14 Electrophoresis 2012, 33, 14–35

Véréna Poinsot1 Review

Marie-Anne Carpéné1
Jalloul Bouajila1
Pierre Gavard1
Bernard Feurer 2
Franc-ois Couderc1
1
Université Paul Sabatier, IMRCP,
UMR 5623, Toulouse, France
2
Picometrics, Toulouse, France
Received July 4, 2011
Revised August 14, 2011
Accepted August 15, 2011
Recent advances in amino acid analysis
by capillary electrophoresis
This paper describes the most important articles that have been published on amino acid
analysis using CE during the period from June 2009 to May 2011 and follows the format
of the previous articles of Smith (Electrophoresis 1999, 20, 3078–3083), Prata et al. (Elec-
trophoresis 2001, 22, 4129–4138) and Poinsot et al. (Electrophoresis 2003, 24, 4047–4062;
Electrophoresis 2006, 27, 176–194; Electrophoresis 2008, 29, 207–223; Electrophoresis 2010,
31, 105–121). We present new developments in amino acid analysis with CE, which are
reported describing the use of lasers or light emitting diodes for fluorescence detection,
conductimetry electrochemiluminescence detectors, mass spectrometry applications, and
lab-on-a-chip applications using CE. In addition, we describe articles concerning clinical
studies and neurochemical applications of these techniques.

Keywords:
Amino acid / LIF / MS DOI 10.1002/elps.201100360

1 Introduction article we will focus on new developments of CE, specially

In the last 2 years many papers concerning CE separations
of amino acid (AA) were reported. Among them, some
describe new CE methods, others separation of enantio-
mers, clinical applications, neurochemistry, and food
chemistry. During this past 2-year period, the enhancement
of sensitivity and reproducibility served to increase the
variety of CE applications in AA separation, especially with
respect to CE/MS and enantio-separations. This review is a
continuation of regular review articles on this topic [1–4].
Since our last review article concerning the recent
applications of AAs [5], some review articles summarized
applications, such as the use of micellar electrokinetic
chromatography (MEKC) [6], neurochemistry applications
[7], and food chemistry were also described [8] and recent
studies regarding chirality in food chemistry [9]. In this
Correspondence: Professor Franc-ois Couderc, Université Paul
Sabatier, Laboratoire des IMRCP, UMR5623, 31062 Toulouse
Cedex 9, France
E-mail: couderc@chimie.ups-tlse.fr
Fax: 133-561558155
Abbreviations: AAs, amino acids; ACE, affinity CE; Br-BQCA,
3-(4-bromobenzoyl)-2-quinolinecarboxaldehyde; CBQCA,
3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde; CF,
cyclofructans; CMA, coronamic acid; Dns-Cl, dansyl
chloride; DOPA, 3,4-dihydroxy-L-phenyl-alanine; DTAF,
5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein; GABA, g-
amino butyric acid; GSH, glutathion; Hcy, homocysteine;
Hp-b-CD, hydroxypropyl-b-CD; MAB, moving affinity
boundary; NBD-F, 4-fluoro-7-nitro-2,1,3-benzoxadiazole;
NDA, naphthalene-2,3-dialdehyde; PEO, poly(ethylene
oxide); pyCDCl, mono-6-deoxy-6-pyrrolidine-b-CD chloride;
SDME, single drop microextraction; TPN, theoretical plate
number
those concerning the novelties in detection methods and on
chirality; then the different biological applications will be
described. The first part on the new developments will insist
on the analytical conditions while the second part, on
applications, will focus on their description; for both of them
a large table (Table 1) summarizes the analytical conditions.
2 New CE methods
Sebastiano et al. [10] proposed two new methods to obtain
reduced EOF by using (i) N-methylpolyvinylpyridinium,
which reverses EOF in acidic conditions (50 mM ammo-
nium formate, pH 4.0, 20 kV) and (ii) deposition of
divalent barium at alkaline pH values (50 mM boric acid,
10 mM barium hydroxide, and 10 mM sodium hydroxide,
pH 9.4, 120 kV), which reduces EOF. In these conditions,
S-adenosylmethionine and its minute impurities, two
diastereoisomers of S-adenosyl-(50 )-3-methylthiopropyla-
mine (resulting from the decarboxylation of S-adenosyl-
methionine) were separated using CE/UV (254 or 210 nm,
respectively). Following the first acidic conditions the
separation of the diastereoisomers was poor because the
large amount of the main compound prevents from getting
a good resolution. Using the barium hydroxide separation, it
is possible to inject large amounts of S-adenosylmethionine
sample, because it prevents the migration of the negatively
charged major component further into the capillary, while
the positively charged decarboxylated species migrate in the
same direction as the reduced EOF.
A number of studies have described the determination
of thiols in biological samples [11]. A new application on
biological thiol detection is presented by Zinellu et al. [12],
Colour Online: See the article online to view Fig. 4 in colour.

& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
 Table 1. Table summarizing some experimental conditions for CE analysis

Sample Labelling BGE Detection Capillary/chip Voltage Ref.

Quantitation of Hcy and derivates No 40 mM Tris (pH 1.6) UV at 190 and 232 nm 30 cm, 75 mm id 15 kV [12]
Determination of intracellular 40 mM borate solution 20 mM borate (pH 10.2), 3.0 mM CL Microchip of poly(dimethylsilox- 2.5 kV [13]
Cys, GSH, and haemoglobin in containing 30 mM Na2S2O8, luminal ane) (PDMS), separation
red blood cells (pH 11.5) channel, 6.5 cm
Lys determination in beverages No 20 mM boric acid (pH 5.3), 5 mM UV at 232 nm Hybrid quartz/printed circuit 1.8 kV [14]
‘‘cupric cation’’ (sic), 0.015% board, microfluidic electro-
Electrophoresis 2012, 33, 14–35

Tween 20 phoresis device (HQPM),

separation channel, 7.5 cm,
50 mm id
Trp and melatonine in milk No 20 mM phosphate (pH 9) UV at 232 nm Hybrid quartz/polymethylmeth- 1.8 kV [15]
acrylate, microfluidic electro-
phoresis device (HQM),
separation channel, 7.5 cm,
50 mm id

& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Ni(II) for selective detection of No 50 mM Na2HPO4-NaH2PO4 (pH UV at 208 nm 44 cm, 75 mm id 30 kV [16]
His in human urine 6.0), 1.0 mM NaCl
Indolamines in urine and serum No 2% Dextran sulphate, 100 mM LIF 266 nm 10 cm, 75 mm id 15 kV [20]
Tris (pH 9.0)
Brine sample from Saline Valley Pacific Blue EDTA adjusted at 75 mM borate buffer (pH 9.5) LIF 425 nm Microchip PMDS, separation 1 kV [23]
water highly acidic sample 50 mM channel 23.6 cm
from the Rı́o Tinto
Mammalian decomposition fluids No 70 mM boric acid (pH 9.5), 32% UV at 200 nm 56 cm, 75 mm id 30 kV [24, 122]
methanol
Cultured cells, cerebrospinal FITC 18 mmol/L phosphate (pH 11.6) LIF 520 nm 50 cm, 75 mm id 28 kV [27]
fluid, saliva, vitreous humor
samples
Quantitation of Glu and Asp in NDA 10 mM sodium phosphate (pH 7.5) Fluorescence diode 20 cm, 25 mm id 20 kV [28]
planaria 445 nm
Asp and Glu in rat 5-Carboxyfluorescein-N- 25 mM borate, 120 mM boric acid LIF 488 nm 50 cm, 50 mm id 25 kV [30]
periaqueductal grey matter succinimidyl ester (pH 8.50)
Isoelectrofocusing separation of 4-Amino-1,8-naphthalimides 50 mM phosphate (pH 7) Fluorescence 431 nm 56 cm, 50 mm id 25 kV [31]
Leu, Ser, Gly, Glu, Asp dye called N-hydroxysuc-
cinimide-UR-431
Mixture of three AA FITC L-Asn NBD-F epinephr- 10 mM borate buffer (pH 9.1) LED-IF 520, 530, 550 nm 48 cm, 75 mm id 26 kV [34]
ine CBQCA L-Leu
AA determination in ascites NDA/KCN 1.5% PEO, 10 mM sodium tetra- LED-IF 410 nm 50 cm, 75 mm id 20 kV [35]
borate (pH 9.3)
Human plasma and vitreous (30:1) Br-BQCA (pH 8):amine 120 mM boric acid (pH 9.1), LIF 488 nm 50 cm, 75 mm id 22.5 kV [36]
CE and CEC

perfusate samples 15-fold KCN/amine 551C, 38.5 mM SDS 19% ACN

50 min

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15
 16
Table 1. Continued
Sample Labelling BGE Detection Capillary/chip Voltage Ref.

AA and catecholamines in (30/1) Br-BQCA (pH 8)/amine 120 mM boric acid (pH 9.15), LIF 488 nm 50 cm, 75 mm id 22.5 kV [37]
HEK293 and PC12 cell samples 15-fold KCN/amine 551C, 38.5 mM SDS 17% v/v ACN
50 min
Chiral separation of D/L-Trp No 1.0 mM phosphate (pH 2.5), 0.35% UV laser 266 nm 5 cm, 50 mm id 4 kV [38]
V. Poinsot et al.

highly sulphated b-CD

Pineapple leaf diurnal changes in No 1 M formic acid ESI/MS 100 cm, 50 mm id 26.5 kV [42]
Asp, Asn, and citrate concen-
trations
Non-protein amino acids in Butanol on dry residue 801C, 0.1 M formic acid (pH 2.0) MS 60 cm, 50 mm id 25 kV [43]
vegetable oils 30 min
Ornithine determination in beer FITC 50 mM ammonium carbonate (pH ESI1/MS/MS transition 100 cm, 50 mm id 25 kV [44]
10.0), 0.75 mM g-CD m/z 456Zm/z 390
Separation of Asp, Glu, Ala, Asn, FITC 50 mM ammonium hydrogen LIF 85 cm, 50 mm id 30 kV [45]
and Arg enantiomers carbonate (pH 8.0), 0.5 mM
3-mono-deoxy-3-monoamino-

& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

b-CD
Glycinates in premix or feed No 20 mM ammonium acetate (pH ICP-MS 55 cm, 50 mm id 30 kV [46]
samples 7.4) (ICP), 50 mM ammonium ESI-Qq-TOF-MS/MS
acetate (pH 7.4) (ESI)
Blood plasma, urine, saliva, and No 0, 5, 4, 8 M acetic acid Capacitively coupled 18 cm, 25 mm id 20 kV [48]
cerebrospinal fluid samples contactless conduc-
tivity detection (C4D)
Separation of the enantiomers of No 10 mM Tris (pH 7.35), 4.4 mM C4D 55 cm, 25 mm id 15 kV [49]
the a-hydroxy acids in milk and maleic acid, 0.03 mM CTAB
yoghurt 5 mM vancomycin
Glyphosate, glufosinate and No 12 mM His, 8 mM MES, 75 mM of C4D 35 cm, 50 mm id 20 kV [50]
aminophosphonic in drinking CTAB (pH 6.3)
water
Gly concentration in periaque- No 1.7 M acetic acid 2% PEG C4D 30 cm, 75 mm id 20 kV [53]
ductal grey matter of rats
Pro, pipemidic acid, and Pro, 5 mM Ru(bpy)3Cl2 50 mM 70 mM phosphate (pH 8) Electrochemilumines- 50 cm, 25 mm id 15 kV [56]
tetracaine enoxacin in human phosphate buffer (pH 9.6) cence 1.15 mV
urine in detector cell
Lysate of mice single fibrosarco- 30 mM NaHCO3-NaOH 30 mM borate solution (pH 10.0), CL Microchip PMDS, separation 1.8 kV [57]
ma cell (S180) (pH 10.5) 50 mM H2O2 2.5 mM luminal, 0.45mM CuSO4 channel 6.5 cm
Rat hepatocytes 50 mM NaHCO3 0.8 mM 20 mM Na2HPO4 (pH 10.0), CL Microchip PMDS, separation 1.8 kV [58]
K3Fe(CN)6 (pH 12.5) 2.5 mM luminal, 40 mM NaBr channel 6.5 cm
Neurotransmitters dopamine, No 25 mM MES-sodium acetate (pH Electrochemically Microchip PMDS, separation 3 kV [59]
epinephrine, and L-DOPA 5.57) channel 3.5 cm
Enantioseparation of 9-Fluorenylmethyl chloro- 100 mmol/L Tris (pH 6.0), 2 mmol/L UV at 214 or 254 nm Poly(dimethylacrylamide) 15 kV [65]
environmental sample formate (FMOC) vancomycin (PDMA)-coated capillary
29 cm, 50 mm id

