CHAPTER 2

ASSAY OF SULFONAMIDE DRUGS*A Brief review

2.1 ASSAY OF SULFONAMIDES

The quantitative determination of the sulfonamide drugs

is based on the chemical property as well as the nature of

the functional groups in the drug. One of the

official methods for the quantitative determination is

that of nitritometry. This method is based on the ability

of the drugs to get diazotized. NaN02 is used as the

standard titrant. The end point is detected with

Iodine-starch paper as an external indicator or with the

aid of tropeolin oo asinternal indicator. The

titration is also carried out potentiometrlcally. The

reaction can be represented as follows.

/-- \ H NaN02 + H
h2n-( O /- S°2-N R + -----31
HCl
N Cl

2NaN02 + 2KI + 4HC1 -- * I2 + 2NaCl + 2KCl + 2NOf +2^0

The other methods used include

a) Neutralization with NaOH using thymolphthaplein

as indicator. The imide group hydrogen is neutralized

with the alkali in this method.

H2n/o\s02A + NaOH -- > n2n/o\so2 - h - R + h20

b) The sodium salts of the sulfonamides (imide hydrogen

being replaced by sodium) are titrated with a standard acid

solution in alchohol-acetone medium in presence of methyl

orange as indicator

9
 a H
H2N
-{oy
c)
so2k
R + HCl

The drugs are also

-- > H2N

determined
S02ft - R + NaCl

by bromatometric

method using KBrC>3 as titrant. The titration is carried out

in acetic acid medium in presence of KBr. Excess Br2

is determined Iodometrically

KBr03 + 5KBr + 6HC1—»3Br2 + 6KC1 + 3H20

By'

Br2 + 2KI -- > I2 + 2KBr

I2 + 2Na2S203 --- > 2 Na I + Na2S40g

d) Photo colorimetric methods too are also used for

the determination. These methods are basically based on the

ability of the drugs to yield

i) Colored product (Schiff’s base) on reaction

with carboryl compounds,ii) to form colored salts with

heavy metal salts like those of Cu and Co. Azo dye

formation reactions involving diazotization followed by

coupling with phenols or amines are also employed in the

determinations. For example, CUSO4 , CoCl2 and dimethyl

p-amino benzaldehyde are respectively used in the

determinations by the methods described above.

10
2.2 BRIEF REVIEW OF PAST WORK
The assay methods based on the reactions mentioned
above as well as by other methods reported in the
literature during the last two decades are briefly
reviewed in the pages that follow
i) Methods based on azo-dye formations
Azo-dye formation involving the diazotization of the

free amino group of the drug and the subsequent

coupling of the diazonium salt formed with different
organic compounds has been extensively utilized for the

assay of sulfonamides. A brief account of the

important reports made in the literature is presented
below.
Ivakhnenko, P.N. et al [3] reported a photometric
method for the determination of drugs containing primary
aromatic amino group. This involved diazotization of
the amino group in presence of ethacridine lactate.
Zoltan and Co workers 143 reported that sulfonamides can
be determined by Nitritometric methods. In these
methods, the drugs were diazotized and coupled with
Tropeolin oo. A photometric method of determination of
sulfanilamide in complex dosage forms through the
treatment of the drug with Na NO2 and JJ - naphthol in

alkaline medium was reported by POPOV, D.M and other [51.

R.T. Sane, V.G. Nayak, and V.B. Maler [6] reported that
the sulfa drugs can be colorimetrically determined
by measuring the absorbance of the orange-yellow species

11
produced, when the drugs are coupled with diazotized p-

amino ethyl benzoate in trimethyl amine medium. The

absorbance was measured at 460 nm. Sane, R.T., Hishra,

P.D., Lodage, K.D., Kothukar R.M., and Dighe, V.V [7]

proposed that sulfamoxole in tablets can be determined by

diazotisation and coupling reaction using 2-naphthylamine

or N-l- naphthyl ethelene diamine. The absorbance was

measured at 540 or 520 nm as the cage may be. Marguez,

M; Silver, M., and Perez - Bendito, D [8] reported a

kinetic method for the determination of sulfonamides by

"continuous addition of a reagent" technigue. This

involved the formation of an azodye between 1-naphthol and

the diazonium salt formed in the reaction between the drug

and NaH02 in weakly acid medium. The reaction was

monitored via the initial rate of change of the absorbance

of the azo dye at 470 nm. Al-Abachi., et. at 19] reported

an oxidative coupling reaction method for the determination

of sulfonamides. Promethazine was used in the

reaction. A spectrophotometric method for the

micro determination of sulfonamide drugs in aqueous

solutions based on diazotization and coupling with Indole

in alkaline medium was reported by Al-Abachi.. et.al

[10], The intense yellow colored water soluble azo dye

with an absorbance maximum at 445nm was formed in the

reaction. Esteve Romero, J.S., Ramis Ramo, G, Forteza

Coll, P, and Cerda Martin. V til] proposed a flow

injection spectrophotometric determination method of

12
sulfonamide by dlazotization and coupling in micellar

medium. The coupling was affected with N-{1, naphthyl)

ethylene diamine.

