Determination of nitrite as pentafluorobenzyl derivative by RP-HPLC and UV detection with and without ion-pair extraction

Summary A reversed-phase high-performance liquid chromatographic (RP-HPLC) method with UV detection (270 nm) for the determination of nitrite as its pentafluorobenzyl derivative with and without ion-pair extraction is described. Ion-pair extraction of nitrite from aqueous solutions was performed by using a 1 mol/l solution of the liquid ion exchanger methyltrioctylammonium chloride in toluene. The residue of the ion-pair extraction or an aliquot of an aqueous nitrite solution or of a biological fluid (100 l) were treated with 400 l of acetone and 10 l of pentafluorobenzyl bromide. Nitrite was converted into its pentafluorobenzyl derivative by heating at 50°C for 90 min. After evaporation of acetone the aqueous phases were diluted with 100 to 400 l of methanol, and up to 100 l were injected into the RP-HPLC system. The method allows accurate analysis of nitrite in the presence of nitrate directly in aqueous solutions and biological fluids in concentrations down to 2.0 mg/l. The method is also applicable to the determination of nitrate following its reduction to nitrite by cadmium.     

A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection

A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.     

The analysis of nitrate in environmental samples by reversed-phase HPLC

A method is presented for the analysis of nitrate in natural waters and waste water by high-performance liquid chromatography (HPLC) with a reversed-phase octadecyl column, aqueous phosphoric acid/dihydrogen phosphate mobile phase, and UV detector. The optimum nitrate concentration is 0.3 to 3 mg/L as N with linear detector response below 3 mg/L. The detection limit is 0.007 mg/L. Relative standard deviations in the optimum range are consistently less than one percent. Several potential interferences have been investigated; nitrite and organic chromophores are resolved from nitrate and do not interfere. Hexavalent chromium and sulfate are slight positive interferences, negligible at typical environmental concentrations. The method produces results in agreement with the accepted chromotropic acid method except in samples from eutrophic lakes, for which evidence is presented indicating that the chromotropic acid is inaccurate.     

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).     

Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation

Nitrite and nitrate are main stable products of nitric oxide, a pivotal cellular signaling molecule, in biological fluids. Therefore, accurate measurement of the two ions is profoundly important. Nitrite is difficult to be determined for a larger number of interferences and unstable in the presence of oxygen. In this paper, a simple, cost-effective and accurate HPLC method for the determination of nitrite and nitrate was developed. On the basis of the reaction that nitrite is oxidized rapidly to nitrate with the addition of acidic potassium permanganate, the determination of nitrite and nitrate was achieved by the following strategy: each sample was injected twice for HPLC analysis, i.e. the first injection was to measure nitrate, and the second injection was to measure total nitrate including initial nitrate and the nitrate from the conversion of nitrite with the addition of acid potassium permanganate in the sample. The amount of nitrite can be calculated as difference between injections 2 and 1. The HPLC separation was performed on a reversed phase C₁₈ column for 15 min. The mobile phase consisted of methanol–water (2:98 by volume); the water in the mobile phase contained 0.60 mM phosphate salt (potassium dihydrogen and disodium hydrogen phosphate) and 2.5 mM tetrabutylammonium perchlorate (TBAP). The UV wavelength was set at 210 nm. Additionally, we systemically investigated the effects of the concentration of phosphate salt and TBAP in the mobile phase, the pH of the mobile phase, and the amount of acidic potassium permanganate added to the sample on the separation efficacy. The results showed that the limits of detection (LOD) and the limit of quantitation (LOQ) were 0.075 and 0.25 μM for nitrate (containing the oxidized nitrite), respectively. The linear range was 1–800 μM. This developed approach was successfully applied to assay nitrite/nitrate levels in cell culture medium, cell lysate, rat plasma and urine.

     

Simultaneous determination of caffeine, theobromine, and theophylline by high-performance liquid chromatography

This work relates the development of an analytical methodology to simultaneously determine three methylxanthines (caffeine, theobromine, and theophylline) in beverages and urine samples based on reversed-phase high-performance liquid chromatography. Separation is made with a Bondesil C18 column using methanol-water-acetic acid or ethanol-water-acetic acid (20:75:5, v/v/v) as the mobile phase at 0.7 mL/min. Identification is made by absorbance detection at 273 nm. Under optimized conditions, the detection limit of the HPLC method is 0.1 pg/mL for all three methylxanthines. This method is applied to urine and to 25 different beverage samples, which included coffee, tea, chocolate, and coconut water. The concentration ranges determined in the beverages and urine are: < 0.1 pg/mL to 350 microg/mL and 3.21 microg/mL to 71.2 microg/mL for caffeine; < 0.1 pg/mL to 32 microg mL and < 0.1 pg/mL to 13.2 microg/mL for theobromine; < 0.1 pg/mL to 47 microg/mL and < 0.1 pg/mL to 66.3 microg/mL for theophylline. The method proposed in this study is rapid and suitable for the simultaneous quantitation of methylxanthines in beverages and human urine samples and requires no extraction step or derivatization.     

Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation

Nitrite and nitrate are main stable products of nitric oxide, a pivotal cellular signaling molecule, in biological fluids. Therefore, accurate measurement of the two ions is profoundly important. Nitrite is difficult to be determined for a larger number of interferences and unstable in the presence of oxygen. In this paper, a simple, cost-effective and accurate HPLC method for the determination of nitrite and nitrate was developed. On the basis of the reaction that nitrite is oxidized rapidly to nitrate with the addition of acidic potassium permanganate, the determination of nitrite and nitrate was achieved by the following strategy: each sample was injected twice for HPLC analysis, i.e. the first injection was to measure nitrate, and the second injection was to measure total nitrate including initial nitrate and the nitrate from the conversion of nitrite with the addition of acid potassium permanganate in the sample. The amount of nitrite can be calculated as difference between injections 2 and 1. The HPLC separation was performed on a reversed phase C₁₈ column for 15 min. The mobile phase consisted of methanol–water (2:98 by volume); the water in the mobile phase contained 0.60 mM phosphate salt (potassium dihydrogen and disodium hydrogen phosphate) and 2.5 mM tetrabutylammonium perchlorate (TBAP). The UV wavelength was set at 210 nm. Additionally, we systemically investigated the effects of the concentration of phosphate salt and TBAP in the mobile phase, the pH of the mobile phase, and the amount of acidic potassium permanganate added to the sample on the separation efficacy. The results showed that the limits of detection (LOD) and the limit of quantitation (LOQ) were 0.075 and 0.25 μM for nitrate (containing the oxidized nitrite), respectively. The linear range was 1–800 μM. This developed approach was successfully applied to assay nitrite/nitrate levels in cell culture medium, cell lysate, rat plasma and urine.     

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.     

基于量子点增敏化学发光同时测定尿液中的亚硝酸盐和硝酸盐

采用铜镉柱还原硝酸盐,与CdTe量子点增敏过氧亚硝酸-碳酸钠体系的化学发光信号相结合,开发了快速在线同时分析亚硝酸盐和硝酸盐的新方法.对流动注射、化学发光等实验参数条件进行优化,在Na2CO3的浓度为0.2 M、H2O2的浓度为0.03 M、Na2EDTA的浓度为1×10-3 M、CdTe量子点粒径为2.84 nm的条件下,过氧亚硝酸-碳酸钠体系可以获得最优的化学发光信号.该方法检测亚硝酸盐的线性范围为0.3~75μM,检测限可达0.12μM,其相对标准偏差为1.9%;硝酸盐的线性范围为1.0~100μM,检测限可达0.26μM,其相对标准偏差为1.5%.此方法无需衍生和分离,可以实现同时、准确、快速和高选择性地检测人体尿液中亚硝酸盐和硝酸盐的含量,回收率分别为94%~105%和96.6%~110.4%.     

Simultaneous determination of nitrite and nitrate in dew, rain, snow and lake water samples by ion-pair high-performance liquid chromatography

A simple, fast, sensitive and accurate reversed-phase ion-pair HPLC method for simultaneous determination of nitrite and nitrate in atmospheric liquids and lake waters has been developed. Separations were accomplished in less than 10 min using a reversed-phase C₁₈ column (150 mm × 2.00 mm i.d., 5 μm particle size) with a mobile phase containing 83% 3.0 mM ion-interaction reagent tetrabutylammonium hydroxide (TBA-OH) and 2.0 mM sodium phosphate buffer at pH 3.9 and 17% acetonitrile (flow rate, 0.4 mL/min). UV light absorption responses at 205 nm were linear over a wide concentration range from 100 μg/mL to the detection limits of 10 μg/L for nitrite and 5 μg/L nitrate. Quantitation was carried out by the peak area method. The relative standard deviation for the analysis of nitrite and nitrate was less than 3.0%. This method was applied for the simultaneous determination of nitrite and nitrate in dew, rain, snow and lake water samples collected in southeast Massachusetts. Nitrate was found being present at 4.79-5.99 μg/mL in dew, 1.20-2.63 μg/mL in rain, 0.32-0.60 μg/mL in snow and 0.12-0.23 μg/mL in lake water. Nitrite was only a minor species in dew (0.62-0.83 μg/mL), rain (<0.005-0.14 μg/mL), snow (0.021-0.032 μg/mL) and lake water (0.12-0.16 μg/mL). High levels of nitrite and nitrate observed in dew water droplets may constitute an important source of hydroxyl radicals in the sunny early morning.     

Nitrite and nitrate measurement in human urine by capillary electrophoresis.

Nitrite and nitrate have been widely used as markers for nitric oxide (NO) formation in vivo and represent the major NO oxidation products in biological fluids. In the present study, the use of capillary electrophoresis (CE) in the measurement of nitrite and nitrate in human urine is described. Urine samples were electrophoresed in an extended light path fused-silica capillary (104 cm; 75 microm ID) at an applied negative potential of 30 kV, and UV detection at 214 nm. Using electrokinetic sample injection (-6 kV x 20 s), we found that urine concentration, pH, sodium and chloride interfered with nitrite and nitrate detection. The detection of nitrite and nitrate was decreased when hydrodynamic sample injection was used (30 mbar x 60 s). However, basal levels of urinary nitrite (0.25 +/- 0.05 microM) and nitrate (591 +/- 115 microM) were detected and no interference by variations in urine concentration and pH was noted when hydrodynamic sample injection was used. Thus, hydrodynamic sample injection is convenient for the measurement of urinary nitrite and nitrate and avoids the effect of variations in urine matrices and pH on nitrite and nitrate detection.     

Quantification of arecoline (areca nut alkaloid) in neonatal biological matrices by high-performance liquid chromatography/electrospray quadrupole mass spectrometry

A high-performance liquid chromatography (HPLC) method with mass spectrometric detection is described for determination of arecoline in newborn meconium, urine and cord serum, using pilocarpine as internal standard. The analytes were extracted from neonatal biological matrices with chloroform/isopropanol (95:5, v/v) at alkaline pH. Extracts were analyzed by HPLC coupled to an electrospray (ESI) interface and a quadrupole mass spectrometer. Chromatography was performed on a C(8) reversed-phase column using 10 mM ammonium acetate (pH 4.3)/acetonitrile (90:10, v/v) as mobile phase. The mass spectrometer was operated in selected ion monitoring mode. The method was validated over the concentration range 0.005-1.00 micro g/g meconium, 0.004-1.00 micro g/mL cord serum and 0.001-1.00 micro /mL urine. Mean recoveries ranged between 86.5 and 90.7% for arecoline in the different biological matrices, with precision always better than 10%. The quantification limits of arecoline were 0.005 micro g/g meconium, 0.004 micro g/mL cord serum, and 0.001 micro g/mL urine. The method was applied to the analysis of neonatal biological matrices to assess eventual fetal exposition to arecoline. Two newborns from Asian mothers who declared areca nut consumption presented arecoline in meconium with concentrations in the range 0.006-0.008 micro g/g; also the urine from one neonate tested positive for the drug.     

