Journal of Chromatography B (v.1055-1056, #C)

Reversed-phase HPLC with derivatization using o-phthalaldehyde (OPA) and sulfite allows electrochemical detection of γ-aminobutyric acid (GABA) in microdialysis samples. However, OPA/sulfite derivatives have been reported to produce lower fluorescent yield than OPA derivatives using organic thiols as the nucleophile. To overcome this limitation we examined excitation and emission spectra, reaction time, pH, and concentration of reagents in the derivatization solution. Optimal detection parameters were determined as λ ex  = 220 nm and λ em  = 385 nm for maximal fluorescence. The derivatization reaction occurred immediately and the product was stable up to 10 min. A pH of 10.4 for the borate buffer used in the derivatization solution was significantly better than lower pH. Increasing the amount of sulfite combined with diluting the derivatization solution in borate buffer resulted in complete separation of the GABA peak from contaminants without any loss in signal. Controlling the temperature of the detector at 15 °C significantly improved sensitivity with a detection limit of approximately 1 nM. To validate this assay, we performed microdialysis in the dorsal striatum and ventral tegmental area (VTA) of adult Long Evans rats. GABA concentrations in dialysates were determined using external standards and standard additions, in order to further confirm interfering peaks were not present in biological samples. Within the dorsal striatum (n  = 4), basal GABA concentrations were 12.9 ± 2.2 and 14.5 ± 2.2 nM (external and additions, respectively). Respective basal GABA concentrations in the VTA (n  = 3) were 4.6 ± 1.1 and 5.1 ± 0.6 nM. Thus, we have developed a novel, sensitive fluorescence method to determine GABA in microdialysates using HPLC of an OPA/sulfite derivative.
Keywords: Fluorescence; GABA; HPLC; Microdialysis; OPA; Sulfite;

Development of a derivatization method for the quantification of hydrogen sulfide and its application in vascular calcification rats by Xiao-Xin Tan; Kao-Qi Lian; Xiang Li; Nan Li; Wei Wang; Wei-Jun Kang; Hong-Mei Shi (8-14).
Hydrogen sulfide (H2S) plays major functional and structural roles in diverse physiological functions and the pathogenesis of a variety of disorders in biological matrices. The significance of H2S has prompted the development of sensitive and selective methods to determine its concentration in biological samples. The fluorescent reagent monobromobimane (MBB) has been widely used to measure various thiol-containing species through alkylation. MBB may prevent the oxidation of sulfide and the reaction of sulfide with several different species (such as superoxide radicals, hydrogen peroxide and peroxynitrite). An isomers of MBB, 3-(bromomethyl)-2, 6, 7-trimethyl-1H, 5H-pyrazolo [1,2-a] pyrazole-1, 5-dione (MMB), is cheaper than MBB and its use in the analysis of H2S has not previously been reported. In the present study, we compared the derivatization reactions of hydrogen sulfide with MMB and MBB and developed a sensitive method to quantify H2S in blood. In our method, H2S was incubated in the dark with excess MMB in 0.1 M Tris-HCl buffer (pH 10.1) at 50 °C for 120 min. 50 μL aliquots of the derivatized product were analyzed using HPLC system with gradient elution of 0.1% (v/v) formic acid-acetonitrile. The limit of detection for the derivatized product was 0.03 nmol/mL. The derivatization reaction was suitable for detecting low concentrations of H2S. The derivate product is stable over time, permitting batch storage and analysis.
Keywords: Hydrogen sulfide; Monobromobimane; Isomers; Derivatization; HPLC; Vascular calcification;

After ultrasonic-assisted extraction, four lycoris radiata alkaloids: galanthamine, homolycorine, lycorenine, and tazettine were determined by capillary electrophoresis electrochemiluminescence. Polyvinylpyrrolidone was added to the running buffer (RB) to obtain better resolution. Experimental conditions influencing the determination were examined, including the additives, detection potential, separation voltage, injection voltage and time, and RB pH and concentration. Under optimal experimental conditions, the baseline separation of the four alkaloids occurred within 16 min. The proposed method displayed the following linear ranges (in ng/mL): galanthamine [60–5000], homolycorine [40–5000], lycorenine [5.0–1500], and tazettine [8.0–2500]. The detection limits in ng/mL, (S/N = 3), were galanthamine [14], homolycorine [11], lycorenine [1.8], and tazettine [3.1]. Intra-day and inter-day RSDs for the four alkaloids of the six replicates were less than 2.7% and 3.1%, respectively. The recoveries in% were: tazettine [102.5], lycorenine [98.20], galanthamine [97.30], and homolycorine [98.33].
Keywords: Capillary electrophoresis; Electrochemiluminescence; Ultrasonic extraction; Lycoris radiata; Alkaloid;

