A comparative study of the use of powder X-ray diffraction, raman and near infrared spectroscopy for quantification of binary polymorphic mixtures of piracetam

Diffraction and spectroscopic methods were evaluated for quantitative analysis of binary powder mixtures of FII(6.403) and FIII(6.525) Piracetam. The two polymorphs of Piracetam could be distinguished using powder X-ray diffraction (PXRD), Raman and near-infrared (NIR) spectroscopy. The results demonstrated that Raman and NIR spectroscopy are most suitable for quantitative analysis of this polymorphic mixture. When the spectra are treated with the combination of multiplicative scatter correction (MSC) and second derivative data pretreatments, the partial least squared (PLS) regression model gave a root mean square error of calibration (RMSEC) of 0.94 and 0.99 % respectively. FIII(6.525) demonstrated some preferred orientation in PXRD analysis, making PXRD the least preferred method of quantification

Solid-state transformations of sulfathiazole polymorphs: the effects of milling and humidity

The effect of milling on the solid-state transitions of sulfathiazole polymorphs in the absence and presence of solvent and excipients was monitored by X-ray powder diffraction (XRPD), attenuated total reflectance infrared (ATR-IR) and near-infrared (NIR) spectroscopy. Sulfathiazole forms FII-FV undergo a transformation toward the metastable FI, which involves an intermediate amorphous stage upon milling at ambient temperature. Milling FIII with catalytic amounts of solvent converts FIII to FIV or to mixtures of FI and FIV depending on the solvent used. Pure FIV can be easily prepared from FIII by this method. The physical stability of pure sulfathiazole forms in the presence of different levels of relative humidity (RH) was also investigated. At low RH all sulfathiazole forms are stable but at RH levels above 70% FII, FIII and FIV remain stable while FI and FV transform to mixtures of FII and FIV without any apparent change in the external form of the crystals. Co-milling FIII with a range of excipients gave results which depended on the excipient used and co-milling with cellulose gave samples which had an amorphous content that was stable at 10% RH for at least nine months at ambient temperature

Analysis of the size distribution of Cdc45-containing protein complexes during the cell cycle and after UV damage.

<p>Asynchronous (Asn) UVC-treated (+UVC, 5 J/m², 1 h post-treatment), G1/S transition or S phase synchronized HeLa S3 cells stably expressing eGFP-Cdc45 were lysed and normalized for protein content and separated by gel filtration chromatography analysed by western blotting using antibodies raised against Cdc45, Mcm5 and RPA 32. (panels a, b, c, and d, respectively). Theoretical molecular weight (kDa) and Stoke's radius (Å) of protein standards are overlayed. RPA32 acts as a marker for DNA damage response following UVC treatment. FACS analysis is provided for asynchronous (Asn), G1/S transition and S phase synchronized cells (e) and asynchronous cells treated with 5 J/m², 1 h post-treatment (f).</p

HeLa S3 cells stably expressing eGFP-Cdc45.

<p>Panel a, schematic diagram of eGFP-Cdc45 protein encoded by Cdc45L ORF cloned into pIC113gw vector. Panel b, total cell extract HeLaS3 cells (-ve) and Hela S3 cells stably expressing eGFP-Cdc45 (eGFP-Cdc45) normalized for protein content and analysed by western blotting using antibodies raised against Cdc45 and β-Actin, which serves as a loading control. Panel c, western blot analysis of immunoprecipitation of eGFP-Cdc45 using GFP-Trap IP from HeLa S3 cells transiently expressing eGFP-Cdc45. Verification of purification of eGFP-Cdc45 and co-immunoprecipitation of Mcm7 was carried out using antibodies raised against Mcm7 and Cdc45. Input (L), unbound (FT), mock IP (-ve) and IP from cells expressing eGFP-Cdc45 (eGFP) indicate yield of the IP and co-immunoprecipiation. Antibody light chain acts as a loading control.</p

Diffusion coefficient of eGFP-Cdc45 at G1 to S phase transition and in S phase and after UV damage.

<p>Mean and standard deviations obtained from at least 15 cells.</p

Association of Cdc45 with chromatin synchronized HeLa S3 cells and after DNA damage.

<p>Panel a, chromatin-associated lysate from 1×10⁶ Hela S3 cells synchronized at various cell cycle stages by two consecutive thymidine block analysed by western blotting using antibodies raised against Cdc45, Mcm7, Mcm5, Lamin B1, P261 and P125 of Pol ε and <i>δ</i>, respectively. The latter serves as a loading control. Asynchronous control cells (Asn) or cells analysed at times ranging from 0 to 12 h following release from the second thymidine block (TdR 0 to TdR 12) were analysed in parallel by FACS. Corresponding FACS profiles for relevant timepoints are also shown. Panel b, western blot of chromatin-associated Cdc45 following UVC treatment. HeLa S3 cells treated with 5 J/m² UVC harvested at indicated timepoints post treatment with untreated cells (UT) acting as a control. Chromatin-associated lysates normalized for protein content were analysed by western blotting using antibodies raised against Cdc45 and Lamin B1, which serves as a loading control.</p

Auto-correlation curves of eGFP-Cdc45.

<p>The figure shows typical auto-correlation curves of eGFP-Cdc45 (□) in asynchronous HeLa S3 cells stably expressing eGFP-Cdc45. In the upper panel the solid black line corresponds to a two-component free diffusion model and in the lower panel the gray line is the residual of the fit.</p

Solid-State Transformations of Sulfathiazole Polymorphs: The Effects of Milling and Humidity

The effect of milling on the transitions of sulfathiazole polymorphs in the absence and presence of solvent and excipients was monitored by X-ray powder diffraction (XRPD), attenuated total reflectance infrared (ATR-IR), and near-infrared (NIR) spectroscopy. Sulfathiazole forms FII–FV undergo a transformation toward the metastable FI, which involves an intermediate amorphous stage upon milling at ambient temperature. Milling the commercial form (FC) with catalytic amounts of solvent converts it to pure FIV or to mixtures of FI and FIV depending on the solvent used. Pure FIV can be easily prepared from FC by this method. The physical stability of nonmechanically activated pure sulfathiazole forms in the presence of different levels of relative humidity (RH) was also investigated. At low RH, all sulfathiazole forms are kinetically stable, but at RH levels above 70% FII, FC and FIV remain stable, while FI and FV transform to mixtures of FII and FIV without any apparent change in the external form of the crystals. Comilling FC with a range of excipients gave results that depended on the excipient used, and comilling with cellulose gave samples that had an amorphous content that was stable at 10% RH for at least nine months at ambient temperature