Analyzing Cleaning Validation Samples:

What Method?
By Herbert J. Kaiser, Ph.D.
and
Maria Minowitz, M.L.S.
Steris Corporation

Cleaning validations are very difficult to perform. They can be made easier if an
appropriate method for analyzing the samples is used. The method used should be based
on the previously established residue limits of the active and cleaning agents. There are
many choices of analytical techniques that can potentially be used. This article will
describe various analytical technologies available for use, particularly for cleaning agent
residues. References are provided to guide the reader to more in-depth information.
Cleaning validation in the pharmaceutical industry is of critical importance. 1,2,3 There are
many analytical techniques available that can be used in cleaning validations. 4 The choice
of the technique used in analyzing a particular sample is very important in cleaning
validation. The technique must be appropriate for measuring the analyte at and below the
acceptance residue limit. Todays analytical chemist has a wide variety of techniques
available for use. These choices include specific and nonspecific methods. Many methods
are complementary to each other. The pros and cons of each technique will be examined.
Validating the methods will be discussed, as well. The references included with this paper
can be used to provide more in-depth information to the reader and act as guides to the
available literature.
Choosing the appropriate analytical tool depends on a variety of factors. 5,6 The most
important factor is determining what species or parameter is being measured. 7 Is it an
organic compound or inorganic compound? The next question is measurement. How is this
compound going to be measured? Is it going to be swabbed from a surface or determined
from a rinse water sample? If it is going to be swabbed from a surface, where will this
swabbing occur? Another important factor in choosing an analytical tool is establishing the
limits of the residue. The limit should always be established prior to selecting the analytical
tool.8,9 The limits should not be established solely based on detection limits of a particular
method. Yet, another important factor in choosing an analytical tool is whether or not the
method can be validated. If the method cant be validated, then another technique needs to
be chosen.

Sampling Technique
The sampling technique plays a large role in determining which analytical technique to
use. Some techniques are more applicable for swab samples, and other techniques are
more applicable for rinse water sampling. The acceptable sampling techniques include
direct surface sampling (swab) and rinse water samples.10 The rinse water sample is a
direct measure of potential contaminants, but the analysis should not just be a compendial
test for water. Rinse water analyses should be directed toward responses peculiar to the
possible contaminants. A questionable form of sampling is placebo sampling. The placebo
method sampling is when the product, not containing the active ingredient, is processed in
the specific piece of equipment. This is analyzed for any active that may have been picked
up from the equipment. A problem with placebos is the potential lack of uniformity. The
contaminant may not be evenly distributed throughout the placebo. Another problem is the
analytical power of the tools that are used to analyze the samples. The residue levels may
be extremely low if in fact the contaminant is evenly distributed throughout the sample. The
use of placebos is only acceptable if used with swab or rinse water data. Therefore,
placebos are generally not used because of the additional work involved.

Another important factor to consider in choosing an analytical method is the type of

residue being analyzed. Residues can be drug actives, formulation components, cleaning
agents, organic, inorganic, water soluble, water insoluble, particulate, microbial, and/or
endotoxins. If the residue being detected is a drug active, and the method used for
detection is the same method that is used for quality control purposes of the final formula,
it must be established that the active has not changed its chemical nature during the
cleaning process. That is, it must be established that the active is still detectable and
quantifiable using the analytical method. This can easily be established by performing
forced degradation studies. Exposing the active to the cleaning compound at an elevated
temperature and then analyzing that sample will help determine the compatibility of the
cleaner with the active. If the active has indeed changed its chemical nature during the
cleaning process, a new technique will need to be established for its analysis.
Limit of Detection and Quantitation
Before choosing a method, some definitions need to be established. The Limit of
Detection (LOD) is the lowest amount of a compound that can be detected. The Limit of
Quantitation (LOQ) is defined as the lowest amount of a compound that can be quantified.
The LOD is usually lower than the LOQ, but is never higher. The LOD should never be
used to establish residue acceptance limits. The residue acceptance limit should be well
above the LOQ so that it can be accurately quantitated.
Specific and Nonspecific Methods
A specific method is a method that detects a unique compound in the presence of
potential contaminants. Some examples of specific methods are High Performance Liquid
Chromatography (HPLC), ion chromatography, atomic absorption, inductively coupled
plasma, capillary electrophoresis, and other chromatographic methods. It should be noted
that HPLC is not inherently specific. What is meant is that the conditions in an HPLC
measurement can usually be adjusted to separate out known potential contaminants.
Nonspecific methods are those methods that detect any compound that produces a
certain response. Some examples of nonspecific methods are Total Organic Carbon
(TOC), pH, titrations, and conductivity. A very interesting and sensitive nonspecific
technique is dynamic contact angle.11 Titrations may be specific for acids or bases, but they
are not specific for particular acids or bases. There are, however, specific titrations for
classes of surfactants.12
Interferences
A good nonspecific strategy that could be followed is to first identify possible
interferences. These interferences can be either positive or negative. The nonspecific
property is then measured, and the residue is calculated as if all of the measured property
is due to that residue. For example, if the cleaning agent was the analyte and TOC was the
method used, all of the TOC would be assumed to have come from the cleaning agent and
calculated as such. This would then provide a worst-case upper-limit value.
There are many possible sources of interferences. Cleaning agents and compounds can
be a source of interferences, for example. Active agents and their byproducts, water
system components, maintenance materials, and the atmosphere can all be sources, as
well as people, if samples are not handled properly. The materials used to perform the
analytical method can also be a source of interference. For example, if a swab that has a
high TOC value is used to sample, it could increase the level of TOC detected.
For specific methods, there should be no interference if the method is properly designed.
Again, it should be stressed that the method must be able to follow the analyte after
exposure to the cleaning environment. It is necessary to establish that the cleaning

environment or the cleaning process does not change the analyte. For nonspecific
methods (which measure a nonspecific property), any compound with the property that is
introduced into the sample will interfere. For example, if the method being used is TOC,
atmospheric carbon that may enter the sample could cause interference. With all
nonspecific methods, there is a need to identify potential sources of interference.
High Performance Liquid Chromatography
The first technique that will be discussed is HPLC. Almost every pharmaceutical company
has an HPLC instrument. HPLCs utilize a variety of detectors. These include ultraviolet
(UV), fluorescence, electrochemical, refractive index, conductivity, evaporative light
scattering, and many others. The ultraviolet detector is by far the most common. However,
Evaporative Light Scattering Detection (ELSD) may be the most appropriate detector for
cleaning agents. We will discuss the use of both UV and ELSD detectors in depth.
Ultraviolet Detectors
There are many advantages of using UV detectors. Many compounds have chromophores
and therefore, they can be easily detected by UV. Many instruments are equipped with
diode array spectral capabilities. This allows for easy detection of impurities or potential
contaminants within peaks. Ultraviolet detection usually requires no additional reagents or
post column or pre-column reactions. UV detectors are not harmful to the sample, if that is
important. They are generally inexpensive and readily available. Also, molar absorptivities
are generally not affected by temperature and therefore, there is no need for heating or
cooling the detector.
While there are many advantages of UV detectors, there are also some significant
disadvantages. UV detectors cannot detect all types of compounds and therefore are not
considered to be universal. All compounds do not have chromophores. This is particularly
true of surfactants that are used in the pharmaceutical industry. Dirty cells, air bubbles, and
the use of gradients can affect baseline drift and detection capability. The limits of
detection can be higher than other detector types due to background interferences.
Evaporative Light Scattering Detection
In ELSD, the compound is separated on an HPLC column as usual, and then enters a
nebulizer that is combined with a gas stream and passed through a heated column. The
heated column evaporates the mobile phase leaving the solid analyte in the column. The
solid analyte then passes through a detector that consists of a laser or light source. The
laser or light source is scattered when it hits the solid analyte. The detector then picks up
this scattering.
There are many advantages associated with evaporative light scattering detectors. ELSD
is claimed to be universal. It is called universal because it can detect any type of
compound. ELSDs are simple, versatile, and rugged in use. Since it is a mass detector, all
compounds produce similar responses. Additionally, there is no baseline drift due to mobile
phase effects.
There are two primary disadvantages of ELSD. First, there is a very limited choice of buffer
salts that can be used. Recall that the mobile phase is evaporated or removed, leaving the
analyte. Any buffers that will not evaporate will also produce solid particles that will then be
detected and cause interferences. The second disadvantage is that the nebulizer and
detector must produce consistent particle sizes. This requires careful cleaning and
monitoring of the nebulizer.
Actives and Detergent
There are many types of residues that can be analyzed using HPLC techniques. These

include both actives and detergent residues. When dealing with detergent residues, it is
important to identify what is being analyzed: surfactant, builder components, chelating
agents, etc. The separation and quantitation of surfactants at low levels is difficult, at best.
Industry literature is full of references for surfactant analyses using HPLC. The vast
majority of techniques described in the literature are for the determination of surfactants in
concentrated products.13,14 Therefore, the limits of quantitation and the limits of detection
are rather high. There are also references for the analysis of surfactants related to the
environment.15,16 In environmental analysis, the sample is pre-concentrated so that the
limits of quantitation are very low. The pre-concentration can be up to one thousand fold.
Suggested Reading
Authors Lin, et. al., compared the analysis of anionic, cationic, and amphoteric surfactants
containing n-dodecyl groups using HPLC and capillary electrophoresis. 17 They found that
HPLC was best for all classes of surfactants, especially for formulated surfactants. Authors
Carrer, et. al., utilized ELSD for amphoteric type surfactants. 18 Amphoteric surfactants are a
class of surfactants that display cationic behavior in an acidic solution and anionic behavior
in an alkaline solution. The lowest calibration standard that they utilized was 50 ppm, but
they probably could have gone much lower. Authors Guerro, et. al., obtained a limit of
quantitation of 0.49 ppm for alkyl polyethylene glycol ethers using ELSD. 19
Capillary Electrophoresis
An interesting method of analysis is Capillary Electrophoresis (CE). There are many
different types of CE. Capillary Zone Electrophoresis (CZE) is by far the most common. CE
instrumentation is fairly simple, consisting of a high voltage source, a capillary, and a
detector. The high voltage source is used to apply a potential across two solutions. One of
the solutions contains the analyte, and the potential applied to the solutions causes the
analyte to migrate through the capillary, through the detector, and into the other solution.
The column or capillary is typically composed of fused silica with a polyimide coating. The
diameter of the capillary is typically 25-75mm in diameter. The capillary has a polyimide
coating simply to make it more rugged. All common detection techniques (UV,
fluorescence, etc.) can be used in capillary electrophoresis detection. The capillary itself
serves as the detector cell. A small portion of the polyimide coating is scraped off prior to
use, and the bare portion of the capillary is placed in the light path. This detection is
different from that seen in HPLC because the detection occurs while the separation is
taking place, rather than after separation has been completed. Using a Z-cell can increase
the sensitivity of the technique. This is accomplished by using a special accessory that
bends the capillary, causing the source radiation to penetrate lengthwise through the
capillary rather than a cross-sectional sampling. This, in effect, increases the path length of
the cell. The Z-cell can be used in all types of CE where UV detection is used.
CE can be used for many different types of analyses. Surfactants can be determined
quite readily using this technique.20,21 However, detection limits typically are higher than with
HPLC. This can be overcome by pre-concentrating the samples on the capillary itself. A
voltage is applied to the capillary in a manner that allows the compounds to collect at one
end of the capillary without flowing through to the detector. An advantage that capillary
electrophoresis holds over HPLC is the ease with which indirect detection can take place.
Indirect detection is where a highly UV-absorbing material is included in the mobile phase.
As the analyte is eluted or travels along the capillary through the detector, a negative peak
is seen for the analyte. This typically is done for compounds that display low UV
absorption. In addition to being useful for the analysis of surfactants, capillary
electrophoresis can be used to analyze organic acids, inorganics, 22 and trace drug

residues.23
Suggested Reading
Vogt, et. al., provided a good overview of the separation of cationic, anionic, and nonionic
surfactants using capillary electrophoresis. 24 They indicated that one can easily adjust the
parameters of the separation to coelute or separate oligomers. Coelution of the oligomers
increased the sensitivity at the expense of increasing the potential for coeluting positive
interferences. Direct UV detection could be used for UV-absorbing materials and indirect or
non-UV absorbing materials.
Heinig, et. al., utilized micellar electrokinetic capillary chromatography for the separation
of non-ionic alkylphenol polyoxyethylene type surfactants. 25 However, the use of this
method was limited because of insufficient peak resolution and relatively low detection
sensitivity. Heinig, et. al., also compared HPLC and CE analyses of surfactants. 26 The
surfactant types they studied were linear alkylbenzenesulfonates,
nonylphenolpolyethoxylates, cetylpyridinium chloride, and alkylsulfonates. For the CE
analyses, they utilized UV detection either in the direct or indirect modes, depending on the
nature of the surfactant. For the HPLC analyses, they utilized either direct UV detection or
conductivity detection. Anionic surfactant samples were pre-concentrated one thousand
fold through the use of solid phase extraction. This allowed for detection limits in the parts
per billion range to be obtained.
Kelly, et. al., utilized CE with indirect detection to determine sodium dodecylsulfate
concentrations.27 They also indicated that it is important to look at the absorption of the
surfactants onto filters if the samples are indeed filtered prior to analysis. This is most
important in dilute solutions. Filtering large volumes of sample can minimize this. Again,
appropriate studies need to be done to determine if this indeed is a problem.
Altria, et. al., examined the use of CE in the analysis of sodium
dodecylbenzenesulphonate.28 They obtained a limit of quantitation of 0.6 ppm and a 0.3
ppm limit of detection. They utilized direct UV detection. Shamsi, et. al., utilized CE with
indirect detection for the determination of cationic and anionic surfactants. 29 The authors
obtained limits of detection of 0.25 and 0.5 ppm, respectively. Heinig, et. al., also utilized
CE in the analysis of cationic surfactants using indirect UV detection. 30 They compared this
with HPLC. They obtained a limit of quantitation for CE of 4.0 ppm; and for HPLC, they
obtained a limit of quantitation of 5.0 ppm.
Total Organic Carbon
TOC is used widely in the pharmaceutical industry. 31,32,33 The TOC is determined by the
oxidation of an organic compound into carbon dioxide. This oxidation can occur through a
number of mechanisms depending on the instrument being used. Some typical methods
are persulfate, persulfate/UV oxidation, and direct combustion. The carbon dioxide that is
produced from these oxidations is either measured using conductivity or infrared
techniques. Instruments generally measure the inorganic carbon content of a sample. The
inorganic carbon consists of carbon dioxide, bicarbonate, and carbonate. They then
determine the total carbon content of the sample. The TOC is then computed by
subtracting the inorganic carbon concentration from the total carbon concentration of the
sample.
There are two primary advantages associated with TOC. The first is that it does not take
long to develop a method. There are not a lot of variables in the actual analysis. The
second advantage is that it is relatively quick. A third potential advantage (which can also
be a disadvantage) is that it will detect and analyze any compound containing carbon.
As with most techniques, there are disadvantages in using TOC. A significant
disadvantage is that the compound or the analyte must be water soluble. This does not

