Determination of cadmium in blood, plasma, and urine by electrothermal atomic absorption spectrophotometry after isolation by anion-exchange chromatography

The determination of cadmium in whole blood, urine, or plasma by atomic absorption using electrothermal atomization is described. In preparation for atomic absorption analysis, cadmium was concentrated on an anion-exchange column, significantly lowering the limit of detection and allowing for the first time the accurate and precise determination of plasma cadmium concentrations in persons/animals with low-level cadmium exposures. Recovery of 109Cd from spiked whole blood, plasma, and urine into supernatants of nitric acid-deproteinated samples averaged 99, 100, and 95%, respectively. Anion-exchange isolation of the anionic chlorocadmium complex removed 99.8% of the major elements associated with a deproteinated whole blood sample. The recovery of 109Cd from the anion-exchange column was 92.2 +/- 0.9% (mean +/- SE, N = 35). The separation of cadmium from constituents in blood, urine, or plasma in this manner allowed comparison of unknown samples to aqueous standards with a defined acid matrix using commercially available acids. The mean intra-assay coefficient of variation (CV) was 12 +/- 3% (mean +/- SE, N = 6) for blood, plasma, and urine samples having cadmium concentrations of 0.1-0.8 microgram/liter. The interassay CV was 13% (N = 7) for a blood sample containing 0.6 microgram Cd/liter. The recovery of known amounts of cadmium added to blood, plasma, and urine in the range of 0.2 to 5.0 micrograms Cd/liter was 97 +/- 6% (mean +/- SE, N = 4).     

Unimpaired formation of hormone-stimulated inositol trisphosphate in human mesangial cells under hyperglycemic conditions.

The relationship between bulk cellular myo-inositol content and phosphatidylinositol metabolism was evaluated in a human mesangial cell line under euglycemic and hyperglycemic conditions. Mesangial cells maintained in high glucose medium displayed a concentration-dependent fall in myo-inositol as measured by gas-liquid chromatography. Measurements of phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate mass revealed slight but statistically insignificant increases in cells exposed to high glucose containing medium. CDP-diacylglycerol: myo-inositol 3-phosphatidylinositol transferase activity, measured in plasma membranes from mesangial cells grown under control and hyperglycemic conditions, was kinetically similar with Michaelis constants (Km values) for myo-inositol of 2.9 and 2.1 mM, respectively. Finally, hormone-stimulated intracellular calcium mobilization and myo-inositol 1,4,5-trisphosphate mass was measured from mesangial cells grown under normal and hyperglycemic conditions. Both intracellular calcium and inositol trisphosphate formation were unchanged in cells previously exposed to high glucose conditions (400 mg/dl) compared to cells grown under normal glucose concentration (100 mg/dl). These data indicate that bulk changes in myo-inositol induced by hyperglycemia are neither associated with alterations in basal levels of inositol containing glycerolipids nor with changes in hormone-stimulated calcium mobilization and inositol trisphosphate formation under conditions of short term changes in extracellular glucose.     

96-Well liquid-liquid extraction liquid chromatography-tandem mass spectrometry method for the quantitative determination of ABT-578 in human blood samples

We report here a quantitative method for the analysis of ABT-578 in human whole blood samples. Sample preparation was achieved by a semi-automated 96-well format liquid-liquid extraction (LLE) method. Aluminum/polypropylene heat seal foil was used to enclose each well of the 96-well plate for the liquid-liquid extraction. A liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method with pre-column regeneration was developed for the analysis of sample extracts. Selective reaction monitoring (SRM) of the mass transitions m/z 983-935 and m/z 931-883 was employed for the detection of ABT-578 and internal standard, respectively. The ammonium adduct ions [M + NH(4)](+) generated from electrospray ionization were monitored as the precursor ions. The assay was validated for a linear dynamic range of 0.20-200.75ng/ml. The correlation coefficient (r) was between 0.9959 and 0.9971. The intra-assay CV (%) was between 1.9 and 13.5% and the inter-assay CV (%) was between 4.7 and 11.3%. The inter-assay mean accuracy was between 86.4 and 102.5% of the theoretical concentrations.     

