Journal of Chromatography B (v.1029-1030, #C)

The direct detection of sulfate conjugates of anabolic androgenic steroids (AAS) can be a powerful tool in doping control analysis. By skipping the solvolysis step analysis time can be reduced, and due to long term sulfate metabolites the detection time can be significantly extended as demonstrated for some AAS. This study presents the successful identification of sulfate metabolites of the doping agents oxandrolone and danazol in excretion urines by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS). The sulfate conjugate of 17β-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one could be identified as a new metabolite of oxandrolone. Sulfate conjugates of the danazol metabolites ethisterone and 2α-hydroxymethylethisterone were identified in an excretion urine for the first time. In addition, these sulfate conjugates were synthesized successfully. For a confirmation analysis, the number of analytes can be increased by additional sulfate conjugates of danazol metabolites (2-hydroxymethyl-1,2-dehydroethisterone and 6β-hydroxy-2-hydroxymethylethisterone), which were also identified for the first time. The presented validation data underline the suitability of the identified sulfate conjugates for doping analysis with regard to the criteria given by the technical documents of the World Anti-Doping Agency (WADA).
Keywords: Doping analysis; Oxandrolone; Danazol; Sulfate conjugates; Liquid chromatography–mass spectrometry;

The chromatographic behavior of 28 plant secondary metabolites belonging to four chemically similar classes (alkaloids, flavonoids, flavone glycosides and sesquiterpenes) was studied by normal-phase thin-layer chromatography (NP-TLC) under 5 different chromatographic systems commonly used in plant drug analysis with the aim to explore whether the retention properties of these metabolites can determine the chemical group they belong to. The use of RM values as the retention parameter is implemented as a relatively new approach in plant analysis. Principal component analysis (PCA), hierarchical clustering heat maps and discriminant analysis (DA), were used for statistical evaluation of the chromatographic data and extraction of similarities between chemically related compounds. The twenty eight metabolites were classified into four groups by principal component analysis. The heat map of hierarchical clustering revealed that all metabolites were clustered into four groups, except for caffeine, while linear discriminant analysis showed that 96.4% of metabolites are predicted correctly as the groupings identified by chemical class in original and cross-validated data. The main advantage of the approach described in current paper is its simplicity which can assist with preliminary identification of metabolites in complex plant extracts.
Keywords: Multivariate; NP-TLC; Retention data; Principal component analysis; Discriminant analysis; Hierarchical clustering heat map;

A new method of analysis has been developed for UV inactive drug carisoprodol using FTIR spectroscopy. These methods were validated for various parameters according to ICH guidelines. The proposed method has also been successfully applied for the determination of the drug concentration in a tablet formulation. The method proved to be accurate (mean percentage recovery between 95 and 105%), precise and reproducible (relative standard deviation < 2%), while being simple, economical and less time consuming than other methods and can be used for routine estimation of carisoprodol in the pharmaceutical industry. The developed method also implicates its utility for other UV inactive substances. The stability of the drug under various stress conditions was studied and the drug was found to be particularly susceptible to alkaline hydrolysis. Degradation products of the alkaline hydrolysis were detected by RP-HPLC and tentatively identified by ESI–MS.
Keywords: Carisoprodol quantitative estimation; FTIR method development; UV inactive drug; Forced degradation; Degradant identification;

Nonmedical use of prescription stimulants such as phentermine (PT) has been regulated by law enforcement authorities due to its euphorigenic and relaxing effects. Due to high potential for its abuse, reliable analytical methods were required to detect and identify PT and its metabolite in biological samples. Thus a dilute and shoot liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for simultaneous determination of PT, N-hydroxyphentermine (NHOPT) and mephentermine (MPT) in urine. A 5 μL aliquot of diluted urine was injected into the LC–MS/MS system. Chromatographic separation was performed by reversed-phase C18 column with gradient elution for all analytes within 5 min. Identification and quantification were based on multiple reaction monitoring (MRM) detection. Linear least-squares regression with a 1/x2 weighting factor was used to generate a calibration curve and the assay was linear from 50 to 15000 ng/mL (PT and MPT) and 5 to 750 ng/mL (NHOPT). The intra- and inter-day precisions were within 8.9% while the intra- and inter-day accuracies ranged from −6.2% to 11.2%. The limits of quantification were 3.5 ng/mL (PT), 1.5 ng/mL (NHOPT) and 1.0 ng/mL (MPT). Method validation requirements for selectivity, dilution integrity, matrix effect and stability were satisfied. The applicability of the developed method was examined by analyzing urine samples from drug abusers.
Keywords: Phentermine; N-Hydroxyphentermine; Mephentermine; Urine; LC–MS/MS;

Serum metabonomics study of anti-depressive effect of Xiao-Chai-Hu-Tang on rat model of chronic unpredictable mild stress by Zhili Xiong; Jie Yang; Yue Huang; Kuo Zhang; Yunhai Bo; Xiumei Lu; Guangyue Su; Jie Ma; Jingyu Yang; Longshan Zhao; Chunfu Wu (28-35).
Xiao-Chai-Hu-Tang (XCHT) has been proven to be effective for the clinical treatment of depression. However, the mechanisms of definite antidepressant-like effects and detailed metabolic biomarkers were still unclear in this prior study. Here, we have investigated the metabolic profiles and potential biomarkers in a chronic unpredictable mild stress model after treatment with XCHT. Metabonomics based on ultra-high performance liquid chromatography coupled with mass spectrometry was used to profile the metabolic fingerprints of serum obtained from a rat model with chronic unpredictable mild stress with and without XCHT treatment. The model rats showed a significant decrease in sucrose preference and food consumption, and these depression-like symptoms were significantly improved by XCHT. Through principal component analysis (PCA), nine potential biomarkers of tryptophan, uric acid, phenylalanine, cholic acid and lysophosphatidylcholine (C18:0 LPC, C16:0 LPC, C16:1 LPC, C18:1 LPC, C20:4 LPC) were characterized as potential biomarkers involved the pathogenesis of depression. The therapeutic effect of XCHT on depression may involve in amino acid metabolism, lipid metabolism, oxidative stress and inflammation response. The present investigation highlights that metabonomics is a valuable tool for studying the essence of depression as well as evaluating the efficacy of the corresponding drug treatment.
Keywords: Xiao-Chai-Hu-Tang; Antidepressant; CUMS; Metabolomics; Multivariate analysis;

