See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/225907237

Analysis of Catharanthus roseus alkaloids by HPLC

Article  in  Phytochemistry Reviews · June 2007

DOI: 10.1007/s11101-006-9036-y

CITATIONS

48

READS

2,240

2 authors, including:

Mario Jolicoeur

Polytechnique Montréal

120 PUBLICATIONS   1,764 CITATIONS   

SEE PROFILE

All content following this page was uploaded by Mario Jolicoeur on 25 January 2014.

The user has requested enhancement of the downloaded file.

Analysis of Catharanthus roseus alkaloids by HPLC

Steve Hisiger Æ Mario Jolicoeur

Received: 22 June 2005 / Accepted: 5 October 2006 / Published online: 13 March 2007� Springer Science+Business Media B.V. 2007

Abstract Catharanthus roseus is a medicinal

plant from which secondary metabolites used in

chemotherapy to treat diverse cancers are ex-

tracted. The well known high value metabolites

vincristine and vinblastine are just 2 of 130

alkaloids that can be found in C. roseus. However,

only few (~11) of this high number of chemical

entities are frequently analyzed and even fewer

(~8) are available commercially. For more than

30 years, different analytical techniques have

been developed to isolate and identify C. roseus

metabolites, and then allowing revealing the

therapeutic potential of C. roseus metabolites.

Among few approaches, high performance liquid

chromatography (HPLC) technique is still widely

used for the separation and analysis of secondary

metabolites such as those from C. roseus. This

article thus reviews the most recent developments

in HPLC analysis of alkaloids from C. roseus.

Diverse considerations that are crucial to the

efficiency of secondary metabolites separation and

identification steps, such as biomass manipulation,

extraction phase and protocols, HPLC separation

and analysis protocols are reviewed in details.

Examples of spectra obtained using the most

common detectors are also shown and suggestions

are made on how to proceed in developing

efficient separation and identification methods at

the analytical and semi-preparative scales.

Keywords Semi-preparative HPLC � Reversed-

phase HPLC � Fluorescence � UV � Vincristine �Serpentine � Vinblastine

Abbreviation

HPLC high performance liquid

chromatography

TLC thin layer chromatography

SPE solid phase extraction

PDA photo diode array

FW fresh weight

DW dry weight

MS mass spectrometry

TIA terpene indole alkaloid

TCA trichloroaceticacid

TEA triethylamine

TFA trifluoroacetic acid

NMR nuclear magnetic resonance

CD circular dichroism

Introduction

Catharanthus roseus is known to biosynthesize

more than 130 alkaloids (van der Heijden et al.

S. Hisiger � M. Jolicoeur (&)Canada Research Chair on the Development ofMetabolic Engineering Tools, Bio-P2 Research Unit,Department of Chemical Engineering, EcolePolytechnique de Montreal, P.O. Box 6079, Centre-ville Station, Montreal, Quebec, Canada H3C 3A7e-mail: mario.jolicoeur@polymtl.ca

123

Phytochem Rev (2007) 6:207–234

DOI 10.1007/s11101-006-9036-y

2004). Although this rich molecular pool has been

screened for therapeutics since few decades, only

five alkaloids from C. roseus (serpentine, ajmal-

icine and the bisindole alkaloids vinblastine,

vincristine and 3¢,4¢-anhydrovinblastine) are mar-

keted. Ajmalicine and serpentine are prescribed

for the treatment of hypertensia, whereas the

bisindoles vinblastine, vincristine and 3¢,4¢-anhy-

drovinblastine are used for their antineoplastic

activity in the treatment of many cancers (see van

der Heijden et al. 2004 for review). These

secondary metabolites are end-products usually

found in plant extracts (Fig. 1), and the biosyn-

thetic pathways leading to these compounds are

already described in literature (van der Heijden

et al. 1989, 2004; Facchini 2001; De Luca and

St-Pierre 2000; De Luca and Laflamme 2001). In

vitro cultures of plant cell suspension and of

diverse tissues (such as hairy roots) have been

widely studied for the production of these high

value and complex molecules, but with limited

commercial success. A major bottleneck is that

some of the alkaloids showed to be tissue-specific

and cannot be obtained in undifferentiated cell

culture. Vincristine and vinblastine require both

aerial and root parts of a plant to be synthesized

(De Luca and Laflamme 2001). Horhammericine,

tabersonine, lochnericine, 19-hydroxytaberso-

nine, 19-O-Acetyl-horhammericine and echitove-

nine are mostly synthesized in roots (Shanks et al.

1998; Laflamme et al. 2001; Rodriguez et al.

2003), and are not always detected in suspension

cells (Kutney et al. 1980; Jolicoeur unpublished

data). In addition, the natural bisindole alkaloids

vingramine and methylvingramine have been

seen in seeds of C. roseus (Jossang et al. 1998),

but their presence in other tissues has not

been demonstrated yet. In the last decades, a

significant research effort has focused on remov-

ing the metabolic limitations that are encountered

in in vitro culture as compared to plants (Ver-

poorte et al. 1999; Verpoorte and Memelink 2002;

Hughes et al. 2004). Research in metabolic engi-

neering and cell line selection have then signifi-

cantly contributed to the improvement of

analytical methods, and most of the efforts have

been placed on chromatographic technologies

which now show to be highly efficient, fast,

accurate and high-throughput for the analysis of

plant alkaloids (McCalley 2002; Drager 2002;

Houghton 2002; Molyneux et al. 2002; Stockigt

et al. 2002; Chen et al. 2004). Thin-layer chroma-

tography (TLC) (De Luca et al. 1986; Asada and

Shuler 1989; Monforte-Gonzales et al. 1992) and

high-performance TLC (Klyushnichenko et al.

1995) were previously suggested for a rapid

screening of plants or cell lines. However, these

methods show a low precision level for the

identification and quantification of alkaloids

from complex mixtures such as crude plant

extracts, which contain hundreds of different

compounds. High-performance liquid chromatog-

raphy (HPLC) then represents a powerful capac-

ity of separation of alkaloids and is particularly

adapted for secondary metabolites fingerprinting.

Automation of HPLC allows high-throughput

analyses and collection of the different fractions

after separation (Stockigt et al. 2002).

Among the molecules illustrated in Fig. 1,

current HPLC methods mainly allow for the

separation and the identification of ajmalicine,

serpentine, vinblastine, vincristine, vindoline,

catharanthine, tabersonine, lochnericine, secolog-

anin, strictosidine, tryptophan and tryptamine.

Reason for this limited list of alkaloids relies on

the fact that no standards are commercially

available (to our knowledge) for the other alka-

loids found in C. roseus. Due to their relative low

abundance in plant and cell extracts, unidentified

alkaloids usually appear as weak peaks that are

‘‘lost in the forest’’ of a chromatogram; a situation

which can bias the identification and the quanti-

fication of the compounds of interest. In the

pathway leading from tabersonine to vindoline,

which occurs in the aerial parts of the plant (De

Luca et al. 1988; St-Pierre et al. 1999; De Luca

and St-Pierre 2000), four intermediates have been

identified as 16-hydroxytabersonine, 16-meth-

oxytabersonine, 16-methoxy-2,3-dihydro-3-hydro-

xy-N-methyltabersonine and deacetylvindoline

(De Luca and Laflamme 2001). Strictosidine

derivatives as 4,21-dehydrogeissoschizine, cathen-

amine, epicathenamine, and stemmadenine and

its derivatives (akuammicine, condylocarpine)

leading to catharanthine and tabersonine (Ste-

vens et al. 1993; El-Sayed et al. 2004) can co-elute

with compounds of interest (see above). There-

fore, the efficiency of a separation method will

208 Phytochem Rev (2007) 6:207–234

123

strictosidine

cathenamine ajmalicine

secologanin

NH

N

CO2Me

H

O

NH

N

CO2Me

H

OH

stemmadenine tabersonine lochnericine

19-hydroxytabersonine catharanthine vindoline

COOMeN

H

N

OH

H

N

N

CO2Me

H

OH

HCHO

MeO CO2Me

COOMeN

H

N

OH

H

N

N

CO2Me

H

OH

H

Me

MeO CO2Me

N

N

CO2Me

H

OH

H

Me

MeO CO2Me

COOMe

N

H

N

CH2CH3

NH

N

CO2Me

H

MeOOC

N

N

H

CH2OH

H

MeOOC

H

NN+

HH

OH

MeOOC

H

NN

HH

OMeOOC

H

NN

HH

O

H

MeOOC

H

NN+

H

O

H

OMeOOC

H

OGlc

NN

HH

H

NH

NH2

O

OMeOOC

H

HOGlc

H

NH

N

CO2Me

H

OH

O

tryptamine

4,21-dehydrogeissoschizine serpentine

hörhammericine

vinblastine vincristine

Fig. 1 Simplified pathways of biosynthesis for the TIAs from Catharanthus roseus. The molecules in bold are thoseseparated and identified by HPLC. Pathways adapted from Meijer et al. (1993) and Tikhomiroff and Jolicoeur (2002)

Phytochem Rev (2007) 6:207–234 209

123

rely on its capacity to result in pure peaks for the

known metabolites as well as for the unknowns.

This constrain of a clear peak separation is not

obvious and is highly demanding on the analytical

equipments and protocols required to detect,

separate, identify and quantify intermediates

and end metabolites. On the one hand, the

development of a large spectral analytical method

allowing for the identification of most of C. roseus

secondary metabolites is required for molecular

screening purposes. On the other hand, with the

recent perspectives of engineering plant cells

metabolism to enhance productivities in second-

ary metabolites from field and in vitro cultures

(Verpoorte et al. 1999, Verpoorte and Memelink

2002; Rao and Ravishankar 2002; Hughes et al.

2004; Capell and Christou 2004), it is crucial to be

able to identify and quantify the fluxes controlling

the secondary metabolic pathways. Quantification

of the alkaloids and of the indole and iridoid

precursors is then required. The indoles trypto-

phan and tryptamine can be analyzed by HPLC

using fluorescence or UV-detection (Dagnino

et al. 1996; Tikhomiroff and Jolicoeur 2002). It

has been established that the biosynthesis of

secologanin is a limiting step for alkaloids accu-

mulation in cell culture of C. roseus (Moreno

et al. 1993). Collu et al. (1999) have then devel-

oped a HPLC method for analyzing the precur-

sors geraniol and 10-hydroxygeraniol in an in

vitro enzymatic assay with detection at their

maximum UV-absorbance (210 nm). In addition,

there are now synthetic binary compounds

derived from natural alkaloids ant that need to

be separated and quantified. For instance, vinde-

sine can be derived from vinblastine and vincris-

tine (Barthe et al. 2002). Coupling vindoline to

catharanthine by chemical reaction has led to

synthetic antitumorals anhydrovinblastine, vino-

relbine and vinfluvine (Potier et al. 1975; Kruc-

zinsky and Hill 2001; Fahy et al. 2002). Barthe

et al. (2002) have developed a HPLC method to

separate these synthetic compounds.

Separation of alkaloids by HPLC analysis is

not only essential for plant and cell line screening,

but also for the design and the validation of

product recovery and purification processes at the

industrial scale. Therefore, efficiency of the har-

vesting procedure as well as the accuracy of

separation methods will rely on the detectors’

sensitivity. This article thus reviews what has been

shown to work with C. roseus, from the early steps

of plant cell and tissue harvesting, to the identi-

fication of the secondary metabolites and their

quantification in extracts.

Finally, the emergence of new techniques in

combinatorial chemistry for massive synthetic

synthesis and rapid identification of potential

drugs has led to the development of impressive

high-throughput screening (HTS) techniques

which can be obviously applied to natural com-

pounds (Strege 1999). HTS platforms require

rapid separation, detection, identification and

collection of alkaloids from thousands of samples

within a day (van Elswijk and Hirth 2003). From

these platforms, UV, MS-MS and NMR spectra

libraries have been established ensuring efficient

recognition of already identified compounds from

crude extracts (Abel et al. 2002). Nevertheless,

the generation of massive sets of chromatograms

from HTS platforms requires efficient automatic

treatment of the data in order to prevent any loss

of crucial information (Hendriks et al. 2005).

Moreover, in such HTS platforms, HPLC sepa-

ration technology is a crucial step enabling

biochemical detection in a continuous flow assay

to achieve isolation of a lead compound which

interacts with a therapeutic target (Ingkaninan

et al. 2000; van Elswijk and Hirth 2003). Using

such systems, functional and identification assays

can be run simultaneously. Despite the emer-

gence of new techniques for the analysis of

alkaloids such as capillary electrophoresis (CE)

(Chu et al. 1996; Barthe et al. 2002) this review

focuses mainly on HPLC methods because it can

be scaled-up and used to obtain milligrams of

pure compounds.

Detection of Catharanthus roseus alkaloids and

precursors

HPLC can be efficiently coupled to different

detectors such as Photo Diode Array (PDA),

fluorescence, ESI-MS, MS-MS, NMR and CD

(Bringmann et al. 2002) for precise identification

and/or quantification of alkaloids. Naaranlathi

et al. (1989) have also proposed electrochemical

210 Phytochem Rev (2007) 6:207–234

123

detection for analyzing cell culture samples.

