Journal of Chromatography B (v.879, #26)

Design of ionic liquids for lipase purification by Sónia P.M. Ventura; Sílvia G. Sousa; Mara G. Freire; Luísa S. Serafim; Álvaro S. Lima; João A.P. Coutinho (2679-2687).
Aqueous two-phase systems (ATPS) are considered as efficient downstream processing techniques in the production and purification of enzymes, since they can be considered harmless to biomolecules due to their high water content and due to the possibility of maintaining a neutral pH value in the medium. A recent type of alternative ATPS is based on hydrophilic ionic liquids (ILs) and salting-out inducing salts. The aim of this work was to study the lipase (Candida antarctica lipase B – CaLB) partitioning in several ATPS composed of ionic liquids (ILs) and inorganic salts, and to identify the best IL for the enzyme purification. For that purpose a wide range of IL cations and anions, and some of their combinations were studied. For each system the enzyme partitioning between the two phases was measured and the purification factors and enzyme recoveries were determined. The results indicate that the lipase maximum purification and recovery were obtained for cations with a C8 side alkyl chain, the [N(CN)2] anion and ILs belonging to the pyridinium family. However, the highest purification parameters were observed for 1-methyl-3-octylimidazolium chloride [C8mim]Cl, suggesting that the IL extraction capability does not result from a cumulative character of the individual characteristics of ILs. The results indicate that the IL based ATPS have an improved performance in the lipase purification and recovery.
Keywords: Candida antarctica lipase B; Ionic liquid; Purification process; Partition process;

The renin–angiotensin–aldosterone system (RAAS) is an essential body fluid maintenance system that controls pressure in the human body. The conversion of angiotensin I to angiotensin II by angiotensin-converting enzyme (ACE) is a key process in the RAAS because angiotensin II causes the vasoconstriction association with hypertension. Because of its effectiveness as an ACE blocker, quinipril is widely used for clinical treatment of hypertension and chronic congestive heart failure. Matrix-assisted laser desorption/ionization coupled with time-of-flight analyzer (MALDI-TOF) is a high throughput instrument for biological sample analysis. This study developed a micro-scale approach for using MALDI-TOF to detect quinapril in biological samples. A micro-liquid-liquid-extraction strategy combined with ion-pair interaction successfully extracted quinapril from aqueous layer to organic layer. Quinolones were then used as matrix additives to suppress undesired substances in plasma produce signals. Several factors affecting extraction efficiency were investigated in a biosample with a volume of only 10 μL. This method is successful to monitor quinapril in the clinical therapeutic range. The proposed method proved effective for monitoring the trace amounts of quinapril typically used for clinical therapy. The relative standard deviation (R.S.D.) and relative error (R.E.) used for evaluating within- and between-day assays of quinapril in plasma consistently remained below 15%.
Keywords: Quinapril; Matrix; Time-of-flight; Mass spectrometry; Quinolones;

Ion-exchange chromatography (IEC) is the most widely used method for amino acid analysis in physiological fluids because it provides excellent separation and reproducibility, with minimal sample preparation. The disadvantage, however, is the long analysis time needed to chromatographically resolve all the amino acids. To overcome this limitation, we evaluated a novel liquid chromatography tandem mass spectrometry (LC–MS/MS) method, which utilizes aTRAQ® reagents, for amino acid analysis in urine. aTRAQ® reagents tag the primary and secondary amino groups of amino acids. Internal standards for each amino acid are also labeled with a modified aTRAQ® tag and are used for quantification. Separation and identification of the amino acids is achieved by liquid chromatography tandem mass spectrometry using retention times and mass transitions, unique to each amino acid, as identifiers. The run time, injection-to-injection, is 25 min, with all amino acids eluting within the first 12 min. This method has a limit of quantification (LOQ) of 1 μmol/L, and is linear up to 1000 μmol/L for most amino acids. The Coefficient of Variation (CV) was less than 20% for all amino acids throughout the linear range. Method comparison demonstrated concordance between IEC and LC–MS/MS and clinical performance was assessed by analysis of samples from patients with known conditions affecting urinary amino acid excretion. Reference intervals established for this method were also concordant with reference intervals obtained with IEC. Overall, aTRAQ® reagents used in conjunction with LC–MS/MS should be considered a comparable alternative to IEC. The most attractive features of this methodology are the decreased run time and increased specificity.
Keywords: Amino acids; Liquid chromatography–tandem mass spectrometry; aTRAQ; Amino acid analyzer; Ion-exchange chromatography;

