CN109187394A - A kind of measuring method of cholesterol concentration - Google Patents
A kind of measuring method of cholesterol concentration Download PDFInfo
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- CN109187394A CN109187394A CN201811212619.3A CN201811212619A CN109187394A CN 109187394 A CN109187394 A CN 109187394A CN 201811212619 A CN201811212619 A CN 201811212619A CN 109187394 A CN109187394 A CN 109187394A
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Abstract
The invention discloses a kind of measuring methods of cholesterol concentration, comprising: (1) pipettes cholesterol solution centrifugation to be measured, 95% ethyl alcohol is added after centrifugation and 50%KOH solution is saponified in hot bath；(2) after being cooled to room temperature, petroleum ether is added, extraction is shaken in water bath with thermostatic control shaking table；(3) it is transferred completely into separatory funnel, and with 95% ethyl alcohol rinse, stands liquid separation, collect petroleum ether layer in beaker；(4) organic reagent is evaporated in water-bath in draught cupboard；(5) dehydrated alcohol dissolution residual substance, the ultrasonic dissolution in hot bath are used；(6) constant volume obtains cholesterol lysate；(7) cholesterol standard curve is drawn；(8) cholesterol lysate is pipetted in test tube, OPA reagent is added, is stood, is slowly added to the concentrated sulfuric acid along test tube wall in a cold water bath, is vortexed to solution and clarifies, and is stood in cold bath；It surveys OD value at spectrophotometer 550nm, substitutes into calibration curve equation, calculate to obtain the final product.This method is low in cost, safety, easy to operate, is suitble to be widely popularized.
Description
Technical field
The present invention relates to norcholesterol detection fields, and in particular to a kind of measuring method of cholesterol concentration.
Background technique
Cholesterol is the derivative of cyclopentanoperhy drophenanthrene, is a kind of waxy, squamous chemical substance, because being originally found in gallbladder
Stone and gain the name.Cholesterol is nutritional ingredient necessary to human survival, has important physiological function, it is both to constitute cell
Important component, and can convert to form steroid hormone in vivo, and be the raw material of synthesis of vitamin d 3 and the precursor of bile acid,
But if cholesterol level is excessively high in vivo will also result in harm.Now, with internal cholesterol excessively related artery sclerosis, hat
The cardiovascular and cerebrovascular diseases such as heart trouble, headstroke, have seriously threatened human health.Since the initial stage eighties, as a kind of possibility
The food component being detrimental to health --- cholesterol has been increasingly becoming the hot topic of domestic and international food medical field debate.Gallbladder is solid
Alcohol be present in animal institute it is organized in, be that animal body maintains necessary to normal physiological activity.But excessive gallbladder is solid in human body
Alcohol will cause hyperlipidemia, and cause a series of cardiovascular diseases in turn, the intake of middle cholesterol so we will keep on a diet, because
The content of this detection Food Cholesterol, it is very necessary for establishing a kind of measuring method of fast and accurately cholesterol level.
Currently, the quantitative analysis method of cholesterol mainly has colorimetric method, enzyme process, gas chromatography and high performance liquid chromatography
Method.Traditional colorimetric method is since operating performance is many and diverse, especially extraction fatty in the pre-treatment of sample and sample, and required
Reagent is more, causes the complication of the deviation and calculated result of Colorimetric results, and at high cost, to define the extensive of this method
It uses.Enzyme process is since technology content is higher, high specificity, and other two methods (gas chromatography and high performance liquid chromatography)
Special equipment is needed again, so being all difficult to promote the use of.
Summary of the invention
In order to overcome the above-mentioned deficiencies of the prior art or defect, the purpose of the present invention is to provide a kind of cholesterol concentrations
Measuring method.
