British Pharmacopoeia Volume I & II

Monographs: Medicinal and Pharmaceutical

Substances

Isoniazid

General Notices

(Ph. Eur. monograph 0146)

C6H7N3O    137.1    54-85-3

Action and use

Antituberculosis drug.

Preparations

Isoniazid Injection

Isoniazid Oral Solution

Isoniazid Tablets

Ph Eur

DEFINITION

Isoniazid contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent

of pyridine-4-carbohydrazide, calculated with reference to the dried substance.

CHARACTERS

A white or almost white, crystalline powder or colourless crystals, freely soluble in water, sparingly

soluble in alcohol.

IDENTIFICATION

First identification  A, B.

Second identification  A, C.

A. Melting point ( 2.2.14 ) : 170 °C to 174 °C.

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

obtained with isoniazid CRS.

C. Dissolve 0.1 g in 2 mL of water R and add 10 mL of a warm 10 g/L solution of vanillin R. Allow

to stand and scratch the wall of the test tube with a glass rod. A yellow precipitate is formed,

which, after recrystallisation from 5 mL of alcohol (70 per cent V/V) R and drying at 100 °C to

105 °C, melts ( 2.2.14 ) at 226 °C to 231 °C.

TESTS

Solution S

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

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 8.0.

Hydrazine and related substances

Examine by thin-layer chromatography ( 2.2.27 ) , using silica gel GF254 R as the coating substance.

Test solution  Dissolve 1.0 g of the substance to be examined in a mixture of equal volumes of

acetone R and water R and dilute to 10.0 mL with the same mixture of solvents.

Reference solution  Dissolve 50.0 mg of hydrazine sulfate R in 50 mL of water R and dilute to

100.0 mL with acetone R. To 10.0 mL of this solution add 0.2 mL of the test solution and dilute to

100.0 mL with a mixture of equal volumes of acetone R and water R.

Apply separately to the plate 5 µL of each solution and develop over a path of 15 cm using a

mixture of 10 volumes of water R, 20 volumes of acetone R, 20 volumes of methanol R and 50

volumes of ethyl acetate R. Allow the plate to dry in air 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 (0.2 per

cent). Spray the plate with dimethylaminobenzaldehyde solution R1. Examine in daylight. An

additional spot, corresponding to hydrazine, appears in the chromatogram obtained with the

reference solution. Any corresponding spot in the chromatogram obtained with the test solution is

not more intense than the spot corresponding to hydrazine in the chromatogram obtained with the

reference solution (0.05 per cent).

Heavy metals ( 2.4.8 )

2.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 2 mL of

lead standard solution (10 ppm Pb) R.

Loss on drying ( 2.2.32 )

Not more than 0.5 per cent, determined on 1.00 g by drying in an oven at 105 °C.

Sulfated ash ( 2.4.14 )

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

ASSAY

Dissolve 0.250 g in water R and dilute to 100.0 mL with the same solvent. To 20.0 mL of the

solution add 100 mL of water R, 20 mL of hydrochloric acid R, 0.2 g of potassium bromide R and

0.05 mL of methyl red solution R. Titrate dropwise with 0.0167 M potassium bromate, shaking

continuously, until the red colour disappears.

1 mL of 0.0167 M potassium bromate is equivalent to 3.429 mg of C6H7N3O.

British Pharmacopoeia Volume III

Formulated Preparations: Specific Monographs

Isoniazid Tablets

General Notices

Action and use

Antituberculosis drug.

DEFINITION

Isoniazid Tablets contain Isoniazid.

The tablets comply with the requirements stated under Tablets and with the following

requirements.

Content of isoniazid, C6H7N3O

95.0 to 105.0% of the stated amount.

IDENTIFICATION

Shake a quantity of the powdered tablets containing 0.1 g of Isoniazid with 10 mL of ethanol (96%)

for 15 minutes, centrifuge and decant the supernatant liquid. Extract the residue with two further

10-mL quantities of ethanol (96%) and evaporate the combined extracts to dryness. The infrared

absorption spectrum of the residue, Appendix II A, is concordant with the reference spectrum of

isoniazid (RS 196).

TEST

Dissolution

Comply with the requirements for Monographs of the British Pharmacopoeia in the dissolution test

for tablets and capsules, Appendix XII B1.

TEST CONDITIONS

(a) Use Apparatus 1, rotating the basket at 100 revolutions per minute.

(b) Use 900 mL of water, at a temperature of 37°, as the medium.

PROCEDURE

After 45 minutes withdraw a 10 mL sample of the medium and measure the absorbance of the

filtered sample, suitably diluted if necessary, at the maximum at 263 nm, Appendix II B.