In this study, we delineated the importance of MS resolving power on the ion-peak quantification of lipids using an Orbitrap Fusion instrument and established a liquid chromatography-based, high-performance lipidomics platform. The ion-peak recognition of several lipids in human plasma, such as LPC(15:0), LPE(22:5), and PC(35:0), was clearly improved by increasing the MS resolving power. In addition, we evaluated the impact of resolving power on the quantitative detection of lipids by automatic ion-peak recognition with calculation of the coefficient of variance (CV). The extracted ions obtained from human plasma were automatically annotated by Compound Discoverer software with manual confirmation of standards or MS2/MS3 fragments (class- and acyl side chain-specific ions and neutral losses). Quantitative evaluation of 499 lipids in human plasma in terms of their CV values clearly demonstrated an improvement in the quantitative performance by enriching the resolving power. Moreover, we evaluated our new lipidomics platform with enriched MS resolving power (setting of 240,000, full width at half maximum at m/z 200). Because automatic annotation by TraceFinder software overlooks several lipid ions, we further manually annotated additional lipid ions, which were confirmed by standards or MS2/MS3 fragments. Eventually, our platform detected 967 lipids encompassing 34 lipid classes, which were confirmed with standards or MS2/MS3 fragments. Of these lipids, 922 scored <20% of the CV values. Taken together, enriching the resolving power improved ion-peak quantification on our novel lipidomics platform, which enabled us to detect broad-spectrum lipids from human plasma.
Keywords: Lipidomics; Mass spectrometry; Orbitrap; Global semi-quantification; Resolving power;

Targeted mass spectrometry: An emerging powerful approach to unblock the bottleneck in phosphoproteomics by Nerea Osinalde; Kerman Aloria; Miren J. Omaetxebarria; Irina Kratchmarova (29-38).
Following the rapid expansion of the proteomics field, the investigation of post translational modifications (PTM) has become extremely popular changing our perspective of how proteins constantly fine tune cellular functions. Reversible protein phosphorylation plays a pivotal role in virtually all biological processes in the cell and it is one the most characterized PTM up to date. During the last decade, the development of phosphoprotein/phosphopeptide enrichment strategies and mass spectrometry (MS) technology has revolutionized the field of phosphoproteomics discovering thousands of new site-specific phosphorylations and unveiling unprecedented evidence about their modulation under distinct cellular conditions. The field has expanded so rapidly that the use of traditional methods to validate and characterize the biological role of the phosphosites is not feasible any longer. Targeted MS holds great promise for becoming the method of choice to study with high precision and sensitivity already known site-specific phosphorylation events. This review summarizes the contribution of large-scale unbiased MS analyses and highlights the need of targeted MS-based approaches for follow-up investigation. Additionally, the article illustrates the biological relevance of protein phosphorylation by providing examples of disease-related phosphorylation events and emphasizes the benefits of applying targeted MS in clinics for disease diagnosis, prognosis and drug-response evaluation.
Keywords: Phosphorylation; Mass spectrometry; Phosphoproteomics; Targeted MS; Disease;

Isolation of new polyacetylenes from the roots of Eurycoma longifolia via high-speed counter-current chromatography by Minglong Wang; Huijuan Zou; Qibin Chen; Jianguo Cao; Haji Akber Aisa; Guozheng Huang (39-44).
Eurycoma longifolia is a tropical plant of diverse applications in folk medicine, which occurs in Southeast Asia. In this study, pre-purified fraction (0.86 g) of the crude extracts from the roots of E. longifolia, was subjected to preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of hexane–ethyl acetate–methanol–water (HEMWat) at a volume ratio of 5:2:5:2 (v/v). Longifolione A (1, 19 mg, purity 96.0%) and longifolione C (3, 317 mg, purity 96.2%), together with longifolione B (2, purity 77.6%) were isolated in one run. The whole mobile and stationary phase was then blown out, concentrated in vacuo, and subjected to second HSCCC purification. Using HEMWat at a volume ratio of 6:1:6:1.2 (v/v), this fraction yielded two more new polyacetylenenes, longifolione D (4, 5 mg purity 94.5%) and longifolione E (5, 33 mg purity 96.3%). All of these five compounds are new natural products and isolated from E. longifolia for the first time. The established protocol for large-scale isolation of these polyacetylenes from E. longifolia was simple, efficient, and economical.
Keywords: Polyacetylene; HSCCC; Eurycoma longifolia; Tongkat Ali; Debilisone; Longifolione;