Simple and rapid quantitative high-performance liquid chromatographic analysis of plasma amino acids

A simple and rapid high performance liquid chromatographic method for the determination of plasma amino acids was developed. The method uses minimal sample volume and automated online precolumn derivitization of amino acids with o-phthalaldehyde and fluorescent detection. Amino acids are separated by a simplified gradient without column heating. The assay is linear from 5 to 1000 micromol/L for all amino acids. Recovery of amino acids was between 91 and 108%, intra-assay coefficient of variation (CV) was 1-7%, and inter-assay CV was 2-12%. The simple sample preparation and minimal sample volume make the method useful for the quantitation of amino acids in both patient and experimental animal samples.     

Anion exchange chromatographic separation of inositol phosphates and their quantification by gas chromatography

The direct measurement of mass of inositol trisphosphate from biologic samples is described. Separation of inositol monophosphate, bisphosphate, trisphosphate, and inositol tetrakisphosphate was achieved using anion exchange chromatography with a sodium sulfate gradient. In addition, separation of the isomers of each inositol phosphate was performed using HPLC procedures. The individual inositol phosphate fractions were subsequently dephosphorylated and desalted. The myo-inositol from each fraction was then derivatized to the hexatrimethylsilyl derivative and the myo-inositol derivatives were quantified by a novel gas chromatographic analysis using the hexatrimethylsilyl derivative of chiro-inositol as an internal concentration reference. This method is a reproducible and relatively rapid procedure for the direct quantification of inositol phosphate mass which overcomes many of the problems associated with the use of radiolabeled precursors. The method is a significant improvement over existing procedures for the quantitative determination of the mass of inositol phosphate by virtue of improved recovery, sensitivity, and technical simplicity. The applicability of this method is illustrated by the quantitative determination of inositol trisphosphate in response to norepinephrine stimulation of adult canine myocytes and cerebral cortical brain slices and by measurement of the isomers of inositol trisphosphate in isolated myocytes.     

A new derivative for oxosteroid analysis by mass spectrometry

Here we report a new method for oxosteroid identification utilizing “tandem mass tag hydrazine” (TMTH) carbonyl-reactive derivatisation reagent. TMTH is a reagent with a chargeable tertiary amino group attached through a linker to a carbonyl-reactive hydrazine group. Thirty oxosteroids were analysed after derivatisation with TMTH by electrospray ionization mass spectrometry (ESI-MS) and were found to give high ion-currents compared to underivatised molecules. ESI-tandem mass spectrometry (MS/MS) analysis of the derivatives yielded characteristic fragmentation patterns with specific mass reporter ions derived from the TMT group. A shotgun ESI-MS method incorporating TMTH derivatisation was applied to a urine sample.     

Simultaneous measurement of urinary polyols using gas chromatography/mass spectrometry

In the present study, we simultaneously measured several polyols, such as adonitol, arabitol, dulcitol, glucose, myo-inositol, mannitol, sorbitol, and xylitol, in urine by gas chromatography/mass spectrometry-positive chemical ionization. We also examined possible relationship between the levels of these metabolites and age in normal individuals. In order to proceed to its quantification by GC/MS, 200 microL of a urine sample were diluted with 3 ml of distilled water, lyophilized, acetylated, and then analyzed them. Using this method, we were able to quantify as little as 0.5-1.0 ng/microL, and we made the calibration curves to be linear from 0.25 to 250 ng/microL (r(2)>0.991). Analytical recoveries were over 89.4%, and the inter-day and intra-day variability for accuracy and reproducibility was less than 20%. In the normal urine sample, the levels of polyols were gender-differentiated and age-related. This simple GC/MS method is sensitive and allows the measurement of wide ranges of polyols using small amounts of urine. We conclude that the quantitation of urinary polyols using GC/MS appears to be a clinically useful method for assessing polyol-pathway activity.     

Simultaneous quantification of 17α-OH progesterone, 11-deoxycortisol, Δ4-androstenedione, cortisol and cortisone in newborn blood spots using liquid chromatography-tandem mass spectrometry

