ANÁLISIS DE

MEDICAMENTOS,
DOPING Y DROGAS
DE ABUSO.

TRABAJO PRÁCTICO N°1

MATERIAS PRIMAS

1
OBJETIVO:

En el transcurso de la presente práctica, el estudiante llevará a cabo la evaluación de materias

primas destinadas a la elaboración de medicamentos, conforme a los criterios establecidos en la
farmacopea correspondiente. A continuación, se detallan los fármacos objeto de estudio, así como
los análisis requeridos para cada uno de ellos.

Fármaco Análisis solicitado Farmacopea Página

● Identificación (A y C)
Nicotinamida BP 2003 3
● Valoración

● Identificación (A)
Ácido
● Límite de ácido salicílico libre USP 30 6
acetilsalicílico
● Valoración

Farmacopea
● Identificación (B, C y D)
Fenobarbital Internacional, 4ªed., 9
● Valoración
2008

● Pureza cromatográfica
Cafeína USP 29 10
● Valoración

A continuación se presenta la información disponible de cada farmacopea de los fármacos a analizar.

Página 2
Browse: 2003 British Pharmacopoeia CD-ROM
British Pharmacopoeia Volume I & II
Monographs: Medicinal and Pharmaceutical substances
Nicotinamide

Nicotinamide
General Notices
(Ph Eur monograph 0047)

C 6H 6N 2O 122.1 98-92-0

Ph Eur

Definition

Nicotinamide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of pyridine-3-carboxamide, calculated with reference to the dried substance.

Characters

A white, crystalline powder or colourless crystals, freely soluble in water and in ethanol.

Identification

First identification

A, B.

Second identification

A, C, D.

A. Melting point (2.2.14): 128°C to 131°C.

B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum

obtained with nicotinamide CRS.

C. Boil 0.1 g with 1 ml of dilute sodium hydroxide solution R. Ammonia is evolved which is
recognisable by its odour.

D. Dilute 2 ml of solution S (see Tests) to 100 ml with water R. To 2 ml of the solution, add 2 ml of
cyanogen bromide solution R and 3 ml of a 25 g/l solution of aniline R and shake. A yellow colour
develops.

Tests

Solution S

Dissolve 2.5 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent.

©Crown Copyright 2003 1

Appearance of solution

Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY7 (2.2.2,
Method II).

pH (2.2.3)

The pH of solution S is 6.0 to 7.5.

Related substances

Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF254 plate R.

Test solution

Dissolve 0.4 g of the substance to be examined in a mixture of equal volumes of alcohol R and
water R and dilute to 5.0 ml with the same mixture of solvents.

Reference solution

Dilute 0.5 ml of the test solution to 200 ml with a mixture of equal volumes of alcohol R and water
R.

Apply to the plate 5 μl of each solution. Develop over a path of 10 cm using a mixture of 4 volumes
of water R, 45 volumes of ethanol R and 48 volumes of chloroform R. Allow the plate to dry and
examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution,
apart from the principal spot, is not more intense than the spot in the chromatogram obtained with
the reference solution (0.25 per cent).

Heavy metals (2.4.8)

Dilute 12 ml of solution S to 18 ml with water R. 12 ml of the solution complies with limit test A for
heavy metals (30 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R.

Loss on drying (2.2.32)

Not more than 0.5 per cent, determined on 1.00 g by drying in vacuo for 18 h.

Sulphated ash (2.4.14)

Not more than 0.1 per cent, determined on 1.0 g.

Assay

Dissolve 0.250 g in 20 ml of anhydrous acetic acid R, heating slightly if necessary, and add 5 ml of
acetic anhydride R. Titrate with 0.1M perchloric acid, using crystal violet solution R as indicator until
the colour changes to greenish-blue.

1 ml of 0.1M perchloric acid is equivalent to 12.21 mg of C6H 6N2O.

Ph Eur

Action and use

Component of vitamin B.

©Crown Copyright 2003 2

Preparations

Nicotinamide Tablets

Vitamins B and C Injection

©Crown Copyright 2003 3

Aspirin USP 30

Aspirin

C9H 8O 4 180.16

Benzoic acid, 2-(acetyloxy)-.

Salicylic acid acetate [50-78-2].

» Aspirin contains not less than 99.5 percent and not more than 100.5
percent of C9H8O4, calculated on the dried basis.
Packaging and storage— Preserve in tight containers.

USP Reference standards 11 —

USP Aspirin RS .

Identification—

A: Heat it with water for several minutes, cool, and add 1 or 2 drops of ferric chloride TS: a
violet-red color is produced.

