CN106855545A - Liposoluble vitamin simultaneously in detection feed and the method for water soluble vitamin - Google Patents
Liposoluble vitamin simultaneously in detection feed and the method for water soluble vitamin Download PDFInfo
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Abstract
The invention discloses a kind of while the method for the liposoluble vitamin and water soluble vitamin in detection feed, comprises the following steps：（1）Prepare more than at least two in multivitamin standard liquid, including following vitamin：VB1、VB3、VB6、VB2、VB7、VB12, vitamine A acetate, vitamin D3, vitamin e acetate.Using HPLC MS/MS examination criteria solution, calibration curve equation is obtained.（2）5 10g samples are weighed to 100 milliliters of brown volumetric flasks in volumetric flask, are preferably used, is added and is extracted the 30min of solution ultrasonic extraction 10, cooling constant volume, centrifugation, with 0.22 μm of organic phase filter membrane, machine in filtering, test is analyzed using with step 1 identical gradient elution program, the MS detection parameters.（3）Measurement result according to step 1 and step 2 calculates the multivitamin content in sample.The vitamin ingredients in the various sources in complicated mixed feed composition are realized disposable continuous detection and analysis treatment by detection method of the invention.
Description
Technical field
The present invention relates to a kind of high performance liquid chromatography (HPLC) detection method, more particularly to a kind of high performance liquid chromatography joint
Mass spectrometry-mass spectrometry detects multivitamin method in feed simultaneously, including while liposoluble vitamin and water soluble vitamin
Detection.
Background technology
Because water soluble vitamin and liposoluble vitamin dissolving sex differernce are big, its report of method for measuring simultaneously is less.
Semahat Kucukkolbasi etc. are reported and are determined water soluble vitamin and fat in B B-complex tablet simultaneously
The method of soluble vitamin content.The method extracts B B-complex tablet using trifluoroacetic acid-methyl alcohol (TFA-MeOH) system
In vitamin, TFA-MeOH systems be eluent gradient wash-out, in Phenomenex Gemini C18 posts separate dimension life
Element, vitamin B is detected using PDAD (DAD) at different wave length1、B3、B9、B12、VC, p- aminobenzoic acids
(PABA)、A、D3And K, fluorescence detector (FLD) detects vitamin E and B at different wave length2.The method in 40 minutes simultaneously
Have detected 11 kinds of vitamins, it is adaptable to the detection of vitamin in B B-complex tablet.But, it is low for vitamin content
The measure of Feed Sample needs further research.
Lu Yan etc. uses double ternary high performance liquid chromatography many column technologies of many valves, while analyzing 21 kinds of vitamin in sample.
The method takes water soluble vitamin in sample using water extraction, and methanol dichloromethane system extracts liposoluble vitamin in sample.Water
Soluble vitamin enters Acclaim PA chromatographic columns, is with phosphate buffer, acetonitrile-phosphate buffer, methyl alcohol-methyl butyl ether
Eluent gradient is eluted；Liposoluble vitamin enters Acclaim C18 chromatographic columns, is flowing with methanol-acetonitrile, methyl butyl ether
Phase gradient is eluted.VWD 3400RS UV-detectors detect vitamin at different wave length.Actual sample analysis result shows, is somebody's turn to do
Method is applied to the detection of the vitamin supplement medicine rich in vitamin etc., but, in the beverage, due to its deliquescent limit
System, only detects several water soluble vitamins, similarly, in chicken feed sample also has part low content vitamin not detect.
Raw material composition is complicated in mixed feed, and vitamin source is more, and content is relatively low, cannot with two methods of the above possibly
Detection.A kind of new method is urgently developed, to detect the water soluble vitamin in the low vitamin content samples such as feed simultaneously
And liposoluble vitamin.
The content of the invention
It is an object of the invention to overcome in the prior art for complicated component in mixed feed, vitamin source is more,
Content is relatively low, it is impossible to the deficiency for detecting completely simultaneously is realized in one-time detection, there is provided a kind of while detecting the liposoluble in feed
The method of property vitamin and water soluble vitamin.The inventive method is more in can completing mixed feed during one-time detection
The detection and analysis of kind of vitamin, with monitoring analysis efficiency it is high the characteristics of, can greatly simplify multiple for mixed feed
Detection and analysis.
In order to realize foregoing invention purpose, the invention provides following technical scheme：
It is a kind of at the same detect feed in liposoluble vitamin and water soluble vitamin method, comprise the following steps：
(1) according to detection needs, multivitamin standard liquid is prepared, (the vitamin standard liquid of preparation) includes following
In vitamin more than at least two：VB1、VB3、VB6、VB2、VB7、VB12, vitamine A acetate, vitamin D3, vitamin E second
Acid esters (standard liquid).The particularly each at least one situation of water soluble vitamin and liposoluble vitamin.Preferably, have
It is for body, water soluble vitamin：VB1、VB3、VB6、VB2、VB7、VB12At least one of, and liposoluble vitamin：Vitamin A
Acetate, vitamin D3, at least one of vitamin e acetate.The standard of at least one of i.e. above-mentioned water soluble vitamin
Solution, and at least one of above-mentioned liposoluble vitamin standard liquid.According to the actual corresponding mark of testing goal adjustment
The preparation situation of quasi- solution, makes simultaneously containing water soluble vitamin and liposoluble vitamin standard liquid, or many
The standard liquid of individual different vitamin.
Using HPLC-MS/MS examination criteria solution, calibration curve equation is obtained.
Use gradient elution in detection process.Mobile phase includes mobile phase A：Methanol aqueous solution phase, and Mobile phase B：First
Aqueous acid phase.
Wherein mobile phase A：The volume ratio of methyl alcohol is more than 95v% in methanol aqueous solution；Mobile phase B：In aqueous formic acid
The content of formic acid is less than 1wt%.
(2) sample is weighed, 5-10g samples are preferably weighed, is placed in volumetric flask, preferably use 100 milliliters of brown volumetric flasks,
Add and extract solution ultrasonic extraction 10-30min, preferably preferably 15-25min, 20min；0.22 μ is used in cooling constant volume, centrifugation
The upper machine of m organic phase filter membranes filtering, using with step 1 identical chromatographic parameter (including gradient elution program) and the MS detection parameters
It is analyzed test.
The extraction solution is, by the component of extraction solution first and the extraction component of solution second according to 25-35：65-75 bodies
The solution of product ratio mixed preparing.
The extraction component of solution first is 0.03-0.07v% ammonia spirits (volume ratio).
The extraction component of solution second is 0.1-0.3%BHT methanol solutions (m/vol, quality percent by volume, g/L).
(3) testing result according to step 1 and step 2 calculates the multivitamin content in sample.
Detection method of the invention is devised with targetedly extraction scheme, will be many in complicated mixed feed composition
The vitamin ingredients for planting source are fully extracted, and then in conjunction with the gradient elution solution combination for being adjusted optimization, are realized for many
Plant the disposable continuous detection and analysis treatment of vitamin so that the multivitamin composition quickly continuously detection point in mixed feed
Analysis, can significantly optimize the problem of the multivitamin detection efficiency and accuracy rate in existing mixed feed, reduce inspection
Survey analysis to take, improve the efficiency of detection and analysis, it is to avoid the problem of existing detection and analysis intricate operation.
Further, the extraction solution is：0.05v% ammonia spirits：0.2 (g/L) %BHT methyl alcohol=25-35：65-75
The mixed solution that volume ratio is configured to.Most preferably, the extraction solution is：0.05% ammonia spirit：0.2%BHT methyl alcohol=
30：70 solution.It is exemplified below, the BHT methanol solutions are that the BHT of 20mg is dissolved in the middle of 100mL methyl alcohol to obtain.
Further, during using gradient elution, mobile phase has two kinds, is respectively：Mobile phase A：Containing 0.01-0.03v%
The 96-99v% methanol aqueous solutions of ammoniacal liquor, Mobile phase B：0.01-0.03wt% aqueous formic acids.During gradient elution, first
It is that main solution is eluted with Mobile phase B, is then gradually transitions mobile phase A for main solution is eluted, finally recovers
It is main wash-out solution to Mobile phase B.
