CN108663448A - Detection method in relation to substance in a kind of Amino Acid Compound Injection - Google Patents
Detection method in relation to substance in a kind of Amino Acid Compound Injection Download PDFInfo
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- CN108663448A CN108663448A CN201810457668.7A CN201810457668A CN108663448A CN 108663448 A CN108663448 A CN 108663448A CN 201810457668 A CN201810457668 A CN 201810457668A CN 108663448 A CN108663448 A CN 108663448A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The present invention provides the detection method in relation to substance in a kind of Amino Acid Compound Injection, this method is using 5 hydroxytryptophans and L kynurenins as pointer impurity, the various amino acid contained in amino acid injection and the separation in relation to substance and baseline stability are optimized by trifluoroacetic acid hyptafluorobutyric acid Ion-pair chromalography method, detection sensitivity is high, accuracy and favorable reproducibility disclosure satisfy that the requirement of related substance control in Amino Acid Compound Injection.
Description
Technical field
The present invention relates to related in the detection method in relation to substance, especially Amino Acid Compound Injection in a kind of injection
The detection method of substance.
Background technology
Amino acid is the basic unit and body synthetic antibody, hormone, enzyme and its hetero-organization for constituting human body protein
Raw material.Amino Acid Compound Injection, which is generally used for the amino acid such as protein insufficiency of intake, malabsorption, cannot meet organism metabolism
The patient needed also is used to improve the nutrition condition of patients after surgery.However, the uncharged amino acid injection of pharmacopoeia of each country
The related substance detecting method of liquid.Although there is pharmacopeia to record the detection method in relation to substance in amino acid starting material, these are related
Substance is remaining process contaminants, mostly Endogenous Amino Acids during amino acids production, by other ammonia in compound preparation prescription
Base acid starting material is covered, to cannot be satisfied the requirement of related substance detection in Amino Acid Compound Injection.
Invention content
The present invention provides the detection method in relation to substance in a kind of Amino Acid Compound Injection, this method determines compound
Pointer impurity in amino acid injection, detection sensitivity is high, accuracy and favorable reproducibility, disclosure satisfy that amino acid is noted
Penetrate the requirement of related substance control in liquid.
The present invention relates to the detection method in relation to substance in a kind of Amino Acid Compound Injection, this method includes following step
Suddenly：
(1) preparation of test solution and reference substance solution：
Amino Acid Compound Injection is taken to be directly used as test solution；
Prepare the 5HTP reference substance solution of at least three various concentration and the L- dogs urine of at least three various concentration
Propylhomoserin reference substance solution；
(2) it takes the 5HTP reference substance solution of various concentration to inject high performance liquid chromatograph, records 280nm wavelength
The chromatogram of the 5HTP reference substance solution of lower various concentration, with a concentration of abscissa of 5HTP, with this
Peak area corresponding to concentration is ordinate, makes standard curve or regression equation；The L- kynurenins of various concentration are taken to compare
Product solution injects high performance liquid chromatograph, records the chromatography of the L- kynurenin reference substance solutions of various concentration under 365nm wavelength
Figure, using the peak area corresponding to the concentration as ordinate, is made standard curve or returned with a concentration of abscissa of L- kynurenins
Return equation；
(3) take test solution inject high performance liquid chromatograph, record 280nm wavelength under and 365nm wavelength under test sample
The chromatogram of solution, by calibration curve method, with the peak area root of 5HTP in test solution 280nm wavelength chromatograms
According to the amount of 5HTP in standard curve or regression equation calculation test solution, Amino Acid Compound Injection must not exceed
The 0.2% of middle tryptophan labelled amount, with the peak area of L- kynurenins in test solution 365nm wavelength chromatograms according to standard
The amount of L- kynurenins, must not exceed tryptophan in Amino Acid Compound Injection in curve or regression equation calculation test solution
The 0.2% of labelled amount；
Wherein, contain tryptophan in the Amino Acid Compound Injection.
