Factorial design applied to LC-ESI-QTOF mass spectrometer parameters for untargeted metabolomics.

Investigations of untargeted metabolomics are based on high-quality data acquisition usually from multiplatform systems that include high-resolution mass spectrometry equipment. The comprehensive set of results is used as data entry for bioinformatics and machine learning sciences to access reliable metabolic and biochemical information for clinical, forensic, environmental, and endless applications. In this context, design of experiments is a powerful tool for optimizing data acquisition procedures, using a multivariate approach, which enables the maximization of a high-quality amount of information with reduced number of tests. In this study, we applied a 33 Box-Behnken factorial design with central point triplicate for optimizing the ionization of an HPLC-ESI-QTOF method used for screening urine samples. Nozzle voltage (V), fragmentor voltage (V) and nebulizer pressure (psig) were the factors selected for variation. The response surface methodology was applied in the molecular features extracted at each level, resulting in a statistical model that helps evaluating the synergic interaction between these factors. Together with the qualitative analysis of the resulting total ion chromatograms, we came across a reproducible (6.14% RSD) and highly efficient method for untargeted metabolomics of human urine samples. The proposed method can be useful for applications in several urine-based metabolomics-driven studies, as the factorial design can be applied in the development of any analytical protocol considering different LC-MS setups.

Determination of creatinine in urine and blood serum human samples by CZE-UV using on-column internal standard injection.

Creatinine is a well-stablished biomarker for kidney malfunctions and for normalization parameter of urinary quantitative information. Recently, metabolic studies have been discovering other functionalities for creatinine tests in human urine and blood serum. In this work we present an enhanced capillary electrophoresis (CE) based protocol for determination of creatinine. CE is a high-throughput separation technique that have been getting attention through the last decades and might be considered to be adopted as an analytical instrumentation for clinical purposes. In the proposed method, we performed a short injection program with on-column addition of internal standard. Additionally, the method allows a simultaneous screening of non-proteinogenic amino acids that could be considered for metabolomics purposes. We design a pilot study that successfully estimated the creatinine value in 100 urine samples with (2.85 ± 1.78) mg dL-1 LOD; (8.24 ± 5.93) mg dL-1 LOQ and 82.4% accuracy. Considering that serum creatinine is also included in the clinical laboratory routines for estimated Glomerular Filtration Rate dosage, the method was complementary applied to 10 blood serum samples, which resulted in a model with (0.4 ± 0.2) mg dL-1 LOD; (2.0 ± 0.6) mg dL-1 LOQ and 83.8% of accuracy. All results were in agreement with reference values. The proposed method promotes a great analytical frequency and reproducibility with enhanced specificity compared with the ongoing protocol by Jaffe's reaction, thereby proving to be useful as an alternative for creatinine exams that might help complete a diagnosis of a series of health-related issues.

Determination of antimalarial drugs in pharmaceutical formulations and human blood by liquid chromatography: a review.

Malaria is a life-threatening disease being treated by oral medication. This is the best treatment to reduce morbidity and mortality, prevent disease progression to the most severe form, lower the transmission of the disease and hinder the appearance of strains resistant to antimalarials. According to the World Health Organization, the most common antimalarial drugs are chloroquine, primaquine, mefloquine, lumefantrine, artemether, and artesunate in single dosage forms or fixed-dose combination. Within this context, the present review aims to show the evolution of different analytical methods that have been applied to the determination of these antimalarial drugs in pharmaceutical formulations and human blood by liquid chromatography in the last 10 years, along with statistical analyses of the methods.

Simultaneous separation of artesunate and mefloquine in fixed-dose combination tablets by CZE-UV.

A novel method was proposed for simultaneous determination of artesunate (ATS) and mefloquine (MFQ) in fixed-dose combination tablets by capillary zone electrophoresis with simultaneous direct and indirect detection by ultraviolet (CZE-UV). The background electrolyte, consisting of 30/15 mmol L-1 TRIS/3,5-dinitrobenzoic acid buffer at pH 8.2, a chromophore buffer, was selected taking into account a detailed study involving the effective mobility vs. pH curves of the analytes and electrolyte compounds in association with the very low molar absorptivity of ATS. Suitable separation conditions, considering voltage, temperature and buffer concentration as factors, were achieved through the 33 Box-Behnken design investigation. The optimum baseline separation conditions were: injection pressure of 30 mbar for 10 s, cartridge temperature of 22.5 °C and positive voltage of +30 kV. The method proved to be rapid (5 minutes), simple, selective, linear (r2 > 0.98), precise (relative standard deviation (RSD): ATS < 2.9% and MFQ < 2.2%) and accurate (recoveries: ATS 98.13-102.96% and MFQ 98.75-106.77%), proving to be suitable for routine quality control analysis.

