Development and validation of an HPLC method for

Development and validation of an HPLC method for the determination of fluorouracil in polymeric nanoparticles Ana Cristina de Mattos, Najeh Maissar Khalil, Rubiana Mara Mainardes* Department of Pharmacy, Midwestern State University/UNICENTRO, Guarapuava, Paraná, Brasil

The objective of this work was to develop and validate a rapid high performance liquid chromatography Chromatographic analyses were performed on an RP C18 column with a mobile phase consisting of

because the maximum relative standard deviation was 3.51%. The method is robust relative to

application to the quantitative analysis of 5-FU in polymeric nanoparticles. Uniterms: Fluorouracil/determination. Nanoparticles. High performance liquid chromatography/ quantitative analysis/method validation.

Unitermos: análise quantitativa/validação de método.

INTRODUCTION

bility that 5-FU pharmacomodulation markedly increases

in the treatment of solid tumors. This drug, an analog of the natural pyrimidine uracil, must be converted to the nucleo-

Groeningein, 1991). The mechanism of 5-FU cytotoxicity is complex because the drug is activated through different pathways oxyuridine monophosphate, which inhibits thymidylate

with well-known antineoplastic properties (Pinedo, Peters, 1988). One explanation for this phenomenon is the possi*Correspondence: R. M. Mainardes. Departamento de Farmácia, Universidade Estadual do Centro-Oeste/UNICENTRO. Rua Simeão Camargo Varela de Sá 03, 85040-080 - Guarapuava - PR, Brasil. E-mail: [email protected]

The 5-FU presents a narrow therapeutic index, and many papers have reported greater variability in pharma-

Article

Brazilian Journal of Pharmaceutical Sciences vol. 49, n. 1, jan./mar., 2013

118

A. C. Mattos, N. M. Khalil, R. M. Mainardes

cokinetics than other anticancer drugs. For these reasons, some inter-patient differences in terms of toxicity and kinetic parameters, especially in the area under the time vs. et al. et al. problem with 5-FU therapy is its toxicity to the bone marrow and the gastrointestinal tract (Tanaka et al. Guo, 2011). Therefore, 5-FU represents an interesting drug model to be improved by nanotechnology. Numerous investigators have shown that the biologi-

5-FU, but the investigators used a C8 column. Zheng et al. a mixture of methanol and 3.6% acetic acid (80:20, v/v), but details such as the retention time, peak characteristics and validation data were not described. The objective of this work was therefore to develop nanoparticles.

nanoparticle delivery systems (Moghimi et al.,

MATERIAL AND METHODS

et al., 2005). The nanoencapsulation of drugs has many advantages for the protection from premature degradation and interaction with the biological environment, enhancement of absorption into a selected tissue, bioavailability, retention time and improvement of intracellular penetration. However, polymeric nanoparticles, when used intravenously, are removed from systemic circulation by the cells of the mononuclear phagocyte system (MPS). Several methods are used to modify the surface of the nanoparticles to avoid recognition and capture by the cells of the MPS and promote a long plasma circulating time

Materials

coating nanoparticles with hydrophilic polymers such as polyethylene glycol (PEG) is the most popular because the

Instrumentation

(MW 85-160 kDa), polyethylene glycol (10 kDa) and Methylene chloride was purchased from FMaia® HPLC-grade acetonitrile was purchased from JTBaker® ® lipore solvents and chemicals were analytical or HPLC grade.

et al., 2008). meric nanoparticles fully, suitable and validated quantitation methods are required to assess pharmaceutical parameters such as drug content. Several methods are described in the literature for the determination of 5-FU in samples of biological matrices using gas chromatography/mass specet al. (Badea et al., 2002), high performance liquid chromatoget al., 1999), hydrophilic inter-

system was equipped with a column compartment with temperature control, an on-line degasser, a quaternary

(Pisano et al., 2005) or liquid chromatography tandem

Preparation of standard and sample solutions

et al., 2010). The analytical determination of 5-FU in pharmaceutical dosage forms such as nanoparticles has been performed by several authors using et al. Zhu et al. et al. et al. et al. et al., 2011), but few studies report the use of HPLC methods for this deter-

analysis, and reporting were performed using Empower analysis was conducted using a RP C18 column (Xterra ® eter and 250 mm length.

was prepared in water and subsequent dilutions were carried out to obtain eight standard solutions (0.1, 0.5, 1.0, solutions were obtained by serial dilutions of a 5-FU stan125.0, 150.0, and 200.0 ng/mL) to determine the limit of

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method. The samples were appropriately diluted in water. (1 and 2) were used according to ICH (2005): Chromatographic conditions

Chromatographic analysis was performed in the isocratic mode. The mobile phase consisted of a mixture of acetonitrile and water (10:90, v/v), which was pumped

the slope of the calibration curve. Robustness was evaluated by deliberately varying

nm. The method run time was 4 min, and all experiments were performed at 25°C.

rate (0.9 and 1.1 mL/min) and using a similar C18 column ).

®

Method validation

The HPLC method was validated according to the lines (2005). The following characteristics were considThe specificity was evaluated by comparing the representative chromatograms of samples containing possible interfering substances and samples containing 5-FU. stress studies (i.e., light stability, pH variation, temperature and oxidation). Linearity was determined by calculating a regression line from the plot of peak area vs. concentration for the eight standard solutions in water (i.e., 0.1, 0.5, 1.0, 2.0, methodology. The accuracy was tested by calculating the percent recovery of the mean concentration of 5-FU at three different concentration levels, and the relative standard deviation (RSD) was determined. The mean concentration value obtained for each level was compared to the theoretical value, which was considered to be 100%. Precision was assessed at two levels: repeatability or intra-day variability and intermediate precision or inter-day variability. The repeatability was assessed by testing three different standard solutions on the same day and using a standard solution with concentration of 1.0 reported as RSD. The intermediate precision was evaludifferent days. The results were reported as the standard deviation (SD) and RSD. -

Method applicability Preparation of 5-FU-loaded PLA or PLA-PEG blended nanoparticles The nanoparticles were obtained using the double emulsion solvent-evaporation technique (Zabaux et al.,

either with or without PEG at room temperature. This Ultrasonic Mixing, mod. DES 500, equipped with a 4 mm probe, Unique Group, ®

tion for 5 min, resulting in a water-in-oil-in-water emulsion g, 30 min, twice with water to remove the surfactant. The nanoparticles were dispersed in the cryoprotectant sucrose, and the result-

dispersity index were determined by dynamic light scattering (BIC 90 plus, Brookhaven Instruments Corp.). The analyses were performed at a scattering angle of 90° and a temperature of 25 °C. For each sample, the mean particle diameter, polydispersity and standard deviation for ten determinations were calculated. Determination of the 5-FU encapsulation The amount of 5-FU incorporated into the nanopar-

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ticles was determined indirectly (Das Neves et al., 2010). The analyte was the supernatant, which contained free 5-FU separated from solid nanoparticles by ultracentrifugation. sample was injected into the HPLC system, and the drug concentration in the supernatant was obtained by comparing the concentration obtained to a previously constructed lipore). The amount of 5-FU entrapped in the nanoparticles was obtained by subtracting the amount in the supernatant from the total amount used during the preparation. These analyses were performed in triplicate.

RESULTS AND DISCUSSION Method development

Due to the high aqueous solubility of 5-FU, hydro-

FIGURE 1 -

for the column and thus obtain short retention times. Initially, the methodology described in the United States Pharmacopoeia USP 34 (2010) was tested, describing ultrapure water as mobile phase, with a rate of 1.0 mL/min. However, for samples, irregular peaks were observed usbecause of instrumental or column differences. improve the resolution of chromatographic peaks of water in many proportions, in isocratic mode and with the proportions of acetonitrile:water ranging from 10:90 shape of the 5-FU peak were observed when the proportion of acetonitrile was higher than the water. Increasing the water proportion in the mobile phase, the 5-FU peak became more regular, and, when the proportion of acetonitrile:water of 10:90 (v/v) was used, a regular and

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symmetric peak was observed. Under these conditions, the 5-FU peak was detected in approximately 3.5 min (Figure 1B). Method validation Specificity

of supernatant obtained by ultracentrifugation of an aquethe chromatogram of the 5-FU standard (Figure 1B) and the 5-FU retention time was observed in the chromatogram it does not interfere with the quantitative determination of

FIGURE 2 -

of 5-FU nanoparticles (B).

standard solution in water (as usual). The percent recovery Tests were also performed under four stress conditions (i.e., temperature, visible light, pH and oxidation) to detect the occurrence of possible interfering peaks at 265 nm resulting from the degradation of 5-FU. These tests are regarded as helpful tools in establishing degradation pathways and the inherent stability of the molecule and help validate the power of the method for studying the drug stability (Das Neves et al., 2010). The percent recovery under stress conditions also revealed that 5-FU was not affected, except at high pH values (4.23%). The results obtained are presented in Table I, showing no alterations in 5-FU retention time. Linearity Linearity was evaluated at eight concentration levels

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TABLE I -

Conditions

Percentage of Recovery ± RSD 1.0 µg/mL 10.0 µg/mL 104.26 ± 3.82 103.90 ± 0.49 105.36 ± 0.81 103.20 ± 1.00 10.36 ± 38.05 2.33 ± 38.63 109.06 ± 3.61 103.13 ± 1.05 106.23 ± 1.46 104.60 ± 0.02

0.1 µg/mL 99.60 ± 1.80 104.46 ± 3.50 103.30 ± 2.65

Reference UV light Congealment High pH Low pH Oxidation

100.46 ± 0.32 100.46 ± 0.61

the method of least squares:

Mean 102.81 ± 2.11 105.28 ± 2.13 103.95 ± 1.48 104.22 ± 1.66

intermediate precision. Three concentrations of 5-FU (0.1, 4

Eq. 3

-value near 1 indicates

on one day or two different days to evaluate intra-day or inter-day variation, respectively. The RSDs of responses were calculated in each case and are shown in Table III, indicating that precision was obtained because the maximal RSD obtained was 3.51%.

linearity in the proposed range. TABLE III - Precision results for different levels of 5-FU in the

sis of variance, which showed that the linear regression

standard solutions

Standard solution (µg/mL)

Measured concentration ± SD RSD (%) (µg/mL)

Accuracy

recovery and the RSD of the mean concentration of the analyte at three different concentrations. Three standard Detailed results for these three tested concentration levels are presented in Table II. The mean percent recovery of The results show agreement between experimental and theoretical values. TABLE II -

standard solutions Standard solution (µg/mL)* 0.1 1.0 10.0

Recovery (%)

RSD (%)

0.1 1.0 10.0 Day 1 0.1 1.0 10.0 Day 2 0.1 1.0 10.0

0.09 ± 0.02 0.98 ± 0.00

0.09 ± 0.00 0.95 ± 0.02

1.46 2.11

0.09 ± 0.00 1.04 ± 0.04

1.66 3.51 0.63

99.55

The instrumental precision is a measure of the relative errors inherent in the equipment and is expressed as the

Precision The precision is a measure of the relative errors of the method, expressed as the RSD for repeatability and

Limits of quantification and detection The lowest concentration at which an analyte can

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precision and accuracy, in the present study, was calculated from the SD of the response and the slope obtained from linear regression of a specific calibration curve (50-200 ng/mL) in the low-end region of the proposed range (ICH, 2005). The method was linear in this range

Range The working range of the method, defined as the range that possesses the required linearity, accuracy and

containing these concentration levels may therefore be assayed using the proposed HPLC method.

Method applicability

The proposed analytical method was used to evalusupernatant containing free 5-FU from the solid nanoparticles by ultracentrifugation, was used. The supernatant test, no alterations to the chromatograms or unusual peaks The method of double emulsion-solvent evaporation was adequate to obtain the nanoparticles containing 5-FU. The main characteristics of the nanoparticles are described -

Robustness

changes in the analytical procedures/parameters on the response. The evaluation of robustness was based on the percent recovery and RSD values obtained using differtemperature and using a similar C18 column. The method is robust concerning these alterations in chromatographic parameters (Table IV). The maximum RSD obtained was 3.48%.

formulations presented a bimodal distribution profile. Nanoparticles can be considered potential carriers for 5-FU delivery. Our objective was to develop a fast, simple and efliterature describes mainly spectrophotometric methods

TABLE IV -

Changes to original method*

Percentage of Recovery ± RSD 0.1 µg/mL

1.0 µg/mL

10.0 µg/mL

None

Mean 99.28 ± 1.10

Flow rate: 0.9 mL/min

104.80 ± 3.48

103.53 ± 2.22

102.52 ± 2.48

Flow rate: 1.1 mL/min

102.40 ± 2.30

100.40 ± 1.05

Similar column

105.50 ± 0.06

106.56 ± 0.65

Column Temperature: 35 °C

98.43 ± 0.00

103.13 ± 0.24

105.90 ± 0.04

* 1.0mL/min, column at 25 °C and mobile phase (acetonitrile:water 10:90, v/v). TABLE V - Nanoparticle characteristics

Encapsulation

Nanoparticle 0.116 305.80 ± 3.62

55.53 ± 9.21

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et al.

et al. et al. et al., et al., 2011), but these methods are not as convenient as HPLC methods considering the sensitivity. The only example in the literature of an HPLC-UV method applied to 5-FU determination in nanoparticles was the method cited by Zheng et al only the mobile phase (methanol:3.6% acetic acid - 80:20, v/v) and did not give any information about validation data Li et al.

(2002) developed an HPLC-UV method for quantitation of

REFERENCES

on poly(g-glutamic acid) with l-phenylalanine as a protein carrier. J. Control. Release, v.108, n.2-3, p.226-236, 2005.

O.C. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol. Pharm., v.5, n.4, p.505-515, 2008.

The mobile phase was comprised of 0.05 M ammonium acetate (pH 6.5), and the results showed a retention time J. Chromatogr. B, v.688, n.1,

method was sensitive and could be applied to 5-FU, but a C8 column was used, so the method does not correspond method developed and validated in this work represents an alternative to spectrophotometric methods for the -

determination of 5-fluorouridine in nanoparticulate formulations. J. Pharm. Biomed. Anal. 866, 2002.

method could be applied not only for the determination also for other assays, such as determining the in vitro 5-FU

derivative spectrometry. J. Pharm. Biomed. Anal., v.30,

CONCLUSION photodiode array detection for determining the encapsunanoparticles has been developed and validated according ments to be considered a reliable and feasible method, including specificity, linearity, precision, accuracy,

5-FU nanoparticles with factorial design-based studies. Farmaco, v.60, n.10, p.840-846, 2005.

. Pharmacol. Res.,

has a chromatographic run time of 4 min, which allows time. The low percentage of acetonitrile used is important because it reduces the cost of solvent and the damage to the environment. This method was found to be suitable for

M.F. Development and validation of a rapid reversed-phase HPLC method for the determination of the non-nucleoside reverse transcriptase inhibitor dapivirine from polymeric nanoparticles, J. Pharm. Biomed. Anal.

nanoparticles.

ACKNOWLEDGMENTS

Simple and sensitive determination of 5-fluorouracil in plasma by high-performance liquid chromatography. J. Chromatogr. B,

125

cancer - status of the art. Crit. Rev. Oncol. Hemat., v.30,

of tegafur, 5-fluorouracil, gimeracil and oxonic acid in human plasma using liquid chromatography-tandem mass spectrometry. J. Pharm. Biomed. Anal., v.52, n.4, p.550556, 2010.

Correlation between uracil and dihydrouracil plasma ratio, fluorouracil (5-FU) pharmacokinetic parameters, and tolerance in patients with advanced colorectal cancer: a potential interest for predicting 5-FU toxicity and determining optimal 5-FU dosage. J. Clin. Oncol., n.4, p.1105-1110, 1999.

circulating and target specific nanoparticles: theory to practice. Pharmacol. Rev., v.53, p.283-318, 2001.

COLLINS, J.M. (Eds.) Cancer chemotherapy: principles and practice. Philadelphia: J.B. Lippincott, 1990. p.180224.

Ann. Oncol.,

nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Deliver. Rev

and pharmacology. J. Clin. Oncol., v.6, n.10, p.1653-1664, 1988.

tissues. J. Pharm. Biomed. Anal. wild-type p53 gene delivery results in sustained antiproliferative activity in breast cancer cells. Mol. Pharm., v.1, n.3, p.211-219, 2004.

chitosan-g-poly(N-vinylcaprolactam) nanoparticles as Carbohyd. Polym.,

Int. J. Pharm., v.404,

of chitosan nanoparticles as drug delivery systems for . Carbohyd. Polym.,

application. Int. J. Biol. Macromol., v.48, n.1, p.98-105, 2011b.

of a sensitive and selective LC MS/MS method for the determination of -fluoro- -alanine, 5-fluorouracil and capecitabine in human plasma. J. Chromatogr. B, n.11-12, p.1040-1046, 2009.

derivative chemotherapeutic agents. Curr. Pharm. Biotech.,

5-Fluorouracil-loaded self assembled ph-sensitive nanoparticles as novel drug carrier for treatment of malignant tumors. Chinese J. Chem. Eng., 382, 2006.

formulary: NF 29: by authority of the United States Pharmacopeia Convention prepared by the council of experts and its expert committees. 29.ed. Rockville: United

126


5-Fluorouracil delivery. J. Colloid Interf. Sci., v.354, n.1, p.202-209, 2011.

VIGNERON, C. Influence of experimental parameters on the characteristics of poly(lactic acid) nanoparticles prepared by a double emulsion method. J. Control. Release, v.50, n.1-3, p.31-40, 1998.

of chitosan and polyaspartic acid sodium salt: Preparation, Eur. J. Pharm. Biopharm.

Received for publication on 28th May 2012 th October 2012

coated magnetic nanoparticles as carriers of 5-Fluorouracil: Coll. Surf. B, v.68, n.1, p.1-6, 2009.