A gas-liquid chromatographic method for the analysis of diphenoxylic acid in urine

Summary A gas-liquid chromatographic method has been developed and evaluated for the measurement of diphenoxylic acid in urine. This method uses base hydrolysis to liberate diphenoxylic acid from compounds conjugated in urine, followed by removal of interfering substances in urine by column chromatography on alumina. Quantitation was carried out using p-chlorodiphenoxylic acid as an internal standard.     

Application of over-run thin-layer and gas-liquid chromatography in the separation of closely related C19O2 steroids.

An improved method for the separation of epimeric C19O2 steroids and their related allylic alcohols is described. In this method, the steroids are first separated by over-run thin-layer chromatography, and the unresolved groups are further analysed as free or as trimethylsilyl ether derivatives by gas-liquid chromatography. The behaviour of twenty-one C19O2 steroids was investigated by thin-layer chromatography in four systems and by gas-liquid chromatography in four liquid phases. All steroid pairs of similar polarity were resolved by the combination of these two fractionation procedures.     

Quantitation of zearalenone by gas-liquid chromatography on capillary glass columns.

A procedure is described for the quantitation of zearalenone from corn by gas-liquid chromatography. An internal standard, zearalanone, is first mixed with the finely ground corn, followed by extraction with ethyl acetate, purification by thin-layer chromatography and formation of trimethyl silyl ether (TMS) and methyl oxime (MOX) derivatives. Positive identification of zearalenone is based upon the retention times of the TMS and MOX-TMS derivatives being identical with those of the standards, comparison of chromatograms from both derivatives and characteristic twin peaks of zearalenone MOX-TMS. The detection limit of 100 parts per 10(9) zearalenone in corn could only be improved by modification of the described procedure.     

Improvement of the analysis of dansylated derivatives of polyamines and their conjugates by high-performance liquid chromatography.

The paper described a method for improving the hydrolysis of conjugated polyamines in PH fraction, isolated from the lichen Evernia prunastri, as well as the optimization of dansylation procedure of these polyamines on the basis of the pH value to which derivatization is achieved. Dansylated polyamines have been later separated by high-performance liquid chromatography (HPLC) using a gradient elution. Hydrolysis of conjugates requires acid treatment at room temperature rather than at 110 degrees C, as usually described. Dansylation is improved at high pH values, whereas removal of phenolics (mainly evernic acid), from the conjugates requires low pH values.     

Gas-liquid chromatography of serum bile acids using glass capillary columns

The proposed combination of a rather simple procedure for sample preparation with capillary gas-liquid chromatography using a barium carbonate/polyethyleneglycol 20,000 column and a Grob-type on-column injector permits measurement of bile acids in serum with high separation efficiency, short analysis time and complete separation of bile acids from cholesterol. The method can be adapted to combined capillary gas-liquid chromatography - mass spectrometry.     

Short-term changes in carbon isotope composition of soluble carbohydrates and starch: from canopy leaves to the root system

Changes in the 13C discrimination of current leaf photosynthesis might have profound impacts on root respiratory substrates. Therefore, the aim of this study was (1) to refine a method for the isolation of root and leaf starch and soluble sugars (neutral fraction) for stable carbon isotope analysis and (2) to assess the short-term temporal variability of the C isotope composition (delta13C) of starch and of the neutral fraction of beech roots and leaves at different canopy heights. An existing method for isolating starch for stable C isotope analysis based on enzymatic hydrolysis was modified to account for the low starch content of the samples. This was achieved by removing the enzyme (alpha-amylase) by ultrafiltration after the hydrolysis, resulting in very low carbon blanks. The neutral fraction was separated from organic acids and cations by ion-exchange chromatography. An anion-exchange resin in the [HCO3]--form was chosen that ensured high precision of C blanks. Beech leaves at 5, 10 and 20 m above the forest floor as well as roots were sampled six times during a day/night cycle in July 2003. Delta13C values of bulk material, starch and the neutral fraction increased from the lower to the higher canopy with mean differences between 5 and 20 m of 3.8, 3.4 and 2.7 per thousand for the delta13C values of starch, neutral fraction and bulk foliage, respectively. The delta13C value of foliar starch increased from the morning to the afternoon and decreased during the night, but diurnal differences (up to 3.1 per thousand) were only statistically significant for leaves sampled at 5 and 10 m height. In roots, no diurnal variation in the delta13C of starch was observed during the short time frame of one day and the delta13C of the neutral fraction did not differ between samples taken at 16:30 and 22:00. Calculated delta13C values of starch, which was mobilised during the night, were more positive than the total starch (all sampling times pooled) in leaves. Furthermore, the delta13C values of mobilised starch were approximately 5 per thousand more positive than that of the mobilised neutral fraction. Hence, the delta13C of potential sources for export from canopy leaves to roots varied considerably in their C isotope composition.     

Quantitative reversed-phase high-performance liquid chromatography of major and modified nucleosides in DNA

Improved, highly accurate high-performance liquid chromatographic methods for the measurement of the major and modified nucleosides in enzymatic digests of DNA using a single column are described. Four high resolution separation protocols (isocratic, binary, ternary and high speed) with specifically improved selectivity for 5-methyldeoxycytidine (m5dCyd) from Ade, dIno and Guo are presented. From a detailed study of the various factors contributing to the precision and accuracy of the measurement, optimized conditions and quantitative protocols were established. The ternary buffer allows for the first time the determination of N6-methyldeoxyadenosine (m6dAdo) in the same chromatographic analysis with the other deoxyribonucleosides. The binary system allows quantitation of the absolute amounts of each ribo- and deoxyribonucleoside as well as the mole % of each as the second buffer elutes 5'dA and the internal standard 8-bromoguanosine. The isocratic system allows precise quantitation of the mole % of each ribo- and deoxyribonucleoside while eliminating the need for buffer change valves, buffer cycling and column re-equilibration. Also, a high-speed isocratic system is described which permits separation of the deoxyribonucleosides in 6 min. The quantitative, enzymatic hydrolysis of DNA was evaluated by comparing a 40-h, three-enzyme system with a 4-h, two-enzyme procedure. The latter protocol proved to be an excellent hydrolysis method. These high resolution liquid chromatography techniques provide the most precise, sensitive and accurate measurement of m5dCyd available, in a straightforward method using as little as 1 microgram of DNA, and have allowed us to demonstrate: the existence of tissue-specific differences in levels of m5dCyd in DNA of humans, monkeys, rats and mice; that m5dCyd levels in DNA change during fetal development; that genomic undermethylation of DNA is correlated with cancer and the presence of m6dAdo in DNA of thermophilic organisms.     

A method for the gas chromatographic determination of vitamin D and related structures.

A procedure is described for the separation of Vitamin D and related compounds by gas-liquid chromatography using flame ionization detection. The method involves a two-step conversion: isomerization to the corresponding (all trans) isotachysterol(s) followed by methyl ether derivatization of the alcohol group(s). The procedure provides a means for identification as well as a possible basis for quantitation of Vitamin D and analogs.     

Gas-liquid chromatographic method for the measurement of methyl urea in blood and urine.

A technique for the measurement of methyl urea in biological fluids is described based upon gas-liquid chromatography of its trifluoroacetyl derivative. The method requires 10 ml of either blood or urine and is capable of measuring methyl urea at concentrations of less than 5 mg/l.     

Determination of reducing end sugar residues in oligo- and polysaccharides by gas-liquid chromatography

Reducing end sugar residues in maltodextrins and arabinoxylans are determined as alditol acetates by gas-liquid chromatography following reduction, acid hydrolysis and acetylation of the samples. After this conversion to alditol acetates, the reducing end sugars are thus separated from their acetylated aldose counterparts. The method allows to identify individual reducing end sugars quantitatively and is a good alternative for colorimetric reducing sugar assays and 1H-NMR analysis. To demonstrate the advantages of the method, an application in a study of enzymic solubilisation and degradation of water unextractable arabinoxylan from a flour squeegee fraction is described.     

Determination of hydrochloric acid in organic solutions by gas chromatography

Summary A method is described for the determination of small quantities of hydrochloric acid in two chlorinated organic solvents (CHCl₃ and CCl₄). An excess of gaseous ethylene oxide is added to a liquid sample; the 2-chloroethanol formed is then analyzed by gas chromatography. The procedure is simpler and more sensitive in comparison with other conventional methods. It can be modified for other organic solvents.D.G.R.C.S.T. grant.     

Assay of Sodium and Potassium Activated Adenosine Triphosphatase in Submicrogram Fragments of Renal Tubules

Abstract

An improved method for measuring Na, K-ATPase in submicrogram fragments of single renal tubules approximately one millimeter long is described. The activity is determined by coupling ATP hydrolysis stoichiometrically to pyruvate kinase and the oxidation of NADH by lactic dehydrogenase. NADH oxidation is followed fluorimetrically using an instrument specially modified for increased sensitivity and stability.     

Absolute Configuration OP Secondary Alcohols: Refinement and Extension of a Gas-Chromatographic Modification of Horeau's Method

Abstract

In our adaptation of Horeau's method of partial kinetic resolution, excess α-phenylbutyric anhydride is converted to diastereomeric amides for determination of its optical composition by gas-liquid chromatography. A modified procedure allows the use of as little as 1 μmole of substrate. The validity of configurational assignments based on these methods has been explored for a wide range of chiral alcohols, including a number of alkaloids. Important areas of potential application of the new procedures are delineated.     

über die gas-chromatographische Bestimmung von DiÄthylcarbonat in DiÄthyldicarbonat

The determination is based on the preceeding hydrolysis of the sample, whereby DEDC is quantitatively decomposed. DEC remains unchanged and is determined by gas chromatography using a flame-ionization deteotor. The DEC value obtained does not correspond exactly to the amount originally present, as a small quantity of DEC is formed in addition by ethanolysis occurring besides the hydrolysis. This additional amount depends on the ethanol concentration of the hydrolysis solution and, therefore, on the quantity of DEDC taken as sample. An exact calculation of the additional amount of DEC permitting a correction of the value found by gas chromatography is possible by the following relation:% DEC formed=0.26 × vol-% of ethanol in hydrolysis solution. Samples of DEDC containing more than 0.01% of DEC can be analysed reliably by the procedure described (standard deviation 0.015).     

Determination of trimethylsilyl methylated nucleic acid bases in urine by gas-liquid chromatography.

A method for the determination of the urinary excretion level of methylated nucleic acid bases by gas-liquid chromatography (GLC) has been developed. A clean-up procedure prior to GLC analysis consisted of hydrolysis, filtration, charcoal adsorption, and anion exchange. Studies to determine optimum derivatization conditions for conversion of the methylated bases to their trimethylsilyl derivatives and to evaluate GLC parameters and columns to obtain the best separation were conducted. Application of the method was shown by determining the excretion levels of methylated bases in urine of normal controls and of patients with various types of malignancy.     

Quantitation of l-alpha-acetylmethadol and its metabolites in human serum by capillary gas-liquid chromatography and nitrogen detection

A procedure is described for the simultaneous measurement of l-alpha-acetylmethadol and its two pharmacologically active metabolites: noracetylmethadol and dinoracetylmethadol. In the method an intramolecular conversion reaction of the two metabolites to their amide configuration is utilized. The reaction is performed while the metabolites are still in the serum. Following solvent extraction the samples are analyzed by capillary gas-liquid chromatography coupled with nitrogen detection. Quantitation is achieved by internal standardization. The lower limit of sensitivity is 5 ng/ml in serum. Absolute sensitivity is 0.1 ng for all three compounds. The advantages over other procedures are: speed due to the single extraction step; increased recovery of noracetylmethadol and dinoracetylmethadol due to decreased polarity of the amides; greater stability of the metabolites in the amide configuration; better chromatographic quantitation and separation because detector response for the amides is greater than it is for the original configuration of the metabolites and the area of the chromatographic tracing is free of interfering substances.     

Size characterization of barley starch granules by gravitational field-flow fractionation: a rapid,low-cost method to assess the brewing capability of different strains

Cereal starch occurs as two types of micrometer-sized granules, large and small. Large starch granules are more susceptible to enzymatic hydrolysis. When cereal starch is used for fermentation processes, as in brewing of barley malt, the barley strains with the highest content of large starch granules should be preferred. Gravitational field-flow fractionation (GFFF) is a separation method able to fractionate starch samples at low cost and short analysis time. In this work, the search for the best GFFF conditions for the analytical separation of barley starch within an inter-laboratory approach is presented. For different barley strains cultivated under monitored conditions the size distributions of starch granules is here quickly monitored and characterized by GFFF. As a consequence, dimensional characterization of barley starch can allow for the selection of the most suitable strains with the lowest content of non-degradable starch.     

Method for the measurement of plasma dehydroepiandrosterone by gas-liquid chromatography with electron capture detection.

A method is described for the measurement of plasma dehydroepiandrosterone, as the iodomethyldimethylsilyl ether derivative, by gas-liquid chromatography and electron capture detection using the relatively new and highly stable stationary phase Dexsil 300. Preliminary purification of the plasma extract was required and alumina column chromatography was utilised, both before and after derivatization of the steroid extract. Specificity, precision, sensitivity and accuracy were all satisfactory. The method was used to study the relationship between age and the level of plasma dehydroepiandrosterone in a group of normal women. A significant negative correlation was observed.     

An integrated procedure for the measurement of total dietary fibre (including resistant starch), non-digestible oligosaccharides and available carbohydrates

A method is described for the measurement of dietary fibre, including resistant starch (RS), non-digestible oligosaccharides (NDO) and available carbohydrates. Basically, the sample is incubated with pancreatic α-amylase and amyloglucosidase under conditions very similar to those described in AOAC Official Method 2002.02 (RS). Reaction is terminated and high molecular weight resistant polysaccharides are precipitated from solution with alcohol and recovered by filtration. Recovery of RS (for most RS sources) is in line with published data from ileostomy studies. The aqueous ethanol extract is concentrated, desalted and analysed for NDO by high-performance liquid chromatography by a method similar to that described by Okuma (AOAC Method 2001.03), except that for logistical reasons, d-sorbitol is used as the internal standard in place of glycerol. Available carbohydrates, defined as d-glucose, d-fructose, sucrose, the d-glucose component of lactose, maltodextrins and non-resistant starch, are measured as d-glucose plus d-fructose in the sample after hydrolysis of oligosaccharides with a mixture of sucrase/maltase plus β-galactosidase.     

Preparation of starch and other carbon fractions from higher plant leaves for stable carbon isotope analysis.

The measurement of the carbon isotope composition of starch and cellulose still relies on chemical isolation of these water-insoluble plant constituents and subsequent elemental analysis by isotope ratio mass spectrometry (EA/IRMS) of the purified fractions, while delta(13)C values of low-molecular-weight organic compounds are now routinely measured by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Here we report a simple and reliable method for processing milligram quantities of dried plant material for the analysis of the carbon isotope composition of lipids, soluble sugars, starch and cellulose from the same sample. We evaluated three different starch preparation methods, namely (1) enzymatic hydrolysis by alpha-amylase, (2) solubilization by dimethyl sulfoxide (DMSO) followed by precipitation with ethanol, and (3) partial hydrolysis by HCl followed by precipitation of the resulting dextrins by ethanol. Starch recovery for three commercially available native starches (from potato, rice and wheat) varied from 48 to 81% for the techniques based on precipitation, whereas the enzymatic technique exhibited yields between 99 and 105%. In addition, the DMSO and HCl techniques introduced a significant (13)C fractionation of up to 1.9 per thousand, while the carbon isotope composition of native starches analyzed after enzymatic digestion did not show any significant difference from that of untreated samples. The enzymatic starch preparation method was then incorporated into a protocol for determination of delta(13)C signatures of lipids, soluble carbohydrates, starch and crude cellulose. The procedure is based on methanol/chloroform/water extraction of dried and ground leaf material. After recovery of the chloroform phase (lipid fraction), the methanol/water phase was deionized by ion exchange (soluble carbohydrate fraction) and the pellet treated with heat-stable alpha-amylase (starch fraction). The remaining insoluble material was subjected to solvolysis by diglyme (cellulose fraction). The method was shown to be applicable to foliar tissues of a variety of different plant species (spruce, erect brome, maize and soybean).