• 29
    Dec
  • Stability of Norepinephrine Solutions: RESULTS

drug stability

Accelerated Degradation and Assay Validation

Degradation of norepinephrine with heat and base (pH 8.264) occurred relatively quickly, such that less than 1% of the initial norepinephrine concentration remained after 37 min. As the norepinephrine concentration decreased, degradation products eluted at 1.3, 1.7, 2.1, 2.5, 2.8, and 4.8 min. These degradation products were well separated from norepinephrine, which eluted at 5.5 min (Figure 1). Degradation appeared to occur in a first-order fashion, with a half-life of 5.5 min (r = 0.9969, n = 8). Degradation of norepinephrine with heat and acid (pH 1.977) occurred more slowly, such that about 80% of the initial norepinephrine concentration remained after 142 h. As a result of the chromatographic separation of these degradation products from norepinephrine, and the similarity of the UV spectrum (200-320 nm) between a fresh norepinephrine sample and norepinephrine in a degraded sample, it was concluded that this analytical method was stability-indicating.

Assay validation demonstrated that the absolute deviation from the known concentration for quality control samples and standards on any day averaged 2.1%. Replicate error within a day averaged less than 0.6% for standards and 0.5% for quality control samples.
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Analysis of accuracy and reproducibility during the study period indicated that the norepinephrine concentration was measured accurately and reproducibly. Accuracy, based on the mean of duplicate determinations of standards over the study period, showed that there was less than 1.5% absolute deviation from the expected concentration. Replicate error within a day (as measured by the coefficient of variation) averaged less than 0.5% for standards. Between-day variation, as measured by the coefficient of variation in terms of the slope of the standard curve from day to day, averaged 4.2%. This was similar to the observed standard deviation of regression for percent remaining of 3.95%. This result indicates that differences of 10% or more could be confidently detected with acceptable error rates19,20 with duplicate analysis. System suitability criteria were developed on the basis of daily calculations of theoretical plates, tailing, retention time, and accuracy observed during the validation period and were used to ensure continued chromatographic performance during the study period. On each day, the mobile phase was prepared to ensure a retention time for norepinephrine between 4.9 and 5.2 min.

Figure 1. Accelerated degradation

Figure 1. Accelerated degradation of norepinephrine. A: Solution of norepinephrine 40 mg/L in water at pH 8.264 (time 0). B: After 10 min at 80°C, 26% of the initial concentration remained. Norepinephrine eluted at 5.5 min. Arrows indicate unidentified degradation products eluting at 1.3, 1.7, and 2.1 min. Additional degradation products, not evident at this scale, eluted at 2.5, 2.8, and 4.8 min. All of the degradation products were well separated from norepinephrine.

Stability of Norepinephrine

For each set of storage conditions, the initial concentration and the percent remaining on each study day during the study period is reported (as mean of replicate analyses) in Table 1. Rep­resentative chromatograms for D5W solutions on days 0 and 61 showed very small amounts of degradation products (Figure 2).
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Table 1. Stability of Norepinephrine during Storage, Expressed as Percentage of Original Concentration Remaining

D5W

NS

Protected from Light

Exposed to Light

Protected from Light

Exposed to Light




Study day




RT




4°C




RT




4°C




RT




4°C




RT




4°C

Observed initial


concentration





(mg/L)




70.06





64.46




66.18





66.67





66.79





63.26





65.29





65.84




0




100.00




100.00




100.00




100.00




100.00




100.00




100.00




100.00




1




103.20




102.71




103.49




103.22




103.35




103.16




105.16




101.92




2




95.85




103.94




97.59




93.97





98.28




100.24




100.04





95.03




3




99.79





98.50




102.79




100.16




103.20




102.62




103.26




101.80




4




103.25




103.61




104.35




103.54




103.67




104.66




106.36




104.02




8




102.97




102.00




104.49





99.63




100.31




103.79




104.70




101.79




9




104.37




104.39




105.89




102.15




106.53




106.21




105.70




104.60




10




102.19




102.12




105.22





99.56




104.82




105.16




107.32




102.23




11




102.82




102.43




104.74





99.53




104.72




104.96




106.93




103.30




14




105.61




104.90




106.02




102.46




106.75




108.16




106.01




102.14




18




102.67




103.39





93.46





95.68




97.14




11
5.62t




96.11




98.16




21




101.30





99.22




91.90




92.75




1
10.89t




103.21





98.75




99.16





23




100.69




104.11





98.02





94.79




100.31




103.72





92.22





97.78




25





118.42t




102.05




106.36




94.91





84.83t




108.73




101.35




102.57





28




101.54





94.69





85.75t




93.03





95.05




103.94





94.42





96.32





30




101.30




102.42




92.64





94.82





99.95




103.34




93.18





96.79




36




104.24




102.36





95.48





94.60




102.68




104.13





95.97





96.74





42




102.94




102.66





96.36




93.16




102.88




103.46





94.57




97.21




61




104.54




103.94





93.46




94.14




103.84




105.06





94.46





98.00


SD from





regressiont





4.274





2.535




5.176




2.873





5.607




3.444





3.885




2.604





Degradation rate (%/day)t§




-0.0596




-0.0728




-0.3711




-0.2485




-0.2019




-0.0720





-0.3486




-0.1719





T-90 (days)§4




167.9




137.5





26.9





40.2




49.5




138.8





28.7





58.2

% remaining§**





Day


14





99.2





99.0





94.8





96.5





97.2





99.0




95.1





97.6





Day


28





98.3





98.0





89.6




93.0





94.3





99.0





90.2





95.2





Day


61





96.4





95.6





77.4




84.8





87.7





95.6





78.7





89.5

% remaining with storage

At 4°C for





28 days




97.96





93.04




97.98




95.19





At 4°C for





38 days





97.23





90.56





97.26




93.47

For 1 additional





day at 23°Ctt




97.1
8tt





90.19tt





97.06tt




93.1
2tt

At 4°C for





60 days





95.64





85.09





95.68





89.69

For 1 additional





day at 23°Ctt





95.58tt





84.72tt





95.48tt





89.34tt

Analysis of variance revealed differences in percent remaining due to study day (p < 0.001) and protection from or exposure to light (p < 0.001), but revealed no differences due to diluent (p = 0.06) or temperature (p > 0.99).
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Figure 2. Chromatograms obtained during

Figure 2. Chromatograms obtained during the 61-day stability study. A: Solution of norepinephrine 64.5 mg/L in 5% dextrose in water (D5W) on study day 0. B: Solution of norepinephrine 64.5 mg/L in D5W after 61 days of storage at 23°C, protected from light, with undetectable loss. C: Solution of norepinephrine 64.5 mg/L in D5W after 61 days of storage at 23°C with no protection from light; 93% of the original concentration remains. D = degradation products (see arrows). These degradation products were identical in retention time with those observed in the accelerated degradation study (Figure 1). It was apparent that 5-hydroxy methyl furfural, a degradation product of dextrose that was observed only in solutions prepared with D5W, increased over the course of the study and increased to a greater extent in solutions that were unprotected from light.

Inspection of the fastest degradation rates observed with 95% confidence (Table 1) revealed that solutions protected from light had slower degradation rates and took 2 to 8 times longer to reach 90% of the initial concentration (T-90) than solutions stored without protection from ambient fluorescent light. Inspection of the concentration remaining on day 61, based on the fast degradation rates with 95% confidence (Table 1), revealed that all solutions stored with protection from light lost between 3.6% and 12.3% (mean 6.2%) of their initial concentration, regardless of storage temperature. In contrast, solutions exposed to ambient fluorescent light lost between 10.5% and 22.6% (mean 17.4%) of their initial concentration.

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