|LC/MS Method Using Column Switching for the Analysis of Acrylamide in Foods|
|National Institute of Health Sciences, Division of Foods|
Standards：acrylamide, for electrophoresis (Kanto Chemical Co., Inc., Tokyo, Japan), purity 99.9% ; internal standard, acrylamide-1-13C (AA-1-13C) (CDN ISOTOPES Co., Quebec, Canada), 99 atom% 13C.
Calibration standard solution：Aliquots of AA standard solutions (equivalent to 0, 50, 100, 500, 1,000, 2,000 and 5,000 ng of AA) were mixed with 20 μL of internal standard solution (100 μg/mL AA-1-13C) and made up to 10 mL with water.
Bond Elut C18 (500 mg/3 mL), Bond Elut Jr-PSA, (500 mg) and Bond Elut ACCUCAT (600 mg/3 mL) : Bond Elut C18 was conditioned with 2 mL of methanol and successively with 4 mL of water before use. The other columns were conditioned only with 4 mL of water before use.
Acetone: high quality for pesticide residue analysis. Dichloromethane, methanol and water: HPLC grade.
Four columns were used and switching valves were inserted between column-1 and -2, and column -2 and -3.
Liquid chromatograph/mass spectrometer：model 2690 separation module liquid chromatograph (Waters, Co.) coupled with a model Platform LCZ mass selective detector (Micromass UK Ltd.)
Degasser: ERC-3215α (ERC Inc., Kawaguchi, Japan)
Switching valve: LabPRO (Rheodyne) and EV700-100 (GL Sciences)
Pump: LC-10AD and LC-10ADvp ( Shimadzu Co.)
Homogenizer：Polytron (KINEMATICA AG, Littau, Switzerland)
Concentrator: Turbo Vap 500 (Zymark Co)
3. LC/MS conditions
Column： column-1, Hypercarb（50×2.1 mm i.d., particle size 5 μm, Thermo Hypersil-Keystone）; column-2, Shodex MSpak GF-310 4B (50×4.6 mm i.d., particle size 6 μm, Shoko. Co. LTD.); column-3, Atlantis dC18 (150×2.1 mm i.d., particle size 5 μm, Waters Co.); column-4, Develosil RPAQUEOUS-AR-3 (35×2.0 mm i.d., particle size 3 μm, Nomura Chemical Co.)
Column oven temp.: 40°C for the column-1, -2 and -3, and room temperature for the column-4
Mobile phase composition: 0.1% acetic acid-methanol (95:5)
Flow rate; 0.25 ml/min
Sampling time from column-1 to column-2: 1.75〜2.05 min
Sampling time from column-2 to column-3 : 4.66〜5.15 min
AA eluting time from column-4: 8.5 min
Source block temp.: 120°C, Desolvation temp.: 350°C, Gas: nitrogen, flow rate, ca.300 L/hr, Capillary voltage: 3.50 eV, Ionization mode: positive ion electron spray, Measuring mode: selected ion recording（SIR）, Monitoring ion and corn voltage : m/z 72 (AA) and m/z 73 (AA-1-13C ) at 24 V of corn voltage and m/z 55 (AA) and m/z 56 (AA-1-13C ) at 40 V of corn voltage, Injection volume: 10 mL
4. Sample preparation
Sample (5.0 g) was weighed into a 250-mL glass tube and 20〜25 mL of water and 100 μL of internal standard solution (100 μg/mL AA-1-13C solution) were added to the sample. The mixture was allowed to stand for 30 min with occasional shaking. Acetone (50 mL) was added and the mixture was homogenized for 1 min with a homogenizer. After filtrating the homogenate through a glass filter (GA-100), the residue on the filter was washed with 30 mL of acetone. The filtrate was concentrated to less than 15 mL by a concentrator at 35℃. The extract was transferred in a 50-mL centrifugal tube, washed twice with each 20 mL of dichloromethane by shaking for 1 min and centrifuged at 2,700 rpm for 5 min. The extract was made up to 15 mL with water.
An aliquot of the extract (3 mL, equivalent to 1.0 g of sample) was passed through a C18 cartridge column with following washing with 2 mL of water. All the eluate was collected and made up to 5 mL with water. An aliquot of the eluate (2 mL, equivalent to 0.4 g of sample) was passed through an ACCUCAT column connected to PSA column with following washing with 2 mL of water. All the eluate was collected and made up to 4 mL with water to prepare the test solution.
5. Determination and confirmation of AA
AA was determined by LC/MS(SIR). The quantification was performed by the comparison of the peak area ratios of m/z 72 (AA) and m/z 73 (AA-1-13C) for the test solution with those for the calibration standard. The peak area ratio of m/z 55 (AA) and m/z 56 (AA-1-13C) was used for the qualification. The recovery of internal standard was calculated from the peak areas of AA-1-13C in the test solution and the calibration standard.
The calibration standard (AA=0, AA-1-13C=200 ng/mL) contained a small quantity of AA as an impurity of AA-1-13C. Therefore, the limit of detection (LOD) was defined as 3 times the SD of the AA peak area of calibration standard (AA=0, AA-1-13C=200 ng/mL). Similarly, the limit of quantification (LOQ) was defined as 10 times the SD.