A simple high-pressure liquid chromatographic assay for the N-hydroxy derivatives of phenacetin,acetaminophen, 2-acetylaminofluorene,and other hydroxamic acids

A sensitive high-pressure liquid chromatographic assay is reported for the quantitation of hydroxamic acids including the N-hydroxy derivatives of phenacetin, acetaminophen, and 2-acetylaminofluorene. In this assay ferric chloride is present in the elution solvent and the hydroxamic acids are detected as their respective ferric cholates at 546 nm. With this assay 25 ng of N-hydroxyphenacetin can be accurately quantitated. N-Acetylarylamines are not detected in the assay whereas some ortho-substituted phenol derivatives formed weakly absorbing colored complexes with ferric ions which in all cases studies eluted before the corresponding N-hydroxy-N-acetylarylamine. Other phenolic derivatives of N-acetylaryl-amines which were tested are not detectable in the assay. With this assay it was demonstrated that phenacetin was N-hydroxylated by hamster liver microsome at rates similar to that previously described by a radiochromatographic assay.     

High-pressure liquid chromatographic method for the determination of 25-hydroxycholecalciferol in cow plasma

A high-pressure liquid chromatographic (hplc) procedure was developed for the determination of 25-hydroxycholecalciferol (25-OH-D₃) in cow plasma or serum. The procedure involved extraction with an ethanol-ethyl ether mixture, separation of the aqueous phase, solvent partitions, column chromatography on silica gel, and, finally, determination by reversed phase hplc on a C₁₈-bonded microparticulate silica column. The identity of the drug in the extract was confirmed by comparison with a standard by liquid, thin-layer, and gas-liquid chromatography as the free steroid and the heptafluorobutyrate and by the uv spectra and also from the mass spectrum of the heptafluorobutyrate. Twenty-four samples from cows on normal diet (dry, lactating, and pregnant) were analyzed. The normal circulating levels of 25-OH-D₃ ranged from 40 to 58 ng/g; mean 48 ± 5.0 ng/g. The procedure was used to analyze a limited number of human and hog samples. Human serum contained 10–20 ng/g which was in agreement with literature values. Hog serum contained 18 ng/g.     

Enzymatic generation of N-[4-dimethylamino)phenyl]acetohydroxamic acid by the action of pyruvate decarboxylase on 4-(dimethylamino)nitrosobenzene

The established ability of pyruvate decarboxylase to catalyze the conversion of nitroso aromatics to hydroxamic acids was utilized to generate the previously unknown hydroxamic acid 2. Although 2 could not be isolated in pure form from enzymatic reactions, evidence for its production is presented in this study. Under the conditions of the enzymatic reaction, 2 undergoes a slower reduction to give the corresponding acetanilide 3, which was isolated and characterized. The isomeric hydroxamic acid 4 was synthesized and its stability compared to that of 2. The much greater reactivity of the hydroxamic acid 2, particularly evidenced by its facile reduction to 3, was explained on the basis of the potential for the formation of the N-acylquinonediimine cation, 9.     

Simple inhibitors of histone deacetylase activity that combine features of short-chain fatty acid and hydroxamic acid inhibitors

Butyric acid and trichostatin A (TSA) are anti-cancer compounds that cause the upregulation of genes involved in differentiation and cell cycle regulation by inhibiting histone deacetylase (HDAC) activity. In this study we have synthesized and evaluated compounds that combine the bioavailability of short-chain fatty acids, like butyric acid, with the bidentate binding ability of TSA. A series of analogs were made to examine the effects of chain length, simple aromatic cap groups, and substituted hydroxamates on the compounds' ability to inhibit rat-liver HDAC using a fluorometric assay. In keeping with previous structure-activity relationships, the most effective inhibitors consisted of longer chains and hydroxamic acid groups. It was found that 5-phenylvaleric hydroxamic acid and 4-benzoylbutyric hydroxamic acid were the most potent inhibitors with IC₅₀'s of 5 μM and 133 μM respectively.     

Liquid chromatography/mass spectrometry of fatty acids as their anthrylmethyl esters

The mass spectra of a series of saturated and unsaturated fatty acids have been recorded as their anthrylmethyl esters using a liquid chromatographic mass spectrometric interface. The spectra show an intense peak for the aromatic nucleus, and a molecular ion. The liquid chromatographic/mass spectrometric separation was performed on a reverse phase column using a solvent system of acetone + acetonitrile. While a complete separation of the fatty acids known to occur in man was not achieved, the recognition of all of these acids is possible using a scanning mode or by ion monitoring.     

Analysis of (3-phenyl,2-thio)hydantoin amino acids by high-performance liquid chromatography: Comparison of three programs with particular reference to the glutamic and aspartic derivatives

(3-Phenyl,2-thio)hydantoin-amino acids are readily separated and quantitated by high-performance liquid chromatography (hplc). Three reversed-phase column procedures were investigated. We found the aspartic and glutamic acid derivatives difficult to separate and quantitate by any of the three until we determined the effects on hplc of several injection solvents and the pH of the elutrient buffers. We found 90% acetic acid or acetonitrile: 90% formic acid (8:2, v:v) satisfactory as injection solvents. Each of the three hplc programs also required pH changes for optimal separation of the aspartic and glutamic acid derivatives from the other (3-phenyl,2-thio)hydantoin-amino acids; none of these had retention times that varied with pH in the ranges investigated. Using the three modified systems, the retention times of 19 commonly found (3-phenyl,2-thio)hydantoin-amino acids were compared. In addition, the N-terminal residues of two model human plasma proteins were chromatographed, fibrinogen and plasminogen. In all of these, the aspartic and glutamic acid derivatives were clearly separable without diminishing resolution of the other amino acids.     

Differential separation of thromboxanes from prostaglandins by one and two-dimensional thin layer chromatography

[¹⁴C]-labelled thromboxane B₂ and hydroxy fatty acids were isolated using thin layer and gas chromatographic procedures from human platelets incubated with [1-¹⁴C]-arachidonic acid. A number of TLC solvent systems were evaluated for differential separation of thromboxanes and hydroxy fatty acids from prostaglandins E₂, A₂, D₂ and F_2α. Chromatographic properties in nine different solvent systems are tabulated. Two dimensional TLC procedures suitable for complete resolution of mixtures of these compounds on a single plate were developed. The systems were used to demonstrate conversion of [1-¹⁴C]-arachidonic acid to thromboxane B₂ and prostaglandin E₂ by human lung fibroblasts in tissue culture.     

Simultaneous determination of all-trans-, 13-cis-, 9-cis-retinoic acid and their 4-oxo-metabolites in plasma by high-performance liquid chromatography

A gradient reversed-phase high-performance liquid chromatographic technique is described for the easy separation and quantification of some retinoids; all-trans-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid and their corresponding 4-oxometabolites, in plasma. The method involved a diethyl ether-ethyl acetate (50:50, v/v) mixture extraction at pH 7 with acitretin and 13-cis-acitretin as internal standards. A Nova-Pak C₁₈ steel cartridge column was used. The mobile phase was methanol-acetonitrile (65:35, v/v) and 5% tetrahydrofuran (solvent A) and 2% aqueous acetic acid (solvent B) at 1 ml/min. The gradient composition was (only the percentages of solvent B are mentioned): I, 25% solvent B at the time of injection; II, 12% solvent B at 11 min until 30 min; III, 25% solvent B and maintenance of 25% solvent B for 10 min until a new injection. Total time between injections was 40 min. Detection was by absorbance at 350 nm. The precision calculated for plasma concentrations ranging from 2 to 250 ng/ml was better than 15% and the accuracy was less than 12%. The linearity of the method was in the range of 2 to 400 ng/ml of plasma. The limit of quantification was 2 ng/ml for each of the compounds. The HPLC method was applied to plasma specimens collected from animals receiving single dose administrations of all-trans-retinoic acid, 13-cis-retinoic acid and 9-cis-retinoic acid.     

HRP-catalyzed bioactivation of carcinogenic hydroxamic acids. The greater reactivity of glycolyl- versus acetyl-derived hydroxamic acids

An analysis of the hydroxamic acid oxidation reaction by H2O2 and horseradish peroxidase (HRP) was made with three pairs of hydroxamic acids. Each pair consisted of the aceto- and glycolhydroxamic acid derivatives from one of three different arylhydroxylamines. The parent arylhydroxylamines were the known carcinogens, N-hydroxy-2-aminofluorene and N-hydroxy-4-aminobiphenyl and the noncarcinogen 4-chlorophenyl-hydroxylamine. All the hydroxamic acids appeared to be converted to products that were expected on the basis of the previously-proposed mechanism of this peroxidative reaction. Each acetohydroxamic acid gave the corresponding nitroso compound and O-acetyl ester of the starting material in approximately equal amounts. The glycolhydroxamic acids gave the corresponding nitroso compound and a relatively unstable product that was proposed, by analogy, to be the O-glycolyl ester of the starting material. A comparison of the initial rates of reaction of each hydroxamic acid pair showed that the glycolhydroxamic acid was much more susceptible to the peroxidation reaction than was the corresponding acetohydroxamic acid. The initial rate of the reaction was also highly dependent upon the nature of the aromatic ring in the order fluorene greater than biphenyl greater than 4-chlorophenyl. The relative degree of HRP-catalyzed covalent binding to DNA of the aceto- and glycolhydroxamic acids in the fluorene series was studied and found to parallel the relative rates of reaction of these substrates in the H2O2/HRP system. It was proposed that glycolhydroxamic acids are likely to be more genotoxic than are acetohydroxamic acids when subjected to peroxidative bioactivation conditions.     

Enrichment and high-performance liquid chromatography analysis of tryptophan metabolites in plasma

Tryptophan metabolites with an indole ring are enriched by adsorption either as an ion pair with a trichloroacetic acid anion or as its undissociated form on porous polystyrene polymer (TSK 2000 S) from strongly acidic plasma deproteinized by trichloroacetic acid, and after washing with water, they are eluted with a 90% methanol solution. Following the removal of the solvent, the residue is dissolved in a small amount of water and then subjected to high-performance liquid chromatography (hplc) analysis. Using 0.2 ml of adsorbent, the recovery of the 500 pmol added for each of the tryptophan metabolites into 1.5 ml of deproteinized plasma is above 70%. This method is used for the analysis of normal rabbit and rat plasma. The hplc analysis, with native fluorescence detection, shows several peaks corresponding to tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindole-3-acetic acid, indole-3-acetic acid, and indole-3-propionic acid. Peak identification and cross reactivity were checked by the retention time with two hplc systems, fluorometric characterization, and electrochemical characterization. This method is easy and is simple enough for routine analysis.     

Gas chromatographic–mass spectrometric analysis of hydroxylamine for monitoring the metabolic hydrolysis of metalloprotease inhibitors in rat and human liver microsomes

A gas chromatographic–mass spectrometric (GC–MS) method was developed for the analysis of hydroxylamine (HA) in supernatants obtained from liver microsomes. HA monitoring was used to determine the metabolic hydrolysis of two hydroxamic acid-based matrix metalloprotease inhibitors in rat and human liver microsomes. The hydrolysis of the hydroxamic acids to their corresponding carboxylic acids releases HA as a common metabolic product. HA was derivatized to acetone oxime by addition of acetone to the liver microsomal supernatant, followed by direct injection of the supernatant into the GC–MS, with detection of the oxime by selected-ion-monitoring. The method is simple, reproducible, and sensitive for the determination of the hydrolysis of hydroxamic acid compounds, where hydrolysis is the major metabolic pathway. The methodology can be used for rank ordering and selecting hydroxamic acid analogs based on their susceptibility to hydrolysis.     

Analysis of abscisic acid by high-performance liquid chromatography.

Methodology for the ready analysis of abscisic acid (ABA) in plant tissues based upon application of high-performance liquid chromatography (hplc) has been developed. The method involves isolation of the acid fraction, preparation of the methyl esters with diazomethane, and hplc using a combination procedure of two columns: (1) reversed-phase C₁₈, and (2) porous silica in the absorption mode. Only isocratic elution is required so the method is readily adaptable to laboratories having limited hplc capability. Measured recoveries are 70% and the use of an internal standard allows quantification of ABA levels to 1 ng/g of tissue with minimum absolute detectable levels of ABA of 20 ng. The method is illustrated by analysis of ABA concentration in potato tubers at various times postcutting.     

Purification of intracellular phospholipase A2 from rat spleen supernatant by reverse-phase high-performance liquid chromatography

Intracellular phospholipase A₂ was purified to homogenity from rat spleen supernatant by reverse-phase high-performance liquid chromatography with a trifluoroacetic acid-acetonitrile solvent system. The method simplified the purification procedure, which includes three consecutive chromatographic steps. The recovery of the enzyme activity was greater than 70% with an about 23,000-fold purification. The solvent system did not affect the catalytic properties of the enzyme. Phospholipases A₂ from rat spleen, human pancreatic juice, and porcine pancreas were eluted in that order from a column of octadecasilyl silica gel in a similar concentration range of acetonitrile. This result suggests that the phospholipases A₂ examined have similar hydrophobicities. This method may be applicable to the purification of phospholipases A₂ from other sources.     

A gas chromatography-mass spectrometry method for the quantitation of N-nitrosoproline and N-acetyl-S-allylcysteine in human urine: Application to a study of the effects of garlic consumption on nitrosation

Biomarkers in urine can provide useful information about the bioactivation of chemical carcinogens and can be used to investigate the chemoprotective properties of dietary nutrients. N-Nitrosoproline (NPRO) excretion has been used as an index for endogenous nitrosation. In vitro and animal studies have reported that compounds in garlic may suppress nitrosation and inhibit carcinogenesis. We present a new method for extraction and sensitive detection of both NPRO and N-acetyl-S-allylcysteine from urine. The latter is a metabolite of S-allylcysteine, which is found in garlic. Urine was acidified and the organic acids were extracted by reversed-phase extraction (RP-SPE) and use of a polymeric weak anion exchange (WAX-SPE) resin. NPRO was quantified by isotope dilution gas chromatography-mass spectrometry (GC-MS) using [¹³C₅]NPRO and N-nitrosopipecolinic acid (NPIC) as internal standards. This method was used to analyze urine samples from a study that was designed to test whether garlic supplementation inhibits NPRO synthesis. Using this method, 2.4 to 46.0 ng NPRO/ml urine was detected. The method is straightforward and reliable, and it can be performed with readily available GC-MS instruments. N-Acetyl-S-allylcysteine was quantified in the same fraction and detectable at levels of 4.1 to 176.4 ng/ml urine. The results suggest that 3 to 5 g of garlic supplements inhibited NPRO synthesis to an extent similar to a 0.5-g dose of ascorbic acid or a commercial supplement of aged garlic extract. Urinary NPRO concentration was inversely associated with the N-acetyl-S-allylcysteine concentration. It is possible that allyl sulfur compounds found in garlic may inhibit nitrosation in humans.     

The structure of the O-antigenic side chain of the lipopolysaccharide of Vibrio cholerae 569B (Inaba)

Mineral acid hydrolysis of the lipopolysaccharide from Vibrio cholerae 569B (Inaba) gives an oligosaccharide fraction which was shown, by use of ¹³C NMR and chemical methods, to be a regular α-(1 → 2) linked chain of d-perosamine (4-amino-4,6-dideoxy-d-mannose) units. This chain represents the O-antigen of the lipopolysaccharide, in which the amino functions are acylated with 3-hydroxypropionyl groups. The chromatographic properties of some hydroxamic acids are described and used to characterize these acyl groups.     

Determination of pyridoxal 5′-phosphate as the semicarbazone derivative using high-performance liquid chromatography

A high-performance liquid chromatographic (hplc) procedure is described for the determination of pyridoxal 5′-phosphate (PLP) in animal tissues. The procedure is based on extraction with perchloric acid and treatment with semicarbazide to form PLP-semicarbazone. This derivative is quantitatively determined using hplc with an octylsilica column, an acidic phosphate mobile phase buffer, and fluorometric detection. The validity of the method was confirmed by inspection of the fluorescence spectra of the PLP-semicarbazone hplc peaks of semicarbazide-treated standards and tissue extracts. Preparative chromatography for extract purification is not required because of the hplc efficiency and detection specificity. This method provides a simple technique for the rapid, direct assay of PLP in animal tissues.     

In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-¹⁴C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C_32:0 mycolic acid by radio-gas-liquid chromatographie (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-¹⁴C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C_34:0 + C_34:1) mycolic acids and the other to (C_36:0 + C_36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C_32:0 mycolic acid synthesized by [1-¹⁴C]palmitic acid was pyrolyzed at 300 °C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-¹⁴C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from ¹⁴C-palmitate whereas cerulenin, a specific inhibitor of β-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-¹⁴] fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.     

Participation of liver aldehyde oxidase in reductive metabolism of hydroxamic acids to amides

The liver enzyme responsible for the reduction of aromatic and heterocyclic hydroxamic acids to the corresponding amides was investigated with salicylhydroxamic acid, benzohydroxamic acid, anthranilhydroxamic acid, and nicotinohydroxamic acid. Rabbit liver cytosol exhibited significant reductase activities toward the hydroxamic acids under anaerobic conditions when supplemented with an electron donor of aldehyde oxidase. Similarly, rabbit liver aldehyde oxidase reduced these compounds to amides in the presence of its own electron donor, indicating that the reductase activities observed in the liver cytosol are due mainly to the cytosolic molybdoflavin enzyme. Furthermore, a significant reduction of salicylhydroxamic acid and nicotinohydroxamic acid was also observed, when an electron donor of aldehyde oxidase was added, with liver cytosols from hamsters, guinea pigs, rats, and mice. The cytosolic reductase activities toward salicylhydroxamic acid were markedly inhibited by menadione, an inhibitor of aldehyde oxidase.     

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.     

Purification of chicken testicular müllerian inhibiting substance by ion exchange and high-performance liquid chromatography

A highly purified protein molecule was obtained from the secretory proteins of 8-week-old chicken testes using ion-exchange column chromatographic procedures, including DEAE Bio-Gel A, CM Bio-Gel A, wheat germ lectin columns, and high-performance liquid chromatographic (hplc) separation techniques. This protein molecule has a molecular weight of 74,000 Da (74K protein). The isolated 74K protein induces regression of chicken Müllerian ducts grown in vitro. The 74K protein does not cause regression of cultured embryonic intestine or Wolffian duct. When the total testicular secretory proteins are resolved in a two-dimensional gel electrophoresis, approximately 120 polypeptides are obtained. The purified 74K protein has a pI of 6.1. Analysis of amino acid composition indicates that the 74K protein is relatively acidic in nature with a ratio of acidic to basic amino acids of 1.93.