Detection of Carbamazepine and Its Metabolites in Blood Samples by LC-MS/MS / 法医学杂志

OBJECTIVES@#To establish a method for the detection of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS).@*METHODS@#The blood samples were treated with 1-butyl-3-methylimidazolium hexafluorophosphate as an extraction solvent. The samples were extracted by ultrasound-assisted extraction and separated by ZORBAX Eclipse Plus C18, 95Å column. The mobile phase A aqueous solution containing 0.1% formic acid and 10 mmol/L ammonium acetate, and mobile phase B mixed organic solvent containing acetonitrile/methanol (Vacetonitrile∶Vmethanol=2∶3) were used for gradient elution at the flow rate of 1.00 mL/min. An electrospray ion source in positive mode was used for detection in the multiple reaction monitoring.@*RESULTS@#The linearities of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples were good within the corresponding range, with correlation coefficients (r) greater than 0.995 6. The limits of detection were 3.00, 0.40 and 1.30 ng/mL, respectively. The limit of quantitation were 8.00, 1.00 and 5.00 ng/mL, respectively. The extraction recoveries ranged from 76.00% to 106.44%. The relative standard deviations of the intra-day and inter-day precisions were less than 16%. Carbamazepine and its main metabolite 10,11-dihydro-10,11-epoxycarbamazepine were detected in blood samples of death cases with a mass concentration of 2.71 μg/mL and 252.14 ng/mL, respectively.@*CONCLUSIONS@#This method has high sensitivity and good selectivity, which is suitable for the detection of carbamazepine and its metabolites in blood samples, and can be used for carbamazepine-related forensic identifications.

Identification of the New Psychoactive Substance Eutylone / 法医学杂志

OBJECTIVES@#To establish a method to identify unknown sample based on the combined use of Fourier transform infrared spectroscopy (FTIR), gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOF-MS), ultra-high performance liquid chromatography-linear ion trap quadrupole-orbitrap mass spectrometry (UPLC-LTQ-Orbitrap MS) and 1H-nuclear magnetic resonance spectroscopy (1H-NMR) technique.@*METHODS@#The unknown sample was directly analyzed by FTIR. The unknown sample was dissolved in methanol solution containing internal standard SKF525A and the supernatant was detected by GC-QTOF-MS and UPLC-LTQ-Orbitrap MS. The unknown sample was dissolved in methanol-d4 solution for structural analysis of 1H-NMR.@*RESULTS@#The characteristic absorption peaks of FTIR spectra obtained from unknown sample were 1 682 (C=O bond), 1 503, 1 488, 1 436, 1 363, 1 256, 1 092, 1 035, 935, 840 and 800 cm-1, the characteristic fragment ions (m/z) of GC-QTOF-MS were 86.096 4 (base peak), 58.065 1, 149.023 5, 121.028 6 and 65.038 6, the accurate mass [M+H]+ detected by UPLC-LTQ-Orbitrap MS was 236.127 7. The sample was identified as synthetic cathinone new psychoactive substance Eutylone by 1H-NMR.@*CONCLUSIONS@#The method established in this study can be used for structural confirmation of Eutylone.

Determination of aconitine in biological human sample by UPLC-MS/MS / 中国法医学杂志

Objective To develop a simple and fast UPLC-MS/MS method for determination of aconitine in biological human sample. Methods The biological human sample was treated by acetonitrile precipitation, and then analyzed on a UPLC C18(2.1mm×50mm, 1.7μm)column. The mobile phase consisted of acetonitrile-0.1% formic acid with gradient elution, at a flow rate of 0.4 mL/min, at 40℃. UPLC-MS/MS was performed in ESI source with MRM mode for quantification. Results The linear range of the concentration were 0.5~500ng/mL in blood and 1~1000ng/mL for aconitine in blood for aconitine (r>0.995). The relative recoveries of aconitine were in the range of 91.3%~110.2%, and the extraction recoveries were in the range of 72.8%~83.5%. The RSDs of intra-days and inter-day were both less than 14%. Conclusion The method is a simple, fast and could be used for determination of aconitine in biological human sample.

Simultaneous determination of three drugs in saliva by UPLC-MS/MS / 中国法医学杂志

Objective A method of determination of methamphetamine, ketamine and morphine in saliva was established by super high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS). Methods The saliva samples after precipitation protein, in the ACQUITY UPLC BEH Phenyl(100mm×2.1mm,1.7μm) column chromatography, after the acetonitrile: 0.3% Formic acid solution gradient elution and through the ESI+ ionization mode MRM analysis and inspection. Results The linearity was estadlished in saliva between 4μg/L and 200μg/L, LOD was 0.2μg/L, LOQ was 4μg/L. the recovery was in the range of 87%-128%. Conclusion The method is simple, rapid and accurate for determination of methamphetamine, ketamine and morphine in saliva.

The change of blood alcohol concentration in vivo and its influencing factor analysis / 中国法医学杂志

Objective To explore the changes of blood alcohol concentration (BAC) and its influencing factors in alcohol consumption, and establish a mathematical model of BAC metabolism. Methods The BAC was measured by using the gas chromatograph and the internal standard curve method, and the data was analyzed by SPSS20.0 and R and the mathematical model was established. Results On average women BAC elimination rate is 9.54mg/100mL/h, the average male BAC elimination rate is 12.19mg/100mL/h, women elimination rate less than men, and BAC elimination rate is related to gender of medium and related to the weight of strong, has nothing to do with age. According to the results of the mixed effects model, the mixed effect model can predict the BAC accurately, the mean absolute error (MAE) is 1.60mg/100mL, and the data is analyzed by the decision tree method, and MAE is 9.99mg/100mL. Conclusion BAC elimination rate was associated with sex and weight after drinking, and the random-effect mixing model could be accurately inferred by time, alcohol consumption, sex and weight.

Distribution of methamphetamine concentration in saliva and urine samples and the analysis of the initial screening / 中国法医学杂志

Objective To obtain methamphetamine concentration profiles in saliva and urine samples of drug addicts and to screen the colloidal gold strip. Methods Methamphetamine concentration in saliva and urine samples of drug addicts was determined by liquid chromatography tandem mass spectrometry. The initial screening was obtained by colloidal gold strip test. The results were compared and analyzed. Results using the method of protein and fluid MRM scan method to detect direct precipitation, saliva is linear in the range of 1~100ng/mL, the linear correlation coefficient is 0.9987, the detection limit is 0.1ng/mL, the limit of quantification was 1ng/mL, the urine is linear in the range of 1~100ng/mL, the linear correlation coefficient is 0.9943, the detection limit is 0.5ng/mL, the limit of quantification was 1ng/mL. Saliva and urine samples diluted, the concentration in the linear range. Saliva and urine samples of four types of methamphetamine colloidal gold reagent strip were screened directly, and the results were judged visually. Conclusion the detection rate of colloidal gold strip is about 79%, the detection rate of saliva is about 81%, and the detection rate can be increased to more than 93% by using two reagent strips. Combined with the initial screening results and the instrument confirmation concentration, it can be found that the gray zone setting and sensitivity setting have certain influence on the detection rate, and it is suggested to improve the sensitivity to meet the needs of screening.

