Simultaneous separation and determination of 20 potential adulterant antigout and antiosteoporosis pharmaceutical compounds in herbal food products using LC with electrospray ionization MS/MS and LC with quadrupole-time-of-flight MS.

An analytical method for the simultaneous and reliable determination of 20 antigout and antiosteoporosis pharmaceutical compounds in adulterated health food products was developed using liquid chromatography with electrospray ionization tandem mass spectrometry and liquid chromatography with quadrupole-time-of-flight mass spectrometry. The method was validated through the determination of specificity, linearity, limit of detection, and limit of quantification, method detection limit, method quantitation limit, precision, accuracy, recovery, and stability. The matrix effect was also determined. The validation results of the developed method are as follows: for solid and liquid blank samples, limits of detection ranged from 0.05 to 5.00 ng/mL and limits of quantification ranged from 0.15 to 15.00 ng/mL. Linearity was acceptable, and the correlation coefficients (R2 ) were ≥0.99 for all target compounds. Both intra and interday precision were less than 9.16% RSD, and accuracies ranged from 95.31 to 116.68%. Mean recoveries for different types of dietary supplements classified as powders, liquids, tablets, and capsules were found to be 80.81 to 117.62% with less than 15.00% relative standard deviation. The stability of the standard mixture solution was less than 11.72% relative standard deviation after 48 h. By the proposed method, the presence of dexamethasone was determined in seized herbal food products at concentrations that ranged from 126 to 215 µg/g.

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.

An LC-MS/MS- and hURAT1 cell-based approach for screening of uricosuric agents.

Urate anion exchanger 1 (URAT1) expressed in the proximal renal tubules is responsible for about 90% of the reabsorption of uric acid. URAT1 is identified as an important target of uricosuric drugs. Here we present an LC-MS/MS-based approach, combined with URAT1-transgenic MDCK cells, for the assessment of uric acid. Cell lysis was executed with 50 mM NaOH to release uric acid. 1,3-15N2 uric acid was employed as the internal standard. The harvested uric acid, along with the stable isotope-labeled uric acid, was analyzed by LC-MS/MS in multiple reactions monitoring and negative modes. Validation, i.e. determination of selectivity, precision, accuracy, extraction recovery, and matrix effect, and feasibility was evaluated by use of the approach developed. The linearity was observed in the range of 1.0-250 µM (r = 0.9960) with limit of detection of 50 nM and limit of quantitation of 200 nM. The precision and accuracy were found to be RSD ≤ 20% and 80-120% of the nominal value, respectively. Uric acid uptake showed concentration and time dependency in URAT1-transgenic cells. The observed inhibitory effects of three URAT1-targeted uricosuric drugs were consistent with those reported in literature. The stable isotope dilution-based approach was proven to be selective, sensitive, and convenient, which is a good in vitro model for URAT1-targeted drug candidate screening.

GLC determination of the polyvalent saluretic uricosuric agent (2-cyclopentyl-6,7-dichloro-2-methyl-1-oxo-5-indanyloxy)acetic acid in biological fluids.

A specific and quantitative GLC method was developed for the determination of (2-cyclopentyl-6,7-dichloro-2-methyl-1-oxo-5-indanyloxy) acetic acid, a novel saluretic uricosuric agent, in biological fluids. The procedure involves the addition of an internal standard to the biological specimens followed by extraction of the acids into benzene at pH 1. The extracted acids, following back-extraction into base and reextraction into methylene chloride at an acidic pH, are converted to the respective methyl esters by reaction with ethereal diazomethane. The sensitivity of the method is such that 2 microgram of material can be detected per aliquot of plasma or urine. In the 2.5-50-microgram/ml range, recoveries were 98.1 +/- 9.6% (plasma, n = 157) and 99.3 +/- 6.4% (urine, n = 181). GLC-mass spectrometric techniques established analysis specificity.

GLC determination of a novel polyvalent saluretic agent, (6,7-dichloro-2-methyl-1-oxo-2-phenyl-5-indanyloxy)acetic acid, in biological fluids.

Highly specific and sensitive GLC methods were developed for the determination of (6,7-dichloro-2-methyl-1-oxo-2-phenyl-5-indanyloxy)acetic acid, a novel saluretic-uricosuric agent, in biological fluids. The procedures involve the addition of an internal standard, (6,7-dichloro-2-cyclopentyl-2-methyl-1-oxo-5-indanyloxy)acetic acid, to the biological specimens followed by extraction of the acids into benzene at pH 1. The indanones are back-extracted into sodium hydroxide and reextracted into methylene chloride under acidic conditions. The acids are subsequently converted to the methyl esters for GLC analysis by reaction with diazomethane. The sensitivity of the method is such that 1.0 microgram of material/ml of plasma can be analyzed using a flame-ionization detector. When the derivatized samples are analyzed using a 63Ni-electron-capture detector, the sensitivity is such that 2.5 ng of compound can be detected. These levels are suitable for the analysis of samples obtained following a therapeutic dose. A recovery of 98.8 +/- 11.9% was obtained using the electron-capture method for plasma (n = 322). Recoveries using flame ionization were 99.1 +/- 4.4% (plasma, n = 207) and 99.8 +/- 4.9% (urine, n = 163). Quantitation of the major ring-hydroxylated metabolite (in chimpanzee and human) was accomplished following silylation of the methyl esters.

The dual actions of Paederia scandens extract as a hypouricemic agent: xanthine oxidase inhibitory activity and uricosuric effect.

Hyperuricemia is associated with a number of pathological conditions, such as gout. Lowering of elevated uric acid levels in the blood could be achieved by xanthine oxidase inhibitors and inhibitors of renal urate reabsorption. Some natural compounds isolated from herbs used in traditional Chinese medicine have been previously demonstrated to act as xanthine oxidase inhibitors. In the present investigation, Paederia scandens (Lour.) Merrill (Rubiaceae) extract (PSE; 4.5, 2.25, and 1.125 g/kg) orally for 14 days was demonstrated to possess in vivo potent hypouricemic activity in hyperuricemic rats pretreated with potassium oxonate. In addition, PSE was also demonstrated to be an inhibitor of xanthine oxidase. Lineweaver-Burk analysis of the enzyme kinetics indicated that the inhibition of PSE was of a mixed type. Using an oxonate-induced hyperuricemic rat model, PSE was indeed shown to exhibit uricosuric action in vivo, which could explain, at least in part, the observed hypouricemic effect of PSE in these rats. The potential application of this compound in the treatment of conditions associated with hyperuricemia is discussed.

Micellar electrokinetic capillary chromatographic method for the quantitative analysis of uricosuric and antigout drugs in pharmaceutical preparations.

A simple MEKC method is described for the separation and quantification of seven widely used uricosuric and antigout drugs, including allopurinol (AP), benzbromarone (BZB), colchicine (COL), orotic acid (OA), oxypurinol (OP), probenecid (PB), and sulfinpyrazone (SPZ). The drugs were separated in a BGE of borate buffer (45 mM; pH 9.00) with SDS (20 mM) as the micellar source and the separated drugs were directly monitored with a UV detector (214 nm). Several parameters affecting the separation and analysis of the drugs were studied. Based on the normalized peak-area ratios of the drugs to an internal standard versus the concentration of the drugs, the method is applicable to quantify BZB, COL, and SPZ (each 5-200 microM), AP, OA, OP, and PB (each 10-200 microM) with detection limits (S/N = 3, 0.5 psi, 5 s injection) in the range of 0.6-4.0 microM. The precision (RSD; n = 5) and accuracy (relative error; n = 5) of the method for intraday and interday analyses of the analytes at three levels (30, 120, and 180 microM) are below 4% (n = 3). The method was demonstrated to be suitable for the analysis of AP and COL in commercial tablets with speed and simplicity.