Investigation of Range Independent Deviations at High End of the Curve Koen van der Drift (Presenter) Darthmouth-Hitchcock Medical Center

Authors: Koen van der Drift, Jacqueline Hubbard
Dartmouth-Hitchcock Medical Center

Problem:

During development of a method to quantify opioids via LC-MSMS in urine, we noticed that the highest two calibrators in our standard curve exhibited a downward skew, regardless of concentration. The deviation appears in components with both low and high concentration ranges, and not all components follow the same trend.

Method Information:

• LC-MSMS of 19 opioids plus matched deuterated internal standards

• Standard curve was prepped in drug free urine

o Lowest range: 0.2 – 200 ng/mL

o Highest range: 10 – 10000 ng/mL

• Internal standard mixture was prepared in MeOH

• Sample prep:

o Samples were processed using either:

 Dilute and Shoot (D&S; 1:10 in water)

 Solid Phase Extraction (Waters Oasis MCX PRiME SPE)

 Dual Mode Extraction (Biotage DME+)

o C18 UPLC BEH 2.1x100 mm, 1.7 m

o 8 min gradient @ 30 °C, 0.4 mL/min, 10 L inj

o Mobile phase A: water/0.1% FA; mobile phase B: ACN/0.1% FA

o Waters Acquity TQD in MRM mode, 2 transitions per component, 1 for IS

Troubleshooting Steps:

• Observation was independent of standard curve preparation

o Serial or parallel dilutions

o Logarithmic or evenly spaced calibrators

o Some components are not affected -> suggests curve is prepared properly

• Appears to be more prominent on plate-based sample preps, suggesting sample loss at higher concentrations, maybe due to adsorption to sample handling surfaces

• Less with D&S which is prepped in glass autosampler vials

• Area counts of each compound and the internal standard were monitored

Outcome:

Response at higher concentrations for some components is suppressed in MS. Limit range to 2 logs:

• Lowest range: 1-100 ng/mL

• Highest range: 20-2000 ng/mL

• Glass vials vs plate was red herring; D&S is less concentrated, so less suppression.

Problem

Method Information

Troubleshooting Steps

Outcome