CN106680389A - Method for rapidly separating and determining water-soluble B-vitamins in infant food and dairy products - Google Patents
Method for rapidly separating and determining water-soluble B-vitamins in infant food and dairy products Download PDFInfo
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Abstract
The invention relates to a method for rapidly separating and determining water-soluble B-vitamins in infant food and dairy products. The method comprises the following steps: preparing a standard working solvent: weighing an appropriate amount of vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9 and vitamin B12 standard substances, placing into a brown volumetric flask, reaching a constant volume of 100mL by virtue of ultra-pure water, and freezing storing; preparing a sample working solvent: weighing 3 to 8 g of samples, adding 50 to 70 mL of 0.1mol/L hydrochloric acid, uniformly mixing, sealing, placing into a high-pressure sterilizing pot, keeping for 25 to 35 min at 120 to 125 DEG C, dissolving, and performing pretreatment for the sample by adopting an enzymolysis method; and analyzing by adopting a liquid-phase chromatograph-mass spectrometry. The method can analyze and detect seven water-soluble B-vitamins, ensure the recovery rate of each component and save the detection time and cost.
Description
Technical field
The invention belongs to field of food detection, water-soluble in particularly a kind of rapid separation and determination infant food and dairy products
The method of B family vitamins.
Background technology
B family vitamins are to maintain human normal function and the indispensable water soluble vitamin of metabolic activity, and human body cannot
Voluntarily synthesize, it is necessary to extra to supplement.Because infant's dietary intake amount is limited, the main source of its intake nutriment is to pass through
Infant food and dairy products are obtaining.At present, China is main for the assay method of B family vitamins in infant food and dairy products
Perform according to the respective standard method of GB GB5413 series, it is specific as follows：
Vitamin B1 is according to standard《Tie up in GB 5413.11-2010 national food safety standard infant foods and dairy products
The measure of raw element B1》；Vitamin B2 is according to standard《GB 5413.12-2010 national food safety standard infant foods and breast
The measure of vitamin B2 in product》；Vitamin B3 is according to standard《GB 5413.15-2010 national food safety standard infant eats
The measure of nicotinic acid and niacinamide in product and dairy products》；Vitamin B5 is according to standard《GB 5413.17-2010 food securities country mark
The measure of pantothenic acid in quasi- infant food and dairy products》；Vitamin B6 is according to standard《GB 5413.13-2010 food securities country
The measure of vitamin B6 in standard infant food and dairy products》；FA is according to standard《GB 5413.16-2010 food is pacified
The measure of full national standard infant food and dairy products Folic Acid (folate activity)》；Vitamin B12 is according to standard《GB
5413.14-2010 the measure of vitamin B12 in national food safety standard infant food and dairy products》.Such as according to above-mentioned standard
Method carries out the measure of B family vitamins to infant food and dairy food sample, and its process is complicated, and needs longer detection week
Phase.
When vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6 are determined using liquid-phase chromatography method only
Monocyte sample just needs the chromatography time 94 minutes；FA, vitamin B12 are measured with microbial method, and detection cycle is more
It is to be calculated with number of days.And different detection projects carry out respectively sample pre-treatments, not only complex steps, further increase it is artificial into
This, occupies experimental period.As can be seen here, there is very big room for improvement in the technology.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, there is provided a kind of rapid separation and determination infant food and breast
The method of water-soluble B vitamin in product, it passes through to optimize chromatographic condition, is capable of achieving water-soluble B vitamin, including：Dimension life
Plain B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (nicotinic acid, niacinamide), vitamin B5 (pantothenic acid), vitamin B6
(pyridoxol), FA (folic acid), vitamin B12 (cobalamin) etc. amount to 7 kinds of common vitamin contents carries out detection simultaneously,
Shorten experimental period, greatly improve operating efficiency.
The present invention solves its technical problem and is achieved through the following technical solutions：
A kind of method of water-soluble B vitamin in rapid separation and determination infant food and dairy products, it is characterised in that：Should
Method comprises the steps：
1) preparation of standard working solution：
Weigh appropriate vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, FA, vitamin
B12 standard items, into brown volumetric flask, ultra-pure water is settled to 100mL, and freezen protective, each standard concentration range of linearity is as follows
Table：
| Project | The range of linearity (μ g/L) |
| Cobastab1 | 2-100 |
| Cobastab2 | 5-600 |
| Cobastab5 | 20-400 |
| Cobastab6 | 5-200 |
| Cobastab9 | 5-2300 |
| Cobastab12 | 5-300 |
| Nicotinic acid | 3-100 |
| Niacinamide | 2-150 |
Make standard working solution；
2) preparation of sample working solution：
3-8g samples are weighed, 0.1mol/L hydrochloric acid 50-70mL are added, is sealed after mixing, be put in high-pressure sterilizing pot,
25-35min dissolvings are kept at 120-125 DEG C, is taken out after being cooled to less than 40 DEG C；PH value is adjusted with 2.0mol/L sodium acetate solutions
To 3.5-4.0,1.5-2mL papains and amylase enzyme liquid are added, be placed in after shaking up in 37 DEG C of incubator overnight；By enzyme
Solution liquid is transferred in 100mL volumetric flasks, and constant volume is filtered, and makes sample working solution；
3) it is analyzed using LC/MS：
Standard working solution and sample working solution are injected into liquid chromatography-mass spectrography/mass spectrograph, liquid-phase chromatographic analysis and matter is carried out
Analysis of spectrum, makees to compare with the peak area of each vitamin standard items in above-mentioned standard working solution and determinand peak area in sample working solution
Compared with quantified by external standard method is calculated.
The condition of described liquid-phase chromatographic analysis is：
Chromatographic column：ACQUITY UPLC BEH C182.1 × 100mm, 1.7 μm；Mobile phase：A：0.1% formic acid water B：
0.1% formic acid methyl alcohol；Column temperature：40℃；Automatic sampler temperature：10℃；Run time 6.5min；Sample size：10 μ L, elute bar
Part：Gradient elution, parameter is：0min, flow velocity 0.4mL/min, formic acid water：Formic acid methyl alcohol 99：1；3min, flow velocity 0.4mL/min,
Formic acid water：Formic acid methyl alcohol 99：1；5min, flow velocity 0.4mL/min, formic acid water：Formic acid methyl alcohol 45：55；5.1min, flow velocity 0.4mL/
Min, formic acid water：Formic acid methyl alcohol 99：1；
Described Mass Spectrometry Conditions are：Capillary voltage：3.0kV；Taper hole voltage：30V；Desolvation temperature：350℃；It is de-
Solvent gas flow velocity：700L/Hr；Blowback gas velocity：50L/Hr.
The step 2) in filter include：First Jing filter paper is filtered, and takes filtrate and 13000 turns/min is centrifuged, and 0.22 μ is crossed after 3min
M filter membranes.
In described Mass Spectrometry Conditions, determine that each composition mass spectrometry parameters are：
| Ingredient names | Parent ion (m/z) | Daughter ion (m/z) | Taper hole voltage (V) | Impact energy (V) |
| Cobastab1 | 256.1 | 122.0 | 24 | 17 |
| Cobastab2 | 377.2 | 243.0 | 42 | 22 |
| Cobastab5 | 241.9 | 153.0 | 30 | 15 |
| Cobastab6 | 170.0 | 152.0 | 28 | 14 |
| Cobastab9 | 442.2 | 295.0 | 23 | 17 |
| Cobastab12 | 678.5 | 147.0 | 36 | 34 |
| Nicotinic acid | 123.9 | 80.0 | 38 | 20 |
| Niacinamide | 123.0 | 80.0 | 40 | 20 |
Described ultra-pure water meets《GB/T 6682-2008 water for analytical laboratory use-Specification and test methods》Middle one-level water
Requirement.
The advantages of the present invention are：
1st, the present invention can to common water-soluble B vitamin in infant food and dairy products, including：Vitamin B1 (sulphur
Amine element), vitamin B2 (riboflavin), vitamin B3 (nicotinic acid, niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxol),
FA (folic acid), vitamin B12 (cobalamin) etc. amount to 7 kinds of common vitamin contents carries out detection simultaneously；Only need 6.5
Complete by minute, carry out according to 7 kinds of national standard methods with original 7 kinds of vitamin needses of measure, instrument and equipment analysis time needs
94 minutes (theoretical value) is taken, carries out detecting that incubation time needs compare for 1-3 days using microbial method, significantly reduced point
The analysis time.
2nd, the present invention preferably goes out the general pre-treating method that can be used for that above-mentioned 7 kinds of water soluble vitamins are extracted from sample,
Component to be measured is extracted using same experiment pre-treating method, on the premise of each component recovery to be measured is ensured, greatly
Width saves time and the cost of labor for processing sample respectively.
3rd, the method for the present invention has relatively low detection limit, and with the wider range of linearity, it is linear good.The rate of recovery exists
Between 80.8%~102.3%, relative standard deviation ＜ 10%, it is seen that method has good veracity and precision.
4th, the present invention adopts chromatogram, Mass Spectrometry Conditions after optimization to water-soluble B vitamin in infant food and dairy products
Detection is analyzed, on the premise of each component recovery to be measured is ensured, significantly shortens experimental period, reduce hold facility, section
Save time and the cost of labor for processing sample respectively.
Description of the drawings
Fig. 1 be the present invention vitamin B2, vitamin B5, FA, the total ion current figure of vitamin B12 standard items；
Fig. 2 is vitamin B1, vitamin B3, the total ion current figure of vitamin B6 standard items of the present invention；
Specific embodiment
Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not limit
Qualitatively, it is impossible to which protection scope of the present invention is limited with this.
A kind of method of water-soluble B vitamin in rapid separation and determination infant food and dairy products, it is characterised in that：Should
Method comprises the steps：
1) preparation of standard working solution：
Weigh appropriate vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, FA, vitamin
B12 standard items, into brown volumetric flask, pure water is settled to 100mL, freezen protective, each standard concentration range of linearity such as following table：
The concentration of standard solution scope of table 1
| Project | The range of linearity (μ g/L) |
| Cobastab1 | 2-100 |
| Cobastab2 | 5-600 |
| Cobastab5 | 20-400 |
| Cobastab6 | 5-200 |
| Cobastab9 | 5-2300 |
| Cobastab12 | 5-300 |
| Nicotinic acid | 3-100 |
| Niacinamide | 2-150 |
Make standard working solution；Ultra-pure water meets《GB/T 6682-2008 water for analytical laboratory use-Specification and test methods》
The requirement of middle one-level water.
2) sample working solution：
Pre-treatment is carried out to sample using enzymatic isolation method：5g samples are weighed, 0.1mol/L hydrochloric acid 60mL is added, after fully mixing
Sealed with cotton plug and brown paper, be put in high-pressure sterilizing pot, at 121 DEG C 30min is kept, taken after being cooled to less than 40 DEG C
Go out, jog is for several times；Adjust pH value to 4.0 or so with 2.0mol/L sodium acetate solutions, add 2.0mL papains and amylase enzyme
Liquid, after shaking up, is placed in 37 DEG C of incubator overnight；Enzymolysis liquid is transferred in 100mL volumetric flasks, with water scale is settled to,
Filter paper is filtered, and takes filtrate and 13000 turns/min is centrifuged, and 0.22 μm of filter membrane is crossed after 3min, makes sample working solution.
3) it is analyzed using liquid chromatography：
Standard working solution and sample working solution are injected into liquid chromatography-mass spectrography/mass spectrograph, liquid-phase chromatographic analysis and matter is carried out
Analysis of spectrum, makees to compare with the peak area of each vitamin standard items in above-mentioned standard working solution and determinand peak area in sample working solution
Compared with quantified by external standard method is calculated.
The condition of liquid-phase chromatographic analysis is：
Chromatographic column：ACQUITY UPLC BEH C182.1 × 100mm, 1.7 μm；Mobile phase：A：0.1% formic acid water B：
0.1% formic acid methyl alcohol；Column temperature：40℃；Automatic sampler temperature：10℃；Run time 6.5min；Sample size：10 μ L, elute bar
Part：Gradient elution, parameter is shown in Table 1：
The condition of gradient elution of table 2
It is initial mobile phase to choose 99% formic acid water, can improve vitamin B1, nicotinic acid, niacinamide, vitamin B6 and retain effect
It is really poor, it is to avoid appearance is too fast, can be effectively separated, response signal is good, and without peak type hangover.
Mass Spectrometry Conditions are：Capillary voltage：3.0kV；Taper hole voltage：30V；Desolvation temperature：350℃；Desolventizing gas
Flow velocity：700L/Hr；Blowback gas velocity：50L/Hr, the concrete mass spectrometry parameters of testing sample refer to table 3：
The testing compound mass spectrometry parameters of table 3
| Ingredient names | Parent ion (m/z) | Daughter ion (m/z) | Taper hole voltage (V) | Impact energy (V) |
| Cobastab1 | 256.1 | 122.0 | 24 | 17 |
| Cobastab2 | 377.2 | 243.0 | 42 | 22 |
| Cobastab5 | 241.9 | 153.0 | 30 | 15 |
| Cobastab6 | 170.0 | 152.0 | 28 | 14 |
| Cobastab9 | 442.2 | 295.0 | 23 | 17 |
| Cobastab12 | 678.5 | 147.0 | 36 | 34 |
| Nicotinic acid | 123.9 | 80.0 | 38 | 20 |
| Niacinamide | 123.0 | 80.0 | 40 | 20 |
Analysis of experiments：
The com-parison and analysis of actual sample：
1st, the method range of linearity and detection limit：
Method detection limit is determined with signal to noise ratio S/N=3,7 kinds of water soluble vitamins to be measured are between 2 μ g/L-2300 μ g/L
With good linear relationship.Detection limit is respectively：Cobastab10.020μg/L；Cobastab20.097μg/L；Vitamin
B50.91μg/L；Cobastab60.014μg/L；Cobastab90.23μg/L；Cobastab120.061μg/L；The μ g/L of nicotinic acid 0.12；
The μ g/L of niacinamide 0.13.
2nd, recovery of standard addition and relative standard deviation：
Blank sample mark-on experiment is carried out using enzymatic isolation method, every kind of B family vitamins to be measured carry out three horizontal mark-ons and test, 6
Parallel determination, experiment measures the rate of recovery and relative standard deviation, the results are shown in Table 4：
The rate of recovery of table 4 and relative standard deviation
Above-mentioned experimental data can be seen that this experimental technique and have relatively low detection limit, and with the wider range of linearity,
It is linear good.The rate of recovery between 80.8%~102.3%, relative standard deviation ＜ 10%, it is seen that method has good standard
Exactness and precision.Compared with the method detected to above-mentioned water-soluble B vitamin respectively in GB, this experiment is significantly
Experimental period is shortened, operating efficiency is improved, manpower and materials are saved, experimental cost is reduced.During this method Instrumental Analysis
Between only need 6.5 minutes by complete.
Although disclosed embodiments of the invention for the purpose of illustration, it will be understood to those skilled in the art that not
Depart from the present invention and spirit and scope of the appended claims, various replacements, to change and modifications all be possible, therefore this
Bright scope is not limited to embodiment disclosure of that.
Claims (5)
1. in a kind of rapid separation and determination infant food and dairy products water-soluble B vitamin method, it is characterised in that：The party
Method comprises the steps：
1) preparation of standard working solution：
Weigh appropriate vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, FA, vitamin B12 mark
Quasi- product, into brown volumetric flask, ultra-pure water is settled to 100mL, freezen protective, each standard concentration range of linearity such as following table：
Make standard working solution；
2) preparation of sample working solution：
3-8g samples are weighed, 0.1mol/L hydrochloric acid 50-70mL are added, is sealed after mixing, be put in high-pressure sterilizing pot, in 120-
25-35min dissolvings are kept at 125 DEG C, is taken out after being cooled to less than 40 DEG C；PH value is adjusted with 2.0mol/L sodium acetate solutions extremely
3.5-4.0, adds 1.5-2mL papains and amylase enzyme liquid, is placed in after shaking up in 37 DEG C of incubator overnight；Will enzymolysis
Liquid is transferred in 100mL volumetric flasks, constant volume, is filtered, and makes sample working solution；
3) it is analyzed using LC/MS：
Standard working solution and sample working solution are injected into liquid chromatography-mass spectrography/mass spectrograph, liquid-phase chromatographic analysis and mass spectrum point is carried out
Analysis, is made comparisons with the peak area of each vitamin standard items in above-mentioned standard working solution with determinand peak area in sample working solution,
Quantified by external standard method is calculated.
2. in rapid separation and determination infant food according to claim 1 and dairy products water-soluble B vitamin method,
It is characterized in that：The condition of described liquid-phase chromatographic analysis is：
Chromatographic column：ACQUITY UPLC BEH C182.1 × 100mm, 1.7 μm；Mobile phase：A：0.1% formic acid water B：0.1% first
Sour methyl alcohol；Column temperature：40℃；Automatic sampler temperature：10℃；Run time 6.5min；Sample size：10 μ L, elution requirement：Gradient
Elute, parameter is：0min, flow velocity 0.4mL/min, formic acid water：Formic acid methyl alcohol 99：1；3min, flow velocity 0.4mL/min, formic acid water：
Formic acid methyl alcohol 99：1；5min, flow velocity 0.4mL/min, formic acid water：Formic acid methyl alcohol 45：55；5.1min, flow velocity 0.4mL/min, first
Sour water：Formic acid methyl alcohol 99：1；
Described Mass Spectrometry Conditions are：Capillary voltage：3.0kV；Taper hole voltage：30V；Desolvation temperature：350℃；Desolventizing
Gas velocity：700L/Hr；Blowback gas velocity：50L/Hr.
3. in rapid separation and determination infant food according to claim 1 and dairy products water-soluble B vitamin method,
It is characterized in that：The step 2) in filter include：First Jing filter paper is filtered, and takes filtrate and 13000 turns/min, mistake after 3min is centrifuged
0.22 μm of filter membrane.
4. in rapid separation and determination infant food according to claim 1 and dairy products water-soluble B vitamin method,
It is characterized in that：In described Mass Spectrometry Conditions, determine that each composition mass spectrometry parameters are：
5. in rapid separation and determination infant food according to claim 1 and dairy products water-soluble B vitamin method,
It is characterized in that：Described ultra-pure water meets《GB/T 6682-2008 water for analytical laboratory use-Specification and test methods》Middle one-level
The requirement of water.
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Cited By (9)
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| CN109444293A (en) * | 2018-12-26 | 2019-03-08 | 中国烟草总公司郑州烟草研究院 | The detection method of endogenous water-soluble B vitamin in a kind of fresh tobacco leaves |
| CN109633016A (en) * | 2018-12-29 | 2019-04-16 | 上海微谱化工技术服务有限公司 | The analysis method of key component in a kind of cosmetics quality control |
| CN109709238A (en) * | 2018-12-26 | 2019-05-03 | 江苏佳信检测技术有限公司 | The detection method of vitamin B1 content in vegetables |
| CN110412186A (en) * | 2019-07-16 | 2019-11-05 | 上海康识食品科技有限公司 | Vitamin B in a kind of measurement condensed food1And vitamin B2Method |
| CN110412157A (en) * | 2019-07-17 | 2019-11-05 | 广州金评检测研究院有限公司 | The measuring method of niacin in a kind of niacinamide raw material |
| CN110954624A (en) * | 2019-12-23 | 2020-04-03 | 河南广电计量检测有限公司 | Method for detecting content of nicotinic acid in peanuts and products thereof |
| CN111272912A (en) * | 2020-03-20 | 2020-06-12 | 山东省食品药品检验研究院 | Method for measuring nicotinic acid and nicotinamide in infant rice flour by solid-phase extraction-high performance liquid chromatography |
| CN112129845A (en) * | 2020-08-17 | 2020-12-25 | 广东省农业科学院动物科学研究所 | Method for rapidly detecting content of B vitamins in pig intestinal contents/feces |
| CN114216982A (en) * | 2021-12-14 | 2022-03-22 | 黑龙江飞鹤乳业有限公司 | Vitamin B12Is detected by |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109444293A (en) * | 2018-12-26 | 2019-03-08 | 中国烟草总公司郑州烟草研究院 | The detection method of endogenous water-soluble B vitamin in a kind of fresh tobacco leaves |
| CN109709238A (en) * | 2018-12-26 | 2019-05-03 | 江苏佳信检测技术有限公司 | The detection method of vitamin B1 content in vegetables |
| CN109444293B (en) * | 2018-12-26 | 2023-11-10 | 中国烟草总公司郑州烟草研究院 | Method for detecting endogenous water-soluble B vitamins in fresh tobacco leaves |
| CN109633016A (en) * | 2018-12-29 | 2019-04-16 | 上海微谱化工技术服务有限公司 | The analysis method of key component in a kind of cosmetics quality control |
| CN110412186A (en) * | 2019-07-16 | 2019-11-05 | 上海康识食品科技有限公司 | Vitamin B in a kind of measurement condensed food1And vitamin B2Method |
| CN110412157A (en) * | 2019-07-17 | 2019-11-05 | 广州金评检测研究院有限公司 | The measuring method of niacin in a kind of niacinamide raw material |
| CN110954624A (en) * | 2019-12-23 | 2020-04-03 | 河南广电计量检测有限公司 | Method for detecting content of nicotinic acid in peanuts and products thereof |
| CN111272912A (en) * | 2020-03-20 | 2020-06-12 | 山东省食品药品检验研究院 | Method for measuring nicotinic acid and nicotinamide in infant rice flour by solid-phase extraction-high performance liquid chromatography |
| CN111272912B (en) * | 2020-03-20 | 2022-02-01 | 山东省食品药品检验研究院 | Method for measuring nicotinic acid and nicotinamide in infant rice flour by solid-phase extraction-high performance liquid chromatography |
| CN112129845A (en) * | 2020-08-17 | 2020-12-25 | 广东省农业科学院动物科学研究所 | Method for rapidly detecting content of B vitamins in pig intestinal contents/feces |
| CN114216982A (en) * | 2021-12-14 | 2022-03-22 | 黑龙江飞鹤乳业有限公司 | Vitamin B12Is detected by |
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