Candidate reference measurement procedure based on HPAEC-PAD for the development of certified reference materials for monosaccharides in serum.

To achieve the measurement reliability of monosaccharides used as diagnostic markers in clinical fields, it is essential to establish certified reference materials (CRMs). The purpose of this study is to develop a serum CRM by adopting high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as a new candidate reference measurement procedure for the measurement of glucose and galactose, common diagnostic markers of diabetes and galactosemia, respectively. Using various monosaccharides as internal standards, the accuracy of the HPAEC-PAD method was tested by measuring glucose CRM following treatment with three different deproteinization methods: ultrafiltration, protein precipitation by trichloroacetic acid (TCA), and protein precipitation by acetonitrile. Results showed that ultrafiltration and 5% TCA provided good accuracy with every tested monosaccharide as the internal standard. Accordingly, serum samples in this study were treated by ultrafiltration after adding 2-deoxy-D-glucose and arabinose, which were selected as internal standards for galactose and glucose, respectively. Both intra- and inter-day recovery tests showed good precision and accuracy within 2%. From the serum CRM batches prepared at two levels, 11 units were analyzed by exact-matched calibration methods, and the mass fractions of galactose and glucose were determined via HPAEC-PAD. The between-unit relative standard deviations were not more than 1.5%, showing homogeneity. The expanded uncertainties (%) of galactose and glucose for both levels were less than 3.6% and 2.3% at 95% confidence. The HPAEC-PAD method presented in this study can significantly improve the accuracy and precision of simultaneous monosaccharide analysis, allowing for the development of further serum CRMs for monosaccharides. Graphical abstract.

Evaluation of change in metabolome caused by comprehensive diabetes treatment: A prospective observational study of diabetes inpatients with gas chromatography/mass spectrometry-based non-target metabolomic analysis.

AIMS/INTRODUCTION: Diabetes patients develop a variety of metabolic abnormalities in addition to hyperglycemia. However, details regarding change in various metabolites after comprehensive diabetes treatment remain unknown. This study aimed to identify the short-term change in metabolome in inpatients who were subject to comprehensive diabetes treatment, using gas chromatography/mass spectrometry-based non-target metabolomics techniques. MATERIALS AND METHODS: Participants of the present study were randomly recruited from the patients with type 2 diabetes hospitalized due to problems with glycemic control (n = 31) and volunteers without diabetes (n = 30), both of whom were aged between 20 and 75 years. A metabolomic analysis of fasting plasma samples on the 2nd (pre-treatment) and 16th hospital (post-treatment) day with gas chromatography/mass spectrometry using a multiple reaction monitoring mode was carried out. RESULTS: A principal component analysis showed that metabolome of fasting plasma was different between individuals with and without diabetes. The metabolome of fasting plasma in diabetes patients after treatment was different from that of pre-treatment, as well as individuals without diabetes. Many amino acids (proline, glycine, serine, threonine, methionine, pyroglutamic acid, glutamine and lysine) were significantly increased by >10% after administering the inpatient diabetes treatment. A hierarchical clustering analysis showed that in the case of patients with markedly decreased monosaccharide levels and increased 1,5-anhydroglucitol, the levels of amino acids increased more significantly. CONCLUSIONS: After a 2-week comprehensive treatment, the plasma levels of various amino acids increased in conjunction with the reduction in monosaccharide levels in poorly controlled type 2 diabetes patients.

New application of a traditional method: colorimetric sensor array for reducing sugars based on the in-situ formation of core-shell gold nanorod-coated silver nanoparticles by the traditional Tollens reaction.

