Linear solvent strength model on porous graphitic carbon stationary phase using high temperature liquid chromatographic method for allopurinol related substances analysis.

A high-performance liquid chromatography (HPLC) method was developed for the analysis of Allopurinol and its Ph.Eur. impurities using a porous graphitic carbon (PGC) stationary phase. Retention behavior of solutes was studied across a wide temperature range (30-90 °C) and various gradient times (5-20 min). Analysis of the data revealed distinct retention mechanisms between reversed-phase and PGC phases. However, it was proved that the retention of Allopurinol and its Ph.Eur. impurities on PGC stationary phase can be effectively modeled using the linear solvent strength (LSS) theory. This allows for the utilization of LSS-based method development software to optimize methods under these conditions. By using commercial chromatographic modeling software, separation of Allopurinol and Ph.Eur. impurities was optimized within a large design space. At the optimized operating conditions (pH = 2.0, tG = 6 min, T = 60 °C), all solutes were separated within 6 min with baseline resolution. Comparison between predicted and experimentally measured chromatograms further confirmed the applicability of LSS theory in developing analytical methods for PGC-based HPLC systems. The presented approach offers a general framework for method development on PGC phases.

Extraction optimization, structural characterization and potential alleviation of hyperuricemia by flavone glycosides from celery seeds.

Celery seeds are commonly used as condiments and in herbal teas with high medicinal value. In the present study, we investigated the contents of extracts derived under different extraction conditions and determined the optimal conditions for only extracting flavone glycosides from celery seeds. The compositional analysis identified three primary flavone glycosides in the ethanolic extract, and apiin, graveobioside A, and graveobioside B were isolated. Apigenin, luteolin, and chrsyeriol were obtained by the acid hydrolysis of flavone glycosides under high-temperature conditions. Here we investigated the inhibitory activity of apigenin and apiin on xanthine oxidase by reducing the rate of oxidative cytochrome C and found that both apigenin and apiin reduced cytochrome C production, except for low concentrations of apiin. In vivo analysis with hyperuricemia mice and rats showed that apiin had excellent uric acid-lowering effects and high dose-dependence, while apigenin was relatively slightly uric acid-lowering. In addition, the flavone glycoside extracts from celery seeds exhibited similar effects of reducing uric acid with apiin. Surprisingly, in hyperuricemia rats, the uric acid-lowering effects of high-dose apiin and flavone glycoside extracts were almost comparable to that of allopurinol. Besides, our experimental results showed that apigenin could improve uric acid clearance by increasing the glomerular filtration capacity, which was reflected in reducing the renal function parameters SUN and SCr; also, apiin showed better results. This study also showed that celery seeds have a unique medicinal value in treating hyperuricemia and that the flavone glycoside extracts from celery seeds can be developed as medicine for hyperuricemia.

Developing, optimizing, and assessing a green electrophoretic method for determination of benzbromarone and allopurinol with its active metabolite in biological and pharmaceutical matrices.

A combination of allopurinol and benzbromarone is a common gout treatment protocol. A suboptimal response to allopurinol in patients is very common due to its pharmacokinetics variability. Moreover, the safe doses of benzbromarone is very crucial in patients with hepatic diseases. This raised the inquisitiveness to develop and optimize a capillary zone electrophoresis method for the determination of allopurinol and benzbromarone in their coformulation and in the presence of oxypurinol, the active metabolite of allopurinol, in biological and pharmaceutical matrices. The method greenness profile was assessed using green metric tools the "National Environmental Method Index," the "Analytical Eco-Scale," and the "Green Analytical Procedure Index" by which the method proved to be ecofriendly. The method was successfully applied for the analysis of the pharmaceutical preparation and urine samples spiked with both drugs and the active metabolite. The linearity range was 25.0-250.0 µg/mL for benzbromarone, 50.0-350.0 µg/mL for allopurinol, and 100.0-500.0 µg/mL for oxypurinol. The recoveries were 99.60 ± 0.67, 99.89 ± 0.98, and 98.71 ± 1.18% for benzbromarone, allopurinol, and oxypurinol, respectively. The analysis results indicate potential usefulness of capillary zone electrophoresis as a competitive and greener method of analysis in biological and quality control labs.

UHPLC-fluorescence method for the determination of trace levels of hydrazine in allopurinol and its formulations: Validation using total-error concept.

The present approach poses an interesting way to quantify residues of the genotoxic impurity hydrazine in allopurinol and its pharmaceutical formulations using ultra high performance liquid chromatography coupled to fluorescence detection. Hydrazine was pre-column derivatized through a unique chemistry with o-phthalaldehyde under acidic conditions. Using highly acidic mobile phase the derivative exhibits a strong fluorescence intensity. Derivatization and chromatographic parameters were thoroughly investigated. The validation of the developed method has been carried out in the range of 10 to 200% of the target concentration limit of the analyte using the accuracy profiles as a graphical decision-making tool. The ß-expectation tolerance intervals did not exceed the acceptance criteria of ±20% which means that 95% of future results will be included in the defined bias limits. The variation of the relative bias ranged between -6.0 and 0.5% and the RSD values for repeatability and intermediate precision were lower than 6.9% in all cases. The limit of detection (LOD) and the lower limit of quantification (LLOQ) were satisfactory and found to be 0.3 ng mL-1 (corresponding to 0.03 µg g-1 in solid sample). Experimental designs were constructed to study the robustness of the instrumental method and the derivatization procedure. The developed method has been successfully applied for the analysis of hydrazine in allopurinol API batches and tablets indicating that this methodology could be adopted from QC laboratories.

Automated fluorimetric determination of the genotoxic impurity hydrazine in allopurinol pharmaceuticals using zone fluidics and on-line solid phase extraction.

In the present study we report a fully automated method for the determination of low levels of the genotoxic impurity hydrazine in allopurinol active pharmaceutical ingredient (API) and formulations based on the concept of zone-fluidics. Hydrazine reacts on-line with o-phthalaldehyde in a unique way, that is in acidic medium (pH < 1.5) and in the absence of nucleophilic reagents, to form a highly fluorescent hydrazone (λex/em = 318 / 376 nm). The adaptation of a 120 s long stopped-flow step at elevated temperature (70 °C) offered adequate sensitivity (LOD =0.9 µg L-1 or 0.1 ppm in the solid API) to meet the pharmacopoeia limits for the selected application. The analyte was separated efficiently from the excess of the API by on line solid phase extraction using a Hydrophilic-Lipophilic technology sorbent that provided direct retention of the more hydrophobic API without the need of wetting/conditioning steps. Percent recoveries ranged between 93.3 and 105.8%.

[Simultaneous determination of allopurinol, probenecid, benzbromarone in dietary supplements by ultra high performance liquid chromatography-tandem mass spectrometry].

A method based on QuEChERS purification was developed for the simultaneous determination of allopurinol, probenecid and benzbromarone in anti-gout dietary supplements by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The samples were extracted by acetonitrile mixed with 0.1% (v/v) ammonium hydroxide, and the extracts were purified using primary secondary amine (PSA) and C18 adsorbents. The samples were separated on a C18 chromatographic column with the gradient elution of 0.1% (v/v) formic acid aqueous solution and methanol as mobile phases. The analytes were detected by a electrospray ionization source in the positive or negative ion mode and the multiple reaction monitoring mode. The results showed that the limits of detection of allopurinol, probenecid and benzbromarone were 5, 25 and 25 µg/kg, and the limits of quantification were 17, 80 and 80 µg/kg. The average spiked recoveries of the three chemical drugs in dietary supplements were in the range of 76.8%-116.6% with the relative standard deviations of 2.7%-14.6%. The proposed method was applied for the analysis of 68 dietary supplements, and allopurinol was detected in one of them. This method is simple and sensitive, and can be used for the determination of the allopurinol, probenecid and benzbromarone in anti-gout dietary supplements.

Improved bioautographic assay on TLC layers for qualitative and quantitative estimation of xanthine oxidase inhibitors and superoxide scavengers.

A new agar-free bioautographic assay for xanthine oxidase (XO) inhibitors and superoxide scavengers on TLC layers was developed and validated. Compared to the first version of TLC bioautographic agar overlay method, our bioautographic assay greatly improved the sensitivity and quantification ability. The limit of detection (LOD) of this assay was 0.017ng for allopurinol. Quantitative estimation of XO inhibitors and superoxide scavengers was achieved by densitometry scanning, expressed as allopurinol equivalents in millimoles on a per sample weight basis. This assay has acceptable accuracy (95.37-99.23%), intra-day and inter-day precisions (RSD, 2.56-6.69%), as well as intra-plate and inter-plate precisions (RSD, 2.93-9.62%). Six pure compounds and three herbal extracts were evaluated for their potential XO inhibitory and superoxide scavenging activity by this bioautographic assay on TLC layers. Four active components were separated, located and identified in Astragalus membranaceus var. mongholicus extract by the bioautographic assay after TLC separation. The developed method is rapid, simple, sensitive and stable for screening and estimation of the potential XO inhibitors and superoxide scavengers.

