pISSN 2234-3806eISSN 2234-3814
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 https://doi.org/10.3343/alm.2022.42.2.121
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