CN106770769B - A kind of method of a variety of liposoluble vitamins in detection feed - Google Patents

A kind of method of a variety of liposoluble vitamins in detection feed Download PDF

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CN106770769B
CN106770769B CN201611219813.5A CN201611219813A CN106770769B CN 106770769 B CN106770769 B CN 106770769B CN 201611219813 A CN201611219813 A CN 201611219813A CN 106770769 B CN106770769 B CN 106770769B
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solution
vitamin
mobile phase
variety
feed
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CN106770769A (en
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朱正鹏
梁玉树
何开蓉
易锡斌
张遨然
汪春霞
王虎
周丽
黄李蓉
梁秋菊
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Sichuan New Hope Animal Husbandry Technology Co Ltd
New Hope Liuhe Co Ltd
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Sichuan New Hope Animal Husbandry Technology Co Ltd
New Hope Liuhe Co Ltd
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/04Preparation or injection of sample to be analysed
    • G01N30/06Preparation
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/04Preparation or injection of sample to be analysed
    • G01N30/06Preparation
    • G01N2030/062Preparation extracting sample from raw material

Abstract

The invention discloses a kind of methods of a variety of liposoluble vitamins in detection feed, comprising the following steps: (1) according to detection needs, multivitamin standard solution is prepared, including at least two or more in following vitamin: vitamine A acetate, vitamin D3, vitamin e acetate;Using HPLC-MS/MS examination criteria solution, calibration curve equation is obtained;(2) sample is weighed, into brown volumetric flask, is added and extracts solution ultrasonic extraction 10-30min, cooling constant volume, centrifugation, with 0.22 μm of organic phase filter membrane, machine in filtering carries out analysis test using chromatographic parameter identical with step 1 and the MS detection parameters;(3) the multivitamin content in sample is calculated according to the testing result of step 1 and step 2.The method that a variety of liposoluble vitamins in mixed feed are detected while of the invention, completes the detection and analysis of a variety of different vitamins in one-time detection analytic process.

Description

A kind of method of a variety of liposoluble vitamins in detection feed
Technical field
The present invention relates to a kind of chromatography determination method, in particular to a kind of chromatographic detection method for detecting vitamin, There is good detection and analysis efficiency for liposoluble vitamin, belong to chemical composition analysis technical field.
Background technique
Liposoluble vitamin includes VA, VD, VE, VK, not soluble in water since its chemical structure contains hydrophobic hydrocarbon chain more, It is soluble in organic solvent.Due to deliquescent difference, detection method is also different with water soluble vitamin, especially sample Types of agents used in pre-treatment, should with organic solvent based on.
It is relatively complete about the detection method of water soluble vitamin at present, as titration, colorimetric method, spectrophotometry and High performance liquid chromatography and reversed-phased high performace liquid chromatographic for being mentioned above etc..And for liposoluble vitamin, although In the presence of the method for detection single component, but the method detected simultaneously is relatively fewer.
It establishes high performance liquid chromatography within Wang Haifeng etc. 2006 while detecting vitamin A palm fibre in composite vitamin for injection Glycerin monostearate, vitamin D2, vitamin E, vitamin K1 content, the average recovery rate of each vitamin is respectively 99.7%, 98.4%, 100.8% and 98.5%, method is easy to operate, as a result accurately.
It establishes within Liu Honghe etc. 2005 High performance liquid chromatography-diode-arvay detection (HPLC-DAD) while measuring The content of food vitamins A, vitamin D and vitamin E (alpha-tocopherol).Ethyl alcohol, hydrogen-oxygen is added in food samples by this method Change potassium and pyrogallic acid, 80 DEG C of water-baths saponification 30min, after petroleum ether extracts, nitrogen purging concentration causes to use mobile phase after doing Dissolution, with Supelco C18 column (15cm × 4.6mm, 5um) chromatographic column, using methanol as mobile phase, in 325nm (VA), 265nm (VD), it is detected under 294nm (VE) Detection wavelength.
Detect vitamin A, vitamine D3 and Wei Sheng in Fish tissue simultaneously using single injected sampling within Tan Qingsong etc. 2007 The method of plain E.The liposoluble vitamin in Fish tissue is extracted with petroleum ether, with 5 μ Cl8 90A color of VARIAN Res Elut Column is composed, mobile phase is methanol-water, is able to carry out good separation to 3 kinds of liposoluble vitamins in extract under this condition.Knot Fruit shows that under 292nm wavelength, vitamin A, vitamine D3 and vitamin E linear relationship are good.Fish body group is extracted with petroleum ether Liposoluble vitamin in knitting can simultaneously give birth to dimension in Fish tissue at wavelength 292nm using rp-hplc Plain A, vitamine D3 and vitamin E are preferably separated and are measured.
It establishes within Song Jinchun etc. 2007 to tie up life in 12 kinds of multi-vitamins of hplc simultaneous determination injection The method of plain D2, vitamin A and content of vitamin E.Method is Inertsil (ODS-2) C18 with chromatographic column, and mobile phase is second Nitrile-methanol dichloromethane (80:10:10), the results showed that calciferol, Vitwas E and Retinol Palmitate are examined The range of linearity for surveying concentration is respectively 0.61~1.43 μ g/mL, 222.6-519.4 μ g/mL and 1.21-2.82mg/mL;It is average to return Yield is respectively 99.79%, 99.53%, 101.63%.
The more valve multicolumn technologies of double ternary high performance liquid chromatography are used within Lu Yan etc. 2012, it is raw to water soluble vitamin in same sample The sample that element and liposoluble vitamin distinct methods are handled carries out double sample introductions, can analyze 21 kinds of vitamin in sample simultaneously. The method detection limit of each vitamin can be used for the vitamin of various samples up to 0.002~0.223 μ g.mL-1 range, this method Detection and analysis work.
There is method for measuring while a variety of liposoluble vitamins: Kareti Srinivasa Rao etc. uses efficient liquid phase Chromatography determination determines the content of vitamin A and vitamin E in tablet simultaneously.Wang Haifeng etc. establishes high performance liquid chromatography Retinol Palmitate in composite vitamin for injection, vitamin D are detected simultaneously2, vitamin E, vitamin K1 content side Method.Liu Honghe etc., which establishes High performance liquid chromatography-diode-arvay detection (HPLC-DAD) while measuring, ties up life in food The content of plain A, vitamin D and vitamin E.Ethyl alcohol, potassium hydroxide and pyrogallic acid, water is added in food samples by this method Bath soap, after petroleum ether extracts, nitrogen purging concentration cause it is dry after with flowing phased soln, with Supelco C18 chromatographic column, with methanol For mobile phase, detected under different Detection wavelengths.
Tan Qingsong etc. has detected vitamin A, vitamin D in Fish tissue using single injected sampling simultaneously3And vitamin E. This method extracts the liposoluble vitamin in Fish tissue with petroleum ether, with 5 μ Cl8 90A chromatographic column of VARIAN Res Elut, Methanol-water is mobile phase, under 292nm wavelength, vitamin A, vitamin D3It is good with vitamin E linear relationship.It is above-mentioned a variety of The document measured while liposoluble vitamin is shown, passes through the optimum organization of chromatographic column, mobile phase and detector, part liposoluble Property vitamin while detection be feasible.
With the continuous development of animal husbandry, the gradually scale of livestock and poultry cultivation, in order to meet growing livestock and poultry consumption Demand, the nutrition of livestock and poultry and health more and more attention has been paid to.A kind of object of the vitamin as important maintenance animal body health Matter is indispensable in animal and fowl fodder.The amount of various vitamins in feed must reach the requirement of animal body, ability Guarantee the healthy growth and production product of animal.
Mixed feed/raw material that animal husbandry uses has the characteristics that ingredient compounding, prescription are complicated more, and includes basic former The vitamin ingredients being scattered in contained in material in raw material, and include the mixed vitamin ingredient of artificial actively addition.Add Mixed feed in interaction between various composition influence so that the liposoluble vitamin ingredient in mixed feed is accurate Detection and analysis become very difficult.It is existing to be directed to a variety of liposoluble vitamin detection methods contained in food, meat It is difficult to the situation suitable for this kind of complexity, thus has to carry out mixed feed using the method for detection one by one, independent analysis It tests and analyzes, leads to the very complicated complexity of detection and analysis work of mixed feed, time-consuming, and working efficiency is lower, is unfavorable for raising Expect the quality control of manufacturing enterprise and feed applications enterprise for feed.So same about a variety of liposoluble vitamins at present When detection method require study, accurately, easily measure a variety of liposoluble vitamin content methods in feed and urgently study.
