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Therapeutic drug monitoring of mycophenolic acid and para-cresol in human plasma and dried blood spots: analytical assay development and validation
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- Author / Creator
- Singh, Yashita
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Kidneys are amongst the most transplanted organs in pediatric, adult, and geriatric populations. Post transplantation, the recipients are prescribed a regimen of immunosuppressants to avoid or decrease the chances of graft rejection. Mycophenolic acid (MPA) is one of the widely prescribed immunosuppressants that works as a selective and reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH), thereby causing a reduction in the proliferation of T and B-lymphocytes. p-Cresol is a protein-bound uremic toxin (PBUTs) that is accumulated to high concentrations in patients with renal dysfunction and is primarily metabolized to p-cresol sulfate (pCS) and p-cresol glucuronide (pCG). Both p-cresol and its metabolites are associated with multi-organ toxicities. In this thesis, we have initially conducted a literature review that summarizes various biological matrices (i.e., dried blood spots, saliva, and urine) that have been employed for the concentration monitoring of MPA and it was determined that dried blood spots may be a suitable alternative matrix for MPA therapeutic drug monitoring. The overall hypothesis of this thesis is that it is feasible to conduct MPA and pC TDM using highly sensitive and high throughput UPLC analytical assays in plasma and DBS. The objective of this thesis was to i) develop high-throughput ultra-high performance liquid chromatography assays for measuring MPA and pC in the plasma; ii) to validate these assays in human plasma in accordance with the US Food and Drug Administration (U.S.F.D.A) guidelines, and iii) to conduct a proof-of-concept feasibility study to assess the applicability of our assays using the dried blood spot (DBS) matrix.
The assays for quantifying MPA and p-cresol were developed on a Shimadzu® ultra-high performance liquid chromatography with ultraviolet detector (UPLC, LC-2040C Plus) coupled with a fluorescence detector (RF-20Axs), respectively. Chromatographic separation was obtained by using a reversed-phase Agilent® Eclipse XDB-C18 column (5µm, 4.6×250mm). The detection methods (i.e., absorbance or fluorescence) and chromatographic conditions such as organic solvents (i.e., methanol or acetonitrile), additives (i.e., ammonium acetate or formic acid), mobile phase flow rate, and injection volume were systematically optimized for each
compound. MPA carboxybutoxy ether and 2’,6’-dimethylphenol were utilized as the internal standards for MPA and pC assays, respectively. The MPA assay (final condition consisting of 40:60 acetonitrile: water, 0.1% v/v formic acid, 2 mM ammonium acetate; 0.5 mL/min flow rate; 10 µL injection volume; UV wavelengths 305 & 295 nm; run time of 24 minutes) had a calibration range in plasma between 0.3003 to 10 µg/mL (r2 =0.99). The pC assay (final conditions consisted of 80:20 acetonitrile: water; 0.5 mL/min flow rate; 20 µL injection volume; excitation & emission wavelengths of 268 & 300 nm; run time of 10 minutes) had a calibration range in plasma from 0.723 to 31.25 µg/mL (r2 =0.99). Both analytical assays could capture these analytes at their respective physiological ranges in plasma humans. The accuracy & precision values for these assays were within 15% of the nominal values, and both assays were stable at the following storage conditions: long-term (6-week for MPA and 1-week for pC at -80°C), benchtop (3h at room temperature), freeze-thaw (3 cycles), and autosampler (24h at 4°C). Furthermore, our validated protocols were successfully
translated to the DBS matrix, and the calibration ranges for the MPA (3.794 to 28.57 µg/mL) and pC (0.723 to 31.25 µg/mL) assays were also linear within known physiological concentrations, illustrating a successful proof-of-concept to measure these analytes using this alternative approach for both analytes. However, further optimizations are required for the DBS matrix, including improving the assay sensitivity, precision, accuracy, and recovery for both MPA and pC, prior to further laboratory and clinical validation of our DBS protocols.
In conclusion, we have successfully developed and validated physiologically relevant analytical assays quantifying MPA and p-cresol with acceptable accuracy and precision in the human plasma. These assays have been successfully applied, as a proof-of-concept, to a novel micro sampling matrix, DBS, proving their potential applicability in the clinic. To our knowledge, this is the first report indicating the suitability of measuring p-cresol using the DBS approach. These minimally invasive assays, once further validated, can have significant impacts to transplant patient care. -
- Graduation date
- Fall 2022
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- Type of Item
- Thesis
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- Degree
- Master of Science
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- License
- This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.