The Art and Science of Quality Control Using Microanalytical Methods

The Art and Science of Quality Control Using Microanalytical Methods
December 12, 2008
by Mary Stellmack

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In recent weeks, widespread reports of contaminated food, grains, drugs, medical appliances, and toys have headlined the news. Thus, it is critical in such a fast-growing marketplace, with many of the products being imported, that stringent quality control procedures be adhered to for both imported as well as domestic products.

As the pharmaceutical industry is already highly regulated, and rigid quality control is prevalent throughout the domestic production industry, contamination is usually the exception in legally produced products. However, as in many industries, production contamination requires constant surveillance to ensure that the resultant product is market safe.

Common contaminants
Some of the more common contamination and defect culprits that we have observed in analyzing thousands of different products for the pharmaceutical industry include fibers from cleanroom wipes, production machinery oil, latex glove fragments, metal wear particles from machinery, glass particles, and charred contaminants (e.g., charred hair, rubber from the stopper, or even the active pharmaceutical ingredient [API] itself). Additionally, products that display defects such as streaks or surface irregularities can be a result of excipients or API not being sufficiently dispersed during the drug formulation mixing process.

These contaminants can enter the manufacturing process at any time, but some stages are more vulnerable than others. For example, during heat-intensive sterilization of vials and packaging, or during the depyrogenation process, fibers or hairs left in vials may become charred and transferred to the pharmaceutical product. Defects can sometimes be traced back to materials left on manufacturing equipment from previous processing runs. Despite strict cleanliness regulations for all parts of the drug development and manufacturing process, cleaning of manufacturing equipment between runs is sometimes insufficient to remove 100% of the debris. Therefore, machinery oils and lubricants, as well as drug ingredients or clumps of charred materials from previous manufacturing runs, may be inadvertently transferred to new batches.

The human eye is often the first line of defense in the quality control process. Trained inspectors can see defects in products as small as 50 µm in size, usually considered to be the lower limit for visual detection. Sometimes contaminants can be identified solely on the basis of visual inspection, and steps can be taken immediately to correct the problem. In some cases, however, further analyses are required to identify the contaminants. Often, isolating and analyzing these contaminants may require specialized technical skills and analytical instrumentation that an in-house QC laboratory does not possess. In these cases, most pharmaceutical companies look to independent analytical/microanalysis laboratories that have the experience, skill, and necessary sophisticated instrumentation to identify the contamination and the source of their contamination issues.

First look
After receiving the sample, microanalytical laboratories typically first perform a visual inspection using a stereomicroscope and, in some cases, a polarizing light microscope. Using light microscopy, laboratory scientists can often make an immediate assessment of the contaminant, possibly identify it, or at least make an educated guess after viewing and measuring its physical properties. For example, the presence of fibrous materials may indicate contamination from cleanroom wipes or garments, metal flakes may possibly point to wear particles from production machinery, clumps of embedded granular material in tablets may be normal tablet ingredients in concentrated form, rubber particles may be pieces of vial stoppers or latex gloves, and discoloration could mean thermal degradation of the product.

After viewing the contaminant with a microscope and gathering as much information as possible, it still may be necessary to isolate the contaminant from the product or substrate material to identify and/or confirm the identity of the material using other analytical techniques. In a Class 100 (ISO 5) cleanroom environment, microscopists can routinely locate and isolate solid contaminants (particles) as small as 1–2 µm in size. Isolation of contaminants smaller than 1–2 µm can be performed, but requires special techniques to ensure the integrity of the material. Sometimes these very small particles can be analyzed in situ (while still partially embedded in the sample matrix).

If the contaminant is in a liquid product, the liquid is filtered and the contaminant particles are removed from the filter with fine needles for further analysis. One of the great advantages of microanalytical testing is that an entire sample usually does not need to be sacrificed—only a minute portion is required, and the area from which this portion is removed is often unnoticeable. Once isolated, scientists digitally document the contaminant and prepare it for additional analytical testing to fully characterize and identify it. To ensure that cross-contamination does not occur during the isolation/sample preparation process, it is highly recommended that all sample manipulations be performed in a cleanroom.

Two common microanalytical techniques for contamination analysis
In addition to the light microscope, the two most commonly used analytical techniques for the analysis of pharmaceutical contaminants are infrared spectroscopy (FTIR or micro-FTIR) and scanning electron microscopy (SEM) combined with an energy dispersive X-ray spectrometer (EDS) detector.

The FTIR method can be used to identify organic materials and some inorganic materials. Every substance absorbs light at a different frequency and produces a unique infrared spectrum, which is a chemical fingerprint of the material. To prepare a sample for micro-FTIR analysis, a 10-µm or larger portion of the contaminant is isolated by hand and mounted on a potassium bromide crystal. The micro-FTIR system, a polarizing light microscope interfaced with an infrared spectrometer, shines a beam of infrared radiation through the sample and records the different frequencies at which the sample absorbs the light. McCrone Associates, the analytical service division of The McCrone Group (Westmont, IL), maintains a reference library of thousands of standards and known materials. By comparing the spectra of different sources through an automated computer search with the spectra from the contaminant, scientists can often identify the contaminant. Occasionally, if samples are severely charred or degraded, an exact library match may be difficult or, in a few cases, not possible. This is because significant charring can chemically alter the contaminant to such an extent that the infrared fingerprint is also altered beyond recognition. Although McCrone Associates maintains reference spectra of various charred materials in its infrared library, in some cases additional testing is needed to further characterize the material and arrive at an identification.

Using the SEM-EDS method, two kinds of information are obtained: a high-quality morphological image showing the features of the contaminant, and a spectrum of the elemental constituents present in the sample. The EDS is commonly used for the analysis of inorganic materials to identify contaminants such as metals, glass fragments, and minerals. It can also be used to characterize certain organic materials, particularly if they contain elements other than carbon and oxygen, such as silicone rubber stopper fragments, or tablet materials that contain metal salts. EDS analysis may also provide a clue as to the identity of charred contaminants. For instance, the presence of sulfur may indicate that the charred substance was a component of the API if the API also contained sulfur. The SEM-EDS technique is also very helpful in determining the distribution of specific ingredients within a tablet, comparing sizes and shapes of particles between batches for uniformity, or imaging layers in tablet cross-sections.

Other useful microanalytical techniques
In addition to FTIR and SEM, the analytical laboratory at McCrone Associates has one of the most complete analytical capabilities in the country. Other techniques available that may be of critical help in identifying unknown contaminants in pharmaceutical products or medical devices include Raman spectroscopy, X-ray photoelectron spectroscopy, chromatographic techniques, and X-ray diffraction.

Raman spectroscopy is a complementary technique to infrared analysis, and provides “fingerprints” of many inorganic materials and other opaque samples that are not suitable for IR. Raman spectroscopy can often provide additional information that is not obtainable by infrared analysis. For example, the identification of a black inclusion in a tablet by FTIR analysis could be narrowed down to graphite (e.g., from machine seals) or amorphous char (e.g., from a charred API or excipient). Since both materials consist of carbon, they cannot be distinguished by EDS analysis. However, because these materials have different crystalline forms, they will have different Raman signatures. At McCrone Associates, the micro-Raman system can obtain spectra of samples that are as small as 1 µm in size, making it suitable for analysis of very small contaminant particles.

X-ray photoelectron spectroscopy (XPS, also known as ESCA [electron spectroscopy for chemical analysis]) is the method of choice for detecting thin layers of surface contamination present at trace levels, since it is more surface sensitive than EDS. In the XPS instrument, an X-ray beam is used to generate photoelectrons in the sample, which carry analytical information from only the outermost surface (~5 nm) of the sample. Thus, this technique is well suited for the analysis of thin surface layers and residues on the surface of solid samples. In addition, depth profiles can be performed using XPS to expose and characterize underlying material. XPS has been used successfully to detect low levels of silicon, which might originate from siliconized rubber stoppers on liquid containing vials that otherwise would not be detectable by FTIR or EDS analysis.

Chromatographic methods such as gas chromatography or liquid chromatography can be used to verify the identity of the API, or identify certain absorbed volatile or liquid contaminants in pharmaceutical products. In a chromatographic analysis, the sample is injected into a column, and as it travels through the column, its components are separated and observed as individual peaks on a chromatogram. A mass spectrometer detector used in combination with GC or LC analyzes the fragmentation pattern of each peak and provides a fingerprint of the unknown compound. The resultant mass spectrum is compared to reference spectra of known compounds in order to identify the unknown sample. The GC-MS method has been used successfully to identify plasticizers from vinyl materials and other plastic additives from packaging materials that have been absorbed by pharmaceutical products.

Micro X-ray diffraction (micro-XRD) is useful for identification of inorganic materials. Small amounts of powdered material are placed in a 0.1-mm-diameter glass capillary and irradiated in the diffractometer. Alternatively, individual particles as small as 10 µm are mounted on the tip of a pin and irradiated in the same manner. The resulting diffraction patterns are compared to a database of known compounds. In addition to identifying contaminants, micro-XRD can also distinguish between crystalline phases (polymorphs) of the API.

Project completion
Upon completing the analysis and identification of a contaminant or defect, the laboratory will provide a detailed report to the customer including a summary of the analyses performed, photographs taken during the analysis, raw data (spectra), and a summary of the data evaluation. The report provides the identity of the contaminant and some possible sources of the contaminant based upon its composition and the information gathered from discussions with the client. If the identification of the contaminant leads to changes in the client’s production process, the client should retest its product to ensure that the problem has been eliminated.

Continuing education
Pharmaceutical industry quality control personnel face considerable challenges in keeping abreast of not only the evolving regulatory requirements, but the various microanalytical capabilities available to resolve their problems. There are many educational institutions that cater to this growing need. For instance, The McCrone Group’s College of Microscopy teaches a practicum on specialized isolation and mounting techniques (COM310: Sample Preparation: Pharmaceutical and Medical Devices) as well as the specific analytical approaches to identifying particle contamination for regulatory compliance under FDA rules (COM410: Microscopical Identification of Pharmaceutical Materials and Contaminants).

Ms. Stellmack is a Senior Research Chemist, McCrone Associates, Inc., the analytical division of The McCrone Group, 850 Pasquinelli Drive, Westmont, IL 60559 U.S.A.; tel.: 630-887-7100; fax: 630-887-7417; e-mail: