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Blood Matrix Effects for Male and Female Wistar Rats, in Simultaneous HPLC-UV

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Blood Matrix Effects for Male and Female Wistar Rats, in Simultaneous HPLC-UV Talanta 86 (2011) 233–240 Contents lists available at SciVerse ScienceDirect Talanta j ournal homepage: www.elsevier.com/locate/talanta Blood matrix effects for male and female Wistar rats, in simultaneous HPLC-UV determination of riparin I and III from Aniba riparia (Nees) Mez. (Lauraceae) Sócrates Golzio dos Santos, Karine Formiga Queiroga, Adriana Maria Fernandes de Oliveira, Josean Fechine Tavares, Stanley Juan Chavez Gutierrez, Margareth de Fátima Formiga Melo Diniz, ∗ José Maria Barbosa-Filho, Marcelo Sobral da Silva Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, 58051-900 João Pessoa, PB, Brazil a r t i c l e i n f o a b s t r a c t Article history: Aniba riparia (Nees) Mez. (Lauraceae) is popularly known as “louro”, and is found in Amazonia and in Received 6 July 2011 the Guianas, its distribution extends to the Andes. Alkamide alkaloids were isolated from its green fruit; Received in revised form 4 September 2011 they were denominated riparin I (methyl ether of N-benzoyl tyramine), riparin II (methyl ether of N-2- Accepted 5 September 2011 hydroxy-benzoyl tyramine) and riparin III (methyl ether of N-2,6-dihydroxy-benzoyl tyramine) in tribute Available online 10 September 2011 to the plant. When administered orally and intraperitoneally to mice, riparin I and III are anxiolytic, yet without any sedative or muscle relaxing effects. The present study shows that variables such as extraction Keywords: solvent, centrifugation force, and centrifugation time, are important in the simultaneous liquid–liquid HPLC-UV extraction of riparin I and III from male and female Wistar rat blood in HPLC-UV studies. The study Matrix effect Blood confirms matrix influence on simultaneous recovery and detection of riparin I and III. The effect of rat − − Aniba riparia blood matrix for riparin I was 13.86%, while for riparin III it was 10.94%. The recovery for riparin I was Riparins 82.14%, while for riparin III it was 87.42%. The efficiency of the process was 73.25% for riparin I and 77.81% for riparin III, demonstrating an optimal method for simultaneous recovery of riparins I and III from the blood of rats. The matrix effect for rat blood showed values of 10.25% for riparin I and −83.01% for riparin III. Recovery for riparin I was 113.11%, whereas for riparin III it was 13.65%. The process efficiency of this method for female rat blood was 125.88% for riparin I and 2.58% for riparin III. Simultaneous recovery of riparin I and III from the blood of male and female rats using acetonitrile as the precipitating solvent, while centrifuged at 10,000 × g for 10 min demonstrated the importance of the parameters chosen for the extraction/recovery process of different analytes. © 2011 Elsevier B.V. Open access under the Elsevier OA license. 1. Introduction west as the Andes. Alkamide alkaloids were isolated from the green fruit of this plant, and were called riparin I (methyl ether The family Lauraceae encompasses 52 genera and 3,000 species of N-benzoyl tyramine) (Fig. 1A) and riparin III (methyl ether of dispersed around the world. They are predominantly found in N-2,6-dihydroxy-benzoyl tyramine) (Fig. 1B) [7]. Riparins I and III, tropical and subtropical regions, especially in Central and South when administered orally or intraperitoneally in mice showed anx- American forests, and usually in temperate zones as well [1]. In iolytic effects, yet without any sedative or muscle relaxing effects, Brazil, we find the family represented by 22 genera which are gen- thus eliminating the common side effects associated with classic erally arboreal [2]. benzodiazepines [8,9]. Brazilian Neotropical Lauraceae are economically, and ecolog- Biological matrix effects are undesireable for analyte studies ically important [3], as many of species of the family are used in and can result in either suppression or increases in ionic sig- popular medicine for cutaneous lesions, gastric disturbances [4,5], nal, sensitivity variance over time in analyte detections, baseline as an anti-inflammatory, and for circulatory problems in both west- increases, data imprecision, retention time alterations, peak shape ern and eastern cultures [4], they also have hypoglycemic [5] and distortions and/or chromatographic tails [10–12]. Signal diminu- anxiolytic properties [6]. ition in the matrix recovery is one of the effects of endogenous Aniba riparia (Nees) Mez. (Lauraceae) is popularly known as compound binding, and analyte metabolism in biological fluids “louro” and is found in Amazonia and the Guianas, and as far [13,14]. Clean-up procedures for biological fluids, such as plasma, are used to separate endogenous material from the analyte. The sen- ∗ sitivity and selectivity of an assay are affected by the efficiency of Corresponding author. Tel.: +55 83 3216.7427. E-mail address: [email protected] (M.S. da Silva). the clean-up procedures [15–17]. 0039-9140 © 2011 Elsevier B.V. Open access under the Elsevier OA license. doi:10.1016/j.talanta.2011.09.008 234 S.G. dos Santos et al. / Talanta 86 (2011) 233–240 SPD-10AV vp detector. The separation was carried out with an ACE C18 (250 mm × 4.6 mm, 5 ␮m) column equipped with an ACE C18 (A) (4.6 mm) guard column. The mobile phase used was (20:80, v/v, H −1 N H2O:MeCN), at a flow rate of 1 mL min . The wavelength of the UV detector used for analysis was 225 nm, column temperature was ◦ controlled at 27 C, and the auto-injector temperature controlled ◦ at 4 C. The sample injection volume was 20 ␮l. Under these con- O ditions, riparin I and riparin III have respective retention times of O 7.89 ± 0.2 min and 10.03 ± 0.2 min. HO The samples of rat blood were gently vortexed (AP-56- TECNAL/PHOENIX) and centrifuged utilizing a Sigma Laborven- (B) trisugen 2K15 centrifuge. H N 2.3. Preparation of standard solutions The stock solutions of riparin I and III were prepared separately −1 O OH in H2O:MeCN (50:50, v/v,) at a concentraction of 1 mg mL and ◦ O stored at 4 C. Fig. 1. (A) Structure of riparin I and (B) structure of riparin III. 2.4. Preparation of samples in blood, samples in solution and samples for quality control No bioanalytical methods for riparin I and III determinations −1 −1 Riparin I (1 mg mL ) and III (1 mg mL ) were added to sam- have been previously reported in the literature, and there are no ples of male rat blood (300 ␮l) to obtain a final concentration of published pharmacokinetic studies for these substances since these −1 100 ␮g mL , and the samples were then gently vortexed for 30 s. types of studies require delineation of blood matrix effects [18]. The blank rat blood samples were gently vortexed for 30 s. These The present study demonstrates that centrifugation force procedures were repeated for the female rat blood samples. and time, and the extraction solvent used for simultaneous Samples of a mixture solution of riparin I and III were pre- liquid–liquid extraction of riparin I and III from male and female −1 pared from stock solutions (1 mg mL ) in H2O:MeCN (50:50, v/v) Wistar rat blood are important variables in determinating the −1 to obtain final concentrations of 100 ␮g mL in 300 ␮l of solution, extent of matrix effect when using high-performance liquid chro- and were then gently vortexed for 30 s. The same solution sam- matography with UV detection (HPLC-UV). ples of riparin I and III in H2O:MeCN (50:50, v/v) were prepared for quality control (CQ). 2. Experimental 2.5. Preparation of the calibration curve 2.1. Chemical products, reagents and animais Samples of riparin I and III in H2O:MeCN (50:50, v/v) were pre- − Riparin I and III were obtained by organic synthesis and donated 1 pared at concentrations of 20, 50, 80, 130 and 160 ␮g mL to by PhD Stanley Juan Chavez Gutierrez, and PhD José Maria Barbosa obtain a calibration curve of peak area (y) plotted against theo- − Filho of the Paraiban Federal University of Brazil, in accordance with ␮ 1 retical concentration in g mL (x) with a correlation coefficient the methodology described in the literature [19]. of >0.99, which served to correlate, by linear regression, the peak For all experiments, ultra-pure water (type I) from an Option-Q areas obtained for the evaluation method at the concentration of Purelab labwater-system (Elga, São Paulo, Brazil), both HPLC-grade the test substances. acetonitrile (MeCN) and HPLC-grade metanhol (MeOH) from TEDIA (São Paulo, Brazil), and EDTA supplied by HEMSTAB Laboratory (São 2.6. Method of evaluation Paulo, Brazil) were used. The blood used was collected from male and female Wistar rats Male rat blood samples were spiked with riparin I and III, and (8–10 weeks, weighing 250–300 g), in tubes with EDTA. The ani- were prepared in triplicate. A volume of 600 ␮l of MeOH or MeCN mals were from the Prof. Dr. Thomas George Animal House and was added, and the tubes were gently vortexed for 1 min and kept under standard laboratory conditions: controlled temperature centrifuged at 10,000 × g, 12,000 × g and 14,000 × g, where each ◦ (21 ± 1 C), light/dark cycle (12 h), with pelleted feed and water centrifugation was for 10, 15 and 20 min (Table 1). The same pro- ad libitum. The whole study was approved by the Committee on cedure was performed for female rat blood spiked with riparin I Ethics in Research Animals (CEPA) under protocol No.
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