Table II.

Common Analytical Causes of Mass Imbalance and Suggested Actions

Potential analytical cause of mass imbalance	Practical suggestions to guide mass imbalance investigations
Impurities are not eluted from the HPLC column	• Use gradient HPLC with wide polarity range and longer hold time with strongest mobile phase condition • Analyze sample using reverse phase or normal-phase thin-layer chromatography (TLC) • Analyze sample(s) using hydrophilic interaction liquid chromatography (HILIC) or normal-phase HPLC • Analyze sample(s) using capillary electrophoresis (CE)
Impurities are poorly separated and are “missed”(20)	• Use gradient HPLC with a steep gradient • Use isocratic HPLC with very strong mobile phase condition, with and without a column in place • Use UV spectrophotometry without any analytical separation
Impurities are co-eluting with the parent compound	• Change HPLC stationary phases, solvent, or gradient • Use an orthogonal (different) separation method • Look for peak purity using a PDA-UV detector with a UV homogeneity algorithm • Look for peak purity using LC/MS techniques
Impurities are not detected by the detector	• Use PDA-UV (200–400 nm) detection to increase “universality”; monitor at low wavelength (e.g., 205–210 nm) (20) • Use UV-transparent solvents and buffers • Consider alternative detector e.g., evaporative light scattering detector (ELSD), mass spectrometry (MS), chemiluminescence nitrogen detector (CLND), corona, charged aerosol detector (CAD), refractive index or flame ionization detector (FID) • Analyze sample using alternate/orthogonal detection method • Use reverse phase (RP) or normal-phase (NP) thin-layer chromatography (TLC)  –Use different options / chemistries for developing TLC spots or fluorescent-impregnated TLC plates • Use CE (often can look as low as 190 nm, more universal wavelength)
There is poor analytical recovery of the impurities	• Consider insolubility of impurities in analytical phases • Careful visual observation • Consider different solvents for sample preparation • Isolate solid material and analyze using other technique (e.g., probe-MS) • Consider possibility of reactions with insoluble excipients • Considered volatility (20) • Consider other analytical techniques (e.g., GC-headspace) • Consider adsorption losses • Compare results using different containers (e.g., glass and polypropylene) • Change sample/extraction solvent (e.g., different pH, different solvent) • Consider possibility of instability during the analytical preparation or workup
There is poor analytical recovery of the parent
There is inaccurate quantification due to differences in response factors.	• Examine UV spectra of detected impurities (PDA detector) • Consider alternative detector—evaporative light scattering, MS, Corona CAD, chemiluminescence nitrogen detector (CLND), FID or LC/NMR • Determine response factors (20,21) • Isolate, purify, and determine using conventional means • Use CLND(23) or CAD (without isolation of impurities) • Use quantitative nuclear magnetic resonance (qNMR) (20)

Mass balance can be measured and expressed in a variety of ways, but the concepts of Absolute and Relative Mass Balance has been advanced and discussed in detail by Nussbaum et al. (23). Absolute mass balance deficits (AMBD) can be expressed as the difference between the mass of parent drug consumed and mass of the parent contained in the degradation products recovered. Relative mass balance deficit (RMBD) can be expressed as the AMBD divided by the mass of the parent consumed and is expressed as a percentage