Journal of Chromatography B (v.958, #C)

A novel Indole-3-carbinol derivative (I3C) prodrug, indole-3-carbinol acetate (I3CA), was synthesized and a rapid high-performance liquid chromatography (HPLC) method for the quantification of I3CA, I3C, and the major metabolite of I3C, diindolylmethane (DIM), in mouse plasma, liver and kidney tissues was developed and validated. 4-Methoxy-1-methylindole was used as the internal standard. Chromatographic separation was achieved on a Symmetry® C18 column (75 mm × 4.6 mm, 3.5 μm) and the detection was made at 280 nm. A gradient elution was programmed with the mobile phases of water (A) and acetonitrile (B) and a flow rate of 1 ml/min. The total run time was 15 min. The calibration curves were linear over the range of 0.06–1.6 μg/ml for both I3C and DIM with a correlation coefficient (r 2) higher than 0.997 and the lower limit of quantitation (LLOQ) of 0.06 μg/ml. The calibration curve of I3CA was linear over the range of 0.15–4.0 μg/ml, with a r 2  > 0.995 and LLOQ of 0.15 μg/ml. I3CA, I3C, and DIM intra-day accuracy values of plasma, liver and kidney samples ranged from 90.0 to 101.3%, while the inter-day ones were between 93.3 and 101.9%. Precision evaluated by the relative standard deviation was ranged from 2.0 to 14.8% for intra-day and 1.9 to 14.4% for inter-day variability. I3CA, I3C, and DIM were stable in mouse plasma, liver and kidney samples containing an esterase inhibitor dichlorvos. This method was successfully applied to a pharmacokinetic study in mice following oral and intravenous administration of I3C and I3CA.
Keywords: Indole-3-carbinol; 3,3′-Diindolylmethane; Indole-3-carbinol acetate; HPLC-UV; Mouse plasma; Live tissue; Kidney tissue; Validation; Stability; Metabolism; Pharmacokinetics;

Stable isotope studies offer the opportunity to study the in-depth metabolic pathway of glutamine, citrulline, and arginine amino acids involved in NO synthesis. The use of multiple stable isotopes can be used to elucidate the exact transformation of glutamine to citrulline and arginine de novo synthesis. This novel method provides a purification step using cation exchange resin in combination with a rapid and easy derivatization procedure for a precise and robust measurement of the concentration and isotopic enrichments of NO synthesis-specific amino acids using a liquid chromatography mass spectrometry (LC/MS) ion trap system with high sensitivity and selectivity. The ethyl chloroformate derivatization procedure is beneficial in terms of robustness, velocity, simplicity, and derivative stability. In addition, the ethyl chloroformate derivatization can be performed at room temperature in an aqueous environment without incubation and the isolation of the derivatives from the reaction mixture also serves as a purification step. The concentration and enrichment of NO synthesis-specific amino acids as well as phenylalanine and tyrosine to determine protein turnover, were measured with good inter-day precision for the concentration (<7.4%) and enrichment (<12.7%) in plasma samples at low and high levels. The low limit of quantification was 0.2 μmol/L for most of the amino acids and the purification method showed to have good recoveries between 78% and 98%. No ion-suppression was observed by post-column infusion experiments. In order to develop new nutritional strategies, this novel method can be used to support the elucidation of the effect of administration of specific supplements on the glutamine–citrulline–arginine pathway by using stable isotope studies.
Keywords: Arginine; Citrulline; Glutamine; Isotopic enrichment; Ion trap mass spectrometry; Metabolism;

Mobilization of Cd from human serum albumin by small molecular weight thiols by Thomas T. Morris; Jennifer L.A. Keir; Steven J. Boshart; Victor P. Lobanov; Anthony M.A. Ruhland; Nishita Bahl; Jürgen Gailer (16-21).
Although the toxic metal Cd is an established human nephrotoxin, little is known about the role that interactions with plasma constitutents play in determining its mammalian target organs. To gain insight, a Cd–human serum albumin (HSA) complex was analyzed on a system consisting of size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using phosphate buffered saline (pH 7.4) as the mobile phase, we investigated the effect of 1–10 mM oxidized glutathione (GSSG), l-cysteine (Cys), l-glutathione (GSH), or N-acetyl-l-cysteine (NAC) on the elution of Cd. As expected, GSSG did not mobilize Cd from the Cd–HSA complex up to a concentration of 4 mM. With 1.0 mM NAC, ∼30% of the injected Cd–HSA complex eluted as such, while the mobilized Cd was lost on the column. With 1.0 mM of Cys or GSH, no parent Cd–HSA complex was detected and 88% and 82% of the protein bound Cd eluted close to the elution volume, likely in form of Cd(Cys)2 and a Cd–GSH 1:1 complex. Interestingly, with GSH and NAC concentrations >4.0 mM, a Cd double peak was detected, which was rationalized in terms of the elution of a polynuclear Cd complex baseline-separated from a mononuclear Cd complex. In contrast, mobile phases which contained Cys concentrations ≥2 mM resulted in the detection of only a single Cd peak, probably Cd(Cys)4. Our results establish SEC–FAAS as a viable tool to probe the mobilization of Cd from binding sites on plasma proteins at near physiological conditions. The detected complexes between Cd and Cys or GSH may be involved in the translocation of Cd to mammalian target organs.
Keywords: Albumin; Small molecular weight thiol; SEC; Hyphenation; Cd-specific detection; Target organ;

A procedure based on solid-phase extraction (SPE) followed by ultra-high-performance liquid chromatography (UHPLC) with UV detection has been developed for the analysis of multiple drugs in human urine. The compounds evaluated were aliskiren, prasugrel, rivaroxaban, prednisolone, propranolol, ketoprofen, nifedipine, naproxen, terbinafine, ibuprofen, diclofenac, sildenafil and acenocoumarol. Seventeen different solid phase extraction (SPE) cartridges were tested to evaluate their applicability for the isolation of drugs from human urine. Comparison were recovery of different drugs and reproducibility. The samples were analyzed by UHPLC using a Poroshell 120 EC-C18 column and acetonitrile −0.05% TFA in water as the mobile phase under gradient elution conditions.SPE combined with UHPLC–UV allowed the determination of drugs over a linear range of 0.01–30.0 μg/mL, with limits of detection at 0.003–0.217 μg/mL and precision of 0.8–7.1%. Phenyl (C6H5) sorbent was found to provide the most effective clean-up, removing the greatest amount of interfering substance and simultaneously ensuring analyte recoveries higher than 85.5% with relative standard deviations (RSD) <10%.The method was applied with good accuracy and precision in the determination of drugs in human urine obtained from patients treated with selected drugs.
Keywords: Sorbent comparison; Solid-phase extraction; Drugs; Liquid chromatography;

Dried blood spot (DBS) sampling represents a suitable method for pharmacokinetic studies in rats, particularly if serial sampling is needed. To study the pharmacokinetics of drugs in a rat heart failure (HF) model, we developed and validated a method for the simultaneous determination of bisoprolol, ramiprilat, propranolol and midazolam in DBS samples. Bisoprolol and ramipril are widely used in the treatment of HF, and midazolam and propranolol are markers of hepatic metabolism, which can be altered in HF. A 20 μL sample of rat blood was pipetted onto Whatman 903 Protein Saver Card and allowed to dry. The whole spot was excised and 300 μL of solvent (methanol with 10% ultrapure water and 0.1% formic acid) was added. After mixing and incubating the sample in an ultrasonic bath, a mixture of isotopically labeled internal standards was added. After centrifugation, the extracts were cleaned on an Ostro™ plate and analyzed using liquid chromatography–tandem mass spectroscopy. The method was successfully validated. No significant interference was observed in the retention times of analytes or internal standards. The intraday and interday accuracy and precision were within a ±15% interval. The method was linear in the range 5–250 μg/L and the lower limit of quantification was 5 μg/L for all four analytes. The absolute matrix effect ranged from 98.7% for midazolam to 121% for ramiprilat. The recovery was lowest for ramiprilat and highest for propranolol. Samples were stable at all tested temperatures. The method has been used successfully in a real-time pharmacokinetic study in rats.
Keywords: Dried blood sample; Bisoprolol; Ramiprilat; Propranolol; Midazolam;

