Tadalafil and Sildenafil illicit association: Stability- indicating HPLC method, photodegradation kinetic and toxicological studies

Abstract The illicit market of counterfeit medicines containing sildenafil and tadalafil has been causing serious public health problems. Thus, further studies on this illicit association are needed. A stability-indicating HPLC method was developed for simultaneous determination of tadalafil (TAD) and sildenafil (SIL) using a C18 column (250 x 4.6 mm, 5 µm). Detection was achieved at 284 nm, for TAD, and 292 nm, for SIL. The method was considered to be specific, linear, precise, accurate, robust, and sensitive. In the photodegradation kinetic studies, the drugs showed a first-order reaction rate when isolated, and zero-order when associated. Toxicological assays demonstrated that the photodegraded drugs decreased cell viability in compared to non- degraded drugs, suggesting cytotoxic activity. Additional, mutagenic activity was not observed under the tested conditions. Photodegraded drugs, in association, depicted DNA damage index, suggesting genotoxic effects. The obtained results will be able to support the forensic intelligence laboratories, as well as to alert the population about the risk inherent to consuming counterfeit products.

Forced degradation studies and stability-indicating liquid chromatography method for determination of tirofiban hydrochloride and synthetic impurities / Estudo da degradação forçada e método indicativo da estabilidade por cromatografía líquida para determinação de cloridrato de tirofibana e impurezas sintéticas

SUMMARY This study aimed to develop and validate a stability-indicating liquid chromatography method for the determination of tirofiban hydrochloride and two synthetic impurities (impurity A and impurity C). The method utilizes a RP-18 column (250 mm x 4.6 mm; 5 µm) with the PDA detector for quantitation. A mixture of triethylamine 0.1% (acidified to pH 5.5 with phosphoric acid) and acetonitrile was used as the mobile phase at a flow rate of 1 mL min-1 with gradient elution. The method presented satisfactory linearity, precision, accuracy and robustness, as well as low limits of detection and quantification, which demonstrate sensitivity in the determination of tirofiban and impurities A and C. It was selective for the determination of the drug and impurities analysed, without interference of the degradation products generated under forced conditions, demonstrating the stability-indicating capacity of the proposed method. Tirofiban showed to be practically stable to oxidative (30% H2O2 for 24 h) and thermal (75 °C for 24 h) conditions, but presented degradation to UVA light and acid hydrolysis, obeying the first order kinetics for both. In this way, it can be used as a stability-indicating method in the quality control of the raw material of tirofiban hydrochloride, as well as of the finished product. The obtained results demonstrate the importance of deepening the studies in this area, to guarantee the quality of commercialized pharmaceutical products.

RESUMO Este estudo teve como objetivo desenvolver e validar método indicativo da estabilidade por cromatografía líquida para determinação de cloridrato de tirofibana e duas impurezas de síntese (impureza A e impureza C). O método utilizou coluna de fase reversa RP-18 (250 mm x 4,6 mm; 5 µm) e detector PDA para quantificação. A fase móvel foi composta por uma mistura de trietilamina 0,1% (acidificada com ácido fosfórico para pH 5,5) e acetonitrila, à vazão de 1 mL/min, no modo gradiente. O método apresentou linearidade, precisão, exatidão, robustez, bem como baixos limites de detecção e quantificação, demonstrando sensibilidade na determinação da tirofibana e impurezas A e C. O método apresentou seletividade na determinação do fármaco e das impurezas, sem interferência dos produtos de degradação gerados na degradação forçada da tirofibana, demonstrando sua capacidade indicativa de estabilidade. O fármaco apresentou-se estável a oxidação (H2O2 30% por 24 h) e a degradação térmica (75 °C por 24 h), mas degradou frente à luz UVA e hidrolise ácida, obedecendo cinética de primeira ordem para ambas. Dessa forma, pode ser utilizado como um método indicativo de estabilidade no controle de qualidade da matéria -prima do cloridrato de tirofibana, bem como no produto acabado. Os resultados obtidos demonstram a importância de aprofundar os estudos na área, com intuito de garantir a qualidade dos produtos farmacêuticos comercializados.

Validation of an analytical method by high-performance liquid chromatography and microbiological assay, biological safety and in silico toxicity for danofloxacin

Danofloxacin is a veterinary fluoroquinolone used to treat respiratory and gastrointestinal diseases of birds, pigs and cattle. The literature reviewed shows some analytical methods to quantify this fluoroquinolone, but microbiological and biological safety studies are limited. The analytical methods were validated by the Official Codes. The LC-DAD method was developed and validated using an RP-18 column, mobile phase containing a mixture of 0.3% triethylamine (pH 3.0) and acetonitrile (85:15, v/v). The microbiological assay was performed by agar diffusion method (3 x 3) and Staphylococcus epidermidis as a microorganism test. Forced degradation studies were performed in both methods. The minimum inhibitory concentration (MIC) was performed by test microdilution and toxicity studies were evaluated using in silico study, cell proliferation, cell viability test, micronuclei and comet assay. LC and a microbiological assay proved linear, accurate, precise, and robust to quantify danofloxacin, but only the LC method showed selectivity to quantify the drug in the presence of its degradation products. These results demonstrate that the LC method is suitable for stability studies of danofloxacin, but a microbiological assay cannot be used to quantify the drug due to the biological activity of the photoproducts. Ex-vivo cytotoxicity and theoretical and experimental genotoxicity were also observed.

Evaluation of the influence of fluoroquinolone chemical structure on stability: forced degradation and in silico studies

ABSTRACT Fluoroquinolones are a known antibacterial class commonly used around the world. These compounds present relative stability and they may show some adverse effects according their distinct chemical structures. The chemical hydrolysis of five fluoroquinolones was studied using alkaline and photolytic degradation aiming to observe the differences in molecular reactivity. DFT/B3LYP-6.31G* was used to assist with understanding the chemical structure degradation. Gemifloxacin underwent degradation in alkaline medium. Gemifloxacin and danofloxacin showed more degradation perceptual indices in comparison with ciprofloxacin, enrofloxacin and norfloxacin in photolytic conditions. Some structural features were observed which may influence degradation, such as the presence of five member rings attached to the quinolone ring and the electrostatic positive charges, showed in maps of potential electrostatic charges. These measurements may be used in the design of effective and more stable fluoroquinolones as well as the investigation of degradation products from stress stability assays.

Avaliação qualitativa e quantitativa de formaldeído em produtos cosméticos para alisamento capilar / Qualitative and quantitative evaluation of formaldehyde in cosmetic products for hair straightening

O formaldeído, comercializado em solução aquosa a 37% (p/p), é um líquido incolor com odor forte e irritante. A presença deste agente cancerígeno em cremes cosméticos resulta em graves riscos à saúde tais como irritação, queimaduras na pele, ferimentos nas vias respiratórias e danos irreversíveis aos olhos e cabelos provocados pela inalação e contato com o produto. Seu uso foi permitido em concentração ≤ 0,2% (p/p) como conservante pela Agência Nacional de Vigilância Sanitária (ANVISA; Brasil, 2001) até junho de 2014, e a substância era adicionada a produtos cosméticos utilizados em escovas progressivas com a finalidade de alisar os cabelos. Diante dos riscos verificados com a utilização da substância, objetivou-se determinar qualitativa e quantitativamente, a incidência deste aldeído em produtos para alisamento capilar, a fim de estimar a concentração que os profissionais e usuários estão expostos, para garantir sua segurança. Observouse que 84,6% das amostras analisadas apresentaram alguma irregularidade, sendo que 61,5% das amostras analisadas apresentaram maior quantidade de formaldeído do que era permitido pela legislação (Brasil, 2001) como conservante e 53,8% apresentou mais de 10 vezes o percentual permitido, concentração irritante para a população em geral. Tendo em vista a gravidade dos dados apresentados, é necessária uma rígida fiscalização da qualidade dos alisantes capilares e estratégias de conscientização sobre riscos à saúde do contato com a substância.

Formaldehyde marketed in a 37% (w/w) aqueous solution is a colorless liquid with a strong irritating odor. The presence of this carcinogen in cosmetics creams results in serious health risks, such as irritation, skin burns, airway injury and irreversible damage to the eyes and hair caused by inhalation and contact with the product. Although its use was allowed up to a concentration of 0.2% (w/w) as a preservative by Brazilian Health Surveillance Agency (ANVISA; Brazil, 2001) until June 2014, the substance is added to cosmetic products used in progressive brushes in order to straighten hair. Given the risks seen with the use of the substance, the objective was to determine qualitatively and quantitatively this aldehyde in products for hair straightening in order to estimate the concentration that professionals involved and users are exposed to ensure their safety. It was observed that 84.6% of the samples showed some irregularity, being that 61.5% of the samples have a higher amount of formaldehyde than allowed by law (Brazil, 2001) as preservative and 53.8% had more than 10 times the allowable percentage, irritating concentration for the general population. Considering the severity of the data presented, it is need a strict quality supervision hair straightening products and strategies to increase awareness about health risks from contact with the substance.