Abstract Vancomycin is a glycopeptide antibiotic mainly excreted by glomerular filtration. Therefore, patients undergoing hemodialysis tend to accumulate its crystalline degradation product, which has been associated with cross-reaction in commercial immunoassays. The aim of this study was to assess the performance of two commercial immunoassays for measuring vancomycin levels in patients undergoing hemodialysis. This method-comparison study enrolled patients undergoing hemodialysis at two hospitals in Porto Alegre, Brazil. Vancomycin serum concentrations measured by Chemiluminescent Microparticle Assay (CMIA) and measured by Kinetic Interaction of Microparticles in Solution (KIMS) were compared with Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS). A total of 64 samples from 42 patients and 54 samples from 23 patients were included in CMIA and KIMS groups. Both measurements were highly correlated with LC-MS/MS, with Spearman rank correlation coefficient r = 0.840 (p < 0.001) and r = 0.926 (p < 0.001), respectively. No deviation of linearity was observed (p = 0.81 and p = 0.49, respectively). The mean difference between CMIA and LC-MS/MS was -1.19 μg/mL and between KIMS and LC-MS/MS was -2.28 μg/mL. LC-MS/MS measured levels were, on average, 2.64 % higher than CMIA and 8.81 % higher than KIMS. CMIA and KIMS revealed accurate commercial methods to measure vancomycin serum concentrations in patients undergoing hemodialysis. filtration Therefore product crossreaction cross reaction methodcomparison method comparison Alegre Brazil (CMIA (KIMS LCMS/MS. LCMSMS LC MS/MS . MS (LC-MS/MS) 6 4 5 2 groups LCMS/MS, MS/MS, 0840 0 840 0.84 0.001 0001 001 0926 926 0.92 0.001, , respectively 081 81 0.8 049 49 0.49 respectively) LCMS/MS 1.19 119 1 19 -1.1 μgmL μg mL 2.28 228 28 -2.2 average 264 2.6 881 8 8.8 LCMS MSMS (LC-MS/MS 084 84 0.00 000 00 092 92 0.9 08 0. 04 0.4 1.1 11 -1. 2.2 22 -2. 26 2. 88 8. 0.0 09 9 1. -1 -2 -

We developed and validated an UHPLC-MS/MS method for the quantification of phosphatidylethanol (PEth) in dried blood spots (DBS). Sample preparation was a liquid extraction and chromatographic separation was performed in an Acquity C8 column. The mobile phases were 4 mM ammonium acetate in water (A) and acetonitrile (B) at 24:76 (v/v). The total analytical run time was 6 minutes, with retention time of 3.36 min for PEth and 4.13 min for phosphatidylpropanol. The method was linear from 10 to 3000 ng mL-1, specific, with, precise and accurate. The analyte was stable in DBS stored from -20 to 45 °C for 21 days. Matrix effect was compensated with the internal standard (-5.21% to + 6.09%). The method was applied in the evaluation of alcohol consumption in DBS from 25 chemical dependents. The PEth concentrations ranged from 14.5 to 2380.8 ng mL-1, with significant correlation with the self-report alcohol consumption AUDIT scores (r=0.41). UHPLCMS/MS UHPLCMSMS UHPLC MS/MS MS (PEth DBS. . (DBS) C column A (A B (B 2476 24 76 24:7 v/v. vv v/v v (v/v) minutes 336 3 36 3.3 413 13 4.1 phosphatidylpropanol 1 300 mL1, mL1 mL 1, mL-1 specific accurate 20 -2 2 days 5.21% 521 5 (-5.21 6.09%. 609 6.09% 09 6.09%) dependents 145 14 14. 23808 2380 8 2380. selfreport self report r=0.41. r041 r r=0.41 0 41 (r=0.41) UHPLCMS MSMS (DBS 247 7 24: (v/v 33 3. 4. 30 mL- - 5.21 52 (-5.2 60 6.09 238 r04 r=0.4 (r=0.41 5.2 (-5. 6.0 23 r0 r=0. (r=0.4 5. (-5 6. r=0 (r=0. (- r= (r=0 ( (r= (r