Identification of saponins from sugar beet (Beta vulgaris) by low and high-resolution HPLC–MS/MS by Katarzyna Mikołajczyk-Bator; Alfred Błaszczyk; Mariusz Czyżniejewski; Piotr Kachlicki (36-47).
We profiled triterpene saponins from the roots of sugar beet Beta vulgaris L. cultivars Huzar and Boryna using reversed-phase liquid chromatography combined with negative-ion electrospray ionization quadrupole mass spectrometry. We tentatively identified 26 triterpene saponins, including 17 that had not been detected previously in this plant species and 7 saponins that were tentatively identified as new compounds. All observed compounds were glycosides of five different aglycones, of which gypsogenin and norhederagenin are reported for the first time in sugar beet. Thirteen of the saponins detected in sugar beet roots were substituted with dioxolane-type (4 saponins) or acetal-type (9 saponins) dicarboxylic acids. Among the 26 detected saponins, we identified 2 groups of isomers distinguished using high-resolution mass measurements that were detected only in the Huzar cultivar of sugar beet.
Keywords: Sugar beet (Beta vulgaris L.); Triterpene saponins; Akebonoic acid; Oleanolic acid; Hederagenin; Gypsogenin; Norhederagenin; HPLC–MS/MS;

Ultra-high performance liquid chromatographic determination of levofloxacin in human plasma and prostate tissue with use of experimental design optimization procedures by O. Szerkus; J. Jacyna; P. Wiczling; A. Gibas; M. Sieczkowski; D. Siluk; M. Matuszewski; R. Kaliszan; M.J. Markuszewski (48-59).
Fluoroquinolones are considered as gold standard for the prevention of bacterial infections after transrectal ultrasound guided prostate biopsy. However, recent studies reported that fluoroquinolone- resistant bacterial strains are responsible for gradually increasing number of infections after transrectal prostate biopsy. In daily clinical practice, antibacterial efficacy is evaluated only in vitro, by measuring the reaction of bacteria with an antimicrobial agent in culture media (i.e. calculation of minimal inhibitory concentration). Such approach, however, has no relation to the treated tissue characteristics and might be highly misleading. Thus, the objective of this study was to develop, with the use of Design of Experiments approach, a reliable, specific and sensitive ultra-high performance liquid chromatography- diode array detection method for the quantitative analysis of levofloxacin in plasma and prostate tissue samples obtained from patients undergoing prostate biopsy. Moreover, correlation study between concentrations observed in plasma samples vs prostatic tissue samples was performed, resulting in better understanding, evaluation and optimization of the fluoroquinolone-based antimicrobial prophylaxis during transrectal ultrasound guided prostate biopsy.Box-Behnken design was employed to optimize chromatographic conditions of the isocratic elution program in order to obtain desirable retention time, peak symmetry and resolution of levofloxacine and ciprofloxacine (internal standard) peaks. Fractional Factorial design 24−1 with four center points was used for screening of significant factors affecting levofloxacin extraction from the prostatic tissue. Due to the limited number of tissue samples the prostatic sample preparation procedure was further optimized using Central Composite design. Design of Experiments approach was also utilized for evaluation of parameter robustness. The method was found linear over the range of 0.030–10 μg/mL for human plasma and 0.300–30 μg/g for human prostate tissue samples. The intra-day and inter-day variability for levofloxacine from both plasma and prostate samples were less than 10%, with accuracies between 93 and 108% of the nominal values. The limit of detection and the limit of quantification for human plasma were 0.01 μg/mL and 0.03 μg/mL, respectively. For the prostate tissue, the limit of detection and the limit of quantification were 0.1 μg/g and 0.3 μg/g, respectively. The average recoveries of levofloxacin were in the range from 99 to 106%. Also, the method fulfills requirements of robustness what was determined and proved by Design of Experiments. The developed method was successfully applied to examine prostate tissue and plasma samples from 140 hospitalized patients enrolled into the clinical study, 12 h after oral administration of LVF at a dose of 500 mg. The mean (± SD) LVF concentration in prostate was 6.22 ± 3.52 μg/g and in plasma 2.54 ± 1.14 μg/mL. Due to simplicity of the method and relative small amount of sample needed for the assay, the method can be applied in clinical practice for monitoring of LVF concentrations in plasma and prostate gland.
Keywords: Levofloxacin; Prostate tissue; Human plasma; Design of experiments (DoE); Transrectal ultrasound guided prostate biopsy (TRUS-Bx); UHPLC-DAD;

Roflumilast had shown good efficacy and safety in Caucasian COPD patients after oral administration of 0.5 mg. The main active metabolite of it is roflumilast N-oxide. A reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) quantitation method was developed for the simultaneous determination of them in human plasma with rather low limits of quantitation for roflumilast (0.02 ng/mL) and roflumilast N-oxide (0.04 ng/mL). Human plasma samples were prepared by solid phase extraction (SPE), which ensured high recovery and slight matrix effect for the both analytes. This method showed good linearity, accuracy, precision and stability in the range of 0.02–10 ng/mL and 0.04–50 ng/mL for roflumilast and roflumilast N-oxide respectively. The developed method was successfully applied for the pharmacokinetic research in Chinese healthy volunteers after oral administration of 0.25 mg, 0.375 mg and 0.5 mg of roflumilast tablet.
Keywords: Roflumilast; Roflumilast N-oxide; LC–MS/MS; Human plasma; Chinese healthy volunteers;

