CN101975837A - Method for determining content and purity of L-carnitine in milk powder - Google Patents
Method for determining content and purity of L-carnitine in milk powder Download PDFInfo
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- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 title claims abstract description 111
- 239000000843 powder Substances 0.000 title claims abstract description 45
- 239000008267 milk Substances 0.000 title claims abstract description 42
- 235000013336 milk Nutrition 0.000 title claims abstract description 39
- 210000004080 milk Anatomy 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title abstract description 12
- 239000000243 solution Substances 0.000 claims abstract description 62
- PHIQHXFUZVPYII-LURJTMIESA-N (S)-carnitine Chemical compound C[N+](C)(C)C[C@@H](O)CC([O-])=O PHIQHXFUZVPYII-LURJTMIESA-N 0.000 claims abstract description 60
- 239000000523 sample Substances 0.000 claims abstract description 44
- 239000012488 sample solution Substances 0.000 claims abstract description 31
- 239000012086 standard solution Substances 0.000 claims abstract description 24
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 16
- 238000006243 chemical reaction Methods 0.000 claims abstract description 7
- 238000001212 derivatisation Methods 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 35
- 239000008367 deionised water Substances 0.000 claims description 29
- 229910021641 deionized water Inorganic materials 0.000 claims description 29
- 230000004044 response Effects 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 12
- 239000007853 buffer solution Substances 0.000 claims description 11
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 238000003556 assay Methods 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- 235000008476 powdered milk Nutrition 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 9
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 claims description 6
- 238000005374 membrane filtration Methods 0.000 claims description 6
- 239000012982 microporous membrane Substances 0.000 claims description 6
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 5
- LRVQJCQTPOUJNW-UHFFFAOYSA-N 2-(9h-fluoren-9-yl)ethyl carbonochloridate Chemical compound C1=CC=C2C(CCOC(=O)Cl)C3=CC=CC=C3C2=C1 LRVQJCQTPOUJNW-UHFFFAOYSA-N 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 235000014121 butter Nutrition 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 2
- 238000009210 therapy by ultrasound Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract 2
- 235000013350 formula milk Nutrition 0.000 description 20
- 229960004203 carnitine Drugs 0.000 description 12
- 239000003153 chemical reaction reagent Substances 0.000 description 11
- 241001411320 Eriogonum inflatum Species 0.000 description 6
- 238000009795 derivation Methods 0.000 description 4
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- 235000013305 food Nutrition 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- -1 (+)-FLEC reagent) Chemical compound 0.000 description 1
- SFRVOKMRHPQYGE-UHFFFAOYSA-N 1-(9h-fluoren-9-yl)ethyl carbonochloridate Chemical compound C1=CC=C2C(C(OC(Cl)=O)C)C3=CC=CC=C3C2=C1 SFRVOKMRHPQYGE-UHFFFAOYSA-N 0.000 description 1
- NSMQYFORTWHWAY-WNQIDUERSA-N C[C@H](N)C(O)=O.C1=CC=CC2=CC(N)=CC=C21 Chemical compound C[C@H](N)C(O)=O.C1=CC=CC2=CC(N)=CC=C21 NSMQYFORTWHWAY-WNQIDUERSA-N 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 240000006413 Prunus persica var. persica Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
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- 238000005515 capillary zone electrophoresis Methods 0.000 description 1
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- 238000001556 precipitation Methods 0.000 description 1
- KRIOVPPHQSLHCZ-UHFFFAOYSA-N propiophenone Chemical class CCC(=O)C1=CC=CC=C1 KRIOVPPHQSLHCZ-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a method for determining content and purity of L-carnitine in milk powder, comprising the following steps in sequence: 1) taking the milk powder as a sample for sample treatment; 2) preparing standard solutions respectively by taking an L-carnitine standard substance and a D-carnitine standard substance; 3) correspondingly preparing an L-carnitine standard solution and a D-carnitine standard solution obtained the step 2) into an L-carnitine derivative solution containing gradient concentration and a D-carnitine solution containing gradient concentration; 4) carrying out derivatization reaction on the sample solutions; 5) carrying out high performance liquid chromatography on the products obtained in the step 3) and step 4); 6) acquiring the concentration cL of the L-carnitine and the concentration cD of the D-carnitine; and 7) acquiring the content and the purity of the L-carnitine in the milk powder sample. By adopting the method for determining the content and the purity of L-carnitine in the milk powder of the invention, the detection sensitivity can be greatly improved.
