Quantitation of Tamoxifen, 4-Hydroxytamoxifen,and N-Desmethyltamoxifen in Human Plasma by High Performance Liquid Chromatography

Abstract

A reverse phase high performance liquid chromatography assay used for quantitative analysis of tamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen extracted from human plasma was developed. Plasma samples were spiked with an internal standard, nafoxidine, extracted with 2% butanol in hexane, and activated to fluorescence by exposure to high intensity short-wave ultraviolet (254 nm) light. Aliquots of extracted plasma were then injected onto a C18 reverse phase column and eluted isocratically with a mobile phase of water and triethylamine in methanol. Fluorescence of activated tamoxifen and its metabolites was measured at 266 nm. Peak height ratios were used to quantitate tamoxifen and its metabolites from extracted human plasma specimens.     

Development and validation of an LC-MS-MS method for the simultaneous determination of sulforaphane and its metabolites in rat plasma and its application in pharmacokinetic studies

A highly sensitive and simple high-performance liquid chromatographic-tandem mass spectrometric (LC-MS-MS) assay is developed and validated for the quantification of sulforaphane and its metabolites in rat plasma. Sulforaphane (SFN) and its metabolites, sulforaphane glutathione (SFN-GSH) and sulforaphane N-acetyl cysteine (SFN-NAC) conjugates, are extracted from rat plasma by methanol-formic acid (100:0.1, v/v) and analyzed using a reversed-phase gradient elution on a Develosil 3 μm RP-Aqueous C(30) 140? column. A 15-min linear gradient with acetonitrile-water (5:95, v/v), containing 10 mM ammonium acetate and 0.2% formic acid, as mobile phase A, and acetonitrile-water (95:5, v/v), containing 10 mM ammonium acetate and 0.2% formic acid as mobile phase B, is used. Sulforaphane and its metabolites are well separated. Sulforaphene is used as the internal standard. The lower limits of quantification are 1 ng/mL for SFN and 10 ng/mL for both SFN-NAC and SFN-GSH. The calibration curves are linear over the concentration range of 25-20,000 ng/mL of plasma for each analyte. This novel LC-MS-MS method shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic studies in rats.     

Liquid chromatographic high-throughput analysis of ketamine and its metabolites in human plasma using a monolithic silica column and solid phase extraction

A rapid and sensitive liquid chromatographic (LC) assay was developed for the simultaneous determination of ketamine (KE) and its two main metabolites, namely, norketamine (NK) and dehydronorketamine (DHNK) in human plasma. Each compound together with an internal standard (Labetalol) was extracted from the plasma matrix using solid phase extraction (SPE). The applicability of monolithic LC phases in the field of quantitative bioanalysis has been evaluated. The existing method with UV detection set at 220 nm was successfully transferred from a conventional reversed phase column to a 10 cm × 4.6 mm i.d. monolithic silica column. By simply increasing the mobile phase flow-rate, run times were about six-fold reduced and consumption of mobile phase were about two-fold decreased, while the chromatographic resolution of the analytes remain unaffected. The method was validated over the range 25-2000 ng/mL for KE, 25-1500 ng/mL for NK, and 15-750 ng/mL for DHNK. The method proved to be precise (within-run precision ranges from 2.2 to 7.2% and between-run precision ranges from 3.7 to 8.2%) and accurate (within-run accuracies ranged from 1.3 to 7.2% and between-run accuracies ranged from 1.5 to 8.7%). The mean absolute recoveries were 95.3, 96.9, and 103.9% for KE, NK and DHNK, respectively. The limit of quantitation (LOQ) and limit of detection (LOD) for KE and NK in human plasma were 25 and 12.5 ng/mL, respectively, and for DHNK were 15 and 7.5 ng/mL (S/N = 3). The assay should be suitable for use in routine determination of KE and its metabolites in human plasma.     

A simple and sensitive assay for the quantitative analysis of paclitaxel and metabolites in human plasma using liquid chromatography/tandem mass spectrometry

A sensitive and specific LC-MS/MS assay for the determination of paclitaxel and its 3'p- and 6-alpha-hydroxy metabolites is presented. A 200 microL plasma aliquot was spiked with a 13C6-labeled paclitaxel internal standard and extracted with 1.0 mL tert-butylmethylether. Dried extracts were reconstituted in 0.1 M ammonium acetate-acetonitrile (1:1, v/v) and 25 microL volumes were injected onto the HPLC system. Separation was performed on a 150 x 2.1 mm C18 column using an alkaline eluent (10 mm ammonium hydroxide-methanol, 30:70, v/v). Detection was performed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range for paclitaxel from 0.25 to 1000 ng/mL and metabolites from 0.25 to 100 ng/mL using 200 microL human plasma samples. Validation results demonstrate that paclitaxel and metabolite concentrations can be accurately and precisely quantified in human plasma. This assay is now used to support clinical pharmacologic studies with paclitaxel.     

