CN111965285A - Method for detecting content of tulathromycin - Google Patents
Method for detecting content of tulathromycin Download PDFInfo
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- CN111965285A CN111965285A CN202010851725.7A CN202010851725A CN111965285A CN 111965285 A CN111965285 A CN 111965285A CN 202010851725 A CN202010851725 A CN 202010851725A CN 111965285 A CN111965285 A CN 111965285A
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- GUARTUJKFNAVIK-QPTWMBCESA-N Tulathromycin A Chemical compound C1[C@](OC)(C)[C@@](CNCCC)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](C)C(=O)O[C@H](CC)[C@@](C)(O)[C@H](O)[C@@H](C)NC[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C GUARTUJKFNAVIK-QPTWMBCESA-N 0.000 title claims abstract description 88
- 229960002859 tulathromycin Drugs 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000002347 injection Methods 0.000 claims abstract description 41
- 239000007924 injection Substances 0.000 claims abstract description 41
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 239000003085 diluting agent Substances 0.000 claims abstract description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000000889 atomisation Methods 0.000 claims abstract description 6
- 239000012159 carrier gas Substances 0.000 claims abstract description 6
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims abstract description 5
- GIPAGNAMYQXLIT-UHFFFAOYSA-N [NH4+].OC.CC#N.[O-]C=O Chemical compound [NH4+].OC.CC#N.[O-]C=O GIPAGNAMYQXLIT-UHFFFAOYSA-N 0.000 claims abstract description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000945 filler Substances 0.000 claims abstract description 3
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 3
- 239000000523 sample Substances 0.000 claims description 36
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 239000012488 sample solution Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 10
- 229940048954 tulathromycin a Drugs 0.000 claims description 10
- 230000014759 maintenance of location Effects 0.000 claims description 8
- JEBMHQVCOAWRCT-QPTWMBCESA-N Tulathromycin B Chemical compound C1[C@](OC)(C)[C@@](CNCCC)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](C)C(=O)O[C@@H]([C@](C)(O)[C@H](O)CC)[C@@H](C)NC[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C JEBMHQVCOAWRCT-QPTWMBCESA-N 0.000 claims description 7
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000004811 liquid chromatography Methods 0.000 claims description 2
- 239000012086 standard solution Substances 0.000 claims description 2
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 9
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000012071 phase Substances 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 239000012088 reference solution Substances 0.000 description 6
- 238000007865 diluting Methods 0.000 description 5
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
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- 241001465754 Metazoa Species 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
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- 230000008020 evaporation Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000011550 stock solution Substances 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
- 241000606750 Actinobacillus Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000606807 Glaesserella parasuis Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 241000606860 Pasteurella Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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- 238000007911 parenteral administration Methods 0.000 description 1
- 230000009340 pathogen transmission Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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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
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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
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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
- G01N30/26—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
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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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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Abstract
The invention relates to a method for detecting the content of tulathromycin, which adopts the following chromatographic conditions for detection: the chromatographic column adopts an inverse chromatographic column with octadecylsilane chemically bonded silica as a filler, and the mobile phase is as follows: 20mmol/L aqueous ammonium formate-methanol-acetonitrile; flow rate: 1.0 ml/min; column temperature: 35 ℃; CAD detector atomization temperature: at 45 deg.C, the carrier gas pressure is 60psi, and the flow rate is 4L/min; the sample volume is 20 mu L; the diluent was methanol-0.1% formic acid water (20: 80). The invention provides a method for measuring the content of a tulathromycin injection sample by HPLC and simultaneously using an electrospray detector in an auxiliary mode. The method is verified by a precision test and a linear relation test, and the result shows that the method is accurate and reliable, and has a large detection range, high sensitivity and high accuracy.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting the content of a tulathromycin preparation.
Background
Tulathromycin, alias tulathromycin, english name: tulathromycin with molecular formula of C41H79N3O12Molecular weight 806.08, is a mixture of 2 isomers consisting of 15-membered azalide ring (formula 1A) and 13-membered azalide ring (formula 1B) in a ratio of 9: 1. It is a special macrolides semi-synthetic antibiotic for animals, and belongs to triamine broad-spectrum antibiotics. The currently marketed 10% tulathromycin injection (trade name: revamphenicol) is composed of 13-membered azalide ring isomer CP-547, 272 (content 8.0% -13.0%, tulathromycin B) and 15-membered azalide ring isomer CP-472, 295 (content 87.0% -92.0%, tulathromycin A).
The tulathromycin is a broad-spectrum antibacterial drug, has the characteristics of quick absorption, high bioavailability, low residue, long half life, lasting drug effect, capability of providing whole-course treatment by single parenteral administration and the like, and is mainly used for treating respiratory diseases of pigs and cows caused by actinobacillus, mycoplasma, pasteurella and haemophilus parasuis. The use of tulathromycin reduces pathogen transmission and is convenient for large-scale management. The tulathromycin plays an important role in the production of animal husbandry. In order to ensure the food safety of the livestock breeding industry and the society, it is necessary to establish a simple, accurate and reliable method for controlling the quality of tulathromycin.
The tulathromycin is a mixture of two isomers of a 15-membered azalide ring and a 13-membered azalide ring in a ratio of 9:1, and the tulathromycin does not contain chromophoric groups in the structure, and has no ultraviolet absorption or only ultraviolet terminal absorption. And therefore cannot be measured by conventional high performance liquid chromatography-ultraviolet or fluorescence detectors. The evaporation light detector has poor sensitivity and reproducibility, and the content can be measured only after linear regression after area derivation, so that the popularization and the application of the evaporation light detector are influenced to a certain extent.
The invention provides a method for detecting the content of a tulathromycin injection preparation by HPLC-CAD (high performance liquid chromatography), aiming at overcoming the defects of the prior art. The principle is that an HPLC detector based on atomization-aerosol charges nitrogen through a corona needle, then the charged nitrogen is fully mixed with solute particles in dried eluent in a reverse direction, and the charge of the charged nitrogen is transferred to charge the solute particles, and then the charged solute particles enter an electrometer to detect an electric signal value. The method has the advantages of simplicity, convenience, high efficiency, high precision and high accuracy.
Disclosure of Invention
The invention provides a high performance liquid chromatography-electrospray detector detection method for the content of a tulathromycin injection preparation, aiming at solving the problems in the prior art, so that the method has the advantages of simplicity, convenience, high efficiency, high precision and high accuracy.
The technical scheme adopted by the invention is as follows:
a high performance liquid chromatography-electrospray detector detection method for the content of a tulathromycin injection preparation adopts the following chromatographic conditions for detection: the chromatographic column adopts an inverse chromatographic column with octadecylsilane chemically bonded silica as a filler, and the mobile phase is as follows: 20mmol/L aqueous ammonium formate-methanol-acetonitrile; flow rate: 1.0 ml/min; column temperature: 35 ℃; CAD detector atomization temperature: at 45 deg.C, the carrier gas pressure is 60psi, and the flow rate is 4L/min; the sample volume is 20 mu L; the diluent was methanol-0.1% formic acid water (20: 80).
Preferably, the chromatographic column is Shimadzu Wondasil C18Column (4.6 x 150 mm)5 μm) or Watt Xterra RP (4.6 x 150mm, 3.5 μm). More preferably, the chromatography column is a Watts Xterra RP (4.6 x 150mm, 3.5 μm). The suitable chromatographic column enables the tested sample to have better separation effect.
Preferably, in the mobile phase, the volume fraction of the methanol is 35-45%, and the volume fraction of the acetonitrile is 30-50%; the balance is 20mmol/L ammonium formate aqueous solution; more preferably, the volume ratio of 20mmol/L ammonium formate aqueous solution to methanol to acetonitrile is: 25: 35: 40.
in the present invention, aqueous ammonium formate and methanol/acetonitrile system are used as the mobile phase, and methanol/0.1% formic acid (20:80) is used as the diluent. The tulathromycin injection preparation can be well diluted before liquid phase entering, and a good peak effect can be obtained in a short retention time, so that the detection precision is favorably improved.
Tulathromycin, which is a mixture of 2 isomers of 15-membered azalide ring (formula 1A) and 13-membered azalide ring (formula 1B) in a ratio of 9: 1.
