Sensitive Analysis of Drugs in Complex Samples Using CE-LIF and CE-UV

The use of laser-induced fluorescence or ultraviolet detectors
for sensitive and selective analysis of tobramycin or erythropoietin
in complex samples
Hytham M. Ahmed a,⇑
, Wael B. Ebeid b
a
Pharmaceutical Analysis Department, Faculty of Pharmacy, Damanhour University, Damanhour, Egypt
b
SEDICO Pharmaceuticals, Merck & Co External Partner, 6th of October City, Cairo, Egypt
h i g h l i g h t s
LIF detector is used for tobramycin
analysis in human urine.
Urine samples were injected directly
without pretreatment.
Erythropoietin was analyzed in the
presence of albumin by CE-UV.
EK and discontinuous buffer used to
increase method sensitivity.
g r a p h i c a l a b s t r a c t
Chemical structure of tobramycin (upper) and primary structure of human erythropoietin (lower).
a r t i c l e i n f o
Article history:
Received 22 October 2014
Received in revised form 29 January 2015
Accepted 4 February 2015
Available online 14 February 2015
Keywords:
Laser-induced fluorescence
CZE
MEKC
Urine direct injection
Erythropoietin
Tobramycin
a b s t r a c t
Complex samples analysis is a challenge in pharmaceutical and biopharmaceutical analysis. In this work,
tobramycin (TOB) analysis in human urine samples and recombinant human erythropoietin (rhEPO) ana-
lysis in the presence of similar protein were selected as representative examples of such samples analysis.
Assays of TOB in urine samples are difficult because of poor detectability. Therefore laser induced fluores-
cence detector (LIF) was combined with a separation technique, micellar electrokinetic chromatography
(MEKC), to determine TOB through derivatization with fluorescein isothiocyanate (FITC). Borate was used
as background electrolyte (BGE) with negative-charged mixed micelles as additive. The method was suc-
cessively applied to urine samples. The LOD and LOQ for Tobramycin in urine were 90 and 200 ng/ml
respectively and recovery was 98% (n = 5). All urine samples were analyzed by direct injection without
sample pre-treatment. Another use of hyphenated analytical technique, capillary zone electrophoresis
(CZE) connected to ultraviolet (UV) detector was also used for sensitive analysis of rhEPO at low levels
(2000 IU) in the presence of large amount of human serum albumin (HSA). Analysis of rhEPO was
achieved by the use of the electrokinetic injection (EI) with discontinuous buffers. Phosphate buffer
was used as BGE with metal ions as additive. The proposed method can be used for the estimation of large
number of quality control rhEPO samples in a short period.
Ó 2015 Elsevier B.V. All rights reserved.
Introduction
Pharmaceutical and biopharmaceutical analysis are based on
qualitative and quantitative analysis of traditional and biotech
http://dx.doi.org/10.1016/j.saa.2015.02.025
1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
⇑ Corresponding author.
E-mail address: hmaahmed@yahoo.co.uk (H.M. Ahmed).
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 143 (2015) 12–19
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

drugs. However one of the most important challenges in analysis is
the sensitivity of the analytical methods. This sensitivity is not
needed only for the analysis of low detection substances but also
for low concentrations. Therefore, the use of sensitive hyphenated
analytical techniques, such as capillary electrophoresis techniques
(CE), are increasingly in a wide range of applications. In general,
CE separates the components of a sample on the bases of differences
in their charge-to-size ratio, and then detects the separated compo-
nents using UV or fluorescence based on their properties. However
theses detectors cannot be used for the analysis of low detection
substances. Also they are not sensitive enough for detection and
quantitation of very minute concentrations. Therefore, derivatiza-
tion reactions which give stable derivative are essential in the case
of low detection substances. On the other hand electrokinetic injec-
tion (EK) coupled with discontinuous buffers are used for enhance
sensitivity towards low analyte concentration. In this work, tobra-
mycin (TOB) analysis in human urine samples and recombinant
human erythropoietin (rhEPO) analysis in the presence of similar
protein were selected as representative examples of such samples
analysis. TOB is a member of aminoglycosides antibiotics (Fig. 1).
It exhibits bactericidal activity against a broad spectrum of bacteria
specially Pseudomonas-aeruginosa [1]. However when determina-
tion of the drug was required, particularly in biological fluids, its
detection was complicated because of low detection sensitivity
due to the poor chromophore effects and when chemical derivatiza-
tion was used, poor stability of the determination was found.
