Development and validation of a hydrophilic interaction liquid chromatography-tandem mass spectrometric method for the analysis of paroxetine in human plasma

A sensitive, simple, fast and rugged hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the determination of paroxetine was developed and validated over curve range 0.050-50 ng/mL using only 0.4 mL plasma. This is the first published LC-MS/MS method and the low limit of quantitation of this method is 10-fold lower than previously published methods. A simple liquid-liquid extraction method using methyl-tert butyl ether (MTBE) as the extraction solvent was used to extract paroxetine and the internal standard (IS) fentanyl-d(5) from plasma. The extract was evaporated to dryness, reconstituted and injected onto a silica column using a low aqueous-high organic mobile phase. The chromatographic run time was 2.0 min per injection, with retention times of 1.1 and 1.2 min for paroxetine and IS, respectively. The detection was by monitoring paroxetine at m/z 330 --> 192 and IS at m/z 342 --> 188, respectively. The inter-day precision and accuracy of the quality control (QC) samples were <5.0% relative standard deviation (RSD) and <2.9% relative error (RE). This method can be used for supporting therapeutical drug monitoring and pharmacokinetic or drug-drug interaction studies.     

Determination and pharmacokinetics of periplocin in rat plasma by LC‐MS

A simple and sensitive LC‐MS method for the determination of periplocin in rat plasma was developed and validated. The chromatographic separation was carried out using a reverse‐phase Kromasil C₁₈ column(150 × 4.6 mm, i.d., 5 µm) with a mobile phase composed of methanol–water (76:24, v/v). The flow rate of mobile phase was 0.8 mL/min. The calibration curve was linear within the concentration range 1–1000 ng/mL. The intra‐ and inter‐day precisions across three validation days over the entire concentration range was lower than 9.2% in terms of relative standard deviation. Accuracy determined at three quality control concentrations ranged from ?2.0 to 6.0% in terms of relative error. The validated method was applied to the pharmacokinetic study of periplocin in rat plasma after intravenous and intramuscular administration. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of midazolam and 1'-hydroxymidazolam in human plasma by liquid chromatography with tandem mass spectrometry

A sensitive and simple liquid chromatography-tandem mass spectrometry method for the determination of midazolam and 1'-hydroxymidazolam in human plasma has been developed and validated with a dynamic range of 0.1-250 ng/mL. The analysis was based on semi-automated liquid-liquid extraction followed by evaporation of the extraction solvent, reconstitution and chromatography on a reversed-phase C(18) column. The mobile phase consists of 5 mm ammonium acetate and methanol and runs in gradient at a flow rate of 0.25 mL/min with column temperature of approximately 20 degrees C. The entire column effluent was transferred into the LC-MS/MS interface operated in positive electrospray ionization mode. The chromatographic run time was 4.3 min per injection, with retention times for midazolam, 1'-hydroxymidazolaml and the internal standard, triazolam, of 2.5, 2.3 and 2.1 min, respectively. The intra-day and inter-day precision (RSD %) and accuracy (bias %) of the quality control samples were <15.0% and within +/-13%, respectively. The current method has been applied to a clinical drug-drug interaction study in human.     

超高效液相色谱-串联质谱法测定食品包装材料中全氟辛烷磺酸盐

建立了超高效液相色谱-串联质谱(UPLC- MS/MS)测定食品包装材料中全氟辛烷磺酸盐(PFOS)的方法.采用乙腈作为溶剂,加速溶剂提取法提取食品包装材料中的PFOS.色谱条件:ACQUITY UPLC BEH C₁₈色谱柱（1.7 μm,2.1 mm×50 mm）;柱温:30 ℃;流动相:乙腈/水,梯度洗脱;流速:0.2 mL/min;经UPLC分离后用多级反应监测(MRM)方式测定.用2个子离子的相对丰度定性, 外标法定量.PFOS在0.005～0.500 μg/mL范围内线性良好(R2=0.999),PFOS的回收率为90.0%～101.6%,相对标准偏差RSD为1.5%～3.5%.方法检出限为0.1 μg/m2(S/N≥3).     

高效液相色谱法同时测定苏木中的巴西苏木素和原苏木素B

建立了同时测定苏木中巴西苏木素和原苏木素B的高效液相色谱检测方法。采用Agela Venusil XBP-C18(4.6 mm×150 mm,5μm)色谱柱,以甲醇-0.2%甲酸溶液为流动相,梯度洗脱,流速1.0 mL/min,测定温度为35℃,检测波长285 nm。巴西苏木素和原苏木素B的质量浓度均在0.005～1.0 mg/mL时与色谱峰面积之间线性关系良好,相关系数都为0.9999;回收率范围分别为96.7%～101.4%和99.4%～103.0%,相对标准偏差(RSD)为2.0%和1.3%。该法可以用于苏木药材的鉴定。     

手性高效液相色谱法测定板蓝根中表告依春和告依春含量

采用Chiralpak IC手性柱(250 mm×4.6 mm, 5 μm),建立了正相高效液相色谱测定板蓝根中表告依春(R-告依春)和告依春(S-告依春)含量的方法。考察了流动相组成、流速和柱温对分离度的影响。经优化后的实验条件: 以正己烷-异丙醇(体积比为90:10)为流动相,流速为0.8 mL/min;检测波长为245 nm,柱温为20 ℃。在此优化条件下,表告依春和告依春分离度为3.4,检出限为2.0 mg/L,在0.02～2.0 g/L范围内有良好的线性关系,平均回收率为101%,相对标准偏差(RSD)小于3.0%(n=6)。本方法可将具有抗病毒活性的表告依春与其对映体告依春基线分离并测定,专属性强,能有效控制板蓝根的质量。     

An LC‐MS/MS method for determination of curculigoside with anti‐osteoporotic activity in rat plasma and application to a pharmacokinetic study

A rapid, simple, selective and sensitive LC‐MS/MS method was developed for the determination of curculigoside in rat plasma. The analytical procedure involves extraction of curculigoside and syringin (internal standard, IS) from rat plasma with a one‐step extraction method by protein precipitation. The chromatographic resolution was performed on an Agilent XDB‐C₁₈ column (4.6 × 50 mm, 5 µm) using an isocratic mobile phase of methanol with 0.1% formic acid and H₂O with 0.1% formic acid (45:55, v/v) at a flow rate of 0.35 mL/min with a total run time of 2.0 min. The assay was achieved under the multiple‐reaction monitoring mode using positive electrospray ionization. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over 4.00–4000 ng/mL (R = 0.9984) for curculigoside with a lower limit of quantification of 4.00 ng/mL in rat plasma. The intra‐ and inter‐day precisions and accuracies were 3.5–4.6 and 0.7–9.1%, in rat plasma, respectively. The validated LC‐MS/MS method was successfully applied to a pharmacokinetic study of curculigoside in rats after a single intravenous and oral administration of 3.2 and 32 mg/kg. The absolute bioavailability of curculigoside after oral administration was 1.27%. Copyright © 2013 John Wiley & Sons, Ltd.     

灵芝发酵液中蛋白酶抑制剂GLPIA2的纯化及其特性

采用乙醇分级沉淀、凝胶色谱纯化、阴离子交换色谱分离等步骤从灵芝深层发酵液中提取得到蛋白酶抑制剂GLPIA1 与GLPIA2。其中GLPIA2仅在215 nm处有紫外吸收,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定为单一条带,相对分子质 量为15000。由其氨基酸组成分析谱图可看出,其酸性氨基酸含量较高,碱性氨基酸及芳香族氨基酸含量较低。GLPIA2抑 制剂的底物特异性研究表明,它对天冬氨酸族的胃蛋白酶和酵母蛋白酶A有相对较强的抑制作用。     

Determination of N-methyl-4-isoleucine-cyclosporin (NIM811) in human whole blood by high performance liquid chromatography-tandem mass spectrometry

A liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of N-methyl-4-isoleucine-cyclosporin (NIM811) was developed and validated over the concentration range 1-2500 ng/mL in human whole blood using a 0.05 mL sample volume. NIM811 and the internal standard, d(12)-cyclosporin A (d(12)-CsA), were extracted from blood using MTBE via liquid-liquid extraction. After evaporation of the organic solvent and reconstitution, a 10 microL aliquot of the resulting extract was injected onto the LC-MS/MS system. Chromatographic separation of NIM811 and internal standard was performed using a Waters Symmetry RP-8 (50 x 4.6 mm, 3 microm particle size) column. The mobile phase consists of 10 mm ammonium acetate in water (A) and acetonitrile (B), with 45% B from 0 to 0.2 min, 45 to 85% B from 0.2 to 0.8 min and 85% B from 0.8 to 2.2 min. The total run time was 3.5 min with a flow rate of 0.8 mL/min. The method was validated for sensitivity, linearity, reproducibility, stability, dilution integrity and recovery. The precision and accuracy of quality control samples at low (2.00 ng/mL), medium (20.0 and 400 ng/mL) and high (2000 ng/mL) concentrations were in the range 1.1-4.3% relative standard deviation (RSD) and -2.5-10.0% (bias), respectively, from three validation runs. The method has been used to measure the exposure of NIM811 in human subjects.     

An improved highly sensitive method to determine low oxyresveratrol concentrations in rat plasma and its pharmacokinetic application

Existing methods to determine oxyresveratrol, a trans‐polyphenolic stilbene, lack selectivity, require large plasma sample volumes or have time‐consuming sample preparation and chromatographic isolation. Here an improved highly sensitive liquid chromatography–tandem mass spectrometry method was developed to determine low oxyresveratrol concentrations in rat plasma. The plasma samples were prepared by liquid–liquid extraction with acetoacetate. The analyte s were separated on Venusil hydrophilic interaction chromatography (HILIC) column (2.1 × 50 mm, 5.0 µm) guarded by a HILIC column (4 × 3.0 mm, 5.0 µm). The mobile phase consisted of acetonitrile–water (containing 1 mmol/L ammonium formate) at gradient elution mode with a flow rate of 0.3 mL/min. Resveratrol was used as the internal standard. An electrospray ionization source was applied and operated in the negative multiple reaction monitoring (MRM) mode. Oxyresveratrol and resveratrol were detected on MRM by the transitions from the precursor to the product ion (m/z 243.1 → 175.1 and 227.1 → 143.0). The total running time was 5 min and the retention times of oxyresveratrol and resveratrol were 1.97 and 1.82 min. Chromatograms showed no endogenous interfering peaks with blank samples. The linear calibration curve was obtained over the concentration range of 1–500 ng/mL. The injection volume was 10 μL and the limit of quantification was 1 ng/mL. The extraction recovery varied from 78.2 to 84.3% for low, medium and high quality control samples. At the same time, the intra‐ and inter‐day relative standard deviations were <6.78 and <10.02%, respectively, while the corresponding intra‐ and inter‐day accuracy relative error values fell in the range of 3.75–6.67%. The HPLC‐MS/MS method was successfully applied to a pharmacokinetics study, in which the experimental rats received a single dose of oxyresveratrol (10 mg/kg, intragastric administration). The pharmacokinetic results are presented. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of swertianolin in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

A sensitive and rapid LC‐MS/MS method has been developed and validated for quantifying swertianolin in rat plasma using rutin as an internal standard (IS). Following liquid–liquid extraction with ethyl acetate, chromatographic separation for swertianolin was achieved on a C₁₈ column with a gradient elution using 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B at a flow rate of 0.3 mL/min. The detection was performed on a tandem mass spectrometer using multiple reaction monitoring via an electrospray ionization source and operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 435.1/272.0 for swertianolin and 609.2/300.1 for IS. The lower limit of quantitation was 0.5 ng/mL within a linear range of 0.5–500 ng/mL. Intra‐day and inter‐day precision was less than 6.8%. The accuracy was in the range of ?13.9 to 12.0%. The mean recovery of swertianolin was >66.7%. The proposed method was successfully applied in evaluating the pharmacokinetics of swertianolin after an oral dose of 50 mg/kg Swertia mussotii extract in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

Liquid chromatography/electrospray ionization tandem mass spectrometry for the quantification of mitiglinide in human plasma: validation and its application to pharmacokinetic studies

