Simultaneous analysis of substrates,products, and inhibitors of xanthine oxidase by high-pressure liquid chromatography and gas chromatography

Procedures are presented for the simultaneous analysis of hypoxanthine, xanthine, allopurinol, oxipurinol, and uric acid in standard mixtures and physiological fluids using gas chromatography (gc) or high-pressure liquid chromatography (hplc). Excellent correlation was obtained between the two methods for hypoxanthine, xanthine, oxipurinol, and uric acid. There are advantages and disadvantages to both methods. hplc requires no prior derivatization, uses isocratic elution with a buffer containing no organic solvent, and has 50- to 100-fold greater sensitivity than gc. Simpler methods of prepurification, readily adapted to clinical laboratories, can be used for hplc analysis. Although substances that are found in some urine samples from cancer patients interfere with hplc, separations by gc are not affected by these substances.     

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.     

Antioxidant status and dialysis: Plasma and saliva antioxidant activity in patients with fluctuating urate levels

The present study is concerned with the influence of processes occurring during dialysis on the antioxidant capacity of plasma and saliva. The biological fluids were also tested for uric acid and total protein content. Before hemodialysis, plasma antioxidant status of hemodialyzed patients appears slightly higher than the corresponding status in normal subjects; after hemodialysis it is found unchanged. The result can be explained by a balance between a reduction in uric acid plasma content, due to the dialytic procedure, and an increase in protein content, possibly due to a dialysis-related hemoconcentration. Moreover, pre-dialysis total antioxidant capacity of whole saliva samples is higher than in healthy individuals and drastically decreases towards normal values following dialytic procedure. Our data indicate a certain concentration of the uric acid in the saliva of hemodialyzed patients and evidence that both total protein concentration and uric acid level show a good correlation with saliva total antioxidant capacity, suggesting that proteins are major antioxidants of this fluid. Further observations are needed to assess whether this improved saliva antioxidant ability has any consequence on the periodontal conditions of hemodialyzed subjects.     

Antioxidant status and dialysis: Plasma and saliva antioxidant activity in patients with fluctuating urate levels

The present study is concerned with the influence of processes occurring during dialysis on the antioxidant capacity of plasma and saliva. The biological fluids were also tested for uric acid and total protein content. Before hemodialysis, plasma antioxidant status of hemodialyzed patients appears slightly higher than the corresponding status in normal subjects; after hemodialysis it is found unchanged. The result can be explained by a balance between a reduction in uric acid plasma content, due to the dialytic procedure, and an increase in protein content, possibly due to a dialysis-related hemoconcentration. Moreover, pre-dialysis total antioxidant capacity of whole saliva samples is higher than in healthy individuals and drastically decreases towards normal values following dialytic procedure. Our data indicate a certain concentration of the uric acid in the saliva of hemodialyzed patients and evidence that both total protein concentration and uric acid level show a good correlation with saliva total antioxidant capacity, suggesting that proteins are major antioxidants of this fluid. Further observations are needed to assess whether this improved saliva antioxidant ability has any consequence on the periodontal conditions of hemodialyzed subjects.     

Analysis of purines in urinary calculi by high-performance liquid chromatography

A high-pressure liquid chromatography method has been developed for the analysis in urinary calculi of six purines: uric acid, 2, 8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol, and oxypurinol. Separation was conducted isocratically on a reversed-phase column, using 50 mM phosphate buffer (pH 5.5) / methanol (97/3, v/v) as mobile phase. Limits of detection, depending on compound, ranged from 7 to 28 microg/g stone weight. Hitherto, no reports have appeared on other purines present with uric acid in stones, due to lack of a sensitive and specific analytical method. We have now found that all calculi with more than 4% uric acid also contained 1-methyluric and 7-methyluric acids and trace amounts of hypoxanthine, xanthine, and 2,8-dihydroxyadenine. Accurate identification and quantitation of purines in urinary calculi are important for the diagnosis of rare metabolic diseases leading to urolithiasis (xanthinuria, dihydroxyadeninuria), as well as for prevention of iatrogenic complications during treatment with allopurinol of uric acid urolithiasis. The method may be used for reference purposes in clinical laboratories and for research on the pathogenesis of urolithiasis in disorders of purine metabolism.     