The literature showed a mass spectrometric [2], spectrofluori-
metric [3,4], spectrophotometric and colorimetric methods [5–7]
for TOB analysis. But each of these methods is not ideal to efficient-
ly detect TOB at trace level. Regarding to chromatographic analysis
of TOB, it was analyzed by paper chromatography [8] and thin lay-
er chromatography [9]. Gas liquid chromatography [10]. However,
HPLC is the most common method of analysis of TOB [11,12] But
the major drawback was the toxicity of the reagent and slowness
of reaction. Also, the main disadvantages of the reported HPLC
pre- and post-derivatizations were the instability of the derivatives
or complicated procedures [13–18]. Few trials of separation of TOB
by CE are reported [19–22]. However these methods were unlikely
to give low detection sensitivities. Therefore, derivatization was
done with OPA with 3-mercaptopropionic acid (MPA) and then
separation of the derivatives by capillary zone electrophoresis
(CZE) [23,24] or by MEKC [25] then direct UV detection. However,
instability of the produced derivative was a problem.
The other example used in this work is rhEPO (Fig. 2) which is a
glycoprotein consisting of 165 amino acid residues. rhEPO is used
as erythropoiesis-stimulating agents for renal anemia during dialy-
sis, anemia of prematurity, and cancer related anemia worldwide.
rEPO innovator and biosimilar products have been marketed in
the USA, Japan, the EU and Egypt [26]. For clinical use, highly effi-
cient methods are required to analyze recombinant proteins [27].
CE has been established as an effective analytical separation tool
for a wide variety of analytes, ranging from small inorganic ions
to biological macromolecules [28–31]. Separation and detection
of erythropoietin by CE and CE–MS [32–36]. However, rhEPO either
was alone or formulated with polysorbate 80. Albumin is used as
rhEPO stabilizer and both were good separated by CE however
without good sensitivity [37]. A trial to increase sensitivity was
done by immunochromatographic removal of albumin in erythro-
poietin biopharmaceutical formulations for its analysis by CE [38].
However, this method was complex, expensive and time consum-
ing. The European Pharmacopoeia (Ph. Eur.) monograph for Ery-
thropoietin Concentrated Solution [39] describes a CZE method
for identification of rhEPO and separation of its glycoforms. How-
ever, this method has shown poor reproducibility due to inade-
quate capillary conditioning [33,40]. In CE, EK is a highly
controversial sampling technique. It is a simple mode of sample
introduction which is suitable for on-line preconcentration of the
analytes [41]. The main advantage of EK injection is that sensitivity
of the methods can be by several orders of magnitude higher, and
consequently, the limit of detection (LOD) correspondingly lower
than using conventional hydrodynamic (HD) injection [41]. EK
sampling can be exploited primarily for the separation of compo-
nents of low diffusion coefficient, e.g., proteins, where the number
of theoretical plates is in the order of millions [42]. The presence of
salt, problematic to traditional CE methods and overly abundant in
protein samples. In this work, desalting of samples followed by theFig. 1. Chemical Structure of TOB.
Fig. 2. Primary structure of human erythropoietin (mature hormone).
H.M. Ahmed, W.B. Ebeid / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 143 (2015) 12–19 13

use of discontinuous buffer in CE method were done to solve this
problem.
Therefore, the aim of this work was to develop a new CE method
that can be used for quantitative analysis of TOB in human urine
after derivatization with FITC. The stable fluorescent derivatives
was detected by LIF detector through direct urine injection. Also,
this work aimed to develop a sensitive, selective and reproducible
method for the characterization and quantification of rhEPO glyco-
forms in bulk and finished products.
Experimental
CE systems
LIF instrumentation
A model P/ACE 5510 Beckman capillary electrophoresis instru-
ment (Fullerton, CA, USA) equipped with a 3 mW, 488 nm air-
cooled argon-ion laser (Beckman Laser Module 488) was used.
The fluorescence emission was 520 nm filtered by a band pass fil-
ter, and a notch filter was used to attenuate background radiation.
Uncoated fused silica capillaries (75 lm ID Â 363 lm OD, total
length 75 cm and effective length 60 cm) were obtained from
Supelco (Bellefonte, PA, USA) were accommodated in a Beckman
cartridge configured for LIF detection. The capillaries were kept
at constant temperature using a thermostated liquid coolant. All
operations of the P/ACE unit were controlled by a PC-Pentium
75 MHz compatible computer running Beckman Gold Software.
Nitrogen gas cylinder (BOC, Manchester, UK) was essential for
the sample injection and to flush the capillary.
