CN113009052A - Method for detecting beta-lactam antibiotics in varicella attenuated live vaccine - Google Patents
Method for detecting beta-lactam antibiotics in varicella attenuated live vaccine Download PDFInfo
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Abstract
The invention provides a method for detecting beta-lactam antibiotics in a varicella attenuated live vaccine, which adopts a liquid chromatography-mass spectrometer to establish a method for simultaneously detecting 40 beta-lactam antibiotics in the varicella attenuated live vaccine for the first time. The method realizes high-flux screening of the residual antibiotics, has the characteristics of high speed, accuracy, high sensitivity and short operation time, and reduces the generation of false positives by adopting an MRM scanning mode. The method has the advantages that the simultaneous qualitative and quantitative analysis of 40 beta-lactam antibiotics in the vaccine provides a new idea for establishing a method for detecting other antibiotic residues in the vaccine, and guarantees are provided for guaranteeing the quality of the vaccine. Meanwhile, the antibiotic residues in different vaccine development stages can be grasped and adjusted in time by detecting the antibiotic residues in different vaccine development stages, so that huge loss caused by unqualified final products is avoided.
Description
Technical Field
The invention relates to detection of antibiotic residues in vaccines, in particular to a detection method of beta-lactam antibiotics in varicella attenuated live vaccines.
Background
The vaccine as one kind of preventing product in biological product is prepared with pathogenic microbe and its metabolite and through artificial attenuation, inactivation or gene engineering. In the production and storage processes, in order to ensure the quality of products, some antibiotics are often added to inhibit the infection of bacteria, chlamydia and mycoplasma, so that the antibiotics are remained, and some adverse reactions are caused. The international harmonization conference (ICH) is required to control the residue of antibiotics in vaccines as process impurities by the registration technology of human drugs; the united states pharmacopeia, european pharmacopeia, chinese pharmacopeia, etc. all require restrictions on the use of antibiotics in vaccines.
The beta-lactam antibiotics are relatively common antibiotics, mainly achieve the bacteriostatic effect by inhibiting the synthesis of bacterial cell walls, have high antibacterial rate and can act on G + bacteria and G-bacteria. Allergic reactions are the most common adverse reactions, which are usually independent of the dosage, and in the case of more sensitive people, very small amounts may cause shock or death, while cross-allergies may also exist between various penicillins, and in the case of penicillin-allergic patients, allergic reactions may also occur to other beta-lactam antibiotics. Therefore, neither the Chinese pharmacopoeia nor the United states pharmacopoeia stipulates that the vaccine cannot be added with beta-lactam antibiotics.
Controlling antibiotic residues in vaccines is particularly important to ensure consumer safety. In the appendix of the Chinese pharmacopoeia, a culture method is adopted for the detection method of antibiotic residues, but the method is easily interfered in the detection of biological products with complex components. Enzyme-linked immunosorbent assay is adopted for detecting antibiotic residues under most vaccine items. The method adopts a corresponding kit to carry out antigen-antibody combination, each antibiotic is detected by using one kit, only one or one class of antibiotics can be detected by one detection, and the high-flux detection of the antibiotics cannot be realized. The Jiangyu (2010) adopts an indirect competition ELISA method to detect the residual amount of kanamycin in the vaccine and adopts a kit. When the residues of ampicillin and chloramphenicol in chick embryos for producing influenza vaccines are measured, two corresponding kits are adopted for measurement.
With the development of instrument analysis in recent years, the method has accurate detection result, high separation speed and high separation efficiency, and is more and more widely applied to the fields of environment, food, biology and medicine. At present, the LC-MS technology is a commonly used detection means in compound analysis, not only has the capability of molecular structure analysis, but also can realize one-time scanning and screening of various compounds, and is widely applied to abuse and residue detection of antibiotics. However, no report is found about the detection method of various beta-lactam antibiotics in vaccines at present.
Disclosure of Invention
The invention aims to provide a method for detecting beta-lactam antibiotics in a varicella attenuated live vaccine.
