Identification and determination of the enantiomers of moprolol and their metabolites in human urine by high-performance liquid chromatography and gas chromatography—mass spectrometry

A simple and sensitive high-performance liquid chromatographic (HPLC) method using chiral derivatization was developed to screen and determine the enantiomers of moprolol and their metabolites in human urine. The recovery of (+)- and (−)-moprolol from urine was 70.8–81.1% at different concentrations. The coefficients of variation (C.V.) were less than 3.2 and 6.5% for intra- and inter-assays, respectively. Moprolol could be detected in urine up to 24 h after oral administration of a 50-mg dose of moprolol. Unconjugated and conjugated enantiomers of moprolol and their metabolites were analyzed by gas chromatography (GC). A gas chromatographic—mass spectrometric (GC—MS) confirmatory method was established to identify the metabolites of moprolol. The double derivatization procedure for moprolol and their metabolites with S-(−)-menthyl chloroformate [(−)-MCF] and N-methyl(trimethylsilyl)trifluoroacetamide (MSTFA) gave very good GC—MS properties of the derivatized compounds and provided reliable structural information for their confirmation analysis. This is the first published report on the use of a GC—MS method for the detection of the enantiomers of moprolol and their metabolites in human urine.     

Determination of 2-hydroxypropyl-γ-cyclodextrin in plasma of cynomolgus monkeys after oral administration by gas chromatography–mass spectrometry

This report describes a specific and highly sensitive gas chromatography–mass spectrometry (GC–MS) assay for the analysis of industrially produced 2-hydroxypropyl-γ-cyclodextrin, a heterogeneous mixture of homologues and isomers, in plasma of cynomolgus monkeys. Instead of analyzing the polysaccharide mixture as a whole, in a first step the HP-γ-cyclodextrin mixture, together with the internal standard (2,6-di-O-methyl-β-cyclodextrin), was deuteromethylated, and in a second step hydrolyzed with hydrochloric acid to the respective monosaccharides. The resulting reaction mixture was trimethylsilylated to 1,4-bis(O-trimethylsilyl)-2,3-bis-O-deuteromethyl-6-O-2′-deuteromethoxypropylglucose (representative for HP-γ-CD) and 1,4-bis-(O-trimethylsilyl)-bis-2,6-O-methyl-3-O-deuteromethylglucose (representative for the internal standard), respectively, and analyzed by GC–MS. The limit of quantification of this assay was 20 nmol/l.     

Determination of Guan-Fu Base A, a new anti-arrhythmic, in human plasma by gas chromatography and electron–capture detection

A gas chromatographic–electron capture detection (GC–ECD) method has been developed for determining Guan-Fu Base A (GFA), an experimental anti-arrhythmic, in human plasma. The method was based on one-step liquid–liquid extraction with toluene and chemical derivatization with pentafluoropropionic anhydride followed by GC–ECD. The derivatives of GFA and metoprolol (Met, internal standard) were confirmed by gas chromatography–mass spectrometry (GC–MS) to be dipentafluoropropionyl-GFA and dipentafluoropropionyl-Met. The method was linear over the concentration ranges of 0.1–20.0 and 1.0–30.0 μg/ml with the detection limit of 0.05 μg/ml at S/N=5. The intra- and inter-assay precisions were less than 6 and 10%, and accuracy 99.70±3.30 and 97.60±5.99%, respectively. The absolute recoveries were 81.88, 77.35, 80.79 and 83.85% for GFA at concentrations of 0.5, 1.0, 5.0 and 14.0 μg/ml and 88.24% for Met at 3.0 μg/ml, respectively.     

Quantification method of human hemoglobin adducts from hexahydrophthalic anhydride and methylhexahydrophthalic anhydride

A method was developed for the determination of human hemoglobin (Hb) adducts from hexahydrophthalic anhydride (HHPA) and methylhexahydrophthalic anhydride (MHHPA). The procedure includes lysis of erythrocytes, dialysis of the Hb-solution followed by acid hydrolysis. The released hexahydrophthalic (HHP) acid and methylhexahydrophthalic (MHHP) acid were purified using a combined liquid–liquid and solid-phase extraction procedure followed by derivatization with pentafluorobenzyl bromide. The derivatives were analyzed using GC–MS in negative ion chemical ionization mode with ammonia as moderating gas. As internal standards, deuterium-labeled HHP and MHHP acids were used. The detection limits were 0.3 pmol/g Hb for HHP acid and 0.9 pmol/g Hb for MHHP acid. The between-day precisions for HHP acid were 18% at 2 pmol/g Hb and 10% at 13 pmol/g Hb. For MHHP acid, the precision was 27% at 2 pmol/g Hb and 14% at 22 pmol/g Hb. The method was applicable for analysis of Hb adducts from workers occupationally exposed to HHPA and MHHPA.     

Structural characterization and antioxidant activity of a polysaccharide from the fruiting bodies of cultured Cordyceps militaris

The water-soluble crude polysaccharides were obtained from the fruiting bodies of cultured Cordyceps militaris by hot water extraction followed by ethanol precipitation. The polysaccharides were successively purified by chromatography on DEAE–cellulose-52 and Sephacryl S-100 HR columns, giving main three polysaccharide fractions termed P50-1, P70-1, and P70-2. Structural features of P70-1 were investigated by a combination of chemical and instrumental analysis, such as partial acid hydrolysis, methylation analysis, periodate oxidation – Smith degradation, GC–MS, ¹³C NMR, HPAEC-PAD, and FT-IR. The results indicated that P70-1 has a backbone of (1 → 6)-linked β-d-mannopyranosyl residues, which occasionally branches at O-3. The branches were mainly composed of (1 → 4)-linked -d-glucopyranosyl and (1 → 6)-linked β-d-galactopyranosyl residues, and terminated with β-d-galactopyranosyl residues and -d-glucopyranosyl residues. In the in vitro antioxidant assay, P70-1 was found to possess hydroxyl radical-scavenging activity with an IC₅₀ value of 0.548 mg/ml.     

Chemical composition of the essential oil from leaves of Lippia citriodora H.B.K. (Verbenaceae) at two developmental stages

The chemical composition of the essential oil extracted from fresh leaves of Lippia citriodora (Verbenaceae) was analyzed by GC-FID and GC–MS in May, when growth rates are maximal, and in September, in full bloom. In both samples the main constituents were geranial, neral and limonene constituting 66.3% of the total essential oil yield in May and increasing to 69% in September. Their individual percentage values, however, changed considerably for geranial and neral decreasing from 38.7 to 26.8% and from 24.5 to 21.8%, respectively, and for limonene increasing from 5.8 to 17.7%. All other components remained more or less unchanged both qualitatively and quantitatively. FT-IR spectrometry was also applied for the qualitative determination of the main components.     

Time-resolved fluoroimmunoassay for equol in plasma and urine

We present a method for the determination of the isoflavan equol in plasma and urine. This estrogenic isoflavan, which is formed by the action of the intestinal microflora, may have higher biological activity than its precursor daidzein. High urinary excretion of equol has been suggested to be associated with a reduction in breast cancer risk. The method is based on time-resolved fluoroimmunoassay, using a europium chelate as a label. After synthesis of 4′-O-carboxymethylequol the compound is coupled to bovine serum albumin (BSA), then used as antigen to immunize rabbits. The tracer with the europium chelate is synthesized using the same 4′-O-derivative of equol. After enzymatic hydrolysis (urine) or enzymatic hydrolysis and ether extraction (plasma) the immunoassay is carried out. The antiserum cross-reacted to variable extent with some isoflavonoids. For the plasma method the cross-reactivity does not seem to influence the results, which were highly specific. The overestimation of the values using the urine method (164%) compared to the results obtained by a gas chromatography–mass spectrometry (GC–MS) method is probably due to some influence of the matrix on the signal, and interference of structurally related compounds. It is suggested that plasma assays are used but if urine samples are measured a formula has to be used to correct the values making them comparable to the GC–MS results. The correlation coefficients between the time-resolved fluoroimmunoassay (TR-FIA) methods and GC–MS methods were high; r-values for the plasma and urine method, were 0.98 and 0.91, respectively. The intra-assay coefficient of variation (CV%) for the TR-FIA plasma and urine results at three different concentrations vary between 5.5–6.5 and 3.4–6.9, respectively. The inter-assay CV% varies between 5.4–9.7 and 7.4–7.7, respectively. The working ranges of the plasma and urine assay are 1.27–512 and 1.9–512 nmol/l, respectively.     

An isotope dilution gas chromatographic-mass spectrometric method for the simultaneous assay of estrogens and phytoestrogens in urine

The metabolism of endogenous estrogens is complicated and certain metabolic patterns may reflect an individual risk of estrogen-dependent diseases such as breast cancer. Since the 1960s we have been constantly involved in developing estrogen profiling methods, in the beginning using gas chromatography and later gas chromatography–mass spectrometry (GC–MS) in the selected ion monitoring mode (SIM) and finally utilizing isotope dilution (ID–GC–MS–SIM). The addition of the dietary phytoestrogens to the profile rendered the method even more complicated. The present work presents the final estrogen profile method for 15 endogenous estrogens, four lignans, seven isoflavonoids and coumestrol in one small urine sample (1/150th of a 24 h human urine sample, minimum 2.5–5 ml) with complete validation including investigations as to the precision, sensitivity, accuracy and specificity. The method does not include the minimal amounts of unconjugated estrogens in urine. It may also be used for animal (e.g. rat and mouse) urine using a minimum of 2 ml of usually pooled sample. Despite its complexity it was found to fulfill the reliability criteria, resulting in highly specific and accurate results.     

Rapid analysis of phytoestrogens in human urine by time-resolved fluoroimmunoassay

A time-resolved fluoroimmunoassay (TR-FIA), with europium labeled phytoestrogens as tracers, was developed for the quantitative determination of enterolactone, genistein and daidzein in human urine. The aim was to create a method for the screening of large populations in order to assess the possible correlations between the urinary levels and the risk of Western diseases.

After the synthesis of the 5′-carboxymethoxy derivative of enterolactone and 4′-O-carboxymethyl derivatives of daidzein and genistein, the respective compound was coupled to bovine serum albumin and then used as an antigen in the immunization of rabbits. The same derivatives of the phytoestrogen were used in preparing the europium tracers. After the enzymatic hydrolysis, the TR-FIA was carried out using the Victor 1420 multilabel counter. The method has sufficient sensitivity to measure the phytoestrogens at concentrations even below 5 nmol/l. The intra- and inter-assay coefficients of variation, at three different concentrations, varied from 1.9 to 5.3 and from 2.4 to 9.7, respectively.

We measured urinary enterolactone, genistein and daidzein in 215 samples from Finnish healthy women and found that more than 50% of the values ranged between 1 and 7, <0.1 and 0.6 and below 0.6 μmol/24 h, respectively. The TR-FIA method including only a hydrolysis step gave higher values than those measured by gas chromatography–mass spectrometry (GC–MS). However, the assay results by the present method showed strong correlation with those obtained by GC–MS. It is concluded that the TR-FIA is suitable for population screening of urinary phytoestrogens.     

Purification of thymocyte growth peptide (TGP) from sheep thymus. Relationship to FTS/thymulin

Thymocyte growth peptide (TGP) initiates DNA synthesis in immature thymocytes and has previously been characterized as an acidic peptide isolated from calf thymus. We now report the isolation of TGP from sheep thymus and show it to be a nonapeptide with a large N-terminal blocking moiety characterized by high UV absorbance. The amino acid composition is identical to FTS, consisting of 2 Gly, 2 Ser, 2 Glx, 1 Ala, 1 Lys, 1 Asx. In contrast to FTS, TGP is acidic with an apparent isoelectric point of 4.2 and a high UV absorbance at 270–280 nm. Reverse phase chromatography of TGP at an acidic pH results in a change of the molecule and the appearance of two new compounds TGP-A and TGP-B, both with less than 50% of the original TGP activity. Full activity could be restored by the addition of ZnCl₂ to TGP-A. Both TGP-A and B have some amino acid composition and high UV absorbance as native TGP. We propose that TGP consists of a non-peptide moiety bound to the N-terminal of the nonapeptide Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn and that the active molecule is stabilized by Zn²⁺.     

