CN106872633A - A kind of rp-hplc analysis method of recombinant human lysozyme - Google Patents
A kind of rp-hplc analysis method of recombinant human lysozyme Download PDFInfo
- Publication number
- CN106872633A CN106872633A CN201710233245.2A CN201710233245A CN106872633A CN 106872633 A CN106872633 A CN 106872633A CN 201710233245 A CN201710233245 A CN 201710233245A CN 106872633 A CN106872633 A CN 106872633A
- Authority
- CN
- China
- Prior art keywords
- recombinant human
- human lysozyme
- concentration
- sodium chloride
- hplc analysis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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
- G01N30/89—Inverse chromatography
Landscapes
- 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)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of recombinant human lysozyme rp-hplc analysis method, the method using 20~50nm of aperture, 2~10 μm of particle diameter octadecylsilane chemically bonded silica as chromatographic column filler, both can allow this high molecular weight protein of recombinant human lysozyme to have certain retention time on post, the albumen will not be allowed to be combined with filler too strong and lead to not wash-out again；By adding sodium chloride in mobile phase, so as to ensure the dissolubility of the recombinant human lysozyme during rp-hplc analysis, can be with mobile phase complete miscibility, pedal number, symmetry, repeatability, the degree of accuracy of quantitative analysis of chromatographic peak are ensure that, can be used for the analysis of recombinant human lysozyme and quantitative determination.
Description
Technical field
The present invention relates to a kind of rp-hplc analysis method of recombinant human lysozyme.
Background technology
The mobile phase of regulation is pumped into the chromatographic column equipped with filler by high performance liquid chromatography system using high pressure pump, right
Test sample carries out the chromatographic process of separation determination.
The test sample of injection, is brought into post by mobile phase, when in mobile phase carry mixture flow through fixing phase when, its with
Fixing phase interacts.Due to difference of each component in property and structure in mixture, produced between fixing phase
The size of active force, strong and weak difference, with the movement of mobile phase, mixture is alternate by repeated multiple times partition equilibrium two,
So that the time that each component is retained by fixing phase is different, so as to be taken out of by mobile phase in a certain order, each group in mixture is realized
Point separation and sequentially enter detector, detection by integrator or data handling system record and treatment chromatographic signal.
Reversed-phased high performace liquid chromatographic：The polarity of mobile phase more than the polarity of fixing phase, i.e., is mutually solid with non-polar linkage
Determine phase (the silane C18 of Chang Yishi eight, octane C8, methyl C1, phenyl etc. are used as fixing phase)；Water or buffer solution, add methyl alcohol, second
The organic solvent miscible with water such as nitrile, isopropanol, acetone, tetrahydrofuran is mobile phase to the efficient of separation detection related substances
Liquid chromatography.
In biological medicine and protein detection analysis field, reversed-phased high performace liquid chromatographic is widely used in target substance
Analysis and quantitative determination.This protein of recombinant human lysozyme has its special physicochemical property, and inventor is had found with anti-phase
There is problems with when detecting in high-efficient liquid phase chromatogram technique analysis：(1) recombinant human lysozyme molecular weight is 14000Da, molecular weight phase
To larger, analyzed with conventional anti-phase C18 posts and albumen often occur and combined with filler too strong and lead to not elute, it is impossible to normally
Analyze and damage chromatographic column；(2) recombinant human lysozyme is salting-in protein, molten in the sodium-chloride water solution of more than 100mM
Solution property is good, and recombinant human lysozyme solution can be presented suspended state in the sodium-chloride water solution less than 100mM；Conventional anti-phase height
The sodium chloride of high concentration is not used in the mobile phase of effect liquid phase chromatogram, recombinant human lysozyme is in not during this will cause analysis
State is completely dissolved, though there is the chromatographic peak cannot also to accurately reflect its dose-effect relationship, and the albumen for not exclusively dissolving can also be damaged
Bad chromatographic system.
The content of the invention
It is an object of the invention to provide a kind of the anti-phase of the degree of accuracy of repeatability and quantitative analysis recombinant human lysozyme high
HPLC analytical method.
The technical scheme for solving above-mentioned technical problem use is made up of following step：
1st, recombinant human lysozyme standard items are dissolved in the sodium chloride ultra-pure water solution that mass concentration is 0.9%, are prepared
Into recombinant human lysozyme standard solution, standard solution is analyzed using RPLC, chromatography bar
Part：Chromatographic column is C18 reverse-phase chromatographic columns, and filler is 20~50nm of aperture, the octadecylsilane bonded silica of 2~10 μm of particle diameter
Glue, mobile phase A is the ultra-pure water solution that sodium chloride mass concentration is 0.9%, trifluoroacetic acid volumetric concentration is 0.1%, Mobile phase B
It is that acetonitrile and ultrapure water volume ratio are 6:Contain the sodium chloride and volume that mass concentration is 0.9% in 4 mixed liquor and mixed liquor
Concentration is 0.1% trifluoroacetic acid, carries out gradient elution, eluent gradient A:B is by (90~0):(10~100) to (50~0):
(50~100), Detection wavelength is 280nm, and flow velocity is 1mL/ minutes；Chromatogram main peak area is drawn to become with recombinant human lysozyme concentration
The standard curve of change.
2nd, recombinant human lysozyme sample to be measured is dissolved in the sodium chloride ultra-pure water solution that mass concentration is 0.9%, is used
Membrane filtration, collects filtrate, and filtrate is analyzed using RPLC, and chromatographiccondition is identical with step 1.
3rd, the chromatogram main peak area obtained according to step 2, the linear equation of the standard curve determined with reference to step 1 can be obtained
The concentration of recombinant human lysozyme in recombinant human lysozyme sample to be measured.
In above-mentioned steps 1, described preferably 0 to 30 minutes A of eluent gradient:B is by 55:45 to 5:95.
In above-mentioned steps 1, the preferred aperture 30nm of described filler, the octadecylsilane chemically bonded silica of 5 μm of particle diameter.
Beneficial effects of the present invention are as follows：
1st, the present invention uses 20~50nm of aperture, the octadecylsilane chemically bonded silica of 2~10 μm of particle diameter as chromatographic column
Filler, can both allow this high molecular weight protein of recombinant human lysozyme to have certain retention time on post, will not allow the egg again
Combined with filler in vain too strong and led to not wash-out.
2nd, it is of the invention by adding sodium chloride in mobile phase, so as to ensure during rp-hplc analysis
The dissolubility of recombinant human lysozyme, can be with mobile phase complete miscibility, it is ensured that the pedal number of chromatographic peak, symmetry, weight
Renaturation, the degree of accuracy of quantitative analysis.
3rd, first be completely dissolved in sodium chloride in ultra-pure water when Mobile phase B is prepared by the present invention, then miscible with acetonitrile, to keep away
Exempt from that sodium chloride is insoluble in acetonitrile, the proportioning for being used ensure that Mobile phase B good dissolubility and during global analysis
Good compatibility between A, B mobile phase.
4th, the repeatability of the inventive method is good, and the degree of accuracy is high, and detection limit is low, repeatability, system suitability, the hole of filler
Footpath particle diameter selection, the formula of mobile phase meets《Pharmacopoeia of People's Republic of China》The three annex III efficient liquid of B of version in 2015
Phase chromatography requirement, can be used for the analysis of recombinant human lysozyme and quantitative determination.
Brief description of the drawings
Fig. 1 is the relation curve of recombinant human lysozyme concentration and chromatogram main peak area in linearity and range experiment.
Fig. 2 is the relation curve of recombinant human lysozyme concentration and chromatogram main peak area in accuracy test.
Specific embodiment
The present invention is described in more detail with reference to the accompanying drawings and examples, but protection scope of the present invention is not limited only to
These embodiments.
Embodiment 1
1st, accurate weighing recombinant human lysozyme standard items 2896.0mg, wherein 150.0mg containing recombinant human lysozyme, adds matter
Measure the sodium chloride ultra-pure water that concentration is 0.9% to be settled in 50.0mL volumetric flasks, being configured to recombinant human lysozyme concentration is
The standard solution of 3.0mg/mL；Then it is accurate with the sodium chloride ultra-pure water solution that 5.0mL volumetric flasks, mass concentration are 0.9%
8 sample concentrations of dilution, respectively：0.30mg/mL、0.60mg/mL、0.90mg/mL、1.20mg/mL、1.50mg/mL、
1.80mg/mL, 2.10mg/mL, 2.40mg/mL, are divided the standard solution after dilution using RPLC
Analysis, chromatographiccondition：Chromatographic column is C18 reverse-phase chromatographic columns, and filler is aperture 30nm, the octadecylsilane key of 5 μm of particle diameter
Silica gel is closed, mobile phase A is the ultra-pure water solution that sodium chloride mass concentration is 0.9%, trifluoroacetic acid volumetric concentration is 0.1%, stream
Dynamic phase B is that acetonitrile and ultrapure water volume ratio are 6:In 4 mixed liquor and mixed liquor containing sodium chloride that mass concentration is 0.9% and
Volumetric concentration is 0.1% trifluoroacetic acid, carries out gradient elution, and eluent gradient selects 0 to 30 minutes A:B is by 55:45 to 5:
95, Detection wavelength is 280nm, and flow velocity is 1mL/ minutes, and applied sample amount is 20 μ L；Calculate the number of plates successively according to testing result, drag
The tail factor, chromatographic peak main peak area, the results are shown in Table 1.
The recombinant human lysozyme linearity and range chromatogram detection statistics result of table 1
Result of calculation according to table 1 draws standard curve of the chromatogram main peak area with recombinant human lysozyme change in concentration, knot
Fruit sees Fig. 1.The linear equation of standard curve is：Y=2435x+29044, x represents recombinant human lysozyme concentration in formula, and y represents color
Spectrum main peak area, R2=0.99964, linearly well, and pedal number, symmetry meet《Pharmacopoeia of People's Republic of China》2015
Year three requirements of annex III B high performance liquid chromatographies of version.
Accurate weighing recombinant human lysozyme standard items 96.53mg, wherein 5.0mg containing recombinant human lysozyme, adds quality dense
The sodium chloride ultra-pure water solution spent for 0.9% is settled in 5.0mL volumetric flasks, is configured to recombinant human lysozyme concentration for 1.0mg/
The standard solution of mL；Concentration is diluted to the sodium chloride ultra-pure water solution precision that 5.0mL volumetric flasks, mass concentration are 0.9%
Respectively：1.0×10-3mg/mL、1.0×10-4mg/mL、1.0×10-5mg/mL、1.0×10-6Mg/mL, using reversed phase high efficiency
Liquid chromatogram is analyzed to the standard solution after dilution, and chromatographiccondition is same as described above, and testing result is shown in Table 2.
The recombinant human lysozyme test limit of table 2 and quantitative limit chromatogram detection statistics result
From table 2, recombinant human lysozyme concentration is 1.0 × 10-5During mg/mL, main peak retention time is 14.86min, high
It is 0.43mV to spend, and baseline noise is highly about 0.04mV, and signal to noise ratio is about 11:1, recombinant human lysozyme concentration is 1.0 × 10- 6During mg/mL, main peak retention time is 14.86min, is highly 0.26mV, and baseline noise is highly about 0.04mV, and signal to noise ratio is about
It is 6:1, illustrate under the chromatographic condition, recombinant human lysozyme applied sample amount is linear good, recombined human in 6.0~48.0 μ g ranges
Quantifying for lysozyme is limited to 1.0 × 10-5Mg/mL × 20 μ L are 0.2ng, and detection is limited to 1.0 × 10-6Mg/mL × 20 μ L are
0.02ng。
2nd, recombinant human lysozyme sample to be measured is dissolved in the sodium chloride ultra-pure water solution that mass concentration is 0.9%, is used
Membrane filtration, collects filtrate, and filtrate is analyzed using RPLC, and chromatographiccondition is identical with step 1.
