Journal of Chromatography B (v.1051, #C)

Sulfur Mustard (HD) has a 100 year history of use as a chemical warfare agent and recent events in the Middle East are causing it to once again be a potential concern. We report a new high-throughput method for the determination of HD exposure by the analysis of the β-lyase metabolite 1,1′-sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE) in human urine. This method features a hydrogen peroxide (H2O2) oxidative conversion of the β-lyase metabolites to SBMSE, followed by sample extraction and concentration using solid phase extraction in 96-well plate format. Subsequent high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) analysis gave linear quantitation over a calibration range of 0.1–100 ng/mL, with a method detection limit of 0.03 ng/mL. Liquid chromatographic separation was achieved using a hydrophilic interaction liquid chromatography (HILIC) column with an analyte retention time of 0.9 min and method time of 1.5 min (cycle time = 2.0 min). Users of this method could prepare and analyze approximately 650 samples in 24 h which would be important for an emergency response.
Keywords: Sulfur mustards (HD); Exposure; Biomarker; HPCL-MS/MS; High throughput; Oxidation;

Dual-cloud point extraction (dCPE) was successfully developed for simultaneous extraction of trace sulfonamides (SAs) including sulfamerazine (SMZ), sulfadoxin (SDX), sulfathiazole (STZ) in urine and water samples. Several parameters affecting the extraction were optimized, such as sample pH, concentration of Triton X-114, extraction temperature and time, centrifugation rate and time, back-extraction solution pH, back-extraction temperature and time, back-extraction centrifugation rate and time. High performance liquid chromatography (HPLC) was applied for the SAs analysis. Under the optimum extraction and detection conditions, successful separation of the SAs was achieved within 9 min, and excellent analytical performances were attained. Good linear relationships (R2  ≥ 0.9990) between peak area and concentration for SMZ and STZ were optimized from 0.02 to 10 μg/mL, for SDX from 0.01 to 10 μg/mL. Detection limits of 3.0–6.2 ng/mL were achieved. Satisfactory recoveries ranging from 85 to 108% were determined with urine, lake and tap water spiked at 0.2, 0.5 and 1 μg/mL, respectively, with relative standard deviations (RSDs, n  = 6) of 1.5–7.7%. This method was demonstrated to be convenient, rapid, cost-effective and environmentally benign, and could be used as an alternative tool to existing methods for analysing trace residues of SAs in urine and water samples.
Keywords: Dual-cloud point extraction; Sulfonamides; Urine sample; Water samples;

A simple, fast, sensitive and accurate methodology based on a LLE followed by liquid chromatography–tandem mass spectrometry for simultaneous determination of four regioisomers (8-iso prostaglandin F, 8-iso-15(R)-prostaglandin F, 11β-prostaglandin F, 15(R)-prostaglandin F) in routine analysis of human plasma samples was developed. Isoprostanes are stable products of arachidonic acid peroxidation and are regarded as the most reliable markers of oxidative stress in vivo. Validation of method was performed by evaluation of the key analytical parameters such as: matrix effect, analytical curve, trueness, precision, limits of detection and limits of quantification. As a homoscedasticity was not met for analytical data, weighted linear regression was applied in order to improve the accuracy at the lower end points of calibration curve. The detection limits (LODs) ranged from 1.0 to 2.1 pg/mL. For plasma samples spiked with the isoprostanes at the level of 50 pg/mL, intra-and interday repeatability ranged from 2.1 to 3.5% and 0.1 to 5.1%, respectively. The applicability of the proposed approach has been verified by monitoring of isoprostane isomers level in plasma samples collected from young patients (n  = 8) subjected to hyperbaric hyperoxia (100% oxygen at 280 kPa(a) for 30 min) in a multiplace hyperbaric chamber.
Keywords: F2-isoprostanes; Prostaglandins; LC–MS/MS analysis; Weighted linear regression; Plasma;

Cashew immature and ripe peduncles (Anacardium occidentale L.) from orange- and red-colored clones CCP 76 and BRS 189, respectively, were prepared as juice or fibrous fraction and submitted to UPLC–MS analyses, while the soluble fraction was also submitted to enzymatic evaluation. Cinnamoyl glucoside was present in ripe juice samples from both cashew clones, while monogalloyl diglucoside and digalloyl glucoside were present in immature juice samples from both cashew clones. Four compounds were found at immature fiber of both clones, anacardic acids (1, 2, 3) and GA19. The phenolic biosynthetic pathway was evaluated in juice samples and phenylalanine ammonia-lyase activity decreased significantly during the development, although it was much higher in ripe CCP 76. UDP-glycosyltransferases activity differed between clones, however its product cinnamoyl glucoside was a possible chemical marker of ripe juice samples from both clones. Flavonol synthase showed the highest specific activity in both cashew clones and its product, flavonols were identified in cashew apple at immature and ripe stages.
Keywords: Ripening; Anacardic acid; Phenolics; Cashew apple; Enzymes;

