Stability Indicating RP-HPLC Method for Estimation of Impurities of Vitamin D 3 Analogue and Corticosteroid Used as Antipsoriatic Drugs . An Attempt to Characterize Pre-Calcipotriene

A single RP-HPLC method is developed for estimation of isomeric impurities of vitamin D3 analogueCalcipotriol/Calcipotriene (Calci) and impurities of Betamethasone dipropionate (BD). The developed method is capable of separating impurities of Calci and BD, specifically pre-Calcipotriene (Pre-Calci) from other known and unknown impurities. Pre-Calci is isolated and is characterised using few analytical techniques. These impurities are separated using a RP-C18 150 × 4.6 mm, 2.7 μm column maintained at 50 ̊C. The mobile phase consisted of mixture of water, methanol, acetonitrile and tetrahydrofuran eluted in gradient mode. Detection was done at 264 nm and 240 nm for Calci and BD impurities respectively. The method can be used for determining quality of Calci and BD drugs and ointment based drug products. It is stability indicating related substance method for both the drugs and drug products.


Introduction
Psoriasis, a chronic inflammatory dermatosis characterized by scaling, infiltration, and erythema, affects approximately 2% of the worldwide population [1].It is well-known that vitamin D 3 analogues, and Calci, are useful for the treatment of hyper proliferative skin diseases, such as psoriasis vulgaris [2]- [5].Calci is an analogue of vitamin D used for the treatment of chronic plaque psoriasis and has minimal effects on calcium metabolism.The therapeutic efficacy of Calci has been demonstrated in numerous clinical studies [6].BD belongs to the class of adrenocortical steroid and used as topicalcorticosteroid in treatment of different skin disorders.BD-Corticosteroid has broad mechanism of action, and inhabits the synthesis of many inflammatory cytokines, production of prostaglandins and nitric oxides, and the expression of adhesion molecules.BD is believed to have anti-inflammatory and anti-pruritic actions that are characteristic of corticosteroids.Several studies have shown that combined therapy with products containing these two active substances is more effective than mono therapy, the additive clinical effects and reduced skin irritationmay be achieved [7]- [10].
The highlights of public assessment reports and defences from product approval package for the drug products of Calci and Calci + BD are as follows.
• Three isomers are mentioned pre-Calci, trans-Calci, and 24-R-Calci.They are also degradation products or related substances [11].• The release and shelf-life requirements/limits are not identical.Related substances/degradation products and assay are different.Under all three conditions in all batches tested a decrease in assay and an increase in both individual and total related substances/degradation products can be seen [11].• 5% Calci stability overage is added in manufacturing [12].
• The Calcirelated substances should contain the limits and test for 5, 6 trans Calci [13].
In literature separate HPLC methods are available to estimate impurities of Calci and BD.Also, these methods are limited to estimate only Imp B, Imp C, and Imp D of Calci.However information is not available for quantification of pre-Calci in drug and drug products.Therefore the aim of the current study is to understand formation of pre-Calci, isolate and characterise it, extract both the drugs and impurities from drug product and determine quantitatively related substances of Calci, BD in bulk and combined drug products by single RP-HPLC method.Calci is a white to off-white crystalline powder; the substance is practically insoluble in water, freely soluble in ethanol slightly soluble in methalene chloride.A reversible isomerisation to pre-Calci takes place in solution depending on temperature and time.The activity is due to both.It is sensitive to light [14].Pre-Calci as an isomer formed in the drug product by a reversible transition, by a slow process which starts when Calci is dissolved and continues until equilibrium is reached.It is influenced by temperature.Therefore, pre-Calci is not a true impurity, and the activity of Calci is due to both substances.So, assay is calculated as the sum of the assays of the isomers [12].

Instrumentation
Agilent 1100 series HPLC system equipped with quaternary pump with an online degasser, auto sampler, thermostatted column compartment and variable wavelength detector.Waters alliance HPLC system equipped with 2695 separations module and 2489 UV/VIS detector or 2998 Photodiode array detector.Waters Empower 2 software is used for data acquisition, data processing.

Standard Preparation
About 15 mg of pure BD is dissolved in 200 ml of diluent.15 mg of pure Calci is dissolved in 100 ml of diluent.Transferred 0.5 ml of BD standard stock solution, 0.1 ml of Calci standard stock solution into 100 ml volumetric flask diluted to 100 ml with diluent and mixed.The diluted standard is about 0.75 µg/ml and 0.075 µg/ml of BD and Calci respectively.

Sample Preparation
About 2500 mg of ointment sample containing 1.25 mg of BD and 0.125 mg of Calci is transferred into a 100 mL amber colour volumetric flask, to it 10 mL of n-Hexane is added and sonicated for 15 min for complete dispersion, to this 5 mL of Diluent is added and mixed on a vortex mixture for 5 min.Transferred the mixture to 50 mL centrifuge tube, centrifuge at 5000 rpm for 5 min.Clear lower layer was injected for analysis.Samples are prepared fresh ever time.

Preparation of Pre-Calci Reference Solution
Pre-Calci is prepared by placing 2 mg of Calci in a vile and dissolved in 200 µl of solution made from 1 ml triethylamine and 9 ml of chloroform.Closed the vile and kept it in a water bath at 60˚C for 2 hours.

Selection of Diluent
Diluentselection is found to be key factor in extraction of selected drugs from ointment base.It is obvious that ointment base is soluble only in non-polar solvents like n-Hexane and THF, whereas drugs are soluble in polar solvents.The present drug product contains about 96% to 97% w/w of wax based ointment and 0.005% w/w of Calci and 0.0643% w/w of BD.It is difficult to extract all related compounds of drugs at such low concentration.Diluent is finalized by checking recovery of many solvent systems.Highest recovery is found with solvent mixture containing ACN, water at 95:5 v/v.few solvent systems recovery results are presented in Table 1.

Method Validation
System suitability solution is prepared by diluting 1 ml of pre-Calci reference solution to 100 ml with diluent and mixed.USP Resolution between Pre-Calci and Calci shall be about 4.0 (Figure 3).The method is validated as per ICH guidelines Q2 (R1) for pure compounds of Calci and BD.Method is found accurate and precise.Specificity is proved by performing force degradation.Acidic, basic, oxidative, humidity, photolytic and thermal stressed samples are analysed.And is found, all known impurity peaks of Calci and BD are pure as they pass the peak purity.LoD and LoQ of BD found to be 0.003 µg/mL and 0.008 µg/mL respectively.LoD and LoQ of Calci found to be 0.002 µg/mL and 0.006 µg/mL respectively.Linearity is established from LoQ to 1.5 µg/mL for BD and 0.15 µg/mL for Calci, which is 0.6% of Sample concentration.Robustness and intermediate precession are within acceptance limits.

Chiral Separation
To isolate Pre-Calci solid compound, preparative chiral chromatography is chosen.Concentrated solution (about 1 mg/ml) of Calci is prepared with ACN as solvent, the solution is kept in water bath at 80˚C for 5 hours, and few microliter solution is injected on to analytical chiral column to ensure formation of pre-Calci.It is found Calci and Pre-Calci are present in the ratio 70:30 percent composition of compounds (mass ratio).20 ml of isomers mixture is run through preparative chiralpack (Daicel) column, fractions of Pre-Calci are collected.The colour of Pre-Calci solution is observed to be light yellow.The pooled fractions are immediately evaporated under vacuum.Few mg of solid Pre-Calci is formed as residue.Chromatographic purity of isolated solid Pre-Calci is determines as about 96% pure.

Liquid Chromatography-EP Method vs Developed Method
Europe Pharmacopeia (EP) Chemical Reference Standard (EP-CRS) is available from EP which contains mixture of Calci, Calci Impurity-C, Calci Impurity-B and Calci Impurity-D along with respective chromatogram.It is confirmed that the EP method is not capable of separating Pre-Calci from Calci.When EP-CRS mixture is analysed by developed method Pre-Calci is perfectly separated.Pre-Calci peak is confirmed by spiking isolated compound on to EP-CRS solution.Pre-Calci peak is also confirmed by spiking "Pre-Calci reference solution" prepared as per EP on to EP-CRS (Figure 4).

UV Scans
All separated peaks are scanned by Ultra violet light, from 400 nm to 200 nm on HPLC-DAD detector.absorption maxima (λ max ) of Pre-Calci is found to be at 260 nm, The peak is confirmed as Pre-Calci related Substances of Calci as λ max of Calci and its related compounds range from 264 nm to 274 nm.The lowering of λ max for Pre-Calci is because of extended conjunction is part of ring (Figure 5).

NMR
1 H NMR spectrum is obtained by subjecting isolated Pre-Calci and pure Calci, dissolved in CDCl 3 and analysed on a Bruker 400 MHz NMR spectrometer.In pre-Calci spectrum, the chemical shifts of 1.25 corresponding to 1 H at carbon position 19 (-CH 3 ) and chemical shift of 3.55 corresponding to 1 H at Carbon position 1 (-CH-).When compared to spectrum of Calci (Figure 6) the changes in chemical shifts are in support of available Pre-Calci structure (Figure 7).

MS and LC-MS
Calci and isolated Pre-Calci are analysed for its mass fragmentation data on a LC-MS and LC-MS/MS.No significant difference in mass fragmentation is observed.Which is in clear support of the fact that Calci and Pre-Calci are isomers.

FT-IR
IR spectrum of isolated pre-Calci and pure Calci are obtained by holding the compounds in a potassium bromide pellet and scanned from 4000 cm −1 to 500 cm −1 using a Perkin Elmer FTIR spectrometer.A shift in stretching/ vibrational wave number (cm −1 ) corresponds to formation of isomer of Calci, which is supporting its structure.Few wave numbers of Calci and Pre-Calci are compared in Table 2.

Discussion
Theoretically it is predicted, Calci undergo Cis/Trans isomerism at carbon 7 forming Imp-B, Followed by rearrangement of double bonds forming Pre-Calci.From different experiments it is noticed that Imp-B levels are

Conclusion
The separated peak is conformed as Pre-Calci.The developed HPLC method can separate EP-CRS mixture into five peaks-Calci, imp-B, imp-C, imp-D and Pre-Calci; BD, imp-A, imp-B, imp-C of BD from each other.The method is superior to current available methods for BD and Calci.The method can be employed for qualitative and quantitative determination of impurities of Calci and BD drugs and drug products.Many queries and deficiencies with respect to assay, related substances, batch release, shelf life stability, and overages can be addressed by using this method.The developed and validated method shall be a quality control method for release and stability testing.

Figure 4 .
Figure 4. Comparison of zoomed chromatograms of (a) EP CRS in EP method; (b) EP CRS in developed method; (c) Isolated pre-Calci spiked on EP CRS in developed method; (d) Blank.

Figure 5 .
Figure 5. UV scans of Calci and related substance of Calcipotriol.

Figure 9 .
Figure 9. Chromatogram of known and un known impurities of BD and Calci.

Table 1 .
Solvent selection-recovery of BD and Calci.