Simultaneous determination of dipyrone metabolites ... - Future Science

R esearch A rticle For reprint orders, please contact [email protected]

Simultaneous determination of dipyrone metabolites in rat hypothalamus, cerebrospinal fluid and plasma samples by LC–MS/MS Background: After oral administration dipyrone is rapidly hydrolyzed to 4-methylaminoantipyrine, which is absorbed and further metabolized to 4-formylaminoantipyrine and to 4-aminoantipyrine, which is acetylated by a polymorphic N-acetyltransferase system to 4-acetylaminoantipyrine. To evaluate the presence of dipyrone metabolites in different rat matrices after intraperitoneal administration, an analytical method was developed and validated. Methodology: The four main dipyrone metabolites were extracted from plasma, cerebrospinal fluid and hypothalamus samples by LLE prior to LC–MS/MS. Results: Standard calibration graphs for all metabolites were linear (r > 0.99). The intra- and inter-day precision and accuracy values were both inferior to 15%. Conclusion: This method is simple and specific for studying dipyrone metabolites after intraperitoneal administration.

The prodrug dipyrone (DIP) is a water-soluble pyrazolonic derivative with potent antipyretic and analgesic effects. After oral administration, it is rapidly hydrolyzed in the gastrointestinal tract to 4-methylaminoantipyrine (4-MAA), which is absorbed and further metabolized to 4-formylaminoantipyrine (4-FAA) and to 4-aminoantipyrine (4-AA), which is acetylated by a polymorphic N-acetyltransferase system to 4-acetylaminoantipyrine (4-AAA) [1]. After intravenous administration, DIP is detectable for approximately 15 min in the serum, while it is undetectable after oral intake [2]. The mechanisms of analgesic and antipyretic effects of DIP have been investigated for a long time. It is widely accepted that prostaglandin E2 (PGE2), generated in the preoptic area of the anterior hypothalamus, is the main mediator of fever induced by lipopolysaccharide [3], and there are a few studies suggesting that DIP – similar to other nonsteroidal anti-inflammatory drugs – produces an antipyretic effect by inhibiting PGE 2 synthesis [4,5]. Corroborating this hypothesis Souza et al. showed that intracerebroventricular injection of DIP produces antipyresis in mice [6], while Hinz et al. and Pierre et al. demonstrated through in vitro and ex vivo studies that DIP inhibits PGE2 synthesis in human blood [7,8]. Furthermore, in vitro studies showed that only 4-MAA and 4-AA metabolites inhibit PGE2 synthesis in human blood, indicating that they are the active metabolites of DIP [7,8]. However, studies from our laboratory indicate that the

antipyretic mechanism of DIP does not involve inhibition of PGE2 synthesis [9,10]. Corroborating these findings we recently demonstrated that the antipyretic effect of DIP is unrelated to PGE2 synthesis inhibition in the hypothalamus [11]. Additionally, there are studies indicating that DIP produces antipyretic and analgesic effects by acting in the CNS [6,12]. In order to act at the CNS, systemically administered drugs must cross the barrier between the blood and the brain parenchyma proper (blood–brain barrier) and/or the barrier between the blood and the cerebrospinal fluid (CSF; blood–CSF barrier), where their distribution depends on the direction of gradients between CSF and interstitial cerebral fluid. Thus, the extent of permeability of each drug and its accessibility to different areas of the CNS is clearly compound-dependent [13]. Cohen et al. demonstrated that DIP metabolites enter in the CSF after oral administration of DIP in humans [1]. Moreover, there is only one study in which the correlation of serum concentration of DIP metabolites and analgesic effect of DIP has been described [14]. However, there are no data about the relation of DIP metabolites concentration in the plasma, CSF or hypothalamus – the thermoregulatory center of the brain [15] – and its antipyretic effects. For this reason, a sensitive technique for the detection and quantification of DIP metabolites in the plasma, CSF and hypothalamus is required. Considering that DIP is only briefly stable in serum [2], its concentration was not evaluated in other matrices.

10.4155/BIO.13.229 © 2013 Future Science Ltd

Bioanalysis (2013) 5(21), 2631–2645

Fernando A Aguiar ‡1, David do C Malvar ‡2 , Artur de LL Vaz3, Leandro A Calixto2 , Giuliano C Clososki3, Cristiane M de Gaitani1, Glória EP de Souza2 , Valquíria AP Jabor*2 Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil 2 Department of Physics & Chemistry, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil 3 Department of Physics & Chemistry, Research Center for Natural & Synthetic Products, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 14040–14903, Ribeirão Preto, SP, Brazil *Author for correspondence: Fax: +55 16 3602 4880 E-mail: [email protected] 1

These authors contributed equally

‡

ISSN 1757-6180

2631

R esearch A rticle | Key Terms Prodrug: Pharmacological

substance that is administered in an inactive (or less than fully active) form and is subsequently converted to an active pharmacological agent through normal metabolic processes (bioactivation). A prodrug serves as a type of ‘precursor’ to the intended drug.

Dipyrone: Prodrug water-

soluble pyrazolonic derivative with potent antipyretic and analgesic effects.

Hypothalamus: Portion of

the brain that contains a number of small nuclei with a variety of functions. One of the most important functions of the hypothalamus is to link the nervous system to the endocrine system via the pituitary gland (hypophysis).

Cerebrospinal fluid: Clear

colorless bodily fluid produced in the choroid plexuses of the brain. It acts as a cushion or buffer for the cortex, providing a basic mechanical and immunological protection to the brain inside the skull and serves a vital function in cerebral autoregulation of cerebral blood flow.

Plasma: Straw-colored/

pale-yellow liquid component of blood that normally holds the blood cells in whole blood in suspension.

Aguiar, Malvar, Vaz et al.

There are few methods for the quantification of DIP metabolites in different biological fluids or tissues. These mostly include studies involving the application of HPLC with diode-array UV, which requires a larger sample quantity and a longer analysis time [1,16–18]. LC–MS/MS appears to be an interesting alternative for DIP metabolite determination in very low concentrations and in complex matrices [19,20]. The present study describes the development and validation of an easy, selective and reliable LC–MS/MS method to determine DIP metabolites in rat plasma, CSF and hypothalamus samples. The importance of the method is demonstrated by its ability to compare the DIP metabolites concentrations in these matrices after a single intraperitoneal (i.p.) dose of DIP to healthy rats. Experimental Materials & reagents Sodium DIP and the metabolite 4-AA were obtained from Sigma-Aldrich (MO, USA). The metabolites, 4-MAA, 4-AAA and 4-FAA were synthesized at the Organic Synthesis Laboratory of the Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (São Paulo, Brazil) by GC Clososki, as described below. All metabolites were obtained with greater than 99% purity (GC-FID analysis). Methanol and glacial acetic acid for the mobile phase were HPLC grade (J.T. Baker®, Mexico City, Mexico). All other reagents were analytical grade (Merck KGaA, Darmstadt, Germany). HPLC grade water (18 Wm) was obtained by purifying distilled water in a Milli-Q system (Millipore, MA, USA). Mobile phase was degassed by the continuous passage of helium gas. LogP

& pKa calculation The pKa and LogP values of all DIP metabolites (Figure 1) were calculated with MarvinSketch 5.12.3 from Chemaxon Ltd (Budapest, Hungary) and with the Vortex data analysis and visualization software from Dotmatics (London, UK [101]), respectively. The calculated pKa values of DIP and its metabolites were below 1.3 or above 12.5, indicating that molecules are neutral at different pH values. Synthesis

of the DIP metabolites All procedures were carried out using commercial HPLC-grade solvents. Reagents were purchased from Sigma-Aldrich and used without further purification. Reactions were monitored by TLC or GC-FID induction decay analysis 2632

Bioanalysis (2013) 5(21)

in a Shimadzu GC-2014 chromatograph (Kyoto, Japan). Flash chromatography purifications were carried out on silica gel (60 Ä, 40–63 µm) from Sigma-Aldrich (Steinheim, Germany). NMR spectra (Supplementary Figures 1–6) were recorded on a Bruker DPX 400 spectrometer (Rheinstetten, Germany). Mass analyses were carried out on a Shimadzu QP-2010 mass spectrometer (Kyoto, Japan). Synthesis of 4-MAA

In a round-bottom flask, DIP (3.0 g, 90 mmol) was dissolved in aqueous sodium hydroxide (0.1 mol/l, 100 ml) and heated at 100°C for 30 min. Following this, the aqueous phase was extracted with chloroform (2 × 100 ml), dried over magnesium sulfate and concentrated by rotary evaporation. The crude product obtained was then purified through silica-gel column chromatography (ethyl acetate:methanol 95:5). The purified fractions were concentrated in vacuum and afforded 1.8 g (60% yield) of a yellow oily product that was analyzed by GC–MS and NMR spectrometry. 1H NMR (400 MHz-CDCl 3) d2.19 (s, 3H), 2.75 (s, 3H), 2.80 (s, 3H), 2.92 (bs, 1H), 7.14–7.20 (m, 1H), 7.32–7.41 (m, 4H); 13 C (100 MHz-CDCl3) d11.0, 35.3, 37.8, 122.9, 126.0, 129.1, 135.3, 139.8, 162.3 (Supplementary Figures 1&2); GC–MS (m/z, %): 217 (12) [M]+, 207 (1), 191 (3), 123 (13), 96 (6), 83 (27), 56 (100), 50 (5). Synthesis of 4-AAA

4-AA (3.3 g, 16.3 mmol) was added to a mixture of glacial acetic acid (1 g, 16.7 mmol) and 0.2 g of fuming sulfuric acid (the temperature was kept below 50°C with a water bath). The reaction mixture was then heated to 80–90°C and stirred for 30 min. Upon cooling, the mixture solidified and it was then grounded and treated with ammonium chloride. The mixture was suspended in ethyl acetate, the organic phase was separated and the solvent removed in rotatory evaporation. Treatment of the oily product with small amounts of cold ethyl acetate afforded 2.8 g (70% yield) of a light yellow product, which was filtered and analyzed by GC–MS and NMR spectrometry. 1H NMR (400 MHz-CDCl3 ) d 2.06 (s, 3H), 2.21 (s, 3H), 3.10 (s, 3H), 7.30 – 7.38 (m, 3H), 7.43 – 7.49 (m, 2H), 9.56 (s, 1H); 13 C (100 MHz-CDCl3) d 12.5, 23.3, 36.1, 108.7, 125.4, 127.9, 129.8, 134.7, 150.7, 162.4, 170.8 (Supplementary Figures 3 & 4); GC–MS (m/z,%): 245 (30) [M]+, 203 (38), 119 (1), 84 (43), 83 (37), 56 (100). future science group

Simultaneous determination of dipyrone metabolites by LC–MS/MS

SO3Na

N

N

Ph DIP (0.99)

O

N

N

N

O

H

O

H

H N

N O

H

Ph

Ph

4-AA (0.83)

4-MAA (1.32)

N

O

O

H N

H N N

| R esearch A rticle

N

N

Ph 4-AAA (0.79)

O

N

N

Ph 4-FAA (1.32)

Figure 1. Structure and LogP (as shown in brackets) of dipyrone metabolites. 4-AA: 4-aminoantipyrine; 4-AAA: 4-acetylaminoantipyrine; 4-FAA: 4-formylaminoantipyrine; 4-MAA: 4-methylaminoantipyrine; DIP: Dipyrone.

Synthesis of 4-FAA

4-AA (2.0 g, 10 mmol) and formic acid 85% (2.5 g, 146.9 mmol) were stirred in a round bottom flask under reflux for 4 h. After the reaction mixture was neutralized with sodium hydroxide, the product was extracted with ethyl acetate and this solution was allowed to stand at 25°C overnight. The product was filtered, washed with cold ethanol, cold water and then dried in vacuum to afford 1.39 g (60% yield) of yellowish crystals that were analyzed by GC–MS and NMR spectrometry. Double signals were observed in the 1H and 13C NMR spectra, due to the restricted rotation of C–N amide bond [21–23]. Herein we have described the resonance signals of the major isomer in solution. 1H NMR (400 MHz-Methanol-D4) d 2.26 (s, 3H), 3.18 (s, 3H), 7.36–7.47 (m, 3H), 7.49–7.59 (m, 2H), 8.24 (s, 1H); 13C (100 MHz-CDCl3) d 11.0, 35.4, 105.4, 127.2, 129.5, 130.6, 135.2, 152.1, 163.6, 167.7 (Supplementary F igures 5 & 6) ; GC–MS (m/z,%): 231 (12) [M]+, 215 (1), 203 (21), 119 (2), 84 (44), 83 (34), 56 (100), 50 (7). Preparation

of calibration standards Primary stock solutions of 4-MAA, 4-AA and 4-AAA (1.0 mg/ml), and 4-FAA (333.3 µg/ml) and moclobemide (IS 1.0 µg/ml), were prepared in methanol and stored at -20°C. Rat plasma, CSF and hypothalamus calibration standards of DIP metabolites were prepared by spiking appropriate aliquots of the stock solutions of each metabolite to drug-free rat matrices to give final concentrations. QC samples were prepared by adding the appropriate aliquots of the stock solutions to drug-free rat matrices. Sample

preparation Plasma, CSF and hypothalamus samples were prepared by liquid–liquid extraction. The drug-free rat plasma (0.1 ml) and CSF (50 µl) samples were spiked with 25 µl of IS solution future science group

(1.0 µg/ml) and 25 µl of the standard solutions of DIP metabolites. Each hypothalamus sample collected (weighing 90 ± 4.0 mg) was homogenized in 1 ml of water using a Digital 600-w ultrasonic microprocessor cell disrupter (Virsonic 100®, VirTis, NY, USA), and an aliquot of 250 µl was used for extraction. The enrichment of the samples was implemented by adding the standard solution of the DIP metabolites dissolved in methanol, and the solvent had evaporated prior to the addition of the matrices. The samples were made alkaline with 50 µl 1.0 mol/l sodium hydroxide solution, and 3 ml dichloromethane was added. The metabolites were extracted by mechanical shaking for 15 min. After centrifugation at 1800 × g for 15 min at 4 ± 0.5°C, the organic phase was recovered and evaporated under an air flow at room temperature (22 ±1.0°C). The residues were redissolved in 50 µl mobile phase and 20 µl were then injected into the LC–MS/MS system. LC–MS/MS

