Glycogen is a multibranched polysaccharide of glucose produced by cells to store energy and plays a key role in cancer. A previously reported fluorescent probe (CDg4) was shown to selectively bind glycogen in mouse embryonic stem cells, however the molecule was not evaluated in cancer cells. We report the synthesis and biological evaluation of a dual-modality imaging probe based on CDg4, for positron emission tomography (PET) and fluorescence microscopy.[18F]5) for in vivo quantification of total glycogen levels in cancer cells was developed and synthesised in 170 min with a non-decay corrected radiochemical yield (RCY n.d.c) of 5.1 ± 0.9% (n = 4) in > 98% radiochemical purity. Compound 5 and [18F]5 were evaluated in vitro for their potential to bind glycogen, but only 5 showed accumulation by fluorescence microscopy. The accumulation of 5 was determined to be specific as fluorescent signal diminished upon the digestion of carbohydrate polymers with α-amylase. PET imaging in non-tumour bearing mice highlighted rapid hepato-biliary-intestinal elimination of [18F]5 and almost complete metabolic degradation after 60 min in the liver, plasma and urine, confirmed by radioactive metabolite analysis.A fluorine-18 radiolabelled derivative of CDg4, (5 selectively accumulated in glycogen containing cancer cells, identified by fluorescence microscopy; however, rapid in vivo metabolic degradation precludes further investigation of [18F]5 as a PET radiopharmaceutical.Fluorescent compound  Compound 3 was reacted with 4′-aminoacetophenone (6) in an aldol condensation reaction to give chalcone 4 which was challenging to purify and resulted in a low yield (16%). Structural identity of the E-isomer was confirmed using NMR by their characteristic J-values (15.4 Hz). Compound 5 was produced by reacting acryloyl chloride with 4 to install the terminal acrylamide moiety. The photochemical properties (excitation: λex and emission: λem) of 5 were evaluated by UV-Vis and fluorescence spectroscopy  was boc-protected (7) and reacted with 4-((2-hydroxyethyl)(methyl)amino) benzaldehyde to yield chalcone 8 (30% yield). The final radