23
Recovery and Reproducibility
This application used a reconstitution volume of 100 µL which lead to a 10 times concentration of the original 1 mL of urine loaded onto the plate. This allowed for greater sensitivity of every compound analysed. The process of the SPE helped to create a cleaner solution by binding the analytes to the resin while washing away interference compounds present in the urine matrix - a high aqueous wash to clean off any polar compounds and a methanol wash to elute the hydrophobic acidic and neutral interferences. These two washes help create a very clean solution when samples are eluted from the SPE product as minimal interferences should still be present when the elution step is performed.
Recoveries for each of the 12 compounds spiked into the human urine were measured after sample preparation was performed using the Microlute™ CP SCX 96 well plate. Figure 3. shows the average recovery, from urine samples spiked pre-sample preparation, of all the compounds from a sample size of 12 replicates. All recoveries for the opioids were greater than 80% except for meperidine and EDDP.
Recoveries were compared to a commercially available 30 mg loose filled product using the same method as run on the Microlute™ CP SCX plate for a comparison. The comparison of the two products are found in Figure 4. The recovery for the loose filled product showed a decrease in recovery across each analyte when compared to the hybrid polymer. This reduction is proposed to be due to the structure of the hybrid polymer which provides more efficient interactions between the analyte and chromatographic media. As a result, there is an overall reduction of channelling effects of analyte and solvent throughout the Microlute™ CP SCX plate.
Channelling is the process of liquids taking the path of least resistance through the SPE bed. Therefore, when urine is loaded onto the plate it may not fully interact with all of the resin present due to the uneven flow path through the SPE bed. This effectively reduces the loading capacity of the product and can lead to breakthrough of analyte leading to low recoveries. The second issue is that on elution, solvent has less contact with the bound analytes which can lead to lower recoveries and a need for larger elution volumes in comparison to the hybrid polymer’s elution volumes.
Recovery of Opioids from Urine
Figure 3. Mean recovery of opioid compounds from human urine using Microlute™ CP SCX plates. Error bars represent the standard deviation of the recoveries (n=12).
Recovery of Opioids - MicroluteTM CP vs Loose Filled Product
Figure 4. Comparison of recovery of Microlute™ CP SCX 30 mg to a loose filled 30 mg SCX product. Error bars are standard deviation of the recovery results (n=12).
% RSD Comparison of Opioids
Figure 5. % RSD of all 12 opioids and metabolites calculated from recovery of analytes (n=12).
Well-to-well reproducibility is a measure of how close each recovery result is to each other between different wells of the 96 well plate. It is an important measure to allow confidence in results collected. When a
low reproducibility is recorded it can bring doubt into the results collected. For method validation, it is an important metric to look at with suggested guidelines in place for what level is acceptable. Chromatographic
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