4000 qtrap lc ms ms system

The shoots of 21-day-old seedlings grown on Murashige and Skoog plates were collected. The extraction and detection of nucleotide sugars was performed according to Rautengarten et al. (2014) . LC-MS/MS analysis was performed using porous graphitic carbon as the stationary phase on a 1100 series HPLC system (Agilent Technologies) and a 4000 QTRAP LC-MS/MS system (Sciex) equipped with a TurbolonSpray ion source, as described previously (Rautengarten et al., 2014 ).

Plasma TMAO, choline, betaine, carnitine, and butyrobetaine were measured at the Cleveland Clinic using stored samples from baseline (1989-1990 or 1992-1993) and follow-up examination (1996-1997). Measurements were performed with stable-isotope dilution liquid chromatography with tandem mass spectrometry using high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry (4000 QTRAP LC-MS/MS System; SCIEX) and corresponding internal standard of d9(trimethyl)TMAO mass spectrometry (LCMS-8050 Triple Quadrupole Liquid Chromatograph Mass Spectrometer; Shimadzu Corporation).^{23 (link)} Concentrations of TMAO and d9(trimethyl)TMAO were monitored using electrospray ionization in positive ion mode with multiple reaction monitoring of parent ion transition m/z 76→58 and characteristic daughter ion transition m/z 85→66. Laboratory coefficients of variation varied by batch throughout the study period and by metabolite; the maximum coefficients of variation observed were 5.8% for TMAO, 4.1% for choline, 5.7% for carnitine, 7.0% for betaine, and 7.9% for butyrobetaine concentrations.

The shoots of 1-week-old seedlings grown under normal YNS hydroponic culture were used for measuring the content of UDP-Glc and UDP-Gal. The extraction method was referred to the report described previously (Behmüller et al. 2014 (link)). Briefly, samples about 40 mg was ground into fine powder in liquid nitrogen and extracted with 600 µl ice-cold chloroform–methanol (3:7, v/v) at below 20 °C for 2 h. After that, 400 µl ddH₂O was added to the sample and vortexed thoroughly, followed by centrifugation for 5 min at 20,000g under 4 °C. The upper clear phase was collected and repeated the above two steps for another two times. Then the upper clear phase mixed together and freeze-dried for 4 h. Finally, the dry matter was suspended in 200 µl ddH₂O and ready for detection. The UDP-Glc and UDP-Gal standards was purchased from Macklin Biochemical Technology Co. Ltd. (Shanghai, China). The nucleotide sugar detection was performed by liquid chromatography-tandem mass spectrometry (LC–MS/MS) using a 4000 QTRAP LC–MS/MS system (Sciex, CA) and an Agilent 1100 Series Capillary LC System (Agilent Technologies, USA), as described previously (Rautengarten et al. 2016 (link)).

Analysis was performed using an ACQUITY UPLC system with a conditioned autosampler at 4 °C. Chromatographic separation was carried out at 35 °C on an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm). The mobile phase was composed of A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) with a gradient elution: 0–5 min, 87–84% (A); 5–7 min, 84–80% (A); 7–15 min, 80–78% (A) and 15–23 min, 78–70% (A). The flow rate of the mobile phase was 0.2 mL/min, and the injection volume was 2 µL.
The mass spectrometry was performed on a 4000 QTRAP LC-MS/MS system (AB Sciex, Foster City, CA, USA) equipped with ESI interface in negative mode. All instruments were controlled and synchronized by Analyst software (version 1.6, SCIEX, AB Sciex, Foster City, CA, USA). Ion spray voltage was set at (3300) V, turbo spray temperature was 550 °C and interface heater was on. Both nebulizer gas (gas 1) and heater gas (gas 2) were set at 55 psi.

Cecal digesta from Exp 2 and cecal and jejunum digesta from Exp 3 were analyzed for CPT-11 and its metabolites. The digesta were extracted 3 times with 67% methanol overnight and solids were removed by centrifugation. The supernatant was then diluted 6 times with water. Metabolite concentration was quantified using a 4000 QTRAP® LC/MS/MS System (AB Sciex, Canada) following a protocol by Corona et al. [21] (link) with slight modifications. A Kinetex™ 2.6 µm C18 100×3 mm column (Phenomenex, Canada) was used. Assays were performed using LC-MS/MS under ESI-MRM mode. The flow rate was 0.4 mL/min. The acetonitrile gradient increased from 10% to 80 % from 0 to 18 min, maintained at 100% from 18.1 to 19.0 min, and returned to 10% from 19.4 to 25 min. MRM transitions used were: m/z 587→167 for CPT-11, m/z 393→349 for SN-38 (7-ethyl-10-hydroxy-camptothecin), m/z 619→393 for APC (7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin), m/z 519→393 for NPC (7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecine), and m/z 569→393 for SN-38-glucuronide (SN-38G). Quantification was done with standards prepared from pure CPT-11, APC, NPC, SN-38, SN-38G purchased from Tocris Bioscience, UK.

The 4000QTRAP LC-MS/MS system (AB Sciex) was used in multiple-reaction monitoring (MRM) mode using negative ionization. The detailed instrument configuration and compound-dependent parameters for isoprenoid precursors were as previously described^{13 (link)}. LC separation prior to MRM detection was achieved by ion pair reverse-phase chromatography as described previously^{63 (link)}, with 10 mM tributylammonium acetate (pH 5.1-5.5) used as the ion pair reagent and the following modifications: (1) RP-hydro 100 mm × 2.0 mm, 2.5 μm high performance liquid chromatography column (Phenomenex), (2) flow rate of 0.14 mL/min, (3) solvent A of 10 mM tributylammonium acetate in 5% methanol, (4) binary LC gradient (20% solvent B (100% methanol) from 0 to 2.5 min, 30% B for 12.5 min, 80% B for 5 min, and column equilibration for 5 min), and (5) autosampler injection volume of 20 μL. For deoxyxylulose 5-phosphate (DOXP) and methylerythritol cyclodiphosphate (MEcPP) metabolites, one-way ANOVA was used to test for significance (VassarStats). A t-test was used to test for significance between UNT and 1x RCB-185 cytidine diphosphate methylerythritol (CDP-ME) levels (VassarStats). A significance test was not performed for MEP, as its levels were below the limit of detection (12.5 ng/mL) for RCB-185-treated parasites.

Analysis was performed using an Acquity UPLC system with a conditioned autosampler at 4 °C. Chromatographic separation was carried out at 35 °C on an Acquity UPLC HSS T3 column (1.8 um, 2.1 mm × 100 mm). The mobile phase was composed of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) with a gradient elution: 0–4 min, 75–15% (A); 4–8 min, 15–12% (A); 8–10 min, 12–0% (A) and 10–12 min, 0–70% (A), until the end of the run at 12.0 min. The injection volume was 2 μL.
The mass spectrometry was performed on a 4000 QTRAP LC-MS/MS system (AB SCIEX) equipped with an ESI interface in negative mode. All instruments were controlled and synchronized by Analyst software (version 1.6, SCIEX). The ion spray voltage was set at 3300) V, turbo spray temperature was 550 °C, and the interface heater was on. Both nebulizer gas (gas 1) and heater gas (gas 2) were set at 55 psi.