d(GC) - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 6 (2) 196-203 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)
Stricture of Nitrofurantoin
Materials and Methods
Materials: Nitrofurantoin and Losartan
analytical standards were obtained from Biocon
India Pvt Ltd, HPLC grade Acetonitrile and
methanol were obtained J.T. Baker, USA,
Ammonium acetate (GR Grade Merck), Dimethyl
sulphoxide (AR Grade Merck), Orthophosphoric
acid (GR Grade Merck),Glacial acetic acid(GR
Grade Merck ),Water was de-ionized and triple
distilled.
Preparation of Nitrofurantoin standard
solutions: Standard solutions of Nitrofurantoin
Plasma Sample Thaw (30 min)
Centrifuge at 4500 rpm for
10 minutes at 5°C
Collect 1.8 mL of supernatant
Add 50 µL of ISTD
Instrumentation and conditions : The Thermo
Scientific HPLC (Model: Surveyor) and LC-MS/
MS system interfaced to LC Quan Software in a
windows platform The Reverse phase C8
analytical column (Kromosil C 8, Length: 4.6×50
mm, Particle size: 5 µ) was used for the
separation of Nitrofurantoin and internal standard,
the mobile phase consists a mixture of
197
(1.0 mg/ml) was prepared by dissolving an
accurately weighed amount of 10.00 mg of
Nitrofurantoin in 5 ml of Acetonitrile and add 50
µL of Dimethyl sulphoxide, sonicated for 5
minutes and the volume was adjusted to 10 ml
with Acetonitrile. The standard solution was
stored subsequently at 2-8 0C. The appropriate
concentrations of standard solution were
prepared by diluting the stock solution with 80:
20 Acetonitrile: water.
Preparation of Losartan Internal standard
solutions: Standard solution of Losartan (1.0 mg/
ml) was prepared by dissolving an accurately
weighed amount of 10.00 mg of Losartan in 5 ml
of methanol, sonicated for 5 minutes and the
volume was adjusted to 10 ml with methanol. The
standard solution was stored subsequently at 2-
8 0C. 10.00µg/mL Losartan was prepared by
diluting the stock solution with 80: 20 methanol:
water.
Sample preparation
Vortex for 5 min
Vortex (1 min)
Acidfy with 50µL of 10 %
Orthophosphoric acid
Vortex (1 min)
Add 2.5 mL Ethyl
Acetate
Evaporate the supernatant at 40 0 C With Nitrogen
Reconstitute the residue with 5mM Ammonium Acetate: Acetonitrile (50:50%)
(50:50%) and inject 10ul into LC-MS/MS.
Rajaram S. Patil et al
Acetonitrile and 5mM Ammonium Acetate at a
ratio of 60:40 respectively. The mobile phase was
degassed by passing through a 0.22 µm
membrane filter (Millipore, Bedford, MA, USA)
prior to use and operated through a single pump
(Isocratic) at a flow rate of 0.5 ml/min. The injector
was filled with an injector loop of 10 µl. Tandem
mass spectrometric detection and quantification