RP-HPLC Bioanalytical Method Development and Validation of Posaconazole

INTRODUCTION:

Posaconazole is a triazole antifungal drug that works against many different kinds of fungi. It is frequently employed to treat and prevent invasive fungal infections, particularly in individuals with compromised immune systems (1). It can strongly fight fungi of the genus Aspergillus, Candida, and other species that are important to public health (2). For pharmacokinetic study and therapeutic drug monitoring, it's very important to acquire an exact measurement of posaconazole in biological matrices because its therapeutic window is small, its bioavailability changes, and it may interact with other drugs (3). A lot of different diagnostic methods have been recorded for finding out the amount of posaconazole there is, including high-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC–MS/MS) (4). Even though LC-MS/MS techniques are very sensitive, they are often associated with high running costs, difficult sample preparation, and limited availability in clinical and routine quality control labs (5). For bioanalytical uses, though, reverse-phase high-performance liquid chromatography (RP-HPLC) with UV detection is a cheap, reliable, and easy-to-use replacement (6). A simple and dependable RP-HPLC method for measuring posaconazole in human plasma needs to be created so that regular bioanalysis can be done more easily (7). Bioanalytical technique validation must follow regulatory guidelines, which say that selectivity, linearity, accuracy, precision, recovery, robustness, stability under different circumstances, and other factors must be checked (8). Proper sample preparation, such as protein precipitation, makes the extraction process even more efficient and consistent (9). The goal of this work is to come up with a simple, quick, accurate, and precise RP-HPLC method with UV detection that follows normal bioanalytical validation rules so that posaconazole can be measured in human plasma (10). The accepted method is supposed to work for pharmacokinetic studies and routine checks on how well therapeutic drugs are working (11).

Figure 1: Structure of Posoconazole

MATERIALS AND METHODS :

Instruments and Chemicals:

A pure sample of posaconazole was acquired from Brick Pharmaceuticals. Throughout the investigation, analytical-grade water and HPLC-grade acetonitrile were utilized. Every chemical and reagent used was pure enough for chromatographic analysis. The Agilent 1120 Compact LC system was utilized to perform the chromatographic analysis. A Shimadzu UV-1700 UV/Visible spectrophotometer was used to perform UV spectrophotometric measurements. An LC/GC analytical balance was used to weigh the samples, and a Lifecare ultrasonic bath was used to help prepare them.

Conditions for Optimization of Chromatographic Method:

To get the best mobile phase, the resolution, peak symmetry, and peak area for posaconazole were measured. Different mobile phase mixtures were tested, such as acetonitrile (HPLC grade) and water (HPLC grade) in different ratios and methanol and water. Acetonitrile: water at a flow rate of 0.8 ml/min turned out to be the best of these because it gave uniform, well-resolved peaks for posaconazole. Chromatographic detection was done at 262 nm, which is posaconazole's highest absorbance. The chromatogram had a good peak shape and a fair resolution of the standard drug. The tailing factor was within the acceptable range. Posaconazole's retention period was found to be 4.683 minutes. The optimized mobile phase was used to find the system suitability parameters. These were shown to be within the appropriate range (12, 13).

Mobile Phase Preparation:

The mobile phase was HPLC-grade acetonitrile and HPLC-grade water mixed together in a 55:45 (v/v) ratio. It was filtered through a 0.45-µm membrane filter and sonicated in an ultrasonic bath for 15 minutes before being used (14).

Standard Stock Solution Preparation:

After 2.5 mg of the working standard was carefully weighed, it was put into a 25 mL volumetric flask to make a standard stock solution of posaconazole. About 10 mL of HPLC-grade methanol was added, and then the mixture was sonicated for 20 minutes to make sure that everything dissolved completely. Then, 25 mL of HPLC-grade water was added to the mixture to get a final concentration of 100 ppm. The solution that was made was filtered using a nylon-66 membrane filter with a pore size of 0.45 µm. The stock solution was properly mixed with methanol to make the working standard solutions that came next (15-17).

Human Blood Plasma Separation:

The human blood plasma was given by Neon Laboratory. Blood samples were taken using purple-top EDTA tubes. The samples were then spun in a centrifuge for 20 minutes at 4°C at 3000 rpm. After spinning, the plasma was carefully separated using a clean pipetting method. 1.0 mL of plasma was then placed into a 1.5 mL Eppendorf tube that was marked with the right tracking number and labeled as plasma (18, 19).

