Stout P R, Claffey D J, Ruth J A
Department of Molecular Toxicology and Environmental Health Sciences, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Drug Metab Dispos. 1998 Jul;26(7):689-700.
The levels of fentanyl extractable from mouse hair after chronic systemic administration and the suitability of externally loaded hair samples for establishing control and comparison samples were determined. Additionally, the effects of chemical modification of specific polar functionalities within the hair protein matrix on the deposition and recovery of fentanyl in hair subjected to external loading were determined. BALB/c mice entering a second phase of synchronized hair growth were treated ip with fentanyl (0.02, 0.05, or 0.10 mg/kg) on Monday, Wednesday, and Friday for 3 weeks. At that time, fentanyl concentrations in hair, as determined by GC/MS, were 0.025-0.050 ng/mg of hair. Hair samples exposed to fentanyl in phosphate buffer (ionized drug) showed no significant accumulation of drug into the hair, as determined by loss of fentanyl from the loading solution or by extraction of the hair. Hair samples exposed to nonionized fentanyl in methanolic solution (10, 50, and 100 ng/ml) showed significant accumulation of drug in the hair and significant removal of drug from the incubation solution. Fentanyl removal from solution plateaued after 24 hr, suggesting equilibration between fentanyl in solution and fentanyl in the hair. A mass balance between drug lost from the incubation solution and drug recovered from hair samples suggests that 94% of accumulated fentanyl is tightly bound to the hair matrix or resides in water-inaccessible compartments within the hair. These results suggest that fentanyl accumulation after in vivo administration differs, in the nature of storage, from fentanyl accumulation from external solutions and that external spiking of hair may not provide suitable control samples. Chemical modification of hair protein functionalities (reaction with diazomethane to esterify carboxylic acid groups or with acetic anhydride and pyridine to acetylate amine and hydroxyl functionalities) led to reproducible protein structure modification, as demonstrated by Fourier transform-IR and by pH titration. Hair from BALB/c mice was used. The accumulation of fentanyl was examined in hair samples exposed to fentanyl in methanol or methylene chloride solutions (10 ng/ml, 24 hr). Fentanyl was recovered from hair by 24-hr extraction in phosphate buffer, pH 6. Esterification of hair resulted in significantly less uptake of nonionized fentanyl from a methanolic solution and significantly lower recovery of drug from hair, relative to untreated hair, suggesting that carboxylic acid functionalities are necessary for the incorporation of drug. Acetylation of hair resulted in increased removal of fentanyl from methylene chloride solutions and increased recovery of fentanyl. This is consistent with the creation or expansion of a less polar compartment. Fentanyl uptake from a methanolic solution was also greater in acetylated hair. These results demonstrate that solution-accessible ionizable functionalities of hair play a significant role in the accumulation and retention of nonionized fentanyl from organic solutions.
测定了慢性全身给药后从小鼠毛发中可提取的芬太尼水平，以及外部加载毛发样本用于建立对照和比较样本的适用性。此外，还测定了毛发蛋白质基质中特定极性官能团的化学修饰对外部加载毛发中芬太尼沉积和回收的影响。进入同步毛发生长第二阶段的BALB/c小鼠在周一、周三和周五腹腔注射芬太尼（0.02、0.05或0.10mg/kg），持续3周。此时，通过气相色谱/质谱法测定毛发中的芬太尼浓度为0.025 - 0.050ng/mg毛发。通过加载溶液中芬太尼的损失或毛发提取测定，暴露于磷酸盐缓冲液（离子化药物）中的芬太尼毛发样本未显示药物在毛发中有显著积累。暴露于甲醇溶液（10、50和100ng/ml）中未离子化芬太尼的毛发样本显示药物在毛发中有显著积累，且从孵育溶液中显著去除药物。24小时后溶液中芬太尼的去除达到平稳状态，表明溶液中的芬太尼与毛发中的芬太尼达到平衡。从孵育溶液中损失的药物与从毛发样本中回收的药物之间的质量平衡表明，94%积累的芬太尼紧密结合在毛发基质上或存在于毛发中难以接触水的隔室中。这些结果表明，体内给药后芬太尼的积累在储存性质上与外部溶液中芬太尼的积累不同，并且毛发的外部加标可能无法提供合适的对照样本。毛发蛋白质官能团的化学修饰（与重氮甲烷反应使羧酸基团酯化或与乙酸酐和吡啶反应使胺和羟基官能团乙酰化）导致了可重复的蛋白质结构修饰，这通过傅里叶变换红外光谱和pH滴定得到证明。使用的是BALB/c小鼠的毛发。在暴露于甲醇或二氯甲烷溶液（10ng/ml，24小时）中芬太尼的毛发样本中检查了芬太尼的积累情况。通过在pH 6的磷酸盐缓冲液中提取24小时从毛发中回收芬太尼。相对于未处理的毛发，毛发的酯化导致从甲醇溶液中摄取的未离子化芬太尼显著减少，并且从毛发中回收的药物显著降低，这表明羧酸官能团对于药物的掺入是必要的。毛发的乙酰化导致从二氯甲烷溶液中去除芬太尼增加以及芬太尼回收增加。这与形成或扩大极性较小的隔室一致。乙酰化毛发从甲醇溶液中摄取的芬太尼也更多。这些结果表明，毛发中可被溶液接触的可电离官能团在从有机溶液中积累和保留未离子化芬太尼方面起重要作用。
