A sensitive assay of galactocerebroside beta-galactosidase by high-performance liquid chromatography using galactosyl-N-dansyl-sphingosine as substrate

A rapid and sensitive method was devised for determining β-galactosidase activity specific for galactocerebroside. A fluorescent derivative of galactocerebroside, 1-O-galactosyl-2-N-1-dimethylaminonaphthalene-5-sulfonyl-sphingosine, was used as substrate, and the product, 2-N-1-dimethylaminonaphthalene-5-sulfonyl-sphingosine, was taken into organic solvent phase. Quantitative analysis of 2-N-dimethylaminonaphthalene-5-sulfonyl-sphingosine was carried out fluorometrically by use of high-performance liquid chromatography on silica gel column.     

Quantitative analysis of sulpiride in body fluids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the analysis of sulpiride, N-ethyl-2-(2-methoxy-5-sulphonamido-benzamido-methyl)-pyrrolidine, in body fluids is described. A structurally related compound, N-ethyl-2-(2,4-dimethoxy-benzamido-methyl)-pyrrolidine, was used as internal standard.A fluorescence detector with excitation maximum at 299 nm and emission maximum at 342 nm was used for the quantitation. The detection limit was about 10 ng/ml in serum and cerebrospinal fluid and about 200 ng/ml in urine. The experimental error was 5–10% in the concentration range 25–100 ng/ml. Some preliminary data from a pharmacokinetic study in healthy volunteers are presented. The half-life for sulpiride in serum was about 8 h. Sulpiride was also measured in cerebrospinal fluid from five drug-treated psychotic patients.     

Rapid assay for theophylline in clinical samples by reversed-phase high-performance liquid chromatography

A fast, sensitive and highly specific method for the determination of theophylline in human serum is reported. Using a C₁₅-bonded reversed-phase column with an acetonitrile—acetate buffer mobile phase theophylline is completely resolved not only from other dietary xanthines and their metabolites but also from co-administered drugs such as paracetamol and phenobarbitone. Use of β-hydroxyethyltheophylline as internal standard allows a within batch precision of 2.0% and a between batch variation of 3.0%. Factors involved in the development of the method and its performance are discussed.     

Application of high-performance liquid chromatography with a chemiluminescence detection system to determine catecholamines in urine

Chemiluminescence generated with the reaction of bis(2,4,6-trichlorophenyl)oxalate and hydrogen peroxide was applied to a detection system for high-performance liquid chromatography to determine fluorescamine-labeled catecholamines. The sensitivity of the chemiluminescence detection system with 25 fmol of detection limit was approximately 20 times higher than that of a conventional fluorescence detection system. Norepinephrine and dopamine in human urine were determined by the use of the new high-performance liquid chromatography detection system with the coefficient of variation of less than 4.0%. Good correlations (r = 0.998 for norepinephrine and r = 0.999 for dopamine) were obtained between the values by the present method and the conventional method.     

Determination of plasma and urinary cortisol by high-performance liquid chromatography using fluorescence derivatization with dansyl hydrazine

A method is described for the determination of cortisol in human plasma and urine by high-performance liquid chromatography using fluorophotometric detection. After extraction with methylene chloride, cortisol is labelled with dansyl hydrazine, and then separated by high-performance chromatography. The eluate is monitored by a fluorophotometer at 350 nm (excitation) and 505 nm (emission). The optimum conditions for the determination, such as HCl and dansyl hydrazine concentrations, reaction time and reaction temperature, and for the eluent of high-performance liquid chromatography, are discussed. Linearity of the fluorescence intensity (peak height) with the amount of cortisol was obtained between 0.5 and 60 ng. The recoveries for 50 and 100 ng of added cortisol were 98.7 and 95.4% for plasma, and 96.4 and 90.6% for urine, respectively. Comparison with a radioimmunoassay gave a correlation coefficient of 0.978. The proposed method is suitable for the routine analysis of cortisol in plasma and urine.     

Quantitative microanalysis of oligosaccharides by high-performance liquid chromatography

The rapid separation and quantitative determination of per-O-benzoyl oligosaccharides were obtained using high performance, liquid chromatography. Oligosaccharides were completely O-benzoylated without concomitant N-benzoylation of acetamidodeoxyhexoses. Benzoylation prior to analysis allowed a quantitative determination of picomolar amounts because the absorbance at 230 nm of these derivatives is directly proportional to the number of benzoyl groups present. Separation by normal and reversed-phase chromatography was demonstrated, and the best resolution was obtained on an Ultrasphere octyl column. Excellent separations of oligosaccharides containing up to 10 sugar residues present in mannosidosis urine and of malto-oligosaccharides containing up to 15 sugar residues present in Karo syrup were achieved within an analysis time of 30 min. Anomers of maltose, maltotriose, and maltotetraose were separated; for this reason, reduction of complex samples prior to analysis is advisable. The effect of linkage configuration on retention time was tested with reduced, α-linked di- and tri-glucopyranosides. The presence of an acetamidodeoxyhexose residue in an oligosaccharide significantly reduced its retention time, whereas branching had the opposite effect. A linear response was obtained for the injection of 1–600 pmol of raffinose, and the detection limit was 0.5 pmol. Derivatization and analysis of raffinose was shown to yield reproducible results within the range 0.01–1 μmol, and, with special precautions to minimize losses, as little as 100 pmol could be analyzed successfully.     

