Assay techniques for free anticonvulsants

Ultrafiltration using Centrifree Micropartition System is the most common technique for monitoring free drug concentrations in clinical laboratories. Usually, 0.8-1.0 ml of serum is centrifuged for 15-20 min to prepare the ultrafiltrates. Then, free drug concentrations are measured in the protein-free ultrafiltrates. The time of centrifuging to prepare ultrafiltrates is crucial for measuring free drug concentrations. Liu et al. demonstrated that there is a significant difference between measured free valproic acid concentration in ultrafiltrates prepared by centrifuging specimens for 5 versus 10 or 20 min. The measured free concentrations were low if the specimen was centrifuged for 5 min. Therefore, authors recommended centrifugation of specimens for at least 15 min (111). McMillin et al. recently reported that ultrafiltrate volumes were directly proportional to the centrifugation time (15-30 min) and were inversely proportional to albumin concentrations of serum. Although ultrafiltrate volume was significantly increased with increasing centrifugation time, free phenytoin values did not change significantly indicating that equilibrium was maintained between the ultrafiltrate and serum retained in the ultrafiltration device (112). Another issue is the add-on request for free drug on specimens analyzed previously for total drug concentrations. Usually, specimens after analysis are stored for 3-7 days at 4°C. In our experience, if the specimen is removed from a refrigerator and the ultrafiltration is started immediately, the concentrations of free drugs are slightly lower. However, this problem can be easily circumvented if specimens are allowed to equilibrate at the room temperature for at least 15 min before the preparation of ultrafiltrates for free drug measurement (113).

Although immunoassays are commercially available for determination of free phenytoin, valproic acid, and carbamazepine concentrations in protein-free ultra-filtrates, liquid chromatography combined with tandem mass spectrometry can also be applied for determination of free phenytoin concentration in protein-free ultrafiltrate (114).

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