Description
Technical field
The present invention relates to the assay method of L-carnitine content and purity in a kind of milk powder; Be particularly related to the method for L-carnitine content and purity in a kind of column front derivation high effective liquid chromatography for measuring milk powder.
Background technology
The synthesis capability of L-carnitine in infants a little less than, must external source picked-up.Therefore existing in the world a plurality of countries and regions add the L-carnitine in baby's milk powder, domestic also have the baby's milk powder that adds the L-carnitine to go on the market.And carnitine has two kinds of existence forms (L type and D type) at nature, and there is negative interaction in the dextrorotation carnitine to health, and the l-cn of chemosynthesis generally is left-handed and dextrorotation carnitine potpourri, separates then to remove that the dextrorotation carnitine produces.
The detection of l-cn is general in the domestic health products adopts direct high performance liquid chromatography, and directly high performance liquid chromatography can not be distinguished left-handed and the dextrorotation carnitine.Cold peach blossoms etc. " D-and L-carnitine Enantiomers by Capillary Electrophoresis Separation Research " propose to separate the carnitine enantiomorph with capillary zone electrophoresis, and detect with UV-detector, and the Capillary Electrophoresis operation is had relatively high expectations, and are unfavorable for that the laboratory is universal; Zhu Weixia etc. " rp-hplc determination of l-cn in the health products " are middle to be derivative reagent with L-alanine-beta-naphthylamine, and the HPLC UV-detector detects the content of l-cn in the health products; Xu Juanjuan etc. " chiral hplc is measured the content of optical isomer in the l-cn " are middle to be derivative reagent with alpha-brominated Propiophenone, and the HPLC UV-detector detects the content of l-cn.
The context of detection of L-carnitine in milk powder, the analytical approach that the patent No. 02100582.6 " a kind of method of utilizing L-carnitine content in enzyme-colorimetric method for determining milk powder " proposes can not be distinguished the carnitine enantiomorph.
Method with L-carnitine in the column front derivation high effective liquid chromatography for measuring baby milk powder still is not reported.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method of measuring L-carnitine content and purity in the milk powder.
In order to solve the problems of the technologies described above, the invention provides the assay method of L-carnitine content and purity in a kind of milk powder, may further comprise the steps successively:
1), sample preparation:
Earlier milk powder 10～20g is settled to 100ml after with deionized water dissolving, removes the processing (promptly respectively with hydrochloric acid solution and sodium hydroxide solution precipitation butter oil and lactoprotein) of butter oil and lactoprotein more respectively, filter, collected filtrate is sample solution;
2), preparation standard solution:
Take by weighing 50mg L-carnitine standard items,, and be settled to 10ml with deionized water dissolving; Gained solution obtains L-carnitine standard solution through 0.45 μ m membrane filtration;
Take by weighing 50mg D-carnitine standard items,, and be settled to 10ml with deionized water dissolving; Gained solution obtains D-carnitine standard solution through 0.45 μ m membrane filtration;
3) with step 2) the L-carnitine standard solution and the D-carnitine standard solution of gained carry out following operation respectively:
The L-carnitine standard solution of getting 30 μ l is put in the volumetric flask, adds the 0.05M carbonic acid buffer of 80 μ l and the 1-of 80 μ l (9-fluorenyl)-ethyl chloroformate (i.e. (+)-FLEC reagent), the bottle stopper of volumetric flask beyond the Great Wall, and slowly rotated 3～5 seconds; Volumetric flask is put into 45 ℃ of water-baths place 1h, after taking out volumetric flask then and naturally cooling to room temperature, in volumetric flask, add deionized water and be settled to 10ml; Solution behind the constant volume is diluted with deionized water, obtain the series derivatives solution that L-carnitine concentration is respectively the L-carnitine of 0.5,1.0,1.5,2.0,2.5 μ g/ml;
In like manner, the D-carnitine standard solution of getting 30 μ l is put in the volumetric flask, adds the 0.05M carbonic acid buffer of 80 μ l and (+)-FLEC reagent of 80 μ l, the bottle stopper of volumetric flask beyond the Great Wall, and slowly rotated 3～5 seconds; Volumetric flask is put into 45 ℃ of water-baths place 1h, after taking out volumetric flask then and naturally cooling to room temperature, in volumetric flask, add deionized water and be settled to 10ml; Solution behind the constant volume is diluted with deionized water, obtain the series derivatives solution that D-carnitine concentration is respectively the D-carnitine of 0.5,1.0,1.5,2.0,2.5 μ g/ml;
The series derivatives solution of the L-carnitine of gained and the series derivatives solution of D-carnitine are used 0.45 μ m filtering with microporous membrane respectively; Get the L-carnitine derivative solution of gradient concentration and the D-carnitine derivative solution (being used for detecting of following step) of gradient concentration respectively with high performance liquid chromatography;
4), sample solution derivative reaction:
The sample solution of getting 60 μ l step 1) gained adds the 0.05M carbonic acid buffer of 30 μ l and (+)-FLEC reagent of 30 μ l again in volumetric flask, the bottle stopper of volumetric flask beyond the Great Wall, and slowly rotated 3～5 seconds; Volumetric flask is put into 45 ℃ of water-baths place 1h, after the taking-up volumetric flask naturally cools to room temperature from water-bath then, in volumetric flask, add deionized water and be settled to 10ml; Solution behind the constant volume with 0.45 μ m filtering with microporous membrane, is got sample introduction liquid;
5), high performance liquid chromatography detects:
The sample introduction liquid of the D-carnitine derivative solution of the L-carnitine derivative solution of the gradient concentration of step 3) gained and gradient concentration and step 4) gained is carried out high performance liquid chromatography respectively to be detected; Testing conditions is as follows:
Select the C18 chromatographic column for use, chromatographic condition is: moving phase is: the triethylamine phosphate buffer: the volume ratio of acetonitrile=73: 27; Flow velocity: 1ml/min; Detect wavelength: emission wavelength 269nm, detect wavelength 310nm; Sample size: 10 μ l;
Respectively the response peak area, the response peak area of sample introduction liquid of D-carnitine derivative solution of response peak area, gradient concentration of L-carnitine derivative solution of gradient concentration;
6), obtain the concentration c L of L-carnitine and the concentration c D of D-carnitine respectively:
Response peak area with the L-carnitine derivative solution of the gradient concentration of step 5) gained is mapped to concentration, the typical curve of L-carnitine;
Response peak area with the D-carnitine derivative solution of the gradient concentration of step 5) gained is mapped to concentration, the typical curve of D-carnitine;
With the response peak area of the sample introduction liquid of step 5) gained respectively with the typical curve contrast of the typical curve and the D-carnitine of above-mentioned L-carnitine, the result of gained multiply by the extension rate 10*1000/60 of sample solution in the derivatization reaction of step 4), thereby gets the concentration c L of L-carnitine in the sample solution and the concentration c D of D-carnitine respectively;
7), obtain L-carnitine content and purity in the powdered milk sample:
In the formula: cL---the concentration of L-carnitine in the sample solution, μ g/ml;
The quality of m---milk powder, g.
In the formula: cL---the concentration of L-carnitine in the sample solution, μ g/ml;
CD---the concentration of D-carnitine in the sample solution, μ g/ml.
Improvement as the assay method of L-carnitine content and purity in the milk powder of the present invention:
0.05M the preparation method of carbonic acid buffer is as follows: 338mg natrium carbonicum calcinatum and 152mg sodium bicarbonate are dissolved in 100ml water;
The preparation method of triethylamine phosphate buffer solution is as follows: the triethylamine of 6.8ml is separated with 950ml is water-soluble, is 84% phosphate aqueous solution adjusting pH to 2.5 then with volume fraction, and last water is settled to 1000ml.
Further improvement as the assay method of L-carnitine content and purity in the milk powder of the present invention: step 1) is:
Earlier with milk powder with deionized water dissolving after in 50 ℃ of water-bath 15min, ultrasonic Treatment 10min then is settled to 100ml with deionized water again;
The hydrochloric acid solution that adds 0.1mol/L is then regulated pH to 1.8～2.2, uses the nature filter paper filtering after leaving standstill 10min; The NaOH solution that adds 1mol/L in the filtrate of gained is regulated pH to 5.8～6.2, carries out filtration second time with nature filter paper after leaving standstill 10min, and the filtrate that filter gained the second time is sample solution.
The present invention is with 1-(9-fluorenyl)-ethyl chloroformate, i.e. (+)-FLEC is a derivative reagent, connects a fluorophor on the carnitine enantiomorph, thereby separates on chromatographic column, and analyze with fluorescence detector, has improved detection sensitivity greatly.
In the present invention, agents useful for same is commercial product.For example, L-carnitine standard items, D-carnitine standard items, (+)-FLEC reagent can be available from Sigma company.
Method of the present invention has at first been got rid of the interference of fat and albumen in the milk powder, left-handed and the dextrorotation carnitine enantiomter of chiral resolution then, and detect with high-efficient liquid phase chromatogram technique analysis, provide a kind of column front derivation high performance liquid chromatography to detect the method for L-carnitine content and purity in the milk powder.
The present invention utilizes the chirality derivatization method, the carnitine enantiomter is split, and carry out high performance liquid chromatography and detect, to measure the content and the purity of l-cn in the dispensed food for baby, significant to the quality control and the safety detection of dispensed food for baby and milk powder.
Adopt method of the present invention to measure the content and the purity of L-carnitine in the milk powder, have following advantage:
1, the present invention compares with the traditional analysis method, can chiral resolution L type and D type carnitine, reach the purpose of separating and detecting, and high performance liquid chromatograph uses in the laboratory more general;
2, the high performance liquid chromatography fluorometry accuracy of the present invention's employing is good, and separating power is good, and is highly sensitive, and detecting lower limit approximately is 1/10～1/100 of UV-detector, and instrumentation is simple;
3, can get rid of the interference of fat and albumen in the milk powder, effectively detect the content and the purity of L-carnitine.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is a L-carnitine typical curve;
Fig. 2 is a D-carnitine typical curve
Fig. 3 is the powdered milk sample chromatogram of embodiment 1.
Embodiment
Example below in conjunction with L-carnitine content and purity in the column front derivation high effective liquid chromatography for measuring milk powder of the present invention illustrates the specific embodiment of the present invention.
The assay method of L-carnitine content and purity in embodiment 1, a kind of milk powder is characterized in that may further comprise the steps successively:
1), sample preparation:
Accurately take by weighing commercially available ordinary powdered milk sample 16.65g, be dissolved in the 30ml deionized water, 50 ℃ of water-bath 15min, ultrasound wave (frequency 30kHz) 10min fully dissolves milk powder, adds deionized water then and is settled to 100ml.
Regulate pH to 2 with the hydrochloric acid solution of 0.1mol/L then, use the nature filter paper filtering after leaving standstill 10min.Regulate pH to 6 the filtrate of gained with the NaOH solution of 1mol/L, carry out filtration second time with nature filter paper after leaving standstill 10min, the filtrate that filter gained the second time is sample solution.
2), preparation standard solution:
Take by weighing 50mg L-carnitine standard items,, and be settled to 10ml with deionized water dissolving; Gained solution obtains L-carnitine standard solution through 0.45 μ m membrane filtration;
Take by weighing 50mg D-carnitine standard items,, and be settled to 10ml with deionized water dissolving; Gained solution obtains D-carnitine standard solution through 0.45 μ m membrane filtration;
3), with step 2) the L-carnitine standard solution and the D-carnitine standard solution of gained carry out following operation respectively:
The L-carnitine standard solution of getting 30 μ l is put in the volumetric flask, adds the 0.05M carbonic acid buffer of 80 μ l and (+)-FLEC reagent of 80 μ l, the bottle stopper of volumetric flask beyond the Great Wall, and slowly rotated 3～5 seconds; Volumetric flask is put into 45 ℃ of water-baths place 1h, after the taking-up volumetric flask naturally cools to room temperature from water-bath then, in volumetric flask, add deionized water and be settled to 10ml; Solution behind the constant volume is diluted with deionized water, obtain the series derivatives solution that L-carnitine concentration is respectively the L-carnitine of 0.5,1.0,1.5,2.0,2.5 μ g/ml;
In like manner, the D-carnitine standard solution of getting 30 μ l is put in the volumetric flask, adds the 0.05M carbonic acid buffer of 80 μ l and (+)-FLEC reagent of 80 μ l, the bottle stopper of volumetric flask beyond the Great Wall, and slowly rotated 3～5 seconds; Volumetric flask is put into 45 ℃ of water-baths place 1h, after the taking-up volumetric flask naturally cools to room temperature from water-bath then, in volumetric flask, add deionized water and be settled to 10ml; Solution behind the constant volume is diluted with deionized water, obtain the series derivatives solution that D-carnitine concentration is respectively the D-carnitine of 0.5,1.0,1.5,2.0,2.5 μ g/ml;
The L-carnitine series derivatives solution and the D-carnitine series derivatives solution of gained are used 0.45 μ m filtering with microporous membrane (can suitably pressurize) respectively; Get the L-carnitine derivative solution (totally 5 kinds) of gradient concentration and the D-carnitine derivative solution (totally 5 kinds) of gradient concentration respectively;
4), sample solution derivative reaction
Measure the preparation of reagent earlier:
The triethylamine of triethylamine phosphate buffer solution: 6.8ml is separated with 950ml is water-soluble, is that 84% phosphate aqueous solution is regulated its pH to 2.5 with volume fraction, and last dilute with water is settled to 1000ml.
0.05M carbonic acid buffer: 338mg natrium carbonicum calcinatum and 152mg sodium bicarbonate are dissolved in 100ml water.
The sample solution of getting 60 μ l step 1) gained adds the 0.05M carbonic acid buffer of 30 μ l and (+)-FLEC reagent of 30 μ l again in volumetric flask, the bottle stopper of volumetric flask beyond the Great Wall, and slowly rotated 3～5 seconds; Volumetric flask is put into 45 ℃ of water-baths place 1h, after the taking-up volumetric flask naturally cools to room temperature from water-bath then, in volumetric flask, add deionized water and be settled to 10ml; Solution behind the constant volume with 0.45 μ m filtering with microporous membrane (can suitably pressurize), is got sample introduction liquid.
5), high performance liquid chromatography detects
The sample introduction liquid of the D-carnitine derivative solution of the L-carnitine derivative solution of the gradient concentration of step 3) gained and gradient concentration and step 4) gained is carried out high performance liquid chromatography respectively to be detected; Testing conditions is as follows:
Select the C18 chromatographic column for use.Chromatographic condition is: moving phase is the triethylamine phosphate buffer: acetonitrile (73: 27, volume ratio); Flow velocity: 1ml/min; Detect wavelength: emission wavelength 269nm, detect wavelength 310nm; Sample size: 10 μ l.
Respectively the response peak area, the response peak area of sample introduction liquid of D-carnitine derivative solution of response peak area, gradient concentration of L-carnitine derivative solution of gradient concentration.
6), obtain L-carnitine content and purity:
Response peak area with the L-carnitine derivative solution of the gradient concentration of step 5) gained is mapped to concentration, the typical curve of L-carnitine; As shown in Figure 1: L-carnitine, c (μ g/ml)=2*10
-7A+0.0978; A is a peak area value in the formula;
Response peak area with the D-carnitine derivative solution of the gradient concentration of step 5) gained is mapped to concentration, the typical curve of D-carnitine; As shown in Figure 2: D-carnitine, c (μ g/ml)=2*10
-7A+0.1185; A is a peak area value in the formula.
With the response peak area of the sample introduction liquid of step 5) gained respectively with the typical curve contrast of the typical curve and the D-carnitine of above-mentioned L-carnitine, the result of gained multiply by the extension rate 10*1000/60 of sample solution in the derivatization reaction of step 4), thereby gets the concentration c L of L-carnitine in the sample solution and the concentration c D of D-carnitine respectively; Be specially:
The testing result figure of sample introduction liquid sees Fig. 3, and L-carnitine derivative peak area value is 158390, and D-carnitine derivative peak area is 0.Thereby L-carnitine concentration c L=(158390*2*10 in the sample solution
-7+ 0.0978) * 10*1000/60=21.58 μ g/ml; CD=0.
7), obtain L-carnitine content and purity in the powdered milk sample:
(annotate: in the formula: cL---the concentration of L-carnitine in the sample solution, μ g/ml;
The quality of m---milk powder, g.)
(annotate: in the formula: cL---the concentration of L-carnitine in the sample solution, μ g/ml;
CD---the concentration of D-carnitine in the sample solution, μ g/ml.)
Result of calculation is summarized as follows: the content of L-carnitine is 12.95mg/100g in the powdered milk sample, and the D-carnitine does not detect.
