盐酸右美托咪定在肥胖患者体内的临床药代动力学研究

发布时间：2018-05-10 06:31

本文选题：盐酸右美托咪定 + 肥胖患者　；参考：《南方医科大学》2014年硕士论文

【摘要】：盐酸右美托咪定(Dexmedetomidine hydrochloride, Dex)化学名：(R)-4-[1-(2,3-二甲苯基)乙基]-1H-咪唑单盐酸盐,最早于2000年3月在美国上市,2008年进入中国市场,作为一种高选择性的α2-肾上腺素受体激动剂,其亲和力比可乐定高8倍。因具有抑制交感神经活性、镇静催眠、抗焦虑和镇痛等作用,且不良反应少,已广泛应用于围术期镇静、镇痛等领域[1]。关于该药血药浓度的测定方法国内鲜有报道[2-3],国外有采用气相色谱串联质谱法[4]、高效液相色谱串联质谱法(High performance liquid chromatography tandem mass spectrometry, HPLC-MS/MS)[5-7]、放射法[8]等,此类方法能检测的最低浓度为5ng-L-1。样品前处理方法有蛋白沉淀法[2]、液液萃取法[3、7]和固相萃取法[5]等。本试验在此基础上采用Agilent6410建立并验证了样品经液液萃取后测定人血浆样品中Dex浓度的HPLC-MS/MS法。肥胖患者每千克体质量肌肉组织更少,脂肪组织更多。一般情况流入脂肪组织的血流量较少,约占心输出量的5%,而流入内脏的血流则占73%,肌肉为22%[9],如果按照总体重来增加药物的剂量将会导致某些组织灌注血液中药物浓度增加,最终导致血浆药物相对过剩,不良反应增加。到目前为止,尚未见有关Dex在全麻肥胖患者体内的药代动力学特征,因此我们将探索出来的HPLC-MS/MS法用于检测全麻肥胖患者血浆中Dex的浓度,阐明Dex静脉泵注后在肥胖患者体内的药代动力学特性,为临床研究和更合理应用Dex提供参考。目的一、建立HPLC-MS/MS测定人血浆Dex浓度；二、全麻状态下肥胖患者Dex的临床药代动力学特征。方法一、建立HPLC-MS/MS法测定人血浆Dex浓度： ①质谱分析：待测物Dex和内标替米沙坦在正离子检测方式下,采用电喷雾离子源(ESI源),分子扫描分别为m/z201.1和m/z512.2。对准分子离子选择性地进行产物离子全扫描分析,获得Dex的主要碎片为m/z95.1,响应值较高,且比较稳定,背景噪音小,因此被选做多反应监测模式(MRM)定量分析的产物离子,用于定量分析。替米沙坦生产的主要产物碎片离子为m/z276.1,将其作为定量分析时监测的产物离子。探索出来的质谱QQQ条件为：毛细管电压4000V,喷雾气压力45psi,干燥气体流速10L·min-1,干燥气体温度350℃。 ②色谱条件：色谱柱选用美国Agilent Eclipse Plus C18(4.6mm×150mm,3.5μm)；流动相为甲醇：1%甲酸水(75：25,V/V)；流速为0.5ml·min-1;柱温为35℃；进样量为2μ1。 ③血浆样品的处理方法：精密加入血浆样品200μ1置于10ml的玻璃离心管中,加入640ng·ml-1的替米沙坦甲醇溶液10μ1,加0.2mo1·L-1氢氧化钠溶液100μ1,快速振荡1分钟,再加入乙酸乙酯：二氯甲烷(4：1液)1ml加盖密封,旋涡振荡2分钟,2800r·min-1离心10分钟,吸取有机层800μ1置锥形玻璃离心管内,30℃水浴锅氮气挥干。进样前加入残渣溶解液(甲醇：水=80：20)100μl,快速振荡30秒使残渣溶解,吸到1.5ml离心管中,20000r·min-1离心5分钟,吸取上清液2μ1进样。 ④标准曲线：分别配置Dex浓度为0、20、50、100、200、500、1000、2000、4000和6000ng·L-1的血浆样品,按照“血浆样品的处理方法”,经HPLC-MS/MS分析,以Dex峰面积与内标峰面积之比(Y)为纵坐标,血浆中Dex浓度与内标浓度之比(X)为横坐标,得出20-6000ng·L-1浓度范围内的标准曲线。 ⑤方法确证：包括专属性、标准曲线、定量下限、精密度、回收率、介质效应和稳定性研究。专属性：对于色谱法至少要考察6个不同个体的空白生物样本色谱图、空白生物样品外加对照物色谱图(注明浓度)及用药后的生物样品色谱图。标准曲线：一般用回归分析法(如用加权最小二乘法等)所得的回归方程来评价,必须至少用6个浓度建立标准曲线,定量范围要能覆盖全部待测的生物样品浓度范围,不得用定量范围外推的方法求算未知样品的浓度。定量下限：应能满足测定经3-5个消除半衰期时样品中的药物浓度或能检测出Cmax的1/10-1/20的药物浓度范围内,相对标准差(RSD)应小于20%,其准确度应在真实浓度的80%-120%,至少应由5个标准样品测试结果证明；精密度：分别配制低(50ng·L-1)、中(500ng·L-1)和高浓度(4000ng·L-1)的Dex血浆质控样本各15份,分为3批,每批5份,并与每批的标准曲线同时进行,计算质控样品的测得浓度,与配制浓度对照,求得本法的精密度；回收率：空白血浆除了不加内标外,经“血浆样品的处理方法”处理后,再加入相应浓度的Dex和替米沙坦,以每一浓度两种处理方法的峰面积比值计算提取回收率,采用1天的数据,将Dex峰面积和内标峰面积的比值带入随行标准曲线,计算所得浓度和加入浓度比值求得方法回收率；取9管空白血浆各200μl平分为三组,每组3管,除不加标准系列溶液和内标外,经“血浆样品的处理方法”处理后,于残渣中加入残渣溶解液80μl后,分别加入低(1μg·L-1)、中(10μg·L-1)、高浓度(80μg.L-1)的Dex溶液和内标替米沙坦(640ng·ml-1)各10μ1,以此种处理方法测得的Dex和内标峰面积与空白试管中加入80μl残渣溶解液后,再加入相应浓度的Dex和内标各10μ1混匀后测得的峰面积的比值计算百分比,即得介质效应；分别观察了Dex残渣溶解液在-20℃避光放置7天和14天、低中高(50ng·L-1、500ng·L-1、4000ng·L-1)质控血浆样品复溶后室温避光放置4小时、低中高质控血浆样品冻融1次、3次,-20℃下冻存3天和15天的稳定性。二、全麻肥胖患者Dex临床药代动力学 ①受试者选择：本试验经广州军区广州总医院伦理委员会批准,并在ClinicalTrials.gov注册(NCT01864187)。选择肥胖患者8名,年龄18-64岁,BMI-28kg·m-2,签署知情同意书,于试验前在医院接受全面的体格检查,对肝、肾功能进行化验测定,并进行心电图检查,符合条件者入选。受试者无既往病史,平时很少服药,不嗜烟酒,试验前14天内未用任何药物。 ②给药方案及血样采集：入选8名肥胖患者,入室后连接MP30(PHILIPS公司)监护仪监测脉搏血样饱和度(Sp02)、心电图(ECG)、无创血压(NBP)、呼气末二氧化碳分压(PETCO2)。开放外周静脉,面罩给氧去氮,分别采用Marsh、Minto模式静脉靶控输注丙泊酚2-3μg·ml-1、瑞芬太尼3-4ng·m1-1,待意识消失后静脉注射顺式阿曲库铵0.15mg·kg-1麻醉诱导,待肌肉松弛后气管插管,再行颈内中心静脉和足背动脉穿刺。经颈内静脉泵入1μg·kg-1Dex10分钟泵完。分别于注射前(Base)和注射后0、5、10、15、20、25、30、45、60、90、120、180、240、360、480分钟于足背动脉采血5ml,采集的血样置于肝素化的试管中,2小时内3000r·min-1离心10分钟,分离血浆置-20℃储藏,15天内采用已探出的方法进行血药浓度检测。 ③血浆样品分析：未知血浆样品的测定按“血浆样品的处理方法”项操作,每批建立一条标准曲线,同时分析低中高(50ng·L-1、500ng·L-1、4000ng·L-1,各两个样本)的质控样本,并以同批次的标准曲线计算各个时间点样本中Dex和质控样本的浓度,根据质控样本的结果决定当天数据的取舍,质控样本的相对偏差应控制在±15%范围之内。 ④数据处理：将各受试者不同时间点样品的血药浓度数据列表,并计算其平均值与标准差,绘制Dex药物浓度-时间曲线图；然后以个体药代动力学软件DAS2.1.1对每个受试者的血药浓度数据进行处理,计算主要的药动学参数[血药浓度-时间曲线下面积(AUC)、血药峰浓度(Cmax)、分布半衰期(t1/2α)、消除半衰期(tl/2β)、表观分布容积(VI)、清除率(CLz)、平均驻留时间(MRT)],并求出参数的平均值和标准差。t1/2按t1/2=0.693/ke求得；ke是由对数血浆药物浓度-时间曲线末端直线部分的斜率求得。结果一、HPLC-MS/MS测定人血浆Dex浓度 Dex在20-6000ng·L-1范围内线性关系良好(r2=0.9951),最低定量下限为20ng.L-1,提取回收率为82.20%-96.37%,方法回收率97.18%-103.63%,批内精密度为3.26%-8.74%,批间精密度为8.24%-10.11%。介质效应百分比为99.13%-105.27%,可是为无介质效应影响。Dex残渣溶解液在-20℃避光放置7天和14天、低中高(50ng·L-1、500ng·L-1、4000ng·L-1)质控血浆样品复溶后室温避光放置4h、低中高质控血浆样品冻融1次、3次,-20℃下冻存3天和15天的稳定性RSD均小于15%。二、全麻肥胖患者Dex临床药代动力学入组临床8名全麻肥胖患者,采集血液Dex浓度检测样本共计128份。全麻肥胖患者药代动力学过程符合二房室模型,主要的药代动力学参数为：AUC(o-t)123.27μg·L-1-min, AUC(0-∞)138.63μg·L-1·min, t1/2α2.49min, t1/2β163.41min, V1162.96L, CLz40.20L·min-1, MRT(0-t)152.06min。结果表明,本试验研究出来的HPLC-MS/MS法测定人血浆中Dex已取得令人满意的结果。首先,测定血浆中Dex及替米沙坦,每个样品的检测时间仅需3.0分钟和3.6分钟,使得样品通过量得到实质性的提高,有利于后期药代动力学研究的进行；其次MRM技术能够同时检测到母离子和具有特征反应的子离子,母离子被碰撞破裂后,特定的子离子被选择性检测到；方法具有高度的专属性和灵敏度,因而显著降低了背景噪音。此外试验中对样品前处理为液液萃取法,该方法简便、快捷、经济符合药动学高通量的需求,而且防止了内源性物质在色谱柱上的残留,延长了色谱柱使用寿命。结论本文所建立的HPLC-MS/MS分析方法,具有专属性强、灵敏度高、检测时间快速的优点,适用于人血浆中Dex的浓度测定,并成功应用于药物动力学研究。所算出的肥胖患者Dex药代动力学参数符合二房室模型,但值得注意的是肥胖患者总体重中肌肉组织和水分比例小于同龄同性别的正常体质量患者,但脂肪组织比例却增大,且心排出量主要运送至非脂肪组织[10],因此按照患者实际体重给药,可能会导致药物相对过量,造成药物作用时间延长[11],说明用药时必须考虑患者体重因素并相应地减少药物用量,以减少不良反应和防止药物蓄积。因此在临床麻醉中,为了防止和避免药物带来的问题,肥胖患者药物用量应相对减少,这样无论实际工作还是在药品应用经济学上均具有重要意义,对肥胖患者更科学更合理。肥胖患者的剂量标准应该根据肥胖患者身体组成和血流的相应变化来确定。
[Abstract]:Dexmedetomidine hydrochloride (Dex) chemical name: (R) -4-[1- (2,3- dimethylbenzene) ethyl]-1H- imidazole mononsalt. It was first listed in the United States in March 2000 and entered the Chinese market in 2008. As a highly selective alpha adrenaline receptor agonist, its affinity is 8 times higher than that of clonidine. Sensory neuroactivity, sedative hypnotism, anti anxiety and analgesic effect, and less adverse reactions, it has been widely used in the perioperative sedative, analgesic and other areas of [1]. on the determination of blood concentration in the drug [2-3], foreign gas chromatography tandem mass spectrometry ([4]), high effect liquid chromatography tandem mass spectrometry (High performance liquid Chr) Omatography tandem mass spectrometry, HPLC-MS/MS) [5-7], radioactivity [8] and so on. The lowest concentration of such methods is 5ng-L-1. sample pretreatment methods such as protein precipitation method [2], liquid liquid extraction [3,7] and solid-phase extraction [5], etc. based on this experiment, Agilent6410 was established and verified by liquid liquid extraction. The HPLC-MS/MS method of Dex concentration in plasma samples. Obese patients have less mass of muscle tissue per kilogram and more fat tissue. In general, the flow of blood flow into adipose tissue is less, about 5% of the cardiac output, while the flow of inflow to the viscera is 73%, and the muscle is 22%[9]. If the dosage of the drug is increased according to the overall weight, it will lead to some groups. The increase of drug concentration in the blood of the woven blood, which eventually leads to the relative excess of plasma drugs and the increase of adverse reactions. Up to now, the pharmacokinetic characteristics of Dex in general anesthesia patients have not been seen, so we will explore the HPLC-MS/MS method to detect the concentration of Dex in the plasma of obese patients with general anesthesia and clarify the Dex intravenous pump after infusion. The pharmacokinetic characteristics in obese patients provide a reference for clinical research and more rational application of Dex.
objective
First, the concentration of Dex in human plasma was determined by HPLC-MS/MS.
Two, the clinical pharmacokinetic characteristics of Dex in obese patients under general anesthesia.
First, the HPLC-MS/MS method was established to determine the concentration of Dex in human plasma:
