Chromatographic Separation and Characterization of Capsaicinoids and Their Analogues

doi:10.4236/pp.2011.23018

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Pharmacology & Pharmacy, 2011, 2, 136-140

doi:10.4236/pp.2011.23018 Published Online July 2011 (http://www.scirp.org/journal/pp)

Chromatographic Separation and

Characterization of Capsaicinoids and Their

Analogues*

Junlian Wang1, Li Zhou2, Lei Wang2, Zhenghong Peng3, Shengze Zhou4, Xiangfeng Zhou5,

Xiaobin Zhang1, Bixian Peng6#

1Department of Materials Science and Engineering, Zhejiang University, Hangzhou, China; 2Chenguang Engineering Technology

Research Center of Natural Pigments, Hebei, China; 3Beijing TianGongLongYuan International Biochemical Company, Beijing,

China; 4Beijing JiuZhouLongLing Environmental Protection Material Company, Beijing, China; 5Institute of Chemistry, Chinese

Academy of Sciences (CAS), Beijing, China; 6Technical Institute of Physics and Chemistry, CAS, Beijing, China.

Email: #[email protected]

Received December 19th, 2010; revised May 4th, 2011; accepted May 15th, 2011.

ABSTRACT

An attempt was made to establish a chromato graphic separation and analysis method for capsaicinoids and their ana-

logues. A series of factors,such as choice of column and eluents, elu ent composition ,flow rate of eluent and operation al

temperature were correlated, countereacted and optimized to attain appropriate separation efficiency and finalize the

experimentally oprational procedures as a whole, permiting a mixture comprising 8 capsaicinoids including analogues

to be separated and Characterized with an allowed deviations and within a period of 36 minutes via 1 test as well.

Keywords: Capsaicinoid, Chromatography, Separation, Characterization

1. Introduction

In our previous paper [1], it has been introduced that

capsaicin had been effectuated to have shown mutagenic,

anti-proliferative and anticarcinogenic properties toward

human prostate gland’s, lung cancer’s and leukemic cells

[2-10] and highlighted that capsaicinoids differ from

each other in pungency depending on the molecular con-

crete structure involved, leading to a conclusion that

capsaicin and dihydrocapsaicin had been ranked to be the

most pungent ones. Combining the research results

originated and come from completely different 2 sectors-

one is the curative effect of capsaicin displayed in phar-

macology with respect to different cancer cells, the other

is pungency structure dependence exploited in bioor-

ganic chemistry-as a whole, it seems to us to have seen

what a bit light of early dawn and we are hopefully

imagined that the potential biologically curative value

and even the possible clinical administration and obser-

vation of capsaicinoid deserve attention and concerns of

medical researchers in deed. In order to bolster such a

kind of systematic research, first of all, it is necessary to

prepare capsaicinoids, firstly the capsaicin and dihydro-

capsaicin, in sufficient high quality and in ample quanti-

ties as to undertake a systematic as well as multifactorial

studies from different angles and points of view. To our

best knowledge there are two principle routes, one is

traditional by which the capsaicin and dihydrocapsaicin

is separated and manufactured from the extracts ex-

tracted from the dry powders of hot peppers whose na-

tive texture contains capsaicin, dihydrocapsaicin, nordi-

hydrocapsaicin, homocapsaicin, homodihydrocapsaicin

etc., the other is artificial with the help of which individ-

ual capsaicinoid, such as capsaicin, dihydrocapsaicin can

be synthesized by reacting vanillylamine and the corre-

sponding acid chloride as outlined in Equation (1) of our

previous article [1]. As far as the traditional route is con-

cerned, it is advantageous and featured by the case of

raw material available for massive production, the safety

as drugs to be administrated to the patients and the plenty

in supply. However anything in the world is twofold. The

traditional route is likewise disadvantageous and charac-

terized by a complexity of the technological production

*Capsaicinoids are referred to all the pungent members of capsaicin

family present in natural hot pepper. Analogues are referred to those

artificial pungent compounds whose molecular structures are similar to

capsaicinoids but unable to be detected in native hot pepper.

Chromatographic Separation and Characterization of Capsaicinoids and Their Analogues 137

line, long term period of recycle and the extremely high

price of the final products. In a word as compared with

the traditional route, the artificial one had displayed a

few distinguished superiorities , such as a comparatively

shortened and reduced preparation and separation pro-

cedures and much lower production cost of the products.

Based on our experience and practice, all routes,

whether they are traditional, artificial even or enzymatic

as well [11], are required to have a liquid chroma-

tographic analytical method able to separate and charac-

terize the possible analogues of capsaicin or dihydrocap-

saicin originated either from the natural raw materials in

case of traditional procedures, or from the sub-standard

chemical reagent raw material (<95% purity , for exam-

ple, isodecyl alcohol with minor impurities of iso-nonyl

and iso-octyl alcohols, <5%) in case of artificial proce-

dures for dihydrocapsaicin-making.

A survey and investigation of literature enables us to

be aware of a fact that neither published papers nor ap-

proved patents can provide a method or a technology by

which a good many (8 - 10) of capsaicinoids or artificial

analogues of capsaicin and dihydrocapsaicin can be sepa-

rated and characterized by 1 run of chromatographic test

within a short period less than 40 minutes.

A required chromatographically analytical method

comprising column, detector, fixed phase, flow phase etc.

is addressed to separate the possible capsaicinoids such

as capsaicin, dihydrocapsaicin, nordihydrocapsaicin, ho-

mocapsaicin and the artificial capsaicin analogues as

listed in Tab le 1, at which is the right purpose of present

study aimed, presented and discussed.

2. Experiment

2.1. Analogues

The chemical name, chemical formula of 8 synthesized

analogues of capsaicin and dihydrocapsaicin in our labo-

ratory according to the procedures outlined in paper [1]

used for chromatographic separation and analysis are

listed in Table 1.

2.2. Analysis

2.2.1. Re a gent

Acetonitrile, chromatographic grade; Glacial acetic acid,

99.5%, analytical grade; Ethanol, analytical grade manu-

factured by Handan Fine Chemical Company, Province

Hebei, China.

2.2.2. Sampl e S ol ution

Approximately 0.016 g of each analogue sample is

weighed precisely and dissolved in ethanol (100 ml),

from which an aliquot (3 ml) was sucked out to form the

analogues mixture stock solution. 20 μl of the said stock

solution, after passing through a filter membrane (0.45 μ

pore diameter), was injected into the column used.

2.2.3 Seeking for Optimum Separation Procedure

Apparatus

Liquid Chromatography Agileng 1200, Chromatographic

Column A.E. Lichyon, C-18, 5 μm, two columns differ

in length, one is 150 mm, the other 250 mm respectively.

Flow phase

1-st flow phase is methanol plus H2O, whose relative

percentage (%) is prepared to be (methanol: H2O) 50:50,

60:40, 65:35, 70:30 and 75:25.

The 2-nd flow phase is acetonitrile: H2O: glacial acetic

acid whose percentage (%) is consisted of 30:70:0.6,

40:60:0.6, 45:55:0.6 and 50:50:0.6 respectively.

A common normalized conditions (column C-18, 250

mm long, column temperature at 30˚C and the constant

flow rate 1.5 ml/min) is used in an effort to seek for the

optimum flow phase’s experimental conditions beneficial

to improve the separation results

3. Results and Discussion

3.1. Optimization of Analytical Conditions

Column

Prolongation of the column length from 150 mm to

250 mm has brought about an extention of 1 run from 42

minutes to 75 minutes, however created no remarkable

improved separation efficiency, in particular for the ana-

logues with much more closer molecular structures (No.

2, 3, 4 and 5 in Table 1). A shorter column (150 mm) is

preferable for use.

Flow phase

A comparative study of a same solution but with dif-

ferent percentage (%) proportions showed that the solu-

tion with the percentage proportion of ethanol: H2O

(75:25) had a better promotion effect with regard to bet-

tering a separation efficiency.

A similar promotion effect is also with the ternary

system of acetonitrile: H2O: glacial acetic acid with per-

centtage proportion (45:55:0.6). In order to save space all

the 9 chromatogram maps for above 9 proportions (%)

are omitted.

Elution rate

An experimental attempt was made to watch in which

degree an deviation from an usual elution rate of 1.5

ml/min can be affected. Neither a deviation change from

1.5 ml/min to a smaller rate, such as 0.85 ml/min, nor to

a longer rate, for example, to 2.0 ml/min could brought

about a bit improvement. A middle rate is the best of 1.5

ml/min.

From the above-mentioned comparative studies it per-

mits to make a fundamental conclusion that an optimum

xperimental conditions for chromatographic separation e

Chromatographic Separation and Characterization of Capsaicinoids and Their Analogues

138

Table 1. The structure, retention time (RT), peak area and percentage content (%) of capsaicinoids and analogues.

No. Structure RTs RTm A

x %

14.34 14.25 198.4 11.24

22.01 21.87 39.8 2.26

12.97 12.91 75.46 4.28

11.20 11.15 734.3 41.60

15.06 14.99 130.8 7.41

38.28 38.50 99.8 5.66

13.67 13.59 237.7 13.47

8 16.75 16.68 249.2 14.12

RTs: retention time for single sample injection; RTm: for mixture sample injection.

of analogues of capsaicin and (or) dihydrocapsaicin can

be recommended as follows: column C-18, 150 × 4.6

mm, 5 μm, flow phase acetonitrile-H2O-glacial acetic

acid in percentage proportion 45.0:54.4:0.6; flow rate of

eluent 1.5 ml/min and a temperature of column 30˚C.

Under the optimized conditions each individual ana-

logues’s solution (20 μl) was injected alone and the spe-

cific retention time for quantitative analysis was found.

The experimentally found retention time for all the 8

analogues was determined and summarized in the figure

note of Figure 1. From Figure 1 it can be seen that the

retention time of each individual analogue obtained by

single analogue injection (No. 1-8) are in very good co-

incidence with that found by mixture analogue ingection

within an allowed deviations and that among the 8 ana-

logues (No. 1-8 in Table 1) 7 analogues are separated

and characterized quite well with an unique fault that

howed an overlapping of the retention time of No. 7with s

Chromatographic Separation and Characterization of Capsaicinoids and Their Analogues 139

Figure 1. Chromatogram map for separation of No. 1-5 and No. 7-8.

Figure 2. Chromatogram map for separation of No. 1-8.

that of No. 6. In order to avoid and overcome this fault a

fine tuning of the acidity of the ternary eluent system

was made and the 8 analogues’ mixture was basically

separated and quantitatively determined without over-

lapping peaks like No. 6 and No. 7 (Figure 2).

The establishment of chromatographic separation and

characterization is of practical significance. Firstly for

analysis of natural capsaicinoids, in principle, applied

140 Chromatographic Separation and Characterization of Capsaicinoids and Their Analogues

with minor modification, the analyzing scope of capsai-

cinoids may be expanded to the minor members of cap-

saicinoid, such as homohomocapsaicin and nornordi-

hydrocapsaicin etc. whose content in native hot pepper

amount totally less than 2% - 3%; Secondly for analysis

of artificial analogues including those pungent com-

pounds with similar or approximate structures to each

other that are not existed in native hot pepper but are able

to be synthesized in chemical laboratory. It is believed

that the recommended chromatographic procedure may

promote the preparative progress of high-qualified

capsaicinoids and their analogues that hold promise of

being candidate drugs for a potential anticarcinogenic

use.

3.2. Summary

1) An optimized Chromatographically operational

conditions for a apsaicinoids’ mixture separation can be

summarized as follows: column C-18, 250 × 4.6 mm. 5u,

flow phase acetonitrile -H2O- glacial acetic acid in per-

centage (V/V) 45.0; 54.4:0.6, flow rate of eluent 1.5

ml/min and a column temperature at 30˚C.

2) 8 capsaicinoids/analogues are able to be separated

and characterized with an allowed deviations and within

a short period of 36 munites via 1 chromatographic ex-

periment.

3) The suggested chromatographic analytical method

holds promise to be used in principle for separating and

characterizing native capsaicinoids as well as artificial

analogues for medical usage.

4. References

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