Determination of (S)-(-)-cathinone and its metabolites (R,S)-(-)-norephedrine and (R,R)-(-)-norpseudoephedrine in urine by high-performance liquid chromatography with photodiode-array detection.

A high-performance liquid chromatographic (HPLC) procedure with photodiode-array detection (DAD) is described for the determination of (S)-(-)-cathinone (S-CA) and its metabolites (R,S)-(-)-norephedrine (R-NE) and (R,R)-(-)-norpseudoephedrine (R-NPE) in urine. Extraction and clean-up of 1-ml urine samples were performed on a cyano-bonded solid-phase column using (+/-)-amphetamine as internal standard. The concentrated extracts were separated on a 3-microns ODS-1 column with acetonitrile-water-phosphoric acid-hexylamine as the mobile phase. Peak detection was done at 192 nm. The detection limits for S-CA and R-NE/R-NPE in urine were 50 and 25 ng/ml, respectively. The differentiation of the enantiomers of cathinone and norephedrine was achieved by derivatization with (S)-(-)-1-phenylethyl isocyanate to the corresponding diastereomers followed by HPLC-DAD on a 5-microns normal-phase column. The R and S enantiomers of norpseudoephedrine were determined by gas chromatography-mass spectrometry after on-column derivatization with (S)-(-)-N-trifluoroacetylprolyl chloride. Following a single oral dose of 0.5 mg/kg of S-CA, the concentrations found in urine ranged from 0.2 to 3.8 micrograms/ml of S-CA, from 7.2 to 46.0 micrograms/ml of R-NE and from 0.5 to 2.5 micrograms/ml of R-NPE.     

Comprehensive screening procedure for diuretics in urine by high-performance liquid chromatography

A rapid and reliable screening procedure using high-performance liquid chromatography for the detection of 23 diuretics (belonging to five different pharmacological groups) in urine has been developed. Two aliquots of 2-ml urine samples were extracted separately under acidic and basic conditions. The acidic and basic extracts were pooled, evaporated to dryness and reconstituted in methanol. The methanolic extract was injected onto a Hewlett-Packard Hypersil ODS C18 (5 microns) column (column I) and a Hewlett-Packard LiChrosorb RP-18 (5 microns) column (column II; an alternative column). The same gradient mobile phase was used for both columns. A diode array ultraviolet detector was set to monitor the signal to the integrator (Chem Station) at 230 and 275 nm. Recovery studies of the 23 diuretics were performed under acidic and basic conditions. The overall lower limits for detection on column I using both extraction procedures ranged from 0.5 to 1.5 micrograms/ml of urine (average 1.0 micrograms/ml). Amiloride, ethacrynic acid and probenecid could not be detected below 5 micrograms/ml of urine. No interference from the biological matrix was apparent. Amiloride could be detected in urine 4 h after oral administration of 15 mg of amiloride to a healthy volunteer, when the sample was extracted under alkaline conditions. The suitability of the screening method for the analysis of urine samples was tested by studying the variation with time of chlorthalidone, furosemide, probenecid, acetazolamide, quinethazone, spironolactone, bendroflumethiazide, bumetanide, triameterene and hydrochlorothiazide concentrations in the urine of normal human volunteers after minimum single or multiple (probenecid) doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous determination of psychotropic phenylalkylamine derivatives in human hair by gas chromatography/mass spectrometry

A gas chromatography/mass spectrometric (GC/MS) method was developed and validated for the determination of thirteen psychotropic phenylalkylamine derivatives (amphetamine; AP, phentermine; PT, methamphamine; MA, cathinone; Khat, methcathinone; MCAT, fenfluramine; FFA, desmethylselegiline; DSEL, 3,4-methylenedioxyamphetamine; MDA, 3,4-methylenedioxymethamphetamine; MDMA, 3,4-methylenedioxyethylamphetamine; MDEA, norketamine; NKT, mescaline; MES, 4-bromo-2,5-dimethoxyphenethylamine; 2CB) in human hair. Hair samples (20 mg) were washed with distilled water and acetone, cut into small fragments (<1 mm), and incubated in 0.25 M methanolic HCl under ultrasonication at 50 degrees C for 1 h. The resulting solutions were evaporated to dryness, derivatized using trifluoroacetic anhydride (TFAA) at 70 degrees C for 30 min, and analyzed by GC/MS. The linear ranges were 0.02-25.0 ng/mg for AP, PT, Khat, FFA, DSEL, MDMA, and 2CB; 0.05-25.0 ng/mg for MA, MCAT, and MES; 0.05-12.5 ng/mg for MDA; and 0.1-25.0 ng/mg for MDEA and NKT, with good correlation coefficients (r(2) > 0.9985). The intra-day, inter-day, and inter-person precisions were within 12.7%, 14.8%, and 16.8%, respectively. The intra-day, inter-day, and inter-person accuracies were between -10.7 and 13.4%, -12.7 and 11.6%, and -15.3 and 11.9%, respectively. The limits of quantifications (LOQs) for each compound were lower than 0.08 ng/mg. The recoveries were in the range of 76.7-95.6%. The method proved to be suitable for the simultaneous qualification and quantification of phenylalkylamine derivatives in hair specimens.     

