Journal of Chromatography B (v.1038, #C)

Component analysis and target cell-based neuroactivity screening of Panax ginseng by ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry by Jinbin Yuan; Yang Chen; Jian Liang; Chong-Zhi Wang; Xiaofei Liu; Zhihong Yan; Yi Tang; Jiankang Li; Chun-Su Yuan (1-11).
Display OmittedGinseng is one of the most widely used natural medicines in the world. Recent studies have suggested Panax ginseng has a wide range of beneficial effects on aging, central nervous system disorders, and neurodegenerative diseases. However, knowledge about the specific bioactive components of ginseng is still limited. This work aimed to screen for the bioactive components in Panax ginseng that act against neurodegenerative diseases, using the target cell-based bioactivity screening method. Firstly, component analysis of Panax ginseng extracts was performed by UPLC-QTOF-MS, and a total of 54 compounds in white ginseng were characterized and identified according to the retention behaviors, accurate MW, MS characteristics, parent nucleus, aglycones, side chains, and literature data. Then target cell-based bioactivity screening method was developed to predict the candidate compounds in ginseng with SH-SY5Y cells. Four ginsenosides, Rg2, Rh1, Ro, and Rd, were observed to be active. The target cell-based bioactivity screening method coupled with UPLC-QTOF-MS technique has suitable sensitivity and it can be used as a screening tool for low content bioactive constituents in natural products.
Keywords: Panax ginseng; Component analysis; Drug screening; Target cell extraction; UPLC-QTOF-MS; SH-SY5Y cell;

Trimethylamine-N-oxide (TMAO) is produced in the liver from trimethylamine (TMA) and is an important cellular osmolyte and potential atherogenic factor. Taurine is involved in cholesterol metabolism and also serves as a cellular osmolyte. Given their significant biological functions, the development of reliable measurement techniques is crucial to further study their role in health and diseaseA new ultrahigh performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of TMA, TMAO, and taurine in plasma and urine. The method consisted of a deproteinization step using methanol/acetonitrile (15:85) that contained 0.2% formic acid and isotope-labeled internal standards. Samples were separated by centrifugation and injected into the UHPLC system. Quantification was conducted using a triple-quadrupole mass spectrometer detector with electrospray ionization interface in positive mode.The limits of detection ranged from 0.08 to 0.12 μmol/L. The calibration curves were linear (r  ≥ 0.999) over the range examined (0.15–400 μmol/L) for all compounds. The inter- and intra-day coefficients of variations were ≤14.5% for TMA and ≤8% for TMAO and taurine. TMAO and taurine were found to be stable in EDTA plasma for at least 14 months at −70 °C. Mean recoveries ranged from 95% to 109% and the relative matrix effects were ≤4.0%. The method was applied to study physiological and pre-analytical factors in plasma and urine samples.The new UHPLC–MS/MS method has good accuracy, precision, and recovery. The assay combines simple sample processing with a short run time, making it well suited for high-throughput routine clinical or research purposes.
Keywords: Trimethylamine N-oxide; Taurine; Plasma; Urine; Stability; Pre-analytical;

Amphetamine and derivatives in natural weight loss pills and dietary supplements by capillary electrophoresis-tandem mass spectrometry by Vagner Bezerra dos Santos; Daniela Daniel; Melina Singh; Claudimir Lucio do Lago (19-25).
A capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method for amphetamine (AM), phentermine (PTM), methamphetamine (MAM), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), and methylenedioxyethylamphetamine (MDEA) in commercial samples of homeopathic and phytotherapic medicines and dietary supplements is presented. The samples were submitted to a modified QuEChERS extraction procedure (at apparent pH 13) followed by electrophoretic separation in 0.1 mol L−1 formic acid electrolyte (pH 2.4) and detection by ESI–MS/MS. A polyvinyl alcohol coated capillary was employed to prevent the adsorption of the analytes to the capillary wall. The limits of detection and quantitation were from 0.02 to 0.06 μg L−1 and from 0.06 to 0.21 μg L−1, respectively, with recovery ranging from 85 to 123% and the standard deviations were not greater than 6.1%. In addition, the separation occurs in less than six minutes.
Keywords: Capillary electrophoresis; Tandem mass spectrometry; Amphetamines; Amphetamine derivatives; Natural weight loss pills; Illegal drugs;

