CN116008134B - Identification method of malt, roasted malt and scorched malt formula particles - Google Patents
Identification method of malt, roasted malt and scorched malt formula particles Download PDFInfo
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Abstract
The invention belongs to the technical field of chromatographic analysis of traditional Chinese medicine formula particles, and particularly relates to a method for identifying malt, roasted malt and scorched malt formula particles. The identification method adopts 10% methanol to carry out ultrasonic extraction on the formula particles, centrifugates to obtain formula particle extracting solution, and adopts liquid chromatography to identify three formula particles quantitatively. The chromatographic column filler is octadecylsilane chemically bonded silica gel, the specification of the chromatographic column is that the column length is 100mm, the inner diameter is 2.1mm, and the particle size is 1.7 μm. Acetonitrile was used as mobile phase a and 0.1% formic acid solution was used as mobile phase B for gradient elution at a flow rate of 0.3mL per minute. The detection wavelength is 295 nm-300 nm. The identification method can be used for effectively distinguishing three formula particles within 25min by qualitative analysis, and is simple and easy to use and popularize.
Description
Technical Field
The invention belongs to the technical field of chromatographic analysis of traditional Chinese medicine formula particles, and particularly relates to a method for identifying malt, roasted malt and scorched malt formula particles.
Background
The fructus Hordei Germinatus is barley belonging to GramineaeHordeum vulgare L.) The ripe fruits of (C) are processed into processed products by germination and drying. There are various methods for processing malt, and malt is classified into malt (also called raw malt), roasted malt and roasted malt according to the processing method. The processing methods of malt, roasted malt and burnt malt specified in the pharmacopoeia of the people's republic of China (2020 edition) are respectively as follows: malt: soaking wheat grains in water, maintaining proper temperature and humidity, and sun drying or low temperature drying when the buds grow to about 5 mm; malt frying: parching cleaned fructus Hordei Germinatus with clear parching method (general rule 0213) to brown yellow, cooling, and sieving to remove dust; malt preparation: parching fructus Hordei Germinatus with clear parching method (general rule 0213) to brown, cooling, and sieving to remove dust.
The efficacy of different processed products of malt is different due to different processing methods, for example, raw malt is used for strengthening spleen and stomach, soothing liver and promoting qi, and is used for spleen deficiency, anorexia and milk stasis; the roasted malt can promote qi circulation, promote digestion and promote lactation, and is used for treating dyspepsia and female weaning; the scorched malt is used for resolving food stagnation and treating abdominal distention and pain (pharmacopoeia of the people's republic of China (2020 edition)).
The traditional Chinese medicine formula granule is prepared by heating, extracting, separating, concentrating, drying and granulating single traditional Chinese medicine decoction pieces with water, and is prepared according to the clinical prescription of the traditional Chinese medicine under the guidance of the traditional Chinese medicine theory, and is taken by patients. Except for the forming process, the other processes of the traditional Chinese medicine formula granule are basically consistent with the traditional decoction, namely, water is used as a solvent for heating extraction, and solid-liquid separation, concentration, drying, granule forming and other processes are carried out by a physical method for production (the technical requirements for quality control and standard formulation of the traditional Chinese medicine formula granule of the national drug administration). The effective components, the nature and the taste, the channel tropism and the like of the Chinese medicinal decoction pieces are basically consistent with those of the traditional Chinese medicinal decoction pieces, and the Chinese medicinal decoction pieces have the advantages of no need of decoction, direct administration, small dosage, convenience in carrying and storage and the like. Therefore, the traditional Chinese medicine formula particles are rapidly applied in clinic, and the use amount of the traditional Chinese medicine formula particles in part of medical institutions is already more than that of the traditional decoction pieces. Malt formulation granules, roasted malt formulation granules and roasted malt formulation granules are commercially available. However, as the traditional Chinese medicine decoction pieces are extracted and concentrated by the traditional Chinese medicine prescription granule, the appearance characteristics and microscopic characteristics of the traditional Chinese medicine decoction pieces are lost, and the characteristics such as smell and the like of the traditional Chinese medicine decoction pieces are not easy to distinguish, so that the characteristics and microscopic identification are difficult to distinguish. This is an inconvenience of the traditional Chinese medicine formulation granule compared with the traditional decoction pieces.
