Colorimetric determination of isoniazid and its pharmaceutical formulations

COLORIMETRIC DETERMINATION OF ISQMAZID AND ITS PHARMACEUTICAL ~O~~~~ATI~N~

(Recefved 22 Septembw l%% &&ed

30 December 1992, &xp#t?d K? J*uv~wry1993)

Isoniazid (INH), the antitubercular drug has -G Wzw (Machamy and Nagei, Duren). The spots were dettxt@d with W at 254 nrn. brx determined by titrimetric~“J ultraviolet speotrophotometrie,4 Colorimetric,$‘4 fluorim rnetr$~~~polar~graphic,~~ paper or thin layer Fz~t red AL salt solutian, (A&&h Chemical ~~~rn~to~~~~~~~,~~ and HPLC’~ methods The ~rn~~~~ methods for the assay oi Co., InG, USA). A &2% (w/v] aqueous ~~u~~n ~~U~~d, in au~~ti~ and was rE& fr&Iy prepared an&Iprote&ed from invoIve HPI&i4 ~~~rn~~~~ su&ght~ ~~~~~~.l$ Sodium byd~~de~ 0.4% (w/v) and IO% (w/v) In the present work, two s~~t~~~hotornet~~ aqueous sohttions were used. Isoniazid (Analar grade) (BDH, PooIe, Dome& methods for the assay of isoniazid with fast red AL salt and DMBQ were described. The UK). 2,6-Dimethoxp 1&benzoquinone reagent, A methods are sensitive, rapid and adopted for the assay of isoniaxid in pure form and its 0.1% w/v ethanahc solution was used. It pharmaceutical formulations. was synthesized according to Hamblocki6 and Baker’? procedur@s.. Its purity and structure were eonfrrmed by TI,C! ~CHC~~~~H 9: I)* mp ~2~-~~4C~*~~ ‘IR and “If-NMR speotm IR Fig. I) reve&d the presence of ~ra~~~s~~ stretching bands* v&r., C-H (3070 and 294O@n& C+Q (1690 and f64O@m), c--C Infrared speetrophotometer (P&in-Elmer conjugated ~1~~~~~)~ C-Q-C (1260 and 298) using potassium bromide diso, 110S/cm)t in addition to one out of plane bend‘HNMR Spectra Varian EM 360~4(60 MHz). ing band at g80/cm corresponding to tetrasubme ‘II-NMR chemical shifts were measured stituted benzene, ‘H+NMR (Fig. 2) showed the against TMS =O.O ppm and cornpared with presence of six protons at S 3.86 ppm (singlet), TMS = CDC&-7.289 ppm. corresponding to the methoxy functions and M&ing points were determined on ““Buchi- two aromatic protons at 6 5.85 ppm (singlet). C&rat*’ in eapiI&ry tubes and were uneorreeti The Bp5eId shift of the Iatter protons was &er ~o~phy FLCJ was GarzGd att~~u~ to &e ~e~d~~g ~~~~~ effect of out using fhroreseen t sihca get ptates poIygram the o~~bo~~~ function.

1024

NADMM. A. M-z

and

M. EMm

WA

100

Oi 40°0

WELLENZAHL

I

1 Cbi’ I

25oo

I

2000

f

1800

I

1600

,

14on

I

I

1200

1CW

I

600

I

6W

I

4OC

Fig. 1. IR spectrum of DMBQ (KBr disc).

All solvents and reagents were of analytical grade (Merck).

50 mg of isoniazid, 5 mg of nicotinamide and 5 mg of vitamin $ per tablet.

Pharmaceutical preparations

Method

The following isoniazid commercial formulations were used:

Preparation of sample ~oZution~. Isoniazid powder: Weigh 50 mg isoniaxid and dissolve it in water and complete to 50 ml. Dilute this solution stepwise with water to obtain drug concentration 0.1 mg per ml. Use l-ml aliquot of this solution for the procedure (fast red AL salt-isoniazid complex). For DMB~soni~d

(I)-Isocid tablets (CID, Egypt) contain 50 mg of isoniaxid per tablet. (2~Isocid forte tablets (CID, Egypt) contain 200 mg of isoniazid per tablet. (3)-B&ibex tablets (Misr, Egypt) contain

0 OCH3

CH30 I 0

0

Fig. 2. ‘H-NMR

spectrum of DMBQ.

Calorimetric determination of isoniazid and its pharmaceutical formulations

of isoniazid was prepared similarly and diluted with the same solvent to obtain drug concentration 0.05 mg per ml. Use l-ml aliquot of this solution for the procedure. Tablets. Mix the contents of 20 tablets thoroughly. Weigh an amount equivalent to 50 mg of isoniazid into 50 ml calibrated flask, extract with 30 ml water (3 x 10 ml), filter and complete to 50 ml with water. Dilute this solution with water to contain 0.1 mg/ml of the drug. Use 1 ml of this solution for the procedure (for fast red AL salt-isoniazid complex). Similarly, for DMBQ-isoniazid complex, ethanolic solution of isoniazid tablets was diluted to obtain drug concentration 0.05 mg/ml and use 1 ml of this solution for the procedure. complex;

ethanolic

solution

Procedure

1025

06

:,,

‘..

200

I

I

I

I

I

I

I

I

I

I

2,O

260

320

360

400

440

460

520

560

600

Wavelength,

nm

Fig. 3. Absorption spectra of fast red AL salt-isoniazid complex (-) and fast red AL salt (-.-.--), final drug concentration; 8.4 pg/ml.

For fast red AL ~uZt-json~azideo~plex, To a 10 ml calibrated flask, add 1 ml of the sample solution, 2 ml of fast red AL salt solution, mix and stand for 10 min. Add 1.0 ml of sodium hydroxide solution (10% w/v). Make up to the mark with ethanol, mix and measure the absorbance at 510 nm against a reagent blank prepared similarly. For DMBQ-isoniazid

complex

Into 10 ml volumetric flask, transfer 1 ml of the sample solution, 1.5 ml of 2,6-dimethoxy1,~~~oq~none and 0.1 ml of sodium hydroxide solution (0.4% w/v). Mix the contents and leave for 10 min at room temperature (20 + 3°C). Dilute the mixture to volume with ethanol and measure the absorbance at 655 nm against a reagent blank treated similarly. RESULTS AND DISCUSSION

Absorption spectra

Isoniazid reacts with fast red AL salt in presence of sodium hydroxide in an aqueous ethanolic medium to form a red colour (& 510 nm). A green complex is formed when isoniazid is allowed to react with 2,6-dimethoxy- 1,4-benzoquinone in presence of sodium hydroxide in an aqueous ethanolic solution. The absorption

160

242

IL

JO4

f t

366

426

460

Wavelength,

Complex Fast red Al sak-isoniazid DMB~isonia~d

655 655

(1 .nwf~?? cm-‘) 8.5 x w 1.6 x t@

$11 614

i 676

736

600

nm

Fig. 4. Absorption spwtra of DMBQ-isoniazid complex (-) and DMBQ (-~-a-), final drug concentration; 7 Irgw.

spectra of the two complexes are shown in Figs 3 and 4. Their spectral characteristics are s~rn~~ in Table 1. Optimum conditions

To achieve maximum colour development, the reaction mixture of isoniazid and fast red AL salt must allowed to stand for 10 min before

Table 1. Spectral characteristics of the fast red AL salt-INH and DMBQ-INH &la= (nm)

I 552

INH Linear range &/ml) 2-15 t-10

Intera@ 0.0126 0.0105

complexes

Quantitative parameters Slope Correlat. coeff. 0.0589 0.0584

0.9993 0.9988

1026

NAD~AM. A. MAH~~UZand IC.u+rmM. EEMRA Table 4. Effect of diluting solvent on the coloured products

Solvent Methanol Ethanol Acetone Dimethyl formamide Isopropanol Dioxane -

0 55

510 510 505 505 510 495

*Final isoniazid concentration, tFina1 isoniazid concentration,

Fast Red AL salt DMsg

0.378 0.616 0.198 0.510 0.417 0.365

DMBQINH complext 1, A 655 655 660 665 660 650

0.435 0.589 0.230 0.354 0.451 0.073

10 &ml. 5 pg/ml.

‘:

t

Ttme,

rn~n

Fig. 5. Stability time of fast red AL salt-isoniaxid and DMBQ-isoniaxid complexes hnal drug concentrations 9.0 and 5.0 p/ml, respectively. Table 2. Effect of the amount of the reagents on the coloured complexes Volume of reagent (ml)

Fast red AL salt-INH complex* A 510

0.5 1.0 1.5 2.0 2.5 3.0

0.179 0.354 0.512 0.626 0.616 0.600

*Final drug concentration, tFina1 drug concentration,

DMBQINH complext A 655 0.268 0.473 0.585 0.576 0.535 0.515

10 pg/ml. 5 pg/ml.

addition of sodium hydroxide solution. The formed red colour was stable for 20 min at room temperature (20 Ifi 3°C) (Fig. 5). For complete formation of DMBeisoniazid complex, it is necessary to stand for 10 min, in presence of sodium hydroxide, before dilution. The maximum absorbance readings remain constant for -20 min (Fig. 5). Table 3. Effect of sodium hydroxide concentration colour intensity Fast red AL salt-INH complex*

DMBQ-INH complex?

10% sodium hydroxide A ml (ml)

0.4% sodium hydroxide A 6SJ (ml)

0.5 1.0 1.5 2.0 2.5 3.0

Fast red AL saltINH complex* I OzaX A

0.508 0.616 0.600 0.580 0.550 0.520

*Final isoniaxid concentration, tFina1 isoniaxid concentration,

0.025 0.050 0.100 0.150 0.200 0.300 10 pg/ml. 5 pg/ml.

0.501 0.534 0.585 0.568 0.530 0.477

on

It is clear from the data reported in Table 2 that isoniazid needs 2 ml of 0.2% fast red AL salt or 1.5 ml of 0.1% 2,6-dimethoxy-1,4benzoquinone for the reactions to complete. Different alkalies were tested for both methods, sodium hydroxide is the best one and 1 ml of 10% sodium hydroxide and 0.1 ml of 0.4% sodium hydroxide are recommended for the formation of fast red AL salt-isoniazid and DMBQ-isoniazid complexes, respectively (Table 3). Methanol, ethanol, isopropanol, acetone, dimethyl formamide and dioxane were tested as diluting solvents in the two methods. The results revealed that ethanol was the best solvent (Table 4).

No interference was observed from the presence of other drugs, vitamins, commonly encountered excipients and additives when isoniazid was determined by the two methods (Table 5). Quantzpcation Beer’s law holds good over the ranges 2-15 and l-10 pg/ml by fast red AL salt-INH and

Table 5. Determination of isoniaxid in the presence of other drugs, vitamins and excipients Recovery (% f SD)t

Substance

Amount (mg)

Fast red AL salt-INH DMBQ-INH complex complex

5 99.9 f 1.04 100.7 io.95 Pyridoxine 50 99.7 f 1.29 99.9 f 0.85 Nicotinamide 10 100.3 f 1.11 99.6* 1.12 Glucose 10 99.3 f 0.96 99.8 f 0.91 Lactose 10 99.4 f 0.86 100.4 f 0.92 Gum acacia 20 99.9 f 0.55 99.1 f 0.89 Magnesium stearate Microcrystalline cellulose 20 100.0 f 0.78 100.4 f 0.78 20 98.8 f 1.03 99.4 f 0.93 Starch *Added per 10 mg isoniaxid. tAverage of five determinations.

Calorimetric determination of isoniazid and its pharmaceutical formulations Table 6. Assay of isoniazid powder by the suggested and reported calorimetric methods Fast red AL salt-INH complex

Isoniazid, m&t

Table 7. Determination of isoniazid and its pharmaceutical preparations Recovery (% f SD)*

DMBQ-INH complex

Reported method*

10

100.7

98.8

100.9

Sample

:: 40

99.3 98.9 100.2

101.8 99.6 99.9

98.5 98.6 101.1

lsoniazid

101.4 99.8 100.1 iO.84

100.5 100.1 100.1 f0.92

E 99.6; 1.08

z Mean&SD

1027

Fast red AL salt-isoniazid complex

*Ref. 6. tAverage of three experiments.

Isocid forte tablets11

DMBQ-INH complexes, respectively. Correlation coefficient, intercepts and slopes for the calibration data of isoniazid by the two suggested methods are given in Table 1. To examine the precision of both procedures, eight replicate a~~aly~es were performed on the same solution containing 10 and 5 fig per ml of the drug, and relative standard deviations of 1.43*/a and 1.55% were obtained by fast red AL salt-INH and DMBQ-INH complexes, respectively.

o~C-NH-NH 2

tl

I&ibex tablets11

;zSoffive

Official method?

99.8 f 0.88 F$ = 1.12

100.1 f 1.26 Ff = 2.31

99.7 f 0.83

100.8 f§ = f 0.26 1.137 F$ = 1.07 t$=0.19 100.8 & 1.21 F$ = 1.26 rg =0.15 99.2 + 1.29 F$ = 1.05 tg = 0.22

101.0 tg + = 0.69 1.18 F$= 1.00 r$ = 0.07 100.9 f 1.29 F$ = 1.11 14 = 0.02 99.2 f 1.31 F$ = 2.02 t$ = 0.29

100.9 + 1.18

powder

Is&d tablets11

DMBQisoniazid complex

Ffor (4,4) degrees of freedom at P = 0.05 is 6.39. t for 4 degrees of freedom at P = 0.05 is 2.776.

$Tklakd §Tab~t~

i/~ebtis in submental &ion.

APPiications The proposed procedures were used to determine isoniazid in authentic and pharmaceutical

0

l&NET

+

N

II

f

lf===N+NII--N--f-O

O--~

q 0

99.0 + 1.31

dete~inations.

3

I

100.9 + 1.36

I eN-I r,3

. . . . . . .. . . . . . -N-C-O

N

0 III Scheme 1.

0 III

i

1028

NADIAM. A. MAHFOUZand KAMLAM. Em

preparations. The results obtained are comparable to those given by the reported and official methods, Tables 6 and 7. Student t- and F-test show no significant differences between the proposed, and official methods. In addition, the suggested methods have the advantages of rapidity and simplicity.

quinone and hydroquinone (charge-transfer or donor-acceptor complex).‘* In addition, it was reportedi that aryl hydrazines are easily oxidized by benzoquinone (Q) to give aryldiimide and hydroquinone (QHJ”

Reaction involved

Moreover, isoniazid as hydrazine derivative can be attacked by oxidizing agents especially in presence of alkali solution.” Consequently the mechanism of formation of the green coloured product when isoniazid I reacts with DMBQ II may proceed through reduction of II into hydroquinone derivative III and formation of quinone: hydroquinone charge transfer or donor-acceptor complex IV (Scheme 2). A preliminary chemical test to confirm the formation of hydroquinone derivative as a by-product was carried out by addition of few drops of ferric chloride T.S., disappearance of the green colour took place. This indicates the decomposition of the complex with oxidation of the hydroquinone derivative into 2,6dimethoxy-1,Cbenzoquinone with formation of a yellow colour. In addition, the continuous molar variation for the reaction between isoniazid and DMBQ reveals that the interaction between these two compounds occurs at a ratio of 1:2. This provides further evidence for the suggested mechanism shown in Scheme 2. All attempts to separate the green coloured product IV failed.

Fast red AL salt (I), in presence of sodium hydroxide, reacts with isoniazid (II) (molar ratio 1: 1) to form a red colour with maximum absorption at 510 nm in aqueous ethanolic medium. The mechanism of the reaction may be interpreted as shown in Scheme 1. A characteristic green coloured product (L = 655 nm) is formed when isoniazid is allowed to react with DMBQ in presence of sodium hydroxide in aqueous medium. Under the experimental conditions, the suggestion that acid hydrazides form hydrazones with the carbonyl function of the reagent is excluded due to the failure of some aromatic amines, e.g. p-aminophenol, sulfonamide, dapsone, hydroxylamine and semicarbazide to give green colour with DMBQ. This negative response reveals that under a mild reaction condition no azomethine derivatives are formed. However, quinones are easily reduced to hydroquinone derivatives by different reducing agents, e.g. hydrazine. ‘*J’ It was reported” that reduction of p-benzoquinone in alkaline medium give a deep green compound which formed from equimolar amount of p-benzo-

Ph-NHNH, + Q ---, Ph-N = NH + QH2

0

OH N

Scheme 2.

Calorimetric determination of isoniazid and its pharmaceutical formulations REFERENCES 1. U. Muralikrishna and K. Subrahmanyam, Indian Drugs, 1984, 21, 356. 2, B. B. Karamkar, J. Inst. Clrem. (India), 1983, 55, 153. 3. M. Sarwar, A. Malik and U. A. Khan, Anal. Lerr., 1989, 22, 853. 4. B. N. Sarkar. Indian J. Piillrm., 1972.24, 58. 5. Z. H. Mohamed, L. El-Sayed and A. M. Wahbi, Egypt. J. Pharm. Sci., 1989, 30, 43. 6. M. E. El-Kommos and A. S. Yank, A~~fysr(~ndon), 1988, 113, 1091. 7. A. Abou-Ouf, A. Taha, and M. Saidham, J. Pharm. Sci., 1973, 62, 1700. 8. L. P. Pavlyuchenkova and M. A. Veksler, Farmatsiga (Mosco), 1974, 23, 29 through Ref. 17. 9. P. C. Ioannou, Tahmta, 1987, 34, 857. 10. J. J. Vallon, A. Badinand and C. Bichon, Anal. Chim. Acta, 1975, 78, 93.

1029

11. L. Reio, J. Chromatogr., 1970, 47, 60. 12. R. M. De Sagher, A. P. De Leenheer and A. E. Claeys, J. Chromatogr., 1975, 106, 357. 13. J. T. Stewart, I. L. Honigberg, J. P. Brant, W. A. Murray, J. L. Webb and J. B. Smith, J. Pharm. Sci., 1976,65, 1536. 14. United States Pharmacopeia XXII, National Formulary XXZ, pp. 728, 729. US Pharmacopeial Convention, Rockville, MD, 1990. IS. British P~~co~~~ 1988, pp. 317, 812 and 957. HMSO, London, 1988. 16. H. Hamblock, Ph. D. Thesis Bonn 1982. 17. W. Baker, J. Chem. Sot., 1941, 662. 18. H. J. Roth, K. Eger and R. Torschiitz, Arzneistof analyse. Readtivitat-Stubilitat-Analytik., p. 79. George Thieme Verlag, Stuttgart, 1981. 19. Hans-Dieter Becker, in The Chemistry of Quinonoid Compound, S. Patai (ed.) Part 1, p. 403. John Wiley, London, 1974.