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Volume 38 Issue 6
Nov.  2020
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CHENG Caiyi, DENG Dongmei, WANG Xianfeng, YANG Zheng, XING Haiyan. Determination of tigecycline in human cerebrospinal fluid by two-dimensional liquid chromatography and its clinical application[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(6): 543-546. doi: 10.12206/j.issn.1006-0111.202003040
Citation: CHENG Caiyi, DENG Dongmei, WANG Xianfeng, YANG Zheng, XING Haiyan. Determination of tigecycline in human cerebrospinal fluid by two-dimensional liquid chromatography and its clinical application[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(6): 543-546. doi: 10.12206/j.issn.1006-0111.202003040

Determination of tigecycline in human cerebrospinal fluid by two-dimensional liquid chromatography and its clinical application

doi: 10.12206/j.issn.1006-0111.202003040
  • Received Date: 2020-03-08
  • Rev Recd Date: 2020-05-14
  • Available Online: 2020-11-27
  • Publish Date: 2020-11-25
  •   Objective  To establish a two-dimensional high-performance liquid chromatography method for the determination of tigecycline in human cerebrospinal fluid, which can be used for the drug monitoring in patients with intracranial infection.  Methods  The quantification was carried out by an external standard method. The first-dimension column was a Aston SNX5 phenyl chromatographic column (50 mm×4.6 mm, 5 μm) with ammonium phosphate (pH was adjusted with ammonium hydroxide to 7.5)-methanol (45∶55, V/V) as the mobile phase and the flow rate was 1.2 ml/min. The second-dimension chromatographic column was Aston SC5 C18 (275 mm×4.6 mm, 5 μm), with ammonium phosphate (pH was adjusted with ammonium hydroxide to 7.4)-ammonium phosphate (pH was adjusted with ammonium hydroxide to 3.0)- acetonitrile (30∶50∶20, V/V/V) as the mobile phase and the flow rate was 1.0 ml/min. The detection wavelength was 340 nm. The temperature was 40 ℃ and the injection volume was 200 μl.  Results  The calibration curve of tigecycline showed good linearity from 64.5 to 1 290.0 ng/ml in human cerebrospinal fluid (r=0.999 8). The RSD of intra and inter-day precision were less than 5.0% with the detection accuracy of 98.80%−106.51%.  Conclusion  This method is simple, quick, accurate, specific and sensitive. It meets the requirements of tigecycline determination in clinical human cerebrospinal fluid, which offers the individualized therapeutic assurance for patients with intracranial infection.
  • [1] 陈洁, 胡章勇. 替加环素治疗多重耐药菌感染的药物监测[J]. 生命的化学, 2019, 39(3):521-526.
    [2] 梅升辉, 朱乐亭, 杨莉, 等. 替加环素治疗颅内耐药菌感染的研究进展[J]. 中国药房, 2016, 27(14):2005-2008. doi:  10.6039/j.issn.1001-0408.2016.14.45
    [3] SIPAHI O R, MERMER S, DEMIRDAL T, et al. Tigecycline in the treatment of multidrug-resistant Acinetobacter baumannii meningitis: Results of the Ege study[J]. Clin Neurol Neurosurg,2018,172:31-38. doi:  10.1016/j.clineuro.2018.06.008
    [4] WU Y X, CHEN K, ZHAO J W, et al. Intraventricular administration of tigecycline for the treatment of multidrug-resistant bacterial meningitis after craniotomy: a case report[J]. J Chemother,2018,30(1):49-52. doi:  10.1080/1120009X.2017.1338846
    [5] 国家药典委员会. 中华人民共和国药典2015年版四部[S]. 北京: 中国医药科技出版社, 2015: 364.
    [6] 缪文清, 汪硕闻, 范先煜, 等. HPLC法测定人脑脊液中万古霉素浓度及在颅内感染中的应用[J]. 中国药师, 2019, 22(3):399-403. doi:  10.3969/j.issn.1008-049X.2019.03.003
    [7] 嵇金如, 沈萍, 魏泽庆, 等. 国产和进口替加环素体外抗菌活性比较[J]. 中国感染与化疗杂志, 2015, 15(4):330-334. doi:  10.3969/j.issn.1009-7708.2015.04.006
    [8] DOWZICKY M J, PARK C H. Update on antimicrobial susceptibility rates among gram-negative and gram-positive organisms in the United States: results from the Tigecycline Evaluation and Surveillance Trial (TEST) 2005 to 2007[J]. Clin Ther,2008,30(11):2040-2050. doi:  10.1016/j.clinthera.2008.11.006
    [9] RODVOLD K A, GOTFRIED M H, CWIK M, et al. Serum, tissue and body fluid concentrations of tigecycline after a single 100 mg dose[J]. J Antimicrob Chemother,2006,58(6):1221-1229. doi:  10.1093/jac/dkl403
    [10] POLAT M, OZKAYA-PARLAKAY A. Tigecycline salvage therapy for ventriculoperitoneal shunt meningitis due to extensi- vely drug-resistant Acinetobacter baumannii[J]. Eur J Pediatr,2019,178(1):117-118. doi:  10.1007/s00431-018-3271-2
    [11] 邵华, 宋一帆, 何杰, 等. 高剂量替加环素在感染性休克患者体内的药物监测及药代动力学[J]. 中国药科大学学报, 2017, 48(6):721-726. doi:  10.11665/j.issn.1000-5048.20170614
    [12] MURALIDHARAN G, MICALIZZI M, SPETH J, et al. Pharmacokinetics of tigecycline after single and multiple doses in healthy subjects[J]. Antimicrob Agents Chemother,2005,49(1):220-229. doi:  10.1128/AAC.49.1.220-229.2005
    [13] MAZUMDER S, RAMYA B S, BILIGI D S. Utility of urine reagent strips in cerebrospinal fluid analysis: an aid to bedside diagnosis of meningitis[J]. Indian J Pathol Microbiol,2018,61(3):356-359. doi:  10.4103/IJPM.IJPM_821_16
    [14] LI X G, SUN S S, LING X, et al. Plasma and cerebrospinal fluid population pharmacokinetics of vancomycin in postoperative neurosurgical patients after combined intravenous and intraventricular administration[J]. Eur J Clin Pharmacol,2017,73(12):1599-1607. doi:  10.1007/s00228-017-2313-4
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Determination of tigecycline in human cerebrospinal fluid by two-dimensional liquid chromatography and its clinical application

doi: 10.12206/j.issn.1006-0111.202003040

Abstract:   Objective  To establish a two-dimensional high-performance liquid chromatography method for the determination of tigecycline in human cerebrospinal fluid, which can be used for the drug monitoring in patients with intracranial infection.  Methods  The quantification was carried out by an external standard method. The first-dimension column was a Aston SNX5 phenyl chromatographic column (50 mm×4.6 mm, 5 μm) with ammonium phosphate (pH was adjusted with ammonium hydroxide to 7.5)-methanol (45∶55, V/V) as the mobile phase and the flow rate was 1.2 ml/min. The second-dimension chromatographic column was Aston SC5 C18 (275 mm×4.6 mm, 5 μm), with ammonium phosphate (pH was adjusted with ammonium hydroxide to 7.4)-ammonium phosphate (pH was adjusted with ammonium hydroxide to 3.0)- acetonitrile (30∶50∶20, V/V/V) as the mobile phase and the flow rate was 1.0 ml/min. The detection wavelength was 340 nm. The temperature was 40 ℃ and the injection volume was 200 μl.  Results  The calibration curve of tigecycline showed good linearity from 64.5 to 1 290.0 ng/ml in human cerebrospinal fluid (r=0.999 8). The RSD of intra and inter-day precision were less than 5.0% with the detection accuracy of 98.80%−106.51%.  Conclusion  This method is simple, quick, accurate, specific and sensitive. It meets the requirements of tigecycline determination in clinical human cerebrospinal fluid, which offers the individualized therapeutic assurance for patients with intracranial infection.

CHENG Caiyi, DENG Dongmei, WANG Xianfeng, YANG Zheng, XING Haiyan. Determination of tigecycline in human cerebrospinal fluid by two-dimensional liquid chromatography and its clinical application[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(6): 543-546. doi: 10.12206/j.issn.1006-0111.202003040
Citation: CHENG Caiyi, DENG Dongmei, WANG Xianfeng, YANG Zheng, XING Haiyan. Determination of tigecycline in human cerebrospinal fluid by two-dimensional liquid chromatography and its clinical application[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(6): 543-546. doi: 10.12206/j.issn.1006-0111.202003040
  • 颅内感染尤其是多重耐药菌感染具有进展快、反应重、治疗难度大、病死率高的特点,故提高颅内感染治愈率受到患者及临床的广泛关注。替加环素(TGC)作为首个甘氨酰四环素类抗菌药物,具有抗菌谱广、抗耐药活性强的特点,对临床上常见耐药菌(如鲍曼不动杆菌、产碳青霉烯酶肺炎克雷伯菌、耐甲氧西林金黄色葡萄球菌等)均有较好的疗效[1]。在耐药菌治疗药物不足的情况下,临床医生将TGC用于颅内感染的治疗,此外,越来越多的案例报道称,TGC可有效清除致病菌[2-3]

    TGC成功治疗颅内感染与脑脊液中有效药物浓度密切相关,TGC浓度监测对于提高颅内感染治愈率、精准指导临床合理用药具有重要意义[4]。本研究将利用二维液相色谱高效的自动化在线样品处理能力,建立脑脊液中TGC浓度测定方法。该方法与已报道的质谱法相比,具有操作简便、成本低、抗干扰能力强、基质效应小等优势,脑脊液样品无需复杂的前处理,高速离心后直接进样,短时间内即可完成萃取、转移及分析,更易于临床推广应用。

  • 二维高效液相色谱由FLC-2701全自动二维液相色谱耦合仪(湖南德米特仪器有限公司)和LC-20A色谱模块(日本岛津公司)构成;−80 ℃冰箱(美国赛默飞科技公司);十万分之一电子天平(德国赛多利斯仪器公司);涡旋混匀器(美国SI仪器公司);低温高速离心机(美国贝克曼仪器公司)。

  • TGC对照品(北京百灵威科技公司,批号: 10-MWV-62-1);乙腈、甲醇、磷酸为色谱纯;高氯酸、氨水为分析纯;超纯水自制。

  • 入住神经外科且未使用TGC的患者,住院期间需开展脑脊液生化检查时留取或从留置腰大池/脑室引流管中适量抽取。

  • 精密称取TGC对照品1.29 mg,用20 mg/ml精氨酸水溶液定容至100 ml容量瓶中,即得12.90 μg/ml的TGC标准溶液。

  • 预处理方法考察时,取脑脊液样品500 μl,直接离心、分别加入3倍体积甲醇、乙腈、10%高氯酸沉淀蛋白,涡旋混匀5 min,13 000 r/min离心10 min,取上清液进样分析。最终确定预处理方法为: 脑脊液样品13 000 r/min离心10 min后直接进样,采用外标法定量分析。

  • 第一维色谱柱LC1为Aston SNX5苯基色谱柱(50 mm×4.6 mm,5 μm),流动相为磷酸铵(氨水调pH7.5)-甲醇 (45∶55,V/V),第二维色谱柱LC2为Aston SC5 C18色谱柱(275 mm×4.6 mm,5 μm),流动相为磷酸铵(氨水调pH7.4)-磷酸铵(氨水调pH3.0)-乙腈(30∶50∶20,V/V/V),辅助流动相为纯化水。检测波长340 nm,色谱柱温40 ℃,进样量:200 μl,工作原理图见图1,时间程序见表1

    时间(t/min)流速(ml/min)功能
    LC1LC2辅助流动相
    0.01~0.801.001.202.00脑脊液样品于LC1中富集
    0.81~3.491.001.200.01目标物在LC1中初步分离
    3.50~5.501.001.200.01LC1与LC2串联,目标物洗脱至LC2
    5.51~11.001.501.200.01目标物在LC2中洗脱分离,清洗泵反向冲洗LC1
  • 分别制备空白脑脊液样品、含TGC的标准脑脊液样品、患者用药后的脑脊液样品,进行二维液相分析,得到色图谱,观察有无内源性干扰,如图2所示。结果表明,TGC的出峰时间9.38 min,脑脊液的内源性成分对TGC的测定无干扰。

  • 取不同体积TGC标准溶液,加入空白脑脊液定容,配制成浓度为64.5、129.0、387.0、645.0、1 290.0 ng/ml的系列对照溶液,按“2.2”项下操作后进样分析。以TGC浓度为自变量(X),脑脊液中TGC的峰面积为因变量(Y)进行线性回归,得回归方程为:Y=116.32X-3 223.6(r=0.999 8)。结果表明TGC浓度在64.5~1 290.0 ng/ml范围内线性关系良好,最低定量限为64.5 ng/ml。

  • 分别配制含TGC低、中、高及定量下限(225.0、450.0、900.0、64.5 ng/ml)4个浓度的质控(QC)脑脊液样本,按“2.2”项下操作后进样分析。1 d内每个浓度平行测定5份QC样品,连续测定3 d。将测得的峰面积代入“2.4.2”项下,计算各质控样品的浓度,并根据样本实际浓度计算本法的精密度与准确度。结果如表2所示,日内、日间RSD均小于5%,准确度均在98.80%~106.51%之间。可见本实验所建立的二维液相色谱法测定脑脊液中TGC的精密度和准确度符合《中国药典》(2015年版)检测要求[5]

    浓度(ng/ml)日内(n=5)日间(n=3)
    准确度RSD准确度RSD
    225.0100.31 ± 2.832.8298.80 ± 0.950.96
    450.0104.58 ± 2.192.09103.51 ± 2.722.63
    900.0106.51 ± 2.061.94101.82 ± 4.284.20
    64.5105.04 ± 2.472.3599.01 ± 2.052.08
  • 配制“2.4.3”项下低、中、高及定量下限4个浓度的QC样品,每个浓度平行3份,分别考察室温放置4 h、进样器放置6 h、反复冻融3次、冰冻放置1周后的稳定性。结果如表3所示,表明上述条件对TGC的检测结果无明显影响。

    样品浓度(ng/ml)室温放置4 h进样器放置6 h反复冻融3次冰冻放置1周
    平均浓度(ng/ml)RSD(%)平均浓度(ng/ml)RSD(%)平均浓度(ng/ml)RSD(%)平均浓度(ng/ml)RSD(%)
    64.568.67 ± 2.934.2765.52 ± 1.522.3265.01 ± 1.432.2068.56 ± 3.064.46
    225223.16 ± 5.062.27221.62 ± 2.451.11224.72 ± 4.502.00222.99 ± 5.402.42
    450452.44 ± 9.112.01459.04 ± 13.452.93464.03 ± 8.391.81459.68 ± 8.251.80
    900885.14 ± 15.091.70881.32 ± 13.361.52888.13 ± 13.801.55899.79 ± 10.131.13
  • 本实验选取2例接受TGC治疗的多重耐药菌所致的重症颅内感染患者,脑脊液标本药敏试验均报告对TGC敏感。患者1接受说明书推荐剂量,即首剂静脉滴注100 mg,后以50 mg每12 h维持治疗。患者2同样接受静脉给药,剂量为受试者1的2倍。达到稳定药物浓度后,留取两位受试者的脑脊液,按“2.2”项下操作后进样分析。受试者脑脊液中TGC浓度分别为187.42、275.41 ng/ml。

  • 甲醇、乙腈、10%高氯酸是常用蛋白沉淀剂,本研究在空白脑脊液中分别加入3倍体积上述沉淀剂,离心后与不加蛋白沉淀剂组比较,沉淀量无显著差异,且所得上清液与沉淀完全分离。现多项研究认为,因脑脊液中蛋白质含量低,故直接离心更利于生物样品的微量分析[6]。另直接离心法进样,脑脊液中TGC浓度无稀释,也无其他前处理方法造成的误差,更利于临床样本的分析。

  • TGC对G-菌(如流感嗜血杆菌、大肠杆菌、对鲍曼不动杆菌等)MIC90为2~4 mg/L[7-8]。本试验中,无论是常规给药还是加倍剂量给药的患者,稳态脑脊液浓度显然均未起到杀菌作用,导致抗感染治疗效果不佳。这可能与以下因素有关:①TGC相对分子质量大而且水溶性强,很难进入到脑脊液中。有研究报道在血脑屏障正常的志愿者中,脑脊液中TGC浓度仅为血浆的11%。而对于脑膜炎患者,血脑屏障通透性可增至33%~52%,这可能也是静脉注射TGC成功治疗颅内感染的主要原因[9-10],但血脑屏障破坏的程度个体差异较大。②重症感染患者常伴有全身炎症反应、肝肾功能异常、多器官功能障碍综合征等,血药浓度低于正常水平,在同等穿透率的情况下,药物从血液进入脑脊液中的剂量减少[11]。③TGC的蛋白结合率较高,为71%~87%[12]。颅内感染时,脑脊液蛋白水平提高,使游离的药物浓度降低[13]。④ 脑脊液引流量与药物清除率呈正相关,引流量越大,脑脊液中的药物浓度越低[14]

    目前,TGC治疗方案大多参照说明书或文献报道推荐剂量,未考虑血脑屏障、脑脊液引流量、给药途径等患者自身因素的影响,使颅内TGC的浓度不足、疗效不佳。本研究建立的脑脊液中抗菌药物TGC的二维液相色谱分析方法简便、准确、稳定,对促进TGC的合理使用、改善临床治疗结局具有重要意义。临床可根据脑脊液中TGC的浓度,及时调整给药剂量或给药途径,提高治愈率,节省医疗费用。此外,也为研究TGC在颅内的药动学及量效关系奠定了基础。

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