As for the influence of malt processing technology on components, many studies have been conducted by liquid chromatography. Several prior studies are available for reference. He Danxia the contents of 4 flavonoid components (catechin, myricetin, quercetin and kaempferol) in different products of barley were studied. The kaempferol content in the raw malt and the roasted malt is found to be slightly increased compared with the kaempferol content in the grains, but the kaempferol content in the roasted malt is basically the same as the kaempferol content in the grains. The contents of three flavonoid components in different malt preparations are slightly different, but the content difference of the same flavonoid component in the same malt preparation is extremely large; the method can not be used for identifying malt, roasted malt and burnt malt, and is more difficult to identify the formula particles even if the content of the same flavonoid components of different malt preparations are overlapped. In addition, the established analytical method has a kaempferol retention time of approximately 40min, and requires longer chromatographic analysis time (He Danxia, rong Liang, qin Minjian, etc. the analysis of flavonoid compounds in barley kernels, barley malt, malt-grist and malt-grist [ J ]. Chinese medicinal materials, 2012, 35 (11): 5.) for identifying different malt preparations. Yang Huasheng the change rule of the effective components and the ineffective components in the malt frying process is researched, the content change of the ineffective components is closely related to the processing of the fire, and the dynamic change rule of the ineffective components possibly provides scientific basis for evaluating the malt frying process and explaining the efficacy mechanism. The obtained two marking components of 5-hydroxymethyl furfural and acrylamide can be used for malt parching and process evaluation and identification of processed products. The content of the wheat flavone, the quercetin, the catechin and the kaempferol is insensitive to the processing temperature or lacks regularity, and the content of the ferulic acid is also sensitive to the processing temperature to a certain extent. The method provides a certain reference for quality control or identification distinction of malt and traditional Chinese medicine products (including crude drugs, crude products, decoction pieces, prescription granules and the like).
In the aspects of malt and traditional Chinese medicine product detection method construction and identification and differentiation, the following related researches can be referred to. Gao Ying the fingerprint of the malt-roasted formula particle is constructed to obtain 9 common peaks, wherein 3 chromatographic peaks in the 9 common peaks have retention time longer than 25min, and only ferulic acid with the longest retention time is identified (Gao Ying, zhangyun Tian, xuezhi, etc. HPLC fingerprint research of malt-roasted formula particle [ J ]. J.Chinese experimental prescription journal, 2013.). Patent document CN202110730599.4 also establishes a characteristic spectrum of a malt-roasted formula particle, obtaining 4 characteristic peaks, wherein the retention time of S-peak ferulic acid is greater than 40min. The remaining three characteristic peaks are not identified. It can be seen that the characteristic spectrum or fingerprint established for the same malt processed product will be significantly different due to the different chromatographic conditions such as extraction method, mobile phase, stationary phase, elution condition, detection wavelength, etc. Patent document CN201711264566.5 constructs a chromatographic detection method of malt formulation particles, and can detect the content of catechin, ferulic acid and the like in the malt formulation particles by detecting the wavelength of 320 nm. But the retention time of catechin and ferulic acid chromatographic peaks is more than 40min. The catechin and ferulic acid also have (He Danxia, rong Liang, qin Minjian, etc. in the roasted malt, the content of flavonoids in barley grain, raw malt, roasted malt and roasted malt was analyzed [ J ]. Chinese medicinal materials, 2012, 35 (11): 5; CN202110730599.4). Therefore, it is difficult to distinguish malted and scorched malted grains.
As can be seen, there is currently a lack of rapid, qualitative chromatographic methods in the art that effectively identify and differentiate malted formulas (also known as malted formulas), roasted formulas, and roasted formulas. This would be detrimental to the identification and differentiation of the finished product, as well as to market regulation, for formulation particles lacking shape and microscopic differentiation.
Disclosure of Invention
In view of the above problems, it is an object of the present invention to provide a qualitative identification method that can identify and distinguish malting formulas (also called malting formulas), roasted malting formulas, and burnt malting formulas. To achieve the object, the technical scheme of the invention is as follows:
the identification method of malt, roasted malt and scorched malt formula particles is liquid chromatography, and the liquid chromatography comprises the following steps:
a21: grinding the formula particles to be detected, adding methanol with the concentration of 5-50% for ultrasonic extraction, centrifuging, taking supernatant, filtering, and taking the subsequent filtrate as a sample solution;
a22: taking the sample solution obtained in the step A21, and injecting the sample solution into a liquid chromatograph; the chromatographic conditions were as follows:
octadecylsilane chemically bonded silica is used as a filler; acetonitrile is taken as a mobile phase A, and 0.1% formic acid solution is taken as a mobile phase B; the detection wavelength is 295 nm-300 nm; the gradient elution procedure was as follows:
a23: and (3) analyzing the A22 liquid chromatogram, and qualitatively identifying three formula particles according to the retention time of chromatographic peaks or the relative retention time of chromatographic peaks.
Preferably, the methanol concentration in a21 is 10%; the mass volume ratio of the formula particles to be tested to the methanol is 0.2g to 25 mL-0.8 g to 25mL. Further preferably, the mass to volume ratio of the formulation particles to be tested to methanol is 0.5g:25mL.
Preferably, the ultrasonic extraction conditions in a21 are: extracting for 15min at power 400W and frequency 40 kHz.
Preferably, the centrifugation method in a21 is: 1200 rpm and centrifuging for 5min.
Preferably, the chromatographic column parameters in A22 using octadecylsilane chemically bonded silica as a filler are: the column length was 100mm, the inner diameter was 2.1mm, and the particle diameter was 1.7. Mu.m.
Preferably, the flow rate of the A22 mobile phase is 0.3mL/min, the column temperature is 20-30 ℃, the sample injection amount is 1 mu L,
the theoretical plate number should be not less than 5000 as calculated by ferulic acid peak. Further preferably, the column temperature of step a22 is 25 ℃.
Preferably, three formulation particles are qualitatively identified in a23 based on chromatographic peak retention times of the liquid chromatogram:
the liquid chromatogram of the malt formulation granule lacks peak 3 with retention time of 10.127 min-10.197 min; the scorched malt formulation particles lack peak 4 with retention time 14.626 min-14.698 min and/or peak 5 with retention time 15.913 min-15.976 min; the liquid chromatogram of the malt-roasted granule has a peak 3 with retention time of 10.127 min-10.197 min, a peak 4 with retention time of 14.626 min-14.698 min and a peak 5 with retention time of 15.913 min-15.976 min.
Preferably, three formulation particles are qualitatively identified in a23 based on the relative retention times of the chromatographic peaks of the liquid chromatograph, said liquid chromatograph further comprising the steps of: a1, adding 10% methanol into ferulic acid reference substance to prepare 1 mug solution containing ferulic acid per 1mL as reference substance solution; in A22, taking reference substance solution and sample solution of the reference substance, and injecting the reference substance solution and the sample solution into a liquid chromatograph, wherein the sample injection amount is 1 mu L; in A23, the liquid chromatogram takes ferulic acid chromatographic peaks as reference peaks, calculates the relative retention time of each chromatographic peak and ferulic acid chromatographic peak, and identifies malt formulation granule, parched malt formulation granule, and scorched malt formulation granule: the malt formulation granule liquid chromatogram lacks peak 3 with a relative retention time of 0.459 to 0.464; the malted formula particles lack peak 4 with a relative retention time of 0.664-0.669 and/or peak 5 with a relative retention time of 0.722-0.727; the liquid chromatogram of the malt-roasted granule has a peak 3 with a relative retention time of 0.459-0.464, a peak 4 with a relative retention time of 0.664-0.669, and a peak 5 with a relative retention time of 0.722-0.727.
Preferably, the preparation method of the malt formula granule comprises the following steps:
y11, soaking wheat grains in water, maintaining proper temperature and humidity, and sun-drying or low-temperature drying to obtain malt decoction pieces when the buds grow to about 5 mm; y12: taking 10kg of malt decoction pieces, adding water for decoction twice, adding 12 times of water for decoction for 2 hours for the first time, adding 8 times of water for decoction for 1 hour for the second time, merging filtrate, concentrating under reduced pressure to obtain an extract with the relative density of 1.03-1.05, adding auxiliary material maltodextrin for uniform mixing and granulating, and freeze-drying to prepare 1kg of malt formula particles.
Preferably, the preparation method of the malt-roasted formula granule comprises the following steps:
y21: taking malt decoction pieces of Y11, parching to brown, cooling, and sieving to remove ash to obtain parched malt decoction pieces according to the general rule parching method of pharmacopoeia of people's republic of China (2020 edition) 0213; y22: taking 10kg of fried malt decoction pieces, adding water for decoction twice, adding 12 times of water for decoction for 2 hours for the first time, adding 8 times of water for decoction for 1 hour for the second time, merging filtrate, concentrating under reduced pressure to obtain an extract with the relative density of 1.03-1.05, adding auxiliary material maltodextrin for uniform mixing and granulating, and freeze-drying to prepare 1kg of fried malt formula particles.
Preferably, the preparation method of the scorched malt formulation granule comprises the following steps:
y31: taking malt decoction pieces of Y11, parching to brown according to the general rule of processing and parching method of pharmacopoeia of the people's republic of China (2020 edition) 0213, cooling, and sieving to remove ash to obtain malt decoction pieces; y32: taking 5kg of scorched malt decoction pieces, adding water for decoction twice, adding 12 times of water for decoction for 2 hours for the first time, adding 8 times of water for decoction for 1 hour for the second time, merging filtrate, concentrating under reduced pressure to obtain an extract with the relative density of 1.03-1.05, adding auxiliary material maltodextrin for uniform mixing and granulating, and freeze-drying to prepare 1kg of scorched malt formula particles.
The invention adopts an ultrasonic extraction method to extract and treat the formula particles, adopts octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A, 0.1 percent formic acid solution as a mobile phase B, the detection wavelength is 297nm, and adopts a liquid chromatography to qualitatively identify malt formula particles, roasted malt formula particles and scorched malt formula particles, and has the following beneficial effects:
1. the identification method has good precision, repeatability and stability.
2. The identification method can still separate and obtain chromatographic peaks of the malt active ingredient ferulic acid, can be used for auxiliary identification of the active ingredient, is stable and reliable, and is suitable for reference.
3. The identification method has the advantages that the peak time of each chromatographic peak is faster, and the peak time of the chromatographic peak is less than 25min, so that the three formula particles can be rapidly distinguished.
4. The identification method has less identification peaks required for distinguishing the malt formula particles, the roasted malt formula particles and the scorched malt formula particles, only needs peak 3 with retention time of 10.127 min-10.197 min (relative retention time of 0.459-0.464), peak 4 with retention time of 14.626 min-14.698 min (relative retention time of 0.664-0.669 with ferulic acid peak) and/or peak 5 with retention time of 15.913 min-15.976 min (relative retention time of 0.664-0.669 with ferulic acid peak) to distinguish the three formula particles, and is simple and easy to use. The retention time can be used for qualitative identification, so that the workload of calculating the relative retention time is reduced, and the method is simpler, more convenient and easier to use.
Drawings
FIG. 1 is a comparison of liquid chromatograms of malt formulation granules, roasted malt formulation granules, and roasted malt formulation granules of example 1;
FIG. 2 is a comparison of liquid chromatograms of 15 total batches of malt-roasted formula particles of example 2;
FIG. 3 is a comparison of liquid chromatograms of 15 total malt formulation granules of example 3;
FIG. 4 is a comparison of liquid chromatograms of 15 total lots of malt formulations of example 4.
Detailed Description
The method for identifying malt-formulated particles, roasted malt-formulated particles and roasted malt-formulated particles according to the present invention will be described below by way of specific examples. The following examples are illustrative examples and are not to be construed as limiting the scope of protection.
Example 1 liquid chromatogram comparison of malt formulation granule, parched malt formulation granule, and scorched malt formulation granule
1. Instrument and reagents:
waters high performance liquid chromatograph (model 2695, watshi technology (Shanghai)), one percent analytical balance (CN-LQC 10002, kunshan Kovilte electronics Inc.), one ten-thousandth analytical balance (FA 1004, force technology Inc.), one ten-thousandth analytical balance (AUW 220D, shimadzu corporation), digital controlled ultrasonic cleaner (KQ-400D, kunshan ultrasonic instruments Inc.), thermostatic water bath (HH-8, changzhou Deke instruments Inc.), ultrapure water system (EKUP-II-10 p, sichuan Korea pure water equipment Inc.).
Ferulic acid control was purchased from Chengdu Pusi biotechnology Co., ltd; malt control was purchased from chinese food and drug assay institute. Methanol, liquid phase with formic acid and acetonitrile were purchased from Colon Chemicals, inc. of Chemicals, inc. Malt formulation granule, stir-fried malt formulation granule and scorched malt formulation granule are self-made by the company.
2. Preparation of formula particles:
malt medicinal materials are from Shandong province and are identified as gramineous plant barley by food and drug inspection institute of Shandong provinceHordeum vulgare L.) Is a germination dried product of (a). Malt decoction pieces, roasted malt decoction pieces and burnt malt decoction pieces are prepared according to the pharmacopoeia of the people's republic of China (2020 edition).
Malt formulation particles: taking 10kg of malt decoction pieces, adding water for decoction twice, adding 12 times of water for decoction for 2 hours for the first time, adding 8 times of water for decoction for 1 hour for the second time, merging filtrate, concentrating under reduced pressure to obtain an extract with the relative density of 1.03-1.05, adding auxiliary material maltodextrin for uniform mixing and granulating, and freeze-drying to prepare 1kg of malt formula particles.
