尿中有机酸的毛细管气相色谱—质谱轮廓分析—用于苯丙酮尿症的诊断

应用毛细管气相色谱-质谱轮廓分析方法,测定了33例2.5～4.5岁健康儿童尿中有机酸种类及含量和8例拟诊为苯丙酮尿症儿童尿中的有机酸,结果表明患儿尿样中苯丙酮酸、苯乙酸、邻羟基苯乙酸、对羟基苯乙酸高于正常值10～470倍。为苯丙酮尿症的确诊提供了可靠方法。     

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 degrees 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 microgram/ml for BA and HA and 5.0 micrograms/ml for PAA and PA with a 20-microliters injection.     

Studies on steroids. CCXXXXV. Determination of 5 beta-cholestanoic acids in human urine by gas chromatography-mass spectrometry with negative ion chemical ionization detection

A method for the determination of 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) in human urine by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is described. Unconjugated, glycine- and taurine-conjugated DHCA and THCA labelled with 18O and 2H were used as internal standards. 5 beta-Cholestanoic acids in urine were extracted with a Sep-Pak C18 cartridge, separated into the unconjugated, glycine- and taurine-conjugated fractions by ion-exchange chromatography on piperidinohydroxypropyl Sephadex LH-20 and, following alkaline hydrolysis of conjugated forms, derivatization into the pentafluorobenzyl ester-dimethylethylsilyl ethers. Subsequent resolution of each fraction into DHCA and THCA was attained by GC on a cross-linked 5% phenylmethylsilicone fused-silica capillary column where 5 beta-cholestanoic acids were monitored with a characteristic carboxylate anion [M-181]- in the NICI mode using isobutane as a reagent gas. The method was applied to separation and determination of 5 beta-cholestanoic acids in urine from a patient with Zellweger syndrome and from healthy volunteers.     

High-performance liquid chromatographic determination of acetone in blood and urine in the clinical diagnostic laboratory.

A method for the determination of acetone in plasma or urine by high-performance liquid chromatography (HPLC) was developed. Plasma specimens are deproteinized with acetonitrile (1:1, v/v) 2,4-dinitrophenylhydrazine (DNPH) is added to the supernatant or to filtered urine samples, similarly treated with acetonitrile (2:1, v/v) to prevent crystallization of the synthesized phenylhydrazone. An aliquot (20 microliters) of the reaction mixture was subjected to HPLC at ambient temperature using a reversed-phase Pecosphere 3 x 3 C18 column with acetonitrile-water (45:55, v/v) as eluent at a flow-rate of 1 ml/min and detection at 365 nm. Hydroxyacetone and acetoacetate phenylhydrazone derivatives do not interfere. The identification of acetone by its retention time was confirmed by comparison with a laboratory-synthesized acetone DNPH derivative. The concentration of acetone, eluted within 3 min, was determined by the peak-height method. The detection limit was 0.034 mmol/l; the relative standard deviations were less than 5% within run (n = 20) and less than 10% between run (n = 20).     

Simultaneous determination of torasemide and its major metabolite M5 in human urine by high-performance liquid chromatography-electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection is described for the simultaneous determination of the loop diuretic 1-isopropil-3-[4-(3-methylphenylamino)-3-pyridinesulphonyl]urea (torasemide) and its major metabolite M5 in human urine. The assay is simple, fast, and easy. It requires a sample cleanup consisting of a solid-phase extraction under acidic conditions followed by chromatographic separation with a C18 microBondapack column. The use of a water-acetonitrile mobile phase (80:20, v/v, pH 3) ensures total separation from urine-interfering substances, and both compounds can be quantitated amperometrically at a glassy carbon electrode set to +1300 mV versus Ag-AgCl. The method demonstrates linearity for both the parent drug and the metabolite over a wide concentration range (up to 7 microg/mL) and reproducibility with relative standard deviation lower than 2% in intraday and 5% in interday assays. The mean extraction recoveries are 78% for M5 and 60% for torasemide, and the limits of quantitation are 1 and 8 ng/mL, respectively. The method developed is applied to the analysis of healthy volunteers' urine samples collected at different time intervals after the oral ingestion of a single dose of 10 mg torasemide, and the results obtained are in agreement with the pharmacokinetic profile of torasemide.     

Nonaqueous capillary electrophoresis method for the analysis of gleevec and its main metabolite in human urine

The viability of nonaqueous capillary electrophoresis (NACE) was investigated for determination of gleevec and its main metabolite in human urine using a fused-silica capillary. Baseline separation of the studied solutes was obtained using a nonaqueous solution composed of 12 mM ammonium acetate and 87.6 mM acetic acid in methanol-acetonitrile (ACN) (80:20, v:v) providing analysis time shorter than 3 min. Different aspects including stability of the solutions, linearity, accuracy and precision were studied in order to validate the method in the urine matrix. Detection limits of 24 microg L(-1) for gleevec and its metabolite were obtained. A robustness test of the method was carried out using the Plackett-Burman fractional factorial model with a matrix of 15 experiments. The developed method is simple, rapid and sensitive and has been used to determine gleveec and its metabolite at clinically relevant levels in human urine. Before NACE determination, a solid-phase extraction (SPE) procedure with a C18 cartridge was necessary. Real determination of these analytes in two patient urines were done.     

Plasma and cerebrospinal fluid concentration of phenylacetic acid in humans and monkeys

A rapid and reliable mass-fragmentographic method for assay of plasma and cerebrospinal fluid (CSF) concentrations of free and conjugated phenylacetic acid (PAA) is described. The method is used to compare plasma and CSF concentrations of PAA in humans and monkeys. Both packed and capillary columns are used. In humans approximately 45% of total plasma PAA is conjugated in contrast to approximately 60% in monkeys. Both free and conjugated PAA concentrations tend to be higher in monkeys than in humans. Plasma mean concentration of total PAA in humans and monkeys are, respectively, 459.1 and 838 ng/ml. Approximately 55 and 25% of total PAA in the CSF are conjugated in humans and monkeys, respectively. Total PAA mean concentrations in human and monkey CSF are 41.6 and 84.2 ng/ml. Because over 90% of total urine PAA in humans is conjugated, it is concluded that over 50% of urine phenylacetylglutamine may be derived from kidney conjugation of free plasma PAA and/or from the kidney's preferential filtration of conjugated PAA as contrasted with free PAA.     

Simultaneous determination of serotonin and 5-hydroxyindole-3-acetic acid in human urine by automated precolumn derivatization and semi-microbore column liquid chromatography with fluorescence detection.

An improved high-performance liquid-chromatographic (HPLC) assay for the simultaneous determination of serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in human urine is reported. Following the automated precolumn derivatization of urinary 5-HT and 5-HIAA with benzylamine, the derivatives are separated by isocratic elution on a reversed-phase C18 semi-microbore column, and are fluorometrically detected at an excitation wavelength of 345 nm and an emission wavelength of 480 nm. The detection limits of 5-HT and 5-HIAA are 7 and 5 nmol/l in urine (0.7 and 0.5 fmol/20-microl injection). The proposed automatic method permits a highly selective and sensitive determination of 5-HT and 5-HIAA in human urine without any sample purification.     

6-oxy-(acetyl piperazine) fluorescein as a new fluorescent labeling reagent for free fatty acids in serum using high-performance liquid chromatography

A new fluorescein-based fluorescent derivatizating reagent, 6-oxy-(acetyl piperazine) fluorescein (APF), has been designed, synthesized and developed for carboxylic acid labeling. It was used as a pre-column derivatizing reagent for the determination of seven free fatty acids (lauric acid, myristic acid, arachidonic acid, linoleic acid, palmitic acid, oleic acid, and stearic acid) with high-performance liquid chromatography (HPLC). The derivatization reaction of APF with seven fatty acids was completed at 60 degrees C for 1 h using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as the condensing reagent. On a C18 column, the derivatives of APF with seven free fatty acids could be separated completely in 22 min using a mobile phase of methanol-water (88:12, v/v) containing 7 mmol L(-1) pH 6.5 Na2HPO4-H3Cit3 buffer with fluorescence detection at lambdaex/lambdaem=467/512 nm. The detection limits could reach 0.1-6.4 nmol L(-1) (signal-to-noise=3). This reagent was applied to the determination of the free fatty acids in human serum samples with satisfying recovery efficiencies varying from 93 to 105%.     

Dispersive liquid–liquid microextraction as an effective preanalytical step for the determination of estradiol in human urine

In this work, a fast and effective dispersive liquid–liquid microextraction was developed for the isolation and preconcentration of free 17 β‐estradiol, the main human estrogen, from real human urine samples. To optimize the extraction technique, few important parameters such as type and volume of extraction and dispersive solvents, centrifugation conditions, effect of salt addition, and extraction time were studied. Optimal conditions were obtained when injecting 600 μL mixture of tetrachloromethane as extraction solvent and ethanol as dispersive solvent (1:5, v/v) into 2 mL of urine containing 8% NaCl and following centrifugation at 10 000 rpm, thus reaching enrichment factor 28 and extraction recovery 98% for estradiol. Procedure was evaluated by means of high‐performance liquid chromatography with UV detection (λ = 280 nm) using a C‐18 column and methanol/water (60:40, v/v) as the mobile phase. The method was linear within the concentration range 1.0–250.0 mg/L (r  = 0.9997) and provided a limit of detection of 0.25 mg/L. The proposed method was applied to the determination of free estradiol in real human pregnancy urine.     

High-performance liquid chromatographic determination of lansoprazole and its metabolites in human serum and urine.

A simple and sensitive high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous determination of lansoprazole and its metabolites in human serum and urine. The analytes in serum or urine were extracted with diethyl ether-dichloromethane (7:3, v/v) followed by evaporation, dissolution and injection into a reversed-phase column. The recoveries of authentic analytes added to serum at 0.05-2 micrograms/ml or to urine at 1-20 micrograms/ml were greater than 88%, with the coefficients of variation less than 7.1%. The minimum determinable concentrations of all analytes were 5 ng/ml in serum and 50 ng/ml in urine. The method was successfully applied to a pharmacokinetic study of lansoprazole in human.     

Simultaneous determination of cefamandole and cefamandole nafate in human plasma and urine by high-performance liquid chromatography with column switching

A high-performance liquid chromatographic method with column switching has been developed for the simultaneous determination of cefamandole and cefamandole nafate in plasma and urine. The plasma and urine samples were injected onto a precolumn packed with Corasil RP C18 (37-50 microns) after simple dilution with an internal standard solution in 0.05 M phosphoric acid. Polar plasma and urine components were washed out using 0.05 M phosphoric acid. After valve switching, the concentrated drugs were desorbed in back-flush mode and separated by a reversed-phase C8 column with methanol-5 mM tetrabutylammonium bromide (45:55, v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed, and a detection limit of 0.5 microgram/ml. The total analysis time per sample was less than 30 min, and the mean coefficients of variation for intra- and inter-assay were both less than 4.9%. The method has been successfully applied to plasma and urine samples for human volunteers after intravenous injection of cefamandole nafate.     

Enantiospecific analysis of ibuprofen by high performance liquid chromatography: Determination of free and total drug enantiomer concentrations in serum and urine

Summary A reversed-phase high-performance liquid chromatographic (HPLC) assay, based on the indirect approach to enantiomeric analysis, for the determination of ibuprofen in human serum and urine has been developed. Following the addition of (R,S)-flurbiprofen, as internal standard, the enantiomers of ibuprofen were isolated from plasma and urine samples by liquid-liquid extraction at acidic pH. The enantiomers of flurbiprofen and ibuprofen were derivatized with (R)-1-(naphthen-1-yl)ethylamine, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole as coupling reagents, to yield the corresponding diastereoisomeric amides. Chromatographic resolution of the derivatives was achieved using a C₁₈ column (Waters Resolve C₁₈; 5 μm, 150×3.9 mm) using a mobile phase of phosphate buffer (pH 3.5, 0.01 M): acetonitrile (50∶50 v/v) at a flow rate of 1.5 mL min⁻¹ at ambient temperature. Quantification was carried out using a spectrofluorometer with excitation and emission wavelengths of 290 and 330 nm respectively. The use of a semimicrobore column (150×2.1 mm) containing the same stationary phase facilitated the analysis of the free drug enantiomer concentrations following equilibrium dialysis. The derivatization procedure was carried out as described above but with a reduction in the quantities of the reagents used in order to reduce the background noise in the chromatographic analysis. The HPLC methodology for the determination of free drug enantiomer concentrations was validated against a previously reported method employing the radiolabelled drug.     

Fluorescence high-performance liquid chromatographic determination of free and conjugated bile acids in serum and bile using 1-bromoacetylpyrene as a pre-labeling reagent

A fluorescence high-performance liquid chromatographic method is described for the determination of free and conjugated bile acids in serum and bile. Free and conjugated bile acids are extracted from serum or bile using a Sep-Pak C18 cartridge and then fractionated on a piperidinohydroxypropyl Sephadex LH-20 column. Free and glycine-conjugated bile acids are labeled with 1-bromoacetylpyrene in acetonitrile using dicyclohexyl-18-crown-6-ether as catalyst. Taurine-conjugated bile acids are hydrolyzed by cholylglycine hydrolase and then derivatized by the same reagent. Derivatized bile acids are separated stepwise on a reversed-phase column (Radial Pak A) using acetonitrile-methanol-water (A) (100 : 50 : 40) and (B) (100 : 50 : 20) as mobile phase. The eluate is monitored by a fluorophotometer at 370 nm (excitation) and 440 nm (emission). Linearities of fluorescence intensities (peak heights) with the amounts of free and conjugated bile acids were obtained between 50 pmol and 200 pmol for free bile acids and between 25 pmol and 100 pmol for glycine-conjugated bile acids, respectively. Recoveries from serum and bile samples are not less than 90%. This method is sensitive, reliable and useful for the simultaneous determination of free and conjugated bile acids in serum and bile.     

High-performance liquid chromatographic determination of pipotiazine in human plasma and urine

A high-performance liquid chromatographic method has been developed for the determination of pipotiazine in human plasma and urine. After selective extraction, pipotiazine and the internal standard (7-methoxypipotiazine) and chromatographed on a column packed with Spherosil XOA 600 (5 micrometers) using a 7:3 (v/v) mixture of diisopropyl either--isooctane (1:1, v/v + 0.2% triethylamine and diisopropyl ether--methanol (1:1, v/v) + 0.2% triethylamine + 2.6% water. The eluted compounds are measured by fluorescence detection. The sensitivity of the method was established at 0.25 ng/ml pipotiazine in plasma and 2 ng/ml pipotiazine in urine (C.V. less than 5%). The method has been successfully applied to a pharmacokinetic study following a single oral administration of 10 mg of pipotiazine.     

Determination of 1-hydroxypyrene in human urine by HPLC with electrochemical detection at a boron-doped diamond film electrode

A high-performance liquid chromatographic method with electrochemical detection (HPLC-ED) at a boron-doped diamond film electrode with preliminary separation and preconcentration by solid-phase extraction (SPE) has been developed for the determination of 1-hydroxypyrene (1-HP) in human urine. 1-HP is among the most widely used biomarkers of exposure to polycyclic aromatic hydrocarbons. Optimal HPLC-ED conditions have been found: mobile phase methanol-0.05 mol L(-1) phosphate buffer pH 5.0 (80:20, v/v), detection potential +1,000 mV versus Ag/AgCl (3 mol L(-1) KCl), and flow rate 0.8 mL min(-1). For SPE, LiChrolut(?) RP-18 E cartridges were used. The extraction yield was (87.0 ± 5.8)% (n = 5). The concentration dependence of 1-HP was measured in the concentration range from 0.01 to 10 μmol L(-1) (2.18-2,180 μg L(-1)) using methanolic solutions resulting from the SPE pretreatment of spiked human urine samples. The limit of detection (signal-to-noise ratio 3) and the limit of quantification (signal-to-noise ratio 10) of the biomarker were 0.013 μmol L(-1) (2.84 μg L(-1)) and 0.043 μmol L(-1) (9.39 μg L(-1)), respectively, which is sufficient for its determination in the urine of persons exposed to polycyclic aromatic hydrocarbons.     

Determination of cetirizine in human urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of the histamine H1-receptor antagonist cetirizine in human urine was developed. Cetirizine and the internal standard are extracted from acidified (pH 5) urine (0.5 ml) into chloroform and the organic layer is evaporated to dryness. The residue is chromatographed on a Spherisorb 5ODS-2 column using Pic A (5 mM aqueous tetrabutylammonium phosphate)-methanol-tetrahydrofuran (33:65:2, v/v) as the mobile phase with ultraviolet detection (230 nm). The calibration graph is linear from 0.1 to 10 micrograms/ml and using 0.5 ml of urine the detection limit is 20 ng/ml. The within-run relative standard deviation is less than 6% and the accuracy is within 10% of the theoretical value at concentrations between 0.1 and 10 micrograms/ml in urine. There is a good correlation (r = 0.99606) with a previously described capillary gas chromatographic assay.     

Extractionless HPLC Method for the Determination of Naproxen in Human Plasma and Urine

Abstract

A simple and rapid (extractionless) high-performance liquid chromatographic method with ultraviolet detection, at 278 nm, is described for the determination of naproxen in human plasma and urine. Niflumic acid is used as internal standard. The chromatographic system consists of a reversed-phase C18-Spherisorb column with acetonitrile/0.1 M sodium acetate (35:65 v/v, pH 6.14) as the mobile phase. The retention time is 3.0 min for naproxen and 3.8 min for niflumic acid. The total run time is 5 min and the typical assay time is 10 min. The method is sufficiently sensitive for biopharmaceutical studies, after the oral administration of a single sustained release dose.     

HPLC with fluorescence detection of urinary phenol, cresols and xylenols using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a fluorescence labeling reagent.

A simple and sensitive HPLC method for the determination of phenolic compounds, i.e., phenol (Phe), cresols (Cres) and xylenols (Xyls), was developed. After a pre-column fluorescence derivatization of these compounds with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) at 60 degrees C for 30 min, 11 DIB derivatives were successfully separated within 50 min with an ODS column using CH3CN-H2O-CH3OH (25 + 22 + 53, v/v) as the eluent. The detection limits of DIB derivatives at a signal-to-noise ratio of 3 ranged from 0.15 to 1.09 microM (0.2-1.6 pmol per 20 microliters). The precision of the proposed method for both within- and between-day assays of free and total phenol related compounds was satisfactory (RSD < 9.5%). By the proposed method, Phe and p-Cre could be detected in normal urine samples, and the calculated concentrations of free Phe and p-Cre in unhydrolysed urine samples were 1.5 +/- 1.3 and 23.9 +/- 24.3 microM and those of total Phe and p-Cre in hydrolysed urine samples were 87.3 +/- 81.2 and 200.7 +/- 195.4 microM (n = 21), respectively.     

Screening method for organic aciduria by spectrofluorometric measurement of total dicarboxylic acids in human urine based on intramolecular excimer-forming fluorescence derivatization

A simple screening method of organic aciduria by spectrofluorometric measurement of total dicarboxylic acids in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoic acid hydrazide (PBH). Dicarboxylic acids in urine were converted to the corresponding dipyrene-labeled derivatives by reaction with PBH in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and pyridine, and the derivatives afforded intramolecular excimer fluorescence (420-540 nm) which can clearly be discriminated from the normal fluorescence (360-420 nm) emitted from reagent blanks. The technique is so selective that it permits spectrofluorometric measurement of total amount of dicarboxylic acids by the direct derivatization of diluted urine samples. The same reaction mixture has also served as a liquid chromatographic (LC) sample for the separative determination of individual dicarboxylic acids. The spectrofluorometric data did not contradict with the LC data. These methods were usefully applied to preliminary screening test of glutaric aciduria. In conclusion, the present derivatization method allows rapid and direct determination of total amount of dicarboxylic acids in human urine samples.