Federation of the Employment Accidents Insurance Institutions of Germany
(Hauptverband der Berufsgenossenschaften)
Centre for Accident Prevention and Occupational Medicine Alte Heerstrae 111, 53757 Sankt Augustin Expert Committee Chemistry
Carcinogenic substances Order number: ZH 1/120.23E Established methods Issue: September 1992
Methods for the determination of N-nitrosamines Methods tested and recommended by the Berufsgenossenschaften for the determination of N-nitrosamines in working areas after discontinuous sampling. For the assessment of working areas, both personal or stationary sampling are possible: 1 Sampling with a pump and adsorption on a sorbent, gas chromatography after elution. N-Nitrosamine-1-GC (Issue: January 1983, withdrawn September 1992) 2 Sampling with a pump and adsorption on a solid collection phase, gas chromatography after elution. N-Nitrosamine-2-GC (Issue: January 1987, withdrawn September 1992) 3 Sampling with a pump and adsorption on a solid collection phase, gas chromatography after elution. N-Nitrosamine-3-GC (Issue: September 1992) This method replaces methods No 1 and 2 for the determination of nitrosamines. 4 Sampling with a pump and adsorption on a solid collection phase, capillary gas chromatography after elution. N-Nitrosamine-4-GC (Issue: September 1992)
IUPAC name: CAS No: N-nitrosodimethylamine 62-57-9 N-nitrosomethylethylamine 10595-95-6 N-nitrosodiethylamine 55-18-5 N-nitrosodipropylamine 621-64-7 N-nitrosodiisopropylamine 601-77-4 N-nitrosodibutylamine 924-16-3 AirMonitoringMethods,Vol.4 Analytical Methods 150 N-nitrosopiperidine 100-75-4 N-nitrosopyrrolidine 930-55-2 N-nitrosomorpholine 59-89-2 AirMonitoringMethods,Vol.4 151 N-Nitrosamines 3 Sampling with a pump and adsorption on a solid collection phase, gas chromatography after elution This method permits the determination of N-nitrosamines concentrations in working areas averaged over the sampling time after personal or stationary sampling. Principle: With a pump a measured air volume is drawn through an adsorp- tion tube. After elution with a dichloromethane/methanol mixture the adsorbed N-nitrosamines are analysed using a gas chromato- graph equipped with a thermal energy analyser (TEA) detector. Technical data: Table 1. Quantification limit. N-Nitrosamine absolute ng relative 1) g/m 3
1) For 200 L air sample, 1 mL sample solution, 5 L injection volume. Selectivity: The TEA detector system is highly selective in combination with the gas chromatographic separation. Interference may occur with such organic nitrogen compounds which also produce a TEA response. Advantages: Personal and selective measurements are possible. Disadvantages: Sophisticated equipment needed. Apparatus: Pump equipped with gas meter or flow meter, adsorption tubes, gas chromatograph with TEA detector. AirMonitoringMethods,Vol.4 Analytical Methods 152 Detailed description of the method Contents 1 Equipment, chemicals and solutions 1.1 Equipment 1.2 Chemicals 1.3 Solutions 2 Sampling 3 Analytical determination 3.1 Sample preparation 3.2 Operating conditions for gas chromatography 4 Evaluation 4.1 Calibration 4.2 Calculation of the analytical result 5 Reliability of the method 5.1 Accuracy 5.2 Quantification limit 5.3 Selectivity 5.4 Recovery 6 Discussion 7 References 1 Equipment, chemicals and solutions 1.1 Equipment For sampling and sample preparation: Pump (e.g. Du Pont Instruments, supplier in Germany: DEHA-Haan & Wittmer GmbH, Friolzheim) Adsorption tubes (e. g. Thermosorb-
N, Isconlab GmbH, Heidelberg)