Comparação entre métodos de diagnósticos para a quantificação da lipoproteína de baixa densidade em pacientes atendidos em um Hospital Universitário / Comparison between diagnostic methods for the quantification of low-density lipoprotein in patients attended at a University Hospital

Introdução: Estudos relatam uma forte ligação entre as dislipidemias e as ateroscleroses. Por esta razão, exames como o perfil lipídico são realizados rotineiramente com o intuito de prevenção e monitoramento dessas doenças. A lipoproteína de baixa densidade possui grande destaque por apresentar maior relação com fatores de risco para o desenvolvimento de doenças ateroscleróticas. Métodos diretos de obtenção dos valores dessa lipoproteína são confrontados com resultados obtidos usualmente na rotina, através de equações que fornecem valores estimados. Objetivo: Comparar os métodos de diagnósticos utilizados para a obtenção da lipoproteína de baixa densidade através das Fórmulas de Friedewald e Martin com os resultados obtidos por metodologia automatizada, em pacientes atendidos em um Hospital Universitário de Juiz de Fora - MG. Material e Métodos: Foram coletadas amostras de sangue venoso para a obtenção do soro de 208 pacientes. Quantificaram-se os níveis de colesterol total, triglicerídeos e da lipoproteína de alta densidade para a obtenção da lipoproteína de baixa densidade através das equações de Friedewald e Martin. Resultados: Há uma correlação positiva entre os resultados de lipoproteína de baixa densidade calculados pelos métodos de Martin e direto (ρ=0,916), e uma correlação positiva entre os resultados pelos métodos de Friedewald e direto (ρ=0,915). Discussão: Foi observada uma correlação positiva entre os valores de colesterol e de lipoproteína de baixa densidade pelas três metodologias. O método de Bland-Altman foi utilizado para comparação dos resultados obtidos pelas equações e pela metodologia direta. Conclusão: Ainda que as equações de Friedewald e Martin tenham apresentado boa correlação com a lipoproteína de baixa densidade medida por metodologia direta, estudos que relacionam doenças arteriais ateroscleróticas à lipoproteína de baixa densidade devem considerar a quantificação direta desta a fim de abranger os indivíduos com suas diversas especificidades.

Introduction: Studies have reported a strong link between dyslipidemia and atherosclerosis. For this reason, exams such as the lipid profile are routinely performed for the prevention and monitoring of these diseases. Among the lipid indices, low density lipoproteins should be highlighted because due to their greater relation with risk factors for the development of atherosclerotic diseases. Therefore, direct methods of obtaining low density lipoproteins values, considered more accurate, are confronted with results usually obtained in the routine, through equations that provide estimated values. Objective:We compared the diagnostic methods used to obtain low density lipoproteins through the Friedewald and Martin formulas with the results obtained by automated methodology in patients attended at a University Hospital of Juiz de Fora ­ MG. MaterialandMethods: A total of 208 patients were recruited and venous blood samples were collected to obtain serum. The levels of total cholesterol, triglycerides and high density lipoprotein were quantified to obtain low density lipoproteins through the Friedewald and Martin equations. Results:A positive correlation between low density lipoproteins results has calculated by Martin and direct methods (ρ = 0.916), and positive correlation between Friedewald results and direct (ρ = 0.915). Discussion: We observed a positive correlation between the values of cholesterol and low density lipoproteins by the three methodologies. The Bland-Altman method has been used to compare the results obtained in search and methodology. Conclusion:Although the Friedewald and Martin equations have a good correlation with low density lipoproteins as measured by direct methodology, studies that relate atherosclerotic arterial diseases to low density lipoproteins should consider the direct quantification of this lipoprotein in order to cover individuals with their different specificities.

Sub-minute determination of rifampicin and isoniazid in fixed dose combination tablets by capillary zone electrophoresis with ultraviolet absorption detection.

A validated sub minute capillary zone electrophoresis method with direct ultraviolet absorption detection for simultaneous determination of isoniazid and rifampicin in fixed-dose combination tablets was developed. Background electrolyte was defined based on the analytes effective mobility curve and it was composed by 20 mmol/L of sodium carbonate/sodium bicarbonate at pH 10.2. A careful validation procedure considering the main figures of merit was performed. Regression models were satisfactory for isoniazid and rifampicin, showing no lack of fit within 95% significance interval. Interday and intraday precision were evaluated in standard and sample and slight relative standard deviations were achieved for concentration, area, and migration time. Recovery values for accuracy in two levels were 99.97 and 90.08% for isoniazid and 95.45 and 95.12% for rifampicin. The limits of detection for isoniazid and rifampicin were 0.22 and 0.34 mg/L, respectively, and the limits of quantification were 0.74 and 1.13 mg/L, respectively. Method selectivity was verified by injecting diluent, background electrolyte, a standard mixture, and a sample, confirming no interferent peaks. The method proved to be simple, environmentally friendly, sensitive, and was successfully applied for simultaneous quantification of isoniazid and rifampicin in fixed-dose combination tablets.

