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. 2017:2017:7846742.
doi: 10.1155/2017/7846742. Epub 2017 Aug 7.

Chromatographic Characterization and Method Development for Determination of Levetiracetam in Saliva: Application to Correlation with Plasma Levels

Imad I Hamdan  1 , Mervat Alsous  2 , Amira Taher Masri  3
Affiliations

Chromatographic Characterization and Method Development for Determination of Levetiracetam in Saliva: Application to Correlation with Plasma Levels

Imad I Hamdan et al. J Anal Methods Chem. 2017.

Abstract

Levetiracetam (LVT) is a widely used antiepileptic drug (AED). A less invasive sampling method for therapeutic drug monitoring (TDM) would be very useful particularly for children. Saliva has been shown as an adequate sample for TDM of some AEDs. Due to the high hydrophilicity of LVT its separation on common stationary phases is quite a challenge so that previous methods for determination of LVT in saliva employed either gradient high performance liquid chromatographic (HPLC) system or mass spectrometer as a detector. In this study the retention behavior of LVT on some common stationary phases was examined, with C8 being the most retentive. A simple isocratic HPLC method that is based on simple protein precipitation was developed and validated for the determination of LVT in saliva. The method was applied to a sample group of epileptic children for the purpose of assessing potential correlation with plasma LVT levels and to investigate patient's compliance. The results confirmed a reasonable correlation between plasma and salivary levels of LVT (R = 0.9) which supports the use of saliva for TDM of LVT. The study also revealed a significant percentage of epileptic patients having LVT levels below the estimated therapeutic range.

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Figures

Figure 1
Figure 1
Structure of levetiracetam (LVT).
Figure 2
Figure 2
Plot of capacity factor “K” obtained for LVT using different columns against percentage of ACN in the mobile phase.
Figure 3
Figure 3
Chromatograms for saliva samples spiked with DEBA (internal standard, IS) and decreasing levels of LVT according to the direction of the dashed arrow (36, 28, 4, and zero µg/ml). Note the overlapping with the peak of IS and at about 5.8 min. In the sample spiked with 28 µg/ml of LVT no DEBA was added. Column: Synergi Polar RP (150 mm × 4.6 i.d). Mobile phase was 3.5% ACN in phosphate buffer at pH 6 with a flow rate of 1.5 ml/min.
Figure 4
Figure 4
Selected chromatograms for samples of saliva spiked with IS and LVT as follows (according to the direction of the arrow): (1) blank saliva spiked with LVT at LOQ (0.24 µg/ml); (2) blank saliva showing no interference with LVT; (3) and (4) are actual saliva of patients showing different levels of LVT, that is, 11.4 and 24.6 µg/ml. The column was C8 using the finally recommended conditions.
Figure 5
Figure 5
Levels of LVT in saliva determined by the proposed HPLC method and in plasma determined by a standard SPE-HPLC-DAD.
Figure 6
Figure 6
Correlation between LVT levels in saliva determined by the proposed PP-HPLC-UV method and that in plasma determined by SPE-HPLC-DAD.
Figure 7
Figure 7
Plot of the obtained plasma levels for LVT against administered dose (the line is a manual fitting of the data).
Figure 8
Figure 8
A plot of the obtained ratio of LVT in saliva to plasma against the administered dose.
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