CN110806449B - Method for simultaneously detecting content of clomipramine and N-norclomipramine in blood - Google Patents

Method for simultaneously detecting content of clomipramine and N-norclomipramine in blood Download PDF

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CN110806449B
CN110806449B CN201911206887.9A CN201911206887A CN110806449B CN 110806449 B CN110806449 B CN 110806449B CN 201911206887 A CN201911206887 A CN 201911206887A CN 110806449 B CN110806449 B CN 110806449B
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CN110806449A (en
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张国成
贾永娟
倪君君
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Qingdao Hehe Medical Laboratory Co ltd
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Abstract

The invention provides a method for simultaneously detecting the contents of clomipramine and N-norclomipramine in blood. Preparing at least three standard working solutions containing a clomipramine standard substance with known concentration and an N-norclomipramine standard substance; mixing the standard working solution, the internal standard working solution and the blank blood sample, and performing sample pretreatment to obtain a standard solution; respectively detecting each standard solution by using a high performance liquid chromatograph to obtain a chromatogram of each standard solution; fitting to obtain a standard curve equation according to the chromatogram of each standard solution; mixing the internal standard working solution with a blood sample obtained by treating blood to be detected, and performing the same sample pretreatment to obtain a sample to be detected; detecting the sample to be detected to obtain a chromatogram map; and calculating the contents of clomipramine and N-norclomipramine in the blood sample according to the chromatogram of the sample to be detected and each standard curve equation. The scheme can simultaneously detect the contents of clomipramine and N-norclomipramine in blood.

Description

同时检测血液中氯米帕明和N-去甲氯米帕明含量的方法Method for detecting clomipramine and N-desmethylclomipramine content in blood simultaneously

技术领域technical field

本发明涉及临床化学技术领域,特别涉及同时检测血液中氯米帕明和N-去甲氯米帕明含量的方法。The invention relates to the technical field of clinical chemistry, in particular to a method for simultaneously detecting the contents of clomipramine and N-desmethylclomipramine in blood.

背景技术Background technique

抑郁症又称抑郁障碍,以显著而持久的心境低落为主要临床特征,是心境障碍的主要类型。临床可见心境低落与其处境不相称,情绪的消沉可以从闷闷不乐到悲痛欲绝,自卑抑郁,甚至悲观厌世,可有自杀企图或行为;甚至发生木僵;部分病例有明显的焦虑和运动性激越;严重者可出现幻觉、妄想等精神病性症状。抑郁症每次发作持续至少2周以上,长者甚或数年,多数病例有反复发作的倾向,每次发作大多数可以缓解,部分可有残留症状或转为慢性。Depression, also known as depressive disorder, is the main type of mood disorder with significant and persistent low mood as the main clinical feature. It can be seen clinically that the mood is not commensurate with the situation. The depression of mood can range from gloomy to distraught, low self-esteem and depression, even pessimistic world-weariness, and there may be suicide attempts or behaviors; even stupor; some cases have obvious anxiety and motor agitation; In severe cases, psychotic symptoms such as hallucinations and delusions may appear. Each episode of depression lasts at least 2 weeks, or even several years in the elderly. Most cases have a tendency to recur. Most of each episode can be relieved, and some may have residual symptoms or become chronic.

氯米帕明是一种三环抗抑郁药物,主要用于治疗强迫症(OCD),也常用于治疗强迫性神经症、恐怖性神经症。氯米帕明主要作用在于阻断中枢神经系统5-羟色胺的再摄取,其活性代谢产物去甲氯米帕明可阻断中枢神经系统去甲肾上腺素再摄取,从而发挥抗抑郁及抗焦虑作用,亦有镇静和抗胆碱能作用。Clomipramine is a tricyclic antidepressant drug mainly used in the treatment of obsessive-compulsive disorder (OCD), and also commonly used in the treatment of obsessive-compulsive neurosis and phobic neurosis. The main function of clomipramine is to block the reuptake of serotonin in the central nervous system, and its active metabolite desmethylclomipramine can block the reuptake of norepinephrine in the central nervous system, thereby exerting antidepressant and anxiolytic effects , also has sedative and anticholinergic effects.

目前已有测定血液中氯米帕明含量的方法。由于其代谢物同样具有药理活性,同样会影响治疗效果,故检测其活性代谢物很有必要。因此,有必要提出能够同时检测血液中氯米帕明及其代谢物N-去甲氯米帕明含量的方法。There are currently methods for the determination of clomipramine levels in blood. Since its metabolites are also pharmacologically active and will also affect the therapeutic effect, it is necessary to detect their active metabolites. Therefore, it is necessary to propose a method capable of simultaneously detecting the content of clomipramine and its metabolite N-desmethylclomipramine in blood.

发明内容Contents of the invention

本发明提供了同时检测血液中氯米帕明和N-去甲氯米帕明含量的方法,能够同时检测血液中氯米帕明和N-去甲氯米帕明的含量。The invention provides a method for simultaneously detecting the contents of clomipramine and N-desmethylclomipramine in blood, which can simultaneously detect the contents of clomipramine and N-desmethylclomipramine in blood.

为了达到上述目的,本发明是通过如下技术方案实现的:In order to achieve the above object, the present invention is achieved through the following technical solutions:

本发明提供了同时检测血液中氯米帕明和N-去甲氯米帕明含量的方法,包括:The invention provides a method for simultaneously detecting the content of clomipramine and N-desmethylclomipramine in blood, comprising:

配制至少三个标准工作液,其中,标准工作液中含有浓度已知的氯米帕明标准品和N-去甲氯米帕明标准品,不同标准工作液中同一标准品的浓度不同;Prepare at least three standard working solutions, wherein the standard working solutions contain clomipramine standard substances and N-desmethylclomipramine standard substances with known concentrations, and the concentrations of the same standard substance in different standard working solutions are different;

将一定量的标准工作液、内标工作液和空白血液样本混合,进行样本前处理,得到标准溶液,其中,内标工作液中含有浓度已知的内标物;Mix a certain amount of standard working solution, internal standard working solution and blank blood sample, and perform sample pretreatment to obtain a standard solution, wherein the internal standard working solution contains an internal standard with a known concentration;

利用高效液相色谱仪,在一定检测条件下,分别检测至少三个标准溶液,得到各个标准溶液的色谱图;Using a high performance liquid chromatograph, under certain detection conditions, respectively detect at least three standard solutions, and obtain the chromatograms of each standard solution;

根据各个标准溶液的色谱图,拟合得到氯米帕明的标准曲线方程和N-去甲氯米帕明的标准曲线方程;According to the chromatogram of each standard solution, fitting obtains the standard curve equation of clomipramine and the standard curve equation of N-desmethylclomipramine;

将一定量的内标工作液和血液样本混合,进行同样的样本前处理,得到待测样本,其中,血液样本经处理待检测血液而得到;Mixing a certain amount of internal standard working solution with the blood sample, performing the same sample pretreatment to obtain the sample to be tested, wherein the blood sample is obtained by processing the blood to be tested;

利用高效液相色谱仪,在相同检测条件下检测待测样本,得到待测样本的色谱图;Using a high performance liquid chromatograph, detect the sample to be tested under the same detection conditions, and obtain the chromatogram of the sample to be tested;

根据待测样本的色谱图和拟合得到的各个标准曲线方程,计算血液样本中氯米帕明和N-去甲氯米帕明的含量。According to the chromatogram of the sample to be tested and each standard curve equation obtained by fitting, the contents of clomipramine and N-desmethylclomipramine in the blood sample are calculated.

优选地,所述进行样本前处理,包括下述A1至A5:Preferably, the sample pretreatment includes the following A1 to A5:

A1:将混合液涡旋混匀;A1: Vortex the mixture;

A2:加入甲醇,涡旋混匀;A2: Add methanol and vortex to mix;

A3:加入萃取剂,涡旋混匀,离心得到上清液;A3: Add the extractant, vortex and mix, and centrifuge to obtain the supernatant;

A4:移取上清液于离心管中,在常温下用氮气吹干;A4: Pipette the supernatant into a centrifuge tube and dry it with nitrogen at room temperature;

A5:向上清吹干的离心管中加入复溶液,涡旋混匀,离心得到上清液。A5: Add the reconstitution solution to the supernatant-dried centrifuge tube, vortex to mix, and centrifuge to obtain the supernatant.

优选地,所述A1包括:将混合液在转速为1800-2800rpm下涡旋混匀1-2min;Preferably, the A1 includes: vortex mixing the mixed solution at a speed of 1800-2800rpm for 1-2min;

所述A2包括:加入80-120μL甲醇,在转速为1800-2800rpm下涡旋混匀1-2min;The A2 includes: adding 80-120 μL of methanol, and vortex mixing at a speed of 1800-2800 rpm for 1-2 minutes;

所述A3包括:加入800-1200μL萃取剂,在转速为1800-2800rpm下涡旋混匀15-25min后,再在10000-15000rpm的转速下离心3-7min,得到上清液;The A3 includes: adding 800-1200 μL extractant, vortex mixing at a speed of 1800-2800 rpm for 15-25 min, and then centrifuging at a speed of 10000-15000 rpm for 3-7 min to obtain a supernatant;

所述A4包括:移取700-1000μL上清液于离心管中,在常温下用氮气吹干;The A4 includes: pipetting 700-1000 μL supernatant into a centrifuge tube, and blowing dry with nitrogen at room temperature;

所述A5包括:向上清吹干的离心管中加入80-120μL复溶液,在1800-2800rpm转速下涡旋混匀2-4min后,再在10000-15000rpm的转速下离心4-6min,得到上清液。Said A5 includes: adding 80-120 μL of reconstituted solution to a centrifuge tube dried with supernatant, vortexing at 1800-2800 rpm for 2-4 minutes, and then centrifuging at 10000-15000 rpm for 4-6 minutes to obtain the above Serum.

优选地,所述检测条件包括:Waters Xbridge C18色谱柱,色谱柱的长度为150mm、内径为2.1mm、填料粒径为3.5μm。Preferably, the detection conditions include: a Waters Xbridge C18 chromatographic column, the length of the chromatographic column is 150 mm, the inner diameter is 2.1 mm, and the filler particle size is 3.5 μm.

优选地,所述检测条件包括:柱温为50-60℃,流动相A为含浓度为80-120mmol/L的乙酸铵、体积比为0.15-0.25%的甲酸、体积比为0.15-0.25%的三乙胺的水,流动相B为乙腈,流速为0.2-0.4mL/min,进样量为20-40μL,分析时间为12-13min。Preferably, the detection conditions include: the column temperature is 50-60°C, the mobile phase A contains ammonium acetate with a concentration of 80-120mmol/L, formic acid with a volume ratio of 0.15-0.25%, and a volume ratio of 0.15-0.25% triethylamine in water, the mobile phase B is acetonitrile, the flow rate is 0.2-0.4mL/min, the injection volume is 20-40μL, and the analysis time is 12-13min.