B: Infrared Absorption 197K .

Loss on drying 731 — Dry it over silica gel for 5 hours: it loses not more than 0.5% of its
weight.

Readily carbonizable substances 271 — Dissolve 500 mg in 5 mL of sulfuric acid TS: the
solution has no more color than Matching Fluid Q.

Residue on ignition 281 : not more than 0.05%.

Substances insoluble in sodium carbonate TS— A solution of 500 mg in 10 mL of warm

sodium carbonate TS is clear.

Chloride 221 — Boil 1.5 g with 75 mL of water for 5 minutes, cool, add sufficient water to
restore the original volume, and filter. A 25-mL portion of the filtrate shows no more chloride
than corresponds to 0.10 mL of 0.020 N hydrochloric acid (0.014%).

Sulfate — Dissolve 6.0 g in 37 mL of acetone, and add 3 mL of water. Titrate

potentiometrically with 0.02 M lead perchlorate, prepared by dissolving 9.20 g of lead
perchlorate in water to make 1000 mL of solution, using a pH meter capable of a minimum
reproducibility of ±0.1 mV (see pH 791 ) and equipped with an electrode system consisting
of a lead-specific electrode and a silver–silver chloride reference glass-sleeved electrode
containing a solution of tetraethylammonium perchlorate in glacial acetic acid (1 in 44) (see
Titrimetry 541 ): not more than 1.25 mL of 0.02 M lead perchlorate is consumed (0.04%).
[NOTE—After use, rinse the lead-specific electrode with water, drain the reference electrode,
flush with water, rinse with methanol, and allow to dry.]

Heavy metals— Dissolve 2 g in 25 mL of acetone, and add 1 mL of water. Add 1.2 mL of

thioacetamide–glycerin base TS and 2 mL of pH 3.5 Acetate Buffer (see Heavy Metals 231
), and allow to stand for 5 minutes: any color produced is not darker than that of a control made
with 25 mL of acetone and 2 mL of Standard Lead Solution (see Heavy Metals 231 ),
treated in the same manner. The limit is 10 µg per g.

Limit of free salicylic acid— Dissolve 2.5 g in sufficient alcohol to make 25.0 mL. To each of
two matched color-comparison tubes add 48 mL of water and 1 mL of a freshly prepared,
diluted ferric ammonium sulfate solution (prepared by adding 1 mL of 1 N hydrochloric acid to
2 mL of ferric ammonium sulfate TS and diluting with water to 100 mL). Into one tube pipet 1
mL of a standard solution of salicylic acid in water, containing 0.10 mg of salicylic acid per mL.
Into the second tube pipet 1 mL of the 1 in 10 solution of Aspirin. Mix the contents of each
tube: after 30 seconds, the color in the second tube is not more intense than that in the tube
containing the salicylic acid (0.1%).

Organic volatile impurities, Method IV 467 : meets the requirements.

(Official until July 1, 2007)

Assay— Place about 1.5 g of Aspirin, accurately weighed, in a flask, add 50.0 mL of 0.5 N
sodium hydroxide VS, and boil the mixture gently for 10 minutes. Add phenolphthalein TS, and
titrate the excess sodium hydroxide with 0.5 N sulfuric acid VS. Perform a blank determination
(see Residual Titrations under Titrimetry 541 ). Each mL of 0.5 N sodium hydroxide is
equivalent to 45.04 mg of C 9H8O4.

Auxiliary Information— Staff Liaison : Clydewyn M. Anthony, Ph.D., Scientist

Expert Committee : (MDCCA05) Monograph Development-Cough Cold and Analgesics

USP30–NF25 Page 1443

Pharmacopeial Forum : Volume No. 30(4) Page 1164

Phone Number : 1-301-816-8139

 Page 1 of 5

541 TITRIMETRY

Direct Titrations— 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 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 volumetric 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 monograph.

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 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 permanganate 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
 Page 2 of 5

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 oxygen.

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 acetate 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. Furthermore, 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 compounds, 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.

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 solvent, this electrode system functions as predicted by theory.
 Page 3 of 5

For the titration of an acidic compound, two classes of titrant are available: the alkali metal alkoxides and the
tetraalkylammonium hydroxides. A volumetric solution of sodium methoxide in a mixture of methanol and
toluene is used frequently, although lithium methoxide in methanol-benzene solvent is used for those
compounds yielding a gelatinous precipitate on titration with sodium methoxide.

The alkali error limits the use of the glass electrode as an indicating electrode in conjunction with alkali
metal alkoxide titrants, particularly in basic solvents. Thus, the antimony-indicating electrode, though
somewhat erratic, is used in such titrations. The use of quaternary ammonium hydroxide compounds, e.g.,
tetra-n-butylammonium hydroxide and trimethylhexadecylammonium 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 determined by performing a blank titration. The blank should not
exceed 0.01 mL of 0.1 N sodium methoxide VS per mL of solvent.

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 eliminating 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 substituted 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
Type of of bases and their (for differential Basic (for titration (for differential
Solvent 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
 Page 4 of 5

Malachite Green p-Hydroxyazobenzene

Electrodes Glass–calomel Glass–calomel Antimony–calomel Antimony–calomel
Glass–silver–silver Calomel–silver–silver Antimony–glass Glass–calomel
chloride chloride
Mercury–mercuric Antimony–antimony2 Glass–platinum2
acetate
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 analytical reaction is complete, may be
determined is with the use of indicators. These chemical substances, usually colored, respond 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 insensitive to any dissolved species, are immersed in the titrate to
form a galvanic cell, the potential difference between the electrodes 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 oxidation-
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 noted 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

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 titrations and in other titrations involving symmetrical reversible reactions where an
indicator is specified, unless otherwise directed 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.
 Page 5 of 5

Table 2. Potentiometric Titration Electrode Systems

Indicating Reference
Titration Electrode Equation1 Electrode Applicability2
Acid-base Glass E = k + 0.0591 Calomel or silver– Titration of acids and bases
pH silver chloride
Precipitimetric Silver Calomel (with Titration with or of silver involving
E = E + 0.0591
(silver) potassium nitrate halides or thiocyanate
log [Ag +] salt bridge)
Complexometric Mercury– Calomel Titration of various metals (M), e.g.,
E = E + 0.0296
mercury(II) Mg+2, Ca+2 Al+3, Bi+3, with EDTA
(log k′ pM)
Oxidation– Platinum Calomel or silver– Titrations with arsenite, bromine,
E=E +
reduction silver chloride cerate, dichromate, exacyonoferrate
(0.0591/n) × log
(III), iodate, nitrite, permanganate,
[ox]/[red]
thiosulfate
1
Appropriate form of Nernst equation describing the indicating electrode system: k = glass electrode
constant; k′ = constant derived from Hg–Hg(II)–EDTA equilibrium; 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 validity 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 determination. Such a blank correction is usually
obtained by means of a residual blank titration, wherein the required procedure is repeated in every detail
except that the substance being assayed is omitted. In such instances, the actual volume of titrant
equivalent 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 detection is employed, the blank
correction is usually negligible.

Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
General Chapter Horacio N. Pappa, Ph.D. (GC05) General Chapters 05
Senior Scientist and Latin American Liaison
1-301-816-8319
USP32–NF27 Page 179
 International Pharmacopoeia 4th edition, 2008

Phenobarbitalum - Phenobarbital

Molecular formula. C12H12N2O3

Relative molecular mass. 232.2

Graphic formula.

Chemical name. 5-Ethyl-5-phenylbarbituric acid; 5-ethyl-5-phenyl-2,4,6-(1H,3H,5H)-

pyrimidinetrione; CAS Reg. No. 50-06-6.

Description. Colourless crystals or a white, crystalline powder; odourless.

Solubility. Soluble in about 1100 parts of water, in about 10 parts of ethanol (~750 g/l)
TS and in about 15 parts of ether R.

Category. Hypnotic; sedative; anticonvulsant.

Storage. Phenobarbital should be kept in a well-closed container.

Additional information. Phenobarbital may exhibit polymorphism.

Requirements

Definition. Phenobarbital contains not less than 98.0% and not more than 101.0% of
C12H12N2O3, calculated with reference to the dried substance.

Identity tests

• Either test A or tests B, C, and D may be applied.

A. Carry out the examination as described under 1.7 Spectrophotometry in the infrared
region. The infrared absorption spectrum is concordant with the reference spectrum of
phenobarbital.

B. Dissolve 20 mg in 5 ml of ethanol (~750 g/l) TS, add 1 drop of cobaltous chloride TS

and 1 drop of ammonia (~100 g/l) TS; a violet colour is produced.

C. Shake for 3 minutes 0.1 g with 4 ml of sodium hydroxide (0.1 mol/l) VS and 1 ml of
water. Filter and to 2 ml of the filtrate add 4 drops of mercuric chloride (65 g/l) TS; a
white precipitate is formed, which dissolves on the addition of 5 ml of ammonia (~100
g/l) TS.
D. Dissolve 0.1 g in 2 ml of sulfuric acid (~1760 g/l) TS, add about 10 mg of sodium
nitrite R, and warm on a water-bath for 10 minutes; an orange-yellow colour with a
brownish sheen is produced.

