Analytica Chimica Acta 557 (2006) 256261

Simplied method for the determination of N-nitrosamines in rubber vulcanizates

Joseph A. Incavo , Melvin A. Schafer
The Goodyear Tire and Rubber Company, Akron, OH, USA Received 16 August 2005; received in revised form 4 October 2005; accepted 9 October 2005

Abstract A simplied method for the trace determination of N-nitrosamines in carbon black-loaded rubber compounds is described. The extraction of volatile nitrosamines is accomplished by thermal desorption rather than the traditional solvent extraction procedure. The analytes are trapped on Thermosorb/N sorbent and subsequently analyzed by gas chromatography with thermal energy analyzer detection (GC/TEA). Conditions that provide full extraction of nitrosamines from actual rubber compounds were determined to be 30 min at 150 C in vessels dynamically purged with N2 . Method precision was found to be 10% for NDMA at 71 ng/g and 7.3% for NMOR at 248 ng/g. Recoveries for the seven common N-nitrosamines ranged from 94 to 117%. Limits of detection in the rubber matrix are 6.313 ng/g. The technique is found to offer improved recovery of lower molecular weight nitrosamines and it is shown to be simpler and faster than previous techniques. 2005 Elsevier B.V. All rights reserved.
Keywords: Nitrosamines; Rubber; Vulcanizates; Desorption; TEA

1. Introduction Certain N-nitrosamines are known to promote signicant carcinogenic effects in laboratory animals, yet there is no direct evidence exposures cause cancer in humans. The International Agency for Research on Cancer recommends the seven common volatile nitrosamines be treated as carcinogenic to humans [1,2], which has prompted the development of analytical methods to quantitate volatile nitrosamines at the part-per-billion level in various consumer products. In rubber-based articles, vulcanization accelerators are the primary source of the secondary amines, which react with atmospheric or adsorbed nitrogen oxides:

Publication pertaining to nitrosamine determinations in rubber articles includes ambient air methods [2,3], rubber nipple method development [38] and black-loaded rubber method development [912]. The standard air method in the US is NIOSH 2522, which employs Thermosorb/N air sampling car

Corresponding author. E-mail address: jaincavo@goodyear.com (J.A. Incavo).

tridges to quantitatively trap volatile nitrosamines [2]. The analytes are back-ushed with methylene chloride/methanol and directly analyzed by gas chromatography with a thermal energy analyzer detection system (GC/TEA). The TEA offers high selectivity and sensitivity toward N-nitroso compounds by pyrolyzing the NNO bonds into nitrosyl radicals (*NO), which undergo chemiluminescent with ozone to yield an *NO2 emission measured on a photomultiplier tube. Most rubber product methods involve solvent extraction procedures; however, baby bottle nipples and paciers are somewhat of a special case. Those methods use articial saliva and/or organic solvents for extraction followed by Kuderna-Danish or rotary evaporations [48]. The most comprehensive investigation of solvent extraction methodology of rubber stocks was reported by Liekefeld et al. [9]. The work details the steps of methanol Soxhlet extraction and, in addition to the OSHA method evaluation [2], established the efciency and limitations of the Thermosorb/N cartridge for air sampling. More recent work involves extracting volatile nitrosamines directly onto an adsorbent using heat [1214]. Thermal desorption (TD) has been used to mimic fumes generated during mixing or curing rubber compounds [13]. Some brief work has been performed on whole baby bottle nipples [14] but further work is required to assess the applicability to rubber compounds

0003-2670/$ see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.10.018

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containing carbon black. In the present study, we investigate the conditions leading to full extraction of volatile nitrosamines by TD and we validate the method on actual carbon black-loaded rubber compounds. The key issue in this methodology is achieving a quantitative extraction of the nitrosamines from the solid matrix. This presents a challenge because there are not suitable standards available for this type of determination. Additionally, it is often difcult to obtain and measure complete extraction from insoluble, cross-linked composites such as rubber products. The efciency of methanol extraction has been investigated in the past [9] and a 20 h Soxhlet procedure was established. However, for thermal extractions, the conditions that lead to complete extraction have not been investigated. For baby bottle nipples, recoveries were reported for a liquid standard injected onto the surface of the product [14]. This procedure would not necessarily achieve signicant incorporation of the analytes into the solid matrix. Conditions that serve to volatilize these liquids from the rubber surface may not be sufcient to fully extract analytes from the interior points (bulk) of the solid. This yield depends largely on the permeability parameters of the analyte such as diffusivity, solubility, particle size, extraction time and temperature. Another important factor is the concentration of analyte in the vapor phase in contact with the solid. Traditional headspace analysis, whereby only a single equilibration between the solid and vapor phase occurs, is inherently less efcient with dependencies on the partition coefcient and the phase ratio of the system [1517]. The current study employing a thermal desorption technique is a dynamic headspace analysis which extracts by refreshing the surface by means of a owing carrier gas.

block with the efuent routed directly to a Thermosorb\N adsorbent cartridge. Nitrogen is purged through the vessel with gas entering at the bottom and exiting as shown. With the glassware used here, it was more amenable to draw gas through the cell with a pump rather than using a pressurized source. A small ow of nitrogen was introduced at the inlet to provide blanketing with the total ow measured at the exit of the pump. The aluminum block was wrapped in heating tape with the block temperature controlled to 1 C. Glass beads were added to the cell prior to charging the diced sample to help form a more uniform temperature prole across the cell. When the vessel was lowered into the heated block at the start of the analysis an initial 23 C temperature drop occurred in the block, which recovered after 5 min. All tests were performed with 1.000 g samples diced into 3 mm cubes. After desorption is completed the adsorbent tube was capped until further processing. The procedures adopted hereafter are consistent with NIOSH 2522 for nitrosamine determinations in air samples [2]. The cartridge is rinsed with 2.5 mL of solvent mixmethanol/methylene chloride (25/75 v/v)and the nal volume eluted is measured to within 0.1 mL. This solution was directly analyzed using external standardization by liquid autosampler on a capillary column GC with TEA detector as is described below. The solvent extraction analyses were performed on 1.000 g of diced sample Soxhlet extracted for 20 h in methanol. The extract was concentrated at room temperature to 2.0 mL under nitrogen purge. To match the solvent mixture used for GC/TEA calibration, the extract was diluted to 8.0 mL with methylene chloride. This extract was injected directly into the GC/TEA by liquid autosampler. 2.2. Gas chromatography/thermal energy analyzer (GC/TEA) conditions GC: Agilent 5890A with 7673A liquid autosampler; Column: Stabilwax DB (Restek Inc.), 0.53 mm 60 m, 1.0 m d.f.;

2. Experimental 2.1. Thermal desorption apparatus The apparatus assembled for experiments here is shown in Fig. 1. It employs a gas dispersion tube contained in a heated

Fig. 1. Schematic diagram of thermal desorption apparatus.

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Oven cycle: 120 C (2 min), 2 C/min to 130 C (0 min), 7.5 C/min to 210 C (0 min), 35 C/min to 240 C (3 min); Injection port: 200 C, splitless, constant pressure control, 2.0 uL injection; Carrier gas: Helium, 10 mL/min; Detector: Thermal Energy Analyzer, Model TEA 543 (Thermedics Inc.), pyrolysis temperature: 550 C, interface temperature: 250 C [20]. 2.3. GC/TEA calibration A certied standard containing the seven common volatile Nnitrosamines served as the stock solution from which aliquots of 5, 10, 25, 40, 50 L were diluted into 1.0 mL of the solvent mixture (MeOH/MeCl2 25/75 v/v). These external standards provide a six-level calibration ranging from 12.5 to 375 ng. After a clean reagent blank is obtained, the ve-point calibration is established with a correlation coefcient of r2 > 0.9990. Sample sequences are bracketed with a check standard that must fall within 10% of the certied value. If these conditions are not met, recalibration is performed. Curves for a typical calibration are shown in Fig. 2. 2.4. Materials A 30 mL gas dispersion tube, 25 mm 152 mm with 24/40 ground glass joint, lled with ten 3.3 mm OD glass beads; parts cleaned by rinsing with chloroform followed by 30 min drying in an 80 C oven; Heated aluminum block, 51 mm 51 mm 76 mm machined to tightly t gas dispersion tube; wrapped with 25.4 mm Thermolyne heating tape, Scotch electrical tape and fabric insulation; Power supply, 120V/10A Variac power supply controlled by Omega CN300 feedback device capable of maintaining setpoint to within 1 C;

Sampling pump, SKC Model 224-44XR air sampling pump capable of producing 1 L/min ow through the adsorbent tubes; Flow meter/calibrator, Bios DryCal DC-Lite; Thermosorb/N, 6533 air sample cartridges, sold by Thermo Electron, Beverly, MA; cartridges manufactured by Thermedics Detection Inc. (Chelmsford, MA); Centrifuge tubes, conical, graduated centrifuge tubes with 0.1 mL volume marks; Methanol, 99.9% HPLC grade; Methylene chloride, 99.9% HPLC grade; Volatile nitrosamine certied standard, 7-component mixture sold by Chemservice Inc. (West Chester, PA, p/n CRFAN-1M) in iosoctane solvent: 2.5 g/mL N-nitrosodimethylamine (NDMA), 2.5 g/mL N-nitrosodiethylamine (NDEA), 5.0 g/mL N-nitrosodi-n-propylamine (NDPA), 7.5 g/mL N-nitrosodi-n-butylamine (NDBA), 7.5 g/mL N-nitrosopiperidine (NPIP), 7.5 g/mL N-nitrosopyrrolidine (NPYR), 7.5 g/mL N-nitrosomorpholine (NMOR).

3. Results and discussion 3.1. Method precision Since suitable standard materials are not available for this type of determination, a true accuracy study cannot be carried out. Considering that solvent extraction method has been previously validated for nitrosamines in rubber vulcanizates [9], a comparative study using actual rubber compounds is the next best validation approach. In this way, a true measure of recovery can be made since nitrosamines formed during rubber mixing, processing and curing will be thoroughly incorporated into the bulk of the compound. This is especially important when mixing high viscosity materials such as elastomers.

Fig. 2. Calibration curves of nitrosamines by GC/TEA (note: NPIP and NPYR omitted for clarity).

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Fig. 3. Thermal desorption efciency curve for a nitrosamine-containing rubber compound.

Upon exposure to air for extended periods of time, these 102 mm 152 mm 3.2 mm compound tensile sheets were found to slowly lose NDMA and NMOR nitrosamines due to their higher vapor pressures (25% over a 3 month period at 20 C). Therefore, the control compound used for this study was cured between Mylar lms to seal the surfaces and did not show measurable lose over the time period studied. The control was chosen to assess some common nitrosamine-generating accelerators and is a carbon-black loaded cured compound containing TMTD (tetramethylthiuram disulde), ZDMC (zinc dimethylodithiocarbamate), 4,4 -dithiomorpholine and EPDM. Fig. 3 shows results from 150 C isothermal trials on the control compound carried out at varying desorption times. A mean NMOR value of 359 ppb (n = 5, R.S.D. = 10%) was obtained on this compound when analyzed by a 20 h Soxhlet extraction. The plateau region indicates that extraction of NMOR is complete 30 min. Further evidence that complete extraction is accomplished is the values at 30 min match the Soxhlet extraction results. NDMA was also detected in these thermal desorption analyses but no recoveries can be computed because it was not detected in the solvent extraction trials. The NDMA concentration also exhibited a leveling off with increasing desorption times to a value of 71 ppb. This plateau for NDMA occurs at slightly shorter desorption times (20 min) due to its higher volatility. It is noted that using a second Thermosorb/N cartridge connected in series showed no breakthrough at 1.0 L/min ow and this is consistent with the OSHA evaluation (NDMA begins a 5% breakthrough at 100 L volume) [2]. Thermosorb/N tubes are favored over other adsorbents such as Tenax GC [16], not only for trapping efciency but also for inhibiting unintended nitrosamine formation on the cartridge adsorbent. The process used by the vendor is proprietary, but early reports [9,18] mention a supported sulfonic acid at the cartridge head, which binds the nitrosamines by salt formation. The organic acid is also believed to complex the free amines thereby preventing reaction with adsorbed NOx to form additional artifacts. For instance, the presence of aniline in the sample matrix is reported to form phenyl nitrosamines during 20 h methanol extraction [9]. Results

in Fig. 3 show that no additional NMOR or NDMA formation is occurring in the cartridge or during the desorption period under the present conditions. Although this is a higher temperature than applied in methanol reux, the signicantly lower residence time is apparently minimizing this effect. As well, no new nitrosamine species were found to be created by this procedure. The current study pertains to the desorption of NMOR and NDMA from an EPDM rubber compound. Although this is considered to be representative of many rubber products it does not encompass the wide variety of accelerators, polymers, carbon blacks and other ingredients potentially in use. A recommendation to accommodate rubber compounds in general is a 60 min desorption at 150 C. The extended desorption time should allow volatile nitrosamines of higher molecular weights or higher solubilities to fully extract. The current data from 30 to 60 min (Fig. 3) indicates that extended desorption times do not have deleterious effects on the recoveries of the nitrosamines studied here. The NMOR values are comparable between the solvent extraction and thermal desorption methodologies. However, the NMDA values are not, since no NMDA was detected by solvent extraction. The thermal desorption procedure involves trapping the volatiles immediately after extraction whereas solvent extraction necessitates a long evaporation step of 98 mL methanol. In the present study, care was taken to maintain the sample near 25 C during this concentration step even though the original method employed 40 C under reduced pressure. The risk of losing the lightest components is inherent in any solvent evaporation methodology, and this shortcoming has been observed elsewhere for solvent extraction of nitrosamines from rubber [12,14]. 3.2. Method precision and detection limit An additional validation test was performed by injecting liquid nitrosamine standard onto a non-nitrosamine rubber compound and measuring the corresponding recoveries, as was previously performed on baby bottle nipples [14]. Results for these

260 Table 1 Spike recovery results Amount detected (ng) Rubber Compound NDMA NDEA NDPA NDBA NPIP NPYR NMOR Trial 1 NDMA NDEA NDPA NDBA NPIP NPYR NMOR Trial 2 NDMA NDEA NDPA NDBA NPIP NPYR ND ND ND ND ND ND ND 58.6 63.2 134 206 209 193 195 66.5 67.7 142 222 220 909

J.A. Incavo, M.A. Schafer / Analytica Chimica Acta 557 (2006) 256261 Table 3 Detection limits Amount spiked (ng) 0 0 0 0 0 0 0 62.5 62.5 125 188 188 188 188 63.0 63.0 126 189 189 189 Recovery (%) 94 101 108 110 111 103 104 106 107 113 117 117 111 ng/g compound NDMA NDEA NDPA NDBA NPIP NPYR NMOR Based on triplicate runs. 6.3 9.3 13 11 8.8 8.2 9.3 S.D. 0.95 0.74 1.2 0.75 0.14 0.9 0.13

Note: Recoveries based on single experiments.

made on separate 1.000 g samples of an aged rubber compound at 150 C/20 min. Percent relative standard deviations are shown to be within 10% for these components at the 71.4 and 248 ppb levels. The GC/TEA detection limit was established by measuring peaks in the calibration solution at three times the instrument noise level. Results are shown in Table 3 based on a 1.000 g sample basis. Lower detection limits should be achievable from larger sample amounts, but adequate heat transfer to the greater mass of rubber must be ensured. Note that these values quoted here are limits of detection, and limits of quantitation for the method would need to be established by assessing standard deviations at levels near the detection limits [19].

experiments are shown in Table 1. This rubber compound contained no secondary amine accelerators and no nitrosamines were detected in the blank analysis. The results show that when 62.5189 ng amounts of various nitrosamines from a certied standard were injected onto the same rubber compound and subsequently desorbed, full recoveries were obtained to within 20%. It was found that complete recovery of this spiked liquid standard was obtained with 20 min desorption timesconditions that do not yield full recoveries from an actual nitrosamine-containing rubber compound. These results further illustrate, the importance of developing the methodology on rubber products in which the nitrosamines are thoroughly incorporated into the bulk of the compound during mixing and curing, as is accomplished here. Results for the reproducibility of the thermal desorption method are shown in Table 2. These repeat measurements were
Table 2 Method precision results NDMA (ng/g) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 n Mean S.D. % R.S.D. 68.5 71.2 70.4 73.5 83.1 61.7 6 71.4 7.0 10 NMOR (ng/g) 261 246 251 259 256 213 6 248 18.0 7.3

4. Summary A simplied analytical method has been developed and validated for the determination of trace volatile nitrosamines in carbon black-loaded rubber compounds. The method combines the established air analysis procedures using Thermosorb/N sorbent tubes and GC/TEA instrumentation with a simple thermal desorption step for extraction of the analytes. A major advantage identied over the traditional solvent extraction technique is an intrinsically better recovery of the most volatile nitrosamines such as NMDA. Other issues associated with methanol extraction such as potential losses due to precipitation and oil immiscibility are circumvented with the present method. Perhaps, the most signicant benet of the method is its simplicity. This affords a total analysis time reduction from daysfor the solvent extraction and evaporation techniqueto hours for the present technique. Because there is much less operator intervention and sample handling, the potential for operator error is diminished. Eliminating the solvent extraction is also advantageous from a health, safety and environmental standpoint.

Acknowledgements The authors appreciate the laboratory assistance provided by A. Winter, E. Lauck and A. Smith as well as the cooperation of E. Johnson and M. Porter.

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