Accepted Manuscript

The use of laser-induced fluorescence or ultraviolet detectors for sensitive and

selective analysis of tobramycin or erythropoietin in complex samples

Hytham M. Ahmed, Wael B. Ebeid

PII: S1386-1425(15)00177-8
DOI: http://dx.doi.org/10.1016/j.saa.2015.02.025
Reference: SAA 13315

To appear in: Spectrochimica Acta Part A: Molecular and Biomo-

lecular Spectroscopy

Received Date: 22 October 2014

Revised Date: 29 January 2015
Accepted Date: 4 February 2015

Please cite this article as: H.M. Ahmed, W.B. Ebeid, The use of laser-induced fluorescence or ultraviolet detectors
for sensitive and selective analysis of tobramycin or erythropoietin in complex samples, Spectrochimica Acta Part
A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.02.025

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
 1

THE USE OF LASER-INDUCED FLUORESCENCE OR ULTRAVIOLET DETECTORS

FOR SENSITIVE AND SELECTIVE ANALYSIS OF TOBRAMYCIN OR
ERYTHROPOIETIN IN COMPLEX SAMPLES

Hytham M. Ahmed1* and Wael B. Ebeid2

1
Pharmaceutical Analysis Department, Faculty of Pharmacy, Damanhour University of
Pharmacy, Damanhour University, Damanhour, Egypt; 2SEDICO Pharmaceuticals, Merck
& Co External Partner, 6th of October City, Cairo, Egypt
*Corresponding author: [email protected]
Abtract
Complex samples analysis is a challenge in pharmaceutical and biopharmaceutical analysis. In
this work, tobramycin (TOB) analysis in human urin samples and recombinant human
erythropoietin (rhEPO) analysis 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 fluorescence 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 succisively
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 analysed 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.

Keywords: laser-induced fluorescence, CZE , MEKC, urine direct injection, erythropoietin,

tobramycin.

1. Introduction:

Pharmaceutical and biopharmaceutical analysis are based on qualitative and quantitative analysis
of traditional and biotech 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 components using UV or
fluorescence based on their properties. However theses detectors cannot be used for the analysis
 2

of low detection substances. Also they are not sensitive enough for detection and quantitation of
very minute concentrations. Therefore, derivatization reactions which give stable derivative are
essential in the case of low detection substances. On the other hand electrokinetic injection (EK)
coubled with discontinuous buffers are used for enhance sensitivity towards low analyte
concentration. In this work, tobramycin (TOB) analysis in human urin 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 (Figure 1). It exhibits bactericidal activity against a broad spectrum of bacteria
specially Pseudomonas-aeruginosa [1]. However when determination 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 derivatization was used, poor
stability of the determination was found.

Figure 1. Chemical Structure of TOB.

The literature showed a mass spectrometric [2], spectrofluorimetric [3, 4], spectrophotometric
and colorimetric methods [5-7] for TOB analysis. But each of these methods is not ideal to
efficiently detect TOB at trace level. Regarding to chromatographic analysis of TOB, it was
analysed by paper chromatography [8] and thin layer 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 (Figure 2) which is a glycoprotein consisting of
165 amino acid residues. rhEPO is used as erythropoiesis-stimulating agents for renal anemia
during dialysis, 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 efficient 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
 3

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 erythropoietin biopharmaceutical formulations
for its analysis by CE [38]. However, this method was complex, expensive and time consuming.
The European Pharmacopoeia (Ph. Eur.) monograph for Erythropoietin Concentrated Solution
[39] describes a CZE method for identification of rhEPO and separation of its glycoforms.
However, this method has shown poor reproducibility due to inadequate 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 components 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 protien samples. In this work, desalting of samples followed
by the use of discontinuous buffer in CE method were done to solve this problem.

Figure 2 : Primary structure of human erythropoietin (mature hormone)

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 glycoforms in bulk and finished products.
 4

2. Experimental

2.1. CE systems

2.1.1. LIF Instrumentation

A model P/ACE 5510 Beckman capillary electrophoresis instrument (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 filter, and a notch filter was
used to attenuate background radiation. Uncoated fused silica capillaries (75 µm ID x 363 µm 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.

2.1.2. UV Instrumentation

A Beckman Coulter P/ACE™ MDQ Capillary Electrophoresis System (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 μm was used. The column
temperature was controlled at 4 ⁰C.

2.2. 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 highest purity analytical grade available
from (BDH, UK). eCAP Amine capillaries and reagents were purchased from Beckman
Instruments (Fullerton, CA, USA). The wash and conditioning procedures used were those
recommended by the manufacturer.

2.3. Procedures

2.3.1. Analysis of TOB in bulk by CE-LIF after derivatization with FITC

2.3.1.1. Capillary conditioning

Initially a new capillary was treated with 1 M NaOH for 15 min, followed by water for 10 min,
and then running 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.
 5

2.3.1.2. 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 µm membrane filter. 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.

2.3.1.3. 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 standard solutions,
working standard solutions containing TOB in the range of (0.25 - 5 µg/ml) were prepared by
dilution with distilled water.

2.3.1.4. FITC derivatization procedure

300 µl aliquoit of aqueous TOB solution containing (0.25 - 5 µg/ml) was added to 300 µl FITC
(0.35 mM) in ethanol and 200 µl 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 analysed without dilution.

2.3.1.5. 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 ºC. The stability
of the derivatized TOB was monitored by measuring the peak areas of the derivative every 10
minutes up to 90 min.
2.3.1.6. 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 magnetic stirrer and pH meter
and the volume was completed with distilled 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 and on-line detection with the LIF detection
system (Ex 488 nm, Em 520 nm).

2.3.1.7. MECK-LIF Separation of TOB in Spiked Urine after Derivatization with FITC

2.3.1.7.1. Preparation of TOB Spiked Urine (TSU) Samples

Human urine samples were collected from five different volunteers (males and females). The
 6

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 μg/ml. Different drug concentrations ranging from 0.25 to 5 µg/ml were
prepared by serial dilution of TOB in the internal standard urine solution.

2.3.1.7.2. 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 μm ID (66 cm effective length) and BGB consisted 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.

2.3.2. CZE-UV Separation of EPO and HAS:

2.3.2.1. Preparation of rhEPO solution:

Fresh HSA stock solution was prepared immediately prior to analysis in Milli-Q water at a
nominal concentration of 2.0 mg/ml from high purity HSA. 72 µl "1mg/ml" rhEPO
BP_Reference were added to 25 µl HSA. Then 1.9 ml dist water were added. The obtained
solution was mixed very will by rotator for 30 sec. For preparation of CE sample, 900 µl
distelled water were pipetted to CE vial. Then rhEPO and Albumine (or EPO finished product)
were added.Finally, the volume was completed to 2 ml with distelled water.

2.3.2.2. 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 prosedure was done as
follows: centrifugal filtration device “Microcon YM-10” with membrane (Amicon, Beverly,
MA, USA) was used as a means to concentrate the sample. The cetrifugator must be cooled in
the refrigerator before its use for at least 1 hr. All desalting steps were done with the use of cold
distilled water. By the use of micropipette, 4 filtration beds were conditioned with 300 µl cold
distilled water for each at 11500 xg/12500 rpm for 20 minutes. The filtrate was rejected. Then
200 µl EPO sample were pipette to the used beds followed by 250 µl cold distilled water. The
centrifugation was done at 2700 xg for 50 minutes (repeated twice by using 300 µl cold distilled
water each time). The retenate were collected at 3200 xg for 5 min. All retenated were collected
in a 2 ml volumetric centrifugation vial and the volume were completed with cold distilled water.

2.3.2.3. Capillary electrophoresis procedure:

Sodium phosphate buffers (200 mM) pH 4.0 and 9.0 containing 1mM Nickel Chloride
hexahydrate. The prepared buffers were filtered before use. The applied voltage was 10 kV, with
 7

reversed polarity, at 20 °C . eCape Amine capillary was used. Electokinetic injection with 10KV
with reversed polarity, for 10 sec.

2.3.2.4. 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 following procedure
should be used to prevent any sample carry-over from previous separations; several high
pressure (20 psi) rinses of 1N HCl were first done for 5 minutes, then water for 5 minutes,
followed by 1N NaOH for 2 minutes,again water for 5 minutes, and finally with amine
regenerator solution for 5 minutes.

