Quantitation of underivatized branched-chain amino acids in sport nutritional supplements by capillary electrophoresis with direct or indirect UV absorbance detection

Abstract

The branched-chain amino acids (BCAAs) including leucine (Leu), isoleucine (Ile) and valine (Val) play a pivotal role in the human body. Herein, we developed capillary electrophoresis (CE) coupled with conventional UV detector to quantify underivatized BCAAs in two kinds of sport nutritional supplements. For direct UV detection at 195 nm, the BCAAs (Leu, two enantiomers of Ile and Val) were separated in a background electrolyte (BGE) consisting of 40.0 mmol/L sodium tetraborate, and 40.0 mmol/L β-cyclodextrin (β-CD) at pH 10.2. In addition, the indirect UV detection at 264 nm was achieved in a BGE of 2.0 mmol/L Na2HPO4, 10.0 mmol/L p-aminosalicylic acid (PAS) as UV absorbing probe, and 40.0 mmol/L β-CD at pH 12.2. The β-CD significantly benefited the isomeric separation of Leu, L- and D-Ile. The optimal conditions allowed the LODs (limit of detections) of direct and indirect UV absorption detection to be tens μmol/L level, which was comparable to the reported CE inline derivatization method. The RSDs (relative standard deviations) of migration time and peak area were less than 0.91% and 3.66% (n = 6). Finally, CE with indirect UV detection method was applied for the quantitation of BCAAs in two commercial sport nutritional supplements, and good recovery and precision were obtained. Such simple CE method without tedious derivatization process is feasible of quality control and efficacy evaluation of the supplemental proteins.

Determination of Branched-Chain Amino Acids in Food Supplements and Human Plasma by a CE-MS/MS Method with Enhanced Resolution

“…The CE methods used in analysis of BCAAs are typically hyphenated with MS detection [ 22 , 25 , 57 , 58 ], but some papers refer to the use of UV [ 32 ], laser induced fluorescence (LIF) [ 58 ], or capacitively coupled contactless conductivity detection (C 4 D) [ 30 ]. The CE-LIF method required conversion of the analytes into their fluorescently active derivatives using an expensive off-line derivatization procedure [ 58 ].…”

“…The CE-UV methods (direct and indirect UV detection) used for determination of BCAAs in pharmaceutical sample suffered from poor resolution between Ile and Leu (resolution was in the range 0.8 to 1.0). Moreover, there was a need to implement various BGE additives into the separation environment (e.g., cyclodextrines), which made the optimization procedure more challenging [ 32 ]. The published CE-MS methods were characterized by time of analysis in the range of 20–40 min and LOD values from 0.6 to 200 µM.…”

“…To the best of our knowledge, no study has been conducted and published dealing with such an advanced approach in this area. The quality control of a nutritional supplement containing BCAAs has been performed only with the use of capillary zone electrophoresis [ 32 ] or micellar electrokinetic chromatography (MEKC) with UV detection [ 33 , 34 ]. However, the demand for accurate and highly reliable analytical methods capable of ensuring and proving the high quality of food supplements or pharmaceuticals is still a subject of interest.…”

Underivatized Amino Acid Chromatographic Separation: Optimized Conditions for HPLC-UV Simultaneous Quantification of Isoleucine, Leucine, Lysine, Threonine, Histidine, Valine, Methionine, Phenylalanine, Tryptophan, and Tyrosine in Dietary Supplements

“…Few methods have been described for the chromatographic determination of underivatized AAs in different samples such as amino acids of plant extracts using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–MS/MS), amino acids in plasma, urine, and cerebrospinal fluid using LC–MS/MS, and amino acids in serum, plasma, cerebrospinal fluid, and tissue homogenates using ultrahigh-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) . However, fewer methods have been reported for the quantification of AAs in nutritional supplements almost using capillary electrophoresis, HILIC-UV, and in parenteral nutritional solutions. , There are two challenges in the quantitative determination of underivatized AAs: the first is the separation process as most AAs are very close in polarity and structure Figure , all have weak UV absorbance, and basic, acidic, and neutral (zwitterionic) behaviors. The second limitation lies in the detection technology, but the detector development provides new opportunities in native AA analysis …”

Molecular dynamics simulations of amino acid adsorption and transport at the acetonitrile–water–silica interface: the role of side chains

“…Therefore, a good understanding of the protein retention and partition mechanism in LC requires a thorough investigation of the interactions between amino acid molecules or residues and the stationary phase. In the past decades, separation methods for amino acids have developed rapidly including liquid chromatography [11][12][13][14][15] and capillary electrophoresis, [16][17][18] combined with analysis techniques such as mass spectrometry, 12,13,17,19 nuclear magnetic resonance, 20 ultraviolet and uorescence detection, 15,18 and amino acid analyzer. 21 In particular, as the most commonly used separation method in biochemistry, the hydrophilic interaction liquid chromatography (HILIC), which is comprised of polar stationary phase and aprotic mobile phase, is widely utilized to separate the amino acids samples.…”