A sharp, robust, and quantitative method by liquid chromatography tandem mass spectrometry for the measurement of EAD for acute radiation syndrome and its application by Yiwei Zhang; Jian Li; Zhiyun Meng; Xiaoxia Zhu; Hui Gan; Ruolan Gu; Zhuona Wu; Ying Zheng; Jinbin Wei; Guifang Dou (45-50).
17-Ethinyl-3,17-dihydroxyandrost-5-ene (EAD) is an agent designed for the treatment of acute radiation syndrome (ARS). Given its vital role played in the prevention and mitigation of ARS, the development of a sharp, sensitive and robust liquid chromatography tandem mass spectrometry (LC–MS/MS) method to monitor the metabolism of EAD in vivo was crucial. A new method was constructed and validated for the determination of EAD with the internal standard of androst-5-ene-3β,17β-diol (5-AED). The blood samples were precipitated with methanol, centrifuged, from which the supernatant was separated on UPLC with C18 column and eluted in gradient with acetonitrile and Milli-Q water both containing 0.1% formic acid (FA). Quantification was performed by a triple quadrupole mass spectrometer with electro spray ionization (ESI) in multiple reactive monitoring (MRM) positive mode.A good linearity was obtained with R  > 0.99 for EAD within its calibration range from 5 to 1000 ng mL−1 with a lowest limit of quantification (LLOQ) of 5 ng mL−1. Inter- and intra-day accuracy and precision of three levels of quality control (QC) samples were within the range of 15%, while the LLOQ was within 20%. Samples were stable under the circumstances of the experiments. The method was simple, accurate and robust applied to determine the concentrations of EAD in Wistar rat after a single administration of EAD orally at the dose of 100 mg kg−1.
Keywords: LC–MS/MS; 5-AED; EAD; Steroids; ARS; Pharmacokinetics;

To elute or not to elute in immunocapture bottom-up LC–MS by Maren Christin Stillesby Levernæs; Marianne Nordlund Broughton; Léon Reubsaet; Trine Grønhaug Halvorsen (51-60).
Immunocapture-based bottom-up LC–MS is a promising technique for the quantification of low abundant proteins. Magnetic immunocapture beads provide efficient enrichment from complex samples through the highly specific interaction between the target protein and its antibody. In this article, we have performed the first thorough comparison between digestion of proteins while bound to antibody coated beads versus after elution from the beads. Two previously validated immunocapture based MS methods for the quantification of pro-gastrin releasing peptide (ProGRP) and human chorionic gonadotropin (hCG) were used as model systems. The tryptic peptide generation was shown to be protein dependent and influenced by protein folding and accessibility towards trypsin both on-beads and in the eluate. The elution of proteins bound to the beads was also shown to be incomplete. In addition, the on-beads digestion suffered from non-specific binding of the trypsin generated peptides. A combination of on-beads digestion and elution may be applied to improve both the quantitative (peak area of the signature peptides) and qualitative yield (number of missed cleavages, total number of identified peptides, coverage, signal intensity and number of zero missed cleavage peptides) of the target proteins. The quantitative yield of signature peptides was shown to be reproducible in all procedures tested.
Keywords: Protein analysis; Digestion; Biomarker quantification; Magnetic beads; Coverage mapping;

A systematic and critical review on bioanalytical method validation using the example of simultaneous quantitation of antidiabetic agents in blood by Mariana Millan Fachi; Letícia Paula Leonart; Letícia Bonancio Cerqueira; Flavia Lada Degaut Pontes; Michel Leandro de Campos; Roberto Pontarolo (61-71).
A systematic and critical review was conducted on bioanalytical methods validated to quantify combinations of antidiabetic agents in human blood. The aim of this article was to verify how the validation process of bioanalytical methods is performed and the quality of the published records. The validation assays were evaluated according to international guidelines. The main problems in the validation process are pointed out and discussed to help researchers to choose methods that are truly reliable and can be successfully applied for their intended use. The combination of oral antidiabetic agents was chosen as these are some of the most studied drugs and several methods are present in the literature. Moreover, this article may be applied to the validation process of all bioanalytical
Keywords: Bioanalytical method; Validation; Antidiabetic agents; Systematic review;

