CN1963496B - Method for setting up finger-print of pain-eliminate paste and standard finger-print thereof - Google Patents

Abstract

The invention discloses a method for establishing GC and GC/MS fingerprints of Xiaotong paste powder or ointment or extract and its standard fingerprints. The method for controlling the quality of the Xiaotong patch of the present invention is composed of the following steps: 1. Prepare the test solution of the Xiaotong patch, take an appropriate amount of the Xiaotong patch, weigh it accurately, put it in a Soxhlet extractor, add ether to reflux and extract, filter, The filtrate was used as the test solution. 2. Use gas chromatography and gas chromatography-mass spectrometry to identify the reference peak (peak No. 10) among the 18 common peaks in the fingerprint of Xiao Tong Tie. The identification result shows that the peak No. 10 is the characteristic peak of eucalyptol. Three, the characteristic peak of eucalyptol is the reference peak, calculate relative retention time, relative peak area, promptly get the standard fingerprint collection of Xiaotong paste extract (see accompanying drawing) the advantage of the present invention is that method is simple, stable, precision High and reproducible, can grasp the quality of Xiaotong paste from the overall characteristics of the chromatogram, and can be used as a quality control index for Xiaotong paste.

Description

Method for establishing fingerprints of Xiaotongtie and its standard fingerprints

technical field

The invention relates to a method for establishing fingerprints of traditional Chinese medicines, in particular to a method for establishing fingerprints of Xiaotong paste powder or ointment or extracts and standard fingerprints thereof.

Background technique

Xiao Tong Tie is a Tibetan patent medicine produced by Linzhi Qizheng Tibetan Medicine Factory in Tibet. It is a state-secret variety and a protected variety of traditional Chinese medicine. It adopts the natural medicine unique to China’s Tibetan Plateau, and is refined through a special low-temperature vacuum dehydration process. Its main functions are promoting blood circulation and removing blood stasis, reducing swelling and relieving pain. It is used for acute and chronic contusions, bruises, bone hyperplasia, rheumatism and rheumatoid pain. etc. Because of its excellent efficacy, it sells well at home and abroad. At present, in its quality control, only the content determination of curcumin, the unique thin-layer identification and inspection, it is not comprehensive to control the quality of this product with these two indicators alone, and It is easy for unscrupulous traders to take advantage of the gaps in their quality control standards to produce and sell "standard" fake drugs. Now there is no report on the establishment of the fingerprint map of Xiaotong stickers. In order to make up for the above shortcomings, make its quality control technology more perfect, scientific and To provide an available quality control model for its future standardized production, 50 different batches of products for six consecutive years were investigated and compared, and a method for determining the standard fingerprint of the product's volatile component extracts was established.

Contents of the invention

The purpose of the present invention is to find out a kind of quality control method of Xiaotong paste powder or paste or extract through the study of GC and GC/MS fingerprints of Xiaotong paste powder or paste or extract, and establish Xiaotong paste The standard fingerprint method makes up for the deficiencies of the existing technology and makes it more perfect, more scientific and more reasonable.

The present invention is achieved through the following technical solutions:

(1) Preparation of the test solution: remove the appropriate amount of pain plaster or powder or extract, accurately weigh it, add ether or chloroform, put it in a Soxhlet extractor, reflux extraction for 1-12 hours or ultrasonic extraction for 5-60 minutes Or reflux extraction for 0.5-8 hours or microwave extraction for 0.1-4 hours, filter, evaporate the filtrate to dryness, add methanol to constant volume as the test solution.

(2) Determination of fingerprints: chromatographic conditions and system adaptability test use SE-54 or OV-1701 or OV-11 as the stationary liquid; 170°C, keep it constant for 1-15 minutes, then raise it at 1-30°C/min to 170-230°C and keep it constant for 1-30 minutes, the carrier gas is high-purity nitrogen, and the detector is a hydrogen flame detector; accurately absorb the test solution 0.5 μL～5μL, using splitless or split injection method; measured by gas chromatography, the relative retention time and relative peak area of No. 10 chromatographic peak (the characteristic peak of eucalyptol) are 1, and the relative retention time and relative peak area are calculated , that is, the standard fingerprint I of Xiaotongtie is obtained.

(3) According to the preparation method of the test solution and the same chromatographic conditions measured by GC, the total ion chromatogram obtained by GC/MS analysis constitutes the standard fingerprint II of Xiao Tong Tie.

The method is as follows: take an appropriate amount of sample, weigh it accurately, add ether or chloroform, soak in a Soxhlet extractor overnight, reflux extraction for 1-12 hours or ultrasonic extraction for 5-60 minutes or reflux extraction for 0.5-8 hours or microwave extraction for 0.1 ~ 4 hours, filter, evaporate the filtrate to dryness, add methanol to constant volume, filter through a microporous membrane (0.45um), and use it as the test solution of Xiaotong paste powder or ointment or extract; according to Appendix VIE of Chinese Pharmacopoeia 2005 Edition Gas chromatography test, using SE-54 or OV-1701 or OV-11 as the stationary liquid; the chromatographic column is a quartz capillary column, the column temperature is programmed temperature rise, the initial column temperature is 100-170 °C, and after 1-15 minutes of constant 1～30°C/min, rise to 170～230°C and keep constant for 1～30 minutes, the carrier gas is high-purity nitrogen, the detector is a hydrogen flame detector, and the sampling method is splitless or split; the chromatographic peak No. 10 (eucalyptol The relative retention time and relative peak area of the characteristic peak) are 1, and the relative retention time and relative peak area are calculated to obtain the standard fingerprint of Xiaotong plaster extract.

The method is: remove an appropriate amount of pain paste or powder or extract sample, accurately weigh, add ether or chloroform, soak in a Soxhlet extractor overnight, reflux extraction for 1-12 hours or ultrasonic extraction for 5-60 minutes or reflux extraction 0.5 to 8 hours or microwave extraction for 0.1 to 4 hours, filter, evaporate the filtrate to dryness, add methanol to constant volume, filter through a microporous membrane (0.45 μm), and use it as the test solution of Xiaotong plaster powder extract; according to the Chinese Pharmacopoeia 2005 edition one appendix VIE gas chromatography test, using SE-54 as the stationary liquid; the chromatographic column is a quartz capillary column, the column temperature is programmed temperature rise, draw 0.5-2 μL of the test solution, inject it into the gas chromatograph, and the chromatographic peak is No. 10 (The characteristic peak of eucalyptol) has a relative retention time and a relative peak area of 1, and calculates the relative retention time and relative peak area to obtain the Xiao Tong Tie standard fingerprint spectrum I (Fig. 1).

The method is: Xiaotong paste powder or ointment or extract test product solution, determined according to gas chromatography, the chromatographic column is a quartz capillary column, adopts splitless or splitless sampling method, and the number of theoretical plates is calculated by peak No. 10 , not less than 40000.

The method is as follows: the chromatographic column uses SE-54 or OV-1701 or OV-11 as a stationary liquid; the chromatographic column is a quartz capillary column, and the column parameters are φ0.22mm×(20m～35m).

The method is as follows: take 1 g of the sample, accurately weigh it, add ether, put it in a Soxhlet extractor, and extract it under reflux for 12 hours, evaporate the extract to dryness, add methanol to constant volume as the test solution.

The method is as follows: the amount of the test solution added is 0.5 μL.

The method is: carry out GC/MS analysis under the same chromatographic conditions of the preparation method and GC of the described need testing solution to obtain the structural information of the No. 10 reference peak and identify it as the characteristic peak of eucalyptol; The MS total ion chromatogram constitutes the standard fingerprint II of Xiaotongpai (Figure 2).

In the Xiaotongtie standard fingerprint spectrum obtained by the method, there are 18 characteristic fingerprint peaks in total, and the sum of the peak areas accounts for more than 90% of the total peak areas.

With the relative retention time and relative peak area of the chromatographic peak (No. 10 peak) of eucalyptol as 1, the common characteristic peaks in the standard fingerprint spectrum of Xiao Tong Tie obtained are (Fig. 1 or Fig. 2):

Peak No. 1: relative retention time 2.08～3.86, relative peak area 2.06～685.73

Peak No. 2: relative retention time 2.00～3.68, relative peak area 5.88～552.83

Peak No. 3: relative retention time 1.00-2.00, relative peak area 54.04-604.21

Peak No. 4: relative retention time 1.00-2.00, relative peak area 9.60-560.99;

Peak No. 5: relative retention time 0.99-1.99, relative peak area 8.30-604.11;

Peak No. 6: relative retention time 0.89-1.55, relative peak area 10.44-86.90;

Peak No. 7: relative retention time 0.87-1.54, relative peak area 10.31-640.7;

Peak No. 8: relative retention time 0.86-1.53, relative peak area 1.05-20.78

Peak No. 9: relative retention time 0.85-1.51, relative peak area 5.72-218.28;

Peak No. 10 (reference peak): relative retention time 1, relative peak area 1;

Peak No. 11: relative retention time 0.81-1.50, relative peak area 3.90-384.76

Peak No. 12: relative retention time 0.80～1.49, relative peak area 2.84～444.0

Peak No. 13: relative retention time 0.78-1.47, relative peak area 6.98-311.99;

Peak No. 14: relative retention time 0.76-1.45, relative peak area 5.89-80.98;

Peak No. 15: relative retention time 0.64-1.43, relative peak area 0.64-20.99;

Peak No. 16: relative retention time 0.53-1.40, relative peak area 8.98-560.04;

Peak No. 17: relative retention time 0.52-1.38, relative peak area 14.77-523.15;

Peak No. 18: relative retention time 0.48-1.20, relative peak area 22.32-600.34;

Preferably, with the relative retention time and relative peak area of the chromatographic peak (No. 10 peak) of eucalyptol as 1, the common characteristic peak in the standard fingerprint spectrum peak of the Xiaotong paste obtained is:

Peak No. 1: relative retention time 3.78, relative peak area 3.06-585.73

Peak No. 2: relative retention time 2.42, relative peak area 7.88-452.88

Peak No. 3: relative retention time 1.61, relative peak area 14.04～504.20

Peak No. 4: relative retention time 1.33, relative peak area 8.60-460.90;

Peak No. 5: relative retention time 1.22, relative peak area 18.30-504.20;

Peak No. 6: relative retention time 1.21, relative peak area 20.44-146.96;

Peak No. 7: relative retention time 1.06, relative peak area 30.31-540.7;

Peak No. 8: relative retention time 1.03, relative peak area 2.17-8.23;

Peak No. 9: relative retention time 1.02, relative peak area 15.22-163.28;

Peak No. 10 (reference peak): relative retention time 1, relative peak area 1;

Peak No. 11: relative retention time 0.98, relative peak area 10.99-218.24;

Peak No. 12: relative retention time 0.95, relative peak area 11.84-349.75;

Peak No. 13: relative retention time 0.88, relative peak area 12.11;

Peak No. 14: relative retention time 0.86, relative peak area 12.25-40.78;

Peak No. 15: relative retention time 0.82, relative peak area 1.61-9.97;

Peak No. 16: relative retention time 0.78, relative peak area 16.73-460.90;

Peak No. 17: relative retention time 0.72, relative peak area 24.97-425.14;

Peak No. 18: relative retention time 0.60, relative peak area 52.71-486.48;

The invention has the characteristics of simple and convenient method, high stability, high precision, good reproducibility and easy mastery.

Description of drawings

Fig. 1 is the standard fingerprint spectrum I of Xiaotong paste of the present invention.

Fig. 2 is the standard fingerprint spectrum II of Xiaotong paste composed of the GC/MS total ion chromatogram of the present invention.

The present invention is further described below through specific examples, but it does not constitute a limitation to the present invention.

