CN103926350A - Inspection method of rehabilitation liquid formulation fingerprint and standard fingerprint - Google Patents
Inspection method of rehabilitation liquid formulation fingerprint and standard fingerprint Download PDFInfo
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- CN103926350A CN103926350A CN201410183701.3A CN201410183701A CN103926350A CN 103926350 A CN103926350 A CN 103926350A CN 201410183701 A CN201410183701 A CN 201410183701A CN 103926350 A CN103926350 A CN 103926350A
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Abstract
The invention relates to a drug detection method, in particular to an inspection method of a rehabilitation liquid formulation fingerprint and a standard fingerprint. The method is characterized in that the rehabilitation liquid formulation is made into a test sample, and the test sample is separated and detected through a high-efficient liquid-phase chromatography to obtain the fingerprint of the rehabilitation liquid formulation. The method comprises the following steps of a, preparing a reference product solution; b, preparing a test sample solution; and c, preparing the fingerprint. According to the inspection method of the rehabilitation liquid fingerprint and the standard fingerprint, characteristic substances are effectively represented, a method for evaluating the quality of rehabilitation liquid through overall fingerprint information is provided, the limitation for judging the quality of the rehabilitation liquid by only measuring one or two chemical components can be avoided, and the quality of the rehabilitation liquid can be accurate and objective. The fingerprint detection method is good in separation, reasonable in inspection time and good in repeatability and stability.
Description
Technical field
The present invention relates to a kind of detection method of medicine, particularly relate to inspection method and the standard finger-print of Kangfuxin Liquid preparation finger.
Background technology
Kangfuxin Liquid is taking rehabilitation newly as the solution (form of Chinese drug as mixture) of bulk drug through being processed into, and existing operative norm is that office issues " Kangfuxin Liquid WS
3-B-3674-2000(Z) " standard.Its bulk drug rehabilitation is newly the ethanol extract of the dry polypide of blattaria (American cockroach) Periploneta americana.
Former Kangfuxin Liquid quality standard WS
3-B-3674-2000(Z) in, only have pH value to check one to the inspection of Kangfuxin Liquid product, assay also only has one of total amino acid, cannot reflect really the feature of medicine.Traditional Chinese medicine fingerprint refers to that some Chinese crude drug or Chinese medicine preparation, after suitably processing, adopt certain analysis means, the chromatogram that can indicate its chemical feature or the spectrogram that obtain.Traditional Chinese medicine fingerprint is a kind of comprehensive, quantifiable identification of means, and it is to be based upon on the basis of chemical composition of Chinese materia medica systematic study, is mainly used in evaluating authenticity, Optimality and the stability of Chinese crude drug and Chinese medicine preparation semi-manufactured goods quality." globality " and " ambiguity " is its distinguishing feature.
Summary of the invention
The object of the present invention is to provide a kind of inspection method of Kangfuxin Liquid preparation finger, adopt the method can truly reflect the contained property material of Kangfuxin Liquid preparation, there is good separating effect, highly sensitive, the feature of the feature of medicine own can be truly reflected, thereby the true and false of this product can be differentiated.
For achieving the above object, technical scheme of the present invention is as follows:
The inspection method of Kangfuxin Liquid preparation finger, adopts Kangfuxin Liquid preparation is made to test sample, separates detection by high performance liquid chromatography, obtains the finger-print of Kangfuxin Liquid preparation;
Described method comprises following steps:
A. the preparation of reference substance solution: get uracil, hypoxanthine and inosine reference substance appropriate, accurately weighed, add water and make every 1mL and be respectively containing uracil, hypoxanthine and inosine the mixing contrast solution of 10 μ g, 25 μ g and 50 μ g, to obtain final product;
B. the preparation of need testing solution: get Kangfuxin Liquid preparation, filter, get subsequent filtrate, to obtain final product;
C. make finger-print: accurate reference substance solution and the each 10 μ L of need testing solution of drawing respectively, injection liquid chromatography separates and detects, and methanol solution (containing 0.07% the acetic acid)-methyl alcohol that wherein mobile phase is 2%～5%, adopts gradient elution; Obtain the finger-print of Kangfuxin Liquid.
High performance liquid chromatograph separates the condition detecting: filling agent is octadecylsilane chemically bonded silica; Mobile phase is 3% methanol solution (containing 0.07% acetic acid)-methyl alcohol; Adopt gradient elution: 0min → 10min → 20min → 21min → 35min → 42min, methyl alcohol 0% → 0% → 30% → 50% → 100% → 0%; Detecting wavelength is 254 nm; Column temperature is 25 DEG C; Flow velocity is 0.6mL/min; Number of theoretical plate calculates and is not less than 5000 by inosine peak.
As shown in Figure 1, adopt the Kangfuxin Liquid standard finger-print that obtains of the present invention to be characterised in that to have in test sample characteristic spectrum 5 characteristic peaks, wherein 3 identical with corresponding object of reference peak retention time respectively; With corresponding peak, inosine object of reference peak be S peak, the relative retention time of characteristic peak 3 is: 0.95-1.05min; The relative retention time of characteristic peak 4 is 0.969-1.071min; The relative retention time of characteristic peak 5 is 1.064-1.176min.
Advantage of the present invention is as follows:
1, Kangfuxin Liquid fingerprint checking method disclosed by the invention and finger-print thereof, carry out Efficient Characterization for its property material, a kind of method of Kangfuxin Liquid quality being evaluated by overall finger-print information is provided, avoid only measuring the wherein just one-sidedness to the judgement of Kangfuxin Liquid total quality of one or two kind of chemical composition, made Kangfuxin Liquid quality control more accurately objective.
2, fingerprint atlas detection method degree of separation provided by the invention is good, and the supervision time is reasonable, repeatability and good stability.
Brief description of the drawings:
Fig. 1 is the feature comparison collection of illustrative plates of Kangfuxin Liquid;
Fig. 2 is the negative control HPLC figure of Kangfuxin Liquid uracil, hypoxanthine, Inosine Content mensuration;
In figure: peak 1: uracil, peak 2: hypoxanthine, peak 3(S): inosine, peak 4: characteristic peak 4, peak 5: characteristic peak 5.
Embodiment:
Further illustrate the present invention below in conjunction with accompanying drawing, but protection scope of the present invention is not limited by embodiment.
Embodiment 1
(1) instrument and reagent
High performance liquid chromatograph: U.S. waters 2695 liquid chromatography instrument systems, the 1200(of Hewlett-Packard are all furnished with UV-detector, quaternary gradient pump, online degasser, automatic sampler, column oven).Chromatographic column: Wates Sunfire
tMc
18(250 × 4.6nm, 5 μ m) for chromatographic column.
Glacial acetic acid, methyl alcohol are chromatographically pure, and sodium dihydrogen phosphate, glycerine, Sodium Benzoate are that analysis is pure, ultrapure water (self-control).
12 batches of Kangfuxin Liquids: commercially available prod, 3 batches of each manufacturing enterprises.
Uracil, hypoxanthine, inosine lot number are respectively 100469-200401
,140661-200402,140669-200401, provide by Nat'l Pharmaceutical & Biological Products Control Institute.
(2) chromatographic condition and system suitability
Taking octadecylsilane chemically bonded silica as filling agent; Taking 3% methanol solution (containing 0.07% acetic acid), as mobile phase A, methyl alcohol is Mobile phase B, and the regulation according to the form below is carried out gradient elution; Flow velocity is per minute 0.5mL, detects wavelength 254 nm, 25 DEG C of column temperatures.Number of theoretical plate calculates and is not less than 5000 by inosine peak.
Mobile phase gradient is as follows.
Table one pyrimidine base analytic system binary gradient program
(3) linearity
Precision takes uracil, hypoxanthine, inosine reference substance respectively, is diluted to the mixing reference substance solution of seven kinds of variable concentrations, and sample size 10 μ l inject high performance liquid chromatograph.By the test of test income approach, taking peak area integrated value as ordinate, do linear regression taking sample size (μ g/mL) as horizontal ordinate, drawing standard curve, result (in table two～tetra-) show, uracil, hypoxanthine, inosine present linear relationship respectively between 4.92～34.44 μ g/mL, 12.438～87.069 μ g/mL, 24.772～173.406 μ g/mL, meet the requirements.
Table two uracil range of linearity measurement result
Table three hypoxanthine range of linearity measurement result
Table four inosine range of linearity measurement result
(4) precision test
Reference substance solution is repeated to sample introduction 6 times, the results are shown in Table five.Show that instrument precision is good.
Table five contrasts liquid Precision test result
(5) stability test
Choose a batch sample (lot number 10060402), prepare need testing solution.Get with a Kangfuxin Liquid need testing solution and same contrast liquid, interval different time carries out repeatedly sample size, the results are shown in Table six and table seven.Show that test sample and reference substance solution solution are good at 24 hours internal stabilities.
Table six Kangfuxin Liquid stability result (lot number 10060402)
Table seven contrasts liquid stability result
(6) replica test
Choose with a collection of Kangfuxin Liquid sample (lot number: 11022203), measure respectively its content 6 times according to content assaying method, result is as following table.Result shows the favorable reproducibility of method.
Table eight Kangfuxin Liquid replica test result
(7) average recovery test
Get Kangfuxin Liquid sample (lot number 10060603), each precision adds reference substance solution, makes the need testing solution of assay concentration approximately 80%, 100%, 120%, and every kind of concentration is prepared 3 parts, measures.Result is as following table.This law average recovery rate, between 95%～105%, meets the requirements.
Table nine Kangfuxin Liquid average recovery test findings
(8) specificity
Negative control test
The preparation of negative control sample solution: prepare the not solution of American-cockroach-extract-containing (rehabilitation is new) by Kangfuxin Liquid prescription, to obtain final product.
Prepare respectively reference substance solution and need testing solution according to test income approach.
Measure according to test income approach, result shows, with uracil, hypoxanthine, retention time position that inosine reference substance is identical, have no Interference Peaks and occur, show that the auxiliary material in prescription is noiseless to the mensuration of uracil, hypoxanthine, inosine.Contrast with need testing solution, have no obvious peak disappearance.As shown in Figure 2.
(9) serviceability test
A, dissimilar chromatographic column and instrument
Get with a collection of Kangfuxin Liquid sample (lot number: 11083019), the in the situation that of ceteris paribus, examine or check chromatographic column of the same type and the impact of different instrument on content results of different brands.Result (in table ten) shows that the chromatographic column of the same type of different brands and different instrument have no significant effect tested composition measurement result.
The investigation result of the dissimilar chromatographic column of table ten and different instruments
B, different in flow rate
Get with a collection of Kangfuxin Liquid sample (lot number: 11083019), the in the situation that of ceteris paribus, examine or check the impact of flow velocity on measurement result.Result (in table ten one) shows, change in flow has no significant effect tested composition chromatographic peak measurement result.
The investigation result of table ten flow velocity on measurement result impact
| Flow velocity (mL/min) | Uracil (μ g/mL) | Hypoxanthine (μ g/mL) | Inosine (μ g/mL) |
| 1.0ml/min | 22.163 | 44.597 | 59.174 |
| 0.8ml/min | 21.775 | 44.788 | 59.852 |
| 0.4ml/min | 21.519 | 44.803 | 60.446 |
| 0.5ml/min | 21.440 | 43.900 | 60.149 |
| RSD% | 1.50 | 0.95 | 0.91 |
C, column temperature
Get with a collection of Kangfuxin Liquid sample (lot number: 11083019), the in the situation that of ceteris paribus, examine or check the impact of column temperature on measurement result.Result (in table ten two) shows, when 20 DEG C, 25 DEG C, 30 DEG C, 40 DEG C of column temperatures, tested composition measurement result had no significant effect.
Table ten two column temperatures change the investigation on measurement result impact
| Temperature (DEG C) | Uracil (μ g/mL) | Hypoxanthine (μ g/mL) | Inosine (μ g/mL) |
| 20 | 21.865 | 44.820 | 58.786 |
| 25 | 22.014 | 44.911 | 60.045 |
| 30 | 22.163 | 44.597 | 59.174 |
| 40 | 22.411 | 44.722 | 59.332 |
| RSD% | 1.05 | 0.30 | 0.89 |
(10) detect wavelength
The DAD ultraviolet spectrogram of solution per sample, uracil, hypoxanthine and inosine maximum absorption wavelength are respectively 260,250,249nm wavelength place, select 254nm that absorption intensity is larger as detecting wavelength, and impurity disturbs less.
Embodiment 2
The inspection method of Kangfuxin Liquid preparation finger, adopts Kangfuxin Liquid preparation is made to test sample, separates detection by high performance liquid chromatography, thereby obtains the finger-print of Kangfuxin Liquid preparation, carries out as follows:
A. the preparation of reference substance solution: get uracil, hypoxanthine and inosine reference substance appropriate, accurately weighed, add water and make every 1mL and be respectively containing uracil, hypoxanthine and inosine the mixing contrast solution of 10 μ g, 25 μ g and 50 μ g, to obtain final product;
B. the preparation of need testing solution: the Kangfuxin Liquid preparation (lot number is 110915) of the good doctor Pan Xi production in Kangfuxin Liquid preparation (lot number is 10052016), Sichuan of promise production, the Kangfuxin Liquid preparation (lot number is 113020) of capital, Inner Mongol new production are matched in Kangfuxin Liquid preparation (lot number is M110830), the Kunming of getting respectively Hunan Central-South Cologne production, filter, get subsequent filtrate, to obtain final product;
C. make finger-print: accurate reference substance solution and the each 10 μ L of need testing solution of drawing respectively, injection liquid chromatography separates and detects, and wherein filling agent is octadecylsilane chemically bonded silica; Mobile phase is 2% methanol solution (containing 0.07% acetic acid)-methyl alcohol; Adopt gradient elution: 0min → 10min → 20min → 21min → 35min → 42min, methyl alcohol 0% → 0% → 30% → 50% → 100% → 0%; Detecting wavelength is 254 nm; Column temperature is 25 DEG C; Flow velocity is 0.6mL/min; Number of theoretical plate calculates and is not less than 5000 by inosine peak.Adopt gradient elution; Obtain the finger-print of Kangfuxin Liquid.
Embodiment 3
The inspection method of Kangfuxin Liquid preparation finger, adopts Kangfuxin Liquid preparation is made to test sample, separates detection by high performance liquid chromatography, thereby obtains the finger-print of Kangfuxin Liquid preparation, carries out as follows:
A. the preparation of reference substance solution: get uracil, hypoxanthine and inosine reference substance appropriate, accurately weighed, add water and make every 1mL and be respectively containing uracil, hypoxanthine and inosine the mixing contrast solution of 10 μ g, 25 μ g and 50 μ g, to obtain final product;
B. the preparation of need testing solution: the Kangfuxin Liquid preparation (lot number is 110916) of the good doctor Pan Xi production in Kangfuxin Liquid preparation (lot number is 10060301), Sichuan of promise production, the Kangfuxin Liquid preparation (lot number is 110308) of capital, Inner Mongol new production are matched in Kangfuxin Liquid preparation (lot number is M110831), the Kunming of getting respectively Hunan Central-South Cologne production, filter, get subsequent filtrate, to obtain final product;
C. make finger-print: accurate reference substance solution and the each 10 μ L of need testing solution of drawing respectively, injection liquid chromatography separates and detects, and wherein filling agent is octadecylsilane chemically bonded silica; Mobile phase is 3% methanol solution (containing 0.07% acetic acid)-methyl alcohol; Adopt gradient elution: 0min → 10min → 20min → 21min → 35min → 42min, methyl alcohol 0% → 0% → 30% → 50% → 100% → 0%; Detecting wavelength is 254 nm; Column temperature is 25 DEG C; Flow velocity is 0.6mL/min; Number of theoretical plate calculates and is not less than 5000 by inosine peak.Adopt gradient elution; Obtain the finger-print of Kangfuxin Liquid.
Embodiment 4
The inspection method of Kangfuxin Liquid preparation finger, adopts Kangfuxin Liquid preparation is made to test sample, separates detection by high performance liquid chromatography, thereby obtains the finger-print of Kangfuxin Liquid preparation, carries out as follows:
A. the preparation of reference substance solution: get uracil, hypoxanthine and inosine reference substance appropriate, accurately weighed, add water and make every 1mL and be respectively containing uracil, hypoxanthine and inosine the mixing contrast solution of 10 μ g, 25 μ g and 50 μ g, to obtain final product;
B. the preparation of need testing solution: the Kangfuxin Liquid preparation (lot number is 110917) of the good doctor Pan Xi production in Kangfuxin Liquid preparation (lot number is 10060402), Sichuan of promise production, the Kangfuxin Liquid preparation (lot number is 110606) of capital, Inner Mongol new production are matched in Kangfuxin Liquid preparation (lot number is M110901), the Kunming of getting respectively Hunan Central-South Cologne production, filter, get subsequent filtrate, to obtain final product;
C. make finger-print: accurate reference substance solution and the each 10 μ L of need testing solution of drawing respectively, injection liquid chromatography separates and detects, and wherein filling agent is octadecylsilane chemically bonded silica; Mobile phase is 5% methanol solution (containing 0.07% acetic acid)-methyl alcohol; Adopt gradient elution: 0min → 10min → 20min → 21min → 35min → 42min, methyl alcohol 0% → 0% → 30% → 50% → 100% → 0%; Detecting wavelength is 254 nm; Column temperature is 25 DEG C; Flow velocity is 0.6mL/min; Number of theoretical plate calculates and is not less than 5000 by inosine peak.Adopt gradient elution; Obtain the finger-print of Kangfuxin Liquid.
According to all uracils of embodiment 2-5, the data statistics result that hypoxanthine and Inosine Content are measured, each Characteristic chromatographic peak good separating effect, excipient is noiseless, taking inosine as object of reference, in Kangfuxin Liquid, can find 7 total peaks, but dissimilar pillar separates the total peak relative retention time obtaining and differs (exceed ± 10%) bigger than normal, the total peak relative retention time that the analysis of even same chromatographic column different time obtains also has certain difference, what total peak relative retention time was stable only has 4 except three object of reference peaks, 5 chromatographic peaks (as shown in Figure 1), for the etalon operability of this sample from now on, now be decided to be: in test sample characteristic spectrum, should have 5 characteristic peaks, wherein 3 should be identical with corresponding object of reference peak retention time respectively, with corresponding peak, inosine object of reference peak be S peak, the relative retention time at calculated characteristics peak 3～5, its relative retention time should setting ± 5% within.Setting is: 1.00 (peaks 3), 1.02 (peaks 4), 1.12(peak 5).Sample relative retention time the results are shown in Table 13.
The total peak of table ten three Kangfuxin Liquids relative retention time
Claims (3)
1. the inspection method of Kangfuxin Liquid preparation finger, is characterized in that Kangfuxin Liquid preparation to make test sample, separates detection by high performance liquid chromatography, obtains the finger-print of Kangfuxin Liquid preparation;
Described method comprises following steps:
A. the preparation of reference substance solution: get uracil, hypoxanthine and inosine reference substance appropriate, accurately weighed, add water and make every 1mL and be respectively containing uracil, hypoxanthine and inosine the mixing contrast solution of 10 μ g, 25 μ g and 50 μ g, to obtain final product;
B. the preparation of need testing solution: get Kangfuxin Liquid preparation, filter, get subsequent filtrate, to obtain final product;
C. make finger-print: accurate reference substance solution and the each 10 μ L of need testing solution of drawing respectively, injection liquid chromatography separates and detects, and methanol solution (containing 0.07% the acetic acid)-methyl alcohol that wherein mobile phase is 2%～5%, adopts gradient elution; Obtain the finger-print of Kangfuxin Liquid.
2. the inspection method of Kangfuxin Liquid preparation finger according to claim 1, is characterized in that high performance liquid chromatograph separates the condition detecting and is: filling agent is octadecylsilane chemically bonded silica; Mobile phase is 3% methanol solution (containing 0.07% acetic acid)-methyl alcohol; Adopt gradient elution: 0min → 10min → 20min → 21min → 35min → 42min, methyl alcohol 0% → 0% → 30% → 50% → 100% → 0%; Detecting wavelength is 254 nm; Column temperature is 25 DEG C; Flow velocity is 0.6mL/min; Number of theoretical plate calculates and is not less than 5000 by inosine peak.
3. the standard finger-print obtaining according to the inspection method of the Kangfuxin Liquid preparation Kangfuxin Liquid preparation finger described in claim 1,2, it is characterized in that in test sample characteristic spectrum, there are 5 characteristic peaks, wherein 3 identical with corresponding object of reference peak retention time respectively; With corresponding peak, inosine object of reference peak be S peak, the relative retention time of characteristic peak 3 is: 0.95-1.05min; The relative retention time of characteristic peak 4 is 0.969-1.071min; The relative retention time of characteristic peak 5 is 1.064-1.176min.
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| CN109324141A (en) * | 2018-12-18 | 2019-02-12 | 广州市药材公司中药饮片厂 | The characteristic fingerprint pattern and its construction method of a kind of wide dragon or its processed product and application |
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Application publication date: 20140716 |