用液相色谱-串联质谱法测定人血清中加巴喷丁的含量

目的:建立测定人血清中加巴喷丁含量的液相色谱-串联质谱(LC-MS/MS)法。方法:以磺胺二甲异嘧啶为内标,血清用乙腈直接沉淀蛋白质后进样分析。色谱条件:XBridge Phenyl(150 mm×2.1 mm,5μm)为色谱柱,流动相:0.02%甲酸的乙腈溶液-0.02%甲酸的水溶液(15∶85)。电喷雾离子源,正离子MRM扫描分析。结果:加巴喷丁和内标磺胺二甲异嘧啶离子对分别为m/z172→154.1和m/z279→124。加巴喷丁在50~5 000 ng/ml范围内线性关系良好(r=0.999 6),检测限为50 ng/ml。批内、批间RSD均<10%,平均提取回收率为97.7%~108.5%。结论:本方法灵敏度高、专一性好,可用于人血清中加巴喷丁的检测。     

Determination of valdecoxib in serum using a HPLC-diode array detector and its application in a pharmacokinetic study

A simple, sensitive, isocratic and reproducible reversed phase HPLC method for the determination of valdecoxib, a novel specific COX-2 inhibitor in human serum was developed using a diode array detector and celecoxib as internal standard. The system consisted of a C18 column and a detector set at 240 nm. The mobile phase was a mixture of acetonitrile:water acidified to pH 3.2 with orthophosphoric acid (OPA) (60:40) pumped at room temperature and a flow rate of 1 ml/min. The mean absolute recovery value was about 90%, while the intra (n = 5) and inter (n = 5) assay variations were <18%. The calibration was linear over a concentration range of 20 ng/ml to 200 microg/ml with r2 > 0.999. The limit of detection was < or = 10 ng/ml. The method was used to study the pharmacokinetics of valdecoxib after a single dose oral administration to human volunteers.     

Reversed-phase high-performance liquid chromatographic determination of mitomycin C in human serum

A reversed-phase high-performance liquid chromatographic method is presented by which the cancer chemotherapeutic agent, mitomycin C, may be measured in human serum. A mobile phase of methanol:water (35:65) passed through a mu-Bondapak C-18 column at a rate of 1.0 ml/min produced a sharp, symmetrical band for mitomycin C. An improved serum extraction procedure, using a reversed-phase sample preparation cartridge, proved to be efficient and reproducible. Recovery over a concentration range of 10-100 ng/ml was 81.6% with a between-day coefficient of variation of 4.6% (n = 5). The within-day coefficient of variation at 50 ng/ml was 5.6% (n = 10). Ultraviolet detection at a wavelength of 365 nm was sensitive to serum concentrations of 10 ng/ml. Serum concentration-time course data from lung cancer patients receiving mitomycin C by rapid intravenous injection are presented.     

Quantitative analysis of the immunosuppressant CP-690,550 in whole blood by column-switching high-performance liquid chromatography and mass spectrometry detection

A fast and accurate method to quantify the new immunosuppressive JAK3 inhibitor CP-690,550 in whole blood using a dual-pump liquid chromatography-liquid chromatography-mass spectrometry (LC/LC-MS) system was developed and validated in nonhuman primate blood. Before injection, blood samples were prepared by precipitation with a reagent that included methanol and acetonitrile (30:70, vol/vol) along with the internal standard (CP-istd). Column-switching LC/LC-MS analysis used online extraction followed by separation on a C8 analytic column and MS detection of the [M + H] CP-690,550 (m/z = 313.1) and CP internal standard (m/z = 288.1). Linearity was always better than r = 0.99 (n = 7) for CP-690,550 (range 2.5-750 ng/mL), with a lower limit of quantification (LLOQ) of 2.5 ng/mL. The intrarun accuracy and precision ranged from 103.0% to 105.4% and 2.7% to 4.3%, respectively (n = 5), and the interday precision ranged from 8.7% to 11.1%, and the interday accuracy ranged from 98.1% to 103.8% of nominal values (n = 14). The injection repeatability for the method was 1.3% (n = 7). Except for the LLOQ, the intraday accuracy and precision in human blood were also within 15% (n = 5). The combination of simple sample preparation and short analytic run time of this sensitive procedure makes it effective for monitoring the concentration of CP-690,550 in whole blood in organ-transplant recipients.     

SPE-HPLC method for the determination and pharmacokinetic studies on paeoniflorin in rat serum after oral administration of traditional Chinese medicinal preparation Guan-Xin-Er-Hao decoction

A new HPLC method for the determination of paeoniflorin in rat serum with solid-phase extraction (SPE) for preconcentration is introduced. Paeoniflorin and an internal standard (pentoxifylline) were extracted from serum by means of SPE using cartridges with octadecyl chemically bound phase. The HPLC separation was then performed on a reversed-phase C(18) column using acetonitrile-water (18:82, v/v) as eluting solvent system, and UV detection at 230 nm to measure the analyte with a limit of quantitation about 10 ng ml(-1). The calibration curve for paeoniflorin was linear (r=0.9938) in the concentration range of 10-1200 ng ml(-1), both intra- and inter-day precision of the paeoniflorin were determined and their coefficience of variation did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of paeoniflorin from rat serum after oral administration of Guan-Xin-Er-Hao decoction.     

LC—MS／MS法测定人血浆中醋酸甲羟孕酮的浓度

目的建立测定人血浆中醋酸甲羟孕酮浓度的LC．MS／MS法。方法血浆样品经液液提取后,以甲醇：水=（90：10,v／v）为流动相,通过Agilentll00VL型离子阱质谱仪,电喷雾离子源正离子模式,采用多反应离子监测方式测定醋酸甲羟孕酮（MRM,m／z387327）。结果血浆中醋酸甲羟孕酮的线性范围为0．2—30．Ong／ml。定量下限为0．2ng／ml。准确度在85％～115％之间,日内、日间精密度（RSD）在15％之内。结论该方法高效、准确,特异性强,可用于醋酸甲羟孕酮药代动力学研究。     

Quantitative determination of paclitaxel in human plasma using semi-automated liquid-liquid extraction in conjunction with liquid chromatography/tandem mass spectrometry

This paper describes a high-throughput sample preparation procedure combined with LC-MS/MS analysis to measure paclitaxel in human plasma. Paclitaxel and an internal standard were extracted from plasma by a semi-automated robotic method using liquid-liquid extraction. Thereafter compounds were separated on a RP C18 column. Detection was by a PE Sciex API 3000 mass spectrometer equipped with a TurboIonSpray interface. The compounds were detected in positive ion mode using the mass transition m/z 854.6-->286.2 and m/z 831.6-->263.2 for paclitaxel and the internal standard, respectively. The limit of quantitation for paclitaxel was 1 ng/ml with an imprecision of 5.2% following extraction of 0.1 ml of plasma. Linearity was confirmed over the whole calibration range (1-1000 ng/ml) with correlation coefficients higher than 0.99 indicating good fits of the regression models. The inter and intra-day precision was better than 9.5% and the accuracy ranged from 90.3 to 104.4%. The assay was simple, fast, specific and exhibited excellent ruggedness.     

Determination of vincristine in infant plasma by liquid chromatography-atmospheric pressure chemical ionization-mass spectroscopy

An LC-MS method using APCI has been developed and validated for the determination of the anticancer drug vincristine in human plasma, using vinblastine as internal standard. Following solid-phase extraction (SPE) of the sample, the lower limit of quantitation (LLOQ) was 0.18 ng/ml, the lower limit of detection was 0.09 ng/ml, and the linear calibration range was 0.18-180 ng/ml. This method has been used to measure plasma concentrations of vincristine from 0.08 to 24 h post bolus in 29 infants as part of a pharmacokinetic study. Concentrations of vincristine at 24 h were 0.2-1.36 ng/ml.     

Simple and rapid HPLC method for determination of amlodipine in human serum with fluorescence detection and its use in pharmacokinetic studies

A fast, sensitive and specific high performance liquid chromatographic method using fluorescence detection is described for analysis of amlodipine in human serum. Amlodipine is extracted from serum by ethyl acetate and involves precolumn derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl) and reverse-phase chromatography on C18 column. The mobile phase was sodium phosphate buffer (pH 2.5) containing 1 ml/l triethylamine and methanol at flow rate of 2.8 ml/min. Propranolol was used as internal standard. The standard curve was linear over the range 0.25-16 ng/ml of amlodipine in human serum. The within-day and between-day precision studies showed good reproducibility with coefficients of variation less than 12% for all the analytes. The limit of quantification was 0.25 ng/ml of serum. The method has been applied to a bioequivalence study after administration of 10 mg amlodipine in 12 normal subjects.     

A subnanogram API LC/MS/MS quantitation method for depsipeptide FR901228 and its preclinical pharmacokinetics

A highly sensitive and specific atmospheric pressure ionization (API) liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method for the quantitation of depsipeptide FR901228 (NSC-630176, FR), a naturally occurring antitumor agent, was developed and validated. FR was extracted from human or rat plasma along with the internal standard, t-Boc-Met-Leu-Phe (BMLP) with ethyl acetate. Components in the extract were separated on a 5-microm C8 Spherisorb 50 x 4.6 mm i.d. column by isocratic elution with methanol/acetonitrile/12 mM ammonium acetate (60:10:30, v/v/v). The liquid flow was passed through a presource splitter and 5% of the eluate was introduced into the API source. The components were analyzed in the multiple-reaction monitoring (MRM) mode to enhance specificity. Linear calibration curves were obtained in the range of 0.1-100.0 ng/ml with 0.5 ml human plasma and 0.5-100.0 ng/ml with 0.1 ml rat plasma. The limit of quantitation (LOQ) was 0.1 ng/ml using 0.5 ml human plasma and 0.5 ng/ml using 0.1 ml rat plasma. The overall within-day precision was below 12% in human plasma and below 7% in rat plasma; and the between-day precision was below 10.2% in human plasma and 7.2% in rat plasma. The accuracy at low, medium and high levels ranged from 99.3 to 111.7% in human plasma and 96.2-107.3% in rat plasma. The high sensitivity permitted pharmacokinetic study of FR in the rat at a single i.v. dose as low as 1 mg/kg. At this dose, plasma FR levels declined biexponentially with a mean terminal t(1/2) of 187.7 min (n = 6) and were detectable up to 24 h. After an oral dose at 5 mg/kg, plasma FR levels were highly erratic and yielded a mean bioavailability of 1.6% (n = 6). At a higher oral dose of 50 mg/kg, a mean bioavailability of 10.6% was obtained, both being estimated by a non-crossover method.     

Validation of a method for the determination of zolpidem in human plasma using LC with fluorescence detection

A sensitive and selective high-performance liquid chromatography (HPLC) method was developed for the determination of zolpidem in human plasma. Zolpidem and the internal standard (trazodone) were extracted from human plasma that had been made basic. The basic sample was loaded onto a conditioned Bond Elut C18 cartridge, rinsed with water and eluted with methanol. Forty microliters were then injected onto the LC system. Separation was achieved on a C18 column (150 x 4.6 mm, 5 microm) with a mobile phase composed of acetonitrile:50 mM potassium phosphate monobasic at pH 6.0 (4:6, v/v). Detection was by fluorescence, with excitation at 254 nm and emission at 400 nm. The retention times of zolpidem and internal standard were approximately 4.7 and 5.3 min, respectively. The LC run time was 8 min. The assay was linear in concentration range 1-400 ng/ml for zolpidem in human plasma. The analysis of quality control samples for zolpidem (3, 30, and 300 ng/ml) demonstrated excellent precision with relative standard deviations (RSD) of 3.7, 4.6, and 3.0%, respectively (n = 18). The method was accurate with all intraday (n = 6) and overall (n = 18) mean concentrations within 5.8% from nominal at all quality control sample concentrations. This method was also performed using a Gilson Aspec XL automated sample processor and autoinjector. The samples were manually fortified with internal standard and made basic. The aspec then performed the solid phase extraction and made injections of the samples onto the LC system. Using the automated procedure for analysis, quality control samples for zolpidem (3, 30, and 300 ng/ml) demonstrated acceptable precision with RSD values of 9.0, 4.9, and 5.1%, respectively (n = 12). The method was accurate with all intracurve (n = 4) and overall (n = 12) mean values being less than 10.8% from nominal at all quality control sample concentrations.     

Determination of tamsulosin in human aqueous humor and serum by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple, sensitive and selective LC-MS/MS method was developed for the determination of tamsulosin in human aqueous humor and serum to study the recently reported eye-related adverse effects of this alpha(1)-blocker drug. Aqueous humor samples were analyzed by direct injection, after addition of the internal standard, labetalol. Liquid-liquid extraction with ethyl acetate was used for serum sample preparation. The chromatographic separation was performed on a reversed phase column by gradient elution with acetonitrile -0.1% formic acid at a flow-rate of 0.2 ml/min. Detection and quantification of the analytes were carried out with a linear ion trap mass spectrometer, using positive electrospray ionization (ESI) and multiple reaction monitoring (MRM). The limit of quantification was 0.1 ng/ml for both aqueous humor and serum samples and linearity was obtained over the concentration ranges of 0.1-4.7 ng/ml and 0.1-19.3 ng/ml for aqueous humor and serum samples, respectively. Acceptable accuracy and precision were obtained for concentrations within the standard curve ranges. The method has been used for the determination of tamsulosin in aqueous humor and serum samples from patients that were on tamsulosin medication and underwent cataract surgery.     

Rapid analysis of docetaxel in human plasma by tandem mass spectrometry with on-line sample extraction

A simple, rapid, and sensitive analytical method for the measurement of docetaxel in human plasma was developed and validated. The method is based on positive electrospray ionization tandem mass spectrometry (ESI+-MS-MS) with on-line sample extraction. It uses paclitaxel as internal standard for calibration. The on-line sample extraction minimizes sample handling and is readily adopted for automation. Quantitation of plasma docetaxel was done by the multiple reaction monitoring (MRM) mode. The method had a linear calibration range of 1.00-3000 ng/mL with a correlation coefficient >0.9999. The limit of quantitation (LOQ) for docetaxel in plasma was 1.00 ng/mL. The on-line extraction recovery of docetaxel was between 86.1-94.7%, with %CV < or = 6.1%. This method has high accuracy (90.1-96.3%), and excellent intra-assay (0.6-3.8%) and inter-assay (2.0-5.7%) precision. Its applicability to clinical samples was demonstrated by measuring patient plasma samples after treatment of weekly docetaxel at 25 mg/m2 as 60-min infusion.     

Quantification of propiverine by liquid chromatography/electrospray tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy subjects

Here we report on the development and validation of a sensitive and rapid reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of propiverine in human plasma. After adding an internal standard (oxybutynin chloride) to human plasma, samples were extracted using n-hexane/ethylacetate (8:2, v/v). Compounds extracted were analyzed by reversed-phase high-performance liquid chromatography (HPLC) with multiple reaction monitoring (MRM) mode for analyte detection. This method for determination of propiverine proved accurate and reproducible, with a limit of quantitation of 0.5 ng/ml in human plasma. The standard calibration curve for propiverine was linear (r2=0.9988) over the concentration range 0.5-1000.0 ng/ml in human plasma. The intra- and inter-day precision over this concentration range was lower than 8.66% (relative standard deviation, %R.S.D.), and accuracy was between 99.46 and 109.41%, respectively. This method was successfully applied to a bioequivalence study of two propiverine hydrochloride tablet formulations (20 mg) in 24 healthy subjects after a single administration.     

Determination of lovastatin in human plasma by ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry and its application in a pharmacokinetic study

A selective, rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantitative determination of lovastatin in human plasma and its application in a pharmacokinetic study. With mycophenolate mofetil as internal standard, sample pretreatment involved a one-step extraction with tert-butyl methyl ether of 0.2 ml plasma. The analysis was carried out on an ACQUITY UPLCTM BEH C18 column (50 mm x 2.1 mm, i.d., 1.7 microm) with flow rate of 0.35 ml/min. The mobile phase was 20% water and 80% acetonitrile (v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). Linear calibration curves were obtained in the concentration range of 0.08-24.50 ng/ml, with a lower limit of quantification of 0.08 ng/ml. The intra- and inter-day precision (RSD) values were below 15% and accuracy (RE) was -7.6 to 9.3% at all QC levels. The method was applicable to clinical pharmacokinetic study of lovastatin in healthy volunteers following oral administration.     

Analysis and pharmacokinetics of quetiapine and two metabolites in human plasma using reversed-phase HPLC with ultraviolet and electrochemical detection

A sensitive and specific HPLC assay for the measurement of the antipsychotic compound quetiapine in human plasma has been developed and validated. The assay employs a three-step liquid liquid extraction of quetiapine and its 7-hydroxylated and 7-hydroxylated, N-dealkylated metabolites from human plasma, and utilizes ultraviolet (UV) detection of quetiapine and electrochemical detection of the metabolites. The method provides a linear response from a quantitation limit of 2.50 to 500 ng ml(-1) for each analyte using 0.4 ml plasma. The assay is applicable from 500 to 5000 ng ml(-1) by sample dilution with de-ionized water. The inter-assay precision of quetiapine in plasma calibration standards across 4 validation days averaged 11.9% relative standard deviation (RSD) over the range 2.50 to 500 ng ml(-1), with intra-assay precision averaging 16.0% RSD and mean accuracy of 98.6% of theory. Similarly, the inter-assay precision of the 7-hydroxylated metabolite in plasma calibration standards across 4 validation days averaged 13.7% RSD over the range 2.50 to 500 ng ml(-1), with intra-assay precision averaging 17.6% RSD and mean accuracy of 109% of theory. The 7-hydroxylated, N-dealkylated metabolite demonstrated inter-assay precision of 16.2% RSD, intra-assay precision of 19.9% RSD, and mean accuracy of 104% of theory over the range 2.50 to 500 ng ml(-1). The present assay method was used to support a study comparing the pharmacokinetic profile of quetiapine with the time course of dopamine D2 and serotonin 5-HT2 receptor occupancy in the brain using positron emission tomography (PET). We describe in this paper the bioanalytical method and the plasma concentrations of quetiapine and its metabolites resulting from this study.     

