A novel chiral HPLC and LC-MS/MS method development for the triazole antifungal compound

The objective of the present study was to separate and develop a chiral high performance liquid chromatography (HPLC) and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) technique to estimate the (+) and (−) enantiomers of Albaconazole and validate the individual enantiomer of the drug. Albaconazole is used to treat for anti-fungal disease. The stationary phase was reverse phase Chiralpak IG-3 (250 × 4.6 mm, 5 µm) and (100 × 4.6 mm, 3 µm), whereas the isocratic mobile phase was ethanol and diethyl amine (100:0.1% v / v ratio HPLC) Acetonitrile and 10 mM ammonium bicarbonate (90:10 v / v ratio LC-MS/MS) and the flow rate was 1.0 and 0.5 ml/minute. The resolution of the (+) and (−) enantiomers were monitored using HPLC diode array detector (DAD) 240 signal and LC-electrospray ionization-MS/MS in positive transition at 432.0 m / z (M + H) for Albaconazole. The retention time of the (+) and (−) enantiomers of the drug was 6.952 and 9.955 minutes and 2.905 and 3.780 minutes by HPLC and LC-MS/MS. The major benefits of the LC-MS/MS are related to its improved selectivity, precision and accuracy and the lower variability in comparison to the HPLC-DAD. This study provided a rapid, sensitive and novel selective method to evaluate the (+) and (−) enantiomers in active pharmaceutical ingredients by HPLC and LC-MS/MS.


INTRODUCTION
Albaconazole is a triazole antifungal belongs to the class of 7-chloro-3-[(2R, 3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1,2,4-triazol-1-yl) butan-2-yl]quinazolin-4-one (Amjad et al., 2016).Generally, azole compounds inhibit the steroid demethylation and the biosynthesis of a critical component of fungal membrane called ergosterol by blocking a cytochrome P 450 dependent enzyme: lanosterol 14-α-demethylase which is crucial for the conversion of lanosterol to ergosterol.Lack of ergosterol and accumulation of lanosterol-14-α-demethylase will increase the membrane permeability and lead to disruption of several enzymes in the membrane, such as chitin synthase (Maertens, 2004).This does not only inhibit its DNA replication, but also distracts cell growth that causes the death of yeast and fungi.Azoles also decrease the adhesion potential of pathogen cells to host tissues and impede the transformation of yeasts to mycelial form (Ghannoum and Rice, 1999;Sumrra et al., 2022).Therefore, they are widely applied as veterinary drugs (Bhanderi et al., 2009), as fungicides in agriculture (Brauer et al., 2019) and as antifungal agents for both humans and animals (Scorzoni et al., 2017;Zafa et al., 2021).Chirality plays a significant role in determining the pharmacological actions of chiral compounds and vital importance at the drug discovery stage (Ates et al., 2013;Zhang et al., 2005).One-third of all marketed drugs are now sold in a single isomeric form and chirality is now a significant factor in the development of new pharmaceuticals, with regulatory and therapeutic considerations driving the process (Mukherjee and Bera, 2012).The enantiomers of a chiral drug molecule may behave differently after administration, so the pharmaceutical industry places a high value on chiral resolution.To have a therapeutic effect, a molecule must engage a target receptor when it is administered.Drug molecules that are chiral will only fit into this receptor in one of their enantiomers (the eutomer), producing the desired therapeutic effect.A lesser effect could result from the other enantiomer (distomer), interacting or not with the receptor.The distomer can occasionally interact with different receptors, leading to side effects or even toxicity.In order to distinguish the eutomer from the distomer during drug substance identification and impurity determinations, additional research is needed on the enantiomers of active compounds during the development process.Racemates resolution is still difficult because of their similar characteristics in chiral environments, and work on highly specialized separation techniques is ongoing to resolve individual enantiomers (Liu et al., 2015).Based on literatures survey (Azhari et al., 2020;Bhowmick et al., 2021;Gazzinelli et al., 2022;Shekar et al., 2014), revealed that few analytical methods were reported for chiral separation on triazole antifungal drugs by high performance liquid chromatography (HPLC).Furthermore, the reported methods on chiral separation were more retention time and less sensitivity.As a result, our objective of this research is to separate and develop a novel, fast, selective and sensitive method with less retention time for chiral separation and estimation of (+) and (−) enantiomers in active pharmaceutical ingredients using HPLC and liquid chromatography tandem mass spectrometry (LC-MS/MS).

