CN107991408A - Index components content assay method in four taste treasure's layer ice boron eye drops - Google Patents
Index components content assay method in four taste treasure's layer ice boron eye drops Download PDFInfo
- Publication number
- CN107991408A CN107991408A CN201711210212.2A CN201711210212A CN107991408A CN 107991408 A CN107991408 A CN 107991408A CN 201711210212 A CN201711210212 A CN 201711210212A CN 107991408 A CN107991408 A CN 107991408A
- Authority
- CN
- China
- Prior art keywords
- treasure
- taste
- eye drops
- solution
- tyrosylglycine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- G01N30/02—Column chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses the content assaying method of characteristic chemical constituent oligopeptides L tyrosyl-glycines and L tryptophans in four taste treasure's layer ice boron eye drops.It is characterized in that, four taste treasure's layer ice boron eye drops are isolated and purified and Structural Identification, determine the feature sexual element that 7 monomeric compounds including oligopeptides L tyrosyl-glycines and L tryptophans are four taste treasure's layer ice boron eye drops, the content of oligopeptides L tyrosyl-glycines and L tryptophans in four taste treasure's layer ice boron eye drops is measured by using HPLC methods, the content of controlling feature sexual element oligopeptides L tyrosyl-glycines and L tryptophans, it can ensure that four taste treasure's layer ice boron eye drops stable quality are homogeneous, so as to improve the quality controllability of the product.The method of the present invention is easy quick, and specificity is strong, can accurately, stabilization, quickly measure the content of oligopeptides L tyrosyl-glycines and L tryptophans in four taste treasure's layer ice boron eye drops, the application of this method can better control over the quality of the product.
Description
Technical field
The invention belongs to Analysis of Chinese Traditional Medicine field, and in particular to containing for characteristic chemical constituent measures in four taste treasure's layer ice boron eye drops
Determine method.
Background technology
Chinese medicine treatment disease has long history, but the complexity of Chinese medicinal ingredients brings difficulty to traditional Chinese medicine quality control
Topic, hinders Chinese medicine and goes on world market, to the quality of comprehensive control Chinese medicine, it is necessary to carry out the accurate of the content of each component
Control.
Four tastes treasure layer ice boron eye drops of the present invention, its main ingredient are nacreous layer powder；Nacreous layer powder is moved by Unionidae
Thing hydriopsis cumingii Hyriopsis cumingii (Lea), cristaria plicata Cristaria plicata (Leach) or Pteriidae
The processed powder being milled into of nacre of the shell of animal Pinctada martensii Pteria martensii (Dunker), is tradition
Chinese medicine, record in《Chinese Pharmacopoeia》2015 editions one, have it is calming the liver, move back screen and other effects.Nacreous layer powder obtains after extraction
Nacreous layer powder extracting solution is obtained, so that the active ingredient in nacreous layer powder preferably plays a role.According to domestic related nacreous layer powder
The report of chemical composition, the main component of nacreous layer powder is calcium carbonate, and containing a small amount of trace element and amino acid etc., it is changed
Composition Study is learned not go deep into,.By nacreous layer powder extracting solution and four taste treasure's layer ice boron eye drops are isolated and purified and tied
Structure identifies that 7 monomeric compounds including isolated for the first time and definite oligopeptides l-tyrosylglycine and L-Trp are
The feature sexual element of nacreous layer powder extracting solution and ZHENSHIMING DIYANYE, lays to improve four taste treasure's layer ice boron eye drops quality control
Basis.
《Chinese Pharmacopoeia》Version one in 2015 has included discriminating and the content assaying method of four taste treasure's layer ice boron eye drops, so
And the project in assay on main component nacreous layer powder only has nitrogen pool measure, nitrogen pool measure there is not the product composition
Targetedly and specificity, and the digestion of nitriding, distillation, titration etc. are complicated, to testing staff's skilled operation degree
It is required that high, time-consuming for detection, and pollution is big, poor accuracy, it is difficult to the evaluation of science and the quality for controlling nacreous layer powder.Only
Characteristic chemical constituent and its content in nacreous layer powder extracting solution and four taste treasure's layer ice boron eye drops further are determined, could be from root
Ensure the drug effect of four taste treasure's layer ice boron eye drops in sheet.The present invention, is measured few in four taste treasure's layer ice boron eye drops using HPLC methods
The content of peptide l-tyrosylglycine and L-Trp, passes through controlling feature sexual element oligopeptides l-tyrosylglycine and L- color ammonia
The content of acid, can ensure that four taste treasure's layer ice boron eye drops stable quality are homogeneous, so as to improve the quality controllability of the product.
The content of the invention
The object of the present invention is to provide index components content assay method in four taste treasure's layer ice boron eye drops.
