Journal of Chromatography B (v.951-952, #C)

A mixed-mode anion exchange solid phase extraction (SPE) method for extraction and clean up of carbazochrome sodium sulfonate (CSS) and (1S)-(+)-10-camphorsulfonic acid (IS) was optimized for quantification by high-performance liquid chromatography/negative electrospray ionization mass spectrometry. The analytes were extracted from 1 mL of human plasma via SPE on Oasis® WAX cartridge. Chromatographic separation was achieved on a Zorbax SB-Aq (4.6 × 250 mm, 5 μm) column under an isocratic condition. Detection was performed using electrospray ionization in negative ion multiple reaction monitoring (MRM) mode. The deprotonated precursor to product ion transitions monitored for CSS and IS was at m/z 299.0 → 256.0 and m/z 230.9 → 79.8, respectively. The method was fully validated for its selectivity, sensitivity, precision, accuracy, recovery, matrix effect and stability. Linear range was 0.189–37.8 ng/mL with a high square regression coefficient (r  = 0.9995). The intra-and inter-day precision (RSD, %) ranged from 0.95% to 4.17%, and the intra-and inter-day accuracy was between 95.03% and 105.9%. This method was successfully applied to a bioequivalence study of 90 mg CSS formulation in 18 healthy Chinese male subjects under fasting condition.
Keywords: Carbazochrome sodium sulfonate; LC-ESI-MS/MS; Solid phase extraction; Human plasma;

Urinary determination of nicotine metabolites provides an ideal tool for the quantitative assessment of the tobacco use-related nicotine dose, provided that the considered metabolites comprise a large share of the amount taken up. A method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the sensitive, fast and robust analysis of nicotine and 10 major nicotine metabolites (“Nic+10”), including cotinine, trans-3′-hydroxy-cotinine, nicotine-N-glucuronide, cotinine-N-glucuronide, trans-3′-hydroxy-cotinine-O-glucuronide, nornicotine, norcotinine, nicotine-N′-oxide, cotinine-N′-oxide and 4-hydroxy-(3-pyridyl)-butanoic acid. Corresponding deuterated internal standards were spiked prior to a simple and straightforward solid phase extraction (SPE) procedure. Liquid chromatography was performed on a reversed phase C8 column and mass-specific detection was conducted in scheduled-MRM mode. The method was validated according to FDA Guidelines, showing excellent selectivity, precision, accuracy and robustness. The limits of quantification were in the range 0.2–2.3 ng/ml for all analytes. The novel method was applied to human urine samples derived from 25 smoking subjects. Quantitative results were correlated against a previously used LC–MS/MS method and compared to reports from the literature. The relative molar profile of nicotine and its 10 major metabolites was in good agreement with the literature. In addition, correlation amongst the two methods was excellent for almost all analytes, whereas the accordance between both methods was moderate for hydroxy-cotinine-O-glucuronide and norcotinine. These deviations, however, could be explained. The current method allows the simultaneous determination of nicotine and its 10 major metabolites (metabolite coverage about 95% of the absorbed dose) from a small sample volume and within a reasonable amount of time. Due to its wide dynamic range, high sensitivity and high throughput capabilities, this method could serve as a powerful tool for quantifying the nicotine dose of smokers, passive smokers as well as novel tobacco and nicotine product users in clinical and epidemiological studies.
Keywords: Biomarkers of exposure (BoE); LC–MS/MS; Mass spectrometry; Nicotine metabolism; Urine;

Purification of pre-miR-29 by arginine-affinity chromatography by Patrícia Pereira; Ângela Sousa; João Queiroz; Ilídio Correia; Ana Figueiras; Fani Sousa (16-23).
Recently, differential expression of microRNAs, in patients with Alzheimer's disease (AD) suggests that they might have key regulatory roles in this neurodegenerative disease. Taking into account this fact, several studies demonstrated that the miR-29 is significantly decreased in AD patients, also displaying abnormally high levels of β-site APP-cleaving enzyme 1. Thus, RNA biochemical or structural studies often require a RNA sample that is chemically pure and biologically active. The present work describes a new affinity chromatography method using an arginine support to specifically purify pre-miR-29 from other Rhodovulum sulfidophilum small RNA species. Nevertheless, in order to achieve higher efficiency and selectivity, it is essential to characterize the behavior of pre-miR-29 binding/elution. Thus, three different strategies based on increased sodium chloride (280–500 mM), arginine (25 mM) or decreased ammonium sulfate (2–0.1 M) stepwise gradients are described to purify pre-miR-29. In this way, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance. As a matter of fact, by employing elution strategies using sodium chloride or arginine, an improvement in the final pre-miR-29 yields (96.5 and 56.7%, respectively) was obtained. Moreover, the quality control analysis revealed high integrity in pre-miR-29 preparations as well as high purity (90 and 98%, respectively), demonstrated by the scarce detection of proteins. This improved method takes advantage of its simplicity, significant cost reduction, due to the elimination of some complex operations, and speed for large-scale purification of pre-miRNAs suitable for biochemical and structural studies.
Keywords: Alzheimer's disease; Arginine-affinity chromatography; Multiple interactions; pre-miR-29; Rhodovulum sulfidophilum; RNA purification;

