Name: quantification of the immunosuppressant tacrolimus on dried blood spots using lc-ms/ms
Uploaded: 2015-11-08T14:00:00
Description: Watch this Scientific Journal Video about Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS at JoVE.com

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.

De-identified blood samples from healthy individuals were from the University of Colorado Hospital (Aurora, Colorado). The use of de-identified blood bank samples for validation studies as well as for the preparation of calibrators and quality control samples was considered &#8220;exempt&#8221; by the Colorado Multi-institutional Review Board (COMIRB, Aurora, Colorado).
1. Preparation of References and Solutions
<ol>
	<li>Purchase tacrolimus and the internal standard D2,13</s...

Although, as aforementioned, the concept of therapeutic drug and adherence monitoring of tacrolimus based on dried blood spots is attractive, there are analytical challenges that go beyond those typically associated with the LC-MS/MS analysis of tacrolimus in venous EDTA whole blood samples. These include, but are not limited to, the fact that the matrix is capillary whole blood soaked into the cotton linters material of the filter card material used here and the low blood volume (20 &#181;l). Nevertheless, high-throughp...

Tacrolimus is a potent immonosuppressant1-7 that has a macrolide structure8 (Figure 1). Due to cis-trans isomerism of the C-N bonds it forms two rotamers in solution9 that can be separated by reversed phase high-performance liquid chromatography (HPLC) Tacrolimus is lipophilic and soluble in alcohols (methanol: 653 g/L, ethanol: 355 g/L), halogenated hydrocarbons (chloroform: 573 g/L) and ether. It is sparingly soluble in aliphatic hydrocarbons (hexane: 0.1 g/L and water (pH 3: 0.0047 g/L)9. The molecule does not contain any chromophore and its UV-absorption maximum is 192 nm. Tacrolimus acts via inhibition of calcineurin. Its mechanism of action has been reviewed in references10,11. It is currently used in more than 80% of solid-organ transplant patients in the United States12.
The therapeutic index of tacrolimus is considered to be narrow13. In addition, the correlation between tacrolimus doses and blood concentrations is poor and pharmacokinetics is variable14,15. Therapeutic drug monitoring to guide tacrolimus dosing in transplant patients is therefore general clinical practice16-20. The goal is to keep the tacrolimus blood concentrations within a pre-defined therapeutic range. Tacrolimus blood concentrations below the therapeutic range may result in increased activity of chronic or acute allo-immune reactions, while concentrations above the therapeutic window increase the risk for over-immunosuppression, cancer and toxicities, such as nephrotoxicity, neurotoxicity, hypertension, and diabetes. High pharmacokinetic intra-individual variability of tacrolimus may be detrimental to both transplant organ and patient survival21,22. While inter-individual variability of tacrolimus pharmacokinetics is mainly caused by CYP3A5 polymorphisms, reasons for intra-individual variability include, but are not limited to, drug-drug, disease-drug and food-drug interactions14,15. Also lack of adherence to the immunosuppressive therapeutic drug regimen is a contributing factor and a major reason for graft loss23,24.
These considerations suggest that frequent home therapeutic drug and adherence monitoring of tacrolimus whole blood concentrations may be beneficial to ensure that patients have tacrolimus exposure within the desired therapeutic window at all times. However, the logistics and cost of more frequent therapeutic drug monitoring as it is current clinical practice15 is prohibitive. One of the reasons is that the patient has to see a phlebotomist to have the required venous blood sample drawn. Dried blood spots have recently emerged as an attractive concept25-28. After a simple finger stick the patient collects a blood drop on a special filter paper card and after the blood spot has dried, it can be mailed to a central laboratory for analysis of tacrolimus and any other immunosuppressant that the patient may currently be taking. This has become possible due to the development of highly sensitive and specific LC-MS/MS assays for the quantification of tacrolimus and other immunosuppressants in very small blood volumes such as dried blood spots (typically 20 &#181;l of blood)25,29-43. Another advantage is that minimally invasive, low volume sample collection strategies such as dried blood spots greatly facilitate therapeutic drug monitoring and pharmacokinetic studies in small children28.
Tacrolimus is usually measured in venous EDTA whole blood15. Reasons are that tacrolimus extensively distributes into blood cells and that clinical studies have reported better correlation between tacrolimus trough concentrations in blood than in plasma with clinical events15,18. In comparison, the analysis of tacrolimus in dried blood spots is based on capillary blood that is mixed with the filter paper matrix. This presents challenges in terms of solubilization of tacrolimus and potential interferences with the LC-MS/MS analysis. Here we present an established and validated assay based on homogenization of the dried blood spot using a bullet blender in combination with a high-flow online column sample clean up procedure and LC-MS/MS analysis. As of today, this assay has successfully been used for the quantification of more than five thousand tacrolimus dried blood spot samples for adherence monitoring in clinical trials.

<table border="0" cellpadding="0" cellspacing="0">
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			<td>Reference Materials</td>
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			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tacrolimus</td>
			<td>U.S. Pharmacopeial Convention</td>
			<td>1642802</td>
		</tr>
		<tr>
			<td>D2,13C-Tacrolimus</td>
			<td>Toronto Research Chemicals Inc.</td>
			<td>F370002</td>
		</tr>
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			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>Test Materials</td>
			<td></td>
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			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>Red blood cells</td>
			<td>University of Colorado Hospital</td>
			<td>W20091305500 V</td>
		</tr>
		<tr>
			<td>Plasma</td>
			<td>University of Colorado Hospital</td>
			<td>W2017130556300Q</td>
		</tr>
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			<td></td>
			<td></td>
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			<td>Solvents</td>
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			<td>Acetone CHROMASOLV, HPLC, &#8805; 99,9 %</td>
			<td>Sigma-Aldrich</td>
			<td>439126-4 L</td>
		</tr>
		<tr>
			<td>Acetonitrile Optima LC/ MS, UHPLC- UV</td>
			<td>Thermo Fisher Scientific</td>
			<td>A955-4</td>
		</tr>
		<tr>
			<td>Isopropanol 99.9 %, HPLC</td>
			<td>Fisher Scientific</td>
			<td>BP2632-4</td>
		</tr>
		<tr>
			<td>Methanol Optima LC/ MS</td>
			<td>Thermo Fisher Scientific</td>
			<td>A452-4</td>
		</tr>
		<tr>
			<td>Water Optima LC/ MS, UHPLC- UV</td>
			<td>Thermo Fisher Scientific</td>
			<td>W6-4</td>
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			<td></td>
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			<td>Other Chemicals</td>
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			<td></td>
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			<td>Formic acid</td>
			<td>Thermo Fisher Scientific</td>
			<td>A118P-500</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
		</tr>
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			<td>Zinc sulfate</td>
			<td>Thermo Fisher Scientific</td>
			<td>Z68-500</td>
		</tr>
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			<td></td>
			<td></td>
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			<td>Laboratory Instruments and Consumables</td>
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		<tr>
			<td>0.5 &#8211; 10 &#181;l pipet, VoluMate LIQUISYSTEMS</td>
			<td>Mettler Toledo</td>
			<td>17008649</td>
		</tr>
		<tr>
			<td>1,5 mL- Eppendorf tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-682-550</td>
		</tr>
		<tr>
			<td>10 &#8211; 100 &#181;L pipet, VoluMate LIQUISYSTEMS</td>
			<td>Mettler Toledo</td>
			<td>17008651</td>
		</tr>
		<tr>
			<td>10 &#956;L- pipet tips with filter, sterile</td>
			<td>Neptune</td>
			<td>BT 10XLS3</td>
		</tr>
		<tr>
			<td>100 &#8211; 1000 &#181;l pipet, VoluMate LIQUISYSTEMS</td>
			<td>Mettler Toledo</td>
			<td>17008653</td>
		</tr>
		<tr>
			<td>100 &#956;L- pipet tips with filter, sterile</td>
			<td>Neptune</td>
			<td>BT 100</td>
		</tr>
		<tr>
			<td>1000 &#956;L- pipet tips with filter, sterile</td>
			<td>Multimax</td>
			<td>2940</td>
		</tr>
		<tr>
			<td>2 &#8211; 20 &#181;L pipet, VoluMate LIQUISYSTEMS</td>
			<td>Mettler Toledo</td>
			<td>17008650</td>
		</tr>
		<tr>
			<td>2 mL- Eppendorf tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-681-258</td>
		</tr>
		<tr>
			<td>20 &#8211; 200 &#181;L pipet, VoluMate LIQUISYSTEMS</td>
			<td>Mettler Toledo</td>
			<td>17008652</td>
		</tr>
		<tr>
			<td>20 &#956;L- pipet tips with filter, sterile</td>
			<td>GeneMate</td>
			<td>P-1237-20</td>
		</tr>
		<tr>
			<td>200 &#956;L- pipet tips with filter</td>
			<td>Multimax</td>
			<td>2938T</td>
		</tr>
		<tr>
			<td>200 &#956;L- pipet tips with filter, sterile</td>
			<td>Multimax</td>
			<td>2936J</td>
		</tr>
		<tr>
			<td>50 mL- Falcon tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
		</tr>
		<tr>
			<td>300 &#956;L inserts for&#160; HPLC vials</td>
			<td>Phenomenex</td>
			<td>ARO-9973-13</td>
		</tr>
		<tr>
			<td>Balance PR2002</td>
			<td>Mettler Toledo</td>
			<td>1117050723</td>
		</tr>
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			<td>Balances AX205 Delta Range</td>
			<td>Mettler Toledo</td>
			<td>1119343379</td>
		</tr>
		<tr>
			<td>Bullet Blender Homogenizer</td>
			<td>Next Advance</td>
			<td>BBX24</td>
		</tr>
		<tr>
			<td>Centrifuge Biofuge Fresco</td>
			<td>Heraeus</td>
			<td>290395</td>
		</tr>
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			<td>Disposable Wipes</td>
			<td>PDI</td>
			<td>Q55172</td>
		</tr>
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			<td>Glass v ials, 4 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-955-334</td>
		</tr>
		<tr>
			<td>Glass vials, 20 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>B7800-20</td>
		</tr>
		<tr>
			<td>Gloves, nitrile</td>
			<td>Titan Brand Gloves</td>
			<td>44-100S</td>
		</tr>
		<tr>
			<td>HPLC vials, 9 mm, 2 mL, clear</td>
			<td>Phenomenex</td>
			<td>ARO- 9921-13</td>
		</tr>
		<tr>
			<td>Lids for HPLC vials</td>
			<td>Phenomenex</td>
			<td>ARO- 8952-13-B</td>
		</tr>
		<tr>
			<td>Needle, 18G 1.5</td>
			<td>Precision Glide</td>
			<td>305196</td>
		</tr>
		<tr>
			<td>Rack for Eppendorf tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>03-448-11</td>
		</tr>
		<tr>
			<td>Rack for HPLC Vials</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-541-29</td>
		</tr>
		<tr>
			<td>Steel beads 0.9 &#8211; 2 mm</td>
			<td>Next Advance</td>
			<td>SSB14B</td>
		</tr>
		<tr>
			<td>Storage boxes for freezers / refrigerators</td>
			<td>Thermo Fisher Scientific</td>
			<td>03-395-464</td>
		</tr>
		<tr>
			<td>Standard multi-tube vortexer</td>
			<td>VWR Scientific Products</td>
			<td>658816-115</td>
		</tr>
		<tr>
			<td>Whatman Paper, 903 Protein Saver US 100/PK</td>
			<td>GE Whatman&#160;</td>
			<td>2016-05</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Equipment and Columns</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autosampler</td>
			<td>CTC PAL&#160;</td>
			<td>PAL.HTCABIx1</td>
		</tr>
		<tr>
			<td>Binary pump, Agilent 1260 Infinity</td>
			<td>Agilent Technologies</td>
			<td>1260 G1312B</td>
		</tr>
		<tr>
			<td>Binary pump, Agilent 1290 Infinity</td>
			<td>Agilent Technologies</td>
			<td>1290 G4220A</td>
		</tr>
		<tr>
			<td>Micro vacuum degasser, Agilent 1260</td>
			<td>Agilent Technologies</td>
			<td>1260 G13798</td>
		</tr>
		<tr>
			<td>Column oven,&#160; Agilent 1290 with 2 position&#160;</td>
			<td>Agilent Technologies</td>
			<td>1290 G1216C</td>
		</tr>
		<tr>
			<td>Thermostated column compartment with integrated 6 port switching valve</td>
			<td>Agilent Technologies</td>
			<td>1290 G1316C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC pre-column cartridge, Zorbax XDB C8 (5 &#181;m particle size), 4.6 &#183; 12.5 mm</td>
			<td>Phenomenex</td>
			<td>820950-926</td>
		</tr>
		<tr>
			<td>HPLC analytical column, Zorbax Eclipse-XDB-C8 (5 &#181;m particle size), 4.6 &#183; 150 mm</td>
			<td>Phenomenex</td>
			<td>993967-906</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tandem Mass Spectrometer</td>
			<td></td>
			<td></td>
		</tr>
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			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>API5000 MS/MS with TurboIonspray source</td>
			<td>AB Sciex</td>
			<td>4364257</td>
		</tr>
		<tr>
			<td>Mass spectrometry software</td>
			<td>AB Sciex</td>
			<td>Analyst 1.5.1</td>
		</tr>
	</tbody>
</table>

quantification of the immunosuppressant tacrolimus on dried blood spots using lc-ms/ms

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Representative ion chromatograms of a blank sample, a sample spiked at the lower limit of quantification and a patient sample are shown in Figure 3.
Calibration Curves
The lower limit of detection was 0.5 ng/ml and the lower limit of quantification was 1.0 ng/ml. Fifty ng/ml was chosen as the highest calibrator as higher concentrations are unlikely to be reached in the clinic under normal circumstances.
Calibration curves...

Watch this Scientific Journal Video about Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS at JoVE.com

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

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.

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

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