Simple Liquid Chromatography-mass Spectrometry Method Research Articles

On-Line Solid Phase Extraction High Performance Liquid Chromatography Method Coupled With Tandem Mass Spectrometry for the Therapeutic Monitoring of Cannabidiol and 7-Hydroxy-cannabidiol in Human Serum and Saliva.

Cannabidiol is a novel antiseizure medication approved in Europe and the US for the treatment of seizures associated with Lennox-Gastaut syndrome, Dravet syndrome and tuberous sclerosis complex. We describe in this article a new and simple liquid chromatography-mass spectrometry method (LC-MS/MS) for the determination of cannabidiol and its active metabolite 7-hydroxy-cannabidiol in microvolumes of serum and saliva (50 μl), to be used as a tool for therapeutic drug monitoring (TDM) and pharmacokinetic studies. After on-line solid phase extraction cannabidiol, 7-hydroxy-cannabidiol and the internal standard cannabidiol-d3 are separated on a monolithic C18 column under gradient conditions. Calibration curves are linear within the validated concentration range (10–1,000 ng/ml for cannabidiol and 5–500 ng/ml for 7-hydroxy-cannabidiol). The method is accurate (intraday and interday accuracy within 94–112% for cannabidiol, 91–109% for 7-hydroxy-cannabidiol), precise (intraday and interday precision <11.6% for cannabidiol and <11.7% for 7- hydroxy-cannabidiol) and sensitive, with a LOQ of 2.5 ng/ml for cannabidiol and 5 ng/ml for 7-hydroxy-cannabidiol. The stability of the analytes was confirmed under different storage conditions. Extraction recoveries were in the range of 81–129% for cannabidiol and 100–113% for 7-hydroxy-cannabidiol. The applicability of the method to TDM was demonstrated by analysis of human serum and saliva samples obtained from patients with epilepsy treated with cannabidiol.

A simple and easy-to-perform liquid chromatography–mass spectrometry method for the quantification of tacrolimus and its metabolites in human whole blood. Application to the determination of metabolic ratios in kidney transplant patients

Tacrolimus is the cornerstone of immunosuppressive therapy in solid organ transplantation and its blood concentrations are routinely monitored. Tacrolimus is extensively metabolized into metabolites that are supposed to be nephrotoxic. Yet, few analytical methods have been described to simultaneously quantify tacrolimus and its main metabolites. We developed and validated a simple liquid chromatography-mass spectrometry method for the quantification of tacrolimus and its three desmethylated metabolites, 13-O, 15-O, and 31-O-desmethylated tacrolimus (M−I, M−III, and M−II respectively) in human whole blood. Protein precipitation of 50 µL of whole blood with 100 µL methanol and zinc sulfate was used as a single-extraction procedure. Tacrolimus and its metabolites were quantified using electrospray ionization-triple quadrupole mass spectrometry in combination with selected reaction monitoring detection in the positive ionization mode. The method was validated following FDA recommendations. This method was precise (intra- and inter-assay coefficients of variation: 2.88–7.81% and 3.96–12.10% for low and high levels of internal quality controls, respectively) and accurate (intra- and inter-assay biases: −1.67–10.30%, and −0.77–-9.36%, respectively).In adult kidney transplant patients who were treated with tacrolimus prolonged release formulation, the median (10th-90th percentiles) trough concentrations (n = 16) of tacrolimus, M−I, and M−III were 5.85 (3.37–7.09), 0.100 (0.037–0.168), 0.051 (0.03–0.104), respectively. M−II was measured in only 2 trough samples. The metabolic ratios M−I/tacrolimus and M−III/tacrolimus were 0.017 (0.009–0.027) and 0.009 (0.006–0.015) when measured on trough concentration and 0.022 (0.011–0.037) and 0.008 (0.006–0.015) when measured on area under the curves 0–24 h.This method is a suitable and easy-to-perform tool for future pharmacokinetic-pharmacodynamics studies investigating the importance of tacrolimus and its metabolites blood exposure for solid organ graft survival.