We described a reference measurement procedure for amino acid (AA) quantification in blood samples based on deproteinization with 5-sulfosalicylic acid (SSA) and an isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry (LC–MS) method. The serum was deproteinized with 15% v/v SSA and the supernatant was injected directly into the LC–MS system without further processing. Compared with the use of other precipitants and water as a control, five model AAs—valine, isoleucine, leucine, tyrosine, and phenylalanine—in the SSA-treated samples showed ionization enhancement as well as stable background signals without significant ion suppression effects. Five analytes were clearly separated within 3 min using gradient elution and ion-pair chromatography of water and acetonitrile containing 0.1% v/v trifluoroacetic acid. The limit of detection range of this method was 2–52 fmol, and the RSDs of accuracy and precision from intra- and inter-day assays were within 2.7%. The method was applied to various blood samples including serum, whole blood and plasma, with no reasonable measurement bias revealed. The quantification accuracy of this method was then assessed using commercially available plasma certified reference material (CRM) for AA, and the results agreed well within certified values. We finally applied this method to the determination of candidate serum CRM. The optimized protocol was found to be suitable for the accurate quantification of five AAs in serum, and may satisfactorily serve as a primary method for AA measurement in various blood matrices.
Keywords: Amino acid; Blood; Reference measurement procedure; Protein precipitation; 5-sulfosalicylic acid; Isotope dilution mass spectrometry;

In allogeneic hematopoietic cell transplantation (HCT) it has been shown that over- or underexposure to conditioning agents have an impact on patient outcomes. Conditioning regimens combining busulfan (Bu) and fludarabine (Flu) with or without clofarabine (Clo) are gaining interest worldwide in HCT. To evaluate and possibly adjust full conditioning exposure a simultaneous analysis of Bu, F-ARA-A (active metabolite of Flu) and Clo in one analytical run would be of great interest. However, this is a chromatographical challenge due to the large structural differences of Bu compared to F-ARA-A and Clo. Furthermore, for the bioanalysis of drugs it is common to use stable isotope labelled standards (SILS). However, when SILS are unavailable (in case of Clo and F-ARA-A) or very expensive, standard addition may serve as an alternative to correct for recovery and matrix effects. This study describes a fast analytical method for the simultaneous analysing of Bu, Clo and F-ARA-A with liquid chromatography-tandem mass spectrometry (LC–MS/MS) including standard addition methodology using 604 spiked samples. First, the analytical method was validated in accordance with European Medicines Agency guidelines. The lower limits of quantification (LLOQ) were for Bu 10 μg/L and for Clo and F-ARA-A 1 μg/L, respectively. Variation coefficients of LLOQ were within 20% and for low medium and high controls were all within 15%. Comparison of Bu, Clo and F-ARA-A standard addition results correspond with those obtained with calibration standards in calf serum. In addition for Bu, results obtained by this study were compared with historical data analysed within TDM. In conclusion, an efficient method for the simultaneous quantification of Bu, Clo and F-ARA-A in plasma was developed. In addition, a robust and cost-effective method to correct for matrix interference by standard addition was established.
Keywords: Busulfan; Clofarabine; Fludarabine; LC–MS/MS; Standard addition;

Development and validation of a UHPLC-ESI-MS/MS method for the simultaneous quantification of mammal lysophosphatidylcholines and lysophosphatidylethanolamines in serum by Susana Suárez-García; Lluís Arola; Aïda Pascual-Serrano; Anna Arola-Arnal; Gerard Aragonès; Cinta Bladé; Manuel Suárez (86-97).
Recent investigations based on non-targeted metabolomics have proposed lysophospholipids (Lyso-PLs) as biomarkers of different diseases. In particular, lysophosphatidylcholines (Lyso-PCs) and lysophosphatidylethanolamines (Lyso-PEs) have been associated with serious lipid pathologies. Methods to determine the different molecular species in a biological sample and to quantify even less abundant species are required for the evaluation of the Lyso-PL pattern as a novel comprehensive biomarker of dyslipidemia. This study describes the development and validation of an ultra-high-performance liquid chromatography coupled to tandem mass spectrometry assay for the determination of a large number of Lyso-PCs and Lyso-PEs in biological samples. The method was validated in rat serum using two simple methanol-based extractions with low sample volumes (5–50 μL) that covered the wide concentration range of these metabolites. In total, thirty-one Lyso-PLs were separated and quantified with low method limits of detection and quantification, reaching values of 0.2 and 0.8 nM, respectively. The method was subsequently applied in the identification of Lyso-PL-related changes produced by the chronic intake of a cafeteria diet. The results showed alterations in the majority of Lyso-PCs and Lyso-PEs in rat serum. Furthermore, multivariate analysis indicated that the comprehensive evaluation of serum Lyso-PLs could be an excellent indicator of the nutritional phenotype associated with an increased risk of lipid disorders.
Keywords: Ultra-high-performance liquid chromatography–tandem mass spectrometry; Glycerophospholipid; Lysophosphatidylcholine; Lysophosphatidylethanolamine; Biomarker; Cafeteria diet;