Sample Solution Preparation:

The sample solutions were made by mixing 100 µL of working standard solutions of posaconazole at 0.150, 0.5, 1.5, 2.5, 3.5, and 5 ppm with 0.90 mL of rat plasma. 1.0 mL of acetonitrile was added to the mixture as a precipitating agent to make plasma proteins form, and it was then vortexed for a long time. The samples that were centrifuged for 15 minutes at 3000 rpm at 2–4°C for 15 minutes showed end concentrations of 1.5, 5, 15, 25, 35, and 50 ppm. Once the clear supernatant was properly sorted, chromatographic analysis was done (20).

Spiking of Posaconazole in plasma

To achieve the necessary calibration concentrations, posaconazole was spiked in plasma by adding suitable volumes of the posaconazole working standard solutions to blank plasma. To guarantee that the medication was distributed evenly, precisely measured volumes of the standard solutions were combined with plasma samples. Following centrifugation and protein precipitation with acetonitrile, the spiking plasma samples were collected for RP-HPLC analysis of the clear supernatant (21, 22).

Table 1: Human Blood Plasma Spiking of Posaconazole

Sr. No	Concentration (ppm)	Volume of Spiking (ml)	Volume of Plasma (ml)	Final volume (ml)	Final concentration (ppm)
1	05	0.1	0.9	1	0.5
2	10	0.1	0.9	1	1.0
3	15	0.1	0.9	1	1.5
4	25	0.1	0.9	1	2.5
5	35	0.1	0.9	1	3.5
6	50	0.1	0.9	1	5.0

Method Development and Validation:

When bioanalytical methods are validated, it means that the data they give us about biological things is correct. Selectivity, sensitivity, accuracy, precision, consistency, recovery, linearity, and stability are critical validation features. To show that the method used is correct, it is necessary to confirm each substance that is measured in a biological matrix. The current RP-HPLC bioanalytical method was created and validated after its selectivity, accuracy, precision, recovery, calibration curve linearity, and analyte stability in treated plasma samples were all tested (23).

Selectivity: Blank plasma samples from at least six different sources were tested to see if they would interfere with the selection test. The retention time of posaconazole showed no peaks that could have interfered, which means that the method had enough specificity (24).

Calibration Curve: Calibration curves over a concentration range of 5–100 ppm were possible by plotting the peak area against the standard concentration. The data, which showed strong linearity over the tested range, are summed up in Table 2 (24).

Quality Control Samples Preparation: Quality control (QC) samples were made with three different amounts of concentration: low quality control (LQC, 1.5 ppm), medium quality control (MQC, 25 ppm), and high quality control (HQC, 50 ppm) (24).

Accuracy and Precision: The method's precision was checked by repeating measurements of the LQC, MQC, and HQC samples three times each. Both intraday and interday accuracy were tested. a. To find out how accurate and precise the three different amounts were within the same batch, they were each measured twice on the same day. b. To check inter-batch accuracy and precision, the same amounts were analyzed twice on different days (24).

Recovery: Comparing the analytical reactions of extracted plasma samples at low, medium, and high QC levels with those of unextracted reference solutions—which indicated 100% recovery—was how recovery investigations were carried out (24).

Stability Studies

Stability of posaconazole in plasma was assessed under different conditions, including freeze–thaw, short-term, and long-term stability. For freeze–thaw stability, samples underwent three cycles and were analyzed after the third cycle. Short-term stability was evaluated by keeping three aliquots each of LQC and HQC samples at room temperature for 8 hours, followed by analysis. Long-term stability was determined by storing at least three aliquots of LQC and HQC samples under specified conditions for 15 days and subsequently analyzing them (9).

RESULT AND DISCUSSION::

Posaconazole in plasma was estimated using the developed RP-HPLC bioanalytical technique, which underwent a rigorous validation process for selectivity, linearity, accuracy, precision, recovery, and stability. The acquired results verify that the technology is appropriate for regular bioanalysis.

Chromatographic Performance and Selectivity

The RP-HPLC chromatogram of posaconazole (Figure 2) demonstrated a well-resolved and symmetrical peak with a consistent retention time, indicating adequate chromatographic separation. Analysis of blank plasma samples (Figure 4) showed no internal interference at the retention time of posaconazole, confirming the selectivity of the method. Furthermore, the chromatogram of posaconazole spiked in blank plasma (Figure 5) exhibited clear and distinct peaks without matrix interference, validating the method’s specificity.

Figure 2: Posaconazole RP-HPLC chromatogram showing its retention time (RT).

Linearity and Calibration Curve

The method's repeatability was tested in the range of 0.5 to 5.0 ppm. The calibration curve formed a good correlation coefficient and a linear reaction when peak area and nominal concentration were plotted against each other. The regression results in Table 2 show that the peak area increases in direct proportion to the posaconazole concentration. The linear link is backed up by the posaconazole calibration curve spiked in plasma (Figure 3), which shows that the method can be used for quantitative analysis in the range that was looked at.

Table 2: Standard Posaconazole Calibration Data for Linearity

Sr. No	Concentration (ppm)	Peak Area (mAU)	Standard Deviation (SD)
1	0.5	296797	0.215
2	1.0	724593	0.0325
3	1.5	1129352	0.0227
4	2.5	1603231	0.0743
5	3.5	1992591	0.0518
6	5.0	2511290	0.0725

Figure 3: Posaconazole Calibration curve of spiked in plasma

Figure 4: Blank Chromatogram of plasma

Figure 5: Chromatogram of the Posaconazole with Blank

Accuracy and Precision:

Quality control samples at three concentration levels—LQC (0.5 ppm), MQC (2.5 ppm), and HQC (5.0 ppm)—were used to test how accurate and precise the work was. Intra-batch (within-day) accuracy and precision data can be seen in Table 3. The %CV values were within the right range, and the %precision values ranged from 97.12% to 99.92%, demonstrating that the method could be used multiple times with consistent results. The inter-batch (between-day) precision and accuracy results in Table 4 showed that the method worked the same on all days. The accuracy rates were between 96.56% and 98.98%, and the CV rates were well within the standards set by the government. These results indicate that the method can accurately and precisely measure posaconazole in plasma.

Table 3: Data of Accuracy and Precision for posaconazole in plasma

Quality Control Sample	Amount. Added (ppm)	Peak Area	Amount found (ppm)	% Accuracy	% C.V
LQC	0.5	296796	0.49	98.15	0.2280
	0.5	296489	0.48	97.12
	0.5	296199	0.49	98.15
MQC	2.5	1129352	2.46	98.43	0.4407
	2.5	1139126	2.47	98.88
	2.5	1139489	2.48	98.82
HQC	5	2511290	4.96	99.92	0.6179
	5	2512671	4.89	98.47
	5	2509612	4.87	97.94

Table 4: For Posaconazole in plasma Inter batch Accuracy and Precision

Quality Control Sample	Amount. Added (ppm)	Peak Area*	Amount found (ppm)	% Accuracy	% C.V
LQC	0.5	269796	0.48	98.00	0.2391
	0.5	296482	0.47	98.45
	0.5	296191	0.49	96.90
MQC	2.5	1129352	2.49	96.56	0.2449
	2.5	1139484	2.46	96.98
	2.5	1139216	2.47	98.80
HQC	5	2511296	4.89	98.98	0.6052
	5	2512671	4.87	98.81
	5	2509612	4.86	98.44

Recovery

Posaconazole recovery from plasma was assessed at low, medium, and high concentrations. According to Table 5, the extraction efficiency was constant across the tested concentrations, with the mean recovery ranging from 85.26% to 87.31%. The reproducible recovery values indicate that the protein precipitation method using acetonitrile is suitable for plasma sample preparation.

Table 5: For Posaconazole in plasma Recovery study

Conc. (ppm)	Peak Area (Extracted)	Peak Area (Un-extracted)	% Recovery
0.5	296796	339896	87.31%
2.5	1129352	1318789	85.63%
5	2511290	2945278	85.26%

Stability Studies

To maintain analyte integrity during analysis, the stability of posaconazole in plasma was evaluated under various handling and storage circumstances. After three freeze-thaw cycles, posaconazole remained stable, according to freeze-thaw stability results (Table 6), with %CV within acceptable bounds and % purity values near nominal concentrations. Studies of short-term stability carried out for eight hours at room temperature (Table 7) showed no change in concentration, suggesting sufficient stability during standard sample handling. Posaconazole was stable in plasma for 15 days, with % purity values reaching 98% and %CV values within acceptable bounds, according to long-term stability tests (Table 8).

