Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection for Quantitative Analysis of Dried Blood Spots with Unknown Blood Volume.

Abstract

Blood volume in dried blood spot (DBS) analysis is assumed to be constant for DBS punches with a fixed area. However, blood volume in the punch is dependent on several factors associated with the blood composition and is preferentially normalized by off-line analysis for quantitative purposes. Instead of using external instrumentation, we present an all-in-one approach for the simultaneous determination of exact blood volume in the DBS punch and the quantitation of target analytes. A DBS is eluted with 500 μL of elution solvent in a sample vial, and the eluate is directly subjected to an automated analysis by capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D). The capillary blood volume in the eluate is calculated from the concentrations of the inorganic blood constituents (K+, Na+, or Cl-) determined by CE-C4D, which are linearly proportional to the blood volume originally sampled onto the DBS card. Alternatively, conductivity of the DBS eluate can be used for the blood volume determination by using C4D in a nonseparation flow-through mode. The methods are suitable for the determination of blood volume in unknown DBS samples by punching out the entire DBS or by subpunching a small section of a large DBS with variations of the true vs the determined volume ≤5.5%. Practical suitability was demonstrated by the simultaneous CE-C4D determination of K+ and Na+ (for DBS volume calculation) and amino acids (target analytes) in unknown DBS samples. Quantitative analysis of selected amino acids (related to inborn metabolic disorders) in the unknown DBS was compared with a standard analytical procedure using wet-blood chemistry, and an excellent fit was obtained. The use of CE-C4D represents an important milestone in quantitative DBS analysis since the detection technique is universal, and the separation technique enables the determination of cations and/or anions and the use of multiple detectors, which further enhance selectivity/sensitivity of the analysis and the range of detectable analytes.