Analysis of Adenosine and Other Adenine Compounds in Patients With Immunodeficiency Diseases

Abstract

Adenine compounds can be measured in picomole amounts using liquid chromatography of the fluorescent 1, N⁶-etheno derivatives. The limit of detection for the etheno derivatives in tissue extracts, however, is tissue-dependent due to interference by nucleotides and fluorescent components which are normally present. Prior to derivatization nucleotides were partially removed from extracts of lymphocytes and erythrocytes by treatment with Dowex AG1-X2 anion exchange resin. Samples were analyzed using either a Partisil PXS 10/25 SCX column eluted with 100 mM NH₄H₂PO₄, pH 4.5, at a flow rate of 2 ml/min; or using two μBondapak/C₁₈ reversed-phase columns eluted with 5 mM KH₂PO,₄:25% methanol (V/V) pH 7.5, at a flow rate of 1 ml/min. Adenosine was found to be 0.07 nmole/ml in normal adult human plasma. The urine of a child with severe combined immunodeficiency disease associated with absence of adenosine deaminase contained a normal amount of adenosine (5–6 nmole/ml), but contained a high level (～60 nmole/ml) of deoxyadenosine. Deoxyadenosine was not detected (<0.01 nmole/ml) in normal adult urine. Because of its sensitivity and selectivity, this method of analysis should be applicable to studies of the physiological roles of adenine compounds.     

在线固相萃取–液相色谱法测定水中3种微囊藻毒素

建立了在线固相萃取–液相色谱法测定水中3种微囊藻毒素的方法。采用Acclaim PA Cartridge在线固相萃取小柱(10 mm×4.3 mm,5μm),以KH2PO4缓冲溶液–甲醇为富集流动相,进样体积为10 m L。以KH2PO4缓冲溶液–乙腈为洗脱流动相,采用二极管阵列检测器进行检测,检测波长为238 nm。微囊藻毒素MC–LR,MC–RR在0.1~10.0μg/L范围内线性关系良好,MC–YR线性范围则为0.1~5.0μg/L,相关系数r2不小于0.998,3种微囊藻毒素MC–LR,MC–RR,MC–YR的检出限均为0.1μg/L(S/N=3),水源水加标回收率为96.9%~105.4%,测定结果的相对标准偏差为3.55%~6.46%(n=12)。该方法适用于水源水及饮用水中3种微囊藻毒素的快速测定。     

Adducts of N-terminal valines in hemoglobin with isoprene diepoxide, a metabolite of isoprene

Isoprene (2-methylbuta-1,3-diene) is a multi-site carcinogen in rodents. To evaluate the role of the diepoxide metabolite (1,2:3,4-diepoxy-2-methylbutane) in carcinogenesis, measurements of in vivo doses of the diepoxide are needed. The in vivo dose may be inferred from levels of reaction products with hemoglobin (Hb adducts). This report presents in vitro studies of the adduct formation by the diepoxide of isoprene with valinamide and oligopeptides as model compounds of N-terminal valines in hemoglobin (Hb). In the reaction with valinamide it was shown that isoprene diepoxide forms as the main product a ring-closed adduct, which is a pyrrolidine derivative [N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valinamide, MPyr-Val]. The analysis was performed by gas chromatography/mass spectrometry (GC/MS) (EI and PICI) after acetylation. The ring-closed adduct was also identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) as the main product in the reaction between isoprene diepoxide and standard hepta- or (2H8)octapeptides, corresponding to the N-terminal peptides of the alpha-chains in mouse and rat Hb. These peptides, alkylated with isoprene diepoxide, to be used as internal standards and calibration standards for quantification of MPyr-adduct levels in vitro and in vivo, were analyzed with respect to the degree of MPyr-alkylation by two independent methods, amino acid analysis and HPLC-UV; similar results were obtained using these methods. A method for measurement of Hb adducts as modified peptides, used earlier to measure a similar adduct to N-terminal valines in Hb from the diepoxide of 1,3-butadiene, has in the present work been tested for application to isoprene diepoxide. The method is based on tryptic degradation of globin and LC/ESI-MS analysis of N-terminal Pyr-heptapeptides of the Hb alpha-chain enriched by HPLC. MPyr-adduct levels in isoprene diepoxide alkylated hemolysate from mouse erythrocytes incubated with different concentrations of isoprene diepoxide (2 and 10 mM) for 1 h were quantified. The adduct level was about 50 nmol/g alpha-chain Hb per mM x h. From the adduct levels the rate constant of isoprene diepoxide for reaction with N-terminal valine was calculated to be about 1.6 times faster than for diepoxybutane.     

胶束扫集毛细管电泳快速测定止咳露中的麻黄碱和可待因

采用胶束扫集毛细管电泳, 建立了快速测定止咳露中麻黄碱和可待因含量的方法, 并通过日间实验、柱间实验等对方法的稳定性进行了考察研究.胶束扫集电动色谱缓冲体系含60 mmol/L 十二烷基磺酸钠, 10 mmol/L NaH2PO4 (pH 2.20), 18%乙腈(V/V), 分离电压-14 kV, 测量波长200 nm. 讨论了pH、 SDS浓度、样品溶剂等对分离效果的影响. 在优化条件下, 麻黄碱和可待因均在5 min内出峰, 方法检出限(μg/mL)、线性范围(μg/mL)、相关系数分别为: 麻黄碱 0.433、 1.73～27.7、 0.9997, 可待因0.833、 3.33～50.3、 0.9996, 回收率在96.7%～103.5%之间. 峰面积日内RSD≤4.2% (n=5), 日间RSD≤8.0% (n=5), 柱间实验RSD≤2.3% (n=3).     

Analytical determination of specific 4,4′-methylene diphenyl diisocyanate hemoglobin adducts in human blood

4,4′-Methylene diphenyl diisocyanate (MDI) is one of the most important isocyanates in the industrial production of polyurethane and other MDI-based synthetics. Because of its high reactivity, it is known as a sensitizing agent, caused by protein adducts. Analysis of MDI is routinely done by determination of the nonspecific 4,4′-methylenedianiline as a marker for MDI exposure in urine and blood. Since several publications have reported specific adducts of MDI and albumin or hemoglobin, more information about their existence in humans is necessary. Specific adducts of MDI and hemoglobin were only reported in rats after high-dose MDI inhalation. The aim of this investigation was to detect the hemoglobin adduct 5-isopropyl-3-[4-(4-aminobenzyl)phenyl]hydantoin (ABP-Val-Hyd) in human blood for the first time. We found values up to 5.2 ng ABP-Val-Hyd/g globin (16 pmol/g) in blood samples of workers exposed to MDI. Because there was no information available about possible amounts of this specific MDI marker, the analytical method focused on optimal sensitivity and selectivity. Using gas chromatography–high-resolution mass spectrometry with negative chemical ionization, we achieved a detection limit of 0.02 ng ABP-Val-Hyd/g globin (0.062 pmol/g). The robustness of the method was confirmed by relative standard deviations between 3.0 and 9.8 %. Combined with a linear detection range up to 10 ng ABP-Val-Hyd/g globin (31 pmol/g), the enhanced precision parameter demonstrates that the method described is optimized for screening studies of the human population.     

离子色谱法测定饮用水中6种阴离子

建立生活饮用水中F~–,Cl~–,PO_3^(3–),PO_4^(3–),SO_3^(2–),SO_4^(2–)6种阴离子的离子色谱检测法。采用戴安DX–120型离子色谱仪及Ion Pac AS9–HC(250 mm×4.0 mm)阴离子交换柱,以5.0 mmo L/L Na_2CO_3–0.6 mmo L/L NaHCO_3溶液为淋洗液,流量为1.2 mL/min。6种阴离子的质量浓度与其色谱峰面积呈良好的线性关系,相关系数为0.999 3~0.999 8,检出限为0.026 4~0.741 4μg/mL(S/N=3)。水样的加标回收率为91.3%~110.0%,测定结果的相对标准偏差为1.65%~3.07%(n=6)。该方法具有操作简单,选择性、准确性好等优点,能够满足饮用水中F~–,Cl~–,PO_3^(3–),PO_4^(3–),SO_3^(2–),SO_4^(2–)6种阴离子的测定要求。     

Determination of the ultraviolet absorbance and radioactivity of purine compounds separated by high-performance liquid chromatography. Application to metabolic flux rate analysis.

