3‐(4‐Carboxybenzoyl)quinoline‐2‐carboxaldehyde labeling for direct analysis of amino acids in plasma is not suitable for simultaneous quantification of tryptophan, tyrosine, valine, and isoleucine by CE/fluorescence

Abstract

Capillary electrophoresis coupled to LED-induced fluorescence detection is a robust and sensitive technique used for amino acids (AA) analysis in biological media, after labeling with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA). We wanted to quantitate in plasma tryptophan (Trp), tyrosine (Tyr), valine (Val), and isoleucine (Ile). Among the different labeled AA-CBQCA, Trp has the lowest fluorescence yield, which makes its detection and quantification very difficult in biological samples such as plasma. We tried to improve Trp analysis by CE/LED-induced fluorescence detection to its maximal sensitivity by using large volume sample stacking as a preconcentration step in our analytical protocol. At pH 9.5, this step caused a drop in resolution during the separation of the four AAs and it was therefore necessary to work at pH 10. We have found that Tyr, Val, Ile, and Trp are detected and well separated from the other AAs, but Trp cannot be quantified in plasma samples, mainly because of the low fluorescence yield of the Trp-CBQCA derivative. The recorded LOD is 0.18 μM for Trp-CBQCA in standard solution with a resolution between Trp and Tyr of 1.2, while the LOD is 6 μM in plasma with the same resolution. Trp, Tyr, Val, and Ile are, however, efficiently quantified when using a 3 M acetic acid electrolyte and CE associated with capacitively coupled contactless conductivity detection, which also has the advantage of not requiring derivatization or large volume sample stacking. This article demonstrates, for the CE user, that quantitative analysis of these four AA in mouse plasma can be performed by CE-fluorescence after CBQCA labeling, with the exception of Trp. It can be advantageously replaced by CE/capacitively coupled contactless conductivity detection, the only efficient one for Trp, Tyr, Val, and Ile quantification. In this case, the LOD for Trp is 2 μM. The four AAs are separated with resolution with neighbors above 1.5.

Application of capillary electrophoresis with capacitively contactless conductivity detection for biomedical analysis

“…The majority of AAs do not produce signals in UV/VIS photometric or fluorescence detection and therefore it is very favorable to employ universal capacitively coupled contactless C 4 D. The application of CE with C 4 D for analysis of AAs in biological fluids did not require derivatization of the analyte [34, 109,110]. Many research articles investigating the applications of CE with C 4 D for determination of AAs in various biological fluids reported in scientific literature [89,[111][112][113][114][115][116][117][118][119][120][121][122][123][124][125][126][127]. Summaries of these applications are shown in Table 3.…”

“…The baseline of HAc is found to be stable; therefore, HAc BGEs have been applied in many applications for the separation of AAs as well as biogenic amines [111][112][113][114][115][116][117][118][119][120][121][122][123][124][125][126][127]. The use of HAc with hydroxypropyl-βcyclodextrin and tween at pH 2.6 and with dextran for the analysis of carnitine and acylcarnitine [119] and heparin [122]. The details on analyses of these compounds with CE-C 4 D can be found in Table 3.…”

“…Underivatized AAs 2.3 M HAc and 0.1% HEC 9.1 and 29 mM [111] AAs 1.7 M HAc and 0.1% HEC, pH 2.15 1.5 mM arginine and 6.7 mM aspartic acid [112] Proteinogenic AAs 1.7 M HAc and 0.1% HEC, pH 2.2 4.3-42.9 mM [113] 3-Methyl-histidine (3-MH) and 1-methylhistidine (1-MH) BGE I (500 mM HAc, 20 mM MCE 26.4-18.3 mM [114] NH 4 OH and 0.1% m/v HEC, pH 3.4 and BGE II 100 mM MES, 25 mM LiOH, and 0.1% m/v HEC, pH 5.5 G-Hydroxybutyric acid (GHB) 20 mM of arginine, 10 mM maleic acid, 30 mM of CTAB 5 mM vancomycin 2 mg/mL [115] AAs 0.5 M HAc, pH 2.5 0.75-2.5 mM [116,117] AAs 2.5 M HAc 0.15-10 mM [118] Carnitine and acylcarnitines 5.0 mM HAc, 1.0 mM HPβCD, and 0.05% Tween, pH 2.6. 1.0 and 3.2 mM [119] 28 Biogenic AAs 0.5-10 M HAc 0.1-1.7 mM [120] Sarcosine 10 mM TEA LOD = 0.034 mM and LOQ = 0.069 mM [121] Heparin 0.8 M HAc and 1% (w/v) dextran (100 kDa) 1.3 µM [ 122] Trp, Tyr, Val, Ile 3.2 M HAc 2-4.3 µM [123] Asymmetric and symmetric dimethylarginine 750 mM HAc/pH 2 45/PAMAPTAC-coating 16-22 nM [124] Cysteine, homocysteine, glutathione, methionine 4.0 M HAc 0.15-1.5 ng/mL [125] Phe, Tyr CE 1.6 M HAc/1 mM 18C6/0.1% Tween 20/pH 2.1 0.46-0.59 µM [ 126] Branched chain amino acids (BCAAs) 3.2 M HAc and 13% v/v methanol 0.7-0.9 µmol/L [127] Abbreviations: BGE, background electrolytes; CTAB, cetyltrimethylammonium bromide; HAc, acetic acid; HEC, hydroxyethyl cellulose; HPβCD, hydroxypropylβ-cyclodextrin; LOD, limit of detection; LOQ, limit of quantitation; MES, morpholinoethanesulfonic acid; TEA, triethylamine.…”

“…Further the same research group [119] demonstrated the analysis of 28 biogenic AAs in short capillary within 6 min, with LODs in range of 0.1–1.7 µmol/L. The baseline of HAc is found to be stable; therefore, HAc BGEs have been applied in many applications for the separation of AAs as well as biogenic amines [111–127]. The use of HAc with hydroxypropyl‐β‐cyclodextrin and tween at pH 2.6 and with dextran for the analysis of carnitine and acylcarnitine [119] and heparin [122].…”

Applications of capillary electrophoresis in the fields of environmental, pharmaceutical, clinical, and food analysis (2019–2021)

“…With the continuous development of pretreatment techniques, CE combined with different detection methods has been widely used in various fields. The researchers investigate the implementations and advancements of CE in recent three years and found that CE was mainly coupled with UV detection [15], fluorescence detection [16], MS [17][18][19], and electrochemical detection [20] including conductivity detection (CD) [21,22] and amperometric detection (AD) [23] for the analysis of some substances in different fields. These fields mainly consist of environmental analysis [24], pharmaceutical analysis [25][26][27], clinical analysis [28,29], and food analysis [30,31].…”