Summary of the invention
It is an object of the invention to overcome a variety of rouge for lacking be directed in mixed feed under complex situations in the prior art The method that soluble vitamin tests and analyzes simultaneously, so that testing and analyzing mixed feed, time-consuming, the deficiency of low efficiency, provides one The method of a variety of liposoluble vitamins in kind detection feed.The method of the present invention is using high performance liquid chromatography tandem mass spectrum/mass spectrographic Determination method (HPLC-MS/MS), in conjunction with the composition characteristic of mixed feed and the physicochemical property of liposoluble vitamin, if The process for counting corresponding pretreatment of raw material and sample detection analysis realizes disposable detection and completes a variety of different vitamins Analysis effect.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
A kind of method of a variety of liposoluble vitamins in detection feed, comprising the following steps:
(1) according to detection needs, prepare multivitamin standard solution, including at least two in following vitamin with It is upper: vitamine A acetate, vitamin D3, vitamin e acetate.
Using HPLC-MS/MS examination criteria solution, calibration curve equation is obtained.
Chromatographic condition is as follows:
Chromatographic column: C18 column;
Mobile phase includes mobile phase A: methanol aqueous solution phase and Mobile phase B: aqueous formic acid phase.
Wherein mobile phase A: the volume ratio of methanol is greater than 95% in methanol aqueous solution, Mobile phase B: first in aqueous formic acid The content of acid is lower than 1wt%.
Using gradient elution, gradient condition: 0~2min, mobile phase A 50~100%;2~9min, mobile phase A 100%;9~9.5min, mobile phase A 100~50%.
(2) sample is weighed, 5-10g sample is preferably weighed, into brown volumetric flask, it is preferable to use 100 milliliters of brown capacity Bottle is added and extracts solution ultrasonic extraction 10-30min, preferably 20min, cooling constant volume, centrifugation, with 0.22 μm of organic phase filter membrane, Machine in filtering carries out analysis survey using chromatographic parameter (including gradient elution program) identical with step 1 and the MS detection parameters Examination.
The extraction solution is by the first component of extraction solution and to extract the second component of solution according to 25-35:65-75 body Solution made of product ratio mixed preparing.
The first component of the extraction solution is 0.03-0.07% ammonia spirit (weight ratio, wt%).
The second component of the extraction solution is 0.1-0.3%BHT methanol solution (m/vol, g/L).
(3) the multivitamin content in sample is calculated according to the testing result of step 1 and step 2.
Detection method of the invention devises ammonia spirit and solution is extracted in the mixing of methanol solution, has specific aim and choosing It selects, effectively multivitamin ingredient in mixed feed complicated ingredient sufficiently can be extracted, then pass through the side of gradient elution Formula disposably tests and analyzes a variety of rouge required for reaching so that several different liposoluble vitamins quickly separate The purpose of soluble vitamin.It overcomes and in the prior art a variety of different liposoluble vitamins is difficult to analyze in one-time detection The middle deficiency for completing high efficiency, high-accuracy detection, reduces and tests and analyzes time-consuming, avoid present in existing national standard detection method The defect of intricate operation.
Further, prepared by standard solution: first dissolving vitamin with methanol, is configured to the standard substance that mass concentration is 1g/L Stock solution, then diluted composition standard working solution.0.2wt%2,6- di-t-butyl -4- first is added in Standard Stock solutions Base phenol.Preferably, what is applied in dilution is to extract solution to be diluted.Solution and sample to be tested after dilution The closest accuracy for being conducive to improve testing result of the property of product.
Further, the first component of the extraction solution is: 0.05% ammonia spirit;The second component of the extraction solution is: 0.2% (m/vol, g/L) BHT methanol.Preferably, extract the first component of solution is with the volume ratio for extracting the second component of solution 30:70.
Further, during using gradient elution, there are two types of mobile phases, is respectively: mobile phase A: ammonia containing 0.01-0.03% The 96-99% methanol aqueous solution of water, Mobile phase B: 0.01-0.03% aqueous formic acid.
Further, gradient elution program are as follows: mobile phase A: 98% methanol aqueous solution containing 0.02% ammonium hydroxide, Mobile phase B: 0.02% aqueous formic acid, flow velocity 0.3mL/min.
Further, in chromatographic condition, chromatographic column is one of the following: 3 μm of 2.0mm I.D. of C18-MG II (pH 2-10) × 3 μm of 150mm column, 3 μm of 2.0mm I.D. × 150mm columns of C18-MG III (pH 2-10), ADME (pH 2-10) 2.1mm I.D. × 150mm column, Agilent ZORBAX Eclipse 2.1 × 50mm of XDB-C18 column, Waters ACQUITY UPLC HSS 1.8 μm of 3.0 × 50mm columns of T3, Waters ACQUITY UPLC 1.7 μm of 2.1 × 50mm columns of BEH.Preferred column is Capcell PAK ADME 2.1mm I.D × 150mm, 3 μm (i.e. 3 μm of 2.1mmI.D. × 150mm columns of ADME (pH 2-10), It is 2-10 using pH range).
Further, Mass Spectrometry Conditions are ESI cation scan pattern: multiple-reaction monitoring (MRM) parameter: dry temperature degree 325 DEG C, dry gas stream speed 11L/min, atomization gas pressure 45psi, 300 DEG C, flow velocity 9L/min of sheath temperature degree, capillary voltage 4000V, spray nozzle voltage 500V.
Further, during mass spectrograph tests and analyzes, multivitamin detection parameters condition such as table under MRM monitoring pattern Shown in 2.
Compared with prior art, beneficial effects of the present invention:
1. the method for a variety of liposoluble vitamins while the present invention in detection mixed feed, according to mixed feed sample The vitamin source situation of middle complexity and the physicochemical property of liposoluble vitamin devise corresponding extraction extracting process and HPLC- The chromatographic condition and Mass Spectrometry Conditions of MS/MS completes the detection and analysis of a variety of different vitamins in one-time detection analytic process.
2. the extraction solution used in detection method of the invention is high-efficient for the selective extraction of liposoluble vitamin, It can be improved the recovery rate of the liposoluble vitamin in test sample, so that result is more accurate.
3. mobile phase used in HPLC chromatogram is for a variety of different liposoluble vitamins in detection method of the invention Good separating effect, it is more accurate reliable that the sample after chromatographic isolation analyzes result in a mass spectrometer, can be realized more high detection Analysis precision, the either qualitative test of liposoluble vitamin or quantitative analysis all have significant progress promotion.
Detailed description of the invention:
Fig. 1 is the ion flow graph of VA full scan.
Fig. 2 is the ion flow graph of VE full scan.
Fig. 3 is VD3The ion flow graph of full scan.
Fig. 4 is the MRM map for detecting three kinds of liposoluble vitamins simultaneously.
Specific embodiment
Below with reference to test example and specific embodiment, the present invention is described in further detail.But this should not be understood It is all that this is belonged to based on the technology that the content of present invention is realized for the scope of the above subject matter of the present invention is limited to the following embodiments The range of invention.
1 instrument and reagent
(1) Agilent 1260-6460 liquid chromatogram-triple quadrupole bar tandem mass spectrum combined instrument matches electric spray ion source ESI (Agilent company of the U.S.);
(2) AB-135 type electronic analytical balance (Mei Teletuo benefit Shanghai Co., Ltd).
(3) LD5-2B centrifuge (system in Beijing Jing founds centrifuge Co., Ltd).
(4) KQ-500DE type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).
(5) swirl mixing device MS3 (German IKA).
(6) second eyeball, methanol (chromatographically pure, Merck & Co., Inc..
(7) formic acid, ammonium acetate, ammonium hydroxide (analyzing pure, Tianjin Ke Miou chemical reagent Co., Ltd).
(8) sodium hydroxide, sodium chloride (analyzing pure, Shanghai Chinese medicines group).
(9) vitamine A acetate (C22H32O2, molecular weight: 328.5).
(10) vitamin e acetate (C31H52O3, molecular weight: 472.74).
(11) vitamine D3 (C27H44O, molecular weight: 384.63).
2 extract the preparation of solution and three kinds of liposoluble vitamin standard solution
2.1 extract the preparation of solution
Extract solution: 0.2% (m/V) BHT+0.05% ammonium hydroxide methanol solution is as sample extracting solution.
The preparation of 2.2 liposoluble vitamin standard solution
Standard substance stock solution: the vitamine A acetate, vitamin e acetate, vitamin D of 100mg are accurately weighed3 Standard items, being configured to mass concentration with methanol (being added 0.2%2,6- di-tert-butyl-4-methy phenol (BHT)) is 1000.0mg/ The standard substance stock solution of L.
Standard substance work intermediate fluid: accurately pipetting suitable standard substance stock solution respectively, is prepared with above-mentioned 2.1Extract solution, it is configured to vitamine A acetate, vitamine D3 mass concentration is 100mg/L, vitamin e acetate mass concentration For the standard work intermediate fluid of 2mg/L.
Standard substance working solution: each standard substance work intermediate fluid of 1mL is drawn respectively, is prepared with 2.1Extract solution, match The hybrid standard for being 20 μ g/L with vitamine A acetate, vitamin D concentrations 1mg/L, vitamin e acetate concentration at concentration Substance working solution.
The preparation of standard series: hybrid standard substance working solution 2,4,10,20mL is taken to be respectively placed in 100mL brown volumetric flask In, it is prepared with 2.1Extract solution, it is diluted in terms of vitamin e acetate, the standard of 0.4,0.8,2,4,20 μ g/L of concentration Series.
3 sample treatments
5-10g sample is weighed into 100 milliliters of brown volumetric flasks, is added and extracts solution ultrasonic extraction 20min, it is cooling fixed Hold, centrifugation, with 0.22 μm of organic phase filter membrane, machine in filtering.
4 chromatographies and Mass Spectrometry Conditions
4.1 chromatographic condition
Chromatographic column: Capcell PAK ADME (2.1mm I.D × 150mm, 3 μm);Mobile phase A: containing 0.02% ammonium hydroxide 98% methanol aqueous solution, Mobile phase B: 0.02% aqueous formic acid, flow velocity 0.3mL/min are run 14min, are washed using gradient De- program, gradient condition is as shown in table 1.
1 mobile phase gradient of table
4.2 Mass Spectrometry Conditions
ESI cation scan pattern: multiple-reaction monitoring (MRM) parameter: dry 325 DEG C of temperature degree, dry gas stream speed 11L/ Min, atomization gas pressure 45psi, 300 DEG C, flow velocity 9L/min, capillary voltage 4000V of sheath temperature degree, spray nozzle voltage 500V, His mass spectrum optimal conditions are as shown in table 2.
The instrument optimal conditions of the lower three kinds of liposoluble vitamins of 2 MRM monitoring pattern of table
5 results and analysis
Influence of 5.1 different solvents to 3 kinds of liposoluble vitamin testing results
It is molten to compare methanol, 0.2% (m/Vol, g/L) alkali protease methanol solution, 0.2% (m/V, g/L) BHT methanol Liquid, 0.2% several extractants of (m/V, g/L) BHT+0.05% ammonium hydroxide methanol solution are respectively to the effect of sample extraction.Upper machine examination Survey can be separated well, but there were significant differences for reproducibility in the daytime, wherein 0.2% (m/V, g/L) BHT+0.05% ammonium hydroxide Methanol (V/V) solution favorable reproducibility.However, when 0.2% (m/V, g/L) BHT+0.05% ammonium hydroxide methanol solution is as mobile phase, Its each component response is optimal, therefore selects to be mentioned with 0.2% (m/V, g/L) BHT+0.05% ammonium hydroxide methanol solution as sample Take liquid.
After selected final sample extracts solution, the method summary of sample pretreatment is as follows: weighing 5~10g of Feed Sample In 100ml volumetric flask, 0.2% (m/V, g/L) BHT+0.05% ammonium hydroxide methanol solution is added, ultrasonic extraction 20min is cooling fixed Hold, centrifugation, with 0.22 μm of organic phase filter membrane, machine in filtering.
Influence of the selection of 5.2 different chromatographic columns and mobile phase to three kinds of liposoluble vitamin testing results
It uses respectively:
3 μm of 2.0mm I.D. × 150mm columns of C18-MG II (pH 2-10),
3 μm of 2.0mm I.D. × 150mm columns of C18-MG III (pH 2-10),
3 μm of 2.1mm I.D. × 150mm columns of ADME (pH 2-10),
Agilent ZORBAX Eclipse 2.1 × 50mm of XDB-C18 column,
1.8 μm of 3.0 × 50mm columns of Waters ACQUITY UPLC HSS T3,
Waters ACQUITY UPLC 1.7 μm of 2.1 × 50mm columns of BEH,
Compare its separating effect, obtains 3 μm of 2.1mm I.D. × 150mm columns of ADME (pH 2-10) to liposoluble vitamin It is effectively maintained.In experimentation, it has been found that: when the general C18 chromatographic column of selection carries out a variety of liposoluble vitamin inspections When surveying analysis, a variety of liposoluble vitamin separating effect performances are more general, and the result precision of detection and analysis cannot reach To optimal desired effect.It is final preferred, when discovery is using ADME (pH 2-10) 3 μm of 2.1mmI.D. × 150mm columns, The accuracy of chromatography effect and detection and analysis is best, and 3 μm of 2.1mmI.D. × 150mm columns of ADME (pH 2-10) can be with As optimal chromatographic condition, it is the chromatography column condition that the present invention most preferably applies, is all made of the chromatography in following experimentation Column system carries out separation detection.
