High-performance liquid chromatographic assay for the derivatized enantiomers of propranolol and 4-hydroxypropranolol in human plasma

A stereospecific method for the analysis of propranolol and 4-hydroxypropranolol in human plasma employing fluorescence detection has been developed using the homochiral derivatizing agent 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (TAGIT). The use of fluorescence detection provided enhanced sensitivity and cleaner chromatograms for the analysis of plasma samples, when compared to UV detection. Furthermore, parameters such as TAGIT concentration, vortex time and reaction time were examined to optimize conditions for maximum derivatization recoveries. The analyses of S(-)- and R(+)-propranolol from plasma were linear over the concentration range of 2.0-200 ng ml-1, while S(-)- and R(+)-4-hydroxypropranolol were linear from 5.0 to 200 ng ml-1.     

Validated HPLC-MS/MS method for determination of quetiapine in human plasma

A validated, highly sensitive and selective high-pressure liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitative determination of quetiapine (QUE) in human Na2EDTA plasma with mass spectrometry (MS) detection. Clozapine (CLO) was employed as an internal standard. Samples were extracted using solid phase extraction (SPE). Oasis HLB cartridges and the concentration of quetiapine was determined by isocratic HPLC-MS/MS. The SRM mode was used for MS/MS detection. The method was validated over a concentration range of 1.0-382.2 ng/mL. Inter- and intra-day precision and accuracy of the proposed method were characterized by relative standard deviation (R.S.D.) and the percentage of deviation, respectively; both were lower than 8%. The developed method was employed in the pharmacokinetic study of quetiapine.     

Simple high-performance liquid chromatographic determination of buspirone in human plasma

A simple, rapid and sensitive high-performance liquid chromatographic method for the determination of buspirone in plasma was developed. Buspirone was isolated from plasma using protein precipitation by acetonitrile and the recovery was complete. Citalopram was used as internal standard. The chromatographic conditions were as follows: analytical 125 x 4 mm, i.d. Nucleosil C18 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 1.0 ml min(-1), UV detection at 235 nm. The quantification limit for buspirone in plasma was 0.5 ng ml(-1).The calibration curve was linear over the concentration range 0.5-10 ng ml(-1). The inter- and intra-day assay coefficients of variation were found to be less than 8%. The present validated method was successfully used for bioequivalence studies of buspirone in human subjects.     

A sensitive liquid chromatography and mass spectrometry method for the determination of posaconazole in human plasma

Posaconazole is a novel extended-spectrum triazole that has favorable in vitro, in vivo and clinical activity against a number of yeasts and moulds. Posaconazole is available as an oral suspension. The dosage found to result in monitored plasma levels that correlate with clinical evidence of good antifungal activity is 800 mg/day in divided doses. A liquid chromatographic/mass spectrometric method (LC-MS/MS) that can be used by clinicians wishing to quantitate, and thereby monitor, plasma levels of posaconazole in certain patients was validated. The method utilized semi-automated 96-well protein precipitation with gradient chromatographic separation of analytes using a Varian Polaris C-18A (2.0 mm x 50 mm, 5-microm particle size) column. The approximate retention time of posaconazole was 2.0 min. Analytes were detected by using tandem mass spectrometry. Sample introduction and ionization was performed by atmospheric pressure chemical ionization in the positive-ion mode. This method has been proven suitable for routine quantitation of posaconazole over the concentration range of 5.00-5000 ng/mL. Inter-run precision based on percent relative deviation for replicate quality controls was < or = 6.2%. Inter-run accuracy expressed as %DIFF was +/-4.0%. Posaconazole quality controls were stable in human plasma for up to five freeze-thaw cycles, when frozen at -20 degrees C for at least 105 days and when kept at room temperature for 24 h. The lower limit of quantitation was 5.00 ng/mL for a 100-microL sample aliquot. These data indicate that the LC-MS/MS method described is suitable for the rapid measurement of posaconazole over the concentration range of 5.00-5000 ng/mL.     

High-performance liquid chromatographic analysis of pterostilbene in biological fluids using fluorescence detection

A method of analysis of pterostilbene [trans-3,5-dimethoxy-4'-hydroxystilbene] is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in foodstuffs. A novel and simple high-performance liquid chromatographic method was developed for determination of pterostilbene in rat serum. Serum proteins (0.1 mL) are precipitated with cold acetonitrile after addition of the internal standard, pinosylvin. Separation was achieved on a Phenomenex C18 column (250 mm x 4.60 mm) with fluorescence excitation at 330 nm and emission at 374 nm. The calibration curves were linear ranging from 0.5 to 100 microg/mL. The mean extraction efficiency was >99%. Precision of the assay was <15% (CV), and was within 14% at the limit of quantitation (0.5 microg/mL). Bias of the assay was lower than 14%, and was within 9% at the limit of quantitation. The assay was applied successfully to the study of pterostilbene pharmacokinetics in rats.     

HPLC法测定人血清及尿中美洛培南浓度

目的　建立测定人血清样品及尿样品中美洛培南的定量分析方法。方法　采用高效液相色谱法 ,以 μ- Bondapak C1 8柱 (3.9mm× 30 0 mm,10 μm)为色谱柱 ;甲醇∶ 5 mmol/ L 磷酸二氢钾缓冲液 (17∶ 83,v/ v)为流动相 ,p H2 .5 ,检测波长 30 8nm。结果　血清中美洛培南的回收率平均为 97.5 4 % (n=6 ) ;日内 ,日间RSD≤ 3.80 % ,在 1～ 10 0 mg/ L 血药浓度范围内线性关系良好 ,r=0 .9997。尿中美洛培南的平均回收率为96 .90 % (n=6 ) ;日内、日间 RSD<3.5 0 % ,在 5～ 10 0 mg/ L 尿药浓度范围内线性关系良好 ,r=0 .9994。结论该方法灵敏准确 ,适用于临床药代动力学的研究。     

High-performance liquid chromatography assay for moxifloxacin: pharmacokinetics in human plasma

A sensitive high-performance liquid chromatographic (HPLC) method for the determination of moxifloxacin in human plasma using fluorescence detection was developed. The drug and an internal standard (norfloxacin) were subjected to precolumn derivatization with 4-chloro-7-nitrobenzodioxazole (NBD-CI). The chromatographic separation was achieved by HPLC using a mixture of acetonitrile-10 mM orthophosphoric acid (pH 2.5) (80:20, v/v) as the mobile phase with isocratically system, a C18 column. The derivative is highly fluorescent at 537 nm, being excited at 464 nm. The linear and reproducible calibration curve over the range was 15-2700 ng/mL of moxifloxacin in human plasma. The limits of detection and quantitation were 6 and 15 ng/mL, respectively. This method was applied in pharmacokinetic studies moxifloxacin preparations in healthy volunteers.     

A rapid reversed phase high performance liquid chromatographic method for the determination of docetaxel (Taxotere®) in human plasma using a column switching technique

A rapid, simple and sensitive isocratic high performance liquid chromatography (HPLC) method was developed to measure the concentration of docetaxel in plasma samples with UV detection at 227 nm. The method uses a column switching technique with an Ultrasphere C₁₈ column (75×4.6 mm ID, 3μ, Altex, USA) as clean-up column and a CSC-nucleosil C₈ column (150×4.6 mm ID, 5μ, CSC, Montreal, Canada) as the analytical column. The mobile phase consisted of Phosphate buffer (30 mM, pH=3)-acetonitrile (47:53, v/v) with the flow rates of 1.1 and 1.3 ml min⁻¹ for clean-up and analytical columns, respectively. Paclitaxel was used as an internal standard. The plasma samples were extracted using a solid phase extraction method with Ammonium acetate (30 mM, pH=5)-acetonitrile (50:50, v/v) as final eluent. The extraction method showed a recovery of 92% for docetaxel. In this system, the retention times of docetaxel and Paclitaxel were 7.2 and 8.5 min, respectively. The detection limit of docetaxel in plasma is 2.5 ng ml⁻¹. This analytical method has a very good reproducibility (7.2% between-day variability at a concentration of 10 ng ml⁻¹). It is applicable in clinical and pharmacokinetic studies.     

High performance thin-layer chromatographic method for the determination of sparfloxacin in human plasma and its use in pharmacokinetic studies

A rapid and sensitive high-performance thin-layer chromatographic (HPTLC) method has been developed for the measurement of sparfloxacin in human plasma and its use for pharmacokinetic study has been evaluated. Detection and quantitation were performed without using an internal standard. A single stage extraction procedure was followed for extracting sparfloxacin from plasma and a known amount of the extract was spotted on precoated silica gel 60 F254 plates using a Camag Linomat IV autosampler. Sparfloxacin was quantified using a Camag TLC Scanner 3. The recovery study of authentic analytes added to plasma at 0.1 to 0.8 μg ml⁻¹ was 94.9±0.98% and the lowest amount of sparfloxacin that could be detected was 50 ng ml⁻¹ plasma. The method provides a direct estimate of the amount of sparfloxacin present in plasma. The method was used for the determination of plasma levels as well as pharmacokinetic parameters of sparfloxacin after oral administration of two marketed preparations to healthy volunteers.     

Rapid fluorimetric procedure for the analysis of Fendosal in plasma and data following oral dosing

A simple, rapid, and sensitive procedure is described for the determination of Fendosal in plasma. After extraction of Fendosal from buffered plasma, the drug is determined by measurement of the fluorescence induced by irradiation with short-wave ultra-violet (UV) light. The complete procedure can be completed in less than 2 h; one technician can perform up to 50 assays in one working day. The limit of detection corresponds to 0–1 μg ml^?1 plasma. Drug concentration and induced fluorescence were linearly related over the concentration range 0–8 μg ml^?1.     

反相高效液相色谱法测定人血浆中甲氨蝶呤的浓度

目的:建立反相高效液相色谱法测定人血浆中甲氨蝶呤浓度的方法. 方法:使用岛津LC-10AD高效液相色谱仪,Lichrosphere C18柱(5 μm,4.6 mm×250 mm),流动相为 0.025 mol·L-1 NaH2PO4溶液(pH5.5)-甲醇(72∶28),检测波长 313 nm,流速 1.0 mL·min-1, 柱温35℃,内标为阿魏酸.结果:甲氨蝶呤浓度在0.05～5.00 μg·mL-1范围内线性关系良好,回归方程为Y=3.094 2c 0.006 8(r=0.999 3),日内变异系数小于7%,日间变异系数小于10%;检测限为 20 ng·mL-1.结论:本法操作简便,适用于甲氨蝶呤的血药浓度测定及药代动力学研究.     

高效液相色谱法测定血浆中伊立替康的含量

目的建立血浆中伊立替康含量的测定方法。方法以C18色谱柱、0．05mol／LNa2HPO4(pH=3．0,内含0．05mol／L辛烷基磺酸钠)-乙腈=68：32为流动相分离、荧光检测伊立替康,外标法定量。结果伊立替康浓度在25．0-1000μg／L内,浓度与峰面积之间有良好的线性关系(C=0．00249A-8．15,r=0．9994)。最小检出质量浓度为2．0μg／L。50,100,500μg／L伊立替康的相对回收率(％)分别为96．4、98．3和100．4。三个浓度的平均日内RSD为2．87％,平均日间RSD为4．30％。结论本方法快速、简便、准确,可用于科研和临床工作中伊立替康血药浓度的快速检测。     

A high-performance liquid chromatographic method for the determination of stobadin pharmacokinetics in serum

A high-performance liquid chromatographic method was developed to determine stobadin pharmacokinetics in dog and man. The relative bioavailability of stobadin dipalmitate compared with dihydrochloride was 46.4 per cent in dog. In man peak serum concentrations ranged from 12 to 289 ng ml-1 after a single oral dose of stobadin dipalmitate (0.79 to 2.5 mgkg-1).     

Selective determination of tenofovir in human plasma by LC-MS-MS method

In the present study, we developed and validated a selective, specific and sensitive liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS) method for the determination of tenofovir in human plasma. Entecavir was used as an internal standard, and plasma samples were prepared by solid-phase extraction performed on Phenomenex Strata cartridges (30 mg). The mobile phase consisted of 10 mM ammonium acetate in water and methanol (60:40, v/v). The chromatographic separation was performed isocratically on a Phenomenex C₁₈ (4.6 mm×150 mm, 5 μm), and analytes were analyzed in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI) interface using the respective [M+H]⁺ ions, m/z 288.2→m/z 176.1 for tenofovir and m/z 278.1→m/z 152 for entecavir. The calibration curve (r² = 0.9962) of tenofovir was established within the range of 4.096–1000 μg/L. The intra- and inter-day precisions were less than 10%. This validated method was successfully applied to a pharmacokinetic study in 12 healthy Chinese volunteers after the oral administration of tenofovir disoproxil fumarate.     

Improved reversed-phase liquid chromatographic method combined with pulsed electrochemical detection for the analysis of amikacin

A two-step gradient liquid chromatographic method combined with pulsed electrochemical detection is described for the determination of amikacin and its impurities. The mobile phase is composed of an aqueous solution containing 1.8 g/l sodium 1-octanesulphonate, 14 ml/l tetrahydrofuran, 50 ml/l of phosphate buffer pH 3.0 and sodium sulphate, which was 20 g/l in mobile phase A and 28 g/l in mobile phase B. 0.5 M sodium hydroxide was added post-column to enhance the detection. An investigation of different reversed-phase columns indicated that the Discovery (C18, 5 microm, 250 mm x 4.6 mm i.d.) column was the most suitable. Compared to previously published investigations, the proposed method showed higher sensitivity and efficiency, allowing the separation of the main component amikacin from 16 impurities, 7 of which were of unknown identity. A central composite experimental design was used to assess the robustness. The method showed good repeatability and linearity in the assay range. The method was further applied to analyze some commercial samples.     

A liquid chromatographic method for the determination of danazol in human serum

A liquid chromatographic method for the determination of danazol in human serum has been developed. Reversed-phase C8 and C18 columns were used with a column-switching valve, isocratic elution and UV detection. Sample pretreatment involved extraction of the drug with pentane-methylene chloride. The method enabled the measurement of the drug at a concentration as low as 1 ng ml-1, with precision of 15.0% and accuracy of 8.3%. The method was used to run a two way replicated pharmacokinetic study of danazol. The main pharmacokinetic parameters were (mean of two periods): AUCinf = 480.94 ng x h ml-1, Cmax = 53.2 ng ml-1, tmax = 2.5 h, t0.5 = 18.00 h.     

Simultaneous determination of zidovudine and nevirapine in human plasma by RP-LC

A simple method for the simultaneous determination of zidovudine and nevirapine in human plasma by reversed-phase liquid chromatography with UV detection at 265 nm was developed. A solid–liquid extraction procedure with internal standard was applied to the samples prior to analysis. The system requires a Zorbax SB-C18 column, 250×4.6 mm I.D. and a mobile phase composed of potassium dihydrogen phosphate (10 mM; pH 6.5)-acetonitrile (83:17, v/v). Peak-areas are linear; correlation coefficients are better than 0.999; both inter- and intra-day accuracy and precision are lower than 15%. Extraction recoveries are higher than 90% for both zidovudine and nevirapine. The method proposed was employed to determine the levels of the two retroviral drugs in plasma from HIV infected human subjects.     

A liquid chromatographic method for quantitating amitriptyline in brain tissue

A previously reported method for measuring tricyclic antidepressants (TCA) in plasma was modified to measure TCA, specifically amitriptyline (AMI) and nortriptyline (NOR) in rat brain tissue. Brains obtained from drugfree and AMI-treated rats were extracted and assayed using a Waters high-performance liquid chromatograph. Drug-free brain tissue contained no substances which interfered with the assay of these TCAs. Drug recovery averaged 90±3.4% (x±SEM). Seven intra-run assays of a spiked brain tissue sample yielded coefficients of variation of 2.7% for AMI and 1.8% for NOR. Seven inter-run assays of the same sample varied 4.2% for AMI and 3.5% for NOR. Five separate assays of a brain homogenate sample spiked with 50 ng/ml of drug yielded values of 50±2.1 SEM ng/ml for AMI and 54±1.1 SEM ng/ml for NOR. Standard curves were linear when constructed from samples in a concentration range of 250–3,000 ng/g wet weight tissue (r=0.96, P<0.001).     

High-performance liquid chromatographic determination of ciprofloxacin in plasma samples

A new bioanalytical high-performance liquid chromatographic (HPLC) method for the determination of ciprofloxacin with norfloxacin as an internal standard was developed and validated for plasma samples. Norfloxacin is structural homologue of ciprofloxacin and exhibits similar retention properties. The quality of respective peak separation is strongly influenced by amphoteric character of ciprofloxacin and norfloxacin as well. In previously published HPLC methods on conventional C18 reversed-phase [F. Belal, A.A. Al-Majed, A.M. Al-Obaid, Talanta 50 (1999) 765–786; G. Carlucci, J. Chromatogr. A 812 (1998) 343–367], ion pair reagents were added into the mobile phase to suppress peak tailing. In comparison with endcapped and high purity silica reversed-phase sorbent (Purospher RP-18e, Merck), which yielded symmetrical peaks, separation efficiency was further enhanced in our method. Gradient elution mode using acetonitril and phosphate buffer pH 3 on the pentafluorophenylpropyl stationary phase (250–4.6 mm Discovery^® HS F5, 5 μm, Supelco) was carried out. The resolution of 4.1 for ciprofloxacin–norfloxacin peaks was achieved. Sample preparation by SPE C18 (Supelclean) with recovery 72% was performed. Fluorescence detection with λ_excit = 280 nm, λ_emis = 446 nm was used. After the validation, the bioanalytical HPLC method was applied to pharmacokinetic studies.     

Simultaneous determination of propranolol and 4-hydroxy propranolol in human plasma by solid phase extraction and liquid chromatography/electrospray tandem mass spectrometry

A very sensitive, reliable, reproducible and highly selective assay for the simultaneous determination of free and total (conjugated and unconjugated) propranolol and its equipotent hydroxyl metabolite, 4-hydroxy propranolol, in human plasma was developed and validated. The analytes were simultaneously extracted from 0.300 mL of human plasma using solid phase extraction and detected in positive ion mode by tandem mass spectrometry with a turbo ionspray interface. Deuterium-labeled propranolol and 4-hydroxy propranolol, propranolol-d7 and 4-hydroxy propranolol-d7, were used as internal standards. The method has a lower limit of quantitation (LOQ) of 0.20 ng/mL for both analytes with the limits of detection (LOD) 50 and 100 pg/mL for propranolol and 4-hydroxy propranolol, respectively, based on a signal-to-noise ratio of 5. The assay was linear over a range 0.20–135.00 ng/mL for free propranolol and 0.20–25.00 ng/mL for free 4-hydroxy propranolol and linear over range 1.00–500.00 ng/mL for total propranolol and 1.00–360.00 ng/mL for total 4-hydroxy propranolol, with coefficient of determination greater than 0.99 for both analytes. The extraction recoveries were >96 and >64% on an average for propranolol and 4-hydroxy propranolol, respectively. The analytes were found stable in human plasma through five freeze (−15 °C)–thaw (room temperature) cycles and under storage on bench-top for at least 6.5 h, and also in mobile phase at 10 °C for at least 48 h. The method has shown tremendous reproducibility, with intra- and inter-day precision <11.3% (RSD), and intra- and inter-day accuracy <11% of nominal values, for both analytes, and has proved to be highly reliable for the analysis of clinical samples.