Screening and determination of drugs in human saliva utilizing microextraction by packed sorbent and liquid chromatography–tandem mass spectrometry

This study presents a new method for collecting and handling saliva samples using an automated analytical microsyringe and microextraction by packed syringe (MEPS). The screening and determination of lidocaine in human saliva samples utilizing MEPS and liquid chromatography–tandem mass spectrometry (LC‐MS/MS) were carried out. An exact volume of saliva could be collected. The MEPS C₈‐cartridge could be used for 50 extractions before it was discarded. The extraction recovery was about 60%. The pharmacokinetic curve of lidocaine in saliva using MEPS‐LC‐MS/MS is reported. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a molecularly imprinted polymer based solid-phase extraction of local anaesthetics from human plasma

Molecular imprints selective for a homologous series of local anaesthetics, including bupivacaine, ropivacaine and mepivacaine, were prepared and the resultant polymers were used for solid-phase extraction of human plasma. The template was a structural analogue, pentycaine, which was imprinted in methacrylic acid-ethylene glycol dimethacrylate copolymers. Equilibrium ligand binding experiments using radiolabelled bupivacaine were performed to characterize the imprinted polymers, as well as to identify optimal conditions for selective extraction of plasma samples. Dilution of the plasma prior to extraction with citrate buffer pH 5.0 containing ethanol and Tween 20 was found optimal for selective imprint-analyte binding, and for reduction of non-specific adsorption of lipophilic contaminants to the hydrophobic MIP surface. Wash steps using 20% methanol in water followed by a solvent switch to 10% ethanol in acetonitrile removed contaminants and strengthened the selective imprint-analyte binding. Elution under basic conditions using triethylamine-water-acetonitrile mixtures recovered bupivacaine in 89% yield with superior selectivity over elution under acidic conditions. The final protocol extracted trace levels of ropivacaine and bupivacaine from human plasma and allowed determination of bupivacaine in the range of 3.9-500 nmol L⁻¹ and ropivacaine in the range of 7.8-500 nmol L⁻¹ with inter-assay accuracies of 94-99 and 95-104%, respectively. This present investigation provides an improved understanding of approaches available for optimization of protocols for molecular-imprint based solid-phase extraction of plasma samples.     

Simultaneous determination of bupivacaine, mepivacain, prilocaine and ropivacain in human serum by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method with a rapid and simple sample preparation was developed and validated for the simultaneous determination of the local anesthetics bupivacaine, mepivacaine, prilocaine and ropivacaine in human serum. An external calibration was used. The mass spectrometer was operated in the multiple reaction monitoring mode. A good quadratic response over the range of 1.0-200.0 ng/ml was demonstrated. The accuracy for bupivacaine ranged from 93.2 to 105.7%, for mepivacaine from 96.2 to 104.3%, for prilocaine from 94.6 to 105.7% and for ropivacaine from 94.3 to 104.0%, respectively. The limit of quantification was 1.0 ng/ml for all substances. This method is suitable for pharmacokinetic studies.     

Microextraction by packed sorbent and liquid chromatography–tandem mass spectrometry as a tool for quantification of peptides in plasma samples: determination of sensory neuron‐specific receptors agonist BAM8‐22 and antagonist BAM22‐8 in plasma samples

Microextraction by packed sorbent (MEPS) is a miniaturized, solid‐phase extraction (SPE) technique that works online with gas chromatography (GC) and liquid chromatography (LC). Not only is the automation process with MEPS advantageous, but the much smaller volumes of the samples, solvents and dead space in the system also provide other significant advantages such as the speed and the simplicity of the sample preparation process. In this study MEPS has been evaluated for quantification of sensory neuron‐specific receptors agonist (BAM8‐22). Owing to the instability of BAMs, the focus was on fast extraction and determination of the peptide online using LC‐MS/MS. Sorbents such as C2, C8 and ENV+ (hydroxylated polystyrene–divinylbenzene copolymer) were investigated in the present study. MEPS‐C8 gave the best results compared with C2 and ENV and it was used for the method validation. The calibration curve was obtained within the concentration range of 20.0–3045 nmol/L in plasma. The regression correlation coefficients for plasma samples were ≥0.99 for all runs (n = 6). The between‐batch accuracy and precision for BAM8‐22 ranged from ?13 to ?2.0% and from 4.0 to 14%, respectively. Additionally, the accuracy and precision for BAM22‐8 ranged from ?13 to 7.0% and from 3.0 to 12%, respectively. The present method was used for pharmacokinetic studies for BAMs in plasma samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative determination of metformin,glyburide and its metabolites in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of an LC‐MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87–99%, and that from urine samples was 85–95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100–0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984–1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra‐day and inter‐day assays in plasma and urine samples, and the accuracy was 86–114% in plasma, and 94–105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. Copyright © 2014 John Wiley & Sons, Ltd.     

Novel micro-extraction by packed sorbent procedure for the liquid chromatographic analysis of antiepileptic drugs in human plasma and urine

A method for the simultaneous determination of the antiepileptic drugs, phenobarbital (PHB), phenytoin (PTN), carbamazepine (CBZ), primidone (PRM) and oxcarbazepine (OXC) in human plasma and urine samples by using micro‐extraction in a packed syringe as the sample preparation method connected with LC/UV (MEPS/LC/UV) is described. Micro‐extraction in a packed syringe (MEPS) is a new miniaturized, solid‐phase extraction technique that can be connected online to gas or liquid chromatography without any modifications. In MEPS approximately 1 mg of the solid packing material is inserted into a syringe (100–250 μL) as a plug. Sample preparation takes place on the packed bed. The bed can be coated to provide selective and suitable sampling conditions. The new method is very promising, easy to use, fully automated, inexpensive and quick. The standard curves were obtained within the concentration range 1–500 ng/mL in both plasma and urine samples. The results showed high correlation coefficients (R²>0.988) for all of the analytes within the calibration range. The extraction recovery was found to be between 88.56 and 99.38%. The limit of quantification was found to be between 0.132 and 1.956 ng/mL. The precision (RSD) values of quality control samples (QC) had a maximum deviation of 4.9%. A comparison of the detection limits with similar methods indicates high sensitivity of the present method. The method is applied for the analysis of these drugs in real urine and plasma samples of epileptic patients.     

Synthesis of a molecularly imprinted polymer and its application for microextraction by packed sorbent for the determination of fluoroquinolone related compounds in water

Molecularly imprinted polymer (MIP) has been synthesized by precipitation polymerization using ciprofloxacin (CIP) as template for the analysis of fluoroquinolone antibiotics (FQs). This MIP material was packed as sorbent in a device for microextraction by packed sorbent (MEPS) combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the analysis of selected FQs drugs including CIP, norfloxacin (NOR) and ofloxacin (OFLO) in municipal wastewater samples. In comparison to the new MIP-MEPS procedure, the target compounds were also determined by solid-phase extraction (MISPE) using the new molecular imprinted polymer material to validate the new MIP-MEPS method. The ability of the MIP for molecular recognition of CIP, NOR and OFLO was proved in presence of structurally different environmental relevant substances such as quinolones (Qs), flumequine (FLU), di(methyl)phthalate (DMP), technical 4-nonylphenol (NP), caffeine, Galaxolide^®, Tonalid^®, di(butyl)phthalate (DBP), Triclosan, bisphenol-A (BPA), carbamazepine, di(ethylhexyl)phthalate (DEHP), estradiol and octocrylene. The analysis of wastewater samples revealed the high selectivity of the synthesized polymer which was able to recognize and retain the target analytes by both extraction methods, the offline SPE with MIP material and the semi-automated MEPS packed with MIP material.     

Selective determination of estrogenic compounds in water by microextraction by packed sorbents and a molecularly imprinted polymer coupled with large volume injection-in-port-derivatization gas chromatography-mass spectrometry

A fully automated protocol consisting of microextraction by packed sorbents (MEPS) coupled with large volume injection-in-port-derivatization-gas chromatography–mass spectrometry (LVI-derivatization-GC–MS) was developed to determine endocrine disrupting compounds (EDCs) such as alkylphenols, bisphenol A, and natural and synthetic hormons in river and waste water samples. During method optimization, the extraction parameters as ion strength of the water sample, the MEPS extraction regime, the volume of organic solvent used for the elution/injection step, the type of elution solvents and the selectivity of the sorbents were studied. For optimum in-port-derivatization, 10 μL of the derivatization reagent N,O-bis(trimethylsilyl)triufloroacetamide with 1% of trimethylchlorosilane (BSTFA + 1% TMCS) was used. 17β-Estradiol-molecularly imprinted polymer (MIP) and silica gel (modified with C-18) sorbents were examined for the enrichment of the target analytes from water samples and the obtained results revealed the high selectivity of the MIP material for extraction of substances with estrogen-like structures. Recovery values for most of the analytes ranged from 75 to 109% for the C18 sorbent and from 81 to 103% for the MIP material except for equilin (on C18 with only 57–66% recovery). Precision (n = 4) of the entire analysis protocol ranged between 4% and 22% with both sorbents. Limits of detection (LODs) were at the low ng L⁻¹ level (0.02–87, C18 and 1.3–22, MIP) for the target analytes.     

Efficiency of a miniaturized silica monolithic cartridge in reducing matrix ions as demonstrated in the simultaneous extraction of morphine and codeine from urine samples for quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Presence of matrix ions could negatively affect the sensitivity and selectivity of liquid chromatography‐tandem mass spectrometer (LC‐MS/MS). In this study, the efficiency of a miniaturized silica monolithic cartridge in reducing matrix ions was demonstrated in the simultaneous extraction of morphine and codeine from urine samples for quantification with LC‐MS. The miniaturized silica monolith with hydroxyl groups present on the largely exposed surface area function as a weak cation exchanger for solid phase extraction (SPE). The miniaturized silica cartridge in 1 cm diameter and 0.5 cm length was housed in a 2‐ml syringe fixed over a SPE vacuum manifold for extraction. The cleaning effectiveness of the cartridge was confirmed by osmometer, atomic absorption spectrometer, LC‐MS and GC‐TOFMS. The drugs were efficiently extracted from urine samples with recoveries ranging from 86% to 114%. The extracted analytes, after concentration and reconstitution, were quantified using LC‐MS/MS. The limits of detection for morphine and codeine were 2 ng/ml and 1 ng/mL, respectively. The relative standard deviations of measurements ranged from 3% to 12%. The monolithic sorbent offered good linearity with correlation coefficients > 0.99, over a concentration range of 50–500 ng/ml. The silica monolithic cartridge was found to be more robust than the particle‐based packed sorbent and also the commercial cartridge with regards to its recyclability and repeated usage with minimal loss in efficiency. Our study demonstrated the efficiency of the miniaturized silica monolith for removal of matrix ions and extraction of drugs of abuse in urinary screening. Copyright © 2011 John Wiley & Sons, Ltd.     

A rapid MCM‐41 dispersive micro‐solid phase extraction coupled with LC/MS/MS for quantification of ketoconazole and voriconazole in biological fluids

A rapid dispersive micro‐solid phase extraction (D‐μ‐SPE) combined with LC/MS/MS method was developed and validated for the determination of ketoconazole and voriconazole in human urine and plasma samples. Synthesized mesoporous silica MCM‐41 was used as sorbent in d ‐μ‐SPE of the azole compounds from biological fluids. Important D‐μ‐SPE parameters, namely type desorption solvent, extraction time, sample pH, salt addition, desorption time, amount of sorbent and sample volume were optimized. Liquid chromatographic separations were carried out on a Zorbax SB‐C₁₈ column (2.1 × 100 mm, 3.5 μm), using a mobile phase of acetonitrile–0.05% formic acid in 5 mm ammonium acetate buffer (70:30, v /v). A triple quadrupole mass spectrometer with positive ionization mode was used for the determination of target analytes. Under the optimized conditions, the calibration curves showed good linearity in the range of 0.1–10,000 μg/L with satisfactory limit of detection (≤0.06 μg/L) and limit of quantitation (≤0.3 μg/L). The proposed method also showed acceptable intra‐ and inter‐day precisions for ketoconazole and voriconazole from urine and human plasma with RSD ≤16.5% and good relative recoveries in the range 84.3–114.8%. The MCM‐41‐D‐μ‐SPE method proved to be rapid and simple and requires a small volume of organic solvent (200 μL); thus it is advantageous for routine drug analysis.     

Recent advances in microextraction by packed sorbent for bioanalysis

Microextraction by packed sorbent (MEPS) is a new format for solid-phase extraction (SPE) that has been miniaturized to work with sample volumes as small as 10 μL. The commercially available presentation of MEPS uses the same sorbents as conventional SPE columns and so is suitable for use with most existing methods by scaling the reagent and sample volumes. Unlike conventional SPE columns, the MEPS sorbent bed is integrated into a liquid handling syringe that allows for low void volume sample manipulations either manually or in combination with laboratory robotics. The key aspect of MEPS is that the solvent volume used for the elution of the analytes is of a suitable order of magnitude to be injected directly into GC or LC systems. This new technique is very promising because it is fast, simple and it requires very small volume of samples to produce comparable results to conventional SPE technique. Furthermore, this technique can be easily interfaced to LC/MS and GC/MS to provide a completely automated MEPS/LC/MS or MEPS/GC/MS system. This extraction technique (MEPS) could be of interest in clinical, forensic toxicology and environmental analysis areas. This review provides a short overview of recent applications of MEPS in clinical and pre-clinical studies for quantification of drugs and metabolites in blood, plasma and urine. The extraction of anti-cancer drugs, β-blockers drugs, local anaesthetics, neurotransmitters and antibiotics from biological samples using MEPS technique will be illustrated.     

Water-compatible molecularly imprinted polymers for efficient direct injection on-line solid-phase extraction of ropivacaine and bupivacaine from human plasma

Two novel molecularly imprinted polymers (MIPs) selected from a combinatorial library of bupivacaine imprinted polymers were used for selective on-line solid-phase extraction of bupivacaine and ropivacaine from human plasma. The MIPs were prepared using methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the cross-linking monomer and in addition hydroxyethylmethacrylate to render the polymer surface hydrophilic. The novel MIPs showed high selectivity for the analytes and required fewer and lower concentrations of additives to suppress non-specific adsorption compared with a conventional MIP. This enabled the development of an on-line system for direct extraction of buffered plasma. Selective extraction was achieved without the use of time-consuming solvent switch steps, and transfer of the analytes from the MIP column to the analytical column was carried out under aqueous conditions fully compatible with reversed-phase LC gradient separation of analyte and internal standard. The MIPs showed excellent aqueous compatibility and yielded extractions with acceptable recovery and high selectivity.     

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.     

Quantitation of sunitinib,an oral multitarget tyrosine kinase inhibitor,and its metabolite in urine samples by nonaqueous capillary electrophoresis time of flight mass spectrometry

A rapid, sensitive, and specific method was developed and validated using a nonaqueous‐capillary electrophoresis method with TOF‐MS for determination of sunitinib and N‐desethyl sunitinib in human urine. In order to avoid ionic suppression a urine samples dilution with methanol 1:10 previous step was used. This was the only treatment step to urine samples before the injection. Despite this dilution of the urine, the detection limit was as low as 0.07 mg/L for sunitinib and 0.15 mg/L for N‐desethyl sunitinib. Separation of compounds was achieved with a mixture of 5 mM ammonium formate in methanol. The calibration curves were linear over the range of 0.5–50.0 mg/L for the two analyzed compounds. The within‐run and between‐run precisions were within 5%, while the accuracy ranged from 96.0 to 100.4%. This method can be used in routine clinical practice to monitor sunitinib and N‐desethyl sunitinib drugs in the urine of cancer patients treated with once daily administration.     

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     

Microextraction in packed syringe (MEPS) for liquid and gas chromatographic applications. Part II--Determination of ropivacaine and its metabolites in human plasma samples using MEPS with liquid chromatography/tandem mass spectrometry

A new technique for sample preparation on-line with liquid chromatographic/tandem mass spectrometric (LC/MS/MS) assay was developed. Microextraction in a packed syringe (MEPS) is a new miniaturized, solid-phase extraction technique that can be connected on-line to gas or liquid chromatography without any modifications. In MEPS approximately 1 mg of the solid packing material is inserted into a syringe (100-250 microl) as a plug. Sample preparation takes place on the packed bed. The bed can be coated to provide selective and suitable sampling conditions. The new method is very promising, very easy to use, fully automated, of low cost and rapid in comparison with previously used methods. This paper presents the development and validation of a method for MEPS on-line with LC/MS/MS. Ropivacaine and its metabolites (PPX and 3-OH-ropivacaine) in human plasma samples were used as model substances. The method was validated and the calibration curves were evaluated by means of quadratic regression and weighted by the inverse of the concentration, 1/x, for the calibration range 2-2000 nM. The applied polymer could be used more than 100 times before the syringe was discarded. The extraction recovery was between 40 and 60%. The results showed high correlation coefficients (R(2) > 0.999) for all analytes in the calibration range studied. The accuracy, expressed as a percentage variation from the nominal concentration values, ranged from 0 to 6%. The precision, expressed as the relative standard deviation, at three different concentrations (quality control samples) was consistently about 2-10%. The limit of quantification was 2 nM.     

Quantitative determination of pravastatin and its metabolite 3α‐hydroxy pravastatin in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of a chromatography/tandem mass spectrometry method for the quantitative determination of pravastatin and its metabolite (3α‐hydroxy pravastatin) in plasma and urine of pregnant patients under treatment with pravastatin, as part of a clinical trial. The method includes a one‐step sample preparation by liquid–liquid extraction. The extraction recovery of the analytes ranged between 93.8 and 99.5% in plasma. The lower limits of quantitation of the analytes in plasma samples were 0.106 ng/mL for pravastatin and 0.105 ng/mL for 3α‐hydroxy pravastatin, while in urine samples they were 19.7 ng/mL for pravastatin and 2.00 ng/mL for 3α‐hydroxy pravastatin. The relative deviation of this method was <10% for intra‐ and interday assays in plasma and urine samples, and the accuracy ranged between 97.2 and 106% in plasma, and between 98.2 and 105% in urine. The method described in this report was successfully utilized for determining the pharmacokinetics of pravastatin in pregnant patients enrolled in a pilot clinical trial for prevention of preeclampsia. Copyright © 2015 John Wiley & Sons, Ltd.     

Validation and application of a liquid chromatography-tandem mass spectrometric method for the determination of GDC-0834 and its metabolite in human plasma using semi-automated 96-well protein precipitation

A liquid chromatographic–tandem mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of GDC‐0834 and its amide hydrolysis metabolite (M1) in human plasma to support clinical development. The method consisted of semi‐automated 96‐well protein precipitation extraction for sample preparation and LC‐MS/MS analysis in positive ion mode using TurboIonSpray® for analysis. D6‐GDC‐0834 and D6‐M1 metabolite were used as internal standards. A linear regression (weighted 1/concentration²) was used to fit calibration curves over the concentration range of 1 – 500 ng/mL for both GDC‐0834 and M1 metabolite. The accuracy (percentage bias) at the lower limit of quantitation (LLOQ) was 5.20 and 0.100% for GDC‐0834 and M1 metabolite, respectively. The precision (CV) for samples at the LLOQ was 3.13–8.84 and 5.20–8.93% for GDC‐0834 and M1 metabolite, respectively. For quality control samples at 3, 200 and 400 ng/mL, the between‐run CV was ≤7.38% for GDC‐0834 and ≤8.20% for M1 metabolite. Between run percentage bias ranged from ?2.76 to 6.98% for GDC‐0834 and from ?6.73 to 2.21% for M1 metabolite. GDC‐0834 and M1 metabolite were stable in human plasma for 31 days at ?20 and ?70°C. This method was successfully applied to support a GDC‐0834 human pharmacokinetic‐based study. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative determination of piritramide in human plasma and urine by off- and on-line solid-phase extraction liquid chromatography coupled to tandem mass spectrometry

A method for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) quantification of piritramide, a synthetic opioid, in plasma after conventional off-line solid-phase extraction (SPE) and in urine by on-line SPE-LC/MS/MS in positive electrospray mode was developed and validated. Applicability of the on-line approach for plasma samples was also tested. Deuterated piritramide served as internal standard. For the off-line SPE plasma method mixed cation-exchange SPE cartridges and a 150 x 2 mm C18 column with isocratic elution were used. For the on-line SPE method, a Waters Oasis HLB extraction column and the same C18 analytical column in a column-switching set-up with gradient elution were utilized. All assays were linear within a range of 0.5-100 ng/mL with a limit of detection of 0.05 ng/mL. The intra- and interday coefficients of variance ranged from 1.3 to 6.1% for plasma and 0.5 to 6.4% for urine, respectively. The extraction recovery for the off-line plasma assay was between 90.7 and 100.0%. Influence of matrix effects, and freeze/thaw and long-term stability were validated for both approaches; influence of urine pH additionally for quantification in urine.     

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.