Development and validation of an economical uric acid-Fe3+/Fe2+-ferrozine-based colorimetric assay to estimate uric acid level of pure and biological samples

ABSTRACT

This work presents the development and validation of a simple, rapid, and cost-effective spectrophotometric method for quantitative analysis of uric acid in biological samples. The method relies upon uric acid-led reduction of Fe(III) to Fe(II) of sample/standard solutions which stoichiometrically engages ferrozine to form a magenta-colored complex. Different parameters including pH, metal and chelator concentrations, temperature, etc., were optimized for the maximum intensity and stability of the complex. The uric acid concentrations of synthetic/plasma solutions were determined by comparing the color intensity of Fe(ferrozine)₃ ²⁺ complex produced by test solution with the standard curve formed by known uric acid concentrations. The method was validated in accordance with ICH guidelines and subjected to human plasma analysis. The results obtained were compared with a reference (enzymatic) method which revealed that there was no significant difference between the two methods at 95% confidence level. The method is highly specific, precise, linear, accurate, and robust.     

Chromatographic analysis of griseofulvin and metabolites in biological fluids

A simple and accurate assay for the determination of griseofulvin and its metabolites in biological fluids using high-performance liquid chromatography is described. Using a reversed phase column and a mobile phase solvent of 45% acetonitrile in 0.1 M acetic acid, baseline separation of griseofulvin and several analogues was obtained. The described method allows one to quantitatively determine griseofulvin, 6-demethylgriseofulvin, and griseofulvic acid, a newly identified metabolite in man, in urine and plasma samples. Treatment of plasma samples prior to the analysis is simply made by deproteinizing the samples with an equal volume of acetonitrile. For urine samples, the procedure involves diethyl ether extraction with subsequent evaporation to dryness and reconstitution with the mobile phase solvent.     

Anaerobic degradation of uric acid via pyrimidine derivatives by selenium-starved cells of Clostridium purinolyticum

Clostridium purinolyticum decomposed uric acid via pyrimidine derivatives under selenium starvation conditions. Products were acetate, formate, glycine, ammonia, and CO₂. 4,5-Diaminouracil could be identified as an intermediate after converting the labile substance into 6,7-dimethyllumazine. The breakdown of uric acid was inhibited by EDTA. High-pressure liquid chromatography methods have been developed for the simultaneous determination of uric acid, 4,5-diaminouracil, and 6,7-dimethyllumazine. The significance of the new pathway is discussed.Abbreviation HPLC high-pressure liquid chromatography     

The simultaneous analysis of clofibric acid and probenecid and the direct analysis of clofibric acid glucuronide by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic method for the simultaneous quantitative analysis of clofibric acid and probenecid in plasma and urine and for the direct analysis of clofibric acid glucuronide in plasma is described. Both methods involve direct injection of deproteinised body fluids. Concentrations of as low as 10 mg/l of clofibric acid and probenecid and 1.5 mg/l of clofibric acid glucuronide can be measured by the analysis. The coefficient of variance for these methods ranges from 1–7%.     

High-pressure liquid chromatographic analysis of anaerobic photoproducts of bilirubin-IX alpha in vitro and its comparison with photoproducts in vivo.

To carry out photochemical experiments under conditions similar to those prevailing for neonatal bilirubin metabolism in jaundice phototherapy, we have studied photoproducts produced by the action of light on a bilirubin--albumin solution and further clarified the relationship between the photoproducts obtained from experiments in vitro and in vivo. (1) An accurate and sensitive separation method by high-pressure liquid chromatography for photoproducts of bilirubin under anaerobic irradiation of visible light is described. (2) There were two main photoproducts obtained from experiments both in vivo and in vitro. (3) Exact correspondence of retention time on high-pressure liquid chromatography, diazo-reactivity, thermal reversion and absorption-spectrum maxima was observed between unknown pigment and photobilirubin-IX alpha from biological fluids, and the comparable peaks 2 and 3 from experiments in vitro. (4) The behaviour of photoproducts in various solutions in the absence of light and O2 is described. (5) A lower affinity of photoproducts, especially unknown pigment, for human serum albumin than with bilirubin-IX alpha for the albumin was demonstrated by the gel-filtration method.     

Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID-MS) as a candidate reference method

Uric acid is an important diagnostic marker of catabolism of the purine nucleosides, and accurate measurements of serum uric acid are necessary for proper diagnosis of gout or renal disease appearance. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) has been described. An isotopically labeled internal standard, [1,3-(15)N(2)] uric acid, was added to serum, followed by equilibration and protein removal clean up to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) analyses. (M-H)(-) ions at m/z 167 and 169 for uric acid and its labeled internal standard were monitored for LC/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for uric acid (Standard Reference Materials SRM909b) with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added uric acid. The method performed well against the established reference method of ion-exchange followed by derivatization isotope dilution (ID) gas chromatography mass spectrometry (ID-GC/MS). The results of this method for uric acid agreed well with the certified values and were within 0.10%. The amounts of uric acid recovered and added were in good agreement for the three concentrations. This method was applied to determine uric acid in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.08-0.18% and between-set CVs of 0.02-0.07% for LC/MS analyses. Liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analysis was also performed. The LC/MS and LC/MS/MS results were in very good agreement (within 0.14%). This LC/MS method, which demonstrates good accuracy and precision, and is in the speed of analysis without the need for a derivatization stage, qualifies as a candidate reference method. This method can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

High-pressure liquid chromatography: separation of the metabolites of vitamins D2 and D3 on small-particle silica columns.

The high-pressure liquid chromatographic separation of all of the known metabolites of vitamin D(2) and vitamin D(3) found in biological fluids has been achieved. This technique has been successfully applied to the analysis of vitamin D mixtures, purification of vitamin D metabolites, and identification of radioactive peaks. Some theoretical bases for the observed resolutions are suggested.     

Rapid high-pressure liquid chromatographic determination of methotrexate and its metabolites 7-hydroxymethotrexate and 2,4-diamino-N10-methylpteroic acid in biological fluids

A rapid and sensitive high-pressure liquid chromatographic method for determination of methotrexate and its metabolites 7-hydroxymethotrexate and 2,4-diamino-N¹⁰-methylpteroic acid has been developed. The assay is based on isocratic reversed-phase chromatography with siliceous microparticulate Spherisorb (5 μm, ODS, 15 × 0.4 cm i.d.) as stationary phase and a ternary solvent mixture of citrate-phosphate (0.05 m, pH 3.2)/methanol/tetrahydrofurane (80:16:4, v/v) as eluant. A precolumn of Perisorb (RP2, 30–40 μm, 3 × 0.4 cm i.d.) reasonably protects the analytical column against deterioration by the components of plasma or other biological fluids. Since the samples of plasma, urine, or cerebrospinal fluid are directly injected into the chromatographic system, the method is very rapid. Within 8 min as little as 50 ng of methotrexate and its metabolites per milliliter (10^?7m) can be measured with a precision better than 7%. Structural analogs of methotrexate do not interfere with the determination. There is a good correlation with the results of other methods, e.g., enzyme immunoassay or radioimmunoassay. The applicability for clinical monitoring in patient's plasma and urine is demonstrated.     

Isolation and analysis of human parathyrin in parathyroid tissue and plasma. Use of reversed-phase liquid chromatography.

Reversed-phase liquid chromatography techniques have been used to extract and purify human parathyrin from parathyroid adenomas and to analyse the circulating forms of human parathyrin in plasma. Both the supernatant from tissue homogenates, and plasma were extracted with octadecylsilyl-silica (ODS-silica) in a batch procedure. Extracts were subjected to reversed-phase high-pressure liquid chromatography (h.p.l.c.) employing solvent systems composed of aqueous acetonitrile containing trifluoroacetic acid or heptafluorobutyric acid as hydrophobic ion-pairing reagents. The volatile solvents facilitated the radioimmunoassay, bioassay in vitro and amino acid analysis of column fractions and permitted monitoring for u.v. absorbance at 210nm. Isolated glandular parathyrin was found to be homogeneous by sodium dodecyl sulphate/urea/polyacrylamide-gel electrophoresis, to have an amino acid composition conforming to that of human parathyrin-(1--84)-tetraoctacontapeptide and to be bioactive in both renal adenylate cyclase and cytochemical bioassays. ODS-silica extraction permitted examination of large plasms samples by reversed-phase h.p.l.c., facilitating the resolution of the various circulating molecular forms of parathyrin according to their hydrophobic character. Because of its rapidity, excellent recovery and high resolving power, the methodology utilized is uniquely suited to the purification and analysis of parathyrin in tissues and body fluids.     

High-performance liquid chromatographic determination of 2-hydroxypropyl-gamma-cyclodextrin in different biological fluids based on cyclodextrin enhanced fluorescence

A high-performance size exclusion chromatographic method with analyte enhanced fluorescence detection is described for the analysis of 2-hydroxypropyl-gamma-cyclodextrin (HPGCD) in different biological fluids. The principle of detection was the in situ complexation of 8-anilinonaphthalene-1-sulfonic acid (ANS) by HPGCD. When HPGCD eluted from the column the increased fluorescence was measured at excitation and emission wavelengths of 270 and 512 nm, respectively. Solid-phase extraction cleanup and concentration of samples resulted in higher than 78% recovery of HPGCD for each of the studied biological fluids. Some important details of the method development as well as the validation of the method for rabbit plasma, rabbit aqueous humour, monkey plasma and monkey urine are given. The limits of quantification varied between 1 and 10 nmol/ml (correspond to 1.5-15 microg/ml) depending on the biological matrix used. The method was successfully adapted in another laboratory proving that HPGCD had not absorbed into aqueous humour and plasma after topical application of HPGCD containing eye drop in rabbits.     