3. Results and Discussion

3.1. TOB analysis:
3.1.1. Optimization of derivatization reaction
3.1.1.1. Effect of pH and buffer concentration

Borate buffer solutions of different 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 concentrations 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.

3.1.1.2. 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 ºC. The rate of reaction of TOB with FTIC was
increased as temperature increased up to 70 ºC and after that no increases were observed.
However to ensure rapid and full reaction, 80 ºC was chosen as the best reaction 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 measuring the peak
areas of the derivative every 10 minutes up to 90 min. The results indicate the maximum stability
was one hour at room temperature reaction time of 10 min.
3.1.1.3. Optimization of CE conditions for FITC

borate buffers were examined and borate (boric acid from 1.4 g to 2.8 g and sodium tetraborate
1.05 g to 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.
 8

3.1.2. Validation of FITC Derivatization Method

Amikacin sulphate was added (0.4 g/2L) as internal standard to give concentration of 0.2 mg/ml
as standard solution. 0.1 g TOB was spiked into 100 ml internal standard solution to give
concentration 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 internal standard solution to give concentrations of 100
µg/ml then different concentrations of TOB in the internal standard solution to give different
concentrations in ranging from 0.25 to 5 µg/ml. The best CE conditions are shown in Figure 3.

Figure 3. Representative electropherograms of TOB-FITC and its blank:

CE conditions: capillary length 75cm x 75µm ID and BGE: boric 2.8 g and sodium borate
2.1 g/100 ml water 20 kV injection time 20 sec (Green) blank,. (Blue) TOB-FITC (Red)
Amikacin with tobramycin, detection was LIF Ex 488 nm; Em 520 nm.
 9

The linearity of the CE pre-column FITC derivatization method was examined by using TOB at
six different concentrations (injections in duplicate), ranging from 0.25 to 5 µg/ml. The standard
calibration curve for TOB was linear, and described by the following equation:
y= 0.7804 x - 0.0764 (R2= 0.9991 n= 6).

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.

Table 1. Intra-day and inter-day precision (n=5) for TOB solutions injected twice after LIF-
detection of FITC-derivatization.

Intra-day Inter-day
TOB
(n=5) (n=5)
Concentration
(µg/ml) 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

3.1.3. 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
available 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
(Scheme1). 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 product or the other peaks for degradation or impurities of TOB. This
observation follows a similar behaviour as previously discussed for these TOB-OPA derivatives
when two derivative peaks have been given [24, 48].
 10

Scheme1. Structure of the amino compounds and their derivatization

reaction scheme with FITC.

3.1.4. Mechanism of borate complexation and binding sites

The use of borate buffer to alter selectivity in CE was first investigated in 1988 [49]. At higher
borate concentration in the aqueous phase, tri- to pentameric polyanionic species such as
[B3O3(OH)5]2-, [B4O5(OH)4]2- and [B5O6(OH)4]2- 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.
 11

OH OH
HO

B - B
HO (1)
HO
+
OH
HO OH
-
(B) (B )

H3C
H3C
O .
OH HO . HO
HO
Cn
+ Cn B + 2 H2O (2)
B
HO O .
HO OH HO .
H3C
-
H3C -
(B ) (BL )
(L)

H3C
H3C H3C H3C
O .
. HO . . O
O
HO
Cn
Cn + Cn Cn B + 2 H2O (3)
B
O O .
O . HO . .
HO
H3C
H3C H3C H3C
-
- (BL2 )
(BL ) (L)

Scheme 2. Equilibria between boric acid, borate and diols in water

Where [54] n= 0 or 1, L is polyol ligand and B- represents tetrahydroxyborate, [B(OH)4 ]-. In a

pH range from 8 to 12, aqueous borate solutions contain not only tetrahydroxyborate ions but
also more highly condensed polyanions such as triborate, [B3O3(OH)5]2-, tetraborate,
[B4O5(OH)4]2-. The reported data [55] confirmed that: 1- Polyols can form 1:1 and 1:2
complexes with borate. 2- Not only hydroxyl groups on adjavant 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 .

