CN101424668A - Arachidonic acid content detecting method in milk and milk products - Google Patents
Arachidonic acid content detecting method in milk and milk products Download PDFInfo
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Abstract
The invention relates to a method for checking the arachidonic acid content in milk and milk products. After a sample is processed, a gas-phase spectrum meter is used for check. The sample processing method comprises the following steps: step 1, normal hexane and absolute-methanol-potassium-hydroxide are added into the sample for carrying out etherification reaction; step 2, the solution obtained in step 1 is centrifuged or stands until the organic phase is clarified to extract the supernatant fluid to obtain a solution to be checked. Water is added into the solution after the etherification after step 1 or before step 2. For a solid or liquid sample, the sample is firstly processed as following: step A, ammonia water is added into the sample, and ethanol is added after reaction; step B, ether and sherwood oil are added for layer separation in a standing mode, the organic phase is taken and concentrated until a dried state, and the sample concentrated liquid is obtained. The method of the invention has the advantages of short extracting time, complete extraction, simple and convenient operation and high accurate rate, and is suitable for checking a great number of samples.
Description
Technical field
The present invention relates to the detection method of arachidonic acid content in a kind of milk and milk products, especially utilize vapor-phase chromatography to detect the method for arachidonic acid content in the milk and milk products, particularly detect the improvement of sample preparation step in the arachidonic acid content process in the milk and milk products about vapor-phase chromatography.
Background technology
Arachidonic acid (AA) is to belong to the necessary higher fatty acid of human body, and is essential to keeping each function of organization of human body, can cause a series of symptoms during shortage, comprises growth retardation, sterile, dysnoesia etc.For satisfying the growth demand of human body, from diet, obtain the common recognition that an amount of fatty acid has become people, various interpolation food such as milk powder, beverage arise at the historic moment.Therefore the various fatty acid compositions that add in the food are followed the tracks of quantitatively fast and accurately and extremely be necessary.Since at present increasing to the detection limit of various fatty acid, a kind of technology that can handle sample in a large number urgently sought.
" mensuration of DHA, EPA, linoleic acid, leukotrienes and arachidonic acid (AA) in dispensed food for baby and the milk powder " (" Chinese dairy industry ", 2006 the 34th the 12nd phases of volume, the 46-47 page or leaf, Wang Kexin, Fang Yuguo, Zhang Lihong, Wang Miao Wang Wei) discloses a kind of method of utilizing vapor-phase chromatography to detect AA, and it utilizes ammonia-ethanolic solution to destroy the colloid proterties and the fat globule membrane of breast, non-fat composition is dissolved in ammonia-ethanolic solution, uses ether-Petroleum ether extraction to go out fat again.Fat detects after at room temperature reacting quick esterification with methyl alcohol-potassium hydroxide in normal hexane.
Yet, in the sample preparation step of this method, be esterification and leave standstill the back just directly on machine testing, and experiment showed, further behind the esterification reaction of organic acid that AA does not extract fully in the normal hexane, therefore can influence the accuracy of testing result.
Summary of the invention
The purpose of this invention is to provide a kind of method that detects arachidonic acid content in the dairy products, improve the arachidonic extraction efficiency and the recovery by improving sample treatment.
Purpose of the present invention realizes by following scheme: the detection method of arachidonic acid content in a kind of milk and milk products, and will use gas chromatograph to detect after the sample preparation, the method for wherein said sample preparation is:
1) in sample, adds normal hexane, absolute methanol-potassium hydroxide, carry out esterification;
2) after the solution centrifugal of step 1) gained or leave standstill is treated the organic phase clarification, get supernatant and obtain solution to be measured; After described step 1) and step 2) add water in the normal hexane layer after the forward direction esterification.
Preferably, described step 1) further comprises: add normal hexane in described sample, constant volume, accurately measure the solution behind the constant volume, the ratio of 1:1 adds absolute methanol-potassium hydroxide (2mol/l-5mol/l) by volume, and shake well leaves standstill and carried out esterification in 5-30 minute.
Preferred, the concentration of described absolute methanol-potassium hydroxide is 4mol/l, and time of repose is 20 minutes.
When sample was solid-state or liquid, sample need be prepared into sample concentration liquid, and its preparation method is as follows: A) add ammoniacal liquor in solid or fluid sample, the reaction back adds ethanol; B) add ether, sherwood oil, standing demix is got organic phase and is concentrated near doing, and obtains described sample concentration liquid.
Preferably, described steps A) and step B) further be: A) adding concentration in described sample is the ammoniacal liquor of 25%-28%, returns in 40-70 ℃ of water-bath to add ethanol after heating up in a steamer 10-30 minute, is cooled to room temperature, adds Congo red reagent; B) add ether, mixing adds sherwood oil again, and mixing leaves standstill layering in 15-30 minute, after the perhaps centrifugal layering, isolates organic phase, under nitrogen protection organic phase is concentrated near doing, and obtains described sample concentration liquid.
When described sample is solid, earlier solid sample is dissolved in the 40-70 ℃ of water before the sample preparation.
Preferably, sample treatment is: the accurate weighing 1.0000g solid sample flat bottom flask of packing into, add 1-3ml25%-28% ammoniacal liquor jog with 10ml (40-70) ℃ of water jolting dissolving (or accurate weighing 10g fluid sample is in flask), in 40-70 ℃ of water-bath return heat up in a steamer 10-30 minute after, return with the 10ml alcohol flushing and to heat up in a steamer pipe, be cooled to room temperature.Sample is added in the extracting container, add 2-3 and drip Congo red indicator.With adding in the extracting container about 100 times of shake well behind the 25ml ether flushing flask.Merge washing lotion in the extracting container, about 50 times of jolting after washing flask at twice with the 25ml sherwood oil again.Mixed liquor washing bottle cap with the ether sherwood oil.After leaving standstill layering in 15-30 minute, after the perhaps centrifugal layering, isolate organic phase.Under nitrogen protection that sample concentration is closely dried, obtain sample concentration liquid.
Preferably, described step B) in described concentrate take a step forward comprise the steps: to isolate organic phase after, add absolute ethyl alcohol, ether, sherwood oil again and repeat to extract 1-3 time the merging organic phase.Preferred, the volume ratio of absolute ethyl alcohol, ether, sherwood oil is: 1:3:3.
The used instrument of described standing demix preferably uses Mao Shi liposuction bottle.
Preferably, before using gas chromatograph to detect, described solution to be measured is filtered.
Preferably, above-mentioned detection method further comprises the steps:
The arachidonic acid standard solution for preparing a plurality of variable concentrations, and with described gas chromatograph it is detected respectively, according to measured data, draw the typical curve between arachidonic content and the testing result; With using described gas chromatograph that described solution to be measured is detected testing result and the comparison of described typical curve that obtains, obtain arachidonic content in the sample.
Among the present invention, above-mentioned gas chromatograph chromatographic condition is as follows: flame ionization detector; Sample introduction temperature: 260 ℃; Detector temperature: 300 ℃; Column temperature: heating schedule: intensification is 75-85 ℃ to be kept 2-4 minute, be raised to 170-180 ℃ with 10-15 ℃/minute then after, be warmed up to 210-220 ℃ with 1-3 ℃/minute again, program 30-40 minute altogether.Capillary column: CP-WAX58 or pillar with equal performance.Carrier gas: high pure nitrogen, air and hydrogen.
The present invention compared with prior art has the following advantages:
1. the fundamental purpose that adds normal hexane is fully to dissolve grease, makes itself and the rapid alkaline hydrolysis of methyl alcohol-potassium hydroxide, esterification.After esterification, in the normal hexane layer, add entry, can make fatty acid transfer to the normal hexane layer fast.After finding esterification reaction of organic acid in the experiment, by existing method operation, the content of arachidonic acid methyl esters is not high in the normal hexane; After adding entry and leaving standstill, arachidonic content obviously increases, and the recovery also increases.The amount that adds water can be selected according to actual conditions, and the amount size is convenient to experimental implementation to the recovery and not influence of precision.
Mao Shi liposuction bottle extract more suitable than traditional separating funnel extraction effect, but the former operation is simple, be fit to the detection of batch samples, and favorable reproducibility.
It is short that method of the present invention has extraction time, extracts fully, simple to operation, and the accuracy rate height is fit to a large amount of sample detection.
