A Complete Solution from Welch Materials for the Determination of Neotame in Food

Neotame is a high-intensity functional sweetener that has been widely used in various food products, including confectionery, baked goods, beverages, seasonings, protein powders, solid beverages, meal replacement powders, and sports nutrition products. It has been approved as a food additive in the U.S., European Union, China, among many other regions.

Neotame is not metabolized by the human body, with more than 90% of ingested neotame excreted through urine and feces. However, excessive long-term intake may cause adverse effects such as headaches and nausea. Therefore, its application must strictly comply with the maximum usage limits and permitted food categories specified by relevant regulations. In particular, neotame is prohibited from being used for the preservation of fresh fruits.

To help laboratories achieve efficient and accurate determination of neotame in food samples, Welch Materials has developed a comprehensive SPE-HPLC and SPE-LC-MS/MS workflow solution, covering sample preparation, purification, and chromatographic analysis.

Note: This method is based on the Chinese national food safety standard GB 5009.247-2025 “Determination of Neotame in Foods”.

SPE Procedure:

• Cartridge: Welchrom HLB, 500 mg/6 mL
• Activation: 6 mL methanol followed by 6 mL water
• Loading: Load 10 mL of the sample extract. Discard the effluent.
• Rinse: Wash with 5 mL formic acid-triethylamine buffer. Discard the effluent.
• Elution: Elute with 8 mL mixed elution solution. Collect the eluate.
• Concentration: Nitrogen evaporate the collected eluate in a 60 °C water bath to 1 mL. Dilute to 2 mL with formic acid-triethylamine buffer. Filter through a membrane. Proceed to HPLC analysis.

Chromatographic Conditions:

• Column: Ultisil XB-C18, 5 μm, 4.6×150 mm
• Mobile Phase: A) Methanol; B) 20 mmol/L ammonium acetate
• Flow Rate: 1.0 mL/min
• Injection Volume: 50 µL
• Column Temperature: 30 °C
• Detection Wavelength: 210 nm
• Gradient Profile:
  

  Time (min)	A (%)	B (%)
  0.00	10	90
  4.00	60	40
  20.00	60	40
  20.01	10	90
  25.00	10	90

SPE Procedure:

• Cartridge: Welchrom HLB, 500 mg/6 mL
• Activation: 6 mL methanol followed by 6 mL water
• Loading: Load 10 mL of the sample extract. Discard the effluent.
• Rinse: Wash with 5 mL formic acid-triethylamine buffer. Discard the effluent.
• Elution: Elute with 8 mL mixed elution solution. Collect the eluate.
• Concentration: Dilute the collected eluate to 10 mL with formic acid-triethylamine buffer. Filter through a 0.22 µm membrane. Proceed to LC-MS/MS analysis.

Chromatographic Conditions:

• Column: Ultisil XB-C18, 3 μm, 3.0×150 mm
• Mobile Phase: A) Methanol; B) 5 mmol/L ammonium acetate
• Flow Rate: 0.3 mL/min
• Injection Volume: 2 µL
• Column Temperature: 30 °C
• Gradient Profile:
  

  Time (min)	A (%)	B (%)
  0.00	10	90
  2.00	80	20
  11.00	80	20
  11.01	10	90
  15.00	10	90