Abstract
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.
Highlights
The reliable quantification of endogenous compounds is an essential process in clinical and nonclinical bioanalysis
Using six clinically important endogenous steroids and their respective Stable isotope-labeled (SIL) standards, we demonstrated that response factor (RF) obtained on different liquid chromatography combined with mass spec trometry (LC-MS) platforms were consistent
We developed an ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify a panel of six steroids in human serum using the internal calibration approach with SIL as surrogate analyte
Summary
The reliable quantification of endogenous compounds is an essential process in clinical and nonclinical bioanalysis In this context, the cali bration methodology plays a crucial role in providing accurate and precise results. To achieve the best estimate of the concentration-response relationship, an internal standard (IS) is usually added to all samples (calibration standards and unknown samples). This allows to normalize the instrumental response and attenuate the overall analytical variation in the measurement process resulting from the presence of random errors from sample preparation and/or systematic errors originating from the presence of interfering compounds contained in the matrix [13,14]. Nilsson and Eklund estimated that approximately 20% of the total time for analysis is spent on the preparation and analysis of cali bration standards [17]
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