In order to achieve the object of the present invention, the present invention provides a method for detecting a β -lactam antibiotic in a varicella attenuated live vaccine, comprising:
A. preparing beta-lactam antibiotic mixed standard solutions with different concentrations; wherein the beta-lactam antibiotic is selected from two or more of faropenem, cefadroxil, oxacillin, cefixime, cefepime, moxalactam, cefodizime, penicillin, amoxicillin, cephalothin, flucloxacillin, cefmenoxime, cefotiam, cefpiramide, cephalexin, cefaclor, cefetamet pivoxil, cefuroxime axetil, cefoperazone, cefradine, ceftizoxime, cloxacillin, cefotaxime, cefpirome, mezlocillin, cefoxitin, ampicillin, meropenem, ceftezole, azlocillin, piperacillin, ceftazidime, cefpodoxime proxetil, penicillin V, ticarcillin, cefuroxime, cefmetazole, cefathiamidine, biapenem and cefprozil;
B. respectively adding the beta-lactam antibiotic mixed standard solutions with different concentrations into the blank matrix of the varicella attenuated live vaccine, uniformly mixing to prepare standard sample solutions with series concentrations, and then carrying out liquid chromatography tandem mass spectrometry detection;
wherein the chicken pox attenuated live vaccine blank matrix is a chicken pox attenuated live vaccine without beta-lactam antibiotics;
C. for each beta-lactam antibiotic, drawing a standard curve according to the concentration of the beta-lactam antibiotic in the standard sample solution and the corresponding peak area thereof;
D. adding 0.5mL of varicella attenuated live vaccine to be detected into a centrifuge tube, adding 1.5mL of acetonitrile, whirling for 15s, fully shaking up, centrifuging at 10000rpm at room temperature for 10min, taking supernatant, and performing liquid chromatography tandem mass spectrometry detection according to the same conditions; and according to the detection result, obtaining the concentration of each beta-lactam antibiotic in the varicella attenuated live vaccine to be detected by contrasting the standard curve.
In the foregoing method, step a includes:
a1, preparation of stock solution: precisely weighing 10mg of each beta-lactam antibiotic reference substance, dissolving with a reagent, and preparing a solution with the concentration of 1mg/mL as a stock solution; wherein, the cephalosporin antibiotics are dissolved by methanol aqueous solution, and the penicillin antibiotics are dissolved by water;
a2, preparation of basic working solution: precisely measuring 10 mu L of each antibiotic stock solution, mixing, and diluting with methanol water solution to a concentration of 10 mu g/mL as a basic working solution;
a3, preparation of beta-lactam antibiotics mixed standard solution standard series solutions with different concentrations: the basic working solution was diluted with aqueous methanol to prepare mixed standard solutions of 10000, 5000, 2500, 1000, 500, 200, 100, 50, 10, 5 and 1ng/mL of β -lactam antibiotics, respectively.
Wherein the volume ratio of methanol to water in the methanol aqueous solution is 1: 1.
The concentrations of the β -lactam antibiotic mixture in the spiked sample solution described in step B, of the foregoing methods, were 0.08, 0.4, 0.8, 4, 8, 18, 40, 80, 200, 400, and 800ng/mL, respectively.
Preferably, the liquid chromatography conditions are as follows:
adopting SHISEIDO CAPCELL PAK C18 MG chromatographic column, 150mm × 2.0mm,5 μm; mobile phase A: 0.1% formic acid solution, flow B: 0.1% formic acid acetonitrile solution; the sample volume is 10 mu L; the flow rate is 0.3 mL/min; gradient elution was performed as follows:
preferably, the mass spectrometry conditions are as follows:
an AB QTrap 6500 triple quadrupole-linear ion trap mass spectrometer was used, containing an ESI ion source.
The mass spectrometry adopts a positive ion scanning mode, the scanning mode IS scheduled MRM, and the electrospray voltage (IS): 5500V; ionization temperature: 500 ℃; atomizing gas pressure: 40 psi; auxiliary gas pressure: 40 psi; air curtain pressure: 25 psi; collision gas (CAD): 6V; inlet voltage: 10ev, exit voltage: 11 ev.
The MRM parameters and retention times for each β -lactam antibiotic are shown in table 1.
TABLE 1 MRM parameters and Retention times for each compound
In the method, the limit of the quantification of the beta-lactam antibiotics in the varicella attenuated live vaccine is 0.4-40ng/mL, and the minimum detection limit is 0.08 ng/mL.
The invention also provides the application of the method in the quality control of the varicella attenuated live vaccine product.
The invention adopts a liquid chromatography-mass spectrometer, establishes a method for detecting 40 beta-lactam antibiotics in the matrix of the varicella attenuated live vaccine, and carries out relevant methodology verification. The method adopts a SHISEIDO CAPCELL PAK C18 MG (150mm multiplied by 2.0mm,5 mu m) chromatographic column, and 0.1% formic acid water-0.1% formic acid acetonitrile as a mobile phase; triple quadrupole-linear ion trap mass spectrometer electrospray ionization source (ESI) Multiple Reaction Monitoring (MRM) positive ion scan mode. Acetonitrile precipitation is used as a pretreatment method. The recovery rate of various antibiotics in the varicella attenuated live vaccine is 68.2 to 107.8 percent. The detection limit of each target antibiotic in the varicella attenuated live vaccine is 0.02-4 ng/dose (S/N is more than or equal to 3), and the quantification limit is 0.04-10 ng/dose (S/N is more than or equal to 10); the precision of each antibiotic in the vaccine matrix is less than 12.5% in day and in the daytime, and the accuracy is 80-115%. The method has high sensitivity and good reproducibility, realizes simultaneous qualitative and quantitative analysis of multiple antibiotics in the vaccine, and provides guarantee for guaranteeing the quality of the vaccine.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1
1. Chemicals and reagents
The control products faropenem (batch 130532-201301 with a purity of 80.3%), cefadroxil (batch 130431-, purity 92.5%), cefalexin (batch 130408-201411, purity 94.4%), cefaclor (batch 130481-201606, purity 95.3%), cefazolin (batch 130421-201204, purity 99.0%), cefetamet pivoxil (batch 130512-201202, purity 70.2%), cefuroxime axetil (batch 130492-201703, purity 82.5%), cefoperazone (batch 130420-201105, purity 93.8%), cephradine (batch 130427-201708, purity 88.4%), cefazolin (batch 130504-201503, purity 98.2%), cloxacillin (batch 130423-200903), purity 91.0%), cefotaxime (batch 130483483-201505, purity 92.2%), cefpirome (batch 130701-545, purity 83.0%), mezlocillin (batch 130130519, purity 13093-130904, purity 13093-1303, purity 1303-130410%, purity 86.5%), meropenem (batch No. 130506-, the enterovirus 71 type inactivated vaccine (vero cells) (0.5 mL/dose), the recombinant hepatitis B vaccine (saccharomyces cerevisiae) (1 mL/dose) and the varicella attenuated live vaccine (0.5 mL/dose) are respectively from A, B, C manufacturers; mass spectrometry grade methanol, acetonitrile were purchased from Fisher corporation; mass Spectrometry grade formic acid was purchased from Sigma and water from Guangzhou Drech GmbH.
2. Preparation of solutions
Stock solution: precisely weighing 10mg of each beta-lactam antibiotic reference substance, and respectively dissolving by adopting a certain dissolving agent to prepare a solution with the concentration of 1mg/mL as stock solution. Typical cephalosporin antibiotics use methanol: dissolving in water (v/v ═ 1:1), dissolving penicillin antibiotic in water, and storing at-20 deg.C.
Basic working solution: precisely measuring 10 mu L of each antibiotic stock solution, mixing, adopting methanol: a solution of water (v/v ═ 1:1) was diluted to a concentration of 10 μ g/mL as a basic working solution.
Standard series of solutions: methanol is adopted: diluting the basic working solution with water (v/v is 1:1) to prepare a beta-lactam antibiotic mixed standard solution with a certain concentration (10000, 5000, 2500, 1000, 500, 200, 100, 50, 10, 5 and 1 ng/mL); it is prepared as before use.
3. Arrangement of mobile phase
And (3) taking 0.1% formic acid aqueous solution as the mobile phase A, measuring 1000mL of water, adding 1mL of formic acid, and shaking up and carrying out ultrasonic treatment. And (3) adopting a 0.1% formic acid acetonitrile solution as the mobile phase B, measuring 1000mL of acetonitrile, adding 1mL of formic acid, and shaking up and carrying out ultrasonic treatment.
4. Preparation of Standard sample solution
Adding 10 μ L of enterovirus 71 inactivated vaccine (vero cells) (0.5 mL/dose), 20 μ L of recombinant hepatitis B vaccine (Saccharomyces cerevisiae) (1 mL/dose), and 20 μ L of varicella live attenuated vaccine (0.5 mL/dose) into the vaccine blank matrix, and mixing to obtain standard sample solutions (with concentration of enterovirus 71 inactivated vaccine (vero cells) and recombinant hepatitis B vaccine (Saccharomyces cerevisiae) of 0.02, 0.1, 0.2, 1, 2, 4, 10, 20, 50, 100 and 200 ng/dose, and concentration of varicella live attenuated vaccine of 0.04, 0.2, 0.4, 2, 4, 8, 20, 40, 100, 200 and 400 ng/dose).
5. Pretreatment of samples
Taking enterovirus 71 type inactivated vaccine (vero cells) (0.5 mL/dose), transferring to a 2mL centrifuge tube, adding acetonitrile 0.5mL, vortexing for 15s, fully shaking, centrifuging at 10000rpm for 10min to precipitate protein, and taking supernatant to enter a liquid chromatograph-mass spectrometer for analysis.
Taking recombinant hepatitis B vaccine (saccharomyces cerevisiae) (1 mL/dose), transferring to a 2mL centrifuge tube, adding acetonitrile 1mL, vortexing for 15s, fully shaking, centrifuging at 10000rpm for 10min to precipitate protein, and taking supernatant to enter a liquid chromatograph-mass spectrometer for analysis.
Dissolving varicella attenuated live vaccine (0.5 mL/dose), vortexing for 15s, transferring to a 2mL centrifuge tube, adding acetonitrile for 1.5mL, vortexing for 15s, centrifuging at 10000rpm for 10min to precipitate protein, and taking supernatant to enter a liquid chromatograph-mass spectrometer for analysis.
6. Liquid condition
The liquid phase system was an LC 20AT liquid chromatograph (Shimadzu, Japan) from Shimadzu corporation, comprising a binary pump, a sample injector, a column oven, and a detector. The mass spectrometry system used an AB QTrap 6500 triple quadrupole-linear ion trap mass spectrometer, containing an ESI ion source (AB sciex).
Using SHISEIDO CAPCELL PAK C18 MG (150mm × 2.0mm,5 μm) chromatography column; mobile phase A: 0.1% formic acid solution, flow B: 0.1% formic acid acetonitrile solution, the sample volume is 10 mu L, and the flow rate is 0.3 mL/min; the gradient elution conditions were as follows:
the mass spectrometry adopts a scanning mode of a positive ion scanning mode scheduled MRM, and electrospray voltage (IS): 5500V; ionization temperature: 500 ℃; atomizing gas pressure: 40 psi; auxiliary gas pressure: 40 psi; air curtain pressure: 25 psi; collision gas (CAD): 6V; inlet voltage: 10ev, exit voltage: 11 ev. The mass spectrum conditions of each compound (including parent ion mass-to-charge ratio, declustering voltage (DP), daughter ion mass-to-charge ratio and collision voltage (CE)) were optimized by needle pump injection, and the mass spectrum parameters obtained by the optimization are shown in table 1.
The method is also suitable for detecting the related beta-lactam antibiotics in the recombinant CHO hepatitis B vaccine.
For each beta-lactam antibiotic, a standard curve was plotted according to the concentration of the beta-lactam antibiotic in the spiked sample solution and its corresponding peak area (Table 2); then, performing liquid chromatography tandem mass spectrometry detection on the pre-treated sample to be detected according to the same conditions; and according to the detection result, obtaining the concentration of each beta-lactam antibiotic in the vaccine to be detected by contrasting the standard curve.
TABLE 2 Linear relationship of 40 beta-lactam antibiotics in varicella attenuated live vaccine
Example 2 optimization and efficiency of extraction conditions
In the pretreatment of the vaccine, a method of concentrating the vaccine by using an ultrafiltration centrifugal tube and recovering a filtrate is tried, but the method has an overlarge matrix effect and cannot achieve the aim of extracting the target antibiotic. A simple and rapid pretreatment solvent precipitation method is adopted, a varicella attenuated live vaccine is used as a matrix, the effect of a precipitator containing ethanol, methanol and acetonitrile is explored, when the volume ratio of the precipitator to the vaccine is 1:1, 3 precipitants have higher matrix effect, an obvious matrix interference peak can be seen, the addition amount of the precipitator is tried to be increased, the matrix effect is reduced, and finally the situation that the volume of the precipitator added into the varicella attenuated live vaccine (0.5 mL/dose) is 3 times of the specification is determined, the precipitation phenomenon of alcohol precipitation is unstable in the precipitation process, sometimes obvious white precipitation phenomenon occurs and sometimes does not occur, so that the acetonitrile is finally determined to be used as the precipitator. Meanwhile, the sensitivity of the detection method is considered, and finally the addition amount of acetonitrile of the recombinant hepatitis B vaccine (saccharomyces cerevisiae) and the enterovirus 71 type inactivated vaccine (vero cells) is determined to be 1 time of the specification, and the addition amount of acetonitrile of the varicella vaccine is determined to be 3 times of the specification.
Example 3 optimization of liquid conditions
In this example, the following three types of columns were tried: ACE Excel 5C 18(75 mm. times.2.1 mm,5 μm), Shiseido CAPCSLL PAK C18 MG (150 mm. times.2.0 mm,5 μm), Shiseido CAPCSLL PAK C18 MG (75 mm. times.2.0 mm,5 μm), for optimization of chromatographic conditions. The results show that: when an ACE Excel 5C 18(75mm multiplied by 2.1mm,5 mu m) chromatographic column is adopted, an amoxicillin peak is split, and tailing is serious; when Shiseido CAPCSLL PAK C18 MG (150mm multiplied by 2.0mm,5 μm) is adopted as a chromatographic column, the peak shape of each antibiotic is sharp, and the separation degree is better; in order to shorten the retention time of each antibiotic, a Shiseido CAPCSLL PAK C18 MG (75 mm. times.2.0 mm,5 μm) column was also used, but the peak pattern of each antibiotic under the column was broad; therefore, a Shiseido CAPCSLL PAK C18 MG (150 mm. times.2.0 mm,5 μm) column was finally selected.
For the selection of mobile phase, mobile phase a: 5mM ammonium formate solution, 0.1% aqueous formic acid; mobile phase B: and (3) acetonitrile. By continuously adjusting the gradient of the mobile phase, the result shows that the peak emergence time of each antibiotic is concentrated and the resolution is low when 5mM ammonium formate solution and acetonitrile are used as the mobile phase; when a 0.1% formic acid solution and a 0.1% formic acid acetonitrile solution are adopted, the peak emergence time of each chromatographic peak is relatively dispersed and the peak shape is good. Thus a 0.1% formic acid solution and a 0.1% formic acid acetonitrile solution were finally used as mobile phases for the process. Formic acid is a common LC/MS additive, which can improve ionization efficiency and peak type of chromatographic peak.
For the selection of the elution gradient, in order to meet the requirement that antibiotics with different polarities can have better peak patterns, the lower proportion of an organic phase is adopted for elution at first, when the proportion of the initial organic phase is too large, the peak pattern of amoxicillin is split, when the proportion of the organic phase is continuously reduced, the peak pattern of the antibiotics is improved, when the initial proportion is finally determined to be 1% of the organic phase, the amoxicillin has better peak patterns, and meanwhile, all other antibiotics have better peak patterns under the proportion. Thus, the final gradient elution conditions were determined to be 0.1min, 1% mobile phase B; 4.5min, 60% mobile phase B; 5.5min, 95% mobile phase B; 8.0min, 95% mobile phase B; 8.1min, 1% mobile phase B; 15min, 1% mobile phase B.
Optimizing the mass spectrum condition of each antibiotic, adopting a needle pump sample injection mode to optimize the mass spectrum condition of each antibiotic (1 mu g/mL or 100ng/mL), firstly adopting a Q1 scanning mode to obtain the parent ion mass-to-charge ratio information of a target antibiotic, then adopting a Q1M scanning mode to optimize the de-clustering voltage, then adopting an MS2 scanning mode to obtain the daughter ion information of a compound, selecting 3 pairs of daughter ion pairs with stronger signals for each antibiotic, selecting the ion pair with the strongest signal for the quantitative analysis of the antibiotic, simultaneously detecting the 3 pairs of ion pairs for the qualitative analysis of the antibiotic, and finally adopting an MRM scanning mode to optimize the collision voltage of each pair of ion pairs. Finally, mass spectrum conditions of each antibiotic are determined.
Example 4 method verification
1. The matrix effect refers to the effect of components which flow out together with a sample to be detected in a chromatographic separation system on the ionization process of the sample to be detected, and the effect of ion enhancement or ion inhibition is generated. In the invention, 3 different concentrations (10, 50 and 100 ng/dose of recombinant hepatitis B vaccine (saccharomyces cerevisiae) and enterovirus 71 inactivated vaccine (vero cells) and 20, 100 and 200 ng/dose of varicella attenuated live vaccine) are selected for each vaccine to carry out matrix effect investigation of 40 beta-lactam antibiotics in the vaccine.
The matrix effect is calculated by taking the ratio of the peak area response of the beta-lactam antibiotic in the vaccine to the peak area response in the pure solvent. The formula is as follows:
matrix effect ═ aBeta-lactam antibiotics in vaccines-ABeta-lactam antibiotics in pure solvents)/ABeta-lactam antibiotics in pure solvents×100％
Wherein A isBeta-lactam antibiotics in vaccinesRepresents the peak area response of beta-lactam antibiotics in the vaccine, ABeta in pure solvent-lactam antibioticsShowing the peak area response of the beta-lactam antibiotic in pure solvent.
The matrix effect range is negligible from-20% to 20%, when the matrix effect is less than-20%, the inhibition effect is considered, and when the matrix effect is more than 20%, the enhancement effect is considered. The final three vaccine measurements are shown in tables 3-5. The results show that most antibiotics have varying degrees of potentiating and inhibiting effects in vaccines. And the matrix effect of the same vaccine extract on different target antibiotics is different, and the matrix effect of different vaccine matrixes on the same beta-lactam antibiotics is different. In order to eliminate the influence of matrix effect, a calibration working curve method is adopted in a blank vaccine matrix to calculate the related content.
The detection limit of each target antibiotic of the enterovirus 71 inactivated vaccine (vero cells) and the recombinant hepatitis B vaccine (saccharomyces cerevisiae) is as follows: 0.02-4 ng/dose (S/N is more than or equal to 3), and the quantitative limits are as follows: 0.04-10 ng/dose (S/N is more than or equal to 10); the detection limit of each target antibiotic in the varicella attenuated live vaccine is as follows: 0.04-16 ng/dose (S/N is more than or equal to 3), and the quantitative limits are as follows: 0.2-20 ng/dose (S/N is more than or equal to 10).
2. Accuracy, which is the concentration of the measured sample/the theoretical sample addition concentration, represents how close the result of the sample measured by this method is to the true value of the sample. Precision is the measure of closeness between multiple samples, usually expressed as relative standard deviation. To determine the accuracy and precision of each antibiotic in the vaccine, the concentrations of each antibiotic of interest were tested in an inactivated vaccine of enterovirus 71 (vero cells) and in a recombinant hepatitis b vaccine (saccharomyces cerevisiae) as follows: 10. 50, 100 ng/dose; the concentration of each target antibiotic tested in the varicella attenuated live vaccine is as follows: 20. 100, 200 ng/dose, the results are shown in tables 3 to 5. The accuracy of each antibiotic in the enterovirus 71 type inactivated vaccine (vero cells) is 90-115 percent, and the accuracy of the recombinant hepatitis B vaccine (saccharomyces cerevisiae) is 80-110 percent; the varicella attenuated live vaccine is 85-115%, and the precision of each antibiotic in the three vaccine matrixes is less than 12.5% in day and in the daytime. The results are shown in tables 6 to 8.
The linearity of the method is inspected by adopting a standard working curve method, and the linearity R of various antibiotics in the three vaccine matrixes is more than or equal to 0.9944, which indicates that the method has good linearity.
3. Three different concentrations, an inactivated enterovirus 71 vaccine (vero cells) and a recombinant hepatitis b vaccine (saccharomyces cerevisiae), were investigated in each vaccine for each antibiotic recovery: 10. 50, 100 ng/dose; varicella attenuated live vaccine: 20. 100, 200 ng/dose. The recovery rate of each antibiotic in the enterovirus 71 type inactivated vaccine (vero cells) is 84.5-108.2% (table 3), and the recovery rate of the recombinant hepatitis B vaccine (saccharomyces cerevisiae) is 73-108% (table 4); the recovery rate of most antibiotics in the varicella attenuated live vaccine is 68.2-107.8% (table 5), but the recovery rates of meropenem, cefoxitin, moxalactam, ceftazidime, cefodizime, biapenem, cefadroxil, ticarcillin and cefprozil antibiotics are poor, probably because the auxiliary materials in the varicella vaccine are complex and are combined with the antibiotics, so the recovery rate of the sample is interfered.
TABLE 3 detection limit, quantitation limit, recovery rate, matrix effect of 40 beta-lactam antibiotics in inactivated vaccine of Enterovirus 71 (vero cells)
TABLE 4 detection limit, quantitation limit, recovery rate, matrix effect of 40 beta-lactam antibiotics in recombinant hepatitis B vaccine (Saccharomyces cerevisiae)
TABLE 5 detection limit, quantitation limit, recovery rate, matrix effect of 40 beta-lactam antibiotics in varicella attenuated live vaccine
TABLE 6 Intra-day precision and inter-day precision of 40 beta-lactam antibiotics in Enterovirus 71 inactivated vaccine (vero cells)
TABLE 7 Intra-day precision, Inter-day precision of 40 beta-lactam antibiotics in recombinant hepatitis B vaccine (Saccharomyces cerevisiae)
TABLE 8 Intra-day precision and inter-day precision of 40 beta-amide antibiotics in varicella attenuated live vaccine
The invention adopts an HPLC-MS/MS method to establish 40 beta-lactam antibiotics in three vaccine matrixes of a live attenuated varicella vaccine, namely a vero cell inactivated vaccine (vero cell), a recombinant hepatitis B vaccine (saccharomyces cerevisiae) and a varicella vaccine which can be simultaneously detected for the first time. Acetonitrile precipitation is adopted as a pretreatment, and relevant methodology verification is carried out. The recovery rate of each antibiotic in the enterovirus 71 type inactivated vaccine (vero cells) is 84.5-108.2 percent, and the recovery rate of each antibiotic in the recombinant hepatitis B vaccine (saccharomyces cerevisiae) is 73-108 percent; the recovery rate of most antibiotics in the varicella attenuated live vaccine is 68.2-107.8 percent, and the recovery rate is better. The establishment of the method makes up the defect that the existing vaccine detection method can only detect one or one type of antibiotics every time through enzyme-linked immunosorbent assay, realizes high-flux, efficient and rapid detection of multiple antibiotics in the vaccine, and provides guarantee for guaranteeing the quality of the vaccine and the safety of consumers. Meanwhile, the antibiotic residue in different vaccine development stages can be detected, the antibiotic residue in different stages can be mastered, and adjustment can be made in time, so that the loss caused by unqualified final products can be avoided.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Reference documents:
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Claims (8)
1. The detection method of the beta-lactam antibiotics in the varicella attenuated live vaccine is characterized by comprising the following steps:
A. preparing beta-lactam antibiotic mixed standard solutions with different concentrations; wherein the beta-lactam antibiotic is selected from two or more of faropenem, cefadroxil, oxacillin, cefixime, cefepime, moxalactam, cefodizime, penicillin, amoxicillin, cephalothin, flucloxacillin, cefmenoxime, cefotiam, cefpiramide, cephalexin, cefaclor, cefetamet pivoxil, cefuroxime axetil, cefoperazone, cefradine, ceftizoxime, cloxacillin, cefotaxime, cefpirome, mezlocillin, cefoxitin, ampicillin, meropenem, ceftezole, azlocillin, piperacillin, ceftazidime, cefpodoxime proxetil, penicillin V, ticarcillin, cefuroxime, cefmetazole, cefathiamidine, biapenem and cefprozil;
B. respectively adding the beta-lactam antibiotic mixed standard solutions with different concentrations into the blank matrix of the varicella attenuated live vaccine, uniformly mixing to prepare standard sample solutions with series concentrations, and then carrying out liquid chromatography tandem mass spectrometry detection;
wherein the chicken pox attenuated live vaccine blank matrix is a chicken pox attenuated live vaccine without beta-lactam antibiotics;
C. for each beta-lactam antibiotic, drawing a standard curve according to the concentration of the beta-lactam antibiotic in the standard sample solution and the corresponding peak area thereof;
D. adding 0.5mL of varicella attenuated live vaccine to be detected into a centrifuge tube, adding 1.5mL of acetonitrile, whirling for 15s, fully shaking up, centrifuging at 10000rpm at room temperature for 10min, taking supernatant, and performing liquid chromatography tandem mass spectrometry detection according to the same conditions; and according to the detection result, obtaining the concentration of each beta-lactam antibiotic in the varicella attenuated live vaccine to be detected by contrasting the standard curve.
2. The method of claim 1, wherein step a comprises:
a1, preparation of stock solution: precisely weighing 10mg of each beta-lactam antibiotic reference substance, dissolving with a reagent, and preparing a solution with the concentration of 1mg/mL as a stock solution; wherein, the cephalosporin antibiotics are dissolved by methanol aqueous solution, and the penicillin antibiotics are dissolved by water;
a2, preparation of basic working solution: precisely measuring 10 mu L of each antibiotic stock solution, mixing, and diluting with methanol water solution to a concentration of 10 mu g/mL as a basic working solution;
a3, preparation of beta-lactam antibiotics mixed standard solution standard series solutions with different concentrations: diluting the basic working solution with methanol aqueous solution to prepare mixed standard solutions of the beta-lactam antibiotics with the concentrations of 10000, 5000, 2500, 1000, 500, 200, 100, 50, 10, 5 and 1ng/mL respectively;
wherein the volume ratio of methanol to water in the methanol aqueous solution is 1: 1.
3. The method of claim 1, wherein the concentrations of the β -lactam antibiotic mixture in the spiked sample solution in step B are 0.08, 0.4, 0.8, 4, 8, 18, 40, 80, 200, 400, and 800ng/mL, respectively.
4. The method of claim 1, wherein the liquid chromatography conditions are as follows:
adopting SHISEIDO CAPCELL PAK C18 MG chromatographic column, 150mm × 2.0mm,5 μm; mobile phase A: 0.1% formic acid solution, flow B: 0.1% formic acid acetonitrile solution; the flow rate is 0.3 mL/min; gradient elution was performed as follows:
5. the method of claim 1, wherein the mass spectrometry conditions are as follows:
an AB QTrap 6500 triple quadrupole linear-ion trap mass spectrometer is adopted, and comprises an ESI ion source;
the mass spectrometry adopts a positive ion scanning mode, the scanning mode is scheduled MRM, and the electrospray voltage: 5500V; ionization temperature: 500 ℃; atomizing gas pressure: 40 psi; auxiliary gas pressure: 40 psi; air curtain pressure: 25 psi; collision gas: 6V; inlet voltage: 10ev, exit voltage: 11 ev.
6. The method of claim 5, wherein the MRM parameters and retention times of each β -lactam antibiotic are as follows:
7. the method of any one of claims 1 to 6, wherein the limit of quantitation of β -lactam antibiotics is 0.4 to 40ng/mL and the minimum detection limit is 0.08 ng/mL.
8. Use of the method of any one of claims 1 to 7 for the quality control of a varicella attenuated live vaccine product.
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