Journal of Chromatography B (v.1012-1013, #C)

Microwave-assisted digestion followed by parallel electromembrane extraction for trace level perchlorate detection in biological samples by Hakimu Nsubuga; Chanbasha Basheer; Mohanad Mubashar Bushra; Mohammed Hussain Essa; Mohammed Hussain Omar; Ahsan Mushir Shemsi (1-7).
A simple and parallel electromembrane extraction (pEME) method was developed and used to investigate trace perchlorate ion contamination in seafood. In this method, three different EME units were arranged simultaneously and connected parallel to a single DC power supply. In each unit, the ClO4 ions were electro-kinetically extracted from the microwave digested seafood homogenates into 100 mM NaOH via a supported liquid membrane (1-Hexanol). Influential extraction parameters were carefully investigated. Under optimized conditions, good linearity with a coefficient of determination (R 2) of 0.9949 over a concentration range of 1–125 μg/g was obtained. The limit of detection (LOD) was 0.04 μg g−1. The methods intraday and inter day precision varied between 4.3–5.6% respectively. Mean recoveries were up to 107% (n  = 6, RSD = 0.7–6.8%). This method was applied to different seafood samples to assess its feasibility for real applications and it exhibited an enhanced sample throughput compatible with both microwave and ion chromatography.
Keywords: Microwave assisted digestion; Parallel electromembrane extraction; Enhanced sample throughput analysis; Supported liquid membrane; Seafood samples; Ion chromatography;

Simultaneous detection of flumethasone, dl-methylephedrine, and 2-hydroxy-4,6-dimethylpyrimidine in porcine muscle and pasteurized cow milk using liquid chromatography coupled with triple-quadrupole mass spectrometry by Dan Zhang; Jin-A. Park; Seong-Kwan Kim; Sang-Hyun Cho; Daun Jeong; Soo-Min Cho; Hee Yi; Jae-Han Shim; Jin-Suk Kim; A.M. Abd El-Aty; Ho-Chul Shin (8-16).
HighlightsDisplay OmittedA simple analytical method based on liquid chromatography coupled with triple-quadrupole mass spectrometry was developed for detection of the veterinary drugs flumethasone, dl-methylephedrine, and 2-hydroxy-4,6-dimethylpyrimidine in porcine muscle and pasteurized cow milk. The target drugs were extracted from samples using 10 mM ammonium formate in acetonitrile followed by clean-up with n-hexane and primary secondary amine sorbent (PSA). The analytes were separated on an XBridge™ hydrophilic interaction liquid chromatography (HILIC) column using 10 mM ammonium formate in ultrapure water and acetonitrile. Good linearity was achieved over the tested concentrations in matrix-fortified calibrations with correlation coefficients (R2 ) ≥ 0.9686. Recovery at two spiking levels ranged between 73.62–112.70% with intra- and inter-day precisions of ≤20.33%. The limits of quantification ranged from 2–10 ng/g in porcine muscle and pasteurized cow milk. A survey of market samples showed that none of them contained any of the target analytes. Liquid–liquid purification using n-hexane in combination with PSA efficiently removed the interferences during porcine and milk sample extraction. The developed method is sensitive and reliable for detection of the three target drugs in a single chromatographic run. Furthermore, it exhibits high selectivity and low quantification limits for animal-derived food products destined for human consumption.
Keywords: Flumethasone; dl-Methylephedrine; 2-Hydroxy-4,6-dimethylpyrimidine; Tandem mass spectrometry; Porcine muscle; Milk; Veterinary drugs;

The relationship between chromatographic resolution and amide structure of chiral 2-hydroxy acids as O-(−)-menthoxycarbonylated diastereomeric derivatives on achiral gas chromatography was investigated to elucidate the best diastereomeric conformation for enantiomeric separation of chiral 2-hydroxy acids. Thirteen chiral 2-hydroxy acids were converted into nine different diastereomeric O-(−)-menthoxycarbonylated amide derivatives using the primary, secondary and cyclic amines to achieve complete enantiomeric separation through an achiral column. Each enantiomeric pair of 2-hydroxy acids as O-(−)-menthoxycarbonylated tert-butylamide derivatives was resolved on both the DB-5 and DB-17 columns with resolution factors ranging from 1.7 to 4.8 and 1.7 to 3.4, respectively. The results revealed that the structure of the amide moiety is shown to significantly affect chromatographic resolution. In addition, O-(−)-menthoxycarbonylated tert-butylamide derivatives were shown to be the best diastereomeric conformations for enantiomeric separation of 2-hydroxy acids.When comparing with our previous O-trifluoroacetylated(−)-menthyl ester derivatization method, the present results suggested that size differences between groups attached to the chiral center and conformational rigidity can have stronger effects on resolution than the distance between chiral centers. The elution of R- and S-stereoisomers was affected by the class of amine; i.e., primary, secondary, or cyclic, regardless of the substituents on the amine group, the structure of the 2-hydroxy acid, and the polarity of the column.
Keywords: Enantiomeric separation; 2-Hydroxy acids; (−)-Menthoxycarbonylated tert-butylamides;

Two liquid chromatographic–tandem mass spectrometric (LC–MS/MS) methods have been developed and validated for the quantitative determination of polymyxin B1, polymyxin B2 and polymyxin B1-1 concentrations in human plasma and treated urine. During method development, technical challenges such as the separation of structural isomers polymyxin B1and polymyxin B1-1 and nonspecific binding in urine samples were encountered and overcome. Two automated solid phase extraction methods were used to extract plasma samples (100 μL) and urine samples (200 μL) and the resulting extracts were analyzed using reversed phase LC–MS/MS with an electrospray (ESI) interface and selected reaction monitoring (SRM) in the positive ionization mode. Both methods were validated over a calibration curve range of 5.00–2000 ng/mL with a linear regression and 1/x 2 weighting. The between-run relative standard deviation (%RSD) ranged from 4.5 to 9.5% for the plasma assay and from 1.1 to 7.1% for the urine assay. For the plasma assay, the between-run accuracy ranged from 100.5 to 115.2% of nominal at all QC concentrations including the LLOQ. For the urine assay, the between-run accuracy ranged from 92.0 to 106% of nominal at all QC concentrations including the LLOQ. The extraction recoveries for all polymyxins in both assays were between 54.0 and 64.2%. Long term matrix storage stability for all polymyxins was established at both −20 °C and −70 °C for up to 85 days in human plasma and for up to 55 days in treated human urine. Both assays were used for the measurement of polymyxin B1, polymyxin B2 and polymyxin B1-1 concentrations in human plasma and treated urine for the determination of bioequivalence and toxicokinetic parameters in clinical studies.
Keywords: Polymyxin B1; Polymyxin B2; Polymyxin B1-1; Human plasma and urine; LC–MS/MS; Selected reaction monitoring (SRM);

Affinity purification of egg yolk immunoglobulins (IgY) using a human mycoplasma protein by Xuemei Jiang; Thirumalai Diraviyam; Xiaoying Zhang (37-41).
Egg yolk immunoglobulin (IgY) is a superior functional equivalent to mammalian IgG. However, the preparation of refined and highly purified IgY is still attributed as difficult task. Protein M (a transmembrane protein from human mycoplasma) has been newly demonstrated as an ideal affinity regent for mammalian antibody purification. This study aimed to evaluate the interaction between protein M and IgY. The results showed protein M could be a superior affinity reagent for IgY, scFv as well as IgYΔFc, based on pull down and western blot investigations; in addition, it was found that ∼125 times increase of effective IgY in the elutent was obtained using protein M affinity chromatography column compared with traditional IgY extraction methods. This indicates, the purification strategy of protein M is entirely different to traditional IBPs and the salient purification feature of protein M would be a breakthrough for purifying not only non-mammalian antibodies, but also monoclonal antibodies and engineered antibodies based on variable region.
Keywords: Egg yolk immunoglobulin (IgY); Protein M; Affinity purification;

In this study, a preparative separation method was developed for isolation of eleven polyphenols from water-soluble fraction of Chinese propolis using macroporous absorptive resin (MAR) coupled with preparative high performance liquid chromatography (PHPLC). Water-soluble fraction of Chinese propolis was first “prefractioned” using MAR, which yielded four subfractions. The four subfractions were then isolated by PHPLC with an isocratic elution of methanol–water. Finally, eleven polyphenols were purified from Chinese propolis including caffeic acid, ferulic acid, isoferulic acid, 3,4-dimethoxy cinnamic acid, pinobanksin, caffeic acid benzyl ester, caffeic acid phenethyl ester, apigenin, pinocembrin, chrysin and galangin. The purities of the compounds were determined by HPLC and the chemical structures were confirmed by UV and NMR analysis. The method developed was simple, effective, rapid, scalable and economical, and it was a promising basis for large-scale preparation of multiple components from natural products.
Keywords: Chinese propolis; Water-soluble fraction; Polyphenols; Macroporous absorptive resin; Preparative high performance liquid chromatography;

Direct detection of Mycobacterium tuberculosis in sputum: A validation study using solid phase extraction-gas chromatography–mass spectrometry by Marta P.B. Mourão; Sjoukje Kuijper; Ngoc A. Dang; Elisabetta Walters; Hans-Gerd Janssen; Arend H.J. Kolk (50-54).
Tuberculosis (TB) remains a worldwide health problem, especially in developing countries. Correct identification of Mycobacterium tuberculosis (MTB) infection is extremely important for providing appropriate treatment and care to patients.Here we describe a solid phase extraction-gas chromatography-mass spectrometry method (SPE-THM-GC–MS) for the detection of five biomarkers for M. tuberculosis. The method for classification is developed and validated through the analysis of 112 sputum samples from patients suspected of having TB. Twenty of twenty-five MTB culture-positive sputum samples were correctly classified as positive by our improved SPE-THM-GC–MS method. Eighty-five of eighty-seven MTB culture-negative samples were also negative by SPE-THM-GC–MS. The overall sensitivity of the new SPE-THM-GC–MS method is 80% (20/25) and the specificity is 98% (85/87) compared with culture. The method proved to be reliable and, although complex in principle, easy to operate due to the high degree of automation.
Keywords: Validation; Mycobacterium tuberculosis; Direct detection; Solid Phase Extraction; Gas chromatography–mass spectrometry;

A new analytical method for the determination of ribavirin in chicken muscle using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method and liquid chromatography-tandem mass spectrometry (LC–MS–MS) was developed and validated. Samples were extracted with acidified methanol (methanol:acetic acid, 99:1, v/v). The extract was further purified by QuEChERS method using primary-secondary amine (PSA) and C18. Finally, the extract was dried by nitrogen under 45 °C and reconstituted in water. The separation was performed on a Hypercarb analytical column under a gradient elution. The mobile phase was composed of water buffered with ammonium acetate (2.0 mM) and acetonitrile. The proposed method was validated according to the European Commission Decision 2002/657/EC. The values of the decision limit (CCα) and the detection capability (CCβ) were 1.1 and 1.5 μg/kg, respectively. The mean recoveries of ribavirin ranged from 94.2% to 99.2%. The repeatability (expressed as coefficient of variation, CVr) of the method ranged from 4.5% to 4.9% and the reproducibility (CVR) of the method ranged from 4.8% to 5.4%. The method is demonstrated to be suitable for the determination of ribavirin in chicken muscle in conformity with the current EU performance requirements through validation. The total time required for the analysis of one sample, including sample preparation, was about 45 min.
Keywords: Ribavirin; Chicken muscle; LC–MS/MS; QuEChERS;

UPLC-QTOFMS based metabolomics followed by stepwise partial least square-discriminant analysis (PLS-DA) explore the possible relation between the variations in secondary metabolites and the phylogenetic divergences of the genus Panax by Huy Truong Nguyen; Dong-Kyu Lee; Won Jun Lee; GwangJin Lee; Sang Jun Yoon; Byong-kyu Shin; Minh Duc Nguyen; Jeong Hill Park; Jeongmi Lee; Sung Won Kwon (61-68).
Phylogenetic and metabolomic approaches have long been employed to study evolutionary relationships among plants. Nonetheless, few studies have examined the difference in metabolites within a clade and between clades of the phylogenetic tree. We attempted to relate phylogenetic studies to metabolomics using stepwise partial least squares-discriminant analysis (PLS-DA) for the genus Panax. Samples were analyzed by ultra-performance liquid chromatography–quadrupole time of flight mass spectrometry (UPLC-QTOFMS) to obtain metabolite profiles. Initially, conventional principal component analysis was subsequently applied to the metabolomic data to show the limitations in relating the expression of metabolites to divisions in the phylogenetic tree. Thereafter, we introduced stepwise PLS-DA with optimized scaling methods, which were properly applied according to the branches of the phylogenetic tree of the four species. Our approach highlighted metabolites of interest by elucidating the directions and degrees of metabolic alterations in each clade of the phylogenetic tree. The results revealed the relationship between metabolic changes in the genus Panax and its species’ evolutionary adaptations to different climates. We believe our method will be useful to help understand the metabolite–evolution relationship.
Keywords: Metabolomics; Phylogenetic mapping; Stepwise partial least square-discriminant analysis; Genus Panax; Secondary metabolites;

An alkyl polyglycoside (APG) surfactant was used in ultrasonic-assisted extraction to effectively extract vitexin-2″-O-rhamnoside (VOR) and vitexin (VIT) from Crataegus pinnatifida leaves. APG0810 was selected as the surfactant. The extraction process was optimized for ultrasonic power, the APG concentration, ultrasonic time, soaking time, and liquid–solid ratio. The proposed approach showed good recovery (99.80–102.50% for VOR and 98.83–103.19% for VIT) and reproducibility (relative standard deviation, n  = 5; 3.7% for VOR and 4.2% for VIT) for both components. The proposed sample preparation method is both simple and effective. The use of APG for extraction of key herbal ingredients shows great potential. Ten widely used commercial macroporous resins were evaluated in a screening study to identify a suitable resin for the separation and purification of VOR and VIT. After comparing static and dynamic adsorption and desorption processes, HPD100B was selected as the most suitable resin. After column adsorption and desorption on this resin, the target compounds VOR and VIT can be effectively separated from the APG0810 extraction solution. Recoveries of VOR and VIT were 89.27% ± 0.42% and 85.29% ± 0.36%, respectively. The purity of VOR increased from 35.0% to 58.3% and the purity of VIT increased from 12.5% to 19.9%.
Keywords: Alkyl polyglycoside; Vitexin-2″-O-rhamnoside; Vitexin; Ultrasonic-assisted extraction; Macroporous resin; Crataegus pinnatifida;

Neurotransmitters (NTs) and their metabolites are known to play an essential role in maintaining various physiological functions in nervous system. However, there are many difficulties in the detection of NTs together with their metabolites in biological samples. A new method for NTs and their metabolites detection by high performance liquid chromatography coupled with Q Exactive hybrid quadruple-orbitrap high-resolution accurate mass spectrometry (HPLC–HRMS) was established in this paper. This method was a great development of the applying of Q Exactive MS in the quantitative analysis. This method enabled a rapid quantification of ten compounds within 18 min. Good linearity was obtained with a correlation coefficient above 0.99. The concentration range of the limit of detection (LOD) and the limit of quantitation (LOQ) level were 0.0008–0.05 nmol/mL and 0.002–25.0 nmol/mL respectively. Precisions (relative standard deviation, RSD) of this method were at 0.36–12.70%. Recovery ranges were between 81.83% and 118.04%. Concentrations of these compounds in mouse hypothalamus were detected by Q Exactive LC–MS technology with this method.
Keywords: Q Exactive MS; Quantitative detection; Neurotransmitters (NTs); Mouse; Hypothalamus;

Naphthalene shows carcinogenic properties in animal experiments. As the substance is ubiquitary present in the environment and has a possibly high exposure at industrial workplaces, the determination of naphthalene metabolites in humans is of environmental–medical as well as occupational–medical importance. Here, biomarkers of 1,2- and 1,4-naphthoquinone, as possibly carcinogenic metabolites in the naphthalene metabolism, are of outstanding significance.We developed and validated a liquid chromatography–tandem mass-spectrometric (LC–MS/MS) method for the simultaneous determination of the naphthoquinone mercapturic acids of 1,2- and 1,4-naphthoquinone in human urine samples as a sum of naphthoquinone- and dihydroxynaphthalene-mercapturic acid. Except for enzymatic hydrolysis and acidification, no further sample preparation is necessary. For sample clean-up, a column switching procedure is applied. The mercapturic acids are extracted from the urinary matrix on a restricted access material (RAM RP 18) and separated on a reversed phase column (Synergi Polar RP C18). The metabolites were quantified by tandem mass spectrometry using labelled D5-1,4-NQMA as internal standard. The limits of detection are 3 μg/l for 1,2-NQMA and 1 μg/l for 1,4-NQMA. Intraday- and interday precision for pooled urine (spiked with 10 μg/l and 30 μg/l of the analytes) ranges from 5.9 to 15.1% for 1,2-NQMA and from 2.0 to 10.8% for 1,4-NQMA. The developed method is suited for the sensitive and specific determination of the mercapturic acids of naphthoquinones in human urine. A good precision and low limits of detection were achieved. Application of those new biomarkers in biomonitoring studies may give deeper insights into the mechanisms of the human naphthalene metabolism.
Keywords: Biomarker; Biomonitoring; Naphthalene; Naphthoquinone; Urine; Mercapturic acid;

This research was mainly focused on the effects of food emulsifier on the bioavailability of six priority controlling phthalate acid esters (PAEs) for the further accurate assessment of their toxic effects, using the corresponding phthalate acid monoesters (PAMEs) in rats urine as biomarkers. Glycerin monostearate was chosen as typical food emulsifier. A method was established to determine PAMEs in urine from rats either in experimental group (integrated gavaged with glycerin monostearate and PAEs) or in control group (gavaged with PAEs only), by using solid-phase extraction (SPE) coupled with ultra performance liquid chromatography tandem mass spectrometry (SPE–UPLC–MS/MS). Extraction recoveries were more than 75% for all the PAMEs; the calibration curve was linear in the range of 1.0–1000.0 ng/mL with R 2  > 0.995; the limits of detection (LOD) were 0.30 ng/mL–0.50 ng/mL. In addition, by analysing quality control (QC) urine samples in 3 days, it showed that the method was precise and accurate, for the intra-day and inter-day RSD within 16%, and the accuracy more than 82%. Internal exposure amount of all PAEs in experimental group was significantly higher than that in control group with p values of less than 0.05 except for butyl benzyl phthalates (BBP) (P  = 0.07). The bioavailability of all PAEs ranged from 5.03% to 109.35% with the presence of food emulsifiers glycerin monostearate, observably higher than that without glycerin monostearate (1.12% to 54.39%). It indicated that food emulsifiers increased the bioavailability of PAEs and may lead to potential food safety risk, which should bring awareness and be further studied.
Keywords: Phthalic acid esters; Glycerin monostearate; Bioavailability; Phthalate acid monoesters; Solid-phase extraction; UPLC–MS/MS;

A sensitive and robust method using high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed for quantitation of 13 phytoestrogens and related metabolites in rat serum samples. A new type of column, the Kinetex core–shell C18 column, was applied for rapid separation of the target analytes in 10 min. Two enzymes, sulfatase H-1 and gulcuronidase H-5 from Helix pomatia were compared on the efficiency of releasing the conjugated forms of the target analytes to their free forms in serum samples. The method detection limit (MDL) defined as three times the signal to noise ratio in spiked serum matrix-based solutions was in the range of 0.1–3.5 ng/mL. The linear dynamic calibration was in the broad range of 0.2–500 ng/mL for all target compounds. Thirty-two rat serum samples from the rats that were fed with diets containing either casein or soy protein isolates with various amounts of isoflavones for 8 weeks were analyzed for the target analytes with the developed method. Nine target analytes were detected in the serum samples. Those detectable compounds are all the metabolites of the dietary isoflavones, suggesting that the diet isoflavones were mostly metabolized to their metabolites in rat.
Keywords: High performance liquid chromatography–tandem mass spectrometry; Phytoestrogens and metabolites; Core–shell C18 column; Rapid separation; Rat serum;

