Liquid Chromatographyof Gramicidin

2001, 53, 17-21

J. A. Orwa / C. Govaerts / E. Roets / A.Van Schepdael / J. Hoogmartens*

Katholieke Universiteit Leuven,Faculteit Farmaceutische Wetenschappen, Laboratorium voor Farmaceutische Chemie en Analyse van
Geneesmiddelen,Van Evenstraat4, 3000 Leuven,Belgium

KeyWords
Column liquid chromatography
Optimization
Validation
Gramicidin

Summary
A simple, selective method is described for separation of more than 10 gramicidin components on Spherisorb ODS B, 5 I~m, 250 x 4.6 mm I. D. column, maintained at 50 ~ The mobile phase comprised methanol-water (71:29) at a flow rate of 1.0 mL min 1. Detection was by
UVat 282 nm. Vahne gramicidins A, B and C were very well separated from the isoleucine gramicidins A, B and C. Four new gramicidin components were also resolved and their structures
determined by liquid chromatography/mass spectrometry The names 10-methionine valine
gramicidin C 4-methionine valine gramicidin A, vahne gramicidin A hydroxypropyl and isoleucine gramicidin A hydroxypropyl were proposed. Robustnessof the liquid chromatography
method was evaluated by performing a full factorial design experiment. The method also
showed good repeatabihly, linearilyand sensitivily.

Introduction
Gramicidin, a peptide antibiotic, was first
isolated from Bacillus brevis in 1939 as a
crude complex together with a second
peptide antibiotic known as tyrocidin.
The complex containing on the average a
mixture of 20% gramicidin and 80% tyrocidin is called tyrothricin [1]. Gramicidin
is the most active component of tyrothricin [2]. It is active against gram-positive
organisms except gram-positive bacilli,
and against certain gram-negative organisms, such as the Neisseria. Gramicidin is
mainly used topically, either alone or in

Chromatographia 2001, 53, January (No. 1/2)

Original
0009-5893/00/02

combination with other antibiotics. Gramicidin is incorporated into lozenges for

local throat infection [1].
Gramicidin was described as a family
of related linear N-formylated pentadecapeptide ethanolamides, which terminate
in a formyl-blocked amino group [3]. It is
subdivided into A, B and C on the basis of
amino acid residues. Gramicidins A, B
and C were first isolated by countercurrent distribution [4] and later by droplet
countercurrent chromatography [5] and
in microgram quantities by preparative
LC [6]. The general structure (Figure 1) is
composed of an alternative sequence of

L- and D-amino acid residues, a formyl

group at the N-terminus and an ethanolamine moiety at the C-terminus. Gramicidin A differs from gramicidin B by having
a tryptophan (Trp) moiety substituted by
a phenylalanine (Phe) moiety. In gramicidin C, a tyrosine (Tyr) moiety substitutes
for a tryptophan moiety. In each of the
gramicidins A, B and C two moieties exist,
forming a pair, that differ only in the amino acid located at the end of the chain having the neutral formyl group on it. If that
amino acid is valine, the compound is
either valine gramicidin A (Val A) or valine gramicidin B (Val B) or valine gramicidin C (Val C). If the amino acid is isoleucine, the compound is isoleucine gramicidin, either A (Ile A) or B (Ile B) or C (Ile
C). In the process of our investigation, the
heterogeneity of these components was
further demonstrated. Four new minor
components were proposed from the results of LC/MS analysis [7]. Gramicidin
was reported to contain about 75% gramicidin A, 8% gramicidin B and 17% gramicidin C [8]. The ratio of the major component valine gramicidin, to the minor component isoleucine gramicidin was found to
be about 85:15. Secogramicidin or desformylgramicidin is the major degradation
product formed by hydrolysis. The ultraviolet absorption spectrum of gramicidin
complex shows maxima at 275, 282 and
290 nm. Gramicidin is neutral, non-polar
and practically insoluble in water [1].
The compendium methods of quantitative analysis for gramicidin in the Ph. Eur.
[9] and USP [10] are currently microbiological. It has been shown that gramicidin
components show quantitative differences

17- 05

$ 03.00/0

9 2001 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

17

1
HCO - - W - -

4
Gly--L-Ala -- X --

10
-- D - L e u - - Z - - Y - - L - T r p - -

L-Ala - -

D-Val--

D-Val - - ]

L-Val

~L-Trp -- D-Leu-- L-Trp-- R

Gramicidin

Val A
Ile A
Val B
Ile B
Val C
Ile C
4-Met Val A*
10-Met Val*
Val A hydroxypropyl*
Ile A hydroxypropyl*

L-Val
L-Ile
L-Val
L-Ile
L-Val
L-Ile
L-Val
L-Val
L-Val
L-Ile

D-Leu
D-Leu
D-Leu
D-Leu
D-Leu
D-Leu
Met
D-Leu
D-Leu
D-Leu

D-Leu
D-Leu
D-Leu
D-Leu
D-Leu
D-Leu
D-Leu
Met
D-Leu
D-Leu

L-Trp
L-Trp
L-Phe
L-Phe
L-Tyr
L-Tyr
L-Trp
L-Trp
L-Trp
L-Trp

NH(CH2)2OH
NH(CH2)2OH
NH(CH2)zOH
NH(CH2)zOH
NH(CH2)zOH
NH(CH2)zOH
NH(CH2)zOH
NH(CH2)zOH
NH(CH2)3OH
NH(CH2)3OH

new

Figure 1. Structures of known and new gramicidins. Ala, alanine; Gly, glycine;Ile, isoleucine;Leu,
leucine;Met, methionine; Phe, phenylalanine;Trp, tryptophan; Tyr, tyrosine; Val, valine.

Series II (Hewlett-Packard, Avondale,

PA, USA) and a Merck Hitachi model L4000 variable UV detector set at 282 nm.
The columns (250 4.6mm I.D.) were:
LC-ABZ C-18, 5 ixm and ABZ + Plus C18, 5 ixm (Supelco, Bellefonte, PA, USA),
YMC-Pack Pro C-18, 51xm and YMCPack ODS-AQ, 5 ixm (YMC, Wilmington,
NC, USA), Inertsil ODS-2, 5 ixm (Alltech,
Avondale, PA, USA), X-Terra RP18,
51xm (Waters, Milford, MA, USA),
Spherisorb ODS B, 5 ixm (Phase Separations, Queensferry, UK), Hypersil BDS
C-18, 5 ixm (Shandon, Runcorn, UK). A
Hypersil ODS, 5 ixm (Shandon) column
was packed in the laboratory. The column
temperature was maintained at 50 ~ in a
water bath controlled by means of a Julabo EM thermostat (Julabo, Seelbach,
Germany).

Chromatography
in their antibacterial activity [8] and therefore reliable microbiological assay can not
be performed if the sample and the standard preparations have different component ratios. Liquid chromatography (LC)
methods are capable of determining these
antibiotics rapidly, selectively and accurately.
Few LC methods for gramicidins appeared in literature. Gramicidins A, B and
C were separated on Zorbax ODS, 5 ixm,
250 2.1 mm I. D. at 60 ~ with a mobile
phase of methanol-0.005M ammonium
sulphate (74:26) at a flow rate of
1 mL min 1 with monitoring at 220 nm.
The elution order was, with increasing lipophilicity, Val C < Ile C < Val A < Ile
A < Val B < Ile B, with a typical lot containing 78% A, 8% B and 14% C [8]. Gradient elution with increasing acetonitrile
concentration, on LiChrosorb RP-8, 5 ixm
column was described for the analysis of
commercial gramicidin samples [11].
ixBondapak Phenyl, 10 ixm, 300 3.9 mm
I.D. was used to separate gramicidins
with a mobile phase of methanol-water
(75:25) at a flow rate of 1.2mLmin 1
with monitoring at 254 nm. Elution sequence is C < A < B. For preparative
chromatography, a larger column was
used [6]. Gramicidin content was determined using Hypersil ODS, 5 ixm, 250
4.2mm I. D. column with a mobile phase
of methanol-tetrahydrofuran-0.1M sodium dihydrogen orthophosphate pH 4.5
(180:20: 50) and 280nm detection [12].
Val C, Val A, Val B, Ile A and Ile B, in
that order, were resolved on poly(styrene18

divinylbenzene) (PSDVB), 1000Zk, 8 ixm,

250 4.6 mm I. D. stationary phase maintained at 65 ~ with a mobile phase of tetrahydrofuran-water (38:62). The flow rate
was 1.0mLmin 1 and UV detection at
222 nm. This method was developed for
the assay of a formulation containing
polymyxin, gramicidin and neomycin [13].
Baseline resolution for the valine and isoleucine variants was never obtained in the
methods described above. In this paper a
simple, selective method is optimised with
separation of more than 10 gramicidin
components.

Experimental
Reagentsand Samples
Methanol was from BDH Laboratory
Supplies (Poole, England). Water was distilled twice from glass apparatus. Commercial gramicidin bulk samples were
from Lundbeck (Copenhagen, Denmark),
Gist-Brocades (Delft, The Netherlands)
and Apothekernes Laboratories (Oslo,
Norway).

LCApparatus
and OperatingConditions
The isocratic LC system consisted of an
L-6200 intelligent pump (Merck-Hitachi,
Darmstadt, Germany), a Merck Hitachi
model 655A-40 autosampler set to inject
101xl, an electronic integrator HP 3396
Chromatographia 2001, 53, January (No. 1/2)

Gramicidin test solution was prepared at

a concentration of 1.0 mg mL 1 in methanol-water (7:3). An accurately weighed
amount was first dissolved in methanol
and then water was added. This solution
(10 ixL) was injected for chromatography.
The mobile phase was mixed in situ from
two bottles containing (A) methanolwater (90:10) and (B) methanol-water
(50:50). The composition of the mobile
phase was adjusted to obtain optimal separation of peaks. Optimal conditions
were determined for the different columns
used. The flow rate was 1.0 mL min 1.

Resultsand Discussion
Optimization
of ChromatographicMethod
An LC method for gramicidin earlier developed in this laboratory utilized PSDVB
stationary phase [13]. Though this method
was an improvement on the existing LC
methods described in literature for gramicidin [6, 8, 11, 12], some minor gramicidin
components were still not resolved.
In an effort to further improve the analysis method for gramicidin we examined
the procedures described in literature [6,
8]. Isocratic systems containing methanol0.005M ammonium sulphate (74:26) [8]
or methanol-water (75:25) [6] were evaluated. It was found that the addition of the
salt did not improve chromatography.
Various organic modifiers were examined.
Original

M e t h a n o l was f o u n d to be superior in
terms of selectivity over isopropanol, acetonitrile a n d t e t r a h y d r o f u r a n . A mobile
phase of m e t h a n o l / w a t e r was used for
further work. In order to optimize the sep a r a t i o n conditions, studies were carried
out using various silica-based stationary
phases. R e s o l u t i o n of the m a j o r gramicidin c o m p o n e n t s , valine a n d isoleucine
gramicidins A, B a n d C was o b t a i n e d with
all the columns examined. However, the
best selectivity was o b t a i n e d with Inertsil
O D S 2 a n d Spherisorb O D S B stationary
phases (Table I). Baseline resolution of
valine a n d isoleucine variants was obtained on these two stationary phases. I n
addition, other m i n o r impurities were also
separated. Spherisorb O D S B c o l u m n was
retained for further use because it gave the
best selectivity a n d c o l u m n efficiency.
The c o n c e n t r a t i o n of the organic modifier in the mobile phase a n d c o l u m n temperature were optimized using D r y L a b
software (LC Resources, W a l n u t Creek,
CA, USA). LC conditions finally chosen
comprised Spherisorb O D S B m a i n t a i n e d
at 50 ~ C with a mobile phase of m e t h a n o l water (71:29) at a flow rate of 1 m L m i n 1.
The L C profile of gramicidin c o m p o n e n t s
u n d e r the optimized conditions is s h o w n
in Figure 2. The assignment of the peaks is
according to the elution order reported in
literature [8, 12]. The identity of the peaks
was confirmed by L C / M S analysis.

Robustness

Table I. Influence of different stationary phases on separation parameters.

Stationary phase
(250 x 4.6 mm I. D., 5 ~tm)

%
Symmetry a
CH3OH

X-Terra RP18
Supelcosil ABZ+Plus
Supelcosil LC ABZ
YMC-Pack ODS-AQ
YMC-Pack Pro C-18
Hypersil ODS
Hypersil BDS
Inertsil ODS-2
Spherisorb ODS B

74
73
72
79
79
75
73
76
72

0.8
0.8
1.2
0.9
0.9
1.1
1.1
0.9
0.9

Original

Cb

Ao

Bd

1.2
1.3
1.3
1.9
1.7
1.5
1.8
2.0
2.0

1.0
1.9
1.6
1.9
1.7
1.9
2.3
2.5
2.8

1.5
2.1
1.8
2.9
2.3
2.4
2.6
2.6
3.6

Theoretical k 'f
platese
660
2030
1710
1860
1290
1790
2810
3510
4250

5.0
5.0
4.7
4.7
4.6
4.6
4.4
5.2
5.2

a Calculated for Val A. b Resolution between Val C and Ile C. ~ Resolution between Val A and Ile A.
d Resolution between Val B and Ile B. e Calculated for Val A. f Capacity factor calculated for Val A.

20-

10

12

~_~1

13
0

-r

10

20
30
Time (min)

40

50

Figure 2. Chromatogram of gramicidin bulk drug. Conditions: Spherisorb ODS B, 5 ~tm,

250 4.6 mm I. D. column maintained at 50 ~ mobile phase, methanol-water (71:29) at a flow rate
of 1.0 mL min 1; UV detection at 282 nm. 1 = unknown; 2 = 10-methionine valine gramicidin C;
3 = unknown; 4 = valine gramicidin C; 5 = 4-methionine valine gramicidin A; 6 = isoleucine gramicidin C; 7 = unknown; 8 = valine gramicidin A; 9 = valine gramicidin A hydroxypropyl; 10 = isoleucine gramicidin A; 11 = isoleucine gramicidin A hydroxypropyl; 12 = valine gramicidin B; 13 = isoleucine gramicidin B.
Table II. Nominal values corresponding to

The robustness of the m e t h o d was evaluated by performing a full factorial design

experiment s u p p o r t e d by S T A T G R A P H I C S Version 6.0 software (Manugistics, Rockville, M D , USA). The influence
of the c o n c e n t r a t i o n of m e t h a n o l in the
mobile phase a n d the c o l u m n t e m p e r a t u r e
was examined by applying a full factorial
design at two levels. This involved 22 = 4
different experimental measurements,
c o m b i n i n g the two parameters e x a m i n e d
at two previously fixed extreme levels of
each parameter. One central level was included in the design a n d so 5 measurem e n t s were performed as well as duplicate
experiments. The values of the design are
given in Table II. The estimated effects of
the two c h r o m a t o g r a p h i c parameters with
their second order interactions o n the selectivity between Val A a n d Ile A, between
Val B a n d Ile B a n d between Val C a n d Ile
C as response variables are presented o n
the standardized pareto charts in Figure 3.

Resolution

Chromatographic variable

1, 0 and + 1 levels.

Low level

1)
Methanol (%)
Column temperature (~

70
45

Central level

High level

(o)

(+ 1)

71
50

72
55

F r o m these experiments, it is observed

t h a t the selectivity between valine a n d isoleucine variants is principally influenced
by small changes in the percentage of
m e t h a n o l in the mobile phase. The selectivity decreases with increase of m e t h a n o l
c o n c e n t r a t i o n in the mobile phase. Colu m n t e m p e r a t u r e has significant effect o n
the selectivity of the m o r e retained peak
pair, Val B a n d Ile B (Figure 3 c). A n increase in c o l u m n t e m p e r a t u r e decreases
the selectivity. However, this is n o t a problem since the selectivity remains more
t h a n sufficient. The interaction between
the organic modifier a n d c o l u m n temperature has less i m p o r t a n t b u t significant ef-

C h r o m a t o g r a p h i a 2001,

53, J a n u a r y (No. 1/2)

fect o n the selectivity between Val C a n d

Ile C (Figure 3 a), while this c h r o m a t o graphic interaction has n o significant influence o n the other selectivities examined. There was sufficient separation under all the conditions examined. Response
surface plots were constructed for Val C
a n d Ile C (Figure 4a), for Val A a n d Ile A
(Figure 4 b) a n d for Val B a n d Ile B (Figure 4 c) with capacity factors as a function
of m e t h a n o l c o n c e n t r a t i o n in the mobile
phase a n d c o l u m n temperature. F o r all
the conditions examined there was n o
overlapping, indicating the robustness of
the method.

19

Figure 3. Standardized pareto charts representing the estimated effects of

parameters (A and B) and parameter interaction (AB) on the selectivity
between (a) Val C and Ile C, (b) Val A and Ile A and (c) Val B and Ile B.

Figure 4. Estimated response surface plots for (a) Val C (lower plane) and
Ile C (upper plane); (b) Val A (lower plane) and Ile A (upper plane); (c)
Val B (lower plane) and Ile B (upper plane); constructed with the capacity
factors as a function of concentration of methanol in the mobile phase
and column temperature.

TaMe III. Monoisotopic mass of gramicidin components determined by mass spectrometry (MS) in
both the positive (+ve) and negative ( v e ) ion mode.
Gramicidin

Molecular
formula

Monoisotopic
mass

MS
+ve mode*

MS
~ e mode

Val A
IleA
Val B
Ile B
Val C
Ile C
4-Met Val A
10-Met Val C
Val A hydroxypropyl
Ile A hydroxypropyl

C99H140N20017
C100H142N20017
C97H139N19017
C98H141N19017
C97H139N19018
C98H141N19018
C9sH 138N20017S
C96H137N19018S
ClooH142N20017
ClolH144N20017

1881
1895
1842
1856
1858
1872
1899
1876
1895
1909

1904
1918
1865
1879
1881
1895
1922
1899
1918
1932

1880
1894
1841
1855
1857
1871
1898
1875
1894
1808

The difference in mass between the positive and the negative modes of the gramicidin components
is due to sodium adduct formation noticed in the positive ion mode.

Quantitative Aspectsof LCMethod

The precision of the method was assessed
using six replicate injections of a 1.0mg
m L 1 solution of gramicidin. The relative
standard deviation (RSD) of the peak
area of the main component (Val A) was
0.7%. The calibration curve obtained by
replicate analysis (n = 3) of a series of analyte concentrations corresponding to 0.1,
0.25, 0.5, 0.75, 1.0, 1.25 and 1 . 5 m g m L 1
was subjected to linear regression analysis: y = 0 . 1 4 + 2 3 . 9 3 x , where y = V a l A
peak area 10 -6, x = concentration inmg m L 1; correlation coefficient r =
0.9983, standard error of estimate Sy,x =
0.69. The limit of quantitation (LOQ) was
0.09% (n = 6; R S D = 14%). The limit of
detection (LOD) with a signal to noise ratio of 3 was 0.03%. LOQ and L O D were

20

determined on the area of the main peak

(65.7% of total gramicidins) contained in
a solution of 1 mg m L 1 total gramicidins
(10 ixg injected mass). Preliminary examination showed that gramicidin is stable in
the solutions used in the study at 25 ~ for
well over 72 h period.

Liquid Chromatography/Mass
Spectrometry(LC/MS)Analysis
The liquid chromatographic system described above was adapted for L C / M S
using a 250 2 m m I. D. column (instead
of 4.6 m m I. D.). Mass spectrometric analysis was performed with a LCQ ion trap
mass spectrometer (Finnigan M A T , San
Jose, CA, U S A ) equipped with an electrospray interface operated in the positive
Chromatographia 2001, 53, January (No. 1/2)

and the negative ion mode. The acquisition and interpretation of the mass spectrometry data are described in more detail
elsewhere [7]. The structures of gramicidin
components were elucidated by comparison of the fragmentation patterns of the
unknown minor components with the
fragmentation patterns of the known gramicidin components. The monoisotopic
mass obtained from L C / M S analysis of
the examined gramicidin components is
presented in Table III. The structures of 4
new minor components in gramicidin
commercial sample were elucidated from
the results of L C / M S analysis. The names,
4-methionine valine gramicidin A (4-Met
Val A), 10-methionine valine gramicidin
C (10-Met Val C), N-deshydroxyethyl-N(3-hydroxypropyl)-Val A (Val A hydroxypropyl) and N-deshydroxyethyl-N-(3-hydroxypropyl)-Ile A (Ile A hydroxypropyl)
were proposed. The numbering is from
the amino acid moiety located at the end
of the chain having a formyl group (Figure 1). 4-Met Val A has the same composition as Val A except that the D-leucine at
position 4 is replaced by methionine. 10Met Val C has the same composition as
Val C except that the D-leucine at position
10 is replaced by methionine. The configuration of the methionine moiety was not
elucidated. Val A hydroxypropyl and Ile
A hydroxypropyl each has a propanolamine moiety at the C-terminus instead of
ethanolamine in Val A and Ile A respectively.
Original

Acknowledgement

TaMe IV. Composition (%) of different gramicidin bulk samples.

Sample

10-Met Val C
Val C
4-MetValA
IleC
ValA
Val A hydroxypropyl
IleA
Ile A hydroxypropyl*
ValB
IleB
Total unknown
Total gramicidins

0.3
9.8
0.8
4.1
65.7
1.0
13.2
< 0.09
3.8
0.6
0.7
100

0.5
12.0
0.9
4.4
63.7
1.2
12.2
< 0.09
3.0
0.4
0.7
99

0.5
11.6
1.0
4.3
58.1
1.4
12.7
< 0.09
2.7
0.4
2.3
95

0.5
16.5
<0.09*
2.0
58.6
0.8
7.4
< 0.09
7.1
0.8
0.3
94

0.4
21.9
<0.09*
2.8
49.8
1.0
6.1
< 0.09
6.1
0.6
0.3
89

0.3
13.3
<0.09*
3.2
52.4
0.9
10.0
< 0.09
5.7
0.9
0.3
87

Detected but not quantified. Limit ofquantitation is 0.09%.

SampleComposition
The composition of different commercial
gramicidin samples was compared. The
sample with the highest total area was taken as corresponding to 100% total gramicidins. The total gramicidins content of
the other samples was calculated against
this sample on the basis of total area ratio.
The composition of the samples was calculated by normalization. The result of each
peak was then multiplied by a factor corresponding to the total gramicidin content
divided by 100. Composition of the samples examined is presented in Table IV.
The composition of the major component,
Val A ranged from 50 to 66% of total gramicidins. The Ph. Eur. m o n o g r a p h for
gramicidin does not refer to gramicidin
impurities, only the identification is performed by T L C resulting in two spots or
two groups of spots for tyrothricin, which
is used as the reference substance [9]. These

Original

two principal spots or groups of spots each

represent the gramicidins and tyrocidins.
Liquid chromatography provides a selective method for complete separation of
gramicidin components, useful for determining the relative contents of the components and for examining the purity.

Conclusion
The isocratic LC method described here
allowed the separation of more than 10
gramicidin components. The structure of
6 components was described previously.
The LC method, coupled to MS, allowed
to elucidate the structures of 4 of the minor components. It was noted that the
choice of the column played an important
role in the quality of the separation. This
simple and robust method shows good selectivity, repeatability and linearity.

Chromatographia 2001, 53, January (No. 1/2)

J.A. Orwa thanks A B O S of the Belgian

Government for financial support.

References
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A.; Roets, E.; Hoogmartens, J. Rapid
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[9] European Pharmacopoeia, 3rd edition,
Council of Europe, Strasbourg, France,
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[10] United States Pharmacopeia 24, United

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Received: Aug 7, 2000
Accepted: Sep 4, 2000

21