A simple, rapid and specific method was developed to separate as well as to determine the four phenylethanoid glycosides (PhGs) (echinacoside, tubuloside B, acteoside and isoacteoside) in rat plasma after oral administration of Cistanche salsa extract by reversed phase high performance liquid chromatography using a microemulsion as the mobile phase. The separations were performed on a Zorbax Extend-C18 column at 25 °C. Photodiode-array detector was conducted at 322 nm and with a flow rate of 0.8 mL min−1. The optimized microemulsion mobile phase consisted of 0.3% triethylamine in 20 mM phosphoric acid at pH 6.0, 0.8% (v/v) ethyl acetate as oil phase, 1.5% (v/v) Genapol X-080 as surfactant, 2.5% (v/v) n-propanol as co-surfactant. Under the optimal conditions, the calibration curve for four PhGs was linear in the range of 10–1000 ng mL−1 with the correlation coefficients greater than 0.9994. The intra-day and inter-day precision (RSD) were below 8.64% and the limits of detection (LOD) for the four PhGs were 0.4–1.3 ng mL−1 (S/N = 3). The microemulsion liquid chromatography (MELC) method was successfully applied to separate and determine the four PhGs in rat plasma after oral administration of C. salsa extract.
Keywords: Cistanche salsa; Phenylethanoid glycosides; Microemulsion liquid chromatography; Rat plasma;

An organic–inorganic hybrid molecular imprinting monolith (HMIM) has been prepared, characterized and applied for the determination of 2,4-dichlorophenoxyacetic acid (2,4-D) in rice with high-performance liquid chromatography-photodiodes array detector (HPLC–PAD). By optimizing the polymerization conditions, such as the volume ratio of the inorganic alcoholysate and organic part, the 2,4-D-HMIM was synthesized in a micro pipette tip using acrylamide as the functional monomer, ethylene dimethacrylate as the cross-linker and methanol as the porogenic solvent. The morphology of the monolith was studied by scanning electronmicroscopy and Fourier transform infrared spectra. The imprinted factor of the monolith for 2,4-D reached 3.29. A simple, rapid and sensitive method for the determination of 2,4-D in rice using the HMIM microextraction combined with high-performance liquid chromatography-photodiodes array detector was developed. Some parameters affecting the sample pretreatment were investigated, including the type and volume of eluent, the flow rate and volume of sample solution. The assay exhibited a linear dynamic range of 167–4167 μg/kg with the correlation coefficient above 0.9972. The detection limit (at S/N  = 3) was 50 μg/kg. The proposed method was successfully applied for the selective determination of 2,4-D in rice.
Keywords: 2 ;4-dichlorophenoxyacetic acid; Organic-inorganic hybrid molecular imprinting monolith; Solid-phase microextraction; High-performance liquid chromatography;

In the present study, five metabolites of Cortex Phellodendri Chinensis, an important herbal drug, were identified using liquid chromatography multi-stage tandem mass spectrometric techniques (LC–MS n ). A sensitive and rapid high-performance liquid chromatographic tandem mass spectrometry (LC–MS/MS) method was developed for the quantitation of the five metabolites, utilizing chlorobenzylidine as the internal standard in rat urine. Urine samples were precipitated with acetonitrile. Chromatographic separation was achieved on a Waters C18 analytical column. Detection was performed by a multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source operating in the positive ionization mode. The method was linear over the concentration range of 0.05–1.00c for all components. The intra- and inter-day precision values were less than 14.6% and the deviations ranged from −4.4 to 13.8%. The recoveries at three levels were more than 73.7%. The fully validated method was used to determine the metabolites amount in rat urine to investigate the changes caused by coupling with Atractylodes lancea in Er Miao San preparation on metabolism.
Keywords: Metabolite; Determination; LC–MS/MS; Cortex Phellodendri Chinensis; Er Miao San preparation;

Determination of ptaquiloside and pterosin B derived from bracken (Pteridium aquilinum) in cattle plasma, urine and milk by Paulo César Reis Aranha; Hans Christian Bruun Hansen; Lars Holm Rasmussen; Bjarne W. Strobel; Christian Friis (44-51).
Ptaquiloside (PTA) is a toxin from bracken fern (Pteridium sp.) with genotoxic effects. Hydrolysis of PTA leads to the non-toxic and aromatised indanone, pterosin B (PTB). Here we present a sensitive, fast, simple and direct method, using SPE cartridges to clean and pre-concentrate PTA and PTB in plasma, urine and milk followed by LC–MS quantification. The average recovery of PTA in plasma, urine, and milk was 71, 88 and 77%, respectively, whereas recovery of PTB was 75, 82 and 63%. The method LOQ for PTA and PTB in plasma was 1.2 and 3.7 ng mL−1, 52 and 33 ng mL−1 for undiluted urine and 5.8 and 5.3 ng mL−1 for milk. The method is repeatable within and between days, with RSD values lower than 15% (PTA) and 20% (PTB). When PTA and PTB spiked samples were stored at −18 °C for 14 days both compounds remained stable. In contrast, the PTA concentration was reduced by 15% when PTA spiked plasma was left for 5 h at room temperature before SPE clean-up, whereas PTB remained stable. The method is the first to allow simultaneous quantification of PTA and PTB in biological fluids in a relevant concentration range. After intravenous administration of 0.092 mg PTA per kg bw in a heifer, the plasma concentration was more than 300 ng mL−1 PTA and declined to 9.8 ng mL−1 after 6 h, PTB was determined after 10 min at 50 ng mL−1
Keywords: Bracken fern; Ptaquiloside; Pterosin B; Cattle; LC–MS;

Electrochemical methods are very often used to detect catecholamine and indolamine neurotransmitters separated by conventional reverse-phase high performance liquid chromatography (HPLC). The present paper presents the development of a chromatographic method to detect monoamines present in low-volume brain dialysis samples using a capillary column filled with sub-2 μm particles. Several parameters (repeatability, linearity, accuracy, limit of detection) for this new ultrahigh performance liquid chromatography (UHPLC) method with electrochemical detection were examined after optimization of the analytical conditions. Noradrenaline, adrenaline, serotonin, dopamine and its metabolite 3-methoxytyramine were separated in 1 μL of injected sample volume; they were detected above concentrations of 0.5–1 nmol/L, with 2.1–9.5% accuracy and intra-assay repeatability equal to or less than 6%. The final method was applied to very low volume dialysates from rat brain containing monoamine traces. The study demonstrates that capillary UHPLC with electrochemical detection is suitable for monitoring dialysate monoamines collected at high sampling rate.
Keywords: Monoamine neurotransmitter; Ultra high performance liquid chromatography; Microdialysis; Electrochemical detection;

Chiral separation of a diketopiperazine pheromone from marine diatoms using supercritical fluid chromatography by Johannes Frenkel; Carsten Wess; Wim Vyverman; Georg Pohnert (58-61).
The proline derived diketopiperazine has been identified in plants, insects and fungi with unknown function and was recently also reported as the first pheromone from a diatom. Nevertheless the stereochemistry and enantiomeric excess of this natural product remained inaccessible using direct analytical methods. Here we introduce a chiral separation of this metabolite using supercritical fluid chromatography/mass spectrometry. Several chromatographic methods for chiral analysis of the diketopiperazine from the diatom Seminavis robusta and synthetic enantiomers have been evaluated but neither gas chromatography nor high performance liquid chromatography on different chiral cyclodextrin phases were successful in separating the enantiomers. In contrast, supercritical fluid chromatography achieved baseline separation within four minutes of run time using amylose tris(3,5-dimethylphenylcarbamate) as stationary phase and 2-propanol/CO2 as mobile phase. This very rapid chromatographic method in combination with ESI mass spectrometry allowed the direct analysis of the cyclic dipeptide out of the complex sea water matrix after SPE enrichment. The method could be used to determine the enantiomeric excess of freshly released pheromone and to follow the rapid degradation observed in diatom cultures. Initially only trace amounts of c(d-Pro–d-Pro) were found besides the dominant c(l-Pro–l-Pro) in the medium. However the enantiomeric excess decreased upon pheromone degradation within few hours indicating that a preferential conversion and thus inactivation of the l-proline derived natural product takes place.
Keywords: Chiral separation; Supercritical fluid chromatography; Diketopiperazine; Cyclodextrine; Pheromone;

Sinotecan is a camptothecin analog, currently under clinical testing as an antitumor medication. We developed and validated a rapid, specific and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of sinotecan and its active metabolite, 7-hydroxyethyl-camptothecin (7-HEC), in human blood. Aliquots (200 μL) of heparinized blood samples were processed by deproteinization with 400 μL acetonitrile each. Chromatographic analyte separation used an Agilent Zorbax SB C8 column (4.6 mm × 150 mm, 5 μm) and methanol/10 mM ammonium acetate/formic acid (70/30/0.14, v/v/v) as mobile phase, at a flow rate of 0.60 mL/min. A Thermo Finnigan TSQ Quantum Ultra tandem mass spectrometer was operated in multiple-reaction monitoring mode. The precursor-to-product ion transitions m/z 493 →  m/z (331 + 375) for sinotecan, m/z 393 →  m/z (233 + 261) for 7-HEC, and m/z 396 →  m/z 352 for d3-SN38 (IS) were used for quantification. The method was validated for 1.0–500 ng/mL for sinotecan and 0.5–250 ng/mL for 7-HEC using 200 μL of blood sample. Total time for each chromatograph was ∼6.0 min. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) < 13.8% and the accuracy values ranged from −5.3% to 2.4%. The method was successfully applied to a pharmacokinetic study of sinotecan in cancer patients
Keywords: Sinotecan; 7-Hydroxyethyl-camptothecin; LC-MS/MS; Pharmacokinetics;

An important area within clinical functional metabolomics is in vivo amino acid metabolism and protein turnover measurements for which accurate amino acid concentrations and stable isotopically labeled amino acid enrichments are mandatory not the least when tissue metabolomics is determined. The present study describes a new sensitive liquid chromatography tandem mass-spectrometry method quantifying 20 amino acids and their tracer(s) ([ring-13C6]/D5Phenylalanine) in human plasma and skeletal muscle specimens. Before analysis amino acids were extracted and purified via deprotonization/ion exchange, derivatized using a phenylisothiocyanate reagent and each amino acid was quantitated with its own stable isotopically labeled internal standard (uniformly labeled-13C/15N). The method was validated according to general recommendations for chromatographic analytical methods.The calibration curve correlations for amino acids were on average; r 2  = 0.998. Interday accuracy for amino acids determined in spiked plasma was on average 97.3% and the coefficient of variation (CV) was 2.6%. The ([ring-13C6]/D5Phenylalanine) enrichment CV's for machine reproducibility in muscle tissue fluid and plasma were 4.4 and 0.8%, and the interday variability was 3.4% and the recovery was 90.5%, respectively.In conclusion, we have developed and validated a method for quantitative amino acid profiling that meets the requirements for systemic and tissue human in vivo amino acid and protein turnover kinetics measurements. Moreover, citrulline, ornithine, π-methyl-histidine, τ-methyl-l-histidine, hydroxy-proline and carnitine were analysed but when similar precision and accuray are required an additional stable istopically labeled internal standard for these meatablites should be be added
Keywords: Amino acids; Metabolomics; Liquid chromatography; Quantification; Tracer kinetics; Stable isotopes;

Andrographis paniculata (AP) has been widely used in Asian countries to treat many kinds of diseases for several decades. Hutchison Medipharma Ltd. developed an aqueous ethanol extract of A. paniculata (APE) named as HMPL-004 to treat inflammatory bowel diseases. The representative chemical components of HMPL-004 include andrographolide (AND), neoandrographolide (NAND), 14-deoxyandrographolide (DAND), 14-deoxy-11,12-didehydro-andrographolide (DDAND), apigenin-7-O-β-d-glucuronopyranoside (AODG) and chlorogenic acid (CLA). HM5013620 is the major circulating metabolite of AND. The purpose of this study was to develop a bioanalytical method to determine all seven compounds in rat plasma using liquid chromatography coupled to electrospray tandem mass spectrometry (LC–MS/MS). The assay was fully validated according to FDA guidelines. The LC–MS/MS detection was operated in the negative mode, and the multiple reaction monitoring (MRM) mode was used for the quantification. The analyte extraction was performed by protein precipitation with acetonitrile after adding a small volume (2% of the total volume) of 10% formic acid into plasma to stabilize AND under bench-top condition (ice-bath). The linear ranges of the analytes were 8–2000 ng/mL for DDAND and 4–2000 ng/mL for others. Validation results demonstrate that AND, NAND, DAND, DDAND, CLA, AODG and HM5013620 can be rapidly, accurately, precisely and robustly quantified in rat plasma. Furthermore, the method was successfully applied to characterize the pharmacokinetic profiles of all seven compounds in Sprague-Dawley rats after a single oral administration of 750 mg of HMPL-004.
Keywords: Andrographis paniculata; HMPL-004; Chemical components; LC–MS/MS; Pharmacokinetic study;

A method was developed for determination of amitraz and its metabolites, N-[2,4-(dimethylphenyl)-N′-methylformamidine (DMPF), 2,4-dimethylformamidine (DMF), 2,4-dimethylaniline (DMA) in whole blood. The analytes were extracted by solid-phase extraction (SPE) using dichloromethane, acetonitrile and methanol (2:1:1) mixture as elute solution. Analysis was performed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in the positive ion mode using multiple reaction monitoring (MRM) technique. Collision-induced dissociation (CID) of amitraz at the electrospray source in MS/MS was observed in the analytic conditions. The method was validated in human whole blood spiked at three concentration levels. The low limit of detection (LOD) and the low limit of quantification (LOQ) for all the analytes were below 0.5 μg/L and 2 μg/L, respectively. Recoveries were between 90.2% and 104.5%, Bias and relative standard deviation (RSD) were below 15% (n  = 6). The good linear relationships were obtained in certain concentration ranges of amitraz and its metabolites. The results demonstrated the method is exclusive, sensitive and accurate, and can be applied in forensic toxicology.
Keywords: SPE; LC–MS/MS; Amitraz; Whole blood;

A sensitive and selective LC-MS/MS method has been developed to quantify almorexant and its four primary metabolites M3, M5, M6, and M8 in human plasma samples. The method involved protein precipitation with acetonitrile in the high calibration range and liquid/liquid extraction with ethyl acetate in the low calibration range. Labeled internal standards were available for four analytes. Separation was performed with an Eclipse XDB-C18 (2.1 mm × 150 mm, particle size 3.5 μm) and a XBridge C18 column (2.1 mm × 50 mm, particle size 3.5 μm). The mobile phases were mixtures of acetonitrile, methanol, and water containing 1% formic acid; flow rate was 400 μL/min. The triple stage quadrupole mass spectrometer was operated in ESI mode and the methods were linear over a range of 0.400–100 ng/mL (almorexant, M5, M6), 1.00–100 ng/mL (M3, M8), and 50.0–1000 ng/mL (all analytes). The inter-day coefficients of variation were equal to or smaller than 10.5%. The inter-day accuracies were between 92.1% and 105.2%. The validated method was successfully applied to the pharmacokinetic assessment of almorexant and its metabolites in several phase I studies.
Keywords: Almorexant; Plasma; Assay validation; Pharmacokinetics; LC-MS/MS;

A pipette tip-based molecularly imprinted polymer monolith microextraction (PT–MIPMME) method was developed for the selective extraction of difenoconazole in tap water and grape juice. In this method, molecularly imprinted polymer (MIP) monolith used as the sorbent was synthesized at the tip of a micropipette. This in situ polymerization reaction used difenoconazole as the template and methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker and the mixture of toluene–dodecanol as the porogenic solvent. The pipette tip containing MIP monolith was matched to a syringe for performing the polymer monolith microextraction (PMME). Several parameters affecting the proposed PT–MIPMME method were investigated, including the flow rate, sample volume, pH and salt concentration of sample, the type and volume of eluent. Under the optimal conditions, the PT–MIPMME method showed a low limit of detection of 0.5 μg L−1. The recoveries were in the range of 87.6–95.4% with relative standard deviations less than 4.9%. The results showed that difenoconazole was selectively enriched from tap water and grape juice samples.
Keywords: Molecularly imprinted polymer; Polymer monolith microextraction; Difenoconazole; Tap water; Grape juice;

A simple quantitative method analysing amikacin, gentamicin, and vancomycin levels in human newborn plasma using ion-pair liquid chromatography/tandem mass spectrometry and its applicability to a clinical study by Yuma Bijleveld; Timo de Haan; Jan Toersche; Sona Jorjani; Johanna van der Lee; Floris Groenendaal; Peter Dijk; Arno van Heijst; Antonio W.D. Gavilanes; Rogier de Jonge; Koen P. Dijkman; Henrica van Straaten; Monique Rijken; Inge Zonnenberg; Filip Cools; Debbie Nuytemans; Ron Mathôt (110-118).
Neuroprotective controlled therapeutic hypothermia is the standard of care for newborns suffering perinatal asphyxia. Antibiotic drugs, such as amikacin, gentamicin, and vancomycin are frequently administered during controlled hypothermia, which possibly alters their pharmacokinetic (PK) and pharmacodynamic (PD) profiles. In order to examine this effect an LC–MS/MS method for the simultaneous quantification of amikacin, the major gentamicin components (gentamicin C, C1a and C2), and vancomycin in plasma was developed. In 25 μL plasma proteins were precipitated with trichloroacetic acid (TCA) and detection of the components was achieved using ion-pair reversed phase chromatography coupled with electrospray ionization tandem mass spectrometry. The chromatographic runtime was 7.5 min per sample. Calibration standards were prepared over a range of 0.3–50 mg L−1 for amikacin and gentamicin and 1.0–100 mg L−1 for vancomycin. At LLOQ accuracy was between 103 and 120% and imprecision was less than 19%. For concentrations above LLOQ accuracy ranged from 98% to 102% and imprecision was less than 6%. Process efficiency, ionization efficiency, and recovery were acceptable. Samples and stock solutions were stable during the time periods and at the different temperatures examined. The applicability of the method was shown by analysing plasma samples from 3 neonatal patients. The developed method allows accurate and precise simultaneous quantification of amikacin, gentamicin, and vancomycin in a small volume (25 μL) of plasma.
Keywords: LC–MS/MS; Amikacin; Gentamicin; Vancomycin; Hypothermia; Neonates;

A versatile ultra-high performance LC-MS method for lipid profiling by Oskar L. Knittelfelder; Bernd P. Weberhofer; Thomas O. Eichmann; Sepp D. Kohlwein; Gerald N. Rechberger (119-128).
A new UPLC-based untargeted lipidomic approach using a qTOF hybrid mass spectrometer is introduced. The applied binary gradient enables separations of lipid species including constitutional isomeric compounds and low abundant lipid classes such as phosphatidic acid (PA). Addition of phosphoric acid to the solvents improves peak shapes for acidic phospholipids. MSE scans allow simultaneous acquisition of full scan data and collision induced fragmentation to improve identification of lipid classes and to obtain structural information. The method was used to investigate the lipidome of yeast.
Keywords: Lipidomics; Ultra-performance liquid chromatography; Mass spectrometry; Neutral lipids; Glycerophospholipids;

Pharmacokinetic study of calenduloside E and its active metabolite oleanolic acid in beagle dog using liquid chromatography–tandem mass spectrometry by Meiyun Shi; Yan Yang; Yantong Sun; Longmei Cheng; Sen Zhao; Huibo Xu; J. Paul Fawcett; Xiaobo Sun; Jingkai Gu (129-134).
Aralia mandshrica is a well-known traditional Chinese medicine from Northeast China commonly used to treat digestive, circulatory and immune system disorders. Calenduloside E is one of its bioactive components currently under evaluation as a pure drug. In this study, a highly sensitive and rapid method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous quantitation of calenduloside E and its active metabolite oleanolic acid in beagle dog plasma has been developed and validated. Samples containing the ammonium salt of simvastatin acid as internal standard (IS) were purified by solid phase extraction and separated on a SUPELCO Ascentis-C18 column (50 mm × 4.6 mm i.d., 5 μm) using gradient elution with 0.35% formic acid and acetonitrile. Analytes and IS were detected in a cycle time of 5 min after ionization in the negative ion mode by multiple reaction monitoring of the precursor-to-product ion transitions at m/z 631.4 → 455.4 and m/z 435.4 → 319.0 for calenduloside E and IS respectively and by single ion monitoring of the ion at m/z 455.4 for oleanolic acid. The method was linear over the concentration range 0.4–100 ng/mL for both analytes using 0.5 mL plasma. Inter- and intra-day precisions were both <6.96% with accuracies <6.40%. In the pharmacokinetic (PK) study, beagle dogs were given oral doses of calenduloside E (1.05, 2.10 and 4.20 mg/kg) and an intravenous injection of 2.10 mg/kg. The absolute bioavailability of calenduloside E was only 0.58%. Area under the plasma concentration time curve (AUC(0–t)) for the oral doses of calenduloside E was approximately dose proportional while other PK parameters (t 1/2, T max and MRT) showed no significant differences among the three doses (P  > 0.05). The PK data provide a useful platform on which to base future clinical studies of calenduloside E.
Keywords: Calenduloside E; Oleanolic acid; LC–MS/MS; Pharmacokinetics; Beagle dog;

Combinatorial peptide ligand library (CPLL) was evaluated as an off line step to narrow the differences of protein concentration in human serum prior to the capturing of human fucome from disease-free and breast cancer sera by a multicolumn platform via lectin affinity chromatography (LAC) followed by the fractionation of the captured glycoproteins by reversed phase chromatography (RPC). Two monolithic lectin columns specific to fucose, namely Aleuria aurantia lectin (AAL) and Lotus tetragonolobus agglutinin (LTA) columns were utilized to capture the fucome, which was subsequently fractionated by RPC yielding desalted fractions in volatile acetonitrile-rich mobile phase, which after vacuum evaporation were subjected to tryptic digestion prior to LC-MS/MS analysis. AAL has a strong affinity towards core fucosylated N-glycans and has a weak binding towards fucose in the outer arm while LTA can bind to glycans having fucose present in the outer arm. The combined strategy consisting of the CPLL, multicolumn platform and LC-MS/MS analysis permitted the identification of the differentially expressed proteins (DEPs) in breast cancer serum yielding 58 DEPs in both the LTA and AAL fractions with 6 DEPs common to both lectins. 17 DEPs were of the low abundance type, 16 DEPs of the borderline abundance type, 4 DEPs of the medium abundance type and 15 DEPs of the high abundance type. The remaining 6 DEPs are of unknown concentration. Only proteins exhibiting 99.9% protein identification probability, 95% peptide identification probability, and a minimum of 5 unique peptides were considered in finding the DEPs via scatterplots.
Keywords: Breast cancer serum; Fucosylation; Glycoproteins; Lectin affinity chromatography; Monolithic columns; ProteoMiner™;

A selective chiral high performance liquid chromatographic (HPLC) method was developed and validated to separate and quantify the pioglitazone enantiomers in rat plasma. After extraction of the plasma samples with ethyl acetate, the separation of pioglitazone enantiomers and internal standard (I.S., dexamethasone acetate) was achieved on a cellulose tris (3,5-dichlorophenylcarbamate) column known as Chiralpak IC with a mobile phase of hexane–isopropanol (70:30, v/v) at a flow rate of 1.0 mL/min. The ultraviolet (UV) detection wavelength was set at 225 nm. Baseline separation of pioglitazone enantiomers and I.S., free from endogenous interferences, was achieved in less than 25 min. Ratio of peak area of each enantiomer to I.S. was used for quantification of plasma samples. Linear calibration curves were obtained over the range of 0.25–50 μg/mL in plasma for both enantiomers (R 2  > 0.9990) with quantitation limit of 0.25 μg/mL. The mean extraction recoveries were 82.37–91.38% for pioglitazone enantiomers and 95.76% for I.S. from rat plasma. The mean relative error (R.E. %) of accuracy and the mean relative standard deviation (R.S.D. %) of intra-day and inter-day precision for both enantiomers were <10%. The method was validated with accuracy, precision, recovery and stability and used to determine the pharmacokinetics of pioglitazone enantiomers, after a single oral administration of racemic pioglitazone (30 mg/kg). The differences between the pharmacokinetic parameters C max, AUC 0–24, AUC 0–∞, CL/F of (+)-pioglitazone and (–)-pioglitazone were significant, suggesting that the disposition of pioglitazone in rats may be enantioselective. Moreover, the plasma levels of (+)- and (–)-pioglitazone in female rats were apparently higher than that in male rats, respectively.
Keywords: Pioglitazone; Enantiomer separation; Pharmacokinetics; HPLC;

Comparison of different solid-phase extraction materials for the determination of fluoroquinolones in chicken plasma by LC-MS/MS by Franziska Janusch; Gesine Scherz; Siegrun A.I. Mohring; Jessica Stahl; Gerd Hamscher (149-156).
Fluoroquinolones are synthetic antibiotics which are frequently used in veterinary medicine e.g. for the treatment of poultry. Their specific importance is based on the fact that they are regarded as antibiotics of last resort because of their broad spectrum of action against Gram-negative and -positive bacteria. Here, a new and sensitive method for the simultaneous determination of four fluoroquinolones (marbofloxacin, ciprofloxacin, enrofloxacin and difloxacin) in chicken plasma by LC-MS/MS was developed. Solid-phase extraction was chosen for sample preparation because a selective sample clean-up is combined with an effective extraction. Various solid-phase extraction materials including polymer-based reversed-phase, silica-based reversed-phase and mixed-mode sorbents were compared. Selection criteria were analyte recovery, sample extract purity and economical aspects (analysis time and elution solvent volume). Best recoveries and minimized elution solvent volumes were achieved using polymeric reversed-phase cartridges. However, post-column infusion experiments revealed that the analysis is influenced by co-eluting matrix components. Hence, a combination of a mixed-mode anion-exchange cartridge and a mixed-mode cation-exchange cartridge was used as final extraction method. This method yield slightly lower analyte recoveries compared to polymeric-reversed-phase cartridges but exhibit no matrix effects. Recoveries of spiked chicken plasma ranged from 61.9% to 84.8% with an inter-day precision of generally less than 12%. LODs are between 0.03 and 0.05 μg/L; LOQs are between 0.08 and 0.16 μg/L. Maximum plasma concentrations of chickens medicated with an enrofloxacin dosage of 3 mg/kg bodyweight were 38.9 μg/L for enrofloxacin and 3.3 μg/L for its main metabolite ciprofloxacin.
Keywords: Fluoroquinolones; LC-MS/MS; Solid-phase extraction; Plasma; Chicken;

A hollow fiber liquid phase microextraction (HF–LPME) combined with ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed for the extraction and determination of naloxone (NLX), buprenorphine (BP) and its major metabolite norbuprenorphine (NBP) in human plasma. The optimum extraction conditions of HF–LPME were: the porous of polyvinylidene fluoride (PVDF) hollow fiber was full of component solvent (1-octanol/chloroform/toluene, 2/4/4), the pH of donor phase was 8.7, the extraction time was 30 min and stirring speed was 1000 revolutions per minute (rpm). The UHPLC–MS/MS method was performed with Waters ACQUITY UPLCTM BEH C18, 50 mm × 2.1 mm, 1.7 μm, using methanol–0.2%formic acid as mobile phase with a gradient elution at a flow rate of 0.25 mL/min. The target compounds were detected under a tandem quadrupole mass spectrometer in positive electrospray ionization (ESI) mode, then analyzed in multiple reaction monitoring (MRM) mode and the isotope internal standard method was used for quantification. The results showed that linearities were in the range of 0.1–25 ng/mL (R  > 0.996). The limits of detection (LOD) of BP/NBP/NLX were 0.05/0.05/0.025 ng/mL and the limits of quantitation (LOQ) of BP/NBP/NLX were 0.1/0.1/0.05 ng/mL, respectively. The spiked recoveries were in the range of 92.1–106.0% with relative standard deviation (RSD) values were less than 15%. This method was simple, inexpensive, sensitive and has been successfully used to quantify plasma samples from patients included in a clinical pharmacogenetic study.
Keywords: Buprenorphine; Naloxone; Norbuprenorphine; HF–LPME; UHPLC–MS/MS;

Iriflophenone-3-C-glucoside from Cyclopia genistoides: Isolation and quantitative comparison of antioxidant capacity with mangiferin and isomangiferin using on-line HPLC antioxidant assays by Christiaan J. Malherbe; Elize Willenburg; Dalene de Beer; Susan L. Bonnet; Jan H. van der Westhuizen; Elizabeth Joubert (164-171).
The benzophenone, iriflophenone-3-C-glucoside, was isolated from Cyclopia genistoides using a combination of fluid-fluid extraction, high performance counter-current chromatography (HPCCC) and semi-preparative high performance liquid chromatography (HPLC). The microplate oxygen radical absorbance capacity (ORAC) assay, with fluorescein as probe, was adapted for use in an on-line HPLC configuration. The method was validated using a mixture of authentic standards including iriflophenone-3-C-glucoside, and the xanthones, mangiferin and isomangiferin. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was included in the mixture for calculation of Trolox equivalent antioxidant capacity (TEAC) values. Using the on-line HPLC-ORAC assay, as well as 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS• +) on-line assays, the antioxidant activity of iriflophenone-3-C-glucoside and isomangiferin was demonstrated for the first time. Iriflophenone-3-C-glucoside presented no radical scavenging ability against DPPH•, but scavenged ABTS• + and peroxyl radicals (TEACABTS of 1.04 and TEACORAC of 3.61). Isomangiferin showed slightly lower antioxidant capacity than mangiferin against DPPH• (TEACDPPH of 0.57 vs. 0.62), but higher capacity against ABTS• + (TEACABTS of 1.82 vs. 1.67) and peroxyl radical (TEACORAC of 4.14 vs. 3.69) than mangiferin. The on-line HPLC-ORAC assay was shown to be more sensitive for radical scavengers, but at the same time less selective for rapid radical scavengers than the DPPH• assay.
Keywords: Honeybush; Herbal tea; Benzophenone; HPCCC; Peroxyl radical; (Bio)chemical detection;