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

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

Simultaneous evaluation of benzotriazoles, benzothiazoles and benzenesulfonamides in water samples from the impacted urban Jacarepaguá Lagoon System (Rio de Janeiro, Brazil) by liquid chromatography coupled to electrospray tandem mass spectrometry.

Benzotriazoles, benzothiazoles, and benzenesulfonamides are emerging pollutants stable in aquatic media emitted by anthropogenic sources. Selected compounds of these classes were evaluated in the impacted urban Jacarepaguá Lagoon System (JLS) located in a tropical coastal region of Rio de Janeiro City, Brazil that has experienced a rapid expansion of urban occupation and environmental degradation in recent decades, and it represents a pioneering study of these compounds carried out in Brazilian areas. A method of solid phase extraction followed by ultra-performance liquid chromatography coupled to electrospray tandem mass-spectrometry was implemented to evaluate water samples collected in different water bodies (rivers, lagoons, and channels) of the JLS from March 2017 to May 2018. Limits of quantification (LOQs) ≤ 10.0 ng L-1, method linearity up to 1000 µg L-1, and recoveries between 62 and 121 % at three different levels were obtained. Individual concentrations varied from < LOQ to 5260 ng L-1 (benzotriazole, in May 2018) which also predominated in all river samples. 2-mercaptobenzothiazole predominated in samples taken in lagoons and channels in March 2017, and 2-aminobenzothiazole was never detected. River samples showed total concentrations up to 30 times larger in all sampling campaigns, except March 2017 when the sample taken at Tijuca Lagoon showed the largest total concentration of the compounds studied due to the largest concentration of 2-mercaptobenzothiazole (2505 ng L-1) found in this study. Principal component analysis (PCA) using only composition data was unable to distinguish samples from rivers, and lagoons and channels, but a PCA combining composition data and environmental parameters (pH, Eh, dissolved O2 concentration, temperature, salinity, and conductivity) discriminated the samples according to two groups: rivers and lagoons and channels. The Joá Channel flows directly to the open sea and our data allowed a (preliminary) estimation of the total mass flows of the studied compounds to the open sea, which would vary between 1702 g day-1 (March 2017) to 106 g day-1 (May 2018) and allowed a preliminary estimative based on the geometric mean of input of 87.9 kg year-1, indicating the importance of the drainage area to the contamination of the coastal area, and consequently to ocean pollution.

Simultaneous determination of alpha-, beta- and gamma-hydroxybutyric acids in micro-pulverized human hair by GC-MS: Method development, validation and application.

An analytical method was developed and validated for the simultaneous determination of alpha, beta and gamma-hydroxybutyric acids (AHB, BHB and GHB, respectively) in human hair. Hair samples (10 mg) were pulverized and submitted to extraction using methanol (1 mL) for 10 min. The extract was centrifuged, filtered, and evaporated to dryness at 40 °C under a gentle N2 flow, dissolved in ethyl acetate (0.050 mL) and derivatized using 0.050 mL of BSTFA containing 1% TMCS, at °C for 40 min. The derivatives were determined by GC/MS. Aliquots of natural hair ("blank") samples after water extraction (3 steps) were pulverized, spiked with AHB, BHB, GHB or d6-GHB and used for method validation. Figures of merit showed the method's feasibility. The LOQ values were 1 ng mg-1 for BHB and AHB and 0.6 ng mg-1 for GHB, and mean recoveries were 54%, 69% and 75% for AHB, BHB and GHB respectively. Precision and bias were better than 15% and 20% respectively. Calibration curves (LOQ to 20 ng mg-1 for AHB and BHB and LOQ to 16 ng mg-1 for GHB) obtained in 5 days were pooled for statistical analysis, but the data were heteroscedastic, as demonstrated by the White test. Therefore, weighted linear regression and data transformations were applied and the best results were obtained using the Box-Cox transformation [c' = (cλ-1)/λ] for GHB and log transformation for AHB and BHB. Authentic hair samples were collected from the posterior head vertex of 23 volunteers (16 females and 7 males). Four of them declared having type-2 diabetes (T2D). Only female volunteers reported hair treatments such as dyeing, flat ironing, straightening and/or bleaching. Dyeing appeared not to affect GHB levels in hair, but no data were found in the literature about other cosmetic treatments. GHB levels varied from < LOQ to 1.5 ng mg-1. BHB levels varied from < LOQ to 3.4 ng mg-1 and from < LOQ to 1.8 ng mg-1 in non-diabetic volunteers with or without family history of T2D, respectively. However, in diabetics, BHB levels varied from 3.4 to 5.2 ng mg-1. The levels of AHB varied from < LOQ to 1.2 ng mg-1 in non-diabetic individuals, and from 1.1 to 6.2 ng mg-1 in diabetics. Our results showed that AHB levels in hair have potential to as a biomarker for T2D, but this needs to be further studied with larger groups of volunteers. To our knowledge, this is the first published method for the determination of AHB and BHB in human hair.