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~wry 1993)

Isoniazid (INH), the antitubercular drug has brx determined by titrimetric~“J ultraviolet speotrophotometrie,4 Colorimetric,$‘4 fluorim rnetr$~~~ polar~graphic,~~ paper or thin layer ~~~rn~to~~~~~~~,~~ and HPLC’~ methods The ~rn~~~~ methods for the assay oi ~~U~~d, in au~~ti~ and invoIve HPI&i4 ~~~rn~~~~ ~~~~~~.l$

In the present work, two s~~t~~~hotornet~~ methods for the assay of isoniazid with fast red AL salt and DMBQ were described. The methods are sensitive, rapid and adopted for the assay of isoniaxid in pure form and its pharmaceutical formulations.

Infrared speetrophotometer (P&in-Elmer 298) using potassium bromide diso,

‘HNMR Spectra Varian EM 360~4 (60 MHz). me ‘II-NMR chemical shifts were measured against TMS =O.O ppm and cornpared with TMS = CDC&-7.289 ppm.

M&ing points were determined on ““Buchi- C&rat*’ in eapiI&ry tubes and were uneorreeti

&er ~o~phy FLCJ was GarzGd out using fhroreseen t sihca get ptates poIygram

-G Wzw (Machamy and Nagei, Duren). The spots were dettxt@d with W at 254 nrn.

Fz~t red AL salt solutian, (A&&h Chemical Co., InG, USA). A &2% (w/v] aqueous ~~u~~n was rE& fr&Iy prepared an&I prote&ed from su&ght~

Sodium byd~~de~ 0.4% (w/v) and IO% (w/v) aqueous sohttions were used.

Isoniazid (Analar grade) (BDH, PooIe, Dome& UK).

2,6-Dimethoxp 1 &benzoquinone reagent, A 0.1% w/v ethanahc solution was used. It 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 conjugated ~1~~~~~)~ C-Q-C (1260 and 1 10S/cm)t in addition to one out of plane bend- ing band at g80/cm corresponding to tetrasub- stituted benzene, ‘H+NMR (Fig. 2) showed the presence of six protons at S 3.86 ppm (singlet), corresponding to the methoxy functions and two aromatic protons at 6 5.85 ppm (singlet). The Bp5eId shift of the Iatter protons was att~~u~ to &e ~e~d~~g ~~~~~ effect of the o~~bo~~~ function.

1024

100

NADM M. A. M-z and WA M. EMm

Oi I I f I , I I I I I

40°0 WELLENZAHL 1 Cbi’ I 25oo 2000 1800 1600 14on 1200 1CW 600 6W 4OC

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

All solvents and reagents were of analytical 50 mg of isoniazid, 5 mg of nicotinamide and grade (Merck). 5 mg of vitamin $ per tablet.

Pharmaceutical preparations

The following isoniazid commercial formulations were used:

Method

(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

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

0

CH30

0

OCH3

I

0

Fig. 2. ‘H-NMR spectrum of DMBQ.

Calorimetric determination of isoniazid and its pharmaceutical formulations 1025

complex; ethanolic solution 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.

Procedure

For fast red AL ~uZt-json~azid eo~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-dimethoxy- 1 ,~~~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

06

:,,

‘.. I I I I I I I I I I

200 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.

IL f t I $11 i 160 242 JO4 366 426 460 552 614 676 736 600

Wavelength, 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 complexes

INH &la= Linear range (nm) (1 .nwf~?? cm-‘) &/ml)

Quantitative parameters Complex Intera@ Slope Correlat. coeff.

