Rapid separation of some antiepileptic drugs in serum by HPLC

Summary A fast and easy, iscratic high-performance liquid chromatographic method for the simultaneous determination of primidone, phenobarbital, phenytoin, carbamazepine and thiobutabarbital from serum samples has been developed. The optimal eluent composition is 36% acetonitrile, 24% methanol and 40% phosphate buffer. Using a programmable wavelength detector very low (sub-therapeutic) levels of the drugs can be measured.     

超高效液相色谱-串联质谱法同时测定血清中12种抗癫痫药物

建立了超高效液相色谱-串联质谱同时测定血清中加巴喷丁、拉莫三嗪、普瑞巴林、拉考沙胺、左乙拉西坦、托吡酯、奥卡西平、氯硝西泮、丙戊酸钠、卡马西平、苯巴比妥、苯妥英钠12种抗癫痫药物的方法。将非那西丁和氯唑沙宗分别作为正离子和负离子模式下的内标物，血清经乙腈沉淀蛋白质后，取上清液进样分析。采用ACQUITY UPLC BEH C₁₈柱分离，流动相为水和甲醇（均含10 mmol/L甲酸铵），流速为0.4 mL/min。采用电喷雾电离源，在多反应监测和正、负离子模式下分段进行扫描分析。结果表明，12种抗癫痫药物在各自范围内线性关系良好，相关系数均大于0.992，在3个添加水平下方法的加标回收率为90.80%~114.0%，批内精密度RSD≤13.2%，批间精密度RSD≤14.8%。该方法专属性强，灵敏度高，可用于12种抗癫痫药物临床血药浓度监测及药代动力学研究。     

Analysis of antiepileptic drugs in human plasma using micellar electrokinetic capillary chromatography.

We describe a method for the simultaneous determination of antiepileptic drugs (ethosuccimide, phenytoin, primidone, phenobarbital, carbamazepine and valproic acid) by micellar electrokinetic capillary chromatography using sodium dodecyl sulphate as the micellar phase. Factors affecting the micellar electrokinetic separation were studied for the quantitative determination of these drugs in human plasma. The confirmation of the peaks and the specificity of the method were investigated by combining multiwavelength detection with micellar electrokinetic capillary chromatography.     

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 C₁₈ 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.     

Simple and rapid HPLC method for simultaneous determination of multiple antiepileptic drugs in human serum

Summary A very rapid, sensitive and reproducible HPLC method was developed for simultaneous determination of eight anti-epileptic drugs (AEDs): lamotrigine, primidone, ethosuximide, sulthiame, felbamate, phenobarbital, carbamazepine, phenytoin and oxcarbazepine-metabolite (10-hydroxy-carbazepine) in human serum. Sample purification requires only protein precipitation with an appropriate reagent. Separation was by reversed-phase HPLC, using a C18 column, 20% acetonitrile and 40 mM phosphoric acid buffer as mobile phase. Column temperature was set at 50°C, and measurement was by UV detection at 205 nm. The inter and intra-assay coefficients of variation (CV) ranged 1.13–7.10% and 1.14–8.49%, respectively. The absolute (measured) and relative (analytic) recoveries of the drugs ranged 96.7%–104.4% and 97.3%–106.1%, respectively. No interference with other common antiepileptic drugs and analgesics were observed. The method requires only 100 μl serum or less. It is very fast (sample preparation and analysis time approx. 23 min for all 9 AEDs), and suitable for routine clinical use, especially for epileptic patients on polytherapy.     

Direct serum injection high-performance liquid chromatographic method for the simultaneous determination of phenobarbital, carbamazepine and phenytoin

A method is described for the simultaneous measurement of serum levels of three antiepileptic drugs, phenobarbital, phenytoin and carbamazepine, by direct injection high-performance liquid chromatography on a 25-cm Pinkerton internal surface reversed-phase (ISRP) column. Several commonly available compounds were tested and found not to co-chromatograph with the three drugs of interest or the internal standard, 5-(p-methylphenyl)-5-phenylhydantoin. Results obtained on patients' samples with this method compared well with those from enzyme-multiplied immunoassay technique (EMIT).     

Octakis-6-sulfato-gamma-cyclodextrin as additive for capillary electrokinetic chromatography of dibenzoazepines: carbamazepine,oxcarbamazepine and their metabolites

Single isomer octakis-(2,3-dihydroxy-)6-sulfato-gamma-cyclodextrin used as pseudostationary phase of the background electrolyte interacts with dibenzo[b,f]azepines (consisting of a condensed 3-ring system) and forms negatively charged complexes. Hydroxygroups in position 2 and 3 at carbamazepine increase the extent of interaction, whereas substitution by oxygen at position 10 and/or 11 reduces it. The complex constants for the analytes are ranging from few tens L/mol (10-hydroxycarbamazepine, 10,11-dihydroxycarbamazepine, 10,11-epoxycarbamazepine, oxcarbazepine) to several hundreds L/mol (carbamazepine, 2-hydroxycarbamazepine, 3-hydroxycarbamazepine), and are much larger than those of the analytes with octakis-(2,3-dimethyl-)-6-sulfato-gamma-cyclodextrin. Full enantiomeric separation of the chiral metabolites of carbamazepine and oxcarbazepine is obtained at octakis-(2,3-dihydroxy-)-6-sulfato-gamma-cyclodextrin concentrations of about 10 mM (3 mM borate buffer, pH 8.5). Compared to heptakis-6-sulfato-beta-cyclodextrin, selectivity differs and stereoselectivity is more pronounced.     

高效液相色谱法同时测定血清中茶碱、苯妥英钠、苯巴比妥及卡马西平的药物浓度

 报道了用高效液相色谱法（HPLC）同时测定血清中茶减、苯妥莫纳、苯巴比妥及卡马西平的药物浓度。实验条件:Nova-PahC18柱，流动相为甲醇-水（1:1，V／V），检测波长210nm，流速为1mL／min，萃取液为级访-异丙醇（95:5，V／V）。方法具有灵敏（10-9）、准确（回收率在97％～105％之间）、快速（7min）等特点，对临床血药浓度监测有实际应用价值。     

One-way multiplexed immunoassay strategy for simultaneous determination of multi-analytes by microchip electrophoresis

An integrated microchip electrophoresis (MCE) system with online immunoreaction and laser induced fluorescence (LIF) detection has been developed for simultaneous determination of multi-analytes. In this system, the multiplexed immunoreactions between multiple antibody-immobilized glass beads with analytes and respective fluorescently labeled antigens were performed in a sample reservoir. After online incubation, the immunoreaction solution was injected into a one-way separation channel, and free fluorescently labeled antigens were separated and detected in the separation channel. With the help of glass beads, the immunocomplex can not move into the separation channel, which simplifies the separation of fluorescently labeled antigens. With the use of phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ) and theophylline (Th) as proof-of-principle analytes, the one-way multiplexed immunoassay could be completed within 20 min, resulting in a response curve over the range of 4.0-400 nM for each analyte. Detection limits (S/N = 3) for the drugs tested were in the range of 1.8 × 10(-9) to 2.5 × 10(-9) M. Compared with the conventional immunoassays, this assay is simple, rapid, sensitive and low cost, and provides an accurate procedure for a multiplex immunoassay. The present method has been applied for the simultaneous determination of PB, PHT, CBZ and Th in human serum, which showed a promise of automated clinical application.     

Liquid chromatographic analysis of oxcarbazepine and its metabolites in plasma and saliva after a novel microextraction by packed sorbent procedure

A rapid and reliable analytical method suitable for the simultaneous determination of the antiepileptic drug, oxcarbazepine and its metabolites in human plasma and saliva by means of liquid chromatography with diode array detection (DAD) has been developed. Oxcarbazepine and its metabolites (10,11-dihydro-10-hydroxycarbamazepine, trans-10,11-dihydro-10,11-dihydroxycarbamazepine and 3-hydroxycarbamazepine) were baseline separated within 6.5 min on a reversed-phase C18 column with a phosphate buffer-acetonitrile-triethylamine mixture as the mobile phase. The DAD detector was set at 240 nm. A sample preparation method for biological samples using a microextraction by packed sorbent technique has been implemented, employing a C18 sorbent inserted into a microvolume syringe and using only a small volume (25 μL) of plasma or saliva. The extraction yield values were satisfactory for all analytes (>86.5%) as well as the precision data, which were always in the low percentage of relative standard deviation values (<4.6%). The method was successfully applied to both plasma and saliva samples drawn from psychiatric and neurological patients undergoing treatment with oxcarbazepine (Tolep^®) tablets.     

高效液相色谱法同时测定血清中茶碱、苯妥英钠、苯巴比妥及卡马西平的药物浓度

报道了用高效液相色谱法（HPLC）同时测定血清中茶减、苯妥莫纳、苯巴比妥及卡马西平的药物浓度。实验条件:Nova-PahC18柱，流动相为甲醇-水（1:1，V／V），检测波长210nm，流速为1mL／min，萃取液为级访-异丙醇（95:5，V／V）。方法具有灵敏（10-9）、准确（回收率在97％～105％之间）、快速（7min）等特点，对临床血药浓度监测有实际应用价值。     

Simultaneous extraction and quantification of lamotrigine,phenobarbital, and phenytoin in human plasma and urine samples using solidified floating organic drop microextraction and high‐performance liquid chromatography

A novel and simple method based on solidified floating organic drop microextraction followed by high‐performance liquid chromatography with ultraviolet detection has been developed for simultaneous preconcentration and determination of phenobarbital, lamotrigine, and phenytoin in human plasma and urine samples. Factors affecting microextraction efficiency such as the type and volume of the extraction solvent, sample pH, extraction time, stirring rate, extraction temperature, ionic strength, and sample volume were optimized. Under the optimum conditions (i.e. extraction solvent, 1‐undecanol (40 μL); sample pH, 8.0; temperature, 25°C; stirring rate, 500 rpm; sample volume, 7 mL; potassium chloride concentration, 5% and extraction time, 50 min), the limits of detection for phenobarbital, lamotrigine, and phenytoin were 1.0, 0.1, and 0.3 μg/L, respectively. Also, the calibration curves for phenobarbital, lamotrigine, and phenytoin were linear in the concentration range of 2.0–300.0, 0.3–200.0, and 1.0–200.0 μg/L, respectively. The relative standard deviations for six replicate extractions and determinations of phenobarbital, lamotrigine, and phenytoin at 50 μg/L level were less than 4.6%. The method was successfully applied to determine phenobarbital, lamotrigine, and phenytoin in plasma and urine samples.     

Interaction of carbamazepine and phenobarbital in rabbits

The interaction of carbamazepine and phenobarbital in rabbits was investigated. The drugs were administered to the rabbit orally as a single dose. By simultaneous administration the sequence of drugs was varied, with an interval between doses of 15 min. The doses of carbamazepine and phenobarbital were 100 and 25 mg, respectively. It was established that phenobarbital appears to be an inductor for carbamazepine independently sequence of administration of the drugs. Carbamazepine reveals inductive properties for phenobarbital in the case where phenobarbital enteres in the organism first. It was ascertained also that, by simultaneous administration, these drugs reduce absorption of each other in plasma.     

Modified dispersive liquid‐phase microextraction based on sequential injection solidified floating organic drop combined with HPLC for the determination of phenobarbital and phenytoin

A modified dispersive liquid phase microextraction based on sequential injection solidified floating organic drop was developed for simultaneous separation/preconcentration of trace amounts of phenobarbital and phenytoin. The important factors affecting on the extraction recovery including pH, the volume of extraction solvent, ionic strength, and the number of injections were investigated and optimized by Box–Behnken design and desirability function. Under the optimum experimental conditions, the calibration graph was linear in the concentration range of 1.0–300.0 μg/L (r² = 0.997) for phenobarbital and 2.0–400.0 μg/L (r² = 0.996) for phenytoin. The limit of detection and limit of quantification were 0.35 and 1.2 μg/L for phenobarbital and 0.65 and 2.2 μg/L for phenytoin, respectively. The relative standard deviation for six replicate determinations at 10 μg/L was 3.3 and 4.1% for phenobarbital and phenytoin, respectively. The developed method was successfully applied to the determination of phenobarbital and phenytoin in urine and plasma samples.     

Simple and rapid micellar electrokinetic capillary chromatographic method for simultaneous determination of four antiepileptics in human serum

A very rapid and simple MEKC method was developed for the simultaneous determination of four antiepileptic drugs, ethosuximide (Etho), primidone (Pri), phenytoin (Pht) and carbamazepine (Cbz) in human serum. Sample analysis required only 100 microL of human serum which only needed to be centrifuged, decanted and combined with the running buffer [5.3 mM Na(2)HPO(4)/3.2 mM borax buffer (pH 9.5) containing 55 mM SDS and 3.5% (v/v) acetone]. The analysis was performed in only 10 min into fused-silica capillaries (57 cm total length with 50 microm i.d. and 50 cm to the detector) using the MEKC methodology with diode-array detection at 220 nm. The calibration graphs were established for ethoximide, primidone, phenytoin and carbamazepine between 0 and 20 mg/L. Recoveries were between 85 and 87%. The simplicity of the proposed methodology makes it suitable for routine clinical use, especially for epileptic patients on polytherapy.     

Novel micro-extraction by packed sorbent procedure for the liquid chromatographic analysis of antiepileptic drugs in human plasma and urine

A method for the simultaneous determination of the antiepileptic drugs, phenobarbital (PHB), phenytoin (PTN), carbamazepine (CBZ), primidone (PRM) and oxcarbazepine (OXC) in human plasma and urine samples by using micro‐extraction in a packed syringe as the sample preparation method connected with LC/UV (MEPS/LC/UV) is described. Micro‐extraction in a packed syringe (MEPS) is a new miniaturized, solid‐phase extraction technique that can be connected online to gas or liquid chromatography without any modifications. In MEPS approximately 1 mg of the solid packing material is inserted into a syringe (100–250 μL) as a plug. Sample preparation takes place on the packed bed. The bed can be coated to provide selective and suitable sampling conditions. The new method is very promising, easy to use, fully automated, inexpensive and quick. The standard curves were obtained within the concentration range 1–500 ng/mL in both plasma and urine samples. The results showed high correlation coefficients (R²>0.988) for all of the analytes within the calibration range. The extraction recovery was found to be between 88.56 and 99.38%. The limit of quantification was found to be between 0.132 and 1.956 ng/mL. The precision (RSD) values of quality control samples (QC) had a maximum deviation of 4.9%. A comparison of the detection limits with similar methods indicates high sensitivity of the present method. The method is applied for the analysis of these drugs in real urine and plasma samples of epileptic patients.     

Simultaneous determination of ten antiepileptic drugs in human plasma by liquid chromatography and tandem mass spectrometry with positive/negative ion‐switching electrospray ionization and its application in therapeutic drug monitoring

A simple, rapid, and high‐throughput liquid chromatography with tandem mass spectrometry method for the simultaneous quantitation of ten antiepileptic drugs in human plasma has been developed and validated. The method required only 10 μL of plasma. After simple protein precipitation using acetonitrile, the analytes and internal standard diphenhydramine were separated on a Zorbax SB‐C₁₈ column (50 × 4.6 mm, 2.7 μm) using acetonitrile/water as the mobile phase at a flow rate of 0.9 mL/min. The total run time was 6 min for each sample. The validation results of specificity, matrix effects, recovery, linearity, precision, and accuracy were satisfactory. The lower limit of quantification was 0.04 μg/mL for carbamazepine, 0.02 μg/mL for lamotrigine, 0.01 μg/mL for oxcarbazepine, 0.4 μg/mL for 10‐hydroxycarbazepine, 0.1 μg/mL for carbamazepine‐10,11‐epoxide, 0.15 μg/mL for levetiracetam, 0.06 μg/mL for phenytoin, 0.3 μg/mL for valproic acid, 0.03 μg/mL for topiramate, and 0.15 μg/mL for phenobarbital. The intraday precision and interday precision were less than 7.6%, with the accuracy ranging between –8.1 and 7.9%. The method was successfully applied to therapeutic drug monitoring of 1237 patients with epilepsy after administration of standard antiepileptic drugs. The method has been proved to meet the high‐throughput requirements in therapeutic drug monitoring.     

Simultaneous HPLC Determination of Oxcarbazepine,Carbamazepine and Their Metabolites in Serum

Abstract

We propose a simple procedure for the simultaneous determination of the anticonvulsants oxcarbazepine, carbamazepine and three of their metabolites (10-hydroxy-10, 11-dihydro-carbamazepine, trans-10, 11-dihydroxy-10, 11-dihydro-carbamazepine and 10, 11-epoxy-carbamazepine) in serum or plasma. The alkalinized sample is extracted with ethyl acetate. The extract is evaporated to dryness and taken up with the mobile phase. An aliquot is injected into the liquid chromatograph and eluted with water/methanol/acetonitrile (55/40/5, by vol.) on a 5-μm C-18 reversed-phase column. Eluent is monitored at 254 nm. No interference by other anticonvulsants or by endogenous constituents from the sample is observed. Owing to its good precision, specificity, sensitivity, and selectivity, this method is well adapted to the therapeutic monitoring of oxcarbazepine or carbamazepine treated patients, as well as for pharmacokinetic studies.     

Improved method for the simultaneous determination of phenobarbital, primidone and diphenylhydantoin in patient's serum by gas-liquid chromatography.

A quantitative gas-liquid chromatographic method for the simultaneous determination of phenobarbital, primidone and diphenylhydantoin in human serum is described. A double extraction is employed to improve the removal of interfering substances. The N,N-dimethyl derivatives of the compounds are prepared by "oncolumn" methylation with 50% Methelute. Decomposition of phenobarbital to N-methyl-a-phenylbutyramide was negligible if the contact time with Methelute was less than 10 min. The drugs were stable in serum for at least two weeks. This procedure provides a rapid, sensitive, selective and accurate method for the routine determination of serum concentrations of three of the most commonly prescribed anticonvulsant drugs.     

Simultaneous gas chromatographic analysis for the four commonly used antiepileptic drugs in serum.

We describe a simple, sensitive method for the determination of phenobarbital, diphenylhydantoin, carbamazepine and primidone in serum, by use of gas-liquid chromatography with temperature programming. The methylated derivatives of these anticonvulsants were well resolved, as was 5-(p-methylphenyl)-5-phenylhydantoin, the internal standard. In this procedure we used an ion-exchange resin for separation of the drug from the serum. The proposed procedure requires only 1.0 ml of serum and can be done in less than 1 h. The lower limit of detection for each of the drugs is 0.5 mg/1. Analytical recoveries of drug from serum were excellent and peak height and concentration were linearly related up to twice the toxic concentration for serum.