Example 1: Method for establishing the standard fingerprint I of Qizheng Xiaotong Tiefen

1. Instruments and reagents

1.1 Instrument

Shimadzu GC-17A Gas Chromatograph; Hydrogen Flame Detector; Shimadzu Class-GC10 Workstation

1.2 Reagent

Diethyl ether, analytically pure; Methanol, chromatographically pure; syringe-type microporous membrane filter, organic system, diameter 13mm, 0.45um; Xiaotong paste powder is produced by Tibet Linzhi Qizheng Tibetan Medicine Factory.

2. Methods and results

2.1 Chromatographic conditions

采用不分流进样；进样量0.5μL。Chromatographic column: SE-54, 0.22mm×25m quartz capillary column; the carrier gas is high-purity nitrogen; the detector is a hydrogen flame detector, and the number of theoretical plates is calculated based on the characteristic peak of No. 10 eucalyptol, not less than 40,000. The inlet temperature is 250°C, the detector temperature is 280°C, and the temperature program is:

Splitless injection was adopted; the injection volume was 0.5 μL.

2.2 Determination of fingerprint

2.2.1 Fingerprint preparation of Xiaotong powder

Preparation of the test solution: cancel 1.0g of pain paste powder sample, add appropriate amount of ether, place in a Soxhlet extractor, reflux extraction for 12 hours, dilute to 5.00ml, take 2.00ml, evaporate to dryness, add methanol 0.50ml, filter , and the filtrate was used as the test solution.

Precisely draw 0.5ul of the test solution, inject it into a gas chromatograph, measure it according to gas chromatography, record the chromatogram for 80 minutes, and get final product. Taking eucalyptol (No. 10) chromatographic peak relative retention time and peak area as 1, calculate relative retention time and relative peak area, promptly obtain Xiao Tong Tie standard fingerprint I.

2.2.2 Determination of common peaks

Through the determination of 10 batches of Xiaotong Tiefen fingerprints, compare their chromatograms, and determine that there are 18 peaks in total, of which No. 10 fingerprint peak (the characteristic peak of eucalyptol) is used as a reference, and its peak area exceeds 10% of the total peak area. , list 10 batches of samples common peak relative retention time and relative peak area statistical results are shown in Table 1 and Table 2 below.

Example 2 The establishment method of the standard fingerprint II of Qizheng Xiaotong Tie

1. Instruments and reagents

1.1 Instrument

TRACEMS gas chromatography mass spectrometry; NIST02 mass spectral library.

1.2 Reagent

Diethyl ether, analytically pure; Methanol, chromatographically pure; syringe-type microporous membrane filter, organic system, diameter 13mm, 0.45um; Xiaotongpai is produced by Tibet Linzhi Qizheng Tibetan Pharmaceutical Factory.

2. Methods and results

2.1 Chromatography-mass spectrometry conditions

采用分流进样；进样量0.5μL；分流比为20∶1；电离方式：EI；离子源温度：200℃；电子能量：70eV；扫描质量范围：3～400amu；发射电流：150mA；电子倍增电压：350V。Chromatographic column is SE-54 (0.25μm, 30m×0.25mm) elastic quartz capillary column, injection volume: 0.5μL; inlet temperature: 280°C; temperature program: inlet temperature is 250°C, detector temperature is 280℃, temperature program:

Split injection is adopted; injection volume is 0.5μL; split ratio is 20:1; ionization method: EI; ion source temperature: 200°C; electron energy: 70eV; scanning mass range: 3-400amu; emission current: 150mA; electron multiplication Voltage: 350V.

3. Results

(1) GC/MS total ion chromatogram constitutes Xiaotongtie standard fingerprint II (see Figure 2).

(2) The test product was analyzed by GC-MS, and the computer searched the NIST 02 spectrum library, and compared with the standard spectrum (EPA/N IH Mass Spectral Data Base), the No. 10 common peak was identified (retention time was 21.96min) It is the characteristic peak of eucalyptol.

Table 1: Relative retention time statistics of the common peaks of 10 batches of Xiaotong Tiefen

Table 2: Peak area statistics of common peaks of 10 batches of Xiaotong Tiefen

Claims (8)

1. method of setting up finger-print of pain-eliminate paste is characterized in that this method comprises the steps: the preparation of need testing solution: it is an amount of that the cancellation pain is pasted sample, accurate claim fixed; Add ether or chloroform; Put in the apparatus,Soxhlet's, refluxing extraction 1～12 hour or ultrasonic Extraction 5～60 minutes or microwave abstracting 0.1～4 hour filter; The filtrating evaporate to dryness adds methanol constant volume as need testing solution;

The mensuration of finger-print I: chromatographic condition and system suitability test: with SE-54 or OV-1701 or OV-11 is immobile liquid; Chromatographic column is a quartz capillary column, and column temperature is temperature programme, and initial column temperature is 100～170 ℃; After constant 1～15 minute with 1～30 ℃/minute rise to 170～230 ℃ constant 1～30 minute; Carrier gas is high purity nitrogen, and detecting device is a hydrogen flame detector, adopts and does not shunt or the split sampling mode; Accurate need testing solution 0.5～5 μ L that draws uses gas chromatograph for determination, and the relative retention time of No. 10 chromatographic peaks and relative peak area are 1, calculate relative retention time and relative peak area, promptly obtain the standard finger-print I that disappears and paste bitterly;

The mensuration of finger-print II: measure identical chromatographic condition according to the preparation method of said need testing solution and GC and carry out GC/MS and analyze and obtain No. 10 with reference to the structural information at peak and identify characteristic peak into eucalyptol; Obtain GC/MS total ion current figure simultaneously and constitute the standard finger-print II that the pain that disappears is pasted.

2. the method for setting up finger-print of pain-eliminate paste according to claim 1 is characterized in that: the method for measuring finger-print I is that it is an amount of that the cancellation pain is pasted sample; The accurate title, decide, and adds ether or chloroform, puts in the apparatus,Soxhlet's; Refluxing extraction 1～12 hour or ultrasonic Extraction 5～60 minutes or microwave abstracting 0.1～4 hour filter, the filtrating evaporate to dryness; Add methanol constant volume, 0.45 μ m filtering with microporous membrane is as the need testing solution of the pain subsides that disappear; According to an appendix VIE of Chinese Pharmacopoeia version in 2005 vapor-phase chromatography test, be immobile liquid with SE-54 or OV-1701 or OV-11; Chromatographic column is a quartz capillary column, and column temperature is temperature programme, and initial column temperature is 100～170 ℃, after constant 1～15 minute with 1～30 ℃/minute rise to 170～230 ℃ constant 1～30 minute, carrier gas is high purity nitrogen, detecting device is a hydrogen flame detector; Draw need testing solution 0.5 μ L～5 μ L, inject gas chromatograph, the relative retention time of No. 10 chromatographic peaks and relative peak area are 1, calculate relative retention time and relative peak area, promptly obtain the standard finger-print I that disappears and paste bitterly.

3. the method for setting up finger-print of pain-eliminate paste according to claim 2; It is characterized in that: the pain that disappears is pasted the sample test liquid according to gas chromatography determination, and chromatographic column is a quartz capillary column, adopts and does not shunt or the split sampling mode; Theoretical cam curve is calculated with No. 10 peaks, is not less than 40000.

4. the method for setting up finger-print of pain-eliminate paste according to claim 3 is characterized in that: packed column is an immobile liquid with SE-54 or OV-1701 or OV-11; Chromatographic column is a quartz capillary column, and column parameter is φ 0.22mm * (20m～35m).

5. the method for setting up finger-print of pain-eliminate paste according to claim 1; It is characterized in that: the preparation method of need testing solution is: the cancellation pain is pasted sample 0.5～2g, and accurate the title decides, and adds ether; Put in the apparatus,Soxhlet's; Refluxing extraction 1～12 hour, the extract evaporate to dryness adds methanol constant volume as need testing solution.

