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Kansas City, Missouri 64110. A new LC/MS method has been developed for the simultaneous measurement, in water and wastewater samples, of all species ...
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Environ. Sci. Technol. 2001, 35, 1223-1230

Derivatization LC/MS for the Simultaneous Determination of Fatty Alcohol and Alcohol Ethoxylate Surfactants in Water and Wastewater Samples JOCELYN C. DUNPHY,* DANIEL G. PESSLER, AND STEPHEN W. MORRALL The Procter & Gamble Company, P.O. Box 538707, Cincinnati, Ohio 45253 K. ALEX EVANS Shell Development Company, Westhollow Research Center, Houston, Texas 77251 DAVID A. ROBAUGH Robaugh & Associates, 425 Volker Boulevard, Kansas City, Missouri 64110 GORDON FUJIMOTO AND ANDRE NEGAHBAN Midwest Research Institute, 425 Volker Boulevard, Kansas City, Missouri 64110

A new LC/MS method has been developed for the simultaneous measurement, in water and wastewater samples, of all species contained in commercial samples of linear type of alcohol ethoxylate (AE) surfactants including fatty alcohols. The method requires derivatization of the terminal hydroxyl of each surfactant species with 2-fluoroN-methylpyridinium p-toluenesulfonate, which imparts a permanent cationic charge, allowing all species including the fatty alcohols and those with only one ethoxylate to be effectively detected by electrospray MS. Detection limits of typically 20. In 1998, an estimated 465 million lb (211 million kg) of linear AE was consumed in the United States (1). Although readily biodegraded and highly removed in sewage treatment plants (2), with such high volumes, usage in consumer detergents, and disposal down-the-drain, it is important for accurate risk assessments to be able to accurately monitor any trace concentrations reaching the environment after treatment. Most data existing on the environmental concentrations of AE rely on non- or semi-specific analytical approaches. Only a few studies have addressed the range of components present in the environment in part because the levels are low and because of the difficulty of the analysis (2, 3). The key to refining the environmental risk assessment of linear AE further is an improved understanding of the fate of each species in the mixture, and thus an analytical method was needed that would more effectively quantitate all components. Analytical methods for these materials have progressed over the years. Early methods were based on colorimetric end points were designed for raw material and product quality control and did not distinguish among individual species, although they were adapted to environmental samples (46). LC/UV methods were also developed but required precolumn derivatization and resolved species only by chain length and not ethoxylate number (7, 8). A GC/MS method (9, 10) based on cleavage of the ethoxylate moiety and derivatization of the resulting alcohols with HBr was also developed for total AE but is not capable of providing ethoxylate distribution information. This limitation exists because, first, GC cannot resolve ethoxymers higher than 10 or so, and second, the EI or CI MS detection is not sensitive enough for the trace levels of each species of AE in environmental samples. However, collapsing all the ethoxylates in a single peak per chain length has allowed this method to be used for a variety of environmental safety studies with AE, including a monitoring study in 1995 (11). Lately, liquid chromatography/mass spectrometry (LC/MS) methods have gained increased use due to the gain in selectivity and resolution of all species. Evans et al. reported a thermospray MS method for environmental AE samples (12) that has been used in safety assessments (2) and have done recent work indicating that electrospray ionization can provide increased sensitivity (13). Crescenzi et al. have also reported a similar electrospray method (14). In both electrospray and thermospray, ionization is accomplished by the formation of adducts with hydrogen, ammonium, or sodium cations. The ionization efficiency for each species varies greatly, and for the fatty alcohol and single ethoxylated material is so poor that these approaches cannot be effectively used. It is worth mentioning in this context that a related class of surfactants, alkylphenol ethoxylates (APEs), has been studied in the environment using similar methodologies including GC, LC, and various MS techniques (15). However, APEs are easier to measure at trace levels than AEs because the presence of the phenolic moiety increases UV response and also allows alkylphenol (EO ) 0) to be effectively ionized relative to straight-chain fatty alcohols. Therefore, we could not apply these methods directly to our goal. On the basis of a recent paper by Van Berkel et al. (16) and others before (17), we decided to investigate the derivatization of AE using a reagent (2-fluoro-N-methylpyridinium p-toluenesulfonate (Pyr+)) that would react with the primary alcohol of each AE species and thus impart a permanent charge. The detection by electrospray/MS would VOL. 35, NO. 6, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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thus not rely on the delicate chemistry of in-solution adduction of cations to the analytes of interest. We found this approach to be experimentally facile and successful in derivatizing the entire AE distribution. Method development included optimization of the derivatization procedure, HPLC separation of derivatized species, development of a new solidphase extraction isolation procedure, and optimization of the mass spectrometric conditions. Discussion of our experimental findings during method development is presented here along with results from validation experiments and comparative data across different methods on wastewater samples.

