Dual-column high-performance liquid chromatographic urinary organic acid profiling

Two-dimensional high-performance liquid chromatography (HPLC) is increasingly used for the separation and identification of compounds in biological matrices. Conventional two-dimensional HPLC involves either heart-cutting or column-switching techniques. These techniques work well but are very time consuming when the analysis involves many compounds and requires more than a few minutes to completely elute from the column. We have modified the column-switching technique by utilizing two columns in series, an Aminex HPX-87H organic acid column followed by a 15-cm 5-μm C₁₈ analytical column. Both columns are compatible with the isocratic pH 2.5 H₂SO₄ mobile phase employed in the organic acid profiling. The dual-column system affords better resolution of urinary organic acids than does either column separately. Reversing the column order does not dramatically affect the elution pattern (peak shape, peak height, and R_f values are approximately the same). The dualcolumn HPLC configuration works well as a rapid means of screening urinary carboxylic acids prior to subsequent definitive analyses of abnormal samples.     

Measurement of urinary vanilmandelic acid and homovanillic acid by high-performance liquid chromatography with electrochemical detection following extraction by ion-exchange and ion-moderated partition

An improved protocol has been developed to isolate homovanillic acid (HVA) and vanilmandelic acid (VMA) from urine with strong anion-exchange resin. The sample is diluted with acetate buffer and passed through a disposable column. HVA, uric acid, and many hydrophobic organic acids are removed with 1.0 M acetic acid—ethanol, Then VMA is eluted with 0.5 M phosphoric acid. Two isocratic mobile phases allow rapid high-performance liquid chromatographic measurement of VMA (5 min) and HVA (8 mins) on a 5-μm ODS column. Selective conditions were developed with dual-electrode coulometric detection to permit specific measurement of VMA, HVA, and internal standards, with less than 5% between-run variation.     

Simultaneous determination of volatile and non-volatile acidic fermentation products of anaerobes by capillary gas chromatography

A method previously used for the analysis of organic acids in silage has been applied to the detection and quantification of acidic fermentation products (C₁ to C₆ volatile fatty acids, lactic and succinic acid) of rumen bacteria and anaerobic fungi grown in pure culture. The acids were converted to tertiary butyldimethylsilyl derivatives prior to separation on a 30 m DB1 capillary gas chromatographic column. The quantitative recoveries of formic and succinic acids were found to be comparable to the recoveries of other acids reported in the original study. The quantitative recovery of lactic acid was found to be dependent on storage of the samples at ambient temperature for at least 24 h following derivatization. The simultaneous determination of a wide range of volatile and non-volatile acidic products is an important feature of this method.     

Solid phase extraction procedure for urinary organic acid analysis by gas chromatography mass spectrometry

We have developed a solid phase extraction procedure for the detection of organic acids by GC-MS using a strong anion exchange column (Sep-Pak Vac RC, Accell Plus QMA cartridge). Extraction efficiencies of 25 organic acids were established by analyzing standards in water based solutions. High extraction efficiencies (90 to 100%) were found for many of the compounds studied. We estimated the limit of detection for 48 organic acids and glycine conjugates. They were below 5 nmole with the exception of malonic and oxalic acids and mevalonic acid lactone. This method provides the advantage of higher recoveries for a wide range of compounds of interest and therefore can be a potential alternative to liquid-liquid extraction for organic acid screening. It is especially sensitive for the detection of some polar compounds, such as 3-OH-glutaric and N-acetylaspartic acids.     

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.     

Determination of Steam-Volatile Organic Acids in Fermentation Media by Gas-Liquid Chromatography

Five gas chromatographic liquid phases (25% Carbowax 20 M plus 4% H₃PO₄, 17.5% dioctyl sebacate plus 7.5% sebacic acid, 17.5% dioctyl sebacate plus 7.5% docosanoic acid, 5% Tween 80, and 20% LAC-296 [poly (diethylene glycol adipate)] plus 2% H₃PO₄) were studied with respect to their utility in the separation and quantitation of steam-volatile organic acids commonly produced in fermentation. Optimal operating conditions and column stability for routine analysis were established. An Aerograph Hy-Fi gas chromatograph was used for all work, except the studies with Tween 80 in which an Aerograph A-90-C was employed. Chromatographic traces are presented of volatile fatty acid analyses with each of the liquid phases. Complete separation of all isomers of the fatty acids from C₂ to C₅ was accomplished by the Carbowax 20 M plus H₃PO₄, dioctyl sebacate plus sebacic acid, and dioctyl sebacate plus docosanoic acid columns. The latter two liquid phases were extremely unstable and proved to be unsatisfactory for analysis of aqueous samples. A column of Carbowax 20 M + H₃PO₄ separated steam-volatile organic acids completely. The volatile fatty acid isomers were separated by 5% Tween 80 somewhat less completely, and the peak shapes were not as sharp and symmetrical as that desired for good quantitative work. LAC-296 (20%) plus 2% H₃PO₄ proved to be the most satisfactory of the liquid phases for routine analysis of deproteinated in vitro rumen fermentation media. The column has been used for routine analysis of rumen fermentation fluid and in vitro rumen incubation fluid. All the organic acids from C₂ to C₅, except isobutyric, could be quantitated with this column. Stability of the column with the aqueous solutions was extremely good. The standard deviation of the analysis of each volatile acid component in a fermentation fluid was less than 0.5 molar per cent. The short-chain organic acids (C₂ to C₅) were shown to be extremely stable in aqueous solution for as long as 6 months after preparation for gas chromatographic analysis by protein precipitation with metaphosphoric acid-H₂SO₄ and refrigeration at 4 C in stoppered tubes.     

Quantitation of fourteen urinary alpha-amino acids using isobutane gas chromatography chemical ionization mass spectrometry with 13C amino acids as internal standards

Isobutane chemical ionization gas chromatography mass spectometry of the N-trifluoroacetyl-carboxy-n-butyl ester derivatives of amino acids, using a commercial per-13C-amino acid mixture as internal standards, provided a sensitive and specific method for quantitative analysis of fourteen urinary alpha-amino acids. A computer controlled quadrupole mass spectrometer was used in a selected ion monitoring mode to record the ion current due to the protonated molecular ions of each alpha-amino acid/13C analogue pair. BASIC programmes located peak maxima, and using previously established standard curves, calculated the amino acid content on the bases of both peak height and peak area ratios. Duplicate amino acid analyses are possible on 5 microliter of urine. Instrumental analysis required 25 minutes, automated data processing 10 minutes, and sample preparation 2 hours. Detection limits approached 1 ng with a typical mean standard deviation of 2% for the instrumental analysis.     

A gas-liquid-chromatographic procedure for separating a wide range of metabolites occuring in urine or tissue extracts

1. A gas–liquid-chromatographic procedure is described which permits separation and identification on the same chromatogram of a wide range of substances occurring in urine or tissue extracts. The method uses hydrogen flame ionization, which detects organic compounds whether free or conjugated with no requirement for specific reactive groups. 2. For chromatography, carboxyl groups are quantitatively converted into methyl esters or trimethylsilyl esters. Phenolic, alcoholic and potential enolic groups are converted into trimethylsilyl ethers. Separations are carried out on a 6ft. column of either 10% F-60 (a polysiloxane) or 1% F-60, temperature programming at 2°/min. being used over such part of the temperature range 30°–260° as is required. Propionyl derivatives of hydroxy compounds can also be used, but only on a non-quantitative basis. Derivatives and columns have been selected for optimum range of usefulness when large numbers of samples are examined by using automated gas chromatography. 3. The method is applicable to: fatty acids above butyric acid; di- and tri-carboxylic acids; hydroxy acids and keto acids; polyhydroxy and alicyclic compounds such as glycerol, inositol, quinic acid, shikimic acid, ascorbic acid and sugar alcohols; aromatic hydroxy and acidic compounds, both benzenoid and indolic; sesquiterpenes; steroids; glycine conjugates; mercapturic acids; glucuronides. It is not satisfactory for sulphate conjugates, iminazoles or polypeptides. 4. Methylene units provide an accurate and reproducible parameter for characterizing peak position. Methylene unit values are reported for a large variety of substances occurring in, or related to those occurring in, urine and tissue extracts. 5. The nature of derivatives was confirmed by combining gas chromatography with mass spectrometry. Combined gas chromatography–mass spectrometry gives a diagnostic tool of great power in the evaluation of metabolic patterns, and various uses are discussed.     

