Simple, Fast and Reliable Liquid Chromatographic and Spectrophotometric Methods for the Determination of Theophylline in Urine, Saliva and Plasma Samples

Document Type: Research article


1 Yeditepe University Faculty of Pharmacy

2 Hacettepe University Faculty of Pharmacy


In this study, a high-performance liquid chromatographic method (HPLC) and UV spectrophotometric method were developed, validated and applied for the determination of theophylline in biological fluids.
Liquid- liquid extraction is performed for isolation of the drug and elimination of plasma and saliva interferences. Urine samples were applied without any extraction. The chromatographic separation was achieved on a C18 column by using 60:40 methanol:water as mobile phase under isocratic conditions at a flow rate of 0.75 mL/min with UV detection at 280 nm in HPLC method. UV spectrophotometric analysis was performed at 275 nm.
The results of HPLC analysis were as follows: the limit of quantification: 1.1 µg/mL for urine, 1.9 µg/mL for saliva, 3.1 µg/mL for plasma; recovery: 94.85% for plasma, 100.45% for saliva, 101.39% for urine; intra-day precision: 0.22–2.33%, inter-day precision: 3.17-13.12%. Spectrophotometric analysis results were as follows: the limit of quantitation: 5.23 µg/mL for plasma, 8.7 µg/mL for urine; recovery: 98.27% for plasma, 95.25% for urine; intra-day precision: 2.37 – 3.00%, inter-day precision: 5.43-7.91%.
It can be concluded that this validated HPLC method is easy, precise, accurate, sensitive and selective for determination of theophylline in biological samples. Also spectrophotometric analysis can be used where it can be applicable.


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