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 particle-free water.1 Transfer a volume of particle-free water that is greater than the sample volume to a container, and weigh. Using the sample feeding device, withdraw 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 31). [note—Instruments of this type require a variable tare volume. This is the amount of sample withdrawn before counting.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. ]

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 below at the same flow rate as that selected for calibration of the instrument.

USP 788 specifies the use of dispersions of spherical particles of known sizes between 10 µm and 25 µm in particle-free water. More options follow:

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.

Determine the particle counting accuracy of the instrument, using Method 1 (for sensors requiring the moving window half-count (MWHC) method for calibration), Method 2 (for multichannel sensors), or Method 3 for any instrument (manual comparison to membrane microscope method).

Prepare the test specimens in the following sequence. Outside of the unidirectional airflow cabinet to be used for the test, remove outer closures, sealing bands, but not the sealing closure. If shedding is noted to be an issue, remove or tape over the product labels as well. Place the samples in the test cabinet, and rinse the exteriors of the containers with filtered water as directed under Test Environment. Protect the containers from environmental contamination until analyzed. After proper mixing, open and withdraw, pour or otherwise sample the contents of the containers under test in a manner least likely to generate particles that could enter the test specimen. Contents of containers with removable stoppers may be poured out directly after 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 using their internal diluent, by removing the closure to add supplied product diluent or by injecting filtered water via hypodermic syringe. If test specimens are to be pooled, remove the closure and empty the contents into a clean container.

USP 788 provides the sampling plan according to product volume. For all products, regardless of volume, comprehensive experience regarding the integrity and consistency of the batch is gained throughout development, allowing the proper sampling plans to be applied in commercial production that ensure sample selection is representative of batch quality. All batches must have sampling plans that accommodate desired statistical measures of batch quality and facilitate process control.

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

For all types of product, if the tested material has been diluted to decrease the viscosity, the dilution factor must be accounted for in the calculation of the final test result. For all test results, the particle count 10 µm represents all threshold bin counts.

The microscope optical alignment and illumination are critical for success of this method. Although it is not difficult to differentiate a 10 µm from a 25 µm particle at 100× with reflected light, the decision regarding the boundary at each size category will be difficult with poor equipment, maintenance or optical alignment. Also operator fatigue is caused by poor microscope alignment. We will have to make decisions such as “is this particle 9 µm or 11 µm?” and “is that particle 24 µm or 26 µm?” Optimized system resolution, that is, the ability to discern discrete points of minimal separation, relies upon good optical systems, aligned well. Factors including instrument cleanliness, resolution, e.g., objective N.A.,4 focus of both eyepieces and the graticule will play significant roles in attaining best images. In consideration of optimizing the use of the binocular compound microscope, it is best to utilize operators familiar with the instrument, and comfortable with alignment. The operator conducting the method should align the optics and illumination for their use, with supervisory/trainer approval.

It is recommended to start with alignment of the microscope for a typical transmitted illumination observation using a known sample. Any specimen familiar to the operator will suffice; however, a common particle count reference standard suspension such as the USP PCRS is a recommended selection, since it is also utilized in method system suitability evaluation. A drop of the USP PCRS is placed between a glass microscope slide and cover slip and viewed microscopically.5 With appropriate interpupillary distance and a comfortable sitting position at the microscope, the operator examines the fields of suspended spheres. One should observe the small standard spheres crisply in a combined field (with ease) for both eyes. One attains crisp focus and ease of view after separate focal adjustment of each eyepiece focus for a single point on the specimen.

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.

When the operator is not comfortable using the microscope or does not attain an equivalent crisp focus for each eye in a merged field of view, the counting will become a difficult experience and fatigue and flawed size comparison will result.

Nothing is better for preparing the operator for counting particles than to examine a test membrane as a positive control. Seasoned microscopists may not require this step, but for new operators or individuals conducting many different types of methods in the modern laboratory, familiarization is a prudent exercise. A filter membrane of the type being used for the method, such as a 25-mm color-contrast, plain 0.45-µm nominal pore size, containing particles, is a good choice. This may be a sample from previous method that contains a variety of particle types, or one prepared for familiarization. This positive test control will contain natural particles (flakes, threads, equant particles, various colors/opacity, a range of sizes, etc.) to effectively refresh the operator's sensitivity and facilitate microscope and illumination alignment for optimal viewing.

One would examine the membrane preparation, locate a typical array of particles and first bring the illumination into good alignment:

The USP graticule is specifically fabricated for each microscope. The relative error of the graticule used must be ±2% and is initially 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:

A relative error of ±2% is acceptable and verifies good alignment, focus and proper magnification. Thereafter, a day-of-use verification by the microscope operator with the NIST stage micrometer or commercial stage micrometer is sufficient to demonstrate proper setup.

The basic technique of measurement applied with the use of the circular diameter graticule is to count all particles 10 µm and larger, further categorizing in 10 µm and 25 µm. The circular zone or graticule field of view is a useful zone for active sizing and counting. Particles are compared to the linear scale and/or circles to determine their size in equivalent circular diameter. This is conducted by transforming mentally the image of each particle into a circle and then comparing 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. The intent of comparing particles to an equivalent circular diameter is correlation to the LO particle sizing methodology, for which many manufacturers have extensive databases. In practice, particles with nearly circular areas will correlate well with the graticule circle diameters. For particles with one long axis, such as rods and needles, the conversion to circular area will produce more significant bias to smaller estimated sizes. It may be simpler, and most conservative, to count particles in longest chord. To use an extreme example, the total count of mono-dispersions of fine needle crystals would vary greatly dependant upon the size determination utilized.

In order to properly focus the ocular lenses and attain balanced single-field view, each operator must bring the USP graticule lines into sharp focus by adjusting the eyepiece diopter ring (it helps to have an “infinite” view,or out-of-focus specimen). Next, focus the microscope on a specimen, through this same eyepiece, and then looking only through the other eyepiece, adjust its diopter ring to bring the specimen into sharp focus. The USP graticule and specimen particles are now in focus on a well-balanced illumination field.

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. It is prudent to maintain the liquid volume in the filtration funnel above one-half of the funnel volume between refills, especially if the partial count procedure is to be used (see Enumeration of Particles, Partial Count Procedure, below). [note—This is necessary in order 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 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 non-serrated forceps. Place the filter in the prepared holder and label with sample identification. Allow the filter to air-dry in the unidirectional airflow enclosure with the cover ajar.

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 microscope 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. ]

The microscope method can be tedious (boring), imprecise (poor agreement within and between labs) and particle sizing can be inaccurate for non-spherical or equant particle shapes. Operator fatigue is promoted by poor ergonomic fit (chair height), by poor or imbalanced ocular focus and by excessive eye movement. Restricting the eye movement to a field-defining graticule such as the USP counting graticule restricts eye movement to the central one-third field of view. This significantly limits eye movement and thus fatigue.

Sample size is an important consideration in counting precision. Care must be taken to sample many containers within a batch for good representation of the particle distribution. Accordingly, the portion of the individual package sampled is key. Particles may float or settle. Sampling only the first 25 mL of a LVP or sampling without adequate and recent mixing is a mistake, and will lead to serious under-counting. Sampling whole, well-mixed containers with the particles in suspension is the best approach.

Counting the isolated particles is an important parameter. Counting all of the particles retained on the membrane is certainly the best approach, and then the simple problem is determining the correct size for placement into the threshold bins, 10 µm and 25 µm. This will be increasingly important for methods utilizing additional bins for population determination, such as 5 µm, 50 µm,100 µm, etc. Note that the particle limits for 788 and 789 must be reported as all particles 10 µm and all particles 25 µm. If the lab method has been configured to count in several bins, such as 10–25 µm, 25–50 µm, 50 µm, etc., all bins 10 µm must be added to yield total 10 µm count; all bins 25 µm need to be added to yield total count 25 µm. Using a number of narrow size bins may be beneficial in product improvement efforts to separate particle groups.

In performance of a total count, the graticule field of view (GFOV) is defined by the large circle of the graticule, and the vertical crosshair is used as a counting target. Scan the membrane in paths that cover the effective filtration area (EFA), adjoining but not overlapping previous scan paths. Repeat this procedure, tabulating particle counts minimally in the 10 µm–25 µm and 25-µm thresholds, moving across the membrane until all particles on the membrane within the EFA are counted. Record the total number of particles that are 10 µm–25 µm and the number that are 25 µm or larger.

