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12 BioPharm International eBook July 2021 www.biopharminternational.com an example of the study design. Japanese health authority guidance also recom- mends the column be included in the study design when appropriate. STUDY DESIGN 2: LOD AND LOQ Study design 2 (Table III) consists of two concentrations of a reference standard by six independent determinations for each concentration at or near the estimated limit of detection (LOD) and limit of quantification (LOQ ) limit. STUDY DESIGN 3: SPECIFICITY IDENTIFICATION 2X3 Study design 3 (Table IV) requires two conditions—a positive control and a negative control—by three indepen- dent determinations. STUDY DESIGN 4: SPECIFICITY INTERFERENCE 3X3 Study design 4 is used to evaluate risk assessment with all interfering com- pounds, impurities, excipients, etc. This study design consist of three concentra- tions of the reference standard for every interfering compound spike in represen- tative concentrations of the interfering compound ( Table V). Results are com- pared to the linear curve (unspiked) in study design one. USP <1033> states: "For products or intermediates associated with complex matrices, specificity involves demon- strating lack of interference from matrix components or product-related compo- nents that can be expected to be present. This can be accessed via parallel dilu- tion of the Standard sample with and without a spike addition of the poten- tially interfering compound" (3). STUDY DESIGN 5: STABILITY INDICATING 1X5 St udy design 5 (Table VI) consists of one standard concentration under stressed conditions (temperature or pH) at five time points. STUDY DESIGN 6: ROBUSTNESS Study design 6 deals with robustness, which is a key element of method qualif ication/validation and is nor- mally performed before the qualifica- tion/validation (Table VII). Robustness is included in the validation report. Normally a single analyst, a single instrument, and a single representative Biopharmaceutical Analysis Method Validation Concentration Day Analyst Instrument 40 L1 L1 L2 50 L1 L2 L1 30 L1 L1 L2 60 L1 L2 L2 70 L1 L2 L2 50 L1 L1 L2 40 L1 L2 L1 40 L1 L1 L1 30 L1 L1 L1 30 L1 L2 L1 70 L1 L1 L1 70 L1 L1 L2 60 L1 L1 L1 50 L1 L2 L2 60 L1 L2 L2 70 L2 L1 L1 50 L2 L1 L2 50 L2 L2 L1 60 L2 L1 L1 50 L2 L1 L1 40 L2 L2 L2 30 L2 L1 L2 30 L2 L2 L2 60 L2 L2 L1 30 L2 L2 L1 40 L2 L1 L2 40 L2 L2 L1 70 L2 L2 L2 60 L2 L1 L2 70 L2 L2 L1 Table II. Linearity study design. Reportable result Method of analysis Acceptance criterion Limit of detection SD at concentration near LOD * 3.3 (divided by the slope of the signal and theoretical concentration if the SD is not in units) Repor t only Limit of quantification SD at concentration near LOQ * 10 (divided by the slope of the signal and theoretical concentration if the SD is not in units) Repor t only Table III. Study design 2: Limit of detection (LOD) and limit of quantification (LOQ). SD is standard deviation.