Solubility Reinforcement using Binary Solvent Mixtures and its use in Development and Validation of UV-Spectrophotometric Method for Posaconazole

Tushar G. Rukari^1,2*, Prashant L. Pingale³, Chandrashekhar D. Upasani⁴

¹Research Scholar, Department of Pharmaceutics,

SNJB's Shriman Suresh Dada Jain College of Pharmacy, Chandwad, Maharashtra 423101, India.

²Asst. Professor, Department of Pharmaceutics,

Yashwantrao Bhonsale College of Pharmacy, Sawantwadi, Maharashtra 416510, India.

³Research Guide, Department of Pharmaceutics, M.S. Gosavi College of Pharmaceutical Education and Research, Vidyanagar, College Road, Nashik 422005, India.

⁴Research Centre Head, Department of Pharmacology,

SNJB's Shriman Suresh Dada Jain College of Pharmacy, Chandwad, Maharashtra 423101, India.

*Corresponding Author E-mail: tushar.rukari@gmail.com

ABSTRACT:

Objectives: To develop a method that meets the requirements as per ICH guidelines for validation and can be used as simple, specific, economical, and repeatable for routine quality control analysis of formulations containing Posaconazole. Materials and Methods: The maximum absorbance of Posaconazole was successfully recorded using a precise 1:1 ratio of binary solvent system Methanol: Buffersolution pH 7.4 at 262nm. Results: After a preliminary physicochemical investigation the precipitation of the Posaconazole issue was resolved using a precise 1:1 ratio of binary solvent system Methanol: Buffersolution pH 7.4. Development and validation of the analytical method was done using the same binary solvent system. The maximum absorbance with this solvent system was found at 262nm, while the calibration curve shows a Regression coefficient (R²) of about 0.9984 for its linearity in the concentration range of 2.5 to 15µ/ml. The developed method was validated and meets the requirements as per ICH (International Conference for Harmonization) guidelines where the Limit of Detection (LOD) as well as Limit of Quantification (LOQ) were noted as 0.74µ/ml and 2.25µ/ml. Percentage recovery was within the permissible range (with a % relative standard deviation of less than 2.0). Conclusion: The results from validation parameters specifically accuracy or % recovery and robustness study reveal that the developed method meets the requirements and can be used as simple, specific, economical, and repeatable for routine quality control analysis of formulations containing Posaconazole.

KEYWORDS: Posaconazole, Validation, Spectrophotometric method, ICH Q2(R2).

1. INTRODUCTION:

To collect scientific data about the drug or API almost every researcher tries to begin his scientific work by analyzing preliminary parameters such as organoleptic evaluation, determination of intrinsic properties such as melting or boiling point, solubility profile of the drug or API as well as updating is research work with the compatibility studies with several excipients and concluding its preformulation studies with the development of suitable analytical technique. The developed analytical technique helps the researcher forqualitative and quantitative estimation of analytes. With the same context, in this research, we tried to summarize the preliminary physicochemical investigation and validation of the UV-Spectrophotometric method for Posaconazole (PSZ).

Figure 1. Structure of pure Posaconazole

Posaconazole (Figure 1) is a second-generation triazole drug that exhibits strong and broad antifungal in-vitro action against numerous fungal infections, such as Aspergillus species and Candida species. It shares structural similarities with Itraconazole and blocks lanosterol 14-demethylase (CYP51), impairing cell membrane integrity and accumulating precursors that have fungistatic or fungicidal effects.^1,2Posaconazole's absorption is dose-limited and highly reliant on dietary intake because of its low solubility in aqueous (1 µg/mL), which is almost insoluble,³and possess good solubility in Methanol and Chloroform.Researchers may face challenges in developing dosage forms, particularly when using dissolution testing to assess drug dissolution capabilities in the GI tract.However, due to the limited solubility of PSZ, it might be questionable to use alone buffer solution as a solvent medium for analytical testing of dissolution samples.The remedy for the same is, may the use of co-solvent.⁴While doing a review of the literature, we found that PSZ content determined with the help of phosphate buffer pH 7.4^5–11and researchers used the UV-Spectrophotometric method for sample analysis, but no solubilization was attempted.UV-visible spectrometry is frequently used in the pharmaceutical industry due to its simplicity, precision, focus, and sensitivity.^12–16

At the end of the literature survey, we concluded to develop a simple, specific, and economical UV-visible spectrometry method for estimatingPSZas a bulk or in formulation.

2. MATERIALS AND METHODS:

Posaconazole (PSZ) was obtained as a gift sample from Everest Organics Ltd., Hyderabad, Telangana, India. All other chemicals used for solubilization, or preparation of buffer were procured from a central chemical store of Yashwantrao Bhonsale College of Pharmacy, Sawantwadi.