Technical solution of the present invention：
Characteristic chemical constituent content assaying method in a kind of four taste treasure's layer ice boron eye drops, separates ZHENSHIMING DIYANYE
Purifying and Structural Identification, determine the monomeric compound including oligopeptides l-tyrosylglycine and L-Trp to value bright drop
The feature sexual element of ocular fluid, by controlling feature sexual element oligopeptides l-tyrosylglycine and the content of L-Trp, can ensure that
Four taste treasure's layer ice boron eye drops stable quality are homogeneous, so as to improve Drug safety and quality controllability.
Further, comprise the following steps：
(1) preparation of reference substance solution：L-tyrosylglycine and appropriate L-Trp are weighed, is placed in 100mL volumetric flasks
In, be dissolved in water dilution, shakes up, constant volume, as mixing reference substance storing solution；
(2) preparation of test solution：Four taste treasure's layer ice boron eye drops are drawn, is centrifuged, is taken supernatant, it is molten to obtain test sample
Liquid；
(3) assay method：Carry out quantitative inspection to test solution and reference substance solution respectively using high performance liquid chromatography
Survey.
Further, further included in step (2) any one or more in following characteristics：
(1) dosage for drawing four taste treasure's layer ice boron eye drops is 1.0~20.0mL；
(2) speed of the centrifugation is 10,000~15,000r/min；
(3) centrifuging temperature is 2~30 DEG C.
Further, in step (3), the Eclipse XDB-C of Agilent company of the U.S. product are selected18(3.0 × 150mm,
3.5 μm) separation of the chromatographic column to ZHENSHIMING DIYANYE.
Further, in step (3), full wavelength scanner is carried out to sample using diode array detector, and to each wavelength
Under chromatogram carry out analysis comparison, it is determined that the characteristic peak and peak shape detected under 220nm, obtain chromatographic component information with
The overall picture of reaction system composition.
Further, in step (3), different flow phase system difference gradients is respectively adopted and are tested, with 0.085%TFA+
70% acetonitrile solution -0.1%TFA waterline gradient separations.
Further, in step (3), the quantitative detecting method step includes：Divided using HPLC under identical chromatographic conditions
Not Ce Ding test solution and reference substance solution, by the chromatogram of test solution compared with the chromatogram of reference substance solution, root
According to relative retention time and characteristic peak, the characteristic peak of l-tyrosylglycine and L-Trp in test solution is confirmed.
Further, in step (3), the quantitative detecting method includes step：Divided using HPLC under identical chromatographic conditions
Not Ce Ding test solution and reference substance solution, by external standard method with the l-tyrosylglycine in calculated by peak area test solution
With the content of L-Trp component.
Further, comprise the following steps：
(1) precision weighs l-tyrosylglycine 6.16mg and L-Trp 3.06mg is placed in 100mL volumetric flasks, adds water
Dissolved dilution, shakes up, constant volume, as mixing reference substance storing solution；
(2) above-mentioned reference substance storing solution can further be diluted according to detection needs；
(3) four taste treasure's layer ice boron eye drops 10mL are drawn, 12,000r/min centrifugation 15min, take supernatant spare.
(4) chromatographic column used in HPLC is：Agilent, Eclipse XDB-C18 (3.0 × 150mm, 3.5 μm)；
(5) mobile phase, A phases are 0.1% trifluoroacetic acid (TFA)-water, and B phases are+70% second of 0.085% trifluoroacetic acid (TFA)
Nitrile aqueous solution；
(6) gradient elution is used：
(7) flow velocity：0.1~1.0mL/min；
(8) Detection wavelength：200~300nm；
(9) sample size：5~50 μ L；
(10) column temperature：20~40 DEG C；
(11) theoretical cam curve is calculated by l-tyrosylglycine is not less than 3000.
(12) this product contains nacreous layer powder per ml with l-tyrosylglycine (C11H14N2O4) meter, 0.99 μ g must not be less than,
With L-Trp (C11H12N2O2) meter, 0.46 μ g must not be less than.
Further, comprise the following steps：
(1) precision weighs l-tyrosylglycine 6.16mg and L-Trp 3.06mg is placed in 100mL volumetric flasks, adds water
Dissolved dilution, shakes up, constant volume, as mixing reference substance storing solution；
(2) the accurate reference substance storing solution that measures is appropriate, is diluted with water into every 1ml and contains l-tyrosylglycine and L-Trp
1.150-1.300 μ g, the solution of 0.550-0.660 μ g, to obtain the final product.
(3) four taste treasure's layer ice boron eye drops 10mL are drawn, 12,000r/min centrifugation 15min, take supernatant spare.
