Microscopic and light obscuration procedures for the determination of particulate matter are given herein. Unless otherwise specified in the individual monograph, the article is expected to conform to the limits for the Light Obscuration Particle Count Test. 

All large-volume injections for single-dose infusion and those small-volume injections for which the monographs specify such requirements are subject to the particulate matter limits set forth for the test being applied, unless otherwise specified in the individual monograph. 

Not all injection formulations can be examined for particles by one or both of these tests. Any product that is not a pure solution having a clarity and a viscosity approximating those of water may provide erroneous data when analyzed by the light obscuration counting method. Such materials may be analyzed by the microscopic method. Emulsions, colloids, and liposomal preparations are examples. Refer to the specific monographs when a question of test applicability occurs.  Higher limits are appropriate for certain articles and are specified in the individual monographs. 

In the tests described below for large-volume and small-volume injections, the results obtained in examining a discrete unit or group of units for particulate matter cannot be extrapolated with certainty to other units that remain untested. Thus, statistically sound sampling plans based upon known operational factors must be developed if valid inferences are to be drawn from observed data to characterize the level, of particulate matter in a large group of units. Sampling  plans should be based on consideration of product volume, numbers of particles historically found to be present in comparison to limits, particle size distribution of particles present, and variability of particle counts between units. 

LIGHT OBSCURATION PARTICLE COUNT TEST 

USP Reference Standards:  USP Particle Count RS. 

The test applies to large-volume injections labeled as containing more than 100 mL, unless otherwise specified in the individual monograph. It counts suspended particles that are solid or liquid. This test applies also to single-dose or multiple-dose small-volume injections labeled as containing 100 mL or less that are either in solution or in solution constituted from sterile solids, where a test for particulate matter is specified in the individual monograph. Injections packaged in prefilled syringes and cartridges are exempt from these requirements, as are products for which the individual monograph specifies that the label states that the product is to be used with a final filter. 

Test Apparatus 

The apparatus is an electronic, liquid-borne particie counting system that uses a light-obscuration sensor with a suitable sample feeding device. A variety of suitable devices of this type are commercially available. It is the responsibility of those performing the test to ensure that the operating parameters of the instrumentation are appropriate to the required accuracy and precision of the test result, and that adequate training is provided  
for those responsibles for the technical performance of the test. 

It is important to note that for Pharmacopeial applications the ultimate goal is that the particle counter reproducibly size and count particles present in the injectable material under investigation. The instruments available range  from systems where calibration and other components of standardization must be carried out by manual procedures to sophisticated systems incorporating hardware- or software-based functions for the standardization procedures. Thus, it is not possible to specify exact methods to be followed for standardization of the instrument, and it is necessary to emphasize  the required end result of a standardization procedure rather than a specific method for obtaining this result. This section is intended to emphasize the criteria that must be met by a system rather than specific methods to be used in their determination. It is the responsiblity of user to apply the various methods of standardization applicable to a specific instrument. Critical operational criteria consist of the following. 

Sensor Concentration Limits- Use an instrument that has a concentration limit (the maximum number of particles per mL) identified by the  manufacturer that is greater than the concentration of particles in the test specimen to be counted. The vendor-certified concentration limit for a sensor is specified as that count level at which coincidence counts due to simultaneous presence of two or more particies in the sensor view volume comprise less than 10% of the counts collected for 10 mm particles. 

Sensor Dynamic Range-The dynamic range of the instrument used (range of sizes of particles that can be accurately sized and counted) must include the smallest particle size to be enumerated in the test articles. 

Instrument Standardization 

The following discussion of instrument standardization emphasizes performance criteria rather than specific methods for calibrating or standardizing a given instrument system. This approach is particularly evident in the description of calibration, where allowance must be made  for manual methods as well as those based on firmware, software, or the use of electronic testing instruments. Appropriate user validation of software and firmware systems is essential to performance of the test according to requirements. Since different brands of instruments may be used in the  test, the user is responsible for ensuring that the counter used is operated according to the manufacturer's specific instructions; the principles to  be followed to ensure that instruments operate within acceptable ranges are  defined below. 

The following information for instrument standardization helps ensure that the sample volume accuracy, sample flow rate, particle size response curve, sensor resolution, and count accuracy are appropriate to  performance of the test. Conduct these procedures at intervals of not more than six months. 

