Direct Injection GC–MS Analysis Extractables
There was only one semivolatile extractable peak observed from the water extracts. The peak was identified as BHT and is very
small because of its limited solubility in water.
There were many extractables peaks observed in the isopropanol extracts (Figure 5). The two dominant peaks are BHT (12.18
min) and an unknown hydrocarbon at 13.66 min. Other compounds observed were palmitic acid (15.35 min) and stearic acid (16.62
min). All other peaks appear to be various hydrocarbons.
Leachables were determined by comparing the stressed formulations to the formulation blanks and the stopper extractables profile.
There is no appreciable interference from the formulation blanks for the main extractables peaks. The only leachables observed
was BHT, which was reported in formulation 3 (pH 5.0), formulation 6 (0.1 mM EDTA), and formulation 7 (0.5 mM EDTA). This
result was consistent with headspace GC–MS data. The BHT peak is very small. No other peaks observed in the isopropanol extract
were observed in any of the formulations. This can be explained by the insolubility of these compounds in the aqueous formulations.
There were two additional peaks observed in formulation 1 (pH 6.8 phosphate buffer) and formulation 2 (phosphate buffer with
2% glycerol). The first elutes at 11.27 min and the second elutes at 12.11 min. The peak at 12.11 min is tentatively identified
as 2,4-t-butylphenol, while the peak at 11.27 min was not identified. For all other formulations, the peak at 11.27 min was
not observed. For the peak at 12.11 min, there is a co-eluting interference peak from all the formulation blanks except formulations
1 and 2, so it is not conclusive if the peak observed in formulations 1 and 2 was also present in those formulations. The
source of the two peaks requires further evaluation.
No extractables were observed with LC–MS from the water extracts of rubber stoppers. The isopropanol extract of the stoppers
contained BHT (5.02 min) and three fatty acids (myristic acid, 5.21 min; palmitic acid, 5.80 min; and stearic acid, 6.30 min).
The leachables were evaluated by comparing the stability samples to the formulation blanks and the stopper extractables profile.
There is no appreciable interference from the formulation blanks for the main extractables peaks. There are no leachables
observed in the LC–MS analysis of the 11 formulations. The observation of BHT in the GC–MS analysis but not in the LC–MS analysis
is likely because of the increased sensitivity of GC–MS over LC–MS.
Eleven different typical formulations of biopharmaceutical products were evaluated to determine their effect on the leachables
profile of a common rubber stopper after storage at 40 °C, 75% RH for one month. The key findings are as follows:
- Different formulation excipients have a significant impact on leachables. Especially of note is Tween 80, which significantly
increased the amount of leachables as compared to other formulations.
- The impact of different formulation excipients does not always affect all leachable components to the same extent. For example,
Tween 80 significantly increased the leachables 2-methylpentane, 3-methylpentane, hexane, methylcyclopentane, and cyclohexane.
However, it does not appear to have an impact on BHT. The bulking agents used, such as sucrose, mannitol, and trehalose, lead
to different leachables levels, with trehalose being a superior bulking agent from the standpoint of minimizing leachables.
- The volatility of the extractables in the stopper material and the solubility of those compounds in the formulations are the
determining factors for leachables. All leachables observed in this study were volatile compounds and observed in headspace
GC–MS analysis. Volatile compounds migrate more easily than nonvolatile compounds and are more likely to leach out into formulations.
The relative solubility of leachables in formulation is directly proportional to the amount that will be present in the formulation
as a leachable from the stopper material.
- This study demonstrates that biopharmaceutical excipients, formulation storage conditions, and to some extent the container/closure
material, can have a significant impact on the success of a potential commercial formulation.