DETERMINATION OF CONTENT

Calculate the total content of isoniazid, C6H7N3O, in the medium taking 307 as the value of A(1%,

1 cm) at the maximum at 263 nm.

ASSAY

Weigh and powder 20 tablets. Dissolve a quantity of the powder containing 150 mg of Isoniazid as

completely as possible in water, filter and wash the residue with sufficient water to produce

250 mL. To 50 mL of the resulting solution add 50 mL of water, 20 mL of hydrochloric acid and

0.2 g of potassium bromide and titrate with 0.0167M potassium bromate VS determining the end

point electrometrically. Each mL of 0.0167M potassium bromate VS is equivalent to 3.429 mg of

C6H7N3O.

STORAGE

Isoniazid Tablets should be protected from light.

USP monographs

Isoniazid

(eye'' soe nye' a zid).

C6H7N3O 137.14

4-Pyridinecarboxylic acid, hydrazide.

Isonicotinic acid hydrazide [54-85-3].

» Isoniazid contains not less than 98.0 percent and not more than 102.0 percent of C6H7N3O,

calculated on the dried basis.

Packaging and storage—Preserve in tight, light-resistant containers. Store at 25 , excursions

permitted between 15 and 30 .

USP REFERENCE STANDARDS 11 —

USP Isoniazid RS

Identification—

A: Infrared Absorption 197K .

B: Transfer about 50 mg of it to a 500-mL volumetric flask, add water to volume, and mix. Transfer

10.0 mL of the resulting solution to a 100-mL volumetric flask, add 2.0 mL of 0.1 N hydrochloric

acid, dilute with water to volume, and mix to obtain a 1 in 100,000 solution: the UV absorption

spectrum of the solution so obtained exhibits maxima and minima only at the same wavelengths

as that of a similar solution of USP Isoniazid RS, concomitantly measured.

MELTING RANGE 741 : between 170 and 173 .

PH 791 : between 6.0 and 7.5, in a solution (1 in 10).

LOSS ON DRYING 731 —Dry it at 105 for 4 hours: it loses not more than 1.0% of its weight.

RESIDUE ON IGNITION 281 : not more than 0.2%.

HEAVY METALS, Method II 231 : 0.002%.

Assay—

Mobile phase—Dissolve 4.4 g of docusate sodium in 600 mL of methanol, add 400 mL of water,

adjust with 2 N sulfuric acid to a pH of 2.5, and mix. Make adjustments if necessary (see System

Suitability under Chromatography 621 ).

Standard preparation—Dissolve an accurately weighed quantity of USP Isoniazid RS in Mobile

phase, and quantitatively dilute with Mobile phase to obtain a solution having a known

concentration of about 0.32 mg per mL.

Assay preparation—Transfer about 16 mg of Isoniazid, accurately weighed, to a 50-mL volumetric

flask, dissolve in and dilute with Mobile phase to volume, and mix.

Chromatographic system (see CHROMATOGRAPHY 621 )—The liquid chromatograph is equipped

with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is

about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak

responses as directed for Procedure: the column efficiency determined from the isoniazid peak is

not less than 1800 theoretical plates; the tailing factor for the isoniazid peak is not more than 2.0;

and the relative standard deviation for replicate injections 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 major peaks. Calculate the quantity, in mg, of C6H7N3O in the portion of Isoniazid

taken by the formula:

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, respectively.

Auxiliary Information— Please check for your question in the FAQs before contacting USP.

Topic/Question Contact Expert Committee

Monograph Behnam Davani, Ph.D., M.B.A. Senior Scientific Liaison1-301-816-8394

(SM12010) Monographs - Small Molecules 1

Reference Standards

RS Technical Services1-301-816-8129rstech@usp.org

USP34–NF29 Page 3203

Pharmacopeial Forum: Volume No. 29(6) Page 1912

Chromatographic Column—

ISONIAZID

Chromatographic columns text is not derived from, and not part of, USP 34 or NF 29.

Isoniazid Tablets

» Isoniazid Tablets contain not less than 90.0 percent and not more than 110.0 percent of the

labeled amount of isoniazid (C6H7N3O).

Packaging and storage—Preserve in well-closed, light-resistant containers.

USP REFERENCE STANDARDS 11 —

USP Isoniazid RS

Identification—

A: The retention time of the isoniazid peak in the chromatogram of the Assay preparation

corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

B: Transfer a portion of finely powdered Tablets, equivalent to about 50 mg of isoniazid, to a 500-

mL volumetric flask. Add water to volume, mix, and filter a portion of the mixture. Proceed as

directed in Identification test B under Isoniazid, beginning with “Transfer 10.0 mL of the resulting

solution to a 100-mL volumetric flask.”

DISSOLUTION 711 —

Medium: 0.01 N hydrochloric acid; 900 mL.

Apparatus 1: 100 rpm.

Time: 45 minutes.

Procedure—Determine the amount of C6H7N3O dissolved by employing UV absorption at the

wavelength of maximum absorbance at about 263 nm on filtered portions of the solution under

test, suitably diluted with Dissolution Medium, in comparison with a Standard solution having a

known concentration of USP Isoniazid RS in the same Medium.

Tolerances—Not less than 80% (Q) of the labeled amount of C6H7N3O is dissolved in 45 minutes.

UNIFORMITY OF DOSAGE UNITS 905 : meet the requirements.

Procedure for content uniformity—Transfer 1 finely powdered Tablet to a 500-mL volumetric flask

with the aid of 200 mL of water. Shake by mechanical means for 30 minutes, add water to volume,

and mix. Filter, and discard the first 20 mL of the filtrate. Dilute a portion of the filtrate quantitatively

and stepwise, if necessary, with a 3 in 100 mixture of 0.1 N hydrochloric acid and water to obtain a

solution containing about 10 µg per mL. Dissolve an accurately weighed quantity of USP Isoniazid

RS in a volume of water corresponding to that used to dissolve a similar amount of isoniazid from

the Tablet, and dilute quantitatively and stepwise, if necessary, with a 3 in 100 mixture of 0.1 N

hydrochloric acid and water to obtain a Standard solution having a known concentration of about

10 µg per mL. Concomitantly determine the absorbances of both solutions in 1-cm cells at the

wavelength of maximum absorbance at about 263 nm, with a suitable spectrophotometer, using

water as the blank. Calculate the quantity, in mg, of isoniazid (C6H7N3O) in the Tablet taken by the

formula:

(TC / D)(AU / AS)

in which T is the labeled quantity, in mg, of isoniazid in the Tablet; C is the concentration, in µg per

mL, of USP Isoniazid RS in the Standard solution; D is the concentration, in µg per mL, of

isoniazid in the solution from the Tablet, based on the labeled quantity per Tablet and the extent of

dilution; and AU and AS are the absorbances of the solution from the Tablet and the Standard

solution, respectively.

Assay—

Buffer solution—Prepare a 0.1 M monobasic potassium phosphate solution, adjust with 10 N

sodium hydroxide to a pH of 6.9, add sufficient triethanolamine to obtain a solution having a known

concentration of 0.2 mM of triethanolamine, and mix.

Mobile phase—Prepare a filtered and degassed mixture of Buffer solution and methanol (95:5).

Make adjustments if necessary (see System Suitability under Chromatography 621 ).

Standard preparation—Dissolve an accurately weighed quantity of USP Isoniazid RS in Mobile

phase, and dilute quantitatively, and stepwise, if necessary, with Mobile phase to obtain a solution

having a known concentration of about 0.32 mg per mL.

Assay preparation—Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately

weighed portion of the powder, equivalent to 32 mg of isoniazid, to a 100-mL volumetric flask, add

40 mL of Mobile phase, and sonicate for 10 minutes. Cool to room temperature, dilute with Mobile

phase to volume, and centrifuge for 5 minutes.

Chromatographic system (see Chromatography 621 )—The liquid chromatograph is equipped

with a 254-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is

about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak

responses as directed for Procedure: the capacity factor, k¢, is not less than 2.35; the column

efficiency is not less than 1800 theoretical plates; the tailing factor is not more than 1.5; and the

relative standard deviation for replicate injections is not more than 1.0%.

Procedure—Separately inject equal volumes (about 20 µL) of the Standard preparation and the

Assay preparation into the chromatograph, record the chromatograms, and measure the

responses for the major peaks. Calculate the quantity, in mg, of isoniazid (C6H7N3O) in the portion

of Tablets taken by the formula:

100C(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 obtained from the Assay preparation and the Standard

preparation, respectively.

Auxiliary Information— Please check for your question in the FAQs before contacting USP.

Topic/Question Contact Expert Committee

Monograph Behnam Davani, Ph.D., M.B.A.

(SM12010) Monographs - Small Molecules 1

Topic/Question Contact Expert Committee

Senior Scientific Liaison1-301-816-8394

Reference Standards

RS Technical Services1-301-816-8129rstech@usp.org

711 Margareth R.C. Marques, Ph.D. Senior Scientific Liaison1-301-816-8106

(GCDF2010) General Chapters - Dosage Forms

USP34–NF29 Page 3204

Chromatographic Column—

ISONIAZID TABLETS

Chromatographic columns text is not derived from, and not part of, USP 34 or NF 29.