Determination of metabolites of pyrethroids in human urine using solid-phase extraction and gas chromatography–mass spectrometry

The described method permits the determination of the five most important metabolites of the pyrethroids permethrin, cypermethrin, deltamethrin, λ-cyhalothrin, fenvalerate, phenothrin and β-cyfluthrin in human urine in one run. The major urinary metabolites of these substances are cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl₂CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl₂CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br₂CA), fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA). After acidic hydrolysis to release the conjugated carboxylic acid metabolites, the analytes were separated from the matrix by means of solid-phase extraction using a reversed-phase column. The components of the eluate were converted to their methyl esters and extracted in hexane. Separation and quantitative analysis of the pyrethroid metabolites was carried out by capillary gas chromatography and mass selective detection. 2-Phenoxybenzoic acid served as an internal standard. The detection limits lay between 0.3 and 0.5 μg per litre urine. The relative standard deviations of the within-series imprecision were between 1% and 6%. The relative recovery rates ranged between 90% and 98%. Using this method we determined the elimination of pyrethroid metabolites in 24-h urine samples from eight pest controllers after indoor application of permethrin. The detected concentrations ranged from 1 to 70 μg g⁻¹ creatinine.     

Simultaneous determination of pyrethroid and pyrethrin metabolites in human urine by gas chromatography-high resolution mass spectrometry

A new developed gas chromatographic-high resolution mass spectrometric method for the sensitive simultaneous determination of trans-chrysanthemumdicarboxylic acid, cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid, cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid, 3-phenoxybenzoic acid and 4-fluoro-3-phenoxybenzoic acid in human urine is presented. These metabolites are biomarkers for an exposure to pyrethrum, allethrin, resmethrin, phenothrin, tetramethrin, cyfluthrin, cypermethrin, deltamethrin or permethrin. Therefore, with the help of this method for the first time a complete assessment of exposure to pyrethroid and pyrethrin insecticides is possible. After acid hydrolysis and extraction with tert-butyl-methyl-ether the residue is derivatized with 1,1,1,3,3,3-hexafluoroisopropanol and analyzed by GC/HRMS in electron impact mode (detection limits < 0.1 microg/l) as well as in negative chemical ionization mode (detection limit < 0.05 microg/l urine).     

New gas chromatographic-mass spectrometric method for the determination of urinary pyrethroid metabolites in environmental medicine

We have developed and validated a new, reliable and very sensitive method for the determination of the urinary metabolites of the most common pyrethroids in one analytical run. After acidic hydrolysis for the cleavage of conjugates, the analytes cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl(2)CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl(2)CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br(2)CA), 4-fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA) were extracted from the matrix with a liquid-liquid extraction procedure using n-hexane under acidic conditions. For further clean-up, NaOH was added to the organic phase and the carboxylic acids were re-extracted into the aqueous phase. After acidification and extraction into n-hexane again, the metabolites were then derivatised to volatile esters using N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA). Separation and detection were carried out using capillary gas chromatography with mass-selective detection (GC-MS). 2-Phenoxybenzoic acid (2-PBA) served as internal standard for the quantification of the pyrethroid metabolites. The limit of detection for all analytes was 0.05 microg/l urine. The RSD of the within-series imprecision was between 2.0 and 5.4% at a spiked concentration of 0.4 microg/l and the relative recovery was between 79.3 and 93.4%, depending on the analyte. This method was used for the analysis of urine samples of 46 persons from the general population without known exposure to pyrethroids. The metabolites cis-Cl(2)CA, trans-Cl(2)CA and 3-PBA could be found in 52, 72 and 70% of all samples with median values of 0.06, 0.11 and 0.16 microg/l, respectively. Br(2)CA and F-PBA could also be detected in 13 and 4% of the urine samples.     

Analysis of 3,5,6-trichloro-2-pyridinol in urine samples from the general population using gas chromatography–mass spectrometry after steam distillation and solid-phase extraction

