Quantitative Analysis of Azimilide and One of its Metabolites in Mouse Blood by HPLC
with UV Detection
James J. Bao, P&G Pharmaceuticals, 8700 Mason-Montgomery Road, Mason, OH 45040-9462,
USA*
Abstract
A high performance liquid chromatography (HPLC) method was developed and validated
for the quantitative analysis of azimilide and one of its major metabolites, NE-10171, in mouse
blood. NE-10133 was used as the internal standard. The samples were prepared by freezethawing the mouse blood in an acetone/dry ice bath, followed by protein precipitation in a
mixture of acetonitrile : methanol : ammonium acetate buffer (8:2:1 v/v/v). The supernatant was
separated, dried, and the residue was reconstructed in mobile phase. The reconstituted sample
was ultrafiltered and injected onto the C8 reversed HPLC column. Azimilide and NE-10171
were separated with an isocratic mobile phase and detected at 340 nm. An analyte/internal
standard ratio was used to quantitate azimilide and NE-10171. The lower limits of quantitation
were 19.2 ng/ml for azimilide and 19.8 ng/ml for NE-10171. Three orders of magnitude linear
response curves were obtained for both azimilide (from 19.2 ng/ml to 19.2 g/ml) and NE-10171
(from 19.8 ng/ml to 19.8 g/ml). The method was successfully applied to quantitate azimilide
and NE-10171 in mouse blood in support of an azimilide safety study.
Keywords: Azimilide, HPLC, metabolite
*
Current Address: Advanced Medicine, Inc., 901 Gateway Boulevard, South San Francisco, CA 94080, 001-650808-3715.
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1. Introduction
Azimilide dihydrochloride (commercial name Stedicor, see Frig 1) is being developed as
a new Class III antiarrhythmic and antifibrillatory drug for the treatment of cardiac
arrhythmias[1-5]. The free base, azimilide, selectively prolongs the action potential duration
and increases the effective refractory period of the heart [6-7]. The prolongation of the action
potential duration is thought to result from the blockage or slowing of potassium ion current [8].
Both clinical and non-clinical studies have demonstrated that azimilide can be developed as a
potent yet sate drug. During both pre-clinical and clinical studies, azimilide, when administrated
to either animal or human, undergoes complicated metabolism resulting in several metabolites.
The metabolic profile is different in different species, i.e. human vs. animals. The
pharmacological functions of these metabolites were investigated [4]. One of the major
metabolites, the desmethyl metabolite, NE-10171 (see Fig 1), was present in the blood of both
humans and animals in significant amounts. Therefore, the analysis of this metabolite along with
azimilide itself in biological fluids is of importance.
In order to support the development of azimilide, various analytical methods are needed
to quantitate azimilide and its metabolites in blood and other matrices. Even though high
performance capillary electrophoresis (HPCE) has shown some unique advantages [9], most of
the bioanalytical methods for azimilide are high performance liquid chromatography (HPLC)
based methods. Further, to meet the stringent regulatory requirement, these methods have to be
very well validated before they can be applied to support real sample analyses. Therefore, to
support the dose ranging study of azimilide in mice, developed and validated a HPLC method for
the analysis of azimilide and NE-10171 in mouse blood using NE-10133 as the internal standard
(see Fig 1).
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Azimilide, NE-10171 and NE-10133 were extracted from mouse blood by freeze-thawing
the blood samples in an acetone/dry ice bath, followed by protein precipitation in a mixture of
acetonitrile, methanol, and ammonium acetate buffer. The supernatant was separated and dried.
The residue was reconstituted with mobile phase, and chromatographed via reversed phase
HPLC on a 5-micron C8 column to complete separate these three compounds. The compounds
were detected and quantiated by ultraviolet (UV) absorbance at 340 nm. Quantitation was done
by comparing the ratio of peak areas of azimilide and of NE-10171 to that of NE-10133. The
concentrations of azimilide and NE-10171 in the samples were determined from calibration
curves generated with spiked matrix standards.
The lower limit of quanititation (LLOQ) was
around 20 ng/ml based on a 200 l. One significant achievement of this method was the fact
that, by properly placing the calibration standards along the calibration curve, we were able to
achieve three orders of magnitude of linearity in a single method with the calibration standards
and the quality control (QC) samples satisfying all of the preset requirements. With this method,
levels in mouse blood were quantitated over three orders of magnitude for both azimilide (from
19.2 ng/ml to 19.2 g/ml) and NE-10171 (from 19.8 ng/ml to 19.8 g/ml). Data reported here
demonstrates the specificity, linearity, accuracy and precision, sensitivity, recovery and
robustness, and stability of this method.
This method was applied to the analysis of mouse blood samples under good laboratory
practice (GLP) conditions. The pharmacokinetic data obtained was used to evaluate the safety of
azimilide.
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2. Experimental
2.1 Materials and reagents
Certified reference compounds for azimilide, NE-10171, and NE-10133 were synthesized
by medicinal chemists at Procter & Gamble Pharmaceuticals, Inc (P&GP). Ammonium acetate
(ACS grade), methanol and acetonitrile (HPLC grade) were from Fisher Scientific (Fair Lawn,
NJ). Triethylamine (TEA) was from Kodak (Rochester, NY). Saline solution was USP standard
from McGaw, Inc (Irvine, CA). Blank mouse blood in heparin was obtained from Pel-Freez
Biologicals (Rogers, AR).
The HPLC system consisted of a Gilson XL autosampler and programmable 305 and 306
solvent delivery modules (Gilson, Middleton, WI). The detector was an Applied Biosystem
785A UV detector (Applied Biosystem, Foster City, CA). Exsil octyl analytical columns (5-m,
250x4.6 mm) were purchased from Keystone Scientific, Inc (Bellefonte, PA). The pre-column
filter containing a replacable 2-micron frit was obtained from Upchurch Scientific (Oak Harbor,
WA). A PE/Nelson 900 series digital interface along with TurboChrom software from
Perkin-Elmer Corp (Norwalk, CT) was used to record the data. The Z-spin, a micrcentrifuge
filter unit with 0.2-micron pore size, was purchase from Gelman Sciences (Ann Arbor, MI). An
automatic environmental SpeedVac (AES 2000) with VaporNet was from Savant Instruments,
Inc (Holbrook, NY). A Turbo Vap, LV was from Zymark Corp (Hopkinton, MA).
A Milli-Q reagent water system from Millipore Corp (Bedford, MA) was used for the
purification of deionized (DI) water. An ammomium acetate buffer (32 mM ammonium acetate
with 36 mM TEA, pH 5.0) was prepared in-house. The protein precipitation solvent was a
mixture of acetonitrile : methanol : ammonium acetate buffer (8 : 2 : 1, v/v/v). The mobile phase
was acetate buffer : methanol : acetronitrile (65: 5 : 30, v/v/v). It was filtered through a 0.45-m
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Nylon-66 filter membrane (47 mm), and degassed under vacuum before use. While being used,
the mobile phase was degassed with a slow stream of helium.
2.2 Preparation of aqueous/saline standards
Standard stock solution (1.0 mg/ml) of azimilide and NE-10171 (free base) were
prepared by dissolving proper amounts in a mixture of saline, methanol, and acetonitrile
(80:10:10, v/v/v). Specifically, the samples were first added into a 50 ml volumetric flasks with
5 ml methanol, 5 ml of saline, and 5 ml of acetonitrile. Then, the solution was brought to the
final volume with saline and mix well. Working standards and blood spiking solutions were
prepared from these stock solutions by subsequent dilutions with DI water or saline, respectively.
The primary NE-10133 solution (100 g/ml) was prepared by dissolving a proper amount
of NE-10133 in 50 ml volumetric flasks with 5 ml methanol (10% of the total final volume), 5
ml of acetate buffer (10% of the total final volume), 5 ml of DI water. Then, the solution was
brought to the final volume with DI water. The working NE-10133 solution (10 g/ml) was
prepared by diluting the primary NE-10133 solution with DI water. The instrument validation
solutions were prepared by diluting proper amounts of azimilide, NE-10171, and NE-10133
solutions with the HPLC mobile phase.
2.3 Preparation of blood calibration standards and quality control samples
Calibration standards and QC samples were prepared by spiking blank mouse blood with
the appropriate amount of azimilide and NE-10171 saline solutions. Separate stock solutions
were used to prepare calibration standards and quality control samples.
The blood calibration standard levels were 20, 40, 200, 3,000, 10,000, 15,000, 18,000,
19,000, and 20,000 ng/ml. The blood QC levels were 20, 60, 8,000 and 16,000 ng/ml. Which
were referred to as limit of quantitation (LOQ), low, medium, and high QC samples,
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respectively. In addition, the calibration curve also included matrix blank (matrix only) and zero
standards (matrix plus internal standard). The spiked blood standards and QC samples were
stored, along with the study samples, at –20 ºC until use. For stability evaluation, some of the
spiked high and low QC samples were stored at –70 ºC for comparison.
2.4 Preparation of blood samples
The frozen (-20 ºC) mouse blood samples, calibration standards and QC samples were
thawed in a 40 ºC water bath for 5 minutes and then allowed to come to room temperature. After
vortexing, 250 l each sample was transferred into polypropylene tubes (5 ml) with a Pipetman
digital micropipette (Rainin Instrument Co, Inc., Woburn, MA). The tubes were inserted with
swirling motion into a dry ice/acetone bath to freeze for 30 seconds and then removed and placed
into a water bath at 40 ºC to thaw for 1 min. The same freeze-thaw procedure was repeated a
second time. The internal standard (100 l) was added to each of the samples except the matrix
blank. Next, the protein precipitation solvent (3 ml) was added and the samples were vortexed
on a multi-tube vortex mixer (MTV) for 4 min. Sample tubes were inverted and vortexed
another 4 min on the MTV and then 30 seconds on a Genie vortex. The samples were then
centrifuged at 3000 g for 15 min at room temperature. The supernatants (2-3 ml) were carefully
transferred into clean amber glass vials (4 ml). The sample vials were dried either in a SpeedVac
under vacuum or in a Zymark Turbo Vap at room temperature. The dried samples were
reconstituted with mobile phase (300 l), vortexed gently, and transferred carefully into a low
volume filtration device (Z-spin) for filtration. The Z-spins were placed in an Eppendorf 5415
micro-centrifuge and centrifuged at 10,000 g for 10 min until all liquid passed through the filter
membrane. Finally, the samples were transferred to 350 l inserts, which were placed in 2-ml
wide mouth amber glass sample vials. The vials were capped and loaded onto the Gilson XL
autosampler.
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2.5 Chromatography
An Exsil octyl analytical column (5-m, 250x4.6 mm), along with a pre-column filter
containing disposable 2-micron frits, was used for chromatography at room temperature. A
200-l aliquot of each blank, spiked standard, sample, or appropriate system suitability solution
was injected. An isocratic solution at a flow rate of 1.5 ml/min was sufficient to separate
azimilide, NE-10171 and NE-10133. Chromatograms were recorded and processed by the
TurboChrom program. Data reduction was achieved using an in-house program, CustomStat,
which was based on Microsoft Excel macros. Peak areas for azimilide, NE-10171, and NE10133 were obtained from the chromatograms. The peak area ratios (azimilide/NE-10133 and
NE-10171/NE-10133) were calculated for each standard and sample.
3. Results and Discussion
The quality of analytical data is critical to the success of a drug development program.
The progress of method development and validation has a direct impact on the quality of these
data. Method development is the process to work out conditions suited for the determination of
the compounds of interest. For HPLC method, this translates into finding conditions capable of
separating and quantitating the corresponding compounds from other potential interfering
compounds. Method validation is the process of proving that an analytical method is suitable for
its intended purpose [10-11]. For HPLC method, this is to make sure that the method can meet
certain predetermined acceptance criteria for the calibration standards and the QC samples.
Since method development process has been very well documented, we will focus on the method
validation process in the following discussions.
3.1 How to validate an HPLC method
In general, analytical method involved in regulatory submission must meet all of the
criteria established in the United States Pharmacopeia (USP) [12]. These criteria include
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specificity, linearity, accuracy, precision, range, detection limit, quantitation limit, and
robustness. Usually, different approaches are used to meet these criteria for different methods.
For an HPLC-based bioanalytical method, the method validation usually requires a minimum of
three analytical batches. At least one, and preferably all of these batches must contain the
number of samples similar to the number of study samples that will be analyzed in a batch.
The first batch is the precision and accuracy (PA) batch, which assesses the precision and
accuracy of the spiked standards and the QC samples. This PA batch also checks the specificity
and sensitivity of the method. This very first PA batch contains duplicated calibration standards
and QC samples at each level. Subsequent batches contain only one set of calibration standards
and may include additional QC sample s for stability evaluation. All calibration standards are
included in the regression analysis, rejection outliners where appropriate. The criteria for
rejecting the regression standards were established in our standard operation procedures (SOPs)
and discussed later in this paper (see Linearity section). At least one more PA batch is needed to
determine the precision and accuracy, sensitivity and specificity between batches. Each of the
PA batches contains two pre-assigned QC samples at each level for between batch precision and
accuracy determination.
The last batch is the method evaluation (ME) batch, which determines the within batch
precision and accuracy, the potential of injector carryover, sensitivity, specificity, and recovery.
The ME batch contains 10 QC samples at each level and the within batch precision and accuracy
is determined using all QC samples. These QC samples can also be used to determine the
stability of the analytes on the autosampler. Two pre-assigned QC samples at each level are used
with the QC samples from the previous PA batches to determine between batch precision and
accuracy. Special attention should be given to the sequence of a few specific samples in the ME
batch. The two reagent blanks are followed by the calibration standards, which are randomly
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distributed among all of the samples, except for the six matrix blanks. These blanks are
distributed so that three blanks immediately follow the highest standard and three blanks follow
the second highest standard or the highest QC sample. The three blanks in each group are placed
in consecutive order (see Note b in Table 1).
This HPLC method was validated in multiple batch following the above procedures and
applied for the analysis of azimilide and NE-10171 in mouse blood. Table 1 lists the number of
samples in each batch for this study. The performance of this method was evaluated for
specificity, linearity, accuracy and precision, sensitivity, recovery, ruggedness, and stability of
azimilide, NE-10171 and NE-10133 based on the data obtained from these batches.
3.2 Chromatographic specificity
The chromatographic specificity of an HPLC method requires the absence of interfering
peaks and good resolution. In our study, the absence of interfering peaks required that the blank
had no interfereing peaks at the retention times of azimilide, NE-10171, or NE-10133. In
addition, the reference solution at the beginning of each new run should give baseline resolution
(resolution = 1.5) for the analytes (azimilide and NE-10171) and the internal standard
(NE-10133).
The chromatographic parameters obtained from the separation of azimilide and
NE-10171 in a system suitability solution (Fig 2) are summarized in Table 2. The resolution
between the individual analytes and the internal standard (Fig 2) was larger than 1.5 (Table 2).
Representative chromatograms (Fig 3a-c) of a matrix blank, a zero blank (spiked with internal
standard only), and a calibration standard show litter interference from the matrix blank at the
retention times of the analytes and/or the internal standard. Therefore, this method met all of the
aforementioned specificity requirements.
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3.3 Linearity
The linearity of a method is defined by the relationship between the response ratios of the
analytes to the internal standard and the analyte concentrations in the standards. The response
(peak area) of azimilide and NE-10171 to that of the internal standard were used for quantitation.
Usually, a calibration curve includes one blank (matrix only), 1-2 zero standards (matrix plus
internal standard), and one standard at each level of the calibration curve, with 1-2 standards at
both the lowest and the highest levels. Since the calibration curve covers a broad concentration
range, from 20 ng/ml to 20 g/ml for both azimilide and NE-10171, linear regression with a
weighting factor (1/x2) has to be applied to the non-zero standards. The goodness-of-fit of the
standards to the calibration curve is reflected in the coefficients of determination (r2) obtained
from the weighted regression. The average coefficients of determination (r2) were 0.998 for
azimilide and 0.996 for NE-10171 in these batches.
In addition to r2, based on our SOPs, the acceptability of these calibration curves for the
analyses of study samples was based on two criteria. First, the percent relative error (%RE) of
the individual standards computed from the linear regression curve should be less than 15%
(20% for LOQ standard). Second, the pooled % relative error (%PRE) of all calibration
standards that define the curve should be less than 10%. The %RE of the spiked standard is
defined as:
%RE 
(X - X o ) x 100%
Xo
(Eq. 1)
where X is the computed standard concentration and Xo is the nominal concentration of
the standard.
If a standard was out of the acceptable %RE range, it was rejected and the regression was
performed on the remaining standards until all of the standards met the acceptance criteria for
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individual standards. Table 3 lists the mean values of the computed concentrations of azimilide
and NE-10171 based on the regression results. It is noted that some of the batches had less than
nine acceptable standards. However, all of the curves met the requirement that at least five
different levels of standards were used to construct a calibration curve (Table 4).
The %PRE is the square root of the average of the square of individual differences. Since
one degree of freedom is lost in the square, the average is calculated by using N-1 rather than N,
i.e.
%PRE 

N 1- N
(X i - X io ) 2
(Eq. 2)
N 1
where Xi is the computed ith standard concentration and Xio is the nominal concentration
of the ith standard.
Table 4 lists the %PRE for all the batches. The goodness of fit was demonstrated by the
small %PRE (less than 6% for azimilide and less than 8% for NE-10171 for all batches). These
data demonstrated that the calibration curves were acceptable based on our SOPs.
It is worth to note that the success in obtaining the three orders of magnitude linearity is
attributed to the fact that the calibration standards are distributed within the calibration range
based on statistic errors. It is well known that the variance near the ends of the calibration curve
is usually larger than that in the middle of the calibration curve. This effect becomes significant
when the calibration range is large. The key is to control this phenomenon with proper
distribution of the calibration standards. In previous work, the calibration standards were diluted
three-fold each consecutively from the highest standard to make the calibration curve. Based on
that curve, the range between the highest and the second highest standards is more than 66% of
the full calibration range. There was no standard between these two. Any slight variation in the
highest standard will change the whole curve dramatically. Therefore, the %RE for some of the
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lower calibration standards could be 1000% even the coefficients of determination (r2) was more
than 0.999. When using that calibration curve, the best linearity we could achieve was two
orders of magnitude. Two separate calibration curves had to be used in order to cover three
orders of magnitude. By placing more standards near the ends, we were able to reduce the
variance near the ends of the calibration curve and have better accuracy and precision.
3.4 Accuracy and precision
Azimilide and NE-10171 levels in mouse blood samples were calculated from the above
spiked calibration standard curves. The relatively small %RE of the calibration standards
indicated that this method had good accuracy and precision. To further assess the accuracy and
precision of these calculations, QC samples were included in each batch. QC samples of known
concentrations were prepared from separately prepared standards. Four levels of QC samples
were used. The lowest QC concentration (LOQ QC, 20 ng/ml) was equal to the concentration of
the standard at the lower limit of quantitation (LOQ) of the calibration curve. The low QC
(LQC, 60 ng/ml) was lower than 3xLOQ but higher than the second lowest standard of the
calibration curve. At the same time, Low QC can’t be the same as any standard. The medium
QC (MQC, 8,000 ng/ml) was approximately midway between the low and high QC
concentrations. The high QC (HQC, 16,000 ng/ml) was 70-90% of the highest calibration
standard, yet lower than the second highest standard, but not the same as any standard. Further,
the LOQ QC was used only during the method validation batches while the other QC samples
were used throughout the study. If the QC samples are made in saline instead of DI water, the
corresponding HQC, MQC, and LQC will be called SHQ, SMQ, and SLOQ (Table 1). Saline is
more compatible with blood than water does.
The percent relative recovery (%REC) for QC samples is defined as:
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%REC 
Y x 100
Y0
(Eq. 3)
where Y and Y0 are the measured and the expected concentrations of the QC sample,
respectively. %REC represents the accuracy of the method when used for unknown sample
analysis. Table 5a summarizes the %RECs of QC samples pre-assigned for between batch
accuracy and precision evaluation. The %REC for 2 QC samples at each of three levels between
a total of 10 batches was very high, between 99.4% and 101.1% for azimilide, and between 88.1
and 94.3% for NE-10171, respectively. The relatively small percent coefficient of variation
(%CV) for the QC samples indicates that the recovery was quite consistent. Table 5b indicates
that within batch precision and accuracy for the three QC samples was also very high. The
%REC of the 10 QC samples at each level within the batch was between 108% and 114% for
azimilide and between 104 and 111% for NE-10171.
3.5 Sensitivity
The sensitivity of the method was determined by evaluating the precision and accuracy of
standards near the detection limit [10]. The LOQ of this method was 19.2 ng/ml for azimilide
and was 19.8 ng/ml for NE-10171. A typical chromatogram for a 20 ng/ml calibration standard
shows a peak significantly larger (S/N > 6) than the background noise (Fig 4). When control
blood was spiked with 19.2 ng/ml of azimilide and assayed, the mean was 19.75 ng/ml with a
CV% of 6.68%. Similarly, when control blood was spiked with 19.8 ng/ml of NE-10171 and
assayed, the mean was 19.8 ng/ml with a CV% of 7.1%.
3.6 Recovery and ruggedness
The absolute recovery (AR) of this HPLC method was determined by comparing the peak
area of both the aqueous and the blood samples, i.e.
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%AR 
Signal of the blood sample x 100
Signal of the aqueous sample
(Eq. 4)
The AR through the clean-up steps, estimated from external standard curves, was 56% 77% for azimilide and 49% - 59% for NE-10171 for the spiked calibration standards (Table 6).
The ARs of multiple QC samples (n=8) at the same concentration level (LOQ) were 69.09 ±
5.81, 55.62 ± 3.28, and 62.77 ± 1.34 for azimilide, NE-10171, and NE-10133, respectively.
Different analysts have repeated this method in a different lab and achieved similar
results. Therefore, the performance of this method was rugged enough to transfer to other labs.
However, as this method was applied to the analyses of mouse blood samples from in-house
azimilide studies, several different batches of columns were used. The retention times for some
of these analytes shifted a little due to column variation from batch to batch. Nevertheless, the
quality of the separation remained sufficiently high throughout this study.
3.7 Stability
As part of the method validation, we evaluated the short-term (14 hours at room
temperature) and freeze-thaw (3 cycles between –20 ºC and room temperature) stability of
azimilide and NE-10171 in mouse blood. To ensure that there was no significant change in the
stability of azimilide and NE-10171 during the course of sample analysis for this study, the longterm (182 days at –20 ºC) storage stability of azimilide and NE-10171 in mouse blood was also
evaluated. The long-term stability tests were performed at 1, 3, 9, 17, 24, 60, 99, and 182 days
with two samples at each level for each day. In addition, an autosampler (44 hours at room
temperature) for the processed sample to ensure the stability of azimilide and NE-10171 in the
reconstituted solution was also evaluated.
The data in Figure 5a-b showed that azimilide was stable over the course of this study.
NE-10171 had similar stability (data not shown). The short-term stability was evaluated by
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checking QC samples at room temperature for 0, 2, 4, 6, 8, and 14 hours. Fig 5c indicated that
azimilide low QC samples were stable under the tested conditions. The azimilide high QC as
well as NE-10171 QC samples showed similar stability. The autosampler stability was evaluated
based on the stability of the processed QC samples in the autosampler at room temperature at 0,
2, 4, 8, 12, 18, 24, 30, and 44 hours. Figure 5d showed that the azimilide low QCs were stable
during the period of the sample sitting on the autosampler. Again, azimilide high QC and
NE-10171 QC samples had similar stability. In addition, we also evaluated the freeze-thaw
stability by alternating freezing and thawing the QC samples between –20 ºC and room
temperature for three cycles and analyzing two QC samples after each cycle. The results are
within the expected range.
4. Conclusions
The method described in this paper is specific, accurate, precise, and sufficiently
sensitive for the quantitation of azimilide and its metabolite, NE-10171, in mouse blood. The
sample preparation steps involve only freeze-thaw, protein precipitation, and solvent
evaporation. All of these steps are easy to perform. These procedures are suitable for the
analysis of azimilide in mouse blood samples.
A significant achievement in this method is that a linearity of three orders of magnitude
was achieved from a single calibration curve. This distribution of calibration standards in this
calibration curve was deigned based on the statistics associated with physical measurements. By
having more calibration standards near both ends of the calibration curve, we were able to
overcome some of the problems associated with previous methods, where the calibration
standards were equally diluted three times from one level to the next. Based on these calibration
curves, the %RE of the lower standards could be off dramatically.
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In contrast, good linearity with broad calibration range can be obtained with acceptable
results for the calibration standards when they are distributed according to our calibration curve.
Since the distribution of calibration standards in our method has statistic background, it has
broad applicability.
5. Acknowledgment
The author wishes to thank Dr. N. J. Parekh and Ms. P. Fitzpatrick for their help and
suggestions in completing this work. The author would also like to thank Dr. R. M. Deibel for
her comments on this manuscript.
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Inc., Rocjville, Maryland, 1995), Chapter <1225>, pp. 1982-1984.
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Table 1
Number of analytical samples in each validation batch
Sample
Ragent Blank
PA Batch a
ME Batch b
Sample Analysis
-
2
-
3 or more
3 or more
3 or more
Matrix Blank c
1 or 2
6
0 or 1
Zero Blank c
1 or 2
1
0 or 1
Standard 1 to 10
1 or 2
1
1
HQC/MQC/LQC
4
10
2
LOQ QC
4
10
0
Subject samples
-
-
36-42
Stability: HQC, LQC
0-18
-
0-2
Total blood samples c
26-58
58
58
-
2
2d
Instrument Validation
Saline QC: SHQ/
SMQ/SLOQ
a
The calibration curve contains two standards at each level for the first PA batch and one standard at each level for
the second PA batch. Two of the four QC samples are pre-assigned for computation of between batch precision and
accuracy.
b
The six matrix blanks are to evaluate carryover. The ten QCs at each level are to assess the within batch precision
and accuracy. The two saline QCs at each level are optional for recovery purposes only.
c
The Matrix Blank and Zero Blank are only needed in the first PA batch when a new matrix is used. The number of
samples in each analysis batch may vary depending on whether the matrix, zero, and the stability samples are used.
The stability sample may vary depending on the need. The total number of blood samples in the PA batch and the
sample analysis batch may be less, but no more than, that of the ME batch.
d
These QC samples are included only if the absolute recovery information is needed.
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Table 2
HPLC parameters for the analysis of azimilide and NE-10171
Analytea
Resolution
NE-10133
Retention
Time

9.62
Tailing
Factor
Capacity
Factor
k'
Theoretical
Plates
N
---
1.144
5.212
8983.10
NE-10171
11.96
5.106
1.273
6.715
8934.59
Azimilide
14.57
4.520
1.368
8.399
8057.50
Rs
a
The parameters listed above were obtained from the system suitability solution on one specific column. There
were some migration time shift when columns from different batches were used.
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Table 3
Concentrations of azimilide and NE-10171 in the standard curve
Azimilide
Std 9a
Std 8
Std 7
Std 6
Std 5
Std 4
Std 3
Std 2
Std 1
Nominal
19.2
38.3
192
2874
9580
14370
17244
18202
19160
Mean
20.1
38.1
176
2918
10188
15363
17435
18671
20217
%RE
5.06
-0.60
-7.96
1.53
6.35
6.91
1.11
2.58
5.52
SD
0.42
1.90
8.37
63.8
113
431
586
504
358
CV%
2.10
4.99
4.75
2.19
1.11
2.81
3.36
2.70
1.77
N
9
7
4
8
10
9
10
10
10
NE-10171
Std 9
Std 8
Std 7
Std 6
Std 5
Std 4
Std 3
Std 2
Std 1
Nominal
19.8
39.7
198
2973
9911
14867
17840
18831
19823
Mean
20.5
36.7
187
2893
10152
15483
17687
18843
20142
%RE
3.55
-7.39
-5.87
-2.72
2.43
4.15
-0.86
0.06
1.61
SD
0.60
1.87
27.4
102
247
546
794
565
683
CV%
2.93
5.11
14.7
3.53
2.43
3.53
4.49
3.00
3.39
N
9
8
4
7
10
9
10
10
10
a
Std = standard
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Table 4
Pooled percent relative error (%PRE) of the standards in the calibration
curves for azimilide and NE-10171
Azimilide
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NE-10171
Batch
%PRE
N
%PRE
N
1
2
3
4
5
6
7
8
9
10
3.41
5.94
3.11
2.33
2.57
5.99
4.54
4.93
4.69
5.55
7
9
7
5
8
9
8
9
7
8
5.96
7.00
6.42
4.06
7.94
5.03
6.64
4.26
4.52
4.94
7
9
8
5
9
7
9
8
7
8
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Table 5
a) between batch precision and accuracy of QC samples for azimilide and
NE-10171
Azimilide
NE-10171
Batch
LQC
MQC
HQC
LQC
MQC
HQC
Nominal (ng/ml)
59.3
7904
15808
59.5
7928
15856
Mean (ng/ml)
59.9
7858
15790
56.1
6987
14049
SD (ng/ml)
6.70
277
445
6.52
437
775
CV%
11.2
3.52
2.82
11.6
6.26
5.52
N
20
18
20
20
18
20
%REC
101
99.4
99.9
94.3
88.1
88.6
b) within batch precision and accuracy of QC samples for azimilide and
NE-10171
Azimilide
NE-10171
Sample
LQC
MQC
HQC
LQ
C
MQC
HQC
Nominal (ng/ml)
59.3
7904
15808
59.5
7928
15856
Mean (ng/ml)
64.8
9009
17028
65.9
8778
16476
SD (ng/ml)
11.1
1220
614
5.4
1223.8
648.1
CV%
17.2
13.5
3.60
8.20
13.94
3.93
N
10
10
10
10
10
10
%REC
109
114
108
111
111
104
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Table 6
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Absolute recoveries of azimilide and NE-10171 in calibration standards
Azimilide
% Recovery
NE-10171
% Recovery
NE-10133
% Recovery
STD 9
56.2
48.0
63.4
STD 8
66.9
54.7
63.1
STD 7
65.8
53.8
63.7
STD 6
76.3
57.6
60.6
STD 5
73.2
57.4
64.0
STD 3
70.2
56.5
62.9
STD 2
71.8
58.9
63.9
STD 1
72.5
58.0
60.5
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Figure captions
Figure 1. Structures, names, formulas of azimilide, NE-10171, and NE-10133
Figure 2. A chromatogram showing the separation of azimilide, NE-10171, and the internal
standard, NE-10133 from a system suitability sample. Experimental conditions: 340
nm UV detection; mobile phase: 65% 32 mM ammonium acetate with 36 mM TEA
buffer (pH 5.0), 5% MeOH, and 30% ACN.
Figure 3. Chromatograms of a) blank mouse blood matrix, b) zero blank, and c) the calibration
standard (20,000 ng/ml) in mouse blood. Experimental conditions are the same as in Figure
2.
Figure 4. Chromatogram of LOQ of azimilide, NE-10171, and NE-10133 in mouse blood.
Sample contained 19.2 ng/ml azimilide and 19.8 ng/ml NE-10171 with internal standard
NE-10133. Insert is the whole chromatogram without expansion. Experimental conditions: the
same as in Figure 2.
Figure 5. Stability data of azimilide QC samples. The low and high limits are 2.5 standard
deviation (SD) of the nominal values as determined during the validation of the
method. a) long-term stability of low QC azimilide in mouse blood; b) long-term
stability of high QC azimilide, and c) short term stability of low QC azimilide, and
autosampler stability of low QC in reconstituted buffer, and d) autosample stability of
low QC samples. The measured concentrations of the QC samples are plotted along
with the 2SD limit lines above and below the Mean.
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Figure 1 Structures, names, formulas of azimilide, NE-10171, and NE-10133
O
Cl
CH=N
N
N
O
(CH2)4
N
(CH2)4
N
CH3
N
O
Azimilide
O
Cl
CH=N
N
N
O
N H . 2 HCl
O
NE-10171
O
Cl
CH=N
N
N
O
(CH2)
3
N
N (CH2) O H . 2 HCl
2
O
NE-10133
Azimilide:
1-[[[5-(4-Chlorophenyl)-2-furanyl]methylene]amino]-3-[4-(4-methyl-1-piperazinyl)butyl]-2,4-imidazolidinedione. Empirical formula: C23H28ClN5O3, FW = 457.97. The
commercial name Stedicor refers to azimilide dihydrochloride.
NE-10171:
1-[[[5-4 Chlorophenyl)-2-furanyl]methylene]amino]-3-[4-(4-methyl-1-piperazinyl)
butyl]-2,4-imidazolidinedione dihydrochloride. Empirical formula:
C22H26ClN5O3*2HCl. Molecular weight: 516.86.
NE-10133:
1-[[[5-(4-Chlorophenyl)-2-furanyl]- methylene]amino]-3-[3-[4-(2-roxyethyl)-1piperazinyl)propyl]-2,4-imidazolidinedione dihydrochloride. Empirical formula:
C23H28ClN5O32*2HCl. Molecular weight: 546.89.
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Figure 2
Chromatogram of azimilide, NE-10171, and NE-10133 in a system suitability
solution.
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Figure 3a Chromatogram of blank mouse blood
Figure 3b
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Representative chromatogram for a zero blank mouse blood extract
spiked with NE-10133
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Fig 3c Chromatogram of reference standard of azimilite and NE-10171 at 20,000 ng/ml
analytes along with NE-10133
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Figure 4 Representative chromatogram of LOQ azimilide and NE-1071 mouse blood
sample along with NE-10133
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Figure 5 a) Long-term stability of azimilide low QC in mouse blood
azimilide LQC
80.00
Con. (ng/ ml)
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
1
1
3
3
9
9
17
17
24
24
60
60
99
99
182 182
Days
Figure 5 b) Long-term stability data for azimilide HQC in mouse blood
azimilide HQC
Con. (ng/ ml)
20000.00
15000.00
10000.00
5000.00
182
182
99
99
60
60
24
24
17
17
9
9
3
3
1
1
0.00
Days
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Figure 5
c) Short-term stability data for azimilide LQC in mouse blood
Con. (ng/ ml)
azimilide LQC
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0
0
2
2
4
4
6
6
8
8
14
14
Hours
Figure 5
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d) Autosampler stability data for azimilide LQC in mouse blood
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