Oxypurinol - A novel marker for wastewater contamination of the aquatic environment.

The anti-gout agent allopurinol is one of the most prescribed pharmaceuticals in Germany and is widely metabolized into oxypurinol (80%) as well as the corresponding riboside conjugates (10%) within the human body. To investigate the occurrence of allopurinol and oxypurinol in the urban water cycle an analytical method was developed based on solid phase extraction (SPE) and subsequent liquid chromatography electrospray-ionization tandem mass spectrometry (LC-MS/MS). In raw wastewater concentration levels of oxypurinol ranged up to 26.6 µg L(-1), whereas allopurinol was not detected at all. In wastewater treatment plant (WWTP) effluents, concentrations of allopurinol were

Why do most human liver cytosol preparations lack xanthine oxidase activity?

When investigating the potential for xanthine oxidase (XO)-mediated metabolism of a new chemical entity in vitro, selective chemical inhibition experiments are typically used. Most commonly, these inhibition experiments are performed using the inhibitor allopurinol (AP) and commercially prepared human liver cytosol (HLC) as the enzyme source. For reasons detailed herein, it is also a common practice to perfuse livers with solutions containing AP prior to liver harvest. The exposure to AP in HLC preparations could obviously pose a problem for measuring in vitro XO activity. To investigate this potential problem, an HPLC-MS/MS assay was developed to determine whether AP and its primary metabolite, oxypurinol, are retained within the cytosol for livers that were treated with AP during liver harvest. Differences in enzymatic activity for XO and aldehyde oxidase (AO) in human cytosol that can be ascribed to AP exposure were also evaluated. The results confirmed the presence of residual AP (some) and oxypurinol (all) human liver cytosol preparations that had been perfused with an AP-containing solution. In every case where oxypurinol was detected, XO activity was not observed. In contrast, the presence of AP and oxypurinol did not appear to have an impact on AO activity. Pooled HLC that was purchased from a commercial source also contained residual oxypurinol and did not show any XO activity. In the future, it is recommended that each HLC batch is screened for oxypurinol and/or XO activity prior to testing for XO-mediated metabolism of a new chemical entity.

Physicochemical characterization and solubility enhancement studies of allopurinol solid dispersions / Caracterização físico-química e estudos de solubilidade de melhoria de dispersões sólidas de alopurinol

Allopurinol is a commonly used drug in the treatment of chronic gout or hyperuricaemia associated with treatment of diuretic conditions. One of the major problems with the drug is that it is practically insoluble in water, which results in poor bioavailability after oral administration. In the present study, solid dispersions of allopurinol were prepared by solvent evaporation, kneading method, co-precipitation method, co-grinding method and closed melting methods to increase its water solubility. Hydrophilic carriers such as polyvinylpyrrolidone, polyethylene glycol 6000 were used in the ratio of 1:1, 1:2 and 1:4 (drug to carrier ratio). The aqueous solubility of allopurinol was favored by the presence of both polymers. These new formulations were characterized in the liquid state by phase solubility studies and in the solid state by differential scanning calorimetry, powder X-ray diffraction, UV and Fourier Transform Infrared spectroscopy. Solid state characterizations indicated that allopurinol was present as an amorphous material and entrapped in polymer matrix. In contrast to the very slow dissolution rate of pure allopurinol, the dispersion of the drug in the polymers considerably enhanced the dissolution rate. Solid dispersion prepared with polyvinylpyrrolidone showed highest improvement in wettability and dissolution rate of allopurinol. Mathematical modeling of in vitro dissolution data indicated the best fitting with Korsemeyer-Peppas model and the drug release kinetics primarily as Non-Fickian diffusion. Therefore, the present study showed that polyvinylpyrrolidone and polyethylene glycol 6000 have a significant solubilizing effect on allopurinol.

