Capillary electrophoresis of natural products: 2011-2012.

190 Ria Marni S. Tubaon1 Heide Rabanes1,2,3 Paul R. Haddad1 Joselito P. Quirino1 1 Australian

Centre for Research on Separation Science (ACROSS), School of Physical Sciences-Chemistry, University of Tasmania, Hobart, Tasmania, Australia 2 Chemistry Department, Xavier University, Ateneo de Cagayan, Cagayan de Oro City, Philippines 3 Department of Chemistry, School of Science and Engineering, Loyola Schools, Ateneo de Manila University, Quezon City, Philippines

Received September 29, 2013 Revised October 29, 2013 Accepted October 29, 2013

Electrophoresis 2014, 35, 190–204

Review

Capillary electrophoresis of natural products: 2011–2012 Bioactive natural products are major sources of lead compounds for drug discovery and pharmaceutical development, therefore, innovative and current separation and characterization techniques are important for these compounds. Here, CE methods applied for the analysis of natural products published during 2011–2012 are reviewed. This is an updated version of an earlier review paper in this journal, which highlighted developments during 2006–2010. The major method developments over the review period centered on derivatization, chiral analysis, modes of detection, stacking or on-line sample concentration, and sample preparation (predominantly using extraction methods). The samples analyzed were herbal products, foods, soil, and biological samples. Developments also occurred in the areas of quality control, toxicology assessment, and enzyme-inhibitor screening. A table that summarizes the areas, source of natural product, nature of the bioactive analyte, CE conditions, LODs, and corresponding reference is provided. A short description on the theory of CE and insights on future activities of CE on natural products are also presented. Keywords: CE / CZE / MEKC / Natural products

1 Introduction Natural products are defined as components that are extracted directly from plants and animal products [1]. Over the years, there has been an increasing interest in the advantages and use of natural products, particularly in cosmetics, foods, and medicines. For example, about half of the active pharmaceutical ingredients available at present are derived from natural products. Therefore, the discovery and development of new techniques for screening, extraction, characterization, and identification of natural products represents a wide range of opportunities within relevant commercial industries. Among the techniques used for natural product research, sophisticated separation techniques, such as HPLC, have been developed to aid in characterization and identification [2]. These techniques are normally suitable for analysis of low abundance analytes present in complex sample matrices. Another relevant separation technique is CE that gained popularity because of its ease of use, high separation efficiency, versatility in separation modes, and its low sample Correspondence: Dr. Joselito P. Quirino, Australian Centre for Research on Separation Science (ACROSS), School of Physical Sciences-Chemistry, University of Tasmania, Hobart, Tasmania 7001, Australia E-mail: [email protected] Fax: +61-3-6226-2858

Abbreviations: AChE, acetylcholinesterase; AD, amperometric detection; APTS, 8-aminopyreno-1,3,3-trisulfonic acid; EKS, electrokinetic supercharging; ELCL, electrochemiluminescence; GNP, gold nanoparticle; MSS, micelle-to-solvent stacking; NAIM, naphthimidazole; PLE, pressurized-liquid extraction; RSG, Rhizoma Smilacis Glabrae; TIQ, 1,2,3,4tetrahydroisoquinoline derivative; XOD, xanthine oxidase C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

DOI 10.1002/elps.201300473

and reagent consumption [3]. More recently, the limitation of poor concentration sensitivity in CE has been actively tackled by researchers. However, the applicability of CE for trace analysis of a wide range of samples has been successfully demonstrated [4–6]. The typical modes of CE utilized for natural products are CZE, MEKC, and ITP. CZE separates the compounds in a mixture based on the electrophoretic mobilities of the ionic analytes, thus CZE application is limited to charged analytes [7]. MEKC, on the other hand, expands the applicability of CE to neutral molecules [8]. It also provides additional separation selectivity for charged analytes that may be difficult to separate by CZE. The separation of neutral analytes is dependent on the additive-analyte interaction. Separation of charged species also involves the electrophoretic mobility. In ITP, the sample is injected in between two buffer solutions, the front and rear buffer containing the leading and terminating ions, respectively. The mobility of the sample must lie between those of the leading (high) and terminating (low) ions. When voltage is applied, only ionic analytes order themselves according to their respective mobilities [9]. The aim of this review is to highlight the developments of CE on natural products for 2011–2012. This follows a similar review of Rabanes et al. [10], which covered the same topic from 2006 to 2010. The search of published research and review papers was done via Scopus (www.scopus.com) using the keywords CE, CZE, MEKC, and natural products. It showed 50 research and 15 review papers. These numbers indicated considerable research activity in the area of CE of natural products, approximately 25 research papers a year. The research papers are summarized in Table 1, which provides a comprehensive overview of the research focus of the publication, sample sources, analytes, LODs, CE conditions, and corresponding references. www.electrophoresis-journal.com

CE and CEC


191

Table 1. Applications of CE in natural products research

Area Method development Separation

Stacking/ preconcentration

Sample source

Analyte(s)

LOD

CE conditions

References

Tropical plant Costus speciosus

Rutin Quercitrin Quercetin

0.69 mg/L 0.47 mg/L 0.32 mg/L1

[11]

Bulb of Lycoris radiata

Pseudolycorine

0.46 ng/mL

Urine

Berberine Palmatine Tetrahydropalmatine

14.1 ng/mL 14.0 ng/mL 8.1 ng/mL

Fresh Lanzhou lily

Colchicine

0.25 ␮g/mL

Tap and river water

2-Ethylidene-1,5dimethyl-3,3diphenylpyrrolidine Cocaine Codeine 6-Acetyl-morphine Phytic acid Phosphate

