Spectrophotometric determination of ethionamide in pharmaceuticals using Folin–Ciocalteu reagent and iron(III)-ferricyanide as chromogenic agents

Peer review under responsibility of Taibah University.

Figures & data

Fig. 1 Absorption spectra of the MTB method: (A) Reaction product of ETM (15 μg/ml) with F–C reagent in Na₂CO₃ solution; (B) the blank.

Fig. 2 Absorption spectra of PB method: (A) Reaction product of ETM (2 μg/ml) with iron(III) and ferricyanide in HCl medium; (B) the corresponding blank.

Fig. 3 Possible reaction pathways yielding coloured species.

Fig. 4 (a) Effect of the F–C reagent concentration (ETM, 15 μg/ml). (b) Effect of 20% Na₂CO₃ (ETM, 15 μg/ml).

Fig. 5 (a) Effect of the iron(III) concentration (ETM, 2.0 μg/ml). (b) Effect of the potassium ferricyanide concentration (ETM, 2.0 μg/ml). (c) Effect of the HCl concentration (ETM, 2.0 μg/ml).

Fig. 6 (a) Calibration curve for the Molybdenum-tungsten blue method. (b) Calibration curve for the Prussian blue method.

Table 1 Sensitivity and regression parameters.

Parameter	MTB method	PB method
λmax, nm	760	760
Colour stability	24 h	60 min
Linear range, μg/ml	1–40	0.2–4.0
Molar absorptivity (ɛ), L/(mol·cm)	5.72 × 10^3	3.18 × 10^4
Sandell sensitivity^a, μg/cm^2	0.0291	0.0053
Limit of detection (LOD), μg/ml	0.09	0.01
Limit of quantification (LOQ), μg/ml	0.27	0.04
Regression equation, Y^b
Intercept (b)	0.0468	0.0235
Slope (m)	0.0238	0.1625
Standard deviation of b (Sb)	9.98 × 10^−2	9.88 × 10^−2
Standard deviation of m (Sm)	2.7 × 10^−3	3.3 × 10^−2
Regression coefficient (r)	0.9986	0.9992

a Limit of determination as the weight in μg/ml of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm² and l = 1 cm.

b Y = b + mX, where Y is the absorbance, X concentration in μg/ml, b intercept and m slope.

Table 2 Evaluation of intra-day and inter-day accuracy and precision.

Method	ETM taken, (μg/ml)	Intra-day accuracy and precision (n = 7)			Inter-day accuracy and precision (n = 5)
		ETM found^a (μg/ml)	RSD^b %	RE^c %	ETM Found, (μg/ml)	RSD^b %	RE^c %
MTB	10.0	9.88	0.88	1.20	9.85	1.94	1.50
	20.0	19.84	1.27	0.80	19.69	2.32	1.55
	30.0	30.48	0.96	1.60	29.44	1.89	1.87
PB	1.0	0.98	1.29	2.00	1.00	1.87	2.00
	2.0	1.97	1.36	1.50	1.97	1.47	1.50
	3.0	3.04	1.55	1.33	2.95	2.49	1.67

a Mean value of seven determinations.

b Relative standard deviation (%).

c Relative error (%).

Table 3 Method robustness and ruggedness expressed as intermediate precision (% RSD).

Method	ETM taken, (μg/ml)	Robustness (%RSD)			Ruggedness (%RSD)
		Parameters altered			Inter-analysts (n = 3)	Inter-cuvettes (n = 3)
		Contact time^a	Volume of F–C reagent/iron(III)^b	Volume of Na2CO3/ferricynide
MTB	10	0.76	0.67	0.86	1.23	1.88
	20	0.88	0.88	1.12	1.75	1.65
	30	1.34	1.58	1.00	0.96	2.07
PB	1	1.68	1.85	1.85	1.22	2.31
	2	2.16	1.17	1.46	0.87	1.63
	3	2.58	1.74	1.78	1.38	1.77

a Contact time used: 8, 10 and 12 min in MTB method, 28, 30 and 32 min in PB method.

b Volume of F–C reagent/Na₂CO₃ were: 1.3, 1.5 and 1.7 ml in MTB method; volume of iron(III) and ferricyanide were: 1.8, 2.0 and 2.2 ml in PB method.

Table 4 Results of analysis of tablets by the proposed methods and statistical comparison of the results with the official method.

Tablet brand name	Nominal amount	Found^a (% of nominal amount ± SD)
		Official method	Proposed methods
			MTB method	PB method
Ethide	250	98.64 ± 1.05	99.72 ± 0.86	97.83 ± 1.12
			t = 1.78	t = 1.73
			F = 1.49	F = 1.14
Ethiokox	250	100.12 ± 0.65	101.28 ± 1.32	101.34 ± 1.45
			t = 1.76	t = 1.72
			F = 4.12	F = 4.98
Myobid	250	98.59 ± 1.67	97.96 ± 1.23	100.4 ± 0.93
			t = 0.68	t = 2.12
			F = 1.84	F = 3.22

a Mean value of five determinations.

(Tabulated t-value at the 95% confidence level and for four degrees of freedom is 2.78).

(Tabulated F-value at the 95% confidence level and for four degrees of freedom is 6.39).

Table 5 Results of recovery experiment through standard-addition method.

Method	Tablet studied	ETM in Tablet, μg/ml	Pure ETM added, μg/ml	Total found, μg/ml	Pure ETM recovered Percent ± SD^a
MTB	Ethide 250	8.97	9	18.12	100.90 ± 0.84
		8.97	18	26.88	99.76 ± 0.54
		8.97	27	36.07	100.32 ± 0.77
PB	Ethide 250	0.73	0.75	1.47	99.41 ± 1.23
		0.73	1.50	2.26	101.44 ± 1.42
		0.73	2.25	3.11	104.39 ± 1.65

a Mean value of three determinations.

Table 6 Comparison of the proposed and the existing spectrophotometric methods.

Sl. No.	Reagent/s	Methodology	Linear range (μg/ml)	LOD/LOQ (μg/ml)	Remark	Ref. no.
1	Sodium nitoprusside	Orange coloured product in basic medium measured at 510 nm	_	_	_	20
2	Sodium nitroprusside	Orange-red complex measured at 510 nm	5–32	_	Narrow range	21
3	DCNQ	Orange coloured product in ethanol measured at 440 nm	_	_	_	16
4	DCNQ	Red coloured product formed in the presence of ammonia in alcohol medium measured at 530 nm	_	_	_	17
5	PAR-V(V)	Ternary complex (1:1:1) extracted into chloroform and measured at 560 nm	0.2–20	_	30 min contact time, involves laborious liquid-liquid extraction step	22
6	Iron(III)	Purple-violet colour complex in acid medium measured at 510 nm	0–36	_	Longer contact time	18
7	Iron(III)-PPD	Thionine compound measured at 600 nm	_	_	Multiple step reaction involved	19
8	Osmic acid	Light yellow coloured formed at pH4 measured at 375 nm	0.25–40	_	60 min contact time, pH adjustment required	23
9	NBS-CB	Unbleached dye colour measured in acid medium at 540 nm	0.2–5.0	_	Critical acid conc.; concentration of NBS &CB to be known accurately	25
10	KMnO4	Blue coloured manganate in alkaline medium measured at 610 nm (direct method), absorbance at a fixed time of 20 min measured (kinetic method)	1–10 1–10	_	Critical NaOH conc., reaction rate precariously dependent on experimental variables	26
11	Sodium azide- iodine	Decrease in absorbance at the 5th min measured at 348 nm (kinetic method)	10–100	0.7/–	Reaction rate precariously dependent on experimental condition, narrow range, less sensitive	27
12	(a) F–C reagent (b) Fe(III) ferricyanide	Mo–W blue measured at 760 nm Prussian blue measured at 760 nm	1.0–40 0.2–4.0	0.09/0.27 0.01/0.04	No drastic experimental conditions involved, no extraction step, no critic pH, more sensitive, wide linear dynamic ranges	Present methods

DCNQ, dichloronaphthoquinone; PAR, pyridylazo resorcinol; PPD, p-phenylenediamine; NBS, N-bromosuccinimide; CB, Celestine blue.