Solid Phase Extraction (SPE) Is Liquid Chromatography (LC):

Operate It as Such, Get Better Results, and Learn

Mark Hayward,1 Rick Youngblood,1 Kim Gamble,1 Jonathan Ho,2 Tom Moran,2 Martin Johnson,3 and Matthew Hardison3

1ITSP Solutions Inc (ITSP), 10 South Carolina St., Hartwell GA 30643; 2Shimadzu Scientific Instruments, 19 Schoolhouse Rd, Suite 107,

Someset NJ 08873; 3Assurance Scientific Laboratories, 727 Memorial Dr, Suite 103, Bessemer AL 35022

SPE: Why and the Challenges• Solid-phase extraction (SPE) is a preferred tool for isolating target analytes from complex matrices because the availability of a diverse range of chromatographic sorbents enables targeted approaches based on the specific chemistry of the analytes and matrices. Also, SPE offers the ability to enrich or pre-concentrate analytes in samples by drying and re-dissolving them afterward. Enrichment is valuable because it allows one to match the analyte concentrations to the approach used to measure them. Given these unique capabilities, SPE is considered the “gold standard” in analytical sample SPE is considered the “gold standard” in analytical sample preparation.

• However, SPE using a typical tube/cartridge on a vacuum (or pneumatic pressure) manifold requires proper training and skill, as care must be taken to maintain appropriate liquid flow in real time. Typically , flow is measured visually and held at 1 drop/s with a stopcock style valve. This is a laborious and requires continuous effort because flow changes with liquid level in the tube. It becomes particularly challenging to perform well when one attempts to process multiple SPE tubes in parallel.

SPE: the Challenges of High Throughput• Thus, the use of SPE presents a significant trade-off between quality and quantity. If the quantity of samples is low and the scientist is skilled, high quality can be achieved. However, if the quantity of samples is high, sacrifices are made in the control of flow and SPE performance suffers. Increased variability in parallel flow results in additional variability in and lower overall analyterecovery, requiring the use of internal standards and acceptance of lower quality, specifically, higher LLOQs recovery, requiring the use of internal standards and acceptance of lower quality, specifically, higher LLOQs (lower limits of quantification) and inconsistent assay results. This outcome is multiplied with plate based SPE, where vacuum (or pneumatic pressure) is held constant, because the varying resistance to flow well to well causes even more departure from ideal flow / high recovery. • It is clear that flow is a crucial parameter in SPE and that the negative effects of insufficient flow control only multiply as the number of samples increase.

Typical single use SPE device performanceflow driven pneumatically or by vacuum

• Flow: slower is better and this is counter productive• Flow varies with liquid level and so do recoveries• In plates, parallel flow varies well to well resulting in additional variancein additional variance• Recovery variance increases overall assay variance• Higher cut-offs needed to anticipate lowerrecoveries• Enrichment (pre-concentration)

requires dry down costing more labor and time

Jordan L, LCGC 1993, 11, 634-8for huge SPE cartridges

We are told that SPE is “digital chromatography”

and that we cannot expect LC like performance

despite the fact that we are using LC sorbents.

This poster challenges that assertion.

Testing a new micro-SPE device• The authors of this poster embarked on an effort to

systematically test a new micro-SPE device from ITSP

• Initially, the compelling reasons for interest in this new SPE device were:– Automation using the CTC/PAL autosampler performed at

the same cost as other single use SPE devices

– SPE performed on-line in parallel with LC/MS/MS (or – SPE performed on-line in parallel with LC/MS/MS (or GC/MS/MS) analysis using the CTC/PAL autosampler

– Enrichment of analyte without the need for drying eluantdue to the small size and hence elution volumes that should be possible with this micro-SPE device

• The authors of this poster otherwise fully expected to observe SPE performance just like other single use SPE devices

Hoses connect the SPE cartridge tray and syringe wash station to an ordinary lab solvent waste container. Used cartridges are typically discarded by the PAL into a box under the wash station and LC valve.

Workflow: minimizing cycle timeParallel PAL operation in the inject ahead mode

SPE 1 SPE 2 SPE 3

LC/MS/MS 1 LC/MS/MS 2 ……

……LC/MS/MS 1 LC/MS/MS 2

4.5 min 4.5 min

……

Total cycle time (SPE + LC/MS/MS) = 4.5 min

Automated and Higher Quality SPE Using an Autosampler: How it works

• Central to automated, rapid, and higher -quality ITSP SPE is a patented, single-use micro cartridge (ITSP, Hartwell, Ga.) containing user-defined, packed chromatographic media

• The crimped-on septum and needle guide (upper 80% of cartridge) enable automation by facilitating accurate cartridge transport on a syringe needle

• Automated SPE begins by using the autosampler syringe for cartridge conditioning, sample loading and sample washing

• Automated SPE begins by using the autosampler syringe for cartridge conditioning, sample loading and sample washing over a waste receptacle

• The syringe is used to perform elution over a clean vial or well, and the used cartridge is discarded in a different waste receptacle

• Automation is complete after the syringe mixes the freshly eluted sample and then injects it into the LC/MS/MS or GC/MS/MS that will be used to measure the sample (see the Figure showing the CTC/PAL autosampler).

