| Objectives For the determination of antiepileptic drugs in serum, a suitable pretreatment process is needed before instrument analysis due to the complexity of the matrices and the extremely low concentrations of the analytes. As a convenient, solvent-saving, highperformance and highly compatible sample pretreatment method, solid phase extraction(SPE) is one of the fastest-growing. However, currently commercially available SPE columns are costly and for single-use only. In the present research, a ion-exchange monolithic column was developed as SPE absorbent, and coupled with high performance liquid chromatography(HPLC) for the determination of antiepileptic drugs in serum.Methods 1 The poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-ethylene glycol dimethacrylate)(poly(AMPS-co-EDMA)) monolithic extractor was fabricated via thermally initiated polymerization in the stainless steel column(50 mm×2.1 mm,i.d.). With the composition of the amount of monomer(69~137 mg), the effects of reaction temperature(55-70 ?) and the reaction time(4-20 h) of the monoliths were investigated,and the monolithic column was characterized from the pressure, mechanical strength,retention factor and surface morphology. 2 Afterward, poly(AMPS-co-EDMA) monolithic column prepared with the optimal conditions was used as SPE absorbent in the sample pretreatment for the determination of antiepileptic drugs in serum. The optimization of extraction parameters including loading solvent, eluting solvent, flow rate and chromatographic conditions was conducted. The influence of the loading solvent was tested using water and phosphate-buffered saline, the loading flow rate was tested using0.1 m L/min?0.3 m L/min and 0.5 m L/min. Methanol and acetonitrile were each evaluated for use as the eluting solvent and the elution volume(1.0-4.0 m L) was evaluated. And the mobile phase of separation including the ratio of acetonitrile, the concentration and the p H of buffer was evaluated. 3 The method was validated with specificity, limit of detection,limit of quantization, linear range and recovery. In addition, the reusability and reproducibility of the poly(AMPS-co-EDMA) monolithic extractors were also assessed by calculating the relative standard deviations(RSDs) of the extracted peak area of carbamazepine while performing the SPE.Results 1 On the basis of the results, the monolithic column prepared at 65 ? for 16 h was chosen for the experiment. The scanning electron microscope image demonstrated the distinct continuous porous structure of the monolith. 2 The poly(AMPS-co-EDMA)monolith as a solid phase extraction sorbent combines with high performance liquid chromatography is used to determinationin of antiepileptic drugs in serum. The SPE was achieved using water as the loading solvent and methanol was selected for the eluting solvent. The loading flow rate is 0.1 m L/min and the eluting volume is 2.0 m L. The treated samples were analyzed by HPLC. 3 For the proposed method, a good linear relationships for 4 antiepileptic drugs were achieved over the concentration range of 0.5~80.0 ?g/m L with a correlation coefficient(r) value higher than 0.995. The limits of detection(LOD)were 0.04 ? g/m L and the limits of quantification(LOQ) were 0.13 ? g/m L. Recoveries obtained for the determination of 4 antiepileptic drugs in spiking samples ranged from83.1%~102.9%, and the intra-day and inter-day relative standard deviations(RSD) below5.33%. The monolithic extractor was reusable for more than 150 injections. The RSD of peak area for the first, the 100 th and the 150 th injection was 4.73%. The intra-batch and inter-batch RSDs were 5.26% and 6.91%, respectively.Conclusions The poly(AMPS-co-EDMA) monolithic column developed as a reusable SPE sorbent coupled with HPLC for the simultaneous determination of carbamazepine,phenobarbital, phenytoin and 10,11-dihydro-10-hydroxy-carbamazepine in human serum.The ion exchange monolithic column as a pretreatment adsorbent on preparation is easy to prepare, defectless and highly porous, it has high specific surface area, permeability and biological compatibility and cost-saving since it can be repeatedly used for at least 150 times. |
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