Commentary on "The road to reliable peptide assays is paved with good guidelines".

We note that Maurer et al., in their correspondence,1 have raised some concerns about the preanalytical and analytical validation of the peptide YY (PYY) assay described in our paper ‘The postprandial secretion of peptide YY1–36 and 3–36 in obesity is differentially increased after gastric bypass versus sleeve gastrectomy’.2 The validation and assay characteristics were not the primary objectives of the paper. We focussed on the physiology of PYY secretion in healthy volunteers and patients pre- and post-gastric bypass versus sleeve gastrectomy. Therefore, we did not include a full description and details of the assay validation procedure and history. We agree with the overall principles that Maurer et al. subscribe to and summarise herewith our findings to show that we have followed and fulfilled internationally accepted standards in the validation of our assay. We have verified stability of PYY1–36 and PYY3–36 at preanalytical, analytical and post-analytical stages of the assay. At preanalytical stage the whole blood is collected in prechilled lithium heparin (LH) or ethylenediaminetetraacetic acid (EDTA) tubes and containing evaluated in-house inhibitors (DPP4 inhibitor and Aprotinin). The sample is centrifuged immediately at 4°C and flash-frozen on dry ice or transferred immediately to a −80°C freezer. The samples are kept at −80°C until being thawed once only for analysis. We deemed our protease inhibitors sufficient for maintaining stability of PYY1–36 and PYY3–36 and inhibiting peptide degradation. Our data showed no degradation of PYY1–36 or PYY3–36 for either EDTA or LH plasma at the 3 h time-point at room temperature (19°C) and the 3 h time-point at 4°C, with or without inhibitors. The mean concentration from injections of triplicate QCs (low and high) showed that for spiked samples and samples from a healthy volunteer, the concentrations remained similar regardless of the presence of inhibitors or their absence (with the mean percentage difference for overall and between all tested sample sets ≤10%). Maurer et al. have quoted Toräng et al.'s study on PYY's major cleaving sites but disregarded the outcome from their study of the inhibitory effects of EDTA on metalloendoproteases, neuroendopeptidase (NEP) and Meprin β.3 Toräng et al. demonstrated that the risk of PYY degradation in collected blood is small in collection tubes containing EDTA. We have studied the stability of both PYY species with our inhibitor mix containing DPP4 inhibitor and Aprotinin, where we spiked PYY1–36 and PYY3–36 into plasma EDTA and LH from three different volunteers. We did not detect any significant differences in concentrations of both peptides with only these inhibitors present suggesting that NEP, Meprin β and Am-P activity is negligible under these conditions. Furthermore, we have verified the stability of both species of PYY in both EDTA and LH plasma under the following conditions: (1) freezing and thawing up to a second cycle; (2) incubation at 4°C for up to 4 h; (3) long-term storage (6 months) at −80°C; (4) postextraction, during storage in the autosampler at 4°C. We have verified the stability of our stock and working stock solutions for up to 7 months, stored at 4°C. We would state that it is a matter of standard operating procedure that we store these solutions at −80°C and discard these at the end of the day of the run after thawing, affording a safety margin. Last, our assay has been validated both with plasma-based calibrants and QCs, and cross-validated in a surrogate matrix (20BMA). We also validated matrix effects (ME) in spiked plasma EDTA and plasma LH. Mean percentage ME for PYY1–36 was 100.7% and for PYY3–36, 111% in plasma LH. EDTA ME results were reported previously.2 Maurer et al. suggest that imputation of 更多