(1) mass spectrometric analysis: under positive ion detection, Dex and internal standard telmisartan are detected by the electrospray ion source (ESI source), and the molecular ions are selected by m/z201.1 and m/z512.2., respectively. The main fragments of Dex are m /z95.1, the response value is higher, and the background noise is small. The background noise is small. Therefore, the product ions of the quantitative analysis of the multi reaction monitoring model (MRM) were selected to be used for quantitative analysis. The main product fragments produced by Telmisartan were m/z276.1, which was used as the product ion for monitoring the quantitative analysis. The QQQ conditions were explored by capillary electropressure 4000V, spray gas pressure 45psi, and the flow velocity of 10L. Min in the dry gas. -1, the temperature of the dry gas is 350 degrees centigrade.
(2) chromatographic conditions: Agilent Eclipse Plus C18 (4.6mm x 150mm, 3.5 mu m), and mobile phase of methanol: 1% formate water (75:25, V/V); flow rate of 0.5ml. Min-1; the column temperature is 35 C; the sample volume is 2 1..
(3) the treatment of plasma samples: the precise addition of 200 to 1 in the plasma sample is placed in the glass centrifuge tube of 10ml, adding 640ng / ml-1's telmisartan methanol solution 10 mu 1, and 0.2mo1 L-1 sodium hydroxide solution 100 mu 1, rapidly oscillating for 1 minutes, and then adding ethyl acetate: dichloromethane (4:1 liquid) 1ml seal, vortex oscillation 2 minutes, 2800r min-1 In the centrifuge for 10 minutes, the organic layer 800 mu 1 taper glass centrifuge tube was absorbed, and the nitrogen in the water bath was dried at 30 C. The residue dissolved (methanol: water =80:20) was added to the sample before entering the sample. The residue was dissolved in 30 seconds quickly, sucked into the 1.5ml centrifuge tube, 20000r. Min-1 was centrifuged for 5 minutes, and the supernatant was 2 mu 1.
(4) standard curve: the plasma samples with Dex concentration of 0,20,50100200500100020004000 and 6000ng. L-1 were arranged in accordance with "the treatment method of plasma samples". By HPLC-MS/MS analysis, the ratio of Dex peak area to the internal standard peak area (Y) was the longitudinal coordinate, and the ratio of Dex concentration to the internal standard concentration (X) in plasma was the transverse coordinates, and 20-6000ng. L-1 concentration was obtained. A standard curve within a degree range.
Method confirmation: including specificity, standard curve, quantitative lower limit, precision, recovery, medium effect and stability. Specific properties: at least 6 different individuals' blank biological sample chromatograms, blank biological samples and control chromatograms (marked concentration) and biological sample chromatograms after drug use should be examined by chromatography. Curve: generally, the regression analysis method (such as weighted least square method) is used to evaluate the regression equation. It is necessary to establish a standard curve with at least 6 concentrations. The quantitative range should cover the concentration range of all the biological samples to be measured, and the concentration of the unknown sample can not be calculated by the method of extrapolation from the quantitative range. The relative standard deviation (RSD) should be less than 20% within the concentration range of the drug concentration or the detection of Cmax 1/10-1/20 in the 3-5 half-life periods. The accuracy should be at the true concentration of 80%-120%, at least by the test results of 5 standard samples; precision: low (50NG. L-1), medium (500ng. L-1) and Gao Nong, respectively. The plasma quality control samples of Dex (4000ng. L-1) were divided into 15 samples, divided into 3 batches, 5 copies per batch, and simultaneously carried out with the standard curves of each batch, calculated the measured concentration of the quality control samples, compared with the preparation concentration, and obtained the precision of the method; the recovery rate: the blank plasma was treated with the "plasma sample treatment" and then added to the phase after the treatment was not added to the internal standard. The concentration of Dex and telmisartan were calculated, and the recovery rate was calculated with the ratio of peak area to two treatments per concentration. The ratio of Dex peak area and internal standard peak area was taken into the standard curve with the ratio of Dex peak area to the internal standard peak area. The ratio of the concentration and concentration ratio was calculated and the recovery rate was calculated. The 200 micron l of 9 tubes of blank plasma was divided into three groups. Each group of 3 tubes, with the exception of the standard series solution and the internal standard, after the treatment of the "plasma sample treatment", after the residue is added to the residue dissolved in 80 mu L, adding low (1 mu g. L-1), medium (10, G. L-1), high concentration (80 u g.L-1) Dex solution and internal standard rice Chatain (640ng. ML-1) each 10 mu 1, as measured by the method of Dex and internal standard. After adding 80 l residue dissolved in the peak area and blank test tube, the percentage calculated by the ratio of the ratio of the corresponding concentration of Dex and the internal standard 10 mu 1, that is, the ratio of the ratio of the ratio of the peak area, that is, the medium effect is obtained, and the plasma samples of the Dex residue dissolved in -20 C for 7 and 14 days, and the low middle height (50NG. L-1500ng. L-14000ng. L-1) plasma samples are observed. After the products were redissolved, they were stored at room temperature for 4 hours. Low, medium and high quality plasma samples were freeze-thaw 1 times, 3 times, and stored at -20 C for 3 days and 15 days.
Two, the clinical pharmacokinetics of Dex in general anesthesia patients with obesity
The subjects were selected: the test was approved by the ethics committee of Genenral Hospital of PLA Guangzhou Military Area and registered in ClinicalTrials.gov (NCT01864187). 8 obese patients, aged 18-64 years old, BMI-28kg m-2, signed informed consent, received a comprehensive physical examination in the hospital before the trial, tested the liver and kidney function, and carried out the electrocardiogram. The subjects were selected without any previous medical history. They seldom took medicine and were not addicted to alcohol and tobacco. They did not use any drugs within 14 days before the test.
(2) drug delivery scheme and blood sample collection: 8 obese patients were selected, and the pulse blood sample saturation (Sp02), electrocardiogram (ECG), non-invasive blood pressure (NBP) and end expiratory carbon dioxide partial pressure (PETCO2) were monitored by MP30 (PHILIPS) monitor after admission to the room. The external peripheral vein was open and the mask was given to oxygen to nitrogen, and the intravenous target controlled infusion of propofol was 2-3 by Marsh and Minto mode respectively. G / ml-1 and remifentanil 3-4ng m1-1 were induced by intravenous injection of CIS CIS atracurium 0.15mg. Kg-1 after the consciousness disappeared. After the muscle relaxation, the endotracheal intubation and the puncture of the internal jugular central vein and the dorsal foot artery were performed. The pump was pumped through the internal jugular vein for 1 mu g. Kg-1Dex10 minutes before injection (Base) and 0,5,10,15,20,25,30,45,60,90,12 after the injection, respectively. The blood samples collected from the dorsum of the foot were 5ml in 0180240360480 minutes. The blood samples were collected in the heparinated test tube, 3000r min-1 was centrifuged for 10 minutes in 2 hours. The separated plasma was stored at -20 C, and the blood concentration was detected by the method that had been detected within 15 days.
