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ORIGINAL
A Convenient Approach to Simultaneous Analysis
of a Pharmaceutical Drug and Its Counter-Ion by CE Using
Dual-Opposite End Injection and Contactless Conductivity
Detection
C. Lopez •R. Nehme •B. Claude •Ph. Morin •
J. P. Max •R. Pena •M. Pelissou •J. P. Ribet
Received: 19 July 2011 / Revised: 22 September 2011 / Accepted: 26 September 2011 / Published online: 10 November 2011
ÓSpringer-Verlag 2011
Abstract Capillary electrophoresis (CE) coupled to a
capacitively coupled contactless conductivity detector
(C
4
D) was used for the determination in a single analysis of
a pharmaceutical drug and its counter-ion. Dual-opposite
end injection (DOI) was used to introduce hydrodynami-
cally the analytes at each end of the capillary. No modifi-
cation of the commercial apparatus is required. After
applying the voltage, the cations and anions migrate from
each end of the capillary in opposite directions toward the
detector placed near the cathode outlet. The electrophoretic
conditions were initially developed with three test drugs
(chlorpheniramine maleate, metoprolol tartrate, clomi-
phene citrate) and then applied to two Vinca alkaloids
(catharanthine sulfate, vinorelbine ditartrate). The 10 mM
histidine–50 mM acetic acid buffer (pH 4.1)–methanol
90:10 (v/v) electrolyte was suitable for the analysis of these
high or medium mobile anions by CE–C
4
D due to its low
conductivity background and high buffer capacity. Finally,
the CE procedure developed was successfully validated for
catharanthine sulfate. The method developed herein is fast
(\10 min) and accurate (repeatability on migration
time \0.6% and peak areas \1.3%, n=6).
Keywords Capillary electrophoresis Contactless
conductivity detection Dual-opposite end injection
Counter-ion Drug Vinca alkaloid
Introduction
An estimated 50% of all pharmaceutical molecules used in
medicinal therapy are administered as salts [1]. Inorganic
ions as well as organic acids or bases are frequently used as
counter-ions of ionizable pharmaceutical drugs [2]. If
hydrochloride and sodium remain the preferred counter-ions
for the salt formation of pharmaceutical compounds, the
availability of numerous counter-ions makes the salt-
selection process based on pharmaceutical and ionic con-
siderations rather difficult [1]. Thus, for a basic drug, several
inorganic (hydrochloride, sulfate, phosphate, nitrate, bro-
mide, carbonate) or organic anions (citrate, maleate, mesy-
late, succinate, salicylate, tartrate, gluconate, fumarate) can
be selected as counter-ions [1]. Selecting the correct form of
salt for a drug candidate is important because it affects its
physico-chemical stability and bioavailability.
Therefore, analytical methods (titration, atomic absorp-
tion, ion chromatography and capillary electrophoresis) are
required to confirm the nature of the correct salt form and
to quantify the level of the counter-ion present in each
manufactured batch of drug substances [2]. During the last
decade, capillary electrophoresis (CE) has been successful
for the analysis of small ions [3–8] and the determination
of counter-ion content of a drug substance [9–16]. In
pharmaceutical analysis, we usually deal with small
organic and inorganic anions and small cations or aliphatic
amines. Most of them have no UV absorption (e.g. chlo-
ride, sulfate) and are generally detected by CE-UV using
indirect photometric detection with a rather limited
C. Lopez R. Nehme B. Claude Ph. Morin (&)
Institut de Chimie Organique et Analytique,
Universite
´d’Orle
´ans, CNRS, BP 6759 rue de Chartres,
FR 2708-UMR 6005, 45067 Orle
´ans, France
e-mail: philippe.morin@univ-orleans.fr
J. P. Max R. Pena M. Pelissou J. P. Ribet
De
´partement de Chimie Analytique,
Institut de Recherche Pierre Fabre,
17 avenue Jean Moulin, 81106 Castres, France
123
Chromatographia (2012) 75:25–32
DOI 10.1007/s10337-011-2154-8
sensitivity [17]. The reversed-electroosmotic flow (EOF)
enabled fast co-electroosmotic separation of inorganic
counter-anions having high electrophoretic mobilities by
CE using reverse polarity.
Ten years ago, a capacitively coupled contactless con-
ductivity detector (C
4
D) for CE was introduced [18–21].
This detector is constituted of two cylindrical electrodes
placed around the outer polyimide coating of the capillary
without any direct contact with the electrolytic solution.
A 1-mm detection gap separates the electrodes. When a
high audio or low-oscillation frequency is applied to one of
the electrodes, a capacitive transition occurs between the
first electrode and the background electrolyte (BGE) filling
the capillary. The resulting current crosses the detection
gap and then a second capacitive transition occurs between
the BGE and the second electrode. A positive or negative
signal is produced when the zone of an analyte having a
different conductivity than the BGE’s passes the detection
gap. The main advantage of this detector comes from the
fact that it can be placed anywhere along the capillary, thus
changing the length of migration and the resolution of
separation.
Many publications report the utility of C
4
D for a wide
variety of small inorganic ions [22,23] or organic ions
[24,25]. This detection system is very sensitive and allows
detection of inorganic and organic ions in various samples.
However, it becomes very interesting now to complete
the simultaneous analysis of anions and cations in a sample
using the dual-opposite end injection (DOE) method
[26,27]. This approach is based on the injection of the
sample at both ends of the capillary. During the electro-
phoretic analysis, cations and anions migrate from each end
of the capillary in opposite directions toward the detector
placed near the center of the capillary and are simulta-
neously detected. The electroosmotic flow (EOF) must be
of sufficient magnitude (by selecting the pH) to allow
simultaneous rapid separation of co-EOF migrating inor-
ganic cations along with analysis of high counter-EOF
mobile inorganic anions [28–33].
Recently, the utility of using dual-opposite end injection
and contactless conductivity detection was demonstrated
for the simultaneous analysis of a pharmaceutical drug
(labetalol) and its counter-ion (hydrochloride) [34].
In this study, the DOE–CE–C
4
D method has been first
applied to several pharmaceutical basic drug compounds
(structures given in Fig. 1) having an organic (citrate,
tartrate, maleate) or inorganic (sulfate) counter-ion. The
parameters (pH, ionic strength) of background electrolyte
(BGE) were optimized to obtain moderate EOF allowing a
rapid co-EOF separation of cationic drug and counter-EOF
separation of anionic counter-ion (\10 min).
Finally, this approach was extended to two salts of Vinca
alkaloids (catharanthine sulfate, vinorelbine ditartrate)
involved in the treatment of cancer. Catharanthine and
vindoline are two indole alkaloids present in Catharanthus
roseus which are used as direct precursor of antitumor
molecules [35]. The coupling of these two molecules results
in the formation of binary alkaloids such as vinorelbine
(Fig. 1) recommended in the treatment of breast and
advanced human non-small-cell lung cancer [36–40]. This
CE procedure developed was successfully validated for
catharanthine sulfate.
Experimental
Chemicals Standards
Ultra-pure products were used. Glacial acetic acid (AcOH,
purity C99.99%), L-histidine (L-His, purity C99.5%),
lithium hydroxide (LiOH, purity C99.995%), phosphoric
acid (H
3
PO
4
, purity C99.999%), chlorpheniramine male-
ate, metoprolol tartrate and clomiphene citrate (purity 99%)
were purchased from Sigma–Aldrich (Saint-Quentin Falla-
vier, France). Potassium sulfate (K
2
SO
4
, purity C99%),
tartaric acid (purity 99%), maleic acid (purity 99%) and citric
acid (purity 99%) were purchased from Fluka (St.-Quentin-
Fallavier, France). Methanol (MeOH) was of HPLC
grade and obtained from SDS (Carlo Erba, Val-de-Reuil,
France). Catharanthine sulfate (C
21
H
24
N
2
O
2
1/2(H
2
SO
4
),
Chlopheniramine Metoprolol
(pKa= 9.2) (pKa= 9.6)
Clomiphene
(pKa= 8.7)
Catharanthine Vinorelbine
(pKa= 6.8) (pKa= 7.6; 5.4)
N
H
N
N
N
OH
O
O
OO
O
O
O
N
H
O
N
O
Fig. 1 Chemical structures and properties of investigated drugs (base
form)
26 C. Lopez et al.
123
MW: 385.456 g mol
-1
, purity of 101%, with 1% of water)
and vinorelbine ditartrate (C
45
H
54
N
4
O
8
2(C
4
H
6
O
6
), MW:
1079.106 g.mol
-1
, purity of 99.62%) were synthesized and
purified at the Centre de Recherche Pierre Fabre (Castres,
France). All product bottles were placed in tightly closed
plastic bags to preserve them from aerosol contaminations
and then stored at 4 °C[20]. Bidistilled 18 MXcm water was
purchased from Carlo Erba (Val de Reuil, France). Hydro-
philic polyvinylidenedifluoride (PVDF) Millex-HV Syringe
Filters, pore size 0.45 lm were purchased from Millipore
(Molsheim, France).
Standard Stock Solution Preparation
All solutions were prepared with ultra-pure bidistilled water
and stored at 4 °C when not in use. The background elec-
trolyte (BGE) was a 10 mM histidine–50 mM acetic acid
buffer (pH 4.12)/methanol (90:10 v/v). Its ionic composition
and parameters without methanol part (ionic strength
10.2 mM; conductivity 0.65 mS cm
-1
) were given by
Phoebus software (Analis, Namur, Belgium). BGE was
prepared fresh daily and filtered through a 0.45 lm PVDF
Millex-HV Syringe Filter before use. The BGE solution in
the separation vials was changed every three runs.
Standard solutions of chlorpheniramine maleate
(0.64 mM), metoprolol tartrate (0.36 mM) and clomiphene
citrate (0.42 mM) were prepared in water/MeOH (90:10 v/v).
Stock standard solutions of catharanthine sulfate
(0.65 mM) as well as of vinorelbine ditartrate (0.23 mM)
were prepared by dissolving 10 mg of each alkaloid in
40 mL water/MeOH (90:10 v/v). All working standard
solutions of alkaloids were prepared by dilution of stock
solution in the BGE diluted (1:10) solution-methanol
(90:10 v/v).
Catharanthine sulfate linearity was assessed using six
standard solutions containing catharanthine and sulfate at
concentrations ranging from 15.62 to 130.13 lM for sul-
fate and from 31.23 to 260.26 lM for catharanthine. Each
standard solution was injected three times. To evaluate the
intra-day precision, three standard solutions of catharan-
thine sulfate at 31.23, 93.69, and 145.75 lM for sulfate,
and at 62.46, 187.39, and 291.49 lM for catharanthine
were used. The inter-day precision was assessed by
injecting the same standard solution of catharanthine sul-
fate at 62.46 lM sulfate and 124.92 lM catharanthine over
three successive days.
Electrophoresis Equipment
All experiments were carried out on an HP
3D
CE electro-
phoresis system (Agilent, Waldbronn, Germany) equipped
with an on-capillary C
4
D detector. The Agilent soft-
ware 3D-CE Chemstation (rev A.08.03) was used. The
contactless conductivity detection was performed with a
TraceDec system from Istech (Innovative Sensor Tech-
nologies GmbH, Strasshof, Austria). The C
4
D signal was
acquired with the Tracemon software (Istech, version
0.07a).
