Determination of Phenytoin and Its Major Metabolites in Human Serum by High-Performance Liquid Chromatography with Fluorescence Detection

Abstract

Phenytoin (PHT), 5,5-diphenyl-2,4-imidazolidinedione, has been widely used in the management of patients with epilepsy, generalized convulsion and partial seizure. 1,2 PHT has been well established to have non-linear "dose dependent" pharmacokinetic properties and a narrow therapeutic range of 10 -20 µg/ml in serum. 3,4 Moreover, because PHT is ~90% bound to plasma protein, mainly to albumin 5 , the unbound concentration has been shown to be a better predictor of the therapeutic response than the total concentration. An accurate determination of the unbound-PHT concentration in serum is thus essential for therapeutic drug monitoring.Several methods used up to now to determine the PHT level have been based on either chromatography or immunochemical techniques. Although both an enzyme-multiplied immunoassay or a fluorescence polarization immunoassay have been widely utilized by clinical laboratories because of their speed and simplicity, these methods are interfered with by the PHT metabolites, and consequently tend to overestimate the PHT levels. 6-8 Some high-performance liquid chromatographic (HPLC) methods with ultraviolet (UV) detection have also been developed to determine the PHT level in human serum. 9-11 However, these methods have a limited sensitivity and require a large amount of serum ( > = 2 ml) to determine the presence of unbound-PHT.We previously developed 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H )-quinoxalinone (Br-DMEQ) as a highly sensitive fluorescence derivatization reagent for carboxylic acids in HPLC. 12 The HPLC method with Br-DMEQ has been successfully used to determine biologically important fatty acids 13 , prostaglandins 14 and antitumor agents (5-fluorouracil and 5-fluoro-2′-deoxyuridine). 15 We found that Br-DMEQ also reacts with PHT and its metabolites, 5-(3-hydroxyphenyl)-5-phenylhydantoin (3HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (4HPPH) (Chart 1), having imino hydrogen to produce the corresponding fluorescent derivatives, which are separable by reversed-phase HPLC. We therefore developed a method for the simultaneous determination of PHT and the metabolites in human serum. Drug-free serum spiked with PHT, 3HPPH and 4HPPH was employed as model samples to establish the most suitable conditions for a general ana- A highly sensitive fluorometric high-performance liquid chromatographic method was developed for the simultaneous determination of phenytoin and its major metabolites [5-(3-hydroxyphenyl)-5-phenylhydantoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin]. After extracting these compounds and 5-(4-methylphenyl)-5-phenylhydantoin (MPPH) as an internal standard from serum (50 µl) with ethyl acetate, they were further converted into the corresponding fluorescent derivatives by a reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H )-quinoxalinone in the presence of potassium hydrogen carbonate and dibenzo-18-crown-6 in acetonitrile. The derivatives were separated by reversed-phase chromatography on a YMC-Pack ODS-A column with a mixture of acetonitrile-50 mM phosphate buf...

High-Throughput Screening Assays for the Assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 Metabolism Using Fluorogenic Vivid® Substrates

“…30 Substrate-dependent effects were also observed with phenytoin, exhibiting similar apparent K i values for all 3 polymorphic variants with CYP2C9 green substrate, but more than 10-fold lower affinity for CYP2C9*3 was detected with the red substrate compared to CYP2C9*1. It is important to note that phenytoin has nonlinear and dose-dependent pharmacokinetic properties, has a narrow therapeutic range, 31 and is involved in well-documented cases of clinical toxicity as a result of drug-drug interactions. 32 Therefore, it is possible that the lower affinity of phenytoin observed in the Vivid ® red assay with CYP2C9*3 ( Table 3) could be an indicator of potentially important clinical drug interactions, especially for CYP2C9*3 polymorphic variant carriers, but this remains to be studied.…”

Studies by biointeraction chromatography of binding by phenytoin metabolites to human serum albumin

“…If A is known to bind to a given region on the ligand, this allows the competition of I at that site to also be examined and quantitated. By using different probe compounds as the injected analyte, each with a different binding region on the ligand, the interactions of I at other sites on the same ligand can be examined in the same manner [4,11,12,16,18,19].…”

“…It has non-linear dose-dependent pharmacokinetics and is mostly excreted in bile as inactive metabolites, which are then reabsorbed from the intestinal tract and excreted in urine [3,4]. The primary metabolites of phenytoin are 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) (see Figure 1) [4].…”

“…Like other drug metabolites, m-HPPH and p-HPPH have similar structures to their parent drug, which can cause these to affect the distribution, action, and protein binding by phenytoin [4,5]. Because phenytoin exhibits dose-dependent pharmacokinetics, the apparent half-life of phenytoin changes with its dose and serum concentration.…”

Analysis of Free Drug Fractions Using Near-Infrared Fluorescent Labels and an Ultrafast Immunoextraction/Displacement Assay

“…Phenytoin is an anti-epileptic drug that has significant binding in blood [6,7]. This drug is mainly bound in blood to the protein human serum albumin (HSA), with approximately 90% of phenytoin being complexed at therapeutic levels in adults [8][9][10][11][12][13][14].…”