β-lactam drugs (β-lactamsdrug) refers to a large class of antibiotics containing β-lactam rings in the structure, which can be divided into penicillins, cephalosporins, carbapenems and monocyclic β-lactams according to the differences in the structure of the parent nucleus, of which penicillin drugs and cephalosporins are most widely used in veterinary clinical applications. This section reviews the structure and classification, physicochemical properties, pharmacokinetics, toxicology, domestic and foreign limit requirements, sample pretreatment for residue detection, and instrument determination methods of β-lactam drugs, in order to provide a reference for the comprehensive understanding and residue detection of such drugs.
1 Structure and classification
1.1 Structure
β-lactam drug structures contain a tetragonal ring structure of β-lactam parent nuclei, which are rare in natural products and are representative features of this class of antibiotics. Among them, penicillins and cephalosporins are the most widely used, and their structural characteristics are shown in Figure 5-39.
(1) The basic structure is composed of two parts: the mother nucleus and the side chain. The parent nucleus part contains a tetragonal β-lactam ring, which is fused with a second five- or six-membered ring by a nitrogen atom and three carbon atoms adjacent to it. The fused ring of penicillins is a hydrogenated thiazole ring with a parent nucleus structure of 6-amino penicillinic acid (6-APA); the fused ring of cephalosporins is a hydrogenated thiazole ring, and the parent nucleus structure is 7-amino cephalosporanic acid ( 7-ACA).
(2) The C6 or C7 position contains an amide-based side chain (RCONH-), which is often the connection point of the side chain.
(3) There are 2 fused rings in the parent nucleus structure, penicillin folded along the N1~C5 axis, and cephalosporins folded along the N1~C6 axis.
(4) Penicillin parent nuclei contain 3 chiral carbon atoms, and only the absolute configuration of 2S, 5R, 6R is active in 8 optical isomers, so it is very difficult to synthesize completely; the cephalosporin parent nucleus contains 2 chiral carbon atoms, and the active absolute configuration is 6R and 7R, and their efficacy is also related to the chiral carbon atoms of the RCONH- substituent.
The chemical structure of common β-lactam drugs is shown in Table 5-28.
1.2 Classification
1.2.1 Classification of penicillin drugs
Penicillin drugs are divided into natural penicillin and semi-synthetic penicillin, natural penicillin extracted from microbial fermentation liquid, including penicillin F, penicillin G, penicillin X, penicillin K and dihydropenicillin F5 components, of which penicillin G content is the highest, the best effect; semi-synthetic penicillin is synthesized by changing the side chain structure.
According to the antibacterial spectrum of penicillin drugs, it is divided into five categories, the first group is mainly used to resist the production of β- lactamase gram-positive bacteria including staphylococcus, streptococcus, bacillus and other infections, belongs to the narrow spectrum penicillin, representing antibiotics such as penicillin G (for injection) sodium salt and potassium salt, penicillin V (oral administration), procaine penicillin, benzylcin penicillin, etc.; the second type is aminopenicillin, which has a wide antibacterial spectrum, representing antibiotics such as ampicillin, amoxicillin and so on The third category is penicillin-resistant penicillin, stable to penicillinase, mainly used to resist various infections caused by Staphylococcus production, its antibacterial spectrum is narrow, representing antibiotics such as cloxacillin, dicloxacillin, benzoxicillin, flucloxacillin, etc.; the fourth group is broad-spectrum penicillin that is active against Pseudomonas aeruginosa, representing antibiotics such as carboxycinillin, ticarcillin, piperacillin, aloxiline, melocillin, etc.; the fifth group is penicillin that mainly acts on gram-negative bacteria, which only has a good effect on Enterobacterium bacteria. The antibacterial spectrum is narrow, representing antibiotics such as mexiline, pemecillin, and timoxillin.
1.2.1 Classification of cephalosporins
Cephalosporin drugs are semi-synthetic drugs, the main synthesis method is to introduce different side chain groups at the R1 and R2 positions of the parent nucleus structure, and the cephalosporin drugs can be divided into four generations according to the sequence of synthesis time. The first generation of cephalosporins is stable against penicillinase, but has poor resistance to β-lactamase produced by most gram-negative bacteria, including cefproxin, ceframadine, cefazolin, cefproxine and other drugs. The second generation of cephalosporins include cefuroxime, cefmondol, etc., the drug production is relatively small, such drugs have gradually been used in medical clinics in recent years, but rarely used in animal clinics. The third generation of cephalosporins contains many types, and the commonly used clinical drugs are cefotaxime, cefoperazone, ceftazidime, ceftriaxone, etc., which are stable for β-lactamase and have significant effects on anaerobic bacteria and gram-negative bacteria. The fourth generation of cephalosporins is broad-spectrum, efficient, highly stable for β-lactamase, and is mainly used for the treatment of stubborn infectious diseases, including cefquinolol and cefpirol. Among them, the third generation of cefotafuran and the fourth generation of cefoquinol are animal-specific drugs, and the efficacy is indeed.
2 Physicochemical properties
β-lactam drugs have poor solubility in water and oily organic solvents, so the clinical drugs are mostly potassium salts or sodium salts, salts are soluble in polar solvents such as water, acetonitrile, methanol and ethanol, and the appearance is white crystal or powder, odorless or specific odor, and has different degrees of hygroscopicity. β-lactam drugs mostly have optical rotation, penicillin parent nuclei lack ultraviolet absorption properties, and their ultraviolet absorption usually comes from the benzene ring on the side chain, which is not strong; the parent nucleus structure of cephalosporins contains "O=C-N-C=C" conjugate system, which has strong absorption near 260 nm and is highly characteristic.
Penicillin drugs and cephalosporins contain a carboxyl group in the structure, which belongs to organic acids, and its pKa is generally 25 to 28, showing strong acidity, while the parent ring has a protonizable tertiary amine site, so the molecule generally shows some characteristics of amphoteric compounds. β-lactam ring is the most unstable part of the drug structure, and when it acts with alkalis, acids, heavy metals (oxidants) and enzymes, it is prone to hydrolysis reactions and molecular rearrangement, resulting in drug failure. At present, there are more studies and reports on the degradation reaction of penicillin G, penicillin G can remain stable in the dry state, it is easy to metabolize in aqueous solution, penicillionic acid and other β-lactam drug residues can be generated in the case of acid, and penicillazic acid and other substances can be generated in the case of alkali, alcohol or enzymes. Among them, penicillium thiazole acid can be polymerized into penicillium thiazole acid polymer, which can be combined with peptides or proteins to form a rapid-onset allergen, which is the main cause of allergic reactions in the human body caused by penicillin G.
3 Mechanism of action
The mechanism of action of β-lactam drugs is mostly similar, by inhibiting the production of cell wall mucopeptide synthase, interfering with the synthesis of cell wall peptidoglycans, causing the cell wall of the body to break, and the bacteria to expand and lyse and die. β-lactams can inhibit the synthesis of bacterial cell walls by inhibiting peptidoglycantranspeptidase. Mucide is the main component of the bacterial cell wall, is some of the sugar-containing polypeptides with a reticular structure, is composed of N-acetyl glucosamine and N-acetylcylanemic acid alternating composition of linear glycan chain short peptide, these polymers need to be mucopeptide transpeptide enzyme catalyzed for transpeptide reaction, so that the linear polymer conversion into a joint structure, to complete the synthesis of the cell wall. The effect of β-lactams is to inhibit the activity of mucopeptide transpeptidase. It is similar in structure to the end structure of mucopeptide-D-propanyl D-alanine, and the conformation is also similar, making the enzyme recognize the error. Penicillin competitively covalently binds to the active center of the enzyme, constituting an irreversible inhibitory effect. Due to the lack of enzyme catalysis, short peptides cannot be converted into chain structures and cannot synthesize cell walls. Without cell walls, cells cannot be shaped and subjected to high osmotic pressure within cells, causing lysozyme and bacterial death. This action feature has great superiority, because human cells do not have cell walls, drugs do not work on human cells, have great selectivity, so β-lactam drugs are antibiotics with little toxicity.
Cell wall mucopeptide synthase is a penicillinbindingproteins, PBPs, in recent years researchers have confirmed that the special protein PBPs on the bacterial cell membrane are the target of β-lactam drugs, the number of PBPs on different bacterial cell membranes, the relative molecular mass, the sensitivity to drugs There are differences, in addition, there are reports that such drugs can also have a lethal effect on bacteria by triggering the activity of bacterial autolysin.
Penicillins and cephalosporins contain a wide range of drugs, a wide range of antibacterial spectrum, and are widely used in veterinary clinics for various animal infectious diseases caused by bacteria such as staphylococcus, streptococcus, pneumococcus, Escherichia coli, Salmonella, Haemophilus and other bacteria.
4 Pharmacokinetic studies
4.1 Absorption and distribution of drugs
β-lactam drugs are chemically unstable, easily destroyed by gastric acid and β-lactamase, except for penicillin V, amoxicillin and other acid-resistant drugs can be taken orally, most β-lactam drugs are administered through intramuscular injection, subcutaneous injection or intravenous injection. It is generally believed that the absorption site of most β-lactam drugs is in the stomach and intestines, but there are obvious differences in the absorption mechanism of different drugs in vivo, Bretschneider et al. believe that the difference in absorption mechanism may be related to the difference in lipophilicity and intestinal transporters of the drugs themselves, he studied the transmembrane transport process of 23 β-lactam drugs in the Caco-2 cell monolayer model, It was found that the oligopeptide transporter PepT1 (peptidetransporter1) can transport cefprohydroxylbenzyl, ceframipine, ceframidine, cefixime, ampicillin and other drugs well, but there is no affinity effect on cefpirine and other drugs. In addition, differences in pH in the intestine can have an impact on drug absorption, and it is generally believed that the best pH absorption environment for β-lactam drugs in the mammalian intestine is 61 to 68.
β-lactams are widely distributed in the body, with the highest concentrations in the liver, kidneys and lungs, followed by sebum and muscle, and also in cerebrospinal fluid and milk. Differences in tissue inflammation and mode of administration can lead to an abnormal distribution of drugs in vivo, for example, in the treatment of dairy mastitis in cows, the pH of milk in the lactum is increased, the plasma-milk barrier is broken, β-lactam drugs are prone to ionization into the lactate, and breast perfusion is the injection of therapeutic drugs directly into the milk pool of the infected area, these characteristics lead to a large amount of therapeutic drugs being absorbed by the mammary glands, and with the increase in lactation and absorption increased, there are reports that penicillin G in the treatment of mastitis through breast perfusion, The content of the drug in the milk within 24 hours of administration can reach 42.6% of the injected amount.
4.2 Transformation and excretion of drugs
β-lactams have shorter half-lives in animals and less differences between mammalian species. The elimination half-life of amoxicillin in dairy cows is 1.5h, which is 0.75 to 1.5h in dogs and cats; the elimination half-life of ampicillin in dogs and cats is 0.75 to 1.3h, and in pigs is 1.0h; the half-life of penicillin G intramuscular injection in cows, horses, calves, pigs, and rabbits is 1.02h, 2.6h, 1.63h, 2.56h and 0.52h, and intravenous administration is given in cattle, horses, camels, pigs, sheep, pigs, sheep, The half-lives in dogs and turkeys are 1.2h, 0.9h, 0.8h, 0.7h, 0.7h, 0.5h and 0.5h, respectively. Yang Dawei et al. studied the pharmacokine process of cefoquinoxid in pigs, the absorption was rapid after intramuscular injection administration, the peak of blood concentration was reached at 0.28~0.52h, and the elimination half-life was 1.79~2.77h, and the results were compared with the pharmacokine research results in dairy cows, and the elimination half-life of cefoquinoxid injection administration in dairy cows was 0.3h, and the difference between the pharmacokinetic excretion process in the two was not obvious.
β- lactam drugs into the blood circulation in the body is not easy to be destroyed, mainly in the prototype from the urine excretion, Liang Beibei and other reviews of 21 commonly used β - lactam drugs excretion pathways, beoxicillin, cefoxin and other drugs are mainly excreted through the kidneys in prototype, the elimination half-life is correspondingly prolonged when the kidney function is reduced, piperacillin, cefoperazone and other drugs in addition to excretion through the kidneys, a small part of the prototype through the hepatobiliary system excretion, and can be excreted into the intestine by bile after reabsorption into the blood, the formation of hepatointestinal circulation. In addition, different modes of administration and site of administration will have an impact on the excretion route of the drug, nafcillin in the treatment of dairy cow mastitis, oral administration is mainly excreted through the kidneys and bile, but breast perfusion administration of most of the dose with milk. The US FDA survey shows that the lack of attention to safe medication time is the main cause of veterinary drug residues in milk, and 61% of veterinary drug residues in milk are caused by drug treatment in dairy cows during lactation, and 31% are drug treatments from the dry milk stage of dairy cows. Different routes of administration can cause differences in the duration of drug residue in milk. When penicillin G is treated for mastitis in dairy cows, the residual of penicillin G can be detected in the milk of intramuscular injection of the drug in the milk within 120 hours of discontinuation, while in cows administered by udder perfusion, penicillin G is excreted through lactation for 144 hours. In addition, there are differences in the residual time of different drugs in the milk tissue of dairy cows. In the case of the same administration, cefoxin is excreted with milk for 52 hours, cefoquinme with milk for 84 hours, and o-chloropenicillin with milk for 96 hours.
5 Toxicological studies
β-lactams are generally low in toxicity themselves, but complexes with proteins can be very strong sensitizers, leading to severe allergic reactions ——— allergic reactions.
The main adverse reaction of penicillin drugs in the body is the high rate of allergic reactions, ranking first among all kinds of drugs. According to WHO statistics, the incidence of allergic reactions to penicillin drugs is 07% to 10%. Penicillin allergic reactions include rapid-onset allergic reactions and delayed allergic reactions, the former clinical manifestations of anaphylactic shock, urticaria, angioedema, etc., which are type I-mediated hypersensitivity reactions; the latter clinical manifestations are maculopapular rash, contact dermatitis, serum disease, etc., T lymphocytes may be involved in such reactions. Among them, anaphylactic shock is the most severe, which can be fatal in a short period of time.
Dewndycetal believes that the culprit of allergic reactions is pre-formed allergens such as penicillin-protein conjugates, that remain in the animal's body. According to modern immunological theory, as an allergen must have a specific antigenic determinant and polyvalent and other necessary conditions, the current clinical application of penicillin drugs relative molecular mass is less than 1000, so it has no immunogenicity itself, but by binding with proteins, polypeptides, polysaccharides and other macromolecular carriers to form a complete antigen or self-polymerization into a polyvalent hemiganent, becoming one of the main reasons for inducing allergic reactions. In addition, Brander's study found that penicillin can bind directly to the histocompatibility complex (MHC)-peptide complex molecule on the surface of T lymphocytes, stimulating T lymphocyte proliferation and inducing further responses without the need for the presentation C of antigen-presenting cells. Zhang Jianmin et al. investigated the use of drugs in 8860 hospitalization cases with allergy history, and found that the allergy rate of β-lactam drugs accounted for the largest proportion, of which penicillin drugs had the highest rate of allergic reactions, accounting for 37.8% of the overall proportion, and the incidence of allergic reactions of cephalosporins was 11.3%. In terms of individual drugs, penicillin G has the highest incidence of allergic reactions, accounting for 89.4% of the allergy rate of penicillin-like drugs.
6 Domestic and foreign limited requirements
In order to protect the health and safety of consumers, China, the European Union, the United States, etc. have formulated β - lactam drugs in various animal organizations MRL, Table 5-29 for China, the Eu,β -lactam drugs in animal organizations msl.
7 Detection methods
7.1 Sample pretreatment
The pretreatment of samples mainly includes extraction, purification, concentration (enrichment), derivatization and other processes, which occupies almost 70% of the workload of the whole process of detection and analysis. Compared with other drugs, β-lactam drugs are chemically unstable, susceptible to pretreatment factors such as reagents and temperature, and the concentration of drug residues in the sample is low, so the sample pretreatment technology is of great significance for the analysis and detection of β-lactam drugs.
7.1.1 Extraction of samples
7.1.1.1 Extract
The role of the extract is to extract the target from the sample, and the interference of the matrix needs to be taken into account under the premise of ensuring the extraction efficiency of the target substance. β-lactam drugs are mostly drug prototypes remaining in animal tissues, and the maternal structure contains carboxyl groups and a protonizable tertiary amine site, showing some characteristics of amphoteric compounds, which are moderately polar or polar compounds, easily soluble in polar organic solvents and acidic or alkaline aqueous solutions. Therefore, a suitable extract can be selected according to the nature of the drug, and the extraction solvents commonly used for the residual analysis of β-lactam drugs are acetonitrile, acidified acetonitrile, acidified methanol, acetonitrile - dichloromethane, phosphate buffer and sodium tungstate. The extraction solvent commonly used for β-lactam drug residues in biological samples can be divided into inorganic reagent extract and organic reagent extract.
7.1.1.1.1 Inorganic reagent extraction solution
β-lactam drugs are easily soluble in acidic or alkaline aqueous solutions, and the inorganic reagents selected in the residue analysis are mostly dilute acids and buffer solutions. Liu Yuan et al. selected phosphate buffer to extract amoxicillin from animal tissues such as chicken, pork, pig skin, and pig fat, and the recovery rate of the drug was 70.9% to 86.8% after HPLC testing. Yuko et al. selected sodium tungstate as an extraction solvent to extract 7 penicillin drugs from bovine kidneys and livers, and after extraction, they were detected by LC-MS/MS, and the recovery rate of drugs was higher than 66%. Huang Baifen et al. used 20% lead acetate solution to extract 12 kinds of penicillin drugs and 7 kinds of cephalosporin drugs in milk, and the samples were purified by HLB solid phase extraction column after protein precipitation, and detected with UPLC-MS/MS, and the quantitative limit of 19 drugs was up to 0.034 μg/L, and the recovery rate was 73.4% to 112.7%.
7.1.1.1.2 Organic reagent extract
β-lactam drug residue analysis is widely used methanol, acetonitrile, acetone, dichloromethane, trichloromethane and other organic solvents as extractants, these solvents have good solvation and permeability, fast extraction speed, both deproteination and degreasing advantages, is currently selected more extraction reagents. Methanol is easy to alcoholize β-lactam drugs, affecting the recovery of drugs, and the impurities in methanol extract are high and are currently used less. Acid organic solutions are conducive to improving the recovery rate of some β-lactam drugs, but because the β-lactam ring of most drugs is unstable and easy to degrade in acidic solutions, acidic organic solutions can only be used for the extraction of some stable drugs. In order to increase the selectivity of the extractant, if necessary, it can be extracted with a mixed solvent, such as trichloroacetic acid - methanol, dichloromethane - acetonitrile, acetonitrile - water and the like. Shen Yingbing et al. used acetonitrile solution containing 25% perchloric acid as an extractant to extract cefpiroxam from human plasma, and the recovery rate was 97.6% to 104.5% after HPCE detection. DeAlwis et al. selected methanol-trichloroacetic acid to extract penicillin, ampicillin and other types of antibiotics in the lees, and the drug recovery rate was 50% to 100%. Mieke et al. used acetonitrile and dichloromethane as extracts of β-lactam drugs in biological tissues such as ampicillin, amoxicillin, cefazoline, piperacillin, cefoquinoxime, and meropenem, which increased the types of extracted drugs, but the recovery rate was slightly worse (59.9% to 71.5%). Cmara extracts 9 kinds of β-lactam drugs such as ampicillin, penicillin G, penicillin V, cefotaxine, dicloxacillin, cloxacillin, benzoxicillin, cefazolin, cefoperazone, etc. in goat milk with aceton, and 5mL of goat milk plus 5mL acetonitrile saves the amount of extraction reagent, but the precipitation effect of the extractant is not good, and it is necessary to filter with a 0.45 μm filter membrane before purification with SPE column. In addition, EDTA, phosphate buffer, etc. can also be used for the extraction of β-lactam drugs, DeAlwis tried to use trichloroacetic acid -EDTA to extract penicillin G, ampicillin 2 drugs, the drug recovery rate of 50% to 100.0%. Santos et al. extracted six drugs such as amoxicillin in milk with a 20% solution of trichloroacetic acid, and the recovery rate was above 72%. Li Mu et al. used acetonitrile as an extract agent to extract a variety of cephalosporin drug residues from blood and urine, and the recovery rate was higher than 80% after HPLC detection.
In order to further improve the extraction efficiency of β-lactam drug residues in biological samples, many researchers have also made choices of different physical or chemical extraction techniques, commonly used extraction techniques such as homogenization, oscillation extraction, and ultrasonic auxiliary extraction. The first two extraction methods are conventional techniques, while ultrasonic assisted extraction mainly uses the mechanical effects, cavitation effects and thermal effects of ultrasonic waves to extract the components to be measured in biological samples by increasing the motion speed and penetration of media molecules. When operating, the appropriate amount of biological samples and extraction solvents are mixed, with the help of ultrasonic water bath action for a certain time, select the appropriate ultrasonic intensity and ultrasonic time to achieve the purpose of quantitative extraction, this process is a physical process, no chemical reaction occurs in the entire extraction process, does not affect the physiological activity of most of the active ingredients of the drug, the operation is simple, and multiple samples can be extracted at the same time. Zhang Yin et al. detected the residues of 9 kinds of β-lactam drugs in milk, selected acetonitrile and phosphate buffer solutions as extracts, assisted by 20kHz ultrasound for 5 min, and tested with HPLC after purification, and the recovery rate of all drugs reached more than 71.0%.
7.1.2 Purification of samples
Due to the complex nature of the sample matrix, some coex extractants with similar properties to the components to be measured will be extracted during the extraction process, and these impurities often interfere with spectral detection, increase baseline noise, reduce column efficiency, block chromatography pipelines, contaminate columns and detectors, etc., so it is essential to perform purification operations before detection such as chromatography. Purification treatment methods mainly include liquid-liquid extraction (LLE) and solid phase extraction (SPE) two categories.
7.1.2.1 LLE
LLE is a traditional purification method, the test conditions are not high, is the basic method of purifying the residue of β-lactam drugs. The method is to use the different solubility of different substances in different solutions, so that the analyte is transferred from one phase to another, thereby realizing the purification of the sample. Dichloromethane and trichloromethane are commonly used as liquid-liquid distribution solvents, and sometimes sodium chloride can be added to enhance the ionic strength of the solvent and further improve the transfer efficiency of β-lactam residues in the organic phase. In addition, after extraction from the sample matrix, it is usually degreased with non-polar solvents such as n-hexane and ether. Jank et al. established a method based on the combination of LLE and LC-MS/MS to rapidly detect the residue of β-lactam drugs in milk, extract drugs in milk with acetonitrile, all drugs recovery rate is above 60%, and the detection limit meets the EU standards.
Kukusamude et al. also established a cloud point extraction method for mixed micelles using cetyltrimethylammonium bromide (CTAB), TritonX-114, etc., and conducted a detailed investigation of different pHs of extraction solutions, concentrations of CTAB, extraction time, triton concentrations, salt concentrations, etc. (Figures 5-40).
LLE is a classic purification method that does not require high test conditions, but requires a large number of high-purity solvents, and the extraction time is long, and the recovery rate is not ideal. In order to improve the efficiency of extraction, auxiliary extraction techniques such as pressurized liquid extraction, supercritical fluid extraction, etc. can be used at the same time as using LLE.
Kukusamude et al. used tetrabutylammonium bromide as an ion pair reagent to extract the three drug residues of penicillin G, cloxacillin and benzacillin in beef and milk, and systematically analyzed the effects of different pH, different tetrabutylammonium bromide concentrations, different salt concentrations and other ions on the extraction effect of the reagent, and the upper solution after liquid-liquid extraction was directly detected by LC-PDA, and the detection limit of the method was up to 1~2ng/mL. Kantiani et al. measured the residues of 6 kinds of β-lactam drugs in animal feed by means of "extraction + purification" of "pressurised liquid extraction (PLE) + solid phase extraction", and systematically studied the factors affecting the extraction of pressurized solvents, such as solution composition, temperature, container size, number of extraction cycles, and solution volume; the recovery rate of the method was 71%-115%, and the RSD was less than 13%. The method was used to test samples taken from 14 markets.
7.1.2.2 SPE
SPE is a new type of purification technology developed on the basis of LLE, is the mainstream purification technology of the current sample pretreatment, the principle is to use a solid adsorbent to adsorb the target compound in the liquid sample, so that it is separated from the sample matrix and interference, and then elution or pyrolysis adsorption with the solution to achieve the purpose of separating and enriching the target compound, which is currently a widely used sample purification method. SPE can purify small volume samples, small solvent dosage, high selectivity, according to different analyte selection of different fillers, according to the retention mechanism can be divided into forward stationary phase, reverse stationary phase and ion exchange fixation equal. It mainly includes C18 columns, hybrid ion columns (OasisMAX, CleanertPAX), strong cation columns (ProElutSAX), hydrophilic ester columns (OasisHLB) and so on.
β-lactam drugs contain carboxyl groups, amino groups, usually have polarity, so the class of drugs can choose reversed-phase extraction columns, such as C18 columns and HLB columns, etc.; β-lactam drugs are organic acids, according to the chemical properties of the target substance acidity, some drugs can choose mixed anion column. The selection of SPE columns requires test screening based on the type and nature of the drug being detected. Macarov et al. detected the residues of 8 kinds of β-lactam drugs in animal muscle tissue, compared the purification effects of HLB columns, C18 columns, MAX columns, enV+ columns, and the test results showed that the purification effect of ENV+ columns was the best, the recovery rate of MAX columns was the lowest, and the retention of C18 columns on amoxicillin, ampicillin and penicillin G was the highest, but the recovery rate of o-cloxacillin was significantly lower than that of other extraction columns, and the efficiency of HLB columns was significantly better than that of MAX columns. However, it is still lower than the other two extraction columns; in addition, the experiment studied the effect of pH on the retention capacity of four extraction columns, and found that when the pH is 5 to 9, the SPE column is ideal for β-lactam drugs. Fernandez-Torres et al. compared the purification effect of C18 column and Plexa solid phase extraction column on four types of drugs, including β-lactam drugs, and this method compared the purification effects of different pH, different washing and elution solutions in detail, and the results showed that the Plexa solid phase extraction column was better than that of C18 column. Guo Dehua et al. established SPE of 76 veterinary drug residues in animal-derived foods, the samples were extracted from acetonitrile and citric acid buffer containing magnesium ions, the organic phase was removed and the buffer was resolved, the polymer and cation exchange SPE column were purified in tandem, methanol and methanol-ammonia (95:5) were elutioned step by step, measured with LC-MS/MS, and the recovery rate was 59.4%~115.3% and RSD was 2.6%~27.3%. This method was successfully used to screen samples in the market. Zhang Qi et al. compared the purification effects of HLB column, C18 column and MAX column with the standard mixture of amoxicillin, cefoxin, ampicillin and penicillin V, and the results showed that the HLB column had the best retention ability for the measured drugs, and the recovery rate of the four drugs was higher than 70%. In the multi-residue analysis, there are differences in the properties of the drug, in addition to the selection of SPE columns with a suitable retention mechanism, in order to maximize the retention and purification capacity of the SPE columns, it is also necessary to conduct experimental screening of the purification conditions of the SPE columns.
7.1.2.3 Solid phase dispersion of matrix (MSPD)
MSPD is a purification technique similar to SPE. Barker et al. first proposed this technology in 1989, so that the sample and the reverse bonding silica gel are uniformly combined, ground, column loaded, and then the appropriate eluent is used to wash the column to elute the target compound. This method has high purification efficiency, less time consumption and saves solvents, but it is not easy to automate. Wang Lian et al. established an MSPD purification method for 20 kinds of veterinary drug residues in livestock and poultry meat and milk, mixed the biological samples with C18 fillers evenly, filled with SPE empty tube compaction, placed on the SPE device, elutioned with methanol decompression, eluate dried with 40 °C nitrogen, dissolved by acetonitrile-ammonium acetate (2:8, V/V), filter membrane injection, and the detection limit of the drug was 0.05~3.05g/kg, and the recovery rate reached more than 70%.
7.1.2.4 QuEChERS methods
QuEChERS method is a sample pretreatment technology for rapid detection of agricultural products that has been newly developed internationally in recent years. The method has the advantages of high recovery rate, accurate results, and large sample processing volume, thereby reducing the consumption of reagents and the contact between the tester and the toxic solvent, and can effectively combine the extraction, distribution and purification, further reduce the extraction steps, and improve the extraction efficiency. QuEChERS method is usually mainly reflected in dispersed solid phase extraction, Pérez-Burgos et al. established a QuEChERS method for detecting the residues of 7 cephalosporins in beef, beef samples were extracted by 15mL aqueous methanol solution (8:2, V/V) after shaking, centrifugation, 10mL supernatant was added to 150mgC18 and 900mg anhydrous magnesium sulfate, oscillated for 5min, centrifuged to take 5mL supernatant, blown dry with nitrogen, reconverted after LC-MS/MS detection The quantitative limit of the method is 4~50μg/kg, and the RSD is less than 15%. Karageorgou et al. established a QuEChERS method for detecting 12 lactam residues in milk using the ultrasound-assisted-MSPD method.
7.1.2.5 Molecular imprinting techniques
Molecular imprinting technology uses a molecularly imprinted polymer (MIP) as a stationary phase with a specific ability to recognize the molecule of interest. Wang Hui et al. synthesized penicillin-specific adsorption capacity of the imprinted polymer PenG-MIP, which is used to detect penicillin in milk, and realized the rapid quantitative detection of molecular imprinting technology for penicillin residue in milk, with a detection limit of 5 μg/L, high accuracy of results, simple operation, and PenG-MIP particles can be used repeatedly, which greatly reduces the detection cost.
7.1.2.6 Immunoaffence chromatography (IAC)
IAC is a chromatographic technique based on the specificity and reversible immune binding response of antigen antibodies. The use of an inert matrix stationary phase with antibodies in the purification process has good specificity and high selectivity, which is one of the effective purification methods for biological samples. Zhi et al. established an automatic flow current immunoassay system, quantitatively determined the cephaloxil residue in milk, the detection limit was 1 μg/L, and the quantitative limit was 3 μg/L, which was lower than the MRL of cefotaxin in milk stipulated by the EU standard. This method has a strong selectivity for cefotaxin, and other cephalosporins and penicillin drugs have little interference with its detection, which is suitable for the qualitative and quantitative analysis of cephalexin residues in milk. IAC has good specificity and high selectivity, and has broad prospects in drug analysis, but due to the limitations of immune methods, they are not widely used at present.
7.2 Detection Methods
There are many literature reports on the residue detection technology of β-lactam drugs at home and abroad. At present, the methods used for the detection of β-lactam drug residues mainly include high performance liquid chromatography (HPLC), capillary electrophoresis (CE), liquid chromatography-mass spectrometry (LC-MS), gas chromatography (GC), immunoassay, microbial method, etc.
7.2.1 GC
GC is a chromatographic process using gas as a mobile phase, suitable for gases, low boiling point, easy to vaporize substances, while β-lactam drugs themselves are not easy to vaporize, and derivatization operations are required in the pretreatment process. Meetschen et al. used GC to determine the residues of 7 β-lactam drugs in animal tissues, and used diazomethane for derivatization to form volatile penicillin methyl ester compounds with a recovery rate of 46.0% to 73.0%. GC requires the sample to be vaporized, constrained by the volatility of the sample, most of the β- lactam drugs have poor volatility and thermal instability, although some pretreatment methods can be used, but increase the difficulty of operation, the process is complex, and changed the original form of the sample, not easy to restore, so in the - lactam drug residue analysis, GC has been basically replaced by LC.
7.2.2 HPLC
HPLC is one of the most widely used technologies in the analysis of β-lactam residues, and commonly used detectors include ultraviolet detectors (UVD), fluorescence detectors (FLD), and diode array detectors (DAD).
Since most penicillins do not have a specific UV emission group, their absorption wavelength is generally 200 to 235 nm, and the selectivity in this wavelength range is poor. Due to the low residual amount of penicillins in biological samples, the background interference is often more serious, so it is generally used before or after column derivatization technology, and the derivatization reaction can effectively improve the sensitivity of UVD. Such as the use of imidazole or 1,2,3-triazole catalytic action to form penicillin to form a mercury derivative of penicillinic acid, its maximum wavelength is 325nm, there are few symbiotic components interfering with the determination of the detection wavelength, and the method has a specificity of the compound of the β- lactam ring, using this principle, Bioson, Verdon and others have established a detection method for detecting penicillin drug residues. Cai Yu'e et al. used the HPLC-UVD method to detect the residue of five cephalosporins in biological samples, including cefadroxam, cefaxime, ceframidine, cefotiprobin, and cefotamin, and took phosphoric acid buffer -acetonitrile as the mobile phase, and detected at 270 nm, and the detection limit was less than 9.7 μg/kg, and the recovery rate was 96.5% to 105.0%. DAD belongs to a kind of UVD, the principle is the same as UV, can be scanned at full wavelength, can be used for the detection of substances of different wavelengths in the mixture. Cmara et al. established HPLC for the detection of amoxicillin, penicillin G, penicillin V, amoxicillin, dicloxacillin, cloxacillin, benzoxicillin, cefazolin, cefoperazolelin,β cefoperazone residues in goat milk using DAD, with a detection limit of 3.4 to 8.6 μg/kg and a recovery rate of 79.0% to 96.0%. In the analysis of drug multi-residue, in order to achieve the desired detection effect, HPLC can connect different detectors, Benito-Pena, etc. established penicillin G, penicillin V, nafcillin, benzoxicillin, clozacillin, dicloxicillin, amoxicillin, ampicillin 8 kinds of HPLC-UVD/DAD detection method for β-lactam drug residues, selected 220nm as the detection wavelength, the detection limit was 8.0~ 24.0 μg / kg, and the average recovery rate was 82.0% ～97.0%。
FLD has stronger sensitivity and specificity than UVD, but requires drugs to have fluorescent characteristics, and has some advantages over UVD for accurate detection of single or small drugs. Since β-lactams do not have a fluorescent emission group, derivatization must be used to produce fluorescent chromophores. Luo Daoxu et al. established the HPLC-FLD detection method for ampicillin residues in milk, and used formaldehyde method to derivatize the drug, with excitation wavelength and emission wavelength of 346nm and 420nm, the detection limit was 1.0μg/kg, and the recovery rate was 70.0%-110.0%. Terada et al. used the principle that ampicillin can produce fluorescent derivatives in formaldehyde and trichloroacetic acid solutions, and used FLD to determine the residual amount of ampicillin in milk.
7.2.3 LC-MS
LC-MS is a modern analytical technique that uses LC as a separation method and MS as a detector. LC-MS realizes the combination of LC and MS advantages, combining the high separation ability of LC on complex samples with the advantages of MS high selectivity and high sensitivity, especially tandem mass spectrometry (MS/MS) can provide more detailed relative molecular mass and structure information, making this combined technology widely used in multiple fields such as pharmaceutical analysis, food analysis and environmental analysis.
