Internal Standard Utilization Strategies for Quantitative Paper Spray Mass Spectrometry

Premixing internal standards (ISTD) with liquid samples prior to paper spray mass spectrometry (PS-MS) analysis consumes unnecessary amounts of ISTD and is not feasible for all sample types and applications. Depositing ISTD directly on the paper independently from sample has been successfully employed in the literature but can negatively affect quantitative performance. We evaluated different ISTD utilization strategies using drugs of misuse as test analytes to investigate the sources of irreproducibility and bias. Performance was assessed using both pre- and postdeposited ISTD (relative to sample loading) at different volumes with a constant final mass loading of 1 ng of each ISTD compound. Precision and accuracy were lower when using independently deposited ISTD compared to premixed ISTD (average CV = 18% vs 1% and average |bias| = 61% vs 5% for independently deposited ISTD and premixed ISTD, respectively). The use of a robotic liquid sample handling system to deposit ISTD was compared with results obtained via manually pipetting. Predeposited ISTD performed best at lower deposition volumes when a robotic liquid handler was used (average CV = 8% and 11% for 2 and 10 μL, respectively), but manual pipetting of low volume ISTD depositions performed poorly. Postdeposited ISTD was inferior to predeposited ISTD strategies, favoring larger deposition volumes regardless of deposition method (average CV = 22% and 16% for 2 and 10 μL, respectively). Systematic biases associated with each ISTD utilization strategy were effectively corrected for using strategy-matched calibrations, and AAFS (American Academy of Forensic Sciences) accuracy and precision requirements were achieved in almost all cases.