Study On The Fluorescence Analysis Of Penicillins Antibiotics

Penicillin antibiotics are widely used in clinical medicine, veterinary medicine and feed additives due to their low-cost and efficient peculiarities. However, the irregular and abusive use of antibiotics will lead to drug residues in animal-derived food and finally do harm to human health and entironment. Therefore, it is significant to develop a series of sensitive, reliable and convenient methods for the determination of penicillin antibiotics. In this paper, fluorescence analysis of common penicillins antibiotics, such as benzylpenicillin (BEN), amoxicillin (AMO) and ampicillin (AMP) have been studied. The main contents are as follows:Part 1 Study on the fluorescence spectrometric determination of benzylpenicillin by derivatization reaction with NBD-C1The derivatization reaction of 4-Chloro-7-nitro-2,1,3-benzoxadiazole (NBD-C1) as fluorescence derivatizing agent with benzylpenicillin degradation was investigated by fluorimetry. The results showed that NBD-C1 was prone to react with acidic degradation products of benzylpenicillin to produce stable fluorescent complexes in weak alkaline solution. Thereby the fluorescence property of benzylpenicillin was displayed. A new method for the fluorescence determination of benzylpenicillin has been developed. A linear calibration graph was obtained over the benzylpenicillin concentration range from 0.1～20.0 mg/L with a detection limit of 20μg/L under optimal conditions. Furthermore, the association constant of derivative complex was studied. Standard free energy changes and the mechanism of derivatization reaction were explored. The method has been applied to the determination of benzylpenicillin in milk and the relative standard deviation was 1.32%～2.13%. Spiked recovery tests were also carried out and the recoveries were 88.4%～95.2%.Part 2 Study on the fluorescence spectrometric for the determination of penicillins antibiotics by charge-transfer reactionChapter 1 Study on the fluorescence spectrometric for the determination of benzylpenicillin by charge-transfer reactionThe charge-transfer reaction of benzylpenicillin degradation as the donor with 2,3,5,6-tetrachloro-1,4-benzoquinone (TCBQ) as the acceptor was investigated by fluorimetry. The results showed that TCBQ was prone to react with acidic degradation products of benzylpenicillin to produce stable charge-transfer complexes in a mixture of methanol and water. Fluorescence intensity of the complex was increased significantly relative to benzylpenicillin degradation. Therefore a new method for the determination of benzylpenicillin has been developed. A linear calibration graph was obtained over the benzylpenicillin concentration range from 0.30-8.0 mg/L with a detection limit of 90μg/L under optimal conditions. Furthermore, infrared spectroscopy of charge-transfer complex was studied and the mechanism of charge-transfer reaction was explored. The method has been applied to the determination of benzylpenicillin in milk and spiked recovery tests were carried out. The recoveries were 88.0%-95.4%, and the relative standard deviation was 1.12%～1.46%.Chapter 2β-cyclodextrin enhanced fluorimetric method for the determination of ampicillin and amoxicillin by charge-transfer reactionThe charge-transfer reaction of ampicillin (AMP) and amoxicillin (AMO) as the donor with 7,7,8,8-tetracyanoquinodimethane (TCNQ) as the acceptor was investigated. A new fluorimetric method for the determination of AMP and AMO was developed. The results showed that TCNQ was prone to react with both AMP and AMO to produce n-πcomplexes at 45℃. The fluorescence intensity of the complex was enhanced significantly relative to that of the studied drugs itself. On the basis, the sensitivity and stability of system were further enhanced due to the clathration ofβ-cyclodextrin (β-CD). The linear calibration graphs were obtained over the AMP, AMO concentration range from 0.1～10.0, 0.05～6.0 mg/L with a detection limit of 30μg/L,10μg/L, respectively. Furthermore, the association constants of charge-transfer complex were studied. Standard free energy changes and the mechanism of charge-transfer reaction were explored. The method has been applied to determine the content of AMP and AMO in capsule and spiked recovery tests were carried out. The recoveries were 102.3% and 98.2% for AMP and AMO, respectively. This method is sensitive, easy and accurate, the results are in good agreement with those obtained with the Chinese Pharmacopoeia method.Part 3 Study on the fluorescence scattering spectrometric for the determination of penicillins antibioticsUnder pH 4-5 HC1 solution and heating condition, penicillins antibiotics (PENs) such as ampicillin (AMP), amoxicillin (AMO) and benzylpenicillin (BEN) were hydrolyzed into intermediates to react with copper(Ⅱ) (Cu(Ⅱ)) to form 1:1 binary chelates (PENs-Cu(Ⅱ)), which can further react with triiodide (I3-) to form 1:1:1 ternary ion-association complexes (PENs-Cu(Ⅱ)-I3-). As a result, the resonance Rayleigh scattering (RRS) and non-linear scattering such as second-order scattering (SOS) and frequency doubling scattering (FDS) were enhanced greatly. The increments of scattering intensity (ΔI) were directly proportional to the concentrations of the antibiotics in certain ranges. The detection limits (3σ) of the three PENs for AMP, AMO and BEN systems were 32μg/L,95μg/L and 151μg/L (RRS method),177μg/L,102μg/L and 184μg/L (SOS method) and 85μg/L,68μg/L and 138μg/L (FDS method), respectively. AMP system for RRS method was the most sensitive. RRS method was taken as an example for the studies of the optimum conditions and influence factors.the composition of ion-association complexes and the reaction mechanism were discussed. The effects of foreign substances were tested and it showed that the method has a good selectivity. Based on the ion-association reaction, a new sensitive, simple and rapid method for the determination of penicillin antibiotics has been developed. It can be applied to the determination of penicillin antibiotics in capsule, milk and human urine samples.