Due to the research and development of MS/MS, LC-MS/MS has made great progress in the field of multi-residue detection of β-lactam drugs. Ohmori et al. selected LC-MS/MS to detect the residues of 8 β-lactam drugs in plasma, and their detection limit was 0.01 to 0.5 μg/mL, except for the recovery rates of dolepenem and meropenem of 49.1% and 62.3%, the recovery rate of other drugs exceeded 80.2%. Macarov et al. established an LC-MS/MS detection method for 8 penicillin drugs in cattle, pigs and chicken tissues, including amoxicillin, ampicillin, dicloxacillin, penicillin G, penicillin V, benzoxicillin, and nafcillin, using internal standard quorum, except for amoxicillin recovery rate of only 50.0%, the recovery rate of other drugs exceeds 70.0%, and all drug detection limits are lower than or equal to the MRL stipulated by the European Union. Becker et al. used LC-MS/MS method to determine the residue of 15 β-lactam drugs in milk and other tissues, amoxicillin, ampicillin, cefotaxin, cefotavirine, cefazoline, etc. using ESI+ detection, cefoperazone, penicillin G, penicillin V, benzoxicillin, o-cloxacillin, dicloxacillin and naphthicillin using ESI-detection, using matrix addition to correct matrix effects, in addition to amoxicillin recovery rate of only 57.0%, Recoveries of the other analytes of the target were greater than 81.0%, and all drug testing limits met EU-mandated standards. Lina et al. established the LC-MS/MS method for the residue of 11 β-lactam drugs in the feed, using 0.1% aqueous carboxylic acid solution -0.1% methanol for formate as the mobile phase, separating 18 drugs within 30 min, with an average recovery rate of 71.0% to 115.0% and a detection limit of 0.12 to 3.94 μg/kg.
Carlier et al. used ultra-high performance liquid chromatography (UPLC) instead of HPLC to establish a detection method for the residues of 7 β-lactam drugs in plasma, and the method selected BEHC18 (1.7 μm, 100 mm×2.1 mm) columns to separate the detectors, containing 0.1% formic acid water and acetonitrile as mobile phases, all drugs were detected within 5.5 minutes, greatly shortening the detection time, and the recovery rate of all drugs was 86.8% to 101.5%. Liu et al. established a method of using UPLC-MS/MS to detect penicillin G, amoxicillin and a variety of metabolites (benzylpenicillazide, benzylpenicillium decarboxylthiazolate, benzylpenicillindiol, amoxicillin thiazole acid, amoxicillindione piperazine) in milk, all drugs were detected using ESI+, 8 compounds were successfully isolated within 8min, the quantitative limit of the drug was 2.5 to 5.0 μg/kg, the recovery rate was 85.0% to 108.0%, and the RSD was less than 13%. Tang et al. applied UPLC-MS/MS to determine 23 kinds of veterinary drug residues in milk at the same time, including 7 kinds of β-lactam drugs, 12 kinds of macrolide drugs and 4 other veterinary drugs, all of which had detection limits of less than 5 μg/mL, and the standard recovery rates were 51.5%-100.6%, 51.8%-139.0%, 82.4%-102.5%, and RSD was less than 15.0%, respectively. The use of UPLC has greatly improved the separation ability of chromatography, accelerated the speed of drug analysis, combined with MS/ MS detector can provide detailed molecular quality and structure information of the analyte, making UPLC-MS/MS has a unique advantage in the qualitative and quantitative detection of chemicals, and has developed rapidly in recent years in the field of drug analysis and food detection.
Kantiani et al. used liquid chromatography-linear ionhydrazine-tandem mass spectrometry to determine the drug residues of cefazolin, cefoperazone, cloxacillin, dicloxacillin, penicillin G, penicillin V and other drugs in animal feed. Dubala et al. established an LC-MS/MS method for the simultaneous detection of cefpoxime and clavulanate residues in human plasma, and took methanol-acetonitrile-2mmol/L ammonium acetate solution (25:25:50, V/V, pH3.5) as the mobile phase, and used negative ion detection to select m/z408 [M-(CH3)--2H)]-and m/z198[M-H]-as the parent ion, and sim detection, the chemical structure and full scan of the two drugs are shown in Figure 5-41. Kantiani et al. also investigated the linearity, precision, accuracy, ion inhibition, matrix effect, sensitivity, and stability of the method.
7.2.4 Microbiological Methods
Microbial method, also known as microbial inhibition test, is a detection method that qualitatively or quantitatively determines the amount of drug residue in a sample based on the inhibitory effect of drugs on microbial physiological functions and metabolism. Commonly used microbiological methods are paperdiskmethod(PD), triphenyltetrazole chloride (TTC) method, cup and dish method (cylinderplatemethod, CP), turbidity method, Delvotest-SP detection method, microbial obstruction method and so on.
PD placed positive control pieces of paper and sheets of paper full of milk samples tested on the agar flat dish of inoculated strains, cultured under suitable conditions, and observed the bacteriostatic circle to determine the results. TTC method is a qualitative method for detecting milk drug residues, which was first proposed by Neel and Calbert et al. in 1955, and streptococcus thermophilus was added to the milk as a strain for culture, and the results were judged according to the color change of the TTC indicator. Wang Daju et al. used Garcinia cambogia as an indicator, and used CP to detect ampicillin residues in pig and chicken tissues, and the minimum detection limit could reach 0.00025 μg/mL, which was lower than 0.0009 μg/mL determined by Vilim with Bacillus thermophilus, and the working range of the standard curve was 0.00125~0.02μg/mL, the coefficient of variation was (2.91±0.2)%, and the recovery rate of ampicillin at different concentrations was 83%-107%. Li Yanhua et al. used the national standard TTC method and the AOAC paper method to compare the detection results of β-lactam drug residues in milk, and the AOAC paper method can successfully detect the MRL concentrations of penicillin G, ampicillin, benzicillin hydroxyamicillin, benzoxicillin, cefaciline, cefotamin, cefpirine β and cefpirine residues, and the TTC method cannot successfully detect the above drugs with MRL concentrations, and can only be used for qualitative detection. Due to its susceptibility to the influence of other antibiotics in the tissue, the microbial method has low specificity and low sensitivity, but it is easy to operate, the sample dosage is small, the pretreatment is simple, and it is suitable for the screening of a large number of samples, and is often used by ranches, dairy companies, etc. for screening and testing antibiotics in large batches of milk samples.