Concise synthesis of the major metabolite M8 from ticagrelor and simultaneous determination of ticagrelor and M8 by a novel LC/MS method

Ticagrelor is an oral antiplatelet agent that has been approved for preventing de novo and recurrent acute coronary syndrome. To date, only a few studies have attempted to clarify population differences in the pharmacokinetics and pharmacodynamics of ticagrelor between Caucasians and Asians. Our aim was to develop a simple quantification method for ticagrelor and its pharmacologically active metabolite M8 (AR-C124910XX) in human plasma and urine. First, we concisely synthesized M8 from ticagrelor via a five-step sequence: the hydroxyethyl group of ticagrelor was removed by bromination and subsequent zinc-mediated chemoselective reduction. Then, we developed a simple liquid chromatography–mass spectrometry method using ticlopidine as an internal standard. Ticagrelor, M8, and internal standard were separated with a reverse-phase C18 column, and ticagrelor and M8 were detected at m/ z [+] of 523.25 and 479.25, respectively, with good sensitivity and precision in the concentration ranges of 10 to 100 µM and 5 to 50 µM, respectively. This novel liquid chromatography–mass spectrometry system may be attractive to investigators in private laboratories because it is less costly than liquid chromatography/mass spectrometry/mass spectrometry systems. Our method is expected to accelerate further clinical studies on the disposition and pharmacodynamics of ticagrelor.

Development and Validation of a Simple LC-MS Method for the Quantification of Oxytocin in Dog Saliva.

Oxytocin (OT) is a mammalian neuropeptide with various functions in regulating birth, lactation, parenting, and social recognition. The study of OT became of increasing interest for the petcare industry due to its role in animal behavior and socialization. In the present study, a simple, sensitive, and accurate liquid chromatography-mass spectrometry (LC-MS) method for quantifying OT in dog saliva was developed and validated. OT and its deuterated internal standard (OT-d5) were detected with multiple reaction monitoring (MRM) in a positive ion mode using an AB Sciex 6500+ QTRAP mass spectrometer. Chromatographic separation was achieved by using an ACE Excel C18 column and a gradient elution at a flow rate of 0.5 mL/min over a 5 min run. The mobile phases consisted of 0.1% (v/v) acetic acid in water and 0.1% (v/v) acetic acid in acetonitrile. After development and optimization, the performance of the method was validated to prove its reliability. Calibration curves were linear over the range of 50–20,000 pg/mL and recovery of OT was above 87.8%. The validated method was successfully applied to evaluate OT concentrations in multiple batches of dog saliva samples.

Simple liquid chromatography-mass spectrometry method for quantification of major free oligosaccharides in bovine milk.

Free oligosaccharides (OS) are a significant functional component of milk that are difficult to quantitate. A simple method for quantitative analysis of the major free OS in bovine milk is described. Following a defatting step, protein elimination was performed by ultrafiltration. OS were separated by hydrophilic interaction liquid chromatography (HILIC) and detected by an Orbitrap mass analyzer in negative mode. The method is sensitive [with a limit of detection (LOD) for all representative OS of <0.1 ng] and reproducible, enabling simultaneous quantification of 13 major OS within a single run. Application of this method to the quantification of major OS in 32 milk samples collected after three different feeding treatments allowed us to reveal the relative abundance of different OS species, the variation of the OS content between individual cows, and the correlations between some of the major OS.

Study on pharmacokinetic and tissue distribution of lycorine in mice plasma and tissues by liquid chromatography–mass spectrometry

A fast and simple liquid chromatography-mass spectrometry method for the determination of lycorine in mice plasma and tissues was developed and well used in the pharmacokinetic and tissue distribution study of lycorine after tail vein injection and intraperitoneal administration. Biological samples were processed with ethyl acetate by liquid-liquid extraction, and evodiamine was used as the internal standard. Chromatographic separation was performed on an Amethyst C18 column (4.6 × 150 mm) with a mobile phase consisting of methanol and water. Quantification was performed by selected ion monitoring with m/z 288 [M+H](+) for lycorine and m/z 304 [M+H](+) for the internal standard. Good linearity was observed over the concentration ranges. Limits of quantification were low up to 10.0 ng/mL in plasma samples, 9.0 ng/g for lung, 12.0 ng/g for heart, 18.0 ng/g for spleen and 6.5 ng/g for other tested tissues. The intraday accuracy and precision in plasma and tissues ranged from -7.4% to 9.1%. Recoveries in plasma and tissue were more than 80%. The method was rapid, accurate and fully validated. It was successfully applied to the investigation of the pharmacokinetics and tissue distribution of lycorine in mice.

A New, Validated Wipe-Sampling Procedure Coupled to LC-MS Analysis for the Simultaneous Determination of 5-Fluorouracil, Doxorubicin and Cyclophosphamide in Surface Contamination

A wipe-sampling procedure followed by a simple liquid chromatography-mass spectrometry method was developed and validated for the simultaneous quantification of three cytotoxic drugs [5-fluorouracil (5FU), doxorubicin and cyclophosphamide (CP)] for the determination of surface contamination. After a solid-phase extraction procedure with wiping filter paper, the separation was performed within 30 min using a gradient mobile phase. The method was validated according to the recommendations of the US Food and Drug Administration. Wiping was performed using Whatman(®) filter paper on different surfaces such as stainless steel, polypropylene and glass. The method was linear, between 10 and 500 ng per wiping sample (i.e., 0.1-5 ng/cm(2)) for 5FU and doxorubicin, and between 1-100 ng per wiping sample (i.e., 0.01-1 ng/cm(2)) for CP. The lower limits of detection and quantification were 5 and 10 ng per wiping sample for 5FU and doxorubicin, and 0.5 and 1 ng per wiping sample for CP. This new sensitive methodology for surface contamination studies was successfully applied on commercial vials and different places in a cancer research hospital. This approach is particularly suitable to assess the risk of occupational exposure to cytotoxic drugs and to optimize the cleaning process, especially for the most toxic molecule studied, CP.

A New Rapid and Sensitive LC-MS Assay for the Determination of Sorafenib in Plasma: Application to a Patient Undergoing Hemodialysis

A simple liquid chromatography-mass spectrometry method was developed and validated for quantification of sorafenib (Nexavar) in human plasma. After a solid-phase extraction procedure, the separation was performed within 2 minutes using an isocratic flow of a mobile phase consisting of formic acid/acetonitrile applied on a C18 analytical column. The analyte was detected by mass spectrometry in the single-ion monitoring mode. The method was validated according to the recommendations of the US Food and Drug Administration. The method was linear (r² > 0.99) between 10 and 10,000 ng/mL. The lower limits of detection and quantification were 5 and 10 ng/mL, respectively. Within-day and between-day imprecisions were less than 10.4%, and inaccuracy did not exceed 8.7%. The mean extraction recovery was 92.2%. The method also provided satisfactory results in terms of time stability and dilution integrity. Sorafenib plasma concentrations of the studied patient ranged between 1831 and 3459 ng/mL. This new technique is rapid, sensitive, and was applied to the determination of sorafenib plasma concentrations in a patient undergoing hemodialysis. Our results indicate that sorafenib is not cleared from plasma by hemodialysis, although analysis should be delayed after dialysis to avoid erratic fluctuations.

Quantification of Tacrolimus in Human Skin Samples and Ointment by LC-MS

A sensitive and simple liquid chromatography-mass spectrometry method was developed to determine the immunosuppressant tacrolimus in human skin samples after treatment with the commercially available ointment. Utilizing diffusion cell experiments according to Franz, human skin samples were treated with ointment containing tacrolimus and the extraction procedure of the drug was optimized. The analytical assay was performed using an LC system consisting of a reversed phase C18 column and an isocratic mobile phase, coupled with electrospray ionization mass spectrometry. The detection was performed in the positive selected ion monitoring mode. Mycophenolate mofetil was used as internal standard to control the stability of the electrospray ionization. The calibration curve was linear for tacrolimus over the range of 5–1,000 ng mL−1 (average correlation coefficient of r 2 = 0.9941) with a limit of detection (LOD) of 2 ng mL−1, with a limit of quantification (LOQ) of 5 ng mL−1 and with a precision of 8.70%. The analytical assay described in this paper was successfully applied in order to quantify tacrolimus in human skin samples as well as in the commercially available ointment.