Head-space flow injection for the on-line determination of iodide in urine samples with chemiluminescence detection

A simple head-space (HS) flow injection (FI) system with chemiluminescence (CL) detection for the determination of iodide as iodine in urine is presented. The iodide is converted to iodine by potassium dichromate under stirring in the closed HS vial, and the iodine is released from urine by thermostatting and is carried in a nitrogen flow through an iodide trapping solution. The concomitant introduction of aliquots of iodine, luminol and cobalt(II) solutions by means of a time-based injector into an FI system allowed its mixing in a flow-through cell in front of the detector. The emission intensity at 425 nm was recorded as a function of time. The salting-out of the standard solutions affected the gas-liquid distribution coefficient of iodine in the HS vial. The typical analytical working graphs obtained under the optimized experimental conditions were rectilinear from 0 to 5 mg l(-1) iodine, achieving a precision of 2.3 and a relative standard deviation of 1.8 for ten replicate analyses of 50 and 200 microg l(-1) iodine. However, a second-order process becomes significant at higher iodine concentrations (from 10 to 40 mg l(-1)). The detection limit of the method is 10 microg l(-1) (80 ng) iodine when 8 ml samples are taken. Data for the iodide content of 10 urine samples were in good agreement with those obtained by a conventional catalytic method, and recoveries varied between 101 and 103% for urine samples spiked with different amounts of iodide. The analysis of one sample takes less than 20 min. In the present study the iodide levels found for 100 subjects were 86.8 +/- 19.0 (61-125) microg l(-1), which is lower than the WHO's optimal level (150-300 microg per day).     

Determination of methamphetamine in urine samples with sodium 1,2-naphthoquinone-4-sulphonate

Optimal conditions have been studied for the determination of methamphetamine in urine samples by an extractive-spectrophotometric method with sodium 1,2-naphthoquinone-4-sulphonate (NQS) as reagent. These conditions are: NaHCO₃ pH 10, NQS 6.3 × 10^–3 mol/l and heating for 5 min at 45°C. The accuracy and precision of the method were tested. The detection limits were 0.2 mg/l in the standard and 0.9 mg/l when 5 ml of urine sample were taken. The standard deviation of blank urine was evaluated from 12 different samples. The relative errors found in the determination of methamphetamine in urine were lower than 10% if the methamphetamine-amphetamine ratio was higher than 4.     

Determination of beryllium and selenium in human urine and of selenium in human serum by graphite-furnace atomic absorption spectrophotometry.

For human urine beryllium (Be), each sample (500 microl) was diluted (1+1) with Nash reagent (containing 0.2% (v/v) acetylacetone and 2.0 M ammonium acetate buffer at pH 6.0) and then a 20-microl volume of Triton X-100 (0.4%, v/v) aqueous solution was added. An aliquot (10 microl) of the diluted urine mixture was introduced into a graphite cuvette and was atomized according to a temperature program. The method detection limit (MDL, 3sigma) for Be was 0.37 microg/l in the undiluted urine sample and the calibration graph was linear up to 65.0 microg/l. Calibration graphs were prepared by the standard addition method. Accuracies of 98.6-102% were obtained when testing standard reference material (SRM 2670) freeze dried human urine samples. Precision (relative standard deviation, RSD) for urine Be was < or = 2.3% (withinrun, n = 5) and was < or = 3.0% (between-run, n = 3). For human urine and serum selenium (Se), samples (100 microl) were diluted with HNO3 (0.2%, v/v) to make a (1+1) dilution for urine analysis or a (1+4) dilution for serum analysis. An additional aliquot (10 microl) of Triton X-100 (0.1%, v/v) was added to each 200 microl of (1+1) diluted urine (or 20 microl of the Triton X-100 was added to each 500 microl of (1+4) diluted serum) sample. After the diluted sample mixture (10 microl) was introduced into a graphite cuvette, the corresponding chemical modifier (10 microl, containing Ni2+ + Pd + NH4NO3 in HNO3 (0.2%, v/v)) was added to it and the mixture was atomized. The MDL (3sigma) for Se in urine and in serum was 4.4 and 21.4 microg/l in undiluted sample, respectively, and the calibration graphs were linear up to 150 and 400 microg/l. Accuracies of urine Se were 98.9 - 99.4% by testing SRM 2670 (NIST) urine standards with RSD (between-run, n = 3) within 2.9%; and that of serum Se was 97.2% when testing a certified second-generation human serum (No. 29, #664) with RSD (between-run, n = 3) of 1.4%. The proposed method can be applied easily, directly, and accurately to the measurement of Be and Se in real samples (including six urine Se and four serum Se from patients of Blackfoot Disease in Taiwan).     

