Background

1. Liquid chromatographic-tandem mass spectrometric determination of atenolol following transdermal administration K.B.Ita1, N. Hatsakorzian1, V.V. Tolstikov2 1.College of Pharmacy, Touro University, Mare Island-Vallejo, California 2. Metabolomics Core Facility, Genome Center, UC Davis, California In vitro skin penetration experiments A vertical, three compartment, flow-through diffusion cell system (LG-1088-IC, Laboratory Glass Apparatus, Inc., Berkeley) was used for experiments. Exposed porcine ear skin area was 0.8 cm2. After equilibration, the donor compartment of each cell was charged with 27mg/ml of atenolol. The donor compartment was covered with parafilm.Receiver solution was phosphate buffered saline (pH 7.4). Diffusion cells were kept at 37 o C by Digital One heating bath circulator (Neslab EX-7, Thermo Fisher, USA) and stirred at 100rpm (Variomag, Daytona Beach, FL, USA). Percutaneous transport was monitored for 12 hours. The receptor was constantly perfused at 3ml/min using a peristaltic pump (Ismatec ISM 943) and samples were collected in two-hour increments using a fraction collector (Retriever 500, Teledyne Isco, USA). The cumulative quantity of atenolol transported was determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described above. A representative chromatogram is shown in Figure 1 while a bar plot showing atenolol plasma concentration is shown in Figure 2. Atenolol therapeutic window is shown in Figure 3. Background Figure 3. Atenolol therapeutic range of concentration (Ct) Atenolol (4-[2 hydroxy-3-[(1-methylethyl) amino] propoxy] benzeneacetamide is a β-blocking agent used widely in the treatment of hypertension (Heel et al, 1979). Atenolol is available as a conventional tablet. The tablet is taken twice or three times daily leading to fluctuations in plasma concentration and side effects such as fatigue, dizziness and cold extremities . Absorption of atenolol upon oral administration is rapid but incomplete . Due to incomplete intestinal absorption, systemic bioavailability is about 50-60% in humans. However, hydrophilic atenolol (logP is -1.4) poorly penetrates the stratum corneum. Anroop et al studied transdermal iontophoretic delivery of atenolol prodrugs across porcine skin. Steady state flux of atenolol was low (0.57± 0.18µmol/sq.cm/h). Modamio et al also studied transcutaneous flux of atenolol across abdominal human skin. Atenolol flux was 0.24± 0.11µg/hr/cm2.The two research groups used high performance liquid chromatography for assay. In the study by Modamio et al, the limit of quantification was 0.78µg/ml and no significant amount of atenolol was detected in the first 20 hours of experiment (Modamio et al 2000). Although high-performance liquid chromatography is widely used for drug quantitation following transdermal application, low drug concentrations justify the development of LC-MS/MS techniques to improve sensitivity, precision and accuracy. In the present study, a sensitive liquid chromatography-tandem mass spectrometric method was developed for analysis of atenolol after transdermal administration. Plasma concentration (ng/ml) Conclusions An LC-MS/MS method was developed and validated for atenolol assay. The method is rapid, sensitive and specific with an limit of quantitation of 0.1ng/ml.This technique was successfully used to monitor atenolol following transdermal delivery across porcine ear skin. It was possible to detect atenolol in the receptor compartment of a flow through cell after 2 hours. Experimental flux value was used to calculate atenolol plasma concentration. Results Figure 1. A representative atenolol chromatogram Acknowledgements This research was supported by the College of Pharmacy,Touro University. Special thanks to Drs Katherine Knapp, Paul Goldsmith and Michael Ellerby. Methods References LC-MS/MS Liquid chromatography-tandem mass spectrometry was performed with Waters Acquity UPLC system interfaced to an Applied Biosystems Sciex 4000-QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA). Analyst software (Version 1.42) was used for data acquisition and processing. Separations were carried out on a UPLC HSS T3 C18 column (Waters). The mobile phase was 0.1% formic acid in water and 0.1% formic acid in acetonitrile.The flow rate was 0.6ml/min. Injection volume was 2µl. The retention time was 1.47min. Detection was performed on a QTRAP 4000 LC-MS/MS system equipped with Turbolon electrospray ion source. The LC-MS/MS detector was operated using multiple reaction monitoring (MRM) mode. MRM transition was predetermined for atenolol: Q1 267.00 (parent ion [M+H]+) and Q3 144.9 (daughter ion [M+H]+). R.C. Heel, R.N. Brogden, T.M. Speight, G.S. Avery (1979) Atenolol: a review of its pharmacological properties and therapeutic efficacy in angina pectoris and hypertension, Drugs 17: 425–460. B. Anroop, B.Gosh,V.Parcha, J. Khanam(2009) Transdermal delivery of atenolol:effect of prodrugs and iontophoresis,Current drug delivery, 6, 280-290 P.Modamio C. F. Lastra, E. L. Mariño (2000) A comparative in vitro study of percutaneous penetration of β-blockers in human skin, International Journal of Pharmaceutics, 194:249-259 Figure 2. Atenolol plasma concentration calculated from passive. flux across porcine ear skin. Plasma concentration (ng/ml) Contact Information Turbo spray ion source settings were as follows-curtain gas, gas 1 and gas 2 (nitrogen) 20, 50 and 50 psi respectively; collision activated dissociation (CAD) gas: high. The dwell time was 100 ms while source temperature and ion spray voltage were 500oC and 1500V respectively. Declustering potential (DP) and collision energy (CE) were 50V and 23eV respectively. Collision extraction potential was set at 20V. The analytical range to be validated was chosen on the basis of expected percutaneous concentrations. The lower limit of quantitation was 0.1ng/ml. Kevin Ita, PhD Assistant Professor, Pharmaceutical Sciences College of Pharmacy, Touro University - California 1310 Johnson Lane, Mare Island Vallejo, CA 94592 Phone: 707-638-5994 Email: kevin.ita@tu.edu.