Biomedical Chromatography

Abdel‐Tawab, Muhammed Abdel‐Hamied; Abd El‐Moghny, Muhammad G.; El Nashar, Rasha Mohamed

doi: 10.1002/bmc.5238pmid: 34469609

Sofosbuvir is a direct‐acting antiviral drug that inhibits hepatitis C virus (HCV) NS5B polymerase, which in turn affects the virus replication inside biological systems. The clinical importance of sofosbuvir is based not only on its effect on HCV but also on other lethal viruses such as Zika and severe acute respiratory syndrome coronavirus disease 2019 (SARS‐COVID‐19). Accordingly, there is a continuous shedding of light on the development and validation of accurate and fast analytical methods for the determination of sofosbuvir in different environments. This work critically reviews the recent advances in chromatographic methods for the analysis of sofosbuvir and/or its metabolites in pure samples, pharmaceutical dosage forms, and in the presence of other co‐administered drugs to highlight the current status and future perspectives to enhance its determination in different matrixes.

Subramanian, Velusamy B.; Katari, Naresh Kumar; Ponnam, Vijetha; Konduru, Naresh; Dongala, Thirupathi; Marisetti, Vishnu M.; Vyas, Govind

doi: 10.1002/bmc.5240pmid: 34486750

According to current regulatory guidelines, a stability‐indicating method has been developed to determine the impurities in sacubitril (SCB) and valsartan (VLS) tablet dosage forms and perform robustness studies using the design of experiments approach. The present study was initiated to understand quality target product profile, analytical target profile, and risk assessment for method variables that affect the method response. A reversed‐phase‐HPLC system was equipped with a Phenomenex Gemini‐NX C18 column (150 × 4.6 mm, 3 μm) and a photo diode array detector. A gradient mobile phase was used in this research work. The detection was performed at 254 nm; the flow rate was 1.5 mL/min, and the column temperature was maintained at 30°C. The proposed method was validated per the International Council for Harmonisation Q2 (R1) guidelines. The coefficient of correlation was >0.999 for all impurities. The limits of detection and quantification were evaluated for SCB, VLS, and all impurities. The precision and accuracy were obtained for SCB, VLS, and their related impurities. Intra‐ and inter‐day relative standard deviation values were less than 10.0%, and the recoveries of impurities varied between 90.0 and 115.0%. Based on the validation results, the proposed DoE method can estimate SCB and VLS impurities in the finished dosage form.

Sun, Guozhang; Jiang, Fengling; Hu, Shaoshan; Cheng, Huakun; Qu, Lianlong; Tao, Yu; Ma, Bowen

doi: 10.1002/bmc.5241pmid: 34505712

Spontaneous intracerebral hemorrhage (ICH) accounts for 10–20% of all strokes and contributes to higher mortalities and severe disabilities. The aims of this study were, therefore, to characterize novel biomarkers, metabolic disruptions, and mechanisms involving ICH. A total 30 ICH patients and 30 controls were enrolled in the study, and their clinical characteristics were analyzed. Nontargeted metabolomic analysis was conducted using ultra‐performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry (UPLC/Q‐TOF). Multivariate statistical analysis and receiver operating characteristic curve analysis were used for screening and evaluating the predictive ability of biomarkers. ICH patients showed significantly higher systolic blood pressure, diastolic blood pressure, blood glucose levels, white blood cell counts, neutrophil count, percentage of neutrophils and globulin and a lower albumin/globin ratio when compared with controls. In sum, 11 important metabolites were identified, which were associated with disruption of fatty acid oxidation and sphingolipid and phospholipid metabolism, as well as increased inflammation, oxidative stress, and vascular pathologies. Further multiple logistic regression analyses of these metabolites showed that l‐carnitine and phosphatidylcholine (20:3/22:6) have potential as biomarkers of ICH, and the area under the curve, sensitivity, specificity were 0.974, 90%, and 93%, respectively. These findings provide insights into the pathogenesis, early prevention, and diagnosis of ICH.

Zhang, Ting; Guo, Sheng; Niu, Yang; Huang, Kaidi; Bu, Fanshu; Ren, Hui; Zhang, Yiying; Shang, Erxin; Duan, Jin‐ao; Qian, Dawei

doi: 10.1002/bmc.5257pmid: 34611921

Hui Medicine ZhaLi NuSi Prescription (ZLNS) is described in “Hui Hui Prescription,” and it has been used to treat cerebral infarction in Hui Region, China. In this study, a rapid and reliable ultra‐performance liquid chromatography coupled with mass spectrometry (UPLC–MS/MS) method was established and applied to simultaneously determine geniposidic acid, oxypaeoniflorin, hydroxysafflor yellow A, caffeic acid, magnoflorine, paeoniflorin, ferulic acid, β‐ecdysterone, icariin, rhein, and baohuoside I in rat plasma. The pharmacokinetic parameters of these components and the influence of essential oils (EOs) on them were investigated in normal rats. The results showed that the pharmacokinetic parameters (AUC0 − t, AUC0 − ∞, t1/2, tmax, cmax) of the aforementioned compounds were significantly changed after co‐administering with ZLNS EO. The AUC values of oxypaeoniflorin, paeoniflorin, ferulic acid, and baohuoside I with EOs were decreased significantly. This is the first report for the comparative pharmacokinetic study of ZLNS bioactive components in normal rats, which may provide the basis for drug interaction study in vivo and insight into their clinical applications.

Lu, Jing‐Ze; Ye, Dan; Chen, Long; Ma, Bing‐Liang

doi: 10.1002/bmc.5245pmid: 34532879

This study aimed to compare the pharmacokinetic properties of four preparations (dispersible tablets, ordinary tablets, capsules and granules) of arbidol hydrochloride, a broad‐spectrum antiviral drug, in beagle dogs. Briefly, a single dose of 100 mg of the four preparations of arbidol hydrochloride was orally administered to dogs; blood was then collected from the veins of the foreleg at different times after administration to prepare plasma samples. The plasma concentration of arbidol hydrochloride was measured using a validated liquid chromatography–tandem mass spectrometry (LC–MS/MS). The results showed that when orally administered with dispersible tablets, ordinary tablets, capsules and granules suspended with water, there were no significant differences in the pharmacokinetic parameters (including peak time, peak concentration, elimination half‐life, area under the curve (AUC0–t), and mean retention time) of arbidol hydrochloride. However, in the case of the dispersible tablets, the pharmacokinetics of arbidol hydrochloride was significantly affected by the mode of administration. Compared with direct feeding, peak time [0.50 (0.13, 0.50) vs. 1.00 (0.50, 2.00)] was significantly shortened (P = 0.033) and the AUC0–48 h (8726.5 ± 2509.3 vs. 3650.8 ± 1536.9 ng h/ml) was significantly increased (P = 0.012) when the dispersible tablets were orally administered as water dispersion. In conclusion, the pharmacokinetics of four preparations of arbidol hydrochloride were not significant different in beagle dogs. However, compared with direct feeding, the absorption of arbidol hydrochloride was faster and the bioavailability was better when the dispersible tablets were orally administered as water dispersion.

Dong, Chao; Zhou, Jie; Zuo, Wei; Li, Zhixia; Li, Jing; Jiao, Bining

doi: 10.1002/bmc.5229pmid: 34414593

Phenthoate is a chiral organophosphate pesticide with a pair of enantiomers which differ in toxicity, behavior and insecticidal activity, and its acute toxicity on human health owing to the inhibition of acetylcholinesterase highlights the need for enantioselective detection of enantiomers. Therefore, this study aimed to establish a simple rapid method for separation and detection of phenthoate enantiomers in fruits, vegetables and grains. The enantiomers were separated using reversed‐phase high‐performance liquid chromatography–tandem mass spectrometry for the first time. Rapid chiral separation (within 9 min) of the target compound was achieved on a chiral OJ‐RH column with the mobile phase of methanol–water = 85:15(v/v), at a flow rate of 1 ml/min and a column temperature of 30°C. Acetonitrile and graphitized carbon black were used as the extractant and sorbent for pretreatment, respectively. This method provides excellent linearity (correlation coefficient ≥0.9986), high sensitivity (limit of quantification 5 μg/kg and limit of detection <0.25 μg/kg), satisfactory mean recoveries (76.2–91.0%) and relative standard deviation (intra‐day RSDs ranged from 2.0 to 7.9% and inter‐day RSDs ranged from 2.4 to 8.4%). In addition, a field trial to explore the stereoselective degradation of phenthoate enantiomers in citrus showed that (−)‐phenthoate degraded faster than its antipode, resulting in the relative accumulation of (+)‐phenthoate.

Shrivastava, Suman; Daharwal, Sanjay J.

doi: 10.1002/bmc.5236pmid: 34469592

The aim of this study was to determine the content of rutin in Hemidesmus indicus and to optimize the high‐performance thin‐layer chromatography method. The method was validated in compliance with the International Council for Harmonisation guidelines Q2 (R1) for parameters such as linearity, accuracy, precision, robustness, limit of detection, and limit of quantitation. A Box–Behnken design and response surface methodology has been used to investigate the impact of independent variables on the response. Three independent variables, mobile phase composition (% v/v), mobile phase volume (mL), and duration of saturation (min), were studied. Rutin was verified, and its content was determined using a validated high‐performance thin‐layer chromatography method with good linearity within the range of 200–1000 ng spot–1 with r2 = 0.9998 and correlation coefficient with calibration curve equation y = 0.0297x + 0.0001. The average percentage recovery values varied from 99.03 to 101.15 and 98.88 to 100.12%, respectively, for in‐house and marketed mother tincture). The peak area determination at three different concentration levels shows low values of percentage relative standard deviation (<2%) for inter‐day (0.04–0.06) and intra‐day (0.04–0.05) precision of rutin. The average content of rutin in extract and marketed mother tincture was 229 ± 0.57 and 210 ± 0.57 μg g–1. The proposed method was simple, precise, and accurate for the determination of rutin with frequent quality control assessment of H. indicus.

Ma, Xiao‐fang; Zhao, Qi; Cheng, Yan; Yan, Dong‐Mei; Zhu, Wei‐Feng; Li, Fei

doi: 10.1002/bmc.5239pmid: 34494281

Coumarins are a group of natural compounds commonly found in the families of Rutaceae and Umbelliferae. 7‐Isopentenyloxycoumarin (ISC), auraptene (AUR), and umbelliprenin (UM) belong to prenyloxycoumarins (PYCs), which link isopentenyl, geranyl, and farnesyl group at C7 position, respectively. The substituent of 7‐ethoxycoumarin (ETC) is the ethyl group. In this study, UPLC–ESI–QTOF–MS (ultra‐performance liquid chromatography–electrospray ionization–quadrupole time of flight–MS)‐based metabolomics was used to evaluate the in vivo and in vitro metabolism of PYCs. Results showed that ETC produced 10 known metabolites, and ISC was transformed into 17 metabolites in vivo and in vitro, which were undescribed compounds. A total of 35 AUR metabolites, including 34 undescribed metabolites were identified, and 21 metabolites were reported for the first time in UM. The results indicated that hydroxylation and N‐acetylcysteine conjugation were the common metabolic reactions for PYCs. The metabolic rates of ETC, ISC, AUR and UM were 26%, 36%, 81%, and 38%, respectively, in human liver microsome, while they were 24%, 40%, 80%, and 37%, respectively, in mouse liver microsomes. In addition, recombinant cytochrome P450s (CYPs) screening showed that CYP1A1, 2C19, 3A4, and 3A5 were the major metabolic enzymes involved in the formation of hydroxylation metabolites. Together, these results suggest that the isopentenyl group plays an important role in the metabolism of PYCs.

Gong, Yong; Chen, Jie; Lim, Heng‐Keang; Weng, Naidong; Salter, Rhys

doi: 10.1002/bmc.5242pmid: 34519061

The reported method involves a novel workflow that eliminates the need for authentic reference standards for the quantitation of drug metabolites in biological samples using a single multi‐isotopically labeled compound bearing both radio and stable isotopes. The resulting radio and stable bifunctionalized isotopolog (RADSTIL) of the parent drug is employed as a substrate for in vitro biotransformation to targeted RADSTILs of metabolites as calibrants. Inclusion of a radio label enables both radiometric and mass spectrometric detection. The addition of stable labels ensures the subsequent isotopic interference‐free quantitation of unlabeled metabolites in preclinical and clinical samples. This affords a more accurate quantitation workflow compared with the current semi‐quantitation method, which utilizes isotopic interfering radio isotopologs of metabolites alone as calibrants. The proof‐of‐concept is illustrated with (14C,13C2)‐acetaminophen where in vitro biotransformation produced (14C,13C2)‐sulfate and (14C,13C2)‐glucuronide calibrants. Absolute quantitation of the acetaminophen metabolites was then achieved by liquid chromatography coupled with radiometry and mass spectrometry. Quantitative data obtained by this method fell within 82–86% of the values from conventional LC–MS/MS method.

Liang, Yuan; Ma, Tiancheng; Li, Yuwei; Cai, Na

doi: 10.1002/bmc.5248pmid: 34555192

Vanillic acid, a phenolic compound isolated from Angelica sinensis and green tea, exhibits excellent antioxidant and anti‐inflammatory activities. In this study, a rapid and sensitive ultra‐high‐performance liquid chromatography tandem mass spectrometry method was established and validated for the determination of vanillic acid in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Chromatographic separation was performed on a Zorbax RRHD Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm) with gradient elution at a flow rate of 0.3 ml/min, using mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). Vanillic acid and caffeic acid (internal standard, IS) were quantified by multiple reaction monitoring in negative ion mode. The method was fully validated according to the US Food and Drug Administration guidelines. The calibration curve was linear over the range of 2–1,000 ng/ml with a correlation coefficient of >0.99. The carryover, matrix effect, extraction recovery, dilution effect, intra‐ and interday precision and accuracy were within acceptable limits. The method was then applied to a pharmacokinetic study of vanillic acid in rats. After oral administration at doses of 2, 5 and 10 mg/kg, the plasma concentration reached peaks of 0.42 ± 0.09, 0.73 ± 0.21 and 0.92 ± 0.28 μg/ml at the time of 0.55–0.64 h, respectively. The oral bioavailability was calculated as 25.3–36.2% in rat plasma. The result provided pre‐clinical information for further application of vanillic acid.