[Fatch]

Hello.

I am working on an LCMS acetylfentanyl extraction method, and my lead investigator wants to scale the entire process down. The method calls for 250µL of donor blood, and I've been asked to scale the process back to 100µL of blood per sample. I worked for a company in the private sector for several years where scaling down was never necessary. I have never had to scale a process down and want to make sure I use the correct volumes for the extraction process and calibration curves. Additionally rather than using 1μg/mL acrylfentanyl, I will be using 50μg/mL acetylfentanyl. I'm also not sure how the concentration substitution will affect the creation of my working standard.
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"Working Standards and Controls

Working standards for acrylfentanyl were prepared at 1μg/mL by transferring 20μL of a 1mg/mL standard into 19.98mL of methanol. The working internal standard solution of fentanyl-d5 (2.5 ng/mL) was prepared in acetonitrile by adding 25μL of a 100μg/mL fentanyl-d5 standard into 1L of acetonitrile. Mobile phase A (0.1% formic acid in DI water was prepared by adding 2mL of concentrated formic acid to 2L of DI water. Mobile phase B (0.1% formic acid in acetonitrile) was prepared by 2mL of concentrated formic acid to 2L of acetonitrile.

A calibration curve was prepared in whole blood by spiking 50 μL of a 1μg/mL acrylfentanyl standard into 4,950μL negative whole blood and diluting to achieve the correct curve concentrations. Calibration curve points were 10.0, 5.0, 2.0, 1.0, 0.50, 0.20 and 0.10ng/mL. Three QC specimens (two positive QCs and one negative QC were run with each batch of specimens. The high positive QC specimen (4.0ng/mL) was prepared by adding 200μL of a 1μg/mL acrylfentanyl working standard to 49.8mL of negative blood. The low positive QC (0.40ng/mL) was prepared by diluting the high QDC specimen 1:10 with negative blood.

Extraction

A 250μL aliquot of blood specimen and a 1mL aliquot of working internal standard solution (stored at −10°C were added to a small culture tube, vortex mixed for 1 min and centrifuged at 3,500RPM for 3 min. An 850μL aliquot of the organic supernatant was transferred to a new tube and evaporated to dryness under nitrogen gas flow. The residue was reconstituted in100μL of DI water, vortex mixed for 5s, and transferred to a plastic autosampler vial."
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Thank you in advance for your time and assistance.