SPE method development

SPE Method for Sample Preparation

Solid phase extraction (SPE) methods are based on the 6 steps with an example method as shown below. If you need to develop or optimize an SPE method, information on this page is a good place to begin. Before starting to develop an SPE method, refer to SPE Phase Selection to determine which SPE phase to use for your analyte. For the SPE method itself, you can begin from scratch or start with an existing method.

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Example SPE method

Six Steps of SPE to a clean extract
Compound(s) Niflumic Acid, niflumic acid D3 (IS)
Matrix Human plasma
SPE SOLA WAX 10mg 1mL cartridge 60109-005
Condition 500µL methanol 500µL water
Sample load Load sample at 0.5mL/min
Wash 500µL 25mM ammonium acetate (NH4 +CH3 COO-) buffer in water 500µL methanol
Elute 500µL methanol with 2% ammonium hydroxide (NH4 OH) at 0.5mL/min Dry under nitrogen and reconstitute in 100µL 2% formic acid in water

Generic SPE methods for SOLA and SOLAμ products

Thermo Scientific SOLA SPE cartridges and plates Technical Guide

Generic SOLA product methods designed to be a starting point for most sample extraction protocols

  SOLA HRP
Reverse phase
SOLA SCX
Mixed mode cation exchanger
SOLA SAX
Mixed mode anion exchanger
SOLA WAX
Mixed mode weak anion exchanger
SOLA WCX
Mixed mode weak cation exchanger
Condition 500µL methanol
Equilibrate 500µL water 500µL water with 1% formic acid 500µL water with 1% ammonium hydroxide 500μL water with 1% formic acid 500μL water with 1% ammonium hydroxide
Load 50 to 500µL of sample at 1mL/min 50 to 500µL of sample at 1mL/min containing 1% formic acid 50 to 500µL of sample at 1mL/min containing 1% ammonium hydroxide 50 to 500μL of sample at 1mL/min containing 1% formic acid 50 to 500μL of sample at 1mL/min containing 1% ammonium hydroxide
Wash 1 500µL 5% methanol in water 500µL water with 1% formic acid 500µL water with 1% ammonium hydroxide 500μL water with 1% formic acid 500μL water with 1% ammonium hydroxide
Wash 2   500µL methanol with 1% formic acid 500µL methanol with 1% ammonium hydroxide 500μL methanol with 1% formic acid 500μL methanol with 5% ammonium hydroxide
Elute 200µL - 500µL methanol 200µL - 500µL methanol with 1% ammonium hydroxide 200µL - 500µL methanol with 1% formic 200μL - 500μL methanol with 5% ammonium hydroxide 200μL - 500μL methanol with 1% formic acid

Optimized SPE Methods for HyperSep products

Pharmaceutical/Biotech Applications

Method Using Product ID
Acetaminophen in Calf Serum Using 60mg 3mL HyperSep Retain PEP Extraction Column 60107-203
60mg 3mL HyperSep Retain-CX Extraction Column 60107-303
Acetaminophen Extraction and Cleanup from Hyclone 60mg 6mL HyperSep Retain PEP and -CX Extraction Columns 60107-203 and 60107-308
Acetaminophen in Solution 60mg 3mL HyperSep Retain PEP Extraction Column 60107-203
Benzodiazepines in Serum or Plasma for HPLC Analysis 200mg 6mL HyperSep Verify-CX Extraction Column 60108-722
Benzodiazepines in Urine for GC or GC/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Column 60108-742
Benzodiazepines in Whole Blood for GC or GC/MS Confirmations 100mg 1mL HyperSep Diol Extraction Column 60108-572
Benzodiazepine Screen: Blood, Serum, Urine and Tissue for GC-GC/MS 200mg 6mL HyperSep Verify-CX Extraction Column 60108-722
Benzodiazepine Screen: Blood, Serum, Urine and Tissue for LC/MS/MS 200mg 6mL HyperSep Verify-CX Extraction Column 60108-722
Beta Agonist Analysis 60mg 3mL HyperSep Retain-CX Extraction Column 60107-303
Beta Agonists in Urine for GC/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Column 60108-742
Beta Blockers in Blood, Urine for GC/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Column 60108-742
Caffeine, Theophylline and Theobromine in Blood, Plasma/Serum, and Urine 200mg 10mL HyperSep Verify-CX Extraction Column 60108-742
Cefoperazone in Water 60mg 3mL HyperSep Retain PEP Extraction Column 60107-203
Clenbuterol in Solution 60mg 3mL HyperSep Retain-CX Extraction Column 60107-303
Clonazepam and 7-Aminoclonazepam in Urine for GC/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Column 60108-742
Cyanuric Acid and Melamine in Food Materials for LC/MS/MS 200mg 6mL HyperSep Verify-CX Extraction Column 60108-722
200mg 6mL HyperSep Retain-AX Extraction Column 60107-412
DHEA, Testosterone, and Epitestosterone in Urine for GC or GC/MS Analysis 200mg 10mL HyperSep Verify-CX Extraction Column 60108-742
Doxepin in Rat Serum 60mg 3mL HyperSep Retain PEP Extraction Column 60107-203
Drug Analysis in Serum 60mg 3mL HyperSep Retain PEP Extraction Column 60107-203
Gabapentin in Blood, Plasma/Serum for GC or GC/MS Analysis 100mg 1mL HyperSep C18 60108-302
Gabapentin in Blood, Plasma/Serum for LC/MS Analysis 200mg 6mL HyperSep Verify-CX Extraction Column 60108-722
Ketotifen Fumarate in Solution 60mg 3mL HyperSep Retain PEP Extraction Column 60107-203
Lovastatin in Water 60mg 3mL HyperSep Retain PEP Extraction Column 60107-203
Methylmalonic Acid from Serum or Plasma for GC/MS Analysis 500mg 6mL HyperSep SAX Extraction Column 60108-434
Nadifloxacin in Solution 60mg 3mL HyperSep Retain-AX Extraction Column 60107-403
Nicotine and Continine in Urine or Serum for GC or GC/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Column 60108-742
Olanzapine in Whole Blood 200mg 3mL HyperSep Cyano Extraction Column 60108-747
Oleic Acid and its Metabolites in Blood Plasma 200mg 3mL HyperSep Retain-AX Extraction Column 60107-401
Opiates in Human Urine – Propyl Derivatives for GC or GC/MS Confirmations 200mg 6mL HyperSep Verify-CX Extraction Column 60108-742
Opiates in Urine-oxime TMS Procedure for GC OR GC/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Column 60108-742
Opiates in Urine for GC/MS Confirmations 200mg 6mL HyperSep Retain-CX Extraction Column 60107-314
Paroxetine in Blood, Plasma/Serum and Urine for LC/MS/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Column 60108-742
Propranolol in Rat Serum 60mg 3mL HyperSep Retain PEP Extraction Column 60107-203
Salbutamol in Solution 60mg 3mL HyperSep Retain-CX Extraction Column 60107-303
Sertraline and Desemethylsertraline in Blood, Plasma/Serum for HPLC Analysis 200mg 10mL HyperSep Verify-CX Extraction Column 60108-742
Tacrolimus, Cyclosporin and Rapamycin in Whole Blood 200mg 6mL HyperSep Retain PEP Extraction Column 60107-212
Tricyclic Antidepressants in Plasma/Serum for HPLC 200mg 10mL HyperSep Verify-CX Extraction Column 60108-742)

Webinars/Videos: SPE Method Development 

Helpful tips to remember when performing SPE

  1. Do not allow silica-based sorbent to dry between conditioning steps or before sample application since this can impact recovery levels. Apply each solvent immediately after the previous one. Note that drying is not an issue with polymeric based materials since it stays conditioned after an appropriate solvent is applied.
  2. Prior to elution, fully drying cartridges will ensure optimum analyte recovery
    1. Confirm dryness by touching the sides of cartridge at full vacuum
    2. Cartridge should not feel cool to the touch
  3. Always use daily prepared solvents since certain solvents can lose strength over time.

Optimization of load step—pH considerations

pH of the sample can greatly affect the recovery levels of the analyte. The optimal pH of the sample is that pH that puts the sample in an optimal state for interacting with the sorbent material. Table 1 provides information about the role of analyte pH for different SPE sorbents.

Table 1. Optimization of the sample load step

Sample load in reversed phase interactions

  • Neutral compounds are not affected by pH (no need to adjust the pH of the sample)
  • For charged compounds, use at a pH at which the compound is not charged. Neutralize the molecule according to the following:
    • For basic compounds, the neutral molecule exists at least 2 pH units below the pKa of the compound
    • For acidic compounds, the neutral molecule exists at least 2 pH units above the pKa of the compound

Sample load in normal phase SPE

  • pH is not normally an issue in normal phase interactions, as the solvents used are typically non-polar organic solvents, rather than water.
  • There is no need to verify the sample application pH

Sample load in ion-exchange phase SPE

  • pH and pKa are important considerations
  • Acidic compounds are extracted from a sample solution at least 2 pH units above the pKa of the analyte
  • Basic compounds are extracted from a sample solution 2 or more pH units below the pKa of the analyte

Optimization of wash and elution step

In general, one should prepare various wash and elution solutions to determine the ideal composition of solution to recover analytes. For example, to determine wash and elution solutions for testosterone using HyperSep Retain PEP cartridge, one can sequentially wash the sample with increasing strengths of methanol mixed in water. Based on the data in Figure 1, 50% methanol can be used as a washing solution whereas 90% of methanol can elute testosterone.

Optimization of wash and elution step
Figure 1. Wash and elution step optimization by testing increasing strengths of methanol mixed in water.

What parameters are adjustable for system optimization?

Optimizing the load, wash, and elution steps of the SPE process can deliver higher recovery and cleaner sample extracts resulting is better analytical results and robustness of your analytical instrument. Here you will find tips and tricks for successful SPE information about the parameters that influence SPE along with.

  • Ionic strength
  • Sample volume
  • pH
  • Organic polarity
  • Sorbent mass
  • Cartridge volume
  • Organic strength
  • Sorbent type

Summary of adjustable parameters in SPE that affect recovery levels

pH considerations can make a difference in terms of recovery levels. pH can be optimized using appropriate buffers to ensure that the sample loaded onto the SPE column is in its correct optimized state.

pH levels Wash solvent Elution solvent
  • Sample loading
  • Buffers used
  • Elution
  • Use a wash solvent is selected that does not recover the analyte of interest
  • Select a solvent that is strong enough to remove the compounds of interest and one in which the analytes are soluble.
  • Polarity
  • Solubility
  • Elutropic strength