We developed a UPLC-MS/MS method for the quantification of the ethanol metabolites ethyl-glucuronide (EtG) and ethyl-sulfate (EtS) in blood samples. Sample preparation was a protein precipitation with acetonitrile 0.1% formic acid containing internal standards (IS) 0.5 µg mL-1 EtG-D5 and EtS-D5. Chromatographic separation was performed in a fluoro-phenyl column, mobile phases were 0.1% formic acid in water (A) and acetonitrile (B) in gradient elution from 5% B to 70% B, with 10 min run time. Ionization was performed with electrospray source at 550 °C in negative mode, quantification mass transitions of m/z EtG 221- 75, EtS 125- 80.1, EtS-D5 130- 79.9, and EtG-D5 226- 75.1. The method was linear from 0.1 to 18 µg mL-1 for EtG and 0.02 to 6 µg mL-1 for EtS, specific, with no interfering peaks during the blank samples analyses. The method was precise and accurate, the matrix effect was compensated with the IS (-12% a + 6.1%). EtG and EtS were analyzed in 28 blood samples from trauma patients with positive alcoholaemia. The concentrations of EtG and EtS ranged from 0.25 to 14.11 µg mL-1 and 0.13 to 5.53 µg mL-1, with high correlation with ethanol concentrations, r=0.739 and r=0.817 (p <0,001), respectively. UPLCMS/MS UPLCMSMS UPLC MS/MS MS ethylglucuronide ethyl glucuronide (EtG ethylsulfate sulfate (EtS 01 0 1 (IS 05 5 0. mL1 mL mL- EtGD5 EtGD D5 D EtG-D EtSD5. EtSD5 EtSD D5. fluorophenyl fluoro phenyl column A (A (B 70 time 55 C mode mz m z 221 75 125 801 80 80.1 EtS-D 130 799 79 9 79.9 226 751 75.1 002 02 0.0 specific analyses accurate 12% 12 (-12 6.1%. 61 6.1% . 6.1%) 2 alcoholaemia 025 25 0.2 1411 14 11 14.1 013 13 553 53 5.5 mL1, 1, r0739 r 739 r=0.73 r0817 817 r=0.81 p <0,001, 0001 <0,001 , 001 <0,001) respectively UPLCMS MSMS 7 22 8 80. 79. 75. 00 (-1 6.1 141 14. 5. r073 73 r=0.7 r081 81 r=0.8 000 <0,00 (- 6. r07 r=0. r08 <0,0 ( r0 r=0 <0, r= <0 <

Abstract Depression plays an important role in non-adherence to medical recommendations. Fluoxetine is a first line of depression treatment. This study aimed to evaluate adherence to drug therapy in fluoxetine users by different methods. A cross-section study was conducted with 53 depressed patients on fluoxetine for at least six months. Drug therapy adherence was assessed by validated questionnaires [Brief Medication Questionnaire (BMQ) and Morisky-Green test (MG)] and by the blood concentration of fluoxetine and its active metabolite norfluoxetine. Blood samples were taken before the daily first dose of fluoxetine. The plasmatic concentration of fluoxetine and norfluoxetine indicated that 58.5% volunteers were within the recommended therapeutic range and thus considered adherent to drug therapy. However, questionnaires indicated a non-adherent majority: 41.5% patients had a high degree of adherence in MG and only 13.2% were adherent to pharmacological treatment in BMQ. Most fluoxetine users showed a plasma concentration of fluoxetine and norfluoxetine within the therapeutic range, despite the low adherence to the drug therapy evaluated by the questionnaires. Thus, we suggest that plasma levels of fluoxetine and norfluoxetine could be used as the main method to check adherence to treatment. nonadherence non recommendations methods crosssection cross section 5 months Brief BMQ (BMQ MoriskyGreen Morisky Green (MG) 585 58 58.5 However nonadherent majority 415 41 41.5 132 13 2 13.2 Thus (MG 58. 4 41. 1 13.

Benzene is an important environmental and occupational pollutant, and recognized as human carcinogen. S-phenylmercapturic acid (SPMA) is a highly specific biomarker of exposure to benzene, applied in occupational toxicology to assess low levels of benzene exposure. The objective of this study was to develop and validate a one-step liquid extraction bioanalytical method for the quantification of SPMA in urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was applied to the evaluation of benzene exposure in an occupational setting. The assay was linear from 0.5 to 500 ng mL-1 (r>0.99), accurate (91.4-105.2%) and precise, with CV% between 4.73 and 9.96%. SPMA was stable in urine for 90 days at -20 °C. Airborne benzene concentrations, urinary levels of SPMA and t,t-MA were significantly higher in gas station workers (n=30) in comparison to outdoor workers (n=14) and individuals non-occupationally exposed to benzene (n=34). Benzene airborne levels had higher correlation with urinary SPMA (r=0.532, p<0.001) than with t,t-MA (r=0.338, p=0.04), demonstrating the applicability of this biomarker for monitoring low levels of benzene exposure.

RESUMO OBJETIVOS: Avaliar a sazonalidade da 25(OH)vitamina D3 [25(OH)D3] e fatores associados em homens adultos saudáveis, que se exercitavam ao ar livre pelo menos 50 min duas vezes por semana, das 10 às 16h, em uma região subtropical. MÉTODOS: Sangue foi colhido no fim das estações para medir 25(OH)D3, por cromatografia líquida em tandem com espectroscopia de massas. A radiação ultravioleta foi estimada por radiômetro, calculando diariamente a resposta fotobiológica para sintetizar vitamina D na pele humana (D-VitD). RESULTADOS: A prevalência de 25(OH)D3 <20ng/mL foi sazonal (p=0.000): 8.7% (n=6/69), 1.5% (n=1/66), 0% (n= 0/64), e 21.7% (n=13/60), respectivamente, no final do inverno, primavera, verão e outono. A prevalência, ajustada para comparações múltiplas, foi maior no inverno do que no verão (p=0.026) e no outono do que na primavera (p=0.001) e verão (p=0.000). A 25(OH)D3 não se associou com o índice de massa corporal (p=0.207), gordura corporal (p=0.064) ou fototipo (p=0.485), na análise univariada. Associou-se à D-VitD nos 30 dias antes da coleta de sangue (p=0.000), ajustada para gordura corporal. Houve sazonalidade na prevalência de 25(OH)D3 <30ng/mL (p=0.000): 69.6% (n=48/69), 68.2% (n=45/66), 43.8% (n=28/64), e 88.4% (n=53/60), respectivamente, no inverno, primavera, verão e outono. CONCLUSÕES: Em uma região subtropical, houve sazonalidade na 25(OH)D3 em homens adultos, saudáveis, embora se exercitassem ao ar livre pelo menos 50 minutos duas vezes por semana, usando shorts e camiseta. 25(OH)D3 <20ng/mL foi 21.7% no outono e a D-vitD 30 dias antes da coleta do sangue foi o único fator associado de modo independente à 25(OH)D3.

SUMMARY OBJECTIVE: To evaluate seasonal variation of 25(OH)vitamin D [25(OH)D3] levels, and factors associated with it, in healthy adult men, who exercised outdoors for 50 min., at least twice a week, from 10AM to 4PM, in a Brazilian semitropical region. METHODS: Blood samples were collected at the end of each season for 25(OH)D3, measured by liquid chromatography with tandem mass spectrometry. Ultraviolet irradiation was estimated by radiometer, calculating the daily photobiological response to vitamin D synthesis in human skin (D-VitD). The prevalence of 25(OH)D3 <20ng/mL changed with the seasons (p=0.000): 8.7% (n=6/69), 1.5% (n=1/66), 0 (n=0/64), and 21.7% (n=13/60), respectively, at the end of winter, spring, summer, and autumn. The prevalence, adjusted for multiple comparisons, was higher in winter than summer (p=0.026), and in autumn than spring (p=0.001) and summer (p=0.000). There were no associations of 25(OH) D3 levels with BMI (p=0.207), body fat (p=0.064), and phototype (p=0.485), in univariate analysis. It was associated with D-VitD in the 30 days before blood sampling (p=0.000), after adjustment to body fat. The prevalence of 25(OH)D3 <30ng/mL varied seasonally (p=0.000): 69.6% (n=48/69), 68.2% (n=45/66), 43.8% (n=28/64), and 88.4% (n=53/60), respectively, in winter, spring, summer, and autumn. CONCLUSIONS: In a Brazilian subtropical region, a seasonal variation in 25(OH)D3 was observed in healthy adult males, although they spent at least 50 min outdoors twice a week, wearing shorts and T-shirts. 25(OH)D3 <20ng/mL was 21.7% in autumn; D-vitD 30 days prior to blood sampling was the only factor independently associated with 25(OH)D3 levels.

A highly sensitive ultra-performance liquid chromatography tandem mass spectrometry method for the determination of Docetaxel and metabolites (M1+M3) in human plasma samples was developed and validated. The method employs a liquid-liquid extraction and a reversed phase separation Acquity® C18 column BEH C18 (150 x 2.1 mm, 1.7 µm). Mobile phase was a mixture of formic acid 0.1% (eluent A) and acetonitrile plus 0.1% formic acid (eluent B) in isocratic mode. Total analytical run time was 7 min. The interval of method linearity interval was 50 to 3000 ng mL-1 for docetaxel and 5.0 to 300 ng mL-1, for M1+M3 metabolites. Intra-assay and inter-assay precision was coefficient of variation 2.5-15.7% and 5.9-12.6%, respectively and accuracy was 89.0-114.0%. The method was successfully applied in the measurement of docetaxel plasma levels and area under the curve calculation in 31 cancer patients, demonstrating adequate analytical performance for the clinical application in the therapeutic drug monitoring and pharmacokinetics studies. ultraperformance ultra M1M3 MM M1 M3 M (M1+M3 validated liquidliquid Acquity C C1 150 (15 21 2 1 2. mm 17 1. µm. µm . µm) 01 0 0.1 eluent B mode min 5 mL1 mL mL- 5. 30 mL1, 1, M1+M Intraassay Intra assay interassay inter 2.515.7% 25157 2.5 15.7% 15 2.5-15.7 5.912.6%, 59126 5.9 12.6%, 9 12 6 5.9-12.6% 89.0114.0%. 8901140 89.0 114.0%. 89 114 89.0-114.0% 3 patients studies M1M (M1+M (1 0. 515 2.515.7 2515 25 157 15.7 2.5-15. 912 5.912.6% 5912 59 126 12.6% 5.9-12.6 0114 89.0114.0% 890114 890 89. 1140 114.0% 8 11 89.0-114.0 ( 51 2.515. 251 15. 2.5-15 91 5.912.6 591 12.6 5.9-12. 011 89.0114.0 89011 114.0 89.0-114. 2.515 2.5-1 5.912. 12. 5.9-12 89.0114. 8901 114. 89.0-114 2.51 2.5- 5.912 5.9-1 89.0114 89.0-11 5.91 5.9- 89.011 89.0-1 89.01 89.0-

Polymyxins are a group of antibacterial substances and have remained the drugs of choice for treatment of resistant Gram-negative bacilli. Polymyxin B is administered by intravenous infusion and requires the reconstitution of lyophilized powder with 0.9% saline or 5% glucose solutions. To date, there is little information about polymyxin stability in different infusions solutions, especially at 40 ºC, a temperature that is recommended to study drug stability as it accelerates degradation reactions. Therefore, in this work an analytical method using LC-MS/MS was developed, validated and applied to determine the stability of polymyxin B diluted in 0.9% saline or 5% glucose solutions at 25 ºC and 40 ºC. The stability of polymyxin B solutions was evaluated during 72 hours. Polymyxin B1 and B2 were stable for 24 hours in saline (0.9%) and glucose solution (5%), however a significant degradation of polymyxin B1 and B2 was observed after 48 hours and 72 hours of assay. The reduction of polymyxin content was evidenced in both saline and glucose media, at room temperature as well as at 40 ºC. No significant differences in pH of polymyxin solutions (glucose or saline) were evidenced during stability assay.

