CN104280467B - The quantitative detecting method of plant sterol in dairy products - Google Patents
The quantitative detecting method of plant sterol in dairy products Download PDFInfo
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Abstract
The present invention relates to the quantitative detecting method of plant sterol in dairy products, the method can detect the concrete content of cupreol in raw material and dairy products contained by plant sterol, campesterol, stigmasterol simultaneously.By the disposal route to sample, the action condition etc. of Saponification Conditions, extractant and chromatographic column is optimized, and establishes testing conditions, sets up detection method.The method is easy, quick, accurate, is suitable for basic unit and applies.
Description
Technical field
The invention belongs to analytical chemistry field, specifically, relate to the quantitative detecting method of plant sterol in a kind of dairy products.
Background technology
Plant sterol is the new resource food of health ministry No. 3 bulletin approval in 2010, utilize soybean wet goods vegetable oil cut or Tall oil to be raw material, obtained by techniques such as saponification, extraction, crystallizations, then plant sterol and sunflower seed oil fatty acids are carried out esterification production and obtain phytosterin ester.
Plant sterol is present in natural veterinary antibiotics, beans, nut and cereal, and be the constituent of plant cell membrane, plant sterol can reduce blood cholesterol concentration.This is because the chemical constitution of plant sterol and cholesterol are extremely similar, the hydrolysis of Reverse transcriptase intestines inner cholesterol can be done, and in intestinal epithelial cell, suppress the resterification of free cholesterol, and the position of intestines inner cholesterol can be occupied competitively, reduce the touch opportunity of cholesterol and intestinal mucosa cells, thus hinder small intestine to the absorption of cholesterol, make it directly discharge from ight soil.Composition contained in plant sterol is modal comprises cupreol, campesterol, stigmasterol, is secondly the saturated form of plant sterol, i.e. β-sitostamol and campestanol etc.In recent years, both at home and abroad to the detection method of plant sterol, mainly contain high performance liquid chromatography, vapor-phase chromatography and By Gas Chromatography-mass Spectrometry.China does not still have the detection method standard of plant sterol in dairy products at present.
Summary of the invention
The object of this invention is to provide the quantitative detecting method of plant sterol in a kind of dairy products.
In order to realize the object of the invention, the quantitative detecting method of plant sterol in a kind of dairy products of the present invention, comprises the following steps:
1) process of sample: add 0.667g/mL potassium hydroxide solution 5-10mL and 95% ethanol 5-30mL in 0.1-1.5 gram of sample, shake makes sample dissolution, in 70-100 DEG C of saponification 1-4h; Then add organic solvent extraction 2-3 time, merge organic phase, be washed with distilled water to neutrality; Then with after anhydrous sodium sulfate dehydration, be evaporated to rotary evaporator dry, proceeded to constant volume in 10mL volumetric flask with normal hexane gradation (preferably 3 times), obtain sample; Described organic solvent is normal hexane, sherwood oil or ether etc.;
2) preparation of standard solution: the concentration of standard reserving solution is 1.0mg/mL, the concentration of standard working solution is 50 μ g/mL;
3) stratographic analysis: adopt that specification is the capillary chromatographic column Elite-1 of 30m × 0.25mm × 0.25 μm, capillary chromatographic column Elite-35 that capillary chromatographic column Elite-5 that specification is 30m × 0.32mm × 0.25 μm or specification are 30m × 0.25mm × 0.25 μm;
Chromatographic condition: injector temperature 270-320 DEG C; Detector temperature 270-310 DEG C; Temperature programme 180-290 DEG C; Carrier gas is nitrogen, flow velocity 2mL/min; Hydrogen flow rate 40-45mL/min; Air velocity 400-450mL/min; Sample size 2 μ L; Split ratio 10:1;
4) drafting of typical curve: after plant sterol standard solution sample introduction, with standard specimen concentration, typical curve is done to peak area;
5) mensuration of content of phytosterol in sample: by sample sample introduction, peak area per sample, substitutes into formula (I), calculates the content of plant sterol in sample;
In formula:
X
i---the content of plant sterol in sample;
A
i---the peak area of plant sterol in sample;
A
s---the peak area of plant sterol standard working solution;
C---plant sterol standard working solution concentration;
The constant volume of V---sample;
The quality of m---sample.
