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CN110907546B - HPLC fingerprint detection method of traditional Chinese medicine for tonifying kidney and strengthening bone - Google Patents

HPLC fingerprint detection method of traditional Chinese medicine for tonifying kidney and strengthening bone Download PDF

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CN110907546B
CN110907546B CN201911139919.8A CN201911139919A CN110907546B CN 110907546 B CN110907546 B CN 110907546B CN 201911139919 A CN201911139919 A CN 201911139919A CN 110907546 B CN110907546 B CN 110907546B
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陈鹏
李霞
陈骞
明凯利
熊丽
向阳
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Jianmin Pharmaceutical Groups Corp ltd
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The invention relates to a HPLC fingerprint detection method of a kidney-tonifying and bone-strengthening traditional Chinese medicine, wherein the kidney-tonifying and bone-strengthening traditional Chinese medicine is prepared from traditional Chinese medicinal materials such as prepared rehmannia root, dogwood fruit, east Asian tree fern rhizome, epimedium herb, Chinese yam, Chinese angelica, tree peony bark, oyster shell and the like, the detection method comprises the step of extracting a sample and then detecting the sample by using a high performance liquid chromatograph, the high performance liquid chromatograph contains an ultraviolet detector, the detection wavelength of the ultraviolet detector is set to be 220-250nm, and the stationary phase of the high performance liquid chromatograph is C18Chromatographic column, mobile phase A is acetonitrile, mobile phase B is water solution containing potassium dihydrogen phosphate 0.05-0.3% and sodium octane sulfonate 0.01-0.1%, and gradient elution is adopted. The fingerprint spectrum established by the invention marks 9 common peaks, the peak No. 6 is the peak of icariin, and the 9 common peaks have high stability, repeatability and precision through methodology investigation and can be completely used for guiding production and controlling product quality.

Description

HPLC fingerprint detection method of traditional Chinese medicine for tonifying kidney and strengthening bone
Technical Field
The invention belongs to the field of traditional Chinese medicine detection, and relates to an HPLC fingerprint detection method for a traditional Chinese medicine for tonifying kidney and strengthening bone.
Background
CN 1493325A discloses a Chinese medicine for treating primary osteoporosis with liver and kidney deficiency and its preparation method, the Chinese medicine is composed of eight Chinese medicinal materials of radix rehmanniae Preparata, Corni fructus, rhizoma Cibotii, herba Epimedii, rhizoma Dioscoreae, radix Angelicae sinensis, cortex moutan, and Concha Ostreae preparata, the trade name "Kidney tonifying and bone strengthening Capsule", is a unique variety of Jianmin pharmaceutical industry group limited company, the quality control of the Chinese medicine at present generally adopts the qualitative or quantitative detection of a single component, however, the quality control has great limitation, firstly, the quality of the medicine can not be fully characterized, the phenomena of adulteration and badness of the Chinese medicine are caused, the Chinese medicine can not ensure the curative effect, and the medication safety is seriously threatened, secondly, the method has the defects of many detection times, many instruments, time and labor consumption for detection, low efficiency and the like. Therefore, there is a need to establish a quality detection method with more comprehensive and efficient traditional Chinese medicine.
The fingerprint is a quantifiable method for detecting the traditional Chinese medicinal materials and the traditional Chinese medicinal preparations, and is mainly used for identifying the truth and evaluating the uniformity and the stability of the quality of the raw medicinal materials, semi-finished products and the preparations. Compared with a quality analysis method for measuring the content of index components, the fingerprint can comprehensively reflect the types and the quantity of chemical components of the traditional Chinese medicine, can realize comprehensive evaluation on the internal quality of the traditional Chinese medicine and effective control on the whole substances of the traditional Chinese medicine under the condition that the effective components of a compound preparation of the traditional Chinese medicine are not completely clarified, and is the most effective means for controlling the quality of the traditional Chinese medicine and the preparation thereof at present. The detection method of traditional Chinese medicine fingerprint spectrum includes spectrometry, etc. The chromatography mainly comprises thin-layer chromatography, high performance liquid chromatography and gas chromatography, wherein the high performance liquid chromatography has the characteristics of high efficiency, rapidness, sensitivity and good reproducibility, and is the mainstream method for the traditional Chinese medicine fingerprint spectrum research.
Disclosure of Invention
The invention aims to comprehensively and effectively control the product quality and establish an HPLC fingerprint detection method of a traditional Chinese medicine for tonifying kidney and strengthening bones.
