[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.

Simultaneous Determination of Cefalexin, Cefazolin, Flucloxacillin, and Probenecid by Liquid Chromatography-Tandem Mass Spectrometry for Total and Unbound Concentrations in Human Plasma.

BACKGROUND: Pharmacokinetic studies and therapeutic drug monitoring of antibiotics require a simple, rapid, and reliable analytical method for monitoring the concentrations in plasma, including unbound concentrations for highly protein-bound drugs. The aim of the current work was to develop and validate a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of total and unbound concentrations of 3 widely used ß-lactam antibiotics (cefalexin, cefazolin, and flucloxacillin) and the often coadministered drug probenecid in human plasma, suitable for pharmacokinetic studies and for routine use in ordinary, busy hospital laboratories. METHODS: Unbound drug was separated from bound drug by ultrafiltration. A simple 1-step protein precipitation was used for sample preparation. Cefalexin, cefazolin, flucloxacillin, probenecid, and their corresponding isotopically labeled internal standards were then resolved on a C18 (2) column. All the compounds were detected using electrospray ionization in the positive mode. RESULTS: Standard curves were linear for all compounds over the concentration range of 0.2-100 mg/L (r > 0.99) for total drug in plasma and 0.01-10 mg/L (r > 0.99) for unbound drug in plasma ultrafiltrate. For both total and unbound drugs, bias was <±10%, and intra- and interday coefficients of variation (imprecision) were <10%. The limit of quantification was 0.2 mg/L for total plasma concentrations and 0.01 mg/L for plasma ultrafiltrate concentrations of all drugs. CONCLUSIONS: The method has proven to be simple, rapid, robust, and reliable and is currently being used in clinical pharmacokinetic studies and in the routine clinical service to enhance the effective use of the ß-lactam antibiotics.

Masking and manipulation.

The list of prohibited substances in sports includes a group of masking agents that are forbidden in both in- and out-of-competition doping tests. This group consists of a series of compounds that are misused in sports to mask the administration of other doping agents, and includes: diuretics, used to reduce the concentration in urine of other doping agents either by increasing the urine volume or by reducing the excretion of basic doping agents by increasing the urinary pH; probenecid, used to reduce the concentration in urine of acidic compounds, such as glucuronoconjugates of some doping agents; 5alpha-reductase inhibitors, used to reduce the formation of 5alpha-reduced metabolites of anabolic androgenic steroids; plasma expanders, used to maintain the plasma volume after misuse of erythropoietin or red blood cells concentrates; and epitestosterone, used to mask the detection of the administration of testosterone. Diuretics may be also misused to achieve acute weight loss before competition in sports with weight categories. In this chapter, pharmacological modes of action, intended pharmacological effects for doping purposes, main routes of biotransformation and analytical procedures used for anti-doping controls to screen and confirm these substances will be reviewed and discussed.

Preparative chromatography of furosemide 1-O-acyl-glucuronide from urine using micronized amberiite XAD-2 and its application to other 1-O-acyl-glucuronides.

Furosemide 1-O-acyl glucuronide (Fgnd) was extracted from the urine following oral administration of furosemide. The crude Fgnd was applied to micronized Amberlite XAD-2 column (2.5 cm i.d. x 90 cm length, 75-500 microns particle size). The purified Fgnd was identified by mass spectrometry and beta-glucuronidase treatment. This method was also applicable to the purification of glucuronide of tolmetin (nonsteroidal anti-inflammatory drug, NSAID), suggesting that it was applicable to the other NSAIDs, most of which were known to be metabolized to acyl-glucuronides.

Capillary electrophoresis of diuretics.

The review surveys the application of capillary electrophoresis to the screening, identification and determination of diuretics and probenecid. The number of publications is still limited, but the studies already published clearly show that capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography are excellent alternatives for the investigation of diuretics. High accuracy identifications of diuretics and probenecid, even in urine samples, can be obtained when CZE is used with the marker techniques. This review paper has been written from the viewpoint of practical use and some hints are given for future CE studies on diuretics.

A death involving probenecid.

A death following deliberate ingestion of approximately 75 g of probenecid in a 36-year-old man is described. Tissue concentrations of probenecid were highest in serum (710 mg/L) and liver (550 mg/kg). Probenecid was also detected in vitreous and bile. Ethanol was also detected in blood at 0.13 g/100 mL.

Direct measurement of probenecid and its glucuronide conjugate by means of high pressure liquid chromatography in plasma and urine of humans.