Establishment of Fluorescence Immunochromatographic Assay for Quantitative Detection of Morphine / 中国药学杂志

OBJECTIVE: To establish a high sensitivity method for rapid quantitative detection of morphine in the biological samples including serum, saliva and urine. METHODS: With the immunochromatographic lateral flow strip as reaction method, the luminescent lanthanide europium nanoparticles covalently conjugated with morphine monoclonal antibody were adopted as reporters. The morphine antigen and goat anti-mouse antibody were coated at the nitrocellulose membrane separately as the test line and control line. The strip based on competitive inhibition immunoassay principle was detected by the fluorescent reader for rapid quantitative detection of morphine in the biological samples. RESULTS: After experiment optimization, the improved strip could provide the line range 3-3 000 ng·mL-1; precise quality morphine control materials verified CV<10%. There were no cross reactions with amphetamine, methylamphetamine, ketamine and so on; keep the strips at 37 ℃ for 7 d, the performance of the strips did not decline markedly. CONCLUSION: The preliminary established method shows high linearity, precision, specificity and stability. The method could quantitatively detect the morphine in the biological samples in short time. It is supposed to be applied into the grassroots unit.

Determination of dexamethasone in human plasma by solid phase extraction with ultra performance liquid chromatography-tandem mass spectrometer / 中国法医学杂志

Objective To develop the analytical method to determine the content of dexamethasone in human plasma by solid phase extraction with ultra performance liquid chromatography-tandem mass spectrometer. Methods The human plasma was extracted with a solid phase extraction(SPE) and determined by UPLC-MS/MS. LC-MS/MS was performed in ESI source with MRM mode for quantification. Results The lowest detectable limit was 0.05ng/mL, the linear range was 1~100ng/mL. The absolute recovery was more than 78.1%. The intra- and inter day precision was within 15% at three concentrations. Conclusion Since the procedure proved to be simple, quick and effective, it could be used for the determination of dexamethasone in human plasma.

Study on the pharmacokinetics of clozapine and its metabolites in human blood / 中国法医学杂志

Objective To study the pharmacokinetics and detection window of clozapine and its metabolites in human blood, so as to provide experimental basis for forensic cases of identification of clozapine poisoning. Methods 29 Taiyuan Han people's elbow venous blood was collected after given oral administration of 12.5mg clozapine at different time point, in which clozapine and its metabolites were extracted with solid phase extraction (SPE) and determined by HPLC-MS-MS. The qualitative analysis was based on retention time and MRM ions. The quantitative analysis was based on an internal standard method and calibration curve. Using the 3p97 pharmacokinetic software, pharmacokinetic equation of clozapine in the blood were imitated from the C-T data, and pharmacokinetic parameters were calculated. Results The pharmacokinetics of clozapine met a two compartment open model with a first kinetics absorption. The Tmax of clozapine(CLP), demethylclozapine(DMCLP), N-oxidation-clozapine(NO-CLP) respectively were 2.96±1.32h, 8.65±3.00h, 9.31±26.38h; The Cmax of CLP, DMCLP, NO-CLP respectively were 34.68±9.32ng/mL, 11.16±4.15ng/mL, 9.62±13.88ng/mL;The t1/2 of CLP, DMCLP, NO-CLP respectively were 17.02±23.63h, 27.06±12.58h, 41.27±29.75h; The detection window of CLP, DMCLP, NO-CLP respectively were 81.72±26.19h, 93.21±29.40h and 19.93±14.62h. Conclusion The pharmacokinetics of clozapine in blood of Han people is consistent with two compartment open model with a first kinetics absorption. The pharmacokinetics model and parameters of clozapine can provide expirimental basis for forensic identification of clozapine poisoning cases.

Study on postmortem distribution of paraquat dichloride in rats / 中国法医学杂志

Objective Establish a method for qualitative and quantitative analysis of paraquat dichloride in organs in rat by UPLC-MS/MS and study the rat animal model poisoned by intragastric administration of paraquat dichloride to investigate the postmortem distribution of paraquat dichloride in poisoning death rat. Methods The rats were given an intragastric administration of 1/2LD50 Paraquat dichloride. The rats were dissected at 0.5h, 2h, 4h, 8h, 12h, 24h, 48h, 72h respectively after the intragastric administration. The specimens of -the heart, liver, spleen, lung, kidney, brain, muscle, bladder and stomach-were collected and analyzed immediately. Qualitative and quantitative analysis were performed by UPLC-MS/MS. Results Within 4h, stomach is the main distribution organ. The content of paraquat dichloride is the highest in stomache and relatively low in other organs. The concentration of organization except stomach changed little within 4h. The concentration of stomach has a sharp decline after 4h. The concentration in organs except stomach has a sharp rise after 4h. There is a significant difference(P<0.05) between each organs and brain. Conclusion There was a postmortem maldistribution of paraquat dichloride in poisoning death rats and the concentration in organs changes with time. The analysis method of UPLC-MS/MS and postmortem distribution of paraquat dichloride can be applied to the forensic identification of paraquat dichloride poisoning death and provide direction for delete this part toxicology analysis.

The postmortem distribution of bromadiolone and its metabolite-benzylideneacetone in poisoned dogs / 中国法医学杂志

Objective To study the postmortem distribution of Bromadiolone and its metabolite-Benzylideneacetone in dogs and provide an experimental evidence for the sampling of Bromadiolone poisoning cases. Methods The dogs were given 2LD50 and 4LD50 Bromadiolone by intragastric administration. Anatomy was conducted immediately after death and samples of body fluids and viscera (heart blood; peripheral blood, bile, urine, heart, liver, spleen, lung, kidney, brain, urinary bladder, left leg muscle, stomach, stomach contents, pancreas) were collected and detected after the dogs poisoning death. The Bromadiolon and its metabolite-Benzylideneacetone contents in samples were analyzed by GC/MS. Results Hemorrhagic symptoms came out at 3d after Bromadiolone delivery and deaths occurred at (178.40±20.94)h after intoxication. The postmortem distribution of Bromadiolon and its metabolite-Benzylideneacetone in dogs was as following: 2LD50 Bromadiolon: bile>urine, liver, heart, kidney>heart blood, peripheral blood, spleen, lung and so on. Benzylideneacetone: the content in bile, urine, heart blood, peripheral blood, lung, stomach contents are higher. 4LD50 Bromadiolon: bile, urine>liver, peripheral blood>heart blood, stomach contents and others. Benzylideneacetone:contents in bile, urine and lung are higher. Conclusion The postmortem distribution of Bromadiolon and its metabolite-Benzylideneacetone in dogs is uneven, contents in bile, urine, liver, heart blood and peripheral blood are higher, whichare suggested for forensic toxicological analysis in Bromadiolon poisonig case.

Detection of xylazine and 2,6-xylidine in blood and urine by SPE-HPLC/MS/MS / 中国法医学杂志

Objective To develop an HPLC-MS/MS method for simultaneous determination of xylazine and 2,6-xylidine in body fluid samples.Methods The samples were extracted by HLB SPE,separated by Waters Atlantis dC18 chromatographiccolumn,and then detected in the MRM scan ion mode under positive ionization condition.Results The recoveryrates of this method were between 70.5％ and 79.8％ with the range of RSD rfrom8.2％ to 10.5％.The limits of detection ofxylazine and 2,6-xylidine in blood and urine samples were 0.4 and 0.3 ng/mL,and the limits of quantitation were 1.2 and 1.0 ng/mL,respectively.Conclusion This method is of high sensitivity,good specificity and good reproducibility,and thus could besuitable for accurate quantification of xylazine in blood and urine samples.

Development of LC-MS/MS assay for simultaneous determination of 25 psychotropic drugs in human blood / 中国法医学杂志