An effective and robust colorimetric sensor array for simultaneous detection and discrimination of five reducing sugars (i.e., glyceraldehyde (Gly), fructose (Fru), glucose (Glu), maltose (Mal), and ribose (Rib)) has been proposed. In the sensor array, two negatively charged polydielectrics (sodium polystyrenesulfonate (NaPSS) and sodium polymethacrylate (NaPMAA)), which served as the sensing elements, were individually absorbed on the surface of the cetyltrimethylammonium bromide (CTAB)-coated gold nanorods (AuNR) with positive charges through electrostatic action, forming the designed sensor units (NaPSS-AuNR and NaPMAA-AuNR). In the presence of Tollens reagent (Ag(NH3)2OH), Ag+ was absorbed on the surface of negatively charged NaPSS-AuNR and NaPMAA-AuNRs. When confronted with differential reducing sugars, different reducing sugars exhibited differential levels of deoxidizing abilities toward Ag+, thus Ag+ was reduced to diverse amounts of silver nanoparticles (AgNPs) in situ to form core-shell AuNR@AgNP by the traditional Tollens reaction method, leading to distinct colorimetric response patterns (value of AS/AL (the ratio of absorbance at 360 nm to that at 760 nm in Ag+-NaPMAA-AuNR, and the ratio of absorbance at 360 nm to that at 740 nm in Ag+-NaPSS-AuNR)). These response patterns are characteristic for each reducing sugar, and can be quantitatively distinguished by linear discriminant analysis (LDA) at concentrations as low as 10 nM with relative standard deviation (RSD) of 4.11% (n = 3). The practicability of this sensor array has been validated by recognition of reducing sugars in serum and urine samples. A colorimetric sensor array for reducing sugar discrimination based on the reduction of Ag+ and in situ formation of AuNR@AgNP.

Simultaneous analysis of monosaccharides using ultra high performance liquid chromatography-high resolution mass spectrometry without derivatization for validation of certified reference materials.

Monosaccharide composition of biological samples can reflect an individual's health status. Monitoring the concentration of individual monosaccharides in human serum requires a technique for the simultaneous analysis of multiple monosaccharide molecules. Furthermore, certified reference materials (CRMs) for overall monosaccharide composition of human serum are required in order to validate the performance of clinical laboratory instruments. In the present study, we present a novel method for the simultaneous analysis of numerous monosaccharide molecules without the need for derivatization or post-column treatment. We utilized ultra-high-performance liquid chromatography (UHPLC)-quadrupole/orbitrap mass spectrometry incorporating a hydrophilic interaction chromatography (HILIC) column. We optimized the precursor ions, product ions, mobile phase composition and gradient program, flow rate, and column temperature. Seven monosaccharides (D-Ribose, L-Arabinose, D-Xylose, D-Fructose, D-Mannose, D-Galactose and D-Glucose) were able to be separated and quantified. We validated the method and the seven molecules showed favorable limits of detection and quantification, recovery rates, carry-over effects, intra- and inter-day accuracy and precision, resolution, and measurement uncertainty. We analyzed human serum samples using the method. To avoid ion suppression and D-d2-Glucose peak interference, compounds present at concentrations outside of the calibration range were analyzed from diluted samples. Quantification of serum samples corroborated some previous clinical research, in that increased D-Glucose concentration was associated with increased concentrations of D-Mannose and D-Ribose. We also validated the CRMs, and expect these to have utility as standards for serum monosaccharide profiling, thus contributing to public health.

Low-glycosylated forms of both FSH and LH play major roles in the natural ovarian stimulation.

BACKGROUND: The natural ovarian stimulation is mediated by four gonadotrophin glycoforms: FSHtri with three, FSHtetra with four, LHdi with two, and LHtri with three N-glycans. The aim of the study was to determine the serum concentrations of the four glycoforms and their contents of anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylgalactosamine (SU) residues throughout the menstrual cycle. METHODS: Serum samples were collected from 78 healthy women with regular menstrual cycles. The serum glycoform molecules were identified by their distributions at electrophoreses. Analyses were also performed after removal of terminal SA. The hormones were measured with time-resolved sandwich fluoroimmunoassays. RESULTS: The concentration profiles of the four glycoforms were markedly different. FSHtri, which had a 3-fold higher biopotency than FSHtetra, had peak levels on cycle day 5 and at midcycle and nadirs on cycle days 9 and 21-23. FSHtetra had a raised level on cycle days 5-12, followed by a decrease. LHdi and LHtri had similar patterns, but the peak/nadir ratio was much more pronounced for LHdi than for LHtri, 18 versus 4. The numbers of SA residues per molecule were at a maximum around midcycle when the corresponding numbers of SU were at a minimum. The SU/SA ratio was at a minimum on cycle day 12. CONCLUSION: The results indicate that the LHdi and the FSHtri molecules play major roles in the natural ovarian stimulation. The SU/SA ratios per molecule favoured a prolonged circulatory half-life of all glycoforms at the midcycle phase. The observations may lead to more successful inductions of ovulation in anovulatory women.

