Development and Validation of UV Spectroscopy Method for the Determination of Posaconazole in Bulk and Formulation

 

Shivprasad Patil*, Dr. Ajay Kshirsagar, Kartik Ade, Akash Bharkade,

Madhav Bharkade, Ashish Birkalwar, Mahesh Chandolkar

Department of Pharmaceutical Quality Assurance, D. K. Patil Institute of Pharmacy, Loha, Maharashtra, India.

 

ABSTRACT:

The present study was undertaken to develop a spectrophotometric method for the determination of Posaconazole (PCZ) in pharmaceutical dosage forms. This paper describes a simple, rapid, accurate, and precise UV-spectrophotometric method for the assay of PCZin bulk and marketed dosage forms. The validation of the developed method was carried out according to ICH guidelines concerning linearity, precision, accuracy, specificity, the limit of detection, and the limit of quantification. The diluent is aqueous methanol. Calibration curves were obtained in the concentration range of 04-20µg/ml for PCZ and with good correlation coefficients (R²=0.9981). The precisions of the new method for the drug were less than the maximum allowable limit (%RSD < 2.0) specified by the ICH. Therefore, the method was found to be accurate, reproducible, and sensitive for the analysis of PCZ in pharmaceutical dosage forms.

 

 

 

INTRODUCTION:

Posaconazole, sold under the brand names Noxafil and Posanol is a triazole antifungal medication. It was approved for medical use in the United States in September 2006 and is available as a generic medication. It is also used to treat invasive infections by Candida, Mucor, and Aspergillus species in severely immunocompromised patients.^1-5 Clinical evidence for its utility in the treatment of invasive diseases caused by Fusarium species is limited. It appears to be helpful in a mouse model of naegleriasis.

 

 

Posaconazole works by disrupting the close packing of acyl chains of phospholipids, impairing the functions of certain membrane-bound enzyme systems such as ATPase and enzymes of the electron transport system, thus inhibiting the growth of the fungi. It does this by blocking the synthesis of ergosterol by inhibiting the enzyme lanosterol 14α-demethylase and accumulation of methylated sterol precursors. Posaconazole is significantly more potent at inhibiting 14-alpha demethylase than itraconazole.⁶

 

Posaconazole is used to treat invasive Aspergillus and Candidainfections. Posaconazole is active against the following microorganisms:

Candida spp.

Aspergillus spp.

Zygomycetes spp.

 

Posaconazole is absorbed within three to five hours. It is predominately eliminated through the liver and has a half-life of about 35hours. Oral administration of posaconazole taken with a high-fat meal exceeds 90% bioavailability and increases the concentration by four times compared to the fasting state. Posaconazole’s chemical formula is C₃₇H₄₂F₂N₈O₄ and has a molecular weight of 700.8gm/mol.^1-6

 

Figure 1: Structure of PCZ

 

In the literature review, various methods are available for the determination of PCZ in drugs by UV spectroscopy. The present work is therefore focused on achieving the optimum chromatographic conditions for the determination of PCZ in a formulation by UV spectroscopic method, and validated according to ICH guidelines.^7-8

 

MATERIALS AND METHOD:

Materials:

The drug was obtained from Dr. Reddy's Laboratories Pharma Ltd. Hyderabad, India. All chemicals and reagents used were of analytical grade. The formulation Posoxil GR 100 mg was gifted from Hetero Healthcare Ltd.

 

Instruments:

Analytical balance (Shimadzu AY220),

Sonicator (Microclean-1103),

UV-Visible spectrophotometer (Shimadzu 1800).

 

Methods:

Preparation of standard stock solution:(WS)

An accurately weighed 10mg of Posaconazole API was dissolved in 10ml of aqueous methanol using a 10 ml volumetric flask. The solution was then sonicated for 10min and the final volume was adjusted up to the mark with the diluent, to obtain the final concentration of 1000 µg/ml.

 

Drug Product Sample Preparation:(DP)

Accurately 10 tablets were weighed individually and the average weight was calculated and powdered. The tablet powder equivalent to 10mg of PCZ was transferred into a 10ml volumetric flask, 5ml of diluent was added and sonicated for 10minutes at a controlled temperature to dissolve the powder, further, the volume was made up with diluent and filtered through 0.45µ membrane filter. From this solution, 0.1ml was diluted to 10ml with the same diluent to give a concentration of 10 µg/ml of PCZ.

 

Selection of wavelength:

Posaconazole standard solution (20µg/ml) was prepared by appropriate dilution of standard stock solution and then scanned in the UV range in between 200-400nm. PCZ showed the absorption maxima at 265 nm.

 

RESULTS AND DISCUSSION:

The absorption spectrum shows λ max of PCZat 265nm.

 

Figure 2. UV spectrum of PCZ

 

The proposed method was validated according to ICH Q2R1 guidelines for the validation of the analytical procedure.

 

Range:

The range of analytical procedures is the interval between the upper and lower concentration of analyte in the sample. The linearity of the method was determined at 5 concentration levels ranging from 04-20µg/ml for PCZ.

 

Linearity:

Linearity is evaluated by visual inspection of the plot of the signal as a function of analyte concentration. If there is a linear relationship test results are calculated by regression line by the method of least squares. The linearity of the method was determined at 5 concentration levels ranging from 04-20µg/ml for PCZ. Evaluation of the drug was performed at 265nm, peak area was recorded for all the peaks. The correlation coefficient value of PCZ was 0.9981. The results show that an excellent correlation exists between absorbance and concentration of drug within the concentration range indicated.

 

Table 1: Results of Linearity

 

Figure 3: Calibration curve forPCZ

 

Table 2: Optimization parameters of PCZ

 

Accuracy:

Accuracy is the measure of exactness of an analytical method or the closeness of agreement between the value that is accepted either as a conventional true value or an accepted reference value and the value found. It is measured as the percent of analyte recovered by assay, by spiking samples in a blind study.

 

Tablet Samples were made of 80%, 100%, and 120% that is 8, 10, and 12µg/ml respectively concentrations for PCZ.

 

 

Samples were scanned in triplicate to calculate % RSD.

 

% Recovery was also calculated.

 

Precision:

The closeness of agreement between a series of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision, and reproducibility. Precision intra-day and inter-day precision were performed at concentrations (10µg/ml). The obtained results were found within the limit that is less than 2% RSD. A single WS was prepared as described and 6 absorbance measurements were made from the same WS; checked for %RSD.

 

 

Limit of Detection (LOD):

The limit of detection is defined as the lowest concentration of the analyte in the sample that can be detected, though not necessarily quantitated. Limit tests specify whether an analyte is above or below a certain value. The limit of detection was found to be 1.30µg/ml (Table 5).

 

Limit of Quantification (LOQ):

The limit of quantitation (LOQ) is defined as the detection of the lowest amount of analyte present in the sample. It is the parameter that gives the actual concentration of an analyte in a sample which can be determined with acceptable precision and accuracy under the stated operating conditions of the method during analysis. The limit of quantification was found to be 3.95 µg/ml (table 5).

 

Table 3: Results of Accuracy

 

Table 4: Results of Intra-day Precision

 

 

Table 5: Results of LOD and LOQ

 

ROBUSTNESS:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Changes in wavelength (264nm and 266nm) and concentration (10µg/ml) didn’t affect the results. Robustness is defined as the capacity of a method to remain unaffected by small but deliberate variations in method parameters. The robustness of a method is evaluated via various method parameters which include the percentage of organic solvent, pH, ionic strength, or temperature, and determining the effect on the results of the method.

 

Table 6: Results of Robustness

 

ASSAY:

For the assay, the minimum specified range is from 80-120 % of the target concentration. For an impurity test, the minimum range is from the reporting level of each impurity to 120 % of the specification. The assaywas performed and the % purity was foundto be 98.71.

 

Table 7:  Results of Assay

 

CONCLUSION:

The presented method was found to be simple, precise, accurate, rugged, and reproducible which gives an acceptable recovery of the analyte, which can be directly easily applied to the analysis of pharmaceutical formulation of PCZ. An analytical UV spectrophotometric method was developed and validated thoroughly for the quantitative determination of PCZin bulk drugs and formulation. The presented method was found to be simple, precise, accurate, rugged, reproducible, and gives an acceptable recovery of the analyte, which can be directly easily applied to the analysis of pharmaceutical formulation of PCZ.

 

ACKNOWLEDGEMENT:

The authors are grateful to the Principal and Management for providing the necessary facilities for completing this research work successfully.

 

REFERENCES:

1.      ICH Q2 (R1). Validation of Analytical Procedures: Text and Methodology. ICH Harmonised Tripartite Guidelines, 1994.

2.      Kim H, Likhari P, Lin C, Nomeir A. High-performance liquid chromatographic analysis of the anti-fungal agent SCH 56592 in dog serum. J Chromatogr. B 2000; 738:93- 98.

3.      Zhong W, Yang X, Tong W, Martin GE. Structural characterization of a novel degradant of the antifungal agent posaconazole. J Pharm Biomed Anal. 2012; 66:40-49.

4.      Ekiert RJ, Krzek J. Determination of azole antifungal medicines using zero-order and derivative UV spectrophotometry. Acta Pol Pharm. 2009; 66:19-24.

5.      Chhun S, Rey E, Tran A, Lortholar O, Pon G, Jullien V. Simultaneous quantification of voriconazole and posaconazole in human plasma by high-performance liquid chromatography with ultra-violet detection. J Chromatogr. B 2007; 852: 223-228.

6.      Kim H, Kumari P, Laughlin M, Hilbert M, Indelicato S, Lim J, Lin C, Nomeir A. Use of high-performance liquid chromatographic and microbiological analyses for evaluating the presence or absence of active metabolites of the antifungal posaconazole in human plasma. J Chromatogr. A 2003; 987: 243-248.

7.      Amitkumar J. Vyas, Brijesh H. Patel, Ashok B. Patel, Ajay I. Patel, Nilesh. K. Patel. A brief Review on Q-absorption Ratio Method in UV-Spectrophotometry. Asian Journal of Pharmaceutical Analysis. 2022; 12(4): 281-5.

8.      Sama Venkatesh, Vijaya Laxmi, Rajeswari Chiluka, Yenumula Padmavathi. Development of UV- Spectrophotometric Method for Simultaneous Estimation of Metformin and Piperine in Combined Tablet Dosage Form. Asian Journal of Pharmaceutical Analysis. 2022; 12(4): 221-7.

 

 

 

Received on 26.08.2022       Modified on 31.12.2022

Accepted on 10.06.2023   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2023; 13(3):171-174.