The Establishment And Clinical Application Of The Detection Method For The Quantification Of Risperidone Or Lamotrigine

Background Risperidone is second-generation atypical antipsychotic drugs,it through the double block of dopamine D2 receptor and serotonin receptors to play a role in treating psychotic.It can effectively treat acute and chronic schizophrenia and other adults and children’s mental illness,including broad developmental disorders,autism and attention deficit disorder.Riperidone have a small side effect compared with other drugs,and has been widely applied in clinical treatment.Lamotrigine is a new kind of benzene triazine antiepileptic drugs,mainly for the treatment of various types of epileptic disease,and especially some additional attack or systemic rigidity seizure,monotherapy,or children,pregnant women with epilepsy.It is also applicable to all types of emotional disorders and antidepressant treatment.In recent years,Psychotropic drugs the priority use chromatography or combination techniques,high performance liquid chromatography mass spectrometry instrument（LC-MS/MS）was widely used with high sensitivity and selectivity,make the possible the determination of most psychiatric drugs and their metabolites,become the preferred method of clinical research and drug concentration monitoring.Liquid chromatography（HPLC）is the common use of the monitoring of psychotropic drugs.LC-MS/MS method adopts the principle of tandem mass spectrometry determination of risperidone,HPLC method using the principle of chromatography determination of lamotrigine.LC-MS/MS can be used to measure the quantity of ng,pg,fg level.HPLC are usually can be measured to μg level of quantity,and sometimes also can measure the ng level.Therefore,according to the difference of drug reference range,LC-MS/MS is convenient to detect risperidone,and the content of lamotrigine can be detected by HPLC.Drug concentration monitoring is an effective method for the formulation of drug regimens according to individual patients’ individualized tailoring.It is a very effective tool and is of great value for the precise treatment of mental diseases.Due to the large difference of individual patients,it is necessary to find a more rapid and simple measurement method to monitor the drug concentration,prevent the drug from being ineffective or adverse reactions,and implement the accurate treatment of the patients.Part one:the establishment and clinical application of the LC-MS/MS method for the quantification of risperidone and 9-hydrorisperidone in human plasma.Objectives 1.Establishing a rapid,high sensitive,high selective LC-MS/MS method for the quantification of risperidone and 9-hydrorisperidone in human plasma.2.This LC-MS/MS method was used to evaluate the clinical application value for more than 1,000 samples of risperidone patients,and to provide theoretical basis for the monitoring of clinical psychiatric drug concentration.Methods 1.By looking up the related literature and data,debugging,and many times change liquid chromatography and mass spectrometry-mass spectrometry instrument parameters,set up preliminary LC-MS/MS method for determination of risperidone drug concentration.2.The methodology of the LC-MS/MS approach in this study is a systematic review of the system.3.From October 2015 to October 2017,a total of 1,076 plasma specimens were taken from the general psychiatric department of the second affiliated hospital of xinxiang medical university.There were 285 females,ranging in age from 9 to 73 years,292 males ranging in age from 11 to 66 years.The risperidone concentration was determined by LC-MS/MS method,and the correlation between blood drug concentration and prognosis was analyzed.Results 1.Liquid phase conditions:Chromatographic separation was performed on a ES Industries C18（2）column（100×2.1mm,3μm）using gradientelution.The mobile phase were methanol-water（1:1,v:v）which contain 6 mmol·L^-1 ammonium acetate and acetonitrile with 0.1% formic acid.Analytes were detected using a triple quadrupole mass spectrometer that was operated in positive ionmode with electrospray ionization.ES Industries C18（2）（100×2.1mm,3μm）chromatographic column;The total flow rate is 0.6ml·min-1;the sample volume was 10μL,column temperature was 30℃.2.Multiple reaction-monitoring mode（MRM）was chosen for detection with the transitions of m/z 411.3→191.1 for risperidone,m/z 427.2→207.0 for 9-hydroxyrisperidone and m/z 285.1→193.1 for diazepam.Dwell time is 200 ms.Software Analyst V1.5.2（AB SCIEX）was used for data collection and analysis.3.Methodology evaluation: the method is verified according to the principle of "verification and guiding principle of quantitative analysis of biological samples" in the 2015 edition of Chinese pharmacopoeia.According to ISO15189 the medical laboratoryquality and capability of the special requirements for the calibration of the instrument,the quality control of each link in the laboratory,standardize the work flow,ensure the results are timely and reliable.This method exhibited excellent linearity for all the analytes with regression coefficients higher than 0.999.The limit of quantification（LOQ）values for risperidone and 9-hydroxyrisperidone were 0.16 μg·L^-1and 0.40 μg·L^-1,respectively.The mean accuracy ranged from 97.40% to 115.00% and the RSD of precision were below 10.9%.Both the selectivity and matrix effect meet the requirement.The sample could store in room temperature for 3 days,in refrigerator for one month.Samples could stay stable after freezing and thawing for 3 times.More than one thousand clinical samples wered detected by LC-MS/MS.4.Clinical application: all samples were recommended by the AGNP-TDM expert group to recommend RIS and 9-OHRIS blood drug concentration treatment range of 20⁶0μg·L^-1 for clinical dose adjustment.It turns out that 69.3% of the patients in the 577 patients were in this range,and there were 68.2 percent of the 245 patients that were monitored in this area.Among the patients with multiple monitoring,140 male patients and 105 female patients,among whom 62.1% of the male patients had the recommended therapeutic concentration,while the female patients were 76.2%.We suggested that clinical doctors on the basis of AGNP panel recommended treatment concentration range dose adjustments,clinical monitoring results according to the percentile method（P2.5^P97.5）calculation,it is concluded that the total RIS actual determination of the concentration range of 13.30⁹3.22μg·L^-1.Conclusions 1.We established the LC-MS/MS analysis method for the study and determination of risperidone clinical blood drug concentration.The sample analysis was fast,easy to operate,high sensitivity,good selectivity,and good quality control.2.Monitoring and analysis of 1076 clinical samples using this method,according to the results of laboratory suggest clinicians use ANGP-TDM experts recommend treatment concentration range(20⁶0μg·L^-1)dose adjustments,the actual monitoring clinical results through the percentile method statistical analysis it is concluded that Chinese people always risperdal clinical actual measured concentration range(13.30⁹3.22μg·L^-1),more than this range from panel recommended treatment concentration range wide,and multiple monitoring in patients with female blood drug concentration relative to men tend to be more stable.Part two:the establishment and clinical application of the HPLC method for the quantification of Lamotrigine in human serum.Objectives 1.Establish a simple and rapid HPLC method to determine the drug concentration of lamotrigine in human serum,analyze and summarize the method,and lay a good foundation for the establishment of HPLC method for psychotropic drugs in the future.2.This HPLC method was used to evaluate the clinical application of lamotrigine patients and provide theoretical basis for clinical drug concentration monitoring.Methods Benzoguanamine was used as internal standard,and sample preparation was performed by protein precipitation with methanol and ammonium acetate after addition of internal standard to 150 μL of plasma specimen.chromatographic separation was achieved using mobile phase coposition with UV detection at 207 nm.Chromatographic separation was performed on a ES Industries C18（2）column（50×2.1mm,3μm）using gradient elution.The mobile phase were methanol-water at a flow rate of 0.6m L·min-1.The injection volume was 20μL and the column temperature was maintained at 40℃.Results This method exhibited excellent linearity for the analytes with regression coefficients higher than 0.999.The limit of quantification（LOQ）values for Lamotrigine were 1.18μg·m L^-1.The mean accuracy meet the requirement and the relative standard deviation（RSD）in the quantitative lower limit,lower,medium and high concentration batch were below 13.1%.The sample could store in room temperature for 1 days,in refrigerator for one week.Conclusion A simple and rapid HPLC method was developed and well validated.This method was successfully used to monitor the serum concentration of Lamotrigine.