HybridSPE-PL Method Development: Recovery Optimization

HybridSPE™ - Phospholipid Technology

The HybridSPE-PL stationary phase is comprised of a proprietary patent pending zirconia bonded silica particle. Phospholipids are retained/removed from the sample through a highly selectivity Lewis acid-base interaction between the phosphate group (Lewis base), inherent of all phospholipids, and Zr atoms bonded to the HybridSPE silica surface. The precipitation agents recommended for HybridSPE-PL is comprised of two parts:

  1. An aqueous miscible organic solvent (e.g., MeCN or MeOH) that denatures protein into a precipitant 

  2. A modifier (formic acid, ammonium formate, or citric acid) that inhibits analyte(s) of interest from co-retaining with phospholipids on the Zr-Si HybridSPE stationary phase, potentially resulting in low recovery
Relative Retention Strength of Lewis Bases to Zirconia

When developing new HybridSPE-PL methods, there are three precipitation agents that we recommend for researchers to screen when optimizing analyte recovery.

96-well Primary Method (Formic Acid Modifier)

Recommended for most applications (basic, neutral, acidic compounds) – amenable for most (80%) applications. 

  1. To each well, add 100 µL plasma followed by 300 µL 1% formic acid in MeCN

  2. Mix the sample well (e.g., vortex for 1 min.)

  3. Apply vacuum and collect the resulting eluent for LC-MS analysis

If low recovery is observed, proceed to secondary procedures. An alternative option is to screen both the primary and secondary procedures concurrently during initial method development.


  • Formate ion (HCOO-) is a strong enough Lewis base to inhibit most acidic compounds from interacting with Zr sites, improving recovery of acidic compounds
  • H+ derived from formic acid neutralizes silanol groups (Si-O- => Si-OH), minimizing potential secondary weak cation exchange (WCX) interactions of basic compounds improving analyte

96-well Primary Method (Formic Acid Modifier)

96-well Secondary Method (Basic & Neutral Compounds)

Recommended for low recovery basic & neutral compounds.

  1. To each well, add 100 µL plasma followed by 300 µL 1% ammonium formate in MeOH

  2. Mix the sample thoroughly (e.g., vortex for 1 min.)

  3. Apply vacuum and collect the resulting eluent for LC-MS analysis

  • Methanol combined with ammonium formate is a powerful protein precipitation agent, providing precipitants similar in consistency to acetonitrile protein precipitation
  • Formate ion (HCOO-) is a strong enough Lewis base to inhibit most acidic compounds from interacting with Zr sites, improving recovery of acidic compounds 
  • NH4+ is a stronger counter-ion than H+, able to inhibit a wider range of basic compounds from interacting with exposed silanol groups
96-well Secondary Method (Basic & Neutral Compounds)

96-well Secondary Method (Chelator & Acidic Chelator Compounds):

Recommended for low recovery chelator & acidic chelator compounds

  1. Condition each well with 400 µL 0.5% citric acid in acetonitrile*

  2. To each well, add 100 µL plasma followed by 300 µL 0.5% citric acid in acetonitrile

  3. Apply vacuum and collect the resulting eluent for LC-MS analysis

  • Citrate ion (citric acid) is a stronger Lewis base than formate, inhibiting most chelator compounds from interacting with Zr ions on the HybridSPE stationary phase
  • Note that citrate is not strong enough to disrupt phosphate (phospholipid) binding

Low Recovery Chelation Functional Groups with Example Compounds

HybridSPE-PPT Method Development (Recovery Optimization) Flow Chart

* Note: When 0.5% citric acid in ACN is employed as the precipitation agent, the HybridSPE-PL phase (96-well or 1 mL cartridge) should be conditioned with 400 µL 0.5% citric acid in ACN prior to sample addition.

Materials

     
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