LC–MS–MS Quantitative Determination of Ursolic Acid in Human Plasma and Its Application to Pharmacokinetic Studies

A sensitive and specific liquid chromatography–electrospray ionization-tandem mass spectrometry method has been developed and validated for the identification and quantification of ursolic acid in human plasma using glycyrrhetic acid as an internal standard. The method involves extraction with methyl tert-butyl ether. The analyte was separated on a C₁₈ column and analyzed in multiple reaction monitoring mode with a negative electrospray ionization interface using the [M–H]? ions, m/z 455.4 for ursolic acid and m/z 469.5 → m/z 425.5 for glycyrrhetic acid. The method was validated over the concentration range of 0.86–110.0 μg L^?1. The intra- and inter-day precisions were less than 13.53% relative standard deviation (RSD) and the accuracy was within ?4.76% in terms of relative error (RE). The lower limit of quantification was 0.86 μg L^?1 with acceptable precision and accuracy. There were almost no matrix effects. Recovery of ursolic acid from spiked drug-free plasma was higher than 68%. The method was used to study the pharmacokinetic profile of ursolic acid in human plasma after oral administration of Jieyu capsules.     

LC–MS–MS Method for Simultaneous Analysis of Abacavir and Lamivudine in Human Plasma, and Its Application to a Bioequivalence Study

A rapid and sensitive LC–MS–MS method has been developed and validated for simultaneous analysis of abacavir (ABA) and lamivudine (LAM) in human plasma. The analytes were extracted from human plasma by SPE. Nelfinavir (NEL) and emtricitabine (EMT) were used as the internal standards for ABA and LAM, respectively. An RP18 column enabled chromatographic separation of the analytes. The method involves simple isocratic chromatography and MS detection in positive-ionization mode. Validation of the method showed response was a linear function of concentration in the ranges 100.0–7000.0 ng mL^?1 for ABA and 80.0–5000.0 ng mL^?1 for LAM. At the LOQ levels, inter-run and intra-run precision were within 5.80 and 3.51%, respectively, for ABA and within 4.68 and 3.16%, respectively, for LAM. Overall recovery for ABA and LAM was 59.32 and 105.18%, respectively. Total elution time was 2 min only, which enabled quantification of more than 200 plasma samples per day. This validated method was used successfully for analysis of plasma samples from a bioequivalence study.     

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.     

Simultaneous Determination of Levodopa, Benserazide and 3-O-Methyldopa in Human Serum by LC–MS–MS

For the first time a sensitive, specific and rapid LC–MS–MS assay is presented for the simultaneous determination of levodopa (L-DP), 3-O-methyldopa (3-OMD) and benserazide (BSZ) in human serum. The three compounds were extracted from human serum by protein precipitation followed by dilution of the supernatant with aqueous formic acid. In serum, linearity was observed between 50 and 1,000 ng mL^?1 of L-DP, 3-OMD and BSZ, respectively. Intra-day and inter-day RSD values were below 10.56 and 6.22% at concentrations of 120, 360 and 720 ng mL^?1. The presented method showed excellent specificity and sensitivity compared with other methods reported. It was applied to a pharmacokinetic study and demonstrated its applicability to pre-clinical and clinical pharmacological research.     

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 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.     

Simultaneous Determination of Tamsulosin and Dutasteride in Human Plasma by LC–MS–MS

A sensitive and selective liquid chromatographic tandem mass spectrometric (LC–MS–MS) method was developed for simultaneous identification and quantification of tamsulosin and dutasteride in human plasma, which was well applied to clinical study. The method was based on liquid–liquid extraction, followed by an LC procedure with a Gemini C-18, 50 mm × 2.0 mm (3 μm) column and using methanol:ammonium formate (97:3, v/v) as the mobile phase. Protonated ions formed by a turbo ionspray in positive mode were used to detect analytes and internal standard. MS–MS detection was by monitoring the fragmentation of 409.1 → 228.1 (m/z) for tamsulosin, 529.3 → 461.3 (m/z) for dutasteride and 373.2 → 305.3 (m/z) for finasteride (IS) on a triple quadrupole mass spectrometer. The lower limit of quantification for both tamsulosin and dutasteride was 1 ng mL^?1. The proposed method enables the unambiguous identification and quantification of tamsulosin and dutasteride for clinical drug monitoring.     

Simultaneous Determination of Atenolol and Chlorthalidone by LC–MS–MS in Human Plasma

A simple, sensitive, selective and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous separation and quantitation of atenolol and chlorthalidone in human plasma using metoprolol and hydrochlorothiazide as internal standard. Following solid phase extraction, the analytes were separated by an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in the multiple reaction-monitoring mode (atenolol in positive and chlorthalidone in the negative ion mode). The limit of quantitation for this method was 10 and 15 ng mL^?1 and the linear dynamic range was generally 10–2,050 ng mL^?1 and 15–3,035 ng mL^?1 for atenolol and chlorthalidone, respectively.     

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.

     

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.     

Analysis of Linarin and Its Metabolites in Rat Urine by LC–MS/MS

Liquid chromatography–ion trap mass spectrometry was employed to investigate the metabolism of linarin in rats. Identification and structural elucidation of the metabolites were performed by comparing the differences in molecular masses, retention times, and full scan MS ⁿ spectra between linarin and its metabolites. Six metabolites (acacetin, apigenin, acacetin glucuronide, apigenin glucuronide, acacetin sulfate, apigenin sulfate) were detected in rat urine after oral administration of linarin at the dose of 50 mg kg^?1. Furthermore, a selective and sensitive liquid chromatography–triple quadruple mass spectrometry assay was developed and validated for the simultaneous determination of linarin and acacetin (the major metabolite of linarin) in rat urine. Chromatographic separation was carried out on a C₁₈ column, and mass spectrometric detection was performed using a triple-quadrupole mass spectrometer coupled with an electrospray ionization source in the positive ion mode. Quantitation of linarin and acacetin was performed using selected reaction monitoring of precursor–product ion transitions at m/z 593 → 285 for linarin, 285 → 242 for acacetin, and 303 → 153 for hesperitin (internal standard), respectively. The assay exhibited good linearity (r > 0.9900) for both linarin and acacetin. The intra- and inter-day precisions were <13.4 % and the accuracy was between ?8.1 and 3.1 %. The method was successfully applied to the urinary excretion study of linarin in rats after oral administration of linarin.     

LC–MS–MS Assay for Simultaneous Quantification of Fexofenadine and Pseudoephedrine in Human Plasma

A rapid, sensitive, and simple HPLC–MS–MS method, with electro-spray ionization and cetirizine as internal standard (IS), has been developed and validated for simultaneous quantification of fexofenadine and pseudoephedrine in human plasma. The analytes were isolated from plasma by solid-phase extraction (SPE) on Oasis HLB cartridges. The compounds were chromatographed on an RP 18 column with a mixture of ammonium acetate (10 mm, pH 6.4) and methanol as mobile phase. Quantification of the analytes was based on multiple reaction monitoring (MRM) of precursor-to-product ion pairs m/z 502 → 466 for fexofenadine, m/z 166 → 148 for pseudoephedrine, and m/z 389 → 201 for cetirizine. The linear calibration range for both analytes was 2–1,700 ng mL⁻¹ (r = 0.995), based on analysis of 0.1 mL plasma. Extraction recovery was 91.5 and 80.88% for fexofenadine and pseudoephedrine, respectively. The method was suitable for analysis of human plasma samples obtained 72 h after administration of a drug containing both fexofenadine and pseudoephedrine.     

LC–MS–MS Determination of Rotenone, Deguelin, and Rotenolone in Human Serum

For the first time we report a rapid and sensitive LC–MS–MS method for quantification of rotenone, deguelin, and rotenolone in human serum. The analytical procedure involves extraction with ethyl acetate without further clean-up. The active ingredients were separated on a C₈ reversed-phase column by isocratic elution. Eleven simultaneous transitions of precursor ions were monitored. Excellent selectivity and sensitivity enables quantification and identification of low levels of rotenoids (LOD 2 ng mL^?1, LOQ 5 ng mL^?1) in human serum.     

LC–MS Simultaneous Determination of Itopride Hydrochloride and Domperidone in Human Plasma

A rapid, simple, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for simultaneous quantification of itopride hydrochloride and domperidone in human plasma. Both drugs were extracted by liquid–liquid extraction with ethyl acetate and saturated borax solution. The chromatographic separation was performed on a reversed-phase C18 column with a mobile phase of water–methanol (2:98, v/v) containing 0.5% formic acid. The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The assay exhibited linearity over the concentration range of 3.33–500 ng mL^?1 for itopride hydrochloride and 3.33–100 ng mL^?1 for domperidone in human plasma. The precursor to product ion transitions of m/z 359.1–72.3 and 426.0–147.2 were used to measure itopride hydrochloride and domperidone respectively. The method was found suitable for the analysis of plasma samples collected during phase 1 pharmacokinetics study of itopride HCl 50 mg and domperidone 20 mg in 12 healthy volunteers after single oral doses of the combination drug.     

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.     

Alternative LC–MS–MS Method for Simultaneous Determination of Proguanil, Its Active Metabolite in Human Plasma and Application to a Bioequivalence Study

An alternative rapid and sensitive liquid chromatography–tandem mass spectrometry method has been developed and validated for simultaneous analysis of proguanil (PRO) and cycloguanil (CYC) in human plasma. The analytes were extracted from human plasma by solid phase extraction. Riluzole (RIL) was used as an internal standard for proguanil and cycloguanil. A HyPURITY Advance C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves simple isocratic chromatography conditions and mass spectrometric detection in the positive ionization mode using an API-4000 system. The proposed method has been validated with linear range of 1.5–150.0 ng mL^?1 for PRO and 0.5–50.0 ng mL^?1 for CYC. The inter-run and intra-run precision values are within 2.54, 9.19% for PRO and 1.99, 10.69% for CYC at LOQ levels. The overall recoveries for PRO and CYC were 102.52 and 106.72%, respectively. Total elution time was as low as 2.50 min. This validated method was used successfully for analysis of plasma samples from a bioequivalence study.     

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.

     

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%.