Determination of endothelin antagonist BMS182874 in plasma by high-performance liquid chromatography

A simple and rapid high performance liquid chromatography (HPLC) method was developed for the determination of BMS182874 (BMS) in mouse plasma. The drug was extracted from plasma by a liquid-liquid extraction process. The method consists of reversed-phase chromatography using a Thermo Hypersil-Keystone RP-18 5 microm, 250 x 2.1 mm column and UV spectrophotometer detection at 255 nm. The mobile phase consists of 45% (v/v) acetonitrile: 55% (v/v) trifluoroacetic acid (0.015% v/v; pH 3.0) at a flow rate of 0.6 ml/min. Validity of the method was studied and the method was precise and accurate with a linearity range from 100 ng/ml to 1000 ng/ml. The extraction efficiency was found to be 81, 84 and 87% for 100, 500 and 1000 ng/ml, respectively for spiked drug in plasma. The limit of quantification and limit of detection were found to be 50 and 10 ng/ml, respectively in plasma. Within-day and between-day precision expressed by relative standard deviation was less than 4% and inaccuracy did not exceed 4%. The assay was also used to analyze samples collected during animal studies. The suitability and robustness of the method for in vivo samples were confirmed by analysis of BMS from mouse plasma and tissues dosed with BMS.     

A validated method for the determination of verapamil and norverapamil in human plasma

The aim of the study was to develop a simple and sensitive analytical method to determine verapamil (V) and its metabolite norverapamil (N) in human plasma with use of an HPLC isocratic system with fluorescence detection, following fast extraction of the investigated compounds. Extraction recovery was 92.12% and 89.58% for V and N, respectively. Internal standard in HPLC was propranolol. Its recovery was 82.50% on the average. Limit of detection was 0.924 ng/ml and limit of determination was 3,080 ng/ml for V, what corresponds concentration in plasma 1.232 ng/ml. For N limit of detection was 0.030 ng/ml and limit of determination was 1.001 ng/ml what corresponds 0,4 ng/ml in plasma. Parameters of validation prove that precision of the presented method is very good. The method is fast and one chromatogram separation lasts about 8 minutes. 30-40 manual (without autosampler) analyses per day were done. It was used successfully in pharmacokinetic and bioavailability studies of verapamil administration in drug formulations alternative to tablets: buccal and flotation ones.     

A rapid reversed phase high performance liquid chromatographic method for determination of etoposide (VP-16) in human plasma

A rapid, simple and sensitive isocratic High Performance Liquid Chromatography (HPLC) method was developed to measure the concentration of etoposide in plasma samples with UV detection at 220 nm. The method uses a Bondapac C18 column at 60 degrees C. The mobile phase consists of Methanol: water (45:55 v/v) at a flow rate of 2.8 ml/min. Phenacetin was used as an internal standard. The plasma samples were extracted using ether with the organic layer evaporated under nitrogen. The residue was dissolved in 200 microl methanol with 20 microl injected into the HPLC column. The extraction method showed a recovery of 91.5+/-3% for etoposide. In this system, the retention time of phenacetin and etoposide were 3.3 and 4.4 min, respectively. The limit of detection of etoposide in plasma is 20 ng/ml and the limit of quantitation is 40 ng/ml. This analytical method has very good reproducibility (8.1% between-day variability at a concentration of 50 ng/ml). It is a fast, sensitive and economic method applicable for clinical and pharmacokinetic studies.     

高效液相色谱法测定法莫替丁的血药浓度

目的 :建立HPLC法测定法莫替丁的血药浓度。方法 :采用Nova_PakC18(3 9×150mm ,5μm )色谱柱 ,柱温35℃ ;乙腈 -醋酸铵缓冲液 (6∶94)为流动相 ;流速1 1ml/min ;检测波长266nm。血浆样品经液 -液萃取处理。结果 :法莫替丁的血药浓度在12 5～300ng/ml范围内 ,与其峰面积有良好的线性关系 (γ=0 9996)。最低检测浓度8ng/ml ,方法的平均回收率102 39 % ,日内精密度≤5 00 % ,日间精密度≤6 59 %。结论 :该方法灵敏、准确、经济 ,可用于法莫替丁的药代动力学及生物利用度研究。     

Hydroxyzine from topical phospholipid liposomal formulations: Evaluation of peripheral antihistaminic activity and systemic absorption in a rabbit model

