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Volume 38 Issue 2
Apr.  2020
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Article Navigation >  Journal of Pharmaceutical Practice and Service  > 2020  >  38(2): 148-151

XIN Bo, WAN Lili, WANG Jing, LIU Jinbian, HUO Yan, GUO Cheng. Cisatracurium assay in human plasma by LC-MS[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(2): 148-151. doi: 10.3969/j.issn.1006-0111.201909001
Citation: XIN Bo, WAN Lili, WANG Jing, LIU Jinbian, HUO Yan, GUO Cheng. Cisatracurium assay in human plasma by LC-MS[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(2): 148-151. doi: 10.3969/j.issn.1006-0111.201909001
shu

Cisatracurium assay in human plasma by LC-MS

doi: 10.3969/j.issn.1006-0111.201909001
  • 1.

    Department of Pharmacy, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 200233 Shanghai, China

  • 2.

    Department of Anesthesiology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 200233 Shanghai, China

  • Received Date: 2019-09-01
  • Rev Recd Date: 2020-01-09
    • Available Online: 2020-04-23
  • Publish Date: 2020-03-01
  •   Objective  To establish a LC-MS method of cisatracurium assay in human plasma for clinical therapeutic drug monitoring.  Method  Propafenone Hydrochloride was used as the internal standard. The plasma samples were treated with 2% formic acid aqueous solution and acetonitrile containing the internal standard to precipitate protein. Agilent SB-C18 column was used for gradient elution with the mobile phase of 0.1% formic acid-water and 0.1% formic acid-acetonitrile solution at 35 ℃ and 0.3 ml/min flow rate. The degradation products of cisatracurium m/z 464.6-358.4 and propafenone hydrochloride m/z 342.2-116.2 were identified by ESI positive-ion detection.  Results  There was a linear rage of cisatracurium in 2-500 ng/ml (r=0.996 5) with a detection limit of 2 ng/ml. The intra-day coefficients of variation (CVs) were less than 16.00%, and the inter-day CVs were less than 6.00%. The mean recoveries were in the range of 97.63%-111.93%. The plasma samples were stable for 4 hours at room temperature, 14 days at -80 ℃ and 24 hours after pretreated.  Conclusion  This method was simple, accurate, fast and repeatable for the cisatracurium assay in human plasma.
  • [1] SZAKMANY T, WOODHOUSE T. Use of cisatracurium in critical care: a review of the literature[J]. Minerva Anestesiol,2015,81(4):450-460.
    [2] DEAR G J, HARRELSON J C, JONES A E, et al. Identification of urinary and biliary conjugated metabolites of the neuromuscular blocker 51W89 by liquid chromatography/mass spectrometry[J]. Rapid Commun Mass Spectrom,1995,9(14):1457-1464. doi:  10.1002/rcm.1290091425
    [3] ZHANG H, WANG P, BARTLETT M G, et al. HPLC determination of cisatracurium besylate and propofol mixtures with LC-MS identification of degradation products[J]. J Pharm Biomed Anal,1998,16(7):1241-1249. doi:  10.1016/S0731-7085(97)00262-8
    [4] SAYER H, QUINTELA O, MARQUET P, et al. Identification and quantitation of six non-depolarizing neuromuscular blocking agents by LC-MS in biological fluids[J]. J Anal Toxicol,2004,28(2):105-110. doi:  10.1093/jat/28.2.105
    [5] GAO J Y, YANG T, YE M, et al. High-performance liquid chromatography assay with programmed flow elution for cisatracurium in human plasma: application to pharmacokinetics in infants and children[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2014,955-956:58-63. doi:  10.1016/j.jchromb.2014.02.023
    [6] GUO J R, YUAN X H, ZHOU X F, et al. Pharmacokinetics and pharmacodynamics of cisatracurium in patients undergoing surgery with two hemodilution methods[J]. J Clin Anesth,2017,38:75-80. doi:  10.1016/j.jclinane.2017.01.013
    [7] KISOR D F, SCHMITH V D, WARGIN W A, et al. Importance of the organ-independent elimination of cisatracurium[J]. Anesth Analg,1996,83(5):1065-1071. doi:  10.1213/00000539-199611000-00029
    [8] BŁAZEWICZ A, FIJAŁEK Z, WAROWNA-GRZEŚKIEWICZ M, et al. Determination of atracurium, cisatracurium and mivacurium with their impurities in pharmaceutical preparations by liquid chromatography with charged aerosol detection[J]. J Chromatogr A,2010,1217(8):1266-1272. doi:  10.1016/j.chroma.2009.12.025
    [9] 吴祝峰. 顺苯磺阿曲库铵在先天性心脏病患者中的药动学与药效学研究[D]. 广州: 暨南大学, 2016.
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Cisatracurium assay in human plasma by LC-MS

