Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C₁₈ and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5–21.5 nmol/ml for NEF and 0.4–9.5 nmol/ml for metabolites in serum and 4–86 nmol/ml for NEF and 8–190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

High-performance liquid chromatography–tandem electrospray mass spectrometry for the determination of lidocaine and its metabolites in human plasma and urine

A sensitive, selective and accurate high-performance liquid chromatographic–tandem mass spectrometric assay was developed and validated for the determination of lidocaine and its metabolites 2,6-dimethylaniline (2,6-xylidine), monoethylglycinexylidide and glycinexylidide in human plasma and urine. A simple sample preparation technique was used for plasma samples. The plasma samples were ultrafiltered after acidification with phosphoric acid and the ultrafiltrate was directly injected into the LC system. For urine samples, solid-phase extraction discs (C₁₈) were used as sample preparation. The limit of quantification (LOQ) was improved by at least 10 times compared to the methods described in the literature. The LOQ was in the range 1.6–5 nmol/l for the studied compounds in plasma samples.     

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma by temperature-programmed packed capillary liquid chromatography with on-column focusing of large injection volumes

A miniaturized temperature-programmed packed capillary liquid chromatographic method with on-column large volume injection and UV detection for the simultaneous determination of the three selective serotonin reuptake inhibitors citalopram, fluoxetine, paroxetine and their metabolites in plasma is presented. An established reversed-phase C8 solid-phase extraction method was employed, and the separation was carried out on a 3.5-microm Kromasil C18 0.32x300 mm column with temperature-programming from 35 (3 min) to 100 degrees C (10 min) at 1.3 degrees C/min. The mobile phase consisted of acetonitrile-45 mM ammonium formate (pH 4.00) (25:75, v/v). The non-eluting sample focusing solvent composition acetonitrile-45 mM ammonium formate (pH 4.00) (3:97, v/v) allowed injection of 10 microl or more of the plasma extracts. The method was validated for the concentration range 0.05-5.0 microM, and the calibration curves were linear with coefficients of correlation >0.993. The limits of quantification for the antidepressants and their metabolites ranged from 0.05 to 0.26 microM. The within and between assay precision of relative peak height were in the range 2-22 and 2-15% relative standard deviation, respectively. The within and between assay recoveries were in the 61-99 and 54-92% range for the antidepressants, respectively, and between 52-102 and 51-102% for the metabolites.     

Fluorometric high-performance liquid chromatographic analysis of 10-deazaaminopterin, 10-ethyl-10-deazaaminopterin, and known metabolites

The antifolate compounds 10-deazaaminopterin (10-dAM) and 10-ethyl-10-deazaaminopterin (10-EdAM) are therapeutically superior to methotrexate in transplanted murine tumor systems and in human tumor xenografts growing in immunodeficient "nude" mice. The increased therapeutic index of these analogs correlates with their selective uptake, retention, and polyglutamation within neoplastic cells. We have developed a fluorescence high-performance liquid chromatographic assay applicable to 10-dAM, 10-EdAM, their polyglutamate anabolites, and their 7-hydroxy (7-OH) and deglutamate catabolites. The assay is based upon the high native fluorescence of pteridine-containing compounds which contain carbon in the 10 position. The assay employs a reverse-phase C-18 column and an ascending acetonitrile gradient in 50 mM phosphate, pH 7.0. The compounds are extracted from plasma and urine with 95 +/- 7% and 98 +/- 2% recoveries, respectively, using C-18 Sep-Paks. The linear range of the assay is, for 10-dAM, 2-100 nM, and for 10-EdAM, 1-100 nM. Polyglutamated metabolites of [3H]10-EdAM isolated from L1210 cells have been separated by HPLC with identification of five derivatives (Glu 1-5) confirmed by enzymatic peak shift using serum conjugase and by quantitative correlation of fluorescence intensity, radioactivity, and titration inhibition of dihydrofolate reductase. The assay has been used successfully in pharmacokinetic analyses of plasma and urine samples from patients receiving 10-dAM and 10-EdAM. In patients who had received 10-EdAM, 7-OH-10-EdAM, and the deglutamate catabolite were also detected. This HPLC fluorescence assay is superior to the dihydrofolate reductase inhibition and binding assays with regard to specificity and precision; moreover, it can provide a means for simultaneous assay of the physiologically important anabolites and catabolites of these new antifolates.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10-μm μBondapak phenyl column with an eluting solvent of water—methanol—1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(d-(-)-α-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 ± 6.3% (S.D.) in the concentration ranges of 0.1–20 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/ml plasma. The urine assay was validated over a concentration range of 0.025–5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 μg/ml) using a 0.1-ml urine specimen per assay.The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Simultaneous determination of lansoprazole enantiomers and their metabolites in plasma by liquid chromatography with solid-phase extraction

A simple and highly sensitive high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of lansoprazole enantiomers and their metabolites, 5-hydroxylansoprazole enantiomers and lansoprazole sulfone, in human plasma have been developed. Chromatographic separation was achieved with a Chiral CD-Ph column using a mobile phase of 0.5M NaClO(4)-acetonitrile-methanol (6:3:1 (v/v/v)). The analysis required only 100 microl of plasma and involved a solid-phase extraction with Oasis HLB cartridge, with a high extraction recovery (>94.1%) and good selectivity. The lower limit of quantification (LOQ) of this assay was 10 ng/ml for each enantiomer of both lansoprazole and 5-hydroxylansoprazole, and 5 ng/ml for lansoprazole sulfone. The coefficient of variation of inter- and intra-day assay was <8.0% and accuracy was within 8.4% for all analytes (concentration range 10-1000 ng/ml). The linearity of this assay was set between 10 and 1000 ng/ml (r2>0.999 of the regression line) for each of the five analytes. This method is applicable for accurate and simultaneous monitoring of the plasma levels of lansoprazole enantiomers and their metabolites in the renal transplant recipients.     

Simultaneous determination of quinine and four metabolites in plasma and urine by high-performance liquid chromatography

The determination of quinine, (3S)-3-hydroxyquinine, 2′-quininone and (10R)- and (10S)-10,11-dihydroxydihydroquinine in plasma and urine samples is described. This is the first time the R and S configurations have been correctly assigned to the two metabolites of 10,11-dihydroxyquinine. One hundred microliter-plasma samples were protein precipitated with 200 μl cold methanol. Urine samples were 10–100× diluted and then directly injected into the HPLC. A reversed-phase liquid chromatography system with fluorescence detection and a Zorbax Eclipse XDB phenyl column and gradient elution was used. The within and between assay coefficients of variation of the method for quinine and its metabolites in plasma and urine was less than 13%. The lower limit of quantitation was in the range of 0.024–0.081 μM.     

Automated GC-MS analysis of free amino acids in biological fluids

A gas chromatography-mass spectrometry (GC-MS) method was developed for the quantitative analysis of free amino acids as their propyl chloroformate derivatives in biological fluids. Derivatization with propyl chloroformate is carried out directly in the biological samples without prior protein precipitation or solid-phase extraction of the amino acids, thereby allowing automation of the entire procedure, including addition of reagents, extraction and injection into the GC-MS. The total analysis time was 30 min and 30 amino acids could be reliably quantified using 19 stable isotope-labeled amino acids as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) were in the range of 0.03-12 microM and 0.3-30 microM, respectively. The method was validated using a certified amino acid standard and reference plasma, and its applicability to different biological fluids was shown. Intra-day precision for the analysis of human urine, blood plasma, and cell culture medium was 2.0-8.8%, 0.9-8.3%, and 2.0-14.3%, respectively, while the inter-day precision for human urine was 1.5-14.1%.     

Determination of the antihypertensive agent 1-(2-aminoethyl)-3-(2,6-Dichlorophenyl)thiourea in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific normal-phase (adsorption) high-performance liquid chromatographic (HPLC) assay was developed for the determination of 1-(2-aminoethyl)-3-(2,6-dichlorophenyl)thiourea [I] in plasma and urine. The assay involves the extraction of the compound into methylene chloride from plasma or urine buffered to pH 10, and the HPLC analysis of the residue dissolved in methylene chloride—methanol—heptane (85:10:5). A 10-μm silica gel column was used with methylene chloride—methanol—heptane—ammonium hydroxide (85:10:5:0.1) as the eluting solvent. The effluent was monitored at 254 nm and quantitation was based on the peak height vs. concentration technique. The assay has a recovery of 64.5 ± 4.5% (S.D.) from plasma and 96.0 ± 6.3% (S.D.) from urine in the concentration range of 0.1–2 μg per ml and 2–40 μg per 0.1 ml of plasma and urine, respectively, with a limit of detection of 0.05–0.1 μg [I] per ml of plasma using a 1-ml specimen and 0.1 μg per ml urine using a 0.1-ml specimen, respectively. The assay was applied to the determination of plasma levels and urinary excretion of the compound [I] in dog following the oral administration of 28.8 mg of [I] · maleate per kg body weight.The HPLC assay was also used to determine the stability of [I] and for the measurement of a potential degradation product, clonidine [II] [2-(2,6-dichlorophenylamino)-2-imidazoline] in pooled human plasma stored at ?17°C, and pooled human urine stored at ?17°C and ?90°C, respectively.     

Liquid chromatographic assay for common sunscreen agents: application to in vivo assessment of skin penetration and systemic absorption in human volunteers

The purpose of the present study was to develop a reverse-phase high-performance liquid chromatographic (HPLC) assay for quantifying four common sunscreen agents, namely 2-hydroxy-4-methoxybenzophenone, 2-ethylhexyl-p-methoxycinnamate, 2-ethylhexylsalicylate (octylsalicylate) and salicylic acid 3,3,5-trimethcyclohexyl ester (homosalate) in a range of biological matrices. This assay was further applied to study the skin penetration and systemic absorption of sunscreen filters after topical application to human volunteers. Separation was achieved utilizing a Symmetry C(18) column with methanol-water as the mobile phase. The assay permits analysis of the sunscreen agents in biological fluids, including bovine serum albumin (BSA) solution, plasma and urine, and in human epidermis. The assay was linear (r2 > 0.99) with minimum detectable limits of 0.8 ng for oxybenzone, 0.3 ng for octylmethoxycinnamate, and 2 ng for homosalate and octylsalicylate. The inter- and intra-day variation for the four sunscreens was less than 3% at the upper end of the linear range and less than 6% at the lower end. Recoveries of sunscreens from plasma, 4% (w/v) BSA solution and epidermal membranes were within the range of 91-104%. Recoveries from urine of the four sunscreens, and oxybenzone with its metabolites were more than 86%. Up to approximately 1% of the applied dose of oxybenzone and its metabolites was detected in the urine. Appreciable amounts were also detected in the stratum corneum through tape stripping. The HPLC assay and extraction procedures developed are sensitive, simple, rapid, accurate and reproducible. Results from the preliminary clinical study demonstrate significant penetration of all sunscreen agents into the skin, and oxybenzone and metabolites across the skin.     

Determination of alpha-naphthylisothiocyanate and metabolites alpha-naphthylamine and alpha-naphthylisocyanate in rat plasma and urine by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of alpha-naphthylisothiocyanate (1-NITC) and two metabolites alpha-naphthylamine (1-NA) and alpha-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C(18) 5-microm column, a mobile phase of acetonitrile-water (ACN-H(2)O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95-106 and 97-103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at -20 and -80 degrees C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4 degrees C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague-Dawley rat.     

Determination of cibenzoline in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (Cipralan ^TM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile---phosphate buffer (0.015 mol/1, pH 6.0) (80:20). A 10-μ ion-exchange (sulfonate) column was used with acetonitrile—phosphate buffer (0.015 mol/1, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard.The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10–1000 ng/ml and 50–5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.     

Determination of the catechol-O-methyltransferase inhibitor tolcapone and three of its metabolites in animal and human plasma and urine by reversed-phase high-performance liquid chromatography with ultraviolet detection

Reversed-phase HPLC procedures were developed for the determination of tolcapone (Ro 40-7592) and its metabolites Ro 40-7591, Ro 61-1448, and Ro 47-1669 in plasma and in urine samples. One of the procedures for plasma involved the determination of tolcapone and its metabolite Ro 40-7591 and the other, the determination of the two other metabolites. The urine assay enabled the simultaneous determination of tolcapone and all metabolites in one run. Sample preparation in plasma involved protein precipitation with acetonitrile. Urine was simply diluted. The compounds of interest were monitored in the UV at 270 nm. The limits of quantification were 0.05 μg/ml for each compound (plasma assay) and 0.2 μg/ml for the urine assay. The mean inter-assay precisions (C.V.) were ≤6% (plasma assay) and ≤8% (urine assay). The procedures were successfully applied to the sample analysis of animal pharmacokinetic (rat, dog, mouse, rabbit and cynomolgus monkey) and clinical pharmacology studies.     

Sulforaphane-mediated induction of a phase 2 detoxifying enzyme NAD(P)H:quinone reductase and apoptosis in human lymphoblastoid cells

The effect of sulforaphane on human lymphoblastoid cells originating from a patient of a high cancer risk was studied. Sulforaphane (SFN) is a naturally occurring substance of chemopreventive activity. In our study, changes in cell growth, induction of apoptosis and phase 2 enzymes as well as glutathione level were examined. Apoptosis was tested by confocal microscopy at three stages: change in mitochondrial membrane potential, caspase activation and phosphatidylserine externalization. We show that SFN increases the activity of the detoxification system: it increases quinone reductase activity at low concentration (0.5-1 microM) and raises glutathione level in a dose-dependent manner. At higher doses (2.5-10 microM) sulforaphane is a cell growth modulator, as it caused cell growth cessation (IC50 = 3.875 microM), and apoptosis inducer. The results obtained suggest that sulforaphane acts as a chemopreventive agent in human lymphoblastoid cells.     

Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry

In order to better understand the role of lysophosphatidic acid (LPA) in physiology and pathophysiology, it is necessary to accurately determine the molecular species and amounts of LPA in biological samples. We have developed a stable-isotope dilution, liquid chromatography-mass spectrometry assay for the direct quantitative analysis of 1-acyl-LPA. This method utilizes a deuterium-labeled internal standard, LPA (18:0-d(35)), and a single liquid-liquid extraction with acidic butanol that allows >95% recovery of LPA, followed by online normal-phase liquid chromatography-mass spectrometry. This protocol allows for the accurate, sensitive, and reproducible analysis of the individual 1-acyl-LPA species present in biological samples. The utility of the assay is demonstrated through the analysis of LPA species in plasma and serum from human volunteers. Total LPA in EDTA plasma was 0.61 +/- 0.14 microM in males and 0.74 +/- 0.17 microM in females, which increased to 0.91 +/- 0.23 and 0.99 +/- 0.38 microM after incubation for 24 h at 25 degrees C. Total LPA in serum was 0.85 +/- 0.22 microM in males and 1.57 +/- 0.56 microM in females, which increased to 4.78 +/- 0.89 and 5.57 +/- 0.73 microM after incubation for 24 h at 25 degrees C.     

Determination of picotamide in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of picotamide in human plasma and urine is described. After addition of an internal standard (bamifylline), the plasma and urine samples were subjected to liquid—liquid extraction and clean-up procedures. The final extracts were evaporated to dryness and the resulting residues were reconstituted in 100 μl of methanol—water (50:50, v/v) and chromatographed on a LiChrosorb RP-SELECT B reversed-phase column coupled to an ultraviolet detector monitored at 230 nm. Chromatographic analysis takes about 10 min per sample. The assay was linear over a wide range and has a limit of detection of 0.005 and 0.1 μg/ml in plasma and urine, respectively. It was selective for picotamide, accurate and robust and thus suitable for routine assays after therapeutic doses of picotamide.     

Dietary organic isothiocyanates are cytotoxic in human breast cancer MCF-7 and mammary epithelial MCF-12A cell lines

Organic isothiocyanates (ITCs) are dietary components present in cruciferous vegetables. The purpose of this investigation was to examine the cytotoxicity of 1-naphthyl isothiocyanate (NITC), benzyl isothiocyanate (BITC), beta-phenethyl isothiocyanate (PEITC), and sulforaphane in human breast cancer MCF-7 and human mammary epithelium MCF-12A cell lines, as well as in a second human epithelial cell line, human kidney HK-2 cells. The cytotoxicity of NITC, BITC, PEITC, and sulforaphane, as well as the cytotoxicity of the chemotherapeutic agents daunomycin (DNM) and vinblastine (VBL), were examined in MCF-7/sensitive (wt), MCF-7/Adr (which overexpresses P-glycoprotein), MCF-12A, and HK-2 cells. Cell growth was determined by a sulforhodamine B assay. The IC50 values for DNM and VBL in MCF-7/Adr cells were 7.12 +/- 0.42 microM and 0.106 +/- 0.004 microM (mean +/- SE) following a 48-hr exposure; IC50 values for BITC, PEITC, NITC, and sulforaphane were 5.95 +/- 0.10, 7.32 +/- 0.25, 77.9 +/- 8.03, and 13.7 +/- 0.82 microM, respectively, with similar values obtained in MCF-7/wt cells. Corresponding values for BITC, PEITC, NITC, and sulforaphane in MCF-12A cells were 8.07 +/- 0.29, 7.71 +/- 0.07, 33.6 +/- 1.69, and 40.5 +/- 1.25 microM, respectively. BITC and PEITC can inhibit the growth of human breast cancer cells as well as human mammary epithelium cells at concentrations similar to those of the chemotherapeutic drug DNM. Sulforaphane and NITC exhibited higher IC50 values. The effect of these ITCs on cell growth may contribute to the cancer chemopreventive properties of ITCs by suppressing the growth of preclinical tumors, and may indicate a potential use of these compounds as chemotherapeutic agents in cancer treatment.     

Accumulation of quercetin conjugates in blood plasma after the short-term ingestion of onion by women

Quercetin is a typical flavonoid present mostly as glycosides in plant foods; it has attracted much attention for its potential beneficial effects in disease prevention. In this study, we examined human volunteers after the short-term ingestion of onion, a vegetable rich in quercetin glucosides. The subjects were served diets containing onion slices (quercetin equivalent: 67.6-93.6 mg/day) with meals for 1 wk. Quercetin was only found in glucuronidase-sulfatase-treated plasma, and its concentration after 10 h of fasting increased from 0.04 +/- 0.04 microM before the trial to 0.63 +/- 0.72 microM after the 1-wk trial. The quercetin content in low-density lipoprotein (LDL) after glucuronidase-sulfatase treatment corresponded to <1% of the alpha-tocopherol content. Human LDL isolated from the plasma after the trial showed little improvement of its resistance to copper ion-induced oxidation. It is therefore concluded that conjugated metabolites of quercetin accumulate exclusively in human blood plasma in the concentration range of 10(-7) approximately 10(-6) M after the short-term ingestion of vegetables rich in quercetin glucosides, although these metabolites are hardly incorporated into plasma LDL.     

Microanalysis of the metabolic intermediates of lactose synthesis in human milk and plasma using bioluminescent methods

Sensitive bioluminescent methods were developed to measure the metabolites glucose, glucose 6-phosphate (G6P), glucose 1-phosphate (G1P), UDP-glucose, and UDP-galactose in human milk and lactose and galactose in human plasma. The bioluminescent methods measured NADH produced by coupled enzymatic assays derived from equivalent spectrophotometric methods. We found that the long chain fatty acids in human milk (C10-C16) inhibited the bioluminescent reactions. This inhibition was overcome by adding defatted bovine serum albumin to the reaction mixture containing the bioluminescent enzymes. It also was necessary to modify methods of deproteinizing milk and blood plasma to accommodate small sample volumes. In the development of these assays emphasis was given to simplicity of reagent preparation, minimizing cost, and ease of use. The detection limit for the bioluminescent method for NADH was 0.28 nM for a 20-microliters sample. For the assays of the metabolites, recoveries ranged from 91 to 107%. For sample sizes of 2 to 5 microliters of protein free sample, the detection limits for milk were G1P, 0.09 microM; G6P, 0.05 microM; UDPhexose, 0.07 microM; UDP-Glc, 0.03 microM; glucose, 9 microM; and for plasma, lactose, 0.76 microM, galactose, 0.31 microM. The bioluminescent methods gave equivalent results to spectrophotometric methods for the measurement of blood lactose and milk glucose.     

Qualitative Profiling and Quantification of Neonicotinoid Metabolites in Human Urine by Liquid Chromatography Coupled with Mass Spectrometry

Neonicotinoid pesticides have been widely applied for the production of fruits and vegetables, and occasionally detected in conventionally grown produce. Thus oral exposure to neonicotinoid pesticides may exist in the general population; however, neonicotinoid metabolites in human body fluids have not been investigated comprehensively. The purpose of this study is the qualitative profiling and quantitative analysis of neonicotinoid metabolites in the human spot urine by liquid chromatography coupled with mass spectrometry (LC/MS). Human urine samples were collected from three patients suspected of subacute exposure to neonicotinoid pesticides. A qualitative profiling of urinary metabolites was performed using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) with a database of nominal molecular weights of 57 known metabolites of three neonicotinoid pesticides (acetamiprid, Imidacloprid, and clothianidin), as well as the parent compounds. Then a quantitative analysis of selected urinary metabolites was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a standard pesticide and metabolite, which were detected by the qualitative profiling. The result of qualitative profiling showed that seven metabolites, i.e. an acetamiprid metabolite, N-desmethyl-acetamiprid; three Imidacloprid metabolites, 5-hydroxy-Imidacloprid, 4,5-dihydroxy-imidacloprid, 4,5-dehydro-Imidacloprid; a common metabolite of acetamiprid and Imidacloprid, N-(6-chloronicotinoyl)-glycine; and two clothianidin metabolites, N-desmethyl-clothianidin, N-(2-(methylsulfanyl)thiazole-5-carboxyl)-glycine, as well as acetamiprid, were detected in the urine of three cases. The result of the quantitative analysis showed N-desmethyl-acetamiprid was determined in the urine of one case, which had been collected on the first visit, at a concentration of 3.2 ng/mL. This is the first report on the qualitative and quantitative detection of N-desmethyl-acetamiprid in the human urine. The results suggest that the one case with detection of N-desmethyl-acetamiprid was exposed to acetamiprid through the consumption of contaminated foods. Urinary N-desmethyl-acetamiprid, as well as 5-hydroxy-Imidacloprid and N-desmethyl-clothianidin, may be a good biomarker for neonicotinoid exposure in humans and warrants further investigation.