50 ng/L

Herbal medicine Leonurus cardiaca

Kaempferol Hesperin Apigenin Rutin Hyperoside Quercetin

0.047 mg/L 0.028 mg/L 0.033 mg/L 0.016 mg/L 0.019 mg/L 0.054 mg/L

Water decoction of Clematis hexapetala pall

Naringenin Hesperetin Naringin Hesperidin

2.0 ng/mL 3.2 ng/mL 6.2 ng/mL 6.8 ng/mL

Fused silica of 76 ␮m id and 51.5 cm effective length; BGE, 10 mM phosphate buffer, 10 mM borate, 50 mM SDS; pH 8.5; 20 kV separation voltage; UV detection at 370 nm; injection at 50 mbar for 5 s; temperature of 20⬚C Fused silica capillary (75 ␮m id) with a length of 50 cm; BGE, 10 mM phosphate buffer at pH 7.5; sample injection, 10 kV for 10 s; detection cell, 300 ␮L of 5 mM Ru(bpy)3 2+ dissolved in 50 mM phosphate buffer (pH 9.0) Fused-silica capillary of 50 cm length, 41 cm effective length, 50 ␮m id, BGE, 50 mM phosphate buffer—20% methanol adjusted at pH 7.0; applied voltage, 14 kV; injection time, 10 s; UV detection 225 nm Fused silica capillary of effective length 40 cm; separation voltage, 20 kV; capillary temperature, 25⬚C; detection at 254 nm; BGE, 20 mM borate buffer (pH 10.0) with 66.7% methanol (v/v); sample injection, 50 mbar for 3 s Fused-silica capillary of 50 ␮m id and 53 cm effective length; sample loading, 930 mbar for 30 min; BGE, 80 mM phosphate buffer at pH 3.0; sample pH, 7.2; capillary temperature, 25⬚C; detector set at 200 nm FEP preseparation capillary (90 mm × 0.8 mm id), FEP analytical capillary (160 mm × 0.3 mm id); electrolytes, 5 mM HCl, 17mM glycylglycine, 10.1% HEC (leading), 10 mM citric acid (terminating) and 20 mM citric acid, 10 mM glycylglycine and 0.1% HEC (background); driving currents, 200 ␮A on the preseparation capillary (ITP stage) and 75 ␮A and the analytical capillary (ZE stage) Fused-silica capillary of 50 ␮m id, and 60 cm length; sample injection, 15 kV for 8 s; MEKC: BGE, 5 mM SDS in 90 mM phosphate buffer at pH 7.0; separation voltage, 15 kV CZE: BGE, 40 mM phosphate buffer at pH 8.5; separation voltage, 15 kV Fused-silica capillary of 50 ␮m id and 50 cm effective length; BGE, 30 mM sodium tetraborate at pH 9.5 containing 5% (v/v) methanol; EKI of sample at −10 kV for 130 s; 100 mM 2-CHES as terminator at 0.5 psi for 17 s injection; separation voltage, −20 kV

Barley seeds

C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

140 ng/L 200 ng/L 200 ng/L 20 ng/mL 10 ng/mL

[12]

[13]

[14]

[15]

[16]

[17]

[18]

www.electrophoresis-journal.com

192

R. M. S. Tubaon et al.


Table 1. Continued.

Area

Sample source

Analyte(s)

LOD

CE conditions

References

Urine

Berberine Jatrorrhizine

0.002 ␮g/mL 0.003 ␮g/mL

[19]

Urine

Ephedrine Berberine

0.5 ng/mL 1.1 ng/mL

Traditional Chinese medicines Portulaca oleracea L., Crataegus pinnatifida, and Aloe vera L.

Linolenic acid Lauric acid p-Coumaric acid Ascorbic acid Benzoic acid Caffeic acid Succinic acid Fumaric acid ( −)- Epicatechin (+)-Catechin Rutin Naringenin 4-Hydroxyphenyl acetic acid Hesperetin Syringic acid Ferulic acid Chlorogenic acid p-Coumaric acid Vanilic acid Salicylic acid Myricetin Quercetin p-Hydroxybenzoic acid 7-Hydroxyflavone Morin Apigenin Hesperidin Caffeic acid Gallic acid Protocatechuic acid Flavone

0.003 ␮g/mL 0.06 ␮g/mL 0.006 ␮g/mL 0.003 ␮g/mL 0.0006 ␮g/mL 0.001 ␮g/mL 0.009 ␮g/mL 0.0004 ␮g/mL 31.0 ␮g/L 27.7 ␮g/L 37.3 ␮g/L 17.6 ␮g/L 34.4 ␮g/L

Fused-silica capillary of 50 ␮m id and 50 cm effective length; sample injection, 20.0 psi for a min; sample matrix, 50 mM ammonium acetate, 6% (v/v) acetic acid and 10 mM SDS in redistilled water; BGE, 50 mM ammonium acetate and 6% (v/v) acetic acid in pure methanol; separation voltage, −20 kV; detection at 210 nm Fused-silica capillary of 50 ␮m id and 50 cm effective length; capillary temperature, 18⬚C; BGE, 20 mM H3 BO3 and 20 mM phosphate buffer solution at pH 4.0; micellar sample matrix, 20 mM SDS, 20 mM H3 BO3 and 20 mM phosphate buffer at pH 4.0; cosolvent buffer, 20 mM H3 BO3 and 20 mM phosphate buffer in methanol/water (90:10, v/v); separation voltage, 25 kV; detection at 210 nm Fused-silica capillary of 75 ␮m id and 50 cm effective length; sample injection, 3 psi for 50 s; separation voltage, 22 kV; capillary temperature at 25⬚C; BGE, 40 mM H3 BO3 to 40 mM Na2 B4 O7 at pH 8.7 with 12% ACN

APTS–labeled oligosaccaharides

–

Red wine

Derivatization

Endo-␤-1,3glucanase from Thermoga petrophila (TpLam)


25.7 ␮g/L 51.0 ␮g/L 37.5 ␮g/L 89.0 ␮g/L 24.0 ␮g/L 62.1 ␮g/L 45.3 ␮g/L 43.0 ␮g/L 61.3 ␮g/L 36.2 ␮g/L 19.6 ␮g/L 68.0 ␮g/L 37.7 ␮g/L 87.5 ␮g/L 32.5 ␮g/L 34.0 ␮g/L 17.1 ␮g/L 19.5 ␮g/L

[20]

[21]

Fused-silica capillary of 25 ␮m id and 40 cm effective length; BGE, 25 mM borate buffer of pH 9.0.5 with 35 mM SDS; capillary temperature, 25⬚C; separation voltage, 20 kV; sample injection, 50 mbar for 350 s; UV detection at 280 nm

[22]

Fused-silica capillary of 50 ␮m id and 31 cm total length; separation voltage, 17 kV, 70–100 ␮A; temperature, 2⬚C; APTS-labeled oligomers excited at 488 nm and emission were collected at 520 nm band pass filter

[23]


CE and CEC


193

Table 1. Continued.

Area

Sample source

Analyte(s)

LOD

CE conditions

References

Digestome of the lower termite Coptotermes Gestroi

APTS-labeled oligosaccaharides

–

[24]

Therapeutic antibiodies

APTS-labeled oligosaccaharides

–

Ganoderma lucidum polysaccharide

NAIM-labeled monosaccharides

–

Traditional Chinese medicine Ephedra sinica

D-Mannose (Man),

1.22 ␮M 1.95 ␮M 1.63 ␮M 0.98 ␮M 1.09 ␮M 1.40 ␮M 1.47 ␮M

Fused-silica capillary of 50 ␮m id and 31 cm length; sample injection, 0.5 psi for 0.5 s; electrophoresis conditions, 10 kV/70–100 mA; BGE, 0.1 M phosphate buffer at pH 2.5; capillary temperature, 20⬚C; detection at 520 nm Capillary of 30 cm effective length, 50 ␮m id; BGE, 100 mM Tris-acetic acid buffer at pH 7.0 with 0.05% hydroxypropyl cellulose; applied voltage, −15 kV at 25⬚C, sample injection, 0.5 psi for 15 s Fused-silica capillary of 30 cm effective length, 50 ␮m id; sample injection, pressure of 5 s; operation temperature 30⬚C; detection wavelength, 254 nm Fused silica capillary, 50 ␮m id, effective length 40 cm; sample injection, 0.5 psi for 5 s; capillary temperature, 25⬚C; BGE, 35 mM borate buffer at pH 10.02; applied voltage, 20 kV; detection at 254 nm

Fused-silica capillary of 75 ␮m id and 60 cm effective length; capillary temperature, 18⬚C; sample injection, 0.3 psi for 3 s; BGE, 0.2 mol/L borate buffer at pH 8.5; applied voltage, 18 kV; detector at 210 nm Column of 50 ␮m id × 75-cm long; sample injection, 50 mbar for 12 s; BGE, 20 mM acetic acid/ammonium acetate buffer at pH 5.5; chiral selector solution, 1.0 mM sulfated ␤-CD in CE BGE; voltage, 25 kV; column temperature, 20⬚C

[28]

L-Rhamnose (Rha), D-Glucose (Glc), D-Galactose (Gal), L-Arabinose (Ara), D-Xylose (Xyl), D-Glucuronic acid

[25]

[26]

[27]

(GlcUA), D-Galacturonic

1.12 ␮M

acid (GalUA) Chiral analysis

Detection

Ground grape pomace seed

(+/−)-Catechin (+/−)-Epicatechin

–

In vitro formation of N-methylsalsolinol

R-6,7-Dihydroxy-1methyl-TIQ (salsinol, Sal) S-6,7-Dihydroxy-1methyl-TIQ(salsinol, Sal) (R/S)-1-Benzyl-TIQ (R/S)-NMethylsalsolinol

1.2 ␮M 1.5 ␮M

[29]

Flavanones and flavanone-7-Oglycosides

Naringenin Hesperetin Eriodictyol Homoeriodictyol Isosakuranetin Hesperidin

–

Capillary, 41.5 cm effective length,50 ␮m id.; applied voltage, 25 kV; positive polarity at the inlet, 5 kPa pressure for 4 s; temperature, 20⬚C; detection at 280 ± 10 nm

[30]

Human urine and nasal drops

Ephedrine Pseudoephedrine

4.5 × 10−8 mol/L 5.2 × 10−8 mol/L

[31]

Traditional Chinese medicine Herba Houttuyniae

Rutin Isoquercitrin Quercitrin Chlorogenic acid

41.4 ␮g/mL 31.8 ␮g/mL 38.2 ␮g/mL 65.6 ␮g/mL

BGE, 15 mM PBS-borax at pH 9.4; electrokinetic injection,10 s × 10 kV; separation voltage, 12 kV; detection potential, 1.2 V; ELCL solution, 5 mM Ru(bpy)3 2+ with 50 mM phosphate buffer solution at pH 8.5 40 cm length of 25 ␮m id fused-silica capillary; BGE, 50 mM borate buffer at pH 9.2; separation voltage, 12 kV; sample injection, 12 kV for 6 s


[32]


194



Table 1. Continued.

Area

Sample source

Analyte(s)

LOD

CE conditions

References

Medicinal plant Catharanthus roseus L.

Catharanthine Vindoline Vinblastine

0.1 ␮g/L 0.1 ␮g/L 0.8 ␮g/L

[33]

Extracts of Chelidonium majus L. herb

Protopine Stylopine Allocyptopine Chelidonine Sanguinarine Chelerythrine Coptisine Sophoridine Matrine

0.05 ␮g/mL 0.06 ␮g/mL 0.06 ␮g/mL 0.07 ␮g/mL 0.22 ␮g/mL 1.7 ␮g/mL 5.5 ␮g/mL 5.0 × 10−9 mol/L 1.0 × 10−9 mol/L

Cytoseira spp extract in rat urine

Putative classifiers

–

Traditional Chinese medicine Rhizoma corydalis

Tetrahydropalmatine Palmatine

– –

Herbal plant Saussurea mongolica Franch

Asebotin Kaempferol-3-O-␤-Dglucopyranoside Kaempferol-3-O-␣-Lrhamnopyranoside Kaempferol-7-methoxy3-O-␣-Lrhamnopyranoside Quercetin-3-O-␤-Dglucopyranoside Quercetin-3-O-␣-Lrhamnopyranoside

2.5 ␮g/mL 5.0 ␮g/mL

Fused-silica capillary of 65-cm length and 50-␮m id BGE, 20 mM ammonium acetate with 1.5% acetic acid; separation voltage at 20 kV; injection at 50 mbar for 5 s Polyimide-coated fused-silica capillary of 40 cm effective length, 75-␮m id; BGE, 20 mM phosphate buffer at pH 3.1; sample injection by hydrodynamic flow at a height differential of 10 cm for 10 s; applied voltage, 16 kV, LED excitation wavelength, 280 nm Fused-silica capillary of 40-cm length; detection potential, 1.2 V; EKI, 10 kV for 10 s; separation voltage, 15 kV; BGE, 50 mM phosphate buffer at pH 8.5, 100 mM phosphate buffer at pH 8.5 in the detection cell Fused-silica of 50 ␮m id and 100 cm total length; BGE, 0.8 mol/L formic acid and 10% methanol; sample injection, 50 mbar for 17 s; separation voltage, 30 kV; capillary temperature, 20⬚C Fused-silica capillary of 50 cm × 75-␮m id; BGE, 60 mM disodium hydrogen phosphate and 55% methanol; 24 kV applied voltage; temp, 30⬚C tetrahydropalmatine: running buffer at pH 4.27; 214 nm detection wavelength. Palmatine: BGE at pH 2.80 and detection wavelength at 254 nm Fused silica capillary with effective length of 30 cm,50-␮m id; BGE, 20 mM borate buffer with 5 mg/mL 1B-3MI-TFB at pH 9.00. Voltage, 15kV; temperature, 25⬚C; UV detected at 280 nm

Traditional Chinese medicine Magnolia officinalis bark and Huoxiang Zhengqi liquid

Honokiol Magnolol

1.67 ␮g/mL 0.83 ␮g/mL

[39]

Herba Geranii, a traditional Chinese medicine

Rutin Hyperin Kaempferol Corilagin Geraniin Gallic acid Protocatechuic acid

36.3 ␮g/L 30.9 ␮g/L 21.6 ␮g/L 45.3 ␮g/L 126.9 ␮g/L 232.2 ␮g/L 290.1 ␮g/L

Fused-silica capillary, 75 ␮m id and 40.5 cm effective length; applied voltage, 25 kV; sample injection, 30 mbar for 3 s; detection wavelength at 210 nm; BGE, 16 mM borate buffer at pH 10 with 11% methanol Fused silica capillary of 40 cm length, 25 ␮m id; BGE, 50 mM borate buffer at pH 9.2; separation voltage, 12 kV; detection electrode potential, +0.80 V (vs. Ag/AgCl wire electrode); sample injection, 12 kV for 6 s

Human urine


0.5 ␮g/mL

[34]

[35]

[36]

[37]

[38]

1.0 ␮g/mL

1.0 ␮g/mL 2.0 ␮g/mL

[40]


CE and CEC


195

Table 1. Continued.

