Title: Determination of polyamine metabolome in plasma and urine by ultrahigh performance liquid chromatography–tandem mass spectrometry method: Application to identify potential markers for human hepatic cancer
Journal: Journal of Chromatography B
Published: 2013
Background
Polyamines are low molecular weight aliphatic amines that play essential roles in cellular processes, such as DNA/RNA stabilization, protein and nucleic acid synthesis, and regulation of cell growth and proliferation. Due to their involvement in these biological functions, polyamines have been recognized as important cancer biomarkers. During cancer development, the balance of polyamine biosynthesis and catabolism can be disrupted, leading to altered levels of polyamine metabolome in body fluids. This study aims to develop a UHPLC–MS/MS method to simultaneously quantify polyamine-related metabolites in plasma and urine, to explore their potential as biomarkers for early diagnosis and treatment of hepatic cancer.
Materials & Methods
1. Chemicals and Reagents:
Reference standards of polyamines and related metabolites were obtained from Sigma–Aldrich, including 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, agmatine, N-acetylated polyamines, amino acid precursors, and γ-aminobutyric acid (GABA). 1,6-Diaminohexane was used as the internal standard (IS). Methanol (HPLC grade) was purchased from Fisher Chemicals, and heptafluorobutyric acid (HFBA) from Sigma–Aldrich. All other reagents were of analytical grade. Water used was redistilled and deionized.
2. Biological Samples:
Plasma and urine samples were collected from 20 hepatic cancer patients and 20 age-matched healthy volunteers (equal gender distribution). All samples were stored at −80 °C until analysis. Cancer diagnoses were confirmed via clinical and pathological methods.
3. Instrumentation and Chromatographic Conditions:
Liquid chromatography was performed using a Shimadzu UFLC system with a Shim-pack XR-ODS column (75 mm × 3.0 mm, 2.2 μm). Column temperature was maintained at 30 °C. Mobile phases were 0.05% HFBA in water (A) and methanol (B), with a gradient elution profile over 9 minutes. The flow rate was 0.4 mL/min, and the injection volume was 5 μL.
Mass spectrometric detection was carried out using a QTRAP 4000 system (AB Sciex) with TurboIonSpray in positive ion mode. Multiple reaction monitoring (MRM) was used for detection. Parameters such as DP, EP, CE, and CXP were optimized for each analyte.
4. Preparation of Standards and Calibration Curves:
Stock solutions were prepared in methanol:water (20:80, v/v), stored at 4 °C, and diluted as working standards. Calibration samples were prepared by spiking blank plasma or urine with analytes at various concentrations. Quality control (QC) samples were prepared at low, medium, and high concentrations using pooled plasma or urine.
5. Sample Preparation:
Each 250 μL plasma or urine sample was mixed with 50 μL IS solution and 50 μL methanol:water (20:80), vortexed, deproteinized using methanol with 0.1% acetic acid, centrifuged, and dried under nitrogen. The residue was reconstituted in methanol–water with 0.05% HFBA (20:80) and injected into the UFLC-MS/MS system.
6. Method Validation:
The method was validated based on US-FDA and related guidelines for:
- Linearity & LOQ: Calibration curves were linear with LOQs between 0.125–31.25 ng/mL.
- Precision & Accuracy: Evaluated via intra- and inter-day analysis at three QC levels, within acceptable limits (±15%).
- Recovery & Matrix Effect: Recovery >50%; matrix effects assessed using IS-normalized matrix factor (MF).
- Dilution Integrity: Confirmed by diluting high-concentration samples with blank matrix, maintaining accuracy and precision.
- Stability: Assessed under different conditions, including room temperature (4 h), freeze–thaw (3 cycles), long-term storage (−80 °C for 1 month), and post-preparation stability (12 h at 4 °C).
Results
Chromatographic and Mass Condition Optimization:
Optimization of chromatographic and mass spectrometric conditions was crucial for accurate polyamine metabolite detection. The addition of heptafluorobutyric acid (HFBA) improved retention and peak symmetry by forming ion pairs with cationic compounds and minimizing column interactions. The optimal HFBA concentration of 0.05% in both methanol and water provided the best retention and response. All analytes were effectively separated and detected within 9 minutes.
Sample Preparation:
Due to their high polarity and water solubility, polyamines were extracted using protein precipitation. The best recovery was achieved with methanol containing 0.1% acetic acid. This method minimized interference from endogenous substances, enabling direct detection by mass spectrometry.
Method Validation:
The method was validated following US-FDA and EMA guidelines. Calibration curves were linear with regression coefficients from 0.9904 to 0.9973. The limits of quantification (LOQ) were determined for both plasma and urine. Precision and accuracy tests showed the method's reliability. Absolute recoveries were over 50%, and matrix effects were minimal. Stability tests confirmed the analytes remained stable under various conditions, including multiple freeze-thaw cycles.
Method Application:
The method was applied to plasma and urine samples from hepatic cancer patients and healthy controls. In plasma, putrescine and spermidine levels were significantly higher in cancer patients, suggesting their potential as plasma biomarkers. In urine, agmatine, N-acetylspermidine, spermine, and spermidine were more abundant in cancer patients, highlighting their potential as urine biomarkers. The metabolism of putrescine to N-acetylspermidine was more pronounced in cancer, further supporting the relevance of these metabolites.
Determination of polyamine metabolome in plasma and urine by UHFLC-MS/MS method
The representative MRM chromatograms obtained from standard polyamines (A), plasma of cancer sufferers (B) and healthy volunteers (C) by UHPLC–MS/MS using the optimized method.
The representative MRM chromatograms obtained from standard polyamines (A), urine of cancer sufferers (D) and healthy volunteers (E) by UHPLC-MS/MS using the optimized method.
Reference
- Liu, Ran, et al. "Determination of polyamine metabolome in plasma and urine by ultrahigh performance liquid chromatography–tandem mass spectrometry method: Application to identify potential markers for human hepatic cancer." Analytica chimica acta 791 (2013): 36-45. http://dx.doi.org/10.1016/j.aca.2013.06.044
