Rapid assay of plasma acetate by gas chromatography: evaluation and comparison with two other methods

Human plasma acetate is derived from colonic fermentation of fiber and endogenous metabolism of dextrose and fatty acids. Acetate may have regulatory functions in hepatic carbohydrate metabolism. Intake of dietary fiber is associated with several beneficial effects on carbohydrates and lipids metabolisms. To study theses effects a valid and automated method for routine analysis of acetate in plasma is necessary. After oral administration of lactulose to healthy human volunteers, the concentration of plasma acetate was measured by head space gas chromatography (HS-GC), vacuum distillation gas chromatography (VD-GC) and enzymatic spectrometric method (ES). The method HS-GC was linear to 0.5 mmol.l-1 (n = 5, r = 0.998), the detection limit is 0.005 mmol.l-1. Within-day variation (CV) was 3.60% and day-to-day variation was 4.5% (0.1 mmol.l-1). The coefficients of correlation between CG-ET/CG-DsV and CG-ET/E-M are 0.903 (p = 0.0001) and 0.54 (p = 0.006) respectively, the mean square errors are respectively 0.118 and 0.138 mmol.l-1. The variation curves of plasma acetate measured by GC versus time show peak concentration of 0.323 to 0.380 mmol.l-1 at 120 min.     

GC/MS/MS quantification of 11-dehydrothromboxane B2 in human urine

A fully validated stable isotope dilution method for rapid quantification of 11-dehydrothromboxane B2 by gas chromatography/negative chemical ionization tandem mass spectrometry is reported. The interassay coefficient of variation was 1.3%. The relative recovery of synthetic, underivatized 11-dehydrothromboxane B2 added to urine was 84.6 +/- 2.5% (mean +/- SEM, N = 9). The equation of the regression plot correlating the amounts added and recovered was Y = 0.894X-0.23, with R = 0.9963. The late introduction of the pentafluorobenzyl moiety at C11 afforded distinct advantages in the cleanup stage of the assay and during the GC/MS analysis as well. The pair of daughter fragments at m/z 345 and 349 of the parent fragment ions at m/z 615 and 619 were monitored. Excretion values of 11-dehydrothromboxane B2 in 3 healthy male subjects were 975, 615 and 822 ng/24 h. Excretion was reduced to 751, 387 and 465 ng/24 h, respectively, after administration of a fish oil supplement (15 g/day) for 10 weeks.     

Detection and quantitation of multiple volatile compounds in tissues by GC and GC/MS

Qualitative and quantitative analysis of tissues and body fluids for multiple volatile organic compounds were performed by a combination of packed and open tubular capillary GC and GC/MS. This paper describes methods for such analyses in a case involving the exposure of two persons to methyl ethyl ketone, methyl isobutyl ketone, toluene and the three isomeric xylenes. Tissue and body fluid concentrations of these substances in the two decedents are presented and discussed briefly.     

Quantitative subfemtomole analysis of alpha-tocopherol and deuterated isotopomers in plasma using tabletop GC/MS/MS

A rapid, high-selectivity method with subfemtomole sensitivity is reported for quantification of alpha-tocopherol in plasma-based gas chromatography/tandem mass spectrometry (GC/MS/MS) using a tabletop quadrupole ion trap mass spectrometer. Sample workup is rapid, consisting of protein precipitation followed by liquid/liquid extraction and O-trimethylsilyl derivatization of alpha-tocopherol (alpha-T-TMS) and an internal standard, 2,2,5,7,8-pentamethyl-6-chromanol (PC-TMS). Rudimentary chromatography was carried out using an 8-m DB-5 capillary column resulting in an analyte retention time of 7.2 min. No interferences from the plasma matrix were observed. The assay has a detection limit of 178 amol (89.6 fg) and a lower limit of quantification of 700 amol (350 fg) of derivatized alpha-tocopherol in diluted plasma; < 30 pL of plasma is estimated to yield sufficient alpha-tocopherol for quantitative analysis at typical concentrations found in humans. A calibration curve constructed from National Institute of Standards and Technology serum standards was linear in the working range of 1.9-1073 ng/mL (0.95-0.54 ng). Within- and between-day precision averaged 5.8% and did not exceed 11.3% for three concentrations of quality control (QC) solutions. The overall accuracy for the QC samples was within 7.2%. Storage studies showed that, alpha-T-TMS and PC-TMS are stable under conditions that might be encountered during analyses. In a test study, plasma kinetic curves for alpha-tocopherol-d6 and alpha-tocopherol-d3 were obtained for a catheterized pregnant ewe and her fetus who were simultaneously given a bolus injection of alpha-tocopherol-d6, to the ewe and alpha-tocopherol-d3 to the fetus. These data show that a tabletop GC ion trap can determine alpha-T-TMS and its isotopomers quantitatively at high selectivity in a complex matrix.     

