Automatic on-line fermentation headspace gas analysis using a computer-controlled gas chromatograph

A computer-controlled headspace gas chromatograph was used to monitor the progress of ethanol production from both aerobic batch and anaerobic continuous fermentations. Using an automatic, electropneumatic sampling system, aliquots of fermentation headspace gas were injected directly onto the column for quantitative ethanol determinations every six minutes. A sample volume of 1 mL permitted liquid ethanol concentrations from 2 to 100 g/L to be measured with better than 3% standard deviation on five repeated injections. Provided fermenter liquid temperature and ionic strength were maintained constant, the signal-tohyphen;concentration ratio remained linear to 80 g/L ethanol. This quantitative gas chromatographic (GC) method is suitable for accurate, precise analysis of multiple solvent fermentations, and is limited only by the elution rate and separating capacity of the GC column.     

Application and evaluation of limited mass monitoring with a gas chromatograph mass spectrometer computer system

The technique of scanning a preselected set of ions employing a combined gas chromatography mass spectrometer computer system has been investigated to ascertain the advantages and disadvantages of such a procedure. This technique allows one to determine gas chromatographic retention data with with a high degree of precision and accuracy, in rapid temperature programming operation, due to shortening of the mas spectral scanning interval. Signal-to-noise ratio in ion abundance recordings can be enhanced by increasing the dwell time for as many as 100 ions without lenghtening the scanning interval. The utility of such an approach was demonstrated by analysis of complex mixtures isolated form human urine and cerebrospinal fluid.     

Determination of pyrimidine dimers in DNA by high-performance liquid chromatography/gas chromatography and electron capture detection

Exposure of DNA to uv radiation results in the formation of a number of photoproducts including the cyclobutyl pyrimidine dimers. At low uv fluences the concentrations of these dimeric compounds are only a small fraction of the corresponding DNA pyrimidine concentration (e.g., as low as 0.02% or less of the total thymine content). Sensitive methods of analysis are therefore required for accurate determinations. Analytical methodology based upon HPLC fractionation and electrophore labeling followed by GC/electron capture detection (ECD) has been developed to quantitate these species. Separation of thymine-thymine, thymine-uracil, and uracil-uracil from the monomeric bases and from other constituents present in acid-hydrolyzed DNA is achieved by reversed-phase HPLC. Isolation of the dimeric fractions is followed by off-line derivatization to form pentafluorobenzyl products for analysis by GC/ECD. All active hydrogens are alkylated, yielding products with high response factors and detection limits in the low femtomole range. The overall analytical scheme for the determination of pyrimidine dimers in DNA is presented.     

Design for an accurate and versatile radio gas chromatograph

A convenient method is presented for quantitating disulfide interchange reactions based on the use of a commercially available organomercurial resin. Isotopically labeled thiol is incubated with a disulfide and the reaction is terminated by charging the incubation mixture onto the resin. The thiol is bound as the mercaptide allowing labeled interchange products, as well as the unlabeled interchanges substrate, to be eluted for quantitation. In principle the method is applicable to all monothiol and disulfide pairs and may be more generally applied to other thiol-transfer reactions that result in conjugation of the thiol. Examples of the use of the technique for glutathione transferase-catalyzed reactions are presented.     

Cellulose/water: liquid/gas and liquid/liquid phase equilibria and their consistent modeling

Liquid/liquid and liquid/gas equilibria were measured for the water/cellulose system at 80 degrees C using three different polymer samples. For these experiments we prepared cellulose films of approximately 20-25 microm in thickness and determined their equilibrium swelling in water. Thereafter the polymer concentration in the mixed phase was increased by means of a stepwise removal of the volatile component, and the equilibrium vapor pressures were measured using an automated combination of head space sampling and gas chromatography. Contrary to the usual behavior of polymers, the swelling of cellulose increases as its molar mass becomes larger. The Flory-Huggins interaction parameters calculated from the measured vapor pressures pass a pronounced minimum as a function of composition; for high cellulose contents they are negative, whereas they become positive for water-rich mixtures. All experimental findings are consistently interpreted by means of an approach accounting explicitly for the effects of chain connectivity and for the ability of macromolecules to respond to environmental changes by conformational rearrangement.     

