Journal of Chromatography B (v.881-882, #C)

Implementation of a cost-effective HPLC/UV-approach for medical routine quantification of donepezil in human serum by Ralf Koeber; Hans-Hermann Kluenemann; Reinhold Waimer; Anton Koestlbacher; Markus Wittmann; Regina Brandl; Anett Doerfelt; Tatjana Jahner; Doris Melchner; Ekkehard Haen (1-11).
► We describe the first assay for TDM of donepezil by HPLC/UV in routine analysis. ► Three different UV detection wavelengths were used to check for peak impurities. ► Most of the dementia patients with donepezil treatment are underdosed. ► Off-label doses will be necessary to reach the therapeutic concentrations. ► Such an approach can be supported by TDM as suggested in this paper.A novel, simple, specific and sensitive high performance liquid chromatography (HPLC) assay for the detection and quantification of donepezil in serum of demented patients has been developed and validated. The analytical procedure involves an offline serum preextraction using solid phase extraction (SPE) cartridges (Oasis® HLB, Waters Co). The chromatographic analyses were performed on a Dionex HPLC system with a Phenomenex Luna Phenyl-Hexyl analytical column, and a mobile phase with the two components 0.02 mol/l phosphate buffer and acetonitrile. The flow rate was 0.4 ml/min. For the detection of donepezil three different UV wavelengths were used as an interference-control check. Interference tests between donepezil and 100 of the most commonly used concomitant medications allow quantification of donepezil under the polypharmaceutical conditions of the daily clinical routine. The retention time for donepezil was 12.1 min. The method was validated according to the guidelines of the Society of Toxicology and Forensic Chemistry (GTFCh): The calibration curve was linear over a concentration range from 5 to 160 ng/ml (n  = 8/r 2  > 0.999). No endogenous compounds were found to interfere with the analyte, which was shown by retention times for the comedication most often prescribed to demented patients. The method had an accuracy of >85%. Intra- and inter-assay coefficients of variation were <6% and <8%, respectively, at three different concentrations. The limit of quantification (LOQ) and the limit of detection (LOD) were found to be 6.1 and 1.7 ng/ml for donepezil. Application of the method to patient serum samples discovered that concentrations suggested as “therapeutic” in the literature may only be reached either by high, off-label dosages or by utilization of inhibitory metabolic effects of the comedication.
Keywords: Analytical methods; Dose-related reference range; Individualized dosage; Therapeutic drug monitoring (TDM); Antidementia drugs; Donepezil;

► UPLC–MS/MS method for the simultaneous detection and confirmation veterinary drugs in milk. ► Rapid tool for confirming the presence of 23 veterinary drug residues in milk. ► Extracting more than 30 samples in less than 1 h by using the proposed method.An ultraperformance liquid chromatography–electrospray tandem mass spectrometry (UPLC–MS/MS) method for the simultaneous detection and confirmation of 23 veterinary (multiclass) drugs in milk was developed and validated. The analytes were extracted by acetonitrile, evaporated and injected into the UPLC–MS/MS system on a Waters UPLC HSS T3 column in gradient mode. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two ion transitions per compound to provide a high degree of specificity. Results showed good repeatability, and recoveries for the 12 macrolide, 7 β-lactam and 2 lincosamide antibiotics and 2 other veterinary drugs (morantel, orbifloxacin) used in milk averaged 51.8–139.0%, 51.5–100.6%, 82.4–102.5% and 87.5–99.4%, respectively. The coefficients of variation (C.V.) of the recoveries were less than 15% for intraday and interday precisions. The limits of quantification (LOQs) were all lower than 5 ng/ml. This method was applied to 17 fresh milk samples and only lincomycin was found in milk samples under allowable levels. Overall, this method is a suitable and rapid tool to confirm the presence of 23 veterinary drug residues in milk.
Keywords: Antibiotics; Multiclass analysis; Milk; Tandem mass spectrometry;

