Rapid alcohol determination in plasma and urine by column liquid chromatography with biosensor detection

An enzyme based amperometric biosensor used as a selective and sensitive detection unit in column liquid chromatography for the determination of ethanol and methanol in biological fluids such as plasma and urine is described. The reagentless enzyme electrode is based on the co-immobilisation of alcohol oxidase and horseradish peroxidase in carbon paste. The selectivity of the biosensor was found to vary when four various alcohol oxidase enzyme preparations from Candida boidinii, Pichia pastoris, and Hansenula polymorpha were used in the biosensors described. High sensitivity could be obtained for a number of alcohols, organic acids, and aldehydes. Optimisation regarding the sensitivity and selectivity of the four alcohol oxidase co-immobilised biosensors are outlined. A fast and reliable liquid chromatographic separation system with a PLRP-S polymer based separation column used with a phosphate buffer as the mobile phase was optimised using the best biosensor which was based on alcohol oxidase from P. pastoris and which showed the highest turnover rate for alcohols, as the detector for the determination of ethanol and methanol in human urine and plasma samples. The selectivity and stability of the biosensor were retained by working at an applied potential of -50 mV versus Ag/AgCl, the optimal operational potential, and by the casting of a protective membrane on the electrode surface. High selectivity of the enzyme electrode was also found towards other easily oxidisable interfering species normally present in biological fluids. It was found that stable and reliable determinations of ethanol and methanol in plasma and urine could be performed with only a simple dilution and centrifugation step prior to injection into the liquid chromatographic system. An analysis time of 4 min was required for the assay, with a sample throughput of 13 samples h(-1).     

Automated determination of hypoxanthine and xanthine in urine by high-performance liquid chromatography with column switching

We report a high-performance liquid chromatographic method with column switching for urinary hypoxanthine and xanthine. Analyses were carried out with both a reversed-phase column and an anion-exchange column connected by a column switch and controlled automatically by a computerized system controller. The relationships between standard concentrations and peak heights were linear in a concentration range of 1 to 1000 nmol/ml. The recovery of hypoxanthine added to urine was 101.1%, and that of xanthine was 98.1%. With our method urinary hypoxanthine and xanthine can be measured accurately without any sample preparation other than filtration.     

毛细管柱气相色谱法测定循环洗油中萘

采用DB-1毛细管柱和FID检测器,以毛细管柱气相色谱法测定循环洗油中的萘含量。研究发现:以正己烷为溶剂,在汽化温度为270℃,检测温度为250℃的条件下,洗油中的萘与其他组分完全分开。萘含量在0~10%范围内与峰面积呈良好的线性关系,相关系数为0.9992。方法的检出限为0.015%。考察了方法的准确度和精密度,其相对标准偏差≤3.6%,标准加入回收率在98%~105%之间。外标法定量分析洗油中的萘含量,测定结果允许误差满足国家标准要求。     

Selective and sensitive determination of pamidronate in human plasma and urine by gas chromatography with flame photometric detection

A rapid, selective and sensitive method for the determination of pamidronate in human plasma and urine samples by gas chromatography (GC) has been developed. After deproteinization of the sample with trichloroacetic acid, pamidronate was converted into its N-isobutoxycarbonyl methyl ester derivative and measured by GC with flame photometric detection (FPD), using a HP-1 capillary column. The derivative preparation and GC analysis were accomplished within 30 min. The derivative was sufficiently volatile and stable, giving a single and symmetrical peak, and provided an excellent FPD response. The detection limit of pamidronate, at a signal-to-noise ratio of 3, was ca. 100 pg injected, and the calibration curve for this compound in the range 20-1000 ng was linear and sufficiently reproducible for quantitative determination. This method could be successfully applied to plasma and urine samples without a preliminary clean-up except for deproteinization with trichloroacetic acid, and pamidronate could be measured without any influence from coexisting substances. Overall recoveries of pamidronate added to plasma and urine samples were 93-97%. The coefficients of variation for intra-assay and inter-assay of pamidronate in these samples were 1.0-7.9% (n = 3) and 4.1-8.3% (n = 6), respectively.     

Rapid determination of cholesterol in milk and milk products by direct saponification and capillary gas chromatography

The level and nature of the albendazole residues in milk of treated cows were determined as a function of the time of milking (12-h intervals), and the fate of those residues during cheesemaking, ripening, and storage was examined when the obtained milk was used for making Teleme cheese. Ion-pair liquid chromatographic analysis with fluorescence detection showed that the albendazole sulfoxide metabolite reached its maximum (523 +/- 199 micrograms/kg) at the 1st milking and declined below the detection limit by the 4th milking. The sulfone metabolite attained its highest level (812 +/- 99 micrograms/kg) more slowly (at the 2nd milking) and declined below detection limit by the 13th milking. The 2-aminosulfone metabolite, which was present in the milk obtained at the 1st milking, reached its maximum (128 +/- 36 micrograms/kg) at the 3rd milking, and slowly declined to a level below detection limit by the 15th milking. Whey and cheese analysis revealed that about 70% of each major metabolite initially present in milk could be distributed in the whey. The remaining 30% occurred in the cheese at residue levels higher than those initially present in the milk of the 1st or 2nd milking (688 versus 445 or 450 versus 230 micrograms/kg for albendazole sulfoxide; 890 versus 608 or 1502 versus 783 micrograms/kg for albendazole sulfone; 19 versus 15 or 161 versus 105 micrograms/kg for albendazole 2-aminosulfone). Ripening and storage of the cheeses made from milks from the 1st or 2nd milkings results in a decrease of the sulfoxide metabolite (to 225 or 206 micrograms/kg), an increase of the sulfone metabolite (to 1,181 or 1,893 micrograms/kg), and no effect on the 2-aminosulfone metabolite.     

Procedure for the determination of eight cholesterol oxides in poultry meat using on-column and solvent venting capillary gas chromatography

A procedure for the determination of eight relevant cholesterol oxides in poultry meat has been developed. The method consists of the enrichment of cholesterol oxides by means of the combined use of solid-phase fractionation and thin-layer chromatography. Florisil and silica columns of 10 g permitted the handling of the total cholesterol oxides content included in the lipid bulk obtained after the Folch's extraction of 20 g of muscle meat. The determination of cholesterol oxides under their trimethylsilyl derivatives was performed by using capillary gas chromatography. The use of a fused-silica open tubular capillary column 30 m x 0.25 mm I.D. coated with 5% phenylmethylsilicone and with a film width of 0.25 micron permitted the separation of all the species. Two modes of injection (on-column and solvent venting) were evaluated and compared for the analysis of cholesterol oxides. On-column capillary gas chromatography (cGC) gave better absolute areas relative standard deviation (R.S.D.) values: 3% to 6% vs. 5% to 7% for solvent venting cGC. Regression analysis for each cholesterol oxide was performed for the two modes of injection. The possibility of large volume injection (10 microliters) by using the solvent venting mode was also evaluated in order to increase the sensitivity of the detection of cholesterol oxides. R.S.D. values for absolute areas ranging from 6% to 14% were obtained. The validation of the method was carried out within the range of 0.1-1 ppm. Absolute and relative recovery values ranging from 80% to 100% were obtained. Statistical analysis revealed that the method was reproducible. cGC-mass spectrometry was also used to confirm the peaks detected by cGC: the total ion chromatogram mode was used for the analysis of samples containing concentrations down to 0.1 ppm of cholesterol oxides. The analysis of fresh and cooked chicken meat revealed the presence of cholesterol oxides proceeding from the autoxidation of the cholesterol B-ring. Finally, saponification was found to be not as accurate as the described procedure for cholesterol oxides analysis.     

A convenient derivatization method for gas chromatography/mass spectrometric determination of phenmetrazine in urine using 2,2,2-trichloroethyl chloroformate

The present experiment examined the effects of dopamine receptor antagonism on subjects' motivation to seek food. Rats were trained to discriminate between 2 olfactory cues predicting either the presence (S+) or absence (S-) of food reinforcement in the goal box of a straight-arm runway. Rats learned to traverse the alley quickly when presented with the S+ and much more slowly when presented with the S-. Haloperidol pretreatment was unable to alter this pattern of behavior (i.e., rats still ran quickly when presented with the scent that predicted food availability). Thus, it seems that the same dopamine antagonist treatments that have been shown to disrupt food reinforcement do not prevent the food-seeking behavior produced by presentation of food-predictive cues.     

The determination of trifluoroacetic acid in rat milk samples by 19F-NMR spectroscopy and capillary gas chromatography

In patients with early-stage squamous cell cancer of the vulva, inguinofemoral lymphadenectomy is performed primarily as a diagnostic procedure. The morbidity of this procedure, however, is not negligible. The aim of this study was to evaluate the feasibility of minimally invasive detection of the sentinel inguinofemoral lymph node (SILN) and to investigate whether the histopathology of the SILNs is representative of that of the other non-SILNs. METHODS: Patients with early-stage squamous cell cancer of the vulva, planned for resection of the primary tumor and uni- or bilateral inguinofemoral lymphadenectomy, were eligible for the study. Technetium-99m-labeled nanocolloid was injected intradermally at four locations around the tumor the day before operation. Images were recorded immediately and after 2.5 hr using a gamma camera. SILN locations were marked on the overlying groin skin. The next day, during general anesthesia, blue patent dye was injected intradermally at the same locations around the tumor. During the operation SILNs were identified at the place indicated using a handheld gamma-detection probe. It was noted if SILNs were found by the probe, by blue dye or by both techniques. After resection of the SILNs, a standard inguinofemoral lymphadenectomy was performed. The results of histopathology of the SILNs were compared with those of the non-SILNs. RESULTS: The procedure was well tolerated by 10 of 11 patients. One patient, initially agreeing to participate, refused the injection of tracer because of fear of pain. In all 10 patients, identification of the SILNs was successful. The mean time for identification was 11 min. Identification of SILNs was primarily performed using the hand probe in all patients, whereas in 10 of 18 removed SILNs afferent lymph channels were also blue stained (56%). In 8 patients, pathologic examination showed no metastatic disease in both SILNs and non-SILNs, whereas in 2 patients metastases in the SILNs (one and two metastatic lymph nodes, respectively), as well as in other non-SILNs, were found. CONCLUSION: This study shows that identification of SILNs in squamous cell cancer of the vulva is feasible with preoperatively administered 99mTc-labeled nanocolloid. Intraoperatively administered blue dye was only useful for confirmation of identification with nanocolloid. To date, no false-negative SILNs have been found, but expansion of the study is necessary to determine the possible clinical application of this new diagnostic technique.     

Separation and detection of phencyclidine in urine by gas chromatography

A rapid, sensitive and selective analytical procedure for phencyclidine and one of its metabolites in urine has bee developed. Three techniques have been studied for extraction of the drug from the biological matrix: (a) reversed-phase XAD resin, (b) charcoal absorption, and (c) solvent extraction using chloroform. Temperature-programmed gas chromatography was used to quantitate the illicit drug. Solvent extraction appears to offer the most efficient separation of the drug and its metabolite, as the recovery was 94% and the technique required only 7-8 min per sample. Reversedphase column extraction is also quite useful; although more time-consuming for an indivisual sample, it would be useful for screening purposes.     

Automated urinary steroid profiles by capillary column gas-liquid chromatography and a computing integrator

13 Urinary steroid metabolites have been determined by automated gas-liquid chromatography with a 20-m glass capillary column and a computing integrator. Concentrations up to 2 mg/24 h computed by the integrator compare well with those obtained by peak height measurements. At higher concentrations discrepancies occurred, paticularly for the C21 steroids where falsely low values were calculated using peak heights. Mean excretion by healthy males and females of seven steroid metabolities is presented.     

Determination of triazolam in serum by deactivated metal capillary gas chromatography with electron-capture detection

A method for the determination of trace amounts of triazolam in serum by deactivated metal capillary gas chromatography with electron-capture detection was established. The column used exhibits excellent thermostability in high-temperature analysis and easy handling and a long lifetime of the column and well shaped peaks on the chromatograms are obtained. With the metal capillary column, it was found to be easier to maintain suitable analytical conditions for the routine assay of triazolam than with a fused-silica column. With this method, 0.5 ng/ml of triazolam in serum can be determined. The method is useful for pharmacokinetic and therapeutic purposes.     

Determination of cyanide in whole blood by capillary gas chromatography with cryogenic oven trapping

Cyanide, one of the most important toxic substances, has been found measurable with high sensitivity by capillary gas chromatography (GC) with cryogenic oven trapping upon injection of headspace (HS) vapor samples. The entire amount of cyanide in the HS sample could be cryogenically trapped prior to on-line GC analysis. A 0.5-mL volume of blood in the presence or absence of cyanide and propionitrile (internal standard, IS) was added to a vial containing 0.25 mL of distilled water, 0.3 g of Na2-SO4, 0.2 mL of 50% H3PO4, and 0.1 g of ascorbic acid (when needed), and the mixture was heated at 70 degrees C for 15 min. A 5-mL volume of the HS vapor was introduced into a GC capillary column in the splitless mode at -30 degrees C oven temperature that was programmed up to 160 degrees C for GC analysis with nitrogen-phosphorus detection. A sharp peak was obtained for cyanide under the present conditions, and backgrounds were very clean. The extraction efficiencies of cyanide and IS were 2.89-3.22 (100 or 500 ng/mL) and 2.42%, respectively. The calibration curve showed good linearity in the range of 25-1000 ng/mL and the detection limit was approximately 2 ng/mL. The coefficients of intraday and interday variations were 2.9 and 11.8%, respectively. The mean blood cyanide level measured for actual fire victims was 687 +/- 597 ng/mL (mean +/- SD, n = 9). Endogenous blood cyanide concentration for healthy subjects was 8.41 +/- 3.09 ng/mL (mean +/- SD, n = 6).     