Abstract HIV-infected individuals on chronic use of highly active antiretroviral therapy (HAART) are more likely to develop adipose tissue and metabolic disorders, such as lipodystrophy (LD) and metabolic syndrome (MetS). The development of these phenotypes is known to be multifactorial. Thus, variants in genes implicated in adipogenesis and lipid metabolism may increase susceptibility to LD and MetS. Sirtuin 1 (SIRT1) may influence the outcome of these disturbances due to its role in the regulation of transcription factors involved in energy regulation. Therefore, we genotyped four polymorphisms located in SIRT1 (rs2273773 T>C, rs12413112 G>A, rs7895833 A>G, rs12049646 T>C) in 832 HIV-infected patients receiving HAART by real-time polymerase chain reaction. The prevalence of LD was 55.8% and MetS was 35.3%. Lipoatrophy was the most prevalent subtype in all samples (38.0%) and showed significant difference between white and non-white individuals (P = 0.002). None of the genetic variants investigated in SIRT1 was associated with LD and MetS. White individuals and those in longer time of HAART use were more likely to develop LD. We concluded that these SIRT1 polymorphisms are not predictive factors to the development of lipodystrophy and metabolic syndrome in HIV-infected individuals from Brazil.

RESUMO Introdução: Leucemia mieloide crônica (LMC) é uma desordem genética de células-tronco hematopoiéticas, resultando em uma expansão mieloproliferativa das células sanguíneas. A LMC está associada à presença do cromossomo Philadelphia (Ph), o que gera um oncogene (BCR-ABL). Atualmente, o tratamento de primeira escolha é o mesilato de imatinibe (MI). Objetivo: Correlacionar os níveis séricos de MI com parâmetros hematológicos em pacientes com LMC. Método: Estudo transversal retrospectivo em pacientes com LMC em tratamento. O nível sérico de MI foi determinado por um sistema de cromatografia líquida de alta eficiência com detector de arranjo de diodos (CLAE-DAD), e a análise estatística foi realizada no programa SPSS versão 20.0. Resultados: Foram estudados 55 pacientes - 24 homens (43,6%) e 31 mulheres (56,4%) - com média de idade de 54 anos, portadores de LMC que utilizavam MI. Destes, 45 encontravam-se em fase crônica (81,6%); sete, em fase acelerada (13,1%) e três, em crise blástica (5,2%). Os pacientes em questão receberam uma média de dose do MI de 434 mg/dia. O nível sérico dos pacientes apresentou média de 1.092 ± 617 ng/ml e, ao todo, 47 pacientes (85,4%) apresentaram resposta hematológica (RH). Conclusão: Não houve correlação do número de leucócitos, plaquetas e hemoglobina com o nível sérico de MI, embora exista uma tendência observada com relação à hemoglobina (p = 0,062).

ABSTRACT Introdution: Chronic myeloid leukemia (CML) is a genetic disorder of hematopoietic stem cells, resulting in a myeloproliferative expansion of blood cells. CML is associated with the presence of the Philadelphia chromosome (Ph), generating an oncogene (BCR-ABL). The current treatment of choice is imatinib mesylate (IM). Objective: To correlate serum levels of MI with hematological parameters in patients with CML. Method: A retrospective cross-sectional study in patients treated for CML. Serum level of IM was determined by a high-performance liquid chromatography with diode array detector (HPLC-DAD), and statistical analysis was performed using SPSS version 20.0 software. Results: We studied 55 CML patients - 24 men (43.6%) and 31 women (56.4 %) - with a mean age of 54 years, who used IM. Among these, 45 patients were in the chronic phase (81.6 %); seven, in the accelerated phase (13.1%); and three, in the blast crisis (5.2%). Patients received a mean IM dose of 434 mg/day. Serum levels of the patients presented an average of 1,092 ± 617 ng/ml, and, in all, 47 patients (85.4%) had hematologic response (HR). Conclusion: There was no correlation between the number of leukocytes, platelets and hemoglobin and the serum level of IM, although there is a trend with respect to hemoglobin (p = 0.062).

Carbamazepine, phenobarbital and phenytoin were determined in dried blood spots (DBS) by high performance liquid chromatography, after extraction of 8 mm DBS using a mixture of acetonitrile and methanol. Analytes were separated by reversed-phase chromatography, with a run time of 17 minutes. Intra-assay and inter-assay precisions were in the 5.3 to 8.4% and 3.3 to 5.2% ranges, respectively. Accuracy was in the 98.8 to 104.3% range. The method had sensitivity to detect all analytes at levels below minimum therapeutic concentrations. The analytes were stable at 4 ºC and room temperature for up to 12 days and at 45 ºC for 9 days. The method was applied to 14 paired clinical samples of blood serum and DBS.

Gene therapy attempts the insertion and expression of exogenous genetic material in cells for therapeutic purposes. Conceived in the 1960s, gene therapy reached its first clinical trial at the end of the 1980s and by December 2013 around 600 genuine open clinical trials of gene therapy were registered at NIH Clinical Trials Database. Here, we summarize the current efforts towards the development of gene therapy in Latin America. Our survey shows that the number of scientists involved in the development of gene therapy and DNA vaccines in Latin America is still very low. Higher levels of investment in this technology are necessary to boost the advancement of innovation and intellectual property in this field in a way that would ease both the social and financial burden of various medical conditions in Latin America.

OBJECTIVE: To evaluate the factors associated with plasma concentrations of atazanavir (ATV) in a cohort of well-controlled HIV infected subjects (undetectable viremia). Design: Cross-sectional study where 69 subjects were consecutively enrolled between April and November, 2011. METHODS: Patients had to be on atazanavir for at least six months, undetectable viral load for a period equal to or longer than 12 months, T CD4+ lymphocyte count higher than 200 cells/mm³, and aged between 18 years and 70 years old. Exclusion criteria were pregnancy, any neurologic disease, active opportunistic disease, hepatitis or cancer. Atazanavir plasma levels were measured by ultra-performance liquid chromatography. RESULTS AND DISCUSSION: Overall, 54 patients (mean age of 47 years and 50% women) were included in the analysis. Those without ritonavir (unboosted atazanavir) had statistically lower plasma concentrations than those with ritonavir boosted atazanavir (p = 0.001) and total and indirect bilirubin were statistically associated with plasma concentration of atazanavir (r = 0.32 and r = 0.33 respectively; p < 0.05 in both cases). no statistical association was found among gender, ethnicity, age, weight, body mass index (BMI), lipid profile, and the plasma concentration of atazanavir. CONCLUSION: in summary, as expected, concomitant ritonavir use was the only factor associated with atazanavir plasma levels. prospective studies with a larger sample size might help to observe an association of atazanavir concentrations to other characteristics such as body weight, since the p-value showed to be close to significance (p = 0.068).

Topiramate and the other frequently co-administered antiepileptic drugs carbamazepine, phenytoin and phenobarbital were determined in 100 µL plasma samples by gas chromatography with nitrogen phosphorus detection (GC-NPD), after a one-step liquid-liquid extraction with ethyl acetate, followed by flash methylation with trimethylphenylammonium hydroxide. Total chromatographic run time was 12.5 min. Intra-assay and inter-assay precision was 2.5-7.3% and 1.6-5.2%, respectively. Accuracy was 100.1-104.2%. The limit of quantitation was 1 µg mL-1 for all analytes, proving suitable for routine application in therapeutic drug monitoring of antiepileptic drugs.

INTRODUÇÃO: O interesse pela vitamina D nos últimos anos teve aumento significativo. Estudos epidemiológicos realizados têm demonstrado um crescente aumento da deficiência de vitamina D entre a população. O marcador diagnóstico de escolha para determinar os níveis de vitamina D é a concentração de 25-hidroxivitamina D (25(OH)D), com suas frações D2 (25(OH)D2) e D3 (25(OH)D3). OBJETIVO: Desenvolver uma metodologia analítica empregando cromatografia líquida de alta eficiência com detector de arranjo de diodos (CLAE-DAD) para a determinação de 25(OH)D3 e 25(OH)D2 em plasma. MATERIAIS E MÉTODOS: 25(OH)D3 e 25(OH)D2 foram extraídos das amostras de plasma com hexano, utilizando-se dodecafenona como padrão interno (PI). Utilizou-se coluna analítica ACE 5 C18 com partículas de 5 µm e dimensões de 150 × 4,6 mm, fase móvel metanol-água (80:20; v/v) e quantificação em 265 nm. RESULTADOS: A exatidão foi entre 98,4 e 107,5%. A precisão intraensaios esteve entre 6,5% e 9,2% para 25(OH)D3 e entre 3,7% e 8,7% para 25(OH)D2. A precisão interensaios esteve entre 2,9% e 6% para 25(OH)D3 e entre 4% e 4,5% para 25(OH)D2. O limite inferior de quantificação foi 10 ng/ml. As concentrações encontradas em amostras de 32 pacientes idosos estiveram entre 10,1 e 32,4 ng/ml, caracterizando deficiência de vitamina D nesse grupo. DISCUSSÃO O método foi capaz de quantificar 25(OH)D2 e 25(OH)D3 com uma preparação de amostra relativamente simples e rápida. O método foi seletivo, com separação adequada dos metabólitos e do padrão interno e sem presença de interferentes. CONCLUSÃO: O método desenvolvido apresenta desempenho analítico adequado e pode ser aplicado em condições clínicas.

INTRODUCTION: The interest in vitamin D has increased significantly in recent years. Epidemiological studies conducted over the past 25 years have shown a steady increase in vitamin D deficiency. The diagnostic marker of choice to determine vitamin D levels is the concentration of 25-hidroxy-vitamin D (25(OH)D) in the fractions D2 (25(OH)D2) and D3 (25(OH)D3). OBJECTIVE: To develop an analytical method using high-performance liquid chromatography with diode-array detection (HPLC-DAD) for the determination of 25(OH)D2 and 25(OH)D3 in plasma. MATERIALS AND METHODS: 25(OH)D3 and 25(OH)D2 were extracted from plasma samples with hexane and dodecaphenone was used as internal standard. The separation was performed in an ACE 5 C18 column, with particle size of 5 µm (4,6 × 150 mm), mobile phase methanol-water (80:20, v/v) and quantification at 265 nm. RESULTS: Accuracy was in the range of 98.4 to 107.5%. Intra-assay precision was between 6.5 and 9.2% for 25(OH)D3 and 3.7 and 8.7 for 25(OH)D2. Inter-assay precision was between 2.9 and 6% for 25(OH)D3 and 4 and 4.5 for 25(OH)D2. The limit of quantification was 10 ng/l. Concentrations of 25(OH)D3 in samples from 32 elderly patients were between 10.1 and 32.4 ng/ml, characterizing vitamin D deficiency in this group. DISCUSSION: The method allowed the quantification of 25(OH)D2 and 25(OH)D3. Furthermore, the sample preparation was relatively simple and fast. The method was selective with an adequate separation of metabolites and internal standard with no interfering substances. CONCLUSION: Not only did the developed method show suitable analytical performance, but it may also be applied in clinical conditions.