We have developed a new method for the quantitative trace determination of 3,5,6-trichloro-2-pyridinol (TCPyr). TCPyr is a urinary metabolite specific to the organophosphorus pesticides chlorpyrifos and chlorpyrifos-methyl. After hydrolysis and separation of TCPyr from the urinary matrix using semi-automated steam distillation and solid-phase extraction on a new polystyrol–divinylbenzene copolymer (Isolute™ 101) the analyte was converted into its tert-butyldimethylsilyl derivative by N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA). Separation and quantitative analysis were carried out by capillary gas chromatography and mass selective detection in selected ion monitoring mode. 2,6-Dibromophenol (DBP) was used as the internal standard. The detection limit was 0.05 μg/l; the limit of quantification was 0.1 μg/l urine. The relative standard deviation of the within-series imprecision was 4.2% at a concentration of 3.5 μg/l. The relative recovery was 104%. The new method was used to analyse the urine samples of 12 persons from the general population without known exposure to the above-mentioned pesticides. TCPyr concentrations between 0.27 and 6.6 μg/l urine were detected in all urine samples. This indicates that there is a baseline excretion of TCPyr in the general population. Four urine samples collected from workers who had applied chlorpyrifos were also analysed. In these samples TCPyr was found in concentrations from 4.7 to 7.9 μg/l.     

Estimating occupational exposure to the pyrethroid termiticide bifenthrin by measuring metabolites in urine

The control of subterranean termites in Australia is predominantly through the application of chemical barriers in the soil beneath and surrounding buildings. The chemicals used to repel or kill termites are the organophosphorus insecticide, chlorpyrifos, and the synthetic pyrethroid, bifenthrin. These are applied through surface sprays and subfloor injection by licensed pest control operators. To determine the exposure of these personnel to these pesticides it is most usual to measure airborne concentrations or dermal deposition rates. However, to support information obtained from these methods it is often appropriate to determine the amount of the chemicals absorbed, using biological monitoring techniques including measurement of the chemicals or their metabolites in urine. While there are effective techniques for the monitoring of chlorpyrifos exposure by measuring either the alkyl phosphate or trichloropyridinol metabolites, there have been no published reports of suitable methods to measure bifenthrin metabolites in urine. This paper describes an extraction and HPLC-UV method used to simultaneously measure the urinary excretion of 2-methyl-3-phenylbenzoic acid (MPA), a metabolite of bifenthrin, and 3-phenoxybenzoic acid (PBA), a metabolite of several other common pyrethroid insecticides, with a detection limit for each of 2.5 ng/ml. The paper also describes the pilot application of this method to a study of South Australian pest control operators handling bifenthrin. MPA ranged from 1.8 to 31.9 microg/g creatinine and PBA from 1.3 to 30.0 microg/g in the urine of pest control workers. MPA was detected in urine of control workers without bifenthrin exposure only at low levels (1.0-1.4 microg/g creatinine), but PBA was found in both at higher levels (1.2-61.1 microg/g creatinine).     

Simultaneous detection of 35S- and 32P-labeled proteins on electrophoretic gels

A new method for the determination of oxalic acid in urine, which does not require isolation of oxalic acid, was developed by derivatizing oxalic acid and separating and quantitating the product by automated liquid chromatography. Oxalic acid in urine was reacted with o-phenylenediamine to form the strongly uv-absorbing compound 2,3-dihydroxyquinoxaline. Isolation and quantitation of this derivative were accomplished using a reverse-phase C₈ column, 5% methanol in 0.1 m ammonium acetate buffer (pH 6.6) as eluant, and absorption at 314 nm. The method was linear from 1 to 151 μg oxalic acid/ml of sample and the conversion of oxalic acid to the dihydroxyquinoxaline over this concentration range was 94.9%. The precision of duplicates averaged ±1.1%. Analyses of urine before and after treatment with oxalate decarboxylase were employed to differentiate actual urinary oxalic acid from oxalogenic compounds. Under the conditions employed, no urine was found to contain inhibitors of oxalate decarboxylase. No significant contribution to the method was found in a study of 19 potentially interfering urinary constituents. Levels of oxalic acid found in 27 urine samples from patients by this method averaged 71% of levels found using an earlier colorimetric method.     

Simultaneous determination of urinary tryptophan, tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection

A high performance liquid chromatography method with ultraviolet and fluorimetric detection has been developed for the simultaneous determination of urinary creatinine (Cr), tryptophan (Trp) and three Trp-related metabolites including kynurenine (Kyn), kynurenic acid (Kyna) and 5-hydroxyindole-3-acetic acid (5-HIAA). Samples were pretreated by centrifugation after a freeze-thaw cycle to remove protein and other precipitates. Separation was achieved by an Agilent HC-C18 (2) analytical column and a gradient elution program with a constant flow rate 1mL/min at an ambient temperature. Total run time was 30 min. Cr, Kyn and Kyna were measured by a variable wavelength detector at wavelengths 258 nm, 365 nm and 344 nm respectively. Trp and 5-HIAA were measured by a fluorescence detector with an excitation wavelength of 295 nm and an emission wavelength of 340 nm. This allowed the determination of Kyn/Cr, Kyna/Cr, Trp/Cr and 5-HIAA/Cr concentration ratios in a single run on the same urine sample. Good linear responses were found with correlation coefficient (r)>0.999 for all analytes within the concentration range of physiological level. The limit of detection of the developed method was: Cr, 0.0002 g/L; Kyn, 0.1 μmol/L; Kyna, 0.04 μmol/L; Trp, 0.02 μmol/L and 5-HIAA, 0.01 μmol/L. Recoveries from spiked human urine were: Cr, 93.0-106.4%; Kyn, 97.9-106.9%; Kyna, 98.5-105.6%; Trp, 96.7-105.2% and 5-HIAA, 96.1-99.7%. CVs of repeatability and intermediate precision of all analytes were less than 5%. This method has been applied to the analysis of urine samples from normal subjects.     

Radioimmunoassay of urinary testosterone glucuronoside

A simple method is described for the determination of testosterone glucuronoside in urine without prior hydrolysis or extraction. Appropriate amounts (5 μl male, 50 μ1 female urine) are dried at 100 °C to eliminate nonspecific binding by urinary proteins. The residues are cooled, redissolved in buffer and equilibrated with antiserum to testosterone-17β-glucosiduronate-bovine serum albumin and tritiated testosterone glucuronoside. The unbound steroid is removed with dextran-coated charcoal. The total random theoretical percentage error was calculated as 7 %. The inter and intra assay precision were 18 and 9 % respectively. Daily urine collections from 8 complete menstrual cycles have been analysed and the results related to the peak of urinary LH. In seven subjects, there was a visible peak of testosterone glucuronoside at mid cycle (LH peak ± 1 day), in six a distinct peak in the luteal phase, and in five a smaller peak during the follicular phase. The levels of testosterone glucuronoside in groups of healthy men and women were 273 ± 169 and 19 ± 10 μg/24 hrs. The corresponding range of values by a gas-liquid chromatographic method were 204 ± 102 and 14 ± 8. The values are discussed.     

Liquid chromatography–electrospray tandem mass spectrometry of acidic monoamine metabolites

A new method based on liquid chromatography–tandem mass spectrometry has been developed for the determination of monoamine metabolites, i.e., homovanillic acid (HVA), vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in human urine. Analytes were separated on a C₁₆ amide (5 cm, 5 μm) column and ionized by negative ion electrospray. Operating in the selected-reaction monitoring mode, linearity was established over three-orders of magnitude and limits of detection were in the range 30–70 μg/l. Precision calculated as RSD was within 0.8–5.2% for all intra- and inter-day determinations. The method was applied to the quantitative analysis of monoamine metabolites in 700 urine samples from occupationally (adults) and environmentally (both children and adults) exposed people living in areas with different soil contamination from lead. The urinary excretion of monoamine metabolites was significantly higher (P<0.001) in the subgroup of children living in polluted areas as compared to the control group (HVA, 6.03 vs. 4.57 mg/g creatinine; VMA, 5.33 vs. 4.37 mg/g creatinine; 5-HIAA 3.24 vs. 2.45 mg/g creatinine). In adults belonging to both groups of subjects occupationally and environmentally exposed, no differences were detected in the urinary concentration of monoamine metabolites. However, adults showed lower values of HVA (2.57 mg/g creatinine), VMA (2.17 mg/g creatinine) and 5-HIAA (2.09 mg/g creatinine) as compared to children groups.     

Urinary iodine and sodium concentration and thyroid status in the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil)

ObjectivesTo evaluate urinary iodine concentration (UIC) in civil servants aged 35-74 years of the Brazilian Study of Adults Health (ELSA-Brasil) to analyze its relationship with sociodemographic, clinical risk factors, lifestyle, urinary Na and thyroid status.DesignCross-sectional study in six Brazilian cities.MethodsThis analysis included 792 participants with information about urinary iodine concentration (UIC). Thyroid status was defined by serum levels of TSH/FT4 and the current use of antithyroid drugs for treatment of overt hyperthyroidism or levothyroxine to treat overt hypothyroidism. The determination of UIC was carried out with an inductively coupled plasma mass spectrometer (ICP-MS) and was expressed as median with Interquartile Range (IQR).ResultsIn 792 participants, thereof 52% women, mean age was 51.9 (9.0) years. The median UIC was 219 (IQR, 166-291) for all persons studied, thereof 211 (IQR, 157-276) for women and 231 (IQR, 178-304) for men. According to the WHO classification, for all persons studied, 60% had more than adequate iodine-supply (UIC ≥200 μg/L), 37% were adequately supplied (UIC 100-199 μg/L) and <3% had a deficient iodine status (<100 μg/L). In the 35-44-year age strata, which includes women of childbearing age, 23.2% of women presented less than 150 μg/L of UIC. No differences in UIC were detected according to thyroid status. (P = 0.39) The correlation between Ur-Na and UIC showed a Spearman coefficient of 0.52 (P < 0.0001) and it was also found an association of Ur-Na with UIC: Beta of 1.76 (95% Confidence Interval (95% CI): 1.01 to 2.51. The urinary Na concentration showed a synergy with the UIC, that means medians of 57, 72, 107 and 141 mmol Na/L urine (P < 0.001) in the groups with the four UIC classes according to the WHO grading mentioned above. The very low Na content in the persons exhibiting <100 μg/L UIC seems to reflect also a higher urine volume due to the frequent use of diuretics. The strong relationship between the urinary Na concentration and the UIC points to a dependence of the UIC on the individual consumption of iodized salt, which should be more considered in future studies. The strong relationship between the urinary Na concentration and the UIC points to a dependence of the UIC on the individual consumption of iodized salt, which should be more considered in future studies.ConclusionsEuthyroid persons were dominating by more than four fifths and no significant association was found between UIC and thyroid status. Although most of the persons studied present more than adequate iodine intake it was observed that nearly a quarter of women in childbearing age are iodine deficient.     

Determination of amoxicillin in body fluids by reversed-phase liquid chromatography coupled with a post-column derivatization procedure

Quantitative methods for determination of amoxicillin in body fluids are described. They comprise separation by reversed-phase chromatography (LiChrosorb RP-8, 5 μm) of the aqueous supernatants obtained from plasma or urine after purification steps involving protein precipitation followed by extraction in the case of plasma, or a double extraction procedure in the case of urine, post-column derivatization with air segmentation, and finally measurement of the UV absorbance at 310 nm. The derivatization involves formation of the mercuric mercaptide of penicillenic acid and is specific for compounds with an intact penicillanic acid ring system.Detection limits achieved on injecting 200 μl of plasma and 20 μl of urine are about 25 ng/ml and 200 ng/ml, respectively, but it is possible to improve the sensitivity further by injecting larger volumes. Precisions (s_rel) obtained for determination of 0.10 and 0.45 μg/ml in plasma were 3.72 and 1.40%, respectively.Some problems regarding column stability originating from the injection of biological samples are discussed.     

A chemical method for the quantitative determination of 2-hydroxyestrone in human urine

A highly specific chemical procedure for the quantitative determination of 2-hydroxyestrone in the urine of pregnant women is described. The assay consists of the following steps: 1) Hot acid hydrolysis of 20 ml of urine, 2) purification of 2-hydroxyestrone by “reducing chromatography” on paper and silica gel column, 3) conversion of 2-hydroxyestrone to the phenazine compound, 4) purification of the phenazine derivative by alumina column chromatography, and 5) spectroscopic quantitation of the phenazine. For internal yield correction [4-¹⁴C]2-hydroxyestrone is added after urine hydrolysis. High specificity of the method is especially guaranteed by the specific transformation of 2-hydroxyestrone to a stable phenazine derivative and by rigorous chromatographic purification of the estrogen as well as of the phenazine. The method can be used for the determination of amounts of less than 1 μg of 2-hydroxyestrone/20 ml of urine. From the data obtained the coefficient of variation is calculated to be ±3.7%. The urinary excretion of 2-hydroxyestrone in late pregnancy was found to vary within a wide range of 30–800 μg of 2-hydroxyestrone/24 hours.It seems possible to extend this method to the determination of other 2-substituted estrogens present in urine.     

High-performance liquid chromatographic method for determination of vanillin and vanillic acid in human plasma, red blood cells and urine

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)–acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 μg/ml and the urine method was linear from 2 to 40 μg/ml. The method had a detection limit of 1 μg/ml for vanillin and vanillic acid using 5 μl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.     

Determination of six hydroxyalkyl mercapturic acids in human urine using hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC–ESI-MS/MS)

Mercapturic acids are increasingly used as biomarkers for exposure to certain carcinogenic substances. Glycidol, ethylene oxide, propylene oxide, acrolein and 1,3-butadiene are important intermediates of toxicological concern used in the industrial production of various chemicals. The main urinary metabolites of these alkylating substances are hydroxyalkyl mercapturic acids. Therefore, we developed and validated an analytical method for the simultaneous determination of six hydroxyalkyl mercapturic acids in human urine after solid phase extraction. The mercapturic acids were separated using hydrophilic interaction liquid chromatography (HILIC) and quantified by tandem mass spectrometry using isotopically labelled internal standards. The developed method enables for the first time the determination of 2,3-dihydroxypropyl mercapturic acid (DHPMA), a metabolite of glycidol, in human urine. Additionally, the mercapturic acids of ethylene oxide (hydroxyethyl mercapturic acid, HEMA), propylene oxide (2-hydroxypropyl mercapturic acid, 2-HPMA), acrolein (3-hydroxypropyl mercapturic acid, 3-HPMA) as well as of 1,3-butadiene(3,4-dihydroxybutyl mercapturic acid, DHBMA and monohydroxy-3-butenyl mercapturic acid, MHBMA) can be determined. The limits of detection range from 3.0 to 7.0 μg/L. Intra- and inter-day precision was determined to range from 1% to 9%. Due to the good accuracy and precision and the low limits of detection the developed method is well suited for the determination of occupational exposure to alkylating substances as well as for the determination of background concentrations of the respective mercapturic acids in the general population.     

Ultrasensitive assay for three polyphenols (catechin,quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection

The concentrations of three polyphenols ((+)-catechin, quercetin and trans-resveratrol) in blood serum, plasma and urine, as well as whole blood, have been measured after their oral and intragastric administration, respectively, to humans and rats. The method developed for this purpose utilized ethyl acetate extraction of 100 μl samples and their derivatization with bis(trimethylsilyl)trifluoroacetamide (BSTFA) followed by gas-chromatographic analysis on a DB-5 column followed by mass selective detection employing two target ions and one qualifier ion for each compound. Total run time was 17 min with excellent resolution and linearity. The limits of detection (LOD) and quantitation (LOQ) were an order of magnitude less than for any previously published method, being 0.01 μg/l and 0.1 μg/l, respectively, for all compounds. Recovery at 1 μg/l and 10 μg/l was >80% in all instances but one, and was >90% in 50%. Imprecision was acceptable at 0.25 and 1.0 μg/l, concentrations below the LOQ of previous methods. Aglycones released from conjugates after hydrolysis were easily measurable. Optimal conditions for hydrolysis were established. After oral administration of the three polyphenols to humans, their conjugates vastly exceeded the concentrations of the aglycones in both plasma and urine. Concentrations peaked within 0.5–1.0 h in plasma and within 8 h in urine. During the first 24 h, 5.1% of the (+)-catechin and 24.6% of the trans-resveratrol given were recovered in the urine (free plus conjugated). This method can be proposed as the method of choice to assay these polyphenols and their conjugates in biological fluids.     

Determination of urinary glucuronide conjugates by high-performance liquid chromatography with pre-column fluorescence derivatization

A simple and highly sensitive high-performance liquid chromatographic method for the direct determination of urinary glucuronide conjugates is described. The method is based on the direct derivatization of the glucuronic acid moiety in glucuronide conjugates with 6,7-dimethoxy-1-methyl-2 (1 H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 0–37°C. The resulting fluorescent derivatives are separated on a C₁₈ column using methanol—acetonitrile—0.5% triethylamine in water (1:1:2, v/v) as mobile phase, and are detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise RATIO = 3) for the glucuronides are 13–48 fmol for an injection volume of 10 μl (130–480 fmol per 5 μl of human urine). The method was applied to the measurement of etiocholanorone-3-glucuronide and androsterone-3-glucuronide in human urine. The method is simple and rapid without conventional liquid—liquid extraction of the glucuronides from urine.     

Über die Gewöhnung von Ziegen an eiskaltes Tränkwasser

90 urine samples obtained in three lamb trials and one experiment using adult wethers were analyzed for their contents of orotic acid and creatinine. The average daily excretion of orotic acid accounted for 0.5 mg to 1.5 mg (35 μg to 130 μg/W^0.75) with a high individual variation. Correlation coefficients between orotic acid and other urinary constituents were low indicating an entirely different response to metabolic variations. There was only a weak relationship to live weight, protein retention and rumen fluid traits. Defaunation reduced the orotic acid excretion (significant in the adult wethers) whereas the addition of rumen‐protected lysine as well as the use of different dietary carbohydrate sources were without effect. The urinary excretion of creatinine increased with live weight and age from 0.4 g/d in the 20 kg lambs to 1.7 g/d in the adult 53 kg wethers. The correlations with live weight were close whereas the apparently negative correlation with protein retention was not real as could be evaluated by calculation of the partial correlations. There was a close correlation of creatinine with total N, urea and allantoin. Neither defaunation nor rumen‐protected lysine and the kind of carbohydrate source had significant effects on creatinine. The use of orotic acid and creatinine as indicators of metabolic disorders were discussed. Easy application in practical diagnosis without quantitative urine collection might be possible by the determination of orotic acid in the milk of cows and of the creatinine/N ratio in urine.     

