**Background:** Advanced Glycation End-products (AGEs) are stable compounds formed from reactions between reducing sugars and free amino groups in proteins, lipids, or nucleic acids. They accumulate with age and are implicated in diseases like diabetes, atherosclerosis, uremia, Alzheimer's disease, and cataracts. Carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) are two key AGEs closely linked to aging and diabetic complications. Accurate quantification methods are needed for clinical research. This study aimed to develop a rapid, sensitive LC-MS/MS method for measuring CML and CEL in human plasma and to evaluate their association with aging in a large clinical sample.
**Methods:** Human plasma samples (50 μL) were incubated with 5 mmol/L perfluorovaleric acid (NFPA), precipitated with trichloroacetic acid, hydrolyzed with 6 mol/L HCl at 110°C for 20 hours, dried under nitrogen, and reconstituted in NFPA. Cotinine-D3 was used as internal standard. Separation used an Agela Venusil ASB C18 column (50 mm × 4.6 mm, 5 μm) with a mobile phase of 5 mmol/L NFPA and acetonitrile/water (60:40 v/v with 0.15% formic acid) in gradient elution over 6 minutes. Detection was by triple quadrupole mass spectrometry in positive electrospray ionization mode with multiple reaction monitoring (MRM). Ion pairs were m/z 205.2>84.1 (quantitative) and 205.2>130.0 for CML; m/z 219.1>84.1 (quantitative) and 219.1>130.1 for CEL; and m/z 180.1>80.1 for cotinine-D3. The method was validated for linearity, lower limit of quantification (LLOQ), accuracy, precision, recovery, and specificity. A total of 1196 clinical plasma samples from the Institute of Geriatrics, Beijing Hospital, were analyzed. Samples were grouped by age: youth (≤44 years), middle-aged (45-59 years), elderly (60-74 years), and advanced age (≥75 years). ANOVA and Pearson correlation were used to assess differences and associations with age.
**Key Results:** The method achieved linearity from 0.025 to 1.500 μmol/L for both CML and CEL, with LLOQ of 0.025 μmol/L. Intra-day and inter-day relative standard deviations (RSD) were below 9%, and relative errors (RE) within ±4%. Average recoveries were 94.24% for CML and 97.89% for CEL. Retention times were 3.43 min for CML, 3.46 min for CEL, and 4.50 min for cotinine-D3. In clinical samples, for males, mean CML values were: youth 0.96±0.41 μmol/L (n=120), middle-aged 0.85±0.44 μmol/L (n=166), elderly 1.10±0.59 μmol/L (n=213), advanced age 1.11±0.81 μmol/L (n=115) (F=7.38, p<0.001). For females, CML values were: youth 0.98±0.39 μmol/L (n=134), middle-aged 0.96±0.47 μmol/L (n=138), elderly 1.01±0.5 μmol/L (n=201), advanced age 1.11±0.53 μmol/L (n=106) (F=2.47, p=0.06). For CEL in males: youth 0.46±0.20 μmol/L, middle-aged 0.37±0.20 μmol/L, elderly 0.50±0.28 μmol/L, advanced age 0.51±0.32 μmol/L (F=9.75, p<0.001). For females: youth 0.39±0.19 μmol/L, middle-aged 0.34±0.18 μmol/L, elderly 0.44±0.27 μmol/L, advanced age 0.50±0.30 μmol/L (F=10.22, p<0.001). Pearson correlation with age for males: CML r=0.135 (p=0.001), CEL r=0.122 (p=0.002); for females: CML r=0.081 (p=0.053), CEL r=0.170 (p<0.001). After age 40, average increase per decade was 9.06% for CML (10.89% in males, 7.39% in females) and 8.00% for CEL (8.56% in males, 7.76% in females).
**Clinical Implications:** This validated LC-MS/MS method provides a rapid (6 min), sensitive, and reliable tool for quantifying CML and CEL in human plasma, requiring only 50 μL of sample. The large-scale analysis of 1196 samples demonstrates that both CML and CEL levels increase significantly with age, particularly after age 40, with average decadal increases of about 9% and 8%, respectively. These biomarkers may serve as useful indicators for assessing aging-related glucose metabolism changes and overall health status. The sex-specific differences (CML more sensitive in males) suggest potential for personalized monitoring. However, aging is multifactorial, and these markers should be combined with other clinical indicators for comprehensive risk assessment. Further research is needed to validate their clinical utility and understand underlying mechanisms.