An HPLC–MS/MS method for the separation of α-retinyl esters from retinyl esters by Hilary J. Goetz; Rachel E. Kopec; Ken M. Riedl; Jessica L. Cooperstone; Sureshbabu Narayanasamy; Robert W. Curley; Steven J. Schwartz (68-71).
Enzymatic cleavage of the nonsymmetric provitamin A carotenoid α-carotene results in one molecule of retinal (vitamin A), and one molecule of α-retinal, a biologically inactive analog of true vitamin A. Due to structural similarities, α-retinyl esters and vitamin A esters typically coelute, resulting in the overestimation of vitamin A originating from α-carotene. Herein, we present a set of tools to identify and separate α-retinol products from vitamin A. α-Retinyl palmitate (αRP) standard was synthesized from α-ionone following a Wittig-Horner approach. A high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method employing a C30 column was then developed to separate the species. Authentic standards of retinyl esters and the synthesized α-RP confirmed respective identities, while other α-retinyl esters (i.e. myristate, linoleate, oleate, and stearate) were evidenced by their pseudomolecular ions observed in electrospray ionization (ESI) mode, fragmentation, and elution order. For quantitation, an atmospheric pressure chemical ionization (APCI) source operated in positive ion mode was used, and retinol, the predominant in-source parent ion was selected and fragmented. The application of this method to a chylomicron-rich fraction of human plasma is demonstrated. This method can be used to better determine the quantity of vitamin A derived from foods containing α-carotene.
Keywords: α-Retinyl palmitate; α-Retinol; Retinyl esters; Provitamin A carotenoids; Synthesis; HPLC–MS/MS;

In this paper, a novel highly sensitive chemiluminescence immune-affinity 96 spots monolith array was developed to detect deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin (T-2), and fumonisin B1 (FB1) in corn samples. Firstly, the monolith array was prepared through on suit UV-initiated copolymerization using polyethylene glycol diacrylate (PEGDA) as cross-linker, glycidyl methacrylate (GMA) as functional monomer and polyethylene glycol 200 (PEG 200) as the porogen. Subsequently, the four mycotoxins immune-affinity monolith array was prepared by immobilization of DON, ZEN, T-2, and FB1 antibody. The mole ratio of PEGDA/GMA, UV exposure time, and the volume ratio of PEG 200/PEGDA were optimized to improve the performances of the immune-affinity monolith array. For the mycotoxins immune-affinity monolith array based on chemiluminescence detection, the limit of detection was 0.0036 ng/mL (DON), 0.0048 ng/mL (ZEN), 0.0039 ng/mL (T-2), and 0.0017 ng/mL (FB1), respectively. The linear response in the range of 0.01–0.1 ng/mL (R2  = 0.98). The results showed that the proposed four mycotoxins immune-affinity monolith array was a stable, accurate, and highly sensitive method to determine levels of DON, ZEN, T-2, and FB1 in real samples.
Keywords: Mycotoxins; UV-initiated copolymerization; Immune-affinity array; Chemiluminescent detection; Monolith;

A new design of hyperbranched polyglycerol/graphene oxide nanocomposite reinforced hollow fiber solid/liquid phase microextraction (HBP/GO −HF-SLPME) coupled with high performance liquid chromatography used for extraction and determination of ibuprofen and naproxen in hair and waste water samples. The graphene oxide first synthesized from graphite powders by using modified Hummers approach. The surface of graphene oxide was modified using hyperbranched polyglycerol, through direct polycondensation with thionyl chloride. The ready nanocomposite later wetted by a few microliter of an organic solvent (1-octanol), and then applied to extract the target analytes in direct immersion sampling mode.After the extraction process, the analytes were desorbed with methanol, and then detected via high performance liquid chromatography (HPLC). The experimental setup is very simple and highly affordable. The main factors influencing extraction such as; feed pH, extraction time, aqueous feed volume, agitation speed, the amount of functionalized graphene oxide and the desorption conditions have been examined in detail. Under the optimized experimental conditions, linearity was observed in the range of 5–30,000 ng mL−1 for ibuprofen and 2–10,000 ng mL−1 for naproxen with correlation coefficients of 0.9968 and 0.9925, respectively. The limits of detection were 2.95 ng mL−1 for ibuprofen and 1.51 ng mL−1 for naproxen. The relative standard deviations (RSDs) were found to be less than 5% (n = 5).
Keywords: Ibuprofen; Naproxen; Hyperbranched polyglycerol; Graphene oxid;

Short-chain fatty acids (SCFAs) produced by the intestinal bacteria are very critical for the intestinal barrier, mucosal cytoprotection and normal intestinal biology. However, accumulation of SCFAs promoted by the polysaccharides from Chrysanthemum morifolium Ramat remains unknown. Thus, it is necessary to investigate SCFAs in the colonic contents of dextran sulfate sodium (DSS) induced colitis mice after oral administration of the polysaccharides from C. morifolium Ramat which is very helpful to unravel how it works. In this study, a rapid and reliable gas chromatographic method with flame ionization detector (GC-FID) for simultaneous determination of six SCFAs such as acetic acid (AA), propionic acid (PA), butyric acid (BA), isobutyric acid (IBA), valeric acid (VA) and isovaleric acid (IVA) has been developed and validated. Under the optimized chromatographic conditions and sample extraction procedure, good separation for 6 target compounds was obtained on a HP-INNOWAX column within 12 min. Results revealed that polysaccharides from C. morifolium Ramat positively affected the SCFAs intestinal production. The polysaccharides group had greater SCFAs concentration in colonic content than the DSS-treated group (P < 0.05), which was decreased remarkably compared to the normal group (P < 0.01). With the decrease of the polysaccharides dosage, the contents of AA, PA and VA increased gradually, while the change of BA concentration was the opposite. There was no significant difference in the content of IBA at the different administration concentrations. And the content of IVA reached the highest concentration 0.953 mg/g at lower dose of the polysaccharides. Additionally, oral administration of the polysaccharides prominently attenuated the body weight loss, reduced the disease activity index, rectal bleeding and stool consistency, improved colon shortening and macroscopic score of colitis. Our results indicated that the polysaccharides of C. morifolium Ramat might be used as prebiotic agents to prevent gut dysbiosis and inflammatory bowel disease.
Keywords: Short-chain fatty acids; DSS induced colitis; Chrysanthemum morifolium; Colonic contents; GC-FID;

Phellodendrine, a quaternary ammonium alkaloid extracted from the dried bark of Phellodendrom chinensis Schneid and Phellodendrom amurense Rupr, has the effect of suppressing cellular immune response, reducing blood pressure and antinephritis. However, few investigations have been conducted for the pharmacokinetic study of phellodendrine. Thus, a rapid, simple and reliable ultra-high performance liquid chromatography–tandem quadrupole mass spectrometry (UHPLC–QQQ MS/MS) method has been established for quantification of phellodendrine in rat plasma and tissues by using magnoflorine as internal standard. The chromatographic separation was achieved on an Agilent ZORBAX SB-C18 column (4.6 mm × 50 mm, 1.8 μm) by gradient elution using 0.1% aqueous formic acid (A) and methanol (B). Triple quadrupole mass detection with multiple reaction monitoring mode was used to monitor the ion transitions, at m/z 342.20 → 192.20 for phellodendrine and m/z 342.20 → 58.20 for internal standard, respectively. The developed method was fully validated and successfully applied to the pharmacokinetics and tissue distribution study of phellodendrine after intravenous administration. The lower limits of quantification were 0.5 ng/mL for plasma samples, 2.5 ng/g for brain and 1 ng/g for other tested tissues. Precisions and accuracy values were within the Food and Drug Administration acceptance criteria, the recovery and matrix effects were between 87.8–113.5%. The area under the curve (AUC0−t) ranged from 15.58 to 57.41 mg/L min and Cmax were between 1.63–4.93 mg/L. The results showed that phellodendrine was eliminated in 120 min in plasma and most of tissues and the highest concentrations of phellodendrine were found in the kidney. This study may provide a basis for the further study of phellodendrine.
Keywords: Phellodendrine; Pharmacokinetic; Tissue distribution; UHPLC–MS/MS; Cortex phellodendri;

Methylglyoxal (MG) is a highly reactive dicarbonyl compound that promotes the non-enzymatic glycation of proteins to yield irreversible advanced glycated end products, leading to the cross-linking or degradation of proteins. The physiological relevance of MG currently remains unclear because its metabolic behavior has not yet been elucidated in detail. Although several labeling methods that require a HPLC system have been developed and used to measure MG, a standard method to analyze the content of MG in biological samples has not been established. We herein present a practical method based on HPLC with fluorescence detection to measure low MG levels. MG concentrations were also measured in human blood plasma using the present method in order to demonstrate its utility.A calibration curve was produced using freshly purified MG at concentrations ranging between 0.05 and 1.0 μM. The intra-day and inter-day relative standard diviations of the method were 2.55% and 4.03%, respectively. The limit of detection and limit of quantification were 60 fmol and 200 fmol, respectively for MG with a 10-μl injection volume of the derivatized sample solution. When the optimized method was applied to human plasma, the resulting concentrations of MG in the plasma of healthy subjects (n = 23) ranged between 0.024 and 0.258 μM (mean ± SD = 0.098 ± 0.066). Thus, the method developed herein is simple, sensitive, and easy to operate for the measurement of MG in biological samples.
Keywords: Methylglyoxal; Biomarker; HPLC method; Carbonyl stress; Fluorescence detection;

Adsorptive removal of trace sulfonamide antibiotics by water-dispersible magnetic reduced graphene oxide-ferrite hybrids from wastewater by Jianrong Wu; Hongyan Zhao; Rong Chen; Chuong Pham-Huy; Xuanhong Hui; Hua He (106-112).
A one-pot solvothermal synthesis method was developed to prepare reduced graphene oxide (RGO) supported ferrite hybrids using graphite oxide and metal ions (Fe3+) as starting materials. The as-prepared composites were characterized by transmission electron microscopy(TEM), Fourier transform infrared spectrophotometer (FT-IR), X-ray powder diffraction pattern(XRD) and vibrating sample magnetometer (VSM). It was shown that Fe3O4 nanoparticles with a uniform size of ∼35 nm were anchored on RGO nanosheets. Importantly, the obtained nanocomposites are effective adsorbents for the determination of three sulfonamides in wastewater. Several parameters affecting the extraction efficiency were optimized, including amount of adsorbent, extraction time, pH and desorption conditions. Compared with other adsorbents, the as-prepared RGO-Fe3O4 showed the better extraction efficiencies for the SAS due to the large surface area of RGO. A linear range from 1 to 200 ng/mL was obtained with a high correlation coefficient (R2  > 0.9987), and the limits of detection for three SAs ranged from 0.43 to 0.57 ng/mL. This method was successfully applied to the analysis of SAs in environmental wastewater samples, the recoveries in different sample matrices were in the range from 89.1 and 101.7% with relative standard deviations less than 8.6%. It is believed that such composites will find wide applications in the water pretreatment area.
Keywords: Magnetic reduced graphene oxide; Sulfonamide antibiotics; Adsorption; Environmental wastewater samples;

Hydrocarbons are widespread in cyanobacteria, and the biochemical synthetic pathways were recently identified. Intracellular fatty alka(e)nes of cyanobacteria have been detected by liquid–liquid extraction (LLE) coupled to gas chromatography–mass spectrometry (GC/MS). However, whether fatty alka(e)nes can be released to cyanobacterial culture media remains to be clarified. This work develops a sensitive method for analyzing the trace level of extracellular hydrocarbons in cyanobacterial culture media by head space solid-phase microextraction (HS-SPME) coupled to GC/MS. Headspace (HS) extraction mode using polydimethylsiloxane fiber to extract for 30 min at 50 °C was employed as the optimal extraction conditions. Five cyanobacterial fatty alka(e)nes analogs including pentadecene (C15:1), pentadecane (C15:0), heptadecene (C17:1), heptadecane (C17:0), nonadecane (C19:0) were analyzed, and the data obtained from HS-SPME-GC/MS method were quantified using internal standard peak area comparisons. Limits of detection (LOD), limits of quantitation (LOQ), linear dynamic range, precisions (RSD) and recovery for the analysis of extracellular fatty alka(e)nes of cyanobacteria by HS-SPME–GC/MS were evaluated. The LODs limits of detection (S/N = 3) varied from 10 to 21 ng L−1. The correlation coefficients (r) of the calibration curves ranged from 0.9873 to 0.9977 with a linearity from 0.1 to 50 μg L−1. The RSD values were ranging from 7.8 to 14.0% and from 4.0 to 8.8% at 1.0 μg L−1 and 10.0 μg L−1 standard solutions, respectively. Comparative analysis of extracellular fatty alka(e)nes in the culture media of model cyanobacteria Synechocystis sp. PCC 6803 demonstrated that sensitivity of HS-SPME–GC/MS method was significantly higher than LLE method. Finally, we found that heptadecane can be released into the culture media of Synechocystis sp. PCC 6803 at the later growth period.
Keywords: Solid-phase microextraction; Cyanobacteria; Extracellular hydrocarbons; Gas chromatography–mass spectrometry (GC–MS);

Bioactivity-guided identification of antimicrobial metabolites in Alnus glutinosa bark and optimization of oregonin purification by Centrifugal Partition Chromatography by Amin Abedini; Sébastien Chollet; Apostolis Angelis; Nicolas Borie; Jean-Marc Nuzillard; Alexios-Leandros Skaltsounis; Romain Reynaud; Sophie C. Gangloff; Jean-Hugues Renault; Jane Hubert (121-127).
Display OmittedBarks from conifers and broadleaved trees constitute abundant wastes generated from wood harvesting and logging activities. Extracts of such residues obtained from Alnus trees have been reported as interesting resources with potent antibacterial activities. The present study aims to determine the antimicrobial activity of a crude methanol extract prepared from the bark of Alnus glutinosa against a panel of 22 bacteria and yeasts and to optimize a purification method enabling the high production of the most active substances. Fractionation of the crude extract was performed by Centrifugal Partition Chromatography (CPC) using a three-phase solvent system composed of n-heptane, methyl-ter-butyl ether, acetonitrile and water. The major known compounds contained in the fractions produced by CPC were chemically profiled by 13C NMR dereplication, resulting in the unambiguous identification of oregonin, hirsutanonol, betulinic acid, and alusenone 1a. The antibacterial evaluation of the fractions by bioautography on Staphylococcus aureus revealed that oregonin, in addition to being the major metabolite of the crude extract (∼32% w/w), was the most active with an antibacterial inhibitory effect comparable to antibiotics. The purification of oregonin was optimized at the laboratory-scale by CPC. A single injection of 3.7 g of crude extract resulted in a recovery of 72% (850 mg) of the available oregonin at purity higher than 94%.
Keywords: Alnus glutinosa; Oregonin; Antimicrobial activity; Centrifugal partition extraction; NMR-based dereplication;

To more reasonably and effectively control the quality of Sanziguben Granule, chromatographic fingerprinting and serum pharmacochemistry of this traditional Chinese medicine compound were performed. A comprehensive comparison and evaluation of 15 batches of Sanziguben Granule was successfully conducted by using high performance liquid chromatography (HPLC) fingerprint analysis.After administering a set amount of Sanziguben Granule orally to rats, blood samples were collected and tested 4 times at intervals of 30 min, 1 h, 2 h, and 4 h using UPLC-Q-TOF-MS/MS. The blood showed presence of gallic acid and corilagin indicating the pharmacological significance of these two chemical compounds.According to the result, above mentional chemical compounds were designated biomarkers for quality control of Sanziguben Granule. Therefore, a purposeful and efficient method for quality control of Sanziguben Granule was established in the present study.
Keywords: HPLC fingerprint; Serum pharmacochemistry; Sanziguben granule; Quality control;

Conventional C18 silica columns have proven to be useful for the analysis of amino acids (AA) from protein hydrolysates but undesirable peak overlapping is usually found when analyzing body fluids given that a large number of AAs are present in the samples. As an alternative to silica packings, an ethyl-bridged packing for reversed-phase liquid chromatography of derivatized AAs with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) has been evaluated. The new packing material improves the separation efficiency allowing better separations when analyzing biological fluids. Moreover, this packing has advantages for routine AA analysis, such as a decrease in the total running time and an increase in the life-time of the columns. The pH of the mobile phase has a significant effect on the elution behavior of the AQC hydrolysis product (AMQ) and on the AA derivatives. It is not possible to elute AMQ before detecting the first AA derivative, which requires an accurate adjustment of the pH in the range of 5.30–5.35 to obtain good separation and resolution for the most polar compounds. Under the conditions proposed, it is possible to separate all AAs except the Gly-Gln pair, which is not a problem when hydrolyzed samples are analyzed. The AMQ-Ser pair requires either the use of a different mobile phase pH for its baseline separation or the use of fluorescence detection. Two different procedures for protein removal from plasma samples have been evaluated, solvent precipitation and ultrafiltration (UF) and it has been found that UF gives better results as no significant losses of AAs were observed. The validation of the proposed method with UV detection gives method detection limits in the range of 8–12 μM, with repeatability values < 8% (n = 6) and inter-day precision in plasma samples ranging from 4 to 13% (n = 4).
Keywords: Amino acids; Ethyl-bridged packing; Human plasma; Ultrafiltration;

Psoralidin has shown a variety of biological and pharmacological activities such as anti-tumor anti-oxidant, anti-bacterial, anti-depressant and anti-inflammatory activities. Herein, we reported the metabolism of psoralidin among different species and its inhibitory effect against UGTs and CYP450s. Liquid chromatography was used to investigate the inhibitory activity of psoralidin against ten different UGTs and eight distinct CYP450 isoforms. In addition, we characterized the CYP450 isoforms involved in the psoralidin metabolism on the basis of chemical inhibition studies and screening assays with recombinant human cytochrome P450s. In vitro metabolic profiles and metabolites of psoralidin from varying liver microsomes obtained from human (HLMs), monkey (MLMs), rat (RLMs), dog (DLMs), minipig (PLMs) and rabbit (RAMs) were determined by LC–MS/MS. In vivo pharmacokinetic profiles were investigated by injecting psoralidin (2 mg/kg) into the tail vein of Wistar rats. Molecular modeling studies were carried out in order to assess the binding profile and recognition motif between psoralidin and the enzymes. Psoralidin showed potent and noncompetitive inhibition against UGT1A1, UGT1A7, CYP1A2 and CYP2C8 with IC50 values of 6.12, 0.38, 1.81, 0.28 μM, respectively. The metabolism of psoraldin exhibited significant differences among humans, monkeys, dogs, minipigs, rabbits and rats; however, monkeys showed the highest similarity to humans. Furthermore, eleven metabolites were observed among these species and their structures were characterized by LC–MS/MS. CYP2C19 played a key role in the metabolism of psorslidin in human liver microsomes. These findings could be used to advance the understanding of psoralidin.
Keywords: Psoralidin; Cytochrome P450; Glucuronosyltransferase (UGT); Drug-drug interactions; LC–MS/MS;

Analysis of lipid profile in lipid storage myopathy by M’hammed Aguennouz; Marco Beccaria; Giorgia Purcaro; Marianna Oteri; Giuseppe Micalizzi; Olimpia Musumesci; Annmaria Ciranni; Rosa Maria Di Giorgio; Antonio Toscano; Paola Dugo; Luigi Mondello (157-168).
Lipid dysmetabolism disease is a condition in which lipids are stored abnormally in organs and tissues throughout the body, causing muscle weakness (myopathy). Usually, the diagnosis of this disease and its characterization goes through dosage of Acyl CoA in plasma accompanied with evidence of droplets of intra-fibrils lipids in the patient muscle biopsy. However, to understand the pathophysiological mechanisms of lipid storage diseases, it is useful to identify the nature of lipids deposited in muscle fiber. In this work fatty acids and triglycerides profile of lipid accumulated in the muscle of people suffering from myopathies syndromes was characterized. In particular, the analyses were carried out on the muscle biopsy of people afflicted by lipid storage myopathy, such as multiple acyl-coenzyme A dehydrogenase deficiency, and neutral lipid storage disease with myopathy, and by the intramitochondrial lipid storage dysfunctions, such as deficiencies of carnitine palmitoyltransferase II enzyme. A single step extraction and derivatization procedure was applied to analyze fatty acids from muscle tissues by gas chromatography with a flame ionization detector and with an electronic impact mass spectrometer. Triglycerides, extracted by using n-hexane, were analyzed by high performance liquid chromatography coupled to mass spectrometer equipped with an atmospheric pressure chemical ionization interface. The most representative fatty acids in all samples were: C16:0 in the 13–24% range, C18:1n9 in the 20–52% range, and C18:2n6 in the 10–25% range. These fatty acids were part of the most representative triglycerides in all samples. The data obtained was statistically elaborated performing a principal component analysis. A satisfactory discrimination was obtained among the different diseases. Using component 1 vs component 3 a 43.3% of total variance was explained. Such results suggest the important role that lipid profile characterization can have in supporting a correct diagnosis.
Keywords: Lipid storage myopathy; Intra-muscular triglycerides; Fatty acids; Non-aqueous reversed-phase liquid chromatography; Gas chromatography; Mass spectrometry;

Quantification of hypoglycin A as butyl ester by Johannes Sander; Michael Terhardt; Stefanie Sander; Nils Janzen (169-173).
L-α-amino-methylenecyclopropyl propionic acid (Hypoglycin A, HGA) has been found to be the toxic compound in fruits of the Sapindaceae family causing acute intoxication when ingested as food or feed. Clinical symptoms are consistent with acquired multiple acyl-CoA dehydrogenase deficiency (MADD). Ultra performance liquid chromatography-tandem mass spectrometry was used to measure HGA after butylation. Sample volumes were 10 μL for serum and 20 μL for urine. Internal standard for HGA was d3-leucine, samples were plotted on a 7-point linear calibration curve. Coefficients of variation were <15% at 0.01 μmol HGA/L and ≤4.1% at 10 μmol/L. R2 values for linearity were ≥0.995.In order to quantify non-metabolized HGA together with some of its metabolites plus a spectrum of acyl glycines and acyl carnitines typical for acquired MADD in one single analysis HGA measurement was integrated into a method which we previously developed for metabolites of HGA and acyl conjugates.The new method is suitable for biochemical diagnosis of Ackee fruit poisoning or atypical myopathy in horses and for forensic purposes in cases of suspected HGA poisoning.
Keywords: Hypoglycine A; Methylenecyclopropyl acetate; Atypical myopathy of horses; Acquired multiple acyl-CoA dehydrogenase deficiency; Jamaican vomiting sickness;

Ofloxacin is a chiral fluoroquinolone commercialized as racemate and as its enantiomerically pure form levofloxacin. This work presents an integrated liquid chromatography (LC) method with fluorescence detection (FD) and exact mass spectrometry (EMS) developed to assess the enantiomeric biodegradation of ofloxacin and levofloxacin in laboratory-scale microcosms. The optimized enantioseparation conditions were achieved using a macrocyclic antibiotic ristocetin A-bonded CSP (150 × 2.1 mm i.d.; particle size 5 μm) under reversed-phase elution mode. The method was validated using a mineral salts medium as matrix and presented selectivity and linearity over a concentration range from 5 μg L−1 (quantification limit) to 350 μg L−1 for each enantiomer. The method was successfully applied to evaluate biodegradation of ofloxacin enantiomers at 250 μg L−1 by an activated sludge inoculum. Ofloxacin (racemic mixture) and (S)-enantiomer (levofloxacin) were degraded up to 58 and 52%, respectively. An additional degradable carbon source, acetate, enhanced biodegradation up to 23%. (S)-enantiomer presented the highest extent of degradation (66.8%) when ofloxacin was supplied along with acetate. Results indicated slightly higher biodegradation extents for the (S)-enantiomer when supplementation was done with ofloxacin. Degradation occurred faster in the first 3 days and proceeded slowly until the end of the assays. The chromatographic results from LC-FD suggested the formation of the (R)-enantiomer during levofloxacin biodegradation which was confirmed by LC–MS with a LTQ Orbitrap XL.
Keywords: Ofloxacin; Levofloxacin; Macrocyclic antibiotic-based stationary phases; Enantioselectivity; Biodegradation; Activated sludge;

In this study, a novel phosphate-Zr4+ immobilized metal affinity membrane (IMAM) was prepared based on the surface initiated-atom transfer radical polymerization technique for the selective adsorption of phosphoprotein. The adsorption capacity and selectivity of the phosphate-Zr4+ IMAM were evaluated by using the mixture of standard phosphoproteins (β-casein, ovalbumin) and nonphosphoproteins (bovine serum albumin and lysozyme) as model samples. The adsorption isotherms and competitive adsorption results demonstrated that the phosphate-Zr4+ IMAM had higher binding capacity and selectivity for phosphoproteins over nonphosphoproteins. Moreover, the phosphate-Zr4+ IMAM exhibited good re-usability and re-productivity. Finally, the phosphate-Zr4+ IMAM was applied to separate phosphoprotein from real samples with high purity. Therefore, the as-prepared phosphate-Zr4+ IMAM could be a promising affinity material for the efficient enrichment of phosphoprotein from complex bio-samples.
Keywords: Immobilized metal affinity membrane; Phosphoprotein; Adsorption; Surface initiated-atom transfer radical polymerization;

Separation and purification of astaxanthin from Phaffia rhodozyma by preparative high-speed counter-current chromatography by Xiping Du; Congcong Dong; Kai Wang; Zedong Jiang; Yanhong Chen; Yuanfan Yang; Feng Chen; Hui Ni (191-197).
An effective high-speed counter-current chromatography (HSCCC) method was established for the preparative isolation and purification of astaxanthin from Phaffia rhodozyma. With a two-phase solvent system composed of n-hexane-acetone-ethanol-water (1:1:1:1, v/v/v/v), 100 mg crude extract of P. rhodozyma was separated to yield 20.6 mg of astaxanthin at 92.0% purity. By further one step silica gel column chromatography, the purity reached 99.0%. The chemical structure of astaxanthin was confirmed by thin layer chromatography (TLC), UV spectroscopy scanning, high performance liquid chromatography with a ZORBAX SB-C18 column and a Waters Nova-pak C18 column, and ESI/MS/MS.
Keywords: Astaxanthin; Phaffia rhodozyma; High-speed counter-current chromatography; Separation and purification;

The series of macroporous monolithic molecularly imprinted monoliths differed by pore size, column length (volume) and amount of template used for imprinting was synthesized using methacrylic acid and glycerol dimethacrylate as co-monomers and antibiotic ciprofloxacin as a template. The prepared monoliths were characterized regarding to their permeability, pore size, porosity, and resistance to the flow of a mobile phase. The surface morphology was also analyzed. The slight dependence of imprinting factor on flow rate, as well as its independence on pore size of macroporous molecularly imprinted monolithic media was observed. The column obtained at different conditions exhibited different affinity of ciprofloxacin to the imprinted sites that was characterized with Kdiss values in the range of 10−5–10−4  M. The solid-phase extraction of ciprofloxacin from such biological liquids as human blood serum, human urine and cow milk serum was performed using the developed monolithic columns. In all cases, the extraction was found to be 95.0–98.6%. Additionally, the comparison of extraction of three fluoroqinolone analogues, e.g. ciprofloxacin, levofloxacin and moxifloxacin, from human blood plasma was carried out. Contrary to ciprofloxacin extracted with more than 95%, this parameter did not exceed 40% for its analogues.
Keywords: Molecular imprinting; Macroporous polymer monoliths; Artificial receptors; Solid-phase extraction; Ciprofloxacin;

Development and evaluation of a liquid chromatography–mass spectrometry method for rapid, accurate quantitation of malondialdehyde in human plasma by Constance A. Sobsey; Jun Han; Karen Lin; Walter Swardfager; Anthony Levitt; Christoph H. Borchers (205-212).
Malondialdhyde (MDA) is a commonly used marker of lipid peroxidation in oxidative stress. To provide a sensitive analytical method that is compatible with high throughput, we developed a multiple reaction monitoring-mass spectrometry (MRM-MS) approach using 3-nitrophenylhydrazine chemical derivatization, isotope-labeling, and liquid chromatography (LC) with electrospray ionization (ESI)–tandem mass spectrometry assay to accurately quantify MDA in human plasma.A stable isotope-labeled internal standard was used to compensate for ESI matrix effects. The assay is linear (R2  = 0.9999) over a 20,000-fold concentration range with a lower limit of quantitation of 30 fmol (on-column). Intra- and inter-run coefficients of variation (CVs) were <2% and ∼10% respectively. The derivative was stable for >36 h at 5 °C. Standards spiked into plasma had recoveries of 92–98%. When compared to a common LC-UV method, the LC–MS method found near-identical MDA concentrations. A pilot project to quantify MDA in patient plasma samples (n = 26) in a study of major depressive disorder with winter-type seasonal pattern (MDD-s) confirmed known associations between MDA concentrations and obesity (p < 0.02). The LC–MS method provides high sensitivity and high reproducibility for quantifying MDA in human plasma. The simple sample preparation and rapid analysis time (5x faster than LC-UV) offers high throughput for large-scale clinical applications.
Keywords: Malondialdehyde; Plasma; 3-Nitrophenylhydrazine; Derivatization; LC/MRM–MS; Isotope-labeling;

