Life science
Medical & healthcare, Diagnostics, Drug discovery, Environmental, Agriculture

Analysis of nonvolatile molecules in supercritical carbon dioxide using protontransfer-reaction ionization time-of-flight mass spectrometry
Project Research Center for fundamental Sciences, Graduate School of Science
Professor Michisato Toyoda

Researchmap https://researchmap.jp/toyodam

Researcher Toshinobu Hondo

Researchmap hhttps://researchmap.jp/toshi-hondoLife science

Abstract
Proton transfer reaction (PTR) mass spectrometry (MS) has been
applied to the real-time quantitative analysis of trace-level volatileorganic compounds in gaseous samples. The principle of thePTR-MS should be able to detect nonvolatile organic compounds if
we can transfer molecules into a PTR flow tube. Supercritical fluid
extraction (SFE) and chromatography (SFC) is a suitable candidate
method. We have found that the detection of ions up to ivermectin(molecular weight 875) was confirmed, and an extremely low quantification limit of 10-200 attomole was obtained for fatty acids.

▍ Background & Results
Supercritical fluid extraction (SFE) was introduced by Zosel et al.
in the late 1960 to 1970s. A well-known application of SFE is thatit selectively removes caffeine from coffee beans without changing
another component (US Pat. 3969196). This example shows thatSFE is a separation technique that selectively separates a component from a mixture by manipulating temperature and pressure
rather than a simple extraction. In addition, the SFE process can becompleted in a short period than extraction using organic solvents.
We have developed a small-scale SFE-PTR ionization systemprototype that uses a column in-line filter as an SFE vessel (1 μLvolume). A sample was applied on the stainless steel flit and thenplaced into a column filter, then performing SFE at 25 MPa that introduced the solute directory into a PTR tube in a vacuum.
The PTR-MS is capable of high detection sensitivity in fast
response time, the ability to ionize a wide range of organic compounds, and the ease of interpretation of the resulting mass spectrum. Although the PTR ionization has been investigated mainly forthe formation of protonated ions, the deprotonated reaction that thehigh sensitivity obtained for fatty acids.
To explore the method's applicability, we tried to measure various
substances shown in Table 1 and obtained low limit of quantitation(LOQ) for most of the tested compounds. In particular, we notedthat the low LOQs were obtained for fatty acids. Fat and fatty acids have a high affinity for scCO2 and are easy to extract from cellular organisms by SFE. We expect the technique to be applied fortrace level fatty acids monitoring in-situ on the biological tissues.
As summarized in the next section, the measurement of reaction intermediates known as arachidonic acid cascade by SFE/SFC-PTRMS in-situ in localized cells such as diseased cite is expected tolead to the development of physiology and to help cancer patientsin the future.

acids was way lower (better) than our expectations, possibly being
applied to the lipid metabolism study.
For example, a series of metabolites derived from arachidonic
acid, a type of fatty acid produced in the body, has been studied
in relation to pain, fever, cancer, and manic depression. Althoughthese fatty acids are widely distributed in the body, the amount offatty acids present in the body is small, and the extraction of fatty
acids from tissues is complicated, so it is not easy to measurechanges in the amount. If these fatty acids can be rapidly and sensitively quantified, in-situ analysis of diseased sites will be possible.
We hope this will contribute to the development of physiology.
Table 1: SFE-DI-PTR Limit of Quantitation
CompoundAcetaminophen
Phenacetin
Caffeine
PyreneOleic acid
Stearic acid
Linolic acidArachidic acidArachidonic acid
Tetracosanoic acidα-Tocopherol
Vitamine K1
γ-Oryzanol A
Reserpine
Ivermectin (B1a )mass
151.063
179.095
194.080202.078282.256284.272
280.240312.303
304.240368.365
430.381450.350
602.434
608.273874.508

log(P )[1]
2.0
2.0
-1.0
4.6
6.1
6.3
5.9
7.1
6.2
8.7
8.8
9.2
10.2
4.2
5.6

positive ion mode
4.9 fmol[M + H]+
1.4 fmol[M + H]+
0.008 fmol
[M + H]+
9.5 fmol[M + H]+––––––
63 fmol[M + H]+406 fmol[M + H]+–
8.3 fmol[M + H]+
2.3 fmol [M + NH4 ]+negative ion mode
274 fmol [M−H] –203 fmol [M−H] –––0.048 fmol [M−H] –10 fmol [M−H] –0.2 fmol [M−H] –27 fmol [M−H] –19 fmol [M−H] –17 fmol [M−H] –4 fmol [M−H] –41 fmol[M] –530 fmol [M−H] –8 pmol [M−H] ––References[1] Scott A. Wildman and Gordon M. Crippen. Prediction of Physicochemical Parameters by AtomicContributions. J. Chem. Inf. Comput. Sci., 39(5):868–873, September 1999.

1▍ Significance of the research and Future perspective
Since the beginning of this study, we have been supposed toobtain a high sensitivity for nonvolatile organic compounds using
supercritical carbon dioxide (scCO2) as a carrier fluid, which isinert to the PTR. However, the obtained result of LOQ for fatty

P a t e n t Japanese Patent Application No. 2021-017547
Hondo, Toshinobu; Ota, Chihiro; Miyake, Yumi et al. Analysis of nonvolatile molecules in supercritical carbon dioxide using proton-transfer-reaction ionizaTr e a t i s etion time-of-flight mass spectrometry. 2021 May 4; 93(17): 6589-6593. doi: 10.1021/acs.analchem.1c00898
URL https://resou.osaka-u.ac.jp/ja/research/2021/20210512_1
K e y w o r d mass spectrometry, supercritical fluid extraction/chromatography, proton transfer reaction ionization