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Physical Tests / 610 Alternative Microbiological Sampling Methods 263

USP 36

610 ALTERNATIVE
MICROBIOLOGICAL SAMPLING
METHODS FOR NONSTERILE
INHALED AND NASAL PRODUCTS
INTRODUCTION
Proper microbiological sampling of microbiologically susceptible nonsterile products can be difficult because these
products are often filled into unique primary containers that
are designed to protect the product from inadvertent contamination during storage and use. These unique designs
may increase the difficulty of taking an aseptic sample of
sufficient size or volume for microbiological testing. Unless
special approaches are used, products such as inhaled, nasal
liquid, or powder dosage forms can be difficult to sample
without potential exposure to extraneous microbial contamination. This general test chapter provides these special approaches for sampling either low- or high-content inhaled or
nasal dosage forms. Alternative sampling approaches may
provide better ways to sample containers in an aseptic manner. Any alternative methodology should employ aseptic
techniques and should be conducted under environmental
and other conditions that are appropriate for aseptic
sampling.

BULK TESTING FOR LOW-CONTENT INDP

Bulk lot testing may be preferable for low-content INDP in
lieu of finished product testing to allow larger sample sizes
that are representative of the batch, without unduly increasing the risk of inadvertent microbial contamination. Bulk
testing can be performed on the bulk powder or liquid formulation just before filling. If bulk testing is performed in
lieu of finished product testing, then manufacturing
processes following bulk sampling (e.g., filling and packaging) must be validated in accordance with current good
manufacturing practice (CGMP) for their ability to prevent
microbial contamination. For microbial enumeration tests, at
least 10 g or 10 mL of bulk material, or, for specified microorganisms tests, 1 g or 1 mL of bulk material may be sampled. For small batch sizes (i.e., less than 1000 g or
1000 mL), the recommended sample size is 1% of the batch
for both microbial enumeration and specified microorganisms tests.

SAMPLING METHODS FOR HIGH-CONTENT

INDP
Dry Powder Inhalers
DPIs have an internal reservoir that contains a sufficient
quantity of formulation for multiple doses that are metered
by the device itself during activation by the patient. For
DPIs, appropriate validated procedures should be used to
sample a nonsterile drug product container.

INHALED OR NASAL DOSAGE FORMS

Inhalation Aerosols

Low-content inhaled and nasal drug products (low-content INDP) are products that have a target fill of less than
100 mg of powder or 1 mL of liquid formulation per unit
(primary container). Examples are pre-metered inhalation
powders, more commonly known as dry powder inhalers
(DPIs), and single-dose nasal sprays.
High-content INDP are multidose drug products that have
a target fill of more than 100 mg of powder or more than
1 mL of liquid formulation per unit. Examples are aerosols
for inhalation and nasal delivery, known as metered-dose
inhalers (MDIs); device-metered inhalation powders; and
multidose nasal sprays.
The appropriate sample quantity or volume should be
based on the test methodology, including any relevant general test chapters, such as 61 Microbiological Examination of
Nonsterile Products: Microbial Enumeration Tests and 62 Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms. Testing may be performed on the unpackaged bulk dry powder or liquid formulation or the
finished product. If testing is performed on the bulk material
alone, then the process leading from the bulk to the finished product should be validated for its ability to prevent
microbial contamination. Testing should be performed on
the finished product if this process is not validated.

Consider safety issues related to both inhalation of the

drug substance and the potential of a flammability hazard.
Avoid contamination of samples by employing aseptic techniques whenever necessary.

SAMPLE SIZE DETERMINATION

For each microbiological test, sample 10 drug product
containers or units or a number of units that can provide a
minimum of 1 gram of product that are representative of
the batch. For batch sizes smaller than 200 units (e.g.,
batches used in clinical trials), sample size may be reduced
to 1% of the units or 1 unit, whichever is greater. The contents of individual containers may be pooled for testing.

AUTOMATIC ACTUATION METHOD

The contents of the inhalation aerosol containers may be
collected by automatically actuating each aerosol container
and collecting the delivered formulation on a suitable sterile
filter.
ROOM TEMPERATURE METHOD
Disinfect the outside of the test containers with an appropriate disinfectant, and allow the containers to dry in a
controlled environment. Empty the contents of the aerosol
container into a sterile vessel using a needle apparatus or
similar device (e.g., icemaker water line tap). If it has been
demonstrated that the propellant does not inhibit the
growth of microorganisms, the contents of the sterile vessel
may be added directly to the liquid media or buffer for the
test. Otherwise, allow the propellant to evaporate from the
vessel by leaving the vessel at room temperature for several
minutes. Remove any residual gaseous propellant by tilting
the vessel slightly or by allowing a slow stream of microbiologically inert sterile gas to pass over the surface. For some
less volatile propellants such as chlorofluorocarbon (CFC)
11/12 combinations, the vessel may be heated slightly (to
temperatures 45) to assist with evaporation. After the propellant has evaporated, add the liquid media or buffer, and
mix the contents to prepare for testing.
Direct expulsion into the broth media or buffer may be
feasible if a needle apparatus that is thin and strong enough
to puncture the container and to allow slow removal of the
contents is available. In this case, the contents may be expelled into and mixed with the aqueous medium. Layering

Official from May 1, 2013

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264 610 Alternative Microbiological Sampling Methods / Physical Tests

of the propellant and aqueous medium may occur, in which
case a longer time period and slight heating (not to exceed
45) may be required for propellant evaporation.
CHILLING METHOD
Place the disinfected aerosol containers in dry ice, or a dry
ice slurry (ensure the microbial quality of the dry ice and
slurry-forming liquid), or a cryofreezer for the period required to liquefy the contents. Disinfect the outside of the
test containers with an appropriate disinfectant, and allow
the containers to dry in an aseptic environment. Aseptically
open the aerosol containers using an appropriate tool. Be
aware that freezing can affect the viability of microorganisms. For CFC-based products, pour the contents of the containers into sterile vessels. Allow the propellant to escape,
and combine residues with a diluent appropriate for the
drug product. Other drug-specific procedures may also be
employed. For hydrofluoroalkane-based products, pour the
contents of the containers into sterile vessels partially immersed in larger vessels containing dry ice. Drive off the
propellant, for example by allowing a slow stream of sterile,
filtered, oil-free compressed air to evaporate the material to
dryness. Combine residues with a diluent appropriate for
the drug product. An alternative procedure when testing
the entire contents of a previously chilled container is to
pour the contents of the opened container onto a sterile
membrane filtration unit, allow the propellant to escape,
and then rinse with an appropriate amount of sterile
diluent.

Multidose Nasal Sprays

Multidose nasal spray containers usually have a cap that is
screwed on, crimped on, or forms a snap-fit. The container
can be opened by unscrewing the cap, cutting the seal, or
using a decrimping tool, taking care to avoid microbial contamination during the process. Following cap removal, traditional sampling methods as described in general test chapters such as 61 Microbiological Examination of Nonsterile
Products: Microbial Enumeration Tests and 62 Microbiological
Examination of Nonsterile Products: Tests for Specified Microorganisms are typically appropriate for use.

611 ALCOHOL
DETERMINATION
METHOD IDISTILLATION METHOD
Method I is to be used for the determination of alcohol,
unless otherwise specified in the individual monograph. It is
suitable for examining most fluidextracts and tinctures, provided the capacity of the distilling flask is sufficient (commonly two to four times the volume of the liquid to be
heated) and the rate of distillation is such that clear distillates are produced. Cloudy distillates may be clarified by
agitation with talc, or with calcium carbonate, and filtered,
after which the temperature of the filtrate is adjusted and
the alcohol content determined from the specific gravity.
During all manipulations, take precautions to minimize the
loss of alcohol by evaporation.
Treat liquids that froth to a troublesome extent during
distillation by rendering them strongly acidic with phosphoric, sulfuric, or tannic acid, or treat with a slight excess

USP 36

of calcium chloride solution or with a small amount of paraffin or silicone oil before starting the distillation.
Prevent bumping during distillation by adding porous
chips of insoluble material such as silicon carbide, or beads.
For Liquids Presumed to Contain 30% of Alcohol or
LessBy means of a pipet, transfer to a suitable distilling
apparatus not less than 25 mL of the liquid in which the
alcohol is to be determined, and note the temperature at
which the volume was measured. Add an equal volume of
water, distill, and collect a volume of distillate about 2 mL
less than the volume taken of the original test liquid, adjust
to the temperature at which the original test liquid was
measured, add sufficient water to measure exactly the original volume of the test liquid, and mix. The distillate is clear
or not more than slightly cloudy, and does not contain
more than traces of volatile substances other than alcohol
and water. Determine the specific gravity of the liquid at
25, as directed under Specific Gravity 841, using this result
to ascertain the percentage, by volume, of C2H5OH contained in the liquid examined by reference to the Alcoholometric Table in the section Reference Tables.
For Liquids Presumed to Contain More Than 30% of
AlcoholProceed as directed in the foregoing paragraph,
except to do the following: dilute the specimen with about
twice its volume of water, collect a volume of distillate
about 2 mL less than twice the volume of the original test
liquid, bring to the temperature at which the original liquid
was measured, add sufficient water to measure exactly twice
the original volume of the test liquid, mix, and determine its
specific gravity. The proportion of C2H5OH, by volume, in
this distillate, as ascertained from its specific gravity, equals
one-half that in the liquid examined.
Special Treatment
VOLATILE ACIDS AND BASESRender preparations containing
volatile bases slightly acidic with diluted sulfuric acid before
distilling. If volatile acids are present, render the preparation
slightly alkaline with sodium hydroxide TS.
GLYCERINTo liquids that contain glycerin add sufficient
water so that the residue, after distillation, contains not less
than 50% of water.
IODINETreat all solutions containing free iodine with
powdered zinc before the distillation, or decolorize with just
sufficient sodium thiosulfate solution (1 in 10), followed by
a few drops of sodium hydroxide TS.
OTHER VOLATILE SUBSTANCESSpirits, elixirs, tinctures, and
similar preparations that contain appreciable proportions of
volatile materials other than alcohol and water, such as volatile oils, chloroform, ether, camphor, etc., require special
treatment, as follows:
For Liquids Presumed to Contain 50% of Alcohol or Less
Mix 25 mL of the specimen under examination, accurately
measured, with about an equal volume of water in a
separator. Saturate this mixture with sodium chloride, then
add 25 mL of solvent hexane, and shake the mixture to extract the interfering volatile ingredients. Draw off the separated, lower layer into a second separator, and repeat the
extraction twice with two further 25-mL portions of solvent
hexane. Extract the combined solvent hexane solutions with
three 10-mL portions of a saturated solution of sodium chloride. Combine the saline solutions, and distill in the usual
manner, collecting a volume of distillate having a simple
ratio to the volume of the original specimen.
For Liquids Presumed to Contain More Than 50% of
AlcoholAdjust the specimen under examination to a concentration of approximately 25% of alcohol by diluting it
with water, then proceed as directed in For Liquids Presumed
to Contain 50% of Alcohol or Less, beginning with Saturate
this mixture with sodium chloride.
In preparing Collodion or Flexible Collodion for distillation,
use water in place of the saturated solution of sodium chloride directed above.
If volatile oils are present in small proportions only, and a
cloudy distillate is obtained, the solvent hexane treatment

Official from May 1, 2013

Copyright (c) 2013 The United States Pharmacopeial Convention. All rights reserved.