Ultrafast quantification of β-lactam antibiotics in human plasma using UPLC–MS/MS

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Mieke Carlier , Veronique Stove , Jan J. De Waele , Alain G. Verstraete
There is an increasing interest in monitoring plasma concentrations of β-lactam antibiotics. The objective of this work was to develop and validate a fast ultra-performance liquid chromatographic method with tandem mass spectrometric detection (UPLC–MS/MS) for simultaneous quantification of amoxicillin, cefuroxime, ceftazidime, meropenem and piperacillin with minimal turn around time. Sample clean-up included protein precipitation with acetonitrile containing 5 deuterated internal standards, and subsequent dilution of the supernatant with water after centrifugation. Runtime was only 2.5min. Chromatographic separation was performed on a Waters Acquity UPLC system using a BEH C18 column (1.7μm, 100mm×2.1mm) applying a binary gradient elution of water and methanol both containing 0.1% formic acid and 2mmol/L ammonium acetate on a Water TQD instrument in MRM mode. All compounds were detected in electrospray positive ion mode and could be quantified between 1 and 100mg/L for amoxicillin and cefuroxime, between 0.5 and 80mg/L for meropenem and ceftazidime, and between 1 and 150mg/L for piperacillin. The method was validated in terms of precision, accuracy, linearity, matrix effect and recovery and has been compared to a previously published UPLC–MS/MS method.



Ultra-high performance liquid chromatography–tandem mass spectrometry for the quantification of icaritin in mouse bone

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Shuang-Qing Zhang
Icaritin (ICT), a bioactive metabolite of prenylflavonoids from genus Epimedium, has displayed potential benefits for the treatment of osteoporosis, prostate cancer, liver cancer, renal cancer and breast cancer. To investigate the quantity of ICT in bones in vivo, a simple and sensitive ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed. After a rapid one-step liquid–liquid extraction using ethyl acetate with recovery more than 87.2% at four levels (0.1, 0.2, 8 and 15ng/mL), ICT and internal standard coumestrol were analyzed on a C18 column using a gradient elution of acetonitrile and water containing ammonium formate and formic acid at a flow rate of 0.3mL/min. Quantification was performed using selected reaction monitoring mode to monitor precursor-product ion transitions of m/z 367.1→297.1 for ICT and of 267.0→211.1 for coumestrol in the negative ionization mode. A calibration curve with good linearity (r>0.99) within the concentration range of 0.1–20ng/mL for ICT was obtained with the lower limit of quantification of 0.1ng/mL. Matrix effect did not interfere with ICT analysis and ICT was stable under three freeze–thaw cycles, short-term temperature, post-preparative and long-term temperature conditions. The method was successfully applied to a dynamic distribution of ICT in mouse bone after a single intraperitoneal administration to ICT to mice.



Multi-residues determination of antimicrobials in fish tissues by HPLC–ESI-MS/MS method

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Mamdouh R. Rezk , Safa’a M. Riad , Fatma I. Khattab , Hoda M. Marzouk
A rapid, simple, sensitive and specific LC–MS/MS method was developed and validated for the simultaneous quantification of four antimicrobials commonly used in aquaculture, namely ciprofloxacin (CPX), trimethoprim (TMP), sulphadimethoxine (SDM) and florphenicol (FLOR) in fish tissues. The LC–MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Sample preparation involves simple liquid extraction step followed by post-extraction clean-up step with n-hexane. The purified extracts were chromatographed on Agilent Poroshell 120 EC, C18 (50mm×3mm, 2.7μm) column by pumping an isocratic mobile phase consisting of 0.1% formic acid in water:0.1% formic acid in methanol (20:80, by volume) at a flow rate of 0.4mL/min. A detailed validation of the method was performed as per FDA guidelines and the standard curves were found to be linear in the range of 1–100ng/g for both CPX and TMP, 0.5–100ng/g for SDM and 1–50ng/g for FLOR. The intra-day and inter-day precision and accuracy of the results were within the acceptable limits. A run time of 1.5min for each sample made it possible to analyze multiple fish tissue samples per day. The developed assay method was successfully applied for the detection of antimicrobials in real fish tissue samples obtained from different fish farms.



Comprehensive polar lipid identification and quantification in milk by liquid chromatography–mass spectrometry

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Zhiqian Liu , Peter Moate , Ben Cocks , Simone Rochfort
Polar lipids (PLs) are a significant functional component of milk that are difficult to quantitate. A simple method for comprehensive identification and quantitative analysis of all essential PL species using bovine milk is described. The lipid fraction was extracted by a mix of chloroform and methanol and the extract was directly used for PL identification and quantification. PLs were separated by hydrophilic interaction liquid chromatography (HILIC) and detected by an Orbitrap mass analyser in positive mode. The structure of PLs was established or confirmed by tandem MS in both positive and negative modes. The method is sensitive (with a LOD for all PL classes ≤0.1ng) and reproducible, enabling simultaneous quantification of 70 PL species within a run of 45min. Application of this method to the quantification of PLs in 32 bovine milk samples revealed the relative abundance of different PL classes, significant variation of PL content between individual samples and the correlation between the major PL classes. The method provides a tool for investigating the variation and metabolism of important PL components in bovine and human milk and in diverse mammalian species.



LC–MS analysis of the plasma metabolome—A novel sample preparation strategy

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Kasper Skov , Niels Hadrup , Jørn Smedsgaard , Henrik Frandsen
Blood plasma is a well-known body fluid often analyzed in studies on the effects of toxic compounds as physiological or chemical induced changes in the mammalian body are reflected in the plasma metabolome. Sample preparation prior to LC–MS based analysis of the plasma metabolome is a challenge as plasma contains compounds with very different properties. Besides, proteins, which usually are precipitated with organic solvent, phospholipids, are known to cause ion suppression in electrospray mass spectrometry.We have compared two different sample preparation techniques prior to LC-qTOF analysis of plasma samples: the first is protein precipitation; the second is protein precipitation followed by solid phase extraction with sub-fractionation into three sub-samples: a phospholipid, a lipid and a polar sub-fraction. Molecular feature extraction of the data files from LC-qTOF analysis of the samples revealed 1792 molecular features from the protein precipitation procedure. The protein precipitation followed by solid phase extraction procedure with three sub-samples gave a total of 4234 molecular features. This suggests that sub-sampling into polar, lipid and phospholipid fractions enables extraction of more metabolomic information as compared to protein precipitation alone. Chromatography showed good separation of the metabolites with little retention time drift (<1s) and a mass accuracy below 3ppm was observed. The performance of the method was investigated using plasma samples from rats administered the environmental pollutant perfluorononanoic acid.



Rapid determination of fenoldopam in human plasma by UPLC–MS/MS for pharmacokinetic analysis in patients

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Xipei Wang , Zhijie Zheng , Guodong He , Liping Mai , Zhiling Zhou , Shilong Zhong , Qiuxiong Lin , Zhixin Shan , Chunyu Deng , Min Yang , Xiyong Yu
We developed and validated a rapid, selective, and sensitive ultra-performance liquid-chromatography mass-spectrometry (UPLC–MS/MS) method for quantifying fenoldopam in human plasma for pharmacokinetic studies. Fenoldopam and the internal-standard (IS), oxazepam, were isolated from human plasma by liquid–liquid extraction using ethyl acetate after alkalization, and were separated on a 2.1×100mm Acquity UPLC HSS T3 C18 column (inside diameter, 1.8μm) using a mobile phase of water (0.05% formic acid) and acetonitrile gradient elution. The fenoldopam and IS were eluted at 1.07 and 2.32min, respectively. Quantification was performed using positive-ion electrospray-ionization (ESI), and the fenoldopam and IS responses were optimized at the m/z 306.16→107.10 and m/z 287.1→241.01 transitions, respectively. The assay was validated over the linear range of 0.1–40ng/mL fenoldopam with intra- and interassay precision <13.21%. The matrix effect of normal and hemolyzed plasma was 94.9–101.6%. Fenoldopam was stable for ≥34 days at −70°C in normal and hemolyzed plasma containing ascorbic acid as a stabilizer. This method can be successfully applied in pharmacokinetic studies of fenoldopam in hypertensive patients.



A validated method for quantifying hypoglycin A in whole blood by UHPLC–HRMS/MS

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Jérémy Carlier , Jérôme Guitton , Cécile Moreau , Baptiste Boyer , Fabien Bévalot , Laurent Fanton , Jean Habyarimana , Gilbert Gault , Yvan Gaillard
Hypoglycin A (HGA) is the toxic principle in ackee (Blighia sapida Koenig), a nutritious and readily available fruit which is a staple of the Jamaican working-class and rural population. The aril of the unripe fruit has high concentrations of HGA, the cause of Jamaican vomiting sickness, which is very often fatal. HGA is also present in the samara of several species of maple (Acer spp.) which are suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy in Europe, often fatal for horses. The aim of this study was to develop a method for quantifying HGA in blood that would be sensitive enough to provide toxicological evidence of ackee or maple poisoning. Analysis was carried out using solid-phase extraction (HILIC cartridges), dansyl derivatization and UHPLC–HRMS/MS detection. The method was validated in whole blood with a detection limit of 0.35μg/L (range: 0.8–500μg/L). This is the first method applicable in forensic toxicology for quantifying HGA in whole blood. HGA was quantified in two serum samples from horses suffering from atypical myopathy. The concentrations were 446.9 and 87.8μg/L. HGA was also quantified in dried arils of unripe ackee fruit (Suriname) and seeds of sycamore maple (Acer pseudoplatanus L.) (France). The concentrations were 7.2 and 0.74mg/g respectively.



