Development and Validation of a Stability-indicating Capillary Zone ...

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2011 © The Japan Society for Analytical Chemistry

Development and Validation of a Stability-indicating Capillary Zone Electrophoretic Method for the Assessment of Entecavir and Its Correlation with Liquid Chromatographic Methods Sergio Luiz DALMORA,† Daniele Rubert NOGUEIRA, Felipe Bianchini D’AVILA, Ricardo Bizogne SOUTO, and Diogo Paim LEAL Department of Industrial Pharmacy, Federal University of Santa Maria, Santa Maria-RS, Brazil

A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of entecavir in pharmaceutical formulations, using nimesulide as an internal standard. A fused-silica capillary (50 μm i.d.; effective length, 40 cm) was used while being maintained at 25° C; the applied voltage was 25 kV. A background electrolyte solution consisted of a 20 mM sodium tetraborate solution at pH 10. Injections were performed using a pressure mode at 50 mbar for 5 s, with detection at 216 nm. The specificity and stability-indicating capability were proven through forced degradation studies, evaluating also the in vitro cytotoxicity test of the degraded products. The method was linear over the concentration range of 1 – 200 μg mL–1 (r2 = 0.9999), and was applied for the analysis of entecavir in tablet dosage forms. The results were correlated to those of validated conventional and fast LC methods, showing non-significant differences (p > 0.05). (Received May 5, 2010; Accepted December 26, 2010; Published March 10, 2011)

Introduction Hepatitis B virus (HBV) is a hepadnavirus capable of causing serious liver disorders in humans,1 and can lead to liver failure, hepatocellular carcinoma, and to death.2–4 Entecavir (2-amino-1,9-dihydro-9-[(1S,3R,4S)-4-hydroxy-3(hydroxymethyl)-2-methylenecyclopentyl]-6H-purin-6-one), a cyclopentyl guanosine analogue, is a potent inhibitor of HBV DNA polymerase, inhibiting both the priming and elongation steps of viral DNA replication. Entecavir is phosphorylated by human cellular kinases to the active triphosphate form, and is clinically indicated for the management of chronic hepatitis B.2,5,6 The literature describe LC-MS/MS bioanalytical methods for the quantitative analysis of entecavir in human plasma, using lobucavir as an internal standard, and solid-phase extraction.2,7 A reversed-phase liquid chromatography method (RP-LC) was described for the determination of entecavir and its related substances in tablet formulations, using gradient elution at a flow-rate of 1 mL min–1.8 Moreover, RP-LC methods9–11 were also validated for the analysis of pharmaceutical formulations using C18 analytical columns and isocratic elution with UV detection, over concentration ranges of 0.5 – 200, 3 – 200 and 2 – 24 μg mL–1, respectively. Capillary electrophoresis (CE) has emerged as a powerful analytical technique for pharmaceutical analysis, allowing for the determination of active pharmaceutical ingredients and their degraded products and impurities, with some advantages related to the existing methodologies.12–15 However, at the moment To whom correspondence should be addressed. E-mail: [email protected]

†

there is no published CE method for entecavir. The aim of the present study was to develop and validate a stability-indicating capillary zone electrophoresis (CZE) method for the determination of entecavir in solid dosage forms while evaluating the in vitro cytotoxicity of the degraded products, and to correlate the results to the validated conventional and fast LC methods, contributing to establish new alternatives for the quality control of pharmaceuticals.

Experimental Chemical and reagents The entecavir monohydrate reference substance was purchased from Sequoia Research Products (Oxford, UK). A total of eight batches of Baraclude® Bristol-Myers Squibb (Mount Vernon, IN) tablets, containing 0.5 and 1 mg of entecavir were obtained from commercial sources within their shelf-life period, and were identified by Arabic numbers from 1 to 8. Analytical-grade disodium tetraborate decahydrate and monobasic potassium phosphate were acquired from Merck (Darmstadt, Germany). HPLC-grade acetonitrile was purchased from Tedia (Fairfield, OH). All chemicals used were of pharmaceutical or special analytical grade. For all of the analyses, ultrapure water was purified using an Elix 3 coupled to a Milli-Q Gradient A10 system Millipore (Bedford, MA). All solutions were degassed by ultrasonication Tecnal (São Paulo, SP, Brazil) and filtered through a 0.22-μm Millex filter Millipore (Bedford, MA). Apparatus CZE experiments were performed on an Agilent 3DCE apparatus Agilent Technologies (Waldbronn, Germany) consisting of a photodiode array (PDA) detector,

266 a  temperature-controlling system (4 – 60° C) and a power supply able to deliver up to 30 kV. CE ChemStation software was used for instrument control, data acquisition and data analysis. The pH of the solutions was measured using a pH-meter, Thermo Orion Model 420 (Beverly, MA). The LC method was carried out on a Shimadzu LC system (Kyoto, Japan) equipped with an SCL-10AVP system controller, an LC-10 ADVP pump, a DGU-14A degasser, an SIL-10ADVP auto sampler, and an SPD-M10AVP PDA detector. The peak areas were integrated automatically by computer using a Shimadzu Class VP® V 6.14 software program. The fast LC method was performed on a Shimadzu Prominence UFLCTM system (Kyoto, Japan) equipped with a CBM-20A system controller, an LC-20AD pump, a DGU-20A5 degasser, an SIL-20ACHT auto sampler, a CTO-20A oven, and an SPD-M20AVP PDA detector. The peak areas were integrated automatically by computer using a Shimadzu LC Solution V® 1.22 SP1 software program. Solutions Preparation of a reference substance solution. A stock solution of entecavir was prepared by accurately weighing 10.65 mg of the reference substance monohydrate, transferred to an individual 10 mL volumetric flask and diluted to volume with methanol, obtaining a concentration of 1 mg mL–1 of entecavir base. Equally, a stock solution of nimesulide was prepared at a concentration of 1 mg mL–1 using acetonitrile. The stock C, protected from light and daily solutions were stored at 2 – 8° diluted to an appropriate concentration with a background electrolyte solution (BGE). Preparation of sample solutions. To prepare the sample stock solutions, tablets containing, respectively, 0.5 and 1 mg of entecavir were accurately weighed and crushed into a fine powder. An appropriated amount was transferred into an individual 10 mL volumetric flask and diluted to volume with methanol. The mixture was then vortex-mixed for 10 min, sonicated for 20 min, and centrifuged at 5000g for 20 min. The final solution with a concentration of 200 μg mL–1 of the active C, protected from pharmaceutical ingredient was stored at 2 – 8° light, diluted to an appropriate concentration with BGE solution and daily filtered through a 0.22-μm membrane filter. Electrophoretic procedure All experiments were carried out on a fused-silica capillary with 50 μm i.d. and 48.5 cm of total length (effective length 40 cm), thermostatized at 25° C, with detection by PDA set at 216 nm. At the beginning of each working day, the capillary was conditioned by rinsing with 1 M sodium hydroxide for 20 min, followed by water for 10 min, and then with a running electrolyte solution for 20 min. To improve the reproducibility of the migration time between injections, the capillary was conditioned again with water (1 min), and a running BGE solution (3 min). Samples were injected using the pressure mode for 5 s at 50 mbar with a constant voltage of 25 kV (current about 61.5 μA) applied during the analysis. Since electrolysis can change the EOF and affect the migration time, efficiency and selectivity, the running electrolyte was replaced by a fresh solution after each three injections. The BGE consisted of 20 mM disodium tetraborate decahydrate at pH 10, adjusted by adding 1 M sodium hydroxide. Validation of the CZE method The method was validated using samples of pharmaceutical formulation of entecavir with a label claim of 1 mg by determinations of the following parameters: specificity, linearity,

ANALYTICAL SCIENCES MARCH 2011, VOL. 27 range, precision, accuracy, limit of detection (LOD), limit of quantitation (LOQ), robustness, stability, and system suitability test, following the ICH guidelines.16,17 Nimesulide was used as an internal standard to compensate for any injection errors and minor fluctuations of the migration time, thus improving the reproducibility of the CZE method. Forced degradation studies The stability-indicating capability of the CZE method was determined by subjecting a reference substance solution (200 μg mL–1) and a tablet dosage form solution (80 μg mL–1) to accelerated degradation by acidic, basic, neutral, oxidative, and photolytic conditions.16 Working solutions prepared in 1 M hydrochloric acid were used for acidic hydrolysis, and working solutions in 0.1 M sodium hydroxide for the basic hydrolysis evaluation. Both solutions were refluxed at 100° C for 6 h, cooled and neutralized with acid or base, as necessary. For a study under a neutral condition, the reference and sample solutions were diluted in water and heated at 80° C for 6 h. Oxidative degradation was induced by storing the solutions in 10% hydrogen peroxide, at ambient temperature for 24 h, protected from light. Photodegradation was induced by exposing the sample in a photostability chamber to 200 W h m–2 of near-ultraviolet light for 6 h. The solutions were diluted with the electrolyte solution to final concentrations of 50 μg mL–1. The interference of the excipients of the pharmaceutical formulation was determined by the injection of a sample containing only a placebo (in-house mixture of all the tablet excipients), and by the standard addition method, where a calibration curve was constructed by the addition of known amounts of the reference substance to the placebo.18 Then, the specificity of the method was established by determining the peak purity of entecavir in the samples using a PDA detector. Additionally, the entecavir reference substance and the degraded samples were subjected to an in vitro cytotoxicity test. LC procedures LC method. The validated conventional LC method applied for the analysis of entecavir in tablet dosage forms is described elsewhere.8 Briefly, the elution was carried out on a RP Phenomenex (Torrance, CA) Gemini C18 column (150 × 4.6 mm i.d., with a particle size of 5 μm and pore size of 110 Å). A security guard holder (4 × 3 mm i.d.) was used to protect the analytical column. The LC system was operated isocratically at 30° C using a mobile phase of potassium phosphate buffer (0.01 M, pH 4)/acetonitrile (90:10, v/v), run at a flow-rate of 1.0 mL min–1, and using PDA detection at 253 nm. The injection volume was 20 μL of a solution containing 50 μg mL–1 for both the reference substance and the samples. Fast LC method. A fast LC method was developed and validated following the ICH guidelines.16,17 Elution was carried out on a Phenomenex (Torrance, CA) Synergi Fusion-RP column (100 × 3 mm i.d., with a particle size of 2.5 μm and a pore size of 100 Å). A security guard holder was used to protect the analytical column. The Shimadzu UFLC system was operated isocratically at 40° C using a mobile phase of water/acetonitrile (90:10, v/v), run at a flow-rate of 0.3 mL min–1, and using PDA detection at 253 nm. The injection volume was 10 μL. In vitro cytotoxicity test The in vitro cytotoxicity method was performed as described elsewhere,19,20 based on a neutral red uptake (NRU) assay, with the exposure of NCTC clone 929 cell line (mammalian fibroblasts, ATCC number CCL-1) to the degraded samples of entecavir. The pH of the samples was adjusted to 7.0,

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Fig. 1 Typical CZE electropherogram of the entecavir tablet dosage form (50 μg mL–1). Peak: 1, excipient povidone; 2, entecavir; 3, nimesulide.

Fig. 2 Representative CZE electropherograms showing peak 1 = entecavir and peak 2 = nimesulide (IS) of: (a) entecavir reference substance solution (50 μg mL–1) and IS, and after degradation under: (b) neutral hydrolysis, peak 3 = degraded form; (c) basic hydrolysis, peak 3 = degraded form; (d) acidic hydrolysis, peak 3 = degraded form; (e) photolytic condition, peaks 3, 4, 5 and 6 = photodegraded forms.

and  positive and diluent controls, together with the entecavir reference solution were included in the assay. The NRU assay was performed on 96-well microplates, maintained at 37° C in a CO2 incubator for 24 h, with the cell suspension having a density of about 2 × 105 cells mL–1. The neutral red released was evaluated by the addition of an extractant solution, and the absorbance measured at 540 nm using microplate reader Anthos Labtec Instruments (Salzburg, Austria).

Results and Discussion

pH 10.0 due to a better peak symmetry (about 1.07). The tetraborate was evaluated at concentrations of 15 – 35 mM, showing a significant effect on the separation performance through its influence on the EOF and the current produced in the capillary. A 20 mM tetraborate solution was selected due to its low current and non-significant increase on the migration time. The effects of organic modifiers, acetonitrile or methanol, in the concentration range of 5 – 20%, were also evaluated, but no improvement on the electrophoretic conditions was achieved. The temperature effect on the separation was investigated in the C, and a temperature of 25° C was chosen due range of 15 – 35° to the short run time and acceptable current. The effect of the voltage was studied through changes from 15 to 30 kV, showing that a potential of 25 kV yielded a short analysis time with an acceptable current (about 61.5 μA). Sample solutions were injected using a pressure mode at 50 mbar for 5 s. Wavelength detection was evaluated in the range of 190 – 400 nm, and a wavelength of 216 nm was chosen due to better sensitivity and signal-to-noise ratio.

Optimization of the electrophoretic conditions To develop the CZE method, some electrolyte solutions were tested by selecting disodium tetraborate decahydrate. The optimum pH of a BGE solution containing 20 mM disodium tetraborate was investigated in the range of 9.0 – 11.0, selecting

Validation of the electrophoretic method The stability-indicating CZE method was validated for the analysis of entecavir in pharmaceutical formulations, with a migration time of about 2.92 min, as shown in the typical electropherogram in Fig. 1.

Analysis of entecavir in pharmaceutical formulations For the quantitation of entecavir in tablet formulations, the stock solutions were diluted to an appropriate concentration (50 μg mL–1) with a BGE solution or a mobile phase, respectively, for the electrophoretic and chromatographic methods, injected in triplicate and the percentage recoveries calculated against the reference substance.

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Table 1 Inter-day and between-analysts precision data of CZE for entecavir in samples of tablet dosage forms Inter-day Sample

1 2

Day 1 2 3 2 2 3

Between-analysts

Concentration RSD, Concentration RSD, Analyst % founda, % % founda, % 98.66 98.08 97.72 98.62 99.86 100.20

0.48 0.83

A B C A B C

98.15 96.64 98.73 99.99 100.22 100.52

1.10 0.26

a. Mean of three replicates.

The stability-indicating capability of the method21 is indicated in Fig. 2, which shows an electropherogram of an entecavir reference substance and IS (Fig. 2a). Forced oxidative degradation studies exhibited a peak related to hydrogen peroxide with the same migration time as entecavir, demonstrating that it was not susceptible to this condition, as previously published.9 Under neutral hydrolysis, a decrease of the area was observed, with one additional peak at 4.76 min (Fig. 2b). The basic condition resulted in a decrease of the entecavir area with one additional peak at 3.01 min (Fig. 2c). The acidic condition showed a decrease of the area, and only one peak was detected at 4.32 min (Fig. 2d). Under the photolytic condition, the entecavir content exhibited a significant decrease, and four additional peaks were detected at 3.02, 3.08, 3.61 and 4.14 min (Fig. 2e). The specificity of the method towards the drug was established through a determination of the peak purity of the analyte and the IS in the working reference substance solution, by overlaying the spectra captured at the apex, upslope and downslope using a PDA detector. Additionally, the standard addition method was applied to evaluate the interference from formulation excipients. The regression equation was found to be y = 0.0216x + 0.0112, r2 = 0.9999, where, x is the concentration of entecavir, expressed in μg mL–1, and y is the peak-area ratio of entecavir to IS. No significant difference was found between the slopes calculated for the calibration curve and the standard addition method. The data, together with the peak purity index in the range of 0.9999 – 1, showed that the peak was free from any co-migrating peak, with no interference of excipients, thus confirming that the proposed method is specific for the analysis of entecavir. The linearity was determined by constructing three calibration curves, each one with eight concentrations of entecavir reference solution in the 1 – 200 μg mL–1 range, spiked with nimesulide at 50 μg mL–1. The value of the determination coefficient calculated by a least-squares regression analysis (r2 = 0.9999, y = (0.0230 ± 0.0010)x + (0.0083 ± 0.0073), where, x is concentration and y is the peak-area ratio of entecavir to IS) indicated linearity of the calibration curve for the method. The precision of the method, evaluated as the repeatability of the method, was studied by calculating the relative standard deviation (RSD%) of the migration time and the peak-area ratio for eight analyses at a concentration of 50 μg mL–1, performed on the same day and under the same experimental conditions. The obtained RSD values were 0.36 and 1.32% for the migration time and the peak-area ratio, respectively. The intermediate precision was assessed by analyzing two samples of the pharmaceutical formulation on three different days (inter-days), giving RSD values of 0.48 and 0.83%,

