CN113049729B - Control method of pearl quality standard in pearl compound preparation - Google Patents
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Abstract
The invention discloses a control method of pearl quality standard in pearl compound preparation, which aims to ensure the quality of pearl in compound, therefore, in the process of developing ' pearl honey oral liquid ', the invention systematically develops the quality standard research of pearl compound with multi-index detection, breaks through the problem that the traditional pearl Chinese patent medicine only measures calcium carbonate and total nitrogen and has weak standard specificity, establishes a quality standard system for measuring the characteristic glycine content of calcium salt, leucine and total nitrogen and the hydrolysis product of the pearl, effectively controls the internal quality of the pearl in the pearl honey oral liquid ' road and ground medicinal material and provides reference for comprehensively controlling the quality of pearl compound products.
Description
Technical Field
The invention relates to a control method for pearl quality standard in pearl compound preparation, belonging to the technical field of traditional Chinese medicine detection.
Background
Pearl is one of 11 marine Chinese medicinal materials which are carried in the pharmacopoeia of the people's republic of China, and is originally found in Jinge Yuan (urgent prescriptions for elbow backup), nanchong (official collection of Lei Paenii Lun), tang Dynasty screening rights (medicinal theory), li (herbal medicine for sea) and Song Dynasty pearl as a new added medicine to supplement "Kaibao Ben Cao". The pearl has definite drug property, efficacy and main treatment record, wide application, definite curative effect and good development value, and is expected to be developed and applied in the fields of senile diseases, immunodeficiency diseases, health maintenance and rehabilitation and the like.
The seawater pearl has sweet taste, salty taste, cold property, heart and liver meridian homing, and has the functions of nourishing yin, suppressing yang, detoxifying, promoting granulation and the like. Modern pharmacological research: seawater pearl contains abundant microelements and all 10 amino acids necessary for human body, and also contains 5 non-protein amino acids such as taurine, etc.; the hydrolyzed pearl is used to obtain active peptide, which is a cell repairing substance with small molecular weight. Modern pharmacological research proves that the pearl has the effect of enhancing the human body immunity regulation and is suitable for developing new preparations. The Hepu pearl is a marked product in Guangxi province, is representative of seawater medicinal pearls, is a road medicinal material accepted by the traditional Chinese medicine community and first pushed, but has no special detection standard in the compound.
According to the national drug administration database display: (1) the medicines containing pearl or pearl layer powder are approved to be marketed for 33 varieties, the quality standard is used for measuring 33 calcium carbonate or total nitrogen content and 0 amino acid content; 6 of the single components and 27 of the compound components; 29, 3 eye drops and 1 ointment are orally taken; the oral solid variety is prepared by taking 26 raw powder medicines and 0 hydrolyzed extract medicines. (2) In the aspect of pearl processing technology, only compound pearl oral liquid and ginseng pearl oral liquid respectively adopt the hydrochloric acid hydrolysis of pearl layer powder and pearl extract. Specifically, table 1 shows the results.
Table 1 on the market, the formulation of the medicines containing pearl or pearl powder, the number of manufacturers, the processing technique and the quality index
(statistics based on national drug administration database data)
According to the data analysis, pearl is mainly used as a medicine with raw powder, the absorption of organisms is affected, the specificity of the identification index of the pearl compound is not strong, and the quality control standard of the pearl Chinese patent medicine is to be improved.
Disclosure of Invention
The invention aims to develop a 'Zhenmi oral liquid' preparation on the basis of independently developing and acquiring 'Zhenmi tablet' (Chinese osmanthus medicinal preparation Z20140001) registered by a Chinese medicinal national medical institution preparation, develop the quality standard research of a pearl compound with multi-index detection in the aspect of quality control, establish the quality standard of calcium salt identification and leucine multi-component synchronous identification and total nitrogen and glycine content measurement, effectively control the internal quality of the 'Zhenmi oral liquid', and provide basis for further comprehensively controlling the quality of pearl compound products.
The technical scheme adopted by the invention is as follows: a method for controlling quality standard of pearl in pearl compound preparation comprises (1) identifying calcium salt; (2) total nitrogen content determination; (3) leucine TCL thin layer identification; (4) And (3) determining the content of glycine by HPLC, wherein the identification and detection standard is as follows: detecting calcium salt; leucine shows spots with the same color on the positions corresponding to the control medicine chromatogram and the control substance chromatogram, the spots are clear, and the separation effect is good; the total nitrogen content is not less than 10 ug/ml, and the glycine content is not less than 40 ug/ml.
The following technical scheme is a further optimized scheme:
the method for measuring the total nitrogen content is carried out according to a second method under the nitrogen measuring method of the rule 0704 of the fourth edition of the Chinese pharmacopoeia 2015, and after sulfuric acid is dripped into a Kjeldahl flask, hydrogen peroxide solution is added, so that the reaction rate is accelerated, the reaction is complete, and the reaction end point is that the solution is gradually clarified.
The concentration of the hydrogen peroxide solution was 30%, and the addition amount was 3ml.
The leucine TCL thin layer identification method comprises the following steps: taking 25ml of pearl compound preparation, adding 50ml of 6mol/L hydrochloric acid, refluxing, heating, hydrolyzing for 4 hours, cooling to room temperature, filtering, evaporating filtrate to dryness, adding 2ml of 70% ethanol into residue to dissolve, centrifuging, and taking supernatant as a sample solution; taking leucine reference substance, adding 70% methanol to obtain solution containing 0.5mg per 1ml, and taking the solution as reference substance solution; sucking 2 μl of the sample solution and 5 μl of the reference solution, respectively spotting on the same silica gel G thin layer plate, spreading with n-butanol-glacial acetic acid-water of 4:1:1 as developing agent, spreading distance of 7.5cm, taking out, air drying, spraying ninhydrin test solution, and heating at 105deg.C until the spot color is clear; spots of the same color appear in the sample chromatogram at positions corresponding to those of the control chromatogram.
The chromatographic conditions adopted for the HPLC content measurement of glycine are as follows: the chromatographic column takes aminopropyl functional group bonded silica gel as a filler; acetonitrile and methanol and water=6:2:2 are taken as a mobile phase A, a 0.05mol/L sodium acetate aqueous solution is taken as a mobile phase B, and the detection wavelength is 254nm; the sample injection amount is 10 μl; and elution was performed according to the following gradient ratio:
the pearl compound preparation is 'Zhenmi oral liquid', and each bottle of 'Zhenmi oral liquid' contains 1g of pearl powder, 2g of honey, 1g of citric acid and 100ml of water.
Compared with the prior art, the invention has the following beneficial effects: the invention provides a method for accurately and rapidly detecting the quality and the quantity of the pearl in the pearl compound preparation, which is simple to use, combines the quality and the quantity, has strong specificity, can complete the quality standard research of multi-index detection, establishes the measurement standard of the content of the characteristic glycine of the hydrolyzate of the Hepu pearl, synchronously identifies multiple components and checks the total nitrogen limit, and effectively controls the internal quality of the pearl compound preparation; meanwhile, provides reference for quality control of pearls in the compound preparation containing pearls, and has great application prospect.
Drawings
FIG. 1 is a technical roadmap for pearl quality standard of the pearl honey oral liquid in the embodiment of the invention.
FIG. 2 is a schematic diagram of a distillation apparatus; the marks in the figure: a-1000ml round bottom flask, B-safety flask, C-distiller with nitrogen balloon, D-funnel, E-straight condenser, F-100ml Erlenmeyer flask, G, H-rubber tube clamp.
FIG. 3 is a chromatogram of 10. Mu.l of a control drug solution and 2. Mu.l of a test solution with n-butanol-glacial acetic acid-water (8:3:1) as developing agent; wherein 1 is a control medicinal material solution; 2 is the sample solution.
FIG. 4 is a chromatogram of 8. Mu.l each of glutamic acid, leucine, valine, and lysine hydrochloride control solutions and 2. Mu.l each of test solutions using n-butanol-glacial acetic acid-water (4:1:0.5) as developing reagent; wherein 1 is glutamic acid; 2 is leucine; 3 is lysine; 4 is valine; and 5 is a test sample.
FIG. 5 is a chromatogram of 5. Mu.l each of glutamic acid and leucine control solution and 2. Mu.l of test solution with n-butanol-glacial acetic acid-water (4:1:1) as developing agent; wherein 1 is a sample; 2 is glutamic acid; and 3 is leucine.