Kinetics of biomarkers: biological and technical validity of isoprostanes in plasma

Summary. Isoprostanes, non-enzymatic peroxidation products of arachidonic acid, are attractive biomarkers of oxidative stress in research in biology, medicine and nutrition. For the appropriate use of biomarkers it is required that these are both biologically and technically valid. Whereas the biological validity of isoprostanes is well-established, it is technically quite complicated to measure isoprostanes and its metabolites in body fluids, and its rapid disappearance from plasma may hamper practical application. This paper shortly introduces isoprostanes as a biomarker for studies with humans, describes a novel fast and sensitive method for measuring isoprostanes in plasma by high-performance liquid chromatography and tandem mass spectrometry, and provides several examples of the use of the method in studies in humans. By taking care of the biological and technical validity of this biomarker it is possible to establish the antioxidant effects of some food ingredients in studies with human volunteers.     

Analysis of sialyllactoses in blood and urine by high-performance liquid chromatography

A sensitive and highly selective high-performance liquid chromatography (HPLC)-based method has been developed for the analysis of oligosaccharides in biological fluids. In this method, a sample of biological fluid, such as blood serum or urine, is filtered through a 10,000 molecular weight cutoff filter cartridge to remove large molecules such as proteins and lipids. The carbohydrates in the filtrate are then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) as described previously [Anal. Biochem. 180, 351-357, (1989)]. The derivatized carbohydrates are separated by reverse-phase HPLC and monitored by UV absorbance at 245 nm. Quantitative analysis of the carbohydrates can be achieved based on their integration values relative to a standard calibration curve. Since neutral and acidic carbohydrates can be separated by using Dowex 1-X8 anion exchange resin, this method can be used specifically to analyze neutral, acidic, and total carbohydrates in the biological fluids. Because PMP specifically reacts with reducing aldoses, interference from noncarbohydrate components present in the biological fluids is essentially eliminated. This method has proven to be highly sensitive, requiring as little as 5 pmol of analyte for reliable analysis. It has also been used successfully for pharmacokinetic analysis of carbohydrate drugs in human blood and urine samples.     

Rapid determination of doxorubicin and its fluorescent metabolites by high pressure liquid chromatography.

An original method for the separation and quantitation of doxorubicin (DOX) and its metabolites by high-pressure liquid chromatography and fluorometry is described. Doxorubicin and its derivatives are extracted from biological samples in a rapid, non-destructive manner, with a recovery close to 100%. The different compounds are rapidly separated by high-pressure liquid chromatography using an eluant system containing magnesium chloride, and detected quantitatively by fluorometry down to a concentration of 1.5 ng/ml in less than 5 min. Using this method, we have determined doxorubicin and its metabolites in plasma and urine, after an intravenous injection into DBA₂ and NMRI mice.     

Ascorbate prevents prooxidant effects of urate in oxidation of human low density lipoprotein

Uric acid and ascorbic acid are important low molecular weight antioxidants in plasma. Their interactions and combined effect on Cu(2+)-catalysed oxidation of human low density lipoprotein were studied in vitro. It was found that uric acid alone becomes strongly prooxidant whenever it is added to low density lipoprotein shortly after the start of oxidation (conditional prooxidant). Ascorbic acid, which is present in human plasma at much lower concentrations (20-60 microM) than urate (300-400 microM), is in itself not a conditional prooxidant. Moreover, ascorbate prevents prooxidant effects of urate, when added to oxidising low density lipoprotein simultaneously with urate, even at a 60-fold molar excess of urate over ascorbate. Ascorbate appears to have the same anti-prooxidant effect with other aqueous reductants, which, besides their antioxidant properties, were reported to be conditionally prooxidant. Such interactions between ascorbate and urate may be important in preventing oxidative modification of lipoproteins in the circulation and in other biological fluids.     

High-performance liquid chromatographic determination of quinine in rat biological fluids

A high-performance liquid chromatographic (HPLC) method with ultraviolet detection for the determination of quinine in rat biological fluids is described. Due to its selectivity and sensitivity, the proposed method can be used in the case of such rat biological fluids as cerebrospinal fluid (CSF) and perilymph for which the accessible volumes are limited to 100 μl and 10 μl, respectively. Consequently, the assay method has been applied to the measurements of quinine concentration in rat plasma, CSF and perilymph samples.