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Common pharmacopeial calculations in USP monographs

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 STIMULI TO THE REVISION PROCESS
Stimuli articles do not necessarily reflect the policies Pharmacopeial Forum
2 of the USPC or the USP Council of Experts Vol. 31(2) [Mar.–Apr. 2005]

Common Pharmacopeial Calculations in USP Monographs

Behnam Davani, Ph.D., Karen A. Russo, Ph.D., Andrzej Wilk, Ph.D., and Lokesh Bhattacharyya, Ph.D., * United States
Pharmacopeia
Stimuli to the Revision Process

ABSTRACT This article was prompted by questions USP has received pertaining to the formulas used in official mono-
graphs. It attempts to explain most commonly used formulas by citing representative examples, and it analyzes all factors that
have led to the final formula. In many cases, factors such as dilution and/or relative response factors are combined in a single
numerical value. It is not always obvious how the numerical factor was derived until a thorough analysis is completed.
Examples were chosen from three areas: miscellaneous tests such as Loss on drying and Loss on ignition, assays
comprising various procedures, and related compounds determined by HPLC. Recommendations to simplify the format
and express the formulas in a more explanatory manner are included.

INTRODUCTION form the monograph procedure. Second, a thorough under-

Calculations are part of almost every monograph proce-
dure. Monograph procedures use various techniques, ex-
perimental designs, and corresponding formulas to
calculate results. In order to understand the results of a test,
the analyst should have complete comprehension of the in-
terdependence of the formula and the experimental design.
The purpose of this Stimuli article is to present detailed
explanations of many of the most commonly used pharma-
copeial calculations. Three categories of formulas are in-
cluded: Calculations for tests such as Loss on drying and
Residue on ignition are included under miscellaneous tests;
the Assay section includes formulas for titrimetric and spec-
troscopic procedures; and the related compounds and assay
categories include formulas used in quantitative chromato-
graphic [high-performance liquid chromatography (HPLC)
and gas chromatography (GC)] procedures. As applicable,
formulas from USP drug substance and drug product mono-
graphs are used to illustrate various examples. Although
many examples are presented, this article is not meant to
be an exhaustive review of every possible type of compen-
dial formula. For the purposes of this article, calculations for
biological tests are not included.
The result of this review of pharmacopeial calculations is
a list of recommendations for presenting formulas in mono-
graph procedures. This list is offered to the pharmaceutical
industry and to USP as a tool for drafting monograph formu-
las so that they are scientifically sound and useful to the ana-
lyst.
The authors have made a few assumptions in writing this
article. First, the analyst must have the required theoretical
knowledge and practical laboratory training in order to per-
standing of the USP General Notices and applicable Gener-
al Chapters is critical in order to execute the procedures
correctly. Finally, the analyst also should have working
knowledge of the appropriate regulatory guidances and In-
ternational Conference on Harmonization (ICH) guidelines
and the USP ‘‘Guideline for Submitting Requests for Revi-
sion to USP–NF’’ (1).
MISCELLANEOUS CALCULATIONS
Following are sample calculations for tests such as Loss
on drying (LOD) or Loss on ignition (LOI) needed for the
correction of the assay result when appropriate.
Percent LOD
This procedure determines the amount of volatile matter
of any kind that is driven off under the conditions specified
(2). In this simple technique, the sample is weighed, then
heated, and the dried sample is reweighed. The difference
in the sample weight is the volatile content. A typical calcu-
lation is as follows:
Weight of container = C
Weight of container and test sample before drying = T
Weight of container and test sample after final drying
=F
%LOD = [(T – F)/(T – C)]   100

*Correspondence should be addressed to: Lokesh Bhattacharyya, Ph.D.,

Director, Department of Standards Development, U.S. Pharmacopeia,
12601 Twinbrook Parkway, Rockville, MD 20852-1790; tel.
301.816.8201; fax 301.816.8373; e-mail lb@usp.org.

# 2005 The United States Pharmacopeial Convention, Inc. All Rights Reserved.
 STIMULI TO THE REVISION PROCESS
Pharmacopeial Forum Stimuli articles do not necessarily reflect the policies
Vol. 31(2) [Mar.–Apr. 2005] of the USPC or the USP Council of Experts
Percent LOI
This procedure determines the percentage of test material
that is volatilized and driven off under the conditions speci-
fied (3). The sample is initially weighed and then ignited in a
suitable oven. The sample is cooled in a desiccator and ac-
curately reweighed. The percentage loss for the test material
is calculated as follows:
Weight of container = C
Weight of container and test sample before ignition
=T
Weight of container and test sample after final ignition
=F
%LOI = [(T – F)/(T – C)]   100
ASSAY
Assay procedures determine the content of an active in-
gredient in a drug substance or a drug product. The accep-
tance criteria for an assay are indicated in the definition
section of the USP monograph and generally are expressed
as a percent. However, the value calculated in the assay gen-
erally is the quantity (e.g., mg or mg). Therefore, further cal-
culations may be needed to convert the assay result to the
appropriate unit (usually percent) consistent with the Defini-
tion section of the monograph.
Example 1 (Assay ‘‘As Is’’)
Malathion contains not less than 98.0 percent and not
more than 102.0 percent of C10H19O6PS2.
The quantity of Malathion obtained in the assay = Q
The quantity of Malathion taken = T
Assume:
Q = 495 mg
T = 500.0 mg
Calculation: Q/T   100 = 495/500.0   100 = 99.0 per-
cent.
The percent acceptance criteria in the definition for the
article without further qualification generally indicate the
assay result ‘‘as is.’’ However, the acceptance criteria for
the assay frequently indicate limits to be calculated on the
anhydrous basis, on the dried basis, or on the ignited basis.
The following section from the USP General Notices (p.
7) may help analysts understand the common calculations
involving appropriate correction factors:
# 2005 The United States Pharmacopeial Convention, Inc.
3
Where the definition in a monograph states the toler-
ances as being ‘‘calculated on the dried (or anhydrous
or ignited) basis,’’ the directions for drying or igniting
the sample prior to assaying are generally omitted from
the Assay procedure. Assay and test procedures may be
Stimuli to the Revision Process
performed on the undried or unignited substance and
the results calculated on the dried, anhydrous, or ignited
basis, provided a test for Loss on drying, or Water, or
Loss on ignition, respectively, is given in the
monograph.
Example 2 (Assay Result Corrected for Loss on Drying)
Miconazole contains not less than 98.0 percent and not
more than 102.0 percent of C18H14Cl4N2O, calculated on
the dried basis.
Quantity of C18H14Cl4N2O obtained in the assay = Q
Quantity of Miconazole taken = T
Percent LOD = A
Assume:
Q = 295.0 mg
T = 300.0 mg
A = 0.34%
Assay result = [Q/T/(100 – A)/100]   100 = [295.0/
300.0/(100 – 0.34)/100]   100 = 98.7 percent.
Example 3 (Assay Result Corrected for Water)
Acyclovir contains not less than 98.0 percent and not
more than 101.0 percent of C8H11N5O3, calculated on
the anhydrous basis.
Quantity of C8H11N5O3 obtained in the assay = Q
Quantity of Acyclovir taken = T
Percent of water = A
Assume:
Q = 96.0 mg
T = 99.8 mg
A = 3.54 %
Assay result = [Q/T/(100 – A)/100]   100 = [96.0/99.8/
(100 – 3.54)/100]   100 = 99.7 percent
Example 4 (Assay Result Corrected for Loss on Ignition)
Ferric Oxide contains not less than 97.0 percent and not
more than 100.5 percent of Fe2O3, calculated on the ig-
nited basis.
Quantity of Fe2O3 obtained in the assay = Q
Quantity of Ferric Oxide taken = T
Percent LOI = A
All Rights Reserved.
 STIMULI TO THE REVISION PROCESS
Stimuli articles do not necessarily reflect the policies
4 of the USPC or the USP Council of Experts
Assume:
Q = 2.48 mg
T = 2.52 mg
A = 1.24%
Assay result = [Q/T/(100 – A)/100]   100 = [2.48/2.52/
Stimuli to the Revision Process
(100 – 1.24/100)]   100 = 99.6 percent
The USP General Notices also state (p. 7):
Where the presence of moisture or other volatile ma-
terial may interfere with the procedure, previous drying
of the substance is specified in the individual mono-
graph and is obligatory. Throughout a monograph that
includes a test for Loss on drying or Water, the expres-
sion ‘‘previously dried’’ without qualification signifies
that the substance is to be dried as directed under Loss
on drying or Water (gravimetric determination).
Example 5 (Assay Result on Previously Dried Sample)
Ganciclovir contains not less than 98.0 percent and not
more than 102.0 percent of C9H13N5O4, calculated on
the previously dried basis.
Quantity of C9H13N5O4 obtained in the assay = Q
Quantity of Ganciclovir, previously dried, taken = T
Assume:
Q = 10.7 mg
T = 10.5 mg
Assay result = Q/T   100 = 10.7/10.5   100 = 101.9
percent.
Example 6: (Assay Result on Previously Dried Sample)
In some cases, the procedure in the monograph will indi-
cate that the sample should be dried before use.
Adenosine contains not less than 99.0 percent and not
more than 101.0 percent of C10H13N5O4 calculated on
the dried basis.
Assay—Dissolve about 200 mg of Adenosine, pre-
viously dried at 1058 for 2 hours and accurately
weighed, in 50 mL of glacial acetic acid. Titrate with
0.1 N perchloric acid VS, determining the endpoint po-
tentiometrically. Perform a blank determination, and
make any necessary correction. Each mL of 0.1 N per-
chloric acid is equivalent to 26.72 mg of C10H13N5O4.
In the two previous cases, the test sample must be dried
first and then assayed. There is no actual additional calcula-
tion for the dried basis. Rather, the term ‘‘calculated on the
dried basis’’ or ‘‘calculated on the previously dried basis’’
implies that the values calculated for the assay have already
been corrected for the volatile compounds (water and/or sol-
vents) because the test sample was previously dried.
# 2005 The United States Pharmacopeial Convention, Inc.
Pharmacopeial Forum
Vol. 31(2) [Mar.–Apr. 2005]
The acceptance criteria for the assay also can be ex-
pressed on the solvent-free basis or other variations and
combination terms. In this case, the assay result needs to
be corrected for the solvent, if present. A test for specific
residual solvent(s) or a general test for volatile organic im-
purities is usually provided in the monograph.
Example 7 (Assay Result Corrected for Both Water and
Solvent)
Lamivudine contains not less than 98.0 percent and not
more than 102.0 percent of C8H11N3O3S, calculated on
the anhydrous and solvent-free basis.
Quantity of C8H11N3O3S in the portion of Lamivudine
obtained in the assay = Q
Quantity of Lamivudine taken = T
Percent of water = A
Percent of residual solvent = B
Assume:
Q = 26.0 mg
T = 25.9 mg
A = 0.183%
B = 0.281%
Assay result = [Q/T/(100 – A – B)/100]   100 =
[26.0/25.9/ (100 – 0.183 – 0.281)/100]   100 = 100.9
percent.
NOTE—When the assay result in a USP monograph is
corrected for LOD, LOI, water, residual solvent, or
combinations of such factors, not only must the accep-
tance criteria for the assay be met but all other accep-
tance criteria for the monograph also have to be
achieved. In other words, the assay value of 100.9% ob-
tained above is within the tolerance value of 98.0% to
102.0%. In addition, both water and residual solvent
must remain within the acceptance criteria of not more
than 0.2% and 0.3%, respectively, for Lamivudine.
COMMON CALCULATIONS FOR
DETERMINATION OF THE ASSAY RESULT
In this section, common or typical calculations used for
the determination of assay results are discussed. Simple
and more complex calculations for both drug substance
and dosage forms will be presented. Because most of the
assay procedures are based on chromatography, titration,
or spectroscopy, examples using such techniques will be ex-
plored. However, a detailed discussion of the different types
of techniques used is beyond the scope of this Stimuli arti-
cle. It is assumed that the reader has a basic understanding of
the concepts and the principles of chromatographic, titra-
tion, and spectroscopic procedures. As necessary, the reader
All Rights Reserved.
 STIMULI TO THE REVISION PROCESS
Pharmacopeial Forum Stimuli articles do not necessarily reflect the policies
Vol. 31(2) [Mar.–Apr. 2005] of the USPC or the USP Council of Experts
is advised to consult USP General Chapters Chromatog-
raphy h621i (4), Titrimetry h541i (5), Spectrophotometry
and Light-Scattering h851i (6), and books on analytical
chemistry and quantitative analysis (e.g., 7 and 8).
In the following examples, only the relevant portions of
the monograph as related to the assay and the corresponding
calculations are selected. Please refer to the current USP–
NF to review the complete monographs and/or procedures.
Chromatography
Example 1 (HPLC)
Isoniazid contains not less than 98.0 percent and not
more than 102.0 percent of C 6H7N3O, calculated on
the dried basis.
Assay preparation—Transfer about 16 mg of Isoniazid,
accurately weighed, to a 50-mL volumetric flask, dis-
solve in Mobile phase, dilute with Mobile phase to vol-
ume, and mix. Calculate the quantity, in mg, of
C6H7N3O in the portion of Isoniazid taken by the for-
mula:
50C(rU / rS),
in which C is the concentration, in mg per mL, of USP
Isoniazid RS in the Standard preparation; and rU and rS
are the peak responses of isoniazid obtained from the
Assay preparation and the Standard preparation, re-
spectively.
The simple calculation given above is very common in
USP monographs. The quantitation is based on an external
Reference Standard. In addition, a factor is also introduced
to correct for the volume dilution. To further clarify the
equation using the appropriate unit factors:
The quantity, in mg, of C6H7N3O in the portion of Iso-
niazid taken = 50 (mL) C (mg/mL) [rU (area of the ma-
jor peak in assay preparation)/rS (area of the major peak
in the reference standard preparation)].
Example 2 (HPLC)
Benzoyl Peroxide Gel is benzoyl peroxide in a suitable
gel base. It contains not less than 90.0 percent and not
more than 125.0 percent of the labeled amount of ben-
zoyl peroxide (C14H10O4).
Assay preparation—Transfer an accurately weighed
quantity of Gel, equivalent to about 40 mg of benzoyl
peroxide, to a 50-mL volumetric flask. Add 40 mL of
acetonitrile, and shake until the material is thoroughly
dispersed. Sonicate the mixture for 5 minutes, dilute
with acetonitrile to volume, mix, and filter. Pipet 10
# 2005 The United States Pharmacopeial Convention, Inc.
5
mL of the filtrate and 5 mL of Internal standard solution
into a 25-mL volumetric flask, dilute with acetonitrile to
volume, and mix. Calculate the quantity, in mg, of ben-
zoyl peroxide (C14H10O4) in the portion of Gel taken by
the formula:
Stimuli to the Revision Process
125C(RU / RS),
in which C is the concentration, in mg per mL, of ben-
zoyl peroxide in the Standard preparation, and RU and
RS are the peak response ratios of benzoyl peroxide to
ethyl benzoate obtained from the Assay preparation
and the Standard preparation, respectively.
This formula has the same general concept and can be
simplified as:
DC(RU / RS),
D = Dilution factor = 50 (mL)   25 (mL)/10 (mL)
=125.
Example 3 (HPLC)
Atovaquone Oral Suspension contains not less than
90.0 percent and not more than 110.0 percent of the la-
beled amount of atovaquone (C22H19ClO3).
Assay preparation—Transfer approximately 5.2 g of
the well-mixed Oral Suspension, accurately weighed,
to a low-actinic 250-mL volumetric flask. Add 50 mL
of water, swirl for about 5 minutes, add 150 mL of 0.1
M methanolic sodium hydroxide, and sonicate for
about 15 minutes. Allow to cool, dilute with 0.1 M
methanolic sodium hydroxide to volume, and mix. Im-
mediately filter a 20-mL portion, discarding the first 5
mL of the filtrate. Transfer 3.0 mL of the clear filtrate to
a low-actinic 100-mL volumetric flask, dilute with a
mixture of methanol and water (1:1) to volume, and
mix. Calculate the quantity, in mg, of atovaquone
(C22H19ClO3) in each mL of the Oral Suspension taken
by the formula:
(25,000/3)(C/V)(rU / rS),
in which C is the concentration, in mg per mL, of USP
Atovaquone RS in the Standard preparation; V is the
volume, in mL, of Oral Suspension taken to prepare
the Assay preparation; and rU and rS are the atovaquone
peak areas obtained from the Assay preparation and the
Standard preparation, respectively.
This formula has the same general concept and can be
simplified as:
DC(rU / rS),
D = Dilution factor = 250 (mL)   100 (mL)/3 (mL) =
25,000/3.
All Rights Reserved.
 STIMULI TO THE REVISION PROCESS
Stimuli articles do not necessarily reflect the policies
6 of the USPC or the USP Council of Experts
NOTE—In the example above, V is defined as ‘‘the vol-
ume, in mL, of Oral Suspension taken to prepare the
Assay preparation.’’ However, the weight rather than
the volume of Oral Suspension was taken to prepare
the Assay preparation. In other words, the weight, ap-
Stimuli to the Revision Process
proximately 5.2 grams in this case, has to be converted
to volume using the density of the Oral Suspension,
1.04 g per mL as given in the related compound section
of the monograph. The reason for taking the weight
rather than the volume is the difficulty of accurately
measuring the volume of the Oral Suspension directly.
In several USP monographs, the assay calculations are
corrected for the molecular weight ratio. This is usually
done when the RS used and the content determined are in
two different forms (e.g., salt and free base). Therefore, a
correction factor (the molecular weight ratio) is included
in the equation.
Example 4 (HPLC)
Dobutamine Injection is a sterile solution of Dobuta-
mine Hydrochloride in Water for Injection. It contains
an amount of Dobutamine Hydrochloride equivalent to
not less than 90.0 percent and not more than 110.0 per-
cent of the labeled amount of dobutamine (C18H23NO3).
Assay preparation—Transfer an accurately measured
volume of Injection, equivalent to about 25 mg of do-
butamine, to a 50-mL volumetric flask. Dilute with Mo-
bile phase to volume, and mix. Calculate the quantity,
in mg, of C18H23NO3 in the portion of Injection taken by
the formula:
(301.39/337.84)(50C)(rU / rS),
in which 301.39 is the molecular weight of dobutamine
(free base), 337.84 is the molecular weight of dobuta-
mine hydrochloride; C is the concentration, in mg per
mL, of USP Dobutamine Hydrochloride RS in the Stan-
dard preparation; and rU and rS are the peak responses
obtained from the Assay preparation and the Standard
preparation, respectively.
NOTE—The RS used is dobutamine hydrochloride (salt),
but the definition is expressed in terms of percentage of
the labeled amount of dobutamine (free base). There-
fore, the correction factor (the molecular weight ratio)
is included in the equation to calculate the dobutamine
content.
# 2005 The United States Pharmacopeial Convention, Inc.
Pharmacopeial Forum
Vol. 31(2) [Mar.–Apr. 2005]
Example 5 (GC)
Hyoscyamine Tablets contain not less than 90.0 percent
and not more than 110.0 percent of the labeled amount
of C17H23NO3.
Assay preparation—Weigh and finely powder not few-
er than 20 Tablets. Transfer an accurately weighed por-
tion of the powder, equivalent to about 0.86 mg of
hyoscyamine, to a separator containing 5 mL of pH
9.0 buffer, and add, by pipet, 2.0 mL of Internal stan-
dard solution. Proceed as directed under Standard
preparation, beginning with ‘‘adjust with 1 N sodium
hydroxide to a pH of 9.0.’’
Standard preparation—Dissolve about 10 mg of USP
Hyoscyamine Sulfate RS, accurately weighed, in water
contained in a 100-mL volumetric flask, add water to
volume, and mix. Prepare fresh daily. Pipet 10.0 mL
of this solution into a separator, add 2.0 mL of Internal
standard solution and 5.0 mL of pH 9.0 buffer, and ad-
just with 1 N sodium hydroxide to a pH of 9.0. Extract
with two 10-mL portions of methylene chloride, filter
the methylene chloride extracts through 1 g of anhy-
drous sodium sulfate supported by a small cotton plug
in a funnel into a 50-mL beaker, and evaporate under
nitrogen to dryness. Dissolve the residue in 2.0 mL of
methylene chloride.
Procedure—Inject 1-mL portions of the Assay prepara-
tion and the Standard preparation successively into the
gas chromatograph . . . Calculate the ratio, AU, of the
area of the hyoscyamine peak to the area of the internal
standard peak in the chromatogram from the Assay
preparation, and similarly calculate the ratio, AS, in
the chromatogram from the Standard preparation. Cal-
culate the quantity, in mg, of C17H23NO3 in the portion
of tablets taken by the formula:
(289.37/676.83)(W/10)(AU / AS),
in which 289.37 and 676.83 are the molecular weights
of hyoscyamine and anhydrous hyoscyamine sulfate,
respectively; and W is the weight, in mg, of USP Hyos-
cyamine Sulfate RS taken for the Standard preparation.
NOTE—Only 10.0 mL of 100 mL standard solution was
used. Therefore, the correction factor 10 (10/100) is
used in the denominator.
Titration
USP monographs do not provide chemical equations or
reactions used for assay calculations. However, they provide
directions for calculating the assay result. We provide a gen-
eral guideline for such calculations.
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 STIMULI TO THE REVISION PROCESS
Pharmacopeial Forum Stimuli articles do not necessarily reflect the policies
Vol. 31(2) [Mar.–Apr. 2005] of the USPC or the USP Council of Experts
Direct titration is the treatment of a soluble substance,
contained in solution in a suitable vessel (the titrate), with
an appropriate standardized solution (the titrant), the end-
point being determined instrumentally or visually with the
aid of a suitable indicator.
Example 1 (Direct Titration)
Ketoprofen contains not less than 98.5 percent and not
more than 101.0 percent of C16H14O3, calculated on the
dried basis.
Assay—Dissolve about 200 mg of Ketoprofen, ac-
curately weighed, in 25 mL of alcohol. Add 25 mL of
water and several drops of phenol red TS, and titrate
with 0.1 N sodium hydroxide having been standardized
by a similar titration of primary standard benzoic acid.
Perform a blank determination, and make any neces-
sary correction. Each mL of 0.1 N sodium hydroxide
is equivalent to 25.43 mg of C16H14O3.
%Assay: [(V – B)   N   F   100] / [TN   W   (100 –
A)/100]
V: Sample titrant volume (mL)
B: Blank titrant volume (mL)
N: Titrant normality
F: Equivalence Factor (mg sample/mL of TN)
TN: Theoretical normality
W: Sample weight (mg)
A: Assay correction for LOD (decimal)
Assume:
V = 8.5 mL
B = 1.4 mL
N = 0.11
TN = 0.1
W = 202 mg
A = 0.42%
Assay result = [(V – B)   N   F   100] / [TN   W  
(100 – A)/100] =
[(8.5–1.4)   0.11   25.43   100]/ [0.1   202   (100 –
0.42)/100] = 98.7%
Some pharmacopeial assays require the addition of a
measured volume of a volumetric solution in excess of the
volume actually needed to react with the substance being
assayed. The excess of this solution is then titrated with a
second volumetric solution in a direct titration. This consti-
tutes a residual titration and is also known as back titration.
The quantity of the substance being titrated is calculated
from the difference between the volume of the volumetric
solution originally added, corrected by blank, and that con-
sumed by the titrant in the back titration (5).
# 2005 The United States Pharmacopeial Convention, Inc.
7
Example 2 (Residual Titration or Back Titration)
Methenamine, dried over phosphorus pentoxide for 4
hours, contains not less than 99.0 percent and not more
than 100.5 percent of C6H12N4.
Assay—Transfer about 1 g of Methenamine, previously
Stimuli to the Revision Process
dried and accurately weighed, to a beaker. Add 40.0 mL
of 1 N sulfuric acid VS, and heat to a gentle boil, adding
water from time to time if necessary, until the formal-
dehyde has been expelled. Test for the absence of for-
maldehyde by adding a drop of the assay solution to a
glass fiber filter disk, on a watch glass, on which has
previously been placed 3 or 4 drops of Chromotropic
acid spot test solution. Formaldehyde produces a violet
color with this reagent; repeat the test until no violet
color is obtained on the warmed test filter disk upon
comparison with a blank filter disk to which no assay
specimen is added. Cool, add 20 mL of water, then
add methyl red TS, and titrate the excess acid with 1
N sodium hydroxide VS. Perform a blank determina-
tion. Each mL of 1 N sulfuric acid is equivalent to
35.05 mg of C6H12N4.
%Assay: [(B – V)   N   F   100] / [TN   W]
B: Titrant volume (mL) for blank titration
V: Titrant volume (mL) for titration of sample
N: Titrant normality
F: Equivalence Factor = (mg sample/mL of TN)
TN: Theoretical normality
W: Sample weight (mg)
Assume:
B = 43.78 mL
V = 15.10 mL
F = 35.05 mg/mL (given)
N = 0.9993
TN = 1
W = 1007.8 mg (previously dried)
Assay result = (43.78 mL – 15.10 mL)   0.9993  
35.05 mg/mL   100/(1   1007.8 mg) = 99.7%
Determination of Equivalent Factor in Titration-Based
Assays
The titrimetric method is based on a general chemical re-
action as below:
 A + bR ? Products
where   molecules of an analyte (e.g., titrate), A, reacts with
b molecules of reactant (e.g., titrant), R.
Stoichiometric calculations can be made using either
moles (molecular weight) or equivalent (equivalent weight).
The equivalent weight (EW) and molecular weight (MW) are
related by the following equation:
EW = MW/n
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 STIMULI TO THE REVISION PROCESS
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8 of the USPC or the USP Council of Experts Vol. 31(2) [Mar.–Apr. 2005]

where n is the number of electrons transferred in the reac- Reactions:

tion. Thus the value of n depends on the reaction. In any
titration reaction, the same number of equivalents of titrate MnO4– + 8H+ + 5e ? Mn2+ + 4H2O (1)
and titrant react at the endpoint.
H2O2 ? 5O2 + 2H+ + 2e (2)
For example, for simple acid–base reactions:
Stimuli to the Revision Process

H+ + OH– ? H2O; n = 1
CO32– + 2 H+ ? [H2CO3] ? H2O + CO2, n = 2 Multiply equation (1) by 5, equation (2) by 2 and add:
CO32– + H+ ? HCO3–, n = 1
2 MnO4– + 6 H+ + 5 H2O2———
2 Mn2+ + 8 H2O + 5 O2
And the following are examples of redox reactions:
or
MnO4– + 8 H+ + 5 e ? Mn2+ + 4 H2O; n = 5 2 KMnO4 + 3 H2SO4 + 5 H2O2———
H2O2 ? 5 O2 + 2 H+ + 2 e; n = 2 2 MnSO4 + 8 H2O + 5 O2 + K2SO4
Cr2O72– + 14 H+ + 6 e ? 2 Cr3+ + 7 H2O; n = 6

Example 3 (Acid-Base Titration) Number of equivalents KMnO4 = Number of equiva-

Sodium Bicarbonate Tablets contain not less than 95.0
percent and not more than 105.0 percent of the labeled
amount of NaHCO3.
Assay—Weigh and finely powder not fewer than 20
Tablets. Weigh accurately a portion of the powder,
equivalent to about 2 g of sodium bicarbonate, dissolve
in 100 mL of water, add methyl red TS, and titrate with
1 N hydrochloric acid VS. Add the acid slowly, with
constant stirring, until the solution becomes faintly
pink. Heat the solution to boiling, cool, and continue
the titration until the pink color no longer fades after
boiling. Each mL of 1 N hydrochloric acid is equivalent
to 84.01 mg of NaHCO3.
NaHCO3 + HCl ? NaCl + CO2 + H2O
Number of equivalents HCl = Number of equivalents
NaHCO3
VN = number of equivalents NaHCO3 (NOTE—V is in
liters)
1 mL of 1 N HCl = mequivalents NaHCO3 = MW/n =
84.01 mg/1 = 84.01 mg
Example 4 (Redox Titration)
Hydrogen Peroxide Concentrate contains not less than
29.0 percent and not more than 32.0 percent, by weight,
of H2O2.
Procedure: Weigh accurately about 1 mL of Concen-
trate in a tared 100-mL volumetric flask, dilute with
water to volume, and mix. To 20.0 mL of this solution
add 20 mL of 2 N sulfuric acid, and titrate with 0.1 N
potassium permanganate VS. Each mL of 0.1 N potas-
sium permanganate is equivalent to 1.701 mg of H2O2.
lents H2O2
V1N1 = Number of equivalents H2O2
(V is in liters)
NOTE—Equivalent weight (EW) = Molecular weight
(MW)/n
N = 2 for H2O2 because this is the number of elec-
trons transferred from H2O2 in reaction 2 above.
1 mL of 1 N KMnO4 = 34.01/2 mg H2O2 = 17.01
mg H2O2.
But the titration uses 0.1 N KMnO4. Thus, 1 mL of
0.1 N KMnO4 = 1.701 mg of H2O2.
Spectroscopy and Other Methods
USP contains numerous examples of analytical tech-
niques used in the Assay section other than chromatography
or titrimetry. In the simplest cases, the measurement of a sig-
nal is directly proportional to the amount of the material ta-
ken, and the formula contains only the dilution and response
factors.
Example 1 (Acetazolamide—Assay Using IR)
Acetazolamide contains not less than 98.0 percent and
not more than 102.0 percent of C4H6N4O3S2, calculated
on the anhydrous basis.
Assay—Dissolve about 200 mg of Acetazolamide, ac-
curately weighed, in a small volume of pyridine in a 10-
mL volumetric flask, add the solvent to volume, and
mix. Similarly, dissolve an accurately weighed quantity
of USP Acetazolamide RS in pyridine to obtain a Stan-
dard solution having a known concentration of about 20

# 2005 The United States Pharmacopeial Convention, Inc. All Rights Reserved.
 STIMULI TO THE REVISION PROCESS
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Vol. 31(2) [Mar.–Apr. 2005] of the USPC or the USP Council of Experts
mg per mL. Concomitantly determine the absorbances
of both solutions in 0.1-mm cells at the wavelength of
maximum absorbance at about 7.38 mm (1350 cm–1),
with a suitable IR spectrophotometer, using pyridine
as the blank. Calculate the quantity, in mg, of
C4H6N4O3S2 in the portion of Acetazolamide taken by
the formula:
10C(AU /AS),
in which C is the concentration, in mg per mL, of USP
Acetazolamide RS in the Standard solution, and AU and
AS are the absorbances of the solution of Acetazolamide
and the Standard solution, respectively.
In this example IR absorbance, AU, at specified wave-
length is compared to that of the reference standard, AS.
Both values are proportional to the concentration. The quan-
tity, in mg, of the C4H6N4O3S2 in the portion of Acetazola-
mide taken = 10 (mL) C (mg/mL) (AU / AS).
Example 2 (Assay Using UV)
Nalorphine Hydrochloride contains not less than 97.0
percent and not more than 103.0 percent of
C19H21NO3
HCl, calculated on the dried basis.
Assay—Transfer about 25 mg of Nalorphine Hydro-
chloride, accurately weighed, to a 250-mL volumetric
flask, dissolve in water, dilute with water to volume,
and mix. Concomitantly determine the absorbances of
this solution and of a Standard solution of USP Nalor-
phine Hydrochloride RS in the same medium having a
known concentration of about 100 mg per mL in 1-cm
cells at the wavelength of maximum absorbance at
about 285 nm, with a suitable spectrophotometer, using
water as the blank. Calculate the quantity, in mg, of
C19H21NO3
HCl in the Nalorphine Hydrochloride ta-
ken by the formula:
0.25C(AU /AS),
in which C is the concentration, in mg per mL, of USP
Nalorphine Hydrochloride RS in the Standard solution,
and AU and AS are the absorbances of the solution of
Nalorphine Hydrochloride and the Standard solution,
respectively.
NOTE—The factor 0.25 is derived by combination of the
dilution factor (250 mL) and unit conversion (mg to
mg).
250 (mL)   C (mg/mL)   1 mg/1000 mg = 0.25
# 2005 The United States Pharmacopeial Convention, Inc.
9
Example 3 (Cefamandole Nafate—Assay Using Polaro-
graphy)
Cefamandole Nafate has a potency equivalent to not
less than 810 mg and not more than 1000 mg of cefa-
mandole (C18H18N6O5S2) per mg, calculated on the an-
Stimuli to the Revision Process
hydrous basis.
Standard preparation—Transfer about 12 mg of USP
Cefamandole Nafate RS, accurately weighed, to a 50-
mL volumetric flask containing 4 mL of water. Imme-
diately before use, add 30.0 mL of pH 2.3 buffer, dilute
with water to volume, and mix.
Assay preparation—Using Cefamandole Nafate, pre-
pare as directed under Standard preparation.
Procedure—Transfer a portion of the Assay prepara-
tion to a suitable polarographic cell. Deaerate by bub-
bling scrubbed nitrogen through the solution for 5
minutes, and redirect the nitrogen flow to the surface
outlet. Insert the dropping mercury electrode of a suit-
able polarograph (9) capable of measuring a current of
0.5 microampere or appropriate current to maintain on-
scale response, using an average capillary, and a drop
rate of 1 per second. Record the polarogram in the dif-
ferential pulse mode from –0.3 volts to –1.05 volts,
using a saturated calomel reference electrode and plati-
num wire counter electrode. Determine the peak height
obtained, in microamperes, where the peak height is de-
fined as the perpendicular distance from the extrapo-
lated baseline to the highest point of the peak as
compared to the full-scale current range. Similarly, de-
termine the peak current of the Standard preparation.
Calculate the quantity, in mg, of C18H18N6O5S2 in each
mg of the Cefamandole Nafate taken by the formula:
P(WS / WU)(iU / iS),
in which P is the potency, in mg of cefamandole per mg,
of USP Cefamandole Nafate RS, WS and WU are the
quantities, in mg, of USP Cefamandole Nafate RS
and Cefamandole Nafate taken to prepare the Standard
preparation and the Assay preparation, respectively,
and iU and iS are the peak currents, in microamperes,
from the Assay preparation and the Standard prepara-
tion, respectively.
This direct comparison is corrected by the potency factor,
in mg per mg, according to the labeled value for the refer-
ence standard, and the result is expressed in the same units.
All Rights Reserved.
 STIMULI TO THE REVISION PROCESS
Stimuli articles do not necessarily reflect the policies
10 of the USPC or the USP Council of Experts
RELATED COMPOUNDS
A primary goal of the USP is to provide public standards
that ensure the purity, strength, and quality of official arti-
cles. One mechanism for assessing the purity of an article
is the detection and quantitation of related compounds. To
Stimuli to the Revision Process
this end, monographs may include a Related compounds or
Chromatographic purity test. Chromatographic procedures
are commonly accepted means by which impurities can be
separated and quantitated. For this reason, chromatographic
procedures, particularly high-performanc e liquid
chromatography (HPLC), are widely used in the pharma-
ceutical industry and subsequently in USP monographs.
According to ICH guideline Q3A(R), Impurities in New
Drug Substances, there are three classes of impurities: inor-
ganic impurities, residual solvents, and organic impurities.
Inorganic impurities include catalysts, ligands, heavy met-
als, and metal salts. Though it is important to identify and
quantitate these types of impurities, analysis and quantita-
tion typically are not accomplished using chromatographic
procedures. Residual solvents, solvents remaining from the
synthesis and/or purification processes, or formed during
formulation are generally measured via gas chromatography
(GC). Detection and quantitation of residual solvents are ad-
dressed in USP General Chapter Organic Volatile Impurities
h467i (10).
Types of organic impurities include starting materials,
process intermediates, degradants, or reagents. This section
includes an exploration of formulas used in USP mono-
graphs to calculate the levels of organic impurities. USP
monographs include chromatographic procedures for the
quantitation of impurities (specified, unspecified, identified,
and unidentified). If an impurity is characterized, it is known
as a specified impurity. The chemical identity of a specified
impurity may be known or unknown, i.e., identified or uni-
dentified. For monograph purposes, the impurity may be
identified by its official USP reference standard name or
chemical name, by a relative retention time designation, or
by a descriptor such as Impurity 1 or Impurity A.
USP monographs describe procedures for quantitation of
organic impurities or degradants as Chromatographic purity
or Related compounds test; this name generally refers to a
quantitative separation technique such HPLC or GC. Re-
sults for Chromatographic purity or Related compounds
typically are expressed in percent, the most commonly used
units for the acceptance criteria. Acceptance criteria should
be provided for each specified identified impurity, each
specified unidentified impurity, any unspecified impurity
with acceptance criteria of not more than the identification
threshold, and total impurities in compliance with ICH
Q3A(R) guideline (11).
These approaches range from simple formulas for calcu-
lating the percentage of total peak area to complex formulas
that incorporate correction factors. Correction factors are in-
corporated to adjust for differences in concentrations be-
# 2005 The United States Pharmacopeial Convention, Inc.
Pharmacopeial Forum
Vol. 31(2) [Mar.–Apr. 2005]
tween test and reference standard solutions, molecular
weights of different forms of a compound, and detector re-
sponse.
Percent of Total Peak Area
An example of the simplest formula follows:
100(ri / rs),
where ri is the peak response for each impurity observed in
the Test solution and rs is the sum of responses for all peaks
in the Test solution. Multiplying by 100 converts the result
to the desired units of percent. The experimental design uses
data from a test solution only; there is no concomitant
analysis of a reference standard solution. This formula as-
sumes similar detector responses for all peaks in the chro-
matogram. Examples of this formula are found in the drug
substance monographs for Fenoldapam Mesylate and Phe-
niramine Maleate. Execution of this simple experimental
design and formula can also be applied to drug product
monographs as shown in the monographs for Fluoxetine
Capsules and Gadodiamide Injection.
Using Different Concentrations of the Test Sample
The next level of complexity in terms of experimental de-
sign and formula used to quantitate impurities involves the
use of test solutions prepared at different concentrations.
The goal of this design is to maximize the peak response
of impurities by using a concentrated test sample solution.
For quantitation purposes, a less concentrated solution of
the test sample is needed because the response of the major
peak in the concentrated test sample solution typically is off-
scale (overrange in an electronic data system); thus the peak
response of the major peak is inaccurate. This experimental
design is reflected in the formula by accounting for the dif-
ference in the two test sample solutions from which data are
used.
Following is an example from the Fluoxetine Hydro-
chloride monograph. This procedure requires the prepara-
tion and analysis of two fluoxetine hydrochloride
solutions: Test solution 1 is 5.6 mg per mL and Test solution
2 is prepared by a 5-fold dilution of Test solution 1.
100ri(rs + 5rU),
in which ri is the peak response from each impurity obtained
from Test solution 1; rs is the sum of all peak responses, ex-
cluding that of fluoxetine, obtained from Test solution 1, and
rU is the peak response of fluoxetine obtained from Test
solution 2. Test solution 1 is 5 times the concentration of
Test solution 2, so the factor of 5 is needed in the formula
to account for this difference. It should be noted that this
factor is not explained in the monograph along with the
other terms of the equation but rather it is based on knowl-
edge of the experimental design.
All Rights Reserved.
 STIMULI TO THE REVISION PROCESS
Pharmacopeial Forum Stimuli articles do not necessarily reflect the policies
Vol. 31(2) [Mar.–Apr. 2005] of the USPC or the USP Council of Experts
Using a Reference Standard
Moving to the next level of complexity in experimental
design involves examining procedures in which impurities
in the test sample are quantitated against a reference stan-
dard solution. In this type of experiment, impurities may
be quantitated against a reference standard of the parent
compound or against an appropriate related compound ref-
erence standard. The formula for calculating the amount of a
related compound must consider whether or not the determi-
nation is made against a corresponding reference standard of
the impurity.
In Related compounds Test 2 of the Pyrilamine Maleate
monograph, the percentage of each impurity is calculated
against the USP Pyrilamine Maleate RS as follows:
10,000(C/W)(ri/rS),
in which C is the concentration, in mg per mL, of USP Pyr-
ilamine Maleate RS in the Standard solution, W is the
weight, in mg, of the Pyrilamine Maleate taken to prepare
the Test solution, ri is the peak response for each impurity,
and rS is the response of the Standard solution. It should be
noted in this example that although the units of the target
concentration given in the instructions for the preparation
of the Standard solution are in mg per mL, the concentration
term in the formula is defined as mg per mL. Therefore, it is
necessary to convert the concentration of the Standard solu-
tion to mg per mL by dividing the concentration in mg per
mL by 1,000. Another feature of this formula is the term of
10,000 which is not explained in the monograph. Based on
the experimental design, the dimensional analysis shows:
Written in this manner, C is the concentration of the Stan-
dard solution in mg per mL, the response of ri and rS are
represented as areai and areaS, respectively, and W is the
weight, in mg, of the test sample. To complete the calcula-
tion, the term 100 mL is added to the numerator because the
sample weight, W, is diluted to 100 mL according the test
solution preparation. The term 100% is needed to convert
the result to percent. The terms mg, mL, and area cancel,
leaving 100   100, or 10,000.
With some exceptions, multipliers appearing in formulas
are not explained. Readers familiar with USP monographs
are accustomed to seeing factors such as 10,000 or 50,000 in
formulas without a specific explanation. The reason for this
is the understanding that the multiplier reflects a combina-
tion of factors reduced to a single term. Although the pur-
pose of the multiplier has been to simplify the formula for
the reader, this practice may not be optimal because the
derivations of these factors are not always clear. An experi-
enced user of USP monographs can usually derive these
# 2005 The United States Pharmacopeial Convention, Inc.
11
numbers, but often it is a time-consuming exercise. In the
interest of providing useful information, formulas in mono-
graphs should include an explanation of all terms and avoid
condensing terms. One way to avoid unexplained multi-
pliers is to include the concentration terms because the pur-
pose of the multiplier is to account for the dilution factors.
Stimuli to the Revision Process
Examples of this approach include the Mefanamic Acid,
Methylprednisone Hemisuccinate, Phenytoin, Phenytoin
Sodium, Propantheline Bromide Tablets, and Proparacaine
Hydrochloride Ophthalmic Solution monographs. Although
the terms may vary slightly, the format for the formulas for
calculating the percentage of related compounds is:
100(CS / CU)(rU / rS),
in which CS is the concentration, typically in mg per mL, of
the Standard solution and CU is the theoretical concentration
of the Test solution (same units as the Standard solution); rU
and rS are the peak responses from the Test solution and
Standard solution, respectively.
A variation of this format is used when the concentrations
of the Test solution and Standard solution are expressed in
different units, e.g., mg per mL for the Test solution and mg
per mL for the Standard solution. Maintaining the same
straightforward approach, the formula for calculating the
percentage of related compounds becomes:
0.1(CS / CU)(rU / rS),
in which the CS is the concentration, in mg per mL, of the
Standard solution; CU is the concentration, in mg per mL,
of the Test solution; rU and rS are the peak responses of
the Test solution and Standard solution, respectively. In this
case, the multiplier of 0.1 represents the 100% divided by
the factor of 1000 necessary for the conversion of mg per
mL to mg per mL. Examples of this format are included
in the Related compounds procedures for the Hydroxyzine
Hydrochloride and Methsuximide monographs.
Using Relative Response Factors
When the quantitation of an impurity is performed using a
reference standard other than the impurity reference stan-
dard, typically the API reference standard, a relative re-
sponse factor (RRF) may be used. An RRF value, which
is specific to a given set of experimental conditions, com-
pares the responses of the impurity and the API reference
standard and is incorporated into the formula as a means
to correct for differences in detector response.
This technique is used in the following formula for calcu-
lating related compounds in the Zileuton drug substance
monograph.
100F(CS / CU)(ri / rS),
in which F is the relative response factor for each impurity
and is 1.2, 1.4, and 1.7 for peaks with relative retention
times of 0.8, 2.1, and 2.8, respectively; CS is the concentra-
tion, in mg per mL, of USP Zileuton RS in the Standard
solution; CU is the concentration, in mg per mL, of zileuton
All Rights Reserved.
 STIMULI TO THE REVISION PROCESS
Stimuli articles do not necessarily reflect the policies
12 of the USPC or the USP Council of Experts
in the Test solution; ri is the peak response for each impurity
obtained from the Test solution; and rS is the peak response
for zileuton obtained from the Standard solution.
In USP monographs, placement of the RRF in the nu-
merator or denominator depends on how RRF was deter-
mined. If the RRF was calculated using the ratio of the
Stimuli to the Revision Process
impurity response relative to that of the API, then the
RRF appears in the denominator. The RRF appears in the
numerator if it was calculated as the response of the API
to that of the impurity. Currently, there is no USP definition
for determining an RRF value; however USP is working to
develop a definition and incorporate it consistently in all
monographs.
As stated in General Chapter Validation of Compendial
Methods h1225i, in the absence of other information, it
may be necessary to calculate the amount of an impurity
based on a comparison of its response to that of the drug
substance. The ratio of the responses of equal amounts of
the impurity and the drug substance (response factor) should
be used if known. When using a response factor in a for-
mula, one should understand that the value is applicable
only to the experimental conditions defined in the procedure
(12).
When a reference standard is indicated in a procedure, it
may be the same compound as the test sample or it may be
different, e.g., the free base of a compound versus the salt
form. Adjustments for molecular weight differences be-
tween different forms of an impurity or parent drug (free
base versus salt form) are incorporated into the formula
(see the Assay section of this article for examples). Having
the molecular weight terms clearly presented and defined is
crucial to a Related compounds test. The calculation of the
percentage of impurities in the Propoxyphene Hydro-
chloride monograph includes a factor of 1.12, which is the
ratio of the molecular weights of  -d-2-acetoxy-4-dimeth-
ylamino-1,2-diphenyl-3-methylbutane hydrochloride to that
of  -d-2-acetoxy-4-dimethylamino-1,2-diphenyl-3-methyl-
butane free base; the individual molecular weights for these
two compounds are not provided in the monograph.
RECOMMENDATIONS
Based on a review of formulas used in current USP
monographs, the following suggestions are offered in order
to provide the most information and facilitate use of the
monograph:
1. Present formulas so that all terms, including numerical
terms, and their units are included and defined for the
user.
2. Avoid condensing several terms into a single multiplier.
If a condensed multiplier must be used, its origin should
be clearly explained in the monograph.
3. For calculation of related compounds, include a con-
centration term for the sample under test because it in-
corporates dilution factors, thereby reducing the need
for an unexplained multiplier in formulas.
# 2005 The United States Pharmacopeial Convention, Inc.
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Pharmacopeial Forum
Vol. 31(2) [Mar.–Apr. 2005]
4. Include relative response factors and relative retention
times in the monograph if corresponding reference
standard materials are not available.
5. Change the format of formulas in current USP mono-
graphs in accordance with points 1–4 above. Revisions
should be made as early as possible.
6. For the Assay, formulas should be written so that the
final results are expressed in the same units as the assay
limits presented in the definition, unless otherwise spe-
cified in the monograph.
7. Create a new General Chapter for the pharmaceutical
calculations used in the USP monographs. The content
of this Stimuli article, after comments from the industry
and other stakeholders, is expected to be the basis for
this chapter. This General Chapter should provide an
easy reference for further clarification of the calcula-
tions used in USP monographs.
REFERENCES
1. Department of Standards Development. USP Guideline for
Submitting Requests for Revision to USP–NF, ‘‘Chapter 1:
Noncomplex Active Drug Substances and Products.’’ Rock-
ville, MD: U.S. Pharmacopeial Convention, Inc.; 2004:11–
27.
2. USP 27–NF 22, General Chapter Loss on Drying h731i.
Rockville, MD: U.S. Pharmacopeial Convention, Inc.;
2004:2320.
3. USP 27–NF 22, General Chapter Loss on Ignition h733i.
Rockville, MD: U.S. Pharmacopeial Convention, Inc.;
2004:2320.
4. USP 27–NF 22, General Chapter Chromatography h621 i.
Rockville, MD: U.S. Pharmacopeial Convention, Inc.;
2004:2272.
5. USP 27–NF 22, General Chapter Titrimetry h541i. Rockville,
MD: U.S. Pharmacopeial Convention, Inc.; 2004:2229.
6. USP 27–NF 22, General Chapter Spectrophotometry and
Light-Scattering h851i. Rockville, MD: U.S. Pharmacopeial
Convention, Inc.; 2004:2387.
7. Day, RA Jr, Underwood, AL. Quantitative Analysis. 5th ed.,
Englewood Cliffs, New Jersey: Prentice-Hall; 1986.
8. Analytical Chemistry in a GMP Environment: A Practical
Guide. Miller, JM, Crowther, JB, eds. New York: Wiley Inter-
science; 2000.
9. USP 27–NF 22, General Chapter Polarography h801i. Rock-
ville, MD: U.S. Pharmacopeial Convention, Inc.; 2004:2371.
10. USP 27–NF 22, General Chapter Organic Volatile Impurities
h467i. Rockville, MD: U.S. Pharmacopeial Convention, Inc.;
2004:2224.
11. International Conference on Harmonization. Impurities in
New Drug Substances Q3A(R). ICH Harmonised Tripartite
Guideline. Geneva, Switzerland; 2002.
12. USP 27–NF 22, General Chapter Validation of Compendial
Methods h1225i. Rockville, MD: U.S. Pharmacopeial Con-
vention, Inc.; 2004:2622.
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