Multitargeted Internal Calibration for the Quantification of Chronic Kidney Disease-Related Endogenous Metabolites Using Liquid Chromatography-Mass Spectrometry.

Accurate quantitative analysis in liquid chromatography-mass spectrometry (LC-MS) benefits from calibration curves generated in the same matrix as the study sample. In the case of endogenous compound quantification, as no blank matrix exists, the multitargeted internal calibration (MTIC) is an attractive and straightforward approach to avoid the need for extensive matrix similarity evaluation. Its principle is to take advantage of stable isotope labeled (SIL) standards as internal calibrants to simultaneously quantify authentic analytes using a within sample calibration. An MTIC workflow was developed for the simultaneous quantification of metabolites related to chronic kidney disease (CKD) using a volumetric microsampling device to collect 20 µL of serum or plasma, followed by a single-step extraction with acetonitrile/water and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Since a single concentration of internal calibrant is necessary to calculate the study sample concentration, the instrument response function was investigated to determine the best SIL concentration. After validation, the trueness of 16 endogenous analytes in authentic human serum ranged from 72.2 to 116.0%, the repeatability from 1.9 to 11.3%, and the intermediate precision ranged overall from 2.1 to 15.4%. The proposed approach was applied to plasma samples collected from healthy control participants and two patient groups diagnosed with CKD. Results confirmed substantial concentration differences between groups for several analytes, including indoxyl sulfate and cortisone, as well as metabolite enrichment in the kynurenine and indole pathways. Multitargeted methodologies represent a major step toward rapid and straightforward LC-MS/MS absolute quantification of endogenous biomarkers, which could change the paradigm of MS use in clinical laboratories.

Fast neonicotinoid quantification in honey using the one-point internal calibration approach.

Neonicotinoids, a highly effective class of insecticides used worldwide, have been identified as a major cause of concern for biodiversity. To assess the ecological and environmental consequences of neonicotinoids' use, reliable analytical methodologies, including calibration approaches, are needed. Here, we compared the performance of internal calibration (IC) using a single concentration of stable isotope-labeled standard (SIL) with classical multipoint external calibration (EC) for the quantification of six neonicotinoids in honey. IC showed acceptable levels of trueness (86.3% - 116.0%) and precision (1.4% - 20.8%), although slight biases were observed at very low concentrations compared to EC. When applied to 32 original honey samples, both approaches showed strong agreement (R2 > 0.998) with proportional biases lower than 5%. These results highlight the possibility of implementing IC to simplify quantification in liquid chromatography-mass spectrometry-based pesticide applications.

Activation of endogenous glucocorticoids by HSD11B1 inhibits the antitumor immune response in renal cancer.

Although immune-based therapies have revolutionized the management of cancer, novel approaches are urgently needed to improve their outcome. We investigated the role of endogenous steroids in the resistance to cancer immunotherapy, as these have strong immunomodulatory functions. Using a publicly available database, we found that the intratumoral expression of 11 beta-hydroxysteroid dehydrogenase type 1 (HSD11B1), which regenerates inactive glucocorticoids into active glucocorticoids, was associated with poor clinical outcome and correlated with immunosuppressive gene signatures in patients with renal cell carcinoma (RCC). HSD11B1 was mainly expressed in tumor-infiltrating immune myeloid cells as seen by immunohistochemistry in RCC patient samples. Using peripheral blood mononuclear cells from healthy donors or immune cells isolated from the tumor of RCC patients, we showed that the pharmacological inhibition of HSD11B1 improved the response to the immune checkpoint inhibitor anti-PD-1. In a subcutaneous mouse model of renal cancer, the combination of an HSD11B1 inhibitor with anti-PD-1 treatment increased the proportion of tumor-infiltrating dendritic cells. In an intrarenal mouse tumor model, HSD11B1 inhibition increased the survival of mice treated with anti-PD-1. In addition, inhibition of HSD11B1 sensitized renal tumors in mice to immunotherapy with resiquimod, a Toll-like receptor 7 agonist. Mechanistically, we demonstrated that HSD11B1 inhibition combined with resiquimod increased T cell-mediated cytotoxicity to tumor cells by stimulating the antigen-presenting capacity of dendritic cells. In conclusion, these results support the use of HSD11B1 inhibitors to improve the outcome of immunotherapy in renal cancer and highlight the role of the endogenous glucocorticoid metabolism in the efficacy of immunotherapy.

From fundamentals in calibration to modern methodologies: A tutorial for small molecules quantification in liquid chromatography-mass spectrometry bioanalysis.

Over the last two decades, liquid chromatography coupled to mass-spectrometry (LC‒MS) has become the gold standard to perform qualitative and quantitative analyses of small molecules. When quantitative analysis is developed, an analyst usually refers to international guidelines for analytical method validation. In this context, the design of calibration curves plays a key role in providing accurate results. During recent years and along with instrumental advances, strategies to build calibration curves have dramatically evolved, introducing innovative approaches to improve quantitative precision and throughput. For example, when a labeled standard is available to be spiked directly into the study sample, the concentration of the unlabeled analog can be easily determined using the isotopic pattern deconvolution or the internal calibration approach, eliminating the need for multipoint calibration curves. This tutorial aims to synthetize the advances in LC‒MS quantitative analysis for small molecules in complex matrices, going from fundamental aspects in calibration to modern methodologies and applications. Different work schemes for calibration depending on the sample characteristics (analyte and matrix nature) are distinguished and discussed. Finally, this tutorial outlines the importance of having international guidelines for analytical method validation that agree with the advances in calibration strategies and analytical instrumentation.

Straightforward quantification of endogenous steroids with liquid chromatography-tandem mass spectrometry: Comparing calibration approaches.

Different calibration strategies are used in liquid chromatography hyphenated to mass spectrometry (LC-MS) bioanalysis. Currently, the surrogate matrix and surrogate analyte represent the most widely used approaches to compensate for the lack of analyte-free matrices in endogenous compounds quantification. In this context, there is a growing interest in rationalizing and simplifying quantitative analysis using a one-point concentration level of stable isotope-labeled (SIL) standards as surrogate calibrants. Accordingly, an internal calibration (IC) can be applied when the instrument response is translated into analyte concentration via the analyte-to-SIL ratio performed directly in the study sample. Since SILs are generally used as internal standards to normalize variability between authentic study sample matrix and surrogate matrix used for the calibration, IC can be calculated even if the calibration protocol was achieved for an external calibration (EC). In this study, a complete dataset of a published and fully validated method to quantify an extended steroid profile in serum was recomputed by adapting the role of SIL internal standards as surrogate calibrants. Using the validation samples, the quantitative performances for IC were comparable with the original method, showing acceptable trueness (79%-115%) and precision (0.8%-11.8%) for the 21 detected steroids. The IC methodology was then applied to human serum samples (n = 51) from healthy women and women diagnosed with mild hyperandrogenism, showing high agreement (R2 > 0.98) with the concentrations obtained using the conventional quantification based on EC. For IC, Passing-Bablok regression showed proportional biases between -15.0% and 11.3% for all quantified steroids, with an average difference of -5.8% compared to EC. These results highlight the reliability and the advantages of implementing IC in clinical laboratories routine to simplify quantification in LC-MS bioanalysis, especially when a large panel of analytes is monitored.

Internal calibration as an emerging approach for endogenous analyte quantification: Application to steroids.

The use of mass spectrometry methods with triple quadrupole instruments is well established for quantification. However, the preparation of calibration curves can be time-consuming and prone to analytical errors. In this study, an innovative internal calibration (IC) approach using a one-standard calibration with a stable isotope-labeled (SIL) standard version of the endogenous compound was developed. To ensure optimal quantitative performance, the following parameters were evaluated: the stability of the analyte-to-SIL response factor (RF), the chemical and isotopic purities of the SIL, and the instrumental reproducibility. Using six clinically important endogenous steroids and their respective SIL standards, we demonstrated that RFs obtained on different LC-MS platforms were consistent. The quantitative performance of the proposed approach was determined using quality control samples prepared in depleted serum, and showed both satisfactory precision (1.3%-12.4%) and trueness (77.5%-107.0%, with only 3 values outside ±30%). The developed method was then applied to human serum samples, and the results were similar to those obtained with the conventional quantification approach based on external calibration: the Passing-Bablok regression showed a proportional bias of 6.8% and a mean difference of -5.9% between the two methodologies. Finally, we showed that the naturally occurring isotopes of the SIL can be used to provide additional calibration points and increase the accuracy for analytes with low concentrations.

From a single steroid to the steroidome: Trends and analytical challenges.

For several decades now, the analysis of steroids has been a key tool in the diagnosis and monitoring of numerous endocrine pathologies. Thus, the available methods used to analyze steroids in biological samples have dramatically evolved over time following the rapid pace of technology and scientific knowledge. This review aims to synthetize the advances in steroids' analysis, from classical approaches considering only a few steroids or a limited number of steroid ratios, up to the new steroid profiling strategies (steroidomics) monitoring large sets of steroids in biological matrices. In this context, the use of liquid chromatography coupled to mass spectrometry has emerged as the technique of choice for the simultaneous determination of a high number of steroids, including phase II metabolites, due to its sensitivity and robustness. However, the large dynamic range to be covered, the low natural abundance of some key steroids, the selectivity of the analytical methods, the extraction protocols, and the steroid ionization remain some of the current challenges in steroid analysis. This review provides an overview of the different analytical workflows available depending on the number of steroids under study. Special emphasis is given to sample treatment, acquisition strategy, data processing, steroid identification and quantification using LC-MS approaches. This work also outlines how the availability of steroid standards, the need for complementary analytical strategies and the improvement of calibration approaches are crucial for achieving complete steroidome quantification.