Melting range. 174-178°C.

Solution in alkali. Dissolve 1.0 g in 4.0 ml of sodium hydroxide (~80 g/l) TS and add
6.0 ml of water; the solution is clear and colourless.

Sulfated ash. Not more than 1.0 mg/g.

Loss on drying. Dry to constant weight at 105°C; it loses not more than 10 mg/g.

Acidity. Boil 1.0 g with 50 ml of water for 2 minutes, adjust the volume to 50 ml, and
filter. To 10 ml of the filtrate add 0.15 ml of methyl red/ethanol TS; not more than 0.1
ml of sodium hydroxide (0.1 mol/l) VS is required to obtain the midpoint of the indicator
(orange).

Phenylbarbituric acid. Boil 1.0 g with 5 ml of ethanol (~750 g/l) TS for 3 minutes
under a reflux condenser; a clear solution is produced.

Neutral and basic impurities. Dissolve 1.0 g in a mixture of 5 ml of sodium hydroxide

(~80 g/l) TS and 10 ml of water and shake for 1 minute with 25 ml of ether R. Wash the
ethereal layer 3 times, each time with 5 ml of water, evaporate the ether, and dry the
residue at 105°C for 1 hour; the residue weighs not more than 3.0 mg.

Related substances. Carry out the test as described under 1.14.1 Thin-layer
chromatography, using silica gel R2 as the coating substance and a mixture of 80
volumes of chloroform R, 15 volumes of ethanol (~750 g/l) TS, and 1 volume of
ammonia (~260 g/l) TS as the mobile phase. Apply separately to the plate 10 μl of each
of 2 solutions in ethanol (~750 g/l) TS containing (A) 10 mg of the test substance per ml
and (B) 0.20 mg of the test substance per ml. After removing the plate from the
chromatographic chamber, allow it to dry in air, and examine the chromatogram in
ultraviolet light (254 nm). Any spot obtained with solution A, other than the principal
spot, is not more intense than that obtained with solution B.

Assay. Dissolve about 0.20 g, accurately weighed, in 30 ml of dimethylformamide R, add

2 drops of thymolphthalein/dimethylformamide TS and titrate with sodium methoxide
(0.1 mol/l) VS to a blue end-point, as described under 2.6 Non-aqueous titration. Method
B. Each ml of sodium methoxide (0.1 mol/l) VS is equivalent to 23.22 mg of C12H12N2O3.
Methods of Analysis:

1. Physical and Physicochemical Methods:

1.14 Chromatography:

1.14.1 Thin-layer chromatography

In thin-layer chromatography the adsorbent is a thin, uniform layer (usually about 0.24
mm thick) of a dry, finely powdered material applied to a suitable support, such as a
glass plate or an aluminium or plastic foil. The mobile phase is allowed to move across
the surface of the plate (usually by capillary action) and the chromatographic process
may depend upon adsorption, partition, or a combination of both, depending on the
adsorbent, its treatment, and the nature of the solvents used. During the
chromatographic procedure the plate is contained in a chromatographic chamber (usually
made of glass to permit observation of the movement of the mobile phase up the plate),
which is usually saturated with the solvent vapour. Solid supports frequently used are
silica gel, kieselguhr, alumina, and cellulose; to these may be added suitable substances,
for example, calcium sulfate to promote adhesion to the support. The prepared layer may
be impregnated with buffering materials to afford acidic, neutral, or basic layers, or with
other material, such as silver nitrate, designed to modify its properties. In certain cases
the layer may consist of an ion-exchange material. This wide range of possible layers,
used in conjunction with different solvent systems allows an almost infinite variation of
separating power that makes thin-layer chromatography such a useful technique in
pharmaceutical analysis.

As an adjunct to identification, thin-layer chromatography may be used by comparing the

behaviour of the material to be identified with that of a standard substance, usually an
authentic specimen of the substance being examined. If the two substances move
identical distances during the chromatographic process and if the two substances, when
mixed together and then subjected to chromatography, move as a single substance, it
may be presumed that the two substances are identical. This presumption may be
strengthened by repeating the procedure using a different system of chromatography; in
general, if two substances behave identically in as many as three fundamentally different
systems the presumption of identity becomes very strong.

