Journal of Chromatography B (v.1040, #C)

Cellular fatty acid (FA) profiles have been acknowledged as biomarkers in various human diseases. Nevertheless, common FA analysis by gas chromatography mass spectrometry (GC–MS) requires long analysis time. Hence, there is a need for feasible methods for high throughput analysis in clinical studies.FA was extracted from red blood cells (RBC) and derivatized to fatty acid methyl esters (FAME). A method using gas chromatography tandem mass spectrometry (GC–MS/MS) with ammonia-induced chemical ionization (CI) was developed for the analysis of FA profiles in human RBC. We compared this method with classical single GC–MS using electron impact ionization (EI). The FA profiles of 703 RBC samples were determined by GC–MS/MS.In contrast to EI ammonia-induced CI resulted in adequate amounts of molecular ions for further fragmentation of FAME. Specific fragments for confident quantification and fragmentation were determined for 45 FA. The GC–MS/MS method has a total run time of 9 min compared to typical analysis times of up to 60 min in conventional GC–MS. Intra and inter assay variations were <10% for all FA analyzed. Analysis of RBC FA composition revealed an age-dependent increase of the omega-3 eicosapentaenoic and docosahexaenoic acid, and a decline of the omega-6 linoleic acid with a corresponding rise of the omega-3 index.The combination of ammonia-induced CI and tandem mass spectrometry after GC separation allows for high-throughput, robust and confident analysis of FA profiles in the clinical laboratory.
Keywords: Fatty acids; Gas chromatography; Tandem mass spectrometry; Chemical ionization; High-throughput analysis;

Quality assessment of Traditional Chinese Medicine using HPLC-PAD combined with Tchebichef image moments by Xue Wang; Bao Qiong Li; Min Li Xu; Jin Jin Liu; Hong Lin Zhai (8-13).
Traditional Chinese medicines have gotten growing attention and the product quality also became uneven. In this paper, Tchebichef image moments coupled with high performance liquid chromatography with photodiode array detector were proposed to the similarity analysis of the fourteen batches of Pudilan Xiaoyan tablets (Chinese medicine complex), which were based on the grayscale images of three dimensional (3D) fingerprint spectra. Hierarchical cluster analysis was used for displaying the classification results. As a comparison, traditional principal component analysis (PCA) method based on the chromatograms under single-fixed wavelength was carried out on the same samples. The analytical results indicated that the proposed approach could provide more accurate and reasonable results owing to its powerful invariant and multi-resolution capability of image moments.
Keywords: Similarity analysis; Traditional Chinese medicines; Grayscale image; Tchebichef moment; Principal component analysis;

Liquid chromatography-tandem mass spectrometry assay for the quantification of niraparib and its metabolite M1 in human plasma and urine by L. van Andel; Z. Zhang; S. Lu; V. Kansra; S. Agarwal; L. Hughes; M.M. Tibben; A. Gebretensae; H. Rosing; J.H.M. Schellens; J.H. Beijnen (14-21).
Niraparib (MK-4827) is a novel poly(ADP-Ribose) polymerase (PARP) inhibitor currently investigated in phase III clinical trials to treat cancers. The development of a new drug includes the characterisation of absorption, metabolism and excretion (AME) of the compound. AME studies are a requirement of regulatory agencies and for this purpose bioanalytical assays are essential. This article describes the development and validation of a bioanalytical assay for niraparib and its carboxylic acid metabolite M1 in human plasma and urine using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Sample pre-treatment involved protein precipitation for plasma and dilution of urine samples using acetonitrile-methanol (50:50, v/v). Final extracts were injected onto a SunFire C18 column and gradient elution using 20 mM ammonium acetate (mobile phase A) and formic acid:acetonitrile:methanol (0.1:50:50, v/v/v) (mobile phase B) was applied. Detection was performed on an API5500 tandem mass spectrometer operating in the positive electrospray ionisation mode applying multiple reaction monitoring (MRM). The assay was successfully validated in accordance with the Food and Drug Administration and latest European Medicines Agency guidelines on bioanalytical method validation and can therefore be applied in pharmacological clinical studies.
Keywords: Niraparib; MK-4827; HPLC–MS/MS; Bioanalysis; Pharmacokinetics;

Display OmittedThe purpose of this study is the development and validation of a simple, novel, selective and fast off-line microextraction technique combining capillary electrophoresis with in-column field-amplified sample injection (FASI) for the simultaneous determination of capecitabine (CAP) and its active metabolite, 5-Fluorouracil (5-FU), in human plasma. At the moment, there is a lack of using cost-effective CE tool combined with novel miniaturized sample clean-up techniques for analysis of these important anti-cancer agents in plasma samples. This paper intends to fill this gap and describe a simple off-line sample pretreatment by means of AgNPs@MCM-41 reinforced hollow fiber Solid/Liquid phase microextraction (AgNPs@MCM41-HF-SLPME) with subsequent quantitation by FASI-CE. The separation of analytes was performed using a BGE containing 60 mM phosphate-Tris buffer (pH 7) with 10% methanol as an organic modifier. Before sample loading, a short water plug (50 mbar, 3 s) was injected to permit FASI for stacking. Various parameters affecting the off-line microextraction efficiency as well as FASI were optimized. Migration time was found to be 6.6 (±0.1) min for 5-FU and 7.4 (±0.2) min for CAP. The linearity, precision, accuracy, recovery, selectivity, specificity, stability as well as the robustness of the method was evaluated from spiked plasma samples during the course of validation. The results revealed that the presented technique demonstrates acceptable accuracy and precision, miniaturized sample preparation and a reduced need for complicated equipment along with an acceptable analysis time. The validated method was successfully applied to determine CAP and 5-FU in patient’s plasma samples.
Keywords: Capecitabine; FASI-CE; 5-Fluorouracil; HF-SLPME; Validation; Nanotechnology;

In this study, the adsorption properties of nine resins including polyamide resin (30–60), polyamide resin (60–100) AB-8, S-8, D-101, NKA-9, NKA-II, XDA-1 and XDA-4 for enrichment phenolics of the olive waste were investigated. XDA-1 and NKA-II were chosen for further study due to their outstanding adsorption and desorption capacity. XDA-1 and NKA-II had similar adsorption and desorption behaviors for phenolics of olive waste. The adsorption mechanism could be better explained by pseudo second-order kinetics model and Freundlich isotherm model, and the adsorption processes were spontaneously and exothermic. The experiment of gradient elution were carried out through treated XDA-1 resins column, the result indicated the total phenolics were mainly obtained from the 40% and 60% ethanol fraction. The order of antioxidant capacity by DPPH • , ABTS+ • radical and FRAP assay was similar with the content of phenolics from fraction elution. The compositions of phenolics from different elution fractions were determined by reversed phase-HPLC-DAD method. Gallic acid, hydroxytyrosol, tyrosol and ferulic acid were the major constituent in the fraction elute, and the content of hydroxytyrosol reached to the 41.69 mg/g. The above results revealed the synergistic effects of the different phenolics contribute to the antioxidant capacity.
Keywords: Olive waste; Resins; Adsorption; Phenolic compounds; Antioxidants;

Purification of high-purity glycyrrhizin from licorice using hydrophilic interaction solid phase extraction coupled with preparative reversed-phase liquid chromatography by Wenyi Yu; Hongli Jin; Aijin Shen; Liang Deng; Jianlian Shi; Xingya Xue; Yadong Guo; Yanfang Liu; Xinmiao Liang (47-52).
Glycyrrhizin (GA), a major bioactive compound in licorice, has been extensively used throughout the world as a medicine to treat chronic viral hepatitis and allergic dermatitis. In this study, a new method based on hydrophilic interaction solid phase extraction (HILIC-SPE) and preparative reversed-phase liquid chromatography (prep-RPLC) was developed to purify GA with high purity from the complex licorice extract. Via evaluation of retention behavior of GA and flavonoids in different commercially available columns, a hydrophilic column——Click XIon was finally chosen for the purification due to its excellent resolution toward GA and flavonoids under HILIC mode. To optimize the SPE elution conditions, relative factors including water content, pH and ionic strength had been investigated in chromatographic condition. The result indicated that the most appropriate water content was 30% and pH at 4.00, as well as salt concentration should be controlled at 5 mM. In addition, the optimization revealed that GA experiences both hydrophilic interaction and ion-exchange interaction on the Click XIon material. According to the chromatographic evaluation, the optimized conditions were applied to HILIC-SPE to enrich GA from licorice, which leads to an increased content of GA from 13.67% to 64.22%. Finally, prep-RPLC was performed to obtain GA with purity higher than 99.00%,which demonstrating great prospect in large-scale preparation of GA.
Keywords: Glycyrrhizin; Hydrophilic interaction solid phase extraction; Licorice; Preparative reversed-phase liquid chromatography;

Is a deuterated internal standard appropriate for the reliable determination of olmesartan in human plasma? by Edyta Piórkowska; Jacek Musijowski; Katarzyna Buś-Kwaśnik; Piotr J. Rudzki (53-59).
A right choice of the internal standard is one of the most challenging tasks during bioanalytical method development. Surprisingly, among the HPLC–MS methods for the determination of a cardiovascular drug olmesartan in plasma only structural analogues or similar compounds were used as internal standards. We have tried to answer the question whether the stable isotope labelled (deuterated) internal standard, as recommended by regulatory agencies, can be used for the reliable determination of olmesartan in human plasma. An HPLC–MS method using this standard in a simplified liquid–liquid extraction procedure led to accurate and precise results in the linearity range of 5–2500 ng/mL. The method is well suited for pharmacokinetic studies following a single 40 mg oral dose of olmesartan medoxomil in humans. The method was fully validated according to international guidelines and successfully applied in a bioequivalence study in humans. The use of deuterated olmesartan as the internal standard afforded a reliable tool for regulatory bioanalysis that can indirectly contribute to therapy efficacy and improve the safety of patients treated with generic medicines.
Keywords: Olmesartan; Liquid chromatography/mass spectrometry; Stable isotopically labelled internal standard; Bioanalytical method validation; Pharmacokinetics;

Immunochromatographic determination of bacopaside I in biological samples by Sontaya Sookying; Dumrongsak Pekthong; Sarawut Oo-puthinan; Watoo Phrompittayarat; Waraporn Putalun; Hiroyuki Tanaka; Jie Xing; Zhaoqi Zhan; Nantaka Khorana; Nitra Nuengchamnong; Kornkanok Ingkaninan (60-66).
We describe a novel immunochromatographic method for qualitative and quantitative analyses of bacopaside I, a bioactive constituent in Bacopa monnieri (L.) Wettst in biological samples. The assay was performed on polyethersulfone membrane using a polyclonal antibody raised against bacopaside I. The finalised method could quantitatively determine bacopaside I in the range of 31.3-1000.0 ng and the detection and quantification limits were 1.0 and 31.3 ng, respectively. The percentage recoveries of bacopaside I in blood and urine were nearly 100% indicating the accuracy of the extraction. The method was then applied for the determination of this compound in rat serum, urine and feces after an oral dose of 15 mg/kg body weight. About 4% of the ingested dose of bacopaside I was detected in rat feces but none was detected in serum and urine which accorded with results from liquid chromatography tandem mass spectrometry. The accuracy, selectivity, sensitivity of the method are appropriate for in vivo pharmacokinetic studies.
Keywords: Bacopaside I; Metabolism; Immunochromatography; Bacopa monnieri;

The efficacy of phenylephrine (PE) is controversial due to its extensive pre-systemic metabolism through sulfation to form phenylephrine-3-O-sulfate (PES). Hence quantitation of PES is important in order to study the metabolism of PE. There are no published methods available for direction detection of PES. We have developed and validated a hydrophilic interaction liquid chromatography (HILIC) method for the direct detection of PES and simultaneous detection of PE to study the enzyme kinetics and metabolism of PE to enable approaches to reduce the presystemic metabolism of PE. This is the first method which facilitates direct detection of PES and simultaneous detection of PE using a zwitterionic HILIC column with improved sensitivity in a single short run. The observed quantitative ranges of our method for PE and PES were 0.39–200 μM and 0.0625–32 μM (respectively) with a run time of 6.0 min. The method was applied to the determination of PE and PES in LS180 human intestinal cell line, recombinant enzymes and human intestinal cytosol (HIC).
Keywords: Sulfation; Sulfotransferase; HILIC; Phenylephrine; Drug metabolism;

Simple and sensitive HPLC-UV method for determination of bexarotene in rat plasma by Jong Bong Lee; Atheer Zgair; Tae Hwan Kim; Min Gi Kim; Sun Dong Yoo; Peter M. Fischer; Pavel Gershkovich (73-80).
Bexarotene is currently marketed for treatment of cutaneous T-cell lymphoma and there has been growing interest in its therapeutic effectiveness for other cancers. Neuroprotective effects of bexarotene have also been reported. In this study, a simple, sensitive and cost-efficient bioanalytical method for determination of bexarotene in rat plasma was developed and fully validated. The method utilises protein precipitation with acetonitrile and liquid-liquid extraction with n-hexane-ethyl acetate (10:1, v/v). An HPLC-UV system with a Waters Atlantis C18 column and a mobile phase of acetonitrile-ammonium acetate buffer (10 mM, pH 4.1) at a ratio of 75:25 (v/v), flow rate 0.2 mL/min was used. Chromatograms were observed by a UV detector with wavelength set to 259 nm. Intra- and inter-day validations were performed and sample stability tests were conducted at various conditions. The applicability of the method was demonstrated by a pharmacokinetic study in rats. Intravenous bolus dose of 2.5 mg/kg was administered to rats and samples were obtained at predetermined time points. As a result, pharmacokinetic parameters of AUCinf (4668 ± 452 h ng/mL), C0 (6219 ± 1068 ng/mL) and t1/2 (1.15 ± 0.02 h) were obtained. In addition, the developed method was further applied to human and mouse plasma to assess the suitability of the method for samples from other species.
Keywords: Bexarotene; HPLC-UV; Preclinical pharmacokinetics; Rat plasma; Human plasma; Mouse plasma;

