CN109187807A - A kind of method that pre-column derivatization HPLC-MS/MS detects contents of monosaccharides in sweet osmanthus - Google Patents
A kind of method that pre-column derivatization HPLC-MS/MS detects contents of monosaccharides in sweet osmanthus Download PDFInfo
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Abstract
The invention discloses the methods of monosaccharide component in a kind of pre-column derivatization HPLC-MS/MS detection sweet osmanthus.The present invention turns to pre-treatment means with 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) column front derivation, 6 kinds of monosaccharide components in sweet osmanthus are measured simultaneously using high performance liquid chromatography and electrospray ionization mass spectrometry combination, the advantages that method is simple, sensitive, selectivity is high, separative efficiency is high, sample volume is few, analysis time is short, multi-mode measurement, the monosaccharide components research for polysaccharide in sweet osmanthus provide scientific basis.This method is that the research of other natural products is laid a good foundation simultaneously.
Description
Technical field
The invention belongs to polysaccharide component analysis fields, and in particular in a kind of pre-column derivatization HPLC-MS/MS detection sweet osmanthus
The method of monosaccharide component.
Background technique
Sweet osmanthus (Osmanthus fragrans Lour) belongs to Oleaceae (Oleaceae), is a kind of ornamental plant, in
State south and middle part are used as Traditional Folk drug, for treating a variety of diseases.It is thousands of as medicinal plant in China
Year, and there is extensive biological effect, including inflammation can be weakened, it works as antioxidant, prevents aging and inhibit
Melanogenesis.In addition, the primary bioactive components in Flos Osmanthi Fragrantis extract are carotenoid and phenolic acid.It is more from natural drug
The analysis of sugar is extensive.Polysaccharide is started from as drug research in the 1950s, so far, it has been found that hundreds of polysaccharide tools
There is bioactivity.It is living that polysaccharide all plays important biomolecule in terms of anti-inflammatory, antiviral and antitumor, anti-aging, promotion
Property, and polysaccharide is very important one kind organic compound in organism, except the construction for participating in organism, as energy object
Matter, cell recognition informational molecule outside, also participation substance in vivo conversion metabolism is generated as the necessary amino acid of organism, nucleosides
Acid, fatty acid etc..Sweet osmanthus polysaccharide is one of important component of sweet osmanthus, but is concentrated mainly on sweet osmanthus essence to the research of sweet osmanthus at present
Oily aspect, and the monosaccharide components of sweet osmanthus polysaccharide are then seldom had been reported that.
The advantages such as HPLC- tandem mass spectrometry (HPLC-MS/MS) is strong with its specificity, sensitivity is high are in effective component of chinese medicine
Using increasingly extensive in assay.In the past decade, dedicated for the sugared exploitation with the HPLC-MS method of monosaccharide of analysis
Significant progress is achieved in terms of sensitivity and specificity, maintains the speed and simplicity of implementation.However, due to list
The low ionizing efficiency of sugar, HPLC-MS method are limited by loss of sensitivity, and therefore, the derivatization of monosaccharide is for obtaining Gao Ling
Sensitivity detection is essential.1-phenyl-3-methyl-5-pyrazolones ketone (PMP) pre-column derivatization HPLC is anti-
Answer condition milder, for product without alloisomerism, ultraviolet detection sensitivity is higher, therefore obtains relatively broad application.However, PMP
There is not been reported for the application of pre-column derivatization high performance liquid chromatography and electrospray ionization mass spectrometry combination analysis method on sweet osmanthus.
Summary of the invention
PMP derivatization before a kind of six kinds of sugared columns of measurement is provided the purpose of the present invention is overcoming the shortage of prior art
HPLC-MS/MS method.The data of the characteristic fragment ion of monosaccharide derived from six kinds of PMP are had collected.It is reacted by detection
Time and neutrality condition optimize the step of pre-column derivatization reacts.Using this method to single in the sweet osmanthus of three kinds of different cultivars
Sugared content is analyzed, and provides foundation for the development and utilization of Osmanthus Fragrans resources.