Prevalência de resultados positivos de VDRL (Venereal Disease Research Laboratory) e análise das variáveis epidemiológicas em pacientes atendidos no serviço de saúde pública / Prevalence of VDRL (Venereal Disease Research Laboratory) positive results and epidemiological variables analysis in patients assessed on a public health service

Syphilis is an infection caused by Treponema pallidum of sexual, blood and vertical transmission. Despite being easy to diagnose and treat, its incidence has been increasing in Brazil in recent years, and it is being considered a public health problem in the country and worldwide. The aim of this study was to describe the prevalence of positive results for VDRL (Venereal Diseases Research Laboratory) in the city of Juiz de Fora / MG between 2014 and 2016, as well its epidemiological characteristics. 25,735 VDRL results were analyzed from the database of the Central Laboratory (LACEN) of the city hall of Juiz de Fora. The results were then related to the variables year, gender, age, pregnant or not and the region of the city where the test was performed. The prevalence of positive results was 5.55%, with the highest number of cases recorded in 2015. Reactive cases were more prevalent in the central region, in men and a higher prevalence was obtained for the age group of 12 to 18 years. Among women, it was observed higher VDRL positivity in non-pregnant women from August 2015 to December 2016. The data obtained in this study indicates an increase in prevalence of syphilis from 2014 to 2015, followed by a reduction in the following year, among the population served by SUS, where young men represent the population at greater risk.

Estudo comparativo de biodisponibilidade relativa entre duas formulações contendo Clozapina 100 mg em pacientes portadores de esquizofrenia / Comparative study of relative bioavailability between two formulations containing Clozapine 100 mg in patients with schizophrenia

Este estudo teve por objetivo avaliar a bioequivalência entre dois produtos contendo Clozapina 100 mg (produto teste: LifalClozapina® da Lifal ? Laboratório Industrial Farmacêutico de Alagoas S/A. O produto referência: Leponex® do Laboratório Novartis Biociências S/A.) em 40 voluntários. O estudo foi aberto, randomizado, do tipo cross-over, em estado de equilíbrio, com dois períodos (duas sequências), nos quais os voluntários receberam, em cada período, a formulação teste ou a formulação referência. A biodisponibilidade relativa das formulações seguidas à administração oral foi avaliada com base em comparações estatísticas de parâmetros farmacocinéticos relevantes obtidos de dados de amostra sanguínea dos voluntários, sendo as amostras coletadas em período de 24h. A concentração de Clozapina foi medida a partir de método analítico apropriado e válido. As medidas farmacocinéticas utilizadas foram: Cmin, Cmax e ASCt. As diferenças médias (± DP) entre referência e teste foram: 0,1615 ± 0,3404 (ng/mL); -0,0969 ± 0,4131 (ng/mL) e 0,9143 ± 3,9941 (ng*h/mL). Os intervalos de confiança para biequivalência média atenderam aos limites determinados pela Agência Nacional de Vigilância Sanitária (ANVISA) de 80 a 125%, sendo os dois medicamentos considerados bioequivalentes. Assim, produtos teste e referência são considerados intercambiáveis.

This study aimed to evaluate the bioequivalence between two products containing Clozapine 100 mg (testing product: LifalClozapina® from Lifal ? Industrial Pharmaceutical Laboratory of Alagoas S/A and the reference product: Leponex® from the Novartis Biociências S/A. Laboratories) in 40 volunteers. The study was open, randomized, crossover type, in a state of equilibrium, with two periods (two sequences) in which volunteers were given, in each period, the testing formulation or the reference formulation. The relative bioavailability of the formulations following oral administration was evaluated based on statistical comparisons of relevant pharmacokinetic parameters obtained from blood samples in a 24 hours period. The concentration of Clozapine was measured using an appropriate and valid analytical method. The pharmacokinetic measures used were: Cmin and Cmax, and ASCt. The mean differences (± SD) between reference and testing were: 0.1615 ± 0.3404 (ng/mL); -0.0969 ± 0.4131 (ng/mL); and 0.9143 ± 3.9941 (ng*h/ mL). The confidence intervals for medium bioequivalence met the limits determined by the National Agency of Sanitary Surveillance (ANVISA) of 80 to 125%, and both medications were considered bioequivalent. Thus, the testing and reference products are considered interchangeable

Development and validation of an HPLC method for the simultaneous determination of artesunate and mefloquine hydrochloride in fixed-dose combination tablets

The present study developed and validated an HPLC method for the simultaneous determination of artesunate (AS) and mefloquine hydrochloride (MQ) in fixed-dose combination tablets, according to ICH guidelines. The chromatographic separation was carried out on an XBridge C18 (250 x 4.6 mm i.d., 5 µm particle size, Waters) analytical column. The mobile phase included a 0.05 M monobasic potassium phosphate buffer (pH adjusted to 3.0 with phosphoric acid) and acetonitrile (50 + 50, v/v). The flow rate was 1.0 mL/min, and the run time was 13 minutes. A dual-wavelength approach was employed: AS detection was performed at 210 nm and MQ was detected at 283 nm, using a diode array detector. Stability of sample solutions was evaluated for 8 hours after preparation, during which time the solutions remained stable. Youden's test was employed to evaluate robustness. The method proved to be linear (r²>0.99), precise (RSD<2.0%), accurate, selective, and robust, proving to be appropriate for routine drug quality control analysis.

