Non-dispersive Atomic Fluorescence Spectrometry Research Articles

Chemical Vapor Generation Non-Dispersive Atomic Fluorescence Spectrometry Technique for the Determination of Arsenic, Antimony, Selenium, and Mercury in Geological Samples by One-Time Digestion

In this paper, a one-time digestion method for the determination of arsenic (As), antimony (Sb), selenium (Se), and mercury (Hg) in geological samples was established. The optimum digestion conditions were determined with aqua regia-hydrofluoric acid (HF)-H3BO3 as a complexing open system, and the contents of As, Sb, Se, and Hg in the same digestion solution were determined by chemical vapor generation non-dispersive atomic fluorescence spectrometry (CVG-AFS). For the determination of As, Sb, Se, and Hg, the method detection limits were 0.53 mg/kg, 0.047 mg/kg, 0.009 mg/kg, and 0.00047 mg/kg, respectively, Δ log C was -0.043–0.048, and the %RSD values were lower than 8.5%, performing with good accuracy and precision. This digestion method avoids the fractional processing of each element, simplifies the digestion steps, saves pretreatment time, and improves efficiency. The digestion method also showed wide applicability, high sensitivity, and low detection limits, and it can be used as a quick method for monitoring and controlling heavy metal pollution in large-scale geological samples.

Association of Methionine Synthase rs1805087 Polymorphism With Arsenic-Related Skin Pigmentary Changes: A Population-Based Case-Control Study

Objective: Chronic arsenic exposure causes skin lesions including skin cancers, pigmentary changes, and keratosis. Genetic polymorphism in arsenic metabolism may increase susceptibility to the development of arsenic-related skin lesions. This study was performed to determine whether arsenic metabolism-related gene variants are associated with arsenic-related pigmentary changes. Methods: This case–control study involved 189 patients with arsenic-related pigmentary changes and 103 controls. Thirty-eight polymorphisms in 10 genes determined by mass spectrometry assay served as candidate drivers of arsenic-induced pigmentary changes. Urine and plasma arsenic levels were determined by inductively coupled plasma mass spectrometry. Hair arsenic concentrations were measured by nondispersive atomic fluorescence spectrometry. Arsenic metabolites in urine were determined using high-performance liquid chromatography with inductively coupled plasma mass spectrometry. Serum folate was measured using a folate radio assay kit. Analysis of variance, nonparametric test, or the chi-square test was selected according to the data distribution. Spearman correlation analysis was used to determine the correlation between two parameters. Logistic regression was used to estimate the effect of single-nucleotide polymorphisms. Results: The arsenic concentrations in urine, plasma, and hair and the urine arsenic species were not significantly different between patients and controls. Logistic regression revealed that among the polymorphisms, the methionine synthase (MTR) rs1805087 polymorphism showed a protective effect against arsenic-related pigmentary changes. In the codominant model, the adjusted odds ratio for age, sex, and ethnicity was 0.41 (95% confidence interval [CI], 0.21–0.80; P = 0.008) for the AG genotype and 0.11 (95% CI, 0.02–0.60; P = 0.012) for the GG genotype. Conclusion: MTR polymorphism showed a protective effect against arsenic-related pigmentary changes in the logistic regression model. The effect of MTR rs1805087 might be independent of arsenic metabolism and one-carbon metabolism. More studies are needed to clarify the biological function of MTR rs1805087 and its relationship with the etiology of arsenic-related pigmentary changes.

Determination of the Total Content of Arsenic, Antimony, Selenium and Mercury in Chinese Herbal Food by Chemical Vapor Generation-Four-Channel Non-dispersive Atomic Fluorescence Spectrometry.

Food security is related to safe and nutritious food which meets people's dietary needs and food preferences for an active and healthy life. A simple and feasible method was proposed for the simultaneous analysis of trace arsenic (As), antimony (Sb), selenium (Se) and mercury (Hg) in Chinese herbal food by chemical vapor generation coupled non-dispersive atomic fluorescence spectrometry (CVG-NDAFS) in this paper. The operating parameters, such as observation height, carrier and shield gas flow rate, were optimized. The detection limits were obtained under optimal conditions, which were 0.051, 0.034, 0.050 and 0.0058ngmL-1, respectively for As, Sb, Se and Hg. The relative standard deviations were 0.42%, 0.74%, 0.97% and 1.0% (n = 7), respectively (10ngmL-1of As, Sb, Se and 1ngml-1of Hg). The proposed method is verified to simultaneously determine As, Sb, Se and Hg for Chinese herbal food.

Atomic fluorescence spectrometry for ultrasensitive determination of bismuth based on hydride generation – the role of excitation source, interference filter and flame atomizers

A method of highly sensitive bismuth determination by hydride generation coupled with in-house assembled non-dispersive atomic fluorescence spectrometry was developed.

Determination of Ammonium in Aqueous Samples by Gas Phase Light Scattering Using Hydrogen Chloride Gas as a Derivatizing Reagent Followed by Nondispersive Atomic Fluorescence Spectrometry.

A sensitive method was developed for the determination of ammonium in an aqueous solution based on gas phase light scattering. In a stream of carrier gas, the gaseous ammonia from the alkalized solution formed a volatile ammonium chloride derivative by reacting with gaseous hydrogen chloride; the gaseous ammonium chloride was analyzed by nondispersive atomic fluorescence spectrometry. The mechanisms of the method are elucidated based on evaporative light scattering detection. Parameters such as temperature, amount of sodium hydroxide, and carrier gas flow rate were studied. Under optimal conditions, the detection limit of ammonium-nitrogen was 0.045 μg. The method was successfully applied to the determination of ammonium in certified reference materials, and tap and seawater samples.

On-line continuous generation of zinc chelates in the vapor phase by reaction with sodium dithiocarbamates and determination by atomic fluorescence spectrometry

The present study shows for the first time that a volatile zinc chelate species can be generated by the on-line continuous merging of an acidified sample solution with an aqueous sodium diethyldithiocarbamate solution followed by rapid separation using a frit-based bubble gas-liquid separator at room temperature. The operating conditions for the generation of the vaporous zinc chelate were preliminarily investigated by non-dispersive atomic fluorescence spectrometry. The possible mechanism of zinc vapor generation is discussed. The study shows that the volatile species is an intermediate species with very similar properties to diethyldithiocarbamic acid and a very short half-life in the acidic solution. Moreover, this species can only be generated by on-line mixing and rapid separation. The efficiency of generation was 33–85% depending on acidity. Under optimal conditions, the flow rates of the sample and Na-DDTC solution were 1.3mlmin−1, the carrier argon flow rate was 225mlmin−1, the acid concentration of the sample solution and the concentration of Na-DDTC were 0.05M and 0.4% (m/v), respectively, the detection limit of zinc was 0.33 (3σ) ngml−1, and the relative standard deviation (RSD) was 1.3%. The accuracy of the method was verified by the determination of zinc in the plant reference materials GBW10015 (spinach) and GBW10045 (rice). The results were in good agreement with the certified reference values.

Direct determination of arsenic in soil samples by fast pyrolysis–chemical vapor generation using sodium formate as a reductant followed by nondispersive atomic fluorescence spectrometry

This new study shows for the first time that sodium formate can react with trace arsenic to form volatile species via fast pyrolysis – chemical vapor generation. We found that the presence of thiourea greatly enhanced the generation efficiency and eliminated the interference of copper. We studied the reaction temperature, the volume of sodium formate, the reaction acidity, and the carried argon rate using nondispersive atomic fluorescence spectrometry. Under optimal conditions of T=500°C, the volumes of 30% sodium formate and 10% thiourea were 0.2ml and 0.05ml, respectively. The carrier argon rate was 300mlmin−1 and the detection limit and precision of arsenic were 0.39ng and 3.25%, respectively. The amount of arsenic in soil can be directly determined by adding trace amount of hydrochloric acid as a decomposition reagent without any sample pretreatment. The method was successfully applied to determine trace amount of arsenic in two soil-certified reference materials (GBW07453 and GBW07450), and the results were found to be in agreement with certified reference values.

Comparison of masking agents for antimony speciation analysis using hydride generation atomic fluorescence spectrometry

A sensitive atomic spectrometric method for the redox speciation analysis of Sb in water is described. The proposed method is based on the selective generation of stibine from Sb(III) in a continuous flow system using non-dispersive atomic fluorescence spectrometry for detection. The effects of the HCl concentration on the fluorescence intensities of Sb(III) and Sb(V) were investigated. The results indicated that atomic fluorescence emission due to Sb(V) can constructively interfere with the determination of Sb(III). For the determination of Sb(III), four compounds were tested as masking agents to inhibit the generation of stibine from Sb(V). The effects of the concentrations of the masking agents and of HCl on the fluorescence signals from Sb(III) and Sb(V) were studied. The results indicated that citric acid and NaF can successfully suppress hydride generation from Sb(V). To evaluate the developed methodology and the influence of the matrix, the recovery of Sb(III) from natural water that was spiked with different Sb(III) and Sb(V) concentrations was tested.

Simultaneous determination of trace lead, tin and cadmium in biological samples by a chemical vapor generation-four-channel atomic fluorescence spectrometry dual gas–liquid separator system

A novel, accurate, and precise method is described for simultaneous determination of trace lead (Pb), tin (Sn) and cadmium (Cd) in biological samples by chemical vapor generation (CVG) coupled with non-dispersive atomic fluorescence spectrometry. A chemical vapor generation dual gas–liquid separator system for Pb, Sn and Cd determination assembled with atomic fluorescence spectrometry was proposed. The system consists of two peristaltic pumps as the liquid driver, two gas–liquid separators and an atomic fluorescence detector. In the first gas–liquid separator system, the main Cd vapor and some SnH4 were carried out with reductant solution I, and in the second gas–liquid separator system, the main PbH4 and some SnH4 were carried out with reductant solution II. Cadmium vapor generation was based on hydrochloric acid, cobalt chloride, thiourea and KBH4 concentrations of the first system, SnH4 generation was based on hydrochloric acid and KBH4 concentrations, and PbH4 was based on hydrochloric acid, potassium ferricyanide and KBH4 concentrations of the second system. The operating conditions of the instrumentation were optimized, including the flow rates of carrier and shield gas. The conditions of chemical vapor generation for Pb, Sn and Cd were studied in detail. Under optimized experimental conditions, the blank signal was satisfactorily minimized, giving limits of detection of 0.071, 0.058 and 0.002 ng ml−1 for Pb, Sn and Cd respectively. The relative standard deviation (R.S.D.) was respectively 1.3%, 3.4% and 2.9% for 10 ng ml−1 of Pb, Sn and 2 ng ml−1 of Cd (n = 7). Finally, the proposed method was successfully applied to the simultaneous determination of Pb, Sn and Cd in a series of Chinese certified biological reference materials using simple aqueous standard calibration techniques, with analytical results in good agreement with the certified values.

Simultaneous determination of arsenic, antimony, bismuth and mercury in geological materials by vapor generation-four-channel non-dispersive atomic fluorescence spectrometry

Chemical vapor generation (CVG) coupled with non-dispersive atomic fluorescence spectrometry (NDAFS) has been widely used for determination of vapor-forming elements, but most of such works have been focused on single element analysis, and reports dealing with more than three elements simultaneous determination by CVG-NDAFS are rare. In this work, a sensitive and robust analytical procedure for the simultaneous determination of arsenic, antimony, bismuth and mercury in geological materials using vapor generation-four-channel non-dispersive atomic fluorescence spectrometry has been developed. The conditions of instrumentation and vapor generation of arsenic, antimony, bismuth and mercury were optimized. The optimized concentrations of KBH 4 and HCl required for analytes generation were 1.3% (m/v) and 20% (v/v), respectively. The interferences of coexisting ions and mutual hydride interferences were investigated carefully. One thousand milligrams per litre of Fe 3+; 500 mg l −1 of Pb 2+, Zn 2+, Mn 2+; 50 mg l −1 Cu 2+, Ni 2+, Cr 6+, Co 2+; 10 mg l −1 Ag + and 5 mg l −1 Au 3+ does not interfere with the determination of As, Sb, Bi and Hg. Associating a dilution of 1:250 (m/v) in the procedure of sample pretreatment, the tolerant concentrations of As, Sb, Bi and Hg in real geological materials are 2500, 1000, 250 and 5000 ppm, respectively. Under optimal conditions, the detection limits for As, Sb, Bi and Hg were determined to be 0.068, 0.047, 0.037 and 0.008 ng ml −1, respectively. The precisions for seven replicate determinations at the 5 ng ml −1 of As, Sb, Bi and 1 ng ml −1 of Hg were 0.47, 0.60, 0.97 and 0.93% (R.S.D.), respectively. Sample digestion was carried out on 500 mg sample with 3 ml HNO 3 and 10 ml HCl, followed by addition of thiourea solution for the quantitative reduction of As(V), Sb(V) to As(III), Sb(III). The proposed method was successfully applied to the simultaneous determination of As, Sb, Bi and Hg in a series of certified geological reference materials using simple aqueous standard calibration technique. The results obtained are in good agreement with the certified values.

Non-chromatographic speciation analysis of mercury by flow injection on-line preconcentration in combination with chemical vapor generation atomic fluorescence spectrometry

A novel non-chromatographic approach for direct speciation of mercury, based on the selective retention inorganic mercury and methylmercury on the inner wall of a knotted reactor by using ammonium diethyl dithiophosphate and dithizone as complexing agents respectively, was developed for flow injection on-line sorption preconcentration coupled with chemical vapor generation non-dispersive atomic fluorescence spectrometry. With the sample pH kept at 2.0, the preconcentration of inorganic mercury on the inner walls of the knotted reactor was carried out based on the exclusive retention of Hg–DDP complex in the presence of methylmercury via on-line merging the sample solution with ammonium diethyl dithiophosphate solution, and selective preconcentration methylmercury was achieved with dithizone instead of ammonium diethyl dithiophosphate. A 15% (v/v) HCl was introduced to elute the retained mercury species and merge with KBH 4 solution for atomic fluorescence spectrometry detection. Under the optimal experimental conditions, the sample throughputs of inorganic mercury and methylmercury were 30 and 20 h − 1 with the enhancement factors of 13 and 24. The detection limits were found to be 3.6 ng l − 1 for Hg 2+ and 2.0 ng l − 1 for CH 3Hg +. The precisions (RSD) for the 11 replicate measurements of each 0.2 μg l − 1 of Hg 2+ and CH 3Hg + were 2.2% and 2.8%, respectively. The developed method was validated by the analysis of certified reference materials (simulated natural water, rice flour and pork) and by recovery measurements on spiked samples, and was applied to the determination of inorganic mercury and methylmercury in biological and environmental water samples.

Interferences Removal for Cadmium Determination in Samples with Complex Matrices by Hydride Generation Coupled with Non-dispersive Atomic Fluorescence Spectrometry

An intermittent on-line concentration and separation system coupled with HG-AFS was developed to eliminate serious interferences from Cu2+, Pb2+ and Zn2+ on the determination of cadmium. In the present method, the interferences from common coexisting ions, such as Cu2+, Pb2+, Zn2+, Fe3+ and Ni2+, were greatly reduced. Under the optimized conditions, a detection limit of 3 pg ml(-1) (3sigma, n=11) and a precision of 1.9% RSD for 1 ng ml(-1) of Cd were obtained. The method was successfully applied to the determination of cadmium in a series of Chinese Geological Reference Materials (SRMs) and GBW01621 ferronickel alloy using simple aqueous standard calibration technique. The results obtained were in good agreement with the certified values.

Studies on the determination of trace amounts of gold by chemical vapour generation non-dispersive atomic fluorescence spectrometry

Volatile species of Au in 0.72 mol l−1 HCl medium were generated at room temperature by reduction with 0.65% (m/v) KBH4 using a programmable intermittent flow reactor and the detection system was used for the determination of trace amounts of gold by atomic fluorescence spectrometry. It was found that the presence of micro-amounts of DDTC greatly enhanced the generation efficiency of the volatile species and the flow rates of carrier and shield gas had a significant impact on the signal intensity of Au. In addition, high surface/volume ratio and rapid gas–liquid separation are essential for the generation of the volatile gold species. Based upon the results of the investigation, a detection limit of 0.23 ng ml−1 was obtained under optimized conditions and the precision of the measurements for 20 ng ml−1 of Au was 1.7% RSD. The method was successfully applied to the determination of gold in geological certified reference materials: the results were found to be in reasonable agreement with the certified values.

Simultaneous determination of arsenic and selenium in biological samples by HG-AFS

A new method is proposed for simultaneous determination of traces of arsenic (As) and selenium (Se) in biological samples by hydride-generation double-channel non-dispersive atomic-fluorescence spectrometry (HG-AFS) from tartaric acid media. The effects of analytical conditions on fluorescence signal intensity were investigated and optimized. Interferences from coexisting ions were evaluated. Under optimum conditions linear response ranges above 20 microg L(-1) for As and 32 microg L(-1) for Se were obtained with detection limits of 0.13 and 0.12 microg L(-1), respectively. The precision for eleven-fold determination of As at the 4 microg L(-1) level and of Se at the 8 microg L(-1) level were 2.7 and 1.9% (RSD), respectively. Recoveries of 92.5-95.5% for As and 101.2-108.4% for Se were obtained for four biological samples and two certified biological reference materials. The proposed method has the advantages of simple operation, high sensitivity, and high efficiency; it was successfully used for simultaneous determination of As and Se in biological samples.

Simultaneous determination of trace arsenic, antimony, bismuth and selenium in biological samples by hydride generation-four-channel atomic fluorescence spectrometry

A new and sample technique for the simultaneous determination of trace arsenic, antimony, bismuth and selenium in biologic samples by hydride generation-four-channel nondispersive atomic fluorescence spectrometry was development. The conditions of instrumentation and hydride generation of arsenic, antimony, bismuth and selenium were optimized. For reducing hexavalent Se to the tetravalent state was to heat the sample with 6 mol l −1 HCl, and then pre-reducing pentavalent As and Sb to the trivalent state was achieved by the addition of 0.05 mol l −1 thiourea. The interferences of coexisting ions were evaluated. Under optimal conditions, the detection limits for As, Sb, Bi and Se were determined to be 0.03, 0.04, 0.04 and 0.03 ng ml −1, respectively. The precision for seven replicate determinations at the 5 ng ml −1 of As, Sb, Bi and Se were 0.9, 1.2, 1.3 and 1.5% (R.S.D.), respectively. The proposed method was successfully applied to the simultaneous determination of As, Sb, Bi and Se in a series of Chinese certified biological reference materials using simple aqueous standard calibration technique, the results obtained are in good agreement with the certified values.

Studies on the Determination of Mercury by On-line Solvent Extraction and Non-Aqueous Media Hydride Generation Non-Dispersive Atomic Fluorescence Spectrometry

A new method for on-line solvent extraction covalent hydride generation in a non-aqueous extraction phase is proposed. Both liquid–liquid extraction and gas–liquid separation steps are accomplished in an online mode and with AFS as detector. Hydride generation is carried out in an aliquot of metal-complex extraction solution by sodium tetrahydroborate in N,N-dimethylformamide solution and anhydrous acetic acid. An improved U-type gas–liquid separator was used. The working conditions and manifolds scheme of flow injection were optimized. The detection limit attained for mercury was 20 ng L−1. The precision of the determination at a concentration level of around 20 times the detection limit was 5.6%. The proposed method gives improved sensitivity and eliminates interference. The method has been applied to the determination of mercury in certified reference materials with good accuracy and precision.

Concentrations of hazardous heavy metals in environmental samples collected in Xiamen, China, as determined by vapor generation non-dispersive atomic fluorescence spectrometry.

Non-dispersive atomic fluorescence spectrometry (NDAFS) coupled with vapor generation (VG) sample introduction was applied to the determination of the concentrations of hazardous heavy metals, such as arsenic, cadmium, lead and mercury, in seawater, soils and total airborne particulate matter (PM) collected around the Xiamen area in China. Almost 100% sample introduction efficiency was achieved by using thiourea and ascorbic acid for the pre-reduction of As(V) to As(III), K3Fe(CN)6 and tartaric acid for pre-oxidation of Pb(II) to Pb(IV), and masking the interferences arising from the co-existing transition metals to As, Cd, Hg and Pb during their vapor generation process. Moreover, a novel sample pretreatment device was developed to avoid the loss of mercury, lead, cadmium and arsenic during sample pretreatment. With such methods, the detection limit (DL) of arsenic, cadmium, lead and mercury was down to 0.08, 0.03, 0.05, 0.01 ng mL(-1) (3sigma), respectively. The relative standard deviations (RSD, n = 11) for arsenic, cadmium, lead and mercury at 10 ng mL(-1) were 0.9%, 1.6%, 1.3% and 2.0%, respectively. The concentrations of hazardous heavy metals in the environmental samples collected in Xiamen, China are in the range from 0.02 +/- 0.001 ng mL(-1) in seawater to 15.3 +/- 0.2 microg g(-1) in soils. Besides flame/GF-AAS and ICP-AES/MS, VG-NDAFS should be another choice for the determination of hazardous heavy metals in environmental samples.

