intermediate precision has been presented in Table 3.
Table 3: Results of precision
|
Parameters |
%RSD for response area |
%RSD for retention time |
|
Repeatability |
0.435 |
0.225 |
|
Intermediate Precision |
0.862 |
0.094 |
Where, % RSD= Percent relative standard deviation
Figure 3: Linearity curve of calcipotriol
Repeatability
The developed method qualifies the test for repeatability, as percent RSD (0.7%) of the peak area of five replicates of injection was within the limits of 2%.
Intermediate precision
The developed analytical technique possess good intermediate precision, as percent RSD (0.7%) for peak area of five replicates of injection fall within the limits of 2% (Table 3).
Accuracy as recovery
Results of recovery study was found to be acceptable and presented in (Table 4). Low percentage of RSD values of drug recovery from standard drug solution was observed at every concentration point which signifies very good accuracy of the developed analytical technique 14,18.
Table 4: Recovery/Accuracy data for recovery of calcipotriol from solution
|
Level of percentage |
Added amount ((µg/ml) |
Amount recovered |
% Recovery |
%RSD |
|
50 |
10 |
10.101 |
101.01 |
0.420 |
|
10 |
10.086 |
100.86 |
||
|
10 |
10.021 |
100.21 |
||
|
100 |
15 |
15.327 |
102.18 |
0.24% |
|
15 |
15.254 |
101.69 |
||
|
15 |
15.298 |
101.98 |
||
|
150 |
20 |
20.132 |
100.66 |
0.55% |
|
20 |
20.306 |
101.53 |
||
|
20 |
20.099 |
100.49 |
Where, % RSD= Percent relative standard deviation
Limit of detection and limit of quantification
The LOD and LOQ of CP were reported to be 0.599 μg/mL and 1.816 μg/mL respectively. The observed findings suggest that a microgram quantity of the drugs can be determined precisely and accurately.
Robustness
Robustness study was done by deliberately altering the experimental setting such as flow rate from1 mL/min to 0.9 mL/min and 1.1 mL/min. The composition of mobile phase was altered by changing the proportion of methanol by 5%. In both conditions the recovery of CP was examined and the %RSD was reported less than 2% (Table 5).
Table 5: Results of robustness study
|
Method Parameter |
Deliberate Changes |
% RSD of peak area |
Retention time |
|
Flow rate |
0.9 ml/min |
1.09 |
8.9 |
|
1.1 min/mim |
0.97 |
7.6 |
|
|
Mobile phase (Methanol: Water) |
75:25 (v/v) |
1.58 |
9.1 |
|
|
85:15 (v/v) |
1.82 |
7.4 |
Where, % RSD= Percent relative standard deviation
Analysis of Marketed Formulation
A solitary peak was recorded at the retention time of calcipotriol, when the marketed formulation was subjected for chromatography. No interaction among CP and excipients present in the marketed formulation was reported. The content of CP was observed to be 99.87% and the RSD was 0.81%. The low value of %RSD signifies the appropriateness of the developed method for routine analysis of CP in pharmaceutical dosage forms.
CONCLUSION:
Present study has established that the developed HPLC method is accurate, precise and reproducible. The developed analytical method has been observed to be better in comparison with earlier reported methods, due to its wide range of linearity, usage of cost effective and easily accessible mobile phase and utilization of the same mobile phase for column washing. In nutshell the developed analytical method could to be extremely useful in routine analysis of CP in bulk as well as in its dosage forms.
CONFLICT OF INTEREST:
Authors report no conflict of interest.
ACKNOWLEDGEMENT:
Authors want to acknowledge the facilities provided by the Rungta College of Pharmaceutical Sciences and Research, Kohka, Kurud Road, Bhilai, Chhattisgarh. Authors also wish to acknowledge the Science and Engineering Research Board (SERB), New Delhi for providing financial assistance under NPDF scheme File no. PDF/2015/000380.
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Received on 26.10.2018 Modified on 21.12.2018
Accepted on 12.01.2019 © RJPT All right reserved
Research J. Pharm. and Tech 2019; 12(2):579-583.
DOI: 10.5958/0974-360X.2019.00103.3