3rd, the chromatogram main peak area obtained according to step 2, the linear equation of the standard curve determined with reference to step 1 can be obtained
The concentration of recombinant human lysozyme in recombinant human lysozyme sample to be measured.
In order to prove the repeatability and accuracy of analysis method of the present invention, inventor has carried out substantial amounts of laboratory research examination
Test, specific test situation is as follows：
1st, replica test
It is the standard solution of 1.5mg/mL, continuous sample introduction 8 times, each applied sample amount that precision prepares recombinant human lysozyme concentration
20 μ L, are analyzed according to the chromatographic condition of the step 1 of embodiment 1, according to corresponding main peak area numerical value, calculate standard deviation (SD
Value STDEV), arithmetic mean of instantaneous value (Xbar AVERAGE), relative standard deviation (RSD)=standard deviation (SD)/arithmetic mean of instantaneous value
(Xbar) × 100%, the relative standard deviation of its peak area measurement value should be not more than 1.0%.
The recombinant human lysozyme replica test result of table 3
The recombinant human lysozyme replica test result statistical analysis of table 4
From the result of table 4, continuous 8 sample introductions, the relative standard deviation of its main peak area is 0.52715%, is less than
1.0%, illustrate that the repeatability of analysis method of the present invention is good.
2nd, accuracy test
Accurate weighing 2559.7mg test samples (through the recombinant human lysozyme sample (20151125 batches) that content is demarcated, wherein
The mass content of recombinant human lysozyme is for 5.86%), wherein 150.0mg containing recombinant human lysozyme, mass concentration is 0.9% chlorine
Change sodium ultra-pure water solution to be settled in 50.0mL volumetric flasks, be configured to the standard items that recombinant human lysozyme concentration is 3.0mg/mL molten
Liquid；5 sample concentrations are diluted with the sodium chloride ultra-pure water solution that 5.0mL volumetric flasks, mass concentration are 0.9%, respectively：
0.90mg/mL, 1.20mg/mL, 1.50mg/mL, 1.80mg/mL, 2.10mg/mL, according to the chromatographic condition of the step 1 of embodiment 1
It is analyzed, the results are shown in Table 5.
The recombinant human lysozyme standard curve chromatogram detection statistics result of table 5
According to the testing result of table 5, standard curve of the chromatogram main peak area with recombinant human lysozyme change in concentration, knot are drawn
Fruit sees Fig. 2.The linear equation of standard curve is：Y=2306.9x+222118.3, x represents recombinant human lysozyme concentration, y in formula
Represent chromatogram main peak area, R2=0.99924, it is linear good.
The above-mentioned test sample 170.65mg of accurate weighing, wherein 10.0mg containing recombinant human lysozyme, adds the mass concentration to be
0.9% sodium chloride ultra-pure water solution is settled in 10.0mL volumetric flasks, is configured to recombinant human lysozyme concentration for 1.0mg/mL
Need testing solution, 1mL/ branch (every 1.0mg containing recombinant human lysozyme, A value) is distributed into pipette, is distinguished again in every sample
Accurate weighing adds 17.15mg test samples (1.0mg containing recombinant human lysozyme, B value), totally 7 samples, by the step 1 of embodiment 1
Chromatographic condition is analyzed.According to the chromatogram main peak area and standard curve y=2306.9x+222118.3 that measure, actual measurement is tried to achieve
Concentration, obtains measured value C, and recovery of standard addition is calculated according to following formula：
Rate of recovery %=(C-A)/B × 100%
A is recombinant human lysozyme component contained by test sample in formula；B is addition reference substance amount；C is measured value.The results are shown in Table 6.
The recombinant human lysozyme chromatographic detection Accuracy Verification statistics of table 6
From table 6, the degree of accuracy of the inventive method is 95.8%~100.0%, meets Chinese Pharmacopoeia 2015 editions accurate
The requirement 95%~102% is spent, can be used for the detection of recombinant human lysozyme content.
