Zastosowanie elektroForezy kapilarnej do analizy potencjalnych

Zastosowanie elektroForezy kapilarnej do analizy potencjalnych

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:ASTOSOWANIEELEKTROFOREZYKAPILARNEJ
DOANALIZYPOTENCJALNYCHRADIOFARMACEUTYKÌW
DOWCZESNEJDIAGNOSTYKICHOROBY!LZHEIMERA
#APILLARYZONEELECTROPHORETICANALYSISOFRADIOPHARMACEUTICALS
USEDINEARLYDIAGNOSISOF!LZHEIMERlSDISEASE
+AMILA0ADKOWSKA0AWEŒ3ZYMAÊSKI%L˜BIETA-IKICIUK†/LASIK
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5NIWERSYTET-EDYCZNYW|ODZI
Abstract
Streszczenie
A new capillary zone electrophoresis (CZE) method for
determination of compounds that are likely to become radiopharmaceuticals used for early diagnosis of Alzheimer’s disease was developed. These compounds are tacrine
and hynic analogs. Agilent Technologies CE, equipped
with a diode array detection (DAD) system, automatic
injector, along with ChemStation software were used for
tacrine analogs analysis. Studies were carried out in order
to optimize measurement conditions. The method of compound determination was optimized. The best results were
obtained when: fused-silica capillary tubing was 48.5 cm
long (40 cm to the detector) with i.d. of 50 μm, the background electrolyte (BGE) was phosphate buffer (pH 2.6,
50 mM), the separation voltage was 20 kV, the capillary
temperature was 25°C, samples were injected hydro-dynamically for 5 s (with injection pressure 25 mbar) and
detection was carried out at 240 nm (tacrine hydrochloride) and 248 nm (tacrine and hynic analogs). In aqueous
solutions, the detection limits (S/N=3) were 5.7 μg/ml, 6.9
μg/ml, 3.7 μg/ml, and 3.8 μg/ml for 6a, 6b, 6c, and 6d,
respectively. The quantification limit (S/N=3:10) obtained
in aqueous solutions was 16.1 μg/ml for 6a, 16.8 μg/ml for
6b, 11.0 μg/ml for 6c and 11.2 μg/ml for 6d. The method
is suitable for a routine pharmaceutical quality control
analysis of the potential radiopharmaceuticals for early
diagnosis of Alzheimer's disease.
Opracowano i zoptymalizowano nową metodę analityczną z wykorzystaniem strefowej elektroforezy kapilarnej
(CZE) do oznaczania jakościowego i ilościowego czterech
radiofarmaceutyków będących potencjalnymi substancjami diagnostycznymi do wczesnego wykrywania choroby
Alzheimera. Związki te są analogami takryny i hynica.
Do optymalizacji i walidacji metody wykorzystano zestaw elektroforezy kapilarnej CE zaopatrzony w detektor
diodowy (DAD) i automatyczny system nastrzyku próby.
Metoda oznaczania związków została zoptymalizowana.
Najlepsze rezultaty otrzymano stosując następujące warunki: kapilara ze stopionej krzemionki pokrytej otoczką poliamidową, długość całkowita 48,5 cm (długość do
detektora 40 cm), średnica wewnętrzna 50 μm. Detekcja
oznaczanych analogów oraz chlorowodorku takryny miała miejsce przy 240 nm (chlorowodorek takryny) i 248 nm
(analogi takryny). Ostatecznie do oznaczeń analitycznych
wybrano 50 mM bufor fosforanowy o pH 2,6. Podczas
analiz temperatura kapilary wynosiła 25ºC a wartości
przykładanego napięcia 20 kV. W trakcie prowadzonych
badań stosowano iniekcję hydrodynamiczną (czas nastrzyku 5 s, wartość przykładanego ciśnienia 25 mbar).
Określono liniowość uzyskując informację, że metoda jest
liniowa w zakresie stężeń: 0,01 - 0,05 mg/ml dla związków
6a, 6b, 0,01 – 0,06 mg/ml dla związku 6c, 0,02 – 0,08 mg/
ml dla związku 6d. Dla badanych związków otrzymano
następujące wartości stężeń odpowiadające granicy wykrywalności: Związek 6a – 5,7 μg/ml, Związek 6b – 6,9
μg/ml, Związek 6c – 3,7 μg/ml, Związek 6d – 3,8 μg/ml.
