Determination of clarithromycin in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A rapid and sensitive method has been developed for the determination of clarithromycin in human plasma with liquid chromatography-tandem mass spectrometry. Clarithromycin and the internal standard, telmisartan were precipitated from the matrix (50 microl) with 200 microl acetonitrile and separated by HPLC using formic acid:10 mM ammonium acetate:methanol (1:99:400, v/v/v) as the mobile phase. The assay based on detection by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring mode was finished within 2.4 min. Linearity was over the concentration range 10-5000 ng/ml with a limit of detection of 0.50 ng/ml. Intra- and inter-day precision measured as relative standard deviation were <3.73% and <9.93%, respectively. The method was applied in a bioequivalence study of two tablet formulations of clarithromycin.     

Determination of berberine in human plasma by liquid chromatography-electrospray ionization-mass spectrometry

A liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method for the determination of berberine in human plasma using chlorobenzylidine as the internal standard (IS) has been developed and validated. The plasma samples were prepared by LLE and the analytes were chromatographically separated on a Hanbon Lichrospher 5-C18 HPLC column under gradient elution with a mobile phase consisted of acetonitrile and 10mm ammonium acetate buffer containing 0.1% formic acid. Berberine was determined with electrospray ionisation-mass spectrometry (ESI-MS). LC-ESI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at M(+)m/z 336.1 for berberine and M(+)m/z 464.1 for the IS. Calibration curve was linear over the range of 0.020-3.0 ng/ml. The lower limit of quantification (LLOQ) was 0.020 ng/ml. The intra- and inter-run variability values were less than 6.7 and 7.7%, respectively. The method has been successfully applied to determine the plasma concentration of berberine in healthy Chinese volunteers.     

液相色谱-质谱／质谱联用法测定健康志愿者血清中阿德福韦

目的:建立测定人血清中阿德福韦的液相色谱-质谱/质谱联用(LC～MS/MS)方法。方法:血清样品经甲醇沉淀蛋白,上清液吹干,200μL流动相复溶,离心,取40μL进样。色谱柱为 Diamonsil C_(18)柱(250 mm×4.6 mm,5μm),流动相为甲醇-水-甲酸(20:80:0.1,v/v/v),流速0.6 mL·min~(-1),采用电喷雾离子化四极杆串联质谱,多反应监测方式测定样品浓度。监测离子对分别为 m/z274→m/z162(阿德福韦)和 m/z226→m/z135(内标阿昔洛韦)。结果:阿德福韦在1.25～160μg·L~(-1)浓度范围内线性关系良好(r=0.9992,n=5),最低定量限为1.25μg·L~(-1)。低、中、高3种浓度质控样品的日内、日间精密度小于8.64%,方法回收率99.20%～101.98%,阿德福韦提取回收率56.50%～59.26%。结论:该方法灵敏度高,定量准确,适用于阿德福韦酯人体药代动力学研究。     

Quantitation of Armillarisin A in human plasma by liquid chromatography-electrospray tandem mass spectrometry

A rapid and sensitive LC–MS/MS method for quantifying Armillarisin A in human plasma after a single oral dose (40 mg) has been developed and validated. Sample preparation used liquid–liquid extraction with a mixture of diethyl ether–dichloromethane (60:40, v/v) in an acidic environment. The retention times of Armillarisin A and the internal standard, probenecid, were 1.63 and 1.78 min, respectively. The calibration curve was linear over the range 0.15–50 ng/mL with a limit of quantitation of 0.15 ng/mL. The coefficient of variation as a measure of intra- and inter-day precision was <9.3% and the accuracy was in the range 92.5–108.0%. The Armillarisin A concentration–time profile in human plasma was determined after an oral dose of a 40 mg tablet.     

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.     

