MS.NO. 7776-301-SI
Quantification of methotrexate by liquid chromatography
With ultraviolet detection for routine monitoring of plasma levels
Nadia Jebabli, Anis Klouz, Ridha Ben Ali, Emna Gaïes, Issam Salouage,
Mohamed Lakhal and Chalbi Belkahia
Laboratory of Clinical Pharmacology, Centre National de Pharmacovigilance, Tunisia
Adresse
Pr Agrégée Anis KLOUZ
9 Rue Zouhair Essafi
1006, Tunis-Bab Souika,
TUNISIA
Tel : +216 98 646 988
Fax : + 216 71 578 196
E-Mail : anis.klouz@rns.tn
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ABSTRACT
A high-performance liquid chromatographic (HPLC) with ultraviolet detection
incorporating solid phase extraction (SPE) was developed to meet analytical and
metrological requirements for routine serum level monitoring of methotrexate (MTX). In
this study, we optimised several parameters such as temperature, flow rate, composition of
the mobile phase and pH of the buffer solution. The method was validated by linearity,
sensibility and precision studies. Response was found linear over the whole range of the
calibrator set with a correlation coefficient of 0.999. The relative standard error (RSD) of
intra assay were (2.1; 1.3; 2.1%) and (2.2; 2.97; 2.4%) for inter assay. The limit of
quantification and the limit of detection were respectively 0.03µmol/l and 0.0063µmol/l.
The method was validated by linearity and precision studies, and it was successfully used
to analyse clinical samples.
Key words: methotrexate, liquid chromatography, solid-phase extraction
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1 Introduction
Methotrexate (MTX) is a competitive inhibitor of dihydrofolate reductase (Bally et al.,
2007) ; a key enzyme of nucleic acid biosynthesis (Albertioni et al., 1997). Thus, it can
block tumoral cell growth and is widely used as a cytostatic agent (Albertioni et al., 1997).
In cancer treatment, the effectiveness of high-dose MTX therapy followed by leucovorin
(folinic acid) rescue was greatly enhanced by the observation that patients have high risk of
serious toxicity might be detected by monitoring serum methotrexate concentrations (Bally
et al., 2007, Albertioni et al., 1997, Aboleneen et al., 1996 ).
The present study describes an alternative method for the determination of MTX in plasma
using UV detection. The main features of this method include simple pre-treatment and
short run time for assaying the major metabolite hydroxymethotrexate (OH-MTX). This
method was validated and used for the quantification of MTX obtained from patients
receiving treatment with MTX.
2. Materiels and methods
2.1 Chemicals and reagents
Methotrexate, hydroxymethotrexate and 8-chlorothéophylline (used at internal standard IS)
were purchased from Sigma. Potassium dihydrogene phosphate (KH2PO4) was purchased
from Carlo Erba. Methanol, acetonitrile were HPLC grade from Merck. All other
chemicals and solvents used were of analytical grade. The reference of cartridge used for
solid phase extraction (SPE) was (Chromsystems Ref: 7008 lot 212).
2.2 Instrumentation
The LC system, used for method development and validation was from Merck and
consisted of L-6000 pump, L-5025 oven and L-4250 spectra absorbance detector.
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2.3 Sample preparation
The stock solutions of MTX, OH-MTX and 8 chlorothéophylline were prepared separately
by dissolving the appropriate amounts of the substances in diluents.
2.4 Chromatographic conditions
The chromatographic column used was a 250 x 4 mm (Lichrospher®) with 5 µm particle
size which was accompanied with a 1 cm long guard column. The mobile phase was
phosphate buffer pH = 6.1: methanol (80/20, v/v). The flow rate of the mobile phase was
1mL min-1. The column was maintained at 25°C and the detection wavelength was 297
nm. The injection volume was 50 µL.
2.5 Extraction procedure
The coextractive cleanup procedure was performed according to the reported method by
H.Abolenen and al (Aboleneen et al., 1996 ) with a slight modification:
One volume (300 µL) of heparinised plasma with 20 µL of internal standard and two
volumes of methanol were thoroughly mixed. After centrifugation (10 min, 3000 g at 4°C),
the deproteinized supernatant mixture was transferred in to the cartridge extraction. The
cartridges were prepared with 9 mL methanol followed by 3 mL 0.04 M phosphate buffer
(pH 6) draw through by centrifugation or suction discard the effluent. The eluate was dried
under nitrogen stream at 60° C. The residue was reconstituted in 200 µL HCl 0.005 M. A
50 µL aliquot of the reconstituted sample was injected on to the HPLC column.
3. RESULTS
3. 1 Optimized Chromatographic conditions
To optimize separation of the drugs, several mobile phase compositions were investigated.
Initially, phosphate buffer pH = 6.1: methanol (80/20, v/v) was used but MTX, OH-MTX
and IS could not be separated under this condition. Subsequently, phosphate buffer pH= 4:
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methanol (80/20, v/v) was used, all the drugs were separated but the retention time was
long (25 min). When acetonitrile was added to the mobile phase, with adjusting the ratio of
phosphate buffer: acetonitril:methanol (82:12:6, v/v/v), the retention time of the three
drugs increased when acetonitrile and methanol content in the mobile phase were lowered
and raised respectively. The mobile phase of phosphate buffer pH= 4: acetonitrile:
methanol, 82:12:6, v/v/v) were found most suitable and gave good baseline separation.
Effects of varying temperature between room temperature (about 25°C) to 70°C and flow
rate between 0.7 and 1.2 mL min-1 gives us a good separation at temperature and flow rate
equal respectively 40°C and a 1 mL/min (resolution > 1.5). The retention times for MTX,
OH-MTX and IS were 8.77, 10.5 and 12.88 min, respectively; and the chromatograms are
shown in figs 1.
3.2 Method Validation
3.2.1 Precision
The accuracy and precision were determined with six determinations per concentration.
Within-and between-day accuracy and precision values are given in (table 1).
The inter-assay and intra-assay variation of the measured concentration was assessed by
the relative standard error (% RSD) which was calculated as the standard deviation of the
measured concentrations divided by the mean and multiplied by 100. Batches, for which
the recovery did not fall within 80–120%, and/or having a % RSD greater than 5%, were
re-analyzed. Mean recoveries were between 82.5 and 92.5% for inter and intra-assay.
3.3 Linearity
Calibration curves for plasma samples were constructed within the concentration ranges of
(0.03-500 µmol/l) using linear least-squares regression. The correlation coefficient, slope
and intercept for six standard curves constructed for each range, were respectively 0.99,
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0.85 and 0.34. The specificity was determined by comparing 15 plasma samples collected
from patients who didn't receive MTX. Each sample were analysed and we didn’t find any
detectable signal corresponding to the retention times of MTX and IS.
3.4 Limit of Detection and Limit of Quantification
To estimate the limit of detection (LOD) and the limit of quantification (LOQ) replicates of
control plasma samples (blanks) were analyzed by the described procedure. LOD was
calculated as mean blank response plus three times the standard deviation and LOQ was
calculated as mean blank response plus 10 times the standard deviation, according to the
guidance for industry (R Eric). The LOD and LOQ of the described method for MTX in
plasma samples were 0.0063 and 0.02 µmol/L respectively.
4 Application
The method has been applied successfully to determine MTX concentration in 34 samples
drawn from acute lymphocytic leukaemia (LAL) patients (figs 1). Samples were taken 24,
36, 48, 72 and 96 hours following the end of 24 h MTX infusion of a dose ranging from
100 to 500 mg/kg. The MTX concentration range from 0.04 to 1.33 µmol/L (median 0.11
µmol/L). No significant correlation was found between plasma MTX and dose.
Considerable individual variability was observed and regular monitoring would be required
in clinical use. Moreover, further investigations are to asses the clinical utility of
monitoring MTX concentrations and to identify the relation between the pharmacokinetic
parameters of MTX and clinical efficacy and toxicity in Tunisian population.
DISCUSSION
The present study describes a highly sensitive, accurate, and reproductible HPLC method
for determination of MTX in human plasma. The procedure of sample preparation is rapid
used a small plasma volume (300 µl) and less expensive.
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In order to optimise a method we varied several parameters, the solid-phase extraction
(SPE) was essential for plasma samples; in fact without SPE a larger solvent front
obscured the two analyte peaks. This extraction was the preferred method by authors
(Albertioni et al., 1997). This extraction need in the first time a procedure for denatured
protein we used a deproteinization by just precipitating the samples with methanol such
reported in the method of Floridia et al., (Floridia et al., 1999). Other method uses
acetonitrile; trichloroacetic acid or centrifugation (Albertioni et al., 1997 , Kambiz et al.,
2005 , Nadège et al., 20032-7-8).
The determination was based on the internal standard method, as reported in several study
(Albertioni et al., 1997 , Cociglio et al., 1995). The mobile phase reported in our study was
composed of (Phosphate buffer at pH = 4/Methanol/Acetonitril 82/12/6, v/v/v). This phase
was described in other study but in the different proportions (Aboleneen H et al., 1996 ,
Kambiz et al., 2005 , Turci et al., 2000 , Vassault et al., 1999).
Calibration curves were constructed using linear least-squares regression. The
determination was based on the internal standard method, as reported in several study (R
Eric et al., 2007 , Kambiz et al., 2005). The coefficient correlation of our method was R =
0.999 and the overall RSD < 5 %. Within-and between-day accuracy were 3.65 and 3% in
other study they varied between 3 and 4% (Albertioni et al., 1997 , Aboleneen et al., 1996
, Cociglio et al., 1995). The reported value in the literature of quantification ranged from
0.03 µmol/l to 0.08 µmol/l in our assay the limit of quantification was 0.03 µmol/l. The
limit of detection in our study was 0.0063 µmol/l. The assay was validated against
published acceptance criteria for linearity, accuracy.
Conclusion
In conclusion, we have developed, optimised and validated a rapid and sensitive assay of
methotrexate in serum. This method was free from serum matrix interferences and
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determines methotrexate concentration at the lowest levels. The utility of the method was
further demonstrated by measuring methotrexate levels in patient samples and it must be
used to estimate OHMTX in plasma sample.
Table 1: Precision data for methotrexate
Theoretical value
Measured value (mean + SD)
(µmol/L)
(µmol/L)
RSD (%)
Intra-assay (n = 6)
0.4
0.38 + 0.008
2.1
0.8
0.72 + 0.010
1.38
5
4.55 + 0.096
2.11
0.4
0.36 + 0.008
2.2
0.8
0.68 + 0.02
2.94
5
4.91 + 0.12
2.4
Inter-assay (n = 6)
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a- Chromatogram of spiked plasma
b- Chromatogram of patient sample
with MTX and OH MTX
and IS (8-chlorothéophylline)
Figure1: Representative chromatogram of spiked plasma (a) and patient sample (b)
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