J.
CHEM. SOC., CHEM. COMMUN.,
1995
224
1
Rapid Estimation
of
Octanol-Water Partition Coefficients using Deoxycholate Micellesin Capillary Electrophoresis
Maxwell Adlard,a George Okafo,b Eugene Meenanb and Patrick Camilleri*,b
a
School
of
Biological and Health Sciences, University
of
Westminister,
I15
New Cavendish Street, London, UK
WIM
8JS
b
SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Herts, UK
AL6
9AR
Deoxycholic acid micelles have been used successfully in capillary electrophoresis to estimate the octanol-waterpartition coefficient (log
P)
f
a number
of
drugs; the methodology developed is fast and is suitable for thedetermination of this physico-chemical parameter for neutral, basic and weakly acidic molecules with a variety ofstructures.
Octanol is probably the most popular solvent used to modellipid membranes. This choice has been influenced to a largeextent by the greater solubility of most organic compounds inthe latter solvent rather than in other more hydrocarbonaceousmedia such as hexane. The hydroxy group in octanol also givesit hydrogen-bond donating and accepting properties in a mannersimilar to biomembranes. The partition coefficient
(P)
of a drugbetween water and octanol is usually expressed as the logarithmof the ratio
of
its equilibrium concentrations in octanol andwater.l.2 Log
P
s determined using the ‘shake-flask’ techniquewhere a solute is allowed to distribute itself between the twophases of octanol (saturated with water) and water (saturatedwith octanol) before analysis.Although the above procedure for the determination of log
P
can give accurate data, the methodology is both laborious andtime consuming, and the measurement of this parameter for onesingle drug can take more than one day. It is often the case indrug design that speed is more important than absoluteaccuracy. Until recently, reversed-phase high performanceliquid chromatography (HPLC) has been the only indirectapproach that allows a fast estimation of the lipophilicity oforganic molecules.3-5 This technique has been most successfulin determining log
P
values of a set of compounds by comparingcapacity factors with those
of
a structurally related sub-set(usually three or four compounds) whose log
P
has beenpreviously determined by the traditional
‘
hake-flask’ method.In the case of unrelated compounds HPLC leads to veryinaccurate estimations of log
P
values mainly due to variation inthe behaviour of these molecules in their interaction with free(uncapped) silanol groups of the stationary phase.The introduction of micellar electrokinetic capillary chro-matography (MECC),6 one of the modes of operation ofcapillary electrophoresis (CE), has provided other opportunitiesto characterise the hydrophobicity of organic molecules.Recently we have demonstrated7 that using sodium dodecylsulphate
(SDS)
micelles, the capacity factor of a number ofanalytes was statistically and significantly related to theircorresponding log
P
values. Our work was followed by twoother publications on the same subject again using either
SDS
alone8 or an emulsion consisting of this surfactant and a mixtureof butanol and hexane.9
In
the case of
SDS
alone the behaviourof
a
number of compounds containing polar moieties such asnitro- and keto-substituents is anomalous; these moleculesmigrate slower than the corresponding unsubstituted com-pounds. The introduction of butanol corrects for these anom-alies, and may be due to the formation of smaller micellescontaining absorbed alcohol.9Here we show that the capacity factors
(k’)
of a number ofsolutes obtained in MECC using deoxycholic micelles can becorrelated with the corresponding octanol-water partitioncoefficients. This technique is fast, reproducible and avoidscomplications arising from the addition of organic solvents tothe surfactant in the separation buffer.Under the conditions (ionic strength and bile salt concentra-tion) of the present study, bile salts are expected to form smallprimary micelles with an aggregation number of about
3
to
4
monomers. The micellar shape has been postulated toconsist of monomers packed back-to-back with the hydrophobichydrocarbon moiety forming the interior of the aggregate andthe hydrophilic hydroxy groups in contact with the surrounding
Table
1
Literature octanol-water log
P
values and capacity factorsa determined in bile salt micellesCapacity factor
(k’)
Capacity factor
(k’)
Capacity factor
(k’)
Octanol-Water 40 mmol dm-3
DCb
40 mmol dm-3 DCb 40 mmol dm-3 GDCaStandard compounds Log
P
PH 8 PH 9 PH 9benzy lalcoholphenolacetophenonenitrobenzenebenzeneanisolep-nitro-methy benzoateo-toluonitrilem-nitro-toluenetoluene6-naphtholbromobenzenebenzophenonep-xylene1 nitronaphthalenephen ylphenolnaphthalenebu ty lbenzene1.101.461.631.852.132.112.062.082.372.732.842.993.183.153.193.203.304.260.140.2
1
0.340.330.390.500.570.6
1
0.951.362.032.614.114.465.355.027.6431.760.140.280.350.360.420.550.590.641.011.462.682.744.674.474.225.367.2019.870.130.250.290.870.360.460.580.524.921.202.022.343.313.7113.884.395.6916.88
0
Electrophoresis conditions as in Fig. 1 legend.12-a-hydroxy-24-oic acid, respectively.