mean that the compound must be soluble in the hundreds of parts per million range but
soluble in the low parts per million range. Another disadvantage is that organic solvents
cannot be used. If organic solvents were used, the TOC of the solvents would be
measured instead of the residue. There are also many sources of contamination that can
occur using TOC. These sources can include the atmosphere, the swab itself, personnel,
and many other sources. Methods developed using TOC should be written to include
controls and blanks to identify or account for possible contamination. For example, a
common source of contamination is the technique used to cut the handles of the swabs so
that they fit into the TOC vials. Many times, the scissors or utensils are not clean enough
for TOC use. This introduces contamination into the sampling vial when the swab is cut.
Excipients
Some methods/techniques can be used in certain situations to complement each other.
Examples include TOC and HPLC. Consider the case of a drug in the presence of
excipients. The excipients are very soluble in water while the drug active has extremely low
solubility in water. The excipients contribute to the TOC values because they are very
soluble in water; however, the drug active does not show up in the TOC analysis. An HPLC
analysis is performed to monitor the loss of the drug. The excipients are removed much
faster from a surface during cleaning than the drug active is removed. In this case, TOC
analysis is not a good stand-alone method. It is, however, a good complement for the
HPLC assay. The TOC analysis enables the analyst to see what water soluble matter is
left behind, if any.
Suggested Reading
Guazzaroni, et. al., examined the use of total organic carbon for the analysis of
detergents, endotoxins, biological media, and polyethylene glycol. 34 For detergents, they
were able to obtain a 0.7 ppm limit of quantitation. Endotoxins were found to have a 0.2
ppm limit of quantitation. The biological media produced a total organic carbon limit of
quantitation of 20.3 ppm; and the polyethylene glycol produced a 0.5 ppm limit of
quantitation. They examined swab and rinse water recoveries. They were able to obtain
78-101 percent recoveries utilizing swabs, and 93 percent or better for rinse water
recoveries.
There are many examples in the literature that utilize ion chromatography as the method
for analysis of surfactants.35 The surfactants have to be charged in order to be analyzed
using ion chromatography, that is, only anionic or cationic surfactants can be detected.
Pan, et. al., recorded limits of quantitation down to 0.5 ppm for linear alkane sulfates and
sulfonates.36 Takeda, et. al., recorded a limit of quantitation of 0.1 ppm for dodecyl alkyl
sulfates.37 Nair, et. al., separated different sulfate, sulfonate, and cationic type surfactants
using ion chromatography with suppressed conductivity detection. 38 They reported
detection limits at less than 1.0 ppm.
Ion Chromatography
In addition to its use for surfactants, ion chromatography can be used for the analysis of
inorganics and other organic compounds present in cleaners. 39,40,41 Most cleaners contain
sodium and/or potassium. The ion chromatography detection technique of suppressed
conductivity is more sensitive to potassium than it is to sodium. Very low levels of cleaning
agent can be detected using this technique. This assumes that the rinse water used
contains no potassium. Ionizable organic acids are also readily quantitated using ion
chromatography. This includes chelating agents that are often found in cleaning
compounds.

Suggested Reading
In determining surfactants, an excellent review concerning their analysis was done by
Vogt, et. al..42 They compared the use of HPLC, CE, ion chromatography, Liquid
Chromatography-Mass Spectroscopy (LC-MS) and Gas Chromatography-Mass
Spectroscopy (GC-MS). They also discussed pre-concentration of the samples. They
compared the use of solid phase extraction, super critical fluid extraction, Soxhlet
extraction, and steam distillation as means of pre-concentrating samples. They found, by
far, that the best method was solid phase extractions for the pre-concentration of
surfactants. They also examined the use of titrimetric methods of analysis for surfactants.
For detecting anionics, substances like methylene blue, pyridinium azo, and
triphenylmethane dye was used to complex the surfactants prior to photometric
determination. Nonionics were determined indirectly by forming a cationic complex with
barium. This complex was then precipitated by bismuth tetraiodide ion in acidic acid. The
bismuth was then quantified by potentiometric titrations. Cationics were complexed with
anionic dyes such as disulfine blue.
Theile, et. al., brought up an excellent point that surfactants tend to concentrate at
interfaces.43 This can be a problem in extremely dilute solutions of surfactants. The
surfactants can collect at the surface of the containers that they are stored in. This may
cause errors in analysis. Proper controls in studies should be done to determine if this is a
problem. The authors indicated that pre-concentration was required to determine very low
levels of surfactant. Solid phase extraction was the best method for this. They were also
able to obtain detection limits for linear alkylbenzenesulfonates of 2.0 ppb with
fluorescence detection and 10.0 ppb using HPLC with UV detection after preconcentration.
Thin-Layer Chromatography
There are many examples in the literature for the use of Thin-Layer Chromatography (TLC)
for the qualitative determination of surfactants. 44,45 Henrich described the TLC of over 150
surfactants in six different TLC systems.46 This was excellent for identification of the
surfactants, but the author did not attempt to quantify the surfactants. Buschmann and
Kruse combined diffuse reflection infrared spectroscopy and TLC, along with SIMS and
TLC for surfactant identification.47 Although these techniques are tedious and timeconsuming, there is no doubt that these methods could be developed into quantitative
analyses. Novakovic has used high performance TLC for two generic drugs. 48
Other Techniques
Other excellent techniques for inorganic contaminants, and in some cases actives, are
Atomic Absorption (AA)49 and Inductively Coupled Plasma (ICP) atomic emission. These
techniques can detect inorganic contaminants down to extremely low levels. Inorganic
contaminants in a system are often ignored. These can come from rouge that forms in
Water for Injection (WFI) systems. They can also come from the detergent utilized in
cleaning the equipment.
Fourier-Transform Infrared Spectroscopy
Fourier-Transform Infrared (FTIR) spectroscopy is never used as a stand-alone method
for analyzing residues on equipment. This is because of the lack of portability of FTIR
equipment and the semi-quantitative nature of the reflectance techniques used for these
types of analyses. However, it is very useful in performing screening studies and in
evaluating potential cleaning agents. This is done by soiling standard coupons with the
cleaning agent, allowing them to dry, and performing static rinsing studies. These types of
studies can indicate whether or not the cleaning agent is readily removed from surfaces.

The height or area of a particular peak is measured versus the concentration of the
standard coupon.
Bioluminescence
Bioluminescence is quite useful for biologicals. This type of analysis usually uses
Adenosine Triphosphate (ATP) bioluminescence.50 This is based on the reaction of ATP
with Luciferin/Luciferase. This technique is often used in biopharmaceutical facilities. It has
extremely high sensitivity and a very high reproducibility. In many cases, the instruments
can be used at the equipment site. This technique utilizes swabs for surface analyses.
Optically Stimulated Electron Emission
In some cases, a companys established limits of residue are so low that they cannot be
detected by conventional methods. A very sensitive method that may be applicable is
Optically Stimulated Electron Emission (OSEE).51 The instrumentation for OSEE is fairly
portable, and can be readily taken to tank side for analysis. The technique uses a probe
that is placed against a surface, and a UV source illuminates and activates the surface.
When some surfaces are exposed to UV light at certain wavelengths, electrons are emitted
from the surface. The instrument measures the current that is produced. If even small
amounts of residues are present on the surface, the current will be affected. The current
can be affected either in a positive or negative way depending on the nature of the residue.
This is an extremely sensitive technique. It can be used in either a qualitative or
quantitative manner.
Portable Mass Spectrometer
For those companies that require ultrasensitive measurements and identification of the
residues, a technique has been developed Lawrence Livermore National Laboratories
has developed a portable mass spectrometer.52 The unit consists of a gun portion of the
instrument that is connected with cables to vacuum pumps. The gun portion is held against
the surface to be analyzed. A seal is formed, and the surface is heated to volatilize any
compounds that are present. This instrument is used not only to measure how much of
something is present, but also what that something is. This piece of equipment has been
utilized in the aerospace industry. One drawback of the portable mass spectrometer is that
it requires relatively flat surfaces. However, they are currently working on adaptors to be
used on non-flat surfaces.
Additional Techniques
In the biopharmaceutical industry, a wide variety of techniques are utilized. 53 These
include the Enzyme-Linked Immunosorbent Assay (ELISA),54 the Limulus Amoebocyte
Lysate (LAL), and a wide variety of protein determinations. These are all contaminant
specific assays. For example, the LAL test measures the level of endotoxins present.
There is also the anthrone assay that can be used to monitor the levels of carbohydrates
on surfaces. These techniques are usually used in combination with TOC.
The nonspecific techniques of pH, conductivity, and titrations can be used throughout all
areas of pharmaceutical manufacturing. Obviously, these techniques are most often
utilized in rinse water monitoring. The conductivity and pH of rinse water is typically
monitored and compared to the conductivity and pH of the water prior to introduction to the
equipment. If acidic or alkaline materials are being measured, titration is a very useful
technique. In some cases, titration can be more sensitive than performing TOC analyses.
The sample size can be adjusted, and/or the normality of the titrant can be adjusted to
increase the sensitivity of the titration.

Method Validation
It is very important to scientifically establish the residue limit prior to choosing the method
of analysis. This includes the limit in the analytical sample and the limit in the next product.
This will ensure that the method chosen will be able to detect and quantitate the limit
chosen. Once the technique for analysis has been chosen, it is very important to validate
the method used.55-60 The validation of a method is very different than the validation of
recovery. A validated method is one that is rugged and robust enough to measure the
residue limit established. The validation of a recovery is to determine the amount that can
be recovered from a surface. Again, it should be stressed that these are two completely
different validations.
Twos of Validation
A minimum validation requires two different analysts, instruments, columns (if
chromatographic method), days, and prepared standards and samples. 60 These are the
twos of method validation. The point of any method validation is to show that the method
can be utilized by different analysts and/or laboratories, along with the ability to produce
the same results. If a validated method is given to a laboratory, that laboratory must
revalidate the method for their laboratory. It is not sufficient or accurate to assume that
another laboratorys validation will apply in all laboratories. For example, if a surfactant is
being quantitated, typically a low wavelength is used in a UV detector for HPLC. UV
detectors vary in their noise levels at these low wavelengths. A detector used in one
laboratory may have significantly less noise than a detector in another laboratory. The
second laboratory may not be able to detect at the same low level as the first laboratory.
Coupons and Swabs
Coupons can be prepared for recovery studies through the use of aerosol bottles
available through laboratory supply companies. A known weight percent of a solution
containing the analyte can be sprayed fairly evenly over the surface of a coupon. The
coupon can be swabbed using a standard technique. It does not matter how you swab the
coupon, as long as the complete surface is covered and that the coupons are swabbed the
same way each and every time. The type of swabs used in recovery studies must be the
same as those used in the validation protocol. If this is a simulated rinse procedure, then
the coupons are rinsed and the rinse water is analyzed.
For swabs, a desorption process is carried out. This can consist of simply shaking the
sample vial or using an ultrasonic bath. The samples are then analyzed. Recovery studies
are always done below acceptance limits in the test solution. This ensures that the limit will
be (or can be) measured in the analysis. A recovery of greater than 80 percent is good. If
the recovery is greater than 50 percent, it may be acceptable. However, if the recovery is
less than 50 percent, questions arise and the source of the poor recovery should be
investigated. A possible cause of a poor recovery can be that the residue is being too
tightly held by the swab. This can be investigated by spiking a swab with a known amount
of residue, allowing it to dry, trying to desorb the residue, and following up with analysis. If
the analyte is held too tightly by the swab, another type of swab material should be
investigated. The recovery factor should be included in analytical calculations or in the
acceptance limit calculation. It should not be included in both of the calculations.
Containers
The choice of containers used in the analysis of samples is very important. It has been
shown that, in very dilute solutions, surfactants can adsorb onto the surfaces of sample
vials. This will produce artificially low results in the analysis. This, however, typically only