Probenecid with its phase-I metabolites, and phase-II glucuronide conjugate can be analysed by a gradient high pressure liquid chromatographic method. Probenecid glucuronide in plasma with pH 7.4 is not stable and declines to 10% of the original value within 6 h (t1/2 approximately 1 h). Probenecid glucuronide is stable in urine with pH 5.0, moderately unstable at pH 6.0 (t1/2 approximately 10 h), and unstable at pH 8.0 (t1/2 approximately 0.5 h). Probenecid glucuronide is stable in water and 0.01 mol/l phosphoric acid in the autosampler of the high pressure liquid chromatograph. The decrease in concentration in water is 5.5% during 9 h and 0% in diluted acid. Probenecid glucuronide and the phase-I metabolites were not detectable in plasma. The main compound in fresh urine is the phase-II conjugate probenecid glucuronide (62% of a 500 mg dose); the phase-I metabolites are present and only a trace of probenecid is present. The percentage of the dose of the phase-I metabolites varies between 5 and 10, while hardly any probenecid is excreted unchanged (0.33%).

Approach to the analysis of diuretics and masking agents by high-performance liquid chromatography-mass spectrometry in doping control.

The application of thermospray and plasmaspray high-performance liquid chromatography-mass spectrometry to the analysis of diuretics and probenecid has been investigated. The latter method gave better ionization efficiency than the former, and its response was optimized by altering the solvent composition: best results were obtained with water-methanol-acetonitrile-trifluoroacetic acid. Using different proportions of these solvents, three isocratic systems were developed to separate the compounds under study. The principal characteristic of plasmaspray positive-ion mass spectra was a protonated molecular ion and very little fragmentation was evident. In the negative ionization mode, the plasmaspray method gave mass spectra showing more fragmentation, which resulted in additional structural information. The ability of trifluoroacetic acid to form negative cluster ions precluded its use as a mobile phase component. The minimum detectable amounts determined by the analysis in the positive-ion mode was compound-dependent, but generally ca. 10-150 ng. In many cases the compounds could be detected in urine extracts.

Determination of benzylpenicillin and probenecid in human body fluids by high-performance liquid chromatography.

A method for the determination of benzylpenicillin and probenecid concentrations in human body fluids using ion-pair reversed-phase chromatography with UV detection has been developed. For plasma samples two extraction techniques were investigated. Precipitation of the plasma proteins with acetonitrile followed by liquid-liquid extraction offered the best results. The limits of detection were 0.5 microgram/ml for benzylpenicillin and 0.25 microgram/ml for probenecid, which offer sufficient sensitivity for application in pharmacokinetic experiments.

Liquid chromatographic determination of colchicine in pharmaceuticals: collaborative study.

A liquid chromatographic (LC) method for the determination of colchicine in pharmaceutical dosage forms and the bulk drug was evaluated in an interlaboratory study which included 13 participating laboratories. The method involves extraction (or dissolution) of the active ingredient with methanol-water (1 + 1), followed by filtration of the extract and reverse phase LC using an octylsilane bonded phase column and UV detection at 254 nm. The mobile phase consists of a methanol-phosphate buffer mixture (pH = 5.5). Three commercial tablet formulations (0.5-0.6 mg colchicine/tablet), 1 synthetic injectable preparation (0.510 mg colchicine/mL), and 1 bulk drug sample were assayed in duplicate by the proposed method. The reproducibility and repeatability standard deviations based on nonpooled results for each sample ranged from 0.0062 mg/mL to 0.0147 mg/tablet and from 0.0037 mg/mL to 0.0127 mg/tablet, respectively; the corresponding coefficients of variation ranged from 1.21 to 2.54% and from 0.73 to 2.19%, respectively. The mean recovery from the synthetic injectable formulation was 100.0%. The method has been adopted official first action.

Probenecid-induced increase of 5-hydroxytryptamine synthesis in rat brain, as measured by formation of 5-hydroxytryptophan.