Hydroxyzine, an effective but sedating H₁-antihistamine is given orally to treat allergic skin disorders. This study was performed to assess the peripheral H₁-antihistaminic activity and extent of systemic absorption of hydroxyzine from liposomes applied to the skin. Using L-α-phosphatidylcholine (PC), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs) containing hydroxyzine were prepared. Hydroxyzine in Glaxal Base (GB) was used as the control. Using a randomized, crossover design, each formulation, containing 10 mg of hydroxyzine, was applied to the shaved backs of 6 rabbits (3.08±0.05 kg). Histamine-induced wheal tests and blood sampling were performed at designated time intervals up to 24 hours. Compared with baseline, hydroxyzine from all formulations significantly suppressed histamine-induced wheal formation by 75% to 95% for up to 24 hours. Mean maximum suppression, 85% to 94%, occurred from 2 to 6 hours, with no differences among the formulations. The areas of plasma hydroxyzine concentration versus time area under the curve (AUCs) from PC-SUV and PC-MLV, 80.1±20.8 and 78.4±33.9 ng/mL/h, respectively, were lower than that from GB, 492±141 ng/mL/h (P<.05) over 24 hours. Plasma concentrations of cetirizine arising in-vivo as the active metabolite of hydroxyzine, from PC-SUV, PC-MLV, and GB, were similar with AUCs of 765±50, 1035±202, and 957±227 ng/mL/h, respectively (P<.05). Only 0.02% to 0.06% of the initial hydroxyzine dose remained on the skin after 24 hours. In this model, hydroxyzine from SUV and MLV had excellent topical H₁-antihistaminic activity, and minimal systemic exposure occurred. Cetirizine formed in-vivo contributed to some of H₁-antihistaminic activity.     

国产盐酸西替利嗪片的药代动力学研究

目的 :建立测定国产盐酸西替利嗪 (西可韦 )血药浓度的方法并进行人体药代动力学研究。方法 :采用反相高效液相色谱法 ,以盐酸普罗帕酮为内标 ;色谱柱 :WaterssymmetryC18 不锈钢柱 (3 9mm×150mm ,5μm) ;流动相 :乙腈 -0 02mol/L磷酸二氢钠 -三乙胺 (50∶50∶0 16 ,V/V) ,含十二烷基硫酸钠 (SDS)4 0mmol/L ;流速 :1 0ml/min ;检测波长 :229nm。测定11名健康男性志愿者单剂量口服西可韦片10mg 的血浆中药物浓度。结果 :线性范围为12 5～800ng/ml,最低检测限为5ng/ml,提取回收率>75 %。11名志愿者的血药浓度数据经3p87软件拟合 ,符合血管外给药二室模型 ,其主要药代动力学参数为 :Cmax=(429 00±108 80)ng/ml,Tmax= (0 91±0 40)h ,K10= (0 18±0 04)/h ,以梯形法计算的AUC0～36= (3312 72±682 39)ng/(ml·h)。结论 :本方法结果准确 ,灵敏度高 ,能满足人体药代动力学研究的需要 ;西可韦主要药代动力学参数与国内、外文献报道一致 ,可广泛应用于临床     

Liquid chromatography-mass spectrometric analysis of buprenorphine and its N-dealkylated metabolite norbuprenorphine in rat brain tissue and plasma

INTRODUCTION: A specific, accurate, and reproducible liquid chromatography-mass spectrometric (LC/MS) method was developed and validated that allows simultaneous measurement of the centrally acting analgesic buprenorphine and its major metabolite, norbuprenorphine, in rat brain and plasma samples. METHODS: A 96-well plate solid phase extraction (SPE) procedure was developed for buprenorphine and norbuprenorphine using mixed-mode cation-exchange reversed-phase sorbent. An LC method using a C8 column with isocratic mobile phase (80:20 water/acetonitrile with 20 mM ammonium acetate and 0.1% acetic acid) was developed for reproducible and selective separation. A quadrupole mass spectrometer with atmospheric electrospray ionization source under positive ion mode was used for detection. d4-Buprenorphine and d3-norbuprenorphine were used as internal standards. RESULTS: The calibration curves for buprenorphine and norbuprenorphine in plasma and brain tissue were linear within the range of 7 to 8333 ng/ml (plasma) and 5 to 5000 ng/g (brain). The lower limit of quantification for both buprenorphine and norbuprenorphine from brain tissue was 5 ng/g, and from plasma was 7 ng/ml. Assay accuracy and precision of back-calculated standards were within +/-15%. DISCUSSION: This method will be useful for investigation of buprenorphine's mechanism of action and clinical profile.     

