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CE CURRENTS
CE Analysis of Propranolol in
Human Serum Using Dynamic
Capillary Coating
Lilian Clohs and Angelina K. Winstanley,
Cardiome Pharma Corp., Vancouver, British Columbia, Canada.
A capillary electrophoresis method using the CElixir buffer system for the analysis of propranolol
in human serum was compared with a method using phosphate buffer. While both methods
showed good linearity over the concentration range tested, the CElixir method showed better
accuracy and precision at the low concentration end (25–50 ng/mL). The limits of quantitation
and detection of the CElixir method were 25 and 12.5 ng/mL, respectively. Improved migration-
time reproducibility was obtained with the CElixir method, which was probably responsible for
an enhanced overall performance compared with the standard phosphate method.
Introduction
materials (e.g., poly[vinyl alcohol]) to the Capillary electrophoresis (CE) is gaining over a broad concentration range (typically wider acceptance in analytical laboratories 75–10 000 ng/mL for plasma samples), but an improvement in concentration sensitivity separations.10–13 Belder and colleagues routine analysis in the pharmaceutical and biotech industries1. Specifically in the area reproducibility using a permanent coating publications report the use of CE for the pharmacokinetic studies. In our experience, with poly(vinyl alcohol) and glutaraldehyde analysis of drugs in biological matrices. The applications include drug quantifications suffers from poor accuracy and precision. studies,2,3 in vitro drug metabolism A possible cause could be inconsistencies in electrolyte or by rinsing the capillary before studies,4,5 as well as applications in forensic runs with a surface modifier. For example, analysis6 and therapeutic drug monitoring.7 migration times. Because the peak area in CE is proportional to the migration time, variability in the latter parameter could EOF and limit solute adsorption.15 This is analysis of drugs in biological matrices, such as plasma, brain, urine and bile. The ionized silanol groups. A second layer of hydrophobic interactions resulting in a net electrophoretic conditions is used in our positive charge on the capillary surface and laboratories to analyse structurally diverse surface and therefore eliminate or modify Thornton et al.16 to explain the EOF reversal the EOF and reduce the interaction of the produced by alkanesulfonic acids, such as solutes with the capillary wall. Permanent coating can be obtained either by covalent silica capillaries for the analysis of basic bonding of agents such as polyacrylamide, Ethanesulfonic acid has also been reported drugs after a liquid–liquid extraction of polyvinylpyrrolidinone or octadecylsilane to to produce sharper peaks for quinidine and compounds from the biological matrix. The the capillary surface or by adsorption of LC•GC Europe May 2002
CE Currents
In our experience, CE analysis of basic drugs in plasma at
0.1 psi for 10 s. The capillary was washedafter each run with a series of rinses at concentrations less than 75 ng/mL often suffers from poor
20 psi: water (0.5 min), methanol (1 min), accuracy and precision.
water (0.5 min), 0.1 M NaOH (1 min),water (0.5 min) and run buffer (1 min). For wall adsorption of these two basic drugs.17 linearity, accuracy and precision of the two poly(vinylsulfonate) introduced in the run buffer resulted in consistent EOF and good reproducibility, analysis time and efficiency used. The capillary was rinsed prior to each migration-time reproducibility for a series with CElixir initiator solution (A) and 2 min limits were also reported when testing the Experimental
with CElixir accelerator solution (B) (pH 2.5) optical purity of drug enantiomers using a Materials: Human serum and
at 20 psi, and the separation voltage was D,L-propranolol (hydrochloride salt) were through decreased peak tailing because of obtained from Sigma (St. Louis, Missouri, areas (area/migration time) were used for USA). The IS, RSD921 (hydrochloride salt), Varian (Harbor City, California, USA). CElixir CEofix. This system consists of two buffers, for the CE analysis of propranolol in human solution (B), and fused-silica capillaries for capillary surface resulting in uniform EOF standard phosphate and the CElixir buffers New Jersey, USA). All other reagents were (Table 1). The back-calculated concentrations injected first to form a positively charged Standard curve preparation and
solution consisting of polyanions is then extraction procedure: Standard solutions
of propranolol in water (40 µL) were added larger for the analyses performed using the positively charged layer and form a highly final concentrations of propranolol in serum the CElixir buffer. The limit of detection insensitive to pH changes, resulting in a noise ratio of 3:1. No interference from the buffer. Altria reported improved migration- precision of the method. IS (50 µL of a 4 µg/mL solution of RSD921 in water) was electropherograms free of interfering peaks.
with CElixir was also reported in clinical concentrations were prepared and analysed Elut cartridges and extracted with ether dryness under a stream of nitrogen and the with a method using the CElixir system for dry extracts were reconstituted in 40 µL of Figure 1: Structures of (a) propranolol
CE conditions: CE analyses were
that the assay will be transferable to other basic molecules in Cardiome’s library. The Uncoated silica capillaries with 60 cm length antiarrhythmic programme and structurally Objective
The objective of this study was to compare temperature was maintained at 20 °C.
CElixir buffer with our standard assay using Samples were injected by pressure at 1 psi phosphate buffer, for the analysis of a basic for 10 s, with a postinjection water plug of www.lcgceurope.com
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Peak shape was improved with the CElixir method as
was 25 ng/mL using the CElixir buffer.
revealed by the increase in theoretical plate number
calculated for both propranolol and the IS…
resulted in poor accuracy (≤ 23%) andprecision (≤ 23%) at the low concentration (pH 2.5) and the CElixir buffer (pH 2.5).
end (25 and 50 ng/mL), while the accuracy show good accuracy (≤ 16%) and precision precision (4%) using the same buffer. The acceptable at the high (500 ng/mL) QC level.
(≤ 3%) for the two low (25 and 50 ng/mL) limit of quantitation (LOQ) of the method Conventionally, accuracy expressed as ±15% QCs after analysis using the CElixir buffer (±20% at LOQ) deviation from the nominal Table 1: Corrected peak area ratio (CAR) propranolol/IS, line parameters, back-calculated concentrations and deviations for the CE
analysis of propranolol in human serum using 100 mM phosphate (pH 2.5) and CElixir (pH 2.5). Weighting 1/x was used.
100 mM Phosphate Buffer (pH 2.5)
CElixir Buffer (pH 2.5)
Propranolol
Calculated
% Deviation
Calculated
% Deviation
concentration
concentration
from nominal
concentration
from nominal
Table 2: QC performance for the CE analysis of propranolol in human serum using 100 mM phosphate (pH 2.5) and CElixir (pH 2.5).
100 mM Phosphate buffer pH 2.5
CElixir buffer pH 2.5
Concentration
Deviation (%)
Concentration
Deviation (%)
LC•GC Europe May 2002
CE Currents
and precision (expressed as coefficient ofvariation, CV%) of ≤ 15% (≤ 20% at LOQ) Figure 2: CE analysis of propranolol in human serum. Electropherograms of extracted
are accepted when validating bioanalytical calibration standards analysed using (a) 100 mM phosphate buffer (pH 2.5) and (b) CElixir buffer (pH 2.5) (CE conditions: see text).
LOQ for the analysis of propranolol inserum using the standard phosphate using the CElixir buffer compared with the variability in migration times was observed buffer while the CElixir analyses resulted in CElixir buffer also allowed shorter analysis times: the IS (last peak) migrated past the detector at about 13 min while it appeared were obtained despite the fact that a much and colleagues when analysing a series of drugs using a CElixir buffer compared witha regular phosphate buffer method.23 Peaks: 1 ϭ propranolol, 2 ϭ internal standard.
reproducibility for the analyses of thecomplete set of samples (n = 17, 8standards and 9 QC samples) using the Table 3: Migration-time reproducibility for the CE analysis of propranolol in human
two different buffer systems. The migration serum using 100 mM phosphate (pH 2.5) and CElixir (pH 2.5).
Migration Time
100 mM Phosphate
CElixir pH 2.5
Reproducibility
pH 2.5 CV (%)
migration time was considerably better (CV = 0.7%). The CV% was consistently lowfor both the propranolol and the IS peak (CV = 0.2%) as well as for the relativemigration time (CV = 0.2%) when theCElixir buffer was used. Good migration-time precision was also reported by Table 4: Comparison of efficiency obtained for the CE analysis using 100 mM
Altria,20 and Lurie and co-workers24 for a phosphate (pH 2.5) and CElixir (pH 2.5).
series of injections of basic drugs analysed Number of theoretical plates
100 mM Phosphate
Concentration (ng/mL)
Compound
CElixir method as revealed by the increase in theoretical plate number calculated forboth propranolol and the IS compared with Conclusions
The CE method for the analysis of
CElixir buffer system showed good linearity, www.lcgceurope.com
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Mall, Vancouver, British Columbia, V6S 2L2, reproducible. Because peak area is related to the migration time in CE, it is probable that the improved migration-time reproducibility, as well as lower adsorption to the capillary Angelina K. Winstanley is a Research
Associate in the Bio-Analytical Chemistry resulted in enhanced overall performance of “CE Currents” editor Kevin D. Altria is
senior principal scientist in thepharmaceutical development group at Acknowledgement
GlaxoSmithKline R&D, Ware, Hertfordshire, We would like to thank Dr Kevin D. Altria for suggesting the use of the CElixir buffer.
Advisory Board of LC•GC Europe. Directcorrespondence about this column to “CE References
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Lilian Clohs is Associate Director of the
Bio-Analytical Chemistry Department at
LC•GC Europe May 2002
CE Currents
LC•GC Europe May 2002

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