doi: 10.3969/j.issn.1006-0111.201909001
  • 1. Department of Pharmacy, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 200233 Shanghai, China
  • 2. Department of Anesthesiology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 200233 Shanghai, China
  • Received Date: 2019-09-01
  • Rev Recd Date: 2020-01-09
  • Available Online: 2020-04-23
  • Publish Date: 2020-03-01

Abstract:   Objective  To establish a LC-MS method of cisatracurium assay in human plasma for clinical therapeutic drug monitoring.  Method  Propafenone Hydrochloride was used as the internal standard. The plasma samples were treated with 2% formic acid aqueous solution and acetonitrile containing the internal standard to precipitate protein. Agilent SB-C18 column was used for gradient elution with the mobile phase of 0.1% formic acid-water and 0.1% formic acid-acetonitrile solution at 35 ℃ and 0.3 ml/min flow rate. The degradation products of cisatracurium m/z 464.6-358.4 and propafenone hydrochloride m/z 342.2-116.2 were identified by ESI positive-ion detection.  Results  There was a linear rage of cisatracurium in 2-500 ng/ml (r=0.996 5) with a detection limit of 2 ng/ml. The intra-day coefficients of variation (CVs) were less than 16.00%, and the inter-day CVs were less than 6.00%. The mean recoveries were in the range of 97.63%-111.93%. The plasma samples were stable for 4 hours at room temperature, 14 days at -80 ℃ and 24 hours after pretreated.  Conclusion  This method was simple, accurate, fast and repeatable for the cisatracurium assay in human plasma.

XIN Bo, WAN Lili, WANG Jing, LIU Jinbian, HUO Yan, GUO Cheng. Cisatracurium assay in human plasma by LC-MS[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(2): 148-151. doi: 10.3969/j.issn.1006-0111.201909001
Citation: XIN Bo, WAN Lili, WANG Jing, LIU Jinbian, HUO Yan, GUO Cheng. Cisatracurium assay in human plasma by LC-MS[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(2): 148-151. doi: 10.3969/j.issn.1006-0111.201909001
shu

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  • 顺式阿曲库铵（cisatracurium, CA）是一种新型中时效非去极化肌松药，其结构式如图1，因其代谢方式（Hofmann elimination，霍夫曼降解）独特，同时释放较低水平的组胺，在临床麻醉过程中被广泛应用。因与其他非去极化肌松药一样，存在术后肌松残留作用[1]，且已成为麻醉后呼吸系统不良事件的主要原因。因此，为保证患者围术期安全，对顺式阿曲库铵进行血药浓度监测十分必要。

    CA的稳定性主要受温度和pH影响，温度和pH升高可加快其消除，因此离体后的生物样本稳定性是血药浓度监测方法建立的一个难点。国内外文献报道测定人血浆中CA浓度的方法主要为高效液相色谱荧光检测法（HPLC-FD）及高效液相色谱串联质谱法（LC-MS）[2-6]，但样本前处理方法较为复杂。本研究在既往文献报道的基础上，建立了一种更加简便、准确、快速、稳定的LC-MS方法，适用于人血浆中CA的血药浓度测定。

1.   仪器与试药

  • 高效液相色谱仪（Agilent 6410，美国Agilent公司）；Triple Quad三重四极杆质谱仪，配电喷雾（ESI）离子源（美国Agilent公司）；纯水机（Millipore）；分析天平（BP211D，德国Sartorius公司）。对照品顺式阿曲库铵（cisatracurium，CA，批号：C496700，纯度90%，Toronto Reasearch Chemicals Inc公司）；对照品盐酸普罗帕酮（propafenone hydrochloride，PPF，批号：101190-201101，纯度99.9%，中国药品食品检定研究院）；乙腈、甲酸均为质谱纯（美国TEDIA公司）；水为自制超纯水。

2.   方法与结果

    2.1.   分析条件

    2.1.1.   色谱条件

  • Agilent SB-C18色谱柱（2.1 mm×50 mm, 3 µm），柱温35 ℃；流动相A为0.1%甲酸水溶液，B为0.1%甲酸乙腈溶液。线性梯度洗脱：0 ~1 min, 20% B；1~3 min，50% B；3 ~5 min，20% B; 5 ~10 min，20% B。流速0.3 ml/min；进样量2 µl。

    • 2.1.2.   质谱条件

  • 采用正离子工作模式；离子源为ESI源；多反应监测（multiple reaction monitoring, MRM）。毛细管电压4 000 V；干燥器温度350 ℃；干燥气流速12 L/min；雾化器压力35 psi。MRM监测离子对，离子驻留时间、碰撞诱导解离电压及碰撞能量等参数见表1。典型质谱图见图2。

    成分母离子子离子驻留时间（t/ms）碰撞电压(U/V)电子倍增电压( U/V)碰撞能量(U/V)
    CA464.4358.4 2011020020
    PPF342.2166.220013020017

    • 2.2.   对照品及内标溶液配制

  • CA标准储备液：精密称取CA 10 mg置10 ml容量瓶，加入2%甲酸甲醇溶液至刻度，溶解混匀，配制成相当于CA质量浓度为1 mg/ml的储备液，于冰箱（4 ℃）备用。

    CA标准工作液：精密吸取CA储备液适量，用50%甲醇水溶液配制成系列浓度的工作溶液，其中，CA质量浓度分别为5 000、2 500、1 000、500、250、100、20 ng/ml。

    PPF标准储备液：精密称取PPF 0.4 mg置2 ml容量瓶，加甲醇至刻度，溶解混匀，配制成相当于PPF质量浓度为0.2 mg/ml的储备液，于冰箱（4 ℃）备用。

    PPF标准工作液：精密吸取PPF储备液适量，配制成200 ng/ml的含内标乙腈溶液。

    • 2.3.   血浆样品处理

  • 精密吸取血浆30 μl，加入2%甲酸水溶液30 μl，振摇10 s，再加50 μl含内标乙腈溶液后旋涡振荡10 s，12 000 r/ min离心10 min，转移上清液置进样瓶，进样。

    • 2.4.   标准曲线及最低定量限

  • 取空白人血浆，精密加入不同浓度的对照品溶液，配制成CA浓度为500、250、100、50、25、10、2 ng/ml的血浆标准品。以浓度为X轴，样品峰与内标峰的比值为Y轴，进行线性回归，经“1/X”权重得回归方程分别为：Y = 7.43×10–3 + 1.99 × 10–2X (r = 0.996 5)。结果表明，CA在2~500 ng/ml内，线性关系良好。本方法CA的定量下限为2 ng/ml。

    • 2.5.   系统精密度与准确度（回收率）

  • 配制CA浓度分别为8、80、400 ng/ml的血浆样品，日内误差以3个浓度各5个样品的测定结果表示；日间误差以3个浓度样品分别3个批次结果表示。人空白血浆分别加入不同浓度CA工作溶液，按照“2.3”项下方法提取后，相同色谱条件进行浓度测定。同时测定用流动相稀释为相应浓度的无基质溶液的峰面积，以计算绝对回收率。结果如表2所示。

    CA理论浓度（ng/ml）日内日间
    测得浓度（ng/ml）精密度RSD（%）准确度REC（%）测得浓度（ng/ml）精密度RSD（%）准确度REC（%）
    8 8.532.39106.67 8.522.00106.46
    80 89.541.77111.93 88.163.48110.19
    400390.5315.9 97.63398.595.19 99.65

    • 2.6.   稳定性试验

  • 空白血浆分别配制成8、80、400 ng/ml的样品，每批次各3个，分别考察其①室温下放置4 h；②处理后室温放置24 h；③-80 ℃放置14 d的稳定性。结果如表3所示。

    时间样品浓度（ng/ml）REC（%）RSD（%）
    室温4 h低103.50 9.28
    中 98.27 5.26
    高101.33 3.73
    处理后室温24 h低 77.22 5.37
    中112.9510.83
    高110.46 6.80
    –80 ℃ 14 d低 75.40 7.95
    中113.6723.61
    高 98.22 1.69

    • 2.7.   基质效应

  • 取6个不同来源的人空白血浆30 μl，除不加内标外，其余按“2.3”项下操作，取上清液，分别制备成CA浓度为31.25和1 000 ng/ml；PPF浓度为200 ng/ml的溶液，进样测定，记录峰面积。另用流动相将CA稀释成31.25和1 000 ng/ml；PPF浓度为200 ng/ml浓度的溶液，进样测定，记录峰面积。

    基质效应=（空白基质中添加工作溶液峰面积/重组溶液中添加工作溶液峰面积）×100%

    CA的基质效应为52%（RSD 2.09%~15.33%）。PPF的基质效应为96.71%（RSD4.6%）。

    • 2.8.   方法在治疗药物监测中的应用

  • 采用肌松仪监测患者注射苯磺顺式阿曲库铵后神经肌肉阻滞程度。给予4个成串刺激（train of four stimulation，TOF），给药后，T4最先发生衰减，T1最后衰减。当T1消失即仪器显示为0时，肌肉阻滞程度最大，随后逐渐恢复。分别在T0（T1消失至0）、TOFr 50（恢复至50%）、TOFr 90（恢复至90%）时采集输液对侧动脉血3 ml，并及时保存于4 ℃冰箱中。采用建立的方法测定样本血药浓度，结果见表4。同时记录T0（给药结束到T1消失至0）、TOFr50（给药结束到恢复至50%）、TOFr90（给药结束到恢复至90%）发生所需时间，结果见表5、表6。

    TOF (%)浓度（ng/ml）
    受试者1受试者2受试者3受试者4受试者5受试者6
    0445.02525.30253.83165.29150.57309.22
    50 76.58125.65 68.71 68.99 53.18 58.46
    90 44.10 71.80 60.72 71.74 41.12 45.07
    基本信息受试者1受试者2受试者3受试者4受试者5受试者6
    性别女女女男女男
    年龄（岁） 50 49 53 70 36 40
    体重（m/kg） 70 75 60 50 53 80
    身高（l/cm）165165164161165173
    TOF（%）时间（t/min）
    0 101515 2020 20
    50 707065 6565 75
    90105959510085110
3.   讨论

  • CA的代谢途径主要为Hoffman消除[7]，Hoffman消除受温度和pH值的影响。有研究显示[8]，将苯磺顺阿曲库铵注射液稀释于20%的甲醇中，室温下，10~15 min即可检测到大量降解产物N-甲基四氢罂粟碱和单季丙烯酸酯。血浆的pH值在7.4左右。我们发现，在CA血浆样本前处理过程中，如果不加入酸调节pH值，即使在-80 ℃的储存条件下，CA仍然会很快降解（结果未在文中体现）。为此，我们考察了不同比例的甲酸，最终确定了在样本前处理过程中加入2%的甲酸能够保证血浆中CA的稳定性。在既往报道中，多是加入一定浓度的硫酸来调控pH值[2-6]。硫酸为有机酸，对测定仪器尤其是质谱仪的离子源损害很大。同时，加入硫酸并不能保证室温条件下样本的长时间稳定（<4 h）[9]。本研究结果显示，加入2%的甲酸，室温下样本稳定性可保持在24 h以上。此外，我们的样本前处理过程并不需要经过离心-上清液真空浓缩或氮吹浓缩-复溶-离心取上清液等[2-6, 9]复杂过程，更为省时、省力。经验证，本研究建立的方法测定CA血浆样本的精密度、准确度和稳定性均符合方法学要求。该方法更加简便、准确、快速、稳定，可应用于临床CA治疗药物浓度的监测。

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