Biomedical Chromatography

Ali, Imran; Mimouni, Fatima Zohra; Belboukhari, Nasser; Sekkoum, Khaled; Locatelli, Marcello; Demir, Ersin; Yusuf, Kareem

2024 Biomedical Chromatography

doi: 10.1002/bmc.6004pmid: 39237855

Thirteen flavanone racemates were successfully separated using a Chiralpak® IA column and isopropanol‐hexane (50:50, v/v). The mobile phase flow rate and detection wavelength were 0.5 mL/min and 254 nm. The retention times values ranged from 5.50 and 56.45 min. The values of the retention, separation, and resolution factors ranged from 0.63 to 21.67, 1.12 to 2.45, and 0.13 to 11.94. The docking binding energies ranged from −6.2 to −8.2 kcal/mol, showing enthalpy‐determined host‐guest complex formation. The molecular docking results and the experimental data were agreed well. The results showed that S‐enantiomers had stronger bindings with chiral selectors compared to R‐enantiomers. Consequently, the R‐enantiomers eluted first followed by S‐enantiomers. The reported method is highly useful to determine the enantiomeric composition of the reported flavanone in any sample.

Tao, Jiayue; Li, Hanyi; Jin, Mingxuan; Shen, Wenchao; Liu, Siqi; Li, Dan; Hou, Jincai; Wang, Rufeng

2024 Biomedical Chromatography

doi: 10.1002/bmc.5997pmid: 39225114

Yigong San (YGS) is a traditional Chinese medicine formula used for pediatric anorexia, chronic atrophic gastritis, and irritable bowel syndrome. In this study, the excretion of eight main compounds, including liquiritin; isoliquiritin; hesperidin; ginsenosides Rb1, Re, and Rg1; and atractylenolides I and II, in rat urine, feces, and bile, was investigated by ultra‐high performance liquid chromatography–tandem mass spectrometry. The results showed that the cumulative excretion rates of the compounds in rat urine, feces, and bile were 0.018–1.15%, 0.024–19.89%, and 0.0025–0.72%, respectively. Among the eight compounds detected, liquiritin was the richest in urine, and ginsenosides Re and Rg1 and atractylenolide I were mainly found in feces and bile. In summary, the main components of YGS are excreted via multiple approaches. Liquiritin is mainly through urine, whereas isoliquiritin; hesperidin; ginsenosides Rb1, Re, and Rg1; and atractylenolides I and II are mainly through feces. The excretion of these compounds in bile is usually positively correlated with that in feces. This study lays a foundation for further pharmacological research and application of YGS.

Dadge, Shailesh D.; Yadav, Shubhi; Rathaur, Shivam; Gayen, Jiaur R.

2024 Biomedical Chromatography

doi: 10.1002/bmc.6005pmid: 39237854

This study investigates the combination of FGFR inhibitor futibatinib (FTB) and MEK inhibitor binimetinib (BTB) for KRASmt NSCLC therapy. An analytical method was developed and validated for measuring FTB and BTB concentrations in rat plasma, adhering to USFDA guidelines. Using liquid–liquid extraction on 45‐μL plasma samples, a 6.5‐min run time was achieved. The linear calibration curve ranged from 2 to 100 ng/mL. Intra‐day and inter‐day accuracy ranged between 92.06% and 100.08%. Four blank injections post high‐concentration samples resolved significant carryover. Extraction recoveries averaged 92.06% to 102.37% across concentrations. No significant endogenous interference was detected in blank plasma. The LLOQ for both drugs was 2.0 ng/mL. Selectivity, matrix effects, stability, and dilution integrity met the acceptance criteria. The method assessed FTB and BTB interaction potential in combination therapy at 5 mg/kg. The findings provide essential pharmacokinetics insights for future clinical trials.

Li, Jingchu; Liu, Shanshan; Jia, Chenglin; Li, Jiacheng; Zhang, Zhihui; Chen, Jian; Cao, Yongbin; Ma, Chao

2024 Biomedical Chromatography

doi: 10.1002/bmc.6002pmid: 39228060

In this study, a simple and sensitive liquid chromatography tandem mass spectrometric method was developed and validated for the determination of iptacopan and two acyl glucuronidation metabolites in monkey plasma. The plasma sample was precipitated with acetonitrile and then separated on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using 0.1% formic acid and 5 mM ammonium acetate in water and acetonitrile as the mobile phase. The mass spectrometry (MS) detection was performed in positive multiple reactions monitoring (MRM) mode with precursor‐to‐production transitions. The developed assay was validated over the range of 1–2000 ng/mL for three analytes with correlation coefficient (r) more than 0.99. The validation parameters including accuracy, precision, carryover effect, matrix effect, recovery, and stability were all within the acceptable limits. The validated method has been applied to investigate the pharmacokinetics of iptacopan and its two acyl glucuronidation metabolites in monkey plasma. After intravenous administration, iptacopan showed low clearance (2.75 mL/min/kg) in monkey plasma. After oral administration, the bioavailability was 55.43%. The exposure (AUC0−t) of direct acyl glucuronide (AG) of iptacopan accounts for 9.73% of the iptacopan plasma exposure. The AUC0−t of AG of dealkylated metabolite of iptacopan was present at a lower level, accounting for 0.5% of the iptacopan plasma exposure.

Srivastava, Shruti; Dhaneshwar, Suneela; Kawathekar, Neha

2024 Biomedical Chromatography

doi: 10.1002/bmc.5994pmid: 39228079

The present study utilized Analytical Quality by Design (AQbD) approach to develop a stability‐indicating high‐performance liquid chromatography (HPLC) method for estimating evogliptin tartrate using design expert software. The key parameters were methodically optimized, contours were plotted, and stability was evaluated using various forced degradation conditions. Using an Agilent HPLC system with a photo diode array (PDA) detector along with Fortis C18 column (250 × 4.6 mm, 5 μm) effectively separated the drug from its degradants. The mobile phase used was methanol: water (pH adjusted to 3.0, 76:24; v/v) at 0.8 mL/min flow rate. Evogliptin was eluted at 2.98 min, at a detection wavelength of 267 nm. The proposed method was found to be specific, precise, linear and robust. The drug was sensitive to acidic, basic, oxidative, thermal, and photodegradation resolving six degradation products. Thus, the developed AQbD‐based stability‐indicating HPLC method is applicable in analyzing evogliptin in bulk, tablet dosage form and stability samples.

Ettaboina, Santhosh Kumar; Nannapaneni, Satyasree; Jeedimalla, Nagalakshmi; Dongala, Thirupathi; Muchakayala, Siva Krishna; Katari, Naresh Kumar

2024 Biomedical Chromatography

doi: 10.1002/bmc.6001

This study validates a stability‐indicating LC method for detecting organic impurities in the chlorzoxazone dosage form. Using a Waters X‐Select R HSS T3 analytical column, mobile phase of it was made by mixing of water, methanol, and glacial acetic acid in the ratio of 700:300:10 (v/v/v). The drug product and drug substance were subjected to the stress conditions such as acid, base, oxidation, heat, and photolysis as per the recommendations of the International Conference on Harmonization (Q2) methodology. The study revealed the susceptibility of 4‐chloro‐2‐aminophenol to alkaline environments, emphasizing peak homogeneity and stability. The method verification, per ICH guidelines and USP<1225>, established precision, specificity, linearity, accuracy, and robustness for quality control. The mean impurity recovery ranged from 95.5% to 105.2%, the correlation coefficient (r) was greater than 1.000, and the RSD values (n = 6) ranged from 0.6% to 5.1% across the LOQ–150% ranges. Full‐factorial design tested final method conditions, evaluating multiple parameters concurrently. Graphical optimization within the design space defined strong method requirements, ensuring consistent and reliable outcomes. The study develops and validates chlorzoxazone stability‐indicating methods, employing advanced statistical approaches like design of experiments and factorial design, with resilient conditions established through graphical optimization of the design space.

Wu, Jianbang; Wang, Changmao; Zhang, Rong; Du, Pengfei; Wang, Yaqin; Wu, Ping; Chen, Xinyan; Huang, Yunzhe; Jia, Yuanwei; Shen, Jie

2024 Biomedical Chromatography

doi: 10.1002/bmc.5964pmid: 39252549

A liquid chromatography electrospray ionization tandem mass spectrometry method with amoxicillin‐d4 as the stable isotope‐labeled internal standard for simultaneous quick detection of amoxicillin and clavulanic acid in human plasma was developed and validated. Chromatographic separations were performed on a Hedera ODS‐2 column (2.1 × 150 mm, 5 μm). The mobile phases for gradient elution were aqueous solution containing 0.2% acetic acid (AA) (mobile phase A) together with organic phase solution (acetonitrile and methanol mixed solution, mobile phase B). Mass spectrometry was performed using negative electrospray ionization in multiple reaction monitoring mode. The target fragment ion pairs of amoxicillin, clavulanic acid and amoxicillin‐d4 were m/z 364.1 → 223.1, 198.1 → 135.9 and 368.1 → 227.1, respectively. The linear ranges of this method were 40–5,000 ng/ml for amoxicillin and 30–2,500 ng/ml for clavulanic acid, with coefficient of determination > 0.9900. This method validation included selectivity, standard curve, lower limit of quantitation, accuracy, precision, recovery, matrix effect (hemolytic matrix and hyperlipidemic matrix), carryover, stability, dilution reliability and incurred sample reanalysis study. A successful application of this method was realized in a pharmacokinetic study after administration of amoxicillin–clavulanic acid potassium granules.

Ramarao, Bandaru Venkata; Kamalakaran, Anand Solomon

2024 Biomedical Chromatography

doi: 10.1002/bmc.6009pmid: 39251377

This study presents a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for quantifying cabotegravir (CAB) in rat plasma. A novel, sensitive, and rapid LC‐MS/MS method has been developed and validated. Furthermore, protein precipitation technique allowed us to lowered the limit of quantification (LOQ) to nanogram levels, allowing detection of smaller CAB amounts in plasma samples. A review of scientific literature reveals that this method is superior than published methods in terms of runtime, sensitivity, wide linearity, and cost, using LC–MS/MS to quantify CAB in biological samples. CAB reached its maximum concentration (Cmax) of 78.401 μg/mL in rat plasma at 1.50 h (Tmax). Linearity was evaluated across 0.05–1000 μg/mL for CAB using five calibration curves with at least nine standards each with r2 > 0.9997. The intra‐ and inter‐day precision and accuracy results were below 15% and acceptable as per Food and Drug Administration (FDA) guidelines. Stability of compounds were established in a battery of stability studies, that is, benchtop, autosampler, and long‐term storage stability as well as freeze thaw cycles. The validated method can be used as a routine method to support pharmacokinetic studies.