Preparation Of Novel Nanomaterials For Solid Phase Extraction And Its Application In Pretreatment Of Sedative-hypnotics

Benzodiazepines(BZD)have been widely used clinically as anxiolytics,hypnotics,sedatives,anticonvulsants,and antiepileptics.Zopiclone and zolpidem(Z-hypnotics)are structurally different from BZD,but act via the same?-aminobutyric acid receptor.In addition to the therapeutic effects,BZD/Z-drugs can cause synergistic effects when consumed together with other sedatives,neuroleptics,antidepressants,morphine-like substances,and especially alcohol,which can lead to hospitalization and even death.BZD are frequently detected in cases of“driving under the influence of drugs”and in drug facilitated crimes,where misuse of BZD/Z-drugs often is implied.For these reasons,BZD/Z-drugs are commonly analyzed in both clinical and forensic laboratories.Sample pre-treatment is a fundamental step in analytical chemistry,especially when analyses are performed on highly complex matrices.In toxicology,the analysis of complex biological samples remains a challenging task.Matrix components need to be eliminated in order to avoid possible interferences during quantification and to minimize the chance of polluting the analytical instrument.Moreover,a good sample preparation step can result in analyte enrichment and thus a final analytical method with improved sensitivity.The introduction of a thorough sample clean-up step has become inevitable.However,sample preparation is the most important and also vulnerable step in a bioanalytical method for handling of matrix effect and removing sample interferences.In the process of drug analysis,the actual instrument detection accounts for only 30%of the total analysis time,while up to 60%of the time is spent in the process of sample pretreatment.At present,the commonly used separation and enrichment methods in toxicological analysis include protein precipitation,liquid-liquid extraction and so on.Most of them have disadvantages such as time-consuming,labor-intensive and using large amount of organic reagents.The characteristics sought for an ideal extraction method are firstly simplicity and rapidity of execution,in order to be applied on a huge number of samples or for routine analyses.Solid phase extraction(SPE)is one of the most frequently used sample preparation techniques in the biomedical field.The concept of SPE is on the basis of analytes partitioning between the liquid phase and the solid sorbent.The type of sorbent and its structure are the most important factors that affect the extraction efficiency.Current trends of research in SPE are focused on the finding of new materials.Recently,nanomaterials gradually becomes a new SPE adsorbent.The high specific surface area of the nanomaterials can provide a large number of active sites,forming the basis for efficient separation,extraction and enrichment.In this paper,polystyrene nanofibers(PS)and Fe₃O₄@SiO₂-NH₂-G5 magnetic nanoparticles were prepared,and the process of SPE based the two materials were successfully applied for the simultaneous extraction and subsequent HPLC-UV or UHPLC-MS/MS determination of sedative-hypnotics from biosamples.The methods were accurate,sensitive and reliable,which provides a method for sedative hypnotic drug abuse screening,forensic toxicology analysis and clinical treatment of poisoning.And the method can also provide basic data for investigation and evidence collection of forensic cases related to such drugs.The main contents are as follows:Part one Preparation of polystyrene nanofibers for adsorption of clonazepamObjective:To prepare PS nanofibers and study its adsorption properties for BZDs.To provide the basic theory for the use of PS nanofibers as an SPE adsorbent in the pretreatment of BZDs in biosamples.Methods:PS was dissolved in a solution of THF and DMF(1:1 m/m)at a concentration of 15 wt%.The spinning solution was loaded into the syringe,the spinneret was connected to the positive pole of the high voltage source,and an aluminum foil(connected to the negative of the high voltage source)was used as the collector to collect the fiber.The distance between the needle and the collector was 150 mm.The voltages on the needle and on the collector were+16 kV and-7 kV,respectively,and the electrospinning was carried out for 8-12 h with the temperature of 25?.Clonazepam was selected as the representative drug of BZDs.The adsorption conditions such as adsorption time,solution pH and the amount of adsorbent were optimized.The adsorption kinetics and thermodynamic properties of clonazepam on polystyrene nanofibers were studied under the optimized conditions.Results:The kinetic study indicated that the adsorption of the target compound could be well fitted by the pseudo-second-order equation,suggesting that the intra-particle diffusion process was the rate-limiting step of the adsorption process.The adsorption equilibrium data of clonazepam were fitted well to the Freundlich isotherm model,with a maximum adsorption capacity values of 3.2 mg/g.Thermodynamic study revealed that the adsorption process was endothermic and spontaneous in nature.Conclusions:The results showed that electrospun polystyrene nanofibers,as a new type of adsorption material,can effectively adsorb clonazepam and has good potential for separation and purification of benzodiazepines from water-soluble matrix.Part two Development of a simple nanofiber-based solid phase extraction procedure coupled with high performance liquid chromatography analysis for the quantification of eight sedative-hypnotic drugs in human urine samplesObjective:To establish a novel SPE method based on nanofibers for the extraction and purification of eight sedative-hypnotic medications(zaleplon,zolpidem and six benzodiazepines drugs)from urine samples.Methods:A clean-up cartridge was homemade and polystyrene nanofibers were spun and selected as the adsorption material.The factors affecting the extraction efficiency such as the amount of adsorbent and desorption conditions were optimized.After the SPE procedure,the obtained extraction solution was analyzed by high performance liquid chromatography(HPLC).Chromatographic separations were performed at 30? on a Dikma Diamonsil C18(2)(5?m,250×4.6 mm)column,with the mobile phase composed by water with 50 mmol/L KH₂PO₄water solution(A)and acetonitrile(B)in gradient conditions at 1 mL/min.The detection wavelength was set at 220 nm to meet the highest absorbance for the most analytes.The injection volume was 20?L.Results:Under the optimal conditions,good linearity was obtained in the range of 1-10?g/mL with the correlation coefficients(r)above 0.99.The LOD of the 8 analytes were 0.2-0.4?g/mL,and the LOQ were 0.4-1?g/mL.The intra-and inter-day precision(RSD%)values of this method were within the acceptable ranges of 1.3-15.2%and 3.7-15.0%,respectively.The recoveries determined for the eight compounds at the three concentration levels were in the range of 90.4-113.3%.Conclusions:These results showed that the home-made cartridge has an excellent extraction efficiency for the studied compounds.Compared to other common pretreatment method,the proposed method is simple and reliable.Part three Simultaneous determination of 12 sedative-hypnotics in human urine and plasma by polystyrene nanofibers based on solid phase extraction combined with UHPLC-MS/MSObjective:To development and validation of a PS nanofiber based SPE-UHPLC-MS/MS method for the detection of 12 sedative-hypnotic drugs in human urine and plasma samples.Methods:Polystyrene nanofibers were prepared by electrospinning technique,and polystyrene nanofibers were used as adsorbents to prepare SPE cartridges to separate and purify 12 sedative-hypnotic drugs from human urine and plasma.The extraction parameters such as polystyrene fiber dosage,sample loading speed,impurity leaching solution and desorption solvent volume were optimized.Determination was carried out on a Waters ACQUITY UPLC BEH C18 column(100×2.1 mm,1.7?m)with the column temperature of 40?.The mobile phase was composed of acetonitrile-0.1%ammonia solution with gradient elution at flow rate 0.3 mL/min.Mass spectrometry acquisition was done in the positive ion mode and analytes were detected in multi-reactive monitoring mode.Results:The 12 analytes in urine samples have a good linear relationship within the concentration range of 0.1-20 ng/mL,and the correlation coefficient are more than 0.99.Except for lorazepam and clonazepam in plasma samples,the linear range were 1-100 ng/mL,the other 10 analytes have a good linear relationship within the concentration range of 0.5-100 ng/mL,and the correlation coefficient are more than 0.99.In urine samples,besides the intra-and inter-day RSDs were 16.0%and 15.2%for nitrazepam and clonazepam respectively at low concentration level,and the intra-and inter-day RSDs of the other concentration points of 12 analytes were all<15.0%.The intra-and inter-day RSD of the 12 analytes in plasma samples ranged from 1.4-15.7%.The extraction recoveries of the 12 analytes in human urine and plasma were43.82-114.6%and 47.24-98.3%,respectively.The LLOQ of the 12 analytes in urine and plasma were 0.01-0.1 ng/mL and 0.1-1 ng/mL.Conclusions:This method is sensitive,accurate and efficient,and is suitable for the analysis of trace target compounds in complex matrix such as biological samples.It provides a reliable means for the detection of sedativehypnotics,especially suitable for forensic analysis and drug abuse or poisoning screening.Part four Development and validation of a fast magnetic nanoparticles-based magnetic solid phase extraction procedure combined with UHPLC-MS/MS analysis for the quantification of 4benzodiazepines and benzodiazepine-like hypnotics in human urineObjective:To develop a magnetic solid-phase microextraction technique based on composite magnetic nanoparticles as adsorbents and liquid-liquid mass spectrometry to detect diazepam,midazolam,zolpidem,and zaleplon in human urine samples.Methods:.Using Fe₃O₄@SiO₂-NH₂ as the central core,Fe₃O₄@SiO₂-NH₂-G5 was synthesized based on dendrimer(G5)of alternating grafting of melamine and imidazole.The morphology and structure of the magnetic materials were characterized by transmission electron microscopy,Fourier transform infrared spectroscopy,and X-ray diffraction.The key parameters affecting the extraction efficiency were optimized.The magnetic solid-phase extraction of the four drugs was performed at pH 9 of the sample solution,with an extraction time of 120 s,using methanol/acetonitrile(v:v=1:1)as the eluent solvent at 30 s of desorption time to obtain the test solution.For detection,5?L of the test solution was injected into the liquid phase mass spectrometry system.Chromatographic column Waters ACQUITY UPLC BEH C18(1.7?m,2.1×100 mm)was used at 40? column temperature,with 0.1%ammonia solution as mobile phase A and acetonitrile as mobile phase B,for gradient elution at a flow rate of 0.3 mL/min.Results:The proposed method was validated,and the limits of detection of zaleplon,diazepam,zolpidem,and midazolam were 0.05,0.05,0.02,and0.02 ng/mL,respectively.The linear correlation coefficients r of the four analytes were greater than 0.996,the intra-day precision was between 1.4%and 9.4%with the recoveries between 88.3%and 104.8%,and the inter-day precision ranged from 3.9%-15.2%with the recovery in the range of94.1%-108.3%.Conclusions:The magnetic dendritic structure nanomaterial Fe₃O₄@SiO₂-NH₂-G5 was successfully used for the first time to extract sedative-hypnotic drugs from human urine samples.This Fe₃O₄@SiO₂-NH₂-G5-based magnetic solid-phase extraction method eliminates centrifugation,filtration,and blowing dry steps as in conventional extraction.Only one step of vortex dispersion extraction could achieve the separation and purification of the target compounds.The method is simple,rapid,environment-friendly,and suitable for the detection and analysis of sedative-hypnotic drugs in human urine.