Quantification of free polyamines and their metabolites in biofluids and liver tissue by UHPLC-MS/MS: application to identify the potential biomarkers of hepatocellular carcinoma

Abstract

Polyamines and their N-acetylated metabolites are potential biomarkers in the diagnosis and therapeutic evaluation of cancer. Thus, we present here an ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of 6 free, 3 monoacetylated, and 2 diacetylated polyamines without derivatization. The major improvement of this method is the use of 0.2 % perfluoroheptanoic acid methanol in the pretreatment step to achieve protein precipitation and 0.0125 % perfluoroheptanoic acid in the mobile phase to achieve analyte separation within 9 min. The established analytical method was validated with plasma, urine, and liver tissue and applied to determine plasma, urine, and liver tissue samples from healthy rats, hepatocellular carcinoma rats, and administrated rats successfully. Results indicated free polyamines such as putrescine mainly existed in liver tissue but more polar N-acetylated metabolites such as N (1),N (12)-diacetylspermine seemed to exist in biological fluid. After carcinogenesis, the levels of polyamines were increased, but the elevated levels of polyamines and their metabolites tended to decrease when administrated with anticancer drug. The method provided a more versatile manner for clinical application in the diagnosis and therapeutic evaluation of hepatocellular carcinoma.

Spermidine-Activated Satellite Cells Are Associated with Hypoacetylation in ACVR2B and Smad3 Binding to Myogenic Genes in Mice

“…The concentrations of polyamine in plasma were determined according to previous publications, with some modifications. , The standards of spermine (85605, sigma, ≥ 99.5% AT, Shanghai, China), spermidine (85578, sigma, ≥ 99.5% AT, Shanghai, China), and putrescine (P7505, sigma, ≥ 98% TLC, Shanghai, China) were purchased from Sigma. Ultra high-performance liquid chromatography (UPLC) was performed with a reverse-phase column (ACQUITY UPLC BEH C18 Column 2.1 × 100 mm 2 , 1.7 μm, 186002350, Waters, Massachusetts, USA), and the column temperature was set at 21 °C.…”

Gender-Related Differences on Polyamine Metabolome in Liquid Biopsies by a Simple and Sensitive Two-Step Liquid-Liquid Extraction and LC-MS/MS

“…In this sense, free polyamines can be defined as the extractable polyamines without a hydrolysis step. Nowadays, most of the current research focused on biomarker discovery in biological samples pays more attention to free polyamines and related metabolites [12]. Various analytical methods have been developed for the determination of polyamines in liquid biopsies, such as serum or urine samples.…”

“…Some authors have used gas chromatography-mass spectrometry (GC-MS) [13,14], enzyme-linked immunosorbent assays (ELISAs) [15], and liquid chromatography with fluorescence detection (LC-FLD) [16,17] among other methods, but the preferred method for polyamine analysis is liquid chromatography-mass spectrometry (LC-MS). Focusing on the LC-MS methods, the direct determination of polyamines is difficult due to their small size and aliphatic structure; however, it has been achieved mainly with the use of ion-pairing reagents, such as heptafluorobutyric acid [4,[18][19][20][21] or perfluoroheptanoic acid [12]. The use of ion-pairing reagents is not advisable due to potential problems with the analysis in the LC-MS system since it is well known that these reagents cause ion source contamination and signal suppression when using electrospray ionization; this is in addition to causing a reduction in the analytical column performance [22].…”

“…Another highly used option is the use of an organic solvent for protein precipitation, such as methanol (MeOH) or acetonitrile (ACN). MeOH is the preferred option when ion-pairing chromatography is used [4,12,18,19,21], but it has also been used with derivatizing agents [30,31]. ACN is also commonly used before derivatization with diverse reagents [24,28,29,32,33].…”

Elevated Polyamines in Saliva of Pancreatic Cancer

“…We previously observed a change in metabolite concentration in salivary samples collected from PC patients [ 28 ]. Among metabolites, polyamines in non-invasively available biofluids have been reported as possible biomarkers of various cancers [ 29 , 30 , 31 ]. Elevation of urinary polyamines and the positive correlation between their concentrations in PC tissue is well known [ 32 ].…”