SAMPLE VOLUME ACCURACY 

Since the particle count from a sample aliquot varies directly with the volume of fluid sampled, it is important that the sampling accuracy is known to be within a certain range. For a sample volume determination, determine the dead (tare) volume in the sample feeder with Water for Injection or distilled water that has been passed through a filter having a porosity of 1.2 mm or finer. Transfer a volume of Water for Injection that is greater than the sample volume to a container, and weigh. Withdraw through the sample feeding device a volume that is appropriate for the specific sampler, and again weigh the container. Determine the sample volume by subtracting the tare volume from the combined sample plus tare volumes. Verify that the value obtained is within ± 5% of the appropriate sample volume for the  test. Alternatively, the  sample volume may be determined using a suitable Class A graduated cylinder (see Volumetric apparatus) . 
[NOTE: instruments of this type require a variable tare volume. This is theamount of sample withdrawn prior to counting. This volume may be determined for syringe-operated samplers by setting the sample volume to zero and initiating sampling, so that the only volume of solution drawn is the tare. Subtract the tare volume from the total volume of solution drawn in the sampling cycle to determine the sample volume]. 
 

SAMPLE FLOW RATE 

Verify that the flow rate is within the manufacture's specifications for the sensor used. This may be accomplished by using  a calibrated stop watch to measure the time required for the instrument to withdraw and count a specific sample volume (i.e the time between beginning and ending of the count cycle as denoted by instrument indicator lights or other 
means). Sensors may be operated accurately over a range of flow rates. Perform the Test Procedure at the same flow calibration of the instrument. 

CALIBRATION 

Use one of the following methods:

Manual Method- Calibrate the instrument with a minimum of three calibrators, each consisting of near-monosize polystyrene spheres drawing diameters of about 10, 15, and 25 mm, in an aqueous vehicle. The calibrator spheres must have a mean diameter of within 5% of the 10-, 15-, and 25-mm nominal diameters and be standardized against materials traceable to NIST standard reference materials. The number of spheres counted must be within the sensor's concentration limit. Prepare suspension of the calibrator spheres in water at a concentration of 1000 to 5000 particles per mL, determine the channel setting that corresponds to the highest count setting for the spheres distribution. This is determined by using the highest count threshold setting to split the distribution into two bins containing equal number of counts, with the instrument set in the differential count mode (moving window half-count method). Use only the central portion of the distribution in this calculation to avoid inclusing assymetrical portions of the peak. The portion of the dístribution which must be divided equally, is the count window. The window is bounded by threshold settings that will define a threshold voltage window of ± 20% around the mean diameter of the test spheres. The window is intended to include all single spheres taking into account the standard deviation of the spheres and of the sensor resolution, while excluding noise and aggregates of spheres. The value of 20% was chosen based on the worst-case sensor resolution of 10% and the worst-case standard deviation of the spheres of 10%. Since the thresholds are proportional to the area of the spheres rather than the diameter, the lower settings are determined by the equations: 
 
V_L = 0.64V_s 

in which V_L is the lower voltage setting and V_s is the voltage the peak center, and 

V_U = 1.44V_s 

in which V_U is the upper voltage setting. 

Once the center peak thresholds are determined, use these thresholds for the standards to create a regression of log voltage versus log particle size, from which the instrument settings for the 10- and 25-mm sizes can be determined. 
  
Automated Method- The calibration (size response) curve may be determined for the instrument-sensor system by the use of validated software routines offered by instrument vendors; these may be included as part of the instrument software or used in conjunetion with a microcomputer interfaced to the counter. The use of these automated methods is appropriate if the vendor supplies written certification that the software provides a response curve equivalent to that attained by the manual method and is the automated calibration is validated as necessary by the user.

Electronic Method- Using a multichannel peak height analyzer, determine the center channel of the particle counter pulse for each standard suspension. This peak voltage setting becomes the threshold used for calculation of the voltage response curve for the instrument. The standard suspensions to be used for the calíbration are run in order, and median pulse voltages for each are determined. These thresholds are then used to generate the size response curve manually or via software routines. The thresholds determined from the multichannel analyzer data are then transferred to the counter to complete the calibration. If this procedure is used with a comparator-based instrument, the comparators of the counter must be adjusted accurately beforehand. 

SENSOR RESOLUTION 

The  particle size resolution of the instrumental particle counter is dependent upon the sensor used and may vary with individual sensors of the same model. Determine the resolution of the particle counter for 10-mm particles using the monosized 10-mm calibrator spheres. The relative standard deviation of the size distribution of the standard particles used is not more than 5%. 

