Sample and Loading
Q: How much sample can I process per well or 1mL tube?
A: You can process up to 200µL of urine hydrolysate – this sample is then diluted with 133µL of methanol, which will result in a total loading volume of 333µL in each case. (Note: The 133µL of methanol is equivalent to a 40% dilution.)
Q: Do I have to use the 40% methanol dilution?
A: Yes – However, it is possible to load 200µL of the urine hydrolysate (undiluted), followed by a “secondary elution” of 133µL of methanol to achieve the same results. If you want to use less than 200µL of urine lysate, employ the following formula to determine how much methanol you need for dilution: µL Methanol = (2/3)*(X µL Urine Hydrolysate) Loading less than 100µL of urine hydrolysate is not recommended.
Q: How much sample can I expect to get out of the filter?
A: β-Gone tubes and 96-well plates for recombinant enzymes contain 30mg of sorbent and have a corresponding dead volume of ~75µL while the β-Gone tubes and 96-well plates for non-recombinant enzymes contain 45mg of sorbent and their dead volume is approximately ~100µL. For information on how to appropriately calculate recovery, please see “How do I calculate recovery” below.
Extraction and Recovery
Q: How do I calculate recovery?
A: For assessing the performance of this product, it is recommended that you calculate Absolute Recovery. Absolute Recovery is defined as follows:
Or sometimes stated as:
There can be variable amounts of reference sample (blank matrix) lost to dead volume depending on vacuum setting and time spent with vacuum open after passing the sample through the plate/tube. In order to make sure that the Post Extract Spike is spiked with the appropriate level of analyte (i.e. to match the concentration of the prespike) we suggest implementing the following technique:
Remove exactly 150 µL of the reference pass through and spike that to the same concentration as the pre-spike.