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

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).
The effect of paper type on analyte determination was evaluated using Whatman FTA Elute and ID Biological Systems 226 collection cards.
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

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.

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

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 â€˜dirty’ sample, thus unsuitable for analysis.
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.
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.

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.

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