pKa PRO™ System Overview

Disruptive Technologies Product Range and Disruptive Technologies Product Range and Manufacturers RepresentedManufacturers Represented

• Biolgical Sample Preparation– Nucleic acides, protein and small

molecules extraction from hard-to-lyse tissue samples (Pressure Biosciences)

• Dissolution/Formulation– Dissolution baths, friability and

disintegration instruments (Distek)

• Physico Chemistry– HT Log P and pKa analyzer (AATI)– Kinetic solubility instruments (Analiza)

• Rapid microbiology with MicroPRO (AATI)

• Service– Agilent Channel Partner to service their

range of HPLC, UV spectrophotometers and CE systems

• Preparative– 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)

• Analytical– 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)

OutlineOutline

• 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

pKpKaa Values (Acid Dissociation Constants) 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+ + A- Ka = [H+][A-] / [HA]

pH = -log [H+] 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 pKWhy is pKaa Important? 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

0 1262 4 8 10

stomach

blood

small intestines

vinegar orangejuice

milk bleachcola

pharmaceutical products

colonurine

Biologically Relevant pH Range for Pharmaceutical Biologically Relevant pH Range for Pharmaceutical ProductsProducts

How pKHow pKaa Affects Membrane Permeability Affects Membrane Permeability

B + H+

B

BH+

• 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 pKCommon Issues Encountered During pKaa Measurements 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 OverviewCapillary Electrophoresis (CE) Technology Overview

• 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

Time

+ -N+ -Bulk Flow: EOF + Vacuum

UV

+

-

N

Measuring pKMeasuring pKaa by Capillary Electrophoresis (CE) by Capillary Electrophoresis (CE)

Time

+ N

Bases

Time

+ N

Time

N

Acids

Time

N -

Time

N

Time

N -

Acid/Base

Time

+ N

Time

N

Time

N -

Low pH

Intermediate pH

High pH

• 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

Key Advantages of CE for Measuring pKKey Advantages of CE for Measuring pKaa

• 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

pKpKaa PRO PRO™™ System 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 pKPrinciples of pKaa PRO™ PRO™ OperationOperation

• 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

Sample Throughput: pKa PRO™ 96XT12 compounds/h for aqueous 24-point pKa measurement

pKa PRO™ 24HT3 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)

pKpKaa PRO™ System Specifications PRO™ System Specifications

pKpKaa PRO™: Some Equations for pK PRO™: Some Equations for pKaa Measurement Measurement

Effective Mobility (Meff)

Ltot Leff

V(1/ta – 1/tm)Meff =

Ltot = Total length of capillaryLeff = Length to detectorV = Applied voltageta = Migration time of analytetm = Migration time of neutral marker (DMSO)

Meff = m Z

MWX

MW = Molecular weightZ = Compound chargem, 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

Meff =Mb10-pH

10-pKa + 10-pHMonobase:

Ma10-pKa

10-pKa + 10-pHMeff =Monoacid:

Ma, Mb = Meff of completely ionized species

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 pKSample and Buffer Tray Configuration for pKaa Analysis Analysis

The marked well (E7) corresponds to

compound 5 analyzed at pH 6.80

Sample Tray

122 3 4 5 6 7 8 9 10 111

A

H

B

C

D

E

F

G

122 3 4 5 6 7 8 9 10 111

A

H

B

C

D

E

F

G

Compound 1

Compound 2Compound 3

Compound 4

Compound 5Compound 6

Compound 7Compound 8

Analyte

Inlet Buffer Tray

2.10

2.91

3.40

4.40

5.20

6.00

6.84

7.60

8.40

9.20

10.0

010

.83

pH

12 pH Point pKa Analysis (8 Samples)

Experimental User Interface Screen 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) 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) Results for 4-Aminopyridine (monobase)

• 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.

pKa

Results for Benzoic Acid (monoacid) 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) Results for Benzoic Acid (monoacid)

• pKa value: 4.06• Charge (benzoic acid) = -1.07 = monoacid

pKa

Sample and Buffer Tray Configuration for pKSample and Buffer Tray Configuration for pKaa Analysis Analysis

Sample Tray

Acyclovir

Acyclovir4-Aminopyridine

4-Aminopyridine

CefadroxilCefadroxil

QuinineQuinine

Analyte

Inlet Buffer Tray

24 pH Point pKa Analysis (4 Samples)

pH 11.2

A

B

CDE

F

G

H

11112 8910 67 25 34pH 1.7

pH 6.8

pH 6.4

pH 11.2

A

B

CDE

F

G

H

A

B

CDE

F

G

H

11112 8910 67 25 34 11112 8910 67 25 34pH 1.7

pH 6.8

pH 6.4

H

Furosemide

H

A

B

CDEF

G

H

Benzoic Acid

Acyclovir

4-Aminopyridine

11112 8910 67 25 34 11112 8910 67 25 34 11112 8910 67 25 34

H

Furosemide

H

A

B

CDEF

G

H

Benzoic Acid

Acyclovir

4-Aminopyridine

11112 8910 67 25 34 11112 8910 67 25 34 11112 8910 67 25 34 11112 8910 67 25 34 11112 8910 67 25 34 11112 8910 67 25 34

24 Point Results for Cefadroxil (diacid/monobase zwitterion) 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 Results for Cefadroxil (diacid/monobase zwitterion)

• pKa values: 2.56,7.24,9.67• pI value: 4.90• Charge (cefadroxil) = +0.85; -1.66 = monobase/diacid

24 Point buffer series increases resolution and expands measurable pKa range

Exported Excel Report Exported Excel Report for Cefadroxilfor 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)

pKpKaa Results Data Table Results Data Table

• Each saved pKa result is entered into sortable indexed data table

Results Obtained with the pKResults Obtained with the pKaa PRO™ PRO™

• 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

Compound MW Type n pK a PRO™ pK a' (I = 50 mM) SD Literature Values

Acebutolol 336 B 15 9.51 0.09 9.37 - 9.56Acyclovir 225 A/B 13 2.19 0.03 2.16 - 2.34

9.20 0.01 9.04 - 9.314-Aminopyridine 94 B 8 9.22 0.03 9.02 - 9.29

Benzoic Acid 122 A 13 4.07 0.03 3.98 - 4.26Betahistine 136 2B 11 3.88 0.02 3.46 - 5.21

9.97 0.04 9.78 - 10.13Cefadroxil 363 2A/B 13 2.57 0.03 2.47 - 2.86

7.21 0.04 7.14 - 7.419.70 0.05 9.89

Cefuroxime 423 2A 9 2.12 0.01 2.0411.19 0.15 NR

Clomipramine 315 B 9 9.56 0.04 9.17 - 9.38Furosemide 331 2A 24 3.61 0.05 3.35 - 3.74

10.39 0.09 10.15 - 10.90Imipramine 280 B 5 9.60 0.03 9.21 - 9.66

Indomethacin 358 A 13 4.02 0.08 4.06 - 4.51Piroxicam 331 A/B 8 1.87 0.05 2.33 - 2.53

5.35 0.06 4.94 - 5.32Procaine 300 2B 16 2.13 0.09 2.27 - 2.29

9.06 0.04 9.01 - 9.15Quinine 324 2B 18 4.33 0.05 3.95 - 4.24

8.50 0.06 8.35 - 8.60Tyrosine 181 2A/B 10 2.23 0.02 2.18 - 2.20

8.85 0.08 8.94 - 9.2110.05 0.07 9.99 - 10.47

• 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

pKpKaa Analysis of Tyrosine (monobase/diacid zwitterion) Analysis of Tyrosine (monobase/diacid zwitterion)

*

pKpKaa Analysis of Procaine + Impurity Analysis of Procaine + Impurity • A 4-aminobenzoic acid hydrolysis impurity (20%) of procaine was present• The pKa values for both species were determined in the same experiment

4-ABA pKa’ Values: 2.37, 4.38

Procaine pKa’ Values: 2.20, 9.04

++

-300

-250

-200

-150

-100

-50

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 10 11

pH Value

Eff

ecti

ve M

ob

ility

(x

106

cm2 /

V•s

) Procaine

4-ABA

pH 1.78 (Top Left) – pH 6.46 (Bottom Right)

pH 6.82 (Top Left) – pH 11.20 (Bottom Right)

**

**

H-Asp-Phe-OH

pKa Values: 2.13; 3.71; 7.95

pI Value: 2.96

pKpKaa Analysis of a Peptide: Asp-Phe Analysis of a Peptide: Asp-Phe

• Charge-based measurement provides indication of isoelectric point as well as pKa values

pKpKaa Analysis of an Insoluble Compound (Clomipramine) Analysis of an Insoluble Compound (Clomipramine)

• Analyzed at 100 ppm (100 g/ml)• Precipitation from solution at pH 7.7-8.5

• Analyzed at 10 ppm (10 g/ml)• No ppt. observed, pKa’ = 9.54 0.05 (n = 8)

MW: 314.9

Calculated log P: 5.53 ± 0.51

Measured log P: 5.19

Calculated solubility at pH 10.0: 16

g/ml

Values obtained from ACD I-Lab V. 7Low solubility compounds can often be analyzed at lower concentration without the use of cosolvent

ppt

Cosolvent pKCosolvent pKaa Extrapolation of Insoluble Compounds Extrapolation of Insoluble Compounds

* Roses, M.; Bosch, E. J. Chromatogr., A 2002, 982, 1-30.

Method:•pH values of methanol containing buffers were measured using aqueous standards

( pH) and converted to pH values as previously described*

•The pKa’ values are determined for compounds using 30%, 40%, 50% and 60%

(v/v) methanol-containing buffers

• pKa’ values are plotted as a function of solution dielectric constant ( ) and

extrapolated to 0% cosolvent to yield the pKa’ value (Yasuda-Shedlovsky Method)

•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)

ws

ss

ss

ss

ww

pKpKaa Analysis of an Aqueous Insoluble Compound Analysis of an Aqueous Insoluble Compound

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. 7Measured value from Avdeef (2003)

ppt

ppt pH 7.20

pH 6.80

• 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

Tamoxifen

pKpKaa Analysis of Tamoxifen in 30% (v/v) Methanol 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 pKYasuda-Shedlovsky Extrapolated pKaa’ Value for Tamoxifen ’ 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 pKCosolvent pKaa Results for Test Compounds Results for Test Compounds

• 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

Extrapolated wwpKa'

Compound # Runs (Yasuda-Shedlovsky) SD Literature Values Solubility (g/ml) log P Value

Amiodarone# 4 8.78 0.08 8.7 - 9.06 0.005 7.80

Bifonazole 4 6.18 0.02 5.72 - 5.88 4.77

Chlorpromazine* 4 9.21 0.04 9.15 - 9.38 1.7 5.40

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 4.39

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

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

pKpKaa Paper in Collaboration with Pfizer 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 pKCorrelation of Multiplexed CE-UV pKaa Values to Literature Values to Literature

• 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

y = 1.0048x + 0.0266

R2 = 0.9921

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

2.00 4.00 6.00 8.00 10.00

Average pKa Value, This Work

Av

era

ge

pK

a V

alu

e,

Lit

era

ture

pKpKaa Measurement Pre-made Buffer Plates 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

pKpKaa Summary 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)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 = Co / 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 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 octanol-water shake flask

Poole, S. K.; Durham, D.; Kibbey C. J. Chromatogr. B 2000, 745, 117-126.

• 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)

Figure adapted from http://www.ceandcec.com (Author Kevin Altria)

• 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)

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.

)/tt(1t

ttk'

meseof

eofs

Experimental Design for Log PExperimental Design for Log Powow Measurement Measurement

96-Capillary MEEKC Measurement of Log P96-Capillary MEEKC Measurement of Log P

• Migration Order: DMSO, Solute, Dodecylbenzene• 96 samples analyzed simultaneously

Separation of Log P Standard MixtureSeparation of Log P Standard Mixture

DMSO(EOF Marker)

Dodecylbenzene(ME Marker)

1

2

3

4

5

6

• 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 PlotTypical 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 SoftwareLog P Calculator Software

• Advanced data analysis software calculates log P and tabulates results

Long Term (> 8 months) Reproducibility of Log P ValuesLong Term (> 8 months) Reproducibility of Log P Values

2-aminopyridine 34 -0.41 ± 0.01 2.44 0.41 ± 0.01 2.44 0.49 -0.08aniline 36 -0.12 ± 0.02 16.67 0.90 ± 0.02 2.22 0.9 0

benzamide 50 -0.17 ± 0.02 11.76 0.81 ± 0.02 2.47 0.64 0.174-chloroaniline 36 0.62 ± 0.03 4.84 2.16 ± 0.04 1.85 1.88 0.28chlorpromazine 7 2.21 ± 0.04 1.81 4.74 ± 0.06 1.27 5.19 -0.61

coumarin 26 0.22 ± 0.02 9.09 1.48 ± 0.05 3.38 1.39 0.093,5-dimethylaniline 15 0.57 ± 0.03 5.26 2.04 ± 0.05 2.45 2.17 -0.13

ethylbenzoate 38 0.97 ± 0.04 4.12 2.75 ± 0.04 1.45 2.64 0.11hydroquinine 42 1.26 ± 0.06 4.76 3.23 ± 0.10 3.10 3.43 -0.2imipramine 52 1.86 ± 0.08 4.30 4.23 ± 0.08 1.89 4.42 -0.19

indazole 46 0.38 ± 0.03 7.89 1.75 ± 0.08 4.57 1.77 -0.02lidocaine 36 0.89 ± 0.04 4.49 2.62 ± 0.03 1.15 2.26 0.36

3,5-lutidine 14 0.42 ± 0.02 4.76 1.77 ± 0.03 1.69 1.78 -0.01naphthalene 53 1.36 ± 0.07 5.15 3.40 ± 0.09 2.65 3.3 0.1

nefopam 32 1.14 ± 0.05 4.39 3.04 ± 0.04 1.32 3.05 -0.01nicotine 53 0.18 ± 0.02 11.11 1.40 ± 0.02 1.43 1.17 0.23

nitrobenzene 35 0.40 ± 0.02 5.00 1.79 ± 0.04 2.23 1.85 -0.06phenanthrene 13 1.92 ± 0.06 3.13 4.29 ± 0.11 2.56 4.46 -0.17

MMEEKC log k' MMEEKC log Pow

Solute n avg. ± SD %RSD avg. ± SD %RSD Lit. log P OW log P OW

acebutolol 42 0.41 ± 0.03 7.32 1.80 ± 0.04 2.22 1.71 0.09

pyrazine 53 -0.96 ± 0.01 1.04 -0.51 ± 0.03 5.88 -0.26 -0.25pyrene 8 2.21 ± 0.23 10.41 4.75 ± 0.38 8.00 4.88 -0.13

pyrilamine 35 1.18 ± 0.06 5.08 3.11 ± 0.05 1.61 3.27 -0.16pyrimidine 36 -1.05 ± 0.02 1.90 -0.67 ± 0.03 4.48 -0.4 -0.3quinoline 53 0.54 ± 0.03 5.56 2.00 ± 0.04 2.00 2.03 -0.03tetracaine 38 1.42 ± 0.07 4.93 3.52 ± 0.10 2.84 3.73 -0.21

• Good reproducibility and agreement to better than 0.5 log units to literature data

7 – pKa PRO™46*1.25MMEEKC

4

5

6

2-3

100 (per week)

2

18-23

-

30

MEEKC

3415MEKC

1,2320RP-HPLC

ReferenceApproximate Throughput (samples/h)

Average Analysis Time per Sample

(min)Method

Sample Throughput for Indirect Log P Methods Sample Throughput for Indirect Log P Methods

* 4 of 96 capillaries are used for the standard mixture

• 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.

Chiral Separations on the pKa PRO™ System

Chiral Separations with the Chiral Separations with the pKa PRO™pKa PRO™ SystemSystem

• 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-ChloroamphetamineChiral Selector Screening Results for p-Chloroamphetamine

S--CD

S--CD

S--CD

S--CD

S--CD

S--CD

HS--CD

HS--CD

HS--CD

HS--CD

HS--CD

HS--CD

Selector Rs Migration Time (min)

p -Chloroamphetamine 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

PTS Internal Standard

96-Capillary Chiral CE: Mixture of (+/-) Isoproterenol96-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 ExcessMeasurement of Enantiomeric Excess

• 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)

( + )

( - )

PTS

Other Applications on the pKa PRO™ System

Other Potential ApplicationsOther 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 ReferencesLiterature ReferencesReviews 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.

• 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

Key Benefits and SummaryKey Benefits and Summary

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