Indeed, there are physical characteristics

(Table 1) guiding for efficient HPLC detection

of C. roseus secondary metabolites. Since alka-

loids present a structure composed of aromatic

rings, UV absorbance is a method of choice for

their detection. The presence of p-delocalized

electrons in the alkaloids structure enables auto-

fluorescence phenomena allowing for their effi-

cient detection with a reduced baseline back-

ground, especially for the highly fluorescent ones

(e.g. serpentine and ajmalicine). Alkaloids are

also easily ionized by electrospray in positive

mode, so their detection in ESI (+) MS is recom-

mended. However, among the detectors used in

HPLC analysis, UV and fluorescence are partic-

ularly efficient for the quantification of alkaloids,

and these detectors are easily accessible to the

large scientific community.

Detection upon UV absorbance

As stated above, indole alkaloids can be UV-

detected and identified from their specific UV

absorbance spectra after their separation as single

compound. Typical UV spectra are showed in

Fig. 2 (Tikhomiroff and Jolicoeur 2002). If a

single wavelength is available, 218 nm then seems

optimal at maximizing the absorbance level for

most of the alkaloids. However, 254 nm for

serpentine and 340 nm for tabersonine-like com-

pounds will allow higher precision and limit of

detection, with signal-to-noise ratios of respec-

tively 4 and 60 times higher than at 218 nm.

Therefore, since the wavelengths generating max-

imal absorbance do not exactly correspond for all

alkaloids, a PDA detector having multiple UV

channels seems preferable for quantitative studies

of alkaloids mixtures. Indeed, setting the detector

channel to a specific wavelength for a compound

allows enhancing the baseline by suppressing the

noise from low-abundant alkaloids or from other

UV absorbing interfering molecules and mobile

phase. Moreover, as suggested by Shanks et al.

(1998) peak identification may be more reliable

using UV–VIS spectrum obtained by PDA than

just relying upon retention time. Limits of detec-

tion (LOD) for vindoline and catharanthine were

10 and 0.52 lg ml–1, respectively for the method

developed by Naraanlathi et al. (1987) and the

method established by Tikhomiroff and Jolicoeur

(2002). Recent developments in PDA detector

and separation column technologies have con-

tributed to significantly decrease the LOD. How-

ever, the limit of detection under UV can be

significantly affected by the presence of other

compounds at proximity of the peaks of interest

(Chu et al. 1997).

Table 1 Specific properties of C. roseus’ TIAs and precursors for HPLC separation and detection

Alkaloid Fluorescence (max Ex/Em in nm) Absorption UV (nm) Hydrophilicity (compared to serpentine)

Tryptophan 270/370 218, 278 +Tryptamine 270/370 218, 278 +Ajmalicine 270/390 246, 290 +Serpentine 350/450 252, 308, 370 0Vinblastine 297/364 214, 266 –Vincristine 222, 256, 298 –Vindoline 307/357 214, 254, 306 –Catharanthine 290/363a 226, 282 –Tabersonine 222, 302, 326 –Lochnericine 332 –Secologanin 238 +Strictosidine 280/354b 280

a Renaudin (1985) reported that catharanthine could be detected by fluorescence, but it is 9 times less fluorescent thanajmalicine and about 380 times less fluorescent than serpentine (at their maximum of fluorescence). Moreover, in the samearticle it is mentioned that the sensitivity in a HPLC analysis for catharanthine is better in absorbance than in fluorescencedetectionb Pennings et al. (1989) found than the strictosidine fluorescence signal intensity is three times smaller than UV absorbancesignal at 280 nm

Phytochem Rev (2007) 6:207–234 211

123

Detection upon fluorescence

The corynanthe-type alkaloids such as ajmalicine

and serpentine are fluorescent and can be

detected using a fluorescence detector (Fig. 3).

Even if these compounds can either be easily

detected under UV, fluorescence is more sensitive

with a lower limit of detection at the micromolar

level. The precursors tryptophan, tryptamine and

the alkaloid ajmalicine can efficiently be detected

nm200 220 240 260 280 300 320 340 360

0

25

50

75

nm200 220 240 260 280 300 320 340 360

200 220 240 260 280 300 320 340 360

mA

Um

AU

mA

Um

AU

mA

U

mA

U

mA

Um

AU

mA

Um

AU

mA

U

0

200

400

Tryptophan Ajmalicine

nm

nm

nm

nm

nm

0

10

20

30

nm200 220 240 260 280 300 320 340 360

0

200

400

Secologanin Tryptamine

0

200

400

600.

220 240 260 280 300 320 34 360 380 400

0

250

500

750

Vincristine Serpentine

nm

0

500

1000

1500

220 240 260 280 300 320 340 360 380 400220 240 260 280 300 320 340 360 380 400

220 240 260 280 300 320 340 360 380 400

0

500

1000.

Catharanthine Vindoline

nm

0

200

400

600

220 240 260 280 300 320 340 360 380 400220 240 260 280 300 320 340 360 380 400

220 240 260 280 300 320 340 360 380 400

0

250

500

750

Tabersonine Vinblastine

nm

0

10

20

30

Lochnericine

Fig. 2 UV spectra of C. roseus TIAs and precursors. Spectra were recorded using a Beckman-Coulter system Gold 168PDA detector

212 Phytochem Rev (2007) 6:207–234

123

at Ex 270/Em 370 nm, despite the fact that the

maximum excitation wavelength for ajmalicine is

Ex 290 nm, whereas it is Ex 270 nm for both

precursors. Limits of detection for tryptophan

and tryptamine were lower to 0.01 lg ml–1 (Tik-

homiroff and Jolicoeur 2002). Renaudin (1985)

reported that the limit of detection can be

decreased by a factor of six for ajmalicine and

vindoline and even by a factor of 50 for serpen-

tine by using fluorescence detection instead of

traditional UV detection. Combining the use of

fluorescence and absorbance detectors, molecules

that co-elute but which show different physical

characteristics can still be quantified by selecting

appropriate wavelengths. For instance, quantifi-

cation of serpentine performed at 306 nm was

possible while co-eluting with an unidentified

compound (Tikhomiroff and Jolicoeur 2002).

Renaudin (1985) showed that co-elution of ser-

pentine with vinblastine has no effect on serpen-

tine response factor since no quenching occurs;

they can then be differentiated from their respec-

tive fluorescence spectra. Nevertheless, this ap-

proach requires to be validated for each peak

with the identification of the co-eluting com-

pounds and by establishing the detection condi-

tions minimizing interference phenomena.

Adequate sample preparation enhances

analytical steps

An extraction method has to cope with the

starting plant material and the differential in the

physico-chemical properties of the molecules to

be extracted and of those to be removed; their

respective partition coefficient in the extraction

solvent being of the most significant parameters.

Special cares that have to be considered at each

step for the commnonly used methods are pre-

sented below.

Biomass considerations

Secondary metabolites are generally present in a

wide range of concentrations that may even vary

from one lot or batch to another. Therefore, large

amount of biomass may be required to have access

to a large spectrum of compounds each at an

adequate concentration. Usual amounts of bio-

mass sampled from in vitro cultures and whole

plant culture are presented in Tables 2 and 3,

respectively. Freeze-drying of the biomass enables

to crush the cells, to perform extraction steps on

concentrated samples which decrease the volume

to be processed without denaturing alkaloids.

From in vitro plant cell and tissue cultures,

50 mg DW have been reported to be the lower

biomass limit for the efficient quantification of

most of the known alkaloids. This sample size

represents 0.5–1 g FW and thus up to 3 ml of a

plant cell suspension in liquid culture. This is seen

as a minimum and may not always allow for the

detection of all alkaloids, even for the known

ones. Sample concentration can be increased in

different ways such as evaporation of the extrac-

tion phase and re-suspension into a smaller

volume of solvent, or by using solid phase extrac-

tion as for aqueous solution extraction (see

below), and then modulating the elution volume

(Renaudin 1985; Lee-Parsons and Shuler 2002). In

most cases, less than few micrograms of pure

330

370

410

450

490

530

570

330

370

410

450

490

530

570

290

330

Emission Wavelength (nm)

0

500.0

1000

1500

2000

2500

3000

3500

4000

290

330

370

410

450

490

5300

100.0

200.0

300.0

400.0

500.0

600.0

700.0

800.0

290

330

370

410

450

490

5300

325.0

650.0

975.0

1300

1625

1950

2275

2600

290

330

370

410

450

490

530

Exc

itatio

n W

avel

engt

h (n

m)

E

xcita

tion

Wav

elen

gth

(nm

)

0

87.50

175.0

262.5

350.0

437.5

525.0

612.5

700.0

370

410

450

490

530

a b

dc

Fig. 3 2D-Fluorescence spectra of some C. roseus’TIAsand precursors acquired in an aqueous solution pH 5.7(from Hisiger and Jolicoeur 2005). The fluorescencespectra of tryptophan (a), tryptamine (b), serpentine (c)and ajmalicine (d) were recorded with a multichannelfluorescence sensor (Delta Light and Optics, Lyngby,Denmark) using 12.5 mg l–1 standard solutions

Phytochem Rev (2007) 6:207–234 213

123

alkaloids can be obtained from a 50 mg DW

sample. However, milligrams of pure alkaloids are

required for obtaining standards that are required

for the identification of unknowns or for assessing

biological activity of the molecules, and thus kg of

cells and tissues will then be needed.

Selection of an extraction phase

Two major extracting phases are suggested in

literature for the extraction of alkaloids, such as

methanol and ethyl acetate, but ethanol (Balague

and Wilson 1982; Naaranlahti et al. 1987; Moreno

et al. 1993, Singh et al. 2000; Uniyal et al. 2001),

phosphoric acid (Blom et al. 1991), acetic acid

(Renaudin 1985) and acetone for secologanin

(Contin et al. 1998) were also used. Different

ratios of solvent volume-to-biomass have been

tested (Tables 2, 3 and 4). In general, typical ratio

of 0.2–0.6 ml solvent mg–1 DW is used. However,

some authors prefer to perform their extraction at

lower ratio of 0.02–0.1 ml mg–1 DW (from leaves:

Uniyal et al. 2001; Singh et al. 2000; from cell

culture: Dagnino et al. 1996; from hairy roots:

Tikhomiroff and Jolicoeur 2002). Increasing the

volume of the extraction phase over the biomass

amount increases obviously the extraction capac-

ity. However, using large volume will dilute the

compounds and it will be necessary to add

concentration steps prior to analysis. Performing

a single step extraction on 20 mg DW of hairy

roots with 1 ml methanol, Tikhomiroff and Jolic-

oeur (2002) were able to extract virtually 100% of

the secologanine and ajmalicine contents, but the

extraction yields were significantly lower for the

other alkaloids. However, several extraction steps

with fresh solvent are usually required for a better

recovery. Besides classical extraction procedures,

Choi et al. (2002) tested the use of supercritical

fluid (mix of CO2, methanol and triethylamine)

for the recovery of vinblastine and vincristine

from C. roseus leaves as an alternative to pre-

purification steps. However, the extraction yield

was at best 76.6% of that using methanol only.

Table 2 Protocols for alkaloids extraction from C. roseusroots or hairy root

Authors Biomass Exraction phase Procedure

Tikhomiroff andJolicoeur (2002)

200 mg FWhairy roots

Methanol – Lyophilization– Crushing of dry material in a tissue grinder– Extraction with 1 ml for 1 h in a sonicating bath

Morgan et al. (2000) 100 mg DW Methanol – Lyophilization– Crushing of dry material with a mortar– Extraction with 45 ml for 5 h in a sonicating bath– Evaporation of the mobile phase

Sim et al. (1994) 50 mg DW Methanol thenethyl acetate

– Extraction with 3 · 10 ml of MeOH for 30 minin a sonicating bath at 50�C

– Evaporation of the methanol– Resupension in 20 ml 0.1 N HCl– Extraction with 20 ml Ethyl acetate– Adjusting to pH 10– Extraction three times with ethyl acetate– Evaporation– Resupension in methanol

Bhadra et al. (1993) 200 mg DW Methanol – Lyophilization– Extraction with 80 ml for 3 h in a Soxhlet apparatus– Evaporation and dilution in a 5 mM (NH4)2HPO4 solution– Fractionation on a 300 mg-C18 cartridge with three successive

elutions with a mixture of MeOH: (NH4)2HPO4,60:40, 95:5 and 100:0 (v:v)

Hughes et al. (2004) 50 mg DW Methanol – Lyophilization– Extraction with 10 ml of methanol for 1 h in a sonicating bath– Centrifugation for 15 min at 1300g, 15�C– Repetition of the extraction step and combination of the both

extracts– Concentration under vacuum to 2 ml

214 Phytochem Rev (2007) 6:207–234

123

Selection of the extraction phase is crucial and

depends on the solubility of the alkaloids. Acidic

extractive solvents (pH = 1.5) are commonly used

and it seems to increase the stability and the

solubility of the alkaloids (Hallard et al. unpub-

lished results; see caption of Fig. 4 for the

complete reference). Solvent phase selection is

also dependent on the nature of the analysis to be

performed. For instance, using methanol as the

extraction phase and then running a HPLC

method with phosphate buffer should be avoided

because methanol can precipitate phosphate salts

in different HPLC parts (e.g. tubings, column and

pre-column, detectors, etc.). In addition, when

further molecular characterization has to be

performed using MS detector, non-volatile sol-

vents (especially phosphoric acid) should be

proscribed in order to prevent any damage to

the MS detector. For MS analysis, TFA should be

used with precaution because high TFA concen-

tration (0.1% w/v) causes ion-suppression in MS

and peak tailing can then be observed (Gustavs-

son et al. 2001; Annesley 2003). Finally, one may

require performing several successive extractions

using the same solvent or even different ones for

pre-cleaning purposes removing contaminating

compounds. For instance, TCA and acetone are

well known to cause protein precipitation,

whereas alkaloids remain in solution, and TFA

seems to cause precipitation of chlorophyll (Hal-

lard et al. unpublished results; see caption of

Fig. 4 for the complete reference).

