Journal of Chromatography B (v.1036-1037, #C)

The bio-active compounds of ginger (Zingiber officinale Roscoe), the gingerols, are gaining considerable attention due to their numerous beneficial health effects. In order to elucidate the physiological relevance of the ascribed effects their bioavailability has to be determined taking their metabolization into account. To quantitate in vivo generated [6]-, [8]- and [10]-gingerol glucuronides in human plasma and urine after ginger tea consumption, a simultaneous and direct liquid chromatography–tandem mass spectrometry method based on stable isotope dilution assays was established and validated. The respective references as well as the isotopically labeled substances were synthesized and characterized by mass spectrometry and NMR. Selective isolation of gingerol glucuronides from human plasma and urine by a mixed-phase anion-exchange SPE method led to recovery rates between 80.8 and 98.2%. LC–MS/MS analyses in selected reaction monitoring modus enabled a highly sensitive quantitation of gingerol glucuronides with LoQs between 3.9–9.8 nmol/L in plasma and 39.3–161.1 nmol/L in urine. The method precision in plasma and urine varied in the range ± 15%, whereas the intra-day accuracy in plasma and urine showed values between 78 and 122%. The developed method was then applied to a pilot study in which two volunteers consumed one liter ginger tea. Pharmacokinetic parameters like the maximum concentration (cmax), the time to reach cmax (tmax), area under the curve (AUC), elimination rate constant (kel ) and elimination half-life (t1/2) were calculated from the concentration-time curve of each gingerol glucuronide. The obtained results will enable more detailed investigation of gingerol glucuronides as bioactives in their physiologically relevant concentrations.
Keywords: Ginger (Zingiber officinale Roscoe; Gingerol glucuronide; Pharmacokinetics; Metabolism; LC–MS/MS;

Preparative separation of cacao bean procyanidins by high-speed counter-current chromatography by Lingxi Li; Shuting Zhang; Yan Cui; Yuanyuan Li; Lanxin Luo; Peiyu Zhou; Baoshan Sun (10-19).
In this work, an efficient method for preparative separation of procyanidins from raw cacao bean extract by high-speed counter-current chromatography (HSCCC) was developed. Under the optimized solvent system of n-hexane-ethyl acetate-water (1:50:50, v/v/v) with a combination of head–tail and tail–head elution modes, various procyanidins fractions with different polymerization degrees were successfully separated. UPLC, QTOF-MS and 1H NMR analysis verified that these fractions contained monomer up to pentamer respectively. Dimeric procyanidin B2 (purity > 86%) could be isolated by HSCCC in a single run. Other individual procyanidins in these fractions could be further isolated and purified by preparative HPLC. The developed HSCCC together with preparative HPLC techniques appeared to be a useful tool for large preparation of different procyanidins from cacao beans. Furthermore, by antioxidant activity assays, it was proved that both fractions and individual procyanidins possessed greater antioxidant activities compared to standard trolox. The antioxidant activities of procyanidins increase as the increase of their polymerization degree.
Keywords: Antioxidant activity; Cacao bean; High-speed counter-current chromatography; Procyanidin; Separation;

Separation of chlorinated diastereomers of decarboxy-betacyanins in myeloperoxidase catalyzed chlorinated Beta vulgaris L. extract by Sławomir Wybraniec; Karolina Starzak; Edward Szneler; Zbigniew Pietrzkowski (20-32).
A comparative chromatographic evaluation of chlorinated decarboxylated betanins and betanidins generated under activity of hypochlorous acid exerted upon these highly antioxidative potent decarboxylated pigments derived from natural sources was performed by LC-DAD-ESI-MS/MS. Comparison of the chromatographic profiles of the chlorinated pigments revealed two different directions of retention changes in relation to the corresponding substrates. Chlorination of all betacyanins that are decarboxylated at carbon C-17 results in an increase of their retention times. In contrast, all other pigments (the non-decarboxylated betacyanins as well as 2-decarboxy- and 15-decarboxy-derivatives) exhibit lower retention after chlorination. During further chromatographic experiments based upon chemical transformation of the related pigments (decarboxylation and deglucosylation), the compounds’ structures were confirmed. The elaborated method for determination of chlorinated pigments enabled analysis of a chlorinated red beet root extract that was submitted to the MPO/H2O2/Cl system acting under inflammation-like conditions (pH 5). This indicates a promising possibility for measurement of these chlorinated pigments as indicators of specific inflammatory states wherein betacyanins and decarboxylated betacyanins act as hypochlorite scavengers.
Keywords: Betanin; Betalains; Myeloperoxidase; Inflammation; Hypochlorite; Red beet extract;