Acute kidney injury (AKI) is a major global public health problems, as it causes high morbidity and serious injury to renal function. However, the etiology for AKI is not very clear. In this study, a serum metabolite profile analysis was performed to identify potential biomarkers for gentamicin-induced AKI and to investigate the mechanism of action of Amomum compactum (AC) used for treatment. A metabonomics approach by ultra-performance liquid chromatography together with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was applied to perform the analysis. Back propagation (BP) neural network models were established for classifying data from the control, model, and AC-treated groups. Accuracy rate for classification was 91.7% in positive ion mode and 87.5% in negative ion mode. By orthogonal partial least squares discriminant analysis (OPLS-DA), 29 metabolites were identified as potential biomarkers of gentamicin-induced AKI. Most of them are related to phospholipid metabolism. After treatment with AC, the levels of sphingomyelin, sphingosine, phytosphingosine, and arachidonic acid were restored to normal. The results indicate that AC plays a protective role in rats with gentamicin-induced AKI via regulation of the phospholipid metabolic pathway. In this work, early biomarkers of AKI has been identified and underlying therapeutic mechanism of AC has been understood, therefore, AC can be further investigated and tested for clinical application.
Keywords: Amomum compactum; Metabonomics; UPLC–MS; Acute kidney injury; BP neural network algorithm; Gentamicin;

Linezolid is the first approved oxazolidinone antibacterial agent, whereas PH027 is a novel compound of the same class that exhibits good in vitro antibacterial activity. The objective of this study was to develop an UPLC–MS/MS assay for the analysis of linezolid and PH027 in plasma and to apply the method for comparative pharmacokinetic and tissue distribution studies of both compounds.Plasma samples and calibrators were extracted with diethyl ether after addition of the internal standard solution. After evaporation of the ether layer, the residue was reconstituted in mobile phase and injected into UPLC–MS/MS. The mobile phase consisted of 2 mM ammonium acetate buffer solution and acetonitrile (70:30) at a flow rate of 0.2 ml/min. Separation was achieved using UPLC BEH C18 column, and quantitative determination of the analytes was performed using multiple-reaction monitoring (MRM) scanning mode. The method was validated by analyzing quality control tissue homogenate samples, and was applied to analyze tissue homogenate samples obtained following IV injections of linezolid and PH027 in rabbits.The developed UPLC–MS/MS method was linear in the concentration range of 50–5000 ng/ml. Validation of the method proved that the method's precision, selectivity and stability were all within the acceptable limits. Linezolid and PH027 concentrations were accurately determined in the quality control tissue homogenate samples, and analysis of samples obtained following IV administration of the two compounds showed that the tissue to plasma concentration ratio of PH027 was higher than that of linezolid probably due to its higher lipophilicity.The developed UPLC–MS/MS method for the analysis of linezolid and PH027 in rabbit's plasma can accurately determine the concentrations of these compounds in different tissues.
Keywords: UPLC–MS/MS method validation; Linezolid; Oxazolidinone antibacterial agents; Tissue analysis; Tissue distribution;

Display OmittedA high-performance liquid chromatography-fluorescence detection method was developed for the simultaneous determination of thiodicarb and its degradation product methomyl in animal-derived food products, including chicken muscle, beef, pork, table eggs, and milk. Thiodicarb is known to degrade during analysis; therefore, a thorough investigation was carried out, revealing that thiodicarb degrades to methomyl immediately after spiking into a matrix of animal-derived food products. Consequently, thiodicarb was determined as the sum of the parent compound and methomyl. Samples were extracted with acetonitrile and sodium salts, and purified using solid-phase extraction (SPE). The limits of detection (LODs) and quantification (LOQs) were 0.0013 and 0.004 mg/kg, respectively, for both analytes in various matrices. Seven-point external calibration curves were obtained, and they showed excellent linearity with determination coefficients (R2 ) ≥ 0.999 for all tested matrices. The method was validated at three fortification levels (LOQ, LOQ × 2, and LOQ × 10) in triplicate with average recoveries ranging from 84.24 to 112.8% (for methomyl) and relative standard deviations (RSDs) ≤ 6.5% in all matrices. The converted recoveries of thiodicarb in various matrices ranged from 74.80 to 107.80% with RSDs ≤ 4.5%. The identities of both compounds in standard solutions and for recovery were confirmed using liquid chromatography-tandem mass spectrometry (LC–MS/MS). The developed method was further validated by accurate reproduction at another laboratory. Finally, the method was applied to market samples collected from different areas (and, in the case of milk, different brands), and none of the samples tested positive for thiodicarb or methomyl. In conclusion, the developed method can be successfully applied for a single-run analysis of thiodicarb and methomyl in livestock products.
Keywords: Thiodicarb; Methomyl; Degradation product; Quantification; Simultaneous determination; Livestock products; HPLC;

Selectivity evaluation and separation of human immunoglobulin G, Fab and Fc fragments with mixed-mode resins by Ying-Di Luo; Qi-Lei Zhang; Xiao-Ming Yuan; Wei Shi; Shan-Jing Yao; Dong-Qiang Lin (105-111).
Adsorption selectivity is critical important for mixed-mode chromatography with specially-designed ligands. Human immunoglobulin G (hIgG), Fc and Fab fragments were used in the present work to evaluate adsorption behavior and binding selectivity of four mixed-mode resins with the ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. The resins showed an obvious pH-dependent adsorption behavior. High adsorption capacities were found at neutral pH for hIgG, Fc and Fab, and almost no adsorption happened under acidic conditions. An adsorption selectivity index was proposed to evaluate separation efficiency. High specificity of hIgG/Fc was found at pH 8.9 for MEP resin, and for W-ABI resin at pH 8.0 and 8.9. In addition, isothermal titration calorimetry was used to evaluate ligand-protein interactions. Finally, the separation of hIgG and Fc (1:1) was optimized with mixed-mode resins, and the best separation performance was obtained with W-ABI-based resin. Loading at pH 8.0 resulted in the flow through of Fc with purity of 90.4% and recovery of 98.8%, while elution at pH 3.6 provided hIgG with purity of 99.7% and recovery of 86.5%.
Keywords: Mixed-mode chromatography; Adsorption selectivity; Separation; Human immunoglobulin G; Fc fragment; Fab fragment;

