Transcript Slide 1

Use of Synchrotron Radiation to
Study Polymorphs of Pharmaceuticals
Lots of help from Cristian Botez, Ashfia Huq, Yuan-Hon Kiang,
Silvina Pagola, many other people who don’t care to be mentioned.
http://powder.physics.sunysb.edu
[email protected]
Why powders?
Why synchrotron radiation?
Examples:
• Real problem - distinguishing polymorphs with
similar x-ray patterns
• Real problem – determining small concentrations
of a protected polymorph
• Crystal structure solutions
• Proxy for a real problem – determining solid form
of a small amount of API in a finished tablet
How synchrotron radiation?
Single crystal diffraction is regarded as the gold
standard for structure determination. (With ~100m
single crystal, structure determination is “routine”.)
-butDetermination of a crystal structure is only a subset
of the structural information generally desired.
IN THE CONTEXT OF POLYMORPHISM
Many materials first show up as powders.
Many materials are available only as powders.
Measurements of solid mixtures are important,
even independent of the crystal structure.
Real Space - Debye-Scherrer cones
(220)
Incident beam
x-rays or neutrons
(200)
Sample
Typically 1010 grains of 1 m
(109 molecules) each, packed
to 50% density
(111)
Where does a powder diffraction pattern come from?
Instrument –
Is your instrument aligned correctly?
(Can you index simple patterns such as lactose hydrate)
Are relative intensities measured accurately?
(Preferred orientation, illuminated volume, …)
Strong pitch for use of synchrotron radiation
(Not giving an unbiased comparison of available instruments)
Three levels of understanding of a powder diffraction pattern
1. Collection of peaks (fingerprint)
2. PROFILE FIT. Use known lattice (or determine lattice) to
constrain all peak positions. Instead of ~20 peak positions, you
have 1 to 6 lattice parameters. Intensitites are matched to the
data.
3. RIETVELD FIT. Use known structure (or solve structure).
Intensitites determined by crystal structure.
What’s in your pill? (fake)
Example 1
Normalzed X-ray Intensity (counts / sec)
50000
"unknown" sample,
0.6997 Å, capillary
40000
30000
Data taken with very good (~0.007º
FWHM) resolution at NSLS
20000
10000
0
5
7
9
11
13
15
2 theta (degrees)
17
19
21
lact_raw.grf
23
A little work turns up this entry in the Powder Diffraction File
Normalzed X-ray Intensity (counts / sec)
50000
Lactose Monohydrate
0.6997 Å, capillary
40000
Powder Diffraction FIle #27-1947
Lactose Hydrate (1975)
30000
20000
10000
0
5
7
9
11
13
15
2 theta (degrees)
17
19
What are these weak peaks?
The active ingredient?
21
lact_pdf.grf
23
Lattice parameters -> possible peak positions
Space group -> some of those peak positions are not seen
Positions of atoms within the unit cell -> relative intensities
of peaks within each phase
X-ray diffractometer optics -> lineshape parameters
(fundamental parameters on well-characterized instrument)
Crystallite size, internal strain, lattice defects -> lineshape
parameters (not usually very interesting; adjust parameters
to give a good fit to lineshape data)
Rietveld method: look at all of your data. Compare the
profile with a model, not just the intensities of the
diffraction peaks.
Normalzed X-ray Intensity (counts / sec)
50000
Lactose Monohydrate
0.6997 Å, capillary
Rietveld refinement
40000
“Missing” peaks are actually
from lactose monohydrate, not
in PDF!
30000
20000
10000
Difference
0
Powder Diffraction File
Rietveld
Not the best fit in the world, but clear enough
5000
0
-5000
lactgsas.grf
4
6
8
10
12
14
16
2 theta (degrees)
18
20
22
24
Bragg-Brentano
X-ray tube
Electrons,
50 keV
40 mA
Anode
Focus diverging beam. Moderate
resolution, sensitive to sample
displacement, transparency
Debye-Scherrer
Resolution
limited by
divergence,
parallax
Synchrotron Radiation
X-ray brightness
Photons/time/solid
angle/bandwidth
Electrons,
2.8 GeV = 5500 mc2
300 mA
99.9999983% of c
1/ ~ 0.01
Magnetic Field
105
(This is NOT
an x-ray laser)
The sample geometry can have a profound influence on
the measured intensity.
Preferred orientation.
There are various means to minimize issues of
preferred orientation. It is usually best to load
samples in a thin glass tube.
(Not a perfect guarantee.)
Broad beam
Bragg-Brentano
From
storage
ring
GE (111)
analyzer crystal
Parallel,
Monochromatic
X-ray beam
Scintillation
detector
Ion chamber
sample
Si(111) double
monochromator
Powder diffraction station at X3B1 beamline,
National Synchrotron Light Source,
Brookhaven National Laboratory, U. S. A.
(available for general users, rent, or collaboration)
Lab(Sealed Tube) and Synchrotron XRD patterns of Enalapril Maleate
Form I
Cu Ka1
Form II
4
9
14
19
2q
24
29
34
39
Form I
l=1.15Å,
Form II
5
9
13
17
2q
21
25
29
33
Advantages of Synchrotron Radiation for Powder Diffraction
 Intensity
 Sharper peaks
 Distinguish nearby peaks and locate peaks more
precisely
 Better Signal to Background (see weak peaks close to
strong ones)
 For analyzer crystal setup, the resolution does not
degrade with sample size (intact samples)
 For analyzer crystal setup, the resolution does not
degrade with shorter wavelength.
 Freedom from parallax errors due to alignment,
transparency
 Generally easier to use environmental chambers.
 (Tunability can tailor x-ray penetration to the problem
at hand, make use of anomalous dispersion.)
Examples of identification of polymorphs with
synchrotron radiation data
This drug product has two polymorphs that can’t be
quantified except by Rietveld. Compare lab vs.
synchrotron data sets.
Form 1 -- Lab
Form 2 -- Lab
Form 1 -- Synchrotron
Form 2 -- Synchrotron
Detection sensitivity depends on
Two polymorphs of Zantac®,
signal intensity
ranitidine hydrochloride
sharp peaks
low background
All are enhanced by use of synchrotron radiation
Pure Form 1
X-ray Intensity (arb. units)
1000
(max 12K)
0
1.0% Form 2
in Form 1
10000
(max 176K)
0
Pure Form 2
0
8
10
12
14
16
18
20
22
Diffraction angle 2 
24
26
28
30
Structure Solution from powder data.
(PS & Silvina Pagola, Nature 404, 647 (2000); ACA
Transactions (2002); several papers in preparation)
PSSP is open source, simulated annealing software.
Three examples:
2½ polymorphs of 4’ methyl ROY
Form II ranitidine hycrochloride
(interesting challenge for sim. annealing)
Form II enalapril maleate
previously unknown structure
Malaria pigment  hematin, -D-Mannitol anhydride, R(-)-Albuterol
sulfate, 2-Hydroxy-N-[3(5)-Pyrazolyl]-1,4-napthoquinone-4-imine,
N-(p-tolyl)-dodecylsulfonamide, …
Pigments from Xiaorong He and coworkers in Byrn’s group
at Purdue, Dept. of Pharmacy
4 polymorphs of the same molecule - different colors.
1 solved by single crystal diffraction at Purdue
2 solved by S. Pagola from powder data
We’re still working on one.
NC
CH3
N
NO2
S
CH3
red
orange
dark red
light red
H
light red
dark red
orange
Comparison of powder vs.
single crystal solution of DR
Ranitidine hydrochloride form II
Undertake a project like this with very good data
Ranitidine HCl (Zantac®) is a very widely used drug for ulcers,
excess production of stomach acid.
There is an interesting subtlety in its crystal structure.
Space Group : P 21/n
6 Spatial coordinates : position
3 Eulerian angles : orientation
11 Torsions.
20 parameters
Monoclinic, Z=4
a=18.808Å,
b=12.981Å,
c=7.211Å
=95.057°,
Two candidate solutions from PSSP
Two others
All four,
superimposed.
Disorder,
or inability of powder
data to distinguish
a few of the atoms?
Atomic structure of our best Rietveld refinement of a single molecule.
Essentially independent of which solution we start from.
Rwp = 11.12%, 2 = 10.56
Normalized X-ray counts
60000
Rietveld plot of Ranitidine Hydrochloride
single configuration (from pssp)
x5
40000
20000
Difference
(a)
(b)
6000
3000
0
-3000
-6000
5.00
(c)
15.00
25.00
Two Theta (degrees)
35.00
45.00
Rwp = 8.43%, 2 = 4.51
x5
Normalized X-ray counts
60000
Rietveld plot of Ranitidine Hydrochloride
with disorder
40000
20000
Difference
(a)
(b)
6000
3000
0
-3000
-6000
5.00
(c)
15.00
25.00
Two Theta (degrees)
35.00
45.00
Refinement incorporating disorder. 50% occupancy of each of two
sites for N14, C16, C18, O20, and O21.
Rwp =8.39%, 2 = 4.51
This is clearly the
correct solution,
which includes
molecular disorder.
All thermal parameters
independently refined!
Gentle restraints on bond
lengths.
The answer, including disorder,
was already known from single
crystal experiment.
T. Ishida, Y. In, M. Inoue (1990)
The crystallographic problem:

Determine{R j } from{I hkl } using I hkl 
2
f
j
expiGhkl  R j
j
No rigorous argument that any solution we find is correct. We look for
heuristic consistency checks, generally based on getting a “reasonable”
solution, and having redundant data.
1.
Single crystals, small molecules: # of observations >> # of atoms
 Demand reasonable atomic distances, angles, etc.
2.
Proteins (single crystals, data with resolution ~ 2–3 Å ):
 Use sequence data, strong constraints on amino acid structure.
3.
Structures determined from powders by direct methods, etc.:
 Demand reasonable atomic distances, angles, etc.
Structures from powders using direct space: models of known
molecular structure
 Caution: many bond distances and angles are built in, so there is
less redundancy.
Conclusions for Ranitidine HCl
•In this case, the crystal structure contains
subtleties not expected in the starting model.
•Careful monitoring of the progress of the
refinement shows trouble if you ignore the
disorder that is suggesting itself through
nonsense thermal parameters.
•Careful monitoring of the simulated annealing
steps even show that the correct answer is
knocking at the door, even though it was not
originally invited.
Enalapril Maleate is a potent angiotensin converting enzyme
(ACE) inhibitor with two known polymorphs, Form I and Form
II. The single crystal structure of Form I has been known for
almost twenty years. On the other hand, the crystal structure
of Form II has never been reported before because of the
difficulty to obtain single crystals of this polymorph, which is
made by water slurry of Form I.
The crystal structure of Form II is of interest for several
reasons:
1. Form II is the more stable of the two polymorphs.
2. The two forms are structurally similar based on X-ray, IR,
and solid-state NMR.
3. The conformation of ACE inhibitors is important to their
biological activity.
Index measured pattern and
extract intensities
Orthorhombic P212121
a=33.987 b=6.642 c=11.210
LeBail Refinement
Cell indexed using 25 peaks in ITO
Enalapril Maleate
form II
a=17.838
b=6.640
c=11.649
106.29
form I
Monoclinic P21/c
Orthorhombic P212121
a=33.987
b=6.642
c=11.210
23 parameters: 11 enalapril torsions
(+2 maleate) + 12 orientation + position
No Solution from simulated annealing methods
• Powder Solve Crashed
• PSSP : ran for  time without success
• DASH – we haven’t tried it yet.
MC/SA
1
2
3
4
torsion
enalapril maleate
fixed
fixed
refined
fixed
fixed
fixed
fixed
refined
Form I
translation+rotation
enalapril maleate
refined
refined
fixed
fixed
refined
refined
fixed
refined
green:carbon
yellow:nitrogen
red:oxygen
Form II
DOF
12
11
12
8
wRp
%
17.51
14.04
13.69
13.66
Real business problem:
_____ has a patented polymorph of _____ , and
suspects that _____ is selling material that infringes.
It is desired to examine the commercial tablets and
determine the polymorph of the API for potential
litigation.
Proxy:
Examine commercial tablet of Endocet 500/7.5
Gross tablet 607 mg
Acetaminophen 500 mg – known lattice & structure
Oxycodone (as HCl) 7.5 mg – pattern in PDF but lattice
unknown,*
* In general, I’d like to get better info into the
PDF database. Please get in touch if you can help.
X-ray Intensity (counts per 10^6 monitor)
80000
Endocet (intact tablet)
500 mg Acetaminophen
7.5 mg. oxycodone HCl
60000
data: endocet.631
Measured
x 50
Profile fit
40000
20000
0
Acetaminophen
2
4
6
8
10
12
2theta (degrees)
14
16
18
20
Powder patterns of oxycodone hydrochloride from
ICDD Powder Diffraction File.
Strucutures and lattices are not known.
100
PDF 06-0014
Peak Intensity
50
0
100
50
PDF 38-1799
0
0
4
8
12
2q (degrees) at l = 0.70Å
16
20
X-ray Intensity
(counts per 10^6 monitor)
Endocet (intact tablet)
500 mg Acetaminophen
7.5 mg. oxycodone HCl
data: endocet.631
Profile fit
Measured
4000
?
3000
?
2000
1000
0
Oxycodone HCl peaks
from PDF
Acetaminophen
100
0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
2theta (degrees)
12.0
13.0
14.0
15.0
16.0
There are five synchrotron x-ray sources in the US
suitable for experiments like these. ~40 worldwide.
In my humble opinion, the most usable is NSLS at
Brookhaven Lab. 2500 users per year, of whom ~30% are
new.
All of these facilities have active programs to serve the
interests of people who want to use them.
Modes of access:
Write a proposal, wait several months, no charge for xrays, publish results.
Pay ~$300 - $5000/hr for time used, you own the data,
get access within hours to weeks.
http://nslsweb.nsls.bnl.gov
What is the real difference between working at a synchrotron
radiation source vs. with a laboratory x-ray diffractometer?
Acknowledgements :
Ashfia Huq, Silvina Pagola (now at Apotex)
Dept. of Physics & Astronomy, SUNY Stony Brook, 11794-3800
Y.-H. Kiang, Wei Xu
Pharmaceutical Research, Merck & Co., Inc. West Point, PA, 19426
“Anonymous” referees who goaded us into completing the Ranitidine story.
Advertisements:
Workshop on Frontiers of Powder Diffraction at the NSLS user meeting, May 19.
nslsweb.nsls.bnl.gov.
The International Centre for Diffraction Data is planning to run its third Pharmaceutical
Powder X-ray Diffraction Symposium in 2004. See me to get on the mailing list, or browse
www.icdd.com/ppxrd.
References :
Ashfia Huq and P.W. Stephens, “Subtleties in Crystal Structure solution from
powder diffraction data using Simulated Annealing Method”, J. Pharm Sci. 92 244-249.
Y.-H. Kiang, Ashfia Huq, Peter W. Stephens, Wei Xu, “Structure Determination of Enalapril
Maleate Form II from High-Resolution X-ray Powder Diffraction Data,” submitted to Journal
Pharm. Sci.
Pagola S. and P.W.Stephens, Mater. Sci. Forum 2000, 321, 40 (Source code and
documentation for PSSP are available at http://powder.physics.sunysb.edu)
Conclusions:
Think about x-ray diffraction as giving information about
the fundamental structure of your material, not just a
list of peaks.
This is a data-driven enterprise. High quality data is
very important.
I do not want to leave the impression that
synchrotron radiation is prerequisite to
good data. Nor that SR is guaranteed to
provide an important breakthrough. It
certainly helps.
Research carried out in part at the National Synchrotron Light Source at Brookhaven National Laboratory, which is
supported by the US Department of Energy, Division of Materials Sciences and Division of Chemical Sciences. The
SUNY X3 beamline at NSLS is supported by the Division of Basic Energy Sciences of the US Department of Energy
under Grant No. DE-FG02-86ER45231.