The purpose of this study is to establish and validate an UPLC–MS/MS approach to determine 4-caffeoylquinic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid, loganic acid, loganin, sweroside, dipsacoside B and asperosaponin VI from extracts of crude and wine-processed Dipsacus asper in biological samples and apply the approach to a comparative pharmacokinetic study. A Waters BEH C18 UPLC column was employed with acetonitrile/0.2% formic acid-water as mobile phases. The mass analysis was carried out in a triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) with negative scan mode. A one-step protein precipitation by acetonitrile was performed to extract the eight analytes from plasma. Our results revealed that all of the calibration curves displayed good linear regression (r2  > 0.9990). The lower limits of quantification (LLOQ) were determined as 10.0, 9.6, 8.9, 9.1, 9.2, 9.8, 10.1 and 9.8 ng/mL. The intra-day and inter-day precisions (RSD) of the eight compounds at high, medium and low levels were less than 4.94% and the bias of the accuracies ranged from −3.89% to 3.95%.The extraction recoveries of the eight compounds were from 90.4% to 100.2% and the matrix effects ranged from 89.3% to 100.1%. The stabilities of these compounds were investigated by analyzing six replicates of QC samples at three different concentrations following storage at 25 °C for 4 h, −80 °C for 30 days, three-freeze-thaw cycles, and 4 °C for 24 h. All the samples showed satisfactory precision and accuracy after various stability tests. Pharmacokinetic parameters were estimated using a non-compartment model. Compared with the crude group, the parameters of C max and AUC0-t of 4-caffeoylquinic acid, loganic acid, loganin and asperosaponin VI increased remarkably (p<0.05) after oral administration of the aqueous extract of wine-processed Dipsacus asper, indicating that wine-processing could enhance bioavailability of 4-caffeoylquinic acid, loganic acid, loganin and asperosaponin VI.
Keywords: Dipsacus asper; Wine-processed; UPLC–MS/MS; Pharmacokinetics;

Measurement of neosaxitoxin in human plasma using liquid–chromatography tandem mass spectrometry: Proof of concept for a pharmacokinetic application by Roy W.A. Peake; Victoria Y. Zhang; Nina Azcue; Christina E. Hartigan; Aida Shkreta; Jasmina Prabhakara; Charles B. Berde; Mark D. Kellogg (42-49).
Neosaxitoxin, a member of the saxitoxin family of paralytic shellfish poisoning toxins, has shown potential as an effective, long-acting, anesthetic. We describe the development and validation of a highly sensitive method for measurement of neosaxitoxin in human plasma using liquid chromatography tandem mass spectrometry (LC–MS/MS) and provide evidence for its use in a human pharmacokinetic study. Samples were prepared using cation exchange solid phase extraction followed by hydrophilic interaction liquid chromatography and MS/MS detection in positive electrospray ionization mode. Multiple reaction monitoring was used to monitor neosaxitoxin (m/z 316.17 > 220.07) and the internal standard analogue decarbamoylneosaxitoxin (m/z 273.12 > 180.00). The method was validated for lower limit of quantification, precision, accuracy, linearity and matrix effect. The stability of neosaxitoxin in plasma matrix at various storage conditions was also investigated. Standard curves for calibration were linear (r > 0.995) across the assay calibration range, 10 to 1000 pg/mL. The analytical measurable range of the assay was 10–10,000 pg/mL in plasma matrix. This method has demonstrated excellent sensitivity demonstrating a lower limit of quantification in human plasma of 10 pg/mL. The mean, inter-batch variation was <5.2% across the concentration range 30 to 800 pg/mL. This method was successfully used in a phase 1 trial to investigate the pharmacokinetic profile of neosaxitoxin in humans following the intravenous administration of the drug at a range of doses up to 40 μg. We conclude that our high-sensitivity method for measurement of neosaxitoxin in human plasma is capable of supporting future clinical trials.
Keywords: LC–MS/MS; Pharmacokinetcs; Neosaxitoxin;

Citral is a widely used monoterpene aldehyde in aromatherapy, food and pesticide industries. A new validated reverse phase high performance liquid chromatography (RP – HPLC) procedure for the detection and quantification of cis-trans isomers of citral was developed. The RP-HPLC analysis was carried out using Enable C – 18G column (250 × 4.6 mm, 5 μ), with acetonitrile and water (70: 30) mobile phase in isocratic mode at 1 mL/min flow. A photodiode array (PDA) detector was set at 233 nm for the detection of citral. The method showed linearity, selectivity and accuracy for citral in the range of 3–100 μg/mL. In order to compare the new RP-HPLC method with the available methods, one of the commercially available essential oil from Cymbopogon flexuosus was analyzed using new RP-HPLC method and the same was analyzed using GC–MS for the comparison of the method for the detection of citral. The GC–MS analysis was done using mass selective detector (MSD) showed citral content to be of 72.76%; wherein the new method showed to contain that same at 74.98%. To prove the application of the new method, essential oils were extracted from lemongrass, lemon leaves and mosambi peels by steam distillation. The citral content present in the essential and also in the condensate was analyzed. The method was found to be suitable for the analysis of citral in essential oils and water based citral formulations with a very good resolution of its components geranial and neral.
Keywords: RP-HPLC; Geranial and neral; Gas chromatography; PDA detector; Mass selective detector;

Display OmittedOnline abstract Figure: Flow diagram for simultaneous quantification of soman and VX adducts to BChE, their aged methylphosphonic acid adduct and BChE in plasma using an off-column procainamide-gel separation combined with UHPLC–MS/MSThis work describes a novel and sensitive non-isotope dilution method for simultaneous quantification of organophosphorus nerve agents (OPNAs) soman (GD) and VX adducts to butyrylcholinesterase (BChE), their aged methylphosphonic acid (MeP) adduct and unadducted BChE in plasma exposed to OPNA. OPNA-BChE adducts were isolated with an off-column procainamide-gel separation (PGS) from plasma, and then digested with pepsin into specific adducted FGES*AGAAS nonapeptide (NP) biomarkers. The resulting NPs were detected by UHPLC–MS/MS MRM. The off-column PGS method can capture over 90% of BChE, MeP-BChE, VX-BChE and GD-BChE from their respective plasma materials. One newly designed and easily synthesized phosphorylated BChE nonapeptide with one Gly-to-Ala mutation was successfully reported to serve as internal standard instead of traditional isotopically labeled BChE nonapeptide. The linear range of calibration curves were from 1.00–200 ng mL−1 for VX−NP, 2.00–200 ng mL−1 for GD−NP and MeP−NP (R2  ≥ 0.995), and 3.00–200 ng mL−1 for BChE NP (R2  ≥ 0.990). The inter-day precision had relative standard deviation (%RSD) of <8.89%, and the accuracy ranged between 88.9–120%. The limit of detection was calculated to be 0.411, 0.750, 0.800 and 1.43 ng mL−1 for VX-NP, GD-NP, MeP-NP and BChE NP, respectively. OPNA-exposed quality control plasma samples were characterized as part of method validation. Investigation of plasma samples unexposed to OPNA revealed no baseline values or interferences. Using the off-column PGS method combined with UHPLC–MS/MS, VX-NP and GD-NP adducts can be unambiguously detected with high confidence in 0.10 ng mL−1 and 0.50 ng mL−1 of exposed human plasma respectively, only requiring 0.1 mL of plasma sample and taking about four hours without special sample preparation equipment. These improvements make it a simple, sensitive and robust PGS-UHPLC–MS/MS method, and this method will become an attractive alternative to immunomagnetic separation (IMS) method and a useful diagnostic tool for retrospective detection of OPNA exposure with high confidence. Furthermore, using the developed method, the adducted BChE levels from VX and GD-exposed (0.10–100 ng mL−1) plasma samples were completely characterized, and the fact that VX being more active and specific to BChE than GD was re-confirmed.
Keywords: Organophosphorus nerve agents; Retrospective detection; BChE adduct; Chemical warfare agent; UHPLC-ESI-MS; MS; OPNA exposure;

Over the last decades, more and more studies focused on the impact of endocrine disruptors on the environment and human health. Among them, phenolic organohalogens (POHs) are a particular concern because of their structural resemblance with natural hormones. There are different methods that are known to quantify these compounds in human serum, however, the current extraction techniques are long, fastidious and using harmfull chemicals such as diazomethane and sulfuric acid. Consequently, we developed an alternative, sensitive and faster method to simultaneously quantify pentachlorophenol (PCP), tetrabromobisphenol A (TBBPA), 4 bromophenols, 7 hydroxypolychlorinated biphenyls (OH-PCBs) and 3 hydroxy-polybrominated diphenyl ether (OH-PBDEs) in human serum sample. The clean-up and the enrichment of the sample were performed in a single extraction step using strong anion-exchange solid phase cartridge. After a rapid liquid–liquid extraction step to remove acidic traces, the extract was derivatized using trimethylsilyldiazomethane (TMSD) and finally analyzed by a gas-chromatograph coupled with an electron negative capture chemical ionization source combined with a triple quadrupole mass spectrometer (GC-ENCI-MS) operating in single ion monitoring. The whole procedure was validated according to the total error approach. The inter and intra assay precision were demonstrated to be lower than 20% and the relative bias to be lower than 15% in the dosing range of concentrations. The limit of quantification (LOQ) ranged from 2 pg mL−1 and 5 pg mL−1, except for the PCP (44.6 pg mL−1) and for the 2,4,6-tribromophenol (49.6 pg mL−1). Finally, the method was successfully applied to measure the POH background contamination in serum samples collected from 20 Belgian blood donors recruited in CHU Mont-Godinne (Namur, Belgium) aged between 21 and 69 years old.
Keywords: Phenolic organohalogens; Serum; GC-ENCI-MS; Trimethylsilyldiazomethane;