Analysis of phenothiazines in human body fluids using disk solid-phase extraction and liquid chromatography

Seven phenothiazine derivatives, perazine, perphenazine, prochlorperazine, propericiazine, thioproperazine, trifluoperazine, and flupentixol, have been found to be extractable from human plasma and urine samples using disk solid-phase extraction (SPE) with an Empore C18 cartridge. Human plasma and urine (1 mL each) containing the 7 phenothiazine derivatives were mixed with 2 mL of 0.1M NaOH and 7 mL distilled water and then poured into the disk SPE cartridges. The drugs were eluted with 1 mL chloroform- acetonitrile (8 + 2) and determined by liquid chromatography with ammonium formate/formic acid-acetonitrile gradient elution. The detection was performed by ultraviolet absorption at 250 nm. The separation of the 7 phenothiazine derivatives from each other and from impurities was generally satisfactory using a SymmetryShield RP8 column (150 x 2.1 mm id, 3.5 microm particle size). The recoveries of the 7 phenothiazine derivatives spiked into plasma and urine samples were 64.0-89.9% and 65.1-92.1%, respectively. Regression equations for the 7 phenothiazine derivatives showed excellent linearity, with detection limits of 0.021-0.30 microg/mL for plasma and 0.017-0.30 microg/mL for urine. The within-day and day-to-day coefficients of variation for both samples were commonly below 9.0 and 14.9%, respectively.     

Liquid chromatographic determination of sotalol in plasma and urine employing solid-phase extraction and fluorescence detection

A liquid chromatographic method using a solid-phase extraction procedure for the quantification of sotalol in plasma and urine is described. Sotalol is eluted from an extraction column with ethyl acetate-acetonitrile (1:2) and, after separation by reversed-phase high-performance liquid chromatography on a mu Bondapak C18 column, is quantified by fluorescence detection at excitation and emission wavelengths of 240 and 310 nm, respectively. The method has been demonstrated to be linear over the concentration ranges 10-6000 ng/ml in plasma and 0.5-100 micrograms/ml in urine. Mean inter-assay accuracy of the method for plasma ranged from 93 to 100% and for urine from 102 to 114%; precision ranged from 0.5 to 1.6% for plasma over a concentration range of 200-4000 ng/ml and for urine from 0.7 to 2.0% at concentrations of 2-50 micrograms/ml. Mass spectrometry confirmed the presence of sotalol in isolated chromatographic fractions of plasma and urine extracts from subjects given sotalol orally.     

High-performance liquid chromatographic determination of BRB-I-28, a novel antiarrhythmic agent, in dog plasma and urine.

A sensitive reversed-phase high-performance liquid chromatographic (HPLC) technique with ultraviolet detection has been developed to determine the concentration of BRB-I-28 (I), a novel antiarrhythmic agent, in dog plasma and urine. The mobile phase was acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8-triethylamine (50:50:75:0.1, v/v). The compound was extracted from dog plasma and urine with chloroform after alkalinization with sodium hydroxide. The extraction recovery was 83% from plasma and 84% from urine. Good linearity (r > 0.996) was observed throughout the ranges 0.1-12.0 micrograms/ml (plasma) and 0.1-8.0 micrograms/ml (urine). Intra- and inter-assay variabilities were less than 4%. The lower limit of quantitation was 0.08 microgram/ml in either plasma or urine. HPLC analysis of plasma and urine samples from a dog treated with I has demonstrated that the method was accurate and reproducible.     

High-performance liquid chromatographic determination of iothalamic acid in human plasma and urine.

