Journal of Chromatography B (v.1041-1042, #C)

Identification of novel psychoactive substances 25B-NBOMe and 4-CMC in biological material using HPLC-Q-TOF-MS and their quantification in blood using UPLC–MS/MS in case of severe intoxications by Marek Wiergowski; Justyna Aszyk; Michał Kaliszan; Kamila Wilczewska; Jacek Sein Anand; Agata Kot-Wasik; Zbigniew Jankowski (1-10).
This paper describes cases of poisoning caused by new psychoactive substances such as: 25B-NBOMe (2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine) and 4‐CMC (1-(4-chlorophenyl)-2-(methylamino)-1-propanone). The analytical procedure includes rapid and selective method for the extraction and determination of 4-CMC and 25B-NBOMe in blood samples using UPLC–MS/MS technique. To the best of our knowledge, this is the first report, that involves a fully validated method for quantification of new-designer drug – 4-CMC in postmortem blood samples. The biological material was also analyzed with the use of routine analytical methods: immunochemical techniques, gas chromatography with flame ionization detection and gas chromatography with electron impact mass spectrometry. The results of real samples analyses correspond to possible toxicological effects: death resulting from 25B-NBOMe - mediated hallucinations (661 ng/mL of 25B‐NBOMe and 0.887 ng/mL of 4-CMC), fatal overdose of 25B-NBOMe and 4-CMC (66.5 ng/mL of 25B‐NBOMe and 2.14 ng/mL of 4-CMC) and non-fatal intoxication of these drugs (38.4 ng/mL of 25B NBOMe and 0.181 ng/mL of 4-CMC). Additionally, O-demethylathed O, O-bis-demethylathed and glucuronidated metabolites of 25B-NBOMe in biological specimens were detected.
Keywords: 25B-NBOMe; 4-CMC; Quadrupole time of flight mass spectrometry; Tandem mass spectrometry; Novel psychoactive substances; Drugs;

Genipin-1-o-glucuronic acid and genipin-monosulfate are two major metabolites from geniposide and genipin. Based on diabetic rat model, we developed a simultaneous quantification method to investigate their comparative pharmacokinetics by online mircrodialysis-ultra performance liquid chromatography–mass spectrometry (MD-UPLC–MS/MS) without their standard compounds. Online microdialysis sampling could avoid unexpected contamination or degradation of the analytes during the storage and transfer steps. Combined with good sensitivity, selectivity and selectivity of UPLC–MS/MS, online MD-UPLC–MS/MS method could real-timely monitor metabolites in rat blood for quantitative analysis. Our research found that AUC0→t of genipin-1-o-glucuronic acid and genipin-monosulfate in blood of diabetic group were 17.68 and 7.58 times than those in normal group, respectively, and AUC0→t of genipin-1-o-glucuronic acid was 2.28 times than that of genipin-monosulfate in blood of diabetic group, which revealed the effect of diabetes on the pharmacokinetic properties of the two metabolites. This study not only provides an approach for pharmacokinetic studies for various metabolites from herb medicines, but also can predict druggability of their bioactive metabolites. The insight obtained should facilitate drug development and toxicity research.
Keywords: Metabolites; Comparative pharmacokinetics; Diabetic rat model; Online mircrodialysis; Mass spectrometry;

Method development and validation for the quantitation of the complement inhibitor Cp40 in human and cynomolgus monkey plasma by UPLC-ESI-MS by Alexandra Primikyri; Malvina Papanastasiou; Yiannis Sarigiannis; Sophia Koutsogiannaki; Edimara S. Reis; Joel V. Tuplano; Ranillo R.G. Resuello; Bo Nilsson; Daniel Ricklin; John D. Lambris (19-26).
Cp40 is a 14-amino acid cyclic analog of the peptidic complement inhibitor compstatin that binds with sub-nanomolar affinity to complement component C3 and has already shown promise in various models of complement-related diseases. The preclinical and clinical development of this compound requires a robust, accurate, and sensitive method for quantitatively monitoring Cp40 in biological samples. In this study, we describe the development and validation of an ultra-high performance liquid chromatography electrospray mass spectrometry method for the quantitation of Cp40 in human and non-human primate (NHP) plasma. Isotope-labeled Cp40 was used as an internal standard, allowing for the accurate and absolute quantitation of Cp40. Labeled and non-labeled Cp40 were extracted from plasma using reversed phase-solid phase extraction, with recovery rates exceeding 80%, indicating minor matrix effects. The triply charged states of Cp40 and isotope-labeled Cp40 were detected at m/z 596.60 and 600.34, respectively, via a Q-TOF mass spectrometer and were used for quantitation. The method was linear in the range of 0.18–3.58 μg/mL (r 2  ≥ 0.99), with precision values below 0.71% in NHP and 0.77% in human plasma. The accuracy of the method ranged from −2.17% to 17.99% in NHP and from −0.26% to 15.75% in human plasma. The method was successfully applied to the quantitation of Cp40 in cynomolgus monkey plasma after an initial intravenous bolus of 2 mg/kg followed by repetitive subcutaneous administration at 1 mg/kg. The high reproducibility, accuracy, and robustness of the method developed here render it suitable for drug monitoring of Cp40, and potentially other compstatin analogs, in both human and NHP plasma samples during pharmacokinetic and pharmacodynamic studies.
Keywords: Compstatin; Absolute quantitation; UPLC-ESI-MS; Method validation; Plasma;

In this work, a series of magnetic and nonmagnetic agarose matrices were fabricated for protein purification. Certain amounts of Fe3O4 nanoparticles were encapsulated in agarose beads to form composite magnetic matrices with enhanced purification efficiency. Structure and morphology of prepared matrices were studied by optical and scanning electron microscopes, FT-IR, and BET-BJH analysis. The prepared matrices had regular spherical shape, followed by a uniform size distribution. By nanoparticles addition, the number of mesopores decreased while population of pores with radius ≤10 nm increased; thus, higher specific area achieved. According to VSM results, magnetization degree was one of the characteristics affected by agarose content of the beads. A dye ligand, Cibacron Blue F3GA (CB), was covalently bound to beads to adsorb Bovine serum albumin. CB concentration was determined by elemental analysis. It was shown that magnetic beads hold higher CB concentrations than nonmagnetic ones due to higher specific area. As a result, magnetic 8%-agarose beads had the highest affinity adsorption capacity in static experiments. Moreover, breakthrough curves were monitored to calculate dynamic binding capacity. And, it was shown that magnetic 4%-agarose had the highest adsorbing amount (6.00 mg/mL). It was implied that pore diffusion in magnetic 4%-agarose may be the reason for higher dynamic capacity. Plus, column efficiency was evaluated. It was revealed that all magnetic beads had lower HETP (0.11, 0.12 and 0.11 cm for magnetic 4, 6, and 8%-agarose beads) than nonmagnetic ones (P-value < 0.05).
Keywords: Agarose beads; Adsorption capacity; BSA; BET-BJH analysis; Cibacrone blue F3GA; Elemental analysis; Fe3O4 nanoparticles; HETP; Magnetization; Spatial hindrance;

Development of an extraction method and LC–MS analysis for N-acylated-l-homoserine lactones (AHLs) in wastewater treatment biofilms by Jinfeng Wang; Lili Ding; Kan Li; Wilhelm Schmieder; Jinju Geng; Ke Xu; Yan Zhang; Hongqiang Ren (37-44).
N-Acylated-l-homoserine lactones (AHLs) play a vital role in Gram-negative bacteria communication by promoting the formation of extracellular polymeric substances (EPS) and biofilms. However, the low concentration of these AHL signals makes the process difficult to understand. A robust and sensitive pretreatment method for AHL detection was developed in this work. Compared with eight different solid-phase extraction (SPE) columns and three various solid extraction method, we found that the UE (ultrasonic extraction) and an Oasis hydrophilic-lipophilic-balanced (HLB) sorbent in column format combined with ultra-performance liquid chromatography linked to tandem mass spectrometry (UPLC–MS/MS) can be successfully used for systematic pretreating moving bed biofilm reactor (MBBR) biological samples to extract AHLs and determine concentration of AHLs in wastewater treatment biofilms. This easy-to-follow protocol makes it ideal for quantitative analyses of AHLs in wastewater treatment biofilms.
Keywords: N-Acylated-l-homoserine lactones (AHLs); Ultrasonic extraction (UE); Solid phase extraction (SPE); Quorum sensing (QS);

The LC–MS-based metabolomics of hydroxytyrosol administration in rats reveals amelioration of the metabolic syndrome by Nikolaos Lemonakis; Hemant Poudyal; Maria Halabalaki; Lindsay Brown; Anthony Tsarbopoulos; Alexios-Leandros Skaltsounis; Evagelos Gikas (45-59).
Hydroxytyrosol (HT), an important component of olive fruit and olive oil, improves the signs of metabolic syndrome in rats following chronic treatment. At a dose of 20 mg/kg/day, HT decreased adiposity and improved cardiovascular and liver structure and function in rats fed with a high-carbohydrate, high-fat diet. An untargeted metabolomics approach has been employed using both UPLC-Orbitrap and -QqTOF methods to identify the changes induced by chronic HT administration on the plasma metabolome. 31 metabolites have been found to be differentially expressed between the examined groups. HT was shown to decrease biosynthesis of unsaturated fatty acids, fatty acid biosynthesis, and the metabolism of linoleic acid, retinol, sphingolipids and arachidonic acid, whereas glycerolipid metabolism is up-regulated. These are plausible mechanisms for the attenuation by HT of cardiovascular, liver and metabolic changes in high-carbohydrate, high-fat diet fed rats.
Keywords: Hydroxytyrosol; Metabolomics; Metabolic pathway analysis; Metabolic syndrome;

Comparison and evaluation of antimuscarinic and anti-inflammatory effects of five Bulbus fritillariae species based on UPLC-Q/TOF integrated dual-luciferase reporter assay, PCA and ANN analysis by Mengge Zhou; Xiaoyao Ma; Guoyu Ding; Zengyong Wang; Dan Liu; Yongling Tong; Hong Zhou; Jie Gao; Yuanyuan Hou; Min Jiang; Gang Bai (60-69).
Many species of Bulbus fritillariae are used as traditional medicines for thousands of years; however, their application is not standardized. To clarify the differences and homologies, the antimuscarinic and anti-inflammatory effects of five BM species were firstly tested and compared at cellular level. With an integrated strategy combining UPLC-Q/TOF MS, PCA and ANN analysis, the active ingredients among 28 different chemical markers were predicted and identified. SB and QB extracts showed the best antimuscarinic effects and several steroidal alkaloids, such as solanidine, contributed to this effects. However, ZB was superior to reduce the inflammatory response. Another five components were responsible by decreasing the expression of NF-κB, including puqiedine, zhepeiresinol, 2-monopalmitin, N-demethylpuqietinone, and isoverticine. More novelty, a new cluster of five BM species based on active ingredients as potential quality markers was depicted to illustrate their functions. These results of the study could make a reference for the medicinal application of BM species in clinic; and the integrated strategy provided an effective method to obtain the quality markers from medical herbs, which was helpful for the quality control of traditional medicinal products.
Keywords: Bulbus fritillariae; M2R antagonists; NF-κB inhibitors; UPLC-Q/TOF MS; Quality markers;

We have utilized an automated acid hydrolysis technology, followed by an abbreviated Soxhlet extraction technique to obtain fat from whole milk for the determination of persistent organic pollutants, namely polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls. The process simply involves (1) pouring the liquid milk into the hydrolysis beaker with reagents and standards, (2) drying the obtained fat on a filter paper and (3) obtaining pure fat via the modified Soxhlet extraction using 100 mL of hexane per sample. This technique is in contrast to traditional manually intense liquid-liquid extractions and avoids the preparatory step of freeze-drying the samples for pressurized liquid extractions. Along with these extraction improvements, analytical results closely agree between the methods, thus no quality has been compromised. The native spike (n = 12) and internal standard (n = 24) precision and accuracy results are within EPA Methods 1613 and 1668 limits. While the median (n = 6) Toxic Equivalency Quotient (TEQ) for polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans and the concentration of the marker polychlorinated biphenyls show a percent difference of 1% and 12%, respectively, compared to 315 previously analyzed milk samples at the same laboratory using liquid-liquid extraction. During our feasibility studies, both egg and fish tissue show substantial promise using this technique as well.
Keywords: POPs; Dioxins; Extraction; Automated; Acid hydrolysis;

A validated LC–MS/MS method for thyroid hormone determination in sea lamprey (Petromyzon marinus) plasma, gill, kidney and liver by Ugo Bussy; Yu-Wen Chung-Davidson; Ke Li; Skye D. Fissette; Ethan G. Buchinger; Weiming Li (77-84).
A UPLC–MS/MS method was developed to provide a reproducible, sensitive and quantitative assay to determine thyroid hormones in sea lamprey tissues and plasma. l-Thyroxine (T4) and its two triiodo-thyronine isomers have been simultaneously quantified and validated for plasma, gill, liver, and kidney matrices. Multiple sample preparation techniques were investigated to achieve optimal sample matrix digestion and clean-up. Enzymatic digestion followed by protein precipitation was selected to process the samples. The developed method exhibited excellent linearity for all analytes with regression coefficients higher than 0.99 for concentrations ranged from 10 to 50,000 pg/mL. The limit of detection (LOD) was under 1 pg/mL while the limit of quantification (LOQ) was estimated as 10 pg/mL. This method was validated according to the FDA guidance and applied to determine thyroid hormone levels in plasma, gill and kidney of sea lamprey exposed to a sex pheromone. With appropriate implementation and further validation, this method could be applied to tissues in other species including humans.
Keywords: T4; T3; rT3; Sea lamprey; UPLC–MS/MS; Thyroid hormones;

A highly sensitive method using ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC–LTQ–Orbitrap–MS) has been developed and validated for the simultaneous identification and quantification of ginkgolic acids and semi-quantification of their metabolites in rat plasma. For the five selected ginkgolic acids, the method was found to be with good linearities (r > 0.9991), good intra- and inter-day precisions (RSD < 15%), and good accuracies (RE, from −10.33% to 4.92%) as well. Extraction recoveries, matrix effects and stabilities for rat plasm samples were within the required limits. The validated method was successfully applied to investigate the pharmacokinetics of the five ginkgolic acids in rat plasma after oral administration of 3 dosage groups (900 mg/kg, 300 mg/kg and 100 mg/kg). Meanwhile, six metabolites of GA (15:1) and GA (17:1) were identified by comparison of MS data with reported values. The results of validation in terms of linear ranges, precisions and stabilities were established for semi-quantification of metabolites. The curves of relative changes of these metabolites during the metabolic process were constructed by plotting the peak area ratios of metabolites to salicylic acid (internal standard, IS), respectively. Double peaks were observed in all 3 dose groups. Different type of metabolites and different dosage of each metabolite both resulted in different Tmax.
Keywords: Ginkgolic acids; Pharmacokinetics; UHPLC–LTQ–Orbitrap–MS; Metabolites;

Existing liquid chromatography – mass spectrometry method for the analysis of short chain carboxylic acids was expanded and validated to cover also the measurement of glycerol from oils and fats. The method employs chloride anion attachment and two ions, [glycerol +35Cl] and [glycerol +37Cl], as alternative quantifiers for improved selectivity of glycerol measurement. The averaged within run precision, between run precision and accuracy ranged between 0.3 − 7%, 0.4 − 6% and 94 − 99%, respectively, depending on the analyte ion and sample matrix. Selected renewable diesel feedstocks were analyzed with the method.
Keywords: Oil; Renewable diesel; Glycerol; LC–MS; Electrospray;

Preparation of ionic liquid-mediated imprinted monolith for selective capture and purification of corilagin by Chao Wang; Xiang-Jie Li; Jian Yang; Yong-Xin Zhao; Zhao-Sheng Liu; Haji Akber Aisa (98-103).
A method for solid-phase extraction (SPE) of corilagin from natural plant extracts based on molecularly imprinted polymers (MIPs) was developed. For the preparation of corilagin-MIP monoliths, 4-vinylpyridine was used as functional monomer, and ethylene glycol dimethacrylate was used as cross-linking monomer, using a mixture of 1-butyl-3-methylimidazoliumtetrafluoroborate (ionic liquid)-N,N-dimethylformamide-dimethyl sulfoxide as a porogen. A morphological characteristic of the corilagin imprinted monolith was further studied by scanning electron microscopy and nitrogen sorption method. The greatest imprinting factor of COR was up to 9. The MIPs were used as solid-phase extraction (SPE) sorbents for purification of COR and the mean recoveries of corilagin was 78.0% with COR purity of 98.0% from the crude extract of phyllanthus urinaria L. The resulting COR-imprinted polymer also displayed the good performance of fragment imprinting polymer for gallic acid with the mean recoveries of 94.0% and purity of 99.7%.
Keywords: Molecularly imprinted polymers; Monolith; Corilagin; Solid phase extraction; Ionic liquid; HPLC;

The Xian-Ling-Gu-Bao capsule (XLGB) is a famous traditional Chinese medicine prescription (TCMP), which has proven effective in osteoporosis treatment. However, due to the lack of a dynamic XLGB profile, the in vivo pharmacokinetics of multiple bioactive components within this medicine remains unknown. In the present study, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) identified a total of eighteen prototypes (using reference standards) in rat serum after oral administration of XLGB. These prototypes were subsequently evaluated to ascertain their effects on the proliferation and alkaline phosphatase activity of UMR106 cells and the adipogenesis of 3T3-L1 cells. Furthermore, a rapid and sensitive UPLC/Q-TOF-MS method was developed and validated for simultaneous quantitative analysis of 11 prototypes in rat serum. Chromatographic separation was achieved using a Waters Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm) and linear gradient elution employing a mobile phase consisting of water and acetonitrile (both containing 0.1% formic acid). All calibration curves showed excellent linearity (r 2  > 0.99) within the sampling ranges considered. The assay was accurate, precise and reproducible, as demonstrated by the obtained intra- and inter-day precisions (less than 12.3%) and accuracies (between −12.7% and 11.0%), and the matrix effects, extraction recoveries and stabilities were all satisfactory. Moreover, pharmacokinetic parameters were calculated from the plasma concentration-time data. Compared to single-compound dosing, significantly enhanced responses were obtained when several analytes were administered simultaneously, indicating possible drug-drug interactions among the complex ingredients of TCMP. This work provides an experimental baseline regarding the clinical applications and medicinal effectiveness of XLGB in the treatment of osteoporosis.
Keywords: Xian-Ling-Gu-Bao capsule; Main prototypes; Bioactivity evaluation; Pharmacokinetics; Rat serum; UPLC/Q-TOF-MS;

This paper develops a highly selective, specific and efficient method for simultaneous determination of ephedrine and methamphetamine by a new carbon coated magnetic nanoparticles (C/MNPs) as a magnetic solid phase extraction (MSPE) adsorbent in biological urine medium. The characterization of synthesized magnetic nano adsorbent was completely carried out by various characterization techniques like Fourier transform infrared (FT-IR) spectroscopy, powder x-ray diffraction (XRD), scanning electron microscopy (SEM) and vibrating sample magnetometer (VSM). Nine important parameters influencing extraction efficiency including amount of adsorbent, amounts of sample volume, pH, type and amount of extraction organic solvent, time of extraction and desorption, agitation rate and ionic strength of extraction medium, were studied and optimized. Under optimized extraction conditions, a good linearity was observed in the concentration range of 100–2000 ng/mL for ephedrine and 100–2500 ng/mL for methamphetamine. Analysis of positive urine samples was carried out by proposed method with the recovery of 98.71 and 97.87% for ephedrine and methamphetamine, respectively. The results indicated that carbon coated magnetic nanoparticles could be applied in clinical and forensic laboratories for simultaneous determination of abused drugs in urine media.
Keywords: Magnetic solid phase extraction; Carbon coated magnetic nanoparticles; Ephedrine; Methamphetamine; Urine; HPLC;

Second generation anticoagulant rodenticides (SGARs), pesticides used worldwide to control rodent populations, exist in two diastereoisomer chemical species because they own two stereogenic centers. A core-shell LC–MS/MS multi-residue method for comprehensive quantitative analysis of the diastereoisomers of five SGARs as well as three first generation anticoagulant rodenticide molecules has been fully validated in liver of rats according to a bioanalytical guideline. A core-shell column (superficially porous particles) has been chosen for its ability to separate the diastereomers of bromadiolone, difenacoum, brodifacoum, flocoumafen and difethialone and for its robustness to rat liver extracts. The highly selective chromatographic separation of the diastereoisomers contributes to good signal to noise ratios and then enhances the sensitivity of the method compared to the ones of fully porous columns. An elution gradient has been optimized with 10 mM ammonium acetate and acetonitrile as aqueous/organic mobile phase respectively. Triple quadrupole mass detector has been used to achieve specifity and LLOQ from 0.92 to 2.2 ng/g for each diastereoisomer, or first generation anticoagulant rodenticides. Then we evidenced diastereoisomeric ratios in liver of rats issued from not controlled exposure of wild rats (Rattus norvegicus) trapped in a French Parisian park through a campaign of rodent eradication. We compared them to diastereoisomeric ratios in SGARs commercial baits that contain both isomers, and showed that one of the two diastereoiomers had nearly disappeared in liver of rats. The proportions of cis-bromadiolone and trans-difenacoum were really lowered compared to the baits: 5/7 and 9/12 rats had only trans-bromadiolone and cis-difenacoum hepatic residues respectively. Liver persistence of the two diastereoisomers of bromadiolone and difenacoum was different due to differences in their pharmacokinetics in wild rats. The new core-shell LC–MS/MS method is particularly well adapted for further exploration of diastereoisomers ratios in rodent and predatory wildlife biological samples in order to evaluate ecological consequences of actual baits, to explore new formulated baits with a good balance between efficacity (ability to kill rodents) and diastereoisomers persistence, and hopefully to mitigate exposure of non-target species.
Keywords: Anticoagulant rodenticides; Diastereoisomers separation; Core-shell LC–MS/MS; Multi-residue validation; Rat liver;

ZSM-5 zeolite-loaded poly(dimethylsiloxane) (PDMS) hybrid thin films were demonstrated for efficient thin-film microextraction (TFME) coupled with gas chromatography–mass spectrometry for analyzing organic volatiles in water. The extraction efficiency for a series of aliphatic alcohols and two aromatic compounds was significantly improved owing to the presence of ZSM-5 zeolites. The extraction efficiency of the hybrid films was increased in proportion to the content of ZSM-5 in the PDMS film, with 20 wt% of ZSM-5 showing the best results. The 20 wt% ZSM-5/PDMS hybrid film exhibited higher volatile organic content extraction compared with the single-component PDMS film or PDMS hybrid films containing other types of zeolite (e.g., SAPO-34). Limits of detection and limits of quantitation for individual analytes were in the range of 0.0034–0.049 ppb and of 0.010–0.15 ppb, respectively. The effects of experimental parameters such as extraction time and temperature were optimized, and the molecular dispersion of the zeolites in/on the hybrid film matrix was confirmed with scanning electron microscopy and atomic force microscopy. Furthermore, the optimized hybrid film was preliminarily tested for the analysis of organic volatiles contained in commercially available soft drinks.
Keywords: Organic volatiles; Thin-film microextraction (TFME); ZSM-5; Poly(dimethylsiloxane); Gas chromatography–mass spectrometry;

We developed and validated an efficient and robust method for the simultaneous quantification of 44 fatty acid species in human plasma via GC-TOF-MS. The method is characterized by its robustness, accuracy and precision covering a wide range of fatty acid species with various saturation degrees including short chain fatty acids (beginning with FA 4:0) and long chain fatty acids (up to FA 32:0). The fatty acids were methylated prior to analyses and subsequently detected as fatty acid methyl esters by means of GC-TOF-MS. A highly substituted polar column allowed the separation of geometrical and positional isomers of fatty acid species. The method was applied to plasma samples of a strictly diet controlled clinical smoking cessation study including 39 smokers followed over the course of three months after having quit. Statistical significant alterations within the fatty acid profile were observed when comparing the baseline (subjects still smoking) with one week, one month and three months of smoking cessation. After 3 months of smoking cessation, a partial recovery of alterations in the fatty acid profile evoked by smoking was observed. In conclusion, the developed fatty acid profiling method using GC-TOF-MS has proven as a reliable tool for the quantitative determination of 44 individual fatty acid species within clinical studies.
Keywords: GC-TOF-MS; Fatty acid profile; Metabolic profiling; Smoking cessation; Metabolomics; Human plasma;

The distribution of fatty acid species at the sn-1/3 position or the sn-2 position of triacylglycerols (TAGs) in natural fats and oils affects their physical and nutritional properties. In fats and oils, determining the presence of one or two regioisomers and the identification of structure, where they do have one, as well as their separation, became a problem of fundamental importance to solve. A variety of instrumental technics has been proposed, such as MS, chromatography–MS or pure chromatography. A number of studies deal with the optimization of the separation, but very often, they are expensive in time. In the present study, in order to decrease the analysis time while maintaining good chromatographic separation, we tested different monomeric and polymeric stationary phases and different chromatographic conditions (mobile phase composition and analysis temperature) using Non-Aqueous Reversed Phase Liquid Chromatography (NARP–LC). It was demonstrated that mixed polymeric stationary bonded silica with accessible terminal hydroxyl groups leads to very good separation for the pairs of TAGs regioisomers constituted by two saturated and one unsaturated fatty acid (with double bond number: from 1 to 6). A Nucleodur C18 ISIS percolated by isocratic mobile phase (acetonitrile/2-propanol) at 18 °C leads to their separations in less than 15 min. The difference of retention times between two regioisomers XYX and XXY are large enough to confirm, as application, the presence of POP, SOP, SOS and PLP and no PPO, SPO, SSO and PPL in Theobroma cacao butter. In the same way, this study respectively shows the presence of SOS, SOP and no SSO, PSO in Butyrospermum parkii butter, POP, SOP, SOS and no PPO, PSO and SSO in Carapa oil and finally POP and no PPO in Pistacia Lentiscus oil.
Keywords: Triacylglycerols; Triglycerides; Regioisomers; Non-Aqueous reversed phase HPLC;

Sex-comparative study of mouse cerebellum physiology under adult-onset hypothyroidism: The significance of GC–MS metabolomic data normalization in meta-analysis by Christoniki Maga-Nteve; Catherine G. Vasilopoulou; Caterina Constantinou; Marigoula Margarity; Maria I. Klapa (158-166).
A systematic data quality validation and normalization strategy is an important component of the omic profile meta-analysis, ensuring comparability of the profiles and exclusion of experimental biases from the derived biological conclusions. In this study, we present the normalization methodology applied on the sets of cerebellum gas chromatography-mass spectrometry metabolic profiles of 124 days old male and female animals in an adult-onset-hypothyroidism (AOH) mouse model before combining them into a sex-comparative analysis. The employed AOH model concerns the monitoring of the brain physiology of Balb/cJ mice after eight-week administration of 1% w/v KClO4 in the drinking water, initiated on the 60th day of their life. While originating from the same animal study, the tissues of the two sexes were processed and their profiles acquired and analyzed at different time periods. Hence, the previously published profile set of male mice was first re-annotated based on the presently available resources. Then, after being validated as acquired under the same analytical conditions, both profiles sets were corrected for derivatization biases and filtered for low-confidence measurements based on the same criteria. The final normalized 73-metabolite profiles contribute to the currently few available omic datasets of the AOH effect on brain molecular physiology, especially with respect to sex differentiation. Multivariate statistical analysis indicated one (unknown) and three (succinate, benzoate, myristate) metabolites with significantly higher and lower, respectively, cerebellum concentration in the hypothyroid compared to the euthyroid female mice. The respective numbers for the males were two and 24. Comparison of the euthyroid cerebellum metabolic profiles between the two sexes indicated 36 metabolites, including glucose, myo- and scyllo-inositol, with significantly lower concentration in the females versus the males. This implies that the female mouse cerebellum has been conditioned to smaller changes in its metabolic activity with respect to the pathways involving these metabolites compared to the male animals. In conclusion, our study indicated a much subtler AOH effect on the cerebellum metabolic activity of the female compared to the male mice. The leaner metabolic profile of the female mouse cerebellum was suggested as a potential factor contributing to this phenomenon.
Keywords: GC–MS metabolomic data validation and normalization; Mammalian brain metabolism; Adult-onset hypothyroidism (AOH); Balb/cJ mouse cerebellum; Metabolic network analysis; Systems biology;

A method for the preparation of curcumin by ultrasonic-assisted ammonium sulfate/ethanol aqueous two phase extraction by Guangkuan Xu; Changchun Hao; Suyang Tian; Feng Gao; Wenyuan Sun; Runguang Sun (167-174).
This study investigated a new and easy-to-industrialized extracting method for curcumin from Curcuma longa rhizomes using ultrasonic extraction technology combined with ammonium sulfate/ethanol aqueous two-phase system (ATPS), and the preparation of curcumin using the semi-preparative HPLC. The single-factor experiments and response surface methodology (RSM) were utilized to determine the optimal material-solvent ratio, ultrasonic intensity (UI) and ultrasonic time. The optimum extraction conditions were finally determined to be material-solvent rate of 3.29:100, ultrasonic intensity of 33.63 W/cm2 and ultrasonic time of 17 min. At these optimum conditions, the extraction yield could reach 46.91 mg/g. And the extraction yields of curcumin remained stable in the case of amplification, which indicated that scale-up extraction was feasible and efficient. Afterwards, the semi-preparative HPLC experiment was carried out, in which optimal preparation conditions were elected according to the single factor experiment. The prepared curcumin was obtained and the purity could up to 85.58% by the semi-preparative HPLC.
Keywords: Curcumin; Aqueous two-phase system; Ultrasonic-assisted; Response surface methodology; Semi-preparative HPLC;

A simple and sensitive UHPLC–MS/MS method was developed and validated to determine the pharmacokinetic profile of 2-(2-hydroxypropanamido) benzoic acid (HPABA) enantiomers and their prodrugs in rat plasma. Separation was performed on a Thermo Syncronis C18 column (50 mm × 2.1 mm, 1.7 μm; Thermo, USA), which was protected by a high pressure column prefilter (2 μm) at a flow rate of 0.4 ml/min. Liquid-liquid extraction with ethyl acetate was used to process plasma samples. The separation of two enantiomers, prodrugs of (R)-/(S)-HPABA and internal standard was obtained within a cycle time of 4.5 min. The lower limit of quantification of (R)-/(S)-HPABA and prodrugs of (R)-/(S)-HPABA in plasma were 0.01 μg/ml and 0.2 μg/ml, respectively. (S)-HPABA showed significantly higher AUC, C max and a longer t 1/2 than (R)-HPABA, indicating higher bioavailability of the (S)-HPABA. Additionally, inversion between HPABA enantiomers was not observed in rats. (R)-/(S)-HPABA showed higher C max and AUC than those of their prodrugs. However, the values of t 1/2 of prodrugs were higher than those of (R)-/(S)-HPABA. Furthermore, the higher V z values of prodrugs might improve the targeting of (R)-/(S)-HPABA in rat tissues.
Keywords: 2-(2-Hydroxypropanamido) benzoic acid; Enantiomers; Prodrugs; UHPLC–MS/MS; Pharmacokinetics; Liquid–liquid extraction;

Brettanomyces/Dekkera produces 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) from hydroxycinnamic acids that affect the wine aroma and overall quality. A simple, cheap, fast and reliable quantitation method is needed for routine quality control of wines. In this work a simple method based on one simple liquid–liquid extraction with pentane/diethyl ether (2:1) and analysis by GC–MS allow to obtain very good recoveries (98–102%) and low quantification limits (24 and 11 μg/L for 4-EP and 4-EG, respectively), well below the sensory threshold for these volatile phenols and with an adequate measurement uncertainty: 70, 1.75 and 78, 1.95 and 1.35 μg/L for levels of 1000, 25 μg/L for 4-EP and 1000, 25 and 10 μg/L for 4-EG, respectively. In addition a screening of eight fining agents (mineral, protein and polysaccharide based) for reducing the levels of these volatile phenols in red wines was performed, and the impact on the physicochemical characteristics of red wines was evaluated. At the levels used, activated carbon was the most efficient fining agent in removing 4-ethylphenol and 4-ethylguaiacol from red wines (57%) resulting in a 75% decrease of headspace concentration of these volatile phenols. Lower reductions were observed when using egg albumin (19%) resulting in a 30% decrease in the headspace concentration. Other fining agents although not reducing the total amount of the volatile phenols present in wine decreased their concentrations in the headspace like isinglass (27%), carboxymethylcellulose (15%) and chitosan (27%). All of these fining agents could be a possibility for treating wine contaminated with 4-ethylphenol and 4-ethylguaiacol.
Keywords: Red wine; 4-Ethylphenol; 4-Ethylguaiacol; Gas chromatography; Liquid-liquid extraction; Fining agents;