Reagents
YMC India private limited gifted pure Albaconazole (+/−) as a working standard.SD fine chemicals and Merck, Mumbai, India supplied the chemicals ammonium bicarbonate and solvents methanol and acetonitrile (HPLC and LC-MS grade).The Milli Q RO system was used to purify the water (Millipore, Bedford, UK).

Instrumentation (HPLC and LC-MS/MS)
HPLC-photo diode array (PDA) chromatographic fingerprints were obtained with an Agilent 1260 Infinity II HPLC instrument (Agilent Technologies, Waldbronn, Germany) equipped with a 1260 Infinity II quaternary pump, a 1260 Infinity II degasser, a 1260 Infinity II vial sampler, a 1260 Infinity II column thermostat, a 1260 Infinity II diode array detector (DAD) HS.PC with the Agilent open lab CDS software for data acquisition.
Ultra fast liquid chromatography coupled with tandem triple quadrupole mass spectrometer (Shimadzu LC-MS/MS, Tokyo, Japan) equipped with interfaced by electrospray ionization (ESI) and solvent delivery system LC-20AD pump, SPD M20 PDA detector, SIL-20AC auto sampler, CTO 20AC column oven, CMB-20 alite controller.Using LC lab solution software, the data acquisition was performed.For the study, optimized factors include heat block temperature, desolvation line, Nebulizer gas, collision energy, etc.The mass spectrometer was run in positive ionization detection mode (M + H) with an ESI source.The nebulizer pressure was set to 345 kPa, ionization temp was set to 300°C, the capillary voltage was 5,000 V and gas flow rate was 11 l/minute.The collision cell gas was ultrapure nitrogen and the ionization source gas was nitrogen.

Chromatographic conditions (HPLC and LC-MS/MS)
The HPLC enantio-selective separation was achieved using a chiral stationary phase as reverse phase (RP) Chiral ART cellulose-SZ (250 × 4.6 mm, 5 µm) and the isocratic mobile phase composition of ethanol: ethanol and diethyl amine (DEA):(100%: 0.1% v/v ratio) at the flow rate of the detection of analyte was 1.0 ml/minute.Injection volume of 20 µl of each sample injects into the system and employed at an ambient column temperature.The total run time for the chiral separation was 20 minutes.The resolution target was detected using Agilent HPLC 1260 infinity II with a DAD detector.
The LC-MS/MS enantio-selective separation was achieved using a chiral stationary phase as RP Chiralpak IG-3 (100 × 4.6 mm, 3 µm) and the isocratic mobile phase composition of acetonitrile and 10 Mm ammonium bicarbonate (90:10 v/v ratio) at the flow rate of the detection of analyte was 0.5 ml/minute.10 µl of injection volume of each sample injects into the system and employed at an ambient column temperature.The total run time for the chiral separation was 5 minutes.The resolution targets was detected using a Shimadzu-8030 triple quadrupole mass / Journal of Applied Pharmaceutical Science 13 (Suppl 1); 2023: 001-008 spectrometer with ESI interfaced with the mass analyzer (Hassan et al., 2022).The molecular ion spectra for albaconazole were found to be at m/z: 432.0 and the most prominent fragmentation peaks were observed at 45.0, 391.0, and 415.0.The most stable fragment of maximum intensity at 391.0 (daughter ion) (Sumrra et al., 2021).The mass spectrometer was run in positive ionization detection mode (M + H) with an multiple reaction monitoring (MRM) mode with the following transitions: m/z 432.0 (parent ion) → m/z 391.0 (daughter ion) for (+/−) Albaconazole, respectively (Figs. 2 and 3).

Standard solution preparation for (+/−) Albaconazole (HPLC and LC-MS/MS)
Working standard of (+/−) Albaconazole 1,000 µg/ ml concentration was prepared by dissolving 10 mg of the enantiomeric drug in a 10 ml volumetric flask with methanol and make up the volume with methanol.A working concentration of 1,000 ng/ml was prepared from the above solution.The calibration curve for (+) and (−) Albaconazole of 10-100 ng/ml enantiomeric drug was prepared using the working standard.