FIG. 6 is a chromatogram of leucine at 5. Mu.l each of three sample solutions at 2. Mu.l each with n-butanol-glacial acetic acid-water (4:1:1) as developing solvent; wherein 1-3 are respectively sample solutions (ZHENMIKOU liquid 20191223, 20191225, 20191227); 4 is leucine.
FIG. 7 is a chromatogram of 2. Mu.l each of the test solution, 5. Mu.l of leucine solution, and 2. Mu.l of negative control solution, with n-butanol-glacial acetic acid-water (4:1:1) as developing agent; wherein 1-3 is sample solution (hydrolyzed south pearl honey oral suspension 20191223, 20191225, 20191227); 4 is leucine; and 5 is a negative control.
FIG. 8 is a chromatogram of 2. Mu.l each of the test solution, 5. Mu.l of leucine solution, and 2. Mu.l of negative control solution at 30 ℃; wherein 1-3 are test samples; 4 is leucine; and 5 is a negative sample.
FIG. 9 is a chromatogram of 2. Mu.l each of the test solution, 5. Mu.l of leucine solution, and 2. Mu.l of negative control solution at 8deg.C; wherein 1-3 are test samples; 4 is leucine; and 5 is a negative sample.
FIG. 10 is a chromatogram showing 2. Mu.l each of the test solution, 5. Mu.l of leucine solution, 2. Mu.l of negative control solution, and 7.5cm in span; wherein 1-3 are test samples; 4 is leucine; and 5 is a negative sample.
FIG. 11 is a chromatogram showing 2. Mu.l each of the test solution, 5. Mu.l of leucine solution, 2. Mu.l of negative control solution, and a span of 8 cm; wherein 1-3 are test samples; 4 is leucine; and 5 is a negative sample.
FIG. 12 is a chromatogram of each solution at a further temperature of 30deg.C, a humidity of 70RH, and a span of 7.5 cm; wherein 1-3 are samples (20191223, 20191225, 20191227) of hydrolyzed south pearl honey oral suspension; 4 is leucine reference substance; and 5 is a negative control of the pearl powder lack.
Detailed Description
In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is apparent that the embodiments described below are only some embodiments of the present invention, not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
1. Pearl compound preparation
The pearl honey oral liquid (hereinafter referred to as the product) contains 1g of pearl powder, 2g of honey, 1g of citric acid and 100ml of water in each bottle.
2. Authentication method
Identification of (one) calcium salts
Because the main component of the product contains a large amount of calcium carbonate salt, the product is subjected to physicochemical identification according to the rule of calcium salt in general identification test of 0301 in the fourth edition of the general edition of Chinese pharmacopoeia 2015.
The specific identification method comprises the following steps: shaking up the product, taking 10ml, adding 2 drops of methyl red indicator liquid, neutralizing with ammonia test liquid, then adding hydrochloric acid dropwise until the mixture is just acidic, and adding ammonium oxalate test liquid to generate white precipitate; the precipitate was separated and insoluble in acetic acid but soluble in dilute hydrochloric acid.
Taking 3 batches of pilot-scale finished products for calcium salt identification test. The test results are shown in Table 2.
Table 23 results of pilot-scale product calcium salt identification test
| Lot number | Authentication result |
| 20191223 | Detection of calcium salt |
| 20191225 | Detection of calcium salt |
| 20191227 | Detection of calcium salt |
(II) determination of total Nitrogen content
The main medicinal materials of the product are pearl powder, wherein the pearl powder contains macro elements, microelements and proteins (18 amino acids can be obtained after hydrolysis, 7 amino acids are essential amino acids of human body), etc.
The raw medicine amount of the pearl powder in each bottle is 1g, and the nitrogen content of the medicinal materials is about 3 mg; after the medicinal materials are hydrolyzed, the nitrogen content of each bottle of the product is about 2.0-2.5 mg. The measurement is carried out according to the second method (semimicro method) under the rule 0704 nitrogen measurement method of the fourth edition of the Chinese pharmacopoeia 2015. During the measurement, the following steps are found: the reaction time of the sample to be tested in a Kelvin flask is too long, and the reaction is incomplete. The product is difficult to turn into clear green color due to the fact that the product contains more honey.