优选地,所述检测条件包括:单柱双泵洗脱模式;Preferably, the detection conditions include: single-column double-pump elution mode;

双泵切换方式为:在10.7min从分析泵切换为清洁泵,在11.2min从清洁泵切换为分析泵;The switching mode of the double pumps is: switch from the analysis pump to the clean pump at 10.7min, and switch from the clean pump to the analysis pump at 11.2min;

洗脱方式为:流动相A和流动相B的体积比,在10.7-11.2min之外的其他洗脱时间范围内为59-61%:41-39%。The elution mode is: the volume ratio of mobile phase A and mobile phase B is 59-61%: 41-39% in other elution time ranges other than 10.7-11.2min.

优选地,所述高效液相色谱仪中的紫外检测器为DAD-3000检测器,检测波长为295-305nm,采集频率为5Hz,带宽为4nm。Preferably, the ultraviolet detector in the high performance liquid chromatograph is a DAD-3000 detector, the detection wavelength is 295-305nm, the collection frequency is 5Hz, and the bandwidth is 4nm.

优选地,所述高效液相色谱仪所使用的在线过滤器为SSI COLPRE-FILTER WATER1/16 0.5M。Preferably, the in-line filter used by the high performance liquid chromatograph is SSI COLPRE-FILTER WATER 1/16 0.5M.

优选地,内标物为亚氨基芪。Preferably, the internal standard is iminostilbene.

优选地,所述标准工作液中,氯米帕明标准品的浓度为500-15000ng/mL,N-去甲氯米帕明标准品的浓度为1000-30000ng/mL,稀释液为40-60%的甲醇水溶液。Preferably, in the standard working solution, the concentration of the clomipramine standard substance is 500-15000ng/mL, the concentration of the N-desmethylclomipramine standard substance is 1000-30000ng/mL, and the diluent is 40-60 % methanol in water.

本发明提供了同时检测血液中氯米帕明和N-去甲氯米帕明含量的方法。配制至少三个含已知浓度的氯米帕明标准品和N-去甲氯米帕明标准品的标准工作液;将标准工作液、内标工作液和空白血液样本混合,进行样本前处理得到标准溶液;利用高效液相色谱仪分别检测各个标准溶液,得到各个标准溶液的色谱图;根据各个标准溶液的色谱图,拟合得到标准曲线方程;将内标工作液和经处理待检测血液而得到的血液样本混合,进行同样的样本前处理得到待测样本;同样检测待测样本得到其色谱图;根据待测样本的色谱图和各个标准曲线方程,计算血液样本中氯米帕明和N-去甲氯米帕明的含量。本发明能够同时检测血液中氯米帕明和N-去甲氯米帕明的含量。The invention provides a method for simultaneously detecting the contents of clomipramine and N-desmethylclomipramine in blood. Prepare at least three standard working solutions containing a known concentration of clomipramine standard and N-desmethylclomipramine standard; mix the standard working solution, internal standard working solution and blank blood sample for sample pretreatment Obtain the standard solution; Utilize high performance liquid chromatography to detect each standard solution respectively, obtain the chromatogram of each standard solution; According to the chromatogram of each standard solution, fit and obtain the standard curve equation; The obtained blood samples are mixed, and the same sample pretreatment is carried out to obtain the sample to be tested; the chromatogram of the sample to be tested is also detected; according to the chromatogram of the sample to be tested and each standard curve equation, the clomipramine and N in the blood sample are calculated. - Desmethylclomipramine content. The invention can simultaneously detect the contents of clomipramine and N-desmethylclomipramine in blood.

附图说明Description of drawings

为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are For some embodiments of the present invention, those skilled in the art can also obtain other drawings based on these drawings without creative work.

图1是本发明一实施例提供的一种同时检测血液中氯米帕明和N-去甲氯米帕明含量的方法的流程图;Fig. 1 is a flow chart of a method for simultaneously detecting the content of clomipramine and N-desmethylclomipramine in blood provided by an embodiment of the present invention;

图2是本发明一实施例提供的氯米帕明的化学结构式;Fig. 2 is the chemical structural formula of clomipramine provided by an embodiment of the present invention;

图3是本发明一实施例提供的N-去甲氯米帕明的化学结构式;Fig. 3 is the chemical structural formula of N-desmethylclomipramine provided by an embodiment of the present invention;

图4是本发明一实施例提供的一标准溶液中氯米帕明标准品、N-去甲氯米帕明标准品及内标物的色谱图;Fig. 4 is the chromatogram of clomipramine standard substance, N-desmethylclomipramine standard substance and internal standard substance in a standard solution provided by an embodiment of the present invention;

图5是本发明一实施例提供的一待测样本中氯米帕明、N-去甲氯米帕明及内标物的色谱图;Figure 5 is a chromatogram of clomipramine, N-desmethylclomipramine and an internal standard in a sample to be tested provided by an embodiment of the present invention;

图6是本发明一实施例提供的氯米帕明的线性关系图;Figure 6 is a linear relationship diagram of clomipramine provided by an embodiment of the present invention;

图7是本发明一实施例提供的N-去甲氯米帕明的线性关系图。Fig. 7 is a linear relationship graph of N-desmethylclomipramine provided by an embodiment of the present invention.

具体实施方式Detailed ways

为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例,基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都属于本发明保护的范围。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments It is a part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the protection of the present invention. scope.

经研究发现,原型(即氯米帕明)、原型的代谢物(即N-去甲氯米帕明)及内标物在血液样本前处理过程中的萃取率不同。举例来说,以亚氨基芪为内标物时,萃取率的试验结果如下述表1所示。It has been found through research that the extraction rates of the prototype (ie clomipramine), the metabolite of the prototype (ie N-desmethylclomipramine) and the internal standard are different during the blood sample pretreatment process. For example, when iminostilbene is used as an internal standard, the test results of the extraction rate are shown in Table 1 below.

表1Table 1

内标物internal standard 代谢物Metabolites 原型prototype 未作前处理时标准溶液中物质的峰面积The peak area of the substance in the standard solution without pretreatment 2.14392.1439 0.36440.3644 0.2420.242 作前处理时标准溶液中物质的峰面积The peak area of the substance in the standard solution during pretreatment 1.22541.2254 0.23820.2382 0.19790.1979 萃取率Extraction rate 57.2%57.2% 65.4%65.4% 81.8%81.8%

表1中,未作前处理,即标准工作液、内标工作液,通过稀释直接配制得到标准溶液并上样分析。作前处理,即标准工作液、内标工作液加空白血后,进行与血液样本前处理相同的前处理操作后,得到标准溶液并上样分析。In Table 1, without pretreatment, that is, standard working solution and internal standard working solution, the standard solution was directly prepared by dilution and loaded for analysis. For pretreatment, that is, after standard working solution, internal standard working solution plus blank blood, the same pretreatment operation as the blood sample pretreatment is performed to obtain the standard solution and load it for analysis.

请参考表1,未作前处理时内标物的峰面积为2.1439,而作前处理时内标物的峰面积为1.2254,可见,前处理过程对内标物的萃取有一定影响,萃取率为57.2%。Please refer to Table 1, the peak area of the internal standard substance is 2.1439 without pretreatment, and the peak area of the internal standard substance is 1.2254 when pretreatment is performed. It can be seen that the pretreatment process has a certain influence on the extraction of the internal standard substance, and the extraction rate was 57.2%.

请参考表1,未作前处理时代谢物的峰面积为0.3644,而作前处理时内标物的峰面积为0.2382,可见,前处理过程对代谢物的萃取有一定影响,萃取率为65.4%。Please refer to Table 1. The peak area of the metabolites without pretreatment is 0.3644, while the peak area of the internal standard is 0.2382 with pretreatment. It can be seen that the pretreatment process has a certain impact on the extraction of metabolites, and the extraction rate is 65.4 %.

请参考表1,未作前处理时原型的峰面积为0.242,而作前处理时内标物的峰面积为0.1979,可见,前处理过程对原型的萃取有一定影响,萃取率为81.8%。Please refer to Table 1, the peak area of the prototype without pretreatment is 0.242, and the peak area of the internal standard is 0.1979 with pretreatment. It can be seen that the pretreatment process has a certain impact on the extraction of the prototype, and the extraction rate is 81.8%.

可以看出,前处理过程中,内标物的萃取率与代谢物的萃取率,以及内标物的萃取率与原型的萃取率,均不同且相差较大。因此,为校正因原型、代谢物与内标物的萃取率不一致而带来的定值误差,获取标准曲线可采用加血前处理的方式,使得根据标准工作液制备标准溶液的前处理过程,与根据血液样本制备待测样本的前处理过程相同,以保证检测准确率。It can be seen that during the pretreatment process, the extraction rate of the internal standard and the metabolite, as well as the extraction rate of the internal standard and the prototype, are all different and quite different. Therefore, in order to correct the fixed value error caused by the inconsistency of the extraction rate of the prototype, metabolites and internal standards, the method of adding blood can be used to obtain the standard curve, so that the pretreatment process of preparing the standard solution according to the standard working solution, It is the same as the pretreatment process of preparing the sample to be tested based on the blood sample to ensure the accuracy of the test.

基于上述内容,如图1所示,本发明实施例提供了同时检测血液中氯米帕明和N-去甲氯米帕明含量的方法,可以包括以下步骤:Based on the above, as shown in Figure 1, the embodiment of the present invention provides a method for simultaneously detecting the content of clomipramine and N-desmethylclomipramine in blood, which may include the following steps:

步骤101:配制至少三个标准工作液,其中,标准工作液中含有浓度已知的氯米帕明标准品和N-去甲氯米帕明标准品,不同标准工作液中同一标准品的浓度不同。Step 101: Prepare at least three standard working solutions, wherein the standard working solutions contain clomipramine standard substances and N-desmethylclomipramine standard substances with known concentrations, and the concentration of the same standard substance in different standard working solutions different.

步骤102:将一定量的标准工作液、内标工作液和空白血液样本混合,进行样本前处理,得到标准溶液,其中,内标工作液中含有浓度已知的内标物。Step 102: Mix a certain amount of standard working solution, internal standard working solution and blank blood sample, and perform sample pretreatment to obtain a standard solution, wherein the internal standard working solution contains an internal standard with a known concentration.

详细地,空白血液样本通常可以为不含氯米帕明、N-去甲氯米帕明和内标物的血清或血浆,该空白血液样本的氯米帕明、N-去甲氯米帕明和内标物的含量检测结果,通常应为未检出。In detail, the blank blood sample can usually be serum or plasma without clomipramine, N-desmethylclomipramine and internal standard substance, and the clomipramine, N-desmethylclomipramine and internal standard of the blank blood sample The content detection result of the internal standard should usually be not detected.

步骤103:利用高效液相色谱仪,在一定检测条件下,分别检测至少三个标准溶液,得到各个标准溶液的色谱图。Step 103: Using a high performance liquid chromatograph, under certain detection conditions, detect at least three standard solutions respectively, and obtain the chromatograms of each standard solution.

步骤104:根据各个标准溶液的色谱图,拟合得到氯米帕明的标准曲线方程和N-去甲氯米帕明的标准曲线方程。Step 104: According to the chromatograms of each standard solution, the standard curve equation of clomipramine and the standard curve equation of N-desmethylclomipramine are obtained by fitting.

步骤105:将一定量的内标工作液和血液样本混合,进行同样的样本前处理,得到待测样本,其中,血液样本经处理待检测血液而得到。Step 105: Mix a certain amount of internal standard working solution with the blood sample, and perform the same sample pretreatment to obtain the sample to be tested, wherein the blood sample is obtained by processing the blood to be tested.

步骤106:利用高效液相色谱仪,在相同检测条件下检测待测样本,得到待测样本的色谱图。Step 106: Using a high-performance liquid chromatograph to detect the sample to be tested under the same detection conditions, and obtain a chromatogram of the sample to be tested.

步骤107:根据待测样本的色谱图和拟合得到的各个标准曲线方程,计算血液样本中氯米帕明和N-去甲氯米帕明的含量。Step 107: Calculate the content of clomipramine and N-desmethylclomipramine in the blood sample according to the chromatogram of the sample to be tested and each standard curve equation obtained by fitting.

本发明实施例用内标法来检测血液样本中氯米帕明及N-去甲氯米帕明的含量,以避免因前处理过程操作失误而引起的定值偏差,故可保证检测准确率。The embodiment of the present invention uses the internal standard method to detect the content of clomipramine and N-desmethylclomipramine in the blood sample, so as to avoid the value deviation caused by the operation error in the pretreatment process, so the detection accuracy can be guaranteed .

请参考图2及图3,图2示出了氯米帕明的化学结构式,图3示出了N-去甲氯米帕明的化学结构式。Please refer to FIG. 2 and FIG. 3 , FIG. 2 shows the chemical structural formula of clomipramine, and FIG. 3 shows the chemical structural formula of N-desmethylclomipramine.

通常情况下,标准曲线方程的建立至少需要三个坐标点,以保证所建立方程的准确性,故需配制至少三个标准溶液,从而可以根据各个标准溶液检测所得到的色谱图,拟合出氯米帕明的标准曲线方程和N-去甲氯米帕明的标准曲线方程。Usually, the establishment of the standard curve equation requires at least three coordinate points to ensure the accuracy of the established equation, so it is necessary to prepare at least three standard solutions, so that the obtained chromatograms can be tested according to each standard solution, and the fitting method can be obtained. Standard curve equation for clomipramine and standard curve equation for N-desmethylclomipramine.

详细地,以氯米帕明为例,拟合得到的氯米帕明的标准曲线方程通常可以为y=k×x+b。其中,x、y这两个变量,分别可以为各个标准溶液的色谱图中,氯米帕明的标准品与内标物的峰面积比值,以及,各个标准溶液中,氯米帕明的标准品与内标物的浓度比值。如此,根据待测样本的色谱图中,氯米帕明与内标物的峰面积比值,以及待测样本中内标物的浓度,代入标准曲线方程即可计算出待测样本中氯米帕明的浓度,从而得到血液样本中氯米帕明的含量。当然,这一实现方式同样适用于N-去甲氯米帕明。In detail, taking clomipramine as an example, the fitted standard curve equation of clomipramine can usually be y=k×x+b. Wherein, these two variables of x, y can be respectively the peak area ratio of the standard substance of clomipramine and the internal standard in the chromatogram of each standard solution, and, in each standard solution, the standard of clomipramine Concentration ratio of product to internal standard. In this way, according to the chromatogram of the sample to be tested, the peak area ratio of clomipramine and the internal standard substance, and the concentration of the internal standard substance in the sample to be tested, the clomipramine in the sample to be tested can be calculated by substituting into the standard curve equation. The concentration of clomipramine was obtained to obtain the content of clomipramine in the blood sample. Of course, this realization is also applicable to N-desmethylclomipramine.

本发明实施例中,血液样本通常可以为血清或血浆,经处理待检测血液而得到。采样到待检测血液后,即可作相应处理以得到血液样本。比如,取待检测血液至少5mL,在离心速度为3500rpm下离心10min,取上清液得血清或血浆,即得到上述血液样本。血清或血浆样本可置于-20℃冷冻下保存至分析前备用。In the embodiment of the present invention, the blood sample can usually be serum or plasma obtained by processing the blood to be tested. After the blood to be tested is sampled, it can be processed accordingly to obtain a blood sample. For example, take at least 5 mL of the blood to be tested, centrifuge at a centrifugation speed of 3500 rpm for 10 min, and take the supernatant to obtain serum or plasma, that is, the above blood sample is obtained. Serum or plasma samples can be stored at -20°C until analysis.

得到血液样本后,经同样的前处理得到待测样本,在相同检测条件下检测待测样本以得到其色谱图。如上所述,基于该色谱图和拟合得到的标准曲线方程,即可得到血液样本中氯米帕明及N-去甲氯米帕明的含量。After the blood sample is obtained, the sample to be tested is obtained through the same pretreatment, and the sample to be tested is detected under the same detection conditions to obtain its chromatogram. As mentioned above, based on the chromatogram and the fitted standard curve equation, the contents of clomipramine and N-desmethylclomipramine in the blood sample can be obtained.

综上所述,本发明实施例提供的这一检测血液中氯米帕明及N-去甲氯米帕明含量的方法,是将内标法与高效液相色谱法相结合,并通过相同的前处理过程来处理标准工作液和血液样本,使干扰因素大大减少,且定量准确、重现性好、特异性强、灵敏度高、检测结果更为准确,且成本低,分析时间短,有利于大通量血液样本的检测。In summary, the method for detecting the content of clomipramine and N-desmethylclomipramine in the blood provided by the embodiment of the present invention is to combine the internal standard method with high performance liquid chromatography, and pass the same The standard working solution and blood samples are processed in the pre-treatment process, which greatly reduces the interference factors, and has accurate quantification, good reproducibility, strong specificity, high sensitivity, more accurate detection results, low cost, and short analysis time, which is beneficial to Detection of high-throughput blood samples.

详细地,在进行英国政府化学家实验室(LGC)的室间比对试验过程中,发现作样本前处理时,若萃取前不存在加甲醇步骤,所得实验结果与真实值偏差较大,而若萃取前存在加甲醇步骤,所得实验结果与真实值偏差较小,具体实验数据请参考下述表2。In detail, during the inter-laboratory comparison test of the British Government Chemist Laboratory (LGC), it was found that when the sample was pretreated, if there was no step of adding methanol before the extraction, the experimental results obtained had a large deviation from the true value, while If there is a step of adding methanol before the extraction, the deviation between the obtained experimental results and the true value is small. For specific experimental data, please refer to the following Table 2.

表2Table 2

Figure BDA0002297118240000081
Figure BDA0002297118240000081

表2中,改进前表示样本前处理中萃取前无加甲醇步骤,改进后表示样本前处理中萃取前有加甲醇步骤。其中,z'score≤2,结果合格。In Table 2, before improvement means that there is no step of adding methanol before extraction in sample pretreatment, and after improvement means that there is a step of adding methanol before extraction in sample pretreatment. Among them, z'score≤2, the result is qualified.

基于上述内容,通过分析认为蛋白结合率偏高应该是导致萃取率偏低的原因,故在样本前处理过程中,优选在萃取前作加甲醇步骤。因此,在本发明一个实施例中,所述进行样本前处理,包括下述A1至A5:Based on the above content, it is believed that the high protein binding rate should be the cause of the low extraction rate through analysis. Therefore, in the sample pretreatment process, it is preferable to add methanol before extraction. Therefore, in one embodiment of the present invention, the sample pretreatment includes the following A1 to A5:

A1:将混合液涡旋混匀;A1: Vortex the mixture;

A2:加入甲醇,涡旋混匀;A2: Add methanol and vortex to mix;

A3:加入萃取剂,涡旋混匀,离心得到上清液;A3: Add the extractant, vortex and mix, and centrifuge to obtain the supernatant;

A4:移取上清液于离心管中,在常温下用氮气吹干;A4: Pipette the supernatant into a centrifuge tube and dry it with nitrogen at room temperature;

A5:向上清吹干的离心管中加入复溶液,涡旋混匀,离心得到上清液。A5: Add the reconstitution solution to the supernatant-dried centrifuge tube, vortex to mix, and centrifuge to obtain the supernatant.

详细地,标准工作液经样本前处理所得到的上清液,即为标准溶液,血液样本经样本前处理所得到的上清液,即为待测样本。In detail, the supernatant obtained by sample pretreatment of the standard working solution is the standard solution, and the supernatant obtained by the sample pretreatment of the blood sample is the sample to be tested.

本发明实施例中,在萃取操作之前可加入甲醇以实现沉淀蛋白操作,以提高实际样本检测的准确度。In the embodiment of the present invention, methanol can be added before the extraction operation to realize the protein precipitation operation, so as to improve the accuracy of actual sample detection.

优选地,在本发明一个实施例中,所述A1包括:将混合液在转速为1800-2800rpm下涡旋混匀1-2min;Preferably, in one embodiment of the present invention, the A1 includes: vortex mixing the mixed solution at a rotation speed of 1800-2800rpm for 1-2min;

所述A2包括:加入80-120μL甲醇,在转速为1800-2800rpm下涡旋混匀1-2min;The A2 includes: adding 80-120 μL of methanol, and vortex mixing at a speed of 1800-2800 rpm for 1-2 minutes;

所述A3包括:加入800-1200μL萃取剂,在转速为1800-2800rpm下涡旋混匀15-25min后,再在10000-15000rpm的转速下离心3-7min,得到上清液;The A3 includes: adding 800-1200 μL extractant, vortex mixing at a speed of 1800-2800 rpm for 15-25 min, and then centrifuging at a speed of 10000-15000 rpm for 3-7 min to obtain a supernatant;

所述A4包括:移取700-1000μL上清液于离心管中,在常温下用氮气吹干;The A4 includes: pipetting 700-1000 μL supernatant into a centrifuge tube, and blowing dry with nitrogen at room temperature;

所述A5包括:向上清吹干的离心管中加入80-120μL复溶液,在1800-2800rpm转速下涡旋混匀2-4min后,再在10000-15000rpm的转速下离心4-6min,得到上清液。Said A5 includes: adding 80-120 μL of reconstituted solution to a centrifuge tube dried with supernatant, vortexing at 1800-2800 rpm for 2-4 minutes, and then centrifuging at 10000-15000 rpm for 4-6 minutes to obtain the above Serum.

举例来说,涡旋转速的取值可以为1800、2000、2200、2400、2600或2800;A1中涡旋时间的取值可以为1、1.2、1.5、1.7或2;甲醇用量的取值可以为80、90、100、110或120;A2中涡旋时间的取值可以为1、1.2、1.5、1.7或2;萃取剂用量的取值可以为800、900、1000、1100或1200;A3中涡旋时间的取值可以为15、17、20、22或25;离心转速的取值可以为10000、11000、12000、13000、14000或15000;A3中离心时间的取值可以为3、4、5、6或7;A4中上清液用量的取值可以为700、80、900或1000;复溶液用量的取值可以为80、90、100、110或120;A5中涡旋时间的取值可以为2、2.5、3、3.5或4;A5中离心时间的取值可以为4、4.5、5、5.5或6。For example, the value of the vortex rotation speed can be 1800, 2000, 2200, 2400, 2600 or 2800; the value of the vortex time in A1 can be 1, 1.2, 1.5, 1.7 or 2; the value of the amount of methanol can be 80, 90, 100, 110 or 120; the value of vortex time in A2 can be 1, 1.2, 1.5, 1.7 or 2; the value of extractant dosage can be 800, 900, 1000, 1100 or 1200; A3 The value of the vortex time in A3 can be 15, 17, 20, 22 or 25; the value of the centrifugal speed can be 10000, 11000, 12000, 13000, 14000 or 15000; the value of the centrifugal time in A3 can be 3, 4 , 5, 6 or 7; the value of supernatant in A4 can be 700, 80, 900 or 1000; the value of complex solution can be 80, 90, 100, 110 or 120; the vortex time in A5 The value can be 2, 2.5, 3, 3.5 or 4; the value of centrifugation time in A5 can be 4, 4.5, 5, 5.5 or 6.

优选地,上述萃取剂可以为正己烷,上述复溶液可以为40-60%的甲醇水溶液。举例来说,复溶液中,甲醇的百分含量可以为40%、45%、50%、55%或60%。Preferably, the above extractant can be n-hexane, and the above complex solution can be 40-60% methanol aqueous solution. For example, in the complex solution, the percentage content of methanol can be 40%, 45%, 50%, 55% or 60%.

本发明实施例采用内标法来检测血液样本中氯米帕明和N-去甲氯米帕明的含量,可以使用如上所述的前处理方式,该前处理方法操作简单,能够避免操作带来的误差,从而能够更准确的定量。此外,由于前处理方法较为简单,故利于本发明实施例的推广应用。The embodiment of the present invention adopts the internal standard method to detect the content of clomipramine and N-desmethylclomipramine in the blood sample, and the above-mentioned pretreatment method can be used. error, so that more accurate quantification can be achieved. In addition, because the preprocessing method is relatively simple, it is beneficial to popularization and application of the embodiments of the present invention.

基于上述内容,在本发明一个实施例中,标准工作液的用量为10μL、内标工作液的用量为10μL,空白血液样本的用量为150-250μL,三者混合后作样本前处理。举例来说,空白血液样本用量的取值可以为150、170、200、220或250。Based on the above, in one embodiment of the present invention, the standard working solution is used in an amount of 10 μL, the internal standard working solution is used in an amount of 10 μL, and the blank blood sample is used in an amount of 150-250 μL. The three are mixed for sample pretreatment. For example, the amount of the blank blood sample can be 150, 170, 200, 220 or 250.

基于上述内容,在本发明一个实施例中,内标工作液的用量为10μL,血液样本的用量为150-250μL,两者混合后作样本前处理。举例来说,血液样本用量的取值可以为150、170、200、220或250。Based on the above, in one embodiment of the present invention, the volume of the internal standard working solution is 10 μL, and the volume of the blood sample is 150-250 μL, and the two are mixed for sample pretreatment. For example, the amount of blood samples can be 150, 170, 200, 220 or 250.

在本发明一个实施例中,所述检测条件包括:Waters Xbridge C18色谱柱,色谱柱的长度为150mm、内径为2.1mm、填料粒径为3.5μm。In one embodiment of the present invention, the detection conditions include: a Waters Xbridge C18 chromatographic column, the length of the chromatographic column is 150 mm, the inner diameter is 2.1 mm, and the filler particle size is 3.5 μm.

在本发明一个实施例中,所述检测条件包括:柱温为50-60℃,流动相A为含浓度为80-120mmol/L的乙酸铵、体积比为0.15-0.25%的甲酸、体积比为0.15-0.25%的三乙胺的水,流动相B为乙腈,流速为0.2-0.4mL/min,进样量为20-40μL,分析时间为12-13min。In one embodiment of the present invention, the detection conditions include: the column temperature is 50-60°C, the mobile phase A is ammonium acetate containing a concentration of 80-120mmol/L, formic acid with a volume ratio of 0.15-0.25%, and a volume ratio of The water is 0.15-0.25% triethylamine, the mobile phase B is acetonitrile, the flow rate is 0.2-0.4mL/min, the injection volume is 20-40μL, and the analysis time is 12-13min.

举例来说,柱温的取值可以为50、52、54、56、58或60;流动相A中,乙酸铵浓度的取值可以为80、90、100、110或120,甲酸及三乙胺的体积比的取值均可以为0.15、0.17、0.20、0.22或0.25;流速的取值可以为0.2、0.25、0.3、0.35或0.4;进样量的取值可以为20、25、30、35或40;分析时间的取值可以为12、12.2、12.4、12.6、12.8或13。For example, the value of column temperature can be 50, 52, 54, 56, 58 or 60; in mobile phase A, the value of ammonium acetate concentration can be 80, 90, 100, 110 or 120, formic acid and The value of the volume ratio of the amine can be 0.15, 0.17, 0.20, 0.22 or 0.25; the value of the flow rate can be 0.2, 0.25, 0.3, 0.35 or 0.4; 35 or 40; the value of analysis time can be 12, 12.2, 12.4, 12.6, 12.8 or 13.

本发明实施例中,基于上述检测条件,分析时间可在12.5min左右,分析时间短,分析效率提高。In the embodiment of the present invention, based on the above detection conditions, the analysis time can be about 12.5 min, which is short and the analysis efficiency is improved.

在本发明一个实施例中,在整个分析时间的目标物分析阶段,流动相A和流动相B的体积比为59-61%:41-39%。举例来说,流动相A和流动相B的体积比可以为59:41、59.5:40.5、60:40、60.5:39.5或61:39。In an embodiment of the present invention, the volume ratio of the mobile phase A to the mobile phase B is 59-61%:41-39% during the target analysis phase of the entire analysis time. For example, the volume ratio of mobile phase A and mobile phase B can be 59:41, 59.5:40.5, 60:40, 60.5:39.5 or 61:39.

详细地,待分析的目标物包括原型、代谢物和内标物。In detail, the target substances to be analyzed include prototypes, metabolites and internal standards.

基于上述内容,在本发明一个实施例中,所述检测条件包括:单柱双泵洗脱模式;Based on the above, in one embodiment of the present invention, the detection conditions include: single-column double-pump elution mode;

双泵切换方式为:在10.7min从分析泵切换为清洁泵,在11.2min从清洁泵切换为分析泵;The switching mode of the double pumps is: switch from the analysis pump to the clean pump at 10.7min, and switch from the clean pump to the analysis pump at 11.2min;

洗脱方式为:流动相A和流动相B的体积比,在10.7-11.2min之外的其他洗脱时间范围内为59-61%:41-39%。The elution mode is: the volume ratio of mobile phase A and mobile phase B is 59-61%: 41-39% in other elution time ranges other than 10.7-11.2min.

本发明实施例中,在10.7-11.2min这段时间,是清洁泵流动相进入色谱柱,以清洗色谱柱,在10.7min之前及11.2min之后的这段时间,是分析泵流动相进入色谱柱,以进行目标物分析。如此,对于分析泵流动相,流动相A和流动相B的体积比为59-61%:41-39%。在本发明一个实施例中,对于清洁泵流动相,流动相A和流动相B的体积比可以为40:60。In the embodiment of the present invention, during the period of 10.7-11.2min, the mobile phase of the cleaning pump enters the chromatographic column to clean the chromatographic column. Before 10.7min and after 11.2min, the mobile phase of the analytical pump enters the chromatographic column. , for target analysis. Thus, for the analytical pump mobile phase, the volume ratio of mobile phase A to mobile phase B is 59-61%:41-39%. In one embodiment of the present invention, for the clean pump mobile phase, the volume ratio of mobile phase A and mobile phase B may be 40:60.

与单泵梯度洗脱模式相比,单柱双泵洗脱模式能在单柱的条件下,最大程度减少分析时间。与此同时,能在一定程度上减少有机试剂的使用量。Compared with the single-pump gradient elution mode, the single-column dual-pump elution mode can minimize the analysis time under single-column conditions. At the same time, the amount of organic reagents used can be reduced to a certain extent.

因此,本发明实施例可以缩短分析时间,提高检测通量,减少有机溶剂用量。Therefore, the embodiment of the present invention can shorten the analysis time, increase the detection throughput, and reduce the consumption of organic solvents.

在本发明一个实施例中,所述高效液相色谱仪中的紫外检测器为DAD-3000检测器,检测波长为295-305nm,采集频率为5Hz,带宽为4nm。举例来说,检测波长的取值可以为295、300或305。In one embodiment of the present invention, the ultraviolet detector in the high performance liquid chromatograph is a DAD-3000 detector, the detection wavelength is 295-305nm, the collection frequency is 5Hz, and the bandwidth is 4nm. For example, the value of the detection wavelength can be 295, 300 or 305.

在本发明一个实施例中,所述高效液相色谱仪所使用的在线过滤器为SSI COLPRE-FILTER WATER 1/16 0.5M。In one embodiment of the present invention, the in-line filter used in the high performance liquid chromatograph is SSI COLPRE-FILTER WATER 1/16 0.5M.