instrument & conditions The LC–MS/MS system included a Shimadzu LC-10AD pump equipped with a Rheodyne 7125 injector (Rheodyne, CA, USA) to which a 20 µl loop was connected, an SLC-10A system controller, a CTO-10AS column oven (both from Shimadzu), and a Quattro LC triple-quadrupole spectrometer (Micromass, Manchester, UK). A XTerra® C18 column 3.5 µm particle size (100 mm × 3.9 mm) preceded by a guard column C18, 4 × 4 mm, 3.5 µm particle size (both from Waters Corp., MA, USA) were used for separation of DIP metabolites and IS. The mobile phase consisted of methanol-water (30:70, v/v) plus 0.5% of glacial acetic acid delivered isocratically at a flow rate of 0.5 ml/min. A Valco valve connection (Restek, PA, USA) was used to split the effluent from the column and a flow rate of approximately 0.2 ml/min was directed into the stainless steel capillary probe of the LC–MS/MS. www.future-science.com

2633

R esearch A rticle |


MS/MS conditions were optimized by direct infusion of all analyte solutions (100 µg/ml), prepared in the mobile phase, at a flow rate of 15 µl/min. The ions were generated by employing ESI and detected in the positive ion mode at a potential of 3.0 kV. The temperatures of the source block and desolvation system were 100 and 350°C, respectively. Nitrogen was used as both drying and nebulizing gas and argon as the collision gas. The cone voltage

was defined at 20 V for DIP metabolites and 23 V for IS. Characteristic mass fragments of the identified precursor ions used for quantification were determined in SRM mode using precursor-product ion combinations. The collision energy was 20 eV for DIP metabolites and 15 eV for IS. Instrumental settings, data acquisition and peak processing were performed by the Micromass MassLynx Software Version 4.1, using an IS calibration method with peak area

A 100 % 0

IS

100 4-AAA % 0

2.00

B IS 100 % 0 100 % 0

6.00

100

2.00

100 4-FAA % 0

4.00

2.00

2.00

4.00

4.00 Time (min)

4.00

6.00

6.00

6.00

4-AAA

100 4-FAA % 0

2.00

2.00

C 100 % 0

IS

100 % 0

4-AAA

2.00

100 4-FAA % 0

100 % 0

2.00

2.00

4.00

4.00 Time (min)

4.00

4.00

4.00 Time (min)

6.00

6.00

6.00

4-MAA

% 0 100 % 0

100 % 0

4-AA

2.00

4.00

6.00

2.00

4.00

6.00

2.00

4.00 Time (min)

6.00

DIP

100 % 0

4-MAA

100 % 0

4-AA

100 % 0

DIP

100 % 0

4-MAA

100 % 0

4-AA

6.00

6.00

DIP

2.00

4.00

6.00

2.00

4.00

6.00

2.00

4.00 Time (min)

6.00

2.00

4.00

6.00

2.00

4.00

6.00

2.00

4.00 Time (min)

6.00

Figure 2. Chromatograms of blank samples from rats. (A) Hypothalamus; (B) cerebrospinal fluid; and (C) plasma. For extraction procedure and chromatographic conditions see ‘Experimental’ section (sample preparation and LC–MS/MS conditions). 4-AA: 4-aminoantipyrine; 4-AAA: 4-acetylaminoantipyrine; 4-FAA: 4-formylaminoantipyrine; 4-MAA: 4-methylaminoantipyrine; DIP: Dipyrone.

2634


future science group

Simultaneous determination of dipyrone metabolites by LC–MS/MS


Table 1. Recoveries of dipyrone metabolites in spiked matrices samples compared with solution calibration standard. Matrix

Spiking level

Hypothalamus 1.1 (ng/mg) 11.1 111.1 Plasma 0.5 (µg/ml) 5.0 10.0 Cerebrospinal 0.5 fluid 5.0 (µg/ml) 20.0

4-AAA

4-MAA

4-AA

Average† recovery

RSD (%)

Average† recovery

RSD (%)

Average† recovery

RSD (%)

90.8 80.9 88.7 38.2 56.4 64.2 53.7 62.1 61.5

12.2 3.5 5.5 0.8 2.8 3.5 4.5 3.5 11.0

110.6 54.9 94.6 61.9 62.3 96.5 78.8 57.5 65.3

4.7 2.3 3.6 8.4 5.2 4.4 1.6 4.8 11.8

31.1 64.9 83.4 81.6 71.5 83.7 79.6 30.9 38.2

1.2 2.1 14.3 9.7 1.6 0.7 9.9 5.1 13.4

Spiking level 0.3 3.3 33.3 0.2 0.8 8.3 0.1 1.0 3.3

4-FAA Average† recovery

RSD (%)

94.0 80.7 92.6 86.4 58.3 52.7 53.2 62.5 62.8

13.1 3.4 8.8 2.4 2.9 1.1 3.2 2.9 11.7

Means of three replicates. 4-AA: 4-aminoantipyrine; 4-AAA: 4-acetylaminoantipyrine; 4-FAA: 4-formylaminoantipyrine; 4-MAA: 4-methylaminoantipyrine. †

ratios. Dwell time was 0.4 s for all analytes. The monitored transitions were m/z 218 >159, 218 >187 and 218 >125 for 4-MAA, m/z 204 >159 and 204 >146 for 4-AA, m/z 246 >228 for 4-AAA, m/z 389 >245 for 4-FAA and m/z 269 >182 for IS. Bioanalytical

method validation Method validation was performed according to the European Medicines Agency Guideline on Validation of Bioanalytical Methods [24]. The calibration curves were constructed using biological matrices (drug-free rat plasma, CSF and hypothalamus) spiked with 25 µl of each diluted standard solution. Each such calibration solution received 25 µl of IS and was submitted to the extraction procedure as described above. For the hypothalamus, the calibration curve covered the range from 0.4 to 220.0 ng/ mg for 4-AAA, 4-MAA and 4-AA, and from 0.13 to 66.7 ng/mg for 4-FAA. For CSF the

calibration curve covered the range from 250 to 150,000 ng/ml for 4-A A A, 4-MA A and 4-A A, and from 83.3 to 50,000 ng/ml for 4-FAA. For plasma, the calibration curve covered the range from 125 to 150,000 ng/ml for 4-AAA, 4-MAA and 4-AA, and from 41.7 to 50,000 ng/ml for 4-FAA. The linear regression equations and the correlation coefficients were obtained from the peak area ratios (analyte/ IS) plotted against nine concentration levels to hypothalamus and 11 concentration levels to CSF and plasma. Stability study of DIP metabolites and IS was carried out. Spiked samples (plasma, CSF and hypothalamus) with high and low concentrations of drugs were subjected to long-term storage conditions, short-term room temperature conditions and three cycles of freeze (-20 ± 1.0°C) and thaw (25 ±1.0°C). Matrix effects were validated using a postextraction spike method. Blank matrix samples

Table 2. Evaluation of stability. Matrix

Nominal concentration

Plasma

0.3 µg/ml 5.0 µg/ml Hypothalamus 1.1 ng/ml 111.1 ng/ml Cerebrospinal 0.5 µg/ml fluid 10.0 µg/ml

Metabolites 4-AA

4-MAA

4-AAA

F-value† p-value‡ F-value p-value

F-value p-value

2.4 2.1 0.8 0.3 0.6 0.01

1.7 2.1 2.5 0.3 0.1 0.7

0.2 0.1 0.6 0.8 0.6 1.0

1.1 0.7 0.8 1.2 0.8 0.3

0.4 0.6 0.6 0.4 0.5 0.8

0.2 0.2 0.1 0.8 0.9 0.6

Nominal concentration 0.1 µg/ml 1.6 µg/ml 0.3 ng/ml 33.0 ng/ml 0.2 µg/ml 3.3 µg/ml

4-FAA F-value p-value 3.2 1.2 0.6 1.5 0.1 2.2

0.1 0.4 0.7 0.3 1.0 0.2

Samples analyzed by one-way ANOVA followed by the Dunett’s test. † Level of significance set at Fcal < Ftab, 95% = 3.59. ‡ p < 0.05 compared with the groups of stabilities with sample recently prepared. 4-AA: 4-aminoantipyrine; 4-AAA: 4-acetylaminoantipyrine; 4-FAA: 4-formylaminoantipyrine; 4-MAA: 4-methylaminoantipyrine.


www.future-science.com

2635



(drug-free) were submitted to the LLE method, and were spiked with 4-MAA, 4-FAA, 4-AA and 4-AAA standard solutions for two concentration levels and IS at 1 µg/ml. These samples

were analyzed in triplicate, and the areas were compared to those obtained from the standards in solution at the same concentration levels. The values were expressed as matrix effect (%).

A 100

100 %

%

IS

0 0.00

2.50

5.00

7.50

DIP

0

10.00

0.00

12.50

100

2.50

5.00

7.50

10.00

12.50

100

%

%

4-AAA

0 0.00

2.50

5.00

7.50

10.00

12.50

100

2.50

B

5.00 7.50 Time (min)

10.00

12.50

100

2.50

5.00

7.50

10.00

12.50

% 2.50

5.00

7.50

10.00

0.00

2.50

5.00

7.50

10.00

12.50

10.00

12.50

4-MAA 0.00

2.50

5.00

7.50

%

10.00

12.50

2.50

C

5.00 7.50 Time (min)

10.00

4-AA

0 0.00

12.50

100

2.50

5.00 7.50 Time (min)

2.50

5.00

10.00

12.50

100

%

%

IS 0.00

5.00 7.50 Time (min)

DIP

0

12.50

4-FAA

0

2.50

100

% 0.00

0.00

0

100 0

12.50

4-AA

0

%

4-AAA 0.00

10.00

100

100 0

7.50

%

IS 0.00

5.00

100

% 0

2.50

%

4-FAA 0.00

0.00 100

% 0

4-MAA

0

2.50

5.00

7.50

10.00

12.50

DIP

0 0.00

7.50

10.00

12.50

100

100 %

%

4-AAA

0 0.00

2.50

5.00

7.50

10.00

2.50

5.00

7.50

10.00

12.50

100

%

%

4-FAA 0.00

0.00

12.50

100 0

4-MAA

0

2.50

5.00 7.50 Time (min)

10.00

4-AA

0 12.50

0.00

2.50

5.00 7.50 Time (min)

10.00

12.50

Figure 3. Chromatograms of samples from rats. (A) Hypothalamus; (B) cerebrospinal fluid; and (C) plasma. All samples were spiked with dipyrone metabolites at LOQ concentration and then extracted. For extraction procedure and chromatographic conditions see ‘Experimental’ section (sample preparation and LC–MS/MS conditions). 4-AA: 4-aminoantipyrine; 4-AAA: 4-acetylaminoantipyrine; 4-FAA: 4-formylaminoantipyrine; 4-MAA: 4-methylaminoantipyrine; DIP: Dipyrone.

2636



Simultaneous determination of dipyrone metabolites by LC–MS/MS In vivo

rat experiments Experiments were performed using male Wistar rats (180–200 g), hosted at 24 ±1°C under a 12:12 h light–dark cycle (lights on at 06:00 AM).


Water and food were available ad libitum until 14 h before the experiment, when only water was made available. The animals were used only once.

A 100

100 %

%

IS

0 0.00

2.50

5.00

7.50

10.00

DIP

0 0.00

12.50

2.50

5.00

7.50

10.00

12.50

100

100 %

%

4-AAA

0 0.00

2.50

5.00

7.50

10.00

4-MAA

0 0.00

12.50

100

2.50

5.00

7.50

10.00

12.50

100

%

%

4-FAA

0 0.00

2.50

B

5.00 7.50 Time (min)

10.00

12.50

4-AA

0 0.00

2.50

5.00 7.50 Time (min)

10.00

12.50

100

100 %

%

IS

0 0.00

2.50

5.00

7.50

10.00

DIP

0 0.00

12.50

2.50

5.00

7.50

10.00

12.50

100

100 %

%

4-AAA

0 0.00

2.50

5.00

7.50

10.00

4-MAA

0 0.00

12.50

100

2.50

5.00

7.50

10.00

12.50

100

%

%

4-FAA

0 0.00

2.50

C

5.00 7.50 Time (min)

10.00

12.50

%

2.50

5.00 7.50 Time (min)

%

IS 0.00

0.00

10.00

12.50

100

100 0

4-AA

0

2.50

5.00

7.50

10.00

DIP

0 0.00

12.50

100

2.50

5.00

7.50

10.00

12.50

100

%

%

4-AAA

0 0.00

2.50

5.00

7.50

10.00

12.50

%

2.50

5.00

7.50

%

4-FAA 0.00

0.00

10.00

12.50

100

100 0

4-MAA

0

2.50

5.00 7.50 Time (min)

10.00

4-AA

0 12.50

0.00

2.50

5.00 7.50 Time (min)

10.00

12.50

Figure 4. Chromatograms of sample from rats after dipyrone administration. (A) Hypothalamus; (B) cerebrospinal fluid; and (C) plasma. All samples were collected 1 h after dipyrone administration (120 mg/kg). For extraction procedure and chromatographic conditions see ‘Experimental’ section (sample preparation and LC–MS/MS conditions). 4-AA: 4-aminoantipyrine; 4-AAA: 4-acetylaminoantipyrine; 4-FAA: 4-formylaminoantipyrine; 4-MAA: 4-methylaminoantipyrine; DIP: Dipyrone.



2637



Rats were treated with DIP, 120 mg/kg i.p. The rat samples were withdrawn, as described below, at 0 h (before the administration of the treatment) and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3.5, 5.5 and 8.5 h after the administration of the DIP. For each time, 6–8 rats were used. Single blood samples (1.0 ml) were collected by cardiac puncture from animals anesthetized by a mixture of ketamine and xylazine (60 and 20 mg/kg, respectively, i.p.). The samples were placed in tubes containing heparin, cooled on ice, protected from light, and centrifuged at 1,300 × g for 15 min at 4°C. The plasma samples

were collected and immediately frozen to -20°C until analysis. Each CSF sample was collected according to the method described by Consiglio et al. [25]. A 25-gauge scalpel connected to a 1.0 ml syringe was inserted vertically and centrally into the cisterna magna, and CSF was gently aspirated, resulting in 50–100 µl samples. CSF samples contaminated with blood were discarded. The CSF samples were immediately frozen to -20°C until analysis. Immediately after CSF collection, the animals were decapitated and their brains rapidly removed. The entire hypothalamus was

Table 3. Linearity and LOQ analysis of dipyrone metabolites spiked to rat hypothalamus samples. Metabolite 4-AAA 4-FAA 4-MAA 4-AA

Range (ng/mg) 0.4–222.0 0.1–66.7 0.4–222.0 0.4–222.0

Linear equation y = 0.076x + 0.3720 y = 0.017x + 0.0289 y = 0.005x + 0.0087 y = 0.047x + 0.0058

Correlation coefficients 0.9974 0.9964 0.9954 0.9972

LOQ Value (ng/mg)

RSD (%) †

RE (%) ‡

0.4 0.1 0.4 0.4

10.9 12.2 11.8 7.4

-11.9 -15.0 19.7 16.5

RSD (%) = RSD[(SD/mean value) × 100]. RE (%) = [(added concentration - recovered concentration)/added concentration] × 100. 4-AA: 4-aminoantipyrine; 4-AAA: 4-acetylaminoantipyrine; 4-FAA: 4-formylaminoantipyrine; 4-MAA: 4-methylaminoantipyrine. † ‡

Table 4. Linearity and LOQ analysis of dipyrone metabolites spiked to rat cerebrospinal fluid samples. Metabolite 4-AAA 4-FAA 4-MAA 4-AA

Range (ng/ml) 250–5000 5000–150,000 83.3–1666.6 1666.6–50,000 250–5000 5000–150,000 250–5000 5000–150,000

Linear equation y = 0.0002x + 0.0378 y = 0.00009x + 2.0092 y = 0.00006x + 0.0032 y = 0.00002x + 0.1768 y = 0.000006x + 0.0001 y = 0.000005x + 0.0705 y = 0.000005x + 0.0004 y = 0.000008x + 0.0072

Correlation coefficients 0.9978 0.9972 0.9972 0.9982 0.9974 0.9975 0.9982 0.9995

LOQ Value (ng/ml)

RSD (%) †

RE (%) ‡

250

9.0

- 14.6

83.3

5.9

- 2.3

250

10.4

1.3

250

14.8

- 13.1

RSD (%) = RSD[(SD/mean value) × 100]. RE (%) = [(added concentration - recovered concentration)/added concentration] × 100. 4-AA: 4-aminoantipyrine; 4-AAA: 4-acetylaminoantipyrine; 4-FAA: 4-formylaminoantipyrine; 4-MAA: 4-methylaminoantipyrine. †

‡

Table 5. Linearity and LOQ analysis of dipyrone metabolites spiked to rat plasma samples. Metabolite Range (ng/ml) Linear Equation 4-AAA 4-FAA 4-MAA 4-AA

125–2500 2500–150,000 41.7–833.3 833.3–50,000 125–2500 2500–150,000 125–2500 2500–150,000

y = 0.0005x + 0.0483 y = 0.0003x + 0.5351 y = 0.0001x + 0.0067 y = 0.0004x + 0.1688 y = 0.00002x + 0.0011 y = 0.00002x + 0.0049 y = 0.000015x + 0.00034 y = 0.000023x + 0.0123

Correlation coefficients 0.9971 0.9977 0.9962 0.9956 0.9974 0.9978 0.9975 0.9972

LOQ Value (ng/ml)

RSD (%) †

RE (%) ‡

125

11.5

-18.3

41.7

7.7

-0.6

125

11.1

9.0

125

6.8

-4.9

RSD (%) = RSD [(SD/mean value) × 100]. RE (%) = [(added concentration - recovered concentration)/added concentration] × 100. 4-AA: 4-aminoantipyrine; 4-AAA: 4-acetylaminoantipyrine; 4-FAA: 4-formylaminoantipyrine; 4-MAA: 4-methylaminoantipyrine. † ‡

2638



Simultaneous determination of dipyrone metabolites by LC–MS/MS dissected from the brain using the following limits: the anterior border of the optic chiasm, the anterior border of the mammillary bodies, and the lateral hypothalamic sulci, with a depth of 2 mm. The total dissection time elapsed from decapitation was less than 2 min [26], and the hypothalamus were immediately frozen to -20°C until analysis. The matrices were collected from the same animal at different time points. Results & discussion Method optimization HPLC method with MS/MS detection is a common method for the analysis of metabolites in biological samples, since it copes with the complexity of the task. In order to obtain clean and interference-free extracts, a LLE procedure was used and different solvents were screened. Thus, the use of dichloromethane, hexane, ethyl acetate, diethyl ether and mix diethyl ether-hexane (1:1, v/v), as well as different pH conditions prior to the extraction procedure, were investigated. Dichloromethane (3.0 ml), under alkaline conditions, provided an adequate recovery after the one-step liquid–liquid extraction in all biological matrices. Moreover, this extraction procedure proved to be precise and reproducible for all analytes. The DIP metabolites were separated on a reversed-phase LC XTerra® C18 column


(100 mm × 3.9 mm, 3.5 µm particle size) using a C18 (4 mm × 4 mm, 3.5 µm particle size) guard column. Under isocratic conditions the mobile phase consisting of methanol:water (30:70, v/v) plus 0.5% of glacial acetic acid, was pumped at a flow rate of 0.5 ml/min. The elution order was as follows, with approximate retention times in parentheses: 4-MAA (2.7 min), 4-AA (3.3 min), 4-FAA (4.2 min), 4-AAA (4.4 min) and IS (4.96 min). Figure 2 shows typical LC–MS/MS chromatograms of hypothalamus extract spiked with DIP metabolites and the IS. The chromatographic profile was the same in all matrices. The sample preparation conditions and analysis procedure resulted in interference-free chromatograms at the retention times of all compounds. Method

validation Absolute recoveries were calculated by comparing peak areas from extracted samples with peak areas of unextracted standards. The recovery is an important parameter in validation for methods requiring sample extraction. However, this parameter is not described or mentioned in the European Medicines Agency guidance [24]. For the analysis of this parameter, the US FDA Bioanalytical Methods Validation guidance was used [27]. Although no acceptance criteria for extraction recovery are recommended by this guide,

Table 6. Intra- and inter-day precision and accuracy analysis of dipyrone metabolites spiked to rat hypothalamus. Metabolite Nominal concentration (ng/mg) 4-AAA

4-FAA

4-MAA

4-AA

1.1 11 111 222 0.3 3.3 33.3 66.6 1.1 11 111 222 1.1 11 111 222

Intra-day†

Inter-day‡

Recovered

RSD (%)

RE (%)

Recovered

RSD (%)

RE (%)

1.2 11.0 115.6 218.1 0.4 3.3 33.8 61.9 1.2 11.1 104.1 217.2 1.1 10.6 115.8 226.8

12.0 4.8 4.0 10.2 10.3 5.4 8.6 10.2 4.2 5.7 10.8 9.2 8.8 12.0 3.4 10.2

12.7 0.2 4.0 -1.8 9,1 0.2 1.3 -7.1 4.5 1.1 -6.3 -2.1 -3.6 -4.0 4.3 2.1

1.2 11.0 119.6 219.8 0.4 3.4 33.9 64.2 1.2 11.3 109.9 222.8 1.1 11.0 113.7 224.0

6.1 5.7 7.3 5.7 8.7 4.9 8.0 9.0 7.6 10.6 11.1 9.8 8.9 8.5 10.6 6.9

5.9 0.04 7.7 -1.0 6.1 0.6 1.8 -3.6 5.5 2.6 -1.8 0.4 -0.4 0.1 2.4 0.9

Six replicates of each concentration. Three consecutive days. 4-AA: 4-aminoantipyrine; 4-AAA: 4-acetylaminoantipyrine; 4-FAA: 4-formylaminoantipyrine; 4-MAA: 4-methylaminoantipyrine. † ‡



2639



Table 7. Intra- and inter-day precision and accuracy analysis of dipyrone metabolites spiked to rat cerebrospinal fluid. Metabolite Range (ng/ml)

4-AAA 4-FAA 4-MAA 4-AA

250–5000 5000–150,000 83.3–1666.6 1666.6–50,000 250–5000 5000–150,000 250–5000 5000–150,000

Intra-day RSD (%)

Inter-day RE (%)

RSD (%)

RE (%)

Max.

Min.

Max.

Min.

Max.

Min.

Max.

Min.

9.0 14.5 5.9 11.3 11.0 14.8 12.2 9.9

2.3 1.9 2.2 3.6 3.3 3.1 3.0 2.3

12.2 3.7 3.5 10.9 13.2 5.7 1.9 2.2

-14.6 -12.9 -7.0 -11.4 -2.4 -13.7 -7.9 -5.6

9.0 12.5 9.8 9.6 13.1 11.9 14.0 9.2

5.4 3.3 3.4 4.0 3.9 3.61 3.1 3.1

12.6 12.6 5.3 12.4 10.5 13.6 7.8 5.5

-16.2† -5.7 -5.7 -7.7 -4.4 -3.4 -4.0 -4.0

LOQ. 4-AA: 4-aminoantipyrine; 4-AAA: 4-acetylaminoantipyrine; 4-FAA: 4-formylaminoantipyrine; 4-MAA: 4-methylaminoantipyrine. †

the extent of recovery of an analyte and of the IS should be consistent, precise and reproducible. A recovery higher than 30% was obtained with a good precision (RSD