The assay method of L-carnitine content and purity in embodiment 2, a kind of milk powder is characterized in that may further comprise the steps successively:
1), sample preparation:
Accurately take by weighing commercially available baby formula milk powder I sample 14.95g, be dissolved in the 30m deionized water, 50 ℃ of water-bath 15min, ultrasound wave (frequency 30kHz) 10min, milk powder fully dissolves, and adds deionized water and is settled to 100ml.
Regulate pH to 1.9 with the hydrochloric acid solution of 0.1mol/L then, use the nature filter paper filtering after leaving standstill 10min.The filtrate of gained is regulated pH to 6.1 with the NaOH solution of 1mol/L, carry out the second time with nature filter paper and filter, the filtrate of filtering gained for the second time is sample solution.
Step 2)～step 6) all is equal to embodiment 1.
Thereby get the concentration c L=(812098*2*10 of L-carnitine in the sample solution respectively
-7+ 0.0978) * 10*1000/60=43.37 μ g/ml, the concentration c D=0 μ g/ml of D-carnitine;
7), obtain L-carnitine content and purity in the sample:
Computing formula is with embodiment 1.
Result of calculation is as follows: the content of L-carnitine is 29.01mg/100g in the baby formula milk powder I sample, and the D-carnitine does not detect.
Step 1)～step 6) all is equal to embodiment 2.
Thereby get the concentration c L=(848302*2*10 of L-carnitine in the sample solution respectively
-7+ 0.0978) * 10*1000/60=44.57 μ g/ml, the concentration c D=0 μ g/ml of D-carnitine;
7), obtain L-carnitine content and purity in the sample:
Computing formula is with embodiment 1.
The result is as follows: the content of L-carnitine is 28.52mg/100g in the baby formula milk powder II sample, and the D-carnitine does not detect.
In order to prove the correctness of conclusion of the present invention, utilize existing high performance liquid chromatography uv analysis method to detecting with the ordinary powdered milk sample among embodiment 1～embodiment 3, baby formula milk powder I sample, baby formula milk powder II sample fully: the result is as follows:
In the ordinary powdered milk sample: the content of L-carnitine is 13mg/100g; In the baby formula milk powder I sample: the content of L-carnitine is 29.1mg/100g; In the baby formula milk powder II sample: the content of L carnitine is 28.6mg/100g.Testing result is consistent with embodiment 1～embodiment 3 results.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (4)
1. the assay method of L-carnitine content and purity in the milk powder is characterized in that may further comprise the steps successively:
1), sample preparation:
Earlier powdered milk sample 10～20g is settled to 100ml after with deionized water dissolving, removes the processing of butter oil and lactoprotein more respectively, filter, collected filtrate is sample solution;
2), preparation standard solution:
Take by weighing 50mg L-carnitine standard items,, and be settled to 10ml with deionized water dissolving; Gained solution obtains L-carnitine standard solution through 0.45 μ m membrane filtration;
Take by weighing 50mg D-carnitine standard items,, and be settled to 10ml with deionized water dissolving; Gained solution obtains D-carnitine standard solution through 0.45 μ m membrane filtration;
3), with step 2) the L-carnitine standard solution and the D-carnitine standard solution of gained carry out following operation respectively:
L-carnitine standard solution/D-carnitine standard solution of getting 30 μ l is put in the volumetric flask, adds the 0.05M carbonic acid buffer of 80 μ l and the 1-of 80 μ l (9-fluorenyl)-ethyl chloroformate; Volumetric flask is put into 45 ℃ of water-baths place 1h, after taking out volumetric flask then and naturally cooling to room temperature, in volumetric flask, add deionized water and be settled to 10ml; Solution behind the constant volume is diluted with deionized water, obtain L-carnitine series derivatives solution/D-carnitine series derivatives solution that L-carnitine/D-carnitine concentration is respectively 0.5,1.0,1.5,2.0,2.5 μ g/ml;
The L-carnitine series derivatives solution and the D-carnitine series derivatives solution of gained are used 0.45 μ m filtering with microporous membrane respectively; Get the L-carnitine derivative solution of gradient concentration and the D-carnitine derivative solution of gradient concentration respectively;
4), sample solution derivative reaction:
