Phospholipid Extractions for Cleanup or Enrichment

By: Carmen T. Santasania, An Trinh, and Mati Sarker, Reporter Volume 27.3


Within the last 5-10 years, hydrophilic polymer SPE phases have become the phase of choice when developing new extraction methods that recover, concentrate, and cleanup small molecules (e.g., pharmaceutical compounds) from complex aqueous sample matrixes (e.g., biological fluids, water, etc.). Unlike traditional reversed phase chemistries such as C18, hydrophilic polymers are typically comprised of a hydrophobic component (e.g., polystyrene and/or divinyl benzene) and a hydrophilic component (e.g., methacrylate, vinylamidazol, N-vinyl pyrillidone, and/or hydroxyl). Because hydrophilic polymer SPE phases contain both nonpolar and polar functional groups, they offer distinct advantages over traditional silica-based reversed phase SPE chemistries. Unlike silica-based, reversed phase SPE chemistries that can only retain moderately polar to nonpolar compounds, hydrophilic polymer phases can retain a much broader range of polar to nonpolar acidic to basic analytes. Once such compounds are retained, they are easily eluted with an aqueous miscible solvent, such as methanol and/or acetonitrile. As a result, hydrophilic polymer phases are highly amenable to generic methodology. In addition, because hydrophilic polymer phases contain polar functionalities, they are very resistant to over-drying, often making associated extraction methods more reproducible/robust. In contrast, alkyl chain collapse can occur for silica-based, reversed phase chemistries if the stationary phase over-dries during conditioning.

In this report, we demonstrate the utility of a new hydrophilic polymer phase, called Supel™-Select HLB SPE (Select HLB), in which we use the phase to extract a range of pharmaceutical compounds and personal care products from both plasma and drinking water. The plasma application describes a generic method used to extract two statin drugs, potent cholesterol-lowering agents. The drinking water method is a modified version of EPA Method 1694: Pharmaceuticals and Personal Care Products in Water, Soil, Sediment, and Biosolids by HPLC/MS/MS. In recent years, there has been increasing evidence to suggest the detrimental effects of pharmaceutical and personal care compounds are finding their way into the public’s drinking water supplies. For example, such effects can possibly lead to the disruption of human hormone metabolic pathways, the emergence of antibiotic-resistant bacterial strains, and increased incidence rates of certain cancer types1. In 2002, the US Geological Survey tested water samples downstream of urban areas, and found pharmaceutical compounds in 80% of the streams sampled2.

What is Supel-Select HLB SPE?

Select HLB SPE is a hydrophilic modified, styrene-based polymer developed for the solid phase extraction of a highly broad range of compounds from aqueous samples. The retention mechanism is predominately based on reversed phase interaction. However, because the phase is hydrophilic modified, it is also selective for more polar compounds (HLB: hydrophilic lipophilic balance). Examples of more polar compounds retained and recovered on Select HLB include (but are not limited to): pyridoxine (logPo/w –0.56), riboflavin (logPo/w –2.02), and biotin (logPo/w 0.11). Tables 1 and 2 describe the specifications and features/benefits of the technology, respectively.

Table 1. Specifications of Select HLB SPE


Phase Chemistry: Hydrophilic modified styrene polymer
pH Compatibility: 0-14
Particle Size: 55-60 μm
MS Suitable: Yes
Surface Area: 400-410 m2/g
Pore Volume: 0.88 mL/g
Pore Size: 87 Å


Table 2. Features & Benefits of Select HLB SPE


Feature Benefit
Hydrophilic modified styrene resin Extract and recover a broad range of analytes (polar to nonpolar, acidic to basic) using a single sorbent
Generic methodology Save time, money, and headache during method development
Greater capacity Allows for smaller bed weights = smaller elution volumes = time savings in sample processing
Resistant to overdrying More robust methodology
Low UV and MS extractables Lower background for greater sensitivity
Stringent production & QC Greater lot-to-lot, tube-to-tube, and well-to-well consistency for improved accuracy and precision

Extraction of Statins from Plasma

In this study, pravastatin and atorvastatin were spiked in rat plasma at the levels of 100 and 5 ng/mL. The spiked rat plasma samples (n=3) were extracted using Select HLB SPE, 30 mg/1 mL (54181-U) and two leading competitor polymer SPE phases, using the method described in Table 3 and analyzed via LC-MS/MS. From the results described in Figure 1, Select HLB SPE offered good sample clean-up and high recoveries for both analytes at the spike concentrations tested, similar to the leading competitors.

Extraction of Pharmaceutical & Personal Care Products in Water

In this study, Select HLB SPE was tested against a leading competitor’s hydrophilic polymer SPE using EPA Method 1694 - Pharmaceuticals and Personal Care Products in Water, Soil, Sediment, and Biosolids by HPLC/MS/MS.3 A calibration solution containing 48 x Group 1 pharmaceutical compounds were prepared at the concentration levels (25 – 25,000 ng/mL) described in EPA Method 1694, Table 11a, column CS-5; and the test sample was prepared by spiking 1 L drinking water with 4 mL of CS-5 calibration solution. The 1 L spiked sample was extracted using the procedure described in Table 4, and analyzed using Ascentis Express C18, 10 cm x 2.1 mm (53823-U) in conjunction with LC-MS/MS conditions described in EPA Method 1694, Tables 2 & 3. Absolute recovery was compared for both Select HLB and Competitor W using two different elution solvents: methanol and methanol:acetonitrile (50:50), see Table 5. For the majority of the compounds tested, recovery values were similar between Select HLB and Competitor. Both products yielded an average absolute recovery of 61-65% recovery for the 48 analytes tested. Note that Select HLB SPE is slightly more hydrophobic than equivalent hydrophilic polymers that are commercially available. As a result, an elution solvent of methanol:acetonitrile (50:50) was tested. A CH3OH:CH3CN elution solvent improved recovery (by >10%) on Select HLB for at least 20 of the 48 compounds tested. However, a neat CH3CN elution provided greater recovery for 10 Group 1 compounds. For optimal recovery using Select HLB, we recommend to first elute with neat methanol followed by methanol:acetonitrile.

Table 3
. Generic Select HLB SPE Procedure for the Extraction of Statins from Plasma


Condition & Equilibrate with methanol (or elution solvent) followed by DI water or buffer
Load Sample  
Wash off or elute interferences buffer and/or 5-10% methanol or acetonitrile
Elute with methanol: acetonitrile (1:1, v/v)


Note: 0.5 mL volumes were used for each Select HLB SPE step.

column: Ascentis Express C18, 5.0 cm x 2.1 mm I.D., 2.7 µm (53822-U)
mobile phase: methanol:0.1% acetic acid (60:40)
flow rate: 0.2 mL/min.
temp.: 35 °C
detection: MS/MS, Applied Bio System 3200 Q TRAP
inj.: 5 µL


Total Ion Chromatogram of Rat Plasma

Figure 1. Total Ion Chromatogram of Rat Plasma Spiked with 5 ng/mL Statins and Extracted by Select HLB SPE

Table 4. EPA Method 1694 SPE Procedure for Group 1 Compounds


SPE: Supel-Select HLB SPE Tube, 200 mg/6 mL (54183-U) & Equivalent Competitor W SPE Product

  1. Condition and equilibrate each SPE cartridge with 20 mL methanol followed by 6 mL DI water and 6 mL DI water, pH 4 (adjusted with 6 M HCl).
  2. Load 1 L spiked water sample, pH 4 (adjusted with 6 M HCl).
  3. Dry cartridge under negative pressure (using vacuum manifold) for 5 minutes.
  4. Elute with 12 mL methanol OR 12 mL methanol:acetonitrile (50:50).
  5. Evaporate SPE eluent to 4 mL under nitrogen at 40 ºC and add 4 mL LC mobile phase.


Absolute Recovery of Group 1 EPA Method 1694 Pharmaceutical Compounds

Table 5. Absolute Recovery of Group 1 EPA Method 1694 Pharmaceutical Compounds


Select HLB SPE is a hydrophilic polymer SPE phase designed for the extraction of a highly broad range of compounds from complex aqueous sample matrices. In this report, we demonstrated the utility of this SPE phase for two applications: 1) statins in plasma and 2) 48 pharmaceutical compounds in drinking water. Select HLB SPE offers similar recovery and selectivity to the leading competitors and high recoveries using generic methodology.




  1. Mesner, M., Pharmaceuticals in Drinking Water: Pharmaceuticals and Personal Care Products Down the Drain, Utah State University
  2. USGS, 2007 Pharmaceuticals, Hormones, and Other Organic Wastewater Contaminants in US Streams. USGS Fact Sheet FS-027-02
  3. US EPA Method 1694: Pharmaceuticals and Personal Care Products in Water, Soil, Sediment, and Biosolids by HPLC/MS/ MS



Agilent – Agilent Technologies; Ascentis, Carbopack, Cleaving, CapSeal Bullet, CHIRALDEX, CHIROBIOTIC, CYCLOBOND, Discovery, Radiello, Separate Source, Sigma-Aldrich, Supel, Supelco, Supeltex, TraceCERT – Sigma-Aldrich Biotechnology LP; Fused-Core – Advanced Materials Technologies, Inc.; GOW-MAC – GOW-MAC Instruments Co.; PerkinElmer – PerkinElmer Inc.; Shortix – Scientifi c Glass Technology B.V.; Tenax – BUCHEM B.V.; Varian – Varian Associates Corp.; VESPEL – E.I. duPont de Nemours & Co., Inc.

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