Journal of Chromatography B (v.976-977, #C)

In this study, two solid-phase recycling method for basic ionic liquid (IL) 1-butyl-3-methylimidazolium acetate ([C4mim]Ac) were studied through a digestion extraction system of extracting biphenyl cyclooctene lignans from Schisandra chinensis. The RP-HPLC detection method for [C4mim]Ac was established in order to investigate the recovery efficiency of IL. The recycling method of [C4mim]Ac is divided into two steps, the first step was the separation of lignans from the IL solution containing HPD 5000 macroporous resin, the recovery efficiency and purity of [C4mim]Ac achieved were 97.8% and 67.7%, respectively. This method cannot only separate the lignans from [C4mim]Ac solution, also improve the purity of lignans, the absorption rate of lignans in [C4mim]Ac solution was found to be higher (69.2%) than that in ethanol solution (57.7%). The second step was the purification of [C4mim]Ac by the SK1B strong acid ion exchange resin, an [C4mim]Ac recovery efficiency of 55.9% and the purity higher than 90% were achieved. Additionally, [C4mim]Ac as solvent extraction of lignans from S. chinensis was optimized, the hydrolysis temperature was 90 °C and the hydrolysis time was 2 h.
Keywords: Basic ionic liquid; Recovery; Schisandra chinensis; Biphenyl cyclooctene lignans; Macroporous resin; Ion exchange resin;

Free arachidonic acid is functionally interlinked with different lipid signaling networks including those involving prostanoid pathways, the endocannabinoid system, N-acylethanolamines, as well as steroids. A sensitive and specific LC-MS/MS method for the quantification of arachidonic acid, prostaglandin E2, thromboxane B2, anandamide, 2-arachidonoylglycerol, noladin ether, lineoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, steroyl ethanolamide, aldosterone, cortisol, dehydroepiandrosterone, progesterone, and testosterone in human plasma was developed and validated. Analytes were extracted using acetonitrile precipitation followed by solid phase extraction. Separations were performed by UFLC using a C18 column and analyzed on a triple quadrupole MS with electron spray ionization. Analytes were run first in negative mode and, subsequently, in positive mode in two independent LC-MS/MS runs. For each analyte, two MRM transitions were collected in order to confirm identity. All analytes showed good linearity over the investigated concentration range (r  > 0.98). Validated LLOQs ranged from 0.1 to 190 ng/mL and LODs ranged from 0.04 to 12.3 ng/mL. Our data show that this LC-MS/MS method is suitable for the quantification of a diverse set of bioactive lipids in plasma from human donors (n  = 32). The determined plasma levels are in agreement with the literature, thus providing a versatile method to explore pathophysiological processes in which changes of these lipids are implicated.
Keywords: LC-MS/MS; Arachidonic acid; Endocannabinoids; Lipids; Steroids; N-acylethanolamines; Palmitoylethanolamide; Prostanoids; Noladin ether;

Analysis of biothiols is still problematic, due to their high polarity, oxidation sensitivity and time-consuming sample preparation. In this paper, a direct, rapid and sensitive method was developed for simultaneous quantification of unbound cysteine (Cys), glutathione (GSH) and phytochelatins (PCs) in rice leaf, stem and root samples by hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry (HILIC–MS/MS). Homogenized samples were extracted with water containing 50 mM dithiothreitol, without derivatization and further clean-up, and the extracts were injected directly onto an Xbridge Amide-HILIC column (3.5 μm, 150 mm × 2.1 mm i.d.). The best chromatographic separation and MS sensitivity was achieved using a linear gradient elution with 10 mM aqueous ammonium formate and acetonitrile as the mobile phase. In MS/MS mode the detection limit (S/N ≥ 3) of seven biothiols was 3–105 nM. Good linearities were observed (r  > 0.995) with linear dynamic range at least over three orders of magnitude. Recoveries for most analytes were within the range of 77–128%, with relative standard deviations less than 18.2%. The intra-day precision (n  = 7) was 6.1–11.7%, and the inter-day precision over 15 d (n  = 15) was 8.5–16.3% for all biothiols. The optimized HILIC–MS/MS method was applied to study the influence of different cadmium (Cd) concentrations (0, 1 and 50 μM) on contents of Cys, GSH and PC2–6 in rice tissue. With increasing Cd concentrations in nutrient solutions, contents of PC2–4 in rice roots increased but contents of Cys and GSH decreased. Contents of PC2–4 in both rice leafs and stems increased markedly at high dose Cd (50 μM) treatment compared with controls, compared with low Cd concentrations (1 μM). However, both PC5 and PC6 were not detected throughout the stress experiment.
Keywords: Biothiol quantification; Rice; Cd stress; Amide-HILIC; ESI-MS/MS;

This study describes the preparation and electrochromatographic application of a new open-tubular capillary column with triethanolamine functionalized stationary phase. The stationary phase was synthesized by in situ grafting polymerization with 3-chloro-2-hydroxypropylmethacrylate based reactive monomer, and followed triethanolamine functionalization. The different 3-chloro-2-hydroxypropylmethacrylate contents on the separation efficiency were studied. The results indicated that the 3-chloro-2-hydroxypropylmethacrylate content (e.g., 15.3 v/v%) on the inner surface of the capillary was very important for final preparation of the polymer stationary phase. The electrochromatographic characterization of the stationary phase was performed using alkylbenzene derivatives. The pH effect on the electroosmotic flow was also investigated. The open tubular column functionalized with triethanolamine allowed to operate in the anodic electroosmotic flow mode in the system. With anodic electroosmotic flow mode, favorable separations of the amino acids and the nucleosides were successfully achieved with high column efficiens ranging from 142 000 to 257 000 plates/m. Good repeatability was gained with relative standard deviation of the migration time and peak areas less than 2.2% for run to run (n  = 5) and less than 3.2% day to day (n  = 3). Furthermore, real sample applicability of this column to the separation of amino acids in white tea sample was demonstrated.
Keywords: Open-tubular CEC; HPMA-Cl; Amino acids; Nucleosides; Triethanolamine; Food analysis;

LC–MS/MS simultaneous quantitation of 2-hydroxyethylated, oxidative, and unmodified DNA nucleosides in DNA isolated from tissues of mice after exposure to ethylene oxide by Fagen Zhang; Michael J. Bartels; Matthew J. LeBaron; Melissa R. Schisler; Yo-Chan Jeong; B. Bhaskar Gollapudi; Nigel P. Moore (33-48).
2-Hydroxyethylated and oxidative DNA nucleosides (DNA adduct biomarkers), such as O6-(2-hydroxyethyl)-2′-deoxyguanosine (O6HEdG), N6-(2-hydroxyethyl)-2′-deoxyadenosine (N6HEdA), 1-(2-hydroxyethyl)-2′-deoxyadenosine (N1HEdA), and 8-hydroxy-2′-deoxyguanosine (8-OHdG), N2,3-etheno-2′-deoxyguanosine (N2,3-ethenodG), α-methyl-γ-hydroxy-1,N2-propano-2′-deoxyguanosine (CrotondG), are important proposed biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) in vivo. A liquid chromatography–tandem mass spectrometric method was developed for the simultaneous determination of O6HEdG, N6HEdA, N1HEdA, 8-OHdG, CrotondG, and N2,3-ethenodG together with regular 2′-deoxyguanosine (dG), and 2′-deoxyadenosine (dA) nucleosides in the DNA extracted from mouse lung tissues for the assessment of exposure to EO after inhalation. The lower limits of quantitation for 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, N1HEdA, N6HEdA, dG, and dA were 0.025, 0.00125, 0.025, 0.00125, 0.025, 0.01, 2342, and 2500 ng/mL, respectively. The linearity of the calibration curves for all analytes were >0.989. The intra-day assay precision relative standard deviation (RSD) values for quality control (QC) samples for all analytes were ≤13.5% with accuracy values ranging from 86.5% to 111%. The inter-day assay precision (RSD) values for all analytes were ≤18.8% with accuracy values ranging from 87.9% to 119%. This method was used for simultaneous determination of the levels of 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, dG, N1HEdA, N6HEdA, and dA in DNA enzymatic hydrolysates from DNA extracted from mouse lung after 12 weeks’ inhalation exposure to EO at atmospheric concentrations of 0, 100, and 200 ppm. Overall, N2,3-ethenodG was not detected in any samples. 8-OHdG, CrotondG, dG, and dA were all quantifiable in all samples. O6HEdG, N1HEdA, and N6HEdA were quantifiable in most samples and the ratio of the corresponding adduct versus their corresponding DNA base (dG or dA) [×10 (e6)] was increased as the EO exposure concentration increased.
Keywords: Simultaneous quantitation; 2-Hydroxyethylated and oxidative DNA nucleoside biomarkers; Ethylene oxide; Liquid chromatography tandem mass spectrometry;

