Development and Validation of a Bioanalytical LC-MS/MS Method for Simultaneous Determination of Sirolimus in Porcine Whole Blood and Lung Tissue and Pharmacokinetic Application with Coronary Stents

Sirolimus is a hydrophobic macrolide compound that has been used for long-term immunosuppressive therapy, prevention of restenosis, and treatment of lymphangioleiomyomatosis. In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of sirolimus in both porcine whole blood and lung tissue. Blood and lung tissue homogenates were deproteinized with acetonitrile and injected into the LC-MS/MS system for analysis using the positive electrospray ionization mode. The drug was separated on a C18 reversed phase column with a gradient mobile phase (ammonium formate buffer (5 mM) with 0.1% formic acid and acetonitrile) at 0.2 mL/min. The selected reaction monitoring transitions of m/z 931.5 → 864.4 and m/z 809.5 → 756.5 were applied for sirolimus and ascomycin (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 0.5–50 ng/mL. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in porcine whole blood and lung tissue homogenates, and all values were within acceptable ranges. The method was applied to a pharmacokinetic study to quantitate sirolimus levels in porcine blood and its distribution in lung tissue following the application of stents in the porcine coronary arteries. It enabled the quantification of sirolimus concentration until 2 and 14 days in blood and in lung tissue, respectively. This method would be appropriate for both routine porcine pharmacokinetic and bio-distribution studies of sirolimus formulations.     

Assessment of Tissue Distribution and Metabolism of MP1, a Novel Pyrrolomycin,in Mice Using a Validated LC-MS/MS Method

MP1 is a novel marinopyrrole analogue with activity in MYCN amplified neuroblastoma cell lines. A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of MP1 in mouse plasma. Analyte separation was achieved using a Waters Acquity UPLC^®BEH C18 column (1.7 µm, 100 × 2.1 mm). Mobile phase consisted of 0.1% acetic acid in water (10%) and methanol (90%) at a total flow rate of 0.25 mL/min. The mass spectrometer was operated at unit resolution in the multiple reaction monitoring (MRM) mode, using precursor ion > product ion transitions of 324.10 > 168.30 m/z for MP1 and 411.95 > 224.15 m/z for PL-3. The MS/MS response was linear over the concentration range from 0.2–500 ng/mL for MP1, correlation coefficient (r²) of 0.988. Precision (% RSD) and accuracy (% bias) were within the acceptable limits as per FDA guidelines. MP1 was stable under storage and laboratory handling conditions. The validated method was successfully applied to assess the solubility, in-vitro metabolism, plasma protein binding, and bio-distribution studies of MP1.     

Eco-Friendly UPLC–MS/MS Method for Determination of a Fostamatinib Metabolite,Tamatinib, in Plasma: Pharmacokinetic Application in Rats

Fostamatinib is a prodrug of the active metabolite tamatinib, which is a spleen tyrosine kinase (Syk) inhibitor used in the treatment of primary chronic adult immune thrombocytopenia and rheumatoid arthritis. A highly sensitive, rapid, reliable, and green method was developed and validated using ultra-performance liquid chromatography and tandem mass spectrometry (UPLC–MS/MS) for quantification of tamatinib in rat plasma. Ibrutinib was used as internal standard and liquid–liquid extraction was applied using tert-butyl methyl ether. The analyte was separated on an Acquity^TM CSH C₁₈ (2.1 mm × 100 mm, 1.7 µm) column using mobile phase consisting of 10 mM ammonium acetate and acetonitrile (10:90) and the flow rate was 0.25 mL/min. Electrospray ionization (ESI) was carried out in positive mode. Quantitation of tamatinib and the IS was performed using multiple reaction monitoring mode with precursor-to-product transitions of m/z 471.1 > 122.0 and m/z 441.1 > 84.0, respectively. The calibration range was 0.1–1000.0 ng/mL and the linearity of the method was ≥0.997. The developed method greenness was investigated. All principal parameters for the method, including linearity, accuracy, precision, recovery, and stability, were within acceptable ranges. Tamatinib pharmacokinetic study in rats was successfully carried out using the developed method.     

