CN109374779A - A kind of rapid detection method of cane sugar content - Google Patents
A kind of rapid detection method of cane sugar content Download PDFInfo
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- CN109374779A CN109374779A CN201811543467.5A CN201811543467A CN109374779A CN 109374779 A CN109374779 A CN 109374779A CN 201811543467 A CN201811543467 A CN 201811543467A CN 109374779 A CN109374779 A CN 109374779A
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- 229930006000 Sucrose Natural products 0.000 title claims abstract description 57
- 229960004793 sucrose Drugs 0.000 title claims abstract description 57
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 title claims abstract description 54
- 238000001514 detection method Methods 0.000 title claims abstract description 44
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 56
- 239000012071 phase Substances 0.000 claims abstract description 50
- 239000005720 sucrose Substances 0.000 claims abstract description 37
- 239000003085 diluting agent Substances 0.000 claims abstract description 31
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 28
- 235000019253 formic acid Nutrition 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 26
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000012086 standard solution Substances 0.000 claims abstract description 19
- 238000010828 elution Methods 0.000 claims abstract description 12
- 235000013305 food Nutrition 0.000 claims abstract description 12
- 239000007791 liquid phase Substances 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 9
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 9
- 239000012898 sample dilution Substances 0.000 claims abstract description 6
- 229940079593 drug Drugs 0.000 claims abstract description 5
- 238000013467 fragmentation Methods 0.000 claims abstract description 3
- 238000006062 fragmentation reaction Methods 0.000 claims abstract description 3
- 150000002500 ions Chemical class 0.000 claims description 29
- 238000010790 dilution Methods 0.000 claims description 24
- 239000012895 dilution Substances 0.000 claims description 24
- 239000000047 product Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 7
- -1 ion ion Chemical class 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 5
- 239000012452 mother liquor Substances 0.000 claims description 5
- 230000005526 G1 to G0 transition Effects 0.000 claims description 4
- 239000012982 microporous membrane Substances 0.000 claims description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- 239000000908 ammonium hydroxide Substances 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 125000003368 amide group Chemical group 0.000 claims description 2
- 230000014759 maintenance of location Effects 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims 1
- 150000001408 amides Chemical class 0.000 claims 1
- 150000001450 anions Chemical class 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 238000001819 mass spectrum Methods 0.000 abstract description 9
- 239000006193 liquid solution Substances 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 description 24
- 239000000243 solution Substances 0.000 description 20
- 238000000034 method Methods 0.000 description 19
- 239000000523 sample Substances 0.000 description 18
- 238000009472 formulation Methods 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 238000005259 measurement Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000004255 ion exchange chromatography Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002203 pretreatment Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 230000005518 electrochemistry Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000105 evaporative light scattering detection Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000012395 formulation development Methods 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 241000208340 Araliaceae Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 239000011260 aqueous acid Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000007910 chewable tablet Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000002706 dry binder Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000002552 multiple reaction monitoring Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 235000019629 palatability Nutrition 0.000 description 1
- 235000010603 pastilles Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004730 pulsed amperometry Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960004016 sucrose syrup Drugs 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 229940054967 vanquish Drugs 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000005550 wet granulation Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The present invention provides a kind of rapid detection methods of cane sugar content.Detection method: the standard solution diluent used with sample dilution is identical, is the aqueous formic acid of 0.05%~3% volume ratio；Utilize the cane sugar content in ultrahigh pressure liquid phase-Mass Spectrometry detection prepare liquid and standard solution；Ultrahigh pressure liquid phase: chromatographic column with hydrophilic function, the flow velocity of mobile phase are 0.1~0.5mL/min, 30~60 DEG C of column temperature, using gradient elution mode；Mobile phase is made of the aqueous formic acid of 10~100mmol/L and the acetonitrile containing 0.05~0.2% formic acid；Mass spectrum uses PRM scan pattern, and sucrose ion parent ion/daughter ion ion pair is 387.08 > m/z of m/z 341.08, fragmentation energies 20eV~50eV.The present invention overcomes prior art accuracy it is low, sensitivity is low, time-consuming and laborious the problems such as, the detection of cane sugar content suitable for any product such as food and drug.
Description
Technical field
The present invention relates to technical field of analytical chemistry, more particularly, to a kind of rapid detection method of cane sugar content.
Background technique
Sucrose is one kind of disaccharide, it is by the hemiacetal hydroxyl of a molecule glucose and the hemiacetal hydroxyl of a molecule fructose
Condensation is dehydrated each other.It is widely used in medicine and field of food, sucrose.In field of medicaments, sucrose is a kind of heavy
The auxiliary material wanted, such as sucrose powder can be used as dry binder, chewable tablets or pastille solubilizer and sweetener；Content be 50%~
67% (W/W) sucrose syrup, for the binder in tablet wet granulation, and the syrup that content is 50%~67% (W/W) is used
It is coated in tablet.In addition, syrup is also widely used in the excipient of oral liquid, to increase palatability or viscosity.In biology
Pharmaceutical field, sucrose play stable albumen, adjust the effects of osmotic pressure often as the auxiliary material in preparation.In field of food, sugarcane
Sugar is widely applied often as a kind of important food additives, and many food such as beverage, candy etc. are all rich in sucrose.
Need to carry out the measurement of cane sugar content in fields such as excipient substance exploitation, food developments.Currently, in field of food
Cane sugar content detection method is mainly national standard method GB/T5009.8-2003 " measurement of sucrose in food " Fei Linshi titration,
Party's law limitation is that pretreatment process is complicated, and titration end-point is not easy to judge, especially to food with low sugar content, (cane sugar content is less than 3g/
100g).In medicine field, Chinese Pharmacopoeia includes the detection method of cane sugar content not yet.In practical application, more it is required to
Separation and measurement while realizing a variety of sugar, and the method for current comparative maturity is high performance liquid chromatography (HPLC) and ion chromatography
(IC).Conventional H PLC chromatography simplifies sample pre-treatments, but sensitivity is low, and need using differential refraction without deriving
(RI) detection, evaporative light-scattering (ELS) or electrochemistry (EC) detection.And the chromatography of ions mostly uses greatly anion exchange separation skill
Art can significantly improve sensitivity, but need special ion chromatography and its matched pulsed amperometry, and sample consumption
It measures also larger.In addition to above two method, gas chromatography applications are than wide, and carbohydrate has to pass through when being detected using this method
It can be detected after derivative, it is when operating cost and cumbersome.
In view of this, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of rapid detection method of cane sugar content, which overcomes existing
There is the problems such as technology accuracy is low, sensitivity is low, time-consuming and laborious, the cane sugar content suitable for any product such as food and drug
Detection.
In order to achieve the goal above, the present invention provides following technical schemes:
A kind of rapid detection method of cane sugar content, including the following steps:
Sample is diluted with diluent, obtains prepare liquid；
With sucrose standard product preparing standard solution, and draw standard curve；The standard solution and the sample dilute institute
Diluent is identical, and the diluent is the aqueous formic acid of 0.05%~3% volume ratio；
The cane sugar content in the prepare liquid and the standard solution is detected using ultrahigh pressure liquid phase-Mass Spectrometry；
Wherein, the chromatographic condition of ultrahigh pressure liquid phase are as follows:
Chromatographic column with hydrophilic function,
The flow velocity of mobile phase be 0.1~0.5mL/min, 30~60 DEG C of column temperature, using gradient elution mode；
Mobile phase is made of mobile phase A and Mobile phase B, and the gradient elution is in for the volume ratio of mobile phase A and Mobile phase B
Change of gradient；
The mobile phase A is the aqueous formic acid of 10~100mmol/L, and pH is 4~6；Mobile phase B be containing 0.05~
The acetonitrile (volume ratio) of 0.2% formic acid.
