Simultaneous determination of nucleosides and nucleotides in dietary foods and beverages using ion-pairing liquid chromatography–electrospray ionization-mass spectrometry

A method using ion-pairing liquid chromatography–electrospray ionization (ESI)-mass spectrometry (MS) was developed for the simultaneous determination of 23 types of purine or pyrimidine nucleosides and nucleotides in dietary foods and beverages. Dihexylammonium acetate (DHAA) was used as an ion-pairing agent and an ultra performance liquid chromatography (UPLC™) system with a reversed-phase column and a gradient program was employed for the separation of nucleosides and nucleotides. Positive-ion ESI-MS was applied for the detection of nucleosides, and negative-ion ESI-MS was used for nucleotides. Lower limits of quantitation ranged from 0.02 μmol/L (UMP and AMP) to 1.3 μmol/L (CDP). The present method was validated, and sufficient reproducibility and accuracy was obtained for the quantitative measurement of the 23 types of nucleosides and nucleotides. The method was subsequently applied to their determination in a range of Japanese foods and beverages that are considered to contain significant amounts of umami flavor compounds. Because dietary purine nucleosides and nucleotides are known to be related to hyperuricemia and gout, the determination of their concentrations in dietary foods is useful for both evaluating umami flavor and assessing the effects of dietary food on purine metabolism.     

Study of polar metabolites in tobacco from different geographical origins by using capillary electrophoresis–mass spectrometry

Many metabolites in plant are highly polar and ionic. Their analysis using gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry can be problematic. Therefore a capillary electrophoresis–mass spectrometry (CE–MS) method with charge-driven separation characteristic was developed to investigate polar metabolites in tobacco. To obtain as many features as possible, extraction of polar metabolites was optimized by the design of experiments and evaluated by univariate statistics. Method validation was carried out to evaluate the analytical characteristics including calibration curve, precision, sample stability and extraction reproducibility. The developed method was successfully applied in studying 30 tobacco leaves obtained from Yunnan and Guizhou provinces in China. A total of 154 polar metabolites were identified based on available database. Multivariate pattern recognition clearly revealed the metabolic differences between the two geographic areas and 43 significantly different metabolites were defined by the non-parametric hypothesis test (Mann–Whitney U test) and false discovery rate. Some key metabolites involved in photosynthesis such as ribulose 1,5-disphosphate, fructose 1,6-diphosphate, glycine, betaine, GABA and serine were found to be susceptible to environmental conditions. This study shows that the metabolic profiling based on CE–MS can clearly discriminate tobacco leaves of different geographical origins and understand the relationship between plant metabolites and their geographical origins.     

Determination of glutathionyl hemoglobin in hemodialysis patients using electrospray ionization liquid chromatography–mass spectrometry

We first detected glutathionyl hemoglobin (Hb) β-chain in hemodialysis patients and healthy subjects using electrospray ionization liquid chromatography–mass spectrometry. The ratio of glutathionyl Hb β-chain to total β-chain was markedly increased in the hemodialysis patients as compared with healthy subjects. Glutathionyl Hb will be used as a new clinical marker of oxidative stress.     

Next-generation capillary electrophoresis–mass spectrometry approaches in metabolomics

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Quantification of dialkylphosphate metabolites of organophosphorus insecticides in human urine using 96-well plate sample preparation and high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry

Organophosphorus (OP) pesticides kill by disrupting a targeted pest's brain and nervous systems. But if humans and other animals are sufficiently exposed, OP pesticides can have the same effect on them. We developed a fast and accurate high-performance liquid chromatography–tandem mass spectrometry method for the quantitative measurement of the following six common dialkylphosphate (DAP) metabolites of organophosphorus insecticides: dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate, (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP). The general sample preparation included 96-well plate solid phase extraction using weak anion exchange cartridges. The analytical separation was performed by high-performance liquid chromatography with a HILIC column. Detection involved a triple quadrupole mass spectrometer with an ESI probe in negative ion mode using multiple reaction monitoring. Repeated analyses of urine samples spiked at 150, 90 and 32 ng/mL with the analytes gave relative standard deviations of less than 22%. The extraction efficiency ranged from 40% to 98%. The limits of detection were in the range of 0.04–1.5 ng/mL. The throughput is 1152 samples per week, effectively quadrupling our previous throughput. The method is safe, quick, and sensitive enough to be used in environmental and emergency biological monitoring of occupational and nonoccupational exposure to organophosphates.     

Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin–fluorescein isothiocyanate conjugates

A protocol using enzymatic digestion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) for the investigation of the binding of the fluorescent contact allergen fluorescein isothiocyanate (FITC) to the 66 kDa large protein bovine serum albumin (BSA), as a model system for protein–hapten binding in the skin, is presented. Mass spectra of BSA–FITC digestions, using trypsin and chymotrypsin, respectively, provided sequence coverage of 97%. To investigate the number of FITC-bound peptides using CE-LIF separation, three different buffer salts at four different pH levels were evaluated. The use of 20 mM sodium citrate pH 6.5 as well as 20 mM sodium phosphate pH 6.5 or pH 7.5 as background electrolyte revealed high numbers of peptides with at least one bound FITC. The effect of the electrolyte counter ion on MALDI-MS was investigated and was found to have effect on the MALDI spectra signal-to-noise (S/N) at 50 mM but not at 10 mM. Of the 60 theoretical FITC-binding sites in BSA this MALDI-MS protocol presents 30 defined, 28 possible and 2 non-binding sites for FITC.     

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and ³⁴S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC–MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r² > 0.99) over a range of concentrations (SAM 0.01–2.0 μM, SAH 0.002–0.10 μM, Met 0.05–4.0 μM, GSH 0.17–20.0 μM, GSSG 0.07–20.0 μM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.     

Quantification of thyrotropin-releasing hormone by liquid chromatography–electrospray mass spectrometry

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography–mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid–histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.     

Confirmation of spectinomycin in milk using ion-pair solid-phase extraction and liquid chromatography–electrospray ion trap mass spectrometry

A confirmation procedure is described for residues of spectinomycin in bovine milk. Spectinomycin is extracted from raw milk using ion-pair reversed-phase solid-phase extraction. The extracts are ion-pair chromatographed on a polymeric reversed-phase column and analyzed on a quadrupole ion trap mass spectrometer equipped with an electrospray interface. MS–MS data are acquired in the scan mode of product ions deriving from m/z 333, the protonated molecular ion. The estimated limit of confirmation is between 0.05 and 0.1 μg/ml. The procedure was validated with control milk, fortified milk (0.1–5.0 μg/ml), and milk from cows dosed with spectinomycin.     

Detection of the isoflavone aglycones genistein and daidzein in urine using solid-phase microextraction–high-performance liquid chromatography–electrospray ionization mass spectrometry

An improved method of detection of the isoflavone aglycones, genistein and daidzein, is reported using solid-phase microextraction–high-performance liquid chromatography–electrospray ionization mass spectrometry (SPME–HPLC–ESI-MS). Extraction of the isoflavonoids from urine using SPME with a Carbowax–templated resin fiber coating allows rapid preconcentration of the analytes without the usual sample preparation required by other methods. Detection of the analytes is accomplished by HPLC–ESI-MS. Analysis of spiked samples of urine resulted in a linear range of 0.25 to 250 ng/ml for daidzein and 0.27 to 27.0 ng/ml for genistein. Limits of detection of daidzein and genistein were measured at 25.4 pg/ml for daidzein and 2.70 pg/ml for genistein. Daidzein and genistein were detected in urine following consumption of a soy drink.     

Characterization of the triacylglycerol profile in marine diatoms by ultra performance liquid chromatography coupled with electrospray ionization–quadrupole time-of-flight mass spectrometry

Diatoms are considered to have great potential as new biofuel sources because they can effectively accumulate triacylglycerols (TAGs). Detailed structure information of TAG in diatoms is much needed not only for the assessment of biofuel quality such as fatty acid chain length and unsaturation degree but also for the tracing of biosynthetic precursors because the biosynthesis of TAG is typically completed by utilizing the diacylglycerol acyltransferase in the cytoplasm. In this report, a comprehensive characterization of TAGs in marine diatoms was performed using ultra performance liquid chromatography–electrospray ionization–quadrupole time-of-flight mass spectrometry. Many types of major TAGs were identified for the first time in these diatoms: 12 TAGs in Chaetoceros debilis, 9 TAGs in Phaeodactylum tricornutum Bohlin, 16 TAGs in Nitzschia closterium f. minutissima, 16 TAGs in Thalassiosira weissflogii, 13 TAGs in Thalassiosira sp., 16 TAGs in Stephanodiscus asteaea and 7 TAGs in Skeletonema costatum. Semi-quantification of TAGs in these diatoms was also carried out, and it was found that the contents of individual TAGs ranged from 0.5?±?0.1 to 217.9?±?8.1 nmol mg^?1 total lipids. In addition, the total lipid contents in diatoms ranged from 143.6?±?16.3 to 201.1?±?16.3 mg g^?1 dry microalgae and the total TAG contents ranged from 36.8?±?9.5 to 793.2?±?54.4 nmol mg^?1 total lipids. By comparative analysis of the compositions and concentrations of major TAGs in the seven algal strains, N. closterium f. minutissima with high abundance of TAGs containing the most monounsaturated fatty acids (mainly palmitoleic acid) was considered as one of the most promising diatom strains for microalgal biofuel production. Additionally, based on the information of sn-2 fatty acid obtained (mainly C16 in the sn-2 position), we propose the hypothesis that TAGs in diatoms are mainly derived from lipids in chloroplasts through the prokaryotic biosynthesis pathway, including monogalactosyldiacylglycerol and digalactosyldiacylglycerol.     

