CN114113399A - Ceftiofur detection method and application thereof - Google Patents

Abstract

The invention provides a method for detecting ceftiofur in blood plasma. The method for detecting ceftiofur in blood plasma comprises the steps of extracting a ceftiofur metabolite de-furylceftiofur (DFC) from a medicine-containing blood plasma sample through an extracting solution-Dithioerythritol (DTE) solution in water bath, wherein the derivatization and solid-phase extraction column treatment of an iodoacetamide solution are not needed, and the extracting solution is centrifuged to prepare a supernatant, and the supernatant is filtered by a filter membrane and then can be directly used for detecting by a high performance liquid chromatograph. The invention has the advantages that: the method simplifies the sample pretreatment steps, reduces the cost, saves the time, has small reagent usage amount and low toxicity, and is suitable for the rapid detection of ceftiofur and metabolites thereof in a large batch of plasma samples in clinical pharmacokinetic experiments.

Description

Ceftiofur detection method and application thereof

Technical Field

The invention belongs to the field of biological sample detection, particularly relates to the field of detection of ceftiofur and metabolites thereof, and particularly relates to detection of ceftiofur metabolites in blood plasma.

Background

Ceftiofur (Ceftiofur) is also known as cefafur, is a veterinary-specific third-generation cephalosporin antibiotic successfully developed in 80 s in the 20 th century in the U.S., and the drug is first marketed in the U.S. in 1988, and is officially approved for the treatment of respiratory diseases of beef cattle, dairy cows, horses, pigs and sheep in some countries in the U.S., canada, japan and europe due to its excellent antibacterial activity and pharmacokinetic characteristics. The chemical name of ceftiofur is (6R,7R) -7- [2- (2-aminothiazole-4-yl) (methoxyimino) acetamido]-3- [ (2-furylcarbonyl) thiomethyl]-8-oxo-5-thia-1-azabicyclo [4.2.0]2-Octen-2-En-2-carboxylic acid, molecular formula C₁₉H₁₇N₅O₇S₃Molecular weight 523.56. Ceftiofur has an amino group and a carboxyl group, and is an amphoteric compound which is easily decomposed under strong acid, strong base and high temperature conditions. The structural formula of Ceftiofur (Ceftiofur) is as follows:

research on the main metabolism and excretion of Ceftiofur in livestock and poultry shows that most of Ceftiofur in animal bodies rapidly removes furan carboxyl on 3-th sulfo group and converts the Ceftiofur into a primary metabolite, namely, Desfuroyl Ceftiofur (DFC) and furoic acid. In plasma, urine and tissues, DFC forms disulfide compounds (cysteine-DFC, glutathione-DFC) with cysteine and glutathione through a thiol group on a substituent at the 3-position thereof, and DFC can also bind to plasma protein or tissue protein to form a DFC-protein complex. Among them, the process of DFC formation of protein complexes and disulfide formation is reversible, and disulfide compounds and protein conjugates can act as a reservoir of DFC in plasma and tissues. DFC has an intact effective antibacterial group beta-lactam ring of ceftiofur, so DFC can be used as an antibacterial active ingredient of ceftiofur in blood plasma for detection. The chemical name of de-furoyl ceftiofur (DFC) is (6R,7R) -7- [ [ (2Z) - (2-amino-4-thiazolyl) (methoxyimino) acetyl]Amino group]-3- (mercaptomethyl) -8-oxo-5-thia-1-azabicyclo [4.2.0]Oct-2-ene-2-carboxylic acid of formula C₁₄H₁₅N₅O₅S₃Molecular weight 429.5, desfuroyl ceftiofur (DFC) formula:

the detection principle of the existing method for detecting ceftiofur in blood or tissue mainly refers to high performance liquid chromatography for detecting ceftiofur residue in animal food No. 1025 bulletin-13-2008 of Ministry of agriculture, and the detection method is that ceftiofur residue in the tissue is cultured together with Dithioerythritol (DTE) solution, so that relevant metabolites of ceftiofur and de-furoyl ceftiofur (DFC) are separated from protein or sulfur-containing compounds to generate DFC. DFC reacts with iodoacetamide to form stable derivatives of DFC acetamide (DCA), which are converted from the prodrug or metabolite. The derivatives were extracted using a C18 solid phase extraction column. Purifying with strong anion exchange (SAX) column, and purifying with strong cation exchange (SCX) column. DCA derivatives were measured by HPLC-UV. The patent CN110412147A discloses a method for detecting pharmacokinetics and elimination of ceftiofur sodium in chicken bodies, which comprises the steps of adding Dithioerythritol (DTE) solution into collected chicken heart blood samples for extraction, standing to room temperature, adding iodoacetamide solution for derivatization, acidifying with phosphoric acid, centrifuging to obtain supernatant, treating with a mixed type cation exchange (MCX) column, and determining the content of DCA by high performance liquid chromatography.

The above methods for treating ceftiofur in the sample all adopt iodoacetamide solution derivatization and solid-phase extraction column treatment, have the defects of more sample pretreatment steps, high cost, long time, more reagents required for sample pretreatment, higher toxicity and the like, and are not suitable for rapid detection of ceftiofur and metabolites thereof in large-batch plasma samples in clinical pharmacokinetic tests.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a detection method of ceftiofur, which comprises sample pretreatment and high performance liquid chromatograph detection, and the detection method comprises the following steps: step one, extracting a metabolite of ceftiofur by a sample through an extracting solution dithioerythritol solution water bath to remove furoyl ceftiofur; and step two, detecting by using a high performance liquid chromatograph.

Preferably, step one does not include the steps of derivatization with iodoacetamide solution and treatment with a solid phase extraction column.

Preferably, the extraction solution is 0.4% dithioerythritol solution by mass.

In any of the above cases, the volume ratio of the sample to the extract solution is preferably 1: 2.

Any one of the above-mentioned preferred methods is that in the step one, the plasma sample is added into the extract, vortexed and mixed, and then water-bathed, then the sample is centrifuged, and the supernatant is filtered by a filter membrane and used for the detection in the step two.

Preferably, in the first step, the water bath conditions are as follows: the temperature is 50 deg.C, the time is 30 min, and the vortex mixing is carried out once every 10 min during the water bath.

Preferably in any of the above, the filter is a 0.22 μm filter.

Preferably, in any of the above steps, the detection conditions of the high performance liquid chromatograph in the second step are: the high performance liquid chromatograph is Agilent 1260 Infinity II; the chromatographic column is ZORBAX SB-C18, 4.6 mm × 250 mm, 5 μm; mobile phase 0.1% trifluoroacetic acid-water: acetonitrile; the detection wavelength was 266 nm.

Preferably, in any of the above cases, the sample is a plasma sample. The detection method is used for detecting ceftiofur and metabolites thereof in blood plasma.

The invention also provides application of the ceftiofur detection method in detection of ceftiofur and metabolites thereof in plasma samples.

In a preferred embodiment of the present invention, a method for detecting ceftiofur in plasma is provided, wherein a drug-containing plasma sample is subjected to water bath extraction of a Dithioerythritol (DTE) solution to obtain ceftiofur metabolite de-furoyl ceftiofur (DFC), and then detection is performed by using a high performance liquid chromatograph; the extracting solution is 0.4 percent of Dithioerythritol (DTE) solution in percentage by mass; the adding volume ratio of the plasma sample to the extracting solution is 1: 2; the water bath conditions were: the temperature is 50 deg.C, and the time is 30 min. Vortex and mix once every 10 min during the water bath.