occurs in static systems. There is no need to worry about the adsorption of the surfactant
on the walls of manufacturing equipment. This is because the agitation that is involved in
cleaning removes the surfactants from the surfaces. This is another matter in sample vials.
Appropriate spiking studies should be performed to ensure that this phenomenon is not
occurring and will not interfere with the analytical method. This includes both HPLC or ion
chromatography sample vials, as well as TOC sample vials. This phenomenon is not
limited to surfactants. Proteins have been shown to adsorb readily onto glass surfaces.
These proteins are much more difficult to remove from surfaces than surfactants.

Specific Versus Nonspecific

The choice of using a specific or nonspecific method can be difficult. If a drug active is
highly toxic, a specific method is always recommended. 61Detergents can be quantitated
either using specific or nonspecific methods; however, care must be taken in choosing
which component is measured. For example, a detergent may contain five percent of a
surfactant and 20 percent of another organic ingredient. Assuming equal sensitivities of the
analytical methods, the limit of quantitation of the whole detergent system will be lowered
by a factor of four if the ingredient present in the greater amount is determined.
If a nonspecific method (i.e., TOC) is used for the same system, a much lower limit of
quantitation could be determined simply because there would be a tremendous amount of
carbon present in the sample. In addition, if detergent systems are combined, such as in
the case of adding a detergent additive to another product, the choice of a specific method
would be made even more difficult. The question would be, Which detergent do I
determine? A disadvantage of using a nonspecific method for the entire cleaning
validation analysis is that, if there is a failure in the future, it would not be known where the
failure originally occurred. The failure could be due to the active, excipients, detergent
system, or even an unknown source.

Conclusion
There are many different analytical techniques available that can be used to detect
residues. These range from simple titrations to more complex LC-MS. The choice of
technique should be based on what equipment is available, the type of residue, and the
scientifically established residue limit. It is important for an analytical chemist to keep
abreast of the literature and what techniques are available. There are techniques available
that will analyze any residue at any level. At the end of the day, however, it is always wise
to choose the simplest technique that can be used to reach the desired goal. o
About the Authors
Herbert J. Kaiser, Ph.D. is Manager Hard Surface Products at STERIS Corporation. He
has 18 years of experience in cleaning and surface technologies, which includes
developing products and methods for the cleaning and analyzing of a wide variety of
surfaces. Dr. Kaiser has developed a wide variety of products for the healthcare, industrial,
and pharmaceutical markets. He is the sole inventor listed in five United States Patents for
various industrial treatment schemes. Dr. Kaiser received his B.A. degree from St. Marys
University in San Antonio, Texas, his M.S.(R) from St. Louis University, and his Ph.D. from
the University of Missouri. He is a member of the American Chemical Society and the
Association for the Advancement of Medical Instrumentation. Dr. Kaiser can be reached by
phone at 314-290-4725, by fax at 314-290-4650, or e-mail at herb_kaiser@steris.com.

Maria Minowitz, M.L.S., Information Associate at STERIS Corporation, has 10 years of

experience in corporate research and development librarianship. She has been
responsible for information management in the disciplines of chemistry, medicine, and
engineering. Minowitz received her A.B. degree from St. Louis University and an M.L.S.
from the University of Missouri-Columbia. She is a member of the Special Libraries
Association, Midcontinental Chapter of the Medical Library Association, and the St. Louis
Medical Librarians.
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