Research Article BIO-ANALYTICAL METHOD DEVELOPMENT AND ITS VALIDATION FOR ESTIMATION OF PHENOBARBITAL IN HUMAN PLASMA USING LIQUID CHROMATOGRAPHY COUPLED WITH TANDEM MASS SMRITI PANDEY b , AJIT KUMAR YADAV* a , SUNIL SINGH a , HEMENDRA GAUTAM a AND SURABHI SHARMA a a Department of Pharmacy, Invertis Institute of Pharmacy, Invertis University, Bareilly-243 123, Uttar Pradesh, India, b Received: 26 July 2012, Revised and Accepted: 08 Sep 2012 Department of Pharmaceutical Chemistry, NRI Institute of Pharmacy, Raisen Road, Bhopal-462021, Madhya Pradesh, India. Email: ajit.y@invertis.org , rssunil29@rediffmail.com ABSTRACT A high throughput and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the estimation of Phenobarbital in human plasma. Phenobarbital was extracted from human plasma using solid-phase extraction technique using Phenytoin Sodium as internal standard. A Beta basic 8 column provided chromatographic separation of analyses followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 251.4 → 98.0 for Phenobarbital. The method involves a simple multiplexing, rapid solid-phase extraction, simple isocratic chromatography conditions and mass spectrometric detection which enable detection at sub-nanogram levels. The proposed method has been validated for a linear range of 5.5 – 10.3 ng/mL with correlation coefficient ≥0.99 79. The precision and accuracy were within 10% for intra-HPLC runs and inter-HPLC runs. The overall recoveries for Phenobarbital were 99.89%. Total MS run time was 4 min. The developed method was applied for the determination of pharmacokinetic parameters of Phenobarbital following a single oral administration of a 30 mg Phenobarbital tablet in human plasma. Keywords: LC/MS/MS, Phenobarbital, HPLC, Validation, Human Plasma. INTRODUCTION Phenobarbital (PBT) chemically 5-ethyl-5-phenylpyrimidine- 2,4,6(1H,3H,5H)-trione 1 anticonvulsant belongs to drugs it also has sedative and hypnotic properties 2 benzodiazepines (Figure. 1) but, as with other barbiturates, has been superseded by the for these indications 3 . Phenytoin Sodium an antiepileptic drug was used as an internal standard. The mass transition ion-pair for Phenobarbital (PBT) m/z 251.4 →98.0. Different methods have been reported in the literature for monitoring plasma levels of PBT 4 , and in dosage forms also 5 . Some other techniques used in individual analysis of PBT from plasma include HPLC with Mass Spectroscopy, UV- Spectroscopy 6,7,8,9 , but these are not sensitive. Since there is no specific method was available in literature for the quantification of Phenobarbitol in human plasma using LC/MS/MS system, the study was done by different literature survey 9,10,11,12,13,14,15 the aim of the study was the development and validation of simple, sensitive, rapid and specific method. Fig. 1: Chemical structure of Phenobarbital MATERIALS AND METHODS Chemicals used Acetonitrile (Merck), Methanol (Merck), Millipore water were used in method development. Instrumentation API 3000 LC/MS/MS was used. Mass Parameters The mass spectrometer was operated in the positive ion mode. The developed mass and LC parameters for the estimation of PBT and Phenytoin Sodium (PTS) as internal standard are given below. The compound dependent parameters for Phenobarbitol was 55eV, Focussing potential was 160eV, Entrance potential 10eV, Collision energy CE 30 psi. For Phenytoin Sodium potential was 50eV, Focusing potential was 190 eV, Entrance potential 10 eV, Collision energy CE 100 V. Source dependent parameters for the method are CUR -15 psi, TEMP -550°c, ISV-5500 V, CAD-18psi. Multi Reaction Monitoring (MRM) The mass transition ion-pair has been followed as m/z 251.4 → 98.0 for PBT and m/z 423.2→ 91.0 for PTS. (a) (b) Fig. 2: Mass spectrum of Phenobarbital (a) Parent ion (b) Daughter ion International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 4, Suppl 5, 2012 A A c c a a d d e e m mi i c c S Sc c i i e e n n c c e e s s