Gas—liquid chromatography of isobutyl ester,N(O)-heptafluorobutyrate derivatives of amino acids on a glass capillary column for quantitative separation in clinical biology

A gas chromatographic method adapted to routine analysis has been developed for quantitative separation on glass capillary columns for free proteic and other known amino acids normally or abnormally found in physiological fluids. The procedure involves ion-exchange chromatography and isobutyl ester, N(O)-heptafluorobutyrate derivatization of free plasma and urine amino acid samples. Derivatized components were ascertained by combined gas chromatography—mass spectrometry. The use of glass for the capillary column is mandatory to achieve qualitative and quantitative analysis of the known occurring amino acids in urine and small plasma samples. Quantitative analysis of several types of human amino acid disorders are presented.     

The use of formic acid in carrier gas : Rapid method for identification and determination of phytanic acid by gas chromatography—mass spectrometry—chemical ionization

A rapid gas chromatographic method to determine phytanic acid in plasma from Refsum's disease is described. After a brief alkaline hydrolysis of lipids, the biological sample is directly injected into a glass pre-column; an acid carrier gas (formic acid in nitrogen) is used to displace the long-chain fatty acids from their sodium salts and from their binding to proteins. Formic acid introduced through the column may also be used as a reagent gas for chemical ionization in combined gas chromatography—mass spectrometry; fatty acids (C₁ to C_16:2 and phytanic acid) are easily identified by their M + 1 (base peak) and M − 17 peaks. The described procedure is also suitable for studying normal fatty acids from plasma lipids.     

Synthesis and spectral studies of 2-alkoxystearic acids — I

A series of 2-alkoxystearic acids was synthesized during dehydrohalogenation of the 2-iodostearic acid with potassium hydroxide in different alcohols. Besides the formation of trans-2-enoic- and 2-hydroxyacids, the reaction provides a convenient method for the preparation of long-chain 2-alkoxyacids in high yield. With (?)-2-butanol and (?)-2-methylbutanol the formation of mixtures of diastereoisomeric 2,1′-methylpropoxy- and 2,2′-methylbutoxystearic acids is evident by gas chromatographic analysis of their methyl esters. The preparation of 2-alkoxystearic acids (I–XI), their separation by column chromatography and structure determination by elementary analysis, thin-layer chromatography (TLC), infrared (IR) and proton magnetic resonance spectroscopy as well as gas chromatography/mass spectrometry are described.     

The trail pheromone of the ant, Lasius fuliginosus: Identification of six components

Hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, and dodecanoic acid are components of the trail pheromone of the ant, Lasius fuliginosus. The acids were extracted from the rectal fluid of dissected worker ants, and identified by the mass spectra and gas chromatographic retention times of the corresponding methyl esters. The same acids could also be detected in the material excreted by the ants on their foraging path.     

Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS

Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of ¹³C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantification of ¹³C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employing equipment with two columns connected parallel to a mass spectrometer, we can double the throughput to the mass spectrometer, reducing the analysis time per sample to 5 min. Palmitate flux measured using this approach agreed well with the GC/C/IRMS method. This HPLC/MS method provides accurate and precise measures of FFA concentration and enrichment.     

The use of negative ion thermospray liquid chromatography/tandem mass spectrometry for the determination of bile acids and their glycine conjugates.

A study was carried out on the negative ion thermospray liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry of a group of bile acids and their glycine conjugates. The liquid chromatographic/tandem mass spectrometric experiments were performed using low-energy collisions on a triple-quadrupole mass spectrometer. It was found that relatively high collision energies and collision gas pressures were required to produce fragmentation and that some unusual fragments were produced.     

Determination of MK-287, a new platelet-activating factor antagonist, in plasma and serum by gas chromatography chemical ionization mass spectrometry

MK-287 is a novel platelet-activating factor antagonist. A sensitive and specific gas chromatographic/mass spectrometric assay has been developed for the determination of the drug in serum and plasma. The assay utilizes an extraction with methyl-t-butyl ether and subsequent trimethylsilylation of the hydroxyl function. The gas chromatographic/mass spectrometric determinations are carried out with temperature-programmed capillary gas chromatography and ammonia negative chemical ionization mass spectrometry. The method has sufficient sensitivity, precision, accuracy and selectivity for the analysis of drug concentrations in clinical samples.     

