Does anyone know what is the current requirements for mass balance calculations for a typical HPLC assay method validation ?

What do you mean with mass balance calculations??

do you mean recovery experiments?

I don't know of a specific regulation or guidance requiring a mass balance.

I work at a pharmaceutical company and the topic of mass balance most often comes up in the context of forced degradation studies for stability indicating assays.

If the parent compound is degraded by 10% you figure out the mass (moles) degraded and make sure the entire amount is accounted for by the increase in the degradant peaks.

The problem is this requires accurate response factors or impurity standards for all degradants. These are often not available in the eariler stages of method validation. For some compounds nitrogen specific detection can uncover some mass defect problems. Mass spec can sometimes be useful.

At a minimum I always perform column recovery, accuracy, and spiking and recovery into matrix to ensure there are no gross problems. Beyond that we do what we can.

It would be nice to hear about how others are doing column recovery, it does not appear to be a trivial problem.

I believe you would look at mass balance in the stress test study where you have no more than 10% degradation as stated by Tom. Usually the criteria is:
potency + related substances must be within 100.0% +/- 2.0%.
In the method validation, I would not look at mass balance only recovery as stated by Tom.

Hans,

For column recovery I make three injections of diluent and integrate everything including flowthrough. Then make three injections of sample again integrating everything. Corrected column area = avg sample area - avg diluent area.

Similarly calculate corrected area w/o column. I use a piece or restriction capillary in place of the column to give some backpressure and provide some bandspreading.

Column Recovery % = (corrected area w/o col.- corrected area with column)/corrected area w/o col. *100%.

If we are doing impurity linearity and accuracy and have collected fractions anyway then I also do column recovery for individual impurities.

I am very interested in learning how much others do or don't do in an attempt to detect a possible mass defect for degradant peaks during forced degradation studies.

Actually, my interest is connected via recovery in ANY chromatography. That is why I have been after calculating the area through absorption coefficients (doing some practicals on that). Flow injection, like you mentioned, Tom, has given immense problems with UV as scattering contributions (air, etc.) have proven non-reproducible. Radio detection is fine in this regard. If one has gamma-emmitters one can measure the gammas remaining on the column (or elsewhere) and get a quick semi-quantitative mass balance.
One big problem is tailing, substance sneaking through the column without being detected. Now one can argue that an internal st. will take care of this, but it should be known, unequivocally, how good all of this works. A mass balance would tell. Since I am talking of recovery, sample workup should be included as well.
The word "recovery" is often used when "relative recovery" is reported (one example discussed in J Chrom B, 678, 137 (1996): Overall recovery was actually ~20%, reported as ~100%, ref 15), more important would be "absolute recovery" which amounts to a mass balance.

Hans