Bioelectrical impedance vector analysis in patients with irritable bowel syndrome on a low FODMAP diet: a pilot study.

BACKGROUND: The aim of this study was to determine the effects of a low fermentable oligo-, di- and monosaccharides and polyols (FODMAP) diet on the nutritional status and body composition, abdominal symptoms, quality of life, anxiety/depression and sleep quality of patients with irritable bowel syndrome (IBS). METHODS: Consecutive patients were given a low FODMAP diet for 8 weeks. At baseline and after 8 weeks, blood tests were taken to evaluate nutritional status and a bioelectrical impedance analysis was performed to assess body composition. Anthropometric data, IBS Symptom Severity Score, results of a bowel habits questionnaire, Bristol Stool Chart classification, SF36, Hamilton Depression Anxiety Scale outcome and Pittsburgh Sleep Quality Index were also recorded. During the 8-week diet period, the patients were phoned periodically by the nutritionist to verify their compliance. RESULTS: Twenty-six IBS patients with a mean age of 46.2 ± 13.8 years were studied. After 8 weeks, there were no abnormalities in anthropometric data, bioelectrical impedance parameters and blood tests. The patients' IBS Symptom Severity Score improved (305.2 ± 84.1 vs 156.3 ± 106.4; p < 0.0001), as did bowel habits, Bristol Stool Chart classification, quality of life and HADS anxiety score, whereas sleeping quality and depression were unchanged. The degree of relief from symptoms and satisfaction with the diet was high. CONCLUSIONS: A low FODMAP diet improved IBS symptoms without effects on nutritional status and body composition.

Prediction of Plasma Concentration-time Profiles of Drugs in Humans from Animals Following Oral Administration: An Allometric Approach.

BACKGROUND: Allometric scaling is regularly used for the prediction of human pharmacokinetic (PK) parameters from animal PK studies. The predicted human PK parameters can also be used for the prediction of plasma concentration-time profiles in humans. OBJECTIVES: The main objective of this work is to predict human concentration-time profiles of drugs (one-compartment model) following oral administration using animal oral pharmacokinetic parameters. METHODS: Six drugs from the literature were chosen that were described by one-compartment model in both humans and animals following oral administration. Pharmacokinetic parameters such as oral clearance, oral volume of distribution of the central compartment, time to reach maximum plasma concentration, absorption rate constant, and half-life in humans were predicted from animals using allometric scaling. These predicted human pharmacokinetic parameters were then used to predict human plasma concentrations-time profiles of drugs. RESULTS: The results of this study indicate that the proposed method can be used to predict human plasma concentrations- time profiles of drugs with reasonable accuracy (≤50% prediction error). CONCLUSIONS: Given the complexity in the pharmacokinetics of oral drugs there remains some uncertainty in this entire exercise. One can minimize the prediction error by experience in allometric scaling, scientific judgment, and unconventional or innovative thinking.

Capillary Electrophoresis of Mono- and Oligosaccharides.

This chapter reports an overview of the recent advances in the analysis of mono- and oligosaccharides by capillary electrophoresis (CE); furthermore, relevant reviews and research articles recently published in the field are tabulated. Additionally, pretreatments and procedures applied to uncharged and acidic carbohydrates (i.e., monosaccharides and lower oligosaccharides carrying carboxylate, sulfate, or phosphate groups) are described.Representative examples of such procedures are reported in detail, upon describing robust methodologies for the study of (1) neutral oligosaccharides derivatized by reductive amination and by formation of glycosylamines; (2) sialic acid derivatized with 2-aminoacridone, released from human serum immunoglobulin G; (3) anomeric couples of neutral glycosides separated using borate-based buffers; (4) unsaturated, underivatized oligosaccharides from lyase-treated alginate.

Simultaneous analysis of neutral monosaccharides, fatty acids and cholesterol as biomarkers from a drop of blood.

AIM: We have developed a method for simultaneous monitoring of more biomarkers from three different classes of compounds by simultaneous analysis of neutral monosaccharides, fatty acids (FAs) and cholesterol as their per-O-methylated derivatives from a drop of blood by GC-MS. This work is a development of our previous results about analysis of neutral monosaccharides from a drop of blood. METHODS & RESULTS: The simultaneous per-O-methylation was obtained by methylation in one step with methyl iodide and NaOH in DMSO. The per-O-methylated derivatives were separated in one chromatogram. The quantitative analysis was reproducible for five monosaccharides, 22 FAs and cholesterol. The results of this method were compared with those of the enzymatic methods using commercial kits. CONCLUSION: This method can avoid the saponification of the FA methyl esters and can analyze for the first time simultaneously neutral monosaccharides, FAs and cholesterol from a drop of blood.

Development of a hydrophilic interaction liquid chromatography-tandem mass spectrometric method for the determination of kinsenoside, an antihyperlipidemic candidate, in rat plasma and its application to pharmacokinetic studies.

Kinsenoside is a major bioactive constituent isolated from Anoectochilus formosanus and is investigated as an antihyperlipidemic candidate. In this study, a rapid, sensitive, and reliable bioanalytical method was developed for the determination of kinsenoside in rat plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The plasma sample was pretreated with 1% acetic acid, followed by protein precipitation with acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC silica column (2.1mm×100mm, 3µm). The mobile phases consisted of 0.1% acetic acid in distilled water (solvent A) and 0.1% acetic acid in acetonitrile (solvent B). A gradient program was used at a flow rate of 0.2mL/min. For mass spectrometric detection, the multiple reaction monitoring mode was used; the MRM transitions were m/z 265.2→m/z 102.9 for kinsenoside and m/z 163.3→m/z 132.1 for the internal standard (IS) nicotine in the positive ionization mode. A calibration curve was constructed in the range of 2-500ng/mL. The intra- and interday precision and accuracy were within 5%. The HILIC-MS/MS method was specific, accurate, and reproducible and was successfully applied in a pharmacokinetic study of kinsenoside in rats.

Determination of Neutral Monosaccharides as Per-O-methylated Derivatives Directly from a Drop of Whole Blood by Gas Chromatography-Mass Spectrometry.

A new analytical procedure was developed for the simultaneous quantification of neutral monosaccharides from a drop of whole blood using gas chromatography-mass spectrometry analysis (GC-MS) of their per-O-methylated derivatives. The per-O-methylation reaction with methyl iodide and solid sodium hydroxide in methyl sulfoxide was used for the first time for analysis of blood monosaccharides. A blood drop volume of 0.6 µL was used without special purification. The elimination of the undesirable components was carried out during methylation in the presence of a strong base and by liquid extraction of the per-O-methylated monosaccharides. The neutral monosaccharides with an anomeric center gave four per-O-methylated isomers, which were well-separated using a capillary column. Identification was done by electron impact mass spectrometry fragmentation, retention times, and library searching. The limits of detection were determined for standards and varied from 2.0 to 2.3 ng mL(-1). Recoveries for human blood samples varied from 99.22% to 99.65%. The RSD values ranged from 1.92 to 2.37. The method is fast, sensitive, reproducible, and an alternative to current methods for quantitative analysis of blood monosaccharides.

Development and validation of a UFLC-MS/MS method for the determination of anhydrosafflor yellow B in rat plasma and its application to pharmacokinetic study.

