CN101975837A - Method for determining content and purity of L-carnitine in milk powder - Google Patents

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

Determination method of L-carnitine content and purity in milk powder

technical field

The invention relates to a method for determining the content and purity of L-carnitine in milk powder; in particular, it relates to a method for determining the content and purity of L-carnitine in milk powder by pre-column derivatization high performance liquid chromatography.

Background technique

The synthesis ability of L-carnitine in infants is weak, so it must be ingested from outside sources. Therefore many countries and regions in the world have added L-carnitine in baby milk powder, and domestic also has the baby milk powder that adds L-carnitine to go on the market. And carnitine has two forms of existence (L-type and D-type) in nature, and D-carnitine has negative effects on human health, and chemically synthesized L-carnitine is generally a mixture of L-carnitine and D-carnitine, and then separates and removes D-carnitine. produced by carnitine.

The detection of L-carnitine in domestic health care products generally adopts direct high-performance liquid chromatography, but direct high-performance liquid chromatography cannot distinguish between L-carnitine and D-carnitine. Leng Taohua et al. "Research on the Capillary Electrophoresis Separation of D- and L-Carnitine Enantiomers" proposed to use capillary zone electrophoresis to separate carnitine enantiomers and detect them with an ultraviolet detector. Capillary electrophoresis requires high operation and is not conducive to laboratories. Popularization; Zhu Weixia et al. "Determination of L-Carnitine in Health Products by Reversed-Phase High Performance Liquid Chromatography" uses L-alanine-β-naphthylamine as a derivative reagent, and HPLC ultraviolet detector detects the content of L-carnitine in health products ; Xu Juanjuan et al. "Determination of the Content of Optical Isomers in L-Carnitine by Chiral High Performance Liquid Chromatography" uses α-bromophenyl ethyl ketone as a derivative reagent, and the HPLC ultraviolet detector detects the content of L-carnitine.

In terms of the detection of L-carnitine in milk powder, the analysis method proposed in Patent No. 02100582.6 "A Method for Measuring L-Carnitine Content in Milk Powder Using Enzyme-Colorimetry" cannot distinguish the carnitine enantiomers.

The method for the determination of L-carnitine in infant milk powder by pre-column derivatization high performance liquid chromatography has not been reported yet.

Contents of the invention

The technical problem to be solved by the invention is to provide a method for determining the content and purity of L-carnitine in milk powder.

In order to solve the above-mentioned technical problems, the invention provides a kind of assay method of L-carnitine content and purity in milk powder, comprises the following steps successively:

1), sample processing:

Dissolve 10-20g of milk powder in deionized water first, and then set the volume to 100ml, and then carry out the treatment of removing milk fat and milk protein respectively (that is, use hydrochloric acid solution and sodium hydroxide solution to precipitate milk fat and milk protein respectively), and filter. The collected filtrate is the sample solution;

2), prepare standard solution:

Weigh 50 mg of L-carnitine standard substance, dissolve it in deionized water, and set the volume to 10ml; the resulting solution is filtered through a 0.45 μm filter membrane to obtain L-carnitine standard solution;

Weigh 50 mg of D-carnitine standard substance, dissolve it in deionized water, and set the volume to 10ml; the resulting solution is filtered through a 0.45 μm filter membrane to obtain D-carnitine standard solution;

3) the L-carnitine standard solution and the D-carnitine standard solution of step 2) gained are carried out as follows respectively:

Take 30 μl of L-carnitine standard solution in a volumetric flask, add 80 μl of 0.05M carbonic acid buffer and 80 μl of 1-(9-fluorenyl)-chloroformic acid ethyl ester (i.e. (+)-FLEC reagent), stopper Put the cork on the volumetric flask and rotate it slowly for 3-5 seconds; put the volumetric flask in a water bath at 45°C for 1 hour, then take out the volumetric flask and let it cool down to room temperature naturally, then add deionized water to the volumetric flask to make up to 10ml; The solution after constant volume was diluted with deionized water to obtain a series of derivative solutions of L-carnitine with L-carnitine concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 μg/ml respectively;

Similarly, put 30 μl of D-carnitine standard solution in a volumetric flask, add 80 μl of 0.05M carbonic acid buffer and 80 μl of (+)-FLEC reagent, plug the stopper of the volumetric flask, and rotate slowly for 3 to 5 seconds; put the volumetric flask in a 45°C water bath for 1 hour, then take out the volumetric flask and cool it down to room temperature naturally, then add deionized water to the volumetric flask to make the volume to 10ml; dilute the volumetric solution with deionized water to obtain A series of D-carnitine derivative solutions with D-carnitine concentrations of 0.5, 1.0, 1.5, 2.0, 2.5 μg/ml;

The resulting L-carnitine derivative solutions and D-carnitine derivative solutions were respectively filtered through a 0.45 μm microporous membrane; L-carnitine derivative solutions with gradient concentrations and D-carnitine derivative solutions with gradient concentrations were respectively obtained. Solution (for the detection of the following steps with high performance liquid chromatography);

4), sample solution derivatization reaction:

Take 60 μl of the sample solution obtained in step 1) in a volumetric flask, add 30 μl of 0.05M carbonic acid buffer solution and 30 μl of (+)-FLEC reagent, plug the stopper of the volumetric flask, and rotate slowly for 3 to 5 seconds; Place the volumetric flask in a 45°C water bath for 1 hour, then take out the volumetric flask from the water bath and let it cool down to room temperature naturally, then add deionized water to the volumetric flask to make the volume to 10ml; Filter to obtain sample solution;

5), high performance liquid chromatography detection:

The L-carnitine derivative solution of the gradient concentration obtained in step 3) and the D-carnitine derivative solution of the gradient concentration and the sample liquid obtained in step 4) are respectively subjected to high performance liquid chromatography detection; the detection conditions are as follows:

Select C18 chromatographic column, the chromatographic conditions are: mobile phase: triethylamine phosphate buffer: acetonitrile = 73:27 volume ratio; flow rate: 1ml/min; detection wavelength: emission wavelength 269nm, detection wavelength 310nm; injection volume: 10μl;

Respectively obtain the response peak area of the L-carnitine derivative solution of the gradient concentration, the response peak area of the D-carnitine derivative solution of the gradient concentration, and the response peak area of the sample solution;

6), obtain the concentration cL of L-carnitine and the concentration cD of D-carnitine respectively:

The response peak area of the L-carnitine derivative solution of the gradient concentration obtained in step 5) is plotted against the concentration to obtain a standard curve of L-carnitine;

The response peak area of the D-carnitine derivative solution of the gradient concentration obtained in step 5) is plotted against the concentration to obtain a standard curve of D-carnitine;

The response peak area of the sample solution obtained in step 5) is compared with the standard curve of the above-mentioned L-carnitine and the standard curve of D-carnitine respectively, and the obtained result is multiplied by the dilution of the sample solution in the derivatization reaction of step 4) The multiple is 10*1000/60, so that the concentration cL of L-carnitine and the concentration cD of D-carnitine in the sample solution are respectively obtained;

7), obtain the L-carnitine content and purity in the milk powder sample:

In the formula: cL—the concentration of L-carnitine in the sample solution, μg/ml;

m——the mass of milk powder, g.

In the formula: cL—the concentration of L-carnitine in the sample solution, μg/ml;

cD—the concentration of D-carnitine in the sample solution, μg/ml.

As the improvement of the assay method of L-carnitine content and purity in the milk powder of the present invention:

The preparation method of 0.05M carbonic acid buffer is as follows: 338mg of anhydrous sodium carbonate and 152mg of sodium bicarbonate are dissolved in 100ml of water;

The preparation method of the triethylamine phosphate buffer solution is as follows: 6.8ml of triethylamine is dissolved in 950ml of water, then the pH is adjusted to 2.5 with a volume fraction of 84% phosphoric acid aqueous solution, and finally the volume is adjusted to 1000ml with water.

As a further improvement of the assay method of L-carnitine content and purity in milk powder of the present invention: step 1) is:

First dissolve the milk powder with deionized water, put it in a water bath at 50°C for 15 minutes, then ultrasonically treat it for 10 minutes, and then dilute to 100ml with deionized water;

Then add 0.1mol/L hydrochloric acid solution to adjust the pH to 1.8-2.2, let it stand for 10 minutes and filter it with natural filter paper; The filter paper is used for second filtration, and the filtrate obtained from the second filtration is the sample solution.

The present invention uses 1-(9-fluorenyl)-ethyl chloroformate, i.e. (+)-FLEC, as a derivative reagent, and a fluorescent group is connected to the carnitine enantiomer, thereby separating on a chromatographic column, and using fluorescent Detector for analysis, greatly improving the detection sensitivity.

In the present invention, all reagents used are commercial products. For example, L-carnitine standard, D-carnitine standard, (+)-FLEC reagent can be purchased from Sigma.

The method of the present invention first eliminates the interference of fat and protein in milk powder, then chirally splits the enantiomers of L-carnitine and D-carnitine, and analyzes and detects them with high-performance liquid chromatography to provide a pre-column derivatization with high efficiency A method for detecting the content and purity of L-carnitine in milk powder by liquid chromatography.

The invention utilizes a chiral derivatization method to separate carnitine enantiomers and perform high-performance liquid chromatography detection to measure the content and purity of L-carnitine in formula food for infants and young children. The quality control and safety testing of milk powder is of great significance.

Adopting the method of the present invention to measure the content and the purity of L-carnitine in the milk powder has the following advantages:

1. Compared with traditional analysis methods, the present invention can chirally split L-type and D-type carnitine to achieve the purpose of separation and detection, and high-performance liquid chromatography is more commonly used in laboratories;

2. The high-performance liquid chromatography fluorescence analysis method adopted in the present invention has good accuracy, good separation ability, high sensitivity, the detection limit is about 1/10 to 1/100 of that of the ultraviolet detector, and the instrument is easy to operate;

3. It can eliminate the interference of fat and protein in milk powder, and effectively detect the content and purity of L-carnitine.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Fig. 1 is L-carnitine standard curve;

Figure 2 is the D-carnitine standard curve

Fig. 3 is the milk powder sample chromatogram of embodiment 1.

Detailed ways

The specific embodiment of the present invention will be described below in conjunction with the example of determining the content and purity of L-carnitine in milk powder by the pre-column derivatization high performance liquid chromatography of the present invention.

Embodiment 1, a kind of assay method of L-carnitine content and purity in milk powder is characterized in that comprising the following steps successively:

1), sample processing:

Accurately weigh 16.65g of a commercially available common milk powder sample, dissolve it in 30ml of deionized water, bathe in 50°C water for 15min, and ultrasonic (frequency 30kHz) for 10min to fully dissolve the milk powder, then add deionized water to make up to 100ml.

Then adjust the pH to 2 with 0.1mol/L hydrochloric acid solution, let it stand for 10 minutes, and then filter it with natural filter paper. The pH of the obtained filtrate was adjusted to 6 with 1mol/L NaOH solution, and after standing for 10 minutes, a second filtration was performed with natural filter paper, and the filtrate obtained from the second filtration was the sample solution.

2), prepare standard solution:

Weigh 50 mg of L-carnitine standard substance, dissolve it in deionized water, and set the volume to 10ml; the resulting solution is filtered through a 0.45 μm filter membrane to obtain L-carnitine standard solution;

Weigh 50 mg of D-carnitine standard substance, dissolve it in deionized water, and set the volume to 10ml; the resulting solution is filtered through a 0.45 μm filter membrane to obtain D-carnitine standard solution;

3), the L-carnitine standard solution and the D-carnitine standard solution of step 2) gained are carried out as follows respectively:

Put 30 μl of L-carnitine standard solution in a volumetric flask, add 80 μl of 0.05M carbonic acid buffer and 80 μl of (+)-FLEC reagent, plug the stopper of the volumetric flask, and rotate slowly for 3 to 5 seconds; Put the volumetric flask in a water bath at 45°C for 1 hour, then take out the volumetric flask from the water bath and let it cool down to room temperature naturally, then add deionized water to the volumetric flask to make the volume to 10ml; dilute the volumetric solution with deionized water to obtain A series of derivative solutions of L-carnitine with L-carnitine concentrations of 0.5, 1.0, 1.5, 2.0, 2.5 μg/ml;

Similarly, put 30 μl of D-carnitine standard solution in a volumetric flask, add 80 μl of 0.05M carbonic acid buffer and 80 μl of (+)-FLEC reagent, plug the stopper of the volumetric flask, and rotate slowly for 3 to 5 seconds; put the volumetric flask in a water bath at 45°C for 1 hour, then take out the volumetric flask from the water bath and cool it down to room temperature naturally, then add deionized water to the volumetric flask to make the volume to 10ml; dilute the volumetric solution with deionized water Dilute to obtain a series of derivative solutions of D-carnitine with D-carnitine concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 μg/ml respectively;

Filter the obtained L-carnitine series derivative solutions and D-carnitine series derivative solutions with 0.45 μm microporous membrane respectively (can be appropriately pressurized); respectively obtain L-carnitine derivative solutions with gradient concentrations (5 kinds in total) and D-carnitine derivative solutions of gradient concentrations (5 kinds in total);

4), sample solution derivatization reaction

The preparation of assay reagents is carried out first:

Triethylamine phosphate buffer solution: Dissolve 6.8ml of triethylamine in 950ml of water, adjust its pH to 2.5 with 84% phosphoric acid aqueous solution, and finally dilute with water to 1000ml.

0.05M carbonate buffer: 338mg of anhydrous sodium carbonate and 152mg of sodium bicarbonate dissolved in 100ml of water.

Take 60 μl of the sample solution obtained in step 1) in a volumetric flask, add 30 μl of 0.05M carbonic acid buffer solution and 30 μl of (+)-FLEC reagent, plug the stopper of the volumetric flask, and rotate slowly for 3 to 5 seconds; Place the volumetric flask in a 45°C water bath for 1 hour, then take out the volumetric flask from the water bath and let it cool down to room temperature naturally, then add deionized water to the volumetric flask to make the volume to 10ml; Filter (can be properly pressurized) to obtain the sample solution.

5), high performance liquid chromatography detection

The L-carnitine derivative solution of the gradient concentration obtained in step 3) and the D-carnitine derivative solution of the gradient concentration and the sample liquid obtained in step 4) are respectively subjected to high performance liquid chromatography detection; the detection conditions are as follows:

Choose C18 chromatographic column. The chromatographic conditions are: mobile phase triethylamine phosphate buffer: acetonitrile (73:27, volume ratio); flow rate: 1ml/min; detection wavelength: emission wavelength 269nm, detection wavelength 310nm; injection volume: 10μl.

The response peak area of the gradient concentration L-carnitine derivative solution, the response peak area of the gradient concentration D-carnitine derivative solution, and the response peak area of the sample solution were respectively obtained.

6), obtain L-carnitine content and purity:

With step 5) the response peak area of the L-carnitine derivative solution of the gradient concentration obtained is plotted against concentration, obtains the standard curve of L-carnitine; As shown in Figure 1: L-carnitine, c (μg/ml) =2*10 ^-7 A+0.0978; where A is the peak area value;

With step 5) the response peak area of the D-carnitine derivative solution of the gradient concentration obtained is plotted against concentration, obtains the standard curve of D-carnitine; As shown in Figure 2: D-carnitine, c (μg/ml) =2*10 ^-7 A+0.1185; where A is the peak area value.

