Determination of the N-methyl-D-aspartate receptor NR2B subunit blocker Ro 63-1908 in rat, cynomolgus monkey and human plasma by high-performance liquid chromatography with column switching and fluorescence detection

A HPLC method with automated column switching was developed and validated for the determination of Ro 63-1908 in rat and cynomolgus monkey plasma. Human plasma was used for calibration and was also included in the validation process. Ro 63-1908 belongs to a class of neuroprotective N-methyl-D-aspartate (NMDA) receptor blockers which were in development for the treatment of stroke and traumatic brain injury. The method involves deproteinisation of plasma samples with ethanol and direct injection of the supernatant (1.4 ml) into the HPLC column-switching system. To prevent a breakthrough of the analyte and the internal standard on the precolumn (Purospher RP-18, 75x4 mm) due to the high ethanol content, the injection solution was diluted, on-line, using an additional pump and a T-piece. 1% ammonium acetate-ethanol (100:2, v/v) was used as mobile phase for injection, as well as for on-line dilution, resulting in pre-concentration of the analyte and the internal standard on the precolumn. As Purospher RP-18 is a non-endcapped stationary phase with a special selectivity for amines, the analyte and the internal standard could then be selectively eluted with 30% acetonitrile (without any buffer in the mobile phase) and transferred to the analytical column [consisting of two coupled columns (125+250x4 mm) packed with Superspher 60 RP-select B], where they were separated by gradient elution and detected by fluorescence detection. Compared to the use of a 125 mm long precolumn and dilution of the supernatant with ammonium acetate prior to injection, the 75 mm precolumn and the on-line dilution procedure allowed about one third shorter run times (21 min) and, therefore, a higher sample throughput. The limit of quantification was 1 ng/ml using 0.4 ml plasma. The method was applied to more than 670 plasma samples from pharmacokinetic and toxicokinetic studies and is also suitable for other matrices and NMDA receptor blockers.     

Determination of the N-methyl- -aspartate receptor NR2B subunit blocker Ro 63-1908 in rat, cynomolgus monkey and human plasma by high-performance liquid chromatography with column switching and fluorescence detection

A HPLC method with automated column switching was developed and validated for the determination of Ro 63-1908 in rat and cynomolgus monkey plasma. Human plasma was used for calibration and was also included in the validation process. Ro 63-1908 belongs to a class of neuroprotective N-methyl- -aspartate (NMDA) receptor blockers which were in development for the treatment of stroke and traumatic brain injury. The method involves deproteinisation of plasma samples with ethanol and direct injection of the supernatant (1.4 ml) into the HPLC column-switching system. To prevent a breakthrough of the analyte and the internal standard on the precolumn (Purospher RP-18, 75×4 mm) due to the high ethanol content, the injection solution was diluted, on-line, using an additional pump and a T-piece. 1% ammonium acetate–ethanol (100:2, v/v) was used as mobile phase for injection, as well as for on-line dilution, resulting in pre-concentration of the analyte and the internal standard on the precolumn. As Purospher RP-18 is a non-endcapped stationary phase with a special selectivity for amines, the analyte and the internal standard could then be selectively eluted with 30% acetonitrile (without any buffer in the mobile phase) and transferred to the analytical column [consisting of two coupled columns (125+250×4 mm) packed with Superspher 60 RP-select B], where they were separated by gradient elution and detected by fluorescence detection. Compared to the use of a 125 mm long precolumn and dilution of the supernatant with ammonium acetate prior to injection, the 75 mm precolumn and the on-line dilution procedure allowed about one third shorter run times (21 min) and, therefore, a higher sample throughput. The limit of quantification was 1 ng/ml using 0.4 ml plasma. The method was applied to more than 670 plasma samples from pharmacokinetic and toxicokinetic studies and is also suitable for other matrices and NMDA receptor blockers.     

填充毛细管液相色谱-毛细管气相色谱在线联用分析八角茴香挥发油

首次将填充毛细管高效液相色谱-毛细管气相色谱在线联用技术(μ-HPLC-CGC)用于分离分析八角茴香果实的挥发油成分。液相色谱选用氰基分析柱(250 mm×0.32 mm i.d.),正己烷-乙腈-二氯甲烷(体积比为80∶8∶12)为流动相,对挥发油样品做族组分分离,得到的5个族组分被依次存放在多位储存接口内,然后不分流分别转入毛细管气相色谱仪做详细分析。气相色谱柱由10 m×0.53 mm i.d.保留间隔柱和30 m×0.53 mm i.d.×1.0 μm SE-54分析柱组成。采用了不分流柱内进样模     

High Performance Liquid Chromatographic Determination of Testosterone Propionate,Progesterone and Estradiol Benzoate in Bovine Implants

Abstract

A Method for the determination of testosterone propionate, progesterone, and estradiol benzoate in bovine implants is presented. The method utilizes a Dupont, zorbax DDS analytical column with a mobile phase consisting of methanol, deionized water, and tetrahydrofuran (65:30:15 v/v). Sample preparation consists of of dissolution of the implant, followed by dilution and injection onto the HPLC system. The procedure is accurate, precise, linear, and specific for the implant steroids with quantitation by peak height, using dipenty 1 phthalate as the internal standard.     

Optimization of dynamic headspace sampling for the analysis of trace volatile components of grape juice: Use of a PTV injector for intermediate trapping

The use of a programmed temperature vaporizing (PTV) injector has been evaluated for the on-line concentration and injection of trace organic compounds either sampled from the head-space above grape juices or purged from solution. The Simplex method was used to improve the sensitivity of the method by optimization of the experimental conditions.     

Azithromycin quantitation in human plasma by high-performance liquid chromatography coupled to electrospray mass spectrometry: application to bioequivalence study

A sensitive and specific liquid chromatography-electrospray ionization mass spectrometry method is developed and validated for the identification and quantitation of azithromycin in human plasma. After the addition of the internal standard and 1.0M sodium hydroxide solution, plasma samples are extracted with a methylene chloride-ethyl acetate mixture (20:80, v/v). The organic layer is evaporated under a stream of nitrogen at 40 degrees C. The residue is reconstituted with 200 microL of the mobile phase. The compounds are separated on a prepacked Shimadzu Shim-pack VP-ODS C18 (5 microm, 150 mm x 2.0 mm) column using a mixture of acetonitrile-water (65:35) (0.5% triethylamine, pH was adjusted to 6.2 with acetic acid) as the mobile phase. Detection is performed on a single quadrupole mass spectrometer by selected ion monitoring mode via electrospray ionization source. The method is fully validated and linear calibration curves are obtained in the concentration ranges from 5 to 2000 ng/mL. The intra- and inter-batch relative standard deviations at four different concentration levels are all < 10%. The limit of detection and quantitation are 2 ng/mL and 5 ng/mL, respectively. The proposed method enables the unambiguous identification and quantitation of azithromycin for pharmacokinetic, bioavailability, or bioequivalence studies.     

On-line extraction coupled with liquid chromatography tandem mass spectrometry for quantitation of pravastatin and its metabolite in human serum

A high-throughput bioanalytical method for simultaneous quantitation of pravastatin and its metabolite (M1) in human serum was developed and validated using on-line extraction following liquid chromatography tandem mass spectrometry (LC-MS/MS). The on-line extraction was accomplished by the direct injection of a 50 microL serum sample, mixed 4:1 with an aqueous internal standard solution, into one of the extraction columns with aqueous 1 mm formic acid at flow rate of 3 mL/min. The separation and analysis were achieved by back-eluting the analytes from the extraction column and the analytical column to the mass spectrometer with an isocratic mobile phase consisting of 62% aqueous 1 mm formic acid and 38% acetonitrile at a flow rate of 0.8 mL/min. The second extraction column was being equilibrated while the first column was being used for analysis, and vice versa. The standard curve range was 0.500-100 ng/mL for pravastatin and M1. The lower limit of quantitation, 0.500 ng/mL for all the analytes, was achieved when 50 microL of human serum was used. The intra- and inter-day precisions were within 7.4%, and the accuracy was between 95 and 103%. The on-line extraction was finished in 0.5 min and total analysis time was 2.5 min per sample.     

Quantitative aspects of directly coupled supercritical fluid extraction–capillary gas chromatography with a conventional split/splitless injector as interface

The quantitative aspects of on-line supercritical fluid extractioncapillary gas chromatography (SFE-GC) with a split/splitless injector as interface were investigated. Special attention was paid to the discrimination behavior and the reproducibility of the split/splitless interface. A simple experimental set-up is proposed that allows accurate quantitation in on-line SFE-split GC. The results obtained in on-line SFE-GC compare favorably with those from conventional GC with split injection. Discrimination was found to be absent when working at sufficiently high interface temperatures. Finally, the effects of the carbon dioxide flow rate, interface temperature and split ratio on both discrimination and reproducibility were studied.     

Determination of ferrocene-A in the blood serum of rabbits using reversed-phase microcolumn HPLC

Ferrocene-A (ferrocenyl-1-phenyl-1-dioxy-1,4-butin-2) has shown significant antitumour and antibacterial activity. To facilitate studies in this field a simple and rapid liquid chromatographic procedure was developed, allowing the determination of ferrocene-A in the blood serum. The procedure was based on a reversed-phase high-performance liquid chromatography (HPLC) method, with UV detection at 210 nm. The mobile phase ethanol-0.01 M KH2PO4 60:40 (v/v) provides the complete separation of ferrocene-A, internal standard and endogenous compounds of serum. The recovery of ferrocene-A from serum using a hexaneisoamyl alcohol mixture was 90%. The proposed method is convenient for pharmacological and pharmacokinetic applications.     

顺序注射在线稀释微量进样技术及其在自动光度法测定水中亚硝酸盐的应用

以测定环境水样中的亚硝酸盐为例,采用顺序注射技术,提出了一种智能化在线微量进样稀释分析系统(SIA系统).将测定过程中分光光度计输出的模拟电压信号,反馈至顺序注射仪,判断是否需要稀释.当获得的电压信号值小于305 mV(约相当于吸光度值0.8)时,SIA系统进入稀释程序,否则,则进入直接测定程序,两种程序均自动完成.直接测定和稀释测定的线性范围分别为1～50μmol·L-1和50～500μmol·L-1NO2-.分别在NO2-浓度水平为10,200μmol·L-1时作精密度试验,测得相对标准偏差(n=11)依次为0.7%和1.0%.该分析系统特别适合于自动分析过程中被测物质浓度变化较大而需要在线稀释的情况.     

Quantitation of glucosamine from shrimp waste using HPLC

This work presents a high-performance liquid chromatography (HPLC) method for the quantitation of glucosamine in chitin. The method includes an acid hydrolysis of chitin. The chromatographic separation is achieved using a Hypersil ODS 5-microm column (250 x 4.6 mm) at 38 degrees C, with precolumn derivatization with 9-fluorenylmethyl-chloroformate and UV detection (lambda = 264 nm). The mobile phase is a mixture of mobile phase A [30 mM ammonium phosphate (pH 6.5) in 15:85 methanol-water (v/v)], mobile phase B [15:85 methanol-water (v/v)], and mobile phase C [90:10 acetonitrile-water (v/v)], with a flow rate of 1.2 mL/min. The HPLC method proposed showed adequate repeatability (relative standard deviation, 5.8%), accuracy (92.7% recovery), and sensitivity, with a detection limit of 2 microg/mL. The method is successfully applied to the quantitation of glucosamine for the determination of the purity of chitin from shrimp waste.     

Validation of the RP–HPLC method for analysis of hydrochlorothiazide and captopril in tablets

A rapid and sensitive reverse-phase high performance liquid chromatography (RP–HPLC) method with ultra-violet (UV) detection for a routine control of hydrochlorothiazide and captopril in tablets was developed. The chromatographic system Hewlet Packard 1100 consisted of a HP 1100 pump, HP 1100 UV–VIS detector and HP ChemStation integrator. The samples were introduced through a Rheodyne injector valve with a 20-L sample loop. The isocratic system consisted of a Beckman Ultrasphere ODS 4.6 mm x 15 cm, 5-m-particle column and a mobile phase containing methanol/water (45:55 v/v). The pH of the mobile phase was adjusted to 3.8 with 85% ortophosphoric acid. Quantitation was accomplished using the internal standard method. At the selected conditions, the other excipients of the tablets did not interfere in the assay of active substances. The developed RP–HPLC method was validated, so linearity, precision, accuracy, robustness, limit of quantitation and limit of detection were investigated. For the robustness test, three factors were considered: the composition of the mobile phase , the pH of the mobile phase, and temperature. With the aid of response surface metodology (RSM), it was possible to precisely define the robustness of the method.     

Separation of the two enantiomers of naproxcinod by chiral normal-phase liquid chromatography

A normal-phase enantioselective high-performance liquid chromatographic method was developed for the enantiomeric resolution of naproxcinod, the most advanced cyclooxygenase-inhibiting nitric oxide donator of anti-inflammatory drugs designed for treatment of osteoarthritis. The enantiomers of naproxcinod were resolved on a Chiralpak AD-H (250 × 4.6 mm, 5 μm) column using a mobile phase system containing n-hexane and 2-propanol (95:5, v/v). The resolution between the enantiomers was found to be more than 2.0. The limit of detection and limit of quantitation of (R)-enantiomer were found to be 5 and 15 ng/mL, respectively, for 20 μL injection volume. The sample solution and mobile phase were found to be stable for at least 48 h. The final optimized method was successfully applied to separate (R)-enantiomer from naproxcinod and was proven to be reproducible and accurate for the quantitative determination of (R)-enantiomer in bulk drugs.     

Determination of carboxylic acids, carbohydrates, glycerol, ethanol, and 5-HMF in beer by high-performance liquid chromatography and UV-refractive index double detection

A high-performance liquid chromatographic method is proposed for the simultaneous separation of main carboxylic acids, carbohydrates, ethanol, glycerol, and 5-HMF in beer by direct injection. A column packed with a sulfonated divinyl benzene-styrene copolymer and an isocratic elution with 0.0045N sulfuric acid and acetonitrile (6%, v/v) are employed. UV and refractive index detectors connected in series are also used to reduce the matrix interference of phenolic compounds. In conditions described, nine compounds are quantitated in a single chromatographic run without any pretreatment except for sample dilution and filtration before injection. Precision, accuracy, linearity of response, limit of detection, and limit of quantitation are also evaluated for each compound. Satisfactory results are obtained to justify the application of this method to all phases of beer production for process and quality control.     

Determination of ampicillin in New Zealand white rabbit plasma using column switching technique and HPLC

Summary The purpose of this study was to develop a simple and fast analytical method for quantitation of ampicillin in rabbit plasma, suitable for analysis of large numbers of samples collected from experimental animals. The concentration of ampicillin in rabbit plasma was determined utilizing ion-pair reverse-phase high performance liquid chromatography (HPLC) with UV detection and a column switching technique. Plasma samples were treated with a perchloric acid solution to precipitate proteins and centrifuged to pellet the precipitated proteins. Cephalexin was used as an internal standard. The C₁₈ precolumn was placed in the injector loop of the Rheodyne injector. Samples were injected with the injector in the load position and the precolumn was washed free from interfering compounds. When the injector was switched to the inject position, the mobile phase was passed through the precolumn taking relatively pure compounds onto the analytical column. The limit of quantitation was established to be 400 ng mL^–1 of plasma. The standard curves were linear over the range of ampicillin concentrations assayed, 400 to 10,000 ng mL^–1 of rabbit plasma, and had a mean regression coefficient of 0.9962 (±0.0043). Intra-day variability was determined using six replicates of controls (low and high) analyzed on a single assay. The percent of relative accuracy for low and high controls were 5.67 and 1.12, respectively. Inter-day variability was determined over a four day period analyzing replicates of each control. The percent of relative accuracy for low and high controls were 4.33 and 1.63, respectively.     

高效液相色谱-荧光检测法测定牛奶中氯霉素的残留量

建立了对牛奶中氯霉素的残留量进行检测的高效液相色谱-荧光检测方法。氯霉素还原后在温和条件下与荧光胺发生衍生化反应,采用十八烷基键合硅胶固定相,以乙腈/四氢呋喃/0.02 mol/L醋酸钠-醋酸缓冲液(pH 6.0)(体积比为16∶8∶76)为流动相,流速1.0 mL/min,柱温40 ℃,荧光检测激发波长为410 nm,发射波长为508 nm。在上述实验条件下,氯霉素检测的线性范围为0.4~800 μg/L (r2=0.9999),检出限为0.2　μg/L。当空白样品中氯霉素添加水平为2~40 μg/L时,该方法的回收率为66.6%~92.8%,相对标准偏差为4.5%~9.4%。该方法适用于牛奶中氯霉素痕量残留的监测,具有干扰小、选择性好、灵敏度高等优点。     

Column switching and high-performance liquid chromatography in the analysis of amitriptyline, nortriptyline and hydroxylated metabolites in human plasma or serum.

A column-switching system for the direct injection of plasma or serum samples, followed by isocratic high-performance liquid chromatography and ultraviolet detection, is described for the simultaneous quantitation of the tricyclic antidepressant amitriptyline, its demethylated metabolite nortriptyline and the E- and Z-isomers of 10-hydroxyamitriptyline and 10-hydroxynortriptyline. The method included adsorption of amitriptyline and metabolites on a reversed-phase C8 clean-up column (10 microns; 20 mm x 4.6 mm I.D.), washing of unwanted material to waste and, after on-line column-switching, separation on a cyanopropyl analytical column (5 microns; 250 mm x 4.6 mm I.D.). The compounds of interest were separated and eluted using acetonitrile-methanol-0.01 M phosphate buffer (pH 6.8) (578:188:235, v/v) within less than 20 min. Various drugs frequently co-administered with amitriptyline or other antidepressants did not interfere with the determinations. In plasma samples spiked with 25-300 ng/ml, the recoveries were between 84 and 112% and the inter-assay coefficients of variation were 3-11%. After a minor modification, as little as 5 ng/ml could be quantitated. There were linear correlations (r greater than 0.99) between drug concentrations of 5-500 ng/ml and the detector signal. The method allows routine measurements of amitriptyline, nortriptyline and hydroxylated metabolites in blood plasma or serum of patients treated with amitriptyline or nortriptyline, and enables the results to be reported within 1 h.     

Simplified high-performance liquid chromatographic determination of residual amprolium in edible chicken tissues

A simplified determining method for the routine monitoring of residual amprolium in edible chicken tissues (muscle and liver) is developed using a high-performance liquid chromatographic (HPLC) method with a photodiode-array detector after sample cleanup by an Ultrafree-MC/PL centrifugal ultrafiltration unit. For the HPLC determination and identification, a Mightysil RP4 GP column and a mobile phase of an ethanol-5 mM 1-heptanesulfonic acid sodium salt solution (35:65, v/v) using an ion-pairing system with a photodiode-array detector are used. Average recoveries (spiked at 0.3-3.0 microg/g) are > 90%. The inter- and intravariabilities are 1.9-2.4%. The limits of quantitation are 0.22 microg/g for muscle and 0.25 microg/g for liver. The total time and solvent required for the analysis of one sample are < 20 min and < 2 mL of ethanol, respectively. No toxic solvents and regents are used.     

Simplified high-performance liquid chromatographic determination of residual amprolium in edible chicken tissues

A simplified determining method for the routine monitoring of residual amprolium in edible chicken tissues (muscle and liver) is developed using a high-performance liquid chromatographic (HPLC) method with a photodiode-array detector after sample cleanup by an Ultrafree-MC/PL centrifugal ultrafiltration unit. For the HPLC determination and identification, a Mightysil RP-4 GP column and a mobile phase of an ethanol-5 mM 1-heptanesulfonic acid sodium salt solution (35:65, v/v) using an ion-pairing system with a photodiode-array detector are used. Average recoveries (spiked at 0.3-3.0 microg/g) are > 90%. The inter- and intravariabilities are 1.9-2.4%. The limits of quantitation are 0.22 microg/g for muscle and 0.25 microg/g for liver. The total time and solvent required for the analysis of one sample are < 20 min and < 2 mL of ethanol, respectively. No toxic solvents and regents are used.     

Simple determination of betamethasone and chloramphenicol in a pharmaceutical preparation using a short monolithic column coupled to a sequential injection system

This contribution describes the use of a new separation method based on a reversed-phase sequential injection chromatography (SIC) technique for simultaneous determination of chloramphenicol and betamethasone in pharmaceutical eye drops. A short monolithic column coupled with a sequential injection analysis (SIA) system enabled separation of two compounds in one step. A Chromolith Flash RP-18e, 25 x 4.6 mm column with a 5 mm precolumn (Merck, Germany) and a FIA1ab 3000 system (USA) with a 6-port selection valve and 5 mL syringe were used for sequential injection chromatographic separations in this study. The mobile phase used was acetonitrile-water (30:80, v/v), flow rate 0.48 mL/min; UV detection was at two wavelengths, i.e., 241 and 278 nm (absorption maxima of betamethasone and chloramphenicol, respectively). The basic validation parameters showed good results: linearity of determination for both compounds including internal standard (propylparaben) >0.999; repeatability of determination (RSD) in the range 0.8-1.7% at two different concentration levels, and detection limits in the range 0.5-1.0 mg/mL. The chromatographic resolution between compound peaks was greater than 2.1 and the analysis time was less than 8 min under optimal conditions. The developed sequential injection chromatography method was compared with the HPLC method and was found to be applicable for routine analysis of active compounds in pharmaceutical preparations.