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 contain ing 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 uni formity. The contaminant may not be evenly distrib uted 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 detect ed. 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 ac cep tance 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 con taminants. 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 par ticular acids or bases. There are, however, specific titrations for classes of surfactants.12
Interferences
A good nonspecific strategy that could be fol lowed is to first identify possible interferences. These interferences can be either positive or negative. The nonspecific property is then measured, and the resi due 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 worstcase upperlimit value.
There are many possible sources of interferences. Cleaning agents and compounds can be a source of
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.
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 interfer ence if the method is properly designed. Again, it should be stressed that the method must be able to fol low the analyte after exposure to the cleaning en viron ment. 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 prop erty that is introduced into the sample will interfere. For example, if the method being used is TOC, atmo spheric carbon that may enter the sample could cause interference. With all nonspecific methods, there is a need to identify potential sources of interference. n 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, elec trochemical, refractive index, conductivity, evapo rative light scattering, and many others. The ultra violet de tector 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 detec tors in depth.
• Ultraviolet Detectors
There are many advantages of using UV detec tors. Many compounds have chromophores and therefore, they can be easily detected by UV. Many in struments are equipped with diode array spec tral capabilities. This allows for easy detection of impurities or potential contaminants within peaks. Ultraviolet detection usually requires no additional reagents or post column or precolumn reactions. UV detectors are not harmful to the sample, if that is important. They are generally inexpensive and read ily available. Also, molar absorptivities are gener ally not affected by temperature and therefore, there
is no need for heating or cooling the detector. While there are many advantages of UV detec tors, 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 evap orative 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 par ticle sizes. This requires careful cleaning and moni toring of the nebulizer.
Actives and Detergent
There are many types of residues that can be ana lyzed 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,
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.
chelating agents, etc. The separation and quantita tion of surfactants at low levels is difficult, at best. Industry literature is full of references for surfactant analyses using HPLC. The vast majority of tech niques described in the literature are for the deter mination of surfactants in concentrated products.13,14
There fore, the limits of quantitation and the limits of detection are rather high. There are also references for the analysis of surfactants related to the environ ment.15,16 In environmental analysis, the sample is
preconcentrated so that the limits of quantitation are very low. The preconcentration
can be up to one thousand fold.
Suggested Reading
Authors Lin, et. al., compared the analysis of anionic, cationic, and amphoteric surfactants con taining ndodecyl groups using HPLC and capillary electrophore sis.17 They found that HPLC was
best for all classes of surfactants, especially for for mulated 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 cali bration 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
n 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 capil lary, 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 ap plied 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 2575µm 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 Zcell 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 crosssectional sampling. This, in effect, increases the path length of the cell. The Zcell can be used in all types of CE where UV detection is used.
CE can be used for many different types of analy ses. 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 preconcentrating 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 dis play low UV absorption. In addition to being useful for the analysis of surfactants, capillary electrophore sis can be used to analyze organic acids, inorganics,22
and trace drug residues.23
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