Alopurinol é fármaco comumente utilizado no tratamento de gota crônica ou hiperuricemia associada com o tratamento em condições diuréticas. Um dos maiores problemas com o fármaco é que este é praticamente insolúvel em água, o que resulta em baixa biodisponibilidade na administração oral. No presente estudo, dispersões sólidas de alopurinol foram preparadas pela evaporação do solvente, pelos métodos de amassamento, de coprecipitação, de comoagem e fusão fechada para aumentar sua solubilidade em água. Transportadores hidrofílicos, como polivinilpirrolidona, polietilenoglicol 6000 foram utilizados nas proporções de 1:1. 1:2 e 1:4 (fármaco: transportador). A solubilidade aquosa do alopurinol foi favorecida pela presença de ambos os polímeros. Estas novas formulações forma caracterizadas no estado líquido pelos estudos de solubilidade de fase e no estado sólido pela calorimetria diferencial de varredura, difração de Raio-X, espectroscopia de UV e de IV com transformada de Fourier. As caracterizações do estado sólido indicaram que o alopurinol estava presente como material amorfo e embebido em matriz polimérica. Ao contrário da velocidade de dissolução lenta do alopurinol puro, a dispersão do fármaco nos polímeros aumentou consideravelmente a taxa de dissolução. A dispersão sólida preparada com polivinilpirrolidona mostrou as maiores melhorias na molhabilidade e taxa de dissolução do alopurinol. A modelagem matemática dos dados da dissolução in vitro indicou o melhor ajuste ao modelo de Korsemeyer-Peppas e a cinética de liberação do fármaco primariamente como difusão não-Fickiana. Assim, o presente estudo mostrou que a polivinilpirrolidona e o polietilenoglicol 6000 têm efeito significativo na solubilização do alopurinol.

Simultaneous electrochemical monitoring of metabolites related to the xanthine oxidase pathway using a grinded carbon electrode.

Using a mechanically grinded pyrolytic graphite electrode in edge orientation, a sensitive electrochemical method was developed for simultaneous determination of uric acid (UA), xanthine (XAN), hypoxanthine (HYP) (products of purine catabolism in human), allopurinol (ALO), and oxypurinol (OXY) (a drug used in treatment of purine catabolism disorders and its metabolite, respectively). It is demonstrated that differential pulse voltammetry in connection with this electrode can serve as a simple and efficient tool for monitoring transformation of purine catabolites (HYP --> XAN --> UA) catalyzed by xanthine oxidase (XO) as well as inhibition of this pathway by ALO being enzymatically converted to OXY. Our protocol is based on direct electrochemical measurement of oxidation peaks for each of the substances during in vitro reactions in a single detection step by the same electrode system. In addition, we show that the proposed electrochemical technique can be applied to parallel detection of metabolites involved in the XO pathway excreted in urine without any pretreatment of the clinical samples.

Flow injection determination of xanthine oxidase inhibitory activity and its application to food samples.

The enzyme xanthine oxidase (XOD) has been recognized as a key enzyme causing oxidative injury to tissues by ischemia-reperfusion. For this reason, XOD inhibitor, which effectively suppresses this enzyme, plays an important role in the inhibition of many diseases related to reactive oxygen species (ROS). In order to screen XOD inhibitors rapidly and conveniently, a novel assay using flow injection analysis (FIA) was proposed in the present investigation. To optimize the practical FIA system, we studied the effect of the reagent concentrations and the flow condition on the enzymatic reaction, and then selected the optimum condition as follows: 200-mU/ml XOD concentration, 0.5-mM xanthine concentration, 0.5-ml/min flow rate, and 2-m mixing coil length. Under this condition, a typical XOD inhibitor quercetin was determined in the concentration range 0.1 - 1.5 mM at a sampling frequency of 10 samples/h. Using the optimized FIA method, we determined the XOD inhibitory activity of some food samples: onions, apples and teas, which are the high sources of flavonoids known as the potential XOD inhibitors. Among these samples, tea leaves showed the highest activity, the second was onions and the lowest was apples. Based on the result of the assay, not only quercetin, but also other components in investigated samples, contributed to the XOD inhibitory activity.

Development of a capillary electrophoresis method for the determination of allopurinol and its active metabolite oxypurinol.