Experimental Section Materials and Equipment. Reagents/Solvents. The derivatization reagent Pyr+ was purchased from either Sigma Chemical Co. or Fluka Chemical Co. in the most pure form available (>99%) and was used as received. Solvents were purchased from Burdick and Jackson, Fisher, or Aldrich and were all HPLC grade. These included methanol, dichloromethane, acetone, acetonitrile, tetrahydrofuran, ethyl acetate, triethylamine, and formic acid. Water used in the method work was obtained from a Waters Millipore purification system, referred to as Milli-Q water. Standard and Reference Materials. Development and validation experiments were performed with the following materials: NEODOL 23-1 (commercial grade linear oxo type AE, C12-C13 with average EO of 1), NEODOL 25-9 standard (commercial grade linear oxo type AE, C12-C15 with average EO of 9), NEODOL 25-6 standard (noncommercial sample linear oxo type AE, C12-C15 with average EO of 6), GENAPOL T110 (commercial grade linear AE, C16 and C18 with average EO of 13), andD27-C13E9, (C13 alkyl chain perdeuterated, average EO of 9). All NEODOL samples were obtained from Shell Development Company, Houston, TX. The GENAPOL material was obtained from Clariant Gmbh. Wastewater Samples for Initial AE Method Comparison. Samples of wastewater influent from three treatment plants in southwestern Ohio were collected in 1997 as 3-day composite samples and preserved on-site with formalin. Data on characteristics of the influent were obtained directly from the treatment plant records. These were as follows: biological oxygen demand (BOD), total suspended solids (TSS), nitrite/ nitrate, ammonia, and phosphate. All values were within acceptable and typical limits for these plants. Environmental Matrixes Used in Validation. The water matrixes used in method validation were grab samples taken from the influent and effluent of the Polk Run wastewater treatment plant near Cincinnati, OH. The effluent water samples were collected on two dates: February 11 and April 14, 1999. The influent samples were collected on April 14, 1999. Formalin was added to these samples as a preservative at the time of collection. Solid-Phase Extraction Cartridges Used. Varian Mega Bond Elut C2 (2 g) Part No. 1225-6056, Lot 032811; Varian HF Mega Bond Elut SAX (2 g) Part No. 1425-6021, Lot 780700; Varian Mega Bond Elut SCX (2 g) Part No. 1425-6019, Lot 772209 were used. Derivatization Procedure and Preparation for LC/MS Analysis. To each standard or sample dissolved in 30 mL of organic solvent (typically AcN), a stir bar, 100 µL of triethylamine (TEA), and 0.2 g of solid derivatizing agent (N-methyl2-fluoropyridinium p-toluenesulfonate) were added. The D27C13E9 internal standard was added just before derivatization to help identify a failed reaction and to account for differential mass spectrometer response with species of varying EO number. Samples were then capped and stirred gently a minimum of 2 h without heating. After being stirred, the derivatized sample was taken to dryness under nitrogen at 25 °C (N-Evap), yielding a yellow-brown oily residue. The 1224

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TABLE 1. m/z Values for Derivatized Homologues of Alkyl Ethoxylates and D27-C13E9 Alcohol Ethoxylate Internal Standard alkyl chain length EOa

C12

C13

C14

C15

C16

C18

IS-C13

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

278 322 366 410 454 498 542 586 630 674 718 762 806 850 894 938 982 1026 1070

292 336 380 424 468 512 556 600 644 688 732 776 820 864 908 952 996 1040 1084

306 350 394 438 482 526 570 614 658 702 746 790 834 878 922 966 1010 1054 1098

320 364 408 452 496 540 584 628 672 716 760 804 848 892 936 980 1024 1068 1112

334 378 422 466 510 554 598 642 686 730 774 818 862 906 950 994 1038 1082 1126

362 406 450 494 538 582 626 670 714 758 802 846 890 934 978 1022 1066 1110 1154

319 363 407 451 495 539 583 627 671 715 759 803 847 891 935 979 1023 1067 1111

a

EO, number of ethoxylate units in AE homologue.

residues were reconstituted in 1 mL of 40% AcN/60% H2O and refrigerated until ready for analysis. The reconstituted sample was passed through a 0.2-µm PTFE syringe filter prior to injection into the LC/MS.