In order to achieve the purpose, the invention adopts the following technical means:
an HPLC fingerprint detection method of a traditional Chinese medicine for tonifying kidney and strengthening bone is disclosed, wherein the traditional Chinese medicine for tonifying kidney and strengthening bone is prepared from the following raw materials in parts by weight: 3-8 parts of prepared rehmannia root, 1-5 parts of dogwood, 1-5 parts of rhizoma cibotii, 1-5 parts of epimedium, 1-5 parts of Chinese yam, 1-5 parts of angelica, 1-4 parts of moutan bark and 2-6 parts of oyster, and the detection method comprises the following steps:
(1) preparation of a test solution: grinding the Chinese medicinal materials to be tested, ultrasonically extracting with 30-70% methanol, and filtering to obtain test solution;
(2) precisely absorbing a test sample solution, and detecting by using a high performance liquid chromatograph, wherein the high performance liquid chromatograph comprises an ultraviolet detector, the set detection wavelength of the ultraviolet detector is 220-250nm, and the stationary phase of the high performance liquid chromatograph is C18A chromatographic column, wherein a mobile phase A is acetonitrile, a mobile phase B is an aqueous solution containing 0.05-0.3% of monopotassium phosphate and 0.01-0.1% of sodium octane sulfonate, gradient elution is adopted, the flow rate is 0.5-2ml/min, and the column temperature is 25-40 ℃;
(3) recording a chromatogram of the test solution for 0-50 min, and performing data import, multipoint correction and data matching on the chromatogram of the test solution by using a Chinese medicine chromatogram fingerprint similarity evaluation system of the State pharmacopoeia Committee to obtain a fingerprint and performing similarity analysis.
Preferably, the set detection wavelength of the ultraviolet detector is 235 nm.
Preferably, the mobile phase B is an aqueous solution containing 0.1% of monopotassium phosphate and 0.05% of sodium octane sulfonate.
Preferably, the procedure of the gradient elution is as follows:
TABLE 1 gradient elution procedure
Elution time Mobile phase A Mobile phase B
0～10min 10％ 90％
10～25min 10→28％ 90→72％
25～35min 28％ 72％
35～50min 28→100％ 72→0％
Preferably, the fingerprint has 9 common peaks, wherein the peak No. 6 is a reference peak, namely an icariin peak, and the retention time, relative retention time, peak area and relative peak area of each peak are as follows:
TABLE 29 retention time and Peak area of common peaks
Figure BDA0002280622510000021
Figure BDA0002280622510000031
The invention has the beneficial effects that:
compared with the existing method for measuring and identifying the content of the traditional Chinese medicine for tonifying the kidney and strengthening the bone, the fingerprint spectrum established by the invention can more comprehensively reflect the chemical information contained in the traditional Chinese medicine, can more effectively guide feeding, strictly standardizes production operation, and fundamentally avoids the phenomena of adulteration and inferior quality of the traditional Chinese medicine, thereby ensuring the safety, effectiveness and reliability of clinical medication.
Compared with other quality control methods such as HPLC content determination, thin layer scanning, thin layer identification and the like, the method can realize comprehensive quality control through one-time detection, and has the advantages of convenient and quick operation, few used instruments and the like.
The fingerprint spectrum established by the invention marks 9 common peaks, the peak No. 6 is identified as the peak of icariin, and the 9 common peaks have high stability, repeatability and precision through methodology investigation and can be completely used for guiding production and controlling product quality.
Drawings
FIG. 1 is HPLC standard fingerprint of kidney invigorating and bone strengthening capsule.
FIG. 2 is an HPLC chromatogram of an icariin control.
FIG. 3 is an HPLC chromatogram of a sample to be tested after 50% ethanol ultrasonic extraction.
FIG. 4 is an HPLC chromatogram obtained at a detection wavelength of 205 nm.
FIG. 5 is an HPLC chromatogram obtained at a detection wavelength of 215 nm.
FIG. 6 is an HPLC chromatogram obtained at a detection wavelength of 250 nm.
FIG. 7 is an HPLC chromatogram obtained at a detection wavelength of 300 nm.
FIG. 8 is a HPLC standard fingerprint of acetonitrile-water system as the mobile phase.
FIG. 9 is a HPLC standard fingerprint with mobile phase of acetonitrile-phosphoric acid system.
FIG. 10 is a HPLC standard fingerprint of a mobile phase acetonitrile-triethylamine system.
Detailed Description
The present invention will be described in detail below with reference to specific examples.