Determination of saccharide content in pneumococcal polysaccharides and conjugate vaccines by GC-MSD

A simple and sensitive gas chromatographic method was designed for quantitative analysis of Streptococcus pneumoniae capsular polysaccharides, activated polysaccharides, and polysaccharide conjugates. Pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F polysaccharide or conjugate were subjected to methanolysis in 3N hydrochloric acid in methanol followed by re-N-acetylation and trimethylsilylation. Derivatized samples were chromatographed and detected using gas chromatography with mass selective detector. Gas chromatographic results were compared with colorimetric values with agreement of 92 to 123% over the range of all samples tested. Monosaccharides released during methanolysis included hexoses, uronic acids, 6-deoxy-hexoses, amino sugars, and alditols. Quantitative recovery of monosaccharides was achieved for all serotypes by the use of a single methanolysis, derivatization, and chromatography procedure. Response factors generated from authentic monosaccharide standards were used for quantitation of pneumococcal polysaccharides and conjugates with confirmation of peak assignments by retention time and mass spectral analysis. This method allows saccharide quantitation in multivalent pneumococcal vaccine intermediates and final drug products with low-level detection (10 pg) and peak purity.     

Analysis of tyrosine and deuterium labelled tyrosine in tissues and body fluids

A gas chromatographic mass spectrometric method has been developed to determine tyrosine and deuterium labelled tyrosine in biological samples using alpha-methyltyrosine (alpha-Me Ty) or m-hydroxyphenylalanine as internal standards. With the latter standard both labelled and unlabelled tyrosine as well as alpha-Me Ty can be determined simultaneously, in for example human blood, following administration of alpha-Me Ty to inhibit catecholamine synthesis. After isolation of the amino acids on an ion exchange column (Amberlite IR 120) the butyl ester pentafluoropropionyl derivatives were prepared. In human plasma the precision of the method was determined at a level of 80 nmol tyrosine ml-1 and found to be +/- 5% (coefficient of variation, n = 10).     

O-trimethylsilylquinoxalinol derivatives of aromatic α-keto acids : Mass spectra and quantitative gas chromatography

As an extension of earlier work on aliphatic α-keto acids, a method is described for the quantitative gas chromatographic determination of urinary aromatic α-keto acids. The keto acids are derivatized with o-phenylenediamine to yield the quinoxalinols. These compounds are chromatographed after trimethylsilyation.The aromatic keto acids are stabilized by sodium dithionite (4 mg/ml urine) and storage below 0°. The final derivatives are stable for weeks at room temperature.Low resolution mass spectra are reported. The fragmentation mechanims are elucidated by analysis of O-trimethylsilyl-(TMS)-quinoxalinols, O-(TMS-d₉)-quinoxalinols and O-TMS-6(7)-chloroquinoxalinols.     

Use of t-butyldimethylsilylation in the gas chromatographic/mass spectrometric analysis of physiologic compounds found in plasma using electron-impact ionization

The use of N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide to prepare the t-butyldimethylsilyl derivatives of a number of organic compounds (selected amino acids, alpha-keto acids, ketone bodies, free fatty acids, urea, glycerol, lactate, and pyruvate) is reported. These derivatives are particularly useful for gas chromatographic/mass spectrometric analysis involving the use of stable isotopes and selected ion monitoring, since a peak of sufficient abundance at 57 mass/charge units below the molecular ion was always present, and was the result of the loss of one t-butyl group. In each case, this fragment contained the entire skeleton of the original compound, which permitted easy analysis using electron-impact ionization of these compounds alone or when labeled with stable isotopes in any nonexchangeable position.     

A micromethod for the analysis of free amino acids by gas chromatography and its application to biological systems.

A gas chromatographic procedure was developed for determination of minute amounts of free amino acids in natural waters and laboratory models simulating biological systems. Sample pretreatment included removal of interfering organic substances by chloroform extraction and isolation of amino acids by cation exchange. Amino acids were converted to their N-heptafluorobutyryl isobutyl ester derivatives in glass capillary tubes, permitting considerable concentration of the sample prior to gc injection. The derivatives of 19 amino acids were successfully separated on either a glass column packed with a mixture of OV-101 and OV-17 on Chromosorb W, a glass capillary column coated with OV-101, or a support-coated capillary column supported with SE-30. One to five nanograms of individual amino acids were detected using flame ionization detector. The detection limit was reduced more than 100-fold using the electron capture detector and more than 1000-fold by mass fragmentography. The procedure allowed determination of less than 1 ppb of individual amino acids in lake and river water samples and was used to estimate the exeretion of free amino acids from microbial populations.     

