| Objective:(1) Determining the concentration of ketamine and norketamine in human urine by ultra fast Liquid chromatography-mass spectrometry (UFLC-MS/MS). To develop a simple, rapid, scientific, effective, specificity, high accuracy and stability UFLC-MS/MS to determin urine ketamine and norketamine simultaneously, and establish mean concentration-time curve model of urine ketamine and norketamine of ketamine abusers during drug withdraw, then acquire the curve model equation.(2) Experimental research on drivers’psychology under the effect ketamine effect by PsyTech psychological experiment system. To develop perfect psychological experiments to research on drivers’psychological characteristics under ketamine effect by testing the reaction time, driving speed perception and attention concentration with PsyTech psychological experiment system. To research on ketamine and norketamine concentration effects on driver’s psychological characteristics.Methods:(1) Determining the concentration of ketamine and norketamine in human urine: ①Urine sample preparation:To a 1 mL aliquot of urine sample in 100 μL of internal standard (IS) (100.Ong/mL KET-D4),2.0 mL sodium borate buffer (0.01 mol/L) and 3 mL ethyl acetate were added. The mixture was vortex-mixed thoroughly for 30 s and then centrifuged at 5000 rpm for 15 min. Take supernatant into tube, drying with nitrogen blow, dissolved residue with 100 μL mobile phase (acetonitrile:0.1% formic acid+20 mol/L ammonium acetate solution= 75:25 (v/v)). The mixture was vortex-mixed thoroughly for 30 s and then injected 5L.L into the UFLC-MS/MS system. ②Mass spectrometry:Detection was performed on a Shimadzu LCMS-8040 Mass Spectrometer System (Kyoto, Japan) equipped with a Turbo lonspray interface. Mass spectrometer settings in positive-ion mode (ESI+), produce ions [M+H]+, m/z 238.20, m/z 224.25 and m/z 242.30, respectively. Selective targeting molecular ion [M+H]+ product ion scan, obtain ketamine the main pieces of the m/z 125.25 and m/z 207.25,norketamine the main pieces of the m/z 125.25 and m/z 179.30 to qualitative analysis. Quantification was performed using multiple reaction monitoring (MRM) of the m/z 207.25 for ketamine and m/z 179.30 norketamine. Data acquisition and processing were performed with the Analyst software. ③ Liquid chromatography:Chromatographic separation was performed on an Agilent RRLC online filter (Agilent, Germany).A Shim-pack FC-ODS ultra fast chromatographic column (150 mm x 2 mm I.D.3 um) was employed and maintained at 40 ℃.The mobile phase consisted of 20 mmol/L ammonium acetate (75:25, v/v) an isocratic flow rate of 0.3 mL/min. The injection volume was 5μL.④Subjects selected and sample collected:The study sample consisted of 22 ketamine abusers of randomly selected voluntary detoxification institutions in Guangzhou. Ketamine abusers were required to sign an informed consent form and a questionnaire. The nurse was responsible for collecting clean-caught midstream urine 10 mL at nine o ’clock every morning and stored at-20 ℃ for further analysis. The urine collection time was controlled by the doctors and nurses. ⑤Sample analysis:Unknown urine samples were processed according to urine sample preparations. The standard curve was established every day with quality control samples. The same batch of the quality control samples and unknown urine samples were calculated by the stand curve. The quality control samples should be in the range of ±15%.⑥Data analysis:Listing and drawing the mean urine concentration and time. Then setting up in ketamine and norketamine mean concentration-time curve model by SPSS 17.0 statistical analysis software and calculating curve model equations.(2) Experimental research on driver’s psychological characteristics under the effect of ketamine: ①Subjects selection and psychological test:Regarding ketamine abusers as experimental group, the others as control group. To test and collect data with the driver’s psychological characteristics at 10 a.m, including reaction time, driving speed perception and attention concentration. ②Experiment content:To test and collect data of reaction time, driving speed perception and attention concentration. Reaction time:including simple reaction time (visual simple reaction time and auditory simple reaction time), choice reaction time (visual choice reaction time and auditory choice reaction time), visual reaction time with foot and auditory reaction time with foot. ③Test methods:The testers were delegated after accepting the operating training of PsyTech-EP 2009 psychological experiment system. The tests of driver’s psychological characteristics were face to face in experimental group and control group. ④Simple reaction time test:Visual simple reaction time:Experiment material was a red solid circle, when there wae a red circle on the computer screen, the red reaction key on the second reaction box test would be pressed by subjects, repeat test for 20 times. Auditory simple reaction time: Experiment material is 750 HZ pure tone, when this voice was heard; the red reaction key on the first reaction box would be pressed by test subjects. ⑤Choice reaction time test:Visual choice reaction time:Experiment materials were four different kinds of color solid circles, the color includes red, yellow, green and blue, these may show up randomly. According to the different color, subiect would press the corresponding color key on the first reaction box, repeat test for 20 times. Auditory choice reaction time:Experiment material was three different frequency of pure tone, the treble is 2000 HZ, the tenor is 750 HZ and the bass is 350 HZ, these may show up randomly. According to the different frequency, subjects would press the corresponding key on the second reaction box, repeat test for 30 times. The treble pressed key of plus sign, the tenor pressed key of equal sign and the bass pressed key of minus sign. ⑥Visual reaction time with foot test:Visual reaction time with foot test with the experiment of light reaction with foot. Experiment material was a red solid circle, when there is the red circle on the computer screen, experimental subjects should make a reaction on the foot pedal as soon as possible, repeat testing 10 times. ⑦Auditory reaction time with foot test:Auditory reaction time with foot test with the experiment acoustic response with foot. Experiment material is 750 HZ pure tone, subjects should make a reaction on the foot pedal as soon as possible, repeat testing 10 times. ⑧Speed perceptual test:Speed perceptual test is expressed in absolute error. Subject is estimated the red highlights to the finish time, which is speeding two units per second. After pressed any key on the first reaction box, the error distance of judgment will be presented to adjust the estimated time in the next operation, repeat testing 30 times. ⑨Attention concentration test:Attention concentration test with cancellation test, represented with the work efficiency. Record the number of total letter, cancellation letter of "B", differentiating letter of "B" and the missing letter of "B". ⑩Analyze methods:Overall comparison of the experimental group and control group with two factors repeated measures, comparison between two groups was done with single effect analysis, single factor repeated measures and comparative t-test is used to compare with the difference in the each group. Listing and drawing the mean urine concentration data and each psychological indicator. Then setting up in ketamine and norketamine mean concentration- psychological curve model by SPSS 17.0 statistical analysis software and calculating curve model equations.Results:(1) Quantitative determination of ketamine and norketamine in human urine: The linear calibration curves were obtained in the concentration range of 0.1-1000.0 ng/mL, the detection limit of 0.05ng/mL and the quantitative limit of 0.1 ng/mL for ketamine and norketamine. The intra-day precision values (RSD) were 3.715% and 1.457%,the inter-day precision values (RSD) were 4.873%and 1.813%,the recovery was 98.071-100.197% and the matrix interference was-2.425%--0.437% of ketamine at two concentration levels between 10.0ng/mL and 500.0ng/mL. The intra-day precision values (RSD) were 4.041% and 0.787%,the inter-day precision values (RSD) were 4.749% and 1.788%,the recovery was 98.419%～102.873% and the matrix interference was-3.322%～2.276% of norketamine at two concentration levels between lO.Ong/mL and 500.0ng/mL. Mean concentration-time curve model equations:y,=2331.899(x1-2247) and y2=2812.051 (x2-1.938), y, and y2is representative of the ketamine and norketamine in urine, unit is ng/mL, x1 and x2 is representative of the withdrawal days, unit is day.The concentration in the urine is inversely proportional to time.(2) Experimental research on driver’s psychological characteristics under the effect of ketamine:There were significant differences between the two groups in each factor of driver’s psychological characteristics, the work efficiency of experimental group were significantly higher in control group, others were significantly lower, apart from work efficiency, there were increased during the time. Mean urine concentration-visual simple reaction time curve model equations: y,=222.505+44.3031nx, and y2= 143.880+55.6961nx2,mean urine concentration-auditory simple reaction time curve model equations:y3=764.548-2444.982/x3 and y4=772.264-6358.084/x4, mean urine concentration-visual choice reaction time curve model equations:y5=309.482+64.9101nx5 and y6=196.339+81.0071nx6, mean urine concentration-auditory choice reaction time curve model equations: y7=784.455-1670.499/x7 and y8=787.830-4272.416/x8, mean urine concentration-visual reaction time with foot curve model equations:y9=347.973+17.4261nx9 and yio=317.675+21.7291nx10, mean urine concentration- auditory reaction time with foot curve model equations:y11=320.435+32.8341nx11 and y12=265.521+40.4291nx12,mean urine concentration- absolute error curve model equations:y13=e6.0117-3104/x13 and y14=e6.114-7.578/x14, mean urine concentration- work efficiency curve model equations: y15=691.228x15-0.107 and y16=819.450x16-0.129, y1-y14 were visual simple reaction time, auditory simple reaction time, visual choice reaction time, auditory choice reaction time, visual reaction time with foot, auditory reaction time with foot and absolute error, respectively, y15 and y,6 were work efficiency. In addition,z1,x3,x5,x7,x9,X11,x13 and x15 were mean urine concentration of ketamine, x2,X4,x6,x8,x10,x12,x14 and x16 were mean urine concentration of norketamine.The highest ketamine’s concentration is 4.752 ng/mL and highest norketamine’s is 14.191 ng/mL of normal visual simple reaction time, the recovery time of visual simple reaction time is 16 days. The highest ketamine’s concentration is 6.640 ng/mL and highest norketamine’s is 16.911 ng/mL of normal auditory simple reaction time, the revecory time of auditory simple reaction time is 14 days. The highest ketamine’s concentration is 34.444 ng/mL and highest norketamine’s concentration is 68.905 ng/mL of normal visual choice reaction time, the revecory time is 7 days. The highest ketamine’s concentration is 7.908 ng/mL and highest norketamine’s concentration is 19.907 ng/mL of normal auditory choice reaction time, the revecory time is 13 days. The highest ketamine’s concentration is 5.654 ng/mL and highest norketamine’s concentration is 16.177 ng/mL of normal visual reaction time with foot,the revecory time is 15 days. The highest ketamine’s concentration is 10.166 ng/mL and highest norketamine’s concentration is 25.577 ng/mL of normal auditory reaction time with foot, the revecory time is 11 days. The highest ketamine’s concentration is 7.388 ng/mL and highest norketamine’s concentration is 18.041 ng/mL of normal absolute error, the revecory time is 14 days. The highest ketamine’s concentration is 5.096 ng/mL and highest norketamine’s concentration is 14.438 ng/mL of normal work efficiency, the revecory time is 15 days.Conclusion:The methods were proved to be simple, rapid, scientific, effective, high accuracy, stability and have been successfully utilized for the quantitative determination of ketamine and norketamine in human urine and evaluation of the Residue dynamics of ketamine and norketamine. The psychological experiments for testing the reaction time, driving speed perception and attention concentration by PsyTech psychological experiment system have been successfully utilized for the evaluation of researching on driver’s psychological characteristics under ketamine effect.(1) Studies have shown that the UFLC-MS/MS method for the determination of ketamine and norketamine in human urine has been a satisfactory outcome. First, the efficiency and separation speed has been further improved with Shim-pack FC-ODS ultra fast chromatographic column. Each sample needs only 5 min for simultaneous determination of urine ketamine and norketamine, sample throughput was greatly improved per day. MRM technology can simultaneously detect the parent ion and the product ion. The parent ion was collided and the product ion was selected to monitor. The background noise was significantly reduces and the degree of specificity and sensitivity was high. The lower limit of quantification for ketamine and norketamine were 0.10ng/mL. Furthermore, internal standard is ketamine deuterated can enhance the stability in the process of quantitative detection. In addition, sample preparation was liquid-liquid extraction. This method was simple, fast, economic and satisfactory the needs of high-throughput. According to the results, the concentration of ketamine and norketamine in urine is directly proportional to the abstinent time. The norketamine residues after the ketamine is cleaned.Pay attention to the analysis of metabolites when detections testing the ketamine abusers.(2) Results have shown the satisfatory result of the PsyTech psychological experiment for testing the driver’s psychological characteristics includes reaction time, driving speed perception and attention concentration. Firstly, experimenter is sufficiently trained and tested to ensure the quality of the experiment. Secondly, sufficient exercise has been done before collecting data. The unique name of the subjects ensured that the experimental data are reliable. Besides, the drivers’ psychological characteristics have been widely used to research on the effect of alcohol on the drivers. The method meets the demand of the experiment and has been successfully applied to research on the driver’s psychological characteristics under the ketamine effect. The results show that the longer reaction time of light and notice, the poorer accuracy of speed perception and the lower level of attention concentration under ketamine effect, and the higher concentration the stronger effect.Research shows that the accident repeaters’ reaction time is longer than safe drivers, the mean absolute error of driving speed perception is bigger than safe drivers, the attention concentration is lower than safe drivers. At the same time, the results also show that the visual choice reaction time can be returned to normal level when the ketamine is uncleaned. The auditory reaction time with foot can returned to normal level when the norketamine is cleaned and ketamine is cleaned. The simple visual reaction time, auditory simple reaction time, auditory choice reaction time, visual reaction time with foot,accuracy of speed estimation and attention concentration can not be returned to normal level,although,ketamine and norketamine is cleaned,all. In summary, the drivers are likely to traffic accident occurs under ketamine, the more druging up, the easier it is to happen.After ketamine and norketamine was cleared within three days, driving behavior will be affected. |
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