Api5000 triple quadrupole mass spectrometer

LC-MS/MS was performed using an API 5000 triple quadrupole mass spectrometer (Sciex), coupled to an Agilent 1200 HPLC system (Agilent Technologies). Samples (20 μL) were injected using a CTC PAL Autosampler set at 4°C (CTC Analytics AG, Switzerland). Analytes were resolved through chromatographic separation using a 4-μm Synergi Hydro-RP C18 column (150×2 mm; Phenomenex, Torrance, CA), with column chamber set at 40°C, over a binary gradient with a flow rate of 0.5 mL/min. HPLC gradient conditions were as follows: 0 min, 25:75 A/B; 2.5 min, 20:80 A/B; 7.5 min, 10:90 A/B; 8 min, 0:100 A/B; 10 min, 25:75 A/B; 18 min, 25:75 A/B. Solvent A: 0.1% formic acid in water; Solvent B: methanol. Total run time was 18 min.
LC-MS/MS was performed in positive ion mode, [M + H]⁺, with quantifier and qualifier ion transitions selected for each analyte, at a dwell time of 50 ms. Source parameters were set as follows: positive ion spray voltage, 5000 V; ion source temperature, 500°C; collision gas, 7 psi; curtain gas, 35 psi; nebulizer gas, 25 psi; turbo gas, 45 psi. Transitions were optimized using direct infusion (10 μL/min) with each standard (100 ng/mL). MS/MS parameters are summarized in Table 1. Data were acquired and processed using Analyst^® (Sciex), version 1.6.2.

The samples were prepared, and targeted quantitative metabolomics was measured by LC/MS/MS as reported previously^{15 (link)} or with minor modifications. Briefly, methanol was added to plasma or urine and kidney homogenate for deproteinization and extraction. Each mixture was vortexed and centrifuged. The supernatant was diluted with water. Analyte separation and quantification were performed using a Shimadzu Nexera UHPLC system (Shimadzu, Kyoto, Japan) coupled with an API5000 triple quadrupole mass spectrometer (SCIEX, Framingham, MA, USA) with electrospray ionization operated in positive ion mode. The separation was performed on a Triart C18 column (3.0 × 150 mm, 5 μm, YMC, Kyoto, Japan). Quantification was performed by multiple reaction monitoring. NAD + , NADH, NADP, and NADPH in kidney tissue were measured using the NAD + /NADH Assay Kit (E2ND-100, EnzyChrom) and the NADP + /NADPH Assay Kit (ECNP-100, EnzyChrom), respectively.

Three glucuronide metabolites of labetalol have been detected (Martin et al., 1976 (link); Niemeijer et al., 1991 (link)). Glucuronidation at the phenolic-OH (Gluc-1) by UGT1A1 and at the benzylic-OH (Gluc-2) by UGT2B7 have been previously reported (Jeong et al., 2008 (link)); however, it has not been reported which UGT isoform catalyzes the N-glucuronidation of labetalol (Gluc-3).
Media samples, cell lysate or recombinant reaction mixture (30 µL) were extracted by protein precipitation with methanol (150 µL) containing the stable, isotopically labeled internal standard labetalol-d₃. Samples were vortexed and centrifuged, and 20 µL of the supernatant was mixed with 80 µL of water prior to LC-MS/MS analysis. Chromatographic separation of the three glucuronide metabolites was achieved on a Waters Atlantis T3 (50 × 2.1 mm, 3 µm particle size) analytical column with 0.1% formic acid in water and acetonitrile under gradient conditions. Analytes and internal standards were detected on SCIEX API 5000 triple quadrupole mass spectrometer using TurboIonSpray in the positive ionization mode. Due to the unavailability of analytical standards for labetalol glucuronides, the levels of the three glucuronides were assessed by the peak areas of each labetalol glucuronide (Gluc-1, Gluc-2, and Gluc-3) normalized to the peak area of internal analytical standard.

All samples of tissue homogenate, plasma, cell lysate, and cell culture medium were precipitated with acetonitrile (containing IS, buspirone, 5 ng/mL) followed by quantitation against the corresponding standard curve prepared in the blank biomatrix. The concentration of TET was determined by an LC-MS/MS method with an API 5000 Triple quadrupole mass spectrometer (AB Sciex, Foster City, CA, USA) connected to a Shimadzu LC-20AD HPLC system (Shimadzu, Japan). The chromatographic column was the C18 column (3.0 mm × 50 mm, 2.6 µm, Phenomenex). The mobile phase consisted of water containing 0.1% of formic acid (A) and acetonitrile containing 0.1% of formic acid (B). Separation was achieved following a binary gradient elution procedure: 0–0.3 min B 10%, 0.3–1.4 min B 10%→90%, 1.4–1.8 min B 90%, 1.8–1.9 min B 90%→10%, 1.9–3.0 min B 10%. The volume of each injection was 5.0 µL, and the flow rate was 0.6 mL/min.
TET and IS were detected by multiple reaction monitoring (MRM) in the positive ion mode. The precursor and product ions used for quantification were as follows: m/z 609.0→381.1 for TET and m/z 386.4→122.1 for IS, respectively.

ST-246 concentrations in rat, dog, and monkey plasma were determined using the liquid chromatography–tandem mass spectrometry (LC-MS/MS) method. A total of 125 µL acetonitrile containing IS (20 ng/mL) was added into 25 µL plasma to precipitate plasma protein. After high-speed centrifugation, 20 µL of supernatant was added to 180 µL of 50% acetonitrile solution and injected onto LC-MS/MS. The chromatographic separation was performed using a C18 column (3.0 mm × 50 mm, 2.6 µm, Phenomenex) with a security guard column. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). Separation was achieved with a 3-min run time with the following gradient program: initial conditions start with an increase from 30 to 90% B over 1.2 min, hold at 90% B for 0.5 min, return to 30% B over 0.1 min, and hold at 30% B for 1.2 min. The constant flow rate is 0.6 mL/min. An AB Sciex API 5000 Triple quadrupole mass spectrometer was tuned to the multiple reaction monitoring (MRM) mode to monitor the m/z transitions, 377/173 for ST-246 and 381/177 for the IS, in the positive ion mode.