1. The role of chromatography in physico-chemical characterisation
Shenaz Nunhuck
CASS, GSK

2. Why do we need physchem measurements?
Physicochemical properties of drugs influence their absorption and distribution in vivo
Systemic absorption of drug involves a number of rate processes:
Distribution of the drug in the body
Dissolution of the drug in the body fluids
Permeation across the cell membranes to reach the site of action.
Key physicochemical parameters influencing these processes are lipophilicity, solubility, pKa, permeability
Physicochemical Properties play a major role not only in screening, development and formulation but also in in vivo compound disposition such as absorption, tissue partition, brain penetration, etc
It has been now well recognised that it is important to consider various physicochemical properties very early in the drug discovery process in order to reduce attrition and cost of development of new drugs.
At this early stage we have to assess a large number of compounds, that means we had to set up high throughput and relatively cheap physicochemical profiling measurements.

3. PHYSCHEM ASSAYS LIPOPHILICITY LogD (oct), CHI IONISATION CONSTANT
PLASMA PROTEIN BINDING
AQUEOUS SOLUBILITY
MEMBRANE PERMEABILITY
Within CASS, we have established high throughput methods to measure physicochemical properties that are fundamental to Drug Developability
Lipophilicity (octanol/water partition coefficient, chromatographic hydrophobicity index, CHI)
aqueous solubility
plasma protein binding
ionisation constant
membrane permeability

4. Sample flow process Samples (DMSO plates, solids in vials) booked in
Chemists request assays through PhysWeb website.
Samples are obtained from compound stores
Barcode read and sample information and required tests recorded in Excel report file
Samples are analysed (Solubility, CHI, protein binding, LogD(oct), pKa)
Data processed and results calculated
Chemists notified of completion of assays via PhysWeb
Results posted to the global company database
This shows the process from receiving the samples to sending out the results.

5. Lipophilicity measurements
Chromatographic Hydrophobicity Index (CHI)
Immobilised artificial membrane (IAM) partition
Protein binding (human serum albumin, alpha-1-acid-glycoprotein)
Octanol/water partition coefficient (LogP/D(oct)
The various lipophilicity measurements we perform routinely in our lab are the
CHI assay, IAM partitioning, protein binding assay and octanol/water logP.
The rest of my talk will cover some aspects of these assays.

6. Theoretical basis of using chromatography for measuring lipophilicity
Different compounds travel at different speeds in the chromatographic system.
The differential migration depends on the interaction of compounds between the mobile and stationary phase.
Retention factor is directly related to the chromatographic partition coefficient.
k= number of mol in the stationary phase/number of mol in the mobile phase
k = (tR - t0 )/ t0
log k = log K + log (Vs/Vm)
k is retention factor
log K is the log of the chromatographic partition coefficient
Vs/Vm is constant column parameter
(the ratio of the mobile and stationary phase volumes)
It has been recognised long time ago that chromatographic retention on a non-polar stationary phase using aqueous mobile phase shows good correlation with compound’s lipophilicity. It relates directly to the compound distribution between the mobile and stationary phase.
The retention factor that can be obtained from the retention time is directly proportional to the ratio of the number of molecules in the stationary and mobile phases. The logarithmic retention factor is in linear relationship with the logarithm of the partition coefficient (logK) of the compound in the chromatographic partition system. There is however a column constant, log Vs/Vm that is constant only on one particular column, and can vary from column to column and it is very difficult to determine its value. But with the use of calibration this term can be eliminated.

7. Chromatographic Hydrophobicity Index, CHI
Fast gradient methods: the gradient retention time is proportional to the compound lipophilicity.
Fast gradient retention time obtained on commercially available C-18 stationary phase converted to Chromatographic Hydrophobicity Index (CHI); this is the chromatographic lipophilicity.
The CHI Indices at three different pHs are determined from the gradient retention times obtained by injecting the compound into a HPLC system.
Dynamic range extended by the gradient method.
Can be expressed on a logP/D scale.
(CHIlogD = 0.054*CHI )
We have developed assays to measure lipophilicity using chromatography. Using fast gradient methods, the gradient retention times can be converted to a volume per cent organic phase composition in the mobile phase when the compound elutes from the column. This solvent strength parameter is proportional to the compound lipophilicity.
The gradient chromatographic system is calibrated and using the calibration coefficients the gradient times can be converted to the CHI value.
The other advantage of using gradient HPLC to measure lipophilcity is that we can measure more than just one lipophilcity. We have a system which can provide us with 4 different CHI indices.
With a single gradient run we can measure the lipophilicity range from logD -1 to logD 6.
As chemists prefer the logP scale, we can convert the CHI scale to CHIlogD scale.

