JoVE Journal > Medicine
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Please note that all translations are automatically generated. Click here for the English version.
Medicine

在免疫抑制剂他克莫司对干血斑使用LC-MS / MS定量

Published: November 08, 2015
doi:
10.3791/52424
, , , , , , ,
1iC42 Clinical Research and Development,University of Colorado, Anschutz Medical Campus, 2Division of Clinical Pharmacology,Cincinnati Children’s Hospital Medical Center, 3Food and Drug Administration (FDA),Center of Drug Evaluation Research – Office of Generic Drugs, 4Transplant Clinical Research,University of Cincinnati

Summary

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

Abstract

钙调神经磷酸酶抑制剂他克莫司是在美国实体器官移植后的免疫抑制大多数治疗方案的基石。他克莫司是一个窄的治疗指数的药物，因此需要治疗药物的监测，并根据它的全血谷浓度调整剂量。为方便家庭治疗药物，并坚持监测，干血斑的集合是一个有吸引力的概念。手指棒后，病人收集在滤纸上血滴在家里。血后进行干燥，则邮寄到分析实验室，其中他克莫司是用高效液相色谱法 – 串联质谱法量化（HPLC-MS / MS）在一个简单的手动蛋白质沉淀步骤和在线柱萃取组合。

他克莫司分析，一个6毫米光盘从血液点的饱和点刻。血斑用一颗子弹搅拌机均质第二然后蛋白沉淀，用甲醇/ 0.2M的硫酸锌含有内标D 2，13 C-他克莫司。涡旋和离心后，将100μl上清液注入到一个在线萃取柱中，用5毫升/分钟0.1甲酸/乙腈（7:3，体积比），持续1分钟。此后，切换阀被激活，分析物被反冲洗到分析柱（并用0.1％甲酸/乙腈梯度分离）。他克莫司被量化中使用的串联质谱仪的阳性反应的多模式（MRM）。

该测定是从1至50ng / ml的线性。批间变异性（3.6％-6.1％）和准确度（91.7％-101.6％）的评估20天以上达到验收标准。平均提取回收率为95.5％。有没有相关的结转，基质干扰和基体效应。他克莫司是稳定的干血斑在RT和在4℃下1周。在提取样本自动进样器是稳定的，在4℃下至少72小时。

Introduction

他克莫司是一种强效immonosuppressant 1-7具有一个大环内酯结构8（ 图1）。由于顺 -该CN键的反异构它形成在溶液9两种旋转异构体，可通过反相高效液相色谱法进行分离（HPLC），他克莫司是亲脂性和可溶于醇（甲醇653克/升，乙醇：355克/升），卤代烃（氯仿：573克/升）和乙醚。它是难溶于脂族烃（己烷：0.1克/升和水（pH 3：0.0047克/升）9所述的分子不包含任何发色团并且其紫外吸收最大值是通过抑制神经钙的192纳米的他克莫司的行为。其作用的机制已经在参考文献10,11综述。它是目前使用在80％以上的固体器官移植患者的在美国12。

他克莫司的治疗指数是considered可窄13。此外，他克莫司的剂量和血液浓度之间的相关性差和药代动力学是可变14,15。治疗药物监测指导剂量的他克莫司在移植患者因此一般的临床实践16-20。的目标是保持他克莫司血液中的浓度的预定义治疗范围内。下面的治疗范围他克莫司血液中的浓度可导致慢性或急性同种异体免疫反应的活性增加，而浓度大于治疗窗口增加用于过免疫抑制，癌症和毒性，如肾毒性，神经毒性，高血压和糖尿病的风险。他克莫司的药代动力学高个体内的变化可能是有害的既移植器官和患者存活21,22。而他克莫司的药代动力学的个体间变异主要是由CYP3A5基因多态性，原因个体内变异包括但不限于，药物与药物，疾病-药物和食品药物相互作用14,15。也缺乏坚持免疫抑制治疗药物治疗是一个促进因素，一个重要原因移植损失23,24。

