Determination of amino acids in urine by gas chromatography

This report describes a convenient means of reducing the complex matrix which is responsible for interference during gas chromatographic determination of amino acids in urine. The pre-chromatographic clean-up employs the principle of solid phase extraction using bonded silica incorporating cation exchange groups. This approach avoids the detrimental effects on amino acid recoveries associated with resin-based cation exchangers. In spite of significant reduction in the complexity of chromatograms, only the high efficiency and resolving power offered by the analytical capillary column (e. g. fused silica open tubular, FSOT) is sufficient for quantitative and analysis of amino acids in urine. Reproducibility data from the complete procedure are determined, coefficients of variation (CV) for most amino acids being better than 5% with a mean recovery of 96%.     

Determination of amino acids in the hepatic and brain tissue of the rat by gas chromatography

A procedure is described for the quantitative determination of amino acids in hepatic and brain tissue samples from the rat. Because the presence of certain matrix components in the tissue material led to interference with chromatographic analysis they were removed by a prechromatographic “clean-up” step. Quantitative analysis of amino acids, as their N-heptafluorobutyryl iso-butyl ester derivatives, was achieved by high resolution gas chromatography on an apolar fused silica open tubular column. Reproducibility data from the complete procedure are presented; coefficients of variation for arginine and histidine in hepatic tissue varied between 7.1 and 10.1% whereas those for most other amino acids were better than 5%, with a mean recovery of 90%.     

Gas chromatography of plasma amino acids using a wide bore column: Comparison with packed and capillary columns

A procedure is described in which a wide bore glass capillary column is used as an alternative to the more traditional packed column in the analysis of amino acid levels in plasma. The coefficients of variation for all amino acids (with the exception of aspartic acid) were better than 11% with recoveries ranging from 81% to 122%. The data are compared with the corresponding results obtained using a packed column and show significant differences (p < 0.005) between values for glycine, serine, isoleucine, proline, methionine, aspartic acid, phenylalanine, and lysine. A similar comparison between results from the wide bore and the fused-silica open tubular (FSOT) column shows better agreement. Adjustment of chromatographic conditions for the wide bore analysis yields results in good agreement with those from FSOT analysis but which still differe significantly from the backed column data.     

Determination of amino acids by gas chromatography using a wide bore capillary column

A procedure is described in which a wide bore capillary column is used as an alternative to the more traditional packed column for the quantitative analysis of amino acids as their N-heptafluorobutyryl isobutyl ester (HBB) derivatives. The column, installed in a gas chromatograph previously configured for use with a packed column, is shown to give good reproducibility by repeated determination of amino acid response factors (RSD values for all amino acids are below 3%). A number of problems, encountered during the use of this column, are discussed and suitable techniques to overcome them are reported.     

Capillary gas chromatography of amino acids as their tert-butyldimethylsilyl derivatives in the analysis of serum amino acids in metabolic diseases

Reaction of amino acids with N-methyl-N-(tert-butyldimethylsilyl)trifluoroaceamide (MTbSTFA) in acetonitrile affords good yields of amino acid derivatives with excellent gas chromatographic and mass spectrometric properties. The single-step derivatization procedure is highly reproducible. The TBDMS amino acids are stable at room temperature for at least three days. Only a single peak is observed for each amino acid. The procedure allows simultaneous analysis of asparagine and glutamine together with other serum amino acids. Separation is achieved on a borosilicate glass capillary coated with OV-1. The mass spectra of the TBDMS amino acids possess characteristic diagnostic ions. These properties were used in the sensitive detection by GC-MS and SIM-GC-MS of GABA and pipecolic acid in the serum of a newborn suspected of a Zellweger-type syndrome, which could not be detected by other methods.     

Enantioselective capillary electrophoresis-mass spectrometry of amino acids in cerebrospinal fluid using a chiral derivatizing agent and volatile surfactant

The sensitivity of coupled enantioselective capillary electrophoresis-mass spectrometry (CE-MS) of amino acids (AAs) is often hampered by the chiral selectors in the background electrolyte (BGE). A new method is presented in which the use of a chiral selector is circumvented by employing (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as chiral AA derivatizing agent and ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase for separation of the formed diastereomers. Efficient AA derivatization with FLEC was completed within 10 min. Infusion experiments showed that the APFO concentration hardly affects the MS response of FLEC-AAs and presents significantly less ion suppression than equal concentrations of ammonium acetate. The effect of the pH and APFO concentration of the BGE and the capillary temperature were studied in order to achieve optimized enantioseparation. Optimization of CE-MS parameters, such as sheath-liquid composition and flow rate, ESI and MS settings was performed in order to prevent analyte fragmentation and achieve sensitive detection. Selective detection and quantification of 14 chiral proteinogenic AAs was achieved with chiral resolution between 1.2 and 8.6, and limits of detection ranging from 130 to 630 nM injected concentration. Aspartic acid and glutamic acid were detected, but not enantioseparated. The optimized method was applied to the analysis of chiral AAs in cerebrospinal fluid (CSF). Good linearity (R² > 0.99) and acceptable peak area and electrophoretic mobility repeatability (RSDs below 21% and 2.4%, respectively) were achieved for the chiral proteinogenic AAs, with sensitivity and chiral resolution mostly similar to obtained for standard solutions. Next to l-AAs, endogenous levels of d-serine and d-glutamine could be measured in CSF revealing enantiomeric ratios of 4.8%–8.0% and 0.34%–0.74%, respectively, and indicating the method's potential for the analysis of low concentrations of d-AAs in presence of abundant l-AAs.     

Liquid crystal stationary phases for gas chromatography and supercritical fluid chromatography

Novel monomeric and polymeric liquid crystalline compounds were synthesized as stationary phases for gas chromatography (GC) and supercritical fluid chromatography (SFC). Monomeric liquid crystalline compounds were used in packed column gas chromatography for the separation of isomeric aromatic compounds and insect sex pheromones. Liquid crystalline polymers possess long nematic ranges and a uniform coating was easily achieved in glass and fused silica capillaries, which could stand temperatures up to 250°C in GC and pressures of 200 MPa at 160°C in SFC. The columns provide excellent selectivity and resolution for fused ring aromatic compounds such as the isomers anthracene and phenanthrene or triphenylene and chrysene.     

Separation of some amino acids by supercritical fluid chromatography after a pre-derivatization step with classical reagents

Summary The separation of polar compounds by supercritical fluid chromatography is a difficult problem to solve. In this work, we have used a pre-derivatization method to obtain apolar or less polar compounds. Amino acids have been chosen as model polar compounds of biological interest, and 9-fluorenylmethyl chloroformate (FMOC) and (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) have been used as derivatizating reagents.With this procedure, the amino acids studied have been separated in less than 40 minutes with a good efficiency. Furthermore, with FLEC reagent, the enantiomeric separation was rapid in comparison with liquid chromatography techniques and the selectivities obtained were in the same order of magnitude.     

Determination of amino acid enantiomers in orange juices by chiral phase capillary gas chromatography

Summary The configurations of free amino acids (AAs) in orange juice beverages (commercial products of satisfactory and unsatisfactory quality), an orange juice concentrate (bulk product suspected of being adulterated), and in an orange juice that has been contaminated by addition ofLactobacillus plantarum as a model for microbial spoilage, were determined, after derivatization, by means of gas-liquid chromatography (GC) using fused-silica capillary columns coated with Chirasil-L-Val or Chirasil-D-Val as stationary phases. AAs were isolated from juices by treatment with Dowex WX8 ion-exchanger and were investigated, by GC, as theirN(O)-pentafluoropropionylorN(O)-trifluoroacetyl 1-propyl esters. It was found that the high quality orange juice beverage contained L-AAs exclusively whereas this juice, after fermentation withLactobacillus, contained free D-Ala (32.7%), D-Val (62.3%), D-Phe (20.0%), D-Glu (24.3%), D-Ser (2.6%), D-Asp (0.8%), and significant amounts of D-Pro [% D=100 D/(D+L)]. D-Ala (8.8%) and D-Ser (4.2%) were found in a sensory and analytically unsatisfactory orange juice beverage, whilst D-Ala (27.5%) and D-Ser (14.3%) were detected in the orange juice concentrate suspected of being adulterated.Although capillary GC on chiral stationary phases is regarded as being highly suitable for the determination of AA enantiomers in fruit juice beverages, detection of D-AAs is currently not considered as conclusive proof of fruit juice adulteration caused by addition of AA racemates since a non-microbial origin of D-AAs in the respective juice, or an original occurrence of D-AAs, in either the free, substituted, or peptide-bonded form in the fruits, cannot be excluded with certainty.Presented in part at the Deutscher Lebensmittelchemikertag, Sept. 18–21, 1990, Frankfurt and at the 14. Jahrestagung Deutscher Lebensmitteltechnologen, Nov. 15–17, 1990, Düsseldorf.     

Heliflex chirasil-Val; GC of amino acid enantiomers

Heliflex Chirasil-Val, a modified form of the original Chirasil-Val phase coated on fused silica capillary, has been investigated with regard to its resolution efficiency and the separation profile for various enantiomeric derivatives of amino acid. The best results could be obtained with TFA-Et derivatives by reason of the relatively low column temperature required.     

Synthesis of siloxane stationary phases for capillary gas chromatography and supercritical fluid chromatography

A comparison is made between dichlorosilanes and cyclic siloxanes as starting materials in the synthesis of stationary phases for capillary gas chromatography (CGC) and supercritical fluid chromatography (SFC). Siloxanes containing one or more of the side groups methyl, vinyl, phenyl, and cyanoethyl in various ratios were synthesized and compared. These phases were characterized by chromatographic (gel permeation, GPC), spectroscopic (IR, ¹H NMR, ²⁹Si NMR), and thermal (DSC) methods. Coated fused silica columns were evaluated with respect to polarity, crosslinkability with several free-radical initiators, and thermal stability. A new liquid phase, 7% cyanoethyl, 7% phenyl, 1% vinyl methyl polysiloxane is shown to be more polar than OV-1701, more temperature stable, easily crosslinked and suitable for use in supercritical fluid chromatography.     

Fused silica capillary micro-packed columns in gas chromatography

Summary Flexibility, strength and adsorption inertness of fused-silica capillaries permits their extensive application for the preparation of micro-packed columns in gas chromatography. Decreasing the column diameter (from 0.5 to 0.15 mm) and the diameter of the sorbent particles (from 100 to 5 μm) results in a marked reduction of the height equivalent to a theoretical plate (HETP), as well as in diminishing the dependence of the HETP on the carrier gas flow rate. The chromatographic characteristics of fused-silica capillary micro-packed columns and open-tubular columns are compared. The fused-silica capillary micro-packed column can be used to advantage for performing rapid and trace analyses and have been shown to be adapted for application in gas-solid chromatography. Separation of organic and inorganic compounds on fused-silica capillary micro-packed columns is illustrated by practical examples. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Supercritical fluid chromatography with a slurry-packed capillary column

A versatile system with a slurry-packed capillary column was developed for supercritical fluid chromatography, which is capable of programming both inlet and outlet pressure independently, as well as using a restrictor to apply back pressure. This system revealed the relationships between pressure drop, flow rate, and linear velocity in pressure-programmed supercritical fluid chromatography. In the restrictor system, both the pressure drop and the flow rate increased almost linearly with inlet pressure, while under conditions of constant pressure drop characteristic behavior was observed which depended on the density-viscosity relationships of supercritical fluid. Resolution in the separation of polysiloxane oligomers was found to be increased by increasing the ratio of pressure drop to pressure-programming rate, although the sensitivity decreased due to the increase in peak volume. The system controlling both inlet and outlet pressure has distinct advantages over the restrictor system controlling both inlet and outlet pressure has distinct advantages over the restrictor system in practical in practical operations.     

Preparation,evaluation, and comparison of wide bore (320 μm) and narrow bore (50 μm) cyanosilicone-coated capillary columns for gas chromatography

The preparation of wide bore (320 μm) and narrow bore (50 μm) fused silica capillary columns is described for immobilized cyanopropyl substituted silicones containing 60 and 88% substitution. The effect of high temperature deactivation with cyanopropylcyclosiloxanes was studied with a special test mixture. Curing was achieved with dicumyl peroxide or azo-tert-butane. The columns were evaluated and compared in terms of efficiency, activity, polarity, and temperature stability. Different coating methods were compared for the narrow bore columns. The activity of the 60% cyanopropyl columns that had been immobilized with dicumyl peroxide was significantly larger than for azo-tert-butane immobilized columns. The polarity of polar columns appeared to depend greatly on column temperature and is completely different for wide and narrow bore columns.     

Determination of amino acids in apple extracts by high performance liquid chromatography

Summary A rapid and sensitive method for the simultaneous determination of primary amino acids in apple is described. After sample preparation, amino acids were derivatized with o-phthaldialdehyde/2-mercaptoethanol and separated on a reversed phase column with a gradient of phosphate buffer-tetrahydrofuran-methanol as the mobile phase. Detection was carried out with a fluorescence detector at excitation and emission wavelengths of 340 nm and 425 nm respectively. Recovery studies showed good results for all substances (91–109%) (with coefficients of variation ranging, from 0.1 to 9.0%). This method was applied to the monitoring of amino acids during the ripening of apples.     

Determination of pentane in human breath by gas chromatography

A new procedure, requiring expired sample volumes of approximately 2L, has been developed for the quantitative determination of n-pentane in human breath. The methodology capitalizes on the superior resolving power and separating efficiency of the open tubular capillary column and incorporates an effective prechromatographic cryofocusing technique. Molecular sieve is used to simultaneously remove moisture and carbon dioxide from breath samples which are “spiked” with an internal standard to optimize the validity of analysis. Reproducible data for both internal standard spiking (CV = 5.8%) and sample n-pentane level (CV = 3.2%) have been obtained, together with a mean recovery of 104%. Preliminary data show n-pentane levels in normal female subjects (n = 10) to range from 0.62 to 3.16 nmole/L (mean 1.76 nmole/L) and in males (n = 10) from 3.20 to 8.76 nmole/L (mean 5.66 nmole/L).