LC�CMS�CMS Quantitative Determination of Brivudine in Human Plasma and Its Application to Pharmacokinetic Studies

A sensitive and specific liquid chromatography?Celectrospray ionization?Ctandem mass spectrometry method has been developed and validated for the identification and quantification of brivudine in human plasma using diclofenac as an internal standard. The method involves extraction with ethyl acetate. The analyte was separated on a C₁₈ column and analyzed in multiple reaction monitoring mode with a negative electrospray ionization interface using the [M?CH]^? ions, m/z 332.8??m/z 80.9 for brivudine, m/z 293.6??m/z 249.5 for diclofenac. The method was validated over the concentration range of 5.54?C2,836 ??g L^?1 for brivudine. The intra-and inter-day precisions were less than 8.91% in terms of relative standard deviation (RSD), and the accuracy was within ?4.22% in terms of relative error (RE). The lower limit of quantification (LLOQ) was 5.54 ??g L^?1 with acceptable precision and accuracy. There were almost no matrix effects. Recovery of brivudine spiked in drug-free plasma was higher than 77.17%. The method was used to study the pharmacokinetic profile of brivudine in human plasma after oral administration of brivudine tablets.     

Validated LC–MS–MS Method for Quantitative Determination of Batifiban in Human Plasma and Its Application to a Pharmacokinetic Study

Batifiban is a new platelet GPIIb/IIIa receptor antagonist. In this work, an analytical method based on liquid chromatography and electrospray ionization tandem mass spectrometry has been firstly developed and validated for the quantitative measurement of batifiban in human plasma to support the investigation of this compound. Separation of analyte and the internal standard eptifibatide was performed on a Thermo HyPURITY C18 column (150 × 2.1 mm, 5 μm) with a mobile phase consisting of formic acid 0.1% (v/v)–acetonitrile (40:60, v/v) at a flow rate of 0.25 mL min^?1. The Waters QuattroMicro API triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode via positive electrospray ionization interface using the transition m/z 819.2 → m/z (623.9 + 159.4) for batifiban and m/z 833.4 → m/z (645.7 + 159.3) for IS. The method was linear over the concentration range of 2.45–5,000 μg L^?1. The intra- and inter- day precisions were less than 15% in terms of relative standard deviation, and the accuracy was within 8.5% in terms of relative error (RE). The lower limit of quantification (LLOQ) was identifiable and reproducible at 2.45 μg L^?1 with acceptable precision and accuracy. The validated method offered sensitivity and wide linear concentration range. This method was successfully applied for the evaluation of pharmacokinetics of batifiban afer single oral doses of 55, 110 and 220 μg kg^?1 batifiban to 36 Chinese healthy volunteers.     

LC–MS–MS Determination of Troxerutin in Plasma and Its Application to a Pharmacokinetic Study

A highly sensitive liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the determination of troxerutin in human plasma using tramadol as internal standard (IS) has been developed and validated. Sample preparation involved liquid–liquid extraction with ethyl acetate–isopropanol (95:5, v/v). The analyte and IS were separated by RP–LC with gradient elution using 10 mM ammonium acetate containing 0.1% formic acid and methanol at a flow rate of 0.9 mL min^?1. LC–MS–MS in the positive ion mode employed multiple reaction monitoring of the transitions at m/z 743.2→435.3 and m/z 264.1→58.0 for troxerutin and IS, respectively. The assay was linear in the concentration range 0.01–10 ng mL^?1 with precision and accuracy within assay variability limits as per FDA guidelines. The assay was successfully applied to a pharmacokinetic study involving oral administration of 300 mg troxerutin to eight healthy Chinese volunteers.     

Determination of Asiaticoside in Rat Plasma by LC–MS–MS and Its Application to Pharmacokinetics Studies

A rapid and sensitive LC–MS–MS method was developed and validated for the determination of asiaticoside in rat plasma. Asiaticoside was extracted by protein precipitation with acetonitrile, and separated on a C₁₈ column. The total analytical time was relatively short (4 min), and the limit of quantification was 38 ng mL^?1 using 100 μL of rat plasma. Asiaticoside and the internal standard (felodipine) were monitored in the multi-reaction-monitoring mode as follows: m/z 957.4 → 469.3 and m/z 382.2 → 145.1, respectively. Calibration was linear over a concentration range from 38 to 7,600 ng mL^?1, and the correlation coefficient was greater than 0.998. The recoveries of asiaticoside from plasma were better than 85%, and RSDs of inter-day and intra-day assays were below 10.1%. The method is sensitive and specific, and suitable for pharmacokinetic studies of asiaticoside in rats.     

Quantitative Determination of Lobeline Hydrochloride in Rabbit Plasma by LC–MS–MS and Its Application

A sensitive and selective liquid chromatography tandem mass spectrometry method for quantitative determination of lobeline hydrochloride in rabbit plasma was developed and validated. After addition of triazolam as internal standard, protein precipitation by acetonitrile was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 column with acetonitrile-0.1% formic acid as mobile phase with gradient elution. Electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 338.1 → 315.8 for lobeline hydrochloride and m/z 342.9 → 308.0 for the IS. Calibration plots were linear over the range of 2–500 ng mL^?1 for lobeline hydrochloride in plasma. Lower limit of quantitation for lobeline hydrochloride was 2 ng mL^?1. Mean recovery of lobeline hydrochloride from plasma was in the range 97.5–102.3%. RSD of intra-day and inter-day precision were both <9%. This developed method is successfully used in pharmacokinetic study of lobeline hydrochloride in rabbit.     

Determination of Memantine in Human Plasma by LC–MS–MS: Application to a Pharmacokinetic Study

A sensitive and selective liquid chromatography–tandem mass spectrometry method for the determination of memantine was developed and validated over the linearity range 0.1–25 ng mL^?1 with 0.5 mL of plasma using procainamide as the internal standard. This analysis was carried out on a Cosmosil 5C₁₈-MS column and the mobile phase was composed of methanol: 0.5% formic acid (50:50, v/v). Detection was performed on a triple–quadrupole tandem mass spectrometer using positive ion mode electrospray ionization and quantification was performed by multiple reaction monitoring mode. The MS–MS ion transitions monitored were m/z 180 → 107 and 236 → 163 for memantine and procainamide, respectively. The between- and within-day precision was less than 10.9% and accuracy was less than 2.5%. The lower limit of quantification (LLOQ) was 0.1 ng mL^?1. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of memantine in healthy Chinese volunteers.     

LC-MS–MS Simultaneous Determination of Niacin, Niacinamide and Nicotinuric Acid in Human Plasma LC-MS–MS and Its Application to a Human Pharmacokinetic Study

A highly selective and sensitive liquid chromatographic tandem mass spectrometric (LC-MS–MS) method was developed and validated for the quantitation and pharmacokinetic study of niacin (NA) and its two metabolites niacinamide (NAM) and nicotinuric acid (NUR) in human plasma. Protein precipitation with 14% perchloric acid solution was selected for sample preparation, and ganciclovir was used as an internal standard. Separation was on a Phenomenex Curosil-PFP (250 mm × 4.6 mm, 5 μm) column by a multiple steep steps linear gradient elution with mobile phase consisting of water and methanol, both containing 0.1% formic acid, pumped at a flow rate of 1 mL min^?1. The determination was optimized and carried out with positive electrospray ionization by selective multiple reaction monitoring. The method was linear in the concentration range of 15–2,000 ng mL^?1 for NA, 70–2,000 ng mL^?1 for NAM and 10–2,000 ng mL^?1 for NUR, by standard addition calibration. The application of LC-MS–MS was demonstrated for the specific and quantitative analysis of NA, NAM and NUR in human plasma from a pharmacokinetic study in 12 healthy Chinese volunteers treated with three incremental doses of niacin extended-release/lovastatin tablets and an additional steady-state regime.     

Determination of Dothiepin in Human Plasma by LC–ESI–MS and its Application to Bioequivalence Studies

An LC–MS method for the determination of dothiepin in human plasma was developed and validated. Sample preparation involved extraction with n-hexane:2-propanol (95:5). Separation was on an Ultimate XB C18 column (2.1 × 150 mm, 5 μm). A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H]⁺ ions at m/z 296 for dothiepin and at m/z 278 for the internal standard (amitriptylene). The method demonstrated good linearity from 0.78 ng mL^?1 (the LOQ) to100 ng mL^?1. The mean extraction recovery was 82.4% for dothiepin and and 84.2% for the internal standard. The intra-day and inter-day precision ranged from 8.5 to 11.4% and 9.7 to 12.1% (RSD), respectively. The method was successfully applied to bioequivalence studies of dothiepin hydrochloride tablets to obtain the pharmacokinetic parameters.     

Determination of Poricoic Acid A in Rat Plasma after Oral and Intravenous Administration by LC–MS–MS and Its Application to a Pharmacokinetic Study

A sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed and validated for the quantification of poricoic acid A (PAA) in rat plasma. The plasma samples were precipitated by protein precipitation with methanol. Glycyrrhetic acid was used as the IS. Chromatography was performed on a Dionex C₁₈ 120 Å (4.6 × 250 mm, 5 μm) column with the mobile phase composed of acetonitrile–water (90:10, v/v) at a flow rate of 0.8 mL min^?1. A tandem mass spectrometer equipped with an ESI source was used as the detector and was operated in the negative ion mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions m/z 497.4 → 423.3 and m/z 469.2 → 425.1 for PAA and IS, respectively. The calibration curves were linear over the range of 5–5,000 ng mL^?1 (r ² = 0.9966) and the lower limit of quantification (LLOQ) was 5 ng mL^?1. In this range, RSDs were <10% for intra-assay and inter-assay precisions. The accuracy expressed by deviation (DEV) was <6%, and the extraction recoveries of QC samples were >78%. The validated method was successfully used to study the pharmacokinetics of PAA in rats after intravenous administration at a dose of 1.0, 2.5 and 5.0 mg kg^?1 and oral administration at a dose of 25, 50 and 100 mg kg^?1, respectively. The relative bioavailability of PAA in rats following oral administration was achieved.     

LC–MS–MS Simultaneous Determination of Paracetamol, Pseudoephedrine and Chlorpheniramine in Human Plasma: Application to a Pharmacokinetic Study

A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed for the simultaneous determination of paracetamol, pseudoephedrine and chlorpheniramine in human plasma. Diphenhydramine was used as the internal standard. Analytes were extracted from alkalized human plasma by liquid–liquid extraction (LLE) using ethyl acetate. After electrospray ionization positive ion fragments were detected in the selected reaction monitoring (SRM) mode with a triple quadrupole tandem mass spectrometer. The method was linear in the concentration range of 20.0–10000.0 ng mL^?1 for paracetamol, 1.0–500.0 ng mL^?1 for pseudoephedrine and 0.1–50.0 ng mL^?1 for chlorpheniramine. The intra- and inter-day precisions were below 14.5% and the bias was between ?7.3 and +2.8% for all analytes. The validated LC–MS–MS method was applied to a pharmacokinetic study in which each healthy Chinese volunteer received a tablet containing 300 mg benorylate, 30 mg pseudoephedrine hydrochloride and 2 mg chlorpheniramine maleate. This is the first assay method described for the simultaneous determination of paracetamol, pseudoephedrine and chlorpheniramine in human plasma samples.     

Determination of Fenofibric Acid in Human Plasma by LC–MS/MS and LC–UV

A sensitive, high-throughput and economic liquid chromatographic method for determination of fenofibric acid in human plasma was developed and validated by ultraviolet detection and tandem mass spectrometry, then applied in pharmacokinetic study to investigate Lipanthyl™ 200 mg MC bioavailability under food and fasting conditions. Fenofibric acid with 2-chloro fenofibric acid-d6 (internal standard) was extracted from 100 µL of human plasma by acetonitrile in a single extraction step. 25 and 2 µL from supernatant were injected onto ACE column, 50 mm, 5 micron with 4.6 mm inner diameter for LC–UV and 2.1 mm for LC–MS/MS, and both systems were eluted isocratically by water:methanol:formic acid (35:65:0.1, v/v/v), with a constant flow rate of 1 mL min⁻¹. The established calibration curve was linear between 0.05–20 µg mL⁻¹, and the within- and between-day precisions were all below 13 % in both LC–MS/MS and LC–UV systems during validation, and accuracies ranged between 91 and 112 %. Twenty-eight healthy adult subjects participated in this clinical study, and the pharmacokinetic parameters including coefficient of variation were calculated and discussed. A dramatic decrease in C _max and AUC0-72 (3.63- and 1.85-fold, respectively) were observed for Lipanthyl™ MC under fasting conditions with more variable inter subject measurements comparing to the fed state.

     

LC–MS–MS Determination of Nikethamide in Human Plasma

A sensitive LC–MS–MS method with electrospray ionization has been developed for determination of nikethamide in human plasma. After addition of atropine as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 mm × 2.1 mm, 5 μm particle, Agilent Zorbax SB-C₁₈ column, with 45:55 (v/v) methanol–water containing 0.1% formic acid as mobile phase. LC–MS–MS was performed in multiple reaction monitoring mode using target fragment ions m/z 178.8 → 107.8 for nikethamide and m/z 289.9 → 123.8 for the internal standard. Calibration plots were linear over the range of 20.0–2,000 ng mL^?1. The lower limit of quantification was 20.0 ng mL^?1. Intra-day and inter-day precisions were better than 4.2 and 6.1%, respectively. Mean recovery of nikethamide from human plasma was in the range 65.3–71.1%.     

Quantitative and Qualitative Analysis of CKD-501, Lobeglitazone, in Human Plasma and Urine Using LC–MS/MS and Its Application to a Pharmacokinetic Study

A simple, rapid and sensitive LC–MS/MS method in positive ion mode was developed and validated to determine CKD-501, lobeglitazone, in human plasma and urine using glipizide as an internal standard (IS). Lobeglitazone is a novel thiazolidinedione (TZDs)-based peroxisome proliferator-activated receptor (PPAR) agonist, used for the management of type-2 diabetes. After mixing the IS, dissolved in acetonitrile, with a plasma or urine sample containing lobeglitazone, 10?μL of supernatant was injected into the LC–MS/MS system. Quantification was performed in the multiple reaction monitoring (MRM) mode using transition of 481.5?→?152.2 (m/z) for lobeglitazone and 446.1?→?321.2 (m/z) for the IS. The method showed good linearity over concentration ranges of 0.5–1,000?ng?mL^?1 for plasma and 0.2–250?ng?mL^?1 for urine (r ²?≥?0.9996). The mean percent extraction recovery of lobeglitazone was 90.8?% for plasma and 87.3?% for urine, while the recoveries of the IS were greater than 86.4?% for both. The intra-day and inter-day precision of plasma ranged from 1.1 to 3.7 and 2.5 to 3.3?% (RSD), respectively, and the intra- and inter-day precision of urine ranged from 1.5 to 2.7 and 3.2 to 3.5?%, respectively. This method is simple, sensitive, and applicable for the pharmacokinetic study of lobeglitazone in human plasma. Most of the urine concentrations of lobeglitazone were below the LLOQ because the lobeglitazone is extensively metabolized.     

Quantitative and Qualitative Analysis of CKD-501, Lobeglitazone,in Human Plasma and Urine Using LC–MS/MS and Its Application to a Pharmacokinetic Study

A simple, rapid and sensitive LC–MS/MS method in positive ion mode was developed and validated to determine CKD-501, lobeglitazone, in human plasma and urine using glipizide as an internal standard (IS). Lobeglitazone is a novel thiazolidinedione (TZDs)-based peroxisome proliferator-activated receptor (PPAR) agonist, used for the management of type-2 diabetes. After mixing the IS, dissolved in acetonitrile, with a plasma or urine sample containing lobeglitazone, 10 μL of supernatant was injected into the LC–MS/MS system. Quantification was performed in the multiple reaction monitoring (MRM) mode using transition of 481.5 → 152.2 (m/z) for lobeglitazone and 446.1 → 321.2 (m/z) for the IS. The method showed good linearity over concentration ranges of 0.5–1,000 ng mL⁻¹ for plasma and 0.2–250 ng mL⁻¹ for urine (r ² ≥ 0.9996). The mean percent extraction recovery of lobeglitazone was 90.8 % for plasma and 87.3 % for urine, while the recoveries of the IS were greater than 86.4 % for both. The intra-day and inter-day precision of plasma ranged from 1.1 to 3.7 and 2.5 to 3.3 % (RSD), respectively, and the intra- and inter-day precision of urine ranged from 1.5 to 2.7 and 3.2 to 3.5 %, respectively. This method is simple, sensitive, and applicable for the pharmacokinetic study of lobeglitazone in human plasma. Most of the urine concentrations of lobeglitazone were below the LLOQ because the lobeglitazone is extensively metabolized.

     

Fast LC–MS Electrospray Ionization for the Quantification of Digoxin in Human Plasma and Its Application to Bioequivalence Studies

A fast, sensitive and specific liquid chromatography-mass spectrometry method has been developed for quantification of digoxin in human plasma. The method was optimized to bioequivalence studies aiming higher sensitivity and selectivity than previously published methods, in addition to shorter run time allowing high-throughput sample analyses from volunteers. Chromatographic separation was achieved by an RP-18e column hyphenated to an API 5000 mass spectrometer system set at negative electrospray ionization and operating in the MRM mode. Calibration curve was linear over a wide range of concentration (50.0–6000.0 pg mL⁻¹), with the lower limit of quantification at 50.0 pg mL⁻¹ and without interfering peaks at the retention time of digoxin (2.09 min). Dexamethasone was used as internal standard and samples were cleaned up by liquid-liquid extraction obtaining a mean recovery of 73.8%. Validation results confirmed inter-batch accuracy (−8.66 to 5.78%), precision (4.1–10.6%) and stability, in accordance with the U.S. Food and Drug Administration and the Brazilian National Health Surveillance Agency guidelines. The developed analytical method could be successfully applied to a single oral dose (0.25 mg), one-way, randomized, two-sequence, crossover bioequivalence study validating, up to date, the fastest analysis and the most sensitive and specific method already published for digoxin quantification.     

Fast LC–MS Electrospray Ionization for the Quantification of Digoxin in Human Plasma and Its Application to Bioequivalence Studies

A fast, sensitive and specific liquid chromatography-mass spectrometry method has been developed for quantification of digoxin in human plasma. The method was optimized to bioequivalence studies aiming higher sensitivity and selectivity than previously published methods, in addition to shorter run time allowing high-throughput sample analyses from volunteers. Chromatographic separation was achieved by an RP-18e column hyphenated to an API 5000 mass spectrometer system set at negative electrospray ionization and operating in the MRM mode. Calibration curve was linear over a wide range of concentration (50.0–6000.0 pg mL⁻¹), with the lower limit of quantification at 50.0 pg mL⁻¹ and without interfering peaks at the retention time of digoxin (2.09 min). Dexamethasone was used as internal standard and samples were cleaned up by liquid-liquid extraction obtaining a mean recovery of 73.8%. Validation results confirmed inter-batch accuracy (−8.66 to 5.78%), precision (4.1–10.6%) and stability, in accordance with the U.S. Food and Drug Administration and the Brazilian National Health Surveillance Agency guidelines. The developed analytical method could be successfully applied to a single oral dose (0.25 mg), one-way, randomized, two-sequence, crossover bioequivalence study validating, up to date, the fastest analysis and the most sensitive and specific method already published for digoxin quantification.