RESULTS AND DISCUSSION
Headspace GC–MS Analysis Extractables Evaluation
The water extracts of stoppers yielded seven extractables peaks (Figure 1). Six of the seven peaks were identified as 2-methylpentane
(4.24 min), 3-methylpentane (4.62 min), hexane (5.14 min), methylcyclopentane (6.24 min), cyclohexane (7.93 min), and butylated
hydroxytoluene (BHT, 33.26 min). The peak at 28.60 min was not identified; the GC–MS library search and manual spectral interpretation
did not produce a good match or a tentative identification. The amounts for each peak are summarized in Table 5. The peak
at 28.60 min was quantitated using cyclohexane as the surrogate standard. Because the relative response factor of the unknown
peak against cyclohexane is not determined, the amount reported for this peak is only considered a semi-quantitative estimate.
Table 5. Quantitation of extractables/leachables with headspace GC–MS
Six leachables peaks were observed in the headspace GC–MS analysis from the formulations: 2-methylpentane, 3-methylpentane,
hexane, methylcyclopentane, cyclohexane, and BHT. These leachables peaks correlate to stopper extractables. The unknown extractables
at 28.60 min were not observed as leachables. This may be because of insolubility of the compound in the aqueous media. The
amount of leachables in different formulations is summarized in Table 5. The effect of various formulation ingredients on
the leachables profile is discussed in detail below.
Phosphate Buffer with or without Glycerol Co-Solvent
Five leachables peaks were observed in the pH 6.8 phosphate buffer: 3 methylpentane, hexane, methylcyclopentane, cyclohexane,
and BHT. All of the peaks were very small. Methylcyclopentane was the largest leachables peak, probably because it is more
soluble in aqueous media than the other compounds. The addition of 2% glycerol in the phosphate buffer as a co-solvent did
not significantly affect the amount of leachables observed.
Formulation pH had an impact on leachables. The neutral pH (pH 6.8) provided slightly lower amounts of leachables compared
to the slightly acidic (pH 5.0) and the slightly alkaline (pH 8.2) formulations (Figure 2). The acidic and alkaline formulations
had similar leachable profiles.
EDTA Chelating Agent
Based on the leachables profiles of formulations containing 0, 0.1, and 0.5 mM EDTA, EDTA did not significantly affect the
amount of organic leachables.
The excipient Tween 80 had the most significant effect on leachables compared to other formulation ingredients. The addition
of Tween 80 significantly increased the leached amount of 2-methylpentane, 3-methylpentane, hexane, methylcyclopentane, and
cyclohexane. The leached amounts of these compounds in the formulation with 0.5% Tween 80 is five to 10 times higher than
in formulations without Tween 80. This is because the surfactant increases the solubility of these compounds in the aqueous
formulation (Figure 3). However, the addition of Tween 80 did not affect the amount of leached BHT because no BHT was detected
in the formulations with 0.5% or 0.1% Tween 80.
Bulking Agents: Sucrose, Mannitol, and Trehalose
The use of bulking agents affected the amount of leachables observed. Higher amounts of leachables were observed in the formulation
with 7% mannitol compared to the formulation with 7% trehalose, in which few leachables were observed (Figure 4). The formulation
with 7% sucrose had leachables levels between those seen in formulations with mannitol and trehalose.