Application Note 1106 Note Application Determination of Azodicarbonamide in Flour Liu Lvye,1 Jin Yan,1 Xu Qun,1 and Jeffrey Rohrer2 1Thermo Fisher Scientific, Shanghai, People’s Republic of China; 2Thermo Fisher Scientific, Sunnyvale, CA, USA Key Words Azobisformamide, HPLC, Syncronis Amino Column, Food Additive, Food Safety, Food Analysis Goal Azodicarbonamide has been linked to respiratory issues, allergies, and asthma. Therefore, this efficient high-performance liquid chromatography (HPLC) method was developed to determine azodicarbonamide in flour. Introduction Azodicarbonamide (structure shown in Figure 1) is used as a flour bleaching agent to improve the appearance of the final product. Azodicarbonamide reacts with moist flour as an oxidizing agent; the main reaction product is biurea, a derivative of urea, which is stable during baking.1 The World Health Organization has linked azodicarbonamide to respiratory issues, allergies, and asthma among individuals at workplaces where azodicarbonamide is manufactured or handled in raw form.2 In the United States and China, limited amounts of azodicarbonamide are considered safe and its addition Equipment, Software, and Consumables to flour is allowed up to 45 and 20 mg/Kg, respectively.3,4 • Thermo Scientific™ Dionex™ UltiMate™ 3000 In Australia and Europe, azodicarbonamide is banned Rapid Separation LC (RSLC) system, including: as a food additive.5 Therefore, it is necessary to – LPG-3400RS Quaternary Pump with SRD-3400 establish efficient and sensitive methods to determine Integrated Solvent and Degasser Rack, 4 channels azodicarbonamide in flour, and HPLC with UV – WPS-3000TRS Wellplate Sampler, Thermostatted, detection offers the advantages of method accuracy with 25 µL sample loop and ruggedness.6,7 – TCC-3000RS Thermostatted Column Compartment – DAD-3000RS Diode Array Detector, with 13 µL flow cell • Thermo Scientific™ Dionex™ Chromeleon™ 0 Chromatography Data System software, version 7.1 or above N NH 2 • Thermo Scientific™ Sorvall™ ST 16 Centrifuge H2N N (P/N 75004240) 0 • Thermo Scientific™ Target2™ Nylon Syringe Filters, 0.45 µm, 30 mm (P/N F2500-1) Figure 1. Structure of azodicarbonamide. 2 Reagents and Standards Stock Standard 2 • Deionized (DI) water, 18.2 MΩ-cm resistivity, generated Dilute 1 mL of Stock Standard 1 to 10 mL with ™ ™ ™ from the Thermo Scientific Barnstead GenPure Pro CH3CN/CH3OH (9:1, v/v). The concentration of Stock ultrapure water system (P/N 50131948) Standard 2 will be 100 µg/mL. • Methanol (CH3OH), 99.8%, HPLC Grade (Fisher Standard Solutions for Calibration Scientific P/N AC610090040) For calibration, prepare five working standard solutions with different concentrations by diluting the proper • Acetonitrile (CH3CN), HPLC Grade (Fisher Scientific P/N AC610010040) amount of the stock standard solutions with acetonitrile/ • Dimethylformamide (DMF), HPLC Grade (Fisher methanol (9:1, v/v). The volumes of each solution needed Scientific P/N 50-276-273) to make the calibration standards are shown in Table 1. • Dimethyl sulfoxide (DMSO), HPLC Grade (Fisher Sample Preparation Scientific P/N D-159-4) Flour was purchased in a supermarket in Shanghai, • Ammonium acetate, HPLC Grade (Fisher Scientific People’s Republic of China. P/N A639-500) Add 10 mL of DMF/DMSO (9:1, v/v) to 5 g of flour in a • Azodicarbonamide, 97% (Fisher Scientific 20 mL centrifuge tube. Mix for 10 min to extract, then P/N AC10516-5000) centrifuge for 10 min at 4000 rpm. Prior to injection, filter the supernatant through a 0.45 µm filter.6 Chromatographic Conditions Add 9.8 mL of DMF/DMSO (9:1, v/v) and 200 µL of ™ ™ Column: Thermo Scientific Syncronis Amino, 5 µm, 100 µg/mL azodicarbonamide standard solution (Stock 4.6 × 250 mm (P/N 97705-254630) Standard 2) to 5 g of flour in a 20 mL centrifuge tube. Mobile Phase: Acetonitrile/Methanol, 20 mM ammonium acetate Mix for 10 min to extract, then centrifuge for 10 min (dissolve 0.15 g of ammonium acetate in 1 L of DI at 4000 rpm. Prior to injection, filter the supernatant water without pH adjustment) through a 0.45 µm filter. The spiked concentration of Gradient: 0–7.5 min, 95% acetonitrile, 5% methanol; azodicarbonamide in the flour sample will be 2 µg/mL. 7.5–11.5 min, 80% acetonitrile, 20% ammonium acetate; 11.5–15 min, 95% acetonitrile, 5% methanol Results and Discussion Extraction Solvent Selection Flow Rate: 0.8 mL/min Azodicarbonamide is soluble in DMSO; however, the Injection Volume: 10 µL (partial-loop injection) high viscosity of DMSO (1.996 mPa·s at 25 °C) can Temperature: 25 °C affect the layering between liquid and solid phases when Detection: UV, 272 nm azodicarbonamide is extracted from a flour sample. Therefore, DMF—a solvent with lower viscosity Preparation of Standard Solutions (0.802 mPa·s at 25 °C) and slight solubility for Stock Standard 1 azodicarbonamide—was used as the extraction solvent Dissolve 0.1 g of azodicarbonamide standard in 100 mL together with DMSO. Experiments showed that using a of DMF/DMSO (9:1, v/v). The concentration of Stock mixture of DMF and DMSO solvent (9:1, v/v) can Standard 1 will be 1000 µg/mL. yield good layering and satisfactory extraction results (>85% recovery). Table 1. Preparation of calibration standards. Acetonitrile/Methanol Volume Calibration Std Calibration Std Azodicarbonamide Stock Std Volume (9:1, v/v) Final Volume Final Concn Stock Std (mL) (mL) (mL) (µg/mL) 5.0 5.0 50 2.0 8.0 20 Stock Standard 2 1.0 9.0 10 10 (100 µg/mL) 0.2 9.8 2.0 0.1 9.9 1.0 55 55 60 3 217.50 A 201.51 B C 242.27 % % % -5 -5 -10 190 250 300 350 402 190 250 300 350 402 190 250 300 350 402 nm nm nm Figure 2. The UV spectra of (A) DMF, (B) DMSO, and (C) azodicarbonamide. UV Detection Wavelength and Column Choice 3.5 1 As shown in Figure 2, DMF and DMSO with cutoff wavelengths of ~260 and 240 nm, respectively, were used in preparations of standard solutions and samples. Peaks: 1. DMF The maximum UV absorbance of azodicarbonamide 2. Azodicarbonamide standard (5 µg/mL) is 242 nm, but it does absorb at higher wavelengths. 3. DMSO Therefore, the detection wavelength was set at 272 nm, which is a compromise between removing interference mAU with DMSO/DMF and getting sensitive detection of 2 azodicarbonamide. An amino-type column, the Syncronis Amino column, was chosen for the separation of 3 azodicarbonamide because amino phases are often appropriate for polar compounds.8 Figure 3 shows a chromatogram of an azodicarbonamide standard under 0 the specified chromatographic conditions. –0.5 Reproducibility, Linearity, and Detection Limit 0 5 10 15 Short-term method reproducibility was estimated Minutes by making five consecutive injections of an Figure 3. An azodicarbonamide standard after a blank (no flour) extraction. azodicarbonamide standard with a 10 µg/mL concentration. The retention time reproducibility 3 RSD was 0.1, and the peak area reproducibility RSD y = 0.0547x – 0.0276 was 1.5, demonstrating good short-term precision for 2.5 r² = 0.995 this HPLC method. 2 Calibration linearity for UV detection of 1.5 azodicarbonamide was investigated by making three Area (µS * min) consecutive 10 µL injections of a standard prepared at 1 Peak five different concentrations (i.e., 15 total injections). 0.5 Linearity was observed from 1 to 50 µg/mL when plotting 0 the concentration versus the peak area (Figure 4). The 0 10 20 30 40 50 60 linear regression equation was A = 0.0547c – 0.0276, Concentration (µg/mL) where A represents peak area and c represents the analyte Figure 4. Calibration curve for azodicarbonamide. concentration. The coefficient of determination was 0.995. This calibration curve was used to quantify azodicarbonamide in flour samples. Five replicate injections of an azodicarbonamide standard with a 1 µg/mL concentration were used for estimating the method detection limit (MDL) using a signal-to-noise ratio = 3. The measured MDL of azodicarbonamide was 0.2 µg/mL, equivalent to 0.2 mg/Kg for flour samples, which is much lower than the limit recognized as safe in the United States and People’s Republic of China (up to 45 and 20 mg/Kg, respectively).3,4 Analysis of Flour Samples 4.75 Azodicarbonamide was not found in the flour samples. Peaks: 1. DMF 1106 Note Application Figure 5 shows chromatograms of a flour sample and the 2. Azodicarbonamide standard 3. DMSO same sample spiked with azodicarbonamide. To judge method accuracy, three injections of the flour sample spiked with a 2 µg/mL of azodicarbonamide standard were made. The average recovery was 86%. 1 mAU Conclusion 2 This work describes an efficient HPLC method with 3 B UV detection for the sensitive determination of azodicarbonamide in flour. The advantages of this approach include simple sample preparation and good 0 A method reproducibility. References –0.75 0 5 10 15 1. Joiner, R.; Vidal, F.; Marks, H. A New Powdered Minutes Agent for Flour Maturing. Cereal Chemistry 1963, 40, Figure 5. (A) A flour sample and (B) the same sample spiked with a 2 μg/mL of 539–553. azodicarbonamide standard. 2. Concise International Chemical Assessment Document 16: Azodicarbonamide; World Health Organization: Geneva, 1999. 3. Title 21: Food and Drugs. Code of Federal Regulations; Volume 3, 21CFR172.806; Government Printing Office: Washington, DC, 2012. 4. GB 2760-2011: Standards for the Use of Food Additives; Ministry of Health of the People’s Republic of China: Beijing, 2011. 5. Wikipedia. http://en.wikipedia.org/wiki/ Azodicarbonamide (accessed Aug. 7, 2014). 6. Chen, Y.; Qi, G.J.; Wang, Y.; Zhao, C.J. Determination of Azodicarbonamide in Flour and its Preparation by RP-HPLC.