Human biomonitoring of pyrethrum and pyrethroid insecticides used indoors: determination of the metabolites E-cis/trans-chrysanthemumdicarboxylic acid in human urine by gas chromatography-mass spectrometry with negative chemical ionization

This work describes a gas chromatographic-mass spectrometric method employing negative chemical ionization (NCI) for the determination of E-cis/trans-chrysanthemumdicarboxylic acid (CDCA) in human urine used as a biomarker for the exposure to pyrethrum and/or certain pyrethroids in insecticide formulations applied indoors. Mixed-mode solid phase extraction was utilized for sample cleanup. Extraction recoveries ranged from 92 to 104% (2-9% R.S.D.). The acids were esterified with 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) allowing both their gas chromatographic separation and their sensitive mass spectrometric detection under NCI conditions. Detection limits of ca. 0.05 microg/l urine were achieved.     

Chromatographic analysis of lipoic acid and related compounds

The analysis of lipoic acid and related compounds, such as its reduced form dihydrolipoic acid, its amide form lipoamide and other analogues, in biological and food samples is important in biochemistry, nutritional and clinical chemistry. This review summarizes the chromatographic methods for the determination of lipoic acid and related compounds, and their applications to various samples such as bacteria, tissues, drugs and food. Gas chromatographic methods with flame ionization detection and flame photometric detection are commonly used for the quantification of lipoic acid present as its protein-bound form, after acid or base hydrolysis of these samples. High-performance liquid chromatographic methods with ultraviolet, fluorescence and electrochemical detection are mainly used for the determination of free lipoic acid and related compounds, such as dihydrolipoic acid, lipoamide and other analogues. Moreover, gas chromatography–mass spectrometry and capillary electrophoresis methods are also developed.     

Chromatographic methods for the determination of toxicants in faeces

Modern chromatographic techniques and their application in the determination of toxic compounds in faeces are reviewed. Faecal analysis may be of importance in toxicokinetic studies of xenobiotics in order to determine factors such as metabolism, body burden and major routes of elimination. Compounds of interest include various food constituents, drugs and occupational or environmental factors. Further, various mutagenic or carcinogenic compounds which are excreted by faeces have been indicated to represent risk factors for colorectal cancer. In this context, the chromatographic determination of the endogenously generated fecapentaenes and bile acids, both postulated etiological factors in colorectal carcinogenesis, is reviewed. For fecapentaene determination, several high-performance liquid chromatographic (HPLC) methods are available; however, the applicability of some of these methods is limited owing to insufficient separation of various isomeric forms or discrimination between fecapentaenes and their precursors. For the determination of bile acids in faeces, many chromatographic procedures have been reported, and the characteristics of the most relevant methods are compared and discussed. It is concluded that separation by gas chromatography (GC) in combination with mass spectrometry provides the highest selectivity and sensitivity. A relatively rapid alternative analysis for the determination of total and aqueous faecal bile acids is proposed. Further, methods for the determination of polycyclic aromatic hydrocarbons (PAHs) are reviewed. Although the use of radiolabelled PAHs in animal studies has many advantages, it cannot be applied for human biological monitoring and HPLC and GC provide sensitive alternatives. An HPLC method for the determination of non-metabolized PAHs in faeces is described.     

Comparison of the limulus amebocyte lysate test and gas chromatography-mass spectrometry for measuring lipopolysaccharides (endotoxins) in airborne dust from poultry-processing industries.

The lipopolysaccharide (endotoxin) content in airborne dust samples from three different poultry slaughterhouses was determined with both the chromogenic Limulus amebocyte lysate assay and gas chromatography-mass spectrometry analysis of lipopolysaccharide-derived 3-hydroxy fatty acids. Gram-negative cell walls were also measured by using two-dimensional gas chromatography/electron-capture analysis of diaminopimelic acid originating from the peptidoglycan. The correlation between the results of the Limulus assay and those of gas chromatography-mass spectrometry for determination of the lipopolysaccharide content in the dust samples was poor, whereas a good correlation was obtained between lipopolysaccharide and diaminopimelic acid concentrations with the gas chromatographic methods. The results suggest that it is predominantly cell-wall-dissociated lipopolysaccharides that are measured with the Limulus assay, whereas the gas chromatographic methods allow determination of total concentrations of lipopolysaccharide, including Limulus-inactive lipopolysaccharide, gram-negative cells, and cellular debris.     

Comparison of the limulus amebocyte lysate test and gas chromatography-mass spectrometry for measuring lipopolysaccharides (endotoxins) in airborne dust from poultry-processing industries

The lipopolysaccharide (endotoxin) content in airborne dust samples from three different poultry slaughterhouses was determined with both the chromogenic Limulus amebocyte lysate assay and gas chromatography-mass spectrometry analysis of lipopolysaccharide-derived 3-hydroxy fatty acids. Gram-negative cell walls were also measured by using two-dimensional gas chromatography/electron-capture analysis of diaminopimelic acid originating from the peptidoglycan. The correlation between the results of the Limulus assay and those of gas chromatography-mass spectrometry for determination of the lipopolysaccharide content in the dust samples was poor, whereas a good correlation was obtained between lipopolysaccharide and diaminopimelic acid concentrations with the gas chromatographic methods. The results suggest that it is predominantly cell-wall-dissociated lipopolysaccharides that are measured with the Limulus assay, whereas the gas chromatographic methods allow determination of total concentrations of lipopolysaccharide, including Limulus-inactive lipopolysaccharide, gram-negative cells, and cellular debris.     

Measurement of isotope ratios in organic compounds at picomole quantities by capillary gas chromatography/quadrupole electron impact mass spectrometry

Measurements of isotope ratios in organic compounds at nanomolar concentrations in biological fluids require sensitive and selective gas chromatographic/mass spectrometric methods. The efficiency of sample preparation procedures must be high and gas chromatographic conditions must assure high resolution chromatography producing narrow, intense peaks. Mass spectrometric conditions must create suitable mass fragments, preferably in the high mass range. Detection, controlled by selected ion monitoring (SIM), requires the use of small mass intervals and adequate acquisition times. However, the choice of optimal conditions is limited by the need to acquire multiple data points for the eluting compound. The effects of these variables on the precision of isotope ratio measurements have been investigated to develop an optimal method for isotope ratio measurements in serum bile acids at low concentrations (0.05-8 micromol l-1). With a 25 m X 0.32 mm fused silica capillary OV-1701 column and cold on-column injection, peak widths of 10 s were obtained. Quadrupole mass spectrometry in the electron impact ionization mode (70 eV) and selected ion monitoring (SIM)(mass interval 1/16 u, acquisition time 100 ms) allowed precise (cv less than 1.5%) isotope ratio measurements for chenodeoxycholic, cholic and deoxycholic acid in a single run at quantities as low as 5 pmol injected on the column. SIM switching during the run permitted isotope ratio measurements (cv less than 2.2%) for an additional six bile acids, normally detected in the serum of healthy subjects.     

Determination of chemical composition of Turkish propolis

The aim of the present work is to study the chemical composition of Turkish propolis. Propolis samples were collected from different regions of Turkey (Bursa, Erzurum-Askale, Gumushane-Sogutagil and Trabzon-Caglayan) in 1999. Ethanol extracts of propolis (EEP) were prepared for chemical analysis, using gas chromatograph coupled with mass spectrometry (GC-MS). Our findings show that propolis samples from Trabzon and Gumushane region have a similar chemical composition. In both samples aromatic acids, aliphatic acids and their esters, and also ketone derivatives are the main compound groups. The chemical composition of the single sample that was collected from Erzurum region shows a very different pattern than the other two samples. In this propolis, the main compounds are aromatic acid esters and alcohols. However, it contains a high amount of amino acids compared to the other samples. The other samples collected from three different region of Bursa City are rich with flavavones, aromatic acids and their esters, terpenoids, flavones and ketones.     

Diagnostic methods for the determination of iduronic acid in oligosaccharides

A high-performance liquid chromatography (HPLC) method with pulsed amperometric detection (PAD) was used for the determination of the acid hydrolysis products of L-iduronic acid containing oligosaccharides isolated from biological sources. This HPLC-PAD method was compared with gas chromatographic (GLC) methods. Since acid hydrolysis of oligosaccharides can produce a number of products, several uronic acid derivatives were prepared by chemical synthesis. These well characterized standards in conjunction with mass spectrometry allowed for the identification of most of the products of methanolysis or hydrolysis of glycosamino-glycans, which included chondroitin sulfates A and B (dermatan sulfate), heparin, and hyaluronic acid. (4 M) HCl in methanol 100 degrees C for 24 h was found to be optimum for GLC and 1 M aqueous HCl for 4 h at 100 degrees C for HPLC-PAD. All of the monosaccharides, hexosamines, and uronic acids could be separately identified in a single chromatographic step using either technique. Good resolution, high sensitivity (low microgram samples) and rapid analysis makes these methods particularly useful for the determination of small amounts of glycosaminoglycans and other glycoconjugates found in samples isolated from biological sources. These two techniques are specifically designed to allow the qualitative determination of the carbohydrate content and composition of samples whose carbohydrate composition and content is completely unknown.