ANALYTICAL TECHNIQUES FOR PARTICULATE MATTER IN INTRAVENOUS SOLUTIONS USING MEMBRANE FILTERS

Transcription

1 ANALYTICAL TECHNIQUES FOR PARTICULATE MATTER IN INTRAVENOUS SOLUTIONS USING MEMBRANE FILTERS Ben Trasen Millipore Corporation, Bedford, Mass. The introduction of particulate matter into the bloodstream has been found-both experimentally with animals and clinically with patients-to cause blockages of minor blood vessels and capillaries in key organs of the body. The most common results have been lesions and granulomas in the capillaries of the lung. The particulate matter is generally introduced into the vascular system by means of intravenous injection and infusion. Once introduced, there the contaminant will remain, the vascular system being a highly intricate, closed loop. The particulate matter will circulate in the bloodstream until it eventually finds its way into a blood vessel that is too small for it to pass. The resulting block prevents further blood circulation to the tissues in the area. In addition, there may be many types of reactions to the particulate matter by the body, such as the buildup of giant cells around it, but all result in the closure of the vessel.'-3 All administered intravenous solutions share in the dilemma, since all contain particulate matter in varying amounts, kinds, and sizes. Although the problem is recognized by the pharmaceutical industry, and vast sums are currently being diverted toward the production of particle-free I.V., it is not yet possible to produce completely particle-free solutions. The vast network of production apparatus in the pharmaceutical company precludes the achievement of a 100 percent particle-free product. Pipelines, valves, tanks, threaded connections, airborne matter and, especially, media-migrating fibrous filters all combine to contaminate the bulk product. Added particulate agents contributed by insufficiently cleaned bottles or vials and their rubber stoppers, as well as particles of glass and pieces of rubber, further complicate the problem.4--6 It is not enough to point accusingly at the I.V. solutions themselves. The solutions are administered to the patients with devices that by test have been shown to contribute at least as much and often far greater amounts of matter to the solution. Tests have been performed on administration sets, syringes and needles confirming the findings of others that these devices themselves can frequently be the major antagonists in yielding particle-free I.V. In view of the fact that the solutions and administration devices are most certainly contributing particles to the I.V., quality-control checks on them are 665

2 666 Annals New York Academy of Sciences essential. Tests must be made that will clearly distinguish acceptable from rejectable items and fluids. An effective test must be keyed directly to the physiological reaction to the administered solutions. In other words, solutions and devices handling them should be graded in accordance with the degree of harm of the particulate matter they contain. Aside from questions of tissue incompatibility, harmfulness of particulate matter is determined not only by the number of particles present, but also by their size and ability to become lodged in a blood vessel. This lodging factor is related to the geometric shape and surface characteristics of the particles. It is obvious that a long thin fiber will cause more severe problems by plugging up a minor vessel than will an equivalent piece of plastic, which may be more nearly spherical in shape and thus progress further to block only a single capillary. Quantitatively, then, a test must be able to detect all of the particles that may prove harmful to the body, count them and size them by their true size taking into account their plugging ability. Today, there exist only three analytical procedures that are routinely performed for particulate matter in I.V. solutions. The first is a visual examination with the unaided eye, a sidelight and a black background. The big advantage is in speed and nondestructive test. However, only particles above approximately 2 0 can ~ be seen, and these are much larger than the 7~ diameter of a capillary. The visible particles cannot be counted or sized. The usefulness of a test of this type is its quick-scan ability to detect only obvious rejects. The second technique, whose advantage lies in its rapidity, employs an electronic resistivity counter. However, because it can only detect particle volume, it can only approximate the particle size, and this has no reliable relation to a particle's plugging tendency. Equating all particles to a standard shape and then sizing them by that shape's diameter is meaningless. The third technique is membrane filtration. It is widely acknowledged that qualitative analyses of I.V. samples can be readily performed by means of filtration through a membrane filter. After visual identification, the nature of a contaminant may be determined. This then is an excellent technique of determining which particles may present a danger to the body due to its biological properties. It also enables the technician to track down the source of a specific contaminant.' Quantitative particle evaluations can also make use of this technique. However, present sampling and counting procedures are based on aerospace fluids and specifically tailored to meet their demands. This has resulted in difficult and often tedious test operations. Recently, in response to numerous requests from the pharmaceutical industry, the Millipore Corporation undertook a study aimed at developing a simplified and specialized technique for the testing of pharmaceutical products, primarily intravenous solutions. A new membrane procedure was developed that greatly simplified and improved upon the old methods. The procedure

3 Trasen: Analytical Techniques 667 has been published as AR-2, Analysis of Intravenous Solutions for Particulate Matter. It is my purpose here to describe this analytical procedure and to discuss a few variations for its utilization in the testing of administration sets, syringes, needles, and solutions from intermediate containers.s It is recognized that a great many errors arise because of poor sampling techniques. Basically, either a sample is taken that is not truly representative of the solution to be analyzed or, during sampling, extraneous contamination is accidentally allowed. Even if the sample itself is taken properly, it can still be mishandled during the counting phase, and erroneous counts will result (e.g., holding the sample container for a period to permit air bubbles to escape, but also enabling contamination to settle). In order to keep sampling errors at a minimum, it is important, whenever possible, to sample directly from the final packaged container and to sample the entire volume. This eliminates transferring to an intermediate vessel, and the possible addition of particulate matter from this vessel. The apparatus must be easily ultracleanable, i.e., simple in design and construction. Only with this simplicity can background be maintained at an extremely low level. The new membrane sampling procedure accomplishes all of these. The entire product volume is taken in a completely enclosed system directly from the container and run through ultracleanable apparatus. The bottle closures are not removed, which is hazardous, but are penetrated only a single time, with an I.V. spike for high volume or a needle for vials. This serves to keep coring and incidental rubber scraping of the stoppers to a minimum. For recovery of all the particles, a flush with membrane-filtered water is accomplished very easily. Although flushing can readily be included in the high-volume sampling procedure, it has been found unnecessary because of the rather high volume-to-surface ratio of the container. The effect of the particles that adhere to the surfaces and those contained in the residual liquid is inconsequential. Using vials, the situation is quite the reverse, and a flushing sequence is included in the procedure. In detail, for high-volume I.V. analysis (bottles over 250 ml), two clear PVC tubes are available for sampling from standard one- and two-hole stoppers, and from screw-cap bottles. For the ClJtter-type single-hole closure, either end adapter is cut off and the spike portion of a Cutter I.V. set affixed to the tube. For the Abbott-type screw-cap bottles, the drip chamber portion of an Abbott set is removed and the small adapter end of the sampling tube is inserted into it. For the Baxter or McGaw-type two-hole closure, no modification is required. The other end of the sampling tube is attached to a small polypropylene filter holder, the sampling holder. Inside is a 25 mm diameter black membrane filter with grid marks on the top surface. The filter is rated at 0.8~ pore size, and will remove from solution all the particles that exceed that size. In all analyses, black filters have been shown to be the most effective from two standpoints. Not only are they more relaxing for the eye than

4 668 Annals New York Academy of Sciences FIGURE 1. The high volume bottle to be sampled is connected by means of a tube to the sampling filter holder shown on top of the vacuum flask. bright backgrounds but, more importantly, since almost all the particulate matter found in I.V. is light in color, they provide much greater contrast. Prior to sampling, the bottle is first shaken vigorously. The spike end of the sampling tube is connected to the bottle with the sampling holder held higher than the bottle s liquid level, and the holder is carefully lowered to allow the solution to enter slowly, thus purging it of air. When the solution starts to emerge from the other end, the bottle is hung from a ringstand and the sampling holder placed on a sidearm vacuum bottle. (FIGURE 1.) At 2.5 Hg vacuum, the entire content of a one-liter bottle will drain in two minutes, depositing all particulate matter on the surface of the membrane. When the presence of gelatinous substances is suspected, the vacuum is reduced to below 5 Hg. The gels will remain on the surface in distinct pools. After filtration, the sampling holder is opened, the filter removed with a pair of unserrated tipped forceps and placed in a petri dish grid side up. The petri dish is placed in a small temperature-regulated drying over or on a hotplate, with the cover slightly ajar. After about ten minutes, the dry filters are taken to the microscope area.

5 Trasen: Analytical Techniques 669 To ensure validity of the particle counts, the apparatus is meticulously ultracleaned beforehand. Two kinds of ultracleaning devices are now available. One type uses a glass Guth bottle to dispense solvents through a filter holder located in the neck and containing a 1.2~ filter. The second type is a pressure vessel with a connecting flexible tubing, filter holder, shut-off valve, and nozzle. (FIGURE 2.) For economy, the pressure vessel is filled with water, whereas the glass dispensers use isopropyl alcohol and Freon TF@ or 113. The water at psi does the actual cleaning, whereas the alcohol is miscible with both water and the Freon which evaporates quickly. The test assembly is sprayed with filtered water, followed by the alcohol and Freon rinses, and allowed to dry. The membrane is sprayed with a jet of Freon only, placed on the filter support, and the sampling holder assembled. The apparatus is now ready for sampling. When many bottles are to be run, all the tubes should be precleaned in this manner. Petri dishes, sufficient for each test, are prewashed, covered, and numbered. For the analysis of vialpackaged solutions, an assembly comprising a three-way smooth plastic valve, the same 25 mm sampling filter holder, a smaller cleanup filter holder, and a gauge needle are used. FIGURE 2. Ultracleaning is accomplished by directing a high-velocity stream of membrane-filtered water at the apparatus.

6 670 Annals New York Academy of Sciences The sampling holder again holds a 0.8~ black filter with a gridded upper surface. This holder is connected to the bottom of the valve, so that solution entering from the needle above will pass directly downward through the filter. The smaller filter holder is assembled with a 1.2~ membrane and a backflow support screen, both 13 mm in diameter. A ml disposable syringe is connected to this holder, which is inserted in the side port of the valve. Using a ringstand with two clamps, the apparatus is held, needle up, in the bottom clamp. The sample vial is vigorously agitated, and the stopper pierced by the needle. The vial is then held securely by the upper clamp. Only the top of the needle actually enters the vial. Air is forced into the vial with the syringe. The compressed air drives the solution through the sampling filter. This may be repeated, if necessary, until the vial is almost drained. After filling the syringe with water, the vial is flushed with ultraclean water. The assembly may be shaken as a unit, if desired, to rinse the vial walls thoroughly. When the valve is turned from the off position, the rinse will filter through the sampling filter. The entire sampling time is less than two minutes. Lastly, excess water is removed by injecting some air, the holder disassembled, and the filter placed in a petri dish to be dried. The apparatus is ultracleaned by the piece and then as an assembly. The valve stem is removable to enable thorough ultracleaning of the stem and body. When the assembly has been rinsed and dried, the Freon-flushed 13 mm cleanup filter and the black 25 mm sampling filter are assembled in their holders. When dry, the filter is removed from the petri dish and mounted on a 2 X 3 glass slide. The slide is placed on the stage of a binocular microscope with magnification available up to 200X. For particle counting, only 40X and loox are used. An illuminator is set to one side at a 15 angle, so that the incident light will cause the particles to cast long shadows, facilitating observation. A two-gang counter is placed near the free hand. This basic equipment is set up, ideally, in a laminar flow hood, although there will be little effect if one is not available and a clean bench top is used. To begin with, the low power setting is used and the entire filtering area (EFA) is quickly scanned. The length of the ocular micrometer is used to determine the width of the scanning fields. Several objectives are accomplished immediately. First, a rapid scan at low power will immediately inform the technician of the general contamination level of the sample. Second, the check makes sure that the particles are distributed evenly, which is a requirement for statistical counting. Third, all particles above the true size of 50,u are counted and recorded in the ranges of ~ and over 100~. This low-power scan takes approximately two minutes. Counting at loox is done statistically with seven randomly selected grid squares. Starting at the upper left of the square and sweeping back and forth

7 Trasen : Analytical Techniques 67 1 across it, the micrometer covers the entire square. As the particles pass between the divisions, they are sized and recorded in two ranges, 5-25~ and 25-Sop. A total is obtained for the sum of the particles in the seven squares falling into each range. These totals times 5.2 give the total for the sample. The time at loox will take approximately five minutes, varying with the magnitude of the count. (FIGURE 3.) Although the particle ranges have been chosen generally to simplify the technique, the 5~ power limit is based on a current estimate of the smallest length particle that could block a capillary. If, at some later date, it becomes desirable to count smaller particles, then this figure can be reduced easily to 2.5~. For purposes of a quick go, no-go analysis, a three-minute scan of the entire filter at 40X for all particles over 25p is recommended. FIGURE 3. Particles smaller than 50p are counted in seven representative grid squares. It is recommended that the microscopist check his technique regularly by running a blank count rather than subtract background counts from the filters, which serves as a license not to be careful in the cleaning sequence. Blank counts should be run to give the analyst an indication of the effectiveness of his ultracleaning. The blank is very easily taken in both procedures. For the sampling tube, a 13 mm cleanup filter holder with a 1.2~ filter at the upstream end of the tube is inserted and 100 ml of water is injected through it with a syringe. For the valve assembly, the valve is turned so that when 100 ml of water is injected with a syringe, it passes directly through the sampling holder. In both tests, the water is followed with some air. It should be pointed out that the procedure is totally unaffected by air bubbles. Agglomerated particles are counted individually. Any filterable solution may be analyzed straight from the package

8 672 Annals New York Academy of Sciences including nonelectrolytes. Lastly, the slide-mounted membrane provides a permanent record. The improvements of this technique over the standard membrane method are evident. The membrane filter diameter has been reduced from 47 to 25 mm, thereby reducing the EFA to 1 / 3 and the counting time proportionately. The limit of the EFA is now clearly indicated by the groove around the filter periphery. And of course, the counting procedure has been simplified and based entirely on the sampling of I.V. and the physiological effect of particles entering the bloodstream. The new method now makes available a completely enclosed system for testing administration devices. It has been found by some investigators that the levels of particulate contamination in administration sets can greatly exceed that found in the bottled solutions. After the development of AR-2, attention was turned to these other applications. For testing an I.V. administration set, an ultraclean sampling filter holder with a 25 mm black filter is attached to the needle adapter. In testing the set alone, a supply of ultraclean water is introduced at the upper end. This is most easily accomplished by first ultracleaning a one-liter bottle and filling it with membrane-filtered water. By using a sidearm vacuum flask, as shown in the high-volume procedure, the test can be run in a few minutes. Syringes may be tested just as easily using the same basic procedure. Connect the male end of an ultraclean 25 mm filter holder by means of a % length of ultraclean Tygon tubing to the syringe to be tested. The filter holder contains a 1.2~ membrane filter. Water is drawn into the syringe from a beaker, agitated in the syringe for a few seconds, and then expelled through a second filter containing the analysis membrane. For the testing of needles and pharaceuticals in intermediate containers, e.g., screw-cap jars, test tubes, or beakers, a modification is made to the equipment. Credit for this procedure is given to a major Eastern pharmaceutical company which, in an effort to track down elusive particulate matter in their lines, began taking jar samples at various locations and adapted the AR-2 technique to their own needs. A makeshift funnel is assembled by inserting the barrel of a mi syringe very firmly into the top piece of a 25 mm filter holder. It is then assembled with the base containing the analysis filter. Test samples may be poured into the open funnel and drawn through the membrane with vacuum. Needles are quickly tested by this method. Brand-new needles may harbor very small bits of metal chips, easily identifiable and counted. Tap water is injected through a 1.2~ filter and the attached needle. The effluent from the needle is directed into the aforementioned funnel. In the modified procedures just described, it is optional with the investigator whether the filtration be followed with an ultraclean rinse. It is a simple procedure, and is recommended where the volume of test water is small.

9 Trasen: Analytical Techniques 673 With the new membrane technique briefly described here, many more analyses can be made in a given time than with the older technique. Although the time factor is still longer than ideally desirable, one is amply rewarded with data that are accurate and meaningful-results that no other test provides. Considered alone, sampling time is no longer than with electronic methods, if we include time for flushing of cells, mixing with electrolytes, and calibration. Most of the time is consumed in microscopic particle counting. Visual examination, however, is essential in producing meaningful data. It is hoped that, eventually, the electronics industry will devise an optical scanning device to display the membrane surface, thus enabling investigators to gain complete, quick data on the particulate matter in their products. References 1. GARVAN, J. M. 8r B. W. GUNNER The Harmful Effects of Particles in Intravenous Solution. Med. J. Aust. 2: GROSS, M. A. & CARTER, C. J The Pathogenic Hazard of Particulate Matter in Solutions for Intravenous Use. Proc. FDA Sympos. Washington, D.C. 3. JONAS, A. M Potentially Hazardous Effects of Introducing Particulate Matter into the Vascular System of Man and Animals. Proc. FDA Sympos. Washington, D.C. 4. General Discussion Proc. FDA Sympos.: 85-87, Washington, D.C. 5. PFLAG, S. C Large Volume Parenterai Solutions Procured by the Military. Proc. FDA Sympos. Washington, D.C. 6. SKOLAUT, M. W Particulate Matter in Large Volume Solutions as Viewed by the Hospital Pharmacist. Proc. FDA Sympos. Washington, D.C. 7. SCHMITT, W. M Control and Analysis of Particulate Matter by Membrane Filtration. Bull. Parenteral Drug Assoc. 8. AR-2. Analysis of Intravenous Solutions for Particulate Matter. Millipore Corporation. Bedford. Mass.

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