Analysis of Hydroxyl,Unsaturated, and Cyclopropane Fatty Acids by High Pressure Liquid Chromatography

Abstract

The investigation describes the HPLC separation of hydroxyl fatty acids as their methoxyphenacyl esters. All separations were performed using two types of reverse phase columns, μ-Bondapak C 18 and Fatty Acid Analysis columns. The solvent system consisted of acetonitrile: water gradient. A simple method for the trifluoroacetylation of the hydroxyl group is described which permits the identification of these specific fatty acids and decreases the lower limit in chain length determination for these fatty acids.

A method for the hydrogenation of unsaturated fatty acid mixtures is described, and the effects of variations in time, temperature, pressure, and the amount of catalyst are examined on the percent hydrogenation of unsaturated to the saturated forms of the fatty acids. Further, the effects of the different variables are related to the percent of hydrogenation of the cyclopropane fatty acids. Evidence is provided on the hydrogenation of the cyclopropane which does not agree with previously described ring opening mechanisms which have been proposed from GLC data which may be due to the difference in temperatures under which the chromatographic processes are carried out. These findings indicated the possibility of isolating an intermediate in the process by HPLC.     

Mass Spectrometric Distinction of Free Phenoxy Fatty Acids from Phenols Part I: Application of El-Mass Spectrometry

Abstract

By comparison of electron impact mass spectra of 16 phenoxy fatty acids with their corresponding phenols, it becomes obvious that the major parts of both spectra are identical.

The dominating signal is the phenol ion. It corresponds to the molecule ion for free phenols and to that fragment for the phenoxy fatty acids which is formed by the loss of the acid moiety under proton migration. As the signals of higher masses than the phenol ions are small in most cases, and as these substances have comparable chromatographic properties, correct identification in mixtures is difficult to achieve. The spectra of both classes of compounds are given to allow the selection of specific signals for the differentiation of fatty acids versus phenols, when this is possible.     

Ion Exchange High Performance Liquid Chromatography of Leukotrienes

Abstract

A novel anion exchange liquid chromatographic system has been developed for isocratic separation of leukotrienes. Hydrophobic as well as ionic forces were found to influence the separation. By optimization of solvent strenght, ionic strenght and pH, amphoteric peptidoleukotrienes could be separated simultaneously with hydroxy fatty acids such as leukotriene B₄ and its ω-oxidized metabolites. To obtain a good buffering capacity of the mobile phase at optimum pH, a multicomponent buffer was developed.     

Quantitative HPLC Analysis of Plasma Amino Acids as Orthophthaldialdehyde/Ethanethiol Derivatives

Abstract

A reverse phase high performance liquid chromatographic method for the analysis of plasma amino acids is described. The method employs pre-column derivatization with o-phthaldialdehyde using ethanethiol as the reducing agent. The analysis shows good linearity and reproducibility. An average overall difference of 12% was seen for results obtained by the HPLC method versus those obtained with an amino acid analyzer. The chromatographic parameters of buffer concentration and column temperature were also examined.     

Enantiomeric Separation of N-Protected Non-Protein Amino Acid Esters by Chiral High-Performance Liquid Chromatography

Abstract

We have found that high-performance liquid chromatographic analysis of enantiomeric N-protected amino acid esters on a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase column (Daicel Chiralcel OD) can be utilized as one of the procedures for determining the optical purities of non-protein amino acids. The methyl esters of the N-benzyloxycarbonyl (Z) derivatives of a number of non-protein amino acids showed excellent to good enantiomeric separations using hexane - 2-propanol as a mobile phase. There was a regularity in the elution order of enantiomers: the L-isomer had a shorter retention time than the D-isomer. We have also investigated the effect of the N-protecting groups and the ester groups on the enantiomeric separation. The Z, 4-methoxybenzyloxycarbonyl (Z(OMe)), and 9-fluorenylmethoxycarbonyl (Fmoc) derivatives gave exceptionally good resolutions. By contrast, the formyl and t-butoxycarbonyl (Boc) groups impaired the enantiomeric separation. Almost all the alkyl esters examined and the benzyl ester gave resolutions better than or of the same order as the methyl ester. The resolution of β-amino acids was worse than that of the corresponding α-amino acids.     

Analysis of Free Fatty Acids on the Fingertips by High Performance Liquid Chromatography

Abstract

This investigation studied the efficiency of high performance liquid chromatography in the determination of free fatty acids present on the fingertips, and assessed the quantitative relationship between skin fatty acids and the degree of microbial contamination. Automated surgical scrub was utilized to eliminate the microbial contamination.

The high performance liquid chromatography provided excellent separation of skin fatty acids for evaluation with the bacterial counts. The fatty acid peaks identified ranged in chain length from C12 through C32. All the fatty acids evaluated showed positive correlation with the bacterial counts with the exception of one acid which had an inverse relationship but none were statistically significant. Finally, the surgical scrub chromatograms showed that the straight chain acids C19 and C21 were lower in concentration than C23 and C25; also, C26 was lower in concentration than C28 and C30.

It was evident from the data that fatty acids which have been shown to be bacteriostatic in vitro do not demonstrate the same property on the fingertips. The finding of lower concentration of C19, C21, and C26 than longer chain acids is inconsistent with simple two carbon addition, and indicates there is possible branching at these points in metabolic pathway of fatty acid synthesis.     

The Sampling and Gas Chromato-graphic Analysis of Fatty Acids from Landfill Sites

Abstract

A gas chromatographic technique has been developed and applied to the qualitative and quantitative analysis of fatty acids in the interstitial gases of landfill sites. Acetic, propionic. and butyric acids were those shown to be present in the highest concentrations. The fatty acid concentrations were higher at a site where landfill had been completed two years ago than at a site where operations had finished twenty-five years ago.

The concentrations of fatty acids measured were well above the odour thresholds. These compounds may be responsible for the unpleasant smells frequently associated with landfill gases.     

Isocratic Separation of Fatty Acid Derivatives by Reversed Phase Liquid Chromatography. Influence of the Solvent on Selectivity and Rules for Elution Order

Abstract

The p-bromophenacyl esters of 16 fatty acids (C₁₂-C₂₂) have been separated by isocratic chromatography on a Radial Pak A cartridge (Reverse phase C₁₈ material). The separation factors α were measured using two solvent mixtures of comparable strength and the superiority of methanol-water to acetonitrile-water becomes evident.

Five precise rules are established, which indicates the retention of every fatty acid. They explain the chromatographic process i.e. elution order, resolution and selectivity.     

Direct analysis of volatile organic compounds in foods by headspace extraction atmospheric pressure chemical ionisation mass spectrometry

Rationale

The rapid screening of volatile organic compounds (VOCs) by direct analysis has potential applications in the areas of food and flavour science. Currently, the technique of choice for VOC analysis is gas chromatography/mass spectrometry (GC/MS). However, the long chromatographic run times and elaborate sample preparation associated with this technique have led a movement towards direct analysis techniques, such as selected ion flow tube mass spectrometry (SIFT‐MS), proton transfer reaction mass spectrometry (PTR‐MS) and electronic noses. The work presented here describes the design and construction of a Venturi jet‐pump‐based modification for a compact mass spectrometer which enables the direct introduction of volatiles for qualitative and quantitative analysis.

Methods

Volatile organic compounds were extracted from the headspace of heated vials into the atmospheric pressure chemical ionization source of a quadrupole mass spectrometer using a Venturi pump. Samples were analysed directly with no prior sample preparation. Principal component analysis (PCA) was used to differentiate between different classes of samples.

Results

The interface is shown to be able to routinely detect problem analytes such as fatty acids and biogenic amines without the requirement of a derivatisation step, and is shown to be able to discriminate between four different varieties of cheese with good intra and inter‐day reproducibility using an unsupervised PCA model. Quantitative analysis is demonstrated using indole standards with limits of detection and quantification of 0.395 μg/mL and 1.316 μg/mL, respectively.

Conclusions

The described methodology can routinely detect highly reactive analytes such as volatile fatty acids and diamines without the need for a derivatisation step or lengthy chromatographic separations. The capability of the system was demonstrated by discriminating between different varieties of cheese and monitoring the spoilage of meats.     

Determination of Free Fatty Acids in Natural Oils and Alkyd Resins by High Performance Liquid Chromatography

Abstract

Mixtures of free fatty acids in natural oil and alkyd resin samples have been analyzed using a μBondapak Free Fatty Acid column in conjunction with a ternary mobile phase. Variation of the mobile phase composition allows ‘‘fingerprinting'’ as well as quantitation of the fatty acid components. Samples can be analyzed in ten minutes by this method. The results of the application of this technique to the identification of oil sources of fatty acids as well as the production of fatty acids during alkyd resin synthesis are given. Good agreement is observed for fatty acid compositions determined via HPLC with those obtained by gas chromatographic methyl ester analysis.     

Analysis of free fatty acids by a micro-liquid chromatograph directly coupled with a mass spectrometer through a vacuum nebulizing interface

A liquid chromatograph directly coupled with a quadrupole mass spectrometer through a vacuum nebulizing interface was applied to the analysis of various free fatty acids. Chemical ionization mass spectra of the C₇? C₂₂ free fatty acids were first examined using either methanol or benzene as the reagents. Then the practical compositional analysis of the fatty acids were performed with various biological samples such as bean oil, rape oil, palm oil and milk fat where most of the fatty acids are included as their triglycerides.     