RP-HPLC detection and dosage method for acrylic monomers and degradation products released from implanted medical devices by Lionel Tortolano; Senda Hammami; Katia Manerlax; Bernard Do; Najet Yagoubi (26-33).
Acrylic copolymers are useful in medical therapeutics. As in dental implants or intraocular lenses, acrylics are present in many medical devices or drug adjuvants. Industrial using of acrylics is still important in painting or textile manufacturing. Scientific research background has proved that acrylic suffer for depolymerized and cross-linking mechanisms under heating and photo-oxidative conditions. Those aging processes could lead to release of unreacted monomers and degradation products.We developed a new RP-HPLC method with good resolution, recovery, linearity, detection and quantification limits that is efficient for acrylic monomers quantification in in vitro and in vivo saline solution matrices. This method allows the detection of copolymer and medical devices degradation products too. Both the limit of quantification and the limit of detection for monomers and degradation products are above cytotoxic concentrations for human epithelial cells. Those biological results confirm the interest of the method for dosage of unreacted acrylics after polymerization and for the research of degradation products in body fluids as aqueous humor.
Keywords: Medical devices; RP-HPLC; Polymer; Acrylic monomers; Cytotoxicity;

In this work, the ultrahigh-performance liquid chromatography quadrupole orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was applied to the rapid screening, identification and quantification of the illegal adulterated glucocorticoids in herbal medicines. The mass spectrometer was operated in positive ion mode and Full MS/dd-MS2 (data-dependent MS2) mode, where selected ions were subjected to a dd-MS2 scan with given fragmentation energy following a Full MS scan. The application of 70 000 FWHM mass resolution and narrow mass windows (5 ppm) effectively improve the selectivity of the method, and a single injection was sufficient to perform the simultaneous screening and identification/quantification of 14 glucocorticoids in 15 min. The method validation including selectivity, sensitivity, calibration curve, accuracy, precision, recovery, matrix effect and stability were evaluated. The results of all analytes showed excellent linear relationship while all coefficient of determination (r 2) were > 0.9990 over wide concentration ranges (e.g., 5–1000 ng/mL for hydrocortisone butyrate, r 2  = 1.0000). The recoveries were in the range of 86.1–102.7%, while the matrix effects ranged from 95.8%–105.8%. Accuracies and precisions were performed. The intra- and inter-day accuracies ranged from 90.6% to 108.9%, while the intra- and inter-day precisions were in the range of 0.5% to 8.5%. Finally, the established method was employed to detect illegal adulterated glucocorticoids in herbal medicines. It will provide more reliable technical basis for the drug quality supervision department and ensure public health.
Keywords: Orbitrap; Glucocorticoid; Illegal adulteration; Screening; Identification;

In this study, we describe a simple and rapid method for the determination of the antipsychotic drug clozapine and five commonly co-administered antidepressants – bupropion, mirtazapine, sertraline, clomipramine and citalopram – in serum, plasma and whole blood. Sample preparation includes solid phase extraction of analytes and determination of drug concentrations by gas chromatography–mass spectrometry without any derivatization steps. The method was fully validated according to international criteria and can be successfully applied for routine analyses. Correlation coefficients of calibration curves for the tested drugs in the three specimens were in the range 0.9977–0.9999. Intra-day and inter-day precisions ranged from 0.81–7.85% and 3.60–12.91% respectively for the studied analytes and matrices. Recoveries were satisfactory for different concentrations of each drug in each specimen allowing accurate determinations in the range from sub-therapeutic to toxic levels. The presented method shows acceptable sensitivity, linearity in wide concentration ranges (sub-therapeutic, therapeutic, supra-therapeutic/toxic levels), it is simple and rapid and it is applicable for qualitative and quantitative routine toxicological analyses of clinical and postmortem cases.
Keywords: Clozapine; Antidepressants; Monitoring; GC–MS; Autopsy;

