The spectra of anthracyclines analyzed

Introduction
The identification of 4 anthracycline molecules: Epirubicin (EPI), Daunorubicin (DAUNO), Doxorubicin (DOXO) and Idarubicin (IDA), is non-specific by analysis with the QCRx^® spectrophotometer (in UV - Raman), requiring a systematic double check of the visual identification of the vial. To improve this control, the spectra of these molecules are analyzed by high-pressure liquid chromatography (HPLC) with different temperature and derivatisation, in order to check whether there are any spectral differences.

Materials and methods
Analysis is performed on HPLC (Shimadzu Nexera X2^®) in direct injection, then detected in UV-Visible (190-700nm). A specific spectral library is created using a protocol of 8 samples per anthracycline diluted in 0.9% NaCl at increasing concentration, for 3 temperatures (Ambient-amb, +15°C, +10°C), and 3 derivative modes (without derivative-WD, 1st derivative-1D and 2nd derivative-2D). Samples from the unit’s production are identified using the same protocol by similarity to spectra acquired with the pre-registered spectral library, at a ʎ from 210 to 700 nm. A different sample identification is used to calculate the error rate (ER) per molecule. The % similarity is calculated in the case of conforming identification (1st%), and up to non-conformity (2nd%). Statistical analyses are performed using Excel^® and XLStat^®.

Results
47 samples (DOXO=14, EPI = 14, DAUNO=8, IDA=7) were analyzed for a total of 383 spectral data. The ER obtained, all temperatures and derivatives combined, are: 22% for DOXO, 40% for EPI, 27% for DAUNO and 5% for IDA. Analysis by subcategory shows a ER for IDA of 0% at amb and +15°C, whatever the derivative of the spectrum, and close to 0% for DOXO for spectra at +10°C. The ER is 17% in amb for EPI’s 3 derivatives and at +15°C for DAUNO’s 1D and 2D.
A chi² test revealed no significant difference in error rate per molecule as a function of spectrum derivative and temperature (p-value=0.169).
A repeated-measures ANOVA test on the IDA samples showed a significant difference between the 1st% and the 2nd%. The same test on DAUNO and DOXO-EPI data, on the other hand, was not significant.

Discussion / Conclusion
Analysis of anthracycline HPLC spectral data showed a error rate and significant statistical analysis only for IDA, which is more pronounced in 1D and 2D, making it possible to dispense without visual control of the vial. As the wavelength range of the UV-Visible detector is wider than that of the QCRx^® (190-700nm vs. 200-400nm), it is not possible to extrapolate the results to the control method currently used routinely. Further analysis at more restricted wavelengths will have to be carried out. On the other hand, for DOXO, EPI and DAUNO, a double visual identification check of the vial is still necessary.

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