High-Performance Liquid Chromatographic Determination of SAZ-VII-23, A Novel Antiarrhythmic Agent,in Dog Plasma and Urine

Abstract

A HPLC method has been developed to determine the concentrations of SAZ-VII-23 (3-benzoyl-7-isopropyl-3,7-diazabicyclo[3.3.1]nonane HClO₄), a novel antiarrhythmic agent, in dog plasma and urine. Plasma treated with acetonitrile and alkalinized urine were extracted with chloroform- propanol (9:1). An aliquot was injected on to HPLC system using a C₆ reversed-phase column and acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8 (28.5:28.5:43 v/v) containing 4.0 mM triethylamine as mobile phase. Detection wavelength was 255 nm. The linear range were 0.04–8 μg/ml, and the lower limit of quantitation was 0.04 μg/ml in plasma and urine, respectively. The method was applied to determine plasma and urine concentrations and preliminary pharmacokinetic profiles of SAZ-VII-23 in a dog.     

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.     

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.     

Determination of imipenem (N-formimidoyl thienamycin) in human plasma and urine by high-performance liquid chromatography, comparison with microbiological methodology and stability

High-performance liquid chromatographic (HPLC) methods using ultraviolet (UV) detection have been developed for the assay of the antibiotic imipenem (N-formimidoyl thienamycin) in human plasma and urine. A reversed-phase analytical column is employed in the plasma assay method and a cation-exchange column is used in the urine assay method. Both methods use borate buffer in the mobile phase. The method of preparation of human fluid samples for HPLC injection has been optimized with respect to the stability of imipenem in aqueous buffers, in morpholine buffer--ethylene glycol stabilizer, and in urine and plasma. Preparation of the samples before injection into the HPLC systems involves deproteination/filtration of the plasma/urine samples. The open lactam metabolite and the coadministered dehydropeptidase inhibitor, cilastatin sodium, do not interfere with the 313-nm detection of imipenem in either the plasma or the urine assay. Thienamycin, the precursor of imipenem and an impurity in imipenem formulations, is separated from the drug using both of these methods. Concentrations generated from the HPLC analysis of plasma and urine samples from two healthy volunteers compare favorably with results using a microbiological assay method. Correlation of the two methods gives r greater than or equal to 0.990 for both fluids.     

Determination of calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine by HPLC

A reversed-phase high-performance liquid chromatographic (HPLC) assay for calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine with solid-phase extraction (SPE) was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) at a flow rate of 1.0 mL/min. Detection was set at 280 nm. The limit of quantitation of calycosin-7-O-beta-D-glucopyranoside was 0.2 microg/mL in both plasma and urine. The standard curve was linear from 0.2 to 10.0 microg/mL in plasma, and 0.2 to 5.0 microg/mL in urine. Both intra- and inter-day precision of the calycosin-7-O-beta-d-glucopyranoside were determined and their RSD did not exceed 10%. The method was successfully applied to the analysis of samples obtained from a basic pharmacokinetic study, in which calycosin-7-O-beta-d-glucopyranoside was administered orally to rats.     

Determination of trovafloxacin in human body fluids by high-performance liquid chromatography.

For the quantitative determination of trovafloxacin (a new naphthyridinone antibacterial agent) in serum and urine a simple isocratic HPLC method with fluorimetric detection is described. Serum was deproteinised with a mixture of acetonitrile and perchloric acid. The protein-free extract was separated on a reversed-phase column (Nucleosil 100-5 C18) and quantified by means of fluorescence (excitation 275 nm, emission 405 nm). The mobile phase consisted of a mixture of 250 ml acetonitrile and 750 ml distilled water containing 10 mmol/l tetrabutylammonium phosphate. Urine was diluted with 0.25 mol/l phosphoric acid 1:20 (v/v) which was adjusted to pH 3.6 with sodium hydroxide solution. Diluted urine samples were separated on a cation-exchange column (Nucleosil 100-5 SA) and also detected by means of fluorescence. Trovafloxacin was sufficiently separated from endogenous compounds. Results of validation are given. The method was applied successfully to a study of healthy volunteers.     

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.     

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.