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Validation of a novel UPLC-HRMS method for human whole-blood cyclosporine and comparison with a CMIA immunoassay.

ABSTRACT: Therapeutic drug monitoring is an essential tool when managing the therapeutic use of immunosuppressant cyclosporine A (CsA) in cases with solid organ transplantation. In China, the concentration of CsA is primarily measured using immunoassays. However, existing literature recommends mass spectrometry as the current gold standard for the quantitation of CsA. In the present study, it was attempted to develop a novel application to determine CsA concentrations by using ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS). This technique was then compared with a commercially available chemiluminescent microparticle immunoassay (CMIA) and it was investigated how clinical factors may contribute to quantitation differences between the two methods. An UPLC-Orbitrap-MS method was developed to determine CsA concentrations and this method was validated using guidelines put forward by the Food and Drug Administration from the US. In total, 127 blood samples were acquired from patients undergoing kidney transplantation and analyzed by UPLC-HRMS and CMIA assays. The novel method provided sensitive, accurate and precise results. The mean CsA concentration measured by CMIA was significantly higher than that measured by UPLC-HRMS (85.70±48.99 vs. 67.06±34.56 ng/ml, P<0.0001). Passing Bablok analysis yielded a slope of 1.34 (95% CI: 1.22-1.47) and an intercept of -2.54 (95% CI: -10.29-5.52). A group of samples with a higher metabolic ratio (hydroxylated CsA/CsA>1) exhibited larger discrepancies, while a group of samples taken from patients with a longer post-transplantation time (>10 years) featured narrow 95% CIs from -15.32 to 65.69%, as determined by Bland-Altman analysis. In summary, a reliable, accurate and rapid UPLC-HRMS method for CsA analysis was successfully developed. The measurement of CsA by the CMIA assay in renal transplant patients should be further evaluated with a specific focus on positive bias.

SUBMITTER: Wang X

PROVIDER: S-EPMC7812591 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Validation of a novel UPLC-HRMS method for human whole-blood cyclosporine and comparison with a CMIA immunoassay.

Wang Xiaoxue X Qin Wei W Chen Wenqian W Liu Huifang H Zhang Dan D Zhang Xianglin X Li Pengmei P

Experimental and therapeutic medicine 20210107 3

Therapeutic drug monitoring is an essential tool when managing the therapeutic use of immunosuppressant cyclosporine A (CsA) in cases with solid organ transplantation. In China, the concentration of CsA is primarily measured using immunoassays. However, existing literature recommends mass spectrometry as the current gold standard for the quantitation of CsA. In the present study, it was attempted to develop a novel application to determine CsA concentrations by using ultra-performance liquid chr ...[more]

PMID: 33488800

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Project description:Antiretroviral drugs are used for the treatment and prevention of HIV infection. Non-adherence to antiretroviral drug regimens can compromise their clinical efficacy and lead to emergence of drug-resistant HIV. Clinical trials evaluating antiretroviral regimens for HIV treatment and prevention can also be compromised by poor adherence and non-disclosed off-study antiretroviral drug use. This report describes the development and validation of a high throughput, qualitative method for the identification of antiretroviral drugs using high-resolution mass spectrometry (HRMS) for the retrospective assessment of off-study antiretroviral drug use and the determination of potential antiretroviral therapy (ART) non-compliance.Serum standards were prepared that contained 15 antiretroviral drugs: 9 protease inhibitors (PIs), 4 nucleotide/nucleoside reverse transcriptase inhibitors (NRTIs), and 2 non-nucleoside/nucleotide reverse transcriptase inhibitors (NNRTIs). Analytical separation was achieved on a Hypersil Gold PFP (100×3mm) column and the eluent was analyzed using the Thermo Exactive Orbitrap mass spectrometer (Exactive-MS) operated in full scan mode. Limit of identification, signal intensity precision, retention time analysis, selectivity, and carryover studies were conducted. Concordance with liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods was evaluated using remnant plasma samples from a clinical trial.The limit of identification ranged from 5 to 10ng/ml for 14 drugs (9 PIs, 1 NNRTI, 4 NRTIs) and was 150ng/ml for 1 NNRTI. Precision studies with high and low control mixtures revealed signal intensity coefficients of variation of 3.0-27.5%. The Exactive-MS method was selective for the compounds of interest. Overall, concordance ranged from 89.1% to 100% for the screening of antiretroviral drugs in clinical plasma specimens as compared to LC-MS/MS methods.Using the Exactive-MS, we developed and validated a highly selective, robust method for the multiplexed detection of 15 antiretroviral drugs.

Project description:BackgroundCyclosporine A (CsA) is used as a posttransplantation immunosuppressant drug, and careful monitoring of CsA concentration in whole blood is essential. A new automated electrochemiluminescence immunoassay (ECLIA) for CsA measurement has been assessed in a multicenter evaluation.MethodsResidual EDTA whole blood samples from patients undergoing CsA therapy after organ transplant were used in assay evaluation at 5 clinical laboratories in Europe. Experiments included imprecision according to CLSI EP5-A2 (within-run and intermediate), lower limit of quantification, linearity according to CLSI EP6-A, and recovery of commercial external quality control samples. In addition, comparisons to liquid chromatography-tandem mass spectrometry methods in routine use at each investigational site and to commercial chemiluminescent microparticle immunoassay and antibody-conjugated magnetic immunoassay methods were performed.ResultsImprecision testing gave coefficients of variation of less than 9% in the 30-2000 mcg/L range for both within-run and intermediate imprecision. Lower limit of quantification of 6.8 mcg/L at one investigational site and 1.8 mcg/L at a second site at 20% coefficient of variation were observed. Linearity was measured over the concentration range 0-2000 mcg/L, yielding a deviation of less than ±12%. External quality control sample recovery by ECLIA was 93%-114% of LC-MS/MS sample recovery. Deming regression analysis of ECLIA method comparison to combined LC-MS/MS results yielded a slope of 1.04 [95% confidence interval (CI), 1.03-1.06] and intercept of 2.8 mcg/L (95% CI, 1.5-4.1 mcg/L). Comparison to chemiluminescent microparticle immunoassay yielded a slope of 0.87 (95% CI, 0.85-0.89) and intercept of 1.4 mcg/L (95% CI, -0.89 to 3.7 mcg/L); comparison to antibody-conjugated magnetic immunoassay yielded a slope of 0.96 (95% CI, 0.93-0.98) and intercept of -4.2 mcg/L (95% CI, -7.1 to -1.2 mcg/L).ConclusionsThe data from this multicenter evaluation indicate that the new ECLIA-based cyclosporine assay is fit for its purpose, the therapeutic monitoring of CsA.

Dataset Information

Validation of a novel UPLC-HRMS method for human whole-blood cyclosporine and comparison with a CMIA immunoassay.

Publications

Validation of a novel UPLC-HRMS method for human whole-blood cyclosporine and comparison with a CMIA immunoassay.

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