Amomum longiligulare or Amomum villosum showed oestrogenic activity. In the present study, oestrogen-active components in fructus amomi, the seeds of A. longiligulare were separated by high-speed countercurrent chromatography (HSCCC) using stepwise elution of eight mobile phases with gradient polarity and advanced separation by high performance liquid chromatography (HPLC). The results yielded 17 compounds with the amount of 8–138 mg and a purity of 94.3–99.8% from a 3 g ethanolic extract of fructus amomi. The chemical structures of the compounds were identified by ESI-MS and NMR spectra, in which eight diarylheptanoids were demonstrated as the main oestrogen-active compounds in the fructus amomi. Determination of the diarylheptanoids in fructus amomi from various origins showed that fructus amomi contains more than 0.5% total diarylheptanoids. The results showed that fructus amomi is a diarylheptanoids-rich food resource possessing oestrogen-activity. The combination method of HSCCC and HPLC can be applied for the analysis of bioactive compounds by detecting the corresponding bioactivity in the HSCCC fractions and separating the target compounds with HPLC.
Keywords: Fructus amomi; Oestrogenic activity; Analysis; Diarylheptanoids;

Simeprevir (also known as TMC435 or TMC435350) is a novel hepatitis C protease inhibitor. A validated high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the sensitive and selective quantification of simeprevir in human EDTA plasma is described. During assay development, special attention was given to light instability of the drug in plasma and blood. The method consisted of precipitation of plasma proteins with acetonitrile after which the supernatant was analyzed using electrospray LC–MS/MS. The linearity was confirmed in the concentration range from 2.00 to 2000 ng/mL, with 50-fold dilution extending to 100,000 ng/mL. The precision of this assay, expressed as CV, ranged between 4.4% and 8.5% over the entire concentration range with assay accuracy between −0.3% and 8.5%. The method was applied successfully in many clinical studies to document the pharmacokinetics of simeprevir in plasma from healthy volunteers and patients.
Keywords: LC–MS/MS; Method validation; Antivirals; Hepatitis C; Simeprevir;

Optimization of diclofenac quantification from wastewater treatment plant sludge by ultrasonication assisted extraction by Emel Topuz; Sevgi Sari; Gamze Ozdemir; Egemen Aydin; Elif Pehlivanoglu-Mantas; Didem Okutman Tas (48-54).
A rapid quantification method of diclofenac from sludge samples through ultrasonication assisted extraction and solid phase extraction (SPE) was developed and used for the quantification of diclofenac concentrations in sludge samples with liquid chromatography/tandem mass spectrometry (LC–MS/MS). Although the concentration of diclofenac in sludge samples taken from different units of wastewater treatment plants in Istanbul was below the limit of quantification (LOQ; 5 ng/g), an optimized method for sludge samples along with the total mass balances in a wastewater treatment plant can be used to determine the phase with which diclofenac is mostly associated. Hence, the results will provide information on fate and transport of diclofenac, as well as on the necessity of alternative removal processes. In addition, since the optimization procedure is provided in detail, it is possible for other researchers to use this procedure as a starting point for the determination of other emerging pollutants in wastewater sludge samples.
Keywords: LC–MS/MS; Method; Micropollutants; Pharmaceutical; Solid phase extraction; Ultrasonication;

Cortex Juglandis Mandshuricae is used as a folk remedy for treating cancer, diarrhea and dysentery in traditional Chinese medicine for many years. Six flavonoids (myricitrin, quercitrin, taxifolin, myricetin, quercetin and naringenin), gallic acid and 5,8-dihydroxy-1,4-naphthoquinone are major bioactive components in Cortex Juglandis Mandshuricae extract. In this study, an ultrahigh performance liquid chromatography and tandem mass spectrometry method was developed for simultaneous determination of eight ingredients in rat plasma using chloromycetin as an internal standard. Plasma samples added vitamin C (antioxygen) were acidified with hydrochloric acid and extracted by liquid–liquid extraction with ethyl acetate. Eight ingredients were separated on a Venusil ASB C18 column and detected by multiple reaction monitoring mode using electrospray ionization in the negative ion mode. The method was linear for all analytes over investigated range with all correlation coefficients greater than 0.9900. The validated lower limit of quantification was 20 ng/mL for gallic acid, 5 ng/mL for myricitrin, 3 ng/mL for quercitrin, 10 ng/mL for taxifolin, 6 ng/mL for myricetin, 3 ng/mL for quercetin, 2 ng/mL for naringenin and 1 μg/mL for 5,8-dihydroxy-1,4-naphthoquinone, respectively. Intra- and inter-day precisions (RSD%) were less than 15% and accuracy (RE%) ranged from −6.9% to 6.9%. The validated method was successfully applied to investigate the pharmacokinetics of the eight analytes after oral administration of Cortex Juglandis Mandshuricae extract to rats.
Keywords: Cortex Juglandis Mandshuricae; 5 ,8-dihydroxy-1 Dihydroxy-1,4-naphthoquinone; Flavonoids; Gallic acid; Pharmacokinetics; UHPLC–MS/MS;

Procyanidins have been extensively investigated for their potential health protective activities. However, the potential bioactivities of procyanidins are limited by their poor bioavailability. The majority of the ingested dose remains unabsorbed and reaches the colon where extensive microbial metabolism occurs. Most existing analytical methods measure either native compounds (catechins and procyanidins), or their microbial metabolites. The objectives of this study were to develop a high-throughput extraction and UPLC–MS/MS method for simultaneous measurement of both native procyanidins and their metabolites, facilitating high-throughput analysis of native and metabolite profiles in various regions of the colon. The present UPLC–MS/MS method facilitates simultaneous resolution and detection of authentic standards of 14 native catechin monomers and procyanidins, as well as 24 microbial metabolites. Detection and resolution of an additional 3 procyanidin dimers and 10 metabolites for which standards were not available was achieved. Elution and adequate resolution of both native compounds and metabolites were achieved within 10 min. The intraday repeatability for native compounds was between 1.1 and 16.5%, and the interday repeatability for native compounds was between 2.2 and 25%. Intraday and interday repeatability for metabolites was between 0.6 and 24.1% and 1 and 23.9%, respectively. Observed lower limits of quantification for native compounds were ∼9–350 fmol on-column, and for the microbial metabolites were ∼0.8–12,000 fmol on-column. Observed lower limits of detection for native compounds were ∼4.5–190 fmol on-column, and for metabolites were 0.304–6020 fmol on-column. For native monomers and procyanidins, extraction recoveries ranged from 38 to 102%. Extraction recoveries for the 9 microbial metabolites tested ranged from 41 to 95%. Data from tissue analysis of rats gavaged with grape seed extract indicate fairly high accumulation of native compounds, primarily monomers and dimers, in the cecum and colon. Metabolite data indicate the progressive nature of microbial metabolism as the digesta moves through the lower GI tract. This method facilitates the high-throughput, sensitive, and simultaneous analysis of both native compounds and their microbial metabolites in biological samples and provides a more efficient means of extraction and analysis than previous methods.
Keywords: Procyanidins; Colon; Microflora; Metabolism; Grape seed extract; LC–MS;

A rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantitatively analyzing T-2 toxin and its major metabolites (HT-2 toxin and T-2 triol) in swine biological samples. For all matrices, liquid–liquid extraction (ethyl acetate or acetonitrile) and Varian Bond-Elut MycoSep cartridges for solid phase extraction were used for sample preparation. The analytes were separated via a Zorbax XDB-C18 column and were detected using LC–MS/MS with an electrospray ionization interface in positive ion mode. The resulting calibration curves offered satisfactory linearity (r 2  > 0.992) within the test range. The limits of quantification for T-2 toxin, HT-2 toxin, and T-2 triol were 1 ng/mL (μg/kg), 2 ng/mL (μg/kg), and 5 ng/mL (μg/kg), respectively. The recovery rates in different matrices ranged from 74.3% to 102.4%, and the interday and intraday precisions were all less than 10.2% for the target analytes. The developed method was successfully applied to toxicokinetics, tissue distribution, and excretion studies of T-2 toxin and its major metabolites after intravenous (i.v.) administration in pigs. The results provide important information for evaluating and controlling human exposure to residual T-2 toxin and its major metabolites in animal-derived food.
Keywords: T-2 toxin; LC–MS/MS; Toxicokinetics; Tissue distribution; Excretion; Pigs;

