Validation Of An Lc-ms/ms Method For The Determination Of Quetiapine, Norquetiapine And Quetiapine Sulphoxide In Human Whole Blood Using The Dried Blood Spot Technique

Neil Doleman “ Principal Investigator
Robin Lane “ Senior Analyst
BASi®, Suite 2 Building 500, Abbey Park, Stareton, Kenilworth, Warwickshire, UK

Background

The Dried Blood Spot (DBS) technique has been evaluated at BASi® UK for the determination of quetiapine, norquetiapine and quetiapine sulphoxide in human whole blood. DBS offers a range of benefits over traditional sampling techniques including:

  • 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 validate an analytical method in human whole blood achieving an LLOQ of 0.5 ng/mL for all analytes at BASi® UK prior to transferring the method to our US sites and performing a cross-validation exercise.

Methodology

  • The concentration of quetiapine, norquetiapine and quetiapine sulphoxide in excised paper discs taken from dried blood spots was determined in the presence of deuterated internal standards (IS) and analysed by LC-MS/MS (Sciex API 4000). This approach was adapted from a validated BASi® UK method (liquid/liquid extraction) in human plasma and utilised ID Biological Systems 226 collection cards.
  • Typically, ˜standard™ DBS sample preparation involves the application of 15 µL of blood to a card from which a 3 mm disc is removed and extracted with 100 µL methanol:water (70:30 v/v) containing IS. On this occasion, changes to the ˜standard™ preparation procedure were required to achieve the desired LLOQ.

Changes to the ˜Standard™ Preparation

In an effort to increase assay sensitivity the following changes were necessary:

  1. The card was spotted with 20 µL of blood.
  2. A 5 mm disc was taken for analysis.
  3. An extraction solvent of methanol:water (80:20 v/v) was utilised.
  4. The extraction solvent volume was increased to 250 µL allowing an aliquot of 200 µL to be taken and evaporated to dryness.
  5. Sample extract was reconstituted in 100 µL of mobile phase prior to injection.

Figure 1: Chromatograms of a typical calibration standard at 0.5 ng/mL (LLOQ)
(Reversed phase isocratic HPLC (phenyl-hexyl stationary phase, 3 µm particle size))

Validation Assessments

  • Method performance batches ( n=3)
  • Effect of dilution after extraction
  • Effect of blood volume; 15 and 25 µL assessed
  • Matrix effects
  • Specificity

Results

1. Accuracy and Precision

Overall ˜inter-run™ results, summarising three separate method performance batches, passed the BASi® accuracy and precision acceptance criteria (Table 1). Each of the three individual runs used for this assessment also met these acceptance criteria.

Dilution integrity (1 in 10) was also demonstrated (data not presented here).

Table 1:

2. Robustness “ the effect of blood volume

The results shown in Table 2 demonstrate that the accuracy and precision of the assay was unaffected by changes in DBS volume.

Table 2:

3. Matrix Effects

Table 3 shows the matrix effects observed for each analyte. Only quetiapine sulphoxide appeared to undergo any degree of matrix suppression although this was successfully compensated for by the IS.

Specificity (n=6 individuals) was confirmed for all analytes (data not presented here).

Table 3:

4. Calibration data

Figure 2 represents typical calibration data for each of the three analytes (Range 0.5 - 500 ng/mL)

Figure 2: (click for larger)

Conclusion

The DBS technique was used successfully to determine the concentration of quetiapine, norquetiapine and quetiapine sulphoxide in human whole blood samples. By focusing on the chromatographic conditions and sample preparation the desired LLOQ of 0.5 ng/mL was achieved.

Future research

The technique and analytical method as developed at BASi® UK will be transferred to the BASi® sites in the USA. On completion, validation samples prepared and analysed at BASi® UK will be crossvalidated at these sites