respectively. The between-analysts precision was determined by calculating the RSD for the analysis of two samples of the pharmaceutical formulation by three analysts; the values were found to be 1.10 and 0.26%, respectively, as given in Table 1. The accuracy was assessed from three replicate determinations of three solutions of in-house mixtures of the excipients with known amounts of the drug, containing 40, 50, and 60 μg mL–1, corresponding to 80, 100, and 120% of the analytical concentrations, respectively. The absolute means obtained with a mean value of 100.19% and a bias lower than 1.36%, show that the method is accurate within the desired range.22 The LOD and the LOQ were calculated from the slope and the  standard deviation of the intercept determined by a linear-regression model, as defined by ICH,17 by using the mean values of the three independent calibration curves. The obtained values were 0.38 and 1.27 μg mL–1, respectively. The evaluated experimental LOQ with a precision lower than 5% and an accuracy within ±5%,23,24 was found to be 1 μg mL–1, suitable as an alternative for quality-control analysis. The low sample injection volume and the short optical path-length can be related to the lower sensitivity of the CZE method compared to the chromatographic methods. The robustness of the analytical procedure25,26 was determined by analyzing samples of an entecavir reference solution containing 50 μg mL–1 in triplicate by the one-variable-at-a-time (OVAT) approach. The results and the experimental range of the selected variables evaluated are given in Table 2, together with the optimized values. Additionally, the robustness was also evaluated and compared by the multi-variable-at-a-time (MVAT) approach25 at three levels (1 unit per parameter up or down around optimized values). This procedure gives results for minimum changing of the maximum number of parameters at a time, and is very useful, rapid and efficient for a robustness determination. The results for the OVAT and MVAT procedures were within the acceptable deviation (RSD < 2%), and an analysis of the variance showed non-significant differences (p > 0.05) for the dosage of the sample solutions. The analysis performed with a wider level of variations of the solution pH, temperature and voltage, showed changes of the migration time related to the optimized conditions. Moreover, the peak symmetry values were also evaluated, showing non-significant differences (p > 0.05). The electropherogram pattern was not altered and different capillary batches also indicated robustness under the conditions tested. The stability of entecavir in BGE was assessed after storage of C, and also placed into an the samples for 48 h at 2 – 8° autosampler for 24 h at room temperature, showing non-significant changes ( 0.05), with a mean of IC50 = 48.89 μg mL–1. However, when subjected to

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Table 2 CZE conditions and the range investigated during robustness testing with the one-variable-at-a-time (OVAT) procedure Variable

Range

Entecavira, %

RSD

Migration time

RSD

Symmetry

RSD

Optimized condition

Electrolyte solution pH

9.6 9.8 10 10.2 10.4 16 18 20 22 24 21 23 25 27 29 21 23 25 27 29 3 4 5 6 7 212 214 216 218 220

102.17 100.02 98.46 101.69 104.34 96.18 96.08 97.62 95.06 95.88 97.76 96.83 97.29 97.36 97.68 97.32 97.99 99.46 97.69 98.91 95.48 95.58 97.45 96.44 96.92 97.79 98.71 98.86 97.35 98.99

1.46 0.63 0.18 1.38 2.00 0.94 0.70 0.07 0.22 0.43 1.36 0.49 0.16 0.33 0.67 1.31 0.64 0.58 0.78 1.49 1.16 0.91 0.10 0.54 0.42 0.82 0.87 0.47 0.73 0.93

2.65 2.83 3.02 3.11 3.22 3.05 2.99 2.90 3.15 3.17 3.28 3.16 3.07 2.98 2.89 3.74 3.30 3.07 2.62 2.33 3.05 3.10 3.04 3.14 3.03 2.94 3.06 2.97 2.95 2.98

0.77 0.18 0.07 0.19 0.38 1.56 0.46 0.34 0.31 1.32 0.89 0.64 0.32 0.49 0.51 0.68 0.22 0.02 0.32 0.33 1.20 0.25 0.04 0.39 1.65 1.92 0.52 0.11 0.17 0.29

1.23 1.22 1.13 1.25 1.26 1.21 1.22 1.24 1.18 1.21 1.25 1.24 1.15 1.24 1.25 1.34 1.24 1.06 1.16 1.10 1.11 1.16 1.10 1.14 1.18 1.13 1.15 1.08 1.20 1.17

1.95 0.81 0.33 1.61 1.99 1.95 0.30 0.05 0.53 1.73 1.98 1.75 0.91 1.08 1.92 0.60 0.32 0.17 0.42 0.96 2.00 0.85 0.44 1.17 1.98 1.85 1.82 0.85 1.92 2.00

10

Electrolyte solution concentration/mM

Temperature/° C

Voltage/kV

Time injection/s

Wavelength/nm

20

25

25

5

216

a. Mean of three replicates.

more drastic conditions, such as basic hydrolysis with 1 M sodium hydroxide and photodegradation with UV light for 24 h, increased toxicity was detected with IC50 = 16.25 μg mL–1 and 15.23 μg mL–1, respectively. Validation of the fast LC method Fast and reliable high-throughput analytical methods with high sensitivity and selectivity are required for pharmaceutical analysis. The fast LC method was validated based on an LC method previously studied;9 the results are partially shown. The specificity was proven through degradation studies; they also showed that there was no interference of the excipients of the tablet formulation. The use of an analytical column with a particle size of 2.5 μm combined with a flow-rate of 0.3 mL min–1 and low-volume tubbing allowed separation with a retention time of 3.65 min, enabling the advantages of lower consumption of solvents and higher samples throughput. The calibration curves were found to be linear in the 0.1 – 200 μg mL–1 (r2 = 0.9992) range, with an LOQ of 0.1 μg mL–1, representing an improvement related to the existing methods. The robustness was assessed by small variations of some of the most important parameters, giving results within the acceptable deviation (RSD < 2%). Moreover, the method validation demonstrated acceptable results for the precision, accuracy and system suitability test. The method was also applied to dissolution rate studies of entecavir tablets using USP apparatus 2 (paddle) at a stirring rate of 50 rpm, with potassium phosphate buffer, pH 6.8, as a dissolution medium, showing that the drug was released at higher than 90% after 30 min.

Table 3 Comparison between the electrophoretic and chromatographic methods in an assay of entecavir in tablet dosage forms Label Sample claim/ mg tablet 1 2 3 4 5 6 7 8 Mean SD

1 1 1 1 1 0.5 0.5 0.5

Experimental amount CZEa, RSD, % % 101.28 0.18 104.14 0.96 102.91 0.54 101.93 0.78 101.34 0.64 102.19 0.02 101.19 0.17 99.30 0.46 101.79 1.41 ANOVA Betweenmethods

LC a, %

RSD, %

102.83 0.12 104.88 0.02 102.75 0.02 100.52 0.19 103.48 0.01 99.16 0.03 98.42 0.12 97.34 0.07 101.17 2.70 F calculatedb 0.40

Fast LCa, % 101.50 103.92 101.84 100.34 100.95 100.70 98.75 99.31 100.91 1.60

RSD, % 0.24 0.03 0.04 0.09 0.12 0.14 0.03 0.13

a. Mean of three replicates. b. F critic for p = 0.05.

Methods application The validated CZE method was applied in parallel with the validated conventional and fast LC methods for the determination of entecavir in tablet dosage forms, giving mean acceptable

270 differences of 0.62 and 0.88%, respectively, higher for the CZE, as given in Table 3. The experimental values were compared statistically by an analysis of the variance (ANOVA), showing non-significant differences (p > 0.05). The capability demonstrated for the proposed method can be useful in the determination of entecavir without prior separation of the excipients of the formulation, with the advantages of small sample volumes without the consumption of organic solvents, and a short analysis time. The CZE method was also applied for the analysis of spiked human plasma samples after solid-phase extraction, giving a recovery of 71.88% for the lower concentration analyzed with 5 μg mL–1, without improvements related to the more sensitive published methods.7

Conclusion The results of validation studies show that the CZE method is sensitive for pharmaceutical analysis with an LOQ of 1 μg mL–1, accurate with a mean value of 100.19%, economic, specific and stability-indicating. It possesses significant linearity (r2 = 0.9999) and a precision of the peak-area ratio with a mean RSD of 0.80%, without any interference from the excipients. Therefore, the method was successfully used as an alternative for the quantitative analysis of entecavir in tablet dosage forms, with significant correlations with the liquid-chromatographic methods.

Acknowledgements The authors wish to thank CNPq (Conselho Nacional de  Desenvolvimento Científico e Tecnológico), project 304860/2008-5 for financial support. The authors declared no conflict of interest.

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