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A novel ionization technique named medium vacuum chemical ionization (MVCI) mass spectrometry (MS), which is a chemical ionization using oxonium (H 3 O + ) and hydroxide (OH - ) formed from water, has excellent compatibility with the supercritical fluid extraction (SFE)/supercritical fluid chromatography (SFC). We have studied a method to determine free fatty acids (FFAs) in a small section of bovine liver tissue using SFE/SFC-MVCI MS analysis without further sample preparation. A series of FFA molecules interact with the C18 stationary phase, exhibiting broad chromatographic peaks when using a non-modified CO 2 as the mobile phase. It can be optimized by adding a small content of methanol to the mobile phase as a modifier; however, it may dampen the ionization efficiency of MVCI since the proton affinity of methanol is slightly higher than water. We have carefully evaluated the modifier content on the ion detection and column efficiencies. The obtained result showed that an optimized performance was in the range of 1 to 2% methanol-modified CO 2 mobile phase for both column efficiency and peak intensity. Higher methanol content than 2% degrades both peak intensity and column efficiency. Using optimized SFC conditions, a section of bovine liver tissue sliced for 14 µm thickness by 1 mm square, which is roughly estimated as about 3300 hepatocytes, was applied to determine 18 FFAs amounts for carbon chains of C12-C24. An amount of each tested FFA was estimated as in the range of 0.07 to 2.6 fmol per cell., (Copyright ©2024 Toshinobu Hondo, Yumi Miyake, and Michisato Toyoda.)

A method for the rapid determination of α -tocopherol ( α -T) and its oxidative products in plant tissue has been developed using supercritical fluid extraction (SFE) coupled with supercritical fluid chromatography (SFC) and medium vacuum chemical ionization (MVCI) with tandem mass spectrometry. The method is designed to study changes in levels for α -T and its oxidative products in plant cells during photosynthesis, aiming to observe the light response curves. α -T oxidation is a non-enzymatic self-defense mechanism in plant cells. Unlike enzyme-involved reactions, it cannot be stopped, so the oxidation continues in crude extracts even after extraction. Therefore, a real-time in-situ method is essential for tracking the light response curves. To optimize the selective reaction monitoring method, the reaction mixture of α -T and singlet oxygen ( 1 O 2 ), generated by rose Bengal under light illumination, was used as the source of oxidative products. The relative abundance changes in α -tocopherylquinone and 8a-hydroperoxy tocopherone in Pisum sativum L. (Pea) leaves under excessive light illumination have been preliminarily analyzed as part of the light response curve study. The method archives a throughput of 10-15 minutes for analyzing duplicate leaf samples. This process includes cutting off the leaf, sectioning it, placing the sample in a frozen SFE vessel, and conducting SFE/SFC analysis. Consequently, the average throughput is approximately 5-7 minutes per sample., (Copyright © 2024 Toshinobu Hondo, Yumi Miyake, and Michisato Toyoda.)

We have developed a rapid and sensitive analytical method for α-tocopherol and its oxidative products by combining online hyphenation of supercritical fluid extraction-supercritical fluid chromatography (SFC) with proton transfer reaction (PTR) ionization mass spectrometry (MS). α-Tocopherol is a well-known antioxidant that plays a vital role in the antioxidant defense system in plant cells. However, studies on the cellular mechanisms of α-tocopherol have been limited owing to the lack of a rapid analytical method, which limits the comparison of plant cells incubated in various conditions. Additionally, complex sample preparation and long chromatography separation times are required. Moreover, the majority of the involved molecules are a combination of isomers, which must be separated before applying tandem MS. α-Tocopherol produces the α-tocopheroxyl radical in the first step of its antioxidant function; another ion with the same mass may also be generated from the source. SFC separation effectively distinguished the observed ions from their oxidative products in the sample and those produced during the ionization reaction process. This method enabled the measurement of α-tocopherol and its oxidative products such as α-tocopheroxyl radical and α-tocopheryl quinone in approximately 3 min per sample, including the time required for sample preparation., (Copyright © 2022 Chihiro Ota, Toshinobu Hondo, Yumi Miyake, Hiroshi Furutani, and Michisato Toyoda.)

Boron neutron capture therapy (BNCT) is a cell-selective particle therapy for cancer using boron containing drugs. Boron compounds are accumulated in high concentration of tens ppm level of boron in target tumors to cause lethal damage to tumor tissue. The examination of boron distribution in target tumor and normal tissue is important to evaluate the efficiency of therapy. The matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful tool to visualize the distribution of target analyte in biological samples. In this manuscript, we report a trial to visualize the distribution of a typical BNCT drug, L-4-phenylalanine boronic acid (BPA) in a brain tumor model rat using MALDI-MSI technique. We performed a MALDI-MSI with high mass resolution targeting to [BPA+H] + at m / z 210 in a BPA-treated rat brain section using a spiral orbit-type time of flight (SpiralTOF) mass spectrometer. Several BPA ion species, [BPA+H] + , [BPA-H 2 O+Na] + , [BPA+DHB-2H 2 O+Na] + and [BPA+DHB-2H 2 O+K] + were detected separate from peaks originated from biomolecules or matrix reagent by achieving the mass resolving power of approximately 20,000 (full width at half maximum; FWHM) at m / z 210. The mass images with 60 μm spatial resolution obtained from these BPA ion species in a mass window of 0.02 Da revealed their localization in the tumor region. Additionally, the mass image obtained from [BPA+H] + also likely showed the distribution of BPA inside the tumor. MALDI-MSI with high mass resolution targeting to [BPA+H] + has a great potential to visualize the distribution of BPA in brain tissue with tumor., (Copyright © 2022 Yumi Miyake, Sachie Kusaka, Isao Murata, and Michisato Toyoda.)

Proton-transfer-reaction (PTR) mass spectrometry (MS), a widely used method for detecting trace-levels of volatile organic compounds in gaseous samples, can also be used for the analysis of small non-volatile molecules by using supercritical fluid as a transporter for the molecules. Supercritical fluid extraction (SFE) is a method that permits lipophilic compounds to be rapidly and selectively extracted from complex matrices. The combination of the high sensitivity of PTR MS with the SFE is a potentially novel method for analyzing small molecules in a single cell, particularly for the analysis of lipophilic compounds. We preliminarily evaluated this method for analyzing the components of a single HeLa cell that is fixed on a stainless steel frit and is then directly introduces the SFE extracts into the PTR MS. A total of 200/91 ions were observed in positive/negative ion mode time-of-flight mass spectra, and the masses of 11/10 ions could be matched to chemical formulae obtained from the LipidMaps lipids structure database. Using various authentic lipophilic samples, the method could be used to detect free fatty acids in the sub-femtomole to femtomole order in the negative ion mode, the femtomole to sub-picomole order for fat-soluble vitamins, and the picomole order for poly aromatic hydrocarbons in both the positive and negative ion mode., (Copyright © 2022 Toshinobu Hondo, Chihiro Ota, Kohta Nakatani, Yumi Miyake, Hiroshi Furutani, Takeshi Bamba, and Michisato Toyoda.)