Category: Formulation and Quality
Purpose: The presence of nanoscale particles in suspensions could increase flux through biological membranes for poorly soluble compounds  and as a result improve their oral absorption and bioavailability. Hence, there is a need for in vitro tests that aid in the understanding of nanosuspension behavior. This is challenging as nanosized particles both scatter and absorb light, obscuring in situ fiber optic UV measurements. That can lead to wrong interpretations of UV absorbance data and as a result erroneous concentration estimates. The goal of this study was to continue developing a method of de-convoluting the UV-Vis spectra measured in situ to obtain the concentration of free drug in the presence of light absorbing nanoparticles.
Methods: A method of identifying the spectrum and building the standard curve for nanoparticles consisted of the following steps: (1) determining the saturated concentration (i.e. solubility) of free drug in the presence of light absorbing nanoparticles by Zero Intercept Method (ZIM ); (2) subtracting the absorbance spectrum of free drug at its solubility value from the combined spectrum of free drug+nanoparticles to obtain the spectral characteristics of nanoparticles alone; and (3) building a standard curve corresponding to various nanoparticle concentrations. The µDISS Profiler™ instrument run by AuPRO™ software version 6.0 (Pion Inc.) was used to monitor UV-VIS spectra in situ. ZIM implemented in the AuPRO™ software determined the concentration when the second absorbing component (i.e. nanoparticles) appeared along with dissolved API. The method was based on the shift of the wavelength where the 2nd derivative of the absorbance spectra of the API component crosses through zero of the wavelength axis. During the dissolution/precipitation experiment the spectra of suspensions were acquired and then linear multi-component regression analysis tool of AuPRO™ software was used to extract the concentration of free drug and quantify amount of nanoparticles present in the suspension. Nanosuspension of crystalline naproxen (NPX) was received from Novartis AG (Basel, Switzerland) and used to evaluate the method using aqueous pH 2.0 and pH 5.0 buffers. A solvent shift method was used to form amorphous precipitations of felodipine (FLD) and albendazole (ABZ) in FaSSIF.
Results: The solubility of nanocrystalline NPX (USP pH 2.0 buffer) determined by ZIM was 19.3 µg/mL (Figure 1). Figure 2 illustrates the method of identifying UV spectrum of nanoparticles of NPX: NPX spectrum at 12 µg/mL (blue) is scaled up to its saturated concentration of 19.3 µg/mL (orange) and subtracted from the nanosuspension spectrum at 30 µg/mL (grey) to obtain the spectrum of nanoparticles at 10.7 µg/mL (red). The procedure was evaluated by de-convoluting the spectra of a suspension of a known ratio (1:4) between dissolved NPX and NPX nanoparticles. The measured concentrations of free NPX and nanoparticles were in excellent agreement with the expected values. The sequential aliquots of FLD dissolved at high concentration in the organic solvent (stock solution) were added to the aqueous buffer (pH 6.5). The formation of liquid-liquid phase separation (LLPS, ) was determined by the ZIM as ~ 7.5 µg/mL. The additions of stock solution continued until the total FLD concentration reached 23.2 µg/m. The spectrum of the newly formed liquid phase was characterized and then the spectrum of FLD emulsion was monitored over a period of 16 hours. The concentration of dissolved FLD remained at its amorphous solubility level for more than 4 hours while the concentration of light absorbing (nanosized) particles or droplets reduced from 8 to 2 µg/mL within the first 90 min and remained quasi-stable for another 2.5 hours. Precipitation of FLD to its crystalline solubility value (~1 µg/mL) correlated with the disappearance of additional spectral influence from nanosized droplets indicating the formation of larger scale particles. During dissolution of 200 mg dose of ABZ in 250 mL of FaSSIF it was observed that UV spectrum was different from the spectrum of standard solution of ABZ. It appeared that ABZ formed LLPS at 30 µg/mL while its crystalline solubility in FaSSIF was ~ 1.9 µg/mL. Both dissolved ABZ and dynamically formed colloidal particles were monitored using developed method.
Conclusion: A novel method enabled simultaneous concentration monitoring of dissolved drug and nanosized light absorbing particles that could be present during dissolution/precipitation processes. The method was implemented in the AuPRO™ software and expanded capabilities of in situ fiber optic technique.
Oksana Tsinman– Pion Inc., Billerica, Massachusetts