Cationic amphiphilic drugs (CADs) can induce the hyperaccumulation of phospholipids in cells and tissues. This side effect, which is known as drug-induced phospholipidosis, is sometimes problematic in the development and clinical use of CADs. It is known that CADs generally interact with phospholipids via both hydrophobic and acid-base interactions, and CADs with the larger affinity to phospholipid exhibit the larger induction risk. To develop a chromatographic assay system to predict the phospholipidosis-inducing potential with considering the acid-base interaction between CAD and phosphate group of phospholipid, hydrophilic interaction chromatographic (HILIC) methods were tested in this study. First, a PC HILIC column with phosphocholine groups on a packed material was used. The acid-base or other hydrophilic interactions to the stationary phase differed among basic drugs, and retention to the PC HILIC column did not accurately reflect the induction potential of phospholipidosis. As an alternative HILIC approach, the elution of CADs with the phosphate buffer from an amide column was tested. The elution effect, which is expressed as ratio of retention factors between different phosphate content in the mobile phase, closely correlated with the induction potential. Using the elution effect and retention factor to a reversed-phase HPLC column, the phospholipidosis-inducing drugs were clearly discriminated from the non-inducers. These results suggest that the proposed chromatographic approach can screen phospholipidosis-inducing drugs.
Keywords: Cationic amphiphilic drug; Hydrophilic interaction chromatography; Phosphate; Phospholipidosis; Phosphocholine;

Rapid determination of 54 pharmaceutical and personal care products in fish samples using microwave-assisted extraction—Hollow fiber—Liquid/solid phase microextraction by Yi Zhang; Wen Guo; Zhenfeng Yue; Li Lin; Fengjuan Zhao; Peijin Chen; Weidong Wu; Hong Zhu; Bo Yang; Yanyun Kuang; Jiong Wang (41-53).
In this paper, a simple, rapid, solvent-less and environmental friendliness microextraction method, microwave-assisted extraction-hollow fiber-liquid/solid phase microextraction (MAE-HF-L/SME), was developed for simultaneous extraction and enrichment of 54 trace hydrophilic/lipophilic pharmaceutical and personal care products (PPCPs) from fish samples. A solid-phase extraction material, solid-phase microextraction (SPME) fiber, was synthesized. The SPME fiber had a homogeneous, loose structure and good mechanical properties, and they exhibited a good adsorption capacity for most PPCPs selected. The material formed the basis for the method of MAE-HF-L/SME. A method of liquid chromatography-high resolution mass spectroscopy (LC–HRMS) for analysis of 54 PPCPs. Under optimal synthesis and extraction conditions, the limits of detection (LODs, n  = 3) and the limits of quantitation (LOQs, n  = 10) for the 54 PPCPs were between 0.01–0.50 μg · kg−1 and 0.05 2.00 μg · kg−1, respectively. Percent recoveries and the relative standard deviations (RSDs) in spiked fish samples (n  = 6) were between 56.3%–119.9% and 0.3%–17.1%, respectively. The microextraction process of 54 PPCPs in MAE-HF-L/SME took approximately 12 min. The method has a low matrix interference and high enrichment factor and may be applicable for determination of 54 different PPCPs in fish samples.
Keywords: Microwave-assisted extraction (MAE); Hollow fiber-liquid/solid phase microextraction; Pharmaceutical and personal care products (PPCPs); Solid-phase microextraction fiber; Liquid chromatography–high resolution mass spectroscopy (LC–HRMS);

Pharmaceutical analysis of a novel propargyl-linked antifolate antibiotic in the mouse by John Hoody; Jeremy B. Alverson; Santosh Keshipeddy; Patrick A. Barney; Larissa Walker; Amy C. Anderson; Dennis L. Wright; Nigel D. Priestley (54-59).
Antimicrobial resistance to current antibiotics is a significant public health problem and the need for new antibiotics is a compelling one. We have been developing a new series of antibiotics, propargyl-linked diaminopyrimidines, based on the structure of trimethoprim. To date we have discovered compounds that are effective inhibitors of dihydrofolate reductase (the target of trimethoprim), that are potent antibiotics in vitro against a range of Gram-positive pathogens including methicillin-resistant S. aureus, and that are non-toxic in mammalian cell culture. In this study we report the development of an LC–MS-based protocol for the quantification of our lead antibiotic 37D1-UCP1099 and the application of this assay to follow the concentration of the compound in mouse plasma after intraperitoneal administration. Extraction of 37D1-UCP1099 from mouse plasma was achieved through a liquid-liquid extraction with ethyl acetate. Separation was performed utilizing a reverse-phase C18 column with a ten minute isocratic elution using 47:53 (v/v) 10 mM NH4HCO3:acetonitrile. The lower limit of quantitation for 37D1-UCP1099 was 50 ng mL−1 and the assay showed a dynamic range of 50–4000 ng mL−1 with good linearity (r 2  ≥ 0.996 for all fits). Intra-day and inter-day precision and accuracy were within 11.3% (%RSD) and 6.6% (%RE) respectably. We have demonstrated that the compound is stable under the assay procedures. The compound was shown to have a mean residence time of 26.2 ± 1.0 min and a half-life of 18.2 ± 0.7 min after intraperitoneal delivery at 5 mg kg−1. These studies now form the foundation of our work to develop additional analogs of 37D1-UCP1099 with improved pharmacokinetic properties.
Keywords: Antibiotic; Folate; Propargyl-linked; Trimethoprim; LC–MS; Mouse;

Determination of the interconversion energy barrier of three novel pentahelicene derivative enantiomers by dynamic high resolution liquid chromatography by Pavel Majek; Jan Krupcik; Zachary S. Breitbach; Milan K. Dissanayake; Peter Kroll; Aaron A. Ruch; LeGrande M. Slaughter; Daniel W. Armstrong (60-67).
Dynamic high resolution liquid chromatography (DHPLC) was used to determine the kinetic and thermodynamic activation parameters of interconversion of three novel pentahelicene derivatives {3,5-bis(trifluoromethyl)benzo[i]pentahelicene, naphtho[1,2-i]pentahelicene and 4-methoxybenzo[i]pentahelicene}. DHPLC was performed on a chiral isopropyl − carbamate cyclofructan 6 (LARIHC CF6-P) column under normal phase conditions. Variation of the column temperature and flow rate was used to study the interconversion process. A computer assisted deconvolution method was employed to determine the individual peak areas and the retention times required for the calculation of apparent enantiomerization energy barriers, enthalphy and entropy of the interconvertion of above defined pentahelicene derivative enantiomers. An ab initio quantum chemistry method was used to estimate theoretical kinetic and thermodynamic interconversion parameters and to evaluate experimental data of these three novel pentahelicene derivative enantiomers.
Keywords: Dynamic high performance liquid chromatography; Enantioselective separation; Thermodynamic activation parameters; Interconversion of pentahelicene derivatives;

This investigation reports a simplified approach for the purification of urinary siderocalin known as neutrophil gelatinase-associated lipocalin (NGAL). Urinary NGAL was purified by tangential flow filtration and ion exchange chromatography. Isolated NGAL was analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular mass of NGAL is 23674 Da. Peptide mass fingerprinting of the purified NGAL yielded peptides that partially matched with known sequence of P80188 (NGAL_HUMAN). The tryptic digestion profile of isolated NGAL infers that it may be unique and additive molecule in the dictionary of urinary proteins. This is the first report of purification and validation of urinary NGAL from large volume sample by using tangential flow filtration and peptide sequencing respectively. This cost-effective and simplified approach to purification of NGAL, together with the easy availability of urine sample makes the large-scale production of NGAL possible, allowing exploration of various bioclinical as well as biodiagnostic applications.
Keywords: Acute kidney injury; Ion exchange chromatography; Immunoblotting; NGAL; Tangential flow filtration;

Binding of TEM-1 beta-lactamase to beta-lactam antibiotics by frontal affinity chromatography by Xiu Chen; Yuhua Li; Yan Zhang; Jianting Yang; Liujiao Bian (75-83).
TEM-1 beta-lactamases can accurately catalyze the hydrolysis of the beta-lactam rings in beta-lactam antibiotics, which make beta-lactam antibiotics lose its activity, and the prerequisite for the hydrolysis procedure in the binding interaction of TEM-1 beta-lactamases with beta-lactam antibiotics is the beta-lactam rings in beta-lactam antibiotics. Therefore, the binding of TEM-1 beta-lactamase to three beta-lactam antibiotics including penicillin G, cefalexin as well as cefoxitin was explored here by frontal affinity chromatography in combination with fluorescence spectra, adsorption and thermodynamic data in the temperature range of 278–288 K under simulated physiological conditions. The results showed that all the binding of TEM-1 beta-lactamase to the three antibiotics were spontaneously exothermic processes with the binding constants of 8.718 × 103, 6.624 × 103 and 2.244 × 103 (mol/L), respectively at 288 K. All the TEM-1 beta-lactamases were immobilized on the surface of the stationary phase in the mode of monolayer and there existed only one type of binding sites on them. Each TEM-1 beta-lactamase bound with only one beta-lactam antibiotic and hydrogen bond interaction and Van der Waals force were the main forces between them. This work provided an insight into the binding interactions between TEM-1 beta-lactamases and beta-lactam antibiotics, which may be beneficial for the designing and developing of new substrates resistant to TEM-1 beta-lactamases.
Keywords: TEM-1 beta-lactamase; Beta-lactam antibiotics; Binding; Frontal affinity chromatography;

Oxidative stress is considered as one of the significant causes of DNA damage which in turn contributes to cell death through a series of intermediate processes such as cancer formation, mutation, and aging. Natural sources such as plant and fruit products have provided us with interesting substances of antioxidant activity that could be recruited in protecting the genetic materials of the cells. This study is an effort to discover some of those antioxidants effects in their standard and natural forms by performing an ultrasensitive determination of the products of DNA oxidation using GC–MS/MS. Experiments were used to determine the direct antioxidant activity of the substances contained in the tendrils of Vitis vinifera (var. alphonse) by extracting them and achieving Folin-Ciocalteau and CHROMAC analyses to determine the total phenolic content (TPC) and the antioxidant capacity of the extract, respectively; results revealed a phenolic content of 11.39 ± 0.30 mg Gallic Acid Equivalent (GAE)/g of the plant’s fresh weight (FW) by Folin-Ciocalteau and 8.17 ± 0.49 mg Trolox Equivalent (TE)/g FW by CHROMAC assays. The qualitative analysis of the plant extract by HPLC–DAD technique revealed that two flavonoid glycosides namely rutin and isoquercitrin in addition to chlorogenic acid were contained in the extract. The determination of the DNA oxidation products was performed after putting DNA, rutin and isoquercitrin standard samples with different concentration, and the extract's sample under oxidative stress. Eighteen DNA oxidation products were traced using GC–MS/MS with ultra-sensitivity and the experiments proved a significant decrease in the concentration of the DNA oxidation products when the extract was used as a protectant against the oxidative stress. It is believed by conclusion that the extract of V. vinifera’s (var. alphonse) tendrils has a good antioxidant activity; hence it is recommended to be used as a part of the daily healthy food list if possible.
Keywords: V. vinifera tendrils; GC–MS/MS; Oxidative stress; DNA oxidation; Rutin; Isoquercitrin;

This study demonstrated a novel dispersive micro solid phase extraction (DMSPE) method for extraction of adamantane drugs (amantadine, rimantadine and memantine) in chicken muscle. The adamantane drugs were extracted from chicken muscle using 1% acidic acetonitrile as extraction solvent. The cleanup of fatty matrices from analytes was achieved by the DMSPE technique using magnetic cation exchange polymer as adsorbent. In this procedure, the experimental parameters and conditions were optimized in detail for the improvement of extraction efficiency. The method showed low limit of detection of 0.03 μg/kg and recoveries of the analytes ranged from 87.2% to 109.3% for adamantane drugs. The proposed DMSPE method proved to be simple, effective and suitable for the treatment of adamantane drugs in chicken muscle with a relatively shorter extraction time.
Keywords: DMSPE method; Adamantane drugs; Chicken muscle; UHPLC–HRMS;

The tyrosine, tryptophan, and glutamate metabolic pathways play key roles on pathological state of neuronal functions and the change of their levels in biological systems reflects the progress degree of neuronal diseases. Comprehensive profiling of these metabolites is important to find new biomarkers for diagnosis or prognosis of various neuronal diseases. However, the overall profiling analysis of various neurochemicals in biological sample is confronted with several limitations due to their low concentration and physicochemical properties and the coexistence of matrices. We developed an efficient and feasible method using gas chromatography–tandem mass spectrometry (GC–MS/MS). Wide-bore mixed cation exchange (MCX) SPE process enables a rapid and effective cleanup of 20 neurochemicals even including acidic and basic neurochemicals in a single SPE cartridge by using different composition of eluents. Selective derivatization of various types of metabolites was applied to achieve highly chromatographic separation and sensitive mass detection. Appropriate selection of precursor and product transition ions used in multiple reaction-monitoring (MRM) mode based on the MS/MS fragmentations of the derivatized neurochemicals could be significantly minimized the matrix effects and enhanced the reliability of quantification results. The developed method was validated in terms of linearity, limits of detection, precision, accuracy, and matrix effects. The intra- and inter-assay analytical variations were less than 10%. The overall linearity for all of the targets was excellent (R2  ≥ 0.996). The detection limits ranged between 0.38 and 8.13 ng/mL for the acidic neurochemicals and between 0.02 and 11.1 ng/mL for the basic neurochemicals. The developed protocol will be expected to be a promising tool for the understanding of the pathological state and diagnosis of various neuronal diseases.
Keywords: Neurotransmitters; Acidic hydrolysis; MCX-SPE; Chemical derivatization; GC–MS/MS; Neuronal diseases;

Using UHPLC Q-Trap/MS as a complementary technique to in-depth mine UPLC Q-TOF/MS data for identifying modified nucleosides in urine by Zhiwei Lu; Qing Wang; Meiling Wang; Shuang Fu; Qingqing Zhang; Zhixin Zhang; Huizhen Zhao; Yuehong Liu; Zhenhai Huang; Ziye Xie; Honghong Yu; Xiaoyan Gao (108-117).
Modified nucleosides, metabolites of RNA, are potential biomarkers of cancer before the appearance of morphological abnormalities. It is of great significance to comprehensively detect and identify nucleosides in human urine for discovery of cancer biomarkers. However, the lower abundance, the greater polarity and the matrix effects make it difficult to detect urinary nucleosides. In this paper, an integrated method consisted of sample preparation followed by ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC Q-TOF/MS) detection and primary identification, then ultra-high performance liquid chromatography coupled with hybrid triple quadrupole linear ion trap mass spectrometer (UHPLC Q-Trap/MS) further identification and validation were introduced. Firstly, to enrich the nucleosides and eliminate the urine matrix effects, different sorbent materials of solid phase extraction (SPE) and the elution conditions were screened. Secondly, UPLC Q-TOF/MS was used to acquire mass data in MSE mode. The structural formulas of nucleosides in urine sample were primarily identified according to retention time, accurate mass precursor ions and fragment ions from in-house database and online database. Thirdly, the preliminary identified nucleoside structures lacking of characteristic fragment ions were verified by UHPLC Q-Trap/MS in multiple reaction monitoring trigger enhanced product ion scan (MRM-EPI) and neutral loss scan (NL). At last, phenylboronic acid (PBA)-based SPE was utilized due to its higher MS signal and weaker matrix effects under optimized extraction conditions. Fifty-five nucleosides were primarily identified by UPLC Q-TOF/MS, among which 50 nucleosides were confirmed by UHPLC Q-Trap/MS. Five nucleosides, namely 4′,5′-didehydro-5′-deoxyadenosine, 4′,5′-didehydro-5′-deoxyinosine, isonicotinamide riboside, peroxywybutosine and hydroxywybutosine, were found from urine for the first time. The results will expand the Human Metabolome Database (HMDB).
Keywords: Urinary nucleosides; Solid phase extraction; Ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry; Ultra-high performance liquid chromatography coupled with hybrid triple quadrupole linear ion trap mass spectrometer; Phenylboronic acid;

Separation and identification of positional isomers is an important issue in forensic toxicology, particularly in the context of new psychoactive substances (NPS). Despite the structural similarity, positional isomers often show different pharmacological properties and thus can exhibit dramatic differences with respect to their toxicity. Additionally, besides these pharmacological and toxicological effects, the legal status is also of great importance. We present a sensitive and selective LC–MS/MS method to separate the ortho, meta and para isomers of methylmethcathinone (MMC) and methylethcathinone (MEC) using a core-shell biphenyl analytical column. Reliability of the method was confirmed under consideration of the validation parameters selectivity, linearity, accuracy and precision, analytical limits, processed sample stability, matrix effects and recovery. Linearity was demonstrated over the entire calibration range from 5 to 250 ng/ml with the use of a 1/x2 weighting. Appropriate quantification and detection limits (LLOQ = 5 ng/ml, LOD < 2 ng/ml) could be achieved. Application of the method to real serum samples collected between June 2014 and August 2016 revealed the proof of a recent MMC or MEC consumption, respectively, in eight cases. Isomers of MMC could be detected in three of these eight cases, of which two were positive for 3-MMC and one was positive for 2-MMC. The other samples were tested positively for 3-MEC. In none of the samples 4-MMC, 2-MEC or 4-MEC could be detected. Only substances that were not governmentally controlled at that time could be detected, reflecting the rapid response of the recreational drug market to newly enacting drug laws.
Keywords: Methylmethcathinone (MMC); Methylethcathinone (MEC); Positional isomers; Chromatographic separation; LC-ESI-MS/MS;