Standardization method for measurement of hydroxyurea by Ultra High Efficiency Liquid Chromatography in plasma of patients with sickle cell disease

Sickle cell anemia (SCA) is a recessively inherited disease characterized by chronic hemolytic anemia, chronic inflammation, and acute episodes of hemolysis. Hydroxyurea (HU) is widely used to increase the levels of fetal hemoglobin (HbF). The objective of this study was to standardize and validate a method for the quantification of HU in human plasma by using ultra high performance liquid chromatography (UPLC) in order to determine the plasma HU levels in adult patients with SCA who had been treated with HU. We used an analytical reverse phase column (Nucleosil C18) with a mobile phase consisting of acetonitrile/water (16.7/83.3). The retention times of HU, urea, and methylurea were 6.7, 7.7, and 11.4 min, respectively. All parameters of the validation process were defined. To determine the precision and accuracy of quality controls, HU in plasma was used at concentrations of 100, 740, and 1600 µM, with methylurea as the internal standard. Linearity was assessed in the range of 50-1600 µM HU in plasma, obtaining a correlation coefficient of 0.99. The method was accurate and precise and can be used for the quantitative determination of HU for therapeutic monitoring of patients with SCA treated with HU.A anemia falciforme (AF) é uma doença hereditária recessiva caracterizada por anemia hemolítica crônica, inflamação crônica e episódios agudos de hemólise. Hidroxiureia (HU) é amplamente utilizada para aumentar os níveis de hemoglobina fetal (Hb F). O objetivo consiste em padronizar e validar um método para a quantificação de HU no plasma humano utilizando Cromatografia Líquida de Ultra Alta Eficiência (UPLC), a fim de determinar os níveis de HU em pacientes adultos com AF, tratados com HU. Utilizou-se coluna analítica de fase inversa (Nucleosil C18), fase móvel constituída por acetonitrila/água (16,7/83,3). Os tempos de retenção da HU, uréia e metiluréia foram respectivamente de 6,7, 7,7 e 11,4 minutos. Definiram-se todos os parâmetros do processo de validação. Para determinar a precisão e exatidão dos controles de qualidade utilizaram-se concentrações de 100, 740 e 1600 mM de HU no plasma, empregando como padrão interno a metiluréia. A linearidade foi avaliada no intervalo de 50 1600 mM de HU no plasma, obtendo-se coeficiente de correlação de 0,99. O método foi considerado exato e preciso e pode ser realizado com o propósito de determinação quantitativa de HU para monitorização terapêutica de pacientes com AF tratados com esse fármaco

Methemoglobin measure in adult patients with sickle-cell anemia: influence of hydroxyurea therapy

Introduction: Hemoglobin S (HbS) is unstable hemoglobin that easily oxidizes, causing methemoglobin (MetHb) increased production in patients with sickle-cell anemia (SCA). Objectives: To determine MetHb levels and the influence of hydroxyurea (HU) therapy on this marker in patients with SCA. Materials and methods: Blood samples from 53 patients with SCA at the steady-state, with and without HU therapy, and 30 healthy individuals were collected to evaluate MetHb levels. The MetHb measurement was performed by spectrophotometry. Complete blood count, HU measurements, and fetal hemoglobin (HbF) and HbS concentrations were taken from medical records. Results: MetHb levels were statically higher in patients with SCA when compared to control group (p < 0.001). There was no statistical difference in MetHb level between SCA patients, either using or not HU. We obtained a positive correlation between MetHb measurements and HbS concentration (r = 0.2557; p = 0.0323). Conclusion: HbS presence favored hemoglobin breaking down, and consequently increased MetHb production. Treatment with HU, however, did not influence the levels of this marker

Influence of gestational diabetes mellitus on the kinetic disposition and stereoselective metabolism of labetalol in patients with arterial hypertension,