这些考虑表明，频繁家治疗药物和他克莫司的全血浓度的粘附监测可能是有益的，以确保患者在任何时候所需治疗窗口内的他克莫司的曝光。但是，物流和更频繁的治疗药物监测的成本，因为它是目前的临床实践15是令人望而却步。其中一个原因是，该患者具有看到一个抽血有绘制所需的静脉血样。干血斑最近成为一个有吸引力的概念25-28。经过简单的手指贴在病人聚集在一个特殊的过滤纸卡，后血斑有d血滴里德，它可以邮寄到中心实验室用于他克莫司的分析和任何其他的免疫抑制剂，该患者可能目前服用。这已经成为可能的，因为高度敏感和特异的LC-MS / MS测定法他克莫司和其他免疫抑制剂的定量在非常小的血液量的发展，如干燥血点（通常是20微升血液）25,29-43。另一个优点是，微创，低体积的样品收集策略诸如干血斑大大方便治疗药物的监测和药代动力学研究中的儿童小28。

他克莫司通常以静脉EDTA全血15。原因是他克莫司广泛分布成血细胞和临床研究报道他克莫司谷浓度在血液之间有更好的相关性比血浆的临床事件15,18。相比较而言，Ta的分析crolimus在干燥血点是基于毛细血与该滤纸基质混合。此呈现在他克莫司和潜在的干扰与LC-MS / MS分析的溶解方面的挑战。这里，我们提出基于使用结合的子弹搅拌器具有高流动性的在线列取样清理过程和LC-MS / MS分析的干燥血点的均质建立和验证实验。到今天为止，此法已成功地用于五千余他克莫司干血斑样本的定量分析在临床试验中坚持监测。

Protocol

从健康人不标明身份的血液样本分别来自美国科罗拉多州大学医院（科罗拉多州Aurora）。使用去鉴定血库样本进行验证研究，以及对标准品和质量控制样品的制备被视为“豁免”是由美国科罗拉多州的多机构审查委员会（COMIRB，极光，科罗拉多）。 1.准备参考和解决方案购买他克莫司和内部标准的D 2，13 C-他克莫司从材料清单中所列的供应商。 ?…

Representative Results

空白样品的代表离子色谱，样品掺入在量化和患者样品的下限示于图3。 校准曲线检测的下限为0.5 ng / ml和定量的下限为1.0毫微克/毫升。把15ng / ml的被选为最高校准器更高的浓度不太可能在临床上正常的情况下达成。 校准曲线在每个验证天在人EDTA全血新鲜制备的，在过滤器上卡干燥和用萃取甲醇/ 0.2M的硫酸锌（70…

Discussion

虽然，如上所述，基于干血斑的治疗药物和他克莫司的粘附监测的概念是有吸引力的，也有超出那些通常与他克莫司的静脉EDTA全血样品中的LC-MS / MS分析相关联的分析挑战。这些包括，但不限于一个事实，即基质是毛细管全血浸透到这里使用的过滤卡材料和低血容量（20微升）的棉短绒材料。尽管如此，在中心实验室高通量分析需要快速和可靠的提取方法，其导致缺乏基质干扰和基质效应结合一个…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the United States Federal Drug Administration (FDA) contract HHSF223201310224C and the United States National Institutes of Health/FDA grant 1U01FD004573-01.

Materials

Reference Materials
Tacrolimus U.S. Pharmacopeial Convention 1642802
D2,13C-Tacrolimus Toronto Research Chemicals Inc. F370002
Test Materials
Red blood cells University of Colorado Hospital W20091305500 V
Plasma University of Colorado Hospital W2017130556300Q
Solvents
Acetone CHROMASOLV, HPLC, ≥ 99,9 % Sigma-Aldrich 439126-4 L
Acetonitrile Optima LC/ MS, UHPLC- UV Thermo Fisher Scientific A955-4
Isopropanol 99.9 %, HPLC Fisher Scientific BP2632-4
Methanol Optima LC/ MS Thermo Fisher Scientific A452-4
Water Optima LC/ MS, UHPLC- UV Thermo Fisher Scientific W6-4
Other Chemicals
Formic acid Thermo Fisher Scientific A118P-500
Phosphate-buffered saline (PBS) Sigma-Aldrich D8537
Zinc sulfate Thermo Fisher Scientific Z68-500
Laboratory Instruments and Consumables
0.5 – 10 µl pipet, VoluMate LIQUISYSTEMS Mettler Toledo 17008649
1,5 mL- Eppendorf tube Thermo Fisher Scientific 02-682-550
10 – 100 µL pipet, VoluMate LIQUISYSTEMS Mettler Toledo 17008651
10 μL- pipet tips with filter, sterile Neptune BT 10XLS3
100 – 1000 µl pipet, VoluMate LIQUISYSTEMS Mettler Toledo 17008653
100 μL- pipet tips with filter, sterile Neptune BT 100
1000 μL- pipet tips with filter, sterile Multimax 2940
2 – 20 µL pipet, VoluMate LIQUISYSTEMS Mettler Toledo 17008650
2 mL- Eppendorf tube Thermo Fisher Scientific 02-681-258
20 – 200 µL pipet, VoluMate LIQUISYSTEMS Mettler Toledo 17008652
20 μL- pipet tips with filter, sterile GeneMate P-1237-20
200 μL- pipet tips with filter Multimax 2938T
200 μL- pipet tips with filter, sterile Multimax 2936J
50 mL- Falcon tube BD Falcon 352070
300 μL inserts for  HPLC vials Phenomenex ARO-9973-13
Balance PR2002 Mettler Toledo 1117050723
Balances AX205 Delta Range Mettler Toledo 1119343379
Bullet Blender Homogenizer Next Advance BBX24
Centrifuge Biofuge Fresco Heraeus 290395
Disposable Wipes PDI Q55172
Glass v ials, 4 mL Thermo Fisher Scientific 14-955-334
Glass vials, 20 mL Thermo Fisher Scientific B7800-20
Gloves, nitrile Titan Brand Gloves 44-100S
HPLC vials, 9 mm, 2 mL, clear Phenomenex ARO- 9921-13
Lids for HPLC vials Phenomenex ARO- 8952-13-B
Needle, 18G 1.5 Precision Glide 305196
Rack for Eppendorf tubes Thermo Fisher Scientific 03-448-11
Rack for HPLC Vials Thermo Fisher Scientific 05-541-29
Steel beads 0.9 – 2 mm Next Advance SSB14B
Storage boxes for freezers / refrigerators Thermo Fisher Scientific 03-395-464
Standard multi-tube vortexer VWR Scientific Products 658816-115
Whatman Paper, 903 Protein Saver US 100/PK GE Whatman  2016-05
HPLC Equipment and Columns
Autosampler CTC PAL  PAL.HTCABIx1
Binary pump, Agilent 1260 Infinity Agilent Technologies 1260 G1312B
Binary pump, Agilent 1290 Infinity Agilent Technologies 1290 G4220A
Micro vacuum degasser, Agilent 1260 Agilent Technologies 1260 G13798
Column oven,  Agilent 1290 with 2 position  Agilent Technologies 1290 G1216C
Thermostated column compartment with integrated 6 port switching valve Agilent Technologies 1290 G1316C
HPLC pre-column cartridge, Zorbax XDB C8 (5 µm particle size), 4.6 · 12.5 mm Phenomenex 820950-926
HPLC analytical column, Zorbax Eclipse-XDB-C8 (5 µm particle size), 4.6 · 150 mm Phenomenex 993967-906
Tandem Mass Spectrometer
API5000 MS/MS with TurboIonspray source AB Sciex 4364257
Mass spectrometry software AB Sciex Analyst 1.5.1
DOWNLOAD MATERIALS LIST

