Research Article

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Quantification of dabigatran and indirect quantification of dabigatran acylglucuronides in human plasma by LC–MS/MS Background: An assay for the quantification of dabigatran and its active metabolites, dabigatran acylglucuronides, has not previously been described in detail. Results: For the quantification of total dabigatran concentration (free dabigatran and acylglucuronides), samples were subjected to alkaline hydrolysis. For the quantification of free dabigatran, samples were acidified with ammonium formate. Following acetonitrile protein precipitation, the samples were analyzed by LC–MS/MS using gradient elution to ensure separation of dabigatran from dabigatran acylglucuronides. Mean recoveries ≥98% were achieved. The assay was validated over the range 2.5–1000 ng/ml dabigatran, imprecision was 98% for both sample preparation methods. Some ionization

Table 1. Extraction recovery and matrix effect in six different individuals. Analyte 

Nominal concentration (ng/ml) 

% Recovery  Mean ±SD

IS normalized mean ±SD

% Matrix Effect  Mean ±SD

IS normalized mean ±SD

Free dabigatran sample preparation (without alkaline hydrolysis) LQC

10

104.9 ±11.1

102.2 ±5.8

112.7 ±13.1

99.7 ±4.0

MQC

100

98.0 ±4.9

100.7 ±0.8

116.2 ±7.7

101.0 ±0.5

HQC

1000

99.4 ±10.7

99.4 ±1.4

111.6 ±4.1

99.8 ±0.9

Total dabigatran sample preparation (after alkaline hydrolysis) LQC

10

107.6 ±10.8

111.6 ±6.2

123.8 ±14.0

100.3 ±1.1

MQC

100

98.7 ±4.7

101.5 ±1.0

124.3 ±15.9

100.1±0.5

HQC

1000

97.8 ±6.0

100.1 ±2.9

105.4 ±8.3

99.5±0.8

HQC: High quality control; LQC: Low quality control; MQC: Mid quality control; SD: Standard deviation.

962

Bioanalysis (2015) 7(8)

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Quantification of dabigatran & indirect quantification of dabigatran acylglucuronides

enhancement was observed for dabigatran. However, this was corrected for by the isotopically labeled IS and the IS normalized variation between individuals was