Journal of Chromatography B (v.879, #32)

Determination of tranexamic acid concentration by solid phase microextraction and liquid chromatography–tandem mass spectrometry: First step to in vivo analysis by Barbara Bojko; Dajana Vuckovic; Erasmus Cudjoe; Md Ehsanul Hoque; Fatemeh Mirnaghi; Marcin Wąsowicz; Angela Jerath; Janusz Pawliszyn (3781-3787).
A solid phase microextraction (SPME) method followed by LC–MS/MS analysis was developed to determine the concentration of tranexamic acid (TA) in plasma. The use of a new biocompatible C18 coating allowed the direct extraction from complex biological samples without prior sample preparation; no matrix effect was observed. The results revealed that SPME was suitable for the analysis of polar drugs such as TA; such an application was previously inaccessible because of the limited availability of SPME coatings that can extract polar molecules. The proposed method was validated according to the bioanalytical method validation guidelines. LOD and LLOQ were 0.5 and 1.5 μg/ml, respectively. The recovery for the method was 0.19%, and the accuracy and precision of the method were <9 and <11%, respectively, with the exception of LLOQ, where the values were <16 and <13%, respectively. The performance of the proposed method was also compared against that of the standard techniques of protein precipitation and ultrafiltration. A statistical analysis indicated a clinically significant agreement among all assays. Another advantage of SPME over conventional techniques was the easy automation and feasibility of in vivo analysis; this advantage makes it possible to use the proposed method for an on-site analysis in clinical practice.
Keywords: Solid phase microextraction; Liquid chromatography; Tandem mass spectrometry; Tranexamic acid; Cardiopulmonary bypass; Plasma;

Lewisite metabolites detection in urine by liquid chromatography–tandem mass spectrometry by Igor Rodin; Arkady Braun; Andrey Stavrianidi; Oleg Shpigun; Igor Rybalchenko (3788-3796).
► We study the determination of such lewisite metabolites as CVAA and CVAOA in urine samples by LC–MS/MS. ► The developed assay was based on the use of solid-phase extraction. ► The method have a detection limit (LOD) of 0.5 ng/ml for CVAA and 3 ng/ml for CVAOA. ► Application of this procedure was demonstrated in the lewisite animals exposure model (for 21 days post-exposure).A sensitive and simple method for the quantification and for the detection of 2-chlorovinylarsonous (CVAA) and 2-chlorovinylarsonic (CVAOA) acids was developed. CVAA and CVOA are important biological markers in human and rat urine specific to lewisite (chlorovinylarsonous chloride compounds) exposure. The developed assay was based on the use of solid-phase extraction (SPE) followed by liquid-chromatography coupled to electrospray ionization (negative ion-mode) low-energy collision dissociation-tandem mass spectrometry (ESI-CID-MS/MS). The method demonstrated linearity over at least three orders of magnitude and had a detection limit (LOD) of 0.5 ng/ml for CVAA and 3 ng/ml for CVAOA. The relative standard deviations for the quality control samples ranged from 6 to 11%. Application of this procedure was demonstrated in the lewisite animals exposure model. Rats were exposed intravenously by no lethal doses of lewisite and markers levels in urine samples were analyzed for 21 days post-exposure.
Keywords: Lewisite; Liquid chromatography; Tandem mass spectrometry;

Determination of free desmosine and isodesmosine as urinary biomarkers of lung disorder using ultra performance liquid chromatography–ion mobility-mass spectrometry by Neil A. Devenport; James C. Reynolds; Ved Parkash; Jason Cook; Daniel J. Weston; Colin S. Creaser (3797-3801).
► We have analyzed free DES/IDES levels in urine (Healthy vs. COPD). ► Targeting Free DES/IDES provides a significant reduction in sample preparation. ► Resolution of the free DES/IDES isomers is achieved in 6 min. ► Free DES/IDES levels have been quantified using ion mobility-mass spectrometry. ► Free DES/IDES levels in urine are significantly elevated in COPD patients.The elastin degradation products, desmosine (DES) and isodesmosine (IDES) are highly stable, cross-linking amino-acids that are unique to mature elastin. The excretion of DES/IDES in urine, in the free form and with associated peptide fragments, provides an indicator of lung damage in chronic obstructive pulmonary disease (COPD). A quantitative ion mobility-mass spectrometry (IM-MS) method has been developed for the analysis of free DES/IDES in urine with deuterated IDES as an internal standard. Resolution of DES/IDES isomers was achieved in less than five minutes using ultra performance liquid chromatography (UPLC) combined with ion pairing. The optimized UPLC–IM-MS method provided a linear dynamic range of 10–300 ng/mL and a limit of quantitation of 0.028 ng/mL for IDES and 0.03 ng/mL for DES (0.55 ng and 0.61 ng on column respectively). The method reproducibility (%RSD) was <4% for DES and IDES. The UPLC–IM-MS method was applied to the analysis of urine samples obtained from healthy volunteers and COPD patients. The DES/IDES concentrations in healthy and COPD urine showed an increase in DES (79%) and IDES (74%) in the COPD samples, relative to healthy controls. The incorporation of an IM separation prior to m/z measurement by MS was shown to reduce non-target ion responses from the bio-fluid matrix.
Keywords: Ion mobility-mass spectrometry; Urinary analysis; COPD; Desmosine; Isodesmosine;

