Electromembrane extraction as a new approach for determination of free concentration of phenytoin in plasma using capillary electrophoresis.

PURPOSE: Electromembrane extraction is a new membrane-based extraction method in which charged compounds are extracted by an electric field. So far, this method has been used to extract and isolate a variety of acidic and basic drugs from various samples, including blood and plasma. However, in this procedure, it is not yet clear whether only unbound fraction of a drug is extracted or the total drug. The aim of this study is to reveal the nature of drug extraction in the presence of plasma proteins. METHODS: To determine the nature of the extraction, the electromembrane extraction was performed from plasma solutions of phenytoin with concentrations 0.03 and 1.0 µg/mL, then the result was compared with the values obtained from the electromembrane extraction of ultrafiltrate of the same solutions (free concentration) and protein-free ultrafiltrate of plasma with final concentration of 0.03 and 1.0 µg/mL (total concentration). For this purpose, EME followed by capillary electrophoresis coupled with diode array detection was optimized and validated. RESULTS: The results showed that the electromembrane extraction method was only able to extract the unbound fraction of phenytoin from plasma samples. The method was validated over a concentration range of 0.03-4 µg/mL. The inter and intra-assay precisions were less than 6.7%. The phenytoin protein binding was also determined to be in agreement with the literature data and confirms the validity of this method. CONCLUSION: This sensitive and quick EME approach for determining the free concentration of a phenytoin, can be a good alternative to classic methods for therapeutic drug monitoring and pharmacokinetic studies.

Determination of phenytoin in exhaled breath condensate using electromembrane extraction followed by capillary electrophoresis.

Application of hollow fiber-based electromembrane extraction was studied for extraction and quantification of phenytoin from exhaled breath condensate (EBC). Phenytoin is extracted from EBC through a supported liquid membrane consisting of 1-octanol impregnated in the walls of a hollow fiber, and into an alkaline aqueous acceptor solution inside the lumen of the fiber. Under the obtained conditions of electromembrane extraction, that is, the extraction time of 15 min, stirring speed of 750 rpm, donor phase pH at 11.0, acceptor pH at 13.0, and an applied voltage of 15 V across the supported liquid membrane, an enrichment factor of 102-fold correspond to extraction percent of 25.5% was achieved. Good linearity was obtained over the concentration range of 0.001-0.10 µg/mL (r2 = 0.9992). Limits of detection and quantitation were 0.001 and 0.003 µg/mL, respectively. The proposed method was successfully applied to determine phenytoin from EBC samples of patients receiving the drug. No interfering peaks were detected that indicating excellent selectivity of the method. The intra- and interday precisions (RSDs) were less than 14%.

A Rapid and Sensitive HPLC-DAD Assay to Quantify Lamotrigine, Phenytoin and Its Main Metabolite in Samples of Cultured HepaRG Cells.

A sensitive and fast high-performance liquid chromatography-diode-array detection assay was developed and validated for the simultaneous quantification of 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), phenytoin (PHT) and lamotrigine (LTG) in samples of cultured HepaRG cells. Chromatographic separation of analytes and internal standard (IS) was achieved in ∼15 min on a C18-column, at 35°C, using acetonitrile (6%), methanol (25%) and a mixture (69%) of water-triethylamine (99.7:0.3, v/v; pH 6.0), pumped at 1 mL/min. The analytes and IS were detected at 215 or 235 nm. Calibration curves were linear with regression coefficients >0.994 over the concentration ranges of 0.1-15 µg/mL for HPPH; 0.15-30 µg/mL for PHT and 0.2-20 µg/mL for LTG. The method showed to be accurate (bias value of ±10.5 or ±17.6% in the lower limit of quantification, LLOQ) and precise (coefficient variation ≤8.1 or ≤15.4% in the LLOQ), and the absolute recovery of the analytes ranged from 62.5 to 96.9%. HepaRG cells have emerged as a very promising in vitro model to evaluate metabolic, drug interaction and/or pharmacokinetic studies, and this methodology will be suitable to support subsequent studies involving the antiepileptic drugs PHT and LTG.

Simultaneous analysis method for GHB, ketamine, norketamine, phenobarbital, thiopental, zolpidem, zopiclone and phenytoin in urine, using C18 poroshell column.