在本发明一个实施例中,内标物为亚氨基芪。In one embodiment of the present invention, the internal standard is iminostilbene.

详细地,亚氨基芪是一种用于药物合成的中间体,基本不会存在于患者体内。同使用功效相近的精神类药物或常用药作为内标相比,选用亚氨基芪作为内标具有很大的优势,可以避免联合用药给分析带来的相互干扰。In detail, iminostilbene is an intermediate used in the synthesis of drugs, and basically does not exist in the patient's body. Compared with using psychotropic drugs or commonly used drugs with similar efficacy as internal standard, the use of iminostilbene as internal standard has great advantages, which can avoid the mutual interference brought by combined drugs to the analysis.

基于上述内容,在本发明一个实施例中,所述标准工作液中,氯米帕明标准品的浓度为500-15000ng/mL,N-去甲氯米帕明标准品的浓度为1000-30000ng/mL,稀释液为40-60%的甲醇水溶液。Based on the foregoing, in one embodiment of the present invention, in the standard working solution, the concentration of the clomipramine standard substance is 500-15000ng/mL, and the concentration of the N-desmethylclomipramine standard substance is 1000-30000ng /mL, the diluent is 40-60% methanol water solution.

详细地,可以结合检测的人群、待检测血液的用量,以及人体内氯米帕明及N-去甲氯米帕明的大致含量范围,来设定相应线性范围,以保证大部分的临床样本检测结果落在可报告范围内。In detail, the corresponding linear range can be set in combination with the population to be tested, the amount of blood to be tested, and the approximate content range of clomipramine and N-desmethylclomipramine in the human body, so as to ensure that most clinical samples The test result falls within the reportable range.

比如,优选地,上述至少三个标准工作液中,氯米帕明标准品的浓度可以为500ng/mL、1000ng/mL、2000ng/mL、4000ng/mL、6000ng/mL、12000ng/mL、15000ng/mL中的至少三个,N-去甲氯米帕明标准品的浓度可以为1000ng/mL、2000ng/mL、4000ng/mL、8000ng/mL、12000ng/mL、24000ng/mL、30000ng/mL中的至少三个。优选地,标准工作液的个数为7个。For example, preferably, in the above-mentioned at least three standard working solutions, the concentration of clomipramine standard can be 500ng/mL, 1000ng/mL, 2000ng/mL, 4000ng/mL, 6000ng/mL, 12000ng/mL, 15000ng/mL At least three in mL, the concentration of N-desmethylclomipramine standard can be in 1000ng/mL, 2000ng/mL, 4000ng/mL, 8000ng/mL, 12000ng/mL, 24000ng/mL, 30000ng/mL At least three. Preferably, the number of standard working solutions is seven.

综上所述,本发明实施例提供的这一检测血液中氯米帕明及N-去甲氯米帕明含量的方法,是将内标法与高效液相色谱法相结合,使干扰因素大大减少,且定量准确、重现性好、特异性强、灵敏度高、检测结果更为准确,且成本低,分析时间短,有利于大通量血液样本的检测。In summary, the method for detecting the content of clomipramine and N-desmethylclomipramine in the blood provided by the embodiments of the present invention is to combine the internal standard method with high performance liquid chromatography, so that the interference factors are greatly reduced. It has the advantages of accurate quantification, good reproducibility, strong specificity, high sensitivity, more accurate detection results, low cost, and short analysis time, which is conducive to the detection of large-volume blood samples.

本发明实施例不仅检测了药物的原型,还同时检测了其活性代谢物,如此,可以利用本发明实施例提供的检测方法,在临床治疗中对患者体内的氯米帕明及其代谢物N-去甲氯米帕明的含量进行监测,为氯米帕明及其代谢物N-去甲氯米帕明的个性化给药、减少毒副反应的发生提供实验基础,从而更加利于指导患者用药。The embodiment of the present invention not only detects the prototype of the drug, but also detects its active metabolites at the same time. In this way, the detection method provided by the embodiment of the present invention can be used to detect clomipramine and its metabolite N in the patient's body during clinical treatment. -Monitoring the content of desmethylclomipramine, providing an experimental basis for the personalized administration of clomipramine and its metabolite N-desmethylclomipramine, and reducing the occurrence of toxic and side effects, so as to be more conducive to guiding patients medication.

以下将通过实施例对本发明进行详细描述,但本发明并不仅限于下述实施例。The present invention will be described in detail through examples below, but the present invention is not limited to the following examples.

实施例1Example 1

本发明实施例用于获取标准曲线方程。The embodiment of the present invention is used to obtain the standard curve equation.

1.1标准储备液的配制1.1 Preparation of standard stock solution

标准储备液:精密移取1.0mg/mL的氯米帕明标准品溶液15μL、1.0mg/mL的N-去甲氯米帕明标准品溶液30μL,置于1.5mL离心管,用50%的甲醇水溶液进行稀释,得到标准储备液。所得标准储备液中,氯米帕明标准品浓度为15000ng/mL,N-去甲氯米帕明标准品浓度为30000ng/mL。Standard stock solution: Accurately pipette 15 μL of 1.0 mg/mL clomipramine standard solution and 30 μL of 1.0 mg/mL N-desmethylclomipramine standard solution, put them in a 1.5 mL centrifuge tube, and use 50% Methanol aqueous solution was diluted to obtain a standard stock solution. In the obtained standard stock solution, the concentration of clomipramine standard substance is 15000ng/mL, and the concentration of N-desmethylclomipramine standard substance is 30000ng/mL.

1.2内标储备液的配制1.2 Preparation of internal standard stock solution

内标储备液:精确称取亚氨基芪标准品5mg于5mL容量瓶,用甲醇溶解,并定容至5mL,得到内标储备液。Internal standard stock solution: Accurately weigh 5mg of iminostilbene standard substance into a 5mL volumetric flask, dissolve in methanol, and dilute to 5mL to obtain internal standard stock solution.

1.3检测用仪器1.3 Instruments for testing

赛默飞U3000高效液相色谱仪。Thermo Fisher U3000 high performance liquid chromatograph.

1.4检测条件1.4 Detection conditions

1.4.1色谱柱1.4.1 Columns

Waters Xbridge C18色谱柱,色谱柱的长度为150mm、内径为2.1mm、填料粒径为3.5μm。Waters Xbridge C 18 chromatographic column, the length of the chromatographic column is 150 mm, the inner diameter is 2.1 mm, and the filler particle size is 3.5 μm.

1.4.2流动相1.4.2 Mobile phase

流动相A:含浓度为100mmol/L的乙酸铵、体积比为0.2%的甲酸、体积比为0.2%的三乙胺的水。Mobile phase A: water containing ammonium acetate at a concentration of 100 mmol/L, formic acid at a volume ratio of 0.2%, and triethylamine at a volume ratio of 0.2%.

流动相B:乙腈。Mobile Phase B: Acetonitrile.

1.4.3洗脱方式1.4.3 Elution method

单柱双泵洗脱模式。Single column double pump elution mode.

双泵切换方式如下述表3所示。在0-10.7min及11.2-12.5min内,流动相A和流动相B的体积比为60:40;在10.7-11.2min内,流动相A和流动相B的体积比为40:60。The double-pump switching method is shown in Table 3 below. Within 0-10.7min and 11.2-12.5min, the volume ratio of mobile phase A to mobile phase B is 60:40; within 10.7-11.2min, the volume ratio of mobile phase A to mobile phase B is 40:60.

表3table 3

序号serial number 时间/mintime/min ValveLeftValveLeft 11 {初始时间}{initial time} 10_110_1 22 10.70010.700 1_21_2 33 11.20011.200 10_110_1

表3中,10_1标识分析泵,1_2标识从分析泵切换为清洁泵。In Table 3, 10_1 marks the analysis pump, and 1_2 marks the switch from the analysis pump to the cleaning pump.

清洁泵的控制如下述表4所示。The control of the cleaning pump is shown in Table 4 below.

表4Table 4

Figure BDA0002297118240000141
Figure BDA0002297118240000141

分析泵的控制如下述表5所示。The controls for the assay pumps are shown in Table 5 below.

表5table 5

Figure BDA0002297118240000142
Figure BDA0002297118240000142

1.4.4其他1.4.4 Others

分析时间为12.5min;柱温为55℃;进样量为30μL;流速为0.3mL/min。The analysis time was 12.5 min; the column temperature was 55°C; the injection volume was 30 μL; the flow rate was 0.3 mL/min.

1.4.5检测器1.4.5 Detector

紫外检测器是DAD-3000检测器,其检测波长为300nm,采集频率为5Hz,带宽为4nm。The ultraviolet detector is a DAD-3000 detector with a detection wavelength of 300nm, an acquisition frequency of 5Hz, and a bandwidth of 4nm.

1.4.6在线过滤器1.4.6 Online filter

在线过滤器为SSI COL PRE-FILTER WATER 1/16 0.5M。The in-line filter is SSI COL PRE-FILTER WATER 1/16 0.5M.

1.5标准工作液的配制1.5 Preparation of standard working solution

标准工作液:取适量标准储备液,用50%的甲醇水溶液进行稀释,配制成氯米帕明标准品的浓度分别为500ng/mL、1000ng/mL、2000ng/mL、4000ng/mL、6000ng/mL、12000ng/mL、15000ng/mL,N-去甲氯米帕明标准品的浓度分别为1000ng/mL、2000ng/mL、4000ng/mL、8000ng/mL、12000ng/mL、24000ng/mL、30000ng/mL的标准工作液,并在-80℃条件下保存。Standard working solution: Take an appropriate amount of standard stock solution, dilute it with 50% methanol aqueous solution, and prepare the concentrations of clomipramine standard products as 500ng/mL, 1000ng/mL, 2000ng/mL, 4000ng/mL, 6000ng/mL , 12000ng/mL, 15000ng/mL, the concentration of N-desmethylclomipramine standard is 1000ng/mL, 2000ng/mL, 4000ng/mL, 8000ng/mL, 12000ng/mL, 24000ng/mL, 30000ng/mL standard working solution and stored at -80°C.

可见,七个标准工作液中,氯米帕明标准品及N-去甲氯米帕明标准品的浓度不同且依次递增。It can be seen that among the seven standard working solutions, the concentrations of the clomipramine standard substance and N-desmethylclomipramine standard substance are different and increase sequentially.

1.6内标工作液的配制1.6 Preparation of internal standard working solution

内标工作液:取适量内标储备液,用50%的甲醇水溶液进行稀释,得到亚氨基芪浓度为20μg/mL的内标工作液,内标工作液-80℃条件下保存。Internal standard working solution: Take an appropriate amount of internal standard stock solution and dilute it with 50% aqueous methanol to obtain an internal standard working solution with an iminostilbene concentration of 20 μg/mL, and store the internal standard working solution at -80°C.

1.7标准溶液的配制1.7 Preparation of standard solution

用移液器分别移取10μL标准工作液、10μL内标工作液和190μL空白血清或血浆,分别置于1.5mL离心管中,在转速为2500rpm下涡旋混匀1min后,加入100μL甲醇,并在转速为2500rpm下涡旋混匀1min后,加入1000μL正己烷,并在转速为2500rpm下涡旋混匀20min后,再在14000rpm的转速下高速离心5min,移取900μL上清液放入另一支1.5mL离心管中,在常温下用N2缓慢吹干,向吹干的离心管中加入100μL 50%的甲醇水溶液(甲醇:水为50:50),然后在2500rpm转速下涡旋混匀3min后,再在14000rpm的转速下高速离心5min,取上清液作为待检测的标准溶液。Use a pipette to pipette 10 μL of standard working solution, 10 μL of internal standard working solution and 190 μL of blank serum or plasma respectively, place them in 1.5 mL centrifuge tubes, vortex and mix at a speed of 2500 rpm for 1 min, add 100 μL of methanol, and After vortex mixing at 2500 rpm for 1 min, add 1000 μL of n-hexane, and vortex mixing at 2500 rpm for 20 min, then centrifuge at 14000 rpm for 5 min at high speed, pipette 900 μL of supernatant into another In a 1.5mL centrifuge tube, blow dry slowly with N2 at room temperature, add 100 μL of 50% methanol aqueous solution (methanol:water ratio 50:50) to the dry centrifuge tube, and then vortex mix at 2500rpm for 3min After that, centrifuge at a high speed of 14000rpm for 5min, and take the supernatant as the standard solution to be detected.

如此,针对七个标准工作液,可以得到七个标准溶液。In this way, for seven standard working solutions, seven standard solutions can be obtained.

1.8检测标准溶液,生成标准曲线方程1.8 Detect standard solution and generate standard curve equation

得到各个标准溶液后,即可利用高效液相色谱仪对七个标准溶液分别进行检测,对应得到各个标准溶液的色谱图。After each standard solution is obtained, the seven standard solutions can be detected by using a high-performance liquid chromatograph, and the chromatograms of each standard solution can be obtained correspondingly.

请参考图4,图4示出了一标准溶液中氯米帕明标准品、N-去甲氯米帕明及内标物(即亚氨基芪标准品)的色谱图。Please refer to Fig. 4, Fig. 4 shows the chromatogram of clomipramine standard substance, N-desmethylclomipramine and internal standard substance (ie iminostilbene standard substance) in a standard solution.

以氯米帕明为例,从标准溶液的色谱图中,可以得到氯米帕明标准品的色谱峰面积和亚氨基芪标准品的色谱峰面积,然后结合各个标准溶液中氯米帕明标准品和亚氨基芪标准品的已知浓度,即可得到氯米帕明的标准曲线方程。同理,可以得到N-去甲氯米帕明的标准曲线方程。Taking clomipramine as example, from the chromatogram of standard solution, can obtain the chromatographic peak area of clomipramine standard substance and the chromatographic peak area of iminostilbene standard substance, then combine the clomipramine standard in each standard solution The known concentration of product and iminostilbene standard can obtain the standard curve equation of clomipramine. Similarly, the standard curve equation of N-desmethylclomipramine can be obtained.

请参考图6,图6示出了得到的氯米帕明线性关系图,根据线性关系图,可以得到氯米帕明的标准曲线方程:Y=0.1789×X-0.2293,相关系数R2=0.99939,Y为氯米帕明与内标的浓度比,X为氯米帕明与内标的面积比。Please refer to Figure 6, Figure 6 shows the obtained linear relationship diagram of clomipramine, according to the linear relationship diagram, the standard curve equation of clomipramine can be obtained: Y=0.1789×X-0.2293, correlation coefficient R 2 =0.99939 , Y is the concentration ratio of clomipramine and internal standard, X is the area ratio of clomipramine and internal standard.

可以看出,氯米帕明在25-750ng/mL线性范围内相关系数R2>0.9900,表示线性关系良好,基于该标准曲线方程来计算血液样本中氯米帕明的含量时,准确性高,误差小。It can be seen that the correlation coefficient R 2 >0.9900 of clomipramine in the linear range of 25-750ng/mL indicates a good linear relationship, and the accuracy of calculating the content of clomipramine in blood samples based on the standard curve equation is high , the error is small.

请参考图7,图7示出了得到的N-去甲氯米帕明线性关系图,根据线性关系图,可以得到N-去甲氯米帕明的标准曲线方程:Y=0.1713×X-0.3935,相关系数R2=0.99927,Y为N-去甲氯米帕明与内标的浓度比,X为N-去甲氯米帕明与内标的面积比。Please refer to Fig. 7, Fig. 7 shows the obtained N-desmethylclomipramine linear relationship diagram, according to the linear relationship diagram, the standard curve equation of N-desmethylclomipramine can be obtained: Y=0.1713×X- 0.3935, correlation coefficient R 2 =0.99927, Y is the concentration ratio of N-desmethylclomipramine and the internal standard, X is the area ratio of N-desmethylclomipramine and the internal standard.

可以看出,N-去甲氯米帕明在50-1500ng/mL线性范围内相关系数R2>0.9900,表示线性关系良好,基于该标准曲线方程来计算血液样本中N-去甲氯米帕明的含量时,准确性高,误差小。It can be seen that the correlation coefficient R 2 of N-desmethylclomipramine in the linear range of 50-1500ng/mL>0.9900, indicating that the linear relationship is good, based on the standard curve equation to calculate the N-desmethylclomipramine in blood samples When the content is clear, the accuracy is high and the error is small.

得到标准曲线方程后,即可对血液样本作前处理,以得到待测样本,进而在相同检测条件下,对待测样本进行检测,结合得到的各个标准曲线方程,即可得到血液样本中氯米帕明及N-去甲氯米帕明的含量。〃After the standard curve equation is obtained, the blood sample can be pretreated to obtain the sample to be tested, and then under the same detection conditions, the sample to be tested is tested, combined with the obtained standard curve equations, the clomiphene concentration in the blood sample can be obtained. Peramine and N-desmethylclomipramine content. 〃

实施例2Example 2

本发明实施例用于检测静脉血中氯米帕明及N-去甲氯米帕明的含量。The embodiment of the present invention is used to detect the content of clomipramine and N-desmethylclomipramine in venous blood.

2.1获取血液样本2.1 Obtaining blood samples

血液样本经处理至少5mL待检测血液而得到。得到血液样本后,即可作前处理,以得到相应可直接上样的待测样本。The blood sample is obtained by processing at least 5mL of blood to be tested. After the blood sample is obtained, it can be pre-treated to obtain a corresponding sample to be tested that can be directly loaded.

2.2血液样本前处理2.2 Blood sample pretreatment

用移液枪移取10μL内标工作液于1.5mL的离心管中,然后加入200μL血液样本,在转速为2500rpm下涡旋混匀1min后,加入100μL甲醇,并在转速为2500rpm下涡旋混匀1min后,加入1000μL正己烷,并在转速为2500rpm下涡旋混匀20min后,再在14000rpm的转速下高速离心5min;取900μL上清液放入另一支1.5mL离心管中,在常温下用N2缓慢吹干;向吹干的离心管中加入100μL 50%的甲醇水溶液(甲醇:水为50:50),然后在2500rpm转速下涡旋混匀3min后,再在14000rpm的转速下高速离心5min,得到上清液即为待测样本。Use a pipette gun to pipette 10 μL of internal standard working solution into a 1.5 mL centrifuge tube, then add 200 μL of blood sample, vortex mix at 2500 rpm for 1 min, add 100 μL of methanol, and vortex mix at 2500 rpm After homogenizing for 1 min, add 1000 μL of n-hexane, vortex and mix at 2500 rpm for 20 min, then centrifuge at 14000 rpm for 5 min at high speed; take 900 μL of supernatant and put it into another 1.5 mL centrifuge tube. Blow dry slowly with N2; add 100 μL of 50% methanol aqueous solution (methanol: water ratio 50:50) to the blow-dried centrifuge tube, then vortex mix at 2500 rpm for 3 min, and then vortex at 14000 rpm After centrifugation for 5 minutes, the supernatant obtained was the sample to be tested.

2.3待测样本检测2.3 Detection of samples to be tested

在实施例1的检测条件下,使用同一高效液相色谱仪对待测样本进行检测,得到待测样本的色谱图。Under the detection conditions of Example 1, the same high performance liquid chromatograph was used to detect the sample to be tested, and the chromatogram of the sample to be tested was obtained.

请参考图5,图5示出了待测样本中氯米帕明、N-去甲氯米帕明及内标物(即亚氨基芪标准品)的色谱图。Please refer to FIG. 5. FIG. 5 shows the chromatograms of clomipramine, N-desmethylclomipramine and internal standard (ie iminostilbene standard) in the sample to be tested.

请参考图4和图5,待测样本中氯米帕明的保留时间与标准溶液中氯米帕明标准品的保留时间相一致,待测样本中N-去甲氯米帕明的保留时间与标准溶液中N-去甲氯米帕明标准品的保留时间相一致,以亚氨基芪为内标物,使得目标化合物的识别更为准确,分析时间短、干扰小,内标定量适宜、特异性强、准确度和灵敏度高。Please refer to Fig. 4 and Fig. 5, the retention time of clomipramine in the sample to be tested is consistent with the retention time of clomipramine standard substance in the standard solution, and the retention time of N-desmethylclomipramine in the sample to be tested is Consistent with the retention time of the N-desmethylclomipramine standard in the standard solution, using iminostilbene as the internal standard makes the identification of the target compound more accurate, the analysis time is short, the interference is small, the internal standard is quantitatively suitable, Strong specificity, high accuracy and sensitivity.

此外,上面提到,为规避因原型萃取率、代谢物萃取率与内标萃取率不一致所带来的定值偏差,标准工作液和血液样本会进行相同的前处理过程,使得经相同前处理后,标准溶液及待测样本的色谱图中通常均只有原型、代谢物及内标的色谱峰,色谱柱洗脱阶段的溶剂峰也基本一致,未发现其他明显的杂质。基于此,也说明了上述前处理方法比较合理,未引入基质中的其他物质。In addition, as mentioned above, in order to avoid the value deviation caused by the inconsistency between the extraction rate of the prototype, the extraction rate of metabolites and the extraction rate of the internal standard, the standard working solution and blood samples will undergo the same pretreatment process, so that after the same pretreatment Finally, in the chromatograms of the standard solution and the sample to be tested, there are usually only the chromatographic peaks of the prototype, metabolites and internal standards, and the solvent peaks in the elution stage of the chromatographic column are basically the same, and no other obvious impurities are found. Based on this, it also shows that the above-mentioned pretreatment method is more reasonable and does not introduce other substances in the matrix.