The response peak area of the sample solution obtained in step 5) is compared with the standard curve of the above-mentioned L-carnitine and the standard curve of D-carnitine respectively, and the obtained result is multiplied by the dilution of the sample solution in the derivatization reaction of step 4) The multiple is 10*1000/60, thereby obtaining the concentration cL of L-carnitine and the concentration cD of D-carnitine in the sample solution respectively; specifically:

The detection results of the sample solution are shown in Fig. 3, the peak area value of the L-carnitine derivative is 158390, and the peak area of the D-carnitine derivative is 0. Therefore, the concentration of L-carnitine in the sample solution cL=(158390*2*10 ⁻⁷ +0.0978)*10*1000/60=21.58 μg/ml; cD=0.

7), obtain the L-carnitine content and purity in the milk powder sample:

(Note: In the formula: cL—the concentration of L-carnitine in the sample solution, μg/ml;

m——the mass of milk powder, g. )

(Note: In the formula: cL—the concentration of L-carnitine in the sample solution, μg/ml;

cD—the concentration of D-carnitine in the sample solution, μg/ml. )

The calculation results are summarized as follows: the content of L-carnitine in the milk powder sample is 12.95mg/100g, and D-carnitine is not detected.

Embodiment 2, a kind of assay method of L-carnitine content and purity in milk powder is characterized in that comprising the following steps successively:

1), sample processing:

Accurately weigh 14.95g of commercially available infant formula milk powder No. 1 sample, dissolve it in 30m deionized water, bathe in 50°C water for 15min, ultrasonic (frequency 30kHz) 10min, the milk powder is fully dissolved, add deionized water to make up to 100ml.

Then adjust the pH to 1.9 with 0.1mol/L hydrochloric acid solution, let it stand for 10min, and then filter it with natural filter paper. Adjust the pH of the obtained filtrate to 6.1 with 1 mol/L NaOH solution, and perform a second filtration with natural filter paper, and the filtrate obtained from the second filtration is the sample solution.

Step 2) to step 6) are all the same as in Example 1.

Thereby, the concentration cL=(812098*2*10 ⁻⁷ +0.0978)*10*1000/60=43.37 μg/ml of L-carnitine in the sample solution and the concentration cD=0 μg/ml of D-carnitine were obtained respectively;

7), obtain the content and purity of L-carnitine in the sample:

The calculation formula is the same as in Example 1.

The calculation results are as follows: the content of L-carnitine in No. 1 sample of infant formula milk powder is 29.01mg/100g, and D-carnitine is not detected.

Example 3. The commercially available infant formula milk powder No. I sample in Example 2 was changed to the commercially available infant formula milk powder No. II sample, and 15.63 g of the sample was accurately weighed.

Step 1) to step 6) are all the same as in Example 2.

Thereby, the concentration cL=(848302*2*10 ⁻⁷ +0.0978)*10*1000/60=44.57 μg/ml of the concentration of L-carnitine in the sample solution and the concentration cD=0 μg/ml of D-carnitine were obtained respectively;

7), obtain the content and purity of L-carnitine in the sample:

The calculation formula is the same as in Example 1.

The results were as follows: the content of L-carnitine in No. II sample of infant formula milk powder was 28.52mg/100g, and D-carnitine was not detected.

In order to prove the correctness of the conclusion of the present invention, utilize the existing high-performance liquid chromatography ultraviolet analysis method to be completely the same as the ordinary milk powder sample in embodiment 1～embodiment 3, infant formula milk powder No. 1 sample, infant formula milk powder No. II sample is tested: the result is as follows:

In ordinary milk powder samples: the content of L-carnitine is 13mg/100g; in sample I of infant formula milk powder: the content of L-carnitine is 29.1mg/100g; in sample II of infant formula milk powder: L-carnitine The content of alkali is 28.6mg/100g. The test results are consistent with the results of Examples 1-3.

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the 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.

Images