HPLC-紫外法测定人血浆中芬太尼浓度

目的 :建立高效液相色谱法 -紫外检测器测定人血浆中芬太尼浓度的方法。方法 :本实验采用外标法 ,以Shim -PackCLC -ODS(6 0mm×150mm ,5μm )为固定相 ,含0 015mol/LNaH2PO4 的乙腈 -水溶液 (30∶70 ,v/v)为流动相 ,流速1 5ml/min ,紫外检测波长195nm。结果 :标准曲线在2 0～100ng/ml范围内线性关系良好 (r=0 999) ,最低检测浓度为1ng/ml,方法回收率为(91 70±4 70) % ,提取回收率为 (97 38±3 69) % ,日内变异RSD (6 50±2 79) % ,日间变异RSD (6 70±3 04) %。结论 :本方法简便 ,准确 ,检测浓度低 ,能够满足血浆中低浓度芬太尼的测定及临床药代动力学研究的要求。     

The determination of nadolol in biological samples using high-performance liquid chromatography

A method has been developed for the determination of nadolol in biological samples by reversed-phase high-performance liquid chromatography with fluorimetric detection. The method has been applied to plasma, serum and urine samples, which are prepared by extraction with diethyl ether-dichloromethane (5:2,v/v), evaporation of the organic solvent, and dissolution of the resultant residue in the chromatographic eluent. The sample is then subjected to chromatography on a C(18)-silica column, with an eluent of water-acetonitrile-triethylamine (800:200:1,v/v) adjusted to pH 3.0 with orthophosphoric acid. A single point external standard is used for quantitation. The working ranges were 1-400 ng/ml for plasma/serum, and 0.1-40 mug/ml for urine, although a detection limit of 0.1 ng/ml appears to be readily attainable. The sample size was 0.5 ml, and for both types of sample the method showed good correlation with a previously published fluorimetric method (for plasma, r = 0.9544, n = 70; for urine, r = 0.9919, n = 35).     

样品固相净化及反相高效液相色谱法测定血浆中茶碱

本文用氧化铝固相净化血样以消除血中杂质及某些合并用药的干扰,用Zorbax-C18柱及甲醇-(0.05 mol/L)醋酸钠缓冲液(50:50)为流动相,254 nm波长检测,咖啡因为内标测定了茶碱人体血浆浓度的变化。该法样品处理简便、快速,净化回收率好(80～86%)茶碱的检测限为0.2ng(信噪比3),在血浆中的最低检测浓度为20 ng/ml。标准曲线r为0.9995,日内变异系数1.89%,日间变异系数为2.41%,方法平均回收率为98.44±0.89%。     

Simultaneous determination of dextromethorphan and three metabolites in plasma and urine using high-performance liquid chromatography with application to their disposition in man

A simple, sensitive, and reproducible high-performance liquid chromatrography assay is described for the simultaneous determination of dextromethophan, dextrorphan, 3-hydroxymorphinan, and 3-methoxymorphinan in plasma and urine. A conventional solvent-solvent extraction procedure was used for the isolation of the analytes from plasma and urine samples. The compounds were separated on a cyano column (150 x 4.6 mm, 5-micron particle size) using a mobile phase of acetonitrile/triethylamine/distilled water (17:0.06:82.94, vol/vol), pH 3.0, and then were measured by fluorescence detection. Calibration curves in the range 2-200 ng/ml for plasma and 0.05-10 micrograms/ml for urine were linear and passed through the origin. The precision and accuracy were greater than 90% and the lowest detectable concentrations were 0.5 ng/ml for 3-hydroxymorphinan and 3-methoxymorphinan and 1 ng/ml for dextromethorphan and dextrophan in plasma. The utility of this method is demonstrated in a preliminary study of dextromethorphan metabolism and pharmacokinetics in man.     

LC-MS method for the determination of albuterol enantiomers in human plasma using manual solid-phase extraction and a non-deuterated internal standard

A sensitive enantioselective liquid chromatography–mass spectrometry (LC–MS) assay using a manual solid-phase extraction (SPE) procedure, a non-deuterated internal standard and an ion trap LC–MS was developed to measure (R)- and (S)-albuterol in plasma. Sample extraction from plasma was achieved by a manual SPE extraction procedure with methoxyphenamine added as the internal standard. Chiral separation was achieved using a teicoplanin-based stationary phase and a mobile phase consisting of methanol, acetic acid and 28% (w/v) ammonia (1000:5:1, v/v/v). Samples were analyzed by selected reaction monitoring of product ions from the protonated molecular ions. The detection limit of the assay was 0.1 ng/ml with a conservative lower limit of quantification of 0.25 ng/ml for each enantiomer. Recovery of albuterol enantiomers from plasma spiked at 10 ng/ml of racemate was determined to be 89±5.8% (mean±S.D.). Reproducibility at 10 ng/ml of racemate assessed by the coefficient of variation was found to be 6.5% (n=5). Instrument precision (measured as coefficient of variation) was 1.4% (n=5). The correlation coefficient r² determined from the calibration curve over the range 0.5–50.0 ng/ml racemate in plasma was 0.998. This assay allows adequate sensitivity, recovery and reproducibility for the application to studies of inhaled albuterol.     

A simple and rapid HPLC method for the determination of atorvastatin in human plasma with UV detection and its application to pharmacokinetic studies

A rapid and sensitive high-performance liquid chromatographic method for the determination of atorvastatin (CAS 134523-00-5) in plasma was developed in this study. Atorvastatin was isolated from plasma using protein precipitation by acetonitrile. Diltiazem (CAS 33286-22-5) was used as internal standard. The chromatographic conditions were as follows: analytical 125 x 4 mm (i.d.) Nucleosil C8 column (5 microm particle size), mobile phase consisting of sodium dihydrogen phosphate buffer-acetonitrile (60:40, v/v) adjusted to pH 5.5 at a flow rate of 1.5 ml/min, UV detection at 245 nm. The detection limit for atorvastatin in plasma was 1 ng ml(-1). The calibration curve was linear over the concentration range 20-800 ng ml(-1). The recovery was complete. The inter-day and intra-day assay coefficients of variation were found to be less than 7%. The present validated method was successfully used for pharmacokinetic studies of atorvastatin in human subjects.     

高效液相色谱法测定普罗帕酮血浆浓度

本文建立了高效液相色谱法测定人体内普罗帕酮血浆浓度。以YWG-C_(18)(10μm)为固定相,改性甲醇-乙腈-醋酸盐缓冲液(45.5:19.5:35)为流动相,用达克罗宁作内标在254nm波长处定量测定。方法最低检测限为5ng(S/W=3),血浆中最低检测浓度为25ng/ml,普罗帕酮血浆浓度在50～500ng/ml范围内线性关系良好,方法回收率为100.5％,不同浓度水平测定蛄果的日内和日间变异系数均小于3％。方法重现性好,专一性强,内源性物质不干扰,操作简便,快速,能适合梏床血药浓度监测及药代动力学研究。     

Beagle犬血浆中辛伐他汀的LC-MS测定

建立了LC-MS法测定Beagle犬血浆中的辛伐他汀。采用C_(18)柱,流动相为乙腈-0.1%甲酸溶液(60:40),以洛伐他汀为内标,采用电喷雾离子化法(ESI)采集正离子,单离子方式检测m/z 419(辛伐他汀)和m/z 405(洛伐他汀)。辛伐他汀在0.5～160ng/ml浓度范围内线性良好,检测限为0.25ng/ml。血浆样品的日内、日间RSD均小于7.7%,方法回收率为92.2%～99.2%,提取回收率为92.8%～100.9%。     

Determination of beta2-agonists by ion chromatography with direct conductivity detection

A simple method for the simultaneous detection of four beta2-agonists (salbutamol, fenoterol, clorprenaline, and clenbuterol) using ion chromatography (IC) with direct conductivity detection (CD) based on their ionization in acidic medium without chemical suppression is presented. The mixture of 1.8 mM HNO3 and 2% (v/v) acetonitrile was used as eluent. The method could be applied to the determination of the beta2-agonists in pharmaceutical preparations. The recovery of salbutamol and clenbuterol in tablets was more than 97% (n=3) and the relative standard deviation (n=11) less than 2.8%. With the proposed method, salbutamol could also be successfully detected in human plasma. In a single chromatographic run, the four beta2-agonists can be separated and determined in less than 8 min. The linear ranges were of 7.0-1.4 x 10(3)ng/ml for salbutamol, 34-7.8 x 10(3)ng/ml for fenoterol, 8.0-1.6 x 10(3)ng/ml for clorprenaline, and 25-7.5 x 10(3)ng/ml for clenbuterol. The detection limits were 2.0 ng/ml for salbutamol, 10 ng/ml for fenoterol, 3.0 ng/ml for clorprenaline, and 10 ng/ml for clenbuterol.     

HPLC assay for albendazole and metabolites in human plasma for clinical pharmacokinetic studies

A sensitive and selective HPLC chromatography method using UV detection (295 nm) was developed for the determination of albendazole, albendazole sulfoxide (ABZSO), and albendazole sulfone (ABZSO2) in human plasma. Albendazole, ABZSO, ABZSO2, and the internal standard, oxibendazole, were extracted from human plasma by loading onto a conditioned C(18) SPE cartridge, rinsing with 15% methanol, and eluting with 90% methanol. Samples were evaporated under a stream of nitrogen, reconstituted with mobile phase, 1.25% triethylamine in water-methanol-acetonitrile (72:15:13, v/v/v) (pH* 3.1), and injected onto a Waters muBondapak Phenyl 3.9 x 300 mm HPLC column. Mobile phase flow rate was 1.0 ml/min. The retention times of albendazole, ABZSO, ABZSO2, and the internal standard were approximately 24.4, 7.9, 13.4, and 11.3 min, respectively. Total run time was 30 min. The assay was linear for concentration ranges in human plasma of 20-600 ng/ml for albendazole, 20-1000 ng/ml for ABZSO, and 20-300 ng/ml for ABZSO2. The analysis of quality control samples demonstrated excellent precision. Coefficients of variation for albendazole (20, 400, 600 ng/ml) were 6.7, 8.1 and 7.0%; ABZSO (20, 400, 800 ng/ml) were 6.0, 8.5 and 5.9%; ABZSO2 (20, 150, 300 ng/ml) were 3.1, 3.9 and 2.3%, respectively. The method appears to be robust and has been applied to a pharmacokinetic study of albendazole in healthy volunteers.     

Detection and separation of the S-adenosylmethionine-decarboxylase inhibitor SAM486A in human plasma and urine by reversed-phase ion-pairing high-performance liquid chromatography.

A reversed-phase ion-pairing high-performance liquid chromatography method for the detection and separation of SAM486A in human plasma and urine is described. Precipitation of proteins was used for plasma sample preparation. Enrichment of SAM486A on a 5 micro C18 column using 0.05 M NaH(2)PO(4) and 0.005 M pentan-sulfonic acid (pH 3.0) as eluent was followed by isocratic elution onto a 5 micro C18 analytical column using 0.01 M NaH(2)PO(4) and 0.005 M pentan-sulfonic acid (pH 3.0) as eluent. Analysis time was 23.0 +/- 0.1 min. The separation parameters were: capacacity factor = 6.21; plates/m = 15,002; peak tailing = 2.076. The method is linear between 5 ng/ml (detection limit) and 1000 ng/ml.     

Improved high performance liquid chromatographic analysis of omeprazole in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of omeprazole in human plasma. Omeprazole and the internal standard, chloramphenicol, were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M Na2HPO4-ACN (65:35, v/v) adjusted to pH 6.5. Analysis was run at a flow rate of 1.0 ml/min at a detection wavelength of 302 nm. The method was specific and sensitive with a detection limit of 2.5 ng/ml at a signal-to-noise ratio of 4:1. The limit of quantification was set at 5 ng/ml. The calibration curve was linear over a concentration range of 5-1280 ng/ml. Mean recovery value of the extraction procedure was about 96%, while the within and between day coefficient of variation and percent error values of the assay method were all less than 14%.     

Quantitative analysis of a bis-thiazolium antimalarial compound, SAR97276, in mouse plasma and red blood cell samples, using liquid chromatography mass spectrometry

A sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method has been developed for the determination of a new antimalarial bisthiazolium salt, SAR97276, in mouse plasma and red blood cells (RBCs). A drug of the same chemical series as the test drug, T2, was used as internal standard. The method involved solid phase extraction of the compound and the internal standard from the two matrices using Oasis HLB columns. LC separation was performed on a Zorbax eclipse XDB C8 column (5 microm) with a mobile phase of acetonitrile containing trimethylamine (130 microl/l, solvent A) and 2 mM ammonium formate buffer (solvent B). MS data were acquired in single ion monitoring mode at m/z 227 for SAR97276 and m/z 326 for T2. The matrix had no influence on the detection of either SAR97276 or T2. The drug/internal standard peak area ratios were linked via quadratic relationships to plasma (1.65-1322 ng/ml) and RBC concentrations (3.31-2644 ng/ml). Precision was below 14% and accuracy was 91.4-104%. Dilution of the samples had no influence on the performance of the method. Extraction recoveries of SAR97276 were > or =90% in plasma and > or =60% in RBCs. The lower limits of quantitation were 1.65 ng/ml in plasma and 3.31 ng/ml in RBCs. Stability tests under various conditions were also investigated. The method was successfully used to determine the pharmacokinetic profile of SAR97276 in healthy mouse.