Um método por cromatografia a líquido de alta eficiência para a determinação simultânea de artesunato (AS) e cloridrato de mefloquina (MQ) em comprimidos em dose fixa combinada foi desenvolvido e validado, de acordo com as normas do ICH. A separação cromatográfica foi realizada com uma coluna analítica XBridge C18 (250 x 4,6 mm d.i., partículas de 5 µm, Waters). A fase móvel foi constituída de tampão fosfato monobásico de potássio 0,05 M (pH ajustado para 3,0 com ácido fosfórico) e acetonitrila (50 + 50, v/v). O fluxo da fase móvel foi de 1,0 mL/min e o tempo de corrida foi de 13 minutos. Utilizaram-se dois comprimentos de onda: a detecção do AS foi realizada em 210 nm e a de MQ foi realizada em 283 nm, utilizando-se um detector de arranjo de diodos. A estabilidade das soluções padrão e amostra foi avaliada por 8 horas após sua preparação e as soluções permaneceram estáveis nesse período. O teste de Youden foi empregado para a avaliação da robustez do método. O método se mostrou linear (r²>0,99), preciso (DPR<2,0%), exato, seletivo e robusto, sendo adequado para análises rotineiras de controle de qualidade dos medicamentos.

Determination of ofloxacin in tear by HPLC-ESI-MS/MS method: comparison of ophthalmic drug release between a new mucoadhesive chitosan films and a conventional eye drop formulation in rabbit model.

Ofloxacin, second-generation fluoroquinolone derivative, is one of the most commonly used to treat and prevent superficial ocular infection in animals and human beings. However, poor bioavailability, rapid elimination, and non compliance by patients are several problems associated with ocular route. Ophthalmic controlled drug delivery offers the potential to enhance the efficacy of treatment for pathological conditions, while reducing the side effects and the toxicity associated with frequent applications. Specific analytical methods to determine drugs in eye are needed to analyze and compare the new controlled release ocular devices with those conventional eye drops. The topical eye administration of ophthalmic drugs induces lachrymation, and the tear promotes a drug wash out. Quantify drugs in tear is a good tool to study their kinetic comportment in the eye. A liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantitation of ofloxacin in rabbits' tears was developed and validated. The tear was collected with tear strips, extracted by a liquid extraction procedure and then separated on an ACE C(18) column with a mobile phase composed of 0.15% aqueous formic acid and methanol (60:40, v/v). Calibration curve was constructed over the range of 10-5000 ng/mL for ofloxacin. The mean R.S.D. values for the intra-run and inter-run precision were 5.15% and 4.35%, respectively. The mean accuracy value was 100.16%. The validated method was successfully applied to determine the ofloxacin concentration in tears of rabbits treated with a mucoadhesive chitosan films and a conventional eye drop formulation.

Liquid chromatography-tandem mass spectrometry for the simultaneous quantitation of artemether and lumefantrine in human plasma: application for a pharmacokinetic study.

A liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the simultaneous quantitation of artemether and lumefantrine in human plasma was developed and validated. Artesunate was used as an internal standard (IS). The analytes were extracted by a protein precipitation procedure and separated on a reversed-phase Zorbax SB-Ciano column with a mobile phase composed of methanol and 10mM aqueous ammonium acetate containing 0.2% (v/v) acetic acid and 0.1% (v/v) formic acid. Multiple reaction monitoring was performed using the transitions m/z 316 → m/z 267, m/z 530 → m/z 348 and m/z 402 → m/z 267 to quantify artemether, lumefantrine and artesunate, respectively. Calibration curves were constructed over the range of 10-1000 ng/mL for artemether and 10-18,000 ng/mL for lumefantrine. The lower limit of quantitation was 10 ng/mL for both drugs. The mean R.S.D. values for the intra-run precision were 2.6% and 3.0% and for the inter-run precision were 3.6% and 4.6% for artemether and lumefantrine, respectively. The mean accuracy values were 102.0% and 101.2% for artemether and lumefantrine, respectively. No matrix effect was detected in the samples. The validated method was successfully applied to determine the plasma concentrations of artemether and lumefantrine in healthy volunteers, in a one-dose pharmacokinetic study, over the course of 11 days.