Nanoflow UHPLC-MS3 for sensitive detection of cortisol in a single-strand 1-cm human hair segment

Hair cortisol is a well-validated biomarker that enables retrospective stress profiling through segmented hair analysis. Because human hair grows at roughly 0.6–1.4 cm per month, there is a pressing need for methods that can reliably quantify cortisol in individual 1-cm segments. Here, we developed a nanoflow ultra-high-performance liquid chromatography–tandem mass spectrometry (nanoflow UHPLC-MS3) workflow tailored for this application. Single-strand 1-cm hair segments (∼0.05 mg) were decontaminated with isopropanol and extracted with methanol via ultrasonication (45 min) followed by water-bath incubation (55 °C, 16 h), then centrifuged, concentrated, and reconstituted in 200 μL methanol. The system used a split-based nanoflow interface coupled to a linear ion trap in negative-ion mode, with MS3 acquisition to improve selectivity and signal quality. Compared with microbore UHPLC-MS3, nanoflow UHPLC-MS3 produced a stronger peak-area response and lower LODs, reaching 0.79 fg in neat solution and 0.08 pg in hair extract. Intra- and inter-day precision were 3.33–6.13% and 13.44–15.17%, respectively, with relative errors of −9.97% to 3.68%. Applied to proximal hair segments from six pregnant women, the standard-addition approach yielded extractable cortisol levels of 8.99–113.78 pg mg−1 after normalization to hair mass. Overall, nanoflow UHPLC-MS3 provides a lower-boundary detection platform for cortisol analysis in single-strand hair, with potential application in studies involving limited hair availability, including neonatal samples.