| Sedative hypnotics can improve the patients’tension,anxiety,fear and other negative emotions and inhibit central nervous system directly with anxiolytic effect.Small dose has a sedative effect,while the dose increased to a certain amount,having a hypnotic effect.And sedative hypnotics are mainly for the treatment of insomnia,as well as have muscle relaxation,anticonvulsant and other pharmacological effects,helping to avoid insomnia damage to human health and normal life.At present,sedative hypnotic drugs which are commonly used in the treatment of insomnia,mainly include benzodiazepines(BZDs),such as diazepam(valium),clonazepam,estazolam,alprazolam and other non-benzodiazepines such as barbital,phenobarbital,zopiclone and so on.Sedative,hypnotic,anticonvulsant and anesthetic effects appear with increased dose of Barbiturates.It is possible to suppress breathing up to 10 times of hypnosis,or even death.Even if benzodiazepine drugs having relatively high safety,overcommitting also can cause poisoning and even death.Therefore,sedative hypnotics can meet the insomnia patients effectively.However,some criminals also use sedative hypnotics to commit criminal activities,such as anesthesia rape,anesthesia robbery,anesthesia removal organs,anesthesia murder,etc.and related crimes occurred every year.It is necessary to establish an accurate,efficient and rapid identification method for clinical drug overdose,the detective work of rescue and court of criminal cases related to crimes of abusing of sedative hypnotic drugs.These analysis methods are used to identify which kind of sedative hypnotic in biological samples.As time goes on,the concentration of sedative hypnotic drug in the body will gradually decrease with the metabolism.And the amount of drugs in the blood,urine or other biological samples is low,making it difficult to detect in many cases.Therefore,it is necessary to establish a simple,rapid,sensitive,accurate and high-throughput detection method for the determination of trace sedative hypnotics in biological samples.In this work,high-resolution UPLC-MS/MS method was used to detect sedative hypnotics in biological samples.Orbitrap high-resolution mass spectrometry(Q Excative Focus)combine the separation ability of ultra-high performance liquid chromatography and high resolution,high sensitivity with powerful qualitative function of Orbitrap mass spectrometry.Q Excative Focus is specific,accurate,reliable,fast and sensitive,becoming a qualitative and quantitative powerful analysis tools.In this study,the Orbitrap high-resolution mass spectrometry technique was used to establish a method for the determination of diazepam,clonazepam,estazolam,midazolam,phenobarbital and barbital,providing an efficient,simple,fast and reliable method for the rapid qualitative and quantitative analysis of these drugs.Part 1 Q-Orbitrap High-Resolution Mass Spectrometry Simultaneous Detection of 6 Kinds of Sedative Hypnotics in Human Urine.Objective:To detect 6 kinds of sedative hypnotic drugs including diazepam(Valium),clonazepam,estazolam,midazolam,barbital and phenobarbital in human urine simultaneously with a rapid,sensitive,high throughput and accurate detection method.Methods:The urine samples were extracted with acetonitrile and analyzed by Orbitrap high resolution mass spectrometry.Chromatographic conditions:Column:Hypersil Gold PFP 100×2.1 mm,3μm;Column temperature:30℃;injection volume:5μl;Mobile phase:A:0.1%ammonium acetate-5 mM formic acid-water,B:methanol;flow rate:0.40 mL/min;elution mode:gradient elution.Mass Spectrometer conditions:a heatable electrospray ion source(HESI-Ⅱ);ionization mode:ESI~+&ESI~-while scanning full MS-dd-MS~2mode;scan range:150-1050 aum;atomizing gas:nitrogen(flow rate ESI~+:45 L/h;ESI~-:35 L/h);Auxiliary gas:nitrogen(flow rate:ESI~+:15 L/h;ESI~-:8 L/h);spray voltage:ESI~+(3KV);ESI~-(2.5 KV);capillary temperature:320℃;auxiliary air heating temperature:300℃;secondary mass spectral collision voltage(V):20/30/40;ultra-high performance liquid chromatography controlled by Dionex Chromeleon 7.0 software,mass spectrometry controlled by Tune software.The data processing was performed by TraceFinder3.3.Results:6 sedative-hypnotics including diazepam(Valium),clonazepam,estazolam,midazolam,barbital and phenobarbital satisfy the linear concentration range detection,and correlation coefficients were higher than 0.9909.The linear range of diazepam was 0.78~50 ng/mL and the limit of quantification was 4 pg.The linear range of clonazepam was 0.78~50 ng/m L and the limit of quantification was 4pg.The linear range of estazolam was 0.39~50 ng/mL and the limit of quantification was 2 pg.The linear range of midazolam was 0.39~50 ng/m L and the limit of quantification was 2 pg.The linear range of barbital was 1.56~50 ng/mL and the limit of quantification was 8 pg.The linear range of phenobarbital was 1.56~50 ng/mL and the limit of quantification was 8 pg.Stability,precision and accuracy all meet the requirements in this study.The intra-day and inter-day RSD of the six target drugs tests were less than 15%for accuracy.The recoveries ranged from 81%to 119%and RSD was 3.6%~14.7%.Conclusion:The method can be used for simultaneous detection of 6 sedative hypnotics including diazepam(Valium),clonazepam,estazolam,midazolam,barbital and phenobarbital in urine samples,since it is simple,rapid,selective,high throughput and sensitive.Part II Q-Orbitrap High-Resolution Mass Spectrometry Simultaneous Detection of 6 Kinds of Sedative Hypnotics in Human Plasma Samples.Objective:To detect 6 kinds of sedative hypnotic drugs including diazepam(Valium),clonazepam,estazolam,midazolam,barbital and phenobarbital in human plasma simultaneously with a sensitive,accurate,simple,rapid,and multiple outputting detection method.Methods:The plasma samples were extracted with ethyl acetate and analyzed by Orbitrap high resolution mass spectrometry.Chromatographic conditions:Column:Hypersil Gold PFP 100×2.1 mm,3μm;Column temperature:30℃;injection volume:5μl;Mobile phase:A:0.1%ammonium acetate-5 mM formic acid-water,B:methanol;flow rate:0.40 mL/min;elution mode:gradient elution.Mass Spectrometer conditions:a heatable electrospray ion source(HESI-Ⅱ);ionization mode:ESI~+&ESI~-while scanning full MS-dd-MS~2mode;scan range:150-1050 aum;atomizing gas:nitrogen(flow rate ESI~+:45 L/h;ESI~-:35 L/h);Auxiliary gas:nitrogen(flow rate:ESI~+:15 L/h;ESI~-:8 L/h);spray voltage:ESI~+(3KV);ESI~-(2.5 KV);capillary temperature:320℃;auxiliary air heating temperature:300℃;secondary mass spectral collision voltage(V):20/30/40;ultra-high performance liquid chromatography controlled by Dionex Chromeleon7.0 software,mass spectrometry controlled by Tune software.The data processing was performed by TraceFinder3.3.Results:6 sedative-hypnotics including diazepam(Valium),clonazepam,estazolam,midazolam,barbital,phenobarbital satisfy the wide linear detection range concentration range detection with relative standard deviation over 0.99.The linear range of diazepam was 2~128 ng/mL and the limit of quantification was 10 pg.The linear range of clonazepam was 2~128 ng/mL and the limit of quantification was 10pg.The linear range of estazolam was 1~128 ng/mL and the limit of quantification was 5 pg.The linear range of midazolam was 1~128 ng/mL and the limit of quantification was 5 pg.The linear range of barbital was 4~128 ng/mL and the limit of quantification was 15 pg.The linear range of phenobarbital was 4~128 ng/mL and the limit of quantification was 15 pg.The limit of detection,stability,precision and accuracy all meet the requirements in this experiment.The intra-day and inter-day RSD of the six target drugs were less than 15.0%.The recoveries ranged from 81%~117%and RSD was 2.3%~13.4%.Conclusion:This method is simple and fast,high selectivity and sensitivity.It can be used for simultaneous detection of 6 sedative hypnotics including diazepam(Valium),clonazepam,estazolam,midazolam,barbital,phenobarbital in plasma samples. |
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