Malt-roasted formula particles: taking 10kg of fried malt decoction pieces, adding water for decoction twice, adding 12 times of water for decoction for 2 hours for the first time, adding 8 times of water for decoction for 1 hour for the second time, merging filtrate, concentrating under reduced pressure to obtain an extract with the relative density of 1.03-1.05, adding auxiliary material maltodextrin for uniform mixing and granulating, and freeze-drying to prepare 1kg of fried malt formula particles.
The scorched malt formulation granule: taking 5kg of scorched malt decoction pieces, adding water for decoction twice, adding 12 times of water for decoction for 2 hours for the first time, adding 8 times of water for decoction for 1 hour for the second time, merging filtrate, concentrating under reduced pressure to obtain an extract with the relative density of 1.03-1.05, adding auxiliary material maltodextrin for uniform mixing and granulating, and freeze-drying to prepare 1kg of scorched malt formula particles.
3. Chromatographic conditions
The chromatographic column is a Voltech ACQUITY UPLC BEH octadecylsilane chemically bonded silica chromatographic column (column length is 100mm, inner diameter is 2.1mm, and particle diameter is 1.7 μm); acetonitrile as mobile phase a and 0.1% formic acid solution as mobile phase B, and performing gradient elution according to the elution procedure in table 1; the flow rate is 0.3mL/min; the column temperature is 25 ℃; the detection wavelength was 297nm. The theoretical plate number should be not less than 5000 as calculated by ferulic acid peak.
TABLE 1 Mobile phase elution procedure
4. Preparation of reference solutions
Taking 1g of malt reference medicine, adding 25mL of 10% methanol, weighing, heating and refluxing for 15min, cooling, weighing again, supplementing the lost weight with 10% methanol, shaking uniformly, centrifuging (rotating at 1200 rpm) for 10min, taking supernatant, filtering, and taking subsequent filtrate as reference medicine solution.
And (3) taking a proper amount of ferulic acid reference substance, precisely weighing, and adding 10% methanol to prepare a solution containing 1 mug of ferulic acid per 1mL serving as the reference substance solution of the reference substance.
5. Preparation of test solution
Taking a proper amount of test sample of the prescription granule, grinding, taking about 0.5g, precisely weighing, placing into a conical bottle with a plug, precisely adding 25mL of 10% methanol, sealing, weighing, performing ultrasonic treatment (power 400W, frequency 40 kHz) for 15min, cooling, weighing again, supplementing the lost weight with 10% methanol, shaking uniformly, centrifuging (rotating at 1200 rpm) for 5min, taking supernatant, filtering, and taking subsequent filtrate.
6. Assay
Precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
7. Precision test
The ferulic acid reference solution was sampled 6 times continuously under the conditions of the "3. Chromatography conditions" item of this example, and the relative standard deviation of the ferulic acid retention time and peak area was calculated (relative standard deviation; RSD).
Results: the ferulic acid retention time and RSD of peak area were all less than 0.5%. Indicating that the instrument has good precision.
8. Repeatability test
Taking the malt control medicinal materials, malt formulation particles, roasted malt formulation particles and burnt malt formulation particles in the same batch, according to the methods of "4. Preparation of reference solution" and "5. Preparation of sample solution" in this example, 12 parts of sample solution and 12 parts of reference solution of reference medicinal material were prepared, respectively, and the detection was performed under the conditions of the measurement under the "3. Chromatographic conditions" item in this example. Results: the retention time of ferulic acid and RSD of peak area in malt control medicinal material, malt formulation granule, parched malt formulation granule and scorched malt formulation granule are all less than 1%. Indicating that the method is well reproducible.
9. Stability test
Taking the malt reference medicinal materials, malt formulation particles, fried malt formulation particles and scorched malt formulation particles in the same batch, respectively preparing 12 parts of the sample solution and 12 parts of the reference medicinal material reference solution according to the preparation method of the reference substance solution and the preparation method of the sample solution in the embodiment '4', and respectively detecting under the measurement conditions of the chromatographic conditions in the embodiment '3', under the conditions of 0, 4, 8, 12, 24 and 48 hours. Results: the retention time of ferulic acid and RSD of peak area in malt control medicinal material, malt formulation granule, parched malt formulation granule and scorched malt formulation granule are all less than 1%. The method is shown to have good stability in the corresponding time.