A simple, rapid and sensitive method for the determination of iothalamic acid (IA) in both plasma and urine is reported. After extraction with ethyl acetate, IA was determined by strong anion-exchange high-performance liquid chromatography with ultraviolet detection at 254 nm. The lower limit of detection was 0.5 micrograms/ml. The average recovery was 73 and 57% from plasma and urine, respectively. Linearity was found over the investigated concentration range (up to 500 micrograms/ml for plasma and up to 10.0 mg/ml for urine). The reproducibility of the technique was good (coefficient of variation less than 6%) as was the precision and accuracy (coefficient of variation less than 2.5%). No interference from endogenous substances or any of the common drugs tested was found.     

Determination of tazobactam and piperacillin in human plasma, serum, bile and urine by gradient elution reversed-phase high-performance liquid chromatography

A gradient elution high-performance liquid chromatographic method is described for the analysis of the beta-lactamase inhibitor tazobactam (YTR-830H) and a semi-synthetic parenteral penicillin, piperacillin, in human plasma, serum, bile and urine. The assay for plasma, serum and bile involves deproteinization with acetonitrile and the removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantitation are achieved using a mobile phase based on ion-suppression chromatography on a C18 reversed-phase column with ultraviolet detection at 220 nm. The limit of quantitation for both compounds is 1.0 microgram/ml in plasma, serum and bile using a 0.2-ml sample and 50.0 micrograms/ml in urine using a 0.1-ml sample. The method has been validated by preparing and analyzing a series of fortified samples (range 1.0-200 micrograms/ml for each compound in plasma, serum and bile and 50.0-10,000 micrograms/ml for each compound in urine). Excellent linearity, accuracy, precision and recovery were obtained. The method was not interfered with by other endogenous components, nor by other commonly administered antibiotics such as amoxicillin, mezlocillin, cefometazole and cefotaxime. The assay has been successfully applied to the analysis of samples from pharmacokinetic studies in man and animals.     

Ion-pair extraction and high-performance liquid chromatographic determination of tianeptine and its metabolites in human plasma, urine and tissues

An isocratic reversed-phase ion-pair liquid chromatographic method for the determination of tianeptine and its two main metabolites in plasma, urine and tissues, using an internal standard, is reported. The influence of two stationary phases on the retention of the drugs was studied. The drugs were extracted as ion pairs, using a heptane-octanol-tetraheptylammonium bromide mixture (98:2:0.5, v/v/w) as extraction solvent. This extraction procedure yielded plasma drug recoveries of greater than 60% and allowed UV detection at 220 nm without interference from endogenous components of plasma, urine or tissues. Linear standard curves up to 1.00 micrograms/ml and drug determination down to 0.01 microgram/ml were observed. This method has been successfully applied to the analysis of human plasma and urine samples and of encephales from tianeptine-dosed rats.     

Chromatographic analysis of methylmercaptopurine riboside in human plasma and urine.

An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of methylmercaptopurine riboside (MMPR) in human plasma and urine is reported. Plasma samples were prepared for analysis by addition of internal standard (6-dimethylaminopurine 9-riboside) followed by extraction using disposable C18 cartridges. Urine samples were filtered through a 0.22-micron membrane prior to HPLC separation. The column effluent was monitored at 289 nm and quantitation performed using peak heights. The linear range for MMPR determination was from 10 to 500 ng/ml in plasma and from 0.25 to 50 micrograms/ml in urine. The reported method is convenient, sensitive, and reproducible, illustrating its usefulness for application in pharmacokinetic studies.     

High-performance liquid chromatographic methods for the determination of the penems SCH 29482 and FCE 22101 in human serum and urine.

High-performance liquid chromatographic methods have been developed for the determination of two 6-(1-hydroxyethyl)penems, SCH 29482 (I) and FCE 22101 (II), in serum and urine. Serum samples were combined with an equal volume of methanol to remove proteins and, after centrifugation, an aliquot of the supernatant was analysed by ion-pair chromatography on a reversed-phase C18 column with hexadecyltrimethylammonium bromide as the ion-pairing agent. The compounds were detected by their ultraviolet absorbance at 305 nm for II and 322 nm for I. Urine samples were diluted, filtered and analysed by the same chromatographic procedure. At concentrations of 1-500 micrograms/ml of each compound, the within- and between-day precisions were 1.8-3.6 and 2.6-5.1%, respectively. The detection limit was 0.2 micrograms/ml for I and 0.3 micrograms/ml for II.     

Determination of adriamycin in plasma and tissue biopsies.

