N-Methylformamide and N-methylacetamide in urine

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1 536 N-Methylformamide and N-methylacetamide in urine Matrix: Urine Hazardous substances: N,N-Dimethylformamide and N,N-dimethylacetamide Analytical principle: Gas chromatography with mass selective detection (GC MS) Completed in: May 2010 Overview of the parameters that can be determined with this method and the corresponding hazardous substances: Hazardous substances CAS Parameters CAS N,N-Dimethylformamide (DMF) N,N-Dimethylacetamide (DMA) N-Methylformamide (NMF) N-Hydroxymethyl-Nmethylformamide (HMMF) N-Methylacetamide (NMA) N-Hydroxymethyl-Nmethylacetamide (HMMA) Summary The present method allows for the determination of the main metabolites of N,Ndimethylformamide and N,N-dimethylacetamide in urine: HMMF, NMF, HMMA and NMA, respectively. HMMF and HMMA readily decompose thermally to form N-methylformamide (NMF) or N-methylacetamide (NMA) and formaldehyde. Prior to sample injection, thermolysis is carried out for 2 hours at 120 C in order to transform the metabolites completely into the target analytes NMF and NMA. The urine samples are then diluted with ethanol and analyzed using GC MS. Thus, the sum of HMMF and NMF (DMF metabolites) and the sum of HMMA and NMA (DMA metabolites), respectively, are detected by this procedure. The MAK Collection for Occupational Health and Safety 2016, Vol 1, No 1 DOI: / bi6812e2115b

2 N-Methylformamide and N-Methylacetamide in urine 537 Reliability data of the method N-Methylformamide (NMF) and N-hydroxymethyl-N-methylformamide (HMMF) Within day precision: Standard deviation (rel.) s w = 6.3%, 2.7% and 1.5% Prognostic range u = 14.2%, 6.0% and 3.4% at a spiked concentration of 1.0, 10 and 100 mg NMF per litre urine and where n = 10 determinations Day to day precision: Standard deviation (rel.) s w = 7.2%, 2.9% and 3.1% Prognostic range u = 16.3%, 6.5% and 6.9% at a spiked concentration of 1.0, 10 and 100 mg NMF per litre urine and where n = 10 determinations Accuracy: Recovery rate (rel.) r = 97% and 101% at a nominal concentration of 27 mg NMF and 21.6 mg HMMF per litre urine and where n = 3 determinations Detection limit: 0.1 mg NMF per litre urine Quantitation limit: 0.3 mg NMF per litre urine N-Methylacetamide (NMA) and N-hydroxymethyl-N-methylacetamide (HMMA) Within day precision: Standard deviation (rel.) s w = 3.7%, 4.3% and 2.0% Prognostic range u = 8.4%, 9.8% and 4.5% at a spiked concentration of 1.0, 10 and 100 mg NMA per litre urine and where n = 10 determinations Day to day precision: Standard deviation (rel.) s w = 8.3%, 4.3% and 2.1% Prognostic range u = 18.8%, 9.7% and 4.7% at a spiked concentration of 1.0, 10 and 100 mg NMA per litre urine and where n = 10 determinations Accuracy: Recovery rate (rel.) r = 97% at a nominal concentration of 26 mg NMA per litre urine and where n = 3 determinations Detection limit: 0.1 mg NMA per litre urine Quantitation limit: 0.3 mg NMA per litre urine General information on N,N-dimethylformamide and N,N-dimethylacetamide N,N-Dimethylformamide and N,N-dimethylacetamide are aprotic bipolar solvents for many applications. They are completely miscible with water and most organic solvents. In industry, DMF and DMA are used predominantly in the production and processing of polymers and serve as solvents for polyacrylonitrile and other

3 538 Biomonitoring Methods polymers. In addition, both substances are used as catalysts, paint strippers and extraction agents [IARC 1999, Drexler and Greim 2010]. The annual production volume of DMF and DMA amounts to more than 50,000 tons. The compounds are thus classified as HPV (High Production Volume) chemicals [IARC 1999, OECD SIDS 2001]. Occupational exposure to DMF and DMA occurs mainly when the compounds are used as solvents in the polymer production and processing (open production processes). DMF and DMA itself are mostly produced in closed systems [IARC 1999, OECD SIDS 2001]. Fig. 1 Simplified metabolism/reaction scheme illustrating the formation of NMF and NMA from HMMF and HMMA, respectively. At work, DMF and DMA are readily absorbed both by inhalation and through the skin, and they are rapidly distributed in the body [DFG 2014, Greim 1998]. The metabolism of the two substances is similar: Initially, enzymatic oxidation by cytochrome P450 monooxygenases takes place. In this process, one of the N-methyl groups is hydroxylated. The first urinary metabolites are N-hydroxymethyl-Nmethylformamide and N-hydroxymethyl-N-methylacetamide. The two compounds are readily demethylated to N-methylformamide and N-methylacetamide, respectively (Figure 1). Further oxidation can lead to the formation of formamide and acetamide, respectively. In addition, methyl isocyanate has been postulated as a reactive intermediate following DMF exposure. The mercapturic acid AMCC (Nacetyl-S-(N-methylcarbamoyl)cysteine) is another urinary metabolite, formed by the reaction of the methyl isocyanate with glutathione. Furthermore, methyl isocyanate can react with hemoglobin to yield the adduct N-methylcarbamoylvaline [Drexler and Greim 2010, Drexler and Greim 2007]. After exposure to DMF, up to 50% of the dose is excreted as HMMF and NMF within 72 hours in the urine. The elimination half-life depends on the route of absorption and amounts to 2 3 hours after inhalative and to 4 6 hours after dermal absorption [Drexler and Greim 2007]. The MAK Collection for Occupational Health and Safety 2016, Vol 1, No 1

4 N-Methylformamide and N-Methylacetamide in urine 539 In the case of DMA, almost 70% of the exposure dose is renally eliminated in the form of the main metabolites HMMA and NMA. The biological half-life is about 16 hours; no influence of the route of absorption has been reported [Drexler and Greim 2010, Greim 1998]. Detailed information on the toxicological properties of DMF and DMA is published in the MAK documentations from 1998 (DMA) and 2010 (DMF) [Greim 1998, Hartwig 2010b]. The MAK value for DMF was established at 5 ml/m 3 or 15 mg/m 3. For DMA, a MAK value of 10 ml/m 3 or 36 mg/m 3 was derived [DFG 2014]. An overview of the toxicological classifications of the two substances is given in Table 1. As DMF and DMA penetrate the skin, the substances have additionally been designated with an H. The dermal absorption of the two substances contributes substantially to the exposure at work. Therefore, monitoring of the internal exposure (BAT value) is essential for exposure analysis and assessment [Drexler and Greim 2010, DFG 2014]. In 2006, the BAT value for DMF was set at 35 mg NMF plus HMMF per litre urine. The samples should be taken at the end of exposure or at the end of shift. The BAT value documentation from 2007 points explicitly to the fact that, for correct determination, the complete sum of NMF and HMMF needs to be quantified [Drexler and Greim 2007]. This should also be noted, when monitoring the BAT value for DMA, which has been set at 30 mg NMA plus HMMA per gram creatinine [Drexler and Greim 2010]. This analytical procedure focuses therefore on a complete thermal decomposition of HMMF and HMMA to NMF and NMA, respectively. In the analytical methods published to date, this issue has not been explicitly examined, assuming that thermolysis in the inlet liner of the gas chromatograph is quantitative. During the development of the method described here an interlaboratory comparison was carried out (see Section 9.5), showing that this assumption does not hold for all analytical implementations. Author: W. Will Examiner: M. Bader Table 1 Classifications of DMF and DMA by the Commission [DFG 2014]. Hazardous substances N,N-Dimethyl formamide N,N-Dimethyl acetamide MAK value 5 ml/m 3 (15 mg/m 3 ) 10 ml/m 3 (36 mg/m 3 ) H; S designation Carcinogen category Pregnancy risk group BAT value H B 35 mg/l (NMF plus HMMF) H C 30 mg/g creatinine (NMA plus HMMA) H: danger of percutaneous absorption S: danger of sensitization B, C: see List of MAK and BAT Values [DFG 2014]

5 540 Biomonitoring Methods N-Methylformamide and N-methylacetamide in urine Matrix: Urine Hazardous substances: N,N-Dimethylformamide and N,N-dimethylacetamide Analytical principle: Gas chromatography with mass selective detection (GC MS) Completed in: May 2010 Contents 1 General principles Equipment, chemicals and solutions Equipment Chemicals Calibration standards Specimen collection and sample preparation Specimen collection Sample preparation Operational parameters Gas chromatography Mass spectrometry Analytical determination Calibration Calculation of the analytical result Standardization and quality control Evaluation of the method Within day precision Day to day precision Accuracy Detection and quantitation limits Interlaboratory comparison Sources of error Discussion of the method 550 The MAK Collection for Occupational Health and Safety 2016, Vol 1, No 1

6 N-Methylformamide and N-Methylacetamide in urine References Appendix General principles The analytical procedure described here is a combination and further development of the DMF and DMA biomonitoring methods already published in this series [Will et al. 1996, Knecht et al. 2003]. Unlike the procedures described so far, the release of formaldehyde from the main metabolites N-hydroxymethyl-N-methylformamide and N-hydroxymethyl-N-methylacetamide does not take place in the inlet liner of the gas chromatograph, but in a preceding thermolysis step. The urine sample pretreated this way is then diluted with ethanol and is injected splitless into the GC MS system. The analytes N-methylformamide and N-methylacetamide are analyzed in the Selected Ion Monitoring (SIM) mode. 2 Equipment, chemicals and solutions 2.1 Equipment Gas chromatograph with mass spectrometric detector (e.g. Agilent GC 6890N and MSD 5973N) with gas-tight syringe or auto sampler Capillary gas chromatographic column: stationary phase: polystyrene-divinylbenzene, length: 30 m; inner diameter: 0.32 mm; film thickness: 20 μm (e.g. HP-Plot Q, Agilent No P-QO4) Block heater (e.g. ReactiVap, Pierce) Analytical balance (e.g. Sartorius) Various volumetric flasks Various pipettes and pipette tips (e.g. Eppendorf) 1.8 ml Crimp cap vials (e.g. Macherey-Nagel) 13 ml Glass tubes with teflon-lined screw caps (e.g. Schütt) Thermostatic water bath 2.2 Chemicals N-Methylformamide 99% (NMF, e.g. Aldrich, No ) N-Methylacetamide 99% (NMA, e.g. Aldrich, No. M26305) N-Hydroxymethyl-N-methylformamide >95% (HMMF, custom synthesis Chiro Block GmbH, Wolfen, Germany) Ethanol absolute (e.g. Merck, No )

7 542 Biomonitoring Methods ultrapure water (e.g. Millipore) Helium 5.0 (e.g. Linde) 2.3 Calibration standards Stock solution NMF (5 g/l) 100 mg N-Methylformamide (NMF) are weighed exactly into a 20 ml volumetric flask and the flask is then made up to the mark with ultrapure water. Stock solution NMA (5 g/l) 100 mg N-Methylacetamide (NMA) are weighed exactly into a 20 ml volumetric flask and the flask is then made up to the mark with ultrapure water. Working solution I (WS I, 2000 mg/l) 4 ml each of the stock solutions of NMF and NMA are pipetted into a 10 ml volumetric flask. The flask is then made up to the mark with ultrapure water. Working solution II (WS II, 100 mg/l) 0.5 ml of the working solution I are pipetted into a 10 ml volumetric flask. The flask is then made up to the mark with ultrapure water. Calibration standards The calibration standards are prepared in ultrapure water or pooled urine. For that purpose, the two working solutions are pipetted into 20 ml volumetric flasks according to the pipetting scheme in Table 3 and diluted with ultrapure water or pooled urine. Table 2 Pipetting scheme for the preparation of the calibration standards (WS I = working solution I, WS II = working solution II). Calibration standard Volume of the working solution Final volume of the calibration standard solution [ml] μL WS I μl WS I μl WS I μl WS I μl WS II μl WS II μl WS II μl WS II NMF and NMA level of the calibration standard [mg/l] All solutions are stored in sealed vials at 20 C. Under these conditions they can be kept for at least 6 months. The MAK Collection for Occupational Health and Safety 2016, Vol 1, No 1

8 N-Methylformamide and N-Methylacetamide in urine Specimen collection and sample preparation 3.1 Specimen collection The urine is collected in sealable plastic containers and kept deep frozen at 20 C. 3.2 Sample preparation Prior to analysis the samples are defrosted in a waterbath at about 40 C and they are thoroughly shaken afterwards. After equilibration to room temperature the samples are once more shaken for homogenization. An aliquot of about 2 ml urine is transferred into a 13 ml screw cap glass tube which is sealed and heated for 2 hours at 120 C in the block heater (thermolysis / pre-heating step). After cooling to room temperature an aliquot of 200 μl of the sample is pipetted into a 1.8 ml vial to which 1000 μl ethanol are added. The vial is sealed, thoroughly shaken and passed on for GC MS analysis. 4 Operational parameters 4.1 Gas chromatography Capillary column: Material: Fused Silica Stationary phase: HP-PlotQ Length: 30 m Inner diameter: 0.32 mm Film thickness: 20 μm Temperatures: Column: Initial temperature 100 C, then increase at a rate of 15 C/min to 250 C, hold for 2 min at final temperature Injector: 300 C Transfer line: 280 C Carrier gas: Helium 5.0 constant flow: 1.5 ml/min Injection volume: 2 μl, splitless (0.3 min; then 20 ml/min septum purge flow) All other parameters need to be optimized in accordance with the manufacturer s instructions.

9 544 Biomonitoring Methods 4.2 Mass spectrometry Ionization type: Electron impact ionization (EI) Ionization energy: 70 ev Source temperature: 230 C Quadrupole temp.: 150 C Detection mode: Selected Ion Monitoring (SIM) Dwell time: 50 ms Solvent delay: 5 min All other parameters need to be optimized in accordance with the manufacturer s instructions. 5 Analytical determination For analytical determination 2 μl of the urine samples prepared according to Section 3 are injected into the GC MS system. The time courses of the ion traces listed in Table 3 are recorded in the SIM mode. Table 3 Retention times and detected ions of the analytes. Analyte Retention time [min] Ion trace [m/z] Quantifier Qualifier NMF 9.1 min NMA 10.2 min The retention times shown in the table are intended as a rough guide only. Users of the method must ensure proper separation performance of the capillary column they use and of the resulting retention behaviour of the analytes. The chromatogram of a urine sample of a volunteer exposed to DMF and DMA is shown in the Appendix (Figure 2). 6 Calibration The calibration standards (see Section 2.3) are prepared in the same way as the urine samples (Section 3.2) and analyzed according to Sections 4 and 5 using GC MS. Calibration graphs are obtained by plotting the peak areas of the calibration standards against the spiked concentration. The linearity of the calibration graph was tested and confirmed up to a concentration of 250 mg/l. Figure 3 in the Appendix shows an example of a calibration graph. The MAK Collection for Occupational Health and Safety 2016, Vol 1, No 1

10 N-Methylformamide and N-Methylacetamide in urine Calculation of the analytical result The concentration of the analytes in mg/l urine is determined based on the peak area of the analyte peak. The linear calibration curve is used to quantitate the amount of analyte in the urine sample (compare Section 6). If the determined analyte level of the sample is outside the calibration range, the urine sample is diluted and re-analyzed. Generally creatinine concentrations < 0.3 g/l or > 3.0 g/l are criteria which would exclude the usability of the spot urine [DFG 2014]. 8 Standardization and quality control Quality control of the analytical results is carried out as stipulated in the guidelines of the Bundesärztekammer (German Medical Association) and in a general chapter of the MAK-Collection for Occupational Health and Safety Part IV: Biomonitoring Methods [Bundesärztekammer 2008, Bader et al. 2010]. To check precision, urine spiked with a known and constant analyte concentration is analyzed as control sample within each analytical run. As quality control material is not commercially available, it must be prepared in the laboratory by spiking pooled urine with defined levels of NMF and NMA, covering the relevant concentration range. The quality control material should be divided up into 2 ml aliquots and stored frozen at 20 C. At least one of these samples is processed and analyzed within each analytical run. The nominal value and the tolerance ranges of this quality control material are determined in a pre-analytical period [Bader et al. 2010]. The analyte levels of the control material should lie within the tolerance ranges obtained. 9 Evaluation of the method 9.1 Within day precision To determine the within day precisions, water and pooled urine was spiked with defined quantities of NMF and NMA at three different concentrations. Ten standards each were analyzed without pre-heating step. The results are shown in Table 4. Furthermore, the within day precision was determined by analyzing three urine samples of exposed individuals with and without pre-heating of the samples. The results are shown in Table 5.

11 546 Biomonitoring Methods Table 4 Within day precision data for the determination of NMF and NMA in water and pooled urine (n = 10). Analyte spiked concentration [mg/l] Within day precision (water) Standard deviation (rel.) [%] Prognostic range [%] Within day precision (pooled urine) Standard deviation (rel.) [%] NMF NMA Prognostic range [%] Table 5 Within day precision data for the determination of NMF and NMA in three urine samples of exposed volunteers (with and without pre-heating of the samples; n = 10). Analyte Mean concentration [mg/l] Within day precision (without pre-heating step) Standard deviation (rel.) [%] Prognostic range [%] Within day precision (with pre-heating step) Standard deviation (rel.) [%] NMF NMA Prognostic range [%] 9.2 Day to day precision The day to day precision was determined by analyzing three standard solutions prepared in water or in urine on 10 different days (see Table 6). Furthermore, the day to day precision was also checked by analyzing three urine samples of exposed individuals with and without pre-heating step (see Table 7). The MAK Collection for Occupational Health and Safety 2016, Vol 1, No 1

12 N-Methylformamide and N-Methylacetamide in urine 547 Table 6 Day to day precision data for the determination of NMF and NMA in water and pooled urine (n = 10). Analyte spiked concentration [mg/l] Day to day precision (water) Standard deviation (rel.) [%] Prognostic range [%] Day to day precision (pooled urine) Standard deviation (rel.) [%] NMF NMA Prognostic range [%] Table 7 Day to day precision data for the determination of NMF and NMA in three urine samples of exposed volunteers (with and without pre-heating of the samples; n = 10). Analyte Mean concentration [mg/l] Day to day precision [without pre-heating step] Standard deviation (rel.) [%] Prognostic range [%] Day to day precision [with pre-heating step] Standard deviation (rel.) [%] NMF NMA Prognostic range [%] 9.3 Accuracy The accuracy of the method was examined using urine samples spiked with NMF or NMA. In addition, pooled urine was spiked with HMMF and HMMA to check whether the thermolysis in the GC inlet liner was complete. The synthesized HMMA standard, however, was not sufficiently stable to calculate the relative recovery rates after spiking. The samples were analyzed without pre-heating step. The results are given in Table 8.

13 548 Biomonitoring Methods Table 8 Relative recovery rates for the determination of NMF and NMA in urine. Analyte Spiked concentration Number of determinations NMF 27.0 mg/l NMF mg/l HMMF NMA 26.0 mg/l NMA Mean rel. recovery rate [%] Additionally, the method described here for the determination of NMF in urine (with minor variations and without the pre-heating step) has been used since 1998 in an external quality assessment scheme for toxicological analyses in biological materials (G-EQUAS) which is run by the German Society of Occupational Medicine and Environmental Medicine (Deutsche Gesellschaft für Arbeitsmedizin und Umweltmedizin (DGAUM)). The results obtained were almost all within the tolerance ranges. 9.4 Detection and quantitation limits The detection limits of the method were estimated on the basis of a signal-to-noise ratio of 3 : 1. The quantitation limits were estimated on the basis of a signal-to-noise ratio of 9 : 1 (see Table 9). Table 9 Detection and quantitation limits of the method. Analyte Detection limit [mg/l] Quantitation limit [mg/l] NMF NMA Interlaboratory comparison The validity of the method was examined in several interlaboratory comparison studies. The participants used different analytical procedures and devices for the determination of NMF and NMA. In the first interlaboratory comparison study both urine samples spiked with NMF, NMA, HMMF or HMMA and native urine samples of DMA exposed volunteers were analyzed. All laboratories performed the analysis without the pre-heating step. The results are shown in Table 10. The data show that the results obtained by the four laboratories for the urine samples spiked with NMF and NMA are in quite good agreement, while considerable differences occurred with regard to the samples spiked with HMMF or HMMA and the urine of the DMA exposed person. Laboratory C and D in particu- The MAK Collection for Occupational Health and Safety 2016, Vol 1, No 1