Vine tea (Ampelopsis grossedentata), a widely used healthy tea, beverage and herbal medicine, exhibited strong antioxidant activity. However, systematic purification of antioxidants, especially for those with similar structures or polarities, is a challenging work. Here, we present a novel at-line hyphenation of high-speed countercurrent chromatography with Sephadex LH-20 column chromatography (HSCCC-Sephadex LH-20 CC) for rapid and efficient separation of antioxidants from vine tea target-guided by 1,1-diphenyl-2-picryl-hydrazyl radical-high performance liquid chromatography (DPPH-HPLC) experiment. A makeup pump, a six-port switching valve and a trapping column were served as interface. The configuration had no operational time and mobile phase limitations between two dimensional chromatography and showed great flexibility without tedious sample-handling procedure. Seven targeted antioxidants were firstly separated by stepwise HSCCC using petroleum ether–ethyl acetate–methanol–water (4:9:4:9, v/v/v/v) and (4:9:5:8, v/v/v/v) as solvent systems, and then co-eluted antioxidants were on-line trapped, concentrated and desorbed to Sephadex LH-20 column for further off-line purification by methanol. It is noted that six elucidated antioxidants with purity over 95% exhibited stronger activity than ascorbic acid (VC). More importantly, this at-line hyphenated strategy could sever as a rapid and efficient pathway for systematic purification of bioactive components from complex matrix.
Keywords: HSCCC; Sephadex LH-20 CC; At-line hyphenation; Antioxidant; Vine tea;

Bentysrepinine (Y101), a derivative of repensine (a compound isolated from Dichondrarepens Forst), is a novel phenyalanine dipeptide inhibiting DNA-HBV and cccDNA activities and is currently under development for the treatment of hepatitis B virus (HBV)-infected hepatitis. Our previous study implied that there might be an existence of extensive metabolism of Y101 in rats. Therefore, it is necessary to perform metabolic profiling study to further evaluate its safety and drug-like properties. In this study, the metabolism of Y101 in rats was investigated by a convincible five-step strategy to characterize metabolites in plasma and that excreted into urine, bile and feces. The five-step strategy was realized by using an combined workflow on two different MS platforms, including various scan modes of liquid chromatography with hybrid quadruple-linear ion trap mass spectrometry (LC-QTRAP-MS/MS) and various post-acquiring data mining tools of liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS). QTOF MS/MS was employed as a powerful complementary tool to enable high confidence of metabolites identification using its functions of accurate MS and MS/MS fragmentation. As a result, a total of 30 metabolites were detected, including 25 phase I and 5 phase II metabolites. Among them, four primary metabolites (M6–M9) were further identified by comparing with the authentic standards chemically synthesized. The possible metabolic pathways of Y101 in rats were proposed to be amide hydrolysis, monohydroxylation, dihydroxylation, N-oxidation, demethylation, methylation, glucosidation and glucuronidation. This is the first study of the metabolism of Y101 in rats. The five-step strategy was successfully used to systematically characterize metabolites of Y101 in rats, and it would be generally applied for metabolite identification of new drug candidate.
Keywords: Y101; Metabolite; Identification; Five-step strategy; LC-QTRAP-MS/MS; LC-QTOF-MS/MS;

A selective and rapid method was developed for quantification of curcumin in human plasma and food samples using molecularly imprinted magnetic multiwalled carbon nanotubes (MMWCNTs) which was characterized with EDX and FESEM. The role of sorbent mass, volume of eluent and sonication time on response in solid phase microextraction procedure were optimized by central composite design (CCD) combined with response surface methodology (RSM) using Statistica. Preliminary experiments reveal that among different solvents, methanol:dimethyl sulfoxide (4:1 V/V) led to efficient and quantitative elution of analyte. A reversed-phase high performance liquid chromatographic technique with UV detection (HPLC-UV) was applied for detection of curcumin content. The assay procedure involves chromatographic separation on analytical Nucleosil C18 column (250 × 4.6 mm I.D., 5 μm particle size) at ambient temperature with acetonitrile-water adjusted at pH = 4.0 (20:80, v/v) as mobile phase at flow rate of 1.0 mL min−1, while UV detector was set at 420 nm. Under optimized conditions, the method demonstrated linear calibration curve with good detection limit (0.028 ng mL−1) and R2  = 0.9983. The proposed method was successfully applied to biological fluid and food samples including ginger powder, curry powder, and turmeric powder.
Keywords: Central composite design; Curcumin; HPLC-UV; Magnetic multiwalled carbon nanotubes; Molecularly imprinted polymer;

Effect of Panax notoginseng saponins on the pharmacokinetics of aspirin in rats by Zhihao Tian; Huanhuan Pang; Shouying Du; Yang Lu; Lin Zhang; Huichao Wu; Shuang Guo; Min Wang; Qiang Zhang (136-143).
Aspirin (ASA) is widely used to treat fever, pain, inflammation and cerebral infarction in clinic. Panax Notoginseng Saponins (PNS) is the extracts of Panax Notoginseng (PN)-a traditional Chinese medicine extensively used in cardiovascular diseases. Panax notoginseng saponins and ASA are both widely used to treat cerebral infarction in China. Good results in clinical practice have been achieved when the two drugs were taken together. To investigate the effect of PNS on ASA in vivo, the concentrations of salicylic acid (SA) in blood were measured after oral administration of ASA or ASA combined with PNS by UPLC–MS/MS. Sample preparation was carried out by the protein precipitation technique with an internal Saikosaponin A standard. The separation of two components was achieved by using an ACQUITY UPLC ®BEH C18 Column (1.7 μm 2.1 × 100 mm) by gradient elution using water (containing 0.2% formic acid) and acetonitrile (containing 0.2% formic acid) as the mobile phase at a flow rate of 0.2 mL/min. The pharmacokinetic parameters were determined by using non-compartmental analysis. The results suggested that drug–drug interaction in vivo existed between PNS and ASA. The concentration of the SA was increasing when the two drugs were administered together. The transport of ASA and SA in MDCK −MDR1 cell monolayer was used to verify this conclusion. The values of apparent permeability coefficients (Papp) were significantly increased when the two drugs were used together. This result suggested PNS could increase the gastrointestinal tract absorption of ASA and SA. These findings provide more insight for wise use of two drugs to treat or prevent cardiovascular diseases.
Keywords: Aspirin; Panax notoginseng saponins; Drug-drug interaction; Pharmacokinetic; Transport;

Highly sensitive LC–MS/MS methods for the determination of seven human CYP450 activities using small oral doses of probe-drugs in human by Alexia Grangeon; Sophie Gravel; Fleur Gaudette; Jacques Turgeon; Veronique Michaud (144-158).
Cocktails composed of several Cytochrome P450 (CYP450)-selective probe drugs have been shown of value to characterize in vivo drug-metabolism activities. Our objective was to develop and validate highly sensitive and selective LC–MS/MS assays allowing the determination of seven major human CYP450 isoenzyme activities following administration of low oral doses of a modified CYP450 probe-drug cocktail in patients. The seven-drug cocktail was composed of caffeine, bupropion, tolbutamide, omeprazole, dextromethorphan, midazolam (all administered concomitantly) and chlorzoxazone (administered separately) to phenotype for CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 and 2E1, respectively. Serial plasma and urine samples were collected over an 8 h period. The probe-drugs and their respective metabolites were measured in both human plasma and urine, except for omeprazole (plasma only) and chlorzoxazone (urine only). Samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS) using a Phenomenex Luna PFP (2) analytical column (3 μm PFP(2) 150 × 3 mm) for chromatographic separation. Optimal detection was achieved based on 3 different analytical methods; (1) isocratic elution with a mobile phase consisting of acetonitrile and water both fortified with 0.01% formic acid for the analysis of bupropion, tolbutamide, chlorzoxazone and their respective metabolites; (2) isocratic elution with a mobile phase composed of acetonitrile and ammonium formate (pH 3; 10 mM) for omeprazole, dextromethorphan, midazolam and their metabolites; (3) for caffeine and paraxanthine, gradient elution using acetonitrile and 0.01% formic acid in water was used. All calibration functions were linear for all probe drugs and metabolites in both matrices over wide analytical ranges. The main advantages of our methods are the use of specific probe drugs available in most countries, the administration of small doses of probe drugs, small volume of plasma required for the analyses and simple and rapid extraction procedures. The methods met all requirements of specificity, sensitivity, linearity, precision and accuracy and stability generally accepted in bioanalytical chemistry. Determination of CYP450 phenotype in patients will permit characterization of their capacities to metabolize drugs through CYP450 under specific conditions at a definite time. This tool will be highly clinically relevant since wide intersubject variability observed in drug response is largely explained by variation in drug metabolism; it will be particularly useful in polymedicated patients with multiple comorbidities. So far, our CYP450 cocktail assays have been successfully applied to phenotype CYP450 activities in patients.
Keywords: LC–MS/MS; CYP450 probe-drug cocktail; Drug metabolism; Drug-drug interactions; Phenotyping;

LC–MS using electrospray ionisation (negative ion mode) and low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis, together with the multiple analyte adduct formation with the components of the mobile phase, were applied to analyse valproic acid in human serum with LC–MS3. The CID-fragmentation of the precursor analyte adduct [M+2CH3COONa-H] was applied in the method validation (307.1/225.1/143.0). Chromatographic separation was performed with a Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm column and the elution with a mobile phase consisting of A (H2O/methanol = 95/5, v/v) and B (H2O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and 0.1% acetic acid. A binary flow pumping mode with a total flow rate of 0.400 mL/min was used. The calculated limit of detection/quantification of the method calibrated in the range of 10–200 μg/mL was 0.31/1.0 μg/mL. The sample preparation based on protein precipitation with 1 mL of H2O/methanol solution (3/97, v/v) with 10 mM sodium acetate and 100 mM acetic acid. On the basis of the experiments performed could be demonstrated, that multiple analyte adduct formation can be applied to generate MS3 quantitation of analytes with problematic fragmentation. The presented new strategy makes the analysis of small drugs, which do not produce any stable product ions at all, on the basis of LC–MS3 possible.
Keywords: Adduct formation; Adduct fragmentation; Valproic acid; VPA; LC–MS3;

Quantifying cortisol concentrations in fish eggs is important to understand the effects of environmental conditions on maternal physiological condition and on egg provisioning and quality. Data are particularly relevant to studies of the ecology of threatened species such as lake sturgeon (Aciperser fulvescens) as well as assessments of larval physical and behavioral phenotypes, fish health and caviar quality in sturgeon aquaculture. This study focuses on development of bioanalytical methods for high sensitivity and robust determination of cortisol in sturgeon eggs. Sample preparation was optimized after investigating protein precipitation and liquid-liquid extraction techniques. Ethyl acetate was found to be the most efficient solvent (recovery parameter) and also provided the best sample clean up (matrix effect parameter). The method was determined to be linear for cortisol concentrations between 0.025 and 100 ng/mL. The limits of detection and quantification were 0.025 and 0.1 ng/mL respectively. Intra- and inter-day performances of the method were validated at three concentrations (0.25; 10 and 100 ng/mL). The method was applied to field-collected samples for the determination of endogenous cortisol in lake sturgeon eggs. Cortisol was detected in all egg samples and statistical analysis showed significant differences between fertilized and non-fertilized eggs.
Keywords: Sturgeon; Egg; Cortisol; LC–MSMS; LLE;

A rapid method for sensitive profiling of folates from plant leaf by ultra-performance liquid chromatography coupled to tandem quadrupole mass spectrometer by M.J.I. Shohag; Qianying Yang; Yanyan Wei; Jie Zhang; Farhana Zerin Khan; Michael Rychlik; Zhenli He; Xiaoe Yang (169-179).
Previous published methods for the analysis of folates are time consuming because of lengthy sample extraction, clean-up and total running time. This study details the development and validation of a rapid, sensitive and robust method that combines a simple extraction step with ultra-performance liquid chromatography coupled to tandem quadrupole mass spectrometry. Here, we reported application of a tandem quadrupole mass spectrometer to analyze maximum seven vitamers of folate from plant origin. The analytical performance was evaluated by linearity, sensitivity, precision, recovery test and analysis of certified reference materials. The limit of detection and limit of quantification ranged between 0.003 and 0.021 μg/100 g FW and between 0.011 and 0.041 μg/100 g FW, respectively; the recovery and precession ranged from 71.27 to 99. 01% and from 1.7 to 7.8% RSD, respectively, depending upon folate vitamers. This newly developed and validated method is rapid (a chromatographic run time of 5 min), easy to be performed (no laborious and time consuming clean-up) and can be used to simultaneously analyze seven vitamers of folate from plant sources.
Keywords: Analysis; Extraction; Folate; Ultra-performance liquid chromatography; Tandem quadrupole mass spectrometer; Leaf sample;

Gas chromatography-triple quadrupole mass spectrometry (GC-QqQMS) was applied for the determination of eight organochlorine pesticides (OCPs) in human serum. OCPs were extracted from the serum sample by solid phase extraction (SPE) and analyzed by gas chromatography mass spectrometry (GC–MS) or gas chromatography tandem mass spectrometry (GC–MS/MS). Electron ionization (EI) and negative chemical ionization (NCI) under two data acquisition modes, namely selected ion monitoring (SIM) and multiple reaction monitoring (MRM), were compared. The use of MRM generally provided higher selectivity and sensitivity because less interference from the sample matrix existed. The EI mode is more suitable for less electronegative compounds such as dichlorodiphenyldichloroethanes (DDDs) with detection limits ranging from 0.0060 to 0.060 ng/mL. In the NCI mode, MRM analysis provided good and lower detection limits (0.0011–0.0030 ng/mL) for pesticides containing more chlorines. The methods were validated by analyzing the pesticides in spiked serum at different levels with recoveries ranged from 83% to 116% and relative standard deviations of less than 10%. The developed method was applied for the determination of the OCPs in real human serum samples.
Keywords: Organochlorine pesticides; Serum; Negative chemical ionization; Multiple reaction monitoring;

This work aimed to establish a systematic strategy to enrich and separate quercetin-3-O-β-d-glucuronide (Q3GA) from lotus leaves with macroporous resin and semi-preparative HPLC. Six resins were tested, and LX-5 was chosen as the appropriate resin for Q3GA based on the adsorption and desorption performances. After one-step enrichment, the content of Q3GA increased from 2.15% in crude extract to 52.25% in 30% ethanol fraction with yield of 11.97%. The Q3GA was then isolated from the 30% ethanol fraction by semi-preparative HPLC, and the purity of Q3GA was above 98.00% with yield of 19.76%. These results suggested that the aforementioned strategy was a useful and economic method to enrich and separate Q3GA from lotus leaves. Additionally, the anti-inflammatory effect of Q3GA was evaluated in lipopolysaccharide-treated RAW264.7 macrophages, and the result demonstrated that Q3GA could significantly inhibit LPS-induced NO release in vitro in a dose-dependent manner compared with control group.
Keywords: Lotus leaves; Macroporous resin; Anti-inflammatory; Macrophages;

This paper reports a novel fabric phase sorptive extraction-high performance liquid chromatography-photodiode array detection (FPSE-HPLC-PDA) method for the simultaneous extraction and analysis of twelve azole antimicrobial drug residues that include ketoconazole, terconazole, voriconazole, bifonazole, clotrimazole, tioconazole, econazole, butoconazole, miconazole, posaconazole, ravuconazole, and itraconazole in human plasma and urine samples. The selected azole antimicrobial drugs were well resolved by using a Luna C18 column (250 mm × 4.6 mm; 5 μm particle size) in gradient elution mode within 36 min. The analytical method was calibrated and validated in the range from 0.1 to 8 μg/mL for all the drug compounds. Blank human plasma and urine were used as the sample matrix for the analysis; while benzyl-4-hydroxybenzoate was used as the internal standard (IS). The limit of quantification of the FPSE-HPLC-PDA method was found as 0.1 μg/mL and the weighted-matrix matched standard calibration curves of the drugs showed a good linearity upto a concentration of 8 μg/mL. The parallelism tests were also performed to evaluate whether overrange sample can be analyzed after dilution, without compromising the analytical performances of the validated method. The intra- and inter-day precision (RSD%) values were found ≤13.1% and ≤13.9%, respectively. The intra- and inter-day trueness (bias%) values were found in the range from −12.1% to 10.5%. The performances of the validated FPSE-HPLC-PDA were further tested on real samples collected from healthy volunteers after a single dose administration of itraconazole and miconazole. To the best of our knowledge, this is the first FPSE extraction procedure applied on plasma and urine samples for the simultaneous determination of twelve azole drugs possessing a wide range of logKow values (extending from 0.4 for fluconazole to 6.70 of butoconazole) and could be adopted as a rapid and robust green analytical tool for clinical and pharmaceutical applications.
Keywords: FPSE-HPLC-PDA; Method development; Plasma and urine; Sample preparation; Azoles antifungal drugs;

Vigabatrin, one of the most widely used antiepileptic drugs, is marketed and administered as a racemic mixture, while only S-enantiomer is therapeutically effective. In the present study, diacetyl-l-tartaric acid anhydride was used as an inexpensive and effective chiral derivatization reagent to produce tartaric acid monoester derivatives of vigabatrin enantiomers that could be readily resolved by reversed phase chromatography. Derivatization conditions were statistically optimized by response surface methodology, resulting in an optimal reaction temperature of 44 °C and an optimal reaction time of 30 min. The derivatized diastereomers of vigabatrin and internal standard (gabapentin) were analyzed using ultra-high performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry. For this analysis, an Agilent ZORBAX Rapid Resolution High Definition Eclipse Plus C18 column (100 mm × 2.1 mm, 1.8 μm) was employed for chromatographic separation using 10 mM ammonium formate (pH 3.0) and methanol as mobile phase at a flow rate of 0.2 mL min−1. The established method was validated in terms of specificity, linearity, precision, accuracy, dilution integrity, recovery, matrix effect, stability, and incurred sample reanalysis. It was linear over a range of 0.25–100.0 mg L−1 for both S- and R-enantiomers (R2  ≥ 0.9987 for both). Intra- and inter-day precisions and accuracies were within acceptable ranges. The method was successfully applied to determine the levels of vigabatrin enantiomers in mouse serum after administration of vigabatrin racemate.
Keywords: Vigabatrin; Enantioseparation; Diacetyl-l-tartaric anhydride; Chiral derivatization; Diastereomer;

Detection of dexmedetomidine in human breast milk using liquid chromatography–tandem mass spectrometry: Application to a study of drug safety in breastfeeding after Cesarean section by Rika Nakanishi; Manabu Yoshimura; Manabu Suno; Kento Yamamoto; Haruka Ito; Yohei Uchimine; Takashi Toriumi; Atsushi Kurosawa; Ami Sugawara; Takayuki Kunisawa (208-213).
Several analytical methods for dexmedetomidine (DEX) in human plasma have been published, but quantification of DEX in human breast milk has not been described. In this article, we describe a high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method suitable for quantification of DEX in human breast milk. DEX and an internal standard were extracted in a single liquid–liquid extraction step with diethyl ether from 200 μL of human breast milk. HPLC was performed on a TSK-gel ODS-100V column with isocratic elution at a flow rate of 0.3 mL/min using a mobile phase of 5 mM ammonium formate:0.1% formic acid in acetonitrile (60:40, v/v). Detection was performed using an API4000 mass spectrometer with positive electrospray ionization. The method was validated in the concentration range of 10 pg/mL (lower limit of quantification) to 2000 pg/mL. The intra- and inter-day accuracy were within ±5.8% and precision was <6.31% based on the coefficient of variation. The recoveries of DEX in human breast milk were 82.4–87.9%. Recovery and matrix effects were consistent and reproducible for human breast milk. The method is robust and was successfully used in a study of drug safety in breastfeeding in patients after administration of DEX.
Keywords: Dexmedetomidine; HPLC–MS/MS; Human breast milk; Breastfeeding; Relative infant dose;

Detection of trans-fatty acids by high performance liquid chromatography coupled with in-tube solid-phase microextraction using hydrophobic polymeric monolith by Fangling Wu; Jiabin Wang; Qi Zhao; Nan Jiang; Xucong Lin; Zenghong Xie; Jianhua Li; Qiqing Zhang (214-221).
Based on in-tube solid-phase microextraction (in-tube SPME) using a hydrophobic poly (octadecyl methacrylate-co-ethylene dimethacrylate) [poly (OMA-co-EDMA)] monolith, a simple high performance liquid chromatography (HPLC) method has been developed for detection of trans-fatty acids (TFAs) as their fatty acid methyl esters (FAMEs). The poly (OMA-co-EDMA) monolithic column with high hydrophobicity was specially prepared for simultaneous microextraction, pre-separation and purification for the analytes. The pre-separation selectivity, the extraction efficiency, and the purification effect for FAMEs were investigated respectively. Furthermore, some operation parameters have been optimized in detail with respect to satisfactory extraction efficiency of the target compounds. Under the optimized conditions, the enrichment factors for model FAMEs were ranged from 58.3 to 70.9, wide linear range (0.01-1.00 mg/kg) and low detection limits (LODs) (3.0-7.1 μg/kg) were achieved, respectively. In addition, recoveries of the method were in the range from 83.0 to 106.4% with low relative standard deviations (RSDs) of 3.2-4.7% (n = 4) at spiking levels of 0.05, 0.25 and 0.5 mg/kg, respectively. Finally, the proposed method was successfully applied for detection of TFAs in practical samples, which possesses short time-consuming, low disturbance and high detection sensitivity.
Keywords: HPLC; In-tube solid-phase microextraction; Poly (OMA-co-EDMA) monolithic column; Trans-fatty acids;

Untargeted serum metabolomics reveals Fu-Zhu-Jiang-Tang tablet and its optimal combination improve an impaired glucose and lipid metabolism in type II diabetic rats by Yi Tao; Xi Chen; Hao Cai; Weidong Li; Baochang Cai; Chuan Chai; Liuqing Di; Liyun Shi; Lihong Hu (222-232).
Fu-Zhu-Jiang-Tang tablet, a six-herb preparation, was proved to show beneficial effects on type II diabetes patients in clinical. This study aims to optimize the component proportion of the six-herb preparation and explore the serum metabolic signatures of type II diabetes rats after treatment with Fu-Zhu-Jiang-Tang tablet and its optimal combination. The component proportion of the preparation was optimized using uniform experimental design and machine learning techniques. Untargeted GC–MS metabolomic experiments were carried out with serum samples from model group and treatment groups. Data were normalized, multivariate and univariate statistical analysis performed and metabolites of interest putatively identified. 23 metabolites were significantly changed by Fu-Zhu-Jiang-Tang tablet treatment and the majority of these were decreased, including various carbohydrates (glucose, mannose, fructose, allose and gluconic acid), unsaturated fatty acids (palmitic acid, 9-octadecenoic acid, oleic acid, arachidonic acid), alanine, valine, propanoic acid, 3-hydroxybutyrate, along with pyrimidine and cholesterol. Increased concentrations of oxalic acid, leucine, glycine, serine, threonine, proline, lysine and citrate were observed. In the optimal combination-fed group, 21 metabolites were significantly affected and strikingly, the magnitudes of changes here were generally much greater than that of Fu-Zhu-Jiang-Tang tablet treated rats. 18 metabolites affected in both groups included various carbohydrates (mannose, glucose, allose, fructose and gluconic acid), unsaturated fatty acids (palmitic acid, 9-octadecenoic acid, oleic acid and arachidonic acid), short-chain fatty acids (oxalic acid, 3-hydroxybutyrate), and amino acids (alanine, valine, leucine, glycine, proline and lysine), as well as pyrimidine. Metabolites exclusively affected in optimal combination treated rat included succinic acid, cysteine and phenylalanine, whilst four metabolites (propanoic acid, citrate, serine and threonine) were only altered in Fu-Zhu-Jiang-Tang tablet treated rat. Our investigation demonstrated Fu-Zhu-Jiang-Tang tablet and its optimal combination treatments were able to ameliorate impaired glucose and lipid metabolism, down- regulate the high level of glucose to a lower level and reverse abnormal levels of metabolites in serum of type II diabetes rats. However, the optimal combination treatment was able to maximize the magnitudes of changes in some metabolites. These findings may be helpful in clarifying the anti-diabetic mechanism of FZJT tablet and its optimal combination.
Keywords: Metabolomic; Type II diabetes; GC–MS; Uniform experimental design;

Determination of β-hydroxy-β-methylbutyrate concentration and enrichment in human plasma using chemical ionization gas chromatography tandem mass spectrometry by Dillon K. Walker; John J. Thaden; Agata Wierzchowska-McNew; Marielle P.K.J. Engelen; Nicolaas E.P. Deutz (233-238).
Our objective was to develop a quick and simplified method for the determination of β-Hydroxy-β-methylbutyrate (HMB) and ɑ-ketoisocaproic acid (KIC) concentrations and enrichments by GC/MS/MS to determine the turnover rate of HMB in humans.In experiment 1, we provided a pulse of L-[5,5,5-2H3]leucine to younger adults in the postabsorptive state then collected blood samples over a 4 h time period. In experiment 2, we provided a pulse of [3,4,methyl-13C3]HMB to older adults in the postabsorptive state then collected blood samples over a 3 h time period. Plasma concentrations of KIC and HMB and MPE of KIC and HMB were determined by GC/MS/MS. Plasma enrichment of leucine was determined by LC/MS/MS.To determine plasma enrichment of [5,5,5-2H3]HMB and [3,4,methyl-13C3]HMB, samples were derivatized using pentafluorobenzyl bromide and analyzed using chemical ionization mode. The final methods used included multiple reaction monitoring of transitions 117.3 > 59.3 for M + 0 and 120.3 > 59.3 for M + 3. In experiment 1, peak MPE of Leu peaked at 9.76% generating a peak MPE of KIC at 2.67% and a peak HMB MPE of 0.3%. In experiment 2, the rate of appearance for HMB was  0.66 μmol/kg ffm/h. We calculated that production of HMB in humans accounts for 0.66% of total leucine turnover.
Keywords: HMB; Chemical ionization; Gas chromatography; Tandem mass spectrometry; Turnover;

Hormonal profiling: Development of a simple method to extract and quantify phytohormones in complex matrices by UHPLC–MS/MS by Carolina Delatorre; Ana Rodríguez; Lucía Rodríguez; Juan P. Majada; Ricardo J. Ordás; Isabel Feito (239-249).
Plant growth regulators (PGRs) are very different chemical compounds that play essential roles in plant development and the regulation of physiological processes. They exert their functions by a mechanism called cross-talk (involving either synergistic or antagonistic actions) thus; it is for great interest to study as many PGRs as possible to obtain accurate information about plant status. Much effort has been applied to develop methods capable of analyze large numbers of these compounds but frequently excluding some chemical families or important PGRs within each family. In addition, most of the methods are specially designed for matrices easy to work with. Therefore, we wanted to develop a method which achieved the requirements lacking in the literature and also being fast and reliable. Here we present a simple, fast and robust method for the extraction and quantification of 20 different PGRs using UHPLC–MS/MS optimized in complex matrices.
Keywords: Plant growth regulator; UHPLC; Tandem mass spectrometry; Pinus pinaster; Complex matrix;

