Blood samples were taken at day 42 after separating the sampled broilers as myopathy (WB score > 1) and nonmyopathy (WB = 0) (n = 5 each group for each strain) groups. Plasma was separated from collected blood samples, and samples were analyzed for differential 3-MH expression. Targeted metabolomics of 3-MH was performed on a triple quadrupole MS coupled to an I-class UPLC system (Waters) for differential expression. Procedures for handling plasma samples in a laboratory, data processing, and bioinformatics were carried out as described in the article by Maharjan et al., 2020c (link).
Triple quadrupole ms
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The Triple Quadrupole MS is a mass spectrometry instrument designed for quantitative and qualitative analysis. It consists of three consecutive quadrupole mass analyzers that allow for the isolation, fragmentation, and detection of target analytes with high sensitivity and selectivity.
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6 protocols using triple quadrupole ms
1
Differential 3-MH Expression in Myopathy
2
Quantification of Intracellular M-CoA and MM-CoA
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The concentrations of intracellular M-CoA and MM-CoA in the relevant strains at 48 h and 72 h of incubation were extracted following the method described by Lu et al. (2016 (link)) and determined by LC–MS/MS. For each time point, samples of 25 mL were harvested. One milliliter of culture was collected to quantify the dry cell weight. The liquid cell sample of remaining culture was transferred into a precooled tube containing quenching and extraction solution (acetonitrile/methanol/0.1% glacial acetate at a volume ratio of 45:45:10, − 20 °C). The extraction was performed by repetitive vortexing and cooling on ice for 15 min and centrifuged to collect the supernatant (12,000 rpm, 4 °C, 3 min). Samples were analyzed using an Atlantis BEH C18 (1.7 µm, 2.1 × 100 mm, Waters Co., Milford, MA) on a triple quadrupole MS (Waters). The mobile phase was acetonitrile with 50 mM ammonium hydrogen carbonate (solvent A), 0.1% ammonium hydroxide (solvent B), ddH2O (solvent C) and 0.1% ammonium hydroxide-acetonitrile (solvent D). Elution was performed as follows: 0–3 min 20% A 5% B 75% C, 3–3.5 min 20% A 10% B 30% C 40% D, 3.5–5 min 20% A 80% D, 5–7 min 20% A 5% B 75% C. Quantification was detected in the multiple reaction monitoring mode (MRM) with the m/z parent > m/z daughter (M-CoA 854 > 347, MM-CoA 868 > 361).
3
Quantification of Acrylamide and Harman Compounds
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After centrifugation and dilution, the 300 μl reaction sample were added to 300 μl of an internal standard containing 100 μg/ml 13C3-acrylamide. The identification and quantification of acrylamide, harman, and norharman were also conducted on an Acquity UHPLC system equipped with a triple quadrupole MS (Waters, Milford, MA, USA). The parameters of UHPLC-MS were same as section 2.3. The fragment used for quantification were m/z 72 → 55 (acrylamide); m/z 75 → 58 (13C3-acrylamide); m/z 169 → 115 (norharman).
4
Composition and Analysis of BXHPD Herbal Medicine
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The oriental herbal medicine BXHPD is composed of the following dried raw materials: Pinellia ternata, Poria cocos Magnolia officinalis, Perilla frutescens and Zingiber officinale. These materials were provided by Zequn Traditional Chinese Medicine decoction pieces and identified by Pro Shengjin Liu, Department of Traditional Chinese Medicine Identification, Nanjing University of Traditional Chinese Medicine with voucher specimen number 211012.
120 g Pinellia ternate, 120 g Poria cocos, 90 g Magnolia officinalis, 60 g Perilla frutescens and 150 g Zingiber officinale were exposed to 10 times the amount of water and refluxed for 2 h. After filtration, the drug residue was added with 2 times the amount of water, refluxed for 2 h and further filtered. The filtrates were combined and concentrated to 2 g/mL crude drug dosage at reduced pressure.
All the drugs were dissolved in DMSO and normal saline [with a concentration of DMSO <0.1% (v/v)]. Qualitative analysis of components in the water extract of BXHPD was performed by an HPLC method. The components of BXHPD in hippocampi were determined by the Waters Acquity UPLC system (Waters) consisting of an Xevo Triple Quadrupole MS.
120 g Pinellia ternate, 120 g Poria cocos, 90 g Magnolia officinalis, 60 g Perilla frutescens and 150 g Zingiber officinale were exposed to 10 times the amount of water and refluxed for 2 h. After filtration, the drug residue was added with 2 times the amount of water, refluxed for 2 h and further filtered. The filtrates were combined and concentrated to 2 g/mL crude drug dosage at reduced pressure.
All the drugs were dissolved in DMSO and normal saline [with a concentration of DMSO <0.1% (v/v)]. Qualitative analysis of components in the water extract of BXHPD was performed by an HPLC method. The components of BXHPD in hippocampi were determined by the Waters Acquity UPLC system (Waters) consisting of an Xevo Triple Quadrupole MS.
5
UPLC-MS/MS Analysis of Target Analytes
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A Waters ACQUITY UPLCTM (Milford, MA, USA) system was interfaced to a triple quadrupole MS (TQD, Waters, Manchester, UK) using an orthogonal Z-spray electrospray ionization (ESI) interface. The chromatographic separation was conducted on a BEH C18 column (1.7 µm, 2.1 × 100 mm, Waters Corp.), and the column temperature was set to 40 °C. The mobile phase consisted of methanol (A) and 0.15 mmol/L of (NH4)2CO3. The separation was performed at a flow rate of 0.25 mL/min, with a gradient elution program as follows: 0 min:5％ A; 6 min: 95% A; 8 min: 5% A; 10 min; 5% A. The injection volume was 3.0 µL. The post-time for column re-equilibration was set to 3 min between consecutive injections. Based on the structural properties of target ATs, both the ESI± and ESI- modes were applied. The parameters were as follows: Capillary voltage, +3.5 kV/−2.5 kV; source temperature, 150 °C; desolvation temperature, 400 °C; desolvation gas flow, 800 L/h; and cone gas flow, 50 L/h. Nitrogen was used as the cone and desolvation gas, and argon was used as the collision gas. Optimal collision energies (CEs) and cone voltages selected for each transition and retention time are shown in Table S1 . The MassLynxTM 4.1 software was used for data acquisition and processing.
6
UHPLC-MS/MS Quantification of Amino Acids and Glucose
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After centrifugation and dilution, the 300 μl reaction sample were added to 300 μl of an internal standard containing 100 μg/ml d4-lysine, and 200 μg/ml 13C6-glucose.
The identification and quantification of lysine, asparagine, tryptophan, and glucose were conducted on an Acquity UHPLC system equipped with a triple quadrupole MS (Waters, Milford, MA, USA). The separation of analytes was performed on a Waters Atlantics dC18 column (250 × 4.6 mm i.d, 3.0 μm) using a gradient elution with 0.1% formic acid (pH 6.8) (solvent A) and acetonitrile (solvent B). The solvent composition was 0–0.1 min, 5% A; 0.1–10 min, 5–100% A; 10–10.5 min, 100–5% A; and 10.5%−15 min, 5% A. The flow rate was 0.3 mL/min, the column temperature was 30°C and the injection volume was 10 μL. The capillary voltage was 3.5 kV. The temperature of the ion source and that of the desolvation gas were 130 and 350°C, respectively. The fragment used for quantification were m/z 133 → 74 (asparagine); m/z 147 → 84 (lysine); m/z 151 → 88 (d4-lysine); m/z 151 → 88 (d4-lysine); m/z 383 → 203 (glucose); and m/z 395 → 209 (13C6-glucose).
The identification and quantification of lysine, asparagine, tryptophan, and glucose were conducted on an Acquity UHPLC system equipped with a triple quadrupole MS (Waters, Milford, MA, USA). The separation of analytes was performed on a Waters Atlantics dC18 column (250 × 4.6 mm i.d, 3.0 μm) using a gradient elution with 0.1% formic acid (pH 6.8) (solvent A) and acetonitrile (solvent B). The solvent composition was 0–0.1 min, 5% A; 0.1–10 min, 5–100% A; 10–10.5 min, 100–5% A; and 10.5%−15 min, 5% A. The flow rate was 0.3 mL/min, the column temperature was 30°C and the injection volume was 10 μL. The capillary voltage was 3.5 kV. The temperature of the ion source and that of the desolvation gas were 130 and 350°C, respectively. The fragment used for quantification were m/z 133 → 74 (asparagine); m/z 147 → 84 (lysine); m/z 151 → 88 (d4-lysine); m/z 151 → 88 (d4-lysine); m/z 383 → 203 (glucose); and m/z 395 → 209 (13C6-glucose).
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