CN105181826A - Measuring method for monosaccharide composition in radix glehniae polysaccharides - Google Patents

Abstract

The invention provides a measuring method for monosaccharide composition in radix glehniae polysaccharides. A GC-MS/SIM method is employed to measure monosaccharide composition in radix glehniae crude polysaccharides and homogeneous polysaccharides, characteristic fragment ions of a target compound are detected, impurity interference is eliminated, even if the target compound and impurities do not achieve complete chromatographic separation, detection can be carried out, and detection selectivity, sensitivity and signal to noise ratio are raised greatly.

Description

The assay method of monose composition in a kind of Glehnia Littoralis Polysaccharide

Technical field

The present invention relates to the assay method of monose composition in a kind of Glehnia Littoralis Polysaccharide, belong to technical field of traditional Chinese medicines.

Background technology

Radix glehniae RadixGlehniae derives from samphire glehnia littoralis glehnialittoralisfr.SchmidtexMiq. root, be one of Shandong genunie medicinal materials, polysaccharide is its principal ingredient, and total sugar content reaches more than 70%, and polysaccharide has antitumor, anti-oxidant, raising immunity, anti-ageing multiple biologically active of waiting for a long time.The biologically active of polysaccharide and its structure closely related, the order of connection of monose in polysaccharide, link position, the type of glycosidic bond and the kind of monose, all can affect its biologically active, and the monose composition measuring in polysaccharide is very necessary for its bioactive research.In current Glehnia Littoralis Polysaccharide, the research of monose composition is less, and Ren Haona etc. adopt pre-column derivatization HPLC to determine the monose composition of Thick many candies in radix glehniae, and in radix glehniae, the monose composition research of homogeneous polysaccharide has no relevant report.

In the composition analysis of polysaccharide, at present after many employing hydrolysis, direct use liquid chromatography evaporative light-scattering detector measures, or use liquid chromatography UV-detector, gas chromatography hydrogen flame detector and gas chromatography combined with mass spectrometry total ion current to measure after derivatization, these method signal to noise ratio (S/N ratio)s are lower, selectivity is poor, and target peak is vulnerable to impurity interference, the accuracy of impact analysis result.

Summary of the invention

For above-mentioned prior art, be the monose composition in Accurate Determining Glehnia Littoralis Polysaccharide, the present invention intends adopting GC-MS/SIM method to measure the monose composition in radix glehniae Thick many candies and homogeneous polysaccharide.GC-MS/SIM method adopts the fragments characteristic ion of target compound to detect, and eliminates impurity interference, also can detect, substantially increase the selectivity of detection, sensitivity and signal to noise ratio (S/N ratio) even if target compound and impurity do not reach complete chromatographic resolution.

The present invention is achieved by the following technical solutions:

Gas chromatograph-mass spectrometer condition

Chromatographic condition: chromatographic column is Shimadzu Rxi-5MS quartz capillary column (30m × 0.25um × 0.25mm); Injector temperature 250 DEG C, split ratio: 99:1, temperature programme: initial temperature 160 DEG C, 1 DEG C of min ^-1 be raised to 195 DEG C later, keep 15min, 5 DEG C of min ^-1 be raised to 230 DEG C, keep 10min.Carrier gas done by helium, flow velocity 1mLmin ^-1 , sample size 1 μ L, purge flow rate 6mLmin ^-1 ;

Mass Spectrometry Conditions: EI source, electronics bombarding energy 70ev; Detector gain: 0.7kv, ion source temperature: 200 DEG C; Satellite interface temperature: 280 DEG C; Scan mode: Salbutamol Selected Ion Monitoring (SIM);

Prepared by need testing solution

Precision takes polysaccharide sample 10mg, adds the trifluoroacetic acid 6mL of 2mol/L, seals after inflated with nitrogen, 100 DEG C of hydrolysis 4h, and nitrogen dries up, and add methyl alcohol 1mL, nitrogen dries up, and repeats 5 times, obtains polysaccharide hydrolysate; By hydrolyzation sample P ₂o ₅vacuum drying 12h, adds 10mg oxammonium hydrochloride, 0.6mL pyridine and 4mg inositol, and 90 DEG C of water-bath 1h, let cool after taking-up to room temperature, add 1.6mL acetic anhydride, and 90 DEG C are continued reaction 1h, and pyridine is settled to 5.0mL, to obtain final product;

The preparation of reference substance storing solution

Precision takes corresponding each monose reference substance 10mg, P ₂o ₅vacuum drying 12h, adds 10mg oxammonium hydrochloride, 0.6mL pyridine and 4mg inositol, and 90 DEG C of water-bath 1h, let cool after taking-up to room temperature, add 1.6mL acetic anhydride, and 90 DEG C are continued reaction 1h, and pyridine is settled to 5.0mL, to obtain final product;

Determination method

Get test sample and each 1 μ L of reference substance solution, inject gas chromatograph-mass spectrometer, calculate the amount of each monose in need testing solution from typical curve, calculate amount of substance divided by molal weight.

The present invention establishes the GC-MS/SIM analytical approach of monose composition and content in Glehnia Littoralis Polysaccharide, and this method is highly sensitive, and signal to noise ratio (S/N ratio) is high, and selectivity is good, and result accurately and reliably.The present invention adopts the characteristic ion of its target compound of SIM model selection as monitoring ion, selects abundance comparatively large, and the stronger ion of characteristic, as its quota ion, eliminates the interference of impurity peaks, has more specificity; Under SIM pattern, baseline noise is starkly lower than SCAN pattern, and signal to noise ratio (S/N ratio) is higher; Because SIM pattern only detects selected characteristic ion, therefore under same detection speed, sensitivity is better than SCAN pattern.

Accompanying drawing explanation

Fig. 1 is homogeneous polysaccharide sample total ion current figure;

Fig. 2 is homogeneous polysaccharide sample SIM ion flow graph;

Fig. 3 is reference substance SIM ion flow graph.

Embodiment

Below in conjunction with embodiment to further instruction of the present invention.

Embodiment 1

Gas chromatography mass spectrometry instrument condition

Chromatographic condition: chromatographic column is Shimadzu Rxi-5MS quartz capillary column (30m × 0.25um × 0.25mm); Injector temperature 250 DEG C, split ratio: 99:1, temperature programme: initial temperature 160 DEG C, 1 DEG C of min ^-1 be raised to 195 DEG C later, keep 15min, 5 DEG C of min ^-1 be raised to 230 DEG C, keep 10min.Carrier gas done by helium, flow velocity 1mLmin ^-1 , sample size 1 μ L, purge flow rate 6mLmin ^-1 .

Mass Spectrometry Conditions: EI source, electronics bombarding energy 70ev; Detector gain: 0.7kv, ion source temperature: 200 DEG C; Satellite interface temperature: 280 DEG C; Scan mode: Salbutamol Selected Ion Monitoring (SIM).

Prepared by need testing solution

Precision takes polysaccharide sample 10mg, adds the trifluoroacetic acid 6mL of 2mol/L, seals after inflated with nitrogen, and 100 DEG C of hydrolysis 4h, nitrogen dries up, and add methyl alcohol 1mL, nitrogen dries up, and repeats 5 times, obtains polysaccharide hydrolysate.By hydrolyzation sample P ₂o ₅vacuum drying 12h, adds 10mg oxammonium hydrochloride, 0.6mL pyridine and 4mg inositol, and 90 DEG C of water-bath 1h, let cool after taking-up to room temperature, add 1.6mL acetic anhydride, and 90 DEG C are continued reaction 1h, and pyridine is settled to 5.0mL, to obtain final product.

The preparation of reference substance storing solution

Precision takes each monose reference substance 10mg, P ₂o ₅vacuum drying 12h, adds 10mg oxammonium hydrochloride, 0.6mL pyridine and 4mg inositol, and 90 DEG C of water-bath 1h, let cool after taking-up to room temperature, add 1.6mL acetic anhydride, and 90 DEG C are continued reaction 1h, and pyridine is settled to 5.0mL, to obtain final product.

