• 27
    Dec
  • Stability of Norepinephrine Solutions: METHODS

Liquid Chromatography

The liquid chromatographic system consisted of an isocratic solvent delivery pump (model P4000, Thermo Separation Products, San Jose, California), which pumped a mixture of acetonitrile (OmniSolv; EMD Chemicals Inc, Gibbstown, New Jersey) and 0.05 M phosphoric acid (catalogue no. P286; Fisher Scientific, Toronto, Ontario) containing 1 mg/mL of heptane sulfonic acid (catalogue no. H 2766, lot 61K5431; Sigma Aldrich Canada Ltd, Oakville, Ontario) through a 15 cm x 4.6 mm reversed-phase C-18, 3-pm column (Supelcosil ABZ+Plus, catalogue no. 59194; Supelco, Oakville, Ontario) at 1.0 mL/min. The ratio of acetonitrile to 0.05 M phosphoric acid was 5:95 and was held constant during each chromatographic run. The samples were introduced into the liquid chromatographic system using an autoinjector (WISP 717-Plus; Waters Scientific, Toronto, Ontario).

The column effluent was monitored with a variable- wavelength ultraviolet (UV) detector (UV6000; Thermo Separation Products) at 280 nm. Each signal from the detector was integrated and recorded with a chromatography data system (ChromQuest, version 5.0; Thermo Fisher Scientific Inc). The area under the norepinephrine peak at 280 nm was subjected to least-squares linear regression, and the actual nore- pinephrine concentration in each sample was determined by interpolation from the standard curve.
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Assay Validation

Following development of the chromatographic system for norepinephrine, the suitability of this method for use as a stability-indicating assay was tested by accelerating the degradation of norepinephrine. A 10-mL sample of a 40 mg/L solution of norepinephrine (norepinephrine bitartrate injection USP, Sandoz Canada Inc, Boucherville, Quebec; lot 149812, expiry April 2010) was adjusted to a pH of 8.264 with 0.5 M sodium hydroxide. A second 10-mL sample of a 40 mg/L solution of the same lot of norepinephrine was adjusted to pH 1.977 with 0.5 M hydrochloric acid. Each solution was placed in a glass vial in a water bath at 80°C. Samples were drawn from the vial with the pH 8.264 solution immediately before it was placed in the water bath and at 7 other times (2, 5, 10, 18, 26, 31, and 37 min) over a 37-min study period. Samples were drawn from the vial with the pH 1.977 solution immediately before it was placed in the water bath and at 10 other times (7, 15, 30, 63, 121, 180, 248, 2763, 4553, and 8513 min) over an 8513-min study period (about 142 days). Samples (10 pL) were injected directly into the chromato- graphic system using an autoinjector. The resulting chro- matograms were inspected for the appearance of additional peaks, and the norepinephrine peak was compared between samples for changes in concentration, retention time, peak shape, and UV spectral purity (200-320 nm) relative to a fresh, undegraded sample.

Following this first phase of evaluation and validation, the accuracy and reproducibility of standard curves were tested over 5 days, and system suitability criteria (theoretical plates, tailing, and retention time) were developed to ensure consistent chromatographic performance, according to accepted analytical guidelines. Standard curves were prepared daily by diluting 0.75 mL of a norepinephrine standard (norepinephrine bitartrate injection USP, Sandoz Canada Inc; lot 149812, expiry April 2010) in 5 mL of distilled water. This stock solution (150 mg/L) was further diluted to prepare additional standards with final concentrations of 100, 50, 37.5, 18.75, and 12.5 mg/L. These standards were combined with a blank and the stock solution to construct a standard curve. A 10-pL sample of each solution was chromatographed in duplicate. Also, 2 quality control samples of norepinephrine (concentra­tions 25 and 75 mg/mL) were chromatographed in duplicate each day, and their concentrations were determined and compared with the known concentrations. Intraday and interday errors were assessed by the coefficients of variation of the peak areas of both quality control samples and standards.  generic cialis soft tabs

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