The Development and Validation of a High-Performance Liquid Chromatographic Method for the Determination of Urinary Levels of Etoricoxib After Fabric Phase Sorptive Extraction.

Etoricoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is widely used for the management of pain and inflammation associated with rheumatoid arthritis, osteoarthritis, and other musculoskeletal disorders. Monitoring its urinary concentration provides valuable information for pharmacokinetic and bioavailability studies. 

In this work, a rapid, sensitive, and environmentally friendly high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of etoricoxib in human urine following extraction by fabric phase sorptive extraction (FPSE). The FPSE procedure employed a sol–gel–C18 coated fabric as the extraction medium, enabling efficient analyte enrichment while minimizing sample handling and solvent consumption. 

The chromatographic separation was achieved using a reversed-phase C18 column under isocratic conditions with an optimized mobile phase consisting of methanol and water, and detection was performed at 284 nm using a UV detector. Method validation was conducted according to ICH guidelines, evaluating linearity, accuracy, precision, sensitivity, recovery, and robustness. The method demonstrated excellent linearity across the tested concentration range, with correlation coefficients (R² > 0.999), limits of detection (LOD) and quantification (LOQ) at nanogram levels, and satisfactory intra- and inter-day precision (RSD < 5%). Recovery rates exceeded 95%, confirming the efficiency of FPSE in preconcentrating etoricoxib from urine matrices. 

The validated method was successfully applied to real human urine samples, proving its suitability for routine bioanalytical applications and therapeutic drug monitoring of etoricoxib. 

Materials and Methods

Reagents and Materials

All reagents used were of analytical or HPLC grade.

• Potassium dihydrogen phosphate (KH₂PO₄), phosphoric acid (H₃PO₄, 85% w/w), and HPLC-grade water were obtained from Sigma-Aldrich (St. Louis, MO, USA;
   
• Methanol (MeOH), acetone (ACE), and acetonitrile (ACN) (LC-MS grade) were supplied by Honeywell International Inc. (Morristown, NJ, USA;
   
• Etoricoxib (5-chloro-3-[4-(methylsulfonyl)phenyl]-2-(2-methyl-5-pyridinyl)pyridine) (≥95% purity) was purchased from Merck KGaA (Darmstadt, Germany;
   
• The pharmaceutical formulation used for volunteer administration (90 mg/tablet) was obtained from IASIS Pharma (Kamatero, Greece;
   
• 100% cellulose fabric substrates for FPSE membrane synthesis were sourced from JoAnn Fabric and Craft Stores (Miami, FL, USA;
   
• Carbowax 20M (CW 20M) was obtained from Sigma-Aldrich (St. Louis, MO, USA).
• Methyl trimethoxysilane was purchased from Gelest Inc. (Morrisville, PA, USA;
   
• Additional solvents and reagents used in FPSE synthesis — including acetone, dichloromethane, NaOH, HCl, MeOH, ammonium hydroxide, and trifluoroacetic acid — were obtained from Fisher Scientific (Milwaukee, WI, USA;
   

Stock solutions of etoricoxib (1000 µg/mL) were prepared in acetonitrile and stored at +4 °C. Working standards were freshly prepared daily by dilution with HPLC-grade water.

HPLC Instrumentation and Conditions

Analysis was performed using a Shimadzu HPLC-DAD system (Model 2010, Kyoto, Japan), equipped with dual pumps, an autosampler, a column compartment, a diode array detector (DAD), and a communication bus module
 

Chromatographic separation was achieved on a Kinetex C18 100 Å column (30 × 4.6 mm, 2.6 µm) supplied by Phenomenex (Torrance, CA, USA;
 

The column temperature was maintained at 35 °C. The mobile phase consisted of (A) 0.05 M potassium dihydrogen phosphate buffer (pH 4.2, adjusted with phosphoric acid) and (B) acetonitrile, under the following gradient:

•  70:30 (A:B, 0–1 min), 40:60 (1.5 min), 50:50 (2 min), and 30:70 (2.5 min).
•  A re-equilibration step was maintained for 1.5 min.
•  The flow rate was 1.0 mL min⁻¹, and the injection volume was 5 µL.
•  Detection was carried out at λ = 284 nm.

FPSE Procedure

The FPSE process consisted of four main steps:

1. Activation: The FPSE membrane was preconditioned in a 1 mL methanol:acetonitrile (50:50 v/v) mixture for 5 min and rinsed with ultrapure water.
2. Extraction: The membrane was immersed in 2 mL urine sample in a glass vial with a magnetic stir bar. Samples were stirred at 100 rpm for 35 min at room temperature.
3. Desorption: After extraction, the membrane was rinsed with ultrapure water and transferred to an Eppendorf tube containing 500 µL methanol. The analyte was desorbed for 5 min, and the eluate was transferred to an HPLC vial.
4. Cleaning: The membrane was regenerated by soaking in 1 mL of the same activation solvent mixture for 5 min, then dried and stored for reuse.

Method Validation

Validation of the FPSE–HPLC–DAD method followed FDA bioanalytical guidelines. Parameters assessed included selectivity, linearity, accuracy, precision, recovery, matrix effects, LOD, LOQ, and carryover.

• Calibration curves were prepared by plotting etoricoxib peak area versus concentration (0.10–10.0 µg mL⁻¹).
• Accuracy and precision were determined using spiked quality control levels at 0.10, 0.50, 1.0, and 10.0 µg mL⁻¹ (LLOQ, LQC, MQC, HQC).
• Intra-day and inter-day studies were conducted in triplicate.
• LOD and LOQ were established at signal-to-noise ratios of 3 and 10, respectively.
• Carryover was evaluated by analyzing blank samples following the highest calibration standard.

All data were processed using LabSolutions Software (Shimadzu).