6410 triple quadrupole mass spectrometer

Manufactured by Agilent Technologies

Sourced in United States

The Agilent 6410 triple quadrupole mass spectrometer is a highly sensitive and versatile analytical instrument designed for quantitative and qualitative analysis of a wide range of compounds. It features a triple quadrupole configuration, which enables precise mass selection, fragmentation, and detection of target analytes.

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36 protocols using 6410 triple quadrupole mass spectrometer

Metformin Quantification in Liver Tissue

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Liver pieces were weighed and homogenized using a dounce homogenizer (Kimble Chase) in 500 µl 80% methanol including an internal metformin standard at a final concentration of 2(µg/ml, diluted from Metformin-d6 hydrochloride stock (Santa Cruz, 1mg resuspended in 1 ml DMSO). Samples were diluted prior to analysis as needed to ensure that measurements were within the linear range of measurements determined by a standard curve. Metformin and the internal standard (metformin-d6) were analyzed on an Agilent 6410 triple quadrupole mass spectrometer in MRM mode, coupled to an Agilent 1100 lc system. The transition states monitored were 136.1 −> 60.1 (quantitative transition), and 60.1 (qualitative transition) for the internal standard, and 130 −> 60.1 (quantitative) and 71.1 (qualitative) for metformin. Spray chamber settings were: N₂ drying gas flow = 10L/min, drying gas temp = 350°C, and nebulizer pressure = 30psi. Capillary voltage was set to 4000V. Column used was a phenomenex 2.0 ×50mm Kinetex C18. Mobile phase A = H₂O/40mM heptafluorobutyric acid (HFBA), and mobile phase B=MeOH. Flowrate was 200 µl/min. Gradient was T=0 90:10 to T=5min 5:95, with stop time at 10 minutes, followed by a 4 minute re-equilibration step (post-time). 5 µl was injected. Concentrations were calculated from a 5-point calibration curve. The concentration of metformin was normalized to tissue weight.

Henriksson E., Huber A.L., Soto E., Kriebs A., Vaughan M., Duglan D., Chan A., Papp S., Nguyen M., Afetian M, & Lamia K. (2017). The liver circadian clock modulates biochemical and physiological responses to metformin. Journal of biological rhythms, 32(4), 345-358.

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HPLC-MS/MS Analysis of Resveratrol and Ceramides

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The analysis of PD and resveratrol was performed using an Agilent 1260 infinity HPLC system and a 6490 triple quadrupole mass spectrometer. Bio-samples were extracted as previously reported45 (link). The 5-μl extraction volume was injected onto the column and gradient eluted into MS. The MRM parameters were as follows: resveratrol = 227− > 185 (20.0 eV) and PD = 389− > 227 (20.0 eV).
HPLC-MRM was also applied to the bio-samples for ceramide analysis. Ceramides were extracted from plasma and tissues as reported previously46 (link). Statistical analyses were conducted based on the data acquired using the Agilent 1200 HPLC system and a 6410 triple quadrupole mass spectrometer.

Yan X.D., Wang Q.M., Tie C., Jin H.T., Han Y.X., Zhang J.L., Yu X.M., Hou Q., Zhang P.P., Wang A.P., Zhang P.C., Gao Z, & Jiang J.D. (2017). Polydatin protects the respiratory system from PM2.5 exposure. Scientific Reports, 7, 40030.

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Quantifying PFOA and PFCAs in Environmental Samples

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PFOA and other PFCAs were quantified on an Agilent 1200 HPLC coupled with an Agilent 6410 triple quadrupole mass spectrometer operating in the negative electrospray ionization mode as described previously.^{28 (link),29} Prior to the quantification analysis, methanol extraction was applied to all samples.
The concentration of S₂O₈²⁻ was determined by KI colorimetric method on a Lambda UV spectrophotometer (PerkinElmer Inc.).^{30 (link)}To assess the form of PFOA associated with the PAC surface, PAC that had been used for treatment experiments under circumneutral pH conditions was analyzed using a LEO 439 scanning electron microbe (SEM) coupled with a Princeton Gamma-Tech energy dispersive X-ray (EDX) spectrometer. Before characterization, the PAC samples were extracted with methanol and dried at 50 °C twice to remove all physically adsorbed PFOA from the PAC.

Sun B., Ma J, & Sedlak D.L. (2016). Chemisorption of Perfluorooctanoic Acid on Powdered Activated Carbon Initiated by Persulfate in Aqueous Solution. Environmental science & technology, 50(14), 7618-7624.