Date-rape drugs have the potential to be used in drug-facilitated sexual assault, organ theft and property theft. Since they are colorless, tasteless and odorless, victims can drink without noticing, when added to the beverages. These drugs must be detected in time, before they are cleared up from the biofluids. A simultaneous extraction and determination method in urine for GHB, ketamine, norketamine, phenobarbital, thiopental, zolpidem, zopiclone and phenytoin (an anticonvulsant and antiepileptic drug) with LC-MS/MS was developed for the first time with analytically acceptable recoveries and validated. A 4 steps liquid-liquid extraction was applied, using only 1.000mL urine. A new age commercial C18 poroshell column with high column efficiency was used for LC-MS/MS analysis with a fast isocratic elution as 5.5min. A new MS transition were introduced for barbital. 222.7>179.8 with the effect of acetonitrile. Recoveries (%) were between 80.98-99.27 for all analytes, except for GHB which was 71.46. LOD and LOQ values were found in the ranges of 0.59-49.50 and 9.20-80.80ngmL(-1) for all the analytes (except for GHB:3.44 and 6.00µgmL(-1)). HorRat values calculated (between 0.25-1.21), revealed that the inter-day and interanalist precisions (RSD%≤14.54%) acceptable. The simultaneous extraction and determination of these 8 analytes in urine is challenging because of the difficulty arising from the different chemical properties of some. Since the procedure can extract drugs from a wide range of polarity and pKa, it increases the window of detection. Group representatives from barbiturates, z-drugs, ketamine, phenytoin and polar acidic drugs (GHB) have been successfully analyzed in this study with low detection limits. The method is important from the point of determining the combined or single use of these drugs in crimes and finding out the reasons of deaths related to these drugs.

Temperature-responsive Solid-phase Extraction Column for Biological Sample Pretreatment.

We have developed a novel solid-phase extraction (SPE) system utilizing a temperature-responsive polymer hydrogel-modified stationary phase. Aminopropyl silica beads (average diameter, 40 - 64 µm) were coated with poly(N-isopropylacrylamide) (PNIPAAm)-based thermo-responsive hydrogels. Butyl methacrylate (BMA) and N,N-dimethylaminopropyl acrylamide (DMAPAAm) were used as the hydrophobic and cationic monomers, respectively, and copolymerized with NIPAAm. To evaluate the use of this SPE cartridge for the analysis of drugs and proteins in biological fluids, we studied the separation of phenytoin and theophylline from human serum albumin (HSA) as a model system. The retention of the analytes in an exclusively aqueous eluent could be modulated by changing the temperature and salt content. These results indicated that this temperature-responsive SPE system can be applied to the pretreatment of biological samples for the measurement of serum drug levels.

Influence of organic surface coatings on the sorption of anticonvulsants on mineral surfaces.

Here, we explore the role that sorption to mineral surfaces plays in the fate of two commonly encountered effluent-derived pharmaceuticals, the anticonvulsants phenytoin and carbamazepine. Adsorption isotherms and pH-edge experiments are consistent with electrostatics governing anticonvulsant uptake on metal oxides typically found in soil and aquifer material (e.g., Si, Al, Fe, Mn, and Ti). Appreciable, albeit limited, adsorption was observed only for phenytoin, which is anionic above pH 8.3, on the iron oxides hematite and ferrihydrite. Adsorption increased substantially in the presence of cationic and anionic surfactants, species also commonly encountered in wastewater effluent. For carbamazepine, we propose the enhanced uptake results entirely from hydrophobic interactions with apolar tails of surfactant surface coatings. For phenytoin, adsorption also arises from the ability of surfactants to alter the net charge of the mineral surface and thereby further enhance favorable electrostatic interactions with its anionic form. Collectively, our results demonstrate that although pristine mineral surfaces are likely not major sinks for phenytoin and carbamazepine in the environment, their alteration with organic matter, particularly surfactants, can considerably increase their ability to retain these emerging pollutants in subsurface systems.

First HPLC-UV method for rapid and simultaneous quantification of phenobarbital, primidone, phenytoin, carbamazepine, carbamazepine-10,11-epoxide, 10,11-trans-dihydroxy-10,11-dihydrocarbamazepine, lamotrigine, oxcarbazepine and licarbazepine in human plasma.

A sensitive and fast high-performance liquid chromatographic method coupled with ultraviolet detection is herein reported for the simultaneous determination of human plasma concentration of six antiepileptic drugs frequently used in clinical practice [phenobarbital (PB), primidone (PRM), phenytoin (PHT), carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC)] and some of their main metabolites, carbamazepine-10,11-epoxide (CBZ-E), 10,11-trans-dihydroxy-10,11-dihydrocarbamazepine (trans-diol) and licarbazepine (Lic). Sample preparation consisted of a deproteinization step with methanol followed by a solid-phase extraction procedure. Chromatographic separation was achieved in approximately 15 min on a reversed-phase C18 column using a mobile phase composed by water-methanol-acetonitrile-triethylamine (68.7:25:6:0.3, v/v/v/v; pH 6.5) pumped isocratically at 1.0 mL/min. The detector was set at 237 nm. Calibration curves were linear with regression coefficients greater than 0.992 over the concentration ranges 0.25-100 µg/mL for PB, 0.4-50 µg/mL for PRM, 0.5-50 µg/mL for PHT, 0.1-50 µg/mL for CBZ, LTG and CBZ-E, 0.1-25 µg/mL for OXC, 0.25-10 µg/mL for trans-diol and 0.15-80 µg/mL for Lic. Inter- and intra-day imprecision did not exceed 12.15% and inaccuracy was within ±14.91%. Absolute mean recoveries ranged from 78.49 to 101.04% and no interferences were observed at the retention times of the analytes and internal standard (ketoprofen). This bioanalytical method was successfully applied to real plasma samples from epileptic patients and it seems to be a suitable tool for routine therapeutic drug monitoring and also to support other clinical pharmacokinetic-based studies.

A novel microextraction by packed sorbent-gas chromatography procedure for the simultaneous analysis of antiepileptic drugs in human plasma and urine.

A simple, accurate, and sensitive microextraction by packed sorbent-gas chromatography-mass spectrometry method has been developed for the simultaneous quantification of four antiepileptic drugs; oxcarbazepine, carbamazepine, phenytoin, and alprazolam in human plasma and urine as a tool for drug monitoring. Caffeine was used as internal standards for the electron ionization mode. An original pretreatment procedure on biological samples, based on microextraction in packed syringe using C(18) as packing material gave high extraction yields (69.92-99.38%), satisfactory precision (RSD < 4.7%) and good selectivity. Linearity was found in the 0.1-500 ng/mL range for these drugs with limits of detection (LODs) between 0.0018 and 0.0036 ng/mL. Therefore, the method has been found to be suitable for the therapeutic drug monitoring of patients treated with oxcarbazepine, carbamazepine, phenytoin, and alprazolam. After validation, the method was successfully applied to some plasma samples from patients undergoing therapy with one or more of these drugs. A comparison of the detection limit with similar methods indicates high sensitivity of the present method over the earlier reported methods. The present method is applied for the analysis of these drugs in the real urine and plasma samples of the epileptic patients.

Occurrence and removal of pharmaceuticals and hormones through drinking water treatment.

The occurrence of fifty-five pharmaceuticals, hormones and metabolites in raw waters used for drinking water production and their removal through a drinking water treatment were studied. Thirty-five out of fifty-five drugs were detected in the raw water at the facility intake with concentrations up to 1200 ng/L. The behavior of the compounds was studied at each step: prechlorination, coagulation, sand filtration, ozonation, granular activated carbon filtration and post-chlorination; showing that the complete treatment accounted for the complete removal of all the compounds detected in raw waters except for five of them. Phenytoin, atenolol and hydrochlorothiazide were the three pharmaceuticals most frequently found in finished waters at concentrations about 10 ng/L. Sotalol and carbamazepine epoxide were found in less than a half of the samples at lower concentrations, above 2 ng/L. However despite their persistence, the removals of these five pharmaceuticals were higher than 95%.

Targeted three-dimensional liquid chromatography: a versatile tool for quantitative trace analysis in complex matrices.

Targeted multidimensional liquid chromatography (MDLC), commonly referred to as 'coupled-column' or 'heartcutting', has been used extensively since the 1970s for analysis of low concentration constituents in complex biological and environmental samples. A primary benefit of adding additional dimensions of separation to conventional HPLC separations is that the additional resolving power provided by the added dimensions can greatly simplify method development for complex samples. Despite the long history of targeted MDLC, nearly all published reports involve two-dimensional methods, and very few have explored the benefits of adding a third dimension of separation. In this work we capitalize on recent advances in reversed-phase HPLC to construct a three-dimensional HPLC system for targeted analysis built on three very different reversed-phase columns. Using statistical peak overlap theory and one of the most recent models of reversed-phase selectivity we use simulations to show the potential benefit of adding a third dimension to a MDLC system. We then demonstrate this advantage experimentally by developing targeted methods for the analysis of a variety of broadly relevant molecules in different sample matrices including urban wastewater treatment effluent, human urine, and river water. We find in each case that excellent separations of the target compounds from the sample matrix are obtained using one set of very similar separation conditions for all of the target compound/sample matrix combinations, thereby significantly reducing the normally tedious method development process. A rigorous quantitative comparison of this approach to conventional 1DLC-MS/MS also shows that targeted 3DLC with UV detection is quantitatively accurate for the target compounds studied, with method detection limits in the low parts-per-trillion range of concentrations. We believe this work represents a first step toward the development of a targeted 3D analysis system that will be more effective than previous 2D separations as a tool for the rapid development of robust methods for quantitation of low concentration constituents in complex mixtures.

Enhanced clearance of highly protein-bound drugs by albumin-supplemented dialysate during modeled continuous hemodialysis.

BACKGROUND: In 2006, there were 16 796 toxic exposures attributed to valproic acid (VPA), carbamazepine (CBZ) and phenytoin (PHT) reported to the US Toxic Exposure Surveillance System. Of these, 30% (5046) were treated in a health care facility with 12 cases resulting in death. These drugs are highly protein bound and poorly dialyzable; however, it has been suggested that albumin-supplemented dialysate may enhance dialytic clearance. We investigated whether the addition of albumin to dialysate affects dialytic clearance of VPA, CBZ and PHT. METHODS: VPA, CBZ and PHT were added to a bovine blood-based in vitro continuous hemodialysis circuit, which included a polysulfone or an AN69 hemodialyzer. VPA, CBZ and PHT clearances were calculated from spent dialysate and pre-dialyzer plasma concentrations. VPA, CBZ and PHT clearances with control (albumin-free) dialysate were compared to clearances achieved with 2.5% or 5% human albumin-containing dialysate. The influences of blood flow (180 and 270 mL/min) and dialysate flow (1, 2 and 4 L/h) on dialysis clearance were also assessed. RESULTS: The addition of 2.5% albumin to dialysate significantly enhanced dialytic clearance of VPA and CBZ, but not PHT. Use of 5% albumin dialysate further increased VPA and CBZ clearance. Overall, drug clearance was related directly to dialysate flow but independent of blood flow. CONCLUSION: Continuous hemodialysis with albumin-supplemented dialysate significantly enhanced VPA and CBZ, but not PHT, clearance compared to control dialysate. Continuous hemodialysis with albumin-supplemented dialysate may be a promising therapy to enhance dialytic clearance of selected highly protein-bound drugs.

Nonlinear adsorption isotherm as a tool for understanding and characterizing molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) have frequently been characterized by quantities which are easily determined from experiments but have no theoretical foundation. This makes it difficult to compare different MIP preparations or to transfer MIP based methods to different experimental conditions. Since the adsorption isotherms of MIPs are markedly nonlinear, one can build a better characterization strategy on isotherms as shown by examples in this paper.

Simultaneous determination of phenytoin and dextromethorphan in urine by solid-phase extraction and HPLC-DAD.

A rapid and simple high-performance liquid chromatographic method with photodiode array detection was developed for the separation and the simultaneous determination of phenytoin and dextromethorphan in human urine. Analysis was performed in less than 4.5 min in isocratic mode on a reversed-phase C18 column (5 microm; 150 x 4.6 mm) using a mobile phase composed of acetonitrile-buffer phosphate 0.01 M (60:40, v/v) adjusted to pH 6.0, at 1 mL/min flow rate and UV absorbance at 210 nm. The elution order of analytes was dextromethorphan (DXM), Internal Standard (IS), and phenytoin (PHT). Calibration curves were linear in the 7.5-25 microg/mL range for PHT and in the 10-30 microg/mL range for DXM. Spike recoveries for urine samples prepared at three spiking levels ranged from 97.8 to 102.3% for PHT and from 94.8 to 100.4% for DXM. The detection limit (LOD) values ranged from 0.08 microg/mL for PHT to 0.5 microg/mL for DXM. The quantitation limit (LOQ) values ranged from 0.3 microg/mL for PHT to 1.6 microg/mL for DXM. The sample preparation method involves a rapid and simple procedure based on solid-phase extraction using a C18 reversed-phase column. Validation of the optimised method was carried out according to the ICH guidelines. The method developed in this study allows the reliable simultaneous analysis of PHT and DXM, drugs that were never quantified together in previously reported analytical methods. The described method has the advantage of being rapid and easy and it could be applied in therapeutic monitoring of these drugs in human urine of epileptic patients.

Pimprinine, an extracellular alkaloid produced by Streptomyces CDRIL-312: fermentation, isolation and pharmacological activity.

Pimprinine, an extracellular alkaloid has been isolated from the culture filtrate of Streptomyces CDRIL-312. Pimprinine was subsequently purified using silica gel column chromatography and also by preparatory thin layer chromatography. Some physicochemical properties, antimicrobial activities (in-vitro) and pharmacological activities of pimprinine were studied. Pimprinine showed promising anticonvulsant activity in both minimum and maximum electric seizure threshold test in mice. Its anticonvulsant activity is very much comparable to that of phenyl hydantion sodium. Pimprinine also inhibited effectively tremorine-induced tremors and analgesia in mice.

Glucuronidation of prodrug reactive site: isolation and characterization of oxymethylglucuronide metabolite of fosphenytoin.

BACKGROUND: This investigation was undertaken to identify the structure of a novel immunoreactive metabolite derived from fosphenytoin that has been hypothesized previously as present in sera from renally impaired patients receiving this prodrug. METHODS: The metabolite was isolated from uremic sera using solid-phase extraction and HPLC. Structural analysis was performed using HPLC-tandem mass spectrometry, nuclear magnetic resonance (NMR), deuterium exchange, and chemical derivatization. Immunoreactivity was evaluated using a fluorescence polarization immunoassay. RESULTS: The metabolite had a parent ion at m/z 457 in the negative-ion mode and fragmented to yield the m/z 251 of phenytoin, as well as other mass fragments of phenytoin. Mass fragments associated with glucuronic acid were also present. The chromatographic peak corresponding to this metabolite demonstrated immunoreactivity sufficient to lead to falsely increased reported values for phenytoin immunoassays. The observed immunoreactivity was also proportional to the relative concentration of the metabolite in collected fractions. Analysis by NMR indicated the presence of phenyl groups with chemical shifts identical to those of phenytoin, as well as the presence of a methylene bridge, which was consistent with the same methylene bridge present on the phosphate ester of fosphenytoin. Comparative analysis of serum samples from renally impaired patients receiving phenytoin vs fosphenytoin using multiple reaction monitoring quantification demonstrated that this metabolite was associated with fosphenytoin administration. CONCLUSIONS: A unique immunoreactive oxymethylglucuronide metabolite derived from fosphenytoin has been isolated from sera from uremic patients receiving this prodrug.

Hair analysis for pharmaceutical drugs. I. Effective extraction and determination of phenobarbital, phenytoin and their major metabolites in rat and human hair.

In order to establish an analytical method for the determination of phenobarbital (PB), phenytoin (PPH) and their hydroxylated metabolites in hair, animal model experiments were performed. Five male dark-agouti pigmented rats, aged 5 weeks, were intraperitoneally and orally administered PB or PPH independently at 25 mg/kg once a day for 5 successive days. The growing back hair was collected 15d after the first administration. Four typical extraction methods, using NH4OH-methanol-acetone, TFA-methanol-acetone, 1M sodium hydroxide and proteinase K, were evaluated using the rat hair samples containing PB or PPH. Methanol-acetone-NH4OH (10: 10: 1) was the best extraction method from all aspects, such as high extraction efficiency and low noise. The analytes in the extract were methylated in acetonitrile with 20% tetramethylammonium hydroxide and methyliodide at 70 degrees C for 10 min. After purification with Bond Elut Certify, the methylated products were analyzed by GC-MS. From rat hair, PB, p-hydroxy PB, PPH and p-hydroxy PPH were detected at average concentrations of 26.9, trace, 4.2 and 0.4 ng/mg with an intraperitoneal (i.p.) injection, and at 30.9, trace, 4.0 and 0.4 ng/mg with oral administration, respectively. There was little difference in hair concentrations between i.p. injection and oral administration. This method was applied to the head hair of two patients who orally took toxic amounts of PB and two volunteers who orally took 100 mg of PPH daily for 5 d. The hair concentrations of PB in the two patients were 16.2 and 14.7 ng/mg, and those of PPH in the two volunteers were 3.3 and 0.1 ng/mg.

New calixarene-bonded stationary phases in high-performance liquid chromatography: comparative studies on the retention behavior and on influences of the eluent.

The chromatographic behavior of six calixarene-bonded stationary phases is reported. Varying analyte selectivities (i.e., for phenols, substituted aromatics, polycyclic aromatic hydrocarbons, barbituric acid derivatives, xanthines) exist as a function of the ring-size of the calix[n]arenes (n=4, 6, 8) and the substitution at the "upper rim" with para-tert.-butyl groups. Although eluents with unusually high proportions of water were used, a comparison with conventional reversed-phase (RP) columns shows a predominantly reversed-phase character with remarkable selectivities of these phases. The influences of several organic solvents on retention variations of solutes are compared for RP-C18, phenyl and calixarene phases.

[Chiral separation of racemic 5-(p-hydroxyphenyl)-5-phenylhydantoin by RP-HPLC using eluents containing beta-cyclodextrin].

A chiral RP-HPLC method was developed to assay the 5-(p-hydroxyphenyl)-5-Phenylhydantoin (p-HPPH) enantiomers, the major metabolite of antiepileptic drug phenytoin, in rat hepatic microsomes. A 50 mm FLC-C8 column was used as the analytical column, beta-cyclodextrin (beta-CD) as chiral mobile phase additive and phenobarbital as the internal standard. The detection wavelength was 250 nm. The linear range of p-HPPH enantiomers was 0.5-110 mg/L. The detection limit was 5 ng (S/N = 3). The recoveries of S- and R-p-HPPH were 93.6% +/- 2.8% and 94.7% +/- 1.8% respectively. The RSD within day and between days were less than 2%. The concentration of beta-CD played an important role in separating chiral enantiomers. When the concentration of beta-CD was between 8.8 and 13.2 mmol/L the resolution of p-HPPH enantiomers had the largest value Rs = -1.1. In this work, 8.8 mmol/L beta-CD solution (4 g beta-CD, 6 g urea, and 1.5 g ammonium acetate in 400 mL water) was selected in considering some factors such as column efficiency, solubility of beta-CD etc. Urea can increase the solubility of beta-CD. When urea: beta-CD = 1:1-1.5:1 (g/g), the solubilization of beta-CD was significant. Methanol concentration in mobile phase affected retention time, resolution of p-HPPH enantiomers and solubility of beta-CD.

Optical separation of racemic 5-(p-hydroxyphenyl)-5-phenylhydantoin by reversed phase high-performance liquid chromatography using eluents containing beta-cyclodextrin.

Both S-(-)- and R-(+)-enantiomers of 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), a main oxidative metabolite of the achiral antiepileptic drug phenytoin, could be determined simply, sensitively and accurately using reversed phase high-performance liquid chromatography by using a methanol-monopotassium phosphate eluent containing beta-cyclodextrin. Using this assay procedure, it was determined that an S-(-)-enantiomer was formed predominantly by the oxidation of phenytoin in isolated rat hepatocytes.