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Journal of Liquid Chromatography & Related
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A Combination of Isocratic and Gradient Elution Modes
in HPLC with the Aid of Time-Overlapping Process for
Rapid Determination of Methyldopa in Human Urine
Samy Emara a , Tsutomu Masujima b , Walaa Zarad a , Maha Kamal c , Marwa Fouad d &
Ramzia El-Bagary d
a Department of Pharmaceutical Chemistry , Faculty of Pharmacy, Misr International
University , Cairo , Egypt
b P.I. Lab. Single Cell MS, RIKEN Quantitative Biology Center , Suita , Osaka , Japan
c Department of Pharmaceutical Analytical Chemistry , Faculty of Pharmacy, Modern
Sciences and Arts University , Cairo , Egypt
d Department of Pharmaceutical Chemistry , Faculty of Pharmacy, Cairo University , Cairo ,
Egypt
Accepted author version posted online: 26 Mar 2014.Published online: 10 Oct 2015.
To cite this article: Samy Emara , Tsutomu Masujima , Walaa Zarad , Maha Kamal , Marwa Fouad & Ramzia El-Bagary
(2015) A Combination of Isocratic and Gradient Elution Modes in HPLC with the Aid of Time-Overlapping Process for Rapid
Determination of Methyldopa in Human Urine, Journal of Liquid Chromatography & Related Technologies, 38:2, 153-162, DOI:
10.1080/10826076.2014.896813
To link to this article: http://dx.doi.org/10.1080/10826076.2014.896813
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A Combination of Isocratic and Gradient Elution Modes in
HPLC with the Aid of Time-Overlapping Process for Rapid
Determination of Methyldopa in Human Urine
SAMY EMARA,
1
TSUTOMU MASUJIMA,
2
WALAA ZARAD,
1
MAHA KAMAL,
3
MARWA FOUAD,
4
and
RAMZIA EL-BAGARY
4
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Misr International University, Cairo, Egypt
2
P.I. Lab. Single Cell MS, RIKEN Quantitative Biology Center, Suita, Osaka, Japan
3
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Modern Sciences and Arts University, Cairo, Egypt
4
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
A new rapid time-overlapping high-performance liquid chromatography method using coupled-column double-injection technique
with fluorescence detection has been developed and validated to determine methyldopa (MTD) in human urine. The method was
based on injecting a new sample onto the second column before finalizing the cleanup and the re-equilibration of the first column
for the former sample. A combination of isocratic and gradient elution was employed according to a pre-set program. At the begin-
ning, isocratic step of acetate buffer solution (0.1 M, pH 2.4) was set until 7 min. Subsequently, a gradient elution step using acetate
buffer (0.1 M, pH 2.4) as mobile phase A and acetonitrile as mobile phase B was employed. After the end of each gradient step, the
column was re-equilibrated with 4 mL of the starting isocratic elution system before the next analysis. The overall cycle time was
7 min per each sample. The calibration curve was linear over the concentration range of 0.1–40 mg=mL MTD. The overall mean
recoveries were in the range of 98.29–101.39%. The applicability of the method was successfully evaluated by monitoring the
incremental urinary excretion of MTD in human urine over 12 hr after a single oral administration of 250mg.
Keywords: coupled-column, double injection, fluorescence detection, HPLC, methyldopa, mixed elution modes
Introduction
Methyldopa (MTD) is an old antihypertensive agent, which
is used in the treatment of mild to moderate hypertension. It
is converted to a-methyl norepinephrine in adrenergic nerve
terminals and its antihypertensive action appears to be due
to the stimulation of the central a-adrenoreceptors by this
agent.
[1]
Several analytical procedures have been reported
for the analysis of MTD in pharmaceutical formulations
or biological fluids. These procedures include determination
by titrimetry,
[2]
spectrofluorimetry,
[3]
spectrophotometry,
[4,5]
potentiometry,
[6]
thin-layer chromatography,
[7]
gas liquid
chromatography,
[8]
and high-performance liquid chro-
matography (HPLC)
[9–23]
methods. Furthermore, flow
injection,
[24]
cyclic voltammetry,
[25]
nuclear magnetic reson-
ance spectroscopy, and kinetic methods
[26,27]
have been
reported. Electrochemical methods have been applied to
determine MTD
[28–34]
due to their simplicity, accuracy, and
rapidity.
The urinary and the plasma concentrations of the drug
have been most commonly measured by HPLC using
electrochemical,
[15,16,19–23]
fluorescence,
[10,12,14,20]
UV,
[9,13,14,17,18]
or diode array
[11]
detections. Analytical con-
ditions in the published methods consisted of gradient
[10,14]
or isocratic
[9,11–13,15–23]
elution of the mobile phase
with
[9,10,12,15,16,18–20,22,23]
or without
[11,13,14,17,21]
using of an
ion pair agent. Determination of MTD in biological matrices
could not be performed without appropriate sample
preparation, to remove potentially interfering components,
even when using powerful analytical instruments, such as
liquid chromatography–tandem mass spectrometry (LC–
MS–MS).
[35]
In liquid chromatography, the mobile phase should be
selective for the components, and its composition is one of
the most necessary variables influencing the separation. In
isocratic elution, the mobile phase composition is unchanged
during the separation. However, the disadvantages of an iso-
cratic mode are broadening of the late-eluting peaks to the
point of difficult detection, tailing peaks, and unnecessarily
long separation times. Analysis time of complex mixtures
with a wide range of retention factors can be made much
shorter in the gradient elution chromatography, than in
the isocratic separation of the same mixtures. However,
Address correspondence to: Samy Emara, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Misr Inter-
national University, Km 28 Ismailia Road, Ismailia 41522, Cairo,
Egypt. E-mail: emara_miu@yahoo.com
Journal of Liquid Chromatography & Related Technologies, 38: 153–162, 2015
Copyright #Taylor & Francis Group, LLC
ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2014.896813
Downloaded by [Samy Emara] at 15:56 12 November 2014
repetitive analyses using gradient elution need a long
re-equilibration time to the initial conditions, which can
extend the run time. For method development and optimiza-
tion, reduction of the time needed for re-equilibration in the
gradient elution is very important.
Until now only a few analytical HPLC methods for mea-
suring urinary concentrations of MTD either individually or
in combination with other drugs have been reported in the
literatures.
[15,17,20,22,23]
The main problem in extracting low
drug levels from complex samples, such as urine, is the lim-
ited concentration of the analyte available. MTD in these
complex biological matrices is often present in low concen-
trations, along with extremely high background signals from
the endogenous components, which interfere with the detec-
tion of the analyte. Sample preparation is a key step in the
quantitative bio-analysis, and can potentially be a bottleneck
in the process to develop robust and efficient methodologies.
Combinations of off-line solid phase extraction (SPE) and
cleanup procedures utilizing alumina and=or ion exchange
resin have been described for the analysis of MTD in
urine.
[15,17,20,22,23]
This combined extraction=cleanup strat-
egy has drastically increased the time spent on sample hand-
ling and contributed significantly to the final cost of the
analysis, both in terms of labor and consumption of materi-
als. Minimization of error resulting from the human factor
and the increasing demands for faster methods are major
incentives to improve the classical procedures used for sam-
ple treatment of biological fluids. With a view to find an
alternative method able to provide satisfactory results with-
out the need for any sample pretreatment step, our study
was involved in a research effort aimed to expand the auto-
mation by using a combined isocratic and gradient elution
modes in HPLC together with the aid of time-overlapping
process. This approach enabled us to directly inject and
determine MTD in human urine samples.
The feasibility of the current method has been proven in
conventional liquid chromatography using two gradient
pumps, two identical ODS analytical columns and two
identical injection valves. With a six-port-switching valve,
human urine sample was loaded on one analytical column,
while the second analytical column was re-equilibrated and
conditioned for the next injection. Besides the opportunity
of injecting urine sample directly to improve the precision,
the developed method offered the possibility of removing
the late-eluting endogenous interferences encountered in
human urine in a short time. One of the most relevant
aspects of this method was the time-overlapping process,
i.e., injecting a new sample on the second analytical column
before finalizing the cleanup and the re-equilibration of the
first analytical column for the former sample. This latter
capability was the key to success for enhancing sample
throughput in the analysis of MTD in urine.
Experimental
Instrumentation
The chromatographic analysis was carried out in an HPLC
system from Agilent (Agilent Technologies, CA, USA),
equipped with two quaternary pumps, degassing devices,
and two identical Rheodyne injectors with a 20 mL loop
(Rheodyne, Berkeley, California, USA). A model Agilent
1200 Series G1321A fluorescence detector was used for the
detection of MTD at an excitation wavelength of 270 nm
and an emission wavelength of 320 nm. The chromato-
graphic separation was achieved by means of two identical
Thermo Scientific Hypersil ODS analytical columns
(100 mm 4.0 mm i.d., 5 mm), from Thermo Scientific
(Florida, USA). A model 7010 flow direction column-
switching valve was applied to facilitate the switching
between the two analytical columns (Rheodyne, Berkeley,
California). This instrument has a data station that controls
its operating parameters, runs the desired programs, and
records the detector signal. The measurements were done
with the columns kept at room temperature (20C1C).
General Procedure
The system setup for the rapid analysis consisted of two
positions (A and B) (shown in Figure 1). Analyte was sepa-
rated by reversed-phase HPLC using acetate buffer (0.1 M,
pH 2.4) as mobile phase A, while acetonitrile as mobile
phase B was used for achieving a complete column cleanup.
In position A, preceding the injection of the urine sample,
mobile phase A was brought onto the first analytical column
by pump I at a flow rate of 1 mL=min for equilibration;
meanwhile, pump II equilibrated the second analytical col-
umn with the same mobile phase at a flow rate of 1 mL=
min. The urine sample (20 mL) was injected directly onto
the first analytical column by pump I, and the analyte was
separated from the endogenous components of the human
urine matrix with an isocratic step consisting of 100% of
mobile phase A. After 7 min, the switching valve was
switched to position (B), and while MTD in urine sample
was injected and chromatographed on the second analytical
column, the first analytical column was cleaned-up with a
linear gradient elution from 0% to 100% of mobile phase
B within 0.5 min followed by an isocratic step consisting of
100% of mobile phase B for 2 min, and finally a linear gradi-
ent elution from 100% to 0% of mobile phase B within
0.5 min. The rest of the single run was continued by
re-equilibrating the first analytical column with mobile phase
A for 4 min to be ready for the next injection. After 7 min,
the switching valve was switched back to its original position
(position A), and while urine sample was injected and
chromatographed on the first analytical column, the second
analytical column was cleaned-up and re-equilibrated with
the same gradient elution system. The overall cycle time
was 14 min, including 7 min for column cleanup and
re-equilibration between analyses, which can almost be
halved in the case, where time-overlapping process was used,
i.e., injecting a new sample on the second analytical column
before finalizing the cleanup and the re-equilibration of the
first analytical column for the former sample.
Materials and Reagents
MTD (99.87% purity) was obtained from ADWIC (Cairo,
Egypt). The present method was applied to determine
154 S. Emara et al.
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MTD in urine after the administration of Aldomet tablet:
Aldomet tablet (Batch No. 01918) was manufactured by
KAHIRA PHARM. & Chem. IND. CO., Cairo, Egypt.
Each tablet was claimed to contain 250 mg of MTD. Aceto-
nitrile used was HPLC grade (Sigma-Aldrich, Germany).
Sodium acetate, orthophosphoric acid, and hydrochloric
acid were of analytical grade (Sigma-Aldrich, Germany).
Calibration Standards and Quality Control Samples
A stock solution of MTD at the concentration of 1 mg=mL
was prepared by dissolving appropriate amount of the drug
in 0.01 M hydrochloric acid solution. Appropriate volumes
of the stock solution of MTD were diluted with the same sol-
vent to achieve the concentration ranges of 1–400 mg=mL.
The standard solutions were protected from light by wrap-
ping the containers with aluminum foil. Urine standards
for calibration were freshly prepared. Each standard sol-
ution was diluted tenfold into drug-free human urine to
obtain the concentration range of 0.1–40 mg=mL MTD. Cali-
bration standards were stored at 20C until required for
assay. Prior to assay, frozen human urine samples were
thawed at ambient temperature and centrifuged at 2000 g
for 5 min at 4C to precipitate solids followed by filtration
of the supernatant through 0.45 mm Millipore filters to avoid
the obstruction of the analytical column. An aliquot of
20 mL was injected onto the column for analysis. This
precaution was important for successive analysis of urine
samples without pressure trouble.
Quality control (QC) working solutions of MTD were
prepared following the same procedure as that used for the
preparation of MTD standard solutions. Specifically, the
stock solution was further diluted to obtain three levels of
QC standard working solutions (10, 100, and 300 mg=mL
MTD). The QC standard working solutions were diluted
tenfold into drug-free human urine to obtain the concen-
tration range of 1–30 mg=mL MTD. The concentrations of
the MTD QC samples were 1 mg=mL (low), 10 mg=mL
(medium), and 30 mg=mL (high).
Recovery
Aliquots of 20 mL of the QC urine samples at three different
concentration levels (1, 10, and 30 mg=mL MTD) were sub-
jected to the described procedure. Five replicates of each
QC sample were injected into the column. The recovery of
the drug from the urine sample was assessed by comparing
the peak area of MTD in urine sample to that of the aqueous
solution with the same concentration of the analyte, and the
assay recovery was calculated using the following equation:
%Recovery ¼mean measured concentration
nominal concentration 100
Precision and Accuracy
Both precision and accuracy of the method were determined
by analyzing five replicates of QC urine samples at low,
medium, and high concentrations (1, 10, and 30 mg=mL
MTD) against calibration curve. Intra-assay precision was
calculated as the relative standard deviation (RSD%) of
the mean concentration resulting from the same day.
Inter-assay precision was assessed by the RSD% of the mean
concentration on five consecutive days. The accuracy was
determined by the percent of the relative errors (RE%) of
the mean measured concentrations:
RE% ¼
mean measured concentration
nominal concentration
nominal concentration 100
Fig. 1. Schematic diagram of the time-overlapping process
for the rapid analysis of MTD in urine; the system in the initial
position is ready for sample injection onto the first analytical
column by pump I; meanwhile, pump II equilibrate the second
analytical column (Position (a)); after 7 min, the switching valve
is switched to Position (b), where a new sample is injected onto
the second analytical column by pump II; meanwhile, pump I
cleanup and re-equilibrate the first analytical column from the
former sample. (S.V.: six-port-switching valve).
Combination of Isocratic and Gradient Elution Modes in HPLC 155
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Application
The validated method was applied to monitor the concen-
tration of MTD in five selected male Egyptian volunteers
(aged between 33 and 34 years and weighing between 93
and 95 kg) after a single oral dose of 250 mg MTD (Aldomet
tablet, 250 mg=tablet). The volunteers have not taken any
other medications for at least 2 weeks prior to the study.
Prior to the drug administration, a 10 mL urine sample
was taken as a control sample. Urine samples were collected
(2, 4, 6, 8, and 12 hr after administration) in analytical vari-
ables dark-glass containers, and were immediately frozen at
20C until analysis. Filtration of the thawed urine samples
prior to injection is of great importance. Thawed samples
often contain clots and solid particles, which on injection
will give an immediate increase in the column backpressure
owing to the restrictions of the inlet filter or the column
packing. Frozen urine samples were first allowed to thaw
at room temperature, centrifuged at 2000 g for 5 min at
4C, and then filtered through 0.45 mm Millipore filters.
Results and Discussion
Optimization of the Elution Conditions
Different elution methods ranging from isocratic, gradient,
and combination elution were tried to optimize the separ-
ation of MTD in human urine by HPLC. Initial efforts to
develop an isocratic elution system for the separation of
MTD using methanol-based mobile phase were unsuccessful
and resulted in a poor resolution of the analyte from the
endogenous matrix components. These efforts were subse-
quently shifted toward developing an elution system that
would overcome the chromatographic limitations described
above, and provide baseline separation of MTD. Two
notable points emerged during the development of this
system: (1) complete separation of MTD and the chro-
matographic quality were buffer concentration and pH-
dependent, preferring acetate buffer to phosphate buffer;
(2) this isocratic mode would require a long run time due
to the presence of a series of strongly retained endogenous
components in the human urine. It was observed that, a
mobile phase consisted of acetate buffer (0.1 M, pH 2.4) gave
an acceptable retention time (SD) (5.80 min 0.047) of
standard MTD with a good peak symmetry on conventional
HPLC equipment using a Thermo Scientific Hypersil ODS
analytical column (100 mm 4.0 mm i.d., 5 mm). When this
condition was used for further separation of MTD in
untreated human urine, a longer run time was required for
complete elution (37 min) (Figure 2a and 2b). This can be
explained by the fact that, if the chromatographic conditions
are adjusted for the satisfactory separation of the weakly
retained compound (MTD), the elution of the strongly
retained urine endogenous components will take very long
time. On the other hand, if the chromatographic conditions
are adjusted for the adequate retention of the strongly
retained endogenous components of human urine, the
weakly retained MTD will elute too early as a poorly sepa-
rated band. When total gradient was employed, it possessed
a disadvantage of requiring a time post-gradient to flush the
initial mobile phase composition through the analytical col-
umn to ensure a reproducible retention time of the analyte in
the subsequent injection. Since we were aiming to develop a
method suitable for clinical studies, efforts were shifted to
reduce the analysis time. In order to achieve this, a combi-
nation of isocratic and gradient elution was employed
according to a pre-set program. We consider this gradient
HPLC step, where, after the separation and the quantifi-
cation of MTD (7 min), the solvent strength was increased
in order to rapidly elute the late-eluting endogenous urine
matrix components from the analytical column. At the
beginning, the proportion of the acetate buffer solution
(0.1 M, pH 2.4) was set at 100% until 7 min so that, the
migration velocity of MTD along the analytical column
was decreased, providing a sufficient separation of the early
eluting MTD and the co-eluted endogenous components of
the urine matrix. Subsequently, a gradient elution step using
acetate buffer solution (0.1 M, pH 2.4) as mobile phase A
and acetonitrile as mobile phase B was employed. The pres-
ence of acetonitrile in the gradient elution step was essential
for the rapid cleanup of the analytical column, specifically,
the elimination of the excessive late-eluting endogenous
components of the urine matrix that were strongly retained
under acetate buffer. After the end of each gradient
step, the composition of the mobile phase was set back
to the starting system, and the analytical column was
re-equilibrated with 4 mL of the starting isocratic elution
system (0.1 M acetate buffer, pH 2.4) before the next analy-
sis. The composition of the mobile phase was changed
according to the following time program: 0–7 min: 100%
mobile phase A, 0% mobile phase B, isocratic; 7–7.5 min: lin-
ear gradient from 0% to 100% mobile phase B; 7.5–9.5 min:
100% mobile phase B, isocratic; 9.5–10 min: linear gradient
from 100% to 0% mobile phase B; 10–14 min: 100% mobile
phase A, isocratic.
The ultimate goal of the developed HPLC method was to
obtain acceptable resolution of MTD within a reasonable
analysis time. This goal has led to investigate the limits of
speed in chromatography. In the present method, the analy-
sis time was determined by the cycle time, which was the
sum of the time required for the separation (i.e., isocratic
development time) and the time required to cleanup and
re-equilibrates the analytical column with the initial con-
dition to prepare it for the next injection (i.e., cleanup and
re-equilibration time). The time necessary for the column
cleanup and re-equilibration increased the total run time of
MTD in human urine, which should be as short as possible
for maximum sample throughput. On the other hand, it
should be long enough to reproducibly reestablish the
equilibrium between the analytical column and the initial
chromatographic isocratic condition. For an optimized sep-
aration (in terms of resolution), the isocratic development
time should be considered fixed; therefore, the only way to
minimize the cycle time was to decrease the cleanup and
the re-equilibration time. In order to obtain a time analysis
suitable for the routine use and to avoid the time required
for complete cleanup and re-equilibration, we chose to
156 S. Emara et al.
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modify the instrument by combining two analytical columns
and two injection valves in single system manifold, called as
couple-column double-injection technique. Such procedure
required especially designed gradient elution HPLC instru-
mentation with a six-port-switching valve, two identical ana-
lytical columns, and two identical injection valves. This
system allowed approximately two-times faster analysis than
the single column technique of the same length at the same
operating parameters. With a coupled-column double-injec-
tion technique and six-port-switching valve, human urine
sample was loaded into one analytical column, while the
late-eluting endogenous components of the previously urine
sample were eluted and cleaned-up on the other analytical
column. In other words, the overall cycle time was 14 min
for the system, which could be almost halved in the case,
where time-overlapping process was used, i.e., injecting a
new sample on the second analytical column before finaliz-
ing the cleanup and the re-equilibration of the first analytical
column for the former urine sample. Figure 3a and 3b shows
chromatograms of directly injected urine sample by a
Thermo Scientific Hypersil ODS (100 mm 4.0 mm i.d.,
5mm) analytical column at flow rate of 1 mL=min resulted
from typical blank urine (Figure 3a) as well as the spiked
drug-free human urine sample chromatogram of 10 mg=mL
MTD (Figure 3b). It can be clearly seen from these chroma-
tograms that no interfering endogenous components of the
untreated urine matrix were found in the retention time of
MTD after 7 min isocratic (100% mobile phase A) pro-
cedure. Under this isocratic conditions, endogenous peaks
were observed at 5.10 and 6.74 min; however, they did not
disturb the actual detection of MTD. The reproducibility
of the chromatographic conditions (retention time, peak
Fig. 2. Typical chromatograms obtained from the analysis of MTD in human urine using single column technique with fluorescence
detection. (a) Drug-free human urine; (b) drug-free human urine spiked with MTD (10 mg=mL). MTD was separated and quantified
on a Thermo Scientific Hypersil ODS analytical column (100 mm 4.0 mm i.d., 5 mm) with a mobile phase consisting of acetate
buffer (0.1 M, pH 2.4) at a flow rate of 1 mL=min.
Combination of Isocratic and Gradient Elution Modes in HPLC 157
Downloaded by [Samy Emara] at 15:56 12 November 2014
area) was determined by replicate injections (n ¼5) of spiked
drug-free urine samples with 10 mg=mL MTD. The standard
deviation (SD) of the peak areas of MTD was found to be
0.895 and the mean retention time for MTD SD was
5.80 0.047 min.
Selection of Time-Events for Column-Switching
The time-overlapping process does not have to wait until
complete column cleanup and re-equilibration of the first
analytical column to reintroduce the new urine sample into
the second analytical column. In fact, the next injection pro-
cess can be initiated on one analytical column when the
cleanup stage begins on the other analytical column. In the
initial position, position A (Figure 1); human urine sample
was loaded onto the first analytical column. The switch to
position B should occur when the MTD peak in the
untreated human urine has been eluted and detected. It
has been shown that, MTD in urine could be eluted within
5.80 min for a Thermo Scientific Hypersil ODS analytical
column (100 mm 4.0 mm i.d., 5 mm) using acetate buffer
(0.1 M, pH 2.4). In order to avoid the overlapping of the
highly retained endogenous components of the urine matrix
from the first run with MTD from the second run, the time
point to inject the next sample must be adjusted so that, a
complete cleanup of the late-eluting endogenous compo-
nents of the urine matrix was achieved. All late-eluted
endogenous components of the urine matrix were cleaned-up
Fig. 3. Typical chromatograms obtained from the analysis of MTD in human urine using coupled-column double-injection
technique (time-overlapping process) with fluorescence detection. (a) Drug-free human urine; (b) drug-free human urine spiked
with MTD (10 mg=mL). MTD was separated and quantified on the first analytical column with an isocratic step consisting of acet-
ate buffer (0.1 M, pH 2.4), at a flow rate of 1 mL=min. After 7 min, the switching valve was switched to the second column, and
while urine sample was injected and chromatographed on this column, the first analytical column was cleaned-up and
re-equilibrated with a binary gradient step using acetate buffer (0.1 M, pH 2.4) as eluent A and acetonitrile as eluent B to be ready
for the next injection operation.
158 S. Emara et al.
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in less than 3 min when eluted with a binary gradient elution
system of mobile phase A (0.1 M acetate buffer, pH 2.4) and
mobile phase B (acetonitrile). The six-port valve was there-
fore kept in position B for 7 min before switching back to
position A to achieve complete column cleanup and re-
equilibration with mobile phase A (4 min) prior to the next
sample injection. Since separation on the first analytical
column, as well as, complete column cleanup and
re-equilibration of the second analytical column can take
place simultaneously, a total analysis time of 7 min for each
sample could be achieved.
Merits of the Time-Overlapping Process
In this work, the application of the time-overlapping process
HPLC method has been established for the first time to
determine MTD. Overlapping development stages, com-
bined with fluorescence detection, is regarded as a core tech-
nique for faster analysis of MTD in human urine. Besides
providing a higher resolution and a shorter separation time,
the time-overlapping process has a characteristic advantage
over other conventional gradient HPLC methods in that;
the final stage of cleanup prepares the analytical column
for re-equilibration to begin the next injection operation.
With this in mind, repeated separation–cleanup and
re-equilibration can be performed. In this case, the injection
of a new sample can occur before the previous analytical
column has been cleaned-up and re-equilibrated. Figure 1a
and 1b shows typical repeated separation–cleanup and
re-equilibration operations, where a urine sample can be
injected onto the second analytical column before finaliza-
tion of the cleanup and the re-equilibration of the first
analytical column for the former sample.
Method Validation
Linearity
To determine the linearity of the HPLC detector response,
calibration standard solutions for MTD were prepared as
described in the text. The calibration curve from directly
injected spiked urine samples was constructed by plotting
the measured peak areas against the concentrations of the
drug in the concentration range of 0.1–40 mg=mL. Sample
concentrations were determined by linear regression, using
the formula Y ¼aþbC, where Y ¼peak area, C ¼
concentration of the standard in mg=mL, b ¼the slope of
the curve, and a ¼the intercept with Y-axis. Characteristic
parameters of the linear calibration curve are shown in
Table 1.
Limit of Detection and Quantification
The limit of detection (LOD) and the limit of quantification
(LOQ) were determined according to the ICH guidelines for
validation of analytical procedures
[36]
and were found to be
0.0296 mg=mL and 0.0896 mg=mL, respectively (Table 1).
Recovery, Precision, and Accuracy
Recoveries for MTD in human urine were found to be
98.91%, 98.46%, and 101.39% for QC samples at low
(1 mg=mL), medium (10 mg=mL), and high (30 mg=mL)
concentration levels, respectively. In order to judge the qual-
ity of the elaborated method, precision and accuracy were
validated on the basis of intra- and inter-assays. A summary
of intra- and inter-day precisions and accuracies at QC con-
centrations is shown in Table 2. At three different concentra-
tions, the intra-day reproducibility and accuracy for the
urine samples were excellent, with RSD% being in the range
of 0.22–0.97% and with mean RE% ranging from 1.39% to
1.54%. At the same concentration levels, the inter-day
RSD% was in the range of 0.20–0.91% and the mean RE%
ranged from 0.27% to 1.71%. Repeatability and reproduci-
bility of MTD in urine samples with high and low concen-
tration levels were below the value of 1.80%, indicating a
reliable measurement using the proposed method (Table 2).
Selectivity and Application
The selectivity was investigated by preparing and analyzing
five individual human blank urine samples and samples of
drug-free human urine spiked with MTD (10 mg=mL). Each
urine sample was tested using the described procedure. Rep-
resentative chromatograms of blank human urine sample,
and sample spiked with MTD (10 mg=mL) are shown in
Figure 3a and 3b. Good selectivity for the analyte was
obtained as evidenced by the symmetrical resolution of the
peak. There was no significant chromatographic interference
close to the retention time of the analyte in the untreated
human urine samples. The typical retention time SD for
MTD was 5.80 0.047 min. The total run time was about
7 min.
Table 1. Characteristic parameters for the regression equations
of the proposed method
Parameters MTD
Calibration range (mg=mL) 0.1–40
Detection limit (mg=mL) 0.0296
Quantitation limit (mg=mL) 0.0896
Slope (b) 41.1752
Standard error of the slope 0.2831
Intercept (a) 2.8932
Standard error of the intercept 5.5056
Correlation coefficient (r
2
) 0.9997
Y¼aþbC, where C is the concentration of MTD in mg=mL and Y is the
peak area.
Table 2. Precision and accuracy validation of MTD
Concentration (mg=mL)
Mean
RE (%)Nominal
Mean recovery
a
(% RSD)
Intra-assay
a
1 98.91 0.97 1.09
10 98.46 0.22 1.54
30 101.39 0.36 1.39
Inter-assay
a
1 98.49 0.91 1.51
10 98.29 0.20 1.71
30 100.27 0.63 0.27
a
Average of five determinations.
Combination of Isocratic and Gradient Elution Modes in HPLC 159
Downloaded by [Samy Emara] at 15:56 12 November 2014
A high degree of confidence in the validity of the new
time-overlapping HPLC method was showed in the suit-
ability of this technique to monitor the incremental urinary
excretion of MTD over 12 hr in five selected male Egyptian
volunteers (aged between 33 and 34 years and weighing
between 93 and 95 kg) after a single oral dose of 250 mg
MTD (Aldomet tablet, 250 mg=tablet), as there were no
potential concomitant interferences arising from the
matrices. Figure 4a and 4b shows chromatograms of a blank
urine sample taken from the volunteer before administrating
the drug (Figure 4a), as well as the chromatogram of the
clinical urine sample collected after 8 hr from orally admin-
istering 250 mg MTD (Figure 4b). A summary of the urinary
concentrations of MTD (lg=mL) at 2, 4, 6, 8, and 12 hr after
a single oral dose of 250 mg MTD is shown in Table 3. It was
clearly observed that, urinary concentrations of MTD
showed degrees of variability within and between subjects
in these preliminary experiments. Also, the maximum uri-
nary concentration of unchanged MTD was recovered, in
all volunteers, when urine samples were collected 6 hr after
a single oral dose. The high similarity between age, weight,
and sex of the selected Egyptian volunteers can explain these
results.
The proposed coupled-column double-injection strategy
for the determination of MTD in human urine is superior
to other published HPLC methods with respect to simplicity,
rapidity, and sensitivity. The linearity range was the para-
meter used to compare the sensitivity of the proposed
method with the other reported methods. The reported lin-
earity ranges (mg=mL) of MTD were found to be 5–170
[17]
and 2–500.
[23]
In the developed method, the calibration
curve showed excellent linearity with correlation coefficient
of 0.9997 over the range of 0.1–40 mg=mL. On the other
hand, intensive sample cleanup and enrichment by off-line
SPE and liquid–liquid extraction were necessary to
achieve the required selectivity and sensitivity in the reported
methods.
[15,17,20,22,23]
The sample cleanup procedures limited
the ultimate performance of these methods, especially with
Fig. 4. Chromatograms of typical drug-free human urine sample (a); and clinical urine sample obtained at 8 hr after a single oral
dose of 250 mg MTD (b).
160 S. Emara et al.
Downloaded by [Samy Emara] at 15:56 12 November 2014
regard to ruggedness and reliability. Whereas, the implemen-
tation of the combined modes of isocratic and gradient elu-
tion with the aid of time-overlapping process can utilize
direct injection procedure. Therefore, current method is less
time consuming, and uses small amounts of organic solvents
than other published methods, besides, the possibility of
human error arising during the several pretreatment steps
is considerably reduced. In addition, the current method
provides a precise methodology that overcomes any inter-
ference from the endogenous matrix components, and since
the recovery of MTD is quantitative, the internal standard
could be safely eliminated.
Stability
The stability studies of MTD in urine were carried out to
ensure the reliability of the results in relation to handling
and storing of the urine samples. The studies involve evalu-
ating the freeze and thaw stability, short-term stability, and
long-term stability. The tests of stability were assessed with
two concentrations of QC samples, i.e., 1 and 10 mg=mL.
In the freeze and thaw stability test, the samples were stored
at 20C for 24 hr and thawed at room temperature for
approximately an hour. Triplicate analyses of the samples
at each concentration were quantitated. Samples were
immediately refrozen at 20C for the next study day. This
cycle was repeated for three consecutive days. The short-
term stability was assessed after the storage of the samples
at 5C and at room temperature. Carrying out the experi-
ment after the storage of the samples at 20C for 4 weeks
assessed the long-term stability. The concentration of MTD
after each storage period was related to the initial con-
centration as determined for the samples that were freshly
prepared. Experiments showed that there was no difference
in the mean peak areas after one, two, and three freeze-thaw
cycles and freshly prepared urine samples. Also, MTD in
urine samples exhibited no chromatographic changes when
stored refrigerated at 5C for 3 days, and at 20C for 4
weeks. Urine samples were found to be stable at room tem-
perature upon standing for at least 8 hr.
Conclusion
A new approach for high-throughput monitoring of MTD in
human urine has been developed by modifying a conven-
tional gradient elution HPLC. Most importantly, the novel
instrument configuration substantially reduces the time
needed to re-equilibrate the analytical columns between con-
secutive gradient runs, thereby reducing the total time for
each analysis. The overall cycle time was 14 min for the sin-
gle column analysis, which could be halved in the case time,
where time-overlapping process was used, i.e., injecting a
new sample on the second analytical column before finaliz-
ing the cleanup and the re-equilibration of the first analytical
column for the former sample. No interference in the assay
from any endogenous components or other concurrently
used drugs was observed. The reduced sample handling
and the short run time made it possible to analyze eight
samples per hour. Validity of the method was studied and
the method was precise and accurate within a linearity range
from 0.1 to 40 mg=mL. The high sensitivity and selectivity of
the coupled-column double-injection procedure, makes it a
suitable technique for the analysis of MTD in human urine
samples.
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