Anal. Chem. 2007, 79, 5093-5096
Analysis of Acrylamide in Water Using a Coevaporation Preparative Step and Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry Shaogang Chu and Chris D. Metcalfe*
Worsfold Water Quality Center, Trent University, Peterborough, Ontario, K9J 7B8, Canada
Acrylamide is a probable human carcinogen, and the drinking water quality guideline for this compound is 0.5 mg/L. However, analysis of this compound in water is difficult because of its very high water solubility, which limits the efficiency of sample preconcentration prior to analysis. We developed a robust and sensitive analytical method for the determination of trace quantities of acrylamide in samples of water using a novel preparative technique and isotope dilution liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization as the ion source (LC-APCI-MS/MS). The preparative method involves coevaporation of acrylamide with water at pH 10 using a rotary evaporator, followed by acidification to pH 3.0 and concentration of the sample prior to analysis by LC-APCI-MS/MS. To compensate for the loss of the analyte during sample preparation and signal suppression due to interference from the sample matrix, isotope dilution with acrylamided3 was used for quantitation. Using this method, analyte recoveries ranged from 74 to 103% for acrylamide spiked into water at a concentration of 0.4 ng/mL. The limit of detection and limit of quantification (LOQ) for acrylamide in water were 0.02 and 0.06 ng/mL, respectively. This method was successfully applied to determine trace levels of acrylamide in samples of river water and in runoff from an agricultural field to which municipal biosolids (i.e., sludge) had been applied. Concentrations of acrylamide in these samples ranged from 55 and 75 > 58, respectively. System control and data evaluation were done with Analyst 1.4 software. Quantification was performed using an internal standard method with a five-point calibration curve spanning the range of anticipated analyte concentrations in the samples and using a weighted (1/ concentration) linear regression. RESULTS AND DISCUSSION Sample Preparation. Rufian-Henares and Morales17 reported that acrylamide was largely lost from an aqueous solution when evaporated to complete dryness. It appears that acrylamide can significantly coevaporate with water, although the vapor pressure of acylamide is only 0.009 kPa at 25 °C.1 Our results showed that acrylamide coevaporated with water to a greater extent when it was in a basic aqueous solution, in comparison to evaporation from an acidic solution. When the pH was adjusted to 10, ∼50% of the acrylamide remained in the flask after drying. When an acrylamide aqueous solution was acidified to pH 3, ∼70% of the acrylamide remained in the flask when it was completely dried. Therefore, adjusting the pH by acidifying the condensate before the final concentration step improves analyte recovery. Although it was not possible to collect all of the acrylamide that coevaporated from the boiling flask, it was possible to collect about half of the dissolved acrylamide in the receiving flask of (17) Rufian-Henares, J. A.; Morales, F. J. Food Chem. 2006, 97, 555-562.
Figure 1. MRM chromatograms of acrylamide-d3 (IS) and acrylamide in a water sample prepared from agricultural runoff.
the rotary evaporator. Because the sample had been distilled by this evaporation step, it was relatively free of interferences from the sample matrix in the final sample. Since isotope dilution with acrylamide-d3 was used for quantitative determination, the internal standard was subject to the same recovery efficiencies, as well as analytical matrix effects. The coevaporation process was completed within ∼90 min. Figure 1 shows a typical MRM chromatogram of acrylamide in a sample of agricultural runoff, in which the measured concentration of the analyte was 0.09 µg/L. To test the stability of acrylamide in this process, surface water from a river (i.e., Otonabee River, Peterborough, ON, Canada), which was adjusted to pH 3 and 10, was spiked with acrylamide at a concentration of 100 ng/L and heated at 90 °C for 1 h (n ) 3 per treatment). The samples were then analyzed by LC-MS/ MS. There was no significant difference in the concentrations of acrylamide in the samples before and after heating, which was interpreted as evidence of thermal stability of the analyte. The ratios of the concentrations in the samples before and after heating were 100.2 ( 1.7 and 99.4 ( 1.5% for the samples at pH 10 and pH 3, respectively. Since acrylamide is very soluble in water, enrichment of this compound from water is very difficult using traditional techniques such as liquid-liquid extraction or SPE. In fact, liquid-liquid extraction and SPE have been widely used as cleanup steps for extracts prepared from food samples, as interfering compounds are extracted into the organic phase or are retained on the SPE cartridge, while the acrylamide remains dissolved in the aqueous phase.8-10,12,13,18 Prior to the development of this coevaporation preparative method, different SPE extraction methods were evaluated using Oasis HLB (6 cm3, 500 mg), Oasis MCX (6 cm3, 150 mg), and Oasis MAX (6 cm3, 150 mg) cartridges purchased from Waters (Mississauga, ON, Canada), and ENVI-Carb (graphitized carbon, 6 cm3, 500 mg) purchased from Supelco (Oakville, ON, Canada). For these recovery tests, after the cartridge was conditioned, 50 mL of a distilled water sample spiked with 20 ng of acrylamide was loaded onto the cartridges. Then, the cartridge was eluted with 3 × 3 mL aliquots of a suitable solvent (i.e., methanol for Oasis HLB; 5% ammonium in methanol for Oasis MCX; 4% formic acid in methanol for Oasis MAX; and acetone for Envi-Carb with reversed elution). Then 20 ng of the acrylamide-d3 internal (18) Claus, A.; Weisz, G. M.; Kammerer, D. R.; Carle, R.; Schieber, A. Mol. Nutr. Food Res. 2005, 49, 918-925.
standard was spiked into the eluant. The eluant was evaporated to dryness, reconstituted in 200 µL of water, and analyzed by LCMS/MS as described earlier. The results showed that none of these cartridges could efficiently extract acrylamide from the water sample. The highest recovery was only 10%, which was achieved using the Oasis HLB (i.e., mixed-phase) cartridge. For the ENVICarb cartridge, the acrylamide recovery was only 4%, and for the Oasis MCX and MAX cartridges, the recoveries were below 1%. Because this recovery test was conducted with distilled water spiked with acrylamide, no cleanup was necessary, but for aqueous samples from more complex matrixes (e.g., surface water, wastewater, runoff), a wash step would be necessary after extraction to reduce interference from coextractives in the sample. Therefore, recoveries might be even lower for SPE extraction of actual environmental samples with more complex matrixes. In some methods for the determination of acrylamide in foodstuffs, SPE cartridges have been used to cleanup sample extracts.4,8,13 However, in these methods, the volume of the extract loaded onto the SPE cartridge is only a few milliliters and acrylamide is effectively retained in the cartridge. Acylamide has a very high water solubility, and it is eluted from the cartridge when the sample volume is large, as is the case for aqueous environmental samples. Bermudo8 tested the breakthrough volumes for acrylamide, using Strata-X-C and ENV+ cartridges, and showed that, for the ENV+ cartridge (200 mg), the breakthrough volume was only 3 mL. Kawata et al.15 developed an SPE method for acrylamide in water using cartridges packed with activated carbon fiber felt. They achieved a minimum detectable concentration of 0.02 µg/L for acrylamide in water, but to enrich the analyte, they had to use one C18 cartridge and three activated carbon cartridges in series. It was anticipated that extraction of acrylamide from water using this SPE technique would be time-consuming, and besides, this kind of cartridge is not commercially available. To avoid a preconcentration step, Cavalli et al.16 reported a method to determine trace quantities of acrylamide in drinking water by large-volume direct injection (i.e., up to 500 µL) and ion-exclusion chromatography-mass spectrometry. However, this method seems suitable only for drinking water where there is low interference from the aqueous matrix. Analysis. For HPLC separation, a traditional C18 column is not the optimal column for acrylamide analysis, as the retention time of acrylamide on this column is very short.16 However, since C18 columns are the most common LC column used in analytical laboratories, we evaluated this stationary phase for chromatographic performance and showed that it can be used for rapid acrylamide analysis (i.e., retention time of ∼4 min) if interferences from the sample matrix are largely removed. In the literature, water or an aqueous solution of formic acid (or acetic acid) is typically used as an HPLC mobile phase for acrylamide analysis. Under our analytical conditions, a solution of 0.01% formic acid in water increased the MS signal for acrylamide, but the chromatographic resolution was poor. Addition of methanol and acetonitrile to the aqueous mobile phase decreased the response to acrylamide and did not improve chromatographic resolution. Therefore, HPLC-grade water was used as the mobile phase. LC-MS/MS analysis using an electrospray ionization source (ESI) shows higher sensitivity for acrylamide than APCI. However, Analytical Chemistry, Vol. 79, No. 13, July 1, 2007
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ESI is subject to more signal suppression due to matrix effects. Therefore, APCI was used as the ionization source for the analysis of acrylamide. Under these operating conditions, analytical linearity spanned from the limit of detection to 2 mg/L, and correlation coefficients >0.998 were routinely observed for the calibration curves. Method Validation. The matrix effects for the analyte in agricultural runoff were tested by the standard additions method described by Matuszewski et al.19 In the test set, 50-mL volumes of an agricultural runoff sample (n ) 4) were spiked with 20 ng of acrylamide and prepared for analysis by the methods described above. The final reconstituted sample volume was 100 µL. The sample was then divided into two subsamples, A and B. For subsample A, 40 µL of the subsample was spiked with 40 µL of a standard solution containing an acrylamide concentration of 200 ng/mL. For subsample B, 40 µL of the subsample was diluted with 40 µL of water. Note that subsample B was also used to check recovery. By comparing the responses of the analytes in the two subsamples to the responses of the analytes in an external standard (100 ng/mL in methanol), a matrix effect (ME) value was calculated as
ME (%) ) 100(A - B)/(S)
(1)
where A, B, and S are the peak areas of acrylamide in subsample A, subsample B, and the analytical standard (S), respectively. In the four replicates of the agricultural runoff sample, the ME values ranged from 93 to 105%, with an average of 97%. These data indicated that there was little suppression or enhancement of the signal when analyzing samples prepared from a complex environmental matrix (i.e., agricultural runoff). Thus, it appears that there are few coextractives from the water matrix that are present in the samples. In contrast, it would be expected that SPE extraction would retain significant amounts of coextractives from this sample matrix. Therefore, coevaporation appears to be a superior method for analyzing acrylamide dissolved in complex aqueous matrixes. Limits of detection (LOD) and limits of quantification (LOQ) were estimated as the amount of analyte that produced a signalto-noise ratio of 3:1 and 10:1 in water sample, respectively. The LOD and LOQ for acrylamide in runoff were 0.02 and 0.06 ng/ mL, respectively. These values are comparable to the analytical limits for water analyzed by GC-ECD or GC/MS.5,15 Recovery was determined from trials using replicate analyses (n ) 4) of a runoff sample that was spiked at a level of 0.4 ng/mL. The recoveries ranged from 74 to 103%, with an average of 87% (SD ) 14%) using the isotope dilution method (acrylamide-d3 as internal standard). To validate the analytical method, the method was applied to the quantitative determination of acrylamide in surface water and agricultural runoff samples. Two surface water samples were collected from Otonabee River, ON, Canada, and 12 samples of agricultural runoff were collected from tile drains at various times after a rainstorm event. The concentrations of acrylamide in the surface water samples were below the limit of detection (