2.4待测样本中氯米帕明及N-去甲氯米帕明含量的计算2.4 Calculation of the content of clomipramine and N-desmethylclomipramine in the sample to be tested

根据待测样本的色谱图中,氯米帕明、N-去甲氯米帕明和内标物的色谱峰面积,以及待测样本中内标物的已知浓度,代入上述2个标准曲线方程,即可计算出待测样本中氯米帕明及N-去甲氯米帕明的含量。According to the chromatogram of the sample to be tested, the chromatographic peak areas of clomipramine, N-desmethylclomipramine and the internal standard, and the known concentration of the internal standard in the sample to be tested, substitute the above two standard curve equations , the content of clomipramine and N-desmethylclomipramine in the sample to be tested can be calculated.

实施例3Example 3

本发明实施例用于测定定量限和检测限。The embodiments of the present invention are used to determine the limit of quantification and limit of detection.

选择适当浓度的标准溶液,用空白血液做不同程度的稀释,从而制备得到不同浓度的血液样本稀释液,并按实施例2中的血液样本前处理方式及测定条件,对这些血液样本稀释液进行测定。Select a standard solution with an appropriate concentration, and dilute it to different degrees with blank blood to prepare blood sample dilutions with different concentrations, and perform blood sample dilutions on these blood sample dilutions according to the blood sample pretreatment method and measurement conditions in Example 2. Determination.

经检测发现,氯米帕明的检测限和定量限如下所示:After testing, it was found that the detection limit and quantification limit of clomipramine are as follows:

(1)检测限(LOD):6.25ng/mL。(1) Limit of detection (LOD): 6.25 ng/mL.

(2)定量限(LOQ):12.5ng/mL。(2) Limit of quantification (LOQ): 12.5 ng/mL.

经检测发现,N-去甲氯米帕明的检测限和定量限如下所示:After testing, it was found that the detection limit and quantification limit of N-desmethylclomipramine are as follows:

(1)检测限(LOD):12.5ng/mL。(1) Limit of detection (LOD): 12.5 ng/mL.

(2)定量限(LOQ):20.0ng/mL。(2) Limit of quantification (LOQ): 20.0 ng/mL.

由本实施例可知,氯米帕明的检测限可低至6.25ng/mL,定量限可低至12.5ng/mL,N-去甲氯米帕明的检测限可低至12.5ng/mL,定量限可低至20.0ng/mL,灵敏度高,可以提高低浓度样本的平行性,增强准确度。As can be seen from this embodiment, the detection limit of clomipramine can be as low as 6.25ng/mL, and the limit of quantification can be as low as 12.5ng/mL, and the detection limit of N-desmethylclomipramine can be as low as 12.5ng/mL, quantitatively The limit can be as low as 20.0ng/mL, and the sensitivity is high, which can improve the parallelism of low concentration samples and enhance the accuracy.

由于灵敏度高,故本发明实施例对待检测血液的样本量的要求会更加的宽泛,从而提高样本检测的整体准确度。此外,本发明实施例对氯米帕明及N-去甲氯米帕明含量很低的生物样本也能准确定量,保证了检测方法的高度准确性及广泛适用性。Due to the high sensitivity, the embodiment of the present invention has wider requirements on the sample volume of the blood to be tested, thereby improving the overall accuracy of sample detection. In addition, the embodiments of the present invention can also accurately quantify biological samples with very low content of clomipramine and N-desmethylclomipramine, which ensures the high accuracy and wide applicability of the detection method.

实施例4Example 4

本发明实施例用于测定回收率和精密度。The embodiments of the present invention are used to determine recovery and precision.

取标准工作液,配制成高、中、低3种浓度,进行加样回收率和精密度实验,按实施例2中的检测方法进行测定,分析测定3批次,氯米帕明及N-去甲氯米帕明的回收率和精密度如表6所示。Get standard working solution, be mixed with high, medium and low 3 kinds of concentrations, carry out sample addition recovery rate and precision experiment, measure by the detection method in the embodiment 2, analyze and measure 3 batches, clomipramine and N- The recovery and precision of norclomipramine are shown in Table 6.

表6Table 6

Figure BDA0002297118240000191
Figure BDA0002297118240000191

可以看出,氯米帕明及N-去甲氯米帕明在低、中、高的3个添加水平范围内,平均回收率为93.9%~106.1%,重现性良好,加样回收率良好,精密度为1.1%~2.7%,检测结果的准确度较高,可消除系统误差。It can be seen that the average recovery rate of clomipramine and N-desmethylclomipramine in the low, medium and high ranges of three addition levels is 93.9% to 106.1%, with good reproducibility. Good, the precision is 1.1% to 2.7%, the accuracy of the test results is high, and the system error can be eliminated.

对比例1Comparative example 1

固相萃取-高效液相色谱法同时测定血浆中氯米帕明和4种苯二氮

Figure BDA0002297118240000192
类药物浓度方法的研究,期刊号为:中国药理学通报,2015Apr;31(4):582~5。Simultaneous Determination of Clomipramine and Four Benzodiazepines in Plasma by Solid Phase Extraction-High Performance Liquid Chromatography
Figure BDA0002297118240000192
Research on the concentration method of similar drugs, the journal number is: China Pharmacology Bulletin, 2015Apr; 31(4):582~5.

一、检测对象1. Detection object

对比例1可检测血液中氯米帕明的含量,但未检测血液中N-去甲氯米帕明的含量。而本发明实施例可同时检测血液中氯米帕明及N-去甲氯米帕明的含量。Comparative Example 1 can detect the content of clomipramine in the blood, but does not detect the content of N-desmethylclomipramine in the blood. However, the embodiment of the present invention can simultaneously detect the contents of clomipramine and N-desmethylclomipramine in blood.

对比例1仅检测了药物原型的血药浓度,而根据神经精神药理学治疗药物血药监测共识指南描述,其代谢物也具有药理活性,同样会影响治疗效果。本发明实施例不仅检测了药物的原型,还同时检测了其活性代谢物,如此,可以利用本发明实施例提供的检测方法,在临床治疗中对患者体内的氯米帕明及其代谢物N-去甲氯米帕明的含量进行监测,为氯米帕明及其代谢物N-去甲氯米帕明的个性化给药、减少毒副反应的发生提供实验基础,从而更加利于指导患者用药。In Comparative Example 1, only the blood concentration of the drug prototype was detected, and according to the consensus guidelines for blood drug monitoring of neuropsychopharmacological therapeutic drugs, its metabolites are also pharmacologically active and will also affect the therapeutic effect. The embodiment of the present invention not only detects the prototype of the drug, but also detects its active metabolites at the same time. In this way, the detection method provided by the embodiment of the present invention can be used to detect clomipramine and its metabolite N in the patient's body during clinical treatment. -Monitoring the content of desmethylclomipramine, providing an experimental basis for the personalized administration of clomipramine and its metabolite N-desmethylclomipramine, and reducing the occurrence of toxic and side effects, so as to be more conducive to guiding patients medication.

二、样本前处理2. Sample pretreatment

(1)对比例1中,标准工作液和血液样本的样本前处理不同,而本发明实施例中,标准工作液和血液样本的样本前处理保持一致。(1) In Comparative Example 1, the sample pretreatments of the standard working solution and the blood sample are different, but in the embodiment of the present invention, the sample pretreatment of the standard working solution and the blood sample are consistent.

由于前处理相一致,故本发明实施例能够校正原型萃取率、代谢物萃取率与内标萃取率不一致带来的定值误差,提高检测准确率。Since the pre-treatments are consistent, the embodiment of the present invention can correct the fixed value error caused by the inconsistency between the extraction rate of the prototype, the extraction rate of metabolites and the extraction rate of the internal standard, and improve the detection accuracy.

(2)对比例1中,血液样本前处理过程为:取固相萃取柱BondElut-C1(100mg,1mL)(美国Agilent公司),依次用5mL甲醇、超纯水活化;取500μL血浆,加入1000μL磷酸盐缓冲液(pH7.0),加入内标工作液50μL,涡旋1min混匀后上样至上述C1柱;分别用1mL去离子水、1mL水/甲醇混合液(80∶20,V/V)淋洗1次弃去;用2mL甲醇洗脱,收集洗脱液,45℃氮气吹干,复溶于125μL流动相,离心后取上清液进样。(2) In comparative example 1, the blood sample pretreatment process is as follows: take a solid phase extraction column BondElut-C1 (100mg, 1mL) (Agilent, USA), activate it with 5mL methanol and ultrapure water in turn; take 500μL plasma, add 1000μL Phosphate buffer (pH7.0), add 50 μL of internal standard working solution, vortex for 1 min to mix well, and then load the sample to the above C1 column; use 1 mL deionized water and 1 mL water/methanol mixture (80:20, V/ V) Rinse once and discard; elute with 2 mL of methanol, collect the eluate, dry it with nitrogen at 45°C, redissolve in 125 μL of mobile phase, and take the supernatant for sampling after centrifugation.

本发明实施例的血液样本前处理过程请参考以上相关描述,在此不作赘述。For the pretreatment process of the blood sample in the embodiment of the present invention, please refer to the relevant description above, and details are not repeated here.

可以看出,对比例1虽然同样采用内标法定量,但其前处理方法较复杂,容易因操作误差导致定值结果不准。本发明实施例同样采用了内标法,但前处理方法相对简单,能够避免操作带来的误差,从而能够更准确的定量。It can be seen that although the internal standard method is also used in Comparative Example 1 for quantification, the pretreatment method is more complicated, and the determination result is likely to be inaccurate due to operational errors. The embodiment of the present invention also adopts the internal standard method, but the pretreatment method is relatively simple, and can avoid errors caused by operation, thereby enabling more accurate quantification.

(3)对比例1中,在萃取操作前未加入甲醇以执行沉淀蛋白操作,而本发明实施例中,在萃取操作前,加入甲醇以沉淀蛋白。(3) In Comparative Example 1, no methanol was added to precipitate protein before the extraction operation, but in the embodiment of the present invention, methanol was added to precipitate protein before the extraction operation.