2.1 Organoleptic evaluation:

The API sample received was evaluated for its organoleptic evaluation such as color, appearance, odor, etc.

2.2 Determination of melting point:

Each substance's melting point is an inherent attribute. Melting points are commonly used to describe and assess the chemical purity of both inorganic and organic compounds. A pure substance's melting point is always higher and has a narrower range than an impure substance's or, more broadly, a mix's melting point.The capillary tube method was used to determine the melting point of PSZ.⁶

2.3 DSC study:

The DSC-60 (Make-Shimadzu) was used to conduct the study. The samples (5mg) were subjected to heating at an intensity of 10⁰C/min between 32⁰C and 300⁰C while contained in an aluminum pan. Figure 2 represents the DSC thermogram of Pure Posaconazole.

Figure 2. DSC thermograph of Pure Posaconazole

2.4 Solubility study:

The solubility of Posaconazole in various solvents was tested qualitatively. It was calculated by stirring 10.0 mg of the drug substance in 10.0 ml of various solvents in a tiny test tube for 30 minutes while keeping track of how long it took for the sample to vanish.The sample PSZ shows slight solubility in Phosphate Buffer 6.8, Phosphate Buffer 5.5, Borate Buffer 9.0 and is freely soluble in Chloroform and Methanol while insoluble in Water.

2.5 FTIR Studies:

To derive specific structural information from spectrum data, FTIR studies were employed. Posaconazole's IR absorption spectra (Figure 2) was measured by the KBr disc method using an IR spectrophotometer(Make-Jasco FTIR-4100).

Figure 3. FTIR spectra of Posaconazole

2.6 XRD Study:

Utilizing Cu as a radiation source and an X-ray diffractometer (Bruker D8 ADVANCE, Bruker, USA). The 0-89.6242-degree diffraction angle 2-Theta scan range was used. To measure the XRD pattern, a 40 kV voltage and a 40-mA current were used. Figure 4 displays the pure drug's XRD pattern.

After performing physicochemical characterization, we proceededwith the development of the UV-Spectrophotometric method, where we tried to rectify the problem associated withthe solubilization of PSZ.

Figure 4. XRD diffractogram of pure Posaconazole

3. UV Spectrophotometric Method Development:

3.1 To finalize the solvent system for solubilization of Posaconazole:

As the drug possesses insolubility in water and is freely soluble in methanol, it was the first task to finalize the solvent system. As per the content read out in a number of research articles, we found some researchers used phosphate buffer 7.4, some have pH 6.8 buffer solution in addition to 0.5% SLS, and somewhere we found that the drug is dissolved in a small quantity of methanol and then diluted with phosphate buffer. But practically it was not happening, by following any one of the methods for solubilization of Posaconazole we found the precipitation of the drug. To rectify this issue we tried to add some quantity of methanol and were surprised by the disappearance of precipitation (Figure 5).

Figure 5. Solubility issues observed in solubility and its rectification.

This indicates that there might be some ratio of phosphate buffer 7.4 and methanol at which we can get a completely clear solution. Hence, we tried some ratio of Methanol: Phosphate buffer pH 7.4 till we got a clear solution. For the same, first, we dissolved the 10 mg Posaconazole in 5ml of methanol and then tried to go the addition of phosphate buffer 7.4, the observations are as follows in Table 1.

Table 1. Finalization of Methanol:Phosphatebuffer pH 7.4 ratio for the calibration curve

Sr. No.	Methanol	Phosphate Buffer	Ratio	Observations
1.	5 ml	5 ml	1: 1	Clear solution
2.	5 ml	7.5 ml	1: 1.5	Translucent solution
3.	5 ml	8.5 ml	1: 1.7	Precipitation

The above observation indicates that in the ratio of 1: 1 of methanol: phosphate buffer 7.4 the drug is completely soluble and gives a clear solution. Hence it was decided to use a mixture of methanol and phosphate buffer 7.4 in the ratio of 1: 1.

3.2 Determination of λ_max of Posaconazole:

A stock solution of 10 µg/ml was prepared in a 1:1 ratio of Methanol: Phosphate buffer pH 7.4. This stock was analyzed to determine the λ_maxusing a UV Spectrophotometer (UV-1900, Shimadzu) in the range between 200nm to 400nm. As shown in Figure 6, at 262 nm we found the maximum peak area and the value of absorbance was adjacent tothe value of λ_maxobtained from the literature. Hence λ_max262 nm was considered as the final wavelength of a received sample of Posaconazole and can be used for further estimation of the drug.

Figure 6. λ_maxof a received sample of Posaconazole

3.3 Establishment of calibration curve:

At the time of determination of λ_max, absorbance was found to be about 1.471 at the wavelength of 262nm. Hence, instead of withdrawing 1ml sample we decided to withdraw 0.5ml and diluteup to 10ml with solvent to give a solution of 5µg/ml and so…. and the solution was scanned at 262nm and absorbance was measured computed in Table 3.As the absorbance we found is much more than expected (Table 2), with this observation we decided to decrease the concertation of API. For the same, with slight modifications in the standard procedure where 5 mg of PSZ was dissolved in a solvent to give 50µg/ml stock solution and then withdrawn 0.5 ml and diluted up to 10ml with solvent to give a solution to give 2.5µg/ml and so…. and the solution is scanned at 262nm and absorbance was measured. For the said concentration the absorbance was found to be less than 1 while R²found was 0.9933 (Figure 7).

Table 2. Absorbance of Posaconazole from the calibration curve

Sr. No.	Conc. (µg/ml)	Absorbance (n=3)	Conc. (µg/ml)	Absorbance (n=3)
1.	5	0.025	2.5	0.119
2.	10	0.092	5	0.214
3.	15	0.256	7.5	0.299
4.	20	0.644	10	0.401
5.	25	0.811	12.5	0.454
6.	30	0.953	15	0.512
7.	35	1.149	17.5	0.595
8.	10	1.331	20	0.646
9.	45	1.549	22.5	0.742
10.	50	1.775	25	0.786

Figure 7. Calibration curves of Posaconazole from the concertation 5µg/ml to 50µg/ml vs 2.5µg/ml to 25µg/ml

4. Validation approach:

In accordance with the standards of the International Conference for Harmonization (ICH), the developed method was validated.^17,18

4.1 System Suitability Test (SST):

Concerning ICH guidelines, to determine system applicability, we utilized the same experimental settings for calibration and attempted to run to the spectrum between 200nm and 400nm for the mixture in the solvent Methanol:Phosphate buffer 7.4 (blank). The spectrum ran three times and observed that the solvent system or other factor did not interfere with the spectrum as absorbance was detected.^{12,13,15–28}

4.2 Linearity:

As per ICH recommendations, six representative solutions were produced, with concentrations ranging from 2.5/ml to 15/ml. Three absorbance measurements (n=3) were collected for each concertation, and the average was calculated. Observations are computed in Table 3, Figure 8.^{12,13,15–28}

Table 4. Intra Day Precision

Conc. (µg/ml)	Abs. (n=1)	Abs. (n=2)	Abs. (n=3)	Abs. (n=4)	Abs. (n=5)	Abs. (n=6)	Mean Abs.	SD	% R.S.D.	Acceptance Criteria
15	0.6958	0.6878	0.6784	0.6687	0.6842	0.6754	0.6817	0.0096	1.41	% R.S.D Not more than 2

Table 5. Intermediate precision

Conc. (µg/ml)	Abs. (n=1)	Abs. (n=2)	Abs. (n=3)	Abs. (n=4)	Abs. (n=5)	Abs. (n=6)	Mean Abs.	SD	% R.S.D.	Acceptance Criteria
Day-I
15	0.6958	0.6878	0.6784	0.6687	0.6842	0.6754	0.6817	0.0096	1.41	% R.S.D Not more than 2
Day-II
15	0.6546	0.6562	0.6635	0.6658	0.6529	0.6562	0.6582	0.0052	0.79	% R.S.D Not more than 2

Table 6. % Recovery by spiking known amount of analyte

Targeted conc. (µg/ml)	Abs. measured for accuracy	Analysed conc.	Conc. after spiking (5 µg/ml)	Abs. after spiking a known amount	Analysed conc. after spiking a known amount	% Recovery	Mean	SD	% RSD
2.5	0.1411	2.54	7.5	0.3485	7.30	97.31	97.35	0.19	0.192
	0.1409	2.54	7.5	0.3493	7.32	97.55
	0.1411	2.54	7.5	0.3481	7.29	97.19
5	0.2632	5.34	10	0.4574	9.80	97.96	97.63	0.67	0.65
	0.2685	5.46	10	0.4578	9.81	98.05
	0.2639	5.36	10	0.4527	9.69	96.88
15	0.6852	15.02	20	0.8704	19.27	96.34	96.11	0.21	0.22
	0.6643	14.54	20	0.8668	19.19	95.93
	0.6791	14.88	20	0.8679	19.21	96.06

Table No. 7. Robustness study

Targeted conc. (µg/ml)

% Amount

Mean±SD, (n=6)