The present inventor has found that tryptophan is amino acid most unstable in 20 kinds of amino acid by numerous studies,
For the Amino Acid Compound Injection containing tryptophan, 5HTP, L- kynurenins are earliest appearance, generate speed most
Impurity fast and that amount is most, to which 5HTP and L- kynurenins are determined as the pointer in Amino Acid Compound Injection
Property impurity.
It will be understood by those skilled in the art that the various Amino Acid Compound Injections containing tryptophan can all use the present invention's
Method detects related substance therein, including but not limited to：Amino acid (15) double peptides (2) injection, amino acid note
Penetrate liquid (18AA) (specification includes 5% and 12%), Amino Acid Compound Injection (18AA-I), Amino Acid Compound Injection (18AA-
II) (specification includes 5%, 8.5%, 11.4%), Amino Acid Compound Injection (18AA-III), Amino Acid Compound Injection
(18AA-V), Amino Acid Compound Injection (18AA- VII), Amino Acid Compound Injection (20AA), Amino Acid Compound Injection
(15AA), Amino Acid Compound Injection (14AA), Amino Acid Compound Injection (17AA) etc., it is preferable that amino acid (15)
Double peptide (2) injections.
In one preferred embodiment, in step (1), it is molten further to prepare the control containing test solution
Liquid；In step (2), contrast solution is further taken to inject high performance liquid chromatograph, records the color of contrast solution under 280nm wavelength
Spectrogram；It is single if detecting other chromatographic peaks in test solution 280nm wavelength chromatograms by Self-control method in step (3)
A peak area is by the way that compared with tryptophan peak area in contrast solution, content must not exceed tryptophan in Amino Acid Compound Injection
The 0.5% of labelled amount, total peak area is by the way that compared with tryptophan peak area in contrast solution, content must not exceed amino acid
The 5% of tryptophan labelled amount in injection.
It will be understood by those skilled in the art that various solvents commonly used in the art can be used to prepare 5HTP pair
According to product solution and L- kynurenin reference substance solutions.It is preferred that the solvent of reference substance solution is diluted hydrochloric acid aqueous solution, more preferable concentration
For the aqueous hydrochloric acid solution of 0.1mol/L.
It will be understood by those skilled in the art that various solvents commonly used in the art can be used to prepare pair of test solution
According to solution.It is preferred that the solvent of contrast solution is diluted hydrochloric acid aqueous solution, the aqueous hydrochloric acid solution of more preferable a concentration of 0.1mol/L.
Preferably, 3-7 are prepared, further preferred 3-5, the 5HTP of more preferable 3 various concentrations compares
Product solution.Those skilled in the art have in multiple reference substance solutions that ability is chosen for making standard curve or regression equation
The concentration of 5HTP.Preferably, Cmin should be less than tryptophan labelled amount in Amino Acid Compound Injection
0.1%, maximum concentration should be higher than that 0.4% of tryptophan labelled amount in Amino Acid Compound Injection.For example, for amino acid
(15) double peptide (2) injections, wherein tryptophan labelled amount are 0.95g/500ml, are used to make standard curve or recurrence side at this time
In 3 reference substance solutions of journey the concentration range of 5HTP can be respectively 0.2-0.8 μ g/ml, 1.6-2.4 μ g/ml,
8-10 μ g/ml, for example, the concentration of 5HTP can be about 0.5 μ g/ml, about 2.0 μ g/ respectively in 3 reference substance solutions
Ml, about 9.0 μ g/ml.The method of the present invention, the range of linearity are more than the 0.1-0.4% of tryptophan labelled amount.
Preferably, 3-7 are prepared, further preferred 3-5 is a, the L- kynurenin reference substances of more preferable 3 various concentrations
Solution.Those skilled in the art have L- in multiple reference substance solutions of the ability selection for making standard curve or regression equation
The concentration of kynurenin.Preferably, Cmin should be less than 0.1% of tryptophan labelled amount in Amino Acid Compound Injection, most
Big concentration should be higher than that 0.4% of tryptophan labelled amount in Amino Acid Compound Injection.For example, for amino acid (15) double peptides
(2) injection, wherein tryptophan labelled amount are 0.95g/500ml, are used to make standard curve at this time or 3 of regression equation right
Concentration range according to L- kynurenins in product solution can be 0.2-0.8 μ g/ml, 1.6-2.4 μ g/ml, 8-10 μ g/ml respectively,
For example, the concentration of L- kynurenins can be about 0.5 μ g/ml, about 2.0 μ g/ml, about 9.0 μ g/ respectively in 3 reference substance solutions
ml.The method of the present invention, the range of linearity are more than the 0.1-0.4% of tryptophan labelled amount.
Preferably, the percent by volume that test solution accounts for contrast solution in contrast solution is 1%, i.e., by test solution
100 times are diluted with solvent.If detecting other chromatographic peaks in test solution 280nm wavelength chromatograms, single peak area must not surpass
Cross tryptophan peak area in contrast solution 0.5 times, total peak area must not exceed 5 times of tryptophan peak area in contrast solution.
Preferably, chromatographic condition is：Using trifluoroacetic acid-hyptafluorobutyric acid ion pair chromatography；Chromatographic column is reverse phase C18 colors
Compose column, preferably specification be 4.6mm × 250mm, 3 μm, more preferable reverse phase C18AQ chromatographic columns；Detector is UV detector.
Preferably, mobile phase A is -0.3% trifluoroacetic acid of 5.0mmol/L hyptafluorobutyric acids-water, and Mobile phase B is acetonitrile；It is preferred that
Ground, using gradient elution, elution program see the table below 1；Preferably, flow velocity 0.5-1.0ml/min, further preferred 0.7-
0.9ml/min, more preferable 0.8ml/min.Preferably, column temperature is 35-45 DEG C, more preferable 40 DEG C.
1 gradient elution program of table
The present invention passes through numerous studies, it is determined that 5HTP and L- kynurenins is in Amino Acid Compound Injections
Pointer impurity, by being detected and controlling energy to 5HTP in Amino Acid Compound Injection and L- kynurenins
Enough detect and control the related substance in Amino Acid Compound Injection.In particular, the present invention by trifluoroacetic acid-hyptafluorobutyric acid from
Son optimizes the various amino acid contained in amino acid injection and the separation in relation to substance and baseline stability to chromatographic process
Property, improve detection sensitivity.By methodology validation, it is found that the method for the present invention is sensitive, accurate, reproducibility is preferable, it can
Meet the requirement of the related substance control of Amino Acid Compound Injection.
Description of the drawings
5HTP reference substance solution chromatogram under Fig. 1 280nm wavelength
L- kynurenins reference substance solution chromatogram under Fig. 2 365nm wavelength
Test solution chromatogram under Fig. 3 280nm wavelength
Fig. 4 5HTP canonical plottings
Fig. 5 kynurenin canonical plottings
Fig. 6 280nm wavelength lower open mouths stir 1 hour after test solution chromatogram
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.Furthermore, it is to be understood that after having read recorded content of the invention, this field skill
Art personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within limited range of the present invention.
Detection in relation to substance in 1 Amino Acid Compound Injection of embodiment
1. instrument
3000 type high performance liquid chromatographs of Thermo dionex UltiMate (on-line degassing machine, double ternary gradient pumps,
DIONEX UltiMate 3000Diode Array Detector, DIONEX UltiMate 3000Autosampler,
7.2 chromatographic work station of DIONEX UltiMate 3000Column Compartment, Chromeleon)
(ThermoFisher, the U.S.)；Balance (XP205, METTLER TOLEDO companies, Switzerland)；Milli-Q Superpure water machines
(Integral 10, Merck Millipore companies, Germany)；Water phase syringe filter (polyether sulfone, lot number：9G020320, on
The general scientific instrument Co., Ltd in Hai'an, China).
2. reagent
Acetonitrile (Merck Chemical Engineering Technology (Shanghai) Co., Ltd., HPLC purity 99.9%, lot number：1312001YT)；Trifluoro second
Acid (lark prestige Science and Technology Ltd. of Beijing, 99%, lot number：L490O140)；Hyptafluorobutyric acid (the limited public affairs of Beijing enlightening equine skill
Department, 99.5%, lot number：7281190)；Hydrochloric acid：Beijing Chemical Plant, lot number 20140910, content w/%：38.0；Water is ultra-pure water
(being more than 18.2M Ω cm).
3. reference substance
L- kynurenins (Sigma, HPLC purity 98%, lot number：BCBP8307V), 5- hydroxyls-L-Trp
(Aldrich, HPLC purity 98%, lot number：BCBP2662V).
4. reagent
Amino acid (15) double peptides (2) injection (lot number：14121601, Double-Crane Pharmaceutical Co., Ltd), tryptophan labelled amount is
0.95g/500ml。
5. the detection in relation to substance in Amino Acid Compound Injection
Amino Acid Compound Injection is taken to be directly used as test solution.Precision measures test solution 1ml and is placed in 100ml amounts
In bottle, it is diluted to scale with 0.1mol/L aqueous hydrochloric acid solutions, shakes up, is used as contrast solution.Precision weighs 5HTP,
It is respectively 0.5105 μ g/ml, 2.002 μ g/ml, 9.544 μ to be configured to 5HTP concentration with 0.1mol/L aqueous hydrochloric acid solutions
The 5HTP reference substance solution of g/ml；Precision weighs L- kynurenins, and L- is configured to 0.1mol/L aqueous hydrochloric acid solutions
Kynurenin concentration is respectively the L- kynurenin reference substance solutions of 0.5285 μ g/ml, 2.016 μ g/ml, 9.304 μ g/ml.
Using Capcell PAK C18AQ S3 (4.6mm × 250mm, 3 μm) chromatographic column；Mobile phase A：5.0mmol/L seven
- 0.3% trifluoroacetic acid of fluorine butyric acid-water, Mobile phase B：Acetonitrile, gradient elution, elution program see the table below 2；Flow velocity 0.8ml/min；
40 DEG C of column temperature；Detection wavelength：280nm and 365nm；In 280nm wavelength chromatograms, number of theoretical plate is based on 5HTP peak
Calculate should be not less than 10000,5HTP, L- kynurenins and adjacent chromatographic peak separating degree>1.5.
2 gradient elution program of table
Precision measures test solution, contrast solution and each 10 μ l of two kinds of reference substance solutions, is injected separately into liquid chromatograph,
Record the chromatogram and 365nm of test solution, contrast solution and 5HTP reference substance solution under 280nm wavelength
The chromatogram of test solution and L- kynurenin reference substance solutions under wavelength.A concentration of 2.0 μ g/ml wherein under 280nm wavelength
5HTP reference substance solution chromatogram, the L- kynurenin reference substances of a concentration of 2.0 μ g/ml under 365nm wavelength
The chromatogram of test solution under the chromatogram and 280nm wavelength of solution, see attached drawing 1-3.
It is respectively the 5HTP reference substance solution of 0.5105 μ g/ml, 2.002 μ g/ml, 9.544 μ g/ml with concentration
5HTP peak area is ordinate in 280nm wavelength chromatograms, with a concentration of abscissa of 5HTP, makes mark
Directrix curve (see attached drawing 4), by calibration curve method with the peak area of 5HTP in test solution 280nm wavelength chromatograms
The 5HTP amount contained in test solution is determined according to the standard curve.The lot number detected in the present embodiment is
It is not detected 5HTP peak in 14121601 amino acid (15) double peptides (2) injection, i.e. 5HTP
Amount is no more than 0.2% of tryptophan labelled amount in Amino Acid Compound Injection.
