Separation and purification of short-, medium-, and long-stranded RNAs by RP-HPLC using different mobile phases and C18 columns with various pore sizes.

Nucleic acids, which have been employed in medicines for various diseases, are attracting attention as a new pharmaceutical model. Depending on the target substances, nucleic acid medicines with various nucleic acid chain lengths (several tens of nucleotides [nt] to several thousands of nt) exist. The purification of synthesized nucleic acids is crucial as various impurities remain in the crude product after synthesis. Presently, reversed-phase high-performance liquid chromatography (RP-HPLC) represents an effective purification method for nucleic acids. However, the information regarding the HPLC conditions for separating and purifying nucleic acids of various chain lengths is insufficient. Thus, this technical note describes the separation and purification of short-, medium-, and long-stranded nucleic acids (several tens of nt to thousands of nt) by RP-HPLC with various mobile phases and octadecyl-based columns with various pore sizes, such as normal (9-12 nm), wide (30 nm), and super wide (>30 nm) pores.

Simultaneous separation and identification of all structural isomers and enantiomers of aminobutyric acid using a highly sensitive chiral resolution labeling reagent.

Aminobutyric acid has structural isomers (α-, ß-, and γ-aminobutyric acids) and enantiomers (D/L-forms) with various unique functions. Therefore, a quantitative method for determining the content of each aminobutyric acid must be developed. In general, quantitative simultaneous analysis of multiple compounds is conducted via high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). However, simultaneous separation and highly sensitive detection of all aminobutyric acids are complicated, so highly sensitive analytical methods for the separation and identification of each compound have not yet been established. We previously developed highly sensitive chiral resolution labeling reagents. Herein, we propose a highly sensitive analytical method for the simultaneous separation and identification of all aminobutyric acids via LC-MS and labeling with our original highly sensitive chiral resolution labeling reagent, 1-fluoro-2,4-dinitrophenyl-5-L-valine-N,N-dimethylethylenediamine amide (L-FDVDA). The labeling reagent was completely bound to all aminobutyric acids through incubation overnight (>15 h) at 50 °C. Additionally, the labeled aminobutyric acids could be stored for at least 1 week at 4 °C. Furthermore, we demonstrated simultaneous separation and identification of aminobutyric acids in biological samples and foods through LC-MS using a C18 column after labeling with L-FDVDA. Our method is expected to be adopted for the analysis of the contents of all aminobutyric acids in biological and clinical samples as well as various foods.

Separation and Identification of Isoleucine Enantiomers and Diastereomers Using an Original Chiral Resolution Labeling Reagent.

D-Amino acids, which are present in small amounts in living organisms, are responsible for a variety of physiological functions. Some bioactive/biomolecular peptides also contain D-amino acids in their sequences; such peptides express different functions than peptides composed only of L-form amino acids. Among the 20 amino acids that make up proteins, threonine (Thr) and isoleucine (Ile) have two chiral carbons and thus have two enantiomers and diastereomers. These stereoisomers have been previously analyzed through HPLC using chiral columns or chiral resolution labeling reagents. However, the separation and identification of these stereoisomers are highly laborious and complicated. Herein, we propose an analytical method for the separation and identification of Ile stereoisomers through LC-MS using our original chiral resolution labeling reagent, 1-fluoro-2,4-dinitrophenyl-5-L-valine-N,N-dimethylethylenediamine-amide (L-FDVDA) and a PBr column packed with pentabromobenzyl-modified silica gel. Twenty DL-amino acids including Thr stereoisomers (41 amino acids including glycine) were separated and identified using C18 column. Ile stereoisomers could be separated using not a C18 column but a PBr column. Additionally, we showed that peptides containing Thr and Ile stereoisomers can be accurately detected through labeling with L-FDVDA.

Separation of highly hydrophilic nicotinamide metabolites using a COSMOSIL PBr column.

Highly hydrophilic compounds such as nicotinamide metabolites are very difficult to separate via high-performance liquid chromatography (HPLC) using octadecyl (C18) columns. In general, for the separation of hydrophilic compounds, hydrophilic interaction liquid chromatography (HILIC) columns are used instead of reversed phase chromatography using C18 columns. However, HILIC columns generally obey complex separation mechanisms because ionic interactions are involved in the retention process, which hinders the optimization of the separation conditions. Additionally, the resulting peak shapes are disturbed when large amounts of aqueous samples are injected. This study demonstrates that COSMOSIL PBr columns, in which both hydrophobic and dispersive interactions occur, show high retention for various hydrophilic compounds under similar separation conditions as those used with C18 columns. Specifically, using a COSMOSIL PBr column, 11 nicotinamide metabolites could be separated under simpler conditions than those used previously with C18 columns, affording better peak shape for each compound. The applicability of the method was evaluated using a tomato sample, from which the nicotinamide metabolites were successfully separated. The results show that the COSMOSIL PBr column is a good alternative to the C18 column for a good separation of all the peaks, including impurity peaks.

Separation of amyloid ß fragment peptides with racemised and isomerised aspartic acid residues using an original chiral resolution labeling reagent.

We developed a system to separate and identify racemised and isomerised aspartic acid (Asp) residues in amyloid ß (Aß) by labeling with an original chiral resolution labeling reagent, 1-fluoro-2,4-dinitrophenyl-5-D-leucine-N,N-dimethylethylenediamine-amide (D-FDLDA). The racemised and isomerised Asp residues labeled with D-FDLDA in Aß fragments generated by digesting with trypsin and endoproteinase Glu-C were separated and identified by liquid chromatography-mass spectrometry (LC-MS) under simple gradient conditions. Furthermore, the labeled Aß fragments did not aggregate and remained stable at least for 1 week at 4 °C.

Separation of long-stranded RNAs by RP-HPLC using an octadecyl-based column with super-wide pores.

Messenger ribonucleic acids (mRNAs) have been used in vaccines for various diseases and are attracting attention as a new pharmaceutical paradigm. The purification of mRNAs is necessary because various impurities, such as template DNAs and transcription enzymes, remain in the crude product after mRNA synthesis. Among the various purification methods, reversed-phase high-performance liquid chromatography (RP-HPLC) is currently attracting attention. Herein, we optimized the pore size of the packing materials, the mobile phase composition, and the temperature of the process; we also evaluated changes in the separation patterns of RNA strands of various lengths via RP-HPLC. Additionally, single-stranded (50-1000 nucleotides in length) and double-stranded (80-500 base pairs in length) RNAs were separated while their non-denatured states were maintained by performing the analysis at 60 °C using triethylammonium acetate as the mobile phase and octadecyl-based RNA-RP1 with super-wide pores (> 30 nm) as the column. Furthermore, impurities in a long-stranded RNA of several thousand nucleotides synthesized by in vitro transcription were successfully separated using an RNA-RP1 column. The columns used in this study are expected to separate various RNA strands and the impurities contained in them.

Separation of nicotinamide metabolites using a PBr column packed with pentabromobenzyl group modified silica gel.

Nicotinamide adenine dinucleotide, a coenzyme involved in the activation of sirtuins, contributes to various regulations in vivo. However, highly hydrophilic nicotinamide metabolites are difficult to separate by high-performance liquid chromatography (HPLC) using octadecyl (C18) columns, which operate via hydrophobic interaction. PBr columns packed with silica gel modified with the pentabromobenzyl group having strong dispersion forces show good retention ability for various highly hydrophilic compounds. Additionally, the peak shape obtained with the PBr column did not collapse like that of the HILIC column, even when a large amount of water was injected. Separation of 11 highly hydrophilic nicotinamide metabolites using a PBr column under simple conditions resulted in baseline separation, but separation on a C18 column was not complete. The peak shape for each compound was better than that in previous studies. Furthermore, the separation of nicotinamide metabolites in tomato using a PBr column enable a more sensitive detection than that using a C18 column. SUBJECT CATEGORY: Chromatographic Technique.

Separation and identification of the DL-forms of short-chain peptides using a new chiral resolution labeling reagent.

There are several reports of D-amino acids being the causative molecules of serious diseases, resulting in the formation of, for example, prion protein and amyloid ß. D-Amino acids in peptides and proteins are typically identified by sequencing each residue by Edman degradation or by hydrolysis with hydrochloric acid for amino acid analysis. However, these approaches can result in racemization of the L-form to the D-form by hydrolysis and long pre-treatment for hydrolysis. To address these problems, we aimed to identify the DL-forms of amino acids in peptides without hydrolysis. Here, we showed that the DL-forms in peptides which are difficult to separate on a chiral column can be precisely separated by labeling with 1-fluoro-2,4-dinitrophenyl-5-D-leucine-N,N-dimethylethylenediamine-amide (D-FDLDA). Additionally, the peptides could be quantitatively analyzed using the same labeling method as for amino acids. Furthermore, the detection sensitivity of a sample labeled with D-FDLDA was higher than that of the conventional reagents Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide (L-FDAA) and Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide (L-FDLA) used in Marfey's method. The proposed method for identifying DL-forms of amino acids in peptides is a powerful tool for use in organic chemistry, biochemistry, and medical science.

Comparison of Retention Behavior between Supercritical Fluid Chromatography and Normal-Phase High-Performance Liquid Chromatography with Various Stationary Phases.

The retention behavior of a wide variety of stationary phases was compared in supercritical fluid chromatography (SFC) and normal-phase high-performance liquid chromatography (NP-HPLC). We also attempted to elucidate the retention behavior in SFC by investigating the selectivity of the different stationary phases. SFC separation conditions with polar stationary phases, such as silica gel (SL) and diol (Diol) phases, operate via adsorptions that include hydrophilic and ionic interactions similar to those in NP-HPLC. Moreover, non-polar stationary phases, such as pentabromophenyl (PBr), pyrenylethyl (PYE), and octadecyl (C18), could be used despite the non-polar mobile phase conditions, because the dispersion and π-π interactions were stronger in SFC than in HPLC. These results reflect the selectivity of the stationary phase and its retention factor, thus providing useful information for the selection of appropriate stationary phases for particular analytes.