Direct determination of glucuronide and sulfate of p-hydroxymethamphetamine in methamphetamine users' urine

Two conjugates of p-hydroxymethamphetamine (p-OHMA), p-OHMA-glucuronide (p-OHMA-Glu) and p-OHMA-sulfate (p-OHMA-Sul) have been identified in methamphetamine (MA) users' urine by using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS-MS). The synthesis of p-OHMA-Glu and p-OHMA-Sul, and an LC-MS procedure for the simultaneous determination of MA and its four metabolites, amphetamine (AP), p-OHMA, p-OHMA-Glu and p-OHMA-Sul, in urine have also been established. After deproteinizing urine samples with methanol, LC-MS employing a C(18) semi-micro column with a gradient elution program provided the successful separations and MS determinations of these analytes within 20 min. Based on the established method, p-OHMA-Sul was detected at higher concentrations than p-OHMA-Glu in all of the three urine samples tested. These data suggest that sulfation is a major pathway in the MA phase II metabolism.     

Oral platelet aggregation inhibitor Ro 48-3657: Determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 μl of plasma) and 25 ng/ml (50 μl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.     

Direct determination of glucuronide and sulfate of 4-hydroxy-3-methoxymethamphetamine, the main metabolite of MDMA, in human urine

A sensitive and reliable LC-ESI-MS procedure for the simultaneous determination of MDMA and its five metabolites including 4-hydroxy-3-methoxymethamphetamine (HMMA) conjugates has been established following the synthesis of two HMMA conjugates, 4-hydroxy-3-methoxymethamphetamine-glucuronide (HMMA-Glu) and 4-hydroxy-3-methoxymethamphetamine-sulfate (HMMA-Sul). Pretreatment of urine samples with methanol and LC-MS employing a C(18) semi-micro column with a gradient elution program provided the successful separations and MS determinations of these analytes within 20 min. Upon applying the method to MDMA users' urine specimens, HMMA-Glu and HMMA-Sul have been directly determined, suggesting the superiority of sulfation to glucuronidation in the HMMA phase II metabolism.     

Automated analysis of a novel anti-epileptic compound, CGP 33 101, and its metabolite, CGP 47 292, in body fluids by high-performance liquid chromatography and liquid-solid extraction

Automated procedures for the determination of CGP 33 101 in plasma and the simultaneous determination of CGP 33 101 and its carboxylic acid metabolite, CGP 47 292, in urine are described. Plasma was diluted with water and urine with a pH 2 buffer prior to extraction. The compounds were automatically extracted on reversed-phase extraction columns and injected onto an HPLC system by the automatic sample preparation with extraction columns (ASPEC) automate. A Supelcosil LC-18 (5 μm) column was used for chromatography. The mobile phase was a mixture of an aqueous solution of potassium dihydrogen phosphate, acetonitrile and methanol for the assay in plasma, and of an aqueous solution of tetrabutylammonium hydrogen sulfate, tripotassium phosphate and phosphoric acid and of acetonitrile for the assay in urine. The compounds were detected at 230 nm. The limit of quantitation was 0.11 μml/l (25 ng/mol) for the assay of CGP 33 101 in plasma, 11 μmol/l (2.5 μg/ml) for its assay in urine and 21 μmol/l (5 μg/ml) for the assay of CGP 47 292 in urine.     

Analysis of antibiotics in urine and wipe samples from environmental and biological monitoring--comparison of HPLC with UV-, single MS- and tandem MS-detection

Results of the simultaneous determination of the structurally different antibiotics cefazoline, cefotiame, cefuroxime, chloramphenicol, ciprofloxacin, ofloxacin, sulfamethoxazole and trimethoprim from environmental and biological monitoring using high-performance liquid chromatography with UV, single mass and tandem mass spectrometry were compared. For sample enrichment and clean-up a SPE method using bakerbond C18 cartridges was developed. Mean recovery rates were above 70%. Because of the complex urine matrix, only the wipe samples could be analyzed by UV-detection. However, UV-detection and single MS-detection are useful for control measurements after spillage, e.g. (LOD=1-2 ng/cm(2)). Samples from biological monitoring of occupational uptake should be analyzed by LC-MS/MS. The limits of detection (LOD) in urine ranged from 0.4 to 70 microg/L for LC-MS and 0.01 to 0.9 microg/L for LC-MS/MS detection. The limits of detection in wipe samples ranged from 0.003 to 0.13 ng/cm(2).     

Simultaneous determination of selected veterinary antibiotics in gilthead seabream (Sparus Aurata) by liquid chromatography-mass spectrometry

A method was optimised and validated for simultaneous monitoring of several drugs of different classes of antibiotics such as quinolones (oxilinic acid and flumequine), tetracyclines (oxytetracycline), sulfonamides (sulfadiazine) and trimethoprim in fish muscle and skin. The method is based on solid-liquid extraction without further sample clean up followed by liquid chromatography-mass spectrometry (LC-MS) determination with electrospray ion source (ESI) in positive mode. The limits of quantification (LOQs) were lower than 20 microg/kg for all compounds and repeatability, expressed as relative standard deviations (RSD), were lower than 15%. Therefore, the LC-MS method was successfully applied for the quantitative determination of antibiotics in gilthead sea bream muscle and skin and oxytetracycline in medicated fishes.     

Determination of urinary 5-hydroxyindole-3-acetic acid by high-performance liquid chromatography with electrochemical detection and direct sample injection.

A method for the routine quantitative determination of the major serotonin metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in urine is described. 5-HIAA was analyzed without prior sample cleanup, using an automated high-performance liquid chromatography system with isocratic elution and electrochemical detection (+0.60 V versus a Ag/AgCl reference electrode). The urine samples were mixed with a solution of the internal standard (5-hydroxyindole-3-propionic acid) and centrifuged. The supernatant was transferred to sealed glass vials, and a 2-microliters aliquot was injected directly onto a C18 reversed-phase analytical column, using an automatic sample injector. Samples of urine could be stored for several months at -80 or at +7 degrees C for 2 days without loss of 5-HIAA. However, a gradual decline with time occurred in crude samples stored at room temperature or above, as well as in urine samples diluted with the mobile phase. The detector response was linear in the range of 0-65 mumol/l 5-HIAA, and the intra- and interassay coefficients of variation were about 5 and 7%, respectively (n = 10).     

Automated high-performance liquid chromatographic method for the determination of nedocromil sodium in human urine using bimodal column switching

An automated HPLC method is described for the determination of nedocromil sodium in human urine. An HPLC autosampler is used to inject urine samples onto a short reversed-phase column. This column acts as a concentration column and performs a preliminary extraction. The concentration column is automatically backflushed onto an ion-exchange column where final separation of nedocromil sodium from urine constituents occurs. Recovery, accuracy, precision, sensitivity and specificity were investigated. The method has been applied to urine samples from clinical studies, and the results were compared to those obtained using a radioimmunoassay developed previously.     

Screening and determination of β-blockers, narcotic analgesics and stimulants in urine by high-performance liquid chromatography with column switching

A fast method is described for the screening of eleven β-blockers, two narcotic analgesics and two stimulants in urine by HPLC with column switching. The urine sample (100 μl), buffered tto pH 9–9.5, is injected onto a short extraction column packed with CN stationary phase. The extraction is flushed with water for 2.5 min to elute polar matrix components to waste. The retained components are then backflushed by means of a six-port valve onto the ODS analytical column where they are separated. Phosphate buffer pH 3.0 and acetonitrile were used as mobile phase. Gradient elution was applied in the screening method to improve separation. Detection was performed with diode-array detector at 220, 235 and 300 nm. Recoveries were near 100%, precision was excellent and sensitivity about 0.25 μg/1. The speed up the quantitative analysis, the same method but with isocratic elution was successfully applied to the determination of acebutolol and metoprolol in urine samples collected 4 h after administration of the compounds as single doses.     

Determination by liquid chromatography-mass spectrometry of clomiphene isomers in the plasma of patients undergoing treatment for the induction of ovulation

A rapid, sensitive and selective LC-MS method is described for the simultaneous determination of zuclomiphene and enclomiphene in plasma from patients undergoing treatment with clomiphene citrate for the induction of ovulation. Samples spiked with N-didesmethyltamoxifen, the internal standard, were extracted into methyl tertiary butyl ether. The compounds were separated on a Luna C(18) analytical column, and a mobile phase of methanol-water (70:30 v/v) containing 0.05% trifluoroacetic acid at a flow rate of 1ml/min. The limits of determination were 35pg/ml and 7pg/ml for zu- and enclomiphene, respectively. Within-day coefficients of variation ranged from 2.1% to 7.2%.     