Pathology laboratories are encouraged to participate in External Quality Assurance (EQA) schemes to estimate the degree of possible bias in their test results. In such schemes, typically the median of results reported by laboratories is used as the “target value” for assessment of accuracy. However, the ideal situation is where reference values with low measurement uncertainties are provided by a reference laboratory using reference methodologies. The National Measurement Institute of Australia (NMIA) recently provided reference values for an EQA for testosterone in human serum coordinated by the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP). Reference values were determined using a novel isotope-dilution two-dimensional ultra-performance liquid chromatography tandem mass spectrometry (2D-UPLC–MS/MS) procedure. Samples were subjected to solvent extraction and solid-phase extraction clean-up prior to instrument analysis. A second analytical method employing different sample clean-up and analysis procedures was also developed for confirmatory analyses of samples. In this method, serum extracts were cleaned-up using high performance liquid chromatography (HPLC) and analysed by gas chromatography-high resolution mass spectrometry (GC-HRMS). The availability of two orthogonal methods for sample analysis facilitated the rigorous investigation of bias in the 2D-UPLC–MS/MS method. Reference values were assigned to 6 EQA samples containing testosterone at 0.2 ng/g–5.4 ng/g. The uncertainties in the reference values were low and ranged from 3.3%–9.0% at a level of confidence of 95%. The reference values can be used by laboratories in the EQA to obtain information on bias in their methods and allow an accuracy-based grading of their performance.
Keywords: Two-dimensional liquid chromatography; IDMS; Selectivity; Matrix interference; Measurement bias;

ORKAMBI (ivacaftor-lumacaftor [LUMA]) and KALYDECO (ivacaftor; IVA) are two new breakthrough cystic fibrosis (CF) drugs that directly modulate the activity and trafficking of the defective CFTR underlying the CF disease state. Currently, no therapeutic drug monitoring assays exist for these very expensive, albeit, important drugs. In this study, for the first time HPLC and LC–MS methods were developed and validated for rapid detection and quantification of IVA and its major metabolites hydroxymethyl-IVA M1 (active) and IVA-carboxylate M6 (inactive); and LUMA in the plasma and sputum of CF patients. With a mobile phase consisting of acetonitrile/water:0.1% formic acid (60:40 v/v) at a flow rate of 1 mL/min, a linear correlation was observed over a concentration range from 0.01 to 10 μg/mL in human plasma (IVA R2  > 0.999, IVA M1 R2  > 0.9961, IVA M6 R2  > 0.9898, LUMA R2  > 0.9954). The assay was successfully utilized to quantify the concentration of LUMA, IVA, M1 and M6 in the plasma and sputum of CF patients undergoing therapy with KALYDECO (IVA 150 mg/q12 h) or ORKAMBI (200 mg/q12 h LUMA-125 mg/q12 h IVA). The KALYDECO patient exhibited an IVA plasma concentration of 0.97 μg/mL at 2.5 h post dosage. M1 and M6 plasma concentrations were 0.50 μg/mL and 0.16 μg/mL, respectively. Surprisingly, the ORKAMBI patient displayed very low plasma concentrations of IVA (0.06 μg/mL) and M1 (0.07 μg/mL). The M6 concentrations (0.15 μg/mL) were comparable to those of the KALYDECO patient. However, we observed a relatively high plasma concentration of LUMA (4.42 μg/mL). This reliable and novel method offers a simple and sensitive approach for therapeutic drug monitoring of KALYDECO and ORKAMBI in plasma and sputum. The introduction of the assay into the clinical setting will facilitate pharmacokinetics/pharmacodynamic analysis and assist clinicians to develop more cost effective and efficacious dosage regimens for these breakthrough CF drugs.
Keywords: Ivacaftor; Lumacaftor; LC/MS; Cystic fibrosis; Plasma; Sputum;

