CN105929038A - Method for detecting citrinin in red yeast rice pigment powder - Google Patents
Method for detecting citrinin in red yeast rice pigment powder Download PDFInfo
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- CN105929038A CN105929038A CN201610234733.0A CN201610234733A CN105929038A CN 105929038 A CN105929038 A CN 105929038A CN 201610234733 A CN201610234733 A CN 201610234733A CN 105929038 A CN105929038 A CN 105929038A
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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
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The invention provides a method for detecting citrinin in red yeast rice pigment powder, and belongs to the technical field of chemical detection. The detection method has the advantages of short detection time and high sensitivity. The detection method comprises the following steps: (1) preparing a standard solution; (2) adding 0.5 gram of red yeast rice pigment powder into a 10 mL conical flask, adding 2 mL of formic acid-methanol solution (1%), carrying out ultrasonic extraction (350 W) for 10 minutes at a temperature of 50 DEG C, cooling, filtering, transferring the filtrate to a 25 mL volumetric flask, adding water to the scale, evenly shaking, filtering, making the filtrate go through a small C18 solid phase extraction column at a flowing speed of 1 mL/min, washing the small column by 5 mL of water, discarding, then washing the small column by 5 mL of ethanol, collecting the eluent, blowing the eluent by nitrogen gas at a temperature of 40 DEG C to dry the eluent, precisely adding 1 mL of ethanol to dissolve the obtained solids, filtering the solution by an organic filter membrane with a thickness of 0.22 [mu]m to obtain a sample to be detected, loading the sample on an Agilent 1295-6550C triple quadrupole HPLC-MS, and analyzing the sample.
Description
Technical field
The present invention provides the detection method of a kind of citrinin, the detection method of citrinin in a kind of monascorubin powder；Belong to technical field of chemical detection.
Background technology
Citrinin (citrinin) is isolated from penicillium Penicillium citrinum first in 1931.Its molecular formula is C13H14O5, molecular weight is 250.25.Citrinin has certain nephrotoxicity and liver toxicity, energy teratogenesis, initiation tumor and Mutation induction.And Monas cuspurpureus Went is Chinese tradition medical material, it is to form by distinctive monascus ruber cultivation and fermentation in rice.Having the statin substance of suppression Biosynthesis of cholesterol because some monascus sp bacteria strain can produce, thus Hongqu is developed to state's monascorubin powder and the health food of blood fat reducing.It is found that in monascus product containing after citrinin first at nineteen ninety-five France scholar Blanc etc., the most using citrinin as the strict Con trolling index of food pollution.And in China, Chinese Pharmacopoeia not yet using citrinin as the Con trolling index containing Monas cuspurpureus Went Chinese patent medicine.
Monascorubin powder is documented and measures its citrinin content by RP-HPLC method, and in food, health food, also have the detection method of citrinin: thin layer chromatography, fluorescence spectrophotometry, high performance liquid chromatography etc..
Summary of the invention
For above-mentioned deficiency, it is an object of the invention to provide a kind of highly sensitive, the method for the detection method citrinin of citrinin in the monascorubin powder that analysis time is short.
For solving above-mentioned technical problem, the technical scheme that the present invention provides is as follows:
The detection method of citrinin in a kind of monascorubin powder, described detection method comprises the steps: 1) prepare standard solution；2) monascorubin powder 0.5g, put in 10ml conical flask, add 2ml 1% formic acid-methanol solution, 350W, 50 DEG C of ultrasonic extraction 10min, cooling, filter, filtrate is transferred in 25ml volumetric flask, it is settled to scale with water, filter after shaking up, with 1ml/min flow velocity by C18 solid phase extraction column, use 5ml water wash, discard, eluent is collected again with 5ml ethanol elution, eluent nitrogen at 40 DEG C is blown near dry, the accurate 1ml ethanol that adds dissolves, with the 0.22 organic membrane filtration of μm, obtain testing sample, triple for Agilent1295-6550C on testing sample level Four bar liquid chromatography mass spectrometric combined instruments are detected.
Further, the detection method of citrinin in above-mentioned a kind of monascorubin powder, described standard solution of preparing comprises the steps:
1) preparation of standard solution
Standard reserving solution: precision weighs standard substance 5mg, puts in 5ml volumetric flask, adds ethanol and dissolves and to scale, and making into concentration is 1mg/ml；
Standard dilution: accurate draw standard solution 0.2ml, puts in the volumetric flask of 50ml, adds ethanol dilution to scale, and making into concentration is 2 μ g/ml1；
Standard working curve: take standard dilution liquid and add ethanol in right amount, be configured to respectively concentration be 0.01,2,5,5,20ng/ml standard working solution.
The detection method of citrinin in the most above-mentioned a kind of monascorubin powder, described liquid chromatograph and Mass Spectrometry Conditions chromatographic column: Agilent poroshell 120EC-C18,3.0 × 50mm 2.7micron, 1.8 μm；Mobile phase A is 0.2% phosphoric acid, and Mobile phase B is methanol, uses gradient elution program as follows: 0-3min, A, 75-5%, B, 25-95%；3-5.5min, A, 5%, B, 95%；5.5.-5.6min, A, 75%, B, 25%；Flow velocity 0.2ml/min；Column temperature 40 DEG C；Sample size 2 μ l；Electrospray ionization source ESI, positive ion mode, selects multiple-reaction monitoring MRM, carrier gas temperature: 150 DEG C, carrier gas flux: 15L/min, nebulizer pressure 40psi, sheath temperature 300 DEG C, sheath throughput: 12L/min, capillary voltage 3500V, qualitative, quantitative ion: parent ion m/z=251.2, daughter ion m/z233.2 and m/z 205.2, collision energy is respectively 5V and 20V.
Establish the citrinin liquid chromatography tandom mass spectrometry determination containing Monas cuspurpureus Went state monascorubin powder herein, this method completes the analysis to this material in 6min and measures, in the case of ensureing that qualitative, quantitative is accurately, achieve high-sensitive analysis, and during extracting, decrease the use of the toxic reagents such as toluene, there is the features such as efficient, environmental protection, can be that the citrinin monitoring in state's monascorubin powder provides technical support.
Accompanying drawing explanation
Fig. 1 is 2 μ g/ml citrinin standard solution multiple-reaction monitoring figures.
Fig. 2 is the multiple-reaction monitoring figure of blank sample.
Detailed description of the invention
Below in conjunction with detailed description of the invention; the claim of the present invention is described in further detail; but not constituting any limitation of the invention, the amendment of any limited number of time made at the claims in the present invention protection domain, still within the claims of the present invention.
Embodiment 1
1 material
1.1 instrument
Agilent1295-6550C triple level Four bar liquid chromatography mass spectrometric combined instrument (U.S. Agilent), HLB-SPE post (60mg/3ml, Shanghai ANPEL Scientific Instrument Co., Ltd.), AgilentZORBAX Eclipse Plus (2.1 × 50mm, 1.8 μm；U.S. Agilent), XA205DU electronic balance (Germany Mei Teletuo benefit company), ultrasonic cleaner.
1.2 state's monascorubin powder and reagent
4 batches, state's monascorubin powder, derives from market sale sample.
Citrinin (lot number: 518-75-2-150403, standard substance are purchased from Pribolab, purity ＞ 98%), ethanol and methanol are chromatographically pure, other reagent is analytical pure, and experimental water is the deionized water filtered through ELGA PURELAB ClassicUVF cleaning system.
The percentage concentration that the present invention relates to, is not particularly illustrated, and liquid is volumetric concentration, and solute is the finger mass concentration of solid.
2 methods and result
The preparation of 2.1 standard solution
2.1.1 standard reserving solution: precision weighs standard substance 5mg, puts in 5ml volumetric flask, adds ethanol and dissolves and to scale, and making into concentration is 1mg/ml.
2.1.2 standard dilution: accurate draw standard solution 0.2ml, puts in the volumetric flask of 50ml, adds ethanol dilution to scale, and making into concentration is 2 μ g/ml, and testing result sees Fig. 1.
2.1.3 standard working curve: take standard dilution liquid and add ethanol in right amount, be configured to respectively concentration be 0.01,2,5,5,20ng/ml standard working solution.
2.2 sample pre-treatments
Monascorubin powder 0.5g, puts in 10ml conical flask, adds 2ml 1% formic acid-methanol solution, 350W, 50 DEG C of ultrasonic extraction 10min, cooling, filters, and filtrate is transferred in 25ml volumetric flask, is settled to scale with water, filters after shaking up, stand-by.
2.3 sample purification
By above-mentioned sample pre-treatments liquid, with about 1ml/min flow velocity by C18 solid phase extraction column (activating with 3ml ethanol and 3ml water respectively before use), use 5ml water wash, discard, collecting eluent with 5ml ethanol elution again, eluent nitrogen at 40 DEG C is blown near dry, and the accurate 1ml ethanol that adds dissolves, with the 0.22 organic membrane filtration of μm, obtain testing sample.
2.4 liquid chromatograph and Mass Spectrometry Conditions
Chromatographic column: AgilentZORBAX Eclipse Plus post (2.1 × 50mm, 1.8 μm)；Mobile phase A is 0.2% phosphoric acid, and Mobile phase B is methanol, uses gradient elution program, is shown in Table 1.Flow velocity 0.2ml/min；Column temperature 40 DEG C；Sample size 2 μ l；Electrospray ionization source (ESI), positive ion mode, select multiple-reaction monitoring (MRM), carrier gas temperature: 150 DEG C, carrier gas flux: 15L/min, nebulizer pressure 40psi, sheath temperature 300 DEG C, sheath throughput: 12L/min, capillary voltage 3500V, qualitative, quantitative ion: parent ion m/z=251.2 (M+1), daughter ion m/z233.2 and m/z 205.2, collision energy is respectively 5V and 20V, Frag and is 380V.
Table 1 gradient elution program
Tab 1Gradient elution program
The mensuration of 2.5 actual samples
In this test, substitute into the regression equation calculation of standard series, 4 batches, result state monascorubin powder with quota ion peak area, all do not detect citrinin.
The accuracy of 2.4 methods
Carrying out recovery of standard addition test in not containing the blank sample of two kinds of substrate of citrinin after measured, spiked levels is 5ng/ml.Parallel assay 6 times, the average recovery of standard addition liquid preparation scope obtained is 95%～50%, sees Fig. 2.
Technical scheme use water (containing 0.2% phosphoric acid)+methanol that the present invention provides=75+25 makees flowing phase, flow velocity 0.3ml/min, the chromatographic peak of citrinin is symmetrical, tandem mass spectrum detector is used to detect, owing to tandem mass spectrum has in separation, high resolution and highly sensitive characteristic, multiple-reaction monitoring pattern (MRM) and parent ion and two corresponding daughter ions are coordinated to be monitored, reach the most qualitative and quantitative purpose, simplify the step that flowing is selected mutually, so this method has selected above-mentioned flowing phase and ratio thereof.
nullAfter using C18 solid phase extraction column that the citrinin in sample is purified for understanding this method，The matrix interference situation to Mass Spectrometer Method when measuring，Select a sample not detecting citrinin to carry out extraction by method and make matrix liquid，And add standard series，External standard standard series is carried out mass spectroscopy simultaneously，Regression analysis is carried out with its concentration according to the quota ion peak area that retention time is corresponding，Matrix liquid to add the regression equation of standard series be Y=3 298.522 431,X+4 428.423 341，R=0.9996，Compare with external standard regression equation Y=3 329.186 799,X+4 486.441 631 (r=0.9996)，Both are without substantial differences，Show that C18 solid phase extraction column has obvious purification to sample substrate.
Establish the citrinin liquid chromatography tandom mass spectrometry determination containing Monas cuspurpureus Went state monascorubin powder herein, this method completes the analysis to this material in 5min and measures, in the case of ensureing that qualitative, quantitative is accurately, achieve high-sensitive analysis, and during extracting, decrease the use of the toxic reagents such as toluene, there is the features such as efficient, environmental protection, can be that the citrinin monitoring in state's monascorubin powder provides technical support.
Claims (3)
1. the detection method of citrinin in a monascorubin powder, it is characterised in that described
Detection method comprises the steps: 1) prepare standard solution；2) monascorubin powder 0.5g,
Put in 10ml conical flask, add 2ml 1% formic acid-methanol solution, 350W, 50 DEG C of ultrasound wave
Extracting 10min, cooling, filter, filtrate is transferred in 25ml volumetric flask, is settled to water
Scale, filters after shaking up, and with 1ml/min flow velocity by C18 solid phase extraction column, uses
5ml water wash, discards, then collects eluent with 5ml ethanol elution, and eluent is in 40 DEG C
Lower nitrogen is blown near dry, and the accurate 1ml ethanol that adds dissolves, by 0.22 μm organic filter membrane mistake
Filter, obtains testing sample, by triple for Agilent1295-6550C on testing sample level Four bar liquid
Phase GC-MS detects.
The detection side of citrinin in a kind of monascorubin powder the most according to claim 1
Method, it is characterised in that described standard solution of preparing comprises the steps:
1) preparation of standard solution
Standard reserving solution: precision weighs standard substance 5mg, puts in 5ml volumetric flask, adds
Ethanol dissolves and to scale, and making into concentration is 1mg/ml；
Standard dilution: accurate absorption standard solution 0.2ml, puts in the volumetric flask of 50ml,
Adding ethanol dilution to scale, making into concentration is 2 μ g/ml1；
Standard working curve: take standard dilution liquid and add ethanol in right amount, be configured to concentration respectively
Be 0.01,2,5,5,20ng/ml standard working solution.
The detection side of citrinin in a kind of monascorubin powder the most according to claim 1
Method, it is characterised in that described liquid chromatograph and Mass Spectrometry Conditions chromatographic column: Agilent
Poroshell 120EC-C18,3.0 × 50mm 2.7micron, 1.8 μm；Mobile phase A is
0.2% phosphoric acid, Mobile phase B is methanol, uses gradient elution program as follows: 0-3min,
A, 75-5%, B, 25-95%；3-5.5min, A, 5%, B, 95%；5.5.-
5.6min, A, 75%, B, 25%；Flow velocity 0.2ml/min；Column temperature 40 DEG C；Sample size 2
μl；Electrospray ionization source ESI, positive ion mode, select multiple-reaction monitoring MRM, carrier gas temperature
Degree: 150 DEG C, carrier gas flux: 15L/min, nebulizer pressure 40psi, sheath temperature
300 DEG C, sheath throughput: 12L/min, capillary voltage 3500V, qualitative, quantitative ion:
Parent ion m/z=251.2, daughter ion m/z233.2 and m/z 205.2, collision energy divides
Wei 5V and 20V.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1808117A (en) * | 2006-01-05 | 2006-07-26 | 北京联合大学应用文理学院保健食品功能检测中心 | Method for detecting citrinin content in red koji fermentation product |
| CN102175788A (en) * | 2010-12-27 | 2011-09-07 | 晨光生物科技集团股份有限公司 | Method for detecting contents of citrinin in monascus by liquid chromatograph/mass spectrometer (LC/MS) |
-
2016
- 2016-04-15 CN CN201610234733.0A patent/CN105929038A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1808117A (en) * | 2006-01-05 | 2006-07-26 | 北京联合大学应用文理学院保健食品功能检测中心 | Method for detecting citrinin content in red koji fermentation product |
| CN102175788A (en) * | 2010-12-27 | 2011-09-07 | 晨光生物科技集团股份有限公司 | Method for detecting contents of citrinin in monascus by liquid chromatograph/mass spectrometer (LC/MS) |
Non-Patent Citations (3)
| Title |
|---|
| ZHENG HAN ET AL: "Multianalysis of 35 Mycotoxins in Traditional Chinese Medicines by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Coupled with Accelerated Solvent Extraction", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| 吉小凤 等: "液相色谱—串联质谱法(LC-MS/MS)测定固态红曲米(粉)中橘青霉素", 《浙江农业学报》 * |
| 李燕 等: "高效液相色谱－串联质谱法测定中药中桔青霉素", 《药物分析杂志》 * |
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