Application of solid-phase microextraction and gas chromatography–mass spectrometry for the determination of chlorophenols in urine

This study investigated the feasibility of applying solid-phase microextraction (SPME) combined with gas chromatography–mass spectrometry to analyze chlorophenols in urine. The SPME experimental procedures to extract chlorophenols in urine were optimized with a polar polyacrylate coated fiber at pH 1, extraction time for 50 min and desorption in GC injector at 290°C for 2 min. The linearity was obtained with a precision below 10% R.S.D. for the studied chlorophenols in a wide range from 0.1 to 100 μg/l. In addition, sample extraction by SPME was used to estimate the detection limits of chlorophenols in urine, with selected ion monitoring of GC–MS operated in the electron impact mode and negative chemical ionization mode. Detection limits were obtained at the low ng/l levels. The application of the methods to the determination of chlorophenols in real samples was tested by analyzing urine samples of sawmill workers. The chlorophenols were found in workers, the urinary concentration ranging from 0.02 μg/l (PCP) to 1.56 μg/l (2,4-DCP) depending on chlorophenols. The results show that trace chlorophenols have been detected with SPME–GC–MS in the workers of sawmill where chlorophenol-containing anti-stain agents had been previously used.     

Determination of muscarine in human urine by electrospray liquid chromatographic-mass spectrometric

A liquid chromatography-mass spectrometry based method for determination of muscarine in human urine was developed and validated. The method involved a solid phase extraction of muscarine from urine using Strata X-CW column. Separation of muscarine was achieved within 16.0 min on a reversed phase Gemini C18 analytical column (150 mm × 2.0mm i.d., 5 μm) with a mobile phase consisted of aqueous 8 mmol/L heptafluorobutyric acid and acetonitrile in a gradient mode. Mass spectrometric detection was performed at m/z 174 and m/z 216 for muscarine and acetylmuscarine (internal standard), respectively. The linearity was satisfactory with a coefficient of determination (R(2)) 0.9993 at concentration range from 0.3 ng/mL to 2.0 μg/mL, LOD and LOQ for muscarine was 0.09 ng/mL and 0.3 ng/mL, respectively. The found out recoveries of muscarine were 96% or 95% for concentration 0.3 ng/mL and 0.2 μg/mL or 2.0 μg/mL, respectively. The precision in the intra-assay-study varied from 0.48% to 1.39% and in the inter-assay-study from 2.39% to 5.49%. The accuracy ranged from -3.3% to -6%. The validation results demonstrated that the method fulfilled satisfactory requirements for precision and accuracy across the calibration curve. The applicability of the method has been demonstrated by analyzing clinical urine samples. The method offers the fast objective identification of intoxication by muscarine and can become a routine screening alternative to more difficult microscopic examination of spores in the gastric content in clinical practice.     

Serum zinc levels and goiter in Iranian school children

Iodine deficiency has been shown to have high prevalence in Iran despite sufficient iodine supplementation. Zinc deficiency may also contribute to the pathogenesis of endemic goiter. The aim of this study was to compare serum zinc level in Iranian school children with and without goiter. A cross-sectional study was performed among urban children aged 8-12 years in city of Kerman, Iran. A multistage proportional to size cluster sampling method was used to screen 5500 subjects out of 29,787 students. After the screening phase, serum and urine specimens of randomly selected 165 students were evaluated for serum zinc levels and urinary iodine excretion and compared in goiterous and non-goiterous children. Serum zinc level was 149.5±29.4 μg/l in goiterous children and 141.2±52 μg/l in non-goiterous children but no significant difference was found between the groups (p=0.37). But urinary iodine excretion was significantly (p<0.001) lower in goiterous children (207.5 μg/l in goiterous children and 262.5 μg/l in non-goiterous children). This study showed that serum zinc level in goiterous and non-goiterus children is not different and zinc deficiency is not a risk factor for endemic goiter in this population.