Description of drawings
Fig. 1 is that the arachidonic acid of one embodiment of the present of invention detects collection of illustrative plates.
Embodiment
Below in conjunction with specific embodiments method of the present invention is done more detailed description.It will be appreciated by those skilled in the art that following embodiment all is used for the present invention's scope required for protection is carried out the description of exemplary, summarize the relative scope of each parameter of the present invention, thereby it can not be interpreted as a kind of concrete restriction of the present invention with this.
Used normal hexane is chromatographically pure reagent among the application.
Chromatograph: U.S. Varian, cp3800.
The making of embodiment 1 arachidonic acid (AA) typical curve
Chromatographic condition: flame ionization detector; Sample introduction temperature: 260 ℃; Detector temperature: 300 ℃; Column temperature: heating schedule: kept 2 minutes for 75 ℃, be raised to 175 ℃ with 10 ℃/minute then after, be warmed up to 215 ℃ with 1.5 ℃/minute again, kept 30 minutes.Capillary column: CP-WAX58 or pillar with equal performance.Carrier gas: high pure nitrogen, air and hydrogen.
The used normal hexane of present embodiment is a chromatographically pure reagent.AA Standard solution is available from sigma, purity 〉=99.99%.With AA esterification standard items with n-hexane dissolution after, the concentration sample that is mixed with 10ppm, 50ppm, 100ppm, 200ppm, 400ppm, 800ppm is sample introduction respectively, according to measured data, draws the typical curve between AA content and the testing result, equation is A
Std=263.76C, R=0.9999.Wherein, A
StdThe chromatographic peak area of expression standard solution, C represents concentration of standard solution (μ g/mL).
The detection of AA content in the embodiment 2 fatty acid samples
1) accurate weighing 0.5g fatty acid sample, add normal hexane, use the n-hexane dissolution sample, be settled to the 10ml scale, the solution of accurately measuring behind the 2ml constant volume adds 0.5ml absolute methanol-potassium hydroxide (4mol/l) shake well, leave standstill and carried out esterification in 20 minutes, in hexane solution, add water to 25ml.
2) leave standstill, treat organic phase clarification after, get supernatant, filter, to the gas chromatograph sample detection.
Chromatographic condition: flame ionization detector; Sample introduction temperature: 260 ℃; Detector temperature: 300 ℃; Column temperature: heating schedule: kept 2 minutes for 75 ℃, be raised to 175 ℃ with 10 ℃/minute then after, be warmed up to 215 ℃ with 1.5 ℃/minute again, kept 30 minutes.Capillary column: CP-WAX58 or pillar with equal performance.Carrier gas: high pure nitrogen, air and hydrogen.Resulting typical curve comparison among result that detection is obtained and the embodiment 1, wherein the computing formula of AA content is:
In the formula: X: arachidonic content (mg/100g) in the test sample;
A
Yp, A
Std: the chromatographic peak area that is respectively sample, standard solution;
C: concentration of standard solution (μ g/mL);
M: sample 70 product sampling amounts (g);
10: extension rate;
100,1000: transformation ratio.
The content that calculates AA in the sample is: 10.8mg/100g.
The detection of AA content in embodiment 3 milk powder
A) the accurate weighing 1.0000g milk powder sample flat bottom flask of packing into 65 ℃ of water joltings dissolvings of 10ml, adds 2ml25% ammoniacal liquor, jog, in 65 ℃ of water-baths return heat up in a steamer 15 minutes after, return with the 10ml alcohol flushing and to heat up in a steamer pipe, be cooled to room temperature.It is added Mao Shi take out in the ester bottle, add 2 Congo red indicator.B) take out in the ester bottle with adding behind the 25ml ether flushing flask, carry out jolting 100 times.Merge washing lotion in taking out the ester bottle, jolting 50 times after washing flask at twice with the 25ml sherwood oil again.Mixed liquor washing bottle cap with the ether sherwood oil.After leaving standstill layering in 30 minutes, isolate organic phase.Repeat to extract 1 time with 5ml absolute ethyl alcohol, 15ml ether, 15ml sherwood oil again, after the merging organic phase.Under nitrogen protection that sample concentration is closely dried, obtain sample concentration liquid.
In sample concentration liquid, add normal hexane, promptly concentrate bottle, be settled to 10ml with the normal hexane flushing, accurately measure 2ml solution and add 2ml absolute methanol-potassium hydroxide (4mol/l), shake well leaves standstill and carried out esterification in 20 minutes, adds water to 25ml in the normal hexane layer; Leave standstill, treat organic phase clarification after, draw supernatant, filter, use the gas chromatograph sample detection.
Chromatographic condition: flame ionization detector; Sample introduction temperature: 260 ℃; Detector temperature: 300 ℃; Column temperature: kept 2 minutes for 75 ℃, be raised to 175 ℃ with 10 ℃/minute then after, be warmed up to 215 ℃ with 1.5 ℃/minute again, kept 30 minutes.Capillary column: CP-WAX 58 or pillar with equal performance.Carrier gas: high pure nitrogen, air and hydrogen.Resulting typical curve comparison among result that detection is obtained and the embodiment 1, the content that employing formula (I) calculates AA in the milk powder sample is: 36.3mg/100g.
The detection of AA content in the embodiment 4 children milk
A) accurately weighing 10g children suckle sample in flat bottom flask, add 2ml 25% ammoniacal liquor jog, in 65 ℃ of water-baths return heat up in a steamer 15 minutes after, return with the 10ml alcohol flushing and to heat up in a steamer pipe, be cooled to room temperature.It is added Mao Shi take out in the ester bottle, add 2 Congo red indicator.B) take out in the ester bottle with adding behind the 25ml ether flushing flask, carry out jolting 100 times.Merge washing lotion in taking out the ester bottle, jolting 50 times after washing flask at twice with the 25ml sherwood oil again.Mixed liquor washing bottle cap with the ether sherwood oil.After leaving standstill layering in 30 minutes, isolate organic phase.Repeat to extract 3 times with 5ml absolute ethyl alcohol, 15ml ether, 15ml sherwood oil again, after the merging organic phase.Under nitrogen protection that sample concentration is closely dried, obtain sample concentration liquid.
Concentrate bottle with the normal hexane flushing, be settled to 10ml, accurately measure 2ml solution and add 0.5ml absolute methanol-potassium hydroxide (4mol/l) shake well, leave standstill and carried out esterification in 20 minutes, in the normal hexane layer, add water to 25ml; Leave standstill, treat organic phase clarification after, draw supernatant, filter, use the gas chromatograph sample detection.
Chromatographic condition: flame ionization detector; Sample introduction temperature: 260 ℃; Detector temperature: 300 ℃; Column temperature: kept 2 minutes for 75 ℃, be raised to 175 ℃ with 10 ℃/minute then after, be warmed up to 215 ℃ with 1.5 ℃/minute again, kept 30 minutes.Capillary column: CP-WAX 58 or pillar with equal performance.Carrier gas: high pure nitrogen, air and hydrogen.Resulting typical curve comparison among result that detection is obtained and the embodiment 1, employing formula (I) calculate children and suckle that the content of AA is in the sample: 2.90mg/ml.
The detection of AA content in embodiment 5 baby's milk powder
A) the accurate weighing 1.0000g baby milk powder sample flat bottom flask of packing into 40 ℃ of water joltings dissolvings of 10ml, adds 1ml 28% ammoniacal liquor, jog, in 40 ℃ of water-baths return heat up in a steamer 30 minutes after, return with the 10ml alcohol flushing and to heat up in a steamer pipe, be cooled to room temperature.It is added Mao Shi take out in the ester bottle, add 2 Congo red indicator.B) take out in the ester bottle with adding behind the 25ml ether flushing flask, carry out jolting 100 times.Merge washing lotion in taking out the ester bottle, jolting 50 times after washing flask at twice with the 25ml sherwood oil again.Mixed liquor washing bottle cap with the ether sherwood oil.After leaving standstill layering in 20 minutes, isolate organic phase.Repeat to extract 1 time with 5ml absolute ethyl alcohol, 15ml ether, 15ml sherwood oil again, after the merging organic phase.Under nitrogen protection that sample concentration is closely dried, obtain sample concentration liquid.
