Quantification of bupivacaine hydrochloride and isoflupredone acetate residues in porcine muscle, beef, milk, egg, shrimp, flatfish, and eel using a simplified extraction method coupled with liquid chromatography–triple quadrupole tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-19 Sang-Hyun Cho, Jin-A Park, Weijia Zheng, A.M. Abd El-Aty, Seong-Kwan Kim, Jeong-min Choi, Hee Yi, Soo-Min Cho, Nehal A. Afifi, Jae-Han Shim, Byung-Joon Chang, Jin-Suk Kim, Ho-Chul Shin
In this study, a simple analytical approach has been developed and validated for the determination of bupivacaine hydrochloride and isoflupredone acetate residues in porcine muscle, beef, milk, egg, shrimp, flatfish, and eel using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A 0.1% solution of acetic acid in acetonitrile combined with n-hexane was used for deproteinization and defatting of all tested matrices and the target drugs were well separated on a Waters Xbridge™ C18 analytical column using a mobile phase consisting of 0.1% acetic acid (A) and 0.1% solution of acetic acid in methanol (B). The linearity estimated from six-point matrix-matched calibrations was good, with coefficients of determination ≥ 0.9873. The limits of quantification (LOQs) for bupivacaine hydrochloride and isoflupredone acetate were 1 and 2 ng g−1, respectively. Recovery percentages in the ranges of 72.51–112.39% (bupivacaine hydrochloride) and 72.58–114.56% (isoflupredone acetate) were obtained from three different fortification concentrations with relative standard deviations (RSDs) of <15.14%. All samples for the experimental work and method application were collected from the local markets in Seoul, Republic of Korea, and none of them tested positive for the target drugs. In conclusion, a simple method using a 0.1% solution of acetic acid in acetonitrile and n-hexane followed by LC-MS/MS could effectively extract bupivacaine hydrochloride and isoflupredone acetate from porcine muscle, beef, milk, egg, shrimp, flatfish, and eel samples.
Rapid determination of 18 glucocorticoids in serum using reusable on-line SPE polymeric monolithic column coupled with LC-quadrupole/orbitrap high-resolution mass spectrometer J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-19 Hui Li, Lianfeng Ai, Sufang Fan, Yan Wang, Dianxing Sun
A simple, rapid and sensitive method for the simultaneous determination of 18 glucocorticoids in serum was developed by coupling on-line solid-phase extraction (SPE) polymeric monolithic column to a liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometer. A simple poly(ethylene glycol dimethacrylate) monolith column (10 mm × 2.1 mm i.d.) was fabricated, and the morphology, surface area and extraction performance of the monolithic column were characterized. Serum samples were extracted by acetonitrile (ACN). Then, online SPE was achieved on the synthesized monolithic column using a 10 mmol/L ammonium acetate solution as the loading solvent. After the transfer from the monolith into analytical column (Capcell Pak ADME column) using ACN, the adsorbed analytes were separated on the analytical column and detected with a high-resolution hybrid quadrupole/orbitrap mass spectrometer with full scan/ddMS2 scan mode Under optimized conditions, the method was linear with target linear correlation coefficient (R2) higher than 0.995. Detection limits were in range of 0.1–0.6 ng/mL, and the quantification limits were 0.3–1.5 ng/mL. The recovery was between 71.9% and 89.2% in three spike levels with precision (n = 5) of 5.40–12.1%. The serum sample was directly analyzed after a simple extraction procedure, and the on-line SPE and determination were achieved within only 16 min. The method was used to analyze the dynamic contents variation of cortisone and hydrocortisone in serum before and after the surgery.
Characterization of 30 therapeutic antibodies and related products by size exclusion chromatography: feasibility assessment for future mass spectrometry hyphenation J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-19 Alexandre Goyon, Valentina D’Atri, Olivier Colas, Szabolcs Fekete, Alain Beck, Davy Guillarme
Despite the popularity of targeted and immune therapies, the number of studies dealing with the quantitation of aggregates for Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved mAb and related products are still very scarce in literature. In this work, 30 therapeutic proteins including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), Fc-fusion proteins and a bi-specific antibody (bsAb) were investigated using size exclusion chromatography (SEC). Their levels of high molecular weight species (HMWS) were experimentally estimated between 0.1% and 13.1%. Except for blinatumomab, etanercept and pembrolizumab, the HMWS amount for the other antibodies was well below the limit of 5% usually set a specification for therapeutic mAbs in the biopharmaceutical industry. The main chromatographic peak shape of 24 therapeutic antibodies and the NIST mAb  was found suitable (0.8 < As < 1.5) with a generic SEC method involving potassium-based salts mobile phase. Conversely, only acidic therapeutic proteins (pI < 7) could be successfully analyzed with a mass spectrometry (MS) compatible mobile phase containing 100 mM ammonium acetate. This study aimed to provide HMWS data for 30 therapeutic proteins covering a wide range of physico-chemical properties with molecular weights between 54 and 153 kDa, pI values comprised between 6.1 and 9.4 and hydrophobic interaction chromatography (HIC) retention factors ranging from 1.2 to 6.0 for the mAbs.
Fibroin/dodecanol floating solidification microextraction for the preconcentration of trace levels of flavonoids in complex matrix samples1 J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-18 Xuan Chen, Jie Li, Shuang Hu, Xiaohong Bai, Haodong Zhao, Yi Zhang
A new fibroin/dodecanol floating solidification microextraction, coupled with high performance liquid chromatography, was developed and applied for enrichment and quantification of the trace flavonoids in traditional Chinese medicine and biological samples. Also, fibroin sensibilization mechanism was described, and influence of sample matrix to enrichment factor was investigated. In this method, a homogeneous fibroin/dodecanol of dispersed solution was employed as microextraction phase to flavonoids (myricetin, quercetin, isorhamnetin, chrysin, kaempferide), the several critical parameters affecting the performance, such as organic extractant, amount of fibroin in organic extractant, volume of extraction phase, dispersant, salt concentration, pH of sample phase, stirring rate, extraction time, and volume of sample phase were tested and optimized. Under the optimized conditions, enrichment factor of flavonoids ranged from 42.4 to 238.1 in different samples, excellent linearities with r2≥ 0.9968 for all analytes were achieved, limits of detection were less than or equal to 5.0 ng/mL, average recoveries were 92.5% to 115.0% in different samples. The new procedure is simple, fast, low cost, environmentally friendly and high EF, it can also be applied to the concentration and enrichment of the trace flavonoids in other complex matrixes.
Simultaneous determination of cucurbitacin B and cuburbitacin E in rat plasma by UHPLC-MS/MS: A pharmacokinetics study after oral administration of cucurbitacin tablets J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-18 Zhibin Wang, Wenbo Zhu, Mingjie Gao, Chengcui Wu, Chunjuan Yang, Jing Yang, Gaosong Wu, Bingyou Yang, Haixue Kuang
Cucurbitacin B (CuB) and cucurbitacin E (CuE) are tetracyclic triterpene compounds from Cucurbitaceae, and the main bioactive compounds of cucurbitacins tablets that used to treatment of chronic hepatitis. Pharmacological research has been very comprehensive, and there are few studies on pharmacokinetics, especially about CuE. An Ultra High Performance Liquid Chromatography-tandem Mass Spectrometry (UHPLC-MS/MS) method which with high selectivity, simplicity and sensitivity has been used for quantitative analysis of Cucurbitacin B (CuB) and cucurbitacin E (CuE). Plasma samples were pretreatment by Liquid-liquid extraction (LLE) method with dichloromethane. The chromatographic separation was performed on a C18 column (Agilent Eclipse Plus, 4.6 × 150 mm, 5 μm) using gradient elution with water – methanol at a flow rate of 0.3 mL/min and the column temperature was set at 30 °C. The method was validated according to FDA guidelines. Lower limit of quantification (LLOQ) was 1.60 ng/mL for CuB and 1.58 ng/mL for CuE. Correlation coefficients of CuB and CuE were more than 0.99 in rat plasma. All values of intra-day and inter-day precision (RSD %) were not exceeded 15%, the accuracy (RE %) were within −5.57–5.20% for CuB and −3.33–7.37% for CuE. The mean extraction recoveries were more than 80%. Pharmacokinetic parameters were also evaluated use UHPLC-MS/MS method. The results suggestion that this method was successfully applied to pharmacokinetic study of CuB and CuE in rat plasma after oral administration cucurbitacin tablets.
Development of a simple multi-residue determination method of 80 veterinary drugs in Oplegnathus punctatus by liquid chromatography coupled to quadrupole Orbitrap mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-17 Fei Zhao, Xin Gao, Zhixu Tang, Xin Luo, Miaomiao Wu, Jiachao Xu, Xiaoting Fu
A simple, rapid and sensitive multi-residue analytical method was developed and validated for 80 veterinary drugs in Oplegnathus punctatus using ultrahigh performance liquid chromatography-Orbitrap high resolution mass spectrometry (LC-HRMS). The analytes belong to 12 different families include benzimidazoles, β-lactams, lincosamides, macrolides, nitromidazoles, quinolones, sulfonamides and trimethoprim, tetracyclines, triphenylmethane dyes, amphenicols, nonsteroidal estrogens and steroid hormones. The sample preparation was optimized base on QuEChERS (quick, easy, cheap, effective, rugged and safe) procedure. A very simple and sufficient preparation procedure without salting-out and complex clean-up process was studied. It had been proved that water in the extract was helpful for extracting hydrophilic compounds and precipitating the lipids during the subsequent cleaning process. In addition, an appropriate percent of methanol was necessary to some analytes. Finally, a mixture of acetonitrile, methanol and water (3:1:1, v/v/v) which include 1% acetic acid and 10 mM ethylenediaminetetraacetic acid disodium salt 2-hydrate was selected as the extraction solvent, and the clean-up step consisted of a low temperature procedure and two times of high-speed centrifugation to deproteinize and remove lipids. The detection and quantification of all compounds were performed by ultrahigh performance liquid chromatography coupled with electrospray ionization quadrupole Orbitrap high resolution mass spectrometry in positive and negative ion mode. This methodology was validated according to the Commission Decision 2002/657/EC and SANTE/11945/2015. The recoveries ranged from 60.74%–109.85% with relative standard deviations (RSDs) < 20%. The limits of quantification (LOQs) were 0.25–25 ug/kg, for the analytes which the MRL or MRPL had been established in fish tissue, the LOQs were all lower than their own legal tolerances. The values of decision limit (CCα) and detection capability (CCβ) were in the range of 1.91–1001.13 ug/kg and 3.52–1002.26 ug/kg, respectively. This validated method has been successfully applied on the determination of veterinary drugs in real commercial oplegnathus punctatus samples.
