Abstract
This chapter illustrates the usefulness of capillary electrophoresis (CE) for the analysis of amino acids, and both normal and chiral separations are covered. In order to provide a general description of the main results and challenges in the biomedical field, some relevant applications and reviews on CE of amino acids are tabulated. Furthermore, some detailed experimental procedures are shown, regarding the CE analysis of amino acids in body fluids, in microdialysate, and released upon hydrolysis of proteins. In particular, the protocols will deal with the following compounds: (1) underivatized aminoacids in blood; (2) γ-Aminobutyric acid, glutamate, and L-Aspartate derivatized with Naphthalene-2,3-dicarboxaldehyde; (3) hydrolysate from bovine serum albumine derivatized with phenylisothiocyanate. By examining these applications on real matrices, the capillary electrophoresis efficiency as tool for Amino Acid analysis can be ascertained.
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Key words
- Capillary zone electrophoresis
- Micellar electrokinetic chromatography
- Amino acids
- Chiral separations
- Bioanalytical applications
1 Introduction
One of the primary goals of biomedical research is the study of new methods for the early diagnosis of a disease, able to follow its progression and to evaluate the therapeutic efficacy of a treatment. Many diseases, however, have a complex and multifactorial behaviour and their diagnosis and understanding cannot be based only on the evaluation of a single molecular marker. Among the whole small molecules produced by metabolism encompassed in metabolomics , amino acids have a key role. The protein “building blocks” are also essential precursors of important biomolecules including nucleotides and nucleotide coenzymes such as heme, a variety of hormones and neurotransmitters; therefore, given the important role in metabolism, it is possible, by carrying out the analysis of the amino acid profile, to get useful indications on various metabolic processes and the functioning of liver, kidneys, heart, or immune system. Amino acids differing from basal levels, in fact, are often symptoms of diseases, as in phenylketonuria and cystinuria. As a result, amino acids are an important target for the “metabolic profiling,” being frequently present in biological matrices and several methods have been developed to identify the presence and measure the concentration with the aim to perform screening of various diseases. From the analytic point of view, on considering that AAs have different behavior (acidic, neutral, or basic) and many of them lack of a strong chromophore, their determination is both interesting and intriguing and has been widely pursued by many authors with the issue of determining more amino acids in a single run with lower detection limits. Another related boosting analytical field is the chiral separation of amino acids: the structure and symmetry of organic molecules plays a crucial role in different biochemical processes but many methods validated for the quantification of amino acids are not stereoselective. For some time the free amino acids in biological matrices were typically quantified as the sum of their enantiomers, because in terrestrial organisms they exist in the form of stereoisomers L. The amino acids of the series D had been identified only in few peptides , such as those of the cell wall of the bacteria. With the increased understanding of the biological significance of the D-amino acids, interest in enantioselective quantification is increased and more applications of chiral CE to real problems of biomedical importance find attention.
The wealth of papers continuously published on amino acids analysis is continuously reviewed and a review of the reviews could be useful to evidence the peculiar aspects each time tracked. Presently, it is important to mention the general reviews published every 2 years in Electrophoresis [1–3].
Aiming at having a general overview, few significant methods, mainly related to biomedical applications and set up to separate and detect AAs, have been collected together with the most relevant experimental information in two tables handling normal [4–66] and enantiomeric [67–94] separations, respectively. Within each table both capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) have been described. All the referred methods also share the properties of being carried out on simple, commercial instruments, and, as a rule, they could be transferred on every CE system.
Different matrices, as far as the biomedical applications are concerned, have been analyzed: physiological fluids like urine, saliva, or plasma; cells like lymphocyte or erythrocyte; neurotransmitters and different hydrolysates (see column “Matrix” in Tables 1 and 2). Moreover, the hyphenation of microdialysis with the CE apparatus deserves attention as it allows both continuous and in vivo analysis: a fraction of the analytes can diffuse through the membrane dialysis and depending on them, temperature and probe characteristics, a definite recovery is attainable and quantitative measurements can be accomplished. Also fast monitoring of AAs is carried out by hyphenating microdialysis with CE: in particular if LIF is used as detector, thanks to the high separation efficiency and low volume sample requirement of CE and the very low detection limits of LIF detector, the sampling rate has been increased to levels not attainable by other analytical common techniques, like HPLC, and short-lasting changes in AAs concentration could be recorded. Moreover, to decrease the overall cost of the detector, the use of LED sources for fluorescence detectors is possible [24, 35, 90, 91].
As already pointed out, detection is a general concern, common to all the different separation schemes. Indeed, nonaromatic nonderivatized amino acids can only be efficiently detected by means of indirect methods; upon derivatization instead, the selected dye /tag determines the appropriate or most useful detector. Derivatization can be effectively employed to overcome both the lack of a chromophore on many AAs and the interferences caused by extraneous compounds in real samples: it results in both improved detection sensitivity and selectivity. Hence the choice of a derivatization reagent is of crucial importance and high demands are therefore put on its properties. Different approaches have been devised: precapillary and in-capillary . Precapillary derivatization is time consuming as it requires batch procedures but it is affordable and widely diffused (see tables). In-capillary (or on-column) derivatization [24, 63] is classified into either “on-site in-capillary derivatization” or “throughout in-capillary derivatization.” In the former the inlet of a separation capillary is used as a reaction chamber and the reaction is performed by introducing an analyte into the capillary between two plugs of labeling reagent. In the latter, the separations and derivatizations of analytes are performed simultaneously during the electromigration of native analytes in a separation capillary tube filled with a run buffer containing a derivatization reagent.
In the last few years, however, two detection systems have been acknowledged as valuable: contactless conductivity and above all mass spectrometry detection . Both allow detection of free amino acids without derivatization , the former is universal and does not interact with the analytes or separation system, the latter is expensive but offer great selectivity. MS detection for CE is viewed, indeed, as more universal than UV or electrochemical detection. The selectivity and specificity of MS compensate for variations in migration times of the analytes, provides molecular weight and structural information. Most important, it adds a second dimension in separation selectivity for coeluting molecules having different fragmentation patterns. This is of great importance in chiral separation of AAs where this possibility greatly enhances the capability of the technique [80, 89].
As to the background electrolyte , an impressing variety respect to the pH (from about 2 up to 12) or the nature (from acetic or formic acid to borate or phosphate buffers) is found. The electrolyte modification with organic, which improves the separation possibly because of a decrease of EOF , lower solute adsorption to the capillary and Joule heating, or cyclodextrins is often reported and specific example can be found in the tables herein.
Chiral separations of amino acids are achieved by using mainly different and differently derivatizated cyclodextrins and selected antibiotics like vancomycin. Blends of chiral selector demonstrated useful in selected applications [84–86] as well. However, application of ligand exchange CE, the separation of two enantiomer analytes due to the difference in complex stability constants of the two ternary diastereomeric mixed complexes formed by a metal ion (often Zinc or Copper), a chiral selector (one of the L-amino acids), and the analyte is increasing [67–74].
The protocols described in the following paragraphs represent different approaches to the AA analysis and all are related to possible application to biomedical problems.
2 Materials
2.1 Analysis of the Amino Acid Standards and the Blood Samples
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1.
48 % Hydrogen fluoride (Merck, Darmstadt, Germany).
-
2.
Background electrolyte (BGE) : 1 M formic acid solution: dilute 1.90 ml of 98–100 % Suprapur formic acid (Merck) to 50 ml with water in a volumetric flask. Store at room temperature (see Note 1 ).
-
3.
Sheath liquid: 5 mM ammonium acetate (Merck) in methanol/water (50:50, v/v).
-
4.
Preparation of the blood sample: soak a 5 mm diameter blood spot on filter paper in 100 μl of water for 10 min. Then take a 20 μl aliquot of this solution and dilute to 200 μl with a solution of acetonitrile/water/formic acid (49.9/49.9/0.2; v/v).
-
5.
High performance capillary electrophoresis/mass spectrometry system.
-
6.
Uncoated 115 cm long, 20 μm i.d., 150 μm o.d. fused silica capillary (Polymicro Technologies, Inc., Phoenix, AZ).
2.2 Capillary Electrophoresis Combined with Microdialysis: Analysis of Trace Amino Acids Neurotransmitters
-
1.
Ringer solution: 140.0 mM NaCl, 4.0 mM KCl, 1.2 mM CaCl2, 1.0 mM MgCl2, 10 mM NaHCO3 at pH 7.4 (see Note 1 ).
-
2.
Prepare the 1 mM γ-Aminobutyric acid (GABA), glutamate (Glu), and L-Aspartate (L-Asp) (all from Sigma-Aldrich) standard solutions in 0.1 M hydrogen chloride (prepared from 30 % Suprapur hydrogen chloride, Merck) and store at 4 °C.
-
3.
NDA solution: 3.0 mM Naphthalene-2,3-dicarboxaldehyde (NDA) (Buchs, Switzerland) in acetonitrile (hypergrade LiChrosolv, Merck)/water 50:50 v/v
-
4.
Borate/NaCN solution: 0.5 M borate buffer pH 9.2/87 mM NaCN in water (100:20 v/v).
-
5.
Internal standard: 0.1 mM cysteic acid in 0.1 M hydrogen chloride.
-
6.
BGE : 75 mM sodium borate, 10 mM hydroxypropyl-β-cyclodextrin (HP-β-CD), 70 mM sodium dodecyl sulfate (SDS) buffer (pH 9.20). (Sigma-Aldrich) (see Note 2 ).
-
7.
A microdialysis apparatus composed by a microinfusion pump and a microdialysis probe equipped with a polycarbonate ether dialysis membrane having a molecular weight cutoff of 20000 D.
-
8.
High performance capillary electrophoresis equipped with a laser-induced fluorescence detector and Helium Cadmium laser (8 mW, 442 nm).
-
9.
Uncoated 63 cm long, 50 μm i.d. fused silica capillary (Polymicro Technologies, Inc.). Effective length 52 cm.
2.3 Analysis of Protein Hydrolysates
-
1.
Hydrochloric acid solution: 6 M HCl (Suprapur, Merck) containing 0.5 % (w/v) phenol (Merck) (see Note 1 ).
-
2.
Triethylamine solution: mix 2 ml of 99.5 % ethanol, 2 ml of water, and 1 ml triethylamine.
-
3.
PITC solution: mix 70 μl of 99.5 % ethanol, 20 μl of triethylamine, and 10 μl of phenylisothiocyanate (Sigma-Aldrich).
-
4.
Bovine serum albumin (BSA, 607 residues) was obtained from Sigma-Aldrich.
-
5.
BGE : 29 mM phosphate buffer, pH 7.4, 168.3 mM SDS (Sigma-Aldrich) (see Note 3 ).
-
6.
Glass tubes for hydrolysis and derivatization .
-
7.
High performance capillary electrophoresis with UV–vis detection.
-
8.
Uncoated 57 cm long, 50 μm i.d. fused silica (Polymicro Technologies). The length to the detector is 50 cm.
3 Methods
The methods described herein outline the use of different electrophoretic techniques to separate and detect AAs in biomedical applications. In the first example a CE–MS system is effectively used to detect phenylketonuria and tyrosinemia, two metabolic diseases, in blood samples. A sheath-flow interface is used due to its easy and reproducible setup. It also poses less constraints on buffer used in the separation. Pressure-assisted CE also minimizes loss of resolution due to the diffusion of counter ion from the sheath liquid back into the capillary. This hyphenation, as already observed, deserves great attention: the results are interesting and the methods can be further improved, for example, separating AAs after derivatization . MS , indeed, has a greater sensitivity when higher molecular weight compounds are detected, and a simpler tuning of the spectrometer is feasible if the tag represents the main part of the molecule.
CE–LIF demonstrated the method of choice for monitoring simultaneously neurotransmitters. Its sensitivity and the low injected volume, typical of CE, make it an ideal technique for the analysis of biological samples , such as microdialysate from discrete brain areas, whose absolute amounts are very small. No cleanup procedures are required as the dialysis membrane is not crossed by high molecular weight substances like the proteins . By selecting the proper membrane cutoff, different real samples can be analyzed without time-consuming purification procedures. Also, if the perfusate is compatible with the derivatization mixture, the derivatized AAs can be collected and promptly analyzed avoiding batch operation.
The microdialysis-CE-LIF experiment, herein described, permitted to monitor the extracellular concentration of neurotransmitters which have a key role in the understanding of human chemical, physiological, and behavioral events.
The last protocol provides a rapid and sensitive tool for analysis of amino acids in polypeptide or protein hydrolysates, which can find application in different fields, from protein analysis to glue identification. The agreement with the conventional methods and the better sensitivity (the needed amounts are 100–1000 times lower than those used for the ninhydrin-based determinations) made the method valuable for real samples.
3.1 CE-ESI -MS
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1.
Electrophoresis sample to sample holder SI:A1. In some instruments for electrical reasons, the outlet terminal in normal mode becomes the inlet terminal with the external adapter.
-
2.
BGE (2.0 ml) in sample holder position BI:A1.
-
3.
1 M NaOH solution (2.0 ml) in sample holder position B1:D1 and water (2.0 ml) in sample holder position BI:E1; place an empty vial in sample holders BI:C1.
-
4.
Fill the syringe with the sheath liquid solution and place it in its holder on mass spectrometer.
-
5.
Before the run, rinse at high forward pressure (20 psi) the capillary sequentially with NaOH (1 min), water (1 min), and electrophoresis buffer (4 min) (see Note 4 )
-
6.
CE programmed to inject electrophoresis sample for 5 s at low pressure (0.5 psi, 3.45 kPa).
-
7.
The conditions used in the CE were as follows: voltage 30 kV, temperature 25 ± 0.5 °C, pneumatic assistance to classical electrophoretic driving force, 10 psi (see Note 5 ).
-
8.
1.5 kV were applied to the CE outlet/ESI electrode and the heated capillary used in these measurements is kept at 200 °C. The source temperature is maintained at 80 °C and nitrogen is used for both nebulizing (35 l/h) and drying (100 l/h). The sheath liquid flow at a flow rate of 5 μl/min is provided by the mass spectrometer controlled syringe pump (see Note 6 ).
-
9.
Set up the mass spectrometer detector to scan the m/z range between 74 and 250 amu under positive ionization mode at unit mass resolution to monitor free AAs.
-
10.
The UV detector , located 20 cm from the capillary injection end, can be operated continuously at 200 nm for coarse control of analyte migration.
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11.
Figure 1 shows the electropherograms of blood sample of both healthy and afflicted individuals.
3.2 CE-LIF
-
1.
Perfuse the microdialysis probe with the Ringer’s solution at high flow rate (10 μl/min) for 1 h, then lower the flow rate to 2 μl/min and implant the probe. Monitor the basal level of the analytes for at least 30 min before stimulus.
-
2.
Collect the perfusate fraction in microvials every 1 min (2 μl sample volume) and immediately store each of them at −40 °C before derivatization . Stop the fraction collection 30 min after the stimulus.
-
3.
After recovery to room temperature, derivatize the microdialysate as follows: add 0.2 μl of the internal standard, 0.4 μl of the borate/NaCN, and 0.2 μl of the NDA solutions to the sample (2 μl). Let the mixture react for about 1 h (see Note 7 ).
-
4.
Electrophoresis buffer (2 × 2.0 ml) in sample holder position BI:A1 and BO:A1. Electrophoresis sample in sample holder SI:A1. Standard solution in sample holder SI:B1.
-
5.
0.25 M NaOH solution (2.0 ml) in sample holder position B1:D1 and water (2.0 ml) in sample holder position BI:E1; place empty vials in sample holders BI:C1 and BO:B1.
