Journal of Chromatography A, 967 (2002) 1–19

www.elsevier.com / locate / chroma

Review

Analysis of the Cinchona alkaloids by high-performance liquid

chromatography and other separation techniques
David V. McCalley*
Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Frenchay, Bristol BS16 1 QY, UK

Abstract

The Cinchona alkaloids, which include the pharmaceuticals quinine and quinidine, continue to have a wide variety of
important uses. A number of different chromatographic procedures have been developed for the qualitative and quantitative
analysis of these compounds in a variety of sample matrices. Reversed-phase HPLC using ODS columns in combination
with acidic mobile phases, and UV detection, is the most widely used method. Nevertheless, precautions need to be taken
due to the strong silanophilic interactions which can occur with these analytes and the column surface, which can lead to
poor peak shape and resolution. Different selectivity may be achieved in HPLC separations by use of alternative stationary
phases, or by varying mobile phase pH. The specificity of detection systems may be improved by use of photodiode array
UV detectors, or especially mass spectrometers. Thin-layer chromatography (TLC) provides a cheap alternative analytical
method, which is especially useful for qualitative analysis. High-performance TLC, gas chromatography, capillary
electrophoresis and capillary electrochromatography are all methods which after some development, could prove useful for
Cinchona alkaloid separations.
 2002 Elsevier Science B.V. All rights reserved.

Keywords: Reviews; Optimisation; Retention mechanisms; Stationary phases, LC; Alkaloids; Cinchona alkaloids

Contents

1. Introduction ............................................................................................................................................................................ 2
2. High-performance liquid chromatography ................................................................................................................................. 3
2.1. Reversed-phase separations ............................................................................................................................................. 3
2.1.1. General .............................................................................................................................................................. 3
2.1.2. Choice of column ................................................................................................................................................ 5
2.1.3. Choice of mobile phase conditions ....................................................................................................................... 7
2.1.4. Use of competitive amines to mask silanol effects ................................................................................................. 9
2.1.5. Effect of sample size ........................................................................................................................................... 10
2.2. Normal-phase separations ................................................................................................................................................ 10
2.3. Detection of alkaloids in HPLC analysis ........................................................................................................................... 13
3. Gas chromatography................................................................................................................................................................ 14
4. Thin-layer chromatography ...................................................................................................................................................... 15
5. Capillary electrophoresis and capillary electrochromatography ................................................................................................... 16

*Corresponding author. Fax: 144-117-3442-904.

E-mail address: david.mccalley@uwe.ac.uk (D.V. McCalley).

0021-9673 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved.
PII: S0021-9673( 01 )01557-6
2 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19

6. Extraction and purification procedures ...................................................................................................................................... 16

7. Conclusions ............................................................................................................................................................................ 17
References .................................................................................................................................................................................. 18

1. Introduction source of this compound. Analytical procedures are

necessary, for example, to assess the quality of
The Cinchona alkaloids are a group of about 35 Cinchona bark, which is harvested usually after 7–
bases occurring in the bark of Cinchona and Remijia 12 years of growth [2]. Despite the average values
species which are indigenous to the Andes but have quoted above, the alkaloid content of the bark is
been cultivated commercially in South and Central subject to considerable variations, occasionally it can
America, India, Sri Lanka, and the East Indies [1,2]. be as high as 18–25% [3,14] but in other cases,
The principal Cinchona alkaloids are the quinoline much less than the typical levels, giving barks of
alkaloids quinine, quinidine, cinchonine and cin- little commercial value. The alkaloid profile of the
chonidine. The ‘‘average’’ bark contains 7–12% total bark as well as the total alkaloid content affects its
alkaloids, of which quinine accounts for 70–90%, usefulness. More recently, attempts have been made
cinchonidine 1–3%, and quinidine up to 1% [3]. to produce Cinchona alkaloids through cell cultures.
Bark preparations have been used for the treatment This work has been driven by the long lead time
of malaria for centuries, and the history of the early between planting and harvesting of trees and the
uses of quinine and other Cinchona alkaloids, to- rather unpredictable plant to plant variation in al-
gether with their isolation and structural determi- kaloid yield [3]. A considerable number of publi-
nation, have been documented recently [4]. For the cations appeared in the 1980s detailing attempts to
last 50 years or so quinine has suffered competition produce the Cinchona alkaloids by culture methods,
from synthetic anti-malarial drugs such as chloro- and there was much interest in the analytical meth-
quine. However, resistance of the parasite Plas- ods to determine alkaloids in them. However, the
modium falciparum to quinine appears to be sig- yields of cultures proved to be rather low. It is
nificantly less than with chloroquine [5–8], thus possible that the production of alkaloids in Cinchona
quinine is still widely used. Other advantages of can be encouraged by genetic modification, which
quinine are its relatively low price and wide availa- may generate further analytical research on such
bility [3]. Quinine and current synthetic antimalarials samples. In general, both biotechnological proce-
may suffer competition from other drugs or by the dures for the production of Cinchona alkaloids and
development of a vaccine. However, there are also subsequent analytical methods have focused on the
many other uses of the Cinchona alkaloids. Quinine quinoline alkaloids due to the established demand for
is used as a bitter flavouring in drinks and in many quinine and quinidine. Nevertheless, some methods
foodstuffs. Paracetamol tablets commonly contain for analysis of the indole alkaloids have been
quinine as a febrifuge that also imparts an unpleasant published. These contain a five-membered rather
bitter taste discouraging inappropriate use. Quini- than a six-membered heterocycle fused to the ben-
dine, which can be prepared from quinine, is used as zene ring [15]. Analytical procedures have also been
a cardiac anti-arrythmic drug and in the treatment of developed for the assessment of quinine or quinidine
atrial fibrillation. About half of the world market for and their metabolites in human biological fluids,
quinine is used for the synthesis of quinidine [3]. An since these drugs tend to have narrow therapeutic
important use of the principal alkaloids is their wide limits, being toxic in high dose but ineffectual if the
application as catalytic reagents in chiral organic dose is too low [16,17]. The most common method
synthesis [9,10], or to produce high-performance of analysis for all samples has been HPLC in the
liquid chromatography (HPLC) stationary phases for reversed-phase (RP) mode, although other HPLC
chiral separations [11–13]. separation mechanisms, principally normal-phase
Because of the expense of synthetic methods to chromatography on silica columns, have also been
obtain quinine, Cinchona bark remains the primary used. A variety of detection systems such as UV,
 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19 3

fluorescence and mass spectrometry (MS) have been retention times, compatibility with aqueous samples
employed. Thus HPLC analysis will be the major and high column efficiencies. The application of RP
topic of this review. Nevertheless, significant ana- chromatography to the separation of the alkaloids is
lytical work has been performed using thin-layer not straightforward, however, due to their basic
chromatography (TLC), and both gas chromatog- nature and thus possible interaction with silanol
raphy (GC) and capillary electrophoresis / capillary groups. Due to steric effects, a considerable number
electrochromatography offer at least a potential of silanol groups (as many as half [19]) can remain
alternative to HPLC procedures. Earlier applications underivatised on bonded RP packings. End-capping
of these separation methods to analysis of Cinchona procedures, where underivatised silanols are further
alkaloids have been reviewed by Baerheim Svendsen reacted with a small silylating agent such as tri-
and Verpoorte [18]. In the present review, alternative methylchlorosilane, can remove the influence of
procedures to HPLC will receive briefer comment in some of the more active silanols. However, the total
line with the extent of their use. number of reacted silanols is largely unaltered by
Many different HPLC procedures have been pub- this procedure [20]. Quinine (see Fig. 1) has two
lished using a variety of stationary and mobile basic nitrogen atoms of pKa approximately 4.3 and
phases. Many of these papers deal with the analysis 8.5, (the quinoline nitrogen having the lower basici-
of Cinchona alkaloids as a group and are orientated ty) and thus the molecule is likely to be at least
towards use of the method in phytochemical studies, partially protonated over the whole pH range of
as described above. Other papers have a more operation of RP columns (pH 2–8). The underiva-
biomedical orientation and are concerned more with tised column silanol groups have an average pKa of
the pharmaceutical analysis of (particularly) quinine 7.1 [19], although this average conceals a range of
and quinidine and separation of the metabolites of acidities, with some silanols likely to have a pKa ,3.
these compounds produced in body fluids. This Thus, ion-exchange interactions can occur between
review will concentrate on the former application, protonated alkaloid and dissociated silanol groups
that is the separation of the alkaloids themselves. over a wide pH range. These, and possibly other
Relatively few papers (especially in the biomedical interactions such as hydrogen bonding can give rise
group) give much attention to the separation mecha- to tailing peaks, poor column efficiency and irre-
nisms involved in the analysis of the alkaloids. Thus, producible retention times for a variety of quite
rather than merely list all papers referring to analysis complex reasons [20]. Tailing is extremely undesir-
of the alkaloids, it is hoped to instead discuss some able when separating complex mixtures of com-
of the more fundamental principles of the analysis pounds due to loss of resolution, and also due to the
and to direct the reader to those methods which are effects it has on quantitative results. For example,
likely to give optimum results or at least offer the integrators may not be able to establish the end of a
possibility of distinct selectivity effects. Finally, tailing peak with sufficient precision or accuracy.
methods for the extraction of Cinchona alkaloids Retention times may be considerably affected by
prior to analysis will be discussed. sample mass (see below). Irreversible adsorption,
either partial or complete, may occur for some basic
compounds. Supposedly similar columns bonded
2. High-performance liquid chromatography with the same ligand may show completely different
silanol effects due to differences in the base silica,
2.1. Reversed-phase separations the choice of which is discussed below. Worst results
seem to be obtained on the older ‘‘classical’’ RP
2.1.1. General which are made from lower purity silica than more
RP separations on bonded silica have been by far recent phases. It is possible that poorer performance
the most widely used for analysis of the Cinchona may be attributed to their high metal content, par-
alkaloids, probably due to their perceived general ticularly with regard to iron and aluminum which can
advantages, including UV transparency and cheap- become incorporated into the silica structure, in-
ness of the typical mobile phases, reproducibility of creasing the acidity of neighbouring silanol groups
4 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19

Fig. 1. Structures of some Cinchona alkaloids.

[21]. However, it is difficult to attribute the success alternatively, phases of low silanol activity can be
or failure of a given phase to a particular feature, chosen. All silica-based columns will show silanol
because phases differ in so many ways, e.g., nature effects to a greater or lesser degree; different workers
of the base silica, surface area, pore size, nature of have used mostly octadecylsilyl (ODS) columns
bonding reaction and ligand, endcapping, etc. It is (C 18 ) but columns with shorter alkyl chains and
possible to mask the effects of silanol groups by use cyanopropyl columns have also been used for al-
of mobile phase additives ([22–24], see below), kaloid analysis. Most workers have used low pH
 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19 5

(2–3) because here the ionisation of silanol groups (A s ,1.4) and high column efficiency (15 000–
(apart from the most acidic) is largely suppressed, 20 000 plates) given by some of most recently
giving rise to better peak shape. However, it is introduced phases. While the peak shape given by a
possible on modern RP columns to obtain reasonable packing has been shown to be very dependent on
peak shapes for the alkaloids at intermediate pH (pH individual features of the solute such as pKa and
7). The relative merits of these methods will be stereochemistry, that of the four major alkaloids and
discussed below. their dihydro derivatives seems to vary relatively
little amongst themselves, probably due to the simi-
2.1.2. Choice of column larities in their structures [25–31]. The first seven
Table 1 lists retention factor, column efficiency phases in Table 1 are ‘‘classical phases’’ which have
(N) and asymmetry factor (A s ) for 24 columns using been available for many years. In general, these give
a pH 3 phosphate buffer modified with acetonitrile very poor results for the analysis of quinine (see
(these are recommended optimum mobile phase above, [32]). Of these older phases, only m-Bon-
conditions—see below. No silanol masking agents dapak C 18 gives reasonable peak shape. LiChrosorb
were used). It can be seen that there is a considerable RP 8 Select B was amongst the first designed
variation in the peak shape for quinine given by especially for the analysis of basic compounds, and
these different phases, ranging from extreme peak can be seen to give improved results for quinine
asymmetry (A s .5.0) and very poor efficiency (500 compared with the ‘‘classical’’ phases (Table 1, Fig.
plates) for Hypersil ODS to good peak asymmetry 2). The rest of the columns listed are considered as

Table 1
Retention factor, column efficiency and asymmetry factor for quinine on phases using acetonitrile–0.0265 M phosphate buffer, pH 3.0
(15:85, v / v)
Column L3I.D. (cm) dp Surface area (m 2 / g) %C k N N(df) As
m-Bondapak C *18 3030.39 10 330 10 4.3 4500 – 1.8
Novapak C *18 1530.39 4 120 7 8.4 4300 – 2.9
Hypersil C *18 2530.46 5 170 10 6.7 500 – .5.0
Spherisorb ODS-1* 2530.46 5 200 7 12.0 2200 – 3.1
Spherisorb ODS-2* 2530.46 5 200 12 3.1 7200 – 2.7
LiChrosorb RP-18* 2530.46 5 300 16 4.3 1280 – 4.0
Nucleosil 5 ODS* 2530.46 5 100 6 2.2 6200 – 2.3
LiChrosorb RP8 Select B* 2530.46 5 500 11.5 8.3 11 000 – 1.4
Inertsil ODS 2530.46 5 350 18.5 7.3 14 200 12 800 1.0
Inertsil ODS-2 2530.46 5 320 15 3.6 16 700 13 800 1.3
Inertsil ODS-3 2530.46 5 450 15 7.0 13 800 11 400 1.4
Inertsil C8-3 2530.46 5 450 9 5.6 13 700 11 800 1.2
Inertsil cyanopropyl-3 2530.46 5 450 14 0.2 8210 6230 1.3
Kromasil C 18 2530.46 5 340 19 5.6 14 200 10 300 1.4
Kromasil C 8 2530.46 5 340 12 5.5 16 300 12 600 1.4
Symmetry C 18 2530.46 5 330 19.5 5.2 11 100 5130 2.3
SymmetryShield RP-18 2530.46 5 340 17.5 2.2 13 500 12 300 1.2
SymmetryShield C 8 2530.46 5 340 15 3.3 14 600 12 600 1.2
Supelco ABZ1 2530.46 5 170 12 1.4 10 500 7780 1.4
Supelco Discovery C 18 2530.46 5 200 13.5 2.4 13 800 11 500 1.3
Supelco Discovery Amide 2530.46 5 200 12 0.8 19 100 16 500 1.2
Supelco Discovery C 8 2530.46 5 200 7.5 2.7 16 800 14 600 1.2
Purospher 2530.46 5 500 18.5 0.9 4200 2200 2.0
Luna C 18 (2) 2530.46 5 410 17.5 4.9 18 500 13 900 1.5
*5Used 0.1 M phosphate buffer. Results would be expected to be slightly worse for these columns using 0.0265 M phosphate buffer. All
columns 5 mm particle size except where stated otherwise.
N5Column efficiency measured at half-height. N(df)5column efficiency measured according to the Dorsey–Foley procedure [29] which
gives a better indication of true column efficiency.
6 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19

silica surface. In general for basic compounds,

shorter alkyl chain C 8 phases have been found to
give better results than C 18 phases on the same silica
[27]. However, at least for quinine using the mobile
phase shown (Table 1), these advantages seem to be
slight (compare Discovery C 18 / C 8 , Kromasil C 18 /
C 8 , Inertsil 3 ODS and C8-3). Table 1 is slightly
misleading in that it contains a high proportion of
‘‘new generation phases’’. There are many classical
phases still in common use (although not listed)
which would be expected to give very poor results
for the analysis of Cinchona alkaloids. Table 1
should be treated with some caution. It is possible
that manufacturers continually improve some named
phases. Results obtained for some phases several
years ago may give an unduly pessimistic view of
the performance of the same phase purchased more
recently.
While Table 1 is a guide to peak shape, it is not an
infallible guide to resolution of the alkaloids, since
this is also affected by their relative retention.
Retention of basic compounds in RP chromatography
Fig. 2. HPLC separation of alkaloids on the LiChrosorb RP-8 is an extreme complex process which is not com-
Select B column. Peaks: 15cinchonine, 25cinchonidine, 35 pletely understood, and a variety of factors are likely
dihydrocinchonine, 45dihydrocinchonidine, 55quinidine, 65
quinine, 75diydroquinidine, 85dihydroquinine, Detection, UV at
to contribute to the resolution of a mixture of such
220 nm. Flow-rate 1 ml min 21 . Eluent, 15% acetonitrile in 0.1 M compounds. The relative magnitude of each of the
phosphate buffer adjusted to pH 3.0 with phosphoric acid before retention processes may also vary from column to
modifier addition (see Ref. [23]). column, affecting the resolution of different com-
pounds. LiChrosorb RP-8 B has been shown to give
baseline resolution of the eight major alkaloids using
‘‘new generation’’ reversed phases which are based the mobile phase conditions shown. However, res-
on pure silica and / or designed especially for basic olution of the four major alkaloids and their dihydro
compound separation; many give some further im- derivatives has not been studied in detail for all of
provement in the peak shape for quinine compared these phases. In addition, resolution of alkaloids
with LiChrosorb RP-8 Select B. For some phases from matrix components in a particular sample must
based on the same silica, incorporation of an embed- be considered, or indeed from minor alkaloids such
ded polar group in the alkyl chain is seen to improve as are present in Cinchona bark [33]. Further work
performance (compare for example the performance on sample preparation techniques, or the use of mass
of Symmetry C 18 with SymmetryShield RP18, con- spectrometric detection in conjunction with HPLC, is
taining an embedded carbamate group. A smaller necessary to allow checks on chromatographic peak
improvement is shown for Discovery Amide, con- purity or allow selective detection of a compound in
taining an embedded amide functionality, compared the presence of a co-eluting impurity. Alternatively,
with Discovery C 18 [27]). It is possible that phases the selectivity of separations may be usefully man-
with an embedded polar group can trap a deactivat- ipulated by change of column or mobile phase
ing layer of water close to the silica surface, thus conditions (see below).
giving some shielding of the silanol groups. Alter- LiChrospher RP-Select B (a spherical version of
natively, there may be some deactivating effect of the LiChrosorb phase) was also used by Nielsen et
hydrogen bonding between the polar group and the al. for the separation of quinine and its two major
 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19 7

metabolites, (3S)-3-hydroxyquinidine and quinidine considerable influence on peak shape of basic com-
oxide in plasma and urine [16]. pounds. In one study, eight ‘‘new generation’’ RP
A number of alternative RP columns were evalu- columns were compared for the analysis of a variety
ated specifically for separation of Cinchona alkaloids of different bases, including quinine, using acetoni-
[34], particularly with a view to developing LC–MS trile, methanol and tetrahydrofuran at both low and
methods (i.e., without use of buffers or tail reducing intermediate pH [28,29]. However, it appeared that
additives incompatible with MS). The columns used at least at pH 3, the peak shape of quinine is not
included a phenyl bonded silica phase, a mixed mode much influenced by the nature of the modifier. The
C 18 -cation-exchange packing, a column based on plate count (measured at half-height) and asymmetry
alumina rather than silica, and a purely polymeric factor of quinine averaged over the eight columns
column. Due to different mobile phase conditions, it studied was 9200 and 1.5 in methanol modified, pH
is difficult to compare results with those above. Also, 3 buffer, 10 000 and 1.4 with THF as modifier and
it was uncertain whether all of the silica-based 12 600 and 1.5 with acetonitrile. Due to the higher
materials chosen were indeed of the type especially plate count obtained, acetonitrile is probably the first
suitable for basic compounds (i.e., based on pure choice modifier at acidic pH; indeed this result was
silica). However, it was possible to conclude at least found for many other pharmaceutical bases studied.
that separations on the polymer column showed Despite the general recommendation of acetonitrile
disappointing efficiency which is a general charac- as modifier at acidic pH, it is possible that for the
teristic of this type of column. Furthermore, the separation of complex mixtures of alkaloids, useful
alumina based column showed poor selectivity of selectivity effects may be obtained by use of the
separation. At the present time, therefore, it would alternative modifiers. Furthermore, despite the gener-
appear that silica-based columns are still the first al recommendation for acetonitrile at acidic pH, use
choice for RP separations of the Cinchona alkaloids. of THF may be advantageous on columns which
Changing the chemical nature of the ligand in have higher inherent activity towards basic com-
bonded silica RP columns from the normal C 18 or C 8 pounds [28,29].
phases may be a useful tool to alter selectivity. A Mobile phase pH is another important considera-
separation at pH 7 was reported on a cyano column tion, both in terms of optimising peak shape and
based on a traditional silica (Spherisorb CN) in the altering the selectivity of the separation. pH 3
RP mode [35] with phosphate buffer, pH 7 modified (generally accepted as the most acidic pH for long-
with mixtures of acetonitrile, methanol and tetrahy- term stability of silica-based columns) generally
drofuran (THF). The order of elution of the major gives better peak shapes for basic compounds than
alkaloids was quinine, quinidine, cinchonidine and pH 7 (accepted as the general high pH stability limit,
cinchonine—this is different from that on C 18 col- although silica-based columns claiming stability at
umns operated at acidic pH (see Fig. 2). It is unclear considerably higher pH are available). At pH 3, most
whether the different selectivity should be attributed silanol groups are probably undissociated even
to the cyano ligand or to the different pH of though alkaloids are protonated, thus there is limited
operation. However, the fact that reasonable peak possibility for ion-exchange effects [28,29].
shapes were obtained even on this impure silica at However, acceptable peak shapes may be obtained
pH 7 is of interest. Recently, cyano columns based on the newer RP columns even at pH 7, and use of
on much purer silicas have become available (e.g., this higher pH may offer useful selectivity effects, as
Inertsil-3 CN [27]). There are indications of consi- has been shown clearly for the tobacco alkaloids,
derable changes in selectivity for general pharma- which elute in a different order at pH 3 and pH 7
ceutical compounds on this new cyano phase, al- [36]. Preliminary results also indicate this is true for
though whether it will be useful for Cinchona Cinchona alkaloid separations [25]. Column ef-
alkaloid separations remains to be studied. ficiency and asymmetry factors have been recorded
for quinine at pH 7 for eight new generation RP
2.1.3. Choice of mobile phase conditions columns using phosphate buffers modified with
The nature of the organic modifier can give a acetonitrile, methanol and THF [28]. With appro-
8 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19

priate modifier and column selection, efficiencies that retention decreased and peak shape improved in
higher than 40 000 plates m 21 with asymmetry the acid range, in accord with the arguments above.
factor ,2 can be obtained. A recent study on the Increased concentrations of ammonium acetate im-
latest columns [27] showed that for a given column, proved peak shape, presumably due to competition
peak shapes at pH 7 for quinine were often compar- for ion-exchange sites. The effect of buffer con-
able with those obtained at pH 3. For example centration was similar to that reported previously
17 000 plates with asymmetry factor 1.6 were ob- with a potassium phosphate buffer [23]. However, it
tained for quinine on a Discovery C 8 column using should be noted in general that overall buffer con-
acetonitrile–phosphate buffer, pH 7 which compares centrations (at least with inorganic buffers) above 0.1
well with the result for acetonitrile–phosphate buf- M may cause problems of precipitation in combina-
fer, pH 3 (16 800 plates, asymmetry factor 1.2—see tion with organic solvents, or may contribute to
Table 1). A further possibility is to use a pH well pump maintenance problems. It can be seen from
above the alkaloid pKa such that the solute is non- Table 1 that an overall buffer concentration of about
protonated even though silanols are dissociated. 0.02 M is entirely adequate to give good peak shapes
Some results at pH 11 have been reported for quinine with many new generation RP columns at acidic pH;
although special phases which are stable at this pH this concentration is also suitable to maintain pH
are required [27]. Further studies on the effect of pH stability [20]. Despite the absence of a formal
on the selectivity and ruggedness of separations are comparison it does appear that using the same
necessary. For example, although pH 3 seems to give column (LiChrospher RP Select B), phosphate buf-
good peak shapes on many columns (Table 1), this fers at low pH give better separations than with
value is close to the second pKa of the alkaloids, volatile buffers such as ammonium acetate [26,34].
especially when considering that the presence of Isocratic analysis appears to be satisfactory for the
organic solvent may alter both pH and analyte pKa . separation of the four major alkaloids and their
Working at a mobile phase pH close to the analyte dihydro derivatives [26]. However, for simultaneous
pKa may mean that small pH changes which occur analysis of the more strongly retained indole, and
between different batches of buffer could cause other minor alkaloids, gradient elution may be
significant change in the degree of protonation, and necessary. A good separation of 19 compounds,
thus retention factors of the alkaloids. Many of the including the major quinoline alkaloids, together
newer RP columns are claimed by their manufactur- with minor compounds and possible biosynthetic
ers to be stable at pH values of 2 or even lower precursors was reported by Giroud et al. [37], and is
which may give more rugged separations than at pH shown in Fig. 3. A Hypersil ODS column together
3. with a mobile phase gradient of increasing acetoni-
No systematic comparisons of different buffer trile concentration in ammonium formate–formic
salts seem to have been carried out specifically for acid buffer was utilised, these buffer constituents
Cinchona alkaloid analysis. In general for basic being compatible with mass spectrometric detection.
compounds, potassium seems to be a better choice of It was reported that the use of ammonium formate
buffer cation than sodium, due to peak shape effects improved the separation of cinchonine and cin-
caused by the relative strength of ion-exchange chonidine compared to ammonium acetate, another
interactions [20]; most work has been carried out commonly used volatile buffer [38,39]. The alkaloids
with potassium phosphate buffers. Addition of were eluted in three distinct groups with the simple
stronger cation exchangers such as barium ions to the indoles serotonin, tryptamine and 5-methoxytryp-
mobile phase may be beneficial. Ammonium acetate tamine first, followed by the quinoline alkaloids and
was used as the buffer salt by one group [34] due to their dihydro derivatives, the terpenoid indoles
its volatility and thus compatibility with mass spec- quinamine, corynantheal, strictosidine and cin-
trometry. pH was adjusted with ammonia or acetic chonamine next, and lastly the cinchophyllines and
acid over a pH range 3.5 to 7.5. Some caution is 2-keto quinolines (cinchonidinone and quinidinone).
necessary in that this buffer has a poor capacity over The ketoquinolines gave broad peaks which was
some of the stated range. However, it was reported attributed to their keto–enol tautomerism.
 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19 9

using a pH 3 buffer containing 0.05 M hexylamine,

modified with acetonitrile (see Fig. 4). In com-
parison, peak shapes on the same column without
addition of the masking agent were very poor (Table
1), and some alkaloids were shown to be irreversibly
adsorbed. This same classical phase was used suc-
cessfully for the analysis of quinine and its major
metabolite, 3-hydroxyquinine in human plasma and
urine using an acetonitrile–phosphate buffer, pH 2.1
containing tetrabutylammonium bromide [40].
In another study using a Novapak C 18 column,
which is again a silica which has been available for
many years, the asymmetry factor of quinidine and
quinidine could be improved from about 3.0 to 1.0
by incorporation of hexylamine in a mobile phase
buffered at pH 3 [23]. It appeared that longer chain
amines such as hexylamine were more efficacious at
silanol masking than shorter-chain compounds such
as triethylamine. This may merely be due to the
greater hydrophobicity of the former and thus conse-
quently greater adsorption of the masking agent on to

Fig. 3. HPLC separation (UV detection, 280 nm) of simple

indoles, quinolines and indole terpenoid alkaloids. Column, m-
Bondapak C 18 . Mobile phase: gradient from 100 mM ammonium
formate–formic acid–acetonitrile, solvent A (88:4:8, v / v) to
solvent B (64:4:32, v / v) in 1 h [37]. 15Serotonin, 25tryptamine,
355-methoxytryptamine,45cinchonine, 55cinchonidine, 65dihy-
drocinchonine, 75quinidine, 85quinine, 95dihydroquinidine,
105dihydroquinine, 115quinamine, 125corynantheal, 135cory-
nantheol, 145dihydrocorynantheal, 155cinchoni(di)none, 165
strictosidine, 175quini(di)none, 185cinchonamine, 195unknown
(dashed line: separate injection of the 2-ketoquinolines 15117).

2.1.4. Use of competitive amines to mask silanol

effects
For clarity, this topic is given a separate section,
although it can be regarded as an extension to
Section 2.1.3.
Successful analysis of the Cinchona alkaloids can
be obtained even on classical RP-HPLC silicas by
incorporation of amines into the mobile phase which
Fig. 4. HPLC separation of alkaloids on the Hypersil 5 mm ODS
compete with the analytes for column silanol sites.
column. Peak identities as in Fig. 1; 95epiquinidine, 105
For example, Hypersil ODS, was used [22] to give a epiquinine. Mobile phase 5.6% acetonitrile in 0.1 M phosphate
complete separation of the four major Cinchona buffer, pH 3.0 containing 0.05 M hexylamine at 1 ml min 21 (see
alkaloids and their corresponding dihydro derivatives Ref. [22]).
10 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19

the column surface. Thus, it is not entirely clear the stationary phase when k is large. It was shown
whether a higher concentration of triethylamine will that as little as 10 mg of injected quinine can cause a
give an equivalent effect to hexylamine. Some work 50% loss of column efficiency using methanol–phos-
on elucidating the efficacy of different masking phate buffer, pH 3 (65:35, v / v) on a phase which
agents is still being carried out [24]. Amines with gave k56.4 for quinine [42], Fig. 5. As a general
chains longer than hexylamine may give solubility recommendation for bases, some loss of efficiency
problems in mobile phases with low organic modifier and resolution may be experienced if the amount
content, which are necessary at acidic pH to achieve injected at acidic pH exceeds 0.5 mg using acidic
reasonable k for protonated alkaloids [25]. The use mobile phases. However, for the specific case of
of silanol masking agents can however, give rise to quinine it appears that under some circumstances,
some difficulties. For some stationary phases, the this figure may be over cautious [42]. The effect of
addition of amines fails to give significant improve- sample load on peak shape at pH 7 appears to be
ment in peak shape. Another problem is that column more complex. Overload seems to take place much
equilibration is slow and some masking agents may less readily (possibly due to the higher number of
be difficult to remove when changing mobile phases, dissociated silanol groups which exist at pH 7
i.e., they may permanently alter stationary phase compared with pH 3, although other factors may be
characteristics. Furthermore such additives contribute involved). Indeed on some more active packings, the
to the complexity of the method, and can affect peak shape of quinine appears to improve somewhat
detector background signals, which may be impor- as sample load is increased from 0.1 to 20 mg.
tant if for example, mass spectrometric detection is Nevertheless, there are still problems on such pack-
used. Finally it is possible that the masking agent can ings at pH 7. The retention time of quinine (and
chemically react with some alkaloids. For example, it presumably other alkaloids) can decrease significant-
was proposed that corynantheal, a precursor in the ly as the sample load increases, as it does at acidic
biosynthetic route of the major alkaloids which pH. This could lead to wrong assignment of peak
contains a free aldehyde group, undergoes Schiff’s identity if retention time is the sole measure used for
base formation with hexylamine [26]. The use of identification. It is important therefore, to match the
modern RP packings based on pure silica in conjunc- concentration of alkaloids in samples and standards.
tion with low pH mobile phases in any case largely The effects of overloading may be difficult to cope
obviates the need for use of these additives. with, for instance when Cinchona bark is analysed
which may contain large variations in the concen-
2.1.5. Effect of sample size tration of the different alkaloids. In case of doubt,
To obtain the best separations of complex mix- samples should be spiked with standard alkaloids as
tures, it is important that the stationary phase is not an aid to peak assignment.
overloaded either in terms of sample volume or
sample mass [41]. The volume or mass of sample 2.2. Normal-phase separations
which produces overload depends on (amongst other
factors) the diameter of the column and the retention Although the great majority of separations of the
factor of the solute. Volume overload effects are Cinchona alkaloids employ RP separations, some
likely to decrease with increasing k since the injected quite reasonable results have been published using
volume becomes small compared with the peak normal-phase (NP) chromatography, mainly using
volume of the analyte (the volume of mobile phase bare silica columns. Indeed, an acceptable separation
which contains the analyte molecules). It is unlikely on silica of the four major alkaloids was published
that sample volumes of 10 ml or less will cause any more than 25 years ago in the early days of HPLC
problem on standard bore HPLC columns, although [43]. The selectivity of the separations may be
much smaller samples may be required for microbore different from that in RP chromatography, offering
columns. Alternatively, mass overload of the station- an alternative for, e.g., resolution of minor alkaloids.
ary phase is likely to increase with k since a greater In addition, peak shapes can be surprisingly good—it
proportion of the injected sample is associated with was recognised as early as 1982 that basic com-
 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19 11

Fig. 5. (a) Plot of N /N0 , (column efficiency divided by maximum column efficiency) (black squares) and A s /A s (min), (asymmetry factor
divided by minimum asymmetry factor) (white squares) against log (sample mass, mg) for quinine on two different ODS columns using
methanol–0.0321 M phosphate buffer, pH 3.0 (30:70, v / v). (b) Plot of N /N0 (black squares) and A s /A s (min) (white squares) against log
(sample mass, mg) for quinine on two different columns using methanol–0.064 M phosphate buffer, pH 7.0 (65:35, v / v) (see Ref. [42]).
12 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19

pounds can give better column efficiencies on light- these comparative studies were performed on less
ly-loaded or bare silica than on conventional re- pure silicas [44] available at the time. The peak
versed-phase materials based on the same silica [44]. shapes obtained for the alkaloids on ‘‘new gene-
The reason for this observation is unclear, although ration’’ reversed phases can be very good, and this
may be partially due to a ‘‘purer retention’’ mecha- apparent advantage may no longer be significant.
nism (mostly cation-exchange on naked silica col- Nevertheless, some separations on classical bare
umns as opposed to cationic / hydrophobic interac- silicas obtained many years ago still stand com-
tions on RP columns) or to reduced overloading parison with more recent separations on RP packings
effects on the larger population of silanols of a naked [45,46], Fig. 6. As with TLC (see below) the mobile
silica phase. It should be recognised however, that phases consist of non-polar solvents modified with

Fig. 6. (a) HPLC chromatogram of alkaloids on Hypersil silica column. Mobile phase: hexane–dichloromethane–methanol–diethylamine
(66:31:2.6:0.4, v / v) at 1 ml min 21 . Peaks: 15quinidinone, 25quinidine, 35cinchonine, 45dihydroquinidine, 55cinchonidine, 65quinine,
75dihydrocinchonidine, 85dihydroquinine. (b) Analysis of minor alkaloids in an extract of Cinchona ledgeriana bark. Column and other
conditions as in (a) (see Ref. [46]).
 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19 13

polar alcohols to adjust retention time and organic tions, it was shown that the relative standard devia-
amines as tail reducing agents. In the first method tion of the retention times of alkaloid standards
[45], chloroform–isopropanol–diethylamine–water injected over a 12 h period was less than 0.5%,
was used as the mobile phase, and in the second which is probably hardly worse than that obtainable
hexane – dichloromethane – methanol – diethylamine even with the most modern reversed phases.
[46]. In both procedures, baseline separation of the One proposed method involved a rather esoteric
four principal alkaloids was reported, and the elution combination of bare silica and ion-exchange columns
order in each case was quinidine, cinchonine, cin- [48]. However, this method appears to show no
chonidine and quinine, whereas the elution order on particular advantage over the single silica column
reversed phases at acidic pH is generally cinchonine, procedure and indeed resulted in a considerably
cinchonidine, quinidine and quinine. Thus, for com- increased analysis time.
plex separations it might be possible to fractionate a Despite the quality of results obtainable using
mixture by for example, normal-phase chromatog- normal-phase chromatography, the method suffers
raphy and chromatograph the different fractions from a number of disadvantages. These include:
further by reversed-phase chromatography to achieve (a) Non-compatibility of mobile phases (largely
a difficult separation. non-aqueous) with injection of aqueous extracts.
It was recommended [46] that to achieve good (Nevertheless, it is relatively easy to extract the
reproducibility in normal-phase separations on silica alkaloids into suitable organic solvents).
the following precautions should be observed. (b) Problems with gradient elution due to slow
(i) Water should not be used as a polar modifier column equilibration times.
because it is difficult to prepare eluents with re- (c) Irreproducible retention times if the above
producible water content—methanol appears to be a precautions are not observed. Note that difficulty in
good alternative polar modifier. control of the water content of the mobile phase may
(ii) The column should be carefully thermostatted. be a major source of irreproducibility.
Retention of the alkaloids (somewhat surprisingly) (d) Environmental costs / impact in the disposal
was shown to increase with increasing temperature, and use of typical NP solvents.
and significant changes in the retention factor of the Simply due to the general dominance of the RP
alkaloids were obtained even over fairly narrow mechanism in all HPLC separations it seems likely
temperature range corresponding to possible fluctua- that normal-phase chromatography will remain a
tions in room temperature. It is possible that as the minority technique for this application.
temperature is raised, polar modifying agents are
desorbed from the surface giving increasing exposure 2.3. Detection of alkaloids in HPLC analysis
of the surface functionality [46,47]. An additional
factor may be that pKa changes of the solute occur Most authors have used UV detection for alkaloid
with temperature, although study of these phenom- analysis. There is no doubt that the scope of UV
ena are complicated by the presence of the organic detection can be considerably improved by the use of
solvent. Note however, that it is also advisable to photodiode array instruments, which are capable of
thermostat the column in RP methods [25], so this generating complete analyte spectra as well as
factor should not be considered a disadvantage of NP conventional HPLC peaks. The originally poor sen-
methods. sitivity of photodiode array detectors compared with
(iii) As with reversed-phase chromatography, the conventional variable wavelength detectors has been
sample mass can influence the retention times. A overcome to a considerable extent by instrument
small continuous decrease in retention was noted as manufacturers. A problem is the generally broad
sample size was increased from 20 ng to 4 mg. Again nature of UV spectra which makes unequivocal
for analyses where retention time is the principal qualitative identification problematic. Another diffi-
means of qualitative analysis, it is important to use culty is the change in the spectra which can occur for
standards which have concentrations reasonably well these ionogenic compounds with pH. Furthermore
matched to those of the samples. Using these precau- the spectra of the pairs quinine / quinidine and cin-
14 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19

chonine / cinchonidine are identical, although these

pairs of compounds are not difficult to separate by
optimised RP-HPLC procedures [26]. Fluorescence
detection gives much higher sensitivity for quinine
and quinidine, but the low fluorescence or absence of
fluorescence of many other alkaloids has limited the
use of this technique. However, in biomedical studies
which involve analysis of quinine and quinidine and
their metabolic products in body fluids, fluorescence
is a popular method [16,17,40]. MS is the method of
choice for the definitive identification of minor
alkaloids, and indeed for confirmation of the identity
of even the major alkaloids when examining low
levels in cell suspension cultures. Giroud et al. [37]
used a thermospray MS interface as well as conven-
tional UV detection at 280 nm to investigate alkaloid
production in Cinchona shoot and compact globular
structure cultures. All the alkaloids gave intense
quasi-molecular ions (M2H)1 but little fragmenta-
tion, an observation made also by previous workers
[38]. This is disadvantageous for structural elucida-
tion of complete unknowns, but when standards are
available, the combination of authentic retention time
and correct molecular ion is nevertheless a powerful
confirmation of peak identity. Some compounds gave
acetonitrile adducts (M2H2ACN)1 . The presence
of most of the standard alkaloids was confirmed in
the cell suspension cultures. HPLC with photodiode Fig. 7. Gas chromatogram of 15cinchonine, 25cinchonidine,
35quinidine, 45quinine. Column 30 m glass capillary coated
array detection was reported as an efficient and
with RSL-903. Temperature, isothermal 280 8C. Carrier gas,
complementary method. The analysis of alkaloids in hydrogen at 5 ml min 21 (see Ref. [50]).
general using LC–MS techniques was later reviewed
by the same group [49] and covered more recent
interfaces such as electrospray and ionspray; how- olution was reported on RSL-903, the most polar
ever, no specific applications of these newer tech- stationary phase investigated, with the major al-
niques to the determination of the Cinchona al- kaloids eluted in the order cinchonine, cinchonidine,
kaloids was reported. quinidine and quinine. By coincidence, this is the
same elution order as obtained usually with RP-
HPLC at acidic pH, although it is virtually certain
3. Gas chromatography that different selectivities would be obtained for
minor alkaloids, due to large differences in the
Verzele et al. [50] reported the separation of the separation mechanism of these techniques. The com-
four major quinoline alkaloids as underivatised com- pounds were injected using a moving-needle injector;
pounds on an etched soft glass capillary as early as the column was a glass tube coated with sodium
1980 (see Fig. 7). Good peak shapes were obtained chloride dendrites prior to stationary phase coating.
on a range of phases from non-polar to polar. A different group reported that GC with mass
However, the resolution of quinine and quinidine spectrometric detection was less successful than
was zero on a non-polar phase, but increased with HPLC–MS [37]; the quinoline isomers and their
the polarity of the stationary phase. Baseline res- dihydro derivatives were not separated, and it ap-
 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19 15

pears that many alkaloids were not eluted. The stituents, and spectra for alkaloids may be non-
method was based on silylation of the compounds definitive (see above). It is also possible in TLC to
rather than gas chromatography of the free com- visualise alkaloids which remain at the origin and
pounds as reported previously [50], followed by have not moved with the solvent system. Alter-
analysis on a thin film non-polar column. It would natively, complete retention of alkaloids when it
appear that the use of GC for analysis of Cinchona occurs in an HPLC column is not so easily recog-
alkaloids warrants further investigation to explore the nised, unless analysis for that particular compound is
apparent differences in the findings of these two being carried out and standards are available.
groups of workers. It would be expected that modern Another advantage of TLC for qualitative analysis is
techniques such as cold on-column injection and the that two dimensional chromatography can be carried
use of highly inert capillary columns constructed of out to achieve further separations of spots poorly
fused-silica and coated with polar phases might be resolved from the first run. Pound and Sears [43]
expected to yield interesting results. A continuing used acetone–water–25% ammonia (80:20:1) and
difficulty with GC is the genuine and also perceived benzene–diethylamine (1:1) for the separation of the
difficulties in the direct injection of aqueous solu- parent alkaloids and the dihydro bases of quinine and
tions. However, aqueous injection with GC is pos- quinidine. Nevertheless, it should be noted that this
sible if precautions are observed [51]. Alternatively, separation is easily carried out by (one dimensional)
alkaloids could be extracted or transferred into HPLC due to the greater efficiency of the latter
organic solvents, obviating the need for aqueous technique. Most published methods have used nor-
injection. The greater general availability of GC–MS mal-phase TLC, using mobile phases containing
systems in comparison with HPLC–MS and the solvents such as chloroform, acetone, and ethyl
greater structural information which may be obtained acetate to which alcohols such as methanol, propanol
from conventional electron impact (EI) spectra are and butanol are added as polarity adjusters, with
both reasons for further research into GC techniques. diethylamine or ammonia used to reduce spot ‘‘tail-
Higher column efficiency of GC is another potential ing’’. The normal-phase HPLC methods, which use
advantage, although this is counteracted by the ease similar solvent systems, were clearly developed
of manipulation of the selectivity of an HPLC using TLC as a guide [53].
separation by adjusting the composition of the Many interesting applications of TLC to the
mobile phase. analysis of the Cinchona alkaloids have been per-
formed. For instance, Mulder-Krieger et al. were
able to show that some indole alkaloids, previously
4. Thin-layer chromatography identified only in the leaves of Cinchona ledgeriana,
also occurred in the stems and roots of living plants
TLC has fallen somewhat out of popularity in [15]. The presence of the indole alkaloids is explic-
recent years. However, until about 20 years ago, it able since they are precursors in the biosynthetic
was probably the most common method for analysis pathway of the quinoline alkaloids. The typical
of the Cinchona alkaloids. TLC methods have been solvent systems described above, together with silica
extensively reviewed [18,52,53]. They still retain a as a stationary phase, were utilised. Mass spec-
number of important general advantages over HPLC trometry was used as a confirmation of peak identity.
such as simultaneous rather than sequential sample A final advantage of TLC is the comprehensive
analysis, and simple and inexpensive equipment. literature data, especially that published by Verpoorte
Perhaps a more significant advantage is the availabil- and co-workers [18,52] which carefully details meth-
ity of specific spray reagents giving definitive ods for separating the minor alkaloids as well as the
colours with the different alkaloids which can act as major alkaloids, in conjunction with suitable spray
a confirmation of qualitative analysis based on reagents. In contrast, the HPLC literature detailing
retardation factors. In contrast, for HPLC the com- the analysis of the minor compounds is considerably
monly used UV absorption methods are relatively more sparse. Thus, TLC in combination with specific
non-selective between alkaloids and matrix con- spray reagents such as ferric chloride may still be the
16 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19

method of choice, at least for qualitative analysis of phoresis rather than CEC; tailing was still evident in
the indole alkaloids [15]. these separations, which may be caused by detrimen-
The main disadvantages of TLC are the lack of tal interaction with silanol groups as in HPLC. To
automation, the semi-quantitative nature of most our knowledge, there have been no specific attempts
analyses, the lower separation efficiency in com- at separation of the Cinchona alkaloids as a group by
parison with HPLC and the less reproducible nature either CE or CEC; these methods have promise, but
of the stationary phase. No doubt many of these it remains to be seen whether difficulties associated
difficulties could be overcome at least partially by with the analysis of basic compounds can be easily
the application of high-performance TLC (HPTLC) overcome.
methods, although this newer technique does not
appear to have been specifically applied to the
Cinchona alkaloids. 6. Extraction and purification procedures

A number of extraction and purification methods

5. Capillary electrophoresis and capillary have been reported, relating almost entirely to sub-
electrochromatography sequent analysis of the extracts by HPLC, which as
noted above, is the dominant final analytical method.
Capillary electrophoresis (CE) is a technique Many different procedures have been applied to the
useful for the separation of charged compounds, extraction of samples connected with phytochemical
giving often higher efficiencies although often lower studies. However, they have rarely been investigated
selectivity than HPLC. Capillary electrochromatog- in a rigorous fashion to determine their precision and
raphy (CEC) is a hybrid technique which can be accuracy. The commercial extraction of major al-
used for charged and uncharged compounds, com- kaloids from Cinchona bark can be carried out by
bining the high efficiency of capillary electrophoresis pulverising and grinding the bark, making it alkaline
with the high selectivity of HPLC. Analytes are with lime and Soxhlet extracting in hot toluene.
transported through the column by the bulk flow of Other alkaloids may be recovered by further treat-
buffer caused by the electroosmotic flow (EOF), and ments [3]. The Bruxelles standard method for pre-
separation may occur due to both electrophoretic and treatment and extraction of alkaloids from Cinchona
chromatographic mechanisms. A difficulty with the bark for analytical purposes [56] was based on these
application of CEC to the analysis of bases such as commercial extraction procedures and was published
the Cinchona alkaloids has been the rather low EOF in 1950 and widely adopted. This method involved
generated by ‘‘new generation’’ RP materials when grinding and sieving the bark, drying at 110 8C (at
used in CEC [54]. Older generation silicas give good which temperature no loss of alkaloids was re-
EOF but continue to give poor peak shapes for bases ported), followed by treatment with alkali and Soxh-
even in CEC. Recently, Lurie et al. [55] have let extraction with benzene. The method was estab-
demonstrated the analysis of quinine by CEC using lished principally for the subsequent determination of
an acidic phosphate buffer modified with acetonitrile quinine and cinchonidine using classical methods
on a traditional HPLC phase, in conjunction with such as titrimetry and gravimetry. About 25 years
hexylamine as a silanol masking agent. This tech- later, Haznagy [57] claimed that there were problems
nique emulates successful approaches with impure involved in methods similar to the Bruxelles ex-
silicas in HPLC. Although some tailing of quinine traction method in that they gave variations in the
was still reported, an efficiency of 10 000 plates was amount of alkaloids extracted. It was suggested that
recorded on a 25 cm column, which is comparable the preliminary use of trichloroacetic acid in metha-
but somewhat inferior to efficiencies obtainable by nol was necessary, possibly to denature cellulose
HPLC on the latest phases (Table 1).This rather low (although this effect was not proved unequivocally)
efficiency was attributable to significant tailing of and improve extraction efficiency. This was followed
quinine. Different selectivity effects were obtained by treatment with base and benzene extraction as
for the same separations using capillary electro- previously. However, this study was not performed
 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19 17

with the aid of HPLC as a final analytical procedure. behaviour without interfering in the chromatography
Staba and Chung [58] adopted the Haznagy pro- of the extracts is difficult. Further study of all the
cedure for the extraction of alkaloids from cell issues is necessary, especially for ‘‘newer’’ sample
suspension cultures although no evaluation of its matrices such as cell suspension cultures.
efficacy was performed. A later study [26] compared Purification methods for extracts have been ap-
the Bruxelles and Haznagy methods for Cinchona plied, especially to cell suspension cultures con-
bark, but did not appear to show any significant taining low levels of alkaloids. In a typical procedure
increase in the quantity of alkaloids extracted using [59], cell samples were homogenised in 0.2 M
the latter procedure. Simple pretreatment of bark sulfuric acid and washed with chloroform to remove
with calcium hydroxide followed by Soxhlet ex- impurities while the alkaloids are presumed to
traction with toluene or methanol was advocated remain in the acid aqueous phase. The aqueous
[26]. Analysis of a bark sample subjected to this extract is made basic (e.g., with ammonium hy-
method of extraction is shown in Fig. 8. The relative droxide) and again extracted with an organic solvent,
standard deviation of the entire analytical procedure this time to remove the alkaloids. Many variations on
including extraction and HPLC analysis of the this theme have been proposed, although few if any
extract was reported as 4%. Recoveries of spiked procedures report the recovery of cell samples spiked
bark samples were close to 100% for this method, with alkaloids. Thus, although these procedures seem
although it was stated that such additions can never eminently reasonable, and are likely to result in
simulate the exact occurrence of alkaloids in the removal of those interferences which do not mimic
bark. Precision and accuracy of the analysis might be the behaviour of the alkaloids, more work is neces-
improved by the use of internal standards [33], sary to assess their quantitative accuracy and repro-
however, finding compounds which are not naturally ducibility.
present in the samples, and yet simulate analyte In biomedical studies, quinine and quinidine can
be extracted from plasma or urine by making the
sample basic with sodium or ammonium hydroxide
and extracting into a solvents such as dichlorome-
thane or diethyl ether [16,60]. Extracts can be
concentrated by evaporation, and for the usual HPLC
procedures, re-constituted in the mobile phase.

7. Conclusions

High-performance liquid chromatography in the

reversed-phase mode using UV detection, is still the
method of choice for the analysis of the Cinchona
alkaloids. Good results may be obtained using ‘‘new
generation’’ phases based on pure silicas, in conjunc-
tion with an acidic phosphate buffer (pH 3) modified
with acetonitrile. Column efficiencies are variable
due to the variety of complex solute–stationary
phase interactions which can take place. However,
many different packings have been evaluated for
their performance, at least with quinine, allowing an
informed choice of phase. Use of higher pH (e.g., pH
Fig. 8. HPLC chromatogram of Cinchona calisaya bark after 7) may give some useful selectivity effects, and
extraction with toluene / alkali. For other conditions and peak raising the temperature above ambient has been
identities, see Fig. 1 [26]. shown to improve peak shape, especially at pH 7
18 D.V. McCalley / J. Chromatogr. A 967 (2002) 1–19

[61]. Lowering the pH to 2.5 or even 2.0 may further work is necessary in this area for phyto-
improve reproducibility of retention times when chemical work. Precise and accurate procedures need
different batches of buffer are used (providing to be developed, especially for samples other than
column stability is adequate). Silica bonded phases Cinchona bark, for which at least some detailed
with ligands other than the usual C 18 may give investigations have been made.
useful selectivity effects. Analysts should be careful
not to overload columns, otherwise loss of efficiency
and resolution for bases may result with as little as References
0.5 mg of compound, if acidic mobile phases are
used. Columns made with alternative RP materials [1] S. Coffey (Ed.), Rodd’s Chemistry of Carbon Compounds,
such as polymeric materials or alumina phases give Vol. 1V, part G, Elsevier, Amsterdam, 1981.
inferior results compared with silica. Normal phase [2] R. Wijnsam, R. Verpoorte, Cell Culture Somatic Cell Genet.
chromatography on silica gives satisfactory results Plants 5 (1988) 335.
with alternative selectivity to RP separations. HPLC [3] C.S. Hunter, in: Y.P. Bajaj (Ed.), Biotechnology in Agricul-
ture and Forestry, Vol. 4, Springer-Verlag, Berlin, 1988,
with mass spectrometric detection offers the best Chapter 3.
possibility of confirmation of peak identity or identi- [4] F. Eiden, Pharm. Unserer Zeit 27 (1998) 257.
fication of minor alkaloids—however, relatively little [5] M.G. Zalis, L. Pang, M.S. Silveira, W.K. Milhous, D.F.
specialist work has been reported in this area, Wirth, Am. J. Trop. Med. Hyg. 58 (1998) 630.
especially with the more modern interfaces. [6] G. Di Perri, P. Olliaro, S. Nardi, R. Deganello, B. Allegranzi,
B. Stefano, S. Vento, E. Concia, Acta Trop. 70 (1998) 25.
Gas chromatography has received very little atten- [7] G.J. Amabeoku, Cent. Afr. J. Med. 37 (1991) 329.
tion for the separation of Cinchona alkaloids in [8] K. Markwalder, C. Hattz, Schweiz. Med. Wochenshr. 128
recent years, due to the dominance of HPLC tech- (1998) 1313.
niques. However, some re-investigation of this tech- [9] J.L. Margitfalvi, E. Talas, E. Tfirst, C.V. Kumar, A. Gergely,
nique might be beneficial, to take advantage of the Appl. Catal. A 191 (2000) 177.
[10] K. Borszesky, T. Burgi, Z. Zhaohui, T. Mallat, A. Baike, J.
recent developments in column inertness and in-
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jection techniques. The greater availability and [11] M. Laemmerhofer, W. Lindner, J. Chromatogr. A 741 (1996)
cheapness of GC–MS systems compared with 33.
HPLC–MS systems, and the greater structural in- [12] V. Piette, M. Laemmerhofer, K. Bischoff, W. Lindner,
formation available from EI spectra generated from Chirality 9 (1997) 157.
[13] S. Schefzick, W. Lindner, K.B. Lipowitz, M. Jalaie, Chirality
GC–MS is an incentive for further work in this area.
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TLC is still a cheap alternative to HPLC, and can [14] E.H. Smit, Pharm. Weekbl. 119 (1984) 159.
be useful for qualitative analysis, particularly for [15] Th. Mulder-Krieger, R. Verpoorte, A. de Water, M. van
minor alkaloids, due to the wealth of information Gessel, B.C. van Oeveren, A. Baerheim-Svendsen, Planta
published on solvent systems and specific spray Med. 46 (1982) 19.
[16] F. Nielsen, K. Kramer-Nielsen, K. Brosen, J. Chromatogr. B
reagents for detection of these compounds. The use
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of high-performance TLC might further improve the [17] L.K. Pershing, M.A. Peat, B.S. Finkle, J. Anal. Toxicol. 6
attractiveness of this technique, although little if any (1982) 153.
work has been published on its specific application to [18] A. Baerheim Svendsen, R. Verpoorte, in: Chromatography of
the Cinchona alkaloids. Alkaloids. Part A: Thin-Layer Chromatography and Part B:
Gas and Liquid Chromatography, Journal of Chromatog-
Capillary electrophoresis and capillary electro-
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chromatography offer some potential for improve- 1983–1984.
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