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Electrophoresis 2012, 33, 14–35
 Table 1. Continued

Sample Labelling BGE Detection Capillary/chip Voltage Reference

D- and L-Asp in cerebrospinal NDA/CN Anode: 0.6% PEO 150 mM sodium LED-IF 410 nm 50 cm, 75 mm id 8 kV [66]
fluid, beer, and soymilk dodecyl sulphate (SDS), 60mM
Hp-b-CD in capillary: 150 mM
SDS 60 mM Hp-b-CD
Urine and plasma samples NDA/CN Buffer vial: 20 mM Tris-borate, LED-IF 410 nm 40 cm, 50 mm id 10 kV [67]
150 mM SDS, 50 mM hydroxy-
Electrophoresis 2012, 33, 14–35

propyl-b-CD, 0.5% PEO in

capillary: 100 mM Tris-borate,
150 mM SDS, 50 mM hydroxy-
propyl-b-CD
Measurements of D-amino acid No 100.0 mM boric acid (pH 8.2), UV at 214 nm 50 cm, 50 mm id 20 kV [73]
oxidase enzyme kinetic 5.0 mM ammonium acetate,
constant 3.0 mM ZnSO4, 6.0 mM L-Orn
Distribution of D-amino acid No 5 mM ammonium acetate, MS 100 cm, 50 mm id 30 kV [74]

& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

oxidase following rat renal 100 mM boric acid, 7 mM
ischaemia ZnSO4, 14 mM L-Aln (pH 8.2)
Catalytic efficiency of L-amino No 100 mM boric acid, 5 mM ammo- UV at 254 nm 50 cm, 50 mm id 21 kV [75]
acid oxidase nium acetate, 4 mM b-CD,
4 mM ZnSO4, 8 mM L-valine
(pH 8.1)
Serum samples No 100 mM boric acid, 5 mM ammo- UV at 214 nm 50 cm, 50 mm id 20 kV [77]
nium acetate, 3 mM ZnSO4,
6 mM L-Arg (pH 8.4)
Determination of the impurities of No 0.12 M phosphate (pH 2.0), 6 mg/ UV at 200 nm 35 cm, 50 mm id 20 kV [84]
L-DOPA mL sulphated
b-CD
D/L Glu and D/L Asp DTAF DTAF-Asp enantiomers, 12 mM Fluorescence 520 nm 50 cm, 50 mm id 25 kV [86]
cholate, 8 mM sodium borate
(pH 8.9), 0.8% HAS, 10%
methanol DTAF-Glu enantio-
mers:12 mM cholate, 10 mM
sodium borate (pH 9.1), 1.6%
HAS, 5% methanol
Cysteine and glutathione in 10 mL of 0.5 M Tris 2-carboxy- 0.2 M Tris/HCl (pH 2.1) UV at 355 nm 56 cm, 50 mm id 30 kV [91]
orange juice ethylphosphine 2511C for
15 min 50 mL of 100 mM 2-
chloro-1-methylquinolinium
tetrafluoroborate RT for
CE and CEC

5 min 100 mL of 3 M
perchloric acid

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17
 18
Table 1. Continued
Sample Labelling BGE Detection Capillary/chip Voltage Ref.

GABA and alanine in tea In-capillary labelled derivati- 50 mM sodium tetraborate Fluorescence at 495 nm 65 cm, 50 mm id 21 kV [92]
zation o-phthaldialdehyde/ (pH 10)
2-mercaptoethanol
Sugars and amino-acids in tea No 30 mM borate 40 mM phosphate Amperometric 75 cm, 25 mm id 18 kV [93]
V. Poinsot et al.

(pH 8.5) detection Cu disc

electrode
S-Adenosyl-L methionine in fruits No 200 mM glycine 50 mM phos- UV detector at 260 nm 60.2 cm, 50 mm id 25 kV [94]
and fruit juices phate (pH 2.5)
Bacterial metabolites Saccharo- 20 mL of cell extract 2 mL of 10 mM tetraborate (pH 9.3), LIF 488 nm 40 cm, 50 mm id 30 kV [95]
myces cerevisiae 185 mM 4-nitro-7-benzofur- 35 mM sodium deoxycholate,
azan (NBD-F) 551C, 15 min 7.5 mM methyl-b-CD
Inorganic ions in A. thaliana No 2.25 mM pyromellitic acid, 6.5 mM Diode array detection 24.5 cm, 50 mm id 18 kV [96]
shoots sodium hydroxide, 0.75 mM at 350 nm
hexamethonium hydroxide,
1.6 mM triethanolamine

& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

(pH 11.2)
Metabolites in A. thaliana shoots No 1 M formic acid MS (TOF) 100 cm, 50 id 30 kV [96]
Anionic metabolites in rice cells No 50 mM ammonium acetate MS 100 cm, 50 mm id 30 kV [97]
(pH 9.0)
Cationic metabolites in rice cells No 20 mM ammonium acetate MS Polyethylene glycol-coated 20 kV [97]
(pH 6,8) capillary 100 cm, 50 mm id
Nucleotides in rice cells No 50 mM ammonium acetate solu- MS 100 cm, 50 mm id 30 kV [97]
tion (pH 7.5)
Cationic and anionic metabolites No Cation or anion buffer solution MS TOF 80 cm, 50 mm id 30 kV [98]
in rice grain (Human Metabolome
Technologies)
Sugars in rice grain No Basic anion buffer for HPCE Indirect diode array 104 cm, 50 mm id 30 kV [98]
(Agilent Technologies) detection at 390 nm
Seed pulp and leaf of Illicium No Agilent basic anion buffer (part MS TOF 72 cm, 50mm id 25 kV [99]
anisatum number 5064-8209)
Lysate of breast cancer cells NDA/CN 0.5 M TRIS (pH 10.2), 30 mM SDS, LED-IF 405 nm 55 cm, 75 mm id 15 kV [100]
0.1% PEO
Urines No 35 mmol/L SDS, 60 mmol/L H3BO3- C4D and amperometric 86.8 cm, 25 mm id 16 kV [101]
Na2B4O7 (pH 8.2) detection
Rat urine No 25 mM borate (pH 9.5), 75 mM UV between 190 and 40 cm, 50 mm id 15 kV [102]
SDS, 1.435% sulphated b-CD 600 nm
Neurotransmitters in micro- NDA/CN 100 mM borate (pH 9.2) LIF 410 nm 60 cm, 50 mm id 25 kV [104]
dialysates
Neurotransmitters from rats NDA/CN 10 mM sodium borate, 0.9 mM LIF 410 nm Microchip PMDS, separation 720 V/cm [105]
HP-b-CD pH 9.7 channel 11.1 cm

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Electrophoresis 2012, 33, 14–35
 Table 1. Continued

Sample Labelling BGE Detection Capillary/chip Voltage Reference

Hippocampus slices from rats NDA/CN 25 mM phosphate (pH 2.15), 20 LIF 420 nm 45 cm, 25 mm id 21 kV [106]
g/L sulphated b-CD
Brain dialysates from rats NDA/CN 50 mmol/L boric acid (pH 9.6) LIF 442 nm 60 cm, 50 mm id 27.5 kV [107]
(carbamathione)
Rat periaqueductal grey matter 2.5 mL microdialysates 1 mL 25 mM borate 120 mM boric acid LIF 520 nm 50 cm, 50 mm id 25 kV [108]
microdialysates 5 mM 5-carboxyfluorescein (pH 8.50)
N-succinimidyl ester
Electrophoresis 2012, 33, 14–35

Neurotransmitters in microdyali-
sates from hypothalamus of
rats
Mycrodialysates from hypothala-
(CFSE) 4 mL 10 mM borate
(pH 8.50) overnight in the
dark, RT
10 mL of microdialysates 50 mL 15 mM borate (pH 9.0), 4% v/v LIF 520 nm 50 cm, 75 mm id 17.5 kV [109, 110]
of 50 mM borate (pH 9.0) isopropanol, 100 mM SDS,
40 mL of 0.375 mM DTAF 5 mM HP-b-CD and 5 mM DM-
401C, 35 min in the dark b-CD
6 mL of microdialysates 10 mL 15 mM borate (pH 10.2), 70 mM LIF 418 nm 50 cm, 75 mm id 22.5 kV [111]

& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

mus of rats of 5 mM borate (pH 9.6) SDS, 5% methanol, 17.5 mM
4 mL of FITC 1 mM 201C in HP-b-CD and 5 mM DM-b-CD
the dark for 16 h
Cerebral ganglia NDA/CN 20 mM b-CD 50 mM SDS 50 mM LIF 400 nm 86 cm, 50 mm id 20 kV [112]
of borate (pH 9.4)
Human urine 10 mL of analyte solution 22 mM sodium tetraborate (pH LIF 520 nm 40 cm, 75 mm id 25 kV [113]
165 mL of 20 mM Na2CO3 10.5) 32% v/v ACN
20 mM NaHCO3 (pH 9.4)
25 mL of 2 mM FITC placed
in a domestic microwave
oven 700 W, 150 s
Human urine No 3 mM 1,3,5-benzenetricarboxylic UV 240 nm Microchip PPMA with capillary 5 kV [114]
acid (BTA), 1.5 mM TEPA, 45 cm 50 mm id
15 mM TRIS (pH 8.4)
Red blood cell lysates No 1.0 M Formic acid (pH 1.8) for MS ESI1 for cations 80 cm, 50 mm id 30 kV [118]
cationic separation 50 mM MS ESI for anions 30 kV
ammonium acetate (pH 8.5) for
anionic separation
Monoclonal antibody samples No SDS MW gel buffer UV at 214 nm 30 cm, 50 mm id 15 kV [119]
Plasma samples No 1.5 M formic acid MS 79.5 cm, 50 mm id 23 kV [120]
Extracellular retinal samples 2.5 mL of 4-fluro-7-nitrobenz- 34 mM hydroxypropyl-b-CD, LIF 488 nm n.i. 15 kV [123]
2-oxa-1,3-diazole in ACN 165 mM borate (pH 10.2)
(3.6 mM final concentra-
tion; 30 mL final volume)
CE and CEC

601C, 15 min

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19
20 V. Poinsot et al. Electrophoresis 2012, 33, 14–35

which quantitates homocysteine (Hcy), Cys, Hcy thio-

lactone, homocystine, Hcy-cysteine, and cystine. In this
application, the molecules were reduced by the reaction of
[124]

[127]

[128]

[129]

[131]
Ref.

CuSO4 on Hcy and Cys with thiolactone as a standard.

CE/UV (190 and 232 nm) was carried out in a 40 mM Tris
buffer adjusted with phosphoric acid to pH 1.6, 151C, and
15 kV (240 mA) at normal polarity with an overpressure of
Voltage

30 kV
30 kV

30 kV

25 kV

12 kV
2 kV
0.1 psi from inlet to outlet. The LODs are around 1 mM.
Chemiluminescence (CL) detection was used on
microchip analyses for the determination of intracellular
Microchip, separation channel Cys, glutathion (GSH), and haemoglobin (Hb). Cell injec-
tion/loading, cytolysis, electrophoretic separation, and CL
detection were integrated onto a simple cross-microfluidic
chip [13]. The electrophoresis electrolyte was 20 mM borate
100 cm, 50 mm id

solution containing 3.0 mM luminol, pH 10.2; with the CL

50 cm, 50 mm id

50 cm, 50 mm id

20 cm, 50 mm id
Capillary/chip

reaction buffer of 40 mM borate solution containing 30 mM

Na2S2O8, (pH 11.5), which is selective for the detection of
4 cm

these compounds. The authors claim that the average

intracellular contents of Cys, GSH, and Hb were in the
ranges of 26–43 amol/cell, 128–323 amol/cell, and
522–667 amol/cell, respectively, for cancer patients,
compared to 579–609 amol Hb/cell and no detectable Cys
MS TOF ESI

and GSH for healthy subjects. The LODs on regular thiol

MS TOF ESI1
UV at 214 nm

UV at 214 nm

UV at 214 nm

solutions are above micromolar.

LIF 473 nm
Detection

A new disposable electrophoresis microdevice, fixing a

fused-silica capillary with a sampling fracture to a printed
circuit board was proposed [14]. Before putting the 7.5 cm
capillary into the groove, it was cut with a piece of ceramic at
50 mM ammonium acetate (pH

20.0 mM borate (pH 9.2), 2.0 mg/

0.1 M tris-borate buffer (pH 8.5)

0.5 cm from one end and the polymer coating at 0.6 cm

0.05% hydroxypropyl methyl
100 mM HEPES (pH 8), 30 mM

20% v/v ACN, 0.4% glycine,

1.0 M formic acid (pH 1.8) for

8.5) for anionic separation

100 mM phosphate (pH 2.5),

from the other end was removed for UV detection. After the
capillary was fixed in the groove, the cut was ultrasonicated
cationic separation

to form the sampling fracture at the sample reservoir. Cu21

–AA complexes can be separated and detected at 232 nm
using 20 mM boric acid (pH 5.3) containing 5 mM ‘‘cupric
mL Brij35

cellulose

cation’’ and 0.015% Tween 20. Lys, Gln, and Ser were
SDS

completely separated at a sampling time of 2 s at 1210 V

BGE

and a separation voltage of 11800 V with theoretical plate

number (TPN) in the order of 140 000–205 000 plates/m for
the various analytes and LODs in the range 2.6–4.8 mM. The
method was used for Lys determination in beverages. An
FITC microwave oven

identical work was performed using a 20 mM phosphate

buffer (pH 9) for Trp and melatonin in milk [15].
Meng et al. described the development of the concept of
moving affinity boundary in affinity CE (MAB-ACE) using a
Labelling

metal ion [Ni(II)] for selective detection of His [16]. In the

FMOC

optimized conditions, the sample was diluted in the elec-

No

No

No

trolyte (50 mM, pH 6.0, Na2HPO4-NaH2PO4 buffer with

1.0 mM NaCl); then the capillary was filled with the sample.
4-hydroxyproline-2-epimerase
Lenses homogenates from calf

processes YihU protein in E.

The anode vial contained the same electrolyte as above with

CMA in Pseudomas syringae
Determination of metabolic

2 mM NiCl2 added, while the cathode vial contains only the

Enzyme kinetic constant

electrolyte (see Fig. 1A). An experiment shows the selectivity

Table 1. Continued

of the detection of His with 19 other AAs, while Fig. 1B

determination

shows the selectivity of the detection of His in a 1:1 mixture

Enzyme activity

of His/histamine. The LOD of 43 mM was observed with

RSD below 5%. The authors indicate the 48-fold enhance-
Sample

eyes

coli

ment for the determination of His comparing the peak

height of His in CZE.

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Electrophoresis 2012, 33, 14–35
A A
B Poly(diallyldimethylammonium chloride) and titanium
+ tional film on PDMS microfluidic channel surface; this film
Initial MAB
C 100 s in a 3.7 cm long separation channel using a 5.0 mM
+ disodium tetraborate, pH 9.2, buffer with good resolution
ACB MAB
EOF
neutral
Ni(II) His positive other solutes His-Ni complex
negative
B the selective precolumn extraction of indoleamines such as
-1
peak of His
-2
mAU
-3
-4
-5
2 4 6 8
Time/min
Figure 1. (A) Diagram of MAB and MAB-ACE method for
selective isolation of His in sample matrix. (A) Initial arrange-
ment of His and Ni(II) in CE; (B) formation of MAB via the Ni(II)
moving towards the cathode quickly and the receptor molecule
of His migrating slowly towards the cathode due to the electric
field; (C) capture of His via the MAB system under proper affinity
conditions. The arrows indicate the movements of the ions.
(B) Selective isolation of His rather than histamine by the
method of MAB formed with 2.0 mM Ni(II) in the anodic vial and
50 mM His and histamine in the whole capillary [16].
Underivatized AAs were preconcentrated using opti-
mized CE/UV conditions and 50 mmol/L CuSO4–0.05%
acetic acid (pH 4.5), 115 kV. Cu21 ions allow the sweeping
of the AA. The LODs for the analytes ranged from 0.1 to
0.5 mM. Human saliva and green tea were analysed [17].
The LOD for Gly with the multichannel mars organic
analyzer was found to be 6 pM. The multichannel mars
organic analyzer is a portable microchip CE instrument
using a four-layer microchip, containing eight CE analysis
systems integrated with a microfluidic network for autono-
mous fluidic processing, and integrating an improved opti-
cal components using a compact 25 mW 405 nm laser
excitation with a linear-scanning optical system capable of
multichannel real-time fluorescence spectroscopic analysis
[18]. The spectrometer has a 3648-element CCD array
detector with a 600 grooves/mm grating blazed at 500 nm
& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
CE and CEC 21
(detection range of 200–1100 nm). The electrophoretic
separations of 200 nM AA standard (citrulline, Val, Ala, Ser,
Gly, Glu, Asp) was run in 4 mM Li2CO3 (pH 8.5) buffer at
room temperature with 700 V/cm and perfectly resolved.
dioxide nanoparticles were employed to construct a func-
reduces electro-osmotic flow (EOF) and limits adsorption
phenomena. Arg, Phe, Ser, and Thr were separated within
and TPN (TPN>60 000 plates/m for 1200 V for Thr, Phe,
and Ser) and detected with indirect amperometry. The
microchip demonstrated good stability and reproducibility
for run-to-run, day-to-day, and chip-to-chip analyses,
running buffer
respectively [19].
Citrate-capped gold nanoparticles were optimized for
5-hydroxytryptophan, Trp, tryptamine, serotonine, and
5-hydroxyindoleacetic acid to preconcentrate samples.
Enhancements of 48-, 4077-, 985-, 920-, and 4030-fold of this
extraction were obtained relative to regular CE/LIF
(266 nm), The migration buffer was 2% w/v dextran
sulphate, in 100 mM Tris adjusted at pH 9.0 with boric acid,
capillary was 75 mm id, 70/10 cm (total/effective length), the
voltage was 115 kV. After preconcentration on nano-
particles, LODs between 4 pM for Trp and 366 pM for
5-hydroxytryptophan were found. Tryptamine and seroto-
nine were quantitated in urine [20].
On-line sample preconcentration of hydrophobically
labelled AA (Alexa Fluor 488 succinimidyl ester, Bodipy
succinimidyl ester) provides a new development in transient
trapping CE and microchip electrophoresis [21]. Figure 2
shows the principle proposed by Sueyoshi et al. [22]. The
micellar phase (M, 25 mM SDS in 29 mM phosphate buffer,
pH 5.3/10% v/v methanol) and the sample S, which
contains the solutions of negatively charged derivatized AA
(S, diluted in 34 mM phosphate buffer, pH 5.2), were
successively introduced into the separation channel filled
with the BGE (34 mM phosphate buffer, pH 5.2, without
SDS) (Fig. 2A); then the separation voltage was applied
(Fig. 2B). In the S zone, the analytes migrate toward the M
zone. When the analytes reach the boundary between the
S/M boundary, they are strongly incorporated into the
micelle and trapped nearby the S/M boundary due to the
effect of the micellar diffusion from the M to S zones.
Therefore, the analytes cannot penetrate into the M zone
and be focused on the S/M boundary as an extremely
narrow band by the trap mechanism. At the same time, the
concentration of the micelle is gradually decreased upon
increasing the length of the M zone due to the diffusion, the
difference in the velocity of the micelle located near both
ends of the M zone, and difference in the electrophoretic
mobility between a micelle and surfactant monomers. As a
result, the interaction between the analytes and micelles is
also decreased, which allows the analytes trapped on the
S/M boundary to be released into the M zone in the order of
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22 V. Poinsot et al.
the hydrophobicity (Figs. 2C and D). The analytes are
separated by the difference in the release time. After
releasing the analytes, they migrate toward the cathode
(Fig. 2E). Authors compared CZE, MEKC, sweeping MEKC,
and trapping, with the different dyes. FITC is not an espe-
cially good dye, as Phe, Val, Leu, and Ile derivatives of
Bodipy succinimidyl ester result in better resolution and
sensitivity (Figs. 2F and G) with LODs in the 50 pM range.
The work was also applied in microchip CE.
When samples are extremely acidic and saline or
contain polyvalent cations, it is necessary to optimize
sample labelling and sample dilution buffers. As an exam-
ple, AAs were determined in a saturated brine sample from
Figure 2. (A) Schematics of the principle of transient-trapping
MEKC. (A) Initial condition, (B) preconcentration of analytes due
to the trap mechanism, (C) separation of analytes due to the
difference in the release timings of analytes, (D) separation
based on MEKC, and (E) detection. The symbols of vS,S, vS,M,
vS,BGS and vS,B are the apparent velocity of the samples in the S,
M, background solution (BGS) and nearby the boundary B
between the S and M zones, respectively. The symbols of vmc
and vEOF are the apparent velocity of the micelle and the
electroosmotic velocity, respectively. (B) Transient trapping–-
MEKC analyses of (A) native and (B) BODIPY-Phe employing UV
and LIF detection, respectively. Total/effective length, 40.2/
30.0 cm buffer, 34 mM phosphate buffer (pH 5.0); M, 25 mM
SDS in 29 mM phosphate buffer (pH 5.0); tinj,M, 180s; tinj,S, 450 s.
Concentration of S, (A) 100 mM and (B) 50 pM [22].
& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2012, 33, 14–35
Saline Valley (California) and a subcritical water extract of a
highly acidic sample from the Rı́o Tinto (Spain) using a
75 mM borate buffer (pH 9.5) for the Pacific Blue labelling
of the AAs. After labelling, final concentrations of EDTA
were adjusted to 50 mM in samples containing divalent
cations to reduce their negative effects [23].
Few chemometric approaches were reported for AA
separations using CE. Swann et al. proposed to optimize the
separation of tyramine, tryptamine, Trp, Phe, and Tyr in
CE/UV (200 nm), using a 75 mm id capillary of 65/56 cm.
Using peak resolution and total analysis time as response
factors, they measured the influences of the four experi-
mental variables, i.e. pH, boric acid concentration, percen-
tage of methanol, and applied voltage. The optimum
separation conditions were determined to be 70 mM boric
acid at pH 9.5 (adjusted with 0.1 M NaOH) with 32% v/v
methanol at 30 kV; the analytes were separated within
12 min [24].
2.1 Fluorescence detection
Fluorescein isothiocyanate (FITC) was largely used for AA
quantitation [25, 26], but its reaction time is long at ambient
temperature. Zinellu et al. [27] developed a 20 min deriva-
tization procedure at 1001C with a 200 mmol/L Na2HPO4,
pH 9.5, buffer and 12 min AA separations were described
using an 18 mmol/L phosphate, pH 11.6, run buffer and
several biological samples (cultured cells, cerebrospinal fluid,
saliva, vitreous humor) were studied to provide limits of
quantitation (S/N 5 10) of approximately 0.2 mM.
A 445 nm 25 nW diode pumped solid state laser was
used with a 25 mm id capillary (20 cm effective length/25 cm
total length, 120 kV) to get rapid separation of Glu and Asp
labelled by naphthalene-2,3-dialdehyde (NDA) [28], for a
review see [29]. The separation was performed with very
diluted buffer (10 mM sodium phosphate, pH 7.5) to
provide migration times between 51 and 56 s with resolution
superior to 2 and LODs around 300 nM. The work was used
to quantitate these AA in planaria.
5-Carboxyfluorescein-N-succinimidyl ester was used
with CE/LIF (488 nm) for the determination of Asp and Glu
in rat periaqueductal grey matter. It is reported that the
LODs were 0.69 and 0.81 nM, respectively at 125 kV, using
a 25 mM borate and 120 mM boric acid (pH 8.50) buffer and
a 50 mm id capillary 60.2/50 cm) [30].
A new 4-amino-1,8-naphthalimide dye (N-hydro-
xysuccinimide-UR-431) was used for isoelectric focusing in
CE with microchips/fluorescence detection [31]. This
marker is specific for the amine function and is weakly basic
when conjugated to analytes. UR-431 effects only a subtle
change of the pI of proteins (where it reacts mainly on Lys)
compared with common anionic labels and it has a pH-
independent fluorescence emission. It absorbs at 431 nm
with a fluorescence maximum at 537 nm and a weak
quantum yield (approximately 0.1). The on-chip detection
limit of labelled Lys using a mercury-lamp-based fluores-
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Electrophoresis 2012, 33, 14–35
cence microscope was determined as 12 nM. A CE/fluores-
cence separation of Leu, Ser, Gly, Glu, Asp is presented
(buffer: 50 mM phosphate, pH 7; injection: 7 s at 5 kV,
separation: 25 kV in a 56/64.5 cm 50 mm id capillary), LODs
are estimated to be about 0.1 mM.
De Jong and Lucy [32] developed a CE experiment with
an LED as fluorescence excitation source, and additional
work on the use of LEDs has been presented. As an exam-
ple, a lens obtained from a waste DVD-ROM reader was
used to focus a blue concave LED light beam (470 nm) into
an approximately 80 mm spot on a capillary and the collected
fluorescence of FITC labelled Arg and Phe results in LOD of
0.92 mM [33].
Three different LEDs (430, 450, and 480 nm) were
collimated with a focus lens and reshaped with a gradient
index lens group for coupling the light into single-mode
optical fiber to be used for fluorescence detection in CE. The
optical fiber was introduced inside the capillary to excite
molecules near the window and this excitation source
allowed for the simultaneous determination of FITC-label-
led L-Asn (480 nm), 4-fluoro-7-nitro-2,1,3-benzoxadiazole
(NBD-F)-labelled epinephrine (450 nm), and 3-(4-carbox-
ybenzoyl)-quinoline-2-carboxaldehyde (CBQCA)-labelled L-Leu
(430 nm). The LODs were respectively 0.8, 120, and 40 nM.
The fluorescence was recorded by a single photomultiplier
tube (PMT), which measured the emission wavelength of
the three dyes. The migration was obtained using a
50/48 cm, 75 mm id capillary with borate buffer (pH 9.1;
10 mM). Using single LED excitation (corresponding to one
of the three dyes) for the analysis of the mixture of the three
labelled molecules, the electropherograms show the two
main peaks being the dye and the corresponding labelled
AA accurately exited by the wavelength, and a small peak of
the weakly excited other dyes. That is to say at 430 nm,
CBQCA Leu was mainly measured while a small peak of
FITC was observed. At 450 nm NBD-F and NBD-labelled
epinephrine were mainly detected as well as FITC. At
480 nm FITC and FITC Asp were detected while a small
peak of NBD-F was recorded. The authors claim that, the
performance of the developed multi-wavelength LED exci-
tation source was superior to the single-wavelength LED
[34]. NDA/potassium cyanate (KCN) was used to label AA
with an LED (probably 405 nm) for fluorescence detection in
CE of AA determination in ascites. The optimized buffer is
1.5% m/v poly(ethylene oxide) (PEO), 10 mM sodium tetra-
borate, pH 9.3, 120 kV (capillary 75 mm id, 60/50 cm, 5 s
gravimetric injection (30 cm height difference). LODs of
10 nM were reported [35].
3-(4-Bromobenzoyl)-2-quinolinecarboxaldehyde (Br-BQCA)
was synthesized as a fluorogenic reagent for primary
amines. The derivatization was optimized (pH 8, 551C,
50 min, 30:1 reagent:amine, 15-fold KCN/amine) with the
optimal running buffer of 120 mM boric acid, pH 9.1,
38.5 mM SDS, and 19% v/v ACN using a 75 mm id, 60.5/
50 cm capillary and a 22.5 kV, 488 nm laser. Asn, His, Met,
Gln (these AAs were not separated) Ser, Thr, Tyr, Gly, Glu,
Asp, Ala, Tau, g-aminobutyric acid; norepinephrine, Val,
& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
CE and CEC 23
dopamine, Ile, Leu, Phe, Arg containing samples were
analysed (the AAs were not separated). LODs for AA were in
the range of 0.65–5 nM, while for catecholamine the LODs
were 58–73 nM. Human plasma and vitreous perfusate
samples treated or untreated with medication after the
intraocular hypertension were studied using this dye [36].
3-(4-Chlorobenzoyl)-2-quinolinecarboxaldehyde reacts in the
same manner. It was used in CE/LIF (488 nm) experiments
to detect AAs and catecholamines in HEK293 and PC12 cell
samples, using the optimized conditions, 120 mM boric
acid, pH 5 9.15, 38.5 mM SDS, and 17% acetonitrile. LODs
ranging from 1.4 to 100 nM were found, which were a bit
higher than the brominated molecule [37].
A 266 nm UV mode-locked diode-pumped laser with
10 s duration pulse and the repetition rate of 40 MHz was
used with a time-correlated single photon counting device to
detect the chiral separation of D/L-Trp, using CD, sulphated
CD, and highly sulphated g-CD at different pH levels. The
best results were achieved at lower pH values (e.g. pH 2.5
and 4.0). Highly sulphated g-CD (0.35%, pH 2.5, 1 mM
phosphate buffer, 4 kV, 12 kV electrokinetic injection 9 s,
capillary 50 mm id 10/5 cm) was found to be the most
effective chiral selector. The LOD of Trp was found to be
65 pM [38], nine times more sensitive than previous studies,
which used a 4 mW/10 kHz/1 ns pulse laser [39].
2.2 CE and MS
CE-ESI/MS is now widely used for AA analysis [40], and
Desiderio et al. recently reviewed this application [41]. A new
assay using a simple optimized 1 M formic acid buffer was
developed by Wakayama et al. [42], who analysed 34 amines
and AAs simultaneously with 12 organic acids without
derivatization. LODs were in the range of 2.5 mM in positive
mode and 5 mM in negative mode. Using a sheath liquid
consisting in 50% methanol at 10 mL/min and a 25 psi
sheath gas pressure with an applied voltage, 26.5 kV/m, the
ions obtained in ESI1 are [M1H]1 for AAs, while they are
[M-H] ions for ESI (Ala, sarcosine, aminobutiric acid were
not detected). If total ion electropherograms are difficult to
interpret, a reconstituted electropherogram is easily identi-
fied using the AA m/z ratio. A metabolomic analysis of
biological samples, pineapple leaf diurnal changes was
obtained and changes in Asp, Asn, and citrate concentra-
tions were measured.
The butylation of the carboxylic part of AAs improves
ionization efficiencies and detection sensitivity. In addition,
it increases the mass of the esters and provides better mass
selectivity. Sánchez-Hernández et al. [43] used a 30 min
reaction with butanol at 801C on a dry residue of extracted
AA (ornithine, b-alanine, g amino butyric acid (GABA),
alloisoleucine, citrulline, and pyroglutamic acid) from
vegetable oils. Using acidic migration conditions (0.1 M
formic buffer (pH 2.0) and uncoated fused-silica capillary)
and ESI/MS/MS detection, they obtained LODs between
0.04 and 0.19 ng/g.
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24 V. Poinsot et al.
FITC was used to derivatize ornithine, and the derivative
was studied in CE-ESI1/MS/MS. The [M12 H]21 ion is the
predominant ion recorded (m/z 456, doubly labelled orni-
thine); it fragments at m/z 522 (singly protonated FITC-
Ornithine) and at m/z 390 (singly protonated FITC). The
transition m/z 456Zm/z 390 was used for ornithine deter-
mination in beer. The optimized CE buffer was 50 mM
ammonium carbonate at pH 10.0 with 0.75 mM g-CD
(capillary, 50 mm id 100 cm) 1 25 kV. ESI1 conditions: spray
voltage, 4.5 kV; sheath liquid, 50/50 v/v isopropanol/25 mM
ammonium carbonate at 3.3 mL/min; drying gas flow and
temperature, 3 L/min 3501C. The LOD in CE/MS/MS was
2.5 nM (50 times more sensitive than UV detection of the
derivatives), while resolution between the two enantiomers
is about 2.5. The migration times were about 28 min due to
a long capillary [44].
6-Deoxy-6-(1-(2-amino)ethylamino)-b-CD, 6-deoxy-6-
(N-(2-methylamino)pyridine))-b-CD, and 3-mono-deoxy-3-
monoamino-b-CD, as chiral selectors for CE CE-ESI1-TOF-
MS and CE-LIF experiments were investigated to enantio-
separate FITC-labelled Asp, Glu, Ala, Asn, and Arg. The
presence of electrical charges in the selector is usually
advantageous since it can bring additional ionic interactions
with the analyte, and are used at low concentrations
compatible with ESI1. CE/LIF experiment using 50 mM
ammonium hydrogeno-carbonate at pH 8.0 and 0.5 mM of
3-mono-deoxy-3-monoamino-b-CD (130 kV, capillary 50 mm
id, 85 cm length) gave better resolutions (up to 16 for
D/L-Asp) between the different enantiomers (sheath liquid,
water:2-propanol (50:50 v/v) delivered at 0.24 mL/h) and
LODs range from 40 to 90 nM. In CE-MS, resolution values
are between 0.9 (D/L-Arg) and 4 (D/L-Asp) and LOD from 68
to 1870 nM. These studies were performed in vinegar and
soya [45].
Metal (M 5 Zn, Cu, Mn, Fe) complexes with glycine,
which are called glycinates (M(Gly)x(H2O)y(SO4)z)n, have
been characterized by CE/ICP-MS and CE/ESI-Qq-TOF-
MS/MS. The CE buffer was 20 mM ammonium acetate
buffer (pH 7.4), 130 kV, 50mm capillary for CE/ICP-MS
experiments and 50 mM ammonium acetate (pH 7.4) for
ESI. These could be identified and quantitated, as the
authors showed that the glycinate integrity is kept constant
in these CE conditions. LODs in CE-ICP/MS for ZnGly,
CuGly, MnGly are (as the metal) around 0.8–4 mM, suffi-
cient for the determination of glycinates in premix or feed
samples [46].
2.3 CE/contactless conductivity detection
Since the work of Cottet and co-werkers [47], contactless
conductivity measurement is commonly used as a CE
detector for underivatized AAs. It has been also used for the
separation of 28 biogenic AAs in a short capillary with an
effective length of 18 cm [48] in three different electrolytes
0.5, 4, and 8 M acetic acid. It allows the analysis of the 28
AAs, with most of the analytes being completely separated.
& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2012, 33, 14–35
The analysis requests less than 6 min and the LODs vary
from 0.1 to 1.7 mM. Applications to blood plasma, urine,
saliva, and cerebrospinal fluid samples were presented.
D/L Asp, D/L Glu, and five a-hydroxy acids were baseline
separated in CE/contactless conductivity detection using a
25 mm id capillary. Reverse polarity (15 kV) was used with
a buffer consisting of 10 mM Tris, 4.4 mM maleic acid,
0.03 mM Cetyl trimethylammonium bromide (CTAB), and
5 mM vancomycin (pH 7.35); this last component was
employed as chiral selector and could be used with
conductivity detection without having to resort to a partial
filling protocol as needed when this reagent is used with UV
absorbance measurements. A resolution above 1.5 was
measured for the two D/L AAs [49]. Contactless conductivity
was also used for sub-mM concentration determination of
glyphosate, glufosinate, and aminophosphonate in drinking
water [50].
A monolithic capillary column, RP-18, end-capped, with
a length of 150 mm, 100 mm id and a migration electrolyte
20% v/v CH3COOH in 40% v/v methanol (pH 2.25) was
optimized to provide baseline resolution of 500 mM of 12
underivatized AAs in less than 10 min. The AAs were
detected using a capacitively coupled contactless conductiv-
ity built in-house detector, based on two tubular electrodes
of 4 mm length, which are separated by a gap of 1 mm and a
Faraday shield [51, 52].
An extremely low LOD for Gly has been obtained
(0.2 mM) using a capillary of 75 mm id, by adding 75% v/v of
ACN to the samples and increasing the sample zone to 20%
of the overall capillary length (43/30 cm). The optimized
BGE was 1.7 M acetic acid with 2% w/v PEG (Mr 8000, PEG
decreases and stabilizes the EOF). It was used to determine
the Gly concentration in periaqueductal grey matter of rats
[53].
2.4 Miscellaneous
Electrochemiluminescence detection is now increasingly
used with CE [54]. As an example, the coupling of Ru(bpy)21 3
(tris(2,20 -bipyridine)ruthenium(II)) with Pro and pipemidic
acid in human urine was developed to quantitate these
compounds in urine [55]. The electroluminiscence signal is
due to the reaction that occurs in the diffusion layer near an
electrode when the active Ru(bpy)31 3 species were electro-
chemically generated from the inactive Ru(bpy)21 3 at the
electrode surface. A double bond C 5 O on the two
molecules is easily oxidized by the generated Ru(bpy)31 3
resulting in reduced form of Ru(bpy)1 3 , which reacts with
another Ru(bpy)31 21
3 and results in Ru(bpy)3 and a photon.
The optimized separation conditions were 115 kV, the BGE
was 70 mM phosphate buffer. pH 5 8. in a 50 cm 25 mm id
capillary, while the detector cell contains 5 mM Ru(bpy)3Cl2
and 50 mM phosphate buffer, pH 9.6. The voltage of PMT
was set at 800 V while the potential of the electrode was
1.15 mV for collecting the electrochemiluminescence signal.
The LODs were around 200 pM and this technique provides
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Electrophoresis 2012, 33, 14–35
an easy quantitation of both molecules in the range 1–90 mM
with recoveries higher than 95% in urine. An identical
method was developed for Pro, tetracaine, and enoxacin [56].
Ala, taurine, Gly, Trp, Glu, Asp were separated via
microchip CE containing an oxidizer solution reservoir for
CL detection [57]. The running buffer was 30 mM borate
solution (pH 10.0) containing 2.5 mM luminol and 0.45 mM
CuSO4, and the effective separation length was 6.5 cm. The
oxidizer solution was 30 mM NaHCO3-NaOH buffer
(pH10.5) containing 50 mM H2O2. The authors report that
260 pL were injected and that LODs were in the range of
0.26–0.61 mM and an analysis of lysate of mice single
fibrosarcoma cell (S180) was presented.
Trp, Gly, and Ala were determined in single cells using
cell injection, loading, lysing, electrophoretic separation
(running buffer was a 20 mM Na2HPO4 solution containing
2.5 mM luminol and 40 mM NaBr (pH 10.0)) and CL
detection (the reaction buffer was 50 mM NaHCO3 solution
containing 0.8 mM K3Fe(CN)6 (pH 12.5)) on an integrated
microchip. The average intracellular contents in rat hepa-
tocytes of these AAs were 5.15, 3.78, and 3.84 fmol (n 5 12),
respectively [58].
A 150  165  70 mm portable instrument using
microchips and electrophoresis with electrochemical detec-
tion was developed. This instrument uses a 73 kV power
supply and an acquisition system with two channels for dual
amperometric data for the separation of the neuro-
transmitters dopamine, epinephrine, and 3,4-dihydroxy-L-
phenyl-alanine (DOPA). The separation buffer was optimized
(25 mM 2-(N-morpholine) ethane sulfonic (MES)-sodium
acetate, pH 5.57) and provided LODs of 2.6, 2.4, and
12.2 mM, respectively [59].
Three on-capillary detection methods, contactless
conductometric, photometric, and fluorimetric, in an iden-
tical point of detection cell, were developed for CE applica-
tions. Two concentrically positioned contactless
conductometric detection electrodes with a detection gap of
7 mm was designed and 255 and 470 nm LEDs were used as
light sources for photometric and fluorimetric detection.
The fluorescence LOD was 10 nM (fluorescein) and the LOD
for photometric measurements was 6 mM (tartrazine) while
it was 50 mM for magnesium chloride via conductimetry.
Samples containing a mixture of fluorescent and non-
fluorescent dyes and common ions, underivatized AAs, and
a fluorescently labelled digest of bovine serum albumin
could be identified [60].
3 Chirality
Enantioseparations of chiral molecules and AAs in parti-
cular are still an important topic for CE and are described in
some recent review articles; as an example, Waldhier et al.
proposed to compare CE and chromatography in biomedical
applications [61].
Research areas that were studied in the past 2 years
include enantioseparations involving in-capillary precon-
& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
CE and CEC 25
centration, the use of CDs and their derivatives, ligand
exchange CE, and studies about the mechanism of mole-
cular interactions involved in these separations. A few new
separations were presented; as an example, Li et al. inves-
tigated molecularly imprinted polymeric monoliths in
pressurized capillary electrochromatography (CEC) using in
situ thermo-initiated copolymerization of methacrylic acid,
4-vinylpyridine and ethylene glycol dimethacrylate, to sepa-
rate D/L Tyr, D/L Trp, and D/L Phe [62], but more work must
be done to obtain additional chromatographic parameters.
3.1 On-line sample enrichment
Quite few studies concerning enantioseparations report the
efforts to improve the detection sensitivity of the technique;
some approaches use LIF [63] while others use preconcen-
tration. As an example, Kirschner et al. [64], used NDA-
labelled AAs with sulphated b-CD at low pH to obtain LODs
of 100 pM for D/L Ser and Glu. In the same vein, Wang et al.
[65] used vancomycin as the chiral selector for enantio-
separation of trace levels of fenoprofen and six 9-fluorenyl-
methyl chloroformate (FMOC)-AA derivatives using
large-volume sample stacking with EOF as a pump plus
anion-selective exhaustive injection. They used a coated
poly(dimethylacrylamide) capillary where the EOF is very
weak, and reversed polarity. Large volumes of the negatively
charged AAs were injected, which migrated to the detection
side and met the positively charged vancomycin; the AA
sweeps on the chiral selectors resulting in resolutions of D/L
AA greater than 2. Val, Ala, Phe, Ser, Met, Leu were studied
and a 1000-fold enhancement in detection sensitivity
compared to the normal injection was recorded.
Lin et al. [66] used a discontinuous system to stack and
separate D/L-Asp, D/L-His, D/L-Leu, D/L-Ile, D/L-Thr deriva-
tized with NDA [29] 0.6% PEO, 150 mM sodium dodecyl
sulphate (SDS), and 60 mM hydroxypropyl-b-CD (Hp-b-CD)
as anode buffer and 150 mM SDS and 60 mM Hp-b-CD in
capillary solution. PEO changes the viscosity and allows to
stack at the interface sample/anode buffer, and SDS acts as
a sweeping agent and pseudo-stationary phase. Aliquots of
600 nL of NDA-D/L-Asp can be injected without the loss of
chiral resolution in CE/LIF (LED 410 nm). The stacking
mechanism is mainly based on the difference in viscosity
between sample zone and PEO. LOD was reported to be
250 pM, a 100-fold improvement compared to non-stacking
experiments and D/L-Asp were rapidly analysed in cere-
brospinal fluid. A similar study was reported by Tseng et al.
[67], who filled the capillary with a solution of 100 mM Tris-
borate, 150 mM SDS, and 50 mM Hp-b-CD, while the buffer
vials contained 20 mM Tris-borate, 150 mM SDS, 50 mM
Hp-b-CD, and 0.5% w/v PEO. In this case, the separation of
NDA-labelled D/L-Val, D/L-Leu, and D/L-Ile was optimized
and the LODs were in the range of 0.18–0.22 nM. D/L-Leu
was determined in urine and plasma samples; the ratio of
D-Leu in both medium compared to total Leu is approxi-
mately 2.6%.
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26 V. Poinsot et al.
Liu et al. [68] synthetized a novel single isomer of
positively charged b-CD to be used in CE/UV (214 nm): the
mono-6-deoxy-6-((2S,3S)-(1)-2,3-O-isopropylidene-1,4-tetra-
methylenediamine)-b-CD. Ten different dansyl AAs (Asp,
Cys, Leu, Met, Phe, Ser, Thr, Trp, Tyr, Val) and N-acetyl-
phenylalanine (N-Ac-Phe) were studied varying the CD
concentration and pH using 50 mm id capillary (60/50 cm),
100 mM NaH2PO4–Na2HPO4, 10 mM mono-6-deoxy-6-
((2S,3S)-(1)-2,3-O-isopropylidene-1,4-tetramethylenediamine)-
b-CD (pH ranging between 5.5 and 8), 115 kV; the migra-
tion times are greater than 24 min. Most resolutions
between D/L enantiomers are above 1.3 (Trp being the sole
AA below 1).
FITC-AA hydrophobicity can be used to enrich deriva-
tized AA in an in-line single drop microextraction (SDME)
[69]. In this work a drop of a basic aqueous acceptor phase
(pH 9.6) covered with a thin organic layer was formed at the
tip of a capillary by simple combination of the sample-
handling sequences of a CE system. The FITC-AAs are
diluted in an acidic solution and enriched into the drop
through the organic layer. In order to improve the stability
of a drop, the capillary inlet tip surface was hydrophobically
coated with octadecyl trimethoxysilane before the first
extraction of a day; then the organic phase (octanol) was
hydrodynamically injected into the capillary at 6 psi for 13 s;
the analytes in 0.1 M HCl were extracted into the basic
acceptor drop through the octanol layer for 10 min by the
driving force of the pH difference between a BGE contain-
ing 80 mM borate buffer, pH 9.3, 12 mM b-CD, and 18 mM
sodium taurodeoxycholate. In addition, to serve as a label-
ling reagent providing a high fluorescence signal, hydro-
phobic FITC was used as a modifier aiding the extraction of
zwitterionic AAs by blocking the amino groups and
increasing the hydrophobicity, yielding a 220 times increase
in extraction efficiency. Several 100-fold enrichments
were achieved with 10 min SDME, yielding LODs of
30–60 pM and enabling direct analysis of D-AA in a 99%
enantiomeric excess mixture. An identical work was
performed with NBD-F [70]. The NBD-AAs, which are
neutral in the sample at acidic pH (pH 1) were successfully
concentrated in octanol and then into the basic electrolyte
(sodium tetraborate buffer, pH 9.2). Enrichment factors are
above 190 for Ala, Gly, Glu, Asp (AAs with a charged side
chain after NBD-F derivatization were not concentrated via
SDME).
The transient moving chemical reaction boundary was
used for the on-line preconcentration of native D/L-AA in
heart-cutting 2-D CE with multiple detection points using
contactless conductivity detection [71]; 0.8 M, pH 8.56,
ammonium formate was used as the sample matrix buffer
and 2.3 M acetic acid as the electrolyte in the first dimension
were used for the heart-cutting 2-D CE using a 10 mm id
capillary. The second dimension was run with the same
electrolyte with 10 mM (1)-(18-crown-6)-2,3,11,12-tetra-
carboxylic acid. The chiral separation of D/L-Phe, and D/L-Thr
in a mixture of 22 native AAs was achieved and an LOD
(S/N 5 3) of 2 mM was determined for L-Thr.
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Electrophoresis 2012, 33, 14–35
3.2 Ligand exchange CE/UV
The formation of diastereomeric ternary mixed metal
complexes between a chiral selector ligand and an analyte
ligand results in the separation of two enantiomer analytes
due to the difference in complex stability constants of the
two diastereomeric mixed complexes.
The chiral complexes, Zn21-L-alaninamide, Zn21-L-
prolinamide, and Zn21-L-phenylalaninamide, were used as
chiral selectors for the enantioseparation of dansylated D/L
AAs. The optimal separation buffer contains 5 mM ammo-
nium acetate, 100 mM boric acid, 4 mM ZnSO4, and 8 mM
L-amino acylamides at pH 8.2. D-AAs migrate faster than
L-AAs when alaninamide and phenylalaninamide are used
in the buffer; it is reversed with L-prolinamide [72]. In a
similar set of experiments, L-ornithine was evaluated for the
enantiomeric separation of Trp, Phe, and Tyr using 100 mM
boric acid, 5 mM ammonium acetate, 3 mM ZnSO4, and
6 mM L-Orn at pH 8.2, 120 kV (capillary 50 mm id, 57/
50 cm, UV detection 214 nm). Measurements of D-AA
oxidase enzyme kinetic constant were reported using these
separation conditions [73]. Using similar CE conditions
(5 mM ammonium acetate, 100 mM boric acid, 7 mM
ZnSO4 and 14 mM L-Aln, adjusted to pH 8.2 with Tris),
Zn21 concentration and pH were optimized to get the best
enantioseparation of dansylated D/L Met. It was used to
measure the change of the distribution of D-AA oxidase
following rat renal ischaemia [74]. Zn21-L-valine complex
was also used with b-CD for enantioseparation of dansylated
AA [75]; the separation was achieved with a buffer of
100 mM boric acid, 5 mM ammonium acetate, 4 mM b-CD,
4 mM ZnSO4, and 8 mM L-valine at pH 8.1. b-CD was used
to separate most of the 20 D/L enantiomers with resolution
ranging between 0.6 and 2.16 (no enantioseparation is
reported for Lys, Glu, Asp, His). An on-column enzymatic
incubation has been developed to study the catalytic effi-
ciency of L-AA oxidases.
Di-L-valinol–Cu21 was optimized for the enantiose-
paration of D/L-dansylated Ala, Ser, Phe, Tyr, Val, Met by
electrophoretic chromatography in a low molecular weight
organo-gel. The organo-gel is composed of trans-(1S,2S)-1,
2-bis-(dodecylamido) cyclohexane in methanol. The L-vali-
nol–Cu21 complex was compared with other amino alcohol
selectors (leucinol, isoleucinol, phenylglycinol, prolinol) and
found to provide the best resolution. The buffer was 0.01 M
ammonia and 5 mM copper diacetate, the pH was adjusted
to 5 with acetic acid, in this case only electrophoretic force
drives the separation. No enantioseparation was observed
for D/L Ser and D/L Phe [76].
On-column labelling of AA enantiomers using dansyl
chloride (Dns-Cl) was combined with chiral ligand-exchange
CE. It was achieved using a sequential injection of buffer
(100 mM boric acid, 5 mM ammonium acetate, 3 mM
ZnSO4, and 6 mM L-Arg, pH 8.4, 10 s), Dns-Cl solution
(1.5 mM, 3 s), AA solution (24 s), and buffer (10 s) at 0.2 psi.
The reaction time was 35 min in the capillary and 16 pairs of
AA enantiomers were separated in less than 24 min, with
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Electrophoresis 2012, 33, 14–35
LODs reported to be approximately 9 mM using UV detec-
tion (214 nm). Serum samples were analysed [77].
Packed CEC using silica-based ligand-exchange chiral
stationary phases was used for the enantioseparation of
underivatized DOPA, Tyr, and Trp derivatives. L-4-Hydro-
xyproline was chemically bonded to the silica material; and a
simpler way was to use N-decyl-L-4-hydroxyproline for a
dynamically coating of a reversed-phase packed capillary.
Both methods using Cu21 ions were compared, and the
superiority of the chemically bonded phase was shown in
terms of resolution of AAs [78].
40 -octadecylneamine, an amino lipophilic compound,
was used for ligand-exchange–MEKC separations. It served
as micelle-forming surfactant (CMC 5 2.5 mM) and Cu21
was used as the complexing ligand. To perform the enan-
tioseparation of D/L Trp, 1-methyl-Trp, 3,5-diiodo-Tyr, and
1-naphtyl-Ala, the electrolyte was 10 mM 40 -octadecylne-
amine and 5 mM CuSO4, 50 mm id, 56/64.5 cm long capil-
lary (UV detection 220 nm) [79].
3.3 Use of CD and modified CD
A non-aqueous CE/UV (250 nm) consisting of a chiral ionic
liquid (ethylcholine bis(trifluoromethylsulfonyl)imide 5 mM)
with heptakis(2,3-di-O-methyl-6-O-sulpho)-b-CD (1.5 mM)
in a methanolic solution of 0.75 M formic acid and 10 mM
ammonium formate was successfully optimized for the
determination of the enantiomeric purity determination of
R/S 4-amino-2,2-dimethyl-6-ethoxycarbonylamino-3,4-dihy-
dro-2H-1-benzopyran [80]. The method allowed the determi-
nation of 0.1% of each enantiomer in the presence of its
stereoisomer. Four new cationic pyrrolidinium-b-CD deri-
vatives were synthesized (mono-6-deoxy-6-pyrrolidine-
b-CD chloride (pyCDCl), mono-6-deoxy-6-(N-methyl-pyrroli-
dine)-b-CD chloride (N-CH(3)-pyCDCl), mono-6-deoxy-
6-(N-(2-hydroxyethyl)-pyrrolidine)-b-CD chloride (N-EtOH-
pyCDCl), and mono-6-deoxy-6-(2-hydroxymethyl-pyrroli-
dine)-b-CD chloride (2-MeOH-pyCDCl)) and their potential
for enantioseparation were tested with dansyl-AA. pyCDCl
exhibited the greatest resolving ability and the optimal CD
concentration was in the range 5–7.5 mM [81].
Gold nanoparticles chemically modified by thiolated
b-CD (size: 9.572.5 nm) were synthesized to separate
dinitrophenyl-labelled D/L-Val, D/L-Leu, D/L-Glu, D/L-Asp.
Using 1-1.4 mg/mL nanoparticles, TPN up to 250 000
plate/m and separation resolution of 4.7 was obtained. The
optimized buffer consisted of 50 mM sodium tetraborate,
pH 8, for neutral AA and 9 for negative AA (50 mm id
capillary, 50/41 cm, UV detection 360 nm) [82].
A series of chiral stationary phases were prepared by the
immobilization of b-CD (singly substituted b-CD by
-NH2, Asp, -hydroxylpropyl) to the epoxy-activated poly
(glycidyl methacrylate-co-ethylene dimethacrylate) monolith
under optimized mild conditions. It results in capillary
electrochromatography with resolution between 0.8 to
5.3 [83].
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CE and CEC 27
The (S)-enantiomer of 3,4-dihydroxyphenylalanine
(levodopa or L-DOPA), and its enantiomer D-DOPA, and the
impurities 6-hydroxy-DOPA, L-Tyr, 3-methoxy-L-tyrosine
were separated using optimized conditions: 0.1 M phos-
phate buffer, pH 2.0 containing 6 mg/mL sulphated b-CD
(capillary 45.2/35 cm, 120 kV, UV detection, 200 nm).
Different batches of sulphated b-CD were used and show
differences in migration profiles [84].
A CE assay for the simultaneous determination of the
impurities of levodopa that are listed in the European
Pharmacopeia including the (R)-enantiomer was developed
and validated. The optimized BGE consisted of 0.1 M
sodium phosphate buffer, pH 2.0, containing 6 mg/mL
sulphated b-CD (120 kV) with L-phenylalanine used as the
internal standard. The assay was validated in the range of
0.1–1.0% for the impurities at a concentration of levodopa of
2 mg/mL. The effect of different batches of sulphated b-CD
was investigated using levodopa and L-tyrosine as a critical
pair. The method was applied to determine the purity of
several samples of levodopa including the chemical refer-
ence substance of the European Pharmacopeia.
The enantioselectivity of cyclofructans (CF) and their
sulphated derivatives were examined. CFs, consisting of a
crown ether skeleton and fructofuranose residues that are
linked to the crown ether ring in a spiral arrangement and 6
and 7 sulphated cyclic-oligomers, were studied. The native
CF showed no enantioselectivity, while the sulphated CF
showed exceptional selectivity toward racemic amines and
AAs. When 15 mM of sulphated CF was used, enantiomeric
resolution factors ranging from 0.6 to 15.4 were achieved
within an analysis time of 10 min [85].
D/L Glu and D/L Asp labelled with 5-(4,6-dichloro-s-tri-
azin-2-ylamino) fluorescein (DTAF) were separated using an
optimized dual selector systems containing sodium cholate
and human serum albumin (HSA) in less than 10.5 min.
The buffers required for DTAF–Asp enantiomers include
12 mM cholate, 8 mM sodium borate buffer (pH 8.9),
0.8% w/v HSA, and 10% v/v methanol; while for DTAF–Glu
enantiomers, the buffer contains 12 mM cholate, 10 mM
sodium borate buffer (pH 9.1), 1.6% w/v HSA, and 5% v/v
methanol (capillary 50 mm id 62.5/50 cm, 25 kV) [86].
3.4 Studies on interactions with chiral selectors
The possible secondary interactions between balhimycin
and derivatized AAs were studied using 12 N-benzoylated
AA enantiomers: benzoyl leucine, benzoyl-Met, 3,5-dinitro-
benzoyl-Leu, 3,5-dinitrobenzoyl-Met, p-nitrobenzoyl-Leu,
p-nitrobenzoyl-Met, 3,5-dichlorobenzoyl-Leu, 3,5-dichloro-
benzoyl-Met, p-chlorobenzoyl-Leu, p-chlorobenzoyl-Met,
m-chlorobenzoyl-Leu), m-chlorobenzoyl-Met. Their enantio-
separations were compared using a CE method, which
combined the partial filling technique with reversed EOF.
Three vancomycin-type macrocyclic antibiotics, i.e. balhi-
mycin, bromobalhimycin, and dechlorobalhimycin (opti-
mized concentration 4 mM), were diluted in the CE running
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28 V. Poinsot et al.
buffer, consisting of Tris (50 mM) and hexadimethrine
bromide (0.001%, w/v, reverse the EOF) (UV detection
214 nm). The displacement study using a competitive
blocking ligand (N-acetyl-L-Lys-D-Ala-D-Ala) for the aglycone
pocket proved that the N-benzoylated derivatives of AA have
the same binding position on these chiral selectors and
hydrogen bonding is the primary interaction responsible for
the enantiorecognition. It was also found that the size of the
side chain of the AA or tagging reagent has a great influence
on enantioselectivity for compounds. It was suggested that
derivatizing the aromatic ring of the N-tagged benzoyl
moiety alter the p–p interaction and steric effect and thus
the enantioresolution and migration time [87].
Tosyl- and Dns-Cl were used to label and quantitate (S)-
(1)-isomer of 3-isobutyl-GABA. Different concentrations of
30 CD derivatives were used as chiral agents at four different
pH values to study the enantioseparation of the racemate.
6-monodeoxy-6-mono-(3-hydroxy)-propylamino-b-CD hydro-
chloride, trimethylated a- and b-CD systems resulted in the
best separation. The structures of the 1:1 inclusion
complexes of the tosyl-S-enantiomer with b-CD were eluci-
dated using 2-D ROESY NMR experiments [88].
Four n-dodecyl thioglycoside sulphates, (sodium
n-dodecyl-L-thio-b-D-galactopyranoside-6-sulphate, sodium
n-dodecyl-L-thio-b-D-glucopyranoside-6-sulphate, sodium
n-dodecyl-2-deoxy-L-thio-b-D-glucopyranoside-6-sulphate, sodium
n-dodecyl-L-thio-b-D-mannopyranoside-3-sulphate) were chosen
as micelle-forming chiral surfactants to separate D/L hydro-
phobic dansylated-AAs (Val, Met, Leu, Phe, Trp). The
separation conditions consist of 30 mM of each surfactant in
50 mM phosphate buffer (pH 6.5) as running buffer, and a
capillary of 50 mm id, 64.5/56 cm, 120 kV. Baseline separa-
tions of all enantiomers were achieved using the first cited
derivatives that have an equatorially oriented hydroxy group
at C-2 position. Although the enantioseparation was
decreased when the two last derivatives were used, it
suggests that the equatorially oriented hydroxy group at C-2
position of the sugar unit is highly important for the
enantiomeric separation of these dansylated AA [89].
4 Applications of CE to AA analysis
4.1 Food and beverage analysis
Since our last review on AA, three reviews have been
published concerning the use of CE techniques to analyse
AAs in food. The first one focuses on grape-derived
products, comparing different chromatographic methodolo-
gies and several derivatizing reagents for enological
purposes [8]. The second one addresses different CE
methods to characterize foods and beverages via the
enantiomeric separation of AAs, pesticides, and phenols
[9]. The last one focused on electromigration methods for
the analysis of food constituents including AA and biogenic
amines [90].
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Electrophoresis 2012, 33, 14–35
The few articles that have been published on the use of
CE for AAs in food are predominantly devoted to qualitative
analyses. In order to identify adulteration of extra virgin
olive oils with seed oils, a CE-MS method has been devel-
oped on a ion trap analyzer allowing to perform MS/MS
experiments [43]. This methodology enables the determi-
nation of six non-protein AAs (ornithine, b-alanine, GABA,
alloisoleucine, citrulline, and pyroglutamic acid) after buta-
nol derivatization. Two of the AAs (ornithine and alloiso-
leucine) are present in seeds but not in olive oil.
Cysteine and glutathione has been quantified in orange
juices and soft drinks at the micromolar level by using a
disulfide reduction step followed by a quinolinium deriva-
tization of the thiols and a separation by CZE with aceto-
nitrile stacking [91].
Two tea analysis methodologies have been published.
The first one focused on the decrease of the matrix inter-
ference for a better analysis of dilute analytes (GABA and
Ala), without the need for prepurification and preconcen-
tration. An in-capillary o-phtaldialdehyde/2-mercaptoetha-
nol derivatization procedure combined with stacking CE was
used to achieve to the subnanomolar detection level [92].
The second work addressed the simultaneous determination
of active compounds in tea (sugars, AAs, catechins etc.)
using CE coupled to an amperometric detection method
(Fig. 3). The running buffer for this purpose was mildly
alkaline and required the addition of NaOH in the detection
cell [93].
S-Adenosyl-L-methionine is the mostly used in-plant as
a methyl-donor. A fast CE separation has been developed to
analyse S-adenosyl-L-methionine in fruits and fruit juices
without pre-purification step; this method was found to be
50% faster and more sensitive than the conventional HPLC
method [94].
For a better understanding of the role and production of
AAs in yeast, a mutant of Saccharomyces cerevisiae has been
investigated using CE-MS. This study combined with
metabolite profiling of 5000 strains showed that lysine and
lysine-related metabolites were accumulated when riboso-
mal protein encoding genes are absent [95].
Figure 3. Electropherogram of oolong tea infusion sample. Peak
identification: (1) L-theanine; (2) fructose; (3) sucrose; (4) glucose;
(5) EGCg; (6) unknown; (7) L-Glu. [93].
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Electrophoresis 2012, 33, 14–35
4.2 Plant physiology
Arabidobsis thaliana is a plant model that is intensively
studied. A mutant of the molibdate transporter has been
studied to understand the role of this micronutriment
essential for plant growth. Using GC/TOF-MS technology
and CE/TOF-MS, authors demonstrated that the levels of
AAs, sugars, organic acids, and purine metabolites were
significantly increased in this mutant [96].
Rice remains one of the most studied crops, and CE-MS
metabolomic studies have been used in two different
investigations. The first report was done to determine the
response to oxidative stress on rice cells affected in the death
suppression factor (Bax inhibitor: BI-1). CE-MS demon-
strated that dynamic metabolic changes are due to an
accumulation of AAs in oxidatively stressed cells [97].
Another study has been focused on the effect of high
temperatures on storage material deposits and a global
metabolic effect could be characterized as sucrose and AA
derivatives accumulation [98].
Metabolic profiles were also characterized for seeds,
pulp stem, and leaves of Illicium anisatum. About 1000
polar metabolites (including AAs and shikimic acid) were
detected and 58 have been annotated and quantified, indi-
cating that CE-Tof technology could be powerful for drug
discovery purposes [99].
4.3 Clinical analyses
AAs analyses by CE present two areas of use: efficient and
fast diagnostic tools or sensitive and time resoluted
techniques to understand dynamic processes.
4.3.1 Diagnostic
Existing methods have been improved and new approaches
developed for diagnostic of some diseases by the analysis of
AAs.
A sensitive efficient CE-LIF analysis of AAs and cate-
cholamines, labelled with Br-BQCA has been developed and
derivatization conditions have been particularly explored.
This procedure demonstrates a wide range of applications
and has been applied to human plasma and rabbit vitreous
sample analyses [36].
NDA labelled AAs and biogenic amines were simulta-
neously analysed using a CE-LED induced fluorescence,
using PEO and SDS containing running buffers. The tech-
nique allowed the quantitative determination of AAs in
breast cancer cells (MCF-7). Taurine and Gln levels revealed
to be significantly different in the MCF-7 cells compared to
the normal epithelial cells (H184B5F5/M10). These results
demonstrate the potential of this approach for breast cancer
study [100].
For a fast diagnosis of metabolic diseases like phenyl-
ketonuria, a method using a CE with dual electrochemical
detection has been developed. Five aromatic acid
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CE and CEC 29
metabolites could be well separated from urines. High
linear response has been obtained, indicating a good
potential for diagnostic tool [101].
Urine fingerprints from Schistosoma mansoni infected
and control mice were obtained on UPLC-MS and CE-UV
systems and the two techniques found equivalent results.
After 30 days infection, phenyl-acetyl-glycine and hippurate
were selected as main disease markers. Statistical analyses
allowed to correlate only phenyl acetyl-glycine to a Schisto-
soma infection [102].
4.3.2 Neurochemistry
A review article has been published at the end of 2009
concerning analytical techniques for the determination of
neurotransmitters. Separation techniques including HPLC,
CE, enzyme assays, sensors, and MS are coupled with
appropriate sampling methods. This review points out the
speed and sensibility gain obtained in the last few years
[103]. A book chapter also described the methods enabling
nanomolar quantification of monoamines and AAs, includ-
ing CE-LIF [104]. Due to microdialysis technology, CE
analyses of AA remains a powerful tool for the investigation
of neurotransmitters.
An array of plugs segmented by immiscible oil in a
teflon tubing has been employed to collect 2 nL micro-
dialysate fractions, corresponding to 1–20 ms sampling.
Temporal resolution (2 s) was not lost by mixing of diffusion
during the transport. The samples were automatically
analysed by CE-LIF at 50 s intervals. This system has been
applied to study the neurotransmitter dynamics evoked by
injecting l-trans-pyrrolidine-2,4-dicarboxylic acid (glutamate
uptake inhibitor) or potassium into rat striatum. The
neuroactive efflux revealed to be maximal at 20 and 13 s,
respectively [105].
NDA-labelled D-Ser and L-Glu effluxes were temporally
studied during cerebral ischaemia. A brain slice micro-
perfusion device coupled off-line with a CE-LIF (LED
420 nm) system was used to monitor dynamic efflux of the
two neurotransmitters in response to oxygen glucose
deprivation in single acute hippocampus slices with a 2 min
temporal resolution. It appeared that D-Ser efflux precede
the L-Glu efflux by one sampling (2 min). L-Gln efflux
decreased significantly in response to oxygen glucose
deprivation [106].
Disulfiram has been used in the treatment of alcohol
abuse and it appears to be effective in cocaine dependence
treatment. Carbamathione has an effect on N-methyl-D-
aspartic acid receptor and the effect of carbamathione on
brain Glu and GABA has been examined. Nucleus accum-
bens microdialysates were collected after intravenous drug
administration (200 mg/kg), labelled with NDA and
analysed using a CE-LIF system. This study revealed a
carbamathione-induced change in both GABA and Glu
levels [107].
The effect of formalin on trace amounts of two
neurotransmitters (Glu and Asp) in the periaqueductal
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30 V. Poinsot et al.
grey matter has been investigated. For this purpose a
5-carboxyfluorescein N-succinimidyl ester labelling has been
performed before CE-LIF analysis. A significant increase of
the Glu and Asp levels was be correlated to formalin injec-
tion [108].
Middle cerebral artery occlusion has been studied by
microdialysis of extracellular fluid of rat hypothalamus.
Neurotransmitters were derivatized using DTAF and the
results demonstrated that middle cerebral artery occlusion
and reperfusion cause significant increases in the levels of
12 AAs [109, 110].
D-Asp and D-Glu were quantified in rat brain and
human cerebrospinal fluid using a microchip electrophor-
esis device equipped with LIF detection. The precolumn
derivatization used FITC and the chiral separation was
performed on a glass/PDMS hybrid microfluidic chip with
g-CD as chiral selector [111].
Endogenous D-Aspartate is often detected in central
nervous system and endocrine tissues. Its synthesis, release,
and accumulation in the CNS of Aplysia californica were
studied By CE-LIF. The D-form is preferentially accumulated
in the neurone containing ganglia. Ionomycin and elevated
extracellular potassium level stimulates the D-Asp release
from the ganglia. These observations indicate a cell-to-cell
signaling role for D-Asp, similar to the classical neuro-
transmitters [112].
4.4 Body fluids
4.4.1 Urine
Two general types of analyses are commonly performed on
urine: detection of the drugs and their metabolites, or the
metabolic effect of them.
Analysis of AA in urine presents some difficulties due to
the low levels of some of these analytes in a quite complex
matrix. Since a rapid and sensitive determination of His and
its methylated derivatives in human urine by CE-LIF is
limited by the rapidity and efficiency of the FITC labelling of
these compounds, a microwave-accelerated derivation was
developed. The labelling was completed in 2.5 min, which is
considerably shorter than the 4–24 h in a conventional
water-bath derivatization process [113].
In order to analyse highly variable anionic metabolites
in urine samples, a microfluidic chip-CE device has been
designed. A ferrofluid valve is incorporated on-chip to
perform cleanup, conditioning, mixing, dilution, injection,
and finally CE separation. The working range covers all
analyte concentrations commonly found and the chip
provides a recovery of more than 94% of spiked standards
[114].
The determination of tetracaine, Pro, and enoxacin in
human urine was effected using a unique combination of a
CE equipped with an electroluminescent detector. The CE
parameters settings, as well as the luminescence parameters
(pH, Ru(bpy)213 ), drug respective concentrations and flow
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Electrophoresis 2012, 33, 14–35
rates have been studied in significant detail. This method is
linear from 0.2 to 100 mg/L with LODs below 20 mg/L. This
method could be applied to drug analysis in human urine
[115].
Hyperlipidimic rats were treated with atorvastatin to
evaluate abnormal effects of this drug and a global and
targeted metabolic profiling was performed on their
urine using HPLC/MS, CE/MS, or GC/MS. Several
biomarkers including steroids and AAs were discovered
as a result of the study and related to a liver toxicity of
atorvastatin [116].
A MAB-ACE technique, using Ni(II) as metal ion and
His as target has been developed as a model. The MAB
could be created and MAB-CE could specifically capture His
rather than other AAs or metabolites in human urines
(Fig. 4). The so obtained results are in agreement with those
obtained via the standard AA analyzer, indicating that this
method could be potentially applied to target metabolites in
a complex biological matrix [16].
4.4.2 Blood/plasma
During the last 2 years, few publications describe method
developments by CE-MS for blood constituent analyses;
however there appears to be a growing interest for high-
performance affinity chromatography (HPAC) and ACE to
characterize drug–protein interactions. ACE has been used
to examine the binding of indoles other than L-Trp
to the Sudlow site II on the protein HAS. The binding
of these small molecules was examined using columns
that contained immobilized HAS. 3-Acetylindole was
found to be the best candidate for use as an alternative to
L-Trp [117].
A differential metabolomic analysis by CE-MS has been
developed to investigate the efficacy of nutritional inter-
vention to attenuate the oxidative stress induced by stren-
uous exercise. Over a period of 6 h, frequent blood collection
Figure 4. Capture of His by MAB-ACE method in human urine.
(A) Electropherogram of His 50 mM with Ni (II) in anodic vial;
(B) electropherogram of tenfold diluted urine without Ni(II) in
anodic vial; (C) electropherogram of tenfold diluted urine with
Ni(II) in anodic vial. [16].
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Electrophoresis 2012, 33, 14–35
was done before, during, and after the exercise. The
experiment has been performed on the same subject two
times, one with and the other without high-dose oral intake
of N-acetyl-L-Cys prior to the exercise. Filtered red blood cell
lysates were analysed by CE-MS on a time-dependent basis.
Oxidized glutathione, reduced, glutathione, 3-methylhisti-
dine, L-carnitine, O-acetyl-carnitine, and creatine were
quantified to demonstrate that oral N-acetyl-Cys pretreat-
ment is effective to attenuate acute oxidative stress, indi-
cating a possible prophylaxis use [118].
Monoclonal antibodies undergo non-enzymatic hydro-
lysis when stored at 51C. Using CE-SDS, it was determined
that iron combined to His enhanced the IgG cleavage when
incubated at 401C. This degradation affects the molecules
containing a l light chain, but not a k light chain. Studies
on this mechanism are ongoing [119].
A trap CE-MS approach was used to analyse Arg
and its methylated derivatives in human plasma (Fig. 5).
A very simple pretreatment consisting of a simple
protein precipitation with acetonitrile was required
and a field amplified injection was applied to enhance
sensitivity. Due to the so generated stacking effect, the
method can be considered as an on-line preconcentration of
the analytes [120].
4.4.3 Decomposition
CZE coupled to ultraviolet detection (200 nm) has been used
to determine selected aromatic biogenic amines and AAs in
mammalian decomposition fluids and the effect of BGE
concentration, pH, percentage of organic modifier, voltage
have been investigated. The separation revealed efficient and
fast (12 min). Compounds were identified via spiking in
porcine decomposition fluids [121].
& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
CE and CEC 31
4.4.4 Retina
CE remains an efficient tool to investigate the retina.
Synthesis of D-Ser in the retina of the tiger salamander
(Ambystoma tigrinum) has been blocked through brief
exposures to phenazine ethosulphate and CE-LIF methods
have been developed to validate the changes in the tissue
levels of D-Ser. A 10 min exposure decreased the D-Ser level
in retina by about half, as did the contribution of the
N-Methyl D-Aspartate receptor to light response of the inner
retina [122]. The N-Methyl D-Aspartate receptor is also
important in different processes in the central nervous
system, including schizophrenia. The extracellular D-Ser
content was directly measured using CE-LIF and a-amino-
3-hydroxy-5-methyl-4-hydroxazole-propionate receptor was
found to be implied in the D-Ser release in intact mouse
retina. This release was found to be persistent in the
presence of a neuronal inhibitor cocktail, but was blocked
after a glial toxin administration [123].
The role of Cys-Gly in glutathione homeostasis in bovine
eye lenses has been investigated. Intracellular and extra-
cellular thiols content was evaluated using free zone CE. The
impairment of Cys-Gly synthesis correlated with the inhibi-
tion of g-glutamyl transferase, resulting high glutathione
effluxes in the extralenticular domain of the bovine lens [124].
Twenty-nine central retinal vein occlusion patients have
been studied and statistically analysed in comparison to 80
control patients. For this purpose, the levels of the plasma
thiols have been measured using CE-LIF and it was found
that central retinal vein occlusion patients exhibit higher
Cys concentrations and lower Cys-Gly and taurine levels
than controls. In the ischaemic subgroup, Cys, Cys-Gly, and
hCys were significantly higher, where taurine remains low
in the two subgroups (ischaemic and non-ischaemic) [125].
Figure 5. Electropherogram
and MS/MS spectra of Arg
and methylated derivatives in
human plasma. ARG is argi-
nine; 1 is an unknown
compound; ADMA is asym-
metric dimethylarginine;
SDMA is symmetric dimethy-
larginine; D7-ADMA is
2,3,3,4,4,5,5-D7-asymmetric
dimethylarginine [120].
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32 V. Poinsot et al.
Identical analyses have been performed on 40 branch retinal
vein occlusion. In this case, only reduction of Cys-Gly and
taurine could be observed [126].
4.4.5 Enzymatic activity
CE is used for enzymatic activity measurements due to its
high temporal resolution speed and sensitivity. A metabolic
profiling by CE coupled with TOF-MS is used to reveal
cellular metabolic processes. The function of YihU protein
in E. coli was uncharacterized. Using an in vitro metabolic
approach in the presence or absence of purified YihU, an
NADH-dependent reduction of succinic semialdehyde to
g-hydroxybutyrate could be demonstrated [127]. Enzyme
activity and competition assays have been evaluated for
hydroxyproline diastereoisomers and Pro enantiomers using
a tunable platform by CE analyses. The method offers
increased sensitivity and selectivity compared the traditional
enzymatic kinetic measurements [128].
The separation of seven kinds of FITC labeled amino
amides was achieved by MEKC-LIF using PEO lauryl ether
as surfactant. The enzyme kinetic constants of L-aspar-
aginase using L-Asp as substrate were determined (K(m) 5
442.0 mM and V(max) 5 69.9 mM/min) [129]. Finally, the
activities of sulfoxide reductase A and methionine sulph-
oxide reductase B in mouse liver were studied using CE-
DAD (436 nm). The method was compared to the HPLC
procedure for the measurements of hepatic activities in mice
fed with or without selenium source [130].
4.4.6 Miscellaneous
The plant pathogen Pseudomonas syringae produces the
coronatine phytotoxin produced by the coupling of crona-
facic acid, and coronamic acid (CMA). Coronatine and
cronafacic acid can be easily be analysed by HPLC, but CMA
require tedious and time consuming pre-purification.
Therefore, a convenient CZE/UV method has been devel-
oped to quantify CMA [131]. Validation of the hypothesis
that N-acetyl-cysteine protect from the 2-hydroxyethyl-
methacrylate by adduct formation has been performed
through CE-MS measurements. In presence of 10 mM N-
acetyl-cysteine, both intracellular and extracellular hydroxy
ethyl methacrylate concentrations greatly decreased and
adducts implying the two could be detected in presence or
absence of the cells [132].
5 Concluding remarks
In this paper we have presented the new developments
concerning CE methods for the analysis of AAs. There have
been a large number of diverse methods for biological
applications, which demonstrate that there is a great
interest in CE for scientists that are interested in AA
analysis. The analysis of chiral molecules seems to be one of
the most interesting applications developed in CE, because
& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2012, 33, 14–35
it is quite simple to separate the different enantiomers. LIF
detection is becoming more popular, especially ones that
use LEDs as the light source, and we note significant
developments of CE/MS for the analysis of AAs. These two
detection technologies are expected to have great applica-
tions in the future specially for metabolomic studies.
The authors have declared no conflict of interest.
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