For the determination of N-nitrosamines any sampling system is suitable with which the formation (artefacts) or decomposition of N-nitrosamines occurs neither on the adsorption phase during sampling and sample transport nor during subsequent sample preparation. 2 mL Sample vial with 1 mL mark and screw caps containing polytetrafluoroethylene (PTFE) coated septa Molecular sieve filter, CTR gas stream filter (e. g. Isconlab GmbH, Heidelberg) Injection syringes 2 mL Pipette AirMonitoringMethods,Vol.4 153 N-Nitrosamines For analysis: Gas chromatograph (e.g. Bodenseewerk Perkin-Elmer & Co GmbH, berlingen; Hewlett Packard, Bblingen; Siemens AG, Karlsruhe;Varian GmbH, Darmstadt) TEA detector (e. g. Isconlab GmbH, Heidelberg) 1.2 Chemicals Dichloromethane, analytical grade Methanol, analytical grade It must be checked that no interfering amine and nitrosamine compounds are present. Helium, 5.0 (99.999%) Oxygen, 5.0 (99.999%) 1.3 Solutions Dichloromethane/methanol mixture: In a 100 mL volumetric flask 25 mL methanol is diluted to the mark with dichloro- methane. N-Nitrosamine stock solution: Commercial N-nitrosamine standards (e.g. from Promochem GmbH, Wesel; Sigma Chemie GmbH, Mnchen). They are available as solutions (e.g. in ethanol) containing about 100 g/mL N-nitrosamine(s). Calibration solution (external standard): Solution of 10 g/mL N-nitrosamine. In a 10 mL volumetric flask 1 mL of the N-nitrosamine stock solution is diluted to the mark with the dichloromethane/methanol mixture. Even in the dark and in a refrigerator the shelf life of the calibration solutions is limited (about 1 week). 2 Sampling An adsorption tube is opened and connected to the pump. During working hours the pump and adsorption tube are carried by a person or used in a stationary position. The air sample volume is 200 L. The flow rate must not exceed 100 L/h. AirMonitoringMethods,Vol.4 Analytical Methods 154 3 Analytical determination 3.1 Sample preparation The loaded adsorption tube must be eluted with 2 mL of the dichloromethane/methanol mixture within 10 days (storage in the dark at 5 C) in the opposite direction to the air flow during sampling. The eluate (1 mL) is collected in a 2 mL sample vial with a 1 mL mark and analysed (sample solution). It contains all the adsorbed N-nitrosamines. To ensure that no interfering substances are present, an unloaded adsorption tube is eluted with 2 mL of the dichloromethane/methanol mixture and, as described, the first millilitre is analyzed (blank value). Each sample solution and each calibration solution should be analysed twice (autosam- pler). 3.2 Operating conditions for gas chromatography The method was characterized under the following experimental conditions: Apparatus: Varian, model 3700 equipped with TEA detector, model 502/LC Column: Material: Borosilicate glass, deactivated with dichlorodimethylsilane in toluene Length: 1.5 m Internal diameter: 2 mm Stationary phase: 15% Carbowax 20M on Volaspher A2 Particle size: 0.150.18 mm (80/100 mesh) Temperatures: Injector block: 150 C Column temperature programme: Starting temperature: 125 C, 2 min isothermal Heating rate: 10 C/min Final temperature: 170 C, 12 min isothermal Interface: 220 C Pyrolysis furnace: 550 C Injection volume: 5 L Detector conditions: Operating parameters optimized according to the operating in- structions of the manual 4 Evaluation 4.1 Calibration 10, 20, 30 and 50 L of the calibration solutions are each injected into 2 mL sample vials containing exactly 1 mL of the dichloromethane/methanol mixture and the sample vials AirMonitoringMethods,Vol.4 155 N-Nitrosamines are closed. Afterwards analysis is carried out as described in Sect. 3.2. The calibration curve is linear up to about 100 g for each N-nitrosamine. Evaluation is carried out with the external standard method. 4.2 Calculation of the analytical result The quantitative evaluation is carried out with the external standard method. The con- centrations by weight of the individual N-nitrosamines in the air sample in g/m 3 are calculated according to the following equation (1): (1) For the calculation of the concentration by volume c v in L/m 3 (ppb) from c w at 20 C and 1013 hPa: (2) Legend: c w N-Nitrosamine concentration by weight in the air sample in g/m 3