Analysis of beta-agonist residues in bovine hair: Development of a UPLC–MS/MS method and stability study by Luigi Giannetti; Giovanni Ferretti; Valentina Gallo; Francesco Necci; Andrea Giorgi; Francesca Marini; Elisa Gennuso; Bruno Neri (76-83).
Beta-sympathomimetic compounds are widely used in therapy because of the bronchodilator, smooth muscle-relaxant and tocolytic properties. However, their growth promoting and performance enhancing effects are often subject to illegal use. The present work describes the development of a fast and reliable analytical multiresidue method for the confirmation 20 β-agonist compounds in animal hair. The procedure is based on alkaline digestion, LLE with organic solvents, SPE clean up and liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) analysis, and is suitable for the public Official control of β-agonist residues in hair sample.Validation was performed according to Commission Decision 2002/657/EC requirements. Independent samples spiked with the investigated compounds in the range 0.2–10.0 μg kg−1 are showing intra-day and inter-day precision (RSD) lower than 17.8% and 19.7%, respectively. Linearity, measured in the range of 0.1–10.0 μg kg−1, resulted with a Pearson’s r > 0.996. The decision limits (CCα) for the all investigated beta agonists resulted in the range 0.2–1.0 μg kg−1.Furthermore, the method was tested on real hair samples obtained from cattle, known as positive to clenbuterol, in order to check its effectiveness and the β-agonists stability.
Keywords: β-Agonist; UPLC–MS/MS; Validation; Bovine hair; Commission decision 2002/657/EC;

It has been proved that the purine metabolic pathway has been implicated in various biological disorders including gout, diabetes, coronary heart diseases, and neurodegenerative diseases. The analysis of the purine metabolic pathway in organisms reveals important alterations under different physiological and pathological conditions, which contributes to the pathological study, diagnosis, and therapy of related diseases. In the present study, an ultra-high performance liquid chromatography with ultraviolet and tandem mass spectrometry (UHPLC-UV–MS/MS) method was developed for conducting the comprehensive analysis of the metabolite profiles of the purine pathway in rat plasma through a single analysis. The purine metabolites including adenosine-5′-monophosphate, guanosine-5′-monophosphate, adenosine, inosine, guanosine, inosine-5′-monophosphate, deoxyadenosine, deoxyguanosine, deoxyinosine, xanthine, hypoxanthine, and uric acid, were separated and quantified in the short running time of 10 min. After rapid chromatographic separation achieved by an Agilent Zorbax SB-Aq column, high concentration of uric acid and the remaining purine metabolites at lower levels were respectively detected by ultraviolet detector and triple quadruple mass spectrometry within a single analysis. The proposed method was validated by applying charcoal-stripped plasma as a matrix and it was proved to be linear (R2  > 0.982), accurate (with a relative error for accuracy < ± 15% and the relative standard deviation for intra- and inter-run precision <11%) and reproducible (with a matrix effect ranging between 86.49% and 111.44% with a maximum RSD of 8.69%). As a result, the method was successfully applied to the quantification of the endogenous purine metabolites in rat plasma. It was found that the concentration levels of the purine metabolites may keep a physiological balance as an integrated system in normal individuals and the concentration level of uric acid in rat plasma was 64 μM which was more than 200 times greater than the other purine metabolites. The established method and the measurement of the concentration of these purines in normal rat plasma may help the investigation of the action mechanisms between purine disorders and related diseases.
Keywords: Purine metabolic pathway; UHPLC-UV–MS/MS; Quantification;