Development of an LC-MS/MS method for aromatase inhibitor screening

Aromatase (CYP 19A1) is a key steroidogenic enzyme that catalyzes the conversion of androgen to estrogen. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for aromatase inhibitor screening was developed and validated. The substrate androstenedione was incubated with human CYP 19A1 supersomes in the presence of NADPH for 30 min, and estrone formation was determined by LC-MS/MS analysis. Cortisone was used as internal standard. The incubation mixture was extracted using a liquid-liquid extraction method with ethyl acetate. Chromatographic separation was achieved using a C₁₈ column (3.0?×?50 mm, 2.7 μm) with a mobile phase consisting of 0.1 % formic acid/acetonitrile adopting gradient elution at a flow rate of 0.4 mL/min. The mass spectrometer was operated in positive electrospray ionization mode. The precursor-product ion pairs used for multiple reaction monitoring were m/z 287→97 (androstenedione), m/z 271?→?159 (estrone), and m/z 361?→?163 (IS, cortisone). The developed method met the required criteria for the validation of bioanalytical methods. The validated method was successfully applied to evaluate aromatase inhibitory activity of plants extracts of Simaroubaceae.

Development and validation of the quantitative determination of avapritinib in rat plasma by a bioanalytical method of UPLC-MS/MS

Avapritinib, an orally consumed, highly selective inhibitor of platelet-derived growth factor receptor alpha (PDGFRA), is approved in the USA for PDGFRA exon 18 (including D842V) mutant gastrointestinal stromal tumour (GIST). The research conducted investigates an advanced reliable and fast UPLC-MS/MS method that could verify and determine avapritinib concentration in plasma of rats. An addition of acetonitrile was incorporated along with the plasma sample in order to precipitate protein with the analyte separated from the matrix by a gradient elution procedure on a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm). The active stage in mobile phase consists of a mixture of 0.1% formic acid in water and acetonitrile with a 0.40 mL/min flow rate. UPLC-MS/MS detection was done employing a mode of multiple reaction monitoring (MRM), and the ion transitions of avapritinib and imatinib (internal standard, IS) was m/z 499.10 → 482.09, and m/z 494.30 → 394.20, respectively. This method has good linearity within 2–4000 ng/mL of avapritinib calibration range and a lower limit of quantification (LLOQ) of 2 ng/mL verified. Avapritinib precisions in both intra-day and inter-day were below 15% with the determined accuracy of ?12.9% to 12.0%. The recoveries, stabilities, and matrix effect of avapritinib and IS were credible. 30 mg/kg avapritinib was administered as a single dose orally to rats. The determination of avapritinib level in pharmacokinetic studies was accomplished by efficiently applying a newly optimized UPLC-MS/MS assay.     

Development and Verification of a Precolumn Derivatization LC-MS/MS Method for the Pharmacokinetic Study of Houttuynine of Houttuynia Essential Oil

Houttuynia essential oil (HEO) has excellent antiviral, anti-inflammatory, and other pharmacological effects, but the lack of effective analytical methods to quantify HEO in plasma has hindered its better clinical monitoring. Houttuynine (Hou) is one of the main active ingredients and quality control substances of HEO, so the pharmacokinetic study of HEO could be conducted by determining Hou blood concentration. Hou is active and not stable in plasma, which makes its blood concentration difficult to measure. In this work, a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method for Hou determination in rat blood was established that involves Hou being derivatized with 2, 4-dinitrophenylhydrazine to form a stable compound to prevent degradation. Herein, p-Tolualdehyde-2,4-dinitrophenylphenylhydrazone was selected as an internal standard substance and the LC-MS/MS method was evaluated for selectivity, precision, accuracy, calibration limit, matrix effect, recovery, and stability. Good linearity (r² = 0.998) was reached in the range of 2–2000 ng/mL, and the lower limit of quantification of Hou was determined to be 2 ng/mL. The mean intra-assay accuracy ranged from 77.7% to 115.6%, whereas the intra-assay precision (relative standard deviation, RSD) was below 11.42%. The matrix effect value for Hou in rat plasma was greater than 75%, and for the internal standard (IS) it was 104.56% ± 3.62%. The extraction recovery of Hou were no less than 90%, and for the IS it was 96.50% ± 4.68%. Our method is sensitive and reliable and has been successfully applied to the pharmacokinetic analysis of Hou in rats given HEO via gavage and injection.     

Determination of free captopril in human plasma by liquid chromatography with mass spectrometry detection

A new simple, sensitive and selective liquid chromatography coupled with mass spectrometry (LC/MS) method for quantification of captopril after precolumn derivatization with p-bromo-phenacyl-bromide in human plasma was validated. Plasma samples were analysed on a monolithic column (Cromolith Performance-RP 18e, 100 mm × 4.6 mm I.D., 3 μm) under isocratic conditions using a mobile phase of a 40:60 (v/v) mixture of acetonitrile and 0.1% (v/v) formic acid in water. The flow rate was 1 mL/min at the column temperature of 30 °C. In these chromatographic conditions, the retention time was 4.4 min for captopril derivative. The detection of the analyte was in MRM mode using an ion trap mass spectrometer with electrospray positive ionisation. The monitored ions were 216, 253, 255, 268, 270 m/z derived from 415 m/z for derivatized captopril. The sample preparation was very simple and consisted in plasma protein precipitation from 0.2 mL plasma using 0.3 mL methanol after the derivatization reaction was completed. Calibration curves were generated over the range of 10-3000 ng/mL with values for coefficient of correlation greater than 0.993 and by using a weighted (1/y²) quadratic regression. The values for precision (CV %) and accuracy (relative error %) at quantification limit were less than 9.9% and 3.9%, for within- and between-run, respectively. The mean recovery of the analyte was 99%. Derivatized samples demonstrated good short-term, long-term, post-preparative and freeze-thaw stability. This is the first reported LC-MS/MS method for analysis of captopril in human plasma that uses protein precipitation as sample processing procedure. The method is very simple and allows obtaining a very good recovery of the analyte. The validated LC-MS/MS method has been applied to a pharmacokinetic study of 50 mg captopril tablets on healthy volunteers.     

Development and validation of a rapid LC‐MS/MS method to quantify letrozole in human plasma and its application to therapeutic drug monitoring

A selective, rapid, and sensitive liquid chromatography–tandem mass spectrometry(LC‐MS/MS) method was developed and validated for the determination of letrozole (LTZ) in human plasma, using anastrozole as internal standard (IS). Sample preparation was performed by one‐step protein precipitation with methanol. The analyte and IS were chromatographed on a reversed‐phase YMC‐ODS‐C₁₈ column (2.0 × 100 mm i.d., 3 µm) with a flow rate of 0.3 mL/min. The mobile phase consisted of water containing 0.1% formic acid (v/v) and methanol containing 0.1% formic acid (v/v). The mass spectrometer was operated in selected reaction monitoring mode through electrospray ionization ion mode using the transitions of m/z 286.2 → 217.1 for LTZ and m/z 294.1 → 225.1 for IS, respectively. The method was validated for selectivity, linearity, lower limit of quantitation, precision, accuracy, matrix effects and stability in accordance with the US Food and Drug Administration guidelines. Linear calibration curves were 1.0–60.0 ng/mL. Intra‐ and inter‐batch precision (CV) for LTZ were <9.34%, and the accuracy ranged from 97.43 to 105.17%. This method was successfully used for the analysis of samples from patients treated with LTZ in the dose of 2.5 mg/day. It might be suitable for therapeutic drug monitoring of these patients and contribute to predict the risk of adverse reactions. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of GC/MS method for determination of pramipexole in rat plasma