(3) analysis of plasma samples: the determination of the unknown plasma samples was operated according to the "treatment method of plasma samples". A standard curve was established in each batch. The quality control samples of low middle and high (50NG. L-1500ng. L-14000ng. L-1, two samples) were analyzed, and the concentration of Dex and quality control samples in each time point sample was calculated with the standard curve of the same batch. According to the result of the quality control sample, the trade-off between the date and the quality control sample should be determined. The relative deviation of the quality control sample should be within the range of + 15%.
(4) data processing: to list the blood drug concentration data of the samples at different time points of the subjects and calculate the average and standard deviation, draw the Dex drug concentration time curve, and then use the individual pharmacokinetic software DAS2.1.1 to process the blood concentration data of each subject and calculate the main pharmacokinetic parameters [blood drug concentration - time]. The area under the curve (AUC), the concentration of the blood drug peak (Cmax), the half life (t1/2 alpha), the elimination half life (tl/2 beta), the apparent distribution volume (VI), the clearance rate (CLz), the average resident time (MRT)], and the average value of the parameters and the standard deviation.T1/2 according to the t1/2= 0.693/ke; Ke is the slope of the end straight part of the logarithmic plasma drug concentration time curve. Get it.
Result
1. HPLC-MS/MS determination of Dex concentration in human plasma
Dex has a good linear relationship (r2=0.9951) in the range of 20-6000ng L-1. The lowest quantitative lower limit is 20ng.L-1, the recovery rate is 82.20%-96.37%, the recovery rate is 97.18%-103.63%, the intra batch precision is 3.26%-8.74%, the percentage of the inter batch precision is 99.13%-105.27%, but it dissolves.Dex residue for the non medium effect. The liquid was placed at -20 C for 7 days and 14 days. The plasma samples of low medium high (50NG. L-1500ng. L-14000ng. L-1) were dissolved at room temperature and placed 4H at room temperature. The low medium high quality plasma samples were frozen thawing 1 times, 3 times, and the stability of the frozen storage for 3 days and 15 days at -20 C was less than 15%..
Two, the clinical pharmacokinetics of Dex in general anesthesia patients with obesity
In the group of 8 general anesthesia patients, 128 samples of blood Dex concentration were collected. The pharmacokinetics of general anesthesia patients were in accordance with the two atrioventricular model. The main pharmacokinetic parameters were: AUC (O-T) 123.27 mu g. L-1-min, AUC (0- infinity) 138.63 u g. L-1 min, T1 /2. RT (0-t) 152.06min.
The results show that the HPLC-MS/MS method for the determination of Dex in human plasma has achieved satisfactory results. First, the determination of Dex and telmisartan in plasma is only 3 minutes and 3.6 minutes for each sample, which makes the sample increase substantially, and is beneficial to the study of pharmacokinetics in the later period; secondly, M The RM technology can simultaneously detect the mother ion and the sub ion with the characteristic reaction. After the parent ion is collided and ruptured, the specific subions are selectively detected. The method has a high specificity and sensitivity, thus significantly reducing the background noise. In addition, the sample is treated with liquid extraction in the front of the sample. The method is simple, quick, and fast. It meets the high demand of pharmacokinetics and prevents the residue of endogenous substances on the chromatographic column and prolongs the service life of the column.
The HPLC-MS/MS analysis method established in this paper has the advantages of strong specificity, high sensitivity and rapid detection time. It is suitable for the determination of Dex concentration in human plasma and successfully applied to the study of pharmacokinetics. The pharmacokinetic parameters of Dex in obese patients are in accordance with the two atrioventricular model, but it is worth noting that the total weight of the obese patients is in the median muscle. The proportion of meat tissue and water is less than that of normal body mass in the same age, but the proportion of adipose tissue increases, and the amount of cardiac output is mainly transported to the non fat tissue [10]. Therefore, the drug may lead to a relatively excessive drug in accordance with the actual weight of the patient and cause the drug.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R614

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