2.3. Isotope dilution liquid chromatography-tandem mass spectrometry

The prepared samples were analyzed for target isoprostanes using UPLC (Agilent 1290 Infinity II LC System) coupled with Triple Quadrupole mass spectrometer (Agilent 6460) (Santa Clara, CA). The gradient flow of HPLC grade methanol and 0.1% aqueous solution of formic acid (Table S1) were used to separate analytes using an ACQUITY UPLC® Oligonucleotide BEH C18 column (150 mm × 2.1 mm i.d. × 1.7 μm particle size). Target analyte’s peak identification was based on the relative retention time (±0.05 min) to their standard solution, two multiple reaction monitoring (MRM) transitions (Table 1 ) in the negative mode of ionization, and the ratio of the abundance of quantitative to qualitative ions (±20%). The gas temperature, sheath gas temperature, and capillary voltage were optimized to 350 °C, 300 °C, and 4000 V, respectively.

Select figure of merits for the developed analytical method.

The isotopic dilution mass spectrometry method was applied where a known quantity of deuterated isotopes of each target analyte (internal standard) is spiked directly into the sample prior to extraction and analytes were quantified based on the relative response factors of isotopic-labeled internal standard and the corresponding analyte. This method allows for accurate quantification by correcting for the loss of analytes during the sample preparation and instrumental analysis process. However, 2,3-dinor-iPF2α-III was quantified using the internal standard for PGE2, due to lack of commercial availability of its deuterated standard. The seven-to-ten-point calibration curves (10.0 ng/L to 10,000 ng/L) of each target analyte were prepared by plotting the concentration-dependent response factor against the response-dependent concentration factor. The linear or quadratic (PGE2) regression coefficients (r ²) determined using Agilent MassHunter Workstation for the Quantitative Analysis were 0.999 for all analytes.