A double detection system for the determination of adenine metabolism in biological tissues using isocratic ion-pair reversed-phase chromatography is presented. Two isocratic ion-pair separations were used: (i) 10 mM NH4H2PO4, 2 mM tetrabutylammonium phosphate (PIC reagent A) and 18% acetonitrile for the determination of nucleotides and (ii) 50 mM KH2PO4, 1 mM PIC reagent A and 1% acetonitrile for the determination of monophosphorylated nucleotides, nucleosides and nucleobases. The parallel detection of ultraviolet absorbance at 254 nm and the radioactivity of separated purine compounds allows the detection of pool sizes and of the specific radioactivities in tracer kinetic experiments. The high-performance liquid chromatography methods were applied to the determination of flux rates during adenine nucleotide metabolism in suspensions of Ehrlich mouse ascites tumour cells. The pathways of adenine metabolism in cells during the proliferation and plateau phases of tumour growth were compared.     

Separation and determination of aminohalogenbenzophenones by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection has been developed for the determination of three aminohalogenbenzophenones: 2-amino-2',5-dichlorobenzophenone, 2-amino-5-chlorobenzophenone and 2-amino-5-bromo-2'-fluorobenzophenone, metabolites of benzodiazepinooxazoles and other psychotropic drugs. A mobile phase of methanol-water (65:35), containing 5 mM KH2PO4 appeared to be the optimal when a 4-microns, 60-A Nova-Pak C18 column and a flow-rate of 0.75 ml/min (130 bar) were used. The temperature was optimized at 30 degrees C. The amperometric detector, equipped with glassy carbon electrode, was operated at 1.3 V versus Ag/AgCl in the DC mode. The method was applied to the determination of these compounds at two concentration levels: ppm and ppb (ng/cm3) using 2-amino-5-chlorobenzophenone as internal standard. The limit of determination was 750 pg/ml of biological fluid for each compound, and recoveries greater than 97% were obtained for spiked samples of urine and serum, using C18 Sep-Pak cartridges in the sample clean-up procedure.     

Analysis of hemoglobin adducts of acrylamide and glycidamide by liquid chromatography-electrospray ionization tandem mass spectrometry, as exposure biomarkers in French population

The determination of biomarkers of acrylamide exposure in humans from general French population by measurement of hemoglobin adduct levels of acrylamide (AA) and glycidamide (GA) is presented. The analytical procedure included modified Edman degradation and LC-ESI-MS/MS analysis of the final derivatives using deuterated internal standards. Method performances were evaluated in terms of linearity, precision, accuracy, and sensitivity. The method was firstly assessed on rat blood samples and then applied to the study of background adducts levels of AA and GA in 68 human hemoglobin samples, showing mean levels of 33 and 23 pmol/g globin for AA and GA adducts, respectively.     

Monitoring bronchodilators with direct injection

A procedure was developed for the determination of caffeine and theophylline using a C18 column (5 microm, 250 mm x 4.6 mm) and micellar liquid chromatography using hybrid mobile phases containing sodium dodecyl sulfate (SDS) and propanol, butanol or pentanol as modifiers. Detection was performed with a variable wavelength UV-vis detector at 272 nm. After the application of an interpretative strategy for the selection of the optimimum mobile phase, caffeine and theophylline can be resolved and determined in serum samples by direct injection, using a mobile phase made up of 50 mM SDS-2.5% (v/v) propanol-10 mM KH2PO4, pH 7, with an analysis time below 5 min. Calibration was linear in the range 0.05 to 50 microg mL(-1) with r > 0.999. The statistical evaluation of the method was examined by performing intra-day (n = 6) and inter-day calibration (n = 7) and was found to be satisfactory, with highly accurate and precise results. The proposed method was suitably validated and applied to the determination of caffeine and theophylline in serum samples of patients treated with bronchodilators.     

电堆积在线扫集胶束电动色谱法测定苦参中的生物碱

建立了电堆积在线扫集胶束电动色谱法测定苦参碱和槐定碱的新方法.考察了pH值、磷酸二氢钠浓度、CTAB浓度、电压、有机溶剂和进样时间对分离效果的影响.采用未涂层熔融石英毛细管,以20mmol/L磷酸二氢钠-0.8 mmol/L CTAB-100mmol/L Tris(含10%异丙醇,pH 8.9)为缓冲液,在进样电压-1...     

Mass fragmentographic determination of 5-hydroxytryptamine and 5-hydroxyindole-3-acetic acid in brain tissue using deuterated internal standards.

A mass fragmentographic method for the determination of 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in the same extract of rat brain tissue is decribed. Deuterium-labelled analogues were used as internal standards. 5-HT and 5-HIAA were separated by solvent extraction and pentafluoropropionyl derivatives were prepared for the mass fragmentographic analysis. Multiple ion analysis confirmed the identity of 5-HT and 5-HIAA in the rat brain. At the mass numbers used routinely for the determination of 5-HT and 5-HIAA in the rat brain. At the mass numbers used routinely for the determination of 5-HT and 5-HIAA, the experimental error was below 3% (calculated from mean values of 0.05 and 0.24 nmole, respectively). The recovery of the authentic compounds added to brain extracts was more than 95%. The levels of 5-HT and 5-HIAA in the rat brain were 2.95 +/- 0.16 and 0.64 +/- 0.18 nmole/g, respectively. More than 100 samples could be analyzed within 3 days. The presence of 5-hydroxytryptophol in rat brain was also investigated, but none could be detected either as a conjugate or as the free alcohol.     

Trace-level determination of triazines and several degradation products in environmental waters by disk solid-phase extraction and micellar electrokinetic chromatography

An analytical method combining disk solid-phase extraction with micellar electrokinetic chromatography has been developed for the determination of atrazine, simazine, hydroxyatrazine, deisopropylatrazine, deethylatrazine, propazine and prometryn in water samples. The influence of the buffer and sodium dodecyl sulfate (SDS) concentration, pH and organic modifier on the separation has been studied. Baseline separation of the seven triazines was achieved under the following conditions: 10 mM borate buffer, 60 mM SDS, 20% methanol and pH 9.2. C18-bonded silica and poly(styrene-divinylbenzene) (PS-DVB) disks were evaluated for solid-phase extraction of the selected pesticides (11 of water sample). Using two PS-DVB disks, quantitative recoveries were obtained for all pesticides tested. The method was successfully applied for the determination of the seven triazines in drinking and well water at the 0.1 microg l(-1) and 0.5 microg l(-1) concentration levels, respectively. The detection limits for these analytes using the proposed analytical method were within the 0.02-0.06 microg l(-1) range in drinking water and the 0.06-0.30 microg l(-1) range in well water.     

Sample stacking with matrix removal for the determination of paraquat, diquat and difenzoquat in water by capillary electrophoresis

Conditions for the simultaneous determination of paraquat, diquat and difenzoquat by capillary zone electrophoresis using a stacking technique in a chemically modified capillary have been established. To apply the stacking method with sample matrix removal for the analysis of cations, an anodic electroosmotic flow is mandatory. For quats, 50 mM acetic acid-ammonium acetate (pH 4.0) with 5% (v/v) methanol as electrophoretic buffer and the addition of 0.8 mM cetyltrimethylammonium bromide as wall capillary organic modifier was proposed. Field polarity reversal time was optimised for several sample matrices. Detection was carried out at 220 and 255 nm. Detection limits, based on a signal-to-noise ratio of 3:1, were lower than 15 microg l(-1) for standards in Milli-Q water and two to ten times higher for drinking water samples. Run-to-run and day-to-day reproducibility have been established. The method was successfully applied to the determination of the three herbicides in spiked drinking water.     

Investigation of a novel solid-phase chemiluminescent analytical system, incorporating photographic detection, for the measurement of glucose

A method has been developed for the rapid determination of substances by use of solid-phase reagents and a luminescence indicator reaction coupled with photographic detection of the light. The viability of the assay has been demonstrated for glucose estimations. The method uses small sample sizes (5-20 mul) and shows good sensitivity, e.g., detection of glucose down to 28 nmole.     

Analysis of baicalein, baicalin and wogonin in Scutellariae radix and its preparation by microemulsion electrokinetic chromatography with 1-butyl-3-methylimizolium tetrafluoborate ionic liquid as additive

Microemulsion electrokinetic chromatography (MEEKC) using 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) ionic liquid (IL) as additive was developed for the analysis of baicalin, wogonin and baicalein in Scutellariae radix and its preparation. After conducting a series of optimizations, baseline separation was obtained for the analytes within 5min under the optimum conditions (sodium dodecyl sulfate (SDS) 0.88% (m/v) ethyl acetate 0.8% (v/v) butan-1-ol 0.2% (v/v) and the buffer composition were 25% acetonitrile (v/v), 7.5 mM BMIM-BF4 and 10 mM NaH2PO4, pH 8.2, applied voltage 17.5 kV and detection at 254 nm), the method has been successfully applied to the determination and quantification of the analytes in the extracts of S. radix (cooked), S. radix (raw) and Qingfeiyihuowan which was the preparation including S. radix.     

Quantitation of caffeine by capillary zone electrophoresis with end-column amperometric detection at a carbon microdisk array electrode

Capillary zone electrophoresis is employed for the determination of caffeine using end-column amperometric detection with a carbon fiber microdisk array electrode at a constant potential of 1.45 V versus a saturated calomel electrode. The optimum conditions of separation and detection are 0.1 52mM NaH2PO4-0.648mM Na2HPO4 for the buffer solution, 20 kV for the separation voltage, 5 kV for the injection voltage, and 10s for the injection time. The limit of detection is 2.9 x 10(-4)mM or 1.2 fmol (signal-to-noise ratio = 2). The relative standard deviation is 0.68% for the migration time and 2.3% for the electrophoretic peak current. The method is applied to determining caffeine in human serum and a cola drink.     

Fluorimetric determination of kynurenine in human serum by high-performance liquid chromatography coupled with post-column photochemical reaction with hydrogen peroxide

A high-performance liquid chromatographic method involving post-column photochemical reaction and fluorimetric detection has been developed for the determination of kynurenine in serum. Kynurenine was separated on a column of Capcell Pak C18 (resistant to pH 10). The mobile phase consisted of 0.05 M Na2B4O7-0.1 M KH2PO4 buffer (pH 8.5)-ethanol (97:3, v/v) containing 60 mM hydrogen peroxide. The post-column reagent, containing 60% (v/v) ethanol, was mixed with the mobile phase, which was irradiated with ultraviolet light to induce fluorescence. The recovery of kynurenine was 95.9 +/- 5.0% (n = 6). The method allows the determination of as little as 2 pmol of kynurenine.     

Analysis of low‐molecular mass aldehydes in drinking waters through capillary electrophoresis with laser‐induced fluorescence detection

The potential of CZE with LIF detection in the separation and determination of low‐molecular mass aldehydes involving precolumn derivatization with fluorescein 5‐thiosemicarbazide was investigated. Different variables that affect derivatization (pH, fluorescein 5‐thiosemicarbazide concentration, time and temperature) and separation (pH and concentration of the BGE, kind and concentration of surfactants at levels higher and lower than CMC, and applied voltage) were studied. The separation was conducted within 16 min by using borate buffer (60 mM; pH 10) with 10 μM polyethylene glycol tert‐octylphenyl ether as modifier. Good linearity relationships (correlation coefficients ranged from 0.9978 to 0.9994 for aldehydes) were obtained between the peak areas and concentration of the analytes (0.5–100 μg/L). The LODs for aldehydes were achieved at submicrogram‐per‐liter level (0.15–0.35 μg/L), which indicated that the proposed method surpassed other electrophoretric alternatives in terms of LOD, in many cases even at ca. 1000‐fold. The inter‐day precision (RSD, %) of the aldehydes ranged from 5.2 to 8.3%. Finally, the method was successfully applied to bottled drinking‐water samples, and the aldehydes were readily detected at 0.6–4.4 μg/L levels with average recoveries ranging from 99.1 to 103.5%.     

Trace determination of perchlorate using electromembrane extraction and capillary electrophoresis with capacitively coupled contactless conductivity detection

Electromembrane extraction (EME) and CE with capacitively coupled contactless conductivity detection (CE‐C⁴D) was applied to rapid and sensitive determination of perchlorate in drinking water and environmental samples. Porous polypropylene hollow fiber impregnated with 1‐heptanol acted as a supported liquid membrane (SLM) and perchlorate was transported and preconcentrated in the fiber lumen on application of electric field. High selectivity of perchlorate determination and its baseline separation from major inorganic anions was achieved in CE‐C⁴D using background electrolyte solution consisting of 7.5 mM L ‐histidine and 40 mM acetic acid at pH 4.1. The analytical method showed excellent parameters in terms of reproducibility; RSD values for migration times and peak areas at a spiked concentration of 15 μg/L of perchlorate (US EPA recommended limit for drinking water) were below 0.2 and 8.7%, respectively, in all examined water samples. Linear calibration curves were obtained for perchlorate in the concentration range 1–100 μg/L (r²≥0.999) with limits of detection at 1 μg/L for tap water and at 0.25–0.35 μg/L for environmental and bottled potable water samples. Recoveries at 15 μg/L of perchlorate were between 95.9 and 106.7% with minimum and maximum recovery values for snow and bottled potable water samples, respectively.