Formula 1A tulathromycin A
Formula 1B tulathromycin B
The method comprises the following steps:
s1: preparing a control solution with known content concentration of the tulathromycin by using a diluent, detecting under the same chromatographic condition by adopting the chromatographic condition, calculating peak areas of the control solutions with different concentrations under the same retention time, wherein the tulathromycin B firstly generates a peak before the tulathromycin A, and making a standard curve equation y (f) (x) of the concentration x of the control solution by the sum y of the peak areas of the tulathromycin A and the B;
s2: precisely weighing a certain mass of a tulathromycin injection sample, treating the tulathromycin injection sample with the diluent in the invention, obtaining the peak area of the tulathromycin injection sample solution to be detected in the same retention time by adopting the liquid chromatography detection conditions (including chromatographic conditions, retention time, sample injection amount and the like) in the same step S1, and calculating the concentration of tulathromycin in the tulathromycin injection sample to be detected according to the standard curve equation y (f) (x) obtained in the step S1;
s3: according to the density of the sample of the tulathromycin injection to be detected, the content data of tulathromycin in the sample of the tulathromycin injection can be obtained.
In step S1, the concentration x of the standard solution in the standard curve equation y ═ f (x) is in the range of 500-. Further, the standard curve equation y ═ f (x) is a unary linear equation, and the correlation coefficient R is2The fitting degree is more than or equal to 0.999, and a linear equation with high fitting degree can be obtained.
The invention has the following beneficial effects:
the invention provides a method for measuring the content of a tulathromycin injection sample by HPLC and simultaneously using an electrospray detector in an auxiliary mode. The method is verified by a precision test and a linear relation test, and the result shows that the method is accurate and reliable, and has a large detection range, high sensitivity and high accuracy.
Drawings
FIG. 1 is an HPLC detection profile of a tulathromycin control solution in a specific embodiment;
FIG. 2 is a graph of the linearity of the tulathromycin control versus the standard curve equation for the specific example.
FIG. 3 is a chromatogram of the content determination of the tulathromycin injection by different chromatographic columns in example 2; wherein FIG. 3-A is a Shimadzu chromatographic column sample assay chromatogram; 3-B Vortecht chromatographic column sample determination chromatogram; 3-C Agilent chromatographic column sample determination chromatogram
FIG. 4 is a chromatogram of various mobile phase and diluent assay samples from example 3.
Detailed Description
The invention will be further described with reference to the accompanying drawings without limiting the scope of the invention.
Example 1
(1) Chromatographic conditions
Adopting Agilent 1260 high performance liquid chromatograph combined with Corona Ultra electrospray detector, adopting octadecyl silane bonded silica gel as the chromatographic column, and selecting Waters Xterra RP (4.6 × 150mm, 3.5 μm); the mobile phase was 20mmol/L aqueous ammonium formate-methanol-acetonitrile (25: 35: 40); the flow rate is 1.0 ml/min; column temperature was 35 ℃, detector atomization temperature: 45 ℃; the carrier gas pressure was 60psi and the flow rate was 4L/min; the diluent is methanol-0.1% formic acid water (20: 80); the amount of the sample was 20. mu.l.
(2) Obtaining of standard curve equation
Accurately weighing 500mg of a tulathromycin reference substance, placing the tulathromycin reference substance into a 50ml measuring flask, dissolving the tulathromycin reference substance by using a diluent, diluting the tulathromycin reference solution to a constant volume to obtain tulathromycin reference solution serving as tulathromycin reference solution stock solution, then gradually diluting the tulathromycin reference solution stock solution to prepare the tulathromycin reference solution with tulathromycin concentrations of 0.5, 0.8, 1, 1.2 and 1.5mg/ml, respectively injecting the tulathromycin reference solutions with different concentrations into a chromatographic column of the HPLC device which is arranged in the step (1) and enters a working state, respectively calculating the peak area under the same retention time, wherein the tulathromycin B firstly comes out of the peak before the tulathromycin A. Taking the concentration x of the control solution as an abscissa and the sum y of the peak areas of the tulathromycin A and the tulathromycin B as an ordinate, making a standard curve equation of which y is 2626.9x-44.857, and R is2Results showed that tulathromycin had the above linear relationship between peak area and concentration in the range of 500-1500. mu.g/ml.