A simple, rapid and sensitive method for the extraction and high-performance liquid chromatographic analysis of adriamycin in tissue and plasma is described. Tissue (5-100 mg) and plasma (1 ml) samples underwent a C18 Sep-Pak extraction into methanol. Chromatography was performed on a muBondapakphenyl column using a mobile phase of acetonitrile-0.1 M ammonium formate (pH 4.0) with a flow-rate of 2 ml/min. Fluorometric detection was used with an excitation of 480 nm and an emission of 550 nm. The procedure produced a linear curve for the concentration range 25-1000 ng/ml. The development of the assay produced rapid, repeatable and accurate results for both small tissue samples and plasma.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10 micrometer muBondapak phenyl column with an eluting solvent of water--methanol--1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(D-(-)-alpha-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 +/- 6.3% (S.D.) in the concentration ranges of 0.1-20 microgram per 0.2 ml of plasma with a limit of detection equivalent to 0.5 microgram/ml plasma. The urine assay was validated over a concentration range of 0.025-5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 microgram/ml) using a 0.1-ml urine specimen per assay. The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Simultaneous determination of methylprednisolone and methylprednisolone 21-[8-[methyl-(2-sulfoethyl)amino]-8-oxooctanoate] sodium salt in human urine by high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic assay with ultraviolet detection at 243 nm has been developed for the quantitative determination of methylprednisolone (MP) and methylprednisolone 21-[8-[methyl-(2-sulfoethyl)amino]-8-oxooctanoate] sodium salt (MPSO) in human urine following therapeutic doses in humans. The assay procedure involves stabilization of urine samples by addition of disodium ethylenediaminetetraacetic acid (Na2EDTA) and ion-pair extractions of MPSO using tetraethylammonium chloride (TEACl) as the counter ion. After extracting both drugs and internal standard into chloroform, the extract was evaporated to dryness under nitrogen. The resulting residue was reconstituted in 200-500 microliters of mobile phase and chromatographed on an IBM C18 reversed-phase column (5 microns). The mobile phase was a mixture of water-acetonitrile-isopropanol (71.2:18.8:10.0, v/v) containing 75 microliters of 0.1 M hydrochloric acid and 0.450 g of TEACl per liter. Propyl p-hydroxybenzoate was used as an internal standard. The extraction efficiencies of MP and MPSO were greater than 90% using the ion-pairing agent TEACl. The chromatographic responses were linear up to about 200 micrograms/ml for MP and 80 micrograms/ml for MPSO and had sufficient precision and accuracy to provide quantitative data from human urine. The assay detection limit was about 8 ng/ml for MP and 25 ng/ml for MPSO in human urine. Stability studies in urine indicated that without Na2EDTA stabilization and at room temperature, rapid degradation of MPSO occurred in urine. Addition of EDTA to the urine specimen and storage at -70 degrees C increased the stability of MPSO, and little or no degradation was observed in urine stored for more than 60 days. The method has been used in the simultaneous determination of MP and MPSO in urine specimens obtained from a single-dose tolerance study of MPSO in normal male volunteers.     

Simultaneous determination of ampicillin and sulbactam by liquid chromatography: post-column reaction with sodium hydroxide and sodium hypochlorite using an active hollow-fibre membrane reactor

A high-performance liquid chromatographic method has been developed for the simultaneous determination of ampicillin (ABPC) and sulbactam (SBT) in serum and urine. The method involves separation of ABPC and SBT from the background components of serum and urine on a C18 column, post-column reaction with sodium hydroxide and sodium hypochlorite using an active hollow-fibre membrane reactor, and detection at 270 nm. At ABPC and SBT concentrations of 10 and 5 micrograms/ml in urine and serum samples, the precisions (relative standard deviations) were 0.9-2.5% (n = 8). The detection limits were 20 and 5 ng for ABPC and SBT, respectively, at a signal-to-noise ratio of 3.     

Microassay for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine by high-performance liquid chromatography

An improved method for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine using reversed-phase high-performance liquid chromatography with ultraviolet detection is described. Following solid-phase extraction, chromatography was performed using a column containing an octadecylsilica-coated packing, eluted with 6% acetonitrile in phosphate buffer, pH 2.1, and detected at 233 nm. Using 80-microliters samples, the detection limit is 18 ng/ml for benzoylecgonine and benzoylenorecgonine and 35 ng/ml for cocaine and norcocaine. The coefficients of variation range from 3.5% (benzoylecgonine) to 7.0% (norcocaine). The procedure has been applied to samples of guinea pig plasma, urine and amniotic fluid and human urine.     