It is exemplified below：Mobile phase A is prepared, first by first alcohol and water 96-99 by volume:1-4 is well mixed to obtain 96-
The methanol aqueous solution of 99v%, then example is hybridly prepared into ammoniacal liquor volume accounting and is by volume for above-mentioned methanol aqueous solution and ammoniacal liquor
The mixed solution of 0.01-0.03v%, as mobile phase A.
It is exemplified below：Mobile phase B is prepared, and the formic acid for weighing 10-30mg is added in the water of 99.97-99.99g, fully shakes
Even, as Mobile phase B, wherein formic acid weight percent are 0.01-0.03wt%.
Further, gradient elution program is：Mobile phase A：98v% methanol aqueous solutions containing 0.02v% ammoniacal liquor, Mobile phase B：
0.02wt% aqueous formic acids, flow velocity is 0.3mL/min, 25min is run, using gradient elution program, gradient condition：0~
2min, mobile phase A 0.5~0.5%；2~8.5min, mobile phase A 0.5~56%；8.5~8.6min, mobile phase A 56.0~
99.5%；8.6~20min, mobile phase A 99.5~99.5%；20~20.01min, mobile phase A 99.5~0.5%.
Further, during detection and analysis, chromatographic column selects one below：C18-MGⅡ(pH 2-10)3μ
M2.0mmI.D. × 150mm posts, 3 μm of 2.0mmI.D. × 150mm posts of C18-MG III (pH 2-10), 3 μm of ADME
2.1mmI.D. × 150mm posts (pH 2-10) (also referred to as Capcell PAK ADME 2.1mm I.D × 150mm, 3 μm), 5 μm of AQ
2.1mm I.D. × 150mm posts, Agilent ZORBAX Eclipse 2.1 × 50mm of XDB-C18 posts, Waters ACQUITY
1.8 μm of 3.0 × 50mm posts of UPLC HSS T3 and Waters ACQUITY UPLC 1.7 μm of 2.1 × 50mm posts of BEH.It is preferred that
Chromatographic column is：Capcell PAK ADME 2.1mm I.D × 150mm, 3 μm, i.e. 3 μm of 2.1mmI.D. × 150mm posts of ADME
(pH 2-10), uses pH scopes 2-10.
Further, mass spectrograph running parameter is preferably as follows：ESI cation scan patterns；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 of sheath temperature degree, flow velocity 9L/min, hair
Tubule voltage 4000V, spray nozzle voltage 500V.
Further, during mass spectrograph detection and analysis, multivitamin detection Parameter Conditions such as table under MRM monitoring patterns
Shown in 2.
Compared with prior art, beneficial effects of the present invention：
1. of the invention at the same detect feed in various liposoluble vitamins and water soluble vitamin method, according to feeding
Complicated vitamin source situation in material sample, and multivitamin property feature, devise corresponding extraction scheme with
And chromatogram, mass spectrometry parameters so that HPLC-MS/MS can complete various different vitamins during one-time detection analysis
Detection and analysis.
2. in detection method of the invention, the special optimization design vitamin extraction scheme of existing feedstuff, profit
The multivitamin composition for enabling the source in feed complicated with different extraction schemes is all fully extracted, Jin Er
The result tested and analyzed on HPLC-MS/MS instruments is accurately and reliably.
3. in detection method of the invention, the mobile phase and gradient elution program of design are according to the various liposoluble for testing and analyzing
Property and the characteristics of water soluble vitamin, realize efficiently separating, provided for follow-up mass spectrograph tests and analyzes qualitative, paced work
Reliable basic condition.
Brief description of the drawings：
Fig. 1 is 9 kinds of MRM collection of illustrative plates of vitamin in broiler chicken material
Fig. 2 is using 0.02mol/L ammonium acetates (pH=4.0)：0.2% (g/L) BHT+0.05v% ammoniacal liquor methanol solution=
50：50 chromatograms obtained as mobile phase.
Fig. 3 is using 0.05v% ammonia spirits：0.2% (m/vol) BHT methyl alcohol=30：70 solution are obtained as mobile phase
Chromatogram.
Specific embodiment
With reference to test example and specific embodiment, the present invention is described in further detail.But this should not be understood
For the scope of above-mentioned theme of the invention is only limitted to following embodiment, all technologies realized based on present invention belong to this
The scope of invention.
1 instrument, reagent and sample material prepare
(1) Agilent 1260-6460 liquid chromatograies-triple quadrupole bar tandem mass spectrum combined instrument matches somebody with somebody electric spray ion source
ESI (Agilent company of the U.S.).
(2) AB-135 types electronic analytical balance (Mei Teletuo benefits Shanghai Co., Ltd).
(3) LD5-2B centrifuges (system in Beijing Jing founds centrifuge Co., Ltd).
(4) KQ-500DE types numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).
(5) swirl mixing device MS3 (German IKA).
(6) second eyeball, methyl alcohol (chromatographically pure, Merck ＆ Co., Inc..
(7) formic acid, ammonium acetate, ammoniacal liquor (analyzing pure, Tianjin Ke Miou chemical reagent Co., Ltd).
(8) NaOH, sodium chloride (analyzing pure, Shanghai Chinese medicines group).
(9) thiamine (C12H17CLN4OS.HCL, molecular weight：337.3) (content 99.5%.Sigma companies).
(10) riboflavin (C17H20N4O6, molecular weight：376) (content 99.5%.Sigma companies).
(11) nicotinic acid (C6H5NO2, molecular weight：123) (content 99.5%.Sigma companies).
(12) pyridoxol (C8H11NO3.HCL, molecular weight：205.6) (content 99.5%.Sigma companies).
(13) biotin (C10H16N2O3S, molecular weight：244.23) (content 99.5%.Sigma companies).
(14) cobalamin (C63H88CoN14O14P, molecular weight：1355.37) (content 99.5%.Sigma companies).
(15) vitamine A acetate (C22H32O2, molecular weight：328.5).
(16) vitamin e acetate (C31H52O3, molecular weight：472.74).
(17) vitamin D3(C27H44O, molecular weight：384.63).
Fish material, broiler chicken material, baby pig feedstuff, laying hen material, sucking pig material and kind pig feed sample are limited by Sichuan new hope livestock technology
Company provides.
2 extract solution and nine kinds of preparations of mixed vitamin standard liquid
2.1 preparations for extracting solution
Extract solution：0.05% ammonia spirit：0.2% (m/vol) BHT methyl alcohol=30：70 volume ratios are mixed to get extraction
Solution
2.2 9 kinds of preparations of mixed vitamin standard liquid
Water soluble vitamin hybrid standard reserve supply solution：The accurate VB for weighing 5mg1、VB3、VB6；50mg VB2、VB7、
VB12In 500mL brown volumetric flasks, it is 10.0mg/L's to prepare mass concentration with 0.01mol ammonium acetates (pH=4.0) to standard items
Hybrid standard reserve supply solution is (with VB1Meter).
Interstitial fluid in liposoluble vitamin standard substance stock solution and work：The accurate vitamin A acetic acid for weighing 100mg
Ester, vitamin e acetate, vitamin D3Standard items, with methyl alcohol (adding 0.2%2,6- di-tert-butyl-4-methy phenols (BHT))
It is configured to the standard substance stock solution that mass concentration is 1000.0mg/L.Interstitial fluid in each standard substance work of 1mL is drawn respectively,
With the extraction solution prepared in above-mentioned 2.1, concentration is made into vitamine A acetate, vitamin D3Concentration is 1mg/L, vitamin E
Acetate concentration is the hybrid standard material working solution of 20 μ g/L.
