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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 D2,13C-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 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 µl 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.

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 D2,13C-tacrolimus from the vendors listed in the Materials List.
    1. Prepare stock solutions in pure methanol at a concentration of 1 mg/ml for tacrolimus and a concentration of 10 µg/ml for D2,13C-tacrolimus. Make stock solutions of reference materials based on three independent weightings. Aliquot stock solutions and store at -70 °C or below.
  2. Prepare solution to precipitate proteins and extract tacrolimus using methanol / 0.2 M ZnSO4 in water (7:3, v:v). This solution also contains the internal standard D2,13C-tacrolimus at a concentration of 2.5 ng/ml and is used for the extraction of all samples except for the extraction of blank samples (please see 1.3.3).
    1. Prepare this protein precipitation solution freshly on each extraction day and set the expiration of the solution at 12 hr.
  3. Preparation of calibration curve and quality control (QC) samples
    1. Prepare stock solutions of tacrolimus by performing appropriate dilutions of the stock solution using pure methanol.
    2. To prepare calibrators and quality control samples, spike 20 µl of appropriately diluted stock solution into EDTA whole blood, incubate at 37 °C under gentle shaking in a water bath to allow for homogeneous distribution of tacrolimus into the cellular blood components for 20 min and aliquot into 1.5 ml polypropylene tubes with conical bottom and snap-on lids. Ensure that the relative volume of organic solvent does not exceed 5%.
    3. Spot 50 µl of the spiked whole blood into the middle of each circle on the filter cards using a pipette.
    4. Dry the blood spots on filter cards at RT for 3 hr.
    5. Prepare tacrolimus calibration standards in human EDTA whole blood at tacrolimus concentrations of 1, 2.5, 5, 10, 25, and 50 ng/ml. Prepare a blank sample for extraction like the calibration standards with the protein precipitation solution containing the internal standard D2,13C-tacrolimus (“zero sample”).
    6. Prepare QC samples in human EDTA whole blood at concentrations of 0, 2, 4, 20, 40 ng/ml. Prepare a blank sample. In contrast to the QC samples that are extracted with precipitation containing the internal standard D2,13C-tacrolimus, extract this blank sample with protein precipitation solution that does not contain the internal standard D2,13C-tacrolimus (“blank sample”).
  4. Collection of clinical samples
    1. Collect dried blood spots as described in43,44.

2. Extraction of Tacrolimus Dried Blood Spot Samples

  1. Visually inspect the dried blood spot to ensure acceptable sample quality and volume45.
  2. Punch center of the blood spot on the filter card with a 6-mm hole punch.
    Note: The quality of punches may be monitored by weighing. A punched saturated filter disc weighs in average 5.02 mg ± 0.09 mg (range: 4.83- 5.14 mg, n=12).
  3. Place discs into 1.5 ml polypropylene tubes with conical bottom and snap-on lids.
  4. Add 20-30 bullets to each tube.
  5. Add 500 µl of the protein precipitation solution (methanol: 0.2 M ZnSO4, 7:3, v:v with 2.5 ng/ml internal standard) into each tube. For the extraction of blank samples, use protein precipitation solution without the internal standard.
  6. Homogenize the discs in the bullet blender for 1 min (maximum speed, setting “10”).
  7. Shake samples at RT on multi-tube vortex (maximum speed, setting “10”) for 10 min.
  8. Centrifuge samples at 16,000 x g and 4 °C for 10 min.
  9. Transfer the supernatants into glass HPLC vials equipped with a 300 µl insert. Use pre-slit Teflon seals.
    Note: Extracted samples may be stored at -20 °C or below until LC-MS/MS analysis.

3. LC-MS/MS Analysis

  1. Load 100 µl of the supernatant of the extracted sample onto the C8 cartridge extraction column and wash with a 7:3 ratio of 0.1% formic acid in water: acetonitrile at a flow of 5 ml/min for 1 min. The connections of the switching valve are shown in Figure 2 and the gradient run by the extraction pump in Table 1.
  2. Hereafter, activate the switching valve resulting in back-flush of the analytes from the pre-column onto the analytical column.
  3. Set the column thermostat to 65 °C.
  4. Elute the analytes from the analytical column using the flow rates and gradient shown in Table 1.
  5. Connect the analytical column to a tandem mass spectrometer via the turbo electrospray ionization source. Adjust the key parameters of the mass spectrometer according to Table 2.
  6. Detect positive ions ([M+Na]+) in the multiple reaction mode (MRM). Use the following ion transitions for quantification: tacrolimus: m/z (mass/charge) = 826.6 → 616.2 and D2,13C-tacrolimus: m/z= 829.6 → 619.2.
    Note: The total run time is 4.6 min.

4. Quantification

  1. For each run, generate a calibration curve based on the calibrators prepared in 1.3.5 and include in each analytical run.
    1. Generate a calibration curve by plotting nominal concentrations versus response factor of analyte (Peak Area [Analyte] / Peak Area [internal standard]) using the mass spectrometer software.
    2. Fit the calibrators using a quadratic fit in combination with 1/X weighting.
  2. To quantity tacrolimus in the dried blood spots integrate the tacrolimus and internal standard peaks in the extracted MRM chromatograms. Calculate the response factor for tacrolimus (Peak Area [Analyte] / Peak Area [internal standard]) and compare with the calibration curve using the mass spectrometry software.

5. Validation Procedures

  1. Lower limit of detection (LLOD) and lower limit of quantification (LLOQ).
    1. Consider the lowest tacrolimus concentration with a peak-to-noise ratio of 4:1 as the lower limit of detection (LLOD). Define the lower limit of quantification (LLOQ) as the lowest concentration of the calibration curve with an accuracy equal to or better than ± 20% deviation from the nominal concentration and precision equal to or better than 20% (coefficient of variance).
  2. Intra- and inter-day accuracies and precisions.
    1. Test the accuracy and precision at four concentration levels of 2 ng/ml (QC1), 4 ng/ml (QC2), 20 ng/ml (QC3) and 40 ng/ml (QC4).
    2. Prepare the QC samples on each validation day in human EDTA whole blood, dry on filter cards, extract, and analyze as described above.
    3. Determine intra-day accuracy and precision with 6 samples per QC concentration level.
    4. Assess inter-day accuracy and precision over 20 days. Measure each QC level with 4 samples each day.
    5. Analyze two calibration curves together with the QC samples on each day.
    6. Calculate intra-day accuracy as the % of the nominal concentration (six samples per concentration level, please see 5.2.2). Calculate precision as the coefficient of variance (CV%).
    7. Consider intra-day accuracy acceptable if it falls into the acceptance limits 85% to 115% of the nominal concentration. Consider intra-day precision acceptable if it is equal to or better than a CV (coefficient of variance) of 15%.
    8. Calculate inter-day accuracy and precision as the mean for each QC concentration level analyzed over the 20 validation days.
    9. Consider mean inter-day accuracy acceptable if it falls into the acceptance limits 85% to 115% of the nominal concentration. Consider inter-day precision acceptable if it is equal to or better than a CV (coefficient of variance) of 15%.
  3. Exclusion of matrix interferences.
    1. For the exclusion of interferences that may be caused by matrix signals, analyze blank dried blood spots (8 different individuals, preferably 4 males and 4 females).
    2. Visually inspect ion chromatograms. If peaks within the retention time window of tacrolimus are detected, integrate and compare their areas under the curve with those of tacrolimus peaks in blank samples spiked with tacrolimus at the LLOQ. The area of peaks in the blank samples are not supposed to exceed 15% of those of tacrolimus at the LLOQ.
  4. Ion suppression/ion enhancement.
    1. Use a post-column infusion protocol as described45 to assess potential interference of ion suppression/ion enhancement caused by co-eluting matrix components.
    2. Infuse tacrolimus at a concentration of 10 µg/ml dissolved in 0.1% formic acid: methanol (30:70, v/v) post-column at a rate of 10 µl/min.
      1. Connect a syringe pump via T-piece between the analytical column and the electrospray source of the mass spectrometer.
      2. Monitor the MS/MS signal intensity of the MRM transitions for tacrolimus and its internal standard (m/z = 826.6 → 616.2 and m/z = 829.6 → 619.2) after injection of extracted blank samples (n = 8 samples from different individuals).
        Note: In the absence of ion suppression/ion enhancement the continuous signal caused by infusion of the analytes should not be affected by injection of the blank matrix, while ion suppression causes a dip of the signal and ion enhancement a peak.
  5. Carry-over.
    1. Assess potential carry-over by analyzing extracted blank samples after the highest calibrators (50 ng/ml, n = 6).
    2. Visually inspect ion chromatograms. If peaks within the retention time window of tacrolimus are detected, integrate and compare their areas under the curve with those of tacrolimus peaks in blank samples spiked with tacrolimus at the LLOQ. The area of peaks in the blank samples are not supposed to exceed 15% of those of tacrolimus at the LLOQ.
  6. Extraction recoveries.
    1. Determine recoveries by comparing the signals of the analytes after extraction of QC samples at all four concentration levels (n = 6 per concentration) with those of blank dried blood spots spiked with the corresponding amounts of tacrolimus after the extraction.
    2. Prepare four sets of QCs (concentration levels: 2, 4, 20, 40 ng/ml).
    3. Prepare another 4 sets of corresponding “recovery test samples” by spotting 50 µl of blank EDTA whole blood onto the filter paper cards and drying for 2 hr.
    4. Hereafter, for both the QC and blank “recovery test samples”, cut out the entire blood spot on the filter card with scissors and place the resulting discs into a polypropylene tube with conical bottom and snap-on lid.
    5. Extract all samples.
    6. Transfer the supernatants (400 µl) into glass HPLC vials.
    7. Add tacrolimus stock solution to the blank “extracted recovery test samples” to reach concentrations of 2, 4, 20 and 40 ng/ml (4 µl of 200, 400, 2,000, 4,000 ng/ml tacrolimus stock solutions to 400 µl of supernatant).
    8. After LC-MS/MS analysis, compare the signals in both QC samples and “recovery test samples” of the corresponding concentration (recovery% = signal samples spiked before extraction / signal samples spiked after extraction x 100).
  7. Dilution integrity.
    1. Establish dilution integrity using samples spiked with the analytes at 500, 250 and 100 ng/ml.
    2. After extraction, dilute samples using protein precipitation solution (1:10, n = 3 per concentration level).
    3. Calculate deviations from the nominal concentrations. Consider results that fall within 85%-115% of nominal acceptable.
  8. Stabilities.
    1. Investigate stabilities using the QC samples at all four concentration levels (n = 4 per concentration) analyzed at different time-points and under the different storage conditions.
    2. Compare results after storage with the nominal values. Consider results that fall within 85%-115% of nominal acceptable.
    3. Establish sample stability for 1 week at ambient temperature, 1 week at 4 °C, 1 month at -20 °C and 1 month at -80 °C.
    4. Test freeze-thaw stability over three cycles (-20 °C). Test extracted sample and autosampler stability by placing samples into the thermostatted autosampler adjusted to 4 °C. Inject samples after 72 hr.

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...

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

NameCompanyCatalog NumberComments
TacrolimusU.S. Pharmacopeial Convention1642802
D2,13C-TacrolimusToronto Research Chemicals Inc.F370002
Red blood cellsUniversity of Colorado HospitalW20091305500 V
PlasmaUniversity of Colorado HospitalW2017130556300Q
Acetone CHROMASOLV, HPLC, ≥99,9%Sigma-Aldrich439126-4 L
Acetonitrile Optima LC/MS, UHPLC-UVThermo Fisher ScientificA955-4
Isopropanol 99.9%, HPLCFisher ScientificBP2632-4
Methanol Optima LC/MSThermo Fisher ScientificA452-4
Water Optima LC/MS, UHPLC-UVThermo Fisher ScientificW6-4
Formic acidThermo Fisher ScientificA118P-500
Phosphate-buffered saline (PBS)Sigma-AldrichD8537
Zinc sulfateThermo Fisher ScientificZ68-500
0.5 – 10 µl pipet, VoluMate LIQUISYSTEMSMettler Toledo17008649
1.5 ml Eppendorf tubeThermo Fisher Scientific02-682-550
10 – 100 µl pipet, VoluMate LIQUISYSTEMSMettler Toledo17008651
10 μl pipet tips with filter, sterileNeptuneBT 10XLS3
100 – 1,000 µl pipet, VoluMate LIQUISYSTEMSMettler Toledo17008653
100 μl pipet tips with filter, sterileNeptuneBT 100
1,000 μl pipet tips with filter, sterileMultimax2940
2 – 20 µl pipet, VoluMate LIQUISYSTEMSMettler Toledo17008650
2 ml Eppendorf tubeThermo Fisher Scientific02-681-258
20 – 200 µl pipet, VoluMate LIQUISYSTEMSMettler Toledo17008652
20 μl pipet tips with filter, sterileGeneMateP-1237-20
200 μl pipet tips with filterMultimax2938T
200 μl pipet tips with filter, sterileMultimax2936J
50 ml Falcon tubeBD Falcon352070
300 μl inserts for HPLC vialsPhenomenexARO-9973-13
Balance PR2002Mettler Toledo1117050723
Balances AX205 Delta RangeMettler Toledo1119343379
Bullet Blender HomogenizerNext AdvanceBBX24
Centrifuge Biofuge FrescoHeraeus290395
Disposable WipesPDIQ55172
Glass v ials, 4 mlThermo Fisher Scientific14-955-334
Glass vials, 20 mlThermo Fisher ScientificB7800-20
Gloves, nitrileTitan Brand Gloves44-100S
HPLC vials, 9 mm, 2 ml, clearPhenomenexARO- 9921-13
Lids for HPLC vialsPhenomenexARO- 8952-13-B
Needle, 18 G 1.5Precision Glide305196
Rack for Eppendorf tubesThermo Fisher Scientific03-448-11
Rack for HPLC VialsThermo Fisher Scientific05-541-29
Steel beads 0.9 – 2 mmNext AdvanceSSB14B
Storage boxes for freezers / refrigeratorsThermo Fisher Scientific03-395-464
Standard multi-tube vortexerVWR Scientific Products658816-115
Whatman Paper, 903 Protein Saver US 100/PKGE Whatman 2016-05
AutosamplerCTC PAL PAL.HTCABIx1
Binary pump, Agilent 1260 InfinityAgilent Technologies1260 G1312B
Binary pump, Agilent 1290 InfinityAgilent Technologies1290 G4220A
Micro vacuum degasser, Agilent 1260Agilent Technologies1260 G13798
Column oven,  Agilent 1290 with 2 position Agilent Technologies1290 G1216C
Thermostated column compartment with integrated 6 port switching valveAgilent Technologies1290 G1316C
HPLC pre-column cartridge, Zorbax XDB C8 (5 µm particle size), 4.6 · 12.5 mmPhenomenex820950-926
HPLC analytical column, Zorbax Eclipse-XDB-C8 (5 µm particle size), 4.6 · 150 mmPhenomenex993967-906
Tandem Mass Spectrometer
API5000 MS/MS with TurboIonspray sourceAB Sciex4364257
Mass spectrometry softwareAB SciexAnalyst 1.5.1

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Keywords TacrolimusImmunosuppressantLC MS MSDried Blood SpotsTherapeutic Drug MonitoringSolid Organ TransplantationProtein PrecipitationOnline Column ExtractionHPLCMass SpectrometryAssay ValidationStability

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