Sensitive assay for the determination of fluvastatin in plasma utilizing high-performance liquid chromatography with fluorescence detection

The authors describe a rapid, useful, specific, and very sensitive high-performance liquid chromatographic assay for the determination of fluvastatin (FV) level using atorvastatin as the internal standard (IS). After a simple deproteinization of 1.0 mL of plasma with acetonitrile, the drug and IS were extracted with tert-methyl butyl ether (TMBE). An efficient separation was performed using an 8 mm x 10 cm Nova Pak C(18) 4-microm particle size radial compression cartridge. The mobile phase consisted of an aqueous solution containing 20 mmol/L dibasic sodium dihydrogen phosphate with 1 mmol/L sodium lauryl sulfate adjusted to pH 7 with phosphoric acid and acetonitrile (70:30 v/v) delivered at a flow rate of 1.0 mL/min. The compounds of interest were detected using a fluorescence detector with the excitation wavelength set at 305 nm and the emission at 380 nm. Under these conditions, the retention times for FV and IS were 8.8 and 10.6 minutes, respectively. The concentration of FV in plasma was linear (r > 0.999) for the wide range that was examined (0.5-1,000 ng/mL). The recovery ranged from 88% to 96%. This sensitive, rapid, and simple analytical method gives accurate results over the wide range of concentrations examined. This method is used currently for clinical therapeutic monitoring and pharmacokinetic studies of FV in patients with hypercholesterolemia.     

LC-ESI-MS/MS法测定人血浆中卢帕他定的浓度（英文）

目的建立测定人血浆中卢帕他定浓度的LC-ESI-MS/MS方法。方法血浆样品加入内标,碱化后用二氯甲烷：乙酸乙酯（20：80）提取,在37℃真空干燥箱中干燥至干,残渣用200μL流动相溶解后进样。色谱条件为：色谱柱为Agilent Eclipse XDB-C18（4.6mm×150mm,5μL）;流动相为乙腈（含1%甲酸）：20mmol·L^-1醋酸铵（76：24,V/V）,流速为0.6mL·min^-1。质谱条件：采用美国安捷仑1100高效液相色谱系统和离子阱（Agilent MSD Trap XCT）检测仪,质谱条件为电喷雾离子源,检测方式为正离子电离、多离子反应监测（MRM）,用于定量分析的离子为卢帕他定m/z416→309,内标氯雷他定m/z383→337。结果该方法应用于检测20名健康志愿者服药后的血浆样品。线性范围为0.05～14ng·mL^-1（r=0.998）,日内和日间精密度均低于15%,方法回收率为85.1%～114.0%。最低检测限为0.05ng·mL^-1（当n=5时,RSD=9.22%）。结论该方法灵敏、准确、快速,可用于该药药代动力学和生物等效性研究。     

Ion-paired reversed-phase high-performance liquid chromatography assay for determination of ceftriaxone in human plasma and urine

A rapid, sensitive, and specific ion-paired reversed-phase HPLC assay for ceftriaxone in human plasma and urine is described. Small volumes (50 microL) of sample are deproteinized with acetonitrile and are directly injected on a C18 analytical column. The UV absorbance is monitored at 280 nm. The assay is linear between 1 and 125 micrograms/mL of ceftriaxone, with less than 10% coefficient of both intra- and interday variation. Chromatography was specific for ceftriaxone as endogenous compounds and 30 common drugs did not interfere. The assay was used in open heart surgery patients where potential interference from corticosteroids was overcome.     

高效液相色谱法和微生物法测定人血浆中哌拉西林浓度的比较

目的：比较HPLC法和微生物法测定血浆中哌拉西林浓度的差异。方法：6名健康志愿单剂量静脉滴注哌拉西林4g，分别用HPLC法和微生物法测定血浆中药物浓度。结果：HPLC法线性范围为0．5－500μg/ml，回收率为97．40％－100．06％，日内、日间RSD分别为2．58％－3．24％和3．32％－5．59％，微生物法线性范围为0．5－10μg/ml，回收率为98．38％－106．85％，日内，日间RSD分别为3．90％－6．37％和7．07％－9．78％。结论：两种方法回收率和精密度均符合要求，受试各时间点平均血药浓度无显性差异。     

Sensitive electrochemical high-performance liquid chromatography assay for the simultaneous determination of haloperidol and reduced haloperidol

An electrochemical HPLC method for the simultaneous determination of haloperidol and reduced haloperidol in plasma is presented. Chlorohaloperidol serves as the internal standard. A cyanopropyl bond elut column was used for sample preparation. The eluate was evaporated and reconstituted with mobile phase and injected onto a nitrile bonded column. The chromatographic system consisted of an ESA Coulochem detector operated in the screen mode. Intra- and interassay coefficients of variation for both compounds were less than 7%, with a sensitivity limit of 20 pg on the column. A plasma level-time profile is presented to illustrate the sensitivity and applicability of this assay in small animal and human pharmacokinetic studies.     

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.     

High-performance liquid chromatographic assay for the determination of nilotinib in human plasma

A precise and convenient high-performance liquid chromatography (HPLC) method has been established to assay nilotinib in human plasma. Chromatographic separation of nilotinib was performed on a LiChrosphere(?)100 RP-18(e) column (250 mm×4.0 mm, 5 μm) using a mixture of acetonitrile and 0.01 M phosphate buffer (pH 3.0) (42 : 58, v/v) under isocratic conditions at a flow rate of 1.0 ml/min with ultraviolet (UV) detection at 266 nm. The calibration curve showed linearity at concentrations between 250 ng/ml and 5000 ng/ml (r(2)>0.999). The mean±S.D. absolute recovery of nilotinib from plasma was 99.2±3.3%. The coefficients of variation of both intra- and inter-day precision were below 9.1%. These results indicate that this new HPLC-based quantification may be useful for therapeutic drug monitoring of nilotinib to help manage treatment in patients with chronic myeloid leukemia in clinical practice.     

LC/MS/MS法测定人血浆中头孢克肟含量

目的 建立人血浆中头孢克肟的LC/MS/MS法.方法 血浆样品经蛋白沉淀提取,采用C8色谱柱分析,以乙腈:水:甲酸(40:60:0.5,v/v/v)为流动相,三重四级杆质谱检测器,正离子多反应监测模式(MRM),监测离子分别为:m/z 454.3→m/z 285.2(头孢克肟),m/z 282.2→m/z 212.2(...     

高效液相色谱法测定人血浆中伏立康唑浓度

目的：建立一种简单高效的高效液相色谱（HPLC）法用来检测人体中伏立康唑的血药浓度，并应用于临床中伏立康唑用药监测，以促进其个体化用药。方法：色谱柱：Kromasil C₁₈（4.6 mm×150 mm，5 μm），柱温：35℃，流速：1.0 mL·min^-1，流动相：甲醇-水（60：40），检测波长：257 nm，内标：酮康唑。对该方法进行方法学验证。结果：该方法专属性良好，血浆中伏立康唑在0.1~20.0 μg·mL^-1范围内线性良好（r=0.999 6），定量下限为0.1 μg·mL^-1。高、中、低3个浓度提取回收率分别为（90.68±10.32）%、（92.82±8.26）%、（97.47±4.58）%；日内精密度RSD分别为5.87%、7.85%、4.10%；日间精密度RSD分别为5.64%、3.30%、2.74%。对某院20例（男12例，女8例）使用伏立康唑抗真菌治疗的患者运用该方法进行了监测，结果显示浓度范围在0.71~13.51 μg·mL^-1之间。结论：本方法专属性高，操作简便，结果准确，可用于临床上伏立康唑血药浓度的检测，从而促进其个体化用药的推广。     

高效液相色谱法测定人体中羟苯磺酸钙的浓度

高效液相色谱法测定人体中羟苯磺酸钙的浓度@郭栋$Institute of Clinical Pharmacology, Central South University!Changsha 410078, Hunan, China @谭志荣$Institute of Clinical Pharmacology, Central South University!Changsha 410078, Hunan, China @欧阳冬生$Institute of Clinical Pharmacology, Central South University!Changsha 410078, Hunan, China @周宏灏$Institute of Clinical Pharmacology, Central South University!Changsha 410078, Hunan, China…     

Quantitative determination of haloperidol in human plasma by high-performance liquid chromatography

We present a method for the quantitative determination of haloperidol in human plasma. The high-performance liquid chromatographic method of analysis is a significant advancement in terms of ease and speed of haloperidol determination. The drug and the internal standard, chlorohaloperidol, are extracted from 2.0 ml of serum or plasma, back-extracted into the aqueous mobile phase, separated on a C-18 reversed-phase column, and monitored at 254 nm. The potential interference by other drugs was evaluated and was found to be negative. The method is sensitive to at least 5 ng/ml of extracted material and is suitable for drug measurement in the therapeutic range (5-20 ng/ml) and in the toxic range (greater than 50 ng/ml).     

Sensitive determination of itraconazole and its active metabolite in human plasma by column-switching high-performance liquid chromatography with ultraviolet detection

A simple and sensitive column-switching high-performance liquid chromatographic method for the simultaneous determination of itraconazole (ITZ) and its active metabolite, hydroxyitraconazole (HIT) in human plasma is described. ITZ, HIT, and an internal standard, R051012, were extracted from 1 mL of alkalinized plasma sample using n-heptane-chloroform (60:40, vol/vol). The extract was injected onto column I (TSK precolumn BSA-ODS/S, 5 microm, 10 x 4.6 mm ID) for clean-up and column II (Develosil C8-5 column, 5 microm, 150 x 4.6 mm ID) for separation. The mobile phase consisted of phosphate buffer-acetonitrile (68:32 vol/vol, pH 6.0) for clean-up and phosphate buffer-acetonitrile (35:65 vol/vol, pH 6.0) for separation. The peaks were monitored with an ultraviolet detector set at a wavelength of 263 nm, and total time for chromatographic separation was about 24 minutes. The validated concentration ranges of this method were 3 to 500 ng/mL for ITZ and 3 to 1000 ng/mL for HIT. Mean recoveries were 59.7% for ITZ and 72.8% for HIT. Intraday and interday coefficients of variation were less than 4.6% and 5.0% for ITZ, and 4.6% and 4.9% for HIT at the different concentrations. The limit of quantification was 3 ng/mL for both ITZ and HIT. This method was suitable for therapeutic drug monitoring of ITZ and HIT, and was applied to pharmacokinetic studies in human volunteers.     

Fluorometric determination of phenylephrine hydrochloride by liquid chromatography in human plasma

A sensitive method is described for the rapid determination of phenylephrine hydrochloride in human plasma. Phenylephrine was analyzed using high-performance liquid chromatography (HPLC) with fluorecence detection at excitation and emission wavelengths of 270 and 305 nm, respectively. A retention time of approximately 4.25 min was observed on chromatograms. Drug was separated from interfering plasma components by the use of disposable solid-phase extraction columns. Linear standard curves were constructed from drug concentrations of 0.5-5.0 and 2.5-80 ng/mL, for which an average recovery of 76-78% was obtained. Phenylephrine could be quantitatively measured at levels as low as 0.5 ng/mL. An assay precision (CV) of 23.6 and 15.9% was obtained at the lower sensitivities of 0.5 and 1.0 ng/mL, respectively. An example of the method was given for analysis of human plasma levels of drug following topical application of two drops of 2.5% aqueous or 10% viscous opthalmic solution to the eyes of two subjects.     

High-performance liquid chromatography assay for the determination of the HIV-protease inhibitor tipranavir in human plasma in combination with nine other antiretroviral medications

An accurate, sensitive and simple reverse-phase (RP) high-performance liquid chromatography (HPLC) assay has been developed and validated for the simultaneous quantitative determination of tipranavir with nine other antiretroviral drugs in plasma. A liquid-liquid extraction of the drugs in tert-butylmethylether (TBME) from 200 microL of plasma is followed by a reversed phase gradient HPLC assay with UV detection at 210 nm. The standard curve for the drug was linear in the range of 80-80,000 ng/mL for tipranavir; 10-10,000 ng/mL for nevirapine, indinavir, efavirenz, and saquinavir; and 25-10,000 ng/mL for amprenavir, atazanavir, ritonavir, lopinavir, and nelfinavir. The regression coefficient (r(2)) was greater than 0.998 for all analytes. This method has been fully validated and shown to be specific, accurate and precise. Due to an excellent extraction procedure giving good recovery and a clean baseline, this method is simple, rapid, accurate and provides excellent resolution and peak shape for all analytes. Thus this method is very suitable for therapeutic drug monitoring.     

Therapeutic drug monitoring of tacrine: simple and fast high-performance liquid chromatography assay method for its determination in human plasma

A new high-performance liquid chromatography (HPLC) assay method was developed for the therapeutic monitoring of tacrine. The method involved a simple protein precipitation by means of either acetonitrile or cold methanol followed by a fast isocratic separation on a CN column eluted in reversed-phase mode. The entire sample preparation took place in an HPLC vial and no further liquid transfer was required. The validation data showed that the assay method was precise, accurate, and robust. Analysis of more than 1,000 plasma samples collected from patients with Alzheimer disease demonstrates the suitability of the assay.     

An expedient assay for determination of gemcitabine and its metabolite in human plasma using isocratic ion-pair reversed-phase high-performance liquid chromatography

An expedient method is presented for determination in human plasma of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine (dFdU) by ion-pair reversed-phase HPLC. Samples were simply prepared by protein precipitation. Separation was processed on a Thermo Hypersil column (250 x 4.6 mm, 5 microm Hypersil BDS C18) with UV detection at 272 nm. The mobile phase consisted of 17% methanol and 83% phosphate buffer (20 mM, pH 3.1) containing 10 mM sodium 1-heptanesulfonate with a flow rate of 0.8 mL/min. The lower limit of quantification (LLOQ) of gemcitabine was 0.08 microg/mL with linear response over the range 0.08-20.0 microg/mL, and LLOQ of dFdU was 0.1 microg/mL with linear response over the range 0.1-50.0 microg/mL. Assay accuracy for both compounds was within +/- 4%. The coefficient of variation (CV %) for intra- and interday precision for both compounds was <7%. The correlation coefficients (r2) were greater than 0.9996 for all standard curves. The simple method with adequate sensitivity has been successfully used in phase I and II gemcitabine pharmacokinetic and pharmacodynamic studies in an Asian population.     

Sensitive assay for the determination of cefazolin or ceftriaxone in plasma utilizing LC

A rapid, specific and very sensitive liquid chromatographic assay using standard ultraviolet detection has been developed to measure cefazolin (CFZ) or ceftriaxone (CFX) in small samples (200 microl) of plasma using either drug as the internal standard for measurement of the other. A rapid extraction was performed using C18 bonded Sep Pak cartridges with high extraction efficiency for both drugs. The chromatographic system employed the use of a Nova-Pak C18 4-microm cartridge with a radial compression system preceded by a Guard-Pak with a C18 insert. The mobile phase consisted of an aqueous solution containing 10 mM of dibasic potassium phosphate and 10 mM cetyltrimethylammonium bromide (pH 6.5) with acetonitrile (73:27 v/v). The drug and internal standard (CFZ/CFX) were detected using a UV detector set at a wavelength of 274 nm. Assay results were linearly related to the concentration (r > 0.997) for the wide range which was examined (0.005-120 microg/ml) for either drug. We report the precision, accuracy, recovery, linearity, sensitivity and specificity of this assay. The intra-run and inter-run CV was less than 9.02%. This method is currently being used for clinical therapeutic monitoring and pharmacokinetic studies of CFZ and CFX in patients undergoing cesarean section.     

高效液相色谱法测定人血浆中帕瑞昔布和伐地昔布的浓度

目的建立高效液相色谱法测定人血浆中前体药物帕瑞昔布及其产物伐地昔布的浓度。方法色谱柱为Diamonsil C18柱(150 mm×4.6 mm,5μm),柱温30℃,流动相为乙腈-水-磷酸(57∶43∶0.01,V/V/V),流速1.0 mL.min-1,检测波长240 nm;内标为塞来昔布。结果帕瑞昔布的线性方程为Y=0.017 8X-0.022 9(r2=0.999 9),线性范围为1～100 mg.L-1。伐地昔布回归方程为Y=0.315 2X-0.005 1,(r2=0.999 8),线性范围为0.05～5 mg.L-1。帕瑞昔布和伐地昔布日内及日间RSD均小于15%,回收率均大于85%。结论本方法灵敏度高、专一性好、操作简单,可同时检测血浆中帕瑞昔布和伐地昔布,能满足药动学研究需要。     

Sensitive determination of buprenorphine and its N-dealkylated metabolite norbuprenorphine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A highly sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the quantitative determination of buprenorphine and its active metabolite norbuprenorphine in human plasma. Automated solid phase extraction (SPE) on disposable extraction cartridges (DEC) is used to isolate the compounds from the biological matrix and to prepare a cleaner sample before injection and analysis in the LC-MS/MS system. After conditioning, the plasma sample (1.0 ml) is loaded on the DEC filled with octyl silica (C8) and washed with water. The analytes are, therefore, eluted by dispensing methanol containing 0.1% of acetic acid. The eluate is collected and evaporated to dryness. The residue is dissolved in mobile phase and an aliquot is injected in the LC-MS/MS system. On-line LC-MS/MS system using atmospheric pressure chemical ionization (APCI) has been developed for the determination of buprenorphine and norbuprenorphine. The separation is obtained on a RP-18 stationary phase using a mobile phase consisting in a mixture of methanol and 50 mM ammonium acetate solution (50:50, v/v). Clonazepam is used as internal standard (IS). The MS/MS ion transitions monitored are m/z 468-->468, 414-->414 and 316-->270 for buprenorphine, norbuprenorphine and clonazepam, respectively. The method was validated regarding recovery, linearity, precision and accuracy. The limits of quantification (LOQs) were around 10 pg/ml for buprenorphine and 50 pg/ml for norbuprenorphine.