Acceptable methods of determining particie size resolution are (1) manual determination of the amount of peak broadening due to instrument response; (2) using an electronic method of measuring and sorting particle sensor voltage output with a multichannel analyzer; and (3) automated methods. 

Method- Adjust the particle counter to operate in the cumulative  mode or total count mode. Refer to the calibration curve obtained earlier, and determine the threshold voltage for the 10 mm monosized spheres. Adjust 3 channels of the counter tobe used in the calibration procedure as follows: 
  
Channel 1 is set for 90% of the threshold voltage. 
Channel 2 is set for the threshold voltage. 
Channel 3 is set for 110% of the threshold voltage. 

Draw a sample through the sensor, observing the count in Channel 2. When the particle count in that channel has reached approximately 1000, stop counting, and observe the counts in Channels 1 and 3. Check to see if the Channel 1 count and the Channel 3 count 168 ± 10% and 32 ± 10%, respectively, of the count of Channel 2. lf not, adjust Channel 1 and Channel 3 thresholds to meet these criteria. When these criteria have been satisfied, draw a sample of suspension through the counter until the counts in Channel 2 have reached approximately 10,000, or until an appropriate volume (e.g., 10 mL) of the spheres suspension has been counted. Verify that Channel 1 and Channel 3 counts are 168 ± 10% and 32 ± 3%, respectively, of the count in Channel 2.  

Record the particle size for the thresholds just determined for Channels 1, 2, and 3. Subtract the particle size for Channel 2 from the size for Channel 3. Subtract the particle size for Channel 1 from the size for Channel 2. The values so determined are the observed standard deviations on the positive and negative side of the mean count for the 10-mm standard. Calculate the percentage of resolution of the sensor by the formula: 

, 

in which S_o is the highest observed standard deviation determined for the spheres, S_s is the supplier's reported standard deviation for the spheres, and D is the diameter, in mm, of the spheres as specified by the supplier. The resolution is not more than 10%. 

Automated Method- Software is available for some counters that allows for the automated determination of sensor resolution. This software may be included in the instrument or used in conjunction with a microcomputer interfaced to the counter. The use of these automated methods is appropriate if the vendor supplies written certification that the software provides a resolution determination equivalent to the manual method and if the automated resolution determination is validated as necessary by the user. 

Electronic Method- Record the voltage output distribution of the particle sensor, using a multichannel analyzer while sampling a suspension of the l0-mm particle size standard. To determine resolution move the cursor of the multichannel analyzer up and down the electric potential scale from the median pulse voltage to identify a channel on each side of the 10-mm peak that has approximately 61% of the counts observed in the center channel.Use of the counter size response curve to convert the mV values of these two channels to particle sizes provides the particle size at within 1 standard deviation of the 10-mm standard. Use these values to calculate the resolution as described under Manual Method. 

PARTICLE COUNTING ACCURACY 

Determine the particle counting accuracy of the instrument, using Method 1 (for small-volume injections) or Method 2 (for large-volume injections). 

Method I: 
Procedure- Prepare the suspension and blank using the USP Particle Count RS. Set the instrument to count at 10 and 15 mm. Mix the blank by inverting 25 times within 10 seconds, and degas the mixture by sonicating for 30 seconds or by allowing to stand. Remove the closure from the container, and gently stir the contents by hand-swirling or by mechanical means, taking care not to introduce air bubbles or contamination. Stir continuously throughout the analysis. Withdraw directly from the container, three consecutive volumes of not less than 5 mL each, obtain the particle counts, and discard the  data from the first portion.  
[NOTE-Complete the procedure within five minutes.]  
Repeat the procedure, using the suspension in place of the blank. From the averages of the counts resulting from the analysis of the two portions of the suspension at 10 mm and from the analysis of the two portions of the blank at 10 mm, calculate the number of particles in each mL taken by the formula: 

(P_s - P_b)/V 

in which P_s is the average particle count obtained from the suspension, P_b is the average particle count obtained from the blank, and V is the average volume, in mL, of the 4 portions tested. Repeat the calculations, using the results obtained at 15 mm. 
  
Interpretation-The instrument meets the requirements for Particle Counting Accuracy if the count obtained at 10 gin. and the ratio of the counts obtained at 10 mm to those obtained at 15 mm conform to the values that accompany the USP Particie Count RS. If the instrument does not meet the requirements for Particle Counting Accuracy, recalibrate with the remaining suspension and blank. If the results of the second test are within the limits given above, the instrument meets the requirements of the Particle Counting Accuracy test. If the results of the second test are within the limits given above, the instrument meets the requirements of the test for Particle Counting Accuracy. If on the second attempt the system does not meet the requirements of the test, determine and correct the source of the failures, and retest the instrument. 