Simultaneous quantification of VX and its toxic metabolite in blood and plasma samples and its application for in vivo and in vitro toxicological studies by Georg Reiter; John Mikler; Ira Hill; Kendal Weatherby; Horst Thiermann; Franz Worek (2704-2713).
The present study was initiated to develop a sensitive and highly selective method for the simultaneous quantification of the nerve agent VX (O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate) and its toxic metabolite (EA-2192) in blood and plasma samples in vivo and in vitro. For the quantitative detection of VX and EA-2192 the resolution was realized on a HYPERCARB HPLC phase. A specific procedure was developed to isolate both toxic analytes from blood and plasma samples. The limit of detection was 0.1 pg/ml and the absolute recovery of the overall sample preparation procedure was 74% for VX and 69% for EA-2192. After intravenous and percutaneous administration of a supralethal doses of VX in anaesthetised swine both VX and EA-2192 could be quantified over 540 min following exposure. This study is the first to verify the in vivo formation of the toxic metabolite EA-2192 after poisoning with the nerve agent VX. Further toxicokinetic and therapeutic studies are required in order to determine the impact of EA-2192 on the treatment of acute VX poisoning.
Keywords: Organophosphorus; Acetylcholinesterase inhibition; VX; Nerve agents; Large volume injection; Toxicokinetics;

A selective, simple and efficient method-ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for determination of two toxic alkaloids, namely strychnine and brucine in mice plasma. The UPLC separation was carried out using a 1.7 μm BEH C18 column (50 mm × 2.1 mm) with a mobile phase consisting of methanol:0.1% formic acid (25:75, v/v), hence providing high efficiency, high resolution and excellent peak shape for the analytes and internal standard. The method was validated over the range of 2.48–496.4 ng/ml for strychnine and 2.64–528 ng/ml for brucine, respectively. Intra- and inter-day accuracy ranged from 95.0% to 107.9% for strychnine, 93.4% to 103.3% for brucine, and the precisions were within 13.8%. The extraction recoveries of both the two alkaloids exceed 81.9%. With a simple and minor sample preparation procedure and short run-time (<3 min), the proposed method was applicable for the pharmacokinetic and toxicological analysis of strychnine and brucine in vivo.
Keywords: Semen Strychni; Strychnine; Brucine; UPLC–MS/MS;

A high performance liquid chromatography method with ultraviolet and fluorimetric detection has been developed for the simultaneous determination of urinary creatinine (Cr), tryptophan (Trp) and three Trp-related metabolites including kynurenine (Kyn), kynurenic acid (Kyna) and 5-hydroxyindole-3-acetic acid (5-HIAA). Samples were pretreated by centrifugation after a freeze–thaw cycle to remove protein and other precipitates. Separation was achieved by an Agilent HC-C18 (2) analytical column and a gradient elution program with a constant flow rate 1 mL/min at an ambient temperature. Total run time was 30 min. Cr, Kyn and Kyna were measured by a variable wavelength detector at wavelengths 258 nm, 365 nm and 344 nm respectively. Trp and 5-HIAA were measured by a fluorescence detector with an excitation wavelength of 295 nm and an emission wavelength of 340 nm. This allowed the determination of Kyn/Cr, Kyna/Cr, Trp/Cr and 5-HIAA/Cr concentration ratios in a single run on the same urine sample. Good linear responses were found with correlation coefficient (r) > 0.999 for all analytes within the concentration range of physiological level. The limit of detection of the developed method was: Cr, 0.0002 g/L; Kyn, 0.1 μmol/L; Kyna, 0.04 μmol/L; Trp, 0.02 μmol/L and 5-HIAA, 0.01 μmol/L. Recoveries from spiked human urine were: Cr, 93.0–106.4%; Kyn, 97.9–106.9%; Kyna, 98.5–105.6%; Trp, 96.7–105.2% and 5-HIAA, 96.1–99.7%. CVs of repeatability and intermediate precision of all analytes were less than 5%. This method has been applied to the analysis of urine samples from normal subjects.
Keywords: Tryptophan; Kynurenine; Kynurenic acid; 5-Hydroxyindole-3-acetic acid; Urine; High performance liquid chromatography;