% RSD

Analyst I

Analyst

Analyst I

Analyst II

98.87 + 0.45

99.96+0.05

0.459

0.053

Table 8. Summary of the validation approach

Sr. No.	Optical parameters	Observation in binary solvent (Ratio 1:1)
1.	Absorbance maximum (λmax)	262nm
2.	Beer’s Law Limit (μg/ml)	2.5µg/ml to 15µg/ml
3.	Equation	y = 0.0436x + 0.0303
4.	Slope	0.0436
5.	Intercept	0.0303
6.	Regression coefficient	R² = 0.9984
7.	LOD (µg/ml)	0.74
8.	LOQ (µg/ml)	2.25

5. RESULTS AND DISCUSSION:

A preformulation study was performed for the authentication of PSZ. The organoleptic evaluation found an odorless, white, crystalline powder of PSZ which is confirmatory with COA obtained from the manufacturer. Further confirmation of PSZ was confirmed by melting point, the melting of PSZ was found to be 172⁰C and the result is within the range (170⁰C - 172⁰C). authentication of the melting point of PSZ was done by comparing the obtained data with the DSC thermogram. Two different endothermic events were occurring around 100⁰C to 200⁰C. The first endothermic peak denotes the glass transition state while the second peak is observed sharp with maximum area under the curve. The melting temperature from the DSC thermogram was found to be 172.26⁰C matches the standard value as well as confirms the purity as there is no shifting of melting point.

The result from the XRD study shows characteristics peaks at 10.00, 11.050, 12.800, 15.200, 15.600, 17.600 (high intensity), 25.200, 26.000, 27.200, 29.200, 43.210, 43.610 confirms the crystalline nature of Posaconazole. IR spectroscopy was used to assess the structural conformance for the presence of various functional groups. Observed IR frequencies of pure drug compared with standard functional group frequencies and the data reveals characteristic peaks at 2967.91 cm^-1, 1685 cm^-1, 1015.34 cm^-1, 1370.18 cm^-1, 1228.43 cm^-1, 1061.62 cm^-1, 1087.66 cm^-1. These observed frequencies correlatewith the functional groups O-H Stretching, C=O (3 amides) stretching, I-F (Fluro compound), C-N (aromatic amine) Stretching, C-O (alkyl-aryl ether) stretching, C-N (amine) Stretching, C-O Stretching (2 alcohol) stretching correspondingly, and presence of these functional groups was confirmed from the structure.

Before proceeding with the development of the analytical method, the ratio of 1: 1 of methanol: phosphate buffer 7.4 of the solvent system was decided and the same solvent system was continued for further method development and validation. PSZ shows maximum absorbance at the wavelength of 262 nm. The calibration curve shows a correlation coefficient (R²) around 0.9984 proving the linearity within the 2.5 to 15 µg/ml. The developed method was validated and meets the requirements as per ICH (International Conference for Harmonization) guidelines where the Limit of Detection (LOD) as well as Limit of Quantification (LOQ) were noted as 0.74 µ/ml and 2.25 µ/ml.

According to ICH criteria, the newly developed UV spectroscopic method was verified as per the norm observed values tested statically, and % RSD was found less than 02 (from the observation tables) is an indication of validation of the developed method.

6. CONCLUSION:

The preliminary study's authenticated PSZ sample was used to move forward with the development of analytical techniques. The straight forward UV spectrophotometric method was created and verified in accordance with ICH standards for the quantification of PSZ in solvent Methanol:Buffer solution pH 7.4 in the ratio 1:1. We draw the conclusion that the suggested method is straightforward, precise, affordable, and repeatable for routine quality control analysis of formulations including PSZ because it satisfies the ICH validation criteria. At last, we like to conclude our study with the notation that, if any researcher wants to work on PSZ, can use methanol at the time making of volumes or dilution of aliquoted volume removed from dissolution media which reinforces the solubilization instead of purely using aqueous based solvent or buffer solutions.

7. CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 29.10.2023 Modified on 26.02.2024

Research J. Pharm. and Tech 2024; 17(8):3700-3706.

DOI: 10.52711/0974-360X.2024.00576

Conc. (µ/ml)	Abs. (n=1)	Abs. (n=2)	Abs. (n=3)	Mean Absorbance	Observation	Acceptance criteria
2.5	0.1295	0.1313	0.1331	0.1313	Correlation Coefficient Not less than 0.9984	Correlation Coefficient 0.999
5	0.2558	0.2541	0.2581	0.2560
7.5	0.3595	0.3567	0.3581	0.3581
10	0.4641	0.4668	0.4643	0.4651
12.5	0.5864	0.5906	0.5822	0.5864
15	0.6694	0.6806	0.6753	0.6751