References

  1. Goto, T., et al. Discovery of FK506, a novel immunosuppressant isolated from Streptomyces Tsukubaensis. Transplant Proc. 19 (5 Suppl 6), 4-8 (1987).
  2. Kino, T., Hatanaka, H., Miyata, S. FK506, a novel immunosuppressant isolated from a streptomyces. I: Fermentation, isolation and physico-chemical and biological characteristics. J. Antibiotics. 40 (9), 1249-1255 (1987).
  3. Starzl, T. E., et al. FK506 for liver, kidney and pancreas transplantation. Lancet. 2 (8670), 1000-1004 (1989).
  4. . Randomised trial comparing tacrolimus (FK506) and cyclosporin in prevention of liver allograft rejection. European FK506 Multicentre Liver Study Group. Lancet. 344 (8920), 423-428 (1994).
  5. . A comparison of tacrolimus (FK 506) and cyclosporine for immunosuppression in liver transplantation. The U.S. Multicenter FK506 Liver Study Group. N. Engl. J. Med. 331 (17), 1110-1115 (1994).
  6. Mayer, A. D., et al. Multicenter randomized trial comparing tacrolimus (FK506) and cyclosporine in the prevention of renal allograft rejection: a report of the European Tacrolimus Multicenter Renal Study Group. Transplantation. 64 (3), 436-443 (1997).
  7. Pirsch, J. D., Miller, J., Deierhoi, M. H., Vincenti, F., Filo, R. S. A comparison of tacrolimus (FK506) and cyclosporine for immunosuppression after cadaveric renal transplantation. FK506 Kidney Transplant Study Group.. Transplantation. 15 (7), 977-983 (1997).
  8. Tanaka, H., et al. Physicochemical properties of FK506 a novel immunosuppressant isolated from Streptomyces Tsukubaensis. Transplant Proc. 14 ((5 Suppl 6)), 11-16 (1987).
  9. Spencer, C. M., Goa, K. L., Gills, J. C. Tacrolimus. An update of its pharmacology and clinical efficacy in the management of organ transplantation. Drugs. 54 (6), 925-975 (1997).
  10. Clipstone, N. A., Crabtree, G. R. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature. 357 (6380), 695-697 (1992).
  11. Barbarino, J. M., Staatz, C. E., Venkataramanan, R., Klein, T. E., Altman, R. B. PharmGKB summary: cyclosporine and tacrolimus pathways. Pharmacogenet. Genomics. 23 (10), 563-585 (2013).
  12. Christians, U., Benet, L. Z., Lampen, A. Mechanisms of clinically significant drug interactions associated with tacrolimus. Clin. Pharmacokinet. 41 (11), 813-851 (2002).
  13. Christians, U., Pokaiyavananichkul, T., Chan, L., Burton, M. E., Shaw, L. M., Schentag, J. J., Evans, W. e. b. b. Tacrolimus In: Pharmacokinetics and Pharmacodynamics. Principles of Therapeutic Drug Monitoring. , 529-562 (2005).
  14. Holt, D. W., et al. International Federation of Clinical Chemistry/ International Association of Therapeutic Drug Monitoring and Clinical Toxicology working group on immunosuppressive drug monitoring. Ther. Drug Monit. 24 (1), 59-67 (2002).
  15. Holt, D. W., Jones, K., Lee, T., Stadler, P., Johnston, A. Quality assessment issues of new immunosuppressive drugs and experimental experience. Ther. Drug Monit. 18 (4), 362-367 (1996).
  16. Jusko, W. J., et al. Consensus document: therapeutic drug monitoring of tacrolimus (FK-506). Ther. Drug Monit. 17 (6), 606-614 (1995).
  17. Oellerich, M., et al. Therapeutic drug monitoring of cyclosporine and tacrolimus. Update on Lake Louise Conference on cyclosporine and tacrolimus. Clin. Biochem. 31 (5), 309-316 (1998).
  18. Wong, S. H. Therapeutic drug monitoring for immunosuppressants. Clin. Chim. Acta. 313 (1-2), 241-253 (2001).
  19. Kahan, B. D., et al. Low intraindividual variability of cyclosporin A exposure reduces chronic rejection incidence and health care costs. J. Am. Soc. Nephrol. 11 (6), 1122-1131 (2000).
  20. Kahan, B. D., et al. Variable oral absorption of cyclosporine. A biopharmaceutical risk factor for chronic renal allograft rejection. Transplantation. 62 (5), 599-606 (1996).
  21. Kelly, D. A. Current issues in pediatric transplantation. Pediatr. Transplant. 10 (6), 712-720 (2006).
  22. Spivey, C. A., Chisholm-Burns, M. A., Damadzadeh, B., Billheimer, D. Determining the effect of immunosuppressant adherence on graft failure risk among renal transplant recipients. Clin. Transplant. 28 (1), 96-104 (2014).
  23. Taylor, P. J., Tai, C. H., Franklin, M. E., Pillans, P. I. The current role of liquid chromatography-tandem mass spectrometry in therapeutic drug monitoring of immunosuppressant and antiretroviral drugs. Clin. Biochem. 44 (1), 14-20 (2011).
  24. Edelbroek, P. M., van der Heijden, J., Stolk, L. M. Dried blood spot methods in therapeutic drug monitoring: methods, assays, and pitfalls. Ther. Drug Monit. 31 (3), 327-336 (2009).
  25. Meesters, R. J., Hooff, G. P. State-of-the-art dried blood spot analysis: an overview of recent advances and future trends. Bioanalysis. 5 (17), 2187-2208 (2013).
  26. Pandya, H. C., Spooner, N., Mulla, H. Dried blood spots, pharmacokinetic studies and better medicines for children. Bioanalysis. 3 (7), 779-786 (2011).
  27. Koster, R. A., Alffenaar, J. W., Greijdanus, B., Uges, D. R. Fast LC-MS/MS analysis of tacrolimus, sirolimus, everolimus and cyclosporin A in dried blood spots and the influence of the hematocrit and immunosuppressant concentration on recovery. Talanta. 115 (Oct 15), 47-54 (2013).
  28. Hinchliffe, E., Adaway, J., Fildes, J., Rowan, A., Keevil, B. G. Therapeutic drug monitoring of ciclosporin A and tacrolimus in heart lung transplant patients using dried blood spots. Ann Clin. Biochem. 51 (Pt 1), 106-109 (2014).
  29. Koop, D. R., Bleyle, L. A., Munar, M., Cherala, G., Al-Uzri, A. Analysis of tacrolimus and creatinine from a single dried blood spot using liquid chromatography tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.. 926 ((May 1)), 54-61 (2013).
  30. Sadilkova, K., Busby, B., Dickerson, J. A., Rutledge, J. C., Jack, R. M. Clinical validation and implementation of a multiplexed immunosuppressant assay in dried blood spots by LC-MS/MS. Clin. Chim. Acta.. 421 ((Jun 5)), 152-156 (2013).
  31. Li, Q., Cao, D., Huang, Y., Xu, H., Yu, C., Li, Z. Development and validation of a sensitive LC-MS/MS method for determination of tacrolimus on dried blood spots. Biomed. Chromatogr. 27 (3), 327-334 (2013).
  32. Hinchliffe, E., Adaway, J. E., Keevil, B. G. Simultaneous measurement of cyclosporin A and tacrolimus from dried blood spots by ultra-high performance liquid chromatography tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.. 883-884 ((Feb 1)), 102-107 (2012).
  33. Webb, N. J., Roberts, D., Preziosi, R., Keevil, B. G. Fingerprick blood samples can be used to accurately measure tacrolimus levels by tandem mass spectrometry). Pediatr. Transplant. 9 (6), 729-733 (2005).
  34. Keevil, B. G., Fildes, J., Baynes, A., Yonan, N. Liquid chromatography-mass spectrometry measurement of tacrolimus in finger-prick samples compared with venous whole blood samples. Ann. Clin. Biochem. 46 (Pt 2), 144-145 (2009).
  35. Yonan, N., Martyszczuk, R., Machaal, A., Baynes, A., Keevil, B. G. Monitoring of cyclosporine levels in transplant recipients using self-administered fingerprick sampling. Clin. Transpl. 20 (2), 221-225 (2006).
  36. Keevil, B. G., et al. Simultaneous and rapid analysis of cyclosporin A and creatinine in finger prick blood samples using liquid chromatography tandem mass spectrometry and its application in C2 monitoring. Ther Drug Monit. 24 (6), 757-767 (2002).
  37. Hoogtanders, K., et al. Dried blood spot measurement of tacrolimus is promising for patient monitoring. Transplantation. 83 (2), 237-238 (2007).
  38. Heijden, J., et al. Therapeutic drug monitoring of everolimus using the dried blood spot method in combination with liquid chromatography-mass spectrometry. J. Pharm. Biomed. Anal. 50 (4), 664-670 (2009).
  39. Cheung, C. Y., et al. Dried blood spot measurement: application in tacrolimus monitoring using limited sampling strategy and abbreviated AUC estimation. Transpl. Int. 21 (2), 140-145 (2008).
  40. Hoogtanders, K., et al. Therapeutic drug monitoring of tacrolimus with the dried blood spot method. J. Pharm. Biomed. Anal. 44 (3), 658-664 (2007).
  41. Wilhelm, A. J., den Burger, C. J., Vos, R. M., Chahbouni, A., Sinjewel, A. Analysis of cyclosporin A in dried blood spots using liquid chromatography tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877 (14-15), 1595-1598 (2009).
  42. Ostler, M. W., Porter, J. H., Buxton, M. O. Dried blood spot collection of health biomarkers to maximize participation in population studies. J. Vis. Exp. (83), e50973 (2014).
  43. Schäfer, P., Störtzel, M., Vogt, S., Weinmann, W. Ion suppression effects in liquid chromatography-electrospray-ionisation transport-region collision induced dissociation mass spectrometry with different serum extraction methods for systematic toxicological analysis with mass spectra libraries. J. Chromatogr. B. 773 (1), 47-52 (2002).
  44. Peck, H. R., Timko, D. M., Landmark, J. D., Stickle, D. F. A survey of apparent blood volumes and sample geometries among filter paper bloodspot samples submitted for lead screening. Clin. Chim. Acta. 400 (1-2), 103-106 (2009).
  45. Christians, U., et al. Automated, fast and sensitive quantification of drugs in blood by liquid chromatography-mass spectrometry with on-line extraction: immunosuppressants. J. Chromatogr. B. 748 (1), 41-53 (2000).
  46. Clavijo, C., et al. Development and validation of a semi-automated assay for the highly sensitive quantification of Biolimus A9 in human whole blood using high performance liquid chromatography-tandem mass spectrometry. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 877 (29), 3506-3514 (2009).
  47. Mei, J. V., Alexander, J. R., Adam, B. W., Hannon, W. H. Use of filter paper for the collection and analysis of human whole blood specimens. J. Nutr. 131 (5), S1631-S1636 (2001).

Tags

TacrolimusImmunosuppressantLC-MS/MSDried Blood SpotsTherapeutic Drug MonitoringSolid Organ TransplantationProtein PrecipitationOnline Column ExtractionHPLCMass SpectrometryAssay ValidationStability
check_url/52424?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
Shokati, T., Bodenberger, N., Gadpaille, H., Schniedewind, B., Vinks, A. A., Jiang, W., Alloway, R. R., Christians, U. Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS. J. Vis. Exp. (105), e52424, doi:10.3791/52424 (2015).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image