A quantitative LC–MS/MS method for comparative analysis of capture-antibody affinity toward protein antigens by Mark S. Lowenthal; Hugo Gasca-Aragon; John E. Schiel; Nathan G. Dodder; David M. Bunk (2726-2732).
A mass spectrometry-based antibody selection procedure was developed to evaluate optimal ‘capture’ monoclonal antibodies that can be used in a variety of analytical measurement applications. The isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC–MS/MS) methodology is based on the use of multiple-reaction monitoring of tryptic peptide fragments derived from protein antigens. A panel of monoclonal antibodies (mAb) was evaluated based on a quantitative determination of relative binding affinity to human cardiac troponin I following immunoprecipitation. Dissociation constants (K d ) were determined for ‘bound mAb–antigen’ vs. ‘unbound antigen’ using non-linear regression analysis. Relative quantification of both antigen and antibody was based on the use of stable isotope-labeled synthetic peptides as internal standards. Optimal ‘capture’ mAbs were determined through evaluation of relative K d constants of all monitored peptide transitions. A panel of six pre-screened candidate capture mAbs was concluded to consist of two subsets of mAbs, each with statistically equivalent K d constants as determined using NIST Standard Reference Material (SRM) 2921 – Human Cardiac Troponin Complex. This ID LC–MS/MS method is shown to be capable of quantitatively differentiating mAbs based on relative binding affinities. Selection of optimal capture mAbs can be applied toward a number of analytical applications which require metrological traceability and unbiased quantification.
Keywords: Immunoassay; Mass spectrometry; Isotope dilution; Antibody; Troponin;

Validation of a UHPLC-FLD analytical method for the simultaneous quantification of aflatoxin B1 and ochratoxin a in rat plasma, liver and kidney by Laura-Ana Corcuera; María Ibáñez-Vea; Ariane Vettorazzi; Elena González-Peñas; Adela López de Cerain (2733-2740).
A rapid and simple method for the simultaneous quantification of AFB1 and OTA in rat plasma, liver and kidney by UHPLC-FLD has been successfully validated according to the following criteria: selectivity, stability, linearity, precision, accuracy, recovery, robustness and limits of quantification and detection. The extraction method, calibration curves and chromatographic conditions are common for the three matrices. Plasma and homogenized tissue samples (100 μL) were extracted with acetonitrile:formic acid mixture (99:1) (300 μL). Chromatographic separation was performed with a mixture of water and acetonitrile:methanol (50:50), both acidified with 0.5% of formic acid using a gradient profile. The method avoids the use of immunoaffinity columns and allows reduction of sample and solvent volumes as well as toxic wastes. The detection is based on a photochemical reaction which enhances the AFB1 response without affecting the OTA signal before reaching the fluorescent detector. The mycotoxin recovery for each matrix was very efficient, between 93% and 96% for AFB1 and between 94% and 96% for OTA. For both mycotoxins the LOQs were 2 μg/L in plasma and 8 μg/kg in liver and kidney. The method has successfully been applied to rat samples after a single oral administration of a mixture of AFB1 and OTA and it could be a useful tool in toxicokinetic and toxicological studies.
Keywords: Aflatoxin B1; Ochratoxin A; UHPLC-FLD; Validation; Plasma; Tissues;

Affinity adsorption of lysozyme on a macroligand prepared with Cibacron Blue 3GA attached to yeast cells by María del Pilar Ferraris; Guillermo I. Barrera; A. Pérez Padilla; Jorge A. Rodríguez (2741-2745).
The objective of this study was the development of affinity adsorbent particles with the appropriate characteristics to be applied in protein purification using the affinity ultrafiltration method. To prepare affinity macroligands Cibacron Blue 3GA, as a ligand molecule, was immobilized by covalent bonding onto yeast cell walls, the support material or matrix. The maximum attachment of the ligand to the matrix was 212 μmol/g (ligand dry weight/yeast dry weight). Lysozyme was selected as the protein model for the adsorption studies. Its adsorption onto the matrix without ligand and matrix with attached ligand were investigated batch-wise. The adsorption equilibrium isotherms appeared to follow a typical Langmuir isotherm. The maximum adsorption capacity (q m ) of the Cell-Cibacron macroligand for lysozyme was 110 mg/ml of wet macroligand. The adsorbent was also employed for the separation of lysozyme from hen egg white. High purity lysozyme was obtained.
Keywords: Affinity macroligand; Yeast cells; Cibacron Blue 3GA; Lysozyme; Affinity ultrafiltration;

Iturin A, a powerful antifungal surfactant, is a kind of bacterial lipopeptide produced by Bacillus strains. This study addresses the use of an aqueous two-phase system (ATPS) using ethanol/ammonium sulfate to extract iturin A from Bacillus amyloliquefaciens NJN-6 fermentation broth and the quantification of iturin A by HPLC. Baseline separation of iturin A homologues was performed using an RP-C18 column with a mixture of water and acetonitrile. The results showed that the correlation coefficient between integral area and concentration was 0.9961 within the range of 20–140 mg/l. The RSD of the retention time and the peak area were 1.29% and 1.45%, respectively. The effects of some operating parameters in ATPS, e.g., pH, temperature and centrifugation time, were also studied. This method can be successfully used for the rapid quantification of iturin A.
Keywords: Aqueous two-phase extraction; High performance liquid chromatography; Iturin A; Quantitative detection;

This paper aims to investigate the metabolism and pharmacokinetics of curcumin, demethoxycurcumin and bisdemethoxycurcumin in mice tumor. To improve water solubility, nanoparticle formulations were prepared as curcuminoids-loaded solid lipid nanoparticles (curcuminoids-SLNs) and curcumin-loaded solid lipid nanoparticles (curcumin-SLNs). After intragastric administration to tumor-bearing ICR mice, the plasma and tumor samples were analyzed by liquid chromatography with ion trap mass spectrometry. We discovered that curcuminoids were mainly present as glucuronides in plasma, whereas in free form in tumor tissue. A validated LC/MS/MS method was established to determine the three free curcuminoids in tumor homogenate. Samples were separated on a Zorbax SB-C18 column, eluted with acetonitrile–water (containing 0.1% formic acid), and detected by TSQ Quantum triple quadrupole mass spectrometer in selected reaction monitoring mode. The method showed good linearity (r 2  = 0.997–0.999) over wide dynamic ranges (2–6000 ng/mL). Variations within- and between-batch never exceeded 11.2% and 13.4%, respectively. The extraction recovery rates ranged from 78.3% to 87.7%. The pharmacokinetics of curcuminoids in mice tumor fit two-compartment model and first order elimination. For curcumin-SLNs group, the dosing of 250 mg/kg of curcumin resulted in AUC(0–48 h) of 2285 ng h/mL and C max of 209 ng/mL. For curcuminoids-SLNs group, the dosing equivalent to 138 mg/kg of curcumin resulted in higher tumor concentrations (AUC = 2811 ng h/mL, C max  = 285 ng/mL). It appeared that co-existing curcuminoids improved the bioavailability of curcumin.
Keywords: Curcumin; Curcuminoids; LC/MS/MS; Nanoparticle; Pharmacokinetics; Tumor;

Quantitative analysis of myo-inositol in urine, blood and nutritional supplements by high-performance liquid chromatography tandem mass spectrometry by Kit-Yi Leung; Kevin Mills; Katie A. Burren; Andrew J. Copp; Nicholas D.E. Greene (2759-2763).
Myo-inositol plays key physiological functions, necessitating development of methodology for quantification in biological matrices. Limitations of current mass spectrometry-based approaches include the need for a derivatisation step and/or sample clean-up. In addition, co-elution of glucose may cause ion suppression of myo-inositol signals, for example in blood or urine samples. We describe an HPLC–MS/MS method using a lead-form resin based column online to a triple quadrupole tandem mass spectrometer, which requires minimum sample preparation and no derivatisation. This method allows separation and selective detection of myo-inositol from other inositol stereoisomers. Importantly, inositol was also separated from hexose monosaccharides of the same molecular weight, including glucose, galactose, mannose and fructose. The inter- and intra-assay variability was determined for standard solutions and urine with inter-assay coefficient of variation (CV) of 1.1% and 3.5% respectively, while intra-assay CV was 2.3% and 3.6%. Urine and blood samples from normal individuals were analysed.
Keywords: Inositol; Glucose; Mass spectrometry; Urine; Chromatography; Epimers; HPLC–MS/MS;

Simultaneous free and glycosylated pyridinium crosslink determination in urine: Validation of an HPLC-fluorescence method using a deoxypyridinoline homologue as internal standard by Elena Monticelli; Caroline Stéphanie Aman; Maria Letizia Costa; Paola Rota; Daniela Bogdan; Pietro Allevi; Giuliana Cighetti (2764-2771).
Pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosyl-pyridinoline (Gal-Pyr) and glucosyl–galactosyl pyridinoline (GluGal-Pyr) are enzymatic mature pyridinium crosslinks. Generally, only total Pyr and D-Pyr urinary amounts (free + bound forms) are evaluated by HPLC as indices of bone resorption. This report describes the validation of an HPLC-fluorescence method for the simultaneous evaluation of free Pyr and D-Pyr, together with GluGal-Pyr and Gal-Pyr, in urine of healthy women (n  = 20, aged 27–41) and girls (n  = 20, aged 5–10). The use of an unnatural D-Pyr homologue, here proposed for the first time as internal standard, and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr synthesized to be used as primary calibrators, guarantees method specificity and correct crosslink quantification. Urine, spiked with IS, was solid-phase extracted prior to HPLC analysis. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. The HPLC method was validated for selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Both free and total Pyr and D-Pyr as well as GluGal-Pyr and Gal-Pyr amounts were significantly higher in girls than in women (p  < 0.0001), indicating an increased collagen turnover rather than only bone turnover. Gal-Pyr, for the first time evaluated in girls, was under its lower quantification limit (<LLOQ, <21.20 pmol/mL) in women. The measurement of free and glycosylated pyridinium crosslinks might provide more information on the degradation of various types of collagen than only that of total Pyr and D-Pyr. Moreover, this validated method could be a useful non-invasive technique for studying pathological conditions characterized by modified glycosylation enzyme activity and for more clinical investigation on bone fragility.
Keywords: Pyridinoline; Deoxypyridinoline; Glycosylated pyridinoline; HPLC; Fluorescence detection; Human urine;

Comprehensive analysis of the intracellular metabolism of antiretroviral nucleosides and nucleotides using liquid chromatography–tandem mass spectrometry and method improvement by using ultra performance liquid chromatography by Leon Coulier; Henk Gerritsen; Jeroen J.A. van Kampen; Mariska L. Reedijk; Theo M. Luider; Albert D.M.E. Osterhaus; Rob A. Gruters; Lars Brüll (2772-2782).
Nucleoside reverse transcriptase inhibitors (NRTIs) are a key class of drugs for the treatment of HIV infection. NRTIs are intracellularly phosphorylated to their active triphosphate metabolites and compete with endogenous deoxynucleotides (dNTP) for substrate binding. It is therefore important to analyze the intracellular concentrations of these compounds to understand drug efficacy and toxicity. To that purpose an analytical platform was developed that is capable of analyzing 8 NRTIs, 12 phosphorylated NRTIs and 4 dNTPs in small numbers of peripheral blood mononuclear cells, i.e. 1 × 106 cells. The platform consists of two liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods: a reversed-phase method for NRTIs using positive electrospray ionization (ESI) and an ion-pair LC–MS/MS method for the phosphorylated compounds using negative ESI. The methods use the same LC–MS system and column and changing from one method to the other only includes changing the mobile phase. The methods were partially validated, focussing on sensitivity, accuracy and precision. Successful transfer of the methods to ultra performance liquid chromatography (UPLC) led to a significant improvement of speed for the analysis of NRTIs and sensitivity for both NRTIs and phosphorylated NRTIs. The latter was demonstrated by the improved separation by UHPLC of dGTP vs. AZT-TP and ATP which made direct analysis of dGTP possible using the optimal MS/MS transition thereby significantly improving the detection limit of dGTP. Typically LLOQs observed for both the NRTIs and phosphorylated NRTIs were 1 nM, while the mean accuracy varied between 82 and 120% and inter- and intra-assay precision was generally <20%.
Keywords: Antiretroviral drugs; HIV; NRTI; Phosphorylated metabolites; UPLC; Ion-pair LC–MS/MS;

One single HPLC-PDA/(-)ESI-MS/MS analysis to simultaneously determine 30 components of the aqueous extract of Rabdosia rubescens by Jingcheng Tang; Ming Zhao; Yuji Wang; Guifeng Kang; Jianhui Wu; Meiqing Zheng; Shiqi Peng (2783-2793).
In China the leaves of Rabdosia rubescens have been cooked in water and widely drank to treat inflammatory and pain related diseases. To explore the components that were possibly absorbed by people the aqueous extract of the leaves was prepared, and one single HPLC-PDA/(-)ESI-MS/MS analysis was developed to simultaneously determine the components. Using the HPLC-PDA analysis 39 peaks were found in the aqueous extract, while using the (-)ESI-MS/MS analysis we were able to identify 30 peaks represented components, including 5 nucleic acids, 21 phenolic acids and 4 diterpenoids. On mouse models the in vivo anti-inflammation and analgesic actions demonstrate that 0.32 g/kg of the aqueous extract of the leaves of Rabdosia rubescens can effectively inhibit the inflammation-induced chronic pain.
Keywords: Rabdosia rubescens; Components; Anti-inflammation activity; Analgesic activity;

Novel analytical approach to a multi-sugar whole gut permeability assay by Kim van Wijck; Hans M.H. van Eijk; Wim A. Buurman; Cornelis H.C. Dejong; Kaatje Lenaerts (2794-2801).
Many pathophysiological conditions are associated with increased gastrointestinal permeability, reflecting an elevated risk of endotoxaemia, inflammation, and sepsis. Permeability tests are increasingly used in clinical practice to obtain information on gastrointestinal functioning, but tests are often restricted to the small intestine, and require large oral sugar doses. Therefore, a novel multi-sugar assay was developed, allowing assessment of whole gut permeability changes in urinary and plasma samples collected at regular intervals from 10 healthy volunteers at baseline and after intake of monosaccharides (rhamnose and erythritol) and disaccharides (sucrose, lactulose, and sucralose). Samples were analyzed by isocratic cation-exchange LC–MS. Sample preparation and detection conditions were optimized. After centrifugation, chromatographic separation was achieved on an IOA-1000 column set at 30 °C. Column effluent was mixed with ammonia for sugar-ammonium adduct formation. The lower limit of detection was 0.05 μmol/L for disaccharides and 0.1 μmol/L for monosaccharides. Linearity for each probe was between 1 and 1000 μmol/L (R 2: 0.9987–0.9999). Coefficients of variation were <5% in urine, and <9% in plasma. Recovery data were within the 90% to 110% range at all spiked concentrations. This highly sensitive novel LC–MS approach resulted in a significant decrease of the detection limit for all sugar probes, allowing a 5-fold reduction of the commonly used lactulose dose and the addition of sugar probes to also assess the gastroduodenal and colon permeability. In combination with its extended application in plasma, these features make the novel assay a promising tool in the assessment of site-specific changes in gastrointestinal permeability in clinical practice.
Keywords: LC–MS; Lactulose; Intestinal permeability;

Cholesterol ozonolysis products, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (secosterol-B) have been found in atherosclerosis plaques and the brain tissues of Alzheimer's disease patients, implicating them in the pathogenesis of cardiovascular and neurodegenerative diseases. We have recently reported that when cholesterol is oxidized with an ozone-like oxidant generated by activated mouse neutrophils, secosterol-A is generated which is then converted to secosterol-B by an aldol reaction. To investigate further pathophysiological roles of secosterols, we have developed a highly sensitive method to detect secosterol-A and -B as derivatives with 2-hydrazino-1-methylpyridine (HMP) by LC–ESI-MS/MS. The limits of detection for the HMP derivatives of secosterol-A and secosterol-B were 0.05 and 0.01 fmol, respectively, which were approximately 400 and 2000 times better than those for underivatized secosterol-A and -B. We also developed a highly reproducible and accurate method to extract, purify and derivatize secosterol in small volumes of biological specimens. Using this method, we determined the levels of secosterol-A and -B as 1.4 ± 0.7 and 4.3 ± 0.8 nM, respectively, in the plasma of normal C57BL/6 mice, and in the range of 10.4 ± 16.3 to 40.7 ± 20.1 pmol/g and 110.9 ± 10.6 to 161.5 ± 56.3 pmol/g, respectively, in the brain, liver and lung tissues.
Keywords: Secosterol; HMP; Derivatization; LC–ESI-MS/MS;

We developed a reliable and effective method to determine costunolide and dehydrocostuslactone in the root of Saussurea lappa C. B.Clarke using matrix solid-phase dispersion (MSPD) extraction, HPLC separation and diode array detection (DAD). Several extraction parameters for the MSPD were optimized. Florisil was chosen as dispersing adsorbent with methanol as elution solvent. The ratio of Florisil to sample was selected to be 4:1 and no additional clean-up steps were needed. Linearities (r  > 0.9995) were determined to be in the range of 22.5–360.0 μg/mL for costunolide and 25.0–400.0 μg/mL for dehydrocostuslactone. Intra- and inter-day precisions were also determined with a relative standard deviation (RSD) less than 3.2%. The limits of detection were found to be 0.122 μg/mL for costunolide and 0.135 μg/mL for dehydrocostuslactone. The recoveries were in the range of 92.5–99.8% with relative standard deviations ranged from 1.2% to 3.5%. The proposed MSPD method required shorter time and lower solvent volume than maceration–ultrasonic and Soxhlet extraction methods.
Keywords: Costunolide; Dehydrocostuslactone; Sesquiterpene lactones; Saussurea lappa C.B.Clarke, Matrix solid-phase dispersion extraction (MSPD); HPLC-DAD;

Determining the limits and confounders for the 2-pentyl furan breath test by gas chromatography/mass spectrometry by Shrawan Bhandari; Stephen Chambers; John Pearson; Mona Syhre; Michael Epton; Amy Scott-Thomas (2815-2820).
Aspergillus fumigatus produces 2-pentyl furan (2-PF), a volatile compound not produced by many other pathogens or normal human metabolism. 2-Pentyl furan has been detected in the breath of patients with invasive aspergillosis (IA) by SPME pre-concentration coupled with CG/MS providing the possibility of an attractive diagnostic test. The limit of detection (LOD) and quantification (LOQ) for peak integration were assessed both statistically and empirically respectively. 2-Pentyl furan was detected from 10 of 45 food stuffs tested. Levels were highest from soymilk (3 of 3 brands), lower from pumpkin, peanuts, rolled oats 2, Ensure Plus®, tinned asparagus, tinned beans and a vegetable exact (Marmite™). No 2-PF was detectable in anti-fungal medications used to treat IA or commonly used cosmetics tested. There was no difference in 2-PF breath levels between morning and afternoon or fasting and non fasting samples taken from healthy subjects eating a diet without 2-PF rich foods. 2-Pentyl furan levels were present in breath samples immediately after a mouth rinse with soy milk (P  < 0.001), and in some subjects after ingesting soy milk and rinsing their mouth with water. The breath test for 2-PF can be conducted without an overnight fast or at a specified time provided the mouth has been rinsed 30 min or more from when 2-PF containing products have been ingested.
Keywords: Aspergillus fumigatus; Invasive aspergillosis (IA); Breath test; GC/MS; SPME and 2-pentyl furan (2-PF);

Revisited mycolic acid pattern of Mycobacterium confluentis using thin-layer chromatography by Silvia Secanella-Fandos; Marina Luquin; Míriam Pérez-Trujillo; Esther Julián (2821-2826).
The profile of mycolic acids from Mycobacterium confluentis has not been adequately published. However, the definition of the composition of mycolic acids is a critical element for describing new mycobacterial species. Thus, an erroneously published profile can lead to confusing citations. The aim of this article is to make the protocols clear, by using thin layer chromatography as a tool, for defining the discrete pattern of mycolic acids of any newly reported mycobacterial species. By using this method, and corroborated using nuclear magnetic resonance analysis, we demonstrated that M. confluentis contains α-mycolates (type I) and epoxymycolates (type V mycolic acids).
Keywords: Mycobacterium; Mycolic acids; Thin layer chromatography;

A highly sensitive, selective and evaporation free SPE extraction, ESI-LC–MS/MS method has been developed for estimation of misoprostol free acid in human plasma using misoprostol acid-d5 as an internal standard (IS). The analyte was separated using isocratic mobile phase on reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M−H] anions, m/z 367–249 for misoprostol acid and m/z 372–249 for the IS. The total run time was 5.0 min and the elution of misoprostol acid and misoprostol acid-d5 (IS) occurred at 3.6 min. The developed method was validated in human plasma with a lower limit of quantification of 2.5 pg/mL. A linear response function was established for the range of concentrations 2.5–1200 pg/mL (r  > 0.998) for misoprostol acid in human plasma. The intra and inter-day precision values for misoprostol acid met the acceptance as per FDA guidelines. Misoprostol acid was stable in the battery of stability studies viz., bench-top, auto-sampler and freeze/thaw cycles. The developed assay method was applied to an oral pharmacokinetic study in humans.
Keywords: Misoprostol free acid; Human plasma; LC–MS/MS; Pharmacokinetics;

Oxidative stress has been proposed as one of the potential causes for infertility in men. Ascorbic acid and uric acid play important role in protection of spermatozoa against free radicals. A method for the simultaneous determination of ascorbic acid and uric acid in human seminal plasma using HPLC with UV detection and investigation their clinical significance as antioxidants protecting male germ cells against oxidative damage are described. Semen samples were obtained from consecutive male partners of couples presenting for a fertility evaluation. After liquefaction, the samples were centrifuged and the supernatants were diluted with dithiothreitol solution and after a filtration injected onto an analytical column. For the separation, a reverse-phase column MAG 1, 250 mm × 4.6 mm, Labiospher PSI 100 C18, 5 μm, was used. The mixture of ethanol and 25 mmol/L sodium dihydrogenphosphate (2.5:97.5, v/v), pH 4.70 was used as a mobile phase. Analytical performance of this method is satisfactory for both ascorbic acid and uric acid: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked seminal plasma were between 92.1 and 102.1%. We have found no significant differences in both ascorbic acid and uric acid concentration between the smokers and non-smokers (351.0 ± 237.9 μmol/L and 323.7 ± 99.5 μmol/L vs. 444.8 ± 245.5 μmol/L and 316.6 ± 108.9 μmol/L, p  > 0.05). This assay is a simple and reproducible HPLC method for the simultaneous measurement of ascorbic acid and uric acid in human seminal plasma.
Keywords: Ascorbic acid; Uric acid; Dithiothreitol; High-performance liquid chromatography with ultraviolet detection; Protein precipitants; Human seminal plasma;

Microchip fluorescence-enhanced immunoaasay for simultaneous quantification of multiple tumor markers by Ming Shi; Shulin Zhao; Yong Huang; Yi-Ming Liu; Fanggui Ye (2840-2844).
A microchip fluorescence-enhanced immunoassay method was developed for simultaneous detection of carcinoma antigen 125 (CA125) and carbohydrate antigen 15-3 (CA15-3). In this method, CA125 and CA15-3 react with excess amount of fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (Ab*) of CA125 and CA15-3 to form CA 125 -A b 125 * and CA 15 -3-A b 15 -3 * complexes. Microchip electrophoresis (MCE) separation of free A b 125 * , A b 15 -3 * , and CA 125 -A b 125 * , CA 15 -3-A b 15 -3 * complexes were then performed. The separated species were sensitively detected by laser-induced fluorescence detection (LIF). CA125 and CA15-3 were quantified simultaneously by measuring the fluorescence intensity of CA 125 -A b 125 * and CA 15 -3-A b 15 -3 * complexes, respectively. Under the optimum conditions, the limits of detection were 0.23 U/mL for CA125 and 0.09 U/mL for CA15-3. The present MCE-LIF method was applied to the determination of CA125 and CA15-3 in serum from healthy subjects and cancer patients. The levels of CA125 and CA15-3 in these sera samples were found to be in the ranges of 15.6–36.1 U/mL and 13.8–28.4 U/mL for healthy subjects, and 192.5–368.3 U/mL and 63.3–198.4 U/mL for cancer patients.
Keywords: Microchip electrophoresis; Immunoassay; Carcinoma antigen 125; Carbohydrate antigen 15-3; Human serum;