As described above, rapid detection method of the invention only has two steps, i.e. sample treatment and instrument sample detection, does not relate to
And derive and etc., without complicated detector.
Compared with prior art, it is had the advantage that using detection method detection cane sugar content of the invention
1, rate is fast: the pretreatment of sample can be completed in one hour, and instrument can be completed in detection half an hour, consume in total
When be no more than 1.5 hours；
2, accuracy is high: through compareing, the deviation of the theoretical value of detected value and sample is controlled within 10%, standard curve
Related coefficient is 0.99 or more；
3, sample-pretreating method is simple: may only relate to filtering, centrifugation, dilution and etc., avoid complicated treatment process
Bring human error also greatly improves the automation of detection；
4, high sensitivity: on the one hand, adverse effect caused by interference component is reduced by sample dilution, adds superelevation
Hydraulic fluid phase-mass spectrometry high sensitivity, greatly enhances the reproducibility of method sensitivity and method；On the other hand, pass through sample
Dilution avoids the residual quantity of sample in the chromatography column, to reduce residual bring interference；
5, high throughput analysis: the mobile phase aqueous formic acid that this method uses or the acetonitrile holding time containing formic acid is realized
Long, along with being connected using ultrahigh-pressure liquid chromatograph with mass spectrum, consumed mobile phase is few, more conducively realizes high-throughput point
Analysis.
The other conditions of the above detection method can also be further improved, with the accuracy, sensitivity, reproduction of improvement method
Property or shorten detection time, it is specific as follows.
Preferably, the diluent is 0.05%~0.1% aqueous formic acid (volume ratio) or 0.1%~3% first
Aqueous acid, preferably 0.1%~1% aqueous formic acid.
The selection of diluent has a major impact result precision, sensitivity, and selection can suitably reduce uncorrelated ingredient
Interference.In principle, diluent can select any aqueous formic acid in 0.05%~3% volume ratio range, such as
0.05%, 0.1%, 0.15%, 0.2%, 0.5%, 0.7%, 1%, 1.5%, 2%, 2.5%, 3% etc., wherein preferred model
It is with 0.05%~0.1% aqueous formic acid or 0.1%~3% aqueous formic acid, more preferable 0.1%~1% formic acid
Aqueous solution.
Preferably, in the standard solution, maximum concentration is 50 times or more of minimum concentration, 100 times or more, preferably 700
Times or more.
The wider accuracy in the section of concentration of standard solution is higher in principle, but bring simultaneously standard solution quantity it is more, detection
The problems such as heavy workload.According to the additive amount of sucrose in general food and drug, being typically chosen maximum concentration is minimum concentration
The standard solution in 50 times or more sections.
Meanwhile the specific concentration of standard solution can also be according to the content appropriate adjustment of sucrose in sample.
The concentration ranges of the standard solution preferred 0.2mmol/L~200mmol/L, preferably 0.2mmol/L~
100mmol/L, preferably 0.2mmol/L~50mmol/L, preferably 0.2mmol/L~20mmol/L.
When preparing the standard solution, the concentration of mother liquor is preferably 0.1mol/L~1mol/L.
When to mother liquor dilution, 2 times are preferably diluted to 10e6Times.
Detection method of the invention is suitable for arbitrarily listing medicine containing the product of sucrose, including food and drug, the present invention
The detection process of cane sugar content in product.
The present invention does not also limit the physical aspect of sample, such as solid, liquid or emulsus etc..
Preferably, before to sample dilution further include:
The filtering with microporous membrane sample for first using 0.22 μm, takes supernatant after being then centrifuged for；The centrifugation is preferably with 13000
The centrifugation of~15000rpm revolving speed, the preferred 10min or more of centrifugation time；
When carrying out the dilution to sample, diluted multiple is 10 times~200 times, preferably 10 times~100 times.
By being centrifuged and diluting on the one hand removal part interference impurity, the concentration of residual interference object is on the other hand reduced, is mentioned
High result precision and sensitivity.
Preferably, gradient elution described in ultrahigh pressure liquid phase are as follows: using mobile phase A: Mobile phase B=20:80 first increases as starting point
Add the accounting of the mobile phase A, then reduces the accounting of the mobile phase A until mobile phase A when terminal: Mobile phase B=20:
80。
During the gradient elution, the volume ratio peak of the mobile phase A is preferably 90%~95%；
The chromatographic column preferred amide bonded stationary phase hydrophilic chromatographic column, preferred amide bonding organosilicon (such as ethylidene
Bridge hydridization, BEH) stationary phase hydrophilic chromatographic column；
The flow velocity of the mobile phase is preferably 0.2~0.5mL/min, sample volume preferably 1~2 μ L.
Preferably, the change of gradient of mobile phase described in ultrahigh pressure liquid phase is as shown in table 1 below.
Table 1
Preferably, the mobile phase A is the aqueous formic acid of 50~100mmol/L, and pH is 4~6；Mobile phase B is preferably
Acetonitrile containing 0.05~0.1% formic acid；
The pH of the mobile phase A is preferably 4.2~5, it is preferred to use ammonium hydroxide adjusts its pH value.
Preferably, the Mass Spectrometry Conditions are as follows: scanning range m/z 60~400,25~35L/min of sheath gas, ion pass
250~300 DEG C of defeated tube temperature degree, ESI ion source, negative ion mode scanning, resolution ratio are 17500~70000.
Preferably, in ultrahigh pressure liquid phase: the retention time of sucrose is 8.0~10.0min.
In addition, the other conditions of ultrahigh pressure liquid phase are preferred: within detection time 15min, 45~60 DEG C of column temperature, chromatographic column ginseng
Several 1.7 μm, 2.1mm × 150mm.
Other Mass Spectrometry Conditions of LC-MS are preferred are as follows: using multiple reaction monitoring MRM or parallel reaction monitoring PRM acquisition
Data, micro scanning number 1 are automatic to obtain controlling value 3e6, maximum ion sample injection time 20min, auxiliary gas velocity 0, S- prism electromagnetism frequency
Rate level value 50, sucrose ion parent ion/daughter ion ion pair for detection are 387.08 > m/z of m/z 341.08, fragmentation
Energy is 20eV~50eV.
To sum up, compared with prior art, invention achieves following technical effects:
(1) detection rates are fast；
(2) accuracy is high；
(3) sample-pretreating method is simple；
(4) high sensitivity；
(5) favorable reproducibility；
(6) high-throughput.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the sucrose parent ion total ion current figure and mass spectrum of 60 times of dilutions of the preparation 1 that the embodiment of the present invention 1 is tested
Figure；
Fig. 2 is that the sucrose of 60 times of dilutions of the preparation 1 that the embodiment of the present invention 1 is tested quantifies daughter ion extraction ion flow graph
And mass spectrogram；
Fig. 3 is the sucrose parent ion total ion current figure and mass spectrum of 60 times of dilutions of the preparation 2 that the embodiment of the present invention 1 is tested
Figure；
Fig. 4 is that the sucrose of 60 times of dilutions of the preparation 2 that the embodiment of the present invention 1 is tested quantifies daughter ion extraction ion flow graph
And mass spectrogram；
Fig. 5 is the survey that the embodiment of the present invention 1 quantifies that daughter ion extracts the integrated peak areas drafting of ion flow graph according to sucrose
The canonical plotting of examination.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with the drawings and specific embodiments, but
Be it will be understood to those of skill in the art that it is following described embodiments are some of the embodiments of the present invention, rather than it is whole
Embodiment is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.Based on the embodiments of the present invention, ability
Domain those of ordinary skill every other embodiment obtained without making creative work, belongs to guarantor of the present invention
The range of shield.The person that is not specified actual conditions in embodiment, carries out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same
Or production firm person is not specified in instrument, is the conventional products that can be obtained by commercially available purchase.
Embodiment 1
One, solution and mobile phase are prepared
Diluents are prepared: a 50mL centrifuge tube taken, is separately added into 40mL water and 40 μ L formic acid, vortex 30s, sufficiently
It mixes.
The preparation of sucrose standard product: accurately weighing the sucrose of 17.115mg in 50mL volumetric flask, and diluents are added
After dissolution, then diluent is settled to graduation mark, sufficiently shakes up the sucrose standard product mother liquor for obtaining 1mol/L.
The calibration curve solution of sucrose standard product is prepared:
200 μ L of 1mol/L standard solution is taken, the diluent of 800 μ L is added, mixes well the standard up to 200mmol/L
Product solution is named as S1 solution；
The S1 solution of 50 μ L is taken, the diluent of 100 μ L is added, is mixed well molten up to the S2 standard items of 66.666mmol/L
Liquid；
The S2 solution of 50 μ L is taken, the diluent of 100 μ L is added, is mixed well molten up to the S3 standard items of 22.222mmol/L
Liquid；
The S3 solution of 50 μ L is taken, the diluent of 50 μ L is added, is mixed well molten up to the S4 standard items of 11.111mmol/L
Liquid；
The S3 solution of 50 μ L is taken, the diluent of 100 μ L is added, is mixed well molten up to the S5 standard items of 7.407mmol/L
Liquid；
The S5 solution of 50 μ L is taken, the diluent of 50 μ L is added, is mixed well molten up to the S6 standard items of 3.703mmol/L
Liquid；
The S5 solution of 50 μ L is taken, the diluent of 100 μ L is added, is mixed well molten up to the S7 standard items of 2.469mmol/L
Liquid；
The S7 solution of 50 μ L is taken, the diluent of 50 μ L is added, is mixed well molten up to the S8 standard items of 1.235mmol/L
Liquid；
The S7 solution of 50 μ L is taken, the diluent of 100 μ L is added, is mixed well molten up to the S8 standard items of 0.823mmol/L
Liquid；
The S8 solution of 50 μ L is taken, the diluent of 100 μ L is added, is mixed well molten up to the S9 standard items of 0.274mmol/L
Liquid；
The preparation of mobile phase A: 50mL water is added in 200mL beaker in precise formic acid 2.3g, after mixing well, with
Then ammonium hydroxide tune pH value of solution is transferred in 1000mL volumetric flask, rinse liquid will be transferred to volumetric flask after beaker water rinse to 4.28,
It is repeated 3 times the operation, is then settled to volumetric flask graduation mark with water, is prepared after shaking up and obtains 50mmol/L aqueous formic acid.
The preparation of Mobile phase B: taking the mobile phase bottle of 1L, is transferred in mobile phase bottle, adds after accurate measurement 500mL acetonitrile
0.5mL formic acid is prepared after shaking up and obtains the acetonitrile containing volume fraction for 0.1% formic acid.
Two, concrete operation steps
(1) 1 pre-treatment of formulation samples: taking formulation samples 1 one of well known auxiliaries sucrose concentration, takes 100 μ L supernatants to be measured
Liquid is in 1.5mL EP pipe, after 0.22 μm of filtering with microporous membrane, takes supernatant spare after being centrifuged 15min with 13000rpm；Take 20 μ
The diluent of 180 μ L is added in the test formulation supernatant of L, mixes and obtains 10 times of 1 dilutions of preparation；10 times of 50 μ L are taken to make
The diluent of 100 μ L is added in 1 dilution of agent, mixes up to 30 times of 1 dilutions of preparation；30 times of 1 dilutions of preparation of 50 μ L are taken,
The diluent of 50 μ L is added, mixes up to 60 times of 1 dilutions of preparation；30 times of 1 dilutions of preparation of 50 μ L are taken, are added 100 μ L's
Diluent mixes up to 90 times of 1 dilutions of preparation.
