Using LC-MS/MS for determination of low-abundance protein biomarkers from dried blood spots is challenging due to the combination of low biomarker levels (low pM-level) and small sample volumes (typically <50 μL). In the present paper it is demonstrated that use of state-of-the-art nano liquid chromatography triple quadrupole mass spectrometry in combination with immunoaffinity sample clean-up enable determination of the low abundance biomarker human chorionic gonadotropin (hCG) from four different biological matrices (whole blood, serum, plasma and urine) at its upper reference level (low pM). Detection limits for hCG was determined for all matrices from both commercially available non-soluble DBS sampling material (DMPK-C) and the water-soluble material carboxymethyl cellulose (CMC). The detection limits (S/N = 3) were ranging from 5.0 IU/L (14.5 pM; whole blood) to 10.5 IU/L (30.5 pM; urine) for DMPK-C and from 2.1 IU/L (6.1 pM; urine) to 6.4 IU/L (18.6 pM; plasma) for CMC. A brief evaluation was performed for both sampling materials using serum as matrix resulting in sufficient linearity (r2 ≥ 0.93, range 20–1000 IU/mL (58–2900 pM) for DMPK-C and 10–1000 IU/mL (29–2900 pM) for CMC), repeatability (RSD% = 13–31%) and accuracy (95–106%). To demonstrate the applicability of the method to real samples, a serum sample from a patient previously diagnosed with cancer was also analyzed using both sampling materials. The concentration levels found using the two materials were similar (5280± 595 IU/L (15,312 ± 1726 pM, n = 3) in the DMPK-C spot and 5060 ± 430 IU/L (14,674 ± 1247 pM, n = 3) in the CMC spot). All in all this demonstrated that the tools for determination of low abundance biomarkers at upper reference level from dried matrix spots now is available through a combination of immunoaffinity enrichment and state-of-the-art LC-MS/MS.
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