An effective method for determining four commonly detected paraben preservatives (methyl, ethyl, propyl and butyl paraben) in marketed seafood is presented. This method employs matrix solid-phase dispersion (MSPD) before identification and quantification of the paraben preservatives via on-line acetylation gas chromatography–mass spectrometry (GC–MS). Parameters affecting the extraction efficiency of MSPD were optimized through a Box-Behnken design method. Under optimal condition, 0.5-g of freeze-dried seafood was mixed with 0.5-g of anhydrous sodium sulfate, and dispersed with 1.0-g of Florisil using vortex. After that, the blend was transferred to a glass column containing 1.5-g of silica gel + C18 (w/w, 9:1), which acted as clean-up co-sorbents. Then, target analytes were eluted with 12 mL of acetonitrile. The extract was then derivatized on-line in the GC injection-port through reaction with acetic anhydride, and the identity and quantity of the target analytes were determined by the GC–MS system. The limits of quantitation (LOQs) were 0.2 to 1.0 ng/g (dry weight). Preliminary results showed that the total concentrations of four selected parabens ranged from 16.7 to 44.7 ng/g (dry weight).
Keywords: Paraben preservatives; On-line acetylation; Matrix solid-phase dispersion; GC–MS; Biota sample analysis;

Zhi-Zi-Da-Huang decoction (ZZDHD) has been used for treatment of alcoholic liver disease in China for thousands of years. In order to reveal the dynamic biotransformation of the decoction in vivo, a high-throughout, sensitive and special method based on high performance liquid chromatography coupled with diode array detection and time-of-flight mass spectrometry (HPLC–DAD–TOF/MS) and high performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC–QqQ/MS) was developed and validated. 25 parent compounds and 28 metabolites were characterized, among which, two metabolites were found for the first time and tentatively identified by neutral-loss scan and product-ion scan. All the compounds were assigned to iridoids, flavones, anthraquinones, coumarin or p-coumaric acid, and their biotransformation pathways were found to involve glucuronidation, sulfation, reduction and ring cleavage. Glucuronidation occurred as a major metabolic pathway of genipin and flavanone and the conjugates could be detected almost during the whole sampling duration. To compounds such as anthraquinones, coumarin and p-coumaric acid, sulfation is the only transformation pathway and the metabolites were found at 0–12, 4–18, 4–48 h respectively after administration. Reduction and/or ring cleavage of genipin glucuronide and naringin were also observed obviously. The phenomena that parts of parent compounds and metabolites were able to be detected even 48 h after administration implied that the accumulating effect of these constituents in vivo would happen and the potential toxicity of the decoction might appear if multiple dosing is adopted. The strategy used in this paper was proved helpful to offer important information for the clinical safe use of ZZDHD.
Keywords: Dynamic metabolic process; HPLC–DAD–TOF/MS; HPLC–QqQ/MS; Rat urine; Solid phase extraction; Zhi-Zi-Da-Huang decoction;

Determination of urinary biogenic amines’ biomarker profile in neuroblastoma and pheochromocytoma patients by MEKC method with preceding dispersive liquid–liquid microextraction by Natalia Miękus; Ilona Olędzka; Alina Plenis; Piotr Kowalski; Ewa Bień; Aleksandra Miękus; Małgorzata Anna Krawczyk; Elżbieta Adamkiewicz-Drożyńska; Tomasz Bączek (114-123).
The unbalanced secretion of biogenic amines (BAs) is considered to be a relevant biochemical biomarker in the screening for neuroendocrine tumors, such as: neuroblastoma and pheochromocytoma. However, there is still a need to improve the bioanalytical procedures for BA determination in biological samples due to their instability (photo- and thermosensitivity, easy oxidation) and low concentration in the body fluids.In this study, the primary analytical challenge was to optimize the method of extraction of seven compounds from among BAs and their precursors from urine samples. Several methods based on liquid–liquid extraction (LLE) or solid phase extraction (SPE) techniques were tested. By optimization of the extraction and data analysis using chemometric tool, the dispersive liquid–liquid microextraction (DLLME) has been chosen due to its low solvents consumption, high efficiency of isolation, preconcentration and suitable clean-up of biological matrix. Further, α-cyclodextrin-modified micellar electrokinetic chromatography (MEKC) with ultraviolet detection (UV) has been applied for quantification of the analyzed biologically active compounds with limits of detection (LOD) and limits of quantification (LOQ) at 0.15 and 0.5 μg mL−1, respectively. Finally, the optimized and validated DLLME-MEKC-UV method has been employed for the analysis of real urine samples, obtained from 6 children with neuroendocrine tumors and 6 healthy children. It was stated that concentrations of BA could serve to differentiate between the patients and healthy children. This pilot study indicates that the elaborated fast and sensitive DLLME-MEKC-UV method for determination of panel of biomarkers could be successfully applied in everyday clinical practice to help to confirm the clinical diagnosis of neuroendocrine tumors in children.
Keywords: Neuroblastoma; Biogenic amines (BAs); Cancer biomarkers; Urine sample analysis; Micellar electrokinetic chromatography (MEKC); Dispersive liquid–liquid microextraction (DLLME); Hierarchical Cluster Analysis (HCA);

Long-term exposure assessment to phthalates: How do nail analyses compare to commonly used measurements in urine by Andreia Alves; Gudrun Koppen; Guido Vanermen; Adrian Covaci; Stefan Voorspoels (124-135).
Phthalate esters (PEs) are easily metabolized and commonly excreted via urine within 24 h, therefore their bioaccumulation potential is thought to be rather low. In the present study, we developed a sample preparation combined with a new microextraction method to measure seven PE metabolites in nails. The use of whole nails did not result in significantly different levels compared to powdered nails, which makes the method very fast and user friendly. The method was validated using whole nails showing good accuracy, satisfactory precision and low limits of quantification (2–14 ng/g).Although method development was the primary aim of the study, the method was also applied to real samples. PEs were measured in nails of 9 individuals collected at 2 distinct time points (15 days apart) and compared to levels in the respective urine samples (daily morning sample for 15 days). Additionally two volunteers have collected two more urine spots (afternoon and evening) per day.Major metabolites in nails were mono (ethyl hexyl) phthalate (MEHP), monoethyl phthalate (MEP) and sum of mono-n-butyl and mono-isobutyl phthalate (Σ(MnBP, MiBP)) while MEP and Σ(MnBP, MiBP) were the major ones identified in urine. In urine, first void morning urine reflected higher total excretion (sum of PEs of 7.0 μg/g creatinine) for all individuals than the afternoon/evening voids.Participants also filled a questionnaire regarding their life-style. The use of hand care products and consumption of pre-packed food was associated with di-(2-ethylhexyl) phthalate (DEHP) oxidative metabolites, while the use of medical devices with butylbenzyl phthalate (BBzP) exposure. Although the metabolism (rate) and other factors that influence the transfer of the analytes from blood or other body compartments into nails needs further investigation, nails can be used to assess exposure to PEs. From our knowledge, urine reflects the excretion of PEs on ‘daily basis’ while nails show less fluctuation and more stable levels.
Keywords: Nails; Urine spots; Human biomonitoring; Phthalate metabolites; Exposure; Predictors;

Development and validation of ultrafast LC–MS/MS method for quantification of anti-influenza agent camphecene in whole rat blood using dried blood spots and its application to pharmacokinetic studies by Artem D. Rogachev; Olga I. Yarovaya; Sergey V. Ankov; Mikhail V. Khvostov; Tatyana G. Tolstikova; Andrey G. Pokrovsky; Nariman F. Salakhutdinov (136-141).
A fast, selective and sensitive procedure for quantitation of the camphor-based anti-influenza agent camphecene in whole rat blood was developed and validated using dried blood spots and LC–MS/MS. The method was validated according to recommendations of the FDA and EMA in terms of selectivity, linearity, accuracy, precision, recovery, matrix factor, stability, and carry-over. Sample preparation included spotting 20 μL of whole blood taken from the tail vein onto the paper, drying and extracting the analyte, followed by evaporation of the solvent and analysis of the residue. HPLC separations were run on a reversed-phase microcolumn; the time of analysis was less than 2 min. MS/MS detection was performed on a triple quadrupole mass-spectrometer using multiple reaction monitoring (MRM) mode. Transitions 196.4 → 122.2/153.3 and 152.2 → 93.1/107.2 were monitored for camphecene and 2-adamantylamine hydrochloride (internal standard), respectively. The intra- and inter-day precisions and accuracies, matrix factor, carry-over and recovery were within acceptable limits. Despite low extraction recovery (less than 2%), the sensitivity of the method was enough to detect the analyte in the concentration range 50–2500 ng/mL. The application of the method was shown in pharmacokinetic studies of camphecene in rats at a dose of 10 mg/kg.
Keywords: Anti-influenza; Camphecene; Dried blood spots; LC–MS/MS;

TRAIL is a member of the TNF family of cytokines which induces apoptosis of cancer cells via its binding to its cognate receptors, DR5 a high affinity site and DR4 a site of low affinity. Our working group has recently demonstrated that nanovectorization of TRAIL with single wall carbon nanotubes (abbreviated NPT) enhanced TRAIL affinity to the high affinity site DR5 and increased pro apoptotic potential in different human tumor cell lines. In this paper, the DR4 low affinity site was immobilized on a chromatographic support and the effect of temperature on a wide temperature range 1 °C–50 °C was studied to calculate the thermodynamic parameters of the binding of TRAIL and NPT to DR4 and DR5 receptors. For the first time the heat capacity changes for the different binding processes were determined. At a physiological pH (7.4) the heat capacity changes for the binding of NPT to DR4 and DR5 were respectively equal to −0.91 kJ/mol K and −0.28 kJ/mol K and those obtained for the binding of TRAIL to DR4 and DR5 were respectively equal to −1.54 kJ/mol K and −1.05 kJ/mol K. By the use of differential scanning calorimetry (DSC), a phase transition (∼12 °C for DR5, ∼4 °C for DR4) between a disordered (low temperature) and an ordered (high temperature) solid like state visualized in the receptor structure confirmed the temperature dependence of binding affinity enthalpy ΔH for soluble TRAIL and its nanovectorized form to its cognate receptors. In the low temperature domain, the positive ΔH values contribute non-favourably to the free energy of binding, TRAIL and NPT described similar affinities for DR4 and DR5. For the high temperature domain, negative ΔH values indicated that van der Waals interactions and hydrogen bonding are engaged favourably at the ligand – receptor interface. Above 30 °C, their rank-ordered affinities were thus strongly different in the sequence: TRAILDR4  <NPTDR4  <TRAILDR5  < NPTDR5. The nanovectorization of TRAIL enhanced its binding to both DR4 and DR5 receptors at 37 °C and could potentially sensitized cancer cells to TRAIL induced apoptosis through simultaneous activation of DR4 and DR5 as described in this paper for the non-small lung carcinoma cell line (H1703), the two hepatocarcinoma cell lines (SK-Hep1, HUH) and the colon carcinoma cell line (HCT116WT). The excellent linear coefficient (r2  = 1) for the plot comparing NPT cytotoxicity to TRAIL cytotoxicity confirmed a high degree of similarity for the mechanism of cytotoxicity of TRAIL and NPT between these four cell lines expressing DR4 and DR5 receptors. The slope (0.10) and the negative intercept (−1.23) of this plot indicated that NPT was much more efficient in these four cell lines as compared to TRAIL alone. As well, it was shown that the extra cellular acidosis led to the protonation of the TRAIL residue histidine by flipping the His switch to the on position with a concomitant decrease in affinity for DR4 and DR5 receptors. The highest affinity for NPT to DR4 and DR5 receptors observed at low pHs was due to the less accessibility of the His molecular switch to be protonated when TRAIL was immobilized on CNTs.
Keywords: HPLC; DSC; Thermodynamic; Phase transition; Binding; TRAIL; Death receptors; Cell viability;

Spiralin is the most abundant protein of several species of spiroplasmas, helical, motile bacteria pathogenic for arthropods and plants. This amphiphilic protein is anchored to the outer face of the plasma membrane by a lipoylated N-terminal cysteine. Although spiroplasma pathogenicity in mammals is controversial, it was shown that spiralin is highly immunogenic and endowed with immunomodulatory activity. In this paper, we describe a high performance method for the purification of Spiroplasma melliferum spiralin under non-denaturing conditions. The protein was selectively extracted with 3-[(3-cholamidopropyl) dimethylammonio]-1-propyl sulfonate (CHAPS) from the membrane pre-treated with sodium dodecyl-N-sarcosinate (Sarkosyl), and purified to homogeneity by cation-exchange HPLC with an overall yield of ∼60%. Detergent-depleted, water-soluble micelles of spiralin displaying a mean diameter of 170 Å, as evidenced by transmission electron microscopy, were obtained by dialysis detergent removal. Circular dichroism spectroscopy and cross immunoprecipitation assay of the purified spiralin strongly suggested that this purification method could retain the structural characteristics of the native spiralin. The strategy developed to purify spiralin (two successive selective extractions of membrane proteins with mild detergents followed by ion-exchange chromatography) should prove useful for the purification of membrane lipoproteins of other bacteria of the class Mollicutes including different pathogens for humans, animals and plants.
Keywords: CE-HPLC; Detergents; Membrane lipoprotein; Spiralin; Spiroplasma melliferum; Mollicutes;

Rapid identification of bile acids in snake bile using ultrahigh-performance liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry by Jie Zhang; Jiao Peng; Xiaoyong Chen; Yajun Gong; Lesheng Wan; Fei Gao; Suran Gan; Feng Wei; Shuangcheng Ma; Jiachun Chen; Jing Nie (157-169).
Snake bile, a precious traditional Chinese medicine (TCM), was used as the major ingredient of some Chinese patent drugs, such as Shedan Chuanbei powder and Shedan Chenpi powder for hundred years. However, there is still requirement for the comprehensive and definite composition of bile acids in snake bile. In order to rapidly identify the bile acids constituents in snake bile to avoid the adulteration, ultrahigh-performance liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/ESI-QTOF-MS/MS) has been applied to conduct a qualitative analysis on snake bile acids. ESI ion source was used for mass spectra, and data were collected in both positive and negative ion mode. 16 kinds of reference standards, attributed to free bile acids, taurine- and glycine- conjugated bile acids, were detected and their MS behaviors were summarized. In negative ion mode, the diagnostic ions of free bile acids were obtained via the neutral losses of H2O and CO2 molecules; the diagnostic ions of taurine-conjugated bile acids were at m/z 124.0068 ([C2H6NO3S]), m/z 106.9803 ([C2H3O3S]) and m/z 79.9568 ([SO3]); the diagnostic ion of glycine-conjugated bile acids was at m/z 74.0242 ([C2H4NO2]). In positive ion mode, dehydration ions, amide bond cleavage ions, and reversed Diels-Alder at A-ring ions were detected in every kind of reference. These reference MS behaviors were used for identifying bile acids without reference standards in snake bile. As a result, totally 15 compounds, including 4 pairs of isomers, were identified by comparing the retention time, exact molecular mass and fragmentation behaviors with reference standards, respectively. Tauro-3β,7α,12α-trihydroxy-5β-cholenoic acid, Tauro-△8-3β,7α,12α- trihydroxy-5β-cholenoic acid, Tauro-3α,7α,12α,23R-tetrahydroxy-5β-cholenoic acid, and Tauro-3α,7α-dihydroxy-12-oxo-5β-cholenoic acid, Taurocholic acid, Glycocholic acid, Taurochenodeoxycholic acid, Taurodeoxycholic acid and Cholic acid were detected as the common bile acids in snake bile. Tauro-△8-3β,7α,12α-trihydroxy-5β-cholenoic acid, Tauro-3α,7α,9α,16α-tetrahydroxy-5β-cholenoic acid, Tauro-3α,12α,17R,22R-tetrahydroxy-5β-cholenoic acid, and Tauro-△1,8-3β,7β,12α-trihydroxy-5β-cholenoic acid were firstly reported in this study.
Keywords: UPLC/ESI-QTOF-MS/MS; Snake bile; Bile acids; Rapid identification;

Quantitative UPLC–MS/MS assay of urinary 2,8-dihydroxyadenine for diagnosis and management of adenine phosphoribosyltransferase deficiency by Margret Thorsteinsdottir; Unnur A. Thorsteinsdottir; Finnur F. Eiriksson; Hrafnhildur L. Runolfsdottir; Inger M.Sch. Agustsdottir; Steinunn Oddsdottir; Baldur B. Sigurdsson; Hordur K. Hardarson; Nilesh R. Kamble; Snorri Th. Sigurdsson; Vidar O. Edvardsson; Runolfur Palsson (170-177).
Adenine phosphoribosyltransferase (APRT) deficiency is a hereditary disorder that leads to excessive urinary excretion of 2,8-dihydroxyadenine (DHA), causing nephrolithiasis and chronic kidney disease. Treatment with allopurinol or febuxostat reduces DHA production and attenuates the renal manifestations. Assessment of DHA crystalluria by urine microscopy is used for therapeutic monitoring, but lacks sensitivity. We report a high-throughput assay based on ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) for quantification of urinary DHA.The UPLC–MS/MS assay was optimized by a chemometric approach for absolute quantification of DHA, utilizing isotopically labeled DHA as an internal standard. Experimental screening was conducted with D-optimal design and optimization of the DHA response was performed with central composite face design and related to the peak area of DHA using partial least square regression. Acceptable precision and accuracy of the DHA concentration were obtained over a calibration range of 100 to 5000 ng/mL on three different days. The intra- and inter-day accuracy and precision coefficients of variation were well within ±15% for quality control samples analyzed in replicates of six at three concentration levels. Absolute quantification of DHA in urine samples from patients with APRT deficiency was achieved wihtin 6.5 min. Measurement of DHA in 24 h urine samples from three patients with APRT deficiency, diluted 1:15 (v/v) with 10 mM ammonium hydroxide (NH4OH), yielded a concentration of 3021, 5860 and 10563 ng/mL and 24 h excretion of 816, 1327 and 1649 mg, respectively. A rapid and robust UPLC–MS/MS assay for absolute quantification of DHA in urine was successfully developed. We believe this method will greatly facilitate diagnosis and management of patients with APRT deficiency.
Keywords: APRT deficiency; Clinical mass spectrometry; Chemometrics; D-optimal design; Therapeutic drug monitoring;