Fast red Al sak-isoniazid 655 8.5 x w 2-15 0.0126 0.0589 0.9993 DMB~isonia~d 655 1.6 x t@ t-10 0.0105 0.0584 0.9988

1026 NAD~A M. A. MAH~~UZ and IC.u+rm M. EEMRA

0 55

t

- Fast Red AL salt DMsg

‘:

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 Fast red AL DMBQINH of reagent salt-INH complex* complext

(ml) A 510 A 655

0.5 0.179 0.268 1.0 0.354 0.473 1.5 0.512 0.585 2.0 0.626 0.576 2.5 0.616 0.535 3.0 0.600 0.515

Table 4. Effect of diluting solvent on the coloured products

Solvent

Fast red AL salt- DMBQINH INH complex* complext I OzaX A 1, A

Methanol 510 0.378 655 0.435 Ethanol 510 0.616 655 0.589 Acetone 505 0.198 660 0.230 Dimethyl formamide 505 0.510 665 0.354 Isopropanol 510 0.417 660 0.451 Dioxane 495 0.365 650 0.073

*Final isoniazid concentration, 10 &ml. tFina1 isoniazid concentration, 5 pg/ml.

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-1,4benzo- quinone 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).

*Final drug concentration, 10 pg/ml. tFina1 drug concentration, 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 on colour intensity

Fast red AL salt-INH complex*

10% sodium hydroxide (ml) A ml

DMBQ-INH complex?

0.4% sodium hydroxide (ml) A 6SJ

0.5 0.508 0.025 0.501 1.0 0.616 0.050 0.534 1.5 0.600 0.100 0.585 2.0 0.580 0.150 0.568 2.5 0.550 0.200 0.530 3.0 0.520 0.300 0.477

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

Fast red AL Amount salt-INH DMBQ-INH

Substance (mg) complex complex

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

*Final isoniaxid concentration, 10 pg/ml. *Added per 10 mg isoniaxid. tFina1 isoniaxid concentration, 5 pg/ml. tAverage of five determinations.


Table 6. Assay of isoniazid powder by the suggested and Table 7. Determination of isoniazid and its pharmaceutical reported calorimetric methods preparations

Fast red AL Recovery (% f SD)* Isoniazid, salt-INH DMBQ-INH Reported

m&t complex complex method* Fast red AL DMBQ- salt-isoniazid isoniazid Official

10 100.7 98.8 100.9 Sample complex complex method?

:: 99.3 98.9 101.8 99.6 98.5 98.6 lsoniazid 99.8 f 0.88 100.1 f 1.26 99.7 f 0.83 40 100.2 99.9 101.1 powder F$ = 1.12 Ff = 2.31

z 101.4 99.8 100.1 100.5 E Is&d 100.8 f§ = f 0.26 1.137 101.0 tg + = 0.69 1.18 100.9 + 1.18 Mean&SD 100.1 iO.84 100.1 f0.92 99.6; 1.08 tablets11 F$ = 1.07 F$= 1.00

t$=0.19 r$ = 0.07 *Ref. 6. Isocid forte 100.8 & 1.21 100.9 f 1.29 100.9 + 1.36 tAverage of three experiments. tablets11 F$ = 1.26 F$ = 1.11

rg =0.15 14 = 0.02 I&ibex 99.2 + 1.29 99.2 f 1.31 99.0 + 1.31

DMBQ-INH complexes, respectively. Cor- tablets11 F$ = 1.05 F$ = 2.02

relation coefficient, intercepts and slopes for tg = 0.22 t$ = 0.29

the calibration data of isoniazid by the two suggested methods are given in Table 1.

;zSoffive dete~inations.

To examine the precision of both procedures, $Tklakd Ffor (4,4) degrees of freedom at P = 0.05 is 6.39.

eight replicate a~~aly~es were performed on the §Tab~t~ t for 4 degrees of freedom at P = 0.05 is 2.776. i/~ebtis in submental &ion.

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 APPiications AL salt-INH and DMBQ-INH complexes, The proposed procedures were used to deter- respectively.

o~C-NH-NH

tl 2

3 +

N

I

mine isoniazid in authentic and pharmaceutical

0 l&NET

II

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

q O--~ 0 I 0

III

. . . . . . . . . . . . . -N-C-O eN-I r,

3 N

0

III

Scheme 1.

1028 NADIA M. A. MAHFOUZ and KAMLA M. Em

preparations. The results obtained are compar- able 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.

Reaction involved

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-amino- phenol, 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-

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”

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

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 oxi- dation of the hydroquinone derivative into 2,6- dimethoxy-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.

0 OH

N

Scheme 2.


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.

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.

colorimetric determination of isoniazid and its pharmaceutical formulations

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