Claims (3)
1. a kind of rp-hplc analysis method of recombinant human lysozyme, it is characterised in that it is made up of following step：
(1) recombinant human lysozyme standard items are dissolved in the sodium chloride ultra-pure water solution that mass concentration is 0.9%, are configured to weight
Group human lysozyme standard solution, is analyzed, chromatographiccondition using RPLC to standard solution：Color
Spectrum post is C18 reverse-phase chromatographic columns, and filler is 20~50nm of aperture, the octadecylsilane chemically bonded silica of 2~10 μm of particle diameter, flowing
Phase A is the ultra-pure water solution that sodium chloride mass concentration is 0.9%, trifluoroacetic acid volumetric concentration is 0.1%, and Mobile phase B is acetonitrile
It is 6 with ultrapure water volume ratio:It is containing the sodium chloride and volumetric concentration that mass concentration is 0.9% in 4 mixed liquor and mixed liquor
0.1% trifluoroacetic acid, carries out gradient elution, eluent gradient A:B is by (90~0):(10~100) to (50~0):(50~
100), Detection wavelength is 280nm, and flow velocity is 1mL/ minutes；Chromatogram main peak area is drawn with recombinant human lysozyme change in concentration
Standard curve；
(2) recombinant human lysozyme sample to be measured is dissolved in the sodium chloride ultra-pure water solution that mass concentration is 0.9%, uses filter membrane
Filtering, collects filtrate, and filtrate is analyzed using RPLC, and chromatographiccondition is identical with step (1)；
(3) the chromatogram main peak area obtained according to step (2), the linear equation of the standard curve determined with reference to step (1)
Obtain the concentration of recombinant human lysozyme in recombinant human lysozyme sample to be measured.
2. the rp-hplc analysis method of recombinant human lysozyme according to claim 1, it is characterised in that：
In step (1), described eluent gradient selects 0 to 30 minutes A:B is by 55:45 to 5:95.
3. the rp-hplc analysis method of recombinant human lysozyme according to claim 1 and 2, its feature exists
In：In step (1), described filler is aperture 30nm, the octadecylsilane chemically bonded silica of 5 μm of particle diameter.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710233245.2A CN106872633B (en) | 2017-04-11 | 2017-04-11 | A kind of rp-hplc analysis method of recombinant human lysozyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710233245.2A CN106872633B (en) | 2017-04-11 | 2017-04-11 | A kind of rp-hplc analysis method of recombinant human lysozyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106872633A true CN106872633A (en) | 2017-06-20 |
| CN106872633B CN106872633B (en) | 2018-09-14 |
Family
ID=59162169
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710233245.2A Active CN106872633B (en) | 2017-04-11 | 2017-04-11 | A kind of rp-hplc analysis method of recombinant human lysozyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106872633B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109799335A (en) * | 2019-01-31 | 2019-05-24 | 陕西慧康生物科技有限责任公司 | The detection method of Pichia pastoris host protein residual quantity in recombinant human lysozyme |
| CN111751468A (en) * | 2020-07-03 | 2020-10-09 | 上海艾魁英生物科技有限公司 | Method for purifying lysozyme dimer and preparing standard substance thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57122756A (en) * | 1981-01-24 | 1982-07-30 | Eisai Co Ltd | Purification of protein |
| CN1664096A (en) * | 2004-10-21 | 2005-09-07 | 北京双鹭立生医药科技有限公司 | Method for preparing human lysozyme |
| CN102268418A (en) * | 2011-08-18 | 2011-12-07 | 上海杰隆生物工程股份有限公司 | Method for purifying lysozyme from milk |
-
2017
- 2017-04-11 CN CN201710233245.2A patent/CN106872633B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57122756A (en) * | 1981-01-24 | 1982-07-30 | Eisai Co Ltd | Purification of protein |
| CN1664096A (en) * | 2004-10-21 | 2005-09-07 | 北京双鹭立生医药科技有限公司 | Method for preparing human lysozyme |
| CN102268418A (en) * | 2011-08-18 | 2011-12-07 | 上海杰隆生物工程股份有限公司 | Method for purifying lysozyme from milk |
Non-Patent Citations (5)