Direct amidation of non-activated carboxylic acid and amine derivatives catalyzed by TiCp2Cl2

This paper described a mild and efficient direct amidation of non-activated carboxylic acid and amine derivatives catalyzed by TiCp₂Cl₂. Arylacetic acid derivatives reacted with different amines to afford the corresponding amides in good to excellent yield except of aniline. Aryl formic acids failed to react with aniline but smoothly reacted with aliphatic amines and benzylamine in moderate to good yield, fatty acids reacting with benzyl and aliphatic amines give amides in good to excellent yield. Chiral amino acids derivatives were transformed into amides without racemization in moderate yield. The possible mechanism of direct amidation catalyzed by TiCp₂Cl₂ was discussed. This catalytic method is very suitable for the amidation of low sterically hindered arylacetic acid, fatty acids with different low sterically hindered amines except aniline, as well as the amidation of aryl formic acid with benzyl and aliphatic amines.     

Chemometrics‐assisted chromatographic fingerprinting: An illicit methamphetamine case study

The volatile chemical constituents in complex mixtures can be analyzed using gas chromatography with mass spectrometry. This analysis allows the tentative identification of diverse impurities of an illicit methamphetamine sample. The acquired two‐dimensional data of liquid–liquid extraction was resolved by multivariate curve resolution alternating curve resolution to elucidate the embedded peaks effectively. This is the first report on the application of a curve resolution approach for chromatogram fingerprinting to identify particularly the embedded impurities of a drug of abuse. Indeed, the strong and broad peak of methamphetamine makes identifying the underlying peaks problematic and even impossible. Mathematical separation instead of conventional chromatographic approaches was performed in a way that trace components embedded in methamphetamine peak were successfully resolved. Comprehensive analysis of the chromatogram, using multivariate curve resolution, resulted in elution profiles and mass spectra for each pure compound. Impurities such as benzaldehyde, benzyl alcohol, benzene, propenyl methyl ketone, benzyl methyl ketone, amphetamine, N‐benzyl‐2‐methylaziridine, phenethylamine, N ,N ,α‐trimethylamine, phenethylamine, N ,α,α‐trimethylmethamphetamine, N‐acetylmethamphetamine, N‐formylmethamphetamine, and other chemicals were identified. A route‐specific impurity, N‐benzyl‐2‐methylaziridine, indicating a synthesis route based on ephedrine/pseudoephedrine was identified. Moreover, this is the first report on the detection of impurities such as phenethylamine, N ,α,α‐trimethylamine (a structurally related impurity), and clonitazene (as an adulterant) in an illicit methamphetamine sample.     

Direct HPLC Resolution of Racemic Nomifensine Hydrogen Maleate Using a Chiral Beta-Cyclodextrin-Bonded Stationary Phase

Abstract

A direct liquid chromatographic method was developed for the optimization of separation of racemic S, R-nomifensine hydrogen maleate, a psychotropic drug, this was achieved without any derivatization, using a β-cyclodextrin-bonded phase column. A separation factor of 1.46 and resolution of 4.30 were obtained for the enantiomers of nomifensine hydrogen maleate. The effect of pH, temperature and methanol content in mobile phase on retention and enantioselectivity of nomifensine hydrogen maleate was also demonstrated.     

Structural characterization of a vegetable oil‐based polyol through liquid chromatography multistage mass spectrometry

A commercial vegetable oil‐based polyol for rigid polyurethane foams has been characterized by liquid chromatography‐electrospray ionization‐quadrupole ion trap mass spectrometry (LC‐ESI‐QIT‐MS). The absolute molecular weight (MW = 960) was measured by gel permeation chromatography (GPC) equipped with both refractive index (RI) detector and static laser light‐scattering detector (SLSD), which allowed further analysis by LC‐MS. The oligo‐polyol mixture was first separated in two elutes and then investigated by a deep multistage mass spectrometry (MSⁿ) study and completed using NMR. The major constituents identified were regioisomers of propoxylated sucrose (n_PO = 6–12), and the related esters of C16:0, C18:1, and C18:2 fatty acids had a mass ratio of 6:3:1. A comparison of fatty acids composition between the sample and palm oil demonstrated that the sample was initially prepared from the mixture of sucrose and palm oil by direct propoxylation. The MSⁿ fragmentation studies validated the structure of propoxylated sucrose and the related fatty acids derivatives. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 255–262     

Analysis of Polyunsaturated Fatty Acids Using High Performance Liquid Chromatography – Atmospheric Pressure Chemical Ionization Mass Spectrometry

Analysis of polyunsaturated fatty acids using high performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry is described. The standard fatty acid methyl esters from 16 to 22 carbons were analyzed by LC‐MS with APCI. The effect of orifice voltage and total carbon atoms versus number of double bonds in each homologue on the mass spectra is discussed. The correction coefficients for homologues from saturated fatty acids to hexaenoic acid are also mentioned.     

Seed Oil Fatty Acids of Mongolian Compositae: The trans‐Fatty Acids of Heteropappus hispidus,Asterothamnus centrali‐asiaticus and Artemisia palustris

Seed oils from the Compositae plant family are known to contain a variety of unusual fatty acids. Subsequent to the recent discovery of γ‐linolenic acid in Saussurea and Youngia, further Mongolian Compositae species were investigated for their seed oil fatty acid composition. A number of δ3trans‐fatty acids (16 : 1δ3t, 18 : 1δ3t and 18 : 3δ3t, 9c, 12c) were found in the seed oils of Heteropappus hispidus and Asterothamnus centrali‐asiaticus. The latter fatty acid, but not the trans‐monoenes, was also found in one species of Artemisia. These unusual fatty acid isomers were characterized by capillary gas‐liquid chromatographic (GLC) separations in combination with other chromatographic techniques (analytical thin layer chromatography, TLC and preparative argentation TLC), and infrared spectrocsopy (IR). Their identity was further confirmed by co‐chromatography with other seed oils known to contain these trans‐fatty acids. The fact that within the Compositae plant family there are apparently two or three distinct groups of genera containing δ3trans‐fatty acids is discussed.     

An improved derivatization method for sensitive determination of fatty acids by high-performance liquid chromatography using 9-(2-hydroxylethyl)-carbazole as derivatization reagent with fluorescence detection

Summary Tagging techniques with reagents used for fluorescent detection for short and long-chain fatty acids using high-performance liquid chromatography are evaluated in terms of the tagging reactions, handing, flexibility, stability of the reagents. Emphasis is given to the applications of the tagging techniques to relatively high molecular mass fatty acids. The fatty acids or carboxylic compounds were derivatized to their corresponding esters with 9-(2-hydroxy ethyl)-carbazole (HEC) in acetonitrile at 60°C with N, N′-carbonyldiimidazole (CDI) as a coupling agent in the presence of 4-dimethylaminopyridine (DMAP). A mixture of esters of C₁−C₂₀ fatty acids was completely separated with 45 min using gradient elution on a reversed-phase C₁₈ column. The maximum fluorescence emission for the derivatized fatty acids is at 365 nm (λ_ex 293 nm). Studies on derivatization conditions indicated that fatty acids react rapidly and smoothly with HEC in the presence of CDI and DMAP in acetonitrile to give the corresponding sensitively fluorescent derivatives. The application of this method to the analysis of long chain fatty acids in plasma is also investigated. The LC separation shows good selectivity and reproducibility for fatty acids derivatives. The relative standard deviations (n=6) for each fatty acid derivative are <5.0%. The detection limits are at 38–57 fmol levels for C₁₄−C₂₀ fatty acids and lower levels for <C₁₄ fatty acids.     

Quantitative determination of the fatty acid composition of human serum lipids by high-performance liquid chromatography

A high-performance liquid chromatographic method for the analysis of the fatty acid composition of human serum lipids with fluorescence detection was examined. Both free and total fatty acids extracted from serum were derivatized with 9-anthryldiazomethane and were analysed using methanol-water (94.7:5.3) as mobile phase. Twelve kinds of fatty acid were detected, both in the free and total fatty acids, and were well separated. Concentrations of individual fatty acids of serum lipids were estimated from an internal standard, heptadecanoic acid. The results correlated well with those from two other quantitative analyses. These results indicate that the high-performance liquid chromatographic analysis of fatty acids is a reliable method for determining individual fatty acids of human serum lipids. The compositions of free fatty acids and total fatty acids of serum lipids were analysed and compared in 27 normal subjects, 27 diabetics, and 20 angina pectoris patients by this method.     

Determination of 2-Oxoglutarate in the Presence of Citrate and/or Isocitrate After Ion-Exclusion High-Performance Liquid Chromatography

Abstract

A method has been designed for the quantification of 2-oxoglutarate after ion-exclusion high performance liquid chromatography of samples containing high levels of citrate plus isocitrate, which interfere in the quantification of 2-oxoglutarate. The amount of 2-oxoglutarate in a mixed chromatographic peak containing citrate plus isocitrate has been estimated using a diode array detector and calculating the extinction coefficient of these molecules at three wavelengths. The reliability of the method has been ascertained using mixtures of standard organic acids. This procedure has been applied to determine the intracellular content of 2-oxoglutarate in the cyanobacterium Phormidium laminosum.