The sample solution of getting 60 μ l step 1) gained adds the 0.05M carbonic acid buffer of 30 μ l and the 1-of 30 μ l (9-fluorenyl)-ethyl chloroformate again in volumetric flask; Volumetric flask is put into 45 ℃ of water-baths place 1h, after the taking-up volumetric flask naturally cools to room temperature from water-bath then, in volumetric flask, add deionized water and be settled to 10ml; Solution behind the constant volume with 0.45 μ m filtering with microporous membrane, is got sample introduction liquid;
5), high performance liquid chromatography detects:
The sample introduction liquid of the D-carnitine derivative solution of the L-carnitine derivative solution of the gradient concentration of step 3) gained and gradient concentration and step 4) gained is carried out high performance liquid chromatography respectively to be detected; Testing conditions is as follows:
Select the C18 chromatographic column for use, chromatographic condition is: moving phase is: the triethylamine phosphate buffer: the volume ratio of acetonitrile=73: 27; Flow velocity: lml/min; Detect wavelength: emission wavelength 269nm, detect wavelength 310nm; Sample size: 10 μ l;
Respectively the response peak area, the response peak area of sample introduction liquid of D-carnitine derivative solution of response peak area, gradient concentration of L-carnitine derivative solution of gradient concentration;
6), obtain the concentration c L of L-carnitine and the concentration c D of D-carnitine:
Response peak area with the L-carnitine derivative solution of the gradient concentration of step 5) gained is mapped to concentration, the typical curve of L-carnitine;
Response peak area with the D-carnitine derivative solution of the gradient concentration of step 5) gained is mapped to concentration, the typical curve of D-carnitine;
With the response peak area of the sample introduction liquid of step 5) gained respectively with the typical curve contrast of the typical curve and the D-carnitine of above-mentioned L-carnitine, the result of gained multiply by the extension rate 10*1000/60 of sample solution in the derivatization reaction of step 4), thereby gets the concentration c L of L-carnitine in the sample solution and the concentration c D of D-carnitine respectively;
7), obtain L-carnitine content and purity in the powdered milk sample:
In the formula: cL---the concentration of L-carnitine in the sample solution, μ g/ml;
The quality of m---milk powder, g.
In the formula: cL---the concentration of L-carnitine in the sample solution, μ g/ml;
CD---the concentration of D-carnitine in the sample solution, μ g/ml.
2. the assay method of L-carnitine content and purity in the milk powder according to claim 1 is characterized in that:
0.05M the preparation method of carbonic acid buffer is as follows: 338mg natrium carbonicum calcinatum and 152mg sodium bicarbonate are dissolved in 100ml water;
The preparation method of triethylamine phosphate buffer solution is as follows: the triethylamine of 6.8ml is separated with 950ml is water-soluble, is 84% phosphate aqueous solution adjusting pH to 2.5 then with volume fraction, and last water is settled to 1000ml.
3. the assay method of L-carnitine content and purity in the milk powder according to claim 2, it is characterized in that: described step 1) is:
Earlier with milk powder with deionized water dissolving after in 50 ℃ of water-bath 15min, ultrasonic Treatment 10min then is settled to 100ml with deionized water again;
The hydrochloric acid solution that adds 0.1mol/L is then regulated pH to 1.8～2.2, uses filter paper filtering after leaving standstill 10min; The NaOH solution that adds 1mol/L in the filtrate of gained is regulated pH to 5.8～6.2, leaves standstill to carry out second time with filter paper behind the 10min and filter, and the filtrate that filter gained the second time is sample solution.
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| CN112379016A (en) * | 2020-11-02 | 2021-02-19 | 江苏宝众宝达药业有限公司 | Method for measuring content of L-carnitine in 3-O-lauroyl-L-carnitine by high performance liquid chromatography |
| CN113219097A (en) * | 2021-01-15 | 2021-08-06 | 杭州海关技术中心 | Method for splitting and determining carnitine enantiomer in infant formula milk powder based on ultra-high performance synthetic phase chromatography |
| CN113433257A (en) * | 2021-01-15 | 2021-09-24 | 杭州海关技术中心 | Method for separating and determining carnitine enantiomer in health food based on ultra-high performance synthetic phase chromatography technology |
| CN113219097B (en) * | 2021-01-15 | 2022-06-24 | 杭州海关技术中心 | Method for splitting and measuring carnitine enantiomer in infant formula milk powder |
| CN113804790A (en) * | 2021-09-18 | 2021-12-17 | 四川新希望畜牧科技有限公司 | Method for simultaneously detecting additive, L-carnitine and D-carnitine |
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