LC-MS method for the determination of albuterol enantiomers in human plasma using manual solid-phase extraction and a non-deuterated internal standard

A sensitive enantioselective liquid chromatography–mass spectrometry (LC–MS) assay using a manual solid-phase extraction (SPE) procedure, a non-deuterated internal standard and an ion trap LC–MS was developed to measure (R)- and (S)-albuterol in plasma. Sample extraction from plasma was achieved by a manual SPE extraction procedure with methoxyphenamine added as the internal standard. Chiral separation was achieved using a teicoplanin-based stationary phase and a mobile phase consisting of methanol, acetic acid and 28% (w/v) ammonia (1000:5:1, v/v/v). Samples were analyzed by selected reaction monitoring of product ions from the protonated molecular ions. The detection limit of the assay was 0.1 ng/ml with a conservative lower limit of quantification of 0.25 ng/ml for each enantiomer. Recovery of albuterol enantiomers from plasma spiked at 10 ng/ml of racemate was determined to be 89±5.8% (mean±S.D.). Reproducibility at 10 ng/ml of racemate assessed by the coefficient of variation was found to be 6.5% (n=5). Instrument precision (measured as coefficient of variation) was 1.4% (n=5). The correlation coefficient r² determined from the calibration curve over the range 0.5–50.0 ng/ml racemate in plasma was 0.998. This assay allows adequate sensitivity, recovery and reproducibility for the application to studies of inhaled albuterol.     

Liquid chromatographic determination of minocycline in human serum

A rapid, specific, and sensitive liquid chromatographic assay for minocycline in human serum is described. The drug and an internal standard (oxytetracycline) were extracted into ethyl acetate from 0.5 ml of buffered serum (pH 6.5). Chromatographic separation was achieved on a 10-micrometer Lichrospher 100 CH 8 column with acetonitrile--citric acid (0.1 M) as the eluent. The column effluent was monitored at 352 nm. The assay was linear up to 3 micrograms/ml with a mean coefficient of variation of 3.3% (n = 6). An extraction recovery of 89.4 +/- 3.2% (mean +/- SD, n = 17) was obtained over the 0.5--2.6 micrograms/ml range. The detection limit averaged 50 ng/ml. A serum concentration-time profile in humans after oral intake is presented.     

Simple HPLC method for determination of rosiglitazone in sheep plasma and amniotic fluid and its application in a pregnant sheep model

This paper describes the development of a sensitive high performance liquid chromatography (HPLC) method for quantification of rosiglitazone in sheep plasma and amniotic fluid. Samples were prepared by liquid-liquid extraction using tert-butyl methyl ether, and rosiglitazone was quantitated by HPLC using a C18 column and fluorescence detector with an excitation wavelength of 247 nm and emission wavelength of 367 nm. The mobile phase consisted of ammonium acetate (10 mM, pH 5.2) and acetonitrile (56.5:43.5, v/v) with a flow rate of 1 ml/min. Ketoconazole was used as the internal standard (IS). The plasma calibration curve was linear over the range of 2.5-250 ng/ml (mean r2=0.9940±0.0024; n=6) with accuracy of 99.4-102.8% over the calibration range. The intra-day and inter-day coefficient of variation (%CV, percent coefficient of variation) were in the range of 0.01-8.68% in sheep plasma. Similar performance was achieved for amniotic fluid. The described method was successfully applied to quantitate rosiglitazone concentrations in the pregnant ewe and her fetus.     

A sensitive and selective HPLC method for estimation of lamotrigine in human plasma and saliva: application to plasma-saliva correlation in epileptic patients

A sensitive and selective high performance liquid chromatographic (HPLC) method was developed and validated for estimation of lamotrigine (CAS 84057-84-1) in human plasma and saliva. The chromatographic separation was achieved with a reversed phase column and a mobile phase consisting of acetonitrile and 20 mM ammonium acetate buffer pH 6.5 (30:70) with a flow rate of 1 mL/min. The calibration curve was linear within the working range for both plasma and saliva. The validated method has been successfully applied for a study of lamotrigine in human plasma and saliva to establish the correlation between these two matrices. A scatter plot of plasma versus salivary lamotrigine concentrations showed a gold linear relationship between them (Pearson correlation coefficient, r = 0.6832, p < 0.001).     

Improved high performance liquid chromatographic analysis of omeprazole in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of omeprazole in human plasma. Omeprazole and the internal standard, chloramphenicol, were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M Na2HPO4-ACN (65:35, v/v) adjusted to pH 6.5. Analysis was run at a flow rate of 1.0 ml/min at a detection wavelength of 302 nm. The method was specific and sensitive with a detection limit of 2.5 ng/ml at a signal-to-noise ratio of 4:1. The limit of quantification was set at 5 ng/ml. The calibration curve was linear over a concentration range of 5-1280 ng/ml. Mean recovery value of the extraction procedure was about 96%, while the within and between day coefficient of variation and percent error values of the assay method were all less than 14%.     

Analysis of pirprofen in cerebrospinal fluid, plasma, and synovial fluid by high-performance liquid chromatography with electrochemical detection

We describe a high-performance liquid chromatographic method, using electrochemical detection, for the determination of pirprofen in cerebrospinal fluid (CSF), plasma, and synovial fluid (SF). A C-18 column with a mobile phase containing acetonitrile acetate:phosphate buffer (pH 3.0) was employed. Samples were added with phosphoric acid, then extracted into dichloromethane, evaporated, and injected into the chromatograph. A detection potential of +0.85 V was applied on the basis of current-potential curves. Good linearity was found for each fluid in the expected range of therapeutic concentrations. The detection limit was 0.1 ng/mL for CSF, and 1 ng/mL for plasma and SF, with a recovery greater than 96% and intraday coefficient of variation less than 5% in all cases. The main advantages of this method include high specificity and sensitivity which allow the analysis of CSF and the use of small volumes of plasma and SF. The application of the method for the analysis of plasma and SF samples and the kinetic profile in CSF are shown.     

High-pressure liquid chromatographic assay for hydralazine in human plasma

A specific high-performance liquid chromatographic assay for hydralazine in human plasma was developed. Plasma hydralazine is reacted with 10 microliter of p-anisaldehyde for 7 min at room temperature to form hydralazine p-anisaldehyde hydrazone. This derivative is extracted into ethyl acetate, and the solvent is removed by evaporation. The residue is reconstituted in 100 microliter of methanol, and 90 microliter is injected onto a reversed-phase column. The mobile phase is 32% acetonitrile in 0.75 M acetate buffer, pH 3.4, at a flow rate of 2 ml/min. The retention time of hydralazine p-anisaldehyde hydrazone is 6.5 min. The average coefficient of variation over 10-200 ng/ml is 5.5%, and the sensitivity limit is 5 ng/ml. Under the assay conditions, hydralazine pyruvic acid hydrazone, a known plasma metabolite of hydralazine, yields less than 0.1% hydralazine. Detectable plasma hydralazine levels of 5-20 ng/ml were found 10-30 min after a 0.5-mg/kg oral dose of hydralazine hydrochloride was given to a male volunteer.     

Determination of methadone in plasma using high pressure liquid chromatography methods]

High-performance liquid chromatography on the column of LiChrosorb RP-18 was applied to the determination of methadone in plasma, after previous precipitation of proteins with methanol and extraction from alkalized plasma with diethyl ether. Methadone, ion-paired with pentane-1-sulphonic acid added in a mobile phase acetonitrile-methanol-ammonium acetate solution (20:58:22), was monitored at 254 nm. Piritramide was used as an internal standard. The determination range was 0.25-1.25 micrograms/cm3 of plasma and recovery was 88-93%.     

反相高效液相色谱法测定人血浆中二甲胺四环素浓度

目的:建立以反相高效液相色谱法测定人血浆中二甲胺四环素浓度的方法。方法:色谱柱为C18,流动相为乙腈-水-三氟乙酸(15∶85∶0.1),内标为土霉素,检测波长为350nm,血样在pH=6.5缓冲液条件下用乙酸乙酯提取,以二甲胺四环素与内标的峰面积比进行定量。结果:二甲胺四环素检测浓度线性范围为0.05～8μg/ml(r=0.9999),最低检测浓度为0.02μg/ml,平均回收率为101.89%,日内、日间相对标准差均小于5%。结论:本方法简便、快速、灵敏,可用于二甲胺四环素药动学研究。     

高效液相色谱双波长法测定人血浆中舒尼替尼的浓度

目的 建立人血浆中舒尼替尼的高效液相色谱测定方法,用于临床进行药物浓度监测.方法 血浆样品经乙酸乙酯乙酯萃取后,采用高效液相色谱法(HPLC)进行分析.XDB-C18柱分离,流动相采用甲醇-0.02 mol/L磷酸二氢钠溶液,流速为1 ml/min;梯度洗脱在0～6 min甲醇-0.02 mol/L磷酸二氢钠溶液为70:30,6.1～15 min甲醇-0.02 mol/L磷酸二氢钠溶液为80∶20;利用光电二极管阵列检测器对流份进行双波长同时检测(舒尼替尼391 nm,内标300 nm),柱温30℃.结果 内源性物质不干扰测定,舒尼替尼的线性范围为0.5～12mg/L(r =0.996),最低定量限为0.5mg/L,基质效应为93.36％～104.26％(n=5),加样回收率为73.12％ ～86.50％ (n =5).结论 建立的HPLC方法简便、灵敏、准确、所需样本量小,可以用于临床血药浓度测定.     

高效液相色谱法测定人血浆中甲氨蝶呤浓度

目的:建立高效液相色谱测定人血浆中甲氨蝶呤（MTX）浓度的方法。方法:以茶碱（Theophyline）为内标,色谱柱为Nova-pak C₁₈柱（150 mm×3.9 mm,4μm）,流动相:乙腈-甲醇-0.05 mol·L^-1磷酸二氢钾缓冲液pH 6.5（6:2:92）,流速:1.0 ml·min^-1,检测波长303 nm及254 nm,柱温25℃。结果:甲氨蝶呤浓度在0.05～10.0 mg·L^-1范围内线性良好,r=0.999 6。绝对回收率为66.72%,相对回收率为98.35%,日内、日间RSD均<10%。结论:该法快速,简便,灵敏度高,适用于临床常规血药浓度监测。     

蝙蝠葛苏林碱在兔血浆浓度监测及药代动力学蝙蝠葛苏林碱在兔血浆浓度监测及药代动力学

目的建立测定血浆中蝙蝠葛苏林碱浓度的方法,并研究蝙蝠葛苏林碱在家兔体内的药代动力学。方法 以蝙蝠葛碱作为内标。血浆样品经乙腈去蛋白,再用二氯甲烷两次萃取,采用RP-HPLC方法检测。结果血浆药物浓度在0.05～20.00 mg·L^-1线性关系良好(r=0.999 6),最低定量浓度为0.05 mg·L^-1,绝对回收率均大于80%,相对回收率为(101±5)%,精密度试验的日内及日间RSD%分别为1.9%～5.6%和3.5%～6.5%。药代动力学研究表明,家兔蝙蝠葛苏林碱单剂量iv后,其体内血药浓度时间数据符合二房室开放模型。结论本方法专属性强、灵敏度高、回收率高、重现性好,可用于蝙蝠葛苏林碱血药浓度测定及药代动力学研究。     

柱前衍生RP-HPLC测定法半夏中的精氨酸和γ-氨基丁酸

目的采用柱前衍生RP-HPLC法测定法半夏中精氨酸和γ-氨基丁酸的含量。方法在碱性条件下,氨基酸与异硫氰酸苯酯反应生成的衍生物用于检测;采用Venusil-AA氨基酸分析柱(250 mm×4.6 mm,5μm),用二元梯度洗脱方式,流动相A为0.05 mol·L-1醋酸钠溶液(pH6.5),流动相B为乙腈-水(80∶20),检测波长248 nm。结果精氨酸进样量10.20～132.60 ng、γ-氨基丁酸进样量12.01～156.13 ng与峰面积的线性关系良好,加样回收率分别为98.3%、99.1%。结论所用方法结果准确,重复性好。     

Quantification of a novel PPARγ partial agonist (<Emphasis Type="Italic">S</Emphasis>)-2-ethoxy-3-(4-{3-methyl-5-[4-(3-methyl-isoxazol-5-yl)-phenyl]thiophen-2-ylmethoxy}-phenyl)-propionic acid (PAM-1616) in rat plasma using liquid chromatography-tandem mass spectrometry

(S)-2-ethoxy-3-(4-{3-methyl-5-[4-(3-methyl-isoxazol-5-yl)-phenyl]thiophen-2-ylmethoxy}-phenyl)-propionic acid (PAM-1616) is a novel peroxisome proliferators-activated receptor γ (PPARγ) partial agonist with excellent antihyperglycemic activity. It is a promising new drug candidate for the treatment of type-2 diabetes with reduced possibility of edema in vitro/in vivo. In order to evaluate the pharmacokinetics of PAM-1616, a reliable, selective and sensitive highperformance liquid chromatography/electrospray ionization tandem mass spectrometry was developed for the quantification of PAM-1616 in rat plasma. The analytes were extracted from rat plasma with ethyl acetate, separated on an Atlantis dC₁₈ column with a mobile phase of 75% acetonitrile in 10 mM ammonium formate (pH 4.5), and detected by tandem mass spectrometry in the selective reaction monitoring mode. The calibration curve was linear (r ² = 0.999) over the concentration range of 0.05–20.0 μg/mL and the lower limit of quantification was 0.05 μg/mL. The coefficient of variation and relative error at four QC levels were 1.8% to 14.3% and −10.0% to 6.5%, respectively. The present method was successfully applied to the pharmacokinetic study of PAM-1616 after intravenous administration of PAM-1616 potassium at a dose of 1 mg/kg in rats.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of levofloxacin in human plasma

A rapid, sensitive and selective hydrophilic interaction liquid chromatography-tandem mass spectrometric (HILIC-MS/MS) method for the determination of levofloxacin in human plasma was developed. Levofloxacin and ciprofloxacin (internal standard) were extracted from human plasma with dichloromethane and analyzed on an Atlantis HILIC Silica column with the mobile phase of acetonitrile-ammonium formate (100 mM, pH 6.5) (82:18 v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r>0.999) over the concentration range of 10.0-5000 ng/ml. The lower limit of quantification for levofloxacin was 10.0 ng/ml using 20 microl plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 2.9-7.8% and -7.3% to -2.2%, respectively. The recoveries of levofloxacin and ciprofloxacin were 55.2% and 77.3%, respectively. This method was successfully applied to the pharmacokinetic study of levofloxacin in humans.     

HPLC法测定盐酸二氧丙嗪片的含量

目的建立HPLC法测定盐酸二氧丙嗪片含量的方法。方法采用HPLC法,C18柱(46mm×200mm,5μm);流动相为醋酸盐缓冲液(取0.05mol/L的醋酸铵溶液1000mL,加三乙胺1mL,用醋酸调节pH值至6.5)-乙腈(75︰25);检测波长为258nm。结果盐酸二氧丙嗪在2.21～22.08μg/mL浓度范围内线性关系良好(r=0.9999),平均回收率为99.76%,RSD=0.52%。结论本方法简便、快速、准确,专属性强,可作为盐酸二氧丙嗪片的含量测定方法。     

Simultaneous determination of a novel M3 muscarinic receptor antagonist and its active 5-OH metabolite in human plasma using liquid chromatography/tandem mass spectrometry

A sensitive, specific, and robust liquid chromatography (LC)/mass spectrometry (MS)/MS method has been developed and validated for a novel M(3) muscarinic receptor antagonist (I) and its active 5-OH metabolite (II) in human plasma. The assay involves a two-step liquid-liquid extraction of the compounds from human plasma, high performance liquid chromatography (HPLC) separation, and MS/MS for the detection of the analytes. The method provides a linear response from a quantitation limit of 0.05-20 ng/ml for I and 0.1-20 ng/ml for II using 1 ml of plasma. The mean absolute recovery was 85.4% for I and 80.8% for II, respectively. The intra-assay accuracy of I and II averaged from 95.0 to 105.3% with coefficient of variation (CV) values 相似文献   

Novel liquid chromatographic method for simultaneous estimation of pioglitazone and glimepiride in rat plasma by solid phase extraction: application to preclinical pharmacokinetic studies

The need for a reliable bioanalytical method is of primary importance during preclinical studies. The aim of the present study was simultaneous determination of pioglitazone (CAS 111025-46-8) (PIO) and glimepiride (CAS 93479-97-1) (GLM) in plasma of rats. A high-performance liquid chromatographic method has been developed and validated using C18 column and UV detector. A mobile phase composed of acetonitrile and ammonium acetate buffer pH 4.5 in the ratio of 55:45%. The plasma samples clean-up was carried out using solid phase cartridges. The method was in the linear range of 50-8000 ng/mL for PIO and 50-2000 ng/mL for GLM. The coefficient of regression was found to be > or = 0.99. Precision and accuracy were within the acceptable limits, as indicated by relative standard deviation varying from 1.5 to 6.1% for PIO and 3.1 to 7.0% for GLM whereas the accuracy ranged from 97.0 to 106.4% for PIO and 96.5 to 106.4% for GLM. The mean extraction recovery was found to be 90.2 +/- 4.5, 76.8 +/- 2.8 and 85.2 +/- 5.2% for PIO, GLM and internal standard, respectively. Moreover, PIO and GLM were stable in plasma, up to 30 days of storage at -70 degrees C and after being subjected to bench top, auto-sampler, and three freeze-thaw cycles. The developed method was applied for preclinical pharmacokinetic studies.     

Development of a rapid HPLC method for determination of famotidine in human plasma using a monolithic column

A rapid and sensitive HPLC method using a monolithic column has been developed for quantification of famotidine in plasma. The assay enables the measurement of famotidine for therapeutic drug monitoring with a minimum detectable limit of 5 ngml(-1). The method involves simple, one-step extraction procedure and analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 mm x 4.6 mm) column with an isocratic mobile phase consisting of 0.03 M disodium hydrogen phosphate buffer-acetonitrile (93:7, v/v) adjusted to pH 6.5. The wavelength was set at 267 nm. The calibration curve was linear over the concentration range 20-400 ngml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 8%.     

Evaluation of a fluorescence polarization immunoassay procedure for quantitation of methotrexate

A fluorescence polarization immunoassay (FPIA) procedure for measuring methotrexate was evaluated. The dynamic range of the assay is from 0.05 to 810 microM, and the calibration curve can be stored for at least 2 weeks. The FPIA procedure is automated and rapid; one result can be obtained in 18 min and five results in 25 min. There was no interference from hemoglobin (800 mg/dl), triglycerides (500 mg/dl), bilirubin (20 mg/dl), and protein (12.1 g/dl). Cross-reactivity with 7-hydroxy methotrexate and 2,4-diamino-N-methylpteroic acid was 0.6 and 44%, respectively. The coefficient of variation for the within-run and between-run precision was less than 5.0%. For the comparison studies, the samples were divided into four groups. The methotrexate concentrations in group 1 were 0.05-2.1 microM; in group 2, 2.2-9.3 microM; in group 3, 10-80 microM; and in group 4, greater than 80 microM. Linear regression analysis of the results obtained with the FPIA procedure and the enzyme multiplied immunoassay gave a correlation coefficient of at least 0.95 for all groups.