Improved GC/MS analysis of opiates with use of oxime-TMS derivatives

An improved gas chromatographic/mass spectrometric (GC/MS) assay is described for the quantitation of codeine and morphine as trimethylsyl (TMS) derivatives. The TMS derivatization of ketone-containing opiates results in the formation of multiple derivatives. Some of these products have retention times close to those of codeine-TMS and morphine-TMS. When the keto-opiates are present in samples assayed for codeine and morphine in urine, they can interfere with the quantitation of these commonly targeted opiates. The assay was improved with the addition of a pre-BSTFA derivatization step, whereby hydroxylamine was used to convert the keto-opiates into the corresponding oxime derivative. These derivatives were then reacted with BSTFA to form the TMS ethers and TMS oxime derivatives. The oxime step enabled production of single derivatives for hydrocodone and hydromorphone. In addition, the retention times for the oxime-TMS derivatives were increased so that they no longer elute near the targeted drugs of codeine and morphine. The addition of the oxime step does not affect the sylation of codeine and morphine, and the accuracy and precision of this assay were unaffected.     

Establishment of microanalysis of prostaglandin metabolites by GC/MS and its clinical application

We established microdetermination methods of prostaglandin (PG) metabolites by GC-selected ion monitoring (GC-SIM) and applied them to the clinical investigations. At first the microdetermination of delta 17-6-keto-PGF1 alpha, a hydrolyzed metabolite of PGI2, is described. An authentic delta 17-6-keto-PGF1 alpha was prepared from eicosapentaenoic acid (EPA) incubated with a homogenate from the bovine aortic intima. [18O] delta 17-6-Keto-PGF1 alpha was synthesized to obtain an internal standard for GC-SIM of delta 17-6-keto-PGF1 alpha. A good linear response over the range of 10 pg-5 ng was demonstrated. Chromatographic conditions using a MP-65HT column presented nearly baseline separation of delta 17-6-keto-PGF1 alpha and 6-keto-PGF1 alpha. Furthermore, a monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate delta 17-6-keto-PGF1 alpha in the human blood sera. Using the prepared immunoaffinity columns of this antibody, delta 17-6-keto-PGF1 alpha was clearly detected in the human blood sera by GC/MS analysis. We were able to detect delta 17-6-keto-PGF1 alpha of the amount ranging from 6 to 26 pg/ml in the human blood plasma. The present method can be applied to the determination of delta 17-6-keto-PGF1 alpha in the human urine and plasma. Diabetes mellitus induces platelet alterations such as hyperaggregation. Variations in PG production seem to be related to this phenomenon but the changes in PG levels remain unclear. So we microanalyzed the 11-dehydrothromboxane B2 (TXB2) and 2,3-dinor-6-keto-PGF1 alpha, which were stable metabolites of TXA2 and PGI2, in the urine and investigated the relationship between the thromboxane/prostacyclin (TX/PGI) ratio and diabetes mellitus. The TX/PGI ratio in the urine of diabetics was higher than that of healthy volunteers. In murine, the TX/PGI ratio of STZ-induced mice was also higher than that of non-induced mice. The ratio of db/db mice also increased with the progress of diabetes mellitus. Furthermore, we investigated the relationship between the retinal vein occlusion (RVO), a thrombotic disease in which the retinal vein is blocked by blood aggregations, and the TX/PGI ratio. The TX/PGI level in patients with the RVO, who were not combine diabetes, was significantly higher than that in healthy volunteers. One of the causes of the RVO may be due to the variation of thromboxane production. This GC-SIM method can be used to determine the TX/PGI ratio in the urine.     

Theoretical evaluation and comparison of fast chemical shift imaging methods

The effects of chemical (CD) and surgical (SD) deafferentation of the cerebellum on different steps of the metabolism of thiamine (Th), thiamine monophosphate (ThMP) and thiamine pyrophosphate (ThPP) were evaluated in vivo in rats. CD was carried out by i.p. injection of 3-acetylpyridine, followed by harmaline and niacinamide. SD was carried out by complete dissection of the peduncles of the left cerebellar hemisphere. Under steady state condition the radioactivity of Th and its phosphoesters was determined in plasma and whole cerebellum after an i.p. injection of thiazole-[2(14)C]-thiamine (30 micrograms:1.25 micro Ci). Analytical data were processed by using an improved mathematical compartmental model, which allowed the calculation of fractional rate constants (FRC), turnover rates (TR) and turnover times (TT). Both CD and SD caused a significant reduction of TR values for Th phosphorylation to ThPP, dephosphorylation of ThPP to ThMP and Th, and ThMP, but not Th, release. TT for all Th compounds were increased compared to controls, indicating a general slowing of thiamine metabolism in the deafferented cerebellum. These results indicate an imbalance in the thiamine metabolism resulting from the impaired activity of cerebellar neurons. The possible implications of the changes in rate of Th compound turnover with respect to biochemical changes in cerebellar ataxia are discussed.     

Influence of sample preparation on analytical results: drug analysis [GC/MS] on hair snippets versus hair powder using various extraction methods

We investigate the nucleotide sequences of 23 retroelements (4 mammalian retroviruses, 1 human, 3 yeast, 2 plant, and 13 invertebrate retrotransposons) in terms of their oligonucleotide composition in order to address the problem of relationship between retrotransposons and retroviruses, and the coadaptation of these retroelements to their host genomes. We have identified by computer analysis over-represented 3-through 6-mers in each sequence. Our results indicate retrotransposons are heterogeneous in contrast to retroviruses, suggesting different modes of evolution by slippage-like mechanisms. Moreover, we have calculated the Observed/Expected number ratio for each of the 256 tetramers and analysed the data using a multivariate approach. The tetramer composition of retroelement sequences appears to be influenced by host genomic factors like methylase activity.     

Automated analysis of phencyclidine in urine by probability based matching GC/MS

A novel form of selected ion recording mass spectrometry using a microcomputer-managed mass spectrometer was employed to automatically identify and quantitate phencyclidine (PCP) in cyclohexane extracts of urine by Probability Based Matching. Seventy urine samples from known abusers were assayed for PCP content. The positively identified PCP concentrations ranged from 0.01 to 10.5 mug/ml for 65 samples, 26% of which fell in the 0.35-1.0 mug/ml range and 30% in the 1.0-3.4 mug/ml range. Five specimens had no detectable PCP (less than 10 mug/ml). Cyclohexane extraction efficiency for PCP in urine exceeded 95%. Selected ion monitoring was found to be necessary in order to avoid gas chromatographic interferences produced by co-elution of contaminants at the same retention time as PCP.     