Using liquid chromatography–tandem mass spectrometry to quantify monohydroxylated metabolites of polycyclic aromatic hydrocarbons in urine

We present an assay which employs enzyme digestion and solid phase extraction followed by liquid chromatography–tandem mass spectrometry to simultaneously quantify 16 hydroxylated polycyclic aromatic hydrocarbons (OHPAHs) in 3-ml samples of urine. The analytes consisted of 2-, 3-, and 4-ring OHPAHs, namely, 1- and 2-hydroxynaphthalene (1- and 2-OHNAP), 2-hydroxyfluorine (2-OHFLU), 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrene (1-, 2-, 3-, 4-, and 9-OHPHE), 1-hydroxypyrene (1-OHPYR), 1- and 2-hydroxybenzo(a)anthracene (1- and 2-OHBAA), 3- and 6-hydroxychrysene (3- and 6-OHCHR) and 3-, 7-, and 9-hydroxybenzo(a)pyrene (3-, 7-, and 9-OHBAP). The method was validated using urine samples from steel workers and control subjects. The coefficients of variation of the method for the particular analytes were between 7% and 27% and the limits of quantitation were between 0.002 and 0.010 μg/l urine. The 2- and 3-ring OHPAHs were easily quantified in all subjects. However, 1-OHPYR was the only representative of the 4- and 5-ring metabolites that could be quantified. Pairwise correlations showed that all OHPAHs were highly correlated with each other (0.553 ≤ r ≤ 0.910) and with 1-OHPYR (0.614 ≤ r ≤ 0.910), the metabolite most widely accepted as a short-term biomarker of exposure to PAHs. The analyte, 2-OHNAP exhibited the lowest pairwise correlations with the other OHPAHs (0.542 ≤ r ≤ 0.628), presumably due to confounding by smoking. Metabolites of phenanthrene, an abundant PAH and the smallest to possess a bay region, are promising OHPAHs for characterizing both exposures to PAHs and the various metabolic pathways.     

Unlocking the secrets to protein–protein interface drug targets using structural mass spectrometry techniques

Protein–protein interactions (PPIs) drive all biologic systems at the subcellular and extracellular level. Changes in the specificity and affinity of these interactions can lead to cellular malfunctions and disease. Consequently, the binding interfaces between interacting protein partners are important drug targets for the next generation of therapies that block such interactions. Unfortunately, protein–protein contact points have proven to be very difficult pharmacological targets because they are hidden within complex 3D interfaces. For the vast majority of characterized binary PPIs, the specific amino acid sequence of their close contact regions remains unknown. There has been an important need for an experimental technology that can rapidly reveal the functionally important contact points of native protein complexes in solution. In this review, experimental techniques employing mass spectrometry to explore protein interaction binding sites are discussed. Hydrogen–deuterium exchange, hydroxyl radical footprinting, crosslinking and the newest technology protein painting are compared and contrasted.     

Determination of alkylresorcinol metabolites in human urine by gas chromatography–mass spectrometry

Alkylresorcinols (ARs) are phenolic lipids present at high concentrations in the outer parts of rye and wheat kernels and have been proposed as biomarkers for intake of whole grain and bran products of these cereals. AR are absorbed in the small intestine and after hepatic metabolism two major metabolites, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), are excreted in urine either as such or as conjugates. Urine samples from nine individuals were incubated with different enzymes to assess type and extent of conjugates. In comparison with DHBA, which was mostly found in the free form, the less polar DHPPA was conjugated to a greater extent and the major conjugates were glucuronides. In this method, urine samples were hydrolyzed using β-glucuronidase from Helix pomatia and syringic acid was used as internal standard. Samples, silylated with BSTFA, were analyzed by GC–MS utilizing a BP-5 fused silica capillary column and single ion monitoring of molecular ions (m/z 370 [DHBA], m/z 398 [DHPPA]). Recoveries of DHBA and DHPPA were estimated to be 94% and 93%, respectively. The average intra-assay/inter-assay coefficients of variation were 4.9/5.7% for DHBA and 7.6/9.3% for DHPPA.     

Determination of cymipristone in human plasma by liquid chromatography–electrospray ionization-tandem mass spectrometry

A rapid, specific and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry method was developed and validated for determination of cymipristone in human plasma. Mifepristone was used as the internal standard (IS). Plasma samples were deproteinized using methanol. The compounds were separated on a ZORBAX SB C₁₈ column (50 mm × 2.1 mm i.d., dp 1.8 μm) with gradient elution at a flow-rate of 0.3 ml/min. The mobile phase consisted of 10 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadruple tandem mass spectrometer by selective reaction monitoring (SRM) mode via electrospray ionization. Target ions were monitored at [M+H]⁺ m/z 498 → 416 and 430 → 372 in positive electrospray ionization (ESI) mode for cymipristone and IS, respectively. Linearity was established for the range of concentrations 0.5–100 ng/ml with a coefficient correlation (r) of 0.9996. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.5 ng/ml. The validated method was successfully applied to study the pharmacokinetics of cymipristone in healthy Chinese female subjects.     

Determination of betaine metabolites and dimethylsulfoniopropionate in coral tissues using liquid chromatography–time-of-flight mass spectrometry and stable isotope-labeled internal standards

A convenient procedure for determination of seven betaine analogs and dimethylsulfoniopropionate (DMSP) in extracts of coral tissues using LC–MS stable isotope dilution is described. Extraction procedures were optimized for selective extraction of polar metabolites from coral tissues. The LC–MS protocol employed a pentafluorophenylpropyl (PFPP) column for HPLC separation, with chromatographic resolution of isobaric and isomeric zwitterionic metabolites optimized by adjusting the acidity of the mobile phase. A ternary gradient was used to exploit the unusual retention characteristics of cationic metabolites on the PFPP column, with incorporation of ammonium acetate in a later gradient stage promoting elution of more hydrophobic betaines which are retained at high organic content in the absence of ammonium acetate. We demonstrate that the new LC–MS based method provides accurate measurements from nanomolar to high micromolar concentrations, and can be applied for profiling of betaine metabolites and DMSP in corals or other aquatic organisms.     

Simultaneous determination of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam in human serum using gas chromatography–mass spectrometry

A method for the quantitation of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam from human serum capable of monitoring concentrations achieved under therapeutic conditions is presented. The substances were extracted under basic conditions with toluene and the hydroxy metabolites transformed to their tert-butyldimethylsilyl derivatives with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. The samples were measured by gas chromatography–mass spectrometry. The limits of detection are 0.2 ng ml⁻¹ for midazolam and 0.1 ng ml⁻¹ for 1-hydroxy- and 4-hydroxymidazolam. The coefficients of variation are 3.9% at 5 ng ml⁻¹ for midazolam, 6.7% at 2 ng ml⁻¹ for 1-hydroxymidazolam and 8.8% (22.2%) at 0.5 (0.2) ng ml⁻¹ for 4-hydroxymidazolam.     

Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

Optimized analytical method for cyclosporin A by high-performance liquid chromatography–electrospray ionization mass spectrometry

The Micromass Platform LCZ mass detector parameters were optimized for simultaneous recording of the protonated (CsA∼H⁺), sodium adduct (CsA∼Na⁺) and potassium adduct (CsA∼K⁺) of cyclosporin A eluted from a Symmetry Shield RP8 column. The optimized procedure allows a precise analysis of CsA in whole blood or serum without removal of salts prior to analysis. The ratio of the three forms of CsA varied depending on the assay condition and the types of specimens being analyzed. The summation of three ionic forms of CsA detected by LC–ESI-MS is a reliable and simple method to assess CsA concentration in the blood.     

Simultaneous determination of selegiline-N-oxide,a new indicator for selegiline administration,and other metabolites in urine by high-performance liquid chromatography–electrospray ionization mass spectrometry

In order to discriminate selegiline (SG) use from methamphetamine (MA) use, the urinary metabolites of SG users have been investigated using high-performance liquid chromatography (HPLC)–electrospray ionization mass spectrometry (HPLC–ESI–MS). Selegiline-N-oxide (SGO), a specific metabolite of SG, was for the first time detected in the urine, in addition to other metabolites MA, amphetamine (AP) and desmethylselegiline (DM-SG). A combination of a Sep-pak C₁₈ cartridge for the solid-phase extraction, a semi-micro SCX column (1.5 mm I.D.×150 mm) for HPLC separation and ESI–MS for detection provided a simple and sensitive procedure for the simultaneous determination of these analytes. Acetonitrile–10 mM ammonium formate buffer adjusted to pH 3.0 (70:30, v/v) at a flow-rate of 0.1 ml/min was found to be the most effective mobile phase. Linear calibration curves were obtained over the concentration range from 0.5 to 100 ng/ml for all the analytes by monitoring each protonated molecular ion in the selected ion monitoring (SIM) mode. The detection limits ranged from 0.1 to 0.5 ng/ml. Upon applying the scan mode, 10–20 ng/ml were the detection limits. Quantitative investigation utilizing this revealed that SGO was about three times more abundant (47 ng/ml, 79 ng/ml) than DM-SG in two SG users’ urine samples tested here. This newly-detected, specific metabolite SGO was found to be an effective indicator for SG administration.