Simplified high performance liquid chromatographic method for the determination of clonazepam and other benzodiazepines in serum

A convenient and sensitive high performance liquid chromatographic method was developed for determination of clonazepam in serum using a C-18 reverse-phase column, and mobile phase consisting of a 50:35:15 mixture by volume of pH 6.0 phosphate buffer:methanol:acetonitrile. Quantitation was performed at 313 nm with flunitrazepam as the internal standard. Using 1 ml of serum for extraction, the assay is linear for clonazepam concentrations between 10 and 250 ng/ml. The relative recovery averaged 100.3%, and the coefficient of variation for between-day and within-day assays was less than 7%. A simple modification permits analysis of 200 microliter of serum, with little loss of precision and with a detection limit of 20 ng/ml. Only three drugs tested (nitrazepam, methaqualone, and norchlordiazepoxide) interfered with the assay, and none are likely to be used therapeutically with clonazepam. Importantly, carbamazepine and carbamazepine-10,11-epoxide do not interfere under the conditions of the assay. The method is equally suitable for the determination of nitrazepam. By adjusting the mobile phase so that volume ratios of phosphate buffer, methanol, and acetonitrile are 45:35:20, and using nitrazepam as the internal standard, seven other benzodiazepines (demoxepam, oxazepam, chlordiazepoxide, norchlordiazepoxide, temazepam, diazepam, and nordiazepam) can be resolved at 254 nm.     

Determination of benzodiazepines in oral fluid using LC-MS-MS

A rapid, simple, highly sensitive procedure for the simultaneous analysis of bromazepam, alprazolam, clonazepam, lorazepam, oxazepam, diazepam, midazolam, flurazepam, flunitrazepam, nordiazepam, triazolam, temazepam, nitrazepam, and chlordiazepoxide in oral fluid is described, using liquid chromatography coupled to a triple quadrupole mass spectrometer in positive electrospray mode. Benzodiazepines in oral fluid samples (1 mL) were analyzed using the Quantisal collection device, quantified using solid-phase extraction, and detected using liquid chromatography with tandem mass spectrometric detection. For confirmation, two transitions were monitored and one ion ratio determined, which was within 20% of the ratio for known calibration standards. The limits of quantitation ranged from 0.5-5 ng/mL of neat oral fluid; the intraday precision of the assays (n=5) ranged from 2.8-7.29%; and the interday precision ranged from 1.42-6.8% (n=5). The percentage recovery of the drugs from the collection pads ranged from a low of 81.4% for midazolam to the highest of 90.17% for nitrazepam. The procedure can be applied to authentic oral fluid specimens.     

高效毛细管电泳分析血清中头孢曲松钠

建立了一种简单、灵敏的高效毛细管电泳分析犬血清中头孢曲松钠的方法。用乙腈为蛋白沉淀剂,硼酸盐为缓冲体系,以苯甲酸为内标物。电泳条件：检测波长２５４ｎｍ ,非涂渍石英毛细管３７ｃｍ ×７５μｍ（ｉ．ｄ．）,电压１０ｋＶ,自动压力进样５ｓ,电泳时间４ｍ ｉｎ。结果：头孢曲松钠测定的线性范围为１～１０－ ４ｍ ｇ／ｍｌ,最低检测限为５×１０－ ６ｍ ｇ／ｍ ｌ;本法的ＲＳＤ日内和ＲＳＤ日间分别为１．１％ 和１．２％ ;头孢曲松代谢产物及血清中的其他成分对分析无干扰。     

The relationship between peak velocity of saccadic eye movements and serum benzodiazepine concentration.

1 Six healthy male volunteers received single oral doses of 10 mg diazepam, 20 mg temazepam, 15 mg flurazepam, 5 mg nitrazepam, 10 mg desmethyl-diazepam and placebo in a double-blind randomized fashion. 2 Peak velocity of saccadic eye movements, serum benzodiazepine concentration, and subjective ratings of wakefulness and co-ordination were measured at intervals up to 12 h after drug administration. 3 All active treatments produced a statistically significant decrease in peak saccadic velocity. The effect of temazepam and diazepam was generally more pronounced than that of flurazepam, nitrazepam and desmethyl-diazepam. 4 There were log-linear correlations between peak saccadic velocity and serum benzodiazepine concentration after ingestion of temazepam, diazepam and nitrazepam. 5 These results demonstrate a clear relationship between serum benzodiazepine concentration and its effect on a convenient measure of brainstem reticular formation function.     

Determination of temazepam and related compounds in capsules by high-performance liquid chromatography

A reversed-phase, high-performance liquid chromatographic assay method is described for temazepam hard gelatin and soft gelatin capsule analysis. The method is simple, specific, accurate, fast, and stability indicating. A reversed-phase octylsilane (C8) column with a mobile phase composed of methanol:1% acetic acid and detection at 254 nm separated sulfanilamide (internal standard), temazepam, synthetic precursor, and possible degradation products. Detector responses showed linearity to temazepam concentrations over the range 0.075-0.60 mg/mL (r = 0.9999). Mean recovery of temazepam added to capsule excipients was 100.3%. Mean assay results for 15- and 30-mg hard gelatin capsules were 101.5 and 101.3%, respectively. Mean assay results for 10- and 20-mg soft elastic gelatin capsules were 101.1 and 101.5%, respectively.     

A validated high-performance liquid chromatographic method for the determination of glibenclamide in human plasma and its application to pharmacokinetic studies

Glibenclamide is a potent second generation oral sulfonylurea antidiabetic agent widely used for the treatment of type II diabetes melitus. A rapid, sensitive, precise, accurate and specific HPLC assay for the determination of glibenclamide in human plasma was developed and validated. After addition of flufenamic acid as internal standard, the analytes were isolated from human plasma by liquid-liquid extraction. The method was linear in the 10-400 ng/ml concentration range (r > 0.999). Recovery for glibenclamide was greater than 91.5% and for internal standard was 93.5%. Within-day and between-day precision, expressed as the relative standard deviation (RSD%), ranged from 1.4 to 5.9% and 5.8 to 6.6%, respectively. Assay accuracy was better than 93.4%. The assay was used to estimate the pharmacokinetics of glibenclamide after oral administration of a 5 mg tablet of glibenclamide to 18 healthy volunteers.     

反相高效液相法测定血清中茶碱的浓度

目的建立反相高效液相色谱法（RP-HPLC）测定血清中的茶碱浓度的方法。方法以莘巴比妥为内标．血清样品用二氯甲烷提取。采用Venusil XBP-C18柱（4．6×200mm,5μm）分析。流动相为甲醇：水（50：50）。流速1．0mL·min^-1,检潮波长275nm,柱漫25℃。结果本法在6．25～25μg·mL^-1间线性关系良好。血清中茶碱的标准曲线方程为y=1．1386x-0．8262,r=0．9991,RSD 1．18％（n=5）,置低检测质量彀度为0．31μg·mL^-1。日内、日间精密度RSD均小于5％。茶碱的回收率为91．89％～105．15％。结论该方法灵敏度高、准确、重复性好。可以用干常规茶碱血药浓度的测定。     

Validation of a method for the determination of zolpidem in human plasma using LC with fluorescence detection

A sensitive and selective high-performance liquid chromatography (HPLC) method was developed for the determination of zolpidem in human plasma. Zolpidem and the internal standard (trazodone) were extracted from human plasma that had been made basic. The basic sample was loaded onto a conditioned Bond Elut C18 cartridge, rinsed with water and eluted with methanol. Forty microliters were then injected onto the LC system. Separation was achieved on a C18 column (150 x 4.6 mm, 5 microm) with a mobile phase composed of acetonitrile:50 mM potassium phosphate monobasic at pH 6.0 (4:6, v/v). Detection was by fluorescence, with excitation at 254 nm and emission at 400 nm. The retention times of zolpidem and internal standard were approximately 4.7 and 5.3 min, respectively. The LC run time was 8 min. The assay was linear in concentration range 1-400 ng/ml for zolpidem in human plasma. The analysis of quality control samples for zolpidem (3, 30, and 300 ng/ml) demonstrated excellent precision with relative standard deviations (RSD) of 3.7, 4.6, and 3.0%, respectively (n = 18). The method was accurate with all intraday (n = 6) and overall (n = 18) mean concentrations within 5.8% from nominal at all quality control sample concentrations. This method was also performed using a Gilson Aspec XL automated sample processor and autoinjector. The samples were manually fortified with internal standard and made basic. The aspec then performed the solid phase extraction and made injections of the samples onto the LC system. Using the automated procedure for analysis, quality control samples for zolpidem (3, 30, and 300 ng/ml) demonstrated acceptable precision with RSD values of 9.0, 4.9, and 5.1%, respectively (n = 12). The method was accurate with all intracurve (n = 4) and overall (n = 12) mean values being less than 10.8% from nominal at all quality control sample concentrations.     

Adaptation of solid phase extraction to an automated column switching method for online sample cleanup as the basis of a facile and sensitive high-performance liquid chromatographic assay for paclitaxel in human plasma

An improved method for assaying paclitaxel in human plasma by high-performance liquid chromatography (HPLC) with UV detection at 227 nm has been developed by adapting previously reported sample preparation methods and chromatographic conditions to facilitate semi-automated sample cleanup using a column switching technique. Manual sample manipulations were limited to isolating the drug and internal standard from plasma (1.0 ml) by liquid-liquid extraction using tert-butyl methyl ether. The sample extract was initially loaded onto a short cartridge column containing a cyanopropyl stationary phase. During the predetermined time interval that the drug and internal standard eluted from the cartridge, 1.50-2.20 min, a fully automated 6-position switching valve was used to direct the effluent onto an octylsilica analytical column. The same mobile phase, composed of acetonitrile-methanol-ammonium acetate buffer (pH 5.0; 20 mM) (76:19:105, v/v/v) and delivered at flow rate of 1.0 ml/min, was used for both separations. The overall retention times of paclitaxel and the internal standard were 10.9 and 18.1 min, respectively. The analytical method was thoroughly validated for quantitating paclitaxel in plasma at concentrations ranging from 6 to 586 nM (5-500 ng/ml). The lowest concentration of paclitaxel measured with acceptable day-to-day accuracy (100.2%) and precision (RSD 11.7%, n = 21, 5 months) was 6 nM (5 ng/ml). The sensitivity and selectivity of the assay proved to be more than adequate for monitoring steady-state plasma concentrations of the drug when administered to cancer patients as a 96 h continuous intravenous infusion in combination with other anticancer agents, such as doxorubicin and topotecan. Moreover, the heart-cutting procedure prevented the problematic introduction of interfering nonpolar plasma components onto the analytical column, thereby enhancing sample throughput while decreasing the technical demands of the assay. The method was found to be extremely reproducible and robust during extended use for the routine analysis of plasma specimens acquired from several clinical trials.     

The simultaneous determination of diazepam and its three metabolites in dog plasma by high-performance liquid chromatography with mass spectroscopy detection

A fast, sensitive and specific LC/MS/MS method for the simultaneous determination of diazepam and its three metabolites, oxazepam, temazepam and desmethyldiazepam, in dog plasma is described. The method consists of an automated 96-well solid phase extraction procedure and electrospray LC/MS/MS analysis. D(5)-Diazepam is used as the internal standard for all the compounds. Intra-day and inter-day assay coefficients of variations are less than 12.7%. The lower limit of quantitation (LLOQ) is 1 nM for each analyte, based on 0.1 ml aliquots of dog plasma. The analytical run time was 5 min. Linearity is observed over the range of 1--500 nM. This method has been used to support the discovery of pharmacokinetic studies.     

A robotics-based liquid chromatographic assay for the measurement of atovaquone in plasma

A precise and specific robotics-based liquid chromatographic (LC) method for measuring atovaquone concentrations in plasma was developed and validated, and the method was compared with an existing manual LC method. The compound was isolated from plasma by liquid-liquid extraction, separated by reversed-phase LC, and quantitated against an internal standard with UV detection. Least-squares linear regression with l/concentration² weighting was used as the calibration model. The range of the calibration curve for the assay under routine conditions was 0.25–50 μg ml⁻¹. No endogenous interferences with the compound or the internal standard were noted in either untreated human plasma or in plasma from patients enrolled in Phase III clinical trials of atovaquone. The accuracy of the assay (determined as the percent bias) ranged from −4.8% to −9.4% in the validation runs. The intra- and interassay precisions (determined as the relative standard deviation) were less than 6.8% and 6.4%, respectively. The contribution of an internal standard on assay accuracy and precision also was examined. Interassay variability was marginally improved by the incorporation of an internal standard to the assay; accuracy and intra-assay precision were essentially unchanged. A paired t-test between estimates of atovaquone concentrations in healthy volunteer and HIV + patient human plasma samples assayed by the automated and manual methods demonstrated no significant difference (p = 0.31) between the values determined by each method.     

高效液相色谱-质谱联用法测定大鼠血浆中五味子醇乙的浓度及其应用

目的 建立快速、灵敏的高效液相色谱-质谱法测定大鼠血浆中五味子醇乙的浓度,并研究其在大鼠体内的药动学变化规律.方法 以地西泮为内标,血浆样品经乙醚萃取后进行高效液相色谱-质谱分析,采用Hypersil-C18色谱柱(150mm×4.6mm,5μm),甲醇-水[72:28)为流动相,检测离子为399.00(五味子醇乙[M+H-H2O]+),284.90(内标地西泮[M+H]+.结果 血浆标准曲线线性范围为5·0～250 ng/mL(r=0.995 7),最低定量下限为5.0 ng/mL.五味子醇乙的提取回收率不小于78.80%,高、中、低3种浓度的日内、日间精密度RSD均小于15%(n=6),结论该方法选择性强、灵敏度高,适用于大鼠血浆中五味子醇乙浓度测定和临床药动学研究.     

Liquid chromatographic assay of ivermectin in human plasma for application to clinical pharmacokinetic studies

There is a need for an accurate, sensitive and selective high-performance liquid chromatography (HPLC) method for the quantitation of ivermectin in human plasma that separates the parent drug from metabolites. Ivermectin and the internal standard, moxidectin, were extracted from 0.2 ml of human plasma using Oasis HLB solid phase extraction cartridges. After extraction, fluorescent derivatives of ivermectin and moxidectin were made by reaction with trifluoroacetic anhydride and N-methylimidazole. Separation was achieved on a Alltech Ultrasphere C18 5mu column with a mobile phase composed of tetrahydrofuran-acetonitrile-water (40:38:22 v/v/v). Detection is by fluorescence, with an excitation of 365 nm and emission of 475 nm. The retention times of ivermectin and internal standard, moxidectin are approximately 24.5 and 12.5 min, respectively. The assay is linear over the concentration range of 0.2-200 ng/ml of ivermectin in human plasma (r = 0.9992, weighted by 1/concentration). Recoveries of ivermectin are greater than 80% at all concentrations. The analysis of quality control samples for ivermectin 0.2, 25, and 200 ng/ml demonstrated excellent precision with coefficient of variation of 6.1, 3.6 and 2.3%, respectively (n = 6). The method is accurate with all intra-day (n = 6) and interday (n = 12) mean concentration within 10% of nominal values at all quality control sample concentrations. Storage stability for 30 days at -80 degrees C and after three freeze-thaw cycles are within acceptable limits. The method separates ivermectin from multiple less and more polar unidentified metabolites. This method is robust and suitable for clinical pharmacokinetic studies. The analytical procedure has been applied to a pharmacokinetic study of ivermectin in healthy volunteers and to the analysis of plasma specimens from patients with disseminated strongyloidiasis.     

Determination of celecoxib in human plasma by high-performance liquid chromatography

A high performance liquid chromatographic method for the quantitation of celecoxib (CEL) in human plasma is presented. The method is based on liquid-liquid extraction with chloroform and reversed-phase chromatography using a Nucleosil CN column (250 mm x 4.6 mm i.d., 5 microm particle size) and UV spectrophotometer detection at 260 nm. The mobile phase consists of acetonitrile:water (60:40 (v/v)). Flutamide was used as internal standard (IS). The assay was linear in the concentration range of 10-1000 ng/ml when 0.5 ml aliquots of plasma were extracted. Within-day and between-day precision expressed by relative standard deviation is less than 4% and inaccuracy does not exceed 3%. The assay was used to analyze samples collected during human clinical studies.     

Assay of olanzapine in human plasma by a rapid and sensitive gas chromatography-nitrogen phosphorus selective detection (GC-NPD) method: validation and comparison with high-performance liquid chromatography-coulometric detection

A gas chromatography-nitrogen phosphorus selective detection (GC-NPD) method with a simple 1-step sample preparation was developed for the assay of the antipsychotic drug olanzapine in plasma. Within a time of analysis of 7 minutes, an HP-5 fused-silica capillary (25 m x 0.2 mm ID, 0.33-microm film thickness, 0.7 mL N2 as carrier gas) provided selectivity with respect to about 30 psychotropic drugs and the internal standard ethylolanzapine. Calibration was linear between 1 and 50 ng/mL and crossed the origin (LOD = 0.3 ng/mL). Intraday precision was 6.7%, 2.7%, and 1.4% at plasma concentrations of 1, 5, and 50 ng/mL, respectively. Interday precision was 4.6% at 20 ng/mL. Accuracy in commercial interlaboratory tests was 108.7% and 88.5%. The method also provided good accuracy in comparison with an HPLC method for patient samples (slope 1.003, r = 0.953) and spiked samples (slope 0.881, r = 0.998). GC-NPD with a simple sample preparation is regarded as an alternative for the assay of olanzapine plasma concentrations in therapeutic drug monitoring (TDM) and in pharmacokinetic studies. Smokers and patients taking concomitant carbamazepine had reduced plasma concentrations of olanzapine. Women and patients older than 60 years had increased plasma olanzapine concentrations.     

Gas chromatographic assay of diethylcarbamazine in human plasma for application to clinical pharmacokinetic studies

A sensitive and selective gas chromatography method using flame ionization detection was developed for the determination of diethylcarbamazine (DEC) in human plasma. DEC and the internal standard, 1-diethylcarbamyl-4-ethyl piperazine HCl (E-DEC), were extracted from human plasma after loading onto a conditioned C(18) solid phase extraction cartridge, rinsed with water and eluted with methanol. After evaporation under a stream of nitrogen and reconstitution in methanol, 3 microl were injected onto the GC system. Separation was achieved on a A Heliflex(R) AT-35 capillary column (length 30 m, internal diameter 0.32 mm). Gas flow rates were: hydrogen, 35 ml/min; carrier gas (helium), 1.5 ml/min, make-up gas (helium), 25 ml/min; and air 420 ml/min. The retention times of DEC and internal standard were approximately 5.5 and 7.28 min, respectively. The GC run time was 22 min. The assay was linear in concentration range 100-2000 ng/ml for DEC in human plasma. The analysis of quality control samples for DEC (120, 1000, 2000 ng/ml) demonstrated excellent precision with coefficients of variation of 4.5,1.3, and 1.6%, respectively (n=6). The method was accurate with all intra-day (n=6) and inter-day (n=12) mean concentrations within 4.3% from nominal at all quality control sample concentrations. DEC was found to be stable after 3 freeze-thaw cycles, and with storage at -20 degrees C for 12 weeks. The method is currently being used for pharmacokinetic studies of DEC in healthy volunteers.     

LC—ESI—MS／MS法快速测定人血浆中环丙沙星的浓度

建立测定人血浆中环丙沙星浓度的高效液相色谱串联质谱电喷雾法（LC-ESI-S／MS）。方法以Agilent ZORBAX C18反相柱（Agilent ZORBAX TC-C18 column,4．6mm×150mm,5μm）为色谱柱,流动相为乙腈（含1％甲酸）-0．02mol·L^-1甲酸铵水溶液：90：10（V：V）,流速为0．8mL·min^-1,柱温：25℃,采用乙腈沉淀簧白法。样品经电喷雾离子源正离子化后,通过三重四级杆串联质谱仪,采用选择反应监测（SRM）对环丙沙星（rdz332．2—314．2）和洛美沙星（m／z352．2→265．1）进行测定。并用此法测定20例健康受试者单次口服500mg后的血药浓度。结果环丙沙星的高（2500μg·L^-1）、中（250μg·L^-1）、低（10μg·L^-1）3个浓度的平均回收率分别为101．22％、102．31％和93．19％,日内（n=5）、日间（n=3）RSD均小于15％;分析方法的最低定量限为5μg·L^-1。线性范围为：5～5000μg·L^-1,回归方程为：F=30692．75ρ-0．0014,r=0．997（n=8）．权重为1／ρ^2。结论该方法灵敏、准确、简单、快速．可用于临床血浓监测和药动学研究。     

A capillary gas chromatographic assay with nitrogen phosphorus detection for the quantification of topiramate in human plasma, urine and whole blood

An accurate and robust method involving liquid liquid extraction and capillary gas chromatographic (GC) assay with nitrogen phosphorus detection (NPD) was developed and validated for the quantitative determination of topiramate [2,3:4,5-bis-O-(-1-methylethylidene)-beta-D-fructopyranose sulfamate], Topamax, an anticonvulsant drug, in human plasma, urine, and whole blood. The galactopyranose analog of topiramate was used as the internal standard. A DB-5, fused silica capillary column (J&W Scientific, Folsom, CA) was used, yielding typical retention times of 4.95 min for topiramate and 5.32 min for the internal standard in human plasma. The assay involved organic extraction with methyl t-butyl ether (MTBE) from base, a back extraction into acid and a second extraction in MTBE. The organic solvent was evaporated, and the residue was redissolved and injected for analysis. The standard curve was validated from 0.5 to 50 microg/ml(-1) for human plasma and whole blood, and from 1.0 to 50 microg/ml(-1) for urine. Peak area ratios of drug to internal standard were determined and used to construct a standard curve. The resulting chromatograms showed no endogenous interfering peaks with the respective blank human fluids. Chromatograms corresponding to topiramate and the internal standard produced sharp peaks that were well resolved. This assay showed precision and accuracy of < or = 5%. Two minor human metabolites of topiramate did not interfere with the assay. This assay was successfully applied to determine the pharmacokinetics of topiramate during the development of this drug.     

Simultaneous determination of fexofenadine and its related compounds by HPLC

A simple reversed phase liquid chromatographic (RPLC) method has been developed and subsequently validated for the determination of fexofenadine hydrochloride and its related compounds A and B. The method utilizes a C8 column for the separation and determination of meta-isomer (related compound B). The separation was achieved using an Eclipse XDB C8, 5 microm, 4.6 x 150 mm column and a mobile phase comprising 1% triethylamine phosphate (pH 3.7), acetonitrile and methanol in the ratio 60:20:20 (v/v/v). 5-Methyl 2-nitrophenol has been used as internal standard for the purpose of quantitation of fexofenadine. The described method was linear over a range of 0.7-18.7 microg/ml for related compounds A and B and 60-750 microg/ml for assay of fexofenadine. The relative standard deviation (n=3) was 0.5% for the drug and 3.4% for related compounds. The intermediate precision was 0.79% (n=9) for assay and 5.16% (n=9) for related impurities. The mean recovery of both the related compounds were in the range of 94-103%. Limits of detection (LOD) and quantification (LOQ) for the related compounds A and B were 0.18, 0.12 and 0.56, 0.48 microg/ml, respectively. The precision of the method was checked by F-test using a reported method as reference and the calculated value (1.35) was found to be less than the table value at 95% confidence levels. The obtained results confirm that the method is highly suitable for its intended purpose.     

Simultaneous high-performance liquid chromatographic assay of furosemide and propranolol HCL and its application in a pharmacokinetic study

A practical, sensitive, selective and efficient reversed-phase high-performance liquid chromatographic (HPLC) method is reported for the determination of two commonly used antihypertensive drugs, furosemide and propranolol hydrochloride. The drugs were eluted through a Nucleosil C(18) column with a mobile phase composed of 0.02 M potassium dihydrogen phosphate and acetonitrile (80:20, v/v) adjusted to pH 4.5 and the effluent from the column was monitored at 235 nm. The present method enabled simple and isocratic HPLC with UV detection of these drugs in raw materials and in pharmaceutical formulations. These procedures were also applied for the assay of furosemide in rabbits' plasma, using propranolol hydrochloride as an internal standard. The linear concentration range of the assay was 0.1-200 and 5-200 microg ml(-1) for furosemide and propranolol hydrochloride, respectively. The inter and intra-day assay precision and accuracy showed reproducibility and good linearity (r(2)>0.99). The method retained its accuracy and precision when applying the standard addition technique. The results obtained by applying the proposed method was statistically analysed and compared with those obtained by the reported methods.