Simultaneous Quantitative Analysis of Shikonin and Deoxyshikonin in Rat Plasma by Rapid LC–ESI–MS–MS

The purpose of this article was to develop a rapid and robust LC–MS–MS method for quantifying shikonin and deoxyshikonin simultaneously in rat plasma using emodin as internal standard. The LC system consisted of an Agilent ZORBAX SB-C18 (1.8 μm, 250 × 4.6 mm, 20 °C) column. Elution with an isocratic mobile phase consisted of methanol/10 mM ammonium acetate in water/acetonitrile containing 0.05% formic acid (45:10:45, v/v/v) at a flow rate of 0.8 mL min^?1 yielded sharp, high-resolved peaks within 12 min. The lower limits of quantitation were 0.5 ng mL^?1 for shikonin, and 8 ng mL^?1 for deoxyshikonin. Correlation coefficient (r) values for the linear range of two analytes were greater than 0.99. Assay precision was <13% and accuracy was 87–99%. This newly developed method was used to the pharmacokinetic studies of the shikonin analogues in rats after intravenous administration (n = 4).     

LC–ESI–MS Determination of Lovastatin in Human Plasma

A convenient, selective and sensitive liquid chromatographic-electrospary ionization mass spectrometry (LC–ESI–MS) method was developed and validated to determine lovastatin in human plasma. The analyte was extracted from human plasma samples by typical liquid–liquid extraction, separated on a C₁₈ column by using the mobile phase consisting of water–methanol (13:87, v/v). Simvastatin was used as the internal standard (IS). The method was linear within the range of 0.1–20 ng mL⁻¹. The lower limit of quantification (LLOQ) was 0.1 ng mL⁻¹. The intra- and inter-run precision, calculated from quality control (QC) samples was less than 10.2%. The accuracy as determined from QC samples was in the range of 99.3–102.9% for the analyte. The mean recoveries for lovastatin and IS were 84.8 and 88.0%, respectively. The method was successfully applied for evaluation of the pharmacokinetic of lovastatin in healthy volunteers.     

LC–ESI–MS Determination of Flupentixol in Human Plasma

Flupentixol and an internal standard, loperamide were extracted from human plasma by liquid–liquid extraction and analyzed on a Thermo Hypersil HyPURITY C18 column, with 10 mM ammonium acetate–acetonitrile–methanol (26:62:12, v/v/v) as mobile phase, coupled with electrospray ionization mass spectrometry (ESI–MS). The protonated analyte was quantified by selected-ion monitoring (SIM) with a quadrupole mass spectrometer in a positive-ion mode. The calibration curve was linear (r = 0.9990) over the concentration range: 0.039–2.5 ng mL^?1. Intra-day and inter-day precision (RSD%) were less than 13.05%. The established method was successfully applied for the determination of pharmacokinetics of flupentixol in human plasma.     

Determination of Pitavastatin in Human Plasma by LC–MS–MS

A simple and rapid LC–MS–MS assay was developed and validated for the quantitative determination of pitavastatin in human plasma. Sample pretreatment involved simple protein precipitation by addition of acetonitrile. Separation was on an Agilent 1.8 μm Zorbax SB-C18 column (150 mm × 4.6 mm) at 25 °C using isocratic elution with methanol–0.1% formic acid in water (85:15, v/v) at a flow rate of 0.4 mL min^?1. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the ion transitions m/z 422.0 → 290.1 for pitavastatin, and m/z 330.1 → 192.1 for paroxetine (IS). LC–MS–MS was found to improve the quantitation of pitavastatin in plasma and was successfully applied in pharmacokinetic studies.     

Simultaneous Determination of Paracetamol and Caffeine in Human Plasma by LC–ESI–MS

A rapid, selective and convenient liquid chromatography–mass spectrometric method for the simultaneous determination of paracetamol and caffeine in human plasma was developed and validated. Analytes and theophylline [internal standard (I.S.)] were extracted from plasma samples with diethyl ether-dichloromethane (3:2, v/v) and separated on a C₁₈ column (150 × 4.6 mm ID, 5 μm particle size, 100 Å pore size). The mobile phase consisted of 0.2% formic acid–methanol (60:40, v/v). The assay was linear in the concentration range between 0.05 and 25 μg mL^?1 for paracetamol and 10–5,000 ng mL^?1 for caffeine, with the lower limit of quantification of 0.05 μg mL^?1 and 10 ng mL^?1, respectively. The intra- and inter-day precision for both drugs was less than 8.1%, and the accuracy was within ±6.5%. The single chromatographic analysis of plasma samples was achieved within 4.5 min. This validated method was successfully applied to study the pharmacokinetics of paracetamol and caffeine in human plasma.     

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.     

Sensitive and Selective LC–ESI–MS Analysis of Aesculin in Rat Plasma

A rapid and sensitive liquid chromatography–electrospray ionization mass spectrometry method was developed for the determination of aesculin in rat plasma. The analyses were chromatographed on a Zorbax Extend-C18 analytical column (150 × 2.1 mm I.D., 5 µm) with 30:70 (v/v) methanol–0.1% formic acid as mobile phase. Detection was performed by triple-quadrupole tandem mass spectrometry in multi-reaction-monitoring mode with an electrospray ionization source. The method was validated for accuracy and precision, and linearity in the two matrices was good. The assay was linear in the range 12.5–1,800 ng mL^?1. The lower limit of quantification of aesculin (LLOQ) was 12.5 ng mL^?1. The recovery of aesculin and tinidazole (IS) were well above 85%. The within- and between-batch accuracy was 100–104% and 97–109%, respectively. There were no stability-related problems in the procedure for the analysis of aesculin. The method was successfully used in a preclinical study of the pharmacokinetics of aesculin in rats.     

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.     

Determination and Pharmacokinetic Study of 10-O-Dimethylaminoethylginkgolide B in Rat Plasma by LC–MS

To evaluate the pharmacokinetics of a novel analogue of ginkgolide B, 10-O-dimethylaminoethylginkgolide B (XQ-1) in rat plasma in pre-clinical studies, a sensitive and specific liquid chromatographic method with electrospray ionization mass spectrometry detection (LC–ESI–MS) was developed and validated. After a simple extraction with ethyl acetate, XQ-1 was analyzed on a Shim-pack C18 column with a mobile phase of a mixture of 1 μmol L⁻¹ ammonium acetate containing 0.02% formic acid and methanol (55:45, v/v) at a flowrate of 0.3 mL min⁻¹. Detection was performed in selected ion monitoring (SIM) mode using target ions at [M + H]⁺ m/z 496.05 for XQ-1 and m/z 432.10 for the internal standard (lafutidine). Linearity was established for the concentration range from 2 to 1,000 ng mL⁻¹ . The extraction recoveries ranged from 86.0 to 89.9% in plasma at concentrations of 5, 50, and 500 ng mL⁻¹. The lower limit of quantification was 2 ng mL⁻¹ with 100 μL plasma. The validated method was successfully applied to a pharmacokinetic study after intragastic administration of XQ-1 mesylate in rats at a dose of 20 mg kg⁻¹.     

LC–ESI-MS Determination of Hydroxycamptothecin in Rat Plasma

A rapid and sensitive method using liquid chromatography with electrospray ionization mass spectrometric detection (LC–ESI-MS) was developed and validated for the determination of hydroxycamptothecin in rat plasma. Plasma samples were extracted with ether and separated on a C₁₈ column interfaced with a single quadrupole mass spectrometer, with mobile phase consisting of 0.1% formic acid–methanol (45:55, v/v). Detection was carried out by positive electrospray ionization (ESI) in selected ion recording (SIR) mode at m/z 321 and 305 for hydroxycamptothecin and camptothecin (internal standard), respectively. The linearity was obtained ranged from 2.5 to 1,000 ng mL^?1 and the lower limit of quantification (LLOQ) was determined to be at 2.5 ng mL^?1. The intra- and inter-day precision (%RSD) was less than 5.5% and accuracy (% RE) ranged from 3.8 to 5.3%. This method was applied successfully to a preliminary pharmacokinetic study following the intravenous administration of hydroxycamptothecin to rats.     

Determination of Gastrodin and Ligustrazine Hydrochloride in Plasma and Brain Dialysate by LC–Tandem MS

A gradient liquid chromatography-tandem mass spectrometry method has been developed and validated for the determination of gastrodin and ligustrazine hydrochloride in rat plasma and brain dialysates. Zolpidem was used as internal standard. For plasma samples, solid-phase extraction was used and the brain dialysates were collected from freely moving rats using brain microdialysis. Both were followed by HPLC separation and positive electrospray ionization tandem mass spectrometry detection (ESI–MS–MS). Chromatographic separation was achieved on a Symmetry RP-18 column using gradient elution with methanol and water containing 0.5% formic acid and 2 mM ammonium formate. Selected reaction monitoring (SRM) mode was used for quantitation. Good linearities were obtained in the range of 0.05–100 and 0.01–50 μg mL^?1 for gastrodin and ligustrazine hydrochloride in rat plasma, and 0.05–1,000 ng mL^?1 for both in dialysate. The lower limit of quantitation was 0.01 ng mL^?1 for gastrodin and 0.05 ng mL^?1 for ligustrazine. The method is precise and reliable and can be applied to pharmacokinetic studies.     

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%.     

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.     

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.     

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.     

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.

     

Measurement of Acetylcholine in Rat Brain Microdialysates by LC–Isotope Dilution Tandem MS

An LC–MS–MS method was developed for measuring acetylcholine (ACh) in an aqueous medium using reversed-phase ion-pair chromatography, electrospray ionization on a quadrupole ion trap instrument and a tetradeuterated analogue (ACh-1,1,2,2-d₄) as an internal standard. A rapid separation was achieved on a 5-cm long octadecylsilica column (2.1 mm i.d.) by employing heptafluorobutyric acid (0.1% v/v) as an ion-pairing agent and requiring 10% v/v acetonitrile in 20 mM ammonium formate buffer under isocratic elution at 200 μl min⁻¹ flow rate. The instrument’s response was calibrated with samples containing known mole ratios of ACh and ACh-1,1,2,2-d₄ in an artificial cerebrospinal fluid, which afforded the conclusion that analyte concentrations could be determined by multiplying the measured analyte to internal standard ion-current ratio with the known molar concentration of the ACh-1,1,2,2-d₄ added. The rapid and simple assay was tested by measuring the basal neurotransmitter concentration in rat brain microdialysates without the use of a cholinesterase inhibitor upon sample collection.

     

Sensitive Determination of Felodipine in Human and Dog Plasma by Use of Liquid–Liquid Extraction and LC–ESI–MS–MS

Summary A sensitive and selective liquid chromatographic method coupled with electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) has been developed for quantification of felodipine in human and dog plasma. Compounds were separated on a 2.0 mm × 150 mm, 5.0 m particle, C₈ column with 1 m m ammonium acetate–acetonitrile, 20:80, pH 6.0, as mobile phase at a flow rate of 200 L min^–1. Nifedipine was used as internal standard. Plasma samples were extracted with diethyl ether, the centrifuged upper layer was evaporated, the residue was reconstituted with mobile phase, and the reconstituted samples were injected. The analytical column lasted for at least 1000 injections. By use of multiple reaction monitoring (MRM) mode in MS–MS felodipine and nifedipine were detected without severe interference from the human or dog plasma matrix. Felodipine produced a protonated precursor ion ([M + H]⁺) at m/z 384 and a corresponding product ion at m/z 338. And internal standard (nifedipine) produced a protonated precursor ion ([M + H]⁺) at m/z 347 and a corresponding product ion at m/z 315. Detection of felodipine in human and dog plasma was accurate and precise, with a limit of quantification of 0.05 ng mL^–1. The method has been successfully applied to preliminary pharmacokinetic study of felodipine in human and dog plasma.     

Development and Validation of a LC–ESI–MS Assay for Determination of Icariin in Rat Plasma after Administration of Herba Epimedii

This paper reports a sensitive, specific, and stable chromatographic procedure with selective detection (electrospray mass spectrometry in selected-ion-monitoring mode) combined with simple and efficient sample preparation for determination of icariin in rat plasma after administration of Herba Epimedii. Separation of the analyte, possible endogenous compounds, and constituents of Herba Epimedii were accomplished on a 250 mm × 2.0 mm i.d. C₁₈ column by use of a rapid gradient. Ionization of icariin and clarithromycin (internal standard) was achieved by use of the electrospray interface in positive-ion mode. Conditions such as ionization mode, type of organic modifier, eluent additives, and Q-array potential were optimized to achieve good sensitivity and specificity of icariin detection. Response was a linear function of concentration over the range 0.2–20 ng mL⁻¹. The method is accurate and precise; within-batch and between-batch precision (CV) are <15%, and accuracy (RE) is better than ±15%. The method can be used for analysis of icariin in plasma after administration of Herba Epimedii or of traditional Chinese medicinal preparations containing Herba Epimedii.