Objective To establish a LC-MS/MS assay for simultaneous determination of 25 psychotropic drugs in human blood and apply the method to analyze the relationship between(driving under the inference of drugs, DUID)and traffic violations. Methods Acetonitrile was added to the sample for precipitating the protein, the mixture was centrifuged for 10min at 4℃ , 14000r/min, the supernatant was filtered, and then the filtrate was analyzed with LC-MS/MS. The separation was performed on C18 chromatography column (50mm×3.0mm, 2.6μm), the mobile phase was consisted of 0.1% formic acid (phase A), acetonitrile:methanol=1:1 (phase B) with gradient elution. Results The calibration curves for 25 psychotropic drugs were linear over the ranges of 0.05~20ng/mL, R=0.9944~0.9996, the assay recoveries were 83.0%~99.7%, the method recoveries were 80.2%~97.4%, the intraday and interday precious were 1.6%~14% and 3.1%~14% respectively. The method was applied to analyze 3140 human samples stored by Center of Forensic Sciences of Hangzhou. Conclusion The method is sensitive and accurate for fast detection of 25 psychotropic drugs in human blood.

Rapid detection of oleandrin and adynerin in human blood by HPLC-MS/MS / 中国法医学杂志

Objective To establish the analytical method of oleandrin and adynerin in human blood by HPLC-MS/MS. Methods After protein sediment by acetonitril, the concentrations of oleandrin and adynerin in human blood were quantitatively determined by HPLC-MS/MS. The qualitative analysis was conducted based on retention time and MRM ions. Besides, the standard curve method was used for quantification. Results The detection limits of both oleandrin and adynerin were 0.5ng/mL, the linear range was from 1ng/mL to 1mg/mL, with a recovery rate of 75.2%~95.7%. Conclusion The detecting protocol has the advantages of high sensitivity, fast and high accuracy with a relatively wide linear range, which is especially suitable for rapid detection of oleander toxins, alexandrine and adynerin in particular, in human blood in poisoning cases.

Rapid screening of 3 synthetic cannabinoids in blood by direct analysis in real time mass spectrometry / 中国法医学杂志

Objective To establish a method for rapid detection of JWH-018, JWH-250 and AM-2201 in blood by direct analysis in real-time coupled with tandem mass spectrometry. Methods These samples were extracted by acetonitrile-methanol (4:1), using DART 12Dip-it automatic sampling system. They were analyzed by positive ion and MRM mode. Results The detection limits of JWH-018, JWH-250 and AM-2201 were in good linearity in the range of 0.02-5.00μg/mL. The correlation coefficients were above 0.99 and the detection limits were 0.016μg/mL, 0.003μg/ mL and 0.017μg/mL, respectively. The intra-day and inter-day RSD were less than 15%. Conclusion The method has the advantages of high sensitivity and good accuracy. The sample processing is simple and can be analyzed in short time. This method is suitable for the analysis of JWH-018, JWH-250 and AM-2201 in practical cases.

Fast determination of aconitine in biological materials by LC-MS/MS / 中国法医学杂志

Objective To establish a HPLC-MS method for determination of aconitum alkaloids in biological samples. Methods The aconitum alkaloids were extracted from the whole blood by using acetonitrile-methanol (5:1 v/v) and then analyzed using HPLC-MS in multiple reaction monitoring (MRM) mode with positive ionization. The analytical column was Agilent Zorbax SB C18 (2.1mm×50mm, 1.8μm)and the mobile phase were water containing 0. 1 % formic acid : acetonitrile (60 : 40 v/v) in isocratic elution. Results The retention time of detection of the aconitine, hypaconitine and mesaconitine were 0.73 min, 0.77 min and 0.63 min, and the precursor product ion combinations of m/z 646.4 → 586.4, 616.1 → 556.5 and 632.4 → 572.1 were used for quantitative analysis, respectively. Calibration curve was linear within the range of 0.1-250 ng/mL with the LOD was 0.1ng/mL, and the coefficient of variation (CV) less than 5.42 % (n=6). The extraction recoveries of aconitine in blood were more than 90 %.Conclusion The results demonstrated that the present method was reliable and robust for natural drugs.

Simultaneous determination of somedon and its metabolites by GC–MS/MS / 中国法医学杂志

Objective To develop a method of support liquid-liquid extraction (SLE) and simultaneous determination of 4 components in somedon and its 8 metabolites by GC–MS/MS. Methods Somedon and its metabolites were extracted by SLE and determined by GC-MS/MS in MRM mode. The qualitative analysis was based on retention time and ratio of ions. The quantitative analysis was based on internal standard method and calibration curve. Results After SLE and determination of 4 components in somedon and its 8 metabolites, the extraction rate were 37.57%~95.87%, the linear range were 0.12μg/mL~16.00μg/mL, the correlation coefficient(r)were 0.989 6~0.999 7, LOD were 0.08ng/mL~14.48ng/mL, the accuracy were 79.63%~122.90%, the interday and intraday precision were 0.99%~7.43% and 2.19%~10.60% respectively. Conclusion Simultaneous determination of somedon and its metabolites by GC–MS/MS in biological samples, which was rapid, simple, accurate and was high precision and recovery, can be used for qualitative and quantitative analysis in forensic cases.

Simultaneous determination of 18 organophosphorous and carbamate pesticides in human plasma by UPLC-MS/ MS / 中国法医学杂志

Objective To develop a method for determination of 18 organophosphorous and carbamate pesticides in human plasma by UPLC-MS/MS. Methods Following deproteinization by acetonitrile, an aliquot of the biological sample was injected into a C18 column(1.7μm 2.1×50mm) using 5mmol/L Ammonium acetate-methanol as the mobile phase with the flow rate of 0.3mL/min, the injection volume was 10μL. Electro spray ionization(ESI) Indicators source was applied and operated in positive ion mode, and multiple reaction monitoring(MRM) mode was used to quantify. Results The limits of detection(LODs) in human plasma ranged from 0.1 to 40ng/mL, and the limits of quantitation(LOQs) ranged from 0.5 to 50ng/mL. An excellent linearity was observed for these LOQs up to 50ng/mL. The average extraction recoveries were with in 64.3%~111.9%, relative standard deviation(RSD) is 3.9%~10.3%. Conclusion This method is specific, sensitive and accurate, and can be used to detect pesticides in forensic.

Study on the analytic method of synthetic cannabinoid JWH-122 by the high performance liquid chromatography / 中国法医学杂志

Objective To establish an effective method for determination of synthetic cannabinoid JWH-122 by the high-performance liquid chromatography (HPLC), which is controled by China’s Regulations on non-clinical narcotic and psychoactive drug. Methods Methanol-deionized water (50%-50%) was used as mobile phase for gradient elution. In addition to the initial concentration in organic phase, gradient steepness, column temperature, flow rate and other chromatographic conditions, the determine wavelengths were tested so as to ifnd out optimal experimental conditions. Linearity range and speciifcity were tested under optimal conditions, and actual samples were used to verify the method established. Results Under the condition of ultraviolet spectrum detection wavelength at 221nm, initial concentration of 70%, organic phase gradient steepness of 0.5%/min, lfow rate at 1.2 ml/min and column temperature of 30℃,excellent linearity of JWH-122 was observed at 0.002mg/mL-0.1mg/mL and the detection limit (S/N≥3) was 0.1μg/mL. The test of actual samples suggested that JWH-122 was able to be well separated from the sample under the optimal conditions. Conclusion Our method has advantages of rapidity, sensitivity, accuracy and excellent separation efifciency, and is capable of the detection of synthetic cannabinoid JWH-122 of the novel “spice” drugs.

Selection of SPE column for trace pesticides in large volume water / 中国法医学杂志

Objective The selection of SPE column to extract trace pesticides in the water. Methods Investigated the recovery of 6 kinds SPE column (including HLB,GDX403,GDX103,C18,C8,C6) to extract phorate, sulfotep, propoxur, carbofuran, cypermethrin, fenvalerate in the water. Results By using oasis HLB cartridge to extract 21 kinds of pesticides in water, the recoveries were over 60%for the most pesticides. Conclusion The method could be applied to the cases which extract and analyse the trace pesticides in large volume water.