Otrzymano następujące wartości stężeń odpowiadające
granicy oznaczalności dla badanych substancji: Związek
6a – 16,1 μg/ml, Związek 6b – 16,8 μg/ml, Związek 6c
– 11,0 μg/ml, Związek 6d – 11,2 μg/ml. Udowodniono, że
metoda ta może być z powodzeniem wykorzystywana do
rutynowych analiz jakościowych potencjalnych radiofarmaceutyków będących analogami takryny.
Keywords: capillary zone electrophoresis, CZE, Alzheimer’s disease, tacrine analog, hynic analog, radiopharmaceutical
Słowa kluczowe: strefowa elektorforeza kapilarna, CZE,
choroba Alzheimera, analog takryny, analog hynic’a, radiofarmaceutyki
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Introduction
Materials and methods
Alzheimer’s disease (AD) was first mentioned in 1906,
when the German psychiatrist and neuropathologist Alois
Alzheimer gave a speech presenting the clinical symptoms
and pathology of presenile dementia observed in one of his
patients – a 51 year old woman. This was the first time AD
was described. Later Emil Kraeplin (head of the psychiatric
division in Munich University) named that type of dementia
after Alzheimer [1].
The disease currently affects nearly 15 million people
(mainly elderly). Prognosis shows that in 2025 there will
be about 22 million sufferers (which is mostly the effect
of prolonged life expectancy) and by 2050, the amount of
patients will have quadrupled. It is assumed that AD affects about 5 to 10% of society and is the main dementia
type among people above 65 years of age [2]. For the
last two decades it has been observed observe significant
progress in the effective, but symptomatic treatment of
Alzheimer disease [3]. The earliest observable symptoms
are often mistakenly thought to be ‘age-related’ concerns,
or manifestations of stress [4]. Therapy is mainly based
on drugs that increase the level of acetylcholine, or drugs
that improve nerve conductivity [5]. Acetylcholinesterase
inhibitors (IAChE) play the main role in AD treatment
[6, 7].
Studies on the role acetylcholine plays in the learning
and remembering process lead to creation of cholinergic
hypothesis of the disease. The hypothesis assumes that
loss of cholinergic stimulation of brain neurons is one of
the causes of the disease [8]. Thus one of the symptomatic
treatment methods is the inhibition of acetylcholinesterase
to increase the amount of acetylcholine in the synaptic
gap.
Early diagnosis is especially important in the treatment
of Alzheimer’s as an early treatment can, to some extent,
slow down the development of the disease, thus, prolonging
the mental agility of the patient. Having knowledge about
cholinergic theory we can design bifunctional compounds
used as drugs for AD. Moreover, those compounds, after the
introduction of radioactive technetium, can be used for early
diagnosis of this disease. Radiopharmaceuticals that were
developed display particles derived from tacrine – which is
responsible for binding with the enzyme (acetylcholinesterase), and particles derived from hynic (containing radioisotope).
Compared to tacrine they are more selective for AChE
and less selective for BChE. Specifically compounds 6a and
6d show high AChE affinity in enzymatic studies [9].
Those compounds (i.e. 6a, 6b, 6c and 6d) are radio-labelled with 99mTc and given to the patient; then scintigraphic methods are used for imaging and early diagnosis. The
next stage in compound research is establishing analytical
methods that are best suited for qualitative and quantitative
studies. For this purpose analysis using capillary electrophoresis has been chosen, and such analytical technique is
gaining importance in routine-analysis as well as in R&D
laboratories. Further, this technique has proven to be an
excellent choice for the determination of compounds 6a,
6b, 6c and 6d.
Reagents
Tacrine hyrochloride and tacrine and hynic analogs:
6-hydrazino-N-[2-(1,2,3,4-tetrahydroacridin-9-ylamino)
ethyl]nicotinamide hydrochloride later called 6a, 6-hydrazino-N-[8-(1,2,3,4-tetrahydroacridin-9-ylamino)propyl]nicotinamide hydrochloride later called 6b, 6-hydrazino-N-[8(1,2,3,4-tetrahydroacridin-9-ylamino)heksyl]nicotinamide
hydrochloride later called 6c, 6-hydrazino-N-[8-(1,2,3,4tetrahydroacridin-9-ylamino)octyl]nicotinamide hydrochloride later called 6d.
We have synthesized these compounds at the Medical
University in Łodz [9]. Tacrine hydrochloride 9-Amino1,2,3,4-tetrahydroacridine hydrochloride hydrate (THA)
used in the experiment at the University of Łódź was of
analytical grade (Aldrich). Other reagents used: sodium
hydroxide 0.1 M and sodium hydroxide 1 M, acetic acid,
acetone, anhydrous ethanol 99.8% were of analytical grade
(Poch Gliwice), fosforic acid (V) 85.7 % was of analytical grade (Fluka). Solutions were prepared with deionized
water.
Apparatus
An Agilent Technologies CZE system DE 01602844
equipped with a diode array detection (DAD) system, automatic injector, thermostating column cartridge and ChemStation software was used in all our experiments. Fusedsilica capillary type G1600-60232, tubing 48.5 cm long
(40 cm to the detector) with i.d. of 50 μm was used. Spectrophotometric measurements were done on a UV-Vis Carry
100 spectrometer Bio Varian. A pH-meter Metrohm 744 and
Metrohm pH sensitive, combined glass electrode were used
for the pH measurements.
CE measurements and procedure
CZE separations were carried out at 25ºC. The solution of a 50 mM phosphate buffer used throughout this
study, was prepared daily, filtered through 0.22 μm nylon
filters and submitted to Ultrasonic Cleaner (Ultron). The
pH was adjusted with diluted NaOH and/or HCl solutions.
The capillary inlet and outlet vials were replenished after
every three injections. The capillary was washed daily for
15 min. with 0.1 M NaOH, 15 min. with deionized water
and 15 min. with run buffer. In each analysis prewash of
5 min. with 0.1 M NaOH and 2 min. with background
electrolyte (BEG) was used. Absorbance was monitored
at 220 nm. In the preliminary experiments electrokinetic
injection (20 s, 10 kV) was used, then hydrodynamic injection 25 mbar, 5 s was applied in the rest of the experiments.
Sample preparation
Solutions of examined compounds 6a, 6b, 6c and 6d were
prepared using deionized water. Samples were dissolved
with the help of an ultrasonic bath and filtered through 0.22
μm nylon filters. Solutions were prepared on the day of the
experiments.
&ARM0RZEGL.AUK
Fig. 1. CE analytical method. development and optimisation.
Results and calculation
Capillary electrophoresis (CE) is a dynamically developing analytical technique, which plays a more and more
important role in a pharmaceutical laboratory. Fundamental
advantages of CE are: very high efficiency, short analysis
time, ecological character, ease of automation, requirement
of minute amounts of sample. The latter is especially important when the tested compound synthesis is time-consuming
and expensive.
Before starting analysis that leads to the development of
an analytical method that uses CE for a new compound one
should know the solubility of the sample (it is most convenient if the sample is water/buffer soluble, or low conductance solution soluble).
The next step is the determination of the ionic character of the sample. Once this is established one can start the
analysis form using:
- phosphate buffer or acetate buffer, concentration about
25 mM, pH about 2.5 for cations
- borate buffer, concentration about 25 mM, pH about 9.4
for anions
- borate buffer, concentration about 25 mM, pH about 9.4
with addition sodium salt addition for neutral ions
After selecting the base buffer, it should be decided what
CE type to use. If the compound can be determined with
the use of zone CE one should decide what kind, length,
i.d., and temperature of capillary to use; other factors left
to determine are: voltage and injection type. If peaks are
separated correctly (i.e. have good symmetry and sufficient
resolution) it can be concluded that parameters are optimal.
Otherwise modifications to buffer parameters and optimizations of hardware-related parameters should be performed.
Development and optimisation of CE analytical method are
shown schematically in [Fig. 1].
The molecular structures of compounds being analysed
is shown in [Fig. 2]. The absorption spectra of the compounds studied are presented in [Fig. 3]. Detection at 240
nm for THA and 248 nm for other studied compounds were
respectively used as the most sensitive wavelengths for the
determination.
CE analyses were started by determining environment in-
Fig. 2. Molecular structure of the drugs for early diagnosis
of Alzheimer's disease.
For given n equal:
n=2 - Compound 6a
6-hydrazino-N-[2-(1,2,3,4-tetrahydroacridin-9-ylamino)
ethyl]nicotinamide hydrochloride
n=3 - Compound 6a
6-hydrazino-N-[2-(1,2,3,4-tetrahydroacridin-9-ylamino)
propyl]nicotinamide hydrochloride
n=6 - Compound 6a
6-hydrazino-N-[2-(1,2,3,4-tetrahydroacridin-9-ylamino)
heksyl]nicotinamide hydrochloride
n=8 - Compound 6d
6-hydrazino-N-[8-(1,2,3,4-tetrahydroacridin-9-ylamino)
octyl]nicotinamide hydrochloride
Fig. 3. Absorption spectra of the radiopharmaceuticals for
early diagnosis of Alzheimer's disease.
(b) – 6a compound 23.5 ppm,
(c) – 6d compound 24.5 ppm in aqueous solution.
fluence on electrophoretic mobility of examined compounds.
Measurement was oriented towards finding optimal pH and
concentration of background electrolyte i.e. conditions under
which THA gives high, clear signal with correct symmetry.
For that purpose two series of electropherograms were prepared – one for phosphate and one for acetate buffer (which
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Tab. I. Statistical parameters of linear regression obtained with least-square method
6a
1186.679
12.4086
-2.4497
0.4115
0.9986
Slope
Slope standard deviation
Intercept
Intercept standard deviation
Linear correlation coefficient R2
6b
888.5603
7.4947
-1.9274
0.2585
0.9991
6d
THA
6a
10
A[mAU]
8
6
4
2
0
-2
0
2
4
6
8
10
time [min]
Fig. 4. The analysis of mixture THA 25.4 ppm, 6a compound 52.2 ppm, 6d compound 78.4 ppm. Conditions: fused-silica capillary tubing 48.5 cm long (40 cm to
the detector), i.d. of 50 mm, the background electrolyte (BGE): phosphate buffer
(pH 2.6, 50 mM), the separation voltage: 20 kV, the capillary temperature 25°C,
samples were injected hydrodynamically for 5 s (injection pressure 25 mbar).
6a compound repeatability
14
12
A[mAU]
10
8
6
4
2
0
0
2
4
6
time [min]
Validation
Validation was performed with
respect to specificity, linearity, range,
accuracy, the limit of detection
(LOD), limit of quantitation (LOQ),
and repeatability.
Specificity
Water used as a solvent eliminated the necessity of analysis of
its influence on quality of determination. Compounds 6a, 6b, 6c and
6d show similar electrophoretic
mobility and have similar structure.
In analytical conditions that were
checked they were separated with
good resolution [Fig. 4]. Stability
tests under long-term and accelerated storage conditions were carried out, and all four compounds
were well separated.
This method shows a significant
degree of specificity and good selectivity in separation.
16
-2
6d
1202.047
9.9879
-0.1172
0.5673
0.9989
were used as background electrolytes
(BGE)). When the buffer was chosen
next step was finding optimal pH and
electrolyte concentration. Analyses
were performed for phosphate buffer
pH from 2.2 to 3.2 and concentration
from 10 mM to 100 mM. The best
results were obtained when: fusedsilica capillary tubing was 48.5 cm
long (40 cm to the detector) with i.d.
of 50 μm, the background electrolyte
(BGE) was phosphate buffer (pH 2.6,
50 mM), the separation voltage was
20 kV, the capillary temperature was
25°C. Samples were injected hydrodynamically for 5 s (with injection
pressure 25 mbar).
14
12
6c
1065.134
6.7458
-0.7706
0.2685
0.9995
8
10
Fig. 5. Repeatability study of the migration time and normalized peak areas for 6a
compound 50.8 ppm. Conditions: fused-silica capillary tubing 48.5 cm long (40 cm
to the detector), i.d. of 50 μm, the background electrolyte (BGE): phosphate buffer
(pH 2.6, 50 mM), the separation voltage: 20 kV, the capillary temperature 25°C,
samples were injected hydrodynamically for 5 s (injection pressure 25 mbar).
Linearity and calibration curves
Calibration curves were obtained as a result of measurements.
The curve for aqueous solution were
evaluated across 0.01 – 0.05 mg/ml
for 6a and 6b compounds, 0.01 – 0.06
mg/ml for 6c compound and 0.02
– 0.08 mg/ml for 6d compound.
&ARM0RZEGL.AUK
6a
6b
6c
6d
œlepa
14
12
Absorbancja [mAU]
10
8
6
4
2
LOD and LOQ
For LOD determination several solutions with low concentrations were used. The aim was
finding concentration corresponding to signal-to-noise ratio
3:1. The low detection limits in
the aqueous media were 5.7μg/
ml (6a), 6.9μg/ml (6b), 3.7μg/
ml (6c), 3.8μg/ml (6d). The limit
of quantitation was estimated
by establishing the amount of
analyte, which gave a signalto-noise ratio 10:1. The limit of
quantitation in aqueous media
was: 16.1μg/ml (6a), 16.8μg/ml
(6b), 11.0μg/ml (6c), 11.2μg/ml
(6d) [Fig. 6].
0
Conclusions
-2
During the development of the
optimal analytical method for the
Fig. 6. LOQ electropherograms 6a, 6b, 6c, 6d compounds. Conditions: fused-silica determination of tacrine hydrocapillary tubing 48.5 cm long (40 cm to the detector), i.d. of 50 mm, the background chloride and derivatives a number
electrolyte (BGE): phosphate buffer (pH 2.6, 50 mM), the separation voltage: 20 kV, of parameters were determined:
the capillary temperature 25°C, samples were injected hydrodynamically for 5 s (in- kind of background electrolyte
(BGE), its concentration, pH,
jection pressure 25 mbar).
capillary type and length, applied
voltage, capillary temperature and
Linear relation between concentration and peak area was type of injection. Conditions were optimized; thus proving
observed. Parameters obtained with the use of linear regres- CE to be an easy, sensitive, fast and reliable method for the
sion (least squares method) are shown in Table I, in which, a determination of compounds 6a, 6b, 6c and 6d in aqueous
solutions.
very strong linear relationship is indicated.
0
2
4
6
8
10
Czas [min]
Accuracy
Accuracy of the method was demonstrated as percent
recovery in nine samples at three different concentrations
(three samples at each concentration). The percent recovery of 6a compound was found to be between 96.4% and
97.7% (R.S.D.=0.44%) at 0.0291 mg/ml level sample, recovery of 6b compound was found to be between 97.0% and
98.6% (R.S.D.=2.84%) at 0.0264 mg/ml level sample, recovery of 6c compound was found to be between 96.5% and
99.9% (R.S.D.=0.47%) at 0.0224 mg/ml level sample and
6d compound was found to be between 97.5% and 101.4%
(R.S.D.=1.26%) at 0.0179 mg/ml level sample.
Precision
The precision was determined by measuring repeatability of relative migration times and normalized peak areas
for 6a 6b, 6c and 6d compounds in order to determine the
repeatability of the method, replicate injections (n=6), 6a
[Fig.5], 6b, 6c and 6d compounds samples. The relative
standard deviations (R.S.Ds) were calculated for the migration time and the peak area ratio. R.S.D. was better than
1% for both the migration time and the peak area ratio.
Typical variations including different days, analysts and
equipment were studied and confirmed intermediate precision.
Praca współfinansowana przez Nycomed Pharma Sp. z o. o.
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data otrzymania pracy: 19.05.2010 r.
data akceptacji do druku: 27.09.2010 r.
Adres do korespondencji:
Kamila Padkowska
ul. Księstwa Łowickiego 12
99-420 Łyszkowice
tel. 0 503 355 758
e- mail: [email protected]