应用超高效液相色谱 - 高分辨质谱法检测血样中秋水仙碱
庄顺, 林坚, 朱明怀, 林葭, 叶树海
应用超高效液相色谱 - 高分辨质谱法检测血样中秋水仙碱
Determination of Colchicine in Blood by UPLC-Q/Orbitrap HRMS
秋水仙碱是一种强效生物碱，因其具有特殊的结构及药效，受到人们广泛的关注。多项研究都表明秋水仙碱体内血药浓度较低，因此对其检测分析方法的灵敏度要求较高。本文建立了使用超高效液相色谱－高分辨质谱仪（UPLC-Q/Orbitrap HRMS）测定血样中秋水仙碱的检测方法。考察不同流动相和色谱柱对秋水仙碱分离效果的影响，结果表明在甲醇水体系作为流动相条件下，使用Accucore™ Phenyl-Hexyl（2.1 mm×100 mm×2.6 μm）色谱柱，仪器响应值高且分离效果较好。采用乙腈为蛋白沉淀剂，有机微孔过滤膜净化提取液，用UPLC-Q/Orbitrap HRMS对血样中秋水仙碱进行定性定量检验。分析采用Accucore™ Phenyl-Hexyl（2.1 mm×100 mm×2.6 μm）色谱柱，电喷雾离子源正离子（FullMS/ddMS2）模式，使用阶梯碰撞能量：35、60和85 eV，用5 mmol/L甲酸铵水溶液和甲醇作为流动相梯度洗脱。本方法血样中的秋水仙碱定量限为0.5 ng/mL；秋水仙碱在0.5~100 ng/mL 范围内线性良好（R2=0.998 5），3个浓度平均回收率为92.8%~98.3%，日内精密度为1.6%~7.1%（n=6）。 这一定性定量分析方法简易可靠，灵敏度高，适用于血样中秋水仙碱的检验鉴定。
As a potent alkaloid, colchicine has attracted much attention due to its unique structure and pharmacological properties. Colchicine has many medical applications, not only for the prevention and treatment of gouty arthritis, but also for the treatment of tumors, hepatitis and so on. Studies indicated that colchicine had low blood concentrations, so the method of determination of colchicine in blood should be more sensitive. Therefore, the objective of this article is to establish a sensitive method for analysis of colchicine in blood by liquid chromatogram-high resolution mass spectrum (UPLC-Q/Orbitrap HRMS). The effects of different mobile phases and chromatographic columns on the separation of colchicine were investigated. The results showed that the instrument had higher response value and better separation efficiency when Accucore™ Phenyl hexyl analytical column (2.1 mm×100 mm×2.6 μm) was used with methanol-water system as mobile phase. The blood sample (1.0 mL) was extracted with acetonitrile (1.0 mL) to precipitate protein. Then, its well-treated supernatant was separated on a Accucore™ Phenyl hexyl analytical column (2.1 mm×100 mm×2.6 μm) with gradient elution that was run through the mobile phases consisting of 5 mmol/L ammonium formate aqueous solution (mobile phase A) and methanol (mobile phase B). The analytes were analyzed with UPLC-Q/Orbitrap HRMS under the mode of positive-ion ESI in full MS/ddMS2. Step collision energies of 35 eV, 60 eV and 85 eV were used. Colchicine was linear with its calibration curve among the range of 0.5 to 100 ng/mL (R2=0.998 5)，which showed good recovery and inter-day precision less than 7.1%. The method established here is simple, reliable, sensitive and appropriate for determination of colchicine in blood. In addition, colchicine is unstable when exposed to sunlight in solution. Attention should be paid to avoiding sunlight during the experiment so as to make the analysis results more reliable.
法医毒物学 / 超高效液相色谱－高分辨质谱 / 秋水仙碱 / 血样 {{custom_keyword}} /
forensic toxicology / UPLC-Q/Orbitrap HRMS / colchicine / blood sample {{custom_keyword}} /
表1 秋水仙碱的离子质量及其测量准确度和二级碎片离子Table 1 Accurate molecular weight, mass accuracy and fragment ions of colchicine |
| 名称 | 离子质量理论值(M+H) /(m/z) | 离子质量实测值(M+H)/(m/z) | 质量测量准确度/ppm | 二级碎片离子 | |
|---|---|---|---|---|---|
| 1 | 2 | ||||
| 秋水仙碱 | 400.17546 | 400.17487 | 1.47 | 358.16495 | 310.11920 |
表2 样本中秋水仙碱的回收率及精密度(n=6)Table 2 Precision and recovery of colchicine in samples (n=6) |
| 添加浓度/（ng/mL） | 精密度RSD/% | 回收率/% |
|---|---|---|
| 1 | 7.12 | 92.8 |
| 50 | 1.58 | 98.3 |
| 100 | 1.97 | 97.4 |
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A 73-year-old man developed nausea, vomiting, and diarrhea 20-30 min after receiving a 1.0 mg intravenous dose of colchicine for the treatment of severe pain due to gouty arthritis in his physician's office. He was hospitalized 8 h later, and his condition deteriorated as he developed renal and respiratory failure. He subsequently died 10 h later, or a total of 18 h after he received the original 1 mg colchicine injection. The patient received a prescription for oral 0.6 mg colchicine tablets 8 days previously and consumed eight tablets during that period, an average of 0.6 mg/day (42 of 50 tablets remained at the time of death). Colchicine concentrations were measured by liquid chromatography-mass spectrometry in selected ion monitoring mode using positive ionization. Chromatography was performed using an Eclipse XDB C8 analytical column (30 mm x 2.1-mm i.d., 3-microm particle size) and a programmed mobile phase consisting of 50 mM pH 4 ammonium acetate buffer and acetonitrile. Colchicine concentrations were as follows: 50 microg/L in cardiac blood, 10 microg/L in vitreous humor, 575 microg/kg in liver, 12,000 microg/L in bile, and 4.4 microg in 60 g received gastric contents (estimated total gastric contents 100 g). The cause of death was ruled to be "acute colchicine toxicity" and the manner of death "accidental."
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A case of suspected acute and lethal intoxication caused by colchicine has been reported. The woman was hospitalized after her suspicion of suicidal poisoning by a rare autumn crocus (Colchicum autumnale). Suspected colchicine poisoning was confirmed using a novel UHPLC method with a modern reversed-phase stationary phase with a sub 2-micron superficial porous particle size combined with a QTOF mass spectrometer. Sample preparation procedure included the addition of propiverine as internal standard, protein precipitation using methanol and solid phase extraction. High-resolution MS only and targeted MS/MS modes are reported for the qualitative analysis and screening of other potential drugs of abuse in blood samples. All Ion MS mode was used for quantitative determination of colchicine afterward. The concentration of colchicine in the blood sample was approximately 41 ng/mL, and more than 200 μg/mL of the plant extract used for the suicide.© 2018 American Academy of Forensic Sciences.
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徐琛, 李晓飞, 张云峰, 等. UPLC-HRMS快速检测血液中7种鼠药[J]. 刑事技术, 2021, 46(4): 365-369.
目的 建立血液中4类（7种）鼠药（毒鼠强、氟乙酸根、溴敌隆、大隆、杀鼠灵、杀鼠醚、氟鼠灵）的超高效液相色谱-高分辨质谱（UPLC-HRMS）快速检测方法。方法 采用乙腈为蛋白沉淀剂,用PLD<sup>+</sup>净化柱或有机微孔过滤膜净化提取液,用UPLC-HRMS对血液中的4类（7种）鼠药进行定性定量检测。分析采用Luna Omega PS C18(2.1mm×100mm, 3µm)色谱柱,质谱扫描方式为负离子的全扫描/实时二级质谱扫描（Full MS/ddMS<sup>2</sup>）模式,一级全扫描（m/z 50~750）分辨率为 70000,二级扫描（ddMS<sup>2</sup>）分辨率为17500。 结果 毒鼠强、氟乙酸根、溴敌隆、大隆、杀鼠灵、杀鼠醚、氟鼠灵的检出限均为50ng/mL,3个添加水平下的回收率为85.3%~94.7%,日内相对标准偏差（RSD）为3.4%~11%( n=6),日间相对标准偏差（RSD）为5.6%~13% ( n=6)。结论 使用本方法可同时准确测定血液中的4类（7种）鼠药。
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<strong>Objective</strong> To develop an analytical method for simultaneous determination of seven rodenticides (TETS, fluoroacetic acid, bromadiolone, brodifacoum, warfarin, coumatetralyl and flocoumafen) (categorized into 4 major kinds) in whole blood through ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). <strong>Methods</strong> The samples were treated with acetonitrile to precipitate protein, successively extracted through purification of PLD+ polymer column or organic microporous filter membrane. Luna Omega PS C18 chromatographic column (2.1mm×100mm, 3µm) was adopted for chromatographic separation with gradient elution. The analytes were detected under the mode of negative-ion ESI in full MS/ddMS<sup>2</sup>. <strong>Results</strong> Detection limits (LODs) of the seven rodenticides varied among 10~50 ng/mL. The recoveries ranged from 85.3%~94.7% at three spiked levels in whole blood. The intra-day relative standard deviation (RSD) was 3.4%~11% (<em>n</em>=6), with the inter-day’s being 5.6%~13% (<em>n</em>=6). <strong>Conclusion</strong> This method is both qualitative and quantitative, capable of simultaneous and accurate screening of seven rodenticides in whole blood.
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目的 建立N,N-二异丙基-5-甲氧基色胺（5-MeO-DiPT,俗称火狐狸）针剂（无针针筒给药器内液体）的GC-MS定性及GC/FID定量分析方法。方法 疑似含毒品针剂样品用乙酸乙酯提取,乙酸乙酯提取液浓缩至干,残渣溶解后用GC-MS及GC/FID进行检测。结果 测得未知组分的(tR=13.81min)质谱特征碎片峰信息为m/z 114（基峰）、274、174、160、145、130、117、72、43。经与标准物质5-MeO-DiPT的保留时间和质谱图比对,确定为N,N-二异丙基-5-甲氧基色胺。查阅资料对质谱图谱裂解机制进行推断解析。建立GC/FID定量分析方法,线性范围为20~500mg/L,三种添加浓度平均回收率85%以上。结论 文章建立的定性定量分析方法简易可靠,适用于含N,N-二异丙基-5-甲氧基色胺针剂液体的检验鉴定。
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<b>Objective</b> To establish a method for analysis of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DiPT, sometimes called the Foxy, usually kept in aqueous solution within needleless syringe) with GC-MS and GC/FID. <b>Methods</b> The sample, suspected of containing drug in its aqueous solution with a needleless syringe, was extracted with ethyl acetate, having the extract concentrated and afterwards analyzed with GC-MS and GC/FID. <b>Results</b> An unknown component presented characteristic fragment ion peaks at <em>m</em>/<em>z</em> 114 (base peak), 274, 174, 160, 145,130, 117, 72, 43, showing well-matched to the reference substance of 5-MeO-DiPT with both the retention time and characteristic fragment ion peaks. Thus, the drug in the injection was tentatively identified as 5-MeO-DiPT. The mechanism of the above-mentioned fragment of characteristic mass-spectrum peak was inferred by reference to relevant literatures. The standard curve was linear from 20mg/L to 500mg/L with the relating coefficient of 0.9999 and average recoveries more than 85 %. <b>Conclusion</b> The method established here is simple, reliable and suitable for identification of 5-MeO-DiPT in aqueous solution.
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