High-performance liquid chromatographic determination of 3-hydroxykynurenine with fluorimetric detection; comparison of preovulatory phase and postovulatory phase urinary excretion.

A high-performance liquid chromatographic assay for 3-hydroxykynurenine in human urine is described. A fluorescent derivative of 3-hydroxykynurenine was prepared, based on the reaction of the compound with p-toluenesulphonyl chloride in a basic medium. The analytical method for the measurement of the fluorescent compound employed a Tosoh ODS 80 column eluted with 10 mM potassium dihydrogenphosphate (pH 4.5) and acetonitrile (3:2, v/v) and detection at an excitation wavelength of 375 nm (10 nm bandpass) and an emission wavelength of 455 nm (10 nm bandpass). The column temperature was maintained at 25 degrees C. The detection limit was 3 pmol (673 pg) at a signal-to-noise ratio of 5:1. The fluorescent derivative of 3-hydroxykynurenine was eluted at ca. 12.5 min. The technique was applied to the analysis of human urine. The total analysis time was ca. 15 min.     

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).     

HPLC determination of ciprofloxacin, cloxacillin, and ibuprofen drugs in human urine samples

This paper reports, for the first time, a liquid chromatographic method for the simultaneous determination of three frequently co-administered active principles, two antibiotics, ciprofloxacin (CIPRO) and cloxacillin (CLOXA) belonging to the fluoroquinolones and beta-lactam families, respectively, and ibuprofen (IBU), a non-steroidal anti-inflammatory drug. The chromatographic separation was performed on a C-18 analytical column, using isocratic elution with methanol-acetonitrile-pH 3 formate buffer (CT = 0.1 M) (15:12:73, v/v/v) for 3 min and, after that, a linear gradient with methanol-acetonitrile (88:12, v/v) for 8 min. Several absorption spectra were obtained for each peak using a DAD detector. Chromatograms at the maximum absorption wavelength for each analyte, 220 nm for both IBU and CLOXA, and 280 nm for CIPRO were selected as the most suitable. The proposed chromatographic method requires about 15 min per sample. The presence of a urine background was tested and no interference was found. The method was satisfactorily applied to the determination of CIPRO, CLOXA, and IBU, in fortified urine, and in urine samples from a patient undergoing treatment with these three active principles, among others. Limits of quantification in urine were 1.00, 1.70, and 2.87 microg/mL for CIPRO, CLOXA, and IBU, respectively.     

Simultaneous determination of angiotensins II and 1-7 by capillary zone electrophoresis in plasma and urine from hypertensive rats

We describe the procedure developed for the simultaneous detection and quantification of angiotensin II and angiotensin-(1-7), by capillary zone electrophoresis with UV detection by photodiode-array, at a wavelength of 200 nm, in the plasma and urine from hypertensive rats. Optimal separation was achieved with a 100 mM boric acid + 3 mM tartaric acid + 10 fM gold (III) chloride electrolyte solution at pH 9.80. The applied voltage was 30 kV and the capillary temperature was kept constant at 20 °C. The method was over the concentration range of 0.01-500 pmol/mL. All determination coefficients were higher or equal to 0.9985. Limits of detection and quantification for angiotensin II were 0.0110 pmol/mL (S/N = 3) and 0.0195 pmol/mL (S/N = 5), respectively. While for angiotensin-(1-7), the limits were 0.0112 pmol/mL (S/N = 3) and 0.0193 pmol/mL (S/N = 5), respectively. The present method offers a time-saving way to simultaneous determination of angiotensin II and angiotensin-(1-7), since it can be completed in 10 min, compared to other methodologies reported in the literature for capillary electrophoresis and liquid chromatography, which require more than 1 h for analysis of complex matrices, such as plasma and urine. The procedure is illustrated by experiments that quantify simultaneously angiotensin II and angiotensin-(1-7) in plasma and urine from hypertensive and normotensive rats, with and without antihypertensive treatment. The levels of angiotensin II and angiotensin-(1-7) detected in the experimental model, resulted in a recovery of 99.00-106.01% and a reproducibility of less than 10%. The proposed analytical method is a use full tool for the simultaneous detection of angiotensin II and angiotensin-(1-7) implicated in vascular remodeling in pathologies such as hypertension.     

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.     

Determination of midodrine in human plasma by high-performance liquid chromatography with fluorescence detection.

A simple and sensitive fluorometric high-performance liquid chromatographic method was developed for the determination of midodrine in human plasma. After liquid-liquid extraction from plasma, the drug and 2-phenylglycinol (internal standard) were convened into the corresponding fluorescent derivatives by reaction with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, a fluorescence derivatization reagent for amines. The derivatives were separated within 30 min on a reversed-phase column using isocratic elution with acetonitrile-methanol-water (10:30:60, v/v) and were detected spectrofluorometrically at 485 nm with excitation at 400 nm. The detection limit for midodrine was 0.3 pmol (76 pg) per mL plasma at a signal-to-noise ratio of 3.     

An HPLC method for the determination of ethacridine lactate in human urine

An HPLC method was developed and validated for the determination of ethacridine lactate in human urine. Solid-phase extraction cartridges were used to extract urine samples. Separation was carried out on a C(18) column maintained at 30 degrees C with methanol-0.05% sodium dodecylsulfonate (70:30, v/v, pH 3) as mobile phase at a flow rate of 1.0 mL/min. Detection was at UV 272 nm. The calibration curve was linear in the concentration range of 4-4000 ng/mL, with linear correlation coefficient r equal to 0.9998. The limit of detection for the assay was 1.1 ng/mL. The within-day accuracy ranged from 94.8 to 101.6% and precision from 2.3 to 5.4%. The between-day accuracy ranged from 96.8 to 102.6% and precision from 4.0 to 5.3%. The absolute recovery was 95.4-101.2%. Urine samples were stable for at least 15 days if stored in the dark at -20 degrees C. This simple and accurate method allows the sensitive determination of ethacridine lactate in human urine. It was successfully applied to assess the urine level of ethacridine lactate in women received intra-amniotic injection.     

Simultaneous determination of inulin and p-aminohippuric acid (PAH) in human plasma and urine by high-performance liquid chromatography

Inulin and p-aminohippuric acid (PAH) clearances are used for the estimation of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). A simple and rapid high-performance liquid chromatography (HPLC) method with UV detection is described for the simultaneous determination of inulin and PAH in the same chromatogram in the plasma and urine of humans. Plasma and urine samples were hydrolyzed with perchloric acid (0.7%) in boiling water. The mobile phase consisted of 0.01 M potassium dihydrogenphosphate with 0.02 M tetramethylammonium chloride and o-phosphoric acid (pH 3)-acetonitrile (94:6, v/v), pumped at a rate of 1.2 ml min-1 on a C8 reversed-phase column. Tannic acid was used as the internal standard and UV detection at 285 nm was employed. The calibration curves were linear over the concentration range of 12.5-100 mg l-1 for inulin and 6.25-50 mg l-1 for PAH with determination coefficients greater than 0.997. The method is accurate (bias < 13%) and reproducible (intra- and inter-day relative standard deviation less than 11%), with a limit of quantitation of 12.5 mg l-1 and 6.25 mg l-1 for inulin and PAH, respectively. Analytical recoveries from urine and plasma were ranged from 81 to 108% for both compounds. This fully validated method, which allows the simultaneous determination of inulin and PAH clearances, is simple, rapid (total run time < 10 min) and requires only a 200 microliters plasma or urine sample.     

High-performance liquid chromatography-electrochemical detection of 3-methylhistidine in human urine.

A reversed-phase high-performance liquid chromatographic method is described for the determination of 3-methylhistidine content in human urine using pre-column derivatization with phenylisothiocyanate, isocratic elution with 15 mM sodium acetate-acetonitrile (92:8, v/v) and electrochemical detection. The limit of quantitation was 0.1 pmol. The method has been applied in routine analyses of 3-methylhistidine in both clinical and research work.     

Fluorometric determination of khellin in human urine and serum by high-performance liquid chromatography using postcolumn photoirradiation.

For the determination of khellin in urine and serum, fluorometry using HPLC-postcolumn photoirradiation has been developed. Khellin and visnagin of similar structure were separated on a column of Capcell Pak C8. The mobile phase consisted of 40%(v/v) ethanol containing 75 mmol l(-1) H2O2. The postcolumn reagent, 70 mmol l(-1) KH2PO4-NaOH buffer (pH 12.7) containing 50%(v/v) ethanol, were mixed with the mobile phase, which was irradiated with ultraviolet light to induce fluorescence. The fluorescence was monitored with excitation at 378 nm and emission at 480 nm. The calibration graph for khellin was linear over the range of 65 - 2620 ng ml(-1) using an injection volume of 20 microl. The pretreatment of the urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively.     