UV instrumentation
A Beckman Coulter P/ACE™ MDQ Capillary Electrophoresis Sys-
tem (Fullerton, CA) was used in this study. The instrument was
equipped with a UV detector module and all measurements were
made at 200 nm. eCAP Amine capillary with an i.d. of 75 lm was
used. The column temperature was controlled at 4 °C.
Chemicals and materials
TOB and FITC, boric acid, TX-100 and SDS were purchased from
sigma–aldrich, UK. Vials of formulated rhEPO (EPO 2000 IU, EPO
4000 IU) were supplied by SEDICO manufacturer, Egypt. Reference
EPO and high purity HSA was obtained from Miles (Diagnostics
Division, Kankakee, IL, USA). All other chemicals were of the high-
est purity analytical grade available from (BDH, UK). eCAP Amine
capillaries and reagents were purchased from Beckman Instru-
ments (Fullerton, CA, USA). The wash and conditioning procedures
used were those recommended by the manufacturer.
Procedures
Analysis of TOB in bulk by CE-LIF after derivatization with FITC
Capillary conditioning. Initially a new capillary was treated with
1 M NaOH for 15 min, followed by water for 10 min, and then run-
ning buffer for 10 min. Between runs, the capillary was flushed
with 0.1 M NaOH for 2 min followed by running buffer for 2 min.
Preparation of buffer solutions. When buffers were employed, the
salts and/or additives (like SDS) in question were weighed and
transferred to a suitable volumetric flask. The salts and/or additives
were dissolved by addition of some double-distilled water (about
80–90% v/v of total volume) before being made up to volume.
The pH of the buffer was then corrected using an appropriate acid
or alkali solution before filtration through a 0.45 lm membrane fil-
ter. Care was taken to ensure that the pH meter was calibrated
twice daily using freshly prepared commercially available standard
buffers at pH 4.0 and 7.0. All buffers were freshly prepared on a
daily basis.
Preparation of TOB solutions. A standard solution containing 1 mg/
ml of TOB was prepared in deionized water. Further dilutions were
made with water to required concentration. From this stock stan-
dard solutions, working standard solutions containing TOB in the
range of (0.25–5 lg/ml) were prepared by dilution with distilled
water.
FITC derivatization procedure. 300 ll aliquoit of aqueous TOB solu-
tion containing (0.25–5 lg/ml) was added to 300 ll FITC
(0.35 mM) in ethanol and 200 ll buffer (5 mM boric acid adjusted
to pH 7.8 with 2 M KOH) in 2 ml reaction vial. The mixture vial was
capped, homogenized, vortexed and allowed to react at 80 °C in an
oven for 20 min. The derivatization mixtures were analyzed with-
out dilution.
Optimization of derivatization reaction. The effect of the pH and the
concentration of the borate buffer were studied over the ranges
6.5–9.5 and 5–100 mM, respectively. The effect of temperature
was investigated by allowing the reaction to precede at different
incubation temperatures ranging from 40 to 85 °C. The stability
of the derivatized TOB was monitored by measuring the peak areas
of the derivative every 10 min up to 90 min.
CE operating parameters for separation of FITC–TOB derivative. The
BGB consisted of Boric acid 2.8 g, sodium borate 2.1 g dissolved
in 100 ml double-distilled water, 1.75 ml v/v TX-100 and 5.25 g
w/v SDS in deionized distilled water in 100 ml volumetric flask.
The pH was adjusted to 7.8 with 3 M NaOH solution using a mag-
netic stirrer and pH meter and the volume was completed with dis-
tilled water. Sample introduction was performed by hydrodynamic
injection at 50 mbar for 1–10 s. Separations were performed at
room temperature (25 °C) using a separation voltage of 10–30 kV
Fig. 3. Representative electropherograms of TOB–FITC and its blank.
14 H.M. Ahmed, W.B. Ebeid / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 143 (2015) 12–19

and on-line detection with the LIF detection system (Ex 488 nm,
Em 520 nm).
MECK-LIF separation of TOB in spiked urine after derivatization with
FITC.
Preparation of TOB spiked urine (TSU) Samples. Human urine sam-
ples were collected from five different volunteers (males and
females). The urine samples were mixed and a representative 2 L
sample was taken for the preparation of the standard solutions.
Polymyxin B was used (0.4 g/2L) as an internal standard to give
concentration of 0.2 mg/ml as standard solution. Urine TOB
(0.1 g) was spiked into 100 ml urine to give concentration 1 mg/
ml then 10 ml was taken from the last solution to a 100 volumetric
flask and made up to the volume with the internal standard urine
solution to give a concentration of 100 lg/ml. Different drug con-
centrations ranging from 0.25 to 5 lg/ml were prepared by serial
dilution of TOB in the internal standard urine solution.
CE procedure for TSU samples. The derivatization of the spiked urine
samples was performed as above. CE parameters were, capillary
length 75 cm with 75 lm ID (66 cm effective length) and BGB con-
sisted of Boric acid 2.8 g, sodium borate 2.1 g, 1.75% TX-100, 5.25 g
SDS in 100 ml volumetric flask and the volume was completed with
deionized water. The pH was adjusted to 7.8 with 3 M NaOH. The
applied voltage was 10 kV and the injection time was 8 s.
CZE-UV Separation of EPO and HAS
Preparation of rhEPO solution. Fresh HSA stock solution was pre-
pared immediately prior to analysis in Milli-Q water at a nominal
concentration of 2.0 mg/ml from high purity HSA. 72 ll ‘‘1 mg/
ml’’ rhEPO BP_Reference were added to 25 ll HSA. Then 1.9 ml dist
water were added. The obtained solution was mixed very will by
rotator for 30 s. For preparation of CE sample, 900 ll distilled water
were pipetted to CE vial. Then rhEPO and Albumine (or EPO fin-
ished product) were added. Finally, the volume was completed to
2 ml with distilled water.
Desalting procedure. EPO vials (1 ml) contains HSA, Sodium citrate,
Sodium chloride and Citric acid with the following amounts 2.5,
5.8, 5.84 and 0.057 mg respectively. The volume was completed
with double distilled water. Due to this large amount of salt, a
desalting procedure was done as follows: centrifugal filtration
Table 1
Intra-day and inter-day precision (n = 5) for TOB solutions injected twice after LIF-
detection of FITC-derivatization.
TOB Concentration (lg/ml) Intra-day
(n = 5)
Inter-day
(n = 5)
RSD% Recovery% RSD% Recovery%
0.2 2.54 100.54 9.23 98.23
2 0.75 101.60 5.99 100.99
5 0.88 99.75 9.12 99.12
Scheme 1. Structure of the amino compounds and their derivatization reaction
scheme with FITC.
B
O
OH
OH O
CH3
CH3
Cn
.
.
+ OH2
OH
OH
CH3
CH3
Cn
.
.
B
OH
OH
OH OH
+
B
O
OH
OH O
CH3
CH3
Cn
.
. OH
OH
CH3
CH3
Cn
.
. .
.
CH3
CH3
Cn B
O
O
O O
CH3
CH3
Cn
.
.
+
B
OH
OH
OH
+ OH
- B
OH
OH
OH OH
2
+ OH22
(B
-
) (L)
(B
-
)(B)
(BL2
-
)
(BL
-
)
(BL
-
)
(L)
(1)
(2)
(3)
Scheme 2. Equilibria between boric acid, borate and diols in water.

device ‘‘Microcon YM-10’’ with membrane (Amicon, Beverly, MA,
USA) was used as a means to concentrate the sample. The centrifu-
gator must be cooled in the refrigerator before its use for at least
1 h. All desalting steps were done with the use of cold distilled
water. By the use of micropipette, 4 filtration beds were condi-
tioned with 300 ll cold distilled water for each at 11,500Âg/
12,500 rpm for 20 min. The filtrate was rejected. Then 200 ll EPO
sample were pipette to the used beds followed by 250 ll cold dis-
tilled water. The centrifugation was done at 2700Âg for 50 min
(repeated twice by using 300 ll cold distilled water each time).
The retenate were collected at 3200Âg for 5 min. All retenated
were collected in a 2 ml volumetric centrifugation vial and the vol-
ume were completed with cold distilled water.
Capillary electrophoresis procedure. Sodium phosphate buffers
(200 mM) pH 4.0 and 9.0 containing 1 mM Nickel Chloride hexahy-
drate. The prepared buffers were filtered before use. The applied
voltage was 10 kV, with reversed polarity, at 20 °C. eCape Amine
capillary was used. Electrokinetic injection with 10 KV with
reversed polarity, for 10 s.
Capillary regeneration. When the capillary shows signs of reduced
performance or when the percent relative standard deviation
(%RSD) of the absolute migration time is greater than 10%. The fol-
lowing procedure should be used to prevent any sample carry-over
from previous separations; several high pressure (20 psi) rinses of
1 N HCl were first done for 5 min, then water for 5 min, followed by
1 N NaOH for 2 min, again water for 5 min, and finally with amine
regenerator solution for 5 min.
Results and discussion
TOB analysis
Optimization of derivatization reaction
Effect of pH and buffer concentration. Borate buffer solutions of dif-
ferent pH values ranging from 6.5 to 9.5 were examined. Maximum
peak areas (which indicates fluorescence intensity) for derivatized
TOB were at a pH range of 7.5 to 7.9. In fact, at borate buffer con-
centrations over 10 mM, the analytical signal strongly decreases,
which can be ascribed to the formation of aminoglycoside–borate
complexes. These complexes have a negative charge and therefore
an electrostatic repulsion can be expected in their reaction with
the label, with the subsequent decrease in the analytical signal.
Based on these results, the pH in the derivatization medium was
adjusted to 7.8 with 10 mM borate buffer.
Effect of reaction time and temperature. The effect of temperature
was investigated by allowing the reaction to precede at different
incubation temperatures ranging from 40 to 85 °C. The rate of reac-
tion of TOB with FTIC was increased as temperature increased up
to 70 °C and after that no increases were observed. However to
ensure rapid and full reaction, 80 °C was chosen as the best reac-
tion temperature. In order to test the stability of the derivatization
which was suggested earlier as crucial, time studies were carried
out. The stability of the derivatized TOB was monitored by measur-
ing the peak areas of the derivative every 10 min up to 90 min. The
results indicate the maximum stability was one hour at room tem-
perature reaction time of 10 min.
Optimization of CE conditions for FITC. Borate buffers were exam-
ined and borate (boric acid from 1.4 g to 2.8 g and sodium tetrabo-
rate 1.05 g–2.1 g) was found to be the best and neomycin was
examined to be an internal standard. It is however known that,
borate can form a complex with aminoglycosides [19,43]. The
interaction of borate ions with TOB was therefore studied. This
was carried out by using different borate buffer concentrations.
The optimum borate buffer concentration for the assay of TOB
was 2.8 g boric acid and 2.1 sodium tetraborate in 100 ml water
and amikacin was used as an internal standard.
Validation of FITC derivatization method
Amikacin sulfate was added (0.4 g/2L) as internal standard to
give concentration of 0.2 mg/ml as standard solution. 0.1 g TOB
Fig. 4. Laser Induced Fluorescence detection of tobramycin FITC derivative in
human urine.
Table 2
Intra-day and inter-day precision and accuracy for TSU after FITC derivatization
method.
Spiked TOB concentration (lg/ml) Intra-day Inter-day
RSD% Recovery% RSD% Recovery%
0.5 0.86 99.30 2.45 98.5
1.0 2.01 99.67 8.30 99.7
2.0 1.77 99.50 2.20 98.4
4.0 4.89 99.20 1.37 99.2
Fig. 5. Sketch diagram for CE system for rhEPO analysis.

was spiked into 100 ml internal standard solution to give concen-
tration of 1.0 mg/ml then 10 ml was taken from the last solution to
a 100 volumetric flask and completed to the volume with the inter-
nal standard solution to give concentrations of 100 lg/ml then dif-
ferent concentrations of TOB in the internal standard solution to
give different concentrations in ranging from 0.25 to 5 lg/ml.
The best CE conditions are shown in Fig. 3.
CE conditions: capillary length 75 cm Â 75 lm ID and BGE:
boric 2.8 g and sodium borate 2.1 g/100 ml water 20 kV injection
time 20 s (Green) blank, (Blue) TOB–FITC (Red) Amikacin with
tobramycin, detection was LIF Ex 488 nm; Em 520 nm.
The linearity of the CE pre-column FITC derivatization method
was examined by using TOB at six different concentrations (injec-
tions in duplicate), ranging from 0.25 to 5 lg/ml. The standard
calibration curve for TOB was linear, and described by the follow-
ing equation:
y ¼ 0:7804 Â À0:0764ðR2
¼ 0:9991 n ¼ 6Þ:
The LOD and LOQ for TOB were calculated from the mean of the
intercept of five calibration curves. The LOD and LOQ were 70 and
160 ng/ml, respectively for LIF-detection.
The precision of assay was evaluated by determining the intra-
day and inter-day %RSD for five replicates (n = 5) at three different
concentrations of TOB solutions (low, medium and high) with LIF-
detection. The results are summarized in Table 1.
Mechanism of TOB–FITC reaction
It is known that, FITC can react with hydroxyl-containing small
molecules [44,45]. However in the case of compounds containing
both hydroxyl and amino groups, the latter can be the only avail-
able functional group for such derivatization reactions [46,47].
Therefore, the five hydroxyl groups of tobramycin are not expected
to participate in these reactions. It is also unlikely to have two FITC
molecules reacting with one TOB molecule because of the large
size of the molecules and the close proximity of the amino groups.
Therefore, it is likely that, only one amino group of the five distinct
primary amino groups of TOB can react with these reagents due to
steric effects (Scheme 1). At the same time, the presence of more
than one TOB peak in the electrogram could be attributed to the
formation of more than one derivatization product due to different
amino sites on TOB structure. Therefore it is suggested that the
same ring reacts at different sites each time to give another pro-
duct or the other peaks for degradation or impurities of TOB. This
observation follows a similar behavior as previously discussed for
these TOB–OPA derivatives when two derivative peaks have been
given [24,48].
Mechanism of borate complexation and binding sites
The use of borate buffer to alter selectivity in CE was first inves-
tigated in 1988 [49]. At higher borate concentration in the aqueous
phase, tri- to pentameric polyanionic species such as [B3O3(OH)5]2À
,
[B4O5(OH)4]2À
and [B5O6(OH)4]2À
are present [50]. These species can
react with the single hydroxyl groups in molecules to form charged
and mobile complexes [51]. Borate complexation induces changes in
the charge to mass ratios of the ligands. Borate is known to induce
the formation of charged and mobile complexes with 1,3-diols six-
membered hexoses [50]. Tetrahydroxyborate ion, rather than boric
acid, is complexed by the polyols [52] and these complex formation
properties were employed for the characterization of polyols using
CZE and MECK [53]. According to the chemical structure of TOB, it
can be considered as polyol due to the presence of five hydroxyl
groups. The complex formation can be described as shown in
Scheme 2.
Where [54] n = 0 or 1, L is polyol ligand and B- represents
tetrahydroxyborate, [B(OH)4 ]À
. In a pH range from 8 to 12, aque-
ous borate solutions contain not only tetrahydroxyborate ions
but also more highly condensed polyanions such as triborate,
[B3O3(OH)5]2À
, tetraborate, [B4O5(OH)4]2À
. The reported data
Fig. 6. Electropherograms of rhEPO in bulk (upper black bold) and in the presence of albumin (lower black thin).

[55] confirmed that: 1 – Polyols can form 1:1 and 1:2 complexes
with borate. 2 – Not only hydroxyl groups on adjuvant carbon
atoms but also those on alternate carbon atoms are involved in
complexation. 3 – An increase in the number of hydroxyl groups
increases the stabilization. So, in case of TOB, the decrease in its
mobility is not only due to the increase of the ionic strength of
the borate buffer but also due to borate-TOB complexation because
TOB acted as a typical polyol in its reaction with borate in BGE .
Determination of TOB in human urine
TOB in a pooled human urine samples (0.25–5 lg/ml) was
determined by CE, using direct injection (without extraction) after
labeling with FITC in the concentration range from 0.25 to 5 lg/ml.
The derivatized sample was injected directly into the CE-LIF sys-
tem for 8 s and separated at ambient temperature using a constant
voltage 10 kV. Endogenous components present in urine were also
shown not to co-migrate with TOB which appeared as the first two
peaks in the electropherogram. The only suggested reason behind
this is an electrostatic repulsion between the anionic TOB deriva-
tives and the TX100/SDS micelles because both the derivative
and the surfactant are negatively charged. The electrostatic repul-
sion can prevent solubilization of TOB derivative in the mixed
micelle thus causing the optimum resolution between TOB and
the indigenous urine peaks and the internal standard peak. There-
fore the parameters in Fig. 4 are the optimum for this method. The
LOD and LOQ for TOB spiked in human urine were estimated prac-
tically using a signal to noise ratio of 3 and 10 as 90 and 200 ng/ml,
respectively.
CE conditions: capillary length 75 cm Â 75 lm ID and BGE ((0.2
boric acid + 1.75% TX100 + 5.25 g SDS/100 ml water in 100 ml
volumetric flask) the pH adjusted to 7.8 with 3 M NaOH, 10 kV,
8 s inj time, detection was LIF Ex 488 nm; Em 520 nm.
The intra- and inter-day precision and accuracy of the assay
were established by calculating the mean concentration of five
replicates (n = 5) at four different concentrations of TOB spiked
urine (TSU) samples (0.5, 1.0, 2.0 and 4.0 lg/ml) (Table 2).
The recovery of TOB was determined by comparing replicate
(n = 5) peak area ratios of urine spiked with known amounts of
the used drug (0.5, 1.0, 2.0 and 4.0 lg/ml) vs. peak area ratios of
the same concentrations calculated from the resultant regression
line. Five replicate analyses were carried out at each concentration.
The precision, accuracy and recovery data obtained from TSU are
summarized in Table 2.
rhEPO analysis
CE has become a powerful analytical tool for resolving and
quantifying complex protein mixtures as well as different forms
Minutes
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
AU
-0.002
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
AU
-0.002
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
UV - 200nm
UV.10003 9-23-2010 12-17-33 PM.dat
Name
Migration Time
Area
UV - 200nm
uv.10003 9-23-2010 11-53-09 am.dat
Fig. 7. Electropherograms showing the difference of hydrodynamic (blue) and electrokinetic (red) injections of the same conc of rhEPO with the same duration. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of the same protein [56]. The main problem associated with pro-
tein separation is protein adsorption onto the capillary wall [57].
Therefore, eCAP Amine capillaries were used to prevent protein
adsorption. On the other hand, the high amount of salt which is
available in EPO vials, causes problems in CE analysis due to
immoderate Joule heating and electrodispersion. Therefore, desalt-
ing of proteins were done followed by CE with a discontinuous buf-
fer of different pH. The discontinuous buffer was composed of an
phosphate buffer pH 4 and 9. When these acidic and basic buffers
were placed at the cathode and anode, respectively, a sharp pH
junction was formed at the buffer boundary within the capillary.
Proteins were trapped at the junction, resulting in the observed
enrichment.
HSA is generally added to rhEPO formulations as a protein sta-
bilizer. Separation of the two proteins by CZE may be problematic
not only because of their similarity but also due to the presence of
large amounts of HAS. Metal ions are well known to selectively
interact with proteins and modify their electrophoretic mobility
CE separations [58]. The optimum conditions were sodium phos-
phate buffers (200 mM) pH 4.0 and 9.0 containing 1 mM Nickel
Chloride hexahydrate as shown in Fig. 5. The prepared buffers were
filtered before use. The applied voltage was 10 kV, with reversed
polarity, at 20 °C. eCape Amine capillary was used. Electrokinetic
injection with 10KV with reversed polarity, for 10 s. Theses condi-
tions for the separation of which allowed resolution of rhEPO into
four glycoforms as shown in Fig. 6.
These conditions also afforded complete separation of HSA and
rhEPO without affecting the glycoform resolution pattern (Fig. 6).
Typically, the migration time for the main rhEPO glycoform and
HAS peaks were found to be 13 and 14 min respectively. The differ-
ence between HD and EK injections of EPO can be shown in Fig. 7.
Conclusion
A MEKC-LIF using a pre-CE derivatization with FITC has been
used for the determination of TOB. A mixed micelle system com-
posed of SDS and TX-100 as buffer additives improved resolution,
selectivity and sensitivity of the method. The method was applied
successfully for analysis of TOB in human urine samples by direct
injection without sample pre-treatment procedures. The validation
results of this method indicate that it is accurate, precise and sen-
sitive enough to be used for the analysis of TOB in urine samples.
On the other hand, CZE-UV using nickel as metal ion additive to
the background electrolyte were used to overcome difficulties in
assessing protein drug products due to the similarity of their phy-
sicochemical properties particularly in the presence of large
amounts of excipients which interfere with the detection and
separation of the active ingredient leading to complete separation
of the two proteins. Moreover, electrokinetic injection along with
discontinuous buffer was used to increase the method sensitivity.
The method was found to be useful for quantitative estimation of
rhEPO in both bulk and final drug preparations.
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