Influence of the selection of 5.3 different Mass Spectrometry Conditions to 3 kinds of liposoluble vitamin testing results
Test uses the certain density 0.2% prepared single rouge of (m/V) BHT+0.05% ammonium hydroxide methanol solution Soluble vitamin standard working solution carries out parent ion MS2 SCAN to it respectively in the positive-ion mode, finds each object [M+H]+molecular ion peak intensity is all high, and the temperature of ion source has no significant change at 260 DEG C -350 DEG C, therefore selects instrument setting temperature 325 DEG C are spent as ion source temperature.Then it is MS2 SIM optimization Fragmentor, is then Product lon, and optimize Collision Energy obtains qualitative ion and quota ion with feature, obtain ion stream map such as Fig. 1-Fig. 3 (according to Secondary is VA, VE and VD3The ion flow graph of full scan), further to dry gas stream speed, atomization gas pressure, sheath temperature degree, sheath gas Flow velocity, capillary voltage, spray nozzle voltage optimize.So described in final Mass Spectrometry Conditions parameter as above 4.2: dry temperature 325 DEG C, dry gas stream speed 11L/min, atomization gas pressure 45psi of degree, 300 DEG C, flow velocity 9L/min of sheath temperature degree, capillary voltage 4000V, spray nozzle voltage 500V.
5.4 ranges of linearity, quantitative limit, recovery of standard addition and relative standard deviation
(1) range of linearity
Prepare with the hybrid standard series of working liquids of sample to be tested same matrix, so that its concentration is distinguished 0.4,0.8,2,4, 20 μ g/L (are counted) by VE ((in terms of vitamin e acetate)), do standard song to mass concentration with selected quota ion peak area Line, the linear equation and related coefficient of 3 kinds of liposoluble vitamins are as shown in table 3.The results show that existing in 3 kinds of liposoluble vitamins 0.4~20 μ g/L (counts) linearly dependent coefficient (R in range by VE (in terms of vitamin e acetate)2) be 0.9995~ 0.9999, show that linear dependence is good.
3 kinds of liposoluble vitamin standard curves of table and related coefficient
(2) detection limit and quantitative limit
Upper machine after being handled using the method for adding target compound in blank extracting solution by 1.2 sample extractions and purification Analysis, in terms of vitamin e acetate, concentration is the SNR that 0.4 μ g/L is obtained.Detection limit is determined with 3 times of signal-to-noise ratio, is believed with 10 times It makes an uproar than determining lower limit of quantitation, it is as shown in table 4 by detection limit and quantitative limit that 6 kinds of water soluble vitamins are calculated.
4 detection limit of table and quantitative limit
(3) recovery of standard addition and relative deviation
The targeted vitamins standard items of various concentration are separately added into solvent blank, according to sample pre-treatments side in 1.2 Method is handled, its concentration of upper machine testing, calculates recovery of standard addition, scalar quantity, the rate of recovery and relative deviation (n=as shown in table 5 6)。
5 recovery of standard addition of table and relative deviation
The measurement of 5.6 actual samples
Using the above method to fish material, broiler chicken material, baby pig feedstuff, laying hen material, sucking pig material and 3 kinds of liposoluble for planting pig feed sample kind Property vitamin detected, while detect three kinds of liposoluble vitamins map as shown in figure 4, simultaneously detect three kinds of fat-soluble dimensions The MRM map of raw element.
Vitamin D can be detected in several Feed Samples3, vitamin e acetate, retinyl acetate, wherein VD3's Content is that 3731~18939 μ g/kg, VE (in terms of vitamin e acetate) contents are 18576~38674 μ g/kg, vitamin A second The content of acid esters is 12579~105648 μ g/kg.Prove that this method is practical.This method can satisfy mixed feed simultaneously Detect the demand of 3 kinds of liposoluble vitamins.
The result for specifically detecting a variety of different samples is as shown in table 6 below:
6 the method for the present invention of table detects the result of a variety of different samples
6 comparison national standard detection methods
Using the 3 kinds of liposoluble vitamin methods established in this test, in sample pre-treatments, it is only necessary to add 0.2% (m/V) BHT+0.05% ammonium hydroxide methanol solution can reach 3 kinds of liposoluble vitamins while the requirement extracted.It is returned by mark-on The verifying of acceptance test, the extracting method is not only simple and easy, but also can reach the required precision of detection.National Standard Method[1-3]In, VA、VD3With the detection of VE, need that anhydrous ether can be used by extracting again after saponification;But also need rotary evaporation It is concentrated, process is cumbersome, is more toxic, and is also causing environmental pressure, there are certain risks.
