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. 2022 Jun 2;27(11):3587.
doi: 10.3390/molecules27113587.

Development and Validation of an HPLC-UV Method for the Quantification of 4'-Hydroxydiclofenac Using Salicylic Acid: Future Applications for Measurement of In Vitro Drug-Drug Interaction in Rat Liver Microsomes

Hassan Salhab  1 , James Barker  1
Affiliations

Development and Validation of an HPLC-UV Method for the Quantification of 4'-Hydroxydiclofenac Using Salicylic Acid: Future Applications for Measurement of In Vitro Drug-Drug Interaction in Rat Liver Microsomes

Hassan Salhab et al. Molecules. .

Abstract

Salicylic acid is a key compound in nonsteroidal anti-inflammatory drugs that has been recently used for preventing the risk of hospitalization and death among COVID-19 patients and in preventing colorectal cancer (CRC) by suppressing two key proteins. Understanding drug−drug interaction pathways prevent the occurrence of adverse drug reactions in clinical trials. Drug−drug interactions can result in the variation of the pharmacodynamics and pharmacokinetic of the drug. Inhibition of the Cytochrome P450 enzyme activity leads to the withdrawal of the drug from the market. The aim of this paper was to develop and validate an HPLC-UV method for the quantification of 4′-hydroxydiclofenac as a CYP2C9 metabolite using salicylic acid as an inhibitor in rat liver microsomes. A CYP2C9 assay was developed and validated on the reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) using a low-pressure gradient elution programming at T = 30 °C, a wavelength of 282 nm, and a flow rate of 1 mL/min. 4′-hydroxydiclofenac demonstrated a good linearity (R2 > 0.99), good reproducibility, low detection, and quantitation limit, and the inter and intra-day precision met the ICH guidelines (<15%). 4′-hydroxydiclofenac was stable for three days and showed an acceptable accuracy and recovery (80−120%) within the ICH guidelines in a rat liver microsome sample. This method will be beneficial for future applications of the in vitro inhibitory effect of salicylic acid on the CYP2C9 enzyme activity in rat microsomes and the in vivo administration of salicylic acid in clinical trials.

Keywords: HPLC-UV; drug–drug interaction; method development; salicylic acid; validation.

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Conflict of interest statement

The authors declare no conflict of interest, financial or otherwise, for this work.

Figures

Figure 1
Figure 1
Chemical structure of (1) salicylic acid, (2) diclofenac, (3) 4′-hydroxydiclofenac, and (4) 4-hydroxyoctanophenone.
Figure 2
Figure 2
UV–VIS spectrum of salicylic acid, diclofenac, 4′-hydroxydiclofenac, and 4-hydroxyoctanophenone in the CYP2C9 assay.
Figure 3
Figure 3
Calibration curve of 4′-hydroxydiclofenac.
Figure 4
Figure 4
HPLC chromatogram of the negative control blank containing rat liver microsomes.
Figure 5
Figure 5
Typical HPLC chromatogram of the components at 282 nm using gradient elution programming in rat liver microsome. The peaks marked are (1) salicylic acid (100 µM) (RT = 4.006 min), (2) 4′-hydroxydiclofenac (100 µM) (RT = 6.681 min), (3) diclofenac (200 µM) (RT = 9.353 min), and (4) 4-hydroxyoctanophenone (50 µM) (RT = 10.079 min), respectively.
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