Development and validation of an LC-MSMS method to quantify creatinine from dried blood spots

Carlos Torres; Rogers A Muldrow; Anissa R Naranjo; Steven W Cotten; Christina C Pierre; Dina N Greene

doi:10.1016/j.jmsacl.2024.03.001

Development and validation of an LC-MSMS method to quantify creatinine from dried blood spots

J Mass Spectrom Adv Clin Lab. 2024 Mar 6:32:50-59. doi: 10.1016/j.jmsacl.2024.03.001. eCollection 2024 Apr.

Authors

Carlos Torres¹, Rogers A Muldrow¹, Anissa R Naranjo¹, Steven W Cotten², Christina C Pierre^{3

4}, Dina N Greene^{1

5}

Affiliations

¹ LetsGetCheked, Monrovia, CA, USA.
² Department of Pathology and Laboratory Medicine, Chapel Hill, NC, USA.
³ Department of Pathology and Laboratory Medicine, Penn Medicine Lancaster General Hospital, Lancaster, PA, USA.
⁴ Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁵ University of Washington Seattle, Department of Laboratory Medicine and Pathology, Seattle, WA, USA.

Abstract

Introduction: Screening for chronic kidney disease relies on accurate and precise creatinine measurements. Traditionally, creatinine is measured in serum or plasma using high-throughput chemistry analyzers. However, dried blood spots (DBS) can also be utilized to improve testing access.

Methods: Samples were obtained from a 6 mm DBS punch, which was reconstituted in water before undergoing an acetonitrile crash. The resulting supernatant was diluted using an 80:20 acetonitrile: water before injection. Creatinine was identified using an isocratic gradient, and detected using an API 4000 triple quadrupole mass analyzer. Quantification relied on matrix-matched calibrators, with values harmonized to the Roche Cobas enzymatic assay. Validation studies assessing method performance included precision, linearity, accuracy, method comparison, stability, interference, and matrix effects.

Results: The LC-MSMS assay was linear from 0.3 to 20 mg/dL (y = 1.02x-0.11; R² = 0.996). Precision ranged from 5.2 to 8.1 % using matrix-matched controls (n = 4) that spanned the analytical measurement range. LC-MSMS results corresponded to the enzymatic assay (Roche) with a fitted line equation of y = 0.956x-0.07 (R² = 0.995; n = 173). The Siemens and Roche enzymatic assays demonstrated higher accuracy in correlating to the DBS creatinine concentration (n = 40 paired venous/DBS collections) compared to the Beckman Jaffe assay (-2.5 % and -0.8 % versus -6.3 % and -4.1 %, respectively) or the iSTAT (-28.4 % and -27.1 %, respectively). Accuracy was unaffected by hematocrit, blood spot volume, excess IgG or IgA, or hypertriglyceridemia. No matrix effects were observed, and both extraction and processing efficiency were robust.Ambient stability extended to at least 10 days, and exposure to extreme temperature did not affect the creatinine results.

Conclusion: We successfully developed an accurate and precise LC-MSMS method for quantifying creatinine in DBS.

Keywords: Chronic kidney disease screening; Creatinine; Dried blood spots; LC-MS/MS.