An improved staining method of cell cycle analysis with Sybr Green I for fungi: Cryptococcus neoformans and Saccharomyces cerevisiae.

Cryptococcus neoformans is a pathogenic fungus which causes millions of deaths and infections, especially threatening immunocompromised individuals. During the development of new drugs, the ubiquitination has been found to play an important role in the regulation of the virulence and cell cycle of this fungus. Based on this mechanism, ubiquitination-related mutant strains exhibiting cell cycle arrest have been established for drug development for the fungus. However, flow cytometry detection of the cell cycle in fungi is generally difficult because the thick cell wall and capsule of fungi generally contribute to a nonspecific signal of cytometry. In this study, an improved method, derived from Saccharomyces cerevisiae assays, is developed to specifically stain C. neoformans, in whose cell cycle the G1 and G2 peaks are separated enough to be allowed for cell cycle analysis. As a result, the improved method facilitates the detection of the alterations in the cell cycle of C. neoformans with a mutation that results in cell cycle arrest, which distinctly delays the cell division of C. neoformans. Thus, the improved method reported here provides detailed technical information regarding assays on C. neoformans and, more importantly, offers a solution for assessing the cell cycle in other fungi in the future. Abbreviation: PI: propidium iodide.

Charge Retention/Charge Depletion in ESI-MS: Theoretical Rationale.

Gas phase modification in ESI-MS can significantly alter the charge state distribution of small peptides and proteins. The preceding paper presented a systematic experimental study on this topic using Substance P and proposed a charge retention/charge depletion mechanism, explaining different gas- and liquid-phase modifications [Thinius et al. J. Am. Soc. Mass Spec. 2020, 10.1021/jasms.9b00044]. In this work, we aim to support this rational by theoretical investigations on the proton transfer processes from (multiply) charged analytes toward solvent clusters. As model systems we use small (di)amines as analytes and methanol (MeOH) and acetonitrile (ACN) as gas phase modifiers. The calculations are supported by a set of experiments using (di)amines, to bridge the gap between the present model system and Substance P used in the preceding study. Upon calculation of the thermochemical stability as well as the proton transfer pathways, we find that both ACN and MeOH form stable adduct clusters at the protonation site. MeOH can form large clusters through a chain of H-bridges, eventually lowering the barriers for proton transfer to an extent that charge transfer from the analyte to the MeOH cluster becomes feasible. ACN, however, cannot form H-bridged structures due to its aprotic nature. Hence, the charge is retained at the original protonation site, i.e., the analyte. The investigation confirms the proposed charge retention/charge depletion model. Thus, adding aprotic solvent vapors to the gas phase of an ESI source more likely yields higher charge states than using protic compounds.

Determination of propineb and its metabolites propylenethiourea and propylenediamine in banana and soil using gas chromatography with flame photometric detection and LC-MS/MS analysis.

A sensitive and specific method for the determination of propineb and its metabolites, propylenethiourea (PTU) and propylenediamine (PDA), using gas chromatography with flame photometric detection (GC-FPD) and LC-MS/MS was developed and validated. Propineb and its metabolite residue dynamics in supervised field trials under Good Agricultural Practice (GAP) conditions in banana and soil were studied. Recovery of propineb (as CS2), PDA and PTU ranged from 75.3 to 115.4% with RSD (n = 5) of 1.3-11.1%. The limit of quantification (LOQ) of CS2, PDA and PTU ranged from 0.005 to 0.01 mg kg-1, and the limit of detection (LOD) ranged from 0.0015 to 0.0033 mg kg-1. Dissipation experiments showed that the half-life of propineb in banana and soil ranged from 4.4 to 13.3 days. PTU was found in banana with a half-life of 31.5-69.3 days, while levels of PDA were less than 0.01 mg kg-1 in banana and soil. It has been suggested that PTU is the major metabolite of propineb in banana. The method was demonstrated to be reliable and sensitive for the routine monitoring of propineb and its metabolites in banana and soil. It also serves as a reference for the detection and monitoring of dithiocarbamates (DTCs) residues and the evaluation of their metabolic pathway.

Electromembrane extraction of diamine plastic restricted substances in soft drinks followed by capillary electrophoresis with contactless conductivity detection.

Ethane-1,2-diamine (EA) and hexane-1,6-diamine (HA) are two important plastic restricted substances commonly existing in food contact materials. A capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) method has been developed for direct determination of above analytes, and the detection sensitivity has been significantly improved based on electromembrane extraction (EME). Under the optimum conditions, EA and HA could be well separated from their aliphatic diamine homologs as well as the common inorganic cations within 25min. The limits of detection could reach sub-ng/mL level, and good linearity (r>0.998) between peak area and analyte concentration could be obtained at three orders of magnitude. This EME/CE-C4D method provided a novel application for determining these plastic restricted substances in different bottled soft drinks, providing an alternative for the sensitive analyses of diamine substances.

Metabolic profile of 1,5-diaminopentane producing Corynebacterium glutamicum under scale-down conditions: Blueprint for robustness to bioreactor inhomogeneities.

Performance losses during scale-up are described since decades, but are still one of the major obstacles for industrial bioprocess development. Consequently, robustness to inhomogeneous cultivation environments is an important quality of industrial production organisms. Especially, Corynebacterium glutamicum was proven to have an outstanding resistance against rapid changes of oxygen and substrate availability as occurring in industrial scale bioreactors. This study focuses on the identification of metabolic key mechanisms for this robustness to get a deeper insight and provide future targets for process orientated strain development. A 1,5-diaminopentane producing C. glutamicum strain was cultivated in a two compartment scale-down device to create short-term environmental changes simulating industrial scale cultivation conditions. Using multi omics based methods, it is shown, that central metabolism is flexibly rearranged under short-term oxygen depletion and carbon source excess to overcome shortage in NAD+ recycling. In order to balance the redox state, key enzymes for the non-oxygen dependent fermentative NAD+ regeneration were significantly up-regulated while parts of non-essential pathways were down-regulated. The transfer of the cells back into the well aerated zones with low substrate concentration triggers an additional upregulation of genes for the re-assimilation of previously formed side products, showing L-lactate forming and utilizing reactions being active at the same time. Especially L-lactate as reversible and flexible external buffer for carbon and redox equivalents puts C. glutamicum in a robust position to deal with inhomogeneity in large scale processes. Biotechnol. Bioeng. 2017;114: 560-575. © 2016 Wiley Periodicals, Inc.

Preparation and characterization of magnetic molecularly imprinted polymers for the extraction of hexamethylenetetramine in milk samples.

Magnetic molecularly imprinted polymers (M-MIPs) were synthesized as the sorbents for extracting hexamethylenetetramine (HMT) from milk samples. Molecular simulations were used to calculate the interaction energies of the template monomers. The physical properties of M-MIPs were characterized. The adsorption isotherms and kinetics were investigated. Gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) was applied to determine the amount of HMT residue in milk samples. In the optimized method, a linear calibration curve was obtained using a matrix-matched standard in the range of 1.0-50.0µgL-1. The limit of detection (LOD) and limit of quantification (LOQ) was 0.3µgkg-1 and 1.0µgkg-1, respectively. The relative standard deviation (RSD) of the intra-day assay ranged from 2.6% to 5.2%, while that of the inter-day assay ranged from 3.6% to 11.5%. The recovery of HMT in milk samples ranged from 88.7% to 111.4%.

Mechanistic investigation of charge-remote and charge-driven fragmentation processes in 2,5-diphenyl-3,4-ethylenedioxythiophene diamidines.

RATIONALE: Diphenylfuran diamidines represent an important class of DNA minor groove binders of high therapeutic interest as antitumor and antibacterial agents. This study aimed to investigate fragmentation patterns in mass spectra of four diamidine derivatives with significant antitumor activity, in order to gain more insight into the structures and stability of their putative biological metabolites. METHODS: Compounds were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) using low-energy collision-induced dissociation (CID). Density functional theory calculations were performed to confirm the main fragmentation paths. RESULTS: The most abundant ion present in mass spectra is the doubly protonated molecule, whereas singly protonated molecules are present to a lesser extent. In the simplest compound, 2,5-bis(4-amidinophenyl)-3,4-ethylenedioxythiophene, the main fragmentation path was loss of ammonia, followed by loss of HCN where possible. The fragmentation of the N-alkyl derivatives (N-isopropyl-, N-isobutyl-, N-cyclopentyl-) includes competition between loss of alkene and the corresponding amine, followed by loss of another alkene and formation of fragment ions present in the pathway of the parent compound. CONCLUSIONS: The primary sites of fragmentations of investigated compounds are amidine groups, while breaking the core 3,4-ethylenedioxythiophene ring system does not take place. Fragmentation of the singly protonated molecule [M + H](+) occurs primarily on the charged side of the molecule, but a charge-remote process is energetically viable. The fragmentation mechanism of the alkyl derivatives revealed that singly and doubly protonated molecules cleave to the singly and doubly protonated molecules of the parent compound. Once formed, they are gradually transformed into nitrile. Copyright © 2016 John Wiley & Sons, Ltd.

Utilization of (18-crown-6)-2,3,11,12-tetracarboxylic acid as a chiral NMR solvating agent for diamines and ß-amino acids.

The compound (18-crown-6)-2,3,11,12-tetracarboxylic acid was evaluated as a chiral nuclear magnetic resonance (NMR) solvating agent for a series of diamines and bicyclic ß-amino acids. The amine must be protonated for strong association with the crown ether. An advantage of (18-crown-6)-2,3,11,12-tetracarboxylic acid over many other crown ethers is that it undergoes a neutralization reaction with neutral amines to form the protonated species needed for binding. Twelve primary diamines in neutral and protonated forms were evaluated. Diamines with aryl and aliphatic groups were examined. Some are atropisomers with equivalent amine groups. Others have two nonequivalent amine groups. Association equilibria for these systems are complex, given the potential formation of 2:1, 1:1, and 1:2 crown-amine complexes and given the various charged species in solution for mixtures of the crown ether with the neutral amine. The crown ether produced enantiomeric differentiation in the (1) H NMR spectrum of one or more resonances for every diamine substrate. Also, a series of five bicyclic ß-amino acids were examined and (18-crown-6)-2,3,11,12-tetracarboxylic acid caused enantiomeric differentiation in the (1) H NMR spectrum of three or more resonances of each compound.

Enzymatic assay to test diamines produced by vaginal bacteria.

An enzymatic assay was developed to determine the concentration of diamines (DA) in clinical samples of vaginal fluids. Putrescine and cadaverine are DA produced by anaerobic bacteria and are typically present in the vaginal fluids of women with an abnormal microbiota, as occurs in bacterial vaginosis. The vaginal DA (VADA) assay is based on the enzyme diamine oxidase which reacts with putrescine and cadaverine to produce H2O2 in a quantitative manner. H2O2 concentration is measured spectrophotometrically by a chromogenic reaction catalyzed by horseradish peroxidase. The VADA assay proved to be capable of detecting DA concentrations as low as 4 mM and showed a dose-response relationship which was linear over DA concentrations ranging from 4 to 256 mM. Using clinical samples it was possible to show that the VADA assay can be performed on human vaginal swabs and that the mean DA concentration is significantly higher in samples positive for microbial pathogens.

Visual determination of aliphatic diamines based on host-guest recognition of calix[4]arene derivatives capped gold nanoparticles.

Since amine compounds have been widespread pollutants in nature and they are extensively used in pharmaceutical industries and dye manufacturing, it is highly desirable to develop simple, effective and naked-eye available analytical methods for such aliphatic diamines determination. Calixarenes as macrocycles have drawn intensive interests for fields such as biomedicine, supramolecular chemistry and smart materials. Here, instead of the normal complicated modification strategy, a facile and efficient method for one-pot synthesis of calix[4]arene crown ether (CCE4) capped gold nanoparticles (AuNPs) is proposed. The as-prepared CCE4-AuNPs are not only high water dispersity and stability even after storage for 3 months, but also capable of host-guest recognition of diamines in aqueous systems. Size-selective encapsulation of amine group between CCE4 and diamines carry out the aggregation of CCE4-AuNPs. The determination of diamines such as hexamethylenediamine or spermine can be realized by the UV-vis absorbance change and visual color difference.

A convenient fluorescent method to simultaneously determine the enantiomeric composition and concentration of functional chiral amines.

A 1,1'-bi-2-naphthol (BINOL)-based chiral aldehyde in combination with Zn(II) shows a highly enantioselective fluorescent response toward functional chiral amines at λ>500 nm. However, the combination of salicylaldehyde and Zn(II) gives the same fluorescent enhancement for both enantiomers of a functional chiral amine at λ=447 nm. By using the fluorescent responses of the combination of the BINOL-based chiral aldehyde, salicylaldehyde and Zn(II) at the two emission wavelengths, both the concentration and enantiomeric composition of functional chiral amines such as amino alcohols, diamines, and amino acids can be simultaneously determined by a single fluorescent measurement. This work provides a simple and convenient method for chiral assay.

