Biomedical Chromatography

Minz, Sunita; Kaurav, Monika; Sahu, Kantrol Kumar; Mandal, Vivekananda; Pandey, Ravi Shankar

doi: 10.1002/bmc.3444pmid: 25708181

A simple, sensitive, selective and precise high‐performance thin‐layer chromatographic method was developed for determination of lipid A (MPLA) adjuvant as a bulk and in solid fat nanoemulsions. Chromatographic separations were performed on thin‐layer chromatography aluminum plates precoated with silica gel 60 F‐254 as stationary phase and chloroform–methanol–ethyl acetate solution (10:2:4, v/v/v) as mobile phase. With this solvent system, compact spots for MPLA at Rf value 0.80 ± 0.02 were obtained. Densitometric analysis of MPLA was carried out in absorbance mode at 357 nm. Linear regression analysis for the calibration plots showed good linear relationship with r = 0.9996 in the concentration range of 20–100 ng/spot. The mean values (±SD) of slope and intercept were found to be 7.355 ± 0.006 and 109.52 ± 0.170, respectively. Limits of detection (LOD) and quantitation (LOQ) were observed at 3.096 and 9.382 ng/spot, respectively.The method was validated for precision, accuracy, robustness and recovery as per the International Conference on Harmonization guidelines. Statistical analysis proved that the developed method for quantification of MPLA as a bulk and in solid fat nanoemulsions is reproducible, selective and economical. This method could be applied for quantitative assay of MPLA in lipid‐based vaccine formulations. Copyright © 2015 John Wiley & Sons, Ltd.

Cho, Soon‐Kil; Cho, Ji‐Mi; Abd El‐Aty, A. M.; Rahman, Md. Musfiqur; Choi, Jeong‐Heui; Seo, Young‐Jun; Shin, Ho‐Chul; Shim, Jae‐Han

doi: 10.1002/bmc.3446pmid: 25753465

In this study, a simple, rapid, and sensitive method was developed for the extraction of ethephon from homogenized tomatoes that does not require a cleanup procedure. In a syringe filter, three distinct layers – aqueous, acetonitrile, and n‐hexane – are clearly separated after storage at −80 °C for 5–10 min. A Dionex IonPac column was used to separate the analyte before detection using negative‐ion mode liquid chromatography with tandem mass spectrometry (LC/MS/MS). The matrix effect of the tested analyte was negligibly small and the matched calibration showed a good linearity over a concentration range of 0.01–1.0 mg/kg with a correlation coefficient (R2) of 0.9998. The recovery at three fortification levels (0.1, 0.5 and 1.0 mg/kg) was between 82.9 and 108.6% with relative standard deviations (RSDs) <5.0%. The limit of quantification (0.03 mg/kg) was lower than the maximum residue limit (3 mg/kg) set by the Ministry of Food and Drug Safety, Republic of Korea. From a field trial, the method developed herein was applied to calculate the decline pattern and predict the pre‐harvest residue limits of ethephon in tomatoes. In conclusion, the proposed sample preparation is feasible for the detection of hydrophilic analytes in tomatoes. Copyright © 2015 John Wiley & Sons, Ltd.

Zou, Nuo‐Shu; Liang, Qiao‐Li; Li, Ping; Liu, Jie; Liu, Xiao; Kang, An; Deng, Hai‐Shan

doi: 10.1002/bmc.3447pmid: 25731641

Sparstolonin B (SsnB), a spontaneous isocoumarin compound isolated from the tuber of Scirpus yagara Ohwi. (Cyperaceae), possesses potent anti‐inflammatory and antitumor activity. In the present study, a rapid and simple UHPLC/MS/MS method for determination of SsnB in rat plasma was developed and validated. Plasma samples were pretreated by liquid–liquid extraction with ethyl acetate containing rhein as an internal standard and separated on a C18 column at 35 °C, with a gradient mobile phase consisting of acetonitrile and water containing 0.2% (v/v) formic acid within 2.1 min. MS/MS detection was accomplished in multiple reaction monitoring mode with negative electrospray ionization. The precursor–product ion transitions were m/z 266.9 (M–H)− → m/z 211.0 for SsnB and m/z 283.2 (M–H)− → m/z 239.0 for IS. The intra‐ and inter‐day precision (RSD) was <8.98% and the accuracy (RE) ranged from −7.40 to 4.50%. The extraction recoveries ranged from 96.28 to 97.30%. The pharmacokinetic parameters were calculated using Win Nonlin53 software. The absolute bioavailability of SsnB was estimated to be 6.98%. The proposed method was successfully applied to a pharmacokinetic study of SsnB in rats after intravenous administration with a dose of 0.5 mg/kg and oral administration at a dose of 5 mg/kg. Copyright © 2015 John Wiley & Sons, Ltd.

Zhang, Rui; Mei, Yong; Liu, Yanru; Dai, Hao; Xia, Hongfang; Zhang, Xin; Wu, Yukang; Gu, Yingying; Peng, Xiaowu

doi: 10.1002/bmc.3448pmid: 25758294

The abundant production of methyl tert‐butyl ether (MTBE) and its widespread use have led to an increase in the potential for human exposure. This work described a simple, fast, sensitive, reliable and low‐cost method for the simultaneous measurement of MTBE and its metabolite, tert‐butyl alcohol (TBA) in human serum by headspace solid‐phase microextraction gas chromatography–mass spectrometry. Extraction conditions were optimized and 40 °C, 10 min, 250 rpm and 0.3 g NaCl for a 1 mL sample were the optimal conditions. This method showed good analytical performance in terms of sensitivity with limits of detection in serum (1 mL) of 0.03 µg/L for MTBE and 0.05 µg/L for TBA, accuracy (mean recovery values) from 75.8% to 85.8%, precision (relative standard deviations) <10% and sample stability (biodegradation) <10% after 28 days. A verification experiment proved the reproducibility and stability of this method as well. Finally the method was used to detect 212 specimens, and the internal dose levels for MTBE in human serum were presented in China. Copyright © 2015 John Wiley & Sons, Ltd.

Meng, Xiang; Zhang, Ting; Li, Ying; Pan, Qi; Jiang, Juan; Luo, Yongwei; Chong, Liming; Yang, Yang; Xu, Sichong; Zhou, Li; Sun, Zuyue

doi: 10.1002/bmc.3449pmid: 25736727

The vaginal administration route suffers from relatively low absorption efficiency, which may hinder the identification of the toxicokinetics of curdione in pregnant women. A sensitive analytical method for determining the plasma concentration of curdione was developed and applied in the determination of curdione in pregnant Sprague–Dawley rats as a simulated model. Glimepiride was used as an internal standard and chromatographic separation was achieved on a Capcell Pak C18 MGIII column. A gradient elution profile with 0.5% formic acid (A)–0.5% formic acid–acetonitrile (B) was selected as mobile phase. The selected reaction monitoring mode was used for quantification based on the target fragment ions m/z 237.2 to m/z 135.1 for curdione and m/z 491.3 to m/z 352.1 for the glimepiride. The standard curve was linear over the range of 0.5–500 ng/mL for curdione in rat plasma and yielded a consistent peak pattern, even at the lower limit of quantitation of 0.5 ng/mL. The retention times of curdione and IS were 6.55 and 6.59 min, respectively. The mean recovery of curdione in rat plasma was 95.5–101.1%. The intra‐day and inter‐day precisions were between 2.35 and 9.08%. This LC‐MS/MS method provides a simple and sensitive means for determining the plasma concentration. Copyright © 2015 John Wiley & Sons, Ltd.