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Quantitative Analysis of Endocannabinoids

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Sample preparation and instrumental parameters for analysis of endoCBs levels have been previously described in detail [29 (link)]. In brief, prior to the instrumental analysis, samples were extracted with a solution of methanol, chloroform and water. Samples were measured with a reversed phase liquid chromatography technique coupled with the triple quadrupole mass spectrometry (LC–MS/MS), operating in multiple reaction monitoring scanning (MRM). LC–MS/MS analysis was performed using Agilent Technologies 6410 triple quadrupole mass spectrometer coupled to Agilent Technologies 1200 series HPLC system. Separation was achieved using Zorbax Eclipse XDB-C18 (Agilent Technologies) column with an isocratic elution. The flow rate was 0.5 mL/min, and the total run time was 4 min. The mass spectrometer was operated in positive ion mode using MRM for quantification of the analytes. Deuterated internal standards (AEA-d8 and 2-AG-d8) were used for quantification. This method is selective, precise, and accurate for concentrations within a range of 0.4 – 70 nM for N-acylethanolamines and 40 – 11,000 nM for 2-AG.

Della Pietra A., Krivoshein G., Ivanov K., Giniatullina R., Jyrkkänen H.K., Leinonen V., Lehtonen M., van den Maagdenberg A.M., Savinainen J, & Giniatullin R. (2023). Potent dual MAGL/FAAH inhibitor AKU-005 engages endocannabinoids to diminish meningeal nociception implicated in migraine pain. The Journal of Headache and Pain, 24(1), 38.

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Plasma metabolite quantification by LCMS

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25 μL of plasma was extracted with acetonitrile spiked with internal standards, and the supernatant was then mixed with 200 mM 3-nitrophenylhydrazine and 120 mM N-(3-dimethylaminopropyl)-N1-ethylcarbodiimide in a 2:1:1 (v/v/v) ratio. The samples were derivatized at 40°C for 30 min and then injected into an Agilent 6410 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source in negative-ion mode coupled to an Agilent 1290 infinity HPLC system with an Acquity UPLC BEH C18 column (2.1×100 mm, 1.7 μm; Waters, Milford MA). Solvent A was formic acid (0.01%, v/v) in water, and solvent B was formic acid (0.01%, v/v) in acetonitrile.
Quantitation was performed by calibration to internal standards and standard curves on the Mass Hunter quantitative suite version B.06.00 (Santa Clara). All levels are expressed in μM (Han et al., 2015 (link); Mell et al., 2015 (link); Theriot et al., 2014 (link)).

Chakraborty S., Galla S., Cheng X., Yeo J.Y., Mell B., Singh V., Yeoh B., Saha P., Mathew A.V., Vijay-Kumar M, & Joe B. (2018). Salt-Responsive Metabolite, β-Hydroxybutyrate, Attenuates Hypertension. Cell reports, 25(3), 677-689.e4.

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Honey Metabolite Profiling by LC-MS/MS

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The honey extracts were subjected to liquid chromatography/tandem mass spectrometry (LC-ESI-MS/MS). The system was an Agilent 1200 liquid chromatograph and a 6410 triple quadrupole mass spectrometer with an electrospray ionization source. Data were analyzed using the MassHunter software package (Agilent Corporation, MA, and USA). Separation was performed on a Zorbax C18 column (2.1 mm × 50 mm, 0.18 μm particle size) by gradient elution using the following gradient: 0–1 min 5% B isocratic; 1–14 min linear gradient to 100% B; 14–15 min to 5% B and the column was reconditioned at 5% B for 5 min. Eluent A was water with 0.1% formic acid and B was methanol with 0.1% formic acid. The acid used in the analysis improves the chromatographic peak shape and provides a source of proton in reverse phase LC-MS. The column temperature was 30° C. Mass spectrometry was performed on triple quad spectrometer in ESI positive ion mode. High purity nitrogen served as both the nebulising agent and the drying gas, with a drying gas flow 9 l/min and a nebuliser pressure 25 psi at 350°C. The capillary voltage was set at 4,000 V and the source at 300°C.

Anand S., Deighton M., Livanos G., Morrison P.D., Pang E.C, & Mantri N. (2019). Antimicrobial Activity of Agastache Honey and Characterization of Its Bioactive Compounds in Comparison With Important Commercial Honeys. Frontiers in Microbiology, 10, 263.

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HPLC-ESI-MS/MS Analysis of Amino Acids

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The Agilent 1290 Infinity Binary LC system consists of an autosampler, column oven, a degasser, and a binary solvent manager (Agilent Technologies, CA, USA). The Agilent 6410 triple-quadrupole mass spectrometer was equipped with an electrospray ionization (ESI) source. The HPLC-ESI-MS/MS system was controlled by MassHunter Workstation (Agilent Technologies, CA, USA).
Chromatographic separation was performed by an Acquity C₁₈ column (4.6 × 100 mm,3.5 μm) (Agilent Technologies, USA). The mobile phase was composed of water (containing 10 mM ammonium formate and formic acid, pH 3.0) (solvent A) and ACN (solvent B). The gradient profile of B was: 0–10 min, 35–38%; 10–11 min, 38– 60%; 11–17 min, 60–35%B; 17–18 min, 35%. The flow rate was kept at 0.5 mL/min. The column oven was set at 30 °C and the injection volume was 10 μL.
The mass spectrometer was operated in the positive ion electrospray mode with multiple reaction monitoring (MRM). The optimal MS parameters were set at the following parameters: a desolvation gas temperature of 450 °C, desolvation gas flow of 10 L/min, a nebulizer gas of 40 psi and a capillary voltage of 4000 V. The mass spectrometry conditions of AA I, AA II, AA C, AA D, 7-OH AA I and IS are summarized in Additional file 1: Table S1.

Yuan J., Ren G., Liang J., Wang C.Z., Yan Z., Huang Q., Li J., Chen Y., Tang Y., Liu X, & Yuan C.S. (2017). Comparative studies on the multi-component pharmacokinetics of Aristolochiae Fructus and honey-fried Aristolochiae Fructus extracts after oral administration in rats. BMC Complementary and Alternative Medicine, 17, 107.

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Quantification of Compounds by LC-MS/MS

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The two compounds were analyzed on Agilent 6410 triple quadrupole mass spectrometer. Samples were dissolved in acetonitrile and eluted in 60% ammonium acetate and 40% acetonitrile on C18 column at a flow rate of 0.2 mL/min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization.

Baker S.J., Cosenza S.C., Athuluri-Divakar S., Reddy M.V., Vasquez-Del Carpio R., Jain R., Aggarwal A.K, & Reddy E.P. (2020). A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent. Molecular cell, 79(1), 180-190.e4.

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Metabolite Profiling of Hairy Roots

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Dried hairy roots (50 mg) were ground into a fine powder and extracted twice with 25 mL of 80% methanol under sonication for 30 min. After centrifugation, the supernatant was diluted with 80% methanol to a total volume of 50 mL, and filtered through a 0.22 μm organic membrane filter prior to HPLC analysis. HPLC analysis was conducted on an Agilent 1200 series instrument with an Agilent 6410 triple-quadrupole mass spectrometer and an electrospray ionization source (Agilent Corporation, MA, USA). Metabolite separation was achieved on an Agilent ZORBAX SB-C18 column (3.5 μm, 2.1 × 150 mm) and an Agilent C18 guard column (5 μm, 4.0 × 2.0 mm). The mobile phase was acetonitrile: 5 mM ammonium acetate solution (the concentration of acetonitrile was from 5 to 95% in 1.0 min, v/v) with the flow rate of 0.3 mL·min⁻¹ and a total run time of 5 min. Metabolite identification and quantification was achieved in multiple reaction monitoring mode (MRM). Characteristic m/z ions are listed in Table S2. The samples for qRT-PCR and metabolites analysis were the same.

Chen R., Li Q., Tan H., Chen J., Xiao Y., Ma R., Gao S., Zerbe P., Chen W, & Zhang L. (2015). Gene-to-metabolite network for biosynthesis of lignans in MeJA-elicited Isatis indigotica hairy root cultures. Frontiers in Plant Science, 6, 952.

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BPA Glucuronidation in Liver Microsomes

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The incubation mixture contained BPA 100 μM as a substrate, microsome (0.2 mg), 10 mM MgCl2, 5 mM UDPGA in a final volume of 200 μL. Incubation was performed for 30 min at 37°C and terminated by adding adding 100 μL of 15% perchloric acid and vertexing. The samples were centrifuged at 13,000 × g for 15 min and the supernantant was injected in LC-MS/MS analysis. Chromatography separation was performed using a Zorbax Eclipse XDB-C18 (150 × 2.1 mm internal diameter, 3.5 μm particle size). Gradient elution with water (solvent A) and acetonitrile (solvent B) with the following conditions was applied:0% B for 1 min, followed by a linear gradient to 90% B within 5 min and then return to 0% B in 10 min and maintained for 5 min. Flow rate was 0.4 mL/min and run time was 15 min.
An Agilent Technologies 6410 triple quadrupole mass spectrometer equipped with electrospray ionization was used. The following conditions were used for analysis: capillary voltage 4 kV, gas temperature 300°C and gas flow 10 L/min. Samples were analyzed by MRM mode in negative ionization mode. The MRM transitions of m/z 567.4 to 391.3 for CDCA-G, m/z 442.1 to 125 for AZT-G, and m/z 403 to 227 for BPA-G were used for analysis.

Marahatta A., Bhandary B., Jeong S.K., Kim H.R, & Chae H.J. (2014). Soybean greatly reduces valproic acid plasma concentrations: A food–drug interaction study. Scientific Reports, 4, 4362.

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