Liquid Chromatography/Mass Spectrometry Instrumentation. Mass spectrometric experiments conducted during the time in which the method was being initially developed were performed on a Perkin-Elmer Sciex API III LC/MS at The Procter & Gamble Company (positive ion mode) under the general conditions: flow (split postcolumn) was 70 µL/min and orifice voltage was 50 V. Data were taken in full-scan mode, and the mass range scanned was typically 250-1200 amu. LC system included a Waters 600MS pump and a Waters Ultra-Wisp 715 autosampler. Flow rates were 1.0 mL/min, and injection volumes were 30 µL. In the early stage of the project, we used flow injection (no separation column) for feasibility work. For environmental samples, we developed an LC method using SupelcoGel TPR-100, 4.6 mm i.d. column. We used a corresponding guard column in line. The mobile phase was a water/acetonitrile gradient with 0.01 M formic acid (detailed further below). The ions monitored for the MS analysis are presented in Table 1. The LC/MS system used during the validation was a Micromass Quattro I triple quadrupole mass spectrometer at Midwest Research Institute, operating in the positive ion electrospray (ESP) mode. The HPLC system used was an HP 1090 equipped with a variable volume autosampler. The HPLC column used was 10 cm × 2.1 mm SupelcoGel TPR100. Mobile phase consisted of water/acetonitrile with 0.01 M formic acid. The gradient started at 60/40 water/acetonitrile and was held for 5 min, then ramped from 5 to 25 min up to 10/90 water/acetonitrile, and ramped again from 25 to 30 min to 100% acetonitrile. A final step consisted of 15 min at the initial mobile phase composition to reequilibrate prior to the next injection. Flow from the analytical column at 0.2 mL/min was sent to the electrospray probe.

FIGURE 1. (a) NEODOL 23-1 full-scan mass spectrum without derivatization. (b) NEODOL 23-1 full-scan mass spectrum as Pyr+ derivative.

Results and Discussion Derivatization Procedure. Initial work on this method involved optimizing the conditions of the derivatization reaction and verifying complete derivatization of all AE homologues. Using 14C-radiolabeled AE, a combination of TLC with rad detection, 1H NMR spectroscopy, and flowinjection electrospray mass spectrometry were used to monitor the derivatization reaction. Initial derivatization attempts in chloroform solvent (10 mL) with pure AE materials (at 2000 ppm concentrations) were not successful because the ethanol preservative in the chloroform consumed the Pyr+ reagent. Substituting methylene chloride for chloroform as a solvent eliminated this problem. Other variables that were critical to ensure complete derivatization were the amount of derivatization reagent, Pyr+, and base catalyst (triethylamine). Excess Pyr+ and TEA (optimized at 0.2 g and 100 µL, respectively) are important to compensate for impurities such as traces of moisture that may be present in the samples or reagents themselves. The reagent is very hygroscopic, and upon repeated exposure to air can trap

moisture; this is observed by aggregation and stickiness of the solid. A reagent received in this condition or allowed to deteriorate over time was found in our hands to effect almost complete failure of the derivatization reaction. In addition, some grades of this reagent were sold as low as 85% pure. To be safe, we always purchased the highest reagent grade possible (95 or 99%) and made sure it was a free-flowing powder before use. Interestingly, we found that the color of the derivatization solution could be an indicator of success of derivatization. Standard solutions of AE turned a golden yellow, while extracts of influent and effluents turned a faint yellow during the derivatization reaction. If no color change was observed, we nearly always observed very poor (10% or less) derivatization efficiency and often traced the problem back to introduction of excess water. The power and selectivity of the derivatization technique is demonstrated with a sample of NEODOL 23-1 (Shell Chemical Co. a commercial linear-oxo type C12/13 alcohol 1 mol average ethoxylate). Samples of derivatized and neat NEODOL 23-1 were diluted to 22.1 ppm in methanol and VOL. 35, NO. 6, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Total Micrograms and Percent of Each Homologue Lost upon Taking a Solution of AE 25-9 Standard to Dryness Prior to Derivatization drying of AE after SPE and before derivatization C no.