| Title |
|---|
| HIDEKO KANAZAWA 等: "Aqueous chromatographic system for separation of biomolecules using thermoresponsive polymer modified stationary phase", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| 周闺臣 等: "溶菌酶的处方设计前稳定性研究", 《药学实践杂志》 * |
| 常景立 等: "苦瓜溶菌酶的分离纯化及特性研究", 《中国食品学报》 * |
| 庞世琦 等: "高效液相色谱法测定葡萄酒中溶菌酶的含量", 《食品安全质量检测学报》 * |
| 张德强 等: "HPLC法测定盐酸溶菌酶片的含量", 《今日药学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109799335A (en) * | 2019-01-31 | 2019-05-24 | 陕西慧康生物科技有限责任公司 | The detection method of Pichia pastoris host protein residual quantity in recombinant human lysozyme |
| CN109799335B (en) * | 2019-01-31 | 2022-04-01 | 陕西慧康生物科技有限责任公司 | Method for detecting pichia host protein residue in recombinant human lysozyme |
| CN111751468A (en) * | 2020-07-03 | 2020-10-09 | 上海艾魁英生物科技有限公司 | Method for purifying lysozyme dimer and preparing standard substance thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106872633B (en) | 2018-09-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Brocks et al. | Determination of metformin in human plasma and urine by high-performance liquid chromatography using small sample volume and conventional octadecyl silane column | |
| Nedelkov et al. | Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors | |
| CN106383186B (en) | The HPLC analytical method of 14 kinds of vitamin contents is determined simultaneously | |
| Zhang et al. | Determination of eight quinolones in milk using immunoaffinity microextraction in a packed syringe and liquid chromatography with fluorescence detection | |
| CN106872633B (en) | A kind of rp-hplc analysis method of recombinant human lysozyme | |
| CN107315059B (en) | The content assaying method of rifampin and its impurity in a kind of rifampicin capsules | |
| Fekete et al. | Simultaneous determination of polysorbate 20 and unbound polyethylene-glycol in protein solutions using new core–shell reversed phase column and condensation nucleation light scattering detection | |
| Naish et al. | Rapid assay for theophylline in clinical samples by reversed-phase high-performance liquid chromatography | |
| Passoni et al. | Development and validation of a new and rapid HPLC for determination of lyophilized teicoplanin | |
| CN107167535A (en) | A kind of method that reversed phase liquid chromatography detects Raltitrexed enantiomter | |
| Patil et al. | Assay determination of tranexamic acid in pharmaceutical dosage form (tablet) using HPLC and ELS detector | |
| CN110514759A (en) | The detection method of azido compound in a kind of candesartan Cilexetil | |
| Forrest et al. | Simple high-performance liquid chromatographic method for the measurement of amiloride in body fluids | |
| CN104965031B (en) | Content measuring method for compound ketoprofen and omeprazole sustained-release capsules | |
| Kamble et al. | A Review on High Performance Liquid Chromatography (HPLC) | |
| CN104374861B (en) | The method of the related substance of the western croak bulk drug of a kind of HPLC separation determination Leo | |
| Jung et al. | " High-pressure" liquid chromatography of sulfisoxazole and N4-acetylsulfisoxazole in body fluids. | |
| RU2775300C1 (en) | Method for determination of vancomycin b and its related impurities | |
| CN106770777A (en) | HPLC detects method of the desmopressin acetate about material | |
| Mcneilly et al. | Determination of ceftiofur in bovine milk by liquid chromatography | |
| Kabra et al. | Liquid chromatographic determination of cyclosporine in whole blood with the advanced automated sample processing unit | |
| RU2702330C1 (en) | Method for quantitative determination of 2-[(z)-1-(3,5-diphenyl-1,3,4-thiadiazole-2(3h)-ylidene)methyl]-3,5-diphenyl-1,3,4-thiadiazole-3-ium in biological objects | |
| Singh et al. | DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR ESTIMATION OF VORICONAZOLE | |
| Savaşer et al. | Validated LC determination of the piroxicam-β-cyclodextrin inclusion complex in tablets and in human plasma | |
| King et al. | HPLC determination of propofol-thiopental sodium and propofol-ondansetron mixtures |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |