Selective Detection of Diethylene Glycol in Toothpaste Products Using

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Anal. Chem. 2009, 81, 8632–8638

Selective Detection of Diethylene Glycol in Toothpaste Products Using Neutral Desorption Reactive Extractive Electrospray Ionization Tandem Mass Spectrometry Jianhua Ding, Haiwei Gu, Shuiping Yang, Ming Li, Jianqiang Li, and Huanwen Chen* Department of Applied Chemistry, East China Institute of Technology, Fuzhou, Jiangxi Province 344000, P. R. China A rapid, sensitive method based on neutral desorption (ND) reactive extractive electrospray ionization mass spectrometry (EESI-MS) has been established for the selective quantitative detection of diethylene glycol (DEG) in toothpaste products without any sample pretreatment. The sensitivity and specificity of DEG detection were enhanced by implementing selective ion/ molecule reactions in the EESI process, featuring the EESI mass spectra with the characteristic signals of DEG. The method provided a low limit of detection (LOD) (∼0.000 02%, weight percent of DEG in toothpaste), reasonable recovery (97.6-102.4%), and acceptable relative standard deviations (RSD < 8%, n ) 8) for direct measuring of DEG in the spiked toothpaste samples. Trace amounts of DEG in commercial toothpaste products have been quantitatively detected without any sample manipulation. The results demonstrate that nonvolatile compounds such as DEG can be sensitively liberated using the neutral gas beam for quantitative detection from the extremely viscous toothpaste containing solid nanoparticles, showing that ND-EESI-MS is a useful tool for the rapid characterization of highly complex and/or viscous samples at molecular levels. On June 1, 2007, the U.S. Food and Drug Administration (FDA) reported that some commercial toothpaste products were contaminated by toxic diethylene glycol (DEG)1,2 up to 3% in weight.3 Similar alerts came out in Singapore, Spain, and Australia.3 Overdosing DEG could cause headache, pain, and even fatal renal failure.4-6 According to the Scientific Committee on Food of the European Union, the tolerable daily intake (TDI) of DEG for adults is 0.5 mg/kg of the body weight.7 Because of the toxicology of DEG, the long-term exposure of DEG from daily toothpaste usage * Corresponding author. Dr. Huanwen Chen, Department of Applied Chemistry, East China Institute of Technology, Fuzhou, Jiangxi Province 344000, P. R. China. Fax: (86)-794-8258-320. E-mail: [email protected]. (1) Brent, J. Drugs 2001, 61, 979–988. (2) Velez, L. I.; Gracia, R.; Neerman, M. F. J. Emerg. Nurs. 2007, 33, 342– 345. (3) http://www.fda.gov/oc/opacom/hottopics/toothpaste.html, accessed March 2009. (4) Hanif, M.; Mobarak, M. R.; Ronan, A.; Rahman, D.; Donovan, J. J.; Bennish, M. L. Br. Med. J. 1995, 311, 88–91. (5) Marraffa, J. M.; Holland, M. G.; Stork, C. M.; Hoy, C. D.; Hodgman, M. J. J. Emerg. Med. 2008, 35, 401–406.

