CN103091442A - Chromatographic method for determination of vitamin B12 content - Google Patents
Chromatographic method for determination of vitamin B12 content Download PDFInfo
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- CN103091442A CN103091442A CN2012104501293A CN201210450129A CN103091442A CN 103091442 A CN103091442 A CN 103091442A CN 2012104501293 A CN2012104501293 A CN 2012104501293A CN 201210450129 A CN201210450129 A CN 201210450129A CN 103091442 A CN103091442 A CN 103091442A
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Abstract
The invention discloses a chromatographic method for determination of vitamin B12 content. The method comprises the following steps of: (1) preparation of a mobile phase; (2) preparation of a reference solution; (3) preparation of a test sample solution; (4) setting of chromatographic conditions; and (5) injection detection. The chromatographic method for determination of the vitamin B12 content disclosed by the invention selects the new mobile phase and other detection conditions matched with the new mobile phase to accurately determine and calculate the content of trace amounts of vitamin B12 in complex matrix health foods directly, is good in chromatographic peak shapes of detection results and strong in anti-interference ability and meets actual determination requirements.
Description
Technical field
The present invention relates to health care food and medicine field tests, relate in particular to the chromatographic process of measuring mcg vitamin B12 content in health food and medicine.
Background technology
Cobalamin is one of micro-nutrient composition of needed by human, if picked-up is not enough, may cause pernicious anaemia, the marrow sex change, and glossitis, the nerveous system symptom, four limbs are stiff, and easily irriate, sleepy, lassitude etc.The cobalamin of wanting for replenishing needed by human body, present many health foods add cobalamin, but cobalamin content in the health products of food and some matrix components complexity is very low, and this mensuration to cobalamin causes very large difficulty.
The assay method of cobalamin has microbiological analysis, spectrophotometric method and chromatography at present.Microbial method can not accurately be distinguished cobalamin and analog thereof, the measurement result out of true, and waste time and energy, complicated operation is high to personnel's technical requirement; Spectrophotometric method is not suitable for the sample of complex matrices, because a lot of component spectra overlap of peakss are arranged, not simple differentiate several times just can make a distinction; The chromatography of setting up at present is mainly to use high performance liquid chromatography (HPLC), and adopting the methanol-water system is mobile phase, and cobalamin is detected, and detection the cobalamin component occurs and can't go out the peak or go out not obvious can't the reaching in peak to measure the phenomenon that requires.Compare with spectrophotometric method than microbial method, existing HPLC method has improved sensitivity and accuracy in general testing process, but for some complicated components and the low health products of cobalamin content, such as only having several ppm, when measuring, need to shift to an earlier date enrichment to cobalamin, cause to find corresponding peak or go out the peak very little if directly carry out sampling determination, repeatability is not high, thereby does not reach testing requirement.
Summary of the invention
The purpose of this invention is to provide a kind of chromatographic process of measuring cobalamin in health food, by selecting novel mobile phase and supporting other testing conditions with it, realize directly Accurate Determining and calculating to mcg vitamin B12 content in complex matrices health food and medicine.
The present invention is achieved in that a kind of chromatographic process of measuring cobalamin in health food, it is characterized in that comprising the following steps: (1) mobile phase preparation: take a certain amount of potassium dihydrogen phosphate, be placed in volumetric flask, after adding deionized water dissolving, adding 85% phosphoric acid compound concentration is that 0.04 M, pH are 3.5 ± 1.5 potassium phosphate buffer solution, above-mentioned buffer solution and methyl alcohol according to the mixing of the volume ratio of buffer solution: methyl alcohol=69:31, are made mobile phase; (2) reference substance solution preparation: adopt the USP standard items as the cobalamin reference substance, accurately weighed a certain amount of cobalamin reference substance, dissolve with mobile phase, be mixed with the storing solution that concentration is 0.01 mg/mL, dilute storing solution with mobile phase before each test, being mixed with concentration is the standard operation solution of 1 μ g/ml; (3) test sample solution preparation: get appropriate testing sample, be ground into fine powder, the accurately weighed sample powder that approximately contains 25 μ g cobalamins, be placed in the brown volumetric flask of 50mL, precision adds 25 mL mobile phase dissolvings, firmly jolts, extracted 2 minutes, with 0.45 μ m membrane filtration; (4) chromatographic condition is set:
A. chromatographic column: adopt anti-phase C18 chromatographic column, 5 μ m, 250 mm * 4.6 mm; B. flow velocity: 0.5-1.5 mL/min; C. detecting device and detect wavelength: diode array detector, wavelength are 546 nm; D. sample size: 70-150 μ L; E. column temperature: 25-35 ° C; (5) sample detection: by retention time and the solution peak area relation of test sample and reference substance, calculate the content of cobalamin in test sample: ω by following equation
Sample=C
Right* A
Sample* 25/ A
Right* m
Sample, wherein
ω SampleBe the content of cobalamin in test sample, unit is μ g/g;
C RightBe the reference substance normal concentration;
A RightBe reference substance standard solution peak area;
A SampleBe test sample solution peak area;
m SampleBe the weight of test sample, unit is g.
Mobile phase of the present invention also can be used sodium ascorbyl phosphate and other substrate preparation buffer solution.
The present invention is by selecting novel mobile phase and supporting other testing conditions with it, realize direct Accurate Determining and calculating to mcg vitamin B12 content in the complex matrices health food, the testing result chromatographic peak profile is good, and antijamming capability is strong, satisfies the practical measurement requirement.
Description of drawings
The original HPLC method of Fig. 1 reference substance chromatogram.
The original HPLC method of Fig. 2 test sample chromatogram.
Fig. 3 reference substance chromatogram of the present invention.
Fig. 4 test sample chromatogram of the present invention.
Embodiment
With reference to accompanying drawing, a kind of chromatographic process of measuring cobalamin in health food, it is characterized in that comprising the following steps: (1) mobile phase preparation: take a certain amount of potassium dihydrogen phosphate, be placed in volumetric flask, after adding deionized water dissolving, adding 85% phosphoric acid compound concentration is that 0.04 M, pH are 3.5 ± 1.5 potassium phosphate buffer solution, and above-mentioned buffer solution and methyl alcohol according to the mixing of the volume ratio of buffer solution: methyl alcohol=69:31, are made mobile phase; (2) reference substance solution preparation: adopt the USP standard items as the cobalamin reference substance, accurately weighed a certain amount of cobalamin reference substance, dissolve with mobile phase, be mixed with the storing solution that concentration is 0.01 mg/mL, dilute storing solution with mobile phase before each test, being mixed with concentration is the standard operation solution of 1 μ g/ml; (3) test sample solution preparation: get appropriate testing sample, be ground into fine powder, the accurately weighed sample powder that approximately contains 25 μ g cobalamins, be placed in the brown volumetric flask of 50mL, precision adds 25 mL mobile phase dissolvings, firmly jolts, extracted 2 minutes, with 0.45 μ m membrane filtration; (4) chromatographic condition is set: a. chromatographic column: adopt anti-phase C18 chromatographic column, 5 μ m, 250 mm * 4.6 mm; B. flow velocity: 0.5-1.5 mL/min; C. detecting device and detect wavelength: diode array detector, wavelength are 546 nm; D. sample size: 70-150 μ L; E. column temperature: 25-35 ° C; (5) sample detection: by retention time and the solution peak area relation of test sample and reference substance, calculate the content of cobalamin in test sample: ω by following equation
Sample=C
Right* A
Sample* 25/ A
Right* m
Sample, wherein
ω SampleBe the content of cobalamin in test sample, unit is μ g/g;
C RightBe the reference substance normal concentration;
A RightBe reference substance standard solution peak area;
A SampleBe test sample solution peak area;
m SampleBe the weight of test sample, unit is g.Described mobile phase also can be used sodium ascorbyl phosphate and other substrate preparation buffer solution.During concrete enforcement, near the peak of original HPLC method reference substance retention time t=10.2min is that cobalamin is measured the peak, and test sample does not have cobalamin to measure the peak near retention time t=10.2min; Reference substance of the present invention and test sample obvious cobalamin all occurs and measure the peak near retention time t=6.5 min, can realize smoothly and effectively measuring.The present invention also can adopt similar reversed-phase column except adopting the C18 post.
The present invention is further illustrated below in conjunction with specific embodiment:
Embodiment: the mensuration of cobalamin content in sample:
(1) mobile phase is prepared: take 5.44 g KH
2PO
4Being placed in 1 L volumetric flask, after adding deionized water dissolving, is that 0.04 M, pH are 3.5 ± 1.5 potassium phosphate buffer solution with 85% phosphoric acid compound concentration, measure the above-mentioned buffer solution mixing of 310 mL methyl alcohol and 690 mL, with 0.45 μ m filtering with microporous membrane, degassed.
(2) reference substance solution is prepared: adopt the USP standard items as the cobalamin reference substance.Accurately weighed a certain amount of cobalamin reference substance with the mobile phase dissolving, is mixed with the storing solution that concentration is 0.01 mg/mL; Dilute storing solution with mobile phase before each test, being mixed with concentration is the standard operation solution of 1 μ g/ml.
(3) test sample is processed: get and be no less than the sample of 20, be ground into fine powder, the accurately weighed sample powder that approximately contains 25 μ g cobalamins, be placed in the brown volumetric flask of 50mL, precision adds 25 mL mobile phase dissolvings, firmly jolts, extracted 2 minutes, with 0.45 μ m membrane filtration.
(4) sample detection:
Reference substance and test sample chromatogram as shown in Figure 3 and Figure 4, reference substance and near the peak of test sample retention time t=6.5 min are that cobalamin is measured the peak.
By retention time and the solution peak area relation of test sample and reference substance, calculate the content of cobalamin in test sample: ω by following equation
Sample=C
Right* A
Sample* 25/ A
Right* m
Sample, wherein
ω SampleBe the content of cobalamin in test sample, unit is μ g/g;
C RightBe the reference substance normal concentration;
A RightBe reference substance standard solution peak area;
A SampleBe test sample solution peak area;
m SampleBe the weight of test sample, unit is g.
(5) reference substance and test sample chromatogram as shown in Figure 3 and Figure 4, reference substance and near the peak of test sample retention time t=6.5 min are that cobalamin is measured the peak.
The range of linearity of many experiments proof the method is: 0.053 μ g-0.16 μ g, method detects and is limited to: 0.011 μ g, recovery of standard addition is: 82%-96%.Fact proved that the present invention can accurately measure the associated sample cobalamin and reach the mensuration peak of measuring requirement, satisfy the needs that detect, and the chromatogram peak shape is good, and the sample of not processing with the method can't find the mensuration peak or it is very little to go out the peak, can't detect the content of cobalamin.The present invention is not limited only to health products, medicine, dietary supplements, also can be used for the mensuration of biology and goods mcg vitamin B12 thereof, and the invention of mensuration proved is highly sensitive, selectivity good, antijamming capability is strong.
Claims (2)
1. chromatographic process of measuring cobalamin in health food is characterized in that comprising the following steps:
(1) mobile phase preparation: take a certain amount of potassium dihydrogen phosphate, be placed in volumetric flask, after adding deionized water dissolving, adding 85% phosphoric acid compound concentration is that 0.04 M, pH are 3.5 ± 1.5 potassium phosphate buffer solution, above-mentioned buffer solution and methyl alcohol according to the mixing of the volume ratio of buffer solution: methyl alcohol=69:31, are made mobile phase;
(2) reference substance solution preparation: adopt the USP standard items as the cobalamin reference substance, accurately weighed a certain amount of cobalamin reference substance, dissolve with mobile phase, be mixed with the storing solution that concentration is 0.01 mg/mL, dilute storing solution with mobile phase before each test, being mixed with concentration is the standard operation solution of 1 μ g/ml;
(3) test sample solution preparation: get appropriate testing sample, be ground into fine powder, the accurately weighed sample powder that approximately contains 25 μ g cobalamins, be placed in the brown volumetric flask of 50mL, precision adds 25 mL mobile phase dissolvings, firmly jolts, extracted 2 minutes, with 0.45 μ m membrane filtration;
(4) chromatographic condition is set:
A. chromatographic column: adopt anti-phase C18 chromatographic column, 5 μ m, 250 mm * 4.6 mm;
B. flow velocity: 0.5-1.5 mL/min;
C. detecting device and detect wavelength: diode array detector, wavelength are 546 nm;
D. sample size: 70-150 μ L;
E. column temperature: 25-35 ° C;
(5) sample detection:
By retention time and the solution peak area relation of test sample and reference substance, by the content of cobalamin in following equation calculation sample: ω
Sample=C
Right* A
Sample* 25/ A
Right* m
Sample, wherein
ω SampleBe the content of cobalamin in testing sample, unit is μ g/g;
C RightBe the reference substance normal concentration;
A RightBe reference substance standard solution peak area;
A SampleBe the sample solution peak area;
m SampleBe the weight of sample, unit is g.
2. a kind of chromatographic process of measuring cobalamin content according to claim 1 is characterized in that: described mobile phase also can be used sodium ascorbyl phosphate and other substrate preparation buffer solution.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104198598A (en) * | 2014-07-15 | 2014-12-10 | 汤臣倍健股份有限公司 | Determination method for vitamin B12 |
| CN107860847A (en) * | 2017-11-14 | 2018-03-30 | 济南维瑞医药科技开发有限公司 | Vitamin B in one kind detection multivitamin preparation12Method about material |
| CN109917037A (en) * | 2019-03-26 | 2019-06-21 | 广西壮族自治区食品药品检验所 | Gentamicin procaine ties up the HPLC detection method of vitamin B12 content in B12 particle/capsule |
| CN113376285A (en) * | 2021-06-11 | 2021-09-10 | 贵州省产品质量检验检测院 | Liquid chromatography for determining vitamin B12 addition in infant food and milk |
| CN114113359A (en) * | 2021-05-07 | 2022-03-01 | 佛山市南海北沙制药有限公司 | Central control detection method of 7-ACA derivative |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060105392A1 (en) * | 2003-03-13 | 2006-05-18 | Paul Lehmann | Differential diagnosis of vitamin B12, vitamin B6, and folic acid disorders |
| WO2006090428A2 (en) * | 2005-02-28 | 2006-08-31 | Giuseppe Lazzarino | Method for the separation and simultaneous direct determination of compounds belonging to at least one of the groups chosen among purines and pyrimidines, n- acetylated amino acids, mono and dicarboxylic acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes, vitamins of group b, and derivatives ther |
| CN101961342A (en) * | 2010-08-19 | 2011-02-02 | 齐慧 | Water soluble vitamin composition for injection and preparation method thereof |
| CN102072846A (en) * | 2010-12-06 | 2011-05-25 | 贵州神奇药业股份有限公司 | Method for detecting quality of compound capsule prepared from 8 kinds of amino acids and 11 kinds of vitamins |
| CN102226795A (en) * | 2011-03-31 | 2011-10-26 | 宁波双伟制药有限公司 | Method for determining content of vitamin B12 in complex vitamin B and diclofenac sodium tablet through high performance liquid chromatography |
-
2012
- 2012-11-13 CN CN2012104501293A patent/CN103091442A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060105392A1 (en) * | 2003-03-13 | 2006-05-18 | Paul Lehmann | Differential diagnosis of vitamin B12, vitamin B6, and folic acid disorders |
| WO2006090428A2 (en) * | 2005-02-28 | 2006-08-31 | Giuseppe Lazzarino | Method for the separation and simultaneous direct determination of compounds belonging to at least one of the groups chosen among purines and pyrimidines, n- acetylated amino acids, mono and dicarboxylic acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes, vitamins of group b, and derivatives ther |
| CN101961342A (en) * | 2010-08-19 | 2011-02-02 | 齐慧 | Water soluble vitamin composition for injection and preparation method thereof |
| CN102072846A (en) * | 2010-12-06 | 2011-05-25 | 贵州神奇药业股份有限公司 | Method for detecting quality of compound capsule prepared from 8 kinds of amino acids and 11 kinds of vitamins |
| CN102226795A (en) * | 2011-03-31 | 2011-10-26 | 宁波双伟制药有限公司 | Method for determining content of vitamin B12 in complex vitamin B and diclofenac sodium tablet through high performance liquid chromatography |
Non-Patent Citations (8)
| Title |
|---|
| HUA-BIN LI ET AL: "Determination of vitamin B12 in multivitamin tablets and fermentation medium by high-performance liquid chromatography with fluorescence detection", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| SURAPOTE WONGYAI: "Determination of vitamin B12 in multivitamin tablets by multimode high-performance liquid chromatography", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| 刘毅 等: "HPLC测定维生素B12注射液的含量和有关物质", 《中国药学杂志》 * |
| 刘毅 等: "维生素B12有关物质检查方法的探讨", 《药物分析杂志》 * |
| 王虎 等: "高效液相色谱法测定复方维生素胶囊中维生素B12的含量", 《中国药房》 * |
| 莫建光 等: "高效液相色谱法测定保健食品中维生素B12的含量", 《广西科学院学报》 * |
| 裴琳 等: "快速分离液相色谱法测定多维元素片中微量维生素B12的含量", 《中国药师》 * |
| 郝岩平 等: "HPLC法测定婴幼儿配方奶粉复合维生素添加剂中维生素B12方法的研究", 《中国食品添加剂》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104198598A (en) * | 2014-07-15 | 2014-12-10 | 汤臣倍健股份有限公司 | Determination method for vitamin B12 |
| CN104198598B (en) * | 2014-07-15 | 2016-03-16 | 汤臣倍健股份有限公司 | A kind of assay method of cobalamin |
| CN107860847A (en) * | 2017-11-14 | 2018-03-30 | 济南维瑞医药科技开发有限公司 | Vitamin B in one kind detection multivitamin preparation12Method about material |
| CN109917037A (en) * | 2019-03-26 | 2019-06-21 | 广西壮族自治区食品药品检验所 | Gentamicin procaine ties up the HPLC detection method of vitamin B12 content in B12 particle/capsule |
| CN114113359A (en) * | 2021-05-07 | 2022-03-01 | 佛山市南海北沙制药有限公司 | Central control detection method of 7-ACA derivative |
| CN114113359B (en) * | 2021-05-07 | 2024-02-20 | 佛山市南海北沙制药有限公司 | Central control detection method of 7-ACA derivative |
| CN113376285A (en) * | 2021-06-11 | 2021-09-10 | 贵州省产品质量检验检测院 | Liquid chromatography for determining vitamin B12 addition in infant food and milk |
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Application publication date: 20130508 |