CN104614463B - The detection method that in Dieda Wan Chinese patent drug, illegal interpolation coloring agent 808 is scarlet - Google Patents
The detection method that in Dieda Wan Chinese patent drug, illegal interpolation coloring agent 808 is scarlet Download PDFInfo
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Abstract
The present invention relates to the detection method that in a kind of Dieda Wan Chinese patent drug, illegal interpolation coloring agent 808 is scarlet.The ultrasonic extraction of concrete employing methenyl choloride, concentrated evaporate to dryness, that obtain removing a lot of interference component after methyl alcohol quantitatively dissolves and can in the high performance liquid chromatograph being furnished with DAD detecting device, thus reach the scarlet large honeyed bolus very complicated from composition of trace detection composition 808 and extract and detect smoothly accurately by sample introduction smoothly.Application HPLC-DAD is legal, the interior illegal coloring agent 808 added of quantitative measurement Dieda Wan Chinese patent drug is scarlet.The present invention efficiently solve Dieda Wan patent medicine because of prescription large, the extraction that complicated component causes, and measure 808 scarlet time interference many problem, have easy and simple to handle, precision is good, the feature that repeatability is high, is expected to the standard method becoming the control of Dieda Wan Quality of Chinese Traditional Proprietary Medicine.
Description
Technical field
The present invention relates to traditional Chinese medicine quality detection field, be specifically related to the detection method that in a kind of Dieda Wan Chinese patent drug, illegal interpolation coloring agent 808 is scarlet.
Background technology
Dieda Wan is by pseudo-ginseng, Radix Angelicae Sinensis, the root of herbaceous peony, the radix paeoniae rubrathe, peach kernel, safflower, dragon's blood, siphonostegia chinensis, Rhizoma drynariae preparata, teasel root, bush, moutan bark, frankincense (system), myrrh (system), turmeric, stir-baked RHIZOMA SPARGANII with vinegar, windproof, muskmelon seed, the dried immature fruit of citron orange (stir-fry), balloonflower root, Radix Glycyrrhizae, akebi, pyritum ignited, the ground bettle large prescription Chinese medicine preparation that forms of the totally 24 taste prepared slices of Chinese crude drugs.There is promoting blood circulation to remove blood stasis, effect of swelling and pain relieving.For traumatic injury, breaking of muscle and tendon is fractured, blood stasis and swelling pain, the diseases such as acute waist sprain.Its preparation method is that all 24 taste prepared slices of Chinese crude drugs are ground into fine powder respectively, sieves, and mixing, every 100g powder adds refined honey 100 ~ 120g and makes large honeyed bolus.
Dragon's blood in prescription is a kind of rare Chinese medicine, and the resin oozed out for babassu Sanguis Draxonis DaemonoropsdracoBl. fruit is through being processed into.Dragon's blood depends on import for a long time, due to import medicinal material limited source, expensive, some lawless persons are in order to pursue economic interests, often pretend to be dragon's blood with resinous matter such as rosin through dyeing or sell after second-rate dragon's blood is dyeed, and 808 scarlet be one of conventional coloring agents.
808 scarlet (808Scarlet) are the red chemical dyestuff of azo, be usually used in the painted of the chemical products such as coating, ink, paint, leather, and non-food coloring, there is carcinogenic and mutagenicity by force, hypodermis can be caused after eating by mistake to grow sarcoma, bring out cancer, even if 808 of human body absorption minute quantity is scarlet, also can severe damaging body health.In Dieda Wan prescription, dragon's blood accounts for 3.9% of prescription ratio, and the quality of dragon's blood raw medicinal material directly can affect the quality of Dieda Wan patent medicine.
At present, in field of traditional Chinese medicine detection, detect the illegal detected object adding the scarlet method of coloring agent 808 and be single Chinese crude drug.As Zhao Jianghong (TCM investigation .24 (8): 17-19) " high performance liquid chromatography identifies illegal 3 kinds of chemical compositions of adding in 29 batches of dragon's blood ", " State Food and Drug Administration: drug inspection supplement the method for inspection and Interventions Requested approved document dragon's blood (approved document is numbered: 2008004) ", " State Food and Drug Administration: drug inspection supplement the method for inspection and Interventions Requested approved document cinnabar (approved document is numbered: 2008003) ", Zou Yaohua etc. (Chinese Journal of Health Laboratory Technology .20 (11): 2724-2725, 2728) " HPLC-PDA method detect in west safflower and safflower 11 kinds illegally add pigment ", these methods visible are only for detecting Chinese crude drug, but not the illegal coloring agent 808 added is scarlet in Chinese patent drug.Because Chinese patent drug carries out prescription by several kinds of Chinese medicinal materials according to corresponding ratio, and be processed into according to certain production technology, ingredient is more more complex than single Chinese crude drug, and the formulation of Chinese patent drug Dieda Wan is large honeyed bolus, not only prescription flavour of a drug are extremely many, complicated component, and the dragon's blood in Dieda Wan only accounts for 3.9% of prescription ratio, therefore also accurately detection coloring agent 808 is scarlet to be difficult to extraction from Dieda Wan, testing process very easily produces vacation " positive " interference, thus cause cannot result of determination clearly.Obviously how extracting also from Dieda Wan patent medicine, accurately detection coloring agent 808 is scarlet, and ensureing people's drug safety, is the current problem needing solution badly.
Summary of the invention
The object of this invention is to provide the detection method that in a kind of Dieda Wan Chinese patent drug, illegal interpolation coloring agent 808 is scarlet, detect that in Chinese patent drug Dieda Wan, the illegal coloring agent 808 added is scarlet, to make up the deficiencies in the prior art quickly and accurately.
The present invention considers that Dieda Wan is a large prescription Chinese medicine preparation be made up of the 24 taste prepared slices of Chinese crude drugs, chemical composition is very complicated, in addition formulation is large honeyed bolus, refined honey proportion is up to 50%, be the interference pollution source that a ratio is sizable, very serious, during mensuration, very easily occur that vacation " positive " is disturbed.Again, because in Dieda Wan prescription, dragon's blood only accounts for 3.9% of prescription ratio, in the Dieda Wan using dragon's blood of poor quality to make for raw material, 808 scarlet content are very low again, are only 1 μ gg
-1, not carrying out enrichment method cannot detect.
The all kinds of solvent that prior art is selected usually as methyl alcohol, ethanol, the direct ultrasonic extraction 808 of acetonitrile equal solvent is scarlet, then after extraction solution evaporate to dryness, the level of residue of gained is larger, need again to add methyl alcohol residue is dissolved, but the viscosity of gained solution is comparatively large, and high performance liquid chromatography therefore can not be utilized to detect.Methenyl choloride of selecting of the present invention is scarlet to reach the coloring agent 808 very easily dissolved in large honeyed bolus as solvent, and this solvent is extremely low to the solubleness of refined honey in large honeyed bolus, and residue obtained amount few (namely meaning that a lot of not clear interference component obtains having picked out by this step) after extracting solution evaporate to dryness, thereafter dissolve, gained solution just may be used for high performance liquid chromatography and detects.
In addition, also consider that Small Meridian-Activating Pill is large honeyed bolus, its viscosity is very large, and if do not disperseed, the above-mentioned Extraction solvent methenyl choloride chosen also is difficult to penetrate in sample, also just can not fully extract 808 scarlet.Therefore a kind of spreading agent is selected, can ensure to prevent sample from luming, the surface of contact of sample and Extraction solvent can be increased again, can also ensure that Extraction solvent methenyl choloride fully penetrates in sample to go, and fully realize the scarlet extraction completely of coloring agent 808, thus reach the scarlet large honeyed bolus very complicated from composition of trace detection composition 808 and extract and detect exactly smoothly.
Technical scheme of the present invention comprises:
(1) preparation of contrast agents solution: precision takes 808 scarlet contrast agents 5.68mg, is placed in 100mL measuring bottle, adds methanol constant volume to 100mL, makes contrast agents strong solution.Precision measures contrast agents strong solution 5mL again, is placed in 50mL measuring bottle, adds methanol constant volume to 50mL, makes the solution in contrast reagent solution of every 1mL containing 808 scarlet contrast agents 5.68 μ g.
(2) preparation of need testing solution: get Dieda Wan sample 6g, shred, accurately weighed weight, add zeyssatite 3g, grind well, precision adds methenyl choloride 20mL, weighed weight, ultrasonic process 20 minutes, lets cool, add the weight that methenyl choloride supplies less loss again, filter, precision measures subsequent filtrate 10mL, evaporate to dryness, residue adds methyl alcohol and dissolves, and is transferred in 1mL measuring bottle, add methyl alcohol to 1mL, shake up, make need testing solution.
(3) content assaying method: accurate absorption contrast agents solution, each 10 μ L of need testing solution respectively, injects the high performance liquid chromatograph being furnished with DAD detecting device and normally measures.
(4) result judges: as sample presents the chromatographic peak identical with contrast agents retention time, the then absorption spectrum of the chromatographic peak that contrast agents retention time scarlet with 808 is identical and the absorption spectrum of 808 scarlet contrast agents in comparative sample, as absorption spectrum is consistent, then can judge in sample scarlet containing 808.
Obviously, the present invention efficiently solve Dieda Wan patent medicine because of prescription large, the extraction that complicated component causes, and measure 808 scarlet time interference many problem, have easy and simple to handle, precision is good, the feature that repeatability is high, is expected to the standard method becoming the control of Dieda Wan Quality of Chinese Traditional Proprietary Medicine.
Accompanying drawing explanation
Fig. 1: the HPLC-UV chromatogram of 808 scarlet contrast agents of the present invention.
Fig. 2: the HPLC-UV chromatogram of Dieda Wan positive solution of the present invention.
Fig. 3: the HPLC-UV chromatogram of Dieda Wan negative sample solution of the present invention.
Fig. 4: the spectrogram of 808 scarlet contrast agents of the present invention.
Fig. 5: the spectrogram of Dieda Wan positive solution of the present invention.
Embodiment
Provide detailed embodiment below.
The present invention adopts: instrument and reagent: Shimadzu LC-20AT highly effective liquid phase chromatographic system (Japan, Shimadzu Corporation, joins DAD detecting device); Waters2695/QuattroMicro liquid chromatography/series connection quadrupole rod mass spectrometry system (U.S., Waters company); BP211D electronic balance (Germany, Sartoris company); AUTOSCIENCEAS10200BT type ultrasonic cleaner (Tianjin Ao Tesaiensi Instrument Ltd.).Acetonitrile is chromatographic grade reagent, and it is pure that other reagent is analysis; 808 scarlet contrast agents (National Institute for Food and Drugs Control, lot number is: 111940-201201).
Chromatographic condition: chromatographic column: AgilentEclipsePlusC
18(250 × 4.6mm, 5 μm); Take acetonitrile as mobile phase A, 0.02molL
-1ammonium acetate solution is Mobile phase B, gradient elution (0 ~ 15min, 15%B ~ 5%; 15 ~ 25min, 5%B); Column temperature 30 DEG C; Flow velocity 1.0mLmin
-1; Determined wavelength is 516nm; Number of theoretical plate calculates should be not less than 4000 by 808 scarlet chromatographic peak.
The preparation of contrast agents solution: get 808 scarlet contrast agents 5.68mg, accurately weighed (accurate to 0.1mg), is placed in 100mL measuring bottle, adds methanol constant volume to 100mL, make contrast agents strong solution.Precision measures contrast agents strong solution 5mL again, is placed in 50mL measuring bottle, adds methanol constant volume to 50mL, makes the solution in contrast reagent solution of every 1mL containing 808 scarlet contrast agents 5.68 μ g.
The preparation of need testing solution: get Dieda Wan sample 6g, shred, accurately weighed weight (accurate to 1mg), adds zeyssatite 3g, grinds well, precision adds methenyl choloride 20mL, weighed weight (accurate to 0.1g), ultrasonic process (power 300W, frequency 40KHz) 20 minutes, let cool, add the weight (accurate to 0.1g) that methenyl choloride supplies less loss again, filter, precision measures subsequent filtrate 10mL, evaporate to dryness, residue adds methyl alcohol and dissolves, and is transferred in 1mL measuring bottle, adds methyl alcohol to 1mL, shake up, make need testing solution.
