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TCH-006 Development and Validation of 3 Methods – UV Spectrophotometry, Flow Injection Analysis and Liquid Chromatography – For the Control of Nystatin Capsules.
  1. M Herold,
  2. A Nicolas,
  3. I May
  1. CHU – Hôpitaux de Brabois Adultes, Pharmacie, Vandoeuvre Cedex, France

Abstract

Background As an alternative to amphotericin B used for selective digestive decontamination, physicians asked the Hospital Pharmacy for the preparation of nystatin capsules, 500,000 IU.

Purpose Three methods were considered for routine checking: UV spectrophotometry, flow injection analysis (FIA) and high performance liquid chromatography (HPLC).

Materials and Methods Three batches (3x200 capsules) were prepared with nystatin (INRESA) and mannitol (VWR). All other reagents were of analytical grade.

Preparation of stock solutions of nystatin and capsules content was in reagent-grade methanol for FIA and HPLC (nystatin 72 µg/mL). For UV spectrophotometry, a subsequent dilution (1/50 V/V) with acetate ammonium buffer/methanol, 50:50 (V/V) was needed.

For FIA and HPLC, 10 µL were injected. In all cases, absorbance was measured at 305 nm.

UV spectrophotometry used a double beam spectrophotometer (UV mc2 – SAFAS).

FIA used an HPLC device (Ultimate 3000 – Dionex) in which the stationary phase was replaced by a capillary flow of water (1.0 mL/min; 25°C).

HPLC equipment was an ELITE LaChrom (VWR/Hitachi). An end-capped C18 stationary phase was used (30°C). The mobile phase was a mixture of 0.05 M acetate ammonium buffer pH = 6.0/reagent-grade methanol (35/65; V/V). Flow rate was 1.0 mL/min; run time was 25 min.

Results For UV spectrophotometry and FIA, the development took into account the nystatin concentration to obtain absorbance levels suitable for the precision and the range of linearity. HPLC was developed as an isocratic stability indicating method.

The three methods were fully validated (ICH Q2R1). HPLC ruggedness was studied according the adjustments allowed by the Ph. Eur. (2.2.46). Nystatin content (3 batches) assayed by each method complied with the acceptance limit: 90.0–110.0%.

Conclusions For routine checking, UV spectrophotometry or FIA would be the methods of choice (rapid, easy to handle); the HPLC method could be used to perform stability studies.

No conflict of interest.

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