Simultaneous assessment of endogenous thiol compounds by LC–MS/MS by Yao Sun; Tong Yao; Xiucai Guo; Ying Peng; Jiang Zheng (213-221).
Biological thiol compounds are very important molecules that participate in various physiological events. Alteration in levels of endogenous thiols has been suggested as a biomarker of early stage of pathological changes. We reported a chemical derivatization- and LC–MS/MS-based approach to simultaneously determine thiol compounds including glutathione (GSH), cysteine (Cys), N-acetyl cysteine (NAC), homocysteine (Hcy), and cysteinylglycine (CysGly) in biological samples. Thiol-containing samples were derivatized with monobromobimane (mBrB) at room temperature, followed by LC–MS/MS analysis. Assessment of the analytes with baseline separation was completed within 10 min, using a gradient elution on a C18 reversed-phase column. Excellent linearity was observed for all analytes over their concentration ranges of 1–400 μM. The lowest limits of detection (S/N = 3) in a range from 0.31  fmol (for NAC) to 4.98  fmol (for CysGly) were achieved. The results indicate that this approach was sensitive, selective, and well suited for high-throughput quantitative determination of the biological thiols.
Keywords: LC–MS/MS; Simultaneous assessment; Thiol compounds;

Interactions between DPPC as a component of lung surfactant and amorphous silica nanoparticles investigated by HILIC-ESI–MS by Yuliya E. Silina; Jennifer Welck; Annette Kraegeloh; Marcus Koch; Claudia Fink-Straube (222-229).
Display OmittedThis paper reports a rapid HILIC-ESI–MS assay to quantify dipalmitoylphosphatidylcholine (DPPC) as component of lung surfactant for nanosafety studies. The technique was used to investigate the concentration-dependent sorption of DPPC to two-sizes of amorphous SiO2 nanoparticles (SiO2-NPs) in a MeOH:H2O (50/50 v/v) mixture and in cell culture medium. In MeOH:H2O (50/50 v/v), the sorption of DPPC was positively correlated with the nanoparticles concentration. A substantial affinity of small amorphous SiO2-NPs (25 nm) to DPPC standard solution compared to bigger SiO2-NPs (75 nm) was not confirmed for biological specimens. After dispersion of SiO2-NPs in DPPC containing cell culture medium, the capacity of the SiO2-NPs to bind DPPC was reduced in comparison to a mixture of MeOH:H2O (50/50 v/v) regardless from the nanoparticles size.Furthermore, HILIC-ESI–MS revealed that A549 cells internalized DPPC during growth in serum containing medium complemented with DPPC. This finding was in a good agreement with the potential of alveolar type II cells to recycle surfactant components.Binding of lipids present in the cell culture medium to amorphous SiO2-NPs was supported by means of HILIC-ESI–MS, TEM and ICP-MS independently.
Keywords: Amorphous SiO2-NPs; A549 cells; DPPC; Solid phase extraction; HILIC-ESI–MS; Phosphor; TEM; ICP-MS;

A new and facile rhodamine B (RhB)-imprinted polymer nanoshell coating for SiO2 nanoparticles was readily prepared by a combination of silica gel modification and molecular surface imprinting. The RhB-imprinted polymers (RhB-MIPs) were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and UV–vis spectroscopy; the binding properties and selectivity of these MIPs were investigated in detail. The uniformly imprinted nanoparticles displayed a rather thin shell thickness (23 nm) with highly effective recognition sites, showing homogenous distribution and monolayer adsorption. The maximum MIP adsorption capacity (Q m) was as high as 45.2 mg g−1, with an adsorption equilibrium time of about 15 min at ambient temperature. Dynamic rebinding experiments showed that chemical adsorption is crucial for RhB binding to RhB-MIPs. The adsorption isotherm for RhB-MIPs binding could also be described by the Langmuir equation at different temperatures and pH values. Increasing temperature led to an enhanced Q m, a decreased dissociation constant (K' d), and a more negative free energy (ΔG), indicating that adsorption is favored at higher temperatures. Moreover, the adsorption capacity of RhB was remarkably affected by pH. At pH > 7, the adsorption of RhB was driven by hydrogen bonding interactions, while at pH < 7 electrostatic forces were dominant. Additionally, the MIPs also showed specific recognition of RhB from the standard mixture solution containing five structurally analogs. This method was also successfully employed to determine RhB content in red wine and beverages using three levels of spiking, with recoveries in the range of 91.6–93.1% and relative standard deviations lower than 4.1%.
Keywords: Silica nanoparticle; Surface imprinting; Rhodamine B; Solid-phase extraction;

Corrigendum to “Simultaneous determination of itraconazole, hydroxy itraconazole, keto itraconazole and N-desalkyl itraconazole concentration in human plasma using liquid chromatography with tandem mass spectrometry” [J. Chromatogr. B 1020 (2016) 111–119] by Xiaorong Liang; Michael Van Parys; Xiao Ding; Ning Zeng; Luke Bi; Drew Dorshorst; Janine McKnight; Dennis Milanowski; Jialin Mao; Yuan Chen; Joseph A. Ware; Brian Dean; Cornelis E.C.A. Hop; Yuzhong Deng (239).