Cimetidine assay in human plasma by liquid chromatography

An assay for the determination of cimetidine in human plasma is described. Cimetidine was extracted from alkalized plasma with ethyl acetate, washed once over hydrochloric acid, re-extracted into ethyl acetate, and the organic phase was evaporated to dryness. The residue was dissolved in ethanol and injected into a liquid chromatograph.In vitro sulphoxidation was found to occur in whole blood, for which reason the assay was performed in plasma. The accuracy of the method was found to be within 3% and the lower limit for sensitivity was demonstrated to be 0.1 mg/l using 750 μl plasma.Five volunteers received 1 g cimetidine perorally per day given in four doses with various intervals. Blood samples were drawn hourly, five dose intervals over two days. The average minimum concentration of plasma cimetidine was found to correlate significantly with the mean value of the area under the time/concentration curve over a period of three dose intervals (r = 0.96).     

Determination of 6-mercaptopurine and azathioprine in plasma by high-performance liquid chromatography

Using 1-ml plasma samples, levels of 6-mercaptopurine (6MP) as low as 5 ng/ml and azathioprine (AZA) as low as 40 ng/ml can be detected using a high-performance liquid chromatography reversed-phase column procedure following extraction. Both compounds were stable in frozen plasma for seven weeks. AZA stability in blood was temperature dependent; the half-lives of AZA breakdown to 6MP at 37° were 28 and 46 min in blood drawn from two rhesus monkeys. Plasma levels of 6MP were measured in a rhesus monkey following 6MP (1.47 mg/kg) and AZA (3 mg/kg) intravenous administration. 6MP levels were also measured in three renal transplant patients on daily 50- and 100-mg AZA doses. Peak levels (45–75 ng/ml) were reached within an hour and 6MP levels were detected for up to 7 h.     

Measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography

A rapid procedure for the isolation, separation, identification and measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography (HPLC) is presented. The initial isolation of these compounds from urine was accomplished with small disposable ion-exchange columns. HPLC was performed on a silica gel column with a mobile phase composed of methylene chloride, methanol and 1 M aqueous ammonium formate buffer. Peaks were recorded at both 254 nm and 280 nm and the response ratio was used in conjunction with the elution volume for compound identification. The minimum detectable amount (signal-to-noise ratio = 2) ranged from 0.2 ng for uracil to 2.2 ng for cytidine. Linearity and recovery for thymine, uracil, uridine, pseudouridine, orotic acid and orotidine added to urine was demonstrated over almost a 10³ concentration range. The potential application of this method for the study of inborn errors in the urea cycle is discussed.     

Separation of phosphohydroxyamino acids by high-performance liquid chromatography

A high-performance liquid chromatography system has been developed which resolves O-phosphoserine, O-phosphothreonine, and O⁴-phosphotyrosine. Separation is performed on an anion-exchange resin eluted with phosphate buffer of low pH. Detection of the amino acid derivatives is accomplished using O-phthalaldehyde in an in-stream fluorometric system. This highly sensitive method has been applied to the detection of phosphohydroxyamino acids in bovine myelin basic protein phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase and in whole bovine myelin phosphorylated by endogenous kinases.     

Simple,rapid, and highly efficient separation of amino acid phenylthiohydantoins by reversed-phase high-performance liquid chromatography

A rapid and efficient separation of amino acid phenylthiohydantoins by high-performance liquid chromatography has been accomplished by step-gradient elution with use of a mobile phase of acetate-buffered aqueous acetonitrile and an octadecylsilyl stationary phase. Typical analyses are completed in less than 12 min. Peak elution positions in this procedure are highly reproducible (with about 0.2% variance) and allow unambiguous identification of all residues. A procedure for the optimal positioning phenylthiohydantoin-Arg and -His is given. Molar extinction coefficients at 254 nm, which are particularly useful with common fixed wavelength detectors, are given for 25 amino acid phenylthiohydantoins.     

Separation and analysis of azosemide in urine and in serum by high-performance liquid chromatography

A new application of high-performance aqueous gel permeation chromatography was developed for the analysis of human serum lipoproteins. A good combination of columns (TSK GEL, type PW and type SW) was found for the separation of serum lipoproteins: very low-density lipoprotein, low-density lipoprotein and high-density lipoproteins. Analyses of serum lipoproteins from individual normal subjects and pathological subjects were performed by this combination of columns. The effects of pH and salt concentration of the eluent on the separation of lipoproteins were also investigated.