EO

12 12 12 12 13 13 13 13 14 14 14 14 15 15 15 15

EO-0 EO-1 EO-2 EO-3 EO-0 EO-1 EO-2 EO-3 EO-0 EO-1 EO-2 EO-3 EO-0 EO-1 EO-2 EO-3 total µg lost total % lost

a

% of total in stda

% loss upon drying

total loss at 50 µg

0.288 0.328 0.510 0.745 0.299 0.339 0.528 0.771 0.247 0.281 0.437 0.638 0.206 0.234 0.364 0.532

55.2 53.2 17.1 14.4 87.5 38.1 0.0 6.7 58.6 13.7 15.1 0.0 33.8 5.0 0.0 2.5

0.08 0.09 0.04 0.05 0.13 0.06 0.00 0.03 0.07 0.02 0.03 0.00 0.03 0.01 0.00 0.01 0.66 1.31

NEODOL 25-9 standard.

analyzed by electropray/MS using a 80:20 methanol:water mobile phase. Figure 1 contrasts the flow-injection full-scan MS spectra of both samples. The spectrum of the underivatized material is complex, with both ammonium and sodium adduct ions present. However, the lowest mass ions of significant intensity at m/z 292 and 306 correspond to ammonium adducts of C12E3 and C13E3. Ions corresponding to the diethoxy, monoethoxy, and fatty alcohol species are not observed. Significant chemical noise is observed at the higher masses. The mass spectrum of the derivatized NEODOL 23-1 is simpler, and the ions corresponding to all AE homologues are clearly evident. For example, derivatized C12 and C13 alcohols are observed at m/z 278 and 292, respectively. This spectrum of derivatized AE is more representative of a NEODOL 23-1 homologue distribution than the ESI/MS spectrum of underivatized material. Aqueous Sample Preparation. Analyte Loss during Solvent Evaporation. Prior to working out detailed extraction conditions, we investigated a potential pitfall of needing to take our final eluents to dryness before derivatizationsthat of differential analyte loss. It is a common practice in SPE to evaporate the eluted sample to dryness prior to the addition of an internal standard or derivatization step and was potentially needed for us as we could not tolerate water in the final mixture or any other protic solvents we made need to use for sample elution. From previous experience, we were concerned about losing the less polar/more volatile lower ethoxymers during solvent evaporation. To test this concern, duplicate solutions of 50 and 100 µg/mL NEODOL 25-9 in methylene chloride were prepared. One replicate of each concentration was derivatized directly in solution; the other was taken to dryness under N2 and reconstituted in methylene chloride prior to derivatization. Each sample was analyzed by LC/MS, and absolute peak areas were compared between dried and nondried pairs to determine loss during evaporation. Results for the lower ethoxymers (EO ) 0, 1, 2, and 3) indicate that up to >50% of these species can be lost (Table 2). Relative to the total NEODOL 25-9 material, the loss represents only slightly more than 1 wt %. Thus, for less specific analytical methods, such as HBr cleavage with GC/ MS (9, 10), the loss is not expected to significantly bias results. However, for other more specific methods where quantitation 1226

