Studies on the Pretreatment Methods for Testing Drug Residuals in Complex Samples and Their Applications
|Keywords||Substrate removal aquatic two phase system QuEChERS molecular imprintingtechnology chemiluminescence equilibrium dialysis|
With the improvement of people’s living standards, food safety standards received widespreadconcern throughout the world. Special attention was also focused on the determination of drugresidues in food. The most commonly reported methods for the analysis of chemical residues incomplex samples are based on a pre-treatment methods step and a determination step by usinginstruments.With the technology progress, the sensitivity and selectivity of the instruments can meetthe requirements of the general determination. However, the key problem for accurate and effectivedetection of chemical residue in animal food samples is sample preparation. This paper aims atsummaring the existing preparation methods, studying the drugs-protein interaction by fluorescencequenching method, expounding the existing state of drugs in protein matrices samples, furtherstudying the influences of protein denaturation to the pesticide extraction, providing a theoreticalbasis for the extraction of pyrethroid pesticides in protein matrix samples. A novel method for theanalysis of6pyrethroids in fish samples, based on the use of an acetonitrile–salt–H2O aquatic twophase approach and a modified QuEChERS （Quick, Easy, Cheap, Effective, Rugged and Safe）approach followed by gas chromatography （GC） method, has been developed. A novel method wasdeveloped using molecular imprinting technology （MIT） coupled with fow-injectionchemiluminescence （FI-CL） for highly sensitive detection of phenformin hydrochloride （PH）. Theinteraction of phenformin with protein has been studied using a combined technique of equilibriumdialysis with flow-injection chemiluminescence detection.The paper can be divided into five sections:Part one: In this paper, the pretreatment methods of complex samples were reviewed, and thebackground and content and significance of this issue were introduced.Part two：Based on the acetonitrile–salt–H2O aquatic two phase system, an effective samplepretreatment method was developed for the analysis of pyrethroids pesticides residues in fish by gaschromatography （GC）. The fluorescence quenching method and GC were applied to study the formof pyrethroids pesticides in protein matrix of aquatic products. The infuence factors on formation ofacetonitrile–salt–H2O system were discussed in depth. Meanwhile, the influence of the protein onextraction of pyrethroids pesticides from fish was detailedly investigated. The results showed that pyrethroids strongly bound with BSA by the hydrophobic interaction. In the φ（acetonitrile）80%, aslow but full protein denaturation takes place, which causes the unfolding of protein and the releaseof pyrethroids. Then K2HPO4was added to above system to form aqueous two phase. Following thedrug residues was extracted into upper phase with high extraction efficiencies. Furthermore, thepolarity of φ （acetonitrile）80%is greater than φ （acetonitrile）100%, the fat-soluble impurities areextracted less in φ （acetonitrile）80%aqueous solution than that of in φ （acetonitrile）100%asQuEChERS procedure or in hydrophobic solvents as liquid-liquid extraction procedure. Thereforethe purification steps were greatly simplified. The anhydrous magnesium sulfate （MgSO4） andprimary secondary amine （PSA） were added in upper phase to eliminate the moisture and otherimpurities. Then, the pyrethroids pesticides in extract （upper phase） was direct analysis by GC. Thismethod has been applied to the determination of pyrethroids pesticides residues in fish. The detectionlimits were in the range of814ng·mL-1, and the spiked recovery of fish were81.196.4%.Part three：Anovel method for the analysis of6pyrethroids in fish samples, based on the use ofa modified QuEChERS （Quick, Easy, Cheap, Effective, Rugged and Safe） approach followed by gaschromatography （GC） method, is reported. In previous reports, it is surprising found that onlyacetonitrile was used as the original QuEChERS extraction solvent. In the present work, theQuEChERS method was modified by replacing the traditional acetonitrile with isopropanol. Itshowed that the use of isopropanol improved the extraction efficiency of the QuEChERS. For thepyrethroids in the protein-matrix samples, the overall recoveries of75.889.4%for the modifiedQuEChERS method are better than those of68.984.8%for the original QuEChERS method.Fluorescence quenching spectra of BSA with pyrethroids showed that the strong binding interactionbetween pyrethroids pesticides and protein, which decreased the extraction efficiency of pyrethroidsfrom fish samples. While, in the80%（volume fraction） isopropanol aqueous solution, a slow proteindenaturation might take place, which would cause the unfolding of protein and the release ofpyrethroids. This resulted in the high extraction efficiency of pyrethroids from protein matrix samples.The method was used satisfactorily for the determination of6pyrethroid pesticides in spiked fishsamples. This paper proposes a new strategy by slowing down the protein denaturation and releasingbound pesticides to enhance the extraction efficiency of pyrethroids in fish samples.Part four：A novel method was developed using molecular imprinting technology （MIT） coupled with flow-injection chemiluminescence （FI–CL） for highly sensitive detection ofphenformin hydrochloride （PH）. The phenformin imprinted polymer was synthesized withmethacrylic acid （MAA） as a functional monomer and ethylene glycol dimethacrylate （EGDMA） asa cross-linker. Newly synthesized molecularly imprinted polymers （MIPs） particles were packed intoa column as a selective recognition element for determination of PH. A chemiluminescence （CL）method for the determination of PH was developed based on the CL reaction of PH withN-bromosuccinimide （NBS） sensitized by eosin Y （EY） in basic media. The optimization ofdetection conditions was investigated. The CL intensity responded linearly to the concentration ofPH in the range0.092.0μg·mL-1, with a correlation coefficient of0.9920. The detection limit was0.031μg·mL-1. The relative standard deviation for the determination of1.0μg·mL-1PH solution was1.0%（n=11）. The method was applied to the determination of PH in urine samples with satisfactoryresults.Part five：The interaction between phenformin hydrochloride and bovine serum albumin （BSA）was investigated by the methods of chemiluminescence combined with equilibrium dialysistechnique. A novel N-bromosuccinimide （NBS）-eosin Y（EY）chemiluminescence （CL） methodwas established for the determination of phenformin. The mechanism of this chemiluminescencesystem was proposed. Optimization studies were performed to determine the phenformin. Under theoptimal conditions, the CL intensity was linear for a phenformin concentration over the range of4.6×10-8to5.0×10-5g·mL-1. The detection limit was1.5×10-8g·mL-1. The data obtained by thepresent equilibrium dialysis-CL system were analyzed using the Klotz plot and the Scatchardanalysis. The results showed that the Klotz plot and the Scatchard plot are linear with goodcorrelation coefficient, indicating that the phenformin has only one type of binding site on BSA. Thebinding parameters were the number of the binding sites n （1.02） and the estimated associationconstant K (2.66×104L·mol-1). The chemiluminescence system combined with equilibrium dialysisdeveloped in this work demonstrated its use for determination of interaction between drug andprotein by using relatively simple instrument.