Probing the surface calcium binding sites of melanosomes using molecular rulers.

Melanosomes have the capacity to bind significant concentrations of calcium, suggesting there are surface binding sites that enable cations to access the interior of fully pigmented melanosomes. The surface of melanosomes is known to contain significant concentrations of carboxylate groups which likely are the initial biding sites for calcium, but their arrangement on the surface of the melanosome is not known. In various calcium proteins, a bidentate coordination by two carboxylate groups is the most common structure. In this study, we determine the distance between neighboring surface carboxylic acid groups by examining the binding of a series of diamines (+)H3N(CH2)mNH3(+) (m = 1-5) to melanosomes isolated from the ink sacs of Sepia officinalis and bovine choroid tissue. Of these amines, ethylenediamine (m = 2) shows optimal bidentate binding, revealing a narrow distribution of distances between neighboring carboxylic acid groups, ∼480 pm, similar to that found in proteins for calcium binding motifs involving two carboxylate groups.

The interaction of diamines and polyamines with the peroxidase-catalyzed metabolism of aromatic amines: a potential mechanism for the modulation of aniline toxicity.

Synthetic and biological amines such as ethylenediamine (EDA), spermine, and spermidine have not been previously investigated in free-radical biochemical systems involving aniline-based drugs or xenobiotics. We aimed to study the influence of polyamines in the modulation of aromatic amine radical metabolites in peroxidase-mediated free radical reactions. The aniline compounds tested caused a relatively low oxidation rate of glutathione in the presence of horseradish peroxidase (HRP), and H2O2; however, they demonstrated marked oxygen consumption when a polyamine molecule was present. Next, we characterized the free-radical products generated by these reactions using spin-trapping and electron paramagnetic resonance (EPR) spectrometry. Primary and secondary but not tertiary polyamines dose-dependently enhanced the N-centered radicals of different aniline compounds catalyzed by either HRP or myeloperoxidase, which we believe occurred via charge transfer intermediates and subsequent stabilization of aniline-derived radical species as suggested by isotopically labeled aniline. Aniline/peroxidase reaction product(s) were monitored at 435 nm by kinetic spectrophotometry in the presence and absence of a polyamine additive. Using gas chromatography-mass spectrometry, the dimerziation product of aniline, azobenzene, was significantly amplified when EDA was present. In conclusion, di- and poly-amines are capable of enhancing the formation of aromatic-amine-derived free radicals, a fact that is expected to have toxicological consequences.

Buffer gas modifiers effect resolution in ion mobility spectrometry through selective ion-molecule clustering reactions.

RATIONALE: When polar molecules (modifiers) are introduced into the buffer gas of an ion mobility spectrometer, most ion mobilities decrease due to the formation of ion-modifier clusters. METHODS: We used ethyl lactate, nitrobenzene, 2-butanol, and tetrahydrofuran-2-carbonitrile as buffer gas modifiers and electrospray ionization ion mobility spectrometry (IMS) coupled to quadrupole mass spectrometry. Ethyl lactate, nitrobenzene, and tetrahydrofuran-2-carbonitrile had not been tested as buffer gas modifiers and 2-butanol had not been used with basic amino acids. RESULTS: The ion mobilities of several diamines (arginine, histidine, lysine, and atenolol) were not affected or only slightly reduced when these modifiers were introduced into the buffer gas (3.4% average reduction in an analyte's mobility for the three modifiers). Intramolecular bridges caused limited change in the ion mobilities of diamines when modifiers were added to the buffer gas; these bridges hindered the attachment of modifier molecules to the positive charge of ions and delocalized the charge, which deterred clustering. There was also a tendency towards large changes in ion mobility when the mass of the analyte decreased; ethanolamine, the smallest compound tested, had the largest reduction in ion mobility with the introduction of modifiers into the buffer gas (61%). These differences in mobilities, together with the lack of shift in bridge-forming ions, were used to separate ions that overlapped in IMS, such as isoleucine and lysine, and arginine and phenylalanine, and made possible the prediction of separation or not of overlapping ions. CONCLUSIONS: The introduction of modifiers into the buffer gas in IMS can selectively alter the mobilities of analytes to aid in compound identification and/or enable the separation of overlapping analyte peaks.