Determination method

Get test sample and each 1 μ L of reference substance solution, sample introduction measures in accordance with the law, calculates the amount of each monose in need testing solution, calculate amount of substance divided by molal weight from typical curve.

Polysaccharide sample determination

Get radix glehniae homogeneous polysaccharide respectively, 30%, 70%, 95% ethanol alcohol precipitation Thick many candies need testing solution, measure according to instrument condition, the monose composition of each sample is calculated according to Glehnia Littoralis Polysaccharide sample determination result, result is as follows: 30% alcohol precipitation Thick many candies forms primarily of wood sugar, 70% alcohol precipitation Thick many candies is primarily of wood sugar, galactose, glucose, mannose forms, mol ratio is 18.6: 2.07: 1.00: 0.0142, 95% alcohol precipitation Thick many candies is primarily of wood sugar, galactose, glucose, mannose, arabinose forms, mol ratio is 7.79: 3.05: 1.00: 0.0977: 0.334, laboratory is separated obtained homogeneous polysaccharide primarily of wood sugar, galactose, glucose, mannose, arabinose forms, mol ratio is 0.823: 1.32: 1.00: 0.0919: 0.761.

The extracting method of radix glehniae Thick many candies is as follows:

Get radix glehniae medicine materical crude slice 100.0g, put in 1000mL round-bottomed flask, 95% alcohol reflux 3 times, each amount of alcohol added is 600mL, return time is respectively 2.0,1.5,1.0h, to remove the micromolecular compounds such as monose ^[5].Filter, the dregs of a decoction dry.Dregs of a decoction use water heating and refluxing extraction 3 times, each distilled water addition is respectively 800,600,600mL, return time is respectively 2.0,1.5,1.0h.150mL is evaporated to after No. 3 times extract merges, centrifugal (4000r/min, 15min) gets supernatant, carries out 30%, 70% and 95% alcohol precipitation respectively, alcohol precipitation polysaccharide successively with absolute ethyl alcohol, acetone, absolute ether washing, solvent volatilize rear respectively radix glehniae 30%, 70%, 95% alcohol precipitation Thick many candies.

The preparation method of homogeneous polysaccharide is as follows:

Get radix glehniae medicine materical crude slice 100g, 95% alcohol reflux 3 times, monose in removing medicinal material, compound sugar, and glycoside, each amount of alcohol added is 600mL, return time is respectively 2.0, 1.5, 1.0h, discard alcohol layer, the dregs of a decoction dry, extracting in boiling water 3 times, distilled water addition is followed successively by 800, 600, 600mL, time is respectively 2.0, 1.5, 1.0h, extract concentrates and centrifugal 4000r/min, 15min, supernatant adds ethanol and regulates concentration of alcohol to be 30%, spend the night, centrifugal, 4000r/min, 15min, supernatant adds ethanol and regulates concentration of alcohol to be 70%, spend the night, centrifugal 4000r/min, 15min, precipitation absolute ethyl alcohol, acetone, absolute ether respectively washs 3 times, evaporate to dryness obtains 70% alcohol precipitation polysaccharide.70% alcohol precipitation polysaccharide 1.5g distilled water dissolves, and filters, DEAE-cellulose chromatography post (1.6cm × 50cm) loading with 0.45 μm of miillpore filter, and first with deionized water wash-out 150min, obtain neutral sugar part, flow velocity is 1.5mL/min; Again with 0.4mol/LNaCl aqueous solution, about one and half column volumes of wash-out 180min(), flow velocity is 2mL/min, obtains acid sugar.Get gained acid sugar to get 40mg and be dissolved in 500uL distilled water, 0.45 μm of miillpore filter filters, and is splined on Sephacryls300 gel chromatographic columns.With deionized water wash-out 200min, flow velocity is 0.5mL/min, detects, obtain acid homogeneous polysaccharide with differential refraction detector.

The present invention establishes the GC-MS/SIM analytical approach of monose composition and content in Glehnia Littoralis Polysaccharide, and this method is highly sensitive, and signal to noise ratio (S/N ratio) is high, and selectivity is good, and result accurately and reliably.In accompanying drawing, Fig. 1 is that radix glehniae homogeneous polysaccharide sample SCAN pattern total ion current TIC schemes; Fig. 2 is radix glehniae homogeneous polysaccharide SIM pattern ion flow graph; Fig. 3 is mannose, glucose, rhamnose, arabinose, wood sugar, galactose, inositol, fructose (2 peaks) mixed mark SIM pattern ion flow graph.Can find out in Fig. 1, under SCAN pattern, mannose, glucose, arabinose are not separated completely with impurity, have impact on the accuracy of detection, can find out in Fig. 2, select the characteristic ion of each monosaccharide compounds as monitoring ion under SIM pattern, select abundance comparatively large, the stronger ion of characteristic is as its quota ion, eliminate the interference of impurity peaks, have more specificity; Can find out in Fig. 3, under SIM pattern, the mass spectra peak of each monose reference substance reaches and is separated completely, noiseless between component.As can be seen from the contrast of Fig. 1-3, under SIM pattern, baseline noise is starkly lower than SCAN pattern, and signal to noise ratio (S/N ratio) is higher; Because SIM pattern only detects the selected characteristic ion of target peak, therefore do not disturb by impurity; Owing to only detecting a small amount of characteristic ion, therefore under same detection speed, the sensitivity of SIM pattern is better than SCAN pattern.

Claims (1)

1. the assay method that in Glehnia Littoralis Polysaccharide, monose forms, it is characterized in that, concrete grammar is as follows:

Gas chromatograph-mass spectrometer condition

Chromatographic condition: chromatographic column is 30m × 0.25um × 0.25mm Shimadzu Rxi-5MS quartz capillary column; Injector temperature 250 DEG C, split ratio: 99:1, temperature programme: initial temperature 160 DEG C, 1 DEG C of min ^-1 be raised to 195 DEG C later, keep 15min, 5 DEG C of min ^-1 be raised to 230 DEG C, keep 10min; Carrier gas done by helium, flow velocity 1mLmin ^-1 , sample size 1 μ L, purge flow rate 6mLmin ^-1 ;

Mass Spectrometry Conditions: EI source, electronics bombarding energy 70ev; Detector gain: 0.7kv, ion source temperature: 200 DEG C; Satellite interface temperature: 280 DEG C; Scan mode: select SIM ion monitoring;

Prepared by need testing solution

Precision takes polysaccharide sample 10mg, adds the trifluoroacetic acid 6mL of 2mol/L, seals after inflated with nitrogen, 100 DEG C of hydrolysis 4h, and nitrogen dries up, and add methyl alcohol 1mL, nitrogen dries up, and repeats 5 times, obtains polysaccharide hydrolysate; By hydrolyzation sample P ₂o ₅vacuum drying 12h, adds 10mg oxammonium hydrochloride, 0.6mL pyridine and 4mg inositol, and 90 DEG C of water-bath 1h, let cool after taking-up to room temperature, add 1.6mL acetic anhydride, and 90 DEG C are continued reaction 1h, and pyridine is settled to 5.0mL, to obtain final product;

The preparation of reference substance storing solution

Precision takes corresponding each monose reference substance 10mg, P ₂o ₅vacuum drying 12h, adds 10mg oxammonium hydrochloride, 0.6mL pyridine and 4mg inositol, and 90 DEG C of water-bath 1h, let cool after taking-up to room temperature, add 1.6mL acetic anhydride, and 90 DEG C are continued reaction 1h, and pyridine is settled to 5.0mL, to obtain final product;

Determination method

Get test sample and each 1 μ L of reference substance solution, inject gas chromatograph-mass spectrometer, calculate the amount of each monose in need testing solution from typical curve, calculate amount of substance divided by molal weight.