Retinol Analysis in Dried Blood Spots by HPLC

Abstract

There are many advantages to measuring vitamin A in dried blood spots (DBS) from a finger prick as compared to plasma collected by venipuncture. The advantages include easier collection, transport and storage; accessibility to younger and more remote populations; and decreased risk of disease transmission. We describe a method for the extraction of retinol from DBS for analysis by HPLC and initial comparison to plasma retinol. The effects of various buffers, detergents, antioxidants and chelators were evaluated to establish the most effective approach to elute the retinol: retinol binding protein (holo-RBP) complex from the blood collection cards. The process involves ultrasonic agitation to elute holo-RBP into a phosphate buffer containing an antioxidant and metal chelator. The holo-RBP complex was denatured by the addition of ethanol containing additional antioxidants permitting the extraction of free retinol into hexane. Following solvent evaporation, the extract was dissolved in methanol for HPLC analysis. The initial measured retinol levels in freshly collected DBS declined for 6-10 d whether stored at 25, 4 or -20 degrees C, but remained consistent thereafter (homeostatic). By incorporating a "recovery/volume adjustment" factor, measured retinol values in homeostatic DBS were adjusted to the equivalent of plasma retinol. For 17 normal adults, the correlation coefficient was 0.90 between plasma retinol and adjusted DBS retinol in samples that had been stored at -70 degrees C for < 9 mo. The use of this new sample matrix for vitamin A assessment will allow access to previously unavailable populations.

Retinol concentrations in capillary dried blood spots from healthy volunteers: method validation

“…It is not surprising that all circulating retinol concentrations were > 1.05 mol/L (30 g/dL), the cutoff for adequacy in adults (12), because our subjects were healthy adults living in a country with vitamin A-fortified table sugar. However, in a previous trial with paired venous serum and DBS samples from Nepalese mothers, samples with serum retinol concentrations < 0.70 mol/L (20 g/dL) correlated well (R 2 = 0.79) with DBS retinol values, despite an incomplete cold chain (13).…”

“…Although the measurement of DBS retinol is a great stride forward, several authors (14), including those associated with the present study (13,15), have pointed out the limitations of using circulating retinol concentrations as an indicator of individual vitamin A status. However, within these accepted limits, we showed that in healthy subjects DBS retinol is comparable with serum retinol.…”

“…Recently, Craft Technologies (Wilson, NC) developed a procedure to measure blood retinol concentrations in DBS by using HPLC (4,5). The application of this technique to assess vitamin A status in public health surveys could improve subject participation and reduce logistic problems.…”

Characteristics of medication use during lactation

“…Foram retirados 300 µL do sobrenadante (n-hexano), que foi secado sob atmosfera de nitrogênio, e a amostra suspendida em 150 µL de metanol e injetada no HPLC, com volume do injetor (loop) de 100 µL, de acordo com Erhardt et al, 2002e Craft et al, 2000 O resultado da concentração de vitamina A na gota foi calculado pela curva de calibração com padrão externo, de no mínimo três pontos, conferido diariamente. Segundo a WHO (WORLD HEALTH ORGANIZATION, 1996), a hipovitaminose A (<0,70 µmol/L) é um problema de saúde pública: grave -prevalência de 20% ou mais da população; moderada -mais de 10%; ou leve -menos que 10%.…”

Mass spectrometry of blood low-molecular fraction as a method for unification of therapeutic drug monitoring

“…Коэффициент позволяет использовать уже сертифицированные на венозной крови методики ТЛМ в приложении их к методам ТЛМ, использующим капиллярную кровь, путём простого умножения полученных показателей на этот коэффициент. Для некоторых лекарственных препаратов этот коэффициент был получен эмпирическим путем и, как правило, близок к 1 (от 0,84 до 1,13 [57,[62][63][64][65][66][67][68][69][70]). Следует также отметить, что для лекарственных веществ, для которых эмпирически коэффициент пересчета не установлен, можно применять формулу пересчёта, известную как поправку на гематокрит [61,[71][72][73][74][75][76][77].…”