Determination of methylphenidate and its metabolite ritalinic acid in urine by liquid chromatography/tandem mass spectrometry by Sharon M. Paterson; Grant A. Moore; Chris M. Florkowski; Peter M. George (20-26).
► Two Q1/Q3 transitions monitored in SRM for methylphenidate and ritalinic acid in urine. ► No interference with other prescription medications or drugs of abuse. ► Used to monitor compliance as well as screening for abuse in a clinical laboratory setting.Methylphenidate (MPH) is a drug that is licensed for treatment of ADHD and also narcolepsy. Monitoring of the parent drug and its major metabolite ritalinic acid (RA) in urine is considered necessary to ensure compliance with treatment programmes. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) assay was developed for the determination of MPH and its metabolite RA in human urine. After urine was diluted with water, methylphenidate, the major metabolite ritalinic acid, and d6-amphetamine as the internal standard were resolved on a PFP propyl column using gradient elution of 0.02% ammonium formate and acetonitrile. The total analysis time was 13.5 min. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 5–5000 μg/L (r  > 0.997), bias was ≤±20%, intra- and inter-day coefficients of variation (imprecision) were <8% and the limit of detection was 5 μg/L. The limit of quantitation was set at 100 μg/L. Matrix effects were up to 140% but these were accounted for by the internal standard. The assay is being used successfully in clinical practice to enhance the safe and effective use of methylphenidate.
Keywords: Methylphenidate; Ritalinic acid; Urine; LC–MS/MS;

► Human exposure to triclocarben (TCC) may be assessed by measuring the concentrations of TCC and its metabolites in urine or serum. ► A method for measuring trace levels of TCC and its two oxidative metabolites in human urine and serum was developed. ► This method is fully automatic, sensitive, precise, and accurate. In addition, it involves very minimal sample handling. ► This method could be used for the analysis of a large number of samples for epidemiological studies.3,4,4′-Trichlorocarbanilide (triclocarban, TCC) is widely used as an antimicrobial agent in a variety of consumer and personal care products. Because of its widespread use, the potential for human exposure to TCC is high. Human exposure to TCC may be assessed by measuring the concentrations of conjugated or free species of TCC and its two oxidative metabolites, 2′-hydroxy-TCC (2′-OH-TCC) and 3′-hydroxy-TCC (3′-OH-TCC), in urine or serum. To assess human exposure to TCC, we developed a method that uses restricted access materials (RAM) on-line solid phase extraction (SPE) coupled to high performance liquid chromatography-isotope dilution tandem mass spectrometry with peak focusing (HPLC–MS/MS). Sample clean-up by RAM relies on both size exclusion chromatography, to remove the high-molecular matrix components, and reversed phase partition, to extract and pre-concentrate the target analytes. TCC, 2′-OH-TCC and 3′-OH-TCC present in urine or serum were concentrated on the RAM SPE column, back-eluted from the SPE column, diluted through a mixing tee for peak focusing, separated by HPLC, and detected by isotope dilution-MS/MS. The method required a small amount of sample (50 μL) and minimal sample pretreatment. The limits of detection (LOD) ranged from 0.01 to 0.1 ng/mL. The method was applied to measure TCC and its metabolites in 158 urine and 16 serum samples collected from adults with no known exposure to TCC. TCC was detected in 35.4% of the urine samples (range: <LOD to 401 ng/mL). This sensitive method is rugged as well as labor- and cost-effective, and allows for the analysis of a large number of samples for epidemiological studies.
Keywords: Triclocarban; Oxidative metabolite; HPLC–MS/MS; Urine; Serum;

Risedronate is a commonly prescribed bisphosphonate for the treatment of bone disorders. Due to its high polarity and low oral bioavailability, low concentrations of risedronate are expected in human plasma and therefore a sensitive assay is required to serve in pharmacokinetic studies. Here, we describe the development and validation of an LC–MS/MS assay for the measurement of risedronate concentrations in human plasma. Risedronate and the internal standard, risedronate-d4, were derivatized on an anion exchange solid-phase extraction cartridge. Trimethylsilyl-diazomethane which is a thermally stable and relatively non-toxic derivatization agent was used to methylate the risedronate phosphonic acid groups and decrease analyte polarity. Following extraction, the analytes were separated on a Phenomenex Gemini C18 column (150 mm × 2.0 mm, 5 μm), using a gradient of ammonium acetate 10 mM and acetonitrile with a flow rate of 300 μL/min. The assay calibration range was 0.2–25 ng/mL. The calibration curve of risedronate standards spiked in six individual plasma samples was linear (r 2  = 0.9998). Accuracy (percent deviation from nominal) and precision (percent coefficient of variation) at concentrations 0.5, 5 and 20 ng/mL, and at the lower limit of quantification (LLOQ) of 0.2 ng/mL were excellent at <6%. Mean recovery was 54% for risedronate and 51% for the internal standard. Risedronate was stable in human plasma samples for at least 5 h at room temperature, 101 days frozen at −80 °C, 72 h in an autosampler at 10 °C, and for three freeze/thaw cycles. The validated assay method successfully quantified the concentrations of risedronate in plasma samples from informed consenting healthy volunteers administered a single 35 mg risedronate tablet.
Keywords: Risedronate; Bisphosphonate; Liquid chromatography/tandem mass spectrometry (LC–MS/MS); Trimethylsilyldiazomethane; Derivatization; Solid phase extraction (SPE); Plasma; Quantification;

