pKa PRO™ System
Overview
Disruptive Technologies Product Range and
Manufacturers Represented
•
Biolgical Sample Preparation
–
•
–
–
•
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Dissolution baths, friability and
disintegration instruments (Distek)
Physico Chemistry
HT Log P and pKa analyzer (AATI)
Kinetic solubility instruments
(Analiza)
Preparative
–
Nucleic acides, protein and small
molecules extraction from hard-tolyse tissue samples (Pressure
Biosciences)
Dissolution/Formulation
–
•
•
–
•
Analytical
–
–
–
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Rapid microbiology with MicroPRO
–
(AATI)
–
•
Service
–
Agilent Channel Partner to service
their range of HPLC, UV
spectrophotometers and CE
systems
OPLC chromatography solutions for
semi preparative applications
(OPLC systems, pumps, sample
applicator, video imaging and
densitometry instruments, reagent
sprayer (OPLC-NIT)
Flash chromatography (Gyan)
Automated SPE system (HTA)
–
–
HT oligonucleotides purity analyzer
(AATI)
HT proteins analyzer (AATI)
HT DNA analyzer (AATI)
HT Chiral analyzer (AATI)
Spotter for MALDI and tissue MALDI
imaging (SunChrom)
Type-C silica hydride HPLC
columns Flat sorbent beds for OPLC
(MicroSolv)
Accessories and consumables for
CE and HPLC (MicroSolv)
Validation kits for HPLC systems
(MicroSolv)
Outline
• Background and Importance of Measuring pKa Values
• Overview of pKa PRO™ Technology
• Measurement of Aqueous pKa Values
• Cosolvent pKa Extrapolation of Aqueous Insoluble Compounds
• Log P Measurements
• Chiral Separations
• Summary
• Literature References
pKa Values (Acid Dissociation Constants)
• Many drugs are either weak acids or weak bases
• The pKa value is a measure of the ionization ability of a weak acid or base:
HA  H+ + ApH = -log [H+]
Ka = [H+][A-] / [HA]
pKa = -log Ka
pKa = pH – log ([A-] / [HA])
• From the above relationship, it is observed that the pKa value = the pH at
which 50% ionization has occurred ([A-]/[HA] = 1; log 1 = 0)
• Ka is an equilibrium constant; the time scale of this equilibrium is much faster
than the separation process so the compound appears as a single peak
Why is pKa Important?
•
From 80% - 95% of commercial drugs are ionizable by some estimates
•
The pKa value of a compound strongly influences its solubility, ability to
permeate cell membranes, complexation to drug targets, and bioactivity
•
The pKa value is of fundamental importance in early discovery and
development processes for:
 Prediction of ADMET (absorption, distribution, metabolism, excretion, toxicity)
 Assessment of potential challenges in formulation/process development
 Prediction of chromatographic/electrophoretic separation behavior
Earlier assessment of drug physicochemical properties (pKa, log P,
solubility, permeability) helps to reduce compound attrition rates
and shorten development times
Biologically Relevant pH Range for Pharmaceutical
Products
pharmaceutical products
stomach
small intestines
colon
urine
0
2
4
cola
vinegar
orange
juice
6
milk
blood
8
10
12
bleach
How pKa Affects Membrane Permeability
BH+
B + H+
B
• The neutral form (B) of an ionizable drug generally has a higher lipophilicity and membrane
permeability as compared to the ionized form (BH+)
• The neutral form (B) of an ionizable drug is most always of lower aqueous solubility
Common Issues Encountered During pKa Measurements
•
Limited amounts of sample available
 Traditional potentiometric methods require mg amounts of pure compound
•
Purity and/or stability of sample has not been precisely evaluated
 Traditional methods provide “batch” analysis of entire sample and cannot resolve
individual components
•
Relatively low aqueous solubility
 Traditional methods require relatively high sample concentrations, leading to
compound precipitation
•
Number of ionizable groups in pH range of interest unknown
 UV spectrophotometric methods are structurally sensitive and may miss pKa values
for ionizable groups 2-3 bonds or more from chromophore
Capillary Electrophoresis (CE) Technology Overview
-
+
Bulk Flow: EOF + Vacuum
-
+
+
N
-
N
UV
Time
•
Charge-based separation by application of high voltage across capillary filled with
aqueous-based buffer
•
Narrow bore, bare fused silica capillaries (75 m i.d., 200 m o.d.)
•
Electroosmotic flow (EOF) provides bulk flow towards cathode (detector) at pH > 4
•
Application of vacuum provides bulk flow to detector at all pH values
•
Migration time depends on analyte charge-to-mass ratio; neutral compounds migrate with
bulk flow
•
Many publications dating back >15 years describe single capillary CE for the
measurement of compound pKa values
Measuring pKa by Capillary Electrophoresis (CE)
Bases
+
Acids
N
Acid/Base
N
+
N
Low pH
Time
+
N
Time
Time
N
-
N
Intermediate pH
Time
N
Time
N
Time
-
N
-
High pH
Time
Time
• Neutral marker (DMSO) is added to sample
• Plot of migration time difference vs. pH yields titration curve
• pKa value corresponds to inflection point of titration curve
Time
Key Advantages of CE for Measuring pKa
•
Often only small quantities (mg) of relatively impure compounds are
available in early discovery
•
CE Approach:
 Requires only small amounts of material (g range – only ng consumed)
 Sample purity not as critical (CE is separation technique)
 Measurement of migration time (ionic mobility) vs. pH (intuitive)
 No spectral differences between ionic and neutral species required; only UV
absorbance at low UV wavelength
 Sparingly soluble compounds can be investigated with aqueous buffers
 Knowledge of sample concentration not required
• However, conventional single capillary instruments can only
analyze a few samples/day and do not have software for pKa
data analysis
pKa PRO™ System
•
A dedicated 24 or 96-channel CE system developed for performing rapid pKa
measurements
•
Simple user interface and predefined CE methods for ease-of-use and
streamlined operation
•
Advanced, fully integrated data analysis software for pKa calculation and report
generation
•
Designed with feedback from scientists directly involved in
pharmaceutical research and pKa analysis
Principles of pKa PRO™ Operation
•
•
•
•
•
•
•
24 or 96 capillaries are arranged in a linear array at detection window
UV light is passed through capillary array and imaged onto photodiode array detector
Capillary inlets are arranged 8 x 12 for direct sample injection from 96-well micro plates
Capillary outlets are bundled and connected to a syringe pump for buffer filling
Different pH buffers are injected into capillary array prior to CE separation
24 or 96 individual CE-UV separations are performed in parallel
Four samples can be analyzed over 24 pH values in a single experiment
pKa PRO™ System Specifications
Sample Throughput:
pKa PRO™ 96XT
12 compounds/h for aqueous 24-point pKa measurement
pKa PRO™ 24HT
3 compounds/h for aqueous 24-point pKa measurement
Detection:
UV absorbance at 214 nm; other wavelengths available
Detection Sensitivity:
5 g/ml (ppm) depending on chromophore; working concentration 50 g/ml
Sample Required:
Working volume 50 l/well; 24 wells per 24 pH analysis (< 100 g)
Sample Format:
DMSO concentration < 0.2% (v/v); higher DMSO concentrations tolerated at
higher wavelength
pKa Measurement Range:
1.8 – 11.2
Software:
Proprietary pKa PRO™ software for system control/data analysis
Data Export Format:
Microsoft® Excel spreadsheet
Environmental Conditions:
Indoor use, normal laboratory environment; lab temperature 15–25º C
Relative Humidity Range:
< 80% (non-condensing)
Electrical:
100–200 VAC; 50-60 Hz (200–230 VAC; 50–60 Hz available); 15 A
Instrument Dimensions:
Fully configured requires 96” W x 30” D x 39” H
Instrument Weight:
195 lbs. (88.6 kg)
pKa PRO™: Some Equations for pKa Measurement
Effective Mobility (Meff)
Meff =
Ltot  Leff
V
Meff = m
(1/ta – 1/tm)
Ltot = Total length of capillary
Leff = Length to detector
V = Applied voltage
ta = Migration time of analyte
tm = Migration time of neutral marker (DMSO)
Z
MWX
MW = Molecular weight
Z = Compound charge
m, x = Determined empirically by experiment
Charge is Directly Related to Meff!
Charge (# pKa) predicted from Meff and MW
Relationships between Meff, pH, and Apparent pKa
Monoacid:
Monobase:
Meff =
Meff =
Ma10-pKa
10-pKa + 10-pH
Mb10-pH
Ma, Mb = Meff of completely ionized species
10-pKa + 10-pH
Non-linear regression of Meff vs pH plot is performed with appropriate equation to yield pKa
Equations in: J. M. Miller et al. Electrophoresis, 2002, 23, 2833-281.
Sample and Buffer Tray Configuration for pKa Analysis
12 pH Point pKa Analysis (8 Samples)
1
2
3 4
5
6 7
8
9 10 11 12
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
A
B
C
D
Sample Tray
Analyte
E
F
G
H
1
2
3 4
5
6 7
8
9 10 11 12
A
B
C
D
E
F
G
H
pH
2.10
2.91
3.40
4.40
5.20
6.00
6.84
7.60
8.40
9.20
10.00
10.83
Inlet Buffer Tray
The marked well (E7)
corresponds to
compound 5 analyzed at
pH 6.80
Experimental User Interface Screen
• User selects experimental mode (12 or 24 point aqueous, 12 or 24 point co-solvent)
• Compound names, molecular weights and predicted pKa values (if available) are entered
• Buffer pH information file is loaded
• Information is saved for pKa calculation and report generation
Results for 4-Aminopyridine (monobase)
• 4-Aminopyridine (red cursor) is a basic compound; therefore it migrates before the DMSO
neutral marker (black cursor)
• Software automatically selects two highest peaks above threshold
Results for 4-Aminopyridine (monobase)
pKa
• Mobility vs. pH plot yields titration curve; inflection point = pKa value (9.23)
• Software automatically predicts compound charge from MW and Meff
• Charge(4-AP) = +1.10 = monobase.
Results for Benzoic Acid (monoacid)
• Benzoic Acid (red cursor) is an acidic compound; therefore it migrates after the DMSO
neutral marker (black cursor)
Results for Benzoic Acid (monoacid)
pKa
• pKa value: 4.06
• Charge (benzoic acid) = -1.07 = monoacid
Sample and Buffer Tray Configuration for pKa Analysis
24 pH Point pKa Analysis (4 Samples)
Analyte
H
G
F
E
D
C
Sample Tray
B
A
Acyclovir
4-Aminopyridine
Acyclovir
4-Aminopyridine
Benzoic
Acid
4-Aminopyridine
Cefadroxil
Acyclovir
Cefadroxil
Quinine
Furosemide
Quinine
12 11 10 9 8 7 6 5 4 3 2 1
H
p
H
1
1
.2
p
H
6
.4
1
21
11
09 8 7 6 5 4 3 2 1
G
F
E
D
C p
H
6
.8
B
A
p
H
1
.7
Inlet Buffer Tray
24 Point Results for Cefadroxil (diacid/monobase zwitterion)
• At low pH, cefadroxil migrates before DMSO neutral marker
• At high pH, cefadroxil migrates after DMS neutral marker
24 Point Results for Cefadroxil (diacid/monobase zwitterion)
24 Point buffer series increases resolution and expands measurable pKa range
• pKa values: 2.56,7.24,9.67
• pI value: 4.90
• Charge (cefadroxil) = +0.85; -1.66 = monobase/diacid
Exported Excel Report
for Cefadroxil
The Excel report contains:
•
•
•
•
•
•
•
•
•
•
•
•
•
Compound Name
Date of measurement
Analyst information
Assay type
User comments
Measured pKa value(s)
Measured pI value (if applicable)
Titration curve
R2 value (goodness-of-fit)
M.W.
Predicted charge
Buffer information
Structural image file can be
inserted if available
• Electropherogram traces
(separate tab)
pKa Results Data Table
• Each saved pKa result is entered into sortable indexed data table
Results Obtained with the pKa PRO™
Compound
Acebutolol
Acyclovir
MW
336
225
Type
B
A/B
n
15
13
4-Aminopyridine
Benzoic Acid
Betahistine
94
122
136
B
A
2B
8
13
11
Cefadroxil
363
2A/B
13
Cefuroxime
423
2A
9
Clomipramine
Furosemide
315
331
B
2A
9
24
Imipramine
Indomethacin
Piroxicam
280
358
331
B
A
A/B
5
13
8
Procaine
300
2B
16
Quinine
324
2B
18
Tyrosine
181
2A/B
10
pK a PRO ™ pK a' (I = 50 mM)
9.51
2.19
9.20
9.22
4.07
3.88
9.97
2.57
7.21
9.70
2.12
11.19
9.56
3.61
10.39
9.60
4.02
1.87
5.35
2.13
9.06
4.33
8.50
2.23
8.85
10.05
• Literature pKa values were reported at ionic strengths from 0 – 150 mM
• To date, the pKa values for >100 compounds have been measured
• Average SD ± 0.06 units; typical agreement to literature ± 0.2 units or better
SD
0.09
0.03
0.01
0.03
0.03
0.02
0.04
0.03
0.04
0.05
0.01
0.15
0.04
0.05
0.09
0.03
0.08
0.05
0.06
0.09
0.04
0.05
0.06
0.02
0.08
0.07
Literature Values
9.37 - 9.56
2.16 - 2.34
9.04 - 9.31
9.02 - 9.29
3.98 - 4.26
3.46 - 5.21
9.78 - 10.13
2.47 - 2.86
7.14 - 7.41
9.89
2.04
NR
9.17 - 9.38
3.35 - 3.74
10.15 - 10.90
9.21 - 9.66
4.06 - 4.51
2.33 - 2.53
4.94 - 5.32
2.27 - 2.29
9.01 - 9.15
3.95 - 4.24
8.35 - 8.60
2.18 - 2.20
8.94 - 9.21
9.99 - 10.47
pKa Analysis of Tyrosine (monobase/diacid zwitterion)
*
•
•
•
•
pKa Values: 2.21, 8.79, 10.08
pI Value: 5.54
Charge: +0.71, -1.62 = monobase/diacid
–COOH pKa value not observed by UV spectrophotometry
pKa Analysis of Procaine + Impurity
**
Effective Mobility (x 106 cm2/V•s)
• A 4-aminobenzoic acid hydrolysis impurity (20%) of procaine was present
• The pKa values for both species were determined in the same experiment
Procaine
300
250
200
150
100
50
0
-50 1
pH Value
2
3
4
5
6
7
9
-100
-150
-200
-250
4-ABA
-300
pH 1.78 (Top Left) – pH 6.46 (Bottom Right)
Procaine pKa’ Values:
2.20, 9.04
4-ABA pKa’ Values:
2.37, 4.38
+
*
pH 6.82 (Top Left) – pH 11.20 (Bottom Right)
8
+
*
10
11
pKa Analysis of a Peptide: Asp-Phe
H-Asp-Phe-OH
pKa Values: 2.13; 3.71; 7.95
pI Value: 2.96
• Charge-based measurement
provides indication of isoelectric
point as well as pKa values
pKa Analysis of an Insoluble Compound (Clomipramine)
• Analyzed at 100 ppm (100 g/ml)
• Precipitation from solution at pH 7.7-8.5
ppt
MW: 314.9
Calculated log P: 5.53 ± 0.51
• Analyzed at 10 ppm (10 g/ml)
• No ppt. observed, pKa’ = 9.54  0.05 (n = 8)
Measured log P: 5.19
Calculated solubility at pH 10.0: 16 g/ml
Values obtained from ACD I-Lab V. 7
Low solubility compounds can
often be analyzed at lower
concentration without the use of
cosolvent
Cosolvent pKa Extrapolation of Insoluble Compounds
Method:
• pH values of methanol containing buffers were measured using aqueous standards
(ws pH) and converted to s pH values as previously described*
s
• The ss pKa’ values are determined for compounds using 30%, 40%, 50% and 60%
(v/v) methanol-containing buffers
• ss pKa’ values are plotted as a function of solution dielectric constant ( ) and
extrapolated to 0% cosolvent to yield the w pKa’ value (Yasuda-Shedlovsky Method)
w
• Four compounds can be run in parallel over 24 pH values or eight compounds can
be analyzed over 12 pH values (2 - 4 compounds/h)
* Roses, M.; Bosch, E. J. Chromatogr., A 2002, 982, 1-30.
pKa Analysis of an Aqueous Insoluble Compound
pH 6.80
ppt
Tamoxifen
pH 7.20
MW: 371.5
Calculated log P: 7.88 ± 0.75
Calculated solubility: 0.05 g/ml
Measured solubility: 0.01 g/ml
Calculated values from ACD I-Lab V. 7
Measured value from Avdeef (2003)
ppt
• 24-Pt aqueous pKa Analysis at 30 ppm (30 g/ml)
• Precipitation from solution at pH 6.8 – 7.2
• Sample dilution to detection limit = ppt
pKa Analysis of Tamoxifen in 30% (v/v) Methanol
• Tamoxifen stays in solution when analyzed at ~20 g/ml in 30% (v/v) cosolvent buffers
Yasuda-Shedlovsky Extrapolated pKa’ Value for Tamoxifen
• Extrapolated pKa’ value (I = 50 mM):8.53 ± 0.07 (n = 9)
• Literature pKa’ value (I = 150 mM): 8.58 (Avdeef, 2003)
• Software performs entire analysis with minimal input
Cosolvent pKa Results for Test Compounds
w
Extrapolated w pKa'
Solubility (g/ml) log P Value
# Runs
(Yasuda-Shedlovsky)
SD
Literature Values
4
8.78
0.08
8.7 - 9.06
Bifonazole
Chlorpromazine*
4
4
6.18
9.21
0.02
0.04
5.72 - 5.88
9.15 - 9.38
Clomipramine
4
9.35
0.05
9.17 - 9.38
5.19
Clotrimazole
4
5.87
0.03
5.48 - 6.3
5.20
Flufenamic Acid
3
4.02
0.06
3.63 - 4.27
Imipramine
12
9.50
0.09
9.34 - 9.66
Miconazole*
10
6.40
0.06
6.07 - 6.63
0.6
Nortriptyline
4
10.03
0.06
10.02 - 10.19
17.4
4.39
Promethazine
6
8.71
0.13
8.62 - 9.10
11.6
4.05
Quinacrine*
4
7.29
0.06
7.34 - 7.74
9.98
0.12
9.97 - 10.18
Compound
Amiodarone
#
0.005
7.80
1.7
4.77
5.40
4.39
Tamoxifen^
8
8.62
0.1
8.48 - 8.71
0.01
5.26
Terfenadine^
4
9.52
0.05
9.21 - 9.86
0.1
5.52
Trimipramine
6
9.29
0.08
9.15 - 9.37
Verapamil
4
8.68
0.04
8.66 - 9.07
9.7
4.33
• Compounds marked (*) required cosolvent; other compounds could be analyzed with aqueous buffers
(^ tamoxifen, terfenadine analyzed from 40%-60% CS; # amiodarone analyzed at 50%-60% CS)
• Overall, extrapolated pKa’ values agree well with available literature values
• pKa PRO™ requires much less sample (<100 g) than potentiometry (mg)
• pKa PRO™ analysis time much faster than potentiometry
pKa Paper in Collaboration with Pfizer
• An exhaustive study was performed over
several years with two different
generations of technology to validate the
multiplexed CE method for pKa analysis
• Excellent correlation found between pKa
values measured with multiplexed CE-UV
and available literature values, using both
aqueous and cosolvent methods
Shalaeva M, Kenseth J, Lombardo F, Bastin A. 2008. Journal of Pharmaceutical
Sciences, Accepted for Publication.
Correlation of Multiplexed CE-UV pKa Values to Literature
11.00
Average pKa Value, Literature
10.00
y = 1.0048x + 0.0266
2
R = 0.9921
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
2.00
4.00
6.00
8.00
Average pKa Value, This Work
• 98 compounds (>150 pKa values) measured by aqueous buffers
compared to average literature values
• 23 compounds (26 pKa values) measured by co-solvent buffers
compared to average literature values
10.00
pKa Measurement Pre-made Buffer Plates
• Pre-made buffer trays provide savings in customer labor and time, reduce error
• Aqueous and cosolvent buffer plates now commercially available
pKa Summary
• The pKa PRO™ system provides a rapid approach for pKa measurements of drug
compounds
• Reproducible pKa results in good agreement to literature values can be obtained
over a wide range of pH values (1.8 – 11.2)
• Impurities, degradants or UV absorbing counterions can be successfully resolved
from the target compound
• pKa values undetectable by UV spectrophotometry can be successfully measured
• Charge-based separation provides clear, intuitive indication of overall charge state
and compound isoelectric point
• Compound charge can be predicted, allowing for assessment of number of
ionizable groups and detection of closely spaced pKa values
• Insoluble compounds can be analyzed for pKa using methanol cosolvent buffers
and linear extrapolation to 0% cosolvent
Log P Measurements on
the pKa PRO™ System
Octanol-Water Partition Coefficients (log P Values)
• log P is a measure of how well the neutral, unionized form of a drug
partitions between a lipid phase (e.g., n-octanol) and water
• P is defined as the partition coefficient:
P = C o / Cw
If log P = 5, Co / Cw = 100,000:1 at equilibrium!
where Co and Cw are the equilibrium drug concentrations measured in
the n-octanol and water phases, respectively
• Traditional method for determining log P is the shake flask method;
HPLC also widely used
n-octanol
water
Log P Analysis of Neutral/Basic Compounds
• Multiplexed, microemulsion electrokinetic chromatography (MEEKC) was employed for
indirect log Pow evaluation.
• Microemulsion Buffer: 8.0% (w/v) 1-butanol, 1.2% (w/v) n-heptane, 2.0% (w/v) sodium
dodecyl sulfate, with phosphate/borate buffer (pH 10.0). Validated to correspond to octanolwater shake flask
• MEEKC is based on the partitioning of analyte between an aqueous phase and an
immiscible microemulsion (ME) phase comprised of oil droplets + surfactant
• More lipophilic compounds favor the ME phase and migrate slower
• Order of migration: DMSO (EOF marker), Analyte, Dodecylbenzene (ME marker)
Poole, S. K.; Durham, D.; Kibbey C. J. Chromatogr. B 2000, 745, 117-126.
Figure adapted from http://www.ceandcec.com (Author Kevin Altria)
Experimental Design for Log Pow Measurement
• A standard mixture of compounds with known log Pow values is used to calibrate the system
• The standard mixture and other test solutes are dissolved in microemulsion buffer containing
DMSO (EOF marker) and dodecylbenzene (microemulsion marker).
• Capacity factors (log k’ values) are calculated for standards and sample using Equation 1:
(1) k'

ts
teof
teof
(1

ts/t
)
me
where ts, teof, and tme are the migration times of the solute, EOF marker (DMSO), and
microemulsion marker (dodecylbenzene), respectively.
• log k’ values for the standard compounds are plotted vs. literature log P values to calibrate
the system via Equation 2:
log POW = A  log k’ + B
(2)
where A is the slope and B is the y-intercept.
Sample log P is calculated by entering experimental log k’ value into Equation 2.
96-Capillary MEEKC Measurement of Log P
• Migration Order: DMSO, Solute, Dodecylbenzene
• 96 samples analyzed simultaneously
Separation of Log P Standard Mixture
4
DMSO
(EOF Marker)
5
2
Dodecylbenzene
(ME Marker)
6
3
1
• Standards: 1. Pyrazine, 2. Benzamide, 3. Nicotine, 4. Quinoline, 5. Naphthalene, 6. Imipramine
• MMEEKC has also been employed as a generic purity screening approach
Typical Standard Log P Calibration Plot
• Averaged (n = 4) log k’ values for the six standards were used to construct the calibration plot
Log P Calculator Software
• Advanced data analysis software calculates log P and tabulates results
Long Term (> 8 months) Reproducibility of Log P Values
Solute
n
acebutolol
2-aminopyridine
aniline
benzamide
4-chloroaniline
chlorpromazine
coumarin
3,5-dimethylaniline
ethylbenzoate
hydroquinine
imipramine
indazole
lidocaine
3,5-lutidine
naphthalene
nefopam
nicotine
nitrobenzene
phenanthrene
pyrazine
pyrene
pyrilamine
pyrimidine
quinoline
tetracaine
42
34
36
50
36
7
26
15
38
42
52
46
36
14
53
32
53
35
13
53
8
35
36
53
38
MMEEKC log k'
avg. ± SD
0.41 ± 0.03
-0.41 ± 0.01
-0.12 ± 0.02
-0.17 ± 0.02
0.62 ± 0.03
2.21 ± 0.04
0.22 ± 0.02
0.57 ± 0.03
0.97 ± 0.04
1.26 ± 0.06
1.86 ± 0.08
0.38 ± 0.03
0.89 ± 0.04
0.42 ± 0.02
1.36 ± 0.07
1.14 ± 0.05
0.18 ± 0.02
0.40 ± 0.02
1.92 ± 0.06
-0.96 ± 0.01
2.21 ± 0.23
1.18 ± 0.06
-1.05 ± 0.02
0.54 ± 0.03
1.42 ± 0.07
%RSD
7.32
2.44
16.67
11.76
4.84
1.81
9.09
5.26
4.12
4.76
4.30
7.89
4.49
4.76
5.15
4.39
11.11
5.00
3.13
1.04
10.41
5.08
1.90
5.56
4.93
MMEEKC log Pow
avg. ± SD
1.80 ± 0.04
0.41 ± 0.01
0.90 ± 0.02
0.81 ± 0.02
2.16 ± 0.04
4.74 ± 0.06
1.48 ± 0.05
2.04 ± 0.05
2.75 ± 0.04
3.23 ± 0.10
4.23 ± 0.08
1.75 ± 0.08
2.62 ± 0.03
1.77 ± 0.03
3.40 ± 0.09
3.04 ± 0.04
1.40 ± 0.02
1.79 ± 0.04
4.29 ± 0.11
-0.51 ± 0.03
4.75 ± 0.38
3.11 ± 0.05
-0.67 ± 0.03
2.00 ± 0.04
3.52 ± 0.10
%RSD
Lit. log P OW
 log P OW
2.22
2.44
2.22
2.47
1.85
1.27
3.38
2.45
1.45
3.10
1.89
4.57
1.15
1.69
2.65
1.32
1.43
2.23
2.56
5.88
8.00
1.61
4.48
2.00
2.84
1.71
0.49
0.9
0.64
1.88
5.19
1.39
2.17
2.64
3.43
4.42
1.77
2.26
1.78
3.3
3.05
1.17
1.85
4.46
-0.26
4.88
3.27
-0.4
2.03
3.73
0.09
-0.08
0
0.17
0.28
-0.61
0.09
-0.13
0.11
-0.2
-0.19
-0.02
0.36
-0.01
0.1
-0.01
0.23
-0.06
-0.17
-0.25
-0.13
-0.16
-0.3
-0.03
-0.21
• Good reproducibility and agreement to better than 0.5 log units to literature data
Sample Throughput for Indirect Log P Methods
Method
Average Analysis
Time per Sample
(min)
Approximate
Throughput
(samples/h)
Reference
RP-HPLC
20
3
1,2
MEKC
15
4
3
MEEKC
18-23
30
2-3
100 (per week)
2
4
5
6
MMEEKC
1.25
46*
7 – pKa PRO™
•
Lombardo F.; Shalaeva M.Y.; Tupper K.A.; Gao F.; Abraham M.H. J Med Chem 2000, 43, 2922-2928.
•
Lombardo F.; Shalaeva M.Y.; Tupper K.A.; Gao F. J Med Chem 2001, 44, 2490-2497.
•
Smith J.T.; Vinjamoori D.V. J Chromatogr B 1995, 669, 59-66.
•
Mrestani Y.; Neubert R.H.H.; Krause A. Pharm Res 1998, 15, 799-801.
•
Kibbey C.E.; Poole S.K.; Robinson B.; Jackson J.D.; Durham D. J Pharm Sci 2001, 90, 1164-1175.
•
Jia Z.; Mei L.; Lin F.; Huang S.; Killion R.B. J Chromatogr A 2003, 1007, 203-208.
•
Wong, K-S; Kenseth J.R.; Strasburg, R.S. J Pharm Sci 2004, 93, 916-931.
* 4 of 96 capillaries are used for the standard mixture
Chiral Separations on the
pKa PRO™ System
Chiral Separations with the pKa PRO™ System
• The different enantiomeric forms of chiral drugs can often possess dramatically
different potency or toxicity
• CE is an attractive technique for separating the different +/- enantiomers of
chiral molecules:
• Minimal sample and reagent consumption
• Different chiral resolving agents can simply be added to the run buffer to optimize the
separation; no separate columns required as in HPLC
• The pKa PRO™, equipped with the thermoelectric cooling option, can perform
chiral CE separations in parallel with dramatically improved throughput
• Useful for:
• Identifying best chiral selector/condition for achieving best resolution
• Screening chiral reactions for enantiomer excess (EE)
Chiral Selector Screening Results for p-Chloroamphetamine
PTS Internal Standard
p -Chloroamphetamine
HS--CD
S--CD
HS--CD
S--CD
HS--CD
S--CD
HS--CD
S--CD
HS--CD
S--CD
HS--CD
S--CD
Selector
Rs
Migration Time (min)
HS--CD
0.89
23
HS--CD
1.76
26
HS--CD
5.64
60
S--CD
2.09
25
• All samples contained pyrenetetrasulfonate (PTS) internal standard (peak #1)
• Migration time could be reduced by use of vacuum assisted CE
96-Capillary Chiral CE: Mixture of (+/-) Isoproterenol
PTS Normalized Migration Time
(+) Isoproterenol: 0.52% (n = 96)
(-) Isoproterenol: 0.72% (n = 96)
(+)/(-) Normalized Peak Area
0.952 ± 0.028 (RSD = 2.68%)
96 samples analyzed in
< 25 min
Measurement of Enantiomeric Excess
(+)
PTS
(-)
• Sample: 1000 ppm (+) isoproterenol
• BGE: 5% sulfated--CD (Aldrich) in 25 mM H3PO4/TEA pH 2.5
• Contains a minor (-) isoproterenol enantiomer impurity
• Normalized corrected peak area of (-) impurity: 0.030 ± 0.002 (RSD = 6.30%; n = 24)
Other Applications on the
pKa PRO™ System
Other Potential Applications
In addition to :
•
•
•
•
pKa
Log P
Chiral separation
Protein purity and size (Protein PRO)
The pKa PRO can be used for the determination of :
•
•
•
•
Log D
Impurity profile
Drug binding to plasma proteins
Etc ...
Any CE separation can be transferred to the pKa PRO platform to
accelerate throughput
Literature References
Reviews Describing pKa and log P Measurement by CE
Weinberger R: Determination of the pKa of Small Molecules by Capillary Electrophoresis. American
Laboratory 2005, August:36-38.
Jia Z: Physicochemical Profiling by Capillary Electrophoresis. Curr. Pharm. Anal. 2005, 1:41-56.
Poole SK, Patel S, Dehring K, Workman H, Poole CF: Determination of acid dissociation constants by
capillary electrophoresis. J. Chromatogr. A 2004, 1037:445-454.
Poole SK, Poole CF: Separation methods for estimating octanol-water partition coefficients. J.
Chromatogr. B 2003, 797:3-19.
Papers Describing pKa PRO™ Core Technology
Zhou C, Jin Y, Kenseth JR, Stella M, Wehmeyer KR, Heineman WR: Rapid pKa Estimation Using
Vacuum-Assisted Multiplexed Capillary Electrophoresis (VAMCE) with Ultraviolet Detection. J.
Pharm. Sci. 2005, 94:576-589.
Pang H, Kenseth J, Coldiron S: High-throughput multiplexed capillary electrophoresis in drug
discovery. Drug Discovery Today 2004, 9:1072-1080.
Key Benefits and Summary
• Parallel CE-UV technology can be applied to a broad range of high throughput
applications spanning pharmaceutical and biotechnology markets
• Parallel CE provides many benefits:





Significantly increased sample throughput
Improved laboratory efficiency
Lower turnaround times
Decreased reagent and sample consumption
Reduction in labor and operational/maintenance costs
• Many methods previously developed for single capillary CE instruments can be
successfully transferred to a parallel format
• The parallel CE configuration format provides an open, flexible format to vary
capillary length, i.d., # capillaries and/or separation conditions to adjust resolution
or accommodate different applications as needed
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