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Detection of Acrylamide in Chips and Fried Bread Stick Introduction

Application Introduction

Acylamide (CAS RN 79-06-1) is odourless, transparent platy crystals; it is soluble in water, alcohol, acetone, ether and chloroform, slightly soluble in toluene, insoluble in benzene and heptane.



Acylamide (CAS RN 79-06-1) is odourless, transparent platy crystals; it is soluble in water, alcohol, acetone, ether and chloroform, slightly soluble in toluene, insoluble in benzene and heptane. The relative molecular weight is 71.08, and the structural formula is shown in Figure 1. Acrylamide is a recognized neurotoxin and quasi carcinogens, both animal experiment and in vitro cell experiment prove that acrylamide also can lead to changes in the genetic material, and it has been listed as 2A class carcinogen by International Agency for Research on Cancer early in 2005. April 2002, the Swedish National Food Administration found that many foods containing starch will produce high levels of acrylamide after frying, baking, frying and other high-temperature cooking. Many studies suggest that potato products such as chips and crisps have highest content of acrylamide, and are 500 times of the maximum drinking water allowed limit.

Therefore, it is important to monitor the acrylamide in food to ensure the safety of food consumption by effective experimental technological method. This experiment reference to the first method of national standard method, using Cleanert® ACA solid phase extraction cartridge to purify samples and established a quickly and effectively acrylamide in food by LC-MS / MS detection method.

Figure 1 Structural formula of acrylamide


Reagents and Materials

Original taste chips and fried bread stick of a relevant brand;

Formic acid, methanol, and hexane are all chromatographically pure; Watsons water;

Acrylamide standard substance (purity > 99%), dissolved in water;

D3-acrylamide standard solution (CD2=CDCONH2) (concentration of 500 mg/L, acetonitrile as solvent);

The substrate standard working solution;

0.1% formic acid solution: take 100 μL formic acid and add 100 mL water, shake well;

The disposable sterile syringes; Nylon pin type filter (0.22 μm, diameter of 13 mm);

Cleanert® ACA solid phase extraction cartridge: 200 mg/6 mL

Sample preparation

Manual operation

Accurately weigh 1 g crushed sample into 50 mL centrifuge tube, add 200 μL D3 - acrylamide internal standard (1 mg/L), then add 10 mL water, after ultrasonic oscillation 30 min, centrifuge at 8000 r/min for 5 min , transfer the supernatant to 15 mL centrifuge tube, add 5 mL n-hexane, vortex extract 1 min, centrifuge at 6000 r/min for 5 min, remove the upper organic phase, repeat extract again with 5 mL n-hexane, and then quickly take 5 mL water phase as the sample load solution.

Before using, activate Cleanert® ACA solid phase extraction cartridge (200 mg/6 mL) with 5 mL methanol and 5 mL water, load sample into Cleanert® ACA phase extraction cartridge, wash with 5 mL water, vacuum dry column, eluted with 5 ml methanol, collect all the eluate and nitrogen blowing concentrated to nearly dry at 40 °C , constant volume to 1 mL with water for LC-MS/MS detection.

Qdaura® automatic solid phase extraction apparatus operation procedure

First install Cleanert® ACA solid phase extraction cartridge on the instrument, and inject samples into the sample loading tube, automatic operation according to the procedure in the following figure:

Figure 2 Automatic solid phase extraction operation procedure


HPLC column: Venusil® AQ C18, 5 μm, 100 Å, 2.1 × 150 mm;

Mobile phase: 0.1% formic acid aqueous solution;

Column temperature: 26 °C;

Flow rate: 0.2 mL/min;

Injection volume: 2 μL;

AB SCIEX API 4000+Liquid Chromatograph Mass Spectrometer;

Ion source: ESI+;

Electrospray voltage: 5500 V;

Atomization gas pressure: 45 psi;

Air curtain pressure: 10 psi;

Aux Gas Pressure: 45 psi;

Ion source temperature: 330 °C;

Acquisition methods: multiple reactions monitoring (MRM).

Table 1 Mass spectrum parameters of acrylamide

Results and Discussion

Prepare acrylamide standard solution with the concentration of 0.01 mg/L, 0.05 mg/ L, 0.1 mg/L, 0.5 mg/L, 1 mg/L and 3 mg/L respectively (internal standard: 0.1 mg/L) for LCMS/MS detection. Take the injection concentration of each acrylamide standard solution (mg/L) as the abscissa, take the rate of acrylamide peak area and internal standard D3 - acrylamide peak area as the ordinate, draw the standard curve, as shown in figure 3, the fitting parameters are shown in table 2.

Table 2 Standard curve and the detection limit of acrylamide

Figure 3 Standard solution curve of acrylamide


Table 3 indicate that when using Cleanert® ACA solid phase extraction cartridge combined with LCMS/MS method to detect acrylamide, the recovery rate of 0.1 mg/kg and 2 mg/kg acrylamide spiked amount is 100% ~ 110%, which meet the detection requirement.

Table 3 Spiked recovery experiment results of acrylamide (n=3)

  • Cleanert ACA

    Cleanert ACA use coconut charcoal material to concentrate polar substances in water sample, such as acrylamide which could not been adsorbed by C18 or other RP phase material. It could be used in EPA 521 and EPA 522.

  • Venusil AQ C18

    The Venusil AQ C18 column is designed for the separation of polar, medium-polar and non-polar compounds from low to medium pH. This column is more polar than XBP C18, but less polar than ASB C18. With a special surface treatment, Venusil AQ C18 is made to be compatible with 100% aqueous mobile phases.

  • Qdaura Automated SPE System

    The Qdaura Automated SPE system is specifically designed for high throughput sample preparation. The system automates routine SPE procedures from conditioning, sample loading, washing, dry and elution. The Qdaura utilizes positive pressure to ensure more reproducible flow rates, more reproducible recoveries, less sample to sample contamination, and higher precision.

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