Table 6: For Posaconazole in plasma Freeze and Thaw stability

Conc.

(ppm)

Peak Area

Conc. Found

Purity

S.D

% C.V

0.5

296421

0.48

98.72

0.3295

0.2430

2.5

1129322

2.49

99.33

0.2468

0.4486

Table 7: For Posaconazole in plasma Short term temperature stability

Conc.

(ppm)

Peak Area

Conc.

Found (ppm)

Purity

S.D

% C.V

0.5

296221

0.48

98.70

0.2100

0.2114

2.5

1129355

2.48

99.12

0.4661

0.4761

Table 8: For Posaconazole in plasma Long Term Stability

Conc.

(ppm)

Peak Area

Conc.

Found (ppm)

Purity

S.D

% C.V

0.5

296421

0.49

99.1

0.31121

0.2225

2.5

1129322

2.47

98.82

0.2900

0.4982

CONCLUSION:

Following the USFDA's bioanalytical validation rules, a simple, sensitive, and reliable RP-HPLC bioanalytical method for measuring posaconazole in plasma was successfully created and confirmed. The method was able to reliably and accurately distinguish between concentrations in the range that was tested. It also showed good predictability, precision, recovery, and stability. A simple protein precipitation process made it possible to consistently and reliably remove posaconazole from plasma. All of the validation factors were found to be within the limits set by the government, and the validated method was shown to be correct, exact, and able to be used more than once with the same results. The suggested method is good for studying the pharmacokinetics, toxicokinetics, bioavailability, and bioequivalence of posaconazole pill formulations because it works well with plasma samples and is generally reliable. It can also be used to measure the amount of posaconazole in rat plasma.

DECLARATIONS:

Consent for publication:

All the authors approved the manuscript for publication.

Competing interests:

All authors declare no competing interests.

Funding:

Not applicable.

REFERENCES:

1. Choudhary H, Singh S, Singh R, Agarwal R, Kaur H, Ghosh A, Chakrabarti A, Rudramurthy SM. Rapid and simple reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous quantifications of triazole antifungals in human serum. Mycopathologia. 2021 Mar;186(1):27-39.

2. Rama A, Govindan I, Hebbar S, Chaturvedi A, Rani U, Naha A. Advancing posaconazole quantification analysis with a new reverse-phase HPLC method in its bulk and marketed dosage form. F1000Research. 2023 Jun 27;12:468.

3. Jain A, Tikariya K, Mukherjee J. Analytical Method Development and Validation of Posaconazole by QbD Approach. Int. J Pharm. Sci. Rev. Res. 2022;74:109-13.

4. Bayat F, Hashemi Baghi A, Abbasian Z, Dadashzadeh S, Aboofazeli R, Haeri A. Development of an HPLC–UV method for quantification of posaconazole in low-volume plasma samples: design of experiments and machine learning models. BMC chemistry. 2024 Dec 4;18(1):238.

5. Susararla KP, Shelke O, Shorgar N. Quantification of oteseconazole in rat plasma using LC-MS/MS and its application to pharmacokinetic study. Journal of Applied Pharmaceutical Research. 2024 Aug 31;12(4):54-65.

6. Maliyakal J, Patel M. Green chemistry approaches in the analytical validation of fosravuconazole using UV spectrophotometry and HPLC. Green Analytical Chemistry. 2025 Mar 1;12:100215.

7. E. Abdel Aziz S, El-Nakib HE, Schaletzky J, Ahmed NS. Analytical Methodologies for Anti-Infective Orphan Drugs: A Comprehensive Review of FDA Approvals (2013-2023) Part 1. Critical Reviews in Analytical Chemistry. 2025 Jan 28:1-26.

8. Hussain A, Ramzan M, Altamimi MA, Khuroo T. HSPiP and QbD Program–Based Analytical Method Development and Validation to Quantify Ketoconazole in Dermatokinetic Study. AAPS PharmSciTech. 2023 Nov 14;24(8):231.

9. Paul J, Singh K, Pannu S, Pal R, Khan SA, Kumar B, Akhtar MJ. An update on recently developed analytical and bio-analytical methods for some anticancer drugs. Current Pharmaceutical Analysis. 2023 Feb 1;19(2):117-35.

