Development and Validation of High-Throughput Bioanalytical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Newly Synthesized Antitumor Carbonic Anhydrase Inhibitors in Human Plasma

Ahmed M Abdel-Megied; Wagdy M Eldehna; Mohamed A Abdelrahman; Fawzy A Elbarbry

doi:10.3390/molecules25235753

Development and Validation of High-Throughput Bioanalytical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Newly Synthesized Antitumor Carbonic Anhydrase Inhibitors in Human Plasma

Molecules. 2020 Dec 6;25(23):5753. doi: 10.3390/molecules25235753.

Authors

Ahmed M Abdel-Megied^{1

2}, Wagdy M Eldehna³, Mohamed A Abdelrahman⁴, Fawzy A Elbarbry²

Affiliations

¹ Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy Kafrelsheikh University, Kafrelsheikh City 33516, Egypt.
² School of Pharmacy, Pacific University Oregon, Hillsboro, OR 97123, USA.
³ Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh City 33516, Egypt.
⁴ Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo 11829, Egypt.

Abstract

In the present study, a sensitive and fully validated bioanalytical high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantitative determination of three newly synthesized carbonic anhydrases inhibitors (CAIs) with potential antitumor activity in human plasma. The analytes and the internal standard (IS) were extracted using 1.5 mL acetonitrile from only 450 µL aliquots of human plasma to achieve the desired protein precipitation. Chromatographic separations were achieved on Phenomenex Kinetex^® C₁₈ column (100 × 4.6 mm, 2.6 µm) using a binary gradient elution mode with a run time of less than 6 min. The mobile phase consisted of solvent (A): 0.1% formic acid in 50% methanol and solvent B: 0.1% formic acid in acetonitrile (30:70, v/v), pumped at a flow rate of 0.8 mL/min. Detection was employed using triple quadrupole tandem mass spectrometer (API 3500) equipped with an electrospray ionization (ESI) source in the positive ion mode. Multiple reaction monitoring (MRM) mode was selected for quantitation through monitoring the precursor-to-parent ion transition at m/z 291.9 → 173.0, m/z 396.9 → 225.1, m/z 388.9 → 217.0, and m/z 146.9 → 91.0 for AW-9a, WES-1, WES-2, and Coumarin (IS), respectively. Linearity was computed using the weighted least-squares linear regression method (1/x²) over a concentration range of 1-1000, 2.5-800, and 5-500 ng/mL for AW-9a, WES-1, and WES-2; respectively. The bioanalytical LC-MS/MS method was fully validated as per U.S. Food and Drug Administration (FDA) guidelines with all respect to linearity, accuracy, precision, carry-over, selectivity, dilution integrity, and stability. The proposed LC-MS/MS method was applied successfully for the determination of all investigated drugs in spiked human plasma with no significant matrix effect, which is a crucial cornerstone in further therapeutic drug monitoring of newly developed therapeutic agents.

Keywords: LC-MS/MS; antitumor; bioanalytical validation; carbonic anhydrase inhibitors; human plasma; tandem mass.

MeSH terms

Antineoplastic Agents / chemistry
Antineoplastic Agents / pharmacokinetics*
Carbonic Anhydrase Inhibitors / chemistry
Carbonic Anhydrase Inhibitors / pharmacokinetics*
Chromatography, Liquid* / methods
Drug Monitoring
Drug Stability
High-Throughput Screening Assays* / methods
Humans
Molecular Structure
Neoplasms / drug therapy
Reproducibility of Results
Tandem Mass Spectrometry* / methods

Substances

Antineoplastic Agents
Carbonic Anhydrase Inhibitors