(3) The content of tulathromycin in the sample to be tested (tulathromycin injection) is as follows:
accurately weighing a sample to be tested, diluting the sample by using the method disclosed by the invention, diluting the sample according to a proper dilution ratio (the dilution factor is the reciprocal of the dilution ratio) to prepare a proper concentration, shaking up to obtain a sample solution of the tulathromycin injection to be tested, and filtering the sample solution by using a 0.45-micrometer organic filter head. And (3) injecting the sample into the chromatographic column of the HPLC device which is set in the step (1) and enters a working state, wherein the sample injection amount is 20 mu L, calculating the peak area under the same retention time as that in the step (2), substituting the peak area into the standard curve equation obtained in the step (2), calculating the concentration of the sample solution of the tulathromycin injection to be detected, and calculating the tulathromycin content of the sample to be detected according to the sampling amount and the dilution ratio of the sample to be detected. The calculation method is as follows:
weighing 0.52321g (trade name: Rekexin; density of 1.0521g/mL) of a commercially available 10% tulathromycin injection into a 50mL volumetric flask, diluting the volumetric flask to a scale by using a diluent, performing constant volume, filtering the volumetric flask by using a 0.45-micrometer organic filter head, measuring 20-micrometer-liter sample injection to obtain 2499 of the peak area of tulathromycin, substituting a linear equation to calculate that the concentration of the tulathromycin injection to be detected is 0.9684mg/mL, and substituting a calculation formula to obtain 97.4% of the tulathromycin injection.
(4) Precision survey
The same sample to be tested (oral tulathromycin solution) was continuously injected 6 times, and the peak area was shown in Table 1.
TABLE 1
| Number of times | 1 | 2 | 3 | 4 | 5 | 6 |
| Peak area | 2591 | 2588 | 2601 | 2594 | 2585 | 2589 |
The Relative Standard Deviation (RSD) of the peak area of 6 times of sample injection is 0.21 percent, and the result shows that the method for detecting the content of the tulathromycin injection sample by the HPLC method has high precision and good reproducibility.
(5) Recovery test
A proper amount of a tulathromycin sample with known content is precisely weighed, and diluted by a mobile phase to prepare a test sample with the concentrations of 80%, 100% and 120% of the concentration of the sample in the machine of 1mg/ml respectively, so that the tulathromycin concentration in the test sample solution is respectively in the low, middle and high regions of a tulathromycin standard curve, the recovery rate is calculated according to the operation under the item of the invention, the average recovery rate is 99.84%, and the RSD is 0.08%, which shows that the recovery rate of the method is good.
Example 2 comparison of the Effect of different chromatography columns on the determination of the content of tulathromycin injection
Chromatographic conditions are as follows: adopting Agilent 1260 high performance liquid chromatograph combined with Corona Ultra electrospray detector, wherein the mobile phase is 20mmol/L ammonium formate aqueous solution-methanol-acetonitrile (25: 35: 40); the flow rate is 1.0 ml/min; column temperature was 35 ℃, detector atomization temperature: 45 ℃; the carrier gas pressure was 60psi and the flow rate was 4L/min; the diluent is methanol-0.1% formic acid water (20: 80); the amount of the sample was 20. mu.l.
A chromatographic column: shimadzu Wondasil C18Columns (4.6X 150mm, 5 μm), Watts Xterra RP columns (4.6X 150mm, 3.5 μm) and Agilent ZORBAX C18Column (4.6 x 150mm, 5 μm) three chromatographic columns
Sample solution: tulathromycin injection (concentration 1.0mg/mL)
Sequentially measuring the sample solution with Shimadzu, Watershi and Agilent three chromatographic columns according to the above chromatographic conditions, wherein the chromatogram is shown in figure 3. Therefore, the following steps are carried out: the sample solution of the Shimadzu chromatographic column has late peak emergence time, and the tulathromycin A has a little tail; the sample solution of the Wottech chromatographic column has fast peak emergence time and good peak pattern; abnormal peaks of the Agilent chromatographic column sample solution.
Example 3 comparison of the Effect of mobile phase, diluent on the results of the experiment
Chromatographic conditions are as follows: adopting Agilent 1260 high performance liquid chromatograph combined with Corona Ultra electrospray detector, wherein the mobile phase is 20mmol/L ammonium formate aqueous solution-methanol (25: 75); the flow rate is 1.0 ml/min; column temperature was 35 ℃, detector atomization temperature: 45 ℃; the carrier gas pressure was 60psi and the flow rate was 4L/min. The amount of the sample was 20. mu.l.