-
6.
CE programmed to inject electrophoresis sample for 10 s at 0.5 psi.
-
7.
The conditions used in the CE were as follows: voltage, 25 kV, temperature 25 ± 0.5 °C. The excitation was performed with a Helium Cadmium laser (8 mW, 442 nm) whereas the fluorescence emission intensity was recorded at 490 nm.
-
8.
Between runs, rinse at high pressure (20 psi) the capillary sequentially with 0.25 NaOH (30 s), water (1 min), and electrophoresis buffer (1 min).
-
9.
Figure 2 shows the electropherograms relevant to the analysis of a microdialysate obtained from the spinal dorsal horn, a standard solution, and a brain dialysate from a rat striatum.
3.3 CE-UV
-
1.
Hydrolysis of Proteins and Peptides : vacuum dry the solution of proteins or peptides in 5 × 35-mm glass tubes. Then add to each tube 40 μl of hydrochloric solution. Evacuate and flame seal the tubes. Put the tubes in an oven at 110 °C for 24 h. After opening of the tubes, dry with a gentle nitrogen flow (see Note 8 ).
-
2.
Derivatization with phenylisothiocyanate: add to each tube 40 μl of triethylamine solution, vortex shortly and evaporate (see Note 9 ). Then add 3 μl 50 % ethanol to each tube followed by subsequent addition of 7 μl of PITC solution. Vortex and incubate the samples for 30 min at room temperature. Dry the derivatized samples under vacuum overnight in a desiccator. Dissolve the PITC-AAs in water before CE analysis (see Note 10 ).
-
3.
BGE (2 × 2.0 ml) in sample holder position BI:A1 and BO:A1. Electrophoresis sample to sample holder SI:A1.
-
4.
CE programmed to inject electrophoresis sample for 5 s at 0.5 psi.
-
5.
The conditions used in the CE were as follows: voltage, 27 kV, temperature 24 ± 0.5 °C. The online UV detector , located 7 cm from the capillary end, is operated continuously at 200 nm for control of analyte migration.
-
6.
Change the BGE after each run and wash the capillary with the fresh electrolyte at least 5 min.
-
7.
Figure 3 shows the electropherograms of a hydrolysate of BSA.
4 Notes
-
1.
All solutions were prepared in water that has a resistivity of 18.2 MΩ cm and total organic content of less than 5 ppb. An UltraClear system (SG Water, Hamburg, Germany) equipped with an UV lamp was used.
-
2.
Filter the BGE through a 0.2 μm filter to prevent blockage of the CE capillary and for degassing.
-
3.
The running buffer may conveniently be prepared by titration of phosphoric acid (Merck) with NaOH (Sigma-Aldrich). Then dissolve the SDS and filter through 0.2-μm membranes before use. It could be stored at room temperature for at least 6 months.
-
4.
Either a chemical or a mechanical method can be used to sharpen the outlet tip of a new capillary, before mounting it in the cartridge. If nitrile gloves and a fume hood are available, the chemical etching in 49 % hydrofluoric acid could be accomplished by soaking 2–4 mm of the capillary end for 5 min while passing nitrogen through the capillary to minimize the etching of the inner wall of the capillary. Otherwise the tip could also be sharpened mechanically with fine emery paper: in this case pay attention to the debris, not to clog the capillary. Moreover before use new capillaries should be eluted with 1 M NaOH for 2–4 h under constant pressure. At the beginning of each day the capillary should be conditioned by flushing with 1 M NaOH solution (5 min), followed by 5 min flush of water and 30 min of electrolyte solution.
-
5.
If available use an HPLC pump as generally the baseline noise is halved.
-
6.
With different CE equipment the pneumatic assistance, which is used to shorten analysis time, could be not available.
-
7.
NDA is a fluorescent tag not fluorescent itself (in contrast with fluorescein isothiocyanate, for instance) and rapidly reacts to give stable fluorescent derivatives. However, since the internal standard cysteic acid reacts less quickly than Glu, L-Asp, and GABA, a reaction time of 1 h at room temperature is necessary to complete the derivatization reaction.
-
8.
To get rid of the metal ions eventually present in the sample, it is possible to extract the proteic fraction in 6 N NH3 first, then to dry the extract and hydrolyze it. For biomedical applications the glass tube should be pyrolyzed (400 °C, 3–4 h) before use.
-
9.
This step is essential to remove residual hydrolysis acid.
-
10.
Reagent mixtures were made fresh daily, stock PITC was stored at about 20 °C under nitrogen . Triethylamine and 50 % ethanol were stored at +4 °C. PTC–amino acids were stored at −20 °C.
Abbreviations
- ANDA:
-
7-Amino-1,3-naphthalene disulfonic acid
- APC:
-
1-(9-Anthryl)-2-propyl chloroformate
- BMIC:
-
1-Butyl-3-methyl imidazolium chloride
- BrBQCA:
-
3-(4-Bromobenzoyl)-2-quinoline carboxaldehyde
- C4D:
-
Capacitively coupled contactless conductivity detection
- CAPS:
-
3-Cycloexylamino-1-propanesulfonic acid
- CBQCA:
-
3-(4-Carboxy benzoyl) quinoline-2-carboxaldehyde
- CC:
-
2,4,6-Trichloro-1,3,5-triazine
- CFSE:
-
5-Carboxy-fluorescein succinimidyl ester
- ClBQCA:
-
3-(4-Chlorobenzoyl)-2-quinoline carboxaldehyde
- DMAB:
-
4-(N,N′-dimethylamino)benzoic acid
- Dns:
-
5-(Dimethylamino) naphthelene-1-sulfonyl chloride
- DTAF:
-
5-(4 6-Dichloro-s-triazin-2-yl amino) fluorescein
- FITC:
-
Fluorescein isothiocyanate
- FMAC:
-
N-fluorenylmethoxycarbonyl-l-alanyl N-carboxyanhydride
- FNBDA:
-
4-Fluoro-7-nitro-2,1,3-benzoxadiazole
- GABA:
-
γ-Amino-n-butyric acid
- HM-β-CD:
-
Heptakis(2,6-di-O-methyl)-β-CD
- HP-β-CD:
-
Hydroxypropyl-β-cyclodextrin
- IAF:
-
5-Iodoacetamidofluoresceine
- MBA:
-
4-Methylbenzylamine
- MDMA-β-CD:
-
3-Monodeoxy-3-monoamino-β-CD
- MDP-β-CDCl:
-
Mono-6-deoxy-6-(3R,4R-dihydroxypyrrolidine)-β-cyclodextrin chloride
- MGA:
-
N-methyl-d-glucamine
- MHP-β-CD:
-
Mono-6-deoxy-6-mono(3-hydroxy) propylamino-β-CD
- MMI-β-CDCl:
-
Mono-(3-methylimidazolium)-β-CD chloride
- NBD-F:
-
4-Fluoro-7-nitro-2,1,3-benzoxadiazole
- NDA:
-
Naphthalene-2,3-dicarboxaldehyde
- OPA:
-
o-Phthaldehyde
- PITC:
-
Phenyl isothiocyanate
- SAMF:
-
6-Oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl) fluorescein
- THSBE:
-
1,3,5,7-Tetramethyl-8-(N-hydroxy succinimidyl butyric ester) difluoro boradiaza-S-indacene
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Acknowledgement
This work was partly funded by Italian MIUR through the project PON 254/Ric./2011 (Cod. PONa3_00334). Francesca Liuzzi is gratefully acknowledged for compiling tables.
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Fico, D., Pennetta, A., De Benedetto, G.E. (2016). Bioanalytical Application of Amino Acid Detection by Capillary Electrophoresis. In: Schmitt-Kopplin, P. (eds) Capillary Electrophoresis. Methods in Molecular Biology, vol 1483. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6403-1_14
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