Stanozolol is one of the most commonly abused anabolic androgenic steroids (AAS) by athletes and usually detected by its parent drug and major metabolites. However, its metabolic pathway is complex, varied and individually different, it is important to characterize its overall metabolic profiles and discover new and long-term metabolites for the aims of expanding detection windows. High performance liquid chromatography coupled with triple quadrupole mass spectrometer (HPLC–MS/MS) was used to analyze the human urine after oral administration of stanozolol. Multiple reaction monitoring (MRM), one of the scan modes of triple quadrupole mass spectrometer showing extremely high sensitivity was well used to develop a strategy for metabolic profiles characterization and long-term metabolites detection based on typical precursor to product ion transitions of parent drug and its major metabolites. Utilizing the characteristic fragment ions of stanozolol and its major metabolites as the product ions, and speculating unknown precursor ions based on the possible phase I and phase II metabolic reactions in human body, the metabolite profiles of stanozolol could be comprehensively discovered, especially for those unknown and low concentration metabolites in human urine. Then these metabolites were further well structure identified by targeted high resolution MS/MS scan of quadrupole-time of flight mass spectrometry (Q-TOF). Applying this strategy, 27 phase I and 21 phase II metabolites of stanozolol were identified, in which 13 phase I and 14 phase II metabolites have not been reported previously. The 9 out of 48 metabolites could be detected over 15 days post drug administration. This strategy could be employed effectively to characterize AAS metabolic profiles and discover unknown and long-term metabolites in sports drug testing.
Keywords: Stanozolol; Long-term and unknown metabolites; HPLC–MS/MS; Multi reaction monitoring; Doping-control;

Diagram of systematic identification strategy based on LC–MS for ZZHPD in rat.Display OmittedIdentification of metabolic profile of traditional Chinese medicine in vivo is always a challenge task. Usually, screening out and identifying the exogenous compounds manually from total ion chromatograms (TICs) of biologic samples is time-consuming and strenuous. In this study, a systematic identification strategy based on LC–MS was adopted to clarify the metabolic profiling of Zhi-Zi-Hou-Po decoction (ZZHPD) in rat. Bile, urine and feces samples of rat were obtained after oral administration and then analyzed by LC–MS after proper preparation. The xenobiotics were screened out from TICs globally and rapidly by untargeted metabolomics-driven strategy (UMDS) based on the combined of XCMS online (a web-based platform to process LC–MS data), MetAlign (a software to process LC–MS data) and SIMCA-P (a software for data analysis). Most of the xenobiotics were identified by means of series product ions filtering (sPIF), which was based on the database-hit of ZZHPD (including prototype and metabolites). Then the unmatched constituents were identified tentatively and their source and metabolic pathway were clarified by using diagnostic fragment ions strategy (DFIS). As a result, a total of 83 compounds including 44 prototype compounds and 39 metabolites were rapidly identified or tentatively characterized from the biologic samples, and among them, four of which were found for the first time. Further research on the correlations of these prototype compounds and metabolites revealed that glucuronidation is the main metabolic pathways of ZZHPD in rat bile and urine, while prototype compounds were the abundant ingredients detected in rat feces.
Keywords: Systematic identification strategy; Metabolic profile; Zhi-Zi-Hou-Po decoction; LC–MS; Chemometrics;

As a bionic chromatographic method, cell membrane chromatography (CMC) has been used widely in screening active components in traditional Chinese medicine. Nevertheless, few studies have characterized the affinity between drug and receptor by CMC model. In this study, the alpha 1 adrenoreceptor (α1A AR) high expression CMC method, combined with frontal analysis and stoichiometric displacement model respectively, was established for characterizing the affinity of seven alkaloids binding to the α1A AR. The results indicate that the seven alkaloids have similar interaction strengths with tamsulosin hydrochloride (α1A AR antagonist) between them and α1A AR. In addition, electrostatic force is the main intermolecular forces between tamsulosin hydrochloride and seven alkaloids and α1A AR. The study provides a versatile approach for the characterization the affinity between drug and receptor by CMC model.
Keywords: Cell membrane chromatography; α1 Adrenoreceptor; Frontal analysis; Stoichiometric displacement model;

Most of synthetic, modified oligonucleotides are candidates for therapeutics. Consequently, their quick, reliable and sensitive analysis has become a critical challenge for scientists. The main aim of the present study was an investigation of the influence of stationary phase type, mobile phase salt and its concentration on the separation and determination of the selected compounds by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Three different columns, together with ammonium acetate and formate, were applied for this purpose. The separation of mixtures of phosphorothioate oligonucleotides and their synthetic metabolites was successfully performed. Moreover, an attempt to isolate these compounds from human serum samples was also made together with their separation, qualification and quantification by hydrophilic interaction liquid chromatography and tandem mass spectrometry. The method developed during the study appeared to be effective and sensitive, due to the limit of quantification which equaled 142–165 ppb.
Keywords: Chromatographic column; Hydrophilic interaction liquid chromatography; Mass spectrometry; Oligonucleotides; Separation;

Suvorexant (Belsomra®) is a novel sedative hypnotic drug that is prescribed to promote sleep in patients with insomnia. It is the first of a new class of drugs classified as dual orexin receptor antagonists (DORAs). Sedative hypnotics with central nervous system depressant effects feature prominently in forensic toxicology investigations. For this reason, a new analytical method was developed to identify suvorexant in urine using liquid-liquid extraction (LLE) and gas chromatography/mass spectrometry (GC/MS). Due to the absence of a commercially available isotopically labeled internal standard, estazolam-D5 was used due to its azepine, triazole and chlorinated functionality. The limit of detection and limit of quantitation was 10 ng/mL and the linear range of the assay was 10–1000 ng/mL. Accuracy and precision (%CV) were 98–101% and <11% at 30, 250 and 800 ng/mL. Interferences from matrix and fifty common drugs were not present and processed samples were stable for 24 h at room temperature. Suvorexant is a new drug of significant forensic interest due to its hypnotic and central nervous system depressant effects. The absence of commercially available metabolites and its chromatographic properties present some challenges in terms of identification. Nevertheless, a robust, reliable and sensitive assay was developed to identify suvorexant using GC/MS analysis.
Keywords: Suvorexant; Liquid-liquid extraction; Gas chromatography mass spectrometry; Urine; Forensic toxicology;