Quantification of topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces by high-performance liquid chromatographic methods

Sensitive high-performance liquid chromatographic (HPLC) methods have been developed and validated for the simultaneous determination of the antitumor drug topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces. Both compounds are reversibly hydrolysed to their hydroxycarboxylate forms at physiologic pH. Separate HPLC systems have been developed for the determination of lactone and total (lactone plus hydroxycarboxylate forms) concentrations in plasma. The instability of the analytes in plasma requires immediate protein precipitation with ice-cold methanol. The lactone forms of the analytes were stable in the methanol extracts for at least 15 months when stored at −70°C. For the determination of the total levels, the plasma extracts were acidified with 25 mM phosphoric acid to convert the compounds into their lactone forms quantitatively. The sample pretreatment procedure for urine included dilution in methanol while the faecal samples were homogenized in distilled water and then extracted twice with an acetonitrile–ammonium acetate mixture. Separation was achieved on reversed-phase columns (Zorbax SB-C18) and detection was performed fluorimetrically at 380/527 nm. Within-run and between-run precisions were less than 10% and average accuracies were between 90 and 110%. The methods were used in a mass balance study in patients with malignant solid tumors to determine the disposition and routes of elimination of topotecan and N-desmethyltopotecan.     

Development of a high performance liquid chromatography method and a liquid chromatography-tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulfonamides in the foods of animal origin

The residues of sulfonamides (SAs) in the foods of animal origin are of the major concern because they are harmful to the consumer's health and could induce pathogens to develop resistance. Rapid and efficient determination methods are urgently in need. A quantitative high performance liquid chromatography method (HPLC) and a confirmative liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of 18 sulfonamides such as sulfamidinum, sulfanilamide, sulfisomidine, sulfadiazine, sulfapyridine, sulfathiazole, sulfamerazine, sulfadimidine, sulfamethoxypyridazine, sulfamethoxydiazine, sulfisoxazole, sulfachloropyridazine, sulfamethoxazole, sulfamonomethoxine, sulfadoxine, sulfaclozine, sulfadimethoxine, sulfaquinoxaline in the muscles, livers and kidneys of swine, bovine and chicken were developed and validated. The sample preparation procedures included a pressurized liquid extraction (PLE) with acetonitrile conducted at elevated temperature (70°C) and pressure (1400 psi). After clean-up with hydrophilic-lipophilic balance cartridge, the extraction solution was concentrated and analyzed by HPLC and LC-MS/MS analysis. 18 SAs were separated by the HPLC with a Zorbax SB-Aq-C18 column and the mobile phase of methanol/acetonitrile/1% acetic acid with a gradient system. The wavelength of UV for the HPLC detection was set at 285 nm. The LC-MS/MS analysis was achieved with a Hypersil Golden column and the mobile phase of acetonitrile and 0.1% formic acid aqueous solution with two gradient systems. The Limits of detection (LOD) and the limits of quantitation (LOQ) were 3 μg/kg and 10 μg/kg, respectively, for both of the HPLC and LC-MS/MS. Linearity was obtained with an average coefficient of determination (R) higher than 0.9980 over a dynamic range from the LOQ value up to 5000 μg/kg. The recoveries of the methods range from 71.1% to 118.3% with the relative standard derivation less than 13%. The peaks of interest with no interferences were observed throughout the chromatographic run. The sample pretreatment provided efficient extraction and cleanup that enables a sensitive and rugged determination of 18 SAs, the obtained results revealed that PLE, in comparison with other sample preparation methods applied, has significantly higher efficacy for SAs isolation from animal tissues.     

Liquid chromatography-electrospray ionization ion trap mass spectrometry for analysis of mesocarb and its metabolites in human urine

A method is described for the determination of metabolites of mesocarb in human urine by combining gradient liquid chromatography and electrospray ionization (ESI)-ion trap mass spectrometry. Seven metabolites (two isomers of hydroxymesocarb, p-hydroxymesocarb, two isomers of dihydroxymesocarb and two isomers of trihydroxymesocarb) and parent drug were detected in human urine after the administration of a single oral dose 10 mg of mesocarb (Sydnocarb, two tablets of 5 mg). Various extraction techniques (free fraction, enzyme hydrolyses and acid hydrolyses) and their comparison were carried out for investigation of the metabolism of mesocarb. After extraction procedure the residue was dissolved in methanol and injected into the column HPLC (Zorbax SB-C18 (Narrow-Bore 2.1 x 150 mm i.d., 5 microm particles)) with mobile phase (0.2 ml/min) of methanol/0.2 mM ammonium acetate. Conformation of the results and identification of all metabolites are performed by LC-MS and LC-MS/MS. The major metabolites of mesocarb in urine of the human were p-hydroxylated derivative of the phenylcarbamoyl group of the parent drug (p-hydrohymesocarb) and dihydroxylated derivative of mesocarb (two isomers of dihydroxymesocarb). This analytical method for dihydrohymesocarb was very sensitive for discriminating the ingestion of mesocarb longer than the parent drug or other metabolites in human urine. The dihydroxymesocarb was detected in urine until 168-192 h after administration of the drug.     

