Transcript The role of chromatography in physico-chemical characterisation Shenaz Nunhuck CASS, GSK Why do we need physchem measurements?  Physicochemical properties of drugs     influence their absorption and distribution in vivo Systemic.

The role of chromatography in
physico-chemical
characterisation
Shenaz Nunhuck
CASS, GSK
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
PHYSCHEM
ASSAYS
LIPOPHILICITY
LogD (oct), CHI
IONISATION
CONSTANT
PLASMA
PROTEIN
BINDING
AQUEOUS
SOLUBILITY
MEMBRANE
PERMEABILITY
Sample flow process
Chemists request
assays through
PhysWeb
website.
Samples are
obtained from
compound
stores
Samples are analysed
(Solubility, CHI, protein
binding, LogD(oct), pKa)
Results
posted to the
global
company
database
Data
processed
and results
calculated
Samples (DMSO plates,
solids in vials) booked
in
Barcode read and
sample information and
required tests recorded
in Excel report file
Chemists notified
of completion of
assays via
PhysWeb
Lipophilicity measurements
 Chromatographic Hydrophobicity Index (CHI)
 Immobilised artificial membrane (IAM) partition
 Protein binding (human serum albumin, alpha1-acid-glycoprotein)
 Octanol/water partition coefficient (LogP/D(oct)
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)
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 -1.467)
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.
Gradient for CHI determination
110
%B
Column: Luna C18(2), 50 x 3.0 mm id, 5 m
Flow:
1.00 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
90
70
50
30
10
-10 0
1
2
3
Time (mins)
4
5
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.
Compound
CHI7.4
at pH 7.4
18.4
23.6
34.3
43.9
51.7
64.1
72.1
77.4
87.3
96.4
Theophylline
Phenyltetrazole
Benzimidazole
Colchicine
Phenyltheophylline
Acetophenone
Indole
Propiophenone
Butyrophenone
Valerophenone
CHI = (slope x Rt) + intercept
Calibration chromatogram at pH 7.4
700.00
CHI2
at pH 2
17.9
42.2
6.3
43.9
51.7
64.1
72.1
77.4
87.3
96.4
y = 53.907x - 43.978
R2 = 0.9949
CHI calibration at pH 7.4
600.00
500.00
CHI10.5
at pH 10.5
5.0
16.0
30.6
43.9
51.7
64.1
72.1
77.4
87.3
96.4
120.00
100.00
400.00
CHI 7.4
80.00
300.00
200.00
60.00
40.00
20.00
100.00
0.00
1
0.00
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80
1.2
1.4
1.6
1.8
2
tR pH7.4
2.2
2.4
2.6
2.8
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
Schematic diagram of the IAM.PC (CH2)12 stationary
phase surface
Fast HPLC method to measure interaction with
Immobilised Artificial Membrane (IAM), CHI IAM
 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)12 150 x 4.6
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
Flow rate: 2 ml/min
CHI IAM Calibration
Gradient: 0 to 2.5 min 0 to 70% acetonitrile
60
50
Buffer: 50mM NH4AC, pH 7.4
Cycle time: 4 min
CHI IAM
2.5 to 3.3 min 70% acetonitrile
3.3 to 3.5 min 0% acetonitrile
y = 29.144x - 44.502
R2 = 0.999
40
30
20
10
0
1.2
1.7
2.2
2.7
tR pH 7.4
Ref: Valko et al, Rapid gradient HPLC method for measuring drug
interactions with immobilised artificial membrane : Comparison
with other lipophilicity measures. J Pharm Sci 89:1085-1096
3.2
3.7
4-way parallel HPLC system
C-18 pH 10.5
IAM
C-18 pH 7.4
C-18 pH 2
4 chromatograms of one compound by the 4-way HPLC
 Typical 4-way chromatograms of a base
CHI values and the acid/base character
The change of CHI values by changing the pH
CHI
100
90
80
70
pH2
60
pH7.4
50
pH10.5
40
30
20
10
0
Neutral
(Zwitterionic)
Strong acid
Weak acid
Strong base
Weak base
Amphoteric
CHIs measured at 3 pHs provide an automatic way of grouping molecules according
to acid/base character without a need for structural information.
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.





•
•
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.
Datect LC-validator (www.datect.com).
Commercially available 4-way HPLC-MS instrument
from Waters
IAM
pH 2.0
MULTIPLEXED
ELECTROS PRAY
INLET
LC COLUMNS
(0.500 mL/min)
Wa te rs
ZQ
pH 7.4
pH 10.5
4 WATERS 1525
HPLC PUMPS
MULTIPLE INJECTOR
S YS TEM
Wate rs 2777
4 PDA
De te c to rs
2996
S PLITTERS
(0.05 mL/min)
Mas s Lynx
DATA S YS TEM
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)
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
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 92:2236-2248
Calibration and Results
System is calibrated using literature plasma protein binding % data.
HSA calibration
y = 2.2331x + 0.2283
Calibration
compounds
Literature
%binding
2.50
Nizatidine
35
1.50
Bromazepam
60
Carbamazepine
75
Piroxicam
94.5
Nicardipine
95
Warfarin
98
Ketoprofen
98.7
Indomethacin
99
Diclofenac
99.8
LogK
2.00
1.00
0.50
-0.40
-0.20
0.00
-0.500.00
0.20
0.40
-1.00
LogtR
Calculate %Binding
 logK = slope * log(tR) + intercept
 K = %B / (101-%B)
•Results are reported as % bound or logK
0.60
0.80
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
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
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
LogD(oct) =
Log[( Peak area of sample in octanol phase
Peak area of sample in aqueous phase)
x Injn vol. (aqu) ]
Injn vol. (oct)
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)
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
Tecan
50 uM standard in
DMSO
fast gradient generic
HPLC method
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
Solubility of sample =
Peak area of sample
Peak area of standard
X Conc. of standard
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
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.
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.
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.
Acknowledgements




Klara Valko
Chris Bevan
Alan Hill
Pat McDonough
Physicochemical Scientist - GSK
Location:
Harlow, Essex, Southeast England
Salary:
Attractive Pay & Benefits
Start Date:
07/12/2005
Duration:
Permanent
Reference:
103002-82072
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.
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 + 0.41HBDC – 1.41
N=86 r=0.94 s=0.40
where HBC = Hydrogen bond donor count
LogPoct = 0.054CHIlogP + 1.319A – 1.877
N=86 r=0.97 s=0.29
where A = Calculated Hydrogen Bond Acidity