► An XLC–MS/MS assay for salivary cortisol and cortisone has been validated. ► The Spark Holland Symbiosis™ provides fully automated online solid phase extraction. ► We demonstrated excellent precision, accuracy, linearity, sensitivity and specificity. ► For use in the investigation of disorders of the hypothalamic–pituitary–adrenal axis.Salivary cortisol measurements are increasingly being used in the investigation of disorders of the hypothalamic–pituitary–adrenal axis. In the salivary gland, cortisol is metabolised to cortisone by the action of 11β-hydroxysteroid dehydrogenase type 2, and cortisone is partly responsible for the variable interference observed in current salivary cortisol immunoassays. The aim of this study was to validate an assay for the simultaneous analysis of salivary cortisol and cortisone using the Spark Holland Symbiosis™ in eXtraction liquid chromatography–tandem mass spectrometry (XLC–MS/MS) mode for fully automated online solid phase extraction (SPE). Saliva samples were diluted in water with the addition of internal standard (d4-cortisol and d7-cortisone). Online SPE was performed using the Spark Holland Symbiosis™ with HySphere™ C18 SPE cartridges and compounds were eluted onto a Phenomenex® C18 guard column attached to a Phenomenex® Onyx monolithic C18 column for chromatography. Mass spectrometry used the Waters® Xevo™ TQ MS in electrospray positive mode. Cortisol and cortisone eluted with their internal standards at 1.95 and 2.17 min, respectively, with a total run time of four minutes. No evidence of ion-suppression was observed. The assay was linear up to 3393 nmol/L for cortisol and 3676 nmol/L for cortisone, with lower limits of quantitation of 0.75 nmol/L and 0.50 nmol/L, respectively. Intra- and inter-assay imprecision was <8.9% for cortisol and <6.5% for cortisone across three levels of internal quality control, with accuracy and recovery within accepted limits. High specificity was demonstrated following interference studies which assessed 29 structurally-related steroids at supra-physiological concentrations. We have successfully validated an assay for the simultaneous analysis of salivary cortisol and cortisone using XLC–MS/MS and fully automated online SPE. The assay benefits from increased specificity compared to immunoassay and minimal sample preparation which allows high sample throughput and is thus suitable for use in a routine clinical laboratory.
Keywords: Cortisol; Cortisone; Saliva; XLC–MS/MS; Spark Holland Symbiosis™;

Separation of two constituents from purple sweet potato by combination of silica gel column and high-speed counter-current chromatography by Kai He; Xiaoli Ye; Xuegang Li; Hongying Chen; Lujiang Yuan; Yafei Deng; Xin Chen; Xiaoduo Li (49-54).
► A thin layer chromatograph coupling with fluorometric (TLC-F) method for selecting HSCCC solvent system was proposed. ► 6,7-Dimethoxycoumarin and 5-hydroxymethyl-2-furfural were successfully separated from purple sweet potato extracts by successive sample injection for the first time. ► The results of our study suggested that the TLC-F method is useful to select HSCCC solvent systems.It is known that the choice of solvent system for high speed counter-current chromatography separation is of utmost importance. In this study, a simple and rapid thin layer chromatograph coupling with fluorometric (TLC-F) method has been used to determine the partition coefficient of target compounds in HSCCC solvent system. Two components, 6,7-dimethoxycoumarin and 5-hydroxymethyl-2-furfural were successfully separated from purple sweet potato extracts by successive sample injection for the first time, using n-hexane–ethyl acetate–methanol–water (1:2:1:1, v/v/v/v) as the solvent system. Additionally, statistical analysis showed that there was no significant difference in partition coefficient obtained by the TLC-F method and by HPLC, which demonstrated the usefulness of TLC-F method.
Keywords: Purple sweet potato; TLC-F method; High speed counter-current chromatography; Partition coefficient; Solvent system;

► CM1 competitively bind to RAGE with RAGE ligand MG-H1. ► A HPLC method was established for competition binding of CM1 with MG-H1. ► This method was an alternative way for competitive binding of drug to receptor. ► This binding was performed on intact cells.The compound 4,4′-diphenylmethane-bis(methyl) carbamate (CM1) has a protective activity on AGEs-induced endothelial dysfunction on human umbilical vein endothelial cell (HUVEC) in our previous study. It suggested that CM1 which may act as a competitive antagonist to the blockade of AGEs to receptor of AGEs (RAGE) and attenuate the HUVEC damage. In order to testify that hypothesis, the cell membrane chromatography (CMC) combined with high performance liquid chromatography (HPLC) was developed for analyzing the competitive binding properties on RAGE of HUVEC between CM1 and MG-H1, the agonist of RAGE. The results from saturation binding of CM1 and MG-H1 on cells demonstrated that dissociation equilibrium constants (K d) of CM1 and MG-H1 were 3.653 nM and 4.12 nM, respectively; while maximum binding capacity (B max) of CM1 and MG-H1 were 30.08 and 18.72 fmol/mg protein, respectively. In competition experiments, IC50 of CM1 with pre-incubation 10−10  M and 10−9  M MG-H1 were 1.37 × 10−9  M and 4.56 × 10−8  M, respectively. The present findings indicated that CM1 conjugated competitively to cells with RAGE ligand MG-H1. The primary study illustrated that CMC combined with HPLC analysis method could be an alternative, rapid and efficient approach for the interaction of drug molecule and receptor, and that CM1 intervene the AGEs inducing HUVEC damage may via the competitively block the AGEs–RAGE path way.
Keywords: Competitive binding; Cell membrane chromatography; 4,4′-Diphenylmethane-bis(methyl) carbamate; MG-H1; Receptor of advanced glycation end products;

PCR-ready human DNA extraction from urine samples using magnetic nanoparticles by Zhi Shan; Zhongwu Zhou; Hui Chen; Zhiming Zhang; Yi Zhou; Anxiang Wen; Ken D. Oakes; Mark R. Servos (63-68).
► A method for urine DNA extraction using carboxylated magnetic nanoparticles was developed. ► The addition of 10 mM EDTA and pH modification (pH 6.0–7.1) can re-dissolve urine sediments. ► Purified DNA ranged from around 0.1 kb to more than 23 kb. ► DNA quality was validated by its yield, molecular weight, and the ability to serve as PCR templates. ► The developed method proved to be simple, rapid, sensitive and environmentally friendly.Urine-derived human genomic DNA (gDNA) has wide application in a variety of disciplines including clinical medicine, sports, and forensic science. We describe a novel method for gDNA extraction from urine samples using carboxylated magnetic nanoparticles (CMNPs) as solid-phase adsorbents. Sedimentation associated with freezing of urine samples significantly reduces cell capture by CMNPs. However, the addition of 10 mM EDTA and subsequent pH modification (pH 6.0–7.1) can re-dissolve urine sediments. Purified gDNA ranged from around 0.1 kb to more than 23 kb. PCR using specific primers targeting K-ras, GAPDH, CYP3A4 and GDF5 amplified 100% of varying sized gene fragments, verifying the high quality of the isolated DNA. Successful PCR amplifications using DNA isolated from urine samples as small as 50 μl were demonstrated. Enrichment of urine cells and subsequent adsorption of DNA can be achieved with the same CMNPs, greatly simplifying extraction procedures. The CMNP gDNA extraction technique proved to be simple, rapid, sensitive and environmentally friendly, with application for routine laboratory use and potentially within automated urine extraction platforms.
Keywords: Carboxylated magnetic nanoparticles; DNA; Urine sediment; PCR;

► Analysis of volatile secretions from the European stink bug Graphosoma lineatum. ► Analysis using HS-SPME coupled to GC × GC/TOF-MS. ► Identification of 57 compounds, 39 of these are reported for the first time. ► No differences in the composition of the secretions between sexes were found.An efficient method combining the headspace solid-phase microextraction (HS-SPME) sampling procedure and comprehensive two-dimensional gas-chromatography/time-of-flight mass spectrometry (GC × GC/TOF-MS) was established to study the volatile secretion components of stink bugs (Heteroptera: Pentatomidae). The combined power of this approach is illustrated by the identification of fifty-seven compounds in the secretion of a European stink-bug representative, Graphosoma lineatum. (E)-4-oxohex-2-enal and (E)-dec-2-enal were found to be the major components in the adult bug secretions followed by lower amounts of n-alkenal (C5–C12), n-alkenyl acetate (C5–C11), n-alkane (C11–C17) homologs, dienals and other compounds. More than thirty known compounds have been identified that had not been described before in G. lineatum adults. Of these compounds, (E)-4-oxohex-2-enal is of particular interest, since its isolation and identification, while calling some previous reports into question, clearly demonstrates a potential ability of our approach to yield artifact-free secretion profiles.
Keywords: Graphosoma lineatum; Pentatomidae; Volatile secretion; GC × GC/TOF-MS; Profiling; (E)-4-oxohex-2-enal;

Rapid detection of sepsis in rats through volatile organic compounds in breath by Ana V. Guamán; Alba Carreras; Daniel Calvo; Idoya Agudo; Daniel Navajas; Antonio Pardo; Santiago Marco; Ramon Farré (76-82).
► Breath analysis with Ion Mobility Spectrometry (IMS) was used as rapid diagnosis for sepsis. ► High levels of accuracy, specificity and sensitivity were achieved by processing IMS spectra. ► Breath samples were measured with GC/MS as reference technique. ► A pattern of compounds were related to sepsis by analysing chromatograms of GC/MS. ► Results showed that breath analysis can be used as point of care tool for diagnosis of sepsis.Sepsis is one of the main causes of death in adult intensive care units. The major drawbacks of the different methods used for its diagnosis and monitoring are their inability to provide fast responses and unsuitability for bedside use. In this study, performed using a rat sepsis model, we evaluate breath analysis with Ion Mobility Spectrometry (IMS) as a fast, portable and non-invasive strategy.This study was carried out on 20 Sprague-Dawley rats. Ten rats were injected with lipopolysaccharide from Escherichia coli and ten rats were IP injected with regular saline. After a 24-h period, the rats were anaesthetized and their exhaled breaths were collected and measured with IMS and SPME-gas chromatography/mass spectrometry (SPME-GC/MS) and the data were analyzed with multivariate data processing techniques.The SPME-GC/MS dataset processing showed 92% accuracy in the discrimination between the two groups, with a confidence interval of between 90.9% and 92.9%. Percentages for sensitivity and specificity were 98% (97.5–98.5%) and 85% (84.6–87.6%), respectively. The IMS database processing generated an accuracy of 99.8% (99.7–99.9%), a specificity of 99.6% (99.5–99.7%) and a sensitivity of 99.9% (99.8–100%).IMS involving fast analysis times, minimum sample handling and portable instrumentation can be an alternative for continuous bedside monitoring. IMS spectra require data processing with proper statistical models for the technique to be used as an alternative to other methods. These animal model results suggest that exhaled breath can be used as a point-of-care tool for the diagnosis and monitoring of sepsis.
Keywords: Sepsis; Volatile organic compounds; Ion mobility spectrometer; Rat model; Bedside patient systems; Non-invasive detection;

A liquid chromatography tandem mass spectrometry method for simultaneous determination of acid/alkaline phytohormones in grapes by Zheng Han; Gang Liu; Qinxiong Rao; Bing Bai; Zhihui Zhao; Hong Liu; Aibo Wu (83-89).
► A rapid LC–MS/MS for simultaneous determination of acid/alkaline phytohormones was developed. ► The procedures for sample preparation were thoroughly optimized. ► The performances of linearity, sensitivity, precision and matrix effects were satisfactory. ► The method was proved to be capable of rapid multiresidue analysis of multiclass phytohormones.A high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for simultaneous determination of five acid/alkaline phytohormones, i.e., indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic acid (NAA), gibberellic acid (GA3) and isopentenyladenine (2IP), in grapes was developed. After optimization, the samples were extracted with methanol containing 1% formic acid and purified by Oasis HLB SPE cartridges. The analytes were separated on a Thermo Hypersil Gold column (100 mm × 2.1 mm, 3.0 μm) with water and acetonitrile, then determined with Thermo tandem quadrupole mass spectrometer operating in negative electro-spray ionization using selected reaction monitoring (SRM) mode. The established method was further validated by determining the linearity (R 2  ≥ 0.9990), average recovery (82.5–105.4%), sensitivity (0.05–1.00 ng mL−1), precision (RSD ≤ 13.0%) and stability (RSD ≥ 82.0%). Finally, the application of the approach proposed to thirty grape samples convinced its desirable performance for rapid analysis of multiclass phytohormones, supporting its sufficient capability for multiresidue analyses or other analytical system targeting phytohormones in agriculture field.
Keywords: Phytohormone; Multiresidue analysis; Liquid chromatography tandem mass spectrometry;

► LC–MS/MS provides the technological tool for the determination of antimicrobial agents. ► Method was validated at 1 μg/kg. Method's accuracy and precision were satisfactory. Recoveries ranged from 92% to 101%. ► The developed method proved efficient and straightforward. ► The method allows for positive identification and quantitation of the target banned analytes.A sensitive and robust LC–APCI-MS/MS method has been developed for the unambiguous detection and quantitative determination of the antimicrobial agent Carbadox, its metabolite quinoxaline-2-carboxylic acid and methyl-3-quinoxaline-2-carboxylic acid the major metabolite of Olaquindox. The method was aimed for application in the assaying of muscle tissue so the developed sample preparation scheme subjected samples to enzymatic digestion prior to the application of solid phase extraction clean-up. Subsequently the purified extracts were analyzed by reversed-phase LC–MS/MS in positive APCI and multiple reaction monitoring mode. The method was validated at a level of 1 μg/kg. The decision limits CCα and detection capability CCβ ranged from 0.09 μg/kg to 0.24 μg/kg and from 0.12 μg/kg to 0.41 μg/kg, respectively. The accuracy and precision of the method were satisfactory. The recoveries ranged from 92% to 101% for the metabolites and from 60% to 62% for Carbadox, with coefficient of variances (CVs) less than 12%. The developed method proved efficient and straightforward allowing positive identification and quantitation of the target banned analytes and is thus suitable for application in residue control programmes and metabolism studies.
Keywords: Carbadox; Olaquindox; Anti-microbial agents; Food quality control; Liquid chromatography–tandem mass spectrometry (LC–MS/MS);

Metabolism of mequindox and its metabolites identification in chickens using LC–LTQ-Orbitrap mass spectrometry by Qi Shan; Yiming Liu; Limin He; Huanzhong Ding; Xianhui Huang; Fan Yang; Yafei Li; Zhenling Zeng (96-106).
► The metabolism of mequindox in vivo was studied using LC–LTQ-Orbitrap. ► 12 new metabolites were detected and identified for the first time. ► Acetyl hydroxylation and deacetylation were new metabolic pathways of MEQ.Mequindox (MEQ), 3-methyl-2-quinoxalinacetyl-1,4-dioxide, is widely used in Chinese veterinary medicine as an antimicrobial and feed additive. Its toxicities have been reported to be closely related to its metabolism. To understand more clearly the metabolic pathways of MEQ, its metabolism in chickens was studied using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap (LC–LTQ-Orbitrap) mass spectrometry. The structures of the MEQ metabolites and their product ions were easily and reliably characterized based on the accurate MS-squared spectra and known structure of MEQ. Twenty-four metabolites were detected in chicken plasma, bile, faeces, and tissues, of which 12 were detected in vivo for the first time. The major metabolic pathways reported previously for in vitro metabolism of MEQ in chicken microsomes were confirmed in this study, including N → O group reduction, carbonyl reduction, and methyl mono-hydroxylation. In addition, deacetylation and acetyl-hydroxylation of MEQ were shown to be important metabolic pathways. Collectively, these data contribute to our understanding of the in vivo metabolism of MEQ.
Keywords: Mequindox; Metabolism; Metabolites; LC–LTQ-Orbitrap; Chicken;