We describe and validate a sensitive UHPLC-ESI-QTOF-MS method for the simultaneous quantification of seven endocannabinoids and non-endocannabinoids related N-acylethanolamides: N-arachidonoylethanolamide, N-palmitoylethanolamide, N-stearoylethanolamide, N-oleoylethanolamide, N-linoleoylethanolamide, N-α-linolenoylethanolamide and N-eicosapentaenoylethanolamide in several bio-matrices for the purpose of research and clinical application. We examined effects of different liquid–liquid and solid phase extraction on the recovery of endocannabinoids and N-acylethanolamides. Protein precipitation with cooled acetone and extraction with acetonitrile (1% v/v formic acid) using OASIS HLB cartridge gave better results. Separation was performed on a Waters Acquity UPLC HSST3 column using a 9 min elution gradient coupled with high resolution mass spectrometry (QTOF/MS). The high sensitivity of the developed method allow its application on sample with low volumes or low levels of endocannabinoids and N-acylethanolamides and make the method suitable for routine measurement in human bio-matrices, such as plasma, serum (500 μL), urine (1 mL) and tissues (10–30 mg). Its application in clinical research could contribute to unravel pathophysiological roles of these family of lipid mediators and disclose novel diagnostic and prognostic markers.
Keywords: Endocannabinoids; N-acylethanolamides; LC–MS/MS; Anandamide, Bio-matrices; Quantitative analysis;

Identification and comparative quantitation of glycation by stable isotope labeling and LC–MS by Hongcheng Liu; Gomathinayagam Ponniah; Alyssa Neill; Rekha Patel; Bruce Andrien (90-95).
Glycation is a common modification of proteins both in vitro and in vivo. To aid identification and comparative quantitation, a method of stable isotope labeling followed by LC–MS analysis was proposed. The samples were reduced using sodium borohydride or sodium borodeuteride. Reduction of the Schiff base between the amine group and the reducing sugars resulted in a molecular weight increase of 2 Da using sodium borohydride or a molecular weight increase of 3 Da using sodium borodeuteride. The molecular weight difference of 1 Da between peptides containing glycated lysine residue reduced using sodium borohydride or sodium borodeuteride was used to identify glycated peptides and to calculate the glycation difference between samples. The method was used to investigate glycation of a recombinant human IgG1 antibody under native and denaturing conditions. The result demonstrated a good correlation between glycation propensity of lysine residues and their solvent exposure levels.
Keywords: Glycation; Antibody; Sodium borohydride; Mass spectrometry;

Simultaneous determination of creatine phosphate, creatine and 12 nucleotides in rat heart by LC–MS/MS by Jun-mei Wang; Yang Chu; Wei Li; Xiang-yang Wang; Jia-hua Guo; Lu-lu Yan; Xiao-hui Ma; Ying-li Ma; Qi-hui Yin; Chang-xiao Liu (96-101).
A simple, rapid and sensitive LC–MS/MS method was developed and validated for simultaneous determination of creatine phosphate (CP), creatine (Cr) and 12 nucleotides in rat heart. The analytes, ATP, ADP, AMP, GTP, GDP, GMP, CTP, CDP, CMP, UTP, UDP, UMP, CP, Cr, were extracted from heart tissue with pre-cooled (0 °C) methanol/water (1:1, v/v) and separated on a Hypersil Gold AQ C18 column (150 mm × 4.6 mm, 3 μm) using an isocratic elution with a mobile phase consisting of 2 mmol/L ammonium acetate in water (pH 10.0, adjusted with ammonia). The detection was performed by negative ion electrospray ionization in selective reaction monitoring mode (SRM). In the assay, all the analytes showed good linearity over the investigated concentration range (r  > 0.99). The accuracy was between 80.7% and 120.6% and the precision expressed in RSD was less than 15.6%. This method was successfully applied to measure the concentrations of the 12 nucleotides, creatine phosphate and creatine in rat heart for the first time.
Keywords: Nucleotides; Creatine; Creatine phosphate; LC–MS/MS; Rat heart;