The present invention is achieved through the following technical solutions:
A kind of measuring method of cholesterol concentration, includes the following steps:
(1) cholesterol solution centrifugation to be measured is pipetted, 95% ethyl alcohol and 50% KOH solution are added after centrifugation and is saponified in hot bath directly
Completely to saponification, cholesterol crude liquid is obtained；
(2) after being cooled to room temperature cholesterol crude liquid, petroleum ether is added, concussion extraction is until extracted in water bath with thermostatic control shaking table
At obtaining extract liquor；
(3) extract liquor is transferred completely into separatory funnel, and with 95% ethyl alcohol rinse, stands liquid separation, collects petroleum ether layer in burning
In cup；
(4) organic reagent is evaporated in water-bath in draught cupboard, until no liquid residual in beaker；
(5) cholesterol solution is obtained with residue, the ultrasonic dissolution in hot bath in dehydrated alcohol dissolution beaker；
(6) constant volume: cholesterol solution is then transferred in 50mL volumetric flask, and with dehydrated alcohol rinse beaker 2~3 times, washing lotion is simultaneously
Enter in volumetric flask, and be titrated to scale, obtains cholesterol lysate；
(7) cholesterol standard curve is drawn；
(8) o-phthalaldehyde (OPA) reagent is added in clean tube in cholesterol lysate in removing step (6), stands, connects
Be slowly added to the concentrated sulfuric acid along test tube wall in a cold water bath, be vortexed to solution and clarify, stood in cold bath；Then in light splitting light
OD value is surveyed at degree meter 550nm, the OD value measured is substituted into cholesterol standard curvilinear equation, it is dense to calculate cholesterol in solution to be measured
Degree.
Wherein, described to pipette 10~20mL cholesterol solution to be measured in centrifuge tube with pipette in step (1), it is centrifuged
Parameter is 5000rpm, 20 DEG C, 10min；The process conditions of the saponification are as follows: pipette appropriate 10~15mL, 95% second with pipette
Triangle bottle sealing is placed on 60 in triangular flask by pure and mild 3~7mL, 50% KOH solution+Water-bath 25 in 1 DEG C of shaking bath~
30min is saponified；The revolving speed of the shaking bath is 100~120rpm.
Wherein, in step (2), the process conditions that petroleum ether is added are as follows: it is 60~90 that boiling range is added in draught cupboard
DEG C and volume be 8~12mL petroleum ether, then sealed with preservative film, the fixed preservative film of rubber band simultaneously stays 5~8 on film
Hole；The process conditions of extraction are shaken in be set forth in water bath with thermostatic control shaking table are as follows: be put into 25+15 are shaken in 1 DEG C of water bath with thermostatic control shaking table
~20min takes out, and 8~12mL distilled water is added, and continues at 15~20min of concussion extraction in water bath with thermostatic control shaking table, is allowed to abundant
Mixing.
Wherein, in step (3), the process conditions with 95% ethyl alcohol rinse are as follows: all shift the sample in triangular flask
Into 120mL separatory funnel, and with 5~10mL95% ethyl alcohol rinse triangular flask 1~2 time, washing lotion is incorporated in separatory funnel, up and down
It reverse separatory funnel 3~5 times, is allowed to be sufficiently mixed, stands 15~20min and be allowed to be layered；The technique for collecting petroleum ether layer
Condition are as follows: the solution layering in separatory funnel is obvious, i.e., boundary without it is muddy, without milkiness, liquid level is clear and stablizes；Open liquid separation
Funnel stopcock slowly releases lower liquid, and the petroleum ether layer on separatory funnel upper layer is burnt from the funnel 250mL high foot suitable for reading that is poured into
In cup.
Wherein, in step (4), the process conditions of water-bath evaporation organic reagent in the draught cupboard are as follows: petroleum ether will be filled
High foot beaker be placed in draught cupboard, in 80+Petroleum ether is evaporated in 1 DEG C of water-bath, is evaporated to no liquid residual, at this time bottom of the beaker
The main component of the white residue in portion is cholesterol, and the evaporation used time is 15~30min, and the temperature of water-bath is no more than 85
℃。
Wherein, in step (5), the process conditions of the ultrasonic dissolution in hot bath are as follows: ultrasonic water temperature is 80~100
DEG C, ultrasonic power 80W needs ultrasound to the residue of beaker bottom to be completely dissolved, and reduces experimental error.
Wherein, in step (5) and (6), the dehydrated alcohol is that analysis is pure.