Body fluid analysis of a phosphonic acid angiotensin-converting enzyme inhibitor using high-performance liquid chromatography and post-column derivatization with o-phthalaldehyde

A method is described for the extraction of a phosphonic acid angiotensin-converting enzyme inhibitor from either urine or plasma, and subsequent quantitation using high-performance liquid chromatographic (HPLC) analysis and post-column o-phthalaldehyde reagent derivatization. The compound cannot be quantitatively extracted from the body fluids, but use of a fluorinated internal standard allowed for the computation of accurate results. With the use of an internal standard, excellent precision, linearity, and recovery were obtained for analyte response in both urine and plasma. In urine a working range of 0.2-10 micrograms/ml was found, with a limit of detection of 0.1 micrograms/ml. For plasma the working range was found to be 2-500 ng/ml, and the limit of detection was established as 1 ng/ml. Due to the non-polar character of the analyte at low pH values, it was possible to use novel extraction (solid-phase C8 column) and HPLC [poly(styrenedivinyl benzene) HPLC column] conditions to separate and quantitate the compound from plasma and urine.     

Simultaneous determination of the sulphonylurea glimepiride and its metabolites in human serum and urine by high-performance liquid chromatography after pre-column derivatization

A sensitive and selective high-performance liquid chromatographic method has been developed for a new sulphonylurea, glimepiride, and its metabolites. The assay involves extraction with diethyl ether, thermolysis of the sulphonylureas at 100 degrees C and trapping of the resulting amines with 2,4-dinitrofluorobenzene. The derivatives were quantitated on a reversed-phase column by absorbance at 350 nm using a step gradient for the three compounds in serum and an isocratic run for the metabolites in urine. Analogous compounds were used as internal standards. The detection limit was 5 ng/ml for glimepiride and metabolite II and 10 ng/ml for metabolite I using 1 ml of serum. The method has been applied to the analysis of serum and urine samples from pharmacokinetic studies in humans.     

微波萃取-气相色谱法测定尿液中的苯丙胺类毒品

建立了人体尿液中甲基苯丙胺(MA)、3,4-亚甲二氧基苯丙胺(MDA)、3,4-亚甲二氧基甲基苯丙胺(MDMA)的微波萃取-气相色谱(GC)测定方法。分别考察了萃取溶剂种类、用量、pH值以及萃取温度、时间等因素对萃取率的影响。实验结果表明,尿液中MA,MDA,MDMA的最佳提取条件为:调节尿样pH为12,以环己烷为萃取溶剂,于40 ℃下微波提取10 min。在此条件下MA,MDA,MDMA的平均回收率分别为92.25%,85.94%和91.50%,相对标准偏差分别为5.5%,5.5%和6.1% (n=5),提取液经气相色谱-氢火焰离子化检测器(GC-FID)检测,3种药物与基体得到了很好的分离,对尿液中MA,MDA,MDMA的最低检测限分别为10,20和20 ng/mL。该方法未对药物进行衍生化,是一种快速、准确、灵敏度高的同时测定尿液中MA,MDA,MDMA的方法。     

High-performance liquid chromatographic assay of pirlimycin in human serum and urine using 9-fluorenylmethylchloroformate

A reversed-phase high-performance liquid chromatographic (HPLC) assay method has been developed for determining pirlimycin in human serum and urine. The method involves chloroform extraction of pirlimycin free base followed by derivatization with 9-fluorenylmethylchloroformate to form a carbamate ester. The reaction is rapid, reproducible, and quantitative. 9-Fluorenylmethylchloroformate reacts with amines to form derivatives sensitive to both ultraviolet and fluorescence detection. Human serum and urine samples following 50-mg and 500-mg single oral doses of pirlimycin were analyzed. The samples were chromatographed on an RP-18 Spherisorb 5-micron, 250 X 4.6 mm I.D. reversed-phase HPLC column. The eluent for the serum assay was acetonitrile-water (58:42) containing 0.02% acetic acid, and for the urine assay was acetonitrile-methanol-tetrahydrofuran-water (48:2:1:49). Fluoranthene was used as an internal standard. The assay sensitivity by ultraviolet detection (lambda max = 264) was about 5 ng/ml and by fluorescence detection (lambda excitation = 270 nm, lambda emission = 300 nm) was 0.1 ng/ml. Statistical analysis indicates an average drug recovery of 101 +/- 4.2% from serum and 102.0 +/- 2.62% from urine.