High-performance liquid chromatographic assay for metamizol metabolites in rat plasma: Application to pharmacokinetic studies

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

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba

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 , ˜ a , José Raúl Medina López a , Martín Gómez-Hernández a , Marcela Hurtado y de la Pena ˜ c Francisco Javier López-Munoz a

Departamento Sistemas Biológicos, UAM-Xochimilco, Calzada del Hueso 1100, Col. Villa Quietud, 04960 México D.F., Mexico 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 b

a r t i c l e

i n f o

Article history: Received 21 February 2012 Received in revised form 25 May 2012 Accepted 26 July 2012 Available online 4 August 2012 Keywords: Metamizol metabolites Morphine Pharmacokinetics Rats HPLC-method

a b s t r a c t In order to evaluate the pharmacokinetics of metamizol in the presence of morphine in arthritic rats, after subcutaneous administration of the drugs, an easy, rapid, sensitive and selective analytical method was proposed and validated. The four main metamizol metabolites (4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoantipyrine) were extracted from plasma 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 linear within a range of 1–100 ␮g/ml (r2 ≥ 0.99). The intra-day coefficients of variation (CV) were in the 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 range of 0.6–9.6% and the inter-day assay accuracy ranged from 0.9 to 7.5% of relative error. The lower limit of quantification was 1 ␮g/ml for all metabolites using a plasma sample of 100 ␮l. Plasma samples were stable at least for 4 weeks at −20 ◦ C. This method was found to be suitable for studying metamizol 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 properties 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 metabolized 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 4acetylaminoantipyrine (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). However, the high doses used in combination have been selected

∗ Corresponding author. Tel.: +52 55 5483 7254; fax: +52 55 5483 7237. E-mail address: [email protected] (A.M. Domínguez-Ramírez). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.07.029

empirically on the basis of the doses commonly used when the drugs are given alone. In this sense, our group has extensively studied (preclinical studies) the combination of low doses of MET and MOR in a model of arthritic pain in rats and has found that the combination 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.

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-formylaminoantipyrine; 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 chromatographic [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 sensitive 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 pharmacokinetic 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), 4acetylaminoantipyrine (AAA) and 4-formylaminoantypyrine (FAA) were synthesized at the Autonomous Metropolitan University (Xochimilco) by Dr. Olivia Soria. 4-Aminoantipyrine (AA), triethylamine and furosemide (FUR), from Sigma Chem. Co. (St. Louis, MO, USA), were used. Morphine hydrochloride (MOR) was generously 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., Danbury 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 highperformance liquid cromatograph (Berlin, Germany) equipped with a Smartline pump 100, a Smartline PDA detector 2800 and a Smartline autosampler 3950. The chromatographic station ClarityChrom 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 concentrations 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, Deerfield, IL, USA); a Phenomena security guard column (4 × 0.3 mm C18 cartridge, Torrance, CA, USA) was used before the analytical 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 single 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 concentration 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 concentration. 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 different 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 samples, by comparing the means of the measured concentrations with the nominal concentrations of each metabolite, either for standard 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: RE% =

(Added concentration − Recovered concentration) × 100 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 nonextracted standard solutions in mobile phase, at the same concentrations. 2.5.5. Stability QC samples containing the four MET metabolites at a concentration 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 concentrations obtained at the two times (Initial and after 4 weeks at −20 ◦ C) was calculated as RE % as follows: RE% =

(Initial concentration − Concentration at 4th week at − 20◦ C) × 100 Initial concentration

The mean value of RE should be within ±15% of the concentration 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 standard chow (Purina Laboratory Rodent Diet 5001) and water ad libitum. Twelve hours before the experiments food was withheld, 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 laboratory 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, dissolved 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 administration 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 analysis. Plasma samples from pharmacokinetic studies and a duplicate of three levels QC samples were analyzed together with a standard 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 concentration (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

MAA AA AAA FAA

Concentration (␮g/ml) Initiala

4th weeka

RE (%)b

16.5 15.9 15.2 15.7

15.5 16.3 15.8 15.3

6.1 2.5 3.9 2.5

a

n = 5. RE (%) = ((Initial concentration − Concentration at 4th week at −20 ◦ C)/Initial concentration) × 100. b

3. Results and discussion 3.1. Chromatography and extraction procedure HPLC method with UV detection is a common method for the 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 adequately 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 background 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.

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.

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 15.9 31.8 106.0

5.2 16.5 33.1 103.9

4.6 1.3 1.6 3.9

1.1 3.8 4.1 1.9

5.3 15.9 31.8 106.0

4.9 15.4 31.3 107.0

3.9 1.7 6.7 8.4

7.5 3.1 1.6 0.9

AA

5.2 15.6 31.2 104.0

4.7 15.5 33.1 111.0

6.7 3.2 1.8 7.8

9.6 0.6 6.1 6.7

5.3 15.9 31.1 106.0

5.2 16.3 31.5 111.3

7.5 1.5 5.8 7.8

1.9 2.5 1.3 5.0

AAA

5.2 15.6 31.2 104.0

5.1 15.2 34.0 107.1

1.5 5.5 8.2 8.4

1.9 2.6 9.0 3.0

5.1 15.4 30.8 102.0

5.2 15.1 32.4 107.8

6.0 2.9 6.4 1.8

1.9 1.9 5.2 5.6

FAA

5.2 15.5 31.1 104.0

5.3 16.9 31.9 111.2

3.9 4.2 2.4 7.6

1.9 9.0 2.6 6.9

5.2 15.5 31.1 104.0

5.4 16.1 30.5 111.3

2.2 4.9 3.9 7.6

3.8 3.9 1.9 7.1

a b

Nominal concentration (␮g/ml) calculated on the basis of the real weight used to prepare the corresponding stock solution. 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

a

Cmax (␮g/ml) tmax (h)b ABC0–∞ (␮g h/ml)a ABC0−4 h (␮g h/ml)a ke (h−1 ) t1/2 (h) Vd/F (l/kg) Cl/F (l/h/kg)

AA

MET

MET + MOR

95.8 ± 8.3 0.25 (0.25) 198.2 ± 22.5 162.8 ± 18.9 0.55 ± 0.05 1.3 ± 0.1 1.7 ± 0.1 0.9 ± 0.1

105.23 ± 10.0 0.25 (0.0)ns 185.5 ± 17.9ns 153.7 ± 14.9ns 0.51 ± 0.04ns 1.4 ± 0.1ns 2.0 ± 0.2ns 1.0 ± 0.1ns

ns

AAA

MET

MET + MOR

MET

MET + MOR

21.9 ± 2.2 2.0 (2.0) 126.9 ± 17.3 66.8 ± 5.3 0.32 ± 0.04 2.4 ± 0.3 4.7 ± 0.4 1.4 ± 0.2

23.4 ± 1.3 3.0 (2.5)ns 156.2 ± 9.0ns 74.1 ± 3.4ns 0.2 ± 0.02ns 2.9 ± 0.21ns 4.8 ± 0.45ns 1.1 ± 0.06ns

11.8 ± 1.1 4.0 (4.5) ND 23.5 ± 1.9 ND ND ND ND

12.7 ± 2.6ns 4.0 (4.5)ns ND 25.2 ± 2.4ns ND ND ND ND

ns

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 compounds (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 single 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 comparable 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 analytes. 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 recovery 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 containing 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 noncompartmental 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), demonstrating 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 number 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 accuracy 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 pharmacokinetic 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, UAMXochimilco. References [1] M.G. Rockemann, W. Seeling, C. Bischof, D. Börtinghaus, P. Steffen, F.M. Georgieff, Prophylactic use of epidural mepivacaine/morphine, systemic diclofenac and metamizol reduces postoperative morphine consumption after major abdominal surgery, Anesthesiology 84 (1996) 1027–1034. ˜ Surface of synergistic interaction between dipyrone and mor[2] F.J. López-Munoz, phine in the PIFIR model, Drug Dev. Res. 33 (1994) 26–32. [3] G.P. Hernández-Delgadillo, R. Ventura, M.I. Díaz, A.M. Domínguez, F.J. López˜ Metamizol potentiates morphine antinociception but not constipation Munoz, after chronic treatment, Eur. J. Pharmacol. 441 (2002) 177–183. ˜ [4] F.J. López-Munoz, B. Godínez-Chaparro, J.C. Huerta Cruz, U. Guevara-López, A.M. Domínguez-Ramírez, A.R. Cortés-Arroyo, The antinociceptive efficacy of morphine, metamizol, or their combination in an experimental rat model with different levels of inflammatory pain, Pharmacol. Biochem. Behav. 91 (2008) 196–201. ˜ F.J. [5] A.M. Domínguez-Ramírez, A.R. Cortés-Arroyo, M. Hurtado, J.R. de la Pena, ˜ Effect of metamizol on morphine pharmacokiMedina López, López-Munoz, netics and pharmacodynamics after acute and subchronic administration in arthritic rats, Eur. J. Pharmacol. 645 (2010) 94–101. [6] N. Sistovaris, W. Pola, Thin-layer chromatographic determination of major metamizole metabolites in serum and urine, J. Chromatogr. 274 (1983) 289–298.

[7] V.R. Weiss, J. Brauer, U. Geortz, R. Petry, Comparative study on the problem of absorption and metabolism of the pyrazolone derivative metamizole, in man after oral and intramuscular administration, Arzneim.-Forsch. 24 (1974) 345–348. [8] H. Maurer, K. Pfleger, Screening-procedure for detecting anti-inflammatory analgesics and their metabolites in urine using a computerized gaschromatographic mass-spectrometric technique, Fresenius Z. Anal. Chem. 314 (1983) 586–594. [9] E.Z. Katz, L. Granit, D.E. Dryer, M. Levy, Simultaneous determination of dipyrone metabolites in plasma by high-performance liquid chromatography, J. Chromatogr. 305 (1984) 477–484. [10] D. Damm, Simultaneous determination of the main metabolites of dipyrone by high-pressure liquid chromatography, Arzneim. -Forsch 39 (1989) 1415–1417. [11] J.A. Agúndez, C. Martínez, R. Martín, J. Benítez, Determination of aminopyrine, dypirone and its metabolites in urine by high-performance liquid chromatography, Ther. Drug Monit. 16 (1994) 316–322. [12] I. Carretero, J.M. Vadillo, J.J. Laserna, Determination of antipyrine metabolites in human plasma by solid-phase extraction and micelle liquid chromatography, Analyst 120 (1995) 1729–1732. [13] G. Suarez-Kurtz, F.M. Ribeiro, R.C. Estrela, F.L. Vicente, C.J. Struchiner, Limitedsampling strategy models for estimating the pharmacokinetic parameters of 4-methylaminoantipyrine, an active metabolite of dipyrone, Braz J. Med. Biol. Res. 34 (2001) 1475–1485. [14] A. Ojha, R. Rathod, H. Padh, Quantification of 4-methylaminoantipyrine, the active metabolite of dipyrone, in human plasma, Bioanalysis 1 (2009) 293–298. [15] G. Geisslinger, R. Böcker, M. Levy, High-performance liquid chromatographic analysis of dipyrone metabolites to study their formation in human liver microsomes, Pharm. Res. 13 (1996) 1272–1275. [16] S. Itoh, K. Tanabe, Y. Furuichi, T. Suzuka, K. Kubo, M. Yamazaki, A. Kamada, Ion-pair high-performance liquid chromatographic analysis of sulpyrine and its metabolites in rabbit plasma, Chem. Pharm. Bull. 32 (1984) 3194–3198. [17] U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER): Guidance for Industry, Bioanalytical Method Validation. http://www.fda.gov/downloads/ Drugs/GuidanceCompilanceRegulatory Information/Guidances/ucm070107. pdf, May 2001 (accessed 30.01.12). [18] M. Zimmermann, Ethical guidelines for investigations of experimental pain in conscious animals, Pain 16 (1983) 109–110. [19] G. Asmardi, F. Jamali, High-performance liquid-chromatography of dipyrone and its active metabolite in biological fluids, J. Chromatogr. 277 (1983) 183–189.