10. Measurement results
The comparison chart of the liquid phase chromatograms of the reference substance solution of the reference medicinal material, the reference substance solution of the ferulic acid, the malt formulation granule, the roasted malt formulation granule and the scorched malt formulation granule is shown in fig. 1, and the retention time data of peak 1 to peak 7 in fig. 1 are shown in table 2.
TABLE 2 malt formulation granule, parched malt formulation granule, scorched malt formulation granule detection data
Remarks: "/" indicates that no such chromatographic peak was detected.
When fig. 1 and table 2 were analyzed, it was found that: under the conditions of extraction separation and detection in the experiment and given samples, liquid chromatograms of malt formulation particles, roasted malt formulation particles and burnt malt formulation particles all have ferulic acid peaks (peak 7), but the liquid chromatograms of the three formulation particles have significant differences. The malt formulation particles did not detect peak 1, peak 2, peak 3; the scorched malt formulation particles did not detect peak 4, peak 5, peak 6; the malt-roasted formula particles can detect peak 1, peak 2, peak 3, peak 4, peak 5, peak 6 and peak 7. The separation degree of the peak 1, the peak 2, the peak 3, the peak 4, the peak 5 and the peak 7 is more than 10, so that the qualitative identification requirement is met. However, the chromatograms of the single source malt sample prepared formula particles may not be widely representative.
Example 2 liquid chromatogram comparison of the malt-grist formulation granules of batch 15
1. Sample source of malt-roasted formula particles
According to the technical requirements of quality control and standard establishment of traditional Chinese medicine formula granule issued by 2021 in China, 15 batches of malt medicinal materials are taken from Shandong province, shanxi province, inner Mongolian autonomous region, qinghai province and Sichuan province, and are identified as gramineous plant barley by Shandong province food and drug inspection instituteHordeum vulgare L.Is a germination dried product of (a). The malt-roasted decoction pieces are prepared according to the pharmacopoeia of the people's republic of China (2020 edition). The detailed preparation method of the malt-fired formula granule is shown in example 1"2.
2. Detection method of roasted malt formula particles
15 batches of malt-roasted formula particles of different origin were tested by liquid chromatography according to the method of example 1.
Liquid chromatogram comparison of 3.15 batches of malt-roasted formula particles
A comparison of liquid chromatograms of 15 batches of malt-roasted formula particles is shown in FIG. 2, and chromatographic peak retention times are shown in Table 3. The liquid chromatograms of the 15 batches of malt-roasted formula particles each have a peak 1, a peak 2, a peak 3, a peak 4, a peak 5, a peak 6 and a peak 7 in example 1. As shown in Table 3, the standard deviation of retention time of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6 and peak 7 in the liquid chromatogram of 15 batches of the malt-roasted formula particles is less than 0.04min, and RSD is less than 0.5%. The relative retention times (Relative retention time, RRT; i.e. the ratio of the retention time of the other peaks to the retention time of peak 7) for peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7 were calculated based on peak 7 (ferulic acid) as follows: 0.180+ -0.001, 0.307+ -0.001, 0.462+ -0.001, 0.666+ -0.001, 0.724 + -0.001, 0.830+ -0.002, 1.
TABLE 3 chromatographic peak retention time (min) for liquid chromatogram of malt-grist formulation batch 15
Example 3 liquid chromatogram comparison of batch 15 malt formulation particles
1. Sample sources of malt formulation particles
According to the technical requirements of quality control and standard establishment of traditional Chinese medicine formula granule issued by 2021 in China, 15 batches of malt medicinal materials are taken from Shandong province, shanxi province, inner Mongolian autonomous region, qinghai province and Sichuan province, and are identified as gramineous plant barley by Shandong province food and drug inspection instituteHordeum vulgare L.Is a germination dried product of (a). Malt decoction pieces are prepared according to the pharmacopoeia of the people's republic of China (2020 edition). The detailed preparation method of malt formulation granule is described in example 1"2, preparation of formulation granule section.
2. Detection method of malt formula particles
Liquid chromatography was used to examine 15 batches of malt formulation particles of different origin, batches, as per example 1.
Liquid chromatogram comparison of 3.15 batches of malt formulation particles
A comparison of liquid chromatograms of 15 batches of malt formulation particles is shown in FIG. 3. Peak 2 was detected in malt formulation pellet lot MY 12; peak 1 was detected in malt formulation granules with lot numbers MY01, MY12, MY 13. Peak 4, peak 5, peak 6, peak 7 were all detected for 15 batches of malt formulation particles, and Peak 3 was not detected for all. The retention times of peaks 4, 5, 6 and 7 in the liquid chromatogram of 15 batches of malt formulation particles are shown in table 4. The standard deviation of retention time of peak 4, peak 5, peak 6 and peak 7 is less than 0.02min, and RRT of peak 4, peak 5, peak 6 and peak 7 are respectively calculated by taking peak 7 (ferulic acid) as a reference peak: 0.667+ -0.001, 0.725+ -0.001, 0.831 + -0.002, 1.
In combination with examples 1, 2, it is clear that a portion of the malt formulation particle liquid chromatogram can detect the presence of either peak 1 or peak 2, and therefore the presence or absence of either peak 1 or peak 2 cannot be used to qualitatively distinguish the malt formulation particle from the other two but peak 3 can qualitatively distinguish the malt formulation particle from the other two.
TABLE 4 chromatographic peak retention time (min) for liquid chromatograms of malt formulation granules of 15 batches
Example 4 comparison of liquid chromatography of batch Coke malt formulation particles
1. Sample source of malted formula particles
According to the technical requirements of quality control and standard establishment of traditional Chinese medicine formula granule issued by 2021 in China, 15 batches of malt medicinal materials are taken from Shandong province, shanxi province, inner Mongolian autonomous region, qinghai province and Sichuan province, and are identified as gramineous plant barley by Shandong province food and drug inspection instituteHordeum vulgare L.Is a germination dried product of (a). The scorched malt decoction pieces are prepared according to the pharmacopoeia of the people's republic of China (2020 edition). The detailed preparation method of the scorched malt formulation granule is shown in example 1"2.
2. Detection method of scorched malt formula particles
Different sources, batches of malt-based formulation particles were tested by liquid chromatography as in example 1.
3.15 liquid chromatogram comparison of the Coke malt formulation granules
A comparison of liquid chromatograms of 15 batches of malt formulation particles is shown in FIG. 4. Peak 1, peak 2, peak 3 and peak 7 were detected for both 15 lot of malted formulas and 15 lot of roasted formulas of example 2; no peak 4, peak 5, or peak 6 were detected for 15 lots of scorched malt formulation particles.
The retention times of peak 1, peak 2, peak 3 and peak 7 in the liquid chromatogram of 15 batches of scorched malt formulation particles are shown in Table 5. The standard deviation of retention time of peak 1, peak 2, peak 3 and peak 7 is less than 0.03min, and RRT of peak 1, peak 2, peak 3 and peak 7 are respectively calculated by taking peak 7 (ferulic acid) as a reference peak: 0.180+ -0.001, 0.307+ -0.001, 0.462+ -0.001, 1. Combining examples 1, 2, 3, 4, peak 5, peak 6 can qualitatively distinguish malted formula particles from the other two formula particles, but peak 6 is slightly less separated.
The peak heights and peak areas of the malted or malted formulas peak 1, peak 2, peak 3 vary greatly compared to the chromatogram of example 1. Comparing the chromatographic peak heights and peak areas of fig. 1, 2 and 4, the following is found: the malted formulation particles were difficult to distinguish effectively from the malted formulation particles of example 2 by peak heights and peak areas of peak 1, peak 2, peak 3; it is difficult to distinguish effectively by peak 1 to peak 7, peak 2 to peak 7, or peak 3 to peak 7 relative peak areas.
In connection with examples 1, 2, 3, 4, comparison of fig. 1, 2, 3, 4 reveals that: neither 15 lots of malt formulation particles had peak 3 nor 15 lots of scorched malt formulation particles nor 15 lots of roasted malt formulation particles had peak 3, so peak 3 could be used to distinguish malt formulation particles from the other two formulation particles.
The liquid chromatogram of the partial scorched malt formulation particle has a larger chromatographic peak between 17min and 18min of retention time. The liquid chromatogram of the partial scorched malt formulation particle has a smaller chromatographic peak between 19 min and 20min of retention time. Both chromatographic peaks were not common to the 15 batches of malted formulas.
TABLE 5 chromatographic peak retention time (min) for liquid chromatogram of batch of malt formulation particles
Example 5 liquid chromatography identification of malt formulation particles, parched malt formulation particles, scorched malt formulation particles
The chromatographic peak retention times and relative retention time data of examples 1, 2, 3, and 4 were comprehensively analyzed, and the chromatograms of tables 2 to 5 were data analyzed. The results are shown in Table 6, and the retention times of peak 1 and peak 2 detected in the malt formulation particles in example 3 remain within the range of Table 6.
TABLE 6 analysis of retention time and relative retention time data for chromatographic peaks in examples 1-4
a : incorporating the formula particles and the chromatographic peaks of the reference medicinal materials into statistics; the relative retention time was obtained by calculating the ratio of the retention time of each chromatographic peak to the retention time of the ferulic acid chromatographic peak with peak 7 (ferulic acid peak) as a reference peak. -: is not applicable.
By analysis of examples 1 to 4 and table 6, in combination with chromatographic peak separation requirements, the liquid chromatographic qualitative identification criteria for the three formulation particles are summarized as follows:
1. three formulation particles can be qualitatively identified using chromatographic peak retention times
Under the conditions of extraction and detection of examples 1 to 4, the malt formulation granule liquid chromatogram lacks peak 3 with retention time 10.127min to 10.197 min; the scorched malt formulation particles lack peak 4 with retention time of 14.626 min-14.698 min and peak 5 with retention time of 15.913 min-15.976 min (either peak 4 or peak 5 can be identified qualitatively); the liquid chromatogram of the malt-roasted granule has a peak 3 with retention time of 10.127 min-10.197 min, a peak 4 with retention time of 14.626 min-14.698 min and a peak 5 with retention time of 15.913 min-15.976 min (one of the peak 4 and the peak 5 can be identified qualitatively).
2. Three formulation particles can be qualitatively identified relative to the chromatographic peak relative retention time of ferulic acid peak, peak 7
Under the conditions of extraction and detection of examples 1 to 4, the malt formulation granule liquid chromatogram lacks peak 3 with a relative retention time of 0.459 to 0.464; the malted formula particles lack peak 4 with a relative retention time of 0.664-0.669 and/or peak 5 with a relative retention time of 0.722-0.727; the liquid chromatogram of the malt-roasted granule has a peak 3 with a relative retention time of 0.459-0.464, a peak 4 with a relative retention time of 0.664-0.669, and a peak 5 with a relative retention time of 0.722-0.727.
The qualitative identification method has the advantages of few identification peaks, short chromatographic detection time and satisfactory separation degree. The retention time or the relative retention time of the chromatographic peak is adopted for identification, and quantitative identification is not needed, so that the method is simple and easy to implement. In particular, when the retention time identification is adopted, the calculation of the relative retention time is not needed, and the method is simpler and more time-saving. And the method is verified by at least 15 batches of formula particles processed by medicinal materials in different places, and meets the technical requirement of qualitative identification.
Claims (1)
1. The method for identifying malt, roasted malt and scorched malt formula particles is characterized by comprising the following steps of:
a1, adding 10% methanol into ferulic acid reference substance to prepare 1 mug solution containing ferulic acid per 1mL as reference substance solution;
a21, grinding the formula particles to be detected, adding 10% methanol, performing ultrasonic extraction for 15min at the power of 400W and the frequency of 40kHz, centrifuging for 5min at the mass-volume ratio of the formula particles to be detected to the methanol of 0.5g:25mL at the speed of 1200 r/min, taking the supernatant, filtering, and taking the subsequent filtrate as a sample solution;
a22, taking the reference substance solution of the reference substance obtained in the step A1 and the sample solution obtained in the step A21, and injecting the reference substance solution into a liquid chromatograph; the chromatographic conditions were as follows:
2695 type Waters high performance liquid chromatograph, with octadecylsilane chemically bonded silica as filler, the chromatographic column parameters are: the column length is 100mm, the inner diameter is 2.1mm, and the grain diameter is 1.7 mu m; acetonitrile is taken as a mobile phase A, and 0.1% formic acid solution is taken as a mobile phase B; the detection wavelength is 297nm; the gradient elution procedure was as follows:
The flow rate of the mobile phase is 0.3mL/min, the column temperature is 25 ℃, the sample injection amount is 1 mu L, and the theoretical plate number is more than or equal to 5000 according to the calculation of ferulic acid peaks, so as to obtain a liquid chromatogram;
a23: analyzing the liquid chromatogram obtained in the step A22, and qualitatively identifying three formula particles according to chromatographic peak retention time: the liquid chromatogram of the malt formulation granule lacks peak 3 with retention time of 10.127 min-10.197 min; the liquid chromatogram of the scorched malt formulation granule lacks peak 4 with retention time of 14.626 min-14.698 min and/or peak 5 with retention time of 15.913 min-15.976 min; the liquid chromatogram of the malt-roasted granule has a peak 3 with retention time of 10.127 min-10.197 min, a peak 4 with retention time of 14.626 min-14.698 min and a peak 5 with retention time of 15.913 min-15.976 min.
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