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HPLC quantification of doxorubicin in plasma and tissues of rats treated with doxorubicin loaded poly(alkylcyanoacrylate) nanoparticles
Journal of Chromatography B, 887–888 (2012) 128–132
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
HPLC quantification of doxorubicin in plasma and tissues of rats treated with doxorubicin loaded poly(alkylcyanoacrylate) nanoparticles Khairallah Alhareth a,b,1 , Christine Vauthier a,b,∗ , Claire Gueutin a,b , Gilles Ponchel a,b , Fathi Moussa c,∗∗ a b c
Univ Paris Sud, Physico-chimie, Pharmacotechnie et Biopharmacie, UMR 8612, Châtenay-Malabry F-92296, France CNRS, Châtenay-Malabry F-92296, France LETIAM, IUT d’Orsay, EA 4041, Univ Paris Sud. Plateau du Moulon, Orsay 91400, France
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Article history: Received 13 October 2011 Accepted 22 January 2012 Available online 30 January 2012 Keywords: Doxorubicin Idarubicin Doxorubicinol Doxorubicinon Poly(alkylcyanoacrylate) nanoparticles
a b s t r a c t The long-term clinical use of doxorubicin (Dox), one of the most important anticancer agent in use, is limited by dose-related acute cardiotoxicity, myelo-suppression and multidrug resistance developed by cancer cells. To improve the antitumor efficacy and reduce the toxicity of Dox, many drug delivery systems have been developed, including poly(alkylcyanoacrylate) (PACA) nanoparticles. A new formulation of PACA nanoparticles with potential stealth properties were prepared by redox radical emulsion polymerization and associated to Dox in our laboratory. To comparatively investigate the pharmacokinetics and the biodistribution of different formulations of Dox associated PACA nanoparticles, a simple and rapid high performance liquid chromatographic method (HPLC) was developed for the quantification of Dox in plasma and tissues of rats treated with Dox loaded PACA nanoparticle (Dox-PACA). Dox was eluted at 4.4 min and it was well separated from its main metabolites doxorubicinol (Doxl) and doxorubicinon (Doxon) and idarubicin (Ida) used as internal standard (IS). Extraction of Dox from biological media was achieved by liquid–liquid extraction. The recovery of total Dox (i.e. free Dox and Dox associated with nanoparticles) from plasma and tissues (liver, spleen and heart) spiked with Dox-PACA were 71 and 78% for 0.05 and 1 g/mL in rat plasma, respectively, and 73% and 80% for 0.5 and 10 g/g in tissues, respectively. The method is linear from 0.05 to 1.5 g/mL of Dox in plasma. The limit of detection of the method is 0.5 ng of Dox per injection (50 L). The between-day and within-day precisions of the method were 97.1–102.9% and 97.3–101.7% for concentrations ranging from 0.05 to 1 g/mL, respectively. Preliminary data suggested that this method can be applied to determine the pharmacokinetic and biodistribution of Dox associated with PACA nanoparticles after intravenous administration to rats. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Doxorubicin (Dox) is one of the most important anticancer agents in use [1,2]. However, the long-term clinical use of Dox is limited by dose-related acute cardiotoxicity, myelo-suppression and multidrug resistance developed by cancer cells [2–4]. To improve the antitumor efficacy and reduce the toxicity of Dox, many drug delivery systems have been developed, including poly(alkylcyanoacrylate) (PACA) nanoparticles [5]. A first generation of PACA nanoparticles is currently under clinical development
∗ Corresponding author at: Univ Paris Sud, Physico-chimie, Pharmacotechnie et Biopharmacie, UMR 8612, Châtenay-Malabry F-92296, France. Tel.: +33 01 46 83 56 03; fax: +33 01 46 83 53 12. ∗∗ Corresponding author. Tel.: +33 133696131; fax: +33 169193318. E-mail addresses: [email protected] (C. Vauthier), [email protected] (F. Moussa). 1 Present address: International University for Science and Technology, Dara Higway-Ghabagheb, Syria. 1570-0232/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2012.01.025
for treatment of the hepatocarcinoma [6]. Because these nanoparticles are only suitable to target drugs to the liver, a new formulation of PACA nanoparticles with potential stealth properties was developed. It is prepared by redox radical emulsion polymerization (RREP). Conditions for association of Dox were recently reported by our laboratory [7–9]. To comparatively investigate the pharmacokinetics and the biodistribution of the different formulations of Dox associated with PACA nanoparticles (Dox-PACA), it was necessary to develop an accurate quantification method of Dox-PACA in plasma and tissues. The method was expected to quantify the total amount of intact Dox (i.e. free Dox and Dox associated with nanoparticles) in biological samples obtained from animals dosed with Dox-PACA. This was an important condition to achieve to take into account the amount of Dox that can be released from the nanoparticles and made available for a biological activity. Several RP-HPLC methods of quantification of Dox in rodent plasma were described during the last decade [10–24]. Sample preparation in general includes either a single step of protein precipitation [10–12] or solid phase extraction [13,14] or liquid–liquid
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extraction (LLE) [15] before injection into the chromatograph. The separation of Dox from its metabolites and daunorubicin, used as internal standard was achieved on various stationary phases [10,12–16,18–24]. Detection methods include UV [14,16], tandem mass spectrometry [17–20], electrochemistry [13], chemiluminescence [21] and fluorescence [11,12,22–24]. The fluorescence detection is by far the most frequently used method thanks to the high fluorescence yield of Dox [9]. To our knowledge, only two methods were developed to achieve determination of Dox in biological samples of animals having received injections of Dox-PACA. These methods were shortly described in papers reporting the pharmacokinetics and biodistribution of Dox in mice after delivery with the first generation of Dox-PACA [25,26]. Data from the validation of the methods were not reported and details about the extraction methods were missing. The aim of the present work was to develop a fast, specific and sensitive HPLC method coupled with fluorometric detection for the quantification of Dox and the detection of its major metabolites: doxorubicinol (Doxol) and doxorubicinon (Doxon) in plasma and tissues of rat treated with different formulations of Dox-PACA. A rapid LLE method was developed for sample preparation applicable to biological samples containing free Dox (f-Dox) or Dox-PACA. 2. Materials and methods 2.1. Reagents Doxorubicin (Dox) was purchased from Chemos GmbH (Regenstauf, Germany). Doxorubicinol (Doxol), doxorubicinon (Doxon) and idarubicin (Ida) used as internal standard (IS) were purchased from Toronto Research Chemicals (North York, Canada). Tris, trichloro-acetic acid (TCA), sodium dodecyl sulfate (SDS) and dextran (MW 70,000) were provided by Sigma (Saint Quentin Fallavier, France). Isobutylcyanoacrylate (IBCA) was a gift from Henkel Biomedical (Dublin, Ireland). All solvents were of HPLC grade and were purchased from Carlo Erba (Italy). Dox-PACA nanoparticles were prepared as described previously, by redox radical polymerization (Dox-RREP) [9] or by anionic emulsion polymerization (Dox-AEP) [27]. Concentrations of Dox in the suspensions of nanoparticles were 1 mg/mL and 0.78 mg/mL for Dox-RREP and Dox-AEP, respectively. 2.2. HPLC instrumentation HPLC analyses were performed with a Waters LC Module 1 HPLC system connected to a C18 column (Uptisphere C18 , 3 m, 4 mm × 150 mm), operated at 30 ◦ C and protected by a pre-column (10 mm × 4 mm) filled with the same stationary phase. The mobile phase consisted in a mixture of pH 2.5 0.05 M trichloro-acetic acid (TCA) and acetonitrile (60/40, v/v). Fluorimetric detection was performed with a Waters 470 Scanning Fluorescence Detector. Fluorescence detection was carried out at an excitation wavelength (ex ) of 480 nm and an emission wavelength (em ) of 558 nm. Azur software (Datalys, France) was used for HPLC monitoring and data acquisition. For qualification of the auto-sampling device, precision and linearity were regularly checked with standard solutions of methyl paraben (CV < 0.5% and linearity 0.98 < r2 < 1.02).
