This article is republished from The Microscope, Vol 51:1, 3-10 (2003)

Abstract

Most pharmaceutical products are specified to be essentially free of visible particles and there are limits on the number of subvisible particles allowed.  The FDA requires that contamination problems are fully investigated in a timely fashion.  This paper describes an analytical approach that utilizes microscopical examination coupled with sample isolation, preparation, and analytical methods optimized for small particles, to successfully identify particulate contamination for regulatory compliance. 

The analytical approach for the identification of particles will be outlined.  The analysis plan includes sample examination, gathering of background information concerning the sample and particle isolation.  Analytical methods utilized include one or more of the following: polarized light microscopy (PLM), Fourier transform infrared spectroscopy (FTIR), Raman Microscopy, and several types of electron microscopy with EDS and WDS detectors.  Common contaminants in pharmaceuticals include natural and synthetic fibers, silicone, plastics, rubber, metal particles and corrosion products, glass particles and delamination flakes, skin flakes, char particles, detergents, lubricant oils, Teflon^® and graphite. Specific examples of product-related particulates, foreign particles and manufacturing process contamination will be discussed.

Introduction

Identification of small particle contamination is crucial in the pharmaceutical industry.  Most pharmaceuticals are specified to be essentially free of visible foreign particles and there are limits on the number of subvisible particles allowed.  Contaminants are usually first noticed during internal quality control inspection; however, some are detected by the consumer after the material has been released.  The FDA requires that contamination problems and consumer complaints are fully investigated in a timely fashion.  Microscopical analysis is particularly well suited to the analysis of particulate contamination because the particles are usually too small to be analyzed using conventional methods.  Proper methods of sample isolation and preparation are also critical to the successful identification of particulate contaminants. 

The most common pharmaceutical samples that are analyzed by McCrone Associates are filled and unfilled vials (parenteral products) and tablets; however, particles and residues have been isolated from syringes, IV bags and tubing, ampoules, dropper bottles, inhalors and patches.  Foreign particles also may be recovered from raw materials and various types of process filter apparatus.

There are two general types of contaminants, product-related and foreign material.  The contaminant may be related to the active ingredient, excipient materials or colorant.  Particles may be generated from the product container or packaging material.  These types of particles include glass, rubber, aluminum, plastics and paper.  Contamination can also result from the manufacture of the product; examples of these include charred product, detergents and lubricant oils.  Metal and metal corrosion, Teflon, graphite and rubber particles are indications of tank, filter or equipment failure.  Environmental contaminants such as fibers and skin cells are also found.  The most common contaminants in pharmaceuticals are cellulose (cotton and paper) fibers, synthetic fibers, silicone, plastics, rubber, metal particles and corrosion products, glass particles and vial delamination flakes, skin flakes and char particles.

Analytical Method for Identification of Small Particles

The first step in the analysis is to obtain as much background information about the particulate problem as possible.  This is important for both in-house investigations as well as for samples submitted to an outside laboratory.  In some cases, clients are not forthcoming about the details of the problem, because they do not wish to bias the investigator.  This can cause longer turn around time and increased costs.  Communication with the client is critical to defining the analysis.  It is also useful to know the compositional information of the product, as this knowledge may aid in the interpretation of the results. Some information can be obtained from reference volumes, such as the Physicians Desk Reference (PDR) and The Merck Index.  It is impossible to underestimate the importance of gathering all available background information about the sample.

Particle identification begins with microscopical examination.  The sample is examined as received, usually using a stereomicroscope.  The optical examination further defines the analysis, and a plan of action can then be developed.  The next step is to isolate the contaminant and prepare it for further microscopical and chemical analysis.  Obviously, it is very important to avoid introducing any further foreign particles into the sample during isolation.  To avoid this, the sample should be isolated in a cleanroom facility, if possible, or in a laminar flow hood.  McCrone Associates has a 1650 sq. ft cleanroom facility.  The facility is equipped with microscopes, analog and digital camera equipment, Class 100 laminar flow hoods, and a deionized, particle-free water source.  Particles in solution are isolated by filtration.  Polycarbonate membrane filters are normally used because they have a smooth surface from which to observe and pick particles.  Contaminants on or in tablets can usually be recovered by picking directly with a tungsten needle and moving it to a suitable substrate for analysis.  Microextraction techniques are useful for isolating oils from defects.  Information on the manipulation and preparation of small particles can be found in several references (1, 2, 3, 4).

After the particles are isolated, they are prepared for further analysis using one or more methods.  The appropriate method or methods are chosen based on the optical examination of the sample.  Sometimes one method is sufficient, other particles require additional analysis to fully characterize the contamination.  Frequently particles are initially examined using polarized-light microscopy (PLM).  Some particles, such as fibers, may be identified readily using light microscopy; other particles are prepared for additional techniques based on characteristics noted by PLM. 

Fourier transform infrared spectroscopy (FTIR) is a technique that is widely used to identify organic and some inorganic materials.  FTIR microscopy is recommended for particles that appear to be organic during the optical examination.  Organic particles include polymeric material, amorphous residues and particles that appear to be related to active ingredients and excipients.  Some inorganic materials and minerals, such as calcium carbonate or clay, can also be characterized and/or confirmed by FTIR.  Raman microspectrometry is a complimentary method to infrared, but it is an emission technique that is particularly well-suited to the analysis of dark and opaque particles, corrosion products and minerals. It can determine the phases of carbon (char, graphite) and is particularly useful for pigments.    

For elemental analysis, a low vacuum scanning electron microscope that is equipped with an energy dispersive x-ray spectrometer (EDS) detector is utilized.  The low vacuum capability is very useful for contaminants that consist of an organic matrix with inorganic inclusions.  The low vacuum mode can be used to minimize charging effects.  SEM imaging also provides high magnification morphology data, and backscattered electron imaging allows elemental mapping.  For identification of specific elements that may be masked or overlapped by another element, an electron microprobe that has both EDS and wavelength dispersive x-ray detectors (WDS) may be used.  Analytical electron microscopy or transmission electron microscopy may be used for submicrometer particle analysis.

For certain applications, other methods such as secondary ion mass spectrometry (SIMS) may be used.  SIMS is very useful in detecting light elements that cannot be detected by EDS.  Gas chromatography coupled with a mass spectrometer (GC-MS) can be used if the contaminant is soluble in an appropriate solvent and is volatile.   Although not necessarily considered microanalytical techniques, time of flight secondary ion mass spectrometry (TOF-SIMS), direct probe pyrolysis mass spectrometry, and liquid chromatography-mass spectrometry (LC-MS) have been successfully used in situations where there was sufficient sample available.