Determination of N-methyl-1,3-propanediamine in bovine muscle by liquid chromatography with triple quadrupole and ion trap tandem mass spectrometry detection.

Morantel, pyrantel and their drug-related metabolites in food of animal-origin are regulated as sum of residues which may be hydrolysed to N-methyl-1,3-propanediamine (NMPA). In this study, an isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method with pentafluoropropionic acid anhydride (PFPA) derivatization was developed for the determination of NMPA in bovine muscle. A stable isotope labeled internal standard N-methyl-d(3)-3,3'-d(2)-propane-1,3-diamine (NMPA-d(5)) was synthesized as internal standard. NMPA was derivatized with PFPA to form an N,N'-bis (pentafluoroacyl) derivative (NMPA-PFPA) and analyzed by liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS/MS) and liquid chromatography ion trap mass spectrometry (LC-IT-MS/MS) using negative ion electrospray ionization (ESI). Chromatographic behavior of several perfluorocarboxylic acid anhydride derivatives of NMPA and other structurally related diamines on C-18 and perfluorophenyl (PFP) columns was studied. Conversion of the parent drugs to NMPA under various hydrolysis conditions was evaluated. In addition, comparison of the matrix effect and linearity with isotopically labeled internal standard (I.S.) and analogous I.S. were performed and investigated. The method was validated using fortified bovine muscle samples. The apparent recovery (obtained after correction with an isotopically labeled I.S.) was between 89% and 97% and repeatability was less than 10%. The lowest LOD and LOQ (0.42 and 1.39µg/kg, respectively) were obtained with LC-QqQ-MS/MS.

Study of the naphthalene-2,3-dicarboxaldehyde pre-column derivatization of biogenic mono- and diamines in mixture and fluorescence-HPLC determination.

A new fluorescence-HPLC method was developed for the simultaneous determination of eight biogenic monoamines (histamine, methylamine, tyramine, ethylamine, propylamine, tryptamine, 2-phenylethylamine, isoamylamine) and two biogenic diamines (putrescine, cadaverine) in the presence of heptylamine as the internal standard. The amines were pre-column derivatized with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide ion as the nucleophile. The effect of the derivatization reaction conditions on the reaction yield was investigated. The derivatives were separated on an Inertsil ODS-3 column (250 × 4mm i.d., 5 µm) using gradient elution and detected fluorimetrically at excitation and emission wavelengths of 424 and 494 nm, respectively. Limits of detection between 0.002 and 0.4 ng, injected on-column (10-µL loop), were achieved. The within- and between-day relative standard deviations ranged between 0.2-3.4% and 0.3-4.8%, respectively. The utility of the method in assaying biogenic mono- and diamine mixtures in Greek cheeses is demonstrated. Ultrasound-assisted liquid-liquid extraction was applied prior to derivatization.

[Methodical aspects of hygienic safety assessment of polymeric materials in contact with foodstuffs].

It was purposed new technique by capillary gas chromatography (GC) for the low level determination of monomer hexamethylenediamine (HMDA) in food simulants water from polymeric materials in contact with foodstuffs. Hexamethylenediamine, HN2-(CH2)6-NH2, is a monomer used in the manufacture of certain of polyamide plastic materials and articles intended to come into contact with foodstuffs. Compound exhibits all the chemical properties of aliphatic amines, is an irritant, causing dermatitis, can accumulate in the body, the degree of human exposure to HMDA assigned to the 2nd class of hazard - the substance is highly dangerous. There was studied two methodological approaches pre-derivatization of compound for GC determination. The first approach involves conversion of the free diamine using ethyl chloroformate as derivatizing agent followed by analysis of the resulting diurethan by gas chromatography using a flame ionization and mass selective detection (HMDA was quantitated by selective ion monitoring at m/z 102, the lower detection limit of 1 ng). According to second methodological approach the water samples were mixed with sodium chloride and extracted with toluene, then derivatized with trifluoroacetic anhydride (60 min, 55 degrees C) to diamide, 1 M potassium phosphate buffer (pH 7,0) was add to remove excess derivatizing agent, followed by analysis of resulting diamide by gas chromatography with electron capture detection (lower limit value 0,01 ng). Conformation of HMDA levels is carried out by combined gas chromatography/mass spectrometry (HMDA was quantitated by selective ion monitoring at m/z 126, the lower limit value of 0,1 ng). The optimal pre-derivatization of the second approach for the determination of low levels of HMDA in the water extracts. The range of measured concentrations of 0,005-0,5 mg/dm3, recovery 88-101%, the total error of measurement is 16%, the relative standard deviation is 1,85%. The method was tested in the study of aqueous extracts of the 10 random samples intended for food purchased in the consumer market. Shows the corresponding output level hexamethylenediamine requirements for products of this type.

