Perfluoroalkyl-containing compounds show a unique “fluorous” property, which is related to the specific affinity, i.e. fluorophilicity, between perfluoroalkyl groups. Therefore, fluorous compounds can be easily and selectively isolated from non-fluorous compounds utilizing liquid‒liquid extraction, solid phase extraction, and liquid chromatographic techniques via their fluorophilicity. We have also developed novel methods for the selective analysis of biogenic-related compounds using fluorous separation techniques. These methods involve the attachment of perfluoroalkyl groups to target analytes, followed by their selective separation from endogenous biological matrices with appropriate fluorous separation techniques. Because the perfluoroalkylated compounds are generally not included in biological matrices, highly selective analysis of perfluoroalkyl-attached target analytes is possible. This review summarizes our recent works concerning biogenic-related compound analysis methods using fluorous separation techniques.
A novel method for the determination of semi-volatile polycyclic aromatic hydrocarbons (PAHs) in gaseous samples was developed using a partition-based extraction device. The target compounds were collected and concentrated on the basis of partitioning between air and octadecyl (C18) chains bound to macroporous silica particles. The extracted PAHs were rapidly desorbed by just passing 10 mL of acetone. The desorption solvent was then subjected to gas chromatography-mass spectrometry (GC-MS) to determine PAHs. On the basis of the quantitative evaluation of the extraction and desorption conditions for 15 PAHs, the benefits of the partition-based extraction for typical airborne PAHs were clearly indicated. Finally, the developed method was successfully applied for the determination of PAHs in tobacco side-stream smoke.
For the simultaneous determination of phenylalanine (Phe), tyrosine (Tyr) and 3,4-dihydroxyphenylalanine (DOPA) enantiomers, a fully-automated two-dimensional high-performance liquid chromatographic (2D-HPLC) system using pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) has been developed and validated. These three amino acids are related to the aromatic amino acid metabolic pathways, and a sensitive and enantioselective analytical method is required to evaluate their intrinsic amounts and metabolism. For the reversed-phase separation in the 1st dimension, a monolithic ODS column (0.53 mm x 1000 mm) was used and a Pirkle-type column, KSAACSP-001S (1.5 mm x 250 mm), was used for the 2nd dimension. The 2D-HPLC system was applied to the determination of the Phe, Tyr and DOPA enantiomers in the urine of D-amino acid oxidase (DAO) deficient mice and control C57BL mice. As for Phe and Tyr, all of the 4 enantiomers were found in the urine of both groups of mice, and the values of %D-Phe (D-Phe/Phe x 100) were significantly higher in the DAO deficient mice than those in the control mice. Concerning the DOPA enantiomers, no peaks were observed in the urine of the C57BL mice and DAO deficient mice. On the other hand, a significant amount of D-DOPA was found in their urine after the intraperitoneal administration of racemic DOPA. These results indicate that the present 2D-HPLC system is a powerful tool for the determination of aromatic amino acid enantiomers in biological matrices, and the ratio of Phe enantiomers in the urine could be one of the non-invasive indicators of DAO activity.
We have developed a high-resolution chiral derivatization-LC method for the determination of D- and L-amino acids using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as an enantioseparation enhancer. In this method, D- and L-amino acids were derivatized with (S)-(-)-1-(naphthyl)ethylamine (NEA) in the presence of DMT-MM as a condensing agent. These conditions resulted in the amino group of the D- and L-amino acid-NEA derivative being introduced in the triazine unit of DMT-MM, producing sterically bulky diastereoisomers. The resulting derivatives of the D- and L-amino acids could be discriminated and successfully enantioseparated through reversed-phase LC. This enantioseparation enhancement effect was demonstrated when using six other commercially available chiral amines as derivatization reagents. Since triazine modification possesses high ESI-responsivity, they showed higher sensitivities than non-triazine derivatives. The developed derivatization method was successfully applied to highly sensitive quantification of D- and L-amino acids by combining LC-MS/MS analysis.
In this article, we report a simple and rapid immunoassay based on microscale electrophoresis using a microchip equipped with a reagent-release cartridge at the sample reservoir. The cartridge consists of an outer tube, a paper filter retaining dried reagents and an antibody, and inner tubes to fix the paper filter. When a sample solution containing an antigen is introduced into the sample reservoir through the reagent-release cartridge, the reagents and the antibody retained on the filter dissolve immediately into the sample solution. The sample solution and reagents containing the antibody are then mixed, promoting rapid immunoreaction between the antigen and the antibody. After sample introduction into the reservoir, the antigen-antibody complex is rapidly separated from the free antibody by microchip zone electrophoresis. The immunoassays for bovine serum albumin and human IgG were easily performed merely by pouring the sample solution into the cartridge in the newly developed device followed by microchip electrophoresis, resulting in the rapid and clear separation of the immunocomplexes from the free antibodies. These results showed that the developed device was applicable to a rapid and simple immunoassay.
Drug-induced phospholipidosis (DIPL) is a phospholipid-hyperaccumulated state in cells, tissues and organs after treatment with cationic amphiphilic drugs (CADs). The biological meaning of phospholipidosis remains unknown. Although a hypothesis has been proposed that DIPL is a biological defense from CADs, experimental data supporting the hypothesis are poor. In this study, we compared CAD toxicity with RAW264 cells with/without egg phosphatidylcholine (egg PC) treatment to identify the role of phospholipids in DIPL. The 50% inhibitory concentrations of chlorpromazine, imipramine and propranolol were significantly increased by egg PC, in accordance with the hypothesis. Then, DIPL biomarkers, bis(monoacylglycero)phosphate (BMP) and the ratio of phosphatidylinositols (PIs) were measured to study the changes of these biomarkers by the relief of CAD toxicity with egg PC. BMP 22:6_22:6 was not increased by imipramine treatment alone due to imipramine toxicity. Co-treatment of egg PC with imipramine did not decrease BMP 22:6_22:6, which was inconsistent with the relief of toxicity. Conversely, the ratio of PIs increased by imipramine treatment alone, whereas it decreased by co-treatment with egg PC. These results suggest that the ratio of PIs reflects cellular phospholipid accumulation concurrent with CAD toxicity and that it is useful as a DIPL biomarker with high specificity.
Quantitative analysis of a kokumi peptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) in fish sauces fermented with koji (grain steamed and fermented with Aspergillus oryzae) was performed using high performance liquid chromatography / tandem mass spectrometry (LC/MS/MS) after the derivatization with 6-aminoquinolyl-N-hydroxy-succinimidyl-carbamate (AQC). Four brands of commercial fish sauce fermented with koji were analyzed, and γ-Glu-Val-Gly was detected in all four sauces. The γ-Glu-Val-Gly content was below the limit of quantification in one of the four sauces, while the content of this peptide ranged from 1.7 mg/L to 5.7 mg/L in the other three sauces. These results indicate that γ-Glu-Val-Gly may be widely present in fish sauces fermented with koji.