1. Scope
This standard specifies the determination of L-carnitine in the foods for export by HPLC and HPLC-MS/MS.
This standard applies to the L-carnitine detection in milk powder, beverages, and other foods.
Note: lf the D-carnitine is present in the sample, the result of HPLC-MS/MS method is the sum of L-carnitine and D-carnitine.
2. Normative Reference
For the application of this standard, othe following references are of the essence. As for the quoted documents noted date, only the dated edition fits this standard, and the latest edition of the citated document without a date (including all the revisions) fits this standard.
GB/T 6682 Water for analytical laboratory use-Specification and test methods (GB/T 6682, neq ISO 3696:1987)
Method I – HPLC
3. Method Principle
L-carnitine in the sample was extracted by hydrochloric acid, after saponification, the bound L-carnitine is released. And purified by cation exchange SPE(solid-phase extraction), and then with L-propionamide-β-naphthylamine occurs substituted reaction under the chloroformate and triethylamine catalyzed, resulting in strong UV absorption of the compound, for equipped with a DAD (diode array detector) or UV detector high-performance liquid chromatography by external standard method for quantitative analysis.
4. Chemicals Reagents and Materials
All reagents are analytically pure, except as noted. All references to water in this standard refer to water Grade1, as defined in GB/T 6682
4.1 Acetronitrile, chromatographic grade.
4.2 Methanol, chromatographic grade.
4.3 Ammonia, analytical grade.
4.4 Hydrochloric acid, excellent grade.
4.5 Potassium hydroxide, analytical grade.
4.6 Trichloromethane, analytical grade.
4.7 Butyl Chloroformate, analytical grade.
4.8 Triethylamine, analytical grade.
4.9 L-propionamide-β-naphthylamine, analytical grade.
4.10 Sodium bicarbonate, excellent grade.
4.11 Potassium dihydrogen phosphate: excellent grade.
4.12 Phosphoric acid: excellent grade.
4.13 1 mol/L hydrochloric acid solution: take 90 ml hydrochloric acid (4.4), and add water to volume to 1000 mL.
4.14 0.1 mol/L hydrochloric acid solution: take 9 mL hydrochloric acid (4.4), and add water to volume to 1000 mL.
4.15 10 mmol/L hydrochloric acid solution: take 10 mL 0.1mol/L hydrochloric acid solution (4.14), and add water to volume to 100 mL.
4.16 1 mol/L Potassium hydroxide solution: take 56 g Potassium hydroxide (4.5), add water to volume to 1 000 mL.
4.17 4% ammonia solution of methanol: take 4 mL of ammonia (4.3) with methanol (4.2) constant volume to 100 mL.
4.18 Mixed-mode cation ion exchange SPE, MCX1-3 mL, 60 mg or equivalent.
Activate the SPE column with 3 mL methanol (4.2), 3 mL water, and 3 mL 10mmol/L of hydrochloric acid (4.13)successively.
4.19 Derived Reagent: Weigh 0.45 g of L-propionamide-β-naphthylamine, and add acetonitrile to a volume of 100 mL.
4.20 Catalyst l: Weigh 0.50 g of triethylamine and add trichloromethane (4.5) to mix to 100 mL.
4.21 Catalyst ll: Weigh 0.50 g of butyl chloroformate, and add trichloromethane (4.5) to mix to 100 mL.
4.22 50 mmol/L sodium bicarbonate solution: Weigh 0.42 g sodium bicarbonate (4.9), and add water to the dissolved set volume to 100 mL.
4.23 50mmol/L potassium dihydrogen phosphate buffer solution (pH 2.8): Weigh 6.80g potassium dihydrogen phosphate, dissolved in 1 000 mL water, adjusted to pH 2.8 with phosphoric acid.
4.24 Acetonitrile solution (90+ 10) (v/v): take 90 ml of acetonitrile and 10 mL of water, and mix well.
4.25 Standard: L-carnitine, CAS number: 541-15-1, purity greater than 98%
4.26 L-carnitine standard stock solution: weigh the right amount of standard material (4.25) accurately, with acetonitrile solution (4.24) configured to a concentration of 1 mg/mL standard stock solution, The solution is kept away from light and below 4 ℃.
4.27 L-carnitine standard working fluid, remove L-carnitine standard stock solution (4.26) 1.0 ml in 100 mL volumetric flask accurately, diluted with acetonitrile to the mark. Preparation of standard working solution at a concentration of 10.0 ug/mL. The solution is kept away from light and below
4.28 0.22 μm water microporous membrane,
5. Instruments and Apparatus
5.1 HPLC with a UV detector or DAD detector.
5.2 Analytical balance, capable of accurately weighing 0.01 g and 0.1 mg
5.3 Vortex mixer.
5.4 High-speed centrifuge, not less than 15,000 r/min,
5.5 Centrifuge, more than 4,000 r/min.
5.6 Solid phase extraction device.
5.7 Nitrogen blowing concentrator.
5.8 Ultrasonic cleaner.
5.9 Thermostat water bath.
5.10 pH meter, 0.01pH.
6. Sample Preparation and Preservation
6.1 Sample Preparation
Take a representative sample of about 500 g, mix uniformity, put it in a clean and closed vessel, and make labeling.
6.2 Sample Preservation
Store the sample at 4 ℃
NOTE: During this process, it should be protected from contamination and loss of the target analyte.
7. Sample Preparation
7.1 Extraction
7.1.1 Milk powder, liquid beverages (L-carnitine content less than or equal to 1 000 mg/kg sample): Weigh 5.00g sample in a 50 mL volumetric flask, adding 20 mL 0.1 mol/L hydrochloric acid solution (4.14) to it, after vortexing for 1 min and ultrasonic 5 minutes to dissolve the sample, by adding 1 mol/L Potassium hydroxide solution (4.16) for vortex mixing saponified for 30 min in water bath of 60 ℃ and cool to room temperature, adding 5 mL 1 mol/L hydrochloric acid solution (4.13).vortex mixing 1min, using 0.1 mol/L hydrochloric acid solution (4.14) to 50 mL. Take 1 mL of the extract in a 15 mL test tube, after adding 9 mL of acetonitrile, vortex mixing 1min, taking some of it to a5 mL centrifuge tube, centrifuging for 5 mins at 16,000 rpm, the supernatant wait to be purified.
7.1.2 Liquid beverages (L-carnitine content greater than 1000 mg/kg sample). First, dilution of L-carnitine, content to 1 000 mg/kg or less than 1 000 mg/kg, and then extract it according to the method in 7.1.1
7.1.3 Solid beverage category, Weigh the appropriate amount of the sample, dissolve and dilute it with water to make the content of L-carnitine less than or equal to 1000 mg/kg, then extract it according to the method in 7.1.1
7.2 Purification
Take 1 mL of the solution in an activated cationic solid phase extraction column (4.18), the rate of 1 drop/sec column, with 3 mL 10 mmol/L aqueous solution of hydrochloric acid (4.15), discard those solutions, vacuum for 1min, place 2 mL 4% ammonium methanol solution into the syringe(4.17), collect the elution into a 15- mL conical tube, Dry with nitrogen evaporator at 40 ℃. The resulting residue is to be derived.
7.3 Derivation
Add 0.5 mL of derivative reagents(4.19) to the residue, place in an ultrasonic bath for 1min to dissolve and mix homogeneously, and then add 0.5 mL of catalyst I (4.20) and 0.5 mL of catalyst ll (4.21) successively to it, vortex stir for 3 min, stand for 30 mins at room temperature, add 1 mL of 50 mmol/laqueous sodium bicarbonate solution (4.22), end the reaction after vortexing swir it for 3 mins. Centrifuge for 5 mins at 4000 rpm, and take the upper layer of water through 0.22um Milliporefilter (4.28) for liquid chromatography measuring, the determination needs to be completed within 12h.
8. Analysis by HPLC
8.1 HPLC Conditions
a) Column: C18,100 mm x 4.6mm lD, 3.5 μm particle size or equivalent;
b) Mobile Phase: 50 mmol/L potassium dihydrogen phosphate(4.23): acetonitrile =75 : 25;
c) Flow rate: 0.8 mL/min,12 min;
d) Temperature: 35 ℃;
e) Injection Volume:10 μL
f) Absorbance detector: 244 nm
8.2 Analysis
Take 25 μL,50 μL,100 μL,500 μL, and 1000 μL L-carnitine standard working solution (4.27)into centrifuge tubes, respectively, according to the 7.3 derivative method. The concentration of L-carnitine derivatives was 0.25 μg/mL,0.50 μg/mL,1 μg/mL,5 μg/mL, and 10 μg/mL. In the above chromatographic conditions, measuring the standard working solution and sample solution by external standard method for quantitative analysis. The retention time of L-carnitine derivatives was 3.8 min, For the standard chromatogram of L-carnitine Derivatives, refer to fig A.1.in Appendix A.
