LC-MS/MS method for simultaneous analysis of uracil, 5,6-dihydrouracil, 5-fluorouracil and 5-fluoro-5,6-dihydrouracil in human plasma for therapeutic drug monitoring and toxicity prediction in cancer patients

The chemotherapeutic drug 5-fluorouracil (5-FU) is widely used for treating solid tumors. Response to 5-FU treatment is variable with 10-30% of patients experiencing serious toxicity partly explained by reduced activity of dihydropyrimidine dehydrogenase (DPD). DPD converts endogenous uracil (U) into 5,6-dihydrouracil (UH(2) ), and analogously, 5-FU into 5-fluoro-5,6-dihydrouracil (5-FUH(2) ). Combined quantification of U and UH(2) with 5-FU and 5-FUH(2) may provide a pre-therapeutic assessment of DPD activity and further guide drug dosing during therapy. Here, we report the development of a liquid chromatography-tandem mass spectrometry assay for simultaneous quantification of U, UH(2) , 5-FU and 5-FUH(2) in human plasma. Samples were prepared by liquid-liquid extraction with 10:1 ethyl acetate-2-propanol (v/v). The evaporated samples were reconstituted in 0.1% formic acid and 10 μL aliquots were injected into the HPLC system. Analyte separation was achieved on an Atlantis dC(18) column with a mobile phase consisting of 1.0 mm ammonium acetate, 0.5 mm formic acid and 3.3% methanol. Positively ionized analytes were detected by multiple reaction monitoring. The analytical response was linear in the range 0.01-10 μm for U, 0.1-10 μm for UH(2) , 0.1-75 μm for 5-FU and 0.75-75 μm for 5-FUH(2) , covering the expected concentration ranges in plasma. The method was validated following the FDA guidelines and applied to clinical samples obtained from ten 5-FU-treated colorectal cancer patients. The present method merges the analysis of 5-FU pharmacokinetics and DPD activity into a single assay representing a valuable tool to improve the efficacy and safety of 5-FU-based chemotherapy.

A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma.

Free arachidonic acid is functionally interlinked with different lipid signaling networks including those involving prostanoid pathways, the endocannabinoid system, N-acylethanolamines, as well as steroids. A sensitive and specific LC-MS/MS method for the quantification of arachidonic acid, prostaglandin E2, thromboxane B2, anandamide, 2-arachidonoylglycerol, noladin ether, lineoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, steroyl ethanolamide, aldosterone, cortisol, dehydroepiandrosterone, progesterone, and testosterone in human plasma was developed and validated. Analytes were extracted using acetonitrile precipitation followed by solid phase extraction. Separations were performed by UFLC using a C18 column and analyzed on a triple quadrupole MS with electron spray ionization. Analytes were run first in negative mode and, subsequently, in positive mode in two independent LC-MS/MS runs. For each analyte, two MRM transitions were collected in order to confirm identity. All analytes showed good linearity over the investigated concentration range (r>0.98). Validated LLOQs ranged from 0.1 to 190ng/mL and LODs ranged from 0.04 to 12.3ng/mL. Our data show that this LC-MS/MS method is suitable for the quantification of a diverse set of bioactive lipids in plasma from human donors (n=32). The determined plasma levels are in agreement with the literature, thus providing a versatile method to explore pathophysiological processes in which changes of these lipids are implicated.

Development of a rapid column-switching LC-MS/MS method for the quantification of THCCOOH and THCCOOH-glucuronide in whole blood for assessing cannabis consumption frequency

The concentration of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH) in whole blood is used as a parameter for assessing the consumption behavior of cannabis consumers. The blood level of THCCOOH-glucuronide might provide additional information about the frequency of cannabis use. To verify this assumption, a column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and direct quantification of free and glucuronidated THCCOOH in human whole blood was newly developed. The method comprised protein precipitation, followed by injection of the processed sample onto a trapping column and subsequent gradient elution to an analytical column for separation and detection. The total LC run time was 4.5 min. Detection of the analytes was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was fully validated by evaluating the following parameters: linearity, lower limit of quantification, accuracy and imprecision, selectivity, extraction efficiency, matrix effect, carry-over, dilution integrity, analyte stability, and re-injection reproducibility. All acceptance criteria were analyzed and the predefined criteria met. Linearity ranged from 5.0 to 500 μg/L for both analytes. The method was successfully applied to whole blood samples from a large collective of cannabis consumers, demonstrating its applicability in the forensic field.