Quantitative Analysis of Benzodiazepines in Whole Blood by QuEChERS and LC–MS/MS

Common sample preparation methods for biological samples include a protein precipitation step followed by liquid–liquid extraction or solid phase extraction. This application describes an easy, fast, and effective method using QuEChERS for the quantitative analysis of benzodiazepines in whole blood. Benzodiazepines are psychoactive drugs widely prescribed for treating anxiety, insomnia, agitation, seizures, muscle spasms, and alcohol withdrawal.

Extraction/Analytical Materials

Procedure

a) Add 2 mL of MeCN with 0.4% Formic Acid to ECQUUS15CT

b) Add internal standards and 1 mL whole blood

c) Cap and shake for 1 min at 1000 strokes/min using a Spex Geno-Grinder

d) Centrifuge for 5 min at 3000 g

e) Transfer 1 mL of supernatant to CUMPSC18CT

f) Cap and shake for 1 min at 1000 strokes/min using a Spex Geno-Grinder

g) Centrifuge for 5 min at 3000 g

h) Transfer 0.4 mL of the cleaned extract into an autosampler vial. Add 0.4 mL D.I. H₂O and vortex for 30 s

Instrumental

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Conclusion

The matrix effects were found to be minor, ranging from -22 to 18%. This indicated that the QuEChERS method with dSPE cleanup sufficiently removed matrix interferences that may cause significant ion suppression or enhancement. Excellent recoveries (85.5–105%) and relative standard deviations (RSD% ≤ 10.7%) were obtained.

Table I: Linearity and Matrix Effect

Table II: Recovery and RSD% from Whole Blood Spiked at 2 Levels (n=6)

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