The kidney-tonifying and bone-strengthening capsules used in the following examples were provided by the health pharmaceutical industry group, ltd.
Prescription: prepared rehmannia root 508g, cornus officinalis 254g, cibotium rhizome 254g, epimedium 254g, Chinese yam 254g, Chinese angelica 254g, tree peony bark 190g, oyster 381g
The preparation method comprises the following steps: the ten medicines are prepared by performing reflux extraction on dogwood (prepared), angelica and epimedium twice by using 70% ethanol, merging extracting solutions, filtering, concentrating filtrate under reduced pressure to obtain an extract with the relative density of 1.20, distilling moutan bark by using steam to extract paeonol, decocting the residues and other medicines (3/4 parts of calcined oyster) in water for three times, merging decoction, filtering, standing, taking supernatant, concentrating the supernatant to the relative density of 1.20-1.28, merging the supernatant with the ethanol extract, concentrating the supernatant to the extract with the relative density of 1.20-1.30, taking the residual 1/4 calcined oyster powder as a bottom material, spraying the extract and paeonol to granulate, and filling the mixture into 1000 granules.
Example 1
1. Instrument and reagent
A DIONEX P680 high performance liquid chromatograph, a quaternary pump, an online vacuum degassing system, an automatic sample injector, a column incubator and an ultraviolet detector; AB204-E electronic balance (Shanghai Merle-Torledo instruments, Inc.).
10 batches of kidney-tonifying and bone-strengthening capsules (190201-03, 190404-05, 190706-10 in 10 batches); icariin reference substances (for content detection, purchased by China institute for food and drug analysis, batch number: 110737-; acetonitrile is chromatographic grade, water is purified water, and the rest is chemical grade.
2. Method step
(1) Preparation of a test solution: grinding the content of the capsule to be tested for tonifying kidney and strengthening bone, taking 1.5g, precisely weighing, precisely adding 30ml of 50% methanol, weighing, performing ultrasonic treatment for 30 minutes (power 250W and frequency 40kHz), cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, and taking the subsequent filtrate.
(2) Precisely absorbing 10ul of a test solution, and detecting by using a high performance liquid chromatograph, wherein the high performance liquid chromatograph comprises an ultraviolet detector, the set detection wavelength of the ultraviolet detector is 235nm, and the stationary phase of the high performance liquid chromatograph is C18And (3) carrying out a chromatographic column, wherein the mobile phase A is acetonitrile, the mobile phase B is an aqueous solution containing 0.1% of monopotassium phosphate and 0.05% of sodium octane sulfonate, gradient elution is adopted, the flow rate is 1ml/min, the column temperature is 30 ℃, and the number of theoretical plates is not less than 4000.
The elution procedure is shown in table 1, namely 1 sample analysis procedure is completed, and the recording time is 0-50 min.
(3) Methodology investigation: performing precision test, stability test and repeatability test according to the requirement of the fingerprint;
(4) establishing a fingerprint spectrum: respectively taking several batches of kidney-tonifying and bone-strengthening capsule samples, preparing a test solution according to the item in the step (1), determining according to the chromatographic conditions in the step (2), recording a chromatogram for 50min, concentrating all chromatographic peaks within 50min, comparing the chromatograms of the sample batches, determining 9 common peaks, and comparing the chromatogram with a reference substance chromatogram to confirm that the No. 6 icariin is the icariin.
(5) Calculating the similarity of the samples: the 10 batches of chromatograms of the kidney-tonifying and bone-strengthening capsules are led into a 2004 edition of a Chinese medicine chromatogram fingerprint similarity evaluation system issued by the State pharmacopoeia Committee, the width of a time window is selected to be 0.1min, a chromatogram fingerprint common mode is generated through multi-point correction and data matching, and similarity analysis is carried out on the fingerprints of 10 batches of samples.
Example 2 optimization of assay conditions
1. Preparation of test solution
According to the prescription and the preparation characteristics of the kidney-tonifying and bone-strengthening capsule, a sample solution is prepared by respectively considering 50% methanol and 50% ethanol as extraction solvents, and the specific method refers to example 1.
A chromatographic column: agilent ZORBAX SB-C184.6 × 250mm, 5 um; detection wavelength: 235 nm; the sample amount is 10 ul; column temperature: 30 ℃; flow rate: 1 ml/min; mobile phase A: acetonitrile, mobile phase B: an aqueous solution containing 0.1% monopotassium phosphate and 0.05% sodium octane sulfonate, the gradient elution procedure is shown in Table 1.
As can be seen from a comparison between FIG. 1 and FIG. 3, the sample solution obtained by 50% methanol extraction has a larger chromatographic peak area and a better separation effect of three peaks from 40 to 45 minutes, so that 50% methanol was determined as the extraction solvent.
Finally, the preparation method of the sample is determined as follows: grinding the kidney-tonifying and bone-strengthening capsule to be detected, precisely weighing 1.5g, precisely adding 30ml of 50% methanol, weighing, performing ultrasonic treatment for 30 minutes (power 250W and frequency 40kHz), cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, and collecting the subsequent filtrate.
2. Determination of the measurement wavelength
HPLC spectra were obtained by selecting detection wavelengths of 205nm, 215nm, 235nm, 250nm, and 300nm, respectively, and performing the same procedures as in example 1 except that the detection conditions were changed.
From the comparison of fig. 1, 4, 5, 6 and 7, it can be seen that the baseline fluctuation of fig. 4 is large, the chromatographic peak areas No. 1 and No. 2 in fig. 4 and fig. 5 are small, the chromatographic peak area No. 6 in fig. 6 is small, the chromatographic peak shapes No. 7, 8 and 9 are too small and can be ignored, and the peaks No. 1, 2 and 3 in fig. 7 are absent, so that in summary, the fingerprint spectrum with 235nm as the detection wavelength can most fully reflect the components of the preparation, and the peak shape is best, and the other peak separation degrees and the number of chromatographic peaks, the baseline and the signal response are weak. Therefore, 235nm was determined as the optimum detection wavelength.
3. Selection of mobile phase
We have examined a plurality of sets of mobile phases in common, wherein the first set of mobile phases is acetonitrile-water, the second set of mobile phases is acetonitrile-phosphoric acid solution, the third set of mobile phases is acetonitrile-0.1% potassium dihydrogen phosphate (0.05% sodium octane sulfonate) system, and the fourth set of mobile phases is acetonitrile-triethylamine system, and the other conditions are the same as in example 1.
As can be seen from the comparison of FIG. 1, FIG. 8, FIG. 9 and FIG. 10, FIG. 8 has only peaks 4, 5 and 6, and lacks peaks 1, 2, 3 and 7, 8 and 9, peaks 4, 5 and 6 in FIG. 9 have poor separation effect, and a peak is not completely separated at 13min, and a large number of peaks in FIG. 10 cannot be separated at 35-45min, whereas the liquid chromatogram obtained by the system of FIG. 1 acetonitrile-0.1% potassium dihydrogen phosphate (0.05% sodium octane sulfonate) has a smoother baseline, more symmetrical peak shape of chromatographic peaks, and better resolution.
Example 3 methodological examination
1. Stability test
Taking the sample solution, measuring at 0, 4, 8, 12h and 24h according to the conditions of the chromatographic term, and observing the relative retention time of each common peak and the RSD of the relative peak area. The results show that the relative retention time of each spectrum peak is less than 2.0 percent, the relative peak area of each spectrum peak is less than 2.0 percent, and the fingerprint spectrum technical requirements are met. See tables 3-4.
TABLE 3 evaluation of relative retention time of Capsule for tonifying kidney and strengthening bone
Figure BDA0002280622510000061
TABLE 4 relative peak area for stability survey of kidney-tonifying and bone-strengthening capsules
Figure BDA0002280622510000062
2. Repeatability test
Taking 6 parts of samples of the same batch, preparing 6 parts of test solution according to the test solution preparation method under item 1, measuring under the chromatographic conditions, and observing the relative retention time of each common peak and the RSD of the relative peak area. The results show that the relative retention time of each spectrum peak is less than 2.0 percent, the relative peak area of each spectrum peak is less than 5.0 percent, and the fingerprint spectrum technical requirements are met. See tables 5-6.
TABLE 5 repeated examination of relative retention time of kidney-tonifying and bone-strengthening capsules
Figure BDA0002280622510000071
TABLE 6 relative peak area for repeated examination of kidney-tonifying and bone-strengthening capsules
Figure BDA0002280622510000072
3. Precision test
And taking a sample solution to be tested, continuously injecting samples for 6 times according to the conditions under the chromatographic terms, and inspecting the relative retention time of each common peak and the RSD of the relative peak area. The results show that the relative retention time of each spectrum peak is less than 1.0 percent, the relative peak area of each spectrum peak is less than 3.0 percent, and the fingerprint spectrum technical requirements are met. See tables 7-8.
TABLE 7 examination of the relative retention time for the kidney-tonifying and bone-strengthening capsules
Figure BDA0002280622510000081
TABLE 8 examination of relative peak area for precision of kidney-tonifying and bone-strengthening capsule
Figure BDA0002280622510000082
Example 4 creation of fingerprint
10 batches of kidney-tonifying and bone-strengthening capsule samples are respectively taken to prepare a test solution, 50min chromatograms are measured and recorded, the chromatograms of the 10 batches of samples are compared, and 9 common peaks are determined. It was confirmed that No. 6 was icariin peak as a reference peak.
1. Calculating the similarity of the samples: 10 batches of kidney-tonifying and bone-strengthening capsule chromatograms are introduced into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2004 edition) issued by the State pharmacopoeia Committee, the width of a time window is selected to be 0.1min, a sample 3 is adopted to generate a comparison spectrum, a chromatogram fingerprint common mode is generated through multipoint correction and data matching, and 10 batches of sample fingerprint similarity calculation results are shown in a table 9.
TABLE 9 Ten sample similarities
Batch number Degree of similarity
190201 0.992
190202 0.993
190203 0.999
190404 0.989
190405 0.985
190706 0.976
190707 0.992
190708 0.993
190709 0.990
190710 0.989
Comparison fingerprint 1
2. Establishment of standard fingerprint of kidney-tonifying and bone-strengthening capsule
Taking a plurality of batches of kidney-tonifying and bone-strengthening capsules, respectively preparing a test solution according to a preparation method of the test solution, establishing a high performance liquid chromatogram of the kidney-tonifying and bone-strengthening capsules according to a plurality of detection data obtained by detection, determining the peak retention time and the peak area value of the high performance liquid chromatogram, then collecting all detection data, calculating the average value of the relative retention time of each corresponding peak, the average value of the relative peak area, the standard deviation and the like, respectively dividing the average retention time and the average peak area of a reference peak, using the average retention time and the relative peak area value of a standard fingerprint, deriving a calculation result, making standard fingerprint data, drawing the standard fingerprint (the standard fingerprint is shown in a figure 1), and identifying a No. 6 peak as an icariin peak (the figure 2), wherein the result is shown in a figure 10.
TABLE 10 Standard fingerprint data
Figure BDA0002280622510000091
Figure BDA0002280622510000101

Claims (2)

1. An HPLC fingerprint detection method of a traditional Chinese medicine for tonifying kidney and strengthening bone is disclosed, wherein the traditional Chinese medicine for tonifying kidney and strengthening bone is prepared from the following raw materials in parts by weight: 3-8 parts of prepared rehmannia root, 1-5 parts of dogwood, 1-5 parts of rhizoma cibotii, 1-5 parts of epimedium, 1-5 parts of Chinese yam, 1-5 parts of angelica, 1-4 parts of moutan bark and 2-6 parts of oyster, and is characterized in that the detection method comprises the following steps:
(1) preparation of a test solution: grinding the Chinese medicinal materials to be tested, ultrasonically extracting with 50% methanol, and filtering to obtain test solution;
(2) precisely absorbing a sample solution, and detecting by using a high performance liquid chromatograph, wherein the high performance liquid chromatograph comprises an ultraviolet detector, the set detection wavelength of the ultraviolet detector is 235nm, and the stationary phase of the high performance liquid chromatograph is C18A chromatographic column, wherein a mobile phase A is acetonitrile, a mobile phase B is an aqueous solution containing 0.1% of monopotassium phosphate and 0.05% of sodium octane sulfonate, gradient elution is adopted, the flow rate is 0.5-2ml/min, and the column temperature is 25-40 ℃;
(3) recording the chromatogram of the test solution for 0-50 min, performing data import, multipoint correction and data matching on the chromatogram of the test solution by using a Chinese medicine chromatogram fingerprint similarity evaluation system of the State pharmacopoeia Committee to obtain a fingerprint and performing similarity analysis,
the procedure for the gradient elution was as follows:
elution time Mobile phase A Mobile phase B 0～10min 10％ 90％ 10～25min 10→28％ 90→72％ 25～35min 28％ 72％ 35～50min 28→100％ 72→0％
。
2. The HPLC fingerprint detection method of a kidney-tonifying and bone-strengthening traditional Chinese medicine as claimed in claim 1, wherein: the fingerprint has 9 common peaks, wherein the No. 6 peak is a reference peak, namely an icariin peak, and the retention time, the relative retention time, the peak area and the relative peak area of each peak are as follows:
Figure FDA0003277288250000011
Figure FDA0003277288250000021
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