Pharmacokinetics of dronedarone in rat using a newly developed high‐performance liquid chromatographic assay method

A sensitive and selective high‐performance liquid chromatographic method for the determination of dronedarone in rat plasma was developed. Dronedarone was extracted using one‐step liquid–liquid extraction. The separation of dronedarone was accomplished using a C₁₈ analytical column. The mobile phase was composed of a combination of monobasic potassium phosphate and acetonitrile. The UV detection was at 254 nm for ethopropazine, the internal standard, and after its elution, changed to 290 nm for dronedarone detection. The total analytical run time was 20 min. Mean recovery was >80%; the assay had excellent linear relationships (>0.999) between peak height ratios and plasma concentrations; the lower limit of quantification 25 was ng/mL, based on 100 μL of rat plasma. Accuracy and precision were <18% over the concentration range of 25–500 ng/mL. The assay was applied successfully to the measurement of dronedarone plasma concentrations in rats given the drug orally. Copyright © 2014 John Wiley & Sons, Ltd.     

A highly sensitive liquid chromatography tandem mass spectrometry method for simultaneous quantification of midazolam, 1'-hydroxymidazolam and 4-hydroxymidazolam in human plasma

A highly sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of midazolam and its major metabolites 1'-hydroxymidazolam and 4-hydroxymidazolam in human plasma was developed and validated. Stable isotope-labeled midazolam-D(4) and 1'-hydroxymidazolam-D(4) were used as internal standards. Compounds were extracted from 0.5 mL plasma by liquid-liquid extraction with ethyl acetate-heptane (1:4). Chromatography was achieved using a Sunfire C(18) column. The mobile phase was a gradient with 10 m m formic acid in Milli-Q water and methanol at a flow rate of 0.3 mL/min. Total run time was 10 min. Detection was performed using a tandem mass spectrometer with positive electrospray ionization. Calibration curves were linear over the range of 0.10-50.0 ng/mL for midazolam and 0.025-25.0 ng/mL for both metabolites. For all compounds the lower limit of quantification was 0.10 ng/mL. Imprecision was assessed according to the NCCLS EP5-T guideline and was below 10% for all compounds. Mean recoveries were between 94 and 109% for midazolam and its metabolites. The validated method was successfully applied in a pharmacokinetic study investigating in vivo CYP3A-activity in a large cohort of renal allograft recipients using sub-therapeutic doses of midazolam as a drug-probe.     

HPLC Determination of Methylphenidate in Human Plasma

Abstract

A simple, rapid and sensitive method for measuring methylphenidate in human plasma by HPLC has been developed. After the addition of the internal standard, ethylphenidate, the two compounds are extracted under basic conditions. The residue obtained is resuspended in acetonitrile and analysed on an ODS reversed phase column with detection by UV absorbance at 192 nm. The limit of sensitivity is 5 ng/ml and the procedure is linear over the 5–50 ng/ml concentration range.     

Quantification of docetaxel and its main metabolites in human plasma by liquid chromatography/tandem mass spectrometry

Docetaxel is an antineoplastic agent widely used in therapeutics. The objective of this study was to develop and validate a routine assay, using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), for the simultaneous quantification of docetaxel and its main hydroxylated metabolites in human plasma. A structural analogue, paclitaxel, was used as the internal standard. Determination of docetaxel and four metabolites (M1, M2, M3 and M4) was achieved using only 100 microL of plasma. Liquid-liquid extraction was used for sample preparation, with extraction efficiency of at least 90% for all analytes. Detection used positive-mode electrospray ionization in selected reaction monitoring mode. The lower limit of quantification (LLOQ) was 0.5 ng/mL for all analytes. The assay was linear in the calibration curve range 0.5-1000 ng/mL and acceptable precision and accuracy (<15%) were obtained with concentrations above the LLOQ. This method was sufficiently selective and sensitive for quantification of metabolites in plasma from cancer patients receiving docetaxel chemotherapy, and is suitable for routine analyses during pharmacokinetic studies.     

Simultaneous determination of YM-64227, a phosphodiesterase type 4 inhibitor, and its five metabolites in dog plasma by high-performance liquid chromatography with fluorescence detection

We developed and validated a reversed-phase high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of YM-64227 [4-cyclohexyl-1-ethyl-7-methylpyrido(2,3-d)pyrimidin-2-(1H)-one], a novel and selective phosphodiesterase type 4 inhibitor, and its fi ve hydroxylated metabolites in dog plasma. The plasma samples were extracted with tert-butyl methyl ether under alkali conditions. The analytes were well separated on a phenyl ethyl column (5 microm, 250 x 4.6 mm i.d.), opreating at 40 degrees C and using an acetonitrile-acetic acid gradient at a fl ow rate of 1.0 mL/min. The fluorescence signal was monitored at an excitation and emission wavelength of 330 and 400 nm, respectively. No interfering peak was observed at the retention time of YM-64227, its metabolites or the internal standard. The validated quantitation range of the method was 0.4-200 ng/mL for all analytes using 0.5 mL of the plasma sample. The recovery of analytes in the extraction process was more than 65.5%. The intra- and inter-assay precision was less than 5.1 and 12.6%, respectively, and the intra- and inter-assay accuracy ranged from -8.1 to 11.8% and -8.0 to 9.9%, respectively. Using this assay, the plasma concentration of YM-64227 and metabolites can be determined after the oral administration of YM-64227 to beagle dogs.     

An HPLC Assay for a Prostacyclin Analogue,Ciprostene Calcium,in Human Plasma

Abstract

A sensitive assay has been developed for the quantification of the prostacyclin analogue, ciprostene calcium, in human plasma. The method involves solid phase extraction of ciprostene calcium and internal standard, carbacyclin, from a small volume of human plasma. The extract is derivatized with 4-bramamethyl-7-acetoxycoumarin, and the derivatized product extracted with a polar solid phase cartridge and concentrated by evaporation. The final extract is separated by reversed phase HPIC and measured by a fluorimetric detector following post-column alkaline hydrolysis. The overall extraction efficiency is better than 75%, and the assay is linear over the concentration range studied (5–20 ng/ml). The limit of quantification was approximately 5 ng/ml. Ultimate sensitivity was limited by interfering peaks endogenous to the biological matrix. Coefficients of variation at mid-range concentrations are less than 10%.     

A validated high-performance liquid chromatographic method for the determination of moclobemide and its two metabolites in human plasma and application to pharmacokinetic studies

A rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with ultraviolet detection has been developed for the determination of moclobemide and its metabolites, p-chloro-N-(-2-morpholinoethyl)benzamide N'-oxide (Ro 12-5637) and p-chloro-N-[2-(3-oxomorpholino)ethyl]-benzamide (Ro 12-8095), in human plasma. The assay was performed after single liquid-liquid extraction with dichloromethane at alkaline pH using phenacetin as the internal standard. Chromatographic separation was performed on a C(18) column using a mixture of acetonitrile and water (25:75, v/v), adjusted to pH 2.7 with ortho-phosphoric acid, as mobile phase. Spectrophotometric detection was performed at 239 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The quantification limit for moclobemide and Ro 12-8095 was 10 ng/mL, and for Ro 12-5637 was 30 ng/mL. Linearity of the method was confirmed for the range 20-2500 ng/mL for moclobemide (r = 0.9998), 20-1750 ng/mL for Ro 12-8095 (r = 0.9996) and 30-350 ng/mL for Ro 12-5637 (r = 0.9991). Moreover, within-day and between-day precisions and accuracies of the method were established. The described method was successfully applied in pharmacokinetic studies of parent drug and its two metabolites after a single oral administration of 150 mg of moclobemide to 20 healthy volunteers.     

Simple method for the determination of rosiglitazone in human plasma using a commercially available internal standard

To the best of our knowledge, bioanalytical methods to determine rosiglitazone in human plasma reported in literature use internal standards that are not commercially available. Our purpose was to develop a simple method for the determination of rosiglitazone in plasma employing a commercially available internal standard (IS). After the addition of celecoxib (IS), plasma (0.25 mL) samples were extracted into ethyl acetate. The residue after evaporation of the organic layer was dissolved in 750 microL of mobile phase and 50 microL was injected on to HPLC. The separation was achieved using a Hichrom KR 100, 250 x 4.6 mm C(18) with a mobile phase composition potassium dihydrogen phosphate buffer (0.01 m, pH 6.5):acetonitrile:methanol (40:50:10, v/v/v). The flow-rate of the mobile phase was set at 1 mL/min. The column eluate was monitored by fluorescence detector set at an excitation wavelength of 247 nm and emission wavelength of 367 nm. Linear relationships (r(2) > 0.99) were observed between the peak area ratio rosiglitazone to IS vs rosiglitazone concentrations across the concentration range 5-1000 ng/mL. The intra-run precision (%RSD) and accuracy (%Dev) in the measurement of rosiglitazone were <+/-10.69 and <-12.35%, respectively across the QC levels (50-1000 ng/mL). The extraction efficiency was >80% for both rosiglitazone and IS from human plasma. The lower limit of quantitation of the assay was 5 ng/mL. In summary, the methodology for rosiglitazone measurement in plasma was simple, sensitive and employed a commercially available IS.     

Simple determination of huperzine A in human plasma by liquid chromatographic-tandem mass spectrometric method

Huperzine A is a potent, reversible acetylcholinesterase inhibitor. In the present work, a rapid and sensitive LC-MS-MS method for the determination of huperzine A in human plasma using codeine phosphate as internal standard has been developed and validated. The analyte and internal standard were extracted from plasma using ethyl acetate, chromatographed on a C(18) column (5 microm, 150 x 4.6 mm i.d.) with a mobile phase consisting of 1% formic acid-methanol (40:60, v/v), and detected using a tandem mass spectrometer with a TurboIonSpray ionization interface. The run time was only 2 min. Good linearity was achieved in the range 0.126 -25.2 ng/mL and the limit of detection in plasma was 0.064 ng/mL. The average recovery for huperzine A was 83.4% from plasma. The analytical sensitivity and accuracy of this assay is adequate for characterization of huperzine A in human plasma.     

Mass spectrometric characterization of toremifene metabolites in human urine by liquid chromatography-tandem mass spectrometry with different scan modes

The metabolism and excretion of toremifene were investigated in one healthy male volunteer after a single oral administration of 120 mg toremifene citrate. Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were carried out for the characterization of the metabolites in human urine for doping control purposes. The potential characteristic fragmentation pathways of toremifene and its major metabolites were presented. An approach for the metabolism study of toremifene and its analogs by liquid chromatography-tandem mass spectrometry was established. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 72.2, 58.2, 44.2, 45.2, 88.2 relative to five metabolic pathways) in positive ion mode were assessed to recognize the metabolites. Based on product ion scan and precursor ion scan techniques, the metabolites were proposed to be identified as 4-hydroxy-toremifene (m/z 422.4), 4'-hydroxy-toremifene (m/z 422.4), α-hydroxy-toremifene (m/z 422.4), 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2), 3-hydroxy-4-methoxy-toremifene (m/z 456.2), dihydroxy-dehydro-toremifene (m/z 440.2), 3,4-dihydroxy-toremifene (m/z 438.2), N-demethyl-4-hydroxy-toremifene (m/z 408.3), N-demethyl-3-hydroxy-4-methoxy-toremifene (m/z 438.3). In addition, a new metabolite with a protonated molecule at m/z 390.3 was detected in all urine samples. The compound was identified by LC/MS/MS as N-demethyl-4,4'-dihydroxy-tamoxifene. The results indicated that 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2) and N-demethyl-4,4'-dihydroxy-tamoxifene (m/z 390.3) were major metabolites in human urine.     

Determination of methadone and its metabolites by high performance liquid chromatography following solid-phase extraction in rat plasma.

This paper describes a rapid, quantitative liquid chromatographic analysis and extraction of methadone and its two major metabolites from rat plasma, using difenoxin as the internal standard. Using a C18 column, resolution of all sample components and the internal standard is achieved with a mobile phase of 25:75 acetonitrile-0.08% diethylamine in 1000 mL water, pH 2.3, at a flow rate of 1.5 mL/min. The injection volume is 100 microL. Standards are linear over the range 25-100 ng, with a lower limit of detection for methadone of 0.25 ng. Within- and between-run coefficients of variation (CV) are 1.24% and 2.94%, respectively. Extraction of methadone and its metabolites from rat plasma uses a solid-phase extraction technique that is highly efficient. Extraction efficiencies of 90.3%, 99.6%, 85.9% and 93.8% were achieved for methadone, its primary and secondary metabolites, and difenoxin, respectively.     

