General Chapters: <788> PARTICULATE MATTER IN INJECTIONS

788

PARTICULATE MATTER IN INJECTIONS

Particulate matter consists of mobile, randomly-sourced, extraneous substances, other than gas bubbles, that cannot be quantitated by chemical analysis due to the small amount of material that it represents and to its heterogeneous composition. Injectable solutions, including solutions constituted from sterile solids intended for parenteral use, is essentially free from particulate matter that can be observed on visual inspection. The tests described herein are physical tests performed for the purpose of enumerating subvisible extraneous particles within specific size ranges.

Microscopic and light obscuration procedures for the determination of particulate matter are given herein. This chapter provides a test approach in two stages. The injection is first tested by the light obscuration procedure (stage 1). If it fails to meet the prescribed limits, it must pass the microscopic procedure (stage 2) with its own set of test limits. Where for technical reasons the injection cannot be tested by light obscuration, microscopic testing may be used exclusively. Documentation demonstrating that the light obscuration procedure is incapable of testing the injection or produces invalid results is required in each case. It is expected that most articles will meet the requirements on the basis of the light obscuration test alone; however, it may be necessary to test some articles by the light obscuration test followed by the microscopic test to reach a conclusion on conformance to requirements.

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. Excluded from the requirements of this chapter are injections intended solely for intramuscular and subcutaneous administration.

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. Similarly, products that produce air or gas bubbles when drawn into the sensor, such as bicarbonate-buffered formulations, may also require microscopic testing. Refer to the specific monographs when a question of test applicability occurs. Higher limits are appropriate for certain articles and will be specified in the individual monographs.

In some instances, the viscosity of a material to be tested may be sufficiently high so as to preclude its analysis by either test method. In this event, a quantitative dilution with an appropriate diluent may be made to decrease viscosity, as necessary, to allow the analysis to be performed.

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. Products for which the individual monograph specifies that the label states that the product is to be used with a final filter are exempt from these requirements.

Test Apparatus

The apparatus is an electronic, liquid-borne particle 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 responsible 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 responsibility of the 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 particles in the sensor view volume comprise less than 10% of the counts collected for 10-µm 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 instrument qualification 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 filtered distilled or deionized water that has been passed through a filter having a 1.2-µm or finer porosity. Transfer a volume of filtered distilled or deionized water 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 the amount 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 manufacturer's specifications for the sensor used. This may be accomplished by using a calibrated stopwatch 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 rate as that selected for 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 having diameters of about 10, 15, and 25 µm, in an aqueous vehicle.* The calibrator spheres must have a mean diameter of within 5% of the 10-, 15-, and 25-µm 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 suspensions of the calibrator spheres in water at a concentration of 1000 to 5000 particles per mL, and determine the channel setting that corresponds to the highest count setting for the sphere distribution. This is determined by using the highest count threshold setting to split the distribution into two bins containing equal numbers 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 including asymmetrical portions of the peak. The portion of the distribution, 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 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 and upper voltage settings are determined by the equations:

VL = 0.64VS,

in which VL is the lower voltage setting and VS is the voltage at the peak center, and

VU = 1.44VS,

in which VU 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-µm 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 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 response curve equivalent to that attained by the manual method and if 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 response 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 calibration 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-µm particles using the 10-µm calibrator spheres. The relative standard deviation of the size distribution of the standard particles used is not more than 5%. Acceptable methods of determining particle 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.

Manual 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-µm spheres. Adjust 3 channels of the counter to be 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 are 1.68 ± 10% and 0.32 ± 10%, respectively, of the count in Channel 2. If 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 sphere suspension has been counted. Verify that Channel 1 and Channel 3 counts are 1.68 ± 3% and 0.32 ± 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-µm standard. Calculate the percentage of resolution of the sensor by the formula:

in which SO is the highest observed standard deviation determined for the sphere; SS is the supplier's reported standard deviation for the spheres; and D is the diameter, in µm, of the spheres as specified by the supplier. The resolution is not more than 10%.

Automated Method— Software that allows for the automated determination of sensor resolution is available for some counters. 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 10-µm 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-µm 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-µm 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 1—

Procedure— Prepare the suspension and blank using the USP Particle Count RS. With the instrument set to count in the cumulative (total) mode, collect counts at settings of greater than or equal to 10 µm and greater than or equal to 15 µm. Mix the blank by inverting 25 times within 10 seconds, and degas the mixture by sonicating (at 80 to 120 watts) for about 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 greater than or equal to 10 µm and from the analysis of the two portions of the blank at greater than or equal to 10 µm, calculate the number of particles in each mL by the formula:

(PS – PB) / V,

in which PS is the average particle count obtained from the suspension; PB 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 the setting of not less than 15 µm.

Interpretation— The instrument meets the requirements for Particle Counting Accuracy if the count obtained at greater than or equal to 10 µm and the ratio of the counts obtained at greater than or equal to 10 µm to those obtained at greater than or equal to 15 µm conform to the values that accompany the USP Particle Count RS. If the instrument does not meet the requirements for Particle Counting Accuracy, repeat the procedure using 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 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 2—

Procedure— Using standard calibrator spheres having a nominal diameter of 15 to 30 µm, prepare a suspension containing between 50 and 200 particles per mL. Degas the suspension by sonicating (at 80 to 120 watts) for about 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-µm 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 Test. 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. Preferably, the test specimen, glassware, closures, and other required equipment are prepared in an environment protected by high-efficiency particulate air (HEPA) filters, and nonshedding garments and powder-free gloves are worn 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 distilled or deionized 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 distilled or deionized water, using a hand-held pressure nozzle with final filter or other appropriate filtered water source, such as distilled or deionized water passed through a capsule filter having a 1.2-µm or finer porosity.

To collect blank counts, use a cleaned vessel of the type and volume representative of that to be used in the test. Place a 50-mL volume of filtered distilled or deionized water in the vessel, and agitate the water sample in the cleaned glassware by inversion or swirling. [NOTE—A smaller volume, consistent with the article to be counted, can be used.] Degas by sonicating (at 80 to 120 watts) for about 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-µm or greater size, or more than 2 particles of 25-µm or greater size are observed in the combined 10-mL sample, the environment is not suitable for particulate analysis: the filtered distilled or deionized 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

Prepare the test specimens in the following sequence. Outside of the laminar enclosure, remove outer closures, sealing bands, and any loose or shedding paper labels. Rinse the exteriors of the containers with filtered distilled or deionized water as directed under Test Environment. Protect the containers from environmental contamination until analyzed. Withdraw the contents of the containers under test in a manner least likely to generate particles that could enter the sample. Contents of containers with removable stoppers may be withdrawn directly by removing the closures. Sampling devices having a needle to penetrate the unit closure may also be employed. Products packaged in flexible plastic containers may be sampled by cutting the medication or administration port tube or a corner from the unit with a suitably cleaned razor blade or scissors.

Dry or lyophilized products may be constituted either by removing the closure to add diluent or by injecting diluent with a hypodermic syringe having a 1.2-µm or finer syringe filter. If test specimens are to be pooled, remove the closure and empty the contents into a clean container.

The number of test specimens must be adequate to provide a statistically sound assessment of whether a batch or other large group of units represented by the test specimens meets or exceeds the limits. If the volume in the container is less than 25 mL, test a solution pool of 10 or more units. Single small-volume injection units may be tested if the individual unit volume is 25 mL or more. For large-volume injections, individual units are tested. For large-volume injections or for small-volume injections where the individual unit volume is 25 mL or more, fewer than 10 units may be tested, based on the definition of an appropriate sampling plan.

PRODUCT DETERMINATION

Depending upon the dosage form being tested, proceed as directed under the appropriate category below.

Liquid Preparations—

Volume in Container Less than 25 mL— Prepare the containers as directed under Test Preparation. Mix and suspend the particulate matter in each unit by inverting the unit 20 times. [NOTE—Because of the small volume of some products, it may be necessary to agitate the solution more vigorously to suspend the particles properly.] In a cleaned container, open and combine the contents of 10 or more units to obtain a volume of not less than 20 mL. Degas the pooled solution by sonicating for about 30 seconds or by allowing the solution to stand undisturbed until it 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 a minimum of three aliquots, each not less than 5-mL in volume, into the light obscuration counter sensor. Discard the data from the first portion. [NOTE—For some products, a pool of 15 or more units may be necessary to achieve a pool volume sufficient for three 5-mL sample aliquots. Smaller sample aliquots (i.e., less than 5 mL) can be used if the assay result obtained with the smaller aliquots is validated to give an assessment of batch suitability equivalent to that obtained with the 5-mL aliquots specified above.]

Volume in Container 25 mL or More— Prepare the containers as directed under Test Preparation. Mix and suspend the particulate matter in each unit by inverting the unit 20 times. Degas the solution by sonicating for about 30 seconds or by allowing the solution to stand undisturbed until it is free from air bubbles. Remove the closure of the unit or effect entry by other means so that the counter probe can be inserted into the middle of the solution. Gently stir the contents of the unit by hand-swirling or by mechanical means. Withdraw not fewer than three aliquots, each not less than 5 mL in volume, into the light obscuration counter sensor. Discard the data from the first portion.

Dry or Lyophilized Preparations— Prepare the containers as directed under Test Preparation. Open each container, taking care not to contaminate the opening or cover. Constitute as directed under Test Preparation, using the specified volume of filtered water or an appropriate filtered diluent if water is not suitable. Replace the closure, and manually agitate the container sufficiently to ensure dissolution of the drug. [NOTE—For some dry or lyophilized products, it may be necessary to let the units stand for a suitable interval, and then agitate again to effect complete dissolution.] After the drug in the constituted sample is completely dissolved, mix and suspend the particulate matter present in each unit by inverting it 20 times prior to analysis. Proceed as directed for the appropriate unit volume under Liquid Preparations, and analyze by withdrawing a minimum of three aliquots, each not less than 5 mL in volume, into the light obscuration counter sensor. Discard the data from the first portion.

Products Packaged with Dual Compartments Constructed to Hold the Drug Product and a Solvent in Separate Compartments— Prepare the units to be tested as directed under Test Preparation. Mix each unit as directed in the labeling, activating and agitating so as to ensure thorough mixing of the separate components and drug dissolution. Degas the units to be tested by sonicating or by allowing the solution to stand undisturbed until it is free from any air bubbles. Proceed as directed for the appropriate unit volume under Liquid Preparations, and analyze by withdrawing a minimum of three aliquots, each not less than 5 mL in volume, into the light obscuration counter sensor. Discard the data from the first portion.

Products Labeled “Pharmacy Bulk Package Not for Direct Infusion”— Proceed as directed for Liquid Preparations where the volume is 25 mL or more. Calculate the test result on a portion that is equivalent to the maximum dose given in the labeling. For example, if the total bulk package volume is 100 mL and the maximum dose volume is 10 mL, then the average light obscuration particle count per mL would be multiplied by 10 to obtain the test result based on the 10-mL maximum dose. [NOTE—For the calculations of test results, consider this maximum dose portion to be the equivalent of the contents of one full container.]

Calculations

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

PVT / VAn,

in which P is the average particle count obtained from the portions analyzed; VT is the volume of pooled sample, in mL; VA is the volume, in mL, of each portion analyzed; and n is the number of 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 / VA,

in which P is the average particle count obtained from the portions analyzed; V is the volume, in mL, of the tested unit; and VA 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 of Injection 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.

For all types of product, if the tested material has been diluted to effect a decrease in viscosity, the dilution factor must be accounted for in the calculation of the final test result.

Interpretation

The injection meets the requirements of the test if the calculated number of particles present in each discrete unit tested or in each pooled sample tested does not exceed the appropriate value listed in Table 1. If the average number of particles exceeds the limit, test the article by the Microscopic Particle Count Test.

Table 1. Light Obscuration Test Particle Count

	10 µm	25 µm
Small-Volume Injections	6000	600 per container
Large-Volume Injections	25	3 per mL

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 that do not filter readily because of high viscosity (e.g., concentrated dextrose, starch solutions, or dextrans). In performance of the microscopic assay do not attempt to size or enumerate amorphous, semiliquid, or otherwise morphologically indistinct materials that have the appearance of a stain or discoloration on the membrane surface. 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 µm 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 for changes in interpupillary distance by maintaining a constant tube length. The objective and eyepiece combination of lenses must give a magnification of 100 ± 10×. The objective must be of 10× 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 10× magnification. In addition, one 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 that can be adjusted to give incident oblique illumination at an angle of 10

to 20

. 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 daylight filters to decrease operator fatigue during use.

Circular Diameter Graticule— Use a circular diameter graticule (see Figure 1)

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-µm and 25-µm diameters at 100× are provided as comparison scales for particle sizing.

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.

Micrometer— Use a stage micrometer, graduated in 10-µm 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-µm or finer retention rating at a range of pressures from 10 psi to 80 psi, and membrane filters (25 mm or 47 mm nongridded or gridded, black or dark gray, or of suitable material compatible with the product, with a 1.0-µm or finer porosity). Use blunt forceps to handle membrane filters.

Test Environment

Use a laminar flow hood or other laminar airflow enclosure, having a capacity sufficient to envelop the area in which the analysis is prepared, and containing HEPA-filtered air having not more than 100 particles (0.5 µm or larger) per cubic foot. For the blank determination, deliver a 50-mL volume of filtered distilled or deionized water to the filtration funnel. 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 100×. If not more than 20 particles 10 µm or larger in size and 5 particles 25 µm or larger are present within the filtration area, the background particle level is sufficiently low for performance of the microscopic assay.

Throughout this procedure, it is preferable to use powder-free gloves and thoroughly clean glassware and equipment. Prior to conducting the test, clean the work surfaces of the laminar flow enclosure with an appropriate solvent. Glassware and equipment should be rinsed successively with a warm, residue-free solution of detergent, hot water, filtered distilled or deionized water, and isopropyl alcohol. [NOTE—Prior to use, pass the distilled or deionized water and the isopropyl alcohol through filters having a 1.2-µm or finer porosity.] 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.

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 10

to 20

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 particles 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 micrometer. To accomplish this, align the graticule micrometer scale with the stage micrometer 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:

100[(GSD – SMD) / 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-µm 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. Compare the area of the particle being sized to that of the black or transparent circles. Use the area of the clear graticule reference circles to size white or transparent particles. Use the area of the black 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 distilled or deionized 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 distilled or deionized water.

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. Assemble the cleaned filtration apparatus with the diffuser on top of the filtration base, placing the clean membrane filter on top of the diffuser. Place the funnel assembly on top of the filtration base, and lock it into place.

Test Procedure

TEST PREPARATIONS

Proceed as directed for Test Preparation under the Light Obscuration Particle Count Test, beginning with “Prepare the test specimens in the following sequence,” and ending with “For large-volume injections, individual units are tested.” For small-volume injections containing a volume of 25 mL or more and tested individually and for large-volume injections, the entire unit volume is tested. For large-volume injections or for small-volume injections where the individual unit volume is 25 mL or more, fewer than 10 units may be tested, based on the definition of an appropriate sampling plan.

PRODUCT DETERMINATION

Depending upon the dosage form being tested, proceed as directed under the appropriate category below.

Liquid Preparations— Thoroughly mix the units to be tested by inverting 20 times. Open the units in a manner consistent with the generation of the lowest possible numbers of background particles. For products less than 25 mL in volume, open and combine the contents of 10 or more units in a cleaned container. Filter large-volume injection units individually. Small-volume injection units having a volume of 25 mL or more may be filtered individually.

Transfer to the filtration funnel the total volume of a solution pool or of a single unit, and apply vacuum. If the volume of solution to be filtered exceeds the volume of the filtration funnel, add, stepwise, a portion of the solution until the entire volume is filtered. If the partial count procedure is to be used (see Partial Count Procedure under Enumeration of Particles), do not allow the liquid volume in the filtration funnel to drop below approximately one-half of the funnel volume between refills. [NOTE—Use a filter funnel appropriate to the volume of solution if a partial count procedure is to be employed. This is necessary to ensure even distribution of particles on the analytical membrane.]

After the last addition of solution, begin rinsing the walls of the funnel by directing a low-pressure stream of filtered distilled or deionized 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. Maintain the vacuum until all the liquid in the funnel is gone.

Remove the filtration funnel from the filtration base while maintaining vacuum, then turn the vacuum off, and remove the filter membrane with blunt forceps. Place the filter in a Petri dish or similar container, secure in place with double-sided tape, and label with sample identification. Allow the filter to air-dry in the laminar-flow enclosure with the cover ajar.

Dry or Lyophilized Preparations— To test a dry powder vial or similar container of drug powder, constitute the material with an appropriate diluent, using the method least likely to introduce extraneous contamination, as directed for Test Preparation under Light Obscuration Particle Count Test. Using a solution pool of 10 or more units, or the desired number of individual units, proceed as directed for Liquid Preparations.

Products Packaged with Dual Compartments Constructed to Hold the Drug Product and a Solvent in Separate Compartments— Activate each unit as directed in the labeling, agitating the contents sufficiently to ensure thorough mixing of the separate components, and then proceed as directed for Liquid Preparations.

Pharmacy Bulk Packages or Multiple-Dose Containers— For Products Labeled “Pharmacy Bulk Package—Not for Direct Infusion,” or for multiple-dose containers, proceed as directed for Liquid Preparations, filtering the total unit volume.

Calculate the test result on a portion that is equal to the maximum dose given in the labeling. Consider this portion to be the equivalent of the contents of one full container. For example, if the total bulk package volume is 100 mL and the maximum dose listed is 10 mL, the microscopic total unit volume count test result would be multiplied by 0.1 to obtain the test result for the 10-mL dose volume. [NOTE—For calculation of the test result, consider this portion to be the equivalent of the contents of one full container.]

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 membrane are counted. Record the total number of particles that are 10 µm or larger and the number that are 25 µm 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 an individual 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-µm or larger particles in one GFOV at the edge of the filtration area as well as those in the center of the membrane. The number of

10-µm 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 µm or larger and 25 µm 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.]

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 or 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 1 mm 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 edge of 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-µm and

25-µm size ranges is used, calculate the particles per mL by the formula:

PAT / APV,

in which P is the number of particles counted; AT is the filtration area, in mm2, of the membrane; AP is the partial area counted, in mm2, based on the number of graticule fields counted; and V is the 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:

PAT / APn,

in which n is the number of units counted (1 in the case of an individual unit); and the other terms are as defined above.

For all types of product, if the tested material has been diluted to effect a decrease in viscosity, the dilution factor must be accounted for in the calculation of the final test result.

Interpretation

The injection meets the requirements of the test if the number of particles present (actual or calculated) in each discrete unit tested or in each pooled sample tested does not exceed the appropriate value listed in Table 2.

Table 2. Microscopic Method Particle Count

	10 µm	25 µm
Small-Volume Injections	3000	300 per container
Large-Volume Injections	12	2 per mL

* ASTM standard F658-87 provides useful discussions pertaining to calibration procedures applying near-monosize latex spheres.

Auxiliary Information— Staff Liaison : James W. Kelly, Ph.D., Scientist

Expert Committee : (PPI05) Parenteral Products-Industrial 05

USP29–NF24 Page 2722

Pharmacopeial Forum : Volume No. 28(6) Page 1930

Phone Number : 1-301-816-8167


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