Simultaneous determination of trace cadmium and mercury in Chinese herbal medicine by non-dispersive atomic fluorescence spectrometry using intermittent flow vapor generator.

A method was proposed for the simultaneous determination of trace cadmium and mercury by vapor generation non-dispersive atomic fluorescence spectrometry using an intermittent flow system. The effects of the parameters on the performance were studied systematically. The parameters such as acid concentration of the reaction medium, flow rate of the carrier gas and shield gas, the observation height and the atomizer temperature, etc. which affected the sensitivity, were optimized. Ascorbic acid, cobalt ion and thiourea were used as enhancement reagents or masking agents to enhance the generation efficiency of the volatile species of Cd and Hg. The mechanisms of their effects on vapor generation were investigated. In the presence of thiourea and ascorbic acid, the influences of some coexisting elements on the determination of cadmium and mercury were investigated. The detection limits (3sigma) were 0.010 microg l(-1) for Cd and 0.019 microg l(-1) for Hg, respectively. The relative standard deviations for Cd and Hg at 1.00 microg l(-1) were 2.6% and 0.97% (n = 11), respectively. The proposed method has been satisfactorily applied to the determination of trace cadmium and mercury in Chinese herbal medicine.

Determination of mercury by nondispersive atomic fluorescence spectrometry

A nondispersive atomic fluorescence spectrometer for the determination of mercury by the cold-vapor technique has been developed. The conditions for mercury vapor generation in a hydride generator (the concentration and volume of the reagents, reaction and purge times, and argon flow rate) are optimized for fluorescence measurements. An algorithm is proposed for the calculation of the analytical signal that leads to an increase in the signal-to-noise ratio by a factor of 3 as compared to the conventional procedure. On the basis of the results obtained, procedures for the determination of mercury in soil and semolina samples are developed with a detection limit of 60 pg and RSD = 10%. The accuracy of the determinations is verified by an analysis of standard reference samples of soil and semolina.

Electrothermal atomic absorption spectrometric determination of selenium in biological fluids with rhodium modifier compared with hydride generation atomic spectrometric techniques

Analytical procedures for direct electrothermal atomic absorption spectrometric (ET-AAS) determination of selenium in urine, blood serum and solubilized hair, employing rhodium modifier (10–20 μg) in a transverse-heated graphite atomizer (THGA) with integrated pyrolytic graphite-coated platform and longitudinal Zeeman-effect background correction were developed. Results for urine were compared with flow-injection hydride generation with in-atomizer trapping of hydrogen selenide in Ir–Zr treated THGA (FI-HG–ET-AAS) and with continuous flow hydride generation coupled with a non-dispersive atomic fluorescence spectrometry (CF-HG–ND-AFS). Two different bomb decompositions proved useful: (i) urine sample (1 ml) soaked overnight with HNO 3, followed by pressurized digestion with HNO 3 in a microwave oven, then H 2O 2 added, and bomb decomposition completed with HNO 3–H 2O 2, then selenium pre-reduced to Se(IV) in 4.8 M HCl; and (ii) sample (1 ml) soaked overnight with HBr–BrO 3 −, then pressurized digestion in a microwave oven, followed by quantitation by FI-HG–ET-AAS or CF-HG–ND-AFS with standard addition calibration. Several certified reference materials (urine, blood serum and hair) as well as the NIES candidate CRM No. 18 ‘Human Urine’ from the National Institute of Environmental Studies (NIES) (Tsukuba, Japan) were analyzed and the results are in agreement between techniques and with certified values. Preference is given to the direct ET-AAS determination of Se in biological liquids after two- to threefold dilution in the presence of 10 μg of Rh. Pyrolysis and atomization temperatures are within 1100–1200°C and 2100–2200°C, respectively, and the ‘end-capped’ graphite tubes are preferred for better sensitivity and lower background absorbance. The limits of detection of the direct ETAAS are approximately 6 ng ml −1 Se in biological fluids. For 24 paired samples of blood serum and urine from non-exposed individuals (Sofia, Bulgaria), selenium levels are: serum 66.5±15.5 (45.5–116.4) ng ml −1 and urine 16.8±8.1 (6.0–41.9) ng ml −1.