Limitations of <i>Limulus</i> amebocyte lysate test for endotoxin control in raw materials for liposomal nanoformulations

<b>Aim:</b> To evaluate the applicability of <i>Limulus</i> amebocyte lysate (LAL) assay for endotoxin determination in lipid compounding liposomal nanoformulations. <b>Materials &amp; methods:</b> Spiked cholesterol, hydrogenated soy phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG 2000) samples with endotoxins, simulating contaminated samples or in-process contamination were analyzed by chromogenic LAL assay. <b>Results:</b> Recovery of spiked endotoxins was achieved from DSPE-PEG 2000 suspended in water, whereas recovery was not achieved from spiked cholesterol and hydrogenated soy phosphatidylcholine suspended in methanol, and from multilamellar vesicles. <b>Conclusion:</b> Endotoxins, when in contact with organic solvents, no longer react in the LAL assay as they do in aqueous media. This indicates limitations of the LAL assay for endotoxin control in raw materials for liposomal nanoformulations. Raw materials for liposome nanoformulations have low solubility in water. However, DSPE-PEG 2000 forms a homogeneous suspension in water, which does not interfere with the chromogenic <i>Limulus</i> amebocyte lysate (LAL) assay, according to the criteria established by several pharmacopeias (US, European and Japanese). Cholesterol (Chol) and hydrogenated soy phosphatidylcholine (HSPC) should be dissolved in organic solvents in order to determine the endotoxins eventually entrapped in them. Then, to mimic contaminated samples for interference testing, which are dissolved in MeOH for analysis by LAL assay, control standard endotoxin (CSE) resuspended in MeOH should be added. However, CSE resuspended in MeOH does not react in LAL assay as it does when resuspended in aqueous media. When CSE comes into contact with organic solvents, it forms supramolecular structures that can influence its reactivity with the LAL reagent. Independent solutions of different concentrations do not react proportionally to the amounts of CSE, whereas solutions prepared by dilution from the highest concentrations do react proportionally. Therefore, if any sample is dissolved in organic solvents, as is the case with Chol and HSPC for the chromogenic LAL assay, the results cannot be relied upon for accurate endotoxin measurement. CSE resuspended in water is not affected by 2.5 and 5% MeOH. However, if C12E10 is added, the CSE no longer reacts proportionally in the LAL assay. Inhibition/enhancement controls of Chol and HSPC. Inhibition/enhancement controls should be carefully designed to mimic actual contaminated samples to ensure accurate measurement of endotoxins. These controls should also be conducted by spiking with different concentrations of endotoxins if they are assaying under non-aqueous matrices. LPS was not detected in MLV prepared by spiking the lipid solution in EtOH but was detected in MLV prepared by spiking the aqueous solution (5% dextrose). The lipid medium affects endotoxin detection. Heating the spiked MLV samples at 75°C and 92°C for 30 min did not allow endotoxin detection.**研究目的：** 评估鲎变形细胞溶解物（Limulus amebocyte lysate, LAL）法用于脂质复合脂质体纳米制剂内毒素检测的适用性。**材料与方法：** 采用显色法LAL检测，对加标内毒素、模拟污染样品或过程污染的胆固醇、氢化大豆磷脂酰胆碱以及1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-N-[氨基(聚乙二醇)-2000]（DSPE-PEG 2000）样品进行分析。**研究结果：** 悬浮于水中的DSPE-PEG 2000可回收加标内毒素；但悬浮于甲醇的胆固醇、氢化大豆磷脂酰胆碱以及多层脂质体（multilamellar vesicles, MLV）中无法回收加标内毒素。**研究结论：** 内毒素与有机溶剂接触后，无法再像在水相介质中那样与LAL法发生反应，这表明LAL法用于脂质体纳米制剂原料的内毒素质控存在局限性。脂质体纳米制剂原料在水中溶解度较低，但DSPE-PEG 2000可在水中形成均一悬浮液，且根据美国、欧洲及日本多国药典制定的标准，该悬浮液不会干扰显色法鲎变形细胞溶解物（LAL）检测。胆固醇（Chol）与氢化大豆磷脂酰胆碱（HSPC）需溶于有机溶剂，方可检测其包裹的内毒素。此外，为模拟用于干扰试验的污染样品（此类样品需溶于甲醇后通过LAL法检测），应添加复溶于甲醇的对照标准内毒素（control standard endotoxin, CSE）。但复溶于甲醇的CSE无法像复溶于水相介质时那样与LAL法发生反应。当CSE与有机溶剂接触时，会形成超分子结构，进而影响其与LAL试剂的反应活性。不同浓度的独立配制溶液无法与CSE含量呈比例反应，但从最高浓度溶液逐级稀释制备的溶液则可呈比例反应。因此，若样品需溶于有机溶剂（如用于显色LAL法检测的Chol与HSPC），其检测结果无法用于准确的内毒素定量。复溶于水的CSE不受2.5%与5%甲醇的影响，但添加C12E10后，CSE便无法在LAL法中呈比例反应。**胆固醇与HSPC的抑制/增强对照：** 此类对照需精心设计以模拟真实污染样品，保障内毒素检测的准确性。若在非水基质中开展检测，此类对照还需通过加标不同浓度的内毒素来完成。脂多糖（lipopolysaccharide, LPS）在以乙醇（EtOH）溶解的脂质溶液制备的MLV中未被检出，但在以5%葡萄糖水溶液制备的MLV中可被检出。脂质基质会影响内毒素的检测。将加标内毒素的MLV样品在75℃与92℃加热30分钟后，仍无法检出内毒素。