Diluent agent: methanol
A chromatographic column: watts Xterra RP (4.6 mm 150, 3.5 μm)
Sample solution: tulathromycin injection (concentration 1.0mg/mL)
The sample solution was measured according to the above conditions and the chromatogram is shown in FIG. 4. It can be seen that the sample solution showed a fast time to peak, but the tulathromycin A and tulathromycin B were not separated, and the content measurement could not be performed.
Claims (7)
1. A detection method of a high performance liquid chromatography-electrospray detector for the content of a tulathromycin injection preparation is characterized by comprising the following steps: the method adopts the following chromatographic conditions for detection: the chromatographic column adopts an inverse chromatographic column with octadecylsilane chemically bonded silica as a filler, and the mobile phase is as follows: 20mmol/L aqueous ammonium formate-methanol-acetonitrile; flow rate: 1.0 ml/min; column temperature: 35 ℃; CAD detector atomization temperature: at 45 deg.C, the carrier gas pressure is 60psi, and the flow rate is 4L/min; the sample volume is 20 mu L; the diluent was methanol-0.1% formic acid water (20: 80).
2. The detection method of the tulathromycin injection preparation content by the high performance liquid chromatography-electrospray detector as recited in claim 1, characterized in that: the chromatographic column is Shimadzu Wondasil C18Columns (4.6 x 150mm, 5 μm) or Watts Xterra RP (4.6 x 150mm, 3.5 μm).
3. The detection method of the tulathromycin injection preparation content by the high performance liquid chromatography-electrospray detector as recited in claim 2, characterized in that: the column was Watts Xterra RP (4.6 x 150mm, 3.5 μm).
4. The detection method of the tulathromycin injection preparation content by the high performance liquid chromatography-electrospray detector as recited in claim 1, characterized in that: in the mobile phase, the volume fraction of the methanol is 35-45%, and the volume fraction of the acetonitrile is 30-50%; the balance was 20mmol/L aqueous ammonium formate solution.
5. The detection method of the tulathromycin injection preparation content by the high performance liquid chromatography-electrospray detector as recited in claim 1, characterized in that: the volume ratio of 20mmol/L ammonium formate aqueous solution to methanol to acetonitrile is as follows: 25: 35: 40.
6. the detection method of the tulathromycin injection preparation content by the high performance liquid chromatography-electrospray detector as recited in claim 1, characterized in that: the method comprises the following steps:
s1: preparing a control solution with known content concentration of the tulathromycin by using a diluent, detecting under the same chromatographic condition by adopting the chromatographic condition, calculating peak areas of the control solutions with different concentrations under the same retention time, wherein the tulathromycin B firstly generates a peak before the tulathromycin A, and making a standard curve equation y (f) (x) of the concentration x of the control solution by the sum y of the peak areas of the tulathromycin A and the B;
s2: accurately weighing a certain mass of a tulathromycin injection sample, treating the tulathromycin injection sample with a diluent in the invention, obtaining the peak area of the tulathromycin injection sample solution to be detected in the same retention time by adopting the liquid chromatography detection conditions in the same step S1, and calculating the concentration of tulathromycin in the tulathromycin injection sample to be detected according to the standard curve equation y ═ f (x) obtained in the step S1;
s3: according to the density of the tulathromycin injection sample to be detected, obtaining the content data of tulathromycin in the tulathromycin injection sample;
tulathromycin, which is a mixture of 2 isomers of 15-membered azalide ring (formula 1A) and 13-membered azalide ring (formula 1B) in a ratio of 9: 1.
7. The detection method of the tulathromycin injection preparation content by the high performance liquid chromatography-electrospray detector as recited in claim 1, characterized in that: in step S1, the standard solution concentration x of the standard curve equation y ═ f (x) is in the range of 500-.
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| CN202010851725.7A CN111965285A (en) | 2020-08-21 | 2020-08-21 | Method for detecting content of tulathromycin |
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| CN202010851725.7A CN111965285A (en) | 2020-08-21 | 2020-08-21 | Method for detecting content of tulathromycin |
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