COST EFFECTIVE, ROBUST PRECISE & STABILITY INDICATING ANALYTICAL METHOD VALIDATION OF RELATED SUBSTANCES FOR PIMECROLIMUS CREAM BYRP-HPLC

COST EFFECTIVE, ROBUST PRECISE & STABILITY INDICATING ANALYTICAL METHOD VALIDATION OF RELATED SUBSTANCES FOR PIMECROLIMUS CREAM BYRP-HPLC

Neha Sharma * and Mukesh Kumar Gupta

Faculty of Pharmaceutical Sciences, Lords University, Alwar, Rajasthan, India.

ABSTRACT: A new, precise, economical and gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method has been validated for the relative substance determination in pimecrolimus cream. The chromatographic separation was achieved with phenomenax luna, C18, 150 x 4.6mm and 3 µm particle size column. The flow rate was 1.5 mL/min and eluents were detected at 210 nm using PDA detector. The retention time of pimecrolimus and desmethyl pimecrolimus was found to be 31.5 min and 27.5 min respectively. The calibration curves were linear for both pimecrolimus and desmethyl pimecrolimus. The LOQ was 0.50μg/ ml for pimecrolimus and 0.47μg/ml for desmethyl pimecrolimus. The approach has been validated in accordance with the international conference on harmonization's regulatory criteria. The evaluated parameters are precision, linearity, detection limit, quantification limit, specificity, accuracy and robustness. The technique may be applied to stability investigations as well as routine analysis to identify and quantify known and unidentified impurities pimecrolimus in the pharmaceutical dosage form.

Keywords: Pimecrolimus, Desmethyl Pimecrolimus, High-Performance Liquid Chromatography, Validation, Related substances

INTRODUCTION: Pimecrolimus is an ascomycin derivative that belongs to a novel class of immunomodulating macrolactams and is especially effective in treating inflammatory skin conditions ¹. Pimecrolimus has a low risk of systemic immunosuppression and significant anti-inflammatory and immunomodulatory effects, which have received much attention. Pimecrolimus works by preventing T cell activation ^2–5, which is how it works. Similar to other ascomycins, pimecrolimus is an immunophilin ligand that only engages the immunophilin macrophilin-12 cytosolic receptor.

By preventing it from dephosphorylating activated T cells' transcription factor nuclear factor, the Pimecrolimus-macrophilia complex efficiently suppresses the protein phosphatase calcineurin. This causes T-cell signal transduction pathways to be closed off, which limits the generation of inflammatory cytokines, especially those of the Th1 and Th2 types. Pimecrolimus has been shown to prevent mast cell release of cytokines and pro-inflammatory mediators ^6–8.

Its use has successfully treated inflammatory skin conditions like vitiligo, seborrheic dermatitis, oral lichen planus, cutaneous lupus erythematosus, and psoriasis ^8–12. The calcineurin inhibitors pimecrolimus and tacrolimus have been approved by the USFDA for the treatment of skin conditions ^13–14. Desmethyl pimecrolimus has a molecular weight of 796.44 g/mol and an empirical formula of C₄₂H₆₆ClNO₁₁, whereas pimecrolimus has a molecular weight of 810.47 g/mol and C₄₃H₆₈ClNO₁₁ as its empirical formula ^15-17. The principal goal of the current work is to validate a new reversed phase-high performance liquid chromatography (RP-HPLC) method to estimate pimecrolimus and desmethyl pimecrolimus impurity in pharmaceutical dosage forms. Pharmaceutical parameter analysis is a crucial and important step in the entire drug development process. Thus, rapid and easy procedures for testing the quality of commercial formulations are required. In light of this, the authors have developed a new, accurate and efficient technique for determining pimecrolimus and desmethyl pimecrolimus impurity in a pharmaceutical dosage form. The suggested RP-HPLC technique is validated using the following factors: specificity, linearity, precision, accuracy, LOD and LOQ experiments. The validation was performed in compliance with the International Conference on Harmonization's (ICH) requirements for validating analytical procedures ^18-20.

FIG. 1: STRUCTURE OF PIMECROLIMUS AND DESMETHYL PIMECROLIMUS

MATERIALS AND METHODS: Apparatus instruments, chemicals, and experiment reagents are listed in Tables 1 and 2.

TABLE 1: APPARATUS / INSTRUMENTS USED

TABLE 2: NAME OF CHEMICALS / REAGENTS

HPLC Instrumentation and Conditions:

Chromatographic Conditions: Gradient elution was used to achieve the chromatographic separation using an analytical column with the dimensions Phenomenex Luna (C18, 150 x 4.6mm, 3) at a temperature of 60°C. Solutions A and B constitute together the mobile phase. The mobile phase was filtered through a 0.45 m Millipore Nylon 6 membrane filter. Before injection, the column was equilibrated with the mobile phase for at least 30 minutes.

Gradient Programming:

Sample and Standard Solution Preparation:

Preparation of Solution A: Water: Acetonitrile: Methyl Tertiary Butyl ether: Formic acid (650:240:70:0.2).

Preparation of Solution B: Water: Acetonitrile: Methyl Tertiary Butyl ether: Formic acid (200:660:70:0.2).

Preparation of Desmethyl Pimecrolimus Impurity Stock Solution: Desmethyl pimecrolimus impurity (1mg) weighed into a volumetric flask (10ml) then 5ml of acetonitrile added and sonicated to dissolve. Volume made up with acetonitrile.

Preparation of System Suitability Solution: Pimecrolimus working standard (30mg) was weighed and transferred to 50mL volumetric flask. To this 3ml of desmethyl pimecrolimus impurity stock solution in 35ml of acetonitrile and sonicated to dissolve, volume made up by Acetonitrile.

Preparation of Diluted Standard Solution: 30mg of pimecrolimus working standard with 30 ml of diluent transferred in a volumetric flask (50ml). The solution sonicated and volume made up with diluent. Further, 1ml of solution take into100 ml volumetric flask to make 6 ppm solution.

Preparation of Sample Solution: Sample (3gm) with 30 ml of diluent taken in 50mL glass stopped test tube. The solution was sonicated for about 15 minutes at room temperature with intermediate shaking and filtered through a 0.45µ Teflon membrane filter.

Selection of Wavelength: The wavelength was chosen by scanning a solution of pimecrolimus between 200 and 400 nm.

RESULTS AND DISCUSSION: To create an accurate, selective, and precise stability-indicating RP-HPLC method for quantifying Pimecrolimus in stressed samples, various mobile phases with various compositions and flow rates were tested.

The chromatographic conditions were developed and modified after a number of compositions and combinations. An acceptable quantification of Pimecrolimus was observed at 1.5 mL/min with the gradient mobile phase, good peak symmetry, and a steady baseline. A clear baseline at 210 nm and one unique peak with a retention time (RT) of 31.5 min both were observed in the Pimecrolimus. Below is a description of each parameter's specific result.

Specificity: In order to confirm that there is no interference with pimecrolimus elution in standard samples or pharmaceutical formulations, the specificity and selectivity of the technique were assessed by injecting each of the system suitability solution, Standard solution, sample solution, individual impurity solution and spiked sample solution. Peak purity passes for pimecrolimus and desmethyl pimecrolimus impurity peaks, the result of specificity given in Table 3 and Fig. 2 to Fig. 4.

TABLE 3: PEAK PURITY OF STANDARD AND CONTROL SAMPLE

FIG. 2 STANDARD CHROMATOGRAM OF PIMECROLIMUS

FIG. 3 CHROMATOGRAM OF SAMPLE

FIG. 4 CHROMATOGRAM OF SPIKED SAMPLE SOLUTION

Limit of Detection and Quantification: The limit of quantitation (LOQ) can be calculated using the formula: LOQ=10 standard deviation (SD)/S, where the standard deviation is response based on either the SD of the blank or the residual SD of the regression lines or the SD of y=intercepts of regression lines and S=Slop of the linear regression. The LOD and LOQ values of pimecrolimus and desmethyl pimecrolimus are given in Table 4.

TABLE 4: LIMIT OF DETECTION AND QUANTIFICATION FOR PIMECROLIMUS AND DESMETHYL PIMECROLIMUS

Linearity: The linearity of response for pimecrolimus and desmethyl pimecrolimus was determined in the concentration range of the limit of quantitation to 150% of the specification limit. A calibration curve was prepared each for Pimecrolimus and impurity by plotting the concentration on the x-axis and the average peak area on the y-axis. A linear regression analysis was used to construct the regression equation. The acceptance criteria squared correlation coefficient was not less than 0.99. The observations and calibration curve was shown in Table 5 and Fig. 5 and 6.

TABLE 5: LINEARITY STUDIES FOR PIMECROLIMUS AND DESMETHYL PIMECROLIMUS

FIG. 5: CALIBRATION CURVE OFPIMECROLIMUS.

FIG. 6: CALIBRATION CURVE OFDESMETHYL PIMECROLIMUS

Precision: A method's precision is a measure of its ability to produce repeatable results. Six replicate injections of the standard preparation of desmethyl pimecrolimus were used for system precision, whereas, for method precision, six sample solution of Pimecrolimus cream and one sample spiked with known desmethyl pimecrolimus at specification limit was prepared. The precision of the method was determined by repeatability and intermediate precision. Repeatability was examined by performing six determinations of the same concentration of desmethyl pimecrolimus on the same day under the same experimental conditions. The result of repeatability and intermediate precision is given in Table 6.

TABLE 6: SYSTEM AND METHOD PRECISION STUDIES FOR DESMETHYL PIMECROLIMUS

Accuracy: The placebo of pimecrolimus cream was spiked with pimecrolimus and desmethyl pimecrolimus at three different levels: LOQ, 50%, 100% and 150% of the label claim in triplicate. The acceptance criteria for accuracy should be in the range of 90.0% to 110.0% for 50%, 100% and 150% levels. In the case of, LOQ mean recovery should be in the range of 85% to 115%. The recovery of desmethyl pimecrolimus is given in Table 7.

TABLE 7: ACCURACY STUDIES FOR DESMETHYL PIMECROLIMUS

Robustness: The HPLC method for the determination of related substances of pimecrolimus in pimecrolimus cream is robust for small changes in Column temperature, Flow rate and wavelength. The system suitability solution, standard solution, placebo, sample solution and sample spiked with known impurity were injected under different chromatographic conditions. The result of the different condition is mentioned in Table 8 and the system suitability of different variable condition are mentioned in Table 9.

TABLE 8: ROBUSTNESS STUDIES FOR DESMETHYL PIMECROLIMUS

TABLE 9: SYSTEM SUITABILITY FOR PIMECROLIMUS AND DESMETHYL PIMECROLIMUS

System Suitability: The system suitability test is a pre-use test to verify the compatibility and efficacy of a chromatographic system. The system's suitability was tested by injecting the system suitability solution. The process was repeated every day during the validation of the method. The resolution between the pimecrolimus peak and the desmethyl pimecrolimus peak should not be less than 3. Tailing factor for the Pimecrolimus peak in the system suitability solution should not be more than 2 the and theoretical plate should not be less than 2000. The RSD of six replicate injections for a standard should be less than 5%. The system suitability of different validation parameters is given in Table 10.

TABLE 10: SYSTEM SUITABILITY FOR PIMECROLIMUS AND DESMETHYL PIMECROLIMUS

CONCLUSION: A novel gradient RP-HPLC approach has been developed to study related substances in pimecrolimus. The linearity, LOD and LOQ of pimecrolimus and desmethylpimecrolimus were determined. The technique has been proven to be precise. It can simultaneously identify and quantify impurities in pimecrolimus that are known and unknown. It is simple, sensitive, linear and exact. The technique may be applied to the routine and stability examination of dosage forms and pharmaceutical substances.

ACKNOWLEDGMENTS & CONFLICT OF INTEREST: The authors are thankful to the faculty of pharmaceutical sciences, lords University, Alwar, Rajasthan, for providing the necessary facilities & guidance to carry out this research. Authors have no conflicts of interest while preparing the manuscripts.

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    How to cite this article:

    Sharma N and Gupta MK: Cost effective, robust precise & stability indicating analytical method validation of related substances for pimecrolimus cream by RP-HPLC. Int J Pharm Sci & Res 2023; 14(4): 1978-85. doi: 10.13040/IJPSR.0975-8232.14(4).1978-85.

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