Analysis of dialkyl urine metabolites of organophosphate pesticides by a liquid chromatography mass spectrometry technique Sukesh Narayan Sinha, * a B. Venkat Reddy, a Kasturi Vasudev, a M. Vishnu Vardhana Rao, a M. Noor Ahmed, a Shaik Ashu, a Alka Kumari a and Vijay Bhatnagar b A sensitive method for the quantification of six dialkyl phosphate (DAP) urinary metabolites (DMP, DEP, DMTP, DETP, DMDTP, and DEDTP) in urine samples at ng mL 1 concentrations was developed. A solvent extraction followed by selective analysis by a liquid chromatography-mass spectrometric method was used. This method was found to be very accurate and exhibited the limits of detection as follows: DMP, 0.058 ng mL 1 ; DEP, 0.009 ng mL 1 ; DMTP, 0.004 ng mL 1 ; DETP, 0.0148 ng mL 1 ; DMDTP, 0.00861 ng mL 1 ; and DEDTP, 0.0198 ng mL 1 . The limits of quantification were in the range of 0.0287 ng mL 1 to 0.0743 ng mL 1 . The value of the coefficient was found to be $0.999 for all of the metabolites. The percent recovery of the pesticides, at the lowest levels of detection, ranged from 93–107% and the coefficient variation was found in the range of 0.400–7.98, which indicates high degree of accuracy and precision. This method is found to be more cost-effective because the quantity of chemicals involved is much less compared with other methods. This method is also found to be simple because the number of steps where there was a chance of sample spillage were reduced, and it can also be used for quantification of pesticide concentrations in urine samples collected from the general population throughout various parts of the world. Initially, 30 samples were analyzed, and the mean concentration of the metabolites ranged from 0.058 to 118.27 ng mL 1 , which suggests that the children of Hyderabad city were exposed to OP pesticides. 1. Introduction Organophosphates (OPs) are the most commonly used insecti- cides in the world. They have achieved great popularity owing to their effectiveness as insecticides and their lack of persistence in the environment. OPs are used extensively in both agricul- tural and residential contexts worldwide. 1 The currently used pesticides include herbicides and insecticides, especially in India. Exposure to OP pesticides typically occurs through multiple routes (food, air, soil and water); however, the domi- nant route of exposure to individuals varies. Assessing exposure to OPs is not a trivial process. The dietary intake represents the major source of pesticide exposure for children, and this exposure may increase pesticide-related health risks in children compared with adults. 2 In many epidemiologic studies, markers of exposure in biological samples have been measured to esti- mate the absorbed dose. 3–5 The National Children's Study examined approximately 100 000 children across the US and also followed them during prenatal development, through birth and childhood, and into adulthood. 6,7 In 110 urban and suburban children, measurable levels of OP metabolites were found in their urine samples. 8 One of the most common measures of OP pesticide exposure is quantifying six common urinary dialkyl phosphate (DAP) metabolites. Although this measure provides no specic information about the pesticide to which one was exposed, it may potentially represent exposure to both the pesticide itself and its environmental degradation to urinary DAP metabolites. 9 OP pesticides tend to be metabolized relatively quickly and are excreted primarily in the urine. Nearly all OPs metabolize to a dialkyl phosphate moiety consisting of a phosphate and two ethyl or methyl esters. Therefore, there are two biomarkers for the exposure of OPs – cholinesterase enzyme in blood samples and urinary metabolites of OPs (dialkyl phosphate). The urinary dialkyl phosphate (DAP) metabolites have been quantied to estimate the exposure and absorption of OPs in the body. 5,9,10 Currently, 39 OP pesticides are regis- tered for use in agriculture. Of these, approximately 75% are O,O-dimethyl or O,O-diethyl substituted and consist of up to three of the six common dialkyl phosphate (DAP) metabolites. These six metabolites are dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP), dimethyl-dithiophosphate (DMDTP), diethyl phosphate (DEP), diethyl thiophosphate (DETP), and diethyl dithiophosphate (DEDTP). DAP metabolites do not a National Institute of Nutrition, Indian Council of Medical Research, Jamai-Osmania Po, Hyderabad-500 007, A.P, India. E-mail: sukeshnr_sinha@yahoo.com; Fax: +91- 40-27019074; Tel: +91-40-27018405 b Swami Vivekanand Subharti University, NH-58, Meerut-250005, India Cite this: Anal. Methods, 2014, 6, 1825 Received 5th November 2013 Accepted 8th January 2014 DOI: 10.1039/c3ay41958d www.rsc.org/methods This journal is © The Royal Society of Chemistry 2014 Anal. Methods, 2014, 6, 1825–1834 | 1825 Analytical Methods PAPER