Direct injection analysis of oxypurinol and metformin in wastewater by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry.

The increasing global prevalence of gout and diabetes has led to a rise in the use of their respective medications, allopurinol and metformin. These are excreted via urine as oxypurinol and metformin and are discharged into wastewater and the environment. Current environmental monitoring of those two polar chemicals requires labour intensive and potentially inefficient sample pre-treatments, such as using solid-phase extraction or freeze-drying. This study validated a sensitive and simple method using direct-injection LC-MS/MS for the simultaneous measurement of oxypurinol and metformin in wastewater. The final method utilised a hydrophilic interaction liquid chromatography together with simple filtration through 0.2 µm regenerated cellulose filter followed by dilution in acetonitrile with a dilution factor of 10. The developed method was validated with the limit of quantifications (LOQ) of 0.11 and 0.34 µg/L for metformin and oxypurinol, respectively. The new method was applied to 42 influent wastewater samples and 6 effluent samples collected from 6 Australian wastewater treatment plants. Both compounds were detected well above the LOQ at concentrations 29-214 µg/L in influent and 2-53 µg/L in effluent for metformin, and 24-248 µg/L in influent and 4-81 µg/L in effluent for oxypurinol, demonstrating its high applicability.

Identifying the Metabolic Signatures of PPARD-Overexpressing Gastric Tumors.

Peroxisome proliferator-activated receptor delta (PPARD) is a nuclear receptor known to play an essential role in regulation of cell metabolism, cell proliferation, inflammation, and tumorigenesis in normal and cancer cells. Recently, we found that a newly generated villin-PPARD mouse model, in which PPARD is overexpressed in villin-positive gastric progenitor cells, demonstrated spontaneous development of large, invasive gastric tumors as the mice aged. However, the role of PPARD in regulation of downstream metabolism in normal gastric and tumor cells is elusive. The aim of the present study was to find PPARD-regulated downstream metabolic changes and to determine the potential significance of those changes to gastric tumorigenesis in mice. Hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy, nuclear magnetic resonance spectroscopy, and liquid chromatography-mass spectrometry were employed for metabolic profiling to determine the PPARD-regulated metabolite changes in PPARD mice at different ages during the development of gastric cancer, and the changes were compared to corresponding wild-type mice. Nuclear magnetic resonance spectroscopy-based metabolomic screening results showed higher levels of inosine monophosphate (p = 0.0054), uracil (p = 0.0205), phenylalanine (p = 0.017), glycine (p = 0.014), and isocitrate (p = 0.029) and lower levels of inosine (p = 0.0188) in 55-week-old PPARD mice than in 55-week-old wild-type mice. As the PPARD mice aged from 10 weeks to 35 weeks and 55 weeks, we observed significant changes in levels of the metabolites inosine monophosphate (p = 0.0054), adenosine monophosphate (p = 0.009), UDP-glucose (p = 0.0006), and oxypurinol (p = 0.039). Hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy performed to measure lactate flux in live 10-week-old PPARD mice with no gastric tumors and 35-week-old PPARD mice with gastric tumors did not reveal a significant difference in the ratio of lactate to total pyruvate plus lactate, indicating that this PPARD-induced spontaneous gastric tumor development does not require glycolysis as the main source of fuel for tumorigenesis. Liquid chromatography-mass spectrometry-based measurement of fatty acid levels showed lower linoleic acid, palmitic acid, oleic acid, and steric acid levels in 55-week-old PPARD mice than in 10-week-old PPARD mice, supporting fatty acid oxidation as a bioenergy source for PPARD-expressing gastric tumors.

Uric acid analogue as a possible xenobiotic marker of uric acid transporter Urat1 in rats.

The inhibitor of uric acid reabsorptive transporter URAT1 in kidney is drawing attention as a drug target for hyperuricemia. However, it is difficult to evaluate efficacy of URAT1 inhibitors in vivo using laboratory animals due to species difference in uric acid metabolism. In the present study, the usefulness of exogenously administering uric acid analogues resistant to uricase was investigated for in vivo evaluation of transport activity of rUrat1 in rats. Uptake of examined four uric acid analogues by rUrat1-expressing Xenopus oocytes was significantly higher than that by water-injected oocytes. In metabolism studies, disappearance of these compounds was negligible, while uric acid was significantly decreased. When oxypurinol was administered to rats, fractional excretion (FE) was 0.4, suggesting reabsorption of oxypurinol. Moreover, FE of oxypurinol was tended to be increased, but not statistically different, by co-administration of a uricosuric agent FYU-981, while plasma concentration of oxypurinol was not affected. These results suggested that oxypurinol is a potential uric acid analogue, although it was not suitable as a probe of uric acid in in vivo study. Our findings may contribute to discovery and development of novel uricosuric agent targeting URAT1.

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.

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.

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.

[Xanthine urinary calculus in a patient with Lesch-Nyhan syndrome. Apropos of a case]. / Calcul urinaire de xanthine chez un patient porteur d'un syndrome de Lesh Nyhan. A propos d'un cas.

The authors report a case of xanthine stones in a 12-year-old child with Lesh Nyhan syndrome treated by allopurinol at the dose of 10 mg/kg/24 hours. This type of urinary stone is unusual and its structure was confirmed by spectrophotometric analysis. This type of stone, in the context of Lesh Nyhan syndrome, suggests the presence of allopurinol treatment. Discontinuation of this treatment prevents recurrence of xanthine stones.

Prophylactic action of allopurinol against chemotherapy-induced stomatitis--inhibition of superoxide dismutase and proteases.

The activities of superoxide dismutase (SOD) and several proteases were measured in kidney of mice treated with allopurinol in order to elucidate the mechanism of prophylactic action of allopurinol against chemotherapy-induced stomatitis. The following results were obtained. Following 3 day administration of allopurinol 20 mg/day per os (Group C), the concentrations of allopurinol and oxipurinol in the renal tissue were 203.9 +/- 52.1 and 1141.7 +/- 194.8 micrograms/g, respectively. The SOD activity was significantly lower in Group C than in the untreated control group (p < 0.01). The enzyme activities of papain and trypsin were suppressed in Group C. However, the other proteases tested were not affected by the administration of allopurinol, indicating only weak anti-protease action of allopurinol. These results suggest that allopurinol may be effective to prevent chemotherapy-associated stomatitis via both direct and indirect actions to oral mucosa, that include inhibitory actions on xanthine oxidase as well as protease.

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.

Simultaneous determination of allopurinol and oxypurinol by liquid chromatography using immobilized xanthine oxidase with electrochemical detection.

A novel liquid chromatographic method using an immobilized xanthine oxidase reactor and an electrochemical detector was developed for the simultaneous determination of allopurinol and oxypurinol in rat plasma, intestinal wash and bile. Xanthine oxidase was immobilized on 5-microns aldehyde silica (prepacked into a 2 mm x 10 mm cartridge) in a simple procedure. Allopurinol eluted from an analytical column was converted to oxypurinol in the enzyme reactor with the eluent as the reaction medium and detected with high selectivity using an amperometric detector with a glassy carbon electrode at the applied potential of +0.85 V. High specificity of the enzymatic reaction combined with selectivity of the electrochemical detection eliminated the need for an extensive sample preparation. The assay was linear in the range 15-500 ng/ml of rat plasma, intestinal wash and bile with a low limit of detection of 10 pg on-column (signal-to-noise ratio = 4) for both allopurinol and oxypurinol.

Improved survival in intestinal ischemia by allopurinol not related to xanthine-oxidase inhibition.

Allopurinol, a xanthine-oxidase (XO) inhibitor, has been used to improve the resistance to ischemia with disappointing results that have been attributed to administration regimen of the drug. Our aim was to investigate the effect of different administration schedules of allopurinol on the survival in rats undergoing intestinal ischemia testing the blockade of XO. Intestinal ischemia was achieved by 90 min of clamping the superior mesenteric artery (SMA) close to its origin from the aorta. Three groups of animals were evaluated: A-group: only the allopurinol solvent was given; B-group: the full dose of allopurinol (100 mg/k b.w.) was given iv and C-group: the 75% dose was administered orally 24 hr before and the remaining 25% was administered 30 min before. Survival was evaluated at 48 hr and the blockade of XO was assayed by High Efficacy Liquid Chromatography (HELC) in homogenate of intestinal wall. Survival was only improved in the C-group (P = 0.02). Levels of hypoxanthine were significantly increased both in B-group and C-group (P = 0.003) when compared with the A-group. Levels of uric acid in B-group (P = 0.0003) and C-group (P = 0.0009) were significantly decreased with respect to A-group. That means that an effective blockade of XO is achieved whichever the regimen of administration. Allopurinol and oxypurinol levels were significantly increased (P = 0.05 and P = 0.008) in C-group when compared with B-group. We conclude that the protective effect of allopurinol on survival in intestinal ischemia in rats is not related to the blockade of XO but rather to the allopurinol and oxypurinol levels in intestinal wall.

Allopurinol kinetics after massive overdosage.

A 15-year-old girl with normal renal function took 75 tablets of allopurinol 300 mg. She did not suffer any ill-effects. Samples obtained over 84 h showed prolonged elimination of allopurinol, with a half-life of 3.6 h, and oxipurinol, with a half-life of 26 h.

Electrochemically treated glassy carbon electrode for amperometric detection in high-performance liquid chromatography.

The application of an electrochemically pre-treated glassy carbon electrode for the amperometric detection of electroactive components, such as tyrosine and oxipurinol, in biological samples was studied in order to demonstrate the usefulness of a pre-anodized electrode in high-performance liquid chromatography. The electrochemical pre-treatment was carried out in 0.2 M phosphate buffer (potassium dihydrogenphosphate-potassium hydroxide, pH 6.5) at 1900 mV vs. Ag/AgCl for 2 min. The pre-anodized electrode response for the oxidation of lactic acid and pyruvic acid was also studied. The electrochemical treatment enhanced and stabilized the electrode response to the oxidation of tyrosine and both acids.

Quantitative liquid chromatography of allopurinol and oxypurinol in human plasma and urine.

A high performance liquid chromatographic (HPLC) assay is described for allopurinol and oxypurinol determination in human plasma and urine, in the range expected during therapy. The procedure involves addition of trichloroacetic acid to samples, followed by centrifugation. The supernatant is then neutralized and analyzed by reversed-phase HPLC. Characteristics of the method are reported, and data are presented on its application to the pharmacokinetics studies. Separation is optimal with an octadecylsilane (ODS) stationary phase and a sodium acetate mobile phase adjusted to pH 7.2 for plasma and pH 5 for urine.

High-performance liquid chromatography with polarographic and voltammetric anodic detection: simultaneous determination of allopurinol, oxipurinol and uric acid in body fluids.

Allopurinol, oxipurinol and uric acid have been determined in human serum and urine by liquid chromatography with electrochemical detection. In particular the use of a polarographic detector operating in the oxidative mode, whose principle of detection is based on the property of allopurinol, oxipurinol and uric acid to form insoluble anodic films on mercury, is described. The performance of such a detector is compared with that of a glassy carbon wall-jet detector. Different procedures for sample pretreatment have been evaluated.