The technical scheme adopted by the invention is as follows:
Before a kind of column in PMP derivatization HPLC-MS/MS detection sweet osmanthus monosaccharide component method, comprise the following steps that
(1) sample treatment
A, in sweet osmanthus polysaccharide extraction
Accurately weigh 10g sweet osmanthus, the dehydrated alcohol of 200ml is mixed after being put into 500ml round-bottomed flask, under the conditions of 80 DEG C
It is condensed back one hour in thermostat water bath, filter residue is taken to be repeated once this operation after filtering, filtered again and dry；Then
Obtained filter residue is placed on to the water in the round-bottomed flask of 1000ml and being put into 400ml again, in thermostat water bath under the conditions of 90 DEG C
Middle extraction 1.5h, is repeated twice；It filters and obtains filtrate later, merge filtrate twice, filtrate is evaporated in Rotary Evaporators
It is concentrated into 150ml；The dehydrated alcohol of 4 times of volumes is added in the polysaccharide solution of this concentration, is centrifuged 15min by centrifuge
(8000r/min) is dried to obtain sweet osmanthus Thick many candies；Thick many candies dissolution is added in distilled water, adds the Sevage reagent of 10ml
(chloroform: n-butanol=4:1) acutely shakes, is centrifuged 5min (3500r/min) with centrifuge at room temperature；By the anhydrous of 4 times of volumes
Ethyl alcohol is added in the supernatant obtained, precipitating ethyl alcohol and acetone washing, then is filtered, and obtains sweet osmanthus polysaccharide after dry；
B, in sweet osmanthus polysaccharide sour water solution
The sweet osmanthus polysaccharide 10mg for accurately weighing step a acquisition is put into the screw thread ml headspace bottle of 10ml, adds the hydrochloric acid solution of 5ml
(2mol/L) is sealed after mixing, is then hydrolyzed 3 hours at 90 DEG C, after cooling, is centrifuged 5min, is obtained supernatant monosaccharide sample
Hydrolyzate, to derivatization；
C, the PMP derivatization of monosaccharide
Precision takes 100 μ l of sweet osmanthus polysaccharide hydrolysis solution, is placed in 7ml centrifuge tube, and 100 μ l1- phenyl -3- methyl -5- pyrroles are added
Oxazoline ketone methanol solution (0.5mol/L), sufficiently shakes up, and 30min is reacted under 70 DEG C of water bath conditions, and cooling room temperature adds suitable
10% glacial acetic acid solution tune PH is measured to neutrality, after mixing plus 1ml chloroform is for extracting excessive PMP, discards lower liquid, this behaviour
It is repeated 3 times, supernatant is collected in centrifugation 10min (revolving speed 8000r/min) afterwards, finally with 0.22 μm of membrane filtration, is diluted to
It is to be measured after 1000 times；
Precision weighs monosaccharide reference substance 10mg, in 10ml volumetric flask, adds ammonium hydroxide to graduation mark that 1mg/mL mixing is made single
Standard for Sugars solution, according to be measured after above method PMP derivation process, the reference substance is mannose, ribose, glucose, gala
Sugar, xylose, fucose；
Precision weighs monosaccharide reference substance 10.0mg and is put into addition 5ml ammonium hydroxide in 10ml volumetric flask respectively, is made into 2mg/ml
Mixed standard solution；Then it being diluted to obtain series of standards solution, concentration is followed successively by 2 μ g/ml, 200ng/ml,
100ng/ml, 50ng/ml, 20ng/ml, 2ng/ml draw each 100 μ l of standard serial solution, derivative according to above method PMP
It is to be measured after processing；
6 kinds of monosaccharide are as shown in the following chart:
Table 1
| Number | Title | Referred to as |
| 1 | Mannose | Man |
| 2 | Ribose | Rib |
| 3 | Glucose | Glu |
| 4 | Galactolipin | Gal |
| 5 | Xylose | Xyl |
| 6 | Fucose | Fuc |
(2) it is detected with high performance liquid chromatography mass spectrometer:
A, the condition of high-efficient liquid phase chromatogram HPLC be column model Shim-pack VP-ODS6022748 (150L ×
2.0), column temperature and detector temperature: 30 DEG C；Flow velocity: 0.2mL/min；Mobile phase: 5mmol/L ammonium acetate-acetonitrile, gradient elution
Program: 0-5mim17% acetonitrile；5-10min18% acetonitrile；10-15min19% acetonitrile；15-19min21% acetonitrile；19-
25min18% acetonitrile 25-40min15% acetonitrile；Sample volume: 2 μ l；
B, mass spectrum MS/MS condition is ion source: electric spray ion source, detection pattern: positive ion mode；Heat deblocking temperature:
400℃；DL heating tube temperature: 250 DEG C, sweep time: 0.1s, scan pattern: first mass spectrometric Q3 scanning, scanning of the mass spectrum range m/
Z100-1000, second order ms Product ion scans, more reaction detections (multiple reaction monition, MRM) mode
Quantitative analysis, electron spray voltage: 3.5KV, atomization gas: N2, atomization gas flow velocity: 3.0L/min, drier flow: 15.0L/
Min, collision gas: high pure nitrogen collides atmospheric pressure: 230Kpa；
(3) qualitative detection:
The mixed solution for taking step (1) c 6 kinds of monosaccharide reference substances after PMP derivation process, under the conditions of step (2) and such as
Sample introduction measures under the mass spectrometry parameters that table 2 is set；
Sweet osmanthus sample feeding of step (1) c after PMP derivation process is measured under above-mentioned condition:
Sample is after liquid phase separation, mass ions, according to the retention time of every kind of monosaccharide, molecular weight, quasi-molecular ion
The mass spectrometry parameters such as peak (table 2) carry out the confirmation of different monosaccharide and content to sample.
The main mass spectrometry parameters of monosaccharide standard derivatization product under 2 positive ion mode of table
(4) quantitative detection:
Take a series of mixing of 6 kinds of monosaccharide reference substances of different quality concentration of step (1) c after PMP derivation process molten
Liquid, sample introduction measurement respectively, is repeated 6 times, measures its corresponding concentration under conditions of the setting of high performance liquid chromatography mass spectrometer
Chromatographic peak area carries out linear regression, obtains regression equation, related coefficient and the range of linearity are shown in Table 3；With linear equation to sample
It is quantified, the response of monosaccharide should be in the range of linearity that instrument detects in sample；
Table 3 mixes the standard curve of monosaccharide standard, the range of linearity
The beneficial effects of the present invention are:
The present invention with mobile phase be 5mmol/L sodium acetate-acetonihile gradient elution, can be obtained more preferably using gradient elution
Separation, improve detection sensitivity of the sugar in mass spectrum, shorten disengaging time and improve peak type simultaneously.Using HPLC-MS/MS method
6 kinds of monosaccharide components in sweet osmanthus are carried out while being measured, method is simple, sensitive, selectivity is high, separative efficiency is high, sample volume is few,
The advantages that analysis time is short, multi-mode measures, the monosaccharide components research for polysaccharide in sweet osmanthus provide scientific basis.This method is simultaneously
It lays a good foundation for the research of other natural products.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
Detailed description of the invention
Fig. 1 is that the Q3 of each monosaccharide standard of PMP label scans mass spectrogram
Fig. 2 is the Product ion scans mass spectrogram of each monosaccharide standard of PMP label
Fig. 3 is the PMP derivatization process of dextrose standard sample and the dextrose standard sample of PMP label spreads out in the positive-ion mode
The MS fragment pathways of biology.
Fig. 4 is the MRM spectrogram for the mixing monosaccharide standard that PMP is marked under positive ion mode.
1- mannose, 2- ribose, 3- glucose, 4- galactolipin, 5- xylose, 6- fucose
Fig. 5 is the MRM spectrogram for the orange osmanthus sweet osmanthus sample that PMP is marked under positive ion mode.