A simple, rapid and sensitive method has been developed and validated for the determination of pramipexole in rat plasma by using gas chromatography mass spectrometry. The lower limit of quantification (LLOQ) is superior to the other reported LC‐MS/MS methods. After being made alkaline with NaOH, plasma samples (0.1 mL) were subjected to liquid–liquid exteraction using methyl‐t‐butyl ether. Analytes were determined using electron impact ionization in a single quadrupole mass spectrometer. GC/MS was performed in the selected ion monitoring mode using target ions at m/z 211, 212 and 152 for pramipexole and m/z 194 and 165 for caffeine as internal standard. A linear calibration curve was plotted over the range of 20–1000 pg/mL for pramipexole (r² > 0.996). The LLOQ was 20.0 pg/mL, respectively, which offered high sensitivity and selectivity enough for bioanalytical investigation. Inter‐ and intraday precisions ranged from 0.3 to 8.8% and from 0.9 to 11.33%, respectively. The recovery of pramipexole from plasma ranged from 82.4 ± 7.1 to 87.8 ± 5.7%. The method fulfills all standards required for bioanalytical methods and can be successfully applied to a pharmacokinetic study of pramipexole in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Establishment and validation of a UPLC-MS/MS bioassay for the quantification of infigratinib in rat plasma

Infigratinib (INF) is a fibroblast growth factor receptor (FGFR)-specific tyrosine kinase inhibitor for the therapy of advanced cholangiocarcinoma. However, CYP3A4 polymorphisms and CYP3A4 inducers or inhibitors might affect the pharmacokinetics of INF. Clinical evaluation of drug-drug interactions and adverse effects in these patients was necessary with reference to INF levels in vivo. The presently conducted study optimized a reproducible and rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analytic technique, which was validated and applied to determine INF concentrations in Sprague-Dawley (SD) rat plasma and pharmacokinetic studies. Protein was precipitated by adding acetonitrile to plasma samples, followed by gradient elution on a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) for complete chromatographic separation of the analyte and derazantinib (used as internal standard, IS). A gradient elution of 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.30 mL/min was applied as the mobile phase for this analysis. The ion transitions of INF and IS were m/z 599.88 → 313.10 and m/z 468.96 → 382.00 during UPLC-MS/MS detection, respectively, by multiple reaction monitoring (MRM). The methodological validation demonstrated that INF presented high linearity over the concentration of 2–600 ng/mL. The lower limit of quantification (LLOQ) for this experiment was 2 ng/mL, of which the precisions and accuracies were within the permissible levels. Inter-day and intra-day precisions were demonstrated to be within reasonable limits, which were within 15%, and the accuracies were determined to be between 2.2% and 11.4%. Moreover, the values of recovery, stability and matrix effect of INF were within the limits of acceptability. The pharmacokinetics of INF in SD rats was investigated by gavage administration of 10 mg/kg INF, followed by the application of the developed UPLC-MS/MS analytical method to detect the content in plasma and derive the main pharmacokinetic parameters.     

Development and application of an analytical method for curdione quantification in pregnant sprague–dawley rats by LC‐MS/MS