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.     

Pyrocatechol-1-Aldehyde 2-Benzothiazolylhydrazone As Reagent for the Spectrofluorimetric Determination of Nanogram Amounts of Gallium in Urine and Blood Serum

Abstract

A method is developed for the spectrofluorimetric determination of 1–80 ng.ml^?1 of gallium with pyrocatechol-1-aldehyde 2-benzothiazolylhydrazone, in a 50% (v/v) DMSO-Water medium at apparent pH 4.0 (monochloracetic/monochloracetate buffer). Λ_ex = 400nm, Λ_em = 504 nm (corrected). Interferences have been evaluated and the method applied to the determination of gallium in human urine and blood serum samples.     

Liquid chromatographic determination of less polar ginsenosides in processed ginseng

A novel method for the determination of iodide by size exclusion chromatography was established. The method was simple and highly sensitive with good precision. Iodide was converted to iodine, then sequestered with starch, and separated from the matrix using a Shim-pack DIOL-150 (250 x 7.9 mm) size exclusion column with methanol-0.01 mol l(-1) aqueous phosphoric acid (10:90, v/v) as mobile phase at 1.2 ml min(-1) and UV detection at 224 nm. The calibration graph was linear from 1.0 ng ml(-1) to 100.0 ng ml(-1) for iodide with a correlation coefficient of 0.9992 (n=6). The detection limit was 0.2 ng ml(-1). The method was successfully applied to the determination of iodide in seawater and urine. The recovery was from 92% to 103% and the relative standard deviation was in the range of 1.5% to 3.7%.     

Capillary electrophoresis of urinary porphyrins with absorbance and fluorescence detection

Urinary porphyrins are separated in a 72 cm x 50 microns I.D. fused-silica capillary by micellar electrokinetic capillary chromatography with 100 mM sodium dodecyl sulfate and 20 mM 3-(cyclohexylamino)-1-propanesulfonic acid at pH 11. Detection is accomplished by absorbance at 400 nm or fluorescence with excitation at 400 nm and emission at wavelengths above 550 nm. Substantial trace enrichment is found for porphyrins in urine samples or for porphyrin standards prepared without surfactant in the injection buffer. Limits of detection are in the 100 pmol/ml concentration range with an optimized fluorescence system. The method is shown suitable for the determination of porphyrins in clinical urine specimens. Comparisons are made between electrophoretic and chromatographic methods for the separation and detection of urinary porphyrins.     

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.     

Stability-indicating methods for determination of tiapride in pure form, pharmaceutical preparation, and human plasma

Four different stability-indicating procedures are described for determination of tiapride in pure form, dosage form, and human plasma. Second derivative (D2), first derivative of ratio spectra (1DD), spectrofluorimetric, and high-performance column liquid chromatographic (LC) methods are proposed for determination of tiapride in presence of its acid-induced degradation products, namely 2-methoxy-5-(methylsulfonyl) benzoic acid and 2-diethylaminoethylamine. These approaches were successfully applied to quantify tiapride using the information included in the absorption, excitation, and emission spectra of the appropriate solutions. In the D2 method, Beer's law was obeyed in the concentration range of 1.5-9 microg/mL with a mean recovery of 99.94 +/- 1.38% at 253.4 nm using absolute ethanol as a solvent. In 1DD, which is based on the simultaneous use of the first derivative of ratio spectra and measurement at 245 nm in absolute ethanolic solution, Beer's law was obeyed over a concentration range of 1.5-9 microg/mL with mean recovery 99.64 +/- 1.08%. The spectrofluorimetric method is based on the determination of tiapride native fluorescence at 339 nm emission wavelength and 230 nm excitation wavelength using water-methanol (8 + 2, v/v). The calibration curve was linear over the range of 0.2-3 microg/mL with mean recovery of 99.66 +/- 1.46%. This method was also applied for determination of tiapride in human plasma. A reversed-phase LC method performed at ambient temperature was validated for determination of tiapride using methanol-deionized water-triethylamine (107 + 93 + 0.16, v/v/v) as the mobile phase. Sulpiride was used as an internal standard at a flow rate of 1 mL/min with ultraviolet detection at 214 nm. A linear relation was obtained over a concentration range of 2-30 microg/mL with mean recovery of 99.66 +/- 0.9%. Results were statistically analyzed and compared with those obtained by applying the reference method. They proved both accuracy and precision.     

Determination of 10 alpha-methoxy-9,10-dihydrolysergol, a nicergoline metabolite, in human urine by high performance liquid chromatography.

A specific method for the determination of 10 alpha-methoxy-9,10-dihydrolysergol, a nicergoline metabolite (metabolite 2), in urine is described. Metabolite 2 was well separated from the urine components on a reversed phase column, Hypersil ODS 5 microns, using an acetonitrile:pH 3.5 phosphate buffer (40:60, v/v) as the mobile phase at a flow rate of 1 mL/min. UV detection was set up at 220 nm. After addition of a known amount of lysergamide as the internal standard, the compounds were extracted from alkalysed urine on a pre-packed glass column (Extrelut 1) with dichloromethane. With 0.5 mL urine, concentrations down to 0.56 mumol/L could be determined.     

Determination of metoprolol in human plasma and urine by high‐performance liquid chromatography with fluorescence detection

A simple, specific and sensitive HPLC method has been developed for the determination of metoprolol in human plasma and urine. Separation of metoprolol and atenolol (internal standard) was achieved on an Ace C₁₈ column (5 μm, 250 mm×4.6 mm id) using fluorescence detection with λ_ex=276 nm and λ_em=296 nm. The mobile phase consists of methanol–water (50:50, v/v) containing 0.1% TFA. The analysis was performed in less than 10 min with a flow rate of 1 mL/min. The assay was linear over the concentration range of 3 – 200 and 5 – 300 ng/mL for plasma and urine, respectively. The LOD were 1.0 and 1.5 ng/mL for plasma and urine, respectively. The LOQ were 3.0 and 5.0 ng/mL for plasma and urine, respectively. The extraction recoveries were found to be 95.6 ± 1.53 and 96.4 ± 1.75% for plasma and urine, respectively. Also, the method was successfully applied to three patients with hypertension who had been given an oral tablet of 100 mg metoprolol.     

Development of a fast capillary electrophoresis method for determination of creatinine in urine samples

The aim of this work was to develop a fast method using capillary electrophoresis for the determination of creatinine in human urine samples. The pH and constituents of the background electrolyte were selected by inspection of effective mobility of creatinine and candidate urine interferents versus pH curves. The tendency of the analyte to undergo electromigration dispersion and the buffer capacity were evaluated by the Peakmaster software and considered in the optimization of the background electrolyte, composed by 10 mmol L(-1) tris(hydroxymethyl)aminomethane and 20 mmol L(-1) 2-hydroxyisobutyric acid (HIBA) at pH 3.93. Separation was conducted in a fused-silica capillary (32 cm total length and 8.5 cm effective length, 50 microm I.D.), with short-end injection configuration and direct UV detection at 215 nm. The migration time of creatinine was only 22s. A few figures of merit of the method are as follows: good linearity in the concentration interval of 5-70 mg L(-1) (R(2)>0.99), limit of detection of 0.5 mg L(-1), inter-day precision better than 2.7% (n=9) and recovery in the range 99.0-103.7% at three concentration levels (50, 100 and 150 mg L(-1)). Urine samples were prepared by deproteination with acetonitrile (1:3 sample:acetonitrile, v/v), centrifugation and dilution of a deproteinated aliquot with 12.5 mmol L(-1) HIBA (1:4, v/v). Creatinine concentrations between 489 and 1063 mg L(-1) were obtained in the urine of four healthy volunteers.     

Direct enantiomeric resolution of disopyramide and its metabolite using chiral high-performance liquid chromatography. Application to stereoselective metabolism and pharmacokinetics of racemic disopyramide in man

A method for the simultaneous determination of disopyramide and mono-N-desisopropyldisopyramide enantiomers extracted from human plasma and urine is presented. Separation and quantitation were carried out using two columns coupled in series, and UV detection at 254 nm. First, the racemates of the two compounds were separated using a reversed-phase column, and then the enantiomers were separated using a stereoselective column packed with human alpha 1-acid glycoprotein. The mobile phase was 8 mM phosphate buffer, pH 6.20-2-propanol (92:8, v/v). The coefficients of variation (%) for the plasma daily determination were 6.7% for R(-)- and S(+)-disopyramide at drug levels of 1.5 micrograms/ml, and 8.5% and 7.7% for R(-)- and S(+)-mono-N-desisopropyldisopyramide, respectively, at drug levels of 0.375 micrograms/ml. The method has allowed the study of stereoselective metabolism and pharmacokinetics of disopyramide after oral administration as a racemate.