Development of a UPLC-ESI-MS/MS method to measure urinary metabolites of selected VOCs: Benzene, cyanide, furfural, furfuryl alcohol, 5-hydroxymethylfurfural, and N-methyl-2-pyrrolidone.

We report on the development of an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneously measuring eight biomarkers of volatile organic compound (VOC) exposure, with potential application to e-cigarette aerosol biomonitoring. Phenylmercapturic acid (PMA) and trans, trans-muconic acid (tt-MA) are metabolites of benzene; 2-aminothiazoline-4-carboxylic acid (ATCA) is a metabolite of cyanide; N-2-furoylglycine (N2FG) is a metabolite of furfural and furfuryl alcohol; 5-hydroxymethylfuroic acid (HMFA), 5-hydroxymethyl-2-furoylglycine (HMFG), and 2,5-furandicarboxylic acid (FDCA) are metabolites of 5-hydroxymethylfurfural; and 5-hydroxy-N-methylpyrrolidone (5HMP) is a metabolite of N-methyl-2-pyrrolidone. A pentafluorophenyl-modified silica column was used for chromatographic separation. The overall run time for the method is about 6 min per sample injection. The method has low to sub-nanograms per milliliter sensitivity, linearity over 3 orders of magnitude, and precision and accuracy within 15%. The method was used to measure human urine samples. Results showed that people with known benzene exposure (daily cigarette smokers) had higher levels of tt-MA and PMA compared with non-smokers. The method is advantageous for high-throughput analysis of selected VOC metabolites in large-scale, population-based studies such as the National Health and Nutrition Examination Survey (NHANES). Quantifying these urinary biomarkers is important to public health efforts to understand human exposure to VOCs from various sources, including tobacco products and electronic nicotine delivery systems.

Relationship between HMF intake and SMF formation in vivo: An animal and human study.

SCOPE: 5-Hydroxymethylfurfural (HMF) is a furanic compound produced in heat-processed foods by nonenzymatic browning reactions. HMF has been demonstrated to be hepato- and nephrotoxic in animals with a link to its metabolite 5-sulfooxymethylfurfural (SMF). To date little is known about either the formation of SMF from ingested HMF or the formation of DNA adducts in animals or human beings. METHODS AND RESULTS: To assess SMF in vivo formation, we first performed a study in mice treated with high/low doses of oral HMF. We found increased concentrations of SMF in plasma and DNA SMF-adducts in leukocytes, hepatic tissue, and kidneys by means of LC-MS/MS, but no spatial formation in such tissues was observed by MALDI-MS imaging technology due to low sensitivity. In a second experiment, we measured the exposure to HMF in a Spanish preadolescent population. We analyzed the concentration of HMF metabolites (plasma, urine) and measured, for the first time, the presence of SMF in plasma and DNA SMF-adducts in leukocytes. CONCLUSION: This study provides the first evidence that oral HMF is readily transformed into SMF in vivo, giving rise to the formation of DNA adducts in a direct relation with HMF intake, both in animals and human beings.

Determination of metabolites of 5-hydroxymethylfurfural in human urine after oral application.

5-Hydroxymethylfurfural (5-HMF) is a natural occurring substance taken up by everyday food. In former studies it was shown that 5-HMF is completely decomposed in the body after oral or intravenous application resulting in three main metabolites named 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid, and N-(hydroxymethyl)furoyl glycine, and possibly a forth metabolic substance, termed 5-sulphoxymethylfurfural, is formed. Determination is possible via HPLC using a hydrophilic interaction chromatography (HILIC) column with an appropriate gradient system (ACN/ammonium formate 100 mM, pH 2.35). Urine samples were purified by use of an SPE method beforehand working with ScreenA cartridges. This cleaning procedure was validated based on ICH guidelines in terms of linearity, quantification, and detection limit, as well as precision, repeatability, and accuracy. Analysis of real-life samples coming from two healthy probands and one cancer patient, who all received 240 mg 5-hydroxymethylfurfural orally once a day, showed dicarboxylic acid and the glycine conjugate in their urine samples. Recovery of the initial compound in form of transformed metabolites was up to 90% within 48 h. Potentially toxic 5-sulphoxymethylfurfural could not be found.

Determination of 5-hydroxymethylfurfural using derivatization combined with polymer monolith microextraction by high-performance liquid chromatography.

A simple and sensitive method for the determination of 5-hydroxymethylfurfural (HMF) in coffee, honey, beer, Coke, and urine by high-performance liquid chromatography (HPLC) is presented. This method is based on the formation of the 2,4-dinitrophenylhydrazone of HMF and subsequent polymer monolith microextraction (PMME) of this derivative. A poly(methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-co-EGDMA) monolithic capillary column was selected as the extraction medium. Several parameters affecting the derivatization of HMF with 2,4-dinitrophenylhydrazine (DNPH) followed by extraction of the derivative were optimized. The procedure is simple and offers high sensitivity and specificity since the derivative of HMF is well preconcentrated by PMME with poly(MAA-co-EGDMA) monolith and well separated from the other components of the samples under examination. The recoveries in coffee, honey, beer, Coke, and urine samples were in the range of 83.9-110.8% spiked at different levels with HMF. The inter- and intraday precisions were less than 10%. The LOD (S/N = 3) and LOQ (S/N = 10) for HMF were 1.0 ng/mL and 3.4 ng/mL, respectively.

Dietary exposure to 5-hydroxymethylfurfural from Norwegian food and correlations with urine metabolites of short-term exposure.

5-Hydroxymethylfurfural (HMF) is formed in carbohydrate-rich food during acid-catalysed dehydration and in the Maillard reaction from reducing sugars. HMF is found in mg quantities per kg in various foods. HMF is mainly metabolised to 5-hydroxymethyl-2-furoic acid (HMFA), but unknown quantities of the mutagenic 5-sulphoxymethylfurfural (SMF) may also be formed, making HMF potentially hazardous to humans. We determined the HMF content in Norwegian food items and estimated the dietary intake of HMF in 53 volunteers by means of 24h dietary recall. The estimated intakes of HMF were correlated with urinary excretion of HMFA. Coffee, prunes, dark beer, canned peaches and raisins had the highest levels of HMF. The 95th percentile of the estimated daily dietary intake of HMF and the 24h urinary excretion of HMFA were 27.6 and 28.6mg, respectively. Coffee, dried fruit, honey and alcohol were identified as independent determinants of urinary HMFA excretion. Most participants had lower estimated HMF intake than the amount of HMFA excreted in urine. In spite of this there was a significant correlation (r=0.57, P<0.001) between the estimated HMF intake and urinary HMFA. Further studies are needed to reveal alternative sources for HMF exposure.

Analysis of 5-hydroxymethylfurfual in coffee, dried fruits and urine.

5-Hydroxymethylfurfural has become a substance of interest since recent results showed a possible carcinogenic potential in consequence of a metabolic activation by sulfotransferases. 5-Hydroxymethylfurfural is formed either by acid catalysed degradation of reducing sugars or via the Maillard reaction. This work provides an overview of foods potentially containing high amounts of 5-hydroxymethylfurfural. It comprises dried fruits with a high sugar content that were exposed to heat for a long time. The concentration ranges from very low in, e. g. figs (1 mg/kg) to plums that contained up to 2,200 mg/kg. Several types of roasted coffee were analysed that contained from 300 to 2,900 mg/kg of 5-hydroxymethylfurfural. In a small human study with seven healthy volunteers the urine excretion of unmetabolised 5-hydroxymethylfurfural was investigated. After uptake of 20 g of plum jam containing 24 mg of 5-hydroxymethylfurfural, 163 microg (mean) were excreted within 6 h, an equivalent of 0.75% of the ingested 5-hydroxymethylfurfural.

Identification and urinary excretion of metabolites of 5-(hydroxymethyl)-2-furfural in human subjects following consumption of dried plums or dried plum juice.

5-(Hydroxymethyl)-2-furfural (I) is a major breakdown product occurring in solutions with high concentrations of fructose and glucose and is present in many fruit juices, in heat-sterilized parenteral solutions, and in baby cereals. The objective of this study was to characterize and identify 5-(hydroxymethyl)-2-furfural metabolites in human subjects following the consumption of dried plum juice and/or dried plums. Subjects were fasted overnight and blood and urine samples were obtained during the day following consumption. Subjects fed the dried plum juice and dried plums consumed 3944 micromol (497 mg) and 531 micromol (67 mg) of I, respectively. Four presumed metabolites of I were detected in the urine of subjects that consumed dried plum juice. They were tentatively identified using HPLC-MS/MS as (1) N-(5-hydroxymethyl-2-furoyl)glycine (III), (2) 5-hydroxymethyl-2-furoic acid (II), (3) (5-carboxylic acid-2-furoyl)glycine (IV), and (4) (5-carboxylic acid-2-furoyl)aminomethane (V). Total urinary excretion during the 6 h following the consumption of dried plum juice was 168, 1465, 137, and 75 micromoles on the basis of II as a standard for II, III, IV, and V, respectively. The estimated total recovery of I metabolites was 46.2% and 14.2% of the I dose during the first 6 h after consumption of dried plum juice and dried plums, respectively. I seems to be metabolized rapidly to glycine conjugates and other metabolites and excreted in the urine.

Metabolism and excretion of [14C]furfural in the rat and mouse.

The fate of furfural (2-furancarboxaldehyde) was investigated in male and female Fischer 344 (F344) rats given single oral doses of 1, 10 and 60 mg/kg and male and female CD1 mice given 1, 20 and 200 mg/kg [carbonyl-14C]furfural. There was a very high recovery (more than 90% of dose) of radioactivity in all dose groups in 72 hr. The major route of elimination was by the urine, with much smaller amounts present in the faeces and exhaled as 14CO2. The residue in the carcass after 72 hr was less than 1% of the administered dose. Furoylglycine and furanacryloylglycine were identified as the major urinary metabolites by high-performance thin-layer chromatography, radio-HPLC, gas chromatography-mass spectrometry and 1H-nuclear magnetic resonance spectroscopy, by comparison with synthetic reference compounds. There were only subtle differences in the metabolic profile as a function of dose size, sex and species. An additional minor polar metabolite was excreted by male rats and mice, and the parent acids of the glycine conjugates were excreted at the higher doses. The results are discussed in terms of the participation of xenobiotics in the chain elongation reactions of fatty acid biosynthesis.

Comparative metabolism and disposition of furfural and furfuryl alcohol in rats.

The comparative metabolism and disposition of furfural (FAL) and furfuryl alcohol (FOL) were investigated following oral administration of approximately 0.001, 0.01, and 0.1 of the LD50, corresponding to approximately 0.127, 1.15, and 12.5 mg/kg for FAL and 0.275, 2.75, and 27.5 mg/kg for FOL. At all doses studied, at least 86-89% of the dose of FAL or FOL was absorbed from the gastrointestinal tract. FAL and FOL were extensively metabolized prior to excretion. The major route of excretion was in urine, where 83-88% of the dose was excreted, whereas 2-4% was excreted in the feces. Approximately 7% of the dose from rats treated with FAL at 12.5 mg/kg was exhaled as 14CO2. At 72 hr following administration, the pattern of tissue distribution of radioactivity was similar for both FAL and FOL. Liver and kidney contained the highest, and brain the lowest concentrations of radioactivity. Generally, the concentrations of radioactivity in tissues were proportional to the dose. Almost all of the urinary radioactivity was tentatively identified. No FAL or FOL was detected in urine. Furoylglycine was the major urinary metabolite (73-80% of dose), and furoic acid (1-6%) and furanacrylic acid (3-8%) were the minor metabolites following treatment with either FAL or FOL. Therefore, the initial step in the metabolism of FAL and FOL involves the oxidation to furoic acid, which is excreted unchanged and decarboxylated to form 14CO2, conjugated with glycine, or condensed with acetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of 5-methyl-2-furaldehyde in rat. III. Identification and determination of 5-methyl-2-furylmethylketone.

1. Continuous extraction, column chromatography and t.l.c. were employed to isolate a minor metabolite of 5-methyl-2-furaldehyde from rat urine. 2. The metabolite was identified by mass spectrometry and independent synthesis as 5-methyl-2-furylmethylketone. 3. A method for quantitative determination of the metabolite in urine was devised. About 7% of the parent compound was metabolized to 5-methyl-2-furylmethylketone.

Non-enzymatic glycosylation of urinary proteins in type 1 (insulin-dependent) diabetes: correlation with metabolic control and the degree of proteinuria.

In 18 control subjects and in 41 Type 1 (insulin-dependent) diabetic patients (13 with normal proteinuria, group A; 15 with microproteinuria, group B; and 13 with clinical proteinuria, group C), mean blood glucose, glycosylated haemoglobin and non-enzymatic glycosylated serum and urinary proteins, expressed as 5-hydroxymethylfurfural (5-HMF), were measured. In each group of diabetic patients, the levels of mean daily blood glucose, glycosylated haemoglobin and serum 5-HMF/mg protein were higher than in the control subjects. The urinary 5-HMF/mg proteinuria and the urinary/serum 5-HMF concentration ratio values were raised in group A and reduced in groups B and C. Moreover, they showed a negative correlation with 24-h urinary protein excretion in the control subjects and in each group of diabetic patients. The urinary 5-HMF/day in groups A, B and C was greater than in the control subjects. The urinary 5-HMF/day did not correlate with the mean daily blood glucose levels and, only in group A, did it correlate with serum 5-HMF and glycosylated haemoglobin. This suggests that, in this group, functional factors result in the increased renal elimination of 5-HMF and, therefore, of non-enzymatically glycosylated proteins. However, in the other groups of patients, this elimination depends on the degree of proteinuria.