CN106093234A

CN106093234A - One utilizes LC-MS technology for detection biotin, folic acid and vitamin B12method

Info

Publication number: CN106093234A
Application number: CN201610393326.4A
Authority: CN
Inventors: 潘拾朝; 蔡伟江
Original assignee: BY Health Co Ltd
Current assignee: BY Health Co Ltd
Priority date: 2016-06-02
Filing date: 2016-06-02
Publication date: 2016-11-09

Abstract

The present invention relates to analytical chemistry field, disclose one and utilize LC-MS technology for detection biotin, folic acid and vitamin B₁₂Method.Detection method of the present invention is with alkaline water pretreatment testing sample and coordinates ultrasonic extraction, utilizes high performance liquid chromatography mass spectrometric hyphenated technique, determines suitable testing conditions, it is achieved that detect biotin, vitamin B simultaneously₁₂With the purpose of folic acid, and detection method has higher accuracy, precision and sensitivity, reduces inspection cost, improves checkability.

Description

One utilizes LC-MS technology for detection biotin, folic acid and vitamin B12Method

Technical field

The present invention relates to analytical chemistry field, particularly relate to one utilize LC-MS technology for detection biotin, Folic acid and vitamin B₁₂Method.

Background technology

Vitamin is one of nutrient of needed by human, can be divided into water soluble vitamin according to they dissolubilities in aqueous Raw element and fatsoluble vitamin.Water soluble vitamins mainly has: thiamine (vitamin B1), riboflavin (vitamin B2), Ni Ke Acid (vitamin PP, nicotinic acid, cigarette phthalein amine), arsenic tremble alcohol (vitamin B6), cyanogen bore element (vitamin B12), folic acid, pantothenic acid, biotin, Ascorbic acid (vitamin C) etc., readily soluble ten water, it is little to deposit in vivo.Fatsoluble vitamin mainly has vitamin A. D. E With K.As one of necessary nutrient of human body, all of vitamin content in vivo is all little, but in the metabolism of body, life Play an important role during length, growth etc..The common feature of fatsoluble vitamin and water soluble vitamins has:

1, typically can not synthesize in vivo, even if or synthesis (such as vitamin D) in vivo, amount of its synthesis is also little, must Must be provided by food or extraneous supplementary；2, it not the constituent of human body, energy is not the most provided, but there is specific function；3, people Body requirement is the least, but definitely can not lack, if the shortage of certain vitamin reaches to a certain degree, will cause corresponding dimension Raw element deficiency disease.

The supply of normal person's vitamin is mainly derived from food, for special dietary demander or geriatric nutrition disease Patient, chronic heart failure and simultaneous loss of appetite and the patient of liver dysfunction, chronic congestion mental and physical efforts Failure patients, tumor especially progressive stage malignant tumor patient, surgery patients etc., take multicomponent complex medicine or health care Product are a kind of important clinical supplementary meanss.Wherein, biotin, vitamin B₁₂, folic acid be conventional collocation, common is multiple Vitamin products all can contain these 3 kinds of raw materials.

Multivitamin product is required to detect wherein vitamin content, controls product quality, as biotin is adopted Detecting by GB/T 17778-2005 method, folic acid uses GB/T 17813-1999 method to detect, and vitamin B12 is adopted Detect with GB/T 17819-1999.But, with reference to existing detection method, need to be respectively adopted three kinds of schemes to biology Element, folic acid and vitamin B₁₂Detect, detect overlong time, inefficient, and take too much instrument and manpower.Cause This, it is desirable to provide one can detect biotin, folic acid and vitamin B simultaneously₁₂Method, improve the inspection of vitamin products whereby Survey efficiency.

Summary of the invention

In view of this, it is an object of the invention to provide one and utilize LC-MS technology for detection biotin, folic acid and dimension Raw element B₁₂Method so that described method can detect biotin, folic acid and vitamin B simultaneously₁₂, and have good linear Relation, higher precision, higher stability, preferably quantitative limit and detection limit and the higher response rate.

To achieve these goals, the present invention provides following technical scheme:

One utilizes LC-MS technology for detection biotin, folic acid and vitamin B₁₂Method, including:

Step 1, prepare biotin, folic acid and vitamin B respectively₁₂Gradient concentration titer, use HPLC-MS be combined skill Art detects, and with concentration C (μ g/mL) as abscissa, peak area (A) is vertical coordinate, draws standard working curve；

Wherein, HPLC system condition is:

It is good for octadecylsilane and closes the chromatographic column of silicagel column or its equal performance for fixing phase, with ammonium acetate solution for flowing Move phase A, carry out gradient elution with acetonitrile for Mobile phase B；

MS uses ESI⁺Pattern, sample introduction one pin time 10min, each parameter such as Tables 1 and 2:

Table 1

Table 2

Step 2, taking testing sample alkaline water moistening, then add water ultrasonic extraction, centrifugal, filter that to obtain test sample molten Liquid, detects according to HPLC-MS multiple techniques in step 1, is updated in standard working curve obtain biotin, leaf in testing sample Acid and vitamin B₁₂Concentration.

Need respectively to biotin, folic acid and vitamin B for existing detection method₁₂Carry out the defect detected, the present invention By suitable HPLC and MS condition, it is achieved that biotin, folic acid and vitamin B in detection sample simultaneously₁₂Concentration, and reach The standards such as higher precision, linear relationship, the response rate.

Wherein, the strong silicagel column that closes of octadecylsilane of the present invention comprises all C18 posts, such as Agilent ZORBAX SB-C18 post (4.6mm × 100mm, 3.5 μm), Alltima C18 (4.6mm × 100mm, 3.0 μm), described equal performance chromatograph Post i.e. has other chromatographic columns of equal extent performance with above-mentioned two C18 post.

As preferably, the program of described gradient elution such as table 3:

Table 3

Time (min)	Flow velocity (mL/min)	A%	B%
				0	0.4	90	10
0.01	0.4	80	20
				2	0.4	50	50
4	0.4	50	50
				6	0.4	90	10
10	0.4	90	10

As preferably, in described ammonium acetate solution, ammonium acetate concentration is 5-50mmol/L, and pH value is 3.5.Have in the present invention In body embodiment, described ammonium acetate concentration is that 10mmol/L, pH are regulated by acetic acid.

As preferably, described biotin, folic acid and vitamin B₁₂Gradient concentration hybrid working titer be:

Biotin: 0.04 μ g/mL, 0.08 μ g/mL, 0.12 μ g/mL, 0.16 μ g/mL, 0.20 μ g/mL；

Folic acid: 0.4 μ g/ml, 0.8 μ g/mL, 1.2 μ g/mL, 1.6 μ g/mL, 2.0 μ g/mL；

Vitamin B₁₂: 0.008 μ g/mL, 0.016 μ g/mL, 0.024 μ g/mL, 0.032 μ g/mL, 0.040 μ g/mL.

Above-mentioned biotin, folic acid and vitamin B₁₂The concentration combination of gradient concentration correspondence order be mixing work together Make titer.

In addition to suitable HPLC-MS testing conditions, the present invention has also carried out alkaline water wettability treatment to testing sample, and Coordinating the smooth detection of ultrasonic extraction, beneficially testing sample, described alkaline water is sodium carbonate liquor；Preferably, described carbonic acid The concentration of sodium solution is 0.1-1.0mol/L.Preferably, the operating temperature of described ultrasound wave is 35-45 DEG C, more preferably 40 DEG C.

Additionally, the method for the invention also includes step 3:

By the biotin in testing sample, folic acid and vitamin B₁₂Concentration substitute into following formula calculates and obtain in testing sample Biotin, folic acid and vitamin B₁₂Content；

X=C × V × K/M

In formula, X represents biotin in testing sample, vitamin B₁₂Or the content of folic acid, unit mg/100g；M represents to be measured The quality of sample, unit g；V represents the volume of testing sample dilution, Unit/mL；K represents unit conversion factor, K=0.1；C generation Biotin, vitamin B in table testing sample₁₂Or the concentration of folic acid, unit μ g/mL.

The present invention is many-sided checking detection method from methodology, such as linear relationship, precision, stability, Quantitative limit and detection limit, the response rate etc., result shows, precision, the response rate, linear relationship not only comply fully with Standard, And effect is better than GB, and the most also has preferable level.

Meanwhile, the present invention the most respectively with detection biotin, vitamin B₁₂Contrast with the GB detection method of folic acid, knot Fruit display, detection method testing result standard deviation is the least more stable, and three kinds of detection methods in GB are relatively Greatly.

From above technical scheme, the present invention is with alkaline water pretreatment testing sample and coordinates ultrasonic extraction, utilizes HPLC-MS technology, determines suitable testing conditions, it is achieved that detect biotin, vitamin B simultaneously₁₂With The purpose of folic acid, and detection method has higher accuracy, precision and sensitivity, reduces inspection cost, improves Checkability.

Accompanying drawing explanation

Fig. 1 show the linear relationship chart of biotin concentration C and peak area A；

Fig. 2 show vitamin B₁₂Concentration C and the linear relationship chart of peak area A；

Fig. 3 show the linear relationship chart of folic acid concentration C and peak area A.

Detailed description of the invention

The embodiment of the invention discloses one and utilize LC-MS technology for detection biotin, folic acid and vitamin B₁₂Side Method, those skilled in the art can use for reference present disclosure, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all Similar replacement and change apparent to those skilled in the art, they are considered as being included in the present invention. The inventive method is described by preferred embodiment, related personnel substantially can without departing from present invention, spirit and In the range of method described herein and application be modified or suitably change and combine, realize and apply the technology of the present invention.

In the methods of the invention, the hybrid working titer that can prepare gradient concentration again by first preparing Standard Reserving Solution enters The drafting of row working curve；

The preparation of Standard Reserving Solution:

Accurately weigh biotin, vitamin B₁₂, folic acid reference substance about 10mg, in 50mL volumetric flask, add appropriate Water makes dissolving (biotin appropriate 0.1-1.0mol/L sodium carbonate liquor, folic acid adds few drops strong aqua ammonia and dissolves), adds water and is settled to carve Degree, shakes up, makees standard reserving solution, and 4 DEG C of refrigerator stores for future use.

Standard working curve is drawn:

Precision draws biotin, vitamin B respectively₁₂, folic acid Standard Reserving Solution dilute with water, respectively obtain concentration and about make a living Thing element (0.04 μ g/mL, 0.08 μ g/mL, 0.12 μ g/mL, 0.16 μ g/mL, 0.20 μ g/mL；Vitamin B₁₂(0.008μg/mL、 0.016μg/mL、0.024μg/mL、0.032μg/mL、0.040μg/mL；Folic acid (0.4 μ g/ml, 0.8 μ g/mL, 1.2 μ g/mL, 1.6 μ g/mL, the hybrid working titer of 2.0 μ g/mL, through the filtering with microporous membrane of 0.45 μm, sample size is 10 μ L, with concentration C (μ g/mL) is abscissa, and peak area (A) is vertical coordinate, draws standard working curve.

Prepared by sample:

Accurately weigh testing sample in right amount in 50mL volumetric flask, add appropriate (about 2ml) 0.1-1.0mol/L sodium carbonate After solution makes sample wetness, add 35-45 DEG C of ultrasound wave of suitable quantity of water and dissolve extraction, vibration (15-30min, every 5min mono-for several times Secondary), constant volume, centrifugal, after 0.45 μm membrane filtration, i.e. obtain need testing solution, take filtrate sample introduction 10uL.

Hereinafter LC-MS technology for detection biotin, folic acid and vitamin B are utilized with regard to one provided by the present invention₁₂Side Method is described further.

Embodiment 1: linear relationship detects

Precision draws biotin, vitamin B respectively₁₂, folic acid Standard Reserving Solution dilute with water, respectively obtain a series of concentration Biotin (0.0502 μ g/mL, 0.100 μ g/mL, 0.151 μ g/mL, 0.201 μ g/mL, 0.251 μ g/mL), vitamin B₁₂ (0.00742 μ g/mL, 0.0148 μ g/mL, 0.0223 μ g/mL, 0.0297 μ g/mL, 0.0371 μ g/mL), folic acid (0.396 μ g/ Ml, 0.792 μ g/mL, 1.19 μ g/mL, 1.58 μ g/mL, 1.98 μ g/mL) hybrid working titer, through 0.45 μm micropore filter Membrane filtration, sample size is 10 μ L, and with concentration C (μ g/mL) as abscissa, peak area (A) is vertical coordinate, draws standard work song Line, result is shown in Fig. 1-Fig. 3, table 4-table 6, and HPLC-MS parameter is shown in Table 1-table 3, and HPLC system condition is:

With Agilent ZORBAX SB-C18 post (4.6mm × 100mm, 3.5 μm) for fixing phase, with 10mmol/L ammonium acetate Solution (pH3.5) is mobile phase A, carries out gradient elution with acetonitrile for Mobile phase B；

MS uses ESI⁺Pattern, time 10min；

Table 4 biotin linear relationship

Table 5 vitamin B₁₂Linear relationship

Table 6 folic acid linear relationship

Biotin, vitamin B₁₂, the correlation coefficient r of folic acid be respectively 0.9999,0.9994,0.9992, so with this Bright method measures biotin, vitamin B₁₂, the content of folic acid, biotin concentration be 0.0502 μ g/mL to 0.251 μ g/mL it Between, vitamin B₁₂Being between 0.00742 μ g/mL to 0.0371 μ g/mL in concentration, folic acid is 0.396 μ g/mL to 1.98 in concentration All present good linear between μ g/mL, meet the requirement [GB/ of GB/T27404-2008 " Good Laboratory control specification " T27404-2008 requires coefficient R²≥0.99】。

Embodiment 2: precision test

Precision weighs same sample (soup minister is good for multivitamin B race sheet 20150722A again, Tang Chenbeijian company provide) 6 parts, accurately weigh testing sample in right amount in 50mL volumetric flask, add appropriate (about 2ml) 0.1 sodium carbonate liquor and make sample wetness After, add suitable quantity of water and dissolve extraction with 40 DEG C of ultrasound wave, vibrate for several times, constant volume, centrifugal, after 0.45 μm membrane filtration, i.e. obtain confession Test sample solution, takes filtrate sample introduction 10uL.Detection sample size (with reference to embodiment 1 standard curve), calculates its RSD (%), knot Fruit is shown in Table 7-9.

Table 7 biotin Precision test result

Table 8 vitamin B₁₂Precision test result

Table 9 folic acid Precision test result

Biotin, vitamin B in 6 parts of samples₁₂, the RSD (%) of content of folic acid be: 1.2%, 1.5%, 0.6%, its RSD (%) is less than 2.9%, shows that the method has preferable precision, and " Good Laboratory controls rule to meet GB/T27404-2008 Model " requirement [GB/T27404-2008 require RSD (%)≤3.8%].

Embodiment 3: stability test

Take biotin, vitamin B₁₂, folic acid mixing reference substance solution the most at room temperature place respectively 0 hour, 2 hours, 4 little Time, within 8 hours, 12 hours, 24 hours, take sample introduction, sample size is 10uL, calculates relative standard deviation RSD (%) of its peak area, Investigate the stability (standard curve is with reference to embodiment 1) of the method, the results are shown in Table 10.

Table 10 stability test result

Standing time	Biotin peak area	Vitamin B<sub>12</sub>Peak area	Folic acid peak area
				0 hour	53300	3410	147000
2 hours	52700	3420	146000
				4 hours	52500	3430	148000
8 hours	52500	3400	146000
				12 hours	53000	3430	149000
24 hours	52500	3390	147000
				RSD (%)	0.7	0.5	0.8

Biotin, vitamin B₁₂, folic acid mixing reference substance solution the most at room temperature place respectively 0 hour, 2 hours, 4 hours, 8 Hour, 12 hours, 24 hours, the RSD (%) of its peak area is respectively as follows: 0.7%, 0.5%, 0.8%, and its RSD (%) is less than 3.0%, show that the stability of the method for the invention is preferable in 24 hours.

Embodiment 4: detection degree and quantitative limit

Concentrations and the quantitative concentrations of analyzing the inventive method are calculated by signal to noise ratio (S/N), and the method for the invention is joined According to embodiment 1 and embodiment 2.

1, the concentrations of biotin and quantitative concentrations: as S/N=3, the concentrations recording biotin is 0.0025119μg/ml；As S/N=10, the quantitative concentrations recording biotin is 0.008373 μ g/ml.

2, the concentrations of vitamin B12 and quantitative concentrations: as S/N=3, record vitamin B₁₂Concentrations be 0.00074189μg/ml；As S/N=10, record vitamin B₁₂Quantitative concentrations is 0.00247297 μ g/ml.

3, the concentrations of folic acid and quantitative concentrations: as S/N=3, the concentrations recording folic acid is 0.0098992 μ g/ml；As S/N=10, the quantitative concentrations recording folic acid is 0.032997333 μ g/ml.

Embodiment 5: recovery test

The method of the invention is with reference to embodiment 1 and the identical (Tang Chenbei of processing method of embodiment 2, reference substance and sample Strong multivitamin B race sheet 20150722A, is provided by Tang Chenbeijian company).

The process of mark-on sample: precision weighs 9 parts of about 0.10g sample, is placed in 50mL brown capacity, is divided into 3 groups, often group 3 parts, in each group, precision adds biotin, vitamin B respectively₁₂, folic acid standard mixed liquor (biotin is 1.006 μ g/mL, Vitamin B₁₂Being 0.0896 μ g/mL, folic acid is 7.792 μ g/mL) 4mL, 5mL, 6mL, add 0.1mol/L sodium carbonate liquor 2mL, Add appropriate water dissolution again, sample is placed in ultrasonic 25min in 40 degree of water temperatures and makes it dissolve, be settled to after being cooled to room temperature carve Degree, shakes up, through the filtering with microporous membrane of 0.45 μm, as mark-on solution；Take this sample solution and mark-on solution each 10uL sample introduction, The results are shown in Table 11-13.

Table 11 biotin recovery test result

Table 12 vitamin B₁₂Recovery test result

Table 13 folic acid recovery test result

Record reference substance input amount=mark-on sample measured amount-sample measured amount；

The response rate (%)=record reference substance input amount/input amount；

Record biotin, vitamin B₁₂, the average recovery rate of folic acid be respectively as follows: 98.63%, 97.79%, 99.35%, phase Being respectively 1.1%, 0.7%, 1.0% to standard deviation (RSD), " Good Laboratory controls rule to meet GB/T27404-2008 Model " requirement [GB/T27404-2008 require the response rate be 95%～105%].

The above method being only intended to understand the present invention and core concept thereof, it is noted that for the art Those of ordinary skill for, under the premise without departing from the principles of the invention, the present invention can be carried out some improvement and modification, These improve and modify the protection domain also falling into right of the present invention.

Claims

1. one kind utilizes LC-MS technology for detection biotin, folic acid and vitamin B₁₂Method, it is characterised in that including:

Step 1, preparation biotin, folic acid and vitamin B₁₂Gradient concentration hybrid working titer, use HPLC-MS be combined skill Art detect, with concentration as abscissa, peak area as vertical coordinate, draw standard working curve；

Wherein, HPLC system condition is:

It is good for octadecylsilane and closes the chromatographic column of silicagel column or its equal performance for fixing phase, with ammonium acetate solution for the phase that flows A, carry out gradient elution with acetonitrile for Mobile phase B；

Table 1

Table 2

Step 2, taking testing sample alkaline water moistening, then add water ultrasonic extraction, centrifugal, filter and obtain need testing solution, Detect according to HPLC-MS multiple techniques in step 1, be updated in standard working curve obtain biotin, folic acid in testing sample And vitamin B₁₂Concentration.

Method the most according to claim 1, it is characterised in that the program of described gradient elution such as table 3:

Table 3

Method the most according to claim 1, it is characterised in that in described ammonium acetate solution, ammonium acetate concentration is 5-50mmol/ L, pH value is 3.5.

Method the most according to claim 1, it is characterised in that described biotin, folic acid and vitamin B₁₂Gradient concentration mix Closing working standard liquid is:

Biotin: 0.04 μ g/mL, 0.08 μ g/mL, 0.12 μ g/mL, 0.16 μ g/mL, 0.20 μ g/mL；

Folic acid: 0.4 μ g/ml, 0.8 μ g/mL, 1.2 μ g/mL, 1.6 μ g/mL, 2.0 μ g/mL；

Method the most according to claim 1, it is characterised in that described alkaline water sodium carbonate liquor.

Method the most according to claim 5, it is characterised in that the concentration of described sodium carbonate liquor is 0.1-1.0mol/L.

Method the most according to claim 1, it is characterised in that the operating temperature of described ultrasound wave is 35-45 DEG C.

8. according to method described in claim 1-7 any one, it is characterised in that also include step 3:

By the biotin in testing sample, folic acid and vitamin B₁₂Concentration substitute into following formula calculates obtain in testing sample biological Element, folic acid and vitamin B₁₂Content；

X=C × V × K/M

In formula, X represents biotin in testing sample, vitamin B₁₂Or the content of folic acid, unit mg/100g；M represents testing sample Quality, unit g；V represents the volume of testing sample dilution, Unit/mL；K represents unit conversion factor, K=0.1；C represents and treats Biotin, vitamin B in test sample product₁₂Or the concentration of folic acid, unit μ g/mL.