Biomedical Chromatography

Dong, Shiqi; Gu, Yuan; Wei, Guangli; Si, Duanyun; Liu, Changxiao

doi: 10.1002/bmc.4323pmid: 29920712

Insulin is an effective therapeutic for diabetes, and the level of insulin in vivo is directly related to the health of diabetic patients. Traditionally, the concentrations of insulin in vivo are determined by the radioimmunoassay (RIA) method. In this study, we developed an LC–MS/MS method for the quantification of human insulin in dog plasma and directly compared the RIA and LC–MS/MS methods. Our LC–MS/MS method exhibited superior accuracy, efficiency and cost‐effective for the pharmacokinetic (PK) assessment of human insulin. The LC–MS/MS method can quantitate human insulin and canine insulin simultaneously without cross‐reactivity, making the analysis more efficient. The LLOQ of our LC–MS/MS method was 38.5 pg/mL, which was necessary to fully describe the PK profiles of endogenous and exogenous insulin in vivo. The direct comparison of PK data obtained from the two methods demonstrated that LC–MS/MS could be an alternative to the RIA method and should be widely used for the quantification of insulin drugs, especially in preclinical studies.

Yu, Chu‐qin; Chen, Jian‐ping; Zhong, Yan‐mei; Zhong, Xun‐long; Tang, Chun‐ping; Yang, Yi; Lin, Hua‐qing

doi: 10.1002/bmc.4332pmid: 29981286

Hao Jia Xu Re Qing Granules (HJ), is an effective clinically used antipyretic based on traditional Chinese medicine. Although its antipyretic therapeutic effectiveness is obvious, its therapeutic mechanism has not been comprehensively explored yet. In this research, we first identified potential biomarkers which may be relevant for the antipyretic effect of HJ based on urine metabolomics using ultra‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS). A rat model of fever was established using the yeast‐induced febrile response. Total‐ion‐current metabolic profiles of different groups were acquired and the data were processed by multivariate statistical analysis–partial least‐squares discriminant analysis. As envisioned, the results revealed changes of urine metabolites related to the antipyretic effect. Fourteen potential biomarkers were selected from the urine samples based on the results of Student's t‐test, “shrinkage t”, variable importance in projection and partial least‐squares discriminant analysis. N‐Acetylleucine, kynurenic acid, indole‐3‐ethanol, nicotinuric acid, pantothenic acid and tryptophan were the most significant biomarkers found in the urine samples, and may be crucially related to the antipyretic effect of HJ. Consequently, we propose the hypothesis that the significant antipyretic effect the HJ may be related to the inhibition of tryptophan metabolism. This research thus provides strong theoretical support and further direction to explain the antipyretic mechanism of HJ, laying the foundation for future studies.

Liu, Yang; Song, Ling; Yao, Xueting; Wu, Yiwen; Liu, Hongzhong; Zhao, Qian; Jiang, Ji; Shi, Chongtie; Ma, Xifeng; Zhou, Huimin; Liu, Dongyang; Hu, Pei

doi: 10.1002/bmc.4324

Krittanai, Supaluk; Kitisripanya, Tharita; Udomsin, Orapin; Tanaka, Hiroyuki; Sakamoto, Seiichi; Juengwatanatrakul, Thaweesak; Putalun, Waraporn

doi: 10.1002/bmc.4330pmid: 29972702

Pueraria candollei or White Kwao Krua (Leguminosae) is an indigenous plant in Thailand which has long been used in Thai traditional medicine. The tuberous root of this plant is widely used for rejuvenation, particularly in elder women. Among the bioactive compounds in P. candollei, miroestrol and puerarin exhibit estrogenic activity. This study aims to develop an immunochromatographic strip (ICS) with a colloidal gold‐based detection system for the simultaneous detection of miroestrol and puerarin in a one‐step analysis. The developed method is sensitive and specific for the detection of miroestrol and puerarin in raw materials and marketed products. The detection limits of miroestrol and puerarin were 0.15 and 4.5 μg, respectively. In addition, the results from the developed ICS were confirmed with an enzyme‐linked immunosorbent assay and presented a good correlation between these two methods. This is the first report on the development of an ICS that can detect miroestrol and puerarin in one step. The developed ICS provides a simplified method for the detection of miroestrol and puerarin in P. candollei and Pueraria spp.

Dai, Guoliang; Sun, Bingting; Wu, Lei; Gao, Xiaojun; Song, Shanshan; Sun, Hong; Ju, Wenzheng

doi: 10.1002/bmc.4329pmid: 29972688

Acute hepatitis is a severe inflammatory disease with high mortality rates. Dehydrocavidine (DC), berberine (BB) and palmatine (PT), the bioactive components of Yanhuanglian total alkaloids extract (YTAE), may play important roles in its protective effect against acute hepatitis. The present study was designed to compare the pharmacokinetic properties of DC, BB and PT after oral administration of YTAE in normal and acute hepatitis rats. The plasma pharmacokinetic profiles of three major bioactive alkaloids from YTAE were analyzed using an LC‐MS/MS method. It was found that the pharmacokinetic parameters of DC, BB and PT represented a statistically significant difference (p ˂ 0.05) between the normal rats and the acute hepatitis rats after administration of YTAE. The results indicated that the Cmax, T1/2β and AUC of DC, BB and PT in acute hepatitis rats were significantly increased, whereas the CL and Vd values were remarkably decreased (p ˂ 0.05) over those of the normal rats. The results suggested that the pharmacokinetic behavior of the active constituents from YTAE could be changed when it was used in acute hepatitis animals, which provide a meaningful basis for developing a clinical dosage regimen in the treatment of acute hepatitis by YTAE.

Abdelwahab, Nada S.; Farid, Nehal F.; Elagawany, Mohamed; Abdelmomen, Esraa H.

doi: 10.1002/bmc.4346pmid: 30045415

A novel stability‐indicating UPLC and CE method was established and validated for the determination of azelastine hydrochloride (AZL) and its genotoxic impurity, benzohydrazide, in the presence of benzalkonium chloride. The developed UPLC method was based on chromatographic separation using a C18 column as a stationary phase and acetonitrile–(0.1% w/v) aqueous sodium lauryl sulfate (55:45, v/v, pH 5 with phosphoric acid) as a mobile phase with a flow rate of 1.2 mL/min and UV detection at 215 nm. The chromatographic run time was ~2 min. The developed CE method depended on using a stationary phase of Standard Bare Fused Silica Capillaries (75 μm i.d. × 59 cm and 50 cm detection length) and the applied voltage was 30 kV using 40 mm phosphate buffer (pH 2 with aqueous H3PO4); the detection wavelength was 225 nm. The analysis time was about 6 min. The suggested methods were successfully applied for the analysis of AZL in a pharmaceutical preparation. The validity of the developed methods was assessed by applying the standard addition technique and no interference from excipients was observed. The results obtained by the proposed methods were statistically analyzed and compared with the manufacturer's method and no significant difference was found between the compared methods.

Zhang, Xiqian; Li, Ruina; Hu, Wenya; Zeng, Jin; Jiang, Xuehua; Wang, Ling

doi: 10.1002/bmc.4331pmid: 29978489

A rapid, specific, and sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method has been developed and validated to simultaneously quantify N‐acetyl‐p‐benzoquinoneimine (NAPQI), acetaminophen‐glutathione (acetaminophen‐glut) and acetaminophen‐glucuronide (acetaminophen‐gluc) in mouse plasma, liver and kidney homogenates. Analytes were eluted by a binary gradient mobile phase composed of water (phase A) and methanol containing 0.1% formic acid (phase B) at a flow rate of 0.3 mL/min, which was performed on a CAPCELL PAK C18 MG II column. It took 3.2 min to detect three analytes in a single run. Quantification was carried out in positive mode combined with multiple reaction monitoring. The validation of the LC‐MS/MS method consisted of specificity, linearity, precision, accuracy, protein precipitation recovery, matrix effect, dilution integrity and stability. The plasma and tissue homogenate calibration curves were linear over concentration ranges of 0.050–5.00, 0.050–5.00 and 0.100–40.0 μg/mL, with a lower limit of quantification of 0.050, 0.050, and 0.100 μg/mL for NAPQI, acetaminophen‐glut and acetaminophen‐gluc, respectively. The intra‐ and inter‐run precision values were within 12.47% for NAPQI, 12.11% for acetaminophen‐glut and 11.86% for acetaminophen‐gluc at their lower limit of quantitation levels. The samples were stable under all tested conditions. This method was successfully applied to study the pharmacokinetics of NAPQI, acetaminophen‐glut and acetaminophen‐gluc in ICR mice following oral administration of 200 mg/kg of acetaminophen suspension.