(2) 2 pre-treatment of formulation samples: taking formulation samples 2 one of well known auxiliaries sucrose concentration, takes 100 μ L supernatants to be measured
Liquid is in 1.5mL EP pipe, after 0.22 μm of filtering with microporous membrane, takes supernatant spare after being centrifuged 15min with 13000rpm；Take 20 μ
The diluent of 180 μ L is added in the test formulation supernatant of L, mixes and obtains 10 times of 2 dilutions of preparation；10 times of 50 μ L are taken to make
The diluent of 100 μ L is added in dilution agent liquid, mixes up to 30 times of 2 dilutions of preparation；30 times of 2 dilutions of preparation for taking 50 μ L, add
The diluent for entering 50 μ L mixes up to 60 times of 2 dilutions of preparation.
(3) 60 times, 90 times of dilutions of formulation samples 1 are taken, 30 times of formulation samples 2,60 times of dilutions and standard curve
Each 2 μ L of 4~S9 of solution S is analyzed into ultrahigh pressure liquid phase-mass spectrometer, in addition takes the dilution of 2 μ L as blank, equally
Into mass spectral analysis.
The LC-MS instrument is Vanquish ultrahigh pressure liquid phase-QE mass spectrometer；
(4) detection method is ultrahigh pressure liquid phase-Mass Spectrometry, and mass spectrum is scanned using negative ion mode, using parallel anti-
PRM scanning mode should be detected, the chromatographic condition is as follows:
Chromatographic column: Waters Glycan BEH Amide, 1.7 μm, 2.1mm × 150mm；
The flow velocity of mobile phase is 0.2mL/min, and 45 DEG C of column temperature, detection time 15min, sample volume is 2 μ L；
Using gradient elution mode, elution parameters are shown in Table 2.
2 Parameters of gradient elution of table
The sucrose ion parent ion/daughter ion ion pair for detection is 387.08 > m/z341.08 of m/z, broken
Splitting energy is 30eV.
Sucrose parent ion total ion current figure and mass spectrum such as Fig. 1 of preparation 1, the sucrose of preparation 1 quantify daughter ion and extract ion
Flow graph and mass spectrum such as Fig. 2.
Sucrose parent ion total ion current figure and mass spectrum such as Fig. 3 of preparation 2, the sucrose of preparation 2 quantify daughter ion and extract ion
Flow graph and mass spectrum such as Fig. 4.
Daughter ion, which is quantified, according to sucrose extracts the canonical plotting that the integrated peak areas of ion flow graph is drawn, canonical plotting
As shown in Figure 3.
The measurement result of cane sugar content and the comparison result of theoretical value are as shown in table 3 in two kinds of formulation samples.
The measurement result of cane sugar content and the comparison result of theoretical value in 3. two kinds of formulation samples of table
It can be seen that this method accuracy is high from the testing result of preparation 1 and preparation 2, and the control of theoretical value deviation is 10%
Within.
From the point of view of the testing result of blank sample, inspection does not measure sucrose residual, therefore sample to be tested in this method in blank
In sucrose can ignore in the residual of chromatographic column.And sample room will not interact testing result.
The present invention has been used for the formulation samples detection of bio-pharmaceuticals technique formulation development process, the results showed that this method is accurate
Property can satisfy the detection needs of the sucrose auxiliary material content of formulation development technique.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. a kind of rapid detection method of cane sugar content, characterized in that it comprises the following steps:
Sample is diluted with diluent, obtains prepare liquid；
With sucrose standard product preparing standard solution, and draw standard curve；Used in the standard solution and sample dilution
Diluent is identical, and the diluent is the aqueous formic acid of 0.05%~3% volume ratio；
The cane sugar content in the prepare liquid and the standard solution is detected using ultrahigh pressure liquid phase-Mass Spectrometry；
Wherein, the chromatographic condition of ultrahigh pressure liquid phase are as follows:
Chromatographic column with hydrophilic function,
The flow velocity of mobile phase be 0.1~0.5mL/min, 30~60 DEG C of column temperature, using gradient elution mode；
Mobile phase is made of mobile phase A and Mobile phase B, the gradient elution be the volume ratio of mobile phase A and Mobile phase B in gradient
Variation；
The mobile phase A is the aqueous formic acid of 10~100mmol/L, and pH is 4~6；Mobile phase B is containing 0.05~0.2% first
The acetonitrile of acid.
