Development and ICH Validation of a RP-HPLC-UV Method for the Quantification of Thimerosal in Topic Creams

Abstract

A reversed phase high-performance liquid chromatography (RP-HPLC) method for determination of Thimerosal (TMS) in topical creams was optimized and validated according to the ICH guidelines which include accuracy, precision, selectivity, robustness, limit of detection (LOD), limit of quantification (LOQ), linearity and range. For topical creams, sample treatment is often an overwhelming step essentially due to its oily nature. For the first time a simple and robust extraction procedure for TMS using phosphate buffer (pH 5.5, 0.2M) was successfully developed. This method describes the TMS quantitation by HPLC in a topical product containing 0.01% fluocinolone acetonide (FLA) as the active molecule. The HPLC separation was achieved on a Column Symmetry® and a methanol: phosphate buffer (pH 2.5, 0.05M) 70:30 v/v mobile phase and wavelength 218 nm. Results from both standards and samples showed adequate validation parameters. Noteworthy, linearity was within the range 1.2 - 2.8 μg/mL. Additionally, robustness and TMS stability were established after sample extraction. The method provides an efficient and safe quality control tool for determination of TMS in topical creams.

Thiamine Assays—Advances, Challenges, and Caveats

“…As recoveries with sulfuric acid tend to be low, HCl is preferred acid for the hydrolysis step. Some further caution may be warranted in the acid treatment of thiamine, as oxythiamine can be formed under strongly acidic conditions . This was confirmed recently, yielding low levels of oxythiamine in 100 m m acetic acid at elevated temperatures (100 °C for 1 h) .…”

“…At physiological pH, thiamine is a cation with a positive charge on the thiazole N3 nitrogen. With further increase in pH, its behavior is complex, passing through an uncharged pseudobase intermediate to yield its negatively charged thiol form (p K a 2 ≈9.2) (Figure ) …”

“…Other means of acid extraction to release thiamine from sample matrices have included autoclaving homogenized samples at 120 °C in sulfuric acid or 10–100 m m HCl for 10–30 minutes . Some studies observed low recoveries with sulfuric acid, believed to be caused by either the presence of sulfite impurities, which can degrade thiamine by cleavage at the methylene bridge under acidic conditions, or adsorption of thiamine onto formed precipitates . The latter has also been suggested with TCA .…”

Rapid and visual readout of vitamin B1 based on the electrostatic interaction induced aggregation of gold nanoparticles

“…On the other hand, the structure and charge of VB1 varies with the pH values. 14,31 As depicted in Fig. 5, VB1 is positively charged on both the pyrimidine N1 nitrogen (pK a1 z 4.8) and thiazole N3 nitrogen at low pH, and the VB1 presents with a cation that a positive charge appears on the thiazole N3 nitrogen at physiological pH.…”

“…Up to now, various methods have been employed to assay VB1, such as high performance liquid chromatography (HPLC), 10 chemiluminescence (CL) method, 11 absorption spectrometric assay, 12,13 potentiometric assay, 14 uorimetry, [15][16][17][18][19][20] resonance Rayleigh scattering spectral method 21 and so on. Among these analytical techniques, the problems like inevitable use of organic solvents in HPLC and CL, poor reproducibility of potentiometric assay, expensive instruments for resonance Rayleigh scattering spectral method, special competitive reaction between VB1 and uorescent materials with the metal ions in some extent limited their wide applications.…”