A multiresidue quantitative screening method covering 39 antibiotics from 7 different families by ultra-high-pressure-liquid-chromatography–tandem mass spectrometry (UHPLC–MS/MS) is described. Sulfonamides, trimethoprim, tetracyclines, macrolides, quinolones, penicillins and chloramphenicol are simultaneously detected in liver tissue. A simple sample treatment method consisting of extraction with a mixture of acetonitrile and ethylenediaminetetraacetic acid (EDTA) followed by solid-phase extraction (SPE) with a hydrophilic–lipophilic balanced (HLB) cartridge was developed.The methodology was validated, in accordance with Decision 2002/657/EC, by evaluating the following required parameters: decision limit (CCα), detection capability (CCβ), specificity, repeatability and reproducibility. The precision, in terms of the relative standard deviation, was under 22% for all of the compounds, and the recoveries were between 80% and 110%. The CCα and CCβ were determined according to the maximum residue limit (MRL) or the minimum required performance limit (MRPL), when established.
Keywords: Antibiotics; Multiclass; Multidetection; UHPLC–MS/MS; Liver; Validation;

Hydrophilic-interaction liquid chromatography–tandem mass spectrometric determination of erythrocyte 5-phosphoribosyl 1-pyrophosphate in patients with hypoxanthine–guanine phosphoribosyltransferase deficiency by Hiroshi Hasegawa; Yoshihiko Shinohara; Sayako Nozaki; Makiko Nakamura; Koei Oh; Osamu Namiki; Kiyotaka Suzuki; Akihiko Nakahara; Mari Miyazawa; Ken Ishikawa; Takahiro Himeno; Sayaka Yoshida; Takanori Ueda; Yasukazu Yamada; Kimiyoshi Ichida (55-60).
Mutations in the gene encoding hypoxanthine–guanine phosphoribosyltransferase (HPRT) cause Lesch–Nyhan disease (LND) and its variants (LNV). Due to the technical problems for measuring the HPRT activity in vitro, discordances between the residual HPRT activity and the clinical severity were found. 5-Phosphoribosyl 1-pyrophosphate (PRPP) is a substrate for HPRT. Since increased PRPP concentrations were observed in erythrocytes from patients with LND and LNV, we have turned our attention to erythrocyte PRPP as a biomarker for the phenotype classification. In the present work, a method for determination of PRPP concentration in erythrocyte was developed using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with multiple reaction monitoring (MRM). Packed erythrocyte samples were deproteinized by heating and the supernatants were injected into the LC–MS/MS system. All measurement results showed good precision with RSD <6%. PRPP concentrations of nine normal male subjects, four male patents with LND and six male patients with LNV were compared. The PRPP concentrations in erythrocyte from patients with LND were markedly increased compared with those from normal subjects, and those from patients with LNV were also increased but the degree was smaller than those with LND. The increase pattern of PRPP concentration in erythrocyte from patients with HPRT deficiency was consistent with the respective phenotypes and was correlated with the disease severity. PRPP concentration was suggested to give us supportive information for the diagnosis and the phenotype classification of LND and LNV.
Keywords: PRPP; LC–MS/MS; HPRT; Lesch–Nyhan disease;

Morroniside, the most abundant iridoid glycoside in the valuable traditional Chinese medicine Fructus Corni, exhibits various pharmacological activities and biological effects. Intestinal flora plays an important role in the metabolism of drug compounds, which might lead to the variation of ethnopharmacological profile of the medicine. However, little is known of the interactions of the morroniside with human intestinal bacteria. In this study, different pure bacteria were isolated from human feces and their capability to convert morroniside were investigated. The metabolites of morroniside were analyzed by ultra high performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UHPLC–Q-TOF-MS) technique using Metabolynx™ software. Parent compound and three metabolites were detected and tentatively identified based on the characteristics of their protonated ions. The parent is proposed to be metabolized by three main metabolic pathways including deglycosylation, dehydroxylation and methylation. Morroniside was firstly metabolized to its aglycone (M1), and then was further converted to dehydroxylated aglycone (M2) and methylated aglycone (M3). This is the first report of the metabolism of morroniside by human intestinal bacteria. These metabolites might influence the biological activities of morroniside in vivo, which could affect the clinical effects of medicines. Thus, the study on the metabolism of morroniside by human intestinal bacteria is very helpful to unravel how traditional medicines work.
Keywords: Morroniside; Human intestinal bacteria; UHPLC–Q-TOF-MS; Metabolites;

A comprehensive evaluation of mixed mode interactions of HEA and PPA HyperCel™ chromatographic media by J. Pezzini; C. Cabanne; R. Gantier; V.N. Janakiraman; X. Santarelli (68-77).
Mixed mode or multimodal chromatography has been developed for rational use of multiple interactions in a controlled manner, in contrast to non-specific interactions. Indeed, as the term “mixed mode” suggests, these resins allow different types of interactions within a single chromatographic medium. In this paper, HEA HyperCel™, PPA HyperCel™ mixed-mode chromatographic media have been studied. These mixed-mode sorbents typically involve hydrophobic pseudo-affinity interactions for binding and essentially ionic interactions (charge repulsion) for elution. We identified and characterized these different interactions in chromatographic experiments by exploiting specific properties of proteins using protein standards and complex mixtures. We highlighted the major intervention of at least two types of interactions in these media: hydrophobic and electrostatic interactions. We observed the behaviour of these resins at different pH, ionic strength, with different salts and buffers types and in the presence of different organic compounds.
Keywords: Mixed mode chromatography; HEA HyperCel™; PPA HyperCel™; Purification; Proteins;