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of individual AE homologues is a primary goal, additional procedures to improve recovery of the low ethoxymers were needed to avoid biasing results. Extraction Procedures and Results. During the development of the method, we tested several types of SPE cartridges (including C18, C8, and C2). We found that C2 (ethyl) cartridges were consistently the best at retaining the wide range of polarity of analytes that compose commercial AE mixtures. All extractions conducted for the remainder of the results discussed were done with the Varian C2 cartridges. Effluent samples (4 L total volume) were split into two 2-L aliquots, which were extracted with separate C2 SPE cartridges. This was done to reduce the amount of clogging, to minimize the chance of analyte breakthrough, and to facilitate quicker sample preparation. For influent, samples were diluted by a factor of 5 (500 mL of influent:2000 mL of Milli-Q water). The 50-mL aliquots of this diluted influent sample were assayed for AE using the same SPE and elution procedures above except that only one C2 SPE cartridge was used for each sample since a much lower volume sample was required due to higher AE levels. Previous work had shown that a dilution step rather than a small aliquot provided a more representative subsample for analyses of surface active materials (9). As mentioned in the previous section, water was found to quench the derivatization reaction, and finding procedures to keep traces of water to a minimum and yet maximize extraction recoveries represented a significant part of the method development. We investigated several methods of drying the extracts, including pulling air through the extraction cartridges for varying amounts of time once samples had been extracted, baking the cartridges for various time periods at 60 °C, adding sodium sulfate to the organic eluent to remove residual water, and passing the extracts through sodium sulfate cartridges, etc. We found that, of all these procedures, the one that worked consistently the best was to dry the cartridges following extraction by pulling air through them for overnight. This was done by placing on top of each extraction cartridge a blank C18 SPE to minimize any contaminants coming from the air from reaching the sample. It is interesting to note that while drying of solvent-based eluents resulted in losses of the lower ethoxymers, drying the cartridges did not result in similar losses. In fact, using radiolabeled tracers, nearly complete mass balances were obtained. Any material that was not recovered (typically no more than 15% of the total) was found to still be adhered to the cartridge. We hypothesize that the reason the lower ethoxymers are lost upon evaporation but not during cartridge drying has to do with the evaporation loss being due to an azeotropic effect rather than simple volatilization or that the adsorption onto the cartridge is of sufficient strength to overcome the tendency toward evaporation during air-drying. Extraction of 14C-radiolabeled AE and dodecanol spikes (at ppb levels) in Milli-Q water and sewage matrixes revealed that recoveries varied greatly as a function of solvent as well as polarity of individual species. For example, we found that dodecanol gave >85% recovery using both 60/40 methylene chloride/acetone and 100% THF, but the C14EO6 homologue gave 47% recovery in 60/40 methylene chloride and 70% recovery in THF. We concluded following many trials spiking AE into water and extracting that a dual extraction using a 100% organic solvent followed by a very polar solvent mix (including one that contained water) would be optimal for recovery of the total range of ethoxymers present. A twostep elution was developed where the SPE cartridges are first extracted with AcN to remove the lower ethoxymers. This fraction is set aside and not taken to dryness. The second elution is done with 1:1 methanol/ethyl acetate containing 2% water, which is dried to remove the solvent including any

FIGURE 2. (a) Chromatogram of standard NEODOL C25E9 with internal standard at the 20 µg level. (b) Average mass spectrum of the C12 homologue peak from chromatogram above. The large ion at m/z 319 is a background contaminant of unknown origin. residual water. The two fractions, AcN and dried residue, are simply combined and then derivatized as a single sample. Loss of less polar/more volatile ethoxymers is minimized by not taking the first extract to dryness; by taking the second extract, containing the polar ethoxymers, to dryness, the pyridinium reagent is not consumed by reaction with the protic solvent. We used a combination of SCX and SAX cartridges to further directly purify the eluents. Without the additional cleanup through the SCX and SAX cartridges, some samples failed to derivatize completely. It is important to mention the analysis time required. Considering the extraction, derivatization, and MS analysis in total, results can be obtained from an effluent sample approximately 24-36 h after the receipt of the aqueous

sample. The total number of samples that can be processed in this period will depend on equipment available (e.g., number of extraction manifold stations, etc.), but it is practical to analyze >10 samples from, for example, three different WWTPs in a 2-day period. Stability of Derivatized Standards. Standard solutions of C25E9 material were prepared, derivatized, dried under N2, and reconstituted in the initial mobile phase containing 10 mM formic acid and stored for 1 week refrigerated (4 °C). Total ion chromatograms were compared for samples prepared immediately and those after having been stored for 1 week; no changes were observed. LC Method. Our initial method LC development centered on silica-based reversed-phase columns (e.g., ODS). We found that the cationic derivatives are highly retained on silicaVOL. 35, NO. 6, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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based RP-HPLC media and that the addition of a basic modifier, such as TEA to the mobile phase, is needed for chromatographic reasons but causes MS signal suppression problems. When we switched to polymer-based columns, we found good chain-length resolution and good peak shape without using basic modifiers. We had success with both poly(divinylbenzene)/methacrylate (Supelco TPR-100) and poly(vinyl alcohol) columns (Supelco ODP-50). We settled on the use of the TPR-100 column since we found that the separation achieved appeared to be less sensitive to changes in mobile phase composition than with the ODP-50 column, and the separations were more easily reproduced. The addition of 10 mM formic acid to the aqueous/organic mobile phase succeeded in resolving the derivatization agent and byproducts from the AE analytes on the TPR-100 column with good peak shapes. However, derivatization byproducts eluting in the initial portion of the HPLC separation tended to foul the electrospray ionization source and reduced sensitivity. This problem was solved by diverting the HPLC effluent to waste for the first 8-10 min of the separation. Total acquisition times were typically 30 min, and homologues of AE had retention times between 15 and 23 min. We found that the D27-C13E9 internal standard was a useful retention reference to aid in analyte identification. Figure 2a shows a typical TIC (250-1200 Da) mass chromatogram of a 20-µg derivatized NEODOL 25-9 (C25E9) standard. The peaks between 10 and 16 min are the AE derivatives separated by alkyl chain. Little resolution of ethoxymers is observed. Figure 2b is the combined mass spectrum of the C12AE peak. It should be noted here that commercial AE materials we tested are “essentially linear”, typically containing about 20% 2-alkyl-branched fraction of each ethoxymer. In the development of this method, we determined that the monobranched species were at best chromatographically resolved only somewhat from their linear counterparts. The resolution was higher for the species eluting in the early part of the chromatogram (EO ) 0, 1, or 2) but lower or indistinguishable for those later-eluting species. In our integration methods then, we attempted to include in all cases (and certainly did where no resolution was possible), both the completely linear and mono-branched component of each ethoxymer. MS Quantitation. Addition of Pyr+ effectively increases the MW of the corresponding AE homologue by 93 Da. Calibration curves for each species in the standard were developed based on area ratios of the standard homologue and the C13 internal standard homologue with the same degree of ethoxylation. For example, C12E1, C13E1, C14E1, and C15E1 homologues are ratioed against the D27-C13E1 internal standard homologue. Chromatographic resolution of AE homologues by alkyl chain enables the selected ion recording (SIR) function to monitor for C12 and C13 homologues early in the HPLC run, shifting to the higher masses for C16 and C18 AE at ∼18 min for improved sensitivity. The C14, C15, and the D27-C13E9 internal standard AE homologues were monitored throughout the chromatographic separation. With this arrangement only 5 of the 7 chain-length series had to be monitored at any time. Figure 3 shows extracted data specific to C14AE, indicating the retention pattern across the range of ethoxymers. Individual standard curves were constructed for each homologue by plotting the peak area ratios of derivatized AE standards/internal standard versus concentration. The use of an internal standard enables a high degree of linearity (correlation coefficients >0.998) to be obtained across the entire suite of AE species. Without a suitable deuterated internal standard, such matrix effects as ion suppression and multiple charged adduct formation would severely limit quantitation. The concentration range over which a calibration line for a particular AE homologue was constructed is different for 1228

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FIGURE 3. Mass chromatograms of C14AE for different AE homologues from the analysis of a derivatized AE standard solution.

TABLE 3. Average Percent Recoveries and Relative Standard Deviation (n ) 4) of Recoveries (in Parentheses) of AE from Spiked Effluent Samples EO

C12

C13

C14

0 84 (11) 47 (20) 26 (28) 1 53 (18) 40 (45) 36 (34) 2 112 (23) 103 (17) 59 (48) 3 95 (15) 75 (8) 64 (14) 4 61 (9) 82 (21) 75 (7) 5 76 (7) 83 (3) 78 (7) 6 89 (15) 81 (4) 80 (4) 7 78 (2) 78 (3) 61 (33) 8 89 (6) 84 (4) 80 (2) 9 82 (8) 78 (3) 78 (4) 10 91 (10) 85 (4) 80 (3) 11 90 (12) 91 (3) 89 (3) 12 91 (12) 88 (9) 84 (9) 13 100 (14) 81 (6) 90 (12) 14 97 (11) 92 (7) 89 (13) 15 96 (18) 85 (8) 92 (14) 16 95 (9) 84 (6) 91 (17) 17 103 (14) 90 (11) 105 (11) 18 91 (12) 77 (11) 89 (7)

C15

C16

C18

29 (38) 28 (10) int (int)a 31 (44) 52 (6) 76 (10) 74 (5) 63 (11) 78 (2) 73 (5) 84 (3) 86 (3) 82 (6) 77 (5) 90 (13) 85 (15) 89 (12) 97 (15) 93 (13)

43 (12) 60 (34) 31 (114) 29 (72) 49 (17) 46 (15) 53 (6) 38 (66) 49 (22) 56 (13) 63 (12) 69 (7) 68 (7) 64 (7) 70 (10) 77 (8) 72 (8) 93 (13) 89 (13)

39 (12) 41 (9) 39 (11) 38 (16) 35 (13) 41 (7) 37 (8) 29 (20) 38 (13) 44 (13) 49 (17) 49 (18) 24 (15) 55 (12) 58 (19) 62 (19) 62 (23) 65 (26)

a int, interfering components in the effluent blank samples prevented evaluation of the homologue.

each AE component since it depends on the percent composition of that homologue in the NEODOL 25-9 (or GENAPOL T110) used to create the standard solutions. Validation of MS Analysis of AE in Sewage and WWTP Effluent. Two sets of effluent samples were analyzed following the procedures described in the Experimental Section. Each sample was spiked with the 5 µg/L NEODOL 25-9 and 2.5 µg/L of the GENAPOL materials described earlier. The entire analytical procedure of extraction, derivatization, and LC/ MS analyses was performed in duplicate for each effluent. Average recoveries and relative standard deviations of recovery (Table 3) are presented for the AE homologues spiked into the effluent samples. Concentrations of AE in unspiked sample aliquots were subtracted from the total

TABLE 4. Limit of Detection (LOD) of AE in Effluent Samples (ng/L)a EO

C12

C13

C14

C15

C16

C18

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

8.0 2.6 7.2 ? 6.2 3.5 1.9 3.4 4.1 3.7 3.1 2.6 3.8 3.8 2.4 4. 3.9 2.5 2.9 4.3

? 12.2 ? 0.8 10.3 ? 21.7 3.1 2.9 2.9 4.1 2.8 5.0 5.9 3.1 4.8 2.9 3.4 4.6 5.6 5.0 6.1

7.8 ?4.6 ?10.4 2.8 7.5 2.5 2.3 1.7 2.4 1.9 2.8 4.0 3.3 3.6 5.7 4.0 5.3 8.7 6.5

2.4 ?2.9 ?22.7 ?8.0 ?7.7 4.4 1.3 4.7 3.8 2.9 0.6 3.9 8.2 9.0 12.6 4.8 4.4 10.8 6.2

7.5 ?1.3 ?4.9 ?1.8 ?1.7 1.5 0.5 1.9 0.7 1.5 3.2 1.7 3.4 5.6 5.8 3.2 3.9 4.5 2.5

1.8 0.1 1.0 0.7 0.7 0.8 1.1 1.2 1.3 2.0 b 8.8 5.1 14.0 1.9 5.6 4.1 5.2 6.2

TABLE 5. Influent Concentrations (µg/L C25AE) HPLC/ES/MS

Pyr+/LC/MS

HBr/GC/MS

WWTP

(EO ) 2-18)

(EO ) 2-18)

(EO ) 0-20)

(measd EO)

(av EO ) 9)

Lebanon Columbus Xenia

899 915 765

619 843 796

953 1103 1181

882 1210 824

1424 1727 1327

a Values preceded by ? are estimated LODs. Interfering components were present in the effluent samples that prevented accurate measurement of the LODs for these AE homologues. Note that LOQs for each homologue are approximately 3.33 times the corresponding LOD. b Calibration curve unavailable.

