Journal of Analytical Sciences, Methods and Instrumentation, 2012, 2, 194-202
http://dx.doi.org/10.4236/jasmi.2012.24030 Published Online December 2012 (http://www.SciRP.org/journal/jasmi)
Coupling of on-Line Pre-Column Oxidative Cleavage and
Solid-Phase Enrichment with Liquid Chromatography
Using an Eco-Friendly Analytical Procedure to Determine
Low Levels of Methotrexate
Samy Emara1*, Walaa Zarad1, Maha Kamal2, Ramzia EL-Bagary3
1Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University, Cairo, Egypt; 2Pharmaceutical Analyti-
cal Chemistry Department, Faculty of Pharmacy, Modern Sciences and Arts University, 6th of October City Egypt; 3Pharmaceutical
Chemistry Department, Faculty of Pharmacy, Cairo University, Giza, Egypt.
Received September 28th, 2012; revised November 9th, 2012; accepted November 18th, 2012
ABSTRACT
A simple, sensitive and precise green high-performance liquid chromatographic method including on-line pre-column
oxidation combined by column switching with a short Hypersil ODS analytical column (100 mm × 4.0 mm i.d.) for
enrichment and separation was developed and validated to determine low levels of methotrexate (MTX). The method
was based on oxidative cleavage of MTX into highly fluorescence products, 2,4-diaminopteridine-6-carboxaldehyde
and the corresponding 2,4-diaminopteridine-6-carboxylic acid, during the flow of phosphate buffer (0.04 M, pH 3.4)
containing the analyte through the packed reactor of cerium (IV) trihydroxyhydroperoxide (CTH) at a flow-rate of 0.2
mL/min and 40˚C. The fluorescent products were enriched on the head of ODS analytical column for the final separa-
tion. The separation was performed at room temperature using an environmentally friendly mobile phase consisting of
ethanol and phosphate buffer (0.04 M, pH 3.4) in the ratio of 10:90 (v/v). The eluent was monitored at emission and
excitation wavelengths of 463 and 367 nm, respectively. The method was successfully applied, without any interference
from the excipients, for the determination of drug in tablets and vials with a detection limit of 0.06 ng/mL from 500 L
of sample MTX.
Keywords: Cerium (IV) Trihydroxyhydroperoxide; HPLC; Methotrexate; ODS Analytical Column; On-Line
Pre-Column Oxidative Cleavage
1. Introduction
Methotrexate (MTX, 2,4-diamino-4-deoxy-N10-methyl-
pteroglutamic acid) is one of the most widely used anti-
cancer drugs and acts as an antimetabolite of folic acid.
In high doses it is used in treatment of some solid tumors
and leukemia. It is also used in a number of autoimmune
diseases [1].
Many methods have been developed for the determi-
nation of MTX, including spectrofluorimetry [2-4], im-
munoassay [5], capillary electrophoresis [6,7] and high-
performance liquid chromatography (HPLC) with UV
detection [8-13]. Due to its higher sensitivity and selec-
tivity, the measurement of low MTX level was more pre-
cise in the HPLC with fluorescence detection [14-25].
MTX does not show native fluorescence but it can be
derivatized into strongly fluorescent product after variety
of successful analytical schemes. Several methodologies
have been developed for the oxidation of MTX to a highly
fluorescent product, 2,4-diaminopteridine-6-carboxalde-
hyde and the corresponding 2,4-diaminopteridine-6-car-
boxylic acid. These methods were based on photo-oxida-
tive irradiation at 254 nm [14,19,20,23,24], electroche-
mical oxidation [16,17], oxidation with permanganate
[4,18,25] and hydrogen peroxide [22-24]. Also, flow
injection analysis (FIA) of MTX in pharmaceutical for-
mulations has been reported [26,27]. These methods are
based on the oxidation of the MTX into highly fluores-
cent product 2,4-diaminopteridine-6-carboxylic acid by
on-line electrochemical oxidation and acidic potassium
permanganate; respectively. A sequential injection ana-
lysis has also been reported for the determination of
MTX using amperometric biosensor detector [28]. Cerium
(IV) trihydroxyhydroperoxide (CTH) has been introduced
as a packed reactor in a flowing system for conversion of
*Corresponding author.

Coupling of on-Line Pre-Column Oxidative Cleavage and Solid-Phase Enrichment with Liquid
Chromatography Using an Eco-Friendly Analytical Procedure to Determine Low Levels of Methotrexate
195
MTX into a highly fluorescent 2,4-diaminopteridine-6-
carboxaldehyde and the corresponding 2,4-diaminopte-
ridine-6-carboxylic acid (Figure 1) [29]. The United
State Pharmacopoeia for the determination of MTX is
based on HPLC with UV detection at 302 nm [30].
In recent years, more strict regulation related to the
quality control of pharmaceuticals led to increasing de-
mands on automation of the analytical assays carried out
in appropriate control laboratories. Also, greener ana-
lytical methods, which minimize the use of toxic chemi-
cals and/or eliminate the generation of toxic wastes, are
strongly demanded, in order to prevent the environmental
pollution and human hazards. Therefore, our study was
involved in a research effort aimed to expand the auto-
mation by incorporating oxidation and pre-concentration
steps prior to HPLC separation of MTX in an automated
green procedure using column switching technique. A
packed reactor FIA method coupled with on-line solid
phase enrichment (SPEn) on a head of ODS analytical
column for the final separation was established for the
determination of MTX. The on-line pre-column proce-
dure includes injecting MTX into a flowing stream of
0.04 M phosphate buffer (pH 3.4) carried through the
packed reactor of CTH for oxidation. In such a way, the
sample zone meets the CTH packed reactor in a con-
trolled manner while the rest of the system is filled with
phosphate buffer. Accordingly, reagent consumption is
greatly decreased and the system is simplified with fewer
junctions for mixing of reagent, sample and carrier
N
N
N
N
C
H
2
NH
2
H
2
N
N C
O
N
HCH
COOH
CH
2
CH
2
COOH
pH 3.4CTH
N
N
N
N
CHN
O
N
HCH
COOH
CH
2
CH
2
COOH
+
NH
2
H
2
N
CHO
Pteridine Carboxaldehyde
CTH
pH 3.4
N
N
N
N
NH
2
H
2
N
COOH
Pteridine Carboxylic acid
MTX
CH
3
CH
3
Figure 1. Structures of the investigated compounds.
streams. The application of phosphate buffer as a flowing
stream and CTH as a packed reactor are considered to be
the main approaches complying with green analytical
chemistry principles. Also, pre-concentration online be-
fore HPLC separation could enhance concentration de-
tection limits of MTX. A particularly attractive feature of
this method was that a simple green isocratic analytical
mobile phase consisting of ethanol and 0.04M phosphate
buffer (pH 3.4) in the ratio of 10:90 (v/v) could be used
for chromatographic separation of the fluorescent prod-
ucts on a short Hypersil ODS analytical column. The on-
line oxidative cleavage-SPEn-HPLC separation strategy
with fluorescence detection appeared to be a viable ap-
proach for the determination of MTX in pharmaceutical
formulation down to a level of 0.20 ng/mL. The results
were evaluated by parallel experiments and analysis us-
ing the procedure recommended by the USP based on
conventional HPLC method.
2. Experimental
2.1. Reagents
MTX (99.83%) was obtained from Kyowa Hakki (Tokyo,
Japan). The present method was applied to the determi-
nation of MTX in its pharmaceutical formulations: 1)
Methotrexate tablets (Batch No. 1368454) 2.5 mg of
MTX; 2) Methotrexate vials (Batch No. 1236627) 50 mg
of MTX (Orion Corporation, Finland). Ethanol used was
HPLC grade (BDH, Poole, UK). Distilled water was used
for the preparation of all reagents and solutions. Potas-
sium dihydrogen phosphate, ortho-phosphoric acid and
isopropyl alcohol used were analytical grades.
2.2. Instrumentation
The HPLC (Agilent Technologies, CA, USA) apparatus,
illustrated in Figure 2, consisted of two solvent delivery
pumps (Agilent 1100 Series Iso pump G1310A). One
used to deliver the carrier solution at a flow-rate of 0.2
mL/min and the other to deliver isocratic mobile phase at
a flow-rate of 1 mL/min. A model 7125 sample injection
valve (500 µL) and a model 7010 flow direction switch-
ing valve were applied to load the sample onto the CTH
packed reactor and facilitate oxidative cleavage of MTX
into highly fluorescence products and to control the flow
direction switching and isocratic elution, respectively (Rheo-
dyne, Berkeley, CA, USA). This system was equipped with
two columns; one was a short (50 × 7.5 mm i.d.) CTH
oxidant column for oxidative-cleavage of MTX into
highly fluorescent products and the other was an analyti-
cal column of Thermo Scientific Hypersil ODS (100 ×
4.0 mm i.d., 5 µm particle size from Thermo Scientific,
FL, USA). A fluorescence detector monitored the eluents,
(Agilent 1200 series, G1321A) set at an excitation wave-

Coupling of on-Line Pre-Column Oxidative Cleavage and Solid-Phase Enrichment with Liquid
Chromatography Using an Eco-Friendly Analytical Procedure to Determine Low Levels of Methotrexate
196
length of 367 nm and an emission wavelength of 463
nm. Data acquisition was performed on Agilent LC Chem-
Station software. The analytical column temperature
was ambient, while that of the CTH packed reactor was
40˚C.
2.3. Standard Solution and Calibration
Stock standard solution of MTX (10 μg/mL) was pre-
pared by dissolving an accurately weighed amount of
MTX in distilled water. A known volume of the stock
standard solution was diluted with the same solvent to
obtain a concentration of 1 μg/mL MTX (solution A).
The standard solutions for calibration were prepared
daily by serial dilutions of appropriate volumes of solu-
tion A to produce MTX in the final concentration range of
1 - 40 ng/mL. An aliquot of 500 μL was analyzed for
MTX according to the proposed procedure. The stock
standard solutions were stored frozen at −20˚C until used.
S.V. Analytical Column
Detector
Waste
Pump
I
CTH column
Waste Pump
II
S.V.
Waste
Pump
I
CTH column
Waste
Analytica l Column
Detector
Pump
II
Se
p
aration ste
p
(
B
)
Derivatization ste
p
(
A
)
Figure 2. Schematic diagram of the online pre-column
HPLC for the analysis of MTX in pharmaceutical formula-
tion: the system in initial position, ready for sample injec-
tion, derivatization and enrichment steps (A); the separa-
tion step, in which the CTH column is isolated from HPLC
circulation (B) (S.V.: six-port switching valve).
2.4. Tablets
A total of 20 tablets containing MTX as the active ingre-
dient were weighed and finally powdered. A portion of
the powder equivalent to 10 mg of MTX was accurately
weighed and transferred to a 100 mL calibrated flask and
dissolved in about 90 mL of distilled water using an ultra
sonic bath. The solution was diluted to the volume with
the same solvent and then filtered. The first portion of the
filtrate was discarded and the remainder was used as a
stock sample solution (solution A, 100 g/mL). A known
volume of solution A was diluted quantitatively with
distilled water to obtain a concentration of 1 μg/mL
MTX (solution B). A further dilution was carried out to
obtain a final concentration of 20 ng/mL MTX. An ali-
quot of 500 μL was analyzed for MTX according to the
proposed procedure. The standard solutions for calibra-
tion were freshly prepared and stored in dark flask at 5˚C
during use.
2.5. Vials
An accurately volume equivalent to 10 mg MTX was
transferred to a 100 mL calibrated flask and completed to
the mark with distilled water. Serial dilutions as de-
scribed under tablets were made to obtain final concen-
tration of 20 ng/mL. An aliquot of 500 μL was analyzed
for MTX according to the proposed procedure.
2.6. Preparation of CTH Column
The CTH packing materials were suspended in isopropyl
alcohol and degassed under vacuum with continuous
stirring for 10 min. A stainless-steel cylinder (100 mm ×
7.5 mm i.d.) was used as a reservoir for the CTH packing
materials. This reservoir was connected to short column
(50 mm × 7.5 mm i.d.) and the suspended CTH supplied
from the reservoir was packed into the column with the
aid of an HPLC pump at flow-rate of 5 mL/min with
ethanol as a purge solvent (10 min). Pumping must con-
tinue until a constant pressure is reached. The cylinder
was then disconnected and a mixture of ethanol and dis-
tilled water (1:1) was passed through the column at a
flow-rate of 1 mL/min for further 10 min. The column
was then equilibrated with 0.04 M phosphate buffer of
pH 3.4 at a flow-rate of 1 mL/min for 30 min.
2.7. Mobile Phases
Two different mobile phases were employed in the assay
procedure; one was phosphate buffer (0.04 M, pH 3.4),
which was used as a carrier stream (MI) to deliver the
sample to the CTH packed reactor in the oxidation step.
The other was an isocratic solvent system (MII) consist-
ing of ethanol and phosphate buffer (0.04 M, pH 3.4)
(10:90 v/v) which was used to elute the enriched fluo-

Coupling of on-Line Pre-Column Oxidative Cleavage and Solid-Phase Enrichment with Liquid
Chromatography Using an Eco-Friendly Analytical Procedure to Determine Low Levels of Methotrexate
197
rescent products from the head of the Thermo Scientific
Hypersil ODS analytical column to the fluorescence de-
tector for further separation. All mobile phases were
freshly prepared on the day of use, filtered through 0.45
µm filters (Millipore, Billerica, MA), and degassed ul-
trasonically under vacuum.
2.8. General Procedure
A 500 µL aliquot of MTX sample was loaded into the
injection valve and then injected into MI. The moving
zone of MTX passed through the CTH column at a flow-
rate of 0.2 mL/min (pump I). The oxidative cleavage of
MTX occurs during the flow of MI containing the drug
through the CTH column. Pre-concentration was per-
formed by means of the flow of the fluorescent prod-
ucts from the oxidant column to the analytical column
head. After 6 min the valve was switched into position B
(Figure 2). At this position, the MII could pass directly
through the analytical column, where the fluorescent
2,4-diaminopteridine-6-carboxaldehyde and the corre-
sponding 2,4-diaminopteridine-6-carboxylic acid were
then separated. The flow-rate was maintained at 1 mL/
min and the fluorescence intensity of the eluting com-
pounds was monitored at emission and excitation wave-
lengths of 463 and 367 nm, respectively. At 9 min after
injection, the valve was switched into position A (Figure
2).
3. Result & Discussion
In order to apply CTH as a packed oxidant reactor, in the
described manifold (Figure 2), the optimization of de-
veloped system was investigated considering particularly
the effect of the pH, concentration and flow rate of car-
rier stream (MI), sample volume and packed reactor
temperature. During the phase of optimization of the
chromatographic system, analytical mobile phase (MII)
was investigated to evaluate the effect of the solvent
composition and pH on the compounds separation. The
detection wavelengths were chosen using the spectrum
mode of the fluorescence detection with respect to the
maximum sample signals. This mode enabled the deter-
mination of the optimum emission and excitation wave-
lengths in real conditions during measurement. The final
measurement conditions were at emission and excitation
wavelengths of 463 and 367 nm, respectively.
3.1. Effect of pH and Concentration of Carrier
Stream
Adjustment of the carrier stream pH was necessary to
improve the reaction completeness between the analyte
and CTH packed reactor. The effect of this parameter
was studied in the pH range 2.8 - 5 using buffer solutions
from phosphate and acetate. Phosphate buffer gave the
best performance as a carrier stream and was selected in
all further experiments. As shown in Figure 3, the pH of
buffer solution affected the fluorescent derivatization of
MTX severely. The highest intensity was observed in the
narrow range of pH 3.2 - 3.6 (Figure 3). At pH < 3.0, a
low signal response was observed which might suggest
the decomposition of the peroxy groups of the CTH ma-
terials, whereas at pH > 3.8, the detector signals was de-
creased abruptly probably due to the low CTH reactor
efficiency which reduces drastically the oxidative cleav-
age of MTX into highly fluorescent derivative. Accord-
ingly, phosphate buffer solution of pH 3.4 was selected
as the optimum carrier stream.
The variation in the fluorescent intensity of MTX with
CTH packed oxidant was examined using phosphate
buffer of concentrations varying from 0.02 to 0.1 M. Best
analytical signals were verified within the concentration
range 0.02 - 0.05 M (Figure 4). With increasing the con-
centration of buffer, the detector response was drastically
decreased, probably due to greater quenching effect on
the fluorescence signal intensity. Although, 0.02 M phos-
phate buffer showed slight improvement of signal inten-
sity, 0.04 M was chosen as a compromise between de-
tector response and precision.
3.2. Effect of Temperature
The packed reactor temperature also has a critical effect
on the reaction progress. The effect of this parameter on
the derivatization of MTX with CTH packed oxidant into
highly fluorescent derivatives was investigated in the
range of 25˚C - 65˚C. Lower temperatures were inade-
quate whereas elevating the temperature within the range
of 25˚C - 65˚C resulted in an increase in the reaction rate
and subsequently detector response (Figure 5). It was
also observed that heating slightly above 45˚C resulted in
an increase of column pressure and showed a generally
5
7
9
11
13
15
17
19
21
2.833.23.43.63.844.24.44.6 4.85
pH
Detect or r espons e
(Arbitrary unit)
Figure 3. Effect of phosphate buffer pH on the reaction ef-
ficiency of CTH-packed reactor with MTX.

Coupling of on-Line Pre-Column Oxidative Cleavage and Solid-Phase Enrichment with Liquid
Chromatography Using an Eco-Friendly Analytical Procedure to Determine Low Levels of Methotrexate
198
5
7
9
11
13
15
17
19
21
23
0.02 0.04 0.06 0.080.1
Buffer concentration (M)
Detector response
(Arbitrary unit)
Figure 4. Effect of phosphate buffer concentration on the
reaction efficiency of CTH-packed reactor with MTX.
10
12
14
16
18
20
22
24
2530 3540 4550 5560 65
Temperature (°C)
Detector response
(Arbitrary unit)
Figure 5. Effect of temperature on the reaction efficiency of
CTH-packed reactor with MTX.
drastic effect on the CTH packed oxidant life span. Con-
sidering the effective reaction temperature and the packed
oxidant limitations, 40˚C was selected as the optimum
value because under this condition good sensitiveity and
reproducibility were achieved.
3.3. Effect of the Flow Rate
The reaction of CTH column with MTX was highly in-
fluenced by the flow-rate of the carrier stream. The use
of rapid analyte transport into and out of the packed re-
actor would be advantageous for the fast analysis; how-
ever, it was also essential that the flow-rate of the carrier
stream not to be so rapid as to compromise the extent of
the analyte in the packed reactor. The effect of the flow-
rate was checked over the range of 0.2 - 0.8 mL/min.
When the flow-rate was reduced from 0.8 to 0.2 mL/min,
a maximum increase in detector response of 2,4-dia-
minopteridine-6-carboxylic acid was observed (Figure 6).
At the same time, the response ratio of the two products
was also changed. Hence, it could be postulated that any
decrease in the flow-rate of the carrier stream will in-
crease the residence time (reaction time) between the
solid surface of CTH and the moving zone of MTX,
whereas at higher flow-rates less fluorescent products
could be produced and the recorded signal was decreased.
The residence time between the sample zone containing
MTX and the solid-phase reactor is very important for
the reaction to proceed sufficiently and to achieve a sub-
stantial enhancement of the detector response. In the
present work, a lower flow-rate was justified for the de-
termination of MTX because peak enrichment could be
achieved on the top of the analytical column (Hypersil
ODS analytical column). As far as the oxidation reaction
proceeded, 2,4-diaminopteridine-6-carboxaldehyde was
converted into the corresponding 2,4-diaminopteridine-
6-carboxylic acid. Thus, it can be deduced that the signal
of 2,4-diaminopteridine-6-carboxylic acid became more
predominant when a lower flow rate was employed. The
fluorescent products could be accumulated on the top of
the analytical column with a zone width almost inde-
pendent on the flow-rate of carrier stream. A compromise
between analytical signal and sample frequency was es-
tablished by choosing a working flow-rate of 0.2 mL/
min.
As a result, the optimal reaction conditions could be
achieved by using phosphate buffer (0.04 M, pH 3.4) at a
flow-rate of 0.2 mL/min and CTH column temperature of
40˚C.
3.4. Effect of Sample Volume
In an effort to push the concentration detection limit to a
lower level, we attempted to use the analytical column
head to pre-concentrate MTX by loading of a large sam-
ple volume of MTX to the packed CTH reactor. The de-
sign of a switching valve containing a packed reactor for
on-line pre-column derivatization and sample enrichment
was described (Figure 2, position A). Samples were
loaded on to the CTH packed reactor with a carrier mo-
bile phase (MI) and pump I, while pre-concentration was
performed by means of the flow of the fluorescent prod-
ucts from the packed reactor to the analytical column
head. Different injection volumes (50 - 600 µL) were
tested to introduce decreasing concentration of MTX.
The efficiency of enrichment for MTX was evaluated on
the basis of the linearity of calibration graph constructed
over sample volumes (50 - 600 L, at 50 L interval). It
was found that, the CTH packed reactor could tolerate
large volumes of MTX standard solution and the linear
relationship (r2 0.9992) between the peak area and the
injected volumes was observed over the range of 50 -
500 L sample volume. If too large a sample is used
(more than 500 µL), then the linearity of MTX between
the peak area and concentration will be disturbed because

Coupling of on-Line Pre-Column Oxidative Cleavage and Solid-Phase Enrichment with Liquid
Chromatography Using an Eco-Friendly Analytical Procedure to Determine Low Levels of Methotrexate
199
D etector res
p
onse
I
II
0 3
d
0 3
II
I
g
0 3
II
a
I I II
0 3
b
I II
0 3
c
I
II
0 3
e
II
I
0 3
f
Figure 6. Chromatograms obtained after oxidative cleavage of MTX with CTH and fluorimetric detection (20 ng/mL). Peaks:
I: 2,4-diaminopteridine-6-carboxylic acid; II: 2,4-diaminopteridine-6-carboxaldehyde. Flow rates: 0.8 mL/min (a); 0.7 mL/
min (b); 0.6 mL/min (c); 0.5 mL/min (d); 0.4 mL/min (e); 0.3 mL/min (f); 0.2 mL/min (g).
a long residence time was found necessary to get repro-
ducible results upon using large volume of MTX samples.
Accordingly, sample volume of 500 µL MTX sample
was selected as a compromise between the sensitivity
and accuracy. The pre-concentration was effective for the
proposed method, achieving detection limit of 0.06 ng/
mL of MTX for a sample volume of 500 µL with fluo-
rescence detection.
3.5. Optimization of the Chromatographic
System
Optimization studies for the oxidative cleavage of MTX
with CTH were carried out in the presence of phosphate
buffer (0.04 M, pH 3.4) aqueous carrier stream (MI).
Also, HPLC experimental set-up required an optimum
mobile phase (MII) composition to provide the necessary
chromatographic separation of the compounds on the
analytical column. It was noted that the CTH reactor
needed a long equilibration time with MI after passing of
any ethanol containing mobile phase (MII) through it.
Thus, the system manifold was set up with column-
switching technique and two separate pumps to deliver
MI and MII independently in order to eliminate such a
long equilibration time (Figure 2).
The main objectives of the chromatographic step were
to pre-concentrate and separate the highly fluorescent
derivatives, 2,4-diaminopteridine-6-carboxaldehyde and
the corresponding 2,4-diaminopteridine-6-carboxylic acid.
The chromatographic separation was achieved on a short
Thermo Scientific Hypersil ODS analytical column (100
mm × 4.0 mm, 5m) using mixture of phosphate buffer
(0.04 M, pH 3.4)-ethanol as MII. The optimization pro-
cedure was continued by changing the pH of MII as well
as changing the ratios of phosphate buffer and ethanol.
Variation in the pH of mobile phase in the range of 2.0 -
6 weakly affected retention behavior of the fluorescent
products. While variation of ethanol concentration strongly
affected retention behaviors and band broadening. A
green mobile phase of ethanol and phosphate buffer (0.04
M, pH 3.4) (10:90 v/v) was found to give acceptable
separation at a flow rate of 1.0 mL/min and 25˚C column
temperature (Figure 6).
3.6. Method Validation
3.6.1. Linearity
The linearity was evaluated and established by triplicate
analysis of the standard solutions of MTX. The obtained
peak areas were plotted against the corresponding con-
centrations to generate calibration curve. Good linearity
was evident (r2 = 0.9997) over the examined concentra-
tion range 1 - 40 ng/mL. The equation for the best-fit
straight line was determined by the linear regression
analysis as Y = a + bC, where Y is the peak area and C
denotes the concentration in ng/mL of MTX. Character-
istic parameters of the linear calibration curve are shown
in Table 1.
3.6.2. Limit of Detection and Quantification
The limit of detection (LOD), defined as the lowest con-
centration of MTX that can be clearly detected above the
base line signal, is estimated as three-times the signal-
to-noise ratio. The LOD was determined (n = 3) by injec-
tion of MTX in decreasing concentrations. The LOD was
found to be 0.06 ng/mL (Table 1). The LOQ is often
defined as 10 times the signal-to-noise ratio. The LOQ
was determined (n = 3) by injection of MTX in decreas-
ing concentrations. The precision was calculated for each
concentration. Then, the LOQ was calculated as the con-

Coupling of on-Line Pre-Column Oxidative Cleavage and Solid-Phase Enrichment with Liquid
Chromatography Using an Eco-Friendly Analytical Procedure to Determine Low Levels of Methotrexate
200
centration, where the precision was less than or equal to
15% and was found to be 0.20 ng/mL (Table 1).
3.6.3. Precision and Accuracy
The relative standard deviation (RSD %) and the relative
error (RE %) of the mean measured concentration were
served as measures of accuracy and precision for valida-
tion of the assay procedure. The intra- and inter-day as-
say precision and accuracy for MTX are summarized in
Table 2. Within the examined range, the intra-day re-
producibility and accuracy of the assay were excellent,
with RSD % being in the range of 0.23 - 0.28 and with
RE % ranged from −0.33 to −0.49. The inter-day RSD%
were 0.26 - 0.32 and the mean RE % ranged from −0.36
to −0.56. Repeatability and reproducibility of MTX sam-
ples with high and low concentration levels were below
0.60%, indicating a reliable measurement using the pro-
posed method (Table 2). RE was evaluated by back-
calculation and expressed as the percent deviation be-
tween concentration added and concentration found ac-
cording to the following:
concentration foundconcentrationadded
RE 100
concentration added


Table 1. Characteristic parameters for the regression equa-
tions of the proposed methoda.
Parameters MTX
Calibration range (ng/mL) 1-40
Detection limit (ng/mL) 0.06
Quantitation limit (ng/mL) 0.20
Slope (b) 1.4465
Standard error of the slope 0.0104
Intercept (a) 0.2421
Standard error of the intercept 0.2149
Correlation coefficient (r2) 0.9997
aY = a + bC, where C is the concentration of MTX in ng/mL and Y is the
peak area.
Table 2. Precision and accuracy validation of MTX.
Concentration
(ng/mL)
Recovery % ±
RSD Mean RE (%)
Intra-assaya 5 99.54 ± 0.24 −0.46
10 99.65 ± 0.27 −0.35
20 99.67 ± 0.23 −0.33
40 99.51 ± 0.28 −0.49
Inter-assaya 5 99.48 ± 0.26 −0.52
10 99.62 ± 0.31 −0.38
20 99.64 ± 0.26 −0.36
40 99.44 ± 0.32 −0.56
a Average of five determinations.
3.6.4. Interference Studies
In order to examine the selectivity of the proposed
method, the effect of common excipients normally used
in pharmaceutical formulations was studied. Solutions
containing MTX (20 ng/mL) in the presence of more
than 100 folds of common additives such as maize starch,
calcium hydrogen phosphate, magnesium stearate, man-
nitol, red ferric oxide, lactose, methylhydroxypropylcel-
lulose, sodium stearyl fumarate, microcrystalline cellu-
lose, hydrophobic colloidal silica and sucrose were pre-
pared. The undissolved materials were filtered off before
injection. No significant changes were observed on the
results and recoveries in the range of 99.42% - 99.75%
were obtained in all cases.
3.7. Drug Content Analysis
The proposed on-line pre-column oxidative cleavage
method was applied for the determination of MTX in its
commercial formulations together with the official USP
method [30]. As indicated, the assay results obtained by
the proposed method were in accordance with those ob-
tained by the official method. The accuracy and precision
of the developed method were further judged by applying
t- and F-test at 95% confidence level. The experimental
t- and F-values did not exceed the theoretical values,
which support the similar accuracy and precision of the
proposed and official USP methods (Table 3). The accu-
racy and precision of the developed method were further
judged by applying t- and F-test at 95% confidence level.
The experimental t- and F-values did not exceed the
theoretical values, which support the similar accuracy
and precision of the proposed and official USA methods
(Table 3).
Table 3. Determination of MTX in commercial formula-
tions by the proposed and official methods.
Recovery (%)a ± RSD
Commercial formulationProposed method Official method
Tablets (20 ng/mL) 99.84 ± 0.34 99.86 ± 0.30
Student’s t-test 0.09 (2.30)b
F-test 1.22 (6.38)b
Vials (20 ng/mL) 99.85 ± 0.27 99.87 ± 0.29
0.13 (2.30)b
1.14 (6.38)b
Recovery (%)c ± RSD
Tablets 99.48 ± 0.35
Vials 99.50 ± 0.32
aAverage of five determinations; bTheoretical values at p = 0.05; cFor stan-
dard addition of 50% of the nominal content.

Coupling of on-Line Pre-Column Oxidative Cleavage and Solid-Phase Enrichment with Liquid
Chromatography Using an Eco-Friendly Analytical Procedure to Determine Low Levels of Methotrexate
201
Mean value are very close to the theoretical concentra-
tions, showing method % recoveries from 99.48 to 99.50
and RSD from 0.32 to 0.35. These results indicate that
the effects of the common additives and ingredients of
the pharmaceutical formulations do not interfere with the
determination of MTX.
3.8. Robustness
To determine robustness of the proposed method, ex-
perimental conditions such as flow-rate, pH and concen-
tration of the buffer solution used as a carrier stream,
packed reactor temperature and organic content of the
mobile phase were purposely altered and the detector
responses were evaluated. Variation of each parameter
by ±2% did not have a significant effect on the detector
response.
4. Conclusion
In this study, a green on-line pre-column oxidation-SPEn-
HPLC separation strategy using a packed oxidant reactor
of CTH and a short Hypersil ODS analytical column
(100 mm × 4.0 mm i.d.) has been developed to determine
low levels of MTX. The method was a powerful analytic-
cal technique that had excellent sensitivity, sufficient
accuracy and required relatively simple and inexpensive
instrumentation. These advantages made the proposed
method an attractive procedure for the routine quality
control and dosage form analysis of MTX at very low
concentration level, down to 0.20 ng/mL. The applicabil-
ity of this method was evaluated by the determination of
MTX in pharmaceutical formulations. Common excipi-
ents used as additives in pharmaceutical preparations did
not interfere. Furthermore, the proposed method is
worthy contributed to the existing environmental friendly
analytical chemistry due to reducing or elimination of the
use and generation of hazardous substances.
REFERENCES
[1] S. Shen, T. O’Brien, L. M. Yap, H. M. Prince and C. J.
McCormack, “The Use of Methotrexate in Dermatology:
A Review,” Australasian Journal of Dermatology, Vol.
53, No. 1, 2102, pp. 1-18.
[2] M. Bărcă, M. Ilie, D. L. Baconi, A. M. Ciobanu, D. Bălă-
lău and G. T. Burcea, “Spectrofluorimetric Methotrexate
Assay in Human Plasma,” Farmacia, Vol. 58, No. 1, 2010,
pp. 95-101.
[3] S. M. Sabry, M. Abdel-Hady, M. Elsayed, O. T. Fahmy
and H. M. Mahr, “Study of Stability of Methotrexate in
Acidic Solution. Spectrofluorimetric Determination of Me-
thotrexate in Pharmaceutical Preparations through Acid-
Catalyzed Degradation Reaction,” Journal of Pharma-
ceutical and Biomedical Analysis, Vol. 32, No. 3, 2003,
pp. 409-423. doi:10.1016/S0731-7085(03)00239-5
[4] A. Espinosa-Mansilla, I. Durán Merás, A. Zamora Madera,
L. Pedano and C. Ferreyra, “Kinetic Fluorimetric Deter-
mination of the Antineoplastic Methotrexate (MTX) in
Human Serum,” Journal of Pharmaceutical and Biomedi-
cal Analysis, Vol. 29, No. 5, 2002, pp. 851-858.
doi:10.1016/S0731-7085(02)00212-1
[5] H. Hayashi, C. Fujimaki, S. Tsuboi, T. Matsuyama, T.
Daimon and K. Itoh, “Application of Fluorescence Polari-
zation Immunoassay for Determination of Methotrexate-
Polyglutamates in Rheumatoid Arthritis Patients,” The
Tohoku Journal of Experimental Medicine, Vol. 215, No.
1, 2008, pp. 95-101. doi:10.1620/tjem.215.95
[6] H. L. Cheng, S. S. Chiou, Y. M. Liao, C. Y. Lu, Y. L.
Chen and S. M. Wu, “Analysis of Methotrexate and Its
Eight Metabolites in Cerebrospinal Fluid by Solid-Phase
Extraction and Triple-Stacking Capillary Electrophore-
sis,” Analytical and Bioanalytical Chemistry, Vol. 398,
No. 5, 2010, pp. 2183-2190.
doi:10.1007/s00216-010-4152-3
[7] H. L. Cheng, Y. M. Liao, S. S. Chiou and S. M. Wu, “On-
Line Stacking Capillary Electrophoresis for Analysis of
Methotrexate and Its Eight Metabolites in Whole Blood,”
Electrophoresis, Vol. 29, No. 17, 2008, pp. 3665-3673.
doi:10.1002/elps.200800029
[8] S. Fang, C. P. Lollo, C. Derunes and M. J. LaBarre, “De-
velopment and Validation of a Liquid Chromatography
Method for Simultaneous Determination of Three Proc-
ess-Related Impurities: Yeastolates, Triton X-100 and
Methotrexate,” Journal of Chromatography B, Vol. 879,
No. 30, 2011, pp. 3612-3619.
doi:10.1016/j.jchromb.2011.10.003
[9] E. Klapkova, J. Kukacka, K. Kotaska, I. Suchanska, R.
Urinovska and R. Prusa, “The Influence of 7-OH Meth-
otrexate Metabolite on Clinical Relevance of Methotrexate
Determination,” Clinical Laboratory, Vol. 57, No. 7-8,
2011, pp. 599-606.
[10] K. Michail and M. S. Moneeb, “Determination of Meth-
otrexate and Indomethacin in Urine Using SPE-LC-DAD
after Derivatization,” Journal of Pharmaceutical and Bio-
medical Analysis, Vol. 55, No. 2, 2011, pp. 317-324.
doi:10.1016/j.jpba.2011.01.032
[11] L. Hu, Y. Liu and S. Cheng, “Simultaneous Determina-
tion of Six Analytes by HPLC-UV for High Throughput
Analysis in Permeability Assessment,” Journal of Chro-
matographic Science, Vol. 49, No. 2, 2011, pp. 124-128.
doi:10.1093/chrsci/49.2.124
[12] T. Sartori, F. S.Murakami, A. P. Cruz and A. M. de Cam-
pos, “Development and Validation of a Fast RP-HPLC
Method for Determination of Methotrexate Entrapment
Efficiency in Polymeric Nanocapsules,” Journal of Chro-
matographic Science, Vol. 46, No. 6, 2008, pp. 505-509.
[13] D. A. el-Hady, N. A. el-Maali, R. Gotti, C. Bertucci, F.
Mancini and V. Andrisano, “Methotrexate Determination
in Pharmaceuticals by Enantioselective HPLC,” Journal
of Pharmaceutical and Biomedical Analysis, Vol. 37, No.
5, 2005, pp. 919-925. doi:10.1016/j.jpba.2004.07.046
[14] M. Uchiyama, T. Matsumoto, T. Matsumoto, S. Jimi, Y.

Coupling of on-Line Pre-Column Oxidative Cleavage and Solid-Phase Enrichment with Liquid
Chromatography Using an Eco-Friendly Analytical Procedure to Determine Low Levels of Methotrexate
202
Takamatsu, K. Tamura and S. Hara, “Simple and Sensi-
tive HPLC Method for the Fluorometric Determination of
Methotrexate and Its Major Metabolites in Human Plasma
by Post-Column Photochemical Reaction,” Biomedical Chro-
matography, Vol. 26, No. 1, 2012, pp. 76-80.
doi:10.1002/bmc.1628
[15] L. van Haandel and J. F. Stobaugh, “Bioanalytical Method
Development for a Generation 5 Polyamidoamine Folic
Acid Methotrexate Conjugated Nanoparticle,” Analytical
and Bioanalytical Chemistry, Vol. 397, No. 5, 2010, pp.
1841-1852. doi:10.1007/s00216-010-3716-6
[16] S. Chen and Z. Zhang, “Molecularly Imprinted Solid-
Phase Extraction Combined with Electrochemical Oxida-
tion Fluorimetry for the Determination of Methotrexate in
Human Serum and Urine,” Spectrochimica Acta Part A,
Vol. 70, No. 1, 2008, pp. 36-41.
doi:10.1016/j.saa.2007.07.009
[17] H. Li, W. Luo, Q. Zeng, Z. Lin, H. Luo and Y. Zhang,
“Method for the Determination of Blood Methotrexate by
High Performance Liquid Chromatography with Online
Post-Column Electrochemical Oxidation and Fluorescence
Detection,” Journal of Chromatography B, Vol. 845, No.
1, 2007, pp. 164-168. doi:10.1016/j.jchromb.2006.07.026
[18] I. Durán Merás, A. Espinosa Mansilla and M. J. Rodríguez
Gómez, “Determination of Methotrexate, Several Pteridi-
nes, and Creatinine in Human Urine, Previous Oxidation
with Potassium Permanganate, Using HPLC with Photo-
metric and Fluorimetric Serial Detection,” Analytical Bio-
chemistry, Vol. 346, No. 2, 2005, pp. 201-209.
doi:10.1016/j.ab.2005.07.038
[19] E. A. McCrudden and S. E. Tett, “Improved High-Per-
formance Liquid Chromatography Determination of Meth-
otrexate and Its Major Metabolite in Plasma Using a Poly
(Styrene-Divinylbenzene) Column,” Journal of Chromatog-
raphy B, Vol. 721, No. 1, 1999, pp. 87-92.
doi:10.1016/S0378-4347(98)00439-3
[20] Z. Yu, D. Westerlund and K. S. Boose, “Determination of
Methotrexate and Its Metabolite 7-Hydroxymethotrexate
by Direct Injection of Human Plasma into a Column-
Switching Liquid Chromatographic System Using Post-
Column Photochemical Reaction with Fluorimetric De-
tection,” Journal of Chromatography B, Vol. 689, No. 2,
1997, pp. 379-386. doi:10.1016/S0378-4347(96)00357-X
[21] T. Suzuki, H. Hashimoto and N. Ichinose, “Microanalysis
of Methotrexate by High-Performance Liquid Chroma-
tography Using a Fluorescence Detector,” Fresenius Jour-
nal of Analytical Chemistry, Vol. 351, No. 8, 1995, pp.
806-807. doi:10.1007/BF00323644
[22] H. Kubo, Y. Umiguchi, M. Fukumoto and T. Kinoshita,
“Fluorimetric Determination of Methotrexate in Serum by
High-Performance Liquid Chromatography Using in-Line
Oxidation with Hydrogen Peroxide,” Analytical Sciences,
Vol. 8, No. 6, 1992, pp. 789-792.
doi:10.2116/analsci.8.789
[23] J. Šalamoun, M. Smrž, F. Kiss and A. Šalamounová,
“Column Liquid Chromatography of Methotrexate and Its
Metabolites Using a Post-Column Photochemical Reactor
and Fluorescence Detection,” Journal of Chromatogra-
phy B, Vol. 419, 1987, pp. 213-223.
doi:10.1016/0378-4347(87)80279-7
[24] J. Šalamoun and J. František, “Determination of Meth-
otrexate and Its Metabolites 7-Hydroxymethotrexate and
2,4-Diamino-N10-methylpteroic Acid in Biological Fluids
by Liquid Chromatography with Fluorimetric Detection,”
Journal of Chromatography B, Vol. 378, No. 1, 1986, pp.
173-181.
[25] J. A. Nelson, B. A. Harris, W. J. Decker and D. Farquhar,
“Analysis of Methotrexate in Human Plasma by High-
Performance Liquid Chromatography with Fluorescence
Detection,” Cancer Research, Vol. 37, No. 11, 1977, pp.
3970-3973.
[26] S. Chen, Z. Zhang, D. He, Y. Hu, H. Zheng and C. He,
“Flow-Injection-Electrochemical Oxidation Fluorimetry
for Determination of Methotrexate,” Luminescence, Vol.
22, No. 4, 2007, pp. 338-342. doi:10.1002/bio.968
[27] S. Emara, “Determination of Methotrexate in Pharmaceu-
tical Formulations by Flow Injection Analysis Exploiting
the Reaction with Potassium Permanganate,” II Farmaco,
Vol. 59, No. 10, 2004, pp. 827-833.
doi:10.1016/j.farmac.2004.06.005
[28] R. I. Stefan, R. G. Bokretsion, J. F. van Staden and H. Y.
Aboul-Enein, “Simultaneous Determination of L- and D-
Methotrexate Using a Sequential Injection Analysis/Am-
perometric Biosensors System,” Biosensors and Bioelec-
tronics, Vol. 19, No. 3, 2003, pp. 261-267.
doi:10.1016/S0956-5663(03)00210-0
[29] S. Emara, S. Razee, A.-N. El-Shorbagy and T. Masujima,
“Flow Injection Method for the Determination of Meth-
otrexate with a Column-Packed Oxidizing Agent,” Ana-
lyst, Vol. 121, No. 2, 1996, pp. 183-188.
doi:10.1039/an9962100183
[30] United States Pharmacopeial Convention, “The United
States Pharmacopeia and National Formulary (USP 28-NF
23),” The Official Compendia of Standards, Asian Edi-
tion, The United States Pharmacopeial Convention Inc.,
Rockville, 2005.