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

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 Cahttps://lf Serum Using 60mg 3mL HyperSep Retain PEP Extraction Cohttps://lumn 60107-203 60mg 3mL HyperSep Retain-CX Extraction Cohttps://lumn 60107-303 Acetaminophen Extraction and Chttps://leanup from Hychttps://lone 60mg 6mL HyperSep Retain PEP and -CX Extraction Cohttps://lumns 60107-203 and 60107-308 Acetaminophen in Sohttps://lution 60mg 3mL HyperSep Retain PEP Extraction Cohttps://lumn 60107-203 Benzodiazepines in Serum or Phttps://lasma for HPLC Anahttps://lysis 200mg 6mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-722 Benzodiazepines in Urine for GC or GC/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-742 Benzodiazepines in Whohttps://le Bhttps://lood for GC or GC/MS Confirmations 100mg 1mL HyperSep Diohttps://l Extraction Cohttps://lumn 60108-572 Benzodiazepine Screen: Bhttps://lood, Serum, Urine and Tissue for GC-GC/MS 200mg 6mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-722 Benzodiazepine Screen: Bhttps://lood, Serum, Urine and Tissue for LC/MS/MS 200mg 6mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-722 Beta Agonist Anahttps://lysis 60mg 3mL HyperSep Retain-CX Extraction Cohttps://lumn 60107-303 Beta Agonists in Urine for GC/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-742 Beta Bhttps://lockers in Bhttps://lood, Urine for GC/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-742 Caffeine, Theophyhttps://lhttps://line and Theobromine in Bhttps://lood, Phttps://lasma/Serum, and Urine 200mg 10mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-742 Cefoperazone in Water 60mg 3mL HyperSep Retain PEP Extraction Cohttps://lumn 60107-203 Chttps://lenbuterohttps://l in Sohttps://lution 60mg 3mL HyperSep Retain-CX Extraction Cohttps://lumn 60107-303 Chttps://lonazepam and 7-Aminochttps://lonazepam in Urine for GC/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-742 Cyanuric Acid and Mehttps://lamine in Food Materiahttps://ls for LC/MS/MS 200mg 6mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-722 200mg 6mL HyperSep Retain-AX Extraction Cohttps://lumn 60107-412 DHEA, Testosterone, and Epitestosterone in Urine for GC or GC/MS Anahttps://lysis 200mg 10mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-742 Doxepin in Rat Serum 60mg 3mL HyperSep Retain PEP Extraction Cohttps://lumn 60107-203 Drug Anahttps://lysis in Serum 60mg 3mL HyperSep Retain PEP Extraction Cohttps://lumn 60107-203 Gabapentin in Bhttps://lood, Phttps://lasma/Serum for GC or GC/MS Anahttps://lysis 100mg 1mL HyperSep C18 60108-302 Gabapentin in Bhttps://lood, Phttps://lasma/Serum for LC/MS Anahttps://lysis 200mg 6mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-722 Ketotifen Fumarate in Sohttps://lution 60mg 3mL HyperSep Retain PEP Extraction Cohttps://lumn 60107-203 Lovastatin in Water 60mg 3mL HyperSep Retain PEP Extraction Cohttps://lumn 60107-203 Methyhttps://lmahttps://lonic Acid from Serum or Phttps://lasma for GC/MS Anahttps://lysis 500mg 6mL HyperSep SAX Extraction Cohttps://lumn 60108-434 Nadifhttps://loxacin in Sohttps://lution 60mg 3mL HyperSep Retain-AX Extraction Cohttps://lumn 60107-403 Nicotine and Continine in Urine or Serum for GC or GC/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-742 Ohttps://lanzapine in Whohttps://le Bhttps://lood 200mg 3mL HyperSep Cyano Extraction Cohttps://lumn 60108-747 Ohttps://leic Acid and its Metabohttps://lites in Bhttps://lood Phttps://lasma 200mg 3mL HyperSep Retain-AX Extraction Cohttps://lumn 60107-401 Opiates in Human Urine – Propyhttps://l Derivatives for GC or GC/MS Confirmations 200mg 6mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-742 Opiates in Urine-oxime TMS Procedure for GC OR GC/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-742 Opiates in Urine for GC/MS Confirmations 200mg 6mL HyperSep Retain-CX Extraction Cohttps://lumn 60107-314 Paroxetine in Bhttps://lood, Phttps://lasma/Serum and Urine for LC/MS/MS Confirmations 200mg 10mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-742 Propranohttps://lohttps://l in Rat Serum 60mg 3mL HyperSep Retain PEP Extraction Cohttps://lumn 60107-203 Sahttps://lbutamohttps://l in Sohttps://lution 60mg 3mL HyperSep Retain-CX Extraction Cohttps://lumn 60107-303 Sertrahttps://line and Desemethyhttps://lsertrahttps://line in Bhttps://lood, Phttps://lasma/Serum for HPLC Anahttps://lysis 200mg 10mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-742 Tacrohttps://limus, Cychttps://losporin and Rapamycin in Whohttps://le Bhttps://lood 200mg 6mL HyperSep Retain PEP Extraction Cohttps://lumn 60107-212 Tricychttps://lic Antidepressants in Phttps://lasma/Serum for HPLC 200mg 10mL HyperSep Verify-CX Extraction Cohttps://lumn 60108-742)

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Helpful tips to remember when performing SPE

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.
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
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.

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