考虑到蛋白结合率偏高应该会导致萃取率偏低,且没有甲醇步骤时,得到的实验结果与真实值偏差较大,故本发明实施例的检测准确率更高。Considering that a high protein binding rate should lead to a low extraction rate, and when there is no methanol step, the experimental results obtained deviate greatly from the true value, so the detection accuracy of the embodiment of the present invention is higher.

三、血液需求量3. Blood requirement

对比例1中,血清量较大,需要500μL,而本发明实施例的血液样本量较小,需要200μL,故可相应减少患者采血量,提升患者依从性。In Comparative Example 1, the serum volume is relatively large, requiring 500 μL, while the blood sample volume in the embodiment of the present invention is relatively small, requiring 200 μL, so the amount of blood collected by patients can be reduced accordingly, and patient compliance can be improved.

四、内标物的选用4. Selection of internal standard

对比例1所用内标为苯海拉明,适用于过敏及晕车的治疗,应用较为普遍,容易因联合用药导致对样本分析的干扰。The internal standard used in Comparative Example 1 is diphenhydramine, which is suitable for the treatment of allergies and motion sickness. It is widely used, and it is easy to interfere with the sample analysis due to the combination of drugs.

而本发明实施例所用内标为亚氨基芪,是一种用于药物合成的中间体,基本不会存在于患者体内。同使用功效相近的精神类药物或常用药作为内标相比,具有很大的优势,可以避免联合用药给分析带来的相互干扰。However, the internal standard used in the examples of the present invention is iminostilbene, which is an intermediate used in drug synthesis and basically does not exist in the patient's body. Compared with using psychotropic drugs or commonly used drugs with similar efficacy as internal standards, it has great advantages, and can avoid mutual interference brought by combined drugs to the analysis.

五、分析时间5. Analysis time

对比例1中,氯米帕明在13min出峰,分析时间不低于21min。而本发明实施例中,N-去甲氯米帕明和氯米帕明的保留时间在3.75-5min之间,内标物亚氨基芪的保留时间约为10.3min,分析时间为12.5min。In Comparative Example 1, clomipramine peaked at 13 minutes, and the analysis time was not less than 21 minutes. In the embodiment of the present invention, the retention time of N-desmethylclomipramine and clomipramine is between 3.75-5min, the retention time of the internal standard iminostilbene is about 10.3min, and the analysis time is 12.5min.

可见,本发明实施例的分析时间明显缩短,极大地提高了分析效率,有利于大通量血液样本的检测。It can be seen that the analysis time of the embodiment of the present invention is significantly shortened, the analysis efficiency is greatly improved, and it is beneficial to the detection of large-throughput blood samples.

六、检测限、定量限、回收率及精密度6. Limit of detection, limit of quantitation, recovery and precision

对比例1中,氯米帕明在5.0~200μg/L线性范围内呈线性,最低检出浓度为5.5μg/L,定量限为18.5μg/L,方法回收率为84.11%~87.78%,相对标准偏差(RSD)为3.53%~13.21%。In Comparative Example 1, clomipramine was linear in the linear range of 5.0-200 μg/L, the minimum detection concentration was 5.5 μg/L, the limit of quantification was 18.5 μg/L, and the recovery rate of the method was 84.11%-87.78%. The standard deviation (RSD) ranged from 3.53% to 13.21%.

而本发明实施例中,氯米帕明在25-750ng/mL线性范围内呈线性,检测限为6.25ng/mL,定量限为12.5ng/mL,N-去甲氯米帕明在50-1500ng/mL线性范围内呈线性,检测限为12.5ng/mL,定量限为20.0ng/mL,平均回收率为93.9%~106.1%,精密度(RSD)为1.1%~2.7%。And in the embodiment of the present invention, clomipramine is linear in the linear range of 25-750ng/mL, the detection limit is 6.25ng/mL, the limit of quantification is 12.5ng/mL, and N-desmethylclomipramine is 50- It was linear in the linear range of 1500ng/mL, the limit of detection was 12.5ng/mL, the limit of quantification was 20.0ng/mL, the average recovery was 93.9%-106.1%, and the precision (RSD) was 1.1%-2.7%.

可以看出,对于氯米帕明来说,本发明实施例的定量限、回收率及精密度,均优于对比例1,检测限与对比例1基本一致。对于N-去甲氯米帕明来说,本发明实施例的检测限、定量限、回收率及精密度均较佳。It can be seen that for clomipramine, the quantitative limit, recovery rate and precision of the examples of the present invention are all better than those of Comparative Example 1, and the detection limit is basically the same as that of Comparative Example 1. For N-desmethylclomipramine, the detection limit, quantification limit, recovery rate and precision of the embodiment of the present invention are all better.

因此,本发明实施例的检测限和定量限较低,检测方法的灵敏度更高,回收率及精密度更好,使得对样本量的要求可以更加的宽泛,样本检测的整体准确度更高。Therefore, the detection limit and quantification limit of the embodiment of the present invention are lower, the sensitivity of the detection method is higher, the recovery rate and precision are better, so that the requirements for the sample size can be wider, and the overall accuracy of sample detection is higher.

七、检测成本7. Testing cost

对比例1使用固相萃取结合高效液相色谱法来实现检测目的,而本发明实施例使用高效液相色谱法来实现检测目的。与固相萃取法相比,本发明实施例的分析成本较低。Comparative Example 1 uses solid phase extraction combined with high performance liquid chromatography to achieve the detection purpose, while the embodiment of the present invention uses high performance liquid chromatography to achieve the detection purpose. Compared with the solid phase extraction method, the analysis cost of the embodiment of the present invention is lower.

综上所述,与对比例1相比,本发明实施例提供的血液中氯米帕明及N-去甲氯米帕明含量的检测方法,具有样本前处理简单、分析时间短、内标选取合理、检测灵敏度高、回收率和精密度好、检测准确率高、检测成本低等优点,利于大量样本的检测,故在临床检测领域更具优势。In summary, compared with Comparative Example 1, the detection method for the content of clomipramine and N-desmethylclomipramine in the blood provided by the embodiment of the present invention has the advantages of simple sample pretreatment, short analysis time, internal standard The advantages of reasonable selection, high detection sensitivity, good recovery rate and precision, high detection accuracy, and low detection cost are conducive to the detection of a large number of samples, so they have more advantages in the field of clinical detection.

最后需要说明的是:以上所述仅为本发明的较佳实施例,仅用于说明本发明的技术方案,并非用于限定本发明的保护范围。凡在本发明的精神和原则之内所做的任何修改、等同替换、改进等,均包含在本发明的保护范围内。Finally, it should be noted that: the above descriptions are only preferred embodiments of the present invention, and are only used to illustrate the technical solutions of the present invention, and are not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention are included in the protection scope of the present invention.

Claims (5)

1. A method for simultaneously detecting the contents of clomipramine and N-norclomipramine in blood is characterized by comprising the following steps:
preparing at least three standard working solutions, wherein the standard working solutions contain clomipramine standard substances and N-norclomipramine standard substances with known concentrations, and the concentrations of the same standard substance in different standard working solutions are different; the diluent of the standard working solution is 40-60% methanol water solution;
mixing a certain amount of standard working solution, internal standard working solution and a blank blood sample, and performing sample pretreatment to obtain standard solution, wherein the internal standard working solution contains an internal standard substance with known concentration; the dosage of the standard working solution is 10 muL, the dosage of the internal standard working solution is 10 muL, the dosage of the blank blood sample is 150-250 muL,
respectively detecting at least three standard solutions by using a high performance liquid chromatograph under certain detection conditions to obtain chromatograms of the standard solutions;
fitting to obtain a standard curve equation of clomipramine and a standard curve equation of N-norclomipramine according to the chromatogram of each standard solution;
mixing a certain amount of internal standard working solution with a blood sample, wherein the using amount of the internal standard working solution is 10 mu L, and the using amount of the blood sample is 150-250 mu L, and performing the same sample pretreatment to obtain a sample to be detected, wherein the blood sample is obtained by treating blood to be detected;
detecting a sample to be detected by using a high performance liquid chromatograph under the same detection condition to obtain a chromatogram of the sample to be detected;
calculating the contents of clomipramine and N-norclomipramine in the blood sample according to the chromatogram of the sample to be detected and each standard curve equation obtained by fitting;
the sample pretreatment comprises the following A1 to A5:
a1 comprises: mixing the mixed solution at 1800-2800rpm for 1-2min;
a2 comprises: adding 80-120 μ L methanol, and mixing for 1-2min at rotation speed of 1800-2800 rpm;
a3 comprises the following steps: adding 800-1200 μ L of extractant, mixing uniformly at 1800-2800rpm for 15-25min, and centrifuging at 10000-15000rpm for 3-7min to obtain supernatant;
a4 comprises: transferring 700-1000 μ L of supernatant into a centrifuge tube, and blow-drying with nitrogen at normal temperature;
a5 comprises: adding 80-120 μ L of the complex solution into an upward blowing centrifugal tube, uniformly mixing at 1800-2800rpm for 2-4min in a vortex manner, and centrifuging at 10000-15000rpm for 4-6min to obtain a supernatant; the compound solution is 40-60% methanol water solution;
the detection conditions include: a Waters xbridge c18 column having a length of 150mm, an internal diameter of 2.1mm, and a packing particle size of 3.5 μm; the column temperature is 50-60 ℃, the mobile phase A is water containing ammonium acetate with the concentration of 80-120mmol/L, formic acid with the volume ratio of 0.15-0.25% and triethylamine with the volume ratio of 0.15-0.25%, the mobile phase B is acetonitrile, the flow rate is 0.2-0.4mL/min, the sample injection amount is 20-40 mu L, and the analysis time is 12-13 min; the detection wavelength is 295-305nm;
the detection conditions include: single column dual pump elution mode;
the double-pump switching mode is as follows: switching from the analysis pump to the cleaning pump at 10.7min, and switching from the cleaning pump to the analysis pump at 11.2 min;
the elution mode is as follows: the volume ratio of the mobile phase A to the mobile phase B is 59-61% in other elution time ranges except 10.7-11.2 min: 41 to 39 percent.
2. The method of claim 1, wherein the uv detector in the hplc is a DAD-3000 detector with a collection frequency of 5Hz and a bandwidth of 4nm.
3. The method of claim 1, wherein the in-line filter used in the hplc is SSI COL PRE-FILTERWATER 1/16 0.5m.
4. The method of claim 1, wherein the internal standard is iminostilbene.
5. The method according to any one of claims 1 to 4, wherein the concentration of clomipramine standard in the standard working fluid is between 500 and 15000ng/mL and the concentration of N-norclomipramine standard is between 1000 and 30000ng/mL.
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