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