► Protein separation was performed with figure-8 columns in centrifugal CCC. ► Tracing of the elution curve was improved with an on-line dialyzer. ► The highest peak resolution was achieved at 0.01 mL/min flow at 1200 rpm.The performance of protein separation using the figure-8 column configuration in centrifugal counter-current chromatography was investigated under various flow rates and revolution speeds. The separation was performed with a two-phase solvent system composed of polyethylene glycol 1000/potassium phosphate each at 12.5% (w/w) in water and with lysozyme and myoglobin as test samples. In order to improve tracing of the elution curve, a hollow fiber membrane dialyzer was inserted at the inlet of the UV detector. The results showed that the retention of stationary phase (Sf) and resolution (Rs) increased with decreased flow rate and increased revolution speed. The highest Rs of approximately 1 was obtained at a flow rate of 0.01 mL/min under a revolution speed of 1200 rpm with a 3.4 mL capacity column.
Keywords: Centrifugal counter-current chromatography; Figure-8 column; Protein separation; Polymer phase system; Lysozyme; Myoglobin; Hollow fiber membrane dialyzer;

Selection of the derivatization reagent—The case of human blood cholesterol, its precursors and phytosterols GC–MS analyses by David Saraiva; Rute Semedo; Maria da Conceição Castilho; José Manuel Silva; Fernando Ramos (3806-3811).
► Simultaneous derivatization of cholesterol precursors and phytosterols in serum. ► MSTFA/DTE/TMIS was selected as the best silylation reagent. ► Assessment of biomarkers for cholesterol absorption/synthesis ratio determination. ► Better control of hypercholesterolemia. ► Contribution for a better drug prescription and for the elaboration of diets.Phytosterols (PS; β-sitosterol and campesterol) and cholesterol precursors (CP; desmosterol and lathosterol) have been suggested as important biochemical markers of cholesterol intestinal absorption and liver biosynthesis, respectively. Given that these compounds appear in human blood in low amounts, sensitive and accurate methodology is required, such as gas chromatography–mass spectrometry (GC–MS) the most frequently used. One of the most critical factors of the GC analytical determination is the step of derivatization. Thus, the main objective of the present study was compare and select the better (one out of three) silylation mixtures as follows: N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide/ammonium iodide (MTBSTFA:NH4I), N-O-bis-(trimethylsilyl) trifluoroacetamide/trimethylchlorosilane (BSTFA:TMCS), and N-methyl-N-(trimethylsilyl)-trifluoroacetamide/1,4-dithioerythritol/trimethyliodosilane (MSTFA:DTE:TMIS). The results of this study are discussed and accompanied by a brief review on the importance and principles of derivatization process, specifically in silylation reactions in GC–MS sterols analyses. Furthermore, a scrutiny of some published results is presented, along with additional information about mass spectral data of these potentially useful compounds. Interestingly, the results of the study showed that from the three validated methodologies, the selected one, based on the best relation specificity/sensibility, is MSTFA:DTE:TMIS. With this silylation procedure for simultaneous determination of PS and CP in human serum by GC–MS in selected ion monitoring (SIM) mode, good linearity (r 2  ≥ 0.931), precision (repeatability ranging from 0.92 to 3.91 CV and intermediate precision ranging from 5.12 to 6.33) and recoveries (≥93.6%) were obtained. Thus, it proved to be a helpful methodology in the quantification of predominant serum cholesterol origin in each patient.
Keywords: Cholesterol; Desmosterol; Lathosterol; Campesterol; β-Sitosterol; GC–MS;

► This is the first study to describe a sensitive and precise LC–MS assay for cediranib. ► This assay allows small specimen volumes and adequate detectability to use in tissue distribution studies. ► We have employed the assay in a brain distribution study in the mouse, to examine different mechanisms that limit the CNS distribution of cediranib. ► The assay is generally applicable to a variety of pharmacokinetic and in vitro cell culture experiments.A new high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) assay for cediranib, a tyrosine kinase inhibitor for VEGFRs, was developed and validated, for the determination of plasma and brain levels of cediranib in small specimen volumes. Tyrphostin (AG1478) was used as internal standard. Mouse plasma and brain homogenate samples were prepared using liquid–liquid extraction. The assay was validated for a 2.5–2500 ng/mL concentration range for plasma, and for 1–2000 ng/mL range for brain homogenate. For these calibration ranges, within-assay variabilities were 1.1–14.3% for plasma and 1.5–9.4% for brain homogenate; between-assay variabilities were 2.4–9.2% for plasma, and 4.9–10.2% for brain homogenate. Overall accuracy ranged from 101.5 to 107.0% for plasma and 96.5 to 100.2% for brain homogenate, for all target concentrations. The developed assay has been successfully applied for a brain distribution study in mice at an oral dose of 5 mg/kg.
Keywords: Cediranib; Liquid–liquid extraction; Liquid chromatography mass spectrometry; Pharmacokinetics;

► 8-OHdG and 8-NO2Gua were simultaneously determined by capillary electrophoresis with amperometric detection (CE–AD). ► CE–AD apparatus employed is inexpensive and easy to operate. ► The proposed method was rather simple and has been applied to analyze clinical samples. ► The excretion levels of urinary 8-OHdG and 8-NO2Gua of cancer patients were significantly higher than those of healthy persons. With the growth of age, 8-OHdG urinary levels also increase slightly.A sensitive and low-cost analytical method has been developed to determine 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 8-nitroguanine (8-NO2Gua) based on capillary electrophoresis with amperometric detection (CE–AD) after solid phase extraction (SPE). Under optimized condition, these two markers were well separated from other components coexisting in urine, exhibiting a linear calibration over the concentration range of 0.1–50.0 μg/mL with the detection limits ranging from 0.02 to 0.06 μg/mL. The relative standard deviations (RSDs) were in the range of 0.1–2.1% for peak area, 0.1–1.5% for migration time, respectively. The average recovery and RSD were within the range of 100.0–108.0% and 0.1–1.7%, respectively. It was found that the urinary contents of 8-OHdG and 8-NO2Gua in cancer patients were significantly higher than those in healthy ones.
Keywords: Capillary electrophoresis; Amperometric detection; 8-Hydroxy-2′-deoxyguanosine; 8-Nitroguanine; DNA damage marker; Urine;

► We describe an HPLC assay for Palomid 529 in human and mouse plasma and tissues. ► Sample pre-treatment involved liquid–liquid extraction with tert-butyl methyl ether. ► Separation involved reversed phase HPLC and detection by UV at 315 nm. ► The dynamic range was 10–10,000 ng/ml. ► The applicability for in vivo pharmacokinetics studies is demonstrated.Palomid 529 (8-(1-Hydroxy-ethyl)-2-methoxy-3-(4-methoxy-benzyloxy)-benzo[c]chromen-6-one), is a novel non-steroidal small-molecule drug, which inhibits both mTORC1 and mTORC2 assembly, and elicits both anti-angiogenic and direct anti-tumor effects in vivo. We have developed and validated a sensitive and selective method for the quantification of Palomid 529 in human and mouse plasma and in a range of mouse tissue samples. Sample pretreatment involved liquid–liquid extraction with tert-butyl methyl ether yielding a recovery of >75%. Palomid 529 and the internal standard Palomid 545 were separated using a GraceSmart RP18 column (2.1 mm × 150 mm) packed with 5 μm C-18 material and a mobile phase comprised of 50% (v/v) acetonitrile and 50% (v/v) water delivered at a flow rate of 0.2 ml/min, and were detected by UV absorbance at a wavelength of 315 nm. Within the linear range of the calibration curve (10–10,000 ng/ml), acceptable accuracy and precision was achieved for all tested matrices. The validation results show that the method was selective and reproducible. Palomid 529 was stable in plasma upon 3 repeated freeze–thaw cycles and during storage for up to 24 h at ambient temperature. However, pre-treated samples waiting for HPLC analyses need to be kept under dimmed light conditions at ambient temperature since a significant degradation of both Palomid 529 and Palomid 545 was observed when exposed to light. A pilot pharmacokinetic study in mice demonstrated the applicability of this method for pharmacokinetic purposes. Even at a low dose of 5.4 mg/kg this assay was still sensitive enough to determine the drug concentration in plasma samples obtained up to 24 h after administration.
Keywords: Palomid 529; Palomid 545; HPLC; mTOR inhibitor; Photosensitivity;

A LC–MS/MS methodology to determine furaltadone residues in the macroalgae Ulva lactuca by Sara Leston; Margarida Nunes; Andreia Freitas; Jorge Barbosa; Fernando Ramos; Miguel Ângelo Pardal (3832-3836).
► New methodology to detect and quantify the nitrofuran antibiotic furaltadone in the green macroalgae Ulva lactuca. ► Method relies on LC–MS/MS techniques. ► Experimental assay with exposure of U. lactuca to hypothetical prophylactic and therapeutic concentrations. ► Successfully applied to blank spiked and experimental samples.Presently, the rise of new contaminants in the environment has widened the scope of pharmaceutical analyses as to face the demanding new challenges. An increasing tendency for the interconnection and overlap of research fields, such as ecology and biochemistry, is intensifying the demand for new methodologies to be applied to the survey of drugs in unconventional matrices. Integrated in this group are macrophytes, such as the green macroalgae Ulva lactuca, which are under study as to ascertain their ability as indicators of contamination for many substances. Nonetheless, methodologies for extraction and determination of drugs in such matrices are scarce and new studies on the subject are pressing. A new methodology for the determination of the antibiotic furaltadone in U. lactuca by liquid chromatography–tandem mass spectrometric (LC–MS/MS) procedure was developed, optimized and validated following the guidelines of the EC Decision 2002/657. The calibration curves showed linearity above 0.99 (R 2). The relative standard deviations obtained for repeatability, expressed as CV, were between 15.3 and 20.5 and for reproducibility 25.3 and 28.2 whereas accuracy was in the interval of 88.9–95.5 (%). The limit of decision (CCα) and the detection capability (CCβ) were respectively 5.57 μg kg−1 and 10.97 μg kg−1. The method was successfully applied to experimental samples.
Keywords: Antibiotics; Furaltadone; Macroalgae; LC–MS/MS;