惰气熔融-红外法测定钛毛细管中氧含量的制样方法

钛毛细管是一种形状特别的钛合金,由于其内径常小于1 mm,管壁厚度不足1 mm,表面积较大易被污染,而且钛毛细管在加工过程中表面很容易因发热氧化,因此在进行钛毛细管氧含量测定时表面油污及氧化层的处理尤为重要.试验中选择依次使用HNO3-HF混合酸、蒸馏水、丙酮于超声波清洗仪中处理试样,使用惰气熔融-红外法进行了精密度和加标回收试验,结果满意.      

Evaluation of capillary zone electrophoresis and micellar electrokinetic capillary chromatography with direct injection of plasma for the determination of cefotaxime and its metabolite

Quantitative aspects of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were investigated for the determination of cefotaxime (C) and its deacetyl metabolite (DA) in human plasma in a concentration range of therapeutic interest. For CZE, plasma samples spiked with C and DA were injected after deproteinization with acetonitrile, and analytes were separated in a fused silica capillary using a borate buffer at pH 9.2 as electrolyte; no suitable internal standard was found. For MECC, plasma samples spiked with C, DA, and theobromine as internal standard were directly injected after dilution with water and analyzed using a phosphate buffer, pH 8.00, containing 165 mM SDS as separation electrolyte and a fused silica capillary. Both methods gave satisfactory interday precision with respect to migration times (RSD < 1%) and gave linear responses over the concentration ranges investigated (5-100 mg L-1 C and 5-20 mg L-1 DA). For CZE, intraday RSD (n = 4 graphs) between the slopes of the calibration graphs was acceptable (5.7%) for C. The corresponding figures for interday precision (n = 4 days) were fair (16.1%) in comparison to those obtained with MECC, for which the RSD was 1.49% when theobromine was used as internal standard. A satisfactory interday precision between slopes was also obtained with MECC even without the use of an internal standard (RSD = 4.38%), which demonstrated the ruggedness of this method. Detection limits (S/N = 3) were about 2 mg L-1 (CZE) and 1 mg L-1 in plasma (MECC) for C and DA. MECC was shown to be superior with regard to simplicity, rapidity, precision, and sensitivity.     

毛细管柱气相色谱法测定煤焦油中萘

研究了毛细管柱气相色谱法测定煤焦油中萘含量的方法。正辛烷和对四甲基苯分别用作样品的溶剂和内标物,采用内标法定量。对汽化温度、检测温度、气体流速等进行了探讨。方法用于煤焦油中萘含量的分析,标准加入回收率在97.4%~100.4%之间。该法与国家标准方法相比,样品分离效果好,结果准确度高,满足了产品质量检验准确、快速的要求。     

A method for the estimation of 2-phenylethylamine in human urine by gas chromatography

The fine structure of the retinal epithelium has been studied by electron microscopy in the opossum (Didelphis virginiana). The retinal epithelium, over most of the retina, is typical of that in other vertebrates and consists of a single layer of heavily pigmented, cuboidal cells. These cells display extensive basal (scleral) infoldings and numerous apical (vitreal) processes which enclose photoreceptor outer segments. A semicircular area of the retinal epithelium in the superior fundus is further specialized as a tapetum lucidum. The reflecting material consists of a large quantity of lipoidal spheres scattered throughout the epithelial cells. Centrally in the tapetal area very few or no melanosomes are found, indicating a non-occlusible tapetum. Peripherally in be tapetum, the epithelial cells contain both reflecting material and melanosomes. As in the non-tapetal area, the epithelial cells of the tapetum display large amounts of smooth endoplasmic reticulum and numerous mitochondria. Bruch's membrane everywhere displays the usual pentalaminate structure described for most vertebrates. The choriocapillaris is also typical, in that numerous fenestrations are present in the endothelium bordering Bruch's membrane.