A rapid and sensitive LC‐MS/MS method for the determination of osthole in rat plasma: application to pharmacokinetic study

Osthole, a major component isolated from the fruit of Cnidium monnieri (L.) Cusson, has been widely used in traditional Chinese medicine. We developed and validated a rapid and sensitive LC‐MS/MS method for the quantification of osthole in rat plasma. Sample preparation involved simple liquid–liquid extraction by ethyl acetate after addition of imperatorin as internal standard (IS). The analyte was separated using a C₁₈ column with the mobile phase of methanol–0.1% formic acid (80:20, v/v) at a flow rate of 0.4 mL/min. The elutes were detected under positive electrospray ionization in multiple reaction monitoring mode. The method was sensitive with 0.5 ng/mL as the lower limit of detection. Good linearity was obtained over the range of 1.0–500.0 ng/mL. The intra and inter‐batch accuracy for osthole in rat plasma samples ranged from 99.5 to 108.1% and the variation was <8.9%. The stability, extraction efficiency and matrix effect were also acceptable. This method was successfully applied to the pharmacokinetic study of osthole in rat after intravenous and oral administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Pressurized liquid extraction and GC‐MS analysis for simultaneous determination of seven components in Cinnamomum cassia and the effect of sample preparation

A pressurized liquid extraction and GC‐MS method was developed for simultaneous quantitative determination of the seven components, including cinnamaldehyde, copaene, cinnamic acid, coumarin, 2‐methoxycinnamaldehyde, 2‐methoxycinnamic acid and safrole in Cinnamomum cassia. The results showed that methanol and ethanol was not available for extraction of cinnamaldehyde and 2‐methoxycinnamaldehyde due to aldol reaction. The developed method was validated to be sensitive, accurate and simple, and was successfully employed for the analysis of 15 samples of C. cassia. The contents of the investigated components were significantly variant and cinnamaldehyde is the most abundant compound, but safrole was not detected in all samples.     

Determination of three capsaicinoids in Capsicum annuum by pressurized liquid extraction combined with LC‐MS/MS

A new method based on pressurized liquid extraction followed by LC‐MS/MS analysis has been developed for the identification and quantification of three capsaicinoids (capsaicin, dihydrocapsaicin, and nordihydrocapsaicin) in extracts of Capsicum annuum. For the recovery of three capsaicinoids, the efficiency levels of ultrasonic‐assisted extraction, microwave‐assisted extraction, Soxhlet extraction, and pressurized liquid extraction were compared under different conditions. Pressurized liquid extraction resulted in higher yields. Pressurized liquid extractions were performed using methanol; temperature was set at 100°C and pressure at 1500 psi. LC analysis was performed on a Waters XBridge? C₁₈ column (150 × 2.1 mm, id 3.5 μm) eluted by a mobile phase of 0.1% formic acid and ACN. Data acquisition was carried out in multiple reaction monitoring transitions mode, monitoring two‐reaction monitoring transitions to ensure an accurate identification of target compounds in the samples. The proposed method is rapid, simple, and could be utilized for the routine analysis of three capsaicinoids in C. annuum samples.     

A UPLC‐MS/MS method for simultaneous determination of five flavonoids from Stellera chamaejasme L. in rat plasma and its application to a pharmacokinetic study

Stellera chamaejasme L. has been used as a traditional Chinese medicine for the treatment of scabies, tinea, stubborn skin ulcers, chronic tracheitis, cancer and tuberculosis. A sensitive and selective ultra‐high liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for the simultaneous determination of five flavonoids (stelleranol, chamaechromone, neochamaejasmin A, chamaejasmine and isochamaejasmin) of S. chamaejasme L. in rat plasma. Chromatographic separation was accomplished on an Agilent Poroshell 120 EC‐C₁₈ column (2.1 × 100 mm, 2.7 μm) with gradient elution at a flow rate of 0.4 mL/min and the total analysis time was 7 min. The analytes were detected using multiple reaction monitoring in positive ionization mode. The samples were prepared by liquid–liquid extraction with ethyl acetate. The UPLC‐MS/MS method was validated for specificity, linearity, sensitivity, accuracy and precision, recovery, matrix effect and stability. The validated method exhibited good linearity (r ≥ 0.9956), and the lower limits of quantification ranged from 0.51 to 0.64 ng/mL for five flavonoids. The intra‐ and inter‐day precision were both <10.2%, and the accuracy ranged from −11.79 to 9.21%. This method was successfully applied to a pharmacokinetic study of five flavonoids in rats after oral administration of ethyl acetate extract of S. chamaejasme L.     

A rapid UPLC‐MS/MS method for the determination of oleanolic acid in rat plasma and liver tissue: application to plasma and liver pharmacokinetics

A reliable high‐throughput ultra‐high performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for oleanolic acid (OA) determination in rat plasma and liver tissue using glycyrrhetic acid as the internal standard (IS). Plasma and liver homogenate samples were prepared using solid‐phase extraction. Chromatographic separation was achieved on a C₁₈ column using an isocratic mobile phase system. The detection was performed by multiple reaction monitoring mode via positive electrospray ionization interface. The calibration curves showed good linearity (R² > 0.9997) within the tested concentration ranges. The lower limit of quantification for plasma and liver tissue was ≤0.75 ng/mL. The intra‐ and inter‐day precision and accuracy deviations were within ±15% in plasma and liver tissue. The mean extraction recoveries ranged from 80.8 to 87.0%. In addition, the carryover, matrix effect, stability and robustness involved in the method were also validated. The method was successfully applied to the plasma and hepatic pharmacokinetics of OA after oral administration to rats. Copyright © 2015 John Wiley & Sons, Ltd.     

LC–MS/MS method for simultaneous determination of flavonoids and physalins in rat plasma: Application to pharmacokinetic study after oral administration of Physalis alkekengi var. franchetii (Chinese lantern) extract

A rapid and sensitive liquid chromatography with tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous determination of luteolin, luteolin‐7‐O ‐β ‐D‐glucopyranoside, physalin A, physalin D and physalin L in rat plasma. Scutellarein and dexamethasone were used as the internal standards (IS). Plasma samples were prepared by liquid‐liquid extraction with ethyl acetate. The five constituents were separated on an Acquity UPLC BEH C₁₈ column (100 mm × 2.1 mm, 1.7 μm). A gradient elution procedure was used with acetonitrile (A)‐0.1% aqueous formic acid (B). Mass spectrometric detection was performed in negative ion multiple reaction monitoring mode with an electrospray ionization (ESI) source. This method showed good linearity (r ² > 0.997) over a concentration range of 2.0–500 ng/mL with a lower limit of quantification of 2.0 ng/mL for all five compounds. The inter‐ and intra‐day accuracy ranged from 91.7 to 104%, and precisions (RSD) were <6.46% for all analytes. The extraction recoveries of all analytes were >85%. This validated method was successfully applied for the first time to the pharmacokinetic study of five ingredients after oral administration of 70% ethanol extract of Chinese lantern in rats.     

Determination of benzoylurea insecticides in food by pressurized liquid extraction and LC‐MS

A method based on pressurized liquid extraction and LC‐MS/MS has been developed for determining nine benzoylureas (BUs) in fruit, vegetable, cereals, and animal products. Samples (5 g) were homogenized with diatomaceous earth and extracted in a 22 mL cell with 22 mL of ethyl acetate at 80°C and 1500 psi. After solvent concentration and exchange to methanol, BUs were analyzed by LC‐MS/MS using an IT mass analyzer, which achieved several transitions of precursor ions that increase selectivity providing identification. LOQs were between 0.002 and 0.01 mg/kg, which are equal or lower than maximum residue limits established by the Codex Alimentarius. Excellent linearity was achieved over a range of concentrations from 0.01 to 1 mg/kg with correlation coefficients 0.995–0.999 (n=7). Validation of the total method was performed by analyzing in quintuplicate seven different commodities (milk, eggs, meat, rice, lettuce, avocado, and lemon) at three concentration levels (0.01, 0.1, and 1 mg/kg). The recoveries ranged from 58 to 97% and the RSDs from 5 to 19% depending on the compound and the commodity. The combination of pressurized liquid extraction with LC‐MS/MS provides a sensitive and selective method for the determination of BUs in food.     

Separation and characterization of sucrose esters from Oriental tobacco leaves using accelerated solvent extraction followed by SPE coupled to HPLC with ion‐trap MS detection

Sucrose esters (SEs) were successfully extracted from Oriental tobacco leaves using a new methodology based on accelerated solvent extraction followed by hydrophilic–lipophilic balanced cartridge cleanup step. The SEs were detected by HPLC with ion‐trap MS detection using an electrospray interface operated in the positive ion mode. This methodology combines the high efficiency of extraction provided by a pressurized fluid and the highly sensitive characterization offered by ion‐trap MS. Under the optimized conditions, 14 SEs were first identified among a total of 23 SEs found in Oriental tobacco leaves. Under the same conditions, only four new SEs were extracted by using traditional ultrasound‐assisted extraction and liquid–solid extraction methods. The present method might be potentially useful in high‐efficiency extraction and sensitive characterization of SEs from complex matrices such as tobacco leaves.     

Supported liquid extraction in combination with LC‐MS/MS for high‐throughput quantitative analysis of hydrocortisone in mouse serum

A high‐throughput LC–MS/MS bioanalytical method was developed and validated for the determination of hydrocortisone in mouse serum via supported liquid extraction (SLE) in a 96‐well plate format. Although sample extracts from SLE result in similar matrix effects compared with conventional liquid–liquid extraction (LLE), greater analyte extraction recovery and much higher analysis throughput for the quantitative analysis of hydrocortisone in mouse serum were obtained. The current LC‐MS/MS method was validated for a concentration range of 2.00–2000 ng/mL for hydrocortisone using a 0.100 mL volume of mouse serum. The intra‐ and inter‐day precision and accuracy of the quality control samples at low, medium and high concentration levels showed ≤12.9% CV and ?3.4–6.2% bias for the analyte in mouse serum. Copyright © 2009 John Wiley & Sons, Ltd.     

An improved LC‐MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and its active metabolites in human plasma and its application to a pharmacokinetic study in patients

In this study, a sensitive, selective and reproducible liquid chromatography–tandem mass spectrometry method for the simultaneous determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and its active metabolites, 1‐caffeoyl‐5‐feruoylquinic acid and 1,5‐O‐diferuoylquinic acid, in human plasma, using puerarin as internal standard, was developed and validated. Analytes were extracted from plasma samples by liquid–liquid extraction with ethyl acetate, separated on a C₁₈ reversed‐phase column with water containing 5 mM ammonium acetate and acetonitrile as the mobile phase and detected by electrospray ionization mass spectrometry in negative selected reaction monitoring mode. The accuracy and precision of the method were acceptable and linearity was good over the range 1–200 ng/mL for each analyte. In addition, the selectivity, extraction recovery and matrix effect were satisfactory too. The validated LC‐MS/MS method was successfully applied to phase II clinical pharmacokinetic study of 1,5‐DCQA in patients. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of aflatoxins in nonalcoholic beer using liquid–liquid extraction and ultraperformance LC‐MS/MS

Aflatoxins AFB1, AFB2, AFG1, and AFG2 are toxic secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus and posses a potential threat to food safety. In the present work, liquid–liquid extraction and ultraperformance LC‐MS/MS method has been applied for the determination of four naturally occurring aflatoxins AFB1, AFB2, AFG1, and AFG2 in nonalcoholic beer. Aflatoxins extraction from nonalcoholic beer was carried out using liquid–liquid extraction procedure. The effects of solvent‐types were studied to obtain maximum recovery of the target analytes with minimum contamination. Among different solvents, the aflatoxins extraction was best achieved using ethyl acetate. The obtained recoveries were ranged from 85 to 96% with good quality parameters: LOD values between 0.001 and 0.003 ng/mL, linearity of the calibration curve (r² > 0.999), and repeatability (run‐to‐run) and reproducibility (day‐to‐day) precisions with RSDs lower than 5% (n = 5) achieved at 0.50 ng/mL concentration. The optimized liquid–liquid extraction in combination with ultraperformance LC‐MS/MS was applied successfully to the analysis of AFB1, AFB2, AFG1, and AFG2 aflatoxins in 11 nonalcoholic beers and were detected up to 15.31 ng/L in some of the samples.     

Selective detection of phosphatidylethanol homologues in blood as biomarkers for alcohol consumption by LC‐ESI‐MS/MS

A new validated method for the quantitation of the abnormal phospholipid phosphatidylethanol (PEth)—a biomarker for ethanol uptake—has been developed by LC‐ESI‐MS/MS following miniaturised organic solvent extraction and reversed phase chromatography with phosphatidylbutanol (PBut) as internal standard. PEth homologues with two fatty acid substituents—PEth 18 : 1/18 : 1, PEth 16 : 0/16 : 0—were determined in post‐mortem blood collected from heavy drinkers at autopsy and also in whole blood samples from a volunteer after a single 60 g‐dose of ethanol. Furthermore, PEth 18 : 1/16 : 0 or its isobaric isomer PEth—16 : 0/18 : 1 was detected. In comparison to previous high‐performance liquid chromatography (HPLC) methods with evaporative light scattering detection (ELSD), the LC‐MS/MS‐method is more sensitive—with a limit of detection below 20 ng/ml—and more selective for single PEth homologues, while ELSD has been used for detection of the sum of PEth homologues with approximately 10 times less sensitivity. LC‐MS/MS enables monitoring of PEth homologues as biomarkers for harmful and prolonged alcohol consumption as with HPLC/ELSD earlier, where PEth is measurable in blood only after more than 50 g ethanol daily intake for more than 2 weeks. Because of its higher sensitivity, there is a potential to detect single heavy drinking by LC‐MS/MS, when PEth is formed in very low concentrations. This opens a new field of application of PEth to uncover single or multiple heavy drinking at a lower frequency and with a larger window of detection in blood than before by HPLC/ELSD or by use of other direct markers, e.g. ethyl glucuronide or ethyl sulfate. Copyright © 2009 John Wiley & Sons, Ltd.     

A supported liquid extraction LC–MS/MS method for determination of concentrations of GDC‐0425, a small molecule Checkpoint kinase 1 inhibitor,in human plasma

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of GDC‐0425 concentrations in human plasma has been developed and validated. Supported liquid extraction was used to extract plasma samples (50 μL) and the resulting samples were analyzed using reverse‐phase chromatography and mass spectrometry coupled with a turbo‐ionspray interface. The mass analysis of GDC‐0425 was performed using multiple reaction monitoring transitions in positive ionization mode. The method was validated over the calibration curve range of 1.00–1000 ng/mL using linear regression and 1/x² weighting. Within‐run relative standard deviation ranged from 0.8 to 5.1%, while between‐run RSD varied from 1.9 to 4.7% for QCs. The accuracy ranged from 90.0 to 101.0% of nominal for within‐run and from 94.0 to 100.0% of nominal for between‐run. Overall extraction recovery was 87.4% for GDC‐0425 and 87.9% for GDC‐0425‐d₉. Stability of GDC‐0425 was established in human plasma for 374 days at ?20 and ?70 °C and established in reconstituted sample extracts for 88 h when stored at 2–8 °C. Stable‐labeled internal standard was used to minimize matrix effects. This assay was used to characterize the pharmacokinetics of GDC‐0425 in cancer patients.     

Determination of main taxoids in Taxus species by microwave‐assisted extraction combined with LC‐MS/MS analysis

A method based on microwave‐assisted extraction (MAE) has been developed for the determination of paclitaxel and five related taxoids, namely 10‐deacetylbaccatin III (10‐DAB III), cephalomannine, 10‐deacetylpaclitaxel (10‐DAT), 7‐xyl‐10‐ deacetylpaclitaxel (7‐xyl‐10‐DAT), and 7‐epi‐10‐deacetylpaclitaxel (7‐epi‐10‐DAT) in Taxus species in this study. The influential parameters of the MAE procedure were optimized, and the optimal conditions were as follows: extraction solvent 80% ethanol solution, solid/liquid ratio 1:10 (g/mL), temperature 50°C, and three extraction cycles, each cycle 10 min. The method validation for LC‐MS/MS analysis was performed. The LOD and LOQ were 3.16–9.20 and 12.20–30.45 ng/mL, respectively. Repeatability and reproducibility for the six taxiods with RSD ranged from 2.78 to 3.85% and from 5.26 to 6.60%. The recoveries of the method for the six taxoids were 92.6–105.6%. The developed MAE‐LC‐MS/MS method was also successfully applied to determine the contents of six taxoids in different Taxus species.     

Ultra‐fast LC–ESI‐MS/MS method for the simultaneous determination of six highly toxic Aconitum alkaloids from Aconiti kusnezoffii radix in rat plasma and its application to a pharmacokinetic study

A fast, sensitive, and efficient ultra‐fast LC–ESI‐MS/MS method was developed for the simultaneous quantitation of six highly toxic Aconitum alkaloids, that is, aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine, in rat plasma after oral administration of crude ethanol extracts from Aconiti kusnezoffii radix by ultrasonic extraction, reflux extraction for 1 h, and reflux extraction for 3 h, respectively. The separation of six Aconitum alkaloids and aminopyrine (internal standard) was performed on an InertSustain® C₁₈ column, and the quantification of the analytes was performed on a 4000Q ultra‐fast LC–MS/MS system with turbo ion spray source in the positive ion and multiple‐reaction monitoring mode. Absolute recoveries ranged within 65.06–85.1% for plasma samples. The intra‐ and interday precision and accuracy of analytes were satisfactory. The methods were validated with sensitivity reaching the lower LOQ for aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine, which were 0.025, 0.025, 0.050, 0.025, 0.025, and 0.100 ng/mL, respectively. The method was successfully applied to a pharmacokinetic study of six Aconitum alkaloids in rat plasma after oral administration of crude ethanol extracts from the raw root of Aconitum kusnezoffii Reichb. by three different extraction processes.     

Sensitive LC‐MS‐MS method for the determination of tiopronin in human plasma

A highly selective and sensitive LC‐MS‐MS method was developed and validated to quantify tiopronin in human plasma, using fudosteine as the internal standard (IS). L ‐Cysteine and 1,4‐dithiothreitol (DTT) were used as the reducer and the stabilizer to release and stabilify tiopronin from a dimmer and mix forms with endogenous thiols in the treatment of plasma samples. After a simple liquid–liquid extraction with ethyl acetate in acidic condition, the post‐treatment samples were analyzed on a C₁₈ column interfaced with a triple‐quadruple tandem mass spectrometer using negative electrospray ionization. Methanol and water (40:60, v/v) were used as the isocratic mobile phase, with 0.2% formic acid and 1.0 mM tris (hydroxymethyl) aminomethane (Tris) in water. The method was validated to demonstrate the specificity, lower limit of quantification, accuracy and precision of measurements. The assay was linear over the concentration range 0.078–10 μg/mL. The correlation coefficients for the calibration curves ranged from 0.9980 to 0.9990. The intra‐ and inter‐day precisions, calculated from quality control samples, were not more than 10.49%. The method was employed in a pharmacokinetic study after oral administration of 200 mg tiopronin tablets to 24 healthy volunteers. Copyright © 2009 John Wiley & Sons, Ltd     

Rapid simultaneous determination of codeine and morphine in plasma using LC‐ESI‐MS/MS: Application to a clinical pharmacokinetic study

A rapid and sensitive high‐performance LC‐MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid‐liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C₁₈ column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H]⁺ ions, mass‐to‐charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2–100/0.5–250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC‐MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.     

Determination of di‐(2‐ethylhexyl)phthalate in environmental samples by liquid chromatography coupled with mass spectrometry

Di‐(2‐ethylhexyl)phthalate (DEHP) was determined in environmental samples such as water and soil. DEHP was extracted from water samples using SPE, whereas for soils pressurized liquid extraction was applied as extraction method, using hexane/acetone (1:1, v/v) as extractant solvent. The use of HPLC coupled to MS provides the basis of the selective determination of DEHP in the analyzed samples. The extraction procedures were validated and good results were found. Recoveries were ranged from 86.0 to 99.8% with RSD lower than 18% and LODs were 0.02 mg/kg and 0.03 μg/L for soils and water, respectively. Finally, the optimized methods were applied to the analysis of real samples and DEHP was not found above the LOQ (0.05 mg/kg) in soil samples whereas it was detected in water samples at concentrations ranging between 0.19 to 0.88 μg/L.     

Development and validation of a highly sensitive LC‐ESI‐MS/MS method for the determination of hyperoside in beagle dog plasma: application to a pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the analysis of hyperoside in beagle dog plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves extraction of hyperoside and ginsenoside Re (IS) from beagle dog plasma. Chromatographic separation was carried out on an Agilent Zorbax XDB‐C₁₈ (100 × 2.1 mm, 1.8 µm) column by isocratic elution with acetonitrile and water (50:50, v/v) at a flow rate of 0.25 mL/min with a total run time of 2.0 min. The MS/MS ion transitions monitored were 464.4 → 463.4 for hyperoside and 947.12 → 969.60 for IS. Linear responses were obtained for hyperoside ranging from 10 to 5000 ng/mL. The intra‐and inter‐day precisions (RSDs) were <5.38 and 3.39% and the extraction recovery ranged from 94.39 to 100.78% with an RSD <3.82%. Stability studies showed that hyperoside was stable in preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration–time profiles of hyperoside. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of antibacterial agent tilmicosin in pig plasma by LC/MS/MS and its application to pharmacokinetics

A rapid and sensitive high‐performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to quantify tilmicosin in pig plasma. Plasma samples were prepared by liquid–liquid extraction. Chromatographic separation was achieved on a C₁₈ column (2.1 × 30 mm, 3.5 μm) using acetonitrile–water (90:10, v /v; water included 0.1% formic acid) as the mobile phase. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear from 0.5 to 2000 ng/mL (r ² = 0.9998). The intra‐ and inter‐day accuracy and precision were within the acceptable limits of ±10% for all tilmicosin concentrations. The recoveries ranged from 95 to 99% for the three tested concentrations. The LC–MS/MS method described herein was simple, fast and less laborious than other methods, achieved high sensitivity using a small sample volume, and was successfully applied to pharmacokinetic studies of tilmicosin enteric granules after oral delivery to pigs. In comparison with tilmicosin premix, tilmicosin enteric granules slowed the elimination rate of tilmicosin, prolonged its period of action and significantly improved its bioavailability.