Sensitive, Selective, Precise and Accurate LC–MS Method for Determination of Clonidine in Human Plasma Chinmoy Ghosh & , Rajendra P. Singh, Shafi Inamdar, Mandar Mote, Bhaswat S. Chakraborty Cadila Pharmaceuticals Limited, 1389-Trasad Road, Dholka, Gujarat, India; E-Mail: chinmoy.ghosh@cadilapharma.co.in; chinmoy_ghosh@yahoo.com Received: 3 December 2008 / Revised: 23 February 2009 / Accepted: 13 March 2009 Abstract A selective and sensitive liquid chromatography–mass spectrometric method in ESI (+ve) mode was developed and validated completely in human plasma. Clonidine and IS, car- bamazapine, were extracted from human plasma via liquid–liquid extraction with ethyl acetate. Following evaporation under nitrogen, the residue was reconstituted with mobile phase and analyzed using API 4000 LC–MS–MS system. An isocratic program with binary mobile phases (0.1% formic acid in water and acetonitrile) was used to separate interference peaks by a C18 analytical column. Linearity range was 0.49–73.98 ng mL -1 . The intra- and inter-day accuracy and precision were within acceptable limits (15%). This method was successfully applied to a single dose 25 lg tablets BE study of clonidine in healthy male volunteers. Keywords Column liquid chromatography Tandem mass spectrometry Clonidine Introduction Clonidine (N-(2,6-dichlorophenyl)-4,5- dihydro-1H-imidazol-2-amine) (Fig. 1), is a direct-acting alpha-2 (a-2) adrenergic agonist, prescribed historically as an antihypertensive agent in mild to mod- erate hypertension especially in cases of hypertension with kidney diseases. It has found new uses, including treatment of some types of neuropathic pain, opioid detoxification, sleep hyperhidrosis, ana- esthetic use, and off-label and to counter the side effects of stimulant medications such as methylphenidate or amphet- amine. It is becoming a more accepted treatment for insomnia, as well as for relief of menopausal symptoms. Clonidine is being increasingly used in conjunction with stimulants to treat attention-deficit hyperactivity disorder (ADHD), for which it is administered in the late afternoon or evening for sleep, and because it sometimes helps to moderate ADHD-associated impul- sive and oppositional behavior, and may reduce tics [1]. Clonidine can also be used in the treatment of the Tourette syndrome [2]. The main use for this medication is to treat high blood pressure. It works by stimulating a-2 receptors in the brain which decreases cardiac output and peripheral vascular resistance, lowering blood pressure. It has specificity towards the presynaptic a-2 receptors in the vasomotor center in the brainstem. This binding decreases presynaptic calcium levels, and inhibits the release of nor- epinephrine (NE). The net effect is a decrease in sympathetic tone in cardiac muscle and peripheral blood vessels [3]. Clonidine may also reduce drug and alcohol withdrawal symptoms. The sys- tematic review of trials found [4] that clonidine can lead to a small increase in the number of people likely to quit smoking. However, the quality of the trials was poor, which makes the evi- dence less reliable. Main adverse effects of clonidine include a dry mouth and sedation. Clonidine may not be the best DOI: 10.1365/s10337-009-1102-3 Ó 2009 Vieweg+Teubner | GWV Fachverlage GmbH Original