The plant sterol related in the present invention is one or more in cupreol, campesterol, stigmasterol etc.
According to aforesaid quantitative detecting method, in step 1), sample preparation is: in 0.2-1.2 gram of sample, add 0.667g/mL potassium hydroxide solution 5-8mL and 95% ethanol 10-20mL, and shake makes sample dissolution, in 80-100 DEG C of saponification 1-3h; Then normal hexane extraction 2-3 time is added.
According to aforesaid quantitative detecting method, in step 1), sample preparation is: in 0.3-1.0 gram of sample, add 0.667g/mL potassium hydroxide solution 6-9mL and 95% ethanol 15-25mL, and shake makes sample dissolution, in 70-100 DEG C of saponification 1-4h; Then petroleum ether extraction is added 2-3 time.
According to aforesaid quantitative detecting method, specification in step 3), is adopted to be that the capillary chromatographic column Elite-1 of 30m × 0.25mm × 0.25 μm carries out stratographic analysis; Chromatographic condition: injector temperature 300 DEG C; Detector temperature 300 DEG C; Temperature programme: 210 DEG C keep 1min, and 15 DEG C/min rises to 285 DEG C, keep 6min.Carrier gas is nitrogen, flow velocity 2mL/min; Hydrogen flow rate 40mL/min; Air velocity 400mL/min; Sample size 2 μ L; Split ratio 10:1.
According to aforesaid quantitative detecting method, specification in step 3), is adopted to be that the capillary chromatographic column Elite-5 of 30m × 0.32mm × 0.25 μm carries out stratographic analysis; Chromatographic condition: injector temperature 310 DEG C; Detector temperature 300 DEG C; Temperature programme: 210 DEG C keep 1min, and 15 DEG C/min rises to 285 DEG C, keep 6min.Carrier gas is nitrogen, flow velocity 2mL/min; Hydrogen flow rate 45mL/min; Air velocity 450mL/min; Sample size 2 μ L; Split ratio 10:1.
According to aforesaid quantitative detecting method, specification in step 3), is adopted to be that the capillary chromatographic column Elite-35 of 30m × 0.25mm × 0.25 μm carries out stratographic analysis; Chromatographic condition: injector temperature 310 DEG C; Detector temperature 300 DEG C; Temperature programme: 210 DEG C keep 1min, and 15 DEG C/min rises to 285 DEG C, keep 14min.Carrier gas is nitrogen, flow velocity 2mL/min; Hydrogen flow rate 45mL/min; Air velocity 450mL/min; Sample size 2 μ L; Split ratio 10:1.
Details are as follows for quantitative detecting method of the present invention:
1 reagent
All reagent, as unreceipted specification, all refers to that analysis is pure; All experimental waters, as other requirements unreceipted, all refer to tertiary effluent.
1.1 potassium hydroxide solutions: take 400g potassium hydroxide and add 600mL distilled water and dissolve and get final product.
1.295% ethanol
1.3 anhydrous sodium sulfate
1.4 sherwood oil
1.5 normal hexane: chromatographically pure
1.6 the preparation of standard solution
1.6.1 standard items
1.6.1.1 cupreol: purity 98%(sigma company).
1.6.1.2 campesterol: purity 65%(sigma company).
1.6.1.3 stigmasterol: purity 95%(sigma company).
1.6.2 standard items stock solution
1.6.2.1 cupreol Standard Reserving Solution: concentration is 1.0mg/mL.
Take 0.051g cupreol normal hexane dissolve and be settled to 50mL, freezen protective.
1.6.2.2 campesterol Standard Reserving Solution: concentration is 1.0mg/mL.
Take 0.0769g campesterol normal hexane dissolve and be settled to 50mL, freezen protective.
1.6.2.3 stigmasterol Standard Reserving Solution: concentration is 1.0mg/mL.
Take 0.0526g stigmasterol normal hexane dissolve and be settled to 50mL, freezen protective.
1.6.3 hybrid standard working fluid
Draw each 0.5mL of 1.6.2.1,1.6.2.2,1.6.2.3 stock solution respectively in 10mL volumetric flask, use normal hexane constant volume, the concentration of three kinds of working fluids is 50 μ g/mL.
2 instrument and equipments
2.1 gas chromatograph (being furnished with fid detector)
2.2 capillary chromatographic columns: Elite-1(30m × 0.25mm × 0.25 μm).
2.3 water-bath
2.4 rotary evaporator
2.5125mL separating funnel
2.6 screw socket glass tubes (doing the screw-mouth cover of interior pad with teflon): 25mL
2.7 analytical balances: sensibility reciprocal is 0.1mg.
3 operation stepss
3.1 sample preparation
3.1.1 saponification
Take sample 1g in screw socket glass tube, add 5mL potassium hydroxide solution, the shake of 10mL ethanol makes sample fully dissolve, put into boiling water bath saponification 1h, period every 20min shake once, takes out and is cooled to room temperature.
3.1.2 extract with concentrated
All proceeded in 125mL separating funnel by saponification liquor with a small amount of water, fully vibrate after adding 25mL sherwood oil 2min, proceeded to by aqueous phase in another 125mL separating funnel after stratification.Again by above-mentioned steps extraction 1-2 time, combined ether liquid, is washed with distilled water to neutrality.Then with after anhydrous sodium sulfate dehydration, near dry with rotary evaporator evaporation, divide with normal hexane solution and proceeded to constant volume in 10mL volumetric flask 3 times, this is Specimen Determination liquid.
3.2 measure
3.2.1 reference color spectral condition
GC-FID: injector temperature 300 DEG C; Detector temperature 300 DEG C; Temperature programme: 210 DEG C, keeps 1min, is warming up to 285 DEG C with 15 DEG C/min, keeps 6min; Carrier gas (N
2) flow velocity 2mL/min; Hydrogen flow rate 40mL/min; Air velocity 400mL/min; Sample size 2 μ L; Split ratio 10:1.
3.2.2 measure
Respectively by the standard working solution prepared, in sample liquid injection gas chromatography, record corresponding peak area (or peak height), quantified by external standard method.
3.2.3 typical curve
After campesterol, stigmasterol, cupreol standard serial solution respectively sample introduction, with standard specimen concentration, typical curve is done to peak area.Linearly dependent coefficient is more than 0.9990.
3.2.4 by sample sample introduction, according to the chromatographic peak of campesterol, stigmasterol, cupreol in the qualitative sample of the retention time of standard items.Peak area per sample, looks into the content that typical curve draws campesterol, stigmasterol, cupreol.
The statement of 4 analysis results
In 4.1 samples, the content of each sterol presses formula (I) calculating:
In formula:
X
i---the content of each sterol in sample, unit is the every hectogram of milligram (mg/100g);
A
i---the peak area of each sterol in sample;
A
s---the peak area of each sterol standard working solution;
C---each sterol standard working solution concentration, unit is micrograms per millilitre (μ g/mL);
The constant volume of V---sample, unit is milliliter (mL);
The quality of m---sample, unit is gram (g).
In 4.2 samples, the content of three kinds of sterols presses formula (II) calculating:
X=∑X
i(II)
X---the total content of three kinds of sterols in sample, unit is the every hectogram of milligram (mg/100g);
X
i---the content of each sterol in sample, unit is the every hectogram of milligram (mg/100g).
Represent with the arithmetic mean of twice that obtains under repeated condition independent measurement result, result retains three position effective digitals.
5 detection method evaluations
5.1 linear analysis scopes are evaluated
By standard solution according to chromatographic condition, inject gas chromatograph, carries out quantitatively, being depicted as curve with peak area.As campesterol typical curve as shown in Figure 1, Y=(-5139.741822)+(7204.193172) X; R
2=0.999713.Stigmasterol typical curve as shown in Figure 2, Y=(1044.283619)+(4279.128078) X; R
2=0.999927.Cupreol typical curve as shown in Figure 3, Y=(-1094.523253)+(4275.082951) X; R
2=0.999943.
As can be seen from the figure the sensing range of campesterol, stigmasterol, cupreol is within the scope of 5-100 μ g/mL, linearly well, and coefficient R
2more than=0.9997.
The accuracy of 5.2 methods and precision evaluation
The accuracy of the inventive method is verified, by the repeatability of the precision verification method of parallel experiment with Standard entertion recovery experiment.Require processing sample according to method, all samples all takes 1g, add campesterol, stigmasterol, the cupreol mixed solution of 100 μ g, 250 μ g, 500 μ g, 750 μ g, 1000 μ g, five gradients in the sample to which respectively, Simultaneously test blank sample, processes according to method.By sample introduction under the chromatographic condition of above method, the addition of variable concentrations and the mean value of recovery of standard addition are respectively:
5.2.1 when addition is 2mL × 50 μ g/mL=100 μ g, its recovery of standard addition campesterol is 92.32%, 91.58%, 97.66%, 95.50%, and mean value is campesterol 94.27%; Its recovery of standard addition stigmasterol is 97.02%, 101.94%, 95.72%, 101.39%, mean value be 99.02% and its recovery of standard addition cupreol be 97.35%, 97.13%, 91.27%, 97.61%, mean value is 95.84%.
5.2.2 when addition is 5mL × 50 μ g/mL=250 μ g, its recovery of standard addition campesterol is 91.13%, 97.24%, 98.39%, 94.38%, and mean value is campesterol 95.29%; Its recovery of standard addition stigmasterol is 95.59%, 102.62%, 97.89%, 95.66%, mean value be 97.94% and its recovery of standard addition cupreol be 94.40%, 97.28%, 97.43%, 97.08%, mean value is 96.55%.
5.2.3 when addition is 10mL × 50 μ g/mL=500 μ g, its recovery of standard addition campesterol is 97.23%, 96.19%, 97.92%, 95.55%, and mean value is campesterol 96.72%; Its recovery of standard addition stigmasterol is 94.31%, 98.90%, 99.27%, 96.00%, mean value be 97.12% and its recovery of standard addition cupreol be 93.38%, 94.56%, 98.57%, 97.20%, mean value is 95.93%.
5.2.4 when addition is 15mL × 50 μ g/mL=750 μ g, its recovery of standard addition campesterol is 96.54%, 99.14%, 96.93%, 97.75%, and mean value is campesterol 97.37%; Its recovery of standard addition stigmasterol is 96.81%, 95.03%, 99.89%, 99.67%, mean value be 97.85% and its recovery of standard addition cupreol be 98.28%, 97.04%, 98.51%, 99.22%, mean value is 98.26%.
5.2.5 when addition is 20mL × 50 μ g/mL=1000 μ g, its recovery of standard addition campesterol is 98.04%, 98.62%, 96.33%, 95.88%, and mean value is campesterol 97.22%; Its recovery of standard addition stigmasterol is 100.49%, 100.74%, 98.33%, 97.88%, mean value be 99.36% and its recovery of standard addition cupreol be 98.65%, 99.14%, 96.26%, 97.09%, mean value is 97.79%.
Reclaim result from the mark-on of Different adding amount, recovery of standard addition is all more than 94% (generally between 80%-105%), more better close to 100%, illustrates that this detection method recovery of standard addition is fine, in well linear.
Require that the absolute value of the twice independent measurement result obtained under repeated condition must not exceed 10% of arithmetic mean, therefore the precision of the method is very high.
The detect and track evaluation of 5.3 methods
According to the concentration corresponding to 3 times of signal to noise ratio (S/N ratio)s, calculate campesterol, stigmasterol, cupreol detect and be limited to 1mg/kg.
5.4 reappearance
The reappearance of the inventive method is verified by personnel's control experiment.Laboratory technician's first and second adopt same detection method, same detecting instrument, at different time, use 1.6.3 hybrid standard working fluid (50 μ g/mL) to adopt parallel experiment to verify the reappearance of the inventive method respectively.Require preparation hybrid standard working fluid according to method, chromatographic condition sample introduction as stated above, records corresponding peak area.Peak area reappearance is gone out according to the chromatographic peak of three kinds of sterols in the qualitative hybrid standard working fluid of the retention time of single product standard items.Fig. 4 A and B be respectively campesterol in the sample that laboratory technician's first and second detects according to the inventive method, stigmasterol, cupreol go out peak area, peak area comparison result (1-6 represents 6 parallel experiments) as shown in table 1.
6 conclusions
By checking, it is good in (5-100) μ g/mL scope internal linear that campesterol, stigmasterol, cupreol measure concentration, coefficient R
2=0.9997, detect and be limited to 1mg/kg, between recovery of standard addition campesterol 91.58% ~ 99.14%, stigmasterol 94.31% ~ 102.62%, cupreol 91.27% ~ 99.22%, RSD% is in required scope, and result shows that its assay is without significant difference.
The method detects campesterol, stigmasterol, cupreol in dairy products as can be seen from the above data, result accurately and reliably, goes for using in real work the method to detect in plant sterol raw material and the content of campesterol, stigmasterol, cupreol in dairy products (as milk powder and the milk powder adding plant sterol).
The quantitative detecting method of plant sterol in dairy products provided by the invention, the method can detect the concrete content of cupreol in raw material and dairy products contained by plant sterol, campesterol, stigmasterol simultaneously.By the disposal route to sample, the action condition etc. of Saponification Conditions, extractant and chromatographic column is optimized, and establishes testing conditions, sets up detection method.The method is easy, quick, accurate, is suitable for basic unit and applies.
Accompanying drawing explanation
Fig. 1-Fig. 3 is respectively campesterol, stigmasterol, cupreol typical curve.
Fig. 4 A and B be respectively campesterol in the sample that laboratory technician's first and second detects according to the inventive method, stigmasterol, cupreol go out peak area.
Fig. 5-8 is respectively 50 μ g/mL standard items spectrograms in the embodiment of the present invention 1, Milk powder for middle-aged and old people spectrogram, plant sterol raw material spectrogram and adds the milk powder spectrogram of plant sterol.
Fig. 9-Figure 12 is respectively 50 μ g/mL standard items spectrograms in the embodiment of the present invention 2, Milk powder for middle-aged and old people spectrogram, plant sterol raw material spectrogram and adds the milk powder spectrogram of plant sterol.
Figure 13-Figure 16 is respectively 50 μ g/mL standard items spectrograms in the embodiment of the present invention 3, Milk powder for middle-aged and old people spectrogram, plant sterol raw material spectrogram and adds the milk powder spectrogram of plant sterol.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The reagent used in following examples and equipment and instrument, that does not mention especially in an embodiment is mentioned reagent and equipment and instrument.Testing sample has three: the milk powder of plant sterol raw material, Milk powder for middle-aged and old people and interpolation plant sterol.
The quantitative detection of plant sterol in embodiment 1 dairy products
Operation steps is as follows:
1 saponification
Take material sample 0.2g(Milk powder for middle-aged and old people sample 1g) in screw socket glass tube, add 5mL potassium hydroxide solution, 20mL ethanol shake sample is fully dissolved, put into boiling water bath saponification 1h, period every 10min shake once, take out be cooled to room temperature.
2 extract with concentrated
All proceeded in 125mL separating funnel by saponification liquor with a small amount of water, fully vibrate after adding 25mL sherwood oil 2min, proceeded to by aqueous phase in another 125mL separating funnel after stratification.Again by above-mentioned steps extraction 2-3 time, combined ether liquid, is washed with distilled water to neutrality.Then with after anhydrous sodium sulfate dehydration, near dry with rotary evaporator evaporation, divide with normal hexane solution and proceeded to constant volume in 10mL volumetric flask three times, this is Specimen Determination liquid.
3 measure
3.1 reference color spectral conditions
Adopt capillary chromatography Elite-1(30m × 0.25mm × 0.25 μm), GC-FID: injector temperature 300 DEG C; Detector temperature 300 DEG C; Temperature programme: 210 DEG C, keeps 1min, is warming up to 285 DEG C with 15 DEG C/min, keeps 6min; Carrier gas (N
2) flow velocity 2mL/min; Hydrogen flow rate 40mL/min; Air velocity 400mL/min; Sample size 2 μ L; Split ratio 10:1.
3.2 measure
Respectively by the standard working solution prepared, in sample liquid injection gas chromatography, record corresponding peak area (or peak height), quantified by external standard method.
3.3 by sample sample introduction, according to the chromatographic peak of campesterol, stigmasterol, cupreol in the qualitative sample of the retention time of standard items.Peak area per sample, looks into the content that typical curve draws campesterol, stigmasterol, cupreol.
The statement of 4 analysis results
In 4.1 samples, the content of each sterol presses formula (I) calculating:
In formula:
X
i---the content of each sterol in sample, unit is the every hectogram of milligram (mg/100g);
A
i---the peak area of each sterol in sample;
A
s---the peak area of each sterol standard working solution;
C---each sterol standard working solution concentration, unit is micrograms per millilitre (μ g/mL);
The constant volume of V---sample, unit is milliliter (mL);
The quality of m---sample, unit is gram (g).
In 4.2 samples, the content of three kinds of sterols presses formula (II) calculating:
X=∑X
i(II)
X---the total content of three kinds of sterols in sample, unit is the every hectogram of milligram (mg/100g);
X
i---the content of each sterol in sample, unit is the every hectogram of milligram (mg/100g).
5 experiment spectrogram and results
The milk powder spectrogram of 50 μ g/mL standard items spectrograms, Milk powder for middle-aged and old people sample (blank sample) spectrogram, plant sterol raw material spectrogram, interpolation plant sterol respectively as shown in Figure 5-Figure 8.
Contrast with standard spectrogram, blank sample, in corresponding appearance time section, does not have peak to occur, namely campesterol is 0, stigmasterol is 0, cupreol is 0.
According to formula, calculate three plant sterols content in plant sterol material sample as follows: campesterol 8219.14mg/100g, stigmasterol 12876.7mg/100g, cupreol 24365.94mg/100g.
According to formula, calculate three plant sterols content in the powdered milk sample adding plant sterol as follows: campesterol 212.62mg/100g, stigmasterol 220.0mg/100g, cupreol 625.81mg/100g.
The quantitative detection of plant sterol in embodiment 2 dairy products
Operation steps is as follows:
1 saponification: with embodiment 1
2 extract with concentrated: with embodiment 1
3 measure: with embodiment 1
3.1 reference color spectral conditions
Adopt capillary chromatography Elite-5(30m × 0.32mm × 0.25 μm), GC-FID: injector temperature 310 DEG C; Detector temperature 300 DEG C; Temperature programme: 210 DEG C, keeps 1min, is warming up to 285 DEG C with 15 DEG C/min, keeps 6min; Carrier gas (N2) flow velocity 2mL/min; Hydrogen flow rate 45mL/min; Air velocity 450mL/min; Sample size 2 μ L; Split ratio 10:1.
The statement of 4 analysis results: with embodiment 1
5 experiment spectrogram and results
The milk powder spectrogram of 50 μ g/mL standard items spectrograms, Milk powder for middle-aged and old people sample (blank sample) spectrogram, plant sterol raw material spectrogram, interpolation plant sterol is respectively as shown in Fig. 9-Figure 12.
Contrast with standard spectrogram, blank sample, in corresponding appearance time section, does not have peak to occur, namely campesterol is 0, stigmasterol is 0, cupreol is 0.
According to formula, calculate three plant sterols content in plant sterol material sample as follows: campesterol 8776.78mg/100g, stigmasterol 12568.45mg/100g, cupreol 23514.40mg/100g.
According to formula, calculate three plant sterols content in the powdered milk sample adding plant sterol as follows: campesterol 237.81mg/100g, stigmasterol 225.98mg/100g, cupreol 622.94mg/100g.
The quantitative detection of plant sterol in embodiment 3 dairy products
Operation steps is as follows:
1 saponification: with embodiment 1
2 extract with concentrated: with embodiment 1
3 measure
3.1 reference color spectral conditions
Adopt capillary chromatography Elite-35(30m × 0.25mm × 0.25 μm), chromatographic condition: injector temperature 310 DEG C; Detector temperature 300 DEG C; Temperature programme: 210 DEG C keep 1min, are warming up to 285 DEG C with 15 DEG C/min, keep 14min; Carrier gas (N
2) flow velocity 2mL/min; Hydrogen flow rate 45mL/min; Air velocity 450mL/min; Sample size 2 μ L; Split ratio 10:1.
The statement of 4 analysis results: with embodiment 1
5 experiment spectrogram and results
The milk powder spectrogram of 50 μ g/mL standard items spectrograms, Milk powder for middle-aged and old people sample (blank sample) spectrogram, plant sterol raw material spectrogram, interpolation plant sterol is respectively as shown in Figure 13-Figure 16.
Contrast with standard spectrogram, blank sample, in corresponding appearance time section, does not have peak to occur, namely campesterol is 0, stigmasterol is 0, cupreol is 0.
According to formula, calculate three plant sterols content in plant sterol material sample as follows: campesterol 8372.87mg/100g, stigmasterol 12791.05mg/100g, cupreol 24135.41mg/100g.
According to formula, calculate three plant sterols content in the powdered milk sample adding plant sterol as follows: campesterol 233.51mg/100g, stigmasterol 219.34mg/100g, cupreol 622.88mg/100g.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Campesterol in the sample that table 1 laboratory technician first and second detect, stigmasterol, cupreol go out peak area comparison result
Claims (3)
1. the quantitative detecting method of plant sterol in dairy products, is characterized in that, comprise the following steps:
1) process of sample: add 0.667g/mL potassium hydroxide solution 5-10mL and 95% ethanol 5-30mL in 0.1-1.5 gram of sample, shake makes sample dissolution, in 70-100 DEG C of saponification 1-4h; Then add organic solvent extraction 2-3 time, merge organic phase, be washed with distilled water to neutrality; Then with after anhydrous sodium sulfate dehydration, be evaporated to rotary evaporator dry, proceeded to constant volume in 10mL volumetric flask with normal hexane gradation, obtain sample; Described organic solvent is normal hexane, sherwood oil or ether;
2) preparation of standard solution: the concentration of standard reserving solution is 1.0mg/mL, the concentration of standard working solution is 50 μ g/mL;
3) stratographic analysis: adopt that specification is the capillary chromatographic column Elite-1 of 30m × 0.25mm × 0.25 μm, capillary chromatographic column Elite-35 that capillary chromatographic column Elite-5 that specification is 30m × 0.32mm × 0.25 μm or specification are 30m × 0.25mm × 0.25 μm;
Specification is adopted to be that the capillary chromatographic column Elite-1 of 30m × 0.25mm × 0.25 μm carries out stratographic analysis; Chromatographic condition: injector temperature 300 DEG C; Detector temperature 300 DEG C; Temperature programme: 210 DEG C keep 1min, and 15 DEG C/min rises to 285 DEG C, keep 6min; Carrier gas is nitrogen, flow velocity 2mL/min; Hydrogen flow rate 40mL/min; Air velocity 400mL/min; Sample size 2 μ L; Split ratio 10:1;
Specification is adopted to be that the capillary chromatographic column Elite-5 of 30m × 0.32mm × 0.25 μm carries out stratographic analysis; Chromatographic condition: injector temperature 310 DEG C; Detector temperature 300 DEG C; Temperature programme: 210 DEG C keep 1min, and 15 DEG C/min rises to 285 DEG C, keep 6min; Carrier gas is nitrogen, flow velocity 2mL/min; Hydrogen flow rate 45mL/min; Air velocity 450mL/min; Sample size 2 μ L; Split ratio 10:1;
Specification is adopted to be that the capillary chromatographic column Elite-35 of 30m × 0.25mm × 0.25 μm carries out stratographic analysis; Chromatographic condition: injector temperature 310 DEG C; Detector temperature 300 DEG C; Temperature programme: 210 DEG C keep 1min, and 15 DEG C/min rises to 285 DEG C, keep 14min; Carrier gas is nitrogen, flow velocity 2mL/min; Hydrogen flow rate 45mL/min; Air velocity 450mL/min; Sample size 2 μ L; Split ratio 10:1;
4) drafting of typical curve: after plant sterol standard solution sample introduction, with standard specimen concentration, typical curve is done to peak area;
5) mensuration of content of phytosterol in sample: by sample sample introduction, peak area per sample, substitutes into formula (I), calculates the content of plant sterol in sample;
In formula:
X
i---the content of plant sterol in sample;
A
i---the peak area of plant sterol in sample;
A
s---the peak area of plant sterol standard working solution;
C---plant sterol standard working solution concentration;
The constant volume of V---sample;
The quality of m---sample;
Described plant sterol is one or more in cupreol, campesterol, stigmasterol.
2. quantitative detecting method according to claim 1, it is characterized in that, step 1) in sample preparation be: in 0.2-1.2 gram of sample, add 0.667g/mL potassium hydroxide solution 5-8mL and 95% ethanol 10-20mL, shake make sample dissolution, in 80-100 DEG C of saponification 1-3h; Then normal hexane extraction 2-3 time is added.
3. quantitative detecting method according to claim 1, it is characterized in that, step 1) in sample preparation be: in 0.3-1.0 gram of sample, add 0.667g/mL potassium hydroxide solution 6-9mL and 95% ethanol 15-25mL, shake make sample dissolution, in 70-100 DEG C of saponification 1-4h; Then petroleum ether extraction is added 2-3 time.
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