Effects Of Acute Barbiturates Poisoning On The Level Of Neurochemicals Based On LC-MS/MS

Research background:Barbiturates areγ-aminobutyric acid（GABA）agonists,which could combine with the GABA receptor complexes to reduce the excitability of cells.Barbiturates are commonly used for anesthesia and anticonvulsion in clinical.However,overuse of barbiturates would cause depression of central nervous system,resulting in barbiturates intoxication.At present,the amount of non-medical uses of barbiturates are enormous,thus the barbiturates are identified as one of the drugs which affect social security and public health.The major target organ of barbiturates is the central nervous system.When suffered from barbiturates poisoning,some neurochemicals（NCs）,for example neurotransmitter（NTs）,would change along with functional changes in the nervous system.Therefore,developing an efficient and sensitive method for separation and analysis of barbiturates and NCs in biological samples and to study the effects of barbiturates poison on the level of neurochemicals benefit both biomedicine and forensic medicine.Research purpose:（1）A method for determination of barbiturates in biological samples would be developed by combining liquid-phase microextraction（LPME）and liquid chromatography-tandem mass spectrometry（LC-MS/MS）,which would lay the foundation for the subsequent study on the effects of barbiturate poisoning on NCs level;（2）A method for determination of neurochemicals in biological samples would be developed by combining protein precipitation（PP）and LC-MS/MS,which would provide technical support for the subsequent study on the effects of barbiturate poisoning on NCs level;（3）investigating the influences of acute barbiturates poisoning on the level of neurochemicals using poisoned rat model.Research method:（1）In this study,a method（LPME-LC-MS/MS）for the detection of barbiturates（barbital,phenobarbital,and pentobarbital）in biological samples was established by combining LPME and LC-MS/MS.The method was validated using four common biological samples（whole blood,plasma,urine,and liver tissue）in forensic toxicology analysis,respectively.（2）PP method was used to separate 20 neurochemicals.Combined with LC-MS/MS,analytical methods for analysis of 20 neurochemicals in plasma and brain tissue were established（PP-LC-MS/MS）.（3）The acute phenobarbital poisoned rat models with concentration gradient(1/8LD₅₀,1/4LD₅₀,1/2LD₅₀,LD₅₀,2LD₅₀)were constructed.Using LPME-LC-MS/MS and PP-LC-MS/MS,the concentration of phenobarbital in the plasma of poisoned rats and the concentration of NCs in the plasma and brain tissue in the poisoned rats were determined,respectively.On this basis,correlation analysis between the level of NCs and toxic doses based on statistical analysis technique of Spearman rank correlation analysis and trend test was conducted.Research result:（1）For different biological samples,the limit of detection（LOD）of FM-LPME-LC-MS/MS method were 1.2-2.7 ng m L^-1（whole blood）,1.2-26 ng m L^-1（plasma）,0.6-3.6 ng m L^-1（urine）and 5.2 to 10.0 ng g^-1（liver tissue）,respectively.In corresponding linear range,the method displayed good repeatability（5%<RSD<14%）and reliable linearity（R²>0.99）.（2）In PP-LC-MS/MS method,the LODs of NCs in plasma and brain tissue were 0.1-7.4 ng m L^-1 for monoamine-NCs,0.1-3.0 ng m L^-1 for acetylcholine-NCs and carnitine-NCs and 5.0-12.5 ng m L^-1for amino acids,respectively.The method exhibited good linearity in the linear range（R²>0.99）for all the analytes.The precision of the method is less than 20%,and the accuracy is between 80%and 120%.The matrix effect of brain tissue was 80.5-118.3%,and the stability of NCs in brain homogenate was good at-20℃and freeze-thaw.（3）It was found that GABA,glutamate,tryptophan,and tyrosine in brain tissue were negatively correlated with phenobarbital drug concentrations in plasma;however,the concentrations of carnitine,and propionyl carnitine in encephalic regions were positively correlated with phenobarbital concentrations in plasma.The concentrations of acetyl carnitine,propionyl carnitine,and decyl carnitine in plasma were negatively correlated with phenobarbital concentrations in plasma;but concentration of serotonin was positively correlated with phenobarbital concentrations in plasma.Research conclusion:In this work,LPME was used for the separation of barbiturates from common biological samples in forensic toxicology analysis.The proposed LPME-LC-MS/MS methods showed high sensitivity,good repeatability,and low matrix interference,and were successfully used to qualitative and quantitative analysis of barbiturate in biological samples.At the same time,another method（PP-LC-MS/MS）was established for detection and analysis of various NCs in plasma and brain tissue.Finally,the above methods were applied for the determination of barbiturates and NCs in the samples from acute phenobarbital poisoned rat model.The results showed that:（1）Acute phenobarbital poisoning led to the reduction of some amino acids in brain tissue,and the degree of the reduction was negatively correlated with the concentration of phenobarbital in plasma;（2）The concentrations of some carnitine in certain brain regions are positively correlated with the concentration of phenobarbital in plasma;（3）Some carnitine in plasma is negatively correlated with the concentration of phenobarbital in plasma.These results of this study provided new ideas not only for the separation and detection of toxicants in forensic toxicological analysis,but also for the toxicological research of other toxicants.