Analysis of cocaine/crack biomarkers in meconium by LC–MS by Felipe Bianchini D’Avila; Pâmela C. Lukasewicz Ferreira; Fernanda Rodrigues Salazar; Andrea Garcia Pereira; Maíra Kerpel dos Santos; Flavio Pechansky; Renata Pereira Limberger; Pedro Eduardo Fröehlich (113-117).
Fetal exposure to illicit drugs is a worldwide problem, since many addicted women do not stop using it during pregnancy. Cocaine consumed in powdered (snorted or injected) or smoked (crack cocaine) form are harmful for the baby and its side effects are not completely known. Meconium, the first stool of a newborn, is a precious matrix usually discarded, that may contain amounts of substances consumed in the last two trimesters of pregnancy. Analyzing this biological matrix it is possible to detect the unaltered molecule of cocaine (COC) or its metabolite benzoylecgonine (BZE) and pyrolytic products anhydroecgonine methyl ester (AEME) and anhydroecgonine (AEC). A liquid chromatography mass spectrometry (LC–MS) method was validated for meconium samples after solvent extraction, followed by direct injection of 10 μL. Linearity covered a concentration range of 15 to 500 ng/mg with a lower limit of quantification (LLOQ) of 15 ng/mg for all analytes. Matrix effect was evaluated and showed adequate results. Detection of illicit substances usage can be crucial for the baby, since knowing that can help provide medical care as fast as possible. The method proved to be simple and fast, and was applied to 17 real meconium samples.
Keywords: Meconium; LC-MS; Cocaine/crack; Biomarkers; HILIC column;

Liquid chromatography–tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid–liquid extraction by Rolf W. Sparidans; Stephanie van Hoppe; Johannes J.M. Rood; Alfred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen (118-123).
A quantitative bioanalytical liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out assisted liquid–liquid extraction (SALLE) with acetonitrile, magnesium chloride and a stable isotopically labeled internal standard. After dilution, the extract was directly injected into the reversed-phase liquid chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and compounds were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer.The assay was completely validated for plasma in a 0.5–500 ng/ml calibration range with r 2  = 0.995 ± 0.002 (n  = 6) using linear regression with the inverse square of the concentration as the weighting factor for the calibration. Within-run precisions (n  = 18) were 2.7–11.7% and between-run (3 runs; n  = 18) precisions 3.0–14.5%. Accuracies were between 96–109% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully applied to determine plasma drug levels and study pharmacokinetics after oral administration of afatinib to female FVB mice.
Keywords: Afatinib; LC–MS/MS; Mouse; Plasma; SALLE;

UFLC-Q-TOF/MS based screening and identification of the metabolites in plasma, bile, urine and feces of normal and blood stasis rats after oral administration of hydroxysafflor yellow A by Yi Jin; Liang Wu; Yuping Tang; Yujie Cao; Shujiao Li; Juan Shen; Shijun Yue; Cheng Qu; Chenxiao Shan; Xiaobing Cui; Li Zhang; Jin-ao Duan (124-129).
The dried flower of Carthamus tinctorius L. (honghua) is a widely used traditional Chinese medicine in clinics to treat coronary heart disease, hypertension, and cerebrovascular disease due to its functions of ameliorating circulation and removing blood stasis. Hydroxysafflor yellow A (HSYA) is an active marker component of honghua. In this paper, ultra-flow liquid chromatography coupled with quadrupole-time-of-flight mass-spectrometry (UFLC-Q-TOF/MS) was established and successfully applied to the detection and identification of the metabolites in bile, urine, plasma and feces samples of normal and model rats with orally administrated HSYA. A total of 8 metabolites were observed in normal rats, while 7 metabolites were detected in model rats. The distribution of metabolites in the plasma, bile, urine and feces of normal and model rats had obvious differences. The major in vivo metabolic pathways for HSYA included hydroxylation, hydroxylation + methylation, acetylation and glucuronidation, and there were also dehydration, hydrogenation, hydration, and hydroxylation + glucuronidation. All of these metabolites were reported for the first time, and these results are valuable and important for the understanding of the metabolic process and therapeutic mechanism of HSYA and some other pigments in honghua.
Keywords: Safflower; Honghua; Hydroxysafflor yellow A; Metabolite; UFLC;

Trace analysis of sulforaphane in bee pollen and royal jelly by liquid chromatography–tandem mass spectrometry by Ana M. Ares; Irene Ayuso; José L. Bernal; María J. Nozal; José Bernal (130-136).
In this study, we investigate for the first time the presence of sulforaphane (SFN) residues in two of the most currently consumed food/dietary supplements, royal jelly and bee pollen. Chromatography-tandem mass spectrometry (LC–MS/MS) was the method employed, the mass spectrometer consisting of an ion-trap mass analyzer used with electrospray ionization (ESI) in positive ion mode. An efficient sample treatment involving a solvent extraction with methanol, centrifugation, and concentration in a rotary evaporator was proposed. In all cases average analyte recoveries were between 92 and 106%. Chromatographic analysis (16 min) was performed on a core–shell technology based column (Kinetex C18, 150 × 4.6 mm, 2.6 μm, 100 Å). The mobile phase consisted of 0.02 M ammonium formate in water and acetonitrile, with a flow rate of 0.5 mL/min in gradient elution mode. The fully validated method was selective, linear from 8 to 1000 μg/kg (bee pollen), or from 10 to 1250 μg/kg (royal jelly), precise and accurate; relative standard deviation (% RSD) and relative error (% RE) values were below 10%. Low limits of detection (LOD) and quantification (LOQ) were obtained, namely, 3 μg/kg (LOD) and 8 (bee pollen) and 10 (royal jelly) μg/kg (LOQ). The method was applied for SFN analysis in several royal jelly and bee pollen samples. SFN was detected at trace levels in some bee pollen samples (<23 μg/kg) examined, whilst SFN went undetected in the royal jelly samples analyzed.
Keywords: Bee pollen; Liquid chromatography; Mass spectrometry; Royal jelly; Sulforaphane;

