An isotopologue-mixture-based internal standard strategy for LC-MS/MS quantification in plasma

Stable isotope-labeled internal standards (SIL-IS) are the gold standard for liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification in complex biological matrices. Their application can be limited by synthetic complexity, high cost, and the lack of commercially available labeled standards for many bioactive analytes. Here, we establish and evaluate an internal-standard strategy based on hydrogen-deuterium exchange (HDX)-derived deuterated isotopologue mixtures. Using Pd/C, Pt/C, and AlCl_₃ catalytic systems, deuterated isotopologue mixtures (d_₁-d_ₙ) of eight structurally diverse bioactive compounds were prepared by HDX in deuterated methanol and characterized by ^¹H NMR and LC-MS. Isotopologues with a mass difference of Δm ≥ 3 were selected as internal-standard candidates to minimize overlap with analyte M + 1/M + 2 isotopic peaks. Isotopic-distribution screening and chromatographic evaluation showed that eligible d_₃-d_ₙ isotopologues co-eluted with their corresponding analytes (Δt < 0.01 min), with no appreciable chromatographic isotope effect (CIE). In plasma, LC-MS/MS validation showed linearity over 1-500 ng/mL (R^² ≥ 0.995), limits of detection of 0.003-0.080 ng/mL and limits of quantification of 0.01-0.25 ng/mL. Trueness ranged from 85% to 115%, while precision, expressed as relative standard deviation (RSD), was ≤8% intra-day and ≤12% inter-day. Ratio-based matrix effect (ME%) values ranged from 85% to 112%. Using clenbuterol hydrochloride as a model analyte, the isotopologue-mixture-derived internal standard showed calibration performance comparable to that of the commercial deuterated standard clenbuterol-d_₉. Compared with quantification based on external calibration without internal-standard normalization, the proposed approach reduced quantitative deviation from 15 to 25% to <8%. Overall, the results support HDX-derived isotopologue mixtures as practical internal-standard sources and establish a selection-and-validation framework for LC-MS/MS quantification when commercial SIL-IS are unavailable.