A sensitive and specific method was developed and validated for the determination of mitiglinide in human plasma using liquid chromatographic separation with electrospray ionization tandem mass spectrometric detection. Acidified plasma samples were extracted with ethyl acetate. The chromatographic separation was performed on an Agilent Zorbax Eclipse Plus C(18) column with a mobile phase of methanol-10 mm ammonium acetate solution at a flow rate of 0.3 mL/min. Analytes were detected with an Agilent 6410 Triple qudrupole mass spectrometer equipped with an electrospray ionization source in positive multiple reaction monitoring mode: m/z 316.2 (precursor ion) to 298.2 (product ion) for mitiglinide and m/z 318.2 (precursor ion) to 120.2 (product ion) for the internal standard. This method was validated over a linear range of 0.5-4000 ng/mL for mitiglinide in human plasma. The lower limit of quantification (LLOQ) was 0.5 ng/mL, while a relative standard deviation (RSD) was less than 3.9%. The intra- and inter-run precision (as RSD, %) obtained from three validation runs were all less than 15%. The validated method was successfully used to analyze human plasma samples for application in pharmacokinetic studies.     

Determination of cefdinir levels in rat plasma and urine by high‐performance liquid chromatography–tandem mass spectrometry: application to pharmacokinetics after oral and intravenous administration of cefdinir

A selective and sensitive liquid chromatography tandem mass spectrometry method (LC‐MS/MS) was developed and validated for the determination of cefdinir in rat plasma and urine. Following a simple protein precipitation using methanol, chromatographic separation was achieved with a run time of 10 min using a Synergi 4 µ polar‐RP 80A column (150 × 2.0 mm, 4 µm) with a mobile phase consisting of 0.1% formic acid in water and methanol (65:35, v/v) at a flow rate of 0.2 mL/min. The protonated precursor and product ion transitions for cefdinir (m/z 396.1 → 227.2) and cefadroxil, an internal standard (m/z 364.2 → 208.0) were monitored in the multiple reaction monitoring in positive ion mode. The calibration curves for plasma and urine were linear over the concentration range 10–10,000 ng/mL. The lower limit of quantification was 10 ng/mL. All accuracy values were between 95.1 and 113.0% and the intra‐ and inter‐day precisions were <13.0% relative standard deviation. The stability under various conditions in rat plasma and urine was also found to be acceptable at three concentrations. The developed method was applied successfully to the pharmacokinetic study of cefdinir after oral and intravenous administration. Copyright © 2013 John Wiley & Sons, Ltd.     

离子液体作高效液相色谱流动相添加剂测定水杨酸

建立了以离子液体作反相高效液相色谱流动相添加剂测定水杨酸的方法.实验以ZORBAX ODS反相色谱柱为分离柱,采用紫外检测方法,研究了检测波长、离子液体烷基链长度、离子液体溶液的浓度以及pH值等对分离和测定的影响.优化的色谱条件为:以体积比60:40的甲醇-3.0 mmol/L1-丁基-3-甲基咪唑四氟硼酸盐溶液(乙酸...     

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.     

Simultaneous determination of olmesartan medoxomil and irbesartan and hydrochlorothiazide in pharmaceutical formulations and human serum using high performance liquid chromatography

 A simple, selective, sensitive, precise, simultaneous high performance liquid chromatographic analysis of serum samples and commercial tablet formulation containing hydrochlorothiazide, olmesartan medoxomil and irbesartan are reported. Good chromatographic separation was achieved using a μ-Bondapak, C18 column (15 cm×4.6 mm, 5 μm), and a mobile phase consisting of acetonitrile-0.2% acetic acid aqueous solution (50∶50, v/v) at a flow rate of 1.0 mL/min. The ultraviolet detector was set at a wavelength of 260 nm. Hydrochlorothiazide, olmesartan medoxomil, and irbesartan were eluted at 1.2, 3.8, and 4.4 min, respectively. No extraneous materials were found to interfere. The method uses protein precipitation with acetonitrile for the preparation of serum sample. The linear ranges for hydrochlorothiazide, olmesartan medoxomil, and irbesartan were 6.25-18.75, 20-60, and 75-225 ng/mL, respectively. The recoveries of hydrochlorothiazide, olmesartan medoxomil, and irbesartan in spiked samples were all greater than 98%, and their relative standard deviations were less than 2.0%. The limits of detection were 1, 2, and 2 ng/mL for hydrochlorothiazide, olmesartan medoxomil, and irbesartan, respectively, and the limits of quantification were 3 ng/mL, which allow their determination at the expected serum concentration levels.     

A validated UHPLC–MS/MS method for the measurement of riluzole in plasma and myocardial tissue samples

Through blocking the cardiac persistent sodium current, riluzole has the potential to prevent myocardial damage post cardiac bypass surgery. A sensitive UHPLC–MS/MS method was developed and validated for quantitation of riluzole and 5‐methoxypsoralen in human plasma and myocardial tissue homogenate using a liquid–liquid extraction with dichloromethane. The chromatographic separation was achieved using Shimadzu Shim‐pack XR‐ODS III, 2.0 × 50 mm, 1.6 μm column with a gradient mobile phase comprising methanol and ammonium acetate buffer pH 3.6 in purified water. The analyte and internal standard were separated within 3.5 min. Riluzole quantitation was achieved using the mass transitions of 235–138 for riluzole and 217–156 for 5‐methoxypsoralen. The method was linear for riluzole plasma concentrations from 0.2 to 500 ng/mL and myocardial tissue homogenate concentrations from 0.2 to 100 ng/mL. The method developed was successfully applied to a clinical study for patients receiving riluzole while undergoing cardiac bypass surgery.     

A sensitive LC–MS/MS‐based bioanalytical method for quantification of salviaflaside and rosmarinic acid in rat plasma and its application in a pharmacokinetic study

A selective and sensitive liquid chromatography tandem mass spectrometry method was developed for the simultaneous determination of salviaflaside and rosmarinic acid in rat plasma. Sample preparation was carried out through liquid–liquid extraction with ethyl acetate using curculigoside as internal standard (IS). The analytes were determined by selected reaction monitoring operated in the positive ESI mode. Chromatographic separation was performed on an Agilent Eclipse Plus C₁₈ column (100 × 4.6 mm, 1.8 μm) with a mobile phase consisting of methanol–water–formic acid (50:50:0.1, v/v/v) at a flow rate of 0.3 mL/min. The run time was 1.9 min per sample and the injection volume was 5 μL. The method had an LLOQ of 1.6 ng/mL for salviaflaside and 0.94 ng/mL for rosmarinic acid in plasma. The linear calibration curves were fitted over the range of 1.6–320 ng/mL for salviaflaside and 0.94–188 ng/mL for rosmarinic acid in plasma with correlation coefficients (r²) >0.99. Intra‐ and inter‐day precisions (relative standard deviation) were < 13.5%, and accuracies (relative error) were between −8.6% and 14.5% for all quality control samples. The method was validated and applied to the pharmacokinetics of salviaflaside and rosmarinic acid in plasma after oral administration of Prunella vulgaris extract to rats.     

聚丙烯酸的凝胶渗透色谱分离及相对分子质量测定

采用Sepax Nanofilm SEC-150色谱柱和自制窄分布聚丙烯酸标样,考察了不同无机盐及其离子强度、酸度等对不同相对分子质量的聚丙烯酸分离效果的影响.在此基础上确定了凝胶渗透色谱分离聚丙烯酸的最佳条件:以磷酸盐缓冲液(离子强度0.10 mol/L,pH=6.9)为流动相,流速1.0 mL/min,柱温25 ℃...     

Determination of a selective Na+/H+ exchanger inhibitor, 4-cyano(benzo[b]thiophene-2-carbonyl)guanidine (KR-33028) in rat plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC/MS/MS) method was developed for the determination of a selective Na(+)/H(+) exchanger inhibitor 4-cyano(benzo[b]thiophene-2-carbonyl)guanidine (KR-33028) in rat plasma. KR-33028 and the internal standard, linezolid, were extracted from rat plasma with ethyl acetate at neutral pH. The analytes were separated on an XBridge C(18) column with a mixture of methanol-0.1% formic acid (35:65, v/v) as mobile phase and detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r = 0.9998) over the concentration range of 2.0-1000 ng/mL. The coefficients of variation of intra- and inter-assay were 1.3-6.8% and the relative error was 0.8-5.0%. The recoveries of KR-33028 and linezolid were 70.5 and 84.6%, respectively. The lower limit of quantification for KR-33028 was 2.0 ng/mL using 50 microL plasma sample. This method was successfully applied to the pharmacokinetic study of KR-33028 in rats.