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is highly harmful to health, especially for children and people suffering kidney or liver diseases. In July 2007, Chinese authorities banned the use of DEG in toothpastes and thus the detection of DEG is obliged for toothpaste products on the market.8 It has been extremely challenging in analytical science to rapidly and quantitatively detect analytes (such as DEG) incorporated with highly viscous gel mixtures containing nanomaterials, of which toothpaste is a typical representative. Gas chromatography (GC) or its combination with mass spectrometry (GC/MS) has been the best choice available for the determination of DEG in wines and/or human plasma.9-13 More recently, the determination of DEG in pharmaceutical products and toothpastes has been performed using liquid chromatography mass spectrometry (LC/MS).14-16 The methods mentioned above offer satisfactory sensitivity (limit of detection, LOD, as low as 0.005% by weight) and good reproducibility, thus they are regarded as the routine techniques for DEG detection in various matrixes. However, sample preparations such as derivatization procedures required by these methods are normally time-consuming (>40 min) and laborious. A fast alternative to these methods can be attenuated total reflection-Fourier transform-infrared spectroscopy (ATR-FTIR), which has been successfully demonstrated for the detection of adulterant DEG in toothpaste and gel dentifrices, although it lacks specificity.17 There are huge batches of toothpaste samples (6) O’Brien, K. L.; Selanikio, J. D.; Hecdivert, C.; Placide, M. F.; Louis, M.; Barr, D. B.; Barr, J. R.; Hospedales, C. J.; Lewis, M. J.; Schwartz, B.; Philen, R. M.; St. Victor, S.; Espindola, J.; Needham, L. L.; Denerville, K.; the Acute Renal Failure Investigation Team. J. Am. Med. Assoc. 1998, 279, 11751180. (7) Scientific Committee on Food of the European Union. SCF/CS/ADD/ EMU/198 Final, December 4, 2002. (8) http://spscjgs.aqsiq.gov.cn/xxgkml/ywxx/spxgcpjhzp/spxgcpjhzpscxkzbszn/ gzdt/200707/t20070720_33865.htm, accessed March 2009. (9) Lawrence, J. F.; Chadha, R. K.; Lau, B. P. Y.; Weber, D. F. J. Chromatogr. 1986, 367, 213–216. (10) Litchfie, M. H. Analyst 1968, 93, 653–659. (11) Williams, R. H.; Shah, S. M.; Maggiore, J. A.; Erickson, T. B. J. Anal. Toxicol. 2000, 24, 621–626. (12) Gembus, V.; Goulle, J. P.; Lacroix, C. J. Anal. Toxicol. 2002, 26, 280–285. (13) Maurer, H. H.; Peters, F. T.; Paul, L. D.; Kraemer, T. J. Chromatogr., B 2001, 754, 401–409. (14) Hernandez, F.; Ibanez, M.; Sancho, J. V. Anal. Bioanal. Chem. 2008, 391, 1021–1027. (15) Wu, J. G.; Yuan, J. B.; Liu, Q.; Tang, F.; Ding, L.; Tan, J.; Yao, S. Z. J. Sep. Sci. 2008, 31, 3857–3863. (16) Zhou, T.; Zhang, H. Y.; Duan, G. L. J. Sep. Sci. 2007, 30, 2620–2627. (17) Lopez-Sanchez, M.; Dominguez-Vidal, A.; Ayora-Canada, M. J.; Molina-Diaz, A. Anal. Chim. Acta 2008, 620, 113–119. 10.1021/ac9013594 CCC: $40.75  2009 American Chemical Society Published on Web 09/17/2009

on the market, and thus reliable mass spectrometric methods suitable for the high throughput detection, specific identification, and consistent quantification of DEG in toothpaste products are urgently required. Atmospheric pressure ionization (API) techniques, such as desorption electrospray ionization (DESI),18,19 desorption atmospheric pressure chemical ionization (DAPCI),20,21 direct analysis in real time (DART),22,23 atmospheric-pressure solids analysis probe (ASAP),24,25 and atmospheric pressure glow discharge (APGD)26 have been widely utilized for fast detection of analytes on solid surfaces, normally without any sample pretreatments. Many analytes in the gas phase, in solution, or in the form of aerosols can be directly detected by extractive electrospray ionization (EESI) MS without any sample pretreatments.27-33 For example, after the extraction of DEG from toothpaste samples, the DEG mixture was directly infused for EESI-MS analysis,34 resulting in a relatively slow process and low sensitivity due to the offline extraction steps and the low proton affinity of DEG. Obviously, it is necessary to further improve the throughput and the sensitivity as well when a large number of complex samples must be rapidly screened. The newly developed ultrasoundassisted EESI technique is suitable for fast screening of melamine in cow’s milk,32 but the toothpaste sample is too viscous to be nebulized by the ultrasonic transducer. Recently, we have demonstrated that with a neutral desorption (ND) device, various analytes can be liberated from either solid or liquid surfaces for the subsequent EESI analysis.35-40 With the use of a sealable ND device, ND-EESI detects low picograms of analytes on surfaces within seconds.39 These facts make NDEESI a good candidate for the direct detection of DEG from toothpaste samples. However, DEG is difficult to be protonated (18) Cooks, R. G.; Gologan, B.; Wiseman, J.; Talaty, N.; Chen, H.; CotteRodriguez, I. Abstr. Pap. Am. Chem. Soc. 2005, 230, U298–U299. (19) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306, 471–473. (20) Chen, H. W.; Liang, H. Z.; Ding, J. H.; Lai, J. H.; Huan, Y. F.; Qiao, X. L. J. Agric. Food Chem. 2007, 55, 10093–10100. (21) Williams, J. P.; Patel, V. J.; Holland, R.; Scrivens, J. H. Rapid Commun. Mass Spectrom. 2006, 20, 1447–1456. (22) Cody, R. B.; Laramee, J. A.; Durst, H. D. Anal. Chem. 2005, 77, 2297– 2302. (23) Moffat, A. C.; Cody, R. B.; Jee, R. D.; O’Neil, A. J. J. Pharm. Pharmacol. 2007, 59, A26–A26. (24) McEwen, C.; Gutteridge, S. J. Am. Soc. Mass Spectrom. 2007, 18, 1274– 1278. (25) McEwen, C. N.; McKay, R. G.; Larsen, B. S. Anal. Chem. 2005, 77, 7826– 7831. (26) Jecklin, M. C.; Gamez, G.; Touboul, F.; Zenobi, R. Rapid Commun. Mass Spectrom. 2008, 22, 2791–2798. (27) Chen, H. W.; Touboul, D.; Jecklin, M. C.; Zheng, J.; Luo, M. B.; Zenobi, R. N. Eur. J. Mass Spectrom. 2007, 13, 273–279. (28) Chen, H. W.; Venter, A.; Cooks, R. G. Chem. Commun. 2006, 2042–2044. (29) Chen, H. W.; Wortmann, A.; Zhang, W. H.; Zenobi, R. Angew. Chem., Int. Ed. 2007, 46, 580–583. (30) Chingin, K.; Gamez, G.; Chen, H. W.; Zhu, L.; Zenobi, R. Rapid Commun. Mass Spectrom. 2008, 22, 2009–2014. (31) Zhou, Z. Q.; Jin, M.; Ding, J. H.; Zhou, Y. M.; Zheng, J.; Chen, H. W. Metabolomics 2007, 3, 101–104. (32) Zhu, L.; Gamez, G.; Chen, H. W.; Chingin, K.; Zenobi, R. Chem. Commun. 2008, 559–561. (33) Zhu, L.; Gamez, G.; Chen, H. W.; Huang, H. X.; Chingin, K.; Zenobi, R. Rapid Commun. Mass Spectrom. 2008, 22, 2993–2998. (34) Ding, J. H.; Yang, S. P.; Liu, Q.; Wu, Z. Z.; Chen, H. W.; Ren, Y. L.; Zheng, J.; Liu, Q. J. Chem. J. Chin. Univ. 2009, 30, 1533–1537.