For identification purposes it convenient to define the relative distance that the unknown
material moves in relation to the distance moved either by the solvent front or by a
standard reference material. On a developed chromatogram, the ratio of the distance
travelled on the adsorbent by a given compound to that travelled by the leading edge oft
he solvent (or mobile phase), both measured from the point of application of the test
substance, is referred to as the Rf value of the substance in the given chromatographic
system. The ratio of the distances moved by the compound and a stated reference
substance is referred to as the Rr value. In practice Rf values may vary considerably
according to the exact experimental conditions so that the Rr value determined against
the reference substance subjected to chromatography on the same surface gives a more
reliable numerical value. Even more reliable, however, is comparison with an authentic
specimen as described above and this is the procedure usually used for pharmacopoeial
purposes.

To determine the position of a colourless substance on a developed chromatogram it is

usually necessary to treat the chromatogram with a reagent that will either char the
separated substances or convert them to coloured or fluorescent derivatives. A
convenient alternative that is frequently applicable is to carry out the chromatography on
a surface impregnated with a substance that fluoresces strongly when examined under
short-wave ultraviolet light. Areas of the plate occupied by substances absorbing at the
same wavelengthshow as dark spots on the fluorescent background. In special cases,
other means of recognition may be used, for example, the detection of radioactivity
where labelled compounds are being separated or a microbiological response where
antibiotics are concerned.

The most valuable use of thin-layer chromatography in pharmaceutical work is to provide

a means of assessing low levels of impurities in medicinal substances. For this purpose
the substance is applied to the chromatographic surface and, after chromatography, any
secondary spots to be seen in the chromatogram after appropriate visualization are
compared for size and intensity with those of low loadings of expected impurities that
have simultaneously been subjected to chromatography on the same plate. Such a
procedure requires that the expected impurities be available and in certain monographs
the use of authentic specimens of impurities is called for. Frequently, such impurities are
not available and in such cases it is often possible to compare secondary spots arising
from trace impurities with the spot obtained by carrying out chromatography on the
same plate using an appropriately low loading of the substance being examined. This
expedient is not always possible since impurities and the substance being examined may
respond in different ways to the method of revelation used, but it often provides an
acceptable criterion by which the level of impurity in the substance may be judged. A
third procedure that is sometimes advocated is to apply such an amount of the substance
being examined that, after chromatography, no secondary spots will appear if the sample
is acceptably pure. This is the least satisfactory of the three methods since ability to see
a secondary spot is a subjective matter and because the intensity of spots on a
chromatogram may vary considerably from one occasion to another depending on the
exact conditions of chromatography.

For quantitative procedures the spot may be removed from the plate, the substance
eluted with a suitable solvent and then determined by a sufficiently sensitive method,
such as a spectrophotometric measurement, either directly or after a chemical reaction.
In certain cases, quantitation can also be achieved by measurement of the spot intensity
with the aid of a scanning densitometer and subsequent comparison of the intensity with
the intensities obtained from standard amounts of the same substance similarly treated.

Separations effected by thin-layer chromatography may sometimes be improved by

multiple development (when the chromatogram is allowed to dry and then subjected
again to the same system of chromatography), by continuous development (when the
mobile phase is allowed to evaporate continuously from the upper end of the adsorbent
surface), or by two-dimensional chromatography (when the chromatographic plate is
allowed to dry, turned at right angles, and then subjected to further chromatography,
frequently in a different solvent system from that used for the initial chromatography).
Caution should be exercised in interpreting the results of chromatograms where such
intermediate drying processes are used, however, since decomposition, such as
oxidation, of the substance being chromatographed may occur on the plate. The process
of two-dimensional chromatography is especially valuable in judging whether any
chemical change is taking place during the development process. If a mixture of
substances is developed first in one direction and then at right angles with the same
solvent the separated substances will lie in a diagonal line across the plate if no artefacts
are being produced.

In thin-layer chromatography the adsorbent is usually spread in a thin even layer on a

support plate. This may be undertaken in the analytical laboratory but it is also possible
and convenient to obtain commercially prepared chromatographic surfaces that are
attached to glass or to plastic or metal foil. Unfortunately, so sensitive may the
chromatographic process be to minor changes in conditions that these various
commercially available materials are not always interchangeable one with another or with
an apparently similar laboratory-coated plate. A factory-coated silica gel plate prepared
by a given manufacturer may give different separation characteristics from a laboratory-
coated plate prepared using the same manufacturer's coating substance, and instances
exist where a perfectly satisfactory method of separation devised using laboratory-
prepared plates fails when using precoated plates and vice versa. Great caution must,
therefore, be exercised when changing from one type of chromatographic plate to
another and the suitability of a given type of plate should always be assessed before
reliance is placed upon it.

