HT-Sample Preparation Techniques for Bioanalysis

  • Tab. 1: Application of monolithic methacrylate polymer packed 96-tips in bioanalysis [7]Tab. 1: Application of monolithic methacrylate polymer packed 96-tips in bioanalysis [7]
  • Tab. 1: Application of monolithic methacrylate polymer packed 96-tips in bioanalysis [7]
  • Tab. 2: Summary for MEPS applications of drugs and metabolites from biological samples [6]
  • Fig. 1: A scheme of MEPS (the process is fully automated)
  • Fig. 2: Monolithic packed 96-tips

The purpose of sample preparation is the removal of interfering substances and also enrichment of the analytes. The procedure must be highly reproducible with a high recovery of the target analytes. Further, an ideal sample preparation method should involve a minimum number of working steps and it should be fully automated. Because of the low concentration levels of drug in plasma and the variety of the metabolites, the selected extraction technique should be virtually exhaustive.

Nowadays, commonly used sample preparation methods are solid-phase extraction (SPE) and liquid-liquid extraction (LLE). While solid-phase extraction (SPE) gives both high recovery and good chromatography, it takes more time and more steps to perform. The process can be semi-automated and sometimes it is necessary to use strong acids or bases for elution of high polar analytes to get high recovery. For several analytical methods this is not suitable as some detector systems (such as in mass spectrometry) are not capable of high amounts of additives. However, SPE and LLE are the most important sample preparation methods in LC-MS.
The growing number of samples to be analyzed requires high-throughput and fully automated analytical techniques. Recent developments of sample handling techniques are directed, from one side, toward automatization and on-line coupling.

Microextraction by Packed Sorbent (MEPS)
MEPS is a technique for miniaturized solid-phase extraction that can be connected on-line to GC or LC without any modifications. Approximately 2 mg of the solid packing material is used with a 100 or 250 µl syringe (fig. 1A). The plasma sample (100 µl) is drawn through the syringe by an auto­sampler (pumping the sample up and down). When the plasma has passed through the solid support the analytes have been adsorbed on the solid phase. The solid phase is washed once with water (50 µl) to remove the proteins and other interferences. The analytes are then eluted with an organic solvent such as methanol or the LC mobile phase (20-50 µl) directly into the instrument‘s injector (fig. 1B). This entire process can be fully automated.

MEPS differs from commercial solid-phase extraction as the packing is integrated within the syringe and not in a separate column.

Thus, there is no need for a separate robot to apply the sample into the solid phase as with conventional SPE. Also, the packed syringe can be used several times: more than 100 times using plasma or urine samples and more than 400 times for aqueous samples. A conventional SPE column is used only once. MEPS can handle small sample volumes (10 µl of plasma, urine, and water) as well as large volumes (1000 µl) and can be used for GC, LC and CEC applications.

MEPS Applications in Bioanalysis
The sample processing, extraction and injection steps are performed on-line using the same syringe. The performance of MEPS was recently illustrated by online LC-MS and GC-MS assays of drugs and metabolites in urine, plasma, blood and hair samples [1-6]. The combination of MEPS and liquid chromatography mass spectrometry (LC-MS) is a valuable tool for screening and determination of drugs and metabolites in blood, plasma and urine samples. This approach for sample preparation is very promising for many reasons: 1) it is easy to use, 2) it is a fully automated online procedure, 3) it is rapid, 4) it reduces the solvent and sample volumes, and 5) the cost of analysis is minimal compared to conventional SPE.

The MEPS technique has been used to extract a wide range of drugs and metabolites (table 2) in different matrices (urine, plasma, blood). Hence, several drugs such as local anesthetics and their metabolites, anti-cancer drugs roscovitine, olomoucine, busulfan, cyclophosphamide, and AZD3409, β-blockers acebutolol and metoprolol, neurotransmitters dopamine, serotonine, methadone, cocaine and cocaine metabolites have been extracted from biological samples such as blood, plasma or urine samples using MEPS technique [4, 5]. In addition MEPS online with GC-MS was used for the determination of amphetamine in human hair and monoterpene metabolites in human urine [6].

Monolithic Packed 96-Tips
The polymerization mixture of methacrylate monoliths consists of a solution containing glycidyl methacrylate (20 %), ethylene glycol dimethacrylate (15.5 %), butyl methacrylate (3.5 %), AIBN (1 wt.% with respect to monomers), 1-dodecanol (30 %) and cyclohexanol (30 %) was vortexed for 10 min and purged with nitrogen for 10 min in order to remove oxygen. The pipette tips were filled with about 8 mm (6-7 μl) by the capillary action and placed vertically inside the polymerization apparatus. The polymerization using UV light at 254 nm was allowed to proceed first for 60 min with the sharp end of the tip down and at a distance to the lamp of 15 cm, and then for 25 min with the sharp end up and at a distance of 5 cm to the lamp. After completion of polymerization the tips were washed with acetone to remove the porogenic solvents and other compounds remaining in the monolith (fig. 2).

Monolithic packed 96-Tips application in bioanalysis
Evaluation of monolithic packed 96-tips for the extraction of drugs [7] such as anticancer drugs (busulfan, cyclophsphamide, rescovitine), β-blocker drugs (metoprolol, pindolol) and local anesthetics (lidocaine, ropivacaine, bupivacaine) from human plasma or blood samples has been developed and validated (table 1). Utilizing plasma samples the tips could be used several times (5 times) and still get reasonable results. Utilizing blood samples, packed tips could only be used once. The results showed that the method is selective and accurate. It was shown that small sample volumes can be handled, solvent consumption was low and the procedure was very fast (2 min per 96-well plate).

References are available from the author.





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