CN115236259B - High performance liquid chromatography determination method for residual citric acid in Fmoc-amino acid - Google Patents
High performance liquid chromatography determination method for residual citric acid in Fmoc-amino acid Download PDFInfo
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- CN115236259B CN115236259B CN202210860699.3A CN202210860699A CN115236259B CN 115236259 B CN115236259 B CN 115236259B CN 202210860699 A CN202210860699 A CN 202210860699A CN 115236259 B CN115236259 B CN 115236259B
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 title claims abstract description 134
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 23
- 150000001413 amino acids Chemical class 0.000 claims abstract description 23
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 43
- 239000000523 sample Substances 0.000 claims description 32
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- 239000004743 Polypropylene Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- -1 polypropylene Polymers 0.000 claims description 4
- 229920001155 polypropylene Polymers 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 11
- 230000002378 acidificating effect Effects 0.000 abstract description 2
- 239000007853 buffer solution Substances 0.000 abstract description 2
- 239000008346 aqueous phase Substances 0.000 abstract 1
- 238000000354 decomposition reaction Methods 0.000 abstract 1
- 238000004811 liquid chromatography Methods 0.000 abstract 1
- 238000007781 pre-processing Methods 0.000 abstract 1
- 230000001360 synchronised effect Effects 0.000 abstract 1
- 238000011084 recovery Methods 0.000 description 8
- 239000012071 phase Substances 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 5
- 239000000047 product Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 238000004451 qualitative analysis Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- KLBPUVPNPAJWHZ-UUWRZZSWSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UUWRZZSWSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 238000002479 acid--base titration Methods 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 238000007813 chromatographic assay Methods 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 229940064958 chromium citrate Drugs 0.000 description 1
- SWXXYWDHQDTFSU-UHFFFAOYSA-K chromium(3+);2-hydroxypropane-1,2,3-tricarboxylate Chemical compound [Cr+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O SWXXYWDHQDTFSU-UHFFFAOYSA-K 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
Classifications
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N30/16—Injection
-
- 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
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a high performance liquid chromatography determination method for residual citric acid in Fmoc-amino acid, which mainly solves the technical problems that a sample needs to be pretreated and a link is complex and a long time is consumed in the existing high performance liquid chromatography method. The measuring method comprises the following steps: firstly, preparing a measured reagent, secondly, and preprocessing a sample; thirdly, performing high performance liquid chromatography determination, wherein a DAD ultraviolet detector is adopted in the liquid chromatography determination process, and KH is added in the high performance liquid chromatography aqueous phase 2 PO 4 The buffer solution can well ensure that the sample is not disturbed by decomposition under the acidic condition; the invention realizes the undisturbed synchronous detection of the sample to be detected, and provides a reliable and convenient detection means for the field of detection of the residual citric acid in the amino acid series.
Description
Technical Field
The invention relates to a high performance liquid chromatography determination method of amino acid, in particular to a high performance liquid chromatography determination method of residual citric acid in Fmoc-amino acid.
Background
The conventional citric acid content measurement needs to use a visible spectrophotometer, citric acid can be used for reducing Cr6+ into Cr3+ under an acidic condition, a chromium citrate chelate is generated, a product has a characteristic absorption peak at 545 nm, and the content of the citric acid can be quantitatively detected through the change of the absorption value. The operation is troublesome; there are also high performance liquid chromatography methods that require pretreatment of the sample, such as dissolution of the sample in ethyl acetate; firstly, carrying out ultrasonic treatment for 30 min; standing overnight, and adding ethyl acetate to fix the volume; filtering again, taking 25 mL filtrate, and evaporating to dryness; formic acid was added for dissolution to a volume of 10mL, followed by HPLC. The HPLC detection needs a certain synthetic experience, has complex links, long time consumption and long report period of the whole detection, and is troublesome.
The national standard method is to use the pH color-changing range of the phenolphthalein indicator, use 1N equivalent sodium hydroxide standard solution to carry out acid-base titration detection, obtain the content of the citric acid through calculation, and the method is more convenient for detecting pure citric acid products, detects the citric acid remained below 0.5%, has large final result error, is not easy to operate, and the like.
Disclosure of Invention
The invention provides a high performance liquid chromatography determination method for residual citric acid in amino acid, which mainly solves the technical problems that the existing high performance liquid chromatography method is used for sample pretreatment, complicated link and long time consumption. The invention can be used for qualitative analysis and quantitative determination of the content of the residual citric acid in the amino acid, is simple, accurate and reliable, and is a high-performance liquid chromatography analysis method capable of analyzing the residual citric acid in the amino acid in a short time.
The invention adopts the following technical scheme: the high performance liquid chromatography determination method of the residual citric acid in the amino acid comprises the following steps:
step one, preparing a measured reagent, wherein the measured reagent comprises acetonitrile-chromatographic purity, ultrapure water, chemical reagent potassium dihydrogen phosphate and analytically pure potassium dihydrogen phosphate.
Step two: preparing a sample solution, weighing about 0.10g of a sample to be accurate to 0.0001 and g, placing the sample in a 10mL volumetric flask, adding 5mL acetonitrile, dissolving the sample in ultrasonic at room temperature for 3 min, then fixing the volume with water to 10mL, finally filtering the sample by using a 0.22 mu m polypropylene organic filter membrane, performing HPLC analysis, dissolving the sample in acetonitrile to be transparent when the amino acid in the sample is not transparent, and finally calculating the sample solution according to the converted weight.
Preparing a standard solution: accurately weighing standard product 0.010 and g of citric acid (accurate to 0.00001 and g), dissolving with 5ml of pure water, diluting with acetonitrile to 10mL volumetric flask, fixing volume, shaking, preparing 1000 mg/L standard stock solution, and sealing and storing at 25deg.C.
Step three, high performance liquid chromatography determination is carried out; the chromatographic assay procedure was: the chromatographic condition is that the temperature of a sample chamber is normal temperature, the sample injection volume of the automatic sample injector is 10 mu L, and the chromatographic column is: agilent ZORBAX SB phenyl C18 (4.6 mm. Times.250 mm, 5.0 μm), column temperature: 30. DEG C, flow rate: 1.0mL/min, mobile phase 10mmolKH 2 PO 4 The aqueous solution (A) and the pure acetonitrile (B) are subjected to gradient elution program, wherein V (B) is 40 to 60 percent, 20.0 to 20.1 min and V (B) is 60 to 60 percent40% of the initial proportion, 20.1-25 min, and V (B) is balanced from 40% of the system; the non-natural amino acid with Fmoc protection is not separated out in the chromatographic column by controlling the proportion of the mobile phase and the water phase; i.e. the main component of citric acid was collected by the chromatographic peak with retention time of 2.0 min.
The chromatograph used in the liquid chromatograph measurement is Agilent 1260 chromatograph, and the chromatograph adopts a DAD detector.
The invention has the following beneficial effects: the method is simple, convenient, quick and accurate in operation, high in sensitivity (0.01% of citric acid can be accurately detected in the amino acid), good in stability, suitable for the method for detecting the high-performance liquid chromatography of the citric acid residue in the unnatural amino acid with Fmoc protection, simple, quick and accurate in operation, good in stability, suitable for qualitative analysis and quantitative detection of the citric acid residue in the unnatural amino acid with Fmoc protection, and good in recovery rate and recovery effect when the silica gel is adsorbed. The invention adopts the accurate quantification of the automatic sampler of the high performance liquid chromatograph, and the sensitivity of the DAD diode array can have a good response value to 0.1mg/ml citric acid, and the accurate quantification of the concentration of 0.01 percent of residual citric acid in the amino acid is detected at the lowest; simultaneously utilize KH 2 PO 4 The buffer solution prevents citric acid from being dissociated, so that the peak type of the citric acid is symmetrical, the temperature of the sample chamber is set to be normal temperature, the preservation of the sample at the room temperature is facilitated, the temperature of the sample chamber is close to that of a column temperature box, and the stable operation of the whole liquid phase system is facilitated.
Drawings
FIG. 1 is a chromatogram of a citric acid standard of the present invention.
FIG. 2 is a chromatogram of the residual citric acid in an amino acid of the present invention.
Fig. 3 is a graph of the linear range, linear equation, and correlation coefficient of citric acid of the present invention.
Detailed Description
The measurement procedure of the present invention is further described by way of examples.
The main instrument of the experimental part:
agilent 1260 type high performance liquid chromatograph equipped with DAD detector, agilent ZORBAX SB phenyl C column, agilent ZORBAX SB phenyl C column size (4.6 mm×250mm, 5.0 μm); ultrasonic extractor (KQ 5200E, zhengzhou Kotai laboratory equipment Co., ltd.), 0.22 μm polypropylene organic filter (Shanghai Anaesthetic science instruments Co., ltd.), electronic analytical balance (CPA 225D, saidolesco instruments Co., ltd.).
The reagents measured included: reagent grade potassium dihydrogen phosphate, ultrapure water; acetonitrile (chromatographic purity, merck, germany), potassium dihydrogen phosphate (analytical purity, shanghai nationality); ultrapure water (resistivity 18.2M. OMEGA. Cm, U.S. Qu Cengshi).
Preparing a standard solution: accurately weighing standard product 0.010 and g of citric acid (accurate to 0.00001 and g), dissolving with 5ml of pure water, diluting with acetonitrile to 10mL volumetric flask, fixing volume, shaking, preparing 1000 mg/L standard stock solution, and sealing and storing at 25deg.C.
Pretreatment of the sample: about 0.1g (accurate to 0.0001 and g) of the amino acid sample was weighed, placed in a 10mL volumetric flask, 5mL acetonitrile was added, and the sample was dissolved by ultrasonic waves at room temperature for 3 minutes, and the volume was fixed to 100 mL with pure water. Finally, the sample is filtered by a 0.22 mu m polypropylene organic filter membrane and then subjected to HPLC analysis.
Analysis of chromatographic conditions: the temperature of the sample chamber is 25 ℃, the sample injection volume of the automatic sample injector is 10 mu L, and the chromatographic column is: agilent ZORBAX SB phenyl C column size 4.6mm×250mm, 5.0 μm column temperature: 30. DEG C, flow rate: 1.0mL/min, mobile phase 10mmolKH 2 PO 4 An aqueous buffer solution (a) and pure acetonitrile (B); adopting a gradient elution program, wherein V (B) is from 40% to 60%,20.0 to 20.1 min, V (B) is from 60% to 40% of initial proportion, 20.1 to 25 min, and V (B) is from 40% of balance system; the chromatogram of the standard substance is shown in figure 1, and the chromatogram of the actual sample is shown in figure 2. Quantification was performed by peak area contrast, i.e. a chromatographic peak with retention time of 2.0 min.
Selection and optimization of a sample pretreatment method: the present invention relates to a method for preparing citric acid, which is characterized by that the amino acid is (Fmoc-D-Cys (Trt) -OH) produced by Shanghai Ji Feng Biotechnology Co., ltd.A lot of amino acids are on the market, citric acid is mainly used for regulating acidity in Fmoc-D-Cys (Trt) -OH production process, according to the principle of easy dissolution of citric acid in water, the present invention tests the actual condition of citric acid remained in amino acid, and through test practice, 0.1g citric acid remained below 1.0% in non-natural amino acid with Fmoc can be completely dissolved as long as 5ml water is present in a 10ml volumetric flask.
Optimization of instrument conditions: optimization of chromatographic conditions
The invention respectively selects water-acetonitrile and 10mmolKH 2 PO 4 The separation effect was compared with the case of buffer aqueous solution-acetonitrile as mobile phase. When a water-acetonitrile system is adopted, the chromatographic peak shape is asymmetric and is easy to tailing; by 10mmolKH 2 PO 4 The symmetry degree of the peak form of the citric acid reaches the standard when the acetonitrile system is adopted, and the separation degree, the sensitivity and the peak form of the target compound are superior to those of other leaching systems. Therefore, the experiment adopts 10mmolKH 2 PO 4 The buffer aqueous solution (phase A) -pure acetonitrile (phase B) is used as eluent, and the chromatographic separation is carried out by gradient elution.
The invention also compares WatersPuresil C18 (4.6mm×150mm, 5.0 μm) chromatographic columns with Agilent ZORBAX SB phenyl-C18 (4. mm X106 mm, 4.0 μm) and Agilent ZORBAX SB phenyl C18 (4.6mm×250mm, 5.0 μm) when selecting chromatographic columns, and experimental results show that the first two chromatographic columns can also separate peaks, but the half-width is larger, while the Agilent ZORBAX SB phenyl C (4.6mm×250mm, 5.0 μm) chromatographic columns can completely separate the main peak of citric acid from impurities, the peak is symmetrical and the peak is narrower, and the detection limit of target compounds can be reduced. The present invention therefore employs a Agilent ZORBAX SB phenyl C (4.6 mm. Times.250 mm, 5.0 μm) column.
The temperature of the sample chamber is set to be room temperature, which is favorable for the preservation of acetonitrile solvent in the sample chamber, and the temperature of the column temperature box is set to be 30 ℃.
Linear range: and (3) taking 50% volume water without citric acid and 50% volume acetonitrile as blank matrixes, adding different volumes of concentration standard solutions to prepare 200mg/L, 400mg/L, 600mg/L and 800mg/L, measuring according to the chromatographic conditions, and taking the mass concentration (x, mug/L) of the components as an abscissa and the corresponding mass spectrum peak area (y) as an ordinate to obtain a standard curve. The results show that: citric acid has good linear relationship in a certain range. See fig. 3.
Precision and recovery: for comparison of the precision and recovery of the present invention, citric acid recovery was selected and measured as described above for 24 hours, each concentration level was measured separately 2 times, and recovery and relative standard deviation are listed in the following table. The following table shows that the recovery rate of citric acid is 97.8% -98.8% and the relative standard deviation is 0.2-0.3%, so that the method has good recovery rate and repeatability on citric acid, and can meet the analysis requirement of actual samples.
Determination of the actual sample: the detection result of the method for detecting the residual citric acid sample in the unnatural amino acid with Fmoc shows that the method is quick, accurate, high in sensitivity, good in selectivity, short in detection period and practical application value.
The invention establishes a high performance liquid chromatography for determining residual citric acid in amino acid. The method is simple, quick and accurate in operation, good in stability and suitable for qualitative analysis and quantitative detection of the residual citric acid in the amino acid. Provides a new method for detecting the residual citric acid in the amino acid, and has a certain practical significance.
Claims (1)
- A high performance liquid chromatography assay for residual citric acid in fmoc-amino acids, characterized by: the method comprises the following steps: step one, preparing a measured reagent: the reagents measured included acetonitrile-chromatographic purity, ultrapure water, analytically pure potassium dihydrogen phosphate; preparing a sample solution, weighing a sample, placing the sample in a volumetric flask, adding acetonitrile, dissolving by adopting ultrasonic wave at room temperature, adding ultrapure water to fix the volume, and finally using polypropylene for the sampleFiltering with organic filter membrane, and performing high performance liquid chromatography; step three, high performance liquid chromatography determination is carried out: the chromatographic condition is that the temperature of the sample chamber is normal temperature, and the sample injector is automatic; the chromatograph is an Agilent 1260 liquid chromatograph, and the chromatograph adopts DAD as a detector; the chromatographic column is as follows: agilent ZORBAX SB phenyl C18 column, size 4.6mm×250mm, 5.0 μm, column temperature: 30. DEG C, flow rate: 1.0 mL/min; mobile phase 10mmol/LKH 2 PO 4 Aqueous solution (a) and pure acetonitrile (B), using a gradient elution procedure: 0-20 min, the proportion of mobile phase B is 40-60%, 20.0-20.1 min, the proportion of mobile phase B is 60-40% of the initial proportion, 20.1-25 min, and the proportion of mobile phase B is 40% of the balance system; the proportion of the mobile phase and the water phase is controlled, so that the unnatural amino acid is not separated out in the chromatographic column; the retention time of citric acid was 2.0 min.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110118843A (en) * | 2019-06-11 | 2019-08-13 | 南京华盖制药有限公司 | The method for measuring citric acid content in sucking ambroxol hydrochloride solution |
| CN112526051A (en) * | 2020-12-18 | 2021-03-19 | 上海吉奉生物科技有限公司 | Fmoc-lysine high performance liquid chromatography determination method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110118843A (en) * | 2019-06-11 | 2019-08-13 | 南京华盖制药有限公司 | The method for measuring citric acid content in sucking ambroxol hydrochloride solution |
| CN112526051A (en) * | 2020-12-18 | 2021-03-19 | 上海吉奉生物科技有限公司 | Fmoc-lysine high performance liquid chromatography determination method |
Non-Patent Citations (2)
| Title |
|---|
| 色谱法在饲料柠檬酸检测中的应用;林仙军等;《浙江畜牧兽医》(第5期);第11-12页 * |
| 高效液相色谱法检测葡萄酒中柠檬酸;何军风;《食品安全质量检测学报》;第9卷(第10期);第2512-2515页 * |
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