Abstract Certolizumab pegol (CZP) is a Fab’ fragment of the humanized antibody with anti-TNF-α activity that is indicated as therapy for Crohn’s disease and rheumatoid arthritis. Using a BioSep-SEC-S3000 column (300 x 4.6 mm i.d., 5 µm particle size), a size exclusion liquid chromatography (SEC) method was developed. Mobile phase A consisted of 100 mM monobasic sodium phosphate and 200 mM sodium chloride (pH 7.0), while mobile phase B was ethanol (95:5, v/v), and the analysis was performed using a diode array detector (DAD) set to 214 nm and a flow rate of 0.5 ml min-1. In addition, a reversed-phase liquid chromatography (RP-LC) method based on gradient elution was developed on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d., 3.5 µm particle size) kept at 80 °C. Mobile phase A was 0.1% (v/v) TFA in ultrapure water, and mobile phase B was a mixture of propanol, acetonitrile, ultrapure water and TFA (70 + 20 + 9.9 + 0.1, v/v) operated at a flow rate of 1.0 ml min-1, and DAD was applied at 214 nm. CZP elution was achieved with retention times of 5.6 min and 9.0 min for SEC and RP-LC, respectively. (CZP Fab antiTNFα anti TNF α Crohns Crohn s arthritis BioSepSECS3000 BioSepSECS BioSep S3000 S BioSep-SEC-S300 (30 46 4 6 4. id i d i.d. size, , (SEC 10 pH 7.0, 70 7.0 7 0 7.0) 955, 955 95 5, (95:5 v/v, vv v/v v (DAD 21 05 0. min1. min1 1. 1 min-1 addition reversedphase reversed RPLC RP LC (RP-LC 30 C C1 150 (15 35 3 3. 8 °C 01 0.1 (v/v propanol acetonitrile (7 2 99 9 9. min1, 1, 56 5. 90 RPLC, LC, RP-LC respectively BioSepSECS300 S300 BioSep-SEC-S30 (3 i.d 7. (95: min- 15 (1 ( BioSepSECS30 S30 BioSep-SEC-S3 (95 BioSepSECS3 S3 BioSep-SEC-S (9

Verbena litoralis is a plant popularly known as “gervãozinho-do-campo” in Portuguese. It is traditionally used for stomach, liver and gallbladder problems, and as an anti-inflammatory and anthelmintic. The goal of this study was to determine the chemical composition of the crude extract obtained from the aerial parts of V. litoralis by Ultra High Performance Liquid chromatography coupled with Mass Spectrometry in Tandem (UHPLC-MS/MS); assess the anti-inflammatory activity of ethyl acetate fraction and of crude extract; and verify liver, kidney and pancreas damage. In this study, the chemical composition of the extract was identified via UHPLC/MS/MS, assessing the anti-inflammatory activity of the crude extract and the acetate fraction in an induction model of the granulomatous tissue, as well as given liver, kidney and pancreas damage markers. Chlorogenic acid, luteolin, caffeic acid, apigenin, p-coumaric acid, vanillic acid, ferulic acid and quercetin were quantified in the extract. After the seven-day treatment, the granuloma of the animals treated with the plant extract and fraction presented values very close to the positive control (nimesulide). The V. litoralis crude extract and ethyl acetate fraction show anti-inflammatory activity similar to the nimesulide without evidence of liver, kidney and pancreas damage, which attributes the plant’s pharmacological action to the flavonoids found.

Recombinant human interferon beta 1b (rhIFNβ-1b) is clinically used to treat multiple sclerosis. A reversed-phase liquid chromatography (RP-LC) method was carried out on a Jupiter C4 column (250 mm × 4.6 mm i.d.). The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B was acetonitrile with 0.1% TFA run at a flow rate of 1.0 mL/min. A size exclusion liquid chromatography (SE-LC) method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm i.d.). The mobile phase consisted of 1 mM monobasic potassium phosphate, 8 mM sodium phosphate dibasic and 200 mM sodium chloride buffer pH 7.4, run isocratically at a flow rate of 0.8 mL/min. Retention times were 31.87 and 17.78 min, and calibration curves were linear over the concentration range of 1-200 µg/mL (r2 = 0.9998) and 0.50-200 µg/mL (r2 = 0.9999), respectively, for RP-LC and SE-LC, with detection at 214 nm. Liquid chromatography (LC) methods were validated and employed in conjunction with the in vitro bioassay to assess the content/potency of rhIFNβ-1b, contributing to improve the quality control and to ensure the efficacy of the biotherapeutic.

A liquid chromatography method was developed and validated for the determination of phenobarbital in human plasma using phenytoin as internal standard. The drugs were extracted from plasma by liquid-liquid extraction and separated isocratically on a C12 analytical column, maintained at 35 ºC, with water:acetonitrile:methanol (58.8:15.2:26, v/v/v) as mobile phase, run at a flow rate of 1.2 mL/min with detection at 205 nm. The method was linear in the range of 0.1-4 μg/mL (r²=0.9999) and demonstrated acceptable results for the precision, accuracy and stability studies. The method was successfully applied for the bioequivalence study of two tablet formulations (test and reference) of phenobarbital 100 mg after single oral dose administration to healthy human volunteers.

A dissolution test for in vitro evaluation of tablet dosage forms containing 10 mg of rupatadine was developed and validated by RP-LC. A discriminatory dissolution method was established using apparatus paddle at a stirring rate of 50 rpm with 900 mL of deaerated 0.01 M hydrochloric acid. The proposed method was validated yielding acceptable results for the parameters evaluated, and was applied for the quality control analysis of rupatadine tablets, and to evaluate the formulation during an accelerated stability study. Moreover, quantitative analyses were also performed, to compare the applicability of the RP-LC and the LC-MS/MS methods.

A liquid chromatography-tandem mass spectrometry method with atmospheric pressure chemical ionization (LC-APCI/MS/MS) was validated for the determination of etoricoxib in human plasma using antipyrin as internal standard, followed by on-line solid-phase extraction. The method was performed on a Luna C18 column and the mobile phase consisted of acetonitrile:water (95:5, v/v)/ammonium acetate (pH 4.0; 10 mM), run at a flow rate of 0.6 mL/min. The method was linear in the range of 1-5000 ng/mL (r²>0.99). The lower limit of quantitation was 1 ng/mL. The recoveries were within 93.72-96.18%. Moreover, method validation demonstrated acceptable results for the precision, accuracy and stability studies.

An HPLC method was validated to assay lamivudine and zidovudine combined in tablets. The chromatographic separation was carried out using methanol and acetate buffer pH 6.5 (50:50 v/v) and a RP-18 column, as mobile and stationary phase, respectively. The UV detection was at 270 nm. The method was linear in the range of 24 - 36 µg/mL (lamivudine) and 48 - 72 µg/mL (zidovudine). The recovery (accuracy) ranged from 101.35% to 103.04% and the precision (repeatability and intermediate precision) was less than 2%. The method can be also applied to the quantification of these drugs in the dissolution test of tablets containing both drugs.

Realizou-se a avaliação de pirogênios em produtos veterinários de uso parenteral, pelo método da hipertermia em coelhos, calculando-se, para o teste das amostras, doses com concentrações de três a sete vezes superiores à terapêutica. Preconizou-se como resposta positiva o aumento de temperatura de 0,6°C. Utilizou-se também o ensaio do lisado de amebócitos do Limulus (LAL) por geleificação, semiquantitativo, executando o teste de interferentes, validando o procedimento e estabelecendo a máxima diluição válida para a análise de cada produto. Paralelamente, efetuou-se avaliação comparativa de amostras com o método do LAL cromogênico, quantitativo, demonstrando correlação e reprodutibilidade dos resultados. Avaliaram-se vinte e oito produtos de diferentes classes farmacológicas, observando-se que dois não cumpriram as especificações, sendo reprovados. Sugere-se que as especificações estudadas sejam adotadas, contribuindo para aprimorar o controle de contaminantes, garantindo a qualidade e a segurança dos produtos veterinários.

The rabbit pyrogen test was used to evaluate veterinary products, suggesting the temperature rise of 0.6°C as the ending point for the positive results. The test doses were calculated based on the therapeutic dose increased from three to seven times. The semi-quantitative Limulus amoebocyte lysate (LAL) gel clot test was performed and compared to the LAL spectrophotometric chromogenic, quantitative assay. The comparative evaluation of the samples showed correlation and reproducibility of the results. The interference test was carried out, the procedure validated and the maximum valid dilution established for the analysis of the products without Pharmacopoeial specifications. Two of the twenty-eight products of different pharmacological groups evaluated didn't meet the requirements and were reproved. The specifications investigated are suggested to be used for the purity evaluation of the veterinary parenteral products, as a contribution to assure the quality and safety of the products.