Abstract
A candidate reference measurement procedure (RMP) for serum theophylline via isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. With a single-step precipitation pretreatment and a 6-min gradient elution, the method achieved baseline separation of theophylline and its analogs on a C18-packed column. A bracketing calibration method was used to ensure repeatable signal intensity and high measurement precision. The intra-assay and inter-assay imprecisions were 1.06%, 0.84%, 0.72% and 0.47%, 0.41%, 0.25% at concentrations of 4.22 µg/mL (23.40 µmol/L), 8.45 µg/mL (46.90 µmol/L), and 15.21 µg/mL (84.43 µmol/L), respectively. Recoveries ranged from 99.35 to 102.34%. The limit of detection (LoD) was 2 ng/mL, and the lowest limit of quantification (LLoQ) was 5 ng/mL. The linearity range extended from 0.47 to 60 µg/mL (2.61–333.04 µmol/L). No ion suppression and carry-over (< 0.68%) were observed. The relative bias for this candidate RMP that participated in 2023 External Quality Control for Reference Laboratories (RELA) conducted by the International Federation of Clinical Chemistry (IFCC) was within a range of 0.17 to 0.93%. Furthermore, two clinical immunoassay systems were compared with this candidate RMP, demonstrating good correlations. The results of the Trueness Verification Plan indicate significant differences among routine systems, highlighting the need for standardization efforts. The developed candidate RMP for serum theophylline serves as a precise reference baseline for standardizing clinical systems and assigning values to reference materials.
Graphical Abstract
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Acknowledgements
We sincerely appreciate Reference System Center of Maccura Biotechnology Co., Ltd. for value assignment for trueness verification samples. We also appreciate the organization by Guangdong Center of Clinical Laboratory and Chongqing Center of Clinical Laboratory for the Trueness Verification Plan in Southern China.
Funding
This work was supported by the Guangdong Basic and Applied Basic Research Foundation (grant numbers 2021A1515220149 and 2021A1515220114); the Research Fund for Bajian Talents of Guangdong Provincial Hospital of Chinese Medicine (grant number BJ2022KY04); and The Specific Research Fund for TCM Science and Technology of Guangdong Provincial Hospital of Chinese Medicine (grant numbers YN2022QN16 and YN2019QL03).
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Yuanyuan Du: investigation; methodology; data curation; visualization; validation; writing—original draft. Qiaofang Yan: investigation; methodology; data curation; visualization; writing—original draft and editing. Min Zhan, Qiaoxuan Zhang, and Di Huang: investigation; validation; writing—review and editing. Pengwei Zhang and Jun Yan: methodology; data curation, software. Jianbing Wang: validation, supervision writing—review and editing. **anzhang Huang and Liqiao Han: conceptualization; funding acquisition; methodology; supervision; validation; writing—review and editing.
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Du, Y., Yan, Q., Zhan, M. et al. Evaluation and clinical application of a bracketing calibration-based isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for serum theophylline. Anal Bioanal Chem (2024). https://doi.org/10.1007/s00216-024-05415-5
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DOI: https://doi.org/10.1007/s00216-024-05415-5