High‐performance liquid chromatography evaluation of the enantiomeric purity of amino acids by means of automated precolumn derivatization with ortho‐phthalaldehyde and chiral thiols

Abstract

The use of ortho-phthalaldehyde (OPA) for the derivatization of amino acids (AA) is well known. It enables the separation of the derivatives on common reversed phase columns and improves the sensitivity with fluorescence detection. With the use of a chiral thiol an indirect enantioseparation of chiral amines and AAs is feasible. The major drawback of the OPA-derivatization is the poor stability of the products. Here, a method with an in-needle derivatization procedure is optimized to facilitate a quantitative conversion of the AA with OPA and the chiral thiols N-acetyl-L-cysteine or N-isobutyryl-L-cysteine, followed by a subsequent analysis, eluding the stability issue. Both enantiomers of a single AA were separated as OPA-derivatives with a pentafluorophenyl column and a gradient program consisting of 50 mM sodium acetate buffer pH = 5.0 and acetonitrile. Fluorescence detection is commonly used to achieve sufficient sensitivity. In this study, the enantiomeric impurity of an AA can be detected indirectly with common UV spectrophotometric detection with a limit of quantitation of 0.04%. Seventeen different L-AAs were tested and the amount of D-AA for each individual AA was calculated by means of area normalization, which ranged from not detectable up to 4.29%. The recovery of the minor enantiomer of L- and D-AA was demonstrated for three AAs at a 0.04% level and ranged between 92.3 and 113.3%, with the relative standard deviation between 1.7 and 8.2%.

Application of Normal-Phase Silica Column in Hydrophilic Interaction Liquid Chromatography Mode for Simultaneous Determination of Underivatized Amino Acids from Human Serum Samples via Liquid Chromatography–Tandem Mass Spectrometry

“…The gold standard for AA analysis was invented a few decades ago, using post-column derivatization with ninhydrin after ion-exchange chromatography [20]. Most pre-column derivatization techniques rely on reversed-phase liquid chromatography (RP-LC) separation using several derivatization agents, such as ortho-phthalaldehyde [21]. Often, derivatization methods are not convenient and are usually time-consuming.…”

Enantioselective high‐performance liquid chromatography of aryl‐substituted oxazolines as an efficient tool for determination of chiral purity of serine medicinal components

“…Alternatively, serine can undergo derivatization [9] by achiral agents, such as dansyl chloride [10][11][12] (Figure 1A), fluorenyl-containing reagents [13, 14], Sanger's reagent [15] or others [16,17] to enable sensitive detection by UV absorbance or fluorescence -these derivatizations lead to enantiomeric compounds, meaning the enantioseparation is still achieved by separation in a chiral environment. Lastly, derivatization with chiral agents, such as chiral thiols in combination with orthophthaldialdehyde (OPA) [18][19][20][21] (see Figure 1B), Marfey's reagent [22] or novel agents [23] yield diastereomeric compounds that can be separated on achiral columns, while also enabling detection by UV absorbance or fluorescence.…”

A Novel Determination Method of Thirty-Seven<i> o</i>-Phthalaldehyde-Derivatized D/L-Amino Acids with Complementary Use of Two Chiral Thiols by High Performance Liquid Chromatography

“…In general, various methods such as high performance liquid chromatography (HPLC) [4][5][6][7][8][9], gas chromatography (GC) [10][11][12], and capillary electrophoresis (CE) [13][14][15] are used to separate D/L-amino acids. Currently, LC/MS [16][17][18][19] and multi-dimensional LC having two or more dimensions [20][21][22][23][24][25] are used for this purpose because it is difficult to separate the proteinogenic D/L-amino acids under a single separation mode.…”

“…One is separating D/L isomers using a chiral column [17][18][19]26]. The other is converting D/L-amino acids into diastereomers using a chiral derivatizing reagent followed by the commonly used reversed phase separation [2,[4][5][6][7][8][9].…”