1- mannose, 2- ribose, 3- rhamnose, 4- galactolipin, 5- glucose, 6- galactolipin, 7- xylose, 8- fucose
Fig. 6 is the MRM spectrogram for the laurel sweet osmanthus sample that PMP is marked under positive ion mode.
1- mannose, 2- ribose, 3- rhamnose, 4- galactolipin, 5- glucose, 6- galactolipin, 7- xylose, 8- fucose
Fig. 7 is the MRM spectrogram for the gold osmanthus sweet osmanthus sample that PMP is marked under positive ion mode.
1- mannose, 2- ribose, 3- rhamnose, 4- galactolipin, 5- glucose, 6- galactolipin, 7- xylose, 8- fucose
Specific embodiment
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.Provided implementation
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.
[embodiment 1] sample treatment
A, in sweet osmanthus polysaccharide extraction
Accurately weigh 10g sweet osmanthus, the dehydrated alcohol of 200ml is mixed after being put into 500ml round-bottomed flask, under the conditions of 80 DEG C
It is condensed back one hour in thermostat water bath, filter residue is taken to be repeated once this operation after filtering, filtered again and dry；Then
Obtained filter residue is placed on to the water in the round-bottomed flask of 1000ml and being put into 400ml again, in thermostat water bath under the conditions of 90 DEG C
Middle extraction 1.5h, is repeated twice；It filters and obtains filtrate later, merge filtrate twice, filtrate is evaporated in Rotary Evaporators
It is concentrated into 150ml；The dehydrated alcohol of 4 times of volumes is added in the polysaccharide solution of this concentration, is centrifuged 15min by centrifuge
(8000r/min) is dried to obtain sweet osmanthus Thick many candies；Thick many candies dissolution is added in distilled water 50mL, adds the Sevage of 10ml
Reagent (chloroform: n-butanol=4:1) acutely shakes, and is centrifuged 5min (3500r/min) with centrifuge at room temperature；By 4 times of volumes
Dehydrated alcohol is added in the supernatant obtained, precipitating ethyl alcohol and acetone washing, then is filtered, and obtains sweet osmanthus polysaccharide after dry；
B, in sweet osmanthus polysaccharide sour water solution
The sweet osmanthus polysaccharide 10mg for accurately weighing step a acquisition is put into the screw thread ml headspace bottle of 10ml, adds the hydrochloric acid solution of 5ml
(2mol/L) is sealed after mixing, is then hydrolyzed 3 hours at 90 DEG C, after cooling, is centrifuged 5min (3500r/min), is obtained supernatant
Liquid monosaccharide sample hydrolyzate, to derivatization；
C, the PMP derivatization of monosaccharide
Precision takes 100 μ l of sweet osmanthus polysaccharide hydrolysis solution, is placed in 7ml centrifuge tube, and 100 μ l1- phenyl -3- methyl -5- pyrroles are added
Oxazoline ketone methanol solution (0.5mol/L), sufficiently shakes up, and 30min is reacted under 70 DEG C of water bath conditions, and cooling room temperature adds suitable
10% glacial acetic acid solution tune PH is measured to neutrality, after mixing plus 1ml chloroform is for extracting excessive PMP, discards lower liquid, this behaviour
It is repeated 3 times, supernatant is collected in centrifugation 10min (revolving speed 8000r/min) afterwards, finally with 0.22 μm of membrane filtration, is diluted to
It is to be measured after 1000 times；
Precision weighs monosaccharide reference substance 10mg, in 10ml volumetric flask, adds ammonium hydroxide to graduation mark that 1mg/mL mixing is made single
Standard for Sugars solution, according to be measured after above method PMP derivation process, the reference substance is mannose, ribose, glucose, gala
Sugar, xylose, fucose；
Precision weighs monosaccharide reference substance 10.0mg and is put into addition 5ml ammonium hydroxide in 10ml volumetric flask respectively, is made into 2mg/ml
Mixed standard solution；Then it is diluted to obtain a series of standard solution of concentration, concentration is followed successively by 2 μ g/ml, 200ng/
Ml, 100ng/ml, 50ng/ml, 20ng/ml, 2ng/ml draw each 100 μ l of standard serial solution, according to above method PMP
It is to be measured after derivation process.
[embodiment 2] HPLC-MS/MS pre-column derivatization qualitative and quantitative analysis
1, instrument and reagent:
Orange osmanthus sweet osmanthus, gold osmanthus sweet osmanthus, laurel sweet osmanthus (Guangxi) (Hubei Ji Shantang prepared slices of Chinese crude drugs Co., Ltd), acetonitrile (color
Compose pure), methanol (chromatographically pure), glacial acetic acid (chromatographically pure), dehydrated alcohol (analysis pure), acetone (analysis is pure), chloroform (analysis is pure)
(Tianjin Ou Bokai Chemical Co., Ltd.), n-butanol, ammonium hydroxide (analyzing pure, Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd),
Hydrochloric acid (analyzes pure, Xinyang chemical reagent factory), ammonium acetate (chromatographically pure, Tianjin Kermel Chemical Reagent Co., Ltd.), 1-
Phenyl -3- methyl -5- pyrazolone (PMP), D-MANNOSE (>=99.0% (GC) Sinopharm Chemical Reagent Co., Ltd.), D-
Glucose, D- xylose, D- galactolipin (>=99.0% (GC), Aladdin purchase), D-ribose, L-fucose (>=99.0% (GC),
The cruel chemical Science and Technology Ltd. of that), Watson drinking water (Guangzhou Watson food and drink Co., Ltd).
Japanese Shimadzu LC-MS-8040 liquid chromatogram-triple level four bars mass spectrometer is furnished with ESI ion source, binary ladder
Degree pump LC-20ADXR type, column oven CTO-20AC type and system controller CBM-20A type；DZF-6050 type vacuum oven (on
Medical Equipment Plant of Hai Boxun Industrial Co., Ltd.), EE-60 thermostat water bath (Wenzhou Ai Ge instrument Co., Ltd), FA2004B electricity
Sub- balance (Shanghai Yue Ping scientific instrument Co., Ltd), KQ3200DB ultrasonic cleaner (the limited public affairs of city of Kunshan's ultrasonic instrument
Department), RE-5299 Rotary Evaporators (Ya Rong Instrument Ltd. of Zhengzhou City), 80-2 electric centrifuge (Changzhou Xiang day laboratory apparatus
Factory)；SHZ-D (III) multiplex vavuum pump of circulating water type (Zhengzhou Boke experimental instruments and equipment limited).
2, chromatographic condition: column model Shim-pack VP-ODS6022748 (150L × 2.0), column temperature and detector
Temperature: 30 DEG C；Flow velocity: 0.2mL/min；Mobile phase: 5mmol/L ammonium acetate-acetonitrile, gradient elution program: 0-5mim17% second
Nitrile；5-10min18% acetonitrile；10-15min19% acetonitrile；15-19min21% acetonitrile；19-25min18% acetonitrile 25-
40min15% acetonitrile；Sample volume: 2 μ l.
3, mass spectrum MS/MS condition:
Ion source: electric spray ion source, detection pattern: positive ion mode；Heating deblocking temperature: 400 DEG C；DL heats tube temperature
Degree: 250 DEG C, sweep time: 0.1s, scan pattern: first mass spectrometric Q3 scanning, scanning of the mass spectrum range m/z100-1000, second level matter
Compose Product ion scans, more reaction detections (multiple reaction monition, MRM) mode quantitative analysis, electron spray
Voltage: 3.5KV, atomization gas: N2, atomization gas flow velocity: 3.0L/min, drier flow: 15.0L/min, collision gas: high-purity
Nitrogen collides atmospheric pressure: 230Kpa.
4, the multiple reaction detection chromatogram and cracking analysis of monosaccharide standard
Monosaccharide standard after PMP is derivative is diluted to the concentration of 200ng/ml, directly progress HPLC-MS separation analysis.Figure
1, the mass ions peak figure that qualitative analysis obtains is carried out to each monosaccharide in Fig. 2, the quasi-molecular ions in figure is all cation peak, in reality
It tests relatively monosaccharide to find after the response under ESI positive ion mode and negative ion mode, is using 5mmol/L ammonium acetate-
When acetonitrile is as mobile phase, response is very low in the negative ion mode for monosaccharide, and response reaches 10 in the positive-ion mode6, comprehensive
It closes and considers that ESI positive ion mode is selected to be tested and analyzed in 5mmol/L ammonium acetate-acetonitrile as mobile phase.
The first mass spectrometric figure of monosaccharide standard that is marked by PMP under observation positive ion mode, second order ms figure and main
Mass spectrometry parameters (Fig. 1, Fig. 2, table 4), the discovery strongest quasi-molecular ion peak of monosaccharide PMP derivative signal [M+2PMP-H2O+H]+,
And monosaccharide derivatives secondary fragment ions peak homolysis solve m/z175 [PMP+H]+daughter ion, infer the fragment ion of m/z175
May be broken a PMP and a molecule monosaccharide as a result, having consistent cracking regular, specific cracking process is spread out with glucose
Fig. 3 is seen for biology.
This experiment by with standard monosaccharide retention time, existing bibliography and Information in Mass Spectra compare, and confirmation is assorted
Spectral peak.Such as Fig. 4, as can be seen from the figure the chromatographic peak of standard items derivative is symmetrical, trailing phenomenon, instruction sheet does not occur
Sugar is single-minded with PMP derivatives reaction, generates without by-product；And mixing polysaccharide is separated well, illustrates that PMP derived
Journey makes the polarity of sugar that very big change occur, and the hydrophobicity enhancing of derivative products, making sugar, separation is more easier on a column.
The main mass spectrometry parameters of monosaccharide standard derivatization product under 4 positive ion mode of table
5, testing result
(1) investigation of linear relationship
100 μ l sample introduction of mixed standard solution after each concentration PMP derivation process to be measured that embodiment 1 is obtained carries out
HPLC-MS detection, measures the chromatographic peak area of its corresponding concentration.The regression equation of each ingredient, related coefficient, detection limit and quantitative
Limit such as the following table 5.
Table 5 mixes the standard curve of monosaccharide standard, detection limit and quantitative limit
Note: LOQ is quantitative limit, and LOD is detection limit.
(2) sample size measures:
It takes 3 kinds of sweet osmanthus samples to carry out the extraction of polysaccharide to it, hydrolyzes, carry out the PMP derivative reaction preparation of monosaccharide for examination
Product solution, is measured according still further to experiment condition, records peak area and calculates its each contents of monosaccharides.
Data processing:
The concentration of monosaccharide in sweet osmanthus polysaccharide: CX(ug/ml)=(Ax-b)/a
Wherein: Cx is each monosaccharide concentration (ug/ml) in polysaccharide sample, and Ax is the peak area of monosaccharide in polysaccharide sample
(mAU.s), a is the slope of standard curve, and b is the intercept of standard curve.
Contents of monosaccharides in sweet osmanthus polysaccharide: (mg/g)=Cx*V*0.001/Mx
Wherein V is the total volume (ml) of polysaccharide sample solution, and Mx is the quality (mg) of sample.
Contents of monosaccharides (n=6) in 63 kinds of sweet osmanthus polysaccharide of table
According to existing standard control, it can be deduced that in 3 kinds of sweet osmanthus containing mannose, ribose, glucose, galactolipin,
Xylose, fucose.The abundance of cleaved fragment is than carrying out area in the retention time (order of polarity) of each monosaccharide, mass spectrometric data and mass spectrum
Point, it determines containing rhamnose and galacturonic acid in sweet osmanthus, and the orange osmanthus sweet osmanthus sample marked according to PMP under positive ion mode
MRM map (Fig. 5), it can be deduced that rhamnose in orange osmanthus have biggish signal value and peak area value.According to existing standard
Product calculate the concrete content of each monosaccharide in sweet osmanthus, compare each contents of monosaccharides in three kinds of sweet osmanthus, orange osmanthus: galactolipin > grape by table 6
Sugar > xylose > mannose > ribose > fucose, laurel, gold osmanthus: galactolipin > xylose > glucose > mannose > ribose > fucose.It is red
Each contents of monosaccharides ratio is mannose: ribose: glucose: galactolipin: xylose: fucose 0.9:0.7:5.2:5.8:4.8 in osmanthus:
0.1；Each contents of monosaccharides ratio is mannose: ribose: glucose: galactolipin: xylose: fucose 0.5:0.3:2.3 in laurel:
3.6:3.1:0.2；Each contents of monosaccharides ratio is mannose: ribose: glucose: galactolipin: xylose: fucose 0.5 in gold osmanthus:
0.3:1.9:3.2:3.0:0.2。
(3) the multiple reaction detection chromatogram and cracking analysis of sweet osmanthus polysaccharide
By optimizing Mass Spectrometry Conditions, standard items are carried out with the karyoplasmic ratio of screening parent ion and daughter ion, is optimized under MRM mode
Mass Spectrometry Conditions, such as table 7.In conjunction with document, the MRM map analysis of the derivatization product of sweet osmanthus polysaccharide is obtained by water extract-alcohol precipitation and sour water solution
Out, there are 8 kinds and the regular identical quasi-molecular ions (Fig. 5,6,7) of monosaccharide cracking, it is consistent with standard items, it then follows monosaccharide derivatives are split
Solution rule.Monosaccharide group becomes mannose, ribose, rhamnose, galacturonic acid, glucose, galactolipin, xylose, rock in 3 kinds of sweet osmanthus
Algae sugar.
Under 7 positive ion mode of table in sweet osmanthus polysaccharide monosaccharide PMP derivatization product main mass spectrometry parameters
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
In the technical scope disclosed by the present invention, any changes or substitutions that can be easily thought of by anyone skilled in the art,
It should be covered by the scope of protection of the present invention.Therefore, protection scope of the present invention should be with the protection scope of claims
Subject to.
Claims (1)
1. a kind of method that PMP derivatization HPLC-MS/MS detects monosaccharide component in sweet osmanthus before column, comprises the following steps that
(1) sample treatment
A, in sweet osmanthus polysaccharide extraction
Accurately weigh 10g sweet osmanthus, the dehydrated alcohol of 200ml is mixed after being put into 500ml round-bottomed flask, in perseverance under the conditions of 80 DEG C
It is condensed back one hour in warm water bath, filter residue is taken to be repeated once this operation after filtering, filtered again and dry；Then again will
Obtained filter residue is placed on the water in the round-bottomed flask of 1000ml and being put into 400ml, mentions in thermostat water bath under the conditions of 90 DEG C
1.5h is taken, is repeated twice；It filters and obtains filtrate later, merge filtrate twice, filtrate is concentrated by evaporation in Rotary Evaporators
To 150ml；The dehydrated alcohol of 4 times of volumes is added in the polysaccharide solution of this concentration, is centrifuged 15min (8000r/ by centrifuge
Min), it is dried to obtain sweet osmanthus Thick many candies；Thick many candies dissolution is added in distilled water, adds the Sevage reagent (chloroform: just of 10ml
Butanol=4:1), it acutely shakes, is centrifuged 5min (3500r/min) with centrifuge at room temperature；The dehydrated alcohol of 4 times of volumes is added
In the supernatant of acquirement, precipitating ethyl alcohol and acetone washing, then filtered, sweet osmanthus polysaccharide is obtained after dry；
B, in sweet osmanthus polysaccharide sour water solution
The sweet osmanthus polysaccharide 10mg for accurately weighing step a acquisition is put into the screw thread ml headspace bottle of 10ml, adds the hydrochloric acid solution (2mol/ of 5ml
L), seal after mixing, then hydrolyzed 3 hours at 90 DEG C, after cooling, be centrifuged 5min, obtain supernatant monosaccharide sample hydrolyzate,
To derivatization；
C, the PMP derivatization of monosaccharide
Precision takes 100 μ l of sweet osmanthus polysaccharide hydrolysis solution, is placed in 7ml centrifuge tube, and 100 μ l1- phenyl -3- methyl -5- pyrazolines are added
Ketone methanol solution (0.5mol/L), sufficiently shakes up, and 30min is reacted under 70 DEG C of water bath conditions, and cooling room temperature adds appropriate
For 10% glacial acetic acid solution tune PH to neutrality, after mixing plus 1ml chloroform is for extracting excessive PMP, discards lower liquid, this operation
It is repeated 3 times, supernatant is collected in centrifugation 10min (revolving speed 8000r/min) afterwards, finally with 0.22 μm of membrane filtration, is diluted to 1000
It is to be measured after times；
Precision weighs monosaccharide reference substance 10mg, in 10ml volumetric flask, adds ammonium hydroxide to graduation mark that 1mg/mL mixing monosaccharide mark is made
Quasi- solution, according to be measured after above method PMP derivation process, the reference substance is mannose, ribose, glucose, galactolipin, wood
Sugar, fucose；
Precision weighs monosaccharide reference substance 10.0mg and is put into addition 5ml ammonium hydroxide in 10ml volumetric flask respectively, is made into the mixed of 2mg/ml
Standardization solution；Then it being diluted to obtain series of standards solution, concentration is followed successively by 2 μ g/ml, 200ng/ml, 100ng/ml,
50ng/ml, 20ng/ml, 2ng/ml draw each 100 μ l of standard serial solution, according to after above method PMP derivation process to
It surveys；
(2) it is detected with high performance liquid chromatography mass spectrometer:
A, the condition of high-efficient liquid phase chromatogram HPLC is
Column model Shim-pack VP-ODS6022748 (150L × 2.0), column temperature and detector temperature: 30 DEG C；Flow velocity:
0.2mL/min；Mobile phase: 5mmol/L ammonium acetate-acetonitrile, gradient elution program: 0-5mim17% acetonitrile；5-10min18% second
Nitrile；10-15min19% acetonitrile；15-19min21% acetonitrile；19-25min18% acetonitrile 25-40min15% acetonitrile；Sample volume:
2μl；
B, mass spectrum MS/MS condition is
Ion source: electric spray ion source, detection pattern: positive ion mode；Heating deblocking temperature: 400 DEG C；DL heating tube temperature: 250
DEG C, sweep time: 0.1s, scan pattern: first mass spectrometric Q3 scanning, scanning of the mass spectrum range m/z100-1000, second order ms product
Ion scan, more reaction detections (multiple reaction monition, MRM) mode quantitative analysis, electron spray voltage:
3.5KV, atomization gas: N2, atomization gas flow velocity: 3.0L/min, drier flow: 15.0L/min, collision gas: high pure nitrogen,
Collide atmospheric pressure: 230Kpa；
(3) qualitative detection:
The mixed solution for taking step (1) c 6 kinds of monosaccharide reference substances after PMP derivation process, under the conditions of step (2) and as table 1 is set
Sample introduction measures under fixed mass spectrometry parameters；
Sweet osmanthus sample feeding of step (1) c after PMP derivation process is measured under above-mentioned condition:
Sample is after liquid phase separation, mass ions, according to the retention time of every kind of monosaccharide, molecular weight, quasi-molecular ion in table 1
The mass spectrometry parameters such as peak carry out the confirmation of different monosaccharide and content to sample；
The main mass spectrometry parameters of monosaccharide standard derivatization product under 1 positive ion mode of table
(4) quantitative detection:
The mixed solution for taking a series of 6 kinds of monosaccharide reference substances of different quality concentration of step (1) c after PMP derivation process, in
Sample introduction measurement respectively, is repeated 6 times, measures the chromatographic peak of its corresponding concentration under conditions of the setting of high performance liquid chromatography mass spectrometer
Area carries out linear regression, obtains regression equation, related coefficient and the range of linearity are shown in Table 2；Sample is determined with linear equation
It measures, the response of monosaccharide should be in the range of linearity that instrument detects in sample；
Table 2 mixes the standard curve of monosaccharide standard, the range of linearity
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