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04-instrumental.Rmd
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---
bibliography: references.bib
---
# Instrumental analysis
To get more information in the samples, full scan is preferred on GC/LC-MS. Each scan would collect a mass spectrum to cover the setting mass range. If you narrow down your mass range and keep the same scan time, each mass would gain the collection time and you would get a higher sensitivity. However, if you expand your scan range, the sensitivity for each mass would decrease. You could also extend the collection time for each scan. However, it would affect the separation process.
Full scan is performed synchronously with the separation process. For a better separation on chromotograph, each peak should have at least 10 points to get a nice peak shape. If you want to separate two peaks with a retention time differences of 10s. Assuming the half peak width is 5s, you need to collect 10 mass spectrum within 10s. So the drwell time for each scan is 1s. If you use a high resolution column and the half peak width is 1s, you need to finish a scan within 0.2s. As we discussed above, shorter dwell time would decrease the sensitivity. Thus there is a trade-off between separation and sensitivity. If you use UPLC, the separation could be finished within 20 min while you need to calculate if you mass spectrometry could still show a good sensitivity. Recently a study [@cai2021] show 6 points will be enough to generate peaks with 20 points with optimized workflow.
## Column and gradient selection
For GC, higher temperature could release compounds with higher boiling point. For LC, gradient and functional groups of stationary phase would be more important than temperature. Polarity of samples and column should match. More polar solvent could release polar compounds. Normal-phase column will not retain non-polar compounds while reversed-phase will elute polar column in the very beginning. To cover a wide polarity range or logP value compounds, normal phase column should match with non-polar to polar gradient to get a better separation of polar compounds while reverse phase column should match with polar to non-polar gradient to elute compounds. If you use an inappropriate order of gradient, you compounds would not be separated well. If you have no idea about column and gradient selection, check literature's condition. Meanwhile, the pretreatment methods should fit the column and gradient selection. You will get limited information by injection of non-polar extracts on a normal phase column and nothing will be retained on column. This study show improved chromatography conditions will improve the annotation results[@anderson2021]. You can also install polar and non-polar columns and run separation on one column while condition on another one, which could extend the chemical coverage[@flasch2022].
Meta-analysis of chromatographic methods in EBI metabolights and NIH Workbench could be a guide for lab without experience on metabolomics chromatographic methods[@harrieder2022].
This work introduce Sequential Quantification using Isotope Dilution (SQUID), a method combining serial sample injections into a continuous isocratic mobile phase, enabling rapid analysis of target molecules with high accuracy, as demonstrated by detecting microbial polyamines in human urine samples with an LLOQ of 106 nM and analysis times as short as 57 s, thus proposing SQUID as a high-throughput LC–MS tool for quantifying target biomarkers in large cohorts[@groves2023].
## Mass resolution
For metabolomics, high resolution mass spectrum should be used to make identification of compounds easier. The Mass Resolving Power is very important for annotation and high resolution mass spectrum should be calibrated in real time. The region between 400--800 m/z was influenced the most by resolution[@najdekr2016]. Orbitrap Fusion's performance was evaluated here[@barbiersainthilaire2018], as well as the comparison with Fourier transform ion cyclotron resonance (FT-ICR)[@ghaste2016; @huang2021]. Mass Difference Maps could recalibrate HRMS data [@smirnov2019].
## Matrix effects
Matrix effects could decrease the sensitivity of untargeted analysis. Such matrix effects could be checked by low resolution mass spectrometry[@yu2017] and found for high resolution mass spectrometry[@calbiani2006]. Ion suppression should also be considered as a critical issue comparing heterogeneous metabolic profiles[@ghosson2021]. This work discussed the matrix effects after Trimethylsilyl derivatization[@tarakhovskaya2023].The study[@dagan2023] investigated how the complexity of matrices affects nontargeted detection using LC-MS/MS analysis, finding that detection limits for trace compounds were significantly influenced by matrix complexity, with higher concentrations required for detection within the "top 1000" list compared to the first 10,000 peaks, suggesting a negative power law functional relationship between peak location and concentration; the research also demonstrated a correlation between power law coefficient and dilution factor, while showcasing the distribution of matrix peaks across various matrices, providing insights into the capabilities and limitations of LC-MS in analyzing nontargets in complex matrices.