O labetalol, um anti-hipertensivo considerado seguro para uso em gestantes, está disponível na clínica como mistura de dois racematos (quatro estereoisômeros), sendo o isômero (R,R) antagonista e o (S,R) responsável pela atividade bloqueadora. O estudo investiga a influência do diabetes mellitus gestacional (DMG) na disposição cinética e no metabolismo estereosseletivos do labetalol administrado por via endovenosa ou oral. Foram investigadas 30 gestantes hipertensas distribuídas em 04 grupos: não diabéticas tratadas com dose única de 40 mg de cloridrato de labetalol endovenoso (grupo EVH, n=8) ou 100 mg de cloridrato de labetalol via oral (grupo VOH, n=9) e diabéticas tratadas com 40 mg de cloridrato de labetalol endovenoso (grupo EVD, n=6) ou 100 mg de cloridrato de labetalol via oral (grupo VOD, n=7). As amostras seriadas de sangue foram coletadas até 12 h (via oral) ou 15 h (via endovenosa) após a administração do cloridrato de labetalol. Os estereoisômeros do labetalol em plasma foram analisados em coluna de fase quiral Chirobiotic V empregando LC-MS-MS. Os parâmetros farmacocinéticos do labetalol inalterado e labetalol glicuronídeo foram calculados com auxílio do programa WinNonlin e avaliados empregando os testes de Mann-Whitney e Friedman com pós-teste de Dunn (p<0,05). A farmacocinética do labetalol não é estereosseletiva em gestantes diabéticas e não diabéticas tratadas com o fármaco por via endovenosa. No entanto, a administração oral de labetalol resulta em menores valores de área sob a curva concentração plasmática versus tempo (AUC) para o isômero ativo (R,R)-labetalol tanto em gestantes diabéticas (60,9 vs 162,7 vs 157,9 vs 114,6 ng.h/mL, respectivamente para (R,R,); (SR); (S,S,) e (R,S)) quanto não diabéticas (45,6 vs 84,2 vs 89,4 vs 78,3 ng.h/mL, respectivamente para (R,R,); (SR); (S,S,) e (R,S)). O DMG resulta em alterações na disposição cinética dos estereoisômeros do labetalol na administração oral. Os valores de AUC do isômero inativo (S,S)-labetalol (157,9 vs 89,4 ng.h/mL) e para o isômero -bloqueador (S,R)-labetalol (162.7 vs 84.2 ng.h/mL) são maiores (p<0,05) nas gestantes diabéticas do que nas gestantes não diabéticas. As gestantes diabéticas também mostram maior biodisponibilidade oral do isômero (S,R)-labetalol (54,7 vs 24,0 %) explicada pela reduzida eliminação pré-sistêmica conseqüente da menor capacidade de conjugação com o ácido glicurônico (68,4 vs 77,9 %). Os valores de AUC do isômero (S,R) aproximadamente 100 % maiores nas gestantess diabéticas tratadas com o fármaco por via oral (162,7 vs 84,2 ng.h/mL) pode ter relevância clínica considerando a atividade -bloqueadora do referido isômero.Labetalol, a hypertensive agent considered to be safe for use by pregnant women, is clinically available as a mixture of two racemates, with the (R,R) isomer being a antagonist and the (S,R) isomer being responsible for the blocking activity. The study investigated the influence of gestational diabetes mellitus (GDM) on the kinetic disposition and stereoselective metabolism of labetalol administered by the intravenous or oral route. Thirty hypertensive pregnant women were divided into 4 groups: non-diabetic women treated with a single 40 mg dose of intravenous labetalol hydrochloride (IVH group, n=8) or with 100 mg labetalol hydrochloride by the oral route (OH group, n=9) and diabetic women treated with 40 mg intravenous labetalol hydrochloride (IVD), n=6) or with 100 mg labetalol hydrochloride by the oral route (OD, n=7). Serial blood samples were collected up to 12 h (oral route) or 15 h (intravenous route) after the administration of labetalol hydrochloride. The labetalol stereoisomers in plasma were analyzed with a chiral phase Chirobiotic V column by LC-MS-MS. The pharmacokinetic parameters of unchanged labetalol and glucuronide labetalol were calculated using the WinNolin software and analyzed by the Mann-Whitney and Friedman tests followed by the Dunn test (p<0.05). The pharmacokinetics of labetalol was not stereoselective in diabetic or non-diabetic pregnant women treated with the drug by the intravenous route. However, oral administration of labetalol resulted in lower values of the area under the plasma concentration versus time curve (AUC) for the active isomer (R,R)-labetalol both in diabetic (60.9 vs 162.7 vs 157.9 vs 114.6 ng.h/mL, respectively for (R,R,), (SR), (S,S,) and (R,S)) and non-diabetic pregnant women (45.6 vs 84.2 vs 89.4 vs 78.3 ng.h/mL, respectively for (R,R,), (SR), (S,S,) and (R,S)). GDM involves changes in the kinetic disposition of the stereoisomers of labetalol when administered by the oral route. The AUC values for the inactive (S,S)-labetalol (157.9 vs 89.4 ng.h/mL) and for the -blocking (S,R) isomer (162.7 vs 84.2 ng.h/mL) were higher (p<0.05) for the diabetic than the non-diabetic pregnant women. The diabetic pregnant women showed greater oral bioavailability of the (S,R)-labetalol isomer (54.7 vs 24.0 %), explained by the reduced pre-systemic elimination due to the lower capacity for conjugation with glucuronic acid (68.4 vs 77.9 %). The approximately 100 % higher AUC values of the (S,R) isomer for the diabetic pregnant women treated with the drug by the oral route may be of clinical relevance in view of the -blocking activity of the isomer in question

Determination of serum lithium: comparison between atomic emission and absorption spectrometry methods

Introduction: The therapeutic monitoring of lithium, through concentration measurements, is important for individual dose adjustment, as a marker of treatment adherence and to prevent poisoning and side effects. Objectives: Validate and compare two methods - atomic emission and atomic absorption - for the determination of lithium in serum samples. Methodology: Parameters such as specificity, precision, accuracy, limit of detection (LOD) and linearity were considered. The atomic absorption spectrometer was used, operating in either emission or absorption mode. For the quantitative comparison of 30 serum samples from patients with mood disorder treated with lithium, the results were submitted to Student's t-test, F-test and Pearson's correlation. Results: The limit of quantification (LOQ) was established as 0.05 mEq/l of lithium, and calibration curves were constructed in the range of 0.05-2 mEq/l of lithium, using aqueous standards. Sample preparation time was reduced, what is important in medical laboratory. Conclusion: Both methods were considered satisfactory, precise and accurate and can be adopted for lithium quantification. In the comparison of quantitative results in lithium-treated patients through statistical tests, no significant differences were observed. Therefore the methods for lithium quantification by flame atomic absorption spectrometry (FAAS) and flame atomic emission spectrometry (FAES) may be considered similar

Standardization method for measurement of hydroxyurea by Ultra High Efficiency Liquid Chromatography in plasma of patients with sickle cell disease

Sickle cell anemia (SCA) is a recessively inherited disease characterized by chronic hemolytic anemia, chronic inflammation, and acute episodes of hemolysis. Hydroxyurea (HU) is widely used to increase the levels of fetal hemoglobin (HbF). The objective of this study was to standardize and validate a method for the quantification of HU in human plasma by using ultra high performance liquid chromatography (UPLC) in order to determine the plasma HU levels in adult patients with SCA who had been treated with HU. We used an analytical reverse phase column (Nucleosil C18) with a mobile phase consisting of acetonitrile/water (16.7/83.3). The retention times of HU, urea, and methylurea were 6.7, 7.7, and 11.4 min, respectively. All parameters of the validation process were defined. To determine the precision and accuracy of quality controls, HU in plasma was used at concentrations of 100, 740, and 1600 µM, with methylurea as the internal standard. Linearity was assessed in the range of 50-1600 µM HU in plasma, obtaining a correlation coefficient of 0.99. The method was accurate and precise and can be used for the quantitative determination of HU for therapeutic monitoring of patients with SCA treated with HU

Adesão ao tratamento medicamentoso em usuários com transtorno do humor de centro de atenção psicossocial do nordeste do Brasil

O Transtorno de Humor (TH) é uma doença crônica, recorrente, de difícil diagnóstico e tratamento terapêutico. A baixa adesão à terapia relaciona-se a fatores ligados ao paciente, ao medicamento, ao profissional de saúde ou à doença. O trabalho objetivou avaliar a taxa de adesão ao tratamento e ao lítio de pacientes acometidos pelo TH, delineando o perfil farmacoepidemiológico destes. Para tanto, foram utilizadas de janeiro a outubro de 2011, entrevistas e questionários validados para indivíduos com TH assistidos em Centro de Atenção Psicossocial (CAPS). Dos 56 entrevistados, a maioria estava na faixa de 30- 50 anos, era do sexo feminino e solteiro. Quanto ao histórico familiar, 55,4 % possuíam outros membros da família com TH, 60,7 % participaram de gruposCAPS e 62,5 % nunca foram internados. A “Escala de Adesão à Medicação” mostrou que 60,7 %, alguma vez já se esqueceu de tomar o medicamento e do horário de tomá-lo. A maioria não toma o medicamento apenas quando se sente doente e seus pensamentos ficam mais coerentes quando está sob o uso de medicamentos. Do total, 35 pacientes eram bipolares e 23 tomavam lítio, os quais foram entrevistados seguindo o Questionário “Atitudes em Relação ao Lítio”. Destes, cerca de 78 % (n=18) considerou fácil seguir a prescrição, aceitável e importante tomar o lítio por vários anos, apesar dos efeitos colaterais e relataram não tomar o lítio somente quando sente necessidade. Em geral, os entrevistados mostraram boa adesão à terapia medicamentosa, compreendendo que somente através de um tratamento bem estabelecido podem manter a doença estabilizada

Stereoselective Analysis of Labetalol in Human Plasma by LC-MS/MS: Application to Pharmacokinetics

