Two-dimensional LC–MS/MS determination of antiretroviral drugs in rat serum and urine

A simple, rapid, reliable and highly sensitive on-line two-dimensional reversed-phase liquid chromatography–tandem mass spectrometric (2D-LC/MS/MS) method to determine antiretroviral drugs viz., abacavir (ABC), nevirapine (NVP) and indinavir (IDV) in rat serum and urine was developed and validated. The analytes were extracted on-line from rat serum and urine by a restricted access material (RAM) column and back-flushed into the reversed-phase C₁₈ column for separation by LC. Detection was carried out by ESI-MS/MS. The developed method showed good selectivity, accuracy and precision for quantification of the antiretroviral drugs in rat serum and urine. Quantification limits for abacavir and nevirapine were 4.0 ng ml⁻¹, whereas for indinavir 4.7 ng ml⁻¹. The calibration graphs were linear in the range of 4–50 ng ml⁻¹for abacavir, nevirapine and indinavir. The method was successfully applied to study the pharmacokinetics of antiretroviral in rats.     

Determination of dexmedetomidine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection: Application to a pharmacokinetic study

A rapid, sensitive and selective high performance liquid chromatography-electrospray ionization-tandem mass spectrometry method (HPLC-ESI-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of dexmedetomidine (DMED) in human plasma. Dexmedetomidine and the internal standard (ondansetron) were extracted in a single step with diethyl-ether from 1.0 mL of alkalinized plasma. The mobile phase was a mixture of acetonitrile and 0.5% formic acid solution (30:70, v/v) at a flow rate of 0.2 mL min⁻¹. The detection was performed on a triple quadrupole tandem mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]⁺ ions m/z 201.0 → 95.1 for DMED and m/z 294.1 → 170.1 for the IS. The assay exhibited a linear dynamic range of 5–5000 pg mL⁻¹ with the correlation coefficient above 0.9995. The lower limit of quantification (LLOQ) was 5 pg mL⁻¹ with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated HPLC-MS/MS method has been successfully applied to study the pharmacokinetics of three level doses of DMED in Chinese healthy volunteers.     

Optimization of a method for determination of phenolic acids in exotic fruits by capillary electrophoresis

In this work, the separation of nine phenolic acids (benzoic, caffeic, chlorogenic, p-coumaric, ferulic, gallic, protocatechuic, syringic, and vanillic acid) was approached by a 3² factorial design in electrolytes consisting of sodium tetraborate buffer (STB) in the concentration range of 10–50 mmol L⁻¹ and methanol in the volume percentage of 5–20%. Derringer's desirability functions combined globally were tested as response functions. An optimal electrolyte composed by 50 mmol L⁻¹ tetraborate buffer at pH 9.2, and 7.5% (v/v) methanol allowed baseline resolution of all phenolic acids under investigation in less than 15 min. In order to promote sample clean up, to preconcentrate the phenolic fraction and to release esterified phenolic acids from the fruit matrix, elaborate liquid–liquid extraction procedures followed by alkaline hydrolysis were performed. The proposed methodology was fully validated (linearity from 10.0 to 100 μg mL⁻¹, R² > 0.999; LOD and LOQ from 1.32 to 3.80 μg mL⁻¹ and from 4.01 to 11.5 μg mL⁻¹, respectively; intra-day precision better than 2.8% CV for migration time and 5.4% CV for peak area; inter-day precision better than 4.8% CV for migration time and 4.8–11% CV for peak area; recoveries from 81% to 115%) and applied successfully to the evaluation of phenolic contents of abiu-roxo (Chrysophyllum caimito), wild mulberry growing in Brazil (Morus nigra L.) and tree tomato (Cyphomandra betacea). Values in the range of 1.50–47.3 μg g⁻¹ were found, with smaller amounts occurring as free phenolic acids.     

