Journal of Pharmaceutical and Biomedical Analysis 71 (2012) 173–178

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Journal of Pharmaceutical and Biomedical Analysis

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Short communication

High-performance liquid chromatographic assay for metamizol metabolites in

rat plasma: Application to pharmacokinetic studies
Adriana Miriam Domínguez-Ramírez a,∗ , Patricia Carrillo Calzadilla b , Alma Rosa Cortés-Arroyo a ,
Marcela Hurtado y de la Peña a , José Raúl Medina López a , Martín Gómez-Hernández a ,
Francisco Javier López-Muñoz c
a
Departamento Sistemas Biológicos, UAM-Xochimilco, Calzada del Hueso 1100, Col. Villa Quietud, 04960 México D.F., Mexico
b
Maestría en Ciencias Farmacéuticas, DCBS, UAM-Xochimilco, Calzada del Hueso 1100, Col. Villa Quietud, 04960 México D.F., Mexico
c
Departamento Farmacobiología, CINVESTAV Sede Sur, Calzada de los Tenorios 235, Col. Granjas Coapa, 14330 México D.F., Mexico

a r t i c l e i n f o a b s t r a c t

Article history: In order to evaluate the pharmacokinetics of metamizol in the presence of morphine in arthritic
Received 21 February 2012 rats, after subcutaneous administration of the drugs, an easy, rapid, sensitive and selective analytical
Received in revised form 25 May 2012 method was proposed and validated. The four main metamizol metabolites (4-methylaminoantipyrine,
Accepted 26 July 2012
4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoantipyrine) were extracted from plasma
Available online 4 August 2012
samples (50–100 ␮l) by a single solid-phase extraction method prior to reverse-phase high performance
liquid chromatography with diode-array detection. Standard calibration graphs for all metabolites were
Keywords:
linear within a range of 1–100 ␮g/ml (r2 ≥ 0.99). The intra-day coefficients of variation (CV) were in the
Metamizol metabolites
Morphine
range of 1.3–8.4% and the inter-day CV ranged from 1.5 to 8.4%. The intra-day assay accuracy was in the
Pharmacokinetics range of 0.6–9.6% and the inter-day assay accuracy ranged from 0.9 to 7.5% of relative error. The lower
Rats limit of quantification was 1 ␮g/ml for all metabolites using a plasma sample of 100 ␮l. Plasma samples
HPLC-method were stable at least for 4 weeks at −20 ◦ C. This method was found to be suitable for studying metami-
zol metabolites pharmacokinetics in arthritic rats, after simultaneous administration of metamizol and
morphine, in single dose.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction
Metamizol sodium (MET) is a non-steroidal anti-inflammatory
analgesic drug (NSAID) with antipyretic and antispasmodic prop-
erties which belongs to the group of pyrazolones. MET is a pro-drug
which is rapidly hydrolyzed by a non-enzymatic mechanism to the
active moiety 4-methylaminoantipyrine (MAA). The MAA is metab-
olized in the liver by demethylation to 4-aminoantipyrine (AA),
the other active metabolite and by oxidation to form the inactive
metabolite 4-formylaminoantipyrine (FAA). AA is acetylated to 4-
acetylaminoantipyrine (AAA), which is also an inactive compound
(Fig. 1).
The therapeutic benefits of the co-administration of MET and
morphine (MOR) have been previously demonstrated. MET has
reduced the frequency of administration of MOR after major
abdominal surgery [1] and in the treatment of chronic pain in
cancer patients in Spain and Mexico (data not published). How-
ever, the high doses used in combination have been selected
∗ Corresponding author. Tel.: +52 55 5483 7254; fax: +52 55 5483 7237.
E-mail address: adoming@correo.xoc.uam.mx (A.M. Domínguez-Ramírez).
empirically on the basis of the doses commonly used when the
drugs are given alone. In this sense, our group has extensively stud-
ied (preclinical studies) the combination of low doses of MET and
MOR in a model of arthritic pain in rats and has found that the com-
bination that resulted in a maximal antinociceptive potentiation,
between 24 different combinations, was composed of 177.8 mg/kg
of MET and 3.2 mg/kg of MOR [2]. This combination also delays
the development of tolerance to the antinociceptive effect of MOR
without producing an increase in constipation effect [3]. We have
also demonstrated that the optimal MOR and MET combination
is able to produce potentiation of antinociceptive effects during
intense pain [4].
Several pharmacodynamic mechanisms appeared to be involved
in the effects produced by the combination of MET and MOR, as
well, they can also be explained as a result of pharmacokinetic
interactions [4]. The effect of MET on the pharmacokinetics and
pharmacodynamics of MOR in arthritic rats was previously studied
[5]. It was demonstrated that MET significantly increases plasma
concentrations of MOR and that the antinociceptive effect of the
combination MET+MOR can be related to MOR pharmacokinetics.
However, the pharmacokinetics of MET in the presence of MOR and
the role that it plays in the effect produced by the combination has
not been studied.

