CN106226438B - A kind of method of inspection of brain protein hydrolysate oral liquid - Google Patents
A kind of method of inspection of brain protein hydrolysate oral liquid Download PDFInfo
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- CN106226438B CN106226438B CN201610834392.0A CN201610834392A CN106226438B CN 106226438 B CN106226438 B CN 106226438B CN 201610834392 A CN201610834392 A CN 201610834392A CN 106226438 B CN106226438 B CN 106226438B
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Abstract
The present invention discloses a kind of method of inspection of brain protein hydrolysate oral liquid.Including step：A, prepare derivative reagent：It draws 1.0ml anhydrous acetonitriles to be simultaneously added in AQC reagents, then shake 10 seconds, place into 55 DEG C of baking ovens and be heated to powder and be completely dissolved, obtain derivative reagent；B, first two 250 μ l internal lining pipes are respectively put into two 2ml liquid phase sample bottles, amino acid reference substance solution is drawn with liquid-transfering gun and each 10 μ l of brain protein hydrolysate oral liquid are put into liner bottom of the tube, it is separately added into 70 μ l borate buffer solutions again, it is placed on vortex 1min in vortex oscillator, it is separately added into 20 μ l derivative reagents again, is placed in vortex oscillator after vortex 5min, is placed after ten minutes in 55 DEG C of baking ovens, taking-up is cooled to room temperature, draws 10 μ l injection liquid chromatographs respectively and is measured.The method of inspection provided by the present invention can examine the type and content of amino acid, while examine precision high, reproducible.
Description
Technical field
The present invention relates to detection field more particularly to a kind of methods of inspection of brain protein hydrolysate oral liquid.
Background technology
" Cerebrolysin Vial " is the extract through purifying after healthy pig brain tissue's enzymolysis, mainly contains a variety of amino acid, god
It is regulation and control god through the bioactive ingredients such as polypeptide gene, nucleotide, neurotransmitter, neurotrophic factor and cephalin, lecithin
Through the substance for developing, determining neural cellular differentiation and axon direction of extension." Cerebrolysin Vial " can act in many ways
Nervous centralis adjusts and improves the metabolism of neuron, promotes Synaptic formation, the differentiation of Induction of neuronal, and further patron saint
Through cell, the damage of various ischemics, hypoxemia and neurotoxin is protected it from.Purified " Cerebrolysin Vial " easily passes through blood-
Cerebrospinal fluid barrier promotes the synthesis of intracerebral protein, influences respiratory chain, has anti-hypoxemia ability, improves intracerebral energetic supersession, swashs
Adenyl cyclase living and other Hormone systems of catalysis, provide neurotransmitter peptide hormone and coenzyme precursors, have and promote nerve
The class nerve growth factor subfunction of fiber growth improves diseased region function of brain cell, is a kind of to treat cranial nerve function obstacle
Drug.
" Cerebrolysin Vial " main component is to contain free amine group through what proteasome degradation generated again after animal brain degreasing
The mixture of acid and molecular weight in 10000 small-molecular peptides below etc..Study carefully its pharmacology, " Cerebrolysin Vial " is small due to its molecule
Blood-brain barrier can be penetrated, brain cell DNA synthesis can be promoted, repair and regenerate, the synthesis of Stimulation of The Brain albumen influences cranial nerve cell
Breathing coupling；Activated adenyl cyclase is simultaneously catalyzed other Hormone systems；Participate in oxidative deamination and the glucose generation of 2-ketoacid
It thanks, improves brain oxygen and glucose availability, supply conduct neurotransmitter itself or neurotransmitter needed for brain cell reparative regeneration
With the various amino acid of the precursor of alkamines, adjust brain neurological motion, promote transmitter in central nervous system γ-aminobutyric acid, dopamine and
The biosynthesis such as serotonin；It is combined into nontoxic glutamine with free ammonia, reduces blood ammonia；Improve adrenal cortex machine simultaneously
Can, promote general metabolism.It is clinically used for treatment atelencephalia, encephalatrophy, neurasthenia, combined external head injuries, nervous centralis
System infections, meningitis, senile dementia, depression, mental disease, a variety of diseases such as cerebrovascular metabolism disorder, while can be effective
Enhance learning memory and physical stress ability, significant in efficacy and safe without toxic side effect.
It can be seen that the small-molecular peptides and free amine group acid substance that contain in drug are the main components for generating drug effect.For
The effect of raising brain protein hydrolysate oral liquid and quality, need to be measured the content of its free amino acid, to carry
The quality control standard of high drug ensures pharmaceutical effectiveness.But current method of inspection accuracy is poor, and repeatability is bad.
Therefore, the existing technology needs to be improved and developed.
Invention content
In view of above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a kind of inspections of brain protein hydrolysate oral liquid
Proved recipe method, it is intended to solve the problems, such as that existing brain protein hydrolysate oral liquid method of inspection accuracy and repeatability are bad.
Technical scheme is as follows：
A kind of method of inspection of brain protein hydrolysate oral liquid, wherein including step：
A, prepare derivative reagent：It draws 1.0ml anhydrous acetonitriles and is added in AQC reagents, then shake 10 seconds, place into 55
DEG C baking oven is heated to powder and is completely dissolved, and obtains derivative reagent；
B, first two 250 μ l internal lining pipes are respectively put into two 2ml liquid phase sample bottles, amino acid pair is drawn with liquid-transfering gun
It is put into liner bottom of the tube according to each 10 μ l of product solution and brain protein hydrolysate oral liquid, then is separately added into 70 μ l borate buffer solutions,
It is placed on vortex 1min in vortex oscillator, then is separately added into 20 μ l derivative reagents, is placed in vortex oscillator after vortex 5min, in
55 DEG C of baking ovens are placed after ten minutes, and taking-up is cooled to room temperature, are drawn 10 μ l injection liquid chromatographs respectively and are measured；
It is 1.5ml per minute that the wherein described liquid chromatograph, which uses C18 columns, flow velocity, and mobile phase includes mobile phase A, flowing
Phase B and mobile phase C；
The mobile phase A preparation method is as follows：Anhydrous sodium acetate 11.48g is dissolved in water and is diluted to 1000ml, then
PH to 4.45 is adjusted with phosphoric acid,diluted；
The Mobile phase B preparation method is as follows：Volume ratio is 60：40 acetonitrile and water is formulated；
The mobile phase C preparation methods are as follows：Anhydrous sodium acetate 11.48g is dissolved in water and is diluted to 1000ml, use is dilute
Phosphorus acid for adjusting pH is to 4.95.
The method of inspection of the brain protein hydrolysate oral liquid, wherein before the step A, further include：
Pre-production standard curve：250 μ l internal lining pipes are put into 2ml liquid phase sample bottles, amino acid is drawn with liquid-transfering gun
10 μ l of reference substance solution are put into liner bottom of the tube, add 70 μ l borate buffer solutions, are placed on vortex 1 in vortex oscillator and divide
Clock adds 20 μ l derivative reagents, is placed in vortex oscillator after vortex 5min, places after ten minutes, takes out in 55 DEG C of baking ovens
It is cooled to room temperature, draws 1 μ l, 2 μ l, 5 μ l, 10 μ l, 15 μ l, 20 μ l injection liquid chromatographs respectively and be measured, obtain standard song
Line.
The method of inspection of the brain protein hydrolysate oral liquid, wherein further include after the step B：
6 250 μ l internal lining pipes are respectively put into 6 2ml liquid phase sample bottles, draw 6 amino acid respectively with liquid-transfering gun
10 μ l of reference substance solution are put into liner bottom of the tube, then are separately added into 70 μ l borate buffer solutions, are placed on vortex 1 in vortex oscillator
Minute, then 20 μ l derivative reagents are separately added into, it is placed in vortex oscillator after vortex 5min, is placed 10 minutes in 55 DEG C of baking ovens
Afterwards, it takes out and is cooled to room temperature, draw 10 μ l injection liquid chromatographs respectively and be measured, detection method is judged according to measurement result
Precision.