柱前衍生，LC-MS/MS同时测定血浆中的孕二烯酮、依托孕烯和炔雌醇

建立LC-MS/MS同时测定血浆中孕二烯酮、依托孕烯和炔雌醇的方法。血浆样品经液-液萃取、柱前衍生处理后, 采用ESI离子源, 多反应离子监测 (MRM), 正离子扫描进行测定。以炔诺孕酮为内标, 采用C₁₈ (100 mm × 2.1 mm, 5 μm) 柱, 梯度流动相, 梯度流速测定血浆中孕二烯酮、依托孕烯和炔雌醇。结果表明, 孕二烯酮、依托孕烯分别在0.1～20 ng·mL⁻¹、炔雌醇在0.01～2 ng·mL⁻¹时线性关系良好, 精密度小于10.0%、方法回收率在93.6%～110.9%。对复方孕二烯酮和复方依托孕烯透皮给药制剂进行了家兔的药动学研究。本方法灵敏度高 (孕二烯酮、依托孕烯达到100 pg·mL⁻¹, 炔雌醇达到10 pg·mL⁻¹)、重现性好, 适合药动学研究。     

Simultaneous determination of tetrahydropalmatine,protopine, and palmatine in rat plasma by LC-ESI-MS and its application to a pharmacokinetic study

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed and validated for the simultaneous quantification of tetrahydropalmatine, protopine and palmatine in rat plasma using phenacetin as the internal standard (IS). Two hundred microliters plasma samples were extracted by dichloromethane under a strong basic condition. The analytes were separated by a C18 column and detected with a single quadrupole mass spectrometer. The used mobile phase was acetonitrile–water (40:60, v/v) containing 5 mM ammonium acetate and 0.2% glacial acetic acid. Detection was carried out by positive electrospray ionization in selected ion reaction (SIR) mode at m/z 356.6 for tetrahydropalmatine, 354.6 for protopine, 352.6 for palmatine and 180.4 for the IS, respectively. The method was validated over the concentration range of 1.00–500 ng mL⁻¹ and the lower limit of quantification (LLOQ) was 1.00 ng mL⁻¹ for all three analytes. The intra- and inter-day precision values were less than 9% relative standard deviation (R.S.D.), and the relative error ranged from −7.4 to 4.8%. The extraction recoveries were on average 91.42% for tetrahydropalmatine, 84.75% for protopine, 57.26% for palmatine, and 83.18% for IS. The validated method was successfully applied to a pharmacokinetic study of tetrahydropalmatine, protopine and palmatine in rats after oral administration of Rhizoma Corydalis Decumbentis extract.     

Development of ultra fast liquid chromatographic method for simultaneous determination of nitrendipine and carvone in skin diffusate samples

A simple and sensitive reverse phase ultra fast liquid chromatographic (UFLC) method for simultaneous determination of nitrendipine and carvone in skin diffusate samples and microemulsions was developed and validated. The separation was achieved using a gradient mobile phase, on an Onyx column. The eluents were monitored by photodiode array detection. The linearity ranges of proposed method were 0.125–50 μg mL⁻¹ and 0.125–30 μg mL⁻¹ for nitrendipine and carvone respectively. The intra-day and inter-day coefficient of variation and percent error values of the assay method were less than 10%. The method was found to be precise, accurate, and specific during the study. The method was successfully applied for simultaneous estimation of nitrendipine and carvone in ex vivo skin diffusate samples and microemulsions.     

A stability-indicating HPLC assay with diode array detection for the determination of a benzylpenicillin prodrug in aqueous solutions

The aim of this study was to develop a stability-indicating HPLC assay for the determination of penethamate (PNT), an ester prodrug of benzylpenicillin (BP), in aqueous solutions. The method was validated by subjecting PNT to forced decomposition under stress conditions of acid, alkali, water hydrolysis and oxidation. A quenching solution was developed to limit degradation to negligible levels before and during the analysis. Both PNT and BP were simultaneously determined and separated in presence of degradation products on a C₁₈ column using a mobile phase consisting of methanol–acetonitrile–acetate buffer. Different degradation products were formed in the stress conditions. The peak purity indexes of PNT and BP obtained by diode array detection were >0.999, confirming the absence of other co-eluting substances. The assay was linear for both analytes in the concentration range 1–100 μg mL⁻¹. The LOD and LOQ of PNT were 0.03 and 0.09 μg mL⁻¹ respectively. Degradation of PNT followed pseudo-first-order kinetics with t_1/2 of 43.6 min at pH 2.01 and 4.2 min at pH 9.31. In addition, the absence of BP in the acidic solutions of PNT emphasises the futility of monitoring BP to assess the stability of PNT. In conclusion, the assay is rapid and stability-indicating with adequate precision and accuracy, and in conjunction with the quenching solution, can be used for stability studies of PNT with simultaneous quantitation of BP. The degradation studies provide useful information for formulation development of PNT.     

卡维地洛人体药动学与药效学结合模型研究

采用间接药效学模型和效应室模型两种药效学模型分别进行卡维地洛药动学与药效学关系研究, 比较两种药效学模型的拟合程度。高效液相色谱法 (荧光) 测定20名健康志愿者单次口服20 mg卡维地洛片后卡维地洛经时血药浓度, 以DAS 2.0实用药动学计算程序计算卡维地洛药动学参数。同时测定给药前后动脉收缩压和舒张压, 计算降压效果。卡维地洛片主要药动学参数t_1/2为 (4.56 ± 2.56) h, C_max为 (46.29 ± 21.07) ng·mL^-1, AUC_0-∞为 (173.76 ± 87.36) ng·mL^-1·h。间接药效模型主要参数K_in为 (0.41 ± 0.31) % h^-1, K_out为 (0.40 ± 0.26) h^-1, IC₅₀为 (24.40 ± 21.10) ng·mL^-1, AUE为 (3.82 ± 1.46) % h。效应室模型主要参数K_e0为 (0.35 ± 0.27) h^-1, EC₅₀为 (24.30 ± 24.30) ng·mL^-1, AUE为 (5.65 ± 2.54) % h。该方法可用于卡维地洛片人体药动学研究。由AIC值可知, 效应室模型可更好的应用于卡维地洛药动学-药效学结合研究。

     

A validated HPTLC method for determination of terbutaline sulfate in biological samples: Application to pharmacokinetic study

Terbutaline sulfate (TBS) was assayed in biological samples by validated HPTLC method. Densitometric analysis of TBS was carried out at 366 nm on precoated TLC aluminum plates with silica gel 60F₂₅₄ as a stationary phase and chloroform–methanol (9.0:1.0, v/v) as a mobile phase. TBS was well resolved at R_F 0.34 ± 0.02. In all matrices, the calibration curve appeared linear (r² ⩾ 0.9943) in the tested range of 100–1000 ng spot⁻¹ with a limit of quantification of 18.35 ng spot⁻¹. Drug recovery from biological fluids averaged ⩾95.92%. In both matrices, rapid degradation of drug favored and the T_0.5 of drug ranged from 9.92 to 12.41 h at 4 °C and from 6.31 to 9.13 h at 20 °C. Frozen at −20 °C, this drug was stable for at least 2 months (without losses >10%). The maximum plasma concentration (Cp_max) was found to be 5875.03 ± 114 ng mL⁻¹, which is significantly higher than the maximum saliva concentration (Cs_max, 1501.69 ± 96 ng mL⁻¹). Therefore, the validated method could be used to carry out pharmacokinetic studies of the TBS from novel drug delivery systems.     

Fabric phase sorptive extraction coupled with UPLC-ESI-MS/MS method for fast and sensitive quantitation of tadalafil in a bioequivalence study

The current study coupled fabric phase sorptive extraction (FPSE) with ultraperformance liquid chromatography method with electrospray ionization and tandem mass detection (UPLC-ESI-MS/MS) for fast and sensitive determination of tadalafil (TAD) in a bioequivalence study. Fabric phase sorptive extraction allowed direct extraction of TAD from the sample matrix with improved selectivity, repeatability, and recoveries. A sol–gel Carbowax 20 M (CX-20 M) coated FPSE membrane revealed the best extraction efficiency for TAD because of its strong affinity for analytes via intermolecular interactions, high mass transfer rate to FPSE membrane, and high permeability. An automated multiple reaction monitoring (MRM) optimizer was employed for the best selection of the precursor and product ions, ion breakdown profile, the fine adjustment of the fragmentor voltages for each precursor ions, and the collision energies for the product ions. The chromatographic separation was conducted using a mobile phase A: 5.0 mM ammonium acetate with 0.1 % formic acid in water and mobile phase B: formic acid (0.1%) in acetonitrile in ratio (55:45, v/v) through isocratic elution mode on an Agilent EclipsePlus C18 (50 × 2.1 mm, 1.8 μm) column and the flow rate was adjusted at 0.4 mL min^?1. The total run time per sample was 1.0 min. The method was validated by FDA standards for bioanalytical method validation over a concentration range of 0.1–100 ng mL^?1 with a correlation coefficient of 0.9993 and the lower limit of quantitation (LLOQ) was 0.1 ng mL^?1 in rat plasma. Intra- and inter-assay precision (%RSD) were lower than 4.1% and accuracy (%RE) was within 2.4%. The developed FPSE-UPLC-ESI-MS/MS method was effectively used in a randomized, two-way, single-dose, crossover study to compare the bioequivalence of two TAD formulations from different companies in male rats and verified to be bioequivalent.     

沙参麦冬汤中3种有效成分血药浓度的UPLC-MS/MS测定及药动学研究

［］ 目的：建立大鼠血浆中甘草苷、花椒毒酚和甲基麦冬黄烷酮A的UPLC-MS/MS测定方法,并探讨大鼠灌胃沙参麦冬汤后其在大鼠体内的药动学过程。方法：以流动相乙腈-0.1%甲酸水溶液,梯度洗脱,流速0.3ml/min;采用ESI源,正负离子同时检测模式扫描,多反应监测模式(MRM)检测各成分血药浓度,并用DAS 3.0 软件计算药动学参数。结果：甘草苷、花椒毒酚和麦冬黄烷酮A分别在4.92-315.00ng.mL_-1、1.44-92.00 ng.mL_-1 和0.35-22.00 ng.mL_-1线性关系良好,平均回收率均大于76.5%,日内、日间RSD 均小于15%。大鼠灌胃沙参麦冬汤提取物后,甘草苷、花椒毒酚和甲基麦冬黄烷酮A的AUC_0-t 分别为(718.23±185.55),(22.52±7.53),和(13.55±6.03)ng.h.mL_-1 ; t_1/2分别为3.61±2.01,6.93±7.78 和3.51±1.92h。结论: 本法方便、快捷,可用于甘草苷、花椒毒素和甲基麦冬黄烷酮A的体内定量分析。     

Are β-adrenoreceptors involved in the intestinal absorption and tissue distribution of iron in the rat?

⁵⁹Fe was incorporated in vivo into intestinal sacs prepared in iron deficient rats and ⁵⁹Fe counts were detected in blood, spleen, liver, femur and intestine. The i.v. injection of isoproterenol (i.v. 2 μg/rat) or N′,O′-dibutyryladenosine 3′,5′-cyclic monophosphate (10 μM/100 g b.w.) enhanced significantly the iron counts in blood, spleen, liver and femur but not in intestine. (−)-Propranolol (2 mg · kg⁻¹ b.w.i.v.) antagonized the stimulatory effect of isoproterenol. It is suggested that isoproterenol increases iron absorption via the stimulation of β-adrenoreceptors of the intestinal mucosa.     

Assessment of a natural extraction phase in disposable pipette extraction coupled with the sub-minute determination of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in human urine by fast-GC-FID

The compound 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) consists of the major metabolite of tetrahydrocannabinol (THC) in human urine. Analysis of this compound exhibits an important role in forensic scenario; therefore, several analytical methods have been developed for this purpose. In this study, for the first time, a biosorbent-based disposable pipette extraction was developed for the determination of THC-COOH, using a simple and high-throughput sample preparation technique. In addition, a rapid instrumental analysis was achieved by fast-gas chromatography coupled with flame ionization detection (fast-GC-FID). Different biosorbents were examined, including cork, moringa and bract. The method was optimized through univariate and multivariate designs with the conditions comprised of 15 mg of cork as the extraction phase, pH maintained at 2.5 with 8 cycles of extraction of 60 s each, and 1 cycle of 5 s for the desorption step with a mixture of acetonitrile:methanol (ACN/MeOH). Limit of detection was 0.21 ng mL^?1 and the limit of quantification was 0.73 ng mL^?1, with a linear range from 5 to 100 ng mL^?1 and coefficient of determination (R²) 0.9905. The relative recoveries ranged from 96 to 108%, intraday and interday precisions were lower than 13%. Different samples were subjected to the extractions using the method developed with four positive samples for the analyte.     

Green capsule phase microextraction employing hydrophobic monolithic sol-gel octadecyl siloxane platforms for the monitoring of organophosphorus pesticides in environmental water samples

In this study, a novel, facile and green capsule phase microextraction (CPME) method is presented for the extraction and preconcentration of organophosphorus pesticides (i.e., chlorpyrifos, disulfoton, ethoprophos, fenchlorphos, prothiofos, and parathion-methyl) from environmental water samples. Monolithic sol-gel octadecyl siloxane (sol-gel C₁₈) sorbent encapsulated within porous polypropylene capsules was synthesized, characterized, and evaluated for its efficiency towards the adsorption of the target organophosphorus pesticides. CPME was combined with gas chromatography-mass spectrometry (GC-MS) for the monitoring of the target analytes. The method was optimized to ensure high method sensitivity and it was fully validated. The limits of detection of the CPME-GC-MS method for the OPPs were 0.02–0.15 ng mL^?1. The relative standard deviations were 1.5–8.7% for intra-day study and 5.4–9.6% for inter-day study, demonstrating satisfactory precision. Moreover, good method accuracy was obtained, since the relative recoveries were within the range 92.6–107.0% and 90.8–107.6% for intra-day and inter-day (c = 5.00 and 20.0 ng mL^?1), respectively. The absence of interferences in the blank samples demonstrates that the proposed method is selective. The sol-gel C₁₈ sorbent encapsulated CPME media could be reused for at least 25 adsorption/desorption cycles. In addition, the methodology presents advantageous features in comparison to existing methods. The final protocol was used for analyzing four different water sample types (i.e., lake water, river water, pond water and tap water sample).     

Development and validation of a high-performance liquid chromatographic method for determination of pinocembrin in rat plasma: Application to pharmacokinetic study

A sensitive and specific reversed-phase high-performance liquid chromatography with ultraviolet detection (RP-UV-HPLC) method has been developed and validated for the identification and quantification of pinocembrin in rat plasma using chrysin as the internal standard. Following protein precipitation with acetonitrile, the analytes were separated by the mobile phase 0.01 M ammonium acetate (pH 4.0)–methanol (35:65, v/v) with an Agilent TC-C18 column (5 μm, 4.6 mm × 150 mm) at a flow rate of 1 ml/min, column temperature 40 °C and detection wavelength 290 nm. A good linear relationship was obtained in the concentration range studied (0.07–133.33 μg/ml, r = 0.9995). The lowest limit of quantification (LLOQ) was 66.7 ng/ml and the lowest limit of detection (LLOD) was 25 ng/ml. Average recoveries ranged from 93.9 to 97.8% in plasma at the concentrations of 0.33 and 33.33 μg/ml. Intra- and inter-batch relative standard deviations were 0.15–2.03 and 1.18–9.96%, respectively. This method was successfully applied to the pharmacokinetic studies in rats after intravenous administration of pinocembrin.     

Synthesis and anti-HIV activity of some novel lactyl and glycolyl phosphate derivatives.

Novel phosphate triester derivatives of 3'-acetylthymidine, and of the anti-HIV nucleoside analogue AZT have been prepared by phosphorochloridate chemistry. These materials are designed to act as membrane-soluble pro-drugs of the bio-active free nucleotides. In particular, novel glycolate and lactate phosphate derivatives have been prepared. In vitro evaluation revealed the AZT compounds to have a pronounced and selective antiviral effect, the magnitude of which varied considerably with the nature of the phosphate blocking group.     

Determination of higenamine in multi-matrix by gas chromatography-mass spectrometry combined with derivatization technology

Higenamine (HG), a cardioactive component of some foods and medicines, has been listed in the doping category by the International Olympic Committee, which may lead to misuse by athletes. We report the development of a gas chromatography-mass spectrometry (GC–MS) method for determination of HG in various matrix samples (biological samples, different forms of Chinese patent medicine, Chinese herbal medicine) based on acylation derivatization of HG by heptafluorobutyric anhydride. Under optimal conditions, the linearity of HG in the range of 5–200 ng mL⁻¹ was acceptable (R² > 0.999), and the limit of detection (LOD) and limit of quantitation (LOQ) for HG was 1.52 ng mL⁻¹ and 5 ng mL⁻¹, respectively. Low, medium, and high concentrations (25, 100 and 160 ng mL⁻¹) of HG were added to plasma, urine, oral liquid, capsule, watered bolus, honeyed bolus and Chinese herbal medicine samples, with recovery ranging from 82.70 to 109.80%, intra-day and inter-day precisions were both less than 3.39%. The results indicated that the method had sufficient sensitivity for analysis of biological samples, and Chinese patent and herbal medicine.     

Three-phase,liquid-phase microextraction combined with high performance liquid chromatography-fluorescence detection for the simultaneous determination of fluoxetine and norfluoxetine in human plasma

A three-phase, liquid-phase microextraction using a hollow fibre (HF-LPME) combined with high performance liquid chromatography-fluorescence detection (HPLC-FL) was developed for the analysis of fluoxetine (FLX) and its active metabolite, norfluoxetine (NFLX), in human plasma. An HF-LPME system using a disposable 7-cm polypropylene porous hollow fibre, 5 mL of alkaline plasma solution (donor phase), n-hexyl ether (extraction solvent) and 20 mM hydrochloric acid (acceptor phase) was used in the extraction. The method was validated after optimisation of several parameters that influence LPME efficiency. A reverse-phase LiChrospher 60 RP-Select B column (125 mm × 4 mm, 5 μm particle size) was used with 0.005 M sodium acetate buffer (pH 4.5) and acetonitrile at a 50:50 (v/v) as the mobile phase at a flow rate of 0.6 mL min⁻¹. In these conditions satisfactory chromatographic resolution and efficiency for the analytes were obtained. Fluorescence detection at 230 nm excitation wavelength and 290 nm emission wavelength was performed. Linearity over a range of 5–500 ng mL⁻¹, with determination coefficients (R²) of 0.9999 and 0.9962 for FLX and NFLX, respectively, was established. Venlafaxine was used as the internal standard for both analytes. Extraction recoveries from plasma samples were 70.9% for FLX and 59.7% for NFLX. The intra-day coefficients of variation (CVs) were below 5.4%, and inter-day CVs were below 13.0%, for both analytes at concentrations of 20, 80 and 160 ng mL⁻¹. HF-LPME extraction followed by HPLC-FL detection for FLX and NFLX analyses demonstrated excellent sample clean-up and selectivity. This method was simple, cheap, and easy to perform, yielding substantial analytes enrichment. The method was applied to the analysis of samples from 12 patients under fluoxetine treatment and proved suitable for routine therapeutic drug monitoring for this antidepressant.     

Simple and rapid HPLC method for simultaneous determination of atenolol and chlorthalidone in spiked human plasma

A simple, sensitive and rapid chromatographic method was developed and validated for the simultaneous quantification of atenolol and chlorthalidone in human plasma using hydrochlorothiazide as internal standard (IS). The method utilized proteins precipitation with acetonitril as the only sample preparation involved prior to reverse phase-HPLC. The analytes were chromatographed on Shim-pack cyanopropyl column with isocratic elution with 10 mM KH₂PO₄ (pH 6.0) – methanol (70:30, v/v) at ambient temperature with flow rate of 1 mL min⁻¹ and UV detection at 225 nm. The chromatographic run time was less than 10 min for the mixture. The calibration curves were linear over the range of 0.1–10 μg mL⁻¹. The method was validated in terms of accuracy, precision, absolute recovery, freeze–thaw stability, bench-top stability and re-injection reproducibility. The within- and between-day accuracy and precision were found to be within acceptable limits <15%. The analytes were stable after three freeze–thaw cycles (deviation <15%). The proposed method was specific for the simultaneous determination of atenolol and chlorthalidone in human plasma where there was no interference from endogenous biological substances.