Laboratory Products
Evaluation of basic analytes using a novel hybrid reversed
phase method for solid phase extraction by HPLC-MS James Edwards, Application Scientist - Chromatographer. George Whitfi eld, Application Scientist - Chromatographer. Email:
technical@porvairsciences.com
The Microlute™ CP range from Porvair Sciences, offers a new method of solid phase extraction (SPE) using a unique hybrid polymer structure combined with retentive media. This composite design enhances the fl ow-through of samples throughout its porous structure to maximise interactions between analytes and the solid phase to deliver a highly reproducible SPE method. This application note demonstrates the robust SPE LC-MS methodology of the Microlute™ CP Reversed Phase (RP) SPE 30 mg plates on basic analytes by comparing recoveries and reproducibility benefi ts over loose-fi lled products.
Introduction
Solid phase extraction (SPE) is a sample preparation method for the clean-up of samples before analysis with HPLC or GC analysis. SPE offers a number of advantages to the analyst, including less system downtime and troubleshooting, cleaner chromatograms with a reduction of contaminating compounds, and more reproducible analyte recoveries. Traditionally, SPE methods use loose-fi lled resins which can create problems, such as voids in the sorbent beds leading to channelling, inconsistent fl ow-through of solutions, instability at extreme pHs and residual silanol activity. This materialises in less interactions between analytes with the active resin leading to inconsistent results and poor analyte recovery.
Over more recent years, significant advantages have been seen from using polymer-based materials. The processes used to synthesise polymer-based sorbents enable incorporation of numerous chemical functionalities into the porous framework (Figure 1). The ability to generate highly specific and regular functionality gives high retention capacity for different types
of compounds including basic compounds and stability at extreme pHs.[1]
With only 20% of pharmaceutical analytes exhibiting acidic properties, basic analytes accounts for 70% of pharmaceutical compounds making them the most abundant analyte type being analysed via HPLC/GC. Unlike neutral and acidic compounds, basic compounds require less method development to ensure they are sufficiently retained on the sorbent medium.[2]
Retention of basic analytes from polar solutions onto reversed phase SPE materials occurs due to Van der Waal forces or dispersion forces between carbon-hydrogen bonds of the analyte and the functional groups bonded to the sorbent material. Stronger Van der Waal forces lead to greater retention on the reversed phase. Finally, silica-based resins can be sensitive to stationary phase collapse if the sorbent bed becomes dry after the condition step whereas, polymer-based sorbents are less susceptible to drying out. This enables quicker SPE method development and gives the analyst ease of mind when performing SPE. In addition, minor packing differences of sorbents into cartridges and wells can cause significant differences in the flow of solutions when performing SPE steps.
Figure 1. Microlute™ Hybrid Technology. A network of porous channels containing immobilised resins for solid phase extraction.
The outlined advantages of polymer-based sorbents greatly suggest a superior method of solid phase extraction not only in performance but increased reproducibility of recovery and retention of analytes of interest. To demonstrate the sensitivity and robustness of the 30 mg Microlute™ CP RP, an SPE experiment was performed using spiked aqueous sample matrices containing basic analytes and was compared against five commercially available 30 mg loose-filled reversed phase products.
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