For large-volume products, calculate the particle count, in particles per mL, for each unit tested by the formulas:

in which PS10 is the total particle count obtained from all portions analyzed, PS25 is the total particle count obtained from all portions 25 µm analyzed, and V is the volume, in mL, of the solution tested.

For example, the analyst has counted the test samples in four bins: (a) 10–25 µm, (b) 25 µm–50 µm, (c) 50 µm –100 µm, and (d) 100 µm. They would then calculate as:

For small-volume products, calculate the particle count, in particles per container, by the formulas:

in which PS10 is the total particle count obtained from all portions analyzed, PS25 is the total particle count obtained from all portions 25 µm analyzed, and n is the number of units pooled (1 in the case of an individual unit).

When encountering a membrane full of particles, the task of counting all of them, properly, is daunting. Consider that an SVP with an at-limit content of small particles, sampled in a 10-vial pool would have 30,000 10-µm particles on the membrane. Partial or statistical counting of the membrane effective filtration area may be the only means to attain reasonable results. Partial counting should not be used to reduce count times, just as a means to estimate the total load on a high-count isolate. A field-defining device, such as grids on the membrane surface or an ocular graticule field of view have been used reliably. An ocular graticule provides a sharp boundary for area definition. Gridded membrane lines are rather broad and have ink-spatter that may be taken for particulate matter.

Which portions and how much of the EFA should be counted? In consideration of 25-mm membranes, the EFA is 16-mm diameter using typical commercial filtration funnels, and thus (pi × r2) = 201 mm2. Based upon earlier proposals from the HIMA committee and discussion by Draftz (see Reference 3), acceptable confidence intervals (Poisson distribution, 2 standard deviations) dictate that for samples with less than 1000 particles, the imprecision of statistical counting is objectionable. Full count is recommended for such samples. For samples with more than 1000 particles on the isolate membrane, using a 25-mm membrane, a reasonable estimate of particle population is attained using 20 GFOV. If a smaller confidence interval about the result is desired, a larger number of fields and particles may be counted.

For 47-mm membranes, the EFA is 37 mm. These larger diameter membranes may be selected for formulations needing more membrane surface area (having slow flow characteristics through 25-mm membranes) the EFA = (pi × r2) = (pi × 18.5 mm2) = 1075 mm2. Thus, for 47-mm membrane EFAs, many more GFOVs must be counted to attain similar confidence. Using 100 GFOVs for partial counting of 47-mm membranes provides similar statistical confidence to the 20 GFOV/25-mm approach. Accordingly, when a particle load of 1000 or less is present, a full count is recommended.

When 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 at 50× to qualitatively scan for heterogeneity or clumps of particles. If heterogeneity is observed, one should perform a full count on the membrane. Next, count the 10-µm or larger particles in one GFOV at the edge of the filtration area as well as one in the center of the membrane. The number of 10-µm or larger particles in the GFOV with the highest total particle count must not be more than twice that of the GFOV with the lowest particle count. Fully count the membrane failing these criteria.

To perform a partial count of the particles on a membrane, Include all particles 10–25 µm and 25 µm 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 and is a useful counting line. [Note—Make the best possible judgment on particle size without changing the membrane position, microscope magnification or illumination. ]

Start at the center edge of the filtration area and begin counting adjacent GFOVs. When the other 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 method to move 1 mm between GFOVs. To facilitate the latter, adjust the microscope x- and y-method 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-method 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 products, extrapolate the total count of particles per mL by the formulas:

in which PS10 is the total particle count obtained from all fields of view and all size thresholds; PS25 is the total particle count obtained from all fields of view and all size thresholds 25 µm; AT is the filtration area, in mm2, of the membrane (inner filtration barrel diameter); AP is the partial area counted, in mm2, based on the number of graticule fields counted (GFOV area × number of GFOV counted); and V is the volume, in mL, of solution filtered.

For a solution pool (for small-volume product units containing less than 25 mL) or for a single unit of a small-volume product, extrapolate the total count of particles per unit by the formulas:

in which PS10 is the total particle count obtained from all fields of view and all size thresholds, PS25 is the total particle count obtained from all fields of view and all size thresholds 25 µm, and n is the number of units counted (1 in the case of an individual unit). For all types of product, if the tested material has been diluted to decrease viscosity, the dilution factor must be accounted for in the calculation of the final test result.