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Liquid chromatographic-tandem mass spectrometric determination of atenolol following transdermal administration
1
K.B.Ita ,
N.
1
Hatsakorzian ,
V.V.
2
Tolstikov
1.College of Pharmacy, Touro University, Mare Island-Vallejo, California 2. Metabolomics Core Facility, Genome Center, UC Davis, California
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.
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.
Results
Figure 1. A representative
atenolol chromatogram
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.
This research was supported by the College of Pharmacy,Touro
University. Special thanks to Drs Katherine Knapp, Paul Goldsmith
and Michael Ellerby.
References
Plasma concentration (ng/ml)
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.
Conclusions
Acknowledgements
Methods
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]+).
Figure 3. Atenolol
therapeutic range of
concentration (Ct)
Plasma concentration (ng/ml)
Background
In vitro skin penetration experiments
Figure 2. Atenolol plasma
concentration calculated from
passive. flux across porcine
ear skin.
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
Contact Information
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: [email protected].