ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF ASCOMYCIN CONTENT IN TACROLIMUS API BY USING RP HPLC

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF ASCOMYCIN CONTENT IN TACROLIMUS API BY USING RP HPLC

M. Mahesh *, L. Hari Kumar Naik and K. Sai Lakshmi

Department of Pharmaceutical Analysis, JNTUA-Oil Technological and Pharmaceutical Research Institute, Jawaharlal Nehru Technological University Anantapur (JNTUA), Ananthapuramu, Andhra Pradesh, India.

ABSTRACT: A quick and affordable analytical method validation for the determination of Ascomycin content in Tacrolimus API by HPLC UV-detector was developed with respect to the accuracy, precision, linearity, selectivity, robustness, limit of quantification, limit of detection, according to ICH guidelines. The extraction of samples was performed using HPLC –LC Solutions Chemstation system manufactured by WATERS by using X-TERRA, C18 (4.6 x 150mm, 3.0μm) in the Presence of mobile phases A&B composed of 6 ml orthophosphoric acid and Acetonitrile: Tert-butyl Methyl Ether (81:19) in the ratio of 4: 1 and 1: 4. The estimated samples were then analysed using an UV visible code 2487 manufactured by Waters. With a linear calibration curve spanning from the mean recovery of method precision and system precision 140765 and 142973 and %of relative standard density is found to be 0.30% and 0.87%. With retention time of Ascomycin 29.5mins and Tacrolimus 33.4 min. Column oven temperature of 60 ⁰C at a flow rate of 1.5mL/min and wavelength is found at 220 nm the total run time is 60mins with an injection volume of 20μL. The method is validated and is found to be accurate and precise and robustness. The r² is 0.9995 and the LOD is 0.133 μg/ml LOQ value is found to be 0.450μg/ml.

Keywords: Ascomycin, Tacrolimus, Validation, ICH guidelines and HPLC

INTRODUCTION: A substance intended for the diagnosis, mitigation, prevention, or treatment of diseases in humans or animals, as well as for changing any bodily structure or function, is referred to as a drug ¹. By treating ailments, medications help advance human civilisation. Most drugs used nowadays are synthetic in nature. They are generated in large quantities and employed in pharmaceutical formulations for their medicinal benefits ².

In pharmacological therapy, safety and efficacy are two crucial considerations. The pharmacological-toxicological profile of a medicine and the negative effects brought on by contaminants in bulk and dose forms are used to assess the drug's safety. The adverse pharmacological or toxicological effects of medication contaminants frequently outweigh any therapeutic benefit from their administration ³.

Molecular Weight: 792.01g/mol.

FIG. 1: STRUCTURE OF TACROLIMUS

Today, many labs employ assay techniques like liquid chromatography-tandem mass spectrometry (LCMS/MS), which more precisely quantify certain Tacrolimus quantities. Only a few methods, including RP-HPLC, UPLC, and mass spectrometry approaches, have been reported for the detection of tacrolimus in pharmaceutical dosage forms, according to a study of the literature ^4-8.

In the current study, by employing RP HPLC, we hope to produce and validate the ascomycin content in tacrolimus API. Better retention duration, incredibly sharp peak shapes, and symmetrical peak shapes are all advantages of the suggested RP-HPLC method's usage of a low-cost solvent solution. The proposed method has received approval based on ICH criteria. Structure of Tacrolimus shown in Fig. 1.

MATERIALS AND METHODS:

Materials: All the chemicals, reagents, instruments and working standards are mentioned in the Table 1, 2 & 3 which are carried in the present study.

TABLE 1: LIST OF CHEMICALS & REAGENTS

TABLE 2: INSTRUMENT AND EQUIPMENT’S

TABLE 3: LIST OF WORKING STANDARDS

Methods:  Reverse Phase HPLC

Methodology:

Diluent Preparation: Acetonitrile: Water (70:30). Measure accurately 1400 mL of HPLC grade Acetonitrile and 600 mL of HPLC grade water using a glass measuring cylinder and transfer into a 2 L mobile phase reservoir, sonicate for about 15 minutes to degas the diluent and use.

Standard Stock Solution Preparation: (300 µg / mL of Ascomycin): Weigh about 30 mg of Ascomycin working standard and 100 mg of Tacrolimus working standard into a 100 mL volumetric flask, add 50 mL of diluent and sonicate to dissolve completely. Then make the volume up to the mark with the diluent and mix well.

Standard Solution Preparation: (15 µg / mL of Ascomycin): Transfer 2.5 mL of standard stock solution in to a 50 mL volumetric flask, make the volume up to the mark with the diluent and mix well.

Sample Solution Preparation: Weigh about 30 mg of Tacrolimus test sample into a 10 mL volumetric flask to this add 5 mL of diluents and sonicate to dissolve completely. Then make the volume up to the mark with the diluent and mix well.

System Suitability Solution Preparation: (30 µg / mL): Weigh 30mg of Tacrolimus standard in 10 ml volumetric flask add some Diluent and shake well and sonicate to dissolve and make up to the volume. Dilute 1.0 mL of the above solution to 100 mL with diluent. Then transfer to amber colour vial and keep at ambient temperature for 3 hours then inject.

Preparation of Solutions for Linearity: From standard stock solution prepare respective concentrations were mentioned below Table 4.

TABLE 4: LINEARITY LEVELS

Solution A: 6ml ortho phosphoric acid. Solution B: Acetonitrile: Tertiary butyl methyl ether 81:19.

Preparation of Solution A 0.1% Ortho Phosphoric Acid: 6ml ortho phosphoric acid: Pipette out 0.1ml OPA and add 100ml of water into a 100 ml of reagent bottle.

Preparation of Solution B Acetonitrile: Tert. Butyl Methyl Ether (81:19): 810.0ml of acetonitrile and 190.0ml of tertbutylmetyl ether make up the volume for 1000 ml transfer into a reagent bottle.

Preparation of Mobile Phase:

Mobile Phase A: Solution A: Solution B (4:1) 4:1 ratio of solution A and solution B collected in 500.0 ml reagent bottle and sonicate in ultra sonic water bath for 15-30 min.

Mobile Phase B: Solution A: Solution B (1:4) 1:4 ratio of solution A and solution B collected in 500.0 ml reagent bottle and sonicate in ultrasonic water bath for 15-30min.

Mobile Phase Optimization: During optimization different composition and proportions of mobile phases are tried such as methanol: water and acetonitrile: water. In these mobile phase system suitability tests was not satisfactory. Gradient programming for Mobile phase A containing solution A: Solution B (4:1) and for mobile phase B containing solution A and B (1:4). And the flow rate was 1.5ml/min details mentioned in Table 5 it gave all System Suitability Test Satisfactory Results so this mobile phase was chosen for analysis of Ascomycin.

TABLE 5: GRADIENT PROGRAM

Method Development: The analytes were conducted on an analytical column X-TERRA, C18 (4.6 x 150mm, 3.0μm) in the Presence of mobile phases A&B composed of 6ml ortho phosphoric acid and Acetonitrile: Tert-butyl Methyl Ether (81:19) in the ratio of 4:1 and 1:4. The diluent is used as Acetonitrile: water (70:30).

The estimated samples a retention time of Ascomycin 29.5 min and Tacrolimus 33.4 min shown in Fig 2. Sample temperature and column oven temperature of 2°C and 60 °C at a flow rate of 1.5 mL/min and wavelength is found at 220 nm the total run time is 60 min with an injection volume of 20μL.

FIG. 2: METHOD DEVELOPMENT CHROMATOGRAM

RESULTS AND DISCUSSION

Results: Method Validation Parameters:

Specificity: Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. The separation between the peak of Ascomycin and Tacrolimus and any blank interference is studied as specificity by injecting all the components individually and mixed at the specification limit. The results were tabulated in 3 and chromatogram shown in 3 & 4.

TABLE 6: PEAK NAME WITH RETENTION TIME

FIG. 3: BLANK CHROMATOGRAM

FIG. 4: CHROMATOGRAM OF TACROLIMUS

System Suitability: System suitability parameters are performed by injecting prepared standard solution in six times and measured the parameters like theoretical plates, retention time, tailing factor and %RSD. The results show in Table 6.

TABLE 7: RESULTS OF SYSTEM SUITABILITY

Linearity: It is ability of developed method to obtain test results shown in Table 8 that are directly proposed to the sample concentration over a given range, Calibration standard of covering the range 3-22.5µg/ml were prepared with the suitable dilution made from stock solution. The calibration curve was plotted between peak response and concentration of the sample and calibration curve shown Fig. 5.

TABLE 8: RESULTS OF LINEARITY

Calibration Curve:

FIG. 5: CALIBRATION CURVE OF ASCOMYCIN

Accuracy: The accuracy was performed for this method by conducting the recovery studies were carried out by adding three different concentration level 50%, 100%, 150% respectively.

The % recovery was found and tabulated in 3.4 to be in the range 95.29-100.29%. Chromatograms of different concentration levels show in Fig. 6, 7 and 8.

TABLE 9: RESULTS OF ACCURACY

FIG. 6: CHROMATOGRAM SHOWING 50% ACCURACY RECOVERY 

FIG. 7: CHROMATOGRAM SHOWING 100% ACCURACY RECOVERY

FIG. 8: CHROMATOGRAM SHOWING 150% ACCURACY RECOVERY

Precision: Method precision is determined by analysing a sample solution and six sample preparations from a homogenous mixture to which the Ascomycin impurity is spiked at the specification limit.

The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. The results shown in the Table 10.

TABLE 10: METHOD PRECISION RESULTS

Limit of Detection & Limit of Quantification: Limit of detection (LOD) and limit of quantification (LOQ) of Dacomitinib were determined from the calibration curve method. The results were tabulated in 3.6.

TABLE 11: RESULTS FOR DETECTION AND QUANTIFICATION LIMITS

Robustness: Robustness of the method was determined by small deliberate changes in the method parameters and measuring the effect on the method by monitoring system suitability test. The obtained results shown in Table 12.

TABLE 12: RESULTS OF ROBUSTNESS STUDIES

CONCLUSION: The Reverse phase HPLC Method for the determination of Ascomycin content in Tacrolimus is simple, precise, specific, and accurate and less time consumption could be recorded for analysis. The method was reliable in terms of system suitability, linearity, precision, accuracy, LOD, LOQ, recovery, and robustness. All the verification parameters were within the range according to ICH guidelines. So, this method can be applied to stability-indicating studies. In this manner, future works can be useful to carry out Impurity profile studies and Pharmacokinetic studies.

ACKOWLEDGEMENT: We are grateful to Teyro labs private limited for providing gift sample.

Authors Contribution Statement: Mahesh designed the whole study including Selection of drug, literature collection, and plan of work at JNTUA-Oil Technological and Pharmaceutical Research Institute and prepared the manuscript. Harikumar Naik conducted method development and validation in JNTUA-Oil Technological and Pharmaceutical Research Institute. Sai Lakshmi prepared the part of the manuscript. All the authors read and approved the final version of the manuscript.

CONFLICTS OF INTEREST: Conflict of interest declared none.

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    How to cite this article:

    Mahesh M, Naik LHK and Lakshmi KS: Analytical method development and validation of ascomycin content in tacrolimus API by using RP HPLC. Int J Pharm Sci & Res 2024; 15(1): 145-52. doi: 10.13040/IJPSR.0975-8232.15(1).145-52.

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