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Table. 4.

Table. 4.

Method development checklist. Note that these components do not include optimization, pre-validation, or validation exercises

Sub-section Method development checklist Notes
Analyte Analyte sourced Reliable manufacturer?
Second source of analyte Two separate lots to be assessed for general agreement.
Quality of analyte confirmed Certificate of analysis review available?
Analyte in solution sub-aliquoted for ongoing Stability testing Stability must be started early.
IS IS sourced Reliable manufacturer?
IS confirmed for labeling Appropriate number of isotopes relative to the molecular weight of analyte(s)?
IS confirmed for lack of analyte Is the IS pure?
MS MS/MS provisional parameters established Checked for in-source dissociations or adducts? All MS/MS transitions retained?
MS/MS transitions reviewed for possible liabilities Aware of facile neutral losses that present specificity concerns?
LC Provisional LC mode established Reversed-phase or hydrophilic-interaction LC/ion exchange?
Solvents determined for LC development Best solvents for maximal MS response?
Columns sourced for LC development Various stationary phases should be assessed to provide data for “best column.”
Injection solution addressed Check for adsorptive loss/stability/peak shape/max injection volume.
Initial injections on LC Good signal? Value in revisiting MS conditions to increase signal?
Column screening Stationary phases exhibit acceptable retention and peak shape? Back-up columns recorded?
Location of known issues Location of problematic species identified in the chromatogram relative to measurand(s)?
Interferences Identification of possible isobaric species Thorough literature and database searches for possible confounders were done?
Location of isobaric species Commercially available species injected and relative retention time to analyte(s) confirmed?
Pre-sample preparation On-column detection limit determined Final extract requires dilution or concentration relative to measurement range?
Precision of IS/analyte demonstrated Appropriate IS concentration and most precise transition pairs empirically determined?
Sample preparation Provisional sample preparation procedure Acceptable analyte recovery in matrix?
IS equilibrium IS shown to be fully equilibrated in sample before extraction?
Recovery Does the extraction allow for the intended measurement range in the test matrix?
Robustness Does the extraction recovery equivalently for the IS across many discrete samples?
Precision Does the extraction work reproducibly over time?
Calibration Calibration selection Range and number of points determined?
Calibration matrix selection Reliable sources of matrix? Possibility of interferences/endogenous content in matrix?
Calibration matrix stability Analytes fortified to known concentrations and stability initiated?
Calibration matrix commutability Analyte in calibration matrix and human matrix measure equivalently? Equivalent matrix effects?
QC QC selection Number and concentration sufficient to observe analytical and clinical errors?
QC matrix stability Is long-term stability of measurands in QC matrix possible?
QC matrix commutability Do the QCs address errors that may be observed in a human matrix?
Matrix effects Ionization suppression Adequate IS recovery across many patient samples?
Analyte/IS equilibrium Has this been stressed across many samples?
Chromatographic matrix effects Has the column been stressed with abnormal samples?

Abbreviations: IS, internal standard; MS, mass spectrometry; QC, quality control; LC, liquid chromatography.

Ann Lab Med 2022;42:121~140

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