**4.6. Capillary electrophoresis**

**TECHNIQUE MOBILE PHASE COLUMN**

water/ acetonitrile

HPLC not informed Zorbax SB-C18

HPLC not informed not informed

5% ethyl acetate in chloroform

water to 50% methanol in water

TECHNIQUE MOBILE PHASE COLUMN

Acetonitrile and water containing 0.05% trifluoroacetic acid Flow: 1 mL/min

water to 50% methanol; 30% acetonitrile in water to 50% acetonitrile MPLC Flow: 6 mL/min

**Table 4.** Separation by Pressure Liquid Chromatography

Octadecyl-silane (ODS) (250×10mm)

Michel-Miller column (200 g Silica gel)

RP-18 (25–40 mm particle size, 460 × 36 mm I.D., 460 × 15 mm I.D.)

Lichrospher C18 (5mm, 250 mm × 4.6 mm I.D.)

LiChroprep C-18 (45 × 3.5 cm) and Shimpack C-18 (10 μm, 45 cm × 250 mm I.D.)

HPLC

170 Column Chromatography

LPLC

MPLC

HPLC

MPLC and HPLC

**4.5. Counterflow**

**SPECIES/ PLANT MATERIAL**

*Plumeria acutifolia* / leaves

*Plumeria rubra* L. cv. acutifolia/ flowers

*Allamanda neriifolia*/ aerial parts

*Allamanda schottii*/ stem

> *Himatanthus sucuuba*/ latex

SPECIES/ PLANT MATERIAL

*Himatanthus sucuuba*/ bark and latex

> *Himatanthus sucuuba/* latex

Protoplumericin and plumieride can be extracted from the methanol extract of *Allamanda neriifolina* stems [117] by droplet counter-current chromatography. Crude extract, ob‐ tained from 1.6 kg of plant material, was successively partitioned with benzene, chloro‐

methanol extract

ethyl acetate fraction

> ethanol extract

> ethanol extract

methanol/ water (1:1)

methanol/ water (1:1)

aqueous fraction and fractions from this procedure

**SAMPLE IRIDOID REF**

15-demethylplumieride [71]

plumieridin A and plumieridin B [120]

allamandin and allamcin [47]

plumieride [73]

[52]

[73]

[70]

allamanoid, plumieride and protoplumericin

SAMPLE IRIDOID REF.

plumieride, isoplumieride and demethylplumieride

15-demethylisoplumieride, 15 demethylplumieride, plumieride and isoplumieride

For analytical purposes, iridoids can be analyzed by capillary electrophoresis. A method to separate nine iridoids described in [131] uses a Hewlett-Packard (HP3D CE) capillary electro‐ phoresis system coupled to a photodiode array detector (210 nm and 230 nm) and equipped with a fused-silica capillary tube (90 cm × 75µm I.D.). The distance to the detector was 81.5 cm. Other conditions: sample injection at 50 mbar for 3 s and further deionized water injection at 50 mbar for 3 s; constant voltage, 16 kV (positive to negative); cartridge temperature, 30 °C; electrolyte (buffer), 50 mM sodium borate and 30 mg/mL 2,6-di-*O*-methyl-*β*-cyclodextrin (DMβ-CD); run time, 32 min. Before the analyses, the capillary column was sequentially purged with 0.5 M NaOH, 0.1 M NaOH, deionized water and buffer solution. The iridoids studied eluted in the following order: geniposide, loganin, shanzhiside, aucubin, catalpol, harpago‐ side, gardenoside, geniposidic acid and loganic acid. All were commercially purchased and only loganin, loganic acid and gardenoside were described for the Apocynaceae family. Several conditions of analyses were studied, including different pH, surfactants, concentra‐ tions of sodium borate and the addition of cyclodextrins (CD) to the buffer, and it was concluded that the less polar DM-*β*-CD added to 50 mM borate solution was the most suitable running buffer. In this condition, only aucubin and catalpol could not be separated, even with the addition of organic solvents and/or valine, urea and barium ion. The greatest advantage of capillary electrophoresis compared to HPLC analyses (the most commonly used technique) is its speed.

**Author details**

Deborah Q. Falcão2

**References**

1426.

Ana Cláudia F. Amaral1

– FIOCRUZ, Manguinhos, Brazil

, Aline de S. Ramos1

1 Laboratório de Plantas Medicinais e Derivados, Depto de Produtos Naturais, Farmanguinhos

[1] Bianco A, De Luca A, Mazzei RA, Nicoleit M, Passacantilli P, Limas RA. Iridoids of

[2] Dewick PM. Medical Natural Products: A biosynthetic Approach. 2º Edition. John

[3] Sampaio-Santos MI, Kaplan MAC. Biosynthesis Significance of Iridoids in Chemo‐

[4] Oudin A, Courtois M, Rideau M, Clastre M. The Iridoid Pathway in *Catharanthus*

[5] Eisenreich W, Bacher A, Arigoni D, Rohdich F. Biosynthesis of Isoprenoids Via the Non-Mevalonate Pathway. Cellular and. Molecular. Life Sciences. 2004; 61 1401–

[6] Hunter WN. The Non-Mevalonate Pathway of Isoprenoid Precursor Biosynthesis.

[7] Contin A, Van der Heijden R, Lefeber AWM, Verpoorte R. The Iridoid Glucoside Se‐ cologanin is Derived from the Novel Triose Phosphate/Pyruvate Pathway in a *Ca‐*

[8] Eichinger D, Bacher A, Zenk MH, Eisenreich, W. Analysis of metabolic pathways via quantitative prediction of isotope labeling patterns: a retrobiosynthetic 13C NMR

systematics. Journal of the Brazilian Chemical Society 2001; 12(2) 144-153.

*roseus* Alkaloid Biosynthesis. Phytochemical Reviews 2007; 6 259–276.

3 Laboratório de Cromatografia – Depto. de Química – UFAM, Japiim, Manaus, Brazil

4 Instituto de Pesquisas Biomédicas, Hospital Naval Marcílio Dias, Brazil

*Rauwolfia grandiflora.* Phytochemistry 1994; 35(6) 1485-1487.

Journal of Biological Chemistry 2007; 282 21573–21577.

*tharanthus roseus* Cell Culture. FEBS Letters 1998; 434 413–416.

study on the monoterpene loganin. Phytochemistry 1999; 51 223–236.

, Bianca O. da Silva4

5 Fac. de Ciências Farmacêuticas – UFAM, Manaus, Brazil

\*Address all correspondence to: acamaral@fiocruz.br

2 Faculdade de Farmácia – UFF, Niterói, Brazil

Wiley & Sons, LTD; 2002.

, José Luiz P. Ferreira1,2, Arith R. dos Santos1

A General Description of Apocynaceae Iridoids Chromatography

, Debora T. Ohana1,5 and Jefferson Rocha de A. Silva3

,

173

http://dx.doi.org/10.5772/55784

According to [132], capillary electrophoresis can be used to analyze a mixture of eleven iridoid glycosides: unedoside, harpagide, methyl catalpol, morroniside, asperuloside, griselinoside, catalpol, ketologanin, verbenalin, loganin and 10-cinnamoyl catalpol. Only loganin was found in the Apocynaceae family. For the analyses, iridoids were diluted in purified water. A Hewlett-Packard 3DCE system coupled to a diode array detector and equipped with an aircooling device was used. The fused-silica capillary tube measured 80 cm in length, 50 µm in I.D. and 375 µm in O.D. Distance to the detector was 71.5 cm only for UV detection (197 nm, 235 nm, 239 nm and 283 nm). When coupled to a mass spectrometer system (Bruker ESQUIRE) with an electrospray ionization source, the drying gas was nitrogen at 200 ºC and flow-rate 100 L/h. In this case, the distance between injector and UV detector was 20 cm. Other condi‐ tions: sample injection at 50 mbar for 5 s (only UV detection) or 25 s (with MS system); voltage, +20 kV; cartridge temperature, 25 °C; electrolyte solution, 20 mM ammonium acetate with 100 mM sodium dodecyl sulfate (SDS), pH 9.5; sheath liquid, 1 mM lithium acetate mixture to water/methanol (1:1 v/v) at a flow rate of 200 µL/h. When the MS system was used: scan range, 100-550 *m/z*; cut-off, 80 *m/z*; glass capillary exit, 95 V; skimmer, 32 V; electrospray voltage for the capillary, -4.0 kV; for the cylinder, -1,8 kV; for the end plate, -3.5 kV. In the comparison among the counterions sodium dodecyl sulfate (SDS), ammonium dodecyl sulfate and lithium dodecyl sulfate, diluted in water and running buffer, the best resolution for separating iridoid glucosides, lower noise in the MS system, and better repeatability and sensitivity were found with SDS in the running buffer. The volatility of ammonium acetate in buffer enables MS analyses, and concentrations higher than 20 mM did not represent better resolution. Quite the contrary, higher SDS concentrations furnished better results. In the study of the influence of pH, the best one was 9.5, although its influence in the range of 8.7-10.0 was lower than the SDS effect. Good linearity was observed for all the iridoids glucosides analyzed, but in different ranges.

The literature describes chromatographic techniques related to the characterization, isolation and purification of iridoids. Most reports show the open column technique as the principal technique used to isolate this class. Also, there have been few studies on counterflow and capillary electrophoresis chromatographies. In general, there has been little scientific invest‐ ment in the area of obtaining iridoids of the Apocynaceae family, despite the great pharma‐ cological importance of this class of constituents.