The vaginal administration route suffers from relatively low absorption efficiency, which may hinder the identification of the toxicokinetics of curdione in pregnant women. A sensitive analytical method for determining the plasma concentration of curdione was developed and applied in the determination of curdione in pregnant Sprague–Dawley rats as a simulated model. Glimepiride was used as an internal standard and chromatographic separation was achieved on a Capcell Pak C₁₈ MGIII column. A gradient elution profile with 0.5% formic acid (A)–0.5% formic acid–acetonitrile (B) was selected as mobile phase. The selected reaction monitoring mode was used for quantification based on the target fragment ions m/z 237.2 to m/z 135.1 for curdione and m/z 491.3 to m/z 352.1 for the glimepiride. The standard curve was linear over the range of 0.5–500 ng/mL for curdione in rat plasma and yielded a consistent peak pattern, even at the lower limit of quantitation of 0.5 ng/mL. The retention times of curdione and IS were 6.55 and 6.59 min, respectively. The mean recovery of curdione in rat plasma was 95.5–101.1%. The intra‐day and inter‐day precisions were between 2.35 and 9.08%. This LC‐MS/MS method provides a simple and sensitive means for determining the plasma concentration. Copyright © 2015 John Wiley & Sons, Ltd.     

Geissospermiculatine,a New Alkaloid from Geissospermum reticulatum Bark

A new alkaloid, geissospermiculatine was characterized in Geissospermum reticulatum A. H. Gentry bark (Apocynaceae). Here, following a simplified isolation protocol, the structure of the alkaloid was elucidated through GC-MS, LC-MS/MS, 1D, and 2D NMR (COSY, ROESY, HSQC, HMBC, ¹H-¹⁵N HMBC). Cytotoxic properties were evaluated in vitro on malignant THP-1 cells, and the results demonstrated that the cytotoxicity of the alkaloid (30  μg/mL) was comparable with staurosporine (10  μM). Additionally, the toxicity was tested on zebrafish (Danio rerio) embryos in vivo by monitoring their development (0–72 h); toxicity was not evident at 30  μg/mL.     

Phenolic compounds,essential oil composition,and antioxidant activity of Angelica purpurascens (Avé-Lall.) Gill

In this study, methanol extracts (MEs) and essential oil (EO) of Angelica purpurascens (Avé-Lall.) Gill obtained from different parts (root, stem, leaf, and seed) were evaluated in terms of antioxidant activity, total phenolics, compositions of phenolic compound, and essential oil with the methods of 2,2-azino-bis(3ethylbenzo-thiazoline-6-sulfonic acid (ABTS•⁺), 2,2-diphenyl-1-picrylhydrazil (DPPH•) radical scavenging activities, and ferric reducing/antioxidant power (FRAP), the Folin–Ciocalteu, liquid chromatography−tandem mass spectrometry (LC−MS/MS), and gas chromatography-mass spectrometry (GC−MS), respectively. The root extract of A. purpurascens exhibited the highest ABTS•⁺, DPPH•, and FRAP activities (IC₅₀: 0.05 ± 0.0001 mg/mL, IC₅₀: 0.06 ± 0.002 mg/mL, 821.04 ± 15.96 µM TEAC (Trolox equivalent antioxidant capacity), respectively). Moreover, EO of A. purpurascens root displayed DPPH• scavenging activity (IC₅₀: 2.95 ± 0.084 mg/mL). The root extract had the highest total phenolic content (438.75 ± 16.39 GAE (gallic acid equivalent), µg/mL)). Twenty compounds were identified by LC−MS/MS. The most abundant phenolics were ferulic acid (244.39 ± 15.64 μg/g extract), benzoic acid (138.18 ± 8.84 μg/g extract), oleuropein (78.04 ± 4.99 μg/g extract), and rutin (31.21 ± 2.00 μg/g extract) in seed, stem, root, and leaf extracts, respectively. According to the GC−MS analysis, the major components were determined as α-bisabolol (22.93%), cubebol (14.39%), α-pinene (11.63%), and α-limonene (9.41%) among 29 compounds. Consequently, the MEs and EO of A. purpurascens can be used as a natural antioxidant source.     

Phytochemical Constituents,Antioxidant Activity,and Toxicity Assessment of the Aerial Part Extracts from the Infraspecific Taxa of Matthiola fruticulosa (Brassicaceae) Endemic to Sicily

In a project designed to investigate the specific and infraspecific taxa of Matthiola endemic to Sicily (Italy) as new potential sources of bioactive compounds in this work, the infraspecific taxa of Matthiola fruticulosa were studied, namely, subsp. fruticulosa and subsp. coronopifolia. HPLC–PDA/ESI–MS and SPME–GC/MS analyses of hydroalcoholic extracts obtained from the aerial parts of the two subspecies led to the detection of 51 phenolics and 61 volatile components, highlighting a quite different qualitative–quantitative profile. The antioxidant properties of the extracts were explored through in vitro methods: 1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing power and Fe²⁺ chelating activity assays. The results of the antioxidant tests showed that the extracts possess a different antioxidant ability: particularly, the extract of M. fruticulosa subsp. fruticulosa exhibited higher radical scavenging activity than that of subsp. coronopifolia (IC₅₀ = 1.25 ± 0.02 mg/mL and 2.86 ± 0.05 mg/mL), which in turn displayed better chelating properties (IC₅₀ = 1.49 ± 0.01 mg/mL and 0.63 ± 0.01 mg/mL). Lastly, Artemia salina lethality bioassay was performed for toxicity assessment. The results of the bioassay showed lack of toxicity against brine shrimp larvae for both extracts. The data presented indicate the infraspecific taxa of M. fruticulosa as new and safe sources of antioxidant compounds.     

LC‐MS/MS methods for the determination of edaravone and/or taurine in rat plasma and its application to a pharmacokinetic study

Three liquid chromatography–tandem mass spectrometry (LC‐MS/MS) methods were respectively developed and validated for the simultaneous or independent determination of taurine and edaravone in rat plasma using 3‐methyl‐1‐p‐tolyl‐5‐pyrazolone and sulfanilic acid as the internal standards (IS). Chromatographic separations were achieved on an Agilent Zorbax SB‐Aq (100 × 2.1 mm, 3.5 µm) column. Gradient 0.03% formic acid–methanol, isocratic 0.1% formic acid–methanol (90:10) and 0.02% formic acid–methanol (40:60) were respectively selected as the mobile phase for the simultaneous determination of two analytes, taurine or edaravone alone. The MS acquisition was performed in multiple reaction monitoring mode with a positive and negative electrospray ionization source. The mass transitions monitored were m/z [M + H]⁺ 175.1 → 133.0 and [M + H]⁺ 189.2 → 147.0 for edaravone and its IS, m/z [M ? H]^? 124.1 → 80.0 and [M ? H]^? 172.0 → 80.0 for taurine and its IS, respectively. The validated methods were successfully applied to study the pharmacokinetic interaction of taurine and edaravone in rats after independent intravenous administration and co‐administration with a single dose. Our collective results showed that there were no significant alterations on the main pharmacokinetic parameters (area under concentration–time curve, mean residence time, half‐life and clearance) of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible. Copyright © 2014 John Wiley & Sons, Ltd.     

New UPLC–MS/MS method for simultaneous determination of irbesartan and hydrochlorthiazide in human plasma

Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) is a preeminent analytical tool for rapid biomedical analysis with the objective of reducing analysis time and maintaining good efficiency. In this study a simple, rapid, sensitive and specific ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated for quantification of the angiotensin II receptor antagonist, irbesartan and hydrochlorthiazide in human plasma. After a simple protein precipitation using methanol and acetonitrile, irbesartan, hydrochlorthiazide and internal standard (IS) telmisartan were separated on Acquity UPLC BEH? C18 column (50 × 2.1 mm, i.d. 1.7 μm, Waters, USA) using a mobile phase consisting of acetonitrile:10 mM ammonium acetate:formic acid (85:15:0.1 % v/v/v) pumped at a flow rate of 0.3 mL/min and detected by tandem mass spectrometry with negative ion mode. The ion transitions recorded in multiple reaction monitoring mode were m/z 427.2 → 193.08 for irbesartan, m/z 295.93 → 268.90 for hydrochlorthiazide and m/z 513.2 → 287.14 for IS. The assay exhibited a linear dynamic range of 30–500 ng/mL for irbesartan and 1–500 ng/mL in human plasma with good correlation coefficient of (0.996) and (0.997) and with a limit of quantitation of 30  and 1 ng/mL for irbesartan and hydrochlorthiazide, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 10.13 % for irbesartan and 11.14 % for hydrochlorthiazide. The proposed UPLC–MS/MS method is simple, rapid and highly sensitive, and hence it could be reliable for pharmacokinetic and toxicokinetic study in both animals and humans.     

A quantitative determination of fluorochloridone in rat plasma by UPLC‐MS/MS method: application to a pharmacokinetic study

A precise, high‐throughput and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed for the determination of fluorochloridone (FLC) in rat plasma. The extraction of analytes from plasma samples was carried out by protein precipitation procedure using acetonitrile prior to UPLC‐MS/MS analysis. Verapamil was proved as a proper internal standard (IS) among many candidates. The chromatographic separation based on UPLC was well optimized. Multiple reaction monitoring in positive electrospray ionization was used with the optimized MS transitions at: m/z 312.0 → 292.0 for FLC and m/z 456.4 → 165.2 for IS. This method was well validated with good linear response (r² > 0.998) observed over the investigated range of 3–3000 ng/mL and with satisfactory stability. This method was also characterized with adequate intra‐ and inter‐day precision and accuracy (within 12%) in the quality control samples, and with high selectivity and less matrix effect observed. Total running time was only 1.5 min. This method has been successfully applied to a pilot FLC pharmacokinetic study after oral administration. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantitative determination of major platelet activating factors from human plasma

Platelet activating factor (PAF) is a potent lipid mediator that is involved in many important biological functions, including platelet aggregation and neuronal differentiation. Although an ELISA assay has been used to measure PAF levels, it cannot distinguish between its isoforms. To achieve this, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used instead. However, isobaric lysophosphatidylcholine (lyso PC), which is often present in large amounts in complex biological samples and has similar retention times in many LC conditions, can affect the accurate measurement of PAF. The present study examined the fragmentation behavior of major PAF and lyso PC during various MS/MS conditions. Fragment ions at m/z 184 and at m/z 104 were abundantly observed from MS/MS of lyso PCs. PAF provided a dominant fragment ion at m/z 184, but a fragment ion at m/z 104 was almost never produced, regardless of the collision energy. Thus, the two fragment ions at m/z 184 and m/z 104 were used to accurately measure PAF levels. First, the fragment ion at m/z 184 and the retention time of PAF in LC-MS/MS were used to identify and quantitate PAF. However, if there were small retention time shifts, which are common in multiple sample runs, and lipid composition in a sample is very complicated, the fragment ion at m/z 104 was used to confirm whether the fragment ion at m/z 184 belonged to PAF. This novel method accurately determined the major PAF (C16:0 PAF, C18:0 PAF, and C18:1 PAF) levels in human plasma.     

Development and validation of a sensitive LC–MS/MS assay for quantification of bruceine D in rat plasma

A sensitive LC–MS/MS method for the determination of bruceine D in rat plasma was developed. The analyte and IS were separated on a Luna C₁₈ column (2.1 × 50 mm, 1.7 μm) using a mobile phase of acetonitrile and 0.1% formic acid in water (40:60, v/v) at a flow rate of 0.25 mL/min. The selected reaction monitoring mode was chosen to monitor the precursor‐to‐product ion transitions of m/z 409.2 → 373.2 for bruceine D and m/z 469.2 → 229.3 for IS using a negative ESI mode. The method was validated over a concentration range of 0.5–2000 ng/mL for bruceine D. Total chromatography time for each run was 3.5 min. The method was successfully applied to a pharmacokinetic study of bruceine D in rats. Copyright © 2016 John Wiley & Sons, Ltd.