Studies On TOF-MS Analysis Methodology Of Antibiotic Residues And Degradation Rule Of Penicillin

The time-of-flight mass spectrometry was widely used for rapid screening of drug and research on its degradation and metabolism, with fast acquisition quality, high sensitivity, high accuracy, high resolution and wide range of m/z. The role of antibiotics in animal husbandry was very important. Abuse of antibiotics would lead to the drug residues, and bring great harm to human health. In recent years, antibiotics screening has gradually become a hot spot. A few years ago, The events about "melamine" in the dairy have caused human high attention. Because penicillin and other antibiotics were abused in the dairy animal husbandry, their residues have potential risks. Therefore, detection of antibiotic residues in feed and food and identification of milk without antibiotics were of extreme importance to human health. The abuse of beta lactamase to eliminate penicillin in milk products would result in perplex for accuracy of the determination of penicillin residue and identification whether milk containing antibiotics. This topic research established a new, high throughput, multi-glass, multi-residue LC-TOF-MS method for rapid screening and accurate quantification of antibiotics in feed and food. The degradation of penicillin with β-lactamase as well as the inhibitory effect of sulbactam and clavulanic acid on β-lactamase in solution were investigated. The rule of degradation of penicillin and β-lactamase was verified, and the degradation pathways of penicillins in milk in the presence of β-lactamase was developed for the first time. Degradation products penilloic acids were prepared using preparative chromatography, and characterized by infrared spectrogram, MS, and nuclear magnetic resonance spectrum. They could be used as the analytical standard for quantitative analysis and as the landmark for detecting whether penicillin was used in milk. This dissertation provided methodology base for food detection and safety evaluation, with important theoretical significance and practical value.The main contents of this dissertation were described as follows:1. The basic theory, instrument assembly and performance of time-of-flight mass spectrometry were described, and the application of time-of-flight mass spectrometry in food analysis, antibiotics degradation research, environmental monitoring, and biological macromolecules study were reviewed.2. The conditions of accelerated solvent extraction for extracting 23 antibiotics veterinary drugs from chicken feed, pig feed and fish feed were optimized. Under the optimized UPLC- QTOF- MS conditions, an accurate-mass database containing retention time, calculated mass, mass error, elemental composition, and isotope information was established. The feed samples were scanned using UPLC-QTOF-MS instruction. On the basis of the database, using the information in Mass Lynx Chromalynx Software, the antibiotics were screened and identified, and further identified for the founded positive target compounds by comparing them with the standards or by comparing their secondary mass spectra. The established ASE-UPLC-QTOF- MS method for the determination of 23 orbidden or limited use of antibiotic drugs in feeds had good linearity, detection limit, precision, and accuracy. The results found that all the purchased veterinary drugs were a mixture of three or more antibiotics and there was difference between the detected veterinary drug component and the labeled effective antibiotics component. Some of forbidden drugs such as furazolidone, ribavirin and dimetridaozole were found. This method could be used for rapid screening, identification and quantification of antibiotics in feeds.3. A simple and rapid multi-class multi-residue analytical method was developed for the screening and quantification of veterinary drugs in honey by ultra performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS). A total of 90 veterinary drugs were investigated belonged to more than 14 families such as lincomycins, macrolides, sulfonamides, quinolones, tetracyclines, β-agonists, β-lactams, sedatives, β-receptor antagonists, sex hormones, glucocorticoids, nitroimidazoles, benzimidazoles, nitrofurans, and the others. A modified quick, easy, cheap, effective, rugged, and safe(QuEChERS) procedure was used for the sample preparation without solid-phase extraction step. ACQUITY UPLC ? BEH C18 analytical column and gradient elution employed with 0.1%(V/V) formic acid aqueous solution and acetonitrile were used for the separation of 90 target analytes. Under the optimized UPLC- QTOF- MS conditions, an accurate-mass database for the 90 analytes was established using Analyst ? TF1.6 software along with 4 second spectra for each drug. The drugs in honey were screened and identified. The linearity, sensitivity, accuracy, repeatability, and reproducibility of the method were fully validated. The matrix-matched calibration curves were linear for each target compounds, in wide concentration range(at least, two orders of magnitude),with correlation coefficient(R2) of 0.9921?0.9999. The detection limit and quantification limit were 0.10?5.41μ g/kg and 0.32?18.03μ g/kg, respectively. The repeatability(RSD, n=6) and reproducibility(RSD, n=6) were 10.89% and 13.45%, respectively. The average recoveries ranged from 70.63%?114.62%, with relative standard deviation of 1.87%~9.57% at three concentration levels. The applicability of the screening method was verified by applying to real royal jelly samples, and certain veterinary drugs were detected in some cases。The method was simple, rapid, efficient, and high throughput,so it was suitable for screening and quantification of multi-glass multi-residue of veterinary drugs in honey.4. The degradation reactions of penicillin G, ampicillin, amoxicillin and penicillin V in the presence of β-lactamase in aqueous solution were studied by high performance liquid chromatography(HPLC)- time of flight mass spectrometry. The influence of temperature, time and PH on the degradation reaction was discussed. The results showed that the reaction in the milk and in aqueous solution was similar. The amount of β-lactamase for degradation of 10μg penicillin G, penicillin V, ampicillin and amoxicillin was 10U、10U、11U and 7U, respectively. When the penicillin and β-lactamase reacted completely, the most appropriate degradation temperature was from 20 ℃ to 30 ℃, and it taken 2.5 hours for penicillin V and amoxicillin, 2 hours for penicillin G, and 1.5 hours for ampicillin. When the temperature is below 40 ℃, their degradation products mainly were penicilloic acids,and when the temperature more than 40 ℃, the degradation products mainly were penilloic acids. the optimum pH for the degradation reaction of penicillin and β-lactamase was 6 ~ 7 in aqueous solution, and percentage of penicilloic acids and penilloic acids were different under different acidity. The results would have a certain guiding significance for clinical medication of penicillin and reasonable use of β-lactamase, and provide a research design and methodology base for investigating penicillin degradation in dairy products in the presence of β-lactamase. The β-lactamse-inhibitory rate of clavulanic acid and sulbactam was studied in aqueous solution using high performance liquid chromatography(HPLC)- time of flight mass spectrometry(TOFMS). The result showed that the β-lactamase-inhibitory rate of clavulanic acid was stronger than that of sulbactam both in aqueous solution. The β-lactamase inhibitory rate of clavulanic acid in combination with amoxicillin was very significantly higher than that of penicillin G, penicillin V, and ampicillin, The inhibitory rates were determined when clavulanic acid and sulbactam reacted for 25 min respectively. When the proportion of clavulanic acid and antibiotics reached 1:1 in aqueous solution, the β-lactamse-inhibitory rates of clavulanic acid reached 100%; when the proportion of sulbactam and antibiotics reached 8:1, the inhibitory rate of sulbactam reached 100%.5. The degradation of penicillin G, penicillin V, ampicillin, and amoxicillin in milk was investigated by high-performance liquid chromatography coupled with electrospray ionization-time-of-flight mass spectrometry. The degradation pathways of penicillins in milk in the presence of β-lactamase were developed. The influence of factors on the degradation was investigated, including β-lactamase dosage, temperature, time, and acidity. The law of penicillin degradation in milk in the presence of β-lactamase was similar to that in aqueous solution at pH 6?7. The ratio of the two degradation products(penicilloic G acid/penilloic G acid; penicilloic V acid/penilloic V acid; ampicillin penicilloic acid/ampicillin penilloic acid; amoxicillin penilcilloic acid/amoxicillin penilloic acid) was different at different temperatures and pH. Penicilloic acid was the dominant species obtained at pH=6 under 40°C, but, being unstable, it could not be used as a standard for accurate analysis of penicilloic acid, and also could not be used as landmark for identification of penicillins in milk. Penicilloic acid began to translate into penilloic acid at pH=2 above 40°C, and it was stable and could be used as a standard for quantitative analysis and as landmark for detecting whether penicillins were used in milk.6. A method was developed for preparation of penilloic G acid、penilloic V, ampilloic acid and amoxilloic acid by preparative reversed-phase high performance liquid chromatography. At the same time, the product was characterized by infrared, nuclear magnetic, and mass spectrometry. The purity of penilloic G acid, penilloic V acid, ampilloic acid and amoxilloic acid achieved to 94%, 92%, 91%, and 89%, respectively. The results showed that the preparation method of penilloic acid was convenient and feasible, which could be used as a standard for quantitative analysis and as landmark for detecting whether penicillins were used in milk.