Adrenal steroid profiling, including 17α-OH progesterone (17OHP), 11-deoxycortisol (S), Δ4-androstenedione (Δ4-A) and cortisol (F) in blood spots by tandem mass spectrometry, is used for newborn screening to detect congenital adrenal hyperplasia (CAH). Pre-analytical sample processing is critical for assay specificity and accuracy; however, it is laborious and time-consuming. This study describes the development and validation of a new Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method for the simultaneous quantification of five steroids: 17OHP, S, Δ4-A, F and cortisone (E) in blood spots from newborns. Whole blood was eluted from a 5.00 mm dried blood spot by an aqueous solution containing the deuterium-labeled internal standards d8-17OHP and d4-cortisol. The steroids extracted from blood spot into aqueous solution were subsequently purified via Extelut mini NT1 column using diethylether. The extracts were evaporated and quantified using LC-MS/MS. The detection limit was 0.25 ng/mL for 17OHP and S, 0.4 ng/mL for Δ4-A and 0.5 ng/mL for F and E. The limit of quantification was 0.5 ng/mL for 17OHP, S and Δ4-A and 1 ng/mL for F and E. Precision for 17OHP, S, Δ4-A at concentrations of 0.5, 2, and 8 ng/mL (n=5) in fortified steroid free serum samples was 1.3-3.5% (intra-assay CV) and 7-14.8% (inter-assay CV). Precision for F and E at concentrations of 5 and 20 ng/mL was 1.5-4.8% (intra-assay, CV%) and 6-15% (inter-assay, CV%). Accuracy was calculated at concentrations of 0.5, 2, and 8 ng/mL for 17OHP, S and Δ4-A and ranged from -0.3 to 0.2%, while for F and E it ranged from -3.2 to 0.2%. Relative recoveries at concentration 2 ng/mL and 8 ng/mL for 17OHP, S, Δ4-A and at 5 ng/mL and 20 ng/mL for F and E ranged from 55% to 80%. Reference intervals were estimated for all steroids in newborns (on day 3). The steroid profile assay herein described is sensitive, specific and accurate and involves a simple pre-analytical sample manipulation; it is therefore suitable for routine analysis and provides data for samples within normal range as well as those with elevated levels. For the first time to our knowledge, cortisone levels are reported in dried blood spots from newborns.     

A sensitive bioluminescence assay for myo-inositol

A specific, sensitive bioluminescence assay for myo-inositol is described employing myo-inositol dehydrogenase linked to a commercial NADH bioluminescence kit. Optimum conditions providing a linear response over 4 orders of magnitude are presented with reproducibility of 6% CV and a sensitivity of 1 pmol. The assay is suitable for the measurement of inositol at low concentrations or in small volumes.     

The simple and sensitive measurement of malondialdehyde in selected specimens of biological origin and some feed by reversed phase high performance liquid chromatography

A method for the determination of malondialdehyde (MDA) concentrations in specimens of animal tissues and feed has been developed using high performance liquid chromatography. The MDA concentration in acidified urine samples was determined after its conversion with 2,4-dinitrophenylhydrazine (DNPH) to a hydrazone (MDA-DNPH). Samples of blood plasma, muscle, liver and feed were prepared by saponification followed by derivatisation with DNPH to MDA-DNPH. The MDA concentration in chicken and hen feed samples was analysed after saponification and derivatisation followed by extractions with hexane. The free MDA in plasma samples was determined after deproteinization followed by derivatisation of MDA with DNPH. The chromatographic separation of MDA-DNPH samples was conducted using Phenomenex C(18)-columns (Synergi 2.5 μm, Hydro-RP, 100 ?, the length of 100mm) with an inner diameter of 2 or 3mm. MDA in processed biological samples was analysed using a linear gradient of acetonitrile in water, and the photodiode detector was set to 307 or 303 nm for detection. The current method that was utilised was based on the high-efficient derivatisation of MDA and was more sensitive compared to previously used methods. The selective and sensitive photodetection of the column effluent was found to be suitable for the routine analysis of MDA in urine, plasma, muscles and liver of animals and some feed samples. Because urine or blood plasma samples can be derivatised in a simple manner, the proposed method can also be suitable for the routine, non-invasive evaluation of oxidative stress in animals and humans.     

Isolation and characterization of 1-O-alpha-2-acetamido-2-deoxy-D-galactopyranosyl-myo-inositol from pregnancy urine

A new neutral glycoside of myo-inositol was isolated from the pregnancy urine of a single donor. Its structure was investigated by 1H-NMR spectroscopy and mass spectroscopy. It was identified as 1-O-alpha-2-acetamido-2-deoxy-D-galactopyranosyl-myo-inositol. No such structure or sequence has previously been reported in either myo-inositol or glucose glycosides.     

myo-Inositol Metabolism in 41 A3 Neuroblastoma Cells: Effects of High Glucose and Sorbitol Levels

