Quantitation of monohydroxy fatty acids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic (HPLC) procedure for the separation of hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecanoic acids (HODEs) after derivatization of the hydroxy group with 1-anthroylnitrile is described. Anthroyl esters of HETEs were separated from those of HODEs by reversed-phase HPLC. The positional isomers of the HETEs and HODEs were well separated by normal-phase HPLC. The fluorimetric HPLC method has a high sensitivity and naturally occurring HETEs can be quantitatively analyzed at the picomolar level. The amount of 5-HETE in A23187-stimulated polymorphonuclear leukocytes (PMNLs) was determined by the present method. PMNLs produced approximately 150 ng of 5-HETE per 10⁷ cells at 5 min stimulation. The amount of 5-HETE determined by fluorimetric detection was consistent with that determined by ultraviolet detection (235 nm).     

Liquid chromatographic analysis of sebum lipids and other lipids of medical interest

A technique is described for the high-pressure liquid chromatographic (HPLC) analysis of sebum lipid classes. The lipid classes present in sebum are separated by gradient elution HPLC from a microparticulate silica column and detected using a moving-wire detector. The system described can be linked to a computer. Quantitation can be carried out by comparing peak areas obtained with those of an internal standard. Peak trapping for further investigations of the separated components, for example by gas chromatography—mass spectrometry, is very easy.Sebum lipids are separated into the following lipid classes: hydrocarbons and squalene, cholesterol esters and wax esters, fatty acids as their methyl esters, triglycerides, 1,3-diglycerides, 1,2-diglycerides, free cholesterol, monoglycerides and other polar materials. Besides to sebum, the method has been successfully applied to other lipid mixtures, such as serum lipids. Examples of other applications are shown.     

Surface lipid composition of C3 and C4 plants

The characteristic surface lipid compositions of several C₃ and C₄ plants are discussed. C₄ plants produce surface lipids (epicuticular waxes) made up of the ubiquitous classes of aliphatic compounds: free fatty acids, aldehydes, primary alcohols, alkanes and aliphatic linear esters. C₃ plants synthesize surface lipids comprising the ubiquitous classes and either of the two following groups of compound: (i) lβ-diketones, hydroxy lβ-diketones, alkan-2-ol esters; (il) ketones and secondary alcohols with the functional group in the middle of the hydrocarbon chain. These features are suggested to represent physioIogical characteristics of the plant and to be related to ecological adaptations. Wax class compositions might also be an ancillary method for defining the C₃ or C₄ mechanism of CO₂ assimilation in cases where uncertainty exists.     

THE MAIN NONPOLAR CHLOROPHYLL c FROM EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) IS A CHLOROPHYLL c 2-MONOGALACTOSYLDIACYLGLYCERIDE ESTER: A MASS SPECTROMETRY STUDY

The main nonpolar chlorophyll c  -like pigment was extracted from Emiliania huxleyi (Lohm.) Hay et Mohler (strain CCMP 370) cultures and isolated by preparative column chromatography and HPLC. The pigment, whose visible spectrum closely resembled that of chlorophyll c  ₂, was studied by low-resolution fast atom bombardment mass spectrometry, showing a very high mass molecular ion (m/z 1313). The fragment ions, either in the direct spectrum or obtained by tandem mass spectrometry with collision-induced dissociation of the molecular ion, were compatible with the consecutive losses of two fatty acids (14:0 and 18:4), glycerol, and a hexose, leaving a chlorophyll c  ₂ backbone, suggesting the molecule consists of a chlorophyll c  ₂ residue linked, via an ester bond, to the sugar moiety of a monohexosyldiacylglycerol. The identities of the two fatty acid residues (14:0 and 18:4n-3) were subsequently corroborated by gas chromatography of the corresponding methyl esters. Chemical hydrolysis–derivatization–gas chromatography–mass spectrometry demonstrated the occurrence of glycerol and that galactose is the constituent sugar. The porphyrin obtained on acid hydrolysis showed chromatographic and visible spectral properties identical to pheoporphyrin c  ₂. This evidence led us to propose a tentative structure whose molecular formula, C₇₆H₉₆O₁₄N₄Mg, was supported by the values of exact mass measurements by high-resolution fast atom bombardment mass spectrometry. This novel structure represents the highest molecular weight natural chlorophyll described to date.     

Assessment of fatty acids in cardiac tissue as 9-anthryldiazomethane esters by high-performance liquid chromatography

A high-performance liquid chromatographic technique for the rapid assessment of fatty acids in cardiac tissue is described. A level of 50.4 ± 14.9 nmol fatty acids per g wet weight of rat myocardial tissue could be monitored. The content of the individual fatty acids C_14:0, C_16:0, C_16:1, C_18:0, C_18:1, C_18:2 and C_20:4 amounted to 1.9, 13.5, 0.6, 14.4, 6.1, 6.5 and 7.2 nmol/g wet weight, respectively. A comparison of this method with a well established gas chromatographic technique yielded good agreement. In contrast with time-consuming gas chromatographic techniques, there is no need to isolate (unesterified) fatty acids from the other lipid classes with column chromatography or thin-layer chromatography, because the derivatizing reagent 9-anthryldiazomethane reacts highly specifically with fatty acids.     

High pressure liquid chromatography methods for separation of omega- and (omega-1)-hydroxy fatty acids: their applications to microsomal fatty acid omega-oxidation

Fatty acids (C12-C18) and their omega- and (omega-1)-hydroxy derivatives, when converted to p-bromophenacyl (PBP) esters, can be completely separated from one another by high pressure liquid chromatography (HPLC) on a silicic acid column using 0.5% (v/v) isopropanol in n-hexane. In this system, fatty acid PBP esters are eluted at the solvent front, whereas the retention times of the omega- and (omega-1)-hydroxy derivatives are 14-20 and 24-29 min, respectively. The PBP esters can also be separated by reverse phase HPLC on a muBondapak C18 column, a method which has been developed by Fan et al. (Fan, L. L., Masters, B. S. S., and Prough, R. A. (1976) Anal. Biochem. 71, 265-272) for separation of methyl esters of fatty acids and their omega- and (omega-1)-hydroxy derivatives. In the latter method, however, the retention times of omega- and (omega-1)-hydroxy derivatives are only about 2 min apart and an increase in the solvent polarity is needed for elution of the esters of unmodified fatty acids. Fatty acid PBP esters, however, can be obtained as independent peaks which are not disturbed by the solvent front. An application of the former method to measure fatty acid omega oxidation by liver microsomes and by a reconstituted monooxygenase system containing purified cytochrome P-450 is described.     

The synthesis and some biological properties of N-(6-purinyl)peptides

The chemical synthesis and biological properties of N-(6-purinyl)peptides are described. N-(6-Purinyl)amino-acid derivatives were synthesized and condensed with amino acid esters and peptide esters using the dicyclohexylcarbodiimide/N-hydroxysuccinimide method. The products were isolated via gel filtration on Sephadex G-10 in 0.05M NH4HCO3 followed by either ion exchange chromatography on SP-Sephadex or by preparative HPLC. The methyl esters were saponified and the tert-butyl ester group was removed by treatment with trifluoroacetic acid without damaging the purinyl residue. N-(6-Purinyl)peptides were characterised by chromatographic and spectroscopic methods. Acid hydrolysis of N-(6-purinyl)-L-amino acids caused the racemization of the neighbouring L-amino acid. Model studies were performed with N-(6-purinyl)-L-alanine, N-(6-purinyl)-D-alanine, N-(6-purinyl)-L-alanyl-L-leucine and N-(6-purinyl)-D-alanyl-L-leucine. After acid hydrolysis the N-(6-purinyl)amino acids were totally racemized and the N-(6-purinyl)dipeptides formed 14% of the enantiomer of alanine. The N-(6-purinyl)-omega-amino acids and the N-(6-purinyl)peptides were screened in a limited number of tests as immunomodulators (antibody-secretion, phagocytosis, cytostatic activity of macrophages) and as cytotoxic agents.     

Epicuticular wax of Juniperus scopulorum

Epicuticular wax from Juniperus scopulorum contains hydrocarbons (16%), esters (11%), free acids (1%), nonacosan-10-ol (27%), nonacosane-diols (7%) and estolides (16%). The major hydrocarbon is tritriacontane; the principal esters are C₃₄–C₄₆, mainly octyl and decyl esters of C₂₈-C₃₆ acids; and the diols consist of nonacosane-4,10-diol (57%),5,10-diol (28%), 7,10-diol (11%) and 10,13-diol (4%). Hydrolysis of the estolides gave a mixture of acids, ω-hydroxy acids, , ω-diols, alcohols and hydroxy acids. The hydroxy acids are a new class of C₂₈–C₃₆ acids with the OH attached to either the eighteenth or twentieth carbon from the terminal methyl end; the major component is 13-hydroxydotriacontanoic acid. Syntheses of this acid and of nonacosane-4,10-dione and nonacosane-4,10-diol are described.     

Capillary gas-liquid chromatography of glycine-conjugated bile acids without prior hydrolysis

A method is described for the gas-liquid chromatographic (GLC) analysis of intact glycine conjugates of the major bile acids present in human plasma. It is, therefore, now possible to analyze glycine-conjugated and unconjugated bile acids together on a single GLC column without the necessity for a hydrolytic step. A large number of derivatives of bile acid glycine conjugates were examined, but only acetate- and silyl ether-derivatives of carboxylic acid methyl esters were found initially to be suitable. It was not possible to make acetates consistently, and trimethylsilyl ethers did not allow resolution of the glycine conjugates of cholic and chenodeoxycholic acids. Dimethylethylsilyl ether methyl ester derivatives were subsequently found to give the best results. Chromatographic conditions for successful analysis of these derivatives were examined and it was found to be necessary to use wall-coated capillary columns of thin film thickness (0.12 micron) and very high carrier gas flow rates (ca. 20 ml/min hydrogen). Using acetonitrile and Bond Elut extraction, fractionation on Sep-Pak SIL cartridges, and derivatization as dimethylethylsilyl ether methyl esters, the capillary gas-liquid chromatography of intact glycine-conjugated bile acids from human plasma was demonstrated for the first time.     

Chromophoric determination of putrescine, spermidine and spermine with dabsyl chloride by high-performance liquid chromatography and thin-layer chromatography

A fast and sensitive method for the determination of putrescine, spermidine, spermine and ammonia by high-performance liquid chromatography (HPLC) with dabsyl chloride is described. These compounds are converted to their chromophoric dabsyl derivatives and are separated by a normal-phase chromatographic column (μPorasil, 10 μm) with 2% acetone in chloroform as isocratic mobile phase. The sensitivity of the method is 20 pmoles. The present method was shown to be a straightforward procedure for estimating polyamines in various rat tissues.The chromophoric derivatives of polyamines are also well separated by thin-layer chromatography (TLC) on silica gel, and the combination of the HPLC and TLC procedures provides a reliable method for qualitative and quantitative analysis of polyamines.     

A gas-liquid chromatographic method for steric analysis of epoxy acids

A gas-liquid chromatographic method for determination of the absolute configuration of the two chiral carbon atoms of epoxy fatty acids was developed. The method involved (1) conversion of the saturated epoxy ester into a pair of regioisomeric allylic alcohols by consecutive treatments with selenophenoxide anion and hydrogen peroxide, (2) oxidative ozonolysis performed on the (-)-menthoxycarbonyl derivatives of the allylic alcohols, and (3) steric analysis of the resulting two 2-hydroxy acids (methyl esters, (-)-menthoxycarbonyl derivatives) by gas-liquid chromatography using appropriate reference compounds. Application of the method for steric analysis of several synthetic epoxyoctadecanoates as well as (+)-vernolic acid derived from Vernonia galamensis is described.     

Covalent immobilization of pure isoenzymes from lipase of Candida rugosa

Covalent immobilization of pure lipases A and B from Candida rugosa on agarose and silica is described. The immobilization increases the half-life of the biocatalysts ( ) with respect to the native pure lipases ( ). The percentage immobilization of lipases A and B is similar in both supports (33–40%). The remaining activity of the biocatalysts immobilized on agarose (70–75%) is greater than that of the enzymatic derivatives immobilized on SiO₂ (40–50%). The surface area and the hydrophobic/hydrophilic properties of the support control the lipase activity of these derivatives. The thermal stability of the immobilized lipase A derivatives is greater than that of lipase B derivatives. The nature of the support influences the thermal deactivation profile of the immobilized derivatives. The immobilization in agarose (hydrophilic support) gives biocatalysts that show a greater initial specific reaction rate than the biocatalysts immobilized in SiO₂ (hydrophobic support) using the hydrolysis of the esters of (R) or (S) 2-chloropropanoic and of (R,S) 2-phenylpropanoic acids as the reaction test. The enzymatic derivatives are active for at least 196 h under hydrolysis conditions. The stereospecificity of the native and the immobilized enzymes is the same.     

Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites

A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of hydroxytestosterone metabolites. The method combines a Hypersil BDS C₁₈ analytical column (10 cm×0.46 cm) and a linear mobile phase (1.25 ml/min) gradient of tetrahydrofuran–acetonitrile–water (10:10:80, v/v) changing to tetrahydrofuran–acetonitrile–water (14:14:72, v/v) over 10 min then remaining isocratic for 3 min. The total run time for the chromatographic separation of eight metabolites of testosterone is 15 min. Detection by UV is linear between 300 ng/ml and 10 μg/ml with a limit of detection on column of 300 ng/ml. A method for the direct HPLC analysis of liver microsomal incubates of [¹⁴C]testosterone is also briefly described and when combined with the HPLC method, offers a distinct advantage over previously reported methods for the rapid screening of testosterone hydroxylase activity in rat and human liver microsomes.     

The synthesis and biochemical evaluation of new A1 selective adenosine receptor agonists containing 6-hydrazinopurine moieties

The synthesis and SAR of a series of novel derivatives of N-aminoadenosine is described, along with their in vitro effects in biochemical assays. The rat brain A₁ adenosine receptor binding of these compounds is very dependent upon the purine 2-substituent. The novel agonist, 2-chloro-N-[4-(phenylthio)-1-piperidinyl]adenosine, exhibits a L_i value for A₁ receptor binding of <1 nM.     

Gas chromatography and detection of microquantities of gibberellins and indoleacetic acid as their fluorinated derivatives

Gas chromatographic analysis of halogenated derivatives of the plant hormones IAA and gibberellins is described, employing an electron capture detector. Heptafluorobutyryl (HFB) and trifluoroacetyl (TFA) derivatives of the gibberellin and IAA methyl esters have excellent chromatographic properties and allow quantitative and qualitative analysis on the nanogram to picogram level. The methods have been used in analysis of apple seed hormones.     

High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids.

A sensitive negative chemical ionization (NCI) gas chromatography-mass spectrometry (GC-MS) method for the detection of pentafluorobenzyl (PFB) esters of deuterated fatty acids is described. Deuterated linoleic [18:2n-6 2H4-9,10,12,13] and linolenic [18:3n-3 2H5-17,17,18,18,18] acids were converted to chain-elongated and desaturated products during incubations with homogenates prepared from rat liver. The extracted fatty acids were derivatized with pentafluorobenzyl bromide and analyzed in the negative ion mode by GC-MS. The detection limit of the PFB esters in NCI using selected ion monitoring was below 10 femtograms. In general, detection of the PFB derivatives using the negative ion mode was more than three orders of magnitude more sensitive than using a positive chemical ionization (PCI) method with methyl ester derivatives. The PFB esters of the 2H4-18:2n-6 metabolites eluted with their unlabeled analogues, whereas the PFB esters of the 2H5-18:3n-3 metabolites were resolved from the unlabeled compounds on polar capillary FFAP columns. Isotope ratios of the 2H4-18:2n-6 metabolites were used to quantify the deuterated compounds from standard dilution curves generated from the ion abundances of the unlabeled fatty acids. The 2H5-18:3n-3 metabolites were quantified similarly using 18:3n-3. This method is feasible for the study of the in vivo metabolism of deuterated essential fatty acids in whole animals.     

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.     

A one-vial method for routine extraction and quantification of free fatty acids in blood and tissue by HPLC

A one-vial procedure has been developed to quantify free fatty acids in human blood serum and rat heart tissue. To allow routine analysis the system has been constructed to allow simultaneous processing of nine samples (Praechromat). The free fatty acids are extracted with Freon 11 and then derivatized to coumarin esters prior to HPLC. The Freon 11 extraction of the free fatty acids is rapid and complete. Neither hydrolytic degradation of natural fatty acid esters nor oxidative damage of unsaturated fatty acids was observed. Fifteen free fatty acids (FFA) were routinely quantified by isocratic elution with high reproducibility (SD less than 4%) and good recovery (0.1 mM FFA: 98-100%, 0.02 mM: 91-105%). The free fatty acids could be determined in the range from 20 pmol to 20 nmol.     

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.     

Cloning, Expression, and Pharmacology of Four Human 5-Hydroxytryptamine4 Receptor Isoforms Produced by Alternative Splicing in the Carboxyl Terminus

Abstract: We report here the molecular cloning of three new splice variants of the human serotonin 5-hydroxytryptamine₄ (h5-HT₄) receptor, which we named h5-HT_4(b), h5-HT_4(c), and h5-HT_4(d). The sequence following the splicing site at Leu₃₅₈ in the C-terminal tail of h5-HT_4(b) displays a 74% protein identity with the same region in the long form of the rat 5-HT₄ receptor (r5-HT_4L) but is shorter by 18 amino acids compared to its rat counterpart. The splice variants h5-HT_4(c) and h5-HT_4(d) are the first of their kind to be described in any animal species. The C terminus of h5-HT_4(c) displays a high number of putative phosphorylation sites. The h5-HT_4(d) isoform corresponds to an ultrashort form of the receptor, with a truncation two amino acids after the splicing site. Tissue distribution studies revealed some degree of specificity in the pattern of expression of the different isoforms within the human body. The four splice variants transiently expressed in COS-7 cells displayed an identical 5-HT₄ pharmacological profile and showed a similar ability to stimulate adenylyl cyclase activity in the presence of 5-HT. The stimulatory pattern of cyclic AMP formation in response to the 5-HT₄ agonist renzapride was found to be significantly different between h5-HT_4(a) and the other h5-HT₄ isoforms, indicating that the splice variants may differ in the way they trigger the signal transduction cascade following receptor activation.