6. the method for setting up finger-print of pain-eliminate paste according to claim 2 is characterized in that the common characteristic fingerprint peaks has 18 among said disappear pain accurate finger-print I of decals or the II, and its peak area summation accounts for more than 90% of total peak area.

7. the method for setting up finger-print of pain-eliminate paste according to claim 2 is characterized in that among said disappear the pain accurate finger-print I of decals or the II, is 1 with the chromatographic peak relative retention time and the relative peak area of eucalyptol, and the common characteristic fingerprint peaks is selected from:

No. 1 peak: relative retention time 2.08～3.86, relative peak area 2.06～685.73;

No. 2 peaks: relative retention time 2.00～3.68, relative peak area 5.88～552.83;

No. 3 peaks: relative retention time 1.00～2.00, relative peak area 54.04～604.21;

No. 4 peaks: relative retention time 1.00～2.00, relative peak area 9.60～560.99;

No. 5 peaks: relative retention time 0.99～1.99, relative peak area 8.30～604.11;

No. 6 peaks: relative retention time 0.89～1.55, relative peak area 10.44～86.90;

No. 7 peaks: relative retention time 0.87～1.54, relative peak area 10.31～640.7;

No. 8 peaks: relative retention time 0.86～1.53, relative peak area 1.05～20.78;

No. 9 peaks: relative retention time 0.85～1.51, relative peak area 5.72～218.28;

No. 10 the peak is with reference to the peak: relative retention time 1, relative peak area 1;

No. 11 peaks: relative retention time 0.81～1.50, relative peak area 3.90～384.76;

No. 12 peaks: relative retention time 0.80～1.49, relative peak area 2.84～444.0;

No. 13 peaks: relative retention time 0.78～1.47, relative peak area 6.98～311.99;

No. 14 peaks: relative retention time 0.76～1.45, relative peak area 5.89～80.98;

No. 15 peaks: relative retention time 0.64～1.43, relative peak area 0.64～20.99;

No. 16 peaks: relative retention time 0.53～1.40, relative peak area 8.98～560.04;

No. 17 peaks: relative retention time 0.52～1.38, relative peak area 14.77～523.15;

No. 18 peaks: relative retention time 0.48～1.20, relative peak area 22.32～600.34.

8. the method for setting up finger-print of pain-eliminate paste according to claim 2 is characterized in that among said disappear pain decals accurate finger-print I, the II, is 1 with the chromatographic peak relative retention time and the relative peak area of eucalyptol, and the common characteristic fingerprint peaks is selected from:

No. 1 peak: relative retention time 3.78, relative peak area 3.06～585.73;

No. 2 peaks: relative retention time 2.42, relative peak area 7.88～452.88;

No. 3 peaks: relative retention time 1.61, relative peak area 14.04～504.20;

No. 4 peaks: relative retention time 1.33, relative peak area 8.60～460.90;

No. 5 peaks: relative retention time 1.22, relative peak area 18.30～504.20;

No. 6 peaks: relative retention time 1.21, relative peak area 20.44～146.96;

No. 7 peaks: relative retention time 1.06, relative peak area 30.31～540.7;

No. 8 peaks: relative retention time 1.03, relative peak area 2.17～8.23;

No. 9 peaks: relative retention time 1.02, relative peak area 15.22～163.28;

No. 10 the peak is with reference to the peak: relative retention time 1, relative peak area 1;

No. 11 peaks: relative retention time 0.98, relative peak area 10.99～218.24;

No. 12 peaks: relative retention time 0.95, relative peak area 11.84～349.75;

No. 13 peaks: relative retention time 0.88, relative peak area 12.11;

No. 14 peaks: relative retention time 0.86, relative peak area 12.25～40.78;

No. 15 peaks: relative retention time 0.82, relative peak area 1.61～9.97;

No. 16 peaks: relative retention time 0.78, relative peak area 16.73～460.90;

No. 17 peaks: relative retention time 0.72, relative peak area 24.97～425.14;

No. 18 peaks: relative retention time 0.60, relative peak area 52.71～486.48.

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