2. rapid detection method according to claim 1, which is characterized in that the diluent is 0.05%~0.1%
Aqueous formic acid or 0.1%~3% aqueous formic acid, preferably 0.1%~1% aqueous formic acid.
3. rapid detection method according to claim 1, which is characterized in that in the standard solution, maximum concentration is most
50 times or more of low concentration, 100 times or more, preferably 700 times or more；
The concentration ranges of the standard solution preferred 0.2mmol/L~200mmol/L, preferably 0.2mmol/L~100mmol/L,
It is preferred that 0.2mmol/L~50mmol/L, preferably 0.2mmol/L~20mmol/L；
When preparing the standard solution, the concentration of mother liquor is preferably 0.1mol/L~1mol/L；
When to mother liquor dilution, 2 times are preferably diluted to 10e6Times.
4. rapid detection method according to claim 1, which is characterized in that the sample is drug or food.
5. rapid detection method according to claim 1 or 4, which is characterized in that also wrapped before to sample dilution
It includes:
The filtering with microporous membrane sample for first using 0.22 μm, takes supernatant after being then centrifuged for；The centrifugation preferably with 13000~
The centrifugation of 15000rpm revolving speed, the preferred 10min or more of centrifugation time；
When carrying out the dilution to sample, diluted multiple is preferably 10 times~200 times, more preferable 10 times~100 times.
6. rapid detection method according to claim 1, which is characterized in that gradient elution described in ultrahigh pressure liquid phase are as follows:
Using mobile phase A: Mobile phase B=20:80 first increases the accounting of the mobile phase A as starting point, then reduces the mobile phase A
Accounting mobile phase A when terminal: Mobile phase B=20:80；
During the gradient elution, the volume ratio peak of the mobile phase A is preferably 90%~95%；
The chromatographic column preferred amide bonded stationary phase hydrophilic chromatographic column, preferred amide are bonded organosilicon stationary phase hydrophilic chromatographic
Column；
The flow velocity of the mobile phase is preferably 0.2~0.5mL/min, sample volume preferably 1~2 μ L.
7. rapid detection method according to claim 6, which is characterized in that the gradient of mobile phase described in ultrahigh pressure liquid phase
Variation is as shown in the table:
8. rapid detection method according to claim 1, which is characterized in that the mobile phase A is 50~100mmol/L's
Aqueous formic acid, pH are 4~6；Mobile phase B is preferably the acetonitrile for containing 0.05~0.1% formic acid；
The pH of the mobile phase A is preferably 4.2~5, it is preferred to use ammonium hydroxide adjusts its pH value.
9. rapid detection method according to claim 1, which is characterized in that the Mass Spectrometry Conditions are as follows: scanning range m/z
60~400,25~35L/min of sheath gas, 250~300 DEG C of ion transfer tube temperature, ESI ion source, anion scans mould
Formula, resolution ratio are 17500~70000；
Preferably, the Mass Spectrometry Conditions are as follows: parallel reaction monitoring or repeatedly reaction monitoring scan pattern, the sucrose of detection from
Primary and secondary ion/daughter ion ion pair is 387.08 > m/z of m/z 341.08, and fragmentation energies are 20eV~50eV.
10. rapid detection method according to claim 1, which is characterized in that in ultrahigh pressure liquid phase: the retention time of sucrose
For 8.0~10.0min.
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