Marijuana metabolites in urine of man. X. Identification of marijuana use by detection of delta 9-tetrahydrocannabinol-11-oic acid using thin-layer chromatography

Marijuana use can be determined by detecting delta 9-tetrahydrocannabinol-11-oic acid (THC-11-oic acid) in urine. For this, we describe a procedure for its chemical detection by using sequential thin-layer chromatography on a single plate for rapid isolation and identification. A volume of urine containing 50 mg of creatinine is concentrated by evaporation to 10 ml, the concentrate is enzymically hydrolyzed for 30 minutes and extracted with ether, and the extract is purified by treatment with NaHCO3, then chromatographed in an alkaline and an acidic solvent sequence. The plate is sprayed with Fast Blue Salt B, and THC-11-oic is identified by its characteristic mobility and its characteristic colour reaction. The sensitivity is 0.5 microgram. THC-11-oic acid has been detected in urines collected after the smoking of one standard cigarette containing 16-18 mg of delta 9-tetrahydrocannabinol and in 34 of the first 100 tests of spontaneously collected urines of patients in a hospital drug-abuse treatment program. Multiple samples are easily carried through this extraction procedure. Evaporative concentration takes about 20 min per sample, and the analysis of eight concentrated samples take about 5.5 h.     

Marijuana metabolites in urine of man. XI. Detection of unconjugated and conjugated delta 9-tetrahydrocannabinol-11-oic acid by thin-layer chromatography

A method for separating unconjugated and conjugated delta 9-tetrahydrocannabinol-11-oic acid, the major urinary metabolite of delta 9-tetrahydrocannabinol in man, by liquid-liquid extraction and detection of both forms by thin-layer chromatography is described. The unconjugated form of the metabolite is extracted with hexane-diethylether (65:35), and the conjugated form (which remains in the aqueous phase) is extracted with ether after enzymic hydrolysis. The residue of each extract is chromatographed in an alkaline and an acidic solvent sequence, and the metabolites are detected with Fast Blue Salt B.     

Development of a high-performance liquid chromatographic method with electrochemical detection for the determination of hyperforin

An HPLC method with electrochemical detection for the determination of hyperforin extracts without using additional sample precleaning has been developed and validated. The hyperforin solutions were separated isocratically using a mobile phase consisting of 10% ammonium acetate buffer (0.5 M, pH 3.7)-MeOH-acetonitrile (10:40:50, v/v) at a flow rate of 0.8 mL/min. Hyperforin was detected amperometrically with a glassy carbon electrode at a potential of +1.1 V versus Ag/AgCl/3 M potassium chloride reference electrode. Under these conditions, a plot of integrated peak area versus concentration of hyperforin was found to be linear over the range of 0.054-5.4 microg/mL, with a relative standard deviation of 2.2-8.6%. The limit of detection was 0.050 ng on column. The determination of the hyperforin content in a commercially available St. John's Wort preparation exhibited a mean content of 1.56 mg. Recovery experiments led to a mean recovery rate of 97 +/- 5.8%. The proposed method is not time-consuming, sensitive and reproducible and is therefore suitable for routine analysis of hyperforin in herbal medicinal products.     

Direct injection high-performance liquid chromatographic method with electrochemical detection for the determination of ethanol and methanol in plasma using an alcohol oxidase reactor

A highly sensitive reversed-phase high-performance liquid chromatographic assay for ethanol and methanol in plasma, using a post-column enzymic reactor with electrochemical detection, has been developed. The alcohols, separated on the column, were converted by immobilized alcohol oxidase into their respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, detected via oxidation at a platinum electrode. As the chromatographic column, two glass cartridges (150 mm x 3 mm I.D.) in series, packed with 10 microns HEMA-S 1000 packing, were used. Alcohol oxidase from Candida boidinii was immobilized onto HEMA-BIO 1000 VS-L (10 microns), packed in a 30 mm x 3 mm I.D. glass cartridge. The reaction product, hydrogen peroxide, was detected with an amperometric detector with a platinum electrode, operated at +500 mV vs. an Ag/AgCl reference electrode. A 20-microliters volume of ten-fold diluted plasma was injected without any pre-treatment. Under the described conditions, methanol and ethanol were well resolved from each other and from the "front" of the chromatogram. The limit of detection was ca. 2.5 nmol for ethanol and 0.6 nmol for methanol in plasma, at a signal-to-noise ratio of 3. Excellent linearity was observed for ethanol, in the range 0.125-4 micrograms injected (r = 0.9999). In contrast, the response for methanol was markedly non-linear above 500 micrograms injected, presumably owing to progressive saturation of the reactor. The precision and accuracy of the assay were satisfactory, as was the reactor life (one month).     

Column-switching high-performance liquid chromatographic determination of clarithromycin in human plasma with electrochemical detection

A column-switching HPLC method was described for the direct analysis of clarithromycin in human plasma using electrochemical detector without sample pre-purification step. Plasma samples were diluted with washing solvent, i.e. acetonirile-methanol-0.05 M potassium phosphate buffer (pH 7.0) (5:2:93, v/v) and then, injected to the precolumn. After plasma proteins had flowed out from the precolumn, clarithromycin and internal standard (roxithromycin) were eluted to a Luna 2 C(18) column and separated with acetonitrile-methanol-0.05 M potassium phosphate buffer (pH 7.0) (41:6:53, v/v). Electrochemical oxidation of clarithromycin occurred at 0.87 V vs. Ag/AgCl reference electrode with glassy carbon electrode. The calibration curve was linear in the concentration range 0.1-4 mug ml(-1) with correlation coefficient of 0.998. This method showed excellent precision (RSD 3.8% at 0.1 mug ml(-1)) and accuracy (+/-2%) with the total analysis time per sample of 30 min. The present method was successfully applied to the pharmacokinetic study of clarithromycin in volunteers receiving a single oral administration of clarithromycin.     

Automated column-switching high-performance liquid chromatographic determination of flumequine and oxolinic acid in extracts from fish

Two methods for automated analysis of extracts from edible muscle tissue of Atlantic salmon are described. Oxolinic acid and flumequine are extracted with phosphate buffer pH 9, and the extracts are analysed by high-performance liquid chromatography using a column-switching system. One method applies on-line concentration and clean-up of the extracts on a precolumn packed with polystyrene-divinylbenzene. This method was useful for the analysis of oxolinic acid and flumequine in the microgram/g range. The other method was based on on-line dialysis and concentration of the dialysate on the polymeric precolumn. This method was shown to be a reliable method for residue analysis, and the limit of detection was 2 ng/g for oxolinic acid and 3 ng/g for flumequine with fluorescence detection.     

Direct and simultaneous high-performance liquid chromatographic assay for the determination of p-aminobenzoic acid and its conjugates in human urine

Procedures based on high-performance liquid chromatography (HPLC) were developed for identifying and measuring p-aminobenzoic acid (PABA) and its conjugate metabolites in human urine after oral doses of PABA. p-Aminohippuric acid (PAH), PABA, p-acetamidohippuric acid (PAHA) and p-acetamidobenzoic acid (PADB) in urine were resolved and determined by HPLC simultaneously and directly without extraction. A mobile phase consisting of 3% (v/v) acetonitrile in distilled water containing 0.005 M 1-heptanesulphonic acid in glacial acetic acid (PIC-B7) at pH 3.3 was eluted at 1 ml/min through a C18 Spherisorb column, followed by UV detection at 280 nm. After hydrolysis of urine samples at 37 degrees C for 3 h with beta-glucuronidase, the amounts of PABA-glucuronide and PADB-glucuronide were also determined. The retention times of PAH, a dominant peak which disappeared after hydrolysis, PABA, DABA (3,5-diaminobenzoic acid, as the internal standard), PAHA and PADB were 11.8, 14, 15, 18, 24 and 46 min, respectively. The 24-h urinary recoveries of PAH, PAHA, PADB, PADB-glucuronide, PABA and PABA-glucuronide after separate oral doses of 200 and 800 mg of PABA in one healthy subject were 43.4 and 48.1, 7 and 29.1, 11.2 and 11.8, 34.8 and 6.6, 0.2 and 0.3, and 1.0 and 2.4%, respectively. It is interesting that at high dose (800 mg) saturation of glucuronidation of PADB (N-acetylated PABA) appeared to occur, which resulted in an increase in the formation of PAHA, the glycine conjugate of PADB. Over 90% of the oral dose was accounted for by 8 h after administration.     

Direct injection analysis of ketoprofen enantiomers in plasma using column-switching high-performance liquid chromatography system

A high-performance liquid chromatography system was developed for the stereoselective determination of ketoprofen enantiomers in human plasma following direct sample injection. The system comprised of a pretreatment column and a chiral separation column connected in a series via a switching valve. When a 200 microliter portion of human plasma containing a therapeutic level of ketoprofen was directly applied to the system, ketoprofen was adsorbed in the pretreatment column, while plasma proteins were excluded. After the elution of proteins from the pretreatment column, the valve was switched and ketoprofen was desorbed and transferred to the chiral separation column where the enantiomers were separated and determined by ultraviolet-absorption. The mobile phase conditions for the pretreatment and chiral separation were optimized, which enabled rapid and complete recovery followed by satisfactory separation of the enantiomers. The calibration line for each enantiomer showed good linearity in the range of 0.25-5 micrograms/ml with a detection limit of 0.02 micrograms/ml (signal to noise ratio (S/N) greater than 3), which was sufficient for practical demands. The precision test indicated that the coefficient of variation for five repeated determinations of (-) ketoprofen was 5.4% at 0.1 microgram/ml and 1.4% at 1 microgram/ml.     

Column-switching high-performance liquid chromatographic detection of pholcodine and its metabolites in urine with fluorescence and electrochemical detection.

A sensitive and selective method for the detection of pholcodine and its metabolite morphine in urine using high-performance liquid chromatography is described. It involves on-line clean-up of urine on a trace enrichment column packed with a polymeric strong cation-exchange material. Pholcodine and its metabolites were separated on two analytical columns with different selectivities. Pholcodine was detected by a fluorescence detector and morphine was detected electrochemically. One system, based on reversed-phase chromatography, applied a polystyrene-divinylbenzene column and gradient elution. The other system was based on normal-phase chromatography with a silica column and isocratic elution. Morphine was confirmed to be a metabolite of pholcodine by reversed-phase chromatography and electrochemical detection. Two unidentified metabolites of pholcodine were separated from pholcodine by normal-phase chromatography and detected by fluorescence detection.     

Sequential analysis of malic acid and both enantiomers of lactic acid in wine using a high-performance liquid chromatographic column-switching procedure.

A liquid chromatographic column-switching method for the sequential determination of malic acid and both enantiomers of lactic acid in wine is described. The procedure involves the heart cutting of lactic acid enantiomers from a reversed-phase high-performance liquid chromatography chromatogram, retaining them, and back-flushing them through a chiral ligand-exchange column in which they are separated. The method is used to determine the concentration of lactic acid enantiomers in commercial wines. The results are in satisfactory agreement with those of other methods. The malic acid contents of various wines are also determined. The total analysis time for one experiment is approximately 10 min.     

Simultaneous determination of uric acid and creatinine in biological fluids by column-switching liquid chromatography with ultraviolet detection

A column-switching liquid chromatographic method for the simultaneous determination of uric acid and creatinine in human serum and urine was developed. Creatinine and uric acid were separated by size-exclusion chromatography on a hydrophilic gel column (C1) and creatinine eluted from Cl was separated from proteins by filtration through a longer hydrophilic gel column (C2). The creatinine fraction eluted from C2 was transferred to a weakly acidic cation-exchange column (C3) and then to a strongly acidic cation-exchange column (C4). Uric acid eluted from Cl after creatinine was transferred to an anion-exchange column (C5) and then to a hydrophilic gel column (C6). The mobile phase was a mixed buffer of pH 5.1 (propionic acid-succinic acid-NaOH, 60:15:60 mmol/1 in water). Diluted serum and urine could be injected onto C1, and Cl was backflushed after the transfer of uric acid from Cl to C5.

Creatinine and uric acid in the eluate were determined by measuring their ultraviolet absorption at 234 and 290 nm, respectively. The recovery of uric acid and creatinine added to diluted serum (20-fold dilution, concentration 20 and 5 μmol/1, respectively) was 98.9±0.56% and 100.9±1.29%, respectively. The recovery of uric acid and creatinine added to diluted urine (100-fold dilution, concentration 50 and 100 μmol/l, respectively) was 99.4±0.72% and 98.7±1.45%, respectively (mean±R.S.D., n=6).     

Determination of delta 9-tetrahydrocannabinol in plasma using solid-phase extraction and high-performance liquid chromatography with electrochemical detection

A method for the determination of nanogram amounts of delta 9-tetrahydrocannabinol (THC) in plasma and serum is described. THC was quantitatively isolated by solid-phase extraction after addition of an aqueous solution of urea and methanol to the sample. The extracts were analysed by high-performance liquid chromatography with electrochemical detection in the oxidizing mode. The detection limit of THC is ca. 100 pg for a signal-to-noise ratio of 3. With this method, levels of 2 ng/ml of THC in plasma can be measured.     

Determination of trimethoprim metabolites including conjugates in urine using high-performance liquid chromatography with combined ultraviolet and electrochemical detection

A high-performance liquid chromatographic method for the determination of trimethoprim metabolites in pig urine was developed. The metabolites-glucuronic acid and sulphuric acid conjugates of phenolic metabolites formed by demethylation of trimethoprim-were quantitated after treatment of urine with beta-glucuronidase (Escherichia coli). The sulphuric acid conjugate was not susceptible to enzymatic hydrolysis and was therefore assayed as the conjugate by use of ion-pair chromatography on the reversed-phase column. In order to find suitable conditions for enzymatic hydrolysis of the glucuronides, the conjugates were obtained by gel chromatography of urine from a [14C] trimethoprim-treated pig.