Method validation for Albaconazole
The optimized HPLC and LC-MS/MS method was validated in accordance with the ICH guidelines in the aspects of specificity and carry-over, limit of quantification (LOQ), limit of detection (LOD), linearity, accuracy and precision and robustness, etc. (ICH, 1996).

Accuracy and precision
The recovery of the method was used to define the accuracy of the method.According to ICH guideline, the accuracy of the proposed HPLC and LC-MS/MS method was evaluated from the three levels of quality control samples by analyzing the six replicates.The recovery of the precision was carried out and the percent RSD was recorded.

Specificity and carry over
The ability to clearly assess the analyte in the presence of components that might be anticipated to be present is known as specificity.Typically, these could be degradants, impurities, etc.

LOD and LOQ
LOD and LOQ were determined by signal to noise ratios (S/N) of 3:1 (LOD) and 10:1 (LOQ), respectively, in accordance with ICH guidelines a method is considered sensitive if it can detect incredibly low concentrations.

Robustness
The robustness of the developed method was determined by altering experimental conditions are mobile phase, flow rate and injection volume, etc. was studied.

RESULTS AND DISCUSSION
In this study, RP direct chiral HPLC and LC-MS/ MS technique has been developed and validated for the chiral resolution of (+/−) Albaconazole.In order to separate the enantiomers, develop a simple, sensitive and effective HPLC   / Journal of Applied Pharmaceutical Science 13 (Suppl 1); 2023: 001-008 and LC-MS/MS method for (+/−) Albaconazole were carried out by selected optimal conditions such as, the resolution factor, theoretical plates, tailing factor, peak area, and peak asymmetry factor.For HPLC, RP Chiral Art Cellulose-SZ (250 × 4.6 mm, 5 µm) as the chiral stationary phase and ethanol: DEA: (100%: 0.1% v/v ratio) as the isocratic mobile phase with a flow rate of 1.0 ml/minute constitute the optimal chromatographic conditions.The total chromatographic resolution run time was 20 minutes.The (+) enantiomeric retention time was found to be 6.952 minutes and the (−) enantiomeric retention time was found to be 9.955 minutes, respectively (Fig. 1).For LC-MS/MS RP-Chiralpak IG-3 (100 × 4.6 mm, 3 µm) as the chiral stationary phase and acetonitrile and 10 Mm ammonium bicarbonate (90:10 v/v ratio) as the isocratic mobile phase with a flow rate of 0.5 ml/minute constitute the optimal chromatographic conditions.The total chromatographic resolution run time was 5 minutes.The (+) enantiomeric retention time was found to be 2.905 minutes and the (−) enantiomeric retention time was found to be 3.780 minutes, respectively (Fig. 4).

Specificity
During the elution time of the individual enantiomers for both the methods, no potential interference peaks were noticed.As a result, the method was found to be specific and highly sensitive.

LOD and LOQ
Based on the S/N ratio and minimum level of peak area and the LOD (4 µg/ml) and (3 ng/ml) as well as LOQ (10 µg/ml) and (10 ng/ml) were determined for the (+) and (−) enantiomers for the developed method by HPLC and LC-MS/MS.

System suitability
As per ICH guidelines, a system suitability study were conducted to determine the system suitability parameters are resolution factor, retention time (R T ), tailing factor and theoretical plates (N).The results were found to be within the limits (Tables 4  and 8)./ Journal of Applied Pharmaceutical Science 13 (Suppl 1); 2023: 001-008

CONCLUSION
In conclusion although both methods here described are reliable, and fast to perform, the major benefits of the LC-MS/MS are related to its improved selectivity, precision and accuracy and the lower variability in comparison to the HPLC-DAD.As per ICH guidelines a sensitive direct chiral reverse HPLC and LC-MS/MS method for chiral separation for novel triazole antifungal compounds was developed and validated.As it provides good sensitivity and reproducibility.In spite of the elevated instrumentation cost, the LC-MS/MS method, here presented, is simple and rapid and therefore it could be applied to routine analysis of oxidative stress in clinical chemistry.This method will be useful for pharmaceutical, pharmacokinetics and bioequivalence study.

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