measured concentrations in calculating recovery. Very similar recoveries and RSDs were obtained from spiked sewage influent samples, although the total AE level is approximately 100 times greater than in corresponding effluent. Interferences of unknown origin in some of the chromatograms made it difficult to identify the AE in some of the samples. MS/MS could be useful in discerning between the chemical background and the derivatized AE homologues, particularly for the lower ethoxylates (EO ) 0, 1, or 2). However, in actual analyses, it is advisable to perform a lab or field spike for LOQ and LOD determination as well as sample recovery. In this case, MS/MS becomes unnecessary because peak size increase in the spiked sample facilities correct peak identification. Influent samples were not subject to these interferences because of the higher overall levels of AE present. For the purposes of this study, the limit of detection (LOD) and limit of quantitation (LOQ) were determined from assays of effluent spiked with AE at a concentration above the expected LOQ. A total of 5.0 µg/L Neodol 25-9 was spiked along with 2.5 µg/L Genapol. In addition, because we expected disproportionately higher levels of alcohols, an additional 0.038 µg/L of each alcohol was also included in the spike. These spike levels were chosen because they were in the range of what we expected to see in most effluent samples (1-10 µg/L total AE) and were enough above our expected limit of detection to be able to accurately determine both recoveries and extrapolated LODs/LOQs as described below. Since the limiting factor in determining the LOD and LOQ for the AE in wastewater and effluent was not the instrument detection limit but rather the presence of endogenous background interferences that can distort the AE peak of interest, each individual LOD and LOQ was determined separately by extrapolating the signal at the spiked concentration to the corresponding signal-to-background of 3:1 (LOD) and 10:1 (LOQ). While these values may vary from sample to sample, we believe they provide a good benchmark, relative to other methods, the concentration of analyte that provides a response sufficiently precise to yield a satisfactory estimate of an unknown concentration. Table 4 provides a representative set of calculated LODs (in ng/L) from an

FIGURE 4. (a) EO distribution of C12AE in Lebanon WWTP influent vs C25EO9OH standard. (b) EO distribution of C13AE in Lebanon WWTP influent vs C25EO9OH standard. effluent sample used in the validation study. LOQs are approximately 3.33 times the LOD value. An LOQ of 1.7 µg/L total AE in effluent was determined by summation of all LOQs for the individual homologues. Note that this “total AE concentration” LOQ is only valid for the AE distribution (NEODOL 25-9 and GENAPOL T110) and sample matrix tested. Pyr+LC/MS Method vs HBr and ESI LC/MS Methods Relative to AE Distribution Measured. The Pyr+LC/MS method has the advantage of measuring a greater portion of commercial AE distribution relative to the HBr GC/MS (9, 10) and ESI LC/MS (13, 14) methods. However, when all methods are examined based on their capabilities, the Pyr+LC/MS is consistent with these methods. To demonstrate this, we analyzed three different wastewater samples (Lebanon, Columbus, and Xenia in Ohio) by each of the three methods referenced (Table 5). Each sample was analyzed only once; however, our method development had indicated a variance of approximately 10% for influent samples. The ESI LC/MS method determined AE homologues with alkyl chains from 12 to 15 carbons in length, which had from 2 to 18 EO groups. A valid comparison can be made between the two electrospray-based methods by summing only these same C12-15E2-18 homologues. Derivatization with HBr integrates all EO homologues by alkyl chain length. Using an assumed EO distribution (average ) 9), the total AE concentrations calculated from the GC/MS data are high. When the actual (measured) EO distribution is used (approximately VOL. 35, NO. 6, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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4), the calculated values are similar to those determined by Pyr+/LC/MS when summing all ethoxymers. AE homologue distributions span a wide range of molecular weight, solubility, and volatility. It is possible that the AE composition in the environment may not reflect any one commercial material due to both physical and biological processes in the sewer and beyond. It is also possible that measured AE distributions have contributions from related surfactants, such as alcohol sulfate (AS) or alcohol ethoxysulfate (AES), for which commercial blends typically have lower average EO values than AE surfactants and which can undergo sulfate hydrolysis to the alcohol in-sewer. As an example, see Figure 4 in which an ethoxylate distribution for C12 and C13 chain lengths found in the Lebanon wastewater (influent) has a higher proportion of lower EO homologues than the AE standard used for the analysis. One explanation is that these lower EO homologues come from AS or AES. As stated above, the other methods compared here would be unable to draw this conclusion as definitively. Development of the Pyr+/LC/MS methodology has provided the capability to obtain more complete AE distribution data than previous methods. The method has recently (1999/2000) been used to survey a variety of environmental concentrations of AE in WWTP influents and effluents around the United States, to be reported separately (18). The data are being used to refine the environmental risk assessments for AE, and should form the basis for a combined risk assessment for the class of related ethoxylated surfactants.

Acknowledgments We thank Bill Begley, Manuel Cano, Bill Eckhoff, Ron Herzog, Eddy Matthijs, Staci Simonich, Jeff Seeley, David Wernery, and Andrew Sherren for helpful discussions during planning and execution of this work.

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Received for review July 17, 2000. Revised manuscript received November 8, 2000. Accepted December 20, 2000. ES001491Q