Method II: 

Procedure- Using standard calibrator spheres having a nominal diameter of 15 to 30 mm, prepare a suspension containing between 50 and 200 particles per mL. Degas the suspension by mild sonication (at 8 mW or less) for 30 seconds or by allowing to stand. Properly suspend the particles by stirring gently, and perform five counts on 5-mL volumes of the suspension, using the particle counter 10-mm size threshold. Obtain the mean cumulative particle count per mL. Pipet a volume of this suspension containing 250 to 500 particles into a filter funnel prepared as described for Filtration Apparatus under Microscopic Particle Count. After drying the membrane, count the total number of standard spheres collected on the membrane filter. This count should be within 20% of the mean instrumental count per mL for the suspension. 

Test Environment 
  
Perform the test in an environment that does not contribute any significant amount of particulate matter. Specimens must be cleaned to the extent that any level of extraneous particles added has a negligible effect on the outcome of the test. The test specimen, glassware, closures, and other required equipment preferably are prepared in an environment protected by high-efficiency particulate air (HEPA) filters. Nonshedding garments and powder-free gloves preferably are wormí throughout the preparation of samples. 

Cleanse glassware, closures, and other required equipment, preferably by immersing and scrubbing in warm, nonionic detergent solution. Rinse in flowing tap water, and then rinse again in flowing filtered water. Organic solvents may also be used to facilitate cleaning.  
[NOTE-These steps describe one way to clean equipment; alternatively, particulate-free equipment may be obtained from a suitable vendor].  
Finally, rinse the equipment in filtered water, using a hand-held pressure nozzle with final filter or other appropriate filtered water  source, such as distilled water passed through a capsule filter. The filter used should have a porosity of 1.2 mm or finer. 

To collect background counts, use a cleaned vessel of the type and volume representative of that to be used in the test. Place a 20-mL volume of filtered water in the vessel, and agitate the sample in the cleaned glassware by inversion or swirling. Degas by sonicating for 30 seconds or by allowing to stand. Swirl the vessel containing the water sample by hand or agitate by mechanical means to suspend particles. Withdraw and obtain the particle counts for three consecutive samples of not less than 5 mL each, disregarding the first count. If more than 10 particles of 10 mm or greater size, or more than 2 particles of 25 mm or greater size are observed in the combined 10-mL sample, the environment is not suitable for particulate analysis: the filtered water and glassware have not been properly prepared or the counter is generating spurious counts. In this case, repeat the preparatory steps until conditions of analysis are suitable for the test. 

Test Procedure 

TEST PREPARATION 

For containers having volumes of less than 25 mL, test a solution pool of 10 or more units. Single units of small-volume injections may be tested individually if the individual unit volume is 25 mL or greater. 

Prepare the test specimens in the following sequence. Remove outer closures, sealing bands, and any loose or shedding paper labels. Rinse the exterior of containers with filtered distilled water as described under Test Environment, and dry, taking care to protect the containers from environmental contamination. Withdraw the contents of the containers in the normal or customary manner of use, or as instructed in the package labeling, except that containers with removable stoppers may be sampled directly by removing the closure, or if test specimens are being pooled, by removing the closure and emptying the contents into a clean container. 

DETERMINATION 

Liquid Fill (where the contents of each unit are less than 25 mL): Mix each unit by inverting it 20 times to resuspend an particles.  
[NOTE-Because of the small volume of some products, it may be necessary to agitate the solution more vigorously in order to suspend the particles completely and homogeneously]. Open and combine, in a cleaned container, the contents of 10 or more units to obtain a volume of not less than 20 mL. Degas by sonicating for 30 seconds or by allowing to stand until the solution is  free from air bubbles. Gently stir the contents of the container by hand-swirling or by mechanical means, taking care not to introduce air bubbles or contamination. Withdraw not less than 3 aliquot portions, each not less than 5 mL in volume, into the light obscuration counter sensor. Obtain the particle counts, and discard the data from the first portion. 

Liquid Fill (where the contents of each unit are 25 mL or more, and where the option of testing individual units is selected): Mix 1 unit by inverting it 20 times. Degas the solution by sonicating or by allowing it to stand until the solution is free from air bubbles. Remove the closure, and insert the counter probe into the center of the solution. Gently agitate the contents of the unit by hand-swirling or by mechanical means. Withdraw not less than 3 aliquot portions, each not less than 5 mL in volume, into the light obscuration counter sensor. Obtain the particle counts, and discard the data from the first portion. 

Dry or Lyophilized Fill: Open the container, taking care not to contaminate the opening or cover. Constitute with a suitable volume of filtered water, or with the appropriate filtered diluent if water is not suitable. Replace the closure, and manually agitate the container to dissolve the drug. Allow to stand until the drug is completely dissolved. Prior to analysis, gently stir the contents of the containers by hand-swirling or by mechanical means, taking care not to introduce air bubbles or contamination. Pool or test individually the appropriate number of units, and withdraw not less than 3 aliquot portions, each not less than 5 mL in volume into the light obscuration counter sensor. Obtain the particle counts, and discard the data from the first aliquot.  

Solid Drugs Packaged with Diluents: For products packaged in containers that are constructed to hold the drug products and a solvent in separate compartments, mix each unit as directed in the labeling, activating and agitating each unit so as to ensure thorough mixing of the separate components. Analyze the solutions as described under Liquid Fill.  

Multiple-dose Containers:  For products labeled Pharmacy Bulk Packages, proceed for each unit as directed under Test Preparation, calculating the results on the basis of a sample volume that is equal to the maximum dose stated in the labelling.  For the calculations below, consider a maximum-dose volume to be the equivalent of the contents of one full container. 

Calculations: 

Pooled Samples (Small-volume Injections)- Average the counts from the 2 or more aliquot portions analyzed. Calculate the number of particles in each container by the formula:  

PV_t/V_an 

in which P is the average particle count obtained from  the portion analyzed, V_t is the volume of pooled sample, in mL, V_a is the volume, in mL, of each portion analyzed, and n is the number containers pooled. 

Individual Samples (Small-volume Injections)- Average the counts obtained for the 5-mL or greater aliquot portions from each separate unit analyzed, and calculate the number of particles in each container by the formula: 

PV/V_a, 

in which P is the average particle count obtained from the portions analyzed, V is the volume, in mL, of the tested unit, and V_a is the volume, in mL, of each portion analyzed. 

Individual Unit Samples (Large-volume Injections)- Average the counts obtained for the two or more 5-mL aliquot portions taken from the solution unit. Calculate the number of particles in each mL taken by the formula: 

P/V, 

in which P is the average particle count for an individual 5 mL or greater sample volume, and V is the volume, in mL, of the portion taken. 

Interpretation: 

The injection meets the requirements of the test if the average numbers of particles present in the units tested do not exceed the appropriate value listed in Table 1. lf the average number of particles exceeds the limit, test the article by the Microscopic Particle Count Test. 

Table 1. Light Obscuration Test Particle Count. 
 

MICROSCOPIC PARTICLE COUNT TEST 

The microscopic particulate matter test may be applied to both large-volume and small-volume injections. This test enumerates subvisible, essentially solid, particulate matter in these products on a per-volume or per-container basis, after collection on a microporous membrane filter. Some articles cannot be tested meaningfully by light obscuration. In such cases, individual monographs specify only this microscopic assay. Solutions exempted from analysis using the  microscopic assay are identified on a monograph basis. Examples are solutions of viscosity too high to filter readily (e.g., concentrated dextrose, starch solutions, or dextrans). Similarly, products known to contain amorphous semiliquid or otherwise morphologically indistinct materials should be tested by the microscopic method. These materials show little or no surface relief and present a gelatinous or film-like appearance. Since in solution this material consists of units on the order of 1 mm or less, which may be counted only after aggregation or deformation on an analytical membrane, interpretation of enumeration may be aided by testing a sample of the solution by the light obscuration particle count method. 

Test Apparatus 

Microscope- Use a compound binocular microscope that corrects changes in interpupillary distance by maintaining a constant tube length. The objective and eyepiece combination of lenses must give a magnification of 100 ± 10 X. The objective must be of 10X nominal magnification, a planar achromat or better in quality, with a minimum numerical aperture of 0.25. In addition the objective must be compatible with an episcopic illuminator attachment. The eyepieces must be of 10X magnification, with a field number of >l5 (widefield). In addition, eyepiece must be designed to accept and focus an eyepiece graticule. The microscope must have a mechanical stage capable of holding and traversing the entire filtration area of a 25-mm or 47 mm membrane filter.

Illuminators- Two illuminators are required. One is an external, focusable auxiliary illuminator adjustable to give incident oblique illumination at an angle of lOº to 20º. The other is an episcopic brightfield illuminator internal to the microscope. Both illuminators must be of a wattage sufficient to provide a bright, even source of illumination and may be equipped with blue day-filters to decrease operator fatigue during use. 

Diameter Graticule-Use a circular diameter graticule (see Figure 1) matched to the microscope model objective  and eyepiece such that the sizing circles are within 2% of the stated size at the plane of the stage. 

Fig. 1.- Circular diameter graticule. The large circle divided by crosshairs into quadrants is designated the graticule field of view (GFOV). Transparent and black circles having 10-um 25-mm diameters at 100X are, provided as comparison scales for particle sizing. 

Micrometer-Use a stage micrometer, graduated in 10-mm increments, that is certified by NIST. 

Filtration Apparatus- Use a filter funnel suitable for the volume to be tested, having a minimum diameter of about 21 mm. The funnel is made of plastic, glass, or stainless steel. Use a filter support made of stainless steel screen or sintered glass as the filtration diffuser. The filtration apparatus is equipped with a vacuum source, a solvent dispenser capable of delivering solvents filtered at 1.2 mm or finer retention rating at a range of pressures 10 psi to  80 psi, and membrane filters (25 mm or 47 mm nongridded or gridded, black or dark gray, of mixed cellulose ester with a porosity of 1.0 mm or finer). Use blunt forceps to handle membrane filters.

Test Environment 

A laminar flow hood or other laminar airflow enclosure, having a capacity sufficient to envelope the area in which the analysis is prepared, with HEPA-filtered air having not more than 100 particles (0.5 mm or smaller) per cubic foot. For the blank determination, deliver from the pressure dispenser a volume of filtered water equal to the sample volume into the Etration Apparatus. Apply vacuum and draw the entire volume of water through the membrane filter. 
Remove the membrane from the filter funnel base, and place atop a strip of double-sided tape in a petri slide or petri dish. After allowing the membrane to dry, examine it microscopically at a magnification of 100X. If not more than 10 particles 10 mm or larger in size and no particles 25 mm or larger are present within the filtration area, the background particle level is sufficiently low for performance of the microscopic assay. 

Preparation of Test Apparatus 

Throughout this procedure, preferably use suitable powder-free gloves, and thoroughly clean glassware and equipment that have been rinsed successively with a warm, residue-free solution of detergent, hot water, filtered distilled or deionized water, and isopropyl alcohol.  
[NOTE-Filfter the distilled or deionized water and the isopropyl alcohol prior to use, using filters having a porosity of 1.2 mm or finer.] Perform the rinsing under the laminar flow enclosure equipped with HEPA filters. Allow the glassware and filtration apparatus to dry under the hood, upstream of all other operations. Preferably, the hood is located in a separate room that is supplied with filtered air-conditioned air and maintained under positive pressure with respect to the surrounding areas. 
  
Prior to conducting the test, clean the work surfaces of the laminar flow enclosure with an appropriate solvent. Prepare the test specimens in the following sequence. Remove outer closures, sealing bands, and any loose or shedding paper labels. Rinse the exterior of containers with filtered Purified Water. Withdraw the contents of the containers in the normal or customary manner of use or as instructed in the package labeling, except that containers with removable stoppers may be sampled by removing the closure and emptying the contents into a clean container or into the filtration funnel. 

The number of samples chosen must be adequate to provide a statistically sound assessment of whether a batch or other large group of units represented by the samples meets or exceeds the limit. Test a solution pool of 10 or more units, or test individual units. For large-volume injections, individual units are tested. 

MICROSCOPE PREPARATION 

Place the auxiliary illuminator close to the microscope stage, focusing the illuminator to give a concentrated area of illumination on a filter membrane positioned on the microscope stage. Adjust the illuminator height so that the angle of incidence of the light is 10º to 20º with the horizontal. Using the internal episcopic brightfield illuminator, fully open the field and aperture diaphragms. Center the lamp filament, and focus the microscope on a filter containing particles. Adjust the intensity of reflected illumination until particles are clearly visible and show pronounced shadows. Adjust the intensity of episcopic illumination to the lowest setting, then increase the intensity of episcopic illumination until shadows cast by particies show the least perceptible decrease in contrast. 

OPERATION OF CIRCULAR DIAMETER GRATICULE 

 The relative error of the graticule used must initially be measured with an NIST-certified stage mierometer. To accomplish this, align the graticule micrometer scale with the stage mierometer so that they are parallel. (Compare the scales, using as large a number of graduations on each as possible). Read the number of graticule scale divisions, GSD, compared to stage micrometer divisions, SMD. Calculate the relative error by the formula: 

(GSD - SMD) 100 SMD. 

A relative error of ± 2% is acceptable. The basic technique of measurement applied with the use of the circular diameter graticule is to transform mentally the image of each particle into a circle and then compare it to the 10- and 25 mm graticule reference circles. The sizing process is carried out without superimposing the particle on the reference circles; particles are not moved from their locations within the graticule field of view (the large circle) for 
comparison to the reference circles. Use the inner diameter of the clear graticule reference circles to size white and transparent particles. Use the outer diameter of the black opaque graticule reference circles to size dark particles. 

Rotate the graticule in the right microscope eyepiece so that the linear scale is located at the bottom of the field of view, bringing the graticule into sharp focus by adjusting the right eyepiece diopter ring while viewing an out-of-focus specimen. Focus the microscope on a specimen, looking through the right eyepiece only. Then, looking through the left eyepiece, adjust the left eyepiece diopter to bring the specimen into sharp focus. 

PREPARATION OF FILTRATION APPARATUS 

Preferably, wash the filtration funnel, base, and diffuser in a solution of liquid detergent and hot water. Rinse with hot water. Following the hot water rinse, apply a second rinse with filtered Purified Water, using a pressurized jet of water over the entire exterior and interior surfaces of the filtration apparatus. Repeat the pressurized rinse procedure using filtered isopropyl alcohol. Finally, using the pressurized rinser, rinse the apparatus with filtered Purified Water. 

Assemble the cleaned filtration apparatus with the diffuser on top of the filtration base, placing the clean membrane filter on top of the diffuser. Remove a membrane filter from its container using ultracleaned blunt forceps. Use a low pressurized stream of filtered Purified Water to wash both sides of the filter thoroughly by starting at the top and sweeping back and forth to the bottom. Place the funnel assembly on top of the filtration base, and 
lock it into place. 

TEST PREPARATIONS 

Liquid Fill (Large or Small-Volume Injections): Thoroughly mix the units to he tested by inverting 20 times. Clean the outer surface of the solution container thoroughly with pressurized filtered Purified Water. Open the units in a manner consistent with generation of lowest possible numbers of background particles. In a cleaned container, open and combine the contents of not less than 10 containers, or filter the contents of individual containers. 

Dry Powder Vials: Constitute the material with an appropriate diluent using the method least likely to introduce extraneous contamination. Add the constituted solütion to the filter funnel atop a sufficient volume of filtered water to bring the total volume to be filtered to about 100 mL, or use a smaller funnel. Pool the desired number of units, and proceed as directed under Test Procedure. 

Drug-Diluent Products: For products packaged in containers that are constructed to hold the drug product and a solvent in separate compartments, mix each unit as directed in the labeling, activating and agitating each unit in order to ensure thorough mixing of the separate components. Proceed as directed under Test Procedure. 

  
Multiple-dose Containers:  For products labeled as Pharmacy Bulk Packages, proceed as directed under Liquid Fill, and calculate the results on a sample volume that is equal to the maximum dose given in the labeling. For the calculations below, consider this portion to be the equivalent of the contents of one full container. 

  
Test Procedure:  Fill the filtration funnel with test solution and apply vacuum.  
[NOTE-For large-volume injections, do not allow the fluid volume in the filtration funnel to drop below 100 mL, between refills]. After the last addition of solution, begin rinsing the walls of the  funnel by directing a low pressure stream of filtered Purified Water in a circular pattern along the walls of the funnel, and stop rinsing the  funnel before the volume falls below about one-fourth of the fill level if a partial count procedure is to be performed. Maintain the vacuum until all the liquid in the funnel is gone. Turn the vacuum off, and remove the filtration funnel from the filtration base, removing the filter with blunt forceps to a petri slide. Secure in place with double stick tape, and label with sample identification. Allow the filter to air-dry in the clean bench with the cover ajar. 

[NOTE-If a small-volume injection unit of 25 mL or a solution pool of less than 25 mL total volume is to be tested and the partial count procedure is to be used, a volume of diluent sufficient to bring the total volume to about 100 mL may be added or, alternatively, a smaller funnel may be used. After adding the pooled material, apply vacuum and proceed with fíltration as specified above for a liquid solution unit. 

Enumeration of Particles:  The microscopic test described in this section is flexible in that it can count, in particles per mL, specimens containing 1 particle per mL as well as those containing significantly higher numbers of particles per mL. 
This method may be used where all particles on an analysis membrane surface are counted or where only those particles on some fractional area of a membrane surface are counted. 

TOTAL COUNT PROCEDURE 

In performance of a total count, the graticule field of view (GFOV) defined by the large circle of the graticule is ignored and the vertical crosshair is used. Scan the entire membrane from right to left in a path that adjoins but does not overlap the first scan path. Repeat this procedure, moving from left to right to left until all particles on the embrane are counted. Record the total number of particles that are 10 um or larger and the number that are 25 mm or larger. For large-volume injections calculate the particle count, in particles per mL, for the unit tested by the formula: 

P/V,  

where P is the total number of particles counted, and V is the volume, in mL, of the solution. For small-volume injections calculate the particle count, in particles per container, by the formula: 

P/N 

in which P is the total number of particles counted, and N is the number of units pooled (1 in the case of a single unit) 

PARTIAL COUNT PROCEDURE 

If a partial count of particles on a membrane is to be performed the analyst must first ensure that an even distribution of particles is present on the membrane. This is assessed by rapid scanning in order to look for clumps of particles. None should be present. Count the 10-mm or larger particles in one GFOV at the edge of the filtration area as well as those in the center of the GFOV. The number of >10-mm or larger particles in the GFOV with the highest total particle count is not more than twice that of the GFOV with the lowest particle count. Reject a filter failing these criteria, and prepare another if a partial count procedure is used or, alternatively, analyze this membrane by the total count method. 

The normal number of GFOV counted for a partial count is 20. If a smaller confidence interval about the result is desired, a larger number of fields and particles may be counted. Count all particles that have a circular area diameter of 10 mm or larger and 25 mm or larger within the GFOV and those that are in contact with the right side of the GFOV circle. Do not count particles outside of the GFOV. Ignore those that touch the left side of the GFOV circle. The dividing line between right and left sides of the GFOV circle is the vertical cross hair. [NOTE- Make the best possible judgment on particle size without changing the microscope magnification or illumination].
  
The analyst may increase or decrease the number of fields counted to achieve a desired confidence interval about the count obtained. Calculate the confidence interval about the number of 10-um or larger and 25-mm or larger particles per mL by the formula: 

in which P is the number of particles counted. In the event that filtration of a solution results in a particle count too high to enumerate accurately in a GFOV, a single quadrant of the GFOV may be counted with a 47-mm filter and a fractional aliquot of the unit. 

To perform a partial count of the particles on a membrane, start at the right center edge of the filtration area and begin counting adjacent GFOVs. When the left edge of the filtration area is reached move one GFOV toward the top of the filter and continue counting GFOVs by moving in the opposite direction. 
Moving from one GFOV to the next can be accomplished by one of two methods. One method is to define a landmark (particle of surface irregularity in the filter) and move over one GFOV in relation to the landmark. A second method is to use the vernier on the microscope stage to move one millimeter between GFOVs. To facilitate the latter, adjust the microscope x and y stage positioning controls to a whole number at the starting position at the center right edge of the filtration area, then each GFOV will be one whole division of movement of the x stage positioning control. If the top of the filtration area is reached before the desired number of GFOVs is reached, begin again at the right center edgeof the filtration area one GFOV lower than the first time. This time move downward on the membrane when the end of a row of GFOVs is reached. Continue as before until the number of GFOVs is complete. 

For large-volume injections, if a partial count procedure for the >10 mm and >25mm size ranges is used, calculate the particles per mL, by the formula: 

PA_t/A_pV, 

in which P is the number of particles counted, A_t is the filtration area in mm² of the membrane, A_p is the partial area counted in mm² based on the number of graticule fields counted, and V is volume,in mL, of solution filtered. For a solution pool (for small-volume injection units containing less than 25 mL) or for a single unit of a small-volume injection, calculate the number of particles per unit by the formula: 

pA_t/A_pn 

in which n is the number of units counted (1 in the case of a single unit), and the other terms are as defined above. 

Interpretation 

The injection meets the requirements of the test if the average number of particles present in the units tested does not exceed the values listed in Table 2. 

Table 2. Microscopic Method Particle Count.