Labetalol is clinically available as a mixture of two racemates (four stereoisomers). The stereoisomer (R,R) has as main activity the beta(1)-antagonism and the stereoisomer (S,R) is highly selective for the alpha(1) adrenoceptor and is responsible for most of the alpha-blocker activity. In the present investigation, a method for the analysis of labetalol stereoisomers in human plasma was developed and applied to pharmacokinetic studies. Plasma samples (0.5 ml) were extracted with methyl tert-butyl ether at pH 9.5. The four labetalol stereoisomers were analyzed by LC-MS/MS on a Chirobiotic (R) V column using a mobile phase consisting of methanol, acetic acid, and diethylamine, with a recovery of more than 90% for all four. The quantitation limit was 0.5 ng/ml and linearity was observed at 250 ng/ml plasma for each stereoisomer. Studies of precision and accuracy presented coefficients of variation and percentage inaccuracy of less than 15%, indicating that the method is precise and accurate. The method was applied to the study of the kinetic disposition of labetalol over a period of 12 h after oral administration of a single 100 mg dose to a hypertensive pregnant woman. The clinical study revealed stereoselectivity in the pharmacokinetics of labetalol, with a lower plasma proportion for the active stereoisomers (R,R)-labetalol and (S,R)-labetalol. The stereoselectivity observed after oral administration is due to the hepatic metabolism and the first pass effect, with an AUC((R,R))/AUC((S,S)) ratio of 0.5. Chirality 21:738-744, 2009. (C) 2008 Wiley-Liss, Inc.Fundacao de Amparo A Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Apoio a Pesquisa e Assistencia do HCFMRP-USP (FAEPA)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Metformin pharmacokinetics in nondiabetic pregnant women with polycystic ovary syndrome

The use of metformin throughout gestation by pregnant women with polycystic ovary syndrome (PCOS) significantly reduces the number of first trimester spontaneous abortions and the rate of occurrence of gestational diabetes. The objective of this study was to investigate the pharmacokinetics and the placental transfer of metformin in pregnant women with PCOS. Eight pregnant women with PCOS taking 850 mg metformin every 12 h during the third trimester of pregnancy were evaluated. Maternal blood samples were collected at steady state during the dose interval (0-12 h). Maternal and umbilical cord blood samples were also obtained at delivery. Metformin plasma concentrations were analyzed by high-performance liquid chromatography, and pharmacokinetic parameters were determined using a non-compartmental model. Data are reported as median and minimum and maximum values. Metformin pharmacokinetic parameters were: t(A1/2), 3.8 (2.8-5.4) h; t(max), 2.0 (0.5-3.0) h; C(max), 1.4 (0.5-2.1) mg/L; C(mean), 0.5 (0.2-0.9) mg/L; AUC(0-12), 6.4 (1.1-9.2) mg h/L; Cl/f, 105 (60-274) L/h; Vd/f, 551 (385-1173) L; median fluctuation, 89 (79-95)%. Umbilical/maternal metformin plasma concentration ratios were 0.7 (0.4-1.3). Metformin oral clearance (Cl/f) had increased in our patients relative to nonpregnant healthy volunteers or diabetic patients. Therefore, lower plasma metformin concentrations were observed for nondiabetic pregnant women with PCOS. Future studies should be conducted to demonstrate the therapeutic efficacy of metformin during pregnancy. Caution is warranted as umbilical/maternal metformin plasma concentrations ratios of around 0.7 require metformin dosage adjustment.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Apoio a Pesquisa e Assistencia do HCFMRP-USP (FAEPA)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Simultaneous Analysis of Tramadol, O-Desmethyltramadol, and N-Desmethyltramadol Enantiomers in Rat Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry: Application to Pharmacokinetics

Tramadol (T) is available as a racemic mixture of (+)-trans-T and (-)-trans-T. The main metabolic pathways are O-demethylation and N-demethylation, producing trans-O-desmethyltramadol (M1) and trans-N-desmethyltramadol (M2) enantiomers, respectively. The analgesic effect of T is related to the opioid activity of (+)-trans-T and (+)-M1 and to the monoaminergic action of (+/-)-trans-T. This is the first study using tandem mass spectrometry as a detection system for the simultaneous analysis of trans-T, M1, and M2 enantiomers. The analytes were resolved on a Chiralpak (R) AD column using hexane: ethanol (95.5:4.5, v/v) plus 0.1% diethylamine as the mobile phase. The quantitation limits were 0.5 ng/ml for trans-T and M1 and 0.1 ng/ml for M2. The method developed and validated here was applied to a pharmacokinetic study in rats. Male Wistar rats (n = 6 at each time point) received a single oral dose of 20 mg/kg racemic trans-T. Blood samples were collected up to 12 h after drug administration. The kinetic disposition of trans-T and M2 was enantioselective (AUC((+)/(-)) ratio = 4.16 and 6.36, respectively). The direction and extent of enantioselectivity in the pharmacokinetics of trans-T and M2 in rats were comparable to data previously reported for healthy volunteers, suggesting that rats are a suitable model for enantioselective studies of trans-T pharmacokinetics. Chirality 23: 287-293, 2011. (C) 2010 Wiley-Liss, Inc.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq