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

Xiaoyan Wang, UCT, LLC

Benzodiazepines are psychoactive drugs widely prescribed for treating anxiety, insomnia, agitation, seizures, muscle spasms, and alcohol withdrawal. Common sample preparation methods to monitor for this drug class in whole blood include a protein precipitation step followed by either a time-consuming
liquid–liquid extraction (LLE) or solid-phase extraction (SPE) procedure. This featured application note describes an easy, fast, and effective method using the QuEChERS approach to provide for their quantitative analysis.

QuEChERS Extraction

• Add 2 mL of acetonitrile with 0.4% formic acid to a 15 mL centrifuge tube containing 400 mg MgSO4 and 100 mg NaOAc.

• Add internal standards (IS), and appropriate amounts of benzo spiking solution to fortified samples.

• Add 1 mL of the negative whole blood into the 15-mL tubes.

• Cap and shake for 1 min at 1000 strokes/min using a Spex 2010 Geno-Grinder.

• Centrifuge at 3000 g for 5 min.

dSPE Cleanup

• Transfer 1 mL of the supernatant to a 2-mL dSPE tube containing 150 mg MgSO4 and 50 mg PSA and 50 mg C18.

• Shake 1 min at 1000 strokes/min using the Spex 2010 Geno‑Grinder.

• Centrifuge at 3000 g for 5 min.

• Transfer 0.4 mL of the cleaned extract into a 2-mL auto-sampler vial, add 0.4 mL of reagent water, and vortex for 30 s.

• The samples are now ready for LC–MS–MS analysis.

Instrumental

LC–MS–MS:         Agilent 1200 Binary Pump SL / AB Sciex API 4000 Qtrap MS–MS

Column:                100 × 2.1 mm, 3-μm

                      UCT Selectra® DA LC column,

Guard column:     10 × 2.1 mm, 3-μm

            UCT Selectra® DA guard column,

Injection volume:  10 μL

Mobile phase A:    0.1% formic acid in D.I. H2O

Mobile phase B:    0.1% formic acid in methanol

Column flow rate: 0.30 mL/min

Results

Conclusion

Excellent recoveries (85.5–105%) and relative standard deviations (RSD% ≤ 10.7%) were achieved. Matrix effects were found to be minor, ranging from -22% to 18%. This indicates that the QuEChERS technique with dSPE cleanup quickly and effectively removes unwanted matrix interferences that may cause significant ion suppression or enhancement.

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