Metabolite Profiling of Alangium salviifolium Bark Using Advanced LC/MS and GC/Q-TOFTechnology
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents and Materials
2.2. Workflow
2.3. Collection of Plant Material and Extraction Procedure
2.4. Fraction Collection
2.5. Multiple LC/Q-TOF Chromatographic Analysis Conditions
2.6. GC/Q-TOF Conditions
2.7. Data Analysis
3. Results and Discussion
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Parameters | Aqueous Extract | Organic Extract | ||
---|---|---|---|---|
Mobile phase | Mobile phase A: Water + 10 mM ammonium acetate Mobile phase B: 100% Acetonitrile | Mobile phase A: 95:5 Water:methanol with 0.1% formic acid and 5 mM ammonium formate Mobile phase B: 65:30:5 Isopropanol:methanol:water with 0.1% formic acid and 5 mM ammonium formate | ||
Flow rate | 1.2 mL/min | 1.2 mL/min | ||
Injection volume | 1 mL | 0.3 mL | ||
Thermostat autosampler | 4 °C | 4 °C | ||
Temperature TCC | 25 °C | 25 °C | ||
DAD | 210 and 254 nm | 210 and 254 nm | ||
Peak width | >0.05 min | >0.05 min | ||
Fraction collection mode | Time based | Time based | ||
Total time | 13 min | 13 min | ||
Column | ZORBAX SB-C18(9.4 × 50 mm, 5 µm, p/n: 846975-202) | ZORBAX SB-C18(9.4 × 50 mm, 5 µm, p/n: 846975-202) | ||
Time slices | 0.292 min/well | 0.292 min/well | ||
Gradient | Time (min) 0.0 1.0 8.0 8.1 10.0 10.1 12.0 | % Solvent B 5 5 35 95 95 5 5 | Time (min) 0.0 1.0 8.0 11.0 11.1 12.0 | % Solvent B 60 60 100 100 60 60 |
Parameter | Aqueous Fractions Analysed Using ZORBAX SB-AQ Column | Aqueous Fractions Analysed Using ZORBAC HILIC Column | ||
---|---|---|---|---|
Ionization mode | Positive MS and positive AutoMSMS | Negative MS and Negative AutoMSMS | Positive MS and positive AutoMSMS | Negative MS and Negative AutoMSMS |
Mobile phase | Mobile phase A: Water with 0.2% acetic acid Mobile phase B: Methanol with 0.2% acetic acid | Mobile phase A: Water with 1 mM ammonium fluoride acetic acid Mobile phase B: 100% Acetonitrile | Mobile phase A: 90:10 of Acetonitrile: 50 mM ammonium acetate Mobile phase B: 50:40:10 of acetonitrile:water:50 mM ammonium acetate | Mobile phase A: 90:10 of Acetonitrile:50 mM ammonium acetate Mobile phase B: 50:40:10 of acetonitrile:water:50 mM ammonium acetate |
LC gradient | Time (min) | % mobile phase B | Time (min) | % mobile phase B |
1.00 | 5.0 | 3.00 | 0.0 | |
10.0 | 35.0 | 10.0 | 100.0 | |
11.0 | 95.0 | 13.0 | 100.0 | |
13.0 | 95.0 | 13.10 | 0 | |
13.1 | 5.0 | 17.00 | 0 | |
15.0 | 5.0 | |||
Parameter | Organic fractions analysed using ZORBAX EP-C18 | Organic fractions analysed using ZORBAX EP Phenyl Hexyl | ||
Ionization mode | Positive MS and positive utoMSMS | Negative MS and Negative AutoMSMS | Positive MS and positive AutoMSMS | Negative MS and Negative AutoMSMS |
Mobile phase | 95:5 of water: Methanol with 0.1% formic acid and 5 mM ammonium formate | 65:30:5 of Isopropanol:methanol: water with 0.1% formic acid and 5 mM ammonium formate | 95:5 of water: Methanol with 0.1% formic acid and 5 mM ammonium formate | 65:30:5 of Isopropanol:methanol: water with 0.1% formic acid and 5 mM ammonium formate |
LC gradient | Time (min) | % of mobile phase B | Time (min) | % of mobile phase B |
1.00 | 60.0 | 1.00 | 60.0 | |
8.0 | 100.0 | 8.0 | 100.0 | |
11.0 | 100.0 | 11.0 | 100.0 | |
11.10 | 60.0 | 11.10 | 60.0 | |
14.00 | 60.0 | 14.00 | 60.0 |
GC Conditions | |
Column | DB-5 ms: 30 m × 0.25 mmID × 0.25 μm, Guard Length: 10 m (Part No. 122-5532G) |
Injection volume | 1 µL |
Split mode and ratio | Split 10:1 |
Split/Splitless inlet temperature | 250 °C |
Oven temperature program | 60 °C for 1 min 10 °C/min to 325 °C, 10 min hold |
Carrier gas | Helium at 1.2798 mL/min constant flow |
Transfer line temperature | 290 °C |
QTOF Conditions | |
Ionization mode | EI |
Source temperature | 230 °C |
Quadrupole temperature | 150 °C |
m/z scan | 50 to 600 m/z |
Spectral acquisition rate | 5 spectra/s, 2679 transients/spectrum, collecting both in centroid and profile modes |
Metabolite | CAS/KEGG | Formula | Mass Difference (ppm) | Overall Score | Confirmed by MSC |
---|---|---|---|---|---|
Ankorine | 13849-54-2 | C19 H29 N O4 | −2.56 | 99.94 | YES |
Deoxytubulosine | C11817 | C29 H37 N3 O2 | −0.69 | 98.97 | YES |
Ipecac (Cephaeline) | 483-17-0 | C28 H38 N2 O4 | −0.28 | 80.06 | YES |
Lacinilene C 7-methyl ether | 56362-72-2 | C16 H20 O3 | −1.84 | 98.95 | YES |
Tubulosine | 2632-29-3 | C29 H37 N3 O3 | 2.86 | 97.31 | YES |
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Siddaiah, C.; Kumar BM, A.; Deepak, S.A.; Lateef, S.S.; Nagpal, S.; Rangappa, K.S.; Mohan, C.D.; Rangappa, S.; Kumar S, M.; Sharma, M.; et al. Metabolite Profiling of Alangium salviifolium Bark Using Advanced LC/MS and GC/Q-TOFTechnology. Cells 2021, 10, 1. https://doi.org/10.3390/cells10010001
Siddaiah C, Kumar BM A, Deepak SA, Lateef SS, Nagpal S, Rangappa KS, Mohan CD, Rangappa S, Kumar S M, Sharma M, et al. Metabolite Profiling of Alangium salviifolium Bark Using Advanced LC/MS and GC/Q-TOFTechnology. Cells. 2021; 10(1):1. https://doi.org/10.3390/cells10010001
Chicago/Turabian StyleSiddaiah, Chandranayaka, Anil Kumar BM, Saligrama Adavigowda Deepak, Syed Salman Lateef, Saurabh Nagpal, Kanchugarakoppal S. Rangappa, Chakrabhavi D. Mohan, Shobith Rangappa, Madan Kumar S, Minaxi Sharma, and et al. 2021. "Metabolite Profiling of Alangium salviifolium Bark Using Advanced LC/MS and GC/Q-TOFTechnology" Cells 10, no. 1: 1. https://doi.org/10.3390/cells10010001