3.1.5. Determination of TOB in human urine

TOB in a pooled human urine samples (0.25 to 5 µg/ml) was determined by CE, using direct
injection (without extraction) after labeling with FITC in the concentration range from 0.25 to 5
µg/ml.The derivatized sample was injected directly into the CE-LIF system for 8 sec 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 derivatives and the TX100/SDS micelles because both the
derivative and the surfactant are negatively charged. The electrostatic repulsion can prevent
solubilization of TOB derivative in the mixed micelle thus causing the optimum resolution
 12

between TOB and the indigenous urine peaks and the internal standard peak. Therefore the
parameters in Figure 4 are the optimum for this method. The LOD and LOQ for TOB spiked in
human urine were estimated practicaly using a signal to noise ratio of 3 and 10 as 90 and 200
ng/ml, respectively.

Figure 4. Laser Indused Flourescence detection of tobramycin FITC derivative in human urine.

CE conditions: capillary length 75 cm x 75 µm ID and BGE ((0.2 boric acid + 1.75% TX100 +
5.25 gm 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 µg/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 µg/ml) vs. peak area ratios of
the same concentrations calculated from the resultant regression line. Five replicate analyses
 13

were carried out at each concentration. The precision, accuracy and recovery data obtained from
TSU are summarised in Table 2.
Table 2. Intra-day and inter-day precision and accuracy for TSU after FITC derivatization
method.

Spiked TOB Intra-day Inter-day

concentration
RSD% Recovery % RSD% Recovery %
(µg/ml)

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

3.2. rhEPO analysis:

CE has become a powerful analytical tool for resolving and quantifying complex protein
mixtures as well as different forms of the same protein [56]. The main problem associated with
protein 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, desalting of proteins were done followed by CE with a
discontinuous buffer 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 stabilizer. 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 phosphate buffers (200 mM) pH 4.0 and 9.0 containing 1mM Nickel Chloride
hexahydrate as shown in figure 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.
Electokinetic injection with 10KV with reversed polarity, for 10 sec. Theses conditions for the
separation of which allowed resolution of rhEPO into four glycoforms as shown in figure 6.
 14

These conditions also afforded complete separation of HSA and rhEPO without affecting the
glycoform resolution pattern(Figure. 6). Typically, the migration time for the main rhEPO
glycoform and HAS peaks were found to be 13 and 14 min respectively. The difference between
HD and EK injections of EPO can be shown in figure 7.

Figure 5. schech diagram for CE system for rhEPO analysis

Figure 6. Electropherograms of rhEPO in bulk (upper black bold) and in the presence of albumin
(lower black thin)
 15

UV - 200nm UV - 200nm
UV.10003 9-23-2010 12-17-33 PM.dat uv.10003 9-23-2010 11-53-09 am.dat
Name
0.009 0.009
Migration Time
Area

0.008 0.008

0.007 0.007

0.006 0.006

0.005 0.005

0.004 0.004
AU

AU
0.003 0.003

0.002 0.002

0.001 0.001

0.000 0.000

-0.001 -0.001

-0.002 -0.002

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Minutes

Figure 7. Electropherograms showing the the difference of hydrodynamic (blue) and

elecktrokinetic (red) injections of the same conc of rhEPO with the same duration.

4. Conclusion
A MEKC-LIF using a pre-CE derivatization with FITC has been used for the determination of
TOB. A mixed micelle system composed of SDS and TX-100 as buffer additives improved
resolution, selectivity and sensitivity of the method. The method was applied succefully 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 sensitive
enough to be used for the analysis of TOB in urine samples. On the other hand, CZE-UV using
nikel as metal ion additive to the background electrolyte were used to overcome difficulties in
 16

assessing protein drug products due to the similarity of their physicochemical 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.

References
[1] R.D. Meyer, L.S. Young, Armstron.D, Applied Microbiology 22 (1971) 1147-&.
[2] E. Kaale, C. Govaerts, J. Hoogmartens, A. Van Schepdael, Rapid Communications in Mass
Spectrometry 19 (2005) 2918-2922.
[3] M. Omar, H. Ahmed, M. Hammad, S. Derayea, Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy 135 (2014) 472-478.
[4] A. Csiba, The Journal of pharmacy and pharmacology 31 (1979) 115-116.
[5] J.A. Ryan, Journal of Pharmaceutical Sciences 73 (1984) 1301-1302.
[6] V. Dasgupta, Journal of Clinical Pharmacy and Therapeutics 13 (1988) 195-198.
[7] S.S. Sampath, D.H. Robinson, Journal of Pharmaceutical Sciences 79 (1990) 428-431.
[8] R.L. Hussey, Journal of Chromatography 92 (1974) 457-460.
[9] A.K. Dash, Tobramycin in Analytical profiles of drug substances and excipients,24, 579-613, Pub.
Academic Press, 1996.
[10] J.W. Mayhew, S.L. Gorbach, Antimicrobial Agents and Chemotherapy 14 (1978) 851-855.
[11] A. Cabanes, Y. Cajal, I. Haro, J.M.G. Anton, M. Arboix, F. Reig, Journal of Liquid Chromatography
14 (1991) 1989-2010.
[12] J. Marples, M.D.G. Oates, Journal of Antimicrobial Chemotherapy 10 (1982) 311-318.
[13] B.G. Keevil, S.J. Lockhart, D.R. Cooper, Journal of Chromatography B-Analytical Technologies in
the Biomedical and Life Sciences 794 (2003) 329-335.
[14] C.H. Feng, S.J. Lin, H.L. Wu, S.H. Chen, Journal of Chromatography B 780 (2002) 349-354.
[15] M.H. Mashat, Pharmaceutical analysis, pharmacokinetic and in-vitro aerodynamic
characterisation of nebulised tobramycin. PhD Thesis, School of Pharmacy, University of Bradford,
Bradford, 2006, p. 378.
[16] M. Mashat, H. Chrystyn, B.J. Clark, K.H. Assi, Journal of Chromatography B 869 (2008) 59-66.
[17] D.M. Barends, C.L. Zwaan, A. Hulshoff, Journal of Chromatography 225 (1981) 417-426.
[18] H. Fabre, M. Sekkat, M.D. Blanchin, B. Mandrou, Journal of Pharmaceutical and Biomedical
Analysis 7 (1989) 1711-1718.
[19] C.L. Flurer, Journal of Pharmaceutical and Biomedical Analysis 13 (1995) 809-816.
[20] H.H. Yeh, S.J. Lin, J.Y. Ko, C.A. Chou, S.H. Chen, Electrophoresis 26 (2005) 947-953.
[21] P.D. Voegel, R.P. Baldwin, Electroanalysis 9 (1997) 1145-1151.
[22] P. Srisom, B. Liawruangrath, S. Liawruangrath, J.M. Slater, S. Wangkarn, Journal of
Pharmaceutical and Biomedical Analysis 43 (2007) 1013-1018.
[23] H. Fonge, E. Kaale, C. Govaerts, K. Desmet, A. Van Schepdael, J. Hoogmartens, Journal of
Chromatography B 810 (2004) 313-318.
[24] E. Kaale, S.A. Van, E. Roets, J. Hoogmartens, Electrophoresis 23 (2002) 1695-1701.
[25] F. Wienen, U. Holzgrabe, Electrophoresis 24 (2003) 2948-2957.
[26] W.M. Ebied, H.M. Ahmed, F.A. Elbarbry, Biosimilars 4 (2014) 11-22.
[27] A. Pantazaki, M. Taverna, C. Vidal-Madjar, Analytica Chimica Acta 383 (1999) 137-156.
[28] F. Kvasnicka, Electrophoresis 28 (2007) 3581-3589.
[29] M. Sniehotta, E. Schiffer, P. Zurbig, J. Novak, H. Mischak, Electrophoresis 28 (2007) 1407-1417.
 17

[30] Y. Li, X.B. Yin, X.P. Yan, Anal. Chim. Acta 615 (2008) 105-114.
[31] V. Garcia-Canas, A. Cifuentes, Electrophoresis 29 (2008) 294-309.
[32] B. Yu, H.L. Cong, H.W. Liu, Y.Z. Li, F. Liu, Trac-Trends in Analytical Chemistry 24 (2005) 350-357.
[33] J. Zhang, U. Chakraborty, A.R. Villalobos, J.M. Brown, J.P. Foley, Journal of Pharmaceutical and
Biomedical Analysis 50 (2009) 538-543.
[34] I. Lacunza, P. Lara-Quintanar, G. Moya, J. Sanz, J.C. Diez-Masa, M. de Frutos, Electrophoresis 25
(2004) 1569-1579.
[35] P. Dou, Z. Liu, J. He, J.-J. Xu, H.-Y. Chen, Journal of Chromatography A 1190 (2008) 372-376.
[36] E. Gimenez, F. Benavente, C. de Bolos, E. Nicolas, J. Barbosa, V. Sanz-Nebot, Journal of
Chromatography A 1216 (2009) 2574-2582.
[37] H.P. Bietlot, M. Girard, Journal of Chromatography A 759 (1997) 177-184.
[38] P. Lara-Quintanar, I. Lacunza, J. Sanz, J.C. Diez-Masa, M. de Frutos, Journal of Chromatography A
1153 (2007) 227-234.
[39] European Pharmacopoeia 2 (2005) 1528–1529.
[40] V. Sanz-Nebot, F. Benavente, A. Vallverdu, N.A. Guzman, J. Barbosa, Analytical Chemistry 75
(2003) 5220-5229.
[41] Z. Krivacsy, A. Gelencser, J. Hlavay, G. Kiss, Z. Sarvari, Journal of Chromatography A 834 (1999)
21-44.
[42] H. Engelhardt, M.A. Cunatwalter, Journal of Chromatography A 717 (1995) 15-23.
[43] C.L. Flurer, K.A. Wolnik, Journal of Chromatography A 663 (1994) 259-263.
[44] Y. Kaneo, S. Hashihama, A. Kakinoki, T. Tanaka, T. Nakano, Y. Ikeda, Drug Metabolism and
Pharmacokinetics 20 (2005) 435-442.
[45] D.A. Wicks, P.C.H. Li, Analytica Chimica Acta 507 (2004) 107-114.
[46] D. Letourneur, D. Logeart, T. Avramoglou, J. Jozefonvicz, Journal of Biomaterials Science-
Polymer Edition 4 (1993) 431-444.
[47] N.A. Stearns, S. PrigentRichard, D. Letourneur, J.J. Castellot, Analytical Biochemistry 247 (1997)
348-356.
[48] F. Lai, T. Sheehan, Journal of Chromatography 609 (1992) 173-179.
[49] R.A. Wallingford, A.G. Ewing, Journal of Chromatography A 441 (1988) 299-309.
[50] P. Schmitt-Kopplin, N. Hertkorn, A.W. Garrison, D. Freitag, A. Kettrup, Analytical Chemistry 70
(1998) 3798-3808.
[51] A.L. Revilla, J. Havel, P. Jandik, Journal of Chromatography A 745 (1996) 225-232.
[52] J.G. Dawber, G.E. Hardy, Journal of the Chemical Society. Faraday Transactions I 80 (1984) 2467-
2478.
[53] H.H. Thanh, Journal of chromatography. A 678 (1994) 343-350.
[54] M. Makkee, A.P.G. Kieboom, H. van Bekkum, Recueil des travaux chimiques des Pays-Bas 104
(1985) 230–235.
[55] S. Hoffstetterkuhn, A. Paulus, E. Gassmann, H.M. Widmer, Analytical Chemistry 63 (1991) 1541-
1547.
[56] Y. Chen, W. Lü, X. Chen, M. Teng, Central European Journal of Chemistry 10 (2012) 611-638.
[57] C.J. Booker, K.K.C. Yeung, Analytical Chemistry 80 (2008) 8598-8604.
[58] H. Kajiwara, Journal of Chromatography A 559 (1991) 345-356.
 Highlights

• LIF detector is used for tobramycin analysis in human urine.

• Urine samples were injected directly without pretreatment.
• Erythropoietin was analysed in the presence of albumin by CE-UV.
• EK and discontinuous buffer used to increase method sensitivity.
Graphical Abstract

Chemical structure of tobramycin (upper) and primary structure of human

erythropoietin (lower)