(4) chromatographic column used in HPLC is：Agilent, Eclipse XDB-C18 (3.0 × 150mm, 3.5 μm)；
(5) mobile phase, A phases are 0.1% trifluoroacetic acid (TFA)-water, and B phases are+70% second of 0.085% trifluoroacetic acid (TFA)
Nitrile aqueous solution；
(6) gradient elution is used：
Gradient elution program table
(7) flow velocity：0.4mL/min；
(8) Detection wavelength：220nm；
(9) sample size：20μL；
(10) column temperature：30℃；
(11) theoretical cam curve is calculated by l-tyrosylglycine is not less than 3000.
(12) this product contains nacreous layer powder per ml with l-tyrosylglycine (C11H14N2O4) meter, 0.99 μ g must not be less than,
With L-Trp (C11H12N2O2) meter, 0.46 μ g must not be less than.
Compared with prior art, the present invention has the advantages that：
The method of the present invention measures oligopeptides l-tyrosylglycine and L- colors in four taste treasure's layer ice boron eye drops using HPLC methods
The content of propylhomoserin, this method are easy quick, and specificity is strong, can accurately, stabilization, quickly measure four taste treasure's layer ice boron eye drops
The content of middle oligopeptides l-tyrosylglycine and L-Trp.This method not only facilitates the quality of four taste treasure's layer ice boron eye drops
Control, improves the quality controllability of the product.
The invention is analyzed：
The main ingredient nacreous layer powder of ZHENSHIMING DIYANYE is by Unionidae animal hydriopsis cumingii Hyriopsis cumingii
(Lea), the processed powder being milled into of nacre of the shell such as cristaria plicata Cristaria plicata (Leach), is tradition
Chinese medicine, record in《Chinese Pharmacopoeia》2015 editions one, have it is calming the liver, move back screen and other effects.Nacreous layer powder obtains after hydrolysis
Pearl layer powder hydrolysate is obtained, so that the active ingredient in nacreous layer powder preferably plays a role.Therefore, in order to more preferable
Using nacre animal drugs resource, the chemical foundation of its drug effect is illustrated, it is necessary to which systematic research is carried out to its chemical composition.This
Experiment determines in nacre containing chemical analysis such as oligopeptides substantially by research.
ZHENSHIMING DIYANYE is isolated and purified inventor and Structural Identification, and acquisition includes oligopeptides l-tyrosylglycine
With 7 monomeric compounds including L-Trp, for the feature sexual element of ZHENSHIMING DIYANYE, bright drop is valued to better control over
The quality of ocular fluid, establishes characteristic component content assay method in ZHENSHIMING DIYANYE.
1. the selection of liquid-phase chromatographic column is the core of liquid-phase chromatographic analysis.This experiment has determined in chromatographic condition
Under the conditions of, different chromatographic columns is compared, the results showed that：The Eclipse XDB-C that Agilent company of the U.S. is produced18(3.0
× 150mm, 3.5 μm) chromatographic column is preferable to the separating effect of ZHENSHIMING DIYANYE.
2. carrying out full wavelength scanner to sample using diode array detector, and the chromatogram under each wavelength is divided
Analysis is compared, it is determined that and the number at the peak detected under 220nm is more, and each peak separation is good, and characteristic peak is obvious and peak shape is preferable,
The overall picture that chromatographic component information is formed with reaction system can be obtained as far as possible from collection of illustrative plates.
3. different flow phase system difference gradients are respectively adopted to be tested, the results showed that with 0.085%TFA+70% second
Nitrile aqueous solution -0.1%TFA waterline gradient separations effects are best, and baseline is most steady.
Brief description of the drawings
Fig. 1 is l-tyrosylglycine reference substance chromatogram.
Fig. 2 is L-Trp reference substance chromatogram.
Fig. 3 is l-tyrosylglycine and L-Trp reference substance chromatogram.
Fig. 4 is four taste treasure's layer ice boron eye drops assay chromatogram.
Fig. 5 is l-tyrosylglycine minimum detection limit chromatogram.
Fig. 6 is L-Trp minimum detection limit chromatogram.
Fig. 7 is the minimum quantitative limit chromatogram of l-tyrosylglycine.
Fig. 8 is the minimum quantitative limit chromatogram of L-Trp.
Embodiment
Embodiment 1
The assay of oligopeptides l-tyrosylglycine and L-Trp in four taste treasure's layer ice boron eye drops, four tastes treasure's layer
Ice boron eye drops is made of following component：Nacreous layer powder extracting solution, natural borneol, boric acid, borax tune pH value to 7.4, sodium chloride
Infiltration is adjusted to be depressed into 0.29Osmol/kg, ethanol, Phenoxyethanol and water for injection.
1. laboratory apparatus and reagent
High performance liquid chromatograph (1260, U.S.'s Agilent)；YC-800 chromatography assays refrigerator (XMT608, Beijing AsiaSat instrument
Development in science and technology Co., Ltd of section)；Micro tabletop refrigerated centrifuge (FRESCO 17, Thermo Fischer Scient Inc.)；Ultrasound Instrument
(KQ-300E, Kunshan Ultrasonic Instruments Co., Ltd.)；Acetonitrile (chromatographically pure, U.S. world Co., Ltd)；Methanol (chromatographically pure, on
Starfish can high pure solvent Co., Ltd)；Trifluoroacetic acid (TFA) (chromatographically pure, Aladdin)；Water for injection (applicant)；Chromatographic column
(Eclipse XDB-C18, U.S.'s Agilent)；Guard column (Utimate XB-C18, Shanghai Yue Xu companies)；Liquid-transfering gun (200 μ
L, 1000 μ L, Ai Bende companies of the U.S.).
2. chromatographic condition
Chromatographic column is Agilent, Eclipse XDB-C18 (3.0 × 150mm, 3.5 μm).Mobile phase is A phases (0.1%
TFA- water), B phases (0.085%TFA+70% acetonitrile solutions), flow velocity as shown in table 2 below using gradient elution program：
0.4mL/min, sample size：20 μ L, column temperature：30 DEG C, Detection wavelength：220nm.
2 four taste treasure's layer ice boron eye drops gradient elution program table of table
3. prepared by reference substance solution
Precision weighs l-tyrosylglycine 6.16mg and L-Trp 3.06mg is placed in 100mL volumetric flasks, adds water-soluble
Solution dilution, after shaking up, constant volume, its mass concentration is respectively 61.6 μ g/mL, 30.6 μ g/mL, as mixing reference substance storing solution.
Precision measurement reference substance storing solution is appropriate, is diluted with water into every 1ml and contains l-tyrosylglycine and L-Trp
1.232 μ g, the solution of 0.612 μ g, to obtain the final product.
4. prepared by test solution
Four taste treasure's layer ice boron eye drops 10mL prepared by the above-mentioned prescription of each batch, 12,000r/min centrifugations are drawn respectively
15min, takes supernatant spare.
5. specificity is tested
5.1 blank auxiliaries influence experiment
This product blank auxiliary (borax, boric acid etc.) is weighed by prescription, is put in certain volume volumetric flask, is dissolved in water and dilutes
To scale, shake up, filter, take subsequent filtrate to draw 20 μ L injection liquid chromatographs as test solution；As a result blank auxiliary exists
In there are no peak appearance in main peak appearance time under this chromatographic condition, show that this product auxiliary material does not interfere with assay.
5.2 mobile phases influence experiment
Take 20 μ L mobile phases to inject liquid chromatograph, record chromatogram, as a result there are no peak in main peak appearance time occurs,
Show that mobile phase is noiseless to assay.
6. test limit and quantitative limit
Precision weighs reference substance, is dissolved in water and a series of gradient concentration solution from high to low are made in equimultiple dilution, point
Other sample introduction.Sample introduction concentration when to take signal-to-noise ratio be 3 (S/N=3) is minimal detectable concentration, and lowest detection is drawn by 20 μ L of sample size
Limit is (see Fig. 5 and Fig. 6).As a result l-tyrosylglycine and the minimum detection limit of L-Trp are respectively：0.091 μ g/mL,
0.1052μg/mL。
It is minimum quantitative limit to take sample introduction concentration during S/N=10 (see Fig. 7 and Fig. 8).As a result l-tyrosylglycine and L-
The minimum quantitative limit of tryptophan is respectively：0.1365 μ g/mL, 0.1925 μ g/mL.
7. linear relationship
Accurate respectively to draw mixing reference substance storing solution 1.0mL, 1.5mL, 2.0mL, 2.5mL, 3.0mL put 100mL capacity
In bottle, be dissolved in water dilution, and after shaking up, constant volume, is made serial standards solution, its concentration is respectively l-tyrosylglycine：
0.616 μ g/mL, 0.924 μ g/mL, 1.232 μ g/mL, 1.540 μ g/mL, 1.848 μ g/mL；L-Trp：0.306 μ g/mL,
0.459 μ g/mL, 0.612 μ g/mL, 0.765 μ g/mL, 0.918 μ g/mL.Tested by 2. lower chromatographic conditions, record chromatography
Figure.
Using peak area Y as ordinate, with l-tyrosylglycine or L-Trp sample introduction concentration (μ g/mL) for abscissa,
Obtain regression equation, l-tyrosylglycine：Y1=63.866X2+ 0.8193, r2=0.9998；L-Trp：Y2=450.39X3-
1.68 r2=0.9997.
The result shows that：L-tyrosylglycine and L-Trp mass concentration respectively in 0.616~1.840 μ g/mL,
0.306~0.918 μ g/mL have good linear relationship.
8. precision test
Same 6 parts of test solutions of batch are taken, by above-mentioned 2. lower chromatographic conditions, continuous sample introduction 6 times, records L- tyrosyls
The peak area of glycine and L-Trp, calculates the RSD of its peak area, as a result：The RSD of l-tyrosylglycine and L-Trp
Respectively 0.64%, 1.33%, show that instrument precision is good.
9. repetitive test
Same 6 parts of test solutions of batch are taken, are measured by above-mentioned 2. lower chromatographic condition sample introductions, record peak area, measure
The content of component to be measured, as a result the RSD of 6 assay values of l-tyrosylglycine and L-Trp 0.80%~
0.93%, repeatability is good.
10. stability test
Same 6 parts of test solutions of batch are taken, by above-mentioned 2. lower chromatographic conditions, respectively at 0h, 2h, 4h, 8h, 12h,
24h sample introductions, measure the peak area of l-tyrosylglycine and L-Trp, calculate its peak area RSD values, as a result RSD exists
1.10%~1.37%, show test solution interior stabilization when 24h is small.
11. sample recovery rate is tested
(actual content is l-tyrosylglycine for l-tyrosylglycine and L-Trp content known to precision measurement：
1.320 μ g/mL, L-Trp：0.630 μ g/mL) same a collection of 6 parts, every part of 5mL of four tastes treasure layer ice boron eye drops, it is accurate respectively
Add above-mentioned 7. lower mixed reference substance solution (l-tyrosylglycine：1.232 μ g/mL, L-Trp：0.612μg/mL)
5mL, is measured with sample determination method, calculates the rate of recovery：The rate of recovery (%)=(measured amount-background amount)/addition × 100%.Knot
Fruit, sample recovery rate measure item meet pharmacopoeial requirements.
3 l-tyrosylglycine of table and the experiment of L-Trp sample recovery rate
12. sample measures
The four taste treasure layer ice each 2mL of boron eye drops for taking 3 batches of the present embodiment prescriptions to prepare respectively, by above-mentioned 2. lower chromatostrips
Part sample introduction measures, and every batch of sample parallel determination 2 times, 3 batches of sample measurement results see the table below 4.
4 embodiment of table, 1 four taste treasure's layer ice boron eye drops sample measurement result
Embodiment 2
The assay of oligopeptides l-tyrosylglycine and L-Trp in four taste treasure's layer ice boron eye drops, four tastes treasure's layer
Ice boron eye drops is made of following component：Nacreous layer powder extracting solution, natural borneol, boric acid, borax tune pH value to 7.3, sodium chloride
Infiltration is adjusted to be depressed into 0.29Osmol/kg, ethanol, Phenoxyethanol and water for injection.
1. laboratory apparatus and reagent, chromatographic condition, reference substance solution are prepared with embodiment 1
2. prepared by test solution
Four taste treasure's layer ice boron eye drops 10mL prepared by the above-mentioned prescription of each batch, 12,000r/min centrifugations are drawn respectively
15min, takes supernatant spare.
3. specificity is tested
3.1 blank auxiliaries influence experiment
This product blank auxiliary (borax, boric acid etc.) is weighed by prescription, is put in certain volume volumetric flask, is dissolved in water and dilutes
To scale, shake up, filter, take subsequent filtrate to draw 20 μ L injection liquid chromatographs as test solution；As a result blank auxiliary exists
In there are no peak appearance in main peak appearance time under this chromatographic condition, show that this product auxiliary material does not interfere with assay.
Take 20 μ L mobile phases to inject liquid chromatograph, record chromatogram, as a result there are no peak in main peak appearance time occurs,
Show that mobile phase is noiseless to assay.
4. test limit is with quantitative limit, linear relationship with embodiment 1
5. precision test
Same 6 parts of test solutions of batch are taken, by above-mentioned chromatographic condition, continuous sample introduction 6 times, records l-tyrosylglycine
With the peak area of L-Trp, the RSD of its peak area is calculated, as a result：The RSD of l-tyrosylglycine and L-Trp is respectively
1.66%, 1.73%, show that instrument precision is good.
6. repetitive test
Take same 6 parts of test solutions of batch, by above-mentioned chromatographic condition sample introduction measure, record peak area, measure it is to be measured into
Point content, as a result the RSD of 6 secondary peak area estimation value of l-tyrosylglycine and L-Trp is 0.73%~1.02%, weight
Renaturation is good.
7. stability test
Same 6 parts of test solutions of batch are taken, by above-mentioned 2. lower chromatographic conditions, respectively at 0h, 2h, 4h, 8h, 12h,
24h sample introductions, measure the peak area of l-tyrosylglycine and L-Trp, calculate its peak area RSD values, as a result RSD exists
0.097%~1.16%, show test solution interior stabilization when 24h is small.
8. sample recovery rate is tested
(actual content is l-tyrosylglycine for l-tyrosylglycine and L-Trp content known to precision measurement：
1.295 μ g/mL, L-Trp：0.600 μ g/mL) same a collection of 6 parts, every part of 5mL of four tastes treasure layer ice boron eye drops, it is accurate respectively
Add 7. lower mixed reference substance solution (l-tyrosylglycine in embodiment 1：1.232 μ g/mL, L-Trp：0.612μg/
ML) 5mL, is measured with sample determination method, calculates the rate of recovery：The rate of recovery (%)=(measured amount-background amount)/addition × 100%.
As a result, sample recovery rate measure item meets pharmacopoeial requirements.
5 l-tyrosylglycine of table and the experiment of L-Trp sample recovery rate
9. sample measures
The four taste treasure layer ice each 2mL of boron eye drops for taking 3 batches of the present embodiment prescriptions to prepare respectively, by above-mentioned chromatographic condition sample introduction
Measure, every batch of sample parallel determination 2 times, 3 batches of sample measurement results see the table below 6.
6 embodiment of table, 2 four taste treasure's layer ice boron eye drops sample measurement result
In addition, l-tyrosylglycine reference substance chromatogram is as shown in Figure 1.L-Trp reference substance chromatogram such as Fig. 2 institutes
Show.L-tyrosylglycine and L-Trp mixture reference substance chromatogram are as shown in Figure 3.Four taste treasure's layer ice boron eye drip liquid hold-up
It is as shown in Figure 4 to measure chromatogram.
It should be noted that described above is presently preferred embodiments of the present invention, the invention is not limited in above-mentioned
Embodiment, as long as it reaches the technique effect of the present invention with identical means, should all belong to protection scope of the present invention.
Claims (10)
1. characteristic chemical constituent content assaying method in a kind of four taste treasure's layer ice boron eye drops, it is characterised in that to four taste treasure's layer ice
Boron eye drops is isolated and purified and Structural Identification, determines the monomer chemical combination including oligopeptides l-tyrosylglycine and L-Trp
Thing is the feature sexual element of ZHENSHIMING DIYANYE, and it is sweet to measure oligopeptides L- tyrosyls in four taste treasure's layer ice boron eye drops using HPLC methods
The content of propylhomoserin and L-Trp, passes through controlling feature sexual element oligopeptides l-tyrosylglycine and the content of L-Trp, energy
Ensure that four taste treasure's layer ice boron eye drops stable quality are homogeneous, so as to improve the quality controllability of the product.
2. according to the method described in claim 1, it is characterised in that it includes following steps：
(1) preparation of reference substance solution：L-tyrosylglycine and appropriate L-Trp are weighed, is placed in 100mL volumetric flasks, adds
Water dissolved dilution, shakes up, constant volume, as mixing reference substance storing solution；
(2) preparation of test solution：Four taste treasure's layer ice boron eye drops are drawn, is centrifuged, is taken supernatant, obtain test solution；
(3) assay method：Carry out quantitative detection to test solution and reference substance solution respectively using high performance liquid chromatography.
3. according to the method described in claim 2, it is characterized in that, any one in following characteristics is further included in step (2)
It is or multinomial：
(1) amount for drawing four taste treasure's layer ice boron eye drops is 1.0~20.0mL；
(2) speed of the centrifugation is 10,000~15,000r/min；
(3) centrifuging temperature is 2~30 DEG C.
4. the according to the method described in claim 2, it is characterized in that, Eclipse XDB- that selection Agilent company of the U.S. is produced
C18Separation of (3.0 × 150mm, the 3.5 μm) chromatographic column to four taste treasure's layer ice boron eye drops.
5. according to the method described in claim 2, it is characterized in that, all-wave length is carried out to sample using diode array detector
Scanning, and analysis comparison is carried out to the chromatogram under each wavelength, it is determined that the characteristic peak and peak shape detected under 220nm, is obtained
The overall picture for taking chromatographic component information to be formed with reaction system.
6. according to the method described in claim 2, tried it is characterized in that, different flow phase system difference gradients are respectively adopted
Test, with 0.085%TFA+70% acetonitrile solution -0.1%TFA waterline gradient separations.
7. according to the method described in claim 2, it is characterized in that, in step (3), the quantitative detecting method step includes：
Test solution and reference substance solution are measured using HPLC respectively under identical chromatographic conditions, by the chromatogram of test solution with
The chromatogram of reference substance solution compares, and according to relative retention time and characteristic peak, confirms the sweet ammonia of L- tyrosyls in test solution
The characteristic peak of acid and L-Trp.
8. according to the method described in claim 2, it is characterized in that, in step (3), the quantitative detecting method includes step：
Test solution and reference substance solution are measured under identical chromatographic conditions using HPLC respectively, supplied by external standard method with calculated by peak area
The content of l-tyrosylglycine and L-Trp component in test sample solution.
9. according to the method described in claim 2, it is characterized in that：
(1) precision weighs l-tyrosylglycine 6.16mg and L-Trp 3.06mg is placed in 100mL volumetric flasks, is dissolved in water
Dilution, shakes up, constant volume, as mixing reference substance storing solution；
(2) above-mentioned reference substance storing solution can further be diluted according to detection needs；
(3) four taste treasure's layer ice boron eye drops 10mL are drawn, 12,000r/min centrifugation 15min, take supernatant spare.
(4) chromatographic column used in HPLC is：Agilent, Eclipse XDB-C18 (3.0 × 150mm, 3.5 μm)；
(5) mobile phase, A phases are 0.1% trifluoroacetic acid (TFA)-water, and B phases are+70% acetonitrile water of 0.085% trifluoroacetic acid (TFA)
Solution；
(6) gradient elution is used：
(7) flow velocity：0.1~1.0mL/min；
(8) Detection wavelength：200~300nm；
(9) sample size：5~50 μ L；
(10) column temperature：20~40 DEG C；
(11) theoretical cam curve is calculated by l-tyrosylglycine is not less than 3000.
(12) this product contains nacreous layer powder per ml with l-tyrosylglycine (C11H14N2O4) meter, 0.99 μ g must not be less than, with L-
Tryptophan (C11H12N2O2) meter, 0.46 μ g must not be less than.
10. according to the method described in claim 9, it is characterized in that:
(1) precision weighs l-tyrosylglycine 6.16mg and L-Trp 3.06mg is placed in 100mL volumetric flasks, is dissolved in water
Dilution, shakes up, constant volume, as mixing reference substance storing solution；
(2) the accurate reference substance storing solution that measures is appropriate, is diluted with water into every 1ml and contains l-tyrosylglycine and L-Trp
1.150-1.300 μ g, the solution of 0.550-0.660 μ g, to obtain the final product.
(3) four taste treasure's layer ice boron eye drops 10mL are drawn, 12,000r/min centrifugation 15min, take supernatant spare.
(4) chromatographic column used in HPLC is：Agilent, Eclipse XDB-C18 (3.0 × 150mm, 3.5 μm)；
(5) mobile phase, A phases are 0.1% trifluoroacetic acid (TFA)-water, and B phases are+70% acetonitrile water of 0.085% trifluoroacetic acid (TFA)
Solution；
(6) gradient elution is used：
Gradient elution program table
(7) flow velocity：0.4mL/min；
(8) Detection wavelength：220nm；
(9) sample size：20μL；
(10) column temperature：30℃；
(11) theoretical cam curve is calculated by l-tyrosylglycine is not less than 3000.
(12) this product contains nacreous layer powder per ml with l-tyrosylglycine (C11H14N2O4) meter, 0.99 μ g must not be less than, with L-
Tryptophan (C11H12N2O2) meter, 0.46 μ g must not be less than.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711210212.2A CN107991408B (en) | 2017-11-27 | 2017-11-27 | Method for measuring content of index component in four-flavor Zhenzhi eye drops |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711210212.2A CN107991408B (en) | 2017-11-27 | 2017-11-27 | Method for measuring content of index component in four-flavor Zhenzhi eye drops |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107991408A true CN107991408A (en) | 2018-05-04 |
| CN107991408B CN107991408B (en) | 2020-06-30 |
Family
ID=62032216
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711210212.2A Active CN107991408B (en) | 2017-11-27 | 2017-11-27 | Method for measuring content of index component in four-flavor Zhenzhi eye drops |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107991408B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110470779A (en) * | 2019-09-27 | 2019-11-19 | 江西珍视明药业有限公司 | The method for building up and its finger-print of four taste treasure's layer ice boron eye drops finger-print |
| CN113049729A (en) * | 2021-03-10 | 2021-06-29 | 广西中医药大学 | Control method for pearl quality standard in pearl compound preparation |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101788537A (en) * | 2009-09-28 | 2010-07-28 | 贵州益康制药有限公司 | Method for measuring content of ellagic acid ingredients in euscaphis japonica medicinal materials |
| CN101837072A (en) * | 2010-04-29 | 2010-09-22 | 贵州益康制药有限公司 | Method for detecting quality of medicinal preparation for curing hepatitis |
| CN102621265A (en) * | 2012-03-27 | 2012-08-01 | 贵州景峰注射剂有限公司 | Method for measuring contents of multiple components in Shenxiong glucose injection |
| CN102901783A (en) * | 2012-11-02 | 2013-01-30 | 江苏吉贝尔药业有限公司 | Content determination method for pranoprofen eye drops |
