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HybridSPE-Troubleshooting & Frequently Asked Questions

HybridSPE™ - Phospholipid Technology

Can I use HybridSPE-PL with smaller plasma volumes (e.g., 20-40 µL plasma)?

Yes. The HybridSPE-PL Small Volume plate (52794-U) is designed for processing plasma/ serum volumes between 20-40 µL. Larger sample volumes of 100-300 µL should be processed on the standard HybridSPE-PL plate (575656-U).

Why is acetonitrile and formic acid used as a precipitating agent in the HybridSPE-PL method?

Acetonitrile is a commonly used protein precipitation agent when prepping plasma samples for LC-MS analysis. The resulting precipitated protein is easily filtered using the “In-well Precipitation” method, and forms easily removed protein pellets when centrifugation is preferred (“Off-Line Precipitation” method – required for HybridSPE-PL 1 mL cartridge format).

The addition of 1-2% formic acid to the acetonitrile precipitating agent is critical because: 1) formic acid is a stronger Lewis base than most carboxyl (-COOH) groups found in acidic pharmaceutical compounds (inhibiting analyte retention on the HybridSPE phase), but not as strong a Lewis base as the phosphate moiety found in phospholipids; and 2) the low pH environment neutralizes residual silanol activity on the silica surface, thereby eliminating secondary cation-exchange interaction with basic compounds of interest.

What if my analyte(s) of interest are not soluble in acetonitrile?

Although some analytes may not be soluble in acetonitrile, after protein precipitation, the HybridSPE eluent will consist of 75% acetonitrile (w/ formic acid) and 25% aqueous (from the biological sample). The aqueous content of the sample should provide adequate solubility prior to LC-MS analysis.

Alternatively, 1% ammonium formate in methanol may be used in place of 1% formic acid in acetonitrile. Ammonium formate in methanol provides increased solubility of polar compounds and precipitates proteins as well as acetonitrile, allowing for both “Off-Line” and “In-well” precipitation methods.

Can I increase assay sensitivity by either increasing sample volume and/or concentrating (evaporation and reconstitution) of the HybridSPE eluent?

Biological sample volumes of >300 µL can be applied; however, some phospholipid breakthrough may occur with analytes of interest. 98-100% of biological phospholipids are removed when <300 µL plasma is applied to the HybridSPE phase. When increasing sample volume, be sure to increase the volume of the precipitating agent accordingly. A 1:3 (v/v) plasma:precipitating agent ratio is necessary for optimal performance.

Another strategy for increasing sensitivity is through evaporation of the HybridSPE eluent, followed by reconstitution in a smaller volume of LC-MS mobile phase. The acetonitrile portion of the HybridSPE eluent greatly aids the evaporation process. On average it takes less than 10 minutes to evaporate 300-400 µL of HybridSPE eluent under nitrogen at 37 °C.

Why can ion-suppression still be evident during LC-MS analysis after HybridSPE-PL?

HybridSPE technology will only remove phospholipids and gross levels of precipitated protein from biological samples. Other chemical entities common to biological samples can lead to ion-suppression if not removed prior to LC-MS-MS analysis. It is important to identify the ion-suppression causing component to facilitate troubleshooting. It may be necessary to adjust chromatographic conditions to separate analytes of interest from interfering matrix components.

Examples of non-phospholipid chemicals that can lead to ion-suppression include: 

  • sodium citrate which is an anti-coagulant used to prepare plasma from blood 
  • phthalates, plasticizers and other mold release agents found in plastic ware 
  • polyethylene glycol which is a common dosing vehicle for many drugs 
  • extractables from o-rings, plastic ware, and seals used to store biological samples

Why is the resulting HybridSPE-PL eluent lower in volume than what was applied to the HybridSPE packed bed? What are the effects of conditioning the phase?

The dead volume for the HybridSPE packed bed is ~80 µL. Also, there is an evaporation effect on the eluent when using a vacuum manifold. When applying ~15 in Hg vacuum to the HybridSPE plate for 3 min. (time taken to pass the sample through the well plate), 10-20 µL of the volume of the SPE eluent can be lost due to evaporation during processing. Therefore, when processing a 400 µL precipitated sample (100 µL plasma + 300 µL precipitating agent) through the unconditioned HybridSPE phase, the resulting eluent will be a volume of ~300 µL. Although the volume is reduced during SPE processing, the final analyte concentration of the eluent does not appear to be affected. Nevertheless, addition of an I.S. is recommended prior to HybridSPE processing (which is standard for most sample prep techniques). If the analyst chooses to condition the HybridSPE phase with >80 µL of solution prior to sample addition, there could be a dilution effect. The final eluent volume will be ~80 µL greater than it should be. As a result, absolute recovery will appear lower than it actually is. If an increase in signal response is necessary during LC-MS analysis, we recommend evaporating the eluent and reconstituting in a smaller known volume of LC mobile phase prior to LC-MS analysis.

Why am I experiencing absolute recovery values > 100%?

The resulting HybridSPE eluate contains acetonitrile (volatile solvent). After the precipitated sample completely passes through the HybridSPE packed bed, the vacuum should be disengaged immediately. Further vacuum application can evaporate the eluate, thereby erroneously giving misleadingly high analyte responses during subsequent LC-MS analysis.

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