Sample preparation

Cell membrane and debris as well as precipitated

macromolecules have to be removed from the

solvent after sample extraction. Liquid sample

clarification is usually achieved by centrifugation

at high speed (5 min, 15,000g) and/or by filtration

(see Table 3 for details). The liquid sample has to

be filtered through a submicron filter. A nylon or

PTFE membrane is adequate for solvents sug-

gested in literature (e.g. methanol or acetonitrile).

This step is essential to extend the HPLC column

life-time as well as the global performance of the

HPLC, including that of the injector. Even when

using SPE columns (Solid Phase Extraction),

filtration/centrifugation is a pre-requisite step

before cartridge loading.

Table 3 Protocols for alkaloids extraction from C. roseus leaves

Authors Biomass Extractionphase

Procedure

Uniyal et al.(2001)

5 g of oven-driedtissue

Ethanol – Oven-drying of the leaves for 48 h at 60�C– Extraction (3 · 30 ml) overnight at room temperature– Filtration and concentration in vacuo at 4�C– Dissolution in ethanol and dilution in 10 ml water

and 10 ml 3 mM HCl– Washing with hexane (3 · 30 ml)– pH 8.5 adjusting of the aqueous phase and cool down at 10�C– Extraction with chloroform (3 · 30 ml)– Washing with water– Evaporation of the chloroform phase– Resuspension in 1 ml of chloroform– Fractionation on SPE cartridge and elution with a

chloroform:methanol 90:10 solution– Evaporation then resuspension in Methanol

Singh et al.(2000)

5 g DW Ethanol – Extraction with 3 · 30 ml ethanol (12 h each at room temp.)– Filtration and concentration to 10 ml– Add 10 ml of water and 10 ml of 3% HCl– Washing three times with hexane– Alkalinization of the aqueous phase to pH 8.5– Extraction with chloroform (3 · 30 ml)– Washing with water– Concentration in vaccu-oven– Dissolution in 10 ml methanol

Phytochem Rev (2007) 6:207–234 215

123

Extraction protocols

Extraction of alkaloids from plant tissues or cells,

from culture medium and from adsorbent resins is

well documented. Commonly used techniques for

TIAs and precursors are summarized in the

following.

From biomass

Alkaloids extraction can be performed starting

with hard tissues such as plant leaves, stems,

roots (including hairy roots) and cell suspension.

After harvesting, plant material is frozen in

liquid nitrogen, ground at cold, freeze-dried and

then extracted using selected organic solvent.

Solvent volume and the number of extraction

steps need to be optimized in order to maximize

extraction yield. Since studies generally focus on

a particular type of biomass (e.g. cell suspension

or hairy roots or plants), extraction protocols are

generally biomass type specific. However, differ-

ent tissues can be extracted using a unique

protocol, as shown by Favali et al. (2004) ana-

lyzing leaves, stems and roots from plants

infected with phytoplasmas. In these works,

tissues were extracted in a Soxtec system using

methanol at 190�C for 2 h. However, the result-

ing extraction efficiency may be then lower

overall.

Table 4 Protocols for alkaloids extraction from cell suspension of C. roseus

Authors Biomass Extraction phase Procedure

Dagnino et al. (1996)(apolar alkaloids)

50 mgDW

Dichloromethane – Freeze-drying of the biomass– Wetting with 0.5 ml of water– Extraction with 5 ml of dichloromethane (repeat one more

time)– Drying and resuspension in acetonitrile

Dagnino et al. (1996)(alkaloids precursors)

50 mgDW

Methanol – Extraction with 5 ml of methanol of the dichloromethaneextracted biomass (repeat one more time)

– Drying and resupension in 0.5 ml 1 M phosphoric acidRenaudin (1985) 1–3 g

FWAcetic acid – Freezing and thawing of the fresh cells

– Extraction four times with 15 ml 0.01% (v/v) acetic acid(pH 4.0)

– Centrifugation 4200g for 5 min– Adjusting the pH between 7.3 and 7.5 with 1 M NaOH– Loading a SPE C18 cartridge– Rinsing with 60 ml methanol–25 mM ammonium dihydrogen

phosphate (pH 7.3) (10/90, v/v)– Elution with 2 ml methanol–25 mM ammonium dihydrogen

phosphate (pH 4.7) (85/15, v/v)Lee-Parsons et al. (2004) 250 mg

DWMethanol – Lyophilization

– Extraction with 25 ml methanol (one more time) for 3 h– Evaporation to dryness– Dissolution in an aqueous solution 10% acetonitrile + 0.1%

TFA– Loading on SPE C18– Elution with 100% acetonitrile plus 0.1% TFA– Evaporation and dissolution in aqueous 20% acetonitrile

plus 0.1% TFAMonforte-Gonzales et al.

(1992)1 g DW Methanol then

ethyl acetate– Lyophilization– Extraction with 50 ml methanol for 2 min in a Polytron

homogenizer– Incubation for 2 h at 55�C– Filtration– Evaporation and dissolution in 15 ml H2SO4

– Washing with 15 ml ethyl acetate (three times)– Adjusting the pH to 9.5– Extracting with 20 ml ethyl acetate (three times)– Evaporation and dissolution in 1 ml methanol

216 Phytochem Rev (2007) 6:207–234

123

From a liquid medium

Some authors analyze directly the alkaloids con-

tent of a culture medium without any preliminary

steps (Dagnino et al. 1995; Whitmer et al. 2002;

El-Sayed et al. 2004). However, the level in

alkaloids that are secreted in the medium is

generally too low to allow for a good detection

upon direct HPLC injection. It then becomes

essential to concentrate TIAs in the liquid sample.

Several methods can be used such as evaporation

at low temperature (e.g. lyophilization) and

liquid–liquid extraction (Asada and Shuler 1989;

Zhao et al. 2001; Satdive et al. 2003). The quicker

and most efficient method is a solid phase extrac-

tion using commercial cartridges with C18 packing

(Renaudin 1985; Hisiger and Jolicoeur 2005). In

addition to sample concentration, the use of SPE

cartridge allows to ‘‘clean’’ a sample. Based of the

strength of the solvent used for the elution step,

SPE cartridge can help to fractionate the sample.

It then becomes possible to generate different

fractions, based on the affinity of the compound

for the stationary phase, as for solid-chromatog-

raphy techniques (Bhadra et al. 1993; Renaudin

1984). If the aim is to recover all the alkaloids and

precursors within a unique liquid phase, elution

with a strong solvent such as 100% methanol or

100% acetonitrile is then recommended. Some

previous SPE methods used diatomite or cation-

exchange as solid phase (see the nice review from

van der Heijden et al. 1989). Now-a-days, octade-

cylsilyl is preferred for alkaloids preparation (Lee

and Shuler 2000; Renaudin 1985; Uniyal et al.

2001; Bhadra et al. 1993; McCalley 2002).

From adsorbent phases

Many research groups refer to the use of a solid

extraction phase in situ in the culture medium of

C. roseus suspension cells (Payne et al. 1988;

Asada and Shuler 1989; Lee-Parsons and Shuler

2002; Wong et al. 2004) and hairy roots (Sim et al.

1994). The most documented use of a solid

extraction phase in plant cell culture is the

Amberlite XAD-7 resins (but also XAD-4 and

others are used). This resin is made of a neutral

polymeric adsorbent that has shown a fast

adsorption rate and a weak selectivity among

alkaloids. XAD-7 resins were successfully em-

ployed to enhance the catharanthine and ajmal-

icine production levels in cell culture. Liquid

extraction phases have also been used in situ in

two-liquid-phase bioreactors, such as silicon oil

(Byun and Pedersen 1994; Tikhomiroff et al.

2002) and trycaprylin (Collins-Pavao and Chin

1996). However, liquid extraction phases usually

show high selectivity for some of the alkaloids.

Finally, after separation from the medium and

the cells, alkaloids are extracted from adsorbents

resins using methanol (Asada and Shuler 1989;

Wong et al. 2004) or methanol:HCl (95:5, v:v).

Similar protocols can be applied extracting alka-

loids from liquid extraction phases (Tikhomiroff

et al. 2002).

Estimation of the extraction yield

There is no universal method which can be

applied for the extraction of secondary metabo-

lites from C. roseus because TIAs have different

hydrophobicity levels. The selection of the extrac-

tion phase is then determinant to reach high

extraction yields and every step should be care-

fully optimized. A typical way in approximating

the extraction efficiency consists in spiking the

Fig. 4 UV chromatogram registered at 254 nm of astandard mixture of C. roseus alkaloids using the methoddeveloped by Hallard D, Vera Rocha RA, Sajjadi SE, vander Heijden R and Verpoorte R (unpublished results).Reproduced with the kind permission of Dr Verpoorte. 1,tryptamine; 2, perivine; 3, vindolinine; 4, yohimbine; 5,strictosidine; 6, ajmalicine; 7, serpentine; 8, catharanthine;9, vincristine; 10, vindoline; 11, vinblastine, 12, anhydro-vinblastine. The chromatographic conditions are describedin details in Table 7

Phytochem Rev (2007) 6:207–234 217

123

sample with a known amount of standard material

as soon as at the first step of the extraction

process; while crushing the tissues. However, this

method only gives an approximation because

there is a bias caused by the interaction of the

spiked alkaloids with the intracellular matrix, a

phenomena which can hardly be characterized.

Indeed, it has been suggested than some alkaloids

can form complexes with phenolics (Renaudin

et al. 1982; Renaudin 1989). Tikhomiroff and

Jolicoeur (2002) have reported low extraction

yields (<60%) when using a single methanol

extraction step for recovering intracellular ser-

pentine, vincristine, vindoline, cathranthine,

tabersonine and tryptophan from plant cells

whereas the yield was more than 85% for

vinblastine, tryptamine and ajmalicine. Interest-

ingly, Naaranlathi et al. (1987) found a 100%

extraction yield when spiking their leaves samples

with vindoline and catharanthine after extraction

with ethanol. Multiple successive extractions of

plant cells using acetone revealed an efficiency of

recovery for pure secologanin of only 7.5%

(Contin et al. 1998). Matrix effect can also hap-

pen during HPLC quantification. The use of an

internal standard, added to the sample, is thus

suggested to asses for matrix effect, extraction

efficiency and injection accuracy. It is then nec-

essary to choose a molecule which is absolutely

not present in the liquid sample, exhibits the same

behavior as the target compound(s) without co-

eluting which would interfere with the HPLC

analysis. 5-Methoxytryptamine has been used as

internal standard by Naaranlathi et al. (1987)

although the use of stable isotopic analogues

(deuterium labeled) can be even more efficient

when an MS detector is available (Auriola et al.

1991).

Pseudo-quantification methods

In the worst case when no standards are available,

one can estimate the total alkaloid content of a

sample selecting a wavelength at which most of

the alkaloids show a maximal response in absor-

bance or fluorescence. According to Monforte-

Gonzalez et al. (1992), total alkaloids can be

monitored by measuring the absorbance at

280 nm using a spectrophotometer. However,

the major problem here relies on the fact that

alkaloids and their precursors respond in absor-

bance or fluorescence at different levels. For

instance, tryptamine is 3 times more fluorescent

than tryptophan and 6 times more than ajmalicine

at 270/370 (Fig. 3). The alkaloids concentration

distribution in plant, plant cells and tissues is also

not similar neither constant with time, so it

becomes highly hazardous to establish a calibra-

tion between the signal in absorbance or fluores-

cence and the total alkaloid content of a sample.

This method is thus highly pseudo-quantitative

and is thought to be mostly useful for a quick pre-

screening of plant or cell lines. In addition, the

samples have to be thoroughly cleaned (see

Sample preparation section) to avoid any inter-

ference from the other metabolites that absorb in

UV or do fluoresce.

In the case of C. roseus, only few of the

required standards can be found commercially

(Table 5). Therefore, it is difficult to obtain most

of the standards. It is then feasible again to

proceed by approximation performing external

calibration using an available and quantifiable

alkaloid as the reference. Indeed, horhammeri-

cine and lochnericine can be quantified, based on

the response factor of tabersonine as they possess

Table 5 Some of commercially available C. roseus alka-loids

Compound Supplier

Vinblastine SIGMA (USA)Spectrum chemicals (USA)Vinkem Labs (India)

Vincristine SIGMA (USA)Eli Lilly (USA)Synnad Chemical Co (China)Southern Herbals Ltd (India)

Catharanthine Dayang Chemicals (China)Chemos GmBH (Germany)Kang’ai biological Products (China)ZYF Pharm Chemical (China)

Serpentine Apin chemicals (UK)Ajmalicine Extrasynthese (France)Yohimbine Extrasynthese (France)Vindoline Kang’ai biological Products (China)Tryptamine SIGMA (USA)

Note: It is not possible to screen for all the suppliersaround the world for C. roseus molecules, but this tablemay be useful to find quickly a supplier for a desiredstandard

218 Phytochem Rev (2007) 6:207–234

123

similar UV spectra and molecular extinction

coefficients (Rijhwani and Shanks 1998; Morgan

et al. 2000). In a similar way, coronaridine was

quantified upon the response factor of catharan-

thine (Morgan and Shanks 2002). Compounds

which have not the same response factor could

nevertheless be quantified, proceeding as follow.

It is then important to consider the molecular

extinction coefficient ratio for both molecules

independently quantified, in the mobile phase at

their respective retention time, and for the same

molar amount loaded on the HPLC column.

Thus, this suggests an initial calibration step using

external standards to assess for analytical bias

such as the matrix effect which can play on co-

eluting alkaloids.

Designing, selecting and transposing HPLCmethods

Numerous HPLC methods have been developed

for the analysis of alkaloids and precursors

content in C. roseus extracts (Tables 6 and 7).

In spite of several studies using C8 columns

(Gorog et al. 1977; Pennings et al. 1989) and

Phenyl columns (van der Heijden et al. 1987),

there is now a consensus for reverse phase

chromatography using C18 columns.

HPLC methods for the analysis of TIAs

TIAs are generally more hydrophobic than their

indole and iridoid precursors. It is then preferable

to develop a set of two HPLC separation meth-

ods, one for hydrophilic (precursors) and one for

hydrophobic (TIAs) molecules. A more polar

elution is then performed when analyzing precur-

sors as compared to alkaloids. The different

methods that have been developed are presented

in Table 7. It can be seen that some methods are

highly similar and may only differ because of

slight differences in equipment performance or

because of specific lab research goals. However,

one can obviously start developing a separation

method from published works. Bhadra and

Shanks (1997) modified a method previously

reported by Bhadra et al. (1993) changing the

C18 lBondapak 100 · 8 mm column (Waters) forTa

ble

6H

PL

Cm

eth

od

sfo

rth

ea

na

lysi

so

fth

eC

.ro

seu

sp

recu

rso

rs

Au

tho

rsC

om

po

un

ds

focu

sed

Re

ten

tio

nti

me

(min

)C

olu

mn

Mo

bil

eP

ha

seD

ete

ctio

nE

luti

on

gra

die

nt

an

dfl

ow

-ra

te

Tik

ho

mir

off

an

dJo

lico

eu

r(2

00

2)

Try

pto

ph

an

,3

.2Z

orb

ax

XD

BC

185

lm

,2

50

·4

.6m

m(A

gil

en

t)A

ceto

nit

rile

:10

0m

MH

3P

O4

(pH

2)

(15

:85

,v

:v)

Flu

ore

sce

nce

Ex

27

0/E

m3

70

Iso

cra

tic,

1.5

ml

min

–1

try

pta

min

e,

3.5

seco

log

an

in,

7.2

ajm

ali

cin

ea

12

.2D

ag

nin

oe

ta

l.(1

99

5)

Lo

ga

nic

aci

d,

5.5

LiC

hro

sph

er

60

RP

Se

lect

B5

lm2

50

·4

mm

(Me

rck

)1

%fo

rmic

aci

d–

ace

ton

itri

le–

tric

hlo

roa

ceti

ca

cid

(10

0:1

0:0

.25

v:v

:w)

PD

AIs

ocr

ati

c,1

.2m

lm

in–1

log

an

in,

13

.5se

colo

ga

nin

,2

2.5

try

pto

ph

an

,2

5.5

try

pta

min

e3

0.5

Pe

nn

ing

se

ta

l.(1

98

9)

Str

icto

sid

ine

,5

.6L

iCh

roso

rbR

P-8

Se

lect

B,

7lm

25

0·

4m

m(M

erc

k)

7m

MS

DS

an

d2

5m

MN

aH

2P

O4

inm

eth

an

ol:

wa

ter

(pH

6.2

)(6

8:3

2,

v:v

)

PD

AIs

ocr

ati

c,1

ml

min

–1

try

pta

min

e(f

rom

en

zym

ati

ca

ssa

y)

7

aT

his

alk

alo

idw

as

qu

an

tifi

ed

usi

ng

the

me

tho

ds

for

TIA

sp

recu

rso

rs

Phytochem Rev (2007) 6:207–234 219

123

Ta

ble

7H

PL

Cm

eth

od

sfo

rth

ea

na

lysi

so

fa

lka

loid

sfr

om

C.

rose

us

Au

tho

rsC

om

po

un

ds

focu

sse

dR

ete

nti

on

tim

e(m

in)

Co

lum

nM

ob

ile

Ph

ase

De

tect

ion

Elu

tio

ng

rad

ien

t

Mo

ren

o-

Va

len

zue

lae

ta

l.(1

99

8)

Ajm

ali

cin

e,

21

.8U

ltra

sph

ere

OD

S,

4lm

25

0·

4.6

mm

(Be

ckm

an

)

Ace

ton

itri

le:1

0m

M(N

H4) 2

HP

O4

(43

:57

,v

:v)

UV 2

80

nm

Iso

cra

tic,

1.5

ml

min

–1

yo

him

bin

e5

.9

Re

na

ud

in(1

98

4)

Ajm

ali

cin

e,

14

lBo

nd

ap

ack

C18

10

lm

30

0·

3.9

mm

(Wa

ters

)

Me

tha

no

l:5

mM

(NH

4) 2

HP

O4

(pH

7.3

)(6

7:3

3,

v:v

)

UV 2

54

nm

Iso

cra

tic,

1m

lm

in–1

vin

do

lin

e,

9.5

cath

ara

nth

ine

,1

6te

tra

hy

dro

als

ton

ine

22

Re

na

ud

in(1

98

4)

Ajm

ali

cin

e,

12

lBo

nd

ap

ack

C18

10

lm

30

0·

3.9

mm

(Wa

ters

)

Me

tha

no

l:5

mM

(NH

4) 2

HP

O4

(pH

7.3

)(7

1:2

9,

v:v

)

UV 2

54

nm

Iso

cra

tic,

1m

lm

in–1

cath

ara

nth

ine

,1

3v

inb

last

ine

,1

6se

rpe

nti

ne

19

Bh

ad

rae

ta

l.(1

99

3)

Ajm

ali

cin

e,

12

lBo

nd

ap

ack

C18

10

lm

10

0·

8m

m(W

ate

rs)

Me

tha

no

l:5

mM

(NH

4) 2

HP

O4

(pH

no

tm

en

tio

ne

d)

(67

:33

,v

:v)

UV 2

54

nm

Iso

cra

tic,

0.8

ml

min

–1

serp

en

tin

e,

59

.2ca

tha

ran

thin

e,

14

.5v

ind

oli

ne

,8

.5v

inb

last

ine

nd

Rij

hw

an

ia

nd

Sh

an

ks

(19

98

)

Ajm

ali

cin

e,

serp

en

tin

e,

tab

ers

on

ine

,h

orh

am

me

rici

ne

,lo

chn

eri

cin

e

N/A

C18

bo

nd

clo

ne

RP

10

lm

30

0·

3.9

mm

(Ph

en

om

en

ex

)

Me

tha

no

l:a

ceto

nit

rile

:5

mM

(NH

4) 2

HP

O4

(32

:32

:36

,v

:v:v

)

PD

AIs

ocr

ati

c,1

ml

min

–1

Le

ea

nd

Sh

ule

r(2

00

0)

Ajm

ali

cin

e,

19

.67

lBo

nd

ap

ack

C18

10

lm

30

0·

3.9

mm

(Wa

ters

)

Ace

ton

itri

le:

0.1

%(w

:v)

TF

Ain

wa

ter

(22

:78

,v

:v)

UV 2

54

nm

Iso

cra

tic,

1m

lm

in–1

cath

ara

nth

ine

,2

1.6

0se

rpe

nti

ne

23

.14

El-

Sa

ye

de

ta

l.(2

00

4)

Ste

mm

ad

en

ine

,7

.2V

yd

ac

C1

8R

P2

50

·4

.6m

m(V

yd

ac)

Ace

ton

itri

le:0

.1%

(w:v

)T

FA

inw

ate

r(2

1:7

9,

v:v

)

PD

AIs

ocr

ati

c,1

ml

min

–1

cath

ara

nth

ine

,2

1.7

tab

ers

on

ine

23

.5

Sim

et

al.

(19

94

)C

ath

ara

nth

ine

,a

jma

lici

ne

N/A

lBo

nd

ap

ack

C18

10

lm

30

0·

3.9

mm

(Wa

ters

)

Me

tha

no

l:a

ceto

nit

rile

:5

mM

(NH

4) 2

HP

O4(p

H7

.3)

(30

:40

:30

,v

:v:v

)

UV 2

98

nm

Iso

cra

tic,

1m

lm

in–1

220 Phytochem Rev (2007) 6:207–234

123

Ta

ble

7co

nti

nu

ed

Au

tho

rsC

om

po

un

ds

focu

sse

dR

ete

nti

on

tim

e(m

in)

Co

lum

nM

ob

ile

Ph

ase

De

tect

ion

Elu

tio

ng

rad

ien

t

Un

iya

le

ta

l.(2

00

1)

Vin

cris

tin

e,

14

.62

Sy

mm

etr

yC

18

5lm

15

0·

4m

m(W

ate

rs)

So

lven

tA

(pH

7.0

):M

eth

an

ol:

ace

ton

itri

le:

5m

MC

H3C

OO

NH

4:

trie

thy

lam

ine

(13

:32

:55

:0.2

,b

yv

olu

me

);S

olv

ent

B(p

H7

.0):

Me

tha

no

l:a

ceto

nit

rile

:5m

MC

H3C

OO

NH

4:t

rie

thy

lam

ine

(19

:46

:35

:0.2

,b

yv

olu

me

)

PD

A

020406080100

120

010

2030

Tim

e (m

in)

% B

40

0-15

min

: 0.

4 m

L m

in-1

15-2

0 m

in :

0.5

mL

min

-1

20-2

5 m

in :

0.7

mL

min

-1

25-3

0 m

in :

0.9

mL

min

-1

30-3

5 m

in :

1.2

mL

min

-1

cath

ara

nth

ine

,1

5.9

2v

ind

oli

ne

,1

9.6

vin

bla

stin

e1

8.0

2

Ch

ue

ta

l.(1

99

7)

Vin

cris

tin

e,

14

.8H

yp

ers

ilO

DS

,5

lm2

00

·4

.6m

m(A

gil

en

t)

So

lven

tA

:2

5m

MC

H3C

OO

NH

4in

wa

ter;

So

lven

tB

:2

5m

MC

H3C

OO

NH

4in

me

tha

no

l

ES

I-M

S

020406080

010

203 0

Tim

e (m

in)

% B

1 m

L m

in-1

vin

bla

stin

e2

1.3

Ch

oi

et

al.

(20

02

)V

inb

last

ine

,1

8Z

orb

ax

Bo

nu

sR

P-1

8,

5l

m1

50

·2

.1m

m(A

gil

en

t)

So

lven

tA

:2

0m

MC

H3C

OO

NH

4;

So

lven

tB

:A

ceto

nit

rile

ES

I-M

S

020406080

015

3045

Tim

e (m

in)

% B

0.2

mL

min

-1

vin

cris

tin

e2

1.5

Phytochem Rev (2007) 6:207–234 221

123

Ta

ble

7co

nti

nu

ed

Au

tho

rsC

om

po

un

ds

focu

sse

dR

ete

nti

on

tim

e(m

in)

Co

lum

nM

ob

ile

Ph

ase

De

tect

ion

Elu

tio

ng

rad

ien

t

Na

ara

nla

hti

et

al.

(19

87

)

Vin

do

lin

e,

18

Sp

he

ri-5

RP

-18

,5

lm2

20

·4

.6m

m(B

row

nle

e)

So

lven

tA

:0

.2%

TE

Ain

25

mM

CH

3C

OO

NH

4;

So

lven

tB

:0

.2%

TE

Ain

am

ixo

fm

eth

an

ol:

ace

ton

itri

le(2

9:7

1,

v:v

)

PD

A

020406080

010

2030

Tim

e (m

in)

% B

0-17

min

: 1

mL

min

1

17-3

0 m

in :1

.5 m

L m

in1

cath

ara

nth

ine

,2

0se

rpe

nti

ne

,1

3v

inb

last

ine

,2

4.5

vin

cris

tin

e,

21

.5a

jma

lici

ne

21

Zh

ao

et

al.

(20

01

)S

erp

en

tin

e,

ajm

ali

cin

e,

cath

ara

nth

ine

N/A

Nu

cle

osi

l5

C18

5l

m2

50

·4

.6m

m(A

gil

en

t)

Me

tha

no

l:a

ceto

nit

rile

:2

5m

MC

H3C

OO

NH

4:

TE

A(1

5:4

0:4

5:0

.1,

by

vo

lum

e)

PD

AIs

ocr

ati

c,1

ml

min

–1

St-

Pie

rre

an

dD

eL

uca

(19

95

)

16

-me

tho

xy

-ta

be

rso

nin

e(f

rom

en

zym

ati

ca

ssa

y)

30

.5N

ov

a-P

ak

C18,

4l

m3

00

·3

.9m

m(M

illi

po

re)

Me

tha

no

l:w

ate

r:T

EA

(75

:25

:0.1

,v

:v:v

)P

DA

Iso

cra

tic,

0.6

ml

min

–1

Zh

ou

et

al.

(20

05

)V

ind

oli

ne

,v

ind

oli

din

e,

vin

cris

tin

e,

vin

bla

stin

e

N/A

Dir

ect

flo

win

ject

ion

Me

tha

no

lE

SI-

MS

/M

SIn

ject

ion

flo

w-r

ate

of

the

sam

ple

3ll

min

–1

Zh

ou

et

al.

(20

05

)1

9S

-vin

do

lin

ine

,1

3.9

Zo

rba

xE

clip

seX

DB

C8,

5lm

,1

50

·4

.6m

m(A

gil

en

t)

So

lven

tA

:W

ate

r,0

.1%

TE

A;

So

lven

tB

:M

eth

an

ol

UV 2

80

nm

,E

SI-

MS

/M

S

020406080100

020

4060

Tim

e (m

in)

% B

0.8

mL

min

-1

vin

do

lin

ine

,1

6.6

vin

do

lin

e,

21

.6v

ind

oli

din

e,

25

.1ca

tha

ran

thin

e2

7.1

vin

cris

tin

e,

28

.2v

inb

last

ine

36

.5

222 Phytochem Rev (2007) 6:207–234

123

Ta

ble

7co

nti

nu

ed

Au

tho

rsC

om

po

un

ds

focu

sse

dR

ete

nti

on

tim

e(m

in)

Co

lum

nM

ob

ile

Ph

ase

De

tect

ion

Elu

tio

ng

rad

ien

t

Ae

rts

et

al.

(19

94

)C

ath

ara

nth

ine

,v

ind

oli

ne

,ta

be

rso

nin

e

N/A

Hy

pe

rsil

OD

S,

5l

m2

00

·2

.1m

m(A

gil

en

t)

So

lven

tA

:W

ate

r,0

.1%

TE

A;

So

lven

tB

:A

ceto

nit

rile

,0

.1%

TE

A

PD

A

020406080

010

203 0

Tim

e (m

in)

% B

Ba

rth

ee

ta

l.(2

00

2)

Vin

do

lin

e,

6.0

XT

err

aR

P-1

8,

5l

m2

50

·4

.6m

m(W

ate

rs)

Ace

ton

itri

le:w

ate

r:b

ori

ca

cid

(pH

10

)(5

5:4

5:3

.1,

v:v

:w)

UV 2

14

nm

Iso

cra

tic,

1m

lm

in–1

vio

relb

ine

,1

4.6

an

hy

dro

vin

bla

stin

e,

20

.8v

infl

un

ine

,1

0.4

cath

ara

nth

ine

8.8

Au

rio

lae

ta

l.(1

98

9)

Try

pta

min

e,

2l

Bo

nd

ap

ak

C18,

10

lm3

00

·3

.9m

m

Ace

ton

itri

le:0

.1M

CH

3C

OO

NH

4

(pH

7.2

)(4

9:5

1,

v:v

)

UV 2

80

nm

,T

SP

-MS

Iso

cra

tic,

1m

lm

in–1

ajm

ali

cin

e,

5.5

serp

en

tin

e,

8ca

tha

ran

thin

e,

10

tab

ers

on

ine

18

To

ivo

ne

ne

ta

l.(1

99

1)

Ajm

ali

cin

e,

cath

ara

nth

ine

,ta

be

rso

nin

e

N/A

Hy

pe

rsil

OD

S,

5lm

10

0·

2.1

mm

(Ag

ile

nt)

So

lven

tA

:1

4m

MT

EA

inC

H3C

OO

NH

4;

So

lven

tB

:1

4m

MT

EA

inM

eth

an

ol:

ace

ton

itri

le(5

0:5

0,

v:v

)

PD

A

0204060

010

2030

40

Tim

e (m

in)

% B

0.4

mL

min

-1

Phytochem Rev (2007) 6:207–234 223

123

Ta

ble

7co

nti

nu

ed

Au

tho

rsC

om

po

un

ds

focu

sse

dR

ete

nti

on

tim

e(m

in)

Co

lum

nM

ob

ile

Ph

ase

De

tect

ion

Elu

tio

ng

rad

ien

t

Tik

ho

mir

off

an

dJo

lico

eu

r(2

00

2)

Ca

tha

ran

thin

e,

14

.5Z

orb

ax

XD

BC

18

5lm

25

0·

4.6

mm

(Ag

ile

nt)

So

lven

tA

:5

mM

KH

2P

O4

(pH

6);

So

lven

tB

:A

ceto

nit

rile

PD

A

020406080

010

203 0

Tim

e (m

in)

% B

2 m

L m

in-1

serp

en

tin

e,

7.8

vin

do

lin

e,

13

.5v

incr

isti

ne

,1

3.1

vin

bla

stin

e,

15

.2ta

be

rso

nin

e2

0.8

Sin

gh

et

al.

(20

00

)C

ath

ara

nth

ine

,1

2.3

2lB

on

da

pa

ckC

18,

10

lm

30

0·

3.9

mm

(Wa

ters

)

Ace

ton

itri

le:0

.1M

Ph

osp

ha

teb

uff

er:

gla

cia

la

eti

ca

cid

(pH

4.1

4)

(38

:62

:0.3

,v

:v:v

)

PD

AIs

ocr

ati

c,0

.6m

lm

in–1

vin

do

lin

e,

13

.54

vin

cris

tin

e,

10

.15

vin

bla

stin

e1

1.3

4

Le

e-P

ars

on

se

ta

l.(2

00

4)

Try

pta

min

e5

Lu

na

C18

5lm

15

0·

4.6

mm

(Ph

en

om

en

ex

)

So

lven

tA

:W

ate

r,S

olv

ent

B:

Ace

ton

itri

le,

So

lven

tC

:F

orm

ica

cid

(ke

pt

at

0.1

%o

fth

eto

tal

elu

tio

nv

olu

me

)

UV 2

54

nm

0510152025

010

2030

4050

Tim

e (m

in)

% B

1 m

L m

in-1

ajm

ali

cin

e,

34

serp

en

tin

e3

7

Sch

rod

er

et

al.

(19

99

)

Ta

be

rso

nin

e,

16

-hy

dro

xy

-ta

be

rso

nin

e,

16

-me

tho

xy

-ta

be

rso

nin

e

26

.7N

ucl

eo

sil

C18

5lm

20

0·

4m

m(A

gil

en

t)

So

lven

tA

:0

.2%

Na

OH

inw

ate

r,S

olv

ent

B:

Ace

ton

itri

le

PD

A

020406080

010

2030

Tim

e (m

in)

% B

1 m

L m

in-1

13

.02

6.2

224 Phytochem Rev (2007) 6:207–234

123

Ta

ble

7co

nti

nu

ed

Au

tho

rsC

om

po

un

ds

focu

sse

dR

ete

nti

on

tim

e(m

in)

Co

lum

nM

ob

ile

Ph

ase

De

tect

ion

Elu

tio

ng

rad

ien

t

Ha

lla

rde

ta

l.,

(un

pu

bli

she

d)

Try

pta

min

e,

3.5

‘‘L

ow

TF

A’’

C18

5lm

25

0·

4.6

mm

(Vy

da

c)

So

lven

tA

:T

FA

:wa

ter:

ace

ton

itri

le(0

.01

:79

:21

);S

olv

ent

B:

TF

A:w

ate

r:a

ceto

nit

rile

(0.0

1:5

:95

)

UV

25

4n

m,

ES

I-M

S

05

10

15

01

02

03

04

0

Tim

e (m

in)

% B

1 m

l min

-1

seco

log

an

in,

4.0

pe

riv

ine

,4

.2v

ind

oli

nin

e,

4.9

yo

him

bin

e,

5.2

stri

cto

sid

ine

,5

.9a

jma

lici

ne

,8

.1se

rpe

nti

ne

,8

.6a

lsto

nin

e,

9.2

cath

ara

nth

ine

,9

.3v

incr

isti

ne

,1

2.5

tab

ers

on

ine

,1

3.5

vin

do

lin

e,

16

.3ca

tho

va

lin

e,

18

.8v

inb

last

ine

,2

1.1

an

hy

dro

-v

inb

last

ine

26

.4

Phytochem Rev (2007) 6:207–234 225

123

a C18Bondclone, 10 lm, 300 · 3.8 mm column

(Phenomenex), and using a gradient flow

mode with a mixture of methanol and 5 mM

(NH4)2HPO4 (pH 7.3) (58:42, v:v). Using the

modified method, pure peaks were obtained for

ajmalicine, serpentine and tabersonine but cath-

aranthine could not be quantified due to UV

interfering compounds. With the same Bondclone

column, quantification of ajmalicine, serpentine,

tabersonine, horhammericine and lochnericine

was successful using an isocratic elution at

1 ml min–1 (Table 7). Alkaloids metabolism in

wounded seedlings was studied by Vazquez-Flota

et al. (2004) using the method previously devel-

oped by Aerts et al. (1994) in a study on the effect

of methyl jasmonate vapor on C. roseus seedlings.

The method developed by Tikhomiroff and

Jolicoeur (2002) for screening secondary metab-

olites in hairy roots, was recently used for the

analysis of the accumulation of alkaloids in

different plant cultivars (Dutta et al. 2005).

There are multiple approaches when develop-

ing a HPLC separation method and the following

basic considerations have to be considered. In the

case of TIAs analysis, there is no consensus on

mobile phase selection. An effective separation

can be achieved using a saline buffer with

potassium phosphate or ammonium acetate as

aqueous phase, and methanol or acetonitrile as

organic phase (Bhadra et al. 1993; Moreno-Val-

enzuela et al. 1998; Choi et al. 2002; Tikhomiroff

et al. 2002). These methods seem to be inspired

from the early works of Renaudin (1984) on the

separation of ajmalicine, catharanthine, serpen-

tine, vindoline and vinblastine. Other studies are

suggesting the use of ion-pairing additives such as

TEA or TFA, which are commonly used for

masking non-derivatized silanol groups (Aerts

et al. 1994; St-Pierre and De Luca 1995; Lee and

Shuler 2000; Zhao et al. 2001; Uniyal et al. 2001;

El-Sayed et al. 2004). These HPLC methods

using ion-pairing reagents have been first devel-

oped by Naaranlathi et al. (1987). With a neutral

pH, ion-exchange interactions and hydrogen

bonding can occur between dissociated silanol

and protonated (or partially protonated) alka-

loids (Nawrocki 1997; McCalley 2002), which

usually results in peak tailing. TEA allows com-

peting alkaloids for the silanol sites. It is usually

observed that an old column (hopefully after

running few thousands of samples) offers a

weaker retention of alkaloids with low pKa (e.g.

ajmalicine) but a stronger retention of com-

pounds with high pKa (as tryptamine and ser-

pentine) than a new column (Renaudin 1984).

This phenomenon is thought to be due to the

degradation of the hydrophobic stationary phase

resulting from the uncapping of the silanol groups

following the disruption of the C18 alkyl chains.

The presence of amine modifier agents in the

mobile phase modulates the interaction of the

stationary phase with basic analytes (e.g. alka-

loids) and can thus change the elution sequence.

It then becomes tedious to compare the methods.

Competitive amines can be used to enhance

peaks shape at neutral pH and reducing peak

tailing (Nawrocki 1997; McCalley 2002). Mobile

phase pH is an important parameter and acidic

pH (pH 3–4) enables better peak shape for basic

alkaloids. At low pH, most of silanol groups are

undissociated and ion-exchange interactions are

then limited, even if alkaloids are protonated

(McCalley 2002). Alkaline pH (pH 10) was only

tested by Barthe et al. (2002), to our knowledge.

Multiple columns can be found commercially if

one chooses to work at extreme alkaline pH but it

should be noted that past-generation columns

(e.g. C8, Phenyl and C18) cannot be used at high

pH because the destruction of the stationary

phase may be induced. In every cases (acidic or

basic mobile phases), methods should employ a

pH which is far from the pKa of the target

compounds, since weak variations in the mobile

phase preparation can change the degree of

protonation of the alkaloids, and thus influence

the elution pattern and the reproducibility in the

retention times (Uniyal et al. 2001; McCalley

2002). Uniyal et al. (2001) observed that the

elution time pattern changed from catharanthine,

vincristine, vinblastine and vindoline at pH 6, to

vincristine, vindoline, catharanthine and vinblas-

tine at pH 7.5. Using a phosphate buffer at pH 6

with acetonitrile as mobile phase, Tikhomiroff

and Jolicoeur (2002) obtained the same elution

sequence than that obtained at pH 7.5 by Uniyal

et al. (2001). This discrepancy may be explained

because Uniyal et al. (2001) used TEA and a

different column, resulting in a different silanol

226 Phytochem Rev (2007) 6:207–234

123

activity of the column packing. Barthe et al.

(2002) found that the elution sequence for vind-

oline and cathranthine was reversed changing the

mobile phase pH from 5 to 10. Therefore, a

judicious combination of buffer and mobile phase

pH can be helpful when designing a separation

method focusing on specific peaks. For instance,

adjustment of the mobile phase pH at 4.7 instead

of 3.9 allowed separating strictosidine and non-

polar alkaloids from an extract of Tabernaemon-

tana divaricata (Dagnino et al. 1995). Using an

isocratic elution, satisfactory separation of alka-

loids can be achieved (Table 7). Bhadra et al.

(1993) and Rijhwani and Shanks (1998) were able

to separate up tofive alkaloids within a unique run

for C. roseus metabolites. Van der Heijden et al.

(1987) used an isocratic HPLC method previ-

ously developed for Tabernaemontana species.

Furthermore, separation of the alkaloids can be

greatly improved by using a gradient elution

(Naaranlahti et al. 1987; Tikhomiroff et al. 2002).

It allows achieving a good separation without

extending HPLC run time. A typical UV-chro-

matogram obtained from the separation of a

standard mixture of 12 C. roseus secondary

metabolites using the gradient method developed

by Hallard et al. (unpublished results reproduced

with the kind permission of Dr. Verpoorte; see

Fig. 4 caption for the complete list of authors), is

presented in Fig. 4. Some co-eluting peaks cannot

be accurately quantified by UV, but they were

discriminated using an ESI-MS detector in single

ion recording mode, based on the mass of the

protonated molecule (not shown). Analysis of a

crude extract of C. roseus hairy roots using the

gradient method developed by Tikhomiroff and

Jolicoeur (2002) is presented in Fig. 5a. All the

alkaloids are eluted within a 23 min run. The

examples presented in this review show the

advantage of a gradient method for the separation

of complex mixtures of C. roseus alkaloids which

allow separating up to twelve known compounds

in the same run.

HPLC methods for the analysis of precursors

For the analysis of indole and iridoid precursors

(Table 6), Tikhomiroff and Jolicoeur (2002)

adapted the method of Dagnino et al. (1996).

For both indole and iridoid precursors, an acidic

aqueous mobile phase (pH 1.8 or 2) was used to

preserve an adequate peak shape for secologanin

(Dagnino et al. 1996). Modulating TCA concen-

tration in Dagnino’s method allowed varying the

retention time of tryptophan and tryptamine.

Using a similar C18 HPLC column, Tikhomiroff

and Jolicoeur (2002) were able to achieve the

separation of the indole precursors and secolog-

anin in less than 8 min by increasing the flow rate

without using an ion-pairing molecule. Trypt-

amine can also be analyzed in the same run than

TIAs (Auriola et al. 1989; Moreno et al. 1993;

Lee-Parsons et al. 2004).

Scaling-up a method

As a litany stated along this review, the major

limitation in developing effective separation and

identification methods relies in the lack of stan-

dards for most of C. roseus secondary metabo-

lites. However, if a lab is conducting metabolic

studies or is screening for therapeutics, it may

become essential to have access to those unavail-

able standards. Transfer between academics is a

simple and efficient way but could be difficult

when several milligrams of pure compounds are

asked. A lab may then be obliged to upscale a

separation method from the analytical to the

semi-preparative (semi-prep) scale. It is thus

important to consider this issue already when

developing an analytical method because some

early precautions may save a lot of time and

efforts at the further developmental stages,

including industrial. The important parameters

not to forget are the column type and the HPLC

system supplier. The choice of the column is

important because a rigorous scale-up can be

done more easily using a larger diameter column

having the same length and packing that the one

used at analytical scale. Total pressure of the

system is also an important parameter to consider

preventing from packing bed deterioration. Pres-

sure is building-up in the column and the guard-

column and is amplified by the viscosity of the

mobile phase. It is also highly helpful to select a

flow rate range for the analytical method enabling

the use of the same HPLC pump for the semi-

prep method.

Phytochem Rev (2007) 6:207–234 227

123

An example of an analytical method that has

been scaled-up to a semi-prep method is given in

the following. The analytical method developed

for TIAs by Tikhomiroff and Jolicoeur (2002) was

slightly modified. First, the phosphate buffer was

changed for ammonium acetate buffer, which is

more volatile and can thus be easily used in

combination to the MS detector for further

characterization of the fractions collected. Sec-

ond, the flow rate was divided from 2 to 1 ml min–

1. Since the diameter of the semi-prep column was

2 times that of the analytical one, it was necessary

to increase the flow rate to 4 ml min–1 for the

semi-preparative run, to preserve similar

chromatographic conditions. The run was 7 min

longer but the separation remained satisfactory

and the pressure limit of the HPLC system was

not exceeded. The chromatograms obtained for

the analytical and semi-prep methods shown to be

highly similar (Fig. 5). The analytical and semi-

prep methods used injection volumes of 20 and

250 ll, respectively. The use of a PDA detector

with a larger cell adapted for semi-prep flow

rates explains the similar absorbance in both

injections. This semi-prep method was designed

for the recovery of standards, for both the known

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0

mA

Um

AU

mA

Um

AU

0

1200

200

400

600

800

1000

200

400

600

800

1000

0

200

400

600

800

10001 4 a

3

2 6 75

0 0

200

400

600

800

1000

12001 4 b

3

25 6 7

Minutes

Minutes

Fig. 5 Analytical method (a) and semi-prep method (b)developed from the modification of the Tikhomiroff andJolicoeur (2002) method. Mobile phase was CH3COONH4

50 mM pH 6 (solvent A) and acetonitrile (solvent B). Theflow rate was 1 ml min–1 for the analytical method and4 ml min–1 for the semi-prep method The eluent profile(volume solvent A: volume solvent B) was the same forboth methods: 0–20 min, linear gradient from 80:20 to

20:80; 20–30 min, isocratic elution with a 20:80 mixture; 30–35 min: isocratic elution 80:20 for column equilibration.1,serpentine; 2, unknown T1; 3, catharanthine; 4, lochneri-cine; 5, unknown T3; 6, tabersonine; 7, unknown T4. Thealkaloids extract was pre-purified using a Strata C18-T SPEcartridge (Phenomenex, Torrance, USA) (unpublisheddata)

228 Phytochem Rev (2007) 6:207–234

123

molecules as well as for some unidentified alka-

loids. A fraction collector was used and each peak

was collected based on the slope of the absor-

bance signal at 296 nm. The collected fractions

could then be further analyzed for purity through

MS-MS or 1H-NMR detection. Since hundreds

of molecules elute from a single injection, some

compounds may unfortunately co-elute. Thus,

the use of semi-prep columns may be of inter-

est for pre-fractionation of a complex mixture

(Mans et al. 2000). This method allows ‘‘cutting’’

the chromatogram into several fractions contain-

ing, hopefully, one to ten alkaloids each.

Then, the fractions can be separated under

modified HPLC conditions, and used for bioac-

tivity screening.

Other useful analytical techniques

While TLC was historically seen as an interesting

low cost technique, capillary electrophoresis (CE)

and CZE (capillary zone electrophoresis) is now

gaining in interest. The reader is invited to consult

the review of Stockigt et al. (2002) on the use of

CZE for the analysis of alkaloids. CE allows

separation of charged molecules based on the

mass-to-charge ratio in an electric field. Due to

their high pKa, alkaloids are present in their

protonated form when using an acidic buffer.

CZE was used for the separation of the bisindole

vincristine and vinblastine from a crude leaf

extract (Chu et al. 1996). Barthe et al. (2002)

have used a non-aqueous electrophoretic buffer

for separating 11 Vinca alkaloids in less than

10 min. The running buffer consisted in 50 mM

ammonium acetate and 0.6 M acetic acid in an

organic mixture of methanol–acetonitrile (75:25,

v:v). A constant voltage of 25 kV was applied and

the temperature of the capillary was kept at 20�C.

Unfortunately, this non-aqueous capillary elec-

trophoresis method has not been tested on plant

extract.

Preparative-scale continuous separation of

enantiomers by CE has also been reported

(Spanik 2002) but scale-up potential of CE for

phytochemicals has still to be demonstrated

(Suntornsuk 2002). Indeed, CE may be an alter-

native for the analysis of indole alkaloids in crude

extracts. This technique combines the selectivity

of HPLC stationary phase with that resulting

from the application of an electric field (McCalley

2002). It has been reported that a C18 stationary

phase with a charged surface allows effective

electroosmotic flow and an efficient separation

(Zimina et al. 1997). However, the limit of

detection for HPLC analysis is still lower that

that for CE and this is probably due to the low

sample volume injected in CE (Barthe et al. 2002;

Drager 2002).

Future developments

HPLC methods and equipments

There are many improvements that can be

achieved to make HPLC an even more powerful

analytical technique. We think that the objective

for improving HPLC technique should be to

simplify method scale-up procedures and enhance

the capacity for integrating more powerful detec-

tors. In addition to UV and fluorescence spectra

determination, measurement of the molecular

weight for each peak can be highly useful to

analyze plant or cell extracts. Molecular weights

of C. roseus metabolites have been recently

presented by van der Heijden et al. (2004) in a

review inspired from the Dictionary of Natural

Products. The use of MS detection, as TSP-MS

(Thermospray), ESI-MS (Electrospray Ionization

Mass Spectrometry) or MALDI-TOF (Matrix

Assisted Laser Desorption with Time-of-Flight)

can enhance the identification of low abundant

compounds and of non-pure peaks with a higher

precision than UV spectra. However, as discussed

previously, because of the silanol activity of

HPLC packing, an acidic mobile phase is used

to limit peak tailing and ensure batch to batch

reproducibility. Although amine modifiers can be

included in the mobile phase as masking agents,

their use is not recommended for ESI-MS detec-

tion since an ion-suppression phenomenon can be

generated and then greatly reduce the sensitivity

of the instrument (Gustavsson et al. 2001; Anti-

gnac et al. 2005). The introduction on the market

of columns integrating a carbamate internal

group in bonded phase seemed to have solved

this problem. Peak tailing showed to be reduced

Phytochem Rev (2007) 6:207–234 229

123

for basic compounds as compared to traditional

C18 alkyl packing (O’Gara et al. 1999). The

choice of different MS interfaces for the analysis

of alkaloids has been reviewed (Verpoorte and

Niessen 1994). The use of LC-TSP-MS allowed

quantification of tryptamine, ajmalicine, serpen-

tine, catharanthine and tabersonine (Auriola

et al. 1989). Hallard (unpublished data, see cap-

tion of Fig. 4 for complete reference) developed a

LC-ESI-MS method allowing quantification of 10

alkaloids and 19 precursors. Using a HPLC

separation system coupled to an ESI interface,

Chu et al. (1997) detected vincristine and vin-

blastine in plant extracts at a limit of detection of

0.2 lg ml–1. The sensitivity for these compounds

was 2–3 times higher in ESI-MS rather than with

UV detection. In addition of increasing the

sensitivity, ESI-MS will help to achieve the

identification of the numerous compounds which

can not be actually identified from a UV detector

because some secondary metabolites possess

highly similar UV-spectra. Zhou et al. (2005)

were able to analyze vindoline, vindolidine, vin-

cristine and vinblastine in a commercial prepara-

tion by direct injection ESI-MS-MS. This is

obviously shortening the time for the identifica-

tion of these molecules. However, the authors

mentioned that HPLC separation is required,

followed by ESI-MS-MS to discriminate catha-

ranthine from the two isomers vindolinine and

19S-vindolinine, which all have a m/z ratio of 337.

Maldi-TOF analysis of crude extract represents a

quick technique for fast screening of different

strains and cell lines for their vinblastine or

vincristine contents, as proposed by Contin and

van der Heijden (unpublished results, cited in van

der Heijden et al. 2004). However, single quad-

rupole MS detector may not be sufficient for

absolute peak identification because many metab-

olites posses the same molecular weight. For

instance, 13 alkaloids from C. roseus have a

similar molecular weight of 352.432 g mol–1.

Identification of such molecules should be

achieved after molecular fragmentation in a

tandem mass spectrometer and/or by 1H-NMR

analysis. Transposition of HPLC methods devel-

oped for UV or fluorescence detector to MS

detector is possible if the methods are using a

volatile buffer as mobile phase. However, most

common HPLC methods are based on non-

volatile buffers, such as phosphate buffer, which

are not compatible with MS detection (Law and

Temesi 2000). Also, since the elucidation of the

structure of many alkaloids is achieved by 1H-

NMR, the association of such detection with the

power of separation of HPLC could be highly

efficient. Such an approach was presented by

Bobzin et al. (2000) for the determination of

marine alkaloids as inhibitors of an enzyme

implicated in the type II diabetes. HPLC-NMR

was also used for the screening of plant extracts

(Bringmann et al. 1999, 2000, 2002; Zhao et al.

1999). To our knowledge, however, this method

has never been used with C. roseus crude extracts.

Coupling NMR detection to HPLC is though to

be the unique method for the elucidation of

isomers.

Conclusion

This article has presented a review on the

significant effort of the scientific community to

develop efficient and reliable HPLC methods for

the analysis of C. roseus alkaloids. There are

some other analytical techniques such as TLC and

CE that have also been presented but emphasis

has been placed on HPLC because it can be easily

scaled-up. This article has then reviewed the most

recent developments in HPLC analysis of alka-

loids from C. roseus. The diverse considerations

and the crucial steps to achieve an efficient

separation and identification of secondary metab-

olites were enumerated and discussed. Biomass

manipulation, choice of an extraction phase and

of the extraction protocols as well as HPLC

separation and analysis protocols were then

reviewed in details. Furthermore, examples of

spectra obtained using most common detectors

were also shown. Finally, suggestions were made

on how to proceed for developing efficient sep-

aration and identification methods, based on

literature.

The screening of C. roseus secondary metabo-

lites represents a challenge for the pharmaceuti-

cal industry. The synthesis of the bisindole

alkaloids vinblastine and vincristine may be

performed industrially through chemical synthesis

230 Phytochem Rev (2007) 6:207–234

123

or from genetically modified plant cells in a near

future. The modification of C. roseus metabolic

pathways or the insertion of C. roseus genes in

other plant species may also result in an increase

of the biodiversity of the alkaloids obtained (van

der Heijden et al. 2004). Thus, many novel

unknown alkaloids of therapeutic interest may

then be obtained in the future and these new

compounds will have to be separated, identified

and tested using HPLC. The development of a

successful HPLC method is thus a crucial step in

the process of identifying the therapeutic

potential of phytochemicals such as those from

C. roseus.

Acknowledgements Authors wish to thank Dr.Verpoorte for his kind permission to include in thispaper his unpublished work shown as Fig. 4.

References

Abel U, Koch C, Speitling M, Hansske FG (2002) Modernmethods to produce natural-product libraries. CurrOpin Chem Biol 6:453–458

Aerts RJ, Gisi D, Decarolis E, Deluca V, Baumann TW(1994) Methyl jasmonate vapor increases the devel-opmentally controlled synthesis of alkaloids in Cath-aranthus and Cinchona seedlings. Plant J 5:635–643

Annesley TM (2003) Ion suppression in mass spectrome-try. Clin Chem 49:1041–1044

Antignac JP, Brosseaud A, Gaudin-Hirret I, Andre F,Bizec BL (2005) Analytical strategies for the directmass spectrometric analysis of steroid and corticoste-roid phase II metabolites. Steroids. Web released

Asada M, Shuler ML (1989) Stimulation of ajmalicineproduction and excretion from Catharanthus roseus:effects of adsorption in situ, elicitors and alginateimmobilization. Appl Microbiol Biotechnol 30:475–481

Auriola S, Naaranlati T, Lapinjoki SP (1991) Determinationof Catharanthus roseus alkaloids by high-performanceliquid-chromatography–isotope-dilution thermospraymass spectrometry. J Chromatogr 554:227–231

Auriola S, Ranta VP, Naaranlahti T, Lapinjoki SP (1989)Thermospray liquid-chromatographic mass-spectro-metric analysis of Catharanthus alkaloids. J Chroma-togr 474:181–185

Balague C, Wilson G (1982) Growth and alkaloid biosyn-thesis by cell suspensions of Catharanthus roseus in achemostat under sucrose and phosphate limitingconditions. PhysiolVeg 20:515–522

Barthe L, Ribet JP, Pelissou M, Degude MJ, Fahy J,Duflos A (2002) Optimization of the separation ofvinca alkaloids by nonaqueous capillary electropho-resis. J Chromatogr A 968:241–250

Bhadra R, Shanks JV (1997) Transient studies of nutrientuptake, growth, and indole alkaloid accumulation inheterotrophic cultures of hairy roots of Catharanthusroseus. Biotechnol Bioeng 55:527–534

Bhadra R, Vani S, Shanks JV (1993) Production of indolealkaloids by selected hairy root lines of Catharanthusroseus. Biotechnol Bioeng 41:581–592

Blom TJM, Sierra M, Vanvliet TB, Frankevandijk MEI,Dekoning P, Vaniren F, Verpoorte R, Libbenga KR(1991) Uptake and accumulation of ajmalicine intoisolated vacuoles of cultured-cells of Catharanthusroseus (L.) G. Don and its conversion into serpentine.Planta 183:170–177

Bobzin SC, Yang S, Kasten TP (2000) Application ofliquid chromatography–nuclear magnetic resonancespectroscopy to the identification of natural products.J Chromatogr B 748:259–267

Bringmann G, Messer K, Wohlfarth M, Kraus J, DumbuyaK, Ruckert M (1999) HPLC-CD on-line coupling incombination with HPLC–NMR and HPLC–MS/MSfor the determination of the full absolute stereostruc-ture of new metabolites in plant extracts. Anal Chem71:2678–2686

Bringmann G, Wohlfarth M, Heubes M (2000) Observa-tion of exchangeable protons by high-performanceliquid chromatography–nuclear magnetic resonancespectroscopy and high-performance liquid chroma-tography–electrospray ionization mass spectrometry:a useful tool for the hyphenated analysis of naturalproducts. J Chromatogr A 904:243–249

Bringmann G, Wohlfarth M, Rischer H, Schlauer J, RetoB (2002) Extract screening by HPLC coupled to MS-MS, NMR, and CD: a dimeric and three monomericnaphthylisoquinoline alkaloids from Ancistrocladusgriffithii. Phytochemistry 61:195–204

Byun SY, Pedersen H (1994) 2-Phase airlift fermenteroperation with elicitation for the enhanced productionof benzophenanthridine alkaloids in cell-suspensionsof Eschscholtzia californica. Biotechnol Bioeng 44:14–20

Capell T, Christou P (2004) Progress in plant metabolicengineering. Curr Opin Biotechnol 15:148–154

Chen X, Yi C, Yang X, Wang X (2004) Liquid chroma-tography of active principles in Sophora flavescensroot. J Chromatogr B 812:149–163

Choi YH, Yoo KP, Kim J (2002) Supercritical fluidextraction and liquid chromatography–electrospraymass analysis of vinblastine from Catharanthus roseus.Chem Pharm Bulletin 50:1294–1296

Chu IH, Bodnar JA, Bowman RN, White EL (1997)Determination of vincristine and vinblastine inCatharanthus roseus plants by high performanceliquid chromatography electrospray ionization massspectrometry. J Chromatogr Relat Technol 20:1159–1174

Chu IH, Bodnar JA, White EL, Bowman RN (1996)Quantification of vincristine and vinblastine in Cath-aranthus roseus plants by capillary zone electropho-resis. J Chromatogr A 755:281–288

Phytochem Rev (2007) 6:207–234 231

123

Collins-Pavao N, Chin CK (1996) Taxol partitioning intwo-phase plant cell cultures of Taxus brevifolia. JBiotechnol 49:95–100

Collu G, Bink HHJ, Moreno PRH, Van Der Heijden R,Verpoorte R (1999) Determination of the activity ofthe cytochrome p450 enzyme geraniol 10-hydroxylasein plants by high-performance liquid chromatography.Phytochem Anal 10:314–318

Contin A, Van Der Heijden R, Lefeber AWM, VerpoorteR (1998) The iridoid glucoside secologanin is derivedfrom the novel triose phosphate/pyruvate pathway ina Catharanthus roseus cell culture. FEBS Lett434:413–416

Dagnino D, Schripsema J, Verpoorte R (1995) Terpenoidindole alkaloid biosynthesis and enzyme-activities intwo cell-lines of Tabernaemontana divaricata. Phyto-chemistry 39:341–349

Dagnino D, Schripsema J, Verpoorte R (1996) Analysis ofseveral iridoid and indole precursors of terpenoidindole alkaloids with a single HPLC run. Planta Med62:278–280

De Luca V, Balsevich J, Tyler RT, Eilert U, Panchuk BD,Kurz WGW (1986) Biosynthesis of indole alkaloids:developmental regulation of the biosynthetic pathwayfrom tabersonine to vindoline in Catharanthus roseus.J Plant Physiol 125:147–156

De Luca V, Fernandez JA, Campbell D, Kurz WGW(1988) Developmental regulation of enzymes ofindole alkaloid biosynthesis in Catharanthus roseus.Plant Physiol 86:447–450

De Luca V, Laflamme P (2001) The expanding universeof alkaloid biosynthesis. Curr Opin Plant Biol 4:225–233

De Luca V, St-Pierre B (2000) The cell and developmentalbiology of alkaloid biosynthesis. Trends Plant Sci5:168–173

Drager B (2002) Analysis of tropane and related alkaloids.J Chromatogr A 978:1–35

Dutta A, Batra J, Pandey-Rai S, Singh D, Kumar S, Sen J(2005) Expression of terpenoid indole alkaloid bio-synthetic pathway genes corresponds to accumulationof related alkaloids in Catharanthus roseus (L) GDon. Planta 220:376–383

El-Sayed M, Choi YH, Frederich M, Roytrakul S,Verpoorte R (2004) Alkaloid accumulation in Cath-aranthus roseus cell suspension cultures fed withstemmadenine. Biotechnol Lett 26:793–798

Facchini PJ (2001) Alkaloid biosynthesis in plants: bio-chemistry, cell biology, molecular regulation andmetabolic engineering applications. Annu Rev PlantPhysiol Plant Mol Biol 52:29–66

Fahy J, Du Boullay VT, Bigg DCH (2002) New method ofsynthesis of vinca alkaloid derivatives. Bioorg MedChem Lett 12:505–507

Favali MA, Musetti R, Benvenuti S, Bianchi A, PressaccoL (2004) Catharanthus roseus L. plants and explantsinfected with phytoplasmas: alkaloid production andstructural observations. Protoplasma 223:45–51

Gorog S, Herenyi B, Jovanovics K (1977) High-perfor-mance liquid chromatography of Catharanthus alka-loids. J Chromatogr A 139:203–206

Gustavsson SA, Samskog J, Markides KE, Langstrom B(2001) Studies of signal suppression in liquid chroma-tography–electrospray ionization mass spectrometryusing volatile ion-pairing reagents. J Chromatogr A937:41–47

Hendriks MMWB, Cruz-Juarez L, Bont DD, Hall RD(2005) Preprocessing and exploratory analysis ofchromatographic profiles of plant extracts. Anal ChimActa 545:53–64

Hisiger S, Jolicoeur M (2005) Plant cell culture monitoringusing an in situ multiwavelength fluorescence probe.Biotechnol Prog 21:580–589

Houghton PJ (2002) Chromatography of the chromoneand flavonoid alkaloids. J Chromatogr A 967:75–84

Hughes EH, Hong SB, Gibson SI, Shanks JV, San KY(2004) Expression of a feedback-resistant anthranilatesynthase in Catharanthus roseus hairy roots providesevidence for tight regulation of terpenoid indolealkaloid levels. Biotechnol Bioeng 86:718–727

Ingkaninan K, De Best CM, Van Der Heijden R, HofteAJP, Karabatak B, Irth H, Tjaden UR, Van DerGreef J, Verpoorte R (2000) High-performance liquidchromatography with on-line coupled UV, massspectrometric and biochemical detection for identifi-cation of acetylcholinesterase inhibitors from naturalproducts. J Chromatogr A 872:61–73

Jossang A, Fodor P, Bodo B (1998) A new structural classof bisindole alkaloids from the seeds of Catharanthusroseus: vingramine and methylvingramine. J OrgChem 63:7162–7167

Klyushnichenko VE, Yakimov SA, Tuzova TP, SyagailoYV, Kuzovkina IN, Wulfson AN, Miroshnikov AI(1995) Determination of indole alkaloids from R.serpentina and R. vomitoria by high-performanceliquid-chromatography and high-performance thin-layer chromatography. J Chromatogr A 704:357–362

Kruczynski A, Hill BT (2001) Vinflunine, the latest Vincaalkaloid in clinical development: a review of itspreclinical anticancer properties. Oncol/Hematol40:159–173

Kutney JP, Choi LSL, Kolodziejczyk P, Sleigh SK, StuartKL, Worth BR, Kurz WGW, Chatson KB, ConstabelF (1980) Alkaloid production in Catharanthus roseuscell cultures: isolation and characterization of alka-loids from one cell line. Phytochemistry 19:2589–2595

Laflamme P, St-Pierre B, De Luca V (2001) Molecular andbiochemical analysis of a madagascar periwinkle root-specific minovincinine-19-hydroxy-O-acetyltransfer-ase. Plant Physiol 125:189–198

Law B, Temesi D (2000) Factors to consider in thedevelopment of generic bioanalytical high-perfor-mance liquid chromatographic–mass spectrometricmethods to support drug discovery. J Chromatogr B748:21–30

Lee CWT, Shuler ML (2000) The effect of inoculumdensity and conditioned medium on the productionof ajmalicine and catharanthine from immobilizedCatharanthus roseus cells. Biotechnol Bioeng 67:61–71

Lee-Parsons CWT, Erturk S, Tengtrakool J (2004)Enhancement of ajmalicine production in Catharan-

232 Phytochem Rev (2007) 6:207–234

123

thus roseus cell cultures with methyl jasmonate isdependent on timing and dosage of elicitation. Bio-technol Lett 26:1595–1599

Lee-Parsons CWT, Shuler ML (2002) The effect ofajmalicine spiking and resin addition timing on theproduction of indole alkaloids from Catharanthusroseus cell cultures. Biotechnol Bioeng 79:408–415

Mans DRA, Da Rocha AB, Schwartsmann G (2000) Anti-cancer drug discovery and development in Brazil:targeted plant collection as a rational strategy toacquire candidate anti-cancer compounds. The Oncol-ogist 5:185–198

McCalley DV (2002) Analysis of the Cinchona alkaloids byhigh-performance liquid chromatography and otherseparation techniques. J Chromatogr A 967:1–19

Meijer AH, Verpoorte R, Hoge JHC (1993) Regulation ofenzymes and genes involved in terpenoid indolealkaloid biosynthesis in Catharanthus roseus. J PlantRes 3:145–164

Molyneux RJ, Gardner DR, James LF, Colegate SM(2002) Polyhydroxy alkaloids: chromatographic anal-ysis. J Chromatogr A 967:57–74

Monforte-Gonzales M, Ayaora-Talavera T, Maldonado-Mendoza IE, Loyola-Vargas VM (1992) Quantitativeanalysis of serpentine and ajmalicine in plant tissuesof Catharanthus roseus and hyoscyamine and scopol-amine in root tissues of Datura stramonium by thinlayer chromatography–densitometry. PhytochemAnal 3:117–121

Moreno PRH, Van der Heijden R, Verpoorte R (1993)Effect of terpenoid precursor feeding and elicitationon formation of indole alkaloids in cell-suspensioncultures of Catharanthus roseus. Plant Cell Rep12:702–705

Moreno-Valenzuela OA, Galaz-Avalos RM, Minero-GarciaY, Loyola-Vargas VM (1998) Effect of differentiation onthe regulation of indole alkaloid production in Catha-ranthus roseus hairy roots. Plant Cell Rep 18:99–104

Morgan JA, Barney CS, Penn AH, Shanks JV (2000)Effects of buffered media upon growth and alkaloidproduction of Catharanthus roseus hairy roots. ApplMicrobiol Biotechnol 53:262–265

Morgan JA, Shanks JV (2002) Quantification of metabolicflux in plant secondary metabolism by a biogeneticorganizational approach. Metabol Eng 4:257–262

Naaranlahti T, Nordstrom M, Huhtikangas A, LounasmaaM (1987) Determination of Catharanthus alkaloids byreversed-phase high-performance liquid chromatog-raphy. J Chromatogr A 410:488–493

Naaranlahti T, Ranta V-P, Jarho P, Nordstrom M,Lapinjoki SP (1989) Electrochemical detection ofindole alkaloids of Catharanthus roseus in high-performance liquid chromatography. Analyst114:1229–1331

Nawrocki J (1997) The silanol group and its role in liquidchromatography. J Chromatogr A 779:29–71

O’Gara JE, Walsh DP, Alden BA, Casellini P, Walter TH(1999) Systematic study of chromatographic behaviorvs. alkyl chain length for HPLC bonded phasescontaining an embedded carbamate group. AnalChem 71:2992–2997

Payne GF, Payne NN, Shuler ML, Asada M (1988) In situadsorption for enhanced alkaloid production of Cath-aranthus roseus. Biotechnol Lett 10:187–192

Pennings EJM, Verpoorte R, Goddijn OJM, Hoge JHC(1989) Purification of tryptophan decarboxylase froma Catharanthus roseus cell-suspension culture. J Chro-matogr 483:311–318

Potier P, Langlois N, Langlois Y, Gueritte F (1975) Partialsynthesis of vinblastine-like alkaloids. J Chem Soc16:670–671

Rao SR, Ravishankar GA (2002) Plant cell cultures:chemical factories of secondary metabolites. Biotech-nol Adv 20:101–153

Renaudin JP (1985) Extraction and fluorimetric detectionafter high-performance liquid chromatography ofindole alkaloids from cultured cells of Catharanthusroseus. Physiol Veg 23:381–388

Renaudin JP (1984) Reversed-phase high-performanceliquid chromatographic characteristics of indole alka-loids from cell suspension cultures of Catharanthusroseus. J Chromatogr A 291:165–174

Renaudin JP (1989) Different mechanisms control thevacuolar compartmentation of ajmalicine in Catha-ranthus roseus cell cultures. Plant Physiol Biochem27:613–621

Renaudin JP, Brown SC, Guern J (1982) Compartmenta-tion mechanisms of indole alkaloids in cell suspensionof Catharanthus roseus. Phyisol Veg 20:533–547

Rijhwani SK, Shanks JV (1998) Effect of elicitor dosageand exposure time on biosynthesis of indole alkaloidsby Catharanthus roseus hairy root cultures. BiotechnolProg 14:442–449

Rodriguez S, Compagnon V, Crouch NP, St-Pierre B, DeLuca V (2003) Jasmonate-induced epoxidation oftabersonine by a cytochrome p-450 in hairy rootcultures of Catharanthus roseus. Phytochemistry64:401–409

Satdive RK, Fulzele DP, Eapen S (2003) Studies onproduction of ajmalicine in shake flasks by multipleshoot cultures of Catharanthus roseus. BiotechnolProg 19:1071–1075

Schroder G, Unterbusch E, Kaltenbach M, Schmidt J,Strack D, De Luca V, Schroder J (1999) Light-induced cytochrome P450-dependent enzyme in in-dole alkaloid biosynthesis: tabersonine 16-hydroxy-lase. FEBS Lett 458:97–102

Shanks JV, Bhadra R, Morgan J, Rijhwani S, Vani S(1998) Quantification of metabolites in the indolealkaloid pathways of Catharanthus roseus: implica-tions for metabolic engineering. Biotechnol Bioeng58:333–338

Sim SJ, Chang HN, Liu JR, Jung KH (1994) Productionand secretion of indole alkaloids in hairy root culturesof Catharanthus roseus – effects of in-situ adsorption,fungal elicitation and permeabilization. J FermentBioeng 78:229–234

Singh D, Maithy A, Verma R, Gupta M, Kumar S (2000)Simultaneous determination of Catharanthus alka-loids using reversed phase high performance liquidchromatography. J Liquid Chromatogr Relat Technol23:601–607

Phytochem Rev (2007) 6:207–234 233

123

Spanik I, Lim P, Vigh G (2002) Use of full-columnimaging capillary isoelectric focusing for the rapiddetermination of the operating conditions in thepreparative-scale continuous free-flow isoelectricfocusing separation of enantiomers. J ChromatogrA 960:241–246

St-Pierre B, De Luca V (1995) A cytochrome p-450monooxygenase catalyzes the first step in the conver-sion of tabersonine to vindoline in Catharanthusroseus. Plant Physiol 109:131–139

St-Pierre B, Vazquez-Flota FA, De Luca V (1999)Multicellular compartmentation of Catharanthus ro-seus alkaloid biosynthesis predicts intercellular trans-location of a pathway intermediate. Plant Cell 11:887–900

Stevens LH, Giroud C, Pennings EJM, Verpoorte R(1993) Purification and characterization of strictosi-dine synthase from a suspension culture of Cinchonarobusta. Phytochemistry 33:99–106

Stockigt J, Sheludko Y, Unger M, Gerasimenko I, War-zecha H, Stockigt D (2002) High-performance liquidchromatographic, capillary electrophoretic and capil-lary electrophoretic–electrospray ionisation massspectrometric analysis of selected alkaloid groups. JChromatogr A 967:85–113

Strege M (1999) High-performance liquid chromato-graphic–electrospray ionization mass spectrometricanalyses for the integration of natural products withmodern high-throughput screening. J Chromatogr B725:67–78

Suntornsuk L (2002) Capillary electrophoresis of phyto-chemical substances. J Pharm Biomed Anal 27:679–698

Tikhomiroff C, Allais S, Klvana M, Hisiger S, Jolicoeur M(2002) Continuous selective extraction of secondarymetabolites from Catharanthus roseus hairy roots withsilicon oil in a two-liquid-phase bioreactor. BiotechnolProgr 18:1003–1009

Tikhomiroff C, Jolicoeur M (2002) Screening of Catha-ranthus roseus secondary metabolites by high-perfor-mance liquid chromatography. J Chromatogr A955:87–93

Toivonen L, Ojala M, Kauppinen V (1991) Studies on theoptimization of growth and indole alkaloid productionby hairy root cultures of Catharanthus roseus. Bio-technol Bioeng 37:673–680

Uniyal GC, Bala S, Mathur AK, Kulkarni RN (2001)Symmetry C18 column: a better choice for the analysisof indole alkaloids of Catharanthus roseus. PhytochemAnal 12:206–210

Van Der Heijden R, Verpoorte R, Ten Hoopen JG (1989)Cell and tissue cultures of Catharanthus (L.)G. Don: aliterature survey. Plant Cell Tiss Org 18:231–280

Van Der Heijden R, Jacobs DI, Snoeijer W, Hallared D,Verpoorte R (2004) The Catharanthus alkaloids:pharmacognosy and biotechnology. Curr Med Chem11:607–628

Van Der Heijden R, Lamping PJ, Out PP, Wijnsma R,Verpoorte R (1987) High-performance liquid chro-matographic determination of indole alkaloids in asuspension culture of Tabersnaemontana divaricata. JChromatogr 396:287–295

Van Elswijk DA, Irth I (2003) Analytical tools for thedetection and characterization of biologically activecompounds from nature. Phytochem Rev 1:427–439

Vazquez-Flota F, Carrillo-Pech M, Minero-Garcia Y, deLourdes Miranda-Ham M (2004) Alkaloid metabo-lism in wounded Catharanthus roseus seedlings. PlantPhysiol Biochem 42:623–628

Verpoorte R, Memelink J (2002) Engineering secondarymetabolite production in plants. Curr Opin Biotech13:181–187

Verpoorte R, Niessen W (1994) Liquid chromatographycoupled with mass spectrometry in the analysis ofalkaloids. Phyotchem Anal 5:217–232

Verpoorte R, Van Der Heijden R, Ten Hoopen HJG,Memelink J (1999) Metabolic engineering of plantsecondary metabolite pathways for the production offine chemicals. Biotechnol Lett 21:467–479

Whitmer S, Van Der Heijden R, Verpoorte R (2002)Effect of precursor feeding on alkaloid accumulationby a strictosidine synthase over-expressing transgeniccell line s1 of Catharanthus roseus. Plant Cell Tiss OrgCult 69:85–93

Wong PL, Royce AJ, Lee-Parsons CWT (2004) Improvedajmalicine production and recovery from Catharan-thus roseus suspensions with increased product re-moval rates. Biochem Eng J 21:253–258

Zhao Y, Nookandeh A, Schneider B, Sun X, Schmitt B,Stockigt J (1999) Lignans from Torreya jackiiidenti-fied by stopped-flow high-performance liquid chro-matography–nuclear magnetic resonancespectroscopy. J Chromatogr A 837:83–91

Zhao J, Zhu WH, Hu Q (2001) Enhanced catharanthineproduction in catharanthus roseus cell cultures bycombined elicitor treatment in shake flasks andbioreactors. Enzyme Microbiol Technol 28:673–681

Zhou H, Tai YP, Sun CR, Pan YJ (2005) Rapid identifica-tion of Vinca alkaloids by direct-injection electrosprayionisation tandem mass spectrometry and confirmationby high-performance liquid chromatography–massspectrometry. Phytochem Anal 16:328–333

Zimina TM, Smith RM, Myers P (1997) Comparison ofODS-modified silica gels as stationary phases forelectrochromatography in packed capillaries. J Chro-matogr A 758:191–197

234 Phytochem Rev (2007) 6:207–234

123

View publication statsView publication stats