Area

Sample source

Analyte(s)

LOD

CE conditions

References

Tea

Caffeine Epigallocatechin Epigallocatechin gallate Epicatechin Gallic acid Epicatechin gallate Galocatechin gallate ␤-Glucan

–

Quartz capillary with an external polyimide coating, 75 ␮m inner diameter, 50 cm effective length; BGE, 25 mM phosphate buffer solution at pH 7 with 25 mM SDS; sample injection, 30 mbar for 10 s; separation voltage, 25 kV Neutral uncoated capillary, 50 ␮m id; BGE, 20 mM phosphate buffer at pH 6.5; sample injection, 0.5 psi for 5 s; separation voltage, 23.5 kV

[41]

Amygladin Paeoniflorin Calycosin-7-O-␤-Dglycoside Salvianolic acid B Decursin

10 mg/L 20 mg/L 10 mg/L

[43]

Mannitol Sucrose Glucose Fructose

0.66 ␮M 1.15 ␮M 0.70 ␮M 0.86 ␮M

Fused-silica capillary of 50 ␮m id, 42 cm effective length; sample injection at 50 mbar for 5 s; applied voltage of 25 kV and 25⬚C column temperature; BGE, 40 mM sodium borate and 60 mM SDS containing 10% methanol; pH 9.5; DAD detector at 250 nm Fifty centimeter length of 25 ␮m id and fused-silica capillary; BGE, 50 mM NaOH; injection and separation voltage, 9 kV; injection time of 6 s

3-Acetylcoumarin 6-Hydroxycoumarin Cinnamic acid Ferulic acid 4-Hydroxycoumarin 4-Hydroxycinnamic acid Caffeic acid Rosmarinic acid Salidroside Tyrosol

1.10 mg/mL 0.27 mg/mL 0.29 mg/mL 0.34 mg/mL 0.26 mg/mL 0.27 mg/mL 0.32 mg/mL 1.00 mg/mL 0.24 ␮M 0.28 ␮M

Fused-silica capillary of 75 mm id, and 50 cm effective length; temperature of capillary, 25⬚C; sample injection, 3.45 kPa for 5 s; separation voltage, 25 kV; BGE, 20 mM sodium tetraborate buffer at pH 9.2; detection at 203 nm

[45]

[46]

Vegetable oils

Glycine betaine Trigonelline Proline betaine Carnitines

0.075 ng/g 0.050 ng/g 0.075 ng/g 0.050 ng/g

Rhizoma Smilacis Glabrae, traditional Chinese medicine

Astilbin Taxifolin 5-O-caffeoylshikimic acid Shikimic acid Trans-resveratrol 5-Hydroxymethyl furaldehyde Geniposidic acid Chlorogenic acid Paeoniflorin 20-Hydroxyecdysone Coptisine Berberine Luteolin Glycyrrhizic acid

–

Fused-silica capillary of 40 cm length, 25 ␮m id; BGE, 50 mM borate buffer (pH 9.8); separation and injection voltage, 12 kV; injection time, 6 s; detection potential, +0.8 V (vs. Ag/AgCl wire) Fused-silica capillary of 50 ␮m id with a total length of 85 cm; capillary temperature, 25⬚C; BGE, 0.1 M formic acid at pH 2.0; sample injection at 50 mbar for 15 s; applied voltage, 25 kV; detection at 195 nm Fused-silica capillary of 50 ␮m id, 50 cm effective length; sample injection, 0.5 psi for 10 s; BGE, 20 mM borax at pH 9.4; separation voltage, 25 kV; temperature, 25C; detection at 214 nm

Oat flours

Application Quality control

Traditional Chinese herbal medicine BoYang-Hwan-O-Tang

Traditional Chinese medicine Folium Lysium Chinensis, dried leaf of Lysium chinensis Mill Tea extracts and medicinal plants

Traditional Chinese medicine, Rhodiola, the dried roots of Rhodiola rosea L.

Herbal medicinal preparation, “samgiumgagambang”


–

150 mg/L 20 mg/L

10 mg/L 20 mg/L 2 mg/L 25 mg/L – 5 mg/L 2 mg/L 2 mg/L 20 mg/L

Fused-silica capillary (50 ␮m id, 52 cm effective length; sample injection, 50 mbar for 5 s; BGE, 70 mM borate buffer with 10% MeOH at pH 9.5; applied voltage, 25 kV; capillary temperature, 25⬚C; detection at 230 nm

[42]

[44]

[47]

[48]

[49]


196



Table 1. Continued.

Area

Sample source

Analyte(s)

LOD

CE conditions

References

Traditional Chinese herbal medicine Radix Aconiti

Aconitine

5.62 × 10−8 mol/L 2.78 × 10−8 mol/L 3.50 × 10−8 mol/L

Fused silica of 50 cm length × 50 ␮m id; 25 mM borax buffer as BGE at pH 9.15 with 20 mM EMImBF4 ; 50 mM phosphate buffer as detection buffer at pH 9.15 with 5 mM Ru(bpy)3 2+ ; electrokinetic injection at 17 kV for 8 s; separation voltage of 15 kV Fused silica capillary, 50 cm effective length, 50-␮m id; BGE, 40 mM ammonium acetate of pH 3.64; capillary temperature, 20⬚C; separation voltage, 25 kV; detection at 203 nm

[50]

Mesaconitine Hypaconitine

Strychnine Brucine Strychnine N-oxide Brucine N-oxide

0.83 ␮g/mL 0.92 ␮g/mL 2.10 ␮g/mL 2.50 ␮g/mL

Charged metabolites

–

Mast cell degranulating peptide Apamine Phospholipase A2 Melittin

1.7 ␮g/g

Oleoropein glycoside Tyrosol Hydroxytyrosol Cinnamic acid Luteolin Apigenin Ferulic acid Caffeic acid p-Coumaric acid Vanilic acid 3,4-Dihydroxybenzoic acid Gallic acid p-Hydroxybenzoic acid

0.15 mg/L 0.22 mg/L 0.09 mg/L 0.07 mg/L 0.85 mg/L 0.59 mg/L 0.10 mg/L 0.08 mg/L 0.05 mg/L 0.08 mg/L 0.04 mg/L

Traditional Chinese medicine

L-Tyrosine Indole quinine

–

Natural extracts

Thiocholine Acetylthiocholine

–

Extracts of medicinal plants

Nicotinamide adenine dinucleotide (␤-NAD+) ␤-Nicotinamide adenine dinucleotide (reduced disodium salt) (␤-NADH)

–

Traditional Chinese medicine, Nux vomica, the dried ripe seed of Strychnos nux-vomica L. Toki-ShakuyakuSan, Chinese herbal medicine

Honeybee venom

Extra-virgin olive oil

Screening inhibitors


1.2 ␮g/g 8.3 ␮g/g 4.6 ␮g/g

[51]

Fused-silica capillaries of 50 ␮m id and 100 cm total length; BGE, 1 M formic acid at pH 1.8; sample injection, 50 mbar for 3 s; separation voltage, 30 kV; capillary temperature, 20⬚C Fused-silica capillary of 56 cm effective length, 75 ␮m id; sample injection, 50 mbar for 7 s; separation voltage, 15 kV; UV–DAD detector at 220 nm

[52]

Fused-silica capillary of 50 ␮m id × 60 cm effective length; BGE, 50 mM of boric acid; pH 10.2; 30 kV; 25⬚C, injection of 50 mbar for 25 s; reverse voltage at −30 kV for 5s, 210 nm detection

[54]

Fused silica capillary of 50 ␮m id, 60 cm length (8.5 cm to the detector); sample injection, enzyme 50 mbar for 10 s, substrate 50 mbar for 5s; BGE, 20 mM borate buffer at pH 9.0; separation voltage, 25 kV; capillary temperature, 37⬚C, detection at 214 nm Fused-silica capillary of 75 ␮m id, 50 cm effective length; separation voltage, 25 kV; capillary temperature at 37⬚C; BGE, 20 mM Tris-HCl at pH 8.0; sample injection, 0.5 psi, 10 s; incubation time, 2 min; detection at 230 nm 50 ␮m id, 29.5 effective length fused-silica capillary; detection at 254 nm; sample injection, substrate solution at 40 mbar for 5s; BGE, 25 mM borate buffer (pH 9.0); voltage applied, 28 kV; column temperature, 25⬚C;

[55]

[53]

0.13 mg/L 0.08 mg/L

[56]

[57]


CE and CEC


197

Table 1. Continued.

Area

Toxicology assessment

Sample source

Analyte(s)

LOD

CE conditions

References

Natural extracts from traditional Chinese medicine

4-Aminopyrazolo[3,4d]pyrimidine Xanthine oxidase Uric acid

– –

[58]

Brachiaria brizantha plant

Citric acid Aspartic acid Malic acid Oxalic acid Pyruvic acid Tartaric acid Formic acid Lactic acid Succinic acid Acetic acid

0.0349 mmol/L 0.0371 mmol/L 0.1084 mmol/L

Fused-silica capillary of 50 ␮m id, and 34.5 cm total length; sample injection, 20 mbar for 5 s; micellar solution, 15 mM; BGE, 25 mM borate buffer at pH 8.0; capillary temperature, 35⬚C; incubation time, 20 s Fused-silica capillary of 75 ␮m id, and 40 cm effective length; sample injection, 25 mbar for 2 s; detection at 240 nm; BGE, 20 mM phthalic acid, 0.8 mM CTAB; separation voltage, −15 kV; temperature, 25⬚C

Human urine

Ephedrine

4.5 × 10−9 g/mL

Fused-silica capillary of 25 ␮m id and length of 40 cm; sample injection, 10 s at 12 kV; separation voltage of 12.5 kV; BGE, 25 mM phosphate buffer solution at pH 8.0; detection potential, 1.15 V; 60 mM PB at pH 8.5 containing 5 mM Ru(bpy)3 2+ in the detection cell

[60]

0.01 mM

[59]

UV, ultraviolet; Ru(bpy)3 2+ , Tris(2,2 -bipyridyl)ruthenium(II); FEP, fluorinated ethylene-propylene; HEC, 2-hydroxyethyl cellulose; EKI, electrokinetic injection; APTS, 8-Aminopyreno-1,3,3-trisulfonic acid; NAIM, naphthimidazole; ELCL, electrochemiluminescence; 1B-3MI-TFB, 1-ethal-3-methylimidazolium tetrafluoroborate.

The research articles are grouped depending on their research focus. Method development in CE wherein natural products were used as sample sources comprised about 63% of the total papers reviewed. Within method development, subsections include sample preparation (8%), stacking or preconcentration (16%), derivatization (10%), chiral analysis (6%), and detection (22%). Specific applications employing natural products as sample sources constituted 37% of papers. CE was used in quality control (24%), toxicology assessment (4%), and screening of enzyme inhibitors (8%). The research articles on method development and applications are further discussed in Sections 3 and 4, respectively, while the reviews are covered in Section 2.

2 Previous reviews There were two recent reviews that are closely related to this work. Chen et al. [61] outlined the development of CE since 2006 and CEC since 2009. However, the review was more focused on phytochemicals as analytes in herbal medicines. Zhao et al. included CE as one of the advanced techniques in the elucidation of phytochemicals in food and medicine in China [62]. Reviews detailing CE as one of the hyphenated techniques coupled with MS detection employed for metabolomic studies [63], complex sample characterization [64], and natural product analysis [65] have appeared. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Other reviews were generally directed to specific applications where CE was one of the techniques used. CE for the analysis of the constituents of tea [66]; polyphenolic compounds in fruits and vegetables [67]; amino acid enantiomers [68]; antimicrobial food components [69]; methylxanthines [70]; optical resolution of flavanones, alkaloids, essential oils, amino acids [71]; foodomic methodologies [72]; and analytical profiling of bioactive constituents of herbal products [28, 73] has been reviewed.

3 Method development This section will describe developments on sample preparation, stacking/preconcentration, derivatization, chiral analysis, and detection. Sample preparation has focused mainly on extraction procedures, with several efforts to reduce or eliminate the use of organic solvents also appearing. Stacking techniques were by field enhancement, micelle to solvent stacking (MSS), and electrokinetic supercharging (EKS). One paper described on-line SPE. Derivatization was with common reagents such as 8-aminopyreno-1,3,3-trisulfonic acid (APTS) and 2,3 naphthalenediamine. Chiral analysis employed cyclodextrin derivatives as selectors to enable enantiomeric separation. Lastly, detection was mainly by UV, but also included LIF, electrochemiluminescence (ELCL), MS, and amperometric detection (AD). www.electrophoresis-journal.com

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Figure 1. Typical electropherograms of colchicine (A) normal injection of 50 ␮g/mL colchicine without extraction. (B) Distilled water spiked with 50 ␮g/mL colchicines enriched by IL-SDME. The inset figure shows the enlarged view of normal injection.


Figure 2. Electropherogram of EKS–CE for Clematis hexapetala pall extract. Separation of four flavonoids (1, naringerine; 2, hesperetin; 3, naringin; 4, hesperidin). CE conditions: EKI of sample at −10 kV for 130 s; hydrodynamic injection of 100 mM CHES at 0.5 psi for 17 s; UV detection at 254 nm.

3.1 Sample preparation Prior to analysis in any CE mode, the desired components must be transferred into forms that are compatible with the method of measurement. Newly designed approaches of extraction, such as directly suspended droplet microextraction [13], and far-infrared light irradiation extraction [44], were successfully utilized for carbohydrates and alkaloids, respectively. Single-drop microextraction was developed for the determination of the toxic alkaloid colchicine found in Lanzhou lily [14]. However, instead of using organic solvent in the extraction process, this investigation employed the ionic liquid 1-alkyl-3-methylimidazolium hexafluorophosphate [C4 MIM][PF6 ]. Using a sample volume of 600 ␮L, a single-drop extraction volume of 2.4 nL, and an extraction time of only 10 min, this novel method produced a lower LOD compared to other CE methods. However, when applied to the extract of Lanzhou lily, colchicine was not detected and thus the method needs to be improved for real sample analysis. A typical electropherogram is shown in Fig. 1. Chang et al. [11] successfully developed a novel pressurized liquid extraction method (PLE) using surfactants, SDS, and Triton X-100 as extraction solvents. This eliminates the use of toxic organic solvents. PLE followed by MEKC was applied to the analysis of flavonoids from a widely used veterinary medicine, Costus speciosus. PLE with SDS was superior to the use of Triton X-100, and was comparable with Soxhlet extraction with a methanol/water mixture.

3.2 Stacking/preconcentration Stacking improves detection sensitivity in CE by increasing the sample load. MSS was successfully applied for the precon-


centration of different charged analytes in CE [74]. MSS–CZE was applied for berberine and ephedrine in urine. Utilization of cosolvents (methanol/water) to induce the micelle collapse in order to reverse the effective electrophoretic mobility of the charged alkaloids was investigated [20]. Enhancement factors of 300 and 1036 were achieved for ephedrine and berberine, respectively. MSS was also implemented in CZE with a nonaqueous solvent for the analysis of berberine and jatrorrhizine in urine [19]. Sharp peaks for the alkaloids were detected within 20 min. The enhancement factors were 128 and 153 for berberine and jatrorrhizine, respectively. EKS was successfully utilized by Zhong et. al [18] for the detection of four flavonoids in an aqueous extract of Clematis hexapetala pall. EKS is a combination of field amplified or enhanced sample injection and transient ITP. Sodium tetraborate buffer was used as the leading electrolyte, with CHES as the terminating electrolyte. The enhancement factors were 8–16 times better than the factors attained in other CE methods employing on-line concentration for separation and analysis of flavonoids. Examples of these preconcentration methods are field-enhanced sample injection in reversemigration MEKC, anion selective electrokinetic injection, and water plug sweeping in a reverse-migrating microemulsion and micellar EKC. Figure 2 depicts the electropherogram of a water decoction of C. hexapetala pall applying a developed EKS method. Four flavonoids, naringenin, hesperetin, naringin, and hesperidin were detected in the sample. Lastly, in-line SPE was developed for the investigation of naturally occurring illicit drugs in tap and river water [15]. Waters Oasis HLB sorbent and methanol were employed for the enrichment and elution steps, respectively. The analytes investigated were 2-ethylidene-1,5-dimethyl-3,3diphenylpyrrolidine, cocaine, codeine, and 6-acetyl- morphine.


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3.3 Derivatization

3.5 Detection

Derivatization is an approach that enhances detection and/or improves the separation of target analytes in separation techniques. During this review period, several sugars were processed and derivatized prior to CE with UV and MS detection. CZE of the hydrolysis products of APTS-labeled laminarihexaose was used for the characterization of an endo-␤1,3-glucanase enzyme from Thermotoga pertophila (TpLam), a Gram negative hyperthermophilic bacterium [23]. The degradation of the labeled sugar was monitored by CZE of the fluorescent products (dimers, trimers, and tetramers). The study shed light into the mode of enzymatic reaction of TpLam and will advance the use of the bacterium in the biotechnology sector. CZE was used for the analysis of degradation products of APTS-labeled oligosaccharides in crude body extracts from Coptotermes gestroi, a lower termite [24]. The study concluded that the termite possessed the ability to cleave almost all the glycosidic bonds of the tested sugar substrates at a wide pH range. CZE was applied to the determination of the fluorescent naphthimidazole (NAIM) derivatives formed from the labeling by 2,3-naphthalenediamine of monosaccarides in Ganoderma lucidum [26]. UV detection was used because of the absence of a fluorescence detector coupled with the instrument. The CE analysis resulted in the quantification of the saccharides depicted in Fig. 3, which coincided with the outcome of a study done on trimethylsilylated monosaccharide derivatives by GC–MS analysis. However, the CE study detected new components in the sample, namely ribose, arabinose, and glucuronic acid. Labeling with 2,3-naphthalenediamine was also successfully applied to saccharides containing carboxylic acid and N-acetyl groups in heparin disaccharide.

The success and advancement of CE as a powerful separation technique is also dependent strongly on the development and progress of the sensitivity of detectors; UV detectors were the most commonly used in CE systems (comprising 64% of the total). This is simply because the majority of bioactive compounds in natural products absorb in the UV region. LIF and ELCL gained popularity because of their high detection sensitivity. LIF detection was used for the analysis of APTS-labeled oligosaccharides [24, 25, 42]. Two of these papers specifically used argon LIF. Moreover, excitation and emission wavelengths at 488 and 520 nm were employed, respectively. ELCL, on the other hand, has a simpler instrumentation design than fluorescence. However, successful derivatization is the major limiting factor in the performance of ELCL. When the appropriate derivatizing chemistry is attained, the sensitivity of ELCL detection in CE is on par with that of fluorescence. Tris(2,2 -bipyridyl)ruthenium(II) (Ru(bpy)3 2+ ) is the most common luminophore for ELCL [12, 35, 50, 60]. There were five articles pertaining to the use of ELCL as the detection method in CE [12, 31, 35, 50, 60] and the two papers that caught our attention are discussed below. First was the work by Liu et al. [35] where they immobilized the Ru(bpy)3 2+ in a Nafion/perylenetetracarboxylic dianhydride (PTC-NH2 ) composite film on a modified Pt disk working electrode. The immobilization approach improved the ELCL signals significantly. Second, Bao et al. found that addition of ionic liquid, for example, 1-ethyl-3methylimidazolium tetrafluorborate (EMImBF4 ), enhanced ELCL sensitivity threefold [50]. The performance of the method in terms of sensitivity, linearity, selectivity, and repeatability for the characterization of alkaloids in the Chinese herbal medicine Radix Aconiti was noteworthy. One paper reported the use of native UV-LED-induced fluorescence (LEDIF) for the sensitive analysis of seven alkaloids in the medicinal herb, Chelidonium majus L. [34]. This light source was operated at 280 nm and the emission was at the range 341–600 nm. With the proposed CE method that employed direct introduction of the filtered methanolic extract, the cytotoxic potential of each alkaloid was effectively determined. MS provides a high level of detection and also identifies and confirms the identities of compounds in complex mixtures. There were five CE–MS applications involving the determination of betaines in vegetable oils [47], anticancer alkaloids [33], enantiomeric neurotoxins [29], components of urine for metabolic fingerprinting [36], and chemical compositions contained in Toki-Shakuyaku-San [52]. AD has a number of attractive features, such as high sensitivity, low cost and power demands, and relatively easy coupling with CE. There are four reports using this form of detection [32, 40, 44, 46] and three of them employed the use of graphene to improve the sensitivity of AD. Specialized composite electrodes incorporating graphene included graphene/PMMA [40], polystyrene/graphene [32], and

3.4 Chiral analysis The chiral selectors sulfated ␣-CD, (2-hydroxy propyl)-␥-CD, sulfated-␤-CD were used for the CE separation of monosaccharide NAIMs, catechin and epicatechin enantiomers, and 1,2,3,4-tetrahydroisoquinoline derivatives (TIQs), respectively. In a study by Kuo et al., the cyclodextrin derivative provided a suitable cavity for the hydrophobic moiety of NAIM [26]. The authors were able to determine D-Man-NAIM and D-Glc-NAIM levels of 73.2 and 26.8%, respectively, in G. lucidum. The epimerization or racemization of catechin and epicatechin enantiomers during the fermentation process of winemaking was investigated by Rockenbach [28]. (+)-Catechin and (−)-epicatechin were identified in all grape seed samples, while the opposite enantiomers were not detected. A CE-MS/MS method was used by Wu to determine the enantioselective neurotoxicity of TIQs [29]. TIQs are neurotoxins that induce Parkinsonism. The proposed method allowed the identification and separation of four NMSal isomers for the first time. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


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Figure 3. CE analysis of the mono saccharide-NAIM derivatives in Ganoderma Lucidum polysaccharide. Identification of the peaks was in comparison with standard monosaccharide NAIMs. CE conditions: fused-silica capillary of 30 cm length and 50 ␮m id; 100 mM borate buffer (pH 9.0) running buffer; 12 kV applied voltage; sample loading by pressure for 3 s; column temperature at 25⬚C; and UV detection at 254 nm.

graphene/poly(urea-formaldehyde) [46]. This type of electrode allowed for high sensitivity detection of phenolic compounds in Herba geranii, bioactive constituents in Herba houttuyniae, and salidroside and tyrosol in Rhodiola.

4 Applications Presented in this section are the applications that utilize CE for bioactive metabolites. CE was widely used for the quality control of herbal drugs during this review period. Also found were innovative CE techniques for biological and toxicological assays, for example screening of enzyme inhibitors, doping in sports, and soil toxicity.

4.1 Quality control This section discusses the quality control of herbal drugs by CE. There are several approaches considered: first, the identification and assay of known active ingredient/s; second, chemical profiling of known and unknown compounds; and third, identification or detection of adulterants. There are four papers published related to the identification and assay of known active ingredient/s. First, the concentrations of mannitol, sucrose, glucose, and fructose in Folium Lysium Chinensis were determined by CZE–AD [44]. Higher amounts of these carbohydrates indicated a better quality of the herbal drug. Far-IR-assisted extraction was also developed to shorten the total analysis time. In another investigation, CZE–AD was used to ensure that the salidroside content was not less than 0.5% in the traditional Chinese herbal medicine, Rhodiola [46]. In the next work, CE–ELCL was applied to monitor the concentration of toxic diester-diterpenoid alkaloids in Radix Aconiti [50]. Lastly, CZE–UV was employed for the simultaneous determination of strychnine, brucine, strychnine N-oxide, C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

and brucine N-oxide in crude and fermented nux vomica [51]. It is noted that for human consumption, crude nux vomica containing toxic strychnine and brucine must be processed by fermentation to their less toxic N-oxide derivatives. Six research papers were published in the area of chemical profiling of known and unknown compounds. First, a CE–DAD fingerprint method was developed for quality evaluation and species differentiation of Rhizoma Smilacis Glabrae (RSG) [48]. RSG has a similar Chinese name to two other medicinal herbs, Rhizoma Smilacis Chinae and Rhizoma Heterosmilacis, and therefore there is a need to differentiate between these herbs. From the nine peaks found in the electropherograms for RSG, five were identified, that is, astilbin, taxifolin, 5-O-caffeoylshikimic acid, shikimic acid, and transresveratrol. However, only astilbin was identified in Rhizoma Smilacis Chinae, while it is absent in Rhizoma Heterosmilacis, shown as peak 2 in Fig. 4. The next study involved CE as well as HPLC of modified Bo-Yang-Hwan-O-Tang [43]. The modified Bo-Yang-Hwan-O-Tang contained five additional major compounds that add to the herbal drug’s biological activity. No comparable difference was obtained between the results by the CE and HPLC methods. In another study, CE with TOF MS was performed to determine the polar metabolites of Toki-Shakuyaku-San that is composed of six varieties of herbal medicines of various pharmacological functions [52]. The presence of metabolites was related to the pharmacological effect by multivariate analysis. Kokot and et al. developed a CE–UV method to profile the honeybee venom content in two bee strains [53]. Several honeybee venom components were separated for the first time. Principal component analysis was used to show differences of constituents that were found dependent on the bee strain type. A CZE method was developed for the analysis of phenolic compounds of Melissa officinalis L. [45]. The investigated samples of commercial M. officinalis L. in a survey carried out in different herbal shops in Argentina corresponded to the bioactivity of Nepeta cataria. The developed method www.electrophoresis-journal.com


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Figure 4. (A). CE electropherograms of Rhizoma Smilacis Chinae and Rhizoma Heterosmilacis. (B). CE electropherogram of Rhizoma Smilacis Glabrae concentrate extract product. Peak 2 was identified as astilbin. CE conditions: fused-silica capillary of 50-cm length and 50 ␮m id; running buffer of 20 mM borax at pH 9.4; sample injection at 0.5 psi for 10 s; separation voltage of 25 kV; UV detection at 214 nm; column temperature of 25⬚C.

shortened the analysis time compared to previous studies of M. officinalis L. Moreover, the method was simple, accurate, and precise, thus makes it suitable for routine quality control. A multivariate statistical technique employed in CZE was proposed by Ballus et al. for the characterization of 13 phenolic compounds in olive oil [54]. Method validation showed satisfactory results for official requirements in the quantification of the phenolic compounds. Lastly, a CE–DAD method was developed for the identification of nine marker components in herbal medicinal preparation, “samgiumgagambang,” and compared with HPLC [49]. Five major markers were recommended to be used for the routine quality control of the herbal drug. There was no significant difference in the outcomes of both separation methods. A single article was found describing the identification or detection of adulterants. A CE–ESI–MS2 method was developed for the sensitive detection of glycine betaine, proline betaine, trigonelline, and total content of carnitines in extra virgin olive oils [47]. Carnitines were proposed to serve as novel markers in detecting adulterations of extra virgin olive oils with seed oils.


4.2 Screening of enzyme inhibitors Four innovative CE-based methods for screening enzyme inhibitor compounds were reported. Two publications were based on EMMA and the other two were based on immobilizing enzymes inside the capillary. In EMMA, the capillary is used as a microbioreactor where the substrate and the enzyme are successively introduced. The same capillary is also used for the separation of substrates and products. All relevant steps, such as reaction, separation, and quantitation, are combined in one miniaturized and fully automated system [75–77]. The first report on EMMA was by Zhang et. al [58] for the screening of xanthine oxidase (XOD) inhibitors in 15 natural extracts. XOD catalyzes the formation of uric acid, which can cause hyperuricemia and gout. Employing 4-aminopyrazolo 3,4-d-pyrimidine as the inhibitor, the percentage of inhibition was determined based on the peak area of uric acid. One of the 15 natural extracts, Cortex Phellodendri, was found to be positive for XOD inhibition as evidenced by the reduction of the peak area of the product. Typical electropherograms for the blank and Cortex Phellodendri are


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inlet of the capillary forming an accurate and reliable GNPmediated enzyme bioreactor. Employing perphenazine as the model inhibitor, inhibition percentages of 37, 45, and 53% were obtained for three natural extracts out of 25 extract samples tested.

4.3 Toxicology

Figure 5. Electropherograms for XOD inhibitor screening. (A) blank; (B) Cortex Phellodendri. CE conditions: fused-silica capillary of 34.5 cm length and 50 ␮m id; running buffer of 25 mM borate buffer (pH 8.0) containing 15 mM SDS; sample injection at 20 mbar for 5 s; separation voltage at 15 kV; concentration of XOD was 0.5 ␮g/mL; concentration of substrate was 1.0 mM; UV detection at 295 nm; column temperature of 35⬚C.

shown in Fig. 5. The developed method was also applied to estimate the IC50 value of the XOD inhibitor. The other study on EMMA was for screening and identification of tyrosinase inhibitors from metal complexes [55]. Tyrosinase enzyme is responsible for the browning of fruits and vegetables, and for the formation of dermatological disorders, such as freckles, melasma, ephedlide, and senile lentigines. The percentage of inhibition was calculated from the peak area of indole quinine, which is the enzymatic product. Four out of the 21 Chinese herbs showed inhibitory characteristic to tyrosinase. Min et al. immobilized acetylcholinesterase (AChE) with the aid of polycationic polyethylenimine, calcium chloride, and chitosan to form an in-capillary enzyme reactor [56]. This reactor was used to screen AChE inhibitors. Enhanced activity of AChE may lead to neurological disorder diseases, such as Alzheimer’s, glaucoma, ataxia, and myasthenia gravis. The substrate, acetylthiocholine, was mixed with known inhibitors (i.e. Huperzine) or natural extracts before injection into the capillary. Fourteen of the 30 natural extracts tested exhibited AChE inhibition. The percentage inhibition range was 31.1–89.0%. Another novel approach introduced by Zhao et al. [57] used L-glutamic dehydrogenase immobilized onto 38 nm-diameter gold nanoparticles (GNPs). The functionalized GNPs were then assembled onto the inner wall at the C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

There were two reports of the use of CE to monitor toxicity. The toxicity of the alkaloid ephedrine was assessed by monitoring its level in urine [60]. This alkaloid is strictly monitored by the medical commission of the international Olympic committee as one of the prohibited substances in the doping list because of its properties as a stimulant of the central nervous system. The developed CE method was coupled with Ru(bpy)3 2+/– ELCL detection. The toxicity of aluminum in the soil on plants was measured indirectly by the CZE determination of organic acids in plant extracts [59]. These organic acids are known to inhibit toxicity. Aided by a chemometric approach, the optimized methodology was applied successfully for the simultaneous determination of citric, malic, and aspartic acid in roots and leaf extracts of Brachiaria brizantha. It was shown that the presence of aluminum encourages the excretion of citric and malic acids in both roots and leaves.

5 Concluding remarks Although CE of natural products is an application-driven research area, it has also stimulated innovative approaches to sensitive detection via modification of detector systems, reduction in the use of harmful organic solvents, and to the design of highly specific miniaturized in-capillary assays. These approaches can be adapted readily to other natural products and thereby to further widen the applications of CE in this field. CE is an attractive tool for natural product analysis because of its known assets, such as low sample and reagent consumption, ease of use, and short analysis time. The improvement in CE–MS technology in the recent years, as well as its ability for qualitative and quantitative analysis should further attract its application in natural products. In the moderately regulated herbal drug industry, CE is preferable over other separation techniques, such as HPLC. The role of MCE is yet to be realized in this field. R. M. S. T. is funded by the Tasmania Graduate Research Scholarship (TGRS) of the University of Tasmania and the Australian Research Council Future Fellowship FT100100213 to J. P. Q. H. R. R. is supported by the Commission on Higher Education (CHED), Philippine Government. The authors have declared no conflict of interest. www.electrophoresis-journal.com


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Capillary electrophoresis.

Capillary electrophoresis.

Multidimensional capillary electrophoresis.

High performance capillary electrophoresis.

Capillary electrophoresis of microbial aggregates.

Stacking and analysis of melamine in milk products with acetonitrile-salt stacking technique in capillary electrophoresis.

Separation of some triazine herbicides and their solvolytic products by capillary zone electrophoresis.

Assay of melamine in milk products with a pH-mediated stacking technique in capillary electrophoresis.

Micro-injector for capillary electrophoresis.

Low pH capillary electrophoresis application to improve capillary electrophoresis-systematic evolution of ligands by exponential enrichment.

Separation of natural and synthetic heparin fragments by high-performance capillary electrophoresis.

Characterization of fruit products by capillary zone electrophoresis and liquid chromatography using the compositional profiles of polyphenols: application to authentication of natural extracts.

Characterization of plasma apolipoproteins by capillary electrophoresis.

Application of capillary electrophoresis in peptide research.

High-performance capillary electrophoresis of gangliosides.

High performance capillary electrophoresis of calmodulin.

Applications of capillary electrophoresis for industrial analysis.

Application of cyclodextrins in chiral capillary electrophoresis.

Capillary electrophoresis of quantum dots: minireview.

Observation of DNA molecules undergoing capillary electrophoresis.

Screening of epidermal growth factor receptor inhibitors in natural products by capillary electrophoresis combined with high performance liquid chromatography-tandem mass spectrometry.

Theoretical evaluation of capillary electrophoresis performance.

Capillary electrophoresis of hemoglobins and globin chains.

Capillary electrophoresis: present state of art.