Simultaneous quantification of dextromethorphan and its metabolites dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan in human plasma by ultra performance liquid chromatography/tandem triple-quadrupole mass spectrometry

A rapid and sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method has been developed and validated for the simultaneous quantitative determination of dextromethorphan (DM) and its metabolites dextrorphan (DX), 3-methoxymorphinan (3MM) and 3-hydroxymorphinan (3HM), in human lithium heparinized plasma. The extraction involved a simple liquid–liquid extraction with 1 ml n-butylchloride from 200 μl aliquots of plasma, after the addition of 20 μl 4% (v/v) ammonium hydroxide and 100 μl stable labeled isotopic internal standards in acetonitrile. Chromatographic separations were achieved on an Aquity UPLC^® BEH C₁₈ 1.7 μm 2.1 mm × 100 mm column eluted at a flow-rate of 0.250 ml/min on a gradient of acetonitrile. The overall cycle time of the method was 7 min, with elution times of 1.3 min for DX and 3HM, 2.8 min for 3MM and 2.9 min for DM. The multiple reaction monitoring transitions were set at 272 > 215 (m/z), at 258 > 133 (m/z), at 258 > 213 (m/z) and at 244 > 157 (m/z) for DM, DX, 3MM and 3HM, respectively. The calibration curves were linear (r² ≥ 0.995) over the range of 0.500–100 nM with the lower limit of quantitation validated at 0.500 nM for all compounds, which is equivalent to 136, 129, 129 and 122 pg/ml for DM, DX, 3MM and 3HM, respectively. Extraction recoveries were constant, but ranged from 39% for DM to 83% for DX. The within-run and between-run precisions were within 11.6%, while the accuracy ranged from 92.7 to 110.6%. The applicability of the bioanalytical method was demonstrated and is currently implemented in a clinical trial to study DM as probe-drug for individualized tamoxifen treatment in breast cancer patients.     

Determination of chamaechromone in rat plasma by liquid chromatography-tandem mass spectrometry: application to pharmacokinetic study

A rapid, simple and accurate method was developed for the determination of chamaechromone in rat plasma using liquid chromatography tandem mass spectrometry (LC–MS–MS). Rosuvastatin was used as the internal standard. The plasma samples were extracted by liquid–liquid extraction with ethyl acetate. Chromatographic separation was performed on Xbridge™ C₁₈ column (2.1 mm × 50 mm, 3.5 μm) with linear gradient elution using water and methanol, both of which were acidified with 0.1% aqueous formic acid. The flow rate was 0.4 mL/min and the total run time was 6 min. Detection was performed on a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 543.3 → 198.9 and 481.9 → 258.3 for chamaechromone and rosuvastatin, respectively. Good linearity was observed over the concentration range of 8–6400 ng/mL in 0.1 mL of rat plasma. The lowest concentration (8 ng/mL) in the calibration curve was estimated as LLOQ with both deviation of accuracy and RSD of precision <20% (n = 6). Intra-assay and inter-assay variability were less than 11% in plasma. This method was successfully applied to a pharmacokinetic study of chamaechromone in rats after intravenous (5 mg/kg) and oral (100 mg/kg) administration. Following oral administration the concentration–time curve of chamaechromone exhibited a biphasic absorption profile. The maximum mean concentration in plasma (C_max, 795.9 ± 14.6 ng/L) was achieved at 11.3 ± 0.8 h (T_max) and the area under curve (AUC_0–60) was 6976.7 ± 1026.9 ng h/L. After single intravenously administration of chamaechromone, the essential pharmacokinetic parameters C_max, AUC_0–48 were 4300.7 ± 113.6 ng/L and 3672.1 ± 225.4 ng h/L, respectively. The result showed that the compound was poorly absorbed with an absolute bioavailability being approximately 8.9%.     

Liquid chromatography–tandem mass spectrometry for the simultaneous quantitation of artemether and lumefantrine in human plasma: Application for a pharmacokinetic study

A liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) method for the simultaneous quantitation of artemether and lumefantrine in human plasma was developed and validated. Artesunate was used as an internal standard (IS). The analytes were extracted by a protein precipitation procedure and separated on a reversed-phase Zorbax SB-Ciano column with a mobile phase composed of methanol and 10 mM aqueous ammonium acetate containing 0.2% (v/v) acetic acid and 0.1% (v/v) formic acid. Multiple reaction monitoring was performed using the transitions m/z 316 → m/z 267, m/z 530 → m/z 348 and m/z 402 → m/z 267 to quantify artemether, lumefantrine and artesunate, respectively. Calibration curves were constructed over the range of 10–1000 ng/mL for artemether and 10–18,000 ng/mL for lumefantrine. The lower limit of quantitation was 10 ng/mL for both drugs. The mean R.S.D. values for the intra-run precision were 2.6% and 3.0% and for the inter-run precision were 3.6% and 4.6% for artemether and lumefantrine, respectively. The mean accuracy values were 102.0% and 101.2% for artemether and lumefantrine, respectively. No matrix effect was detected in the samples. The validated method was successfully applied to determine the plasma concentrations of artemether and lumefantrine in healthy volunteers, in a one-dose pharmacokinetic study, over the course of 11 days.     

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.     

Chromatography–tandem mass spectrometry method for the simultaneous quantitation of metoprolol succinate and simvastatin in human plasma

The liquid chromatographic–tandem mass spectrometry method was developed for the accurate quantitation of metoprolol succinate (MET) and simvastatin (SIM) in human plasma which were obtained from the pharmacokinetic (PK) study. The sample purification and pre-concentration was performed by protein precipitation technique using propranolol hydrochloride as working internal standard (WIS). The chromatographic separation was achieved using an isocratic mobile phase consisting of a mixture of acetonitrile and 0.5% formic acid (90:10 (v/v), pH 3.5) flowing through C18 column at a flow rate of 0.2 ml/min. Electro spray ionization (ESI) with multiple reaction monitoring (MRM) was used to acquire mass spectra. Ions were monitored in positive mode and the mass transitions measured were m/z 268.1 → m/z 103.2, m/z 441.3 → m/z 325.1 and m/z 260.0 → m/z 129.5 for MET, SIM and WIS, respectively. An extensive pre-study method validation was carried out in accordance with USFDA guidelines. The linearity for the calibration curve in the concentration range of 1.0–500.0 and 0.1–20 ng/ml for MET and SIM, respectively and the lower limits of quantitations (LLOQ) were 1.0 and 0.1 ng/ml for MET and SIM, respectively. The method was successfully applied to a PK study on fixed dose combination (FDC) tablet containing MET and SIM in healthy human subjects.     

Determination of a peroxisome proliferator-activated receptor γ agonist, 1-(trans-methylimino-N-oxy)-6-(2-morpholinoethoxy-3-phenyl-1H-indene-2-carboxylic acid ethyl ester (KR-62980) in rat plasma by liquid chromatography–tandem mass spectrometry

A novel peroxisome proliferator-activated receptor γ (PPARγ) agonist, KR-62980, was determined by liquid–liquid extraction with ethyl acetate and liquid chromatography–tandem mass spectrometry (LC/MS/MS) in rat plasma. In order to evaluate the pharmacokinetics of KR-62980, a reliable, selective and sensitive high-performance liquid chromatographic method with electrospray ionization tandem mass spectrometry was developed for the quantification of KR-62980 in rat plasma. KR-62980 and imipramine (IS) were separated on Hypersil GOLD C18 column with a mixture of acetonitrile–ammonium formate (10 mM) (80:20, v/v) as mobile phase. The ion transitions monitored were m/z 437.2 → 114.2 for KR-62980, m/z 281.3 → 86.1 for imipramine in multiple reaction monitoring (MRM) mode. The percent recoveries of KR-62980 and imipramine were 90.1 and 98.4% from rat plasma, respectively. The linear dynamic range extended from 0.01 to 10 μg/ml with a correlation coefficient (R²) greater than 0.99 and the lower limit of quantification was 0.01 μg/ml. The mean of intra- and inter-assay precisions was 2.1 and 9.3%. The method was validated and successfully applied to the pharmacokinetic study of KR-62980 in rat.     

Effect of pH on absorption of sufentanil citrate in a portable pump reservoir during storage and administration under simulated epidural conditions

Sufentanil (5g/ml as citrate) was investigated for its stability when diluted with sodium chloride 0.9%, in 100 ml polyvinyl chloride portable pump reservoirs during administration under simulated epidural conditions at 32°C for 48 h. Sufentanil was absorbed into the polyvinyl chloride, resulting in a reduction of 10.9% of the concentration after 48 h. The absorption of sufentanil (5g/ml as citrate), alone and in combination with bupivacaine hydrochloride (2 mg/ml), was investigated when diluted with sodium chloride 0.9% in combination with a citrate buffer (pH 4.6), in the same reservoirs under similar conditions. There was no loss of sufentanil after 48 h in both experiments. The effect of the pH on the absorption of sufentanil in polyvinyl chloride was investigated at different pH values. After storage for 21 days at 32°C there was 5.1% loss of sufentanil at pH 4 and 80.6% loss at pH 6. The citrate buffer at the optimum pH (4.6) has a low, acceptable buffer capacity for epidural administration.     

Quantitation of the tacrine analogue octahydroaminoacridine in human plasma by liquid chromatography–tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the determination of octahydroaminoacridine in human plasma using tramadol as internal standard (I.S.). Sample preparation involved pH adjustment with sodium carbonate followed by solvent extraction with dichloromethane:ethyl ether (40:60, v/v). Chromatographic separation was achieved on a Venusil MP-C18 column (5 μm, 100 mm × 4.6 mm) using acetonitrile:10 mM ammonium acetate:formic acid (30:70:1, v/v/v) as mobile phase. Detection utilized an API 4000 system operated in the positive ion mode with multiple reaction monitoring of the analyte at m/z 203.1 → 175.1 and of the I.S. at m/z 264.1 → 58.0. The method was linear in the range 0.01–10 ng/ml with a lower limit of quantitation of 0.01 ng/ml. Intra- and inter-day precisions measured as relative standard deviation were <3.15% and <5.01%, respectively. The method was successfully applied to a pharmacokinetic study involving oral administration of a tablet containing 4 mg octahydroaminoacridine succinate to healthy volunteers. Pharmacokinetic parameters for octahydroaminoacridine include C_max 1.19 ± 0.53 ng/ml, T_max 0.77 ± 0.17 h, AUC_0−t 3.42 ± 1.01 ng h/ml and t_1/2 2.89 ± 0.56 h.     

Determination of gemifloxacin in different tissues of rat after oral dosing of gemifloxacin mesylate by LC–MS/MS and its application in drug tissue distribution study

A simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to evaluate the accumulation of gemifloxacin in different tissues of Wister albino rat. The analytical method consists of the homogenization of tissues followed by simple liquid–liquid extraction and determination of gemifloxacin by an LC–MS/MS. The analyte was separated on a Peerless basic C₁₈ column (33 mm × 4.6 mm, 3 μm) with an isocratic mobile phase of methanol–water containing formic acid (1.0%, v/v) (9:1, v/v) at a flow rate of 0.6 ml/min. The MS/MS detection was carried out by monitoring the fragmentation of m/z 390.100 → 372.100 for gemifloxacin and m/z 332.100 → 314.200 for ciprofloxacin (internal standard; IS) on a triple quadrupole mass spectrometer. The validated method was accurate, precise and rugged with good linearity in all tissue homogenates. The accuracy and precision value obtained from six different sets of quality control samples of all tissues and serum analyzed in separate occasions within 91.833–102.283% and 0.897–5.291%, respectively. The method has been successfully applied to tissue distribution studies of gemifloxacin. The present study demonstrates that the highest tissue concentration of gemifloxacin was obtained in lung (11.891 ng/g), followed by liver (10.110 ng/g), kidney (10.095 ng/g), heart (4.251 ng/g), testis (3.750 ng/g), stomach (3.182 ng/g), adipose tissue (1.116 ng/g) and brain (0.982 ng/ml) in 3 h after multiple oral dosing of 200 mg gemifloxacin mesylate for 7 days. This method may also be used for gemifloxacin tissue distribution modeling study in rat tissues and antibiotic residue analyses in other animal tissues.     