Interstitial fluid in the work of mixed vitamin standard substance：Accurately pipette 1mL water soluble vitamin hybrid standard reserve supplies
Solution, 1mL vitamine A acetates, vitamin e acetate, vitamin D3Interstitial fluid in standard substance work, holds in 100mL browns
In measuring bottle, with the 70% methanol aqueous solution constant volume of 0.2%BHT, being configured to concentration is：The μ g/L of water soluble vitamin concentration 100 (with
VB1 count), the mixed vitamin standard working solution of the μ g/L (in terms of VE) of liposoluble vitamin 20.
Hybrid standard work series：Take hybrid standard material working solution 2,4,10,20mL and be respectively placed in 100mL brown capacity
In bottle, it is diluted in terms of VE (vitamin e acetate), concentration is the standard series of 0.4,0.8,2,4,20 μ g/L.
3 sample treatments
Weigh 5-10g samples and extract solution (extraction prepared in above-mentioned 2.1 is molten in 100 milliliters of brown volumetric flasks, adding
Liquid) ultrasonic extraction 20min, cool down constant volume, centrifugation, with 0.22 μm of organic phase filter membrane, machine in filtering.
4 chromatograms and Mass Spectrometry Conditions
4.1 chromatographic conditions
Chromatographic column：Capcell PAK ADME (2.1mmI.D × 150mm, 3 μm)；Mobile phase A：Containing 0.02% ammoniacal liquor
98% methanol aqueous solution, Mobile phase B：0.02% aqueous formic acid, flow velocity is 0.3mL/min, runs 25min, is washed using gradient
De- program, gradient condition is as shown in table 1.
The mobile phase gradient of table 1
4.2 Mass Spectrometry Conditions
ESI cation scan patterns：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 of sheath temperature degree, flow velocity 9L/min, capillary voltage 4000V, spray nozzle voltage 500V, its
His mass spectrum optimal conditions are as shown in table 2.
The instrument optimal conditions of the lower 9 kinds of vitamin of the MRM monitoring patterns of table 2
5 results and discussion
5.1 different solvents are to 9 kinds of influences of mixed vitamin testing result
In order to be able to there is simple, fast method analysis sample, following two composite solutions are compared as extractant scheme：
Scheme one：0.02mol/L ammonium acetates (pH=4.0)：0.2% (m/vol) BHT+0.05v% ammoniacal liquor methanol solution=
50：50.
Scheme two：0.02mol/L ammonium acetates (pH=4.0)：0.2% (m/vol) BHT+0.05% ammoniacal liquor methanol solution=
30：70.
Respectively as shown in Figures 2 and 3, wherein Fig. 2 is using 0.02mol/L ammonium acetates (pH=4.0) to result：0.2% (g/
L) BHT+0.05% ammoniacal liquor methanol solution=50：50 chromatograms obtained as mobile phase (scheme one).Fig. 3 is to use 0.05%
Ammonia spirit：0.2% (m/vol) BHT methyl alcohol=30：The chromatogram (scheme two) that 70 solution are obtained as mobile phase.
As can be seen that liposoluble vitamin can be separated preferably in the case of two kinds.But, when extract solution pH or
Salinity, ion contain ammonium acetate ratio it is big when, the peak shape for the vitamin in the HPLC separation processes of vitamin has
Larger adverse effect.Due to salt large percentage in scheme one, water soluble vitamin occurs in that fat head peak, and the peak type for obtaining is not
It is good.Ought particularly there are problems that salt timesharing peak type can be caused to broaden, separating degree；Work as pH<When 7, liposoluble can be caused
Property vitamin A peak type is bad.
Therefore employ 0.05% ammonia spirit：0.2% (m/vol) BHT methyl alcohol=30：The mobile phase of 70 or so ratios is to sample
Product are processed, and can so meet the separation of water soluble vitamin, can also meet the separation of liposoluble vitamin.Particularly,
Preferably use 0.05% ammonia spirit：0.2% (m/vol) BHT methyl alcohol=30：70 solution as sample extracting solution, each detection group
Point response is optimal.
5.2 different chromatographic columns are to 9 kinds of influences of mixed vitamin testing result
Respectively with having attempted carrying out same analysis test job using different chromatographic columns in following 7：
3 μm of 2.0mmI.D. × 150mm posts of C18-MG II (pH 2-10),
3 μm of 2.0mmI.D. × 150mm posts of C18-MG III (pH 2-10),
3 μm of 2.1mmI.D. × 150mm posts of ADME (pH 2-10),
5 μm of 2.1mm I.D. × 150mm posts of AQ,
Agilent ZORBAX Eclipse 2.1 × 50mm of XDB-C18 posts,
Waters ACQUITY UPLC HSS 1.8 μm of 3.0 × 50mm posts of T3
With Waters ACQUITY UPLC 1.7 μm of 2.1 × 50mm posts of BEH.
Analysis is compared to test result, it is found that the separating effect of different chromatographic columns has certain difference.Wherein
The separation to 6 kinds of water soluble vitamins and 3 kinds of liposoluble vitamins and retention time with 5 μm of 2.1mmI.D. × 150mm posts of AQ
Control is optimal.So, the solution of the present invention preferably uses 5 μm of 2.1mm I.D. × 150mm posts of AQ and carries out chromatography.
5.3 different mobile phases and gradient are to 9 kinds of influences of mixed vitamin testing result
Because water soluble vitamin is more stable in acid condition, and fat-soluble A does not have in acid condition
Activity, therefore use mobile phase A：98v% methanol aqueous solutions containing 0.02v% ammoniacal liquor, Mobile phase B：0.02wt% aqueous formic acids,
Gradient elution is carried out, the separation of water soluble vitamin can be so met, the separation of liposoluble vitamin can be also met.
5.4 different Mass Spectrometry Conditions are to 9 kinds of influences of mixed vitamin testing result
Experiment uses 0.05% ammonia spirit using certain density：0.2% (m/vol, g/L) BHT methyl alcohol=30：70 solution
The single vitamin standard working solution for preparing carries out parent ion MS2 SCAN to it respectively in the positive-ion mode, hair of being not difficult
Now [M+H]+molecular ion peak intensity of each object is all high, and the temperature of ion gun has no significant change at 260 DEG C -350 DEG C, therefore
325 DEG C of instrument design temperature is selected as ion source temperature, MS2 SIM optimization Fragmentor are then, Product is then
Lon, and optimize Collision Energy, draw qualitative ion and quota ion with feature.Then proceed to join mass spectrum
Number：Flow velocity, atomization gas pressure, sheath temperature degree, sheath gas, capillary voltage, spray nozzle voltage etc. are optimized.
5.5 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 testing sample same matrix, make its concentration for respectively 0.4,0.8,2,4,
20 μ g/L are in terms of vitamin e acetate), standard curve, 6 kinds of water solubilitys are done to mass concentration with the quota ion peak area selected
The linear equation and coefficient correlation of vitamin and 3 kinds of liposoluble vitamins are as shown in table 3.Result shows, in 6 kinds of water soluble vitamin lifes
Element and linearly dependent coefficient (R of 3 kinds of liposoluble vitamins in the range of 0.4~20 μ g/L (in terms of vitamin e acetate)2) be
0.9982~0.9999, show that linear dependence is good.
3 nine kinds of vitamin standard curves of table and coefficient correlation
(2) detection limit and quantitative limit
Above-mentioned steps " 3 sample treatment " are pressed using the method that target compound is added in blank extract solution, after being processed
Upper machine analysis, in terms of vitamin e acetate, concentration is the SNR that 0.4 μ g/L draw.Detection limit is determined with 3 times of signal to noise ratios, with 10
Times signal to noise ratio determines lower limit of quantitation, by calculate 6 kinds of water soluble vitamins why bother 3 kinds of detection limits of liposoluble vitamin and
Quantitative limit is as shown in table 4.
(3) recovery of standard addition
The targeted vitamins standard items of various concentrations are separately added into solvent blank, it is right according to step " 3 sample treatment "
Sample is processed, upper machine testing its concentration, calculates recovery of standard addition, mark-on amount, the rate of recovery and relative deviation (n as shown in table 4
=6).
