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