GB 5009.247-2016Method for the determination of Neotame in foods -- High-performance liquid chromatography (English PDF)

3.1 Reagents

3.1.1 Acetonitrile (CH3CN). chromatographically pure.

3.1.2 1-Octanesulfonic acid sodium salt (C8H17NaO3S). chromatographically pure.

3.1.3 Phosphoric acid (H3PO4).

3.1.4 Formic acid (CH3COOH). chromatographically pure.

3.1.5 Methanol (CH3OH). chromatographically pure.

3.1.6 Triethylamine (C6H15N). chromatographically pure.

3.2 Preparation of reagents

3.2.1 Mixed extract. absorb 0.8 mL of formic acid and 2.5 mL of triethylamine; add water to make up to 1,000 mL. The pH is about 4.5.

3.2.2 Ion pair reagent buffer solution. weigh 2.00 g of 1-Octanesulfonic acid sodium

salt; use 500 mL of water to dissolve; add 1.0 mL of phosphoric acid; add water to make up to 1,000 mL.

3.3 Standard substance

Neotame (C20H30N2O5, CAS no.. 165450-17-9), of purity >= 99.0%.

3.4 Preparation of standard solutions

3.4.1 Standard stock solution. weigh accurately 0.100 0 g of neotame standard

substance; add mixed extracting solution to dissolve and make up to 100 mL. The neotame content of this solution is 1.00 mg/mL.

3.4.2 Standard working solutions. absorb an appropriate amount of neotame standard

stock solution; use mixed extracting solution to prepare serial standard working solutions of 0.2 µg/mL, 1.0 µg/mL, 5.0 µg/mL, 10.0 µg/mL, 50.0 µg/mL and 100.0

µg/mL.

3.5 C18 solid phase extraction column. 6 mL, 500 mg or equivalent and activated before use, using 5 mL of methanol and 10 mL of water in succession.

3.6 Filter membrane 0.45 µm, organic system.

5.1 Sample preparation

5.1.1 Solid sample

Weigh 10 g (accurate to 0.01 g) of sample pulverized evenly to place into a stoppered plastic centrifugal tube of 50 mL; add 30 mL of mixed extract; carry out vortex oscillation for 10 min and ultrasonic extraction for 30 min; use mixed extracting solution to make up to scale. If the solution is turbid, filter it after centrifugation for 10 min at not lower than 4,000 r/min.

5.1.2 Liquid sample

Weigh accurately 10.0 mL of sample to place into a stoppered plastic centrifugal tube of 50 mL; add 30 mL of mixed extracting solution; carry out vortex oscillation to mix up.

After ultrasonic extraction for 15 min, use mixed extracting solution to make up to scale.

If the solution is turbid, filter it after centrifugation for 10 min at not lower than 4,000 r/min.

NOTE. First slightly heat gas-containing sample such as carbonated beverages and aerated waters; stir to remove carbon dioxide in sample or carry out ultrasonic degassing; then accurately measure and take sample.

5.2 Sample purification

Absorb 10.0 mL of filtrate to pass the solid phase extraction column at the flow rate of

1 mL/min ~ 2 mL/min; after the filtrate is fully discharged, use 5 mL of mixed extracting solution to elute the extraction column at the flow rate of 1 mL/min ~ 2 mL/min; discard all effluent; use 5 mL of methanol to elute at the flow rate of 1 mL/min; use the nitrogen blowing concentrator for the concentration of eluant on a water bath at 40°C; use mixed extracting solution to make up to 2.0 mL; filter it using filter membrane of 0.45 µm before using it as the solution to be tested on the liquid chromatograph.

5.3 Instrument reference conditions

5.3.1 Chromatographic column. C18 chromatographic column, 5 µm, 250 mm × 4.6 mm

(inner diameter) or equivalent.

5.3.2 Column temperature. 30°C.

5.3.3 Moving phase. phase A. acetonitrile; phase B. ion pair reagent buffer solution.

For the gradient elution procedures see Table 1.

5.3.4 Flow rate. 1.0 mL/min.

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