The analysis of linear and monomethylalkanes in exhaled breath samples by GC×GC-FID and GC–MS/MS

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Alexandra Hengerics Szabó , Peter Podolec , Viktória Ferenczy , Róbert Kubinec , Jaroslav Blaško , Ladislav Soják , Renáta Górová , Gabriela Addová , Ivan Ostrovský , Jozef Višňovský , Václav Bierhanzl , Radomír Čabala , Anton Amann
A new arrangement of the INCAT (inside needle capillary adsorption trap) device with Carbopack X and Carboxen 1000 as sorbent materials was applied for sampling, preconcentration and injection of C6C19n-alkanes and their monomethyl analogs in exhaled breath samples. For the analysis both GC–MS/MS and GC×GC-FID techniques were used. Identification of the analytes was based on standards, measured retention indices and selective SRM transitions of the individual isomers. The GC–MS/MS detection limits were in the range from 2.1pg for n-tetradecane to 86pg for 5-methyloctadecane. The GC×GC-FID detection limits ranged from 19pg for n-dodecane to 110pg for 3-methyloctane.



An LC–MS/MS method for the determination of five erectile dysfunction drugs and their selected metabolites in hair

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Sooyeun Lee , Boyeon Choi , Jihyun Kim , Sanghwan In , Seungkyung Baeck , Seung Min Oh , Kyu Hyuck Chung
The abuse of sildenafil and its analogous, accelerated by their inappropriate or illegal distribution, is a serious social issue globally. However, no studies have been conducted to monitor these drugs simultaneously in hair, which can provide valuable information on chronic drug use. In the present study, an LC–MS/MS method was developed for the simultaneous determination in hair of five erectile dysfunction drugs having a high risk for abuse (mirodenafil, sildenafil, tadalafil, udenafil and vardenafil) and their selected metabolites (SK3541, desmethylsildenafil, DA8164 and desethylvardenafil). The novel method was fully validated after optimizing matrix effects and extraction efficiency. The optimized sample preparation included acidic methanol extraction followed by solid phase extraction using C18 mixed mode strong cation exchange polymeric cartridges. The prepared samples were analyzed by LC–MS/MS with electrospray ion source in the positive ionization mode. The validation results proved the method to be selective, sensitive, accurate and precise, with acceptable linearity within calibration ranges. LODs ranged from 0.05 (DA8164) to 1ng/10mg hair (tadalafil). LOQs were 1ng/10mg hair except for DA8164 and vardenafil, of which they were 2.5ng/10mg hair. No significant variations were observed by different sources of matrices in both human and rat hair, except for tadalafil, for which a stable isotope-labeled internal standard was effective. The animal study suggested hair pigmentation was a major factor for the incorporation of the drugs and metabolites into hair. However, a wide variation of the sildenafil-to-desmethylsildenafil ratios was observed in human hair samples. The developed method will be very useful for monitoring the abuse of erectile dysfunction drugs for both legal and public health aspects.



Simultaneous determination of six flavonoids from Paulownia tomentosa flower extract in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Bin Dai , Zhiqiang Hu , Haiyan Li , Chong Yan , Liwei Zhang
A simple, rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of six components including apigenin, quercetin, apigenin-7-O-β-d-glucoside, quercetin-3-O-β-d-glucoside, 3′-methoxyluteolin-7-O-β-d-glucoside, and tricin-7-O-β-d-glucopyranoside in rat plasma using formononetin as the internal standard (IS). The plasma samples were pretreated by a one-step liquid–liquid extraction with dichloromethane. The chromatographic separation was carried out on a ZORBAX SB-Aq column with a gradient mobile phase consisting of acetonitrile and 2mM aqueous ammonium acetate. All analytes and IS were quantitated through electrospray ionization in negative ion multiple reaction monitoring mode. The mass transitions were as follows: m/z 269.1→117.2 for apigenin, m/z 301.2→151.2 for quercetin, m/z 431.3→311.2 for apigenin-7-O-β-d-glucoside, m/z 463.2→300.2 for quercetin-3-O-β-d-glucoside, m/z 461.3→283.1 for 3′-methoxyluteolin-7-O-β-d-glucoside, m/z 491.3→313.1 for tricin-7-O-β-d-glucopyranoside, and m/z 267.2→252.2 for IS, respectively. All calibration curves exhibited good linearity with correlation coefficient (r)>0.995. The intra-day and inter-day precisions (RSD) at three QC levels were both less than 14.0% and the accuracies ranged from 89.8% to 113.8%. The extraction recoveries of six compounds ranged from 82.3% to 92.5%. The validated method was successfully applied to pharmacokinetic study of the six components in male rat plasma after oral administration of Paulownia tomentosa flower extract.



Simultaneous determination of ten active constituents of Yankening Capsule in rat plasma by ultra high performance liquid chromatography with tandem mass spectrometry

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Jin Wang , Qian Pang , Weijian Cen , Pingchuan Zhu , Yuanjin Xu
An ultra high performance liquid chromatography with tandem mass spectrometry (U-HPLC–MS/MS) method was developed for simultaneous determination and pharmacokinetic study of ten active constituents, phellodendrine, coptisine, jatrorrhizine, berberine, palmatine, baicalin, wogonoside, baicalein, wogonin and emodin in rat plasma after oral administration of Yankening Capsule. After mixing with two internal standards tetrahydropalmatine and rutin, plasma samples were pretreated by protein precipitation with anhydrous ethanol–acetonitrile (9:1, v/v). The U-HPLC separation was carried on a ZORBAX RRHD Eclipse Plus C18 column (2.1mm×50mm, 1.8μm) with gradient elution using a mobile phase composed of methanol and water (containing 0.3% formic acid) at a flow rate of 0.3mLmin−1. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via electrospray ionization source with positive–negative ionization mode. The calibration curves of ten analytes showed good linearity (r>0.9979). The lower limits of quantification of phellodendrine, coptisine, jatrorrhizine, berberine, palmatine, baicalin, wogonoside, baicalein, wogonin and emodin were 0.50, 0.50, 0.30, 0.30, 0.30, 10, 3.0, 8.0, 1.0, 8.0μgL−1, respectively. The relative standard deviation of intra-day precision and inter-day precision were in the range from 1.13% to 5.96% and from 0.65% to 8.85%, respectively. The matrix effects of all analytes were found to be within the acceptable range with a range of 89.99–109.3%. The method is reliable and rapid and has been applied successfully to pharmacokinetic study of the ten active constituents in rat plasma after oral administration of Yankening Capsule.



Highly sensitive analysis of nucleic acids using capillary gel electrophoresis with ultraviolet detection based on the combination of matrix field-amplified and head-column field-amplified stacking injection

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Dong-Sheng Lian , Shu-Jin Zhao
To develop a highly sensitive method for analyzing nucleic acids using capillary gel electrophoresis with ultraviolet detection (CGE-UV), we combined matrix field-amplified with head-column field-amplified stacking injection (C-FASI) to employ the advantages of two methods. Without diminishing the resolution, a limit of detection of 0.13ng/ml (signal/noise=3) in a 300,000-fold diluted sample was obtained, the sensitivity is 102,308 times higher than that achieved with normal pressure injection, 3077 times that with normal electrokinetic injection, 154 times that with pressure field-amplified sample stacking injection, and 31 times that with matrix field-amplified stacking injection. After establishing the method, we tested the detection of a φX174-Hae III digest DNA product without purification and with a high ionic strength. At the lowest dilution of 5000-fold, sample at a concentration of 10ng/ml was enriched and detected. The relative standard deviations for migration time and peak area (n=3) were 0.03–1.15 and 0.72–6.42, respectively. To further validate C-FASI was applicable for real sample, a 400bp PCR product without purification was directly detected with a limit of detection at the concentration of 6000-fold dilution (signal/noise=3), The relative standard deviations for migration time and peak area (n=6) were 0.44 and 4.8, respectively. These results indicated that C-FASI had good qualitative and quantitative detection abilities and CGE-UV based on C-FASI is easy to perform, practical, highly-sensitive and robust for nucleic acid detection, which makes it a highly valuable tool for genetic diagnostics based on nucleic acid analysis.



Determination of urinary cortisol, cortisone and 6-sulfatoxymelatonin using dilute and shoot ultra-high pressure liquid chromatography–tandem mass spectrometry

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Anna Maria Sniecinska-Cooper , Ajit Jesang Shah , Dagmara Dimitriou , Ray Kruse Iles , Stephen Andrew Butler , Richard Bayford
Human sleep is a natural part of every individual’s life. Clear relationship between sleep and endocrine system has been already established. In particular, melatonin and cortisol are known to affect and regulate sleep/wake patterns. Here we report the development of an ultra-high pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for simultaneous measurement of 6-sulfatoxymelatonin (MT6s), cortisol and cortisone in urine. A separate method was developed for measurement of creatinine in urine. These levels were used to normalise the levels of analytes. First void morning urine samples were collected from 24 healthy volunteers. Samples were diluted 1:1 in water prior to injection onto reversed-phase C18 column and analysed using UHPLC–MS/MS method. Linear calibrations were obtained for all analytes with correlation coefficient in the range 0.998–0.999. The observed concentration was found to be in the range 92–105% for cortisol, 92–107% for cortisone and between 93 and 120% for MT6s of the reference levels. The total run time of 6min with all peaks of interest eluting within 3min was obtained. This demonstrates the feasibility of utilising the method for large multi-scale studies, where high throughput is required for studying the circadian rhythm of melatonin and cortisol secretion. These hormones play significant role in circadian rhythm and sleep/wake cycle; therefore it is important to monitor the levels of these endocrine markers in individuals suffering from sleep disorders. It is also beneficial with clinical applications to analyse melatonin and cortisol simultaneously in order to assess their interrelationships of these substances, such as their effect on diurnal rhythm and sleep.



Quantification of 5,7-dimethoxyflavone in mouse plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and its application to a pharmacokinetic study

Publication date: 26 January 2015
Source:Journal of Chromatography B, Volumes 978–979
Author(s): Di Bei , Guohua An
5,7-Dimethoxyflavone (5,7-DMF), a natural flavonoid abundant in many plants, has been reported to have many beneficial pharmacological effects, including strong chemopreventive and chemosensitizing properties, anti-oxidant, cardiovascular protectant, and anti-inflammatory activities. However, to date 5,7-DMF was evaluated mainly in vitro and its pharmacokinetics (PK) in vivo remains largely unknown. In addition, current available quantification methods of 5,7-DMF all lack sufficient sensitivity (lower limit of quantification >800ng/mL). The purposes of our study are to establish a sensitive quantification method of 5,7-DMF using LC–MS/MS and evaluate the PK profile of 5,7-DMF in mouse. Our method was fully validated and all of the fundamental parameters in method validation, including accuracy, precision, sensitivity, selectivity, recovery and stability were evaluated thoroughly in mouse plasma. The calibration curve covered 2–1000ng/mL with the lower limit of quantification (LLOQ) of 2ng/mL. The inter-run and intra-run precision and accuracy were less than 15% of nominal concentrations. The matrix effect and recovery yield were within ±15% of nominal concentrations. In the PK study, 5,7-DMF was detectable in mouse plasma up to 21h, with a terminal half-life of 11.5h. The clearance of 5,7-DMF (CL/F) is 22.3L/h/kg and area under the curve (AUCinf) is 449hng/mL. In conclusion, a fast, accurate, sensitive and selective quantification method of 5,7-DMF was established and the developed method was successfully applied to a PK study of 5,7-DMF following oral administration of 5,7-DMF in mice.



Electrochemical Sensors and Biosensors Based on Nanomaterials and Nanostructures

Analytical ChemistryDOI: 10.1021/ac5039863



Dielectrophoretically controlled Fresnel zone plate

Lab Chip, 2015, Advance ArticleDOI: 10.1039/C4LC01213E, PaperA. F. Chrimes, I. Khodasevych, A. Mitchell, G. Rosengarten, K. Kalantar-zadehWe present a novel switchable Fresnel zone plate, created using nanoparticle suspensions which are controlled by d…



Counting and Dynamic Studies of the Small Unilamellar Phospholipid Vesicle Translocation with Single Conical Glass Nanopores

Analytical ChemistryDOI: 10.1021/ac5029243



Versatile Biosensing Platform for DNA Detection Based on a DNAzyme and Restriction-Endonuclease-Assisted Recycling

Analytical ChemistryDOI: 10.1021/ac5034903



Microfluidic vapor-diffusion barrier for pressure reduction in fully closed PCR modules

Lab Chip, 2015, Advance ArticleDOI: 10.1039/C4LC01115E, PaperG. Czilwik, I. Schwarz, M. Keller, S. Wadle, S. Zehnle, F. von Stetten, D. Mark, R. Zengerle, N. PaustSchematic view of fluidic structures with a liquid-air mixture a) the vapor-liquid equili…



Mass Spectrometry Imaging of Biomolecular Information

Analytical ChemistryDOI: 10.1021/ac504543v