Use of post-column fluorescence derivatization to develop a liquid chromatographic assay for ranitidine and its metabolites in biological fluids

Ranitidine and its main metabolites, ranitidine N-oxide and ranitidine S-oxide, were determined in plasma and urine after separation using reversed-phase liquid chromatography. The mobile phase consisted of an initial isocratic step with 7:93 (v/v) acetonitrile–7.5 mM phosphate buffer (pH 6) for 8 min, followed by a linear gradient up to a 25:75 (v/v) mixture over 1 min. Detection was carried out by a post-column fluorimetric derivatization based on the reaction of the drugs with sodium hypochlorite, giving rise to primary amines that reacted with o-phthalaldehyde and 2-mercaptoethanol to form highly fluorescent products. The calibration graphs, based on peak area, were linear in the range 0.1–4 μg/ml for all drugs. The detection limits were 30, 41 and 32 ng/ml (8.6, 12.5 and 9.1 pmol) for ranitidine S-oxide, ranitidine N-oxide and ranitidine, respectively. Chromatographic profiles obtained for plasma and urine samples showed no interference from endogenous compounds.     

Spectrophotometric assay of phenolpthalein in biological fluids.

An assay for phenolphthalein in biological fluids has been developed utilizing methods previously applied to the assay of bromosulphalein and to the deconjugation of steroidal compounds in urine. Intestinal perfusate, serum, and urine samples containing phenolphthalein are deproteinized with acidified acetone, the samples dried, and the phenolphthalein redissolved in ethanol. Color is developed with 0.5 m glycine buffer, pH 12, and the samples read at 550 nm after blanking the spectrophotometer with one of the replicates to which acidic glycine buffer is added. To measure conjugated phenolphthalein in urine, the sample is incubated overnight with β-glucuronidase/arylsulphatase prior to phenolphthalein determination as noted above. This method gives an accurate assay of phenolphthalein to 10^?5m concentrations with coefficients of variation between 2 and 8% and with no resulting interference from hemoglobin or bilirubin.     

Improved method for routine determination of nicotine and its main metabolites in biological fluids

Extrelut^R extraction and glass capillary gas chromatography were applied to the routine determination of nicotine and its metabolites cotinine, nicotine-1′-N-oxide and cotinine-1-N-oxide in urine and plasma. After extraction of nicotine and cotinine both N-oxides and phendimetrazine-N-oxide (used as internal standard) were reduced to their bases by SO₂ on-column and eluted by a mixture of diethyl ether and dichloromethane. The minimum detectable concentrations are 0.03 μg/ml for urinary nicotine and cotinine and 0.1 μg/ml for the N-oxides. In plasma samples the corresponding values are 5 ng/ml and 15 ng/ml, respectively, with sample values as small as 2 ml. The advantage of the direct determination of all four compounds of interest in one sample reduced the amount of plasma required. The straightforward and rapid extraction and reduction procedure as well as the long-term stability of the gas chromatographic separation system make the method suitable for routine application.     

High-performance liquid chromatographic analysis of sulfinpyrazone and its metabolites in biological fluids

A rapid, sensitive, and specific high-performance liquid chromatographic method is described for the quantitative analysis of sulfinpyrazone and its sulfone and p-hydroxy metabolites in plasma and urine. The method uses two different procedures for sample preparation: (1) a rapid and convenient procedure using a single extraction with 1-chlorobutane and subsequent back-extraction into sodium hydroxide solution for the analysis of sulfinpyrazone and its sulfone metabolite, and (2) a more time consuming procedure using triple extraction with ethylene dichloride, a buffer wash, and back extraction into the base for the additional analysis of the p-hydroxy metabolite. The lower limit of sensitivity for sulfinpyrazone is 50 ng/ml. Concentrations of sulfinpyrazone between 0.05 to 0.1 and 50 μg/ml were measured with an average coefficient of variation of 3.9%, ranging from 1.5 to 6.1%.     

Quantitation of chlordiazepoxide and its metabolites in biological fluids by thin-layer chromatography

Chlordiazepoxide and its 4 major metabolites were assayed after separation by thin-layer chromatography following extraction from biological fluids. The compounds become intensely fluorescent in the presence of red, fuming nitric acid. The resulting compounds are quantitated with a spectrodensitometer with a fluorescent attachment. The sensitivity varies between 0.05 and 0.1 μg. The coefficient of variation is 1.4% for assays in urine and 6.4% in serum.     

Chromatographic-mass spectrometric methods for the quantification of L-arginine and its methylated metabolites in biological fluids

L-Arginine (Arg) and its methylated metabolites play a major role in the synthesis of the cell signaling molecule nitric oxide (NO). Arg serves as a substrate for the enzyme NO synthase (NOS), which produces NO, whereas monomethylarginine (L-NMMA) and asymmetric dimethylarginine (ADMA) act as competitive inhibitors of NOS. Symmetric dimethylarginine (SDMA) has virtually no inhibitory effect on NOS activity, but shares the pathway for cell entry and transport with Arg and ADMA. Accurate and reliable quantification of these substances in various biological fluids is essential for scientific research in this field. In this review, chromatographic-mass spectrometric methods for Arg and its methylated metabolites ADMA and SDMA are discussed. Mass spectrometric detection provides an intrinsic higher selectivity than detection by means of UV absorbance or fluorescence. Taking advantage of the high selectivity, approaches involving mass spectrometric detection require less laborious sample preparation and produce reliable results. A consensus emerges that the concentration values in plasma of young healthy volunteers are about 65 microM for Arg, 0.4 microM for ADMA and 0.5 microM for SDMA.     

High-performance liquid chromatographic determination of ibuprofen and its major metabolites in biological fluids

A sensitive and selective high-performance liquid-chromatographic assay for ibuprofen and its major metabolites in biological fluids is described. To ensure good chromatographic separation the drug and metabolites were run on a gradient elution system and detected with a variable wavelength detector set at 220 nm. A second, more rapid, isocratic system is also described for the detection of only ibuprofen.     

High-performance liquid chromatographic determination of licochalcone A and its metabolites in biological fluids

A high-performance liquid chromatographic method has been developed for the analysis of the novel antiparasitic agent, licochalcone A (Lica), and three of its glucuronic acid conjugates in plasma and urine. The high-performance liquid chromatography assay was performed using gradient elution and UV detection at 360 nm. The proposed technique is selective, reliable and sensitive. The limits of quantification for Lica are 0.2 μg/ml in plasma and 0.14 μg/ml in urine, 1.2 μg/ml for the 4′-glucuronide in plasma and 1.4 μg/ml in urine, and 2.0 μg/ml for the 4-glucuronide in plasma and 3.2 μg/ml in urine. The reproducibility of the analytical method according to the statistical coefficients is 7% or below. The accuracy of the method is good, that is, the relative error is below 10%. The stability of Lica and its glucuronides in urine and plasma samples has been assessed during storage in the autosampler and freezer. The applicability of the assay for determining Lica and its intact glucuronide conjugates in biological fluids was shown using a single dose study in rat.     

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.     

Identification and estimation of endogenous digoxin in biological fluids and tissues by TLC and HPLC.

A procedure for estimation of digoxin in biological samples after adding a known quantity of digoxin followed by extraction, separation by TLC and HPLC is described. The identity of digoxin thus extracted from rat brain has been established by reaction with digoxin antibody and by its inhibition of Na(+)-K+ ATPase activity. The method could be a better substitute to the routine radioimmunoassay as interfering substances are removed by TLC and HPLC.     

A radiometric microbiological assay for pantothenic acid in biological fluids

A semi-automated radiometric-microbiological assay (RMA) was developed and applied for the analysis of pantothenic acid in human blood and milk. The RMA is based on the measurement of 14CO2 produced from the metabolism of L-[1-14C]methionine or L-[1-14C]valine by the yeast Kloeckera brevis in the presence of pantothenic acid. The assay was shown to be sensitive and specific for the analysis of this vitamin in biological fluids.     

Surfactant ion-pair high-performance liquid chromatography of tryptophan and some of its metabolites in biological fluids

A rapid, sensitive assay for tryptophan and some of its metabolites in urine, plasma and saliva has been developed using sodium dodecylsulphate as a pairing ion in a surfactant ion-pair high-performance liquid chromatography technique. The method is highly selective for tryptophan which is separated from its main indoleamine metabolites, 5-hydroxytryptophan, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, and from kynurenine. The usefulness of the assay has been demonstrated in plasma level and urinary excretion studies of orally administered tryptophan.     

Simultaneous determination of moricizine and its sulphoxidation metabolites in biological fluids by high-performance liquid chromatography

A simultaneous assay for moricizine, its two sulphoxidation metabolites, morizine sulphoxide and moricizine sulphone, using high-performance liquid chromatography (HPLC) is described. The drug and metabolites and clozapine (internal standard) in biological fluids were extracted using pentanesulphonic acid into diethyl ether. The ethereal extract was evaporated to dryness and the residue was redissolved in the mobile phase (methanol-water-triethylamine, 65:35:0.5, v/v). The analyses were performed on a μBondapak reversed-phase C₁₈ column housed in a Waters Z-module, linked to a C₁₈ pre-column, with a run-time of 12 min. The retention times were 2.7, 3.5, 6.2 and 9.7 min for moricizine sulphone, moricizine sulphoxide, moricizine and clozapine, respectively. The recovery of the compounds from plasma ranged from 89.9% for the sulphoxide to 98.1% for clozapine. The limits of detection of the assay for moricizine, moricizine sulphoxide and moricizine sulphone were 20, 10 and 5 ng/ml, respectively.     

Simultaneous determination of oxidative hair dye p-phenylenediamine and its metabolites in human and rabbit biological fluids

A liquid chromatography with an electrochemical detector method has been developed for the quantitative measurement for three diamine derivatives (p-phenylenediamine, N,N(')-p-phenylenebisacetamide, and 4-aminoacetanilide) in human urine and rabbit blood, urine, and feces. The detection cell consisted of a glassy carbon electrochemical signal obtained with a supporting electrolyte containing 20% methanol-5mM octylammonium orthophosphate (pH 6.30) as the mobile phase. A comparison of the results obtained from HPLC-UV shows agreement.     

Synthesis and biological activity of some known and putative duloxetine metabolites

Several putative phase I duloxetine metabolites, 4-hydroxy-, 5-hydroxy-, 6-hydroxy-, 5-hydroxy-6-methoxy-, 6-hydroxy-5-methoxy-, 5,6-dihydroxy-, and 4,6-dihydroxyduloxetine were synthesized, and their phase II metabolite as glucuronide or sulfate conjugates were also synthesized. Their in vitro binding activities were compared to that of parent compound duloxetine.