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 />
was performed using multiple reaction monitoring<br />
(MRM).<br />
Results <strong>and</strong> Discussion<br />
Development conditions for rapid extraction<br />
<strong>of</strong> Nitr<strong>of</strong>urantoin from Human Plasma:<br />
Reported extraction techniques for determination<br />
<strong>of</strong> nitr<strong>of</strong>urantoin from human plasma [3-6, 8-11,<br />
14-19] is more laborious <strong>and</strong> involves more<br />
tedious process like derivatisation. More over,<br />
they involve cost efforts <strong>and</strong> in addition to this,<br />
they are time consuming. While developing a<br />
method, one needs to look at the<br />
chromatographic conditions <strong>and</strong> extraction<br />
process for minimizing the disadvantages <strong>of</strong> older<br />
techniques or methods. In the process, mobile<br />
phase selection <strong>and</strong> optimization, column<br />
selection is critical to minimize run time, solvent<br />
consumption <strong>and</strong> injection volumes. It was<br />
observed that, a novel method can be developed<br />
with advantages that will eliminate derivatisation,<br />
more solvent consumption, high run time <strong>and</strong><br />
rising costs. The new method should <strong>of</strong>fer the<br />
benefits <strong>of</strong> using small columns, reducing plasma<br />
<strong>and</strong> solvent consumption to arrive at an extraction<br />
procedure which will have more extraction<br />
recoveries. Conditions for simple <strong>and</strong> rapid HPLC<br />
separation with MS/MS Electro-spray Negative<br />
ionization mode were developed using an<br />
isocratic elution with a mobile phase composed<br />
<strong>of</strong> Acetonitrile <strong>and</strong> 5mM Ammonium acetate at a<br />
ratio <strong>of</strong> 60:40% v/v. Thus the ions formed for drug<br />
<strong>and</strong> Internal st<strong>and</strong>ard in ESI Negative mode due<br />
to the addition <strong>of</strong> Hydroxyl ion (OH- ) in carbonyl<br />
function (C=O) present in the drug (Fig.1). These<br />
conditions gave a well defined, sharp peak <strong>of</strong><br />
Nitr<strong>of</strong>urantoin <strong>and</strong> Losartan (ISTD) with a<br />
retention time <strong>of</strong> approximately 1.21 <strong>and</strong><br />
1.51minutes. Under these conditions an amount<br />
<strong>of</strong> Nitr<strong>of</strong>urantoin as low as 1ng/mL could be<br />
detected. With these retention times, analysis<br />
could be completed in about 3.0 minutes.<br />
Method validation<br />
Linearity: The quantification <strong>of</strong> the<br />
chromatogram was performed using the peak<br />
198<br />
area ratio <strong>of</strong> Nitr<strong>of</strong>urantoin <strong>and</strong> Losartan (ISTD).<br />
Nine st<strong>and</strong>ard solutions were prepared.<br />
(10.248, 25.621, 51.242, 102.484, 256.209,<br />
457.515, 667.906, 861.814 ng/mL <strong>and</strong> 1013.899<br />
ng/mL) <strong>and</strong> subjected analyses by HPLC-MS/<br />
MS. Three precision <strong>and</strong> accuracy (P&A) batches<br />
were injected. The peak area ratio was<br />
determined <strong>and</strong> plotted versus the concentration<br />
<strong>of</strong> Nitr<strong>of</strong>urantoin. Statistical analysis using least<br />
square regression analysis indicated excellent<br />
linearity for Nitr<strong>of</strong>urantoin with the concentration<br />
range studied as shown in Table 1. In constructing<br />
the st<strong>and</strong>ard curve, samples <strong>of</strong> Nitr<strong>of</strong>urantoin in<br />
Human Plasma identical to those in the st<strong>and</strong>ard<br />
solutions were prepared <strong>and</strong> the Nitr<strong>of</strong>urantoin<br />
response ratios were plotted against the<br />
concentrations <strong>of</strong> Nitr<strong>of</strong>urantoin in ng/mL as<br />
shown in Fig. 2. The linearity <strong>of</strong> the concentration<br />
<strong>and</strong> response relation was established over the<br />
range <strong>of</strong> 10.248 – 1013.899 ng/mL (R 2 = 0.9898).<br />
Fig. 3 shows the LC-MS/MS chromatograms <strong>of</strong><br />
pure drug. (Nitr<strong>of</strong>urantoin), Fig. 4 shows the LC-<br />
MS/MS chromatograms <strong>of</strong> drug-free Human<br />
plasma <strong>and</strong> Fig. 5 shows the LC-MS/MS<br />
chromatograms <strong>of</strong> st<strong>and</strong>ard Plasma sample<br />
containing the drug at a concentration <strong>of</strong><br />
10.248ng/mL.<br />
Accuracy <strong>and</strong> precision: The intra-day<br />
accuracy <strong>and</strong> precision <strong>of</strong> the assay was<br />
evaluated by analyzing six replicates <strong>of</strong> the<br />
Plasma containing Nitr<strong>of</strong>urantoin at three<br />
different concentrations. The intra-day precisions<br />
<strong>of</strong> the analyzed samples are determined by<br />
Relative St<strong>and</strong>ard Deviation (RSD) range from<br />
2.11% to 11.01%, while the intra-day accuracy<br />
ranged from 83.61% to 107.16%. The inter-day<br />
precision <strong>of</strong> the assay was measured by<br />
analyzing six replicates <strong>of</strong> Nitr<strong>of</strong>urantoin Plasma<br />
samples for three consecutive days. The interday<br />
precision <strong>of</strong> the analyzed samples as<br />
determined by Relative St<strong>and</strong>ard Deviation<br />
(RSD) range from 6.48% to 12.37%, while the<br />
inter-day accuracy ranged from 93.13to 103.02%.<br />
Nitr<strong>of</strong>urantoin in Human Plasma by using Liquid Chromatography / T<strong>and</strong>em Mass Spectrometry