► Highly sensitive and accurate LC/MS/MS method was developed for measuring digoxin. ► The developed method achieved higher sensitivity than previously reported methods requiring only 25 μL of the sample fluid. ► The key successful factor was to employ negative ionization to completely avoid competitive adduct formation. ► The unique, one-step sample pretreatment method may provide advantages over existing methods, such as the small required sample volume, no need for extensive sample cleanup, safety, and low cost of the procedure, among others.Highly sensitive and accurate liquid chromatography–tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for measuring digoxin (DGX), a typical P-glycoprotein probe, in human plasma, rat plasma, and rat brain. We extracted DGX and deuterium-labeled DGX (as internal standard) from sample fluids under basic conditions using acetonitrile and sodium chloride-saturated 0.1 mol/L sodium hydroxide. The upper organic layer was diluted with distilled water, and the resulting solution was injected into an LC/MS/MS system in negative ionization mode. Chromatographic separation was achieved on a C18-ODS column in the gradient mobile phase, which comprised 0.05% (w/v) ammonium carbonate (pH 9.0) and methanol at a flow rate of 0.7 mL/min. Regardless of the type of biological matrix, intra-day and inter-day validation tests demonstrated good linearity of calibration curves within ranges of 0.1–10 ng/mL for plasma and 0.5–50 ng/g for rat brain and gave excellent accuracy and precision of quality control samples at 4 concentration levels. Unlike existing methods, our approach uses negative ionization to avoid competitive adduct formation of DGX. Our method showed higher sensitivity and wider applicability to various types of biological matrices than existing methods. Our method will support clinical and preclinical investigation of in vivo P-glycoprotein functionality using DGX.
Keywords: Digoxin; P-glycoprotein; Tandem mass spectrometry; Analytical method; Adduct formation;

High-performance liquid chromatographic determination of tiapride and its phase I metabolite in blood plasma using tandem UV photodiode-array and fluorescence detection by Milan Nobilis; Zuzana Vybíralová; Barbora Szotáková; Květa Sládková; Martin Kuneš; Zbyněk Svoboda (3845-3852).
New bioanalytical SPE–HPLC–PDA–FL method for the determination of the neuroleptic drug tiapride and its N-desethyl metabolite was developed, validated and applied to xenobiochemical and pharmacokinetic studies in humans and animals. The sample preparation process involved solid-phase extraction of diluted plasma spiked with sulpiride (an internal standard) using SPE cartridges DSC-PH Supelco, USA. Chromatographic separation of the extracts was performed on a Discovery HS F5 250 mm × 4 mm (Supelco) column containing pentafluorophenylpropylsilyl silica gel. Mobile phase (acetonitrile–0.01 M phosphate buffer pH = 3, flow rate 1 ml min−1) in the gradient mode was employed in the HPLC analysis. Tandem UV photodiode-array → fluorescence detection was used for the determination of the analytes. Low concentrations of tiapride and N-desethyl tiapride were determined using a more selective fluorescence detector (λ(exc.)/λ(emiss.) = 232 nm/334 nm), high concentrations (500–6000 pmol ml−1) using a UV PDA detector at 212 nm with a linear response. Each HPLC run lasted 15 min. Lower limits of quantification (LLOQ) for tiapride (N-desethyl tiapride) were found to be 8.24 pmol ml−1 (10.11 pmol ml−1). The recoveries of tiapride ranged from 89.3 to 94.3%, 81.7 to 86.8% for internal standard sulpiride and 90.9 to 91.8% for N-desethyl tiapride.
Keywords: Benzamide neuroleptics; Tiapride; N-desethyl tiapride (metabolite); Human pharmacokinetics; HPLC with UV photodiode-array and fluorescence detection;

Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system by José Murillo P. Barbosa; Ranyere L. Souza; Alini T. Fricks; Gisella Maria Zanin; Cleide Mara F. Soares; Álvaro S. Lima (3853-3858).
► ATPS could be used to selectively purify lipase from the crude culture filtrates. ► The best purification factor was 201.53 fold. ► The enzyme partitioning is governed by entropy. ► Lipase by Bacillus sp. ITP-001 purified in PEG/potassium phosphate ATPS has 54 kDa.This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000 g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4 °C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS–PAGE. According to the results the best combination for purifying the enzyme is PEG 8000 g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4 °C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution.
Keywords: Lipolytic enzyme; Purification; Aqueous two-phase system;

► It was demonstrated that GC–IDMS is suitable for the accurate absolute quantification of metabolites in microbial cultures. ► The described method exhibits certain attractive advantages over established LC–IDMS protocols. ► Certain metabolites were subjected to absolute quantification by GC–IDMS for the first time. ► Examination of derivatization conditions and sample matrix effects resulted in distinct improvements as well as practical recommendations. ► First-time use of commercially available U–13C-labeled algal cells as a convenient source for the generation of isotope labeled internal standards.In the field of metabolomics, GC–MS has rather established itself as a tool for semi-quantitative strategies like metabolic fingerprinting or metabolic profiling. Absolute quantification of intra- or extracellular metabolites is nowadays mostly accomplished by application of diverse LC–MS techniques. Only few groups have so far adopted GC–MS technology for this exceptionally challenging task. Besides numerous and deeply investigated problems related to sample generation, the pronounced matrix effects in biological samples have led to the almost mandatory application of isotope dilution mass spectrometry (IDMS) for the accurate determination of absolute metabolite concentrations. Nevertheless, access to stable isotope labeled internal standards (ILIS), which are in many cases commercially unavailable, is quite laborious and very expensive. Here we present an improved and simplified gas chromatography–isotope dilution mass spectrometry (GC–IDMS) protocol for the absolute determination of intra- and extracellular metabolite levels. Commercially available 13C-labeled algal cells were used as a convenient source for the preparation of internal standards. Advantages as well as limitations of the described method are discussed.
Keywords: Isotope dilution MS; GC–MS; Yeast; Metabolites; Absolute quantification; U–13C-labeled algae;