Volumetric Equipment Vials and Fluid Handling
Polyethylene olefin snap-caps as well as polypropylene
flasks, containers and CE vials were used. Before use, vials
and pipetting material were rinsed with the corresponding
solution. After use, all volumetric equipment, vials and
snap-caps were rinsed with water (18 MXcm), and then
soaked overnight in water. Sample solutions were prepared
in large volumes to minimize aerosol contamination.
Nitrile (powder free) gloves were used for manipulation to
avoid any contamination from perspiration.
Electrophoresis Conditions
The different experimental conditions used throughout this
study are summarized in Table 1.
CE analyses were performed in uncoated silica capil-
laries (Polymicro Technologies, Phoenix, AZ, USA) of
70 cm total length and 50 lm internal diameter (i.d.). The
C
4
D detector was placed at 18 cm from the cathode. Thus,
the effective length for the detection of cations and anions
was respectively 52 and 18 cm.
New capillaries were initially conditioned by rinsing
with 0.1 M LiOH (15 min), water (5 min), 0.05 M H
3
PO
4
(15 min), water (5 min) and then BGE (20 min). Acidic
H
3
PO
4
is used to remove any adsorbed Li
?
on the capillary
wall and to prevent any possible contamination during
subsequent analyses. Between runs, the capillary was
Table 1 Experimental conditions used all through this study for
simultaneous determination of a drug and its counter-ion by DOE–
CE–C
4
D
Capillary Total length =70 cm; i.d. =50 lm
C
4
D detector: l
cat
=52 cm; l
an
=18 cm
Pre-separation rinse BGE (950 mbar, 4 min)
Dip capillary ends in water
Injection Anode: sample (50 mbar 910 s); i.e. volume =12.3 nL
Anode: BGE (50 mbar 910.2 s); i.e. volume =12.5 nL
Dip capillary ends in water
Cathode: sample (50 mbar 910 s); i.e. volume =12.3 nL
BGE 10 mM His–50 mM AcOH (pH 4.12, I =10.2 mM) /
MeOH (90:10 v/v)
Separation Voltage : ?25 kV
Temperature : 25 °C
Detection C
4
D: frequency medium, voltage 0 dB, gain 100%,
offset 30, filter: frequency 1/3 and cut-off 0.05
A Convenient Approach to Simultaneous Analysis of a Pharmaceutical Drug 27
123
rinsed for 5 min with the BGE. At the end of a workday,
the capillary was rinsed with 0.05 M H
3
PO
4
(10 min)
followed by water (20 min) to ensure good reproducibility.
All rinse cycles were carried out at 3 bar (using an external
pressure pump).
Separations were performed by applying ?25 kV volt-
age. The temperature of the capillary was set to 25 °C.
For the contactless conductivity detector, the following
parameters were selected: frequency medium; voltage 0 dB;
gain 100%; offset 30; filter: frequency 1/3 and cut-off 0.05.
For the dual-opposite injection mode, sample injection
plugs were obtained by applying a pressure of 50 mbar (0.7
psi) for a period of 10 s (i.e. volume =12.3 nL). Between
the two sample injection zones, a plug of BGE was
obtained by applying the same pressure for a longer period
10.2 s (volume =12.5 nL) to avoid any loss of the first
sample injection zone and to obtain reproducible results.
The same sample vial was used for injection at both ends of
the capillary. To remove any retained components on the
outside of the capillary, capillary ends were dipped in
water before each sample injection (Table 1).
The apparent mobility of an anionic counter-ion (l
app
)
was determined as following: l
app
=L
d
L
t
/Vt
m
, where L
d
is the length from the cathode inlet to the detector
(L
d
=18 cm), L
t
is the total length of the capillary
(70 cm), Vthe applied voltage and t
m
the migration time.
Results and Discussion
Optimization of Experimental Conditions—BGE
and Sample Solvent Selection
Generally, the best C
4
D sensitivity is obtained with a BGE
whose co-ion has an electrophoretic mobility very different
from the analyte’s. Moreover, a BGE with co- and counter-
ion of weak mobility will engender a low background noise
and better detection limits [40]. However, CE requires
BGE co-ion with mobility close to the analyte’s mobility in
order to limit the electromigration peak dispersion.
Therefore, amphoteric buffers are generally well suited for
CE–C
4
D analyses [42].
The BGE composition and the nature of sample solvent
were optimized by selecting catharanthine sulfate as mol-
ecule test. Regarding the BGE, we first used an aqueous
buffer (pH 4.12) composed of 10 mM histidine and 50 mM
acetic acid as previously reported in ref. [34].
This BGE has a sufficiently weak ionic strength
(10.2 mM) to ensure low conductivity (0.65 mS cm
-1
),
good buffer capacity (19 mM/pH unit) and reduced EOF
mobility. This background electrolyte allows rapid co-EOF
separation of cationic drugs and counter-EOF separation of
anionic counter-ions. Furthermore, EOF was low enough
(?15.10
-5
cm
2
v
-1
s
-1
) at pH 4.12, so no EOF reversal
was necessary.
If the sample was dissolved in water, clogging of the
capillary was observed few runs after, which is probably
due to a low solubility of catharanthine in water.
Since Vinca alkaloids and other investigated drugs are
hydrophobic (2 \log P\6) and weakly soluble in water
(-3\log S\-6) organic molecules [43], the presence
of methanol in the sample solvent is expected to increase
their solubility. The sample was prepared in a 1:10 diluted
BGE–methanol (90:10 v/v) mixture to have a weaker
conductivity in the injected sample zone.
The aqueous 10 mM histidine–50 mM acetic acid BGE
used above was first tested. However, a continuous shift in
migration times and in peak areas was observed, resulting
in poor repeatability. This may be due to adsorption of
catharanthine to the capillary wall [44,45]. Therefore,
methanol was added to the BGE to improve the solubility
of the analyte and reduce the adsorption of hydrophobic
and cationic alkaloids onto the inner capillary surface via
electrostatic and hydrophobic interactions. As a result,
hydro-organic mixture of 10 mM His–50 mM AcOH buf-
fer (pH 4.12) and methanol (90:10 v/v) was tested as a
separation BGE.
Application of DOE–CE–C
4
D Method to the Analysis
of Different Pharmaceutical Drugs
The DOE–CE–C
4
D method has been applied to three phar-
maceutical basic drugs (chlorpheniramine, metoprolol, clo-
miphene) having maleate (l
app
*19.10
-5
cm
2
V
-1
s
-1
),
tartrate (l
app
*17.10
-5
cm
2
V
-1
s
-1
) and citrate
(l
app
*10.10
-5
cm
2
V
-1
s
-1
) as respective counter-ion.
Their structures are given in Fig. 1.
The dual-opposite end injection of a sample is explained
in Fig. 2. This injection process took place in three steps.
During step 1, the sample was injected hydrodynamically
(50 mbar 910 s) at the anodic end of the capillary. In the
step 2, a larger BGE volume (50 mbar 910.2 s) was
C
4
D
Inlet vial
Outlet vial
cationsanions
EOF
BGE
BGE
sample
sample
Fig. 2 Principle of simultaneous determination of cationic drug and
its anionic counter-ion in CE using dual-opposite end injection (DOE)
28 C. Lopez et al.
123
injected at the anodic side. The step 3 consisted in injecting
the sample at the cathodic end of the capillary (50 mbar 9
10 s). Thus, during the step 3, the BGE plug was expelled
at the anodic side whereas the first sample injection zone
(step 1) was preserved. Injecting a volume of BGE larger
than the sample is essential to obtain reproducible results.
When a separation voltage was applied, cations and
anions will migrate in opposite directions toward the
detector placed almost in the center of capillary (Fig. 2).
The simultaneous determination of one cationic drug
and its anionic counter-ion was successfully achieved by
DOE–CE–C
4
D for the three pharmaceutical drugs studied,
as shown in Fig. 3. Good peak shapes were obtained for
these different drugs and counter-ions.
Then, the DOE–CE–C
4
D method developed was applied
to two Vinca alkaloids (catharanthine, vinorelbine) and their
respective anionic counter-ion, sulfate (l
app
*42.10
-5
cm
2
V
-1
s
-1
) and tartrate (l
app
*17.10
-5
cm
2
V
-1
s
-1
).
Figure 4shows well-resolved separations of 0.22 mM
catharanthine sulfate (a) and 0.08 mM vinorelbine ditar-
trate (b) in less than 10 minutes by DOE–CE–C
4
D
approach. Under these conditions of BGE and analyte
solvent, good baseline stability was observed and satisfying
peak shape was obtained. Moreover, no migration time
shift was observed resulting in good repeatability.
All these results prove that DOE–CE–C
4
D is an easy, fast,
and universal technique to quantify a drug and its counter-ion
in the same CE run. Subsequently, the validation of this
method has been assessed for catharanthine sulfate.
Validation of the DOE–CE–C
4
D Method for the
Simultaneous Analysis of Catharanthine and Sulfate
The calibration curves were determined over the following
concentration ranges, 15.62–130.13 lM for sulfate and
31.23–260.26 lM for catharanthine. Six concentration
maleate
chlorpheniramine
tartrate
metoprolol
citrate
clomifene
a
b
c
Fig. 3 Simultaneous determination of different pharmaceutical drugs
and their counter-ions using C
4
D and performing dual-opposite end
injection (DOE) in capillary electrophoresis. Sample achlorphenir-
amine maleate (0.64 mM), bmetoprolol tartrate (0.36 mM), cclomi-
phene citrate (0.42 mM). Experimental conditions are described in
Table 1
sulfate
catharanthine
tartrate
vinorelbine
a
b
Fig. 4 Analysis by DOE–CE–C
4
Dofacatharanthine sulfate
(0.22 mM), bvinorelbine ditartrate (0.08 mM). Experimental condi-
tions are described in Table 1
A Convenient Approach to Simultaneous Analysis of a Pharmaceutical Drug 29
123
levels were tested, namely 15.62, 26.03, 52.05, 78.08,
91.09 and 130.13 lM for sulfate, and 31.23, 52.05,
104.10, 156.16, 182.18 and 260.26 lM for catharanthine.
Three independent analyses were performed at each
concentration.
The response function was examined by plotting the
corrected-peak areas versus respective concentrations of
sulfate and catharanthine.
The homoscedasticity was checked by applying the
Cochran’s test. The values of the calculated C-parameter
(C
c
) were lower than the theoretical value (C
th(6;2)
=
0.6161) for both catharanthine and sulfate (Table 2) which
proves the homogeneity of the variances all over the cali-
bration range.
The linearity was then assessed by least squares
regression. The determination coefficient (r
2
) was C0.997
for both catharanthine and sulfate. For the F1-test, the
calculated F1-values (F1
c
) were higher than the critical
theoretical value (F1
th(1;16)
=4.49) which indicated that
the slope of the regression line was significant (Table 2).
The lack of fitness (F2-test) was then applied. The calcu-
lated F2-values (F2
c
) were smaller than the theoretical
value (F2
th(4;12)
=3.26) proving that the linear model is
valid (Table 2).
The Student test was subsequently performed to verify if
the y-intercept of the calibration curves is significantly
different from zero. The y-intercept was equal to zero for
both catharanthine and sulfate since calculated t
c
were
lower than the theoretical value t
th
(respectively, t
c
=0.025
and 1.19 \t
th(16)
=2.12). Thus, only one calibration
solution is needed for quantification.
Besides, the limits of quantification (LOQ) were about
102 lgL
-1
for tartrate and 120 lgL
-1
for metoprolol,
93 lgL
-1
for maleate and 175 lgL
-1
for chlophenir-
amine, respectively.
The precision was evaluated in terms of repeatability
and inter-day precision of migration times and of cor-
rected-peak areas for catharanthine and sulfate. It is
expressed as relative standard deviation (RSD%).
To evaluate the intra-day precision of the method, three
standard solutions of catharanthine sulfate at three con-
centration levels (31.23, 93.69, and 145.75 lM for sulfate
and 62.46, 187.39, and 291.49 lM for catharanthine) were
analyzed six times successively. Results are reported in
Table 3. The RSDs obtained were less than 0.9% (n=6)
for migration times and less than 1.3% for corrected-peak
areas for both sulfate and catharanthine.
The inter-day precision was assessed by injecting the
same standard solution of catharanthine sulfate containing
62.46 lM sulfate and 124.92 lM catharanthine over three
successive days. Within the same day, the catharanthine
sulfate was analyzed three times successively under
repeatability conditions. The RSDs for sulfate and catha-
ranthine analysis were less than 0.5% for migration times
(0.48 and 0.21% for sulfate and catharanthine, respec-
tively) and less than 0.5% for corrected-peak areas
Table 2 Linearity of the detector response for catharanthine sulfate
Catharanthine Sulfate
Calibration range (lM) 31.23–260.26 15.62–130.13
Calibration points 6 6
Linear regression
Determination coefficient 0.9991 0.997
Slope 0.3741 0.7344
y-intercept 0.0107 -0.9122
Statistical tests
Cochran’s test (C
c
) 0.306 0.329
Fisher’s test (F1
c
) 17581 5349
Fisher’s test (F2
c
) 3.25 2.69
Comparison of intercept
with zero (Student test) (t
c
) 0.025 1.19
All statistical tests were performed at a level of confidence of 95%
(P=0.05%). F
1th (1; 16)
=4.49; F
2th (4;12)
=3.26; C
th (6;2)
=0.616.
F
1
-test and F
2
-test were realized at N-2 and N-kdegrees of freedom,
respectively, with Nnumber of experiments (18), knumber of cali-
bration points (6). Student test: t
th
(16) =2.12
Table 3 Intra-day precision of migration times and corrected-peak
areas for catharanthine and sulfate analysis
Concentration (lM) Migration time (min) Peak area
Sulfate (n=6)
31.23
Average 2.199 22.445
RSD (%) 0.12 0.75
93.69
Average 2.255 72.428
RSD (%) 0.16 0.48
145.75
Average 2.265 98.534
RSD (%) 0.32 1.26
Catharanthine (n=6)
62.46
Average 9.153 22.552
RSD (%) 0.58 0.51
187.39
Average 8.722 73.0901
RSD (%) 0.86 0.28
291.49
Average 8.823 102.864
RSD (%) 0.57 0.34
RSD (%) corresponded to the relative standard deviation
30 C. Lopez et al.
123
(0.45 and 0.12% for sulfate and catharanthine, respec-
tively). All these results indicate a good accuracy of the
method.
As previously reported [34], these results prove that
dual-opposite end injection in CE–C
4
D allows a fast,
simple, and accurate determination of inorganic counter-
ion content in a pharmaceutical compound.
Conclusion
CE–C
4
D with dual-opposite end injection (DOE) allowed
the determination in a single analysis of a basic drug and its
anionic counter-ion. Its versatility was demonstrated by the
analysis of three test drugs (chlorpheniramine maleate,
metoprolol tartrate, clomiphene citrate) and two Vinca
alkaloids (catharanthine sulfate, vinorelbine ditartrate).
The selected background electrolyte was 10 mM
histidine–50 mM acetic acid buffer (pH 4.12)–methanol
(90:10 v/v).
In these conditions, the analytical method developed was
successfully validated for catharanthine sulfate (repeat-
ability on migration time \0.9% and peak areas \1.3%,
n=6).
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