• SPE performed in parallel after SPE of first sample in list

The heart of the ITSP form of SPE is the patented single use cartridge containing customer-defined packed chromatographic media

The crimped-on septum and needle guide (upper 80% of cartridge) enable 80% of cartridge) enable automation by facilitating accurate cartridge transport on the PAL syringe needle

The small (16 µµµµl belolwsorbent) extra-column volume facilitates low volume elution (50-100 µµµµl)

Development of an Automated SPE MethodResulted in Detailed SPE Flow Studies

• To test the ITSP form of micro-SPE, the authors set out to develop a SPE clean-up of urine and oral fluid samples for drug analysis using UCT 50 µm C18 end capped silica sorbent

– See poster P-T-0612 here at HPLC 2016 for full details

• Included in the method development was a thorough flow optimization study intended to measure cost/benefit in time/recovery

– The authors fully expected to observe a sigmoidal curve as reported – The authors fully expected to observe a sigmoidal curve as reported by Jordan L, LCGC 1993, 11, 634-8

• The result was a 20 data point U-shaped curve showing that flow of 5 µµµµl/s resulted in 100% recovery

• Skeptical, the flow study was repeated, then again measuring 94 data points, then again measuring load and elute steps separately while holding the other at 5 µµµµl/s

• All of these produced the same U-shaped curve and they all looked similar to a van Deemter curve

How to plot a van Deemter curve for SPE• The conventional measures of SPE (recovery) differ from

conventional measures of GC and LC (retention time and peak width [2σ])

• Yet the processes are the same (diffusion, adsorption [or not], and desorption [or delayed]), the random walk model applies, and the van Deemter equation is a variance (σ2) equation [Giddings, JC, Unified Separation Science, Wiley 1991, p 92-101]

• Practical insights in the use of computerized chromatography data to evaluate separation performance [%RSD method for plate

•

to evaluate separation performance [%RSD method for plate height calculation in Neue, UD, HPLC Columns: Theory, Technology, and Practice, Wiley, 1997, p12-13] offers a simple view of the relative nature of σ and how to address SPE data

• %Recovery is a measure of deviation (σ) like LC peak width and thus, (100% - measured %Recovery)2 is a measure of variance (σ2)

• Hence, plotting (100% - measured %Recovery)2 should yield a typical van Deemter curve shape if van Deemter processes govern the dispersion of molecules in SPE

van Deemter Curves for RP SPEusing 2 different particle sizes

% R

eco

very

8010 um 50 um

Methadone

Flow optimization for ITSP SPE:just like a LC column

Load and elute steps

optimized separately

behave identically

% R

eco

very

Flow (µµµµl/s)

100

0 5 10 15 20

94 data points per curve measured by LC/MS/MS and fit to van Deemter equation in Excel

Methadone

Flow Optimization: Outcome and Impact

• ITSP SPE cartridges behave like LC columns due to:

• Accurate flow control from PAL autosampler (syringe

pump)

• Sorbent is packed

• Low extra-column volume• Low extra-column volume

• Benefits:

• 100% recovery is systematically achieved (within the

measurement precision of the LC/MS/MS ±3%

• Same tools used to increase speed and performance

for LC can be used for SPE (eg. smaller particles)

Further Study• Rapid progress in developing the urine and

oral fluid method led to a variety of method development efforts expanding the range analytes in more complex matrices (blood/tissues/food)

To address the more complex matrices this led • To address the more complex matrices this led to the use of more selective sorbents:

– Cation exchange for drugs

– Anion exchange for lipids

– Chelation for phospholipids and phosphopeptides

• As might expected, this led to more SPE flow studies

Flow optimization for CationExchange SPE (50 µµµµm particles)

80

SCX %-Recovery in 20% Water

SCX %-Recovery in 80% Water

SCX %-Recovery in 50% Water

Reverse Phase

% R

eco

ve

ry

100

0 1 2 3 4 5 6 7 8

Reverse Phase

% R

eco

ve

ry

Flow (µµµµl/s)

Load and elute steps

optimized separately

behave identically

n-propyl linked phenyl-SO3-

on silicaMolecules without cationic sites have an optimal flow of 5 µµµµl/s (i.e. RP)

Oxycodone

Ionic SPE Flow Optimization: Outcome and Impact

• Outcomes:– Cation exchange SPE has an optimum flow of 1.2 µl/s (1/4 that

of reverse phase, 50 µm particles)

– In addition, the acceptable flow range for high recovery widens with additional water content

– Preliminary results suggest anion exchange and chelation SPE have the same van Deemter curves as cation exchangehave the same van Deemter curves as cation exchange

• Benefits:– 100% recovery is systematically achieved (within the

measurement precision of the LC/MS/MS ±3%) [rare with these chemistries]

– Knowledge of the chemical preference for more water (less solvent) leads to more predictable and consistent outcomes• Use silica (not polymer) based particles to minimize solvent levels

(n-propyl phenyl linker still gives sufficient mixed mode [RP] behavior)

• Use strong miscible solvents (IPA/THF) to maximize water content

A possible explanation for water dependence on ionic adsorption / desorption

• It seems likely that ionic sorbent sites will have a layer around them that is more concentrated with water (rather than solvent)

• This is the likely reason cation, anion, and chelation

exc

ha

ng

e s

orb

en

t

Water layer with lower solvent

content

cation, anion, and chelationhave the same optimum flow while having different binding energies (mass transfer regulated by transport through the water layer)

• The amount of solvent present regulates the thickness of the water layer

Ca

tio

ne

xch

an

ge

so

rbe

nt

-SO3-

Water layer with higher solvent

content

Summary and Conclusions• SPE is gradient liquid chromatography

– All existing knowledge about gradient liquid chromatography can be applied to SPE

• Use of smaller particles for RP SPE significantly increases speed without impact on performance

– Applying this knowledge produces significantly better results than the alternatives

• 100% recovery can be systematically achieved• 100% recovery can be systematically achieved

• Adsorption and desorption is a reversible equilibrium

– Separate measurement of load and elute flow behavior produces the same van Deemter curves

• SPE using single use devices can achieve high quality even in high throughput applications

– Given the same price point, it is hard to see any rationale for continued use of single use SPE devices that utilize loose sorbent and/or vacuum / pneumatic driven flow