Fasted, resting morning venous blood samples were collected at baseline and postintervention from each participant's antecubital vein at local pathology clinics around Melbourne, and sent to a central NATA-accredited pathology laboratory for processing. Total serum cholesterol, HDL cholesterol, and triglycerides were assessed using an enzymatic, colorimetric method (Roche Cobas c701 analyzer). LDL cholesterol was calculated by using the Friedewald formula. Serum aliquots were stored at −70°C for the assessment of serum 25(OH)D by a validated LC/MS/MS method, using a Shimadzu Nexeria ultra performance LC system for separation, and a Sciex Triple Quad 5500 system for detection, at Monash Health Pathology. All samples were assessed in the same batch (in duplicate) at the completion of the study. The calibrators for the LC/MS/MS 25(OH)D method are traceable to the National Institute of Standards and Technology Standard Reference material 972a. The intra-assay CV was 5.7% at 32.3 nmol/L, 3.9% at 60.6 nmol/L, and 2.7% at 101.8 nmol/L, and the interassay CV was 7.8% at 41.1 nmol/L and 7.0% at 113 nmol/L.
Triple quad 5500 system
Manufactured by AB Sciex
Sourced in United States
The Triple Quad 5500 System is a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument designed for analytical laboratories. It provides accurate and sensitive quantitative analysis of a wide range of analytes. The system combines a triple quadrupole mass analyzer with a variety of ionization sources and chromatographic options to enable versatile and reliable quantitative analysis.
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Lab products found in correlation
Cobas c701 analyzer, by Roche (1 mentions)
Prominence uflc, by Shimadzu (1 mentions)
Advia centaur cp immunoassay system, by Siemens (1 mentions)
Cobas 8000, by Roche (1 mentions)
Xn 9000, by Sysmex (1 mentions)
Analyst software, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation buffer, by Nacalai Tesque (1 mentions)
Inertsustain c18 column, by GL Sciences (1 mentions)
Agilent 7890b gas chromatograph, by Agilent Technologies (1 mentions)
Pegasus ht mass spectrometer, by Leco (1 mentions)
Nexera xr uplc system, by Shimadzu (1 mentions)
Prominence uflc xr system, by Shimadzu (1 mentions)
Elisa kit, by ALPCO (1 mentions)
Xt 2000iv, by Sysmex (1 mentions)
Tba 120fr, by Toshiba (1 mentions)
Acquity uplc, by AB Sciex (1 mentions)
Aphera nh2 polymer, by Merck Group (1 mentions)
Nexera uplc system, by Shimadzu (1 mentions)
11 protocols using triple quad 5500 system
1
Serum Lipid and Vitamin D Assessment
2
Serum Estradiol Measurement by LC-MS/MS
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Serum estradiol levels were measured at the Brigham Research Assay Core Laboratory using LC-MS/MS on the Triple Quad 5500+ system (SCIEX).12 (link),13 (link) Further details are provided in the eMethods in Supplement 2 .
3
Pregabalin Pharmacokinetic Study Design
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For Cohort A (target n = 8), the schedule was designed as an open, one-sequence, two-period crossover study to obtain pregabalin PK information for both formulations. Subjects were administered a single dose of Y75T (Period 1) or Y150T (Period 2). A four-day washout interval was established between the two periods. The single-sequence design could be justified because the objective was not to compare the results from both periods but to generate information to compare the results from Cohort B. In Cohort B (target n = 8), subjects received Y75T, but 150 mg pregabalin was not administered to minimize safety risk due to increased exposure. To measure the pregabalin concentration, blood samples were collected pre-dose (0 hours) and 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 24, 48, and 72 hours post-dose, regardless of the cohort and period. The subjects were hospitalized during PK sampling, provided with a standard meal 30 min before dosing, and completed their meal within 20 min. Blood samples were handled and analyzed using validated liquid chromatography (Prominence UFLC, Shimadzu, Kyoto, Japan)-tandem mass spectrometry (Triple Quad 5500+ system, SCIEX, Framingham, MA, USA.), using the method by Mandal et al. [15 ]. The lower limit of quantification was set at 20 ng/mL.
4
Plasma Biomarkers for B12 Assessment
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Plasma was analyzed for the B12 biomarkers MMA, B12, and holoTC concentrations at the Department of Clinical Biochemistry at Aarhus University Hospital, Denmark. All samples from each participant were analyzed in one run. MMA was quantified by Liquid Chromatography–Tandem Mass Spectrometry on the AB SCIEX Triple Quad 5500 System (AB SCIEX). Plasma B12 was measured on the Advia Centaur CP Immunoassay System (Siemens) after a 1:2 dilution in 0.9% NaCl. HoloTC was determined by an in-house sandwich ELISA after removal of unsaturated transcobalamin with B12-coated magnetic beads [16 (link)]. Out of the cohort, some of the plasma samples contained a limited volume not sufficient for measurement of all three biomarkers. In these cases, we prioritized the analysis of the biomarkers in the following order: MMA, B12, and holoTC. The number of samples measured are indicated in figures, tables, and text, whenever relevant.
Hemoglobin, mean red blood cell volume (MCV), and creatinine were measured at the Department of Clinical Biochemistry at Bispebjerg Hospital, Denmark. Hemoglobin and MCV were determined on the Sysmex XN 9000 (Sysmex). Creatinine was determined on the Cobas 8000 (Roche).
Hemoglobin, mean red blood cell volume (MCV), and creatinine were measured at the Department of Clinical Biochemistry at Bispebjerg Hospital, Denmark. Hemoglobin and MCV were determined on the Sysmex XN 9000 (Sysmex). Creatinine was determined on the Cobas 8000 (Roche).
5
Mass Spectrometric Analysis of Dansylated Metabolites
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After
derivation, all samples were analyzed by a positive ion electrospray
tandem mass spectrometric method as previously described.32 (link) Briefly, LC-MS/MS was performed by using an
Agilent 1200 series nano flow LC system (Agilent Technologies, Aanta
Clara, CA) coupled to an AB SCIEX Triple Quad 5500 System (AB SCIEX,
Framingham, MA). The liquid chromatography separation was carried
out with a 100 mm × 3 mm i.d. Waters BEH C-18 column packed with
1.7 μm particles (Waters, Milford, MA) and maintained at 40
°C. Multiple reactions monitoring transitions were selected as
follows: 534.4–171.2 for the detection of dansylated MeOE1 and 538.4–171.2 for dansylated MeOE1-d4.
Quantitation was performed using the Analyst software (Applied Biosystems,
Forster City, CA), and data were normalized to the E2 control
treatment in each independent experiment.
derivation, all samples were analyzed by a positive ion electrospray
tandem mass spectrometric method as previously described.32 (link) Briefly, LC-MS/MS was performed by using an
Agilent 1200 series nano flow LC system (Agilent Technologies, Aanta
Clara, CA) coupled to an AB SCIEX Triple Quad 5500 System (AB SCIEX,
Framingham, MA). The liquid chromatography separation was carried
out with a 100 mm × 3 mm i.d. Waters BEH C-18 column packed with
1.7 μm particles (Waters, Milford, MA) and maintained at 40
°C. Multiple reactions monitoring transitions were selected as
follows: 534.4–171.2 for the detection of dansylated MeOE1 and 538.4–171.2 for dansylated MeOE1-d4.
Quantitation was performed using the Analyst software (Applied Biosystems,
Forster City, CA), and data were normalized to the E2 control
treatment in each independent experiment.
6
Quantifying Thyroid Hormones in Mice
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Organ-specific T4, T3, and TRIAC contents were measured using LC-MS/MS.18 (link) We mechanically homogenized 50 mg of either the liver or cerebrum of a mouse in 300 μL radioimmunoprecipitation buffer (Nacalai Tesque) and left the homogenate on ice for 30 min. Supernatants were centrifuged at 10,000 g for 10 min at 4°C and collected in microtubes. We added 300 μL methanol and 600 μL chloroform, mixed with vortex mixer, and centrifuged at 15,000 g for 2 min at 4°C. The upper water/methanol phase was collected in new microtubes. We injected 6 μL of each sample into a Triple Quad 5500+ system and QTRAP Ready (AB SCIEX). Chromatography was performed using InertSustain C18 column (2.1 × 150 mm, 5 μm; GL Sciences, Tokyo, Japan) maintained at 40°C. A gradient of mobile phase A (0.5 mM ammonium fluoride in water) and mobile phase B (methanol) was used. The general conditions were as follows: 40% methanol (0–1 min), 40–90% methanol linear gradient (1–10 min), 90% linear gradient (10–15 min); flow rate of 0.2 mL/min. The following MS settings were adopted: curtain and collision gas pressure of 40 and 8 psi, respectively; ion spray voltage of −4500 V; temperature of 500°C; and ion source gas 1 and 2 pressure of 80 and 70 psi; correspondingly. T4, T3, and TRIAC were all measured with ESI in negative mode by MRM. The limits of quantification of T4, T3, and TRIAC were all 0.01 ng/mL.
7
Comprehensive Metabolomics Analysis by HILIC-LC-MS/MS and GC-MS
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HILIC-LC-MS/MS analyses were performed using the NEXERA XR UPLC system (Shimadzu, Columbia, MD, USA), coupled with the Triple Quad 5500 System (AB Sciex, Framingham, MA, USA) as described in Tolstikov et al., 2014 [30 (link)]. GC-MS analyses were performed as described in Tolstikov et al., 2014 [30 (link)] using an Agilent 7890B gas chromatograph (Agilent, Palo Alto, CA, USA) interfaced to a time-of-flight Pegasus HT mass spectrometer (Leco, St. Joseph, MI, USA). Automated injections were performed using an MPS2 programmable robotic multipurpose sampler (Gerstel, Muhlheim an der Ruhr, Germany). The GC system was fitted with a Gerstel temperature-programmed injector, cooled injection system (model CIS 4). An automated liner exchange (ALEX) (Gerstel, Muhlheim an der Ruhr, Germany) was used to eliminate cross-contamination from the sample matrix that was occurring between sample runs.
8
Salivary CoQ10 Levels: LC-MS/MS Analysis
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Resting saliva (1–1.5 ml) was collected from each subject into a 2-ml sample tube before and after the study intervention, and the samples were stored at −80°C until analysis. The total salivary CoQ10 level was measured by Kaneka Techno Research Co., Ltd. using liquid chromatography with tandem mass spectrometry (LC/MS/MS) [20 ]. In brief, 0.7 mL of the extraction solvent (isopropanol containing 2 mg/mL of 1, 4-benzoquinone and 20 ng/mL of CoQ9) was added to 0.1 mL of saliva and mixed. After centrifugation, the supernatant was filtered through a membrane filter and used as the sample for LC/MS/MS, which was performed using an AB Sciex Triple Quad 5500 system (AB Sciex, Framingham, MA, USA).
9
Comprehensive Iron Biomarker Analysis
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Hematological indices were measured by an automated hematology analyzer (XT-2000iV; Sysmex, Hyogo, Japan). Serum iron levels as well as unsaturated iron binding capacity (UIBC) and total iron binding capacity (TIBC) were measured using an automatic biochemistry analyzer (TBA-120FR, Toshiba Medical Systems, Tochigi, Japan). Serum hepcidin level was measured by liquid chromatography/electrospray ionization tandem mass spectrometry using an AB Sciex Triple Quad 5500 system (AB Sciex, Foster City, CA, USA) equipped with a Prominence UFLCXR system (Shimadzu Corporation, Kyoto, Japan) as reported previously [11 (link)]. Serum ferritin levels were determined by ELISA kit (ALPCO, Salem, NH, USA).
10
Enzymatic Activity Determination of XimA
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For determination of enzymatic activity, we used 100 µl of the reaction mixture containing 50 mM Tris-HCl buffer (pH 7.5), 5 mM MgSO4, 5 mM ATP, 10 µg 3, 10 mM L-threonine and 1 mg XimA. After incubation at 30°C for 12 h, the reaction was quenched by adding 1 ml methanol. The protein was removed by centrifugation at 13,000 g for 10 min, and the supernatant was then evaporated at 50°C. The remaining residue was freeze-dried for 24 h and then dissolved in 100 µl methanol. Enzymatic products were analyzed by UPLC-Q-TOF-MS as described above. The control assay was carried out under the same conditions with heat-inactivated enzyme.
Reactions to determine the Km of XimA toward xiamenmycin B contained 50 mM Tris-HCl buffer (pH 7.5), 5 mM MgSO4, 5 mM ATP, 10 mM L-threonine, 40 µM XimA and various concentrations of xiamenmycin B ranging from 0.2 µM to 345 µM. The reaction products were detected by Ultra Performance Liquid Chromatography and a Triple Quadrupole Mass Spectrometer (Waters ACQUITY UPLC, AB SCIEX SelexION Triple Quad 5500 System).
Reactions to determine the Km of XimA toward xiamenmycin B contained 50 mM Tris-HCl buffer (pH 7.5), 5 mM MgSO4, 5 mM ATP, 10 mM L-threonine, 40 µM XimA and various concentrations of xiamenmycin B ranging from 0.2 µM to 345 µM. The reaction products were detected by Ultra Performance Liquid Chromatography and a Triple Quadrupole Mass Spectrometer (Waters ACQUITY UPLC, AB SCIEX SelexION Triple Quad 5500 System).
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