c v N-Nitrosamine concentration by volume in the air sample in L/m 3 (ppb) w s Weight of the calibration standard in ng F s Peak area of the N-nitrosamine standard F p Peak area of the N-nitrosamine sample V Air sample volume in L V mol Molar volume in L (24.1 L at 20 C and 1013 hPa) w mol Molecular weight in g 5 Reliability of the method 5.1 Accuracy In the analytical determination, under the operating conditions described a relative standard deviation of 5.5% was obtained for a N-nitrosodimethylamine weight of 5 ng per 5 l injection volume and 25 single measurements. The mean variation was 11%. 5.2 Quantification limit The quantification limits for the N-nitrosamines are listed in Table 2: c F F w V w p s s . . w V c c w . v mol mol AirMonitoringMethods,Vol.4 Analytical Methods 156 Table 2. Quantification limit. absolute ng relative 1) g/m 3
N-Nitrosodimethylamine 0.1 0.1 N-Nitrosomethylethylamine 0.1 0.1 N-Nitrosodiethylamine 0.1 0.1 N-Nitrosodipropylamine 0.1 0.1 N-Nitrosodiisopropylamine 0.1 0.1 N-Nitrosodibutylamine 0.2 0.2 N-Nitrosopiperidine 0.1 0.1 N-Nitrosopyrrolidine 0.2 0.2 N-Nitrosomorpholine 0.2 0.2 1) For 200 L air sample, 1 mL sample solution, 5 L injection volume. 5.3 Selectivity The TEA detector system is highly selective in combination with gas chromatographic separation. Substance groups such as organic nitrites and nitrates, nitro compounds and C-nitroso compounds may also produce a TEA response. 5.4 Recovery 20 L of the calibration solution was injected into an open adsorption tube and pure ni- trogen (40 mL/min) drawn through the adsorbent to remove the solvent. After that it was eluted and analysed as described in Sect. 3. The recovery rate was >
0.9 for the various N-nitrosamines. 6 Discussion Ethyl formate can be used instead of the dichloromethane/methanol mixture. The method is unsuitable for determining N-nitrosomethylphenylamine and N-nitro- soethylphenylamine [1]. 7 References [1] Mitteilung der Arbeitsgruppe Analytik im Fachausschu Chemie (1990) Zur Problematik der Konzentrationsbestimmung von N-Nitrosomethylphenylamine und N-Nitrosoethyl- phenylamine in Luft. Staub-Reinhalt. Luft 50 No 2: 6970. AirMonitoringMethods,Vol.4 157 N-Nitrosamines 4 Sampling with a pump and adsorption on a solid collection phase, capillary gas chromatography after elution This method permits the determination of the N-nitrosamine concentrations in working areas averaged over the sampling time after personal or stationary sampling. Principle: With a pump a measured air volume is drawn through an adsorp- tion tube. After elution with a dichloromethane/methanol mixture the adsorbed N-nitrosamines are analysed by a gas chromato- graph equipped with a thermal energy analyser (TEA) detector. Technical data: Table 1. Quantification limit. N-Nitrosamine absolute ng relative 1) g/m 3
1) For 200 L air sample, 1 mL sample solution, 3 L injection volume. Selectivity: The TEA detector system is highly selective in combination with gas chromatographic separation. Interference may occur with or- ganic nitrogen compounds which also produce a TEA response. Advantages: Personal and selective measurements are possible. Disadvantages: Sophisticated equipment needed. Apparatus: Pump equipped with gas meter or flow meter, adsorption tubes, gas chromatograph with TEA detector. AirMonitoringMethods,Vol.4 Analytical Methods 158 Detailed description of the method Contents 1 Equipment, chemicals and solutions 1.1 Equipment 1.2 Chemicals 1.3 Solutions 2 Sampling 3 Analytical determination 3.1 Sample preparation 3.2 Operating conditions for gas chromatography 4 Evaluation 4.1 Calibration 4.2 Calculation of the analytical result 5 Reliability of the method 5.1 Accuracy 5.2 Quantification limit 5.3 Selectivity 5.4 Recovery 6 References 1 Equipment, chemicals and solutions 1.1 Equipment For sampling and sample preparation: Pump (e.g. Du Pont Instruments, supplier in Germany: DEHA-Haan & Wittmer GmbH, Friolzheim) Adsorption tubes (e. g. Thermosorb-
N, Isconlab GmbH, Heidelberg)
For the determination of N-nitrosamines any sampling system is suitable with which the formation (artefacts) or decomposition of N-nitrosamines occurs neither on the adsorption phase during sampling and sample transport nor during subsequent sample preparation. 2 mL Sample vial with 1 mL mark and screw caps containing polytetrafluoroethylene (PTFE) coated septa Molecular sieve filter, CTR gas stream filter (e. g. Isconlab GmbH, Heidelberg) Injection syringes 2 mL Pipette For analysis Capillary gas chromatograph (e. g. Bodenseewerk Perkin-Elmer & Co GmbH, berlin- gen; Hewlett Packard, Bblingen; Siemens AG, Karlsruhe;Varian GmbH, Darmstadt) TEA detector (e. g. Isconlab GmbH, Heidelberg) AirMonitoringMethods,Vol.4 159 N-Nitrosamines 1.2 Chemicals Dichloromethane, analytical grade Methanol, analytical grade It must be checked that no interfering amine and nitrosamine compounds are present. Helium, 5.0 (99.999%) Oxygen, 5.0(99.999%) 1.3 Solutions Dichloromethane/methanol mixture: In a 100 mL volumetric flask 25 mL methanol is diluted to the mark with dichloro- methane. N-Nitrosamine stock solution: Commercial N-nitrosamine standards (e.g. from Promochem GmbH, Wesel; Sigma Chemie GmbH, Mnchen). They are available as solutions (for example in ethanol) containing about 100 g/mLN-nitrosamine(s). Mixed standards: The N-nitrosamines concentrations range from 1020 g/mL (e.g. from Promochem GmbH, Wesel). The mixed standards contain the following N-nitrosamines: N-Nitrosodimethylamine N-Nitrosodiethylamine N-Nitrosodipropylamine N-Nitrosodibutylamine N-Nitrosopiperidine N-Nitrosopyrrolidine N-Nitrosomorpholine Individual standards: The dissolved N-nitrosamine is available as solution containing about 100 g/mL (e.g. from Promochem GmbH, Wesel). Individual standards in pure form are also available in a special safety bottle (Isopack) from Sigma Chemie GmbH, Mnchen. Solutions with a concentration of 10 mg/mL are obtained by appropriate dilution. Nitrosodiisobutylamine is used as an internal standard. N-Nitrosamine calibration solutions: 10, 20, 40 and 100 L of the N-nitrosamine mixed standard are transferred to a 2 mL sample vial. If necessary other N-nitrosamines can be added as individual standards (stock solution) (e.g. 2.5, 5, 7.5 and 15 L N-nitrosodiisopropylamine). The solution is diluted to the 1 mL mark with the dichloromethane/methanol mixture and as an internal standard 5 L N-nitrosodiisobutylamine is added to each solution. These solutions con- tain about 0.1, 0.2, 0.4 and 1 g/mL of each of the N-nitrosamines of the mixed standard and, if necessary, corresponding concentrations of the individual standards. AirMonitoringMethods,Vol.4 Analytical Methods 160 2 Sampling An adsorption tube is opened and connected to the pump. During working hours the pump and adsorption tube are carried by a person or used in a stationary position. The air sample volume is 200 L. The flow rate must not exceed 100 L/h. 3 Analytical determination 3.1 Sample preparation The loaded adsorption tube must be eluted with 2 mL of the dichloromethane/methanol mixture within 10 days (storage in the dark at 5 C) in the