10. Zarad W, El-Gendy H, Bazan L, Ali A, Aboulella Y, Kamal M, Emara S, Shawky A. Bio-analytical liquid chromatographic-based method with a mixed mode online solid phase extraction for drug monitoring of fluconazole in human serum. Journal of Chromatography B. 2021 Dec 15;1187:123045.

11. Gunawan U, Ibrahim S, Ivansyah AL, Damayanti S. Separation and analysis of triazole antifungal in biological matrices by liquid chromatography: a review. Pharmacia. 2023 Nov 1;70:1265-81.

12. Chokshi A, Gajjar A, Bhanushali P, Desai P. Quantification of antileukemic drug Dasatinib in human plasma: Application of a sensitive liquid chromatographic method. Journal of Chemical Metrology. 2021 Jul 1;15(2).

13. Marakatham S, Shanmugapandiyan P. Stability indicating bioanalytical validation of elexacaftor, ivacaftor and tezacaftor using HPLC in human plasma. Research Journal OF Pharmacy and Technology. 2023;16(8):3780-6.

14. Gandi A, Amgoth KM. Stability-indicating RP-HPLC method using Box–Behnken design for simultaneous estimation of Abiraterone and Niraparib in bulk and combined dosage form with application to dissolution studies. Accreditation and Quality Assurance. 2025 Dec;30(6):677-91.

15. Chanduluru HK, Sugumaran A. Assessment of greenness for the determination of voriconazole in reported analytical methods. RSC advances. 2022;12(11):6683-703.

16. Honour JW. Development and validation of a quantitative assay based on tandem mass spectrometry. Annals of clinical biochemistry. 2011 Mar;48(2):97-111.

17. Khan SR, Dadge S, Rathaur S, Gayen JR. Analytical quality by design (AQbD) assisted RP-HPLC technique for quantification of Picroside II in bulk and pharmaceutical dosage form. Future Journal of Pharmaceutical Sciences. 2025 Sep 22;11(1):125.

18. Sheikh S, Beg AE, Baig MT, Ibrahim S, Huma A, Jabeen A, Anwar Z. High-performance liquid chromatographic method for determination of voriconazole in pure and gel formulation. PloS one. 2024 Dec 16;19(12):e0315704.

19. Lin SC, Liu HY, Lin SW, Yao M, Wu UI, Kuo HP, Kuo CH. Simultaneous determination of triazole antifungal drugs in human plasma by sweeping-micellar electrokinetic chromatography. Analytical and Bioanalytical Chemistry. 2012 Jul;404(1):217-28.

20. Patel V, Pandya C, Patel Z, Patel D, Pandya A. Isocratic RP-UHPLC method development and validation of stability-indicating for simultaneous determination of teneligliptin and metformin in fixed-dose combination. Curr Chem Lett. 2021 Mar 22;10:503-16.

21. Campestre C, Locatelli M, Guglielmi P, De Luca E, Bellagamba G, Menta S, Zengin G, Celia C, Di Marzio L, Carradori S. Analysis of imidazoles and triazoles in biological samples after MicroExtraction by packed sorbent. Journal of Enzyme Inhibition and Medicinal Chemistry. 2017 Jan 1;32(1):1053-63.

22. Sharma A, Jaiswal S, Shukla M, Lal J. Dried blood spots: concepts, present status, and future perspectives in bioanalysis. Drug testing and analysis. 2014 May;6(5):399-414.

23. Heinle L, Sulaiman K, Olson A, Ruterbories K. A homologous series of internal standards for near universal application in the discovery LC-MS/MS bioanalytical laboratory. Journal of Pharmaceutical and Biomedical Analysis. 2020 Oct 25;190:113578.

24. Morikawa G, Fukami K, Moriiwa Y, Okazawa K, Yanagida A. Evaluation of the clinical and quantitative performance of a practical HPLC-UV platform for in-hospital routine therapeutic drug monitoring of multiple drugs. Journal of Pharmaceutical Health Care and Sciences. 2023 Oct 1;9(1):29.

RP-HPLC Bioanalytical Method Development and Validation of Posaconazole

Article Information:

Abstract:

Keywords:

Article :

INTRODUCTION:

MATERIALS AND METHODS :

RESULT AND DISCUSSION::

CONCLUSION:

REFERENCES:

Article Citation

APA

MLA

Chicago

Harvard

Vancouver