NOE


Table 1-2: NOE Spiking Study with Buff ers at 2-8°C Results (EU/mL)


Sample Buff er B


T0 (initial timepoint)


Buff er A 55 61


T1 (1 day)


46 54


T2 (2 days)


46 53


T3 (1 week)


44 42


T4 (2 weeks)


39 45


Table 1-3: NOE Spiking Study with Buff ers at -80°C Results (EU/mL)


Sample Buff er B


T0 (initial timepoint)


Buff er A 55 61


T1 (1 day)


42 54


T2 (1 month)


50 51


T3 (2 months)


49 52


T4 (3 months)


46 52


T5 (3 weeks)


41 48


directly after NOE spiking, consideration must be taken as to whether the sample matrix has an immediate eff ect on endotoxin recovery. To demonstrate that there is no sample matrix interference, an identical spike into LAL reagent water (LRW) is performed. Table 1-4 illustrates the use of this LRW spiking confi rmation. Three diff erent NOE spiking levels were used in this study, indicated by the target NOE spike amount. The actual amount recovered in EU/mL is indicated in the columns under test sample and LRW, are well within the LAL assay variability, and indicate that there is no interference from the sample matrix.


Table 1-4: LRW Spiking Confi rmation


Target NOE spike 50 EU/mL 100 EU/mL


200 EU/mL


32 67


Test sample (EU/mL) LRW (EU/mL) 30 47


124 111


The importance of performing this spiking confi rmation in LRW for the initial time point is clearly illustrated in the following study (Table 1-5). NOE was spiked into a test sample and assayed immediately to determine the starting NOE concentration. NOE was spiked in three diff erent concentrations, indicated by the target NOE spike. The endotoxin recovery was below the limit of detection of the assay at the dilution tested for all three spike levels. However, because an LRW confi rmation was not performed, it is diffi cult to determine whether there was a dilution or assay error, or actual matrix or product interference.


Table 1-5: Test Sample NOE Spiking without LRW Confi rmation Target NOE spike


Test Sample (EU/mL)


50 EU/mL 100 EU/mL


200 EU/mL


< 50 < 50


< 50


The study was repeated at two diff erent spiking levels, with an LRW confi rmation sample performed side-by-side with the test sample (Table 1-6). The addition of the LRW confi rmation sample clearly illustrates that there is sample matrix interference, leading to lack of endotoxin detection in the test sample.


In this case, multiple studies were performed to determine the cause of the sample matrix interference. The use of laboratory-prepared NOE


23 www.americanpharmaceuticalreview.com


Table 1-7: Test Sample NOE Spiking at Low EU/mL Concentrations Target NOE spike


5 EU/mL 20 EU/mL


40 EU/mL 4


Test sample (EU/mL) LRW (EU/mL) 4


15 27 20 29


Preparations from several diff erent organisms were used as well, adding robustness and variability to the data collected. This highlights an additional benefi t of using in-house preparations of NOE, since stocks from multiple organisms can be quickly prepared and used when desired.


Table 1-8 shows some of the data generated using the same test sample from Tables 1-5 and 1-6, with spikes from a high EU/mL NOE producer (S. marcescens) and a low EU/mL NOE producer (A. genomospecies). Both were spiked at a target of 200 EU/mL and showed similar recovery, further indicating that the test sample was interfering with the recovery of endotoxin, rather than any assay errors or issues with the a particular source of endotoxin.


Table 1-8: Spiking with Multiple NOE Producers LRW


NOE used S. marcescens


A. genomospecies Conclusion


The data presented in this report is used to illustrate the importance of testing various matrices, temperatures and storage times for interference in endotoxin recovery for samples that will be held in the laboratory for any length of time prior to testing. It is especially important to understand the interaction between endotoxin and the sample matrix at the initial time point, since any matrix inhibiton that may be present would be identifi ed at this time. The use of laboratory-prepared NOE stocks greatly increased the fl exibility, effi ciency and range of testing options available when performing this testing or troubleshooting matrices that demonstrated inhibition when NOE was spiked into the test sample matrix. When encountering matrix inhibition at the initial time point, alternate testing methods would need to be developed, and NOE is extremely useful in this case as well, since confi rmation of successful endotoxin recovery is critical to any alternate method that would be implemented.


Target NOE spike


200 EU/mL 200 EU/mL


Test Sample (EU/mL)


62 < 50


(EU/mL) 121 175


T5 (3 weeks)


41 48


stocks led to increased effi ciency in the laboratory’s ability to perform multiple studies in short periods of time, and allowed for the use of spiking concentrations from 5 EU/mL up to 3500 EU/mL. Table 1-7 shows data from a study with a diff erent test sample where the NOE spike was very low (5-40 EU/mL). In this case, endotoxin recovery was consistent between the test sample and the LRW confi rmation sample indicating no matrix interference.


Target NOE spike 100 EU/mL 200 EU/mL


Table 1-6: Test Sample NOE Spiking with LRW Confi rmation Test Sample (EU/mL) LRW (EU/mL)


< 50 < 50


73 250


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