It is respectively the L- kynurenin reference substance solutions of 0.5285 μ g/ml, 2.016 μ g/ml, 9.304 μ g/ml with concentration
L- kynurenin peak areas are ordinate in 365nm wavelength chromatograms, and with a concentration of abscissa of L- kynurenins, it is bent to make standard
Line (see attached drawing 5), by calibration curve method with the peak area of L- kynurenins in test solution 365nm wavelength chromatograms according to this
Standard curve determines the L- kynurenin amounts contained in test solution.The lot number that is detected in the present embodiment is 14121601 to answer
L- kynurenins peak is not detected in square amino acid (15) double peptides (2) injection, i.e. the amount of L- kynurenins is no more than compound amino
The 0.2% of tryptophan labelled amount in acid injection.
Other chromatographic peaks detected in test solution 280nm wavelength chromatograms, it is molten that single peak area is no more than control
0.5 times of tryptophan peak area in liquid, total peak area are no more than 5 times of tryptophan peak area in contrast solution.
The Amino Acid Compound Injection of above-mentioned same lot number is placed in room temperature opening, it is unstable because fully investigating oxidation
Property, it sets and stirs 1h on magnetic stirring apparatus, the related substance wherein contained is detected according to above-mentioned same method, find it
Test solution detects 5HTP peak (see Fig. 6) at a wavelength of 280 nm, and it is a concentration of to calculate its according to calibration curve method
1.078μg/ml；L- kynurenins are detected under 365nm wavelength, its a concentration of 0.5907 μ g/ml is calculated according to calibration curve method.
2 methodology validation of embodiment
Reference《Chinese Pharmacopoeia》Four annex of version in 2015 carry out methodological study to the detection method of the present invention.Wherein,
Used-amino acid (15) double peptides (2) injection (lot number：14121601,14121701,14121801, from double crane medicines
Industry)
1. specificity is tested
Blank solvent (0.1mol/L aqueous hydrochloric acid solutions), 5HTP reference substance solution (a concentration of 40.72 are taken respectively
μ g/ml), L- kynurenins reference substance solution (a concentration of 60.80 μ g/ml), test solution, test solution:5- hydroxyl colors
Propylhomoserin positions solution (a concentration of 400 μ g/ml):It is 1 that L- kynurenins, which position solution (a concentration of 400 μ g/ml),:1:1 mixing
Solution injects liquid chromatograph, and chromatographic condition records the chromatogram under 280nm wavelength with embodiment 1, finds blank solvent and confession
Other amino acid in test sample solution are noiseless to the measurement of 5-HTP and L- kynurenins, show that the present invention's is special
Attribute is good.
2. system suitability
Prepare containing 5HTP, L- kynurenin concentration be respectively 3.802 μ g/ml, 3.722 μ g/ml reference substance
Solution, injects liquid chromatograph, and chromatographic condition records the chromatogram under 280nm, 365nm wavelength, 5- hydroxyl colors with embodiment 1
The theoretical cam curve of propylhomoserin is that the theoretical cam curve of 456909, L- kynurenins is 323966, and the separating degree of the two is 8.52.
3. repeatability is investigated
6 parts of parallel preparation is respectively 3.802,3.722 μ g/ml containing 5HTP, L- kynurenin concentration, contains 20%
The mark-on test solution of test solution is injected separately into liquid chromatograph, and chromatographic condition is the same as embodiment 1,5HTP
(under 280nm wavelength), L- kynurenins (under 365nm wavelength) peak area RSD are respectively 0.30%, 1.89%.
4. quantitative limit
Prepare containing 5HTP, L- kynurenin concentration be respectively 0.8144,1.216mg/ml quantitative limit solution,
Liquid chromatograph is constantly diluted and injects, chromatographic condition is with embodiment 1, until signal-to-noise ratio S/N >=10.5HTP
The quantitative limit of (under 280nm wavelength), L- kynurenins (under 365nm wavelength) is respectively 0.1629 μ g/ml, 0.2432 μ g/ml.
5. detection limit
Prepare containing 5HTP, L- kynurenin concentration be respectively 0.8144,1.216mg/ml quantitative limit solution,
Liquid chromatograph is constantly diluted and injects, chromatographic condition is with embodiment 1, until signal-to-noise ratio S/N >=3.5HTP
The detection limit of (under 280nm wavelength), L- kynurenins (under 365nm wavelength) is respectively 0.08144 μ g/ml, 0.1216 μ g/ml.
6. precision is investigated
Take contrast solution (test solution 0.1mol/L aqueous hydrochloric acid solutions dilute 100 times) and containing 5HTP,
L- kynurenin concentration is respectively the reference substance solution injection liquid chromatograph of 3.802,3.722 μ g/ml, respectively repeatedly sample introduction 6
Secondary, chromatographic condition is with embodiment 1,5HTP (under 280nm wavelength), L- kynurenins (365nm waves in reference substance solution
Under length) peak area RSD is respectively 0.10%, 0.33%, and tryptophan (under 280nm wavelength) peak area RSD is in contrast solution
0.15%.
7. linear test
Prepare concentration containing 5HTP be respectively 814.4,407.2,203.6,40.72,8.144,4.072,
1.629,0.8144,0.3258,0.1629 μ g/ml, L- kynurenin concentration be respectively 1216,608.0,304.0,60.80,
12.16, the control series solution of 6.080,2.432,1.216,0.4864,0.2432 μ g/ml injects liquid chromatograph, chromatography
Condition is with embodiment 1, and the linear relationship of 5HTP concentration and peak area is good, regression equation：Y=0.2858X-
0.0011, correlation coefficient r=1.0000, the method range of linearity：0.1629~814.4 μ g/ml；L- kynurenins concentration and peak face
Long-pending linear relationship is good, regression equation：Y=0.2014X-0.2513, correlation coefficient r=0.9999, the method range of linearity：
0.2432~1216 μ g/ml.
8. test solution stability
Take be placed at room temperature for 0 hour, 3 hours, 7 hours, 14 hours, 16 hours, 20 hours, 21 hours, 24 hours for examination
Product solution and concentration containing 5HTP are respectively the reference substance solution of 0.5105,2.002,9.544 μ g/ml, the urine of dog containing L-
Propylhomoserin concentration is respectively the reference substance solution of 0.5285,2.2016,9.304 μ g/ml, is injected separately into liquid chromatograph, chromatostrip
Part measures the peak area of 5-OH-Trp and L-KY in test sample sample with embodiment 1.In the test sample placed in 24 hours not
5-OH-Trp and L-KY is detected, Amino Acid Compound Injection has good stability under room temperature.
9. accuracy
Precision measures the test sample (lot number of 2.0mL：14121701) it 9 parts, is respectively placed in the volumetric flask of 10mL, essence is added
The close reference substance about 1.0 for measuring (a concentration of 19.92,19.82 μ g/ml of 5HTP, L- kynurenins), 2.0,4.0mL
Each 3 parts, scale is diluted to 0.1mol/L HCl, be made mass concentration be about 1.90 μ g/ml, 3.80 μ g/ml, 7.60 μ g/ml
Solution be injected separately into liquid chromatograph as accuracy determination solution, chromatographic condition with embodiment 1,5HTP
Average recovery rate is 98.86%, RSD=0.78% (n=9), and the average recovery rate of L- kynurenins is 104.8%, RSD=
1.78% (n=9).
10. serviceability test
Prepare containing 5HTP, L- kynurenin concentration be respectively 3.802,3.722 μ g/ml reference substance solution,
It is 14121701 test sample to take lot number, and filtration is injected separately into liquid chromatograph, chromatographic condition investigates flow velocity ± 0.1ml/ respectively
Min, under the conditions of column temperature ± 5 DEG C, 5HTP, the influence of L- kynurenin peak areas and test sample are molten in reference substance solution
The influence of 5HTP, L- kynurenin peak areas in liquid.
The results show that (flow velocity ± 0.1ml/min, column temperature ± 5 DEG C) change testing conditions in a certain range, to 5-
Hydroxytryptophan, the determination influences of L- kynurenins are little.
More than, embodiments of the present invention are illustrated.But the present invention is not limited to the above embodiments.It is all
Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in the guarantor of the present invention
Within the scope of shield.
Claims (10)
1. the detection method in relation to substance in a kind of Amino Acid Compound Injection, which is characterized in that this method comprises the following steps：
(1) preparation of test solution and reference substance solution：
Amino Acid Compound Injection is taken to be directly used as test solution；
Prepare the L- kynurenins of the 5HTP reference substance solution and at least three various concentration of at least three various concentration
Reference substance solution；
(2) it takes the 5HTP reference substance solution of various concentration to inject high performance liquid chromatograph, records under 280nm wavelength not
With the chromatogram of the 5HTP reference substance solution of concentration, with a concentration of abscissa of 5HTP, with the concentration
Corresponding peak area is ordinate, makes standard curve or regression equation；Take the L- kynurenin reference substances of various concentration molten
Liquid injects high performance liquid chromatograph, records the chromatogram of the L- kynurenin reference substance solutions of various concentration under 365nm wavelength, with
A concentration of abscissa of L- kynurenins makes standard curve or recurrence side using the peak area corresponding to the concentration as ordinate
Journey；
(3) take test solution inject high performance liquid chromatograph, record 280nm wavelength under and 365nm wavelength under test solution
Chromatogram, by calibration curve method, with the peak area of 5HTP in test solution 280nm wavelength chromatograms according to mark
The amount of 5HTP, must not exceed color in Amino Acid Compound Injection in directrix curve or regression equation calculation test solution
The 0.2% of propylhomoserin labelled amount, with the peak area of L- kynurenins in test solution 365nm wavelength chromatograms according to standard curve
Or in regression equation calculation test solution L- kynurenins amount, must not exceed in Amino Acid Compound Injection tryptophan and indicate
The 0.2% of amount；
Wherein, contain tryptophan in the Amino Acid Compound Injection.
It is preferred that the Amino Acid Compound Injection is amino acid (15) double peptides (2) injection, Amino Acid Compound Injection
(18AA) (specification includes 5% and 12%), Amino Acid Compound Injection (18AA-I), Amino Acid Compound Injection (18AA-II)
(specification includes 5%, 8.5%, 11.4%), Amino Acid Compound Injection (18AA-III), Amino Acid Compound Injection (18AA-
V), Amino Acid Compound Injection (18AA- VII), Amino Acid Compound Injection (20AA), Amino Acid Compound Injection (15AA), multiple
Square amino acid injection (14AA), Amino Acid Compound Injection (17AA).
It is preferred that the Amino Acid Compound Injection is amino acid (15) double peptides (2) injection.
2. according to the method described in claim 1, it is characterized in that, in step (1), further prepares and contain test solution
Contrast solution；In step (2), contrast solution is further taken to inject high performance liquid chromatograph, records and compareed under 280nm wavelength
The chromatogram of solution；In step (3), by Self-control method, if detecting its allochromatic colour in test solution 280nm wavelength chromatograms
Spectral peak, single peak area is by the way that compared with tryptophan peak area in contrast solution, content must not exceed Amino Acid Compound Injection
The 0.5% of middle tryptophan labelled amount, total peak area is by the way that compared with tryptophan peak area in contrast solution, content must not exceed multiple
The 5% of tryptophan labelled amount in square amino acid injection.
3. method according to claim 1 or 2, which is characterized in that the solvent for preparing reference substance solution is that dilute hydrochloric acid is water-soluble
Liquid, the aqueous hydrochloric acid solution of more preferable a concentration of 0.1mol/L.
4. method according to any one of claim 1-3, which is characterized in that the solvent of contrast solution is that dilute hydrochloric acid is water-soluble
Liquid, the aqueous hydrochloric acid solution of more preferable a concentration of 0.1mol/L.
5. according to the described method of any one of claim 1-4, which is characterized in that 3-7 are prepared, further preferred 3-5 is a,
The 5HTP reference substance solution of more preferable 3 various concentrations.
It is preferred that the Cmin of 5HTP is answered in multiple reference substance solutions for making standard curve or regression equation
Less than 0.1% of tryptophan labelled amount in Amino Acid Compound Injection, maximum concentration should be higher than that color in Amino Acid Compound Injection
The 0.4% of propylhomoserin labelled amount.
It is preferred that Amino Acid Compound Injection is amino acid (15) double peptides (2) injection, tryptophan labelled amount is 0.95g/
500ml, the concentration range of 5HTP is respectively in 3 reference substance solutions for making standard curve or regression equation
0.2-0.8 μ g/ml, 1.6-2.4 μ g/ml, 8-10 μ g/ml, it is further preferred that in 3 reference substance solutions 5HTP concentration
It is about 0.5 μ g/ml, about 2.0 μ g/ml, about 9.0 μ g/ml respectively.
6. method according to any one of claims 1-5, which is characterized in that 3-7 are prepared, further preferred 3-5 is a,
The L- kynurenin reference substance solutions of more preferable 3 various concentrations.
It is preferred that in multiple reference substance solutions for making standard curve or regression equation the Cmin of L- kynurenins answer it is low
The 0.1% of tryptophan labelled amount in Amino Acid Compound Injection, maximum concentration should be higher than that color ammonia in Amino Acid Compound Injection
The 0.4% of sour labelled amount.
It is preferred that Amino Acid Compound Injection is amino acid (15) double peptides (2) injection, tryptophan labelled amount is 0.95g/
500ml, the concentration range of L- kynurenins is respectively in 3 reference substance solutions for making standard curve or regression equation
0.2-0.8 μ g/ml, 1.6-2.4 μ g/ml, 8-10 μ g/ml, it is further preferred that the concentration of L- kynurenins is divided in 3 reference substance solutions
It is not about 0.5 μ g/ml, about 2.0 μ g/ml, about 9.0 μ g/ml.
7. according to the method described in any one of claim 1-6, which is characterized in that test solution accounts for control in contrast solution
The percent by volume of solution is 1%, i.e., test solution solvent is diluted 100 times；Test solution 280nm wavelength chromatograms
If other chromatographic peaks are detected in, single peak area must not exceed 0.5 times of tryptophan peak area in contrast solution, and total peak area is not
Obtain 5 times more than tryptophan peak area in contrast solution.
8. according to the described method of any one of claim 1-7, which is characterized in that use trifluoroacetic acid-hyptafluorobutyric acid ion
To chromatography.
It is preferred that chromatographic column be reverse phase C18 chromatographic columns, further preferred specification be 4.6mm × 250mm, 3 μm, more preferable reverse phase
C18AQ chromatographic columns.
It is preferred that detector is UV detector.
9. according to the method described in any one of claim 1-8, which is characterized in that mobile phase A is seven fluorine fourths of 5.0mmol/L
Sour -0.3% trifluoroacetic acid-water, Mobile phase B is acetonitrile；Preferably, using gradient elution, elution program see the table below：
10. according to the method described in any one of claim 1-9, which is characterized in that flow velocity 0.5-1.0ml/min, preferably
0.7-0.9ml/min, more preferable 0.8ml/min.
It is preferred that column temperature is 35-45 DEG C, more preferable 40 DEG C.
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| CN112697934A (en) * | 2020-12-10 | 2021-04-23 | 武汉久安药业有限公司 | Method for detecting content of pyroglutamic acid in compound amino acid injection |
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| CN112946099A (en) * | 2021-01-25 | 2021-06-11 | 石家庄四药有限公司 | Method for detecting related substances in amino acid glucose injection |
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