Online SPE

Development and validation of protocols for solid-phase extraction coupled to LC and LC-MS have been described [136]. Hennion [137] has reviewed online SPE-HPLC coupling followed by various detection modes. 

Pesticides Triazines, phenylureas, OPs, chloroacetanilides, thiocarbamates River water Online SPE... 

Features that make online SPE more attractive compared to off-line SPE consist of ... 

Case II Analysis of Fenofibric Acid Using Dual Online SPE for Clinical... 

Direct injection of pretreated biological samples (also called online sample cleanup) greatly simplified sample preparation for LC/MS/MS analysis. The normal process involves sample aliquot steps, internal standard addition, and centrifugation. Compared to traditional off-line LLE and SPE sample preparation procedures, online methods are easier and faster. Two types of online SPE columns are commercially available. One is the restricted access media (RAM) column. The other is the turbulent flow chromatography (TFC) column. 

Instrumentation — The parallel LC/MS/MS system for this application was operated under the dual online SPE mode. Figure 2.7 is a flow diagram for the dual stream and Figure 2.8 depicts the staggered timing scheme. Figure 2.9 shows the autosampler setup in action. 

FIGURE 2.7 Flow diagram for dual-stream online SPE. 

Online SPE — One HPLC system was used to perform both SPE and HPLC separation. A column switching valve was used to direct the flow from the extraction column to waste (off-line) or the analytical column (online). A second column switching valve directed flow from the analytical column to the mass spectrometer or to waste. 

FIGURE 2.9 Autosampler set-up for performing dual-stream online SPE (A) overall view, (B) zoomed view of the valving configuration. 

Commercial Online SPE/LC Systems with Disposable Cartridges.285... 

COLUMN SWITCHING ONLINE SPE LC/MS/MS SYSTEMS 10.2.1 Basic Concepts... 

The feasibility of online SPE LC/MS/MS has been tested since the introduction of thermospray ionization. In an early research paper by Lant and Oxford (1987), a prototype online SPE LC/MS system was set up and successfully applied for the measurement of labetalol, a hypertension drug and a- and /J-adrenergic receptor, in plasma. This system was set up by coupling an advanced automated sample processor (AASP, Varian, Walton-on-Thames, UK) with a reversed-phase column, a ten-port switching valve, and an MS equipped with thermospray interface (Vestec, Houston, Texas) (Blakley et al. 1980, Blakley and Vestal 1983). 

After overcoming the instability of the thermospray ionization source, a sensitivity of 2 ng/mL was achieved with a calibration range of 10 to 103 ng/mL for human plasma samples. In recent years, the online SPE LC/MS/MS technology has matured and is now easy to build and use. It is used widely in the pharmaceutical industry (Jemal et al. 2000 Hsieh 2004 Hennion 1999). 

Figure 10.1 shows a basic online SPE LC/MS/MS system, a column switching system (Kahlich et al. 2006) that includes a six-port switching valve, an injection valve, an SPE cartridge, an analytical column, SPE washing and HPLC pumps, and MS detector. The online process has three steps ... 

Online SPE after conditioning of the SPE cartridge, the sample solution is flushed into the cartridge by the washing solvent after a positional switch of the injection valve. A large volume of washing solvent is used to clean up the sample. 

FIGURE 10.1 Online SPE-LC/MS/MS (A) sample injection (B) online SPE (C) LC gradient elution and MS detection. (Source Adapted from Kahlich. R. et al., Rapid Commun Mass Spectrom 2006, 20, 275. With permission.)... 

RAMs packed in small cartridges are often used for online SPE of samples in biological matrixes. First developed by Boos et al. (1991), these alkyl-diol-silica (ADS) materials consist of large size particles (20 to 50 /.an compared to 3.5 to 5 /an for analytical columns) with hydrophilic electron-neutral surfaces that do not retain proteins and hydrophobic internal pore surfaces that allow only small molecules to enter and bind. 

In addition to online filters and precolumns, a simple protein precipitation step often precedes online SPE LC/MS/MS to prolong cartridge life. Protein precipitation can also reduce analytical interference and shorten chromatographic separation time. Since an internal standard (IS) solution is often added to plasma samples and centrifugation is used to remove possible particles before loading into the autosampler, protein precipitation does not add labor to the process. 

Sometimes orthogonal offline SPE steps were used prior to online SPE LC/MS/MS. These preparation steps were used to remove interference and concentrate samples. In an application to measure urinary N7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a biomarker for exposure to polyaromatic hydrocarbons (PAHs), a two-step offline SPE was first performed using Sep-Pak C8 (Waters, Milford, Massachusetts) and Strata SCX (Phenomenex, Torrance, California) cartridges to obtain high sensitivity (Chen et al. 2005). The extracts were applied to an online reversed phase SPE LC/MS system. The lower limit of detection was 2.5 fmol/mL when 10 mL of urine was used. 

Gundersen and Blomhoff (1999) used online dilution with online SPE to measure vitamin A (retinol) and other active retinoids in animal plasma. The intention of online dilution in this application was on optimizing SPE extraction conditions rather than on peak focusing during analytical separation. An SPE cartridge packed with Bondapak C18 materials (37 to 53 jt/M, 300 A, Waters, Milford, Massachusetts) and a reversed-phase analytical column (250 x 2.1 mm inner diameter, Superlex pkb-100, Supelco, Bellefonte, Pennsylvania) were controlled by a six-port switching valve (Rheodyne, Cotati,... 

The application from van der Hoeven et al. (1997) used an ADS cartridge online SPE to measure cortisol and prednisolone in plasma and arachidonic acid in urine. A precolumn packed with a C18 alkyl-diol support (LiChrosphere RP-18 ADS, 25 /an, Merck) was used. To reduce run time, column switching was programmed as heart-cut , diverting only the analyte fraction into the analytical column. Another LiChrosphere column (125 x 4 mm inner diameter, Merck) handled separation. After the injection of 100 fiL plasma, the lower limit of detection for prednisolone was 1 ng/mL while cortisol was readily quantitated at its endogenous level of 100 ng/mL. The run time was 5 min. For arachidonic acid, a Hypersil ODS column (200 x 3.0 mm inner diameter, 5 /.an) was used. The injection volume was 200 //I. and run time was 9.5 min. The detection limit was 1 ng/mL and recovery was 77%. 

An interesting idea was to use a monolith column to perform dual functions of online SPE and chromatographic separation. Because of the porous structure of a monolith column and its very low backpressure, plasma or diluted plasma can be directly injected. Plumb et al. (2001) used this approach to quantitate an isoquinoline drug and 3 -azido-3 -deoxy thymidine (AZT). Diluted plasma samples (plasma water 1 1) were injected directly into a Chromolith Speed ROD RP-18e column... 

COMMERCIAL ONLINE SPE/LC SYSTEMS WITH DISPOSABLE CARTRIDGES... 

Commercially available fully automated online SPE systems include Prospekt, Prospekt-2, and Symbiosis systems (Spark Holland, the Netherlands) and Merck s OSP-2 (Darmstadt). Figure 10.2 shows a Symbiosis (Kuklenyik et al. 2005) two-cartridge, single analytical column online SPE... 

---