In addition, this test detects vitamin using the method for mass spectrometry, by qualitative/quantitative ion pair come really Recognize vitamin object, compared with National Standard Method, the attainable detection limit of method institute and quantitative limit of this test are lower, such as 7 institute of table Show, more conducively the detection of the vitamin sample of low content.
Compared with 7 the method for the present invention of table is limited to quantitative limit with the detection of National Standard Method
Unit: μ g/kg
In conclusion in contrast with National Standard Method, the method for the present invention substantially reduces time and the difficulty of sample pre-treatments, It is convenient to provide for reagent detection work, greatly improves working efficiency.Meanwhile extraction reduces poisonous and harmful reagent (methanol) It uses, plays a very good protection to personnel.
Conclusion: this test establish HPLC-MSMS and meanwhile detect fish material, broiler chicken material, baby pig feedstuff, laying hen material, sucking pig material and The analysis method of 3 kinds of liposoluble vitamins in the mixed feeds such as kind pig feed, and adding for 3 kinds of liposoluble vitamins is detected with this method Marking the rate of recovery is 89.7%~98.8%, and relative standard deviation (n=6) is 4.5%~9.8%, the inspection of each liposoluble vitamin Rising limit is 0.12~25.80 μ g/kg, and lower limit of quantitation is 0.38~86.20 μ g/kg.This method is quick, easy to operate, energy-saving ring Guarantor, result are accurate, meet the requirement of relevant laws and regulations, meet routine testing needs.
Remarks: herein various vitamin contents based on mass fraction, if you need to be converted to international unit (IU), conversion coefficient It is as follows:
1 international unit VA (IU)=0.344 μ g retinyl acetate.
1 international unit VE (IU)=1mg vitamin e acetate.
1 international unit VD3 (IU)=0.025 μ g but Vitamin D3.
BHT:2,6- di-tert-butyl-4-methy phenol.
National standard [1-3] refers to:
The measurement high performance liquid chromatography GB/T17817-2010 of vitamin A in feed;
Vitamin D in feed3Measurement high performance liquid chromatography GB/T17818-2010;
The measurement high performance liquid chromatography GB/T17812-2008 of vitamin E in feed.

Claims (8)

1. a kind of method of a variety of liposoluble vitamins in detection feed, comprising the following steps:
(1) multivitamin standard solution is prepared, including at least two or more in following vitamin: vitamine A acetate, dimension Raw element D3, vitamin e acetate;
Using HPLC-MS/MS examination criteria solution, calibration curve equation is obtained;
Chromatographic condition is as follows:
Chromatographic column: C18 column;The C18 column is: 3 μm of 2.1 mm I.D. × 150mm column of ADME pH 2-10;
Mobile phase includes mobile phase A: methanol aqueous solution phase and Mobile phase B: aqueous formic acid phase;
Wherein mobile phase A: the volume ratio of methanol is greater than 95% in methanol aqueous solution, Mobile phase B: formic acid in aqueous formic acid Content is lower than 1wt%;
Using gradient elution, gradient condition: 0 ~ 2min, mobile phase A 50 ~ 100%;2 ~ 9min, mobile phase A 100%;9~ 9.5min, mobile phase A 100 ~ 50%;
Mass Spectrometry Conditions are ESI cation scan pattern: multiple-reaction monitoring parameter: dry 325 DEG C of temperature degree, dry gas stream speed 11L/min, atomization gas pressure 45psi, 300 DEG C, flow velocity 9L/min, capillary voltage 4000V of sheath temperature degree, spray nozzle voltage 500V;
(2) sample is weighed, into brown volumetric flask, is added and extracts solution ultrasonic extraction 10-30min, cooling constant volume is centrifuged, uses 0.22 μm of organic phase filter membrane, machine in filtering, carries out analysis survey using chromatographic parameter identical with step (1) and the MS detection parameters Examination;
The extraction solution is by the first component of extraction solution and to extract the second component of solution according to 25-35:65-75 volume ratio Solution made of example mixed preparing;
The first component of the extraction solution is 0.03-0.07% ammonia spirit;
The second component of the extraction solution is 0.1-0.3% BHT methanol solution;
(3) the multivitamin content in sample is calculated according to the testing result of step (1) and step (2).
2. detecting the method for a variety of liposoluble vitamins in feed as described in claim 1, which is characterized in that standard solution system It is standby: first to dissolve vitamin with methanol, be configured to the standard substance stock solution that mass concentration is 1g/L, then diluted composition standard Working solution.