On the basis of the scheme, the method for detecting ceftiofur in blood plasma comprises the following steps:

1) sample pretreatment: taking 200 μ l of plasma sample, adding 400 μ l of extractive solution (0.4% DTE w/v), mixing by vortex, water bathing at 50 deg.C for 30 min, and mixing by vortex once every 10 min. After completion of the water bath, the sample was centrifuged at 10000 r/min at 4 ℃ for 10 min, and the supernatant was filtered through a 0.22 μm filter and transferred to a sample vial for chromatographic analysis. And a water bath step, wherein ceftiofur remaining in the plasma is cultured together with a Dithioerythritol (DTE) solution, so that ceftiofur and Desfuroylceftiofur (DFC) related metabolites are separated from protein or sulfur-containing compounds to generate DFC. The protein as main interference substance in the plasma sample can be denatured and precipitated by water bath at 50 ℃ for 30 min, and most of the denatured and precipitated protein interference substance can be removed by centrifugation. The supernatant can be further purified by filtration through a 0.22 μm filter membrane to remove impurities such as proteins without centrifugation. The plasma sample is processed through water bath, centrifugation and filtration, so that the specificity of the sample can be ensured, and the specificity test of methodology shows that the plasma sample can be used for subsequent research and detection.

2) Determination conditions of high performance liquid chromatograph

High performance liquid chromatograph: agilent 1260 Infinity II;

a chromatographic column: ZORBAX SB-C18, (4.6 mm X250 mm, 5 μm);

mobile phase: 0.1% trifluoroacetic acid-water (a): acetonitrile (89: 11, V: V)

Detection wavelength: 266 nm;

flow rate: 1.0 ml/min;

column temperature: 35 ℃;

sample introduction amount: 50 μ l.

On the basis of the scheme, the method for detecting ceftiofur in blood plasma is applied to detecting ceftiofur and metabolites thereof in a large batch of blood plasma samples in clinical pharmacokinetic experiments.

Compared with the prior art, the invention has the beneficial effects that:

(1) the plasma sample is simple to pretreat, only water bath, centrifugation and filtration are needed, and iodoacetamide solution derivatization and solid phase extraction column treatment are not needed, namely, the extract obtained by water bath at 50 ℃ for 30 min in the step one is not needed to be added with iodoacetamide solution and then is subjected to water bath derivatization to obtain DCA. The liquid obtained after the filtration of the 0.22 mu m filter membrane in the step one can be directly used for the subsequent chromatographic detection without carrying out solid phase extraction and other subsequent steps; one part of blood plasma sample only needs 400 mul of extracting solution, the time is 40 min, the method simplifies the sample pretreatment step, reduces the cost, saves the time, has less reagent usage amount and low toxicity, and is suitable for the rapid detection of ceftiofur and metabolites thereof in a large batch of blood plasma samples.

(2) The specificity, detection limit and quantification limit, standard curve, extraction recovery rate, precision, accuracy and stability of the method are verified, and all the results are in accordance with the guiding principle of veterinary drug chemical clinical pharmacokinetic experiment (ministry of agriculture bulletin 1247). The method disclosed by the invention is successfully applied to clinical pharmacokinetic research of veterinary drug chemicals.

Drawings

FIG. 1 is a chromatogram (10. mu.g/ml) of a DFC standard in preferred embodiment 1 of the present invention;

FIG. 2 is a chromatogram of a sample of white pig plasma according to a preferred embodiment 1 of the present invention;

FIG. 3 is a sample chromatogram of plasma-supplemented DFC from white pig (10. mu.g/ml) according to preferred embodiment 1 of the present invention;

FIG. 4 is a chromatogram of a plasma sample of a pig after administration in accordance with a preferred embodiment 1 of the present invention (4 hours after administration of ceftiofur injection a produced in a certain country);

FIG. 5 is a chromatogram of a plasma sample of a pig after administration in accordance with a preferred embodiment 1 of the present invention (4 hours after administration of an imported ceftiofur injection);

fig. 6 is a pharmacokinetic test drug-time curve (5 mg/kg b.w.) of ceftiofur in swine in a preferred embodiment 2 of the present invention;

in fig. 6, a: a certain domestic ceftiofur injection B, B: a certain domestic ceftiofur injection a, C: an imported ceftiofur injection.

Detailed Description

The present invention will be more clearly and completely described in the following embodiments, but the described embodiments are only a part of the embodiments of the present invention, and not all of them. The examples are provided to aid understanding of the present invention and should not be construed to limit the scope of the present invention.

Example 1 detection method of ceftiofur in porcine plasma

1. Preparation of the principal solution

0.05 mol/L boric acid buffer: 19 g of boric acid and 3.7 g of potassium chloride are weighed, and the volume is up to 1000 ml after the boric acid and the potassium chloride are dissolved in water.

Extract (0.4% DTE w/v): 1.0 g Dithioerythritol (DTE) was weighed out and dissolved in 250 ml borate buffer and ready to use.

0.1% trifluoroacetic acid-water solution: 1 ml of trifluoroacetic acid is added into 1000 ml of ultrapure water and mixed evenly.

1 mg/ml DFC standard stock: approximately 10 mg of DFC standard was weighed into a 10 ml brown volumetric flask, dissolved in methanol and brought to volume, and stored at-70 ℃ for further use.

2. Sample pretreatment

Taking 200 μ l of plasma sample, adding 400 μ l of extractive solution (0.4% DTE w/v), mixing by vortex, water bathing at 50 deg.C for 30 min, and mixing by vortex once every 10 min. After completion of the water bath, the sample was centrifuged at 10000 r/min at 4 ℃ for 10 min, and the supernatant was filtered through a 0.22 μm filter and transferred to a sample vial for chromatographic analysis.

3. Determination conditions of high performance liquid chromatograph

High performance liquid chromatograph: agilent 1260 Infinity II;

a chromatographic column: ZORBAX SB-C18, (4.6 mm X250 mm, 5 μm);

mobile phase: 0.1% trifluoroacetic acid-water (a): acetonitrile (89: 11, V: V);

detection wavelength: 266 nm;

flow rate: 1.0 ml/min;

column temperature: 35 ℃;

sample introduction amount: 50 μ l.

4. Methodology validation

The methodology of the detection method is verified according to the guiding principle of clinical pharmacokinetic experiments of veterinary drug and chemical drugs (bulletin 1247 of Ministry of agriculture), and the verification contents comprise specificity, detection limit, quantification limit, standard curve, extraction recovery rate, precision, accuracy and stability.

4.1, specificity

Respectively taking 200 mul of blank pig plasma; adding 20 mul of DFC standard working solution with the concentration of 100 mug/ml into 180 mul of blank pig plasma, and preparing the DFC standard working solution into a pig plasma sample with the mass concentration of 10 mug/ml; plasma samples of pigs given ceftiofur injection 4 h; the samples were tested after being treated with each of the 6 parallel samples according to the sample pretreatment method.

4.2, detection and quantitation limits

Each 180. mu.l of blank porcine plasma was added with 20. mu.l of standard working solutions of DFC at concentrations of 0.1, 0.2, 0.5, 1.0 and 2.0. mu.g/ml, respectively, to prepare porcine plasma samples at DFC mass concentrations of 0.01, 0.02, 0.05, 0.1 and 0.2. mu.g/ml, each concentration being repeated 5 times. And (4) respectively processing the samples according to a sample pretreatment method, and then detecting. The S/N ratio is greater than or equal to 3 as the detection Limit (LOD), and S/N is greater than or equal to 10 as the quantification Limit (LOQ).

4.3, Standard Curve

Each 180. mu.l of blank porcine plasma was taken and added with 20. mu.l of DFC standard working solutions at concentrations of 0.5, 1, 5, 10, 50, 100 and 200. mu.g/ml, respectively, to prepare porcine plasma samples having DFC mass concentrations of 0.05, 0.1, 0.5, 1, 5, 10 and 20. mu.g/ml. Operating according to sample pretreatment method, taking drug DFC concentration as abscissa and peak area as ordinate, and adopting weighting (1/x)²) The least square method performs linear regression, and calculates a regression equation and a correlation coefficient.

