Supel-Select HLB SPE for Pharmaceutical Analysis

Introduction

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 fl uids, 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 non-polar and polar functional groups, they offer distinct advantages over traditional silica-based reversedphase SPE chemistries. Unlike silica-based reversed-phase SPE chemistries that can only retain moderately-polar to non-polar compounds, hydrophilic polymer phases can retain a much broader range of polar to non-polar 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 modifi ed 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 fi nding 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 types.¹ In 2002, the US Geological Survey tested water samples downstream of urban areas, and found pharmaceutical compounds in 80% of the streams sampled.²

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.

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), 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.

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

Conclusions

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.