The method of inspection of the brain protein hydrolysate oral liquid, wherein further include after the step B：
6 250 μ l internal lining pipes are respectively put into 6 2ml liquid phase sample bottles, draw 6 test samples respectively with liquid-transfering gun
10 μ l of solution are put into liner bottom of the tube, then are separately added into 70 μ l borate buffer solutions, are placed on vortex 1 minute in vortex oscillator,
It is accurate respectively again that 20 μ l derivative reagents are added, it is placed in vortex oscillator after vortex 5min, is placed after ten minutes in 55 DEG C of baking ovens,
Taking-up is cooled to room temperature, draws 10 μ l injection liquid chromatographs respectively and is measured, the weight of detection method is judged according to measurement result
Renaturation.
Advantageous effect：The method of inspection provided by the present invention can not only effectively examine ammonia in brain protein hydrolysate oral liquid
The type and content of base acid, while examining precision high, reproducible, be conducive to fast and accurately examine Cerebrolysin Vial
Oral solution.
Specific implementation mode
The present invention provides a kind of method of inspection of brain protein hydrolysate oral liquid, to make the purpose of the present invention, technical solution
And effect is clearer, clear, the present invention is described in more detail below.It should be appreciated that specific implementation described herein
Example is only used to explain the present invention, is not intended to limit the present invention.
A kind of method of inspection of brain protein hydrolysate oral liquid provided by the present invention comprising step：
S1, prepare derivative reagent：It draws 1.0ml anhydrous acetonitriles and is added in AQC reagents, then shake 10 seconds, place into
55 DEG C of baking ovens are heated to powder and are completely dissolved, and obtain derivative reagent；
S2, first two 250 μ l internal lining pipes are respectively put into two 2ml liquid phase sample bottles, amino acid is drawn with liquid-transfering gun
Reference substance solution and each 10 μ l of brain protein hydrolysate oral liquid are put into liner bottom of the tube, then are separately added into 70 μ l boric acid salt buffers
Liquid is placed on vortex 1min in vortex oscillator, then is separately added into 20 μ l derivative reagents, is placed on vortex 5min in vortex oscillator
Afterwards, it is placed after ten minutes in 55 DEG C of baking ovens, taking-up is cooled to room temperature, draws 10 μ l injection liquid chromatographs respectively and is measured；
It is 1.5ml per minute that the wherein described liquid chromatograph, which uses C18 columns, flow velocity, and mobile phase includes mobile phase A, flowing
Phase B and mobile phase C；
The mobile phase A preparation method is as follows：Anhydrous sodium acetate 11.48g is dissolved in water and is diluted to 1000ml, then
PH to 4.45 is adjusted with phosphoric acid,diluted；
The Mobile phase B preparation method is as follows：Volume ratio is 60：40 acetonitrile and water is formulated；
The mobile phase C preparation methods are as follows：Anhydrous sodium acetate 11.48g is dissolved in water and is diluted to 1000ml, use is dilute
Phosphorus acid for adjusting pH is to 4.95.
Brain protein hydrolysate oral liquid can be differentiated as follows：Brain protein hydrolysate oral liquid 10ml is taken to add water
20ml dissolves, as test solution.
(1) test solution 5ml is taken, adds ninhydrin solution few drops, is heated, solution should show bluish violet.
(2) test solution 2ml is taken, adds sodium hydroxide solution (6 → 10) 2ml, adds alkaline copper citrate test solution 0.2ml,
Mixing is placed at room temperature for 15 minutes, and solution should show bluish violet or purple.
The pH value of brain protein hydrolysate oral liquid should be 6.0~8.0.
Liquid chromatograph in the present invention is high performance liquid chromatograph.
It is specifically filler with octadecylsilane chemically bonded silica【AcclaimC18,120A,(4.6*150nm*5μm)】；
Flow velocity is 1.5ml per minute；Column temperature is 37 DEG C；Detection wavelength is 248nm.Gradient such as the following table 1：
1 gradient of table
The preparation of reference substance solution：Precision weighs glutamic acid, valine, arginine, glycine, threonine, serine, dried meat
Propylhomoserin, tyrosine, methionine, leucine, phenylalanine, isoleucine, cystine, lysine, histidine, L-aminobutanedioic acid,
Alanine, each 10mg of tryptophan product add water or 0.1mol/l hydrochloric acid solutions to dissolve and be diluted to 10ml, then inhale respectively respectively
It takes 0.5ml to set in a volumetric flask, is diluted with water to 10ml, the solution for respectively containing 50 μ g in every 1ml is made.