7.2.5 EC
CE, or high-performance capillary electrophoresis (HPCE), is a kind of liquid phase separation technology that uses high-voltage electric field as the driving force and capillary as the separation channel to achieve separation according to the difference in electrophoresis intensity and distribution behavior between sample components. Compared with HPLC, HPCE has the characteristics of high column efficiency, good selectivity, small injection volume and wide sample analysis range, and is one of the more active technologies in analytical chemistry in recent years. Due to the organic acid base groups of β-lactams, the method of electrophoresis in capillary regions is also common. Bailón-Pérez et al. used HPCE-UV/DAD method to detect clavulanic acid, amoxicillin, ampicillin, penicillin G, penicillin V, nafcillin, benzoxicillin, chlooxacillin, dicloxacillin, piperacillin, dicloxacillin, piperacillin 10 kinds of β-lactam drug residues in environmental sewage and feed, and the detection limits in water samples and feed were 0.04 to 0.06 μg/L and 0.80 to 1.4 μg/L, with recovery rates of 82.9%-98.2% and RSD less than 9.0%, respectively. The chromatogram of the 10 drugs is shown in Figures 5-42. Sérgio et al. established ce for the simultaneous detection of ampicillin, amoxicillin, penicillin, cloxacillin, tetracycline, chloramphenicol in milk, taking KH2PO4 and Na2B4O7 solutions at pH8.0 as the mobile phase, and UVD detection at 210 nm at a voltage of 18 kV, and adding recovery tests at 2.5 μg/mL2 and 5.0 μg/mL addition concentrations, with the recovery rate of the method being more than 72% and the RSD less than 5%. Electroosmosis is unstable, resulting in poor reproducibility of HPCE results, low sample carrying capacity, lower sensitivity than HPLC, and limited in applications of residue analysis. Bailon et al. used HPCE-UV/DAD method to detect 7 kinds of β-lactam drug residues in water, and selected offline SPE method to purify and concentrate samples, with a detection limit of 0.08~0.8μg/L, a recovery rate of 94%-99%, and an RSD of less than 10%.
7.2.6 Immunoassays
The advantage of this method is that the operation is simple and the detection is fast, some biological samples such as urine can be directly determined, and it is mainly used for screening and analysis in the detection of β-lactam residues. Immunoassays mainly include ELISA, radioimmunoassay (RIA) and so on. ELISA is currently the most widely used immunoassay method, and has a variety of commercial elisa microplate kits, can be used for a variety of veterinary drug residue detection, such as the total antibiotic (milk) kit produced by the United States Burr (Bioo) company can be used to detect penicillin G, ampicillin, amoxicillin, benzoxicillin and other β-lactam drug residues in milk, the detection limit is 4.0 μg / kg; the penicillin detection kit produced by Beijing Wanger Biotechnology Co., Ltd. can be used to detect milk, honey, Penicillin G in tissues, detection limit is 0.2 μg/kg. Lamar et al. established an ELISA for the detection of β-lactam residues in milk, beef, pork, honey, and eggs using penicillin-binding proteins (PBPs) prepared from recombinant proteins.
Penicillin molecules have multiple antigenic determinants, which can produce multiple types of antibodies in animal immunity, so the synthesis of artificial antigens can be guided according to the structure and analysis purposes of the analyte. In order to obtain antibodies that mainly recognize a certain penicillin drug, the antigenic determinant needs to be dominated by the side chain antigenic determinant, so the side chain structure should be highlighted during antigen synthesis, Edwards et al. successfully crosslinked the protein carrier to the carboxyl site of the penicillin molecule thiazole ring to obtain a specific antibody. Also in order to obtain parent nucleus-specific antibodies that can recognize the entire penicillin class, the parent nucleus structure (6-APA) is highlighted when the antigen is synthesized. Shirazid et al. adopted different immune antigen synthesis methods such as glutaraldehyde method and penicillinization reaction, used mice for immune response, obtained a variety of penicillin cross-reactive monoclonal antibodies, and indirect competition ELISA can simultaneously detect the residues of β-lactam drugs such as ampicillin, penicillin G, oxylanillin, benzoxicillin, dichloropenicillin in milk and livestock products, and the detection limit is 2.5~5ng/mL, and the detection sensitivity is within the EU MRL. DeLewu et al. directly prepared anti-penicillin yolk antibodies by using 6-APA; Diertihc et al. used glutaraldehyde to connect ampicillin side chain amino groups to proteins to prepare nuclear-specific antibodies; Cliuqet et al. used penicillin physiological reactions to synthesize penicillin thiazole protein to prepare nuclear-specific antibodies. These acquired parent-nucleus-specific antibodies have great cross-reactivity with the whole penicillin drug and can be used for penicillin residue analysis.
Zhang Jia et al. established a rapid screening and analysis method for radioactive receptor residues of β-lactam drugs in animal tissues, which was successfully used to detect penicillin G, amoxicillin, ampicillin, dicloxacillin, cloxacillin, cefotafurofuran and other drug residues in pork, beef, chicken, fish and shrimp meat, and the determination of a batch of samples could be completed within 90 minutes, which greatly accelerated the measurement speed and saved the measurement cost. Wei Dong et al. used indirect competition ELISA to detect amoxicillin residues in milk, using synthetic AMX-OVA as the detection antigen and AMX standard as the competitive hemiantant, the minimum detection limit of the method was 3.926ng/mL, and the average recovery rate of sample addition in the range of 0.5 to 2000ng/mL was 91.45%.