Plasma samples were placed on ice, centrifuged at 4°C, separated, and stored at −20°C until assay. Glucose concentrations were measured using a glucose oxidase method (Yellow Springs Instruments, Yellow Springs, OH). Plasma insulin was measured using a chemiluminescence assay with reagents obtained from Beckman (Access Assay; Beckman, Chaska, MN). Plasma glucagon and C-peptide were measured by radioimmunoassay using reagents supplied by Linco Research (St. Louis, MO). Plasma [6,6-2H2]glucose and [1-13C]glucose enrichments were measured using gas chromatography–mass spectrometry (Thermoquest, San Jose, CA) to simultaneously monitor the C-1, C-2, and C-3 to C-6 fragments, as described by Beylot et al. [30 (link)]. [6-3H]glucose specific activity was measured by liquid scintillation counting following deproteinization and passage over anion- and cation-exchange columns.
Gas chromatography mass spectrometry
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Gas chromatography-mass spectrometry (GC-MS) is an analytical technique that combines gas chromatography and mass spectrometry. It is used for the identification and quantification of chemical compounds in complex mixtures. The gas chromatography component separates the individual compounds, while the mass spectrometry component identifies and measures the amount of each compound.
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6 protocols using gas chromatography mass spectrometry
1
Glucose and Isotope Tracer Analysis
2
Insulin Aspart Pharmacokinetics and Safety
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Fat‐free mass was determined by dual‐energy X‐ray absorptiometry. Free serum IAsp concentrations (polyethylene glycol precipitated) were determined using a validated IAsp‐specific enzyme‐linked immunosorbent assay having a lower limit of quantification (LLOQ) of 10 pmol/L.
The PG concentrations were measured using a SuperGL 2 glucose analyser (Dr Müller Gerätebau GmbH, Freital, Germany) using an electrochemical method.
Plasma [6‐3H] glucose specific activity was determined using liquid scintillation counting,14 and plasma enrichment of [1‐13C] glucose and [6,6‐2H2] glucose was determined by gas chromatography‐mass spectrometry (Thermoquest),15 at the Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota.
Plasma glucagon concentrations were determined in plasma using a validated enzyme‐linked immunosorbent assay with an LLOQ of 17.7 pg/mL (Mercodia Glucagon ELISA, Mercodia AB, Uppsala, Sweden).
Safety assessments included adverse events, hypoglycaemic episodes (classified as “severe” according to the American Diabetes Association, ie, requiring third‐party assistance,16 or “confirmed”, ie, documented by PG <3.1 mmol/L, with or without symptoms consistent with hypoglycaemia), laboratory safety variables, physical examination, vital signs and ECG.
The PG concentrations were measured using a SuperGL 2 glucose analyser (Dr Müller Gerätebau GmbH, Freital, Germany) using an electrochemical method.
Plasma [6‐3H] glucose specific activity was determined using liquid scintillation counting,
Plasma glucagon concentrations were determined in plasma using a validated enzyme‐linked immunosorbent assay with an LLOQ of 17.7 pg/mL (Mercodia Glucagon ELISA, Mercodia AB, Uppsala, Sweden).
Safety assessments included adverse events, hypoglycaemic episodes (classified as “severe” according to the American Diabetes Association, ie, requiring third‐party assistance,
3
Intracellular Fatty Acid Analysis
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Intracellular fatty acids were assayed by referring to the method of Zhou et al. [40 (link)]. In detail, BMECs were transfected with miR-193a-5p mimic, inhibitor and negative control. A total of 100 mg BMECs mixed with 2 mg of methanol containing 0.25% sulfuric acid were sonicated by ultrasound and then incubated at 80 °C for 1 h to methylate the fatty acids. The solution, returned to room temperature, was mixed with 2 mL hydrochloric acid solution (0.1 M) and then 800 μL of n-hexane was added, obtaining a mixed solution. The mixed solution was centrifuged at 900× g for 5 min, and the supernatant was transferred to a siliconized glass tube. The supernatant was added to 0.5 g anhydrous sodium sulfate and vortexed. After centrifugation at 13,800× g for 5 min, the supernatant and anhydrous sodium sulfate were separated from the solution. The supernatant was applied to the detected fatty acid composition and content with gas chromatography−mass spectrometry (Thermo Fisher, Waltham, MA, USA) analysis of fatty acid composition and content. The relative content of fatty acid was evaluated using the percentage of peak area for individual fatty acids.
4
Bioactive Phytochemicals Identification in Justicia adhatoda
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For the identification of bioactive phytochemicals in the ethyl acetate fraction of Justicia adhatoda, gas chromatography–mass spectrometry (Thermo Scientific Co.) was used. Identification of active phytochemicals was as per the ‘National Institute of Standards and Technology 2008’ (NIST-2008) database that contained over 62,000 patterns used for interpreting gas chromatography–mass spectrometry mass spectra. A comparison of the spectrum of an unknown component with the spectrum of the known component in the NIST library was performed (Sher et al., 2022 (link)).
5
GC-MS Analysis of Seed Oil and Root Compounds
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The identification of essential fatty acids and bioactive compounds present in seed oil and root extract was carried out by Gas chromatography-mass spectrometry (Thermo Fisher Scientific, Waltham, MA, United States). The instrument was set with GC 1300 gas chromatography, autosampler (TriPlus RSH), TSQ Duo Mass selective quadrupole detector, and Trace Gold TG-5MS column (40 m length, internal diameter 0.15 mm, and film thickness 0.15 m). The analysis of compounds present was done by diluting each sample in n-hexane at a ratio of 1:99. The initial temperature was kept at 60°C for 60 s to 180°C for 3 min and the final temperature was kept at 240°C for 12 min. The ramp rate was maintained constant at a temperature of 10°C/min and the complete analysis was kept for 34 min. The program was run with the linear velocity of the helium carrier gas at the rate of 0.7 mL/min. The electron impact was kept at 70 eV and used as ion source temperature with 250°C of transfer line temperature at 230°C. The sector of the mass analyzer was set to scan from m/z 45 and 450. Gas chromatographic and mass spectrometric data were processed by using Xcalliber software.
6
Bioactive Compounds Analysis of A. aegerita
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The identification of bioactive compounds present in A. aegerita was carried out by Gas chromatography-mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA).The instrument was set with GC 1300 gas chromatography, autosampler (TriPlus RSH), TSQ Duo Mass selective quadrupole detector, and Trace Gold TG5MS column (40 m length, internal diameter 0.15 mm, and film thickness 0.15 m). The compounds present in the extract were diluted in n-hexane (1:99) and the initial temperature was kept at 62 °C to 185 °C for 60 to 180 s, final temperature was kept at 235 °C for 15 min, and in splitless mode, 1 µL of sample (10 µg/mL) was injected with autosampler. The complete analysis was kept for 35 min by maintaining a constant temperature of 10 °C/min. The program was run at the rate of 0.6 mL/min with linear velocity of the helium carrier gas. The electron impact was kept at 65 eV and used as an ion source temperature with 245 °C of transfer line temperature at 225 °C. The sector of the mass analyzer was set to scan from 45 to 450 m/z. Gas chromatographic and mass spectrometric data were processed by using Xcalibur software (Version 10).
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