Zhuang, Jialang; Chen, Jiangying; Wang, Xueding; Pang, Yin; Bi, Huichang; Huang, Lihui; Zeng, Guixiong; Liao, Xiaoxing; Ma, Zhongfu; Chen, Xiao; Zhong, Guoping; Huang, Min; Zhao, Xianglan

doi: 10.1002/bmc.3450pmid: 25776729

A rapid and sensitive liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) method to determine clonidine in human plasma was developed and fully validated. Sample preparation was involved an one‐step extraction with diethyl ether. Donepezil was employed as the internal standard (IS). Chromatographic separation was performed on a Hypersil BDS C18 column (i.d. 2.1 × 50 mm, particle size 3μm) with a mobile phase of methanol–water (containing 0.1% formic acid; 60:40, v/v) at a flow rate of 200 μL/min. The peaks were detected by mass spectrometry using the electrospray ion source in selected reaction monitoring mode. The extraction recovery was 72.53–85.25%. The method was found to be linear in a concentration range of 0.02–6.00 ng/mL and the lower limit of quantification was 0.02 ng/mL. The within‐ and between‐batch precisions at three concentrations were 4.33–16.47 and 7.24–17.24% with accuracies of −2.47–10.91 and 1.86–10.19%, respectively. This validated method was successfully used for a bioequivalence study of two clonidine transdermal patches on healthy volunteers. The results suggested that the test formulation of clonidine patch met the regulatory criterion for bioequivalence to the reference formulation, but a larger sample size should be needed for the estimation of bioequivalence. Copyright © 2015 John Wiley & Sons, Ltd.

Shi, Hai‐Li; Jia, Yan‐Wei; Peng, Shu‐Lin; Liao, Xun; Ding, Li‐Sheng; Liu, Yi‐Ming

doi: 10.1002/bmc.3451pmid: 25753568

Highly active and recoverable nanobioreactors prepared by immobilizing rat liver microsomes on magnetic nanoparticles (LMMNPs) were utilized in metabolic study of Angelica dahurica extracts. Five metabolites were detected in the incubation solution of the extracts and LMMNPs, which were identified by means of HPLC‐MS as trans‐imperatorin hydroxylate (M1), cis‐imperatorin hydroxylate (M2), imperatorin epoxide (M3), trans‐isoimperatorin hydroxylate (M1′) and cis‐isoimperatorin hydroxylate (speculated M2′). Compared with the metabolisms of imperatorin and isoimperatorin, it was found that the five metabolites were all transformed from these two major compounds present in the plant. Since no study on isoimperatorin metabolism by liver microsomal enzyme system has been reported so far, its metabolites (M1′ and M3′) were isolated by preparative HPLC for structure elucidation by 1H‐NMR and MS2 analysis. M3′ was identified as isoimperatorin epoxide, which is a new compound as far as its chemical structure is concerned. However, interestingly, M3′ was not detected in the metabolism of the whole plant extract. In addition, a study with known chemical inhibitors on individual isozymes of the microsomal enzyme family revealed that CYP1A2 is involved in metabolisms of both isoimperatorin and imperatorin, and CYP3A4 only in that of isoimperatorin. Copyright © 2015 John Wiley & Sons, Ltd.

Li, Peng; Chen, Qinhua; Yu, Fei; Xie, Xiaoyu; Wang, Sicen

doi: 10.1002/bmc.3452pmid: 25728326

Camptotheca acuminata Decne is an important medicinal plant that contains various cytotoxic alkaloids, such as camptothecine (CPT) and 10‐hydroxycamptothecine (HCPT). A rapid and sensitive liquid chromatography with fluorescence detection (LC‐FLD) method for the quantification of CPT and HCPT is described. The separation was carried out on a DL‐Cl8 column (4.6 × 150 mm, 5 µm), with the mobile phase of acetonitrile–sodium dihydrogen phosphate buffer (10 mm) using an gradient elution at the flow rate of 0.6 mL/min. The LC‐FLD method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the above components. The lower detection limits of CPT and HCPT were 0.4 and 0.1 ng/mL, respectively. The precision was <1.58% and the mean recovery of the analytes was 96.0–98.6%. The LC‐FLD method was successfully applied to determine CPT and HCPT in real samples including C. acuminate, HCPT injection and rat plasma. Copyright © 2015 John Wiley & Sons, Ltd.