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. 2014 Spring;13(2):431-9.

Simple, fast and reliable liquid chromatographic and spectrophotometric methods for the determination of theophylline in urine, saliva and plasma samples

Mohammad Charehsaz  1 , Aylin Gürbay  2 , Ahmet Aydin  1 , Gönül Sahin  2
Affiliations

Simple, fast and reliable liquid chromatographic and spectrophotometric methods for the determination of theophylline in urine, saliva and plasma samples

Mohammad Charehsaz et al. Iran J Pharm Res. 2014 Spring.

Abstract

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.

Keywords: HPLC; Plasma; Saliva; Theophylline; UV spectrophotometry; Urine.

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Figures

Figure 1
Figure 1
Chemical structure of theophylline
Figure 2
Figure 2
Chromatograms of theophylline (20 µg/mL): (A) in methanol solution, (B) in plasma sample, (C) in saliva sample and (D) in urine sample
Figure 3
Figure 3
Chromatogram obtained with blank urine which was spiked with theobromine (30 µg/mL), theophylline (50 µg/mL) and caffeine (50 µg/mL).
Figure 4
Figure 4
Chromatogram obtained with blank saliva which was spiked with theobromine (30 µg/mL), theophylline (50 µg/mL) and caffeine (50 µg/mL).
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