Examples of Contamination

Fibers

Fibers are a commonly encountered contaminant.  Fibers are usually identified using polarized light microscopy alone, but FTIR is sometimes used to confirm the chemical identification.  Sometimes, SEM is used to examine the fiber morphology.  Most often, paper, cotton, and colorless polyester fibers are observed in pharmaceuticals.  These fibers are related to cleanroom wipes and garments.  Sometimes, a variety of fibers is found within the same sample, and this is an indication of a more serious contamination issue.  Figure 1 shows some typical fibers recovered from a pharmaceutical product.

click image to enlarge (228K) FIGURE 1 Examples of paper, cotton and polyester fibers recovered from a pharmaceutical product.

Glass

Glass is another contaminant that is often observed during quality control screening.  Glass particles can be generated by fracture of the vial neck or opening, from external sources such as other vials, glassware and lighting, and from delamination of the inner vial surface. Because of their density, glass particles sink rapidly to the bottom of the vial when the liquid is agitated.  Glass delamination flakes are extremely thin and may be missed during visual examination.  If a larger number are present, a “twinkling” effect in the solution is observed.  The “twinkling” is best observed using a fiber optic light source.   Delamination flakes do not sink to the bottom of the vial.  Glass delamination usually occurs when a highly acidic, highly basic or sodium chloride solution is stored in the wrong type of container. 

click image to enlarge (164K) FIGURE 2 Glass particles recovered from a pharmaceutical product, as they appear with oblique illumination.

Glass particles have distinctive morphology and may be identified by microscopical analysis.  Figure 2 shows several glass particles on a polycarbonate membrane filter.  The glass particles are shiny, refractive and have a characteristic conchoidal fracture.  When examined using polarized light, glass is isotropic.  Refractive index measurement and elemental analysis can be used to determine the type of glass and to compare it to suspect sources.  The elements lithium and boron are present in some types of glass, and these light elements can not be detected using EDS detectors; if detection of these elements is needed, the particles can be analyzed using secondary ion mass spectrometry (SIMS).

click image to enlarge (224K) FIGURE 3 Glass delamination flakes recovered from a pharmaceutical product, as they appear on a polycarbonate filter membrane. Note the small holes or pits on the flakes. Image was taken using episcopic illumination.

click image to enlarge (301K) FIGURE 4 Glass delamination on inside of vial wall. This image shows the delamination at the bottom of the vial. Image was taken using episcopic illumination.

click image to enlarge (290K) FIGURE 5 Glass delamination on inside of vial wall. The fill line of the vial is evident. Image was taken using episcopic illumination.

Glass delamination flakes as they appear on the filter membrane are shown in Figure 3.  Using correct illumination is critical.  Due to their extreme thinness, the flakes would not be observed on a membrane filter using oblique or side light; they are only seen using episcopic illumination or top light.  If glass delamination flakes are detected, the interior of the sample container should also be examined microscopically.  The beginnings of delamination are observed as small pitted areas resembling circles and doughnuts on the interior glass surface. A vial interior exhibiting a severe case of delamination is presented in Figure 4.  The heaviest delamination tends to occur at the bottom sides of the vial, and lessens further up the vial sides.  In Figure 5, the fill line of the vial is evident. 

Silicone

Silicone is used in many products and it is frequently found in pharmaceutical products.  When it is used as a lubricant for rubber stoppers and plungers, it very easily “sloughs off” and ends up in the product.  Silicone can interact with protein based drugs or active ingredients producing particles or residue.  Thermal degradation of the silicone can occur if the product is autoclaved.  Silicone oil can be observed in liquid products as oil droplets. Sometimes it occurs as fine droplets that give the solution a hazy appearance.  After filtration of the product, silicone oil appears as “cleared” areas on the filter that can be observed using transmitted light.  Figure 6 shows the cleared zones on a polycarbonate filter.  Degraded silicone, or silicone that has interacted with protein or active ingredient, forms a semi-solid particulate residue that appears as stringy “fiber-like” particles in solution.  Stringy silicone particulate, as it appears on the membrane, is shown in Figure 7.  It is essential to use the correct type of illumination (epi-illumination in this case), as this type of residue often cannot be seen with transmitted or oblique illumination. 

click image to enlarge (271K) FIGURE 6 Cleared areas on a polycarbonate filter membrane caused by silicone. Image was taken using transmitted light.

click image to enlarge (348K) FIGURE 7 Stringy, degraded silicone particulate residue as it appears on a polycarbonate filter membrane. The image was taken using episcopic illumination.

Suspect silicone residues are prepared for analysis using FTIR to confirm the identification.  The silicone oil can be recovered from the filters with oily or cleared zones by extracting a small portion of the filter with nonane.  This procedure is performed onto a polished salt plate for FTIR analysis.  Stringy residues can be scraped off the filter using a fine tungsten needle and prepared for FTIR.  Small amounts of silicone residue can be prepared for analysis using special replication techniques.  Infrared spectra of silicone oil and stringy silicone particulate from pharmaceutical products are compared in Figure 8, and a typical spectrum of silicone/protein residue is presented in Figure 9.  

click image to enlarge (98K) FIGURE 8 FTIR spectrum of (top) silicone oil extracted from a polycarbonate filter, (middle) stringy degraded silicone particulate residue, and (bottom) a silicone reference.

click image to enlarge (63K) FIGURE 9 FTIR spectrum of silicone/protein residue.

Vials

Particles and residues can occur as a result of processing and sometimes are observed on the vials prior to filling.  Figure 10 shows a residue from an unfilled vial.  FTIR analysis indicated that the material was similar to the processing detergent (Figure 11).

click image to enlarge (49K) FIGURE 10 Light brown residue in an unfilled vial.

click image to enlarge (78K) FIGURE 11 FTIR spectrum of the light brown residue from an unfilled vial (Figure 10) compared to a spectrum of the processing detergent.

Occasionally, a vial will be submitted that was thought to contain a dark particle.  Optical examination indicates that the defect or particle is, in fact, embedded in the glass or is contained entirely within the wall of the vial.  Figure 12 shows black material that is embedded in the vial glass.  The material was identified as hematite (iron oxide or rust) using Raman microscopy (Figure 13).  The presence of iron and oxygen can be confirmed using EDS.  Raman has the benefit of giving compound information that complements the elemental data.

click image to enlarge (61K) FIGURE 12 Black material embedded in glass vial wall.

click image to enlarge (78K) FIGURE 13 Raman spectrum of the black material embedded in glass vial wall (Figure 12) compared to a Raman spectrum of hematite (iron oxide).