Determination of bergenin in human plasma after oral administration by HPLC-MS/MS method and its pharmacokinetic study

A highly sensitive, simple and selective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and applied to the determination of bergenin concentration in human plasma. Bergenin and the internal standard (IS) thiamphenicol in plasma were extracted with ethyl acetate, separated on a C(18 )reversed-phase column, eluted with mobile phase of acetonitrile-water, ionized by negative ion pneumatically assisted electrospray and detected in the multi-reaction monitoring mode using precursor --> product ions of m/z 327.1 --> 192 for bergenin and 354 --> 185.1 for the IS, respectively. The linear range of the calibration curve for bergenin was 0.25-60 ng mL(-1), with the lowest limit of quantification of 0.25 ng mL(-1), and the intra/inter-day relative standard deviation (RSD) was less than 10%. The method is suitable for the determination of low bergenin concentration in human plasma after therapeutic oral doses, and has been first and successfully used for its pharmacokinetic studies in healthy Chinese volunteers.     

A stereospecific high-performance liquid chromatographic assay for the determination of ketoconazole enantiomers in rat plasma

A stereospecific high-performance liquid chromatographic assay was developed for the quantitation of ketoconazole enantiomers (KTZ) in rat plasma. After protein precipitation of 100 microL plasma using acetonitrile, a wash step was performed using hexane. The supernatant was removed and KTZ enantiomers and amiodarone, the internal standard, were extracted using liquid-liquid extraction with tert-butyl methyl ether. After transfer and evaporation of the organic layer, the residue was reconstituted in mobile phase and injected into the HPLC through a chiral column. The mobile phase consisted of hexane:ethanol:2-propanol with diethyl amine, pumped at 1.5 mL/min. All components eluted within 18 min. KTZ enantiomers were baseline resolved and peaks were symmetrical in appearance with no interferences. Calibration curves were linear over the range 62.5-5000 ng/mL of enantiomer. The intraday and interday CV% assessments were 相似文献   

Solid phase extraction of oxcarbazepine and its metabolites from plasma for analysis by high performance liquid chromatography.

A rapid, sensitive and simple-to-operate high performance liquid chromatographic method for the simulataneous determination of oxcarbazepine, 10-hydroxycarbazepine and 10,11-dihydro-10,11-trans-dihydroxy-carbamazepine in plasma is described. The drug and its metabolites were extracted from plasma using commercially available reversed phase octadecylsilane bonded-silica columns (Bond Elut C18, 1 mL capacity). Chromatographic separation of oxcarbazepine and its metabolites was achieved using a mobile phase consisting of acetonitrile/methanol/water (13:25:62 by volume) at a flow rate of 1.2 mL/min in conjunction with a Waters Associates Nova-Pak C18 column. The analytical column, in Radial-Pak cartridge form, was used in combination with a LiChrospher 5 microns C18 guard column. By measuring the UV absorbance at 214 nm, plasma levels in the region of 50-100 ng/mL for the drug and its metabolites can be detected with only 100 microL of plasma. The method has been applied to pharmacokinetic studies of oxcarbazepine and its metabolites in children with epilepsy; preliminary pharmacokinetic findings in two patients at steady-state are presented.     

Reverse Phase HPLC Determination of Azq in Biological Fluids

Abstract

A sensitive and specific reverse phase HPLC method employing a simple sample preparation procedure and utilizing an internal standard was developed to measure the new antitumor agent AZQ in biological fluids. A single chloroform extraction gave drug recoveries of greater than 88% from plasma, urine and CSF in the range of expected physiological concentrations (20–800 ng/ml). Isocratic reverse phase HPLC with UV detection at 340 nm resulted in a limit of quantisation of 5 ng/ml although smaller amounts of the drug could be detected. This assay was successfully applied to determine the single dose plasma pharmacokinetics of AZQ in rats. The potential of this method for determining AZQ disposition and pharmacokinetics in human subjects was demonstrated by analysis of patient CSF.     

Determination of oxprenolol in human plasma by high-performance liquid chromatography, in comparison with gas chromatography and gas chromatography-mass spectrometry

A high-performance liquid chromatographic method for the quantitative assay of oxprenolol in human plasma is described. After addition of alprenolol as internal standard, the compounds are extracted from plasma at alkaline pH into an organic phase and back-extracted into an acidic aqueous phase. Separation of the plasma components and metabolites was achieved on a reversed-phase column. Concentrations down to 66 nmol/l (20 ng/ml) can be determined with UV detection at 222 nm. This technique compares favourably with gas chromatographic and gas chromatographic-mass spectrometric methods.