4.4 extraction recovery

Each 180. mu.l of blank porcine plasma was added with 20. mu.l of standard working solutions of DFC at concentrations of 0.5, 10 and 200. mu.g/ml, respectively, to prepare porcine plasma samples with low, medium and high DFC concentrations of 0.05, 1.0 and 20. mu.g/ml, respectively, each concentration being repeated 5 times. And (4) respectively processing the samples according to a sample pretreatment method, and then detecting. The extraction recovery of DFC in porcine plasma samples was calculated.

4.5 precision and accuracy

Internal precision: the low (0.05. mu.g/ml), medium (1.0. mu.g/ml) and high (20. mu.g/ml) DFC mass concentrations were prepared in terms of extraction recovery, and the intra-batch coefficient of variation CV was calculated by repeating the above 5 times for each concentration.

Batch precision: samples were prepared according to the intra-batch precision, 3 batches of samples were prepared and tested continuously on different days, and the inter-batch coefficient of variation CV was calculated.

4.6, stability

Quality control samples with low (0.05. mu.g/ml), medium (1.0. mu.g/ml) and high (20. mu.g/ml) DFC were prepared according to the extraction recovery rate, and the stability of the samples after standing at room temperature for 4 h and standing in an automatic sample injector for 24 h after pretreatment of the samples and freeze-thaw cycling at-70 ℃ for 3 times was examined.

5. Analysis of results

5.1, specificity

Under the selected detection conditions, the DFC in the sample can be effectively separated from other endogenous impurities in the porcine plasma, the drug peak shape is better, the baseline is stable, the interference with the impurity peaks is avoided, the retention time of the DFC in the porcine plasma is about 20 min, and the retention time is more stable, as shown in figures 1-5.

5.2, detection and quantitation limits

The signal to noise ratio S/N is more than or equal to 3 as the detection Limit (LOD), S/N is more than or equal to 10 as the quantification Limit (LOQ), and the detection limit of ceftiofur in porcine plasma is determined to be 0.02 mu g/ml, and the quantification limit is determined to be 0.05 mu g/ml.

5.3 Standard Curve

The concentration of DFC in pig plasma is in the range of 0.05-20 mug/ml, 3 repeated standard curves and correlation coefficients (R)²) All are more than 0.99, the DFC drug concentration and the peak area have good linear relation, and the results are shown in Table 1.

5.4 extraction recovery

The DFC concentrations of low, medium and high quality were 0.05, 1.0 and 20. mu.g/ml, respectively, and each concentration was repeated 5 times. The recovery rate of DFC in the pig plasma is 94.74% -106.51%, and is more than 70%, see table 2.

5.5 precision and accuracy

DFC low (0.05. mu.g/ml), medium (1.0. mu.g/ml), high (20. mu.g/ml) mass concentrations were repeated 5 times each, and the intra-and inter-batch coefficient of variation CV was calculated, with the results of less than 15% both for intra-and inter-batch coefficient of variation, as shown in Table 3.

5.6, stability

And compared with the concentration of a freshly prepared sample, the concentration of the sample measured by placing the quality control sample with low (0.05 mu g/ml), medium (1.0 mu g/ml) and high (20 mu g/ml) DFC at room temperature for 4 h, placing an automatic sample injector after sample pretreatment for 24 h and freezing and thawing at-70 ℃ for 3 times, wherein the deviation is less than 15%, and the detection requirement is met.

EXAMPLE 2 comparative pharmacokinetic testing of three ceftiofur injections in pigs

The weight of the healthy long-white binary hybrid pig is 18, and the weight is 90-110 kg. The animals are raised for 1 week before the test according to a conventional method, and the feed is daily ration without any antibiotic. The pigs for the test are screened before the test, and the ages, the weights and the fat conditions are as consistent as possible. By adopting the test design of single-center, completely random, single-cycle parallel and single administration, 18 healthy long-white binary hybrid pigs are divided into a first group, a second group and three groups, wherein the total number of the groups is three, and each group comprises 6 male and female halves.

Fasting was 12 h before dosing, but sufficient drinking was provided. The pigs were administered by intramuscular injection at an administration dose of 5 mg/kg b.w. (calculated as ceftiofur) on the fasting empty stomach next morning in the first, second and third groups, wherein the ceftiofur injection (ceftiofur injection a produced in some country) was administered in the first group, the ceftiofur injection (ceftiofur injection b produced in some country) was administered in the second group, and the ceftiofur crystal injection (ceftiofur injection imported) was administered in the third group. About 5 ml of blood of the anterior vena cava sinus and anticoagulation of heparin sodium are respectively collected at 20 time points of pre-administration (0 min), 0.25 h, 0.5 h, 0.75 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 120 h, 144 h and 168 h after administration. Centrifuging the blood sample at 4000 r/min for 10 min, sucking the supernatant into another clean centrifugal tube, and storing at-70 ℃ for testing.

Plasma samples were processed according to the sample pretreatment method and analyzed by high performance liquid chromatography, and the mean plasma concentration-time curve is shown in fig. 6, wherein a: a certain domestic ceftiofur injection B, B: a certain domestic ceftiofur injection a, C: an imported ceftiofur injection. Phoenix winnonLin 8.2 software was used to select a non-compartmental model for calculation, and the main pharmacokinetic parameters are shown in Table 4.

Note: "-" indicates no such value.

And (4) conclusion: the specificity, detection limit and quantification limit, standard curve, extraction recovery rate, precision, accuracy and stability of the method all accord with the guiding principle of veterinary drug chemical drug clinical pharmacokinetic experiment (bulletin 1247 of ministry of agriculture), and the method provided by the invention is successfully applied to the detection of large-batch plasma samples in ceftiofur pharmacokinetic experiment.

Claims (8)

1. The detection method of ceftiofur comprises sample pretreatment and high performance liquid chromatograph detection, and is characterized by comprising the following steps of: step one, extracting a metabolite of ceftiofur by a sample through an extracting solution dithioerythritol solution water bath to remove furoyl ceftiofur, and then removing a protein interfering substance of a denaturation precipitate through centrifugation; step two, detecting by using a high performance liquid chromatograph; no solid phase extraction column treatment step is included.

2. The detection method according to claim 1, wherein the extract in the first step is 0.4% dithioerythritol solution by mass.

3. The detection method according to claim 2, wherein the ratio of the sample to the extraction solution added in the first step is 1:2 by volume.

4. The detection method as claimed in claim 3, wherein in the first step, the plasma sample is added into the extracting solution, the plasma sample is evenly mixed by vortex and then is subjected to water bath, then the sample is centrifuged, and the supernatant is filtered by a filter membrane and then is used for the detection in the second step.

5. The detection method according to claim 4, wherein in the first step, the water bath conditions are as follows: the temperature is 50 deg.C, the time is 30 min, and the vortex mixing is carried out once every 10 min during the water bath.

6. The detection method according to claim 4, wherein the filter is a 0.22 μm filter.

7. The detection method according to claim 1, wherein the detection conditions of the high performance liquid chromatograph in the second step are as follows: the high performance liquid chromatograph is Agilent 1260 Infinity II; the chromatographic column is ZORBAX SB-C18, 4.6 mm × 250 mm, 5 μm; mobile phase 0.1% trifluoroacetic acid-water: acetonitrile; the detection wavelength was 266 nm.

8. Use of the assay according to any one of claims 1 to 7 in the detection of ceftiofur and metabolites thereof in a plasma sample.

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