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

The overall goal of the following experiment is to quantify tacrolimus in dried blood spots. The main advantage of this technique over quantification of tacrolimus in blood samples after IV blood draw is that patients can collect samples themselves minimally invasive low volume sample collection strategies such as dried blood spots also facilitate therapeutic drug monitoring and pharmacokinetic studies in small children. After a simple finger stick, the patient collects a blood drop on a special filter paper card, and after the blood spot is dried, it can be mailed to a central laboratory for analysis of tacrolimus and any other immunosuppressant that the patient may be taking.

Quantification of drugs on dried blood spots has become possible due to the development of highly sensitive and specific LCM SM S assays for the quantification of tacrolimus and other immunosuppressants in very small blood volumes such as dried blood spots. First, prepare stock solutions in pure methanol at a concentration of one milligram per milliliter for tacrolimus and a concentration of 10 micrograms per milliliter. For D two 13 C aliquot stock solutions and store at negative 70 degrees Celsius or below on each extraction day, prepare a seven to three ratio of methanol and 0.2 molar zinc sulfate in water to precipitate proteins and extract tacrolimus.

The internal standard stock solution is spiked into the precipitation solution. Set the expiration of the solution at 12 hours. Following this, prepare stock solutions of tacrolimus by performing appropriate dilution of the stock solution using pure methanol to prepare calibrators and quality control samples.

Spike 20 microliters of the appropriately diluted stock solution into EDT. A whole blood incubate at 37 degrees Celsius under gentle shaking in a water bath for 20 minutes to allow for homogeneous distribution of tacrolimus into the cellular blood components. To prepare a larger amount of calibrators and quality control samples spike 2000 microliters of the appropriately diluted stock solution into EDT.

A whole blood incubate at 37 degrees Celsius under gentle shaking in a water bath for 20 minutes to allow for homogeneous distribution of tacrolimus into the cellular blood components. Then aliquot the samples into 1.5 milliliter polypropylene tubes with conical bottoms and snap-on lids. Spot 50 microliters of the spiked whole blood into the middle of each circle on filter cards using a pipette after drying the blood spots at room temperature for three hours, prepare tacrolimus calibration standards in human EDTA whole blood at tacrolimus concentrations of one 2.5 5, 10 25, and 50 nanograms per milliliter.

Prepare a blank sample for extraction like the calibration standards with the protein precipitation solution, not containing the internal standard D two 13 C tacrolimus. Next, prepare quality control samples in human EDTA whole blood at concentrations of 0 2 4 20 and 40 nanograms per milliliter. Prepare a blank sample as well After collecting the dried blood spots, visually inspect each one to ensure acceptable sample quality and volume.

Then punch the center of the blood spot on the filter card with a six millimeter hole punch. Place the discs into 1.5 milliliter polypropylene tubes with conical bottoms and snap on lids. Add 20 to 30 bullets to each tube.

Following this, add 500 microliters of the protein precipitation solution into each tube. Homogenize the discs in a bullet blender for one minute at maximum speed. When finished, shake the samples at room temperature on a multi tube vortex at maximum speed for 10 minutes.

Then centrifuge the samples at 16, 000 GS and four degrees Celsius for 10 minutes. After centrifugation, transfer the SNAT into glass HPLC vials equipped with a 300 microliter insert. At this point, load 100 microliters of the supernat of the extracted sample onto the C eight cartridge extraction column wash with a seven to three ratio of 0.1%formic acid in water and acid nitrile at a flow of five milliliters per minute.

For one minute hereafter activate the switching valve resulting in back flush of the analytes from the pre-cal onto the analytical column. Then set the column thermostat to 65 degrees Celsius. Elute the analytes from the analytical column using the flow rates and gradient shown here.

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 the following table. Next, detect positive ions in the multiple reaction mode.

To quantify tacrolimus in the dried blood spots, compare it with the calibration curve using the mass spectrometry software representative ion chromatograms of a blank sample. A sample spiked at the lower limit of quantification and a patient sample are shown here. A typical calibration curve is shown here.

The mean coefficient of correlation is 0.9994. The accuracy and precision results are shown in detail here. No indications of significant ion suppression or ion enhancement were observed and no relevant carryover resulting in peaks exceeding.

15%of the signal at the lower limit of quantitation were detected. Dilution integrity was investigated by analyzing samples prepared at concentrations above the highest calibrator and diluted in protein precipitation solution after extraction to reach the target concentrations. All dilutions tested met acceptance criteria.

No losses were apparent after one week of storage at room temperature after one week of storage at four degrees Celsius and after one month of storage at negative 20 degrees Celsius. In addition, no losses were observed after one month of storage at negative 80 degrees Celsius after three freeze and thaw cycles, and after 72 hours of extracted samples in the auto sampler at four degree Celsius. Once mastered 20 samples can be extracted in less than one hour.

While attempting this procedure, it is important to remember that the key to reliable quantification is the quality of the dried blood spot and that the disc is punched from a completely saturated area of the filter paper. The techniques and the assay shown in this video provide a platform technology to quantify other immunosuppressants and drugs in dried blood spots.