Sequential injection spectrophotometric determination of trace amounts of iodide by its catalytic effect on the 4,4'-methylenebis(N,N-dimethylaniline)-chloramine-T reaction

A simple and sensitive sequential injection spectrophotometric procedure is proposed for the determination of trace amounts of iodide in pharmaceutical preparations. The method is based on the catalytic effect of iodide on the (tetra base) 4,4′-methylenebis(N,N-dimethylaniline)-chloramine-T reaction in acidic solution. The method involves a sequential aspiration of 255 μl sample/standard followed by 170 μl tetra base and then 128 μl chloramine-T solutions into a carrier stream to be stacked inside a holding coil and flow reversed through a reaction coil towards a detector. The resulting colored compound is measured at 600 nm using an UV/Vis-spectrophotometer. All the parameters that affect the reaction were evaluated and the calibration curve is linear over a range of 0.1–6.0 μg l⁻¹ of iodide concentration with detection limit of 0.05 μg l⁻¹. A sample throughput of 80 samples per hour and relative standard deviation of less than 2.0% was achieved. The method is successfully applied for the determination of iodide in three different samples (tablets).     

Alcohol biomarker analysis: simultaneous determination of 5-hydroxytryptophol glucuronide and 5-hydroxyindoleacetic acid by direct injection of urine using ultra-performance liquid chromatography-tandem mass spectrometry

A direct ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) for simultaneous measurement of urinary 5-hydroxytryptophol glucuronide (GTOL) and 5-hydroxyindoleacetic acid (5-HIAA) was developed. The GTOL/5-HIAA ratio is used as an alcohol biomarker with clinical and forensic applications. The method involved dilution of the urine sample with deuterated analogues (internal standards), reversed-phase chromatography with gradient elution, electrospray ionisation and monitoring of two product ions per analyte in selected reaction monitoring mode. The measuring ranges were 6.7-10 000 nmol/l for GTOL and 0.07-100 micromol/l for 5-HIAA. The intra- and inter-assay imprecision, expressed as the coefficient of variation, was below 7%. Influence from ion suppression was noted for both compounds but was compensated for by the use of co-eluting internal standards. The accuracy in analytical recovery of added substance to urine samples was 96 and 98%, respectively, for GTOL and 5-HIAA. Method comparison with GC-MS for GTOL in 25 authentic patient samples confirmed the accuracy of the method with a median ratio between methods (GC-MS to UPLC-MS/MS) of 1.14 (r(2) = 0.975). The difference is explained by the fact that the GC-MS method also measures unconjugated 5-hydroxytryptophol naturally present in urine. The comparison with data for 5-HIAA obtained by an HPLC method demonstrated a median ratio of 1.05 between the methods. The UPLC-MS/MS method was capable of measuring endogenous GTOL and 5-HIAA levels in urine, which agreed with the literature data. In conclusion, a fully validated and robust direct method for the routine measurement of urinary GTOL and 5-HIAA was developed.     

New extraction procedure and high-performance liquid chromatographic method for analyzing polyethylene glycol-400 in urine.

We describe a new, highly efficient method for extracting polyethylene glycol-400 from urine and for its analysis by isocratic reversed-phase high-performance liquid chromatography. This method is an improvement over previously published methods in that it does not require the use of ion-exchange resins and lyophilization prior to extraction, nor does it require the separation and analysis of the individual polymers of polyethylene glycol. The procedure described in this report entails extraction with a salt-solvent combination of ammonium sulfate and dichloromethane and analysis by reversed-phase high-performance liquid chromatography. The lower limit of detection was approximately 0.25 g/l with a 2-ml urine sample. Analytical recoveries of polyethylene glycol-400 added to urine at 2.5 and 5.0 g/l averaged 97 and 96%, respectively (n = 10). Within- and between-day coefficients of variation were less than 5% at 2.5 and 5.0 g/l. Studies of various urine samples from patients receiving polyethylene glycol-400 revealed no interferences from other urine substances.     

Rapid screening assay of trimethylaminuria in urine with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Trimethyamine (TMA) and trimethylamine N-oxide (TMAO) are the most important urine parameters for diagnosing and monitoring trimethylaminuria. A rapid, simple, and specific method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was developed to determine the presence of TMA and TMAO in urine samples from patients with trimethylaminuria. Formation of the quaternary tetramethylamino iodide by derivatization of TMA with methyl iodide allows measurement of TMA by MALDI-TOFMS. The method is repeatable and reproducible, with coefficients of variance (CVs)<3%. This new method was used for direct determination of TMA and TMAO in urine specimens obtained from normal children and patients. The proposed method allows for rapid and reliable measurements of TMA and TMAO in urine specimens from patients affected by trimethylaminuria.     

Determination of 8-hydroxy-2'-deoxyguanosine in untreated urine by capillary electrophoresis with UV detection

A capillary electrophoresis method with UV detection was developed for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in untreated urine samples. The calibration graph for 8-OHdG in urine is linear in the concentration range 10-500 mg/l. and the detection limit is 5 mg/l (17 microM). 8-OHdG was determined in urine from oncological patients treated by radiation therapy. Its concentrations relative to creatinine were found to be in the range 10-47 microg 8-OHdG/l mg creatinine (4-19 micromol 8-OHdG/mmol creatinine). The overall time of the analysis of a urine sample was less than 15 min.     

Quantitative analysis of urinary C-peptide by liquid chromatography-tandem mass spectrometry with a stable isotopically labelled internal standard

We describe the first results of a quantitative LC-tandem mass spectrometry method for urinary C-peptide with the use of [2H14]C-peptide as internal standard. LC was based on gradient elution of a Hypersil PEP C18 column. Mass spectrometry was performed in the negative electrospray ionization mode and by monitoring of the transitions at m/z 1514/1334 ([2H14]C-peptide) and 1507/1320 (C-peptide). For sample preparation, we applied ultrafiltration. The analytical performance of the method in terms of measurement precision gave an RSD of <2% (n=10). The overall imprecision was investigated from independent analysis of two urine samples in six-fold and resulted in an RSD<5%. The limit of detection, expressed as signal-to-noise ratio 3, was approximately 0.15 ng C-peptide injected. Analysis of 10 random urine samples from laboratory volunteers showed interference-free ion chromatograms at a signal-to-noise ratio of approximately 75 on average. The C-peptide concentrations calculated from quantification by the bracketing calibration technique ranged from 32 to 165 ng/ml.     

Determination of 2-naphthylamine in urine by a novel reversed-phase high-performance liquid chromatography method.

A high-performance liquid chromatographic method for the determination of 2-naphthylamine in urine using fluorescence detection was developed. The method validation analysis showed the method to be in analytical control, i.e. the distribution of the difference between the observed and true values of the method evaluation samples did not deviate significantly from the normal distribution. The recovery of the method was 85%. The entire run time of chromatography was 10 min using isocratic elution (acetonitrile-water, 35:65), and the retention time for 2-naphthylamine was 5.8 min. The relative short time of analysis in combination with the low limit of detection (0.272 nmol/l) makes the method potentially applicable for surveillance of occupational and environmental exposure to 2-nitronaphthalene. The developed method is presently used for measurement of 2-naphthylamine in urine samples from workers employed at factories, characterized by a low airborne exposure level of polycyclic aromatic hydrocarbons, i.e. in general less than 25 micrograms/m3. The urine samples of exposed workers (n = 95) showed a 2-naphthylamine range of up to 9.4 nmol/l, whereas unexposed control individuals (n = 114) showed a range of up to 0.87 nmol/l.     

Determination of iodide in drinking water by isotope dilution analysis

The most suitable way of determination iodine-deficiency is to measure iodine concentrations in water and urine. For this reason, a method that can determine iodide concentrations in drinking water and suitable for routine analysis, is developed. Water samples have been collected from four Aegean localities: Izmir, Salihli, Ödemis and Tire situated in the western Turkey. The method is based on substochiometric isotope dilution analysis. Iodile concentrations vary within 9.86–85.14 μg/l ranges in the analyzed samples. Mean value is 44.92±22.07 μg/l.     

Determination of Mercury in Urine and Seawater by Flow Injection Vapor Generation Isotope Dilution Inductively Coupled Plasma Mass Spectrometry

A simple and inexpensive laboratory-built vapor generator was used with inductively coupled plasma mass spectrometry (ICP-MS) for the determination of mercury in urine and seawater samples. The applications of vapor generation ICP-MS alleviated the non-spectroscopic interferences and the sensitivity problem of mercury determination encountered when the conventional pneumatic nebulizer was used for sample introduction. The concentration of mercury was determined by isotope dilution method. The isotope ratio of mercury was calculated from the peak areas of each injection peak. The repeatability of the peak areas and isotope ratio determinations of seven consecutive injections of 1 ng mL^?1 Hg solution were 2.3% and 2.2%, respectively. This method has a detection limit of 0.07 ng mL^?1 for mercury. This method was applied to determine mercury in a CASS-3 nearshore seawater reference sample, NASS-4 open ocean seawater reference sample, NIST SRM 2670 freeze-dried urine reference sample and several urine and seawater samples collected from National Sun Yat-Sen University. The results for the reference samples agreed satisfactorily with the reference values. Results for other samples analyzed by the isotope dilution method and the method of standard additions agreed satisfactorily. Precision was better than 10% for most of the determinations.     

Gas chromatographic analysis of flunixin in equine urine after extractive methylation

A quantitative method for the analysis of flunixin, 2-(2-methyl-3-trifluoromethylanilino) nicotinic acid, in equine urine by gas chromatography with nitrogen-phosphorus detection has been developed. Flunixin and the internal standard, mefenamic acid, N-(2,3-xylyl) anthranilic acid, were analysed after extractive methylation of the carboxylic acid group using methyl iodide. The extraction and alkylation conditions of flunixin and mefenamic acid have been studied. The detection limit of the method was 0.25 mumol/l flunixin in urine (74 ng/ml). Flunixin was found to be conjugated to 96.5% in equine urine, and the conjugate was spontaneously hydrolysed to free flunixin. This approach can also be used to confirm the presence of flunixin or mefenamic acid in horse urine in the doping control of racehorses.     

Quantitative gas chromatographic measurement of glycosaminoglycan hexosamines in urine and plasma

A method is described for the quantitative determination of urine and plasma glycosaminoglycans (GAGs) by gas chromatography of the acetylated amino sugars. GAGs were first recovered by precipitation from urine with alkyltrimethylammonium bromide and from plasma by mini-column chromatography after papain digestion. Urine samples (24) analysed for total hexosamines by gas chromatography and for uronic acid by colorimetry had a correlation coefficient of 0.85. The within-run coefficient of variation (C.V.) for nineteen samples from a pooled urine was 5.2% for total hexosamines and that for the ratio of galactosamine to total hexosamines was 3.7%. The corresponding C.V. values for twelve plasma samples from a common pool were 6.5 and 3.7%. The mean ratio of galactosamine to total hexosamine in ten pre-breakfast spot urines was 51.5%. The corresponding ratio in the plasma from twenty adolescent blood donors was 76.3% and the mean total hexosamine content of the GAGs was 47.36 mumol/l.     

Determination of iodide in seawater samples by ion chromatography with chemically-bonded poly(ethylene glycol) stationary phase

An ion chromatographic method for the rapid and direct determination of iodide in seawater is reported. Poly(ethylene glycol) (PEG) groups were chemically bonded onto silica gel or C30-bonded silica gel via diol groups. PEG-bonded C30 binary phases allowed determination of iodide in seawater samples without any interference. Effects of eluent composition on retention behavior of inorganic anions have been investigated. Both cation and anion of the eluent affected the retention of analyte anions. The retention time of anions increased with increasing eluent concentration. The detection limit for iodide obtained by injecting 0.2 microl of sample was 13 microg l(-1) (S/N=3) while the limit of quantitation was 43 microg l(-1) (S/N=10). The present method was successfully applied to the rapid and direct determination of iodide in seawater with long-term durability.     

Speciation of halogenides and oxyhalogens by ion chromatography-inductively coupled plasma mass spectrometry

A speciation method utilizing ion chromatography coupled with inductively coupled plasma mass spectrometry is described for simultaneous analysis of eight halogenides and oxyhalogens: chloride, chlorite, chlorate, perchlorate, bromide, bromate, iodide and iodate. The method was applied for the analysis of drinking water samples collected from water treatment plants in areas in Finland, which are known to have high bromine concentrations in ground water. Water samples collected before and after disinfection were analyzed to get information about potential species conversion as a result of purification. Chloride and chlorate were the chlorine species found in these water samples, and iodine existed as both iodate and iodide. In the case of bromine, species conversion had taken place, since total bromine concentrations were increased during disinfection but bromide concentrations were decreased. No bromate was observed in the samples. The detection limits for all the chlorine species studied were 500 μg/l, for bromine species studied 10 μg/l, for iodide 0.1 μg/l and for iodate 0.2 μg/l.     

Routine determination of urinary free catecholamines by high-performance liquid chromatography with electrochemical detection

A simple and reliable high-performance liquid chromatographic method is described for the routine determination of the free catecholamines (norepinephrine, epinephrine and dopamine) in urine. The catecholamines are isolated from urine samples using small affinity chromatography columns prepacked with immobilised m-aminophenylboronic acid, separated by ion-pair reversed-phase liquid chromatography and quantified by electrochemical detection. Total analysis, including sample preparation time, is achieved in less than 30 min with analytical recoveries of 92-96% for all three catecholamines. Long-term stability and reproducibility of the liquid chromatographic system is attained by selection of optimised conditions for chromatographic separation with a formate mobile phase and produces detection limits of 1.4, 1.8 and 2.2 nmol/l for norepinephrine, epinephrine and dopamine, respectively, in urine samples and day-to-day coefficients of variation of less than 6%. Furthermore, the affinity isolation gels can be reused a minimum of ten times providing a rapid and cost-effective means of sample preparation.     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) method for the measurement of 2,4-dichlorophenoxyacetic acid in human urine

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoxyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline containing 0.05% Tween and 0.02% sodium azide, with analysis by a 96-microwell plate immunoassay format. No clean up was required as dilution step minimized sample interferences. Fifty urine samples were received without identifiers from a subset of pesticide applicators and their spouses in an EPA pesticide exposure study (PES) and analyzed by the ELISA method and a conventional gas chromatography/mass spectrometry (GC/MS) procedure. For the GC/MS analysis, urine samples were extracted with acidic dichloromethane (DCM); methylated by diazomethane and fractionated by a Florisil solid phase extraction (SPE) column prior to GC/MS detection. The percent relative standard deviation (%R.S.D.) of the 96-microwell plate triplicate assays ranged from 1.2 to 22% for the urine samples. Day-to-day variation of the assay results was within ±20%. Quantitative recoveries (>70%) of 2,4-D were obtained for the spiked urine samples by the ELISA method. Quantitative recoveries (>80%) of 2,4-D were also obtained for these samples by the GC/MS procedure. The overall method precision of these samples was within ±20% for both the ELISA and GC/MS methods. The estimated quantification limit for 2,4-D in urine was 30 ng/mL by ELISA and 0.2 ng/mL by GC/MS. A higher quantification limit for the ELISA method is partly due to the requirement of a 1:5 dilution to remove the urine sample matrix effect. The GC/MS method can accommodate a 10:1 concentration factor (10 mL of urine converted into 1 mL organic solvent for analysis) but requires extraction, methylation and clean up on a solid phase column. The immunoassay and GC/MS data were highly correlated, with a correlation coefficient of 0.94 and a slope of 1.00. Favorable results between the two methods were achieved despite the vast differences in sample preparation. Results indicated that the ELISA method could be used as a high throughput, quantitative monitoring tool for human urine samples to identify individuals with exposure to 2,4-D above the typical background levels.     

Matrix interference free determination of perchlorate in urine by ion association-ion chromatography-mass spectrometry

Quantitative measurement of perchlorate in biological fluids is of importance to assess its toxicity and to study its effects on the thyroid gland. Whenever possible, urine samples are preferred in toxicologic/epidemiologic studies because sample collection is non-invasive. We present here a pretreatment method for the determination of perchlorate in urine samples that lead to a clean matrix. Urine samples, spiked with isotopically labeled perchlorate, are exposed to UV to destroy/decompose organic molecules and then sequentially treated with an H⁺-form cation exchange resin to remove protolyzable compounds, with ammonia to raise the pH to 10-11 and finally passed through a mini-column of basic alumina to remove the color and other organic matter. After filtration through a 0.45 μm syringe filter, the sample thus prepared can be directly injected into an ion chromatograph (IC). We use ion association-electrospray ionization-mass spectrometry (ESI-MS) to detect and quantify perchlorate. The proposed sample preparation method leads to excellent limits of detection (LOD's) for perchlorate since there is essentially no dilution of sample and the matrix effects are eliminated. Results of urine samples from both men and women volunteers are reported for perchlorate, as well as for iodide and thiocyanate, which are generally present at much higher concentrations and for which a “dilute and shoot” approach is adequate. The limit of detection (S/N = 3) for iodide, thiocyanate and perchlorate by the present method was 0.40, 0.10 and 0.080 μg l⁻¹, respectively.     

反相高效液相法测定血浆及尿液中的异烟肼

 建立了血浆及尿液中异烟肼的高效液相快速测定方法 ,以满足临床药物分析和法医学鉴定的需要 ,提高对血浆及尿液中异烟肼浓度检测的准确性。以香草醛为衍生化试剂 ,将异烟肼经柱前衍生为异烟肼 香草醛腙 ,直接对处理后样品中的腙进行定性、定量分析。以在空白人体液样本中定量添加标准异烟肼的方法考察了样品的前处理方法、仪器条件、线性范围、精密度、回收率等 ,并对健康受试者血液中的异烟肼浓度进行了监测。结果表明 ,方法的线性范围为 0 2mg/L～ 1 2 0mg/L ;检测限为 0 2mg/L ;日内、日间精度均小于 4 0 % (n =5) ;回收率在96 3 %以上。