Wherein, the drafting of step (7) standard curve is as follows:
After taking 15 clean tubes number, reagent is sequentially added according to the form below 1: with 2mL pipette absorption 0.00mL, 0.20 mL,
0.30 mL、0.40 mL、0.45 mL、0.55 mL、0.60 mL、0.65 mL、0.70 mL、0.80 mL、0.85 mL、0.90
ML, 1.00 mL, 1.10 mL, 1.20 mL cholesterol standard stock solutions are respectively placed in test tube, are then drawn with 2mL pipette
Glacial acetic acid complements to 2mL(note respectively: according to document, cholesterol level is closed in 0~4 mg/mL, with OD value in good linear
System), 4mL O-phthalic aldehyde reagent (OPA reagent) then is drawn with 5mL pipette and is separately added into each test tube, is placed at room temperature for
It after 10min, is drawn in the cold bath of the 4mL concentrated sulfuric acid with 5mL pipette and is slowly added to along test tube wall, shaken 30s with oscillator, make it
After mixing well, 10min is stood under room temperature in dark.Colorimetric is identified under 550nm, records OD value, is cross with cholesterol concentration
Coordinate, OD value are that ordinate makes standard curve, and calculates its equation of linear regression, do parallel laboratory test twice.
Wherein, cholesterol standard stock solution (1.0 mg/mL) prepare it is as follows:
Assay balance is opened, cholesterol standards 0.10g(is accurately weighed and is accurate to 0.1mg) in the beaker of dried and clean, it uses
Dehydrated alcohol stirring and dissolving in beaker, then the cholesterol lysate in beaker is all transferred to along glass bar and uses nothing in advance
In the 100mL volumetric flask of water-ethanol rinse, cholesterol residual solution in beaker is all transferred to capacity by rinse beaker 2-3 times
It in bottle, is titrated with disposable rubber head dropper, and be settled to scale, turning upside down mixes solution, is 0.45 with aperture after mixing
μm filtering with microporous membrane degerming, cholesterol standard stock solution is poured into brown reagent bottle, is closed the lid, glass bottle opening pad one layer
Preservative film, 4 DEG C of sealings stand overnight spare, can store half a year.
Preparing for O-phthalic aldehyde reagent (OPA reagent) is as follows:
After assay balance zeroing, a kind of measuring method of cholesterol concentration according to claim 1, it is characterised in that: step
Suddenly OPA reagent is O-phthalic aldehyde reagent in (7), and precise 0.05~0.07g o-phthalaldehyde is molten with about 30mL glacial acetic acid
Solution, is transferred in 100mL volumetric flask, and be settled to scale with glacial acetic acid.Experimental day is limited to use.
1 OPA method of table measures the design of the Specification Curve of Increasing of cholesterol
Wherein, in step (8), the concentrated sulfuric acid is that analysis is pure, to prevent carbonization from the measurement that black impurity interferes cholesterol occur,
Should the concentrated sulfuric acid be slowly added to along test tube wall in a cold water bath.
Wherein, in step (8), described be vortexed to solution is clarified, the process conditions stood in cold bath are as follows: the concentrated sulfuric acid is added
Afterwards, be vortexed 30~45s immediately, until mixed solution the clear, (pale red~brown red of test tube inner cholesterol mixed liquor discoloration at this time
Color), it then continues to be put into cold water and radiate, stand 10~30min, OD value is then surveyed at spectrophotometer 550nm, every group is real
Test three in parallel.
Compared with prior art, the present invention having the following beneficial effects:
The present invention replaces n-hexane as solvent using 50%KOH solution and using petroleum ether, and uses water bath with thermostatic control shaking table generation
For the saponification mode of water-bath, saponification, extraction can be made more thorough；Entire petroleum ether layer is used to test using separatory funnel
The branch mode of organic layer, experimental error caused by cholesterol can be reduced may being unevenly distributed in organic layer；Using thermostatted water
Bath evaporation replaces the drying mode that is dried with nitrogen, does not need specific apparatus and can be completed to be evaporated, and be avoided that nitrogen cylinder uses
It is improper and caused by security risk；Using the ultrasonic technique condition of special ultrasonic temperature, it can speed up cholesterol dissolution, shorten
The dissolution time of cholesterol residue；Using the concentrated sulfuric acid is slowly added to along test tube wall in a cold water bath, make the fast prompt drop of test tube wall
Temperature prevents concentrated sulfuric acid carbonization from the measurement and unnecessary scald of black impurity interference cholesterol occur, guarantees entire experiment safety
It carries out.After optimizing to existing method for determination of cholesterol, low in cost, safety is implemented, it is easy to operate, without high
Expensive detection device can accurately be completed to detect, and be suitble to be widely popularized.
Detailed description of the invention
Fig. 1 is cholesterol standard curve graph of the present invention.
Specific embodiment
Further illustrate that the present invention, following embodiment are the specific embodiment party of the present invention below by specific embodiment
Formula, but embodiments of the present invention are not limited by following embodiments.
Embodiment 1:
A kind of measuring method of cholesterol concentration, includes the following steps:
(1) pipette pipettes 10mL cholesterol-MRS solution (cholesterol theoretical concentration is 0.2mg/mL) in centrifuge tube, is centrifuged
(5000rpm, 20 DEG C, 10min)；Centrifuged supernatant is poured slowly into 250mL triangular flask, pipettes 95% second of 10mL with pipette
Pure and mild 3mL 50%KOH solution muffles triangle bottleneck in triangular flask, with preservative film, and fixes preservative film with rubber band, is placed in 60+1 DEG C, revolving speed is that water-bath 25min carries out saponification until saponification completely, obtains cholesterol crude liquid in the shaking bath of 100rpm；
(2) after being cooled to room temperature cholesterol crude liquid in a cold water bath, it is 60~90 DEG C and volume that boiling range is added in draught cupboard
It for the petroleum ether of 8mL, is then sealed with preservative film, the fixed preservative film of rubber band simultaneously stays 6 holes on film；It is put into 25+1 DEG C of perseverance
15min is shaken in tepidarium shaking table, is taken out, 8mL distilled water is added, and is continued at concussion extraction 15min in water bath with thermostatic control shaking table, is made
Be sufficiently mixed until extraction complete, obtain extract liquor；
(3) extract liquor is transferred completely into 120mL separatory funnel, and with 95% ethyl alcohol rinse triangular flask of 8mL 1~2 time, washing lotion
It is incorporated in separatory funnel, turns upside down separatory funnel 3~5 times, be allowed to be sufficiently mixed, stand 15min and be allowed to be layered；Separatory funnel
In solution layering it is obvious, i.e., boundary without it is muddy, without milkiness, liquid level is clear and stablizes；Separatory funnel stopcock is opened by lower layer
Liquid is slowly released, and the petroleum ether layer on separatory funnel upper layer is poured into 250mL high foot beaker from funnel is suitable for reading；
(4) the high foot beaker for filling petroleum ether is placed in draught cupboard, in 80+The interior evaporation petroleum ether of 1 DEG C of water-bath be (water-bath
Temperature is no more than 85 DEG C), it is evaporated to no liquid residual, the main component of the white residue of beaker bottom is solid for gallbladder at this time
Alcohol, evaporation used time are 25min, until no liquid residual in beaker；
It (5) is 85 DEG C with residue, the ultrasonic dissolution in hot bath, ultrasonic water temperature in dehydrated alcohol dissolution beaker, ultrasonic power
For 80W, ultrasound to the residue of beaker bottom is needed to be completely dissolved, reduces experimental error and obtain cholesterol solution；
(6) constant volume: cholesterol solution is then transferred in 50mL volumetric flask, and with dehydrated alcohol rinse beaker 2~3 times, washing lotion is simultaneously
Enter in volumetric flask, and be titrated to scale, obtains cholesterol lysate；
(7) it draws cholesterol standard curve: weighing 0.05g o-phthalaldehyde, dissolved with about 30mL glacial acetic acid, be transferred to 100mL
In volumetric flask, and scale is settled to glacial acetic acid；
(8) liquid-transfering gun pipettes the cholesterol lysate in 2mL step (6) in clean tube, and 4mL O-phthalic aldehyde reagent is added
(OPA reagent) stands 10min, is then slowly added to the 4mL concentrated sulfuric acid along test tube wall in a cold water bath, vortex 45s is clear to solution
Clearly, 10min is stood in cold bath；Then OD value is surveyed at spectrophotometer 550nm, and the OD value measured is substituted into cholesterol standard
Curvilinear equation calculates cholesterol concentration in solution to be measured.
Embodiment 2:
(1) pipette pipettes 15mL cholesterol-MRS solution (cholesterol theoretical concentration is 0.2mg/mL) in centrifuge tube, is centrifuged
(5000rpm, 20 DEG C, 10min)；Centrifuged supernatant is poured slowly into 250mL triangular flask, pipettes 95% second of 12mL with pipette
Pure and mild 5mL 50%KOH solution muffles triangle bottleneck in triangular flask, with preservative film, and fixes preservative film with rubber band, is placed in 60+1 DEG C, revolving speed is that water-bath 27min carries out saponification until saponification completely, obtains cholesterol crude liquid in the shaking bath of 110rpm；
(2) after being cooled to room temperature cholesterol crude liquid in a cold water bath, it is 60~90 DEG C and volume that boiling range is added in draught cupboard
It for the petroleum ether of 10mL, is then sealed with preservative film, the fixed preservative film of rubber band simultaneously stays 6 holes on film；It is put into 25+1 DEG C
18min is shaken in water bath with thermostatic control shaking table, is taken out, 8mL distilled water is added, and continues at concussion extraction 18min in water bath with thermostatic control shaking table,
It is allowed to be sufficiently mixed until extraction completion, obtains extract liquor；
(3) extract liquor is transferred completely into 120mL separatory funnel, and with 95% ethyl alcohol rinse triangular flask of 8mL 1~2 time, washing lotion
It is incorporated in separatory funnel, turns upside down separatory funnel 3~5 times, be allowed to be sufficiently mixed, stand 20min and be allowed to be layered；Separatory funnel
In solution layering it is obvious, i.e., boundary without it is muddy, without milkiness, liquid level is clear and stablizes；Separatory funnel stopcock is opened by lower layer
Liquid is slowly released, and the petroleum ether layer on separatory funnel upper layer is poured into 250mL high foot beaker from funnel is suitable for reading；
(4) the high foot beaker for filling petroleum ether is placed in draught cupboard, in 80+The interior evaporation petroleum ether of 1 DEG C of water-bath be (water-bath
Temperature is no more than 85 DEG C), it is evaporated to no liquid residual, the main component of the white residue of beaker bottom is solid for gallbladder at this time
Alcohol, evaporation used time are 15min, until no liquid residual in beaker；
It (5) is 90 DEG C with residue, the ultrasonic dissolution in hot bath, ultrasonic water temperature in dehydrated alcohol dissolution beaker, ultrasonic power
For 80W, ultrasound to the residue of beaker bottom is needed to be completely dissolved, reduces experimental error and obtain cholesterol solution；
(6) constant volume: cholesterol solution is then transferred in 50mL volumetric flask, and with dehydrated alcohol rinse beaker 2~3 times, washing lotion is simultaneously
Enter in volumetric flask, and be titrated to scale, obtains cholesterol lysate；
(7) it draws cholesterol standard curve: weighing 0.06g o-phthalaldehyde, dissolved with about 30mL glacial acetic acid, be transferred to 100mL
In volumetric flask, and scale is settled to glacial acetic acid；
(8) liquid-transfering gun pipettes the cholesterol lysate in 2mL step (6) in clean tube, and 4mL O-phthalic aldehyde reagent is added
(OPA reagent) stands 15min, is then slowly added to the 4mL concentrated sulfuric acid along test tube wall in a cold water bath, vortex 40s is clear to solution
Clearly, 20min is stood in cold bath；Then OD value is surveyed at spectrophotometer 550nm, and the OD value measured is substituted into cholesterol standard
Curvilinear equation calculates cholesterol concentration in solution to be measured.
Embodiment 3:
(1) pipette pipettes 20mL cholesterol-MRS solution (cholesterol theoretical concentration is 0.2mg/mL) in centrifuge tube, is centrifuged
(5000rpm, 20 DEG C, 10min)；Centrifuged supernatant is poured slowly into 250mL triangular flask, pipettes 95% second of 12mL with pipette
Pure and mild 5mL 50%KOH solution muffles triangle bottleneck in triangular flask, with preservative film, and fixes preservative film with rubber band, is placed in 60+1 DEG C, revolving speed is that water-bath 27min carries out saponification until saponification completely, obtains cholesterol crude liquid in the shaking bath of 110rpm；
(2) after being cooled to room temperature cholesterol crude liquid in a cold water bath, it is 60~90 DEG C and volume that boiling range is added in draught cupboard
It for the petroleum ether of 10mL, is then sealed with preservative film, the fixed preservative film of rubber band simultaneously stays 6 holes on film；It is put into 25+1 DEG C
18min is shaken in water bath with thermostatic control shaking table, is taken out, 10mL distilled water is added, and is continued at and is shaken extraction in water bath with thermostatic control shaking table
18min is allowed to be sufficiently mixed until extraction completion, obtains extract liquor；
(3) extract liquor is transferred completely into 120mL separatory funnel, and with 95% ethyl alcohol rinse triangular flask of 8mL 1~2 time, washing lotion
It is incorporated in separatory funnel, turns upside down separatory funnel 3~5 times, be allowed to be sufficiently mixed, stand 20min and be allowed to be layered；Separatory funnel
In solution layering it is obvious, i.e., boundary without it is muddy, without milkiness, liquid level is clear and stablizes；Separatory funnel stopcock is opened by lower layer
Liquid is slowly released, and the petroleum ether layer on separatory funnel upper layer is poured into 250mL high foot beaker from funnel is suitable for reading；
(4) the high foot beaker for filling petroleum ether is placed in draught cupboard, in 80+The interior evaporation petroleum ether of 1 DEG C of water-bath be (water-bath
Temperature is no more than 85 DEG C), it is evaporated to no liquid residual, the main component of the white residue of beaker bottom is solid for gallbladder at this time
Alcohol, evaporation used time are 30min, until no liquid residual in beaker；
It (5) is 100 DEG C with residue, the ultrasonic dissolution in hot bath, ultrasonic water temperature in dehydrated alcohol dissolution beaker, ultrasonic power
For 80W, ultrasound to the residue of beaker bottom is needed to be completely dissolved, reduces experimental error and obtain cholesterol solution；
(6) constant volume: cholesterol solution is then transferred in 50mL volumetric flask, and with dehydrated alcohol rinse beaker 2~3 times, washing lotion is simultaneously
Enter in volumetric flask, and be titrated to scale, obtains cholesterol lysate；
(7) it draws cholesterol standard curve: weighing 0.06g o-phthalaldehyde, dissolved with about 30mL glacial acetic acid, be transferred to 100mL
In volumetric flask, and scale is settled to glacial acetic acid；
(8) liquid-transfering gun pipettes the cholesterol lysate in 1mL step (6) in clean tube, and 2mL O-phthalic aldehyde reagent is added
(OPA reagent) stands 10min, is then slowly added to the 4mL concentrated sulfuric acid along test tube wall in a cold water bath, vortex 30s is clear to solution
Clearly, 12min is stood in cold bath；Then OD value is surveyed at spectrophotometer 550nm, and the OD value measured is substituted into cholesterol standard
Curvilinear equation calculates cholesterol concentration in solution to be measured.
Table 2 compares embodiment 1-3 experimental result
As can be seen from Table 2, due to the sampling amount OD that is different, therefore being measured with same method of cholesterol solution in experiment
Value is also different.Therefore, OD average value is substituted into curvilinear equation (y=69.09025x-0.01104, R2=0.99606) it, is converted into molten
Cholesterol concentration in liquid.The standard deviation tested three times is very close to final cholesterol concentration (0.19mg/mL) and theoretical cholesterol are dense
It is approximately equal to spend (0.2mg/mL), therefore experimental program is feasible.
Comparative example 1:
It is molten to include the following steps: that step (1) pipette pipettes 10mL cholesterol-MRS for a kind of measuring method of cholesterol concentration
Liquid, cholesterol theoretical concentration are 0.2mg/mL, other are the same as embodiment 1；
After lamination occurs in separatory funnel in step (3), 0.4mL petroleum ether organic layer is pipetted in clean tube, other
With embodiment 1；
The test tube in step (3) is placed in draught cupboard in step (4), in 80+Petroleum ether is evaporated in 1 DEG C of water-bath, is evaporated to
No liquid residual, other are the same as embodiment 1；
Test tube after being evaporated is directly added into 2mL glacial acetic acid and supplies volume, add o-phthalaldehyde OPA reagent that 4mL now matches into
Row dissolution, stands 10min, is then slowly added to the 4mL concentrated sulfuric acid along test tube wall in a cold water bath, and vortex 35 seconds clear to solution
Clearly, 10min is stood in cold bath.Then OD value is surveyed at spectrophotometer 550nm, and the OD value measured is substituted into cholesterol standard
Curvilinear equation calculates cholesterol concentration in solution to be measured；Three parallel groups are done in experiment.
Table 3 compares comparative example 1 and the cholesterol determination result of embodiment 1
Find out from upper table 3, relatively, but the standard deviation of comparative example 1 is much big for the OD average value of embodiment 1 and comparative example 1
In the standard deviation of embodiment 1, illustrate that three groups of data discrete degree of comparative example 1 are big, i.e., selected after liquid separation all petroleum ether layers into
The effect of row experiment is got well than directly pipetting the effect of 0.4mL petroleum ether layer.
