Pharmaterials Ltd
Unit B
5 Boulton Road
Reading, RG2 0NH
UK
Tel: +44 (0) 118 9070650
Fax: +44 (0) 118 9310679
Industrial Perspective on Detection and Quantification of
Amorphous Content
Dr Mark Hooper and Mridul Majumder
April 2009
Summary
Amorphous content (non-crystalline solid material) can directly affect the bio-availability and stability
of both APIs and excipients. Therefore it is a factor that must be controlled and quantified in
pharmaceutical applications, especially for inhalation products. Many techniques can be used to
detect and quantify amorphous content including XRPD, DSC, INC, SolCal, Raman (Mapping), GVS,
ss-NMR. The choice of the best technique to use is often material specific i.e. the magnitude of the
measured response for each technique varies between materials. INC has often been used for single
component systems (validated and accepted by regulatory authorities). Sensitivity is often a problem
in measuring amorphous content of APIs in final formulations. Raman mapping (microscopic) has
been successfully applied to cases where other techniques (macroscopic) have failed to distinguish
amorphous/ crystalline material.
Choosing which technique is suitable for your needs depends on balancing the following factors:
•
why you are analysing e.g. detection, quantification, validation, desired LOD/LOQ
•
single component (e.g. API or excipient) or formulated product (e.g. mixture post granulation)
•
how much material you have available
•
cost (!) and time of the analyses
•
complexity of the analysis e.g. is it heavily software analysis dependant? Is this a problem
(e.g. for understanding, patent cases, repeatability)?
Pharmaterials has experience in applying all the techniques in real applications on both pure
APIs/excipients and also in more difficult final formulations/ mixtures. We can suggest which
techniques would be suitable. We would then rapidly establish how to provide the specific answers
that you need for your project, whether it be very early stage ‘checking’ of crystallinity levels, to final
product monitoring and validation of a technique to quantify the levels of amorphous content. The
discussion attached outlines the various analysis techniques, the principals behind how they should
work,
and
some
real
practical
advantages
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and
limitations
of
each
technique.
Pharmaterials Ltd
Unit B
5 Boulton Road
Reading, RG2 0NH
UK
Tel: +44 (0) 118 9070650
Fax: +44 (0) 118 9310679
Definitions
XRPD – X-Ray Powder Diffraction
DSC – Differential Scanning Calorimetry
INC – Isothermal Nanocalorimetry (also referred to as IMC, Isothermal Microcalorimetry)
SolCal – Solution Calorimetry
GVS – Gravimetric Vapour Sortion, aka DVS (Dynamic Vapour Sorption), GMS (Gravimetric Moisture
Sorption)
ss-NMR – solid state Nuclear Magnetic Resonance
microscopic (analysis technique) – focuses analysis on one point of the sample
macroscopic (analysis technique) –analysis applied to all/most of the sample – ‘bulk’ techniques
API – active pharmaceutical ingredient
Amorphous content – What is it and why is it important?
Crystalline solid materials (e.g. APIs or excipients) can exhibit regions with significant disorder in the
crystal lattice. This is referred to as amorphous content. Amorphous materials have significantly lower
lattice energy which can increase the dissolution rate and therefore bio-availability. The amorphous
form can also often have higher hygroscopicity (i.e. hold onto water more) and is often significantly
less stable. Whilst the increased dissolution rate (bio-availability) can be advantageous, there is a
definite need to control, detect and quantify amorphous content to ensure consistency in
pharmaceutical performance (efficacy and stability). Amorphous content can be produced during
precipitation or processing of the APIs or excipient, or during the formulation processing. Processing,
such as milling, sieving, granulation or compression often affects the surface of the particles, and
therefore amorphous content can often be important for products which depend on the surface
characteristics of the materials e.g. inhalation products.
Techniques to detect and/or quantify Amorphous Content
These include XRPD, DSC, INC, SolCal, Raman (Mapping), GVS, ss-NMR. Less common techniques
include density measurements, DMA (Dynamic mechanical analysis) and IGC (Inverse gas
chromatography). A brief summary of each common technique and some advantages/ limitations is
given below. This is not intended as a definitive guide, but as a starting point for discussion of what
type of analysis is suitable for you.
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Pharmaterials Ltd
Unit B
5 Boulton Road
Reading, RG2 0NH
UK
Tel: +44 (0) 118 9070650
Fax: +44 (0) 118 9310679
XRPD
This is the traditional technique to detect crystallinity, or lack of it. Crystalline materials give sharp
diffraction peaks and amorphous materials give an ‘amorphous halo’. Amorphous material can be
detected by looking for evidence of the amorphous halo. However, it is possible that some ‘fully
crystalline’ materials can appear to have a halo effect. Quantification of amorphous content is
possible, often requiring a fully crystalline sample and a fully amorphous sample. A software program
is then used to calculate the proportion of contribution from the reference diffractograms that makes
up the analysed sample’s diffractogram.
In real terms, this technique works well for single component systems where 100% amorphous and
crystalline samples are available, calibration mixtures of crystalline and amorphous blends can be run
and all the data are collected at the same time and on the same machine. However, the analysis
relies heavily on the software analysis, which can lead to mis-assignment (e.g. due to chemical or
polymorphic impurities or background noise). The technique can be used for mixtures but this reduces
the accuracy and sensitivity, and again, care must be taken in using the software analysis which is
often designed for single component systems. The technique is quick to run, can use small amounts
of samples (~10-50mgs) and is relatively cheap. One limitation is that it is a bulk analysis technique,
and therefore can have difficulty detecting changes in APIs at low concentrations in formulations.
DSC
This technique is often used for detecting amorphicity, and sometimes for quantifying levels. The
technique measures energy responses to thermal events during heating of the sample. The common
events monitored to detect amophicity are the ‘glass-transition’ or recrystallisation of the amorphous
material. The observed energy change is proportional to the amount of amorphous material present.
Various modified DSC techniques can be used to increase the sensitivity and reliability, including
Hyper-DSC (very fast scan rates e.g. 500°C/min) and step-scan-DSC/ MTDSC.
DSC can be very quick, uses small amounts of material (~1-5mgs) and is relatively cheap. However,
there is often high variability between repeat analyses, and overlapping/ concurrent thermal events
that can make DSC unsuitable for quantifying (low levels of) amorphous content. Again a limitation is
that it is a bulk analysis technique using small amounts of sample, and therefore can have difficulty
detecting changes in APIs at low concentrations in formulations. Another consequence of the small
samples used is significant errors in generating suitable calibration samples for low levels of
amorphous content.
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Pharmaterials Ltd
Unit B
5 Boulton Road
Reading, RG2 0NH
UK
Tel: +44 (0) 118 9070650
Fax: +44 (0) 118 9310679
INC
This has been the most widely used and accepted technique for quantifying (and validating)
amorphous content. The technique measures the (exothermic) energy change when amorphous
material recrystallises, usually due to exposure of the sample to a solvent vapour (either at constant
or variable vapour pressure). The technique relies on a thorough understanding of the recrystallisation
process and conditions. The magnitude of the recrystallisation energy measured should be
proportional to the amorphous content.
There is an initial set up effort (time and cost!) to understand the recrystallisation conditions, and the
technique requires good control of instruments and conditions. However, the results are very
consistent and can give good sensitivity for single component systems (less than 10% amorphous
content quantified, sometimes as low as 0.1%). The technique can be validated and subsequent
analyses are more routine (i.e. quicker and cheaper). Therefore, whilst this technique is not routinely
used for detecting amorphous content, it has been the analysis of choice for quantification. There is
also very little reliance on complicated software-based analysis, which increases the robustness of the
results. Varying amounts of material can be used (50-100mg sample). However, it can be difficult to
detect amorphicity of APIs at low concentrations in formulations using INC, unless a system can be
found that causes recrystallisation of any amorphous API that is present (possible, but requires time
and effort, and therefore cost!).
SolCal
This technique is similar in scope to INC. The analysis measures the heat of dissolution of the sample
and, all other factors being equal (e.g. solvation energy), the measured value is proportional to the
lattice energy, therefore proportional to the amorphous (crystalline) content.
The sensitivity of this technique depends strongly on difference in heat of solution between
amorphous and crystalline samples, i.e. is material specific. However, it has been used for
quantification and can be validated. Again, there is also very little reliance on complicated softwarebased analysis, which increases the robustness of the results.
Analysis of finished formulations or mixtures can be difficult due to the increased number of
components contributing to the measured solvation energy which can mask the amorphicity of the
API. Sample sizes can vary but are typically ~100mg.
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Pharmaterials Ltd
Unit B
5 Boulton Road
Reading, RG2 0NH
UK
Tel: +44 (0) 118 9070650
Fax: +44 (0) 118 9310679
Raman – spectroscopy and mapping
Raman spectra show peaks that are characteristic of the chemical and physical structure of the
material analysed. Therefore differences can be observed between amorphous and crystalline forms
of the same material. These differences can be used to detect and sometimes quantify amorphous
content. Raman spectroscopy can be applied in two ways: in macroscopic/ bulk mode with average
Raman signal spectra being analysed for the whole material; or via Raman mapping – using a
focused Raman beam as a microscope to determine the Raman spectra from one ‘small’ point on the
sample. These two techniques give different data, and have different advantages and limitations.
Average spectra. This technique is similar in concept to XRPD and ss-NMR. Reference Raman
spectra are taken of 100% amorphous and crystalline material, and then software analysis is used to
calculate the proportion of contribution from the reference spectra that makes up the analysed
sample’s spectrum. This analysis indicates the amount of amorphous material present. Alternatively, if
well separated and distinct peaks are observed between the amorphous and crystalline spectra, the
intensity (or area) of peaks can be used to calculate the amount of amorphous material present. This
technique can give fast and simple (i.e. relatively cheap) data for detection and quantification of
amorphous content. However, there can be variations in the levels detected depending on which part
the of sample is analysed, and sometimes there are insufficient differences in the reference Raman
spectra to allow good sensitivity.
Raman mapping. This technique is a microscopic technique. This uses a focused Raman
microscope to detect the physical form of the material at one small spot on the surface of the sample.
By comparison to reference spectra of fully amorphous and crystalline, each spot can be assigned.
The spot is then shifted and analysis is repeated. In this way a 2-dimensional map is developed of the
sample. By comparing the ratios of amorphous and crystalline material detected, the amorphous
content can be quantified. This technique is especially useful for detecting and quantifying
amorphous (and crystalline) content of APIs in final formulations, as the microscope can focus in
on small amounts of the desired component within the formulation and therefore detect levels of
amorphous content that are generally impossible to detect using macroscopic/ bulk techniques.
GVS
This technique relies on the tendency of amorphous material to absorb more water (or solvent vapour)
than crystalline material. Therefore, the difference in the amount of water absorbed (or adsorbed) can
lead to a difference in the measured mass of the samples at a certain relative humidity (RH). The
difference in measured mass can be proportional to the amount of amorphous content.
The sensitivity of this technique depends on the difference in hygroscopicity between crystalline and
amorphous state. One weakness is that it is measuring an indirect effect of amorphous content, and
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Pharmaterials Ltd
Unit B
5 Boulton Road
Reading, RG2 0NH
UK
Tel: +44 (0) 118 9070650
Fax: +44 (0) 118 9310679
in must be assumed (or proved) that other factors don’t interfere (e.g. change in chemical or physical
form such as recrystallisation, or especially surface area of the material which can directly affect the
hygroscopicity). The initial investigation of conditions can be time consuming (some cost), but in ideal
cases good sensitivity can be shown. Sample sizes are typically ~ 25-50mg.
Analysis of finished formulations or mixtures can be difficult as the changes of hygroscopicity due to
small components are often masked by the majority excipients and other factors that contribute to
vapour sorption for formulations.
ss-NMR
This technique is normally run on
13
C CPMAS NMR. It relies on differences in the NMR spectra of
amorphous and crystalline materials. In general, amorphous spectra are broadened versions of the
sharper peaks seen for crystalline materials. Samples of 100% crystalline and amorphous materials
are run as reference, and then software analysis is used to calculate the proportion of contribution
from the reference spectra that makes up the analysed sample’s spectrum. This analysis indicates the
amount of amorphous material present.
This technique is similar in concept to XRPD analysis for the data handling. It requires specialised
equipment (NMR specifically set up for solids), and can be expensive. As in other techniques (i.e.
XRPD, Raman), the analysis relies heavily on the software analysis, which can lead to misassignment (e.g. due to chemical or polymorphic impurities or background noise). One limitation is
that it is a bulk analysis technique, and therefore can have difficulty detecting changes in APIs at low
concentrations in formulations, unless ‘unique’ signals can be found that can be assigned to the
component of interest (API).
Selected References for further information
General: G. Buckton, P. Darcy, Assessment of disorder in crystalline powders – a review of analytical techniques and their
application, Int. J. Pharm. 179 (1999) 141-158
V.P. Lehto, M. Tenho et al, The comparison of seven different methods to quantify the amorphous content of spray dried
lactose, Powder Technology 167 (2006) 85-93 (Good review, except ss-NMR)
INC: G. Buckton, P. Darcy, A.J. Mackellar, The use of isothermal microcalorimetry in the study of small degrees of amorphous
content of powder, Int. J. Pharm. 117 (1995) 253-256
XRPD: P. Bergese, I. Colombo, D. Gervasoni, L. E. Depero, Assessment of the X-ray diffraction absorption method for
quantitative analysis of largely amorphous pharmaceutical composites, J. Appl. Cryst. 36, (2003), 74-79
ss-NMR, DSC: R. Lefort, A. De Gusseme, J.-F. Willart, F. Danède, M. Descamps, Solid state NMR and DSC methods for
quantifying the amorphous content in solid dosage forms: an application to ball-milling of trehalose, Int. J. Pharm. 280 (2004)
209–219.
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