Abstract
Phenytoin (diphenylhydantoin) is an anticonvulsant drug that has been used for decades for the treatment of many types of seizures. The drug is highly protein bound and measurement of free-active form of the drug is warranted particularly in patients with conditions that can affect drug protein binding. Here, we describe a LC/MS/MS method for the measurement of free phenytoin. Free drug is separated by ultrafiltration of serum or plasma. Ultrafiltrate is treated with acetonitrile containing internal standard phenytoin d-10 to precipitate proteins. The mixture is centrifuged and supernatant is injected onto LC-MS-MS, and analyzed using multiple reaction monitoring. This method is linear from 0.1 to 4.0 μg/mL and does not demonstrate any significant ion suppression or enhancement.
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References
Broussard LA (2007) Monitoring anticonvulsants concentrations: general considerations. In: Hammett-Stabler CA, Dasgupta A (eds) Therapeutic drug monitoring data. AACC Press, Washington, D.C., pp 41–75
Moyer TP, Shaw LM (2006) Therapeutic drugs and their management. In: Burtis CA, Ashwood ER, Bruns DE (eds) Tietz textbook of clinical chemistry and molecular diagnostics, 4th edn. Elsevier Saunders, St. Louis, MO, pp 1237–1285
Biddle DA, Wells A, Dasgupta A (2000) Unexpected suppression of free phenytoin concentration by salicylate in uremic sera due to the presence of inhibitors: MALDI mass spectrometric determination of molecular weight range of inhibitors. Life Sci 66:143–151
Wolf GK, McClain CD, Zurakowski D, Dodson B, McManus ML (2006) Total phenytoin concentrations do not accurately predict free phenytoin concentrations in critically ill children. Pediatr Crit Care Med 7:434–439, quiz 440
Bardin S, Ottinger JC, Breau AP, O’Shea TJ (2000) Determination of free levels of phenytoin in human plasma by liquid chromatography/tandem mass spectrometry. J Pharm Biomed Anal 23:573–579
Godolphin W, Trepanier J (1984) Gas chromatography versus immunoassay (EMIT) for analysis of free phenytoin in serum ultrafiltrate. Ther Drug Monit 6:374–375
Joern WA (1981) Gas-chromatographic assay of free phenytoin in ultrafiltrates of plasma: test of a new filtration apparatus and specimen stability. Clin Chem 27:417–421
Lin PC, Hsieh YH, Liao FF, Chen SH (2010) Determination of free and total levels of phenytoin in human plasma from patients with epilepsy by MEKC: an adequate alternative to HPLC. Electrophoresis 31:1572–1582
May T, Rambeck B (1990) Fluctuations of unbound and total phenytoin concentrations during the day in epileptic patients on valproic acid comedication. Ther Drug Monit 12:124–128
Van Langenhove A, Costello CE, Biller JE, Biemann K, Browne TR (1980) A mass spectrometric method for the determination of stable isotope labeled phenytoin suitable for pulse dosing studies. Biomed Mass Spectrom 7:576–581
Van Langenhove A, Costello CE, Biller JE, Biemann K, Browne TR (1981) A gas chromatographic/mass spectrometric method for the simultaneous quantitation of 5,5-diphenylhydantoin (phenytoin), its para-hydroxylated metabolite and their stable isotope labelled analogs. Clin Chim Acta 115:263–275
Garg U, Peat J, Frazee C 3rd, Nguyen T, Ferguson AM (2013) A simple isotope dilution electrospray ionization tandem mass spectrometry method for the determination of free phenytoin. Ther Drug Monit 35:831–835
Datta P (1997) Oxaprozin and 5-(p-hydroxyphenyl)-5-phenylhydantoin interference in phenytoin immunoassays. Clin Chem 43:1468–1469
Datta P, Scurlock D, Dasgupta A (2005) Analytic performance evaluation of a new turbidimetric immunoassay for phenytoin on the ADVIA 1650 analyzer: effect of phenytoin metabolite and analogue. Ther Drug Monit 27:305–308
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Peat, J., Frazee, C., Garg, U. (2016). Quantification of Free Phenytoin by Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS). In: Garg, U. (eds) Clinical Applications of Mass Spectrometry in Drug Analysis. Methods in Molecular Biology, vol 1383. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3252-8_25
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DOI: https://doi.org/10.1007/978-1-4939-3252-8_25
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3251-1
Online ISBN: 978-1-4939-3252-8
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