高效液相色谱法同时测定人血清中茶碱与苯巴比妥、苯妥英、卡马西平的含量

本文报道茶碱与苯巴比妥（ＰＢ）、苯妥英（ＤＰＨ）、卡马西平（ＣＢＺ）两类（４种）不同药物血浓度的ＨＰＬＣ同时测定法．采用国产色谱柱ＹＷＧＣ１８（４．６×２５０ｍｍ），检测波长为２５４ｎｍ，流动相为甲醇－水（５０：５０，ｖ／ｖ），流速：１ｍｌ／ｍｉｎ，以４－氨基安替匹林作内标，各药物的平均回收率分别为茶碱９９．０２％，ＰＢ１００．７３％，ＤＰＨ１０１．０％，ＣＢＺ１０１．７７％。对各药血清标准液的峰高比测量的变异系数（ｎ＝１０），分别为茶碱（１０μｇ／ｍｌ）６．３％，ＰＢ（２０μｇ／ｍｌ）６．１％，ＤＰＨ（２０μｇ／ｍｌ）４．０％，ＣＢＺ（１０μｇ／ｍｌ）５．２％。本法具有快速、准确、适用性广的特点，用于治疗药物的监测，效果满意，大大提高了实验室的工作效率。     

高效毛细管电泳法分析血样尿样中巴比妥类药物

建立了血、尿样品中巴比妥类药物的高效毛细管电泳分离测定方法，测定条件为：运行缓冲液配比：１００ｍｍｏｌ／Ｌ十二烷基硫酸钠－１００ｍｍｏｌ／Ｌ磷酸二氢钠－甲醇－水＝７０∶１５∶５∶１０（ｐＨ值调至７．５５±０．０５），操作电压１７ｋＶ，检测波长２００和２８５ｎｍ，２０ｍｉｎ内７种巴比妥类药物全部得到分离。考察并选择了体液样品的预处理方法，测定了血浆和尿液中６种巴比妥类药物浓度（苯巴比妥、甲基苯巴比妥、异戊巴比妥、硫喷妥、戊巴比妥和速可眠）。测定血药浓度的线性范围为５．０～３５μｇ／ｍｌ，尿样药物浓度线性范围为１．０～８．０μｇ／ｍｌ，最低检测浓度为１．０μｇ／ｍｌ，方法重现性为ＲＳＤ小于１３％。     

固相萃取用于血浆中抗癫痫药物的提取

目的：探讨固相萃取法用于血浆中抗癫痫药物的提取。方法：以活化的ＯＤＳ柱（４０ｕｍ）为固相萃取柱,二氯甲冠为洗脱剂,洗脱液浓缩后以ＨＰＬＣ法测定药物的浓度。结果：苯巴比妥、苯妥英在５．００～６０．００ｕｇ／ｍｌ范围内,卡马西平在２．５０～２０．００ｕｇ／ｍｌ范围内线性关系良好,ｒ分别为０．９９８０,０．９９９９,０．９９９０;绝对回收率分别为５９．６０％～６８．７％,５８．９％～６８．０％,５３．８     

高效液相色谱法监测苯妥英血药浓度回顾性分析

目的：对苯妥英血药浓度进行分析。方法：对１９８９年１１月至１９９６年１１月间应用高效液相色谱法监测苯妥英血药浓度的数据进行统计。结果：在监测的１５８８８份样本中，血药浓度低于１０μｇ·ｍｌ－１占８０．８９％，高于２０μｇ·ｍｌ－１的占４．７７％，在有效血药浓度１０～２０μｇ·ｍｌ－１范围内仅占１４．３４％。结论：监测苯妥英血药浓度具有临床意义。     

固相萃取用于血浆中抗癫痫药物的提取

目的：探讨固相萃取法用于血浆中抗癫痫药物的提取。方法：以活化的ＯＤＳ 柱（４０μｍ） 为固相萃取柱,二氯甲烷为洗脱剂,洗脱液浓缩后以ＨＰＬＣ 法测定药物的浓度。结果：苯巴比妥、苯妥英在５ ．００ ～６０ ．００μｇｍｌ 范围内,卡马西平在２ ．５０ ～２０ ．００μｇｍｌ 范围内线性关系良好,ｒ 分别为０ ．９９８０ ,０ ．９９９９ ,０ ．９９９０ ;绝对回收率分别为５９ ．６０ ％～６８ ．７ ％ ,５８ ．９ ％ ～６８ ．０ ％ ,５３ ．８ ％ ～５７ ．３ ％ ;相对回收率分别为９２ ．２ ％ ～９８ ．６ ％ ,９７ ．１ ％ ～１０２ ．６ ％ ,９７ ．９ ％ ～１０２ ．０ ％ ,ＲＳＤ＜ ６ ％ ;血浓监测结果与经传统的液液萃取后所得结果非常接近。结论：固相萃取法操作简单、省时、提取干净,可用于血浆中抗癫阅药物的提取。     

反相高效液相色谱法同时测定血浆中氯胺酮,苯巴比妥和阿托品的浓度

本文采用ＺｏｒｂａｘＣ８色谱柱，甲醇－磷酸盐缓冲液（６９∶３１）为流动相，２２０ｎｍ为检测波长，非那西丁为内标，建立了同时测定人血浆中氯胺酮、苯巴比妥和阿托品浓度的反相高效液相色谱法。本法简便、快速、灵敏、重现性好。氯胺酮、苯巴比妥和阿托品的平均回收率分别为９９．２０％，９９．１３％和９７．６２％；不同浓度水平测定结果的日内和日间相对标准偏差均小于５％；其检测限分别为０．２μｇ／ｍｌ，０．０５μｇ／ｍｌ和０．５μｇ／ｍｌ（Ｓ∶Ｎ＝３∶１）。     

差示分光光度法测定血清苯巴比妥浓度

目的：改进差分分光光度法测定血清苯巴比妥浓度。方法：以二氯甲烷在酸性条件下二次提取,用苯巴比妥在不同ＰＨ值的介质中吸收度之差值,按差示分光光度法测定血清中苯巴比妥浓度。结果：５－２０μｇ．ｍｌ＾－１与２５－１００μｇ．ｍｌ＾－１的苯巴比妥标准溶液之间稳定性差异显著性。     

反相高效液相色谱法同时测定苯巴比妥和卡马西平血药浓度

目的：建立同时测定微量血浆中苯巴比妥和卡马西平浓度的方法。方法：采用ＲＰ－ＨＰＬＣ法,以艾司唑仑为内标,同时测定微量血浆中苯芭巴比妥和卡马西平浓度。色谱柱ｓｈｉｍａｄｚｕ ｓｈｉｒｍｐａｃｋ ＣＬＣ－Ｃ１８不锈钢柱,流动相为醇－水（６０：４０）,流速０．８ｍｌ．ｍｉｎ＾－１,检测波长２５４ｎｍ。结果：苯巴比妥在４～６０μｇ．ｍｌ＾－１浓度范围内线性良好（ｒ＝０．９９９８）,卡马西平在２～１６μｇ．ｍｌ＾－１浓度范围内线性良好（ｒ＝０．９９９５）,最低检测限分别为１１．５７ｎｇ．ｍｌ＾－１和４．９２ｎｍ．ｍｌ＾－１,两者高、中、低３种浓度的平均回收率分别为９９．９２％,１０１．３０％,９７．９２％和９９．４１％,１０１．５２％,９８．２２％（ｎ＝９）,日内ＲＳＤ分别为３．１％,２．６％,３．８％和１．９％,１．６     

反相高效液相色谱法同时测定血浆中苯妥英钠及其主要代谢产物的浓度

以非那西丁为内标，乙酸乙酯萃取血浆药物，甲醇－乙腈－水（４０：１０：５０，Ｖ／Ｖ）为流动相，紫外检测波长为２０３ｎｍ的色谱条件下，反相高效液相色谱法同时测定血浆中苯妥英钠及其主要代谢产物（对羟基苯妥英钠）。结果，血浆苯妥英钠及对羟基苯妥英钠在５￣３５μｇ／ｍｌ和２．５￣３０μｇ／ｍｌ的浓度范围内标准曲线线性关系良好，检测限分别为２．５μｇ／ｍｌ和１．５μｇ／ｍｌ（Ｓ／Ｎ＝３：１）。还测定了家兔口服     

反相高效液相色谱法测定血浆中左旋四氢巴马汀浓度

本文建立了测定血浆中左旋四氢巴马汀浓度的反相高效液相色谱法。采用国产ＹＷＧ－Ｃ１８填充柱，以甲醇－水（７０：３０）为流动相，安定作内标，在紫外检测器２８１ｎｍ波长处进行监测，按内标法定量。血样用二氯甲烷提取。最低检测浓度为０．０６μｇ／ｍｌ，线性范围０．６２５～４０μｇ／ｍｌ，日内日间测定的ＲＳＤ小于６．９％。     

高效液相色谱法同时测定苯巴比妥、苯妥英、卡马西平的血药浓度

目的:建立以高效液相色谱法同时测定苯巴比妥、苯妥英、卡马西平血药浓度的方法。方法:取患者血清,经二氯甲烷提取后在C18柱上分析,流动相为甲醇-水(57:43),柱温为30℃,检测波长为254nm,流速为0.8ml/min。结果:苯巴比妥、苯妥英、卡马西平分别在2.5-40、2.5-40、1.25-20μg/ml范围内线性关系良好,日内和日间RSD<10％(n=5)。结论:本方法简便、稳定,用于苯巴比妥、苯妥英、卡马西平的血药浓度监测效果良好。     

反相高效液相色谱法同时测定茶碱和3种抗癫痫药血药浓度

目的建立可同时测定茶碱和苯巴比妥、苯妥英钠、卡马西平血药浓度的反相高效液相色谱法。方法色谱柱为Promosil C18柱(150 mm×4.6 mm,5μm),流动相为甲醇-水(60∶40),检测波长205 nm,流速0.7 mL/min,柱温25℃。结果该色谱条件下,茶碱、苯巴比妥、苯妥英钠、卡马西平分离良好,血药浓度线性范围茶碱为0.30～180.00μg/mL(r=0.999 7),苯巴比妥为0.42～169.60μg/mL(r=0.999 9),苯妥英钠为0.40～170.00μg/mL(r=0.999 2),卡马西平为0.20～140.00μg/mL(r=0999 8),平均回收率分别是97.3%,98.0%,103.1%,99.3%,日内及日间精密度RSD均小于6%。结论该方法操作简便、快速、准确,4种药物互不干扰,可用于临床血药浓度监测。     

Evaluation of therapeutic drug level monitoring of phenobarbital, phenytoin and carbamazepine in Iranian epileptic patients

OBJECTIVE: The antiepileptic drugs phenobarbital, phenytoin and carbamazepine are widely used for the treatment of partial and tonic-clonic seizures. Large inter-individual differences in pharmacokinetics of these drugs, and the intermittent nature of epileptic attacks, increase the need for therapeutic drug level monitoring of these drugs. MATERIAL AND METHODS: In this study, data from the therapeutic drug monitoring of phenobarbital, carbamazepine and phenytoin in 328 epileptic patients were evaluated. Serum levels of drugs were determined in a University Department of Pharmacology by high-performance liquid chromatography. RESULTS: In this study, approximately 56% of patients were treated with 1; 30% with 2; and 14% with 3 antiepileptic drugs. In patients receiving 1 antiepileptic drug, the percentages treated with phenobarbital, carbamazepine and phenytoin were 41, 38 and 21%, respectively. In patients who received carbamazepine, serum levels in 40% of the patients were in the range of 4-8 microg/ml and more than in the range 4-12 microg/ml in 74% of the patients. In phenobarbital-treated patients, serum levels in 73% of the patients were in therapeutic range of 10-40 microg/ml, and about 44% of phenytoin-treated patients had serum levels in therapeutic range of 10-20 microg/ml. Approximately 50% of carbamazepine- and phenytoin-treated patients and approximately 70% of phenobarbital-treated patients were completely controlled. The frequency of concentrations within the therapeutic ranges decreased in patients using more than 1 antiepileptic drug. In patients who received both phenobarbital and sodium valproate, serum levels of phenobarbital were significantly (p < 0.05) greater than in patients who were taking this drug in combination with carbamazepine or phenytoin. CONCLUSION: Our results indicate that serum levels of antiepileptic drugs, and the percentage of patients with complete seizure control are comparable with results obtained in other populations in previous studies.     

Relationship between plasma high-density lipoprotein cholesterol and anticonvulsant levels in epileptics

Plasma high-density lipoprotein (HDL) cholesterol is a recognized negative risk factor for coronary heart disease. In this study, therapy with phenytoin alone, carbamazepine alone, and phenytoin in combination with phenobarbital was associated with elevated plasma HDL cholesterol concentrations. The highest HDL cholesterol levels were seen in subjects treated with the combination of phenytoin and phenobarbital. Plasma HDL cholesterol levels were proportional to the serum phenobarbital and carbamazepine concentrations. In subjects treated with phenytoin alone, low plasma HDL cholesterol levels were associated with low drug concentrations. The results suggest direct links running from the serum anticonvulsant levels to the extent of hepatic microsomal enzyme induction, and further to the plasma HDL cholesterol concentrations.     

HPLC法同时测定苯巴比妥、苯妥英、卡马西平和茶碱血药浓度

目的建立同时测定血清中茶碱（TPH）、苯巴比妥（PBB）、苯妥英（PHT）和卡马西平（CBZ）浓度的HPLC法。方法取患者血清,经二氯甲烷提取后岛津LC-20A Prodigy ODS C18柱（4.6mm×250mm,5μm）上分析,流动相为甲醇-水（48：52）,流速为1.0mL.min-1,检测波长为254nm,柱温40℃。结果 TPH,PB,PT和CBZ的线性范围和相关系数分别为0.26～80.88（0.9994）,1.0～80.56（0.9992）,1.02～81.6（0.9990）,0.27～40.22（0.9996）μg.mL-1,最低检测1浓度分别为0.09,0.20,0.24,0.03μg.mL-1,方法回收率分别为99.0%,99.4%,100.3%,99.6%日内、日间RSD均〈3.0%,绝对回收率分别为91.9%,95.4%,92.3%,95.9%,RSD均〈2%。结论该方法简便、灵敏、实用、有良好的分离效果,适用于治疗药物监测及药物动力学实验。     

Microdialysis sampling of carbamazepine,phenytoin and phenobarbital in subcutaneous extracellular fluid and subdural cerebrospinal fluid in humans: an in vitro and in vivo study of adsorption to the sampling device

The purpose of the study was to determine if binding of the drugs to the sampling equipment during microdialysis would influence the results for carbamazepine, phenytoin and phenobarbital. In vitro experiments with microdialysis catheters and separate parts of catheters were performed to estimate the degree of drug binding to the dialysis equipment. A mathematical model to calculate drug binding and recovery is proposed. In vivo protein unbound carbamazepine concentrations in subcutaneous extracellular fluid at different flow rates (6 patients), unbound carbamazepine (1 patient) and unbound phenobarbital (I patient) in subdural cerebrospinal fluid and subcutaneous extracellular fluid were estimated and the in vivo data were compared to the in vitro results and data generated by the mathematical model. Binding to the soft outlet polyurethane tubing was extensive and variable for phenytoin, which precluded in vivo testing, but limited and more predictable for carbamazepine and phenobarbital. None of the three compounds bound to the hard internaltubing. Phenytoin and phenobarbital did not bind to the dialysis membrane, while a small degree of binding may be present for carbamazepine. In vivo estimates of carbamazepine protein unbound subcutaneous extracellular concentrations by microdialysis, adjusted for binding to the plastic tubing, were 81% of protein unbound plasma concentrations. In single case studies, subdural cerebrospinal fluid and subcutaneous extracellular levels of carbamazepine and phenobarbital were similar and when corrected for binding to the plastic tubings they were also close to protein unbound plasma concentrations. Microdialysis can be used for reliable estimations of protein unbound carbamazepine and possibly phenobarbital concentrations when drug binding to the plastic tubing is considered. Reliable estimation of unbound phenytoin is not possible at present.     

比较RP-HPLC法与荧光偏振免疫法测定苯巴比妥、卡马西平、苯妥英钠在癫痫患者的血药浓度

目的 建立同时测定人血清苯巴比妥、卡马西平、苯妥英钠(抗癫痫药)浓度的RP-HPLC法,并与荧光偏振免疫法测定方法比较.方法 以乙酸乙酯-二氯甲烷(4:1)为提取溶剂、氯硝西泮为内标,固定相为Diamonsil C18柱(250mm×4.6 mm,5 μm),流动相为乙腈-水(35:65),紫外检测波长285 nm,流量1.0 mL·min-1;收集患者服药后稳态谷浓度样本,分别用2种方法测定,考察2种方法的相关性.结果 3种药物均具有良好的线性范围,日内、日间RSD均小于8%;苯妥英钠、苯巴比妥2种方法的测定值无统计学差异(P>0.05);而卡马西平2种测定方法比较有统计学差异(P<0.01),卡马西平RP-HPLC法测定值较低.结论 RP-HPLC法适用于治疗药物浓度监测.     

Effect of 4-(4-bromophenyl)-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione on the anticonvulsant action of different classical antiepileptic drugs in the mouse maximal electroshock-induced seizure model

The aim of this study was to determine the effects of 4-(4-bromophenyl)-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (TP4-a new S-triazole derivative possessing anticonvulsant properties in preclinical studies) on the protective action of four different classical antiepileptic drugs (carbamazepine, phenobarbital, phenytoin and valproate) against maximal electroshock-induced seizures in mice. Results indicate that TP4 administered intraperitoneally at doses of 75 and 100 mg/kg significantly elevated the threshold for electroconvulsions in mice. TP4 at doses of 12.5, 25, 37.5 and 50 mg/kg had no impact on the threshold for electroconvulsions in mice. Moreover, TP4 (50 mg/kg) significantly enhanced the anticonvulsant activity of carbamazepine, phenobarbital and valproate, but not that of phenytoin in the maximal electroshock seizure test in mice. TP4 at 25 mg/kg significantly potentiated the anticonvulsant action of carbamazepine, but not that of phenobarbital, phenytoin and valproate in the mouse maximal electroshock-induced seizure model. Pharmacokinetic experiments revealed that TP4 significantly elevated total brain concentrations of carbamazepine and valproate, having no impact on total brain concentrations of phenobarbital in mice. In conclusion, the enhanced anticonvulsant action of phenobarbital by TP4 was probably pharmacodynamic in nature and, therefore, the combination of TP4 with phenobarbital is worthy of consideration while extrapolating the results from this study into clinical settings. The enhanced anticonvulsant action of carbamazepine and valproate by TP4 in the mouse maximal electroshock-induced seizure model was associated with pharmacokinetic increases in total brain concentrations of the antiepileptic drugs in mice. The combination of TP4 with phenytoin was neutral from a preclinical point of view.     

Microdialysis Sampling of Carbamazepine,Phenytoin and Phenobarbital in Subcutaneous Extracellular Fluid and Subdural Cerebrospinal Fluid in Humans: An in vitro and in vivo Study of Adsorption to the Sampling Device

Abstract: The purpose of the study was to determine if binding of the drugs to the sampling equipment during microdialysis would influence the results for carbamazepine, phenytoin and phenobarbital. In vitro experiments with microdialysis catheters and separate parts of catheters were performed to estimate the degree of drug binding to the dialysis equipment. A mathematical model to calculate drug binding and recovery is proposed. In vivo protein unbound carbamazepine concentrations in subcutaneous extracellular fluid at different flow rates (6 patients), unbound carbamazepine (1 patient) and unbound phenobarbital (1 patient) in subdural cerebrospinal fluid and subcutaneous extracellular fluid were estimated and the in vivo data were compared to the in vitro results and data generated by the mathematical model. Binding to the soft outlet polyurethane tubing was extensive and variable for phenytoin, which precluded in vivo testing, but limited and more predictable for carbamazepine and phenobarbital. None of the three compounds bound to the hard internal tubing. Phenytoin and phenobarbital did not bind to the dialysis membrane, while a small degree of binding may be present for carbamazepine. In vivo estimates of carbamazepine protein unbound subcutaneous extracellular concentrations by microdialysis, adjusted for binding to the plastic tubing, were 81% of protein unbound plasma concentrations. In single case studies, subdural cerebrospinal fluid and subcutaneous extracellular levels of carbamazepine and phenobarbital were similar and when corrected for binding to the plastic tubings they were also close to protein unbound plasma concentrations. Microdialysis can be used for reliable estimations of protein unbound carbamazepine and possibly phenobarbital concentrations when drug binding to the plastic tubing is considered. Reliable estimation of unbound phenytoin is not possible at present.     

Chronically administered fluoxetine enhances the anticonvulsant activity of conventional antiepileptic drugs in the mouse maximal electroshock model

Interactions between chronically administered fluoxetine and valproate, carbamazepine, phenytoin, or phenobarbital were studied in the maximal electroshock test in mice. Fluoxetine administered for 14 days at doses up to 20 mg/kg failed to affect the electroconvulsive threshold. Nevertheless the drug (at 15 and 20 mg) enhanced the anticonvulsant activity of valproate, carbamazepine, and phenytoin. When applied at 20 mg/kg, it potentiated the protective action of phenobarbital. Fluoxetine, antiepileptic drugs, and their combinations did not produce significant adverse effects evaluated in the chimney test (motor coordination) and passive-avoidance task (long-term memory). Chronically applied fluoxetine significantly increased the brain concentrations of valproate, carbamazepine, phenobarbital and phenytoin, indicating a pharmacokinetic contribution to the observed pharmacodynamic interactions. In conclusion, long-term treatment with fluoxetine exhibited some favorable effects on the anticonvulsant properties of conventional antiepileptic drugs, resulting, however, from pharmacokinetic interactions.