LC determination of praziquantel in human plasma

A simple high-performance liquid chromatographic (HPLC) method for the determination of praziquantel in human plasma was developed and validated. The present method was described by adding drop-wise 0.2 M Zinc sulfate and acetonitrile to plasma sample for deproteinization. This method used a reversed-phase Spherisorb ODS 2 column (5 microm), 250 x 4.6 mm i.d. as a stationary phase with a mobile phase consisting of acetonitrile- methanol-water (36:10:54, v/v/v), a flow rate of 1.5 ml/min and UV detection wavelength of 217 nm. Diazepam was used as internal standard. The standard calibration curve was linear over the concentration range of 100-2000 ng/ml (r=0.999). The equation of a linear regression line was y=8.05E-04+7.25E-04x with slope and intercept values of 0.0007 and 0.0008, respectively. The limit of detection was 12.25 ng/ml and the limit of quantification was set at 100 ng/ml. The intra- and inter-day assay coefficients of variation (CV) were 3.0+/-1.7 and 6.3+/-1.9%, respectively. The percentage of recovery was 102.1+/-5.6. Therefore, the HPLC method described here was simple, rapid and reproducible since it did not require extraction and evaporation processes in sample preparation, which will reduce time-consuming or expensive sample preparation.     

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.     

Stereoselective determination of pyridoglutethimide enantiomers in serum with a chiral cellulose-based high-performance liquid chromatographic column using solid phase extraction and UV detection

A sensitive method for the separation and determination of R(+)- and S(-) enantiomers of pyridoglutehimide in serum by high performance liquid chromatography (HPLC) with UV detection was developed. The assay involves the use of a solid-phase extraction for serum sample clean-up prior to HPLC analysis using a C18 Bond-Elute column. Chromatographic resolution of the enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OD-R, 250 x 4.6 mm I.D.) under isocratic conditions using a mobile phase of 25:75 v/v acetonitrile-0.3 M aqueous sodium perchlorate (pH 6.2 adjusted with perchloric acid) at a flow rate of 0.8 ml/min. Recoveries for R(+)- and S(-)-pyridoglutethimide enantiomers were in the range 86-91% at 300-900 ng/ml level. Intra-day and inter-day precision calculated as %R.S.D. were in the ranges of 2.9-3.9 and 1.5-4.7% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percentage error were in the ranges of 1.9-3.3 and 1.5-3.9% for both enantiomers, respectively. Linear calibration curves in the concentration ranges of 100-1500 ng/ml for each enantiomer show correlation coefficient (r) of more than 0.9995. The limit of quantification (LOQ) of each enantiomer was 100 ng/ml using 1 ml of serum. The detection limit (LOD) for each enantiomer in serum using a UV detection set at 257 nm was 50 ng/ml (S/N = 2).     

A rapid, simple, specific liquid chromatographic-electrospray mass spectrometry method for the determination of finasteride in human plasma and its application to pharmacokinetic study

A fast, accurate, sensitive, selective and reliable method using reversed-phase high-performance liquid chromatography-mass spectrometry coupling with an electrospray ionization interface was developed and validated for the determination of finasteride in human plasma. After deprotienation with acetonitrile, centrifugation, evaporation to dryness and dissolving in mobile phase, satisfactory separation was achieved on a Hypersil-Keystone C(18) reversed-phase column using a mobile phase consisting of acetonitrile-water (46:54, v/v), 0.1% acetic acid and 0.1% trifluoracetic acid. Carbamazepine (IS) was used as internal standard. This method involved the use of the [M+H](+) ions of finasteride and IS at m/z 373 and 237 with the selective ion monitoring (SIM) mode. The calibration curve was linear in the range of 0.2-120 ng ml(-1). The limit of quantification for finasteride in plasma was 0.2 ng ml(-1) with good accuracy and precision. The intra-assay precision and accuracy were in the range of 2.1-11.2% and -1.3% to 8.5%, respectively. The inter-assay precision and accuracy were in the order of 3.4-12.1% and -1.5% to 11.5%, respectively. The mean sample extract recoveries of the method were higher than 85% and 74% for finasteride and internal standard (IS), respectively. The assay has been successfully used to estimate the pharmacokinetics of finasteride after oral administration of a 5mg tablet of finasteride to 24 healthy volunteers.