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ethics were institutionally approved. Rats were caged individually and allowed to acclimate 1 week before treatment. Nine rats were randomly divided into three groups of three rats and were administered intravenously with f-Dox or Dox-RREP or Dox-AEP at a single dose of 6 mg of equivalent Dox per kg of body weight (bw). Twenty-four hours after treatment, urine was collected and animals were sacrificed under anaesthesia (50 mg/kg bw of Pentobarbital) by cardiac puncture for blood and organs (heart, liver and spleen) collection. Blood was collected on heparinized tubes and centrifuged at 2000 × g for 10 min. To preserve Dox from degradation [24], all samples were stored at −20 ◦ C until analysis. 2.4. General procedure of sample preparation for doxorubicin analysis in plasma and tissue samples Plasma (100 L) or tissue (liver, spleen, heart, 100 mg homogenized in 100 L of 0.1 M SDS aqueous solution) was spiked with 100 L of IS (aqueous solution of IS at 1 g/mL for plasma samples and 10 g/mL for tissue samples). After adding 100 L of pH 8.8, 1.0 M Tris buffer solution, the extraction of anthracyclins was performed twice by adding 3 mL of a chloroform/methanol (9/1, v/v) mixture and stirring during 3 min. After centrifugation, the organic phases were collected and evaporated to dryness at 30 ◦ C under a flow of nitrogen. Dry residues from plasma and tissues were dissolved in 100 L or 1000 L of mobile phase, respectively, and 50 L of the resulting solution was injected into the chromatograph. For plasma samples with Dox concentrations lower than the limit of quantification, higher volumes of plasma (up to 1 mL) were used and then extracted by adding 30 volumes of the chloroform/methanol mixture. The dried residue was then dissolved in 100 L of mobile phase prior to the injection into the chromatograph. 2.5. Preparation of calibration standards and quality control samples Aqueous stock solutions (100 g/mL) of Dox, Doxol and Doxon were prepared and stored at −20 ◦ C until use. An aqueous solution of Ida (IS) was also prepared at 1 g/mL. For preliminary separations, a mixture of Dox, Doxol, Doxon, and Ida, was prepared giving a final concentration of 1 g/mL for each compound. Stock solutions of Dox used for the calibration of the method were prepared three times. The Dox stock solution was diluted to give a series of substock solutions with concentrations ranging from 0.5 g/mL to 15 g/mL. Standard solutions were prepared by appropriate dilution of sub-stock solutions with untreated rat plasma and liver homogenates. Final concentrations of calibration samples used to draw the calibration-curves in plasma ranged from 0.05 to 1.5 g/mL (0.05, 0.1, 0.2, 0.6, 1.0, and 1.5 g/mL). Quality control samples (QCs) were prepared by spiking untreated rat plasma and tissue samples with Dox-RREP nanoparticles giving final Dox concentrations of 0.05, 0.3, and 1 g/mL for plasma samples, 0.5, 5.0, and 10 g/g for liver tissues and 10 g/g for spleen and heart tissues. In order to preserve Dox from degradation [24], all standard solutions for calibration and QCs were frozen and stored at −20 ◦ C until analysis on the HPLC system. 2.6. Validation of the method
2.3. Animals Experiments were performed on Wistar male rats (Janvier, Le Genest Saint Isle, France) weighing 250 ± 20 g. All experiments were carried out in compliance with the guidelines of the European Community (Recommendation 2007/526/EC). The protocol
The linearity of the method was checked in plasma from 0.05 to 1.5 g/mL and in tissues from 0.5 to 10 g/g with samples prepared as described in Section 2.5. The extraction recovery of Dox was determined by spiking samples collected from untreated animals with Dox (Dox-RREP) at two
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concentrations (0.05 and 1 g/mL in plasma; 0.5 and 10 g/g in liver tissue, n = 6). Internal standard was used at 1 g/mL for plasma samples and at 10 g/mL for tissue samples. Extraction was performed as described in Section 2.4. Recoveries were calculated by comparing the peak areas obtained after extraction with those obtained after direct injection into the chromatograph of the corresponding pure solutions. For plasma and liver, the precision of the method was assessed by analyzing the intra- and inter-day variability of QCs samples on the same day (n = 5) and on three different days. For the other tissues, a partial validation was performed using QCs at 10 g/g (Table 1). Accuracy was calculated by comparing the added and the determined concentration of QCs. Precision was determined by calculating the RSD % for each QC concentration. 2.7. Statistics Statistical comparisons were performed using two-sided t test of unequal mean performed by Origin (Origin Lab corp., USA) software, p values of ≤0.05 were considered significant. 2.8. Qualitative analysis of Dox in urines For qualitative analysis, urine of untreated and treated rats were collected and filtered on a 0.22 m Millipore® filter. The filtrate was mixed with ACN (1/1, v/v) and 50 L of the resulting mixture was injected into the HPLC system. Urine from untreated rats was spiked with the solution of Dox and Doxol prior to filtration. 3. Results and discussion 3.1. Method development and validation The pKa of Dox being 8.2 and 9.3, to reach the full cationic form of Dox or its full anionic form, the pH of the mobile phase must be adjusted to 6.2 or 11.3, respectively. Considering that protonated Dox is soluble in aqueous phases and that high pH can shorten the lifetime of usual chromatographic octadecyl silica bonded solid phases, it comes obvious that best conditions for HPLC analysis should use a pH lower than 6. However, in biological media it was necessary to adjust the pH of the mobile phase to 2.5 in order to efficiently separate Dox and its metabolites as well as IS. Thus, we decided to use an endcapped stationary phase stable at low pH (with high carbon content = 18%). Preliminary separations were performed on standard mixtures and plasma from untreated rats spiked with a mixture of Dox, Doxol, Doxon, and Ida, giving final concentrations of 1 g/mL for each compound. Fig. 1 shows the chromatographic profiles of plasma from untreated rats before and after spiking with a mixture of Dox, Doxol, Doxon and IS (Ida). The ratios between the log of retention factors between Dox and IS were constant over a range of composition of the mobile phase in ACN of 30–40%. Under our chromatographic conditions, the best and the fastest separation for plasma and tissue samples was achieved with a mobile phase containing 40% of acetonitrile. With this mobile phase, Dox was eluted at 4.4 min while all other peaks of interest including the internal standard and Dox metabolites were eluted in less than 10 min (Fig. 1). Mobile phase of the same composition was also suitable for Dox analysis of tissue samples (data not shown). For urine samples, a better separation of interfering substances was obtained with a mobile phase containing 35% of acetonitrile (Fig. 2). The total time required for one analysis was reduced by 30% compared to the time required with other methods using similar types of equipment [12,20,21]. The method was further developed for quantitative analysis in plasma, liver, spleen and heart tissues.
Fig. 1. Chromatograms of plasma from (1) untreated rat and (2) untreated rat spiked with a mixture of doxorubicin (Dox) and its metabolites: doxorubicinol (Doxol), doxorubicinon (Doxon) and idarubicin used as IS (Ida). Final concentration of each compound = 1 g/mL (mobile phase ACN:TCA 40:60).
Fig. 3 shows the chromatographic profiles of tissue samples collected from treated rats, 24 h after administration of Dox-RREP. Chromatograms of liver, spleen, heart, and plasma samples, collected from untreated animals, did not exhibit any peak interfering with Dox, metabolites and IS (Fig. 3). The recovery of Dox was 71 ± 5.8 and 78 ± 2.7% for plasma and 73 ± 4.1 and 80 ± 3.5% for liver tissue for low and high concentrations, respectively. The extraction recovery of IS was 79 ± 2.1% for plasma and 82 ± 3.4% for liver. From spleen and heart samples, extraction recoveries of Dox were 77 ± 4.3 and 76 ± 3.4% and those of IS were 85 ± 3.5 and 85 ± 2.7%, respectively. Recoveries calculated with respect to the IS exceeded 90% in all types of samples. The linearity of the method was checked for plasma samples from 0.05 to 1.5 g/mL (y = 1.003x − 0.008; n = 6, r = 0.999; RSD of the slope = 7%, RSD of the intercept = 36%; where y is the area ratio, Dox/IS, and x the concentration of Dox in the extract). Intra- and inter-day variability was assessed through analysis of QCs on the same day and on three different days (Table 1). For liver samples, the linearity was checked from 0.5 to 10 g/g (y = 0.9986x − 0.0204; n = 4, r = 0.999). As the slopes and the intercepts of the calibration curves was not significantly different from that obtained with plasma samples, and as the recoveries of Dox from plasma and tissue samples were not significantly different, we used the calibration curves obtained with spiked plasma to quantify Dox in all tissues. The accuracy and the precision of the method for liver, spleen, and heart samples are summarized in Table 1. These results indicate that the method is accurate and reproducible and
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Table 1 Precision and accuracy of the HPLC assays for doxorubicin determination in rat plasma and tissues: (A) full validation (n = 5) and (B) partial validation (n = 3). Spiked sample
Spiked concentration
Concentration determined Intra-day assays
A
Plasma (g/mL)
Liver (g/g)
B
Spleen (g/g) Heart (g/g)
0.05 0.3 1 0.5 5 10 10 10
Inter-day assays
Mean
Accuracy (D/A) × 100
Precision (RSD%)
Mean
Accuracy (D/A) × 100
Precision (RSD%)
0.05 0.297 0.989 0.50 4.91 10.03
101.0 98.9 98.9 101.7 98.2 100.3
6.2 2.6 2.8 5 3 1.9
0.05 0.293 0.984 0.51 4.85 10.08
100 97.8 98.4 102.9 97.1 100.8
6.9 2.5 2.8 5.8 2.5 2.1
98.7 97.3
1.4 1.3
nd nd
nd nd
nd nd
9.87 9.73
nd: not determined.
can be applied for quantitative determination of Dox in plasma and tissues of rats. The limit of quantification (LOQ) was determined experimentally from the lowest concentration which was included in the range of linearity and had a signal to noise ratio superior to 10. The LOQ was found to be 0.05 g/mL, the corresponding accuracy was 104 ± 2% and the precision was 6 ± 1%. The limit of detection (LOD) for a signal to noise ratio equal to 3 was found to be 0.010 g/mL, i.e. 0.005 g (5 ng) per injection. The LOQ and LOD were comparable to those given in the literature based on a fluorescence detection method [11,12,24]. 3.2. Determination of Dox in plasma and tissues of rats treated with different formulations The aim of the work was to develop an analytical method of Dox suitable to study the pharmacokinetic and biodistribution of polymeric nanoparticle formulations of this drug. Developing an
Fig. 2. Chromatograms of urine from untreated rat (A) and of urine from rat after 24 h of iv administration of Dox-RREP (B) (mobile phase ACN:TCA 35:65).
HPLC method for the determination of Dox in plasma and tissue was only part of the work as it was also necessary to have a suitable method to extract Dox from plasma and tissues containing Dox-loaded nanoparticles. It was suggested to use a liquid–liquid extraction method with a high ratio of organic to aqueous solvent. The rational behind this choice was that it was necessary to dissolve PACA nanoparticles to release the associated Dox. Results obtained from the validation of the extraction method showed that the proposed extraction procedure allowed extracting Dox from biological samples with a satisfactory recovery yield and a good reproducibility including in those containing Dox formulated as nanoparticles. The validation of the whole method was demonstrated by performing a preliminary investigation of the in vivo distribution of Dox administered to rats as different formulations including the free Dox and Dox associated with two types of PACA nanoparticles. Table 2 summarizes the concentrations of Dox in plasma and tissues, 24 h after injection of three formulations of Dox (f-Dox,
Fig. 3. (A) Chromatograms of (1) untreated rat liver tissue; (2) untreated liver tissue spiked with Dox. (B) Chromatograms of (1) plasma, (2) liver, (3) spleen, and (4) heart tissue after 24 h of iv administration of Dox-RREP (finale concentration 1 g/mL or 10 g/g). IS = Ida at concentration of 1 g/mL (mobile phase ACN:TCA 40:60).
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Table 2 Tissue and plasma distribution after 24 h of i.v. administration of three formulations of Dox to rats: free Dox (f-Dox), Dox loaded RREP PACA nanoparticles (Dox-RREP) and Dox loaded AEP PACA nanoparticles (Dox-AEP). Formulation
Plasma* (g/mL) Mean ± S.D
Liver (g/g) Mean ± S.D
Spleen (g/g) Mean ± S.D
Heart (g/g) Mean ± S.D
f-Dox Dox-RREP Dox-AEP
0.015 ± 0.001 0.022 ± 0.003a 0.011 ± 0.003b , c
1.6 ± 0.2 2.0 ± 0.3 3.7 ± 0.5b , c
5±1 4.9 ± 0.4 4.3 ± 0.9
3.6 ± 0.6 4.1 ± 0.3 2.4 ± 0.3b , c
* a b c
The extraction was performed on a volume up to 1 mL of plasma. p < 0.05 Dox-RREP vs f-Dox. p < 0.05 Dox-AEP vs Dox-RREP. p < 0.05 Dox-AEP vs f-Dox.
Dox-RREP and Dox-AEP). To visualize the heterogeneity of organ Dox distribution, Dox determinations were made in triplicates, CVs did not exceed 10% in all instances. Dox concentrations found in tissues depended on the type of formulation injected to rats. Chromatograms showed peaks of Dox and internal standard while they did not reveal presence of metabolites (Fig. 3B). In general, high concentrations of Dox were found in tissues 24 h after treatments by i.v. injections of the formulations while concentrations of Dox found in plasma were very low. These preliminary results clearly indicated that the amounts of Dox found in the different organs varied as a function of the type of formulation. In agreement with previous reports, nanoparticle formulations gave a different profile of accumulation of Dox in organs compared with that of the free Dox [26,28]. Dox AEP accumulated mainly in the liver as expected from data of the literature [26]. In contrast, Dox from the Dox-RREP did not accumulated in the liver but was found to distribute towards other organs. These results were conformed to those expected from evaluations of interactions between nanoparticles and blood proteins including measurement of the capacity of those nanoparticles to activate the complement system [29,30]. They also showed that the HPLC method is suitable to be used to evaluate the pharmacokinetic and biodistribution of Dox formulated in PACA nanoparticles after intravenous administration to rats. The qualitative analysis of urine of treated rats performed with the chromatographic method showed the presence of Doxol and of Dox (Fig. 2B). This suggested that Dox was partially metabolized before excretion in urine. 4. Conclusion A rapid, sensitive and accurate HPLC method of determination of doxorubicin was developed. Compared with other methods with similar separation performance, sensitivity and domain of the linear range, the time required for one analysis was reduced by 30% with the present method as well as the solvent consumption hence the cost of the analysis. Because of the simplicity of the material used, the method can be applied in every lab equipped with a basic HPLC system. The extraction method proposed in this work was suitable to extract Dox from biological samples including those containing Dox associated with PACA nanoparticles. Thus, the method can be used to quantify the total amount of Dox in biological samples of animal dosed with Dox loaded PACA nanoparticles including
both the free Dox and the Dox associated with the nanoparticles. The HPLC method developed in the present work was successfully applied to the evaluation of Dox in biological samples from rats treated by i.v. injection of free Dox and Dox associated with two types of nanoparticles. As shown by preliminary data, the method is ready to be applied to study pharmacokinetics and biodistribution of formulations of Dox in polymeric nanoparticles. Acknowledgements The authors wish to thank Ministry of Health of Syria for financial support as a scholarship to Khairallah Alhareth. The authors also would like to thank Henkel Biomedical (Dublin, Ireland) for constant support in providing the isobutylcyanoacrylate monomer used in the synthesis of the nanoparticles. References [1] R.B. Weiss, Semin. Oncol. 19 (1992) 670. [2] C. Carvalho, R.X. Santos, S. Cardoso, S. Correia, P.J. Oliveira, M.S. Santos, P.I. Moreira, Curr. Med. Chem. 16 (2009) 3267. [3] I.C. Henderson, E. Frei 3rd., Am. Heart J. 99 (1980) 671. [4] P.A. Speth, Q.G. van Hoesel, C. Haanen, Clin. Pharmacokinet. 15 (1988) 15. [5] C. Vauthier, D. Labarre, G. Ponchel, J. Drug Target. 15 (2007) 641. [6] Bioalliance Pharma, http://www.bioalliancepharma.com/fre/R-D/Projets, search for Livatag® , accessed on 9 October 2011. [7] C. Chauvierre, D. Labarre, P. Couvreur, C. Vauthier, Pharm. Res. 20 (2003) 1786. [8] I. Bertholon-Rajota, D. Labarre, C. Vauthier, Polymer 46 (2005) 1407. [9] K. Alhareth, C. Vauthier, C. Gueutin, G. Ponchel, F. Moussa, J. Appl. Polym. Sci. 119 (2011) 816. [10] A. Kummerle, T. Krueger, M. Dusmet, C. Vallet, Y. Pan, H.B. Ris, L.A. Decosterd, J. Pharm. Biomed. Anal. 33 (2003) 475. [11] A.M. Al-Abd, N.H. Kim, S.C. Song, S.J. Lee, H.J. Kuh, Arch. Pharm. Res. 32 (2009) 605. [12] S.R. Urva, B.S. Shin, V.C. Yang, J.P. Balthasar, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 877 (2009) 837. [13] R. Ricciarello, S. Pichini, R. Pacifici, I. Altieri, M. Pellegrini, A. Fattorossi, P. Zuccaro, J. Chromatogr. B: Biomed. Sci. Appl. 707 (1998) 219. [14] T. Hu, Q. Le, Z. Wu, W. Wu, J. Pharm. Biomed. Anal. 43 (2007) 263. [15] J. Van Asperen, O. van Tellingen, J.H. Beijnen, J. Chromatogr. B: Biomed. Sci. Appl. 712 (1998) 129. [16] D.L. Chin, B.L. Lum, B.I. Sikic, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 779 (2002) 259. [17] R.D. Arnold, J.E. Slack, R.M. Straubinger, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 808 (2004) 141. [18] R. DiFrancesco, J.J. Griggs, J. Donnelly, R. DiCenzo, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 852 (2007) 545. [19] Y. Liu, Y. Yang, X. Liu, T. Jiang, Talanta 74 (2008) 887. [20] C. Sottani, P. Rinaldi, E. Leoni, G. Poggi, C. Teragni, A. Delmonte, C. Minoia, Rapid Commun. Mass Spectrom. 22 (2008) 2645. [21] S. Ahmed, N. Kishikawa, K. Ohyama, M. Wada, K. Nakashima, N. Kuroda, Talanta 78 (2009) 94. [22] P. De Bruijn, J. Verweij, W.J. Loos, H.J. Kolker, A.S. Planting, K. Nooter, G. Stoter, A. Sparreboom, Anal. Biochem. 266 (1999) 216. [23] C.M. Gilbert, R.P. McGeary, L.J. Filippich, R.L. Norris, B.G. Charles, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 826 (2005) 273. [24] G. Wei, S. Xiao, D. Si, C. Liu, Biomed. Chromatogr. 22 (2008) 1252. [25] S. Gibaud, J.P. Andreux, C. Weingarten, M. Renard, P. Couvreur, Eur. J. Cancer 30 (1994) 820. [26] N. Chiannilkulchai, N. Ammoury, B. Caillou, J.P. Devissaguet, P. Couvreur, Cancer Chemother. Pharmacol. 26 (1990) 122. [27] C.E. Soma, C. Dubernet, D. Bentolila, S. Benita, P. Couvreur, Biomaterials 21 (2000) 1. [28] L. Harivardhan Reddy, R.S.R. Murthy, Biomed. Papers 148 (2004) 161. [29] C. Vauthier, P. Lindner, B. Cabane, Colloids Surf. B: Biointerf. 69 (2009) 207. [30] C. Vauthier, B. Persson, P. Lindner, B. Cabane, Biomaterials 32 (2011) 1646.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
HPLC quantification of doxorubicin in plasma and tissues of rats treated with doxorubicin loaded poly(alkylcyanoacrylate) nanoparticles Khairallah Alhareth a,b,1 , Christine Vauthier a,b,∗ , Claire Gueutin a,b , Gilles Ponchel a,b , Fathi Moussa c,∗∗ a b c
Univ Paris Sud, Physico-chimie, Pharmacotechnie et Biopharmacie, UMR 8612, Châtenay-Malabry F-92296, France CNRS, Châtenay-Malabry F-92296, France LETIAM, IUT d’Orsay, EA 4041, Univ Paris Sud. Plateau du Moulon, Orsay 91400, France
a r t i c l e
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Article history: Received 13 October 2011 Accepted 22 January 2012 Available online 30 January 2012 Keywords: Doxorubicin Idarubicin Doxorubicinol Doxorubicinon Poly(alkylcyanoacrylate) nanoparticles
a b s t r a c t The long-term clinical use of doxorubicin (Dox), one of the most important anticancer agent in use, is limited by dose-related acute cardiotoxicity, myelo-suppression and multidrug resistance developed by cancer cells. To improve the antitumor efficacy and reduce the toxicity of Dox, many drug delivery systems have been developed, including poly(alkylcyanoacrylate) (PACA) nanoparticles. A new formulation of PACA nanoparticles with potential stealth properties were prepared by redox radical emulsion polymerization and associated to Dox in our laboratory. To comparatively investigate the pharmacokinetics and the biodistribution of different formulations of Dox associated PACA nanoparticles, a simple and rapid high performance liquid chromatographic method (HPLC) was developed for the quantification of Dox in plasma and tissues of rats treated with Dox loaded PACA nanoparticle (Dox-PACA). Dox was eluted at 4.4 min and it was well separated from its main metabolites doxorubicinol (Doxl) and doxorubicinon (Doxon) and idarubicin (Ida) used as internal standard (IS). Extraction of Dox from biological media was achieved by liquid–liquid extraction. The recovery of total Dox (i.e. free Dox and Dox associated with nanoparticles) from plasma and tissues (liver, spleen and heart) spiked with Dox-PACA were 71 and 78% for 0.05 and 1 g/mL in rat plasma, respectively, and 73% and 80% for 0.5 and 10 g/g in tissues, respectively. The method is linear from 0.05 to 1.5 g/mL of Dox in plasma. The limit of detection of the method is 0.5 ng of Dox per injection (50 L). The between-day and within-day precisions of the method were 97.1–102.9% and 97.3–101.7% for concentrations ranging from 0.05 to 1 g/mL, respectively. Preliminary data suggested that this method can be applied to determine the pharmacokinetic and biodistribution of Dox associated with PACA nanoparticles after intravenous administration to rats. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Doxorubicin (Dox) is one of the most important anticancer agents in use [1,2]. However, the long-term clinical use of Dox is limited by dose-related acute cardiotoxicity, myelo-suppression and multidrug resistance developed by cancer cells [2–4]. To improve the antitumor efficacy and reduce the toxicity of Dox, many drug delivery systems have been developed, including poly(alkylcyanoacrylate) (PACA) nanoparticles [5]. A first generation of PACA nanoparticles is currently under clinical development
∗ Corresponding author at: Univ Paris Sud, Physico-chimie, Pharmacotechnie et Biopharmacie, UMR 8612, Châtenay-Malabry F-92296, France. Tel.: +33 01 46 83 56 03; fax: +33 01 46 83 53 12. ∗∗ Corresponding author. Tel.: +33 133696131; fax: +33 169193318. E-mail addresses: [email protected] (C. Vauthier), [email protected] (F. Moussa). 1 Present address: International University for Science and Technology, Dara Higway-Ghabagheb, Syria. 1570-0232/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2012.01.025
for treatment of the hepatocarcinoma [6]. Because these nanoparticles are only suitable to target drugs to the liver, a new formulation of PACA nanoparticles with potential stealth properties was developed. It is prepared by redox radical emulsion polymerization (RREP). Conditions for association of Dox were recently reported by our laboratory [7–9]. To comparatively investigate the pharmacokinetics and the biodistribution of the different formulations of Dox associated with PACA nanoparticles (Dox-PACA), it was necessary to develop an accurate quantification method of Dox-PACA in plasma and tissues. The method was expected to quantify the total amount of intact Dox (i.e. free Dox and Dox associated with nanoparticles) in biological samples obtained from animals dosed with Dox-PACA. This was an important condition to achieve to take into account the amount of Dox that can be released from the nanoparticles and made available for a biological activity. Several RP-HPLC methods of quantification of Dox in rodent plasma were described during the last decade [10–24]. Sample preparation in general includes either a single step of protein precipitation [10–12] or solid phase extraction [13,14] or liquid–liquid
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extraction (LLE) [15] before injection into the chromatograph. The separation of Dox from its metabolites and daunorubicin, used as internal standard was achieved on various stationary phases [10,12–16,18–24]. Detection methods include UV [14,16], tandem mass spectrometry [17–20], electrochemistry [13], chemiluminescence [21] and fluorescence [11,12,22–24]. The fluorescence detection is by far the most frequently used method thanks to the high fluorescence yield of Dox [9]. To our knowledge, only two methods were developed to achieve determination of Dox in biological samples of animals having received injections of Dox-PACA. These methods were shortly described in papers reporting the pharmacokinetics and biodistribution of Dox in mice after delivery with the first generation of Dox-PACA [25,26]. Data from the validation of the methods were not reported and details about the extraction methods were missing. The aim of the present work was to develop a fast, specific and sensitive HPLC method coupled with fluorometric detection for the quantification of Dox and the detection of its major metabolites: doxorubicinol (Doxol) and doxorubicinon (Doxon) in plasma and tissues of rat treated with different formulations of Dox-PACA. A rapid LLE method was developed for sample preparation applicable to biological samples containing free Dox (f-Dox) or Dox-PACA. 2. Materials and methods 2.1. Reagents Doxorubicin (Dox) was purchased from Chemos GmbH (Regenstauf, Germany). Doxorubicinol (Doxol), doxorubicinon (Doxon) and idarubicin (Ida) used as internal standard (IS) were purchased from Toronto Research Chemicals (North York, Canada). Tris, trichloro-acetic acid (TCA), sodium dodecyl sulfate (SDS) and dextran (MW 70,000) were provided by Sigma (Saint Quentin Fallavier, France). Isobutylcyanoacrylate (IBCA) was a gift from Henkel Biomedical (Dublin, Ireland). All solvents were of HPLC grade and were purchased from Carlo Erba (Italy). Dox-PACA nanoparticles were prepared as described previously, by redox radical polymerization (Dox-RREP) [9] or by anionic emulsion polymerization (Dox-AEP) [27]. Concentrations of Dox in the suspensions of nanoparticles were 1 mg/mL and 0.78 mg/mL for Dox-RREP and Dox-AEP, respectively. 2.2. HPLC instrumentation HPLC analyses were performed with a Waters LC Module 1 HPLC system connected to a C18 column (Uptisphere C18 , 3 m, 4 mm × 150 mm), operated at 30 ◦ C and protected by a pre-column (10 mm × 4 mm) filled with the same stationary phase. The mobile phase consisted in a mixture of pH 2.5 0.05 M trichloro-acetic acid (TCA) and acetonitrile (60/40, v/v). Fluorimetric detection was performed with a Waters 470 Scanning Fluorescence Detector. Fluorescence detection was carried out at an excitation wavelength (ex ) of 480 nm and an emission wavelength (em ) of 558 nm. Azur software (Datalys, France) was used for HPLC monitoring and data acquisition. For qualification of the auto-sampling device, precision and linearity were regularly checked with standard solutions of methyl paraben (CV < 0.5% and linearity 0.98 < r2 < 1.02).
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ethics were institutionally approved. Rats were caged individually and allowed to acclimate 1 week before treatment. Nine rats were randomly divided into three groups of three rats and were administered intravenously with f-Dox or Dox-RREP or Dox-AEP at a single dose of 6 mg of equivalent Dox per kg of body weight (bw). Twenty-four hours after treatment, urine was collected and animals were sacrificed under anaesthesia (50 mg/kg bw of Pentobarbital) by cardiac puncture for blood and organs (heart, liver and spleen) collection. Blood was collected on heparinized tubes and centrifuged at 2000 × g for 10 min. To preserve Dox from degradation [24], all samples were stored at −20 ◦ C until analysis. 2.4. General procedure of sample preparation for doxorubicin analysis in plasma and tissue samples Plasma (100 L) or tissue (liver, spleen, heart, 100 mg homogenized in 100 L of 0.1 M SDS aqueous solution) was spiked with 100 L of IS (aqueous solution of IS at 1 g/mL for plasma samples and 10 g/mL for tissue samples). After adding 100 L of pH 8.8, 1.0 M Tris buffer solution, the extraction of anthracyclins was performed twice by adding 3 mL of a chloroform/methanol (9/1, v/v) mixture and stirring during 3 min. After centrifugation, the organic phases were collected and evaporated to dryness at 30 ◦ C under a flow of nitrogen. Dry residues from plasma and tissues were dissolved in 100 L or 1000 L of mobile phase, respectively, and 50 L of the resulting solution was injected into the chromatograph. For plasma samples with Dox concentrations lower than the limit of quantification, higher volumes of plasma (up to 1 mL) were used and then extracted by adding 30 volumes of the chloroform/methanol mixture. The dried residue was then dissolved in 100 L of mobile phase prior to the injection into the chromatograph. 2.5. Preparation of calibration standards and quality control samples Aqueous stock solutions (100 g/mL) of Dox, Doxol and Doxon were prepared and stored at −20 ◦ C until use. An aqueous solution of Ida (IS) was also prepared at 1 g/mL. For preliminary separations, a mixture of Dox, Doxol, Doxon, and Ida, was prepared giving a final concentration of 1 g/mL for each compound. Stock solutions of Dox used for the calibration of the method were prepared three times. The Dox stock solution was diluted to give a series of substock solutions with concentrations ranging from 0.5 g/mL to 15 g/mL. Standard solutions were prepared by appropriate dilution of sub-stock solutions with untreated rat plasma and liver homogenates. Final concentrations of calibration samples used to draw the calibration-curves in plasma ranged from 0.05 to 1.5 g/mL (0.05, 0.1, 0.2, 0.6, 1.0, and 1.5 g/mL). Quality control samples (QCs) were prepared by spiking untreated rat plasma and tissue samples with Dox-RREP nanoparticles giving final Dox concentrations of 0.05, 0.3, and 1 g/mL for plasma samples, 0.5, 5.0, and 10 g/g for liver tissues and 10 g/g for spleen and heart tissues. In order to preserve Dox from degradation [24], all standard solutions for calibration and QCs were frozen and stored at −20 ◦ C until analysis on the HPLC system. 2.6. Validation of the method
2.3. Animals Experiments were performed on Wistar male rats (Janvier, Le Genest Saint Isle, France) weighing 250 ± 20 g. All experiments were carried out in compliance with the guidelines of the European Community (Recommendation 2007/526/EC). The protocol
The linearity of the method was checked in plasma from 0.05 to 1.5 g/mL and in tissues from 0.5 to 10 g/g with samples prepared as described in Section 2.5. The extraction recovery of Dox was determined by spiking samples collected from untreated animals with Dox (Dox-RREP) at two
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concentrations (0.05 and 1 g/mL in plasma; 0.5 and 10 g/g in liver tissue, n = 6). Internal standard was used at 1 g/mL for plasma samples and at 10 g/mL for tissue samples. Extraction was performed as described in Section 2.4. Recoveries were calculated by comparing the peak areas obtained after extraction with those obtained after direct injection into the chromatograph of the corresponding pure solutions. For plasma and liver, the precision of the method was assessed by analyzing the intra- and inter-day variability of QCs samples on the same day (n = 5) and on three different days. For the other tissues, a partial validation was performed using QCs at 10 g/g (Table 1). Accuracy was calculated by comparing the added and the determined concentration of QCs. Precision was determined by calculating the RSD % for each QC concentration. 2.7. Statistics Statistical comparisons were performed using two-sided t test of unequal mean performed by Origin (Origin Lab corp., USA) software, p values of ≤0.05 were considered significant. 2.8. Qualitative analysis of Dox in urines For qualitative analysis, urine of untreated and treated rats were collected and filtered on a 0.22 m Millipore® filter. The filtrate was mixed with ACN (1/1, v/v) and 50 L of the resulting mixture was injected into the HPLC system. Urine from untreated rats was spiked with the solution of Dox and Doxol prior to filtration. 3. Results and discussion 3.1. Method development and validation The pKa of Dox being 8.2 and 9.3, to reach the full cationic form of Dox or its full anionic form, the pH of the mobile phase must be adjusted to 6.2 or 11.3, respectively. Considering that protonated Dox is soluble in aqueous phases and that high pH can shorten the lifetime of usual chromatographic octadecyl silica bonded solid phases, it comes obvious that best conditions for HPLC analysis should use a pH lower than 6. However, in biological media it was necessary to adjust the pH of the mobile phase to 2.5 in order to efficiently separate Dox and its metabolites as well as IS. Thus, we decided to use an endcapped stationary phase stable at low pH (with high carbon content = 18%). Preliminary separations were performed on standard mixtures and plasma from untreated rats spiked with a mixture of Dox, Doxol, Doxon, and Ida, giving final concentrations of 1 g/mL for each compound. Fig. 1 shows the chromatographic profiles of plasma from untreated rats before and after spiking with a mixture of Dox, Doxol, Doxon and IS (Ida). The ratios between the log of retention factors between Dox and IS were constant over a range of composition of the mobile phase in ACN of 30–40%. Under our chromatographic conditions, the best and the fastest separation for plasma and tissue samples was achieved with a mobile phase containing 40% of acetonitrile. With this mobile phase, Dox was eluted at 4.4 min while all other peaks of interest including the internal standard and Dox metabolites were eluted in less than 10 min (Fig. 1). Mobile phase of the same composition was also suitable for Dox analysis of tissue samples (data not shown). For urine samples, a better separation of interfering substances was obtained with a mobile phase containing 35% of acetonitrile (Fig. 2). The total time required for one analysis was reduced by 30% compared to the time required with other methods using similar types of equipment [12,20,21]. The method was further developed for quantitative analysis in plasma, liver, spleen and heart tissues.
Fig. 1. Chromatograms of plasma from (1) untreated rat and (2) untreated rat spiked with a mixture of doxorubicin (Dox) and its metabolites: doxorubicinol (Doxol), doxorubicinon (Doxon) and idarubicin used as IS (Ida). Final concentration of each compound = 1 g/mL (mobile phase ACN:TCA 40:60).
Fig. 3 shows the chromatographic profiles of tissue samples collected from treated rats, 24 h after administration of Dox-RREP. Chromatograms of liver, spleen, heart, and plasma samples, collected from untreated animals, did not exhibit any peak interfering with Dox, metabolites and IS (Fig. 3). The recovery of Dox was 71 ± 5.8 and 78 ± 2.7% for plasma and 73 ± 4.1 and 80 ± 3.5% for liver tissue for low and high concentrations, respectively. The extraction recovery of IS was 79 ± 2.1% for plasma and 82 ± 3.4% for liver. From spleen and heart samples, extraction recoveries of Dox were 77 ± 4.3 and 76 ± 3.4% and those of IS were 85 ± 3.5 and 85 ± 2.7%, respectively. Recoveries calculated with respect to the IS exceeded 90% in all types of samples. The linearity of the method was checked for plasma samples from 0.05 to 1.5 g/mL (y = 1.003x − 0.008; n = 6, r = 0.999; RSD of the slope = 7%, RSD of the intercept = 36%; where y is the area ratio, Dox/IS, and x the concentration of Dox in the extract). Intra- and inter-day variability was assessed through analysis of QCs on the same day and on three different days (Table 1). For liver samples, the linearity was checked from 0.5 to 10 g/g (y = 0.9986x − 0.0204; n = 4, r = 0.999). As the slopes and the intercepts of the calibration curves was not significantly different from that obtained with plasma samples, and as the recoveries of Dox from plasma and tissue samples were not significantly different, we used the calibration curves obtained with spiked plasma to quantify Dox in all tissues. The accuracy and the precision of the method for liver, spleen, and heart samples are summarized in Table 1. These results indicate that the method is accurate and reproducible and
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Table 1 Precision and accuracy of the HPLC assays for doxorubicin determination in rat plasma and tissues: (A) full validation (n = 5) and (B) partial validation (n = 3). Spiked sample
Spiked concentration
Concentration determined Intra-day assays
A
Plasma (g/mL)
Liver (g/g)
B
Spleen (g/g) Heart (g/g)
0.05 0.3 1 0.5 5 10 10 10
Inter-day assays
Mean
Accuracy (D/A) × 100
Precision (RSD%)
Mean
Accuracy (D/A) × 100
Precision (RSD%)
0.05 0.297 0.989 0.50 4.91 10.03
101.0 98.9 98.9 101.7 98.2 100.3
6.2 2.6 2.8 5 3 1.9
0.05 0.293 0.984 0.51 4.85 10.08
100 97.8 98.4 102.9 97.1 100.8
6.9 2.5 2.8 5.8 2.5 2.1
98.7 97.3
1.4 1.3
nd nd
nd nd
nd nd
9.87 9.73
nd: not determined.
can be applied for quantitative determination of Dox in plasma and tissues of rats. The limit of quantification (LOQ) was determined experimentally from the lowest concentration which was included in the range of linearity and had a signal to noise ratio superior to 10. The LOQ was found to be 0.05 g/mL, the corresponding accuracy was 104 ± 2% and the precision was 6 ± 1%. The limit of detection (LOD) for a signal to noise ratio equal to 3 was found to be 0.010 g/mL, i.e. 0.005 g (5 ng) per injection. The LOQ and LOD were comparable to those given in the literature based on a fluorescence detection method [11,12,24]. 3.2. Determination of Dox in plasma and tissues of rats treated with different formulations The aim of the work was to develop an analytical method of Dox suitable to study the pharmacokinetic and biodistribution of polymeric nanoparticle formulations of this drug. Developing an
Fig. 2. Chromatograms of urine from untreated rat (A) and of urine from rat after 24 h of iv administration of Dox-RREP (B) (mobile phase ACN:TCA 35:65).
HPLC method for the determination of Dox in plasma and tissue was only part of the work as it was also necessary to have a suitable method to extract Dox from plasma and tissues containing Dox-loaded nanoparticles. It was suggested to use a liquid–liquid extraction method with a high ratio of organic to aqueous solvent. The rational behind this choice was that it was necessary to dissolve PACA nanoparticles to release the associated Dox. Results obtained from the validation of the extraction method showed that the proposed extraction procedure allowed extracting Dox from biological samples with a satisfactory recovery yield and a good reproducibility including in those containing Dox formulated as nanoparticles. The validation of the whole method was demonstrated by performing a preliminary investigation of the in vivo distribution of Dox administered to rats as different formulations including the free Dox and Dox associated with two types of PACA nanoparticles. Table 2 summarizes the concentrations of Dox in plasma and tissues, 24 h after injection of three formulations of Dox (f-Dox,
Fig. 3. (A) Chromatograms of (1) untreated rat liver tissue; (2) untreated liver tissue spiked with Dox. (B) Chromatograms of (1) plasma, (2) liver, (3) spleen, and (4) heart tissue after 24 h of iv administration of Dox-RREP (finale concentration 1 g/mL or 10 g/g). IS = Ida at concentration of 1 g/mL (mobile phase ACN:TCA 40:60).
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Table 2 Tissue and plasma distribution after 24 h of i.v. administration of three formulations of Dox to rats: free Dox (f-Dox), Dox loaded RREP PACA nanoparticles (Dox-RREP) and Dox loaded AEP PACA nanoparticles (Dox-AEP). Formulation
Plasma* (g/mL) Mean ± S.D
Liver (g/g) Mean ± S.D
Spleen (g/g) Mean ± S.D
Heart (g/g) Mean ± S.D
f-Dox Dox-RREP Dox-AEP
0.015 ± 0.001 0.022 ± 0.003a 0.011 ± 0.003b , c
1.6 ± 0.2 2.0 ± 0.3 3.7 ± 0.5b , c
5±1 4.9 ± 0.4 4.3 ± 0.9
3.6 ± 0.6 4.1 ± 0.3 2.4 ± 0.3b , c
* a b c
The extraction was performed on a volume up to 1 mL of plasma. p < 0.05 Dox-RREP vs f-Dox. p < 0.05 Dox-AEP vs Dox-RREP. p < 0.05 Dox-AEP vs f-Dox.
Dox-RREP and Dox-AEP). To visualize the heterogeneity of organ Dox distribution, Dox determinations were made in triplicates, CVs did not exceed 10% in all instances. Dox concentrations found in tissues depended on the type of formulation injected to rats. Chromatograms showed peaks of Dox and internal standard while they did not reveal presence of metabolites (Fig. 3B). In general, high concentrations of Dox were found in tissues 24 h after treatments by i.v. injections of the formulations while concentrations of Dox found in plasma were very low. These preliminary results clearly indicated that the amounts of Dox found in the different organs varied as a function of the type of formulation. In agreement with previous reports, nanoparticle formulations gave a different profile of accumulation of Dox in organs compared with that of the free Dox [26,28]. Dox AEP accumulated mainly in the liver as expected from data of the literature [26]. In contrast, Dox from the Dox-RREP did not accumulated in the liver but was found to distribute towards other organs. These results were conformed to those expected from evaluations of interactions between nanoparticles and blood proteins including measurement of the capacity of those nanoparticles to activate the complement system [29,30]. They also showed that the HPLC method is suitable to be used to evaluate the pharmacokinetic and biodistribution of Dox formulated in PACA nanoparticles after intravenous administration to rats. The qualitative analysis of urine of treated rats performed with the chromatographic method showed the presence of Doxol and of Dox (Fig. 2B). This suggested that Dox was partially metabolized before excretion in urine. 4. Conclusion A rapid, sensitive and accurate HPLC method of determination of doxorubicin was developed. Compared with other methods with similar separation performance, sensitivity and domain of the linear range, the time required for one analysis was reduced by 30% with the present method as well as the solvent consumption hence the cost of the analysis. Because of the simplicity of the material used, the method can be applied in every lab equipped with a basic HPLC system. The extraction method proposed in this work was suitable to extract Dox from biological samples including those containing Dox associated with PACA nanoparticles. Thus, the method can be used to quantify the total amount of Dox in biological samples of animal dosed with Dox loaded PACA nanoparticles including
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