Determination of urinary valproylcarnitine by gas chromatography—mass spectrometry with selected-ion monitoring

A modified method for the determination of valproylcarnitine in urine samples of patients receiving sodium valproate by gas chromatography—mass spectrometry with selected-ion monitoring is described. The chemically analogous internal standard 2-ethylpentanoylcarnitine was added to the urine samples. Valproic acid and its metabolites were removed by extraction with chloroform at pH 5.0. The samples were then applied onto a C₁₈ Sep-Pak column. Inorganic and water soluble compounds were washed out with water. Valproylcarnitine and internal standard were eluted with methanol and were derivatized to the corresponding acyl-containing lactones by heating at 100°C for 60 min in dimethylformamide. Urinary valproylcarnitine levels of epileptic patients receiving valproate were determined according to the present method. The data obtained might be useful for diagnosis of carnitine deficiency.     

Rapid and simple method for the determination of urinary benzoic and phenylacetic acids and their glycine conjugates in ruminants by reversed-phase high-performance liquid chromatography

A simple, rapid and reproducible reversed-phase high-performance liquid chromatographic method for the simultaneous determination of benzoic acid (BA), phenylacetic acid (PAA) and their respective glycine conjugates hippuric acid (HA) and phenaceturic acid (PA) in sheep urine is described. The procedure involves only direct injection of a diluted urine sample, thus obviating the need for an extraction step or an internal standard. The compounds were separated on a Nova-Pak C₁₈ column with isocratic elution with acetate buffer (25 mM, pH 4.5)—methanol (95:5). A flow-rate of 1.0 ml/min, a column temperature of 35°C and detection at 230 nm were employed. These conditions were optimized by investigating the effects of pH, molarity, methanol concentration in the mobile phase and column temperature on the resolution of the metabolites. The total analysis time was less than 15 min per sample. At a signal-to-noise ratio of 3 the detection limits for ten-fold diluted urine were 1.0 μg/ml for BA and HA and 5.0 μg/ml for PAA and PA with a 20-μl injection.     

Dexamethasone quantification in dried blood spot samples using LC-MS: The potential for application to neonatal pharmacokinetic studies

A high-performance liquid chromatography (LC-MS) method has been developed and validated for the determination of dexamethasone in dried blood spot (DBS) samples. For the preparation of DBS samples whole blood spiked with analyte was used to produce 30μl blood spots on specimen collection cards. An 8mm disc was cut from the DBS sample and extracted using a combination of methanol: water (70:30, v/v) containing the internal standard, triamcinolone acetonide. Extracts were centrifuged and chromatographic separation was achieved using a Zorbax Eclipse Plus C18 column using gradient elution with a mobile phase of acetonitrile and water with formic acid at a flow rate of 0.2ml/min. LC-MS detection was conducted with single ion monitoring using target ions at m/z 393.1 for dexamethasone and 435.1 for the internal standard. The developed method was linear within the tested calibration range of 15-800ng/ml. The overall extraction recovery of dexamethasone from DBS samples was 99.3% (94.3-105.7%). The accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤15% at all concentrations. Factors with potential to affect drug quantification measurements such as blood haematocrit, the volume of blood applied onto the collection card and spotting device were investigated. Although a haematocrit related effect was apparent, the assay accuracy and precision values remained within the 15% variability limit with fluctuations in haematocrit of ±5%. Variations in the volume of blood spotted did not appear to affect the performance of the developed assay. Similar observations were made regarding the spotting device used. The methodology has been applied to determine levels of dexamethasone in DBS samples collected from premature neonates. The measured concentrations were successfully evaluated using a simple 1-compartment pharmacokinetic model. Requiring only a microvolume (30μl) blood sample for analysis, the developed assay is particularly suited to pharmacokinetic studies involving paediatric populations.     

Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken

A high-performance liquid chromatography (HPLC) method with UV detection was developed for the simultaneous determination of arsanilic acid, roxarsone, nitarsone, and carbarsone in the feeds of swine and chicken. Feed samples were extracted with methanol/1% acetic acid (90:10, v/v) in an ultrasonic bath and the protein was precipitated with 2% Cu(2)SO(4). The samples were further purified by solid phase extraction (SPE) on SAX cartridges. Separation was performed on a Zorbax SB-Aq C18 HPLC column using an isocratic procedure with methanol and 1% acetic acid (3:97, v/v) at a flow-rate of 0.7 mL min(-1), and the UV detector was set at a wavelength of 260 nm. The recoveries of organoarsenic compounds spiked at levels of 2, 20 and 200 μg g(-1) ranged from 81.2% to 91.3%; the inter-day relative standard deviation values were less than 7.0%. The limits of quantification for four organoarsenic compounds were 1.0-2.0 μg g(-1). This simple and fast method could be applied to the determination of multi-residues of organic arsenic compounds in animal feeds.     

Column-switching high-performance liquid chromatographic assay for determination of apigenin and acacetin in human urine with ultraviolet absorbance detection

A high-performance liquid chromatographic (HPLC) method is described for the determination of apigenin and the 4′-methylated derivative acacetin in human urine using column-switching and ultraviolet (UV) absorbance detection. Urine samples were enzymatically hydrolysed and solid-phase extracted prior to injection onto the HPLC system. Prior to elution of apigenin and the internal standard, 5,7,8-trihydroxyflavone, from the first column used for sample clean-up, the six-port valve was switched to the second column for analysis with UV detection. Detection of apigenin was precise and reproducible, with a limit of quantification of 10 ng ml⁻¹ urine. Detection and quantification of acacetin was linear down to 70 ng ml⁻¹ urine. The method has been successfully applied to determine the level of apigenin in 100 human urine samples from an intervention study with parsley.     

Structures of (-)-epicatechin glucuronide identified from plasma and urine after oral ingestion of (-)-epicatechin: differences between human and rat

(-)-epicatechin is one of the most potent antioxidants present in the human diet. Particularly high levels are found in black tea, apples, and chocolate. High intake of catechins has been associated with reduced risk of cardiovascular diseases. There have been several reports concerning the bioavailability of catechins, however, the chemical structure of (-)-epicatechin metabolites in blood, tissues, and urine remains unclear. In the present study, we purified and elucidated the chemical structure of (-)-epicatechin metabolites in human and rat urine after oral administration. Three metabolites were purified from human urine including (-)-epicatechin-3'-O-glucuronide, 4'-O-methyl-(-)-epicatechin-3'-O-glucuronide, and 4'-O-methyl-(-)-epicatechin-5 or 7-O-glucuronide, according to 1H- and 13C-NMR, HMBC, and LC-MS analyses. The metabolites purified from rat urine were 3'-O-methyl-(-)-epicatechin, (-)-epicatechin-7-O-glucuronide, and 3'-O-methyl-(-)-epicatechin-7-O-glucuronide. These compounds were also detected in the blood of humans and rats by LC-MS. The presence of these metabolites in blood and urine suggests that catechins are metabolized and circulated in the body after administration of catechin-containing foods.     

Determination of raloxifene and its glucuronides in human urine by liquid chromatography-tandem mass spectrometry assay

A selective, sensitive, accurate and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of raloxifene and its three glucuronides: raloxifene-6-β-glucuronide (M1), raloxifene-4'-β-glucuronide (M2), raloxifene-6,4'-diglucuronide (M3) in urine samples is presented in this paper. To our knowledge the developed analytical method is the first fully validated method capable of simultaneous determination of raloxifene and its glucuronides in real urine samples. Moreover, for the first time a method for determination of raloxifene diglucuronide in relevant biological samples was introduced. Metabolites were obtained by a bioconversion process of raloxifene to its glucuronides using the microorganism Streptomyces sp. and were used as standards for validation. Urine samples were introduced to a simple solid phase extraction prior to the analysis by LC-MS/MS. The method was linear in a wide range with high determination coefficient (r(2) > 0.997). The limits of quantification achieved were 1.01, 1.95, 2.83 and 4.69 nM for raloxifene, M1, M2 and M3, respectively. The recoveries were higher than 92.5%, the accuracy was within 100 ± 8.8% and the precision was better than 12% for all compounds. The developed method was successfully applied to the real urine samples and showed to be appropriate for use in further research of still not completely discovered raloxifene pharmacokinetics. Furthermore, the presented method could also serve for a potential application in anti-doping analysis.