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6268 á531ñ / Chemical Tests USP 41

CU = nominal concentration of thiamine in the appropriate Sample solution (mg/mL)

For products containing thiamine mononitrate, calculate the percentage of the labeled amount of thiamine mononitrate
(C12H17N5O4S) in the portion of sample taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100

rU = peak response of thiamine from the appropriate Sample solution

rS = peak response of thiamine from the Standard solution
CS = concentration of USP Thiamine Hydrochloride RS in the Standard solution (mg/mL)
CU = nominal concentration of thiamine mononitrate in the appropriate Sample solution (mg/mL)
Mr1 = molecular weight of thiamine mononitrate, 327.36
Mr2 = molecular weight of thiamine hydrochloride, 337.27

ADDITIONAL REQUIREMENTS
• USP REFERENCE STANDARDS á11ñ
USP Thiamine Hydrochloride RS

á541ñ TITRIMETRY

DIRECT TITRATIONS

Direct titration is the treatment of a soluble substance, contained in solution in a suitable vessel (the titrate), with an appro-
priate standardized solution (the titrant), the endpoint being determined instrumentally or visually with the aid of a suitable
indicator.
The titrant is added from a suitable buret and is so chosen, with respect to its strength (normality), that the volume added is
between 30% and 100% of the rated capacity of the buret. [NOTE—Where less than 10 mL of titrant is required, a suitable
microburet is to be used.] The endpoint is approached directly but cautiously, and finally the titrant is added dropwise from
the buret in order that the final drop added will not overrun the endpoint. The quantity of the substance being titrated may
be calculated from the volume and the normality or molarity factor of the titrant and the equivalence factor for the substance
given in the individual monograph.

RESIDUAL TITRATIONS

Some Pharmacopeial assays require the addition of a measured volume of a volumetric solution, in excess of the amount
actually needed to react with the substance being assayed, the excess of this solution then being titrated with a second volu-
metric solution. This constitutes a residual titration and is known also as a “back titration.” The quantity of the substance being
titrated may be calculated from the difference between the volume of the volumetric solution originally added, corrected by
means of a blank titration, and that consumed by the titrant in the back titration, due allowance being made for the respective
normality or molarity factors of the two solutions, and the equivalence factor for the substance given in the individual mono-
graph.
General Chapters

COMPLEXOMETRIC TITRATIONS

Successful complexometric titrations depend on several factors. The equilibrium constant for formation of the titrant-analyte
complex must be sufficiently large that, at the endpoint, very close to 100% of the analyte has been complexed. The final
complex must be formed rapidly enough that the analysis time is practical. When the analytical reaction is not rapid, a residual
titration may sometimes be successful.
In general, complexometric indicators are themselves complexing agents. The reaction between metal ion and indicator
must be rapid and reversible. The equilibrium constant for formation of the metal-indicator complex should be large enough
to produce a sharp color change but must be less than that for the metal-titrant complex. Indicator choice is also restricted by
the pH range within which the complexation reaction must be carried out and by interference of other ions arising from the
sample or the buffer. Interfering ions may often be masked or “screened” via addition of another complexing agent. (The
masking technique is also applicable to redox titrations.)

OXIDATION-REDUCTION (REDOX) TITRATIONS

Determinations may often be carried out conveniently by the use of a reagent that brings about oxidation or reduction of
the analyte. Many redox titration curves are not symmetric about the equivalence point, and thus graphical determination of

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USP 41 Chemical Tests / á541ñ 6269

the endpoint is not possible; but indicators are available for many determinations, and a redox reagent can often serve as its
own indicator. As in any type of titration, the ideal indicator changes color at an endpoint that is as close as possible to the
equivalence point. Accordingly, when the titrant serves as its own indicator, the difference between the endpoint and the
equivalence point is determined only by the analyst's ability to detect the color change. A common example is the use of per-
manganate ion as an oxidizing titrant since a slight excess can easily be detected by its pink color. Other titrants that may
serve as their own indicators are iodine, cerium (IV) salts, and potassium dichromate. In most cases, however, the use of an
appropriate redox indicator will yield a much sharper endpoint.
It may be necessary to adjust the oxidation state of the analyte prior to titration through use of an appropriate oxidizing or
reducing agent; the excess reagent must then be removed, e.g., through precipitation. This is nearly always the practice in the
determination of oxidizing agents since most volumetric solutions of reducing agents are slowly oxidized by atmospheric oxy-
gen.

TITRATIONS IN NONAQUEOUS SOLVENTS

Acids and bases have long been defined as substances that furnish, when dissolved in water, hydrogen and hydroxyl ions,
respectively. This definition, introduced by Arrhenius, fails to recognize the fact that properties characteristic of acids or bases
may be developed also in other solvents. A more generalized definition is that of Brönsted, who defined an acid as a substance
that furnishes protons, and a base as a substance that combines with protons. Even broader is the definition of Lewis, who
defined an acid as any material that will accept an electron pair, a base as any material that will donate an electron pair, and
neutralization as the formation of a coordination bond between an acid and a base.
The apparent strength of an acid or a base is determined by the extent of its reaction with a solvent. In water solution all
strong acids appear equally strong because they react with the solvent to undergo almost complete conversion to oxonium
ion and the acid anion (leveling effect). In a weakly protophilic solvent such as acetic acid the extent of formation of the ace-
tate acidium ion shows that the order of decreasing strength for acids is perchloric, hydrobromic, sulfuric, hydrochloric, and
nitric (differentiating effect).
Acetic acid reacts incompletely with water to form oxonium ion and is, therefore, a weak acid. In contrast, it dissolves in a
base such as ethylenediamine, and reacts so completely with the solvent that it behaves as a strong acid. The same holds for
perchloric acid.
This leveling effect is observed also for bases. In sulfuric acid almost all bases appear to be of the same strength. As the acid
properties of the solvent decrease in the series sulfuric acid, acetic acid, phenol, water, pyridine, and butylamine, the bases
become progressively weaker until all but the strongest have lost their basic properties. In order of decreasing strength, the
strong bases are sodium 2-aminoethoxide, potassium methoxide, sodium methoxide, and lithium methoxide.
Many water-insoluble compounds acquire enhanced acidic or basic properties when dissolved in organic solvents. Thus the
choice of the appropriate solvent permits the determination of a variety of such materials by nonaqueous titration. Further-
more, depending upon which part of a compound is the physiologically active moiety, it is often possible to titrate that part by
proper selection of solvent and titrant. Pure compounds can be titrated directly, but it is often necessary to isolate the active
ingredient in pharmaceutical preparations from interfering excipients and carriers.
The types of compounds that may be titrated as acids include acid halides, acid anhydrides, carboxylic acids, amino acids,
enols such as barbiturates and xanthines, imides, phenols, pyrroles, and sulfonamides. The types of compounds that may be
titrated as bases include amines, nitrogen-containing heterocyclic compounds, oxazolines, quaternary ammonium com-
pounds, alkali salts of organic acids, alkali salts of weak inorganic acids, and some salts of amines. Many salts of halogen acids
may be titrated in acetic acid or acetic anhydride after the addition of mercuric acetate, which removes halide ion as the
unionized mercuric halide complex and introduces the acetate ion.

General Chapters
For the titration of a basic compound, a volumetric solution of perchloric acid in glacial acetic acid is preferred, although
perchloric acid in dioxane is used in special cases. The calomel-glass electrode system is useful in this case. In acetic acid sol-
vent, this electrode system functions as predicted by theory.
For the titration of an acidic compound, two classes of titrant are available: the alkali metal alkoxides and the tetraalkylam-
monium hydroxides. A volumetric solution of sodium methoxide in a mixture of methanol and toluene is used frequently, al-
though lithium methoxide in methanol-benzene solvent is used for those compounds yielding a gelatinous precipitate on titra-
tion with sodium methoxide.
The alkali error limits the use of the glass electrode as an indicating electrode in conjunction with alkali metal alkoxide ti-
trants, particularly in basic solvents. Thus, the antimony-indicating electrode, though somewhat erratic, is used in such titra-
tions. The use of quaternary ammonium hydroxide compounds, e.g., tetra-n-butylammonium hydroxide and trimethylhexade-
cylammonium hydroxide (in benzene-methanol or isopropyl alcohol), has two advantages over the other titrants in that (a)
the tetraalkylammonium salt of the titrated acid is soluble in the titration medium, and (b) the convenient and well-behaved
calomel-glass electrode pair may be used to conduct potentiometric titrations.
Because of interference by carbon dioxide, solvents for acidic compounds need to be protected from excessive exposure to
the atmosphere by a suitable cover or by an inert atmosphere during the titration. Absorption of carbon dioxide may be deter-
mined by performing a blank titration. The blank should not exceed 0.01 mL of 0.1 N sodium methoxide VS per mL of sol-
vent.

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6270 á541ñ / Chemical Tests USP 41

The endpoint may be determined visually by color change, or potentiometrically, as indicated in the individual monograph.
If the calomel reference electrode is used, it is advantageous to replace the aqueous potassium chloride salt bridge with 0.1 N
lithium perchlorate in glacial acetic acid for titrations in acidic solvents or potassium chloride in methanol for titrations in basic
solvents.
Where these or other mixtures are specified in individual monographs, the calomel reference electrode is modified by first
removing the aqueous potassium chloride solution and residual potassium chloride, if any, by rinsing with water, then elimi-
nating residual water by rinsing with the required nonaqueous solvent, and finally filling the electrode with the designated
nonaqueous mixture.
In nearly all cases, except those where silver ion might interfere, a silver-silver chloride reference electrode may be substitu-
ted for the calomel electrode. The silver-silver chloride electrode is more rugged, and its use helps to eliminate toxic mercury
salts from the laboratory. Generally, a salt bridge may be used to circumvent interference by silver ion.
The more useful systems for titration in nonaqueous solvents are listed in Table 1.
Table 1. Systems for Nonaqueous Titrations
Acidic (for titration Relatively Neutral Relatively Neutral
of bases and their (for differential Basic (for titration (for differential
Type of salts) titration of bases) of acids) titration of acids)
Solvent1 Glacial Acetic Acid Acetonitrile Dimethylformamide Acetone
Acetic Anhydride Alcohols n-Butylamine Acetonitrile
Formic Acid Chloroform Pyridine Methyl Ethyl Ketone
Propionic Acid Benzene Ethylenediamine Methyl Isobutyl Ketone
Sulfuryl Chloride Toluene Morpholine tert-Butyl Alcohol
Chlorobenzene
Ethyl Acetate
Dioxane
Indicator Crystal Violet Methyl Red Thymol Blue Azo Violet
Quinaldine Red Methyl Orange Thymolphthalein Bromothylmol Blue
p-Naphtholbenzein p-Naphtholbenzein Azo Violet p-Hydroxyazobenzene
Alphezurine 2-G o-Nitroaniline Thymol Blue
Malachite Green p-Hydroxyazobenzene
Electrodes Glass–calomel Glass–calomel Antimony–calomel Antimony–calomel
Glass–silver–silver chloride Calomel–silver–silver chlor- Antimony–glass Glass–calomel
Mercury–mercuric acetate ide Antimony–antimony2 Glass–platinum2
Platinum–calomel
Glass–calomel
1 Relatively neutral solvents of low dielectric constant such as benzene, toluene, chloroform, or dioxane may be used in conjunction with any acidic or basic
solvent in order to increase the sensitivity of the titration end-points.
2 In titrant.

INDICATOR AND POTENTIOMETRIC ENDPOINT DETECTION

The simplest and most convenient method by which the equivalence point, i.e., the point at which the stoichiometric ana-
General Chapters

lytical reaction is complete, may be determined is with the use of indicators. These chemical substances, usually colored, re-
spond to changes in solution conditions before and after the equivalence point by exhibiting color changes that may be taken
visually as the endpoint, a reliable estimate of the equivalence point.
A useful method of endpoint determination results from the use of electrochemical measurements. If an indicator electrode,
sensitive to the concentration of the species undergoing titrimetric reaction, and a reference electrode, whose potential is in-
sensitive to any dissolved species, are immersed in the titrate to form a galvanic cell, the potential difference between the elec-
trodes may be sensed by a pH meter and used to follow the course of the reaction. Where such a series of measurements is
plotted correctly (i.e., for an acid-base titration, pH versus mL of titrant added; for a precipitimetric, complexometric, or oxida-
tion-reduction titration, mV versus mL of titrant added), a sigmoid curve results with a rapidly changing portion (the “break”)
in the vicinity of the equivalence point. The midpoint of this linear vertical portion or the inflection point may be taken as the
endpoint. The equivalence point may also be determined mathematically without plotting a curve. However, it should be no-
ted that in asymmetrical reactions, which are reactions in which the number of anions reacting is not the same as the number
of cations reacting, the endpoint as defined by the inflection of the titration curve does not occur exactly at the stoichiometric
equivalence point. Thus, potentiometric endpoint detection by this method is not suitable in the case of asymmetric reactions,
examples of which are the precipitation reaction,
2Ag+ + CrO4–2

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USP 41 Chemical Tests / á541ñ 6271

and the oxidation-reduction reaction,

5Fe+2 + MnO4–.

All acid-base reactions, however, are symmetrical. Thus, potentiometric endpoint detection may be employed in acid-base ti-
trations and in other titrations involving symmetrical reversible reactions where an indicator is specified, unless otherwise direc-
ted in the individual monograph.
Two types of automatic electrometric titrators are available. The first is one that carries out titrant addition automatically and
records the electrode potential differences during the course of titration as the expected sigmoid curve. In the second type,
titrant addition is performed automatically until a preset potential or pH, representing the endpoint, is reached, at which point
the titrant addition ceases.
Several acceptable electrode systems for potentiometric titrations are summarized in Table 2.
Table 2. Potentiometric Titration Electrode Systems
Titration Indicating Electrode Equation1 Reference Electrode Applicability2
Acid-base Glass E = k + 0.0591 pH Calomel or silver–silver Titration of acids and bases
chloride
Precipitimetric (silver) Silver E = E° + 0.0591 log [Ag +] Calomel (with potassium ni- Titration with or of silver in-
trate salt bridge) volving halides or thiocya-
nate
Complexometric Mercury–mercury(II) E = E° + 0.0296(log k¢ − pM) Calomel Titration of various metals
(M), e.g., Mg+2, Ca+2 Al+3,
Bi+3, with EDTA
Oxidation–reduction Platinum E = E° + (0.0591/n) × log Calomel or silver–silver Titrations with arsenite, bro-
[ox]/[red] chloride mine, cerate, dichromate,
exacyonoferrate(III), iodate,
nitrite, permanganate, thio-
sulfate
1 Appropriate form of Nernst equation describing the indicating electrode system: k = glass electrode constant; k¢ = constant derived from Hg–Hg(II)–EDTA equi-

librium; M = any metal undergoing EDTA titration; [ox] and [red] from the equation, ox + ne ¬ ® red.
2 Listing is representative but not exhaustive.

BLANK CORRECTIONS

As previously noted, the endpoint determined in a titrimetric assay is an estimate of the reaction equivalence point. The val-
idity of this estimate depends upon, among other factors, the nature of the titrate constituents and the concentration of the
titrant. An appropriate blank correction is employed in titrimetric assays to enhance the reliability of the endpoint determina-
tion. Such a blank correction is usually obtained by means of a residual blank titration, wherein the required procedure is repea-
ted in every detail except that the substance being assayed is omitted. In such instances, the actual volume of titrant equiva-
lent to the substance being assayed is the difference between the volume consumed in the residual blank titration and that
consumed in the titration with the substance present. The corrected volume so obtained is used in calculating the quantity of
the substance being titrated, in the same manner as prescribed under Residual Titrations. Where potentiometric endpoint de-
tection is employed, the blank correction is usually negligible.

General Chapters

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6272 á551ñ / Chemical Tests USP 41

á551ñ VITAMIN E ASSAY

INTRODUCTION
The following liquid chromatographic procedures are provided for the determination of vitamin E as an active pharmaceutical
ingredient, as a dietary supplement ingredient, or as a component in compendial dosage forms in the forms of alpha toco-
pherol (C29H50O2), alpha tocopheryl acetate (C31H52O3), or alpha tocopheryl acid succinate (C33H54O5).
Throughout this assay, protect solutions containing, and derived from, the test specimen and the Reference Standard from the
atmosphere and light, preferably by the use of a blanket of inert gas and low-actinic glassware.
Where vitamin E (alpha tocopherol, alpha tocopheryl acetate, or alpha tocopheryl acid succinate) is specified in the following
procedure, use the chemical form present in the formulation and the relevant USP Reference Standard.

ASSAY
• PROCEDURE 1
• This procedure can be used to determine vitamin E in:
• Oil-Soluble Vitamins Tablets
• Oil-Soluble Vitamins Capsules
• Oil-Soluble Vitamins with Minerals Tablets
• Oil-Soluble Vitamins with Minerals Capsules
• Oil- and Water-Soluble Vitamins Tablets
• Oil- and Water-Soluble Vitamins Capsules
• Oil- and Water-Soluble Vitamins with Minerals Tablets
• Oil- and Water-Soluble Vitamins with Minerals Capsules
• This is a neutral procedure that involves the use of dimethyl sulfoxide to dissolve the excipients in the sample, fol-
lowed by a liquid–liquid extraction of vitamin E with hexane. The hexane extract is then evaporated in vacuum to
dryness, and the residue is reconstituted in methanol prior injection into the chromatograph.
• Unless specified in the individual monographs, the System suitability solution, Standard solution, Sample solutions, and
reagent solutions are prepared as follows.
Solution A: Phosphoric acid solution (1 in 100) in water
Mobile phase: Methanol and Solution A (19:1)
System suitability solution: Prepare a 0.65-mg/mL solution of USP Ergocalciferol RS in methanol. Transfer 1.0 mL of this
solution to a 100-mL volumetric flask containing 100 mg of USP Alpha Tocopheryl Acetate RS. Dissolve in 30 mL of metha-
nol, with the aid of sonication if necessary, and dilute with methanol to volume. Store this solution in a refrigerator.
Standard solution: 2 mg/mL of USP Alpha Tocopherol RS, USP Alpha Tocopheryl Acetate RS, or USP Alpha Tocopheryl
Acid Succinate RS in methanol
Sample solution for Tablets: Finely powder NLT 20 Tablets. Transfer a portion of the powder typically equivalent to 20
mg of the vitamin E form under testing but not exceeding 7.5 g of the powder, to a centrifuge tube having a polytef-lined
screw cap. Add about 2 mL of dimethyl sulfoxide per each g of powdered Tablets, and about 3 mL of n-hexane each per g
of powdered Tablets, and shake for 45 min on a shaker in a water bath maintained at 60°. [NOTE—Set up the shaker to
ensure that the contents of the container are mixed vigorously and thoroughly.] Centrifuge at 3000 rpm for 10 min, and
transfer the hexane layer by means of a pipet to a volumetric flask. [NOTE—Volumetric flask size: NLT 20 mL.] Add 3 mL of
n-hexane per each g of powdered Tablets to the dimethyl sulfoxide layer, shake thoroughly for 5 min, and transfer the
hexane layer by means of a pipet to the same volumetric flask. Repeat this extraction with three additional portions of n-
General Chapters

hexane. Dilute the extracts in the volumetric flask with n-hexane to volume. Transfer NLT 20 mL of this solution to a suita-
ble container, and evaporate in vacuum at room temperature to dryness. Transfer the residue with the aid of methanol to a
suitable volumetric flask, and dilute with methanol to volume to obtain a concentration of 2 mg/mL of alpha tocopherol,
alpha tocopheryl acetate, or alpha tocopheryl acid succinate.
Sample solution for Capsules: Transfer the contents of NLT 20 Capsules to a suitable container, mix, and weigh. Transfer
a portion of the mixture, typically equivalent to 20 mg of the vitamin E form under testing but not exceeding 7.5 g of the
mixture, to a centrifuge tube having a polytef-lined screw cap. [NOTE—For hard gelatin Capsules, remove, as completely as
possible, the contents of NLT 20 Capsules by cutting open the Capsule shells, transferring the shells and their contents to a
suitable container, and triturating to a homogeneous mass. Transfer a portion of the mass, typically equivalent to 20 mg of
the vitamin E form under testing but not exceeding 7.5 g of the mixture to a centrifuge tube having a polytef-lined screw
cap.] Add about 2 mL of dimethyl sulfoxide per each g of Capsule contents, and about 3 mL of n-hexane per each g of
Capsule contents, and shake for 45 min on a shaker in a water bath maintained at 60°. [NOTE—Set up the shaker to ensure
that the contents of the container are mixed vigorously and thoroughly.] Centrifuge at 3000 rpm for 10 min, and transfer
the hexane layer by means of a pipet to a volumetric flask. [NOTE—Volumetric flask size: NLT 20 mL.] Add 3 mL of n-hex-
ane per each g of Capsule contents to the dimethyl sulfoxide layer, shake thoroughly for 5 min, and transfer the hexane
layer by means of a pipet to the same volumetric flask. Repeat this extraction with three additional portions of n-hexane.
Dilute the extracts in the volumetric flask with n-hexane to volume. Transfer NLT 20 mL of this solution to a suitable con-

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