The chromatographic chamber in which chromatography takes place should be protected

from light if it is suspected that the materials to be examined may be unstable in light. In
any case, the chromatographic chamber should always be in a position where the direct
rays of the sun cannot fall on it since the rays may be refracted to different degrees
owing to imperfections in the glass walls of the chamber. This may give rise to areas of
elevated temperature on the chromatographic plate and result in erratic flow of the
mobile phase.

Recommended procedure

The method given below assumes the use of a laboratory-prepared chromatographic

plate but a precoated plate, activated if necessary, may be used provided that it has
been shown to be suitable for the particular application.

The equipment consists of:

• a device for spreading on plates a uniform layer of coating substance of the desired
thickness;

• plates 200 mm long and wide enough to accommodate the required number of
solutions to be examined and the reference solutions;

• a chromatographic chamber of transparent material, usually glass, with a tightly fitting

lid, of a size suitable for the plates used.

Prepare a slurry of the coating substance and, using the spreading device, coat the
carefully cleaned plates with a layer about 0.25 mm thick, unless otherwise specified in
the monograph. Allow the coated plates to dry in air and heat to activate, unless
otherwise specified in the monograph, at 110 °C for 30 minutes, then allow to cool. If the
plates are not to be used immediately, store them in a desiccator containing silica gel,
desiccant, R. Remove a narrow strip (2-5 mm) of the coating substance from the vertical
sides of the plate.

Unless otherwise specified in the monograph, work under saturated chamber conditions.
To achieve such conditions, line the chromatographic chamber with filter-paper and pour
into the chamber a sufficient quantity of the mobile phase to saturate the filter-paper and
form a layer about 5 mm deep. Close the chamber and allow to stand for at least 1 hour
at room temperature.

Method

All operations during which the plate is exposed to the air should preferably be carried
out at a relative humidity of 50-60%. Apply the volume of the solution as specified in the
monograph as a compact spot, preferably not more than 4 mm in diameter. Application
may be made using a micropipette, a syringe, or other suitable means. The spot should
be placed about 1.5 cm from the lower edge and not less than 2 cm from the vertical
sides of the plate. Where more than one chromatogram is run on the same plate, the
spots should be placed not less than 1.5 cm apart and form a line parallel with the lower
edge of the plate. When the solvent has evaporated, place the plate in the
chromatographic chamber, ensuring that the plate is as nearly vertical as possible and
that the starting points are above the level of the mobile phase. Close the chamber and
maintain it at a constant temperature. Allow the mobile phase to ascend, usually 10-15
cm, remove the plate, mark the position of the solvent front and dry as specified in the
monograph.
Methods of Analysis:

2. Chemical methods:

2.6 Non-aqueous titration

Acids and bases have long been defined as substances that, when dissolved in water,
furnish hydrogen and hydroxyl ions, respectively. This definition, introduced by
Arrhenius, fails to recognize the fact that properties characteristic of acids or bases may
also be developed in other solvents. A more generalized definition is that of Brönsted,
who defined an acid as a proton donor, and a base as a proton acceptor. 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 base is determined by the extent of its reaction with
a solvent. In aqueous solution all strong acids appear equally strong because they react
with the solvent to undergo almost complete conversion to hydronium ion (H3O+) and the
acid anion. In a weakly protophilic solvent such as acetic acid, the extent of formation of
the acetonium ion (CH3COOH2+) due to the addition of a proton provides a more sensitive
differentiation of the strength of acids and shows that the order of decreasing strength
for acids is perchloric, hydrobromic, sulfuric, hydrochloric, and nitric.

Acetic acid reacts incompletely with water to form hydronium 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.

This so-called levelling 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, bases dissolved
in them become progressively weaker and the differences between bases are
accentuated. In order of decreasing strength, strong bases of value for non-aqueous
titrations are potassium methoxide, sodium methoxide, lithium methoxide, and
tetrabutylammonium hydroxide.

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 non-aqueous titration. Further, depending
upon which part of a compound is physiologically active, 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,
quarternary ammonium compounds, alkali salts of organic acids, alkali salts of 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. In the case of hydrochlorides of weak bases
not containing acetylatable groupings it is also possible to titrate in acetic anhydride
without the addition of mercuric acetate and using an indicator such as malachite green
or crystal violet. Titrations carried out in the presence of an excess of acetic anhydride
must be applied cautiously, however, since any reaction of the anhydride with the
substance being titrated may give rise to low results.

