Analysis of Serum Exosome Metabolites Identifies Potential Biomarkers for Human Hepatocellular Carcinoma.

Currently, the clinical cure rate for primary liver cancer remains low. Effective screening and early diagnosis of hepatocellular carcinoma (HCC) remain clinical challenges. Exosomes are intimately associated with tumor development and their contents have the potential to serve as highly sensitive tumor-specific markers. A comprehensive untargeted metabolomics study was conducted using exosome samples extracted from the serum of 48 subjects (36 HCC patients and 12 healthy controls) via a commercial kit. An ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) strategy was used to identify the metabolic compounds. A total of 18 differential metabolites were identified using the non-targeted metabolomics approach of UPLC-QTOF-MS/MS. Pathway analysis revealed significant alterations in the arachidonic acid metabolism, linoleic acid metabolism, and unsaturated fatty acid metabolism pathways. ROC analysis indicated that three metabolites with AUC values exceeding 0.900 were selected as potential biomarkers: caprylic acid and linoleic acid were upregulated in the HCC group, whereas pentadecanoic acid was downregulated. Linoleic acid, caprylic acid, and pentadecanoic acid are potential biomarkers for diagnosing HCC. The significant alterations in these three metabolic pathways offer new insights into the mechanisms underlying HCC formation and progression.

Quantification and analyses of seven tyrosine kinase inhibitors targeting hepatocellular carcinoma in human plasma by QuEChERS and UPLC-MS/MS.

Tyrosine kinase inhibitors (TKIs) are commonly used to treat various cancers. Literature suggests that the blood concentration of TKIs strongly correlates with their efficacy and adverse effects. Therefore, establishing a Therapeutic Drug Monitoring (TDM) methodology for TKI drugs is crucial to improving their clinical efficacy and minimizing the treatment-related adverse effects. However, quantifying their concentrations in the plasma using existing methods to avoid potential toxicity is challenging. Herein, seven TKIs, namely sorafenib tosylate, axitinib, erlotinib, cediranib, brivanib, linifanib, and golvatinib, were successfully analyzed in human plasma by following a quick, easy, cheap, effective, rugged, and safe (QuEChERS) pretreatment method combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Briefly, biological samples were extracted using 1 mL of methanol, followed by the sequential addition of 250 mg of anhydrous magnesium sulfate and 25 mg of N-propylethylenediamine (PSA) for salinization and purification by adsorption, respectively. In this study, dovitinib was used as the internal standard. The seven TKIs were detected by the gradient elution method for 4 min in the positive ion electrospray mode. The mobile phase comprised methanol (phase A) and 0.1 % aqueous formic acid solution (phase B) on the Agilent Zorbax RRHD Stablebond Aq, (2.1 × 50 mm; 1.8 µm). Brivanib, linifanib, axitinib, sorafenib tosylate, and golvatinib exhibited good linearity in the range of 5-500 ng/mL, and erlotinib and cediranib exhibited good linearity in the range of 10-1000 ng/mL, with linear correlation coefficients (R2) ≥ 0.99. The limits of detection and quantification were 0.60-0.18 ng/mL and 5-10 ng/mL, respectively. The intraday and interday accuracy values ranged from -6.12 % to 7.31 %, with a precision (RSD) of ≤ 10.57 %. The method was rapid, accurate, specific, simple, reproducible, and suitable for the quantitative determination of the seven TKIs in human plasma.

Determination of nine cardiovascular drugs in human plasma by QuEChERS-UPLC-MS/MS.

To determine the concentrations of nine drugs used in the treatment of cardiovascular diseases in human plasma through QuEChERS pre-treatment combined with ultra-performance liquid chromatography-tandem mass spectrometry. Plasma samples were extracted with 3 mL of acetonitrile, 400 mg of anhydrous magnesium sulfate as a salting agent, and 20 mg of C18 as a sorbent. An Agilent Poroshell 120 EC-C18 column (4.6 × 100 mm, 2.7 µm) was selected and methanol-0.1 % water was used as the mobile phase, and ESI positive ion detection mode was selected. Results: The plasma concentrations of nisoldipine, metoprolol, and prazosin exhibited good linearity within the range of 0.05-4.0 ng/mL (r > 0.99), while atenolol, bisoprolol, propranolol, rosuvastatin, and atorvastatin showed linearity within the range of 0.5-40 ng/mL (r > 0.99). Fluvastatin showed good linearity within the range of 5.0-400 ng/mL. The accuracy of the method ranged from 94.15 to 110.62 %, while the recovery levels were in the range of 85.23 %-115.13 %. The matrix effects were observed between-6.54 % and 12.43 %. The intra-day and inter-day RSD was <15 % for the three concentrations of low, medium, and high. Conclusion The proposed method is rapid, accurate, specific, simple, reproducible, and suitable for the simultaneous measurement of the concentrations of nine drugs used in the treatment of cardiovascular diseases in human plasma.

QuEChERS-Based Approach to the Extraction of Five Calcium Channel Blockers from Plasma Determined by UPLC-MS/MS.

Here, a QuEChERS (quick, easy, cheap, effective, rugged, and safe) pretreatment method was combined with UPLC-MS/MS to facilitate the rapid and reliable simultaneous detection of five calcium channel blockers (CCBs) in human plasma. For this approach, samples were treated with 1 mL of acetonitrile, 350 mg of magnesium sulfate, and 70 mg of PSA adsorbent prior to centrifugation. Supernatants then underwent gradient elution for 8 min with an Agilent C18 column using an acetonitrile-water solution supplemented with 5 mmol⋅L-1 of ammonium acetate. This technique exhibited a good linear response in the 1-800 ng⋅mL-1 range for the analyzed drugs, with an R2≥ 0.9921, an accuracy of 87.54-113.05%, a matrix effect (ME) of 91.21-116.39%, a precision of 0.19-11.64%, and stability of no more than 10.05%. This time-saving QuEChERS reagent-based pretreatment technique thus allowed for the simultaneous and accurate detection of five CCBs in human plasma samples, providing a promising new basis for therapeutic drug monitoring in patients with hypertension.

Rapid Determination of 9 Tyrosine Kinase Inhibitors for the Treatment of Hepatocellular Carcinoma in Human Plasma by QuEChERS-UPLC-MS/MS.

A reliable and rapid method employing QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) pretreatment coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was successfully developed and validated for the analysis of nine tyrosine kinase inhibitors (TKIs) in human plasma. Biological samples were extracted with acetonitrile and salted out with 350 mg of anhydrous magnesium sulfate (MgSO4), followed by purification with 40 mg of ethyl enediamine-N-propylsilane (PSA) adsorbents. All analytes and internal standards (IS) were separated on the Hypersil GOLD VANQUISH C18 (2.1 mm × 100 mm, 1.9 µM) column using the mobile phases composed of acetonitrile (phase A) and 0.1% formic acid in water (phase B) for 8.0 min. Detection was performed by selection reaction monitoring (SRM) in the positive ion electrospray mode. Lenvatinib, sorafenib, cabozantinib, apatinib, gefitinib, regorafenib, and anlotinib rendered good linearity over the range of 0.1-10 ng/ml, and 1-100 ng/ml for tivantinib and galunisertib. All linear correlation coefficients for all standard curves were ≥ 0.9966. The limits of detection (LOD) and the limits of quantitation (LOQ) ranged from 0.003 to 0.11 ng/ml and 0.01-0.37 ng/ml, respectively. The method was deemed satisfactory with an accuracy of -7.34-6.64%, selectivity, matrix effect (ME) of 90.48-107.77%, recovery, and stability. The proposed method is simple, efficient, reliable, and applicable for the detection of TKIs in human plasma samples as well as for providing a reference for the clinical adjustment of drug administration regimen by monitoring the drug concentrations in the plasma of patients.