A sensitive ultrafast liquid chromatography coupled with triple quadrupole mass spectrometric (UFLC-MS/MS) method for the quantification of anhydrosafflor yellow B (AHSYB), a major active water-soluble pigment from Carthamus tinctorius, in rat plasma has been developed and validated. Sample preparation was achieved by protein precipitation of plasma with four volumes of methanol. Rutin was used as the internal standard (IS). The analytes were separated using a C18 column with an 8min gradient elution, followed by mass spectrometric detection using negative electrospray ionization (ESI(-)) in multiple reaction monitoring (MRM) mode. The method was linear in the concentration range of 25-10,000ng/mL for AHSYB. Intra-day and inter-day precision variation was less than 6.5%. The relative error of accuracy was within ±9.4%. The mean recovery of AHSYB was higher than 70.9%. The established method was successfully applied to the pharmacokinetic study after intravenous (2.5mg/kg) and oral (30mg/kg) dosing of AHSYB in normal rats. And the pharmacokinetic properties of AHSYB in rats with acute blood stasis and the differences between normal and acute blood stasis syndrome rats were also investigated. The results showed that the compound was poorly absorbed (∼0.3%) and the AUC0-t, AUC0-∞ and F were all significantly lower (P<0.05) in acute blood stasis syndrome rats, suggesting that disease condition may alter the body metabolism by enhancing metabolite enzyme activity.

Analyte-Size-Dependent Ionization and Quantification of Monosaccharides in Human Plasma Using Cation-Exchanged Smectite Layers.

Smectite, a synthetic inorganic polymer with a saponite structure, was subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Typical organic matrix molecules 2,4,6-trihydroxyacetophenone (THAP) and 2,5-dihydroxybenzoic acid (DHBA) were intercalated into the layer spacing of cation-exchanged smectite, and the complex was used as a new matrix for laser desorption/ionization mass spectrometry. Because of layer spacing limitations, only a small analyte that could enter the layer and bind to THAP or DHBA could be ionized. This was confirmed by examining different analyte/matrix preparation methods and by measuring saccharides with different molecular sizes. Because of the homogeneous distribution of THAP molecules in the smectite layer spacing, high reproducibility of the analyte peak intensity was achieved. By using isotope-labeled (13)C6-d-glucose as the internal standard, quantitative analysis of monosaccharides in pretreated human plasma sample was performed, and the value of 8.6 ± 0.3 µg/mg was estimated.

Pharmacokinetic Interaction of astragaloside IV with atractylenolide I and prim-O-glucosylcimifugin in male Sprague Dawley rats.

Astragaloside IV, atractylenolide I, and prim-O-glucosylcimifugin are main medicinal components of the traditional Chinese medicine prescription Yu-ping-feng which is composed of three herbs: Astragalus membranaceus, Atractylodes macrocephala, and Saposhnikovia divaricata. This study is aimed to assess the influence of atractylenolide I and prim-O-glucosylcimifugin on the pharmacokinetic profile of astragaloside IV so as to investigate the pharmacokinetic mechanisms of the Yu-ping-feng prescription. Fifteen Sprague Dawley rats were randomized to three groups; astragaloside IV, astragaloside IV plus atractylenolide I, and a combination of astragaloside IV, atractylenolide I, and prim-O-glucosylcimifugin were respectively administered to rats of these three groups via intragastric gavage. Serum samples were collected at different times after drug administration, and serum concentrations of astragaloside IV and atractylenolide I were simultaneously detected using HPLC-electrospray ionization-MS. Compared with administration of astragaloside IV alone, concentrations of astragaloside IV in the serum were significantly increased when it was given in combination with atractylenolide I or atractylenolide I+prim-O-glucosylcimifugin, with higher values for Cmax (p = 0.019 and p = 0.033 compared with astragaloside IV + atractylenolide I and astragaloside IV + atractylenolide I + prim-O-glucosylcimifugin groups, respectively) and AUC (p = 0.0052 and p = 0.0047 compared with astragaloside IV + atractylenolide I and astragaloside IV + atractylenolide I + prim-O-glucosylcimifugin groups, respectively). Improvement in mean oral Cmax and mean systemic serum exposure because of the pharmacokinetic interaction between astragaloside IV and atractylenolide I might explain the rationale for the use of multiple herbs in Yu-ping-feng and of combinations of A.membranaceus and A. macrocephala.

[Studies on effects of calycosin-7-O-ß-D-glucoside on prim-O-glucosylcimifugin and cimifugin in vivo pharmacokinetics].

