Li Yubo, Shi Zhihong, Zhang Hongyi
(College of Chemistry and Environmental Science; Hebei University; Key Laboratory of Analytical Science and Technology of Hebei Province，Baoding 071002)

Abstract a new pre-column derivatization-HPLC method for the determination of acrylamide was presented in this paper, including the preparation of derivative reagent 2 - hydroxy -3 - iodo-1 ,4- naphthoquinone and the optimization of the derivatization conditions. The derivative reagent and the derivative product of acrylamide were separated effectively on a Diamonsil C₁₈ (150mm × 4.6mm i.d., 5mm) column by using a mixture of acetonitrile and water (80:20, v / v) as mobile phase.
Keywords Acrylamide, pre-column derivatization, HPLC, determination

1. INTRODUCTION                         
Report of high concentrations of acrylamide in common heated foodstuffs by the Swedish National Food Administration and researchers from Stockholm University in April 2002 attained worldwide concern, since acrylamide is classified as a probable human carcinogen by the International Agency for Research on Cancer [1]. Hence the detection of acrylamide is of great importance.   
    For the analysis of acrylamide in food samples, there are primarily two approaches, based either on gas chromatography with mass spectrometric detection (GC–MS)[2-3] or liquid chromatography with tandem mass spectrometric detection (HPLC–MS–MS)[4-7]. However, due to the relatively high cost of GC-MS and LC-MS-MS, the application of these methods might be beyond the means of some laboratories. Compared with GC-MS and LC-MS-MS techniques, high-performance liquid chromatographic (HPLC) techniques possess the advantages of easy generalization, lower cost and strong maneuverability [8]. Being a polar molecule, acrylamide shows poor retention on conventional RP-HPLC columns, to conquer poor retention on conventional RP-HPLC column and to avoid the interference of co-extractives, derivatization of acrylamide may be a good solution.
    In this paper, 2 - hydroxy -3 - iodo-1 ,4- naphthoquinone was used as derivative reagent to react with acrylamide under a nitrogen atmosphere while heating to reflux, good separation was achieved between derivative reagent and the derivative product on C₁₈ column.

2. EXPERIMENTAL
2.1 Chemicals and materials
Acrylamide (>99.9%) was purchased from Amresco (Solon, Ohio, USA). Lawsone (99%) was obtained from New Jersey, USA. Morpholine (99.5%) was obtained from West Chester, USA. Iodine and Pd(OAc)₂ were of analytical grade and obtained from Tianda chemical laboratorial plant, China. DMF was of analytical grade and purchased from Dingsheng chemical plant in Tianjin.
    Stock solution of acrylamide was prepared by dissolving the compound in doubly-distilled water.
2.2 Instrumentation
HPLC analyses were performed on an LC-10ATvp plus liquid chromatograph (Shimadzu, Japan) which consisted of an LC-10ATvp plus pump, a Rheodyne model 7725i injection valve (sample loop 20m l) and an SPD-10Avp plus multi-wavelength detector. The chromatographic data were recorded and processed with a CBM-10Avp plus LC Solution Lite software. The analytical column was a Diamonsil C18 (150mm × 4.6mm i.d., 5mm) column.
2.3 Preparation of the derivative reagent
To a mixture of lawsone (0.22g) and K₂CO₃ (0.42g) in an Erlenmeyer flask, 10mL of doubly-distilled water was added，while the mixture of a solution of iodine(0.05g) in benzene(8mL) and morpholine (0.03mL) was added in small portions every 15 min during 2 hours. The solution was stirred at room temperature for an additional hour and then the mixture was filtered to remove any solids present. The filtrate was cooled in an ice bath for 30min, and then it was acidified with 25% H₃PO₄ until the pH was approximately 2. The mixture was allowed to age for 60min to eliminate any complex remaining and to form a bright yellow precipitate. The solid was filtered off and washed with cold water. The product was dried under vacuum overnight [9].
2.4 Derivatization of acrylamide
To a 50 mL round-bottom flask (with stir-bar), 0.15g of iodolawsone was added with 5 mg of Pd(OAc)₂ and 0.346mg of K₂CO₃. The flask was capped with a septum and 5 mL of deoxygenated water was transferred into the flask via a canula under nitrogen gas protection. 0.071g acrylamide was added and the dark red mixture was stirred under a nitrogen atmosphere while heating to reflux. After 6 hr, the reaction mixture was cooled and the black Pd metal precipitate was filtered off. The red filtrate was cooled in an ice bath and was acidified with H₃PO₄ (25% aqueous solution) until pH ~2. During the acidification, the solution changes color to bright yellow or orange and a precipitate forms. The solid was filtered off by vacuum filtration and the product was dried in a vacuum [10].

3. RESULTS AND DISCUSSION
3.1 Optimization of derivatization
3.1.1 The effect of heating temperature on the formation of derivative product
Temperature is an important factor for this reaction. From Fig 1, it could be seen that the peak area of derivative product increased along with the rise of heating temperature. The peak area of the derivative product reached the maximum value at 100℃ and remained constant after 100℃. So 100℃was selected as the heating temperature for the derivative reaction.

Fig. 1 The effect of heating temperature on the formation of derivative product

3.1.2 The effect of heating time on the formation of derivative product
Experimental results showed that the peak area of derivative product increased along with the increase of heating time. When the heating time was 6 hr, the peak area of derivative product reached the maximum value, but it decreased when the heating time was prolonged. So the optimum heating time was selected to be 6 hr for the derivative reaction.
3.2 Separation and determination of derivative reagent and the derivative product of acrylamide
Acetonitrile/water mixture was employed as the mobile phase. When the volume ratio of acetonitrile to water was 80:20, good separation was obtained and the retention time of derivative reagent and product were 18.2 min and 14.2 min, respectively (Figure. 2).
    Spectrum scanning shows that the derivative product of acrylamide has the maximum absorption at 270nm, so 270nm was set as the detection wavelength.

Fig.2 Chromatogram of derivative reagent and derivative product

4. CONCLUSION
This paper discussed a new pre-column derivatization-HPLC method for the determination of acrylamide. The derivatization conditions were optimized and effective separation between 2 - hydroxy -3 - iodine-1,4 - naphthoquinone and 2 - hydroxy -3 - acrylamido-1,4 - naphthoquinone was obtained. The method will be useful for the determination of acrylamide in food samples.

ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (20575016) and the Natural Science Foundation of Hebei Province China (B2006000953) are gratefully acknowledged.

REFERENCES
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丙烯酰胺的新型柱前衍生-高效液相色谱检测方法的初步研究
石志红，李玉波，张红医
（河北大学化学与环境科学学院，河北省分析科学技术重点实验室， 保定 071002）
摘要 本文探讨了一种新型的丙烯酰胺柱前衍生-高效液相色谱检测方法，包括衍生试剂的制备过程、衍生反应条件的优化等，在所选色谱操作条件下实现了衍生试剂与衍生产物的有效分离。
关键词 丙烯酰胺 柱前衍生 高效液相色谱