Figure 1. Schematic diagram of the reactive neutral desorption EESI source. Note that the distances a, b, and the angles R, β were experimentally optimized to achieve better sensitivity. The dimensions are not proportionally scaled. The inset shows a picture (top view) of the toothpaste sample after being sampled for 10 min, with a small cavity in the center of the toothpaste sample surface.

due to its low proton affinity. Similar to reactive DESI,41-43 reactive EESI29 has been developed in this study by implementing selective ion/molecule reactions in the EESI process to improve the sensitivity and specificity of EESI for DEG detection. Therefore, fast quantification of DEG in toothpaste products was achieved using ND-EESI-MS without any sample preparations, providing low LOD values (∼0.000 02%, weight percent of DEG in toothpaste) for actual sample analysis. EXPERIMENTAL SECTION Experimental Setup. In this report, a C-shaped glass cell (i.d., 20 mm) was utilized to cover air-tight the sampling area (∼10 mm2) and the ND device (as shown in Figure 1). The surface of the toothpaste gel (∼0.2 g) was impacted by a pure nitrogen gas beam (room temperature, velocity 300 m/s, flow 2.7 mL/s) ejected from an aperture (i.d. 100 µm) for desorption sampling. The distance between the ND gas emitter and the gel surface was 1.5 mm. The desorbed analytes were sampled as an aerosol flow (velocity 3.4 m/s, flow 2.7 mL/s) into the EESI source using the sample transfer line (STL) (Teflon tube, i.d. 3 mm). A homedesigned EESI source was coupled to a Thermo Scientific LTQXL mass spectrometer (San Jose, CA) for the direct analysis of sampled DEG mixture. An angle β of 60° was formed between the sample outlet and the electrospray beam. The angle R between the sample outlet and the heated capillary of the LTQ instrument was 150°, which was equivalent to the one formed by the electrospray beam and the heated capillary of the LTQ instrument. The interaction region between the ESI plume and the ND gas outlet was coaxially mounted to the heated capillary of the LTQ instrument. The distance (a) between the inlet of the LTQ instrument and the gas outlets was 10 mm. The distance (b) between the two spray tips was 2 mm. (35) Chen, H. W.; Yang, S. P.; Wortmann, A.; Zenobi, R. Angew. Chem., Int. Ed. 2007, 46, 7591–7594. (36) Chen, H. W.; Zenobi, R. Nat. Protoc. 2008, 3, 1467–1475. (37) Chen, H. W.; Wortmann, A.; Zenobi, R. J. Mass Spectrom. 2007, 42, 1123– 1135. (38) Chen, H. W.; Zenobi, R. Chimia 2007, 61, 843–843. (39) Chingin, K.; Chen, H. W.; Gamez, G.; Zhu, L.; Zenobi, R. Anal. Chem. 2009, 81, 123–129. (40) Chen, H. W.; Hu, B.; Hu, Y.; Huan, Y. F.; Zhou, J. G.; Qiao, X. L. J. Am. Soc. Mass Spectrom. 2008, 20, 719–722. (41) Huang, G. M.; Chen, H.; Zhang, X. R.; Cooks, R. G.; Ouyang, Z. Anal. Chem. 2007, 79, 8327–8332. (42) Nyadong, L.; Green, M. D.; De Jesus, V. R.; Newton, P. N.; Fernandez, F. M. Anal. Chem. 2007, 79, 2150–2157. (43) Song, Y.; Cooks, R. G. J. Mass Spectrom. 2007, 42, 1086–1092.

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The reagent solvent (i.e., 10 µmol/L ammonium acetate in methanol solution) was electrosprayed at 5 µL/min with a high voltage (+4 kV, for positive ion detection mode). The temperature of the heated capillary was optimized to be 275 °C. A mass range between 50 and 400 Da was scanned for each measurement. The default values of voltages for the heated capillary, ion optics, and the detectors were used without further optimization. Collisioninduced dissociation (CID) experiments were done by applying 18-35% (arbitrary units defined by the LTQ instrument) of the collision energy to the precursor ions isolated with a window width of 1.6 mass/charge (m/z) units. Sample Manipulation. To prepare the standard series of DEG toothpaste samples, seven portions of DEG-free toothpaste (2.0 g each) were put into seven beakers (10 mL, Tianjin Tianbo Glass Instrument Co., Ltd., Tianjin, China). A total of 400 µL of DEG aqueous solutions of different concentrations (1000, 500, 50, 5, 0.5, 0.05, and 0.005 mg/mL) were added into those seven beakers, respectively. Samples were stirred to achieve the homogeneous distribution of DEG inside the toothpaste samples. These mixtures were partially dried in a vacuum (1 mTorr, 298 K) until the final mass loss was 400 mg for each beaker. As a results, a series of toothpaste standards containing DEG at different levels such as 20%, 10%, 1%, 0.1%, 0.01%, 0.001%, and 0.0001% were prepared. These standard toothpaste samples were used to make the calibration curve for quantification of the DEG contents in actual toothpaste samples. For recovery experiments, a similar procedure was used to prepare the toothpaste samples with spiked DEG at different levels. For example, a certain amount of DEG pure compound (e.g., 1 g) was added into toothpaste samples (e.g., 4 g) containing various amounts of DEG. In order to evenly distribute the DEG in the toothpaste sample, 10 mL water was successively added into the DEG-toothpaste mixture and then homogenized by stirring the mixture for 20 min. The homogenized DEG-toothpaste mixture was then placed in a vacuum (1 mTorr, 298 K) to vaporize the water added (10 g in weight for this case), resulting in a toothpaste sample containing 20% of DEG in weight. Following these procedures, toothpaste samples spiked with different concentrations (e.g., 10%, 1%, etc.) of DEG in weight were prepared. For each measurement, 0.2 g of each spiked toothpaste sample were placed under the ND probe and sampled for EESI ionization. Reagent and Materials. Chemicals such as diethylene glycol and ammonium acetate were bought from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China); methanol was imported from Fisher Scientific (Fair Lawn, NJ). Water used was deionized, which was provided by the Chemistry Department facility at East China Institue of Technology. The commercial toothpaste products containing DEG produced before in March 2007 were collected by the local division of food safety administration from a local supermarket in July 2007 and stored at 3-4 °C before use. The DEG-free toothpaste products were bought from local supermarkets. All the actual samples were directly used without further treatment. RESULTS AND DISCUSSION Reactive ND-EESI Mass Spectrum of Toothpaste. It is wellknown that toothpaste products on the markets are extremely viscous complex matrixes (∼500 kcP), consisting of majorly fine abrasive particles in the low micrometer range, thickening agent, 8634

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foaming agent, antifreezer, surfactant, water, and so on. The thickening agent, such as sodium magnesium silicate or colloidal fumed silica nanoparticles, tends to form random networks, which provide both fluidity and stability of the product. DEG and other ingredients such as organic compounds of low molecular weights can be embedded inside these networks. Up to date, the detection of DEG incorporated with such a complex matrix has been a complicated and laborious task. For chromatographic techniques, a single run including the extraction of DEG out of toothpaste samples, separation, and preconcentration requires more than 40 min. Liquid-liquid EESI can be applied to analyze those exacts without further sample treatment but still takes quite a few minutes for each sample.33 As demonstrated in this work, DEG incorporated with toothpaste was directly analyzed by ND-EESIMS without any sample preparations. Since DEG molecule contains no chemical groups that could promote its sensitive detection under positive/negative ion detection mode, reactive EESI method with electrospraying 10 µmol/L ammonia acetate (CH3COONH4) in methanol solution was applied to all the measurements in this study. The molecule of DEG has two hydroxyl groups which are active in forming compounds with NH4+ ions present in the charged microdroplets. As shown in parts a and b of Figure 2, mass spectra taken with ND-EESI and reactive ND-EESI sources do not show peaks of the protonated DEG (m/z 107). The peak (m/z 124) dominating the mass spectra collected using reactive EESI is ascribed to the ionic complex of [DEG + NH4]+, which exclusively loses NH3 and water in MS2 (Figure 2c) and MS3 (Figure 2d), respectively. The absolute count of m/z 124 ([DEG + NH4]+) in reactive EESI spectrum is much larger than that of the protonated DEG (m/z 107) obtained using a conventional EESI source.33 Other major peaks such as m/z 129 and m/z 145 in the MS spectrum are corresponding to the complexes of [DEG + Na]+ and [DEG + K]+, respectively. This can be rationalized by the high affinities of the DEG molecule to the metal ions.14 These assignments were confirmed by the MS/MS data shown in parts e and f of Figure 2, correspondingly, in which the precursor ions show the same fragmentation patterns by the loss of H2O, CO, CH3OH, and CH3CH2OH, successively. The absence of the protonated molecules in their CID spectra can be explained by the strong affinity between the metal ion and the DEG molecule (fragments). The signal abundance of [DEG + K]+ complex is much lower than that of the [DEG + Na]+ complex. As the sodium and potassium salts were equally added into the electrospray solution, the different signal intensities are likely attributed to the differential DEG affinities of potassium from sodium. All the findings above suggest that the affinity of DEG molecules to different cations is listed as following: NH4+ > Na+, K+ > H+. The characteristic signal patterns of DEG molecules caused by H+, Na+, K+, and NH4+ can be utilized as the molecular markers for the DEG presence while a MS equipment without tandem capability is used. Optimization of the ND-EESI Source. As described above, with the use of reactive EESI, the signal of DEG (m/z 124) was abundantly detected, so the reactive EESI conditions were optimized using the signal at m/z 124. Note that every parameter except the inherent EESI source settings (e.g., the electrospray voltage, the temperature of ion transfer capillary, the pressure of

Figure 2. Mass spectra of toothpaste obtained using ND-EESI-MS: (a) neutral desorption EESI mass spectrum recorded using 10% acetic acid in methanol spray solution, showing no abundant signals of DEG; (b) reactive ND-EESI mass spectrum of the same toothpaste recorded by spraying 10 µmol/L ammonium acetate in methanol solution, showing the characteristic signal patterns of DEG; (c) MS/MS spectrum of the signal at m/z 124 detected using reactive EESI; (d) MS/MS/MS spectrum of the product ions of the precursor ions (m/z 124) detected using reactive EESI; (e) MS/MS spectrum of the signal at m/z 129 detected using reactive EESI; (f) MS/MS spectrum of the signal at m/z 145 detected using reactive EESI.

nitrogen gas for ND sampling, the composition, and the flow rate of the spray solvent) was optimized automatically by the LTQ instrument. In our experiment, the nitrogen gas flow for neutral desorption was critical. The signal intensity of m/z 124 increased with elevated pressure of the nitrogen gas for neutral desorption between 0.8 and 1.4 MPa (Figure 3a). While the nitrogen flow was too gentle (e1.0 MPa), only volatile compounds released from the toothpaste sample was sampled for EESI ionization/detection, resulting in a mass spectrum featured with signals irrelevant to DEG. Higher gas flow would be beneficial for desorption of DEG out of viscous toothpaste samples. When the gas pressure was set to 1.2 MPa, DEG signals (m/z 124, etc.) could be detected with a good signal-to-noise ratio, but the DEG signals decreased quickly (within 1 min) down to the noise level. It was noticed that some signals were detected with constant signal levels; probably, because they were volatile compounds and thus were easily sampled using such a gentle ND gas flow. Therefore, it is reasonable to have a strong gas flow for the ND process to liberate sticky compounds (e.g., DEG) incorporated within the toothpaste. However, after the toothpaste is impacted using the gas beam for awhile, the soft surface of the toothpaste shrank slightly to

make the toothpaste out of the right position for liberation of DEG from the surface. Similar phenomenon was also observed using sheath gas assisted ambient ionization techniques such as DESI and DAPCI. For example, no DEG signal could sustain at a stable level for more than 2 min using DESI/DAPCI-MS. It has been demonstrated that signals generated by either DESI18,42,44 or DAPCI21,45-47 are sensitive to the position of the surface. The active spot used for the DESI/DAPCI and ND sampling process is small (∼10 mm2). The indentation of the toothpaste caused by the sheath gas impacting could push the sample surface out of the phase and thus be responsible for the signal dropping. With the use of a stronger stream of nitrogen (∼1.4 MPa) for the ND process, the signal of DEG was stably (44) Chen, H. W.; Talaty, N. N.; Takats, Z.; Cooks, R. G. Anal. Chem. 2005, 77, 6915–6927. (45) Chen, H. W.; Lai, J. H.; Zhou, Y. F.; Huan, Y. F.; Li, J. Q.; Zhang, X.; Wang, Z. C.; Luo, M. B. Chin. J. Anal. Chem. 2007, 35, 1233–1240. (46) Chen, H. W.; Zheng, J.; Zhang, X.; Luo, M. B.; Wang, Z. C.; Qiao, X. L. J. Mass Spectrom. 2007, 42, 1045–1056. (47) Yang, S. P.; Ding, J. H.; Zhu, L.; Hu, B.; Li, J. Q.; Chen, H. W.; Zhou, Z. Q.; Qiao, X. L. Anal. Chem. 2009, 81, 2426–2436.

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Figure 3. Optimization of ND-EESI working conditions: (a) effects of the nitrogen gas pressure for neutral desorption on the EESI signal levels; (b) effects of the temperature of the heated capillary (ion entrance) on the EESI signal intensities; and (c) effects of the electrospray voltage on the EESI signal levels. The error bars show the measured errors for each data point.

detected for a long time (g20 min). As a result of the ND sampling for more than 5 min, a tiny cavity (shown in the inset of Figure 1) caused by the force of the impinging gas jet was formed on the toothpaste surface. The DEG signal would decrease again eventually when the outer surface of the cavity could not be efficiently desorbed by the ND gas beam. This suggested the ND process was a necessity to generate the DEG signals. It was also noticed that some paste residue was found on the inner wall of the C-glass shell if the ND gas flow was higher than 1.8 MPa. This was because the big particles of the toothpaste sample, which were created by the strong ND gas jet, deposited on the inner 8636

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side of the C-glass shell. This indicated that the heterogeneous liquid mixture was sampled as a portion of the mixture, in which the nonvolatile compounds such as DEG were most likely sampled as aerosols rather than gas-phase vapors. As a reference experiment, pure DEG ice (cooled by dry ice at about -70 °C) was sampled using a nitrogen gas (room temperature) for EESI-MS analysis, resulting in good signals of DEG. At the low temperature (∼-70 °C), DEG was unlikely to be sampled as a gas-phase vapor. Secondary electrospray ionization (SESI), a technique reported early by Hill et al.48,49 and being developed by de la Mora and co-workers,50-52 was primarily used to ionize the gas-phase vapors. However, SESI was also used to detect analytes such as explosives in solutions,49 but the analytes were transferred into the gas phase by all means such as severe heating.49 The data reported previously35-37,39,53 and in this study as well have shown that NDEESI samples mixtures on surfaces to form aerosols for extractive ionization by the electrospray plume. Thus, EESI features sensitive detection of compounds under ambient conditions without generating gas-phase vapors and facilitates detection of heat sensitive compounds such as biological molecules. The peak intensity of m/z 124 kept increasing when the temperature of the heated capillary of the LTQ instrument was increased from 50 to 275 °C, possibly due to the better desolvation effects achieved at a higher temperature (Figure 3b). However, extremely high temperature (>300 °C) would cause thermal dissociation of DEG molecules while the ions were being transferred into the mass analyzer, and thus a signal decline was observed. As shown in Figure 3c, the signal levels of DEG were also increased when the electrospray voltage was increased from +1 to +4 kV, because more primary ions were created when a higher voltage was used. The signal level of the DEG was not further improved when the voltage was raised to +5 kV, probably the efficiency of the primary ion generation was not accordingly enhanced. The analyte ions were immediately accelerated by the high voltage once they were created by the EESI process. However, the ions that gained energy more than necessary could not be collected with proper efficiency, and thus no voltage higher than 5 kV was tested in this study. Alternatively, the signal intensity of m/z 124 became more abundant along with the increasing of the electrospray flow rate from 2 to 5 µL/min, because more primary ions were created using a high flow rate. The signal intensity of m/z 124 was stable with the flow rate of spray solvent between 5 and 7 µL/min, and then decreased slightly when a high flow rate was used. The signal drop was probably because an insufficient desolvation of the analyte ions. Therefore, as shown in Figure 3, the best signal of m/z 124 appeared when the temperature of the ion entrance capillary was set at 275 °C; the pressure of the nitrogen gas for desorption was 1.4 MPa; the electrospray voltage was 4 kV; and the flow rate of ESI solvent was 5 µL/min using 10 µmol/L ammonia acetate in methanol solution. Wu, C.; Siems, W. F.; Hill, H. H. Anal. Chem. 2000, 72, 396–403. Tam, M.; Hill, H. H. Anal. Chem. 2004, 76, 2741–2747. Martinez-Lozano, P.; de la Mora, J. F. Anal. Chem. 2008, 80, 8210–8215. Martinez-Lozano, P.; de la Mora, J. F. J. Am. Soc. Mass Spectrom. 2009, 20, 1060–1063. (52) Martinez-Lozano, P. Int. J. Mass Spectrom. 2009, 282, 128–132. (53) Chen, H. W.; Hu, B.; Hu, Y.; Huan, Y. F.; Zhou, J. G.; Qiao, X. L. J. Am. Soc. Mass Spectrom. 2009, 20, 719–722. (48) (49) (50) (51)

Figure 4. Calibration curve in logarithmic scales for detection of DEG in toothpaste samples. Each data point designates 8 measurements; the RSD values are in the range of 3.8-7.2% for all the data points; the error bar shows the measured error of each data point.

Quantification of DEG Spiked into Toothpaste Samples. Trace amounts of DEG were spiked into the blank toothpaste samples following the procedures described in the Experimental Section. No notable morphology change was found in these spiked toothpaste samples, which suggests negligible matrix change caused by adding the DEG solutions into the blank toothpaste samples. Each measurement only took less than 2 s, much faster than those reported in previous literature,8-15,33 which is suitable for high-throughput applications. Currently, the hindrance of further speeding up of the measurements is the loading of individual samples, which could be solved by utilizing automatic sampler in the future. To reduce the possibility of a false positive signal, the fragment ion (m/z 107) of m/z 124 ([M + NH4]+) was proposed as the signal for the quantification of DEG in toothpaste samples. The duration of active CID was 30 ms for each mass analysis scan. The averaged signal intensity of m/z 107 obtained using 50 scans were actually used for quantification. The relationship between the measured intensities and the concentrations of spiked DEG in toothpaste samples was drawn in Figure 4, showing a linear regression equation lg y ) 0.32 lg x (%) + 3.4 (R2 ) 0.999) and a dynamic range from 0.0001 to 20%. The signal response was not linearly dependent on the concentration of DEG in the toothpaste, probably because the total amount of DEG could not be linearly released from the toothpaste mixtures due to the comprehensive interactions that occurred at the molecular level. The LOD of this method was calculated to be 0.000 02% (weight percent of DEG in toothpaste, S/N ) 3) using the following equation based on measurements of a series of DEG-spiked toothpaste samples. LOD )

c3σ S

(1)

where c is the DEG concentrations in the toothpaste sample, σ is the standard deviation of all the measurements (n ) 8), and S is the mean value of the 8 signals measured. The LOD was much lower than the legally allowed TDI of DEG. The low value of LOD and the high specificity and sensitivity achieved by reactive tandem EESI-MS combined with no need of sample pretreatment make our method an ideal candidate for routine quality monitoring of toxic DEG in toothpaste. One important point is that no memory effect was observed except for the cases when extremely high DEG concentration (∼10%) was spiked into the toothpaste. Since

toothpaste is a representative of extremely high viscous gel mixtures with nanomaterials (such as sodium magnesium silicate), the ability of detecting organic molecules (such as DEG) embedded inside toothpaste would make reactive neutral-desorption EESI potentially attractive to the rapid characterization of other complex mixtures with nanomaterials and high viscosity. Recovery Efficiency and Standard Deviation. Eight measurements were performed to obtain the DEG concentrations in the toothpaste samples spiked with different amounts of DEG. The concentrations of DEG were derived by fitting the acquired intensities to the calibration curve of DEG in Figure 4. The corresponding values are shown in Table 1. The recovery of DEG (0.1-10%) ranged from 97.6% to 102.4%. The relative standard deviation (RSD) for eight measurements on the same sample was below 5%. The relatively low RSD values after eight consecutive measurements confirmed the absence of memory effect during our experiments. Another set of measurements performed on 10 toothpaste samples spiked with the same amount of DEG (2%) gave a low RSD value of 4.3%, demonstrating that the acceptable precision for multiple measurements was obtained using the reported method. Validation of the Method for Rapid Actual Sample Analyses. A total of 11 commercial toothpaste products including two formulas for children were quantitatively analyzed using this method. Eight measurements were performed on the 0.2 g sample from each of the toothpaste products by following the procedures described before. As listed in Table 2, significant amounts of DEG were determined in 5 out of 11 samples. Positive toothpaste samples contained DEG by 0.95-10.99%. The blind test results were in good agreement with the quantitative analysis data obtained by the local division of food safety administration using a conventional GC/MS method.54 In order to confirm these measured values, various amounts of DEG were added into the samples according to the measured DEG levels. For example, an amount of 1% DEG was added to the samples containing DEG less than 2%, while an amount of 10% DEG was added to the samples containing DEG between 2% and 11%. The recoveries were in the range of 90.0∼97.3% in all cases (Table 2), demonstrating the robustness of this method for the rapid detection of DEG in actual toothpaste products. The method has been validated for the quantitative detection of DEG in various commercial toothpaste products. Furtherer, the relatively good recovery (90.0∼97.3%) derived from the standard addition measurements implies that the calibration curve shown in Figure 4 can be applied to the detection of DEG in many commercial products, although their matrixes might not be the same. In reactive ND-EESI-MS, the sensitivity was significantly improved for rapid detection of DEG, which was poorly protonated in normal EESI-MS without selective ion/molecule reactions.34 Also, a few minutes were required for extracting DEG from toothpaste samples, which hinders the high-throughput applications using conventional liquid-liquid EESI methods for rapid analysis of highly viscous and heterogeneous samples. In this study, the experimental data show the LOD (∼0.000 02%) of reactive ND-EESI-MS is much lower than the previously reported data obtained using Chinese authority standard methods (0.05 (54) National Standards of the People’s Republic of China. GB/T 21842-2008, 2008.

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Table 1. Recovery and Precision for DEG Measurements concentration of DEG detected (weight percent)

concentration of DEG added (weight percentage, %) 0.1 1 10

intensity detected

mean value (weight percent, %)

RSD (%)

recovery (%)

1310, 1370, 1280,1260, 1410, 1380, 1220, 1350 2630, 2880, 2700, 2630, 2780, 2920, 2820, 2920 5950, 5840, 5740, 5500, 5550, 5330, 6100, 5930

0.0976 1.024 9.89

5.0 4.7 4.6

97.6 102.4 98.9

Table 2. Quantitative Detection of DEG in Commercial Toothpaste Productsa

sample

measured DEG (weight percentage, %)

mean value of DEG measured (%)

amount of DEG added (%)

total amount of DEG found after standard addition (%)

recovery (%, n ) 8)

1 2 3 4 5 6 7 8 9 10 11

0.95; 0.96; 0.94; 0.98; 0.93 4.41; 4.22; 4.21; 4.15; 4.32 11.01; 10.98; 10.99; 11.10; 10.89 4.95; 4.57; 4.77;4.84; 4.69 1.33; 1.24; 1.35; 1.39; 1.36 -

0.95 4.26 10.99 4.76 1.33 -

1 1 1 10 10 10 1 1 1 1 1

1.82 0.91 0.92 13.48 20.42 14.03 0.92 0.91 0.91 2.18 0.90

93.3 91.0 92.0 94.5 97.3 95.1 92.0 91.0 91.0 93.6 90.0

a

- means that no DEG was detectable using the method reported here.

g/kg for GC and 0.03 g/kg for GC/MS).54 The fast analysis speed, high specificity, acceptable reproducibility, and sensitivity make the reactive ND-EESI-MS a useful tool for the high throughput detection of trace analytes in heterogeneous samples of extremely high viscosity. CONCLUSIONS A technique combining neutral desorption and reactive EESI has been developed and demonstrated to be a rapid and sensitive approach for the specific detection and quantification of trace amounts of toxic diethylene glycol in toothpaste without any sample pretreatment, providing a successful example for rapid characterization of extremely viscous, heterogeneous liquid mixtures. Highly viscous liquid mixtures are commonly seen in daily life and play important roles in many areas of modern science and technology but challenge analytical chemistry for rapid, sensitive analysis, especially when the samples are highly heterogeneous such as gel mixtures that contain nanomaterials. In this work, with the use of toothpaste as a representative sample, a single sample analysis has been completed within 2 s, providing

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a LOD of ∼0.000 02% for DEG in MS/MS experiments. Acceptable RSD (