The invention thus makes the following improvements:
1. after sulfuric acid is added into the Ke flask in a dropwise manner, 3ml of 30% hydrogen peroxide solution is added, so that the oxidative decomposition of the to-be-detected sample can be accelerated, the reaction speed can be accelerated, and the reaction can be completed.
2. The end point of the reaction is changed from the solution to "clear green" to the solution "gradually clear (with little precipitation)".
The final determination method comprises the following steps: the distillation apparatus is shown in FIG. 2. Connecting a distillation device, adding a proper amount of water into a bottle A and a plurality of drops of methyl red indicator liquid, adding dilute sulfuric acid to make the bottle A acidic, adding glass beads or zeolite particles, adding about 50ml of water from a funnel D, closing a clamp G, opening condensed water, boiling the water in the bottle A, removing a fire source when vapor is condensed from the tip of a condensing tube, closing the clamp H, reversely pumping the water in a bottle C into a bottle B, opening the clamp G, discharging the water in the bottle B, closing the bottle B and the clamp G, inserting the tip of the condensing tube into about 50ml of water, reversely pumping the water from the tip of the condensing tube into the bottle C, and pumping the water into the bottle B. The inside of the instrument was washed 2 to 3 times.
Shaking up the product, precisely weighing 10ml, placing the product in a dry 50ml Kjeldahl flask, adding 0.3g of potassium sulfate (or anhydrous sodium sulfate) and 5 drops of 30% copper sulfate solution, then adding 2.0ml of sulfuric acid dropwise along the wall of the flask, finally adding 3ml of 30% hydrogen peroxide solution, and shaking up fully; a small funnel is placed at the mouth of a Ke's flask, the flask is inclined at 45 degrees, the solution is kept below the boiling point by slow heating with small fire, the boiling is stopped, the firepower is gradually increased, the solution is boiled until the solution is gradually clarified (a little sediment exists), and then the heating is continued for 10 minutes, and the solution is cooled.
10ml of 2% boric acid solution is taken and placed in a 100ml conical flask, 5 drops of methyl red-bromocresol green mixed indicator solution are added, and the tip of a condensing tube is inserted under the liquid surface of a person. Then, the content in the Ke's flask was transferred to a C distillation flask via a D funnel, the Ke's flask and the funnel were rinsed with a small amount of water several times, 10ml of 40% sodium hydroxide solution was further added, the funnel was rinsed with a small amount of water several times, the G clamp was closed, the A flask was heated to conduct steam distillation until the boric acid solution started to turn from reddish wine to bluish-green, distillation was continued for 10 minutes, the tip of the condenser tube was lifted out of the liquid surface, the steam was continuously rinsed for about 1 minute, and the distillation was stopped after rinsing the tip with water.
The distillate was titrated with sulfuric acid titration (0.005 mol/L) until the solution changed from bluish green to grey purple, and the result of the titration was corrected with a blank test. Each 1ml of sulfuric acid titration (0.005 mol/L) corresponds to 0.1401mg of N.
Sample measurement:
three pilot runs (20191223, 20191225, 20191227) were taken 2 bottles each and assayed according to the finalization method. The results are shown in Table 3.
Table 33 results of total Nitrogen determination for pilot plant products
As a result, the maximum amount of the sample in the 3 batches was 2.45 mg/bottle, the minimum amount was 2.33 mg/bottle, and the average amount was 2.40 mg/bottle. Considering the complexity of medicinal material sources and the stability of hydrolysate, the limit is adjusted downwards according to the average value, and the total nitrogen (N) in each bottle of the product is set to be not less than 1mg.
(III) leucine TCL thin layer identification
1. Experimental conditions
Sample: zhenmi oral liquid, lot number: 20191223, 20191225, 20191227
Domestic silica gel G prefabricated plate: qingdao ocean chemical Co., ltd.
Control medicinal material and control: pearl control drug (121022-201604, national food and drug verification institute); glutamic acid reference substance (140690-201604, national food and drug verification institute); leucine control (140690-201604, national food and drug verification institute); valine control (140690-201604, national food and drug verification institute); lysine hydrochloride control (140673-201009, china food and drug testing institute).
The reagents used were all analytically pure.
2. Preparing test solution, control medicinal material solution, control solution and negative control solution
2.1 preparation of sample solution: shaking up the product, taking 25ml, adding 50ml of 6mol/L hydrochloric acid, refluxing and heating for hydrolysis for 4 hours, cooling to room temperature, filtering, evaporating filtrate to dryness, adding 2ml of 70% ethanol into residues to dissolve, centrifuging, and taking supernatant as a sample solution.
2.2 preparation of control drug solution: taking 20mg of reference medicinal material, adding 10ml of dilute hydrochloric acid, soaking for 20 minutes, adding 50ml of 6mol/L hydrochloric acid, refluxing, heating and hydrolyzing for 4 hours, cooling to room temperature, filtering, and evaporating filtrate to dryness. The residue was dissolved in 2ml of 70% ethanol to prepare a control solution.
2.3 preparation of control solution: taking glutamic acid, leucine, valine and lysine hydrochloride, and respectively adding 70% methanol to prepare a reference substance solution of 0.5 mg/mL.
2.4 preparation of negative control solution: taking a negative sample without pearl powder, and preparing a negative control solution according to a preparation method of a sample solution.
3. Inspection of reference medicinal materials and reference substances
3.1 investigation of control herbs
10 μl of the control medicinal material solution and 2 μl of the test solution are sucked and respectively spotted on the same silica gel G thin layer plate, and n-butanol-glacial acetic acid-water (8:3:1) is used as developing agent for developing. And (5) taking out. Air-dried, sprayed with ninhydrin test solution, and heated at 105deg.C until the spots develop clearly (see FIG. 3).
Results: the sample solution is prepared by using a sample prepared from the Hepu pearl, one main spot in the reference medicinal material cannot be detected in the sample, and the main spot possibly is caused by the complexity of the medicinal material and possibly related to the production place and the variety, so the reference medicinal material is considered not to be used as a reference standard for detection, but is used for thin-layer identification; in addition, the spectrum unfolding and separating effects are not ideal, and the proportion of the unfolding agent needs to be adjusted.
3.2 inspection of control
3.2.1 sucking 8. Mu.l each of glutamic acid, leucine, valine and lysine hydrochloride control solution and 2. Mu.l each of the test solution, spotting on the same silica gel G thin layer plate, and developing with n-butanol-glacial acetic acid-water (4:1:0.5) as developing agent. And (5) taking out. Air-dried, sprayed with ninhydrin test solution, and heated at 105deg.C until the spots develop clearly (see FIG. 4).
Results: in the chromatogram of the sample, spots with the same color are displayed on the positions corresponding to the chromatogram of the reference sample, and spots with the same color are not displayed on valine and lysine, so that the glutamic acid and leucine are considered to be reserved for further investigation; in addition, the chromatographic separation of the sample is not clear enough, and the proportion of the developing agent can be further optimized.
3.2.2 sucking 5. Mu.l each of glutamic acid and leucine reference solution and 2. Mu.l of test solution, respectively spotting on the same silica gel G thin layer plate, and developing with n-butanol-glacial acetic acid-water (4:1:1) as developing agent. And (5) taking out. Air-dried, sprayed with ninhydrin test solution, and heated at 105deg.C until the spots develop clearly (see FIG. 5).
The results show that: in the chromatogram of the sample, leucine spots are in the same color and clear at the positions corresponding to the chromatogram of the reference sample, and glutamic acid is not in the corresponding spots.
3.2.3 sucking leucine 5 μl, and adding 2 μl of each of the three sample solutions onto the same silica gel G thin layer plate, and spreading with n-butanol-glacial acetic acid-water (4:1:1) as spreading agent. And (5) taking out. Air-dried, sprayed with ninhydrin test solution, and heated at 105deg.C until the spots develop clearly (see FIG. 6).
The results show that: in the chromatogram of the sample, leucine spots have the same color and are clear at the positions corresponding to the chromatogram of the reference sample, and the thin-layer separation effect is good.
4. Investigation of specificity
Sucking 2 μl of each sample solution, 5 μl of leucine solution and 2 μl of negative control solution, respectively spotting on the same silica gel G thin layer plate, and developing with n-butanol-glacial acetic acid-water (4:1:1) as developing agent. And (5) taking out. Air-dried, sprayed with ninhydrin test solution, and heated at 105deg.C until the spots develop clearly (see FIG. 7).
The results show that: in the chromatogram of the sample, leucine shows spots with the same color on the positions corresponding to the chromatograms of the control medicinal materials and the control substance, the negative control substance is interfered, the specificity is strong, the spots are clear, and the separation effect is good.
5. Method durability investigation
Mu.l of each of the test solution and 5. Mu.l of the leucine solution were aspirated, and 2. Mu.l of the negative control solution were developed and developed at 30℃and 8℃respectively, and observed (see FIG. 8 and FIG. 9).
The results show that: the temperature has little influence on the unfolding effect.
6. Spread investigation
Mu.l of each sample solution, 5. Mu.l of leucine solution and 2. Mu.l of negative control solution were pipetted onto the same silica gel G thin layer plate with a spreading distance of 7.5cm and 8cm, respectively, and developed, developed and observed (see FIG. 10, FIG. 11).
The results show that: when the span is 8cm, the reference substance spots and the test sample spots at corresponding positions are not aligned, so that the identification span is determined to be 7.5cm.
7. Final method
Shaking up the product, taking 25ml, adding 50ml of 6mol/L hydrochloric acid, refluxing and heating for hydrolysis for 4 hours, cooling to room temperature, filtering, evaporating filtrate to dryness, adding 2ml of 70% ethanol into residues to dissolve, centrifuging, and taking supernatant as a sample solution. Taking leucine reference substance, adding 70% methanol to obtain solution containing 0.5mg per 1ml, and making into reference substance solution. According to a thin layer chromatography (China pharmacopoeia 2015 edition four general rules 0502), 2 μl of the test solution and 5 μl of the control solution are sucked, the test solution and the control solution are respectively spotted on the same silica gel G thin layer plate, the mixture is spread by using n-butanol-glacial acetic acid-water (4:1:1) as a developing agent, the spreading distance is 7.5cm, the mixture is taken out, dried, and sprayed with ninhydrin test solution, and the mixture is heated at 105 ℃ until the spot color development is clear. Spots of the same color appear in the sample chromatogram at positions corresponding to those of the control chromatogram.
Three batches of samples were identified (see FIG. 12).
HPLC (high performance liquid chromatography) determination of Glycine content
The method for measuring the glycine content of the product by adopting the high performance liquid chromatography is established, and the separation of each peak is good, which shows that the method is not interfered by other components, has strong specificity, good reproducibility and stability, and simple and convenient method, and can achieve the aim of better controlling the internal quality of the product. Therefore, the glycine content of the product is measured by a high performance liquid chromatography method and is used as an index for controlling the quality of the product. The experimental results are as follows:
1. selection of experimental conditions
1.1 selection of chromatographic columns
The separation is carried out by using amino chromatographic columns of different brands such as Shimadzu, ultimateXB, venusilAA and the like, and the glycine peak and the adjacent impurity peak in the HPLC chromatogram of the sample liquid can be well separated, and the column plate number is more than 4000, as shown in Table 4. Therefore, the theoretical plate number (n) should be not lower than 4000 in terms of glycine peak.
TABLE 4 test results of the applicability of the present system
1.2 selection of mobile phases
Acetonitrile was compared separately: methanol: water (6:2:2) mobile phase a, acetonitrile: methanol: water (4:1:1) is mobile phase a, acetonitrile: methanol: water (3:2:1) as mobile phase a, resulting in acetonitrile: methanol: the measured component glycine peak shape was good with water (6:2:2) as mobile phase a as mobile phase and baseline separation was achieved with the other components.
1.3 selection of detectors
An ultraviolet detector is used.
2. Methodology investigation and determination of samples
2.1 instruments, reagents and samples
Instrument: the device comprises an Shimadzu LC-10AT high performance liquid chromatograph, an Shimadzu SPD-10Avp ultraviolet detector and a Wimatron chromatographic data workstation.
Glycine control: provided by the Chinese medicine biological product verification (batch number: 140689-201103 for content determination).
Reagent and sample: acetonitrile and methanol are chromatographic purity, water is distilled water, 10 batches of pearl honey oral liquid and 1 batch of negative control products without pearl powder.
2.2 methods and results
2.2.1 chromatographic conditions
Chromatographic column: aminopropyl functional group bonded silica gel is used as a filler; mobile phase: acetonitrile: methanol: water (6:2:2) as mobile phase A,0.05mol/L sodium acetate aqueous solution (pH value adjusted by glacial acetic acid is 6.50+/-0.05) as mobile phase B, and gradient elution is carried out according to the specification in the following table; wavelength: the detection wavelength is 254nm; sample injection amount: 10 μl.
TABLE 5 gradient elution ratio
2.2.2 preparation of stock solutions
Glycine stock solution: precisely weighing glycine reference substance 0.90mg, placing in 25ml volumetric flask, adding 70% methanol, shaking, and diluting to scale.
Phenyl isothiocyanate solution: 500. Mu.l of phenyl isothiocyanate was fixed to a volume of 20ml with acetonitrile to obtain a 0.2mol/L phenyl isothiocyanate solution.
Triethylamine solution: 1.4ml of triethylamine was fixed to 10ml with acetonitrile to obtain a 1.0mol/L triethylamine solution
2.2.3 preparation of control solution
Precisely weighing 4ml glycine use solution, placing into a 10ml volumetric flask, and precisely adding 2ml phenylisothiocyanate solution and triethylamine solution respectively. Shaking up, reacting for 1h at room temperature, and then adding water to the scale. Shaking to obtain the final product.
2.2.4 preparation of sample solutions
Shake the product, precisely measure 25ml, place in a flat bottom flask, and add 3 parts. Then, 6mol/L of hydrochloric acid 40, 50 and 60ml was added precisely. Precisely weighing. And heating and refluxing for 4 hours. Cool to room temperature. The weight loss was made up with 6mol/L hydrochloric acid. Shaking and filtering. The subsequent filtrate was measured in an evaporation dish with a precision of 50ml and evaporated to dryness. The residue was taken up in a 25ml volumetric flask with 70% methanol. Precisely weighing 4ml, placing in a 10ml volumetric flask, and precisely adding 2ml of each of the phenylisothiocyanate solution and the triethylamine solution. Shaking up, reacting for 1h at room temperature, and then adding water to the scale. Centrifuging the solution, and collecting supernatant.
TABLE 6 extraction solvent Screen results
| Extraction of |
40 | 50 | 60 |
| Glycine content (mg/g) | 0.750 | 0.843 | 0.833 |
2.2.5 linear relationship investigation
Precisely sucking the glycine reference substance solutions 2, 4, 8, 10, 12 and 14 mu L, carrying out sample injection analysis according to the chromatographic conditions, measuring the peak area, and carrying out linear regression on the sample injection quantity (mu g) (X) according to the peak area (Y), wherein regression equations are respectively obtained: y= 7389814X-15308, r=0.9998. The experimental results show that glycine is in the range of 0.0288-0.2016 μg, and the peak area (Y) and the sample injection amount (μg) (X) have good linear relationship (n=6).
2.2.6 precision test
The content of the same sample solution (lot number: 20191223) was measured 6 times continuously under the planned chromatographic conditions. Results the average of the 6 measurements is: 1058455, rsd=0.50% (n=6). See Table 7 for details. Experiments show that the method has good precision.
TABLE 7 Glycine precision test results
2.2.7 repeatability test
For the same batch (batch number: 20191223), 6 samples were measured in parallel according to the proposed method, and the results are shown in Table 8. The average of 6 measurements is: 865 μg/g, rsd=0.59% (n=6). The results show that the repeatability of the method is better.
TABLE 8 Glycine repeatability test results (measurement results are average of two samples)
2.2.8 stability test
The same sample solution (lot number: 20191223) was measured at regular intervals 6 times. The results of 6 determinations have a peak area average of 987982, rsd=0.68% (n=6), as detailed in table 9. The results indicated that the test solution was stable over 24 hours.
