High Performance Liquid Chromatography (HPLC)

Shimadzu, Japan

The High Performance Liquid Chromatography (HPLC) system consisted of two pumps (LC-20AD), Photo diode array detector (SPD-M20A), Flourescence detector (RF20A), column oven (CTO10AS VP) and auto sampler (SIL-20AC HT) with built in system controller. Lab Solutions is used for data collection and acquisition. The analytical column used for different assays are C18, C8, Atlantis T3 and Cyano columns, protected by compatible guard columns. This instrument is highly sensitive and is used to quantitate anti-TB drugs in mirco volume plasma samples in few minutes. Methods have been developed and validated for estimation of first line and second line anti-TB drugs (Rifampicin, Rifabutin, Isoniazid, Pyrazinamide, Ofloxacin, Moxifloxacin, Levofloxacin, Ethionamide, Cycloserine), Verapamil, Metformin and Glybenclamide.

Principle

HPLC is a separation technique that involves the injection of a small volume of liquid samples into a tube (column) packed with silica particles called the stationary phase and where individual components of the sample are moved down the column with a liquid (mobile phase) forced through the column by high pressure delivered by a pump. The main principle of separation is adsorption.

Two ml of blood in heparinized vacutainer has to be collected. Mix gently and centrifuge the blood at 5000 rpm for 5 minutes. Separate the plasma (10 microliters of 5% ascorbic acid to be added for rifampicin and Rifabutin assay) and store at -20 o C until transportation. Plasma samples should be transported to the Department of Biochemistry & Clinical Pharmacology, NIRT in dry ice within a week.

Applications

Following drug assays can be done: Rifampicin, Rifabutin, Isoniazid, Pyrazinamide, Ofloxacin, Moxifloxacin, Levofloxacin, Ethionamide, Cycloserine, Verapamil, Metformin and Glybenclamide.

Sequencing application charges