Determination of malonaldehyde in biological materials by high-pressure liquid chromatography

A new one-dimensional agarose gel electrophoresis method for the quantitation of glycosaminoglycans in biological samples has been described. In this procedure, concanavalin A, suspended in agarose gel, interacts with glycosaminoglycans such that rocket-like precipitin lines are formed. The area of the rocket is directly proportional to the glycosaminoglycan content of the sample. This procedure permits measurement of glycosaminoglycans in amounts as low as 0.5 nmol uronic acid equivalents with a coefficient of variation of only 8%. The described method has been applied to the determination of free heparan sulfate in plasma. This method can also be used to measure all high-charge glycosaminoglycans of biological interest.     

Determination of uric acid in biological fluids by high-pressure liquid chromatography

A high-pressure liquid chromatographic assay for uric acid in biological fluids has been developed. Blood uric acid can be analyzed in as little as 20 μl of plasma. The mean and range of plasma uric acid concentrations in healthy adults determined by high-pressure liquid chromatography were similar to these obtained by enzymatic analysis. One of the advantages of the present method is that naturally occurring metabolites in biological fluids or drugs do not interfere with the analysis. Data are presented for blood and urine specimens obtained from mice fed a known uricase inhibitor, potassium oxonate. Comparisons are made between the present method and methods previously employed for uric acid determination.     

Determination of adenosine kinase activity by high-pressure liquid chromatography

High-pressure liquid chromatography (reverse-phase mode) was used to assay adenosine kinase in cell and tissue extracts. The method is optimized for a rapid and selective analysis using 6-methylthiopurine riboside as substrate, isocratic elution and detection at 300 nm. A complete separation of substrate and product is achieved in about 3 min with no interference by other UV-absorbing compounds; the limit of detection is 20 pmoles.     

Determination of plasma oxalic acid by high-pressure liquid chromatography

A new specific and sensitive method for determination of oxalic acid in plasma by High Performance Liquid Chromatography (HPLC) is described. The plasma sample is deproteinized by ultrafiltration. The oxalic acid in the ultrafiltrate is purified by precipitation with CaCl2, new dilution of calcium oxalate precipitate, oxalic acid extraction with diethyl-ether and total dryness of the sample. The losses of oxalic acid during this process are evaluated by the addition of oxalic acid (U-14C) before the precipitation step. The dried samples are redissolved in mobile phase (o-H3PO4, 0.05 M) and injected into a HPLC chromatograph, with reversed phase column (Lichrosorb RP-8, Merck). Oxalate peak is detected spectrophotometrically at 220 nm with a retention time of 3.20 minutes. The method shows a mean recovery value of 82.11, with an intra-run and between-run CV values of 2.54 and 6.95 respectively. The oxalic acid measured in plasma by this method is 291 +/- 89 micrograms/100 ml plasma ultrafiltrate, in 16 normal subjects.     

Quantitative determination of allantoin in biological fluids by reversed-phase high-pressure liquid chromatography

Allantoin is quantitatively determined in biological fluids by reversed-phase high-pressure liquid chromatography. The proteins of blood plasma are precipitated by perchloric acid. Urine can be analyzed directly. The reproducibility was over 99%. The average recovery of added allantoin in blood was 96% and in urine 98%. The retention factor k′ was 0.235.     

Determination of tissue UDP-glucuronic acid levels by high-pressure liquid chromatography

A new and sensitive method to measure UDP-glucuronic acid extracted from as little as 25 mg wet weight tissue has been developed. This procedure employs high-pressure liquid chromatography and liquid scintillation spectrophotometry to measure p-[¹⁴C]nitrophenylglucuronide generated enzymatically from p-[¹⁴C]nitrophenol and UDP-glucuronic acid. The reaction was catalyzed by UDP-glucuronyltransferase obtained from rat liver microsomes. The tissue levels of UDP-glucuronic acid assayed were 2 to 20 μmol/100 g wet wt, which are well below the levels detectable by the widely used spectrophotometric method.     

Analysis of the free nucleotide pools of mammalian tissues by high-pressure liquid chromatography

Perchloric acid extracts of tissues were neutralized with tri-N-octylamine and, after removal of ClO₄^?, subjected to preliminary purification on a Cu²⁺-loaded column of Chelex 100. A high-pressure liquid chromatographic (HPLC) anion-exchange procedure was developed and gave good resolution of the naturally occurring free nucleotides on a single column. Where heterogeneous peaks eluted, an effective supplementary analysis was achieved by reverse-phase HPLC. An HPLC paired-ion technique was also evaluated for use in nucleotide analysis. Although anion-exchange was best for overall separation of nucleotides, both reverse-phase and paired-ion chromatography gave excellent separation of cyclic nucleotides. Reduced pyridine nucleotides were detected and measured in the form of their acid-decomposition products. The recovery of nucleotides was examined throughout the described analytical techniques and shown to be quantitative.     

Determination of tissue folate composition by affinity chromatography followed by high-pressure ion pair liquid chromatography

A recent report from this laboratory described the use of affinity chromatography for the isolation of pure folates from tissue extracts (J. Selhub, B. Darcy-Vrillon, and D. Fell (1988) Anal. Biochem. 168, 247-251). The present study was undertaken to develop chromatographic procedures for quantitative analysis of the individual folates in the affinity-purified mixture. Methods were devised whereby mixtures containing pteroylglutamates (PteGlu1-7) were batch reduced to the dihydro, H2PteGlu1-7, and tetrahydro, H4Pte-Glu1-7, forms. The 5-methylH4PteGlu1-7 and the 10-formylH4PteGlu1-7 series were prepared from H4Pte-Glu1-7. These compounds were used to calibrate a liquid chromatographic system for the resolution of folate mixtures. This system included reverse-phase ion pair chromatography and a diode array detector. A mixture containing oxidized and reduced PteGlu1-7, a total of 35 derivatives, was separated into seven clusters arranged in an order of increasing number of glutamate residues. Each cluster was represented by two or more peaks which were due to folates that differed in the pteridine ring structure but had the same number of glutamate residues. In clusters containing mono and diglutamyl derivatives the 10-formyltetrahydro-, the tetrahydro-, and the dihydrofolate forms appeared as separate peaks while those representing folic acid and 5-methyl-tetrahydrofolate derivatives eluted in coinciding peaks. This hierarchy was maintained in the following clusters except for increasing tendency of the former three forms of folates to elute in the same peak. The number of glutamate residues of any eluting folate can be determined on the basis of retention time in relation to those of the clusters. The pteridine ring structure of that same folate can be determined on the basis of its elution position within that cluster and spectral characteristics determined by the diode array detection system. If that position is common for more than one derivative then identification is based on differential spectral properties. Using uv absorption signals at 280 nm to determine indiscriminate folate activity, absorption signals at 350 nm are used to identify folic acid and dihydrofolate derivatives and signals at 258 nm are used to identify 10-formyltetrahydrofolate derivatives. These principles were incorporated into mathematic expressions which were used for quantitative resolution of simulated mixtures containing oxidized and reduced PteGlu5 and for the analysis of folate composition in rat liver, human milk, and cows milk.     

Determination of dimethylamine in biological samples by high-performance liquid chromatography

A reversed-phase HPLC method for the quantification of dimethylamine in serum and urine is presented. Dimethylamine (DMA) is converted into a stable fluorescent product by precolumn derivatization with fluorenylmethylchloroformate. The DMA derivative is resolved from derivatives of other amines and amino acids by gradient elution with a total run-time of 15 min. The lower limit of determination in biological samples is 0.1 μmol/1. Recoveries from spiked serum samples were 99–107%. Within- and between-run precision were better than 6%. Concentrations of DMA in serum from normal human subjects (n=8) and from continuous ambulatory peritoneal dialysis patients (n=15) were 3.3±1.5 and 29.2±12.1 μmol/1, respectively.     

Determination of trimethoprim in biological fluids by high-performance liquid chromatography

A rapid, sensitive, and specific high-performance liquid chromatographic assay was developed for the determination of trimethoprim in blood, plasma, and urine using normal-phase (adsorption) chromatography on a microparticulate silica column and UV monitoring at 280 nm. Trimethoprim is selectively extracted from the biological sample matrix at alkaline pH with chloroform, providing nearly quantitative extraction (>95%) and a sensitivity limit of 0.01 to 0.02 μg/ml blood or plasma, without interference from sulfonamides.     

Determination of adenosine deaminase activity using high-pressure liquid chromatography

A method of assaying adenosine deaminase, using high-pressure liquid chromatography to isolate products, has been developed, which has several advantages over available procedures. The method has inherently low background values, affording high sensitivity. Ten picomoles of product can be reliably detected, so that as little as 0.008 unit of enzyme can be determined. Up to six samples per hour can be assayed. The procedure has been applied to erythrocytes and lymphocytes.     

Determination of N-methyl-d-aspartate in tissues of bivalves by high-performance liquid chromatography

The natural occurrence of N-methyl-d-aspartate (NMDA) is limited to the foot muscle of Scapharca broughtonii; it is a well known compound for its neuroexitatory activity. This paper describes a high-performance liquid chromatographic (HPLC) method for the determination of NMDA in biological extracts. The method involves removal of neutral and basic substances by anion-exchange chromatography and removal of acidic primary amino acids by treatment with o-phthalaldehyde before derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate, followed by HPLC with isocratic elution with a selected mobile phase that separates the two diastereomers formed. The identity of the detected NMDA has been confirmed by a procedure using (−)-1-(9-fluorenyl)ethyl chloroformate as a derivatizing agent. The identification has been further supported by the disappearance of the peak of the NMDA derivative by pretreatment of the sample with d-aspartate oxidase. Application of the method has shown the presence of NMDA in several tissues of S. broughtonii and Scapharca subcrenata.     

Analysis of leukotrienes by high-pressure liquid chromatography

A method is described for the partial synthesis of saturated mixed-chain phosphatidylcholines of a high degree (typically 99 mol%) of purity. This procedure has been designed to eliminate the contamination of the mixed-chain product by symmetric chain phosphatidylcholine and the mixed-chain isomer of the desired product, the two principal impurities introduced by previous techniques. This high degree of purity is obtained by employing a method designed for the complete enzymatic hydrolysis of the C-2 fatty acyl moiety in saturated symmetric phosphatidylcholines and a new technique for the acylation of lysophosphatidylcholines employing the catalyst 4-pyrrolidinopyridine. The versatility of this new procedure is illustrated with the synthesis of several saturated mixed-chain phosphatidylcholines.     

Determination of isofezolac in biological fluids by reversed-phase liquid column chromatography

A rapid, sensitive, and specific reversed-phase high-performance liquid chromatography assay was developed for the determination of 1,3,4-triphenylpyrazole-5-acetic acid (isofezolac) in plasma and urine. The assay involves extraction into diethyl ether from plasma buffered at pH 4.4. The organic phase is evaporated and the residue, dissolved in the mobile phase [acetonitrile—water—0.2 M phosphate buffer (pH 3) (65 : 15 : 20)] is chromatographed at a flow-rate of 1.5 ml/min. The drug is detected by its UV absorption (detection limit 100 ng/ml) or its very intense fluorescence (detection limit 10 ng/ml). Absolute analytical recoveries for isofezolac varied from 92.9 to 100.4%. The accuracy is ca. 1%. Each separation requires about 6 min. This method was applied successfully to the determination of isofezolac in humans for pharmacokinetic studies.     

Determination of cephalosporins in biological material by reversed-phase liquid column chromatography

Seven cephalosporins (β-lactam antibiotics), viz. cefazolin, cephalotin, cefoxitin, cefotaxime, cefamandole, cefuroxime and cefoperazone (T 1551) were determined in biological material. The compounds were extracted from acid-treated body fluids into chloroform—1-pentanol (3:1) and re-extracted into a small volume of an aqueous phase at pH 7, which was injected into the chromatographic column. The chromatographic support was μBondapak C_1a (10 μm) and the mobile phase was a mixture of 0.01 M acetate buffer (pH 4.8) and methanol or acetonitrile. Detection limits are about 50 ng/ml for extractions from 1 ml of serum and have permitted pharmacokinetic studies of the seven cephalosporins.     

Determination of methenamine in biological samples by gas—liquid chromatography

Methenamine (hexamethylenetetramine), a urinary disinfectant, was determined in human plasma and urine by gas—liquid chromatography with a short (10 m) open-bone glass capillary column (split ratio 1:20) and nitrogen-selective detector. An almost quantitative recovery (92.1%) was achieved by simple dilution of water-containing samples (0.5 ml) with acetone (4.5 ml). After centrifugation and aliquot (2 μl) of the supernatant was injected into the gas chromatograph. Selectivity and sensitivity of the nitrogen detector allowed the quantitation of unchanged methenamine in plasma and urine up to 24 h after a single therapeutic dose of 1 g.Reproducibility of the method was 7.6 and 2.1% (C.V.) in serum and urine, respectively. The time required for the analysis of one sample was approx. 2 min. Due to the simple extraction and short analysis time it was possible to analyze the samples concurrently with sample taking. Absorption of standard tablets and an enterosoluble preparation of methenamine hippurate was compared.