Study on the effects of Astragali Radix main active flavone calycosin-7-O-ß-D-glucoside on Saposhnikoviae Radix main active ingredients prim-O-glucosylcimifugin and cimifugin, a UPLC-MS/MS method for simultaneous determination of prim-O-glucosylcimifugin and cimifugin in rat plasma was established, and the comparative pharmacokinetics of prim-O-glucosylcimifugin and cimifugin after oral administration of prim-O-glucosylcimifugin and calycosin-7-O-ß-D-glucoside-prim-O-glucosylcimifugin to rats were carried out, which might be conductive in exploring the rationality of Astragali Radix - Saposhnikoviae Radix herb couple. Twelve male SD rats were divided into two groups. Prim-O-glucosylcimifugin and cimifugin in rat plasma of different time points after oral administration of prim-O-glucosylcimifugin and calycosin-7-O-ß-D-glucoside - prim-O-glucosylcimifugin to rats were determinated. And the main pharmacokinetic parameters were investigated using DAS 3. 2. 4. The established method was rapid, accurate and sensitive for simultaneous determination of prim-O-glucosylcimifugin and cimifugin in rat plasma. The analysis was performed on a Waters Acquity BEH C18 column (2.1 mm x 100 mm, 1.7 µm) with the mixture of acetonitrile and 0.1% formic acid/water as mobile phase, and the gradient elution at a flow rate of 0.3 mL x min(-1). The analytes were detected by tandem mass spectrometry with the electrospray ionization (ESI) source and in the multiple reaction monitoring (MRM) mode. Compared with prim-O-glucosylcimifugin group, the AUC(0-t)., and AUC(0-∞) of p-O-glucosylcimifugin as well as the C(max) of cimifugin significantly increased (P < 0.05) in calycosin-7-O-ß-D-glucoside-prim-O-glucosylcimifugin group. Calycosin-7-O-ß-D-glucoside could enhance the absorption of prim-O-glucosylcimifugin and cimifugin and improve the bioavailability, explaining preliminarily the rationality of Astragali Radix-Saposhnikoviae Radix herb couple.

Comparative pharmacokinetics of prim-O-glucosylcimifugin and cimifugin by liquid chromatography-mass spectrometry after oral administration of Radix Saposhnikoviae extract, cimifugin monomer solution and prim-O-glucosylcimifugin monomer solution to rats.

A sensitive and reliable liquid chromatography-mass spectrometry method has been developed and validated for simultaneous determination of cimifugin and prim-O-glucosylcimifugin in rat plasma after oral administration of Radix Saposhnikoviae (RS) extract, prim-O-glucosylcimifugin monomer solution and cimifugin monomer solution. Plasma samples were pretreated by protein precipitation with acetonitrile containing the internal standards puerarin and daidzein. LC separation was achieved on a Zorbax SB-C(18) column (150 × 4.6 mm i.d., 5 µm) with 0.1% formic acid in water and methanol by isocratic elution. The detection was carried out in select-ion-monitoring mode with a positive electrospray ionization interface. The fully validated method was successfully applied to the pharmacokinetic study of the analytes in rats. A bimodal phenomenon appeared in the concentration-time curve of prim-O-glucosylcimifugin and cimifugin after oral administration of RS extract. Prim-O-glucosylcimifugin mainly transformed to cimifugin when it was absorbed into blood. Both absorption and elimination of cimifugin after oral administration of RS were longer than after administration of single cimifugin. The pharmacokinetic parameters (AUC(0-t) , AUC(0-∞) and t(1/2) ) of prim-O-glucosylcimifugin and cimifugin by giving cimifugin monomer solution, prim-O-glucosylcimifugin monomer solution and RS extract had significant differences (P < 0.05).

Novel analytical approach to a multi-sugar whole gut permeability assay.

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.

[Glycemia and free sugar levels of the goby Perccottus glehni depending on period before the beginning and after the end of hibernation].

Goby Perccottus glehni is one of the most winterhardy species of fresh-water eurythermal fish. Study of grounds of biochemical adaptation of these animals to hibernation under conditions of ice, which are currently absent, are of undoubted for understanding of the nature of hypometabolic states. This work deals with a study of changes in the content of glucose and other free sugars in goby blood and muscle tissue under different physiological states: active, prehibernation, and arousal after experimental cooling to negative near-zero temperature. (1) A relatively high glycemia level with fluctuation amplitude from 9.8 +/- 2.1 to 24.4 +/- 2.4 mmol/ml is revealed. The minimal value of these fluctuations is recorded at the period almost coinciding with beginning of hibernation, while the maximal value--at the period of artificial termination of the three-month hibernation in ice at -1.5 degrees C. The high blood glycemia level correlating with that in the muscle tissue might probably be due to the protector role of this sugar in adaptive mechanisms of the studied fish not much to winter hypothermia, but rather to the winter hibernation on ice. (2) The level of disaccharide maltose in muscle tissue that is maximal in April is predominantly in reciprocal dependence on the fructose content that is maximal in July. (3) Dynamics of changes in the mono- and disaccharide content depending on the stage of preparation of hibernation basically coincided with the previously revealed dynamics for the fresh-water mollusc Lymnaea stagnalis, which indicated homology of mechanisms of the low-temperature adaptation for animals of different phylogenetic levels.

Individual variability in the human metabolism of an arsenic-containing carbohydrate, 2',3'-dihydroxypropyl 5-deoxy-5-dimethylarsinoyl-beta-D-riboside, a naturally occurring arsenical in seafood.

We report studies on the variability in human metabolism of an oxo-arsenosugar involving the ingestion of a chemically synthesized arsenosugar and quantitative determination of the arsenic metabolites in urine and serum by HPLC coupled with arsenic-selective mass spectrometric detection (ICPMS, inductively coupled plasma mass spectrometry). The total, four-day, urinary excretion of arsenic for six volunteers ranged widely from ca. 4-95%. The arsenic metabolites present in the urine also showed great variability: high arsenic excretion was accompanied by almost complete biotransformation of the ingested oxo-arsenosugar into a multitude of metabolites (>10), whereas the subjects that excreted low amounts of arsenic produced low quantities of metabolites relative to unchanged oxo-arsenosugar and its thio-analogue. Major arsenic urinary metabolites were dimethylarsinate (DMA) and possible intermediates in the degradation of arsenosugar to DMA, namely, dimethylarsinoylethanol (DMAE) and dimethylarsinoylacetate (DMAA) present both as their oxo- and thio-analogues. Thio-DMAE and thio-DMAA were also found in blood serum indicating that these species were formed in the liver rather than on storage of the urine in the bladder. The large variability in the way individuals metabolize arsenosugars has implications for risk assessment of arsenic intake from seafood.

Determination of helicidum and its metabolites in dog plasma by LC/UV/MS/MS and its application to pharmacokinetic studies.

A simple, rapid and reliable method was developed for the identification and quantification of helicidum and its metabolites in beagle dog plasma by liquid chromatography/ultra-violet/electrospray ionization-ion trap mass spectrometry (LC/UV/ESI-ITMS). Two metabolites were identified by MS: formylphenyl-O-beta-d-pyranosyl alloside (I) and hydroxylmethylphenyl-O-beta-d-pyranosyl alloside (II). UV was used for concentration determination with the wavelength of 270 nm. Liquid-liquid extraction was used and the extraction recovery exceeded 90%. Kromacil C(18) column (5 microm, 4.6mm i.d. x 250 mm) was used as the analytical column. Linear detection responses were obtained for helicidum concentration ranging from 1.76 x 10(-4) to 70.4 x 10(-4) micromol/mL (0.050-2.00 microg/mL). The precision and accuracy data, based on intra- and inter-day variations over 3 days, were less than 5%. The limit of determination and quantitation (LOD, LOQ) for helicidum was 0.010 and 0.030 microg/mL, respectively. Pharmacokinetic data of helicidum and the two metabolites were obtained with this method after administration of intravenous injection and a single oral dose of tablets to six beagle dogs, respectively.