9. Black experiment
Refer to the above procedure, but without adding the sample.
10. Calculation
10.1 The calculation of the result is carried out by the data processor or according to the formula(1) (The result of the calculation is deducted from the blank value).
where:
X – Concentration of the analytes in the sample, in mg/kg:
c – Final concentration of the analytes in the sample solution, in μg/mL;
n – Dilution factor;
V – The final volume of the sample solution, in mL;
m – Weigh of the sample in the final solution, in g.
10.2 Limit of Detection (LOD) and Limit of Quantitation (LOQ)
The detection limit for this method is 10 mg/kg and the limit of quantitation is 25 mg/kg.
11. Recover
Liquid chromatography was carried out in an indoor recovery experiment with different samples as blank matrices for three concentration levels of additive recovery experiments, The three concentrations included a quantitative low limit, the minimum allowable additions, and the maximum allowable additions, and each concentration level conducted 10 times repeated experiments, the measured range of L-carnitine recovery are shown in Appendix B in Table B.1
Method II HPLC-MS/MS
12. Method Principle
L-carnitine is extracted by dilute hydrochloric acid from samples, after saponification, the bound L-carnitine is released. And purified by cation exchange SPE, the extract is subjected to HPLC-MS/MS.
13. Reagent and materials
All reagents are analytically pure, except as noted. All references to water in this standard refer to water Grade 1, as defined in GB/T 6682.
13.1 Acetonitrile (same as 4.1)
13.2 Methanol (same as 4.2)
13.3 Ammonia (same as 4.3)
13.4 Hydrochloric acid (same 4.4)
13.5 Formic acid, Chromatographic grade
13.6 0.1 mol/L hydrochloric acid solution (same as 4.14)
13.7 10 mmol/L hydrochloric acid solution (same as 4.15)
13.8 4% ammonia solution of methanol (same as 4.17)
13.9 Mixed-mode cation ion exchange SPE (same as 4.18)
13.10 Acetonitrile solution (90+10) (v/v) (same as 4.24)
13.11 0.1% formic acid solution: slowly add 1 mL of formic acid (13.5) to 1000 mL of water
13.12 Standard (same as 4.25)
13.13 Isotope internal standard substance: L-carnitine d3, the purity of it is greater than 98%
13.14 L-carnitine standard stock solution(same as 4.26)
13.15L-carnitine d3 standard working solution: accurately weigh the appropriate amount of L-carnitine deuterium isotope internal standard substance (13.13), with acetonitrile-water solution (90 + 10) (13.10) configured into a concentration of 1mg/mL internal standard working solution, The solutions kept away from light and below 4℃
13.16 0.22 μm organic microporous membrane.
14. Instrument and Apparatus
14.1 HPLC-MS/MS: ESI
14.2 Other instruments and apparatus: Same as 5
15. Sample Preparation and Preservation
The Preparation and Preservation of the sample is the same as 6
16. Sample Preparation
16.1 Extraction
Weigh 1.00g in a 50 mL volumetric flask, and add 50 μl of internal standard solution (13.15), the rest of the steps is the same as 7.1
16.2 Purification
The purification is the same as 7.2. Add 1.0 mL 0.1% aqueous solution of formic acid (13.11) to the obtained residue accurately, disperse the residue in the ultrasonic bath for 1min for dissolution, and filter it through 0.22 um millipore filter(13.16) before HPLC-MS/MS analysis.
17. Analysis by HPLC-MS/MS
17.1 Chromatographic Conditions
Chromatographic Conditions:
a) Column: HlLlC column, 100 mm x 2.1 mm (ID), 3.5 μm particle size or equivalent;
b) Mobile Phase: A.0.1% formic acid water, B: acetonitrile, gradient elution conditions are shown in Table 1;
c) Temperature: 35℃;
d) Injection Volume:2 μL;
e) Flow Rate: 0.30 mL/min.
| time/min | A% | B% |
|---|---|---|
| 0.00 | 10 | 90 |
| 2.00 | 10 | 90 |
| 6.00 | 55 | 45 |
| 6.10 | 10 | 90 |
| 10.00 | 10 | 90 |
17.2 MS/MS conditions
MS/MS conditions:
a) ESl mode: ESl+
b) Scan mode: MRM;
c) Dry gas temperature and flow rate: 300 ℃,10 L/min;
d) Sheath gas temperature and flow rate: 325 ℃,8 L/min;
e) Capillary voltage: 3500V;
f) L-carnitine and its internal standard of quantitative and qualitative ions, collision energy, and fragmentation voltage parameters are shown in Table 2.
| Compound | Parent ion (m/z) | Daughter ion (m/z) | Collision energy (V) | Cracking voltage (V) |
|---|---|---|---|---|
| L-carnitine | 162.1 | 60.4 * | 18 | 100 |
| 102.8 | 16 | |||
| L-carnitine d3 | 165.1 | 63.2 * | 17 | 100 |
| 85.1 | 17 |
* ions for quantification
17.3Quantitative analysis
L-carnitine content is less than or equal to 500 mg/kg, The sample and standard working solution are determined according to the above liquid chromatography-mass spectrometry/mass spectrometry conditions. The chromatographic peak area was quantified by the internal standard method (L-carnitine linear concentration range: 0.001 μg/mL to 1,000 μg/mL), and L-carnitine content greater than 500 mg/kg samples, only confirms the analysis. Reference retention time is 3.65 min, standard solution total ion flow chart and multiple reaction monitoring (MRM) chromatograms are shown in Appendix C, Figures C.1, C.2.
17.4 Qualitative analysis
Analyze the samples and working standard solution under the above chromatographic conditions. Under the same experiment condition, the deviation between the retention time of the analytes and standard solution is under ±2.5%; Comparing the deviation of relative abundance of the analytes qualitatives to that of the working standard solution, it is confirmed that the analyte is contained in sample if the standard solution is under the range defined as Table 3.
| Relative ion abundance (%) | >50 | >20 to 50 | >10 to 20 | ≤10 |
| Maximum allowable misalignment (%) | ±20 | ±25 | ±30 | ±50 |
18. Blank experiment
Refer to the above procedure, but without adding samples.
19. Calculation
19.1 The calculation of the result is carried out by the data processor or according to the formula (2) (The result of the calculation is deducted from the blank value).
where:
X – Concentration of the analytes in the sample, in mg/kg:
c – Final concentration of the analytes in the sample solution, in μg/mL;
n – Dilution factor;
V – The final volume of the sample solution, in mL;
m – Weigh of the sample in the final solution, in g.
19.2 Limit of Detection (LOD) and Limit of Quantitation (LOQ)
The detection limit for this method is 0.05 mg/kg and the limit of quantitation is 0.2 mg/kg.
20. The range of recovery
HPLC-MS/MS was carried out in an indoor recovery experiment with different samples as a blank matrix for three concentration levels of additive recovery experiments, The three concentrations included a quantitative low limit, the minimum allowable additions, and the maximum allowable additions, and each concentration level conducting 10 times repeated experiments, the measured range of L.carnitine recovery are shown in Appendix B in Table B.2
Annex A
(informative appendix) Chromatogram of L-carnitine Derivatives
Annex B
(informative appendix) additive recovery
| Sample Name | Additive Concentration had (mg/kg) | Recovery (%) |
|---|---|---|
| Modified Milk Powder | 25 | 82.9 – 90.4 |
| 50 | 81.4 – 86.9 | |
| 400 | 80.8 – 87.8 | |
| Milk Beverage | 25 | 81.7 – 89.3 |
| 600 | 86.3 – 90.6 | |
| 3000 | 83.1 – 89.2 | |
| Flavor Drinks | 25 | 84.8 – 92.0 |
| 600 | 86.6 – 90.5 | |
| 3000 | 80.4 – 86.9 | |
| Fruit-vegetable juice beverage | 25 | 83.2 – 89.2 |
| 600 | 84.8 – 89.8 | |
| 3000 | 80.0 – 86.4 | |
| Special Purpose beverage | 25 | 80.5 – 89.8 |
| 100 | 83.2 – 92.8 | |
| 1000 | 88.9 – 92.5 | |
| Solid drink | 25 | 82.8 – 90.6 |
| 6000 | 85.8 – 90.0 | |
| 30000 | 80.5 – 88.4 |
| Sample Name | Additive Concentration had (mg/kg) | Recovery (%) |
|---|---|---|
| Modified Milk Powder | 0.20 | 80.6 – 91.5 |
| 50 | 82.4 – 90.6 | |
| 400 | 90.9 – 93.6 |
Annex C
(informative appendix) Determination of L-carnitine by LC-MS/MS
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