Fully automated high-performance liquid chromatography : A new chromatograph for pharmacokinetic drug monitoring by direct injection of body fluids

A new fully automated high-performance liquid chromatograph is described which detects drugs from directly injected plasma (urine, saliva) without sample pretreatment. The apparatus consists of a programmable automatic sampling unit, which is connected via two alternating working pre-columns to an analytical column (“alternating pre-column sample enrichment”). The new device is able to operate with directly injected body fluids like an auto-analyzer and is especially useful for pharmacokinetic and clinical studies, where drug concentration have to be determined from plasma, urine or saliva.     

Validated semiquantitative/quantitative screening of 51 drugs in whole blood as silylated derivatives by gas chromatography-selected ion monitoring mass spectrometry and gas chromatography electron capture detection

A comprehensively validated procedure is presented for simultaneous semiquantitative/quantitative screening of 51 drugs of abuse or drugs potentially hazardous for traffic safety in serum, plasma or whole blood. Benzodiazepines (12), cannabinoids (3), opioids (8), cocaine, antidepressants (13), antipsychotics (5) and antiepileptics (2) as well as zolpidem, zaleplon, zopiclone, meprobamate, carisoprodol, tizanidine and orphenadrine and internal standard flurazepam, were isolated by high-yield liquid-liquid extraction (LLE). The dried extracts were derivatized by two-step silylation and analyzed by the combination of two different gas chromatographic (GC) separations with both electron capture detection (ECD) and mass spectrometry (MS) operating in a selected ion-monitoring (SIM) mode. Quantitative or semiquantitative results were obtained for each substance based on four-point calibration. In the validation tests, accuracy, reproducibility, linearity, limit of detection (LOD) and limit of quantitation (LOQ), selectivity, as well as extraction efficiency and stability of standard stock solutions were tested, and derivatization was optimized in detail. Intra- and inter-day precisions were within 2.5-21.8 and 6.0-22.5%, and square of correlation coefficients of linearity ranged from 0.9896 to 0.9999. The limit of quantitation (LOQ) varied from 2 to 2000 ng/ml due to a variety of the relevant concentrations of the analyzed substances in blood. The method is feasible for highly sensitive, reliable and possibly routinely performed clinical and forensic toxicological analyses.     

High pressure liquid chromatograph ic separation of C18 AND C19 steroids

High Pressure liquid chromatographic procedures for the resolution of mixtures of C₁₈ and C₁₉ steroids of biologic importance are described. On-line monitoring of the absorbance and refractive index of the eluate was found to be suitable for the quantitative measurement of the amounts of eluted steroids. Using a high sensitivity absorbance detector, reliable measurements of as little as 5 ng Δ⁴?3 keto steroids were possible.     

Efficient high performance liquid chromatograph/ultraviolet method for determination of diclofenac and 4'-hydroxydiclofenac in rat serum

A rapid and sensitive high-performance liquid chromatographic method was developed for determination of diclofenac and its major metabolite, 4'-hydroxydiclofenac, in serum from rats treated with diclofenac. The method is simple with a one-step extraction procedure, isocratic HPLC separation, and UV detection at 280 nm. Use of N-phenylanthranilic acid as the internal standard provided good accuracy without interference by endogenous compounds or 5-hydroxydiclofenac, another metabolite of interest. Limits of detection for diclofenac and 4'-hydroxydiclofenac were 0.0225 and 0.0112 microg/ml, respectively. Average extraction efficiencies of diclofenac, 4'-hydroxydiclofenac, and the internal standard were >/=76%. The method was applied to serum collected at 3h after rats were treated with an experimentally useful dosage range of 3, 10 and 50mg/kg diclofenac. Recovery (as a percentage of dose) for the 4'-hydroxy metabolite in serum was found to consistently average from 0.10 to 0.12% following each dosage, whereas recovery of diclofenac in serum declined from 0.45 to 0.37%. Thus, the method is suitable for measurement of a major diclofenac metabolite in experimental studies.     

A pilot study of gas chromatograph/mass spectrometry-based serum metabolic profiling of colorectal cancer after operation

Colorectal cancer (CRC) is mainly depended on the radical operation, the changing energy metabolism after operation reflects the extent, the magnitude, and the degree of surgical trauma. The aim of this study was to analyse the biochemical perturbation in the serum of CRC after operation and to evaluate their involvement in the progression of CRC. Gas chromatograph-mass spectrometry (GC-MS) in combination with pattern recognition techniques (Partial least squares discriminant analysis, supervised clustering analysis) was used to analyze serum metabolome in 30 CRC patients. A 34 endogenous metabolites included amino acid, fatty acid, carbohydrate and other intermediate metabolites were identified. Partial least squares discriminant analysis based on these metabolites discriminated preoperative from postoperative CRC group. Compared with preoperative CRC patients group, decreases in l-valine, 5-oxo-l-proline, 1-deoxyglucose, d-turanose, d-maltose, arachidonic acid and hexadecanoic acid levels and increases in l-tyrosine levels were observed in postoperative CRC patients group. The result demonstrated the GC-MS technique is an valuable tool for the characterization of the metabolic perturbation, and the metabolomic study will certainly benefit for monitoring the nutrition state of CRC patients, the prognosis and therapy evaluation of CRC patients after operation.     

