Acessibilidade / Reportar erro

Brasil

Brazilian Journal of Pharmaceutical Sciences

Español English

Brasil

Español English

sumário « anterior atual seguinte »

Sumário

Article • Braz. J. Pharm. Sci. 60 • 2024 • https://doi.org/10.1590/s2175-97902024e23126 copy

Direct spectrofluorimetric methods as alternatives to compendial ones used for the quality control of biopharmaceuticals: development, validation and application

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

A simple, rapid, precise, accurate and sustainable spectrofluorimetric method (SFM) was developed, validated and applied for the determination of 4-aminobenzoic acid and aromatic amino acids (phenylalanine, tryptophan and tyrosine). These compounds are used in biopharmaceutical formulations and therefore must be analyzed by quality control laboratories to meet the criteria established in pharmacopoeias. In general, potentiometric titration (PT) is described in the compendia as the official analytical technique. However, this method showed low sensitivity and selectivity, and moreover was performed with a non-aqueous solvent (acetic acid), which led to higher consumption of reagents and consequently to the formation of residues. Therefore, the SFM was developed in aqueous medium at pH 7.2 using phosphate buffer. It was successfully validated according to the ICH guidelines and showed good linearity range (r>0.999), specificity, accuracy and precision (within and between days) and robustness. The test results were compared between the SFM and PT using raw material samples, while according to the F- and t-tests at 95% confidence level, no statistical difference was found between the methods.

Keywords:
4-aminobenzoic acid; Analytical validation; Aromatic amino acids; Fluorescence spectroscopy; Raw material

Analyte	Weight (mg)	Titrand solution	Titrant solution
PABA	100	50 mL of water + heating*	NaOH 0.1 mol L^-1
PHE	100	3.0 mL of formic acid + 30 mL of acetic acid	HClO₄ 0.1 mol L^-1
TRP	150	3.0 mL of formic acid + 50 mL of acetic acid	HClO₄ 0.1 mol L^-1
TYR	150	5.0 mL of formic acid + 30 mL of acetic acid	HClO₄ 0.1 mol L^-1

Compounds	Molar weight (g mol^-1)	Solubility* (g kg^-1)	pKa₁	pKa₂	pKa₃	pI
4-aminobenzoic acid	137.14	5.40	2.50	4.79	-	3.69
L-phenylalanine	165.19	27.9	1.83	9.13	-	5.48
L-tryptophan	204.23	13.2	2.83	9.39	-	5.89
L-tyrosine	181.19	0.46	2.20	9.11	10.07	5.66

Parameters	PABA	PHE	TRP	TYR
λ_EX / λ_EM (nm)	304/336	261/283	288/352	275/312
Linearity range (mg L^-1)	0.050 to 2.5	10 to 135	0.025 to 5.0	0.25 to 10
Correlation coefficient (r)	1.000	0.999	0.999	0.999
Shapiro-Wilk test^a	0.793 (0.788)^b	0.937 (0.842)^d	1.184 (0.818)^c	0.855 (0.788)^b
Cochran test^a	0.242 (0.616)^b	0.282 (0.445)^d	0.338 (0.516)^c	0.277 (0.616)^b
Regression significance^a (ANOVA F-test)	5,859 (4.45)^e	3,853 (4.18)^f	2,451 (4.28)^g	2,168 (4.45)^e
LOD (mg L^-1)	0.013	1.8	0.008	0.061
LOQ (mg L^-1)	0.045	5.4	0.025	0.18
Target concentration (mg L^-1)	1.0	50	2.0	1.0
Calibration curve (mg L^-1)	0.80 to 1.2	40 to 60	1.6 to 2.4	0.8 to 1.2
Intercept (a)	4.89	-29.9	2.53	-13.1
Slope (b)	135	8.40	362	209
Correlation coefficient (r)	0.997	0.999	1.000	1.000

Parameters	PABA	PHE	TRP	TYR
Precision (%RSD; n=3)
Repeatability (intra-day)
80% TC	2.4	3.4	1.7	1.6
100% TC	1.4	3.2	2.1	0.79
120% TC	1.8	1.5	2.5	1.0
Intermediate precision (inter-day)
80% TC	0.92	1.1	0.07	0.78
100% TC	2.6	0.38	3.6	1.0
120% TC	2.0	1.5	2.4	1.2
Accuracy (%recovery mean±SD; n=3)
80% TC	96.8 ± 1.6	100.7 ± 3.3	103.2 ± 0.9	99.3 ± 2.4
100% TC	99.8 ± 2.5	100.0 ± 3.0	103.5 ± 1.6	100.4 ± 1.6
120% TC	100.0 ± 2.8	99.5 ± 1.4	102.1 ± 1.0	99.9 ± 2.3
Robustness (%recovery mean±SD; n=9)
Instrumental (λ _EM ± 1 nm)
80% TC	96.8 ± 1.7	99.8 ± 4.4	102.9 ± 1.1	99.0 ± 4.9
100% TC	99.8 ± 2.1	99.5 ± 4.3	103.1 ± 1.9	99.1 ± 4.9
120% TC	100.0 ± 1.9	99.7 ± 3.6	102.1 ± 1.2	98.6 ± 5.4
Experimental (pH 7.2 ± 0.2)
80% TC	97.7 ± 3.4	99.2 ± 2.1	102.2 ± 1.2	102.0 ± 2.4
100% TC	101.0 ± 0.4	99.5 ± 0.9	101.6 ± 1.4	101.8 ± 2.2
120% TC	101.1 ± 1.7	97.3 ± 4.8	100.8 ± 2.2	102.6 ± 2.8

Samples and standards	Water and volatile compounds^a (%)		Assay (% on dry basis)		Acceptance criteria^a.b (%)
	(mean ± SD)	Acceptance criteria^a	Spectrofluorimetric method (mean ± SD)	Potentiometric method^a (mean ± SD)
PABA-Sample 1	0.19 ± 0.006^c	≤0.2%	99.9 ± 0.2	98.7 ± 0.7	BP and Ph. Eur.: 99.0-101.0
PABA-Sample 2	0.19 ± 0.006^c		100.1 ± 1.9	99.7 ± 0.8	USP: 98.0-102.0
PABA-Sample 3	0.18 ± 0.008^c		100.4 ± 0.1	99.0 ± 0.1
PABA-CRS	-		100.6 ± 0.6	-
PHE-Sample 1	0.12 ± 0.006^d	≤0.5%	100.0 ± 1.2	100.3 ± 0.6	BP and Ph. Eur.: 98.5-101.0
PHE-Sample 2	0.03 ± 0.000^d		100.1 ± 0.8	99.3 ± 1.1	JP: ≥98.5
PHE-Sample 3	0.39 ± 0.006^d		100.1 ± 0.9	99.7 ± 0.6	USP: 98.5-101.5
PHE-Sample 4	0.01 ± 0.000^d		100.0 ± 0.6	99.9 ± 0.8
PHE-CRS	-		99.4 ± 0.4	-
TRP-Sample 1	0.37 ± 0.001^d	≤0.5%	100.0 ± 0.8	99.9 ± 0.3	BP: 98.5-101.0
TRP-Sample 2	0.38 ± 0.006^d		99.9 ± 1.2	100.1 ± 0.9	JP and Ph. Eur.: 99.0-101.0
TRP-Sample 3	0.00 ± 0.000^d		100.3 ± 0.6	100.1 ± 0.8
TRP-CRS	-		100.0 ± 0.8	-
TYR-Sample 1	0.27 ± 0.006^d	≤0.5%	99.9 ± 1.2	99.3 ± 0.2	BP: 99.0 - 101.0
TYR-Sample 2	0.00 ± 0.010^d		99.8 ± 0.4	99.6 ± 0.6	Ph. Eur.: 98.5-101.0
TYR-Sample 3	0.20 ± 0.008^d		99.5 ± 0.4	99.3 ± 0.2	JP: ≥98.5
TYR-CRS	-		100.4 ± 0.7	-	USP: 98.5-101.5

Samples	Comparison between samples by the SFM and PM		Comparison between sample and CRS by SFM
	t-test^a	F-test^b	t-test^a	F-test^b
PABA-Sample 1	2.4	1.3	2.0	12
PABA-Sample 2	1.2	1.5	0.29	1.4
PABA-Sample 3	1.5	5.7	1.5	17
PHE-Sample 1	0.33	4.5	1.0	11
PHE-Sample 2	1.0	2.1	2.3	5.1
PHE-Sample 3	0.87	2.3	1.7	6.4
PHE-Sample 4	0.29	1.7	2.6	3.1
TRP-Sample 1	0.02	13	0.46	2.0
TRP-Sample 2	0.29	1.8	0.45	2.1
TRP-Sample 3	0.57	2.2	0.29	2.0
TYR-Sample 1	0.80	1.8	2.0	1.1
TYR-Sample 2	0.97	3.3	2.2	1.8
TYR-Sample 3	2.2	3.3	2.3	3.8

Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br

Acompanhe os números deste periódico no seu leitor de RSS