Lc/ms/ms Bioanalysis Of Acidic And Basic Compounds With Double Liquid-liquid Extraction By Changing Ph In Between

Rachel Sun, Gary Overdorf
Bioanalytical Systems, Inc., 2701 Kent Avenue, West Lafayette, IN 47906


  • Sample clean-up is very important for bioanalytical analysis
  • Liquid-liquid extraction is widely used: clean, fast, low cost
  • Different chemicals are extracted at different conditions
  • Liquid-Liquid extraction at basic condition for HCD
  • Liquid-Liquid extraction at acidic condition for MPA
  • What is the extraction method to quantitate both compounds from one sample?
    Double liquid-liquid extraction by changing pH in between
  • Tomtec automation
  • Positive-negative ion switch during detection


Extraction procedure (Double liquid-liquid extraction)

  • Add 100 µL sample to sample plate and 50 µL IS (HCD-d6 and MPA-d3)
  • Add 50 µL saturated borate
  • Add 650 µL MTBE
  • Vortex, centrifuge, and tranfer organic to a clean plate then blow down to dryness
  • Add 50 µL 10% formic acid to the same sample plate
  • Add 650 µL MTBE
  • Vortex, centrifuge, and tranfer organic to the same extract plate then blow down to dryness
  • Reconstitute the extract plate and inject on LC/MS/MS

HPLC conditions

Column: Gemini C18, 50 x 2.0, 3μ, #408886-5
Mobile Phase A: 0.1% formic acid in water
Mobile Phase B: 100% Acetonitrile
Run time: 7.5 minutes
Retention time: 2.5 minutes for HCD; 4.2 minutes for MPA
Gradient: Time (min) %B
  0 5
  1 5
  3 50
  5 82
  5.05 90
  6.5 90
  6.55 5

Tandem mass spectrometry

Mass spectrometer: API 4000
Source: Turbo Ionspray
Resolution: Unit/Unit
Ion Monitored: 0 to 3.2 minutes
  HCD and HCD-d6, +300.1/199, +306.2/202,
  3.2 to 6.0 minutes
  MPA and MPA-d3, -319/191, -322/191,



Calibration standard statistics



QC statistics




Representative calibration curve

Typical chromatogram

Blank matrix

Low calibration standard

0.200 ng/mL for HCD and 50.0 ng/mL for MPA



The recovery for liquid-liquid extraction is variable with different extraction conditions.

The presence of drugs, their metabolites and co-administered drugs often require quantitation of a set of chemicals with different chemical properties, such as pKa. One solvent or pH extraction condition could prove difficult in achieving adequate recovery for all the interested compounds in a single extraction. Double liquid-liquid extraction gives the flexibility to change extraction pH, as demonstrated above, or extraction solvents to greatly enhance analyte recovery.


This novel methodology enlarges the scope of a liquid-liquid extraction sample preparation method. The method could be adjusted according to analytes' pKa. Acidic, basic or neutral conditions could be combined to extract compounds with different chemical identities, such drug and metabolites.