Formats for Rapid andor High Throughput Solid phase Extraction of Drugs in Biological Matrices

With the advent of fast analytical techniques such as LC/MS/MS, 96-well plate formats gained preference around 1995 to cater to the high-throughput sample preparation needs of bioanalysis. The historical development of these 96-well plate formats was well documented by Wells42 and will not be detailed here. In this well format, the sorbent is packed at the bottom of the plate with popular bed mass sizes ranging from 10 to 500 mg. Further refinements of this 96-well flow-through system include miniaturization of the plate and well geometry to accommodate as little as 2 mg of sorbent in a particle bed or disk (laminar, sintered, glass fiber, or particle-loaded membranes) that allows the use of very small elution volumes (e.g., 25 ^L). Other modifications consist of modular geometries

Desloratadine 3-OH desloratadine

FIGURE 1.7 Structures of desloratadine and 3-hydroxydesloratadine.111 (Reproduced with permission from Elsevier.)

FIGuRE 1.8 Comparison of SCX, MCX, and MP1 for retention of phosphatidylcholine.111 (Reproduced with permission from Elsevier.)

with removable well plates containing 10 to 100 mg of sorbent. Increased well plate formats (384 to 1536) are also available for very high volume turnarounds in sample processing.

Liquid handling systems such as the Tomtec Quadra Model 320 or Packard Multiprobe II EX (HT) are used to automate the solid phase extraction process. The former carries 96 pipette tips for simultaneous delivery of liquid into all 96 wells, while the latter is designed with 8 tips. Processing of a well plate using the Tomtec takes 10 min. Multiprobe processing requires 30 to 60 min to complete SPE on a 96-well plate. This compares favorably in terms of time and labor to manual SPE of a 96-well plate that requires more than 5 hr for completion of one extraction. Other popular liquid handling systems include the Sciclone Advanced Liquid Handler Workstation (Zymark), Cyberlab (Gilson, Inc.), Multimek (Beckman Coulter), and Personal Pipettor (Apricot Designs), all of which use 96-tip channel pipettors. Among the 4- to 8-channel pipettors, SPE 215 (Gilson), Genesis (Tecan), Biomek 2000 (Beckman Coulter), and Microlab (Hamilton) are widely used. Wells42 contains detailed accounts of these automated liquid stations; they are not discussed here due to spatial considerations.

A few examples from the latest literature will be presented to illustrate the use of the 96- and higher well formats and pipette tip formats for high-throughput sample preparation. An interesting example of orthogonal extraction chromatography and ultra-pressure liquid chromatography (UPLC) of plasma samples of desloratadine and 3-hydroxy-desloratadine (see Figure 1.7 for structures) was recently reported.112 Sample clean-up was achieved in a 96-well plate containing 10 mg of the MCX mixed mode polymeric sorbent. After conditioning with 400 mL of methanol and then 400 mL of 2% formic acid, a sample solution of the analytes (250 mL of plasma spiked with the metabolites diluted with 500 mL of 2% formic acid) was loaded. Washing was done with 400 mL of 2% formic acid and then 400 mL of methanol:acetonitrile (1:1 volume %). Elution with two 200 mL aliquots of methanol:acetonitrile:water:ammonia (45:45:10:4% v/v/v/v%) solution followed. After concentration under nitrogen, the eluate was reconstituted in the mobile phase (A = 10mM ammonium formate/0.2% formic acid; B = 10mM ammonium formate in methanol with 0.2% formic acid, A:B = 80:20). Experiments to evaluate extraction efficiencies showed that 4% ammonium hydroxide was optimal (5% ammonia reduced extraction yield by about 15%, while 2 or 3% ammonia showed about 6% lower recoveries). It was hypothesized that higher concentration of ammonia in the eluent co-eluted the phospholipids or the excess ammonium ions caused ion suppression.

The presence of 10% water in the eluent minimized variations. Two different LC modes were used for the analysis of the extracts: a smaller (50 x 2.1 mm) Atlantis C18 column (5 mm particle size) on a Shimadzu liquid chromatograph and a UPLC column (Acquity C18, 1.7 mm particle size, 50 x 2.1 mm) on a Waters Acquity system. In the Shimadzu experiment, a gradient from 0.5 min

■ XIC of +MRM (4 pairs): 311.2/259.1 amu from Sample 10 (MP1 Blank) of 05_04_04 Ion Suppression Tests.wiff (Turbo Spray) j 7e5 Max. 1.7e5 cds

■ XIC of +MRM (4 pairs): 311.2/259.1 amu from Sample 10 (MP1 Blank) of 05_04_04 Ion Suppression Tests.wiff (Turbo Spray) j 7e5 Max. 1.7e5 cds

XIC of+MRM (4 pairs): 327.2/275.1 amu from Sample 10 (MP1 Blank) of05_04_04 Ion Suppression Tests.wiff (Turbo Spray)

1.14e5 1.00e5


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