Sensitive analysis and pharmacokinetic study of epalrestat in C57BL/6J mice by Jingqiu Huang; Runbin Sun; Siqi Feng; Jun He; Fei Fei; Haoxue Gao; Yuqing Zhao; Yue Zhang; Huilin Gu; Jiye Aa; Guangji Wang (98-103).
Epalrestat is clinically applied for the management of diabetic peripheral neuropathy, yet its pharmacokinetic properties are not well understood. In this study, a rapid and sensitive LC–MS/MS method was established for assaying epalrestat in bio-samples of mice. The method was validated and it showed a good linearity over the range of 2–5000 ng/mL, a precision of less than 12.3%, and recovery and matrix effects of 112.5–123.6% and 87.9–89.5%, respectively. After administration of a single dose of epalrestat administered, the exposure level of AUC0–∞ was positively dose-dependent and the mean C max, AUC0–12 h, T 1/2, and MRT were 36.23 ± 7.39 μg/mL, 29,086.5 μg/L h, 1.2 h and 1.8 h, respectively. Epalrestat was highly exposed in stomach, intestine, liver and kidney, and only a small amount was detected in brain, urine and feces. Multi-dose of epalrestat significantly increased MRT and apparent volume of distribution (V d) relative to those of a single-dose.
Keywords: LC–MS/MS; Epalrestat; Pharmacokinetics; Tissue distribution; Excretion;

Purification of β-mannanase derived from Bacillus subtilis ATCC 11774 using ionic liquid as adjuvant in aqueous two-phase system by Nur Fazrin Husna Abdul Aziz; Sahar Abbasiliasi; Hui Suan Ng; Pongsathon Phapugrangkul; Mohamad Hafizi Abu Bakar; Yew Joon Tam; Joo Shun Tan (104-112).
The partitioning of β-mannanase derived from Bacillus subtilis ATCC 11774 in aqueous two-phase system (ATPS) was studied. The ATPS containing different molecular weight of polyethylene glycol (PEG) and types of salt were employed in this study. The PEG/salt composition for the partitioning of β-mannanase was optimized using response surface methodology. The study demonstrated that ATPS consists of 25% (w/w) of PEG 6000 and 12.52% (w/w) of potassium citrate is the optimum composition for the purification of β-mannanase with a purification fold (PF) of 2.28 and partition coefficient (K) of 1.14. The study on influences of pH and crude loading showed that ATPS with pH 8.0 and 1.5% (w/w) of crude loading gave highest PF of 3.1. To enhance the partitioning of β-mannanase, four ionic liquids namely 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4), 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim]BF4), 1-butyl-3-methylimidazolium bromide ([Bmim]Br), 1-ethyl-3-methylimidazolium bromide ([Emim]Br) was added into the system as an adjuvant. The highest recovery yield (89.65%) was obtained with addition of 3% (w/w) of [Bmim]BF4. The SDS-PAGE analysis revealed that the β-mannanase was successfully recovered in the top phase of ATPS with the molecular size of 36.7 kDa. Therefore, ATPS demonstrated a simple and efficient approach for recovery and purification of β-mannanase from fermentation broth in one single-step strategy.
Keywords: β-Mannanase; Aqueous two-phase system; Polyethylene glycol; Ionic liquid; Purification;

Simplified HPLC methodology for quantifying biological pterins by selective oxidation by Cristian Valdés; Diego Arauna; Daniel González; Jorge Villaseñor (113-118).
Tetrahydrobiopterin (BH4) has become a potential therapeutic tool to treat cardiovascular diseases, since it is an essential cofactor of nitric oxide synthase. In order to quantify the amount of BH4 and its related biopterins, a procedure that involves differential oxidation is currently used, which measures biopterin (the product of the oxidation of BH4 and BH2) at two different pH conditions to calculate the quantity of BH2 and BH4, using high performance liquid chromatography (HPLC).In this work, a method was established in order to quantify BH4 and BH2 by adapting previously described procedures. Several chromatographic conditions were evaluated to define the most convenient methodology. Four types of mobile phases and two different analytical columns were used for HPLC. Additionally, calibration curves were made in acid and basic pH compatible with the differential oxidation method. Each method was suitable for quantification purposes, but the choice was based on an economic factor. The selected condition was a mobile phase of 95% water/5% methanol using a C18 column at 35 °C at a flow rate of 0.9 mL/min. Then, it was calculated the recovery rate, which was about 80% using the chosen method.The aim of this work was to establish a simplified method of differential oxidation, compatible with matrixes such as cardiac tissue in order to facilitate the assessment of the BH4/BH2 ratio in biological samples.
Keywords: Liquid chromatography; Biopterins; Tetrahydrobiopterin; Heart; Nitric oxide synthase; Oxidation;

Yejuhua (YJH) injection is a traditional Chinese medicine (TCM) injection and has a widely application in clinical practice. However, adverse drug reactions (ADRs) caused by YJH injection, majorly manifested as allergic reactions, have been reported. Hence, Effective and practical method for allergen screening and identification is needed. In this work, a LAD2/CMC model coupled online with HPLC-IT-TOF-MS system was developed to screen, analyze, and identify the allergenic components of YJH injection. A fraction was retained on the LAD2/CMC column, and identified as linarin (LN). Histamine release assay was performed by the multiple reaction monitoring (MRM) method of UPLC-ESI–MS/MS. Results showed that YJH injection and LN were in accord with their allergic effects by increasing histamine release. In conclusion, the LAD2/CMC-HPLC-IT-TOF-MS system developed in this study could be used to screen allergenic components in complex systems.
Keywords: LAD2 cell membrane chromatography; HPLC-IT-TOF-MS; Allergic components; Linarin; Yejuhua injection;

A liquid chromatography–tandem mass spectrometry method to measure fatty acids in biological samples by Milene Volpato; Jade A. Spencer; Amanda D. Race; Alessandra Munarini; Andrea Belluzzi; Andrew J. Cockbain; Mark A. Hull; Paul M. Loadman (125-134).
As pre-clinical and clinical research interest in ω-3 polyunsaturated fatty acids (PUFA) increases, so does the need for a fast, accurate and reproducible analytical method to measure fatty acids (FA) in biological samples in order to validate potential prognostic and predictive biomarkers, as well as establishing compliance in ω-3 PUFA intervention trials. We developed a LC–ESI-MS/MS method suitable for high throughput development to measure FAs and validated it in the context of treatment with the ω-3 PUFA eicosapentaenoic acid (EPA). Uniquely we directly compared the LC–ESI-MS/MS method to a GC–MS protocol. We demonstrated the LC–ESI-MS/MS method is accurate and reproducible, with coefficients of variation consistently below 15% for each PUFA analysed. The relative FA content values correlated well with those obtained by GC–MS (r 2  = 0.94, p  < 0.001 for EPA) in vitro. The data obtained following analysis of FA content of liver tissues from mice fed an eicosapentaenoic acid enriched diet showed similar results to that of published studies in which GC–MS was used. The LC–ESI-MS/MS method allows concomitant analysis of unesterified (free, unbound) and esterified (bound) FAs in biological samples, allowing investigation of different PUFA pools in cells and tissues.
Keywords: Cancer; Mass spectrometry; Omega-3 fatty acids; Eicosapentaenoic acid;

Wine-processing, which is sauteing with rice wine, will change the inclination and direction of herbs’ actions. After being wine-processed, the effects of nourishing liver and kidney of Dipsacus asper will be strengthened. However, the underlying mechanism remains elusive. The following study is to establish and validate an UHPLC–MS/MS approach to determine six bioactive constituents in tissue samples, including loganin, loganic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid, 4-caffeoylquinic acid and asperosaponin VI and apply the approach to a comparative tissue distribution study of raw and wine-processed Dipsacus asper in rats. A Shimadzu UHPLC system coupled with triple quadrupole mass spectrometer was employed for analysis of the six analytes using multiple reaction monitoring (MRM) mode. A one-step protein precipitation by methanol was employed to extract the six analytes from tissues. Chloramphenicol and glycyrrhetinic acid were selected as internal standards. The proposed approach was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery. Our results revealed that all of the calibration curves displayed good linear regression (r2  > 0.9991). Intra- and inter-assay variability for all analytes ranged from −4.62 to 4.93% and from −4.98 to 4.92%, respectively. The recovery rates for each analytes were determined to be 88.3–100.1%. All the samples showed satisfactory precision and accuracy after various stability tests, including storage at 25 °C for 4 h, −80 °C for 30 days, three-freeze-thaw cycles, and 4 °C for 24 h. Tissue pharmacokinetic parameters including AUC 0-t, t 1/2, T max and C max were calculated. Collectively, the parameters of C max and AUC 0-t of the six analytes in wine-processed group were remarkably elevated (p <  0.05) in the rat liver and kidney as compared with those of the raw group. But in the rat heart and spleen, the C max and AUC 0-t of asperosaponin VI was decreased as compared with those of the raw group. The accumulation of bioactive constituents in liver and kidney tissues after wine-processing will contribute to the enhancement of liver and kidney nourishing effects.
Keywords: Dipsacus asper; Wine-processed; Tissue distribution; UHPLC–MS/MS;

Mitochondrial membrane fragments from human platelets and monkey skeletal muscles were successfully immobilized onto immobilized artificial membrane chromatographic support for the first time, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. These columns were validated by characterization of translocator protein (TSPO), where multiple concentrations of dipyridamole were run and the binding affinities (K d) determined. Further, the relative ranking data of TSPO ligands was consistent with previously reported rankings for both, the platelet (MMAC-Platelet) and the skeletal muscle (MMAC-Muscle) column (dipyridamole > PK11195 > protoporphyrin IX > rotenone). The functional immobilization of the F-ATPase/ATP synthase was demonstrated on MMAC-Muscle column. Online hydrolysis of ATP to ADP and synthesis of ATP from ADP were both demonstrated on the MMAC-Muscle column. Hydrolysis of ATP to ADP was inhibited by oligomycin A with an IC50 of 40.2 ± 13.5 nM (∼60% reduction in ATP hydrolysis, p < 0.001), similar to previously reported values. Additionally, the Michaelis-Menten constant (Km) for ADP was found to be 1525 ± 461 μM based on the on column dose-dependent increase in ATP production.
Keywords: Mitochondrial membrane affinity chromatography; Translocator protein; ATPase; ATP synthase; Platelets; Skeletal muscle;

Production of pure fractions of immunoglobulin G subclass autoantibodies against tumor necrosis factor by Fedor D. Kireev; Julia A. Lopatnikova; Sergey V. Sennikov (149-157).
Autoantibodies directed against cytokines are important effector molecules regulating the biological activity of cytokines. There is experimental evidence indicating that autoantibodies belonging to different immunoglobulin G (IgG) subclasses may have different functional activity. The purpose of this work was to develop a protocol for the purification of fractions of IgG subclass antibodies directed against tumor necrosis factor (TNF). We developed a series of steps, including gel filtration, positive and negative affinity chromatography, and ultrafiltration, to achieve this goal. Our protocol purified IgG subclass autoantibodies directed against TNF from a human immunoglobulin preparation. The isolation of these anti-TNF autoantibodies will enable evaluation of the effect of TNF-specific antibodies on TNF biological activity. Our newly developed technique for purifying subclasses of anti-TNF autoantibodies may be important for both basic research on the functional activity of these autoantibodies and for clinical immunology.
Keywords: Autoantibodies; Immunoglobulin G; Subclasses; Chromatography; Tumor necrosis factor;

Preparative separation of monoclonal antibody aggregates by cation-exchange laterally-fed membrane chromatography by Pedram Madadkar; Rahul Sadavarte; Michael Butler; Yves Durocher; Raja Ghosh (158-164).
Cation exchange (CEX) chromatography is widely used for large-scale separation of monoclonal antibody (mAb) aggregates. The aggregates bind more strongly to CEX media and hence elute after the monomeric mAb in a salt gradient. However, monomer-aggregate resolution that is typically obtained is poor, which results in low product recovery. In the current study we address this challenge through the use of cation-exchange laterally-fed membrane chromatography (LFMC). Three different LFMC devices, each containing a bed of strong cation-exchange (S) membranes were used for preparative-scale removal of mAb aggregates. Trastuzumab (IgG1) biosimilar derived from human embryonic kidney 293 (293) cells was used as the primary model mAb in our study. The other mAbs investigated were Chinese hamster ovary (CHO) cell line derived Alemtuzumab (Campath-1H) and a heavy chain chimeric mAb EG2-hFc. In each of these case-studies, aggregates were well-resolved from the respective monomer. The separated and collected monomer and aggregate fractions were analyzed using techniques such as hydrophobic interaction membrane chromatography (HIMC), native polyacrylamide gel electrophoresis (or PAGE), and size-exclusion high-performance liquid chromatography (SE-HPLC). The high efficiency of separation obtained in each case was due to a combination of the small membrane pore size (3–5 μm), and the use of LFMC technology, which has been shown to be suitable for high-resolution, multi-component protein separations. Also, the LFMC based separation processes reported in this study were more than an order of magnitude faster than equivalent resin-based, cation exchange chromatography.
Keywords: Monoclonal antibody; Aggregates; Membrane chromatography; Device; Protein purification; Bioseparation;

Dynamic lipid profile of hyperlipidemia mice by Yu-Lian Chen; Chuan-Hao Xiao; Zhi-Xiong Hu; Xiao-Shan Liu; Zhiguo Liu; Wei-Nong Zhang; Xiu-Ju Zhao (165-171).
Biomarkers of serum fatty acids in hyperlipidemia need to be elucidated. 90 SPF KM male mice were randomly divided into 18 groups (n = 5/group), control groups, and high fat diet (HFD) groups at 9 time points. On day 7, 10, 15, 18, 21, 24, 28, 31, and 35, the mice were sacrificed; blood was collected into tubes from the eyes, serum samples for clinical biochemistry assays and gas chromatography–mass spectroscopy were attained after centrifugation, and the contents of serum fatty acids were detected with GC–MS. Sections of livers were taken and stored in formalin solution for histological assessments. No species differences existed in all these groups. The contents of C16:1, C18:1, C22:6 were significantly different between HFD groups and the corresponding controls; meanwhile, the proportion of fatty acids, especially the monounsaturated degree, the polyunsaturated degree, changed significantly and regularly (P <  0.05). Thus the three unsaturated fatty acids C16:1, C18:1, C22:6 and the monounsaturated/polyunsaturated unsaturated degrees may be as potential biomarkers of hyperlipidemia.
Keywords: Hyperlipidemia; Biomarkers of serum fatty acids; GC–MS;

Liquid chromatography–tandem mass spectrometry quantification of acetamiprid and thiacloprid residues in butterbur grown under regulated conditions by Han Sol Lee; Sung-Woo Kim; A.M. Abd El-Aty; Hyung Suk Chung; Md. Humayun Kabir; Md. Musfiqur Rahman; Ji Hoon Jeong; Byung-Joon Chang; Ho-Chul Shin; Jae-Han Shim (172-177).
Display OmittedAn analytical method was developed to quantify the residual levels of the neonicotinoid insecticides, acetamiprid and thiacloprid, in field-incurred butterbur samples using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Samples were extracted with acetonitrile and partitioned with dichloromethane. After partitioning, purification was conducted using a Florisil® cartridge. Linearity of a matrix-matched calibration curve of the two compounds over a concentration range of 0.004–0.4 μg/g was excellent, with determination coefficients (R2 ) ≥ 0.9998. The limits of detection (LOD) and quantitation (LOQ) for both acetamiprid and thiacloprid were 0.0006 and 0.002 mg/kg, respectively. The average recoveries for acetamiprid and thiacloprid at two spiking levels (0.02 and 0.1 mg/kg, i.e., 10 × LOQ and 50 × LOQ) were between 78.23 to 82.17%, with relative standard deviations (RSDs) ≤ 7.22%. The method was successfully applied to field-incurred samples treated with a commercial pesticide product, either once (zero or 7 days before harvest) or twice (0 and 7, 7 and 14, or 14 and 21 days before harvest). The highest and lowest residues were obtained for the 7 and 0 days’ treatment and the 21 and 14 days’ treatment, respectively. The developed method is simple and accurate and can be extrapolated to other leafy vegetables.
Keywords: Residual levels; Acetamiprid; Thiacloprid; Liquid chromatography–tandem mass spectrometry; Leafy vegetables;

A method to determine the lignin monomers (p-hydroxybenzaldehyde, vanillin and syringaldehyde) in plant cell wall of wheat internode was developed and validated using a high-throughput nitrobenzene oxidation step and ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) for quantification. UPLC analyses were carried out using an reversed phase C18 column (ACQUITY UPLC BEH, 1.7 μm, 2.1 × 100 mm) and gradient elution with water and acetonitrile. This method was completely validated in terms of analyzing speed, linearity, sensitivity, limits of detection (LODs) and limits of quantification (LOQs).The three lignin monomers were successfully separated within 6 min and only 2 min were required to regain its equilibrium. The method linearity with regression coefficients values (R2) greater than 0.997. Additionally, LODs ranged from 0.21 to 0.89 μg L−1 and LOQs ranged from 0.69 to 2.95 μg L−1. The applicability of this analytical approach for determining the three lignin monomers was confirmed by the successful analysis of real samples of wheat stem internodes. The nitrobenzene oxidation method was used for the analysis of lignin monomers. We have optimized the treatment temperature (170 °C, 1 h) and realized the high-throughput using the microwave digestion instrument. Recovery of this extraction method ranged from 68.4% to 77.7%. The analysis result showed that the guaiacyl unit (G) was the major component of lignin and there was a higher content of the syringyl unit (S) than that of the hydroxybenzyl unit (H).
Keywords: UPLC–MS/MS; Nitrobenzene oxidation; Lignin monomer; Wheat straw;