TABLE 9 Glycine stability test results
2.2.9 negative test
Taking a negative control sample of the pearl powder, and preparing the negative control solution according to a preparation method of a sample solution. Measured according to the established chromatographic conditions. The negative control showed no absorption peak at the glycine peak position, indicating that other ingredients in the formulation had no interference with the assay.
2.2.10 measurement of recovery rate of sample addition
About 25ml of the same batch of samples (batch number: 20191223) with known content is taken, 9 parts are taken, the samples are precisely weighed and divided into 3 groups, and each group is respectively 1 according to the content of the samples and the amount of the reference substances: 0.8, 1:1. 1:1.2 adding glycine reference substance, and determining according to the preparation method and chromatographic conditions of the sample solution, wherein the average recovery rate of glycine is 99.33% and RSD is 1.04%. (n=9) meets the quantitative analysis requirements, as detailed in table 10.
TABLE 10 Glycine recovery measurement (measurement is average of two samples)
2.2.11 sample assay
The glycine content of 10 samples of the product was determined according to the proposed content determination method, and the results are shown in Table 11, wherein the highest 1331 mug/25 ml and the lowest 1208 mug/25 ml of 10 samples have an average content of 1269 mug/25 ml. Considering the complexity of medicinal materials, the limit is about 30% of the average value, and the original standard is kept that each bottle of the product contains 100ml glycine (C) 2 H 5 NO 2 ) And not less than 3000. Mu.g. Namely 1ml of glycine (C) 2 H 5 NO 2 ) Not less than 30. Mu.g.
TABLE 11 sample measurement results
Claims (2)
1. A control method of pearl quality standard in pearl compound preparation is characterized in that the method comprises (1) calcium salt identification, (2) total nitrogen content measurement, (3) leucine TLC thin layer identification and (4) glycine HPLC content measurement; the identification detection standard is as follows: detecting calcium salt; leucine shows spots with the same color on the positions corresponding to the control medicine chromatogram and the control substance chromatogram, the spots are clear, and the separation effect is good; the total nitrogen content is not less than 10ug per ml, and the glycine content is not less than 40ug per ml;
the pearl compound preparation is 'pearl honey oral liquid', and each bottle of 'pearl honey oral liquid' contains 1g pearl powder, 2g honey, 1g citric acid and 100ml water;
the method for measuring the total nitrogen content is carried out according to a second method under the rule 0704 nitrogen measurement method of the fourth edition of the Chinese pharmacopoeia 2015, and after sulfuric acid is added into a Kjeldahl flask in a dropwise manner, hydrogen peroxide solution is added, so that the reaction rate is accelerated, the reaction is complete, and the reaction end point is that the solution is gradually clarified; the addition amount of the sulfuric acid is 2ml, the concentration of the hydrogen peroxide solution is 30%, and the addition amount is 3ml;
the chromatographic conditions adopted for the HPLC content measurement of glycine are as follows: the chromatographic column takes aminopropyl functional group bonded silica gel as a filler; acetonitrile and methanol and water=6:2:2 are taken as a mobile phase A, a 0.05mol/L sodium acetate aqueous solution is taken as a mobile phase B, and the detection wavelength is 254nm; the sample injection amount is 10 μl; and elution was performed according to the following gradient ratio:
2. the method for controlling the quality standard of pearl in a compound pearl preparation according to claim 1, wherein the method for identifying the leucine TLC thin layer is as follows: taking 25ml of pearl compound preparation, adding 50ml of 6mol/L hydrochloric acid, refluxing, heating, hydrolyzing for 4 hours, cooling to room temperature, filtering, evaporating filtrate to dryness, adding 2ml of 70% ethanol into residue to dissolve, centrifuging, and taking supernatant as a sample solution; taking leucine reference substance, adding 70% methanol to obtain solution containing 0.5mg per 1ml, and taking the solution as reference substance solution; sucking 2 μl of the sample solution and 5 μl of the reference solution, respectively spotting on the same silica gel G thin layer plate, spreading with n-butanol-glacial acetic acid-water of 4:1:1 as developing agent, spreading distance of 7.5cm, taking out, air drying, spraying ninhydrin test solution, and heating at 105deg.C until the spot color is clear; spots of the same color appear in the sample chromatogram at positions corresponding to those of the control chromatogram.
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