DC
and GDC represent the potassium
salts
ofdeoxycholic acid and 3-~-D-glucopy- anosyl-5-f3-cholan-
D o w n l o a d e d b y U n i v e r s i t y o f M i s s o u r i - C o l u m b i a o n 1 9 S e p t e m b e r 2 0 1 0 P u b l i s h e d o n 0 1 J a n u a r y 1 9 9 5 o n h t t p : / / p u b s . r s c . o r g | d o i : 1 0 . 1 0 3 9 / C 3 9 9 5 0 0 0 2 2 4 1
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2242
J.
CHEM. SOC., CHEM. COMMUN.,
1995
aqueous medium.10.12 The interaction of water with thesemicelles bears some semblance to that of either octanol-waterpartitioning
or
of
SDS
micelles in the presence of butanol.9Using deoxycholic acid and an (3-glycosylated f0d3of thisbile acid we have measured the capillary electrophoreticmigration
of 35
analytes, made up
of
18
simple organicmolecules and
17
drug substances. The
k’
values werecalculated from migration times by eqn.
(1),6
where
tr, to
and
(1)
tr
-
oto
(1
-
r/trnc>
I%=
t,,
are the migration times of the analyte, the electroosmoticfront and the micelle, respectively. The value of
to
wasmeasured from the migration time of methanol which
is
excluded from the micelle whereas
tmc
was measured from themigration time of the antimalarial drug, halofantrine whichcompletely associates with micelles.
14
As
a matter
of
interestwe calculated (Pomona Software) the log
P
value for halofan-trine. The high value of
8.3
obtained again justified its use as amarker for
t,,.
Capacity factors determined for the
18
standard compoundsunder different conditions are presented in Table
1.
Fordeoxycholic acid micelles
k’
values are similar at pH’s
8
and
9,
despite the fact that some of the analytes are weak acids withexpected pKa values around
9.
Moreover under both conditionsthe order
of
migration of these analytes is the same and overall,
1
0
‘a
m
-
-1
-2
,&
‘-0
0
1
2 3
4
Fig.1
Correlation of octanol-water log
P
with log
k’
for 32 analytes
(0,
standard compounds and
0,
rug substances). MECC conditions as in Fig.2, except that separation buffer was carried out at pH
8.
Log
P
k’
increases with the corresponding octanol-water partitioncoefficient. It is noteworthy that, unlike the case
of SDS
micelles,7*8 nitro- and keto- derivatives migrate before theunsubstituted solutes, indicating that partitioning across de-oxycholate micelles occurs predominantly by hydrophobic
0.020
r
(a)
-
.016
E
1
g0.012
,
3,4
a,
tl
d
D
>
3
0.004
0.000
I
4.0
5.5
7.0
8.5
10.0
11.5 13.0
14.5 16.017.5
19.0
0.030
c
(b
0.0250.020
cu
v
i7
2
14
-
4.05.57.0
8.5
10.0 11.5 13.0
14.5
tlmin
Fig.
2
Electropherograms of a number drug substances analysed usingmicelles made up of
(a)
DC and
(h)
GDC:
(1) acetaminophen, (2)mephenytoin, (3) lidocaine, (4) cromakalim
(5)
olbutamide, (6) warfarin,(7) dexamethasone, (8) ropinirole, (9) testosterone, (10) nabumetone,
(1
1)
prazepam, (12) fluoxetine, (13) progesterone, (14) halofantrine, (1
5)
sumatriptan, (16) fenoldopam and (17) propranolol. Buffer consisted of 50mmol dm-3 boric acid adjusted
to
pH 9 with potassium hydroxide andcontaining 40 mmol dm-3 bile salt; capillary, 50 mm i.d.
X
57 cm (50 cmto detector); voltage, 22
kV;
etection, 214 nm; 25 “C. Samples
(
10-3-10-5mol dm-’), dissolved in aqueous methanol or a 4% aqueous solution of bilesalt, were applied by pressure injection for
1
s.
Table
2
Comparison of measured or calculated log
P
values with estimates obtained with deoxycholate micellesBiological MECC at pH 8.0 MECC at pH 9.0 Octanol-Water
Drug
indication log
P
log
P
log
P
acetaminophen analgesic 1.6 1.7 0.510mephenytoin anticonvulsant 1.9 1.9 1.74sumatriptan antimigraine 2.4 2.3 0.93warfarin anticoagulant 2.4 2.4 2.52dexamethazone antiinflammatory 2.6 2.6 2.58ropinirole antiparkinsonian 2.6 2.8 2.5
1
nabumetoneantiinflammatory
3.5
3.4 3.08tolbutamide antidiabetic 2.4 2.4 2.34testosterone androgen 3.3 3.3 3.32lidocaine anesthetic 2.1 2.1 2.26fluoxetine antidepressant 3.7 3.6 4.05progesterone progestogen 3.8 3.8 3.87cromakalim antihypertensive 2.1 2.1 2.32fenoldapam renal blood flowenhancer 2.5 2.2 2.39propranolol antihypertensive 3.4 3.3 3.56prazepam antidepressant 3.5 3.5 3.73
D o w n l o a d e d b y U n i v e r s i t y o f M i s s o u r i - C o l u m b i a o n 1 9 S e p t e m b e r 2 0 1 0 P u b l i s h e d o n 0 1 J a n u a r y 1 9 9 5 o n h t t p : / / p u b s . r s c . o r g | d o i : 1 0 . 1 0 3 9 / C 3 9 9 5 0 0 0 2 2 4 1
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