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C18 SPME Fiber Probes for LC Applications

By: Robert Shirey, Craig Aurand, Reporter US Volume 28.3

Product Description

Traditional solid phase microextraction (SPME) fibers were developed with common GC type phases, which were primarily designed for extraction of volatile or semi-volatile compounds using thermal desorption as the means of introducing sample into the chromatographic column. In this article, the development of a new SPME fiber coating is being introduced specifically for extraction of polar and nonpolar analytes using solvent desorption for LC applications. The newly developed fiber probes contain C18 silica particles embedded in a proprietary, non-swelling, biocompatible polymer. The benefit of this design enables minimized binding of macromolecules such as proteins and phospholipids, but allows extraction of most smaller analytes of interest.

The fiber core is made from a flexible metal alloy with shape memory properties and a diameter larger than typical SPME fibers. The larger diameter enables the fiber to be used both as the plunger and fiber core, thus simplifying the device construction and reducing the cost of the probe. The fiber is also sealed into a hypodermic needle with an attached hub to allow movement of the fiber for exposure and retraction into the needle. Figure 1 shows the configuration of the fiber design. This design enables the fiber assembly to be pierced into a catheter shunt, vial septa or the fiber can be exposed for direct immersion into a liquid sample.


Figure 1. C18-SPME Fiber Probes

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Extraction of Drugs from Plasma

To demonstrate the properties of the LC fiber assembly, extraction studies were conducted using propranolol and the 4-hydroxy metabolite (4-HP) in both buffered water and plasma. Extraction fibers were then desorbed with solvent and analyzed by LC-MS-MS. The extraction and chromatographic conditions are detailed in Table 1.


Table 1. Conditions for Extraction and Analysis of Propranolol and Hydroxypropanolol (4-HP)(57281-U - product reference)

Because SPME is primarily concentration dependent and not volume dependent, only small sample volumes are necessary to perform the extractions. In this study, both 100 μL and 500 μL sample volumes were evaluated with only minimal difference in analyte response. This ability to perform extractions on such a small sample size greatly reduces the need for larger sample volumes to be collected for analysis.

Because the new SPME fiber coating is designed to be biocompatible, the fiber can be inserted directly into plasma or other biological fluids without the need for performing protein precipitation or other tedious sample prep steps. In this study, only the pH of the plasma was adjusted to increase the extraction efficiency. Figure 2 (pg. 4) depicts the response for propranolol and the 4-hydroxy metabolite obtained for the extraction from buffer and plasma.


Figure 2. Linearity of Extractions of Propranolol and 4-HP from 100 μL Samples

The results show that the analytes can be extracted out of a small volume of either buffer or plasma with good linearity at low concentration levels. The lower response for the 4-hydroxy metabolite is primarily associated with a lower ionization efficiency than the parent drug under the LC-MS conditions. Also, the recovery of both analytes is lower from plasma when compared to buffer. This is primarily due to drug-protein binding in the plasma. It was shown that it could take multiple hours for the binding equilibrium between the drugs and protein to be met. If the extraction occurs immediately after spiking the drugs into the plasma, the difference in recovery between buffer and plasma is much smaller. Better linearity is obtained if full protein binding equilibrium is obtained.

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Advantage of Biocompatibility

Phospholipids (PL) are large molecules that can interfere with the analysis of drugs by suppressing ionization in LC-MS electrospray ionization. The most common sample prep technique in bioanalysis is protein precipitation using an organic solvent to crash the proteins from the plasma sample. After filtration or centrifugation, the resulting supernatant is analyzed directly. Using this technique, PL are co-extracted with the analytes causing ion-suppression of the analytes resulting in irregular quantitation. To demonstrate the biocompatible properties of the C18 fibers, LC-MS-MS comparison of samples prepared using protein precipitation versus samples extracted using the C18 fibers were conducted. Comparisons were based upon total analyte response and phospholipid content. Figure 3 depicts the MS-MS chromatograms for phospholipids along with propranolol and 4-hydroxymetabolite.


Figure 3. LC-MS Analysis of Drugs in Plasma: Comparison of SPME Extraction to Direct Injection on the Matrix Background and Detection of the Drugs

The results show the SPME fiber coating minimized the extraction of phospholipids as compared to the protein precipitation technique. The response of propranolol and the 4-hydroxy metabolite from the 10-minute SPME extraction was comparable to the response from direct injection following protein precipitation. This demonstrates that the large molecules are not retained by the fiber and do not inhibit the extraction of the analytes of interest.

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In-Vivo SPME Using Fiber Probes

Biocompatibility of the SPME assembly not only refers to the fiber coating resisting macromolecules, but it also refers to the materials used in the coating and used to make the fiber probe. All of the materials in the fiber probe are inert and approved for medical use. Because of the biocompatibility of the probes, it is possible to use them for in-vivo animal studies.

NoAb Biodiscoveries in Mississauga, ON, Canada in conjunction with the University of Waterloo in Waterloo, ON, have developed a method utilizing SPME in pharmacokinetic (PK) studies. In typical PK studies, a drug is administered to multiple mice or other animals. At various time intervals, blood is removed from the animal and the amount of the parent drug in the blood is measured at specific time intervals. Usually there are 6-7 time intervals in a study, ranging from time 0 minutes to 18 hours. The problem encountered is that usually 1 mL of plasma must be obtained for the SPE extraction or precipitated with acetonitrile followed by direct injection. To obtain 1 mL of plasma requires that approximately 1.5 mL of blood be removed from the mouse. In many cases, this amount of blood loss is detrimental to a mouse and may result in death. So in a typical PK study, 18-21 mice are sacrificed since there are triplicate reps at each time point. This can be extremely costly, especially if transgenic mice are used in the study. Also, the metabolism varies between mice which affects the breakdown of the drug and results in variability in the data.

PK studies utilizing SPME consist of an arterial catheter connected to a specially designed shunt device inserted into the carotid artery or jugular vein of mice or rats. This design enables the blood to pass through a port in the shunt. The SPME fiber probe is inserted into the port, the fiber exposed and the blood is pumped over the fiber at a specific rate for a given time period between 1-2 min. After extraction, the fibers are then rinsed in water and stored in a freezer for analysis at a later time.

In a preliminary study conducted by the University of Waterloo and NoAb, a comparison was made between terminal blood draw followed by extraction using SPE, to in vivo extraction using SPME with the shunt device. A 2 mg/Kg dose of carbamazepine (CBZ) was given to the mice and the level of the drug in the blood stream was monitored for 18 hr. In this study triplicate reps were made at each time point. This results in 3 mice being used for the SPME study and 18 mice being sacrificed in the terminal blood draw study, significantly reducing the number of animals harmed. Figure 4 compares the results of the average response for CBZ at each time point between in vivo SPME and the terminal blood draw/SPE method.


Figure 4. Comparison of SPME in vivo Pharmokinetics (PK) Study of Carbamazepine (CBZ) from Mice Whole Blood to Extracts of Plasma Removed from Mice

The results show that there is good correlation for the response of CBZ in the whole blood between in vivo SPME testing and blood drawn followed by SPE testing. In addition to the benefits of in vivo SPME, some others were observed. One benefit is that SPME only measures the free form of the drug, the active form. Also, it was observed that when using in vivo SPME the metabolite of CBZ was detected. The metabolite was not observed when using SPE.

Validation studies are ongoing at NoAb Biodiscoveries to determine if in vivo SPME can be used as an alternative to terminal blood draw. Once these studies are completed in vivo SPME could be used for drug discovery studies.

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In this report the utility and application of newly developed C18 Silica Fiber Probes has been demonstrated. The goal was to offer an SPME fiber device specifically designed for solvent desorption instead of thermal desorption. This enables new possibilities for microextraction, where traditional SPME is not capable. These options could include monitoring of pesticides in plants, the uptake of contaminants in animal tissues and organs, and other applications that simply require extraction out of a vial followed by solvent desorption and LC analysis.

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  1. Ines DeLannoy and Brad Gien - NoAb BioDiscoveries, Inc, Mississauga, ON L5N 8G4 Canada
  2. Dajana Vuckovic and Janusz Pawliszyn –Department of Chemistry, University of Waterloo, Waterloo, ON N2L3G1 Canada

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  1. Vuckovic, Dajana; Shirey, Robert; Chen, Yong; Sidisky, Len; Aurand, Craig; Stenerson, Katherine; Pawliszyn, Janusz. In vitro evaluation of new biocompatible coatings for solid-phase microextraction: Implications for drug analysis and in vivo sampling applications. Analytica Chimica Acta (2009), 638(2), 175-185.

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