Name: validated lc-ms/ms panel for quantifying 11 drug-resistant tb medications in small hair samples
Uploaded: 2020-05-19T13:00:00
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作者要感谢开普敦大学肺研究所的基尔坦·迪达教授、阿里·伊斯梅尔博士和玛丽特杰·普雷托利斯教授为这项研究收集头发样本提供便利。作者还感谢本研究参与者的贡献。

所有患者在收集头发样本前都提供书面知情同意。我们获得了开普敦大学和加州大学旧金山分校的机构审查委员会的批准。
1. 头发取样
<ol><li>获得书面知情同意。</li>
	<li>使用干净的剪刀从尽可能靠近头皮的区域切割大约20-30个头皮发丝。</li>
	<li>在头发的远端放置胶带，以指示方向性。将头发样本折叠成铝箔正方形，并在室温下存放。标记头发的远端，以避免因对近端进行...

<ol>
	<li>Kurbatova, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictors+of+poor+outcomes+among+patients+treated+for+multidrug-resistant+tuberculosis+at+DOTS-plus+projects.">Predictors of poor outcomes among patients treated for multidrug-resistant tuberculosis at DOTS-plus projects.</a> Tuberculosis (Edinb). 92, 397-403 (2012).</li><li>Dheda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology,+pathogenesis,+transmission,+diagnosis,+and+management+of+multidrug-resistant,+extensively+drug-resistant,+and+incurable+tuberculosis.">The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis.</a> Lancet Respiratory Medicine. , (2017).</li><li>Berg, K. M., Arnsten, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+and+conceptual+challenges+in+measuring+antiretroviral+adherence.">Practical and conceptual challenges in measuring antiretroviral adherence.</a> Journal of Acquired Immunodeficiency Syndromes (JAIDS). 43, 79-87 (2006).</li><li>Kagee, A., Nel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+association+between+self-report+items+for+HIV+pill+adherence+and+biological+measures.">Assessing the association between self-report items for HIV pill adherence and biological measures.</a> AIDS Care. 24 (11), 1448-1452 (2012).</li><li>Haberer, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherence+to+antiretroviral+prophylaxis+for+HIV+prevention:+a+substudy+cohort+within+a+clinical+trial+of+serodiscordant+couples+in+East+Africa.">Adherence to antiretroviral prophylaxis for HIV prevention: a substudy cohort within a clinical trial of serodiscordant couples in East Africa.</a> PLoS Medicine. 10 (9), 1001511 (2013).</li><li>Pullar, T., Kumar, S., Tindall, H., Feely, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+to+stop+counting+the+tablets.">Time to stop counting the tablets.</a> Clinical Pharmacology &amp; Therapeutics. 46 (2), 163-168 (1989).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+study+of+multiple+measures+of+adherence+to+HIV+protease+inhibitors.">A comparison study of multiple measures of adherence to HIV protease inhibitors.</a> Annals of Internal Medicine. 134 (10), 968-977 (2001).</li><li>Wendel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barriers+to+use+of+electronic+adherence+monitoring+in+an+HIV+clinic.">Barriers to use of electronic adherence monitoring in an HIV clinic.</a> Annals of Pharmacotherapy. 35, 1010-1101 (2001).</li><li>Ruiz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+voriconazole+plasma+concentrations+on+treatment+response+in+critically+ill+patients.">Impact of voriconazole plasma concentrations on treatment response in critically ill patients.</a> Clinical Pharmacology &amp; Therapeutic. , (2019).</li><li>Saktiawati, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+sampling+strategies+for+therapeutic+drug+monitoring+of+first-line+tuberculosis+drugs+in+patients+with+tuberculosis.">Optimal sampling strategies for therapeutic drug monitoring of first-line tuberculosis drugs in patients with tuberculosis.</a> Clinical Phamacokinetics. , (2019).</li><li>Podsadecki, T. J., Vrijens, B. C., Tousset, E. P., Rode, R. A., Hanna, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="White+coat+compliance"+limits+the+reliability+of+therapeutic+drug+monitoring+in+HIV-1-infected+patients.">"White coat compliance" limits the reliability of therapeutic drug monitoring in HIV-1-infected patients.</a> HIV Clinical Trials. 9 (4), 238-246 (2008).</li><li>Cuypers, E., Flanagan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interpretation+of+hair+analysis+for+drugs+and+drug+metabolites.">The interpretation of hair analysis for drugs and drug metabolites.</a> Clinical Toxicology. 56 (2), 90-100 (2018).</li><li>Knitz, P., Villain, M., Crimele, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+analysis+for+drug+detection.">Hair analysis for drug detection.</a> Therapeutic Drug Monitoring. 28 (3), 442-446 (2006).</li><li>Barroso, M., Gallardo, E., Vleira, D. N., Lopez-Rivadulla, M., Queiroz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair:+a+complementary+source+of+bioanalytical+information+in+forensic+toxicology.">Hair: a complementary source of bioanalytical information in forensic toxicology.</a> Bioanalysis. 3 (1), 67-79 (2011).</li><li>Gandhi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atazanavir+concentration+in+hair+is+the+strongest+predictor+of+outcomes+on+antiretroviral+therapy.">Atazanavir concentration in hair is the strongest predictor of outcomes on antiretroviral therapy.</a> Clinical Infectious Diseases. 52 (10), 1267-1275 (2011).</li><li>Koss, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+concentrations+of+antiretrovirals+predict+viral+suppression+in+HIV-infected+pregnant+and+breastfeeding+Ugandan+women.">Hair concentrations of antiretrovirals predict viral suppression in HIV-infected pregnant and breastfeeding Ugandan women.</a> AIDS. 29 (7), 825-830 (2015).</li><li>Pintye, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+Report:+Lopinavir+Hair+Concentrations+Are+the+Strongest+Predictor+of+Viremia+in+HIV-Infected+Asian+Children+and+Adolescents+on+Second-Line+Antiretroviral+Therapy.">Brief Report: Lopinavir Hair Concentrations Are the Strongest Predictor of Viremia in HIV-Infected Asian Children and Adolescents on Second-Line Antiretroviral Therapy.</a> Journal of Acquired Immune Deficiency Syndromes (JAIDS). 76 (4), 367-371 (2017).</li><li>Baxi, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nevirapine+Concentration+in+Hair+Samples+Is+a+Strong+Predictor+of+Virologic+Suppression+in+a+Prospective+Cohort+of+HIV-Infected+Patients.">Nevirapine Concentration in Hair Samples Is a Strong Predictor of Virologic Suppression in a Prospective Cohort of HIV-Infected Patients.</a> PLoS One. 10 (6), 0129100 (2015).</li><li>Gandhi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiretroviral+concentrations+in+hair+strongly+predict+virologic+response+in+a+large+HIV+treatment-naive+clinical+trial.">Antiretroviral concentrations in hair strongly predict virologic response in a large HIV treatment-naive clinical trial.</a> Clinical Infectious Diseases. 5, 1044-1047 (2019).</li><li>Gerona, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+analysis+of+11+medications+for+drug+resistant+TB+in+small+hair+samples+to+quantify+adherence+and+exposure+using+a+validate+LC-MS/MS+panel.">Simultaneous analysis of 11 medications for drug resistant TB in small hair samples to quantify adherence and exposure using a validate LC-MS/MS panel.</a> Journal of Chromatography B. 1125, 121729 (2019).</li><li>Metcalfe, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+anti-tuberculosis+drug+concentration+in+hair+and+treatment+outcomes+in+MDR-+and+XDR-TB.">Association of anti-tuberculosis drug concentration in hair and treatment outcomes in MDR- and XDR-TB.</a> European Respriatory Journal Open Research. 5 (2), (2019).</li><li>Metcalfe, J. Z., O'Donnell, M. R., Bangsberg, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+Beyond+Directly+Observed+Therapy+for+Tuberculosis.">Moving Beyond Directly Observed Therapy for Tuberculosis.</a> PLoS Medicine. 12 (9), 1001877 (2015).</li></ol>

我们在这里报告我们开发和验证的方法的协议，用于量化11种抗结核病药物，用于治疗使用LC-MS/MS的小头发样本中的DR-TB。以前没有其他方法来量化头发中的这11种药物，但已经开发、验证和发布。我们的方法可以量化亚纳米克的药物水平，只有20-30发丝长约3厘米（约2毫克），并已经过验证22。头发分析的低重量意味着参与研究的患者可以谨慎参与，并有可能返回重复测试，而不?...

在21世纪，耐药结核病（DR-TB）是本已薄弱的国家结核病控制计划中不断演变的灾难，仅在过去5年中，确诊病例就翻了一番，占全球抗微生物药物耐药性死亡总数的近三分之一,。与治疗对药物敏感的结核病相比，成功治疗DR-TB通常要求更长、毒性更大的二线治疗方案。此外，DR-TB患者在依从性方面往往存在重大挑战，导致最初出现耐药性。
与HIV感染不同，病毒载量可用于监测治疗，结核病治疗反应的代理终点在个别4级延迟且不可靠。监测患者依从性是亚治疗抗结核药物浓度和治疗失败的重要预测器，也是一项挑战。自我报告的依从性有召回偏见和取悦供应商的愿望55，6。6丸数和药物事件监测系统（MEMS）可以更客观7，但不测量实际药物消耗88，9，10。9,10生物基质中的药物水平可以提供依从性和药代动力学数据。因此，血浆药物水平通常用于治疗药物监测11，12。11,然而，在药物依从性监测方面，血浆水平代表短期接触，在确定适当的依从性参考范围时，受患者体内和患者间变异性的限制。"白衣"效应，在临床或研究访问之前，依从性改善，进一步复杂化血浆水平的能力，提供准确的药物依从模式13。
头发是一种替代生物基质，可以测量长期药物暴露1414，15。15许多药物和内源性代谢物结合到头发蛋白基质从系统循环，因为头发生长。随着头发生长过程中这种动态过程的继续，沉积在头发基质中的药物量取决于药物在循环中的连续存在，使头发成为药物摄入量的极佳时间读出。头发作为生物基质具有另一个优点，即易于收集，无需与血液相比，冷链进行储存和装运。此外，头发是非生物危害的，这提供了额外的可行性优势，在外地。
头发药物水平早已用于法医申请16。在过去十年中，头发抗逆转录病毒（ARV）水平在评估药物在艾滋病毒治疗和预防方面是否得到效用，我们小组对此作出了贡献。头发中的抗逆转录病毒药物水平已被证明是HIV感染治疗结果的最独立预测变量17、18、19、20、21。17,18,19,20,21为了确定DR-TB患者的头发水平在预测治疗结果方面是否具有相同的效用，我们使用LC-MS/MS来开发和验证一种分析小头发样本中11种DR-TB药物的方法。作为对测定性能的初步评估，我们在南非西开普22号对接受直接观察治疗的DR-TB患者的方便样本中测量了DR-TB药物水平。

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2 mL injection vials</td>
			<td style="width:211px;">Agilent Technologies</td>
			<td style="width:161px;">5182-0716</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">250 uL injection vial inserts</td>
			<td style="width:211px;">Agilent Technologies</td>
			<td style="width:161px;">5181-8872</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bead ruptor 24</td>
			<td style="width:211px;">OMNI International</td>
			<td style="width:161px;">19001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bead ruptor tubes (2 mL bead kit, 2.8mm ceramic, 2 mL microtubes)</td>
			<td style="width:211px;">OMNI International</td>
			<td style="width:161px;">19628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bedaquiline</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">B119550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bedaquiline-d6</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">B119552</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Clofazimine</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>C324300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Clofazimine-d7</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>C324302</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable lime glass culture tubes</td>
			<td style="width:211px;">VWR</td>
			<td style="width:161px;">60825-425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethambutol</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>E889800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethambutol-d4</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>E889802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethionamide</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">E890420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethionamide-d5</td>
			<td style="width:211px;">ClearSynth</td>
			<td>CS-O-06597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formic acid</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:161px;">F0507-100mL</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Glass bottles</td>
			<td style="width:211px;">Corning</td>
			<td style="width:161px;">1395-1L</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hot Shaker</td>
			<td style="width:211px;">Bellco Glass Inc</td>
			<td style="width:161px;">7746-32110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC</td>
			<td style="width:211px;">Agilent Technologies</td>
			<td style="width:161px;">Infinity 1260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC grade acetonitrile</td>
			<td style="width:211px;">Honeywell</td>
			<td style="width:161px;">015-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC grade methanol</td>
			<td style="width:211px;">Honeywell</td>
			<td style="width:161px;">230-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC grade water</td>
			<td style="width:211px;">Aqua Solutions Inc</td>
			<td style="width:161px;">W1089-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoniazid</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">I821450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoniazid-d4</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>I821452</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LC column, Synergi 2.5 um Polar RP 100 A 100 x 2 mm</td>
			<td style="width:211px;">Phenomenex</td>
			<td style="width:161px;">00D-4371-B0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LC guard cartridge</td>
			<td style="width:211px;">Phenomenex</td>
			<td style="width:161px;">AJ0-8788</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LC guard cartridge holder</td>
			<td style="width:211px;">Phenomenex</td>
			<td style="width:161px;">AJ0-9000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LC-MS/MS quantitation software</td>
			<td style="width:211px;">Sciex</td>
			<td style="width:161px;">Multiquant 2.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Levofloxacin</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td dir="LTR">1362103-200MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Levofloxacin-d8</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">L360002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Linezolid</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">L466500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Linezolid-d3</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>L466502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro centrifuge tubes</td>
			<td style="width:211px;">E&#38;K Scientific</td>
			<td style="width:161px;">695554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Moxifloxacin</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>M745000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Moxifloxacin-13C, d3</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">M745003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS/MS</td>
			<td style="width:211px;">Sciex</td>
			<td style="width:161px;">Triple Quad 5500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">OPC 14714</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>O667600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pretomanid (PA-824)</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td style="width:161px;">P122500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Prothionamide</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>P839100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Prothionamide-d5</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>P839102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pyrazinamide</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">P840600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pyrazinamide-15N, d3</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">P840602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Septum caps for injection vials</td>
			<td style="width:211px;">Agilent Technologies</td>
			<td style="width:161px;">5185-5862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Turbovap LV evaporator</td>
			<td style="width:211px;">Biotage</td>
			<td style="width:161px;">103198/11</td>
			<td></td>
		</tr>
	</tbody>
</table>

validated lc-ms/ms panel for quantifying 11 drug-resistant tb medications in small hair samples

耐药结核病 （DR-TB） 是一个日益严重的公共卫生威胁，评估治疗药物水平可能具有重要的临床益处。血浆药物水平是当前的黄金标准评估，但需要肾切除术和冷链，并捕获只是最近依从。我们的方法使用头发，一个易于收集并反映长期依从性的矩阵，测试11种抗结核药物。我们小组以前的工作表明，头发中的抗逆转录病毒药物水平与艾滋病毒结果有关。我们的DR-TB药物方法使用2毫克的头发（3厘米接近根），这是粉碎和提取甲醇。用一种LC-MS/MS方法分析样品，在16分钟内量化11种药物。11种药物的定量（LLOQs）下限范围从0.01纳克/毫克到1纳克/毫克不等。 药物存在通过比较两种质谱过渡的比率得到确认。样品使用药物与脱硝、15N-或13C标记药物等值的面积比进行量化。我们使用的校准曲线范围为 0.001-100 ng/mg。 该方法在直接观察治疗（DOT）上从DR-TB患者采集的头发样本的方便样本中的应用表明，在11种药物中的9种（异酰胺、苯丙胺、ethambutol、线胶、levofloacin、莫西氟辛、氟胺、贝达奎林、头头体）的线性动态范围内，头发中的药物水平。没有病人使用蛋白苯酰胺，并且测得的液酰胺水平接近其LLOQ（通过进一步的工作，而不是检查依西酰胺的代谢物是否适合监测暴露）。总之，我们描述了头发DR-TB药物的多机解毒板的开发，作为耐药结核病治疗期间治疗药物监测的技术。

图1显示了所有11种DR-TB药物的确诊水平的色谱图图图。使用不同的仪器和列时，每个护身奶的保留时间可能会更改，因此应单独确定确切的保留时间。
图2显示了一种特定药物（isoniazid，INH）在一种校准器（空白头发样本与DR-TB药物参考标准尖刺）的提取的Ion色谱仪（EIC）。定量器和限定符过渡用于定性地确认药物的存在，因为定量...

Watch this Scientific Journal Video about Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples at JoVE.com

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

目前分析患者是否遵守复杂耐药结核病（DR-TB）方案的方法可能不准确且资源密集。我们的方法分析头发，一个易于收集和储存的基质，浓度为11个DR-TB药物。使用LC-MS/MS，我们可以确定亚纳米格药物水平，可用于更好地了解药物依从性。

经过验证的LC-MS/MS面板，用于在小头发样本中量化11种耐药结核病药物

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...

患者的坚持是非常具有挑战性的，药物代谢可以改变人，所以客观测量药物水平可能证明是重要的，以防止在结核病治疗期间出现耐药性。如果坚持可以更准确和容易量化，那么卫生保健提供者可以制定一个治疗计划，将限制耐药性的发展。我们开发了第一个多分析物质谱仪面板，用于在单个检测中小头发样本中的多种结核病药物。我们的多药面板只需要每个患者一个两毫克的样品，并且既不使用固相提取，也不使用过滤步骤来防止样品量的任何损失。在治疗过程早期测量的药物暴露的客观测量可能会提醒临床医生治疗方案中个别药物的问题。头发样本必须在分析当天进行粉碎，以进行准确的药物浓度测量。要获得头发样本，在获得书面知情同意后，准备好材料，包括一个薄标签，两个参与者标签，一把剪刀，一块展开的铝箔，酒精擦拭和一个塑料袋。用酒精擦拭将剪刀的刀片擦干净。转到头部的背面，如果需要，请使用发夹抬起头发的顶部。从后部收集30缕头发，用手隔离吹毛。把剪刀拿到头皮上，剪头发的毛草。小心地把毛发放在铝箔上。将薄标签贴在毛草的端端上，安全地贴在铝箔上。远端是离头皮最远的一面。折叠在头发吹毛上一块铝箔上，并在铝箔上贴上参与者标签。将折叠的铝箔放入塑料袋中，并在塑料袋上贴上另一个参与者标签。对于从头发样本中提取药物，将每个头发样本的两毫克称重到单独标记的珠管中，将两毫克的空白头发样本重入 16 个附加珠管中。称重后，将所有珠管放入珠磨机均质器中，以 6.95 米/秒的速度均质样品，进行两个 30 秒的循环，每个周期之间休息 15 秒。第二次均质化后，在每个样品中加入500微升适当的内部标准混合物，并在基质空白管中加入500微升甲醇。根据表格将校准曲线管与适当体积的参考标准混合，将所有管放在37度水浴中，轻轻摇动两小时。在孵育结束时，将上经剂从每个珠管转移到新的标记微离心管中，并在珠管中加入500微升甲醇。涡珠管，将甲醇洗涤转移到相应的微离心管。将样品离心，小心地将超自然剂转移到新管中，而不会干扰头发颗粒。将管中的液体蒸发至32摄氏度的干燥，用200微升高压液相色谱级水与1%的甲酸重新组成样品。涡旋后，使用 250 微升刀片将液体样品转移到琥珀小瓶中。对于液相色谱串联质谱法，首先将2至100毫米的柱体安装为2.5微米粒径和100个安格斯孔径，用极端乙醚链接酚珠完全由多孔二氧化硅组成进入柱室。确认该列还安装了制造商推荐的防护盒。复制并粘贴 MDR-TB LCMS 方法转换中包含的复合信息。csv 文件进入质规格窗口中的框中。在电子表格中，创建校准曲线、质量控制、患者样本、校准曲线、质量控制、患者样本、校准曲线和质量控制的批次序列。在跑步的开始和结束以及校准曲线、质量控制和患者样本之前和之后添加溶剂空白注射。然后在校准曲线和高质量控制小瓶中的注射中至少放置8个溶剂空白注射，以减少分析性结转。批次完成后，打开量化软件。滚动转换以确保自动选择的保留时间准确无误，并检查高斯平滑设置为 1.5。接下来，单击显示校准曲线和回归，然后将等待类型设置为超过 x 的类型。单击"确定"并验证校准曲线和质量控制样本，以确保批次成功运行。对于每个限定符参考分析，至少三分之二的校准点必须在 80-120% 内具有精度检查校准曲线上方显示的 R 值是否大于 0.975。如果满足上述所有条件，则批次已通过，样本可以量化。单击工具栏中的"编辑"，然后单击"复制整个表"。然后将表格粘贴到电子表格中，并使用两次样品注射的计算浓度平均值来确定每个样品的报告浓度。图为所有测试的11种耐药结核病药物的色谱图，其确认水平。在这些数字中，可以观察到其中一种校准器中特定药物的离子色谱图。限定符和限定符转换用于定性地确认药物的存在，因为限定符区域与限定符区域之间的比率在样本中保持不变。内部标准也得到监控，以确保每个样品注射都得到规范化。在这项分析中，对南非西开普省直接观察到的治疗条件下服用耐药结核病药物的96名患者的总数中15个头发样本进行了方便抽样评估，以确定每种分析剂最低和最高水平的耐药结核病药物的代表性水平。在没有固相提取或过滤步骤的情况下，在将上流液转移到蒸发管时应小心。任何转移到管子中的东西最终都可以被注射到柱子中。由于珠子猛禽的转子可以飞走，如果没有紧密拧或锁定，请务必在操作前仔细检查转子。与艾滋病毒领域的抗逆转录病毒药物相比，结核病药物药代动力学、坚持和临床结果之间的关系在很大程度上仍未得到研究。我们假设一些结核药物在头发上积累得不好。瞄准这些药物的代谢物，可以更好地了解患者的依从性。

Research

JoVE Journal

Medicine

Protocol for Long Duration Whole Body Hyperthermia in Mice

持续时间较长的小鼠全身热疗协议

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

科研

医学

Orthotopic Small Bowel Transplantation in Rats

大鼠原位小肠移植

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

在免疫抑制剂他克莫司对干血斑使用LC-MS / MS定量

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

A Novel Approach for the Administration of Medications and Fluids in Emergency Scenarios and Settings

应急方案和设置药物和液体管理的新方法

The available routes of administration commonly used for medications and fluids in the acute care setting are generally limited to oral, intravenous, or ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

在耐药性颞叶癫痫患者卵圆孔电极记录的网络分析

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

在女小鼠害保留神经的中期尿道梗阻（NEMO）

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

一种使用加速度计量化上肢表现在日常生活的方法

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

经优化的 LC/ms 方法对囊性纤维化患者 Ivacaftor 及其主要代谢物和 Lumacaftor 的高通量分析

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

在体内MRI 对小鼠股骨骨愈合过程中骨折愈伤组织发育的评价

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Taste Exam: A Brief and Validated Test

味觉考试: 一个简短的验证测试

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement ...