Analysis of polar organic acids and inorganic anions using ion-chromatography coupled to HRMS
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Ion-chromatography coupled with high-resolution mass spectrometry became available as an analytical technique after Thermo Scientific acquired Dionex. This, still rare, technology is by far superior for the analysis of polar organic and inorganic anions than conventionally used HILIC or mixed-mode chromatography or GC-MS analysis of polar compounds after chemical derivatization. Only after our laboratory purchased an ion-chromatograph for Q-Exactive, we were able to carry out reliable, sensitive, and accurate quantitative analysis of polar organic acids and do much more...
Instrumental: Q-Exactive Plus hybrid FT mass spectrometer coupled with Dionex Integrion HPIC ion-chromatograph (IC) (Thermo Scientific). The IC is equipped with an automated eluent (KOH) generator and conductivity detector (CD). Chromatographic separations are carried out using hydroxide-selective Dionex ion-chromatography columns. Such configuration of the instruments allows simultaneous high-resolution accurate mass MS detection and MS/MS analysis of anions together with CD detection. The sensitivity of the mass spectrometer is significantly higher than that of the ion-conductivity detector. However, the CD can be used for detecting ions below m/z 50 (for example, chloride and fluoride) that cannot be detected by MS and for quantifying analytes at higher concentrations because of a much wider linear dynamic range.
Schematic diagram of IC coupled with Q-Exactive
The IC-CD (red) and IC-MS (black) chromatograms of the calibration mixture of organic and inorganic anions. Some anions can not be chromatographically separated and therefore cannot be unequivocally determined using IC-CD. Coeluting analytes, however, can be easily identified and quantified as long as they have different atomic compositions or MS/MS spectra. We often observe the limitations of IC-CD when analyzing biological samples.
Demonstration of IC-MS sensitivity and selectivity. The IC-CD chromatogram (red, no analytes were detected) and extracted-ion MS chromatograms of some analytes recorded in 5 ng/ml calibration sample (volume of injection was 10µl).
Demonstration of IC-MS sensitivity. The limits-of-detections measured for some anions using IC-HRMS (volume of injection was 10µl).
The IC-HRMS analysis is not just more sensitive than IC-CD but it also is more specific and therefore reliable. Analytes are identified using accurate-mass scan and MS/MS data.
For years we tried to analyze polar organic acids using conventional GC-MS methods. Since polar organic compounds are usually non-volatile they cannot be analyzed using the gas chromatography "as is" but must be converted into semi-volatile compounds by chemical derivatization. Here the problems begin, the derivatization rarely gives a 100% yield of the expected product and for some organic acids, due to keto-enol tautomerism, more than one derivatization product may be expected.
Thus, while reliable calibration data can usually be obtained using the IC-CD-HRMS technique, that is not always the case in GC-MS analysis of polar organic acids.
Calibration curves of some organic acids obtained using IC-HRMS technique. There were used α-ketoglutaric acid-13C5, and 2-methylcitric acid-D3 as internal standards.
The GC-MS, - problematical and unreliable way for quantitative analysis of polar organic acids
The EI mass spectra of trimethylsilylated acetoacetic (above) and α-ketoglutaric (below) acids. The molecular ions of trimethylsilylated compounds are often unstable, weak, or not detected, the mass spectra have characteristic abundant noninformative ions of alkyl-silyl cations (here m/z 73, 147) while the use of isotopically labeled internal standards is limited due to the presence of silicon stable isotopes 29Si (4.7%), 30Si (3.1%) which may interfere with m/z of labeled compounds.
The GC-MS EI chromatogram of trimethylsilylated acetoacteic acid. Two isomers were detected after the derivatization reaction due to keto-enol tautomerism. Here are shown calibration curves obtained for both isomers. It can be clearly seen here that the ratio of keto/enol forms is not a stable constant. These calibration curves were obtained under "ideal" conditions working with the standard compounds.
I am wondering what is the scientific value of thousands of publications discussing polar metabolites analyzed using conventional derivatization and GC-MS techniques ...
IC-CD-HRMS analysis results of organic and inorganic anions determined in plasma, urine, and liver extract using. The analytes can be reliably identified and quantified in ranges of concentrations from a few ng/ml to hundreds of µg/ml (samples were diluted in the course of sample preparation, - urine, and liver samples at ratio 1:100, plasma, – 1:10).
Polar organic acids commonly analyzed in TSABAM.
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