14 N-Methylformamide and N-Methylacetamide in urine 549 Table 10 Interlaboratory comparison 1: Determination of NMF and NMA without pre-heating step ( 1 direct injection after dilution with ethanol, GC MS; 2 extraction of the analytes with butanol, GC-NPD). Analyte Sample Nominal value [mg/l] Determined analyte level [mg/l] Lab A 1 Lab B 1 Lab C 1 Lab D 2 NMF spiked with NMF spiked with HMMF spiked with HMMF NMA spiked with NMA spiked with HMMA 23.0 * DMA exposed DMA exposed * The synthetic HMMA standard is not stable and about half of it had already decomposed at that time. The nominal value thus corresponds to the weighed HMMA but not to the actual HMMA level in the sample. lar found considerably lower analyte levels in these samples (60 80% of the nominal value), indicating that the thermolytic decomposition in the inlet liner was not quantitative. Based on these results, a second interlaboratory comparison was carried out, including a pre-heating step in the analysis. Beside NMF and NMA spiked urine samples, each participating laboratory analyzed urine samples of volunteers exposed to DMF and DMA, respectively, with and without pre-heating step. The results are shown in Table 11. Table 11 Interlaboratory comparison 2: Determination of NMF and NMA with and without pre-heating step ( 1 direct injection after dilution with ethanol, GC MS; 2 extraction of the analytes with butanol, GC-NPD). Analyte Sample Preheating Determined analyte level [mg/l] Lab A 1 Lab B 1 Lab C 1 Lab D 2 NMF spiked with NMF (26.6 mg/l) no NMA urine of a DMF exposed volunteer spiked with NMA (22.4 mg/l) Urine of a DMA exposed volunteer no yes no no yes The data show that, with the pre-heating step included, the results of the individual laboratories are in far better agreement. The laboratories C and D in particular, for which incomplete thermolysis in the injector had been suspected, detected clearly higher NMF and NMA levels after pre-heating. Using the pre-heating step,

15 550 Biomonitoring Methods the determined standard deviations of the laboratory results (10 15%) were within the prognostic range of the method (compare Section 9.1 and 9.2). 9.6 Sources of error No matrix interferences were observed, when determining NMF and NMA after the preceding pre-heating step. The calibration standards can be prepared both in pooled urine and in water as it was found that the slope of the calibration curves in both matrices were nearly the same. As the samples, following pre-heating and dilution, are directly injected into the analytical device, the use of an internal standard is not imperatively required either. This is confirmed by the good precision and accuracy of the method. The interlaboratory comparison revealed that the thermolytic decomposition of HMMF and HMMA to NMF and NMA in the inlet liner is not always complete under different instrumental and operational conditions. The included pre-heating step now ensures a device-independent, complete thermolysis. No other analytical interferences were observed. 10 Discussion of the method The analytical procedure described here is a combination and further development of the DMF and DMA biomonitoring methods already published in the MAK-collection [Will et al. 1996, Knecht et al. 2003] and allows for the simultaneous determination of the internal levels of NMF and NMA resulting from external DMF and DMA exposure. The main modification with regard to the previous methods is the thermolysis step, introduced to ensure complete decomposition of the main metabolites N-hydroxymethyl-N-methylformamide (HMMF) and N-hydroxymethyl-N-methylacetamide (HMMA) into the final analytes N-methylformamide (NMF) and N-methylacetamide (NMA). This step is of essential importance for the accuracy of the analysis, as interlaboratory comparison studies had shown that the thermolysis in the inlet liner of the gas chromatograph is not always complete. The introduction of the pre-heating step has improved the accuracy of the method without having any adverse effects on its reproducibility (compare to Sections 9.1 and 9.2). When complete thermolysis is ensured, method calibration can be done using the commercially available standard substances NMF and NMA. During method development the extraction of the analytes with butanol, as recommended in one of the previously published methods, was tested. It could be shown, that the extraction yields, using butanol for the extraction of the readily water soluble metabolites HMMF and HMMA or NMF and NMA, were in the range of 15-25%. In the present method, these analyte losses during sample extraction are avoided by direct sample injection. The MAK Collection for Occupational Health and Safety 2016, Vol 1, No 1

16 N-Methylformamide and N-Methylacetamide in urine 551 Quantitation is performed using mass-selective detection. Alternatively, nitrogen-phosphorus detectors (NPD) also give reliable results. Instrument used: Gas chromatograph GC 6890N with mass spectrometric detector MSD 5973N and autosampler (Agilent, Santa Clara, Calif. USA). 11 References Bader M, Barr D, Göen Th, Schaller KH, Scherer G, Angerer J, Working group Analyses in Biological Materials (2010): Reliability criteria for analytical methods. In: Angerer J and Hartwig A (eds): The MAK-Collection for Occupational Health and Safety Part IV: Biomonitoring Methods, Vol. 12, Wiley-VCH, Weinheim DOI: / bireliabe0012 Bundesärztekammer (2008): Richtlinien der Bundesärztekammer zur Qualitätssicherung laboratoriumsmedizinischer Untersuchungen. Dt. Ärztebl. 105, A DFG (Deutsche Forschungsgemeinschaft) (2014): List of MAK and BAT Values 2014, Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area, Report No. 50, Wiley-VCH, Weinheim DOI: / Drexler H and Greim H (eds) (2007): N,N-Dimethylformamid, Addendum. Biologische Arbeitsstoff-Toleranz-Werte (BAT-Werte), Expositionsäquivalente für krebserzeugende Arbeitsstoffe (EKA) und Biologische Leitwerte (BLW) [BAT Value Documentation in German language], 14th issue, Wiley-VCH, Weinheim DOI: / bb6812d0014 Drexler H and Greim H (eds) (2010): N,N-Dimethylacetamide. The MAK- Collection for Occupational Health and Safety Part II: BAT Value Documentations, Vol. 5, Wiley-VCH, Weinheim DOI: / bb12719e0005 Greim H (ed) (1998): N,N-Dimethylacetamid. Gesundheitsschädliche Arbeitsstoffe, Toxikologisch-arbeitsmedizinische Begründung von MAK-Werten [MAK Value Documentation in German language]. 26th issue, Wiley-VCH, Weinheim DOI: / mb12719d0026 Greim H (ed) (2010b): N,N-Dimethylformamide. The MAK-Collection Part I: MAK Value Documentations, Vol. 26, Wiley-VCH, Weinheim DOI: / mb6812e0026b IARC (International Agency for Research on Cancer) (1999): Re-evaluation of Some Organic Chemicals, Hydrazine and Hydrogen Peroxide: Dimethylformamide. IARC Monographs on the Evaluation of Carcinogenic Risks of Chemikals to Man. 71, Knecht U, Müller G, Working group Analyses in Biological Material : N,N-Dimethylacetamide (DMA) and N-methylacetamide (NMA). In: Angerer J, Schaller KH, Greim H (eds) (2003): Analyses of Hazardous Substances in Biological Materials, Vol. 8, Wiley-VCH, Weinheim DOI: / bi12719e0008

17 552 Biomonitoring Methods OECD SIDS (2001): N,N-Dimethylacetamide. Screening Information Dataset for High Production Volume Chemicals URL: (accessed 19 March 2013). Will W, Schulz G, Lewalter J, Working group Analyses in Biological Material (1996): N,N-Dimethylformamide (DMF). In: Angerer J, Schaller KH, Greim H (eds): Analyses of Hazardous Substances in Biological Materials, Vol. 5, VCH, Weinheim DOI: / bi6812e0005 Author: W. Will Examiner: M. Bader 12 Appendix Fig. 2 Chromatogram of a urine sample of a volunteer exposed to DMF and DMA. Determined analyte level: NMF: 24.6 mg/l; NMA: 22.0 mg/l. The MAK Collection for Occupational Health and Safety 2016, Vol 1, No 1

18 N-Methylformamide and N-Methylacetamide in urine 553 Fig. 3 Calibration graphs of NMF and NMA in water in the range between 1 and 250 mg/l.