Simple method for monitoring enzymatic reactions by direct injection of the medium reaction in a gas chromatograph equipped with packed liner

Reduction of sulcatone, catalyzed by Thermoanaerobium brockii alcohol dehydrogenase in Tris/HCl buffer (pH 7.8) using 2-propanol 20% (v/v) as an NADPH regeneration system could be followed by using a gas chromatograph equipped with a packed liner that allowed the direct injection of the aqueous medium reaction. The time-course curve obtained by the analytical method here described and that obtained by spectrophotometric methods were similar.     

反相高效液相色谱法测定平邑

用高效液相色谱法（ＨＰＬＣ）分析平邑甜茶内源ＡＢＡ、ＩＡＡ,选用Ｓｐｈｅｒｉｓｏｒｂ Ｃ１８反相柱,用ＵＶ检测器进行检测,使ＡＢＡ、ＩＡＡ得到很好的分离,测定方法迅速简便,流动相为甲醇、乙酸和水,系统研究了ｐＨ值、纯化方法对测定结果的影响,确定适合平邑甜茶ＡＢＡ、ＩＡＡ提取的分离条件。方法的精密度为：变异系数分别为２．８３和２．７９,最低检出限（信噪比＝２）分别为３．２７和３．２６ｎｍｏｌ／Ｌ,线     

Electron capture gas chromatographic detection of thromboxane B2

An electron capture gas chromatographic method is described for the detection of thromboxane B₂. Thromboxane B₂ is esterified with diazomethane, followed by treatment with pentafluorobenzylhydroxylamine hydrochloride and silylation with BSA. In pyridine, the free aldehyde form of the acetal ring is favored allowing rapid formation of a novel thromboxane B₂ pentafluorobenzyloxime. The method has been applied to detect thromboxane B₂ formation during aggregation of washed platelets. It must be emphasized that by ordinary analytical standards, the derivatization reproducibility from 50–375 nanograms is poor (±11% – ±42%); however, the improved selectivity of the method and its ability to detect nanogram levels of thromboxane B₂ make it a useful complement to commonly employed bioassay techniques.     

Proteomic analysis of prolactinoma cells by immuno-laser capture microdissection combined with online two-dimensional nano-scale liquid chromatography/mass spectrometry

Background  

Pituitary adenomas, the third most common intracranial tumor, comprise nearly 16.7% of intracranial neoplasm and 25%-44% of pituitary adenomas are prolactinomas. Prolactinoma represents a complex heterogeneous mixture of cells including prolactin (PRL), endothelial cells, fibroblasts, and other stromal cells, making it difficult to dissect the molecular and cellular mechanisms of prolactin cells in pituitary tumorigenesis through high-throughout-omics analysis. Our newly developed immuno-laser capture microdissection (LCM) method would permit rapid and reliable procurement of prolactin cells from this heterogeneous tissue. Thus, prolactin cell specific molecular events involved in pituitary tumorigenesis and cell signaling can be approached by proteomic analysis.     

High throughput quantitative analysis of serum proteins using glycopeptide capture and liquid chromatography mass spectrometry

It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates.     

Simultaneous analysis of substrates,products, and inhibitors of xanthine oxidase by high-pressure liquid chromatography and gas chromatography

Procedures are presented for the simultaneous analysis of hypoxanthine, xanthine, allopurinol, oxipurinol, and uric acid in standard mixtures and physiological fluids using gas chromatography (gc) or high-pressure liquid chromatography (hplc). Excellent correlation was obtained between the two methods for hypoxanthine, xanthine, oxipurinol, and uric acid. There are advantages and disadvantages to both methods. hplc requires no prior derivatization, uses isocratic elution with a buffer containing no organic solvent, and has 50- to 100-fold greater sensitivity than gc. Simpler methods of prepurification, readily adapted to clinical laboratories, can be used for hplc analysis. Although substances that are found in some urine samples from cancer patients interfere with hplc, separations by gc are not affected by these substances.