Study Of Novel Large Volume Injection Liquid Chromatography Methods For Trace Analysis Of Hydrophilic Toxic Compounds

The effective control and prevention of the occurrence of chemical poisoning is of great significance for the maintenance of public health safety.The detection of chemical poisons play a key role,which provides important support for clinical diagnosis,judicial identification and toxicological research.Toxic compounds detection involves complex samples,such as biomaterials(blood and urine),complex matrix food samples and so on.Usually,the content of the toxic compounds is very low,so toxic compounds detection relies on rapid,sensitive and accurate analysis techniques.Hydrophilic toxic compounds usually lack chromophores,have poor volatility,and are difficult to separate and concentrate from water-rich samples,making detection extremely difficult.Nowadays,the most effective analytical technique is high performance liquid chromatography,especially high performance liquid chromatography-mass spectrometry.However,hydrophilic toxic compounds remain weak in the traditional reversed-phase liquid chromatography and the liquid chromatography-mass spectrometry.However,hydrophilic toxic compounds remain weak in the traditional reversed-phase liquid chromatography and the separation effect is unsatisfactory.Therefore,it is urgent to develop efficient sample purification and enrichment techniques to solve the problem of trace analysis of hydrophilic toxic compounds.At present,solid phase extraction is the most common effective purification and concentration method,but it usually requires off-line manual operation.The process is complicated and time-consuming,and there is human error.If it is on-line or automated,an expensive device is required.Large volume Injection method by liquid chromatography can achieve trace enrichment in the sample injection process.The method greatly increases sensitivity,and is easy to operate.It is a technology with good application potential.However,due to the weak retention of hydrophilic toxic compounds and the difficulty of matrix purification,the application of the technology in hydrophilic toxicology testing is greatly limited.In this dissertation,we intend to solve the problem of efficient enrichment,separation,and high sensitivity analysis of hydrophilic toxic compounds in complex water-rich samples through large volume injection method by liquid chromatography.The following two analysis strategies are proposed:First,a large-volume sample injection and enrichment device for automatic FSS-hydrophilic interaction chromatography was developed,which greatly increased the sensitivity and solved the trace analysis of hydrophilic toxic compounds in biological samples.Secondly,a new method of reversed cloud point extraction-large volume injection-ion pair chromatography is developed and systematically studied.A novel "inversed" cloud point extraction method is used to remove the matrix,and a simple and effective preconcentration method for large volume sample column is combined to solve the problem of trace analysis of hydrophilic toxic compounds in milk products.1,Fractionized sampling and stacking(FSS)large volume injection method for hydrophilic interaction chromatographyFSS large volume injection method for hydrophilic interaction chromatography is developed and systematically studied.By improving the liquid chromatography sample introduction device,automatic versatile milliliter-level large-volume injection is realized.The device is low in cost,simple in structure,and has no gradient delay.Then,the method is optimized.When the ratio of mixing is 1/10,the number of sections is 5 or 10,the volume of the mixer is 700 μL,and the initial mobile phase is 98%acetonitrile,the peak shape of the chromatogram is sharp and symmetry.The method was used to analyze the hydrophilic toxic compound(tetrodotoxin)in a complex matrix(plasma and urine)and was validated by methodology.The linear range,LOD,precision,accuracy,recovery,and matrix effect of the method were examined.The calibration curve concentration range was 1 ng/mL to 400 ng/mL.The intraday and interday precisions of plasma were 4.13%to 8.85%and 5.32%to 7.32%,respectively.The intraday and interday precisions of urine were 2.38%to 8.54%and 8.02%to 9.29%,respectively.The accuracy were 93.0%to 106.8%(plasma),91.0%to 106.4%(ueine),respectively.The recovery were 62.1%to 83.6%(plasma),79.2%to 92.3%(urine),respectively.The matrix effect were 81.7%to 123.4%(plasma)、100.9%to 131.7%(urine),respectively.The established analysis method solves the analysis requirements of biological samples.The hydrophilic toxic compounds(paraquat,diquat,melamine,and ethyl glucuronide)are still suitable for this method.When the hydrophilic toxins compound is in rich-water complex samples,the hydrophilic toxic compound can be separated by this method.2.Inversed cloud point extraction-large volume injection-ion chromatographyEfficient separation of trace hydrophilic compounds from complex,water-rich samples is a daunting challenge.In order to solve the bottleneck of the analysis,based on the extraction principle contrary to the traditional cloud point extraction(CPE)principle,a novel inverted cloud point extraction(ICPE)strategy was proposed.Then,based on the retention mechanism of the dynamic ion exchange model,a large volume ion-pair chromatography(LVI-IPC)method was developed and systematically studied.The ICPE-LVI-IPC method combines the advantages of extraction,complex sample purification,and column enrichment in fast and easy phase separation and sample introduction.This method has been successfully applied to the determination of melamine in milk samples,including milk,yogurt and milk powder.Sample preparation without the use of potentially hazardous organic solvents and adsorbents can be completed in 11 minutes,requiring only 1.2 mL of aqueous solution per sample.LVI as an efficient on-column enrichment strategy has greatly improved sensitivity with a detection limit of 0.0028 mg/kg.which is far below the limits set by the Codex Alimentarius Commission.The recoveries of the spiked samples at the four concentrations ranged from 92.6%to 95.8%,and intraday and interday precisions were 1.9%to 3.9%and 1.6%to 5.2%,respectively.Comparison with various effective methods shows the superiority of the ICPE-LVI-IPC method in terms of sensitivity,rapidity,sample and solvent consumption,practicality and yield.