A simple and sensitive capillary zone electrophoresis method with UV absorbance detection is described for the quantitation of allopurinol and its metabolite oxypurinol in aqueous solution. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was systematically investigated; these parameters included the nature and concentration of the separation buffer, pH and applied voltage. A buffer consisting of 15 mM 2-[N-cyclohexylamino]ethanesulfonic acid (CHES) adjusted to pH 8.8 was found to provide a very efficient and stable electrophoretic system for the analysis of these compounds. The optimized method was validated with respect to precision, linearity, limits of detection and quantification, accuracy and robustness. The applicability of the assay was demonstrated by analyzing these compounds in serum and allopurinol in commercial pharmaceutical preparations.

Determination of allopurinol by micelle-stabilised room-temperature phosphorescence in real samples.

A very simple, rapid and highly sensitive method has been developed for the determination of allopurinol. The method is based on the room temperature phosphorescence of allopurinol in sodium dodecylsulphate (SDS) micelles, with thallium (I) providing the external heavy atom and sodium sulphite acting as the oxygen scavenger. Under the optimum experimental conditions, the range of application is 0.25-7.0 microg ml(-1) and the limit of detection is 0.014 microg ml(-1). The most relevant characteristic of this method is its great selectivity, e.g. allopurinol can be determined in the presence of its metabolite, oxypurinol. The results of the analysis of several pharmaceutical preparations were satisfactory. The clinical applicability of this procedure has been tested by analysing allopurinol in urine samples.

Anionic polysaccharides. A class of substances with hepatoprotective and antiadhesive properties in rat liver preservation.

In liver preservation, the substitution of the anion Cl(-) by lactobionic acid (LB) prevents reperfusion edema and extends the preservation time for human livers. We studied the effect of compounds that are structurally related to lactobionic acid: anionic polycarbohydrates (sulfated anionic polysaccharide, SAP, and pentosan polysulfate, PPS) on liver function and leukocyte-endothelial cell interaction in isolated perfusion and liver transplant models. Rat livers, cold-stored (24 h) in a Cl(-) -containing control solution, became edematous during 1 h of reperfusion. Substitution of Cl(-) by either LB, SAP, or PPS decreased reperfusion edema in a Cl(-) concentration-dependent fashion. Reperfusion edema was abolished completely after preservation in 100 mM SAP solution or PPS solution. Also hepatic lactic dehydrogenase (LDH) and aspartate aminotransferase (ASAT) release was lowest after preservation in those solutions. After preservation in LB or anionic polycarbohydrate solutions, portal venous resistance was significantly higher than after preservation in Cl(-)-containing control solution. Capillary blood flow was 391 +/- 83 pl/s and 398 +/- 174 pl/s after preservation in SAP solution (SAPs) and PPSs, and 803 +/- 117 pl/s and 641 +/- 219 pl/s after preservation in LB or Cl(-)-containing control solution. The number of leukocytes sticking to the vascular wall was lower ( P < 0.05) after preservation in SAPs or PPSs (109 +/- 31 cells/mm(2) and 108 +/- 60 cells/mm(2), respectively), when compared with preservation in Cl(-)-containing control or LB solutions (429 +/- 63 cells/mm(2) and 277 +/- 59 cells/mm(2)). In rat liver preservation, anionic polysaccharides are antiedematous compounds, with a higher potency than LB and additional antiadhesive properties.

Determination of raffinose and lactobionic acid in ViaSpan by anion exchange chromatography with pulsed amperometric detection.

An anion exchange chromatographic method has been developed to quantify raffinose and lactobionic acid in ViaSpan, an organ preservation product. Separation was accomplished using an aqueous sodium hydroxide/acetate solvent system (pH 13) on a Carbopac PA1 column. Detection was performed using a pulsed amperometric detector equipped with a gold working electrode. The method was able to resolve raffinose,lactobionic acid and other ingredients in the ViaSpan product. Validation testing of the method for routine use produced excellent linearity, precision and accuracy. The limits of detection for raffinose and lactobionic acid were 1.0 ng (1.7 x 10(-12) mol, S/N = 3) and 2.0 ng (5.6 x 10(-12) mol, S/N = 3) respectively. The total analysis time is less than 15 min.

Stability of acetazolamide, allopurinol, azathioprine, clonazepam, and flucytosine in extemporaneously compounded oral liquids.

The stability of drugs commonly prescribed for use in oral liquid dosage forms but not commercially available as such was studied. Acetazolamide 25 mg/mL, allopurinol 20 mg/mL, azathioprine 50 mg/mL, clonazepam 0.1 mg/mL, and flucytosine 10 mg/mL were prepared in 1:1 mixture of Ora-Sweet and Ora-Plus (Paddock Laboratories), a 1:1 mixture of Ora-Sweet SF and Ora-Plus (Paddock Laboratories), and cherry syrup and placed in polyethylene terephthalate bottles. The sources of the drugs were capsules and tablets. Six bottles were prepared per liquid; three were stored at 5 degrees C and three at 25 degrees C, all in the dark. A sample was removed from each bottle initially and at intervals up to 60 days and analyzed for drug concentration by stability-indicating high-performance liquid chromatography. At least 94% of the initial drug concentration was retained in all the oral liquids for up to 60 days. There were no substantial changes in the appearance or odor of the liquids, or in the pH. Acetazolamide 25 mg/mL, allopurinol 20 mg/mL, azathioprine 50 mg/mL, clonazepam 0.1 mg/mL, and flucytosine 10 mg/mL were stable for up to 60 days at 5 and 25 degrees C in three extemporaneously compounded oral liquids.

Simultaneous determination of allopurinol and oxipurinol in human plasma and urine by high-performance liquid chromatography.

The uricostatic drug allopurinol (CAS 315-30-0) is used for treatment of hyperuricaemia and is mainly bio-transformed to the active metabolite oxipurinol (CAS 2465-59-0) in humans. A new assay was developed for the simultaneous determination of both compounds in plasma and urine using ultrafiltration and ion exchange purification steps for plasma and urine, respectively. Reversed-phase high-performance liquid chromatography with ultraviolet detection was applied for the separation and quantitation of both compounds. The limit of detection was 0.1 microgram/ml for both compounds in plasma and 0.2 and 0.5 microgram/ml for allopurinol and oxipurinol, respectively, in urine. Within-run and day-to-day precision of 3-5% and 5-7% was determined for plasma and 6-8% and 8-10% for urine analysis. The assays were further validated using liquid chromatography with photodiode array detection and by comparison with methods using protein precipitation as the purifying step. The high analytical recoveries, selectivity, sensitivity, accuracy and reproducibility were adequate for the measurement of both compounds in pharmacokinetic studies and for drug monitoring in patients on allopurinol therapy.

Allopurinol and oxypurinol in human breast milk.

To pregnant or breast feeding women drugs should be given with caution. We report the case of a 5-week-old breast-fed infant whose mother was taking 300mg allopurinol/day for 4 weeks. Allopurinol and oxypurinol were detected by HPLC in maternal plasma and breast milk with a method first described here. In infant's plasma taken 2 h after breast feeding oxypurinol was found; allopurinol was below the limit of detection. The milk/plasma ratio in the mother 2 h (4 h) after drug ingestion was 0.9 (1.4) for allopurinol and 3.9 (2.4) for oxypurinol. The average daily dose for the baby of allopurinol was 0.14-0.20 mg/kg and that of oxypurinol 7.2-8.0 mg/kg by ingestion of breast milk after oral intake of allopurinol by the mother.

[Studies on purine-pyrimidine metabolism (1)--Quantitation of purine-pyrimidine metabolites and allopurinol-oxipurinol in biological fluids].

A reversed-phase high-performance liquid-chromatography method for determining simultaneous quantitation of purine-pyrimidine metabolites, allopurinol and oxipurinol in plasma and urine samples was studied. Separation was optimal with phosphate buffer (10 mmol/l, pH 5.0) containing 1% methanol as an eluent and mu Bondapak C18 as a column. An isocratic separation of a standard mixture of 13 compounds was achieved within 40 minutes with adequate reproducibilities (coefficient of variation: 2.49% for 1.63 mumol/l orotidin-0.12% for 50 mumol/l uridine). A simple ultrafiltration of plasma yielded quantitative recoveries (uric acid: 101.7-107.5%, hypoxanthine: 90.4-102.8%, xanthine: 95.9-99.5%, oxipurinol: 104.4-107.1%, allopurinol: 97.4-103.4%). Compounds were identified by their retention times, absorbance ratios, co-elution with standards and enzymic shifts. In addition to the above compounds, simultaneous quantitation of pseudouridine, uridine, adenine and inosine in the plasma would be possible under the same conditions.