The recovery of standard addition of table 4, relative deviation, detection limit and quantitative limit (μ g/kg)
The measure of 6 actual samples
Fish material, broiler chicken material, baby pig feedstuff, laying hen material, sucking pig material and kind pig feed sample are detected using the above method, it is several
VB can be detected in kind Feed Sample1、VB2、VB3、VB6、VB7、VB12, wherein VB1Content be 3731~8939 μ g/kg,
VB2Content is 8860~14880 μ g/kg, VB3Content be 6744~14720 μ g/kg, VB6Content be 2293~7136 μ g/
Kg, VB7Content is 356~457 μ g/kg, VB12Content is 339~485 μ g/kg, vitamin D3Content be 3731~18939 μ
G/kg, vitamin e acetate content is 18576~38674 μ g/kg, and the content of vitamine A acetate is 12579~105648 μ
g/kg.Prove that the method is practical.This method disclosure satisfy that mixed feed detects 9 kinds of demands of vitamin simultaneously.
The inventive method of table 5 detects the result of various different samples
7 brief summaries
This experiment establishes HPLC-MSMS while detecting fish material, broiler chicken material, baby pig feedstuff, laying hen material, sucking pig material and planting pig feed
Deng 3 kinds of liposoluble vitamins and 6 kinds of water soluble vitamins in mixed feed, totally 9 kinds of analysis methods of vitamin, and use the method
9 kinds of recovery of standard addition of vitamin of detection are 82.7%~102.6%, and relative standard deviation (n=6) is 3.9%~9.1%,
The detection of each liposoluble vitamin is limited to 0.11~30.00 μ g/kg, and lower limit of quantitation is 0.38~99.80 μ g/kg.The method is fast
Prompt, simple to operate, energy-conserving and environment-protective, result are accurate, meet the requirement of relevant laws and regulations, meet routine testing needs.
Remarks：
Various vitamin contents need to be converted to international unit (IU) in terms of mass fraction, such as herein, and conversion coefficient is as follows：
1 international unit VA (IU)=0.344 μ g retinyl acetates；
1 international unit VE (IU)=1mg vitamin e acetates；
1 international unit VD3(IU)=0.025 μ g but Vitamin D3.
BHT：2,6- di-tert-butyl-4-methy phenols, butylated hydroxytoluene, are commonly called as, antioxidant 264.
Claims (10)
1. a kind of at the same detect feed in liposoluble vitamin and water soluble vitamin method, comprise the following steps：
（1）According to detection needs, more than at least two in multivitamin standard liquid, including following vitamin are prepared：VB1、
VB3、VB6、VB2、VB7、VB12, vitamine A acetate, vitamin D3, vitamin e acetate；
Using HPLC-MS/MS examination criteria solution, calibration curve equation is obtained；
Use gradient elution in detection process, mobile phase includes mobile phase A：Methanol aqueous solution phase, and Mobile phase B：Formic acid water
Solution；
Wherein, mobile phase A：The volume ratio of methyl alcohol is more than 95%, Mobile phase B in methanol aqueous solution：Formic acid in aqueous formic acid
Content is less than 1wt%；
（2）Sample is weighed in volumetric flask, is added and is extracted solution ultrasonic extraction 10-30min, cool down constant volume, centrifugation, with 0.22 μm
The upper machine of organic phase filter membrane filtering, test is analyzed using with step 1 identical chromatographic parameter and the MS detection parameters；
The extraction solution is, by the component of extraction solution first and the extraction component of solution second according to 25-35：65-75 volume ratios
The solution of example mixed preparing；
The extraction component of solution first is 0.03-0.07v% ammonia spirits；
It is described to extract the BHT methanol solutions that the component of solution second is 0.1-0.3% g/L；
（3）Testing result according to step 1 and step 2 calculates the multivitamin content in sample.
2. as claimed in claim 1 while the method for the liposoluble vitamin and water soluble vitamin in detection feed, its feature
It is, step 1 to prepare multivitamin standard liquid, including：
Water soluble vitamin：VB1、VB3、VB6、VB2、VB7、VB12At least one of,
With
Liposoluble vitamin：Vitamine A acetate, vitamin D3, at least one of vitamin e acetate.
3. as claimed in claim 1 while the method for the liposoluble vitamin and water soluble vitamin in detection feed, its feature
It is that the extraction solution is：0.05% ammonia spirit：0.2% BHT methyl alcohol=25-35：The mixing that 65-75 volume ratios are configured to
Solution.
4. as claimed in claim 3 while the method for the liposoluble vitamin and water soluble vitamin in detection feed, its feature
It is that the extraction solution is：0.05v% ammonia spirits：BHT methyl alcohol=30 of 0.2% g/L：70 solution.
5. as claimed in claim 1 while the method for the liposoluble vitamin and water soluble vitamin in detection feed, its feature
It is that during gradient elution, mobile phase has two kinds, is respectively：Mobile phase A：96- containing 0.01-0.03v% ammoniacal liquor
99v% methanol aqueous solutions, Mobile phase B：0.01-0.03wt% aqueous formic acids.
6. as claimed in claim 5 while the method for the liposoluble vitamin and water soluble vitamin in detection feed, its feature
It is that gradient elution program is：Mobile phase A：98v% methanol aqueous solutions containing 0.02v% ammoniacal liquor, Mobile phase B：0.02wt% formic acid
The aqueous solution, flow velocity is 0.3mL/min, 25min is run, using gradient elution program, gradient condition：0 ~ 2min, mobile phase A 0.5
~0.5%；2 ~ 8.5min, mobile phase A 0.5 ~ 56%；8.5 ~ 8.6min, mobile phase A 56.0 ~ 99.5%；8.6 ~ 20min, mobile phase
A 99.5~99.5%；20 ~ 20.01min, mobile phase A 99.5 ~ 0.5%.
7. as claimed in claim 1 while the method for the liposoluble vitamin and water soluble vitamin in detection feed, its feature
It is that during detection and analysis, chromatographic column selects one below：C18-MGⅡ(pH 2-10) 3μm 2.0mmI.D.×150mm
Post, 3 μm of 2.0mmI.D. × 150mm posts of C18-MG III (pH 2-10), 3 μm of 2.1mmI.D. × 150mm posts of ADME（pH 2-
10）, 5 μm of 2.1mm I.D. × 150mm posts of AQ, Agilent ZORBAX Eclipse 2.1 × 50mm of XDB-C18 posts,
1.7 μm of Waters 1.8 μm of 3.0 × 50mm posts of ACQUITY UPLC HSS T3 and Waters ACQUITY UPLC BEH
2.1 × 50mm posts.
8. as claimed in claim 7 while the method for the liposoluble vitamin and water soluble vitamin in detection feed, its feature
It is that chromatographic column is：Capcell PAK ADME 2.1 mm I.D × 150 mm, 3 μm.
9. as claimed in claim 1 while the method for the liposoluble vitamin and water soluble vitamin in detection feed, its feature
It is that mass spectrograph running parameter is preferably as follows：ESI cation scan patterns；Multiple-reaction monitoring parameter：325 DEG C of temperature degree is dried,
Dry gas stream speed 11L/min, atomization gas pressure 45psi, 300 DEG C of sheath temperature degree, flow velocity 9L/min, capillary voltage 4000V, spray
Mouth voltage 500V.
10. as claimed in claim 1 while the method for the liposoluble vitamin and water soluble vitamin in detection feed, its feature
It is in step 2, to add and extract solution ultrasonic extraction 15-25min.
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| CN110068643A (en) * | 2019-03-01 | 2019-07-30 | 山东越品检验认证集团有限公司 | A method of based on vitamin in Liquid Chromatography-Tandem Mass Spectrometry measurement medical food |
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| CN115308346A (en) * | 2022-09-02 | 2022-11-08 | 汤臣倍健股份有限公司 | Method for extracting and detecting content of multiple vitamins in blood |
| CN115598256A (en) * | 2022-10-28 | 2023-01-13 | 杭州汉库医学检验所有限公司（Cn） | High-throughput serum full-spectrum vitamin detection instrument and detection method |
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