Determination of semicarbazide in flour and its products LC-MS/MS method

1. Scope

This standard specifies the method of determination of semicarbazide in flour and its products for export by LC-MS/MS method
This standard applies to the determination of semicarbazide in wheat flour, fry powder, fine-dried noodles, bread crumbs, noodles, steamed bread, Fried flour, egg yolk pie, biscuits, instant noodles, bread, wheat flour, and its products for export by LC-MS/MS method

2. Normative reference

It is essential to cite the following documents in applying the standard. For all the cited documents with date, only the dated references apply to this standard. For undated references, the latest edition ( including all amendments) of the normative document is applicable to the standard. GB/T6682 Water for analytical laboratory Specification and test methods.

3. Method Summary

The semicarbazide in the sample reacted with o-nitrobenzaldehyde in acidic conditions. The derivatization product was purified by the HLB column under neutral conditions and then determined by LC-MS-MS, quantified by external standard method.

4. Reagents and Materials

Unless specified, all the reagents used should be analytical grade, “water” was the first level water described in GB/T 6682.

4.1 Acetonitrile: HPLC grade.

4.2 Methanol: HPLC grade.

4.3 Hydrochloric acid: GR.

4.4 Formic acid: 98% of purity, HPLC grade.

4.5Ethyl acetate: HPLC grade.

4.6 Tripotassium phosphate trihydrate (K₃PO₄ · 3H₂0).

4.7 Sodium hydroxide (NaOH).

4.8 O-nitrobenzaldehyde.

4.9 0.2 mol/L Hydrochloric acid solution: measure 17mL hydrochloric acid (4.3) accurately, dilute with water to a volume of 1 L, mix, and reserve.

4.10 2.0 mol/L NaOH solution: weigh 20 g NaOH (4.7) accurately, dilute with water to a volume of 250mL, mix and reserve.

4.11 0.3 mol/L K₃PO₄ solution: weigh 79.8 g K₃PO^₄ · 3H₂0 (4.6) accurately, dilute with water to a volume of 1 L, mix and reserve.

4.12 0.1 mol/L O-nitrobenzaldehyde solution: weigh 1.5 g 0-nitrobenzaldehyde (4.8) accurately, dilute with methanol to a volume of 100 mL, mix and reserve.

4.13 0.1% Formic acid solution: measure 1 mL formic acid (4.4) accurately, dilute with water to a volume of 1 L, mix, and reserve.

4.14 Semicarbazide hydrochloride standard substance: purity > 95.0%.

4.15 Semicarbazide isotope internal standard (iS) stock solution (Semicarbazide hydrochloride [1,2-15N213C):concentration 100 μg/mL.

4.16 Semicarbazide standard stock solution(100 μg/mL): weigh 14.9 mg semicarbazide hydrochloride (4.14) into 50 mL water, dilute with methanol to a volume of 100 mL. it should be stored below 4 ℃ and protected from light for 3 months.

4.17 Semicarbazide intermediate standard solution(1 μg/mL): measure 1.0 mL semicarbazide standard stock solution(4. 16), and dilute with methanol to a volume of 100 mL. lt should be stored below 4 ℃ and protected from light for 1 month.

4.18 Semicarbazide working standard solution(0.02 μg/mL): measure 2.0 ml semicarbazide inter-mediate standard solution (4.17), dilute with methanol to a volume of 100 mL. lt should be stored below 4 ℃ and protected from light, freshly prepared just before use.

4.19 Semicarbazide isotope internal working standard solution (IS 0.1 μg/mL): Semicarbazide isotope internal standard stock solution (4.15) dilute with methanol to the concentration of 0.1 ug/mL. It should be stored below 4 ℃ and protected from light, freshly prepared just before use.

4.20 50 mL centrifuge tubes with sealed caps.

4.21 SPE column: HLB, 60mg, 3 mL, or equivalent: activate with 3 mL methanol and 3 mL water before use.

4.22 Membrane: 0.22 μm, the organic solvent film.

4.23 pH test strips: range 5-8.

5. Instruments and Equipment

5.1 Liquid chromatograph/ tandem mass spectrometer (LC-MS/MS): A triple quadrupole equipped with an electrospray ionization (ESl) source.

5.2 Analytical balance: a sensitivity of 0.0001 g and 0.01 g.

5.3 Shaker.

5.4 Calorstat.

5.5 Nitrogen evaporator.

5.6 Refrigerated centrifuge: 4 ℃, speed 10 000 r/min.

5.7 Vortex mixer.

6. Preparation and storage of test samples

6.1 Wheat flour, fry powder, and other powder samples

Take 500g representative samples from the total taken samples. Put into a clean container after homogenization, seal, and label.

6.2 Fine-dried noodles, bread crumbs

Put all the samples (500g) into a clean container after homogenization using a food grinder, seal and label.

6.3 Noodles, steamed bread, Fried flour, egg yolk pie, biscuits, instant noodles, bread

Cut the sample into pieces (the sample with a jam center, filling, or cream, should be removed first), then put it into a clean container after homogenization using a food grinder, seal, and label.

7 Procedure

7.1 Hydrolysis and derivation

Weigh 2.0 g (accurate to 0.01 g) sample in a 50 ml centrifuge tube with sealed caps, and add 50 μl Semicarbazide IS working solution(4.19), set aside for 5 minutes, then add 10 mL hydrochloric acid solution (4.9), intermixed. Add O-nitrobenzaldehyde solution (4.12) 100 μL, vortexed for 1min. Shake for 10 min, react at 37 ℃ for 16 hours.

Wait for the sample to cool to room temperature after derivatization, add 3 mL K₃PO₄ solution (4.11) with NaOH solution (4.10) and adjust pH to 7(±1). Centrifuge at 4 ℃ 1000 rpm for 10 min. Take the supernatant for purification.

7.2 Purification

Transfer the extract solution (7.1) to the HLB column (4.21), wash with 6 mL water, dry. Elute with 3 mL ethyl acetate. Control the liquid to pass the column at a steady rate of 2 mL/min~3 mL/min. Dry the eluent with N₂ under 40 ℃. Dissolve the residue with 1 mL formic acid solution (4.13), vortex to mix uniformly, then filtrate through 0.22 μm membrane (4.22) for LC-MS/MS determination.

7.3 Standard operation curves

Take six 50 ml centrifuge tubes with caps, add semicarbazide standard working solution (4.18) 0 μL, 25 μL, 50 μL, 250 μL, 500 μL, 1000 μL respectively, then add 50 μL semicarbazide lS solution (4.19). The next operations of preparation of the standard solution are the same as 7.1 and 7.2.

7.4 Determination

7.4.1 LC conditions

LC conditions:

a) column: Waters BEH C₁₈, 50 mm x 2.1 mm, 1.7 μm, Or equivalent;

b) column temperature: 30℃;

c) flow rate: 0.3 mL/min;

d) Mobile phase and elution gradient are shown in Table 1.

7.4.2 MS operation conditions

MS operation conditions:

a) Ion source: ESl ion source;

b) Scan mode: Positive ion;

c) Detect mode: Multiple reaction monitoring (MRM);

d) Other conditions refer to appendix A.

7.4.3 Qualitative determination

Test the samples and standard solution according to the above conditions. lf the retention time of the target peak was within ±2.5% deviation of standards, and the deviation of ion abundance ratio between the sample and standard is no more than the ranges shown in Table 2, the semicarbazide can be determined exist, in the above conditions, the reference retention time of the semicarbazide derivatives is 6.3 min, The MRM mass chromatogram of the standard solution is shown in Appendix B.

7.4.4 Quantitative determination

Test the same volume of standard and sample solution, according to the above conditions. With the concentration of semicarbazide in the standard solution as the abscissa, and the ratio of the quantitative ion peak area to the IS-ion as the ordinate, calculate the concentration of semicarbazide in the sample solution in the internal standard curve method.

8. Calculation and the result

Calculate the content of semicarbazide in the sample by LC-MS/MS data processor or according to the formula (1).

Where:
X – the concentration of semicarbazide in the test sample, μg /kg;
c – the concentration of semicarbazide in the test sample by Standard curve( ng/mL );
c₀ – the concentration of semicarbazide in the black test by Standard curve( ng/mL );
V – the final volume of sample solution, mL;
m – the corresponding mass of the test sample in the final sample solution, g.
The results are expressed in the arithmetic mean of parallel tests with the three valid digits.

9. Limit of quantitation(L0Q) and recovery

9.1 Limit of quantitation

LOQ of this method: 0.5 μg/kg.

9.2 Recovery and accuracy

The recovery range is between 73%~112%, in adding different concentrations of 0.5 μg/kg ~ 20 μg/kg. RSD <10%. Refer to Appendix C Table C. 1.

Annex A

(Informative Appendix) MS operating conditions

MS operating conditions are as follows:

a) Capillary voltage: 3.5 kV;

b) Source temperature: 150℃;

c) Desolvation gas and temperature: N₂, 350 ℃;

d) Cone gas and flow: N₂, 50 L/h;

e) Desolvent gas and flow: N₂, 600 L/h;

f) Collision gas and pressure: Ar, 3.2 x 10^-4 mbar.

Annex B

(Informative Appendix) Semicarbazide and its iS derivatives MRM chromatograms

Annex C

(Informative Appendix) Recovery and precision of urea in 10 substrates