High-performance liquid chromatographic assay for 1-aminocyclopropanecarboxylic acid from plasma and brain.

A reversed-phase high-performance liquid chromatographic method for the analysis of 1-aminocyclopropanecarboxylic acid (ACPC) from plasma or brain tissue is described. Samples were deproteinized with perchloric acid, centrifuged, alkalinized with potassium hydroxide and recentrifuged. The supernatants were derivatized with o-phthaldialdehyde and injected onto a C18 3-microns column (100 mm x 4 mm I.D.) pumped with 1 ml/min methanol-acetonitrile-0.1 M sodium phosphate buffer pH 6.0 (28:5:67, v/v). The retention times for ACPC and the internal standard were 15 and 31 min, respectively. The minimum detectable amount of ACPC was 0.08 nmol. The extraction recovery of ACPC (2.7-270 nmol) from spiked plasma or brain tissue ranged from 88 to 109%. The intra- and inter-day coefficients of variation for 27 nmol ACPC were 3.9 and 4.9%, respectively. This method was utilized to obtain preliminary pharmacokinetic parameters following ACPC administration to mice.     

Quantification of myrislignan in rat plasma by solid-phase extraction and reversed-phase high-performance liquid chromatography

A rapid and simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for determination of myrislignan in rat plasma after intravenous administration. The analytes extracted from plasma samples by solid-phase extraction were successfully carried out on a Diamonsiltrade mark ODS C(18) column (250 x 4.6 mm i.d., 5 microm) with an RP(18) guard column (8 x 4.6 mm i.d., 5 microm) and a mobile phase of MeOH-H(2)O (4:1, v/v). The UV detector was set at a single wavelength of 270 nm. The linear ranges of the standard curves were 0.5-30.0 microg/mL with the correlation coefficients greater than 0.9992. The lower limits of detection and quantification were 0.1 and 0.3 microg/mL for myrislignan. Intra- and inter-day precisions were 2.4-7.5 and 1.3-5.7%, respectively. The extraction recovery from plasma was more than 90%. This assay method has been successfully used to study the pharmacokinetics of myrislignan in rats.     

Direct injection HPLC micro method for the determination of voriconazole in plasma using an internal surface reversed-phase column

A direct plasma injection liquid chromatographic method has been developed for the determination of a new triazole antifungal agent, voriconazole, using an internal surface reversed phase column. Therapeutic drug monitoring of voriconazole is relevant for patient management, especially in the case of drug-drug interaction. The method is easy to perform and requires 10 microL of a plasma sample. The chromatographic run time is less than 9 min using a mobile phase of 17:83 v/v acetonitrile-potassium dihydrogen phosphate buffer, 100 mM, pH 6.0 and UV detection at 255 nm. The fl ow rate was 1 microL/min. A linear response was observed over the concentration range 0.5-10 microg/mL (r2 = 0.977). A good accuracy (bias < or = 7.5%) was achieved for all quality controls, with intra-day and inter-day variation coefficients inferior to 6.7%. The lower limit of quantitation was 0.2 microg/mL, without interference of endogenous components. The stability of voriconazole in plasma stored at different temperatures was checked. Finally, the possibility of direct injection of plasma samples into the column permits a reduction in reagent consumption and in analytical steps, and hence in analytical error.     

Determination of erythromycin ethylsuccinate by liquid chromatography.

A method is described for the determination of erythromycin ethylsuccinate by liquid chromatography. A C18 reversed-phase column (25 cm x 4.6 mm I.D.) was used with acetonitrile-0.2 M tetrabutylammonium sulphate (pH 6.5)-0.2 M phosphate buffer (pH 6.5)-water [x:5:5:(90-x)] as mobile phase. The proportion of acetonitrile (x) has to be adapted to the type of stationary phase used. For RSil C18 LL, 42.5% was used. The column was heated at 35 degrees C, the flow-rate was 1.5 ml/min and UV detection was performed at 215 nm. The main component, erythromycin A ethylsuccinate, was separated from all other components which were present in commercial samples. The main impurities were erythromycin A and the ethylsuccinate esters of erythromycin B and C. The amide N-ethylsuccinyl-N-demethylerythromycin A was shown to be present in all the samples examined. The method was successfully applied to the analysis of specialities.     

Determination of human plasma protein binding of baicalin by ultrafiltration and high-performance liquid chromatography

A simple and selective HPLC assay was developed and utilized for determination of human plasma protein binding of baicalin. The method involved solid-phase extraction and reversed-phase chromatographic separation with a mobile phase of acetonitrile-0.02 mol/L phosphate buffer (pH 2.5; 25:75, v/v) and UV detection at 276 nm. The standard curve for baicalin was linear over the concentration range 0.1-20 microg/mL and the limit of detection was 0.02 microg/mL. The absolute recovery was greater than 76%. The intra-day and inter-day variations were less than 10%. Ultrafiltration technique was applied to determining the plasma protein binding of baicalin in human plasma. Results show the plasma protein binding of baicalin was in the range 86-92% over all the concentrations studied and the protein binding association constant was determined to be 1.21 x 10(5) L/mol at 4 degrees C.     

A high-performance liquid chromatographic method for determination of the niguldipine analogue DHP-014

A simple and reliable reversed-phase high-performance liquid chromatography method was developed and validated for the determination of DHP-014, a niguldipine analogue with potent P-glycoprotein inhibitory and negligible calcium channel blocking properties, in rat plasma. DHP-014 and niguldipine hydrochloride (the internal standard) were extracted from rat plasma by liquid extraction using hexane. DHP-014 was then separated by HPLC on a C18 column and quantified by ultraviolet detection at 238 nm. The mobile phase consisted of acetonitrile-aqueous 5 mM phosphate buffer (65:35, v/v) containing 0.4% (v/v) triethylamine adjusted to pH 7.0. The mean extraction efficiency of DHP-014 was 109.0 +/- 12.9, 97.7 +/- 8.0 and 102.9 +/- 7.5% for DHP-014 concentrations of 10, 50 and 100 nM, respectively (n = 5). The method was linear over the concentration range 2.5-200 nM with a regression coefficient of 0.998. The limit of detection of DHP-014 in rat plasma was 1.0 nM. The intra- and inter-day coefficients of variation for DHP-014 in rat plasma were 4.7-7.9 and 6.9-9.9%, respectively. The intra- and inter-day accuracy was 98.2-99.5 and 97.9-103%, respectively. The bioanalytical technique was used to determine DHP-014 in plasma samples in a pharmacokinetic study of DHP-014 administered to female Sprague-Dawley rats.     

A dual-column HPLC method for the simultaneous determination of DHPG (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) and its mono and diesters in biological samples

A convenient method for the simultaneous determination of various DHPG species present in biological samples is presented. This method utilizes a cation exchange column (25 cm X 4.6 mm i.d.) coupled in series to a short reversed-phase column (5 cm X 4.6 mm i.d.). The mobile phase consists of methanol:0.005M ammonium phosphate buffer, pH 2.5. There is a large polarity difference between DHPG and its esters due to the non-polar side chain of the ester moiety. The simultaneous determination of the diesters, monoesters, and DHPG in these samples using only the cation exchange or the reversed-phase column is not possible without time-consuming gradient elution. In the reversed-phase mode alone, the esters are highly retained relative to DHPG, whereas the esters are only slightly retained on a cation exchange column and are insensitive to changes in pH and ionic strength of the mobile phase. However, a combination of these two columns provides interesting selectivity for these compounds and offers a unique way of controlling the retention times of these species relative to each other. The retention time of esters can be selectively altered (with respect to DHPG) by changing the composition of methanol in the mobile phase. In contrast, the retention time of DHPG is controlled by changing the buffer strength and pH of the mobile phase.     

High-performance liquid chromatographic determination of usnic acid in plasma.

A high-performance liquid chromatographic method for the determination of usnic acid in human plasma using diclofenac sodium as internal standard is described. Plasma proteins were precipitated with methanol. A 250 mm x 4 mm I.D. Nucleosil. C18 (5 microns) column with a mobile phase consisting of methanol-phosphate buffer (pH 7.4) (70:30, v/v) was used. Chromatography was performed at ambient temperature with flow-rate of 1 ml min-1 and ultraviolet detection at 280 nm. Each analysis required no longer than 7 min. Quantification was achieved by measurement of the peak-height ratio and the absolute recovery varied from 93.8 to 97.3%. The limit of quantitation of usnic acid in plasma was 0.25 micrograms ml-1. The intra-day relative standard deviation (R.S.D.) ranged from 1.24 to 4.53% and the inter-day R.S.D. from 2.23 to 8.25% at three different concentrations. The method was applied to the determination of plasma levels of usnic acid after intravenous and oral administration to study its disposition in a healthy male rabbit.     

Determination of paeoniflorin in rat plasma by a liquid chromatography-tandem mass spectrometry method coupled with solid-phase extraction

A rapid, sensitive and selective liquid chromatography-tandem spectrometry method was developed and validated for determination of paeoniflorin in rat plasma using geniposide as the internal standard. The samples were pretreated with solid-phase extraction using Extract-Clean cartridges. Separation of paeoniflorin and IS was achieved on a reversed-phase C18 column (50x4.6 mm i.d.) with a mobile phase made up of acetonitrile and 0.05% formic acid (25:75, v/v) at a flow rate of 0.5 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer by multiple-reaction monitoring and an electrospray ionization source was employed as the ionization source. The lower limit of quantification obtained was 4 ng/mL (n=6) using 200 microL plasma with an accuracy of -3.67% (relative error) and a precision of 4.13% (relative standard deviation). A good linearity was found in the range of 4-1000 ng/mL. The intra- and inter-day relative standard deviations in the measurement of quality control samples 10, 150 and 800 ng/mL ranged from 3.73 to 4.94% and from 4.31 to 6.56%, respectively. The accuracy was from -3.93 to -1.11% in terms of relative error. The analyte and IS were stable in the battery of stability studies. This method was successfully applied to a pharmacokinetic study of paeoniflorin after a single oral administration of 53.36 mg/kg paeoniflorin to rats.     

Simultaneous determination of estramustine phosphate and its four metabolites in human plasma by liquid chromatography-ionspray mass spectrometry

A sensitive and selective method, using liquid chromatography-ionspray mass spectrometry, was developed and validated for the simultaneous determination of Estracyt (estramustine phosphate) and its four metabolites, estramustine, estromustine, estrone and estradiol, in human plasma. Deuterated internal standards were available for all analytes. The five compounds were extracted from plasma by protein precipitation with acetonitrile. The chromatographic separation was performed using a Zorbax SB C18, (150 x 4.6 mm i.d., 5 microm) reversed-phase column under gradient conditions with a mobile phase containing 2 mm ammonium acetate buffer (pH 6.8) and acetonitrile. MS detection was by electrospray ionization with multiple reaction monitoring in the positive ion mode for estramustine phosphate, estromustine and estramustine, and in the negative ion mode for estrone and estradiol. The limit of quantitation was 10 ng/mL for estramustine phosphate, 3 ng/mL for estromustine, estramustine and estrone and 30 ng/mL for estradiol. Linearity was verified from these LLOQs up to about 4000 ng/mL for the parent drug and 2000 ng/mL for the metabolites. Inter-day precision and accuracy values were all less than 15%. This assay was applied successfully to the routine analysis of human plasma samples collected in cancer patients administered estramustine phosphate intravenously.     

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.     

Selective and sensitive determination of bis(7)-tacrine, a high erythrocyte binding acetylcholinesterase inhibitor, in rat plasma by high-performance liquid chromatography-tandem mass spectrometry

The current study aims to develop a specific and sensitive LC-MS/MS method for determination of bis(7)-tacrine (B7T) in rat plasma. A 100 microL plasma sample was extracted with ethyl acetate. B7T and the internal standard (IS), pimozide, in the samples were then analyzed with LC-MS/MS in positive electrospray ionization condition. Chromatographic separation of B7T and IS was achieved in a C(18) reversed-phase HPLC column (150 x 2.1 mm i.d.) by isocratic elution with a mobile phase consisting of 0.05% formic acid in water and acetonitrile (1:1, v/v) at a flow rate of 0.35 mL/min. Multiple-reaction monitoring (MRM) mode was employed to measure the ion transitions: m/z 247 to 197 for B7T and m/z 462 to m/z 328 for IS, respectively. The method was linear over the studied ranges of 100-5000 and 10-100 ng/mL. The intra-day and inter-day variations of the analysis were less than 6.8% with standard errors less than 9.0%. The detection limit of B7T in rat plasma was 1 ng/mL. The developed method was successfully applied to the pharmacokinetic study of B7T after intravenous administration of 1 mg/kg B7T and further proved to be readily utilized for determination of B7T in rat plasma samples.     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of wogonoside in rat serum after oral administration of traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction

A high-performance liquid chromatographic method for the determination of wogonoside in plasma of rats administrated orally with the traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction was developed. Sample preparation was carried out by protein precipitation with a mixture of acetonitrile and methanol (1:1, v/v). The extracted sample was separated on a Hypersil C(18) (150 x 5 mm i.d., 5 microm) analytical column by linear gradient elution using 0.05% (v/v) phosphoric acid (containing 5 mm sodium dihydrogen phosphate) and acetonitrile as mobile phase at a flow rate of 1.5 mL/min. The eluate was detected using a UV detector at 276 nm. The assay was linear over the range 0.109-7.0 microg/mL (R(2) = 0.9999, n = 5). Mean recovery was determined as 98.39%. Intra- and inter-day precisions (RSD) were < or =7.59%. The limit of quantitation was 0.109 microg/mL. After validation, the HPLC method developed was applied to investigate the preliminary pharmacokinetics of wogonoside in rat after oral administration of Huang-Lian-Jie-Du decoction.     

Validation and pharmacokinetic application of a method for determination of doxazosin in human plasma by high-performance liquid chromatography

A simple high-performance liquid chromatographic method for the determination of doxazosin in human plasma was developed and validated. Prazosin was used as internal standard. After extraction twice with ethyl acetate, chromatographic separation of doxazosin in human plasma was carried out using a reversed-phase Apollo C18 column (250 x 4.6 mm, 5 microm) with mobile phase of methanol-acetonitrile-0.04 m disodium hydrogen orthophosphate (22:22:56, v/v/v) adjusted to pH 4.9 with 0.9 m phosphoric acid and quantified by fluorescence detection operated with an excitation wavelength of 246 nm and an emission wavelength of 389 nm. The lower limit of quantification (LLOQ) of this assay was 1 ng/mL using 500 microL human plasma. Linearity was established over the range 1-25 ng/mL (r2 > 0.9994). The intra- and inter-day accuracy ranged from 90.5 to 104.4% and the coefficient of variation were not more than 8.6% for both intra- and inter-day precision, over the range of the calibration curve. The absolute recoveries of doxazosin and prazosin from human plasma were more than 91%. Doxazosin demonstrated acceptable short-term, long-term and freeze-thaw stability in human plasma. The assay has been successfully applied to plasma sample ana-lysis for pharmacokinetic study.     

Simultaneous determination of free and bound adriamycin, adriamycinol, adriamycinone and duanorubicin in plasma using column-switching technique and protein-coated pre-columns

Adriamycin, adriamycinol, adriamycinone and duanorubicin were simultaneously determined by the development of an on-line plasma clean-up system. A short protein-coated Lichrosorb, RP-8, RP-2, CN and muBondapak phenyl as well as ODS silica have been examined for their performance as pre-columns. The drugs and metabolites were separated from weakly retained plasma components through two steps; phosphate buffer saline, pH 7.4 and 15% acetonitrile in 0.1 M sodium dihydrogen phosphate, pH 3. The chromatographic conditions were: ODS/TM column, flow rate 1 ml/min, 35% acctonitrile in 0.1 M sodium dihydrogen phosphate (pH 3) containing 0.3% heptafluorobutyric acid as mobile phase. The detection was carried out using fluorescence monitor operated at an emission 555 nm and excitation 460 nm. Good resolution was obtained within 13 min. This method is reproducible for analysis of drugs and metabolites (99.3-100.1%, CV < 2%) in plasma.     

High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man.

High-performance liquid chromatographic assay procedures have been developed for naproxen, ibuprofen and diclofenac in human plasma and synovial fluid samples. A single liquid-liquid extraction procedure was used to isolate each compound from acidified biological matrix prior to the quantitative analysis. A Spherisorb ODS column (12.5 cm x 4.6 mm I.D.) was used for all the chromatography. Naproxen was eluted with a mobile phase of methanol-S?rensen's buffer at pH 7 (37:63, v/v). Ibuprofen and diclofenac were eluted using mobile phases of methanol-water at pH 3.3 (65:35, v/v and 63:37, v/v, respectively). Diphenylacetic acid was used as the internal standard for the assay of naproxen and flurbiprofen was used in the analysis of ibuprofen and diclofenac. Inter- and intra-day coefficients of variation were less than 7%. The assays were used in clinical studies of the three drugs in osteo- and rheumatoid arthritis patients.     

Optimization via experimental design of an SPE-HPLC-UV-fluorescence method for the determination of valsartan and its metabolite in human plasma samples

A chemometric approach was applied for the optimization of the extraction and separation of the antihypertensive drug valsartan and its metabolite valeryl-4-hydroxy-valsartan from human plasma samples. Due to the high number of experimental and response variables to be studied, fractional factorial design (FFD) and central composite design (CCD) were used to optimize the HPLC-UV-fluorescence method. First, the significant variables were chosen with the help of FFD; then, a CCD was run to obtain the optimal values for the significant variables. The measured responses were the corrected areas of the two analytes and the resolution between the chromatographic peaks. Separation of valsartan, its metabolite valeryl-4-hydroxy-valsartan and candesartan M1, used as internal standard, was made using an Atlantis dC18 100 mm x 3.9 mm id, 100 angstroms, 3 microm chromatographic column. The mobile phase was run in gradient elution mode and consisted of ACN with 0.025% TFA and a 5 mM phosphate buffer with 0.025% TFA at pH 2.5. The initial percentage of ACN was 32% with a stepness of 4.5%/min to reach the 50%. A flow rate of 1.30 mL/min was applied throughout the chromatographic run, and the column temperature was kept to 40+/-0.2 degrees C. In the SPE procedure, experimental design was also used in order at achieve a maximum recovery percentage and extracts free from plasma interferences. The extraction procedure for spiked human plasma samples was carried out using C8 cartridges, phosphate buffer (pH 2, 60 mM) as conditioning agent, a washing step with methanol-phosphate buffer (40:60 v/v), a drying step of 8 min, and diethyl ether as eluent. The SPE-HPLC-UV-fluorescence method developed allowed the separation and quantitation of valsartan and its metabolite from human plasma samples with an adequate resolution and a total analysis time of 1 h.     

毛细管区带电泳法测定血浆中的苯妥英钠

 建立了以毛细管区带电泳测定血浆中苯妥英钠含量的方法。此法具有良好的重现性和线性关系 ,日内、日间的平均相对标准偏差分别为 3.1%和 4 .7% ,平均回收率大于 95 % ,标准曲线的相关系数为 0 9985 ,是一种简便、快速、准确、灵敏的测定方法 。     

Quantitative determination of p-aminosalicylic acid and its degradation product m-aminophenol in pellets by ion-pair high-performance liquid chromatography applying the monolithic Chromolith Speedrod RP-18e column

An ion-pair high performance liquid chromatographic method was developed for the simultaneous determination of p-aminosalicylic acid (PAS) and its degradation product m-aminophenol (MAP) in a newly developed multiparticular drug delivery system. Owing to the concentration differences of PAS and MAP, acetanilide and sulfanilic acid were used as internal standards, respectively. The separation was performed on a Chromolith SpeedROD RP-18e column, a new packing material consisting of monolithic rods of highly porous silica. The mobile phase composition was of 20 mm phosphate buffer, 20 mm tetrabutylammonium hydrogen sulphate and 16% (v/v) methanol adjusted to pH 6.8, at a flow-rate of 1.0 mL/min, resulting in a run-time of about 6 min. Detection was by UV at 233 nm. The method was validated and proved to be useful for stability testing of the new dosage form. Separation efficiency was compared between the new packing material Chromolith SpeedROD RP-18e and the conventional reversed-phase cartridge LiChroCART 125-4 (5 microm). A robustness test was carried out on both columns and different separation parameters (retention, resolution, run time, temperature) were determined.     

固相萃取-高效液相色谱法测定人血浆中的川芎嗪

建立了高效液相色谱测定人血浆中川芎嗪浓度的方法。色谱条件:分析柱为Luna C18(150 mm×4.6 mm i.d.,5 μ m),流动相为甲醇-乙腈-醋酸盐缓冲液(pH 5.0)(体积比为50∶8∶42),流速1.0 mL/min,柱温40 ℃,检测波长280 nm。 血浆样品预处理采用C8固相小柱萃取法。方法的线性范围为25～5000 μg/L,线性相关系数为0.9999。高、中、低浓度 的川芎嗪在标准血浆样品中的平均提取回收率为96.72％～100.90％,日内和日间相对标准偏差(RSD)小于8.64％,准确度 为99.59%~103.26%,检测限为10 μg/L。该方法的各项效能指标符合生物样品的分析要求,可用于川芎嗪制剂的人体药 代动力学研究。