Evaluation Of The Dried Blood Spot Technique For The Quantification Of Diltiazem In Human Blood

Neil Doleman
BASi®, Suite 2 Building 500, Abbey Park, Stareton, Kenilworth, Warwickshire, UK

Background

There is renewed interest in the development of the Dried Blood Spot technique (DBS) for determining drug levels in blood samples. The benefits of this sampling technique for pre-clinical and clinical bioanalytical assays have been recognised by BASi®.

  • Refinement - elimination/reduction of rodent warming
  • Reduction - reduced volumes enables serial sampling from one rodent reducing animal numbers and eases sampling in paediatric studies
  • Reduced costs - animal numbers, test substance, simplified procedures (collection and laboratory), shipping and storage

Objective

To evaluate the use of DBS to determine diltiazem in human blood.

Methodology

  • The concentration of diltiazem in excised paper discs taken from dried blood spots was determined incorporating a deuterated internal standard and analysed by LC-MS/MS. This approach was adapted from a validated BASi® UK method for the quantitation of diltiazem in human plasma.
  • Multiple spots/sample were made each of 40µL/spot. Up to three discs were taken manually from individual blood spots and two types of paper with different coatings were compared (example paper shown in figure).

Assessments

  1. Homogeneity and selectivity of DBS was assessed using 6 lots of individual blood applied at 40µL/spot. One disc was taken from each DBS calibration standard (range 1.0 - 500 ng/mL) and 3 discs taken from each QC DBS. Acetonitrile:water (10:90 v/v) containing internal standard was added and the discs extracted (Table 1). The effect of excising only one disc from the centre of each QC DBS was also evaluated (Table 2).
  2. The effect of paper type on analyte determination was evaluated using Whatman FTA Elute and ID Biological Systems 226 collection cards.
  3. In an effort to increase the percentage of blood taken with each disc, the amount of blood/spot was reduced to 15µL and a single disc excised (Table 3).

Results

  1. Overall results passed the BASi® accuracy and precision acceptance criteria (Table 1). Although, some individual results were inconsistent, possibly due to the non uniform nature of the DBS.

  2. Table 1: 40µL/spot evaluation - triplicate discs from One DBS
    Concentration (ng/mL)
    1.00
    3.00
    150
    400
    Run 1
     

    Intra-run Mean
    Intra-run SD
    Intra-run %CV
    Intra-run %Bias
    n

    1.07
    0.143
    13.4
    7.0
    6
    2.85
    0.254
    8.9
    -5.0
    6
    140
    11.5
    8.2
    -6.7
    6
    398
    34.6
    8.7
    -0.5
    6
    Run 2
     
    Intra-run Mean
    Intra-run SD
    Intra-run %CV
    Intra-run %Bias
    n
    1.03
    0.0709
    6.9
    3.0
    6
    3.02
    0.264
    8.7
    0.7
    5
    150
    8.13
    5.4
    0.0
    6
    423
    43.5
    10.3
    5.8
    6
    Run 3
     
    Intra-run Mean
    Intra-run SD
    Intra-run %CV
    Intra-run %Bias
    n
    1.03
    0.162
    15.7
    3.0
    6
    3.16
    0.25
    7.9
    5.3
    6
    133
    12.5
    9.4
    -11.3
    6
    389
    24.2
    6.2
    -2.8
    6
             
    Mean Concentration (ng/mL)
    1.05
    3.01
    141
    403
     
    Intra-run SD
    Intra-run %CV
    Intra-run %Bias
    n

    0.125
    11.9
    5.0
    18

    0.274
    9.1
    0.3
    17
    12.7
    9.1
    -6.0
    18
    36
    8.9
    0.8
    18

  3. The ID Biological Systems 226 collection cards were not able to retain the blood from the highly aqueous solution used in the extraction. In consequence, the resulting extractions appeared red in colour producing a ‘dirty’ sample, thus unsuitable for analysis.

  4. The overall results from the single excised disc experiment again passed the BASi® accuracy and precision acceptance criteria (Table 2). However, again some individual results were inconsistent and further investigation is required.

  5. Results indicate that increasing the percentage of sample taken for analysis increases the accuracy of the DBS technique (Table 3). Further experimentation will be done to confirm this finding.

  6. Table 2: 40µL/spot evaluation - Individual disc from one DBS
    Concentration (ng/mL)
    1.00
    3.00
    150
    400
    Run 1
     

    Intra-run Mean
    Intra-run SD
    Intra-run %CV
    Intra-run %Bias
    n

    1.03
    0.118
    11.5
    3.0
    6
    2.76
    0.284
    10.3
    -8.0
    6
    144
    9.54
    6.6
    -4.0
    6
    448
    34.5
    7.7
    12.0
    6
    Run 2
     
    Intra-run Mean
    Intra-run SD
    Intra-run %CV
    Intra-run %Bias
    n
    1.15
    0.108
    9.4
    15.0
    6
    2.8
    0.275
    9.8
    -6.7
    6
    145
    9.00
    6.2
    -3.3
    6
    441
    45.4
    10.3
    10.3
    6
    Run 3
     
    Intra-run Mean
    Intra-run SD
    Intra-run %CV
    Intra-run %Bias
    n
    0.966
    0.0918
    9.5
    -3.4
    6
    3.07
    0.190
    6.2
    2.3
    6
    145
    8.42
    5.8
    -3.3
    6
    458
    22.4
    4.9
    14.5
    6
             
    Mean Concentration (ng/mL)
    1.05
    2.88
    145
    449
     
    Intra-run SD
    Intra-run %CV
    Intra-run %Bias
    n

    0.126
    12.0
    5.0
    18

    0.277
    9.6
    -4.0
    18
    8.46
    5.8
    -3.3
    18
    34.0
    7.6
    12.3
    18

    Table 3: 15µL/spot evaluation - 1 disc per spot
    Concentration (ng/mL)
    1.00
    3.00
    150
    400

    Intra-run Mean
    Intra-run SD
    Intra-run %CV
    Intra-run %Bias
    n

    1.00
    0.0741
    7.4
    0.0
    6
    2.83
    0.168
    5.9
    -5.7
    6
    144
    13.7
    9.5
    -4.0
    6
    362
    20.9
    5.8
    -9.5
    6

Conclusion

The DBS technique was used successfully to determine the concentration of diltiazem in human blood samples. The reliability of the technique for 40µL blood spots will require further investigation but increasing the percentage of sample taken for analysis, by only spotting 15µL of blood, increased its precision.