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

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

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 D_2,¹³C-tacrolimus. After vortexing and centrifugation, 100 µl 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 °C for 1 week. Extracted samples in the autosampler are stable at +4 °C for at least 72 hr.

Introduction

Tacrolimus is a potent immonosuppressant^1-7 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 ac....

Protocol

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 “exempt” by the Colorado Multi-institutional Review Board (COMIRB, Aurora, Colorado).

1. Preparation of References and Solutions

1. Purchase tacrolimus and the internal standard D_2,¹³C-tacrolimus from the vendors listed in the Materials List.
   1. Prepare stock solutions in pure methanol at a concentration of 1 mg/ml for ....

Results

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.......

Discussion

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 µl). Nevertheless, high-throughp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tacrolimus	U.S. Pharmacopeial Convention	1642802
D2,13C-Tacrolimus	Toronto Research Chemicals Inc.	F370002
Red blood cells	University of Colorado Hospital	W20091305500 V
Plasma	University of Colorado Hospital	W2017130556300Q
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
Formic acid	Thermo Fisher Scientific	A118P-500
Phosphate-buffered saline (PBS)	Sigma-Aldrich	D8537
Zinc sulfate	Thermo Fisher Scientific	Z68-500
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 – 1,000 µl pipet, VoluMate LIQUISYSTEMS	Mettler Toledo	17008653
100 μl pipet tips with filter, sterile	Neptune	BT 100
1,000 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, 18 G 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
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