Stable isotope labeled internal standard testosterone-2,3,4-13C3, were purchased from IsoSciences (King of Prussia, PA). Testosterone (T) pure standards was purchased from Steraloids (Newport, RI), and hydroxylamine hydrochloride, ultrapure methanol, and water (Chromasolv®) from SigmaAldrich (St. Louis, MO). Internal T-13C3 (0.5 ng) standard was added to mouse sera. An Agilent 6495 triple quadruple mass spectrometer (Agilent, Santa Clara, CA) equipped with a Jet Stream electrospray ion source (ESI, Agilent), a 1290 Infinity ultrahigh performance liquid chromatography system (Agilent) and MassHunter Workstation software (Agilent) was used to quantify testosterone in this study. Chromatographic separation of steroid oximes was conducted with a Chromolith C18 reverse phase column (50–2 mm) with a matching Chromolith guard column (5–2 mm). Testosterone oxime was introduced into ESI source and analyzed in the positive ion mode. Molecular ions for T (m/z 304.2) and for 13C3 internal standard T (m/z 307.2) were selected in the first quadrupole and quantified using product ion for T (m/z 124.1) and for internal standard T (m/z 127.1). The lower limit of quantification on column for T was 2.5 fg.
1290 infinity ultrahigh performance liquid chromatography system
Manufactured by Agilent Technologies
Sourced in United States
The 1290 Infinity ultrahigh performance liquid chromatography system is a high-performance liquid chromatography (HPLC) instrument designed for analytical separation and quantification of chemical compounds. It features advanced technology to deliver high-resolution separations, increased sensitivity, and faster analysis times. The system is capable of operating at pressures up to 1200 bar, enabling the use of narrow-bore and sub-2 μm particle size columns for improved chromatographic performance.
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Lab products found in correlation
Testosterone pure standards, by Steraloids (2 mentions)
Chromasolv, by Merck Group (2 mentions)
Masshunter workstation software, by Agilent Technologies (2 mentions)
Uhplc q tof ms, by Agilent Technologies (1 mentions)
Ab triple tof 6600 mass spectrometer, by AB Sciex (1 mentions)
Agilent 1290 infinity lc system, by Agilent Technologies (1 mentions)
Qualitative analysis software, by Agilent Technologies (1 mentions)
Zorbax rrhd sb c18 column, by Agilent Technologies (1 mentions)
6540 quadrupole time of flight q tof mass spectrometer, by Agilent Technologies (1 mentions)
Dual ajs esi, by Agilent Technologies (1 mentions)
4 protocols using 1290 infinity ultrahigh performance liquid chromatography system
1
Quantitative Testosterone Assay by LC-MS/MS
2
Quantitative Testosterone Assay by LC-MS/MS
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Stable isotope labeled internal standard testosterone-2,3,4-13C3, were purchased from IsoSciences (King of Prussia, PA). Testosterone (T) pure standards was purchased from Steraloids (Newport, RI), and hydroxylamine hydrochloride, ultrapure methanol, and water (Chromasolv®) from SigmaAldrich (St. Louis, MO). Internal T-13C3 (0.5 ng) standard was added to mouse sera. An Agilent 6495 triple quadruple mass spectrometer (Agilent, Santa Clara, CA) equipped with a Jet Stream electrospray ion source (ESI, Agilent), a 1290 Infinity ultrahigh performance liquid chromatography system (Agilent) and MassHunter Workstation software (Agilent) was used to quantify testosterone in this study. Chromatographic separation of steroid oximes was conducted with a Chromolith C18 reverse phase column (50–2 mm) with a matching Chromolith guard column (5–2 mm). Testosterone oxime was introduced into ESI source and analyzed in the positive ion mode. Molecular ions for T (m/z 304.2) and for 13C3 internal standard T (m/z 307.2) were selected in the first quadrupole and quantified using product ion for T (m/z 124.1) and for internal standard T (m/z 127.1). The lower limit of quantification on column for T was 2.5 fg.
3
Metabolomic Analysis of MitoQ-Treated Cells
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The cells were treated using MitoQ, and after collection, the cells were washed with pre-cooled phosphate buffered saline (PBS) three times and removed from the supernatant. Pre-cooled methanol: acetonitrile: water (2:2:1, v/v) solution was added to each sample to precipitate protein. The samples were pulverized by the ultrasonic wave at 0 °C for 30 min, incubated at −20 °C for 10 min, and then centrifuged at 4 °C, 14,000× g, for 20 min. The supernatants were transferred into new 1.5 mL tubes and dried inside a centrifugal vacuum evaporator, then reconstituted using a 100 μL acetonitrile: water (1:1, v/v) mixture for further analysis. The metabolites in the samples were detected with Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China) using UHPLC-Q-TOF MS. which is a 1290 Infinity Ultra-high Performance Liquid Chromatography system (Agilent, Santa Clara, CA, USA) coupled with AB Triple TOF 6600 Mass Spectrometer (AB SCIEX, Radio Road Redwood City, CA, USA).
4
UPLC-MS/MS Analysis of OMT and MT
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The following UPLC conditions were used to analyze OMT and MT: systems, Agilent 1290 Infinity LC system (Agilent Technologies, Santa-Clara, CA, USA), which consisted of a solvent degasser, a binary pump, an autosampler, and a column oven; column, Agilent ZORBAX RRHD SB-C18 column (100 mm ×3 mm, 1.8 μm); mobile Phase B, 100% acetonitrile; mobile Phase A, 0.1% formic acid in water; flow rate, 0.3 mL/min; gradient, 95%–85% A for 0–1.8 min, 85%–64% A for 1.8–4.0 min, 64%–55% A for 4.0–4.5 min, 55%–5% A for 4.5–5.0 min, 5%–5% A for 5.0–6.5 min, 5%–95% A for 6.5–7.5 min, and 95%–95% A for 7.5–8.0 min; wavelengths, 210 nm for OMT and MT, and 243 nm for TES; injection volume, 10 μL. The MS/MS detector used was an Agilent 6540 quadrupole-time of flight (Q-TOF) mass spectrometer in combination with an Agilent 1,290 Infinity ultrahigh-performance liquid chromatography system. Samples were analyzed using Dual AJS ESI (Agilent Technologies) in the positive model. The main working parameters were set as follows: capillary voltage, 3,500 V; temperature of the dry heater, 300°C; nebulizer voltage, 35 (psig); dry gas, 8.0 L/min; sheath gas temperature, 350°C; and sheath gas flow, 11 L/min. Data were collected and analyzed by the Qualitative Analysis software (version B.06.00, Agilent Technologies).
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