0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jpba.2012.07.029
174 A.M. Domínguez-Ramírez et al. / Journal of Pharmaceutical and Biomedical Analysis 71 (2012) 173–178
Fig. 1. Structure and biotransformation of metamizol in man and in rat. MET: metamizol; MAA: 4-methylaminoantipyrine; AA: 4-aminoantipyrine; FAA: 4-formylamino-
antipyrine; AAA: 4-acetylaminoantipyrine.
There are several methods for the quantification of MET and/or
its metabolites in biological fluids and/or tissues. These include
thin-layer chromatography [6] spectrophotometric [7], gas chro-
matographic [8] and high-performance liquid chromatographic
(HPLC) methods with UV detection [9–12]. However, some of this
methods only quantify MAA or MAA and AA [9,13,14], while others
use large plasma samples, multiple liquid–liquid extraction steps
[9,12,15], short concentration intervals [15,16] or long analysis
time (≥60 min).
As repeated blood sampling is required in pharmacokinetic
studies in small species (rats), it is necessary to utilize a sensi-
tive and selective method and reduce the total volume of plasma
extracted from the animals in order to avoid serious impairment
to its physiological state. When studying the interaction of highly
metabolized drugs, as is the case of MET, the determination of most
of the main metabolites is required.
In this study we propose an easy, selective and reliable
chromatographic method for the quantification of MET main
metabolites, MAA, AA, AAA and FAA, from a small volume of rat
plasma (50–100 ␮l). The potential importance of the assay was
demonstrated by the application of this method to a pharmacoki-
netic study of MET administered in combination with MOR in single
dose, to arthritic rats.
2. Experimental
2.1. Chemicals, materials and instrumentation
Metamizol sodium was kindly supplied by RETECMA, S.A. de
C.V., México. The metabolites, 4-methylaminoantipyrine (MAA), 4-
acetylaminoantipyrine (AAA) and 4-formylaminoantypyrine (FAA)
were synthesized at the Autonomous Metropolitan University
(Xochimilco) by Dr. Olivia Soria. 4-Aminoantipyrine (AA), triethy-
lamine and furosemide (FUR), from Sigma Chem. Co. (St. Louis,
MO, USA), were used. Morphine hydrochloride (MOR) was gen-
erously supplied by the Mexican Secretary of Health, México City,
México. Methanol for the mobile phase was chromatographic grade
(J.T.Baker, México). All other reagents were analytical grade (E.
Merck KGaA, Darmstadt, Germany). HPLC grade water (18 ) was
obtained by purifying distilled water in a Milli-Q filtration system
(Millipore, Bedford, MA, USA). Mobile phase was filtered through
0.45 ␮m pore size nylon membranes (Millipore, Bedford MA, USA)
and degassed in an ultrasonic bath (Branson Ultrasonic Corp., Dan-
bury CT, USA).
Sep-Pak C18 cartridges (Waters Milford, MA, USA) and a vacuum
device (Spe-ed Mate-10, Applied Separations, Allentown, PA, USA)
were used for solid-phase extraction.
The chromatographic system consisted of a Knauer high-
performance liquid cromatograph (Berlin, Germany) equipped
with a Smartline pump 100, a Smartline PDA detector 2800 and
a Smartline autosampler 3950. The chromatographic station Clari-
tyChrom V2.6.xx software, was used for acquisition and processing
of data.
2.2. Preparation of calibration standards and quality control
samples
Primary stock solutions of MAA, AA, AAA, FAA (1 mg/ml) and
FUR(400 ␮g/ml), were prepared in methanol and stored at −4 ◦ C.
Rat plasma calibration standards of metamizol metabolites were
prepared by spiking appropriate aliquots of the stock solutions of
each metabolite to drug-free rat plasma to give final concentrations
ranging from 1 to 100 ␮g/ml. Quality control (QC) samples at con-