Simultaneous quantitative analysis of fasudil and its active metabolite in human plasma by liquid chromatography electro-spray tandem mass spectrometry

A fast and sensitive method to quantify fasudil hydrochloride (FH) and its active metabolite hydroxyfasudil (M3) in human plasma using HPLC–MS/MS has been developed and validated in present study. The method involved simple sample preparation with methanol as protein precipitation (3:1, v/v) and ranitidine as an internal standard (IS). The analytes and IS were separated using a gradient elution procedure on the analytical column ZORBAX StableBond-C18 (5 μm, 150 mm × 4.6 mm). Detection was performed by an AB 3200 QTRAP tandem mass spectrometer equipped with a Turbo IonSpray ionization source set in positive ion mode. Multiple reaction monitoring (MRM) using the precursor to product ion was m/z 292.2/99.2 for fasudil, m/z 308.2/99.2 for M3 and m/z for 315.3/176.2 for IS. The linear range of the method was from 0.4 to 250 ng/mL for both fasudil and M3. The lower limit of quantification was 0.4 ng/mL for both fasudil and M3. The intra- and inter-day relative standard deviation over the entire concentration range was less than 7.11% for fasudil and 10.6% for M3, respectively. The validated method was successfully applied for the evaluation of pharmacokinetic of fasudil hydrochloride after administration of 30 mg fasudil hydrochloride by continuous intravenous infusion over 30 min in 12 healthy Chinese volunteers.     

LC–MS/MS determination of carbamathione in microdialysis samples from rat brain and plasma

A selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed for the determination of S-(N, N-diethylcarbamoyl) glutathione (carbamathione) in microdialysis samples from rat brain and plasma. S-(N, N-Diethylcarbamoyl) glutathione (carbamathione) is a metabolite of disulfiram. This metabolite may be responsible for disulfiram's effectiveness in the treatment of cocaine dependence. Chromatographic separations were carried out on an Alltech Altima C-18 (50 mm long × 2.1 mm i.d., 3 μm particles) analytical column at a flow rate of 0.3 ml/min. Solvent A consisted of 10 mM ammonium formate, methanol, and formic acid (99:1:0.06, v/v/v). Solvent B consisted of methanol, 10 mM ammonium formate and formic acid (99:1:0.06, v/v/v). A 20 min linear gradient from 95% aqueous to 95% organic was used. Tandem mass spectra were acquired on a Micromass Quattro Ultima “triple” quadrupole mass spectrometer equipped with an ESI interface. Quantitative mass spectrometric analysis was conducted in positive ion mode selected reaction monitoring (SRM) mode looking at the transition of m/z 407–100 and 175 for carbamathione and m/z 392–263 for the internal standard S-hexyl glutathione. The simultaneous collection of microdialysate from blood and brain was used to monitor carbamathione concentrations centrally and peripherally. Good linearity was obtained over a concentration range of 0.25–10,000 nM. The lowest limit of quantification (LLOQ) was determined to be 1 nM and the lowest limit of detection (LLOD) was calculated to be 0.25 nM. Intra- and inter-day accuracy and precision were determined and for all the samples evaluated, the variability was less that 10% (R.S.D.).     

Development and validation of a LC–MS/MS method for determination of bivalirudin in human plasma: Application to a clinical pharmacokinetic study

A simple, sensitive and rapid LC–MS/MS method has been developed and validated for the identification and quantification of bivalirudin in human plasma using nafarelin as the internal standard. Following protein precipitation with methanol, the analytes were separated on a C₁₈ column interfaced with a triple-quadrupole tandem mass spectrometer using positive electrospray ionization. Quantification of bivalirudin was conducted by multiple reaction monitoring (MRM) of the transitions of m/z 1091.4 → (356.4 + 227.4) for bivalirudin and m/z 662.4 → 328.5 for IS. The lower limit of quantification was 1.25 ng/ml, and the assay exhibited a linear range of 1.25–500 ng/ml. The developed assay method was successfully applied to a pharmacokinetic (PK) study in healthy volunteers after intravenous administration of bivalirudin.     

Simultaneous determination of 3-chlorotyrosine and 3-nitrotyrosine in human plasma by direct analysis in real time–tandem mass spectrometry

A novel method for the simultaneous determination of 3-nitrotyrosine (NT) and 3-chlorotyrosine (CT) in human plasma has been developed based on direct analysis in real time–tandem mass spectrometry (DART–MS/MS). Analysis was performed in the positive ionization mode using multiple reaction monitoring (MRM) of the ion transitions at m/z 216.2/170.1 for CT, m/z 227.2/181.1 for NT and m/z 230.2/184.2 for the internal standard, d³-NT. The assay was linear in the ranges 0.5–100 μg/mL for CT and 4–100 μg/mL for NT with corresponding limits of detection of 0.2 and 2 μg/mL. Intra- and inter-day precisions and accuracies were respectively <15% and ±15%. Matrix effects were also evaluated. The method is potentially useful for high throughput analysis although sensitivity needs to be improved before it can be applied in clinical research.KEY WORDS: 3-Nitrotyrosine, 3-Chlorotyrosine, Determintion, DART–MS/MS, Human plasma     

Quantification of tamoxifen and three of its phase-I metabolites in human plasma by liquid chromatography/triple-quadrupole mass spectrometry

In view of future pharmacokinetic studies, a highly sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method has been developed for the simultaneous quantification of tamoxifen and three of its main phase I metabolites in human lithium heparinized plasma. The analytical method has been thoroughly validated in agreement with FDA recommendations. Plasma samples of 200 μl were purified by liquid-liquid extraction with 1 ml n-hexane/isopropanol, after deproteination through addition of 50 μl acetone and 50 μl deuterated internal standards in acetonitrile. Tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and endoxifen were chromatographically separated on an Acquity UPLC^® BEH C18 1.7 μm 2.1 mm × 100 mm column eluted at a flow-rate of 0.300 ml/min on a gradient of 0.2 mM ammonium formate and acetonitrile, both acidified with 0.1% formic acid. The overall run time of the method was 10 min, with elution times of 2.9, 3.0, 4.1 and 4.2 min for endoxifen, 4-hydroxy-tamoxifen, N-desmethyl-tamoxifen and tamoxifen, respectively. Tamoxifen and its metabolites were quantified by triple-quadrupole mass spectrometry in the positive ion electrospray ionization mode. The multiple reaction monitoring transitions were set at 372 > 72 (m/z) for tamoxifen, 358 > 58 (m/z) for N-desmethyl-tamoxifen, 388 > 72 (m/z) for 4-hydroxy-tamoxifen and 374 > 58 (m/z) for endoxifen. The analytical method was highly sensitive with the lower limit of quantification validated at 5.00 nM for tamoxifen and N-desmethyl-tamoxifen and 0.500 nM for 4-hydroxy-tamoxifen and endoxifen, which is equivalent to 1.86, 1.78, 0.194 and 0.187 ng/ml for tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and endoxifen, respectively. The method was also precise and accurate, with within-run and between-run precisions within 12.0% and accuracy ranging from 89.5 to 105.3%. The method has been applied to samples from a clinical study and cross-validated with a validated LC-MS/MS method in serum.     

An HPLC-MS/MS method for the quantitative determination of 4-hydroxy-anethole trithione in human plasma and its application to a pharmacokinetic study

A selective, rapid and sensitive method for the quantitation of 4-hydroxy-anethole trithione (ATX) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed and validated. Paracetamol was used as the internal standard (I.S.). After liquid–liquid extraction of 500 μL plasma with ethyl acetate, ATX and the I.S. were chromatographed on an Inertsil^® ODS-3 column. The mobile phase was consisted of methanol–water (75:25, v/v) with a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The calibration curve was linear over the range of 0.452–603 ng/mL (r² ≥ 0.99) with a lower limit of quantitation (LLOQ) of 0.452 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 13% and the accuracy (relative error, R.E.) was from −2.7% to −7.5% at three quality control levels. The assay herein described was successfully applied to a pharmacokinetic study of anethole trithione (ATT) tablet in healthy volunteers after oral administration.     

Simultaneous analysis of mono-, di-, and tri-ethanolamine in cosmetic products using liquid chromatography coupled tandem mass spectrometry

Alkanolamines such as monoethanolamine (MEA), diethanolamine (DEA), and triethanolamine (TEA) are used as wetting agents in shampoos, lotions, creams, and other cosmetics. DEA is widely used to provide lather in shampoos and maintain a favorable consistency in lotions and creams. Although DEA is not harmful, it may react with other ingredients in the cosmetic formula after extended storage periods to form an extremely potent carcinogen called nitrosodiethanolamine (NDEA), which is readily absorbed through the skin and has been linked to the development of stomach, esophagus, liver, and bladder cancers. The purpose of this study was to develop a simultaneous quantification method for measurement of MEA, DEA, and TEA in cosmetic products. Liquid chromatography coupled tandem mass spectrometry (LC–MS/MS) was performed using a hydrophilic interaction liquid chromatography (HILIC) column with isocratic elution containing acetonitrile and 5 mM ammonium formate in water (88:12, v/v). Identification and quantification of alkanolamines were performed using MS/MS monitoring to assess the transition from precursor to product ion of MEA (m/z, 61.1 → 44.0), DEA (m/z, 106.1 → 88.0), TEA (m/z, 150.1 → 130.0), and the internal standard triethylamine (m/z, 102.2 → 58.0). Alkanolamines extractions were simplified using a single extraction with acetonitrile in the cosmetic matrix. Performance of the method was evaluated with quality parameters such as specificity, carry-over, linearity and calibration, correlation of determination (R²), detection limit, precision, accuracy, and recovery. Calibration curves of MEA (2.9–1000 ppb), DEA (1–1000 ppb), and TEA (1–1000 ppb) were constructed by plotting concentration versus peak-area ratio (analyte/internal standard with a correlation coefficient greater than 0.99). The intra- and inter-assay accuracy ranged from 92.92 to 101.15 % for all analytes. The intra- and inter-assay precision for MEA, DEA, and TEA showed all coefficients of variance were less than 9.38 % for QC samples. Limits of detection and limits of quantification were 2.00 and 15.63 ppb for MEA, 0.49 and 1.96 ppb for DEA, and 0.49 and 1.96 ppb for TEA, respectively. This novel quantification method simplified sample preparation and allowed accurate and reproducible quantification of alkanolamines in the ng/g cosmetic weight (ppb) range for several cosmetic products.     

Simultaneous quantitation of levodopa and 3-O-methyldopa in human plasma by HPLC-ESI-MS/MS: application for a pharmacokinetic study with a levodopa/benserazide formulation

A sensitive and simple method was developed for the quantitation of levodopa and its metabolite 3-O-methyldopa, in human plasma, after oral administration of tablet formulations containing levodopa (200 mg) and benserazide (50 mg). The analytes were extracted by a protein precipitation procedure, using carbidopa as an internal standard. A mobile phase consisting of 0.2% formic acid and acetonitrile (94:6, v/v) was used and chromatographic separation was achieved using ACE C₁₈ column (50 mm × 4.6 mm i.d.; 5 μm particle size). Selected reaction monitoring was performed using the fragmentation transitions m/z 198 → m/z 107, m/z 212 → m/z 166 and m/z 227 → m/z 181 for levodopa, 3-O-methyldopa and carbidopa, respectively. Calibration curves were constructed over the range 50.0-6000.0 ng/mL for levodopa and 25.0-4000.0 ng/mL for 3-O-methyldopa. The method shown to be specific, precise, accurate and provided recovery rates higher than 85% for all analytes. No matrix effect was detected in the samples. The validated method was applied in a pharmacokinetic study with a levodopa/benserazide tablet formulation in healthy volunteers.     

Identification and determination of phase I metabolites of propafenone in rat liver perfusate

Propafenone (PF) is a class 1C antiarrhythmic agent. To study the mechanisms of PF interactions with dietary nutrients in isolated, perfused rat livers, metabolites of PF in liver perfusate were identified and an analytical method was developed for these metabolites plus parent drug. Identification of phase I metabolites was performed using HPLC/MS equipped with a Lichrospher RP-18 column and tandem mass spectrometry (MS/MS) with electrospray and atmospheric pressure chemical ionizations. Three major metabolite peaks, whose protonated molecular ions were m/z 358, 358 and 300, were identified as a propafenone derivative hydroxylated in the ω-phenyl ring (ω-OH-PF), 5-hydroxypropafenone (5-OH-PF), and N-despropylpropafenone (N-des-PF). The levels of ω-OH-PF, 5-OH-PF, N-des-PF and PF were determined simultaneously by HPLC with UV detection at 210 nm and a mobile phase of 0.03% triethylamine and 0.05% phosphoric acid in water-acetonitrile-methanol (45:20:35, v/v/v) after extraction with 5 ml diethyl ether at pH 10.0 and evaporation of solvent under nitrogen. The results revealed that ω-OH-PF, which was not found in humans, was the major metabolite of PF in rat liver perfusate, not 5-OH-PF which is the major metabolite in human plasma.     

Bioanalytical method for the quantification of sunitinib and its n-desethyl metabolite SU12662 in human plasma by ultra performance liquid chromatography/tandem triple-quadrupole mass spectrometry

A rapid and sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method has been developed and validated for the quantitative determination of sunitinib and its n-desethyl metabolite SU12662, in 100 μl aliquots of human potassium EDTA plasma with deuterated sunitinib as internal standard. As sunitinib was found to be extremely sensitive to light causing rapid conversion of the Z (cis)-isomer to the E (trans)-isomer, the sample extraction and cleaning-up were performed under sodium-light and in amber vials. The extraction involved a simple liquid–liquid extraction with tert-butyl methyl ether. Chromatographic separations were achieved on an Aquity UPLC^® BEH C₁₈ 1.7 μm, 2.1 mm × 50 mm column eluted at a flow rate of 0.250 ml/min on a gradient of acetonitrile. The overall cycle time of the method was 4 min, with elution times of 1.05, 1.43, 0.95, and 1.34 min, for the E (trans)- and Z (cis)-isomers of sunitinib and the E (trans)- and Z (cis)-isomers of SU12662, respectively. The multiple reaction monitoring transitions were set at 399 > 326 (m/z), at 371 > 283 (m/z) and at 409 > 326 (m/z) for sunitinib, SU12662 and the internal standard, respectively. The calibration curves were linear over the range of 0.200 to 50.0 ng/ml with the lower limit of quantitation validated at 0.200 ng/ml for both sunitinib and SU12662. The within-run and between-run precisions were within 11.7%, while the accuracy ranged from 90.5 to 106.8%.     

Determination of oxaceprol in rat plasma by LC–MS/MS and its application in a pharmacokinetic study

A sensitive method for the quantification of oxaceprol in rat plasma using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. Sample pretreatment involved a simple protein precipitation by the addition of 60 μL of acetonitrile–methanol (1:2, v/v) to 20 μL plasma sample volume. Separation was achieved on a Dikma ODS-C18 (5 μm, 150 mm × 4.6 mm) reversed-phase column at 40 °C with acetonitrile/0.1% formic acid–4 mM ammonium acetate in water (35:65,v/v) at a flow rate of 0.6 mL/min. Detection was performed using an electrospray ionization (ESI) operating in negative ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 172 → 130 (oxaceprol) and m/z 153 → 109 (protocatechuic acid, internal standard). The calibration curve of oxaceprol in plasma showed good linearity over the concentration range of 1.25–800 ng/mL. The limit of detection and limit of quantification were 0.400 ng/mL and 1.25 ng/mL, respectively. Intra- and inter-day precisions in all samples were within 15%. There was no matrix effect. The validated method was successfully applied to a preclinical pharmacokinetic study of oxaceprol in rats. After oral administration of 20 mg/kg oxaceprol to rats, the main pharmacokinetic parameters T_max, C_max, T_1/2, V_z/F and AUC_0–t were 1.4 h, 1.2 μg/mL, 2.3 h, 19.7 L/kg and 3.4 mg h/L, respectively.