Methodology in the study of organic acids in children

A method is described for the extraction of urinary organic acids in children, their conversion to TMS and oxime TMS derivatives and their separation by gas liquid chromatography on a packed column of 10% OV 101. The compounds are identified by mass spectrometry. Profiles of urinary organic acids in normal neonates and in several metabolic disorders are presented.     

Studies on mineral phosphate solubilization by cyanobacteria Westiellopsis and Anabaena

Two diazotrophic cyanobacteria, Westiellopsis prolifica and Anabaena variabilis were evaluated for elucidating the possible mechanism of mineral phosphate solubilization. Phosphate starved cyanobacteria evaluated for the presence of organic acids, extracellular compounds or enzymes that might have been produced and promoted the mineral phosphate solubilization with Mussorie Rock Phosphate and Tricalcium Phosphate as substrates. Both the cultures did not reveal production of organic acids throughout the incubation period when checked for decrease in pH of the media and thin layer chromatography Thin layer chromatography of culture filtrates showed the presence of hydrocarbon like compound. Further analysis of the culture filtrates with gas liquid chromatography, a single peak near to the retention time of 7.6 was observed in all extracts of culture filtrates irrespective of phosphate source. UV-visible spectra of culture filtrates revealed the absorption maxima of 276 nm. Gas Chromatographic-Mass Spectrometric analysis of culture filtrates showed most intense peak in the electron impact (EI) ionization was at m/z 149 and molecular ion peaks at m/z 207 and 167, inferring the presence of phthalic acid. Among the mechanisms in mineral phosphate solubilization, it was evident that these cyanobacteria used phthalic acid as possible mode of P solubilization.     

Quantitative Method for the Gas Chromatographic Analysis of Short-Chain Monocarboxylic and Dicarboxylic Acids in Fermentation Media

A method for the preparation and gas chromatographic analysis of the butyl esters of volatile (C-1-C-7) and nonvolatile (lactic, succinic, and fumaric) acids in microbial fermentation media is presented. Butyl esters were prepared from the dry salts of the acids. The esters were separated by temperature programming on a column of Chromosorb W coated with Dexsil 300 GC liquid phase and analyzed with a flame ionization detector. Apparent recoveries with butanol-HCl or butanol-H2SO4 as butylating agents were 80 to 90% for most acids. Chromatographic profiles of the butyl esters demonstrated that both volatile and nonvolatile acids can be detected and separated in 24 min on a single column. Standard calibration curves (peak area versus concentration) of the butyl esters were linear in the range of 5 to 40 mumol of acid per ml. The advantages of using an internal standard (heptanoic acid) for quantitating fatty acids in a mixture are given. Chromatograms of butylated fermentation media in which rumen anaerobic bacteria were grown illustrated that this method is useful for determining short-chain volatile and nonvolatile acids of toxonomic significance.     

Analysis of major tomato xylem organic acids and PITC-derivatives of amino acids by RP-HPLC and UV detection

Major amino acids and organic acids in xylem exudates of tomato plants were separated by reversed phase high performance liquid chromatography (RP-HPLC) and quantified by UV detection. Before separation, amino acids were converted into their phenylisothiocyanate (PITC) derivatives. In a single run, Asp, Glu, Ser, Gln, His, Thr, Ala, Tyr, Val, Met, Cys, Ile, Leu, Phe, and Lys could be separated and detected down to the pmol level. Unresolved peaks were obtained for Asn and Gly and for Arg and Pro. For organic acid analysis, exudates were pre-treated by perfusion over a prepacked Adsorbex SCX cation exchange column, to eliminate exudate amino acids. Elution recoveries for organic acids were close to 100%. The exudate organic acids were separated by ion suppression RP-HPLC chromatography, and peaks could be resolved for L-malic acid, malonic acid, maleic acid, citric acid and fumaric acid, down to the pmol level. UV signals for exudate ascorbic acid, and succinic acid were below the limits of detection. Determination of oxalic acid and tartaric acid was impossible, due to the presence of the exudate salt peak in the chromatogram. The results indicate the potential of the methods applied, and show the applicability of RP-HPLC analysis for the determination of both amino acids and organic acids in xylem exudates.     

Concurrent quantification of quinolinic, picolinic, and nicotinic acids using electron-capture negative-ion gas chromatography-mass spectrometry.

Quinolinic, picolinic, and nicotinic acids and nicotinamide are end products of the kynurenine pathway from l-tryptophan and are intermediates in the biosynthesis of nicotinamide adenine dinucleotide. These compounds are involved in complex interrelationships with inflammatory and apoptotic responses associated with neuronal cell damage and death in the central nervous system. To facilitate the study of these compounds, we have utilized gas chromatography-mass spectrometry in electron capture negative ionization mode for their concurrent trace quantification in a single sample. Deuterium-labeled quinolinic, picolinic, and nicotinic acids were used as internal standards and the compounds were converted to their hexafluoroisopropyl esters prior to chromatography. Nicotinamide was readily quantified after conversion to nicotinic acid using gas-phase hydrolysis-a process which did not affect the deuterated internal standards. The on-column limit of quantification was less than 1 fmol for each of the analytes and calibration curves were linear. A packed column liner was used in the gas chromatograph inlet to effectively eliminate sample interference effects in the analysis of trace (femtomolar) levels of quinolinic acid. The method enables rapid and specific concurrent quantification of quinolinic, picolinic, and nicotinic acids in tissue extracts and physiological and culture media.     

Application of amino acid analysis by high-performance liquid chromatography with phenyl isothiocyanate derivatization to the rapid determination of free amino acids in biological samples

An amino acid analysis by reversed-phase high-performance liquid chromatography after precolumn derivatization with phenyl isothiocyanate was adapted to the determination of free amino acids in plasma or other biological fluids and in tissue homogenates. Preparation of samples included deproteinization by 3% sulphosalicylic acid, and careful removal under high vacuum of residual phenyl isothiocyanate after derivatization. A Waters Pico-Tag column (15 cm long) was used, immersed in a water-bath at 38°C. In rat or human plasma, separation of 23 individual amino acids, plus the unresolved pair tryptophan and ornithine, was obtained within 13 min. Including the time for column washing and re-equilibration, samples could be chromatographed at 23-min intervals. Variability was tested for each amino acid by calculating the coefficients of variation of retention times (less than 1% in the average) and peak areas (less than 4% for both intra-day and inter-day determinations). The linearity for each standard amino acid was remarkable over the concentration range 3–50 nmol/ml. The mean recovery of amino acid standards added to plasma prior to derivatization was 97 ± 0.8%, except for aspartate (82%) and glutamate (81%). This method is rapid (almost three samples per hour can be analysed, more than in any other reported technique), with satisfactory precision, sensitivity and reproducibility. Therefore, it is well suited for routine analysis of free amino acids in both clinical and research work.     

Measurement of Hydroxy and Hydroperoxy Fatty Acids by a High Pressure Liquid Chromatography with a Column-switching System

Hydroxy and hydroperoxy fatty acids were labeled with 9-bromomethylacridine at room temperature. They were separated from the degradation products and less polar fatty acid derivatives on an octyl silicagel column, and put on an octadecyl silicagel column by on-line column switching. By this method, picomolar levels of the derivatives were measured with good reproducibility. The detection limit of 16-hydroxy-hexadecanoic acid as a representative was 0.9 pmol (S/N =3) and the relative standard deviation of its peak areas was 2.5% (18.5 pmol, n = 7). The method was used for the measurement of hydroxy fatty acids derived from hydroperoxy fatty acids and phosphatidylcholine (PC) hydroperoxides spiked in human plasma. By incubation at 37°C for 4h with human plasma, the hydroperoxy fatty acid was reduced to the corresponding hydroxy fatty acid. In this condition, the PC hydroperoxides showed a considerable decrease, however, a small portion (2.5–3%) of PC hydroperoxides decomposed gave the corresponding hydroxy fatty acids.     

Gas chromatographic detection of sulphur compounds as methyl esters after growth of anaerobic bacteria in broth media

The use of a highly polar capillary column permits gas chromatographic analysis of organic acids as methyl esters. This method has found use in the study of end products of anaerobic bacteria. In addition, it also permits the detection of nonvolatile sulphur compounds which are neglected on the usual packed columns. These compounds have been identified by gas chromatography-mass spectrometry as methyl esters of 3-(methylthio)-propanoic acid and 4-(methylthio)-butanoic acid. When certain species were grown in a medium supplemented with 0·4% (w/v) DL-methionine, the relative amounts of both acids increased significantly. These nonvolatile sulphur compounds may serve as markers for specific bacteria.     

Gas chromatographic profiling and pattern recognition analysis of urinary organic acids from uterine myoma patients and cervical cancer patients

An efficient organic acid profiling and pattern recognition method is described for the correlation between urinary organic acid profiles and uterine cervical cancer. After methoximation of keto acids in alkalinized urine samples, all free organic acids were recovered by a dual solid-phase extraction procedure, followed by conversion to tert.-butyldimethylsilyl derivatives for the profiling analysis by dual-capillary column gas chromatography (GC) with subsequent screening for acids by retention index (I) library matching. A total of 50 organic acids were positively identified in urine samples (0.25 ml) from 12 uterine myoma (benign tumor group) and 14 uterine cervical cancer (malignant tumor group) patients studied. When the GC profiles were simplified to their corresponding organic acid I spectra in bar graphical form, characteristic patterns were obtained for each average of benign and malignant tumor groups. Stepwise discriminant analysis performed on the GC data selected 16 acids as the variables discriminating between the two groups. Canonical discriminant analysis applied to these 16 variables correctly classified 26 urine samples into two separate clusters according to tumor types in the canonical plot.     

Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples

High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-peak with reliable retention time (Rt.). However, in the current study, we have found that under the same LC-MS conditions, the Rt. and shape of LC-peaks of bile acids in urine samples from animals fed dissimilar diets differed significantly among each other. To verify this matrix effect, 17 authentic bile acid standards were dissolved in pure methanol or in methanol containing extracts of urine from pigs consuming either breast milk or infant formula and analyzed by LC-MS/MS. The matrix components in urine from piglets fed formula significantly reduced the LC-peak Rt. and areas of bile acids. This is the first characterization of this matrix effect on Rt. in the literature. Moreover, the matrix effect resulted in an unexpected LC behavior: one single compound yielded two LC-peaks, which broke the rule of one LC-peak for one compound. The three bile acid standards which exhibited this unconventional LC behavior were chenodeoxycholic acid, deoxycholic acid, and glycocholic acid. One possible explanation for this effect is that some matrix components may have loosely bonded to analytes, which changed the time analytes were retained on a chromatography column and interfered with the ionization of analytes in the MS ion source to alter the peak area. This study indicates that a comprehensive understanding of matrix effects is needed towards improving the use of HPLC and LC-MS/MS techniques for qualitative and quantitative analyses of analytes in pharmacokinetics, proteomics/metabolomics, drug development, and sports drug testing, especially when LC-MS/MS data are analyzed by automation software where identification of an analyte is based on its exact molecular weight and Rt.     

Mass Fragmentographic Method for the Determination of Trace Amounts of Putative Amino Acid Neurotransmitters and Related Compounds from Brain Perfusates Collected In Vivo

Abstract: A mass fragmentographic method for the determination of trace amounts of amino acid neurotransmitter candidates from brain perfusates is described. The analytical procedure includes the measurements of glycine, β-alanine, γ-aminobutyric acid, proline, aspartic acid, and glutamic acid; αalanine, leucine, and sarcosine, undergoing gas chromatographic coelution, are detected simultaneously. Amino acids extracted from dried perfusate residues are converted to the corresponding N -pentafluoropropionyl hexafluoroisopropyl esters by a single-step procedure. Gas chromatographic separation of the amino acid derivatives is achieved on a packed glass column filled with trifluoropropylsilicone as stationary phase. The limit of detection for the different derivatives (signal-to-noise, 3:1) ranges from 50 femtomol to 1 picomol. Deuterium-labeled amino acid analogues are used as internal standards for quantitative measurements. The mass spectral characteristics of the derivatives are compared and discussed. The technique has been applied to the assay of amino acids released in vivo within the pigeon optic tectum, demonstrating the capabilities of the present analytical approach.     

Gas Chromatographic Separation and Determination of Optical Isomers of Chrysanthemic Acid

Separation and determination of the optical isomers of chrysanthemic acid has been carried out by gas chromatography. The diastereoisomeric esters of chrysanthemic acid with l-menthol were separated on a column of 2% QF-1 coated on Chromosorb W. d-trans, l-trans and dl-cis Chrysanthemic acids were resolved but d-cis and l-cis acids were not separatable from one another on any column tested. These isomers of chrysanthemic acid were not isomerized during esterification and gas chromatographic operation, and their ratios were determined from their peak area ratios. The l-bornyl esters of the isomers of chrysanthemic acicf were not so easily separatable as the l-menthyl esters.