centrations of 5, 15, 30 and 100 ␮g/ml were prepared by adding the
appropriate aliquots of the stock solutions to drug-free rat plasma.
The QC samples were aliquoted (100 ␮l) into polypropylene tubes
and stored at −20 ◦ C until analysis.
2.3. Sample preparation
Cartridges were preconditioned by flushing with 6 ml of
methanol and 6 ml of distilled water. 50–100 ␮l of sample
 A.M. Domínguez-Ramírez et al. / Journal of Pharmaceutical and Biomedical Analysis 71 (2012) 173–178
(calibration standards or QC samples and 50–100 ␮l of blank
plasma) were directly loaded into the cartridge and added with
100 ␮l of the internal standard (IS) solution (FUR, 40 ␮g/ml). The
sample was allowed to stand for 5 min, washed with 0.4 ml of water
and then dried under vacuum. The analytes and the IS were eluted
with 3 ml of methanol, at a flow rate of 1 ml/min. The eluate was
evaporated to dryness in a water bath at 45 ± 5 ◦ C under a gentle
stream of nitrogen. The residue was reconstituted in 50–100 ␮l of
mobile phase and 20 ␮l were injected onto the HPLC system for
analysis.
2.4. HPLC conditions
The separation was performed on an Alltech Platinum
C18 column (5 ␮m, 250 × 4.6 mm; Alltech Associates, Deer-
field, IL, USA); a Phenomena security guard column (4 × 0.3 mm
C18 cartridge, Torrance, CA, USA) was used before the ana-
lytical column. The mobile phase consisted of a mixture
of water–methanol–triethylamine–acetic acid (70.9:27.7:0.9:0.5,
v/v/v/v) at pH 5, degassed before use, and the flow rate was
1 ml/min. Detection wavelength was set at 254 nm. All analysis
were carried at room temperature (25 ◦ C).
2.5. Method validation
2.5.1. Selectivity
To determine the selectivity of this method, blank plasma
obtained from rats, alone and spiked with known amounts of MET;
MET metabolites, MAA, AA, AAA, FAA; FUR and MOR, were analyzed.
In addition, plasma samples of rats administered with MOR in sin-
gle dose (3.2 mg/kg, s.c), were extracted and injected into the HPLC
system to test the potential interference of MOR and its metabolites.
2.5.2. Calibration curves and linearity
Three calibration curves for each metabolite in a concentra-
tion range of 1–100 ␮g/ml were determined. Standard calibration
curves were generated for each metabolite by plotting peak area
ratio of metabolite/furosemide vs. metabolite plasma concentra-
tion. A least-squares linear regression analysis was performed
to determine slope, intercept, 95% confidence interval (CI95% ) for
intercept and determination coefficient (r2 ).
2.5.3. Intra-day and inter-day precision and accuracy and lower
limit of quantification
For the intra-day variation determination, sets of five replicates
of QC samples of each metabolite at four concentration levels, along
with a standard plasma curve were analyzed on the same day. For
the inter-day validation, three replicates of each concentration level
were analyzed along with a standard curve in plasma on three dif-
ferent days. The coefficient of variation (CV) served as a precision
measure. The CV should be less than 15%, except at the lower limit
of quantification (LLQ) where it should not exceed 20% [17].
The accuracy of the assay was determined on the above sam-
ples, by comparing the means of the measured concentrations with
the nominal concentrations of each metabolite, either for stan-
dard samples (intra-day accuracy) or for QC samples (inter-day
accuracy). The percentage deviation of the mean from true values,
expressed as relative error (RE) served as a measure of accuracy. RE
was calculated as follows:
(Added concentration − Recovered concentration) × 100
RE% =
Added concentration
The mean value of RE should be within ±15% of the nominal
value, except for the LLQ where it should not exceed 20% [17].
175
2.5.4. Recovery
The absolute recovery of MET metabolites by the proposed
method was determined by extracting five replicates of QC
plasma samples at 5, 20 and 40 ␮g/ml. The peak areas obtained
were compared to those obtained after direct injection of non-
extracted standard solutions in mobile phase, at the same
concentrations.
2.5.5. Stability
QC samples containing the four MET metabolites at a concen-
tration of 15 ␮g/ml were stored at −20 ◦ C and analyzed by the
previously described method in five replicates, at zero time, and
after 4 weeks. The relative difference between mean values of con-
centrations obtained at the two times (Initial and after 4 weeks at
−20 ◦ C) was calculated as RE % as follows:
(Initial concentration − Concentration at 4th week at − 20◦ C) × 100
RE% =
Initial concentration
The mean value of RE should be within ±15% of the concentra-
tion at zero time.
2.6. Pharmacokinetic studies
Male Wistar rats [Crl:(WI)fBR] weighing 180–220 g, from our
own breeding (UAM-Xochimilco, México), were used in this study.
Rats were maintained under controlled environmental conditions
at 22 ◦ C, under a 12 h light/dark cycle and provided with stan-
dard chow (Purina Laboratory Rodent Diet 5001) and water ad
libitum. Twelve hours before the experiments food was with-
held, but animals had free access to water. Experiments were
performed during the light phase and animals were used only
once. All experimental procedures were approved by the local
Institutional Animal Care and Use Committee and complied with
the Mexican federal regulations for the care and use of labora-
tory animals NOM-062-ZOO-1999 (Mexican Ministry of Health)
and the Guidelines on Ethical Standards for Investigations of
Experimental Pain in Animals [18]. Two groups of six rats were
used in this pharmacokinetic study of MET administered alone or
concomitantly with MOR to analyze a possible pharmacokinetic
interaction. The day of the study, rats were lightly anaesthetized
with diethyl ether and the caudal artery was cannulated with
PE-10 cannula (Clay Adams, Parsippany, NJ, USA) connected to
a PE-50 cannula. The cannula was kept patent with heparinized
saline solution and stoppered with a needle. Rats were allowed
to recover from anaesthesia and a dose of 177.8 mg/kg of MET or
the combination of 177.8 mg/kg of MET +3.2 mg/kg of MOR, dis-
solved in saline solution, was subcutaneously administered. Blood
samples (100–150 ␮l) were withdrawn from the caudal artery at
0 h (before the administration of the treatment) and at 0.25, 0.5,
0.75, 1, 1.25, 1.5, 2, 2.5, 3, 4, 8 and 24 h after the administra-
tion of the drug(s), and transferred to heparinized polypropylene
tubes. The total volume of blood taken from each animal did
not exceed 1.8 ml. Plasma was separated by centrifugation at
3000 rpm for 10 min at 25 ◦ C and stored at −20 ◦ C until analy-
sis.
Plasma samples from pharmacokinetic studies and a duplicate
of three levels QC samples were analyzed together with a stan-
dard curve in plasma prepared the day of the analysis. Assays were
acceptable if the accuracy of QC samples were within ±15% of the
nominal value.
Pharmacokinetic parameters: maximum plasma concentra-
tion (Cmax ), time to Cmax (tmax ), area under the curve from
time 0 to infinite (AUC0–∞ ), area under the curve from time
0 to 4 h (AUC0–4 ), elimination constant (ke ), half-life time
(t1/2 ), volume of distribution (Vd/F) and plasma clearance (Cl/F),
were calculated as previously reported [5]. Differences between
176 A.M. Domínguez-Ramírez et al. / Journal of Pharmaceutical and Biomedical Analysis 71 (2012) 173–178

Table 2
Stability of metamizol metabolites in plasma samples stored at −20 ◦ C.

Metabolite Concentration (␮g/ml)

Initiala 4th weeka RE (%)b

MAA 16.5 15.5 6.1

AA 15.9 16.3 2.5
AAA 15.2 15.8 3.9
FAA 15.7 15.3 2.5
a
n = 5.
b
RE (%) = ((Initial concentration − Concentration at 4th week at −20 ◦ C)/Initial
concentration) × 100.

3. Results and discussion

3.1. Chromatography and extraction procedure

HPLC method with UV detection is a common method for the

Fig. 2. Chromatograms of: (A) rat blank plasma, (B) rat plasma spiked with MET
metabolites FAA (5.62 min), AAA (5.88 min), AA (10.2 min), MAA (12.95 min) and
FUR (19.37 min) and (C) rat plasma sample taken 1.5 h after administration of a
single dose of MOR (3.2 mg/kg, s.c).
pharmacokinetic parameters (except tmax ) were assessed by
unpaired Student’s t-test. Cmax and AUC data were analyzed after
logarithmic transformation. Mann–Withney test was applied for
tmax comparisons. A p < 0.05 was considered significant.
analysis of MET metabolites in biological samples. However, many
published papers describe the need for multiple extraction and
complex chromatographic systems to ensure reproducibility and
improve the recovery of analytes [15,16]. Sample preparation in
this study consists in a modification of the solid-phase extraction
(SPE) method proposed by Carretero et al. [8] for the separation of
MET metabolites, including a single step extraction in solid-phase
while reducing the sample volume to 50–100 ␮l.
Good sensitivity and adequate retention times were obtained
with the isocratic HPLC system, which is similar to that proposed
by Agúndez et al. [11] for the determination of MET metabolites
in urine. Under our conditions, all metabolites were analyzed in
21 min (Fig. 2) and the reproducibility on the retention times (n = 5)
was better than 1.9%.
Separations of spiked plasma gave good resolution for peaks,
except for FAA and AAA (Fig. 2). Although FAA and AAA peaks were
not completely resolved, the corresponding peak areas were ade-
quately integrated by the software used, with good reproducibility
and accuracy.
No interfering peaks were observed around the retention times
of these compounds with only one-step extraction when every
drug-free plasma sample was treated. The chromatographic back-
ground after extraction was clean enough that low concentrations
of the metabolites could be detected. The detection limit based on
a signal:noise ratio of 3:1, was 0.1 ␮g/ml for all metabolites.

Table 1
Intra- and inter-day precision and accuracy for analysis of metamizol metabolites spiked to rat plasma.

Metabolite Intra-day (n = 5) Inter-day (3 days; n = 3)

Addeda (␮g/ml) Recovered (␮g/ml) CV (%) REb (%) Addeda (␮g/ml) Recovered (␮g/ml) CV (%) REb (%)

MAA 5.3 5.2 4.6 1.1 5.3 4.9 3.9 7.5

15.9 16.5 1.3 3.8 15.9 15.4 1.7 3.1
31.8 33.1 1.6 4.1 31.8 31.3 6.7 1.6
106.0 103.9 3.9 1.9 106.0 107.0 8.4 0.9

AA 5.2 4.7 6.7 9.6 5.3 5.2 7.5 1.9

15.6 15.5 3.2 0.6 15.9 16.3 1.5 2.5
31.2 33.1 1.8 6.1 31.1 31.5 5.8 1.3
104.0 111.0 7.8 6.7 106.0 111.3 7.8 5.0

AAA 5.2 5.1 1.5 1.9 5.1 5.2 6.0 1.9

15.6 15.2 5.5 2.6 15.4 15.1 2.9 1.9
31.2 34.0 8.2 9.0 30.8 32.4 6.4 5.2
104.0 107.1 8.4 3.0 102.0 107.8 1.8 5.6

FAA 5.2 5.3 3.9 1.9 5.2 5.4 2.2 3.8

15.5 16.9 4.2 9.0 15.5 16.1 4.9 3.9
31.1 31.9 2.4 2.6 31.1 30.5 3.9 1.9
104.0 111.2 7.6 6.9 104.0 111.3 7.6 7.1
a
Nominal concentration (␮g/ml) calculated on the basis of the real weight used to prepare the corresponding stock solution.
b
RE (%) = ((Added concentration − Recovered concentration)/Added concentration) × 100.
 A.M. Domínguez-Ramírez et al. / Journal of Pharmaceutical and Biomedical Analysis 71 (2012) 173–178 177

Table 3
Pharmacokinetic parameters for MAA, AA, AAA obtained after s.c. administration of a single dose of metamizol 177.8 mg/kg alone (MET) and in combination with 3.2 mg/kg
of morphine (MET + MOR) in arthritic rats.

Parameter MAA AA AAA

MET MET + MOR MET MET + MOR MET MET + MOR

Cmax (␮g/ml) a
95.8 ± 8.3 105.23 ± 10.0 ns
21.9 ± 2.2 23.4 ± 1.3ns
11.8 ± 1.1 12.7 ± 2.6ns
tmax (h)b 0.25 (0.25) 0.25 (0.0)ns 2.0 (2.0) 3.0 (2.5)ns 4.0 (4.5) 4.0 (4.5)ns
ABC0–∞ (␮g h/ml)a 198.2 ± 22.5 185.5 ± 17.9ns 126.9 ± 17.3 156.2 ± 9.0ns ND ND
ABC0−4 h (␮g h/ml)a 162.8 ± 18.9 153.7 ± 14.9ns 66.8 ± 5.3 74.1 ± 3.4ns 23.5 ± 1.9 25.2 ± 2.4ns
ke (h−1 ) 0.55 ± 0.05 0.51 ± 0.04ns 0.32 ± 0.04 0.2 ± 0.02ns ND ND
t1/2 (h) 1.3 ± 0.1 1.4 ± 0.1ns 2.4 ± 0.3 2.9 ± 0.21ns ND ND
Vd/F (l/kg) 1.7 ± 0.1 2.0 ± 0.2ns 4.7 ± 0.4 4.8 ± 0.45ns ND ND
Cl/F (l/h/kg) 0.9 ± 0.1 1.0 ± 0.1ns 1.4 ± 0.2 1.1 ± 0.06ns ND ND

Cmax , maximum plasma concentration; tmax , time to Cmax ; AUC0–∞ , area under the curve from time 0 to infinite; AUC0–4 , area under the curve from time 0 to 4 h; ke , elimination
constant; t1/2 , half-life time; Vd/F, volume of distribution; Cl/F, plasma clearance; ns, p > 0.05 compared MET vs. MET + MOR; ND = not determined because experiment was
finalized before beginning of elimination phase.
a
Geometric mean ± SEM (n = 6).
b
Median (range); all other data expressed as arithmetic mean ± SEM (n = 6).

3.2. Method validation
3.2.1. Selectivity
The extraction method allowed the adequate separation of MET,
MET metabolites and FUR from possible endogenous plasma com-
pounds (Fig. 2). Neither MOR nor MOR metabolites were detectable
in samples of rats treated with a single dose of MOR.
MOR concentrations expected after administration of a sin-
gle dose of 3.2 mg/kg, s.c., are below detectable concentrations
by HPLC–UV methods. Even more, pH conditions used with SPE
method are unfavorable for the extraction of MOR from plasma
samples. Consequently, this method can measure MET and its
metabolites in low plasma volume with clean chromatogram and
sufficient limit of quantification without interference from MOR
due to its specific extraction procedure.
3.2.2. Calibration curves and linearity
A linear relationship was found when the peak area
ratio of the metabolite/IS was plotted against metabolite
plasma concentration. Regression lines for metabolites data
were: y = 0.069x − 0.058 (r2 = 0.999) for MAA; y = 0.076x − 0.0036
(r2 = 0.999) for AA; y = 0.064x + 0.037 (r2 = 0.999) for AAA and
y = 0.051x + 0.02 (r2 = 0.999) for FAA. Linear regression of the data
was significant for the range of concentrations studied (p < 0.001)
with an intercept equal to zero at 95% confidence level.
3.2.3. Precision, accuracy and lower limit of quantification
Table 1 shows a summary of intra-day and inter-day precision
and accuracy of the method. CV values ranged from 1.3 to 8.4% and
from 1.5 to 8.4% respectively, for all metabolites demonstrating the
good precision of the method. Precision of the method is compara-
ble to published methods that use higher volume of plasma sample
[14,15].
The intra-day RE values assessed by quintuplicate analysis of QC
samples ranged from 0.6 to 9.6%. Inter-day accuracy, assessed by
the triplicate analysis of QC samples at four concentrations, in three
different days, gave RE values from 0.9 to7.5% (<15%). These results
demonstrate the accuracy of the method (Table 1).
The LLQ was 1 ␮g/ml for all metabolites. Plasma concentrations
of MET metabolites can be accurately quantified up to 1 ␮g/ml
(20 ng injected) with a CV ranged from 2.3 to 5.6% and RE from
3.5 to 9.6%. The sensitivity of this method is equivalent to methods
previously employed in other pharmacokinetic studies in humans,
using 1 ml of plasma sample [11,12,19].
3.2.4. Recovery
An adequate recovery after the one-step SPE was obtained
by washing samples with 0.4 ml of water, previously to the
elution with methanol. The final solvent volume used to elute the
compounds was 3 ml, without sacrificing the recovery of the ana-
lytes. Absolute recoveries, calculated by comparing peak areas from
extracted samples with peak areas of unextracted standards, were
between 99.7 and 108.1% for MAA, 89.1 and 100.2% for AA 97.6 and
102% for AAA, and 97.4 and 103.2% for FAA, with a good precision
(CV < 8%), independently of the concentration studied. The recov-
ery of the method was higher than other methods that use larger
plasma samples or at least two-step LLE [9,12,14,19]. One of the
advantages of SPE is that it allows a better recovery of the analytes,
from the biological matrix, than LLE, and allows multiple samples
to be processed at the same time.
3.2.5. Stability
From the stability study, it was found that plasma samples con-
taining 15 ␮g/ml of all metabolites were stable for at least 4 weeks
at −20 ◦ C (Table 2). Furthermore, stock solutions of metabolites
in methanol, stored at −4 ◦ C, were stable for at least two weeks.
Reconstituted extracted samples in mobile phase were stable for
24 h at room temperature (data not shown).
3.3. Pharmacokinetic studies
The validated HPLC method was used to analyse plasma MET
pharmacokinetics in rats after single administration of 177.8 mg/kg
of the drug alone and after simultaneous administration of MET
and MOR (177.8 + 3.2 mg/kg, s.c.). QC samples in each analytical
run were within 15% of the nominal value. No interfering peaks
were found during the analysis of the samples obtained for the
pharmacokinetic study.
Pharmacokinetic parameters were calculated by non-
compartmental analysis from metabolites concentration data
found in rat plasma samples. Results are summarized in Table 3.
No differences in the pharmacokinetic parameters for MAA, AA,
and AAA between both treatments were found (p > 0.05), demon-
strating that MOR does not modifies the pharmacokinetics of MET
when administered together in single dose, under the conditions
studied. FAA pharmacokinetic parameters were not calculated
because plasma concentrations of this metabolite were under the
LLQ.
The present method proved to be useful for the determination of
plasma levels of the main MET metabolites (MAA, AA, AAA and FAA)
in rats, in a small sample volume (50–100 ␮l). So, a sufficient num-
ber of samples can be obtained from the same animal in order to
define the pharmacokinetics of MET, without any impairment to its
physiological state. The selectivity, sensitivity, precision, and accu-
racy obtained with this method make it suitable for the purpose of
the present study. In conclusion, the proposed method is easy and
178 A.M. Domínguez-Ramírez et al. / Journal of Pharmaceutical and Biomedical Analysis 71 (2012) 173–178
fast to perform; it is also characterized with an adequate accuracy,
precision, selectivity, and stability, using a small plasma volume
(50–100 ␮l). The method was successfully applied to a pharma-
cokinetic study of MET administered in combination with MOR in
arthritic rats.
Acknowledgements
This work was partially supported by grant PIFI.3.3 (PROMEP).
The authors wish to thank Ericka Zavala L. and Laura Benavides
G. for their technical assistance. Patricia Carrillo C. is a CONACYT
fellow and a student of Maestría en Ciencias Farmacéuticas, UAM-
Xochimilco.
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