Neeraj Upmanyu
1, Pawan Kumar Porwal
2,3*1 School of Pharmacy & Research, People's University, By-Pass Road, Bhanpur, Bhopal (M.P.)-462037, India.
2 Department of Pharmaceutical chemistry, SNJB’s SSDJ College of Pharmacy, Chandwad (Maharashtra)-423 101, India.
3 Department of Quality Assurance, ISF College of Pharmacy, Moga, Punjab-142001, India.
Abstract
Purpose: Desmopressin acetate (DDAPV), a synthetic
analogue of vasopressin, has been recommended to be used in diabetes insipidus,
mild forms of hemophilia and Von Willebrand disease. The DDAPV is available for
adminstration via different routes viz. oral, parenteral and nasal, however
its dose is very less in case of nasal sprays (20 µg) and parenteral route (4
µg) compared to oral route (0.1 to 0.3 mg in tablet). A sensitive and selective
method is needed to be developed and validated for assay of low concentrations
of DDAPV in its pharmaceutical dosage form i.e. nasal spray.
Methods: Simple and specific HPLC-Fluorescecne method
has been proposed for the quantitation of DDAPV at nanogram level in nasal
formulations for the first time. DAPV, DDAPV EP impurity-B, chlorobutanol,
benzalkonoium chloride were successfully derivatised with Ortho-Phthalaldehyde
(OPA) and co-eluted on a C8 (50×2.1 mm, 3.5 µm particle size, 120Å) with mobile
phase composed of 0.1% trifluroacetic acid, acetonitrile and Isopropyl alcohol
in ratio of 70:25:5. The emission was measured at 450nm and flow rate was
0.8ml/min. The reaction was optimized in the terms of pH, stability of formed
fluorophore and time consumed during the reaction.
Results: The maximal fluorescence intensity was reached
when the solutions were mixed for 3 min, and remained constant for at least 30
min at 20-25ºC. The calibration curve was found linear from 50 to 5000 ng/ml
with weight of 1/X2. The limit of detection was 10ng/ml and
precision was less than 2.0.
Conclusion: The
developed HPLC-fluorescence assay method was successfully applied for
quantitation of DDAPV in nasal spray. HPLC-Fluorescence method was specific,
sensitive, precise and accurate for determination of DDAPV. The method was able
to quantify DDAPV at 50ng/ml with sufficient accuracy and precision. The
validated HPLC-Fluorescence was successfully applied.