Determination of N,N-dimethyltryptamine in Mimosa tenuiflora inner barks by matrix solid-phase dispersion procedure and GC–MS by Alain Gaujac; Adriano Aquino; Sandro Navickiene; Jailson Bittencourt de Andrade (107-110).
► It is new the use of MSPD procedure and GC–MS method for the determination of N,N-dimethyltryptamine in Mimosa tenuiflora barks. ► Use of an analytical standard of DMT isolated from M. tenuiflora. ► The proposed method uses small amounts of sample and low demand for reagents and solvents. N,N-dimethyltryptamine (DMT) is a potent hallucinogen found in beverages consumed in religion rituals and neo-shamanic practices over the world. Two of these religions, Santo Daime and União do Vegetal (UDV), are represented in countries including Australia, the United States and several European nations. In some of this countries there have been legal disputes concerning the legalization of ayahuasca consumption during religious rituals, a beverage rich in DMT. In Brazil, even children and pregnant women are legally authorized to consume ayahuasca in a religious context. A simple and low-cost method based on matrix solid-phase dispersion (MSPD) and gas chromatography with mass spectrometric detection (GC–MS) has been optimized for the determination of N,N-dimethyltryptamine in Mimosa tenuiflora inner bark. The experimental variables that affect the MSPD method, such as the amounts of solid-phase and herbal sample, solvent nature, eluate volume and NaOH concentration were optimized using an experimental design. The method showed good linearity (r  = 0.9962) and repeatability (RSD < 7.4%) for DMT compound, with detection limit of 0.12 mg/g. The proposed method was used to analyze 24 samples obtained locally. The results showed that concentrations of the target compound in M. tenuiflora barks, ranged from 1.26 to 9.35 mg/g for these samples.
Keywords: N,N-Dimethyltryptamine; Mimosa tenuiflora; Experimental design; MSPD; GC–MS;

A fast liquid chromatography–tandem mass spectrometry with electrospray ionization method was developed and validated for simultaneous quantification of lidocaine and its active metabolite MEGX in 10 μL of plasma of neonates with seizures. The sample preparation consists of an easy protein precipitation sample pre-treatment with methanol. Chromatographic separation was achieved on a Alltima HP C18-EPS 150 mm × 2.1 mm column with an isocratic mobile phase of 0.1% (v/v) ammonium acetate in purified water–0.1% (v/v) formic acid in acetonitrile (70:30, v/v). The analytes were detected with a Thermo Scientific triple quadrupole Quantum Access with positive ionization. Ions monitored in the selected reaction monitoring (SRM) mode were m/z 235.2 → 86.6 for lidocaine (at 3.35 min), m/z 207.1 → 58.8 for MEGX (at 2.75 min) and 280.1 → 86.7 for 3-nitrolidocaine (internal standard, at 3.20 min). The method was validated over a linear range of 0.2–18.0 mg/L for lidocaine and MEGX, using 3-nitrolidocaine as the internal standard. The lower limit of quantification (LLQ) was 0.2 mg/L for lidocaine and MEGX. The within-run and between-run CV (%) were lower than 6.9% for both lidocaine and MEGX. Recoveries were in the range of 99.4% to103.6%. Observed LC–MS/MS matrix effects were −6.2% for MEGX (ion suppression) and were negligible for lidocaine and the internal standard (i.e. <0.1%). Compared to other bioanalytical articles published in medical literature (PubMed) during the last 15 years that described LC–MS/MS methods for quantification of lidocaine in human plasma, our method uses less plasma, has a shorter and more simple sample pre-treatment and has a short run time.
Keywords: Lidocaine; MEGX; Metabolite; Liquid chromatography; Tandem mass spectrometry; LC–MS/MS; Neonatal seizures; Therapeutic drug monitoring;

Thiopurines are S-substituted antimetabolites that are widely used in the treatment of hematological malignancies and as immunosuppressants. Because of extensive inter-individual variation in drug disposition and the significant toxicity associated with thiopurine therapy, there is a need for improved individualized treatment. We here present a fast and sensitive method for quantifying the pharmacological end-point of thiopurines, 6-thioguanine (TG) in chromosomal DNA. Purine nucleobases are released from DNA, etheno-derivatized with chloroacetaldehyde, separated by HILIC and quantified by tandem mass spectrometry using endogenous chromosomal guanine as internal standard. The method is linear up to at least 10 pmol TG/μg DNA and the limit of detection and quantification are 4.2 and 14.1 fmol TG/μg DNA, respectively. The matrix (DNA) had no effect upon quantification of TG. SPE recovery was estimated at 63% (RSD 26%), which is corrected for by the internal standard resulting in stable quantification. The TG levels found were above the LOQ in 18 out of 18 childhood leukemia patients on 6-mercaptopurine/methotrexate maintenance therapy (median 377, range 45–1190 fmol/μg DNA) with intra- and inter-day RSDs of less than 11%. The method uses 2 μg DNA/sample, which can easily be obtained from these patients.
Keywords: Thiopurines; Acute lymphoblastic leukemia; 6-Mercaptopurine; DNA-TGN; Maintenance therapy; LC–MSMS;

► In this work, we developed a new sample pre-processing method, salting-out homogeneous liquid–liquid extraction (SHLLE), for determination of entecavir in human plasma by LC–MS. ► In this study, we got a LLOQ at pg/mL lever of entecavir in human blood. ► It is the first time to introduce SHLLE as sample preparation for bioanalysis of entecavir and it will be useful for pre-clinical and clinical study of entecavir.A convenient, robust, economical and selective sample preparation method for the quantitative determination of entecavir in human plasma by LC–MS was developed and validated. Entecavir and the internal standard of acyclovir were extracted from 500 μL of human plasma by a salting-out homogeneous liquid–liquid extraction approach (SHLLE) with acetonitrile as the organic extractant and magnesium sulfate as the salting-out reagent. They were analyzed on a Hanbon® Lichrospher RP C18 HPLC column (150 mm × 2.0 mm; 5 μm) with gradient elution. The mobile phase comprised 0.1% acetic acid–0.2 mmol ammonium acetate in water (mobile phase A) and acetonitrile (mobile phase B). The flow rate is 0.2 mL/min. The analytes were detected by a LC–MS 2010 single quadrupole mass spectrometer instrument equipped with an electrospray ionization interface using selective ion monitoring positive mode. A “post cut” column switch technique was incorporated into the method to remove interferences of earlier and later eluting matrix components than entecavir and internal standard, including salting-out reagent used in sample pre-processing. The method was validated over the concentration range of 0.05–20 ng/mL. The intra-day and inter-day precision of the assay, as measured by the coefficient of variation (%CV), was within 3.59%, and the intra-day assay accuracy was found to be within 4.88%. The average recovery of entecavir was about 50% and the ion suppression was approximately 44% over the standard curve. Comparison of matrix effect between SHLLE and SPE by continuous post column infusion showed that these two methods got similar, slight ion suppression. The SHLLE method has been successfully utilized for the analysis of entecavir in post-dose samples from a clinical study.
Keywords: Entecavir; Matrix effect; SHLLE; SPE; LC–MS;

Determination of Bis(9)-(−)-Meptazinol, a bis-ligand for Alzheimer's disease, in rat plasma by liquid chromatography–tandem mass spectrometry: Application to pharmacokinetics study by Xin-xing Ge; Xiao-lin Wang; Pan Jiang; Ying Xie; Tao Jiang; Zheng-xing Rong; Qi-zhi Zhang; Qiong Xie; Zhui-bai Qiu; Hao Wang; Hong-zhuan Chen (126-130).
► We develop and validate a HPLC–MS/MS method of a new anti-Alzheimer's dimer B9M. ► Mobile phase buffered at pH 9.8 obtains good peak shape and high sensitivity. ► The method is characteristic of short running time and simple preparation process. ► We evaluate preliminary PK profile of B9M in rat after iv and sc administration.A rapid, simple and sensitive LC–MS/MS method was developed and validated for the determination of Bis(9)-(−)-Meptazinol (B9M) in rat plasma. Protein precipitation method was used for sample preparation, using five volumes of methanol as the precipitation agent. The analytes were separated by a Zorbax Extend-C18 column with the mobile phase of methanol-water (containing 5 mM ammonium formate, pH 9.8) (95:5, v/v), and monitored by positive electrospray ionization in multiple reaction monitoring (MRM) mode. Retention time of IS (Bis(5)-(−)-Meptazinol) and B9M were 1.9 min and 3.3 min, respectively. The limit of detection was 0.1 ng/ml and the linear range was 1–500 ng/ml. The relative standard deviation (RSD) of intra-day and inter-day variation was 4.4–6.2% and 6.2–8.9%, respectively. The extraction recoveries of B9M in plasma were over 95%. The method proved to be applicable to the pharmacokinetic study of B9M in rat after intravenous and subcutaneous administration.
Keywords: Bis(9)-(−)-Meptazinol; Alzheimer's disease; HPLC–MS/MS; Pharmacokinetics; Rat plasma;