opposite direction to the air flow during sampling. The eluate (1 mL) is collected in a 2 mL sample vial with a 1 mL mark, 5 L of the internal standard is added and the solution is analysed as described in Sect. 3.3 (sample solution). It contains all the adsorbed N-nitrosamines. To ensure that no interfering substances are present an unloaded adsorption tube is eluted with 2 mL of the dichloromethane/methanol mixture, the internal standard is added and the first millilitre is analysed (blank value). Each sample solution and each calibration solution should be analysed twice. 3.2 Operating conditions for gas chromatography The method was characterized under the following experimental conditions: Apparatus: Capillary gas chromatograph SIEMENS SiChromat 1-4 equipped with split/splitless injector, TEA detector, model 543 Column: Material: Quartz capillary (fused silica, wide bore) Length: 25 m Internal diameter: 0.53 mm Stationary phase: CP-WAX 52 CB Film thickness: 1.0 m (available from Chrompack, Frankfurt) Temperatures: Injector block: 160 C Column temperature programme: Starting temperature: 60 C, 1 min isothermal Heating rate: 4 C/min Final temperature: 140 C Interface: 210 C Pyrolysis furnace: 510 C Injection volume: 3 L, splitless AirMonitoringMethods,Vol.4 161 N-Nitrosamines Carrier gas: Helium: 6 mL/min Detector conditions: Operating parameters optimized according to the operating instructions of the manual. When using the TEA detector with capillary columns it is necessary to reduce the dead volumes of the TEA detector considerably, especially the internal diameter of the PTFE tubing [1]. 50 cm deactivated unloaded capillary columns (internal diameter 0.53 mm) must be connected in front and behind the chromatography column. The first column prevents rapid pollution of the chromatography column while the second one prevents the vacuum in the detector causing a pressure decrease in the chromatography column. Under the above conditions N-nitrosodibutylamine and N-nitrosopiperidine are not sep- arated. If one of these nitrosamines is present in the sample, the separation conditions have to be changed for differentiation (e.g. heating rate 2 C/min). 4 Evaluation 4.1 Calibration 3 L of each of the calibration solutions (cf. Sect. 1.3) are injected into the gas chromatograph and analysed. The calibration function is linear in the range of 0.33 ng (absolute). The calibration factor f N (response factor) is calculated by using the peak areas of the N-nitrosamines and of the internal standard (N-nitrosodiisobutylamine) in the different calibration solutions according to equation (1): (1) Legend: f N Response factor of the particular N-nitrosamine F Ci Peak area of the internal standard in the calibration solutions F CN Peak area of the particular N-nitrosamine in the calibration solutions C Ci Concentration of the internal standard in the calibration solutions in g/mL C CN Concentration of the particular N-nitrosamine in the calibration solutions in g/mL f N is nearly the same for all concentrations. For the calculation of the analytical result the mean value - f N must be used. 4.2 Calculation of the analytical result The N-nitrosamine concentration by weight in the sample air in g/m 3 is calculated ac- cording to equation (2): . . F F C C f N Ci Ci CN CN AirMonitoringMethods,Vol.4 Analytical Methods 162
(2) Legend: c w Concentration by weight of the particular N-nitrosamine in the sample air in g/m 3
F N Peak area of the N-nitrosamine in the sample solution F i Peak area of the internal standard in the sample solution w i Weight of the internal standard in the sample solution in g V Air sample volume in m 3
- f N Mean response factor For the calculation of the concentration by volume c v in L/m 3 (ppb) from c w at 20 C and 1013 hPa: (3) Legend: c v Concentration by volume of N-nitrosamine in the air sample in L/m 3 (ppb) V mol Molar volume in L (24.1 L at 20 C and 1013 hPa) w mol Molecular weight in g 5 Reliability of the method 5.1 Accuracy For this method the relative standard deviations and mean variations of eight N-nitrosa- mines were determined in two concentration ranges. To calculate the standard deviation, the nitrosamines were transferred to ThermoSorb-N tubes with a microliter syringe, 200 L laboratory air were drawn through the tubes and analysed as described in Sect. 3. For the number of the individual determinations (n), concentrations (c(solution) and c(air)), relative standard deviations (s rel ) and the mean variations (u) cf. the Table 2. c w . . . N N F F w f V i i w V c c w . v mol mol AirMonitoringMethods,Vol.4 1 6 3
N - N i t r o s a m i n e s
Table 2. Standard deviation (rel.) s and mean variation u. N-Nitrosamines
n Concentration of the individual N-Nitrosamine in the solution g/mL Concentration of the individual N-Nitrosamine in the air 1) g/m 3
Standard deviation (rel.) s
% Mean variation u
%
n Concentration of the individual N-Nitrosamine in the solution g/mL Concentration of the individual N-Nitrosamine in the air 1) g/m 3
Standard deviation (rel.) s
% Mean variation u
% N-Nitrosodimethylamine 16 0.066 0.330 6 13 10 0.330 1.65 5 11 N-Nitrosodiethylamine 16 0.040 0.201 12 25 10 0.201 1.01 8 17 N-Nitrosodiisopropylamine 16 0.104 0.522 14 30 10 0.522 2.61 6 13 N-Nitrosodipropylamine 16 0.059 0.293 16 34 10 0.293 1.47 5 11 N-Nitrosodibutylamine 16 0.035 0.177 11 24 10 0.177 0.89 5 11 N-Nitrosopiperidine 16 0.048 0.241 16 34 10 0.241 1.21 6 14 N-Nitrosopyrrolidine 16 0.040 0.199 17 36 10 0.199 1.00 3 8 N-Nitrosomorpholine 16 0.053 0.263 17 36 10 0.263 1.32 8 19 1) For 200 L sample air, 1 mL sample solution and 3 L injection volume. A i r M o n i t o r i n g M e t h o d s , V o l . 4 Analytical Methods 164 5.2 Quantification limit The quantification limits of the N-nitrosamines are listed in Table 3 below: Table 3. Quantification limit. N-Nitrosamines absolute ng relative 1) g/m 3
N-Nitrosodimethylamine 0.019 0.03 N-Nitrosomethylethylamine 0.021 0.03 N-Nitrosodiethylamine 0.023 0.04 N-Nitrosodipropylamine 0.036 0.06 N-Nitrosodiisopropylamine 0.032 0.05 N-Nitrosodibutylamine 0.051 0.08 N-Nitrosopiperidine 0.028 0.05 N-Nitrosopyrrolidine 0.028 0.05 N-Nitrosomorpholine 0.037 0.06 1) For 200 L sample air, 1 mL sample solution and 3 L injection volume. 5.3 Selectivity The TEA detector system is highly selective in combination with the gas chromatographic separation. Substance groups such as organic nitrites and nitrates, nitro and C-nitroso compounds may also produce a TEA response. The possible formation of artefacts by nitrosation of amines collected on the collection phase was checked as follows: 50 g each of dimethylamine and morpholine were trans- ferred to a gas collecting vessel, connected to a ThermoSorb-N tube and 100 L laboratory air were drawn through this tube. In all cases (n = 6) no N-nitrosamine could be detected. 5.4 Recovery 4 L (about 50 ng nitrosamine) and 20 L (about 250 ng nitrosamine) of the mixed standard were injected into an open adsorption tube: after that 200 L air (100 L/h) were drawn through the adsorption phase, eluted and analysed as described in Sect. 3. The calibration solutions served as a reference. The recovery rates of the various N-nitrosamines ranged between 0.95 and 1.05. 6 References [1] Borwitzky H (1986) Some improvements in the determination of volatile nitrosamines by fused silica-capillary GC in combination with a thermal energy analyser. Chromatographia 22 (16): 6572. AirMonitoringMethods,Vol.4