Influence of acute renal failure on FPIA rapid serum assay of midazolam and its main metabolite

We recently developed a simple and fast assay technique, providing the possibility of monitoring of midazolam (M) during sedation. We compared HPLC vs FPIA for the measurement of the sum M plus alpha 1-hydroxymidazolam (OM), its main and pharmacologically active metabolite, in the serum of sedated ICU patients; this activity referred to as M-like. We identified certain patients in whom M-like activity appeared abnormally high in comparison with HPLC assays. Their common denominators were: long-term sedation with M, and seriously impaired renal function. Further, the conjugates of OM (OMG) accumulated in patients with acute renal failure could contribute to the sedation. We compared the metabolic and analytic behavior of M, OM, and OMG in 2 groups of sedated patients either presenting with normal renal functions (group 1) or with a picture of acute renal failure (group 2). Blood samples were assayed by HPLC and by FPIA and analysis was performed before and after hydrolysis of OMG. Before hydrolysis there was a dramatic accumulation of OMG in the patients of group 2, HPLC vs FPIA results were not different within group 1, while in group 2 the FPIA response exceeded that of HPLC. After hydrolysis, measurement by HPLC was greatly increased in group 2, in each group (vs HPLC) and from one group to another, the FPIA signal (the M-like activity) showed a significant increase. It would be important to take OMG into account as a coprotagonist in sedation whenever circumstances predispose to its accumulation.     

Rat brain beta-galactosidases: observations on cerebroside-beta-galactosidase catalysed transgalactosylation and comparison of assay methods for sphingolipid hydrolysis

Report is given of cryobiological principles, technique and results of cryotherapy for eyelid or conjunctival tumors in 181 cases. In the majority of cases they were basaliomas. In all cases the therapy was successful. No eyelid contractions or tear duct stenosis were noted. The follow-up of 4,5 years showed only 2 recidives. Histologically, cryonecrosis showed capillary ectasis with hyperemia.     

Job applicant training and work experience evaluation: An empirical comparison of four methods.

Assessed the reliability, quasi validity, and scoring time required for each of 4 training and experience (TAE) evaluation methods for 1st-line supervisory positions in 3 substantially different occupational areas. The TAE scoring procedures were applied to application forms by 4 undergraduates who received formal training regarding TAE methods. Ss were assigned to pairs, and application packets for each of 3 target jobs (TJs) were randomly split into 2 groups of equal size and distributed to Ss, so that each pair rated half the application forms from each of the TJs. Results indicate that the greatest amount of variation was obtained for the behavioral consistency method, the 2nd most for the task-based method (TBM), the 3rd most for the point method, and the least for the grouping method (GM). The TBM yielded significantly higher reliability coefficients, as compared with the other 3 methods, and the GM yielded significantly lower reliability coefficients. However, the TBM indicated high interrater reliability coefficients due to the straightforward nature of the scoring procedure. (13 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

GC/MS分析铝箔轧制基础油组成

用GC／MS技术分析铝箔轧制基础油,得到了基础油的组成成分。并提出了采用计算机检索和参考标准物质的相对保留时间,结合各物质的物理化学性质的方法来解析石油产品色谱图。     

A comparison of two methods of evaluation of teat skin pathology

A split-plot design with repeated measures was used to test the relationship between visual teat skin condition score, the degree of transepidermal water loss from the skin, and the colonization by Staphylococcus aureus on experimentally chapped and inoculated teats of 20 lactating Holstein cows. Visual teat skin chapping score and the number of S. aureus colonies obtained from a teat skin swab were correlated (r = 0.53). Transepidermal water loss and S. aureus count were not correlated (r = 0.02). Results indicated that visual teat skin evaluation is superior to measurements of transepidermal water loss in the prediction of the susceptibility of teat skin to colonization by S. aureus.     

Evaluation of assay methods of glycohemoglobin: reference method and parameter

In this paper, the characteristics of the analytical procedures allowing the measurement of glycated hemoglobin products are presented. With respect to their respective performances, the authors recommend the specific measurement of HbA1c and the more reliable procedure, high pressure liquid chromatography (HPLC).     

In vitro evaluation of cell/biomaterial interaction by MTT assay

The nonlinear propagation of ultrasound was demonstrated using Doppler transducers on two commercial duplex machines. The influence of nonlinear propagation on Doppler measurements was studied on both a flow phantom and a string phantom. It was found that although the pulse waveforms showed clearly different degrees of nonlinear distortion, no effect due to the nonlinearity could be detected on the received Doppler spectrum both in terms of the maximum frequency or underlying Doppler spectral profile.