Determination of degradation products of nerve agents in human serum by solid phase extraction using molecularly imprinted polymer

Analytica Chimica Acta 435 (2001) 121–127

Determination of degradation products of nerve agents in human serum by solid phase extraction using molecularly imprinted polymer Meng Zi-Hui∗ , Liu Qin Beijing Institute of Pharmacology and Toxicology, Beijing 100850, PR China Received 17 October 2000; received in revised form 15 January 2001; accepted 22 January 2001

Abstract Molecularly imprinted polymers (MIPs) for pinacolyl methylphosphonate, ethyl methylphosphonate and methane phosphonic acid were prepared. By taking the advantage of their cross selectivity, the MIPs could recognize not only the print molecule, but also the degradation products of the other nerve agents. The absorbed degradation products could be quantitatively extracted using distilled water. The pinacolyl methylphosphonate-imprinted polymer was used as a solid phase extraction sorbent for all possible degradation products of nerve agents from human serum. Following the solid phase extraction procedure, accurate analysis for the degradation products was carried out using capillary electrophoresis directly. A detection limit of 0.1 ␮g/ml and an R.S.D. of <9.12% were obtained. A good linearity (r > 0.99, n = 4) in the concentration range of 0.1–10 ␮g/ml was also obtained. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Molecular imprinting; Chemical warfare; Nerve agents; Solid phase extraction; Capillary electrophoresis

1. Introduction Among the lethal chemical warfare (CW) agents, the nerve agents have played dominant role since the second World War. When absorbed through the skin or via respiration, they are highly toxic and have rapid effects. Nerve agents can be manufactured using fairly simple chemical techniques. However, nerve agents have been banned from use, and the production and stockpiling of such weapons should have been prohibited. However, some countries are known to have ∗ Corresponding author. Present address: Department of Pure and Applied Biochemistry, Chemical Center, University of Lund, P.O. Box 124, S 221 00 Lund, Sweden. Tel.: +46-46-2220676; fax: +46-46-2224611. E-mail address: [email protected] (M. Zi-Hui).

manufactured and stockpiled such weapons and toxins. Growing concerns over possible contamination and injury caused by nerve agents has prompted the desire for more sensitive and accurate methods of analysis for these nerve agents [1–4]. Ordinarily, the proto nerve agents can be identified by analysis of their degradation products. Standard gas chromatographic techniques have been established for these analyses [2,3]. However, these approaches are challenged by complex sample matrices containing many interfering components, such as human serum. And degradation products must be derivatized with a tedious procedure before GC analysis. Solid phase extraction (SPE) can be used to isolate and preconcentrate the analytes in complex samples. The materials routinely used in SPE are usually based on the non-specific binding of the

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targets. Molecularly imprinted polymers (MIPs) offer the possibility of achieving selective extraction, analogous to those achieved by immuno-based extraction systems, and thus may represent an advance on conventional SPE materials [5–9]. MIPs are highly cross-linked polymers synthesized in the presence of print molecules. After removal of the print molecule, recognition sites suitable for the selective rebinding of the print molecule are obtained. MIPs also offer several other advantages such as being stable at high temperatures and in organic solvents [10–13]. In this work, we report for the first time the extraction of all possible degradation products of the nerve agents from human serum using an MI-SPE method. Following their extraction, the degradation products were analyzed directly using CE without derivatization procedure.

monomer (MAA, 20 mmol) and acetonitrile (12 ml) were added to a 25 ml glass tube. The mixture was allowed to stand overnight at 4◦ C in order to form the stable complex between print molecule and monomer [6]. The crosslinker (20 mmol TRIM) was then added, followed by 0.4 mmol of the initiator ABCHC. The mixture was sonicated and purged with nitrogen for 5 min, and then polymerized under UV (366 nm) irradiation in an ice bath for 48 h. MIPs A, B and C were prepared using PMPA, EMPA and MPA as print molecule, respectively. Blank polymer D was prepared in the absence of any print molecule. The bulk polymers were ground and sieved through a 120 mesh sieve. The particles obtained were washed with methanol/acetic acid (9/1, v/v) until no print molecule could be detected by CE.

2. Experiments

CE analysis was carried using a BiFocus 3000 capillary electrophoresis system. The applied voltage was −18 kV at temperature 25◦ C. A constant negative pressure of 15 kPa was applied during a 3 s injection. Fused silica capillaries (70 cm × 75 ␮m i.d.) were obtained from Polymicro Technologies (Phoenix, AZ, USA). The detection wavelength was set at 210 nm. The running buffer was 200 mM boric acid–5 mM phenylphosphonic acid–0.1 mM didecyldimethylammonium bromide (DDAB)–0.2% Triton X-100 (pH 3.55). KH2 PO4 was used as an internal standard for the quantitative analysis.

2.1. Materials Pinacolyl methylphosphonate (PMPA, degradation product of Soman), ethyl methylphosphonate (EMPA, degradation product of VX), isopropyl methylphosphonate (IMPA, degradation product of Sarin), cyclohexyl methylphosphonate (CMPA, degradation product of GF), isobutyl methylphosphonate (BMPA, degradation product of a new nerve agent named as Russian VX developed by the former Soviet Union) and methylphosphonic acid (MPA, the final degradation product of all nerve agents) were provided by The Research Institute of Chemical Defense, People’s Liberation Army of China, with purities of up to 97%. The molecular structures of these degradation products are shown in Fig. 1. Trimethylolpropane trimethacrylate (TRIM), 1,1 -azobis(cyclohexanecarbonitrile) (ABCHC) and methacrylic acid (MAA) were purchased from Aldrich (Millwaukee, WI). Other chemicals were purchased from various sources and were all of analytical grade. 2.2. Preparation of MIPs The preparation of polymers has been previously reported [14–17]. The print molecule (5 mmol),

2.3. Capillary electrophoretic analysis

2.4. Binding and extraction assays An amount of 5 ml of standard acetonitrile solution spiked with PMPA (0.075 mM), IMPA (0.087 mM), EMPA (0.2 mM), CMPA (0.082 mM), BMPA (0.18 mM) and MPA (0.095 mM) was applied to the cartridges packed with 500 mg MIPs A, B, C and D 10 times, respectively, and then analyzed by CE. The absorptivities of each degradation products were determined as (Astd − Aleft )/Astd , where Astd is the peak area of degradation product in the standard sample and Aleft is the peak area of degradation product in the standard solution after applying to the cartridge. The cartridges were extracted five times using 2 ml of distilled water, the

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Fig. 1. The molecular structures of the degradation products of nerve agents. Pinacolyl methylphosphonate (PMPA), ethyl methylphosphonate (EMPA), isopropyl methylphosphonate (IMPA), cyclohexyl methylphosphonate (CMPA) and isobutyl methylphosphonate (BMPA) are the degradation products of Soman, VX, Sarin, GF and Russian VX, respectively. Methylphosphonic acid (MPA) is the final degradation product of all nerve agents.

recoveries of each degradation product were determined as Aeluent /Astd , where Aeluent is the peak area of degradation product in the eluent. The recoveries are shown in Table 1. Table 1 The recoveries (%) of the absorbed degradation products eluted by water (5 × 2 ml) from the cartridges packed with MIPs A, B and C, respectivelya MIPb

PMPA

EMPA

MPA

IMPA

BMPA

CMPA

A B C

83.3 50.8 70.2

98.9 86.5 94.4

97.4 79.8 88.4

99.2 97.5 89.4

97.5 97.5 89.2

78.5 81.3 87.3

n = 3; R.S.D. < 7% in all instances. MIPs A, B and C were imprinted with PMPA, EMPA and MPA, respectively. a

b

2.5. Solid phase extraction of the degradation products from the human serum SPE cartridges were made by packing 500 mg MIP A particles into a home-made cartridge. The cartridge was successively conditioned with 5 ml HCl (30%), 50 ml distilled water and 5 ml acetonitrile at a flow rate of 1.5 ml/min. An amount of 1 ml human serum spiked with PMPA, IMPA, EMPA, CMPA, BMPA and MPA was acidified to pH 1 with HCl, then 1 ml Triton X-100 and 1 ml 10N NaOH were added to separate the aqueous and acetonitrile layers [2]. After centrifugation for 5 min, the supernatant was extracted twice with 3 ml of acetonitrile. The extracts were combined, dried over anhydrous Na2 SO4 , and then repeatedly

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Fig. 2. CE electrophorogram of sample eluted from the PMPA-MI-SPE cartridge. An amount of 1 ml human serum spiked with PMPA, IMPA, EMPA, CMPA, BMPA and MPA (0.1 mM each) was applied to the MI-SPE procedure, the elution from the SPE cartridge was analyzed by CE. CE conditions were: column, 70 cm × 75 ␮m i.d. fused silica capillaries; running buffer, 200 mM boric acid–5 mM phenylphosphonic acid–0.1 mM DDAB–0.2% Triton X-100 (pH 3.55); sample injected, 15 kPa for 3 s; detection, λ = 210 nm; internal standard, 10 ␮M KH2 PO4 . Peak designation: (1) MPA; (2) EMPA; (3) IMPA; (4) BMPA; (5) CMPA; (6) PMPA.

applied to the SPE cartridge 10 times. The cartridge was eluted five times with 2 ml distilled water. The eluents were combined and concentrated to 1 ml using a rotary evaporator, and finally analyzed by CE. The electrophorogram of the degradation products is shown in Fig. 2. For the control experiment, we analyzed the acetonitrile extract by CE directly without using the MI-SPE cartridge, and the electrophorogram is shown in Fig. 3.

3. Results and discussion 3.1. Performance of the MIPs In binding assays, MIPs A and B could absorb all the degradation products completely from the standard solution; while the blank polymer D could not absorb any of them. This difference is believed to arise from the imprinting effect. Increasing the percentage

Fig. 3. The CE electrophorogram of the acetonitrile extract of the human serum containing 0.1 mM degradation products. CE conditions were the same as in Fig. 2.

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Fig. 4. The correlation between the absorptivity of PMPA on MIP A and the water content (%, v/v) in the standard solution. The concentration of PMPA was 0.15 mM, the water content in acetonitrile was varied from 0 to 50%.

of water in the standard solution caused the absorptivities of the degradation products decrease significantly, presumably due to the hydrogen bonds formed between the water molecules and the recognition sites. The decreasing absorptivity of PMPA on MIP A with the increase in water content in the standard solution is shown in Fig. 4. The absorptivities of the degradation products on MPA-imprinted MIP C were as follows: MPA 100%, IMPA 90%, PMPA 84%, EMPA 88%, BMPA 82% and CMPA 88%. MIP C could completely absorb the print molecule MPA. MPA is the final degradation product of all nerve agents, and it is the smallest and the most polar molecule in the all degradation products of the nerve agents. The other degradation products have much bigger alkyl chain of the phosphonate esters (see Fig. 1), it seemed that they have difficulties in diffusing into the recognition sites formed by MPA because of steric hindrance and hydrophobicity. Resultantly, MIP C could not completely absorb the other degradation products. The extraction recoveries of the degradation products eluted by water from the cartridges are also important for the development of a successful SPE procedure. Actually, most of absorbed degradation products could be extracted using distilled water with high recovery (Table 1), especially for the degradation products absorbed by the MIP A cartridge. The relative low recovery of PMPA from the MIP A cartridge is mainly due to the strong binding of the print molecule to the recognition sites formed during

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the imprinting procedure; while the low recovery of PMPA from the other two cartridges may arise from the strong and non-specific hydrophobic interactions between the PMPA and the matrices of the MIPs. When we washed the absorbed degradation products from MIP A cartridge using methanol, no PMPA could be extracted, and the recoveries for three other degradation products (MPA 90.6%, EMPA 71%, IMPA 24.7%) are lower than those obtained using water. The capacity of each MIP was roughly evaluated by applying 1 ml of acetonitrile solution spiked with 1 mg/ml print molecules 10 times to cartridges packed with MIPs A, B and C, respectively. The absorptivities of the print molecules on each cartridge was MIP A: PMPA 76.8%, MIP B: EMPA 66.9% and MIP C: MPA 94.5%. So the capacity of each MIP for its print molecule is MIP A: PMPA 0.081 mmol/g dry polymer, MIP B: EMPA 0.12 mmol/g dry polymer and MIP C: MPA 0.20 mmol/g dry polymer. All of the MIPs prepared in this research showed significant cross-selectivity. They could recognize not only the print molecule but also the degradation products of the other nerve agents because the degradation products of all nerve agents differ only in the alkyl chain of the phosphonate esters. Since the aim of our research is to bind the degradation products of all nerve agents, regardless of the interferents in the practical samples, the cross-selectivity is actually advantageous. In fact, if the MIPs prepared in this research showed high selectivity to the print molecule alone, they could not be used for entrapping the whole group of degradation products.

3.2. Extraction of degradation products of nerve agents from the human serum From the experiment results obtained from the binding and extraction assays, PMPA-MI-SPE cartridge was selected for practical application, and acetonitrile and distilled water were used as loading and eluting solvents, respectively. By comparing the electrophorogram shown in Figs. 2 and 3, we can see that when we analyze the acetonitrile extract from the serum directly, most of the peaks of the degradation products are obscured by the interferents, and analysis of the degradation products not possible in such a situation. After applying the acetonitrile extract of the serum to

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analyzed using CE. Using the concentration C of the degradation products as the abscissa, the ratio A between the corrected peak area (A/tr ) of the degradation products and the internal standard as the ordinate, the standard curves for A against the concentration C were plotted. The linear regression equations, correlation coefficients and the detection limits for each degradation products are shown in Table 3. In this concentration range, good linearity (r > 0.99) was obtained for each degradation product. The detection limit for the print molecule PMPA for its cartridge is 0.1 ␮g/ml, while for the other degradation products the detection limits are lower than 0.5 ␮g/ml. In order to evaluate the average recovery and the reproducibility of the method, standard serum samples with concentration of 0.8, 3 and 10 ␮g/ml of each degradation product were extracted using the PMPA-MI-SPE cartridge and analyzed by CE. The average recoveries and the relative standard deviation (R.S.D.) values (n = 4) are shown in Table 4. The average recoveries are ranged from 94.6 to 104%, and R.S.D. ranged from <1 to 9%. In conclusion, the preliminary results obtained during the course of this research clearly demonstrate for the first time the value of MI-SPE methods for the absorption and determination of the degradation products of nerve agents extracted from complex matrices, such as serum. By taking advantage of the cross selectivity of the MIPs, PMPA-MI-SPE cartridge could be used to extract the possible degradation products of all nerve agents from the human serum with extraction recoveries of up to 90.5%. The interfering components for the CE analysis were successfully removed. Detection limits of 0.1 ␮g/ml and R.S.D. of <9.12% were obtained. In practice, the MI-SPE procedure could be simplified by applying the acetonitrile extract to the SPE cartridge only twice within 10 min.

Table 2 The quantitative recoveries of 2.0 and 0.5 ␮g/ml degradation products in serum for the MI-SPE procedurea,b Degradation products

EMPA IMPA BMPA CMPA PMPA a b

Recovery (%) 2.0 ␮g/ml

0.5 ␮g/ml

81.7 81.2 86.7 89.8 83.0

82.1 81.7 88.9 85.2 90.5

n = 4; R.S.D. < 9% in all instances. SPE cartridge was packed with 500 mg MIP A particles.

the PMPA-MI-SPE cartridge followed by elution with distilled water, the individual degradation product can be identified. Thus, the entire group of degradation products is absorbed by the SPE cartridge, while the interferents did not interfere. We were able to obtain a satisfactory CE electrophorogram from the eluted sample, which enables the quantitative analysis to be easily performed. The quantitative extraction recovery of this method was determined by extraction and analysis of 1 ml serum samples containing 0.5 and 2.0 ␮g/ml degradation products, respectively, and the results are shown in Table 2. The recoveries for the degradation products are in the range of 81.2–90.5%, which is high enough to guarantee the validity of the MI-SPE procedure. 3.3. Quantitative analysis of degradation products by CE Standard serum samples containing 0.2–10 ␮g/g of each degradation product were prepared using KH2 PO4 as an internal standard. The samples were then extracted using PMPA-MI-SPE cartridge and

Table 3 Linear regression equation, correlation coefficient (r) and detection limit of each degradation products for the quantitative analysis methoda,b Degradation product EMPA IMPA BMPA CMPA PMPA a b

Linear regression equation Y Y Y Y Y

= 5.03 = 9.09 = 1.70 = 1.15 = 1.49

× × × × ×

10−2 X 10−2 X 10−1 X 10−1 X 10−1 X

10−3

+ 9.8 × + 3.59 × + 5.95 × + 1.81 × + 3.04 ×

10−2 10−2 10−2 10−2

r

Detection limit (␮g/ml)

0.996 0.995 0.998 0.999 0.998

0.5 0.3 0.1 0.2 0.1

n = 4. Serum samples containing 0.2–10 ␮g/ml degradation products were extracted using MI-SPE procedure, and then analyzed using CE.

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Table 4 The average recoveries and R.S.D. of the quantitative analysis methoda,b Degradation product

EMPA IMPA BMPA CMPA PMPA a b

0.8 ␮g/ml

3.0 ␮g/ml

10.0 ␮g/ml

Recovery (%)

R.S.D. (%)

Recovery (%)

R.S.D. (%)

Recovery (%)

R.S.D. (%)

98.1 98.4 98.1 102 94.6

5.18 5.51 3.85 6.59 7.18

103 101 100 104 102

9.12 7.71 3.06 3.67 1.93

96.4 98.2 102 101 102

2.86 7.33 2.47 0.93 2.11

n = 4. Serum samples containing 0.8–10 ␮g/ml degradation products were extracted using MI-SPE procedure, and then analyzed using CE.

The MI-SPE cartridge developed in this research has also been applied to the rice and soil samples containing degradation products of nerve agents. A detection limit of 50 ng/ml, linear range of 0.5–5.0 ␮g/ml, and an R.S.D. of <6.2% were obtained for the rice sample. In order to make this method much more practical, more research will be carried out to simplify the SPE procedure, lower the detection limit and expand the linearity range. Acknowledgements The authors are grateful to Dr. Scott Mcniven, Dr. Lei Ye and Dr. Yihua Yu (Lund University) for scientific and language suggestion. References [1] A.L. Jenkins, O.M. Uy, G.M. Murray, Anal. Chem. 71 (1999) 373.

[2] M. Katagi, M. Nishikawa, M. Tatsuno, H. Tsuchihashi, J. Chromatogr. B 689 (1997) 327. [3] E. Bonierbale, L. Debordes, L. Coppet, J. Chromatogr. B 688 (1997) 255. [4] Z.H. Meng, Y.X. Zhou, Chin. J. Anal. Chem. 28 (2000) 432. [5] J. Olsen, P. Martin, I.D. Wilson, G.R. Jones, Analyst 124 (1999) 467. [6] W.M. Mullett, E. Lai, Anal. Chem. 70 (1998) 3636. [7] W.M. Mullett, E. Lai, B. Sellergren, Anal. Commun. 36 (1999) 217. [8] N. Masque, R.M. Marce, F. Borrull, P.A.G. Cormack, D.C. Sherrington, Anal. Chem. 72 (2000) 4122. [9] L.I. Andersson, Analyst 125 (2000) 1515. [10] M. Kempe, K. Mosbach, J. Chromatogr. A 694 (1995) 3. [11] A.M. Klibanov, Nature 374 (1995) 596. [12] B. Sellergren, TRAC 18 (1999) 164. [13] Z.H. Meng, Hua Xue Jin Zhan 11 (1999) 358 (in Chinese). [14] Z.H. Meng, J.F. Wang, L.M. Zhou, Anal. Sci. 15 (1999) 141. [15] Z.H. Meng, L.M. Zhou, J.F. Wang, Biomed. Chromatogr. 26 (1999) 1251. [16] M. Kempe, Anal. Chem. 68 (1996) 1948. [17] C. Yu, K. Mosbach, J. Org. Chem. 62 (1997) 4057.