Uplc ms ms system

The 25(OH)D₂ and 25(OH)D₃ levels in the serum were measured using a modified LC‐MS/MS method. Briefly, 100 μl aliquots of calibrators or serum samples were spiked with 500 μl isotope‐labeled internal standard solution. Samples were vortexed for 1 min and centrifuged for 5 min at 9,250 g and 4°C. After centrifugation, 400 μl of sample supernatant was transferred into a 96‐deep well plate and then dried under nitrogen at 60°C. Using 100 μl acetonitrile solution of PTAD, we performed derivatization for 60 min and then added 50 μl methanol for reconstitution. Subsequently, the residuals were injected into the UPLC‐MS/MS system (Waterscorporation). Chromatographic separation on the Waters ACQUITY UPLC I‐class was performed under binary gradient conditions with a mobile phase A of water with 0.1% formic acid (volume fraction) and 5 mM ammonium formate and mobile phase B of methanol with 0.1% formic acid. The column temperature was maintained at 40°C. Xevo TQD was used for MS/MS detection in positive electrospray ionization mode and multiple reaction monitor (MRM) mode. The accuracy of this method was validated by analyzing the National Institute of Standards and Technology SRM 972a. Comparing the measurement results with the reference values of SRM 972a, the accuracy of this method was 90%–115% and 95%–110% for 25(OH)D₂ and 25(OH)D₃, respectively.

DEHP/MEHP concentrations were measured in serum samples form the Bio-Bank of the Medical Research Department, E-DA Hospital. Before analysis, serum samples were thawed at 4°C for 24 h (Supplementary Table 2). Serum (100 μL) was added to 200 μL of MeOH containing 75 ng/mL of DEHP-d₄ and ¹³C₄-MEHP, placed in a 1.5-mL microcentrifuge tube, and vortexed vigorously for 5 min to precipitate proteins. The mixture was then centrifuged at 15,000 × g at 4°C for 10 min, and the supernatant was transferred to a 500 μL sample vial for DEHP and MEHP analysis.
We used a Waters UPLC-MS/MS system to separate and detect DEHP and MEHP. An ACQUITY UPLC Isolator (2.1 × 50 mm) was applied for elimination of background phthalates, and an ACQUITY UPLC^® BEH C18 column (2.1 mm × 50 mm, 1.7 µm) was used for separation. Five μL of prepared sample were injected for analysis at a flow-rate of 0.5 mL/min. The mobile phases were MeOH (mobile phase A) and 5 mM of NH4Ac in Milli-Q water (mobile phase B). The initial condition (0 min) was 20% mobile phase A and 80% mobile phase B, with a gradient from 0.5–2.5 min bringing mobile phase A to 90% and mobile phase B to 10%. The column and sample tray temperatures were 45°C and 4°C, respectively.

The plasma concentration of F-VCR and T-VCR and the urine VCR concentration was determined using the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system (Waters Corporation, Milford, MA, United States) with vinblastine sulfate used as the internal standard (IS) as previously described by our team (Yang et al., 2013 (link), 2015 (link)). The compounds were detected as doubly charged ions and the multiple reaction monitor (MRM) transitions of VCR and the IS were m/z 413.2 → 353.2 and m/z 406.2 → 271.6, respectively. F-VCR was separated from liposomal form in plasma using SPE method; plasma T-VCR and urine VCR were extracted using liquid-liquid extraction method. F-VCR and T-VCR were identified and quantified over a theoretical concentration range of 0.2–50 ng/ml for F-VCR in plasma, 0.5–400 ng/ml for T-VCR in plasma and 0.5–100 ng/ml for VCR in urine, respectively. Assay specificity was assessed using blank sample from six different lots to verify the absence of interference at retention time. Quantitation was made using peak area ratios of analyte/IS, and back-calculated concentrations were determined using a weighted (1/ × 2) linear regression (y = ax + b).

Kyn and Trp were separated by ultra-performance liquid chromatography and measured by tandem mass spectrometry (UPLC-MS/MS) system (Waters, Milford, MA, USA). The selected plasma samples were shipped at or below -70 °C to the Amsterdam University Medical Centers for analysis. Upon analyses, samples were thawed rapidly at room temperature and a mixture of stable isotope-labeled internal standards was added to 50 μl of plasma. The samples were deproteinized using acetonitrile, dried under nitrogen, and reconstituted in 100 μl of 0.1% heptafluorobutyric acid. Aliquots (10 μl) of the extracts were injected into the UPLC–MS/MS system comprising of Acquity Xevo TQ-XS system operated in positive ESI mode using multiple reaction monitoring (MRM) for preselected analytes and an overall run time was 6 min. The MRM transition which gave the most intense signal was chosen for quantification. IDO activity was calculated as the ratio of measured kynurenine concentration to measured tryptophan concentration (K/T ratio). In order to capture all metabolites in the Trp and Kyn pathways, we measured other metabolites including anthranilic acid, 3OH-kynurenine, 3OH-anthranilic acid, quinolinic acid, kynurenic acid and xanthurenic acid (Supplementary Fig. 1). We also calculated the ratio of the sum of all determined kynurenine metabolites to Trp concentration (Sum/T ratio).

MGO and 3-DG were determined, which were derivatized with OPD. To perform derivatization, 200 µL of the extract was mixed with 100 µL of 0.1 mM 2,3-hexanedione internal standard and 200 µL of 5 mM OPD. Derivation was performed at 4 °C for 12 h. The quinoxaline derivatives of 3-DG and MGO were determined by using the Waters UPLC-MS/MS system (Waters, Milford, MA, USA). The separation was conducted on a C18 column (2.1 × 100 mm, 3.5 μm; Waters X-Bridge) with a flow rate of 0.3 mL/min at 35 °C and with 5 µL of the samples. The mobile phase was (A) acetonitrile and (B) 1% formic acid. The gradient elution condition was as follows: A: 10%, 0–0.1 min; 20%,1 min; 70%, 6 min; 100%, 6.3–7.5 min; and 10%, 8–10 min. Positive ESI ionization and MRM scan modes were used. The parent ion and daughter ion were as follows: m/z 145 → 77 for MGO (cone, 35 v; collision, 25 v), m/z 235 → 199 for 3-DG (cone, 30 v; collision, 12 v), and m/z 187 → 77 for 2,3-hexanedione (cone, 32 v; collision, 35 v).