Determination and pharmacokinetic study of amlodipine in human plasma by ultra performance liquid chromatography-electrospray ionization mass spectrometry

A novel, specific and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination and pharmacokinetic study of amlodipine in human plasma. The analysis was carried out on an ACQUITY UPLC BEH C(18) column (50 mm x 2.1 mm, i.d., 1.7 microm) with gradient elution at a flow-rate of 0.35 ml/min. The mobile phase was water and acetonitrile under gradient conditions (both containing 0.3% formic acid) and nimodipine was used as the internal standard. Detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via Turbo ion spray ionization (ESI). Linear calibration curves were obtained over the concentration range 0.15-16.0 ng/ml, with a lower limit of quantification of 0.15 ng/ml. The intra- and inter-day precision (R.S.D.) values were below 15% and the accuracy (R.E.) was -2.3% to 6.9% at all three QC levels. The method was used to support clinical pharmacokinetic studies of amlodipine in healthy volunteers following oral administration.     

Quantification of nitrated tryptophan in proteins and tissues by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry

Aromatic amino acids are targets of reactive nitrogen species (RNS) such as peroxynitrite (ONOO(-)) and nitrogen dioxide. It is known that tryptophan (Trp) as well as tyrosine is nitrated, generated isomers. However, no quantitative method to determine nitrotryptophan (NO(2)Trp) in proteins has been developed so far. In this study, we have developed a method for the quantification of Trp and NO(2)Trp isomers, 2-, 4- and 6-NO(2)Trp, which uses liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In order to confirm the applicability of our method to in vitro and in vivo system, we measured protein-bound NO(2)Trp levels in ONOO(-) treated bovine serum albumin (BSA) and in liver of B6C3F1 mice at 2, 4, and 8h after administration of 300 mg/kg acetaminophen (APAP). A mass spectrometer equipped with an electrospray ionization source using a crossflow counter electrode and ran in the positive ion mode (ESI(+)) was used for multiple reaction monitoring (MRM) of transitions 205-->188, 250-->130, 250-->159 and 250-->233 for Trp, 2-, 4- and 6-NO(2)Trp, respectively. The recoveries from mice liver samples were 98.3-105.9% for each compound. The limits of quantification were 50, 3.0, 10 and 4.0 nM for Trp, 2-, 4- and 6-NO(2)Trp, respectively. In in vitro experiments demonstrated that all isomers of NO(2)Trp were detectable from BSA treated with ONOO(-) and the amount generated decreased in the order of 6-, 4- and 2-NO(2)Trp. In in vivo experiments, 4- and 6-NO(2)Trp were detected in the liver of mice administered APAP. The concentration range of 4- and 6-NO(2)Trp per mol of Trp in the sample was 2.24-3.92 and 26.96-32.71 nmol/mol of Trp, and its existence in vivo was confirmed for the first time with our method. The LC-ESI-MS/MS method was able to determine protein-bound NO(2)Trp in a small amount of tissue sample, and is therefore applicable not only as a biomarker of RNS, but also as a mean to clarify novel mechanisms underlying RNS-related tissue damage.     

Determination of loratadine and its active metabolite in human plasma by high-performance liquid chromatography with mass spectrometry detection

A new sensitive and selective liquid chromatography coupled with mass spectrometry (LC/MS/MS) method for quantification of loratadine (LOR) and its active metabolite descarboethoxyloratadine (DSL) in human plasma was validated. After addition of the internal standard, metoclopramide, the human plasma samples (0.3 ml) were precipitated using acetonitrile (0.75 ml) and the centrifuged supernatants were partially evaporated under nitrogen at 37 degrees C at approximately 0.3 ml volume. The LOR, DSL and internal standard were separated on a reversed phase column (Zorbax SB-C18, 100 mmx3.0 mm i.d., 3.5 microm) under isocratic conditions using a mobile phase of an 8:92(v/v) mixture of acetonitrile and 0.4% (v/v) formic acid in water. The flow rate was 1 ml/min and the column temperature 45 degrees C. The detection of LOR, DSL and internal standard was in MRM mode using an ion trap mass spectrometer with electrospray positive ionisation. The ion transitions were monitored as follows: 383-->337 for LOR, 311-->(259+294+282) for DSL and 300-->226.8 for internal standard. Calibration curves were generated over the range of 0.52-52.3 ng/ml for both LOR and DSL with values for coefficient of determination greater than 0.994 by using a weighted (1/y) quadratic regression. The lower limits of quantification were established at 0.52 ng/ml LOR and DSL, respectively, with an accuracy and precision less than 20%. Both analytes demonstrated good short-term, long-term, post-preparative and freeze-thaw stability. Besides its simplicity, the sample treatment allows obtaining a very good recovery of both analytes, around 100%. The validated LC/MS/MS method has been applied to a pharmacokinetic study of loratadine tablets on healthy volunteers.     

Determination of azelnidipine in human plasma by liquid chromatography-electrospray ionization-mass spectrometry

A liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method for the determination of azelnidipine in human plasma was established. Nicardipine was used as the internal standard (IS). After adjustment to a basic pH with sodium hydroxide solution (0.1 M), plasma samples were extracted with cyclohexane-diethyl ether (1:1, v/v) and separated on a C(18) column with a mobile phase of 20 mM ammonium acetate solution-methanol-formic acid (25:75:0.5, v/v). The electrospray ionization was employed in a single quadrupole mass spectrometer for the determination. The method was linear over the concentration range of 0.05-40 ng/ml. The lower limit of quantification (LLOQ) was 0.05 ng/ml. The intra- and inter-run standard deviations were less than 9.5% and 11.0%, respectively. The method was successfully applied to study the pharmacokinetics of azelnidipine in healthy Chinese volunteers.     

Determination of limonin in rat plasma by liquid chromatography-electrospray mass spectrometry

A simple, sensitive and selective liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) method for determination of limonin (LM) in rat plasma has been developed and validated. The method had advantages of a single liquid–liquid extraction with ether and high sensitivity. Analyses were conducted at a flow rate of 0.2 ml/min by a gradient elution. The detection utilized selected ion monitoring in the negative ion mode at m/z 460.00 and 423.15 for the deprotonated molecular ions of LM and the internal standard, respectively. The quantitation limit for LM in rat plasma was 1.0 ng/ml. The linearity was also excellent over the concentration range of 1.9–500 ng/ml of LM. The intra- and inter-day precision (relative standard deviation (R.S.D.%)) was lower than 10% and accuracy ranged from 90 to 110%, showing a good reproducibility. This developed method was successfully applied to analysis of LM in biological fluids.     

Liquid chromatography tandem mass spectrometry assay to determine the pharmacokinetics of aildenafil in human plasma

A simple, sensitive and specific liquid chromatography/tandem mass spectrometry method for the quantitation of aildenafil, a new phosphodiesterase V inhibitor, in human plasma is presented. The analyte and internal standard, sildenafil, were extracted by a one-step liquid-liquid extraction in alkaline conditions and separated on a C(18) column using ammonia:10mM ammonium acetate buffer:methanol (0.1:15:85, v/v/v) as the mobile phase. The detection by an API 4000 triple quadrupole mass spectrometer in multiple-reaction monitoring mode was completed within 2.5 min. The calibration curve exhibited a linear dynamic range of 0.05-100 ng/ml with a 10 pg/ml limit of detection. The intra- and inter-day precisions measured as relative standard deviation were within 8.04% and 5.72%, respectively. This method has been used in a pharmacokinetic study of aildenafil in healthy male volunteers each given an oral administration of one of the three dosages.     

Quantitative assay for bradykinin in rat plasma by liquid chromatography coupled to tandem mass spectrometry

An assay to quantify bradykinin in rat plasma has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Sar-D-Phe(8)-des-Arg(9)-bradykinin was used as internal standard. Aprotinin was added to rat plasma to inhibit the activity of proteinases. Recoveries for solid-phase extraction (SPE) on Strata X reversed phase were greater than 80%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with an electrospray source (ESI), operating in the positive ion-mode, was used for detection. The assay was validated and stability was explored. Bradykinin (10-500 ng/mL) was quantified with accuracy values (% RE) below 10% and intra- and inter-day precisions (% RSD) below 12 and 16%, respectively, for all concentrations. The method was successfully applied to several plasma samples from low levels kallikrein rats (LKRs) compared with normal kallikrein rats (NKRs).     

高效液相色谱-串联质谱法测定人血清中替加环素的浓度

目的：建立人血清中替加环素浓度的高效液相色谱-串联质谱法（HPLC-MS/MS）测定方法。方法：以[叔丁基-d9]替加环素为内标,将待测血清样品经氢氧化钠碱化后,加乙酸乙酯-二氯甲烷（4∶1,V/V）提取,上清液经真空干燥,得到的残渣经流动相复溶后进样。进样液经Agilent Eclipse plus C₈（4.6 mm×100 mm,5 μm）色谱柱分离,甲醇-水（30∶70,V/V,含0.005 mol·L^-1甲酸铵和0.25%甲酸）为流动相,流速0.5 mL·min^-1,进行洗脱,采用电喷雾电离源（ESI）,以多反应监测（MRM）方式进行正离子检测。用于定量分析的离子分别为替加环素m/z586.4→513.3和[叔丁基-d9]替加环素m/z595.4→514.3。结果：血清中替加环素的浓度与峰面积比在10~2 000 ng·mL^-1范围内线性关系良好,定量下限为10 ng·mL^-1,提取回收率大于70.04%,日内日间精密度小于4.54%。结论：该法简单、灵敏、快速,分离效果良好,可用于人体血药浓度测定和PK/PD研究。     

高效液相色谱-质谱/质谱联用法测定人血浆中莫沙必利

建立测定人血浆中莫沙必利的高效液相色谱-质谱/质谱联用法。取血浆样品经液-液萃取后，以乙腈为有机相，0.3%甲酸水溶液为水相，采用梯度洗脱的方式，用C₁₈柱分离，通过电喷雾离子化，以多反应监测(MRM)方式进行正离子检测。莫沙必利线性范围为0.17~68.00 ng·mL^-1，定量下限为0.17 ng·mL^-1，每个样品测试时间仅2.8 min，日内、日间精密度(RSD)均小于13%，准确度(RE)在±6.3%范围内。应用此法研究了20名志愿者单剂量口服枸橼酸莫沙必利片后的药代动力学特点。该方法、灵敏、准确、快速，适用于莫沙必利的药代动力学及生物等效性研究。     

Simultaneous determination of alfentanil and midazolam in human plasma using liquid chromatography and tandem mass spectrometry

A fast, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of alfentanil and midazolam in human plasma has been developed and validated. Alfentanil and midazolam were extracted from plasma using a mixed-mode cation exchange solid phase extraction method, with recoveries of both compounds greater than 80% at 3 different concentrations (1, 10 and 100ng/ml). Compounds were analyzed on a C(18) column with a water and methanol mobile phase gradient with acetic acid as an additive, at a flow rate of 0.3ml/min. The working assay range was linear from 0.25 to 100ng/ml for each compound. The signal to noise ratio was 80 and 40 for alfentanil and midazolam, respectively, at the lowest concentration calibration standard, with less than 10% matrix suppression by human plasma at this concentration. Alfentanil and midazolam were stable in human plasma during storage at -80°C, processing, and analysis. The procedure was validated and applied to the analysis of plasma samples from healthy human subjects administered oral and intravenous alfentanil and midazolam.     

Determination of olprinone in human plasma utilizing liquid chromatography tandem mass spectrometry

A sensitive and rapid method was developed for quantification of olprinone in human plasma utilizing liquid chromatography tandem mass spectrometry (LC–MS/MS). An aliquot of 1 mL plasma sample was extracted with ethyl acetate–dichloromethane. Separation of olprinone and the milrinone (internal standard, IS) from the interferences was achieved on a C₁₈ column followed by MS/MS detection. The analytes were monitored in the positive ionization mode. Multiple reaction monitoring using the transition of m/z 251 → m/z 155 and m/z 212 → m/z 140 was performed to quantify olprinone and IS, respectively. The method had a total chromatographic run time of 3 min and linear calibration curves over the concentration range of 0.5–60 ng/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL. The intra- and inter-day precisions were less than 16.3% for low QC level, and 7.1% for other QC levels, respectively. The intra- and inter-day relative errors were ranged between −12.2% and 3.7% for three QC concentration levels. The validated method was successfully applied to the quantification of olprinone concentration in human plasma after intravenous (i.v.) administration of olprinone at a constant rate of infusion of 2 μg/(kg min) for 5 min in order to evaluate the pharmacokinetics.     

Determination of benzodiazepines in human urine using solid-phase extraction and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the determination of benzodiazepines, on the market in Norway, and/or their metabolites in human urine. The following compounds were included: 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, alpha-hydroxyalprazolam, oxazepam, 3-OH-diazepam, and n-desmethyldiazepam. The method includes hydrolysis of urine samples (0.5 mL) with beta-glucuronidase at 60 degrees C for 2 h before solid-phase extraction with a polymer-based mixed-mode column. The analytes were quantified in multiple reaction monitoring mode using two transitions. Deuterated analogues were used as internal standards for all analytes except 7-aminonitrazepam and alpha-hydroxyalprazolam, which were quantified using 7-aminoclonazepam-d(4) and alprazolam-d(5), respectively. The concentration range was 0.1-8.0 microM for 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, and alpha-hydroxyalprazolam and 0.5-40 microM for the other compounds. The average recovery for the different analytes ranged from 56% to 83%. The between-day precision of the method was in the range of 3-12%. The limits of quantification were found to be between 0.002 and 0.01 microM for the different compounds. Comparison with other analytical methods was performed for method validation, using approximately 500 samples provided by the routine laboratory at the Norwegian Institute of Public Health. The LC-MS-MS method has proven to be robust and specific for the determination of benzodiazepines in urine. It has been routinely used for approximately 1800 samples in the past 7 months.     

High sensitive analysis of rat serum bile acids by liquid chromatography/electrospray ionization tandem mass spectrometry

Sensitive liquid chromatography (LC)/electrospray ionization (ESI) tandem mass spectrometry (MS) can be used to analyze the bile acid composition of rat serum. This method can analyze eight common bile acids and their glycine and taurine conjugates in 100 μl rodent serum by gradient elution on a reversed-phase column using a mixture of 20 mM ammonium acetate buffer (pH 8.0), acetonitrile and methanol as a mobile phase. Selected reaction monitoring analysis under negative ion detection mode allowed the achievement of a high sensitive assay with a simple solid phase extraction using an ODS cartridge column. We used this method to investigate the effect of a one-day fast on the concentration and composition of serum bile acids in rats. The results suggested that the method described here is useful for the dynamic analysis of serum bile acids in rats.     

Determination of puerarin in rabbit aqueous humor by liquid chromatography tandem mass spectrometry using microdialysis sampling after topical administration of puerarin PAMAM dendrimer complex

To study pharmacokinetic properties of puerarin poly(amido amine) (PAMAM) dendrimer complex, a sensitive liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed and validated to determine puerarin in rabbit aqueous humor using microdialysis sampling. Astilbin was used as the internal standard. The linear range for puerarin was from 2 to 1000 ng/mL (r = 0.9986) based on 20 μL of aqueous humor. The coefficients of variations for intra-day and inter-day precisions were less than 10.0%, and the relative error of accuracy was within ±6.3%. The mean extraction recovery of puerarin varied from 80.4% to 85.5%. Microdialysis provides a complete concentration versus time profile. A significant difference was observed in main pharmacokinetic parameters of C_max, AUC and t_1/2 between puerarin solution and puerarin PAMAM dendrimer complex. Complex formation resulted in an obvious increase in bioavailability of puerarin after topical administration to rabbit according to the above LC-MS/MS assay method.     

Determination of isotretinoin in human plasma by high performance liquid chromatography-electrospray ionization mass spectrometry

A rapid, sensitive and specific high performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for the quantification of 13-cis-retinoic acid (isotretinoin) in human plasma has been developed. Acitretin was employed as the internal standard (IS). The analytes were chromatographically separated on a Shimadzu Shim-pack VP-ODS C18 column (150 mm × 2.0 mm I.D.) with a mobile phase consisting of acetonitrile and water (90:10, v/v). Detection was performed on a single quadrupole mass spectrometer using an electrospray ionization interface with the selected-ion monitoring (SIM) mode. The method showed excellent linearity (r = 0.9989) over the concentration range of 10-1500 ng/mL with good accuracy and precision. The intra- and inter-batch precisions were within 10% relative standard deviation. The recoveries were more than 80%. The validated method was successfully applied to a preliminary bioequivalence study of isotretinoin in 20 Chinese healthy male volunteers.