Quantification of dapaconazole in human plasma using high-performance liquid chromatography coupled to tandem mass spectrometry: Application to a phase I study by Fernanda C. de Moraes; Samara F. Bittencourt; Elisa Perissutti; Francesco Frencentese; André M.M. Arruda; Lu Shi Chen; Tainah Babadópulos; Gilberto De Nucci (102-107).
A simple, selective and sensitive method based on high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS) has been developed for the determination of dapaconazole in human plasma using tioconazole as internal standard. The drugs were extracted from plasma by liquid–liquid extraction with ether/hexane (80/20, v/v). The chromatography separation was performed on a Genesis® C18 reversed phase analytical column 4 μm (100 × 2.1 mm i.d.) with a mobile phase of methanol/acetonitrile/water (80/10/10, v/v/v) + ammonium acetate (0.5 mM). Dapaconazole was quantified using a mass spectrometer with an electrospray source in the ESI positive mode (ES+) configured for multiple reaction monitoring (MRM) to monitor the transitions 415.1 > 159.2 and 387.0 > 131.0 for dapaconazole and tioconazole, respectively. The method had a chromatography run time of 3.8 min and a linear calibration curve over the range 0.2–100 ng/mL (r  = 0.9998). The lower limit of quantification (LLOQ) was 0.2 ng/mL. The precision and accuracy values of the assay were within ±10%. The stability tests indicate no significant degradation under the conditions of the experiment. This method was used for a phase I study of topical administration of dapaconazole tosylate in healthy human male volunteers.
Keywords: Antifungal; Imidazole; Healthy volunteers; HPLC; MS/MS; Blood;

HPLC/ESI-MS n method for non-amino bisphosphonates: Application to the detection of tiludronate in equine plasma by M.A. Popot; P. Garcia; C. Hubert; A. Bolopion; L. Bailly-Chouriberry; Y. Bonnaire; D. Thibaud; J. Guyonnet (108-116).
Tiludronate is a non-nitrogen-containing biphosphonate drug approved in equine veterinary medicine for the treatment of navicular disease and bone sparvin in horse. Its hydrophilic properties and its strong affinity for the bone have made the control of its use quite difficult. After an initial step of method development in plasma and urine, due to a strong matrix effect and erratic detection in urine, the final method development was conducted in plasma. After addition of (3-trifluoromethylphenyl) thiomethylene biphosphonic acid as internal standard, automated sample preparation consisted of a filtration on a Nexus cartridge followed by a Solid Phase Extraction on an Oasis WAX cartridge with weak anion exchange properties. After methylation of the residue with trimethyl orthoacetate (TMOA), analysis was conducted by HPLC/ESI-MS n on a LTQ mass spectrometer. The method has been validated with a LOD and LOQ of respectively 1 and 2.5 ng/mL. Using a weighting factor of 1/concentration2, a linear model was suitable in the range of 2.5 up to 500 ng/mL. Precision and accuracy data determined at two concentrations were satisfactory (i.e. less than 15%). Carryover would have been a problem but this has finally been fixed using the additional steps of washing during robotised SPE extraction and analysis on both the autosampler and the analytical column. The method was successfully employed for the first time to the quantification of tiludronate in plasma samples collected from horses treated with Tildren™ (Intravenous administration at the dose of 0.1 mg/kg/day for 10 days).
Keywords: Horse doping control; Tiludronate; Quantification; Plasma; SPE; TMOA methylation; LC/MS;

Determination of mesoridazine by liquid chromatography–tandem mass spectrometry and its application to pharmacokinetic study in rats by So Hee Im; Myoung Joo Park; Hyewon Seo; Sung Heum Choi; Sang Kyum Kim; Sung-Hoon Ahn (117-123).
The object of the present study was to develop and validate an assay method of mesoridazine in rat plasma using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Plasma samples from rats were prepared by simple protein precipitation and injected onto the LC–MS/MS system for quantification. Mesoridazine and chlorpromazine as an internal standard (IS) were separated by a reversed phase C18 column. A mobile phase was composed of 10 mM ammonium formate in water and acetonitrile (ACN) (v/v) by a linear gradient system, increasing the percentage of ACN from 2% at 0.4 min to 98% at 2.5 min with 4 min total run time. The ion transitions monitored in positive-ion mode [M + H]+ of multiple-reaction monitoring (MRM) were m/z 387 > 126 for mesoridazine and m/z 319 > 86 for IS. The detector response was specific and linear for mesoridazine at concentrations within the range 0.001–4 μg/ml and the correlation coefficient (R 2 ) was greater than 0.999 and the signal-to-noise ratios for the samples were ≥10. The intra- and inter-day precision and accuracy of the method were determined to be within the acceptance criteria for assay validation guidelines. The matrix effects were approximately 101 and 99.5% from rat plasma for mesoridazine and chlorpromazine, respectively. Mesoridazine was stable under various processing and/or handling conditions. Mesoridazine concentrations were readily measured in rat plasma samples after intravenous and oral administration. This assay method can be practically useful to the pharmacokinetic and/or toxicokinetic studies of mesoridazine.
Keywords: LC–MS/MS; Mesoridazine; Method validation; Rat plasma; Pharmacokinetics; Toxicokinetics;

In this study, an accurate and reliable LC–MS/MS assay was firstly developed and validated for the quantitative determination of trans δ-veniferin (TVN) in rat plasma. Chlorzoxazone was used as the internal standard (IS). After one-step protein precipitation with methanol, the analyte and IS were separated on an ODS column by gradient elution with mobile phase of acetonitrile—0.2% formic acid at a flow rate of 0.3 mL/min. Negative electrospray ionization was performed using multiple reactions monitoring (MRM) mode with transitions of m/z 453.0 > 410.9 for TVN, and m/z 168.0 > 132.0 for IS. Good linearity (R  ≥ 0.996) was observed over the concentration range of 5–5000 ng/mL for TVN with a lower limit of quantification (LLOQ) of 5 ng/mL. The mean recoveries for TVN and IS were 91.05% and 96.68%, respectively. The intra- and inter-day precisions (RSD) were no more than 10.5% and accuracies (RE) were within the range of −6.3% to 2.1%. The validated method was suitable for quantification of TVN and successfully applied to the pharmacokinetic study of TVN after oral and intravenous administration to rats. The oral absolute bioavailability of TVN was 14.2% in rat.
Keywords: Trans δ-veniferin; LC–MS/MS; Pharmacokinetics; Bioavailability;

Determination of urinary aromatic amines in smokers and nonsmokers using a MIPs-SPE coupled with LC–MS/MS method by Jingjing Yu; Sheng Wang; Ge Zhao; Bing Wang; Li Ding; Xiaobing Zhang; Jianping Xie; Fuwei Xie (130-135).
Urinary aromatic amines (AAs) could be used as biomarkers for human exposure to AAs in cigarette smoke. A liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the determination of urinary AAs (i.e. 1-naphthylamine (1-NA), 2-naphthylamine (2-NA), 3-aminobiphenyl (3-ABP) and 4-aminobiphenyl (4-ABP)) in smokers and nonsmokers. A molecularly imprinted polymers (MIPs) solid phase extraction (SPE) cartridge was applied to purify urine samples and no derivatization reaction was involved. Each analytes used respective stable isotope internal standards, which could well compensate matrix effect. Lower limit of detections (LODs) for four AAs were obtained and in the range of 1.5–5 ng L−1. Recovery ranged from 87.7 ± 4.5% to 111.3 ± 6.4% and precision were less than 9.9%. The method was applied to analyze urine samples of 40 smokers and 10 nonsmokers. The 24 h urinary excretion amounts of total AAs were higher for smokers compared with nonsmokers. What's more, 1-NA, 3-ABP and 4-ABP excretion amounts showed significant differences (p  < 0.05) between smokers and nonsmokers.
Keywords: LC–MS/MS; Aromatic amines; Urine; Biomarker; MIPs;