Method validation of clonidine hydrochloride in human plasma by LC-MS technique

Clonidine (Fig. 1), an imidazoline derivative, is a centrally acting hypotensive agent but its use is being explored in a number of other indications such as anesthesia and the management of opiate with drawl (1). Clonidine belongs to a class of drugs called central alpha-adrenergic agonists. Clonidine has been in clinical use for over 40 years, which is used to treat hypertensive disorders, hyperactivity disorder, anxiety disorders, migraine, menopausal flushing and certain pain conditions (1). It has been proven by clinical studies that fluctuation of clonidine plasma concentration is responsible for itsside effects. Hence a transdermal patch formulation of clonidine was developed (1) to maintain clonidine plasma concentration in a steady state over a prolonged period of time and reduce the risk of adverse effects. Then a sensitive andreliable analytical method needed to be established to describe the determination of clonidine in human plasma. Several bioanalytical methods are reported to determine clonidine in different biological matrices like plasma (2,3), serum (4,5,7-8), urine (4), and cerebrospinal fluids (2). Sensitive and selective methods based on LC-MS/MS Abstract A simple and sensitive high performance liquid chromatography-electrospray ionization mass spectrometry method has been evaluated for the assignment of clonidine hydrochloride in human plasma. The mobile phase composed of acetonitrile–water 60:40 (v/v), and 0.2% formic acid 20 μL of sample was chromatographically analyzed using a repacked ZORBAX-XDB-ODS C18 column (2.1 mm×30 mm, 3.5 micron). Detection of analytes was achieved by tandem mass spectrometry with electrospray ionization (ESI) interface in positive ion mode was operated under the multiple-reaction monitoring mode (m/z 230.0 → 213). Sample pretreatment involved in a one-step protein precipitation (PPT) with methanol and percholoric acid (HClO4) of 0.15 mL plasma. Standard curve was linear (r = 0.998) over the concentration range of 0.01-10.0 ng/ml and showed suitable accuracy and precision. The limit of quantification (LOQ) was 0.01 ng/ml. The method is rapid, simple, very steady and precise for the separation, assignment, evaluation of clonidine healthy in human plasma.