Specificity is tested: accurate absorption contrast agents solution, each 10 μ L of need testing solution respectively, inject hplc determination, record chromatogram and the spectrogram of each solution, chromatogram is shown in Fig. 1,2,3, and spectrogram is shown in Fig. 4,5.
Result shows, positive presents the chromatographic peak identical with contrast agents retention time, and negative sample is then without corresponding chromatographic peak.In positive the absorption spectrum of the chromatographic peak that contrast agents retention time scarlet with 808 is consistent and the absorption spectrum of 808 scarlet contrast agents consistent.Tested by specificity, can be effectively qualitative 808 scarlet.
Linear relationship is investigated: accurate absorption 808 scarlet contrast agents solution 2,4,6,8,10,15,20 μ L respectively, measure by above-mentioned chromatographic condition, with integrating peak areas value for ordinate, with sample size (μ g) for horizontal ordinate, drawing standard curve, calculate regression equation, obtaining regression equation is Y=3774523.29X – 136.00r=1, and the range of linearity is 0.01136 ~ 0.11360 μ g.
Detectability is investigated: according to solution concentration during 808 scarlet contrast agents solution S/N=3, calculate the detection limit (LOD) of assay method, and 808 scarlet detections are as a result limited to 1ngg
-1.
Precision test: accurate absorption contrast agents solution 10 μ L, continuous sample introduction 6 times, measure integrating peak areas value, the RSD that result records integrating peak areas value is 0.71% (n=6), shows that instrument has good precision.
Replica test: get with a collection of Dieda Wan sample, by the parallel preparation of the preparation method of need testing solution 6 parts of need testing solutions, measures respectively under above-mentioned chromatographic condition, to calculate in Dieda Wan sample 808 scarlet content (μ gg
-1), the RSD of content is 1.24% (n=6).Result shows, this method has good repeatability.
Stability test: get with a need testing solution, under above-mentioned chromatographic condition in 0,1,12,24h sample introduction, measure integrating peak areas value, the RSD of 808 scarlet integrating peak areas values is 0.62% (n=6).Result shows, need testing solution is basicly stable in 24 hours.
Average recovery is tested: get negative sample 6g, shred, and accurately weighed weight (accurate to 1mg), it is 5.68 μ gmL that precision adds concentration
-1808 scarlet contrast agents solution 2mL, make application of sample test liquid 6 parts according to the preparation method of need testing solution.Accurate draw application of sample test liquid 10 μ L, measure as stated above, calculate average recovery, 808 scarlet average recovery rates are 96.82%, RSD=1.06% (n=6) as a result, show that average recovery is good.
More than experiment shows, qualitative and quantitative analysis result of the present invention is accurate, effectively can control the illegal 808 scarlet detections added in Dieda Wan.
In order to confirm containing 808 scarlet positive further, have employed again HPLC/MS/MS method and verifying.
Mass Spectrometry Conditions: chromatographic column: WatersACQUITYUPLCTMBEHC
18(2.1 × 100mm, 1.7 μm); Column temperature: 30 DEG C; Flow velocity: 0.2mLmin
-1; Take acetonitrile as mobile phase A, 5mmolL
-1ammonium acetate solution is Mobile phase B, gradient elution (0 ~ 3min, 70%B; 3 ~ 8min, 70%B ~ 20%; 8 ~ 10min, 20%B ~ 5%; 10 ~ 14min, 5%B; 14.0 ~ 14.5min, 5%B ~ 70%; 14.5 ~ 16min, 70%B); Sample size: 10 μ L.The MS detection parameters: scan mode: ESI+; Detection mode: multiple-reaction monitoring (MRM) pattern; Capillary voltage: 4.0KV; Extraction taper hole voltage: 6V; RFLens:4.0V; Source temperature: 110 DEG C; Desolventizing temperature degree: 350 DEG C; Desolventizing airshed: 600Lh
-1; Taper hole airshed: 50Lh
-1; Collision cell entrance potential :-2; Exit potential 1; MS1 and MS2 high-resolution and low-resolution is 13.0.
