CN114371243A - HPLC detection method of d-biotin - Google Patents
HPLC detection method of d-biotin Download PDFInfo
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- CN114371243A CN114371243A CN202111623458.9A CN202111623458A CN114371243A CN 114371243 A CN114371243 A CN 114371243A CN 202111623458 A CN202111623458 A CN 202111623458A CN 114371243 A CN114371243 A CN 114371243A
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- biotin
- acetonitrile
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- phosphoric acid
- trifluoroacetic acid
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 title claims abstract description 90
- 238000001514 detection method Methods 0.000 title claims abstract description 34
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 15
- 229940068840 d-biotin Drugs 0.000 title claims abstract description 14
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 57
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 46
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 17
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 8
- 150000002500 ions Chemical class 0.000 claims abstract description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 5
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000000741 silica gel Substances 0.000 claims abstract description 5
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 5
- 239000000945 filler Substances 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 69
- 238000010829 isocratic elution Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 10
- 239000007924 injection Substances 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 4
- 239000008213 purified water Substances 0.000 claims description 4
- 238000012856 packing Methods 0.000 claims description 2
- BOFKNJIZADZUTP-UHFFFAOYSA-N CC#N.OP(O)(O)=O.OC(=O)C(F)(F)F Chemical compound CC#N.OP(O)(O)=O.OC(=O)C(F)(F)F BOFKNJIZADZUTP-UHFFFAOYSA-N 0.000 claims 1
- 239000012071 phase Substances 0.000 claims 1
- IZGYIFFQBZWOLJ-CKAACLRMSA-N phaseic acid Chemical compound C1C(=O)C[C@@]2(C)OC[C@]1(C)[C@@]2(O)C=CC(/C)=C\C(O)=O IZGYIFFQBZWOLJ-CKAACLRMSA-N 0.000 claims 1
- 230000014759 maintenance of location Effects 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000003908 quality control method Methods 0.000 abstract description 2
- 229960002685 biotin Drugs 0.000 description 38
- 235000020958 biotin Nutrition 0.000 description 38
- 239000011616 biotin Substances 0.000 description 38
- ZHGNHOOVYPHPNJ-UHFFFAOYSA-N Amigdalin Chemical compound FC(F)(F)C(=O)OCC1OC(OCC2OC(OC(C#N)C3=CC=CC=C3)C(OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C2OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C1OC(=O)C(F)(F)F ZHGNHOOVYPHPNJ-UHFFFAOYSA-N 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 5
- 238000003556 assay Methods 0.000 description 4
- 108090001008 Avidin Proteins 0.000 description 3
- 238000009739 binding Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000004007 reversed phase HPLC Methods 0.000 description 3
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000000825 ultraviolet detection Methods 0.000 description 2
- LLIANSAISVOLHR-GBCQHVBFSA-N 5-[(3as,4s,6ar)-2-oxidanylidene-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 LLIANSAISVOLHR-GBCQHVBFSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 230000037037 animal physiology Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000021523 carboxylation Effects 0.000 description 1
- 238000006473 carboxylation reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
The invention provides a method for detecting d-biotin by HPLC, which takes conventional bonded silica gel as a chromatographic column filler, adopts an ultraviolet detector, takes an acetonitrile-water system as a mobile phase, and simultaneously adds trifluoroacetic acid and phosphoric acid double ions to carry out HPLC detection on a reagent. The method provides a better peak type and high detection sensitivity for the HPLC detection standard of the d-biotin, enhances the retention function, and provides a quality control standard reference for an experiment using the d-biotin.
Description
Technical Field
The invention belongs to the field of chemical analysis, and particularly relates to an HPLC (high performance liquid chromatography) detection method of d-biotin.
Background
d-Biotin (d-Biotin) has the chemical formula C10H16O3N2S and the molecular weight 244, which are widely distributed in nature, are one of vitamins essential to the biological life activity, have irreplaceable effects on the animal physiology, participate in carboxylation and decarboxylation in organisms, play an important role in the synthesis of fatty acid and purine in the metabolic process and the metabolism of fatty acid, and are already abundantThe method is applied to the aspects of medicine, detection and the like.
The very stable, non-covalent interaction between biotin and avidin is one of the most commonly used tools in the fields of chemistry and biology. Biotin has high affinity and binding to avidin, streptavidin, and the like, and the binding is the strongest non-covalent action in nature (Kd 10-15M). In the field of polypeptide protein drugs, the interaction between biotin and avidin has been widely utilized for a variety of purposes such as protein purification, detection, immobilization, drug targeting, protein structure analysis, and the like.
With the wider application of d-biotin in detection and structural analysis, the development of methods for detecting the purity of biotin becomes more and more important, and the methods are required to be accurate and easy to implement, and the detection sensitivity of biotin is required to be improved as much as possible.
Because the biotin molecule has carboxyl and is a weakly acidic compound, part of the biotin molecule exists in an ionic form in a neutral solution, and the polarity is strong, almost no retention exists on a reversed phase chromatographic column, only an acetonitrile-water system is used as a mobile phase, and biotin generates a peak before 3min, almost no retention exists and the peak shape extends forwards and widens no matter what proportion of acetonitrile-water. CN101504393B has certain effect on retention of biotin in a column and improvement of peak shape tailing by selecting a certain amount of trifluoroacetic acid as acetonitrile of an ion pair reagent and a water system as a mobile phase, but can not meet the requirement of sensitivity.
In view of this, the present application is presented.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide an HPLC detection method of biotin, which mainly solves the problem of high-sensitivity d-biotin purity detection under an HPLC method.
In order to solve the technical problem, the invention discloses an HPLC detection method of d-biotin, which takes conventional bonded silica gel as a chromatographic column filler, adopts an ultraviolet detector, takes an acetonitrile-water system as a mobile phase, and simultaneously adds trifluoroacetic acid and phosphoric acid double ions to carry out HPLC detection on a reagent.
Preferably, trifluoroacetic acid aqueous solution with mass concentration of 0.05 percent-acetonitrile-phosphoric acid is selected as the mobile phase.
Specifically, the volume ratio of the trifluoroacetic acid aqueous solution to the acetonitrile to the phosphoric acid in the mobile phase is 60-80: 40-20: 0.5-1, and the flow rate is 0.8-1.2 mL/min.
Preferably, the detection wavelength is 210-220nm, isocratic elution is carried out, the injection volume is 10 mu L, and the column temperature is 30-40 ℃.
In a preferred embodiment, the conventional bonded silica gel is used as a chromatographic column packing, an ultraviolet detector is adopted, 0.05% trifluoroacetic acid aqueous solution-acetonitrile-phosphoric acid is selected as a mobile phase, and the volume ratio of the trifluoroacetic acid aqueous solution to the acetonitrile to the phosphoric acid in the mobile phase is 60-80: 40-20: 0.05-0.1, flow rate 0.8-1.2mL/min, detection wavelength 210 and 220nm, isocratic elution, sample introduction volume of 10 mu L and column temperature of 30-40 ℃.
Preferably, the volume ratio of aqueous trifluoroacetic acid, acetonitrile to phosphoric acid in the mobile phase is 75: 25: 0.1.
preferably, the flow rate is 1.0mL/min, the detection wavelength is 210nm, isocratic elution is carried out, the injection volume is 10 μ L, and the column temperature is 35 ℃.
In a preferred embodiment, the volume ratio of aqueous trifluoroacetic acid, acetonitrile to phosphoric acid in the mobile phase is 75: 25: 0.1; the flow rate is 1.0mL/min, the detection wavelength is 210nm, isocratic elution is carried out, the sample injection volume is 10 mu L, and the column temperature is 35 ℃.
Has the advantages that: the invention selects trifluoroacetic acid and phosphate double ions as mobile phases to acetonitrile and a water system of a reagent, obtains better peak pattern and detection sensitivity, plays a role in enhancing the retention of biotin chromatography, provides better peak pattern and high detection sensitivity for the HPLC detection standard of d-biotin, provides a method for enhancing the retention function, and provides quality control standard reference for experiments using d-biotin.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 is a column effect verification analysis chart of a chromatographic column;
FIG. 2 is a graph showing the analysis of biotin at a concentration of 0.5mg/mL under mobile phase conditions of 0.05% aqueous TFA and acetonitrile (75: 25);
FIG. 3 is a 0.1mg/mL biotin assay under mobile phase conditions of 0.05% aqueous TFA and acetonitrile (75: 25);
FIG. 4 is a 0.05mg/mL biotin assay under mobile phase conditions of 0.05% aqueous TFA and acetonitrile (75: 25);
FIG. 5 is a 0.5mg/mL biotin profile under mobile phase conditions of 0.05% aqueous TFA, phosphoric acid and acetonitrile (75: 0.1: 25);
FIG. 6 is a 0.1mg/mL biotin assay under mobile phase conditions of 0.05% aqueous TFA, phosphoric acid, and acetonitrile (75: 0.1: 25);
FIG. 7 is a 0.05mg/mL biotin concentration profile under mobile phase conditions of 0.05% aqueous TFA, phosphoric acid and acetonitrile (75: 0.1: 25);
FIG. 8 is a 0.5mg/mL biotin assay under mobile phase conditions of 0.05% aqueous TFA, phosphoric acid, and acetonitrile (75: 0.05: 25);
FIG. 9 is a 0.05mg/mL biotin concentration profile under mobile phase conditions of 0.05% aqueous TFA, phosphoric acid and acetonitrile (75: 0.05: 25).
Detailed Description
Instruments and reagents:
high performance liquid chromatograph (Agilent 1260), chromatographic column is Ultimate LP-C18-5um, 4.6 × 250 mm.
Acetonitrile, phosphoric acid and TFA were chromatographically pure, ethanol was analytically pure, water was purified water, and biotin was a product sold by the company Aladdin (cat. No. B105434).
Column effect experiment of chromatographic column:
and (3) sampling and detecting a mixing system stability experiment standard product, and measuring the column efficiency and the separation degree, wherein the result is shown in figure 1, the theoretical plate number is more than 5000, and the separation degree meets the requirement.
And (3) taking biotin samples with the concentrations of 0.5mg/mL and 0.05mg/mL, respectively placing for 2h and 4h, and then carrying out sample detection, wherein the result shows that the peak area and percentage of each peak of a chromatographic peak have no obvious change, and the RSD is within 1%.
Examples
Biotin is colorless needle crystal, slightly soluble in cold water, soluble in ethanol, resistant to high-pressure heating in acidic solution, easily destroyed by heating in alkaline solution, and easily oxidized to lose physiological activity. Therefore, biotin is accurately weighed, a small amount of ethanol is added, purified water is used for dissolving the biotin, the volume is determined to be 1.0mg/mL, the biotin is dissolved by heating in water bath at 40 ℃, the pure water is diluted to be 0.5mg/mL, 0.1mg/mL and 0.05mg/mL after the biotin is filtered by a 0.22um filter membrane, and the biotin is to be detected.
And (3) taking the same dissolving solvent as a reference solution, and scanning a 0.5mg/mL biotin sample by using an ultraviolet spectrophotometer (the wavelength is 190-300 nm) to determine the optimal ultraviolet detection wavelength of the biotin. And finally selecting 210nm as the optimal ultraviolet detection wavelength by scanning and combining the acetonitrile ultraviolet absorption limit value under the condition of ensuring the maximum absorption value.
Respectively detecting three-concentration samples of 0.5mg/mL, 0.1mg/mL and 0.05mg/mL by an Agilent 1260 II reversed-phase high performance liquid chromatography analyzer with ultraviolet as a detector and isocratic elution; the mobile phase comprises 0.05 percent of TFA aqueous solution and acetonitrile (75: 25) by mass percent, and is eluted at equal intervals, the detection wavelength is 210nm, the flow rate is 1.0mL/min, the injection volume is 20ul, and the column temperature is 35 ℃. The results of the analysis are shown in FIGS. 2 to 4.
Respectively detecting 0.5mg/mL, 0.1mg/mL and 0.05mg/mL three-concentration samples by an Agilent 1260 II reverse phase high performance liquid chromatography analyzer, wherein mobile phases comprise 0.05 percent of TFA aqueous solution, phosphoric acid and acetonitrile (75: 0.1: 25) in percentage by mass, isocratic elution is carried out, the detection wavelength is 210nm, the flow rate is 1.0mL/min, the sample injection volume is 20 mu l, and the column temperature is 35 ℃. The analysis results are shown in FIG. 5, FIG. 6 and FIG. 7.
Respectively detecting samples with concentration of 0.5mg/mL and 0.05mg/mL by an Agilent 1260 II reversed-phase high performance liquid chromatography analyzer, wherein mobile phases comprise TFA aqueous solution with mass percent of 0.05%, phosphoric acid and acetonitrile (75: 0.05: 25), isocratic elution is carried out, the detection wavelength is 210nm, the flow rate is 1.0mL/min, the sample injection volume is 20 mu l, and the column temperature is 35 ℃. The analysis results are shown in FIG. 8 and FIG. 9.
From the results of all spectrograms, the biotin detection retention time of the mobile phase of TFA acetonitrile aqueous solution containing 0.05 percent or 0.1 percent of phosphoric acid is obviously superior to that of the mobile phase without phosphoric acid, so that the synchronous detection and monitoring of other reaction substrates and reaction products are convenient during biotin binding reaction, and simultaneously, under a double-ion pair reagent, the whole peak area of biotin is obviously higher than the condition of only using TFA as the ion pair reagent, so that the sensitivity is higher, and the related reaction monitoring is more facilitated.
The area of biotin peak is slightly better than 0.05% when 0.1% phosphoric acid is added into the mobile phase, which indicates that the detection sensitivity of biotin is slightly higher than 0.05% phosphoric acid concentration after 0.1% phosphoric acid is added into the mobile phase. The pH of the aqueous solution containing 0.05% TFA and 0.1% phosphoric acid was already close to 1.5, and no attempt was made to add phosphoric acid at a concentration of more than 0.1% by mass of phosphoric acid in view of the tolerance of the column to the pH range.
The invention makes biotin exist in molecular form by inhibiting the ionization of biotin in the mobile phase of weakly acidic medium, reduces polarity, prolongs the retention time of biotin molecules on a reversed phase column, and improves the shape of biotin efflux peak.
The present invention provides a method and a concept of HPLC of d-biotin, and a plurality of methods and ways for implementing the technical scheme, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (9)
1. An HPLC detection method of d-biotin is characterized in that conventional bonded silica gel is used as a chromatographic column filler, an ultraviolet detector is adopted, an acetonitrile-water system is used as a mobile phase, and trifluoroacetic acid and phosphoric acid double ions are added to carry out HPLC detection on a reagent.
2. The method according to claim 1, wherein 0.05% by mass of aqueous trifluoroacetic acid-acetonitrile-phosphoric acid is used as the mobile phase.
3. The process according to claim 2, characterized in that the volume ratio of aqueous trifluoroacetic acid, acetonitrile and phosphoric acid in the mobile phase is 60-80: 40-20: 0.05-0.1, and the flow rate is 0.8-1.2 mL/min.
4. The method as claimed in claim 1, wherein the detection wavelength is 210-220nm, the isocratic elution is performed, the injection volume is 10 μ L, and the column temperature is 30-40 ℃.
5. The method according to claim 1, characterized in that conventional bonded silica gel is used as a chromatographic column packing, an ultraviolet detector is adopted, trifluoroacetic acid aqueous solution with a mass concentration of 0.05% -acetonitrile-phosphoric acid is used as a mobile phase, and the volume ratio of the trifluoroacetic acid aqueous solution to the acetonitrile to the phosphoric acid in the mobile phase is 60-80: 40-20: 0.05-0.1, flow rate 0.8-1.2mL/min, detection wavelength 210 and 220nm, isocratic elution, sample introduction volume of 10 mu L and column temperature of 30-40 ℃.
6. The process according to claim 5, characterized in that the volume ratio of aqueous trifluoroacetic acid, acetonitrile and phosphoric acid in the mobile phase is 75: 25: 0.1.
7. the method according to claim 5, wherein the flow rate is 1.0mL/min, the detection wavelength is 210nm, isocratic elution is performed, the sample injection volume is 10 μ L, and the column temperature is 35 ℃.
8. The process according to claim 5, characterized in that the volume ratio of aqueous trifluoroacetic acid, acetonitrile and phosphoric acid in the mobile phase is 75: 25: 0.1; the flow rate is 1.0mL/min, the detection wavelength is 210nm, isocratic elution is carried out, the sample injection volume is 10 mu L, and the column temperature is 35 ℃.
9. The method according to any one of claims 1 to 8, wherein d-biotin is added with 30% ethanol by volume before sample injection, dissolved in purified water and made to a volume of 1.0mg/mL, dissolved by heating in a water bath at 40 ℃ and diluted to 0.05-0.5 mg/mL in purified water, and filtered through a 0.22 μm filter for use.
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|---|---|---|---|---|
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-
2021
- 2021-12-28 CN CN202111623458.9A patent/CN114371243A/en active Pending
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|---|---|---|---|---|
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Non-Patent Citations (2)
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| 裘丞军， 任玉琴: "高效液相色谱法测定饲料添加剂 D-生物素的含量", 中国饲料, no. 19, 31 December 2018 (2018-12-31), pages 80 - 84 * |
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