Background: Phenytoin has very challenging pharmacokinetic properties. To prevent its
toxicity and ensure efficacy, continuous therapeutic monitoring is required. It is hard to get a simple,
accurate, rapid, easily available, economical and highly sensitive assay in one method for therapeutic
monitoring of phenytoin.
Objective: The present study is directed towards establishing and validating a simpler, rapid, an accurate,
highly sensitive, novel and environment friendly liquid chromatography/mass spectrometry
(LC/MS) method for offering rapid and reliable TDM results of phenytoin in epileptic patients to physicians
and clinicians for making immediate and rational decision.
Methods: 27 epileptics patients with uncontrolled seizures or suspected of non-compliance or toxicity
of phenytoin were selected and advised for TDM of phenytoin by neurologists of Qilu Hospital Jinan,
China. The LC/MS assay was used for performing of therapeutic monitoring of phenytoin. The Agilent
1100 LC/MS system was used for TDM. The mixture of Ammonium acetate 5mM: Methanol at (35:
65 v/v) was used for the composition of mobile phase. The Diamonsil C18 (150mm×4.6mm, 5μm) column
was used for the extraction of analytes in plasma. The samples were prepared with one step simple
protein precipitation method. The technique was validated with the guidelines of International Conference
on Harmonisation (ICH).
Results: The calibration curve demonstrated decent linearity within (0.2-20 µg/mL) concentration
range with linearity equation, y= 0.0667855 x +0.00241785 and correlation coefficient (R2) of
0.99928. The specificity, recovery, linearity, accuracy, precision and stability results were within the
accepted limits. The concentration of 0.2 µg/mL was observed as lower limit of quantitation (LLOQ),
which is 12.5 times lower than the currently available enzyme-multiplied immunoassay technique
(EMIT) for measurement of phenytoin in epilepsy patients.
Conclusion: A rapid, simple, economical, precise, highly sensitive and novel LC/MS assay has been
established, validated and applied successfully in TDM of 27 epileptics patients. It was alarmingly
found that TDM results of all these patients were out of safe range except two patients. However, it
needs further evaluation. Besides TDM, the stated method can also be applied in bioequivalence,
pharmacokinetics, toxicokinetics and pharmacovigilance studies.