Semi-preparative HPLC separation followed by HPLC/UV and tandem mass spectrometric analysis of phorbol esters in Jatropha seed by Santi Kongmany; Truong Thi Hoa; Le Thi Ngoc Hanh; Kiyoshi Imamura; Yasuaki Maeda; Luu Van Boi (63-72).
Phorbol esters (PEs) are well known as the main toxic compounds in Jatropha curcas Linnaeus (JCL), the seed oil of which has been considered as a major feedstock for the production of biodiesel. In the present study, we investigated a series of PEs extracted from JCL seed kernels with methanol (MeOH), and identified more than seven components contained in the PEs. The isolation of main five components of a series of PEs was revised using a semi-preparative reversed phase HPLC analysis of ODS-3 column. The five peaks of components were successfully isolated, and peaks of J2, J3, J5, and J7 were assigned to be Jatropha factors C1, C2, C3, and C4/5, but J6 was a mixture of Jatropha factor C6 and its isomer based on the data of UV and LC–MS/MS, and J2 was identified using 1H NMR analysis. By characterization using LC–MS/MS analysis, all components of a series of PEs were elucidated to be the 12-deoxy-16-hydroxyphorbol esters composed of isomeric form of dicarboxylic groups with same m/z value of 380.
Keywords: Preparative HPLC; Reverse phase ODS-3 column; Jatropha curcas seeds; Phorbol ester isomers; Tandem mass spectrometry characterization; 1H NHR spectrometry;

Synthesis and characterisation of magnetised Dacron-heparin composite employed for antithrombin affinity purification by Aurenice Arruda Dutra das Mercês; Ricardo de Souza Silva; Karciano José Santos Silva; Jackeline da Costa Maciel; Givanildo Bezerra Oliveira; Davian Martinez Buitrago; José Albino Oliveira de Aguiar; Luiz Bezerra de Carvalho-Júnior (73-79).
Human antithrombin is a blood derivative widely used in the treatment of coagulation dysfunction. Affinity chromatography using heparin (HEP) derivatives is usually used for antithrombin purification. In this study, an affinity procedure based on a magnetic Dacron-HEP composite is proposed. Dacron was firstly converted to Dacron-hydrazide and magnetised by co-precipitation with of Fe2+/Fe3+ (mDAC). HEP was activated by carbodiimide and N-hydroxysuccinimide and covalently linked to mDAC (mDAC-HEP). EDX and infrared spectra analyses confirmed each synthesis step of mDAC-HEP. This composite exhibited superparamagnetism behaviour. Human plasma was incubated with mDAC-HEP (fresh and stored over a long period) and washed with phosphate buffer containing increasing concentrations of NaCl. Human plasma antithrombin activity was reduced by approximately 20% in the presence of the 1.0 M NaCl fraction, and this eluate was able to prolong coagulation time (aPTT) using both preparations. Electrophoresis of the eluates revealed bands corresponding to the expected size of antithrombin (58 kDa). The mDAC-HEP particles are reusable. This method presents the following advantages: easy, low-cost synthesis of the composite, magnet-based affinity purification steps, and reusability.
Keywords: Antithrombin; Heparin; Immobilisation; Magnetic support; Purification;

Most previous assays for thiopental are time-consuming due to laborious sample extraction steps prior to analysis using gas chromatography or high pressure liquid chromatography. Here, we describe the first high-throughput liquid chromatography – tandem mass spectrometry (LC–MS/MS) method for quantification of thiopental concentrations in samples of human plasma. Robotic on-line solid phase extraction (SPE) was used to elute the analytes of interest from samples of human plasma (50 μL) loaded onto C18 SPE cartridges to which were added aliquots (50 μL) of internal standard solution (thiopental-d5 100 ng/mL) and 0.5% formic acid in water (100 μL). Cartridges were washed using 10% methanol in ammonium acetate buffer (50 mM, pH 7) before elution with mobile phase comprising 0.1% formic acid in water and acetonitrile with a flow rate of 0.55 mL/min using a 7.2 min run time. The analytes were separated on a C18 XTerra® analytical column. Mass spectrometry detection was performed using a QTrap 5500 mass spectrometer (AB Sciex) with negative ionisation. The multiple reaction monitoring (MRM) transitions for thiopental and the internal standard were 241 → 58, and 246 → 58, respectively. The calibration curve was linear over a range of 6–600 ng/mL. Thiopental was stable in human plasma samples for at least 36 h in the autosampler, as well as after three cycles of freeze and thaw, and after 3 h storage at room temperature. The absolute recovery and matrix effect were 102% and 6.9%, respectively, and the within-run and between-run precision and accuracy were ≤15%. Our method is fully-validated and satisfies the requirements of the 2012 European Medicines Agency (EMEA) guideline for Bioanalytical Method Validation.
Keywords: Thiopental; LC–MS/MS; Bioanalysis; Method development; Validation; Solid phase extraction;

Individualized therapy is a recent approach aiming to specify dosage regimen for each patient according to its genetic state. Cancer chemotherapy requires continuous monitoring of the plasma concentration levels of active forms of cytotoxic drugs and subsequent dose adjustment. In order to attain optimum therapeutic efficacy, correlation to pharmacogenetics data is crucial. In this study, a specific, accurate and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) has been developed for determination of methotrexate (MTX), 6-mercaptopurine (MP) and its metabolite 6-thioguanine nucleotide (TG) in human plasma. Based on the basic character of the studied compounds, solid phase extraction using a strong cation exchanger was found the optimum approach to achieve good extraction recovery. Chromatographic separation was carried out using RP-HPLC and isocratic elution by acetonitrile: 0.1% aqueous formic acid (85:15 v/v) with a flow rate of 0.8 mL/min at 40 °C. The detection was performed by tandem mass spectrometry in MRM mode via electrospray ionization source in positive ionization mode. Analysis was carried out within 1.0 min over a concentration range of 6.25–200.00 ng/mL for the studied analytes. Validation was carried out according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained. The applicability of the assay for the monitoring of the MTX, MP and TG and subsequent application to personalized therapy was demonstrated in a clinical study on children with acute lymphoblastic leukemia (ALL). Results confirmed the need for implementation of reliable analysis tools for therapeutic dose adjustment.
Keywords: 6-Mercaptopurine; Acute lymphoblastic leukemia; Individualized therapy; LC–MS/MS; Leukemia; Methotrexate;

Identification of absolute conversion to geraldol from fisetin and pharmacokinetics in mouse by Jun Hyeon Jo; Jung Jae Jo; Jae-Mok Lee; Sangkyu Lee (95-100).
Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a flavonoid found in several fruits, vegetables, nuts, and wine and has anti-oxidant, anti-inflammatory, and anti-angiogenic properties. Geraldol is the 3′-methoxylated metabolite of fisetin (3,4′,7-trihydroxy-3′-methoxyflavone). The concentration of fisetin and geraldol in mouse plasma was determined by LC–MS/MS, following direct protein precipitation. These concentrations were determined after administration of fisetin at doses of 2 mg/kg (i.v.) and 100 and 200 mg/kg (p.o.). The method was validated in terms of linearity, accuracy, precision, matrix effect, and stability. The pharmacokinetics parameters of fisetin and geraldol were successfully determined using a validated method in mice. Results indicated that fisetin was very rapidly methylated to geraldol in vivo. Following administration of fisetin, it was observed that the Cmax and AUC values for geraldol were higher than those of fisetin. The absolute bioavailability of fisetin was calculated as 7.8% and 31.7% after oral administration of 100 and 200 mg/kg fisetin, respectively. This method was successfully applied to determine the pharmacokinetic parameters of fisetin and its main metabolite geraldol in mouse plasma. Geraldol was the dominant circulating metabolite after fisetin administration in vivo.
Keywords: Fisetin; Geraldol; Methylation; LC–MS/MS; Pharmacokinetics; Mouse;

LC–MS/MS method for quantitation of seven biomarkers in human plasma for the assessment of insulin resistance and impaired glucose tolerance by Qibo Zhang; Lisa A. Ford; Kelli D. Goodman; Tiffany A. Freed; Deirdre M. Hauser; Jessie K. Conner; Kate E.T. Vroom; Douglas R. Toal (101-108).
Early detection of insulin resistance (IR) and/or impaired glucose tolerance (IGT) is crucial for delaying and preventing the progression toward type 2 diabetes. We recently developed and validated a straightforward metabolite-based test for the assessment of IR and IGT in a single LC–MS/MS method. Plasma samples were diluted with isotopically-labeled internal standards and extracted by simple protein precipitation. The extracts were analyzed by LC–MS/MS for the quantitation of 2-hydroxybutyric acid (0.500–40.0 μg/mL), 3-hydroxybutyric acid (1.00–80.0 μg/mL), 4-methyl-2-oxopentanoic acid (0.500–20.0 μg/mL), 1-linoleoyl-2-hydroxy-sn-glycero-3-phosphocholine (2.50–100 μg/mL), oleic acid (10.0–400 μg/mL), pantothenic acid (0.0100–0.800 μg/mL), and serine (2.50–100 μg/mL). Liquid chromatography was carried out on a reversed phase column with a run time of 3.1 min and the mass spectrometer operated in negative MRM mode. Method validation was performed on three identical LC–MS/MS systems with five runs each. Sufficient linearity (R2  > 0.99) was observed for all the analytes over the ranges. The imprecision (CVs) was found to be less than 5.5% for intra-run and less than 5.8% for inter-run for the seven analytes. The analytical recovery was determined to be between 96.3 and 103% for the seven analytes. This fast and robust method has subsequently been used for patient sample analysis for the assessment of IR and IGT.
Keywords: Biomarkers; LC–MS/MS; Insulin resistance; Impaired glucose tolerance; Validation; Bioanalytical;

Highly sensitive LC–MS/MS methods for urinary biological monitoring of occupational exposure to cyclophosphamide, ifosfamide, and methotrexate antineoplastic drugs and routine application by Mireille Canal-Raffin; Karim Khennoufa; Béatrice Martinez; Yves Goujon; Celia Folch; Dominique Ducint; Karine Titier; Patrick Brochard; Catherine Verdun-Esquer; Mathieu Molimard (109-117).
Highly sensitive ESI-LC–MS/MS methods were developed for urinary biological monitoring of occupational exposure to cyclophosphamide (CP), ifosfamide (IF), and methotrexate (MTX), which are hazardous antineoplastic drugs frequently handled by healthcare professionals. Extraction methods consisted of liquid/liquid extraction for simultaneous urinary CP and IF assays, and of solid phase extraction for the urinary MTX assay. A good linearity (r2 > 0.997), precision (CV < 14.6%), and accuracy (bias < 9.9%) were achieved for all compounds. The limit of detection (LOD) was 10 pg/ml and the lower limit of quantification (LOQ) was 20 pg/ml for all three drugs. Applying these methods in routine, more than 116 healthcare professionals occupationally exposed to antineoplastic drugs were monitored and 635 urines were analysed. Eleven healthcare professionals (9.5%) were found to be contaminated to at least one of the three antineoplastic drugs. Among analysed urines, 22 samples were found positives. The measured concentrations ranged from 20.1 to 1850 pg/ml and, for six samples, concentrations were at CP trace level, between the LOD and LOQ values (10–20 pg/ml). Such efficient analytical tools combining high specificity with high sensitivity are essential for reliable detection and routine biological monitoring of healthcare professionals occupationally exposed to these widely used antineoplastic drugs. These methods allow to monitor the healthcare professionals exposure to antineoplastic drugs in the aim to assess the effectiveness of collective and individual protective measures.
Keywords: Antineoplastic drugs; Urine; Occupational exposure; Biological monitoring; LC–MS/MS; Healthcare professionals;

1H NMR-based metabonomic study on the effects of Epimedium on glucocorticoid-induced osteoporosis by Sina Pan; Ali Chen; Zhihui Han; Yaling Wang; Xin Lu; Yongxia Yang (118-126).
Glucocorticoids are widely used in clinical practice for the treatment of many immune-mediated and inflammatory diseases, and glucocorticoid-induced osteoporosis (GIO) is the most common type of secondary osteoporosis. Epimedium is one of the most commonly used traditional Chinese medicines for treating osteoporosis. In the present study, we systematically analysed the metabonomic characteristics of GIO model rats and elucidated the therapeutic effect of Epimedium by using a 1H NMR-based metabonomic approach in conjunction with multivariate data analysis. Rats in treatment and model groups were injected with dexamethasone (0.1 mg/kg/day) for 5 weeks. Simultaneously, two treatment groups were orally administered Epimedium (10 g/kg/day) or Alendronate (1.2 mg/kg/day) for 5 weeks. In GIO model rats, lipid and lactate levels in serum were increased, while creatine/creatinine, PC/GPC, taurine, glycine and β-glucose levels were decreased. In urine, GIO rats had higher levels of phenylacetylglycine but lower levels of 2-oxoglutarate, citrate, creatine/creatinine, taurine, PC/GPC and hippurate than controls. Epimedium reversed the aforementioned metabolic alterations in multiple metabolic pathways involved in energy, lipid, amino acid and phospholipid metabolism and gut microbiota derangement. Our results indicated that Epimedium had significant effects in the prevention and treatment of osteoporosis. It is concluded that 1H NMR metabonomics is a useful method for studying the metabolic effects of traditional Chinese medicine from a systematic and holistic view.
Keywords: Metabonomics; Osteoporosis; Epimedium; 1H NMR; Multivariate data analysis;

Determination of antipsychotic drugs in hospital and wastewater treatment plant samples by gas chromatography/tandem mass spectrometry by F. Logarinho; T. Rosado; C. Lourenço; M. Barroso; A.R.T.S. Araujo; E. Gallardo (127-135).
The development and performance evaluation of a method for the simultaneous determination of six antipsychotic drugs in hospital effluents and wastewater treatment plants (WWTP) samples are herein presented. The method involves an off-line mixed mode (reversed-phase and strong cation exchange) solid phase extraction (SPE) with gas chromatography (GC) coupled to tandem mass spectrometry (MS/MS). The present methodology was validated following internationally accepted criteria, and the studied parameters included selectivity, linearity, limits of detection (LOD) and quantitation (LLOQ), instrumental limits, precision and accuracy, stability and recovery. The procedure was linear for concentrations ranging from 0.1 to 10 μg/L (0.02 to 2 μg/L for haloperidol), with determination coefficients higher than 0.99 for all analytes. Intra- and inter-day precision was lower than 15% for all analytes at the studied concentrations, while accuracy remained between a ±15% interval. Recoveries ranged from 31% to 83%. Low LODs were achieved, between 2 and 10 ng/L, allowing a reliable identification of all analytes at trace levels, using only 50 mL as sample volume. All studied parameters complied with the defined criteria and the method was successfully applied to gather preliminary results of the determination of antipsychotics on hospital effluents and on influent and effluent of WWTPs, opening perspectives for the study of their fate in the aquatic environment.
Keywords: Antipsychotics; Solid phase extraction; Gas chromatography–tandem mass spectrometry; Wastewaters samples;

Tacrolimus is a commonly used immunosuppressive agent in organ transplant recipients. Therapeutic drug monitoring (TDM) of tacrolimus is essential to adjust the dose and achieve optimal immunosuppression level. Routine TDM is practiced using whole blood samples obtained through venipuncture. However, tacrolimus concentration that is present in oral fluid (OF) can theoretically represent the free or pharmacologically active form of tacrolimus. In this study, we report the development and validation of a rapid, sensitive and selective liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for quantification of tacrolimus in OF. Chromatographic separation were achieved on an Acquity UPLC BEH C18 column by a gradient elution using 2 mM ammonium acetate/0.1% (v/v) formic acid in water (mobile phase A) and in methanol (mobile phase B) with a 2.2 min chromatographic run time. Tacrolimus was extracted from OF with acetonitrile as the precipitating solvent. Both extraction and chromatography was optimized to provide optimal sample cleanness, negligible matrix effect, and optimal specificity. The lower limit of quantification (LLOQ) for the assay was set at 10 pg/mL using a 50 μL aliquot of OF obtained by passive drool. The method demonstrated adequate accuracy and precision with accuracy between 94.5–103.6%, and coefficient of variation ranging from 4 to 9.8%. Tacrolimus was stable in OF for up to one month at −80 °C and the extracted matrix was stable up to 48 h in auto-sampler at 20 °C. The method showed high reproducibility as confirmed by incurred sample reanalysis test. This assay was employed in several clinical pharmacokinetic studies and could successfully measure the concentration of tacrolimus in OF.
Keywords: Assay; Immunosuppressant; LC–MS/MS; Oral fluid; Saliva; Tacrolimus; Therapeutic drug monitoring; Transplantation; Validation;