Divergent regioselective synthesis of 2,5,6,7-tetrahydro-1H-1,4-diazepin-2-ones and 5H-1,4-benzodiazepines.

A novel and simple one-pot synthesis of 3-substituted 2,5,6,7-tetrahydro-1H-1,4-diazepin-2-ones from 1,2-diaza-1,3-dienes (DDs) and N-unsubstituted aliphatic 1,3-diamines is described. Here we also report a procedure to selectively obtain alkyl 5H-1,4-benzodiazepine-3-carboxylates from the DDs and 2-aminobenzylamine. Both processes occur by means of sequential 1,4-conjugated addition followed by regioselective 7-exo cyclization. The behavior of N-methyl- and N,N'-dimethyl-1,3-diaminopropanes toward the DDs furnished pyrazol-3-ones and bis-α-aminohydrazones, respectively.

[Migration of monomers and primary aromatic amines from nylon products].

Migration of 2 kinds of monomer and 21 kinds of primary aromatic amines (PAAs) from 21 kinds of nylon products such as turners, ladles and wrap film were determined. Samples were classified as regards materials by mean of pyrolysis-GC/MS. One sample was classified as nylon 6, 15 samples as nylon 66 and three samples as nylon 6/66 copolymers, while two samples were laminate of nylon 6 with polyethylene or polypropylene. All of the nylon 66 samples contained a small amount of ε-caprolactam (CPL), which is the nylon 6 monomer. Migration levels of monomers and PAAs at 60°C for 30 min into 20% ethanol were measured by LC/MS/MS. CPL was detected at the level of 0.015-38 µg/mL from all samples, excluding one wrap film sample, and 1,6-hexamethylenediamine was detected at the level of 0.002-0.013 µg/mL from all nylon 66 samples and one nylon 6/66 sample. In addition, 0.006-4.3 µg/mL of 4,4'-diaminodiphenylmethane from three samples, 0.032-0.23 µg/mL of aniline from four samples, 0.001 µg/mL of 4-chloroaniline from two samples, and 0.002 µg/mL of 2-toluidine and 0.066 mg/mL of 1-naphthylamine from one sample each were detected. The migration levels at 95 or 121°C were about 3 and 10 times the 60°C levels, respectively.

Urine 1,6-hexamethylene diamine (HDA) levels among workers exposed to 1,6-hexamethylene diisocyanate (HDI).

Urinary 1,6-hexamethylene diamine (HDA) may serve as a biomarker for systemic exposure to 1,6-hexamethylene diisocyanate (HDI) in occupationally exposed populations. However, the quantitative relationships between dermal and inhalation exposure to HDI and urine HDA levels have not been established. We measured acid-hydrolyzed urine HDA levels along with dermal and breathing-zone levels of HDI in 48 automotive spray painters. These measurements were conducted over the course of an entire workday for up to three separate workdays that were spaced approximately 1 month apart. One urine sample was collected before the start of work with HDI-containing paints and subsequent samples were collected during the workday. HDA levels varied throughout the day and ranged from nondetectable to 65.9 microg l(-1) with a geometric mean and geometric standard deviation of 0.10 microg l(-1) +/- 6.68. Dermal exposure and inhalation exposure levels, adjusted for the type of respirator worn, were both significant predictors of urine HDA levels in the linear mixed models. Creatinine was a significant covariate when used as an independent variable along with dermal and respirator-adjusted inhalation exposure. Consequently, exposure assessment models must account for the water content of a urine sample. These findings indicate that HDA exhibits a biphasic elimination pattern, with a half-life of 2.9 h for the fast elimination phase. Our results also indicate that urine HDA level is significantly associated with systemic HDI exposure through both the skin and the lungs. We conclude that urinary HDA may be used as a biomarker of exposure to HDI, but biological monitoring should be tailored to reliably capture the intermittent exposure pattern typical in this industry.