Extraction and purification of IgG by hydrophilic organic solvent salting-out extraction by Yuesheng Dong; Bofeng Pang; Fang Yu; LingLing Li; Wencai Liu; Zhilong Xiu (137-143).
The distribution of IgG in different salting-out extraction systems (SOESs), including ethanol/K2HPO4, Na2CO3, and trisodium citrate, was investigated. The SOESs that were composed of 20% K2HPO4 (w/w)–14% ethanol (w/w), 19% Na2CO3 (w/w)–13% ethanol (w/w) and 25% trisodium citrate (w/w)–19% ethanol (w/w) showed satisfactory results, with IgG recovery rates as high as 97.4%, 93.1%, and 90.2%, respectively. ELISA, CD and IR analyses confirmed the active retention and structural constant of IgG in the K2HPO4–ethanol system. An optimized system that consisted of 14% ethanol (w/w)–20% K2HPO4 (w/w) (pH 7.0) was selected for extracting active plasma proteins directly from pig plasma. As a result, recovery rates as high as 95.7% for IgG and 93.0% for albumin were achieved. In addition, some contaminating proteins were also removed by this system, which may provide a new alternative method for the separation and purification of immunoglobulin.
Keywords: Active retention; Immune globulin; Purification; Recovery; Salting-out extraction systems; Structural constant;

Quantitative structure retention/activity relationships of biologically relevant 4-amino-7-chloroquinoline based compounds by Sandra Šegan; Igor Opsenica; Mario Zlatović; Dušanka Milojković-Opsenica; Bogdan Šolaja (144-152).
The chromatographic behaviour of series of 4-amino-7-chloroquinoline (4,7-ACQ) based compounds was studied by reversed-phase thin-layer chromatography (RPTLC) with binary mobile phases containing water and the organic modifiers, DMSO or acetone. The lipophilicity of the studied compounds was determined by extrapolation of retention parameters R M to pure water content in mobile phase. In order to obtain some basic insight into the chromatographic behaviour and structural features of investigated compounds, PCA was performed on both chromatographic data (R M values) and calculated 2D and 3D structural descriptors. Both QSRR and QSAR models were built by means of the partial least squares (PLS) statistical method. It was found that descriptors which encode hydrophobic (dispersive) interactions have positive influence on retention, while influence of descriptors encoding polar interactions was negative. According to the obtained PLS model for inhibition of botulinum neurotoxin serotype A light chain, hydrophobic interactions influence positively on the mechanism of action of the investigated 4,7-ACQ, while polar interactions are less favoured. Contrary, the results of PLS modelling of activity against Plasmodium falciparum strains (W2, D6 and TM91C235) indicate that higher polarity of 4,7-ACQ contribute to their higher antimalarial activity.
Keywords: 4‑Amino-7-chloroquinoline; Reversed-phase thin-layer chromatography (RPTLC); Lipophilicity; Quantitative structure-retention relationship (QSRR); Quantitative structure activity relationship (QSAR);

Purification of influenza deoxyribonucleic acid-based vaccine using agmatine monolith by D. Bicho; C. Caramelo-Nunes; A. Sousa; F. Sousa; J.A. Queiroz; C.T. Tomaz (153-161).
Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation.
Keywords: Agmatine; Chromatography; Influenza vaccine; Monoliths; pDNA; Transfection;

The counter current salting-out homogenous liquid-liquid extraction (CCSHLLE) joined with the dispersive liquid–liquid microextraction based on solidification of floating organic drop (DLLME–SFO) has been developed as a high preconcentration technique for the determination of different drugs in urine samples. Amphetamines were employed as model compounds to assess the extraction procedure and were determined by high performance liquid chromatography–ultraviolet detection (HPLC–UV). In this method, initially, NaCl as a separation reagent is filled into a small column and a mixture of urine and acetonitrile is passed through the column. By passing the mixture, NaCl is dissolved and the fine droplets of acetonitrile are formed due to salting-out effect. The produced droplets go up through the remained mixture and collect as a separated layer. Then, the collected acetonitrile is removed with a syringe and mixed with 30.0 μL 1-undecanol (extraction solvent). In the second step, the 5.00 mL K2CO3 solution (2% w/v) is rapidly injected into the above mixture placed in a test tube for further DLLME–SFO. Under the optimum conditions, calibration curves are linear in the range of 1–3000 μg L−1 and limit of detections (LODs) are in the range of 0.5–2 μg L−1. The extraction recoveries and enrichment factors ranged from 78 to 84% and 157 to 168, respectively. Repeatability (intra-day) and reproducibility (inter-day) of method based on seven replicate measurements of 100 μg L−1 of amphetamines were in the range of 3.5–4.5% and 4–5%, respectively. The method was successfully applied for the determination of amphetamines in the actual urine samples. The relative recoveries of urine samples spiked with amphetamine and methamphetamine are 90–108%.
Keywords: Counter current salting-out homogenous liquid–liquid extraction; Dispersive liquid–liquid microextraction; Amphetamines; Urine analysis;

An ultra high pressure liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method for the simultaneous quantitation of pravastatin and major metabolites, 3′α-hydroxy-pravastatin, pravalactone and 3′α-hydroxy-pravalactone, in human plasma has been developed and validated. Aliquots of (100 μL) plasma in EDTA were diluted in pH 4.5 (0.1 M buffer) to stabilize the analytes and subjected to hydrophilic lipophilic balance (HLB) solid phase extraction on 96 well μelution plates. Extracted samples were evaporated to dryness and reconstituted in pH 4.5 buffer. Chromatographic separation was performed on a Cortecs™ C18 column (2.1 × 100 mm, 1.8 μm), using gradient elution with a blend of acetonitrile and 10 mM methylammonium acetate buffer (pH 4.5) at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed using multiple reaction monitoring (MRM) switching between positive/negative electrospay ionization (ESI). Pravastatin, 3′α-hydroxy-pravastatin, and internal standards [2H3]-pravastatin, and [2H3]-3′α-hydroxy-pravastatin were monitored in negative ESI mode at ion transitions m/z 423.2 → 321.1 and 426.2 → 321.1, respectively. Positive ESI mode was used for the detection of pravalactone, 3′α-hydroxy-pravalactone, and internal standards [2H3]-pravalactone, and [2H3]-3′α-hydroxy-pravalactone at ion transitions m/z 438.2 → 183.1 and 441.2 → 269.1 respectively. The method was linear for all analytes in the concentration range 0.5–200 nM with intra- and inter-day precisions (as relative standard deviation) of ≤5.2% and accuracy (as relative error) of ≤8.0% at all quality control levels. The method was successfully applied to the investigation of pharmacokinetic properties of pravastatin and its metabolites in children after an oral dose of 20–40 mg.
Keywords: UPLC-MS/MS; Dyslipidemia; Mass spectrometry; Pharmacokinetics; Pediatrics; Pravastatin;

Use of capillary electrophoresis with dual-opposite end injection for simultaneous analysis of small ions in saliva samples from wrestlers undergoing a weight training program by Masanobu Mori; Fumi Ishikawara; Toshihiro Tomoda; Sachiko Yamada; Minako Okamoto; Hideyuki Itabashi; Yoichi Seki; Ryutaro Matsumoto; Yoshifumi Shoho; Larasati Martha; Hiroyuki Sumino; Masami Murakami (178-185).
Capillary electrophoresis–capacitively coupled contactless conductivity detection (CE–C4D), conducted using an in-house-developed polyvinyl alcohol (PVA)-coated capillary system, was applied for the simultaneous analysis of small anions and cations in saliva samples from wrestlers undergoing a weight training program. Use of the PVA capillary for CE provided good reproducible ion separation with minimization of the electroosmotic flow and suppression of protein adsorption onto the capillary wall. Four cations and eight anions were separated in 12 min, using a background electrolyte of 20 mM MES/20 mM histidine and 18-crown-6 ether (pH 6) at 20 kV. The relative standard deviations (n  = 5) of the migration times and peak areas were <1% and <8%, respectively. The detection limit at a signal-to-noise ratio of 3 ranged from 1.6 to 10 μM. Using the optimized CE–C4D system, we investigated the correlations between the concentrations of salivary ions and cortisol, which is commonly used as a stress marker. Analysis of saliva samples from ten wrestlers, who were attempting rapid weight loss before a competition, showed the following trends: (1) all ion concentrations, except for Ca2+, Na+, and Cl, increased between the first and last days of weight loss; (2) Mg2+ increased to 166% (from 0.50 mM to 1.4 mM) between the first and last days of weight loss, being the highest increase of all the ions; and (3) K+, Mg2+, NO3 , and SCN levels were strongly correlated (P  < 0.05) with cortisol. The CE–C4D rapidly produced useful data on saliva ion contents, with good ion recovery as determined by the standard addition method (89–110%).
Keywords: Athlete; Capillary electrophoresis; Ions; Saliva; Stress marker; Weight loss;

Simultaneous detection of five one-carbon metabolites in plasma using stable isotope dilution liquid chromatography tandem mass spectrometry by Antonysunil Adaikalakoteswari; Craig Webster; Ilona Goljan; Ponnusamy Saravanan (186-192).
Disturbance in one-carbon (1-C) cycle occurs due to nutritional deficiencies (vitamin B12/folate) or specific genetic polymorphisms. This leads to altered levels of key 1-C metabolites such as SAM (s-adenosyl methionine), SAH (s-adenosyl homocysteine), methionine, homocysteine and MMA (methyl malonic acid). These 1-C metabolites are determinants of cellular methylation potential and epigenetic modifications of DNA which impairs metabolic pathways in several pathological diseases and developmental programming. Though methods were able to measure these analytes only independently, none of the methods detect simultaneously. Therefore we developed a method to measure these five 1-C metabolites in a single run using liquid chromatography tandem mass spectrometry (LC–MS/MS). We used stable isotopes dilution LC–MS/MS to measure the 1-C metabolites in human plasma. Blood samples were collected from pregnant women (n  = 30) at early gestation in the ongoing, multicentre, prospective PRiDE study. Linearity exhibited across the calibration range for all the analytes with the limit of detection (LOD) of 1.005 nmol/l for SAM, 0.081 nmol/l for SAH, 0.002 μmol/l for methionine, 0.046 μmol/l for homocysteine and 3.920 nmol/l for MMA. The average recovery for SAM was 108%, SAH—110%, methionine—97%, homocysteine—91% and MMA—102%. The inter-assay CV for SAM was 7.3, SAH—5.6%, methionine—3.5%, homocysteine—7.0% and MMA—4.0%. The intra-assay CV for SAM was 8.7%, SAH—4.7%, methionine—5.4%, homocysteine—8.1% and MMA—6.1%. Pregnant women at early gestation with low B12 levels had significantly higher homocysteine, MMA, lower levels of methionine, SAM and SAM:SAH ratio and higher triglycerides. We developed a simple and rapid method to simultaneously quantify 1-C metabolites such as SAM, SAH, methionine, homocysteine and MMA in plasma by stable isotope dilution LC–MS/MS which would be useful to elucidate the epigenetic mechanisms related in the gene–nutrient interactions.
Keywords: Vitamin B12 deficiency; One-carbon metabolism; Epigenetic modifications; Homocysteine;

Sialic acid-specific affinity chromatography for the separation of erythropoietin glycoforms using serotonin as a ligand by M. Meininger; M. Stepath; R. Hennig; S. Cajic; E. Rapp; H. Rotering; M.W. Wolff; U. Reichl (193-203).
Recombinant human erythropoietin (rhEPO) is an important CHO cell-derived glycoprotein and the degree of sialylation of this hormone is crucial for its in vivo bioactivity. In order to improve the purification process serotonin as a potential affinity ligand was tested for preparative chromatographic separation of rhEPO glycoforms into fractions of different degrees of sialylation. Therefore, two chromatographic matrices were prepared by immobilizing serotonin on CNBr- and NHS-Sepharose™. First it was shown both matrices bind rhEPO only in its sialylated form. Results indicate that binding is pH independent between pH 3.5 to 8 suggesting it is not only based on electrostatic interactions. Second, after optimal binding conditions were identified, semi-purified rhEPO was loaded onto both matrices and eluted using a stepwise elution gradient of sodium chloride. For comparison same affinity purification experiments were performed using wheat germ agglutinin-coupled agarose, a lectin known for its affinity towards sialylated glycoproteins. To monitor changes in N-glycan fingerprint, eluate fractions were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (xCGE-LIF). For the serotonin matrices an increasing degree of sialylation was observed from the first to the third elution fraction while purity of rhEPO could be increased at the same time. The late elution fractions of serotonin-coupled CNBr- and NHS-Sepharose™ also showed an overall sialylation degree exceeding that of the starting material. In contrast, for rhEPO bound to wheat germ agglutinin-coupled agarose, no distinct change in the degree of sialylation could be observed after elution. Overall, these encouraging results highlight the potential of serotonin as a chromatographic ligand for the improvement of pharmaceutical purification processes of rhEPO.
Keywords: Erythropoietin Glycoforms; Affinity Chromatography; Serotonin; Sialic Acid; Glycan Analysis; Glycoproteins;

The adsorption kinetics of chymotrypsin, a pancreatic serine protease, onto an alginate-gum guar matrix cross-linked with epichlorohydrin has been performed using a batch-adsorption technique. The effect of various experimental parameters such as pH, salt presence, contact time and temperature were investigated. The pseudo-first-order and pseudo-second-order kinetic models were used to describe the kinetic data which shows that the adsorption of the enzyme followed the pseudo-second-order rate expression. The Langmuir, Freundlich and Hill adsorption isotherm models were applied to describe the equilibrium isotherms, and the isotherm constants were determined. It was found that Hill model was more suitable for our data because the isotherm data showed a sigmoidal behavior with the free enzyme concentration increasing in equilibrium. At 8 °C and at pH 5.0, 1 g hydrate matrix adsorbed about 7 mg of chymotrypsin. In the desorption process 80% of the biological activity of chymotrypsin was recovered under the condition of 50 mM phosphate buffer, pH 7.00–500 mM NaCl. When successive cycles of adsorption/washing/desorption were performed, it was observed that the matrix remained functional until the fourth cycle of repeated batch enzyme adsorption. These results are important in terms of diminishing of cost and waste generation.
Keywords: Alginate; Guar gum; Polyelectrolytes; Chymotrypsin; Adsorption;

Keywords: Arachidonic acid; Biological samples; Eicosanoids; Quantitation; Tandem mass spectrometry; Validation;

Keywords: Arachidonic acid; Biological samples; Eicosanoids; Quantitation; Tandem mass spectrometry; Validation; Correlation; Cortisol;