DEVELOPMENT AND VALIDATION OF RP-HPLC-DAD METHOD FOR DETERMINATION OF NINE DRUGS AND THEIR ELEVEN METABOLITES IN PLASMA AND URINE: PLASMA SAMPLES MEASUREMENTS

Abstract

A new RP-HPLC-DAD method for determination of nine drugs and eleven metabolites in body fluids was developed. The separation of drugs and metabolites (PAR, SOT, MET, ASP, PRO, NIF, CAR, DEX, KET, PAR-S, PAR-G, MET-H, D-MET, SAL, GENT, PRO-S, D-NIF, ODMC, O-DEX, and KET-G) was achieved using Develosil RP-AQUEOUS-AR5 C30 column in 30 min. The obtained MQL values (0.02 µg/mL−0.23 µg/mL) are suitable for urine/plasma measurements. Correlation coefficient (r²) was higher than 0.98 for all analytes. The recoveries ranged from 72.1% to 115.2% (RSD <6.4%). Chromatographic data were obtained with accuracy in the range from 0.3 to 5.6%.

Keywords:

• drugs
• HPLC-DAD
• metabolites
• plasma
• solid phase extraction
• urine

Abbreviations:

• PAR, paracetamol
• PAR-S, paracetamol sulfate
• PAR-G, paracetamol glucuronide
• SOT, sotalol
• MET, metoprolol
• MET-H, α-hydroxymetoprolol
• D-MET, o-desmethylmetoprolol
• ASP, aspirin
• SAL, salicylic acid
• GENT, gentisic acid
• PRO, propranolol
• PRO-S, propranolol sulphate
• NIF, nifedypine
• D-NIF, dehydronifedypine
• CAR, carvedilol
• ODMC, o-desmethylcarvedilol
• DEX, dexamethasone
• ODEX, 17-oxo-dexamethasone
• KET, ketoprofen
• KET-G, ketoprofen glucuronide
• MeOH, methanol
• can, acetonitrile
• TFA, trifluoroacetic acid
• FA, formic acid
• UHPLC, Ultra High Pressure Chromatography
• HPLC, High Performance Chromatography

Notes

^a Only the drugs and metabolites of interest are mentioned.

^a Standard deviation of a slope.

^b Standard deviation of intercept.

The spiking amount for recovery tests was 1 µg/mL for each drug.

*sample acidified to pH 2 with 0.1 M HCl.