Probenecid blocks the efflux of 5-hydroxyindole acetic acid (5-HIAA) from the central nervous system, and has therefore been used for turnover measurements of central 5-hydroxytryptamine (5-HT). This substance also elevates tryptophan (TP) levels in rat brain. In this investigation, the time courses of probenecid and TP levels in rat serum and brain after administration of probenecid were studied. Maximal levels of probenecid were reached within 15 min, followed by 50% decrease of serum TP and a 40% increase of brain TP. Brain levels of probenecid were about ten times lower than those in serum. Because TP level in brain is an important factor in the control of cerebral 5-HT synthesis, the effects of probenecid on 5-HT formation in rat brain were investigated. By means of the aromatic L-amino acid decarboxylase inhibitors Ro 4-4602 and NSD 1015, an enhancement of TP hydroxylation of about 35% was demonstrated. It was concluded that penetration of probenecid into the brain is very limited and that probenecid, in addition to blocking egress of 5-HIAA from the CNS, stimulates 5-HT synthesis.

High pressure liquid chromatographic method for probenecid in flavored oral suspensions containing ampicillin: collaborative studies.

A high pressure liquid chromatographic method for determining probenecid in oral suspensions of amoxicillin was applied to the determination of probenecid in oral suspensions of ampicillin. Three preparations containing various known amounts of probenecid in synthetic mixtures of ampicillin oral suspensions were analyzed by 5 chemists in an intralaboratory study, with satisfactory results. Blind duplicates of 3 prepared oral suspensions were sent to 12 collaborators, who were instructed to analyze the samples in a fixed random order. The standards showed a satisfactory linear response. Average recoveries of probenecid in the interlaboratory study for the 6 mixtures ranged from 95.2 to 99.1%, and the coefficients of variation ranged from 1.63 to 4.9%.

New spectrophotofluorometric assay for probenecid.

A new spectrophotofluorometric assay for probenecid is presented based on conversion of the drug to a fluorescent anthranilic acid derivative. The assay is especially applicable with "clean" biological fluids such as cerebrospinal fluid and offers severalfold greater sensitivity than the commonly used UV method.

Comparison of methods for determining probenecid in tablets and flavored oral suspensions containing ampicillin.

Several methods for quantitating probenecid were compared: USP XIX method for probenecid in tablets, a modified USP extraction method for probenecid in tablets, a new column extraction method for probenecid in tablets and oral suspensions containing ampicillin, and a high pressure liquid chromatographic (HPLC) method for probenecid in tablets and oral suspensions containing antibiotics. The first 3 methods were satisfactory for probenecid in bulks and tablets but proved unsatisfactory for oral suspeonsions containing flavors. The flavors gave positive interference for probenecid in oral suspensions. The HPLC method, although more time-consuming, separated probenecid from excipients, thus eliminating positive interference from flavors.

Probenecid in CSF and plasma of rabbits and dogs measured by radioimmunoassay.

Probenecid (P) in CSF of rabbits and dogs was investigated using a new radioimmunoassay technique. In rabbits, under steady-state conditions, CSF/free plasma concentration ratios of P were found to be similar to those reported in man. The ratios were concentration dependent and less than 0.5 suggesting an inhibition of transport of the drug in CNS. Under nonsteady-state conditions, no clear evidence of a transport system was found in dogs.

Relationship between the transport and toxicity of cephalosporins in the kidney.

Large doses of cephaloridine cause acute necrosis of the proximal renal tubule that can be prevented by probenecid and other organic anions. Although there is little or no net secretion of cephaloridine by the mammalian kidney, the degree of cephaloridine uptake by the cortex of the rabbit kidney is substantial; this uptake is also prevented by probenecid and other organic anions. Cortical concentrations of cephaloridine were measured in control and probenecid-treated animals of different mammalian species. Evidence of cephaloridine trnsport was found in the guinea pig and the rat as well as in the rabbit. The degree of reduction of cortex-to-serum ratios by probenecid (control cortex-to-serum minus probenecid-treated cortex-to-serum ratios) correlated with the sensitivity to the nephrotoxicity of the drug. This degree or reduction was greatest in the rabbit, intermediate in the guinea pig, and least in the rat. In addition, the newborn rabbit, which is more resistant to the toxicity of cephaloridine than the adult, also had significantly lower cortical concentrations of cephaloridine. Finally, the acute tubular necrosis produced by extremely large doses of cefazolin in the adult rabbit was prevented by probenecid. It was concluded (1) that the nephrotoxicity of cephaloridine is related to its renal cortical transport with high intracellular concentrations of drug; and (2) that this relationship between transport and toxicity exists for cefazolin as well, although the toxicity is of a different order of magnitude. The unusual mechanism of cephaloridine transport in the proximal tubule was contrasted with that of the other cephalosporins in an attempt to explain its greater degree of nephrotoxicity.