3. detecting the method for a variety of liposoluble vitamins in feed as claimed in claim 2, which is characterized in that Standard Stock solutions Middle addition 0.2wt%BHT.
4. detecting the method for a variety of liposoluble vitamins in feed as claimed in claim 2, which is characterized in that answered in dilution It is to extract solution to be diluted.
5. detecting the method for a variety of liposoluble vitamins in feed as described in claim 1, which is characterized in that the extraction solution First component is: 0.05% ammonia spirit;The second component of the extraction solution is: 0.2% BHT methanol.
6. detecting the method for a variety of liposoluble vitamins in feed as claimed in claim 5, which is characterized in that extract solution first Component and the volume ratio for extracting the second component of solution are 30:70.
7. detecting the method for a variety of liposoluble vitamins in feed as described in claim 1, which is characterized in that use gradient elution In the process, mobile phase is: mobile phase A: the 96-99% methanol aqueous solution of the ammonium hydroxide containing 0.01-0.03%, Mobile phase B: 0.01- 0.03% aqueous formic acid.
8. detecting the method for a variety of liposoluble vitamins in feed as claimed in claim 7, which is characterized in that gradient elution program Are as follows: mobile phase A: 98% methanol aqueous solution containing 0.02% ammonium hydroxide, Mobile phase B: 0.02% aqueous formic acid, flow velocity 0.3mL/ min。
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CN111595970A (en) * 2017-11-20 2020-08-28 国药控股星鲨制药(厦门)有限公司 Method for detecting compound three-dimensional D-calcium pantothenate syrup vitamin by high performance liquid chromatography
CN108088925A (en) * 2017-12-14 2018-05-29 金华傲农生物科技有限公司 A kind of detection method of concentrated feed liposoluble vitamin content
CN108195961A (en) * 2017-12-27 2018-06-22 浙江海洋大学 A kind of natural cod-liver oil vitamin D and A and related substances detection method
CN108445098B (en) * 2018-02-26 2020-10-16 济南康和医药科技有限公司 Analysis method for detecting impurities in vitamin A palmitate
US10656059B2 (en) 2018-03-07 2020-05-19 Alcala Pharmaceutical, Inc. Method for qualitative and quantitative multiplexing of drug analytes from biological samples
CN108802213A (en) * 2018-04-20 2018-11-13 首都儿科研究所 Vitamin A detection dried blood spot reagent preparation box and detection method
CN109030648A (en) * 2018-08-01 2018-12-18 北京出入境检验检疫局检验检疫技术中心 The method and its sample-pretreating method of liposoluble vitamin content in a kind of detection formula milk
CN109828058A (en) * 2019-03-29 2019-05-31 新疆维吾尔自治区分析测试研究院 Method based on a variety of liposoluble vitamins of liquid chromatography-tandem mass spectrometry

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7358092B2 (en) * 2003-12-09 2008-04-15 Dsm Ip Assets B.V. Method for the determination of 25-hydroxycholecalciferol in feed
CN104155386A (en) * 2014-09-01 2014-11-19 上海迪安医学检验所有限公司 Method for measuring 9 fat-soluble vitamins in blood serum by UPLC
CN105891384A (en) * 2016-06-06 2016-08-24 漳州傲农牧业科技有限公司 Detection method for content of lipid-soluble vitamins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7358092B2 (en) * 2003-12-09 2008-04-15 Dsm Ip Assets B.V. Method for the determination of 25-hydroxycholecalciferol in feed
CN104155386A (en) * 2014-09-01 2014-11-19 上海迪安医学检验所有限公司 Method for measuring 9 fat-soluble vitamins in blood serum by UPLC
CN105891384A (en) * 2016-06-06 2016-08-24 漳州傲农牧业科技有限公司 Detection method for content of lipid-soluble vitamins

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Raphaël Vazquez等.Simultaneous quantification of water-soluble and fat-soluble vitamins in parenteral nutrition admixtures by HPLC-UV-MS/MS.《EJHP Science》.2009,第15卷(第2期), *
高效液相色谱-串联质谱法同时测定奶粉中维生素A、维生素D、维生素E;朱姜等;《中国卫生检验杂志》;20150630;第25卷(第11期);第1733-1737页 *
高效液相色谱法(HPLC)测定复合多维添加剂中脂溶性维生素VA、VD3、VE;付丽华等;《饲料工业》;19951231;第16卷(第12期);第36-38页 *
高效液相色谱法快速测定饲料中脂溶性维生素A、D3、E及其关键点;吕明等;《畜禽业》;20150531;第67页 *

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