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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。
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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。
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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。
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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。
表 1 TGC检测时间程序
时间(t/min) 流速(ml/min) 功能 LC1 LC2 辅助流动相 0.01~0.80 1.00 1.20 2.00 脑脊液样品于LC1中富集 0.81~3.49 1.00 1.20 0.01 目标物在LC1中初步分离 3.50~5.50 1.00 1.20 0.01 LC1与LC2串联,目标物洗脱至LC2 5.51~11.00 1.50 1.20 0.01 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。
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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。
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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]。
表 2 脑脊液中测定TGC浓度的精密度与准确度(%)
浓度(ng/ml) 日内(n=5) 日间(n=3) 准确度 RSD 准确度 RSD 225.0 100.31 ± 2.83 2.82 98.80 ± 0.95 0.96 450.0 104.58 ± 2.19 2.09 103.51 ± 2.72 2.63 900.0 106.51 ± 2.06 1.94 101.82 ± 4.28 4.20 64.5 105.04 ± 2.47 2.35 99.01 ± 2.05 2.08 -
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。
表 3 脑脊液中TGC在不同温度条件下的稳定性
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(%) 64.5 68.67 ± 2.93 4.27 65.52 ± 1.52 2.32 65.01 ± 1.43 2.20 68.56 ± 3.06 4.46 225 223.16 ± 5.06 2.27 221.62 ± 2.45 1.11 224.72 ± 4.50 2.00 222.99 ± 5.40 2.42 450 452.44 ± 9.11 2.01 459.04 ± 13.45 2.93 464.03 ± 8.39 1.81 459.68 ± 8.25 1.80 900 885.14 ± 15.09 1.70 881.32 ± 13.36 1.52 888.13 ± 13.80 1.55 899.79 ± 10.13 1.13 -
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。
Determination of tigecycline in human cerebrospinal fluid by two-dimensional liquid chromatography and its clinical application
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摘要:
目的 建立二维液相色谱法检测人脑脊液中替加环素浓度,用于颅内感染患者的药物浓度监测。 方法 采用外标法定量,第一维色谱柱为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),流速1.0 ml/min。第二维色谱柱为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),流速1.2 ml/min。检测波长340 nm,色谱柱温40 ℃,进样量:200 μl。 结果 脑脊液中替加环素64.5~1 290.0 ng/ml内与峰面积呈良好线性关系(r=0.999 8);日内、日间精密度RSD<5%,准确度为98.80%~106.51%。 结论 该方法操作简便、快速、准确、灵敏,适用于临床人脑脊液替加环素的浓度监测,可为颅内感染患者的个体化给药提供科学依据。 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. -
表 1 TGC检测时间程序
时间(t/min) 流速(ml/min) 功能 LC1 LC2 辅助流动相 0.01~0.80 1.00 1.20 2.00 脑脊液样品于LC1中富集 0.81~3.49 1.00 1.20 0.01 目标物在LC1中初步分离 3.50~5.50 1.00 1.20 0.01 LC1与LC2串联,目标物洗脱至LC2 5.51~11.00 1.50 1.20 0.01 目标物在LC2中洗脱分离,清洗泵反向冲洗LC1 表 2 脑脊液中测定TGC浓度的精密度与准确度(%)
浓度(ng/ml) 日内(n=5) 日间(n=3) 准确度 RSD 准确度 RSD 225.0 100.31 ± 2.83 2.82 98.80 ± 0.95 0.96 450.0 104.58 ± 2.19 2.09 103.51 ± 2.72 2.63 900.0 106.51 ± 2.06 1.94 101.82 ± 4.28 4.20 64.5 105.04 ± 2.47 2.35 99.01 ± 2.05 2.08 表 3 脑脊液中TGC在不同温度条件下的稳定性
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(%) 64.5 68.67 ± 2.93 4.27 65.52 ± 1.52 2.32 65.01 ± 1.43 2.20 68.56 ± 3.06 4.46 225 223.16 ± 5.06 2.27 221.62 ± 2.45 1.11 224.72 ± 4.50 2.00 222.99 ± 5.40 2.42 450 452.44 ± 9.11 2.01 459.04 ± 13.45 2.93 464.03 ± 8.39 1.81 459.68 ± 8.25 1.80 900 885.14 ± 15.09 1.70 881.32 ± 13.36 1.52 888.13 ± 13.80 1.55 899.79 ± 10.13 1.13 -
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