ISSN: (Online) 2321 -4155
ISSN: (Print) 2320 -7000

JOURNAL OF INDIAN RESEARCH
VOLUME : 5, ISSUE : 1, Janurary-March, 2017 (ISSN No. : 2321-4155)
 
DEVELOPMENT AND VALIDATION OF BIO –ANALYTICAL METHOD FOR THE ESTIMATION OF SOME DRUGS IN HUMAN PLASMA BY USING LC-MS/MS.
Lingeswara Rao Punati* Saikishore Vankayalapati**
 
ABSTRACT
A sensitive and selective method based on liquid chromatography – tandem mass spectrometry (LC-MS/MS) has been developed for the quantative determination of Loperamide in human plasma. Automated Solid Phase Extraction (SPE) on Disposable Extraction Cartridge (DEC) is used to isolate the compounds from the biological matrix and to prepare a clear sample before injection and analysis in the Lc-Ms/Ms system. After conditioning, the plasma sample is loaded on the DEC filled with end capped ethyl silica and washed twice with water. The analytes are therefore eluted with by dispensing with methanol. The elute is then collected and with 60% Acetonitril in water solution in order to inject an aliquot of this final extract in the LC-MS/MS System. Online Lc-Ms/Ms system using Atmospheric Pressure Chemical Ionization (APCI) has been developed for the determination of Loperamide. The separation is obtained on an octadecylsilica based stationary phase using a mobile phase consisting in a mixture of Methanol and 0.1% Formic acid in 10mM Ammonium acetate: Acetronitrile (30:70 v/v) Loperamide d6 is used as internal standard (ISTD). The Ms/ Ms ion transitions monitored are m/z 513.50 and 519.54 Loperamide and Loperamide d6, respectively. Calibration curves, which were linear over the range 20.000 to 3003.000 pg/mL (Loperamide), were run contemporaneously with each batch of samples, along with lower limit of quantification (20.000pg/mL), medium (1206.000pg/ mL), and high (2412.000pg/mL) quality control samples, the lower limit of quantification (LLOQ) of Loperamide and Loperamide d6 was about 0.25pg/ml in plasma. The extraction efficiency of Loperamide and Loperamide d6 from human plasma was 72.06 ±1.50 (range 70.7- 73.7%) and 67.97 ± 12.8% (64.9- 88.8%), respectively. The intraand inter-assay variability of Loperamide and Loperamide d6 ranged from 2.1 to 14.5% for the low, medium, and high quality control samples. The method has been validated successfully with respect to ICH guidelines on Q2B parameters.
 
KEYWORD
KEYWORDS: Calibration Curve, Loperamide , Loperamide d6 (ISTD).
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