| CN104257692A (en) * | 2014-09-12 | 2015-01-07 | 江西珍视明药业有限公司 | Quality ensuring method of preparing aseptic four-pearl-layer bingpeng eye drops with non-terminal sterilization |
-
2017
- 2017-11-27 CN CN201711210212.2A patent/CN107991408B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101788537A (en) * | 2009-09-28 | 2010-07-28 | 贵州益康制药有限公司 | Method for measuring content of ellagic acid ingredients in euscaphis japonica medicinal materials |
| CN101837072A (en) * | 2010-04-29 | 2010-09-22 | 贵州益康制药有限公司 | Method for detecting quality of medicinal preparation for curing hepatitis |
| CN102621265A (en) * | 2012-03-27 | 2012-08-01 | 贵州景峰注射剂有限公司 | Method for measuring contents of multiple components in Shenxiong glucose injection |
| CN102901783A (en) * | 2012-11-02 | 2013-01-30 | 江苏吉贝尔药业有限公司 | Content determination method for pranoprofen eye drops |
| CN104257692A (en) * | 2014-09-12 | 2015-01-07 | 江西珍视明药业有限公司 | Quality ensuring method of preparing aseptic four-pearl-layer bingpeng eye drops with non-terminal sterilization |
Non-Patent Citations (2)
| Title |
|---|
| A. F. FELL 等: "Post-column continuous-flow analysis combined with reversed-phase liquid chromatography and computer-aided detection for the characterisation of peptides", 《JOURNAL OF CHROMATOGRAPHY》 * |
| 周平 等: "珍视明滴眼液中硼砂、硼酸及总硼的含量测定", 《中国实验方剂学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110470779A (en) * | 2019-09-27 | 2019-11-19 | 江西珍视明药业有限公司 | The method for building up and its finger-print of four taste treasure's layer ice boron eye drops finger-print |
| CN113049729A (en) * | 2021-03-10 | 2021-06-29 | 广西中医药大学 | Control method for pearl quality standard in pearl compound preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107991408B (en) | 2020-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113702541B (en) | Poria cocos medicinal material characteristic spectrum construction method and poria cocos triterpene component detection method | |
| CN104950052B (en) | A kind of method that Dementholized mint oil dripping pill quality is detected with gas chromatograph | |
| CN108426956A (en) | A kind of method of impurity F in Captopril by HPLC | |
| CN102495167A (en) | Method for detecting lycium barbarum polysaccharide in lycium barbarum polysaccharide extract | |
| CN107356691A (en) | Build the detection method of bent finger-print | |
| CN107991408A (en) | Index components content assay method in four taste treasure's layer ice boron eye drops | |
| CN106338564B (en) | A method of for detecting enantiomter in vildagliptin intermediate | |
| CN107515255A (en) | Utilize high performance liquid chromatograph measure Dapagliflozin and its method about material | |
| CN104914194B (en) | A method of with Determination of menthol in gas chromatograph detection Dementholized mint oil dripping pill | |
| CN110297061A (en) | Survey the methods for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content using one more | |
| CN105974035A (en) | Method for detecting content of vitamin D3 in vitamin D calcium chewable tablet preparation | |
| CN114034797B (en) | Method for measuring content of flower components of dendrobium nobile lindl | |
| CN105572240A (en) | Method for detecting content of pharmaceutic adjuvant carmine by using high performance liquid chromatography | |
| CN104965031B (en) | Content measuring method for compound ketoprofen and omeprazole sustained-release capsules | |
| CN108645933A (en) | The gas-chromatography detection method of residual solvent in Eliquis bulk pharmaceutical chemicals | |
| CN104090056A (en) | Method for determining content of hyodeoxycholic acid in Beiling capsules through HPLC (High Performance Liquid Chromatography)-ELSD (Evaporative Light Scattering Detector) method | |
| CN109932437A (en) | A kind of detection method of pharmaceutical composition | |
| CN104833757B (en) | Method of simultaneously determining multiple chemical drugs illegally added in soft capsule health food | |
| CN107782833A (en) | A kind of method and its finger-print for determining stilbene flat lung particle finger-print in vain | |
| CN104807940A (en) | Method for simultaneously determining multiple illegally-added chemical medicines in health-care food | |
| CN109507334A (en) | The detection method of amino acid content and the grade separation method of tabasheer in tabasheer | |
| CN102539562A (en) | Detection method for kidney tonifying and vigour nourishing mixture | |
| Qun et al. | Simultaneous determination of four active ingredients in Wuji Pellet by HPLC | |
| CN108956835A (en) | A kind of fingerprint atlas detection method of the antipyretic oral drugs of clearing | |
| CN106404960B (en) | The quality determining method of furan lightyellow sophora root berberine tablet |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |