CN117451863A

CN117451863A - Fingerprint detection method for nucleoside and amino acid components in centipede pharmaceutical preparation

Info

Publication number: CN117451863A
Application number: CN202310937552.4A
Authority: CN
Inventors: 江斌; 蔡小兵; 张辉; 谭沛; 郑晓英; 李璐; 梁素仪
Original assignee: China Resources Sanjiu Modern Traditional Chinese Medicine Pharmaceutical Co ltd
Current assignee: China Resources Sanjiu Modern Traditional Chinese Medicine Pharmaceutical Co ltd
Priority date: 2023-07-27
Filing date: 2023-07-27
Publication date: 2024-01-26

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, and discloses a fingerprint detection method for nucleoside and amino acid components in a centipede medicinal preparation. By adopting the fingerprint detection method, the fingerprint of the centipede medicinal preparation can be constructed, the obtained fingerprint has strong characteristics and rich chromatographic information, and the chemical component characteristics of the centipede medicinal preparation can be fully displayed; in addition, the detection method has the advantages of good separation degree, high precision, good stability and repeatability, and can comprehensively and rapidly detect nucleotide components, amino acid components and the content thereof in the centipede medicinal preparation.

Description

Fingerprint detection method for nucleoside and amino acid components in centipede pharmaceutical preparation

Technical Field

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a fingerprint detection method for nucleoside and amino acid components in a centipede medicinal preparation, and further relates to a fingerprint detection method, a content measurement method and a quality control method for nucleoside and amino acid components in the centipede medicinal preparation.

Background

The centipede formula particles are prepared by extracting, concentrating and granulating traditional Chinese medicine centipedes. The Chinese medicine centipede is a dry body of centipede Scolopendra subspinipes mutilans L.Koch of centipede family animals, mainly contains various components such as amino acids, nucleoside compounds and the like, and is commonly used for calming endogenous wind and relieving spasm, dredging collaterals and relieving pain, and counteracting toxic substances and resolving masses.

The quality control of centipede in the 2020 edition of Chinese pharmacopoeia mainly comprises the projects of raw material varieties, decoction piece processing, decoction piece characters, physicochemical identification and the like, and some documents describe chemical components in centipede to indicate that the centipede contains nucleoside, amino acid and other components. However, due to the difference of the properties of the decoction pieces and the pharmaceutical preparation, the quality detection method applicable to the centipede decoction pieces cannot be applicable to the quality detection of the centipede pharmaceutical preparation.

There are also quality detection methods for centipede pharmaceutical preparations in the related art, but these methods generally can detect only a single component, and it is difficult to rapidly detect the quality of the centipede pharmaceutical preparation as a whole.

Disclosure of Invention

In view of the above, the invention provides a fingerprint detection method, a content measurement method and a quality control method for nucleoside and amino acid components in a centipede medicinal preparation, so as to solve the problem that the centipede medicinal preparation is difficult to quickly detect the quality of the centipede medicinal preparation on the whole by utilizing the related technology.

In a first aspect, the invention provides a fingerprint detection method for nucleoside and amino acid components in a centipede pharmaceutical preparation, comprising the following steps:

Taking a sample solution, and detecting by adopting a high performance liquid chromatography; wherein the chromatographic conditions of the high performance liquid chromatography include:

the octadecylsilane chemically bonded silica is used as a filler, methanol is used as a mobile phase A, water is used as a mobile phase B, gradient elution is carried out, and the gradient elution program comprises:

0-15 min, wherein the volume percentage of methanol in the mobile phase is 1%;

15-30 min, wherein the volume percentage of methanol in the mobile phase is 1% -12%;

30-45 min, and the volume percentage of methanol in the mobile phase is 12% -40%.

According to the fingerprint detection method for the nucleoside and amino acid components in the centipede medicinal preparation, octadecylsilane chemically bonded silica is used as a filler, acetonitrile-water is used as a mobile phase for gradient elution, and a specific elution program is selected, so that the fingerprint containing 8 common characteristic peaks can be detected, the number of the common characteristic peaks is remarkably improved, and good separation of the common characteristic peaks is realized; the fingerprint detection method has simple elution program, stable obtained fingerprint baseline, good peak shape of each characteristic peak and high separation degree among each characteristic peak, and can accurately position peak positions of 8 characteristic peaks such as uracil, tyrosine, hypoxanthine, xanthine, phenylalanine, inosine, guanosine, tryptophan and the like, thereby being beneficial to comprehensive quality detection and overall quality control of the centipede medicinal preparation, further being beneficial to improving the use safety and stability uniformity of the medicament and providing basis for quality detection and control of the centipede medicinal preparation.

By adopting the fingerprint detection method, the fingerprint of the centipede medicinal preparation can be constructed, the obtained fingerprint has strong characteristics and rich chromatographic information, and the chemical component characteristics of the centipede medicinal preparation can be fully displayed; in addition, the detection method has the advantages of good separation degree, high precision, good stability and repeatability, and can comprehensively and rapidly detect nucleotide components, amino acid components and the content thereof in the centipede medicinal preparation.

In an alternative embodiment, the chromatographic conditions of the high performance liquid chromatography further comprise at least one of the following conditions:

1) The detection wavelength is 205 nm-215 nm;

2) The column temperature is 23-27 ℃;

3) The flow rate is 0.9 mL/min-1.1 mL/min;

4) The sample injection amount is 5-15 mu L;

5) The chromatographic column is Dikma Platisil ODS-C18 chromatographic column, has an inner diameter of 4.6mm, a column length of 250mm and a particle diameter of 5 μm.

In an alternative embodiment, the gradient elution procedure further comprises:

45-50 min, wherein the volume percentage of methanol in the mobile phase is 40% -45%;

50-55 min, wherein the volume percentage of methanol in the mobile phase is 45%;

55-56 min, wherein the volume percentage of methanol in the mobile phase is 45% -1%;

56-60 min, and the volume percentage of methanol in the mobile phase is 1%.

In an alternative embodiment, the method of preparing the test solution is as follows:

adding solvent into a sample, extracting, performing solid-liquid separation, and collecting liquid to obtain a sample solution;

optionally, the solvent is a methanol aqueous solution, and the volume percentage of methanol in the methanol aqueous solution is 20% -40%;

alternatively, the solvent is added in an amount of 20 mL-30 mL relative to 0.5g of the sample;

optionally, the extraction is ultrasonic extraction, and the ultrasonic time is 15-45 min.

In an optional embodiment, the fingerprint detection method further includes a step of preparing a reference solution by using a hypoxanthine reference and a phenylalanine reference, and a step of detecting the mixed reference solution by using the high performance liquid chromatography to obtain a reference map of the reference;

and/or the fingerprint detection method further comprises the steps of preparing a control medicinal material solution by adopting centipede control medicinal materials, and detecting the control medicinal material solution by adopting the high performance liquid chromatography to obtain a control medicinal material reference map;

optionally, the preparation process of the control medicinal material solution comprises the following steps: taking centipede reference medicinal materials, adding water, heating and refluxing, then carrying out solid-liquid separation, taking liquid, evaporating to dryness, adding solvent into the obtained residues, and taking liquid after extraction and solid-liquid separation.

In an alternative embodiment, the centipede pharmaceutical formulation comprises centipede formula particles.

In a second aspect, the invention provides a method for determining the content of nucleoside and amino acid components in a centipede pharmaceutical preparation, which comprises the following steps:

taking a sample solution and a reference substance solution, and respectively adopting the high performance liquid chromatography in the fingerprint detection method to detect;

wherein the reference substances comprise hypoxanthine reference substance and phenylalanine reference substance;

alternatively, the detection wavelength of hypoxanthine is 249nm, and the detection wavelength of phenylalanine is 210nm.

In a third aspect, the invention provides an application of the fingerprint detection method and/or the content determination method in quality control of nucleoside and amino acid components in centipede pharmaceutical preparations.

In a fourth aspect, the invention provides a quality control method for nucleoside and amino acid components in a centipede pharmaceutical preparation, comprising the steps of obtaining a fingerprint of the centipede pharmaceutical preparation to be detected according to the fingerprint detection method, and comparing the fingerprint with a reference fingerprint;

the control fingerprint is obtained by fitting a fingerprint obtained by using at least one batch of standard products of centipede pharmaceutical preparations according to the fingerprint detection method through an average value or a median method;

And/or the method comprises the step of measuring the content of the nucleoside and amino acid components in the centipede pharmaceutical preparation to be measured according to the content measuring method.

In an alternative embodiment, the control fingerprint comprises 8 common characteristic peaks, wherein peak 1 is uracil peak, peak 2 is tyrosine peak, peak 3 is hypoxanthine peak, peak 4 is xanthine peak, peak 5 is phenylalanine peak, peak 6 is inosine peak, peak 7 is guanosine peak, and peak 8 is tryptophan peak;

taking phenylalanine peak as S peak, wherein the relative retention time of each characteristic peak and the S peak is within + -10% of the specified value, wherein the specified value corresponding to peak 1 is 0.29, the specified value corresponding to peak 2 is 0.43, the specified value corresponding to peak 3 is 0.55, the specified value corresponding to peak 4 is 0.68, the specified value corresponding to peak 6 is 1.19, the specified value corresponding to peak 7 is 1.24, and the specified value corresponding to peak 8 is 1.47;

optionally, the similarity between the fingerprint of the centipede pharmaceutical preparation to be detected and the reference fingerprint is greater than 0.90.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a fingerprint of a sample of 18 batches of Scolopendra formula particles in example 2 of the present invention;

FIG. 2 is a fingerprint after rearrangement of chromatographic peaks in example 2 of the present invention;

FIG. 3 is a graph of a mixed control in example 2 of the present invention;

FIG. 4 is a comparison feature map determined in example 2 of the present invention;

FIG. 5 is a negative control chromatogram of example 3 of the present invention;

FIG. 6 is a chromatogram of a test sample in example 3 of the present invention;

FIG. 7 is an integrity verification chromatogram of example 3 of the present invention;

FIG. 8 is a chromatogram obtained by detection with column 1 in example 3 of the present invention;

FIG. 9 is a chromatogram obtained by detection with column 2 in example 3 of the present invention;

FIG. 10 is a chromatogram obtained by detection with column 3 in example 3 of the present invention;

FIGS. 11 and 12 are chromatograms of a control in example 5 of the present invention;

FIGS. 13 and 14 are chromatograms of a test sample in example 5 of the present invention;

FIG. 15 is a chromatogram obtained by gradient 1 detection in example 6 of the present invention;

FIG. 16 is a chromatogram obtained by gradient 2 detection in example 6 of the present invention;

FIG. 17 is a chromatogram obtained by gradient 3 detection in example 6 of the present invention;

FIG. 18 is a chromatogram obtained by detection at 210nm using mobile phase 0.1% phosphoric acid-methanol in example 6 of the present invention;

FIG. 19 is a chromatogram obtained by measurement at 249nm using mobile phase 0.1% phosphoric acid-methanol in example 6 of the present invention;

FIG. 20 is a chromatogram obtained by detection at 210nm using mobile phase 0.1% formic acid-methanol in example 6 of the present invention;

FIG. 21 is a chromatogram obtained by detection at 249nm with mobile phase 0.1% formic acid-methanol in example 6 of the present invention;

FIG. 22 is a chromatogram obtained by detection at 210nm using mobile phase water-methanol in example 6 of the present invention;

FIG. 23 is a chromatogram obtained by detection at 249nm using mobile phase water-methanol in example 6 of the present invention;

FIG. 24 is a chromatogram of a test sample detected at 249nm in the specific verification in example 7 of the present invention;

FIG. 25 is a chromatogram of a test sample detected at a wavelength of 210nm at the time of specificity verification in example 7 of the present invention;

FIG. 26 is a negative blank chromatogram obtained by detection at 249nm wavelength at the time of specificity verification in example 7 of the present invention;

FIG. 27 is a negative blank chromatogram detected at a wavelength of 210nm at the time of specificity verification in example 7 of the present invention;

FIG. 28 is a linear calibration curve of hypoxanthine when validated for linear relationship in example 7 of the present invention;

FIG. 29 is a linear standard curve of phenylalanine at the time of linear relationship verification in example 7 of the present invention.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

In order to solve the above-mentioned problems in the related art, according to a first aspect of the present invention, there is provided a fingerprint detection method of nucleoside and amino acid components in a centipede pharmaceutical preparation, comprising the steps of:

0-15 min, wherein the volume percentage of methanol in the mobile phase is 1%;

1) The detection wavelength is 205 nm-215 nm;

2) The column temperature is 23-27 ℃;

3) The flow rate is 0.9 mL/min-1.1 mL/min;

4) The sample injection amount is 5-15 mu L;

Illustratively, the detection wavelength may be 210nm, 205nm, 215nm; the column temperature can be 25 ℃, 23 ℃ and 27 ℃; the flow rate can be 1.0mL/min, 0.9mL/min, 1.1mL/min; the sample injection amount can be 10 mu L, 5 mu L and 15 mu L.

In an alternative embodiment, the gradient elution procedure further comprises:

56-60 min, and the volume percentage of methanol in the mobile phase is 1%.

Illustratively, the test solution is prepared as follows: taking a proper amount of the product, grinding, precisely weighing, precisely adding the solvent, weighing, performing ultrasonic treatment, supplementing the reduced weight with the solvent, shaking uniformly, filtering, and taking the subsequent filtrate.

In a particularly preferred embodiment, the method for preparing the test solution is as follows: grinding the product, taking about 0.5g, precisely weighing, precisely adding 25mL of 30% methanol, weighing, performing ultrasonic treatment (power 250W, frequency 53 KHz) for 30 min, adding 30% methanol to the rest weight, shaking, filtering, and collecting the filtrate.

Illustratively, the control medicinal solution is prepared by the steps of: taking centipede reference medicinal material, adding water, heating and refluxing, filtering, evaporating filtrate to dryness, adding solvent into residue, performing ultrasonic treatment, supplementing weight loss with solvent, filtering, and taking subsequent filtrate as reference medicinal material solution.

In a particularly preferred embodiment, the control drug solution is prepared by a process comprising: taking 3.0g of centipede reference medicine, adding 50mL of water, heating and refluxing for 30 minutes, filtering, evaporating filtrate to dryness, adding 25mL of 30% methanol into residues, weighing, performing ultrasonic treatment (power 250W, frequency 53 KHz) for 30 minutes, taking out, supplementing weight loss with an extraction solvent, filtering, and taking the subsequent filtrate as a reference medicine solution.

The centipede medicinal preparation related in the invention can be prepared by the following method:

taking centipede medicinal materials, heating and reflux-extracting for at least 1 time, adding water with the weight being 6-12 times of that of the centipede medicinal materials each time for at least 0.5 hour, filtering, combining filtrate, concentrating the filtrate to the relative density of 1.05-1.10 g/mL at 60 ℃, adding conventional auxiliary materials, and preparing clinically acceptable tablets, capsules, pills, granules, honeyed pills, sustained release preparations, quick release preparations, controlled release preparations, oral liquid preparations or injection preparations according to the conventional process. The pharmaceutically acceptable auxiliary materials comprise at least one of filling agent, disintegrating agent, lubricant, suspending agent, adhesive, sweetener, corrigent, preservative and matrix. The filler comprises at least one of starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose and sucrose; the disintegrating agent comprises at least one of starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose and crosslinked sodium carboxymethyl cellulose; lubricants include magnesium stearate, sodium lauryl sulfate, talc, silica, and the like; the suspending agent comprises: at least one of polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, and hydroxypropyl methylcellulose; the adhesive comprises at least one of starch slurry, polyvinylpyrrolidone and hydroxypropyl methylcellulose; the sweetener comprises at least one of saccharin sodium, aspartame, sucrose, sodium cyclamate and glycyrrhetinic acid; the flavoring agent comprises sweetener and various flavors; the antiseptic comprises at least one of nipagin, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorhexidine acetate, and oleum Eucalypti; the matrix comprises at least one of PEG6000, PEG4000 and insect wax.

The invention is described in further detail below in connection with specific examples which are not to be construed as limiting the scope of the invention as claimed.

The instruments, reagents and reagents involved in the examples are as follows:

1. The preparation method of the centipede formula particle comprises the following steps:

taking centipede, heating and reflux-extracting for 2 times, firstly adding 10 times of water by weight for soaking for 30min, heating and reflux-extracting for 1.0h, filtering, secondly adding 8 times of water by weight for extracting for 1.0h, filtering, combining the two filtrates, concentrating the filtrate to the relative density of 1.05g/mL at 60 ℃, performing spray drying, adding auxiliary material maltodextrin into dry powder, uniformly mixing, performing dry granulation, and preparing the granule.

2. Instrument and apparatus:

high performance liquid chromatograph 1: the Waters e2695 chromatographic system comprises a quaternary gradient infusion pump (ACQUITY Arc), a 120-bit high-performance automatic sampler, an original inlet chromatographic column incubator, a Waters 2998 diode array ultraviolet detector and an Empower chromatographic management system;

high performance liquid chromatograph 2: thermo ultime 3000, including Pump: LPG-3400SD; coLum Compartment: TCC-3000RS; autosubtler: WPS-3000SL; photometer: DAD-3000;

high performance liquid chromatograph 3: the Shimadzu LC-20AT chromatographic system comprises a CTO-20AC quaternary pump, a SIL-20AC autosampler, an SPD type M20A detector and a chromatographic workstation;

other instruments: an electronic balance (FA 1004, a firm of science instruments, seashore, apocynum); one ten million electronic balance (sQP, sidoriscom instruments Co., ltd.); ultrasonic cleaning machine: WB400US Shanghai brand instruments inc;