In the titration of a basic compound, a volumetric solution of perchloric acid in glacial

acetic acid is usually used, although perchloric acid in dioxan may be useful in special
cases. In the titration of an acidic compound, a volumetric solution of lithium methoxide
in a methanol-toluene solvent is often used. For many applications it is convenient to use
a solution of tetrabutylammonium hydroxide in toluene; sodium methoxide, formerly in
wide use, may often give rise to troublesome gelatinous precipitates.

Because of interference by carbon dioxide, solvents for acidic compounds must be

protected from excessive exposure to the atmosphere by a suitable cover or by an inert
atmosphere during the titration. A blank determination should be carried out and the
volume generally should not exceed 0.01 ml of a 0.1 mol/l titrant for each ml of solvent.

The end-point may be determined visually by colour change, or potentiometrically. If the

calomel reference electrode is used, it is advantageous to replace the aqueous potassium
chloride solution in the salt bridge with lithium perchlorate/acetic acid TS for titrations in
acidic solvents, or potassium chloride in methanol for titrations in basic solvents. It
should be recognized that certain indicators in common use (crystal violet, for example)
undergo a series of colour changes and, in establishing a non-aqueous titration method
for a particular use, care should be taken to ensure that the colour change specified as
the end-point of the titration corresponds to the maximum value of dE/dV (where E is the
electromotive force and V the volume of titrant) in a potentiometric titration of the
substance under consideration.

When using titrants prepared with solvents that may have a relatively high coefficient of
expansion, for example, glacial acetic acid, toluene, etc., care should be taken to
compensate for differences in temperature that may exist between the time the titrant is
used and that at which it was standardized.

Recommended procedure

Method A (for bases and their salts)

Prepare a solution as specified in the monograph or dissolve the substance being

examined in a suitable volume of glacial acetic acid R1, previously neutralized to crystal
violet/acetic acid TS, warming and cooling if necessary. Alternatively the titration blank
for the solvent and indicator may be established in a separate determination. When the
substance is a salt of a hydrohalic acid, add 10 ml of mercuric acetate/acetic acid TS.
When the end-point is determined visually by colour change, add 2-3 drops of crystal
violet/acetic acid TS, and titrate with perchloric acid of the specified concentration (mol/l)
to the appropriate colour change of the indicator. When a different indicator is specified
in the monograph, this indicator should also be used for the neutralization of the glacial
acetic acid R1, and mercuric acetate/acetic acid TS, and the standardization of the
titrant.

When the equivalence point is determined potentiometrically, the indicator is omitted and
neutralization of the solution and standardization of the titrant are also carried out
potentiometrically. A glass electrode and a saturated calomel cell (containing potassium
chloride (350 g/l) TS) as reference electrode, are used. The junction between the calomel
electrode and the titration liquid should have a reasonably low electrical resistance and
there should be a minimum of transfer of liquid from one side to the other. Serious
instability may result unless the connections between the potentiometer and the
electrode system are in accordance with the manufacturer's instructions.
When the temperature (t2) at which the titration is carried out differs from the
temperature (t1) at which the titrant was standardized, multiply the volume of the titrant
required by [1 + 0.001(t1 - t2)] and calculate the result of the assay from the corrected
volume.

Method B (for acids)

The titrant, solvent and (in the case where the end-point is determined visually) the
indicator to be used for each substance, are specified in the monograph.

Protect the solution and titrant from carbon dioxide of the atmosphere throughout the
determination. This may conveniently be done by replacing the air above the titration
liquid with nitrogen.

Dissolve the substance being examined in a suitable volume of the solvent previously
neutralized to the indicator, warming and cooling if necessary, or prepare a solution as
specified in the monograph. Titrate to the appropriate colour change of the indicator.
Carry out a blank determination and make any necessary corrections. The titrant is
standardized using the same solvent and indicator as specified for the substance.

When the equivalence point is established potentiometrically, the indicator is omitted and
neutralization of the solution and standardization of the titrant are also carried out
potentiometrically.

A glass electrode and a saturated calomel reference electrode in which the aqueous
potassium chloride (350 g/l) TS has been replaced by a saturated solution of potassium
chloride R in methanol R are used. The junction between the calomel electrode and the
titration liquid should have a reasonably low electrical resistance and there should be a
minimum of transfer of the liquid from one side to the other. Serious instability may
result unless the connections between the potentiometer and the electrode system are
made in accordance with the manufacturer's instruction.
USP 29
Caffeine

C8H10N4O2 (anhydrous) 194.19

1H-Purine-2,6-dione, 3,7-dihydro-1,3,7-trimethyl-.1,3,7-Trimethylxanthine [58-08-2].

Monohydrate 212.21 [5743-12-4].

» Caffeine is anhydrous or contains one molecule of water of hydration. It contains not less than
98.5 percent and not more than 101.0 percent of C8H10N4O2, calculated on the anhydrous basis.

Packaging and storage— Preserve hydrous Caffeine in tight containers. Preserve anhydrous Caffeine in well-closed
containers.

Labeling— Label it to indicate whether it is anhydrous or hydrous.

USP Reference standards 11 — USP Caffeine RS.

Identification—

A: Infrared Absorption 197M .

B: Dissolve about 5 mg in 1 mL of hydrochloric acid in a porcelain dish, add 50 mg of potassium chlorate, and
evaporate on a steam bath to dryness. Invert the dish over a vessel containing a few drops of 6 N ammonium
hydroxide: the residue acquires a purple color, which disappears upon the addition of a solution of 1 N sodium
hydroxide.

Melting range 741 : between 235 and 239 , determined after drying at 80 for 4 hours.

Water, Method III 921 — Dry it at 80 for 4 hours: the anhydrous form loses not more than 0.5% and the hydrous
form not more than 8.5% of its weight.

Residue on ignition 281 : not more than 0.1%.

Heavy metals, Method II 231 : 0.001%.

Readily carbonizable substances 271 — Dissolve 0.5 g in 5 mL of sulfuric acid TS: the solution has no more
color than Matching Fluid D.

Organic volatile impurities, Method I 467 : meets the requirements.

Other alkaloids— To 5 mL of a solution (1 in 50) add mercuric-potassium iodide TS: no precipitate is formed.
Chromatographic purity—

Mobile phase, System suitability preparation, Standard preparation, and Chromatographic system— Proceed as
directed in the Assay.

Test preparation— Use the Assay preparation.

Procedure— Inject a volume (about 10 µL) of the Test preparation into the chromatograph, record the chromatogram,
and measure all the peak responses. Calculate the percentage of each impurity in the portion of Caffeine taken by the
formula:
100(ri / rs),

in which ri is the peak response for each impurity, and rs is the sum of the responses of all the peaks: not more than
0.1%
of any individual impurity is found; and not more than 0.1% of total impurities is found.

Residual solvents 467 : meets the requirements.

Assay—

Mobile phase— Transfer about 1.64 g of anhydrous sodium acetate, accurately weighed, to a 2-L volumetric flask,
dissolve in and dilute with water to volume, and mix. Transfer 1910 mL of this solution to another 2-L volumetric flask,
add 50 mL of acetonitrile and 40 mL of tetrahydrofuran, and mix. Adjust with glacial acetic acid to a pH of 4.5, mix,
filter, and degas.

System suitability preparation— Transfer about 2 mg of theophylline, accurately weighed, to a 100-mL volumetric flask,
add about 50 mL of Mobile phase, shake, and sonicate, if necessary, to dissolve. Dilute with Mobile phase to volume,
and mix.

Standard preparation— Transfer an acccurately weighed quantity of about 5.0 mg of USP Caffeine RS to a 25-mL
volumetric flask, add 5.0 mL of System suitability preparation and 10 mL of Mobile phase, shake, and sonicate, if
necessary, to dissolve. Dilute with Mobile phase to volume, mix, and filter.

Assay preparation— Transfer about 10 mg of Caffeine, accurately weighed, to a 50-mL volumetric flask, add 10 mL of
Mobile phase, shake, and sonicate, if necessary, to dissolve. Dilute with Mobile phase to volume, mix, and filter.

Chromatographic system (see Chromatography 621 )— The liquid chromatograph is equipped with a 275-nm
detector and a 4.6-mm × 15-cm column containing packing L1. The flow rate is about 1 mL per minute. Chromatograph
the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times for
theophylline and caffeine are about 0.69 and 1.0, respectively; the resolution, R, between theophylline and caffeine is
not less than 6.0; the tailing factor for each of the peaks identified in the chromatogram is not more than 2.0; and the
relative standard deviation is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and measure the responses for the caffeine peaks. Calculate the
quantity, in mg, of C8H10N4O2 in the portion of Caffeine taken by the formula:

50C(rU / rS),

in which C is the concentration, in mg per mL, of USP Caffeine RS in the Standard preparation; and rU and rS are the
peak responses for caffeine obtained from the Assay preparation and the Standard preparation, respectively.