Abstract
As an important regulatory factor in the process of embryo culture, amino acids are necessary components of in vitro fertilization medium (IVFM). The accurate determination of their content is of great significance to assisted reproductive technology (ART). The determination of 20 amino acids in IVFM samples with easy pre-treatment is established using the high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method. Chromatographic separation is achieved on a pentafluorophenyl column (150 mm × 2.1 um × 3 um) in a gradient elution mode with acetonitrile and water (both containing 0.1% formic acid) as the mobile phase at a flow rate of 0.2 mL/min. The column temperature is maintained at 40 ℃. All targets are resolved and identified based on the precursor ion, product ion, and retention time in a 12 min run time. The method is evaluated by linearity, limit of detection (LODs), limit of quantitation (LOQs), reproducibility, and precision according to the Chinese pharmacopeia based on the optimized conditions. The results demonstrate that the linear relationships of 20 amino acids are kept in the range of 0.1–2.0 mg/L with a correlation coefficient r ≥ 0.996. The LODs and LOQs are in the range of 0.83–66.67 and 2.76–222.22 ug/L, respectively. Recoveries (n = 3) of 95.9 – 118.8% are obtained at spiking of 0.4, 1.0 and 2.0 mg/L in quality control samples. Intra-day relative standard deviation (RSD) of these spiked samples is in the range of 0.03–6.82% with average relative error (RE) ranging from 0.35% to 18.07%, and the inter-day accuracy and precision results are within the acceptable range (RE ≤ 16.36%, RSD ≤ 11.25%). The proposed method is applied to test the real IVFM samples, and the measured values are within the labeled range, demonstrating its suitability for the quantification of amino acids in IVFM.
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Qin, Sj., Zheng, Gc., Li, F. et al. Simultaneous Determination of Twenty Amino Acids in In Vitro Fertilization Medium by the HPLC–MS/MS Method. Chromatographia 85, 643–654 (2022). https://doi.org/10.1007/s10337-022-04169-5
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DOI: https://doi.org/10.1007/s10337-022-04169-5