Neuroblastoma cells were used to determine the effect of high carbohydrate and polyol levels on myo-inositol metabolism. The presence of elevated concentrations of glucose or sorbitol caused a significant decrease in both inositol accumulation and incorporation into phospholipid. These conditions, however, did not alter the accumulation of the other phospholipid head groups or the growth rate and water content of the cells. Two weeks of growth in either of the modified conditions was necessary to obtain a maximal effect on inositol incorporation. In contrast, growth in elevated concentrations of fructose, mannitol, or dulcitol had no effect on inositol metabolism. The reduced inositol accumulation and incorporation into lipids seen with glucose or sorbitol supplementation resulted in a decrease in the total phosphatidylinositol content of the cell without changing the levels of the other phospholipids. Kinetic analysis of cells grown in the presence of elevated glucose indicated that V'max for inositol uptake was significantly decreased with little change in the K'm. These data suggest that glucose decreases myo-inositol uptake in this system by noncompetitive inhibition. Cells grown in the presence of increased glucose also had elevated levels of intracellular sorbitol and decreased levels of myo-inositol. These results suggest that the high levels of glucose and sorbitol which exist in poorly regulated diabetes may be at least partially responsible for diabetic neuropathy via a reduction in the cellular content of myo-inositol and phosphatidylinositol. This system may be a useful model to determine the effect of reduced inositol phospholipid levels on neural cell function.     

THE SPECIFICITY AND SULFHYDRYL SENSITIVITY OF THE INOSITOL TRANSPORT SYSTEM OF THE CENTRAL NERVOUS SYSTEM

Abstract— The transport of two cyclohexitol stereoisomers, myo-inositol (inositol) and scyllo-inositol (scyllitol), from blood into the CNS in vivo and into the choroid plexus in vitro was studied. In vitro , the uptake of [³H]scyllitol or [³H]inositol by choroid plexuses, isolated from rabbits and incubated in artificial CSF, was measured. Both scyllitol and inositol inhibited [³H]scyllitol or [³H]inositol accumulation by the choroid plexus. Inositol competitively inhibited [³H]scyllitol accumulation and both isomers had a comparable affinity (K_t= 0.1 m m ) for the single cyclohexitol accumulation system. The other 6 stereoisomers tested had an order of magnitude less affinity for the cyclohexitol accumulation system of choroid plexus. Thiol reagents that penetrate cells inhibited inositol accumulation by choroid plexus more effectively than nonpenetrating thiol reagents. In vivo , in unanesthetized rabbits. the transport of unmetabolized [³H]inositol from blood into CSF, choroid plexus and brain was readily saturated by increasing the plasma levels of myo-inositol but not by the stereoisomer d -chiroinositol. Similarly, the transport of unmetabolized [³H]scyllitol into CSF, brain and choroid plexus was readily saturated by increasing the plasma levels of myo-inositol. Beside documenting the stereospecificity and thiol reagent sensitivity of the inositol transport mechanism of the choroid plexus, these results provide further evidence that the choroid plexus is a locus for cyclohexitol transport between blood and CSF. Moreover, they show that scyllitol, which, like inositol, is present at a higher concentration in brain than plasma, can be transported from blood into CSF and brain by the same system that transports inositol.     

myo-Inositol uptake is essential for bulk inositol phospholipid but not glycosylphosphatidylinositol synthesis in Trypanosoma brucei

myo-Inositol is an essential precursor for the production of inositol phosphates and inositol phospholipids in all eukaryotes. Intracellular myo-inositol is generated by de novo synthesis from glucose 6-phosphate or is provided from the environment via myo-inositol symporters. We show that in Trypanosoma brucei, the causative pathogen of human African sleeping sickness and nagana in domestic animals, myo-inositol is taken up via a specific proton-coupled electrogenic symport and that this transport is essential for parasite survival in culture. Down-regulation of the myo-inositol transporter using RNA interference inhibited uptake of myo-inositol and blocked the synthesis of the myo-inositol-containing phospholipids, phosphatidylinositol and inositol phosphorylceramide; in contrast, it had no effect on glycosylphosphatidylinositol production. This together with the unexpected localization of the myo-inositol transporter in both the plasma membrane and the Golgi demonstrate that metabolism of endogenous and exogenous myo-inositol in T. brucei is strictly segregated.     

Studies on avian erythrocyte metabolism. XVII. Kinetics and transport properties of myo-inositol in chicken reticulocytes

The uptake of myo-inositol was determined in a reticulocyte-enriched fraction prepared from chicken blood and compared with uptake in mature erythrocytes. While reticulocytes accumulated inositol at levels more than threefold that of the plasma concentration, erythrocyte levels were only slightly higher than that of the plasma concentration. The rate of uptake in reticulocytes was approximately 66 mumol/ml rbc/h compared to 5 mumol/ml rbc/h in mature erythrocytes when measured at an inositol medium concentration of 250 microM. The kinetic analysis of inositol influx by reticulocytes reveals a two component system: saturable and nonsaturable. The saturable component, which has a Km for inositol of approximately 222 microM, is Na-dependent. This Na-dependent saturable component, which presumably reflects active transport of inositol, accounts for 30-35% of the transport process. The saturable component is completely inhibited by amiloride but to a lesser extent by ouabain and bumetanide. Moreover, in the course of reticulocyte maturation, the saturable component is lost concomitantly with the completion of the synthesis of myo-inositol pentakisphosphate and the drastic decrease in the membrane permeability to inositol. In addition, phloretin and cytochalasin B, which bind to hexose carriers and inhibit hexose sugar transport, also inhibited inositol transport. The uptake of inositol was not affected by excesses of 3-O-methylglucose (100 mM) or by physiological concentrations of D-glucose. Thus, the transport mechanism of myo-inositol appears distinct from that of D-glucose.     

Quantitative measurement of plasma free metanephrines by ion-pairing solid phase extraction and liquid chromatography-tandem mass spectrometry with porous graphitic carbon column

Plasma free metanephrine and normetanephrine are the best biomarkers for diagnosing pheochromocytoma. In the past few years, liquid chromatography-tandem mass spectrometry has become the preferred technology to measure plasma metanephrine and normetanephrine because of its high sensitivity and specificity, as well as fast and simple sample preparation. In this study, we report a liquid chromatography-tandem mass spectrometry method for measuring plasma metanephrine and normetanephrine. A solid phase extraction method using ion-pairing reagent and C18 stationary phase was used for sample preparation. We tested a porous graphitic carbon column and a HILIC column for chromatographic separation, and the former one showed better resolution with no interference from plasma matrix. This method was linear from 7.2-486.8 pg/mL for metanephrine and 18.0-989.1 pg/mL for normetanephrine with an accuracy of 92.2-111.8% and 92.1-115.0%, respectively. Inter-assay and intra-assay CV for metanephrine and normetanephrine at two different concentration levels ranged from 2.0% to 10.9%. In conclusion, this liquid chromatography-tandem mass spectrometry method using ion-pairing solid phase extraction and porous graphitic column was simple and efficient for measuring plasma metanephrines.     

Quantitative analysis of cocaine and its metabolites in whole blood and urine by high-performance liquid chromatography coupled with tandem mass spectrometry

AIM: In forensic toxicology it is important to have specific and sensitive analysis for quantification of illicit drugs in biological matrices. This paper describes a quantitative method for determination of cocaine and its major metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and urine by liquid chromatography coupled with tandem mass spectrometry LC/MS/MS. METHOD: The sample pre-treatment (0.20 g) consisted of acid precipitation, followed by centrifugation and solid phase extraction of supernatant using mixed mode sorbent columns (SPEC MP1 Ansys Diag. Inc.). Chromatographic separation was performed at 30 degrees C on a reverse phase Zorbax C18 column with a gradient system consisting of formic acid, water and acetonitrile. The analysis was performed by positive electrospray ionisation with a triple quadropole mass spectrometer operating in multiple reaction monitoring (MRM) mode. Two MRM transitions of each analyte were established and identification criteria were set up based on the retention time and the ion ratio. The quantification was performed using deuterated internal analytes of cocaine, benzoylecgonine and ecgonine methyl ester. The calibration curves of extracted standards were linear over a working range of 0.001-2.00 mg/kg whole blood for all analytes. The limit of quantification was 0.008 mg/kg; the interday precision (measured by relative standard deviation-%RSD) was less than 10% and the accuracy (BIAS) less than 12% for all analytes in whole blood. Urine samples were estimated semi-quantitatively at a cut-off level of 0.15 mg/kg with an interday precision of 15%. CONCLUSION: A liquid chromatography mass spectrometric (LC/MS/MS) method has been developed for confirmation and quantification of cocaine and its metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and semi-quantitative in urine. The method is specific and sensitive and offers thereby an excellent alternative to other methods such as GC-MS that involves derivatisation.     

High-performance liquid chromatographic assay of metabolites of thioguanine and mercaptopurine in capillary blood

The main metabolites of the cytotoxic drugs thioguanine (6TG) and mercaptopurine (6MP) can be measured conveniently in red blood cells (RBC). Isolation of RBC, however, is laborious and requires some milliliters of blood. This HPLC assay allows measurements of thiopurine metabolites in very small blood samples obtained from the finger-tip. The metabolites, derivatives of 6TG and methylmercaptopurine (6MeMP), were extracted and hydrolized with perchloric acid to liberate the corresponding base. 6MeMP is completely transformed under these conditions to 4-amino-5-(methylthio)carbonyl imidazole. The chromatographic separation of 6TG and this imidazole was performed in a single run under isocratic conditions within 10 min using a 70 mm column. The quantification limit was 0.5 nmol/ml for 6TG and 3 nmol/ml blood for 6MeMP. The accuracy was 83% for 6TG (CV=3%) over the concentration range of 0.5-20 nmol/ml blood and 102% (CV=4%) for 6MeMP over the range of 3-150 nmol/ml blood. The intra-assay CV ranged from 5.4 to 7.4% for 6TG and from 6.2 to 10.6% for 6MeMP. The inter-assay CV was 7.5 and 9.5% in a pooled blood sample. The levels in RBC in whole blood were nearly coincident with those obtained in separated RBC, isolation of RBC therefore is not necessary for these measurements, if the drugs are given per os in the day before blood sampling. The concentration of 6MeMP nucleotides is more dependent on the given 6MP dose than the concentration of 6TG nucleotides. Intraindividual variations were small at unchanged drug doses, interindividual metabolite concentrations were highly variable.     

Simultaneous determination of nicotine and eight nicotine metabolites in urine of smokers using liquid chromatography-tandem mass spectrometry

A method based on liquid chromatography tandem mass spectrometry (LC-MSMS) applying atmospheric pressure chemical ionisation (APCI) in the positive ion mode was developed for the direct determination of nicotine, cotinine, trans-3'-hydroxycotinine, their corresponding glucuronide conjugates as well as cotinine-N-oxide, norcotinine, and nicotine-N'-oxide in the urine of smokers. The assay involves filtration of crude urine, fast liquid chromatography on a reversed-phase column and mass-specific detection using MSMS transitions. Deuterium-labeled nicotine, cotinine, and trans-3'-hydroxycotinine were used as internal standards. Glucuronides used as reference material were either chemically (cotinine-N-glucuronide) or enzymatically synthesized (nicotine-N-glucuronide and trans-3'-hydroxycotinine-O-glucuronide). Precision for the major nicotine analytes at levels observable in urine of smokers was better than 10%. Accuracy expressed in recovery rates in urine matrix for nicotine, cotinine, trans-3'-hydroxycotinine, and cotinine-N-glucuronide ranged from 87 to 113%. Quantitative results for the three glucuronides in urine samples of 15 smokers were compared to an indirect method in which the aglycons were determined with gas chromatography and nitrogen-selective detection (GC-NPD) before and after enzymatic splitting of the conjugates. Good agreement was found for cotinine-N-glucuronide (coefficient of variation, CV: 9%) and trans-3'-hydroxycotinine-O-glucuronide (CV: 20%), whereas the accordance between both methods was moderate for nicotine-N-glucuronide (CV: 33%). The described LC-MSMS method allows the simultaneous determination of nicotine and eight of its major metabolites in urine of smokers with good precision and accuracy. Since the method requires a minimum of sample clean-up and a very short time for chromatography (3 min), it is suitable for determining the nicotine dose in large-scale human biomonitoring studies.     

Gas chromatographic method with mass-selective detection for the determination of 2-isopropoxyphenol in human urine

Human metabolism of the insecticide propoxur yields 2-isopropoxyphenol (IPP) which is excreted conjugated in urine. In this publication a sensitive and selective analytical method is described which permits the determination of IPP as a suitable parameter for biomonitoring. The clean-up of the hydrolysed urine samples consisted of steam distillation and solid-phase extraction using a reversed-phase column. IPP and the internal standard 2-ethoxyphenol were converted to their pentafluorobenzyl ethers. Excess of the derivatisation reagent was removed using deactivated silica gel. Separation and quantitative analysis was carried out by capillary gas chromatography and mass selective detection. Coefficients of variation were below 5% for concentrations from 6 to 300 μg/l. The detection limit was 0.5 μg/l. The method was checked by analysing six urine samples from pest controllers after indoor application of propoxur. The IPP concentrations ranged from 45 to 306 μg/g creatinine. IPP was not detected in urine specimens from 10 non-exposed persons. The sensitivity of the developed method permits the detection of latent exposure to propoxur.