ii) Determination of sulfonamides-color forming reagents

Borodai, I.V and Gaidukevich, A.N. [12]

reported a photometric method of determination for the

sulfa drugs using 9-chloro acridine as the color

generating reagent. The drug was treated with the reagent

in ethanolic solution of HCl (pH=1.5) and the absorbance of

the yellow product formed was determined at 450 nm. A

spectrophotometric method of determination of

sulfonamides using anisaldehyde as the coloring reagent

was reported by Amer, M.M., Hussan, S.M and El. Sharif,

Z.A.I13). The absorbance was measured at 370-388nm.

The optimum temperature for the development was 50°c and

color was stable for 2 hours. El-Obied, H.A, Jado, A.I [14]

reported a colorimetric method for the determination

of sulfonamides through the measurement of the

absorbance of the product formed in the reaction of

sulfonamides with furfural in acidic medium. They stated

that the color was maximum and stable for only 15-45

minutes after the addition of the reagent. A

colorimetric estimation of sulfomethoxazole in pure form and

in pharmaceutical formulations was reported by Patel, P.R.,

Patel, A.A., Patel, M.R., Mankiwala, S.C and Patel, S.N.,

[15]. In this method the drug was treated with p-

(dimethyl amino) benzaldehyde in acidic medium to give

13
 * of
yellow color with maxlmum#45Q nm. Thulus, Rasim., Sungur,

Sidlka 116] reported that sulfonamides can be determined by

treating them with p-dimethyl amino cinnamaldehyde

and measuring the absorbance at 545 nm.of the resulting

colored product. Determination of Sulfathiazole in a

mixture containing sulfadiazine and sulfamerazine by

adopting a simultaneous equation method was reported by

Hassan, S.M., et.at [171. The simultaneous equations

are solved by using, the absorbance data obtained by

%

recording the spectra in different media. Sastry,

C.S.P., and his co-worker [18] reported that primary

amine drugs can be determined by measuring the absorbance

of the chromogen formed in the reaction between the

drug, orthoamino phenol and KIO3 in glycine - HC1 buffer pH

1.7. The absorbance measurements were made at 520nm. A

spectrophotometric method using 3-(c*£,J8- dicarboxyethyl)

rhodamine, NaN02 and HC1 was reported for the

determination of sulfonamides in presence of borate

buffer (pH=12) by Mynka, A.F.,et all. [19]. Kamala

Purkar, G.S., Kamat, G.T., [20] reported that a stable

yellow Schiff's base was formed when sulfamethoxazole

was treated with salicyladehyde in acidic medium. the

product showed absorbance meximum at 380 nm. This reaction

was therefore used for the determination of the drug

coloriometrically. A new rapid, simple, selective and

sensitive method for the spectrophotometric determination of

sulfathiazole was proposed by Agrwal, Y.K., Menon, S.K., and

14
Giridhar, R [21], based on the formation of yellow complex

with Pd (II) in 1M NaOH. The complex shows maximum

absorbance at 410 nm. Yuang, Jimir and CUI.MIN [22]

determined sulfacetamide in eye drops, at pH 5.8 by

measuring the absorbance of the drug at 257 nm.

Sulfapyridazine was determined spectrophotometrlcally

by using the color reaction of the drug with bindone and

measuring the absorbance of the chromogen at 480 nm by

Deryugina, L.T. and Petrenko, V.V. [231. A

spectrophotometric method for the estimation of

sulfacetamide was developed by Rao.G. Ramana et.al 124].

The method was based on the reaction of

sulfacetamide with O.Chloranil at pH.9. The purple

colored chromogen formed was measured at 524nm.

Shukla, I.c.' 125] reported a spectrophotometric

method for the determination of some sulfonamides

in the pure and in the drug samples. In this methods

the drug compounds were treated with the oxidized

nitroprusside reagent and the absorbance of the green

color developed in the reaction was measured at 670 nm.

Sulfonamides react with 7,7,8,8 -tetracyanoquinodimethane at

pH 9 to produce intense blue product with maximum

absorbance at 578nm. The reaction was used by Mohamed

Abdul Hoboud I {263, for the determination of the drug.

The color formed, was stable for at least 90 min. at 25°c.

Sulfonamide reacts with N-chlorosuccinimide in the molar

ratio of 1:2 in presence of acidified KBr to form a

15
colored product. This observation made by Srivastava,

Ashutosh and co-workers 127], helped them to formulate a

method for the determination of the drug for measuring

the absorbance at 324-395 nm at pH 7.4 at room temperature.

Rao.G. Ramana and his co-workers [28] reported that

sulfadoxine and sulfalene can be determined by the reaction

of sulfadoxine and sulfalene with sodium 1,2

napthoquinone -4 sulfonate. The absorbance of the chromogen

was measured at 425-430 nm. A spectrophotometric method

for the determination of sulfadoxine and sulfalene

based on their reaction with metol at pH 2.5 in presence

of Nal(>4 as oxidant was proposed by Rao, G. Ramana and

his co-workers [29]. The absorbance was measured at

520 nm. Rao.G.Ramana Hurthy, S.S.N., Raju., I.R.K.,

[30] reported ' a spectrophotometric method for the

determination of sulfadoxine and sulfalene in combined

dosage forms using O.Chloranil. Paimoo,P [31] reported

that primethamine and sulfadoxine can be simultaneously

determined by differential spectrophotometric method based

on the measurement of the absorbance in various media of

240-250 nm and by solving the simultaneous equations set

up.
An Accurate and simple spectrophotometric method

for the determination of sulfa drugs in presence of

sulfonilic acid using a potassium 1,2 - naphthoquinone 4-

sulfonate in chloroacetic - chloroacetate buffer at pH

3.4 by punta cordero, A., Barragan de la Rosa, F.J., and

16
Guiraum, A., (321 was reported. An extraction technique

with chloroform was employed to eliminate the

interference of sulfonilic acid.

Sulfadiazine was determined by FIA method by Zhang, Xicum

1331. In this, sulfadiazine was dissolved in 6M HC1 and

treated with dimethyl amino benzaldehyde. The absorbance

maximum of the product was at 448nm.

A three component spectrophometric assay method

using drugs sulfacetamide, sulfanilamide and azobenzene at

pH 4 was reported by Ahmad Tauqur., Ahmed Eqbal [341.

Sulfonamides react with 5-nitrobarbituric acid to form

colored chromogen. This reaction reported by Sadlvski.,

V.M., et at [351 was used by them to determine the

drugs by the measurement of the absorbance of the

chromogen at 40(1 nm. Sulfamethoxazole was extracted from

the compound with 6 H HCl and the extract was used to

determine the drug by flow injection analysis at 452 nm by

Guw, Tao., et al [361. Noyanalpan, Ningur., and

co-workers [37] reported a spectrophotometric qualitative

determination of trimethoprim and sulfamethoxazole in

tablets by an absorbance ratio method. They used O.lM

NaOH as the solvent for the above drugs. The

absorbance measurements were made at 256 and 288 nm.

Ray Satyabrata., et al [38] reported a

simultaneous spectrophotometric method for the determination

of trimethoprim and sulfamethoxazole in combined

pharmacutical dosage forms. These compounds were

17
simultaneously determined spectrophotometrically

measuring the absorbance of the solutions between 400 and

200 nm. The absorbance maxima of the compounds were at

287 and 269 nm for trimethoprim and sulfa melthoxazole

respectively. Nikolic, K., and Medenicas, M 1391

reported a colorimetric method for the determination

of sulfisomidine, sulfamethoxydiazine and sulfamoxole using

0.1M H2SO4 - 2M HCl as supporting electrolyte and

methyl orange as indicator. Sulfonamides were

extracted by acid and were made to react with an azodye

and the chromogen thus formed was used to determine the

drug by Krach. A.S. and his co-workers [401.

Sulfadiazine was determined in acidic medium by

Maheswari, Sapna and Co-workers [411 by measuring the

absorbance of ' the colored product formed at 533 nm.

Youssef, AdelF., et al [42] reported'’ that sulfonamide

derivatives of the type benzothiadiazines and

quinazolone form chromogenS when treated with

phenothiazine and N-bromo succinimide. This reaction

was therefore used by them to determine the drugs. They

showed that the SO2 NH2 group was responsible for

coupling with oxidized phenothiazine. The absorbance was

measured at 560 nm.

Spectrophotmetric methods for the determination of

sulfacetamide, sulfaguanidine and sulfamethazine in

pharmacutical compounds and foods were reported by Salinas,

F and co-workers [43] by spectro fluorometric method. A

18
new method for analyzing the binary mixtures of

sulfamethoxazole and trimelthoprim, using a derivative

spectrophotometry was reported by Berzas Nevada, J.J., et

al [44]. The procedure does not require any separation

step. Sastry, C.S.P., et al [45] reported a

spectrophotometric method for the determination of

sulfinpyrazone by measuring the absorbance of the color

formed during the oxidation of the drug with N-

bromo succinamide. A spectrophotometric method of

estimation of sulfadiazine was developed by Raghuveer and

the co-workers [44]. The method was based on the

reaction of sulfadiazine with 4-dimethyl amino

cinnamaldehyde in orthophosphoric acid medium. The

absorbance was measured at 545 nm. Feng, Jianzhang., et at

[47] observed that in 50% (vol/vol) elhylalchohol-water

medium, sulfamethoxazole and p-benzo guinone react to form a

pink color. This was employed by them to determine the

drug. A sensitive spectrophotometric method for the

determination of sulfadoxine based on the drug reaction

with 4 - methyl aminocinnamaldehyde in orthophosphoric

acid medium to form a stable red chromogen was used

by Raghuveer,S., et. al [483.

A quantitative determination of sulfamethoxazole,

trimethoprim in tablets by spectrophometry based on

vierordt method was reported by Atay. OKan and co­

workers [49] by measuring the absorbance at 256 and 288

nm. A solid phase spectrophotometric method was reported

19
by Captln Vallvey, L.Fermln., et.all 150} for the detection

of sulfathiazole in presence of sulfametazine using first

derivative solid phase spectrophotometry. A study on

charge transfer reaction of sulfadiazine with

benzoquinone reagent was reported by Zhou, Xuguang.,

et.al [513. The measurement of absorbance was done at 500

nm. Issa, Y.M. and Amin, A.S [52] proposed a photometric

method based on the charge transfer complex formation of

the drug with alizarin or guinalizarin in alkaline

medium. A colorimetric quantitative determination of

sulfamethoxzole in the tablets, by generating the color

with p-dimethyl amine benzaldehyde was reported by Sun,

Guoxiang and co-workers [53],

A differential spectrophotometric method was reported

by Zhang, Yuqln et.al [54] for the determination of

sulfadimidine and sulfaguanidine in Infantile powders

without interference of the additives,

ii) Determination of sulfonamides by non-colorimetric methods

Sulfonilamide drugs were determined by Koka, I.P

[55], by adding excess Ce(804)2 and subsequent

Iodometric titration of excess Ce(S04)2»Verma, Krishna K,

Gupta, Anil, K., [56] reported that sulfonamides in

pharmaceutical preparations were determined by titrating

with chlorasmine-T in the presence of acidified KBr,,

using methyl red as indicator. They reported that 6 to 4

Br atoms were consumed depending on the substituent

attached to the sulfonamide group. Alekperov, A.F [57]

20
reported that the titration of sulfanilamide in aqueons

HC1 medium with NaN02 solution in the presence of an

indicator increases the accuracy. Frenolon was used • as

the indicator. An A.C. oscilloscope polarographic

titration method using standard NaN02 solution in 6M HCl

containing KBr was reported for the determination

of sulfanamides, sulfamerazine, sulfamethazine,

sulfamonomethoxine and sulfa methoxydiazine by Zhan,

Yongfu., et.al [58]. Nie, L.H.Wang, T.Q, and Yao, S.Z

[59]., reported a piezoelectric sensor method for the

determination of sulfa drugs. This method was based on the

reaction of the drug with Br and the reduction of the

resultant N-bromo derivative with I- to form 12- A

simple titrlmetric method of determination of sulfonamides

by titration with N-N-dibromodimethyl hydantoin was

reported by Kumar, K, Girish., et.all [60]. Sulfanilamide

was treated with NaBrC>3 and the released nitrogen was

determined volumetrically by XIONG, et.al [61]. Pena, H.

Sanchez., et. al [62] employed a photochemically induced

fluorescence method to determine sulfamethazine at room

temperature, in presence of methanol, ethanol, and 2-

propanol. Ivakhnenko, P.N., et.al [63] reported an indirect

titric method.Sulfanilaminde with NaN02 in presence of

bias anal
HCl (pH 0.1-0.5)^treated with KMn04 the excess KMn04 was

determined Iodometrically.

21