In sample concentration liquid, add normal hexane, promptly concentrate bottle, be settled to 10ml with the normal hexane flushing, accurately measure 2ml solution and add 2ml absolute methanol-potassium hydroxide (2mol/l), shake well leaves standstill and carried out esterification in 30 minutes, adds water to 25ml in the normal hexane layer; Leave standstill, treat organic phase clarification after, draw supernatant, filter, use the gas chromatograph sample detection.
Chromatographic condition: flame ionization detector; Sample introduction temperature: 260 ℃; Detector temperature: 300 ℃; Column temperature: kept 4 minutes for 85 ℃, be raised to 180 ℃ with 15 ℃/minute then after, be warmed up to 210 ℃ with 1 ℃/minute again, kept 40 minutes.Capillary column: CP-WAX58 or pillar with equal performance.Carrier gas: high pure nitrogen, air and hydrogen.Resulting typical curve comparison among result that detection is obtained and the embodiment 1, the content that employing formula (I) calculates AA in baby's milk powder sample is: 47.6mg/100g.
The detection of AA content in the embodiment 6 old milk powder
A) the accurate weighing 1.0000g baby milk powder sample flat bottom flask of packing into 70 ℃ of water joltings dissolvings of 10ml, adds 1ml26% ammoniacal liquor, jog, in 70 ℃ of water-baths return heat up in a steamer 10 minutes after, return with the 10ml alcohol flushing and to heat up in a steamer pipe, be cooled to room temperature.It is added Mao Shi take out in the ester bottle, add 2-3 and drip Congo red indicator.B) take out in the ester bottle with adding behind the 25ml ether flushing flask, carry out jolting 100 times.Merge washing lotion in taking out the ester bottle, jolting 50 times after washing flask at twice with the 25ml sherwood oil again.Mixed liquor washing bottle cap with the ether sherwood oil.After leaving standstill layering in 15 minutes, isolate organic phase.Repeat to extract 2 times with 5ml absolute ethyl alcohol, 15ml ether, 15ml sherwood oil again, after the merging organic phase.Under nitrogen protection that sample concentration is closely dried, obtain sample concentration liquid.
In sample concentration liquid, add normal hexane, promptly concentrate bottle, be settled to 10ml with the normal hexane flushing, accurately measure 2ml solution and add 2ml absolute methanol-potassium hydroxide (3mol/l), shake well leaves standstill and carried out esterification in 15 minutes, adds water to 25ml in the normal hexane layer; Leave standstill, treat organic phase clarification after, draw supernatant, filter, use the gas chromatograph sample detection.
Chromatographic condition: flame ionization detector; Sample introduction temperature: 260 ℃; Detector temperature: 300 ℃; Column temperature: kept 3 minutes for 80 ℃, be raised to 170 ℃ with 12 ℃/minute then after, be warmed up to 220 ℃ with 3 ℃/minute again, kept 35 minutes.Capillary column: CP-WAX58 or pillar with equal performance.Carrier gas: high pure nitrogen, air and hydrogen.Resulting typical curve comparison among result that detection is obtained and the embodiment 1, the content that employing formula (I) calculates AA in the old milk powder sample is: 2.79mg/100g.
The detection of AA content in the embodiment 7 fortifications milk
A) accurately weighing 10g fortification milk sample adds 3ml25% ammoniacal liquor jog in flat bottom flask, in 60 ℃ of water-baths return heat up in a steamer 25 minutes after, return with the 10ml alcohol flushing and to heat up in a steamer pipe, be cooled to room temperature.It is added Mao Shi take out in the ester bottle, add 2 Congo red indicator.B) take out in the ester bottle with adding behind the 25ml ether flushing flask, carry out jolting 100 times.Merge washing lotion in taking out the ester bottle, jolting 50 times after washing flask at twice with the 25ml sherwood oil again.Mixed liquor washing bottle cap with the ether sherwood oil.After the centrifugal layering, isolate organic phase.Repeat to extract 3 times with 5ml absolute ethyl alcohol, 15ml ether, 15ml sherwood oil again, after the merging organic phase.Under nitrogen protection that sample concentration is closely dried, obtain sample concentration liquid.
Concentrate bottle with the normal hexane flushing, be settled to 10ml, accurately measure 2ml solution and add 0.5ml absolute methanol-potassium hydroxide (5mol/l) shake well, leave standstill and carried out esterification in 10 minutes, in the normal hexane layer, add water to 25ml; Leave standstill, treat organic phase clarification after, draw supernatant, filter, use the gas chromatograph sample detection.
Chromatographic condition: flame ionization detector; Sample introduction temperature: 260 ℃; Detector temperature: 300 ℃; Column temperature: kept 2 minutes for 75 ℃, be raised to 175 ℃ with 10 ℃/minute then after, be warmed up to 215 ℃ with 1.5 ℃/minute again, kept 30 minutes.Capillary column: CP-WAX 58 or pillar with equal performance.Carrier gas: high pure nitrogen, air and hydrogen.
Obtain measuring collection of illustrative plates as shown in Figure 1.Because complicated component in the milk, preceding several peaks are other material in the fatty acid.Can find out obviously that from spectrogram peak shape and the degree of separation of AA are better.Sample peak degree of separation and reappearance are better in the following detection of the chromatographic condition of the present invention gained collection of illustrative plates.
Resulting typical curve comparison among result that detection is obtained and the embodiment 1, the content that employing formula (I) calculates AA in the fortification milk sample is: 3.72mg/ml.
Embodiment 8 different esterification times contrast the result that influences that milk powder detects the AA recovery
Use the milk powder sample of same batch of the method detection identical with embodiment 3, wherein change the time of leaving standstill esterification, detect the different content that leave standstill AA under the reaction time of esterification, its recovery is as follows, wherein expression is in the milk powder sample, 25 ℃ of results that influence that descend different esterification times to the recovery of room temperature:
From the result, after 20 minutes, the recovery is more stable relatively.The recovery is low slightly before 20 minutes.So can select 5-30 minute as leaving standstill reaction time of esterification, preferably adopt 20 minutes for leaving standstill reaction time of esterification.
The check of embodiment 9 recovery
(1) leaves standstill and add water after the esterification and do not add water and detect effect comparison
Adopt the method for embodiment 2,3 and 5 to detect grease, milk powder, the children sample of suckling respectively, simultaneously to identical sample, cancellation adds the step of water after leaving standstill esterification, directly detect AA content with gas chromatograph, resulting result is as follows, wherein expression is esterification after 20 minutes, adds water and does not add the contrast of the water AA recovery:
Therefore when not adding water after the esterification, the content of arachidonic acid methyl esters is not high in the normal hexane, is fully to dissolve grease because add the fundamental purpose of normal hexane, makes itself and the rapid alkaline hydrolysis of methyl alcohol-potassium hydroxide, esterification.After the experiment esterification, in the normal hexane layer, add entry, can make fatty acid transfer to normal hexane fast.After adding entry and leaving standstill, arachidonic detection level obviously increases, and the recovery also increases.The amount that adds water can be selected according to the experiment situation, experiment showed, the extension rate that how much only influences that adds water, and the not influence of the recovery to detecting.
(2) use the contrast of Mao Shi liposuction bottle and separating funnel separating effect
Adopt the method for embodiment 3 and 5, an amount of AA Standard solution is added in the middle of the sample and sample synchronous processing and detection, the gained sample recovery rate is as follows:
Compare with existing method, the traditional relatively separating funnel of the recovery of standard addition of method of the present invention extracts higher relatively.It is more suitable than traditional separating funnel extraction effect that Mao Shi liposuction bottle extracts, but the former operation is simple, be fit to the detection of batch samples, and favorable reproducibility.
(3) the detection effect under the different solution temperatures of milk powder
The method of employing embodiment 3 detects the AA content in the milk powder, changes the temperature of water dissolving milk powder, detects AA content and calculate recovery rate under the different solution temperatures, and it is as follows to obtain the result:
| The water temperature of dissolving milk powder | 40℃ | 50℃ | 55℃ | 60℃ | 65℃ | 70℃ |
| The recovery | 97.03 | 97.21 | 99.08 | 99.11 | 99.25 | 99.23 |
The solution temperature of milk powder is too high, can destroy nutritional labeling wherein, and viscosity is crossed the low dissolving that is unfavorable for sample, so water temperature when determining its optimal dissolution, determined 40 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ several temperature point, in this temperature range, it is good to detect the recovery.
(4) the detection effect under the different ammonia concns
Adopt the method for embodiment 2,3,4 to detect AA content in fatty acid, milk powder and the children's milk respectively, change the concentration of ammoniacal liquor, detect AA content and calculate recovery rate under the different ammonia concns, the result is as follows:
| Ammonia concn | 25％ | 26％ | 27％ | 28％ |
| The milk powder recovery | 99.25 | 99.08 | 99.11 | 98.79 |
| Children's recovery of suckling | 98.99 | 98.52 | 98.56 | 98.00 |
| The fatty acid recovery | 100.35 | 100.30 | 99.26 | 99.23 |
The concentration that adds ammoniacal liquor in the sample, destruction nutritional labeling wherein that also can be in various degree after testing, is in 25%, 26%, 27%, 28% in concentration range, detects the recovery and reaches 98.79-100.35.
(5) the detection effect under the time of heating up in a steamer is returned in different water-baths
Adopt the method for embodiment 2,3,4 to detect AA content in fatty acid, milk powder and the children's milk respectively, change water-bath and return the time of heating up in a steamer, detect different water-baths and return AA content and calculate recovery rate under the time of heating up in a steamer, the result is as follows:
| Return time | 10 minutes | 15 minutes | 20 minutes | 25 minutes | 30 minutes |
| The milk powder recovery | 96.27 | 99.25 | 99.18 | 99.23 | 99.20 |
| Children's recovery of suckling | 98.78 | 98.99 | 98.78 | 98.87 | 98.97 |
| The fatty acid recovery | 92.03 | 100.35 | 100.23 | 100.33 | 100.32 |
Sample have only fully react with ammoniacal liquor after, could effectively be extracted by ether, sherwood oil, return that to detect the recovery in the scope of heating up in a steamer 10-30 minute all better.
(6) behind ether, Petroleum ether extraction, the separating effect of different time of repose
Adopt the method for embodiment 2,3,4 to detect AA content in fatty acid, milk powder and the children's milk respectively, behind ether, Petroleum ether extraction, change the time of leaving standstill, separating effect is as follows:
| Time of repose | 20 minutes | 30 minutes | 40 minutes |
| Milk powder | Separating effect is general, and the time is shorter | Separating effect is better | Separating effect is better, and the time is longer |
| Children's milk | Separating effect is general, and the time is shorter | Separating effect is better | Separating effect is better, and the time is longer |
| Fatty acid | Separating effect is general, and the time is shorter | Separating effect is better | Separating effect is better, and the time is longer |
Leave standstill behind the ether Petroleum ether extraction, be in order better organic phase to be separated, so relatively 20 minutes, 30 minutes, 40 minutes effect all can reach separation between 20-40 minute, wherein ether sherwood oil organic phase can better be toppled over out more than 30 minutes.
(7) the detection effect under the concentration of different absolute methanol-potassium hydroxide
Adopt the method for embodiment 2,3,4 to detect AA content in fatty acid, milk powder and the children's milk respectively, change the concentration of absolute methanol-potassium hydroxide, detect AA content and calculate recovery rate under the concentration of different absolute methanol-potassium hydroxide, the result is as follows:
| Concentration | 2mol/l | 3mol/l | 4mol/l | 5mol/l |
| The milk powder recovery | 99.00 | 99.00 | 99.08 | 98.11 |
| Children's recovery of suckling | 97.23 | 97.38 | 98.52 | 97.08 |
| The fatty acid recovery | 99.01 | 99.11 | 100.30 | 97.26 |
The concentration that this shows absolute methanol-potassium hydroxide all has the better recovery in the 2-4mol/l scope, under 4mol/l concentration, it is best to detect the recovery.
As seen from the above-described embodiment, method of the present invention is more complete to the extraction of AA, accurately.
Claims (10)
1. the detection method of arachidonic acid content in the milk and milk products will use gas chromatograph detect after the sample preparation, and the method for wherein said sample preparation is:
1) in sample, adds normal hexane, absolute methanol-potassium hydroxide, carry out esterification;
2) after the solution centrifugal of step 1) gained or leave standstill is treated the organic phase clarification, get supernatant and obtain solution to be measured;
It is characterized in that: after described step 1) and step 2) add water in the normal hexane layer after the forward direction esterification.
2. detection method as claimed in claim 1 is characterized in that:
Described step 1) further comprises: add normal hexane in described sample, constant volume is accurately measured the solution behind the constant volume, and the ratio of 1:1 adds absolute methanol-potassium hydroxide (2mol/l-5mol/l) by volume, shake well leaves standstill and carried out esterification in 5-30 minute.
3. detection method as claimed in claim 2 is characterized in that, described time of repose is 20 minutes.
4. as the described detection method of one of claim 1-3, it is characterized in that: described sample is a sample concentration liquid, and the preparation method is as follows:
A) add ammoniacal liquor in the described sample, the reaction back adds ethanol;
B) add ether, sherwood oil, standing demix is got organic phase and is concentrated near doing, and obtains described sample concentration liquid.
5. detection method as claimed in claim 4 is characterized in that: described steps A) and step B) further be:
A) adding concentration in described sample is the ammoniacal liquor of 25%-28%, returns in 40-70 ℃ of water-bath to add ethanol after heating up in a steamer 10-30 minute, is cooled to room temperature, adds Congo red reagent;
B) add ether, mixing adds sherwood oil again, and mixing leaves standstill layering in 15-30 minute, after the perhaps centrifugal layering, isolates organic phase, under nitrogen protection organic phase is concentrated near doing, and obtains described sample concentration liquid.
6. detection method as claimed in claim 5 is characterized in that: when described sample is solid, before the described sample preparation earlier with described sample dissolution in 40-70 ℃ of water.
7. as claim 5 or 6 described detection methods, it is characterized in that: described step B) described concentrate take a step forward comprise the steps: to isolate organic phase after, add absolute ethyl alcohol, ether, sherwood oil again and repeat to extract 1-3 time the merging organic phase.
8. detection method as claimed in claim 7 is characterized in that: the used instrument of described standing demix is a Mao Shi liposuction bottle.
9. as the described detection method of one of claim 1-8, it is characterized in that: the arachidonic acid standard solution that further comprises the steps: to prepare a plurality of variable concentrations, and it is detected respectively with described gas chromatograph, according to measured data, draw the typical curve between arachidonic content and the testing result;
With using described gas chromatograph that described solution to be measured is detected testing result and the comparison of described typical curve that obtains, obtain arachidonic content in the sample.
10. detection method as claimed in claim 9, it is characterized in that: described gas chromatograph heating schedule is as follows: be warming up to 75-85 ℃ and kept 2-4 minute, after being raised to 170-180 ℃ with 10-15 ℃/minute then, again with 1-3 ℃/minute, preferred 1.5 ℃/minute are warmed up to 210-220 ℃, program 30-40 minute altogether.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109030657A (en) * | 2018-08-27 | 2018-12-18 | 广州风行乳业股份有限公司 | The detection method of 5a,6,9,9a-hexahydro-6,9-methano-2,4 pesticide residue in a kind of animal milk |
| CN112748195A (en) * | 2020-12-23 | 2021-05-04 | 沈阳农业大学 | Method for simultaneously detecting fatty acid, amino acid and multifunctional group organic acid by GC-NCI-MS |
| CN113311098A (en) * | 2021-05-21 | 2021-08-27 | 汤臣倍健股份有限公司 | Method for measuring content of free fatty acid in breast milk |
-
2008
- 2008-11-20 CN CNA2008101800278A patent/CN101424668A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109030657A (en) * | 2018-08-27 | 2018-12-18 | 广州风行乳业股份有限公司 | The detection method of 5a,6,9,9a-hexahydro-6,9-methano-2,4 pesticide residue in a kind of animal milk |
| CN112748195A (en) * | 2020-12-23 | 2021-05-04 | 沈阳农业大学 | Method for simultaneously detecting fatty acid, amino acid and multifunctional group organic acid by GC-NCI-MS |
| CN113311098A (en) * | 2021-05-21 | 2021-08-27 | 汤臣倍健股份有限公司 | Method for measuring content of free fatty acid in breast milk |
| CN113311098B (en) * | 2021-05-21 | 2022-07-29 | 汤臣倍健股份有限公司 | Method for measuring content of free fatty acid in breast milk |
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