Testing result: adopt ESI positive ion scan pattern, 808 scarlet easy formation m/z368 [M+H]
+quasi-molecular ion peak, it is tuning with m/z368 to be that parent ion carries out daughter ion, forms the fragment that two abundance of m/z275 and 77 are stronger, form two pairs of ion pairs, carry out the detection of multiple-reaction monitoring (MRM) pattern quantitative and qualitative analysis.Quota ion is 6.5 to the abundance ratio with qualitative ion pair, and scope is ± 30%.In positive chromatogram, with 808 scarlet corresponding chromatographic peaks, there is identical mass spectral characteristic.
To sum up, the High Performance Liquid Chromatography/Mass Spectrometry detection method that the present invention sets up can effectively in qualitative and quantitative analysis Chinese patent drug Dieda Wan the illegal coloring agent 808 added scarlet, avoid vacation " positive " and quantitatively detect inaccurate problem, method operation quick and precisely, not only greatly increase work efficiency, can more comprehensively, more effectively control and the quality of supervisory Chinese patent drug Dieda Wan.
Claims (4)
1. the detection method that in Dieda Wan Chinese patent drug, illegal interpolation coloring agent 808 is scarlet, is characterized in that comprising the following steps:
(1) preparation of contrast agents solution: precision takes 808 scarlet contrast agents 5.68mg, is placed in 100mL measuring bottle, adds methanol constant volume to 100mL, makes contrast agents strong solution; Precision measures contrast agents strong solution 5mL again, is placed in 50mL measuring bottle, adds methanol constant volume to 50mL, makes the solution in contrast reagent solution of every 1mL containing 808 scarlet contrast agents 5.68 μ g;
(2) preparation of need testing solution: get Dieda Wan sample 6g, shred, accurately weighed weight, add zeyssatite 3g, grind well, precision adds methenyl choloride 20mL, weighed weight, ultrasonic process 20 minutes, lets cool, add the weight that methenyl choloride supplies less loss again, filter, precision measures subsequent filtrate 10mL, evaporate to dryness, residue adds methyl alcohol and dissolves, and is transferred in 1mL measuring bottle, add methyl alcohol to 1mL, shake up, make need testing solution;
(3) content assaying method: accurate absorption contrast agents solution, each 10 μ L of need testing solution respectively, injects the high performance liquid chromatograph being furnished with DAD detecting device and measures;
(4) result judges: as sample presents the chromatographic peak identical with contrast agents retention time, the then absorption spectrum of the chromatographic peak that contrast agents retention time scarlet with 808 is identical and the absorption spectrum of 808 scarlet contrast agents in comparative sample, as absorption spectrum is consistent, then can judge in sample scarlet containing 808.
2. the detection method that in Dieda Wan Chinese patent drug as claimed in claim 1, illegal interpolation coloring agent 808 is scarlet, is characterized in that imposing a condition of above-mentioned high performance liquid chromatograph: AgilentEclipsePlusC
18, 250 × 4.6mm, 5 μm of chromatographic columns; Take acetonitrile as mobile phase A, 0.02molL
-1ammonium acetate solution is Mobile phase B, A+B=100%; Gradient elution: 0 ~ 15min, 15%B ~ 5%B; 15 ~ 25min, 5%B; Column temperature is 30 DEG C; Flow velocity is 1.0mLmin
-1; Number of theoretical plate calculates should be not less than 4000 by 808 scarlet chromatographic peak.
3. the detection method that in Dieda Wan Chinese patent drug as claimed in claim 1, illegal interpolation coloring agent 808 is scarlet, is characterized in that the parameter that above-mentioned DAD detecting device detects: determined wavelength is 516nm.
4. the detection method that in Dieda Wan Chinese patent drug as claimed in claim 1, illegal interpolation coloring agent 808 is scarlet, is characterized in that above-mentioned ultrasonic treatment conditions are power 300W, frequency 40KHz.
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