8. Generic Gradient HPLC ( ‘Four minute CHI method’)
CHI is derived directly from a reversed phase chromatographic gradient retention time.
Luna C-18 column, buffer:acetonitrile gradient, pH2
Luna C-18 column, buffer:acetonitrile gradient, pH7.4
Luna C-18 column, buffer:acetonitrile gradient, pH 10.5
Each run time is 4 minutes.
Retention times are converted to CHI lipophilicity values after calibration.
Column: Luna C18(2), 50 x 3.0 mm id, 5 m
Flow: ml/min
Mobile phase
A: 50 mM ammonium acetate pH 7.4/10.5,
0.1M H3PO4
B: Acetonitrile
Gradient: 0-100% B in 2.5 minutes,
hold at 100% B for 0.5 minute,
return to 0% B in 0.2 minute,
equilibrate at 0 % B for 1.8 minutes
Analysis time:4 mins
We use a generic gradient method of 2.5 minutes on C-18 columns to obtain the CHI values. We run the same acetonitrile gradient but using different starting mobile phase pHs. This allows us to obtain CHI values at different pH’s.

9. Derivation of CHI
A set of calibration compounds of known CHI values (determined isocratically) is run.
A plot of Rt v/s CHI gives the calibration curve.
Research compounds are run. The Rt is converted to CHI from the coefficients of the calibration curve.
CHI = (slope x Rt) + intercept
The gradient chromatographic system should be calibrated with a compound mixture covering a wide range of lipophilicity. Hence prior to the analysis of the samples, a CHI test mix of 10 components is injected on the Luna C18 columns. the chromatographic hydrophobicity index of these calibration compounds were obtained from isocratic measurements
These form part of the whole sequence. Their gradient retention times are plotted against the known CHI values as shown in the table. A typical chromatogram of the calibration mixture at pH 7.4 is shown. The slope and intercept for the calibration plot at each different pHs are used to convert the gradient retention times of new compounds to CHI values.

10. Immobilised Artificial Membrane (IAM)
Immobilised Artificial Membrane- phosphatidylcholine (PC) head group with an ester linkage between two acyl chains and the glycerol backbone of the PC molecule.
Phosphatidylcholine (PC) is the major phospholipid found in cell membranes.
IAM stationary phases prepared from PC analogs closely mimic the surface of a biological cell membrane.
CHI IAM are extensively used in GSK for various purposes
Brain penetration models
Hepatoxicity models
In drug discovery the evaluation of drug-membrane interaction is a critical step as
drug activity
toxicity
distribution
absorption
depends on drug-membrane partitioning.
Pidgeon and co first publicised measuring drug-membrane interaction by immobilised artificial membrane (IAM) chromatography.
IAM stationary phase consists of phospholipids immobilised on a silica surface. The immobilised phospholipids mimics the lipid environment of a fluid cell membrane on a solid matrix.
Various immobilised artificial membrane (IAM) hplc stationary phases are available from Regis Technologies.
CHI IAM values are routinely determined at GSK and used for various project specific purposes such as in brain penetration models and hepatoxicity models. It has been shown that compounds binding very strongly to phospholipids, i.e with high CHI IAM values can often be associated with in vivo toxicity problems.
Schematic diagram of the IAM.PC (CH2)12 stationary phase surface

11. Fast HPLC method to measure interaction with Immobilised Artificial Membrane (IAM), CHI IAM
tR 7.4
CHI IAM
Compound
3.284
49.4
Octanophenone
3.167
45.7
Heptanophenone
3.033
41.8
Hexanophenone
2.866
37.3
Valerophenone
2.658 32
Butirophenone
2.415
25.9
Propiophenone
2.093
17.2
Acetophenone
1.893
11.5
Acetanilide
1.648
2.9
Paracetamol
A set of calibration compounds of known CHI IAM values (previously determined isocratically) is run.
A plot of Rt v/s CHI gives the calibration curve.
Research compounds are run. The Rt is converted to CHI from the coefficients of the calibration curve.
CHI = (slope x Rt) + intercept
Column: IAM PC2 (CH2) x 4.6
Flow rate: 2 ml/min
Gradient: 0 to 2.5 min 0 to 70% acetonitrile
2.5 to 3.3 min 70% acetonitrile
3.3 to 3.5 min 0% acetonitrile
Buffer: 50mM NH4AC, pH 7.4
Cycle time: 4 min
We use similar gradient approach as to the CHI at different pH on C-18 column to determine compounds interaction with phospholipids by using Immobilised Artificial Membrane chromatography.
As for the C-18 procedures, the system is calibrated using a mixture 9 components for which the CHI IAM values have been previously determined from isocratic logk measurements.
The calibration coefficients from the curve are used to determine CHI IAM values for research compounds.
Ref: Valko et al, Rapid gradient HPLC method for measuring drug interactions with immobilised artificial membrane : Comparison with other lipophilicity measures. J Pharm Sci 89:

12. 4-way parallel HPLC system
C-18 pH 10.5
C-18 pH 7.4
IAM
C-18 pH 2
CHI measurements are carried out on the 4 way parallel hplc (Agilent HP1100) system shown here. There are 4 separate hplc systems (pump, detector, column thermostat). Three of them use reversed phase columns and we run the same acetonitrile gradient but using different starting mobile phase pHs.
System 1 has an IAM column at pH 7.4, Systems 2 all have Luna C18 columns but at different pH's of 2, 7.4 and Data on each detector is collected at 3 wavelengths: 254 nm (Channel A), 210 nm (Channel B) and 230 nm (Channel C). A single sequence is created in cycle composer and the samples are injected using the CTC autosampler. 40 uL of solution is drawn from each sample and 5 uL is injected into the 4 different systems at the same time.
With this instrumental set-up we can measure one compound lipophilicity at three different pHs and the compound interaction with artificial membrane in 4 minutes.

13. 4 chromatograms of one compound by the 4-way HPLC
Typical 4-way chromatograms of a base
A typical four-way chromatogram is shown on this slide for a basic compound. It can be seen immediately, that the compound has longer retention at high pH as it is neutral, and that it is partially ionised at pH At acidic pH, it is highly ionised and hence elutes faster.

14. CHI values and the acid/base character
CHIs measured at 3 pHs provide an automatic way of grouping molecules according to acid/base character without a need for structural information.
With a simple comparison of the CHI values obtained under the three different starting mobile phase pHs we can group the compounds according to their acid/base character.
Strong acids that are fully ionised at pH 7.4 give the same CHI value at pH 10.5, while they give much higher values at low pH when they loose the charge.
If all the three pH CHI values are different, the compound is most probably partially ionised at pH 7.4, and from the direction of the change we can determine whether the compound has a positive or negative charge.
Weak bases give the same CHI values at pH 7.4 and 10.5 and lower values at pH 2. Again we can determine whether the compound is in its neutral form at pH 7.4 or not.
Neutral compounds have the same CHI values at various pHs
CHIs measured at 3 pHs provide an automatic way of grouping molecules according to acid/base character without a need for structural information.

15. Automated Data Processing
All data from the 4 systems are stored in a single data file
Complex data processing….major rate-limiting step as this requires visual inspection of each chromatogram for identifying the major peak.
Datect LC-validator software is run after data is collected on Chemstation
Chromatograms are automatically inspected and validated by the software.
Of course with the parallel system and improved automation of the sample preparation, a large amount of data is produced. The processing of these data had been a major rate limiting step. Fortunately about 2 years ago we acquired a software , the Datect LC Validator from an American company Datect which helped to solve this problem.
So how does the LC validator work?
LC validator is run after data is collected on Chemstation
Analyses the raw data files (integrates using its own parameters)
Validates the chromatograms (highlights failures according to user set criteria)
Allows quick review of chromatograms
Exports Rt data with compound id and file name to an Excel report

16. Automated review of Chromatograms
Peaks of interest are identified, their shape examined and retention time automatically transferred into excel spreadsheets for further data processing.
Software highlights any anomalies and generates explanatory error messages prompting expert visual inspection.
Customised alerts are set up by the user.
Most of our validation failures occur when the:
Major component’s peak area is less than 80% of total, indicating that the compound is probably impure.
Major peak retention times are not identical at various wavelengths.
Major peak absorbance is weak indicating lack of chromophore or absence of compound.
This is an example of a report generated by the LC validator where it highlights failures according to some set criteria which are set up by the user. Failures for example could be due to the presence of impurities or weak signal.
The retention time data is then used for further processing to obtain the CHI values as explained before.
Datect LC-validator (

17. Commercially available 4-way HPLC-MS instrument from Waters
We have recently purchased a similar 4-way parallel HPLC system with Mass Spectrometer from Waters. Like our Agilent system, it consists of 4 hplc systems in one.
It has been designed to be used for early candidate profiling, i.e, for determination of retention time based properties like CHI and protein binding and for quantitation based properties like solubility and permeability. It is fully automated from quick data acquisition to quick data processing
Calculates the slope and intercept from the calibration curve
Uses these calibration coefficients to calculate CHI values of compounds
Allows a quick review of the raw data
Produces a customised report
Retention Time based properties CHI, IAM, Protein Binding Coefficient
Quantitation based properties Solubility, Membrane Permeability, Protein Binding, Partitioning Coefficient, Stability and Metabolism
ProfileLynx
Supports a range of pre-defined experiment types
3 distinct component areas
Experiment Definition
Retention Time based experiments requiring a Calibration curve
Quantitation based experiments (with/without calibration)

18. Biomimetic hplc stationary phases (HSA, RSA, AGP)
Used to measure the binding affinity of compounds to proteins.
Plasma protein binding affects the unbound (free) drug concentration available to diffuse from the blood and reach the target tissue.
Commercially available human and rat serum albumin and α-acid glycoprotein hplc stationary phases (available from ChromTech Ltd)
One of the great advantage of using hplc technology for lipophilicity determination is that various types of stationary phases can be applied including ones that mimic biologically important constituents such as membranes and proteins.
It is known that high protein binding can reduce brain penetration as it affects the free drug concentration available to reach the target tissue.
It can also cause drug safety issues.
One of these types is stationary phase containing plasma proteins such as human serum albumin, rat serum albumin and alpha- acid glycoprotein attached to a silica support.
We routinely run HSA binding assay in our lab using a fast gradient hplc methods on these commercially available columns.
It has been demonstrated by several publications that the retention factors obtained on immobilised albumin column show good correlation to the %HSA binding obtained by traditional ultra-filtration or dialysis methods.

19. Plasma Protein Binding
Fast generic gradient hplc method based on propan-2-ol gradient and chemically bonded HSA or RSA column.
Warfarin site is the major binding site on HSA.
By injecting a racemic mixture of warfarin on the column, the R and S enantiomer are separated indicating the warfarin site is intact and the column is suitable for use.
Only 6 minutes analysis time
Column: Chromtech HSA 50 x 3 mm
Flow rate: 1.8 ml/min at 300C
Mobile phase: 50 mM ammonium acetate pH7.4/Propan-2-ol
Gradient:
0 - 3 min 0 to 30% propan-2-ol; 3 to 5 min 30% propan-2-ol; 5 to 5.1 min 0% propan-2-ol
Cycle time: 6 min
We use a fast gradient method of isopropanol and aqueous buffer on a chemically bonded HSA column.
To be able to reduce the retention times of strongly bound compounds isopropanol is added to the aqueous pH 7.4 mobile phase up to a concentration of 30%. A fast isopropanol gradient (3 min) up to 30% and a 6-minute run time are used on these columns. In this way compounds that bound more than 99% still can be eluted. We also check that the warfarin site which is the major binding site on HSA is intact by injecting a racemic mixture of warfarin. The column is fit for use if the enantiomenrs are separated.
Warfarin binds to the major drug binding site of HSA and it is stereospecific
Very similar method has been developed for binding measurements using Alpha-acid glycoprotein column as well.
Ref: Valko et al, 2003.Fast gradient HPLC method to determine compounds binding to human serum albumin. Relationships with octanol/water and immobilised artificial membrane lipophilicity. J Pharm Sci 92:

20. Calibration and Results
System is calibrated using literature plasma protein binding % data.
Calibration compounds
Literature %binding
Nizatidine 35
Bromazepam 60
Carbamazepine 75
Piroxicam
94.5
Nicardipine 95
Warfarin 98
Ketoprofen
98.7
Indomethacin 99
Diclofenac
99.8
Calculate %Binding
logK = slope * log(tR) + intercept
K = %B / (101-%B)
When protein stationary phase is used in HPLC, the retention of the compound in the system depends on the proportion of bound molecules. The retention time is directly related to the K partition coefficient of the compound. The volume ratio of the protein phase and the aqueous phase is constant using the same HPLC column. It can change however, from column to column, therefore we need calibration of the retention times.
We calibrate the HPLC system by several drug molecules with known literature plasma protein binding data. Nine calibration compounds as shown above are injected onto the hplc. The slope and intercept obtained from the calibration curve are used to convert the retention times of the discovery compounds to % HSA binding or the log K association constant. LogK is the linear free energy term linearly related to the logarithmic gradient retention time logtR. The use of calibration graph increases the accuracy of the determination as we have noticed that the retention times of the compounds progressively decrease as the column ages.
Results are reported as % bound or logK

21. Plasma Protein Binding
Reproducibility on the column is very good; the gradient retention time is within 0.1 min from day to day
The hplc method is fast, simple and is easily automated.
The use of calibration compensates for any changes in the column properties and hence increases the accuracy of the determination.
The hplc procedure can discriminate easily in the high binding region (better than the traditional ultrafiltration or equilibrium dialysis methods) as the percentage of drug bound to the protein is measured and not the free drug.
Approximately 400 injections per column
Day to day reproducibility on the column is very good
The hplc method is faster, simpler and easy to automate.
Approximately 400 injections per column
The use of calibration compensates for any changes in the column properties and hence increases the accuracy of the determination as we have noticed that the retention times of the compounds progressively decrease as the column ages.
This procedure is more precise in ranking compounds binding and we measure the percentage of drug bound to the protein and not the free drug.

22. Chromatographic methods for quantitative assays
HPLC is a powerful technique for separation and quantification
Suitable approach for determination of compound concentration
Applied as end-point for logP(octanol) and solubility determination
HPLC is a powerful analytical technique that was developed to
separate the components of complex mixtures,
isolate compounds from complex mixtures, and
quantify components in complex mixtures.
The peak height or the peak area is proportional to the amount of compound in the mixture and hence it is a suitable for determination of concentration of a component in a sample. In our lab, hplc coupled with UV detection is used as an end-point in octanol/water partition coefficient partitioning and solubility experiments.

23. LogP(octanol) “shake-flask” determination
Equilibration of the compound between n-octanol and water in 96-well plate
Determination of concentration of the compound in each phase by fast gradient hplc method.
The syringe in the autosampler is set to sample first at the depth of the octanol phase in the well and then at the depth of the aqueous phase without any cross contamination
Ratio of the peak areas obtained from the aqueous and octanol phases directly provides partition coefficients
Lipophilicity can be measured via the equilibrium partitioning of a compound between 1-octanol and aqueous media.
logP- compound measured in its neutral state
logD- compound measured at a given pH
Traditionally logD was measured using shake flask method in a hplc autosampler vial. This has now been scaled down to deep well microtiter plates, hence accelerating the measurements.
The compound as 10 mM DMSO stock solution is dissolved in an aqueous phosphate buffer at ph 7.4. A known volume of n-octanol is added and the plate is equilibrated at room temperature. The plate is placed in a hplc autosampler and the syringe is set to sample first at the depth of the octanol phase in the well and then at the depth of the aqueous. The test compound is quantitated using hplc with UV detection to obtain the ocatnol/water partition coefficient.
LogD(oct) = Log[( Peak area of sample in octanol phase x Injn vol. (aqu) ]
Peak area of sample in aqueous phase) Injn vol. (oct)

24. Why is aqueous solubility important in early drug discovery?
Solubility is a key property for gastrointestinal absorption of orally administered drugs.
Affects bioavailability
Helpful in drug formulation stages for optimal drug delivery route and optimization
Insoluble compounds may compromise screening results.
Various solubilities
DMSO precipitative solubility
Solubility from solids
Solubility in simulated intestinal fluid (SIF)
Solubility influences drug absorption from the GI tract and affects bioavailability. High solubility and moderate lipophilicity are the hallmarks of a well-absorbed compounds. Moreover a drug candidate that has adequate solubility would less likely to have trouble for formulation work later in the development stage. Access to solubility data may help to interpret in vivo screening test results. Hence evaluation of solubility in the early stages of drug discovery is essential.
Various solubility assays are performed at GSK:
The DMSO precipitative solubility used to determine a solubility of compounds starting from 10mM DMSO solutions, that are diluted with buffer at pH7.4. This is the kinetic solubility. In drug discovery the focus is to keep a drug solubilised for an in vitro assay. It is therefore common practice to predissolve compounds put through biological screens in a water-miscible solvent such as DMSO. This assay flags up compounds with low solubility under screening conditions.
Solubility from solids which measures solubility from solid allowed some time to go into solution. Solubility of solids is dependent on crystal form and purity. This assay is labour intensive and suitable only for pure compounds available in crystal form.
Solubility in Simulated Intestinal Fluid (SIF)
This is offered as a bespoke to projects that have compounds with very low buffer solubility in order to assess a potential absorption problem due to low aqueous solubility.

25. HPLC-based Precipitative Aqueous Solubility
incubation
& filtration
Sample 500 uM
in pH 7.4 aqueous buffer
data in GSK database
compounds dissolved in DMSO at 10 mM
fast gradient generic HPLC method
Tecan
How is the solubility determined?
The solute concentration is measured following partial precipitation from DMSO solution.
• Two aliquots of a 10mM solution of the test compound in DMSO are used; the first sample is diluted with phosphate
buffered saline (PBS) at pH7.4 whilst the second is diluted with DMSO-the standard.
• Samples are filtered after 1h standing at room temperature and the solute concentrations remaining in solution determined by HPLC.
All solutions are in 96-well plates.
Compound (10 mM) is initially in DMSO solution
Final solution contains DMSO (5%)
Precipitation rate is usually rapid
Solubility range is narrow 1 to 500 uM
• The rate of precipitation may be affected by the degree of crystallinity (lattice energy) of the precipitate.
• True equilibration is not guaranteed.
50 uM standard in DMSO

26. Quantification by HPLC
Fast automated sample preparation
Gradient HPLC method same as the CHI method
Sample and standard solutions injected next to each other (single point calibration)
Data collected at two wavelengths
Impurities separated
Automated data processing using in-house macro
Macro identifies the peak of interest in the standard solution and matches it with that in the sample solution
Peak area and retention time data exported to excel
All the sample preparation is fully automated using the liquid handling robot Tecan.
After filtration the sample and standard solutions are injected onto hplc with UV detection. A fast gradient method same as the CHI method is used to elute the components of the solutions. Impurities are separated from the main component.
An in-house macro automatically identifies the main component in the standard solution and based on retention time matches this with a component in the sample solution. The peak area and retention time data are exported to excel for further data processing. The solubility is calculated and reported in uM.
Solubility of sample = Peak area of sample X Conc. of standard
Peak area of standard

27. Artificial Membrane Permeability
High throughput assessment of compound intestinal permeability
Cultured cell monolayer with reconstituted lipid membrane
Lipid is egg phosphatidyl choline and cholesterol dissolved in n-decane.
Permeation experiment is initiated by adding the compound to the bottom well and stopped at a pre-determined elapsed time.
Samples are analysed by HPLC/UV or/MS
P = (Vd/a* t) * ln [(R+X)/R(1-X)] * [1/(1+R)]
t is the equilibration time in s
Vd is the volume of donor solution in cm3
Vr is the volume of acceptor solution in cm3
a is the membarne surface area in cm2
R is the Vd/Vr ratio
A is the acceptor side peak area
D is the donor side peak area
X is the A/D peak area ratio
This technique is used to measure the permeability of a compound in a phospholipid bilayer system
The lipid is egg phosphatidyl choline (1.%) and cholesterol (1%) dissolved in n-decane.
This is applied to the bottom of the microfiltration filter inserts in a Transwell plate. Phosphate buffer (50mM Na2HPO4 with 0.5% 2-hydroxypropyl-b-cyclodextrin), pH 7.05 is added to the top and bottom of the plate.
5 uL of lipid added to the plate with 12 seconds shaking
200 uL of buffer added to the top (ACCEPTOR)
900 uL of buffer added to the bottom (DONOR)
30 mins shaking for equilibration of lipid
After 30 mins equilibration, 9 uL of 10 mM DMSO stock solution is added to the bottom well (900 uL)
Performed in duplicate
Plates are shaken for 2 hours
Using the Tecan robot,
Aliquots of 150 uL are removed from the top well and dispensed into a 96 well plate (Acceptor plate)
Aliquots of 150 uL are removed from the bottom well and dispensed into a 96 well plate (Donor plate)
Both plates are analysed on the hplc. The data acquisition is very similar to the solubility experiment; the main component is identified using a macro and the peak areas used to calculate the permeability in nm/s

28. Advantages of using HPLC technique
The compounds retention time can be directly related to the distribution between the stationary and the mobile phase, there is no need for concentration determination.
By changing the stationary phases and the mobile phase composition various types of lipophilic interactions can be investigated.
Impurities do not affect results as they are separated from the main peak and the compound of interest can be identified.
The advantages of the chromatographic technique are that
there is no need for concentration determination since the retention time is independent of the concentration or amount injected on the column.
Various stationary phases can be used including biomimetic phases such as IAM, HSA. This allows different flavours of lipophilicity to be determined.
As hplc is a separation technique impurities are separated from the main component

29. Advantages of using HPLC technique
Only small amount of material is needed.
Parallel systems can be used to lower cost and increase throughput.
With generic gradient hplc method, one method can be used with a variety of compounds; there is no need for individual customised method development.
Provides an excellent platform for computer controlled automated measurements with computerised data acquisition.
small amount of compound is needed for the measurements
With parallelisation the throughput can be increased while lowering the costs.
Using generic gradient methods, no time is wasted developing different methods for different compounds.
Hplc can be easily automated which makes it very attractive for use in an environment where high throughput measurements are needed

30. CONCLUSIONS
HPLC provides an excellent generic platform for measuring lipophilicity, acid/base character and bio-mimetic partition properties.
With the application of gradient methods and system calibration with known compounds, large amounts of reproducible data are obtained covering a wide dynamic range of the property.
The extensive application of automated platforms and parallelised chromatography has enabled hundreds of thousands of determinations to be made per annum with a minimum of labour.
The data are suitable to build local and general models to predict developability properties in early stages of drug discovery.
To conclude, these messages can be taken home:
HPLC is a powerful technique and provides an excellent generic paltform for measuring lipophilicity, acid/base character and bio-mimetic binding.
The use of generic gradient methods and calibration enable a large amount of reproducible data to be produced.
With the help of automation and parallel hplc systems, a large amount of data has been produced with minimum labour.
These data are extensively used to help chemists to progress their projects and to build models for predicting developability properties.

31. Acknowledgements
Klara Valko
Chris Bevan
Alan Hill
Pat McDonough

32. Physicochemical Scientist - GSK
   
Reference:
Permanent
Duration:
07/12/2005
Start Date:
Attractive Pay & Benefits
Salary:
Harlow, Essex, Southeast England
Location:
GlaxoSmithKline is currently recruiting for a Physicochemical Scientist in Harlow - Essex, Southeast England. At GlaxoSmithKline (GSK), one of the world's leading healthcare businesses, we discover, develop and produce products that help people live longer, do more and feel better. Minimum Requirements: You will have a BSc or equivalent experience in Chemistry, Analytical Chemistry or related discipline and have experience within an analytical laboratory environment.

33. Difference between CHIlogP and octanol/water logP
Chromatographic lipophilicity is not the same as the octanol/water lipophilicity
H-bond donor compounds (Series 1) partition more into octanol because the octanol OH groups can interact with the solute H-bond donor group resulting in higher logP values (i.e they look more lipophilic).
The two scales of lipophilicity can be aligned by introducing a H-bond acidity (A) term or a count of H-bond acid groups on the molecules (HBDC)
LogPoct = 0.05CHIlogP HBDC – 1.41
N=86 r= s=0.40
where HBC = Hydrogen bond donor count
LogPoct = 0.054CHIlogP A – 1.877
N=86 r= s=0.29
where A = Calculated Hydrogen Bond Acidity
Chromatographic lipophilicity is not the same as the octanol/water lipophilicity.
The major difference between the octanol/water and the C-18/aqueous acetonitrile water partition system is their sensitivity toward H-bond donor groups. H-bond acidity decreases compounds partition into C-18 stationary phase but does not decrease compounds partition into octanol/water.
H-bond donor compounds (Series 1) partition more into octanol because the octanol OH groups can interact with the solute H-bond donor group resulting in higher logP values (i.e they look more lipophilic). The H-bond donor/acceptor groups will not interact with the C-18 hydrocarbon stationary phase in HPLC, so they will look more hydrophilic.
By comparing the CHI values with the octanol/water partition coefficient values for more than 80 drug molecules we have found that with a single additional term that describes the H-bond donor ability of the molecule, we can approximate the octanol/water partition coefficients from the CHI values reasonable well.
The two scales of lipophilicity can be aligned by introducing the H-bond acidity (A)
The two equations below provides an easy tool to align the CHI lipophilicity to the octanol/water lipophilicity by taking into account the H-bond acidity of the compound (A) or a simple count of H-bond acid groups on the molecules (HBDC)