► Catecholamines and indolamines are the most studied monoamines in neuroscience. ► Time of routine analyses by conventional HPLC and electrochemical detection is too long. ► Use of sub-2 μm particles increases the throughput of analysis using ultra-fast HPLC. ► Brain monoamines can be determined in various tissue samples by this way. ► Potential applications of ultra-fast HPLC in brain dialysis.Electrochemical detection is often used to detect catecholamines and indolamines in brain samples that have been separated by conventional reverse-phase high performance liquid chromatography (HPLC). This paper presents the transfer of an existing chromatographic method for the determination of monoamines in brain tissues using 5 μm granulometry HPLC columns to columns with a particle diameter less than 3 μm. Several parameters (repeatability, linearity, accuracy, limit of detection, and stability of samples) for this new ultrafast high performance liquid chromatography (UHPLC) method were examined after optimization of the analytical conditions. The separation of seven compounds, noradrenaline, dopamine and three of its metabolites, dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine, and serotonin and its metabolite, 5-hydroxyindole-3-acetic acid was analyzed using this UHPLC–electrochemical detection method. The final method, which was applied to brain tissue extracts from mice, rats, and cats, decreased analysis time by a factor of 4 compared to HPLC, while guaranteeing good analytical performance.
Keywords: Monoamine; Metabolite; Ultrafast high performance liquid chromatography; Brain samples; Electrochemical detection;

Multiplex quantification of lamprey specific bile acid derivatives in environmental water using UHPLC–MS/MS by Ke Li; Huiyong Wang; Cory O. Brant; SangChun Ahn; Weiming Li (3879-3886).
► Simultaneous measurement of bile acid derivatives in environmental water and animal conditioned samples. ► Using of MCX cartridge in sample process resulted in excellent recoveries. ► UHPLC/MS/MS accomplished high-resolution separation and provided lower LODs. ► The developed method is an appealing methodology for routine analysis.Larval and adult sea lampreys (Petromyzon marinus) release bile salts and acids into the surrounding aquatic environment. Some of these bile salts and acids, such as petromyzonol sulfate (PZS), 3-keto petromyzonol sulfate (3k PZS), petromyzonamine disulfate (PADS), petromyzosterol disulfate (PSDS), and 3-keto allocholic acid (3k ACA), may function as pheromones. To examine the release and distribution patterns of these metabolites, which this study has termed bile acid derivatives, we developed a novel UHPLC–MS/MS method that was characterized by simple sample preparation, baseline separation, and short analysis time for all studied compounds. These five analytes were separated in 7 min using a reversed-phase C18 column containing 1.7 μm particles and a gradient elution at pH 8.9. Once separated, the analytes were subjected to electrospray ionization-mass spectrometry (negative ion mode) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) using the multiple reaction monitoring (MRM) mode. Deuterated 3k PZS ([2H5]3k PZS) was added as the internal standard (IS) to the sample prior to solid phase extraction (SPE). Among the three types of SPE sorbent tested, mixed-mode cation-exchange and reversed-phase sorbent for bases (MAX) and acids (MCX), and reversed-phase C18 sorbent (Sep-pak), the best recoveries (84.1–99.7%) were obtained with MCX cartridges. The calibration curves of all five analytes were linear between 0.15 and 1200 ng/mL, with R 2  ≥ 0.9997. This method had a precision of relative standard deviation (RSD) ≤9.9% and an accuracy of deviation (DEV) ≥92.5%. The developed method was successfully used to quantify bile acid derivatives found in streams where lampreys spawn (SD < 1.4) and water conditioned with male sea lampreys (SD < 4.8). Utilizing this method provides a routine analysis of lamprey bile acid derivatives and may prove useful for sea lamprey population estimates in future studies and applications.
Keywords: Petromyzon; Pheromone; Solid phase extraction; Tandem mass spectrometry; Ultrahigh performance liquid chromatography;

► We develop a HPLC-ELSD method for species discrimination of Radix Bupleuri. ► We analyze the ten saikosaponins simultaneously by HPLC-ELSD and LC–MS. ► The extraction condition of saikosaponins from Radix Bupleuri was optimized. ► The developed method was fully validated. ► This method was applied to determine saikosaponins in 20 Bupleurum species samples.A simple, rapid and robust high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method was established for the species discrimination and quality evaluation of Radix Bupleuri through the simultaneous determination of ten saikosaponins, namely saikosaponin-a, -b1, -b2, -b3, -b4, -c, -d, -g, -h, and -i. These compounds were chromatographed on an Ascentis® Express C18 column with a gradient elution of acetonitrile and water containing 0.1% acetic acid at a flow rate of 1.0 mL/min. Saikosaponins were monitored by ELSD, which was operated at a 50 °C drift tube temperature and 3.0 bar nebulizer gas (N2) pressure. The developed method was validated with respect to linearity, intra- and inter-day accuracy and precision, limit of quantification (LOQ), recovery, robustness and stability, thereby showing good precision and accuracy, with intra- and inter-assay coefficients of variation less than 15% at all concentrations. Furthermore, a high performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) method was developed to certify the existence of ten saikosaponins, as well as to confirm the reliability of ELSD. The extraction conditions of saikosaponins from Radix Bupleuri were also optimized by investigating the effect of extraction methods (sonication, reflux and maceration) and various solvents on the extraction efficiencies for saikosaponins. Sonication with 70% methanol for 40 min was found to be simple and effective for extraction of major saikosaponins. This analytical method was applied to determine saikosaponin profiles in 20 real samples consisting of four Bupleurum species, namely B. falcatum, B. chinense, B. sibiricum and the poisonous B. longiradiatum. It was found that three major saikosaponin-a, -c and -d were the major constituents in B. falcatum, B. chinense, and B. longiradiatum, while one major saikosaponin (saikosaponin-c) was not identified from B. sibiricum. In addition, no saikosaponin-b3 was detected in B. longiradiatum samples, indicating that the toxic B. longiradiatum may be tentatively distinguished from officially listed Bupleurum species (B. falcatum and B. chinense) based on their saikosaponin profiles. Overall the simultaneous determination of ten saikosaponins in Radix Bupleuri was shown to be a promising tool to adopt for the discrimination and quality control of closely related Bupleurum species.
Keywords: Species discrimination; Radix Bupleuri; Saikosaponins; Bupleurum falcatum; Bupleurum longiradiatum; Evaporative light scattering detection;

Biomimetic affinity purification of Candida antarctica lipase B by Hongyan Yao; Tian Zhang; Hongwei Xue; Kexuan Tang; Rongxiu Li (3896-3900).
► Biomimetic affinity purification of Candida antarctica lipase B. ► One-step purification effect of ligand columns A9-14. ► Active groups of A9-14 were cyclohexylamine and propenylamine. Candida antarctica lipase B (CalB) is one of the most widely used biocatalysts in organic synthesis. The traditional method for purification of CalB is a multi-step, high cost and low recovery procedure. Biomimetic affinity purification had high efficiency purification. We selected 298 ligand columns from a 700-member library of synthetic ligands to screen Pichia pastoris protein extract. Of the 298, three columns (named as A9-14, A9-10, and A11-33) had one-step purification effect, and A9-14 of these affinity ligands, had both high purification and recovery. The one-step recovery of CalB reached 73% and the purification reached 91% upon purification. The active groups of A9-14 were cyclohexylamine and propenylamine. Furthermore, both A9-14 and A9-10 had the same R1 active group of cyclohexylamine which might act the main binding role for CalB. The synthetic ligand A9-14 had a binding capacity of 0.4 mg/mL and had no negative effects on its hydrolytic activity. Unlike a natural affinity ligand, this synthetic ligand is highly stable to resist 1 M NaOH, and thus has great potential for industrial scale production of CalB.
Keywords: Affinity purification; Synthetic ligand; Candida antarctica lipase B; Pichia pastoris;

► Identification of the metabolites of flavonoids from Abelmoschus manihot in intestinal flora. ► Using UPLC–Q-TOF MS with automated data analysis software MetaboLynx™. ► A total of 14 metabolites were identified in incubated solution. ► The results indicated that hydrolysis, hydroxylation and acetylation were the major metabolic pathways of flavonoids in A. manihot extract. Abelmoschus manihot has drawn much attention recently due to its potential beneficial health effects after oral administration. However, the metabolic fate of A. manihot in intestinal flora is not well understood. In this paper, we describe a strategy using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC–Q-TOF MS) with automated data analysis software (MetaboLynx™) for fast analysis of the metabolic profile of flavonoids from A. manihot in intestinal flora. The human and rat incubated samples collected 72 h in the anaerobic incubator were analyzed by UPLC–Q-TOF MS within 10 min. A total of 14 metabolites were identified in human and rat incubated solution compared with blank samples. The results indicated that hydrolysis, hydroxylation and acetylation were the major metabolic pathways of flavonoids in A. manihot extract in vitro. MSE was used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of metabolites. This work demonstrated the potential of the UPLC–Q-TOF MS approach using Metabolynx for fast and automated identification of metabolites of natural product in intestinal flora.
Keywords: UPLC–Q-TOF MS; Intestinal bacteria; Metabolites; Abelmoschus manihot extract; MDF; MSE;

LC–MS/MS-based metabolites of Eurycoma longifolia (Tongkat Ali) in Malaysia (Perak and Pahang) by Lee Suan Chua; Nor Amaiza Mohd Amin; Jason Chun Hong Neo; Ting Hun Lee; Chew Tin Lee; Mohamad Roji Sarmidi; Ramlan Abdul Aziz (3909-3919).
Eurycoma longifolia has different metabolite profiles dependent on the geographical origin. ▶ Leucine based peptide has been identified as marker in the room temperature extracts. ▶ A new hydroxyl methyl β-carboline propionic acid has been identified in the 100 °C extracts. ▶ Eurycomanone and its derivatives present in the highest amount compared to other groups. ▶ The concentration of alkaloids was higher than quassinoids in the 100 °C extracts.A number of three LC–MS/MS hybrid systems (QTof, TripleTof and QTrap) has been used to profile small metabolites (m/z 100–1000) and to detect the targeted metabolites such as quassinoids, alkaloids, triterpene and biphenylneolignans from the aqueous extracts of Eurycoma longifolia. The metabolite profiles of small molecules showed four significant clusters in the principle component analysis for the aqueous extracts of E. longifolia, which had been collected from different geographical terrains (Perak and Pahang) and processed at different extraction temperatures (35 °C and 100 °C). A small peptide of leucine (m/z 679) and a new hydroxyl methyl β-carboline propionic acid have been identified to differentiate E. longifolia extracts that prepared at 35 °C and 100 °C, respectively. From the targeted metabolites identification, it was found that 3,4ɛ-dihydroeurycomanone (quassinoids) and eurylene (squalene-type triterpene) could only be detected in the Pahang extract, whereas canthin-6-one-3N-oxide could only be detected in the Perak extract. Overall, quassinoids were present in the highest concentration, particularly eurycomanone and its derivatives compared to the other groups of metabolites. However, the concentration of canthin-6-one and β-carboline alkaloids was significantly increased when the roots of the plant samples were extracted at 100 °C.
Keywords: LC–MS/MS; Eurycoma longifolia; Quassinoids; Alkaloids; Triterpenes; Biphenylneolignans;

Development and validation of a liquid chromatography–tandem mass spectrometry method for the quantification of opiorphin in human saliva by Lidija Brkljačić; Maja Sabalić; Ivan Salarić; Ivanka Jerić; Ivan Alajbeg; Ina Nemet (3920-3926).
► Opiorphin, the QRFSR-pentapeptide, showed beneficial effects in pain management. ► An LC–MS/MS method for the analysis of opiorphin in human saliva was developed. ► Its level in saliva collected from young individuals ranged from 2.8 to 25.9 ng/ml.Opiorphin, QRFSR-peptide, is a mature product of the PROL1 (proline rich, lacrimal 1) protein that showed beneficial effects in pain management, antidepressant-like actions as well as involvement in colonic motility and erectile physiology. Using opiorphin as a potential biomarker of different pathological states requires the development of robust and sensitive methods. We report a highly sensitive and specific liquid chromatography with tandem mass spectrometric detection (LC–MS/MS) analytical method for the analysis of opiorphin in human saliva. Quantification was based on multiple reaction monitoring using characteristic transitions (m/z 347/120 – as quantifying ion; 347/175 and 347/268 as qualifying ions). The assay was linear in the range of 0–110 ng/ml and the lower limit of quantification reached was 1.0 ng/ml. The intra-day precision and accuracy were between 2.7–5.6% and −2.3 to 3.2%, respectively. The inter-day precision and accuracy were between 10.8–13.7% and −11.0 to 52%, respectively. Mean recovery was 106% and mean matrix effect was 0.97. Opiorphin in TFA treated saliva samples was stable for at least 12 h at room temperature and up to 30 days at −20 °C. Opiorphin levels in human saliva samples collected from young healthy individuals ranged from 2.8 to 25.9 ng/ml.
Keywords: Opiorphin; Saliva; LC–MS/MS; Biomarker;

Surrogate based accurate quantification of endogenous acetylcholine in murine brain by hydrophilic interaction liquid chromatography–tandem mass spectrometry by Liang Peng; Tao Jiang; Zhengxing Rong; Ting Liu; Hao Wang; Biyun Shao; Jian Ma; Lan Yang; Lei Kang; Yifeng Shen; Huafang Li; Hong Qi; Hongzhuan Chen (3927-3931).
► We develop a surrogate-based method to determine endogenous ACh in murine brain. ► Surrogate-based method improved accuracy and precision of quantitation. ► We use surrogate-based method to evaluate the matrix effect in authentic matrix. ► We apply the method to evaluate the effect of huperzine A on ACh in murine brain.Cholinergic dysfunction is known as a hallmark feature of Alzheimer's disease (AD). Measurement of endogenous acetylcholine (ACh) in specific brain regions is important in understanding the pathology of AD and in designing and evaluating novel cholinomimetic agents for the treatment of AD. Since ACh is an endogenous neurotransmitter, there is no real blank matrix available to construct standard curves. It has been a challenging task to determine ACh in complex brain matrices. To overcome these difficulties, we employed a surrogate analyte strategy using ACh-d4 instead of ACh to generate calibration curves and Ch-d9 as internal standard (IS). The brain samples were deproteinized by acetonitrile with IS. Analytes and IS were separated by a HILIC column with the mobile phase composed of 20 mM ammonium formate in water–acetonitrile (30:70, v/v, adjusted to pH 3.0 with formic acid) and monitored in multiple reaction monitoring (MRM) mode using a positive electrospray source. The concentrations of endogenous ACh were calculated based on the peak area ratio of the analyte to the IS using a regression equation for the corresponding surrogate standard (ACh-d4). The lower limit of detection was 0.2 ng/mL and linearity was maintained over the range of 10–1000 ng/mL. Compared to other currently available methods, this approach offers improved accuracy and precision for efficient analysis of ACh. The proposed method was proved successfully by evaluating the action of typical acetylcholinesterase inhibitor huperzine A in senescence accelerated mouse prone 8 (SAMP8).
Keywords: Acetylcholine; Surrogate analyte; HILIC; LC–MS/MS;

► We develop an HPLC-fluorescence method for analysis of thiol compounds. ► The derivatization of coenzyme A, Cys, GSH and N-acetyl-cysteine needs only 6 min. ► Baseline separation is achieved in 6 min using isocratic elution. ► The chromatograms are background-free. ► The detection limits are from 0.13 nM for coenzyme A to 0.25 nM for Cys (S/N = 3).A rapid and simple background-free high-performance liquid chromatographic (HPLC) approach has been developed for simultaneously determining free thiol compounds including coenzyme A (CoA), cysteine (Cys), glutathione (GSH) and N-acetyl-cysteine (NAC) in biological samples by using 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide) difluoroboradiaza-s-indacene (TMPAB-o-M) as fluorogenic reagent. After derivatization under physiological conditions within 6 min, baseline separation was finished in just 6 min using isocratic elution with reversed-phase HPLC and fluorescence detection. Excellent linearity was observed for all analytes over their concentration ranges of 1–500 nM and detection limits ranging 0.13 nM for CoA to 0.25 nM for Cys (S/N = 3) were achieved. The utility of the proposed method has been validated by measuring thiol compounds mentioned above in tissue, fluid and cell samples. The results indicated that this approach was well suited for high-throughput quantitative determination of thiols and study of the physiological role of them.
Keywords: Thiols; 1,3,5,7-Tetramethyl-8-phenyl-(2-maleimide) difluoroboradiaza-s-indacene; HPLC; Fluorescence;

► This is the first study to describe a sensitive and precise LC–MS/MS assay for codeine, ephedrine, guaiphenesin and chlorpheniramine. ► We utilized this method to measure concentration of codeine, ephedrine, guaiphenesin and chlorpheniramine in beagle dog plasma. ► This method supports research of codeine, ephedrine, guaiphenesin and chlorpheniramine and its pharmacokinetics in beagle dog plasma.A rapid and sensitive method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous determination of codeine, ephedrine, guaiphenesin and chlorpheniramine in beagle dog plasma has been developed and validated. Following liquid–liquid extraction, the analytes were separated on a reversed-phase C18 column (150 mm × 2.0 mm, 3 μm) using formic acid:10 mM ammonium acetate:methanol (0.2:62:38, v/v/v) as mobile phase at a flow rate of 0.2 mL/min and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode. The method was linear for all analytes over the following concentration (ng/mL) ranges: codeine 0.08–16; ephedrine 0.8–160; guaiphenesin 80–16,000; chlorpheniramine 0.2–40. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. It is the first time that the validated HPLC–MS/MS method was successfully applied to a bioequivalence study in 6 healthy beagle dogs.
Keywords: Codeine; Ephedrine; Guaiphenesin; Chlorpheniramine; HPLC–MS/MS; Bioequivalence;

► A new LC–MS/MS method for the measurement of R123 in rat plasma has been established. ► The method proved to be specific, highly sensitive and accurate over a concentration range of 1–200 ng/ml. ► Liquid–liquid extraction was more suitable for disposing plasma samples and available for enhancing the sensitivity. ► The method was successfully applied to evaluate the functional activity of P-gp in an absorption experiment in the rat.Rhodamine 123 (R123), as a typical of P-gp substrate, was widely used to quantify P-glycoprotein (P-gp) functional efflux activity in vivo. A new, rapid and sensitive method was developed for quantifying R123 in rat plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS). R123 and Rhodamine 6G (R6G, the internal standard, IS) were extracted from aliquots of plasma with ethyl acetate and dichloromethane (4:1) as the solvent and chromatographic separation was performed using a Zorbax Eclipse Plus C18 column. The mobile phase was composed of A: ammonium formate–formic acid buffer containing 5 mM ammonium formate and 0.1% formic acid and B: methanol (A:B, 5:95, v/v). To quantify R123 and IS respectively, multiple reaction monitoring (MRM) transition of m/z 345.2 → 285.2 and m/z 443.3 → 415.2 was performed. The analysis time was 4 min in positive mode; the calibration curve was linear in the concentration range of 1–200 ng/ml. The lowest limit of quantification (LLOQ) reached 1 ng/ml. The intra and inter-day precision were less than 9.2% for the low quality control (QC) level, and 3.4% for other QC levels, respectively, while the intra and inter-day relative errors ranged between −7.4% and 9.1% for three QC concentration levels. The LC–MS/MS method proved to be simple, accurate, reliable and with a shorter running time and has been successfully applied to evaluate the functional activity of P-glycoprotein in an absorption experiment in the rat.
Keywords: Rhodamine 123; Liquid chromatography–tandem mass spectrometry; Rhodamine 6G; Rat plasma;

Corrigendum to “Rapid UHPLC determination of polyphenols in aqueous infusions of Salvia officinalis L. (sage tea)” [J. Chromatogr. B 879 (2011) 2459–2464] by Benno F. Zimmermann; Stephan G. Walch; Laura Ngaba Tinzoh; Wolf Stühlinger; Dirk W. Lachenmeier (3949).