ABC-BIO cheat sheet

Ligand-binding assays (ELISA) quantify via antibody-antigen binding; calibration curves are typically 4-parameter logistic (4PL) fits of signal vs. log concentration.

LC-MS/MS quantification requires an internal standard: stable-isotope-labeled analog corrects for extraction and ionization variability; matrix-matched calibration accounts for ion suppression.

Lower limit of quantification (LLOQ) must show signal-to-noise >= 5 and accuracy within +/-20% of nominal, with precision CV <= 20%.

Calibration standards: >= 6 non-zero levels; accuracy within +/-15% (+/-20% at LLOQ); >= 75% of standards and >= 67% of each level must pass.

Selectivity is demonstrated by analyzing blank matrix from >= 6 individual lots; interference at analyte retention time must be < 20% of LLOQ signal.

Carryover is assessed by injecting blank after high-concentration sample; residual signal must be <= 20% of LLOQ.

Dilution integrity: dilution QC at 2-5x ULOQ must yield accuracy within +/-15% after dilution.

Extraction recovery = (response of extracted sample)/(response of post-extraction spiked blank) at matched concentration; stable-isotope IS normalizes recovery differences.

Protein precipitation, liquid-liquid extraction, and solid-phase extraction differ in cleanliness vs. throughput; SPE offers selective retention for polar analytes.

Beer-Lambert law: A = epsilonbc; absorbance is linear in concentration only at low c and fixed wavelength; deviations occur at high absorbance (>1) or polychromatic light.

Chromatographic resolution: Rs = 2(t2 - t1)/(w1 + w2); baseline resolution typically requires Rs >= 1.5.

Van Deemter equation: HETP = A + B/u + C*u; minimum plate height occurs at an optimum linear velocity u.

Retention factor k = (tR - t0)/t0; selectivity factor alpha = k2/k1; resolution depends on N, alpha, and k.

pH and pKa: Henderson-Hasselbalch pH = pKa + log([A-]/[HA]); buffer capacity is maximal at pH = pKa.

Acid-base titration equivalence point determined by stoichiometry; indicator chosen so its transition range brackets the equivalence pH.

Gravimetric analysis: mass of precipitate relates to analyte via stoichiometric gravimetric factor (molar mass analyte / molar mass precipitate).

Signal-to-noise improvement scales with sqrt(n) scans for random noise; signal averaging does not remove systematic drift.

Standard addition compensates for matrix effects: spiked response extrapolated to zero signal gives original concentration.