## 4 Discussion
Although the procedure is generally considered safe, it also carries potential surgical complications, especially in the case of total thyroidectomy. The more extensive surgical procedures for thyroid cancer, which often require removal of lymph nodes and resection of potentially aggressive tumor material, contribute to an increased risk of complications. Specific postoperative complications of thyroid cancer surgery include transient or permanent injury of the laryngeal nerve and the occurrence of hypoparathyroidismism. The usually temporary injury of the recurrent laryngeal nerve leads to hoarseness or swallowing difficulties and occurs in 4.5% of cases after total thyroidectomy and in 3.3% of cases after hemithyroidectomy [30]. Although hypoparathyroidismism is usually also transient, it can be permanent, occurring in 21.3% of cases after total thyroidectomy and in 1.8% of cases after total thyroidectomy [30]. Other non-specific complications include hematomas, wound infections, impaired wound healing, excessive bleeding, and damage to the surrounding nerves. These complications remain relatively rare, but when they do occur, they have a significant impact on the quality of life of the affected patients. It is therefore necessary to develop an innovative approach to classify indeterminate thyroid nodules, and thus to reduce unnecessary burdens on patients and health-care systems. The first main challenge of this study was to establish a protocol for the extraction of metabolites from the CytoRich(tm) solution of FNAC samples. In our experience, liquid-phase extraction methods do not result in good separation of the metabolites from the fixatives of the CytoRich(tm) solution, so sample preparation for LC-MS/MS analysis was not possible. We therefore tested several approaches based on solid-phase extraction methods. The best sample preparations for LC-MS/MS analyses were achieved with SPME tips. Despite our promising results, more experiments are still needed to further improve the reproducibility of metabolite extraction using SPME tips. In this study, given the very small amount of biological material available (due to FNAC), a single SPME extraction was performed per sample. This extract was used entirely for two LC-MS/MS analyses, i.e., one in positive and one in negative mode. As expected, the MS signals were low, but a large number of ions could be detected. For best reproducibility and sensitivity, the LC-MS/MS analyses of all the samples shown in this study were performed consecutively in a single run. The order in which the samples were subjected to LC-MS/MS analyses was based on their entry number in the biobank and was thus determined randomly. Our standard procedure included regular controls of all MS parameters and equipment throughout the whole acquisition (pump pressure, quality of the spectra, regular prefilter replacement, etc.). No significant variations in the analyses were found with pooled samples from several subjects that were carried out as quality controls. Blank samples were included to subtract background noise from signals (see Results). In addition, we verified that the peaks of the individual ions determined with MZmine could also be identified with another posttreatment software of MS/MS data, Compound Discoverer. Furthermore, this second software predicted if the ion was [M + H]\({}^{+}\) for the positive mode or [M - H]\({}^{-}\) for the negative mode, another adduct, or an isotope (see Supplementary data). The reproducibility of the obtained data was an important issue in this study. These first analyses with small sample sets already indicated the importance of the experimental procedures and sample collection for reproducible identification of the top ions. However, the results obtained are promising, but further studies with a larger number of samples should now be performed to strengthen the robustness of the metabolic signature. Special attention should be paid to the standardization of sample storage after FNAC.