Tryptophan and kynurenine determination in human hair by LIQUID CHROMATOGRAPHY J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-17 Michelli F. Dario, Thamires Batello Freire, Claudinéia Aparecida Sales de Oliveira Pinto, María Segunda Aurora Prado, André R. Baby, Maria Valéria R. Velasco
Tryptophan, an amino acid found in hair proteinaceous structure is used as a marker of hair photodegradation. Also, protein loss caused by several chemical/physical treatments can be inferred by tryptophan quantification. Kynurenine is a photo-oxidation product of tryptophan, expected to be detected when hair is exposed mainly to UVB (290–320 nm) radiation range. Tryptophan from hair is usually quantified directly as a solid or after alkaline hydrolysis, spectrofluorimetrically. However, these types of measure are not sufficiently specific and present several interfering substances. Thus, this work aimed to propose a quantification method for both tryptophan and kynurenine in hair samples, after alkali hydrolysis process, by using high-performance liquid chromatography (HPLC) with fluorimetric and UV detection. The tryptophan and kynurenine quantification method was developed and validated. Black, white, bleached and dyed (blond and auburn) hair tresses were used in this study. Tryptophan and kynurenine were separated within ∼9 min by HPLC. Both black and white virgin hair samples presented similar concentrations of tryptophan, while bleaching caused a reduction in the tryptophan content as well as dyeing process. Unexpectedly, UV/vis radiation did not promote significantly the conversion of tryptophan into its photo-oxidation product and consequently, kynurenine was not detected. Thus, this works presented an acceptable method for quantification of tryptophan and its photooxidation metabolite kynurenine in hair samples. Also, the results indicated that bleaching and dyeing processes promoted protein/amino acids loss but tryptophan is not extensively degraded in human hair by solar radiation.
Development of a highly sensitive and specific ELISA method for the determination of l-corydalmine in SD rats with monoclonal antibody J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-14 Hongwei Zhang, Lan Gao, Menglin Shu, Jihua Liu, Boyang Yu
l-Corydalmine (l-CDL) is a potent analgesic constituent of the traditional Chinese medicine, Rhizoma Corydalis. However, the pharmacokinetic process and tissue distribution of l-CDL in vivo are still unknown. Therefore, it is necessary to establish a simple and sensitive method to detect l-CDL, which will be helpful to study its distribution and pharmacokinetic process. To determine this compound in biological samples, a monoclonal antibody (mAb) against l-CDL was produced and a fast and highly sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed in this study. The icELISA was applied to determine l-CDL in biological samples. The limit of detection (LOD) of the method was 0.015 ng/mL with a liner range of 1–1000 ng/mL (R2 = 0.9912). The intra- and inter-day precision were below 15% and the recoveries were within 80–117%. Finally, the developed immunoassay was successfully applied to the analysis of the distribution of l-CDL in SD rats. In conclusion, the icELISA based on the anti-l-CDL mAb could be considered as a highly sensitive and rapid method for the determination of l-CDL in biological samples. The ELISA approach may provide a valuable tool for the analysis of small molecules in biological samples.
Simultaneous determination of gentiopicroside and its two active metabolites in rat plasma by LC-MS/MS and its application in pharmacokinetic studies J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-14 Kai Xiong, Tingting Gao, Tong Zhang, Zhengtao Wang, Han Han
Gentiopicroside is a natural secoiridoid glycoside that may require metabolic activation to exert pharmacological effects. In this study, two active metabolites of gentiopicroside (M1 and M2) were isolated from rat urines and identified with our previous method. Most importantly, a fast, sensitive and selective ultra high-performance liquid chromatography–tandem mass spectrometry method was developed to simultaneously determine gentiopicroside and its two metabolites in rat plasma. The analytes and internal standard (swertiamarin) were separated on an ACQUITY UPLC® BEH C18 column (2.1 × 50 mm, 1.7 μm) using gradient elution by acetonitrile and 0.1% formic acid at a flow rate of 0.4 mL/min. The mass spectrometry detector was operated in the multiple reaction monitoring with positive ionization mode. The method had a good linearity over the concentration range of 0.2–10000 ng/mL for gentiopicroside and 0.1–5000 ng/mL for the two metabolites. The validated method was successfully applied to the pharmacokinetic study of gentiopicroside and its metabolites after single oral administration of gentiopicroside (150 mg/kg) to rats (n = 8). The pharmacokinetic differences between gentiopicroside and its two metabolites were identified.Results provided the evidence for in vivo metabolism-based activation of gentiopicroside.
Development and validation of carbofuran and 3-hydroxycarbofuran analysis by high-pressure liquid chromatography with diode array detector (HPLC-DAD) for forensic Veterinary Medicine J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-14 Vagner Golçalves Junior, Nicolle Queiroz Hazarbassanov, Adriana de Siqueira, Jorge Camilo Florio, Claudia Helena Pastor Ciscato, Paulo Cesar Maiorka, Andre Fukushima, Helenice de Souza Spinosa
Agricultural pesticides used with the criminal intent to intoxicate domestic and wild animals are a serious concern in Veterinary Medicine. In order to identify the pesticide carbofuran and its metabolite 3- hydroxycarbofuran in animals suspected of exogenous intoxication a high pressure liquid chromatography with diode array detector (HPLC-DAD) method was developed and validated in stomach contents, liver, vitreous humor and blood. The method was evaluated using biological samples from seven different animal species. The following parameters of analytical validation were evaluated: linearity, precision, accuracy, selectivity, recovery and matrix effect. The method was linear at the range of 6.25–100 μg/ml and the correlation coefficient (r2) values were >0.9811 for all matrices. The precision and accuracy of the method was determined by coefficient of variation (CV) and the relative standard deviation error (RSE), and both were less than 15%. Recovery ranged from 74.29 to 100.1% for carbofuran and from 64.72 to 100.61% for 3-hydroxycarbofuran. There were no significant interfering peaks or matrix effects. This method was suitable for detecting 25 positive cases for carbofuran amongst a total of 64 animal samples suspected of poisoning brought to the Toxicology Diagnostic Laboratory, School of Veterinary Medicine and Animal Sciences, University of Sao Paulo.
Determination of quantitative retention-activity relationships between pharmacokinetic parameters and biological effectiveness fingerprints of Salvia miltiorrhiza constituents using biopartitioning and microemulsion high-performance liquid chromatography J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-13 Haoshi Gao, Hongzhang Huang, Aini zheng, Nuojun Yu, Ning Li
In this study, we analyzed danshen (Salvia miltiorrhiza) constituents using biopartitioning and microemulsion high-performance liquid chromatography (MELC). The quantitative retention-activity relationships (QRARs) of the constituents were established to model their pharmacokinetic (PK) parameters and chromatographic retention data, and generate their biological effectiveness fingerprints. A high-performance liquid chromatography (HPLC) method was established to determine the abundance of the extracted danshen constituents, such as sodium danshensu, rosmarinic acid, salvianolic acid B, protocatechuic aldehyde, cryptotanshinone, and tanshinone IIA. And another HPLC protocol was established to determine the abundance of those constituents in rat plasma samples. An experimental model was built in Sprague Dawley (SD) rats, and calculated the corresponding PK parameterst with 3P97 software package.Thirty-five model drugs were selected to test the PK parameter prediction capacities of the various MELC systems and to optimize the chromatographic protocols. QRARs and generated PK fingerprints were established. The test included water/oil-soluble danshen constituents and the prediction capacity of the regression model was validated. The results showed that the model had good predictability.
Systematic Characterization of the Metabolites of Paeonol in Rats Using Ultra Performance Liquid Chromatography Coupled with Electrospray Ionization Quadrupole Time-of-Flight tandem Mass Spectrometry with an Integrative Strategy J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-11 Li-Qin Ding, Tian-Yi Qiu, Zhao-Xi Liu, Li-Xia Chen, Mahmood Brobbey Oppong, De-Qin Zhang, Bo-li Zhang, Gang Bai, Feng Qiu
Paeonol, an active constituent in the root bark of Paeonia suffruticosa Andrews, is used to treat inflammation, headache and other diseases in clinic. Though the data on pharmacological researches of paeonol abounds, its metabolic profile is not so clear. It is essential to systematically characterize the in vivo metabolites in order to better understand its mechanism of action. In this study, ultra performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q/TOF-MS) with an integrative strategy was developed for analysis of paeonol metabolites. As a result, based on seven reference substances isolated or synthesized, twenty-five metabolites were detected and identified in urine, feces, bile and plasma of rats after oral administration of paeonol. To the best of our knowledge, 14 of these metabolites have not been reported previously. In addition, the dominating metabolic fates were oxidation, demethylation, hydrogenation, glucuronic acid and sulfate conjugations, and hydrogenation of paeonol was reported for the first time. This research provides scientific and reliable support for full understanding of the metabolic profiling of paeonol.
A Simple Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry Assay for the Simultaneous Quantification of 15 Antibiotics in Plasma J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-11 S. Lefeuvre, J. Bois-Maublanc, L. Hocqueloux, L. Bret, T. Francia, C. Eleouet-Da Violante, E.M. Billaud, F. Barbier, L Got
Antibiotic (ATB) treatment of critically ill patients with pathophysiological injuries remains a challenge due to the constant increase in antimicrobial resistance. Therapeutic drug monitoring (TDM) is advised for ATB dose adjustments to avoid suboptimal concentrations and dose-related adverse effects. Therefore, a single and reliable analytical method for a broad selection of ATBs was developed using a high-resolution mass spectrometry (HRMS) platform for frequent use in intensive care units.An UHPLC assay coupled to high resolution accurate mass acquisition has been developed for the quantification of penicillins (amoxicillin, oxacillin, piperacillin, and ticarcillin), cephalosporines (cefepime, cefotaxime, ceftazidime, and ceftriaxone), carbapenems (ertapenem, imipenem, and meropenem), lincosamide (clindamycin), quinolones (ofloxacin and ciprofloxacin) and tazobactam. Plasma samples (100 μL) were spiked with an internal standard solution followed by protein precipitation. Separation was achieved on an Accucore C18 column, which enabled sample analysis every 9 min.All compounds were detected in electrospray positive ion mode and quantified with a linear regression between 0.5 and 32 mg/L (r2 > 0.998). Overall precision and accuracy did not exceed 15%. No significant matrix effect was observed for the studied ATBs. Stored stock solutions at −20 °C were stable for 6 months, except for amoxicillin and imipenem. Analytes in plasma were stable for 24 h under ambient conditions as well as in post-preparation in an autosampler, except for amoxicillin and imipenem.This HRMS assay provides the simultaneous quantification of 15 ATB; it fulfills the usual quality criteria and was successfully applied for routine TDM of ATBs. The method is based on a full scan acquisition, and it would be easy to add other compounds to the present panel in the future, as this assay has already been proven to be efficient for different classes of compounds.
Application of ultra-high-performance liquid chromatography coupled with LTQ-Orbitrap mass spectrometry for identification, confirmation and quantitation of illegal adulterated weight-loss drugs in plant dietary supplements J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-09 Qiaoyuan Cheng, Linjun Shou, Cen Chen, Shi Shi, Minghao Zhou
In this paper, an ultra-high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap HRMS) method was developed and validated for identification, confirmation and quantitation of illegal adulterated weight-loss drugs in plant dietary supplements. 13 weight-loss drugs were well separated by the gradient elution of 10 mmol/L ammonium acetate − 0.05% formic acid H2O and acetonitrile at a flow rate of 0.2 mL/min within 12 min. The MS analysis was operated under the positive ion and in full MS/dd-MS2 (data-dependent MS2) mode. The full MS scan with resolution at 60 000 FWHM and narrow mass windows at 5 ppm acquired data for identification and quantitation, and dd-MS2 scan with resolution at 15 000 FWHM obtained product ions for confirmation. The method validation showed good linearity with coefficients of determination (r2) higher than 0.9951 for all analytes. Meantime, all the LOD and LLOQ values were in the respective range of 0.3-2 and 1–9 ng/g. The accuracy, intra- and inter-day precision were in the ranges of −1.7-3.4%, 1.7-5.0% and 1.9-4.4%, respectively. The mean recoveries ranged from 85.4 to 107.1%, while the absolute and relative matrix effect were in the corresponding range of 98.2-108.6% and 2.6-8.7%. Among 120 batches of weight loss plant dietary supplements, sibutramine and fluoxertine or both were positive in 29 samples. In general, LTQ-Orbitrap HRMS technology was a powerful tool for the analysis of illegal ingredients in dietary supplements.
Simultaneous determination of ethyl carbamate and urea in Korean rice wine by ultra-performance liquid chromatography coupled with mass spectrometric detection J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-09 Gyeong-hweon Lee, Dae-young Bang, Jung-hoon Lim, Seok-min Yoon, Myeong-Jai Yea, Young-min Chi
In this study, a rapid method for simultaneous detection of ethyl carbamate (EC) and urea in Korean rice wine was developed. To achieve quantitative analysis of EC and urea, the conditions for Ultra-performance liquid chromatography (UPLC) separation and atmospheric-pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) detection were first optimized. Under the established conditions, the detection limit, relative standard deviation and linear range were 2.83 μg/L, 3.75−5.96%, and 0.01−10.0 mg/L, respectively, for urea; the corresponding values were 0.17 μg/L, 1.06−4.01%, and 1.0−50.0 μg/L, respectively, for EC. The correlation between the contents of EC and its precursor urea was determined under specific pH (3.5 and 4.5) and temperature (4, 25, and 50 °C) conditions using the developed method. As a result, EC content was increased with greater temperature and lower pH. In Korean rice wine, urea was detected 0.19–1.37 mg/L and EC was detected 2.0–7.7 μg/L. The method developed in this study, which has the advantages of simplified sample preparation, low detection limits, and good selectivity, was successfully applied for the rapid analysis of EC and urea.
Determination of chlormequat and mepiquat residues and their dissipation rates in tomato cultivation matrices by ultra-performance liquid chromatography-tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-08 Hongxia Zhang, Zheng Feei Ma, Haiyan Yang, Liming Kong
This study described the development and validation of a simple, rapid, specific and sensitive method for detecting chlormequat chloride (CQ) and mepiquat chloride (MQ) residues in tomato cultivation matrices covering soil, water, seedling samples. The dissipation rates of CQ and MQ in tomato cultivation matrices were also determined in this study. A Hydrophilic Interaction Liquid Chromatography (HILIC) column was used for chromatographic separation. A triple quadrupole mass spectrometer equipped with an electrospray ionisation source in positive ion mode by multiple reaction monitoring was used for detection. Soil samples were extracted with accelerated solvent extraction (ASE) and cleaned up with WCX phase extraction column; water samples were extracted with WCX phase extraction column; seedling samples were extracted with methanol-ammonium acetate solution. LODs and LOQs of CQ and MQ were 0.02 μg/kg and 0.1 μg/kg in soil samples, 0.005 ng/mL and 0.02 ng/mL in water samples, and 0.05 μg/kg and 1.0 μg/ kg in seedling samples, respectively. The mean recovery rate of CQ in soil, water and seedling samples ranged from 76.98% to 111.60%. While the mean recovery rate of MQ in soil, water and seedling samples ranged from 96.90% to 105.40%. The fastest to the slowest metabolising rates of CQ and MQ were as follows: soil samples > seedling samples > water samples. In conclusion, this study provided a new potential method for detecting CQ and MQ in tomato cultivation matrices using ultra-performance liquid chromatography-tandem mass spectrometry.
Sensitivity improvement of the LC-MS/MS quantification of carbidopa in human plasma and urine by derivatization with 2,4-pentanedione J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-08 Nico C. van de Merbel, Kees J. Bronsema, Steven H. Gorman, Ray Bakhtiar
The reliable quantification of carbidopa in biological samples at low concentrations is challenging because of the polar and highly unstable nature of the compound. In this paper, LC–MS/MS methods are described for the determination of carbidopa in 50 μL of human plasma and 25 μL of human urine in the concentration ranges 1-1,000 ng/mL and 100-50,000 ng/mL, respectively. After a simple protein precipitation (plasma) or dilution (urine) step, carbidopa is derivatized at its hydrazine moiety by reaction for one hour with 2,4-pentanedione under acidic conditions and at 40⁰C. The product is a relatively non-polar molecule that is suitable for reversed-phase liquid chromatography (3.5 min run time) with detection by tandem mass spectrometry with electrospray ionization. A stable-isotope labeled internal standard is used for response normalization. Precision, accuracy and selectivity of the methods meet the criteria of international guidelines for bioanalytical method validation. Acidification of urine to pH 1.5 and the addition of two anti-oxidants (5 mg/mL sodium metabisulfite and 1 mg/mL butylated hydroxytoluene) to plasma, in combination with sampling and analysis on ice and under yellow light, ensure sufficient stability of carbidopa. The methods were successfully used to determine plasma pharmacokinetics and urinary excretion of carbidopa in healthy volunteers after a single 37.5 mg oral dose.
Determination of Eight Quinolones in Milk using Immunoaffinity Microextraction in a Packed Syringe and Liquid Chromatography with Fluorescence Detection J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-08 Xinda Zhang, Cuicui Wang, Linyan Yang, Wei Zhang, Jing lin, Cun Li
We have established a new, highly selective, and sensitive method for the determination of eight quinolones (QNs) in milk: danofloxacin, enrofloxacin, orbifloxacin, norfloxacin, ofloxacin, lomefloxacin, fleroxacin, and ciprofloxacin. The method uses immunoaffinity microextraction in a packed syringe and liquid chromatography with fluorescence detection (IA-MEPS-LC-FLD). Traditionally, QN residues are determined by liquid-liquid extraction (LLE) and solid phase extraction (SPE) sample preparation techniques; however, these methods are time-consuming and require large quantities of organic solvents. We thus developed a novel immunoaffinity adsorbent combined with MEPS for QN residue analysis. The syringe was filled with 0.2 g of microbeads bound with a QN monoclonal antibody using glutaraldehyde. The relevant parameters of the IA-MEPS method were optimized and discussed herein. Milk samples were extracted at a flow rate of 3.5 mL/min, 600 μL of methanol-and phosphate-buffered saline (9:1, v/v) was used for elution, and 200 μL of mobile phase was used for reconstitution after the sample was dried with nitrogen. Then, the sample was detected by LC-FLD. For the eight QNs, the limit of detection ranged from 0.05 to 0.1 ng/g, the limit of quantification ranged from 0.15 to 0.3 ng/g, and the intra- and inter-day precision were 3.2%–14.6% and 9.1%–15.8%, respectively. The advantages of the IA-MEPS method includ simple operation, low cost and reduced organic solvent use. Moreover, the sample pretreatment is environmentally friendly because of the reduced solvent volume requirements.
Determination of hydroxyurea in human plasma by HPLC-UV using derivatization with xanthydrol. J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-07 Tiphaine Legrand, Marie-Georgine Rakotoson, Frédéric Galactéros, Pablo Bartolucci, Anne Hulin
Determination of 74 new psychoactive substances in serum using automated in-line solid-phase extraction-liquid chromatography-tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-07 Sabrina Lehmann, Tobias Kieliba, Justus Beike, Mario Thevis, Katja Mercer-Chalmers-Bender
A detailed description is given of the development and validation of a fully automated in-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method capable of detecting 90 central-stimulating new psychoactive substances (NPS) and 5 conventional amphetamine-type stimulants (amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA), 3,4-methylenedioxy-amphetamine (MDA), 3,4-methylenedioxy-N-ethyl-amphetamine (MDEA), methamphetamine) in serum. The aim was to apply the validated method to forensic samples. The preparation of 150 μL of serum was performed by an Instrument Top Sample Preparation (ITSP)-SPE with mixed mode cation exchanger cartridges. The extracts were directly injected into an LC-MS/MS system, using a biphenyl column and gradient elution with 2 mM ammonium formate/0.1% formic acid and acetonitrile/0.1% formic acid as mobile phases. The chromatographic run time amounts to 9.3 min (including re-equilibration). The total cycle time is 11 min, due to the interlacing between sample preparation and analysis. The method was fully validated using 69 NPS and five conventional amphetamine-type stimulants, according to the guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh). The guidelines were fully achieved for 62 analytes (with a limit of detection (LOD) between 0.2 and 4 μg/L), whilst full validation was not feasible for the remaining 12 analytes. For the fully validated analytes, the method achieved linearity in the 5 μg/L (lower limit of quantification, LLOQ) to 250 μg/L range (coefficients of determination > 0.99). Recoveries for 69 of these compounds were greater than 50%, with relative standard deviations ≤ 15%. The validated method was then tested for its capability in detecting a further 21 NPS, thus totalling 95 tested substances. An LOD between 0.4 and 1.6 μg/L was obtained for these 21 additional qualitatively-measured substances. The method was subsequently successfully applied to 28 specimens from routine forensic case work, of which 7 samples were determined to be positive for NPS consumption.
Relationship between the color stability and impurity profile of cefotaxime sodium J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-07 Hua Sun, Xuejun Cui, Baoshu Liu, Junli Zhang
The color grade, mainly introduced in the processes of semisynthesis and storage, is an important index used to evaluate the quality of cefotaxime sodium. Because the drug itself is prone to degradation under susceptible conditions, including those involving moisture, heat, ultraviolet light, acids, alkalis, and oxidants, and a series of degradation products as impurities are generated. In this study, the factors affecting color grade stability and the degradation mechanisms of cefotaxime sodium were investigated by designing different accelerated stability tests under the aforementioned conditions. The degradation extent was studied by using analytical methods, such as a solution color comparison method, ultraviolet spectrophotometry, and HPLC. The relationship between the color grade stability of cefotaxime sodium and its impurity profile has been explored, and a reasonable degradation mechanism has been proposed. The manufacturing conditions of inspection have been optimized, and a scientific basis for drug packaging, storage, and transportation conditions has been established. The results show that the color grade stability of cefotaxime sodium is related to the impurity profile to some degree, and the difference between the actual color and the standard color can reflect the levels of impurities to some extent.
Describing the holistic toxicokinetics of hepatotoxic Chinese herbal medicines by a novel integrated strategy: Dioscorea bulbifera rhizome as a case study J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-06 Ling-Li Wang, Dong-Sheng Zhao, Wei Shi, Zhuo-Qing Li, Zi-Tian Wu, Ping Li, Hui-Jun Li
It is vital to monitor the holistic toxicokinetics of toxic Chinese herbal medicines (CHMs) for safety. Although an integrated strategy based on the area under the curve (AUC) has been proposed to characterize the pharmacokinetic/toxicokinetic properties of CHMs, improvement is still needed. This study attempted to use 50% inhibitory concentration (IC50) as weighting coefficient to investigate holistic toxicokinetics of the major diosbulbins i.e. diosbulbin A (DA), diosbulbin B (DB), and diosbulbin C (DC) after oral administration of Dioscorea bulbifera rhizome (DBR) extract. Firstly, the cytotoxicities of the three diosbulbins on human hepatic L02 cells were evaluated and the IC50 values were calculated. Then, integrated toxicokinetics of multiple diosbulbins based on AUC and IC50 were determined. Finally, correlations between integrated plasma concentrations and hepatic injury biomarkers including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bile acid (TBA) were analyzed. As a result, integrated plasma concentrations were correlated well with TBA and the correlation between TBA and IC50-weighting integrated plasma concentrations was better than that of AUC-weighting integrated plasma concentrations. In conclusion, the newly developed IC50-weighting method is expected to generate more reasonable integrated toxicokinetic parameters, which will help to guide the safe usage of DBR in clinical settings.
High Sensitivity Method Validated to Quantify Estradiol in Human Plasma by LC–MS/MS J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-05 Mônica Siqueira Ferreira, André M.M. Arruda, Giovanni Tieghi Pepi, Aline Cristina Martho, Pâmela Maria Maximiano, Lina S.O.B.O. Ricci, Maria Francesca Riccio, Ana Cláudia Noboli, Pedro Serafim Júnior
17β-estradiol (E2) is an endogenous steroid in the human body. Its measurement is important for health and human biology understanding. However, E2 concentration in human plasma is in the range of pg/mL, which makes it difficult to detect. In this way, LC–MS/MS has been shown the most sensitive tool, although E2 is a weakly ionizable molecule. In this work, we validated a more sensitive and accurate method for E2 quantification in human plasma. Our extraction step ensured a cleaner chromatography, resulting in a precise measurement and highly reproducible method in the range of 2–150 pg/mL. Moreover, we proved a long stability for E2 in several conditions. All results indicate that our developed method is robust and sensitive enough to apply in bioequivalence studies for E2 measurement in human plasma, even at very low concentrations.
Purification, identification and molecular mechanism of two dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from Antarctic krill (Euphausia superba) protein hydrolysate J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-05 Wei Ji, Chaohua Zhang, Hongwu Ji
Dipeptidyl peptidase IV (DPP-IV) played an important role in blood glucose regulation. Inhibition of DPP-IV may improve glycemic control in diabetics by preventing the rapid breakdown of incretin hormones and prolonging their physiological action. In this study, Antarctic krill (Euphausia superba) protein was hydrolyzed using animal proteolytic enzymes. The hydrolysate was purified sequentially by ultrafiltration, gel filtration chromatography and reversed phase high-performance liquid chromatography (RP-HPLC). DPP-IV inhibitory activity of the fractions achieved from Antarctic krill protein was determined by DPP-IV screening reagent kit. Two purified peptides were identified by Xevo G2-XS QTof mass spectrometer (QTOF-MS). One peptide purified was Ala-Pro (AP) with IC50 values of 0.0530 mg/mL, the other Ile-Pro-Ala (IPA) with IC50 values of 0.0370 mg/mL. They both exhibited strong DPP-IV inhibitory activity. The molecular docking analysis revealed that DPP-IV inhibition by AP and IPA was mainly due to formation of a strong interaction surface force with the 91-96 and 101-105 amino acids of the DPP-IV. Our results suggested that the protein hydrolysate from Antarctic krill can be considered as a promising natural source of DPP-IV inhibitory peptides in the management of diabetes.
A cost-effective LC-MS/MS method for identification and quantification of α-amanitin in rat plasma: Application to toxicokinetic study J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-05 Chunlei Li, Fen Wei, Saqib Muhanmmad, Guangde Yang, Sicen Wang, Xinshe Liu
α-Amanitin is the main lethal component of amanita mushrooms, and data on its toxicokinetics are few. The aim of this study was to develop a sensitive and cost-effective method to identify α-amanitin and investigate its toxicokinetic parameters using liquid chromatography-triple quadrupole tandem mass spectrometry. The colchicine was used as the internal standard (IS). The compounds were extracted from plasma samples by protein precipitation with acetonitrile (containing 1% formic acid). The analysis was performed through multiple reactions monitoring. The molecular ions and fragment ions of α-amanitin could be used as characteristic ions to perform qualitative analysis of α-amanitin. The assay was successfully validated by selectivity, linearity, matrix effect, precision and accuracy, recovery and stability according to the U.S. Food and Drug Administration Guidance, and applied to study the toxicokinetic profile of α-amanitin in rats after a single intraperitoneal administration.
Determination of Doping Peptides via Solid-Phase Microelution and Accurate-Mass Quadrupole Time-of-Flight LC–MS J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-05 Darío Cuervo, Cynthia Loli, Gloria Muñoz, Daniel Carreras
A complete analytical protocol for the determination of 15 doping-related peptidic drugs and 2 metabolites in urine was developed by means of accurate-mass quadrupole time-of-flight (Q-TOF) LC-MS analysis following solid-phase extraction (SPE) on microplates and conventional SPE pre-treatment for initial testing and confirmation, respectively. These substances included growth hormone releasing factors (GHRP-1, -2, -4, -5, -6, alexamorelin, hexarelin, anamorelin, ipamorelin, GHRP-2 deamidated and GHRP-4 deamidated), gonadotropin releasing factors (LHRH, leuprolide, buserelin and triptorelin) and anti-diuretic hormones (desmopressin and lypressin), with molecular weights ranging from 540 to 1310 Daltons. Optimal experimental conditions were stablished after investigation of different parameters concerning sample preparation and instrumental analysis. Different SPE sorbents, washing/elution protocols, HPLC columns, chromatographic and spectrometric conditions were assessed during the method development; weak cation exchange SPE followed by C18 HPLC chromatography and accurate mass detection provided the required sensitivity and selectivity for all the target peptides under study. 2 mg SPE on 96-well microplates can be used in combination with full scan MS detection for the initial testing, thus providing a fast, cost-effective and high-throughput protocol for the processing of a large batch of samples simultaneously. On the other hand, extraction on 30 mg SPE cartridges and subsequent target MS/MS determination was the protocol of choice for confirmatory purposes. The validated procedures fulfilled the concentration levels established by the World Anti-Doping Agency (WADA), and can be easily transferred to other laboratories.
LC-MS/MS Quantification of 7α-hydroxy-4-cholesten-3-one (C4) in Rat and Monkey Plasma J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-05 Lijuan Kang, Thomas M. Connolly, Naidong Weng, Wenying Jian
7α-hydroxy-4-cholesten-3-one (C4) is an oxidative enzymatic product of cholesterol metabolism via cholesterol 7α-hydroxylase, an enzyme also known as cholesterol 7-alpha-monooxygenase or cytochrome P450 7A1 (CYP7A1). C4 is a stable intermediate in the rate limiting pathway of bile acid biosynthesis. Previous studies showed that plasma C4 levels correlated with CYP7A1 enzymatic activity and could serve as a biomarker for bile acid synthesis. Here we developed and qualified a simple and robust high-throughput method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to quantify C4 in rat and monkey plasma. As C4 being an endogenous compound, this method used calibration standards in 50/50: acetonitrile/water (v/v). In order to mimic the incurred samples, quality control samples were prepared in the authentic plasma. Stable isotope labeled C4 (C4-d7) was used as the internal standard. The sample volume for analysis was 20 μL and the sample preparation method was protein precipitation with acetonitrile. The average endogenous C4 concentrations, from 10 different lots of rat and monkey plasma, were 53.0 ± 16.5 ng/mL and 6.8 ± 5.6 ng/mL, respectively. Based on these observed endogenous C4 levels, the calibration curve ranges were established at 1–200 ng/mL and 0.5–100 ng/mL for rat assay and monkey assay, respectively. The method was qualified with acceptable accuracy, precision, linearity, and specificity. Matrix effect, recovery, and plasma stability of bench-top, freeze-thaw, and long-term frozen storage were also evaluated. The method has been successfully applied to pre-clinical studies.
Toxicity, Teratogenicity and Antibacterial Activity of Posterior Salivary Gland (PSG) Toxin from the cuttlefish Sepia pharaonis (Ehrenberg, 1831) J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-05 Ramachandran Karthik, Venkatesan Manigandan, Ramachandran Saravanan
Characterization of the changes in eicosanoid profiles of activated macrophages treated with 20(S)-ginsenoside-Rg3 J. Chromatogr. B (IF 2.603) Pub Date : 2017-09-05 Jae Won Lee, Yu Ri Choi, Hyuck Jun Mok, Hyun-A Seong, Dae Young Lee, Geum-Soog Kim, Ji Hye Yoon, Kwang Pyo Kim, Hyung Don Kim
In this study, we used ultra-performance liquid chromatography coupled with tandem mass spectrometry to assess the levels of eicosanoids from RAW264.7 macrophages treated with lipopolysaccharides (LPS) and 20(S)-ginsenoside-Rg3 (Rg3). The production of nitric oxide (NO) and the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were increased in inflammatory macrophages treated with LPS. Rg3 treatment, however, decreased the levels of NO, TNF-α, and IL-6 in activated macrophages. Eicosanoids, known as major metabolites correlated with inflammation, have pro- or anti-inflammatory activities. For a detailed characterization of the eicosanoids altered by treatment with LPS and Rg3, the eicosanoids were profiled by multiple reaction monitoring. A total of 69 macrophage eicosanoids were analyzed and the profiling dataset was statistically analyzed. Principal component and hierarchical cluster analyses differentiated control cells from cells treated with LPS, Rg3, or LPS + Rg3 for 12 or 24 h. Furthermore, 18 differentially regulated eicosanoids were found between macrophages treated with LPS for 24 h and those treated with LPS + Rg3 for 24 h (fold change > 2, p value < 0.05). These results indicate that Rg3 alters eicosanoid metabolism in activated macrophages treated with LPS. Furthermore, we also identified several eicosanoids correlated with the anti-inflammatory activity of Rg3.
Chaihu-Shu-Gan-San regulates phospholipids and bile acid metabolism against hepatic injury induced by chronic unpredictable stress in rat J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-31 Hong-mei Jia, Meng Yu, Li-Yan Ma, Hong-wu Zhang, Zhong-mei Zou
Chaihu-Shu-Gan-San (CSGS) is a famous classic traditional Chinese medicines (TCM) formula for treatment of liver stagnancy recorded in a famous book of traditional Chinese medicine, Jing Yue Quan Shu published in 1624. It has been extensively accepted as an antidepressant in China and its mechanism of action is still not clear. Previously we have found that hepatic injury happens in chronic unpredicted mild stress (CUMS). Thus, the protection of CSGS against hepatic injury induced by CUMS treatment was explored by metabonomics study and gene expression of the rat liver tissue. The results indicated that CSGS improved 8 of the 18 perturbed potential biomarkers in liver tissues of rats treated with CUMS, and involved in regulating phospholipids and bile acid metabolism against hepatic injury induced by CUMS in rat. The expressions of two apoptosis associated genes (Bcl-2 and Bax) and four genes (Pnpla6, Pla2g15, Baat and Gad1) related to the perturbed metabolic pathways were further investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Both metabonomics and studies of genetic influences on metabolites demonstrated that CSGS inhibited hepatocyte apoptosis, and regulated phospholipids and bile acid metabolism against hepatic injury induced by CUMS in rat. Exploring the protection of CSGS against hepatic injury related to depression further clarify the relationship between CUMS-induced depression and hepatic injury, and also provide a novel insight to understand the underlying antidepressive mechanism of CSGS.
Bithionol residue analysis in animal-derived food products by an effective and rugged extraction method coupled with liquid chromatography–tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-31 Weijia Zheng, Jin-A Park, A.M. Abd El-Aty, Seong-Kwan Kim, Sang-Hyun Cho, Jeong-Min Choi, Hee Yi, Soo-Min Cho, H.A. El-Banna, Jae-Han Shim, Byung-Joon Chang, Jing Wang, Jin-Suk Kim, Ho-Chul Shin
Herein, we developed a simple analytical procedure for the quantitation of bithionol residues in animal-derived food products such as porcine muscle, eggs, milk, eel, flatfish, and shrimp using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method coupled with liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS). Samples were extracted with 0.1% solution of formic acid in acetonitrile and the extract was purified using a C18 sorbent. Separation was performed on a Waters XBridge™ C18 reversed-phase analytical column using 0.1% solution of formic acid/acetonitrile as the mobile phase. Six-point matrix-matched calibration indicated good linearity, with the calculated coefficients of determination (R2) being ≥ 0.9813. Intra- and inter-day recoveries (determined at spiking levels equivalent to 1 × and 2 × the limit of quantitation (0.25 μg/kg)) ranged between 80.0 and 94.0%, with the corresponding relative standard deviations (RSDs) being ≤ 8.2%. The developed experimental protocol was applied to different samples purchased from local markets in Seoul, which were tested negative for bithionol residues. In conclusion, the proposed method proved to be versatile and precise, being ideally suited for the routine detection of bithionol residues in animal-derived food products with various protein and fat contents.
Determination of chlorogenic acids and caffeine in homemade brewed coffee prepared under various conditions J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-31 Jong-Sup Jeon, Han-Taek Kim, Il-Hyung Jeong, Se-Ra Hong, Moon-Seog Oh, Kwang-Hee Park, Jae-Han Shim, A.M. Abd El-Aty
HPLC-MS/MS Method for Bioavailability Study of Bruceines D & E in Rat Plasma J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-30 Farahdina Man, Chee-Yan Choo
Bruceines D and E are quassinoids from seeds of Brucea javanica (L.) Merr. exhibiting hypoglycemia effect. The crude drug is used as a traditional medicine by diabetes patients. The aim of this study is to understand the bioavailability and pharmacokinetics of both the bruceines D & E. A rapid and sensitive HPLC-MS/MS method was developed and validated for the quantification of both quassinoids, bruceines D & E in rat plasma. Both the bruceines D & E were separated with the Zorbax SBC-18 column with gradient elution and mobile phase system of acetonitrile and deionized water with 0.1% formic acid at a flow rate of 0.5 mL/min. Analytes were detected in multiple reaction monitoring (MRM) mode with electrospray positive ionization. The quassinoids, namely bruceines D & E were detected with transitions of m/z 411.2 → 393.2 and m/z 395.2 → 377.2, respectively. Another quassinoid, eurycomanone was used as the internal standard with transition of m/z 409.2 → 391.2. The method was validated and conformed to the regulatory requirements. The validated method was applied to pharmacokinetic and bioavailability studies in rats. The pharmacokinetic study indicated both bruceine D and E were rapidly absorbed into the circulation system and reached its peak concentration at 0.54 ± 0.34 h and 0.66 ± 0.30 h, respectively. Bruceine E was eliminated slower than Bruceine D with t1/2 value almost increased two-fold compared to Bruceine D. In conclusion, a rapid, selective and sensitive HPLC-MS/MS method was developed for the simultaneous determination of both the bruceines D and E in rat plasma. Both bruceines D and E displayed poor oral bioavailability.
Simultaneous quantitation of hydrazine and acetylhydrazine in human plasma by high performance liquid chromatography-tandem mass spectrometry after derivatization with p-tolualdehyde J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-30 Lu Song, Dan Gao, Shangfu Li, Yanwei Wang, Hongxia Liu, Yuyang Jiang
A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for simultaneous quantitative analysis of hydrazine and acetylhydrazine in human plasma based on the strategy of p-tolualdehyde derivatization. The derivatization reactions were easily realized by ultrasonic manipulation for 40 min. Good separation of the derivatization products was achieved using a C18 column by gradient elution. The optimized mass transition ion-pairs (m/z) monitored for the two hydrazine derivatives were m/z 237.1 > 119.9 and m/z 176.9 > 117.8, respectively. The limit of detection (LOD) and limit of quantification (LOQ) for hydrazine were 0.002 and 0.005 ng mL−1 separately. And they were 0.03 and 0.05 ng mL−1 for acetylhydrazine, respectively. The linear range was 0.005–50 ng mL−1 for hydrazine and 0.05–500 ng mL−1 for acetylhydrazine with R2 greater than 0.999. The recovery range was determined to be 95.38–108.12% with the relative standard deviation (RSD) in the range of 1.24–14.89%. The method was successfully applied to detect 30 clinical plasma samples of pulmonary tuberculosis patients treated with isoniazid. The concentrations were from 0.04–1.99 ng mL−1 for hydrazine and 0.06–142.43 ng mL−1 for acetylhydrazine. The results indicated that our developed method had the potential for the detection of hydrazine toxicology in complex biological samples. Furthermore, the method has an important significance to clinical treatment with drugs.
Preparative separation of the polar part from the rhizomes of Anemarrhena asphodeloides using a hydrophilic C18 stationary phase J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-30 Jianfeng Cai, Huaxia Xin, Lingping Cheng, YanHui Fu, Dasen Jiang, Jiatao Feng, Qing Fu, Yu Jin, Xinmiao Liang
Semi-preparative high-performance countercurrent chromatography method for the purification of chemically synthesized ATP analogue, ApppI J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-30 Elina Puljula, Petri A. Turhanen
Metabolomic study for monitoring of biomarkers in mouse plasma with asthma by gas chromatography-mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-30 Chan Seo, Yun-Ho Hwang, Hyeon-Seong Lee, Youngbae Kim, Tae Hwan Shin, Gwang Lee, Young-Jin Son, Hangun Kim, Sung-Tae Yee, Ae Kyung Park, Man-Jeong Paik
Asthma is a multifaceted chronic disease caused by an alteration of various genetic and environmental factors that is increasing in incidence worldwide. However, the biochemical mechanisms regarding asthma are not completely understood. Thus, we performed of metabolomic study for understanding of the biochemical events by monitoring of altered metabolism and biomarkers in asthma. In mice plasma, 27 amino acids(AAs), 24 fatty acids(FAs) and 17 organic acids(OAs) were determined by ethoxycarbonyl(EOC)/methoxime(MO)/tert-butyldimethylsilyl(TBDMS) derivatives with GC-MS. Their percentage composition normalized to the corresponding mean levels of control group. They then plotted as star symbol patterns for visual monitoring of altered metabolism, which were characteristic and readily distinguishable in control and asthma groups. The Mann-Whitney test revealed 25 metabolites, including eight AAs, nine FAs and eight OAs, which were significantly different (p < 0.05), and orthogonal partial least-squares-discriminant analysis revealed a clear separation of the two groups. In classification analysis, palmitic acid and methionine were the main metabolites for discrimination between asthma and the control followed by pipecolic, lactic, α-ketoglutaric, and linoleic acids for high classification accuracy as potential biomarkers. These explain the metabolic disturbance in asthma for AAs and FAs including intermediate OAs related to the energy metabolism in the TCA cycle.
High sensitivity HPLC method for determination of the allysine concentration in tissue by use of a naphthol derivative J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-26 Philip A. Waghorn, Bruno L. Oliveira, Chloe M. Jones, Andrew M. Tager, Peter Caravan
Common to all fibrotic and metastatic diseases is the uncontrollable remodeling of tissue that leads to the accumulation of fibrous connective tissue components such as collagen and elastin. Build-up of fibrous tissue occurs through the cross-linking of collagen or elastin monomers, which is initiated through the oxidation of lysine residues to form α-aminoadipic-δ-semialdehyde (allysine). To provide a measure of the extent of collagen oxidation in disease models of fibrosis or metastasis, a rapid, sensitive HPLC method was developed to quantify the amount of allysine present in tissue. Allysine was reacted with sodium 2-naphthol-7-sulfonate under conditions typically applied for acid hydrolysis of tissues (6 M HCl, 110 °C, 24 h) to prepare a fluorescent bis-naphthol derivative of allysine. High performance liquid chromatography was applied for analysis of allysine content. Under optimal reaction and detection conditions, successful separation of AL-NP was achieved with excellent analytical performance attained. Good linear relationship (R2 = 0.994) between peak area and concentration for AL-NP was attained for 0.35–175 pmol of analyte. A detection limit of 0.02 pmol in the standard sample with a 20 μL injection was achieved for AL-NP, with satisfactory recovery from 88–100% determined. The method was applied in the quantification of allysine in healthy and fibrotic mouse lung tissue, with the fibrotic tissue showing a 2.5 fold increase in the content of allysine.
Molecularly Imprinted Polymer-Sol-Gel Tablet toward Micro-Solid Phase Extraction: II. Determination of Amphetamine in Human Urine samples by Liquid Chromatography–Tandem Mass Spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-26 Aziza El-Beqqali, Lars I. Andersson, Amin Dadoun Jeppsson, Mohamed Abdel-Rehim
Amphetamine selective molecularly imprinted sol-gel polymer tablets, MIP-tablets, for solid-phase microextraction of biofluid samples were prepared. An acetonitrile solution of deuterated amphetamine template and silane precursor, 3-(propylmethacrylate) trimethoxysilane, was soaked into the pores of polyethylene tablet substrates and polymerized by an acid-catalysed sol-gel process. Application of the resultant MIP-tablets to extract amphetamine from human urine samples followed by LC-MS/MS analysis was investigated. The extraction protocol was optimised with respect to pH of sample, addition of sodium chloride, extraction time, desorption solvent and desorption time. The final analysis method determined amphetamine in human urine with a limit of detection (LOD) of 1.0 ng/mL and a lower limit of quantification (LLOQ) of 5 ng/mL. Validation demonstrated accuracy of the method was 91.0-104.0% and inter-assay precision was 4.8-8.5% (RSD). Extraction recovery was 80%. The MIP-tablets could be re-used and the same tablet could be employed for more than twenty extractions.
Detection of 8-Hydroxydeoxyguanosine (8-OHdG) as a Biomarker of Oxidative Damage in Peripheral Leukocyte DNA by UHPLC-MS/MS J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-26 Danni Wu, Baodong Liu, Junfa Yin, Tian Xu, Shuli Zhao, Qun Xu, Xi Chen, Hailin Wang
8-Hydroxydeoxyguanosine (8-OHdG) is a widely-used biomarker of oxidative DNA damages. 8-OHdG in peripheral blood leukocyte is associated with mutation and cancer risk. The level of 8-OHdG in peripheral blood leukocytes can indicate a long-term response to oxidative stress rather than that in urine. Accurate identification and quantification of leukocyte 8-OHdG are essential for understanding its mechanism of formation, repair, and biological consequences. In this study, a fast and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated to detect 8-OHdG in human peripheral leukocyte. DNA in blood samples were extracted and digested, then subjected onto UHPLC-MS/MS using an isocratic elution on a Zorbax Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm). Multiple reaction monitoring (MRM) mode was adopted by using [15N5]-8-OHdG as an internal standard. The assay was linear over the concentration range of 1.0–100 nM with R2 = 0.999. The accuracy for spiked samples was 90.9% − 94.8%, and the intra-day precision was within 3.7%. The limit of detection (LOD) is 0.30 nM and limit of quantification (LOQ) is 1.0 nM with 5 μl of sample injection. By the analysis of human leukocyte 8-OHdG (n = 121) using the developed UHPLC-MS/MS method, it demonstrates that the level of leukocyte 8-OHdG of the cancer patients group (n = 46) is significantly higher than that of the health control (n = 75).
Quantification of menadione from plasma and urine by a novel cysteamine-derivatization based UPLC-MS/MS method J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-24 Teng-Fei Yuan, Shao-Ting Wang, Yan Li
A plasma metabonomic analysis on potential biomarker in pyrexia induced by three methods using ultra high performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-24 Ting Liu, Songhe Li, Xiumin Tian, Zhaoqin Li, Yue Cui, Fei Han, Zhiguo Yu, Yunli Zhao
Pyrexia usually is a systemic pathological process that can lead to metabolic disorders. Metabonomics as a powerful tool not only can reveal the pathological mechanisms, but also can give insight into the progression of pyrexia from another angle. Thus, an ultra high performance liquid chromatography combined with Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) metabonomic approach was employed for the first time to investigate the plasma biochemical characteristics of pyrexia induced by three methods and to reveal subtle metabolic changes under the condition of pyrexia so as to explore its mechanism. The acquired metabolic data of the models were subjected to principal component analysis (PCA) for allowing the clear separation of the pyrexia rats from the control rats. Variable importance for project values (VIP) and Student’s t-test were used to screen the significant metabolic changes caused by pyrexia. 52 endogenous metabolites were identified and putatively identified as potential biomarkers primarily associated with phospholipid metabolism, sphingolipid metabolism, fatty acid oxidation metabolism, fatty acid amides metabolism and amino acid metabolism, and related to bile acid biosynthesis and glycerolipid catabolism. LysoPC (14:0), LysoPC (18:3), LysoPC (20:4), LysoPC (16:0), phytosphingosine, Cer (d18:0/12:0), N-[(4E,8E)-1,3-dihydroxyoctadeca-4,8-dien-2-yl]hexade-canamide, oleamide, fatty acid amide C22:1, tryptophan, acetylcarnitine, palmitoylcarnitine and stearoylcarnitine were considered as common potential biomarkers of pyrexia rats induced by three methods: Our results revealed that the UHPLC-FT-ICR-MS-based metabolomic method is helpful for finding new potential metabolic markers for pyrexia detection and offers a good perspective in pyrexia research.
LC-MS/MS based method development for the analysis of florfenicol and its application to estimate relative distribution in various tissues of broiler chicken J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-24 Muhammad Imran, Fazal-e-Habib, Abdul Tawab, Waqar Rauf, Moazur Rahman, Qaiser Mehmood Khan, Muhammad Rafique Asi, Mazhar Iqbal
Florfenicol, a broad spectrum bacteriostatic antibiotic belonging to amphenicol class, is widely used in poultry and livestock for the treatment of various infections. The major metabolite of florfenicol in different animal species is florfenicol amine which is exploited as the marker residue for the determination of florfenicol. Analysis of florfenicol merely by solvent extraction cannot determine the accurate amount of the drug present in incurred tissues (muscle, liver and kidney) of treated birds, as indicated by this study. Thus the methods solely based on solvent extraction may lead to false negative results. A reliable LC-MS/MS based confirmatory method for the analysis of florfenicol and its metabolites in chicken muscle was developed and validated according to the European Union Commission Decision 2002/657/EC. The method was based on acid hydrolysis to liberate non-extractable residues having presumably been covalently bound to tissues, and to convert all the florfenicol residues as well as its metabolites into florfenicol amine. The amine was subsequently recovered with ethyl acetate at pH 10.5, defatted and further cleaned up with dispersive solid phase extraction (dSPE). The LC separation was achieved on reverse phase C-18 column with isocratic elution using acetonitrile/water mobile phase and the analysis was performed on linear ion trap mass spectrometer. Calibration curve was obtained over a concentration range of 25–600 μg/kg for chicken muscles. The accuracy values ranged from 84-101.4% and the precision values for within day and between days ranged from 1.2-11.7%, respectively. Limit of detection (LOD), limit of quantification (LOD), CCα and CCβ values were 0.98, 3.2, 113 and 126 μg/kg, respectively. The developed method was highly robust and was further applied to estimate the relative distribution of solvent-extractable against solvent-non-extractable florfenicol drug residues in muscle, liver and kidney samples of broiler chicken after 5 days of oral dosing.
Bio-dispersive liquid liquid microextraction based on nano rhaminolipid aggregates combined with magnetic solid phase extraction using Fe3O4@PPy magnetic nanoparticles for the determination of methamphetamine in human urine J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-24 Seyed Ammar Haeri, Shahryar Abbasi, Sami Sajjadifar
In the present investigation, extraction and preconcentration of methamphetamine in human urine samples was carried out using a novel bio-dispersive liquid liquid microextraction (Bio-DLLME) technique coupled with magnetic solid phase extraction (MSPE). Bio-DLLME is a kind of microextraction technique based nano-materials which have potential capabilities in many application fields. Bio-DLLME is based on the use of a binary part system consisting of methanol and nano rhaminolipid biosurfactant. Use of this binary mixture is ecologically accepted due to their specificity, biocompatibility and biodegradable nature. The potential of nano rhaminolipid biosurfactant as a biological agent in the extraction of organic compounds has been investigated in recent years. They are able to partition at the oil/water interfaces and reduce the interfacial tension in order to increase solubility of hydrocarbons. The properties of the prepared Fe3O4@PPy magnetic nanoparticles were characterized using Fourier transform infrared spectroscopy and X-ray diffraction methods The influences of the experimental parameters on the quantitative recovery of analyte were investigated. Under optimized conditions, the enrichment factor was 310, the calibration graph was linear in the methamphetamine concentration range from 1 to 60 μg L−1, with a correlation coefficient of 0.9998. The relative standard deviations for six replicate measurements was 5.2%.
An Atmospheric Pressure Chemical Ionisation Liquid Chromatographic-Tandem Mass Spectrometry method for the analysis of Benzodiazepines in Urine J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-24 S. Dunlop, K. Hayes, P. Leavy, D. Cusack, R. Maguire
The objective of this work was to establish an analytical method for the analysis of 7 Benzodiazepines (diazepam, oxazepam, temazepam, nordiazepam, desalkylflurazepam, alprazolam and α-hydroxyalprazolam) in urine specimens taken from drivers suspected of driving under the influence of drugs. The specimen, calibrator and control preparation involved hydrolysis of conjugated benzodiazepines using β-glucuronidase in sodium acetate buffer, with incubation at 60 °C for 2 hours. Specimens were then centrifuged, before being diluted 1 in 5 (total dilution 1 in 10), with 10% acetonitrile in water. Specimens were analysed using a Shimadzu Prominence UPLC coupled to an AB Sciex 4000 QTrap LC-MS-MS. The chromatographic column was a Shim-pack XR ODS 2.2 μm. 3.0 × 50 mm column and the mobile phase was a binary gradient system comprising of mobile phase A which was an ammonium formate/formic acid buffer dissolved in water and mobile phase B which was a ammonium formate/formic acid buffer dissolved in Acetonitrile. APCI was selected as the ionisation technique and the MS was operated in MRM mode, monitoring 2 transitions per analyte. The validation of the method is described. The method was found to be linear, accurate and precise (within day and between day) for diazepam, oxazepam, temazepam, nordiazepam, desalkylflurazepam, alprazolam and α-hydroxyalprazolam. The results of 480 cases are reviewed and show that alprazolam use was found in 35% of cases. Use of benzodiazepines resulting in oxazepam, nordiazepam or temazepam were found ca. 70% of cases analysed.
Micro-matrix solid-phase dispersion coupled with MEEKC for quantitative analysis of lignans in Schisandrae Chinensis Fructus using molecular sieve TS-1 as a sorbent J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-24 Chu Chu, Mengmeng Wei, Shan Wang, Liqiong Zheng, Zheng He, Jun Cao, Jizhong Yan
A simple and effective method was developed for determining lignans in Schisandrae Chinensis Fructus by using a micro-matrix solid phase dispersion (MSPD) technique coupled with microemulsion electrokinetic chromatography (MEEKC). Molecular sieve, TS-1, was applied as a solid supporting material in micro MSPD extraction for the first time. Parameters that affect extraction efficiency, such as type of dispersant, mass ratio of the sample to the dispersant, grinding time, elution solvent and volume were optimized. The optimal extraction conditions involve dispersing 25 mg of powdered Schisandrae samples with 50 mg of TS-1 by a mortar and pestle. A grinding time of 150 s was adopted. The blend was then transferred to a solid-phase extraction cartridge and the target analytes were eluted with 500 μL of methanol. Moreover, several parameters affecting MEEKC separation were studied, including the type of oil, SDS concentration, type and concentration of cosurfactant, and concentration of organic modifier. A satisfactory linearity (R2> 0.9995) was obtained, and the calculated limits of quantitation were less than 2.77 μg/mL. Finally, the micro MSPD-MEEKC method was successfully applied to the analysis of lignans in complex Schisandrae fructus samples.
Development, validation, and clinical pharmacokinetic application of ultra-performance liquid chromatography/tandem mass spectrometry method for simultaneously determining a novel recombinant hirudin derivative (Neorudin) and its active metabolite in human serum J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-24 Xiaona Dong, Zhiyun Meng, Jide Jin, Ruolan Gu, Guifang Dou
Recombinant Neorudin (EPR-hirudin, EH), a novel, low-bleeding anticoagulant fusion protein, has been developed as an inactive prodrug that is converted to an active metabolite, hirudin variant 2-Lys47 (HV2), at the thrombus site and is undergoing Phase I clinical trials in China. The goal of our present research was to establish a novel ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method for simultaneously quantifying EH and HV2 in human serum. Furthermore, the method was used in clinical pharmacokinetic study after validation. The stock and dilute working solutions were dissolved in methanol/water (1/1, v/v) to avoid their adsorption. The internal standard (IS) used, had a similar structure to that of EH. The serum sample pretreatment involved protein precipitation with methanol. The volume ratio of the precipitating solvent to the serum sample was 3:1 (300 μL methanol: 100 μL serum sample). The chromatographic separation was performed using a 300 Å C18 column using a multi-step gradient with a mobile phase consisting of acetonitrile:water containing 0.1% formic acid. The detection was carried out using an ESI source in the positive multiple reaction monitoring (MRM) mode. The within and between run precision were in the range of 3.5%–10.3% for EH and 3.3%–8.8% for HV2, and the accuracy of both EH and HV2 was between −4.6% and 2.1%. The extraction recoveries and matrix effect at three quality control (QC) levels for EH and HV2 were satisfactory. The stabilities of EH and HV2 during the storage, preparation, and analysis were confirmed, and the carryover also proved to be acceptable. This technique was efficiently used in Phase I clinical pharmacokinetic trials of EH following intravenous administration of 0.2 mg/kg to healthy volunteers.
Reverse phase HPLC method for detection and quantification of lupin seed γ-conglutin J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-23 Sharmilee Mane, Scott Bringans, Stuart Johnson, Vishnu Pareek, Ranjeet Utikar
A simple, selective and accurate reverse phase HPLC method was developed for detection and quantitation of γ-conglutin from lupin seed extract. A linear gradient of water and acetonitrile containing trifluoroacetic acid (TFA) on a reverse phase column (Agilent Zorbax 300SB C-18), with a flow rate of 0.8 ml/min was able to produce a sharp and symmetric peak of γ-conglutin with a retention time at 29.16 min. The identity of γ-conglutin in the peak was confirmed by mass spectrometry (MS/MS identification) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The data obtained from MS/MS analysis was matched against the specified database to obtain the exact match for the protein of interest. The proposed method was validated in terms of specificity, linearity, sensitivity, precision, recovery and accuracy. The analytical parameters revealed that the validated method was capable of selectively performing a good chromatographic separation of γ-conglutin from the lupin seed extract with no interference of the matrix. The detection and quantitation limit of γ-conglutin were found to be 2.68 μg/ml and 8.12 μg/ml respectively. The accuracy (precision and recovery) analysis of the method was conducted under repeatable conditions on different days. Intra-day and inter-day precision values less than 0.5% and recovery greater than 97% indicated high precision and accuracy of the method for analysis of γ-conglutin. The method validation findings were reproducible and can be successfully applied for routine analysis of γ-conglutin from lupin seed extract.
Selective solid-phase extraction based on molecularly imprinted technology and simultaneous determination of 20 triazole pesticides in cucumber samples by high-performance liquid chromatography-tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-22 Fengnian Zhao, Yongxin She, Chao Zhang, Xiaolin Cao, Fen Jin, Maojun Jin, Hua Shao, Shanshan Wang, Lufei Zheng, Jing Wang
A selective analytical method for the simultaneous determination of 20 triazole fungicides and plant growth regulators in cucumber samples was developed using solid-phase extraction with specific molecularly imprinted polymers (MIPs) as adsorbents. The MIPs were successfully prepared by precipitation polymerization using triadimefon as the template molecule, methacrylic acid as the functional monomer, trimethylolpropanetrimethacrylate as the crosslinker, and acetonitrile as the porogen. The performance and recognition mechanism for both the MIPs and non-molecularly imprinted polymers were evaluated using adsorption isotherms and adsorption kinetics. Liquid chromatography-tandem quadrupole mass spectrometry was used to identify and quantify the target analytes. The solid-phase extraction using the MIPs was rapid, convenient, and efficient for extraction and enrichment of the 20 triazole pesticides from cucumber samples. The recoveries obtained at three concentration levels (1, 2, and 10 μg L−1) ranged from 82.3% to 117.6% with relative standard deviations of less than 11.8% (n = 5) for all analytes. The limits of quantification for the 20 triazole pesticides were all below 0.5 μg L−1, andwere sufficient to meet international standards.
Online monitoring of astragaloside II metabolism using a homemade cultural device coupled with microdialysis and ultra-performance liquid chromatography-mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-22 Xue Li, Zifeng Pi, Shu Liu, Wei Wang, Zhiqiang Liu, Fengrui Song
A new system was described for the online monitoring of astragaloside II (AII) metabolism in intestinal microbial community. The system was based on a homemade cultural device coupled with microdialysis (MD) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Main improvements include a simplified anaerobic incubator enabling the experiment to be conducted in ambient atmosphere, continuous sampling, and decreased matrix effect. Importantly, our method distinctly decreases the interference of small molecules by adding 20 mg ml−1 of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) to the perfusion fluid. Using the developed method, the metabolism of AII in intestinal bacteria was successfully investigated. Results were then compared with those obtained by conventional incubation and sampling method. We found that the integrated experimental system maintained the proper fermentation environment for bacteria and enabled high chromatography performance. With the advantages of auto-sampling, online detection, non-requirement of expensive fermenting equipment, and negligible matrix interference, the method can greatly contribute to the investigation of the dynamic biotransformation of astragalosides in complicated matrix-based biological samples.
Insulin Glargine and its Two Active Metabolites: A Sensitive (16 pM) and Robust Simultaneous Hybrid Assay Coupling Immunoaffinity Purification with LC–MS/MS to Support Biosimilar Clinical Studies J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-19 Yang Xu, Li Sun, Melanie Anderson, Philippe Bélanger, Vincent Trinh, Patricia Lavallée, Bhavna Kantesaria, Marie-Josée Marcoux, Sheila Breidinger, Kevin P. Bateman, Dina Goykhman, Eric J. Woolf
MK-1293 is a newly approved follow-on/biosimilar insulin glargine for the treatment of Type 1 and Type 2 diabetics. To support pivotal clinical studies during biosimilar evaluation, a sensitive, specific and robust LC–MS/MS assay for the simultaneous quantification of glargine and its two active metabolites, M1 and M2 were developed. Strategies to overcome analytical challenges, so as to optimize assay sensitivity and improve ruggedness, were evolved, resulting in a fully validated LC–MS/MS method with a lower limit of quantification (LLOQ) at 0.1 ng/mL (∼16 pM, equivalent to ∼2.8 μU/mL) for glargine, M1 and M2, respectively, using 0.5 mL of human plasma. The assay employed hybrid methodology that combined immunoaffinity purification and reversed-phase chromatography followed by electrospray-MS/MS detection operated under positive ionization mode. Stable-isotope labeled 6[D10]Leu-glargine and 4[D10]Leu-M1 were used as internal standards. With a calibration range from 0.1 to 10 ng/mL, the intra-run precision (n = 5) and accuracy were <6.21%, and 96.9–102.1%, while the inter-run (n = 5/run for 7 days) precision and accuracy were <9.55% and 96.5–105.1%, respectively, for all 3 analytes. Matrix effect, recovery, analyte stability, and interferences from control matrix, potential concomitant medications and anti-drug antibody were assessed. The assay was fully automated and has been successfully used in support of biosimilar clinical studies. Greater than 94.3% of incurred sample reanalysis (ISR) results met acceptance criteria, demonstrating the robustness of the assay. The strategic considerations during method development and validation are discussed, and can be applied to quantification of other peptides, especially insulin analogs, in the future.
Gas Chromatographic Quadrupole Time-of-Flight Full Scan High Resolution Mass Spectrometric Screening of Human Urine in Antidoping Analysis J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-19 Wadha Abushareeda, Emmanouil Lyris, Suhail Kraiem, Aisha Al Wahaibi, Sameera Alyazidi, Najib Dbes, Arjen Lommen, Michel Nielen, Peter L. Horvatovich, Mohammed Alsayrafi, Costas Georgakopoulos
This paper presents the development and validation of a high-resolution full scan (FS) electron impact ionization (EI) gas chromatography coupled to quadrupole Time-of-Flight mass spectrometry (GC/QTOF) platform for screening anabolic androgenic steroids (AAS) in human urine samples. The World Antidoping Agency (WADA) enlists AAS as prohibited doping agents in sports, and our method has been developed to comply with the qualitative specifications of WADA to be applied for the detection of sports antidoping prohibited substances, mainly for AAS. The method also comprises of the quantitative analysis of the WADA’s Athlete Biological Passport (ABP) endogenous steroidal parameters. The applied preparation of urine samples includes enzymatic hydrolysis for the cleavage of the Phase II glucuronide conjugates, generic liquid-liquid extraction and trimethylsilyl (TMS) derivatization steps. Tandem mass spectrometry (MS/MS) acquisition was applied on few selected ions to enhance the specificity and sensitivity of GC/TOF signal of few compounds. The full scan high resolution acquisition of analytical signal, for known and unknown TMS derivatives of AAS provides the antidoping system with a new analytical tool for the detection designer drugs and novel metabolites, which prolongs the AAS detection, after electronic data files’ reprocessing. The current method is complementary to the respective liquid chromatography coupled to mass spectrometry (LC/MS) methodology widely used to detect prohibited molecules in sport, which cannot be efficiently ionized with atmospheric pressure ionization interface.
The effect of deep eutectic solvent on the pharmacokinetics of salvianolic acid B in rats and its acute toxicity test J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-19 Jue Chen, Qi Wang, Mengjun Liu, Liwei Zhang
Deep eutectic solvent (DES), the benign green solvent with uniquely physical properties, has been widely applied in various fields. Our previous study indicated that DES could improve the stability and extraction efficiency of salvianolic acid B (SAB). In this work, with SAB as a model drug, the feasibility of DES as a drug carrier for oral preparation was investigated by evaluating the influence of DES on the pharmacokinetics of SAB and the toxicity of DES. Acute oral toxicity test illustrated that choline chloride-glycerol (ChCl-GL, molar ratio 1:2) was non-toxic with the median lethal dose of 7733 mg/kg. To comparison the difference of pharmacokinetics between SAB dissolved in ChCl-GL (1:2) and in water, a rapid and sensitive ultra-performance liquid chromatography coupled with mass spectrum was established to determine SAB and its metabolites in rat plasma. The method validation was also tested for the specificity, linearity (r2 > 0.9980 over two orders of magnitude), precision (intra-day relative standard deviation (RSD) < 2.73% and inter-day RSD < 7.72%), extraction recovery (70.96-80.78%) and stability under three different situations. Compared to water, the pharmacokinetic parameters clarified that ChCl-GL (1:2) could promote the absorption of SAB, the peak concentration (Cmax) of 0.308 ± 0.020 mg/L was slightly higher than 0.277 ± 0.024 mg/L (SAB dissolved in water), and the peak time (Tmax) was significantly decreased from 30 min (SAB dissolved in water) to 20 min. There was no significant difference on the metabolites between SAB dissolved in ChCl-GL (1:2) and in water. This is the first report on the pharmacokinetic study of DES as a candidate of drug carrier, and the results provide a meaningful basis for the application of DES in pharmaceutical preparation.
One-step lipid extraction for plasma lipidomics analysis by liquid chromatography mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-19 Yoshinori Satomi, Megumi Hirayama, Hiroyuki Kobayashi
In the past decade, various lipidomics methodologies have been developed using mass spectrometry based analytical technologies, enabling wide coverage lipid detection in a quantitative manner. Hence, lipidomics has become a widely-accepted approach for biomarker discovery and mechanism elucidation in both medical and biology research fields; however, there are still technical challenges. In this study, focusing on the sample preparation procedure, a single step deproteinization by a water-soluble organic solvent, such as methanol (MeOH), ethanol (EtOH), isopropanol (IPA) or acetonitrile (ACN), was evaluated and proved to be satisfactory for lipidomics analysis. Moreover, during this investigation ACN deproteinization was revealed to not be an effective method for lipid extraction because lipid decomposition was observed during the protein precipitation process through lipase activation, potentially due to the insufficient protein denaturation. Therefore, excluding ACN, protein precipitation by alcohol was evaluated as the lipid extraction reagent. Moreover, adding the MTBE-MeOH (mMM) method, one of the major liquid-liquid extraction methods for shotgun lipidomics, these four approaches were compared.Lipids were extracted from mouse plasma by these four methods and used for exhaustive lipid profiling by liquid chromatography mass spectrometry (LC/MS) analysis. Comparison of these four methods revealed that alcohol based protein precipitation was a useful sample preparation procedure for LC/MS based lipidomics analysis. Whereas MeOH extraction was appropriate for hydrophilic lipid species, IPA was effective for hydrophobic lipids such as triacylglycerols (TG). In practice, EtOH extraction is thought to be the best approach to cover wide range of lipid species using a simple preparation procedure.
Comprehensive characterization and identification of antioxidants in Folium Artemisiae Argyi using high-resolution tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-19 Binsong Han, Zhongquan Xin, Shasha Ma, Wenbin Liu, Bingyang Zhang, Lu Ran, Lunzhao Yi, Dabing Ren
Antioxidants from natural sources, such as vegetables and fruits, are attracting more and more interest. In this work, we evaluated the antioxidant potential of Folium Artemisia Argyi, a traditional Chinese herb medicine and food supplement. The total phenolic content, total flavonoid content, and antioxidant ability of the crude extracts and fractions obtained from consecutively partition of n-hexane, ethyl acetate, and n-butanol were measured and compared. Ethyl acetate fraction shows the highest total phenolic and flavonoid contents and highest antioxidant capability with regard to DPPH, ABTS, superoxide anion free radical scavenging ability, and ferric-reducing antioxidant power. In addition, the potential antioxidant components were screened by DPPH –UHPLC–MS experiments and subsequently characterized by using high-resolution tandem mass spectrometry. This work finally identified 45 antioxidants, including organic acids, phenolic compounds, flavonoids, and methoxylated flavonoids. The results suggested that Folium Artemisiae Argyi is a potential inexpensive resource of natural antioxidants.
An efficient analytical method for determination of S-phenylmercapturic acid in urine by HPLC fluorimetric detector to assessing benzene exposure J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-18 Michele P. Rocha Mendes, Josianne Nicácio Silveira, Leiliane Coelho Andre
Benzene is an important occupational and environmental contaminant, naturally present in petroleum and as by-product in the steel industry. Toxicological studies showed pronounced myelotoxic action, causing leukemic and others blood cells disorders. Assessing of benzene exposure is performed by biomarkers as trans, trans-muconic acid (AttM) and S-phenylmercapturic acid (S-PMA) in urine. Due to specificity of S-PMA, this biomarker has been proposed to asses lower levels of benzene in air. The aim of this study was to validate an analytical method for the quantification of S-PMA by High-Performance Liquid Chromatography with fluorometric detector. The development of an analytical method of S-PMA in urine was carried out by solid phase extraction (SPE) using C-18 phase. The eluated were submitted to water bath at 75 °C and nitrogen to analyte concentration, followed by alkaline hydrolysis and derivatization with monobromobimane. The chromatography conditions were reverse phase C-18 column (240 mm, 4 mm and 5 μm) at 35 °C; acetonitrile and 0.5% acetic acid (50:50) as mobile phase with a flow of 0.8 mL/min. The limits of detection and quantification were 0.22 μg/L and 0.68 μg/L, respectively. The linearity was verified by simple linear regression, and the method exhibited good linearity in the range of 10 to 100 μg/L. There was no matrix effect for S-PMA using concentrations of 40, 60, 80 and 100 μg/L. The intra- and interassay precision showed coefficient of variation of less than 10% and the recovery ranged from 83.4 to 102.8% with an average of 94.4%. The stability of S-PMA in urine stored at −20 °C was of seven weeks. The conclusion is that this method presents satisfactory results per their figures of merit. This proposed method for determining urinary S-PMA showed adequate sensitivity for assessment of occupational and environmental exposure to benzene using S-PMA as biomarker of exposure.
Color and alcohol removal for the simultaneous detection of amino acids and sugars in wine by two-dimensional ion chromatography J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-18 Yun Fa, Yinghui Liu, Aihua Xu, Yuexue Yu, Fangfang Li, Huizhou Liu
An effective pretreatment method for wine color removal by a PS-DVB SPE cartridge and online alcohol elimination by valve switching was presented. The optimum parameters for color removal were investigated: 40-μm and 100 Å poly (styrene)-divinylbenzene (PS-DVB) (0.4 g) was selected as the color removal material and 5 mL of ethanol (10%) as the elution solvent for sample pretreatment under given condition. Moreover, an accurate and automated two-dimensional ion chromatography method for the simultaneous detection of amino acids and sugars was achieved with two valves after injection without alcohol interference. The method had a mean correlation coefficient of >0.99 and a repeatability of 0.92%–4.30% for eight replicates. The mean recovery of six red wine samples were 97.6%, 96.6%, 96.1%, 95.9%, 97.3% and 96.4% respectively. And this method successfully analyzed the amino acid and sugar contents of six wine samples of different origins.
Determination of chlorpyrifos and its metabolites in cells and culture media by liquid chromatography-electrospray ionization tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-18 Xiangkun Yang, Xian Wu, Kyle A. Brown, Thao Le, Steven L. Stice, Michael G. Bartlett
A sensitive method to simultaneously quantitate chlorpyrifos, chlorpyrifos oxon and the detoxified product 3,5,6-trichloro-2-pyridinol (TCP) was developed using either liquid-liquid extraction for culture media samples, or protein precipitation for cell samples. Multiple reaction monitoring in positive ion mode was applied for the detection of chlorpyrifos and chlorpyrifos oxon, and selected ion recording in negative mode was applied to detect TCP. The method provided linear ranges from 5–500, 0.2–20 and 20–2000 ng/mL for media samples and from 0.5–50, 0.02–2 and 2–200 ng/million cells for CPF, CPO and TCP, respectively. The method was validated using selectivity, linearity, precision, accuracy, recovery, stability and dilution tests. All relative standard deviations (RSDs) and relative errors (REs) for QC samples were within 15% (except for LLOQ, within 20%). This method has been successfully applied to study the neurotoxicity and metabolism of chlorpyrifos in a human neuronal model.
Enantiomeric separation of five acidic drugs via capillary electrophoresis using streptomycin as chiral selector J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-16 Xuejiao Zhang, Shuhua Qi, Chunye Liu, Xiaoli Zhao
A simple capillary zone electrophoresis (CZE) method was developed to achieve the rapid enantiomeric separation of a set of acidic drugs using streptomycin as chiral selector. The enantiomers of 5 chiral phenyl-containing acidic pharmaceutical compounds were separated excellently by CE using an uncoated silica capillary. Several experimental parameters such as the concentration of streptomycin, buffer concentration and pH, running voltage, and capillary temperature were all investigated systematically in order to optimize the chiral separation. All analytes were got baseline separation within 10 min. And the results showed that streptomycin can be used as a chiral selector to the enantioseparation of five acidic drugs by CZE method.
Silver perchlorate in the mobile phase for rapid separation and determination of a pair of positional isomers in Inula racemosa Hook.f. with RP-HPLC J. Chromatogr. B (IF 2.603) Pub Date : 2017-08-14 Yang Yang, Rui Tu, Wenji Sun, Zhongliang Zhu, Yongmin Zhang
Alantolactone and isoalantolactone isolated from many species of plants are a pair of positional isomers of C C bond. Previously, alantolactone and isoalantolactone have been proved to be good lead compounds for future anticancer agent development. Similarity of their molecular structures increases the separation difficulty for these two isomers on a conventional C18 column. Silver perchlorate (AgClO4) as mobile phase additives with RP-HPLC for improving the separation was developed for rapid determination of the positional isomers in Inula racemosa Hook.f. The effects of the concentration of silver perchlorate on the separation of the analytes were investigated. The composition of acetonitrile and water containing 5.0% silver perchlorate in a 65:35 (v/v) ratio was used as mobile phase, in which they were well separated within a short period of time on the C18 column. The method was successfully applied to determine them in an extract of Inula racemosa Hook.f. root. Silver perchlorate in mobile phase can efficiently improve the separation of the positional isomers and could be applied to rapidly determinate their content in this plant.
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