Comparative example 2:
A kind of measuring method of cholesterol concentration includes the following steps: the white residue of beaker bottom in step (5) with anhydrous
Ethyl alcohol dissolution, water temperature are respectively 85 DEG C, 90 DEG C, 100 DEG C, and ultrasonic power 80W records ultrasonic time, other are the same as embodiment 1.
The lower cholesterol dissolution situation comparison of 4 different condition of table processing
From upper table 4 it can be concluded that, thermostat water bath heating make residue thoroughly dissolve the time be greater than ultrasound bath institute expense
Time, meanwhile, the standard deviation of ultrasonic processed by hot bath is heated lower than water bath with thermostatic control, therefore, uses ultrasonic heat water bath processing ratio
Compared with time-saving and efficiency.
Comparative example 3:
A kind of measuring method of cholesterol concentration includes the following steps: the white residue of beaker bottom in step (5) with anhydrous
Ethyl alcohol dissolution, water temperature are 85 DEG C, ultrasonic power 80W, respectively ultrasound 10min, 30min, 60min, 90min, and record experiment is existing
As other are the same as embodiment 1.
Influence of the different ultrasonic times of table 5 to result
From upper table 5, it can be concluded that, after ultrasonic 10min, cholesterol is not completely dissolved, so that the Lower result measured.And surpass
The experimental result that sound 30min or more is measured is consistent；Therefore, thoroughly whether dissolution influences experimental result to beaker bottom residue.
Comparative example 4:
A kind of measuring method of cholesterol concentration includes the following steps: to rapidly join along test tube wall at room temperature in step (8)
The concentrated sulfuric acid, with embodiment 1, observation experiment variation is as shown in table 6 for other.
Comparative example 5:
A kind of measuring method of cholesterol concentration includes the following steps: to be slowly added to along test tube wall at room temperature in step (8)
The concentrated sulfuric acid, with embodiment 1, observation experiment variation is as shown in table 6 for other.
Experimental phenomena under 6 different condition of table
It can be concluded that, heat release after the concentrated sulfuric acid is added from upper table 6, test tube wall temperature is excessively high to be unfavorable for being vortexed manually uniformly, cold bath
It can make test tube fast cooling, while also be avoided that the concentrated sulfuric acid is carbonized, safe ready.
Comparative example 6:
A kind of measuring method of cholesterol concentration includes the following steps: to replace embodiment 1 using 30%KOH solution in step (1)
The 50%KOH solution, other are the same as embodiment 1.
Influence of the KOH solution of 7 various concentration of table to final result
From upper table 7, it can be concluded that, the OD average value that 30%KOH solution measures is slightly higher, but calculated cholesterol concentration does not have
It changes, illustrates that 30%KOH solution is suitble to this experiment, in line with excessive principle, the concentration of KOH can be suitably turned up, and avoid reality
It tests and causes experimental error because of the amount of KOH deficiency.
Comparative example 7:
A kind of measuring method of cholesterol concentration includes the following steps: to replace described in embodiment 1 in step (1) using water-bath
Water bath with thermostatic control shaking table be saponified, other are the same as embodiment 1.
Influence of the different saponification modes of table 8 to final result
From upper table 8, it can be concluded that, the final result of the two is the same, but the standard deviation after being saponified using water-bath
(1.36%) 0.50% be higher than in embodiment 1, the data after explanation is saponified with water bath with thermostatic control shaking table are more stable.
Claims (9)
1. a kind of measuring method of cholesterol concentration, which comprises the steps of:
(1) cholesterol solution centrifugation to be measured is pipetted, 95% ethyl alcohol and 50% KOH solution are added after centrifugation and is saponified in hot bath directly
Completely to saponification, cholesterol crude liquid is obtained；
(2) after being cooled to room temperature cholesterol crude liquid, petroleum ether is added, concussion extraction is until extracted in water bath with thermostatic control shaking table
At obtaining extract liquor；
(3) extract liquor is transferred completely into separatory funnel, and with 95% ethyl alcohol rinse, stands liquid separation, collects petroleum ether layer in burning
In cup；
(4) organic reagent is evaporated in water-bath in draught cupboard, until no liquid residual in beaker；
(5) cholesterol solution is obtained with residue, the ultrasonic dissolution in hot bath in dehydrated alcohol dissolution beaker；
(6) constant volume: cholesterol solution is then transferred in 50mL volumetric flask, and with dehydrated alcohol rinse beaker 2~3 times, washing lotion is simultaneously
Enter in volumetric flask, and constant volume obtains cholesterol lysate to scale；
(7) cholesterol standard curve is drawn；
(8) o-phthalaldehyde (OPA) reagent is added in clean tube in the cholesterol lysate in removing step (6), stands,
Then it is slowly added to the concentrated sulfuric acid along test tube wall in a cold water bath, is vortexed to solution and clarifies, stood in cold bath；Then in light splitting
OD value is surveyed at photometer 550nm, and the OD value measured is substituted into cholesterol standard curvilinear equation, it is dense to calculate cholesterol in solution to be measured
Degree.
2. the measuring method of cholesterol concentration according to claim 1, which is characterized in that described to use liquid relief in step (1)
Pipe pipettes 10~20mL cholesterol solution to be measured in centrifuge tube, parameter of noncentricity 5000rpm, and 20 DEG C, 10min；The saponification
Process conditions are as follows: pipette appropriate 10~15mL, 95% ethyl alcohol and 3~7mL, 50% KOH solution in triangular flask with pipette,
Triangle bottle sealing is placed on 60+25~30min of water-bath is saponified in 1 DEG C of shaking bath；The revolving speed of the shaking bath
For 100~120rpm.
3. the measuring method of cholesterol concentration according to claim 1, which is characterized in that in step (2), the addition stone
The process conditions of oily ether are as follows: the petroleum ether that boiling range is 60~90 DEG C and volume is 8~12mL is added in draught cupboard, then with guarantor
Fresh film sealing, the fixed preservative film of rubber band simultaneously stay 5~8 holes on film；The work of extraction is shaken in be set forth in water bath with thermostatic control shaking table
Skill condition are as follows: be put into 25+15~20min is shaken in 1 DEG C of water bath with thermostatic control shaking table, is taken out, 8~12mL distilled water is added, and is continued
15~20min of concussion extraction, is allowed to be sufficiently mixed in water bath with thermostatic control shaking table.
4. the measuring method of cholesterol concentration according to claim 1, which is characterized in that described to use 95% in step (3)
The process conditions of ethyl alcohol rinse are as follows: with 5~10mL95% ethyl alcohol rinse triangular flask 1~2 time, washing lotion is incorporated in separatory funnel, up and down
It reverse separatory funnel 3~5 times, is allowed to be sufficiently mixed；The process conditions for collecting petroleum ether layer are as follows: open separatory funnel stopcock
Lower liquid is slowly released, the petroleum ether layer on separatory funnel upper layer is poured into 250mL high foot beaker from funnel is suitable for reading.
5. the measuring method of cholesterol concentration according to claim 1, which is characterized in that in step (4), the draught cupboard
The process conditions of interior water-bath evaporation organic reagent are as follows: the high foot beaker for filling petroleum ether is placed in draught cupboard, in 80+1 DEG C of water
Petroleum ether is evaporated in bath, is evaporated to no liquid residual, the main component of the white residue of beaker bottom is cholesterol at this time,
The evaporation used time is 15~30min.
6. the measuring method of cholesterol concentration according to claim 1, which is characterized in that described in hot water in step (5)
The process conditions of ultrasonic dissolution in bath are as follows: ultrasonic water temperature is 80~100 DEG C, ultrasonic power 80W.
7. the measuring method of cholesterol concentration according to claim 1, which is characterized in that described in step (5) and (6)
Dehydrated alcohol is that analysis is pure.
8. the measuring method of cholesterol concentration according to claim 1, which is characterized in that in step (8), the concentrated sulfuric acid
It is pure to analyze.
9. the measuring method of cholesterol concentration according to claim 1, which is characterized in that in step (8), the vortex is extremely
Solution is clarified, the process conditions stood in cold bath are as follows: after the concentrated sulfuric acid is added, be vortexed 30~45s immediately, until mixed solution is clarified
It is transparent, it then continues to be put into cold water and radiate, stand 10~30min.
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| CN112067506A (en) * | 2020-09-22 | 2020-12-11 | 北京鑫开元医药科技有限公司 | Method for measuring viscosity of bismuth potassium citrate-containing preparation |
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