Determination of the enantiomers of bunolol in human urine by high-performance liquid chromatography on a chiral AGP stationary phase and identification of their metabolites by gas chromatography—mass spectrometry

A high-performance liquid chromatographic method using a chiral AGP column was developed to screen and determine the enantiomers of bunolol in human urine. The recovery of (+)- and (−)-bunolol from urine was 91.79–95.23% at different concentrations. The coefficients of variation (C.V.) were less than 2.1 and 2.3% for intra- and inter-assays, respectively. Urinary metabolites were detected using GC—MS after derivatization with N-methyl(trimethylsilyl)trifluroacetamide. The influences of pH and modifier on a chiral AGP column were studied.     

Preparation and characterization of armadillo submandibular glycoproteins.

The nine-banded armadillo (Dasypus novemcinctus mexicanus Peters) was chosen for this study so that a comparison could be made of the salivary mucus glycoproteins of an ancient mammalian species with those derived from previously studied, more highly evolved species. Two mucus glycoproteins, armadillo submandibular glycoprotein A and armadillo submandibular glycoprotein B, were prepared from the armadillo submandibular gland by a modification of the method of Tettamanti & Pigman (1968) (Arch. Biochem. Biophys. 124, 41-50). The composition of glycoprotein A is the simplest one among the known mucus glycoproteins. Six amino acids constitute 98.5 mol/100mol of the protein of glycoprotein A and 82 mol/100 mol of that of glycoprotein B. These are serine and threonine (which make up 40-50% of the molar amino acid composition), glutamic acid, glycine alanine and valine. Proline is absent from glycoprotein A and comprises only 2.3% of glycoprotein B. For both glycoproteins, the protein content, as determined by the method of Lowry, Rosebrough, Farr & Randall (1951) (J. Biol. Chem 193, 265-275), with bovine serum albumin as standard, was nearly 60% higher than when determined by the sum of the amino acids. The ratios of total mol of amino acid/total mol of carbohydrate are 1:0.63 for glycoprotein A and 1:0.68 for glycoprotein B, N-Acetylneuraminic acid and N-acetylgalactosamine, in a molar ratio of about 0.35:1.00, are the principal carbohydrates present in both glycoproteins. Neutral sugars seem to be absent from glycoprotein A, but galactose and fucose are present in glycoprotein B. The carbohydrate side chains in glycoprotein A are composed of about two-thirds monosaccharide and one-third disaccharide residues, whereas those of glycoprotein B are more complex. For both glycoproteins, essentially all of the N-acetylgalactosamine was attached O-glycosidically to the hydroxyamino acid residues of the protein core. The linkage of N-acetylneuraminic acid glycoprotein A was extremely sensitive to dilute acid and neuraminidase. Glycoprotein B has chemical properties similar to those of glycoprotein A. However, whereas glycoprotein A was susceptible to both Clostridium perfringens and Vibrio cholerae neuraminidases, only the latter enzyme had an effect on glycoprotein B at pH 4.75. Both glycoproteins were homogeneous by cellulose acetate electrophoresis and ultracentrifugal analyses. The apparent mol.wts. of glycoprotein A and glycoprotein B were 7.8 X 10(4) and 3.1 X 10(4) respectively.     