A robust and validated method based on ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS/MS) has been developed for the simultaneous determination of remimazolam, which is a new chemical entity, and its major carboxylic acid metabolite (M1) in human plasma. Plasma samples were pre-purified by protein precipitation procedure and analyzed using an isocratic chromatographic separation over an Acquity UPLC CSH C18 column. The mobile phase consisted of acetonitrile–water containing 10 mM ammonium formate and 0.1% formic acid at a flow rate of 0.4 mL min−1. Positive electrospray ionization was employed as the ionization source in the multiple reaction monitoring (MRM) mode. The analysis time was about 1.5 min. The method was fully validated over the concentration range of 0.5–1000 ng mL−1 for both analytes. The lower limit of quantification (LLOQ) was 0.5 ng mL−1. Inter- and intra-batch precision was less than 8.4% and the accuracy was within 88.8–107.0%. The mean extraction recoveries obtained from three concentrations of QC plasma samples were 96.8%, 98.7% and 98.6% for remimazolam, 98.7%, 99.8% and 101.5% for M1, respectively. Selectivity, matrix effect and stability were also validated. The method was applied to the pharmacokinetic study of remimazolam in Chinese healthy subjects.
Keywords: Determination; Remimazolam; Metabolite; Ultra-performance liquid chromatography–tandem mass spectrometry; Human plasma;

Uveitis is a severe autoimmune eye disease that can cause intraocular inflammation even lead to severe vision loss, and the occurrence of uveitis can be closely associated with abnormal expression of proteins. However, the abnormally expressed proteins involved in uveitis are not well identified. Using liquid chromatography–tandem mass spectrometry technique, we examined the alterations in proteomic expression profiling in rat plasma specimens related to experimental autoimmune uveitis (EAU) versus normal samples. In addition, the experimental verification was further performed using enzyme-linked immunosorbent assay (ELISA) for abnormally expressed proteins in EAU rat plasma. The results indicate that 62 proteins were upregulated and 106 proteins were downregulated in plasma from EAU rats compared with those in saline-treated samples. In the meantime, we observed that the plasma level of complement component 3 in EAU rats was upregulated versus saline-treated rats (from 92.32 μg/mL to 168.92 μg/mL), whereas the level of interleukin-1 receptor accessory protein was downregulated (from 1120.97 pg/mL to 798.39 pg/mL), and these results were highly in agreement with those of mass spectrometry determination. Taken together, our results indicate that liquid chromatography–tandem mass spectrometry analysis possesses a good resolution for peptides in plasma, and the findings will provide the baseline plasma dataset for EAU rats and the relevant information can contribute to future studies on the understanding the mechanism of uveitis.
Keywords: Experimental autoimmune uveitis; Rat; Mass spectrometry; Proteomics; Plasma;

Improved determination of malonaldehyde by high-performance liquid chromatography with UV detection as 2,3-diaminonaphthalene derivative by Sara Panseri; Luca Maria Chiesa; Andrea Brizzolari; Enzo Santaniello; Elena Passerò; Pier Antonio Biondi (91-95).
A rapid, specific and simple procedure is proposed for the determination of free malonaldehyde (MA) contained in fish tissue. The method is the optimization of the reaction of MA with 2,3-diaminonaphthalene to afford a naphtodiazepinium ion that present a UV absorption at 311 nm, useful for MA determination by HPLC with UV detection. The reaction proceeds in the presence of 25% acetonitrile at 37 °C in 20 min at pH 2 using 2,4-pentanedione as internal standard. The method has been applied to homogenized samples of canned mackerel fillets that were treated with 2,3-diaminonaphthalene in an acidic aqueous:acetonitrile mixture. The produced naphtodiazepinium ion was extracted in acetonitrile by a salting-out homogeneous liquid–liquid extraction. A standard calibration was carried out in the range 0.625–10 nmol/g. The reliability of the procedure is demonstrated by linearity (r 2  = 0.998), limit of detection (0.16 nmol/g), limit of quantification (0.22 nmol/g), repeatibility (RSD 5.57%), and intermediate precision (RSD 8.92%).
Keywords: Malonaldehyde; HPLC-UV; 2,3-Diaminonaphthalene; Canned mackerel fillet;