Chromatographic column 1: agilent ZORBAX SB-AQ C18 (4.6 mm. Times.250 mm,5 μm);

chromatographic column 2: DIKMA platisil 5um ODS C18 (4.6 mm. Times.250 mm,5 μm);

chromatographic column 3: welch AQ-C18 (4.6 mm. Times.250 mm,5 μm);

3. reagent:

uracil control (lot number: 100469-201302, purity: 99.6%, purchased from chinese food and drug assay institute);

tyrosine control (lot number: 140609-201914, purity: 99.9%, purchased from chinese food and drug assay institute);

hypoxanthine reference (lot number: 140661-202005, purity: 99.4%, purchased from China food and drug inspection institute);

xanthine reference (lot number: 140662-200802, purity: 100.0%, purchased from China food and drug inspection institute)

Phenylalanine control (lot number: 140676-201706, purity: 100.0%, purchased from chinese food and drug verification institute);

inosine control (lot number: 140669-202007, purity: 99.2%, purchased from China food and drug inspection institute);

guanosine reference (lot number: 111977-201501, purity: 93.6 purchased from China food and drug inspection institute);

tryptophan reference (lot number: 140686-202108, purity: 99.9%, purchased from China food and drug inspection institute);

Centipede control (lot number 400015-202111, available from Shanghai HongYongsheng biotechnology Co., ltd.);

centipede formula particles (lot numbers: 1905001W, 1907001S, 1908001W, 1912002W, 2001004S, 2001002W, 2003001W, 2108001W, 2011003S, 2102003S, 2106002S, 2109002W, 2111001S, 2111002S, 2202002W, 2205001W, 2210002S, 2212002S, from Huarun Sanjiu pharmaceutical Co., ltd., huarun Sanjiu modern Chinese medicine Co., ltd.);

4. reagent: methanol is chromatographic pure, and water is ultrapure water; the other reagents were all analytically pure.

Example 1

The embodiment provides a fingerprint detection method of centipede formula particles (batch number: 1905001W):

(1) Preparing a solution:

preparation of control medicinal material solution: taking 3.0g of centipede reference medicine, adding 50mL of water, refluxing for 30min, filtering, evaporating filtrate to dryness, precisely adding 25mL of 30% methanol, weighing, performing ultrasonic treatment (power is 250W, frequency is 53 kHz) for 30min, taking out, supplementing weightlessness with extraction solvent, filtering, and taking out subsequent filtrate to obtain reference medicine solution;

preparation of a control solution: taking uracil reference substance, tyrosine reference substance, hypoxanthine reference substance, xanthine reference substance, phenylalanine reference substance, inosine reference substance, guanosine reference substance and tryptophan reference substance, adding 30% methanol, and preparing into mixed reference substance solution containing 0.1mg of each reference substance per 1 mL;

Preparation of test solution: grinding the product into fine powder of about 0.5g, precisely weighing, precisely adding 25mL of 30% methanol, weighing, performing ultrasonic treatment (power 250W, frequency 53 kHz) for 30 min, taking out, adding extraction solvent to make up weight loss, filtering, and collecting the subsequent filtrate.

(2) According to high performance liquid chromatography (China pharmacopoeia 2020 edition rule 0512):

chromatographic conditions: octadecylsilane chemically bonded silica is used as filler (column length 4.6mm×250mm, particle size 5 μm); using methanol as mobile phase A and aqueous solution as mobile phase B, and performing gradient elution according to the specification in Table 1; the flow rate is 1.0mL per minute, and the column temperature is 25 ℃; the detection wavelength is 210nm, and the theoretical plate number is not less than 5000 according to the hypoxanthine peak.

Table 1 gradient elution procedure used in example 1

Assay: precisely sucking 10 μl of each of the control medicinal material solution, the mixed reference substance solution and the test sample solution, and measuring with high performance liquid chromatograph. The basic information of each chromatographic peak in the chromatogram obtained by detection, the chromatogram of the mixed reference substance, the chromatogram of the reference medicinal material and the chromatogram of the sample is shown in table 2.

Table 2 mixing the control, control medicinal materials and test results

As can be seen from table 2, 8 characteristic peaks are present in the sample chromatogram and correspond to the retention times of 8 characteristic peaks in the mixed control chromatogram and the control chromatogram, and among the 8 characteristic peaks, peak 1 is uracil peak, peak 2 is tyrosine peak, peak 3 is hypoxanthine peak, peak 4 is xanthine peak, peak 5 is phenylalanine peak, peak 6 is inosine peak, peak 7 is guanosine peak, and peak 8 is tryptophan peak. Taking phenylalanine peak as S peak, wherein the relative retention time of each characteristic peak and the S peak is within + -10% of a specified value, and the specified value is: 0.29 (Peak 1), 0.43 (Peak 2), 0.55 (Peak 3), 0.68 (Peak 4), 1.19 (Peak 6), 1.24 (Peak 7), 1.47 (Peak 8).

Example 2

The present example is used to illustrate the establishment of a control fingerprint for a centipede drug formulation:

the fingerprint spectrum similarity evaluation software '2012 edition of traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system' compiled by the pharmacopoeia committee is adopted, 18 batches of fingerprint spectrum of the representative centipede formula particle samples are adopted to generate a comparison fingerprint spectrum, and the comparison fingerprint spectrum is subjected to positioning and identification by adopting comparison products such as hypoxanthine, phenylalanine and the like. The fingerprint of each batch of centipede formula particle samples was detected as in example 1. The fingerprint of 18 batches of centipede formula particle samples is shown in fig. 1, the batches of S1-18 are 1905001W, 1907001S, 1908001W, 1912002W, 2001004S, 2001002W, 2003001W, 2108001W, 2011003S, 2102003S, 2106002S, 2109002W, 2111001S, 2111002S, 2202002W, 2205001W, 2210002S and 2212002S in sequence; r is the reference medicinal material map.

(1) Characteristic spectrum characteristic peak confirmation and characteristic peak identification

And analyzing detection results of the fingerprints of a plurality of batches of samples, and generating a control characteristic map by adopting fingerprint similarity evaluation software '2012 edition of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system' compiled by pharmacopoeia committee. By identifying and assigning characteristic peaks, the obtained HPLC characteristic spectrum of the centipede formula particle has 8 chromatographic peaks, and the other chromatographic peaks are chromatographic peaks with smaller response or poorer separation degree, so that 8 chromatographic peaks with larger response are selected as common characteristic peaks, and the chromatographic peaks are rearranged according to the sequence of the chromatographic peaks, as shown in figure 2.

According to literature reports, chemical components in centipedes mainly comprise centipede venom (proteins and polypeptides), special small molecular compounds (quinoline alkaloids), nucleosides, amino acids, histamine, total phospholipids and the like, wherein the centipede venom, the special small molecular compounds (quinoline alkaloids), the nucleosides and the amino acids are one of active components. Considering that the centipede venom is mostly protein and polypeptide components, the water solubility is poor, the centipede venom is easy to lose activity after being heated and decocted, and the water solubility of components such as special small molecular compounds (quinoline alkaloids), nucleosides, amino acids and the like is good, the small molecular compounds (quinoline alkaloids), nucleosides and amino acids are selected for analysis and research. Through analysis of special small molecular compounds (quinoline alkaloids), nucleosides and amino acids of the centipede formula particles, the special small molecular compounds (quinoline alkaloids) of the centipede formula particles are detected, and 3, 8-dihydroxyquinoline, nucleoside hypoxanthine, amino acids phenylalanine and the like are detected, so that the quinoline components, the nucleoside components and the amino acids are selected as main development components of the characteristic spectrum of the centipede formula particles.

From the results of peak identification and reference localization, it was confirmed that 8 of the known peaks, respectively, peak 1 (uracil), peak 2 (tyrosine), peak 3 (hypoxanthine), peak 4 (xanthine), peak 5 (phenylalanine), peak 6 (inosine), peak 7 (guanosine), and peak 8 (tryptophan), were each one of the main active ingredients of centipede, wherein tyrosine, phenylalanine, tryptophan were amino acid components, uracil, hypoxanthine, xanthine, inosine, guanosine were nucleoside components, and the characteristic map contained the main chemical components of centipede.

(2) Selection basis of characteristic spectrum characteristic peak S-peak

According to the peak identification and peak selection results of characteristic peaks in centipede formula particles, in the characteristic spectrum of the centipede formula particles, hypoxanthine is highest in response, phenylalanine is second in response and is one of main active ingredients of centipedes, and in consideration of the fact that the detection time of the centipede nucleoside and amino acid characteristic spectrum is 45 minutes, the time interval of each chromatographic peak in the chromatogram is large, the peak outlet time of the phenylalanine chromatographic peak is moderate (retention time is 25 minutes), the peak separation degree is good, and in order to ensure the durability of each characteristic peak relative retention time, phenylalanine is used as an S peak of the characteristic spectrum, and the relative retention time of each characteristic peak is calculated.

(3) Identification of characteristic peaks of characteristic spectrum

By HPLC control localization study, in centipede formula particle HPLC chromatogram, 8 peaks are known chromatographic peaks, namely peak 1 (uracil), peak 2 (tyrosine), peak 3 (hypoxanthine), peak 4 (xanthine), peak 5 (phenylalanine), peak 6 (inosine), peak 7 (guanosine), and peak 8 (tryptophan). Wherein, the spectrum of the mixed reference substance is shown in figure 3, and the LC/MS/MS analysis result is shown in table 3.

TABLE 3 LC/MS/MS analysis results of Scolopendra formula particles

(4) Characteristic peak prescribed value of characteristic map

The relative retention time of the characteristic spectrum is determined according to the research result: the centipede formula particle feature map should show 8 feature peaks, which should correspond to the retention time of the corresponding feature peaks of the control medicinal materials. The finally determined comparison characteristic spectrum is shown in fig. 4, the relative retention time of each characteristic peak and the S peak in the comparison characteristic spectrum is shown in table 4, and the relative peak area of each characteristic peak and the S peak is shown in table 5.

TABLE 4 relative retention times of characteristic peaks and S-peaks in comparison characteristic spectra

TABLE 5 comparison of relative peak areas of characteristic peaks and S-peaks in characteristic spectra

Example 3

In this embodiment, except for specific descriptions, the preparation method of each solution and the used high performance liquid chromatography are the same as those in embodiment 1, and the description thereof is omitted.

(1) Specificity verification

A sample solution (lot number: 1905001W) was prepared and analyzed by high performance liquid chromatography using 30% methanol as a negative control solution, and the results are shown in FIGS. 5 and 6. Wherein, fig. 5 is a negative control chromatogram, fig. 6 is a sample chromatogram, and as can be seen from fig. 5 and 6, the negative is not interfered.

(2) Integrity verification

A sample solution (lot number: 1905001W) was prepared, and analyzed by high performance liquid chromatography, and after peak formation, measurement was performed for a period of time 2 times the fingerprint measurement period. As shown in FIG. 7, after 60min, no chromatographic peak appears in the fingerprint, which indicates that the method can determine the main chromatographic peak of the fingerprint of the centipede formula particle, and the method has better integrity.

(3) Instrument precision verification

Sample solutions of the same centipede formula particles (batch number: 1905001W) are taken and repeatedly injected for 6 times, RSD values of relative retention time of each characteristic peak and S peak are calculated, and the results are shown in Table 6, wherein t is the retention time, and t/ts is the relative retention time. As can be seen from Table 6, the relative retention time of each characteristic peak and S peak has an RSD value of less than 2.0%, which indicates that the method has good instrument precision.

Table 6 results of instrument precision test relative retention time

(4) Repeatability verification

Samples of the same centipede formula particles (lot number: 1905001W) were taken, 6 test solutions were prepared in parallel and HPLC measurements were performed to calculate the RSD values for the relative retention times of each characteristic peak and S peak, and the results are shown in Table 7. As can be seen from Table 7, the relative retention time of each characteristic peak to the S peak has an RSD value of less than 2.0%, indicating good reproducibility of the process.

TABLE 7 relative retention time results of repeatability tests

(5) Intermediate precision (personnel) verification

Samples of the same centipede formula particles (lot number: 1905001W) were taken, test solutions were prepared by three laboratory workers and subjected to HPLC determination, and the RSD values of the relative retention times of each characteristic peak and S peak were calculated, and the results are shown in Table 8. As can be seen from Table 8, the relative retention time of each characteristic peak to the S peak has an RSD value of less than 2.0%, indicating good intermediate precision of the method.

TABLE 8 relative retention time of intermediate precision (personnel)

(6) Stability investigation

Sample solutions of centipede formula particles (1905001W) were taken, HPLC measurements were performed at the 0h, 4h, 8h, 12h, 16h and 24h after the completion of the preparation, and the RSD values of the relative retention times of the characteristic peaks and the S peaks were calculated, and the results are shown in Table 9. As can be seen from Table 9, the relative retention time of each characteristic peak and S peak has an RSD value of less than 2.0%, which indicates that the test sample solution is stable within 24 hours and meets the measurement requirements.

Table 9 stability test relative retention time results

(7) Investigation of different column temperatures

The same sample solution (1905001W) was taken, the column temperature was set at 23℃and 25℃and 27℃respectively, HPLC was performed to examine the effect of different column temperatures on the relative retention time of each characteristic peak in the test sample pattern of the centipede formula particles, and the results are shown in Table 10.

Table 10 results of relative retention time for different column temperatures

(8) Investigation of different flow rates

The same sample solution (1905001W) was taken, the flow rates were set to 0.9mL/min, 1.0mL/min and 1.1mL/min, HPLC measurements were performed, and the influence of different flow rates on the relative retention time of each characteristic peak in the sample pattern of the centipede formula particles was examined, and the results are shown in Table 11.

TABLE 11 relative retention time results for different flow rates

(9) Investigation of different chromatographic columns

The same sample solution of Scolopendra granule (1905001W) was measured with different manufacturer's chromatographic columns (column 1:Agilent ZORBAX SB-AQ C18; column 2:DIKMA platisil 5um ODS; column 3: welchAQ-C18), respectively, as shown in FIGS. 8-10. Wherein, fig. 8 is a chromatogram obtained by detecting the chromatographic column 1, fig. 9 is a chromatogram obtained by detecting the chromatographic column 2, and fig. 10 is a chromatogram obtained by detecting the chromatographic column 3.

As can be seen from FIGS. 8 to 10, the two brands of hydrophilic chromatographic columns Agilent ZORBAX SB-AQ C18 and Welch AQ-C18 have the problems of characteristic peak deletion and poor separation degree, and the detection result of the DIKMA platisil 5um ODS C18 chromatographic column is good.

(10) Inspection of different liquid phase devices

The same sample solution (1905001W) is taken, liquid phase equipment is respectively replaced by Watersarc, siemens U3000 and Shimadzu LC-20AT, HPLC measurement is respectively carried out, the influence of different liquid phase equipment on the relative retention time of each characteristic peak in the centipede formula particle sample map is examined, and the results are shown in Table 12. As can be seen from Table 12, the relative retention time of the characteristic peaks of the characteristic spectrum part of each brand of instrument is within + -10% of the specified value, so that the method has better durability for different instruments.

Table 12 relative retention time results for different brands of instruments

Example 4

Liquid phase fingerprints of 18 batches of centipede formula particles were respectively measured according to the method of example 1, and the measurement results are shown in table 13. As can be seen from Table 13, the characteristic spectra of 18 batches of centipede formula particles all show 8 characteristic peaks, and the relative retention time of peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8 and S peak is within + -10% of the specified value.

Table 13 results of relative retention time for 18 batches of Scolopendra formula particles

Example 5

The present example provides a method for measuring the content of hypoxanthine and phenylalanine in centipede formula particles (batch number: 1905001W):

(1) Preparing a solution:

preparation of a control solution: taking appropriate amounts of hypoxanthine reference substance and phenylalanine reference substance, adding 30% methanol, and making into reference substance solution containing 0.1mg of each reference substance per 1 mL;

chromatographic conditions: octadecylsilane chemically bonded silica is used as filler (column length 4.6mm×250mm, particle size 5 μm); gradient elution was performed as specified in table 14 with methanol as mobile phase a and aqueous solution as mobile phase B; the flow rate is 1.0mL per minute, and the column temperature is 25 ℃; the detection wavelength is 210nm (phenylalanine) and 249nm (hypoxanthine), and the theoretical plate number of phenylalanine and hypoxanthine peak is calculated to be not less than 3000.

Table 14 gradient elution procedure used in example 5

Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into high performance liquid chromatograph for measurement.

The chromatogram of the control obtained by measurement is shown in figures 11-12, the chromatogram of the test sample is shown in figures 13-14, the concentration of hypoxanthine in the control solution is 0.095mg/mL, and the concentration of phenylalanine is 0.095mg/mL; the peak area of hypoxanthine in the chromatogram of the control is 4603659, and the peak area of phenylalanine is 2632491; the peak area of hypoxanthine in the chromatogram of the test sample was 7035633, and the peak area of phenylalanine was 4453594. The content of hypoxanthine in the sample is calculated to be 3.62mg/g, and the content of phenylalanine is calculated to be 4.00mg/g.

Example 6

This example is used to illustrate the screening procedure for detection conditions, and in this example, the detection conditions are the same as in example 5 except for the elution gradient.

(1) Screening of elution gradients

HPLC detection is carried out on the same centipede formula particle (batch number: 1905001W) sample solution according to gradients 1-3 shown in tables 15-17, and the separation degree and system adaptability parameters of hypoxanthine, phenylalanine and the like in the chromatograms obtained by detection under each elution program are examined, and the results are shown in figures 15-17 and tables 18-20. Here, fig. 15 is a chromatogram obtained by the gradient 1 detection, fig. 16 is a chromatogram obtained by the gradient 2 detection, and fig. 17 is a chromatogram obtained by the gradient 3 detection. As can be seen from fig. 15-17 and tables 18-20, the separation is better and the system adaptation parameters are more ideal when using gradient 3.

TABLE 15 gradient 1

TABLE 16 gradient 2

TABLE 17 gradient 3

Table 18 detection results using gradient 1

Table 19 detection results using gradient 2

Table 20 detection results using gradient 3

(2) Screening of different mobile phases

Taking the same part of centipede formula particles (batch number: 1905001W) as a sample solution, performing HPLC detection according to the use of mobile phases of 0.1% phosphoric acid-methanol, 0.1% formic acid-methanol and water-methanol, and examining the elution separation effect of each mobile phase. The results are shown in FIGS. 18 to 23 and tables 21 to 23, wherein FIG. 18 is a chromatogram detected at 210nm using 0.1% phosphoric acid-methanol as a mobile phase, FIG. 19 is a chromatogram detected at 249nm using 0.1% phosphoric acid-methanol as a mobile phase, FIG. 20 is a chromatogram detected at 210nm using 0.1% formic acid-methanol as a mobile phase, FIG. 21 is a chromatogram detected at 249nm using 0.1% formic acid-methanol as a mobile phase, FIG. 22 is a chromatogram detected at 210nm using water-methanol as a mobile phase, and FIG. 23 is a chromatogram detected at 249nm using water-methanol as a mobile phase.

As can be seen from fig. 18-23 and tables 21-23, when water-methanol is used as the mobile phase, the chromatographic resolution is better, the baseline is stable, the peak shape is better, and the system adaptation parameters are more ideal.

TABLE 21 detection results of 0.1% phosphoric acid-methanol using mobile phase

TABLE 22 detection results of 0.1% formic acid-methanol using mobile phase

TABLE 23 detection results with mobile phase Water-methanol

Example 7

In this embodiment, except for specific descriptions, the preparation method of each solution and the used hplc method are the same as those in embodiment 5, and the description thereof is omitted.

(1) Investigation of specificity

Preparing a test solution and a negative blank control solution, performing HPLC analysis according to chromatographic conditions described under the content of hypoxanthine and phenylalanine, and recording chromatograms, as shown in figures 24-27, wherein figure 24 is a test chromatogram detected at 249nm wavelength, figure 25 is a test chromatogram detected at 210nm wavelength, figure 26 is a negative blank control chromatogram detected at 249nm wavelength, and figure 27 is a negative blank control chromatogram detected at 210nm wavelength. As can be seen from fig. 24-27, the negative control test was undisturbed.

(2) Linear relationship verification

Taking appropriate amount of hypoxanthine reference substance, adding 50% methanol to make into linear verification solutions containing 0.00436mg, 0.00872mg, 0.0174mg, 0.0349mg, 0.174mg and 1.74mg of hypoxanthine reference substance, respectively, and precisely sucking the above linear verification solutions with different concentrations 10. Mu.l of each solution was injected into a liquid chromatograph, and the peak area was measured as shown in Table 24. And drawing a standard curve by taking the concentration of the reference substance as an abscissa and the peak area as an ordinate, and obtaining a hypoxanthine regression equation as shown in figure 28: y=30348033.58x+374657.42, r ² =0.9998. Experimental results show that the hypoxanthine has good linear relation in the mass concentration range of 0.00436 mg/ml-1.74 mg/ml.

TABLE 24 hypoxanthine linear relationship results

Taking proper amount of phenylalanine reference substance, adding 50% methanol to prepare each 1mL of linear verification solution containing 0.00300mg, 0.0060mg, 0.0120, 0.0241mg, 0.120mg and 1.20mg of phenylalanine reference substance, respectively, precisely sucking 10 μl of each of the above linear verification solutions with different concentrations, and injecting into a liquid chromatograph to determine peak area, as shown in Table 25. The standard curve is drawn with the concentration of the control on the abscissa and the peak area on the ordinate, as shown in fig. 29, and the phenylalanine regression equation is obtained as follows: y=25860179.10x+27413.41, r ² =1.0000. Experimental results show that the phenylalanine has good linear relation in the mass concentration range of 0.00301mg/ml to 1.20 mg/ml.

TABLE 25 phenylalanine linear relationship results

(3) Precision test

(1) Precision test of instrument

Taking the same sample solution of centipede formula particles (1905001W), repeating the sample injection for 6 times, and measuring peak areas of hypoxanthine and phenylalanine, wherein the peak areas of hypoxanthine and phenylalanine are shown in table 26, and the results show that RSD values of the peak areas of hypoxanthine and phenylalanine are respectively 0.23% and 0.73%, which indicates that the instrument precision is good.

TABLE 26 precision test results of hypoxanthine and phenylalanine

(2) Intermediate precision test (personnel)

The test solution is prepared by the same batch of centipede formula particles (1905001W) by three laboratory workers A, B and C according to the method under the content measurement items of hypoxanthine and phenylalanine, and the contents of hypoxanthine and phenylalanine are calculated, as shown in table 27, and the result shows that the intermediate precision of the method is good.

Table 27 results of intermediate precision test of hypoxanthine and phenylalanine

(4) Repeatability test

6 parts of centipede formula particles (1905001W) to be tested are taken from the same batch, the centipede formula particles are prepared according to a preparation method of a test solution, the contents of hypoxanthine and phenylalanine are respectively measured, and the content result RSD% is calculated. The results are shown in Table 28. The results showed that the hypoxanthine and phenylalanine contents RSD were 1.4% and 1.2%, respectively, indicating that the method was good in reproducibility.

Table 28 results of repeated experiments with the hypoxanthine and phenylalanine methods

(5) Accuracy test

Namely, in a sample collection test, 9 parts of test substances of centipede formula particles (1905001W, the hypoxanthine content is 3.63 mg/g) are precisely weighed, each part is about 0.25g, the test substances are respectively placed in a measuring flask, 3 parts of test substances are used as a group, each group of test substances is precisely added with 1ml of control substance solution containing 0.725mg/ml, 0.921mg/ml and 1.110mg/ml of hypoxanthine, and the mixture is treated according to a test substance preparation method to carry out content measurement. The recovery rate of hypoxanthine was calculated, and the recovery rate results are shown in Table 29. The result shows that the average recovery rate of hypoxanthine is 99.9%, and the RSD values are 2.8% respectively, which indicates that the method has good accuracy.

TABLE 29 results of hypoxanthine accuracy test

9 parts of test substances of centipede formula particles (1905001W, the phenylalanine content is 4.08 mg/g) are precisely weighed, each part is about 0.25g, the test substances are respectively placed in a measuring flask, 3 parts of test substances are used as a group, each group of test substances is respectively and precisely added with 1ml of reference substance solution containing 0.819mg/ml, 1.016mg/ml and 1.231mg/ml of phenylalanine, and the test substances are processed according to a test substance preparation method to carry out content measurement. The phenylalanine recovery rate was calculated and the recovery rate results are shown in Table 30. As a result, the average recovery rate of phenylalanine was 98.8% and the RSD values were 2.2%, respectively, indicating that the method was excellent in accuracy.

TABLE 30 phenylalanine accuracy test results

(6) Durability inspection

(1) Stability test

Sample solutions of the same centipede formula particles (1905001W) are taken and respectively injected for 0h, 6h, 8h, 12h and 24h after preparation is finished, and chromatographic peak areas of hypoxanthine and phenylalanine are measured, and the test results are shown in Table 31. The result shows that the test sample solution is stable within 24 hours, and the RSD values are respectively 0.22% and 0.46%, so as to meet the measurement requirements.

TABLE 31 stability measurement results of sample solutions of hypoxanthine and phenylalanine

(2) Investigation of different column temperatures

The effect of different column temperatures at 23deg.C, 25deg.C and 27deg.C on the hypoxanthine and phenylalanine content of Scolopendra formulation granule (1905001W) was compared. The test results are shown in Table 32, and the experimental results show that the durability of the analysis method is good at different column temperatures, and the results show that the small variation of the column temperatures can meet the requirement of system adaptability.

Table 32 results of durability investigation of different columns Wen Cihuang purine, phenylalanine

(3) Investigation of different flow rates

The effect of different flow rates of 0.9ml/min, 1.0ml/min and 1.1ml/min on the hypoxanthine and phenylalanine content of the centipede formula particles (1905001W) was compared. The test results are shown in Table 33, and the experimental results show that the analysis method has better durability at different flow rates, and the small change of the flow rate can meet the system adaptability requirement.

TABLE 33 durability test results of hypoxanthine and phenylalanine at different flow rates

Example 8

The contents of hypoxanthine and phenylalanine in 18 batches of centipede formula particles were respectively measured according to the method of example 5, and the results are shown in Table 34.

Table 34 transfer rate and range of hypoxanthine content in 18 batches of centipede formula particles

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims

1. The fingerprint detection method of the nucleoside and amino acid components in the centipede pharmaceutical preparation is characterized by comprising the following steps:

0-15 min, wherein the volume percentage of methanol in the mobile phase is 1%;

2. The fingerprint detection method according to claim 1, wherein the chromatographic conditions of the high performance liquid chromatography further comprise at least one of the following conditions:

1) The detection wavelength is 205 nm-215 nm;

2) The column temperature is 23-27 ℃;

3) The flow rate is 0.9 mL/min-1.1 mL/min;

4) The sample injection amount is 5-15 mu L;

3. The fingerprint detection method according to claim 1, wherein the gradient elution procedure further comprises:

56-60 min, and the volume percentage of methanol in the mobile phase is 1%.

4. The fingerprint detection method according to claim 1, wherein the preparation method of the sample solution comprises the following steps:

5. The fingerprint detection method according to claim 1, further comprising a step of preparing a reference solution using a hypoxanthine reference and a phenylalanine reference, and a step of detecting the mixed reference solution using the high performance liquid chromatography to obtain a reference map;

6. The fingerprint detection method according to any one of claims 1-5 wherein the centipede drug formulation comprises centipede formula particles.

7. The method for measuring the content of the nucleoside and amino acid components in the centipede medicinal preparation is characterized by comprising the following steps of:

taking a sample solution and a reference substance solution, and detecting by adopting a high performance liquid chromatography in the fingerprint detection method according to any one of claims 1 to 6;

8. Use of the fingerprint detection method according to any one of claims 1 to 6 and/or the content determination method according to claim 7 for quality control of nucleoside and amino acid components in centipede pharmaceutical preparations.

9. A quality control method for nucleoside and amino acid components in centipede pharmaceutical preparation is characterized in that,

the method comprises the steps of obtaining a fingerprint of a centipede pharmaceutical preparation to be detected according to the fingerprint detection method of any one of claims 1-6, and comparing the fingerprint with a reference fingerprint;

Wherein the reference fingerprint is obtained by fitting a fingerprint obtained by using at least one batch of standard products of centipede pharmaceutical preparations according to the fingerprint detection method of any one of claims 1 to 6 by an average value or a median method;

and/or, the method comprises the step of measuring the content of the nucleoside and amino acid components in the centipede pharmaceutical preparation to be measured according to the content measuring method of claim 7.

10. The quality control method of claim 9, wherein the control fingerprint comprises 8 common characteristic peaks, wherein peak 1 is uracil peak, peak 2 is tyrosine peak, peak 3 is hypoxanthine peak, peak 4 is xanthine peak, peak 5 is phenylalanine peak, peak 6 is inosine peak, peak 7 is guanosine peak, and peak 8 is tryptophan peak;