carbon concentration of the sample.~

Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

Vogt, et. al., provided a good overview of the separation of cationic, anionic, and nonionic surfac­ tants using capillary electrophoresis.24 They indicated

that one can easily adjust the parameters of the sepa­ ration to coelute or separate oligomers. Coelution of the oligomers increased the sensitivity at the ex pense of increasing the potential for coeluting positive interferences. Direct UV detection could be used for UV­absorbing materials and indirect or non ­UV absorbing materials.

Heinig, et. al., utilized micellar electrokinetic cap­ illary chromatography for the separation of non­ionic alkylphenol polyoxyethylene type surfactants.25 How­

ever, the use of this method was limited because of insufficient peak resolution and relatively low detec­ tion sensitivity. Heinig, et. al., also compared HPLC and CE analyses of surfactants.26 The surfactant types

they studied were linear alkylbenzenesulfonates, nonylphenolpolyethoxylates, cetylpyridinium chlo­ ride, 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. An ionic sur­ factant samples were pre­concentrated 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 detec­ tion to determine sodium dodecylsulfate concentra­ tions.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 analy­ sis of sodium dodecylbenzenesulphonate.28 They

ob tained 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 de tection for the determination of cationic and anion­ ic surfactants.29 The authors obtained limits of detec­

tion 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.

n Total Organic Carbon

TOC is used widely in the pharmaceutical indus­ try.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 mecha­ nisms depending on the instrument being used. Some typical methods are persulfate, persulfate/UV oxidation, and direct combustion. The carbon diox­ ide that is produced from these oxidations is either measured using conductivity or infrared techniques. In stru ments 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 sam­ ple. 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 it self, personnel, and many other sources. Methods de veloped using TOC should be written to include controls and blanks to identify or account for possible contamination. For example, a common source of con­ tamination 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.

Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

Some methods/techniques can be used in certain situations to complement each other. Examples in clude 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 ex tremely low solubility in water. The excipients con tribute 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 ex ci pients are removed much faster from a surface during cleaning than the drug active is removed. In this case, TOC analysis is not a good stand­alone 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 or ganic carbon for the analysis of detergents, endo­ toxins, 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 78­101 percent recoveries utilizing swabs, and 93 percent or better for rinse water recoveries.

There are many examples in the literature that uti­ lize 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 sulfo­ nates.36 Takeda, et. al., recorded a limit of quantita­

tion of 0.1 ppm for dodecyl alkyl sulfates.37 Nair, et.

al., separated different sulfate, sulfonate, and cat­ ionic type surfactants using ion chromatography with suppressed conductivity detection.38 They reported

detection limits at less than 1.0 ppm. n Ion Chromatography

In addition to its use for surfactants, ion chro­ matography can be used for the analysis of inor­ ganics and other organic compounds present in

cleaners.39,40,41 Most cleaners contain sodium and/or

potassium. The ion chromatography detection tech­ nique 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 chro­ matography, Liquid Chromatography­Mass Spectro­ scopy (LC­MS) and Gas Chromatography­Mass Spectro scopy (GC­MS). They also discussed pre­con­ centration of the samples. They compared the use of solid phase extraction, super critical fluid extraction, Soxhlet extraction, and steam distillation as means of pre­concentrating samples. They found, by far, that the best method was solid phase extractions for the pre­concentration of surfactants. They also examined the use of titrimetric methods of analysis for surfactants. For detecting anionics, substances like methylene blue, pyridinium azo, and triphenyl­ methane dye was used to complex the surfactants prior to photometric determination. Non ionics 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 pre­concentration was re quired 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 fluo­ rescence detection and 10.0 ppb using HPLC with

Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

UV detection after pre­concentration. n Thin­Layer Chromatography

There are many examples in the literature for the use of Thin­Layer 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 iden­

tification of the surfactants, but the author did not attempt to quantify the surfactants. Buschmann and Kruse combined diffuse reflection infrared spectros­ copy and TLC, along with SIMS and TLC for sur­ factant identification.47 Although these techniques

are tedious and time­consuming, there is no doubt that these methods could be developed into quantita­ tive analyses. Novakovic has used high performance TLC for two generic drugs.48

Other Techniques

Other excellent techniques for inorganic con­ taminants, 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 lev­ els. 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.

n Fourier­Transform Infrared Spectroscopy

Fourier­Transform Infrared (FTIR) spectroscopy is never used as a stand­alone method for analyzing residues on equipment. This is because of the lack of portability of FTIR equipment and the semi­quanti­ tative nature of the reflectance techniques used for these types of analyses. However, it is very useful in performing screening studies and in evaluating po tential 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. n Bioluminescence

Bioluminescence is quite useful for biologicals. This type of analysis usually uses Adenosine Tri­

phosphate (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 repro­ ducibility. In many cases, the instruments can be used at the equipment site. This technique utilizes swabs for surface analyses.

n Optically Stimulated Electron Emission

In some cases, a company’s 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 posi­ tive 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.

n Portable Mass Spectrometer

For those companies that require ultrasensitive measurements and identification of the residues, a technique has been developed – Lawrence Liver­ more National Laboratories has developed a port­ able 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 com­ pounds 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 equip­ ment 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 non­flat surfaces.

Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

In the biopharmaceutical industry, a wide vari­ ety of techniques are utilized.53 These include the

Enzyme­Linked Immunosorbent Assay (ELISA),54

the Limulus Amoebocyte Lysate (LAL), and a wide variety of protein determinations. These are all con­ taminant 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 sur­

faces. 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. Ob viously, these techniques are most often utilized in rinse water monitoring. The conductivity and pH of rinse water is typically moni­ tored and compared to the conduc­ tivity 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.