Analysis and Compliance in Pharmaceuticals

Contents

Introduction

Chapter 1:
Implementing Handheld Raman Spectroscopy Across the
Manufacturing Line

Chapter 2:
Assay by Potentiometric Titration

Chapter 3:
Automatic Water Determination in Pharmaceuticals

Chapter 4:
Ion Chromatography: The All-Rounder in Pharmaceutical
Analysis

Chapter 5:
Near-infrared Spectroscopy in Compliance with Pharma
Regulations

Chapter 6:
Compliance in the Pharmaceutical Industry
Introduction
Pharmaceuticals play a prominent role worldwide. This widespread presence is met with regulatory requirements at each
phase of pharmaceutical development and manufacturing—from inspection of raw materials to quality control of final
products. Consistently producing pharmaceuticals that both continue to satisfy the consumer and adhere to regulatory
mandates, requires analytical methods, instruments, and systems that ensure quality standards are upheld throughout
the pharmaceutical industry. In the following chapters, you will discover relevant analytical techniques, useful industry
examples, and tips for assuring continued compliance with the latest regulations.

One beneficial tool, handheld Raman spectroscopy, is useful across the pharmaceutical manufacturing line. Inspection
of incoming raw materials, assessment of intermediates, and quality control of final products can now occur during
production with little to no sample preparation and provide traceable, 21 CFR Part 11 compliant data recording. In
addition, titration remains a powerful method for pharmaceutical analysis, and automated potentiometric titration,
combined with automated sample preparation, is a mainstay for pharmaceutical development and manufacturing with
its rapid in-process feedback and accurate, repeatable quality control measures. Further, water content for an array of
substances is determined using automated sample preparation processes in conjunction with automated Karl Fischer
titration methods.

Determining concentrations of active pharmaceutical ingredients (APIs) such as gentamicin and impurities like azide is
critical to ensuring quality and safety to the consumer. The array of available ion chromatography options makes IC a
valuable tool for determining safety and efficacy of these materials as described by the US Pharmacopeia (USP). In cases
where a secondary analysis method is necessary, near-infrared spectroscopy (NIRS) offers reliable, real-time analysis at
all phases of pharmaceutical development. NIRS instruments, applications, and software are validated in compliance
with necessary regulations, and are therefore recognized as a dependable, efficient source for secondary analyses.
Finally, you’ll discover how USP <1058> defines analytical instrument qualification (AIQ) and learn about software with
integrated 21 CFR Part 11 compliant features. In each chapter you will find resources for increasing efficiency, reducing
cost, and eliminating waste while upholding the regulations that guide the industry.
Chapter 1:
Implementing Handheld Raman Spectroscopy
Across the Manufacturing Line
Adam J. Hopkins, Ph.D.
Raman spectra provide a data rich signal from which
materials can be identified or verified with high accuracy,
and little or no sample preparation. Direct analysis of
incoming and outgoing materials at the warehouse and
on the manufacturing floor is cost effective and provides
increased traceability, eliminating the need to send
materials to an analytical laboratory. These capabilities
allow handheld Raman to be used throughout the
manufacturing process and result in increased productivity
and lower production costs. Despite these advantages,
smooth implementation of handheld Raman across the
manufacturing line remains a challenge due to the differing
expectations from operators, developers, and facility or
corporate management. Fulfilling the expectations of these
three important groups in the manufacturing environment
is the key to successful implementation of handheld
Raman. This article sheds light on recent advances in
sampling and instrument management that better meet
the needs of all users.
Handheld Raman Instruments
Lab-based spectroscopy techniques; whether FTIR,
Raman, or other technology, require the same traditional
workflow of sampling, labeling, and transporting the
sample to the analytical lab. Traditional lab-based Raman
instruments are expensive and primarily used for research
and development by spectroscopists. Handheld and
portable Raman systems bring this analytical capability
into mainstream activities. Implementing handheld Raman
Incoming
In-process Screening &
Material
Confirmation
Identification
Figure 1: Examples of places where handheld Raman can be implemented in manufacturing.
can significantly streamline quality control measurements
by eliminating this traditional workflow and measuring in
situ.
Since the introduction of the first widely successful
handheld Raman spectrometer, different sectors of the
pharmaceutical and chemical industries have moved to
adopt handheld Raman as a screening tool to replace
traditional lab-based analysis methods. Handheld Raman
instruments are now used as identification, verification,
and screening tools across manufacturing lines for raw
materials, in process samples, and finished products.
(Figure 1)
Measurement traceability and cost-effectiveness are
critical drivers of handheld Raman implementation across
industries as problems with poor quality raw materials,
product adulteration, and counterfeit ingredients
proliferate. In the pharmaceutical industry, traceability
and incoming material inspection is mandated by FDA
regulations. Other sectors are following suit with the
implementation of cGMP and cGLP standards as a strategy
to ensure quality.
Successful Handheld Raman
Implementation
A major factor for successful implementation of handheld
Raman comes with providing training for operators and
Finished Product
Confirmation
gaining an understanding of technical and financial benefits
for method developers and managers. The features and
benefits that appeal to the end user sometimes differ from
features that appeal to spectroscopists doing method
development and upper management. The end-user may
receive hundreds of incoming containers per day in a facility
that may have or process thousands of materials. These
operators may have limited technical education, receive
minimal training, and must maintain a high operational
tempo. In this environment easy to use instruments help
maintain adequate workflow. Method developers are
most concerned with compliance features, instrument
specifications, error rates, and overall result reliability. At
the management level, other requirements such as overall
return on investment, instrument reliability, and the ease
of deploying and managing multiple instruments are
primary concerns.
Technology for Operators
The underlying technology for handheld Raman has
advanced, resulting in systems that do more than verify
raw materials and identify unknowns. They now identify
multicomponent mixtures and can make quantitative
measurements. Even fluorescence, long the bane of
Raman spectroscopy at 785nm, is being mitigated.
The appearance of instruments with longer excitation
wavelengths (above 1000 nm) or multiple wavelength
excitation promise to extend handheld Raman’s reach
into increasingly difficult materials and matrices. Recently
developed 532 nm systems promise to make handheld
Raman more applicable to biopharmaceuticals, which
are often in aqueous solutions. With the recent advent
of spatially offset Raman spectroscopy instruments, rapid
through-package screening is now available. Materials that
should not be opened can now be more easily identified
and qualified.
Despite these technological advances, user-to-user
variation and material/packaging variety are still challenges
encountered when handheld Raman is used along the
manufacturing line. These challenges are mitigated by
a combination of successful sampling strategies and
continual end-user education. To address the challenges
associated with sampling, instrument manufacturers are
constantly improving the feature sets of their hardware and
software to improve the data reliability while maintaining
the usability that has made this family of tools successful.
Examples include the
incorporation of mixture
Orbital-Raster-Scan
deconvolution algorithms,
new sampling methods
A
such as immersion
probes, and even smart
scan technology that
B
automatically optimizes
the signal.
A major challenge for
C
operators can be variability
of the Raman signals due
to sample damage or
material heterogeneity. Figure 2: Orbital Raster Scan Technology,
in which a small diameter laser beam is
Many organic materials
rastered about a sample surface.
and biopharmaceuticals
degrade when exposed
to 100+ mW laser beams with sub millimeter diameters
for 10 to 30 seconds. Since high laser powers are required
to keep integration times short, there are two strategies
for mitigation. One is to emit a large diameter laser
beam, which reduces the power density, but requires
larger collection optics for efficiency. A larger beam
simultaneously compensates for material heterogeneity
by measuring a macroscopic area. The other strategy
is to raster the Raman laser beam across a small area of
the sample at high speed. (Illustrated in Figure 2) This
raster technology prevents the laser from being incident
on any area too long, avoiding damage. It accounts for
heterogeneity by spatially averaging the sample. Because
the spot size remains small, the size of the optical train can
be reduced without sacrificing spectral resolution (1).
The first step to reliable Raman, anywhere in the
manufacturing line, is selection of the correct sampling
accessory (Table 1). Standoff objective lenses, or point and
shoot accessories, are the most commonly used sampling
tools for handheld Raman. Rather than holding the sample
at some specified distance from the instrument, these tools
make direct contact with a container to provide a more
consistent sample exposure. To use them correctly, simply
press the objective against the surface of a bag or bottle
and wait until the analysis is complete. Because the lenses
come in a variety of focal lengths, it is important to choose
the right lens for each application. Short working distance
(SWD) objectives typically have focal lengths around 1mm
and are best used to scan through transparent and thin
 Table 1
Sampling Accessories and Applications
Sampling Accessory Application
SWD Objective RMID through 1-3 layers of plastic bags
LWD Objective RMID through bottles
Vial Holder RMID of liquids when a sample must be pulled
Performance BallProbe ®
RMID of hard to reach areas. Top/middle/bottom sampling.
Tablet Holder Counterfeit detection

packaging, such as plastic bags and drum liners. Long bottles. Sapphire is both scratch and chemical resistant,
working distance (LWD) accessories, sometimes called making it well suited to the manufacturing or warehouse
bottle adapters, have typical working distances of 5-10 floor. Probes such as those developed by MarqMetrix and
mm and place the focal point, where the Raman signal is licensed by Metrohm for handheld Raman have an exposed
strongest, beyond the contact surface. LWD accessories sapphire sphere at the tip, which gives a very short focal
measure inside transparent plastic and glass bottles, length of around 0.250 mm. The small depth of field and
without having to open them. the exposure of the sapphire sphere make this technology
well suited for analyzing solid samples and small amounts
To further improve measurement reliability and consistency of materials on surfaces with which the probe is in direct
with standoff objectives, the use of vial holders and contact. This also enables top-middle-bottom sampling of
tablet holders is recommended. Liquids or powders are containers through a thief.
transferred to disposable glass vials and inserted into a
special holder that reproducibly positions the vial, avoiding
operator error by stabilizing the measurement location.
Tablet holders provide reproducible positioning of a tablet
to the Raman sensor. Because of the wide variety of tablet
shapes and sizes, reproducibly scanning tablets with a
standoff objective can be challenging, although necessary
if the material cannot be removed from a blister pack. Some
tablet holders require manual positioning and tightening of
screws which allow for a high degree of operator control
but may pose challenges for high throughput applications. a b
Other instrument manufacturers provide spring – loaded Figure 3: (a) Immersion probe in use measuring a solid sample.
holders that allow for quicker operation and reproducible (b) Detail of a MarqMetrix immersion probe with a solid sample.
placement.

In applications where direct measurement is preferable,
immersion probes bring the sample into direct contact
with the probe, thereby eliminating human error from
mispositioning the standoff objective. Immersion probes
also minimize sample handling and provide a way to rapidly
evaluate large numbers of samples. Figure 3 shows an
example of an immersion probe in use.
The Raman probes for handheld instruments currently
available have long (20 –36 cm) stainless steel shafts with
sapphire windows that can take direct measurements
inside liquid containers such as barrels, drums, and
Handheld Raman spectrometers with probes may also
be directly inserted into the production lines through a
sample thief or other appropriate provision to provide rapid
spectral data for process monitoring and improvement. For
example, blend homogeneity can easily be monitored with
Raman spectroscopy, and reaction monitoring has been
shown to be a viable application for handheld instruments
(2). While process monitoring can also be accomplished
with viewports and standoff objectives (3), immersion
probes minimize the impact on the manufacturing line.
Figure 4: Spectra collected of lactose monohydrate with a (green) SWD and (blue) LWD lens with a Mira. The LWD data has a much
lower signal to noise ratio.
Technology for
Method Developers
As a method developer, algorithms that work in the lab
must translate into durable operating procedures along
the manufacturing line. This means minimizing operator
driven errors and ensuring consistent instrument function.
For example, both Bruker and Metrohm have added
coded memory chips to their sampling accessories so
that their instruments know which accessory collects
each measurement. These accessories can be locked
to a particular method, ensuring the use of the correct
sampling tool and avoiding errors (Figure 4). The sample
accessory information is recorded in the instrument audit
trail to provide additional measurement traceability.
In addition, data collection and analyses are enhanced by
available automations to sample naming. Batch scanning
allows the naming parameters to be configured and then
left alone on the instrument until the task is complete.
Many of the instruments available today also have
integrated barcode readers. These can be configured to
automatically populate sample name, lot, and batchfields
with information according to how they are configured in
software. Barcode scanning and task automation ensure
that accurate sample information is captured and signed,
reducing post-analysis correction time.
Many industries use materials that do not yet exist in
qualified libraries provided by instrument manufacturers.
With open library structures, developers can create
qualified library elements that make it easier to identify
and build verification methods that capture the variance
of the acceptable raw materials. The robustness of these
methods can be further enhanced by implementing
additional controls on operating procedures. Examples of
enhanced controls include configuration of measurement
time, sample averaging, laser power, and even algorithm
thresholds. These features can ensure that every analysis
is performed exactly as it was in the laboratory, thus
preventing sample degradation or enhancing the spectrum
from a low signal material.
Implementing Handheld Raman
Across Multiple Locations
Higher levels of instrument performance available through
operating procedure controls in newer instruments make
it easier to deploy instruments across multiple sites.
Procedures can be transferred between a development
instrument and others through a validated instrument
method transfer protocol as outlined in USP <1224>.
Operating procedure transfer can overcome the challenges
of calibration survivability and variations in automated
method transferability among manufacturers. Each
measurement no longer has to be independently validated
on each instrument to ensure consistent results across
sites, thus saving time and the overall amount of validation
required.
Conclusion
Software and hardware innovations in sampling and
instrument control are increasing the reliability of handheld
Raman measurements, making the technology suited for a
wider number of users and applications. High quality, simple
sampling accessories optimized for different applications
deliver rapid and reproducible results. Microchips, barcode
readers, and the seamless integration of hardware and
software allow handheld Raman to be implemented by
a variety of users for applications ranging from incoming
materials inspection, to production monitoring, to finished
product inspection.

References
1. W
 atson, M.; Buller, S.; Carron, K. US Patent 8988678
B2. 2011.

2. Padlo, T. and Bakeev, K. Spectroscopy 31(9) p16-22.

3. Hopkins, A. J. “Implementation of Handheld Raman for

Online and at Line Applications.” IFPAC 2017.
Chapter 2:
Assay by Potentiometric Titration
Kerri-Ann Blake, Ph.D.

Introduction to Assay by Modern Assay of APIs

Potentiometric Titration
Analysis of composition and quality is essential at every
stage of the pharmaceutical manufacturing process,
from formulation development to production. This
is underscored by the FDA’s Quality by Design (QbD)
initiative, in which product and process understanding is
applied to preemptively control variation and reduce risk. It
requires the thorough characterization of pharmaceutical
products and the processes by which they are developed
and produced.
Results of in-process tests are used to inform critical decisions
made by formulators, and thus require accurate and timely
results to facilitate efficient formulation development
and production. They also ensure final material quality,
from content to uniformity. Among the many analytical
tests used to characterize a novel formulation, titration
is one of the most economical, fastest, and most reliable
techniques. Both active pharmaceutical ingredients (APIs)
and excipients such as surfactants, edible oils, minerals,
and chelating agents are addressed by titration and
described in the U.S. Pharmacopeia (USP) monograph
for roughly 630 APIs and 110 excipients. Titration also
continues to be a relevant technique for assay and content
uniformity of tablets.
The characterization of a novel formulation requires
distinct and varied analytical tests, both during processing
and on the final material. The results of in-process tests
are used to inform critical decisions made by formulators.
Receiving timely results is key to efficient formulations
development and production, as every tablet, vial, tube,
or bag of product in a batch must have the same active
substance content as stated on the packaging.
Various analytical methods are available for the exact
determination of APIs, defined by the standard operating
procedures in the European Pharmacopoeia and the USP.
Current USP-NF monographs recommend potentiometric
titration for assay of about 630 active pharmaceutical
ingredients in both aqueous and non-aqueous media.
Case study: Sulfanilamide
The purity of sulfanilamide, often employed for treatment
of vaginal yeast infections, can be determined in aqueous
solution by automatic, potentiometric titration using
sodium nitrite as the titrant (Figure 1). Potassium bromide
is added to the solution, as bromide ions act as catalyst for
the diazotization titration. Using the Pt Titrode electrode,

Potentiometric titration in particular is a powerful tool

in many facets of pharmaceutical manufacturing, from
formulation development to process feedback to quality
control of the finished product. The breadth of options in
automation at every step of the titration process allows it
to adapt to the various dosage forms, analytes, and goals
needed to ensure tight control of incoming materials
and high quality of finished pharmaceutical products.
Automated titration offers many benefits over manual
titration, providing more consistency and objectivity at
each step, from sample preparation to determination of
endpoint. It also improves the accuracy and repeatability of
results, reduces waste of time and materials through human
error, and increases the throughput of the analytical lab. Figure 1: Titration of sulfanilamide using the Titrando 905, using
sodium nitrite, c(NaN)2) = 0.1 mol/L as the titrant, hydrochloric acid,
w(HCl) = 20%, and potassium bromide solution c(KBr) = 2.5 mol/L,
with Pt Titrode, and temperature sensor. [Graphics note: Image taken
from 2283144_ AN-t157]
 purity of the sample is determined in as little as three to
five minutes, including time for electrode maintenance.
Samples of fats and oils or components of acid-base
mixtures often cannot be titrated in aqueous media. In
these cases, solvent selection is key to obtaining accurate,
repeatable results, particularly when isolating APIs from
interfering excipients and carriers.
Case study: Ketoconazole
Ketoconazole is an antifungal drug used in the treatment
of fungal infections and is prescribed as a tablet, cream
(for ringworm or cutaneous candidiasis), or shampoo
(for dandruff). Because of its low solubility point, less
than 1 mg/mL, the concentration of ketoconazole can be
determined by non-aqueous acid-base titration in three
to five minutes, or up to 10 minutes including electrode
conditioning time (Figure 2).
Assay of Excipients
Excipients often comprise a large fraction of pharmaceutical
products because they are used as fillers or bulking agents
for formulations containing low concentrations of potent
active ingredients. They also perform important functions
like long-term stabilization and enhancement of the active
ingredient. In the manufacturing process excipients can
facilitate powder flow or non-stick properties, enhance
stability of intermediates, and extend shelf life.
Figure 2: Titration of ketoconazole using the Titrando 907, using
perchloric acid, c(HclO4) = 0.1 mol/L as the titrant, and Solvotrode
easyClean as the electrode. [Graphics note: Image taken from
2073151_A N-t151]
While the most common dosage forms of pharmaceuticals
are tablets and injectables, many formulations are also
delivered as aerosols, capsules, creams, films, foams, and
gels. Excipients play a key role in formulation and need
to be of high purity to meet the needs and regulatory
requirements of the pharmaceutical industry. Although
excipients are listed as inactive ingredients by the FDA,
this class of compounds is often now used to impact the
delivery and/or efficacy of the APIs, from reducing viscosity
or enhancing solubility to facilitating drug absorption.
Excipients as a class are diverse, and often have uses other
than in pharmaceutical applications. A supplier may discover
that their product is being used by the pharmaceutical
industry as an excipient, even if it was not originally
intended for that purpose. In such cases, purity analysis
of key excipients is imperative for successful formulation
and batch to batch reproducibility, driving the need for
accurate and repeatable test methods like potentiometric
titration. Testing of excipients by potentiometric titration
includes both raw material characterization assay and
impurity testing. Current USP- NF monographs recommend
potentiometric titration for assay for about 110 excipients,
including surfactants, edible oils and lubricants, minerals,
and chelating agents.
Surfactants
Surfactants, or surface-active agents, are widely used in
pharmaceutical formulation as dispersing and solubilizing
agents to increase the solubility and bioavailability of APIs.
Surfactants are also used as emulsifying and wetting agents
to maintain the pH or osmolality of liquid formulations.
Additionally, surfactants are used in the pharmaceutical
industry as antioxidants, emulsifying agents, aerosol
propellants, tablet binders, and disintegrants; preventing
aggregation or dissociation; and modulating immunogenic
responses of active ingredients.
The concentration of anionic, cationic, and nonionic
surfactants is a critical parameter and will often vary
across formulations. The development of surfactants with
improved capabilities leads to more complex matrices
that are often challenging to evaluate. Specific surfactant
electrodes are available for assay of each type of
surfactant. As a result, potentiometric titration has grown
to replace the classic, manual Epton titration method. A
sample with a simple matrix or a raw substance can be
analyzed in aqueous solution. Anionic surfactants are
most often titrated using sodium dodecyl sulfate (sodium
lauryl sulfate) as the titrant, buffer solution of pH = 3.0, or
methanol as the reagent. Cationic surfactants are titrated
using sodium dodecyl sulfate or formaldehyde solution as
the reagent.
Nonionic surfactants are typically titrated using sodium
tetraphenylborate (STPB), polyvinyl alcohol protective
colloid, papaverine hydrochloride, sodium hydroxide
solution, boric acid, or hydrochloric acid as the reagent.
Titrants based on STPB are frequently used for the
determination of nonionic surfactants and pharmaceutical
compounds containing additives that significantly reduce
deposition of precipitates formed during the titration
on the electrode to reduce the frequency of electrode
cleaning. Owing to the high surface affinity of cationic
titrants, these must be added to the buret one day before
use to ensure wetting of all glass parts and tubing that
come into contact with the standard solution. Samples
with a more complex matrix or which are not easily soluble
in aqueous solvents are best analyzed with two-phase
titration. Electrodes are available which are resistant to
organic solvents and designed to be maintenance free.
These include models optimized for use with chlorinated
solvents and for samples with a high salt content and
relative low surfactant content or measurements at pH
values greater than 10 (e.g. soaps).
Case study: Lidocaine in ointments
Lidocaine is an API used as an anesthetic and to counter
arrhythmias. It can be assayed via potentiometric
titration with sodium tetraphenylborate using a nonionic
surfactant electrode. Methanol and heat are used to
dissolve or destroy emulsion formulations, then glacial
acetic acid is added to the prepared sample solution
prior to titration with sodium tetraphenylborate. Here,
automated potentiometric titration improves accuracy and
repeatability of results while reducing human error.
Fats and Edible Oils
Fats, edible oils, and fixed oils are widely used in
pharmaceutical formulation. Their rancidity will directly
affect the shelf life and stability of the formulated
products. USP chapter <401> recommends the following
characterization tests for fats, fixed oils, waxes, resins,
balsams, and similar substances, all of which can be
determined by potentiometric titration (Table 1).
Table 1
Acid value
An acid value measurement corresponds to the quantity
of carboxylic acid groups in fatty acids and is given in
mg KOH per gram sample. The older an oil is, the higher
the acid value, as triglycerides are converted into fatty
acids and glycerol upon aging. If the results of a free
fatty acids test are simply reported as acidity, without
further definition this is, by convention, expressed as
oleic acid. If the sample contains mineral acids these are,
by convention, determined as fatty acids.
Ester value
The ester value is given in mg KOH required to react with
the esters in one gram of a fat or oil. The ester value
is the difference between the saponification value and
acid value.
Hydroxyl value
Hydroxyl value is usually given in mg KOH required to
neutralize the acetic acid taken up on acetylation of one
gram of a sample containing free hydroxyl groups.
Iodine value
The determination of the iodine value is based on the
addition of iodine to the double bonds of unsaturated
fatty acids. The result is given as grams of iodine
consumed by 100 g sample and is a measure for the
unsaturation of an oil. Modern laboratories involved
in routine process and quality control of fats and oils
demand high analytical productivity with minimum
operator involvement. Iodine value determination can
be automated using the DIS- Cover technique and
magnesium acetate as catalyst. The addition of the
catalyst reduces the reaction time from 1–2 hours to 5
minutes.
Peroxide value
The peroxide number gives information about the
number of peroxide compounds in the oil and the age
and quality of the edible oil. The lower the peroxide
number the better and/or newer the oil. Peroxide value
is determined by redox titration using sodium thiosulfate
titrant. Determination can be automated using the DIS-
Cover technique.
Saponification value
The saponification value is expressed as the quantity of
KOH in mg required to saponify one gram of fat under
the conditions specified. It contains the information of
the average molecular weight of all fatty acids present.
Pharmaceutical Water
Water is used as a solvent, vehicle, diluent, or filler for
many pharmaceutical products. USP General Chapter
<1231> on water for pharmaceutical purposes explains
in detail the chemical testing needed for purified water,
water for injection, water for hemodialysis, and pure
steam. Chemical tests for these waters include accurate
and reliable determination of pH, conductivity, alkalinity,
hardness, and chloride. Prior to titration, the same
electrode used for titration is often used to measure
pH, and conductivity is often measured via a module
connected to the titration system. An automated system
for full water analysis including automated sample addition,
automated calibration, and automated titer and cell
constant determination—carried out in conjunction with
automated titration systems—enhances reproducibility
and minimizes manual error.
Assay by Potentiometric Titration
with Colorimetric Endpoint
Detection
Potentiometric titration with colorimetric endpoint
detection is commonly used in the pharmaceutical
industry as an alternative to manual visual color change
titration. It offers fast, simple measurements based on a
color change at the equivalence point and can be used
with both aqueous and non-aqueous solvents. Some
surfactants and several metals and metal oxides such as
iron, nickel, cobalt, zinc, zirconium, aluminum, calcium,
and magnesium are used in pharmaceutical formulations
and titrated according to the USP using a photometric
sensor as shown in the following case studies.
Case study: Chondroitin sulfate
Chondroitin sulfate is a component of connective tissues
found in cartilage and bone and is often used to treat
osteoarthritis. It can be determined photometrically
using hexadecylpyridinium chloride as the titrant with the
Optrode at a wavelength of 660 nm (Figure 3).
Minerals and Chelating Agents
Minerals such as zinc, chromium, copper, iron, aluminum,
zirconium, calcium, and magnesium are widely used in
pharmaceutical formulations, over the counter products,
and in dietary supplements. The purity of these minerals
plays an important role, as they are used in pharmaceuticals
Figure 3: Titration of chondroitin sulfate sodium using the Titrando
907, using 1-hexadecylpyridinium chloride as the titrant, with Optrode
(660 nm) as the sensor. (Graphics note: Image taken from 928186_AN-
t083)
(oral, parenteral, and topical formulations) to sequester
ions from solution and to form stable complexes. Both
the minerals and chelating agents’ purity can be easily
determined using selective potentiometric titration with a
colorimetric endpoint.
Case study: Zirconium in pharmaceutical
formulation
Zirconium is used in over the counter formulations with or
without aluminum. Photometric titration offers a simple
and straightforward method for its analysis. Zirconium
is titrated directly with EDTA in acidic aqueous solution
(buffer, pH 1). Eriochrome cyanine R is used as the indicator
for this procedure. The equivalence point is determined
with the Optrode at a wavelength of 520 nm.
Case study: Automated determination of
aluminum and magnesium in mixtures
Aluminum is commonly used in various antacid
formulations. Aluminum can be selectively determined
using photometric titration or potentiometric titration
using copper ISE as an automated method. Mixtures
of aluminum and magnesium ions can be analyzed by
back titration at different pH values. The ion-selective
copper electrode is used as the indicator electrode. First
the aluminum is determined in acidic solution, then the
magnesium in alkaline solution.
Assay and Content Uniformity of Tablets
Tablets are one of the most common dosing forms
for pharmaceuticals, and yet can be one of the most
challenging to analyze. Each tablet in a batch is required
to have the same active substance content as that given
on the packaging and within narrow limits. To achieve
this, analysis is required at several stages. Mixing studies
are performed during solid dosage form development and
in manufacturing; a series of samples representing the
uniformity of the mixers contents is collected and analyzed
to provide feedback on the process and formulation. Post-
manufacturing, tablet content uniformity studies validate
on a statistical basis that every tablet of each batch contains
the same solid dosage. While assays of APIs are often
performed by UV-Vis or HPLC, modern potentiometric
titration systems are a cost-effective option for automated
analyses and provide relatively quick feedback to the
manufacturing process.
To achieve highly accurate results using potentiometric
titration, sample preparation must be carried out
carefully and reproducibly. Depending on the shape and
coating of the tablets, the fillers they contain, and their
API concentration, various sample preparation steps
may be necessary before the analysis can be carried
out. The first sample preparation step always includes
the homogenization of the tablets in a suitable solvent
mixture (Figure 4). Depending on the analysis technique,
further steps such as diluting or pipetting may be required.
Most laboratories carry out these tedious steps manually,
allowing for carryover and contamination of the sample,
leading to inaccurate results.
Figure 4: High frequency homogenizers: short blades are used
to homogenize pastes and suspensions (left), while protruding
blades are used to pulverize tablets (right). [Graphics note:
Image taken from 700953_80006024EN]
These problems can be eliminated by using a completely
automatic system to standardize the sample preparation
procedure. The same automated system can be used for
the determination of the API concentration of a single
tablet and to validate that the quality requirements have
been met for the whole batch through random sampling.
The automation available with modern potentiometric
titration systems ensures high accuracy and reproducibility
of results, increases sample throughput, improves safety in
the laboratory, and reduces reagent consumption.
Complete automated processing of a tablet with
subsequent titrimetric determination of the content of an
ingredient can be performed in as little as eight minutes.
Note that the time required for homogenization depends
on the hardness and solubility of the tablets to be analyzed
and is decisive for the total duration of the analysis. The only
manual steps required are weighing out the sample, filling
in the sample table, and placing the sample on the sample
changer rack. The remaining steps occur automatically.
Automation prevents any changes to the water content of
the sample during preparation, and in some cases, it can
also eliminate the need for toxic solubility promoters.
Frequency of Sampling
The USP monograph on content uniformity stipulates
testing of solid dosage form samples at a frequency of 10
samples per batch. While this provides a solid baseline to
validate dosage, interest has increased in cost-effective,
time-efficient methods for tablet assay and content
uniformity testing as indicated by the FDA’s process
analytical technology (PAT) initiative.
While HPLC is very accurate, analyzing 10 tablets for
content uniformity could take hours – and the results may
not be available to the tablet press operators or for batch
release for many days or even weeks after the tablets are
compressed. Potentiometric titration offers much faster
feedback for the assay of many APIs, facilitating the
analysis of content uniformity in tablets at a rate that is
beneficial to production.
Case Study: Benzbromaron
Benzbromaron is the API used in a common tablet
administered to lower an increased uric acid level in
the blood in the treatment of gout. In addition to
sophisticated and expensive LC-MS and GC-MS methods,
benzbromaron can be effectively determined by titration
with sodium hydroxide solution using a straightforward,
fully automated sample preparation. Benzbromaron is a
weak acid whose pKa (4.50) is comparable to that of acetic
acid (4.75). After the loss of the hydrogen ion, the negative
charge at the oxygen atom is delocalized around the ring
(resonance stabilization). The more stable the ion is, the
more likely it is to form. Hence, titration with strong bases
is a convenient method for benzbromaron determination.
Figure 5: Titration curves for the determination of benzbromaron content with one sample tablet. For reasons of clarity the titration
curves of the 10 individual measurements are shown in two plots. [Graphics Note: Image taken from 700953_80006024EN]

The total determination, including sample preparation, References

takes only eight minutes. A 10-fold determination of
samples containing one tablet produced an active
substance content of 99.2 mg per tablet, in excellent
agreement with the 100 g per tablet value given by the
manufacturer (Figure 5).
Conclusion
Potentiometric titration is the most common approach
for automated titrations. In combination with automated
sample preparation, it has both increased efficiency and
reduced manual work load in many pharmaceutical labs.
Each sample is handled in exactly the same way, increasing
the accuracy of determinations, simplifying overall
workflow, and delivering consistently accurate results. It
has become a powerful technique for the analysis of active
ingredients and excipients in pharmaceutical and personal
care products. These factors have earned potentiometric
titration a place as a go-to technique for analysis of the
composition and quality of pharmaceuticals. By providing
both rapid in-process feedback and accurate quality
control, it will remain a mainstay of the industry as it
advances in its Quality by Design efforts.
AB-268: Potentiometric titration of surfactants and
pharmaceuticals – an overview.
AN-T-109: Automated determination of the iodine value.
8.000.6061: Applications of automated thermometric
titrimetry in routine process and quality control of fats and
oils.
AB-141: Analysis of edible fats and oils.
AN-T-110: Automated determination of the peroxide value.
AN-T-111: Determination of the saponification value.
AN-T-148: Determination of zirconium using automatic
photometric titration.
AN-T-117: Automatic determination of aluminum and
magnesium mixtures with ion-selective copper electrode
(Cu ISE).
Chapter 3:
Automatic Water Determination in
Pharmaceuticals
Kerri-Ann Blake, Ph.D.
Many active pharmaceutical ingredients (APIs) and
adjuvants contain water in an adsorbed form (surface
water) or bound as a hydrate (water of crystallization). The
water content of pharmaceuticals strongly influences their
quality, shelf-life, and stability as well as the release of the
active substances. The determination of water content is,
therefore, vital in pharmaceutical analysis.
The European Pharmacopoeia, 4th Edition (2002), describes
various methods for determining the water content of
pharmaceuticals and highlights the Karl Fischer titration
method. In cases of high water content, the method is
carried out volumetrically (semi-micro determination). For
substances with a very low water content a coulometric
KF titration (micro-determination) is performed. Automatic
Karl Fischer water determination offers advantages over
alternative, manual, time-intensive methods. Here you will
discover how the KF oven method in combination with
the KF coulometer offers automated features that increase
accuracy, repeatability, and efficiency when determining
water content of various low moisture substances. In
addition, you will learn how volumetric KF titration
in combination with a high-frequency homogenizer
determines water content of tablets quickly and accurately.
Karl Fischer Oven Method
Substances that release water slowly, at high temperatures,
or exhibit low solubility in alcohols are not suitable
for a direct Karl Fischer titration. In these cases the KF
oven method can be employed to overcome challenges
associated with traditional methods involving the use of
toxic solvents, extensive sample preparation procedures,
or the release of impurities when using a drying cabinet
or desiccator. With the automated KF oven method, the
substance under investigation is heated in a tube oven and
the released water is transferred by a carrier gas to the
titration cell where it is then determined by Karl Fischer
titration. Only the water enters the KF cell, and the sample
itself does not come into contact with the KF reagent
eliminating unwanted side reactions and matrix effects.
Using the 874 Oven Sample Processor, analytes are
weighed directly into sample vials, sealed tightly, moved
by the turntable to the appropriate position above the
oven, and then lowered automatically into the heating
block. carrier gas, loaded with the released moisture, then
flows through the outlet needle to a heated transfer tube
directly into the titration cell, where the Karl Fischer water
determination takes place (Figure 1). Since the Carrier gas
passes directly through the sample, instead of passing over
it, a more complete, accurate amount of water is released.
Table 1. Instruments, Accessories, and Reagents
874 Oven Sample Processor
851 KF Coulometer, including KF cell without diaphragm
803 Magnetic Stirrer. 6.5617.000 complementary
equipment for automatic reagent exchange 800 Dosino
tiamo 2.5 software for data acquisition, storage, and
reprocessing
Reagents:
Hydranal Coulomat AG Oven
Hydranal Water Standard KF Oven
Nitrogen as inert carrier gas
The following case study highlights the benefits of the
automated KF oven method using the 874 Oven Sample
Processor and the 851 KF Coulometer. Table 1 lists the
equipment and reagents used in the study.
Case Study: KF oven method
Using the KF oven method, approximately 40
pharmaceuticals from the European Pharmacopoeia
were analyzed. The investigated pharmaceuticals were
substances with a defined water content, some of which
undergo side reactions with KF reagents and therefore
cannot be analyzed by direct Karl Fischer titration. Between
15 and 30 mg of the pharmaceutical is weighed directly

Figure 1: Schematic of Moisture Delivery to Karl Fischer Titration Vessel
Using the 874 Oven
into each sample vial, which is then hermetically sealed
with PTFE-coated septa. Here, manual sample preparation
is reduced to a minimum, and significantly less product is
used as compared with traditional methods which require
a sample size of one gram or more. Smaller sample size
also allows for lower reagent consumption during titration.
At least a threefold determination is carried out on each
substance allowing for highly reproducible analysis
conditions and improved repeatability of results. Prior to
each determination the complete system is conditioned
until a constant low drift (approx. 10µg/min) is attained.
During this procedure the needle is located in a special
conditioning vessel on the rack of the Oven Sample
Processor.
To obtain accurate results, the blank (three empty sample
vials) is analyzed to account for moisture adhering to
the vessel walls, vial cap, and septum. In addition, the
complete system is checked at regular intervals with a
certified KF standard.
Analysis Temperatures
To determine the appropriate analysis temperature, the
Oven Sample Processor allows temperature gradients
to be run. Using the recorded water-release curve, the
optimum analysis temperature is determined. Here both
thermal stability (instability) of the substance and the fact
that water is only released at a sufficiently rapid rate at
temperatures above 100C are taken into account. The
oven temperature should be set as high as possible to
ensure short determination times but remain 20 to 30C
below the decomposition temperature.
The analysis temperatures are determined on the basis
of the water release curves that were recorded for all the
investigated pharmaceuticals in the temperature range 50
- 250C. In addition, all the pharmaceuticals were examined
by means of a Kofler microscope, and their melting points
were determined. This instrument allows the substance
to be closely observed during the heating and melting
phases; any alterations such as color changes, sublimation,
or decomposition reactions can be easily recognized.
Figure 2 shows the water release curve and temperature
gradient of metamizole sodium. Metamizole sodium melts
at 220 to 221C with decomposition. Water determination
by direct volumetric or coulometric KF titration is not
possible because the substance is oxidized completely or
partially by iodine. The water release curve was recorded
using a heating rate of 2C/min: the sample was heated
from 50 to 250C in 100min. Both the surface moisture and
the water of crystallization are released within the time
interval 0 - 1600s (50 - 103C). The drift then falls to its
original value of approximately 10µg/min and remains
virtually constant for 3800s. Starting at 5400s (230C)
both curves show a steep increase. Evidently, water is
released by decomposition from this temperature onward.
Figure 2: Water Release Curve and Temperature Gradient of
Metamizole Sodium
A temperature from the central region of the plateau of
the red curve (150C) was selected as the oven temperature
for determining the water content of metamizole sodium,
ensuring that the water is released quickly and completely
without decomposition.
Case Study Results
Table 2 shows the six most important substances among
the 40 investigated pharmaceuticals (the water contents
shown are the mean values of threefold determinations).
For comparison the table also contains the theoretical
(calculated) water contents of the substances and
Table 2: Oven Sample Processor and 851 KF Coulometer Results for Six Most Important Substances
information about the loss on drying given in the European
Pharmacopoeia. Analyses are performed in 10 to 12
minutes as compared to several hours in traditional drying
cabinet methods. Further, the KF oven method precludes
release of other volatile species, thus ensuring accurate
results.
The water contents determined with the 874 Oven
Sample Processor and 851 KF Coulometer all lie within
the ranges specified in the European Pharmacopoeia. The
Pharmacopoeia gives a wide recovery range for the loss on
drying. In the case of quinine sulfate, an antimalarial drug,
a range between 65.2 and 108.7% is specified based on
the theoretical (calculated) water content. The oven system
yields an excellent recovery of 100.7% for this substance.
When all the investigated pharmaceuticals are considered,
the recovery using the KF oven method lies between 90
and 110%. High repeatability was also obtained with
relative standard deviations lying between 0.30 and 2.0%.
Automated Homogenization for
Water Content in Tablets
The quality, hardness, compaction, and shelf-life of
pharmaceuticals depend largely on their water content.
Most pharmacopoeias stipulate thermogravimetry and
Karl Fischer titration for water quantification. The former
method requires labor-intensive sample preparation steps
and leaves considerable margin for error, and tablets often
have limited solubility in Karl Fischer working media. Using
an automated, high-frequency homogenizer that also
serves as a stirrer during titration significantly reduces
manual sample preparation and allows for volumetric KF
titration of released water.
The system for the automated determination of the water
content by volumetric KF titration consists of the 901
Titrando, the 815 Robotic USB Sample Processor XL with
two towers, and Polytron with comminution aggregate
(high-frequency homogenizer). The Polytron homogenizer
is mounted on the robotic titration head of the sample
processor tower and is adjusted to the correct working
height. The second tower is used for emptying the sample
beakers after the determinations, reducing reagent
handling to a minimum. All instruments are controlled by
the tiamo™ software.
Once the system is set up, the dosing devices are pre-
flushed to displace air bubbles and moisture. Four blank
determinations of working solution without sample are
carried out; the first one is the system preparation value,
the latter three provide the mean blank value. The titer
of a commercially available water standard is determined
(n = 3). A defined number of tablets is directly weighed
out into the sample vessel, the samples are then placed on
the sample processor rack, and all relevant data (sample
weight, sample identification) are entered into the tiamo™
software. All sample vessels are sealed with aluminum foil
and a sleeve. The working medium is transferred to the
sample vessel, and the Polytron comminutes the tablets.
Comminution speed and time depend on tablet size and
hardness and were determined in preliminary experiments.
The released water is titrated with KF reagent at a stirring
Figure 3: Linearity Test

Table 3: Water Content Determination of Tablets

speed of 7500 rpm. After each determination a cleaning
step with methanol is performed to avoid sample material
carry-over; in order to prevent cross-contamination, the
methanolic cleaning solution is titrated to dryness.

Linearity
A linearity test in the range of 4…215 mg was performed
with the sodium tartrate dihydrate standard. The
experimentally determined amount of water agreed
very well with the theoretical amount, resulting in an
outstanding coefficient of determination (Figure 3).

Four tablet type samples were analyzed to determine

moisture content using the automated KF homogenizer
system. In addition to determining the system preparation
value, the blank, and the titer, ten determinations were
carried out. The determined water contents were all within
the range expected by the manufacturer and corresponded
to previously validated values (See Table 3).

Conclusion
In cases where direct KF titration is not possible, employing
the KF oven method and using automated homogenizers
for tablets provide solutions for automatically determining
water content of challenging samples. Using an automated
oven sample processor in combination with an automated
KF coulometer reduces sample size, increases sample
throughput, and improves accuracy and repeatability for
many low moisture content pharmaceuticals. Employing an
automated, high-frequency homogenizer in combination
with KF volumetric titration allows for accurate, highly
repeatable water content determination of various tablet
samples.
Chapter 4:
Ion Chromatography: The All-Rounder in
Pharmaceutical Analysis
Stephanie Kappes, Alfred Steinbach, and Katinka Ruth
Ion chromatography is a flexible technique with a wide
variety of practical uses in the pharma sector. Here we take
a look at some pertinent trends and recent advances in its
application.
Testing in a Regulated Environment
High standards have to be met by the pharmaceutical
industry when it comes to drug quality and safety.
These standards are documented in pharmacopoeias as
officially recognized pharmaceutical rules and published
as legal tools of customer protection by authorities such
as governments and medical societies. The identification
of a drug depends on sensitive, reliable instruments,
and methods – as does the determination of the drug’s
compliance with applicable regulations.
Ion chromatography (IC) is the method of choice to
determine active ingredients, excipients, and traces of
impurities, as well as metabolites in the form of organic
and inorganic ions or polar substances, in a number of
pharmaceuticals, pharmaceutical solutions, and even body
fluids. It can determine the presence of several substances
within a very short time in a single analysis and even
distinguish chemically similar analytes. The concentration
of analytes can vary from ng/L up to the percent range.
The large selection of separation columns and elution
systems available makes IC useful for almost any kind of
analyte. Interfering effects caused by the sample matrix
can easily be avoided by using the right sample preparation
or choosing a suitable detection method. Inline sample
preparation is a feature of many modern IC systems, as
the focus of recent advances in IC has been mainly on ease
of use. However, convenience is not the only advantage
brought by the automation of the IC process; reducing
human interference to a minimum also means reducing
the chances of mistakes and contamination.
Depending on the requirements of the analyte and matrix,
a broad range of detection methods is available:
• Conductivity detection with and without suppression
• Electrochemical detection
• Spectrophotometric detection with and without
post-column derivatisation (ultraviolet–visible
spectrophotometry)
• Coupled detection methods such as IC–mass-
spectrometry (MS) and IC–inductively-coupled-plasma-
MS
Pharmaceutical samples come in many different forms
which require different ion chromatographic approaches.
What follows is an overview of frequent sample types with
relevant example analyses.
Pharmaceutical Solutions
The term ‘pharmaceutical solutions’ denotes isotonic,
hemodialysis, or infusion solutions. They contain
anions, cations, carbohydrates, and organic acids, the
concentrations of which frequently differ from one
another by several orders of magnitude. Within the
context of production monitoring and final quality control,
an analysis method is required that can determine these
ingredients with a high degree of precision. In addition,
the analysis should be quick and require minimal effort.
With its intelligent analytical procedure and automatic
inline sample preparation, IC fully accomplishes this task.
Two example analyses of hemodialysis solutions are shown
in Figures 1 – 2. Patients suffering from renal failure require
hemodialysis to compensate for the loss of the kidney’s
blood cleansing function. During the process, the patient’s
blood exchanges solutes with a hemodialysis solution
through a semi-permeable membrane. The exchanged
solutes include, among others, waste products such as
urea and phosphate, which diffuse out of the blood and
into the dialysis solution along the concentration gradient.
 a b
Figure 1: (a) IC measurement on a Metrosep A Supp 7 - 250/4.0 using Na2CO3 gradient elution, followed by sequential suppression
and conductivity detection. Anion standard including acetate and citrate. (b) IC measurement on a Metrosep A Supp 7 - 250/4.0 using
Na2CO3 gradient elution, followed by sequential suppression and conductivity detection. Acetate and citrate in hemodialysis solution.

The composition of dialysis solutions is complex, because
the removal of solutes from the blood changes its osmotic
activity; therefore, it has to take place at a controlled
rate, which is achieved by the right solute concentration.
A strong change in osmotic activity can cause dialysis
disequilibrium syndrome where, due to the low solute
concentration in blood, solutes are washed out from other
body compartments.
Figure 1 shows the simultaneous determination of citrate
and acetate in diluted hemodialysis solution. In part A, an
anion standard was measured; part B shows the sample
determination. Citrate is added to hemodialysis solutions
for its anticoagulant properties and acetate is added
as a buffer substance. It is transferred to the patient’s
bloodstream during hemodialysis and stabilizes the blood’s
pH value. This is necessary because the kidneys of dialysis
patients are not capable of excreting acid components –
therefore, patients are often acidotic.
Besides citrate and acetate, the chromatogram reveals
the presence of a nearly physiological concentration of
chloride. By using physiological solute concentrations, the
concentration gradient is reduced to a minimum, and a
dynamic equilibrium is reached between blood and dialysis
solution. The loss of certain solutes – including chloride –
is thereby prevented. Figure 2 shows the determination
of cations in hemodialysis concentrate after an automated
inline dilution step. Like chloride, the cations are present in
nearly physiological concentrations to avoid their drainage
from patients’ blood by osmosis.
chloride can be determined by IC in accordance with
the regulations of the US Pharmacopeia (USP) and the
European Pharmacopoeia. The requirements regarding
precision, separation, and recovery of the analytes are
described in detail in the pharmacopoeias. Figure 3
depicts the ion chromatogram of an analysis of gentamicin,
an antibiotic belonging to the group of aminoglycosides.
Aminoglycosides are bactericidal antibiotics that block
bacterial protein biosynthesis by binding to ribosomes,
thereby causing mistakes in the translation from
messenger ribonucleic acid to DNA. Gentamicin consists
of several closely related compounds, namely gentamicin
C1, gentamicin C1a, and gentamicin C2, C2a, and C2b.
In spite of their structural similarity, IC achieves a good
separation of the different gentamicin components.
Impurities in Pharmaceuticals
Impurities in pharmaceutical products can also be
determined by IC. Even small concentrations of an
impurity can cause significant side effects. For example,
in the synthesis of the antihypertensive irbesartan, azide
can be detected in traces as an impurity in the product.

Active Pharmaceutical Ingredients

Active pharmaceutical ingredients (APIs) in medicines
such as gentamicin, neomycin, cefadroxil, or bethanechol

Figure 2: Cations in diluted hemodialysis concentrate using the

Metrosep C 4 - 150/4.0 column and non- suppressed conductivity
detection.
 Azide is strongly toxic to humans and its concentration
in irbesartan is therefore subject to rigorous controls.
According to USP 621, the US Pharmacopeia recommends
ion chromatographic azide determination after direct
injection. In this method, a transfer solution consisting
of the IC eluent and suitable organic solvent is used to
remove the API from the analytical column. However,
this procedure is tedious, time consuming, and cannot be
automated. Azide determination is more selective, more
sensitive, and, above all, quicker with the use of inline
matrix elimination. The interfering pharmaceutical matrix is
separated from the target analyte in the course of sample
preparation. The ion chromatogram shows the analysis of
an irbesartan sample spiked with different concentrations
of azide (See Figure 4).
Figure 4: Irbesartan sample spiked with 5-80μg/L azide; column:
Metrosep A Supp 10 - 250/4.0; eluent: 5mmol/L Na2CO3, 5mmol/L
NaHCO3; inline matrix elimination with 70:30(v/v) methanol/water.
High Sensitivity Thanks to Matrix
Elimination
The signal is recorded by a conductivity detector following
sequential suppression. Table 1 lists the average recovery
values of azide that were achieved over three measurements,
the mean conductivity measured by the detector, and the
Table 1. Precision and Recovery of Azide
Peak area
Mean value (μS/cm)
5 µg/L spike ± 5.00
30 µg/L spike ± 0.30
Figure 3: IC determination of the antibiotic gentamicin by pulsed
amperometric detection; column: Polymer Laboratories RP-S; eluent:
60g/L Na2SO4, 1.75g/L sodium octanesulfonate, 1.34g/L NaH2PO4,
8mL/L THF (pH = 3, H3PO4); post-column addition: 300mmol/L NaOH.
relative standard deviation. The determination of azide
in irbesartan with preceding matrix elimination fulfills
all requirements of the regulatory authorities, including
the selectivity of the method, its limits of detection and
quantitation, precision, linearity, accuracy, and robustness.
Thus, it can be used as a quicker and more sensitive alternative
to the proposed determination according to USP 621.
Radio IC
Radio IC aims to determine the radiochemical purity of
radiopharmaceuticals. These are radioactive substances
that are used for medical purposes, mainly in diagnostics,
but also in the treatment and prevention of certain
diseases. [18F] fluorodeoxyglucose and [18F] fluorocholine
are two prominent examples of radiotracers that are used
in diagnostics by positron emission tomography (PET).
They are labeled with the radionuclide [18F] fluorine.
During the radioactive decay of this unstable isotope,
a proton in the nucleus of [18F] fluorine changes to a
neutron. This is accompanied by the emission of a neutrino
and a positron. The latter combines with an electron in
the surrounding tissue, resulting in annihilation of both
particles and emission of two photons (gamma rays) in
opposite directions, each with an energy of 0.511 MeV.
From the data acquired through coincidence detection of
the photon pair, the location of its emission in the patient’s
body is calculated. This location coincides closely with the
Relative standard devitation (%) Recovery (%)
1.96 101.71
0.14 103.38
Table 2. Selection of IC Applications in the Pharmaceutical Industry
Pharmaceutical or excipient Analyte Pharmaceutical or excipient Analyte
Acamprosate calcium Acetate Glycine carbonate, sodium salt Carbonate
Acifluorfen, sodium Acetate Glimepiride Trans-4-methylcyclohexylamine
Adrenaline Adrenaline Guaifenesin Epichlorhydrine
Amisulpride Dimethyl and diethyl sulfate Heparin sodium Glucosamine and galactosamine
Ibandronate, phosphite,
Anticoagulation solution Phosphate, citrate Ibandronic acid sodium
phosphate
Arsenic trioxide Arsenate, arsenite Indinavir sulfate Ethyl sulfate
Atovaquone Acetate Indomethacin sodium 2-ethylhexane acid
Atorvastatin calcium salt Cyanide, tetrabutylammonium Irbesartan Cyanide, azide
Sulfobutylether-ß-cyclodextrin ß-cyclodextrin Ibuprofen Ibuprofen, valerophenone
Bethanechol, sodium, calcium,
Bethanechol chloride Lamotrigine Cyanide
decomposition product (HPTA)
Bromide salt Chloride Lanthanum carbonate Nitrate
Busulfan Methanesulfonic acid Levetiracetam Tetrabutylammonium
Calcium gluconate Oxalate Levofloxacin Fluoride
Calcium salt Borate Linezolid Morpholine
Camphorsulfonic acid Camphorsulfonic acid Losartan potassium Azide
Carbamazepine Chloride, bromide Meropenem EDTA, dimethylamine
Carbidopa EDTA, hydrazine, sodium disulfite Metformin hydrochloride Dimethylamine
Cefadroxil Cefadroxil (Mono)sulfiram (temosol) Cyanide
Cefdinir Iron, EDTA Montelukast sodium Methanesulfonic acid, acetate
Cefepime hydrochloride N-methyl-pyrrolidinium Multivitamin tablets Cations, Vitamin C
Ceftazidime sodium Sodium Mycophenolate mofetil Morpholine
Clopidogrel besylate Anions, carbonate, cations Nebivolol hydrochloride Monomethylamine
Colesevelam Quaternary alkylamines Neomycin sulfate Neomycin
Copovidone EP Acetate, formate Oxaliplatin Chloride
Dasatinib Ethylenediamine Pioglitazone hydrochloride Piperidine
Dextromethorphan HBr Formic acid Piperacillin Chloride
(2,3-Dichlorophenyl)
Cyanide, tetrabutylammonium Piperazine Piperazine, N-methylpiperazine
oxoacetonitrile
Diclofenac sodium Sodium, potassium RA-Thermoseal toothpaste Potassium, zinc
Dicyclopropylmethylamine Dicyclopropylmethylamine Ribitol Ribitol (adonitol)
Doxazosin, methanesulfonic acid Bromide S-Adenosyl methionine Sulfate
Drospirenone Propargyl alcohol Sevelamer Binding capacity of phosphate
Enoxaparin sodium Sulfate Suxamethonium chloride Choline chloride
Esomeprazole magnesium Tartrate Tadalafil Methanolic methylamine
Monomethylamine,
Febuxostat Hydroxylamine Terbinafine hydrochloride
tetrabutylammonium
 Table 2. Selection of IC Applications in the Pharmaceutical Industry
Pharmaceutical or excipient Analyte Pharmaceutical or excipient Analyte
Carbohydrates, sulfate and
Felodipine Silicate, sodium Topiramate
sulfamate
Fenofibrate Sodium lauryl sulfate (SLS) Triclosan Potassium
Ferumoxide (contrast enhancer) Citrate Timolol maleate Chlorite
Fluorouracil (also fluoruracil) Fluoride Varenicline tartrate salt Trifluormethanesulfonic acid
Gabapentin Chloride Voriconazole Camphorsulfonic acid
Gadopentetate dimeglumine Gadolinium Zingisol Potassium and zinc
Gentamicin sulfate (see page 17) Gentamicin Zoledronic acid Phosphite, phosphate

Detection method: conductivity detection with suppression; direct conductivity detection; conductivity detection with and without
suppression; amperometric detection; spectrophotometric detection

location of the original radiotracer molecule, and thus Conclusion

reveals information on its activity.
The purity of radiotracers is of crucial importance. The highly
energetic gamma rays emitted during the combination of a
positron with an electron are harmful to the human body.
By using a pure radiotracer, thereby avoiding the injection
of free [18F] fluorine or other radioactive contaminants,
the amount of radioactive substance administered to the
patient can be kept to a minimum.
Today, IC covers a diverse field of applications in the
pharma industry (See Table 2). The technique has
become extremely versatile to the user with the large
number of available columns, eluent and gradient options,
varied sample preparation techniques, and automation
possibilities.

The quality control of radiotracers is established by

radio IC in the short time between their synthesis and
the recording of the three-dimensional PET scan. The
separation step in radio IC is the same as in regular IC,
except that it happens behind lead doors. What really sets
radio IC apart from conventional IC is the detection step,
in which a radioactivity detector is added to the setup.
The radioactivity chromatogram reveals the presence – or,
ideally, the absence – of radioactive contaminants.
Chapter 5:
Near-infrared Spectroscopy in Compliance
with Pharma Regulations
M. Schilling, N. Rühl
Introduction
Near-infrared spectroscopy (NIRS) is recognized by
common pharmacopoeias as a secondary method for a fast
and reliable, non-destructive analysis in pharmaceutical
manufacturing. Since the 1960’s it has been explored
and used in the pharmaceutical industry for raw material
identification, process control, and quality assurance of
final products. Because quality control plays a central role
in the pharmaceutical industry and is based on regulatory
requirements and guidelines, the need for an approach like
NIRS has become a valuable tool. Its versatility, reliability,
and ability to meet validation requirements make NIRS
“a highly relevant tool for achieving control when built-
in quality is preferred over quality by testing” by the
U.S. Food and Drug Administration (FDA), the Process
Analytical Technologies (PAT) initiative, guidelines by the
European Medicines Agency (EMA), and the International
Conference on Harmonization in the standards (ICH)
Q8(R2), ICH Q9 and ICH Q10 (1). Here, you’ll discover
how Metrohm solutions are applied and validated across
the pharmaceutical industry to comply with regulatory
requirements.
NIRS Applications
NIRS is a versatile analysis method and can be used for a
vast number of applications throughout the pharmaceutical
manufacturing process. Here example applications will be
explored across the manufacturing line, from inspection of
raw materials, to inline/online process control, to product
development, to quality control of finished products.
Incoming Materials Inspection
According to FDA CFR 211.84 and EU GMP 8, all incoming
materials are tested to verify identity and conformity. The
Metrohm spectrometer product portfolio offers suitable
solutions for a convenient inspection of large quantities
and varieties of incoming materials—such as multiple
varieties of medicinal plants used as raw materials—
whether they are analyzed directly in the warehouse, in
the weighing area, or in the QC lab (2, 3).
Inline/Online Process Control
Metrohm NIRS inline/online analyzers offer real-time
monitoring and optimization of residual solvent and water
content in powders and granulates, such as in lyophilized
products. More specifically, inline monitoring of the
blending, granulation, and drying of lactose anhydrous and
starch 1500 with residual methanol, can occur using an
NIRS XDS Process Analyzer fitted with a fiber optic probe.
The NIR SmartProbe Analyzer can be used to determine
the purity of methylene chloride (MeCl2) solvent recovered
for the manufacture of active pharmaceutical ingredients
(APIs) (4). Viable cell density or drying processes can
also be measured in real time which maximizes time
efficiency and reduces material loss due to extensive
sample preparation. With little to no sample preparation,
NIRS acquires information on both chemical and physical
sample properties in each measurement. From the data
acquired in a single measurement, multiple parameters can
be determined – qualitatively or quantitatively. Because of
its short measuring times and the non-destructive nature
of its measurements, the full potential of NIRS unfolds, in
particular, in process control (5-8).
Atline/Offline Process and Product Development
Metrohm NIRS analyzers allow for monitoring of atline and
offline processes like intermediate and product assays or
blending and granulation (4, 7, 9). Furthermore, the content
uniformity in solid dosage forms (tablets and capsules)
can be determined, as well as tablet characteristics like
hardness and stability. The NIRS XDS MasterLab has been
used to assess content uniformity of chlorpheniramine
maleate (CPM), an antihistamine found in common allergy
medications. Multiple tablets are loaded and scanned
simultaneously with results generated in less than five
minutes (9-13).
Quality Assurance of Finished Products
NIRS allows for real-time quality assurance of finished
 products, such as content determination in creams, gels,
tablets, and capsules. In addition, the full transmission
spectrometer Metrohm NIRS XDS MasterLab guarantees
reliable and highly accurate results when investigating APIs
and excipients in tablets (even in blisters) to ensure that
chemical and physical properties are verified and purity
requirements are met (9-13). Examples include assuring
product strength of the anticoagulant heparin sodium,
the dissolution profile of the beta-blocker propranolol,
and solid dosage form of procainamide HCl used in the
treatment of cardiac arrhythmias (10, 11, 14).
Validation of NIRS
Validation processes for use of NIRS ensure compliance
with regulatory authorities. Following the published
guidelines to develop, validate, submit, and maintain NIRS
analytical procedures ensures complete validation and
fulfillment of those requirements. While the validation
process takes some time and effort, it pays off quickly.
Once the software, instrument, and method are validated,
NIRS provides rapid, reproducible, real-time monitoring
that ensures a high return on investment.
Software Validation
Software that complies with applicable regulations, such
as FDA 21 CFR Part 11 (Electronic records; electronic
signatures) and/or EU Annex 11 (Computerized Systems),
is qualified to be used in any GMP/GLP environment.
Metrohm Vis-NIR spectroscopy software Vision Air fulfills
all technical requirements mentioned in the FDA 21 CFR
Part 11 and the EU Annex (See Figure 1). Vision Air
software fulfills requirements for electronic signatures
with both customizable and predefined fields and provides
for unique log-in and password combinations so that each
user maintains appropriate system access. In addition, this
software fulfills requirements for electronic record storage
Figure 1: Report view in Vision Air.
and audit trails with its database design, detailed fields for
recording instrument and procedural changes, provision of
mandatory sample registration fields, and its capability to
automatically print, store, and back up records (15).
Instrument Qualification
The validation process of instruments consists of three
phases according to USP<1058> and GMP/GLP: installation
qualification (IQ), operational qualification (OQ), and
performance qualification (PQ) as described in Table 1.
Table 1.
IQ: According to USP<1119>: “The IQ requirements help
ensure that the hardware and software are installed
according to vendor and safety specifications at the
desired location.” (16).
OQ: According to USP<1119>: “The instrument’s
performance is controlled with respect to external
certified standards to verify that the system operates
within target specifications. The purpose of OQ is to
ensure that an instrument is suitable for its intended
application. (...) Similar to any spectrophotometric
device, NIR instruments need to be qualified for both
wavelength and photometric scale. Maximum and
reduced light-flux noise tests are also included.” (16).
PQ: According to USP<1119>: “A quality to fit to an
initial scan or group of scans included in the operational
qualification is employed. In such an analysis, it is
assumed that reference standard spectra collected on a
new or a newly repaired, properly operating instrument
represent the best ones available. Comparisons of
spectra taken over time on the identical reference
standards form the basis for evaluating the long-term
stability of an NIR measurement system. The objective is
to ensure that no wavelength calibration shift of change
in sensitivity occurs during ongoing analysis.” (16). PQ is
usually performed by laboratory personnel (1).
Metrohm Qualification and Validation
Metrohm’s Analytical IQ for NIRS instruments meets
all requirements mandated by many governing bodies
(USP<1058>, USP<1119>, GAMP, 21 CFR Part 11, PIC/S,
etc.). Metrohm offers professional installation and startup
of new instruments in compliance with IQ and guarantees
that Metrohm NIRS instruments meet OQ requirements,
including complete documentation. Metrohm also provides
instrument performance certification and subsequent
certification for users through work-related training.
Once software and hardware validation are complete, the
methods are then developed and validated. According to
Ciurczak, “(...) the development laboratory must provide
the end user or designated laboratory with the following
documentation:
• A written procedure
• A method validation report
• System suitability criteria” (1).
The necessary documentation guarantees the suitability
of an analytical procedure when each step of the method
development, method validation, and method transfer
are clearly documented. Metrohm’s Vis-NIR spectroscopy
software Vision Air Complete includes features for
method validation. The chemometric software Vision and
supported third party tools such as CAMO’s Unscrambler
and PLS Toolbox by Eigenvector Research allow for
the development and the validation of identification,
qualification, and quantification methods (See Figure 2).
Figure 2: Report of the USP Wavelength Accuracy Test from Vision software (left) and Metrohm NIRS XDS SmartProbe Analyzer with
standard (right).
Conclusion
Near-infrared spectroscopy (NIRS) is an established
secondary analysis method for offline, atline, online,
and inline applications in the pharmaceutical industry.
Once validated, NIRS offers cost effective and time
saving results with little to no sample preparation or
destruction. Metrohm NIRS solutions and software enable
pharmaceutical analyses that are reliable, compliant
with common regulations, and enhanced by support for
method and application development.
References
1. E.W. Ciurczak and B. Igne, Pharmaceutical and Medical
Applications of Near-Infrared Spectroscopy (CRC Press,
Boca Raton, Florida, 2015).
2. 
Analysis of pharmaceuticals using near-infrared
spectroscopy, Application Bulletin AB-410.
3. Identification of 45 aromatic and medicinal plants used
in cosmetic and pharmaceutical industry, Application
Note AN-NIR-027.
4. Monitoring the purity of recovered solvents by NIRS,
Application Note AN-NIR-021.
5. N. Broad, P. Graham, R. Hailey, A. Hardy, S. Holland, S.
Hughes, D. Lee, K. Prebble, N. Salton and P. Warren,
Guidelines for the Development and Validation of Near-
Infrared Spectroscopic Methods in the Pharmaceutical
Industry, Handbook of Vibrational Spectroscopy (John
Wiley & Sons, Chichester, 2002).
6. 
Robert Mattes et al., Monitoring viable cell density
in bioreactors using near-infrared spectroscopy,
BioProcessing Journal, 2010.

7. Near-infrared spectroscopy for monitoring a single-pot

granulator, Application Note AN-NIR-016.

8. A nalysis of residual moisture in a lyophilized

pharmaceutical product by near-infrared spectroscopy
(NIRS), Application Bulletin AB-358.

9. F ollowing the progress of pharmaceutical mixing studies

using near-infrared spectroscopy, Application Note AN-
NIR- 014.

10. N
 ondestructive, single tablet analysis using the NIRS
XDS RapidContent Analyzer, Application Note AN-
NIR-002.

11. 
NIRS “predictive model” for the release of
pharmaceutical active ingredients from solid dosage
forms, Application Note AN-NIR-017.

12. 
Near-infrared (NIR) assay and content uniformity of
tablets, Application Note AN-NIR-018.

13. 
Determination of active ingredients in solid
(pharmaceutical) dosage forms utilizing solid-state
standard additions, Application Note AN-NIR-001.

14. Q
 uantification of USP heparin units using near-infrared
spectroscopy, Application Note AN-NIR-042.

15. FDA 21 CFR Part 11 requirements for NIR spectroscopy,

White Paper WP-018EN.

16. N
 ear-Infrared Spectroscopy, <1119>, USP 39 (2016).
Chapter 6:
Compliance in the Pharmaceutical Industry
Ashlyn Cooper
In the regulated environment of the pharmaceutical
industry, commissioning and use of analytical instruments
in accordance with the latest standards of the U.S. Food
and Drug Administration (FDA) and GLP/GMP guidelines
is mandatory in order to ensure accurate material analysis,
consistent product manufacturing, and ongoing, verified
monitoring of materials and procedures.
Metrohm has developed a modular system for the
installation and qualification of instruments in strict
accordance with the current regulations and provides
Instrument Qualification (IQ), Operational Qualification
(OQ) and Performance Verification (PV) documentation
that satisfy USP 1058, GAMP, and FDA Title 21 CFR part
11 compliance. This chapter will provide a foundation for
understanding the requirements of analytical instrument
qualification (AIQ) to ensure you demonstrate traceable,
guaranteed, and documented system performance.
Implemented in 2008, the United States Pharmacopeia
(USP) is the only pharmacopeia with a chapter specific to
AIQ. The new USP general chapter <1058> was released
in August 2017 and defines AIQ as the collection of
documented evidence that an instrument performs
suitably for its intended purpose (1). This chapter will help
you understand how USP <1058> defines and manages
AIQ risk, why change control processes are important, and
how integrated software facilitates compliance with 21
CFR Part 11.
Quality
control
check
samples
System
suitability tests
Analytical method validation
Analytical instrument qualification
Figure 1: Components of data quality.
Qualification Lifecycle
Data integrity is the fingerprint of a company’s processes
and products. USP <1058> outlines four components
within the quality triangle which are critical to data quality:
AIQ, method validation, system suitability, and quality
control checks (see Figure 1). While the AIQ contributes
to confidence in the validity of data generated; method
validation provides the proof that an analytical procedure
is suitable for intended use. The system suitability test
verifies that the system will perform in accordance with
set criteria, and the quality control check provides ongoing
assurance of suitable performance and accuracy. The base
layer, AIQ, is expanded into the 4Q lifecycle phases for
the defined specifications, installation assessment, and
monitoring of ongoing instrument performance (Table 1)
(1). While there are no regulatory barriers about who
performs laboratory instrument qualification, the person
performing the work must have the proper education,
training, and experience.
Table 1: The 4Q Phases of Analytical Instrument
Qualification
Design Qualification (DQ): Almost always provided
by the instrument manufacturer, DQ documentation
defines the functional and operational use and intended
purpose of the instrument.
Installation Qualification (IQ): Provided by trained
technicians from the instrument manufacturer and
performed on-site, IQ documents that the instrument is
delivered as designed and specified, is properly installed
in the selected environment, and that this environment
is suitable for the instrument.
Operational Qualification (OQ): Also performed in
the laboratory, OQ demonstrates that an instrument
will function according to its operational specification
testing in the selected environment. OQ demonstrates
fitness for the selected use and should reflect the
contents of the DQ document.
Performance Qualification (PQ): Verifies fitness for
purpose under actual conditions of use. PQ plan must
be defined by the user.
Risk Assessment
USP <1058> manages AIQ risk by classifying instruments
based on complexity into three groups: A, B, or C (Table 2).
Table 2: AIQ Groups
Group A: Standard equipment with no measurement
capability or requirement for calibration, where the
manufacturer’s specification of basic functionality is
accepted as user requirement.
Test Level: “Verify by Observation”
Group B: Standard equipment and instruments providing
measured values as well as equipment controlling
physical parameters (such as temperature, pressure, or
flow) that need calibration, where the user requirements
are typically the same as the manufacturer’s specification
of functionality and operational limits.
Test Level: “Verify by Calibration”
Group C: Instruments and computerized analytical
systems, where user requirements for functionality,
operational, and performance limits are specific for the
analytical application.
Test Level: “Verify by Qualification”
Since the same instrument can exist in more than one
group, depending on its use, risk assessment eliminates
duplication of effort and avoids costly practices such as
repeating qualification steps and generating redundant
documentation (1). When an instrument’s intended use
falls into Group C, all elements of qualification, including
software validation, must be considered to ensure proper
functioning of the instrument. Table 2 shows how an
instrument can be grouped and which test strategy is
therefore required to meet qualifications.
Range of Use Functional Test
Design OQ Verifies Operational
Qualification DQ Qualification
Install
Qualification
Figure 2: Instrument Qualification Lifecycle.
Change Control
After impact has been established for the equipment or
system, it is important to develop a change control process
for lifecycle management. The change control process
should include a documented SOP outlining requirements
for instrument installation, failure management, routine
maintenance and qualification frequency (Figure 2).
Metrohm USA offers comprehensive IQ, OQ and PV
packages that comply with the latest regulations and
standards for easy implementation and liability protection.
Metrohm USA recommends performing preventive
maintenance and an operational qualification service
annually to ensure reliable and accurate results. Key
benefits of these packages include the following:
• Modular document structure
• Individual component tests
• Certified engineer and reference instruments
• Documented proof of whole system operation
functionality
• Traceable monitoring of the system performance
through regular requalification and testing
Software Compliance
The Title 21 Code of Federal Regulations Electronic
Records; Electronic Signatures of the U.S. Food and Drug
Administration (21 CFR Part 11) defines the requirements
for electronic documentation and signatures. This rule,
which has been in effect since August 1997, specifies how
User Defined
Application
Performance Qualified
Qualification State
Repair
System
Suitability &
Check
Relocation Standards
the system components, controls, and procedures have to
be designed to ensure the reliability and authenticity of
electronically stored records (2).

Achieving and maintaining full compliance with 21 CFR

Part 11 necessitates standard operating procedures
(SOPs) that support and complement the functionality of
electronic systems. Products with integrated functions
supporting 21 CFR Part 11 requirements ease the task of
achieving and maintaining full compliance. Systems that
include Metrohm’s tiamo™, MagIC Net, Mira Cal, Vision
Air, and OMNIS software platforms have been developed
from the ground up to satisfy these regulations (3).

Conclusion
Compliance with regulations in the pharmaceutical
industry is essential to successful product development
and manufacture. Employing systems that are designed
to fulfill these requirements streamlines the process
for ensuring complete, ongoing adherence to such
regulations. Following guidelines for assessing instrument
risk, developing change control processes, and using
software designed around these regulations is key to
ongoing success.

References
1. U
 nited States Pharmacopeia General Chapter <1058>
“Analytical Instrument Qualification”.

2. http://www.fda.gov/ora/compliance_ref/Part11.

3. h
 ttps://partners.metrohm.com/
GetDocument?action=get_dms_
document&docid=627230.
For more information, please email:
communications@metrohmusa.com

9702.C9.1006.B © 2019 Metrohm USA. Metrohm and design® is a registered trademark of Metrohm Ltd.