Two Tetrahymena G-DNA-binding proteins, TGP1 and TGP3, share novel motifs and may play a role in micronuclear division

G-DNA is a four-stranded DNA structure with diverse putative biological roles. We have previously purified and cloned a novel G-DNA-binding protein TGP1 from the ciliate Tetrahymena thermophila. Here we report the molecular cloning of TGP3, an additional G-DNA-binding protein from the same organism. The TGP3 cDNA encodes a 365 amino acid protein that is homologous to TGP1 (34% identity and 44% similarity). The proteins share a sequence pattern that contains two novel repetitive and homologous motifs flanking an extensively hydrophilic and basic region. A nuclear fractionation experiment showed that TGP1 and TGP3 activities are localized predominantly in the nuclear fraction. To further investigate the biological roles of the proteins in vivo, we have generated separate macronuclear gene knockout (KO) strains (TGP1KO and TGP3KO) for each of the two genes. Southern blot analysis demonstrated that the macronuclear copies of each gene were completely disrupted. Mobility shift assays showed that the corresponding G-DNA-binding activity for each protein was abolished in the KO strains. Growth analysis showed that both KO strains grew at near wild-type rates, indicating that neither of the genes is essential for cell growth. Nevertheless, nuclear staining analysis revealed that both TGP1KO and TGP3KO cells have an increased occurrence (more than 2-fold) of extra micronuclei, implying faulty control of micronuclear division in the KO cells.     

A simple and rapid method for the differentiation of C-13 manoyl oxide epimers in biologically important samples using GC-MS analysis supported with NMR spectroscopy and computational chemistry results

There is confusion in the literature related to the characterization of biologically important C13 manoyl oxide epimers by using their mass spectrometric data. Furthermore a method for calculating the epimeric purity has not been established. In this work mixtures of manoyl oxide C13 epimers and pure ent-13-epi-manoyl oxide have been isolated from plant extracts. The GC–MS analysis allows the characterization of each stereoisomer in a sample of natural origin: the ratio of intensities of peaks m/z 275 : 257 is lower in ent-13-epi-manoyl oxide than in manoyl oxide. NMR spectroscopy is used to give experimental evidence and simple calculations were performed to support the MS data. On the basis of these results the characterization of the two stereoisomers and the calculation of their ratio in plant extracts and essential oils can be done in a routine basis. The biological activity evaluation of mixtures with different epimeric composition of manoyl oxide showed that the ratio of the two epimers is important for their antibacterial activity. Ent-13-epi-manoyl oxide seems to be more active than its epimeric congener against Gram-positive bacteria.     

Assay method for the carboxylic acid metabolite of clopidogrel in human plasma by gas chromatography–mass spectrometry

This paper describes a GC–MS method for the analysis of the carboxylic acid metabolite (SR26334, II) of methyl (+)-(S)--(o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetate hydrogensulfate (clopidogrel, SR 25990, I) in plasma and serum. The analytical procedure involves a robotic liquid–liquid extraction with diethyl ether followed by a solid–liquid extraction on C₁₈ cartridges. The derivatization process was performed using n-ethyl diisopropylethylamine and -bromo-2,3,4,5,6-pentafluoro toluene. A structural analogue (III) of II, was used as internal standard. The 1/X²; weighted calibration curve obtained in the range 5–250 ng/ml was well described by a quadratic equation. The extraction efficiency was better than 48% over the range studied; for the internal standard it averaged 51% at 50 ng/ml. Precision ranged from 3.6 to 15.8%, and accuracy was between 92 and 114%. Dilution has no influence on the performance of the method which could then be used to quantitate plasma samples containing up to 25 000 ng/ml. The limit of quantification was 5 ng/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay methods for use in pharmacokinetic studies.     

Determination of the amino acid sequence in a cyclic lipopeptide using MS with DHT mechanism

A TOF MS/MS method to directly determine the amino acid sequence in a cyclic lipopeptide without its hydrolysis is described. The fragments of the peptide and the hydrocarbon chains were identified through comparing the MS of two analogues of the lipopeptide; the connecting relationship of amino acid residues in the lipopeptide was determined based on the difference of mass to charge ratio between peaks in the MS spectra and the amino acid analysis; and finally, according to the mechanism of double hydrogen transfer(DHT) the C-terminal of peptide and hydroxy aliphatic acid in the lipopeptide was directly determined without the hydrolysis. The determined sequence of amino acid residues in the cyclic lipopeptide is also supported by the rest peaks in the MS spectra grounded on simple fragmenting mechanism. This method can be used to determine the amino acid sequence in any aliphatic acid loop-inlaying cyclic lipopeptides.     

福建云霄地方茶树品种资源生化成分特征分析与评价北大核心CSCD

以福建省云霄县小帽山、梁山和南乌山3个区域共23份地方茶树品种资源为试材,采用高效液相色谱(HPLC)法和超高效液相色谱-质谱联用(UPLC-MS/MS)法测定其儿茶素、嘌呤生物碱及氨基酸组分含量,并进行描述统计分析、差异分析和聚类分析,以筛选生化成分含量特异的茶树品种资源。结果表明:(1)23份茶树品种资源的儿茶素总量变化范围为73.54~260.31 mg·g^(-1),平均值为160.48 mg·g^(-1);表没食子儿茶素-3-O-甲基-没食子酸酯(EGCG3″Me)在所有资源中均检出,含量最高的为小帽山3号(X03),高达33.65 mg·g^(-1)。(2)23份茶树品种资源的嘌呤生物碱总量变化范围为15.28~70.42 mg·g^(-1),平均值为40.95 mg·g^(-1);苦茶碱(TC)在梁山地区的茶树品种资源(LS01-LS07)均检出,其中梁山6号(L06)和梁山7号(L07)的苦茶碱含量高于其咖啡碱含量。(3)23份茶树品种资源的氨基酸总量变化范围为6.40~47.57 mg·g^(-1),平均值为20.83 mg·g^(-1);氨基酸构成丰富,共检测出20种氨基酸物质。(4)差异分析和聚类分析结果显示,云霄县3个区域茶树资源的生化成分具有明显地域特征。其中梁山区域茶树资源富含苦茶碱(TC),小帽山茶树资源的表没食子儿茶素没食子酸酯(EGCG)和表没食子儿茶素-3-O-甲基-没食子酸酯(EGCG3″Me)含量较高,南乌山茶树资源的丙氨酸(Ala)、谷氨酸(Glu)、谷氨酰胺(Gln)含量略高于其他两地。研究发现,云霄地方茶树品种资源的儿茶素、嘌呤生物碱及氨基酸含量差异大,具有丰富的遗传多样性,区域特征明显,筛选出高儿茶素、高EGCG3″Me、高苦茶碱、高茶氨酸等18份优特茶树品种资源,为云霄地方茶树品种资源的系统开发利用奠定了基础。     

三叶虫茶营养成分的分析与评价

通过与老鹰茶虫酿茶和一些品牌绿茶的对比,对三叶虫茶的一般营养成分、茶的生化特征、氨基酸和脂肪酸组成、维生素、矿物质元素进行分析和营养评价,为开发三叶虫茶提供科学依据。方法:用常规方法分析其主要营养素和生化成分;用氨基酸分析仪分析氨基酸组成,并采用WHO推荐的蛋白质模式对蛋白质进行营养评价;维生素和矿物元素含量直接说明其营养功能。三叶虫茶含有36.44%的水浸出物,16.28%的茶多酚、1.39%的氨基酸;氨基酸的种类较为齐全,总含量与传统茶叶相当,9种人体必需氨基酸的总量达到0.722%,是传统茶叶的3~12倍,特别是赖氨酸、蛋氨酸、苏氨酸、色氨酸的含量远远高于常规茶叶,而这4种氨基酸在一般植物食品中都是限制性氨基酸;但其茶氨酸、天门冬氨酸、谷氨酸的含量远远低于常规茶叶,而这3种氨基酸对改善茶汤滋味具有重要作用;三叶虫茶中脂肪酸含量为1.23%,其中多不饱和脂肪酸占35.25%;钙、磷、镁含量较高,铁、铜、锌、锰等的含量丰富;维生素C和维生素E含量与普通茶叶相当。三叶虫茶营养丰富,具备常规茶叶的一些生化特征,可作为茶饮料的代用品;若与普通茶叶混合使用,能在滋味和营养价值上实现互补。