In one specific example, the sampling amount of reference substance：(g)
A concentration of 1.0686mg/ml of reference substance solution.
In step S1, derivative reagent can prepare as follows：Waters AccQ Tag Fluor Reagent Kit
(kit) kit has 5 group reagents, and every group includes 3 reagent bottles, and 1# bottles are pH8.8 borate buffer solutions, and 2A bottles are AQC
Powder, 2B bottles are anhydrous acetonitrile.Before use, anhydrous acetonitrile in 2B bottles of 1.0ml is inhaled (in advance to shake bottle in 2A bottles, make powder
It is completely disposed at bottom of bottle), bottle cap is tightened, is shaken 10 seconds, 55 DEG C of baking ovens is put into and is heated to powder and be completely dissolved and (must not exceed 10 minutes).
Then it is measured according to the method for step S2.
First, it may be determined that the type of brain protein hydrolysate oral liquid amino acid, it is specific as follows：
Take 18 kinds of amino acid reference substances, according to reference substance solution preparation method with method operate, then take test solution according to
Assay method carries out derivative measurement, to the identical position of each corresponding amino acid reference substance retention time in reference substance collection of illustrative plates, altogether
There are 17 peaks.It follows that containing 17 kinds of amino acid, respectively valine, arginine, alanine, sweet ammonia in test sample altogether
Acid, serine, proline, tyrosine, methionine, leucine, phenylalanine, isoleucine, cystine, relies ammonia at threonine
Acid, L-aminobutanedioic acid, histidine, glutamic acid.
In addition, also carrying out specificity judgement to the method for inspection of the present invention, negative controls can be first prepared：Use liquid-transfering gun
10 μ l of extract water are put into liner bottom of the tube, add 70 μ l borate buffers, are placed on vortex 1 minute in vortex oscillator, then essence
20 μ l derivative reagents of close addition after covering tightly bottle cap vortex 5min rapidly, are placed after ten minutes, taking-up is cooled to room in 55 DEG C of baking ovens
Temperature, as negative controls.
Draw 10 μ l injection liquid chromatograph of negative controls to be measured, be in addition blank with mobile phase, and with compare
Product solution is compared.From testing result it is found that negative controls on assay without influence, the specificity of method is good.
Further, before the step S1, further include：
Pre-production standard curve：250 μ l internal lining pipes are put into 2ml liquid phase sample bottles, amino acid is drawn with liquid-transfering gun
10 μ l of reference substance solution are put into liner bottom of the tube, add 70 μ l borate buffer solutions, are placed on vortex 1 in vortex oscillator and divide
Clock adds 20 μ l derivative reagents, is placed in vortex oscillator after vortex 5min, places after ten minutes, takes out in 55 DEG C of baking ovens
It is cooled to room temperature, draws 1 μ l, 2 μ l, 5 μ l, 10 μ l, 15 μ l, 20 μ l injection liquid chromatographs respectively and be measured, obtain standard song
Line, as a result such as the following table 2.
2 standard curve determination result of table
Using peak area as ordinate, standard curve is drawn with a concentration of abscissa of reference substance solution, obtains standard curve Y=
204.39X+2.2435, r=0.9995.Illustrate sample concentration between 0.10686 μ of μ g~2.13720 g, with peak area in good
Good linear relationship.
Further, further include after the step S2：
6 250 μ l internal lining pipes are respectively put into 6 2ml liquid phase sample bottles, draw 6 amino acid respectively with liquid-transfering gun
10 μ l of reference substance solution are put into liner bottom of the tube, then are separately added into 70 μ l borate buffer solutions, are placed on vortex 1 in vortex oscillator
Minute, then 20 μ l derivative reagents are separately added into, it is placed in vortex oscillator after vortex 5min, is placed 10 minutes in 55 DEG C of baking ovens
Afterwards, it takes out and is cooled to room temperature, draw 10 μ l injection liquid chromatographs respectively and be measured, detection method is judged according to measurement result
Precision, peak area the results are shown in Table 3.
3 Precision test result of table
As can be seen from Table 3, the precision of the method for inspection of the present invention is good.
Further, further include after the step S2：
6 250 μ l internal lining pipes are respectively put into 6 2ml liquid phase sample bottles, draw 6 test samples respectively with liquid-transfering gun
10 μ l of solution are put into liner bottom of the tube, then are separately added into 70 μ l borate buffer solutions, are placed on vortex 1 minute in vortex oscillator,
It is accurate respectively again that 20 μ l derivative reagents are added, it is placed in vortex oscillator after vortex 5min, is placed after ten minutes in 55 DEG C of baking ovens,
Taking-up is cooled to room temperature, draws 10 μ l injection liquid chromatographs respectively and is measured, the weight of detection method is judged according to measurement result
Renaturation.Peak area the results are shown in Table 4.
4 repetitive test result of table
As can be seen from Table 4, the repeatability of the method for inspection of the present invention is good.
Further, further include after the step S2：
6 250 μ l glass inner-lining pipes are respectively put into 6 2ml liquid phase sample bottles, have drawn 6 parts respectively with liquid-transfering gun
Know that the 5 μ l of test solution of free aminoacid content (1.055mg/ml) are put into liner bottom of the tube, then accurate be added compares respectively
5 μ l of product solution (1.0686mg/ml).It is separately added into 70 μ l borate buffers again, is placed on vortex 1 minute in vortex oscillator, then
It is accurate respectively that 20 μ l derivative reagents are added, after covering tightly bottle cap vortex 5min rapidly, places after ten minutes, take out cold in 55 DEG C of baking ovens
To room temperature, accurate 10 μ l injection liquid chromatographs of drawing are measured respectively, calculate the rate of recovery.It the results are shown in Table 5.
5 sample recovery rate test result of table
As can be seen from Table 5, the rate of recovery is in 95% or more, RSD% within 3%, the method for inspection of the present invention it is accurate
Property is good.
Ten batches of test samples (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) are finally taken, are divided in this way
Not Ce Ding free amino acid content, the results are shown in Table 6.
6 test sample free aminoacid content result of table
| Serial number | Lot number | Content (mg/ml) | Serial number | Lot number | Content (mg/ml) |
| 1 | (1) | 0.66 | 6 | (6) | 0.96 |
| 2 | (2) | 0.84 | 7 | (7) | 0.94 |
| 3 | (3) | 0.84 | 8 | (8) | 0.96 |
| 4 | (4) | 0.86 | 9 | (9) | 0.90 |
| 5 | (5) | 1.01 | 10 | (10) | 1.04 |
As can be seen from Table 6, the free aminoacid content of ten batches of samples is more than 6.0mg/ branch, so by free amine group
Acid content measurement limit is set to every must not be less than 6.0mg containing free amino acid.
It should be understood that the application of the present invention is not limited to the above for those of ordinary skills can
With improvement or transformation based on the above description, all these modifications and variations should all belong to the guarantor of appended claims of the present invention
Protect range.
Claims (4)
1. a kind of method of inspection of brain protein hydrolysate oral liquid, which is characterized in that including step：
A, prepare derivative reagent：It draws 1.0ml anhydrous acetonitriles and is added in AQC reagents, then shake 10 seconds, place into 55 DEG C
Baking oven is heated to powder and is completely dissolved, and obtains derivative reagent；
B, first two 250 μ l internal lining pipes are respectively put into two 2ml liquid phase sample bottles, amino acid reference substance is drawn with liquid-transfering gun
Solution and each 10 μ l of brain protein hydrolysate oral liquid are put into liner bottom of the tube, then are separately added into 70 μ l borate buffer solutions, are placed on
Vortex 1min in vortex oscillator, then 20 μ l derivative reagents are separately added into, it is placed in vortex oscillator after vortex 5min, in 55 DEG C
Baking oven is placed after ten minutes, and taking-up is cooled to room temperature, is drawn 10 μ l injection liquid chromatographs respectively and is measured；
The wherein described liquid chromatograph use C18 columns, flow velocity be 1.5ml per minute, mobile phase include mobile phase A, Mobile phase B and
Mobile phase C；
The mobile phase A preparation method is as follows：Anhydrous sodium acetate 11.48g is dissolved in water and is diluted to 1000ml, then with dilute
Phosphorus acid for adjusting pH is to 4.45；
The Mobile phase B preparation method is as follows：Volume ratio is 60：40 acetonitrile and water is formulated；
The mobile phase C preparation methods are as follows：Anhydrous sodium acetate 11.48g is dissolved in water and is diluted to 1000ml, uses phosphoric acid,diluted
Adjust pH to 4.95；
When liquid chromatograph is measured sample, sample is eluted by following proportion of mobile phase, 0-3.3min, is flowed
Dynamic matched uses 95% mobile phase A and 5% Mobile phase B, and 8.6min, proportion of mobile phase is using 91.5% mobile phase A and 8.5% stream
Dynamic phase B, 14min, proportion of mobile phase is using 12% Mobile phase B and 88% mobile phase C, and 17min, proportion of mobile phase is using 22%
Mobile phase B and 78% mobile phase C, 28min, proportion of mobile phase is using 35.5% Mobile phase B and 64.5% mobile phase C, 28.5-
30min, proportion of mobile phase use 100% Mobile phase B, and 30.1-32min, proportion of mobile phase is using 95% mobile phase A and 5% flowing
Phase B.
2. the method for inspection of brain protein hydrolysate oral liquid according to claim 1, which is characterized in that the step A it
Before, further include：
Pre-production standard curve：250 μ l internal lining pipes are put into 2ml liquid phase sample bottles, amino acid control is drawn with liquid-transfering gun
10 μ l of product solution are put into liner bottom of the tube, add 70 μ l borate buffer solutions, are placed on vortex 1 minute in vortex oscillator, then
20 μ l derivative reagents are added, are placed in vortex oscillator after vortex 5min, are placed after ten minutes in 55 DEG C of baking ovens, taking-up is cooled to room
Temperature is drawn 1 μ l, 2 μ l, 5 μ l, 10 μ l, 15 μ l, 20 μ l injection liquid chromatographs and is measured, obtains standard curve respectively.
3. the method for inspection of brain protein hydrolysate oral liquid according to claim 1, which is characterized in that the step B it
After further include：
6 250 μ l internal lining pipes are respectively put into 6 2ml liquid phase sample bottles, draw 6 amino acid controls respectively with liquid-transfering gun
10 μ l of product solution are put into liner bottom of the tube, then are separately added into 70 μ l borate buffer solutions, are placed on vortex 1 in vortex oscillator and divide
Clock, then 20 μ l derivative reagents are separately added into, it is placed in vortex oscillator after vortex 5min, is placed after ten minutes in 55 DEG C of baking ovens,
Taking-up is cooled to room temperature, draws 10 μ l injection liquid chromatographs respectively and is measured, the essence of detection method is judged according to measurement result
Density.
4. the method for inspection of brain protein hydrolysate oral liquid according to claim 1, which is characterized in that the step B it
After further include：
6 250 μ l internal lining pipes are respectively put into 6 2ml liquid phase sample bottles, draw 6 test solutions respectively with liquid-transfering gun
10 μ l are put into liner bottom of the tube, then are separately added into 70 μ l borate buffer solutions, are placed on vortex 1 minute in vortex oscillator, then divide
It is inaccurate that 20 μ l derivative reagents are added, it is placed in vortex oscillator after vortex 5min, places after ten minutes, take out in 55 DEG C of baking ovens
It is cooled to room temperature, draws 10 μ l injection liquid chromatographs respectively and be measured, the repeatability of detection method is judged according to measurement result.
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| CN101204576A (en) * | 2007-12-12 | 2008-06-25 | 新乡市奇利生物技术工程有限公司 | Process for preparing cerebroprotein hydrolysate NaCl injection |
| CN102305831A (en) * | 2011-06-03 | 2012-01-04 | 上海实业联合集团长城药业有限公司 | Method for detecting cerebroprotein hydrolysate tablets |
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