Journal of Chromatography B (v.949-950, #C)

In analysis of muramic acid (MA) as bacterial marker, two dominant disturbing factors lead the researchers to use gas chromatography–tandem mass spectrometry (GC–MS/MS) technique instead of gas chromatography–mass spectrometry (GC–MS). These factors are the trace concentration of MA and fundamental disturbance of base line mass channels in GC–MS technique. This study aimed to utilize multivariate curve resolution (MCR) methods combined with GC–MS to improve the analysis of MA. First, the background and noise in GC–MS analysis were corrected and reduced using MCR methods. In addition, the MA overlapped peaks were resolved to its pure chromatographic and mass spectral profiles. Then the two-way response of each component was reconstructed by the outer product of the pure chromatographic and mass spectral profiles. The overall volume integration (OVI) method was used for quantitative determination. The MA peak area was decreased dramatically after the background correction and noise reduction. The findings severely ratify the appropriateness of using MCR techniques combined with GC–MS analysis as a simple, fast and inexpensive method for the analysis of MA in complex mixtures. The proposed method may be considered as an alternative method to GC–MS/MS for thorough analysis of the bacterial marker.
Keywords: Multivariate curve resolution; Gas chromatography–mass spectrometry; Muramic acid;

The current study aims to investigate the pharmacokinetic study of eight caffeic acid derivatives (forsythoside A, isoforsythoside, forsythoside B, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid) following oral administration of Flos LoniceraeFructus Forsythiae herb combination in rats. A rapid and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed to determine the eight caffeic acid derivatives simultaneously in rat plasma. After mixing with the internal standard (IS) tinidazole, plasma samples were pretreated by liquid–liquid extraction with n-butyl alcohol/ethyl acetate (7:3, v/v). The separation was performed on an Acquity UPLC HSS T3 C18 column (100 mm × 2.1 mm, 1.8 μm) at a flow rate of 0.4 mL min−1, and acetonitrile/methanol (4:1, v/v)–0.4% formic acid was used as mobile phase. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) via electrospray ionization (ESI) source with positive and negative ionization modes. All calibration curves had good linearity (r  > 0.991) over the concentration ranges of 1.097–2246 ng mL−1 for neochlorogenic acid, 6.535–6692 ng mL−1 for chlorogenic acid, 2.103–2153 ng mL−1 for cryptochlorogenic acid, 0.5058–129.5 ng mL−1 for 3,5-dicaffeoylquinic acid, 0.3205–82.05 ng mL−1 for 3,4-dicaffeoylquinic acid, 1.002–512.8 ng mL−1 for isoforsythoside, 0.4795–982.1 ng mL−1 for forsythoside A and 0.7587–776.9 ng mL−1 for forsythoside B, respectively. The intra- and inter-batch precisions were all within 15% and the accuracy (relative error, RE%) all ranged from 85.68% to 114.7%. It was shown from pharmacokinetic parameters that the rank order of AUC 0–t , C max and T 1/2k for phenolic acids was chlorogenic acid > neochlorogenic acid ≥ cryptochlorogenic acid > 3,4-dicaffeoylquinic acid ≥ 3,5-dicaffeoylquinic acid (most of them had significant differences), which corresponded to their administration dosages to rats, but that of MRT 0–t and T 1/2z were opposite. Besides, the AUC 0–t , C max, MRT and T 1/2z except T 1/2k of isoforsythoside and forsythoside B had no significant difference, compared to that of forsythoside A though their administration dosages were significantly lower than that of forsythoside A. All results showed that the method was applied to the pharmacokinetic study of the eight caffeic acid derivatives in rat plasma successfully after oral administration of Flos LoniceraeFructus Forsythiae herb combination, and there were significant differences of caffeic acid derivatives even isomers in the pharmacokinetic parameters.
Keywords: Flos LoniceraeFructus Forsythiae herb combination; Caffeic acid derivatives; UPLC–MS/MS; Oral delivery; Pharmacokinetics;

Considering the important complexity of biological samples, a molecularly imprinted polymer (MIP) was applied to the selective extraction of cocaine and its two main metabolites, benzoylecgonine and ecgonine methyl ester from biological samples. The MIP was imprinted with cocaine and it was synthesized in acetonitrile with methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a crosslinker. The selectivity of the MIP was first assessed for the three target analytes in acetonitrile with recoveries higher than 80% on the MIP and lower than 30% on the non-imprinted polymer (NIP). The MIP was then evaluated for the selective extraction of these targets from real aqueous media, i.e. serum and urine samples. The pH adjustment of the sample as well as the optimization of the washing step led to a very selective extraction of cocaine from these media. A LOQ of 0.5 ng/mL was obtained for cocaine in urine. Concerning cocaine metabolites, benzoylecgonine and ecgonine methyl ester, they were first extracted from urine by liquid–liquid extraction and the resulting extract was purified on the MIP. The results obtained with the MIP as compared to the LLE alone showed the great potential of the MIP extraction for the clean-up of the biological matrix. This procedure was tested for the extraction of the analytes from urine samples, leading to a very selective protocol with LOQs of 0.09 ng/mL, 0.4 ng/mL and 1.1 ng/mL for cocaine, benzolecgonine and ecgonine methyl ester respectively in urine samples.
Keywords: Molecularly imprinted polymer; Selective solid-phase extraction; Cocaine; Metabolites; Plasma; Urine;

An inorganic–organic co-functional monomer, methacrylic acid-vinyltriethoxysilan (MAA-VTES) was designed for the synthesis of molecularly imprinted microspheres (MIMs). By virtue of the aqueous suspension polymerization and dummy template (pazufloxacin), the obtained MAA-VTES based MIMs exhibited good recognition and selectivity to fluoroquinolones (FQs), and were successfully applied as selective sorbents of a miniaturized home-made solid phase extraction device for the determination of ofloxacin (OFL), lomefloxacin (LOM) and ciprofloxacin (CIP) in milk samples. Under the optimum conditions of the miniaturized molecularly imprinted solid phase extraction (mini-MISPE) coupled with liquid chromatography-ultraviolet detector (LC-UV), good linearities were obtained for three FQs in a range of 0.2–20.0 μg mL−1 and the average recoveries at three spiked levels were ranged from 87.2% to 106.1% with the relative standard deviation (RSD) less than 5.4%. The presented co-functional monomer based mini-MISPE-LC-UV protocol introduced the rigidity and flexibility of inorganic silicon materials, exhibited excellent extraction performance towards targets, and could be potentially applied to the determination of FQs in milk samples.
Keywords: Inorganic–organic co-functional monomer; Molecularly imprinted microspheres; Home-made solid-phase extraction; Fluoroquinolones; Milk samples;

Ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry for determination of avicularin metabolites produced by a human intestinal bacterium by Min Zhao; Jun Xu; Dawei Qian; Jianming Guo; Shu Jiang; Er-xin Shang; Jin-ao Duan; Jing Yang; Le-yue Du (30-36).
Intestinal bacteria from human were screened to isolate the specific bacteria involved in the metabolism of avicularin. A Gram-positive anaerobic bacterium, strain 46, capable of metabolizing avicularin (quercetin-3-O-arabinoside) was isolated for the first time. Its 16S rRNA gene sequence showed 99% similarity with that of Bacillus. Then strain 46 was identified as a species of the genus Bacillus, and was named to be Bacillus sp. 46. Additionally, the metabolites were analyzed by ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) technique combined with Metabolynx™ software. The structure of these metabolites were proposed and confirmed by comparing the UPLC retention time and MS/MS spectrum with that of authentic standards. Parent compound and six metabolites were detected in the isolated bacterial samples compared with blank samples. Avicularin (M1) was anaerobic metabolized to its aglycone quercetin (M2) and methoxylated avicularin (M3, M4), then quercetin was converted to quercetin glycosides: quercetin-3-O-rhamnoside (M5), quercetin-3-O-glucoside (M6) and quercetin-7-O-glucoside (M7) by Bacillus sp. 46. The metabolic pathway and metabolites of avicularin by the intestinal bacterium Bacillus sp. 46 were reported for the first time.
Keywords: Bacillus sp. 46; 16S rRNA; Avicularin; UPLC/Q-TOF-MS; Metabolism;

Phthalates, parabens and 2-hydroxy-4-methoxybenzophenone or benzophone-3 are thought to act as endocrine disrupting chemicals, being able to disrupt the endocrine balance and therefore able to lead to some hormonal diseases. Numerous large-scale biomonitoring studies have detected the biomarkers of these compounds in more than 75% of the general population. To assess the exposure to these chemicals, we developed an analytical method based on a Solid Phase Extraction (SPE) prior to ultra high pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the simultaneous measurement of seven phthalate metabolites (monobenzyl phthalate, mono-n-butyl phthalate, mono-iso-butyl phthalate, mono-2-ethylhexyl phthalate, mono-2-ethyl-5-hydroxyhexyl phthalate, mono-2-ethyl-5-oxohexyl phthalate, monoethyl phthalate), four parabens (methyl paraben, ethyl paraben, n-propyl paraben, n-butyl parabens) and benzophenone-3 in human urine. The distinction between unconjugated, glucuro- and sulfoconjugated forms was achieved using different enzymatic hydrolyses. The whole procedure was validated according to the total error approach, and was demonstrated to be linear (regression coefficient ranging from 0.987 to 0.998) and accurate (inter and intra assay precision <17.71%, relative bias <5.87%) in the dosing range of concentrations. The limits of quantification (LOQs) obtained ranged between 0.30 and 1.23 ng/ml depending on the analyte. The reliability of the method was proven in passing successfully the German External Quality Assessment Scheme (G-EQUAS). Moreover, the urine from 25 volunteers were analyzed for the determination of glucuro-, sulfo- and free species separately. Phthalate metabolites, parabens and benzophenone-3 were positively detected in almost all urine samples, with detection rates ranging from 40 to 100%. Levels measured ranged from <LOQ to 2207 ng/ml varying widely depending on the compound and the individual. In our small participating population, most of the phthalate metabolites were excreted predominately as glucuroconjugated forms while parabens and benzophenone-3 were detected as glucuro- and sulfoconjugated species in variable proportions according to the target compound.
Keywords: Parabens; Phthalate metabolites; Benzophenone-3; Urine; LC-MS/MS;

Single-domain antibody-based ligands for immunoaffinity separation of recombinant human lactoferrin from the goat lactoferrin of transgenic goat milk by S.V. Tillib; M.E. Privezentseva; T.I. Ivanova; L.F. Vasilev; G.A. Efimov; Y.G. Gursky; G.P. Georgiev; I.L. Goldman; E.R. Sadchikova (48-57).
Single-domain antibody generation technology was applied to make new Sepharose-bound ligands for affinity separation of closely related proteins, such as human and goat lactoferrin. We generated recombinant antibodies that can selectively bind/recognize only lactoferrins having amino acid sequences identical to that of human natural lactoferrin (anti-hLF Ab). Selected and purified histidine-tagged single-domain antibodies were used as ligands, and different lactoferrins were used as analytes in the kinetics analysis of lactoferrin binding to captured anti-hLF Abs using the Bio-Rad ProteOn XPR36 protein interaction array system. The data obtained were consistent with a 1:1 binding model with very high affinity, practically equal in the case of hLF and rec-hLF (calculated K D varied from 0.43 nM to 3.7 nM). Interaction of captured fsdAbs with goat LF was significantly weaker and not detectable under the same analysis conditions. We demonstrated the high efficiency of the recombinant human lactoferrin purification from goat lactoferrin and other proteins using the obtained single domain antibody-based affinity ligands. We believe this approach can be used for the generation of single-domain antibody-based affinity media for the efficient separation/purification of a wide spectrum of other highly homologous proteins.
Keywords: Single-domain antibody; Recombinant human lactoferrin; Immunoaffinity chromatography; Separation of homologous proteins;

HPLC-UV analysis of thymidine and deoxyuridine in plasma of patients with thymidine phosphorylase deficiency by Susan Mohamed; Leonardo Caporali; Roberto De Giorgio; Valerio Carelli; Manuela Contin (58-62).
We present a simple, fast and validated method for the determination of the two nucleosides thymidine (dThd) and deoxyuridine (dUrd) in plasma of patients with symptoms suggestive of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), using high performance liquid chromatography coupled with ultraviolet spectrophotometric detection (HPLC-UV). Plasma sample (100 μL) pretreatment was based on simple deproteinization by 1.2 M perchloric acid, using theophylline as internal standard (I.S.). HPLC-UV analysis was carried out on a Synergi 4 μm Hydro-RP, 150 × 4 mm I.D. column, at room temperature. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (20 mM, pH 4.5) and acetonitrile (95:5, v/v), at an isocratic flow rate of 0.7 mL/min. The UV detector was set at 267 nm. The chromatographic run lasted 19 min. Similar pyrimidine nucleotides and nucleosides do not interfere with the assay. Calibration curves were linear for both dThd and dUrd over a range of 0.5 to 5.0 μg/mL. The limit of quantitation was 0.5 μg/mL for both nucleosides and the absolute recovery was >90% for dThd, dUrd and the I.S. Both intra- and inter-assay precision and accuracy were lower than 10% at all tested concentrations. The proposed method was successfully applied to measure plasma concentrations of dThd and dUrd in two MNGIE patients. This assay simplifies both plasma pretreatment and chromatographic conditions of previously reported procedures and describes the first validated method for the determination of the two nucleotides in human plasma.
Keywords: Thymidine; Deoxyuridine; Mitochondrial neurogastrointestinal encephalomyopathy; High performance liquid chromatography; UV detection;

Simultaneous quantification of idelalisib, fludarabine and lenalidomide in rat plasma by using high-performance liquid chromatography coupled with heated electrospray ionization tandem mass spectrometry by Sridhar Veeraraghavan; Satheeshmanikandan Thappali; Srikant Viswanadha; Santhoshkumar Nalla; Sandhyarani Chennupati; Manikantakumar Golla; Swaroopkumar Vakkalanka; Manivannan Rangasamy (63-69).
A sensitive and reliable high-performance liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of idelalisib, fludarabine and lenalidomide using tolbutamide as an internal standard. Analytes were recovered by liquid–liquid extraction and separated on a reverse phase C18 column (150 mm × 4.6 mm i.d., 5 μm) using methanol:0.1% formic acid buffer (70:30) as mobile phase at a flow rate of 1 mL/min in isocratic mode. Selective reaction monitoring was performed using the transitions, i.e. m/z 416.25/176.48, 286.11/154.10, 260.15/149.15, and 271.14/155.06 to quantify idelalisib, fludarabine and lenalidomide and tolbutamide, respectively. The method was validated over the concentration range of 1.15–576.84 ng/mL for idelalisib, 0.95–476.25 ng/mL for fludarabine and 0.97–486.19 ng/mL for lenalidomide. Intra and inter-day accuracy and precision of validated method were within the acceptable limits of <15%. Coefficients of correlation (r 2) for the calibration curves were >0.998 for all analytes. The method was successfully applied for simultaneous estimation of idelalisib, fludarabine and lenalidomide in a pharmacokinetic study in rats.
Keywords: Idelalisib; Fludarabine; Lenalidomide; LC-MS/MS; Bioanalytical; Pharmacokinetics;

Curcuminoids, a mixture of curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC), have shown a variety of clinical benefits for several human chronic diseases including osteoarthritis, rheumatoarthritis, and type II diabetes. However, the oral bioavailability of curcumin is extremely low due to its avid metabolism to curcumin O-glucuronide (COG), curcumin O-sulfate (COS), tetrahydrocurcumin (THC), and other minor metabolites. This paper reports a unique liquid chromatography/tandem mass spectrometry (LC-MS/MS) method to quantify curcumin, DMC, BDMC, COG, COS, and THC simultaneously in human plasma. These compounds were extracted with ethyl acetate from human plasma, separated on a BetaBasic-8 column, and monitored on a triple quadruple mass spectrometer coupled with API electrospray under a negative ion mode. The linearity of these respective curcuminoids and curcumin metabolites was shown in the range of 2–1000 ng/mL with 85–115% accuracy and ≤20% precision in human plasma. This method was validated according to the US FDA GLP analytic criteria and applied to characterize the pharmacokinetics of curcumin, COG, and COS in human plasma after an oral dose of bioavailable curcumin (nanoemulsion curcumin).
Keywords: Curcuminoids; Liquid chromatography/tandem mass spectrometry (LC-MS/MS); Curcumin metabolites; Pharmacokinetics;

Determination of 26 veterinary antibiotics residues in water matrices by lyophilization in combination with LC–MS/MS by Feng-Yang Hu; Li-Min He; Jian-Wen Yang; Kui Bian; Zong-Nan Wang; Hai-Cui Yang; Ya-Hong Liu (79-86).
A sensitive, simple and reliable multi-residue method was developed for the determination of 26 widely used veterinary antibiotics including 6 macrolides, 2 pleuromutilins, 4 tetracyclines, 2 lincosamides, 6 fluoroquinolones and 6 sulfonamides in different water matrices using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Water samples were lyophilized to dryness. Target compounds were separated on Zorbax SB-Aq column (150 mm × 2.1 mm i.d., 3.5 μm) and determined by LC–MS/MS operating in positive electrospray ionization mode. Spiked at concentration levels of 0.02, 0.4 and 4 μg L−1, recoveries of all target compounds were over 70% except sulfaquinoxaline (59.0% at 0.02 μg L−1) with relative standard deviations below 20%. Limits of detection (LOD) and limits of quantification (LOQ) of 26 drugs ranged from 0.1 to 6.5 ng L−1 and from 0.3 to 19 ng L−1, respectively. The developed method was successfully applied to the analysis of 26 antibiotics residues in fish pond water, groundwater, biogas digester water, and lagoon wastewater samples collected from local pig farms.
Keywords: Antibiotics; Liquid chromatography–tandem mass spectrometry; Environmental water; Lyophilization;

The main aim of the present study was to develop a method for the separation of 5′-monophosphate nucleotides with the use of ion chromatography. Novel dendrimeric stationary phases were used for this purpose. The effects exerted by the type of anion-exchanger support (silica or polymeric) and the number of stationary phase layers on nucleotide retention were studied. A silica-based dendrimeric anion-exchanger was most suitable for analyzing the studied compounds. An increase in the number of layers enhanced nucleotide retention inside the column. The separation efficiency of the studied compounds was tested at various concentrations of the mobile phase buffer. At higher phosphate buffer concentrations, nucleotide resolutions were achieved in 6 min. Three commercially available infant milk formulas were analyzed to verify the applicability of the studied method. Solid phase extraction was used for sample cleanup and concentration. The limit of quantification of nucleotides was 0.40 μg/ml, and the method was linear in the concentration range of 0.40–20.6 μg ml−1.
Keywords: Nucleotides monophosphates; Ion chromatography; Stationary phase; Dendrimer packing materials; Infant milk;

Determination of 3-mercaptopyruvate in rabbit plasma by high performance liquid chromatography tandem mass spectrometry by Michael W. Stutelberg; Chakravarthy V. Vinnakota; Brendan L. Mitchell; Alexandre R. Monteil; Steven E. Patterson; Brian A. Logue (94-98).
Accidental or intentional cyanide poisoning is a serious health risk. The current suite of FDA approved antidotes, including hydroxocobalamin, sodium nitrite, and sodium thiosulfate is effective, but each antidote has specific major limitations, such as large effective dosage or delayed onset of action. Therefore, next generation cyanide antidotes are being investigated to mitigate these limitations. One such antidote, 3-mercaptopyruvate (3-MP), detoxifies cyanide by acting as a sulfur donor to convert cyanide into thiocyanate, a relatively nontoxic cyanide metabolite. An analytical method capable of detecting 3-MP in biological fluids is essential for the development of 3-MP as a potential antidote. Therefore, a high performance liquid chromatography tandem mass spectrometry (HPLC-MS-MS) method was established to analyze 3-MP from rabbit plasma. Sample preparation consisted of spiking the plasma with an internal standard (13C3-3-MP), precipitation of plasma proteins, and reaction with monobromobimane to inhibit the characteristic dimerization of 3-MP. The method produced a limit of detection of 0.1 μM, a linear dynamic range of 0.5–100 μM, along with excellent linearity (R 2  ≥ 0.999), accuracy (±9% of the nominal concentration) and precision (<7% relative standard deviation). The optimized HPLC-MS-MS method was capable of detecting 3-MP in rabbits that were administered sulfanegen, a prodrug of 3-MP, following cyanide exposure. Considering the excellent performance of this method, it will be utilized for further investigations of this promising cyanide antidote.
Keywords: Cyanide antidote; 3-Mercaptopyruvate; Sulfanegen; Liquid chromatography-tandem mass spectrometry;

Identification and quantification of 14 phthalates and 5 non-phthalate plasticizers in PVC medical devices by GC–MS by Pascal Gimeno; Sébastien Thomas; Claudine Bousquet; Annie-Françoise Maggio; Corinne Civade; Charlotte Brenier; Pierre-Antoine Bonnet (99-108).
A GC/MS method was developed for the identification and quantification of 14 phthalates: 8 phthalates classified H360 (DBP, DEHP, BBP, DMEP, DnPP, DiPP, DPP and DiBP), 3 phthalates proposed to be forbidden in medical devices (DnOP, DiNP and DiDP) and 3 other phthalates none regulated (DMP, DCHP and DEP) which may interfere with hormone function. In order to identify and quantify other plasticizers that are commonly used in PVC medical devices such as DEHP substitute, 5 non-phthalate plasticizers (ATBC, DEHA, DEHT, TOTM, and DINCH) were included in this study. Analyses are carried out on a GC/MS system with electron impact ionization mode (EI). The separation of plasticizers is obtained on a cross-linked 5%-phenyl/95%-dimethylpolysiloxane capillary column 30 m × 0.25 mm (i.d.) × 0.25 μm film thickness using a gradient temperature. Compounds quantification is performed by external calibration using an internal standard. Validation elements on standard solutions were determined using the ISO 12787 standard approach. Plasticizers are extracted from PVC medical devices using THF for dissolving the PVC part of the sample followed by precipitation of the PVC by addition of ethanol. The supernatant is injected into a GC/MS system after dilution in ethanol. Different validation elements, including extraction recoveries for all compounds or for DEHP a cross-validation of the extraction process using the European pharmacopoeia monograph 3.1.14 as reference method, are discussed. Results obtained on 61 medical devices in PVC and 12 raw materials used as plasticizers are given.
Keywords: Phthalates; Plasticizers; Medical devices; ISO 12787; Gas chromatography–mass spectrometry;

A simple, efficient and green analytical procedure for monitoring sub ppb amounts of chlorpyrifos (CP) and chlorpyrifos-oxon (CPO) in urine samples was reported. The methodology is based on the solidified floating organic drop microextraction of the analytes with a free microdrop of 2-dodecanol. The parameters those can affect the microextraction efficiency, such as solvent type, extraction solvent volume, extraction time and temperature, salt effect, pH and stirring rate on extraction were optimized. The analytes were extracted from the urine samples by using 10 μL of 2-dodecanol for 40 min at 70 °C and then, the extracts were injected to GC-MS column by applying 100 kPa injection pressure. The regression coefficients relating to linearity were at least 0.99. The accuracy of the developed method was tested upon recovery studies for CP and CPO calculated as 100 ± 7% and 110 ± 9% (at 0.1 ng mL−1 level), respectively. LOD value for CP was found 4.8 ng L−1 and for CPO it was found 3.8 ng L−1. This method can easily be adopted by clinical laboratories for the contribution to policies aiming to reduce exposure of pesticides.
Keywords: Solidified floating organic drop microextraction; Chlorpyrifos; Chlorpyrifos oxon; Pesticide; Gas chromatography; Urine;

Identification of metabolites of deoxyschizandrin in rats by UPLC–Q-TOF-MS/MS based on multiple mass defect filter data acquisition and multiple data processing techniques by Minyan Liu; Shaohua Zhao; Zongquan Wang; Yufeng Wang; Ting Liu; Song Li; Cuicui Wang; Hongtao Wang; Pengfei Tu (115-126).
Deoxyschizandrin is an active lignin ingredient originating from Schisandra chinensis (Turcz.) Baill or Schisandrae Sphenantherae Fructus. In the present study, a novel and efficient strategy was developed for the in vivo screening and identification of deoxyschizandrin metabolites using ultra high performance liquid chromatography combined with triple TOF mass spectrometry (UPLC–TOF/MS/MS). This strategy was characterized by the following: a novel and unique multiple mass defect filter (MMDF) combined with an on-line data acquisition method that is dependent on dynamic background subtraction (DBS) was developed to trace all of the probable metabolites of deoxyschizandrin. The MMDF and DBS methods could trigger an IDA scan for the low-level metabolites that are masked by background noise and endogenous components. A combination of data processing methods including extracted ion chromatography (XIC), mass defect filtering (MDF), product ion filtering (PIF) and neutral loss filtering (NLF) were employed to identify the metabolites of deoxyschizandrin. Next, the structures of the metabolites were elucidated based on an accurate mass measurement, the fragmentation patterns of the parent drug and relevant drug bio-transformation knowledge. Finally, an important parameter Clog  P was used to estimate the retention time of isomers. Based on the proposed strategy, 51 metabolites (including 49 phase I and 2 phase II metabolites) were identified in rats after the oral administration of deoxyschizandrin. Among these metabolites, 41 metabolites were characterized in the rat urine, and 28 metabolites were identified in the rat bile. The results indicated that oxidization was the main metabolic pathway and that the methoxy group and the biphenyl cyclooctene were the metabolic sites. Conjugation with sulfate and cysteine groups produced two phase-II metabolites. This study firstly reported the description of deoxyschizandrin metabolism in vivo. This study provided a practical strategy for rapidly screening and identifying metabolites, and this methodology can be widely applied for the structural characterization of the metabolites of other compounds.
Keywords: Deoxyschizandrin; Metabolites; UPLC–Q-TOF-MS/MS; Identification; Multiple mass defect filter; Multiple data processing;

Opiates play a relevant role in forensic toxicology and their assay in urine or blood is usually performed for example in workplace drug-testing or toxicological investigation of drug impaired driving. The present work describes two new methods for detecting morphine, codeine and 6-monoacethyl morphine in human urine or blood using a single step derivatisation in aqueous phase. Propyl chloroformate is used as the dramatizing agent followed by liquid–liquid extraction and gas-chromatography–mass spectroscopy to detect the derivatives. The methods have been validated both for hydrolysed and unhydrolysed urine.For hydrolysed urine, the LOD and LOQ were 2.5 ng/ml and 8.5 ng/ml for codeine, and 5.2 ng/ml and 15.1 ng/ml for morphine, respectively. For unhydrolysed urine, the LOD and LOQ were 3.0 ng/ml and 10.1 ng/ml for codeine, 2.7 ng/ml and 8.1 ng/ml for morphine, 0.8 ng/ml and 1.5 ng/ml for 6-monoacetyl morphine, respectively. In blood, the LOD and LOQ were 0.44 ng/ml and 1.46 ng/ml for codeine, 0.29 ng/ml and 0.98 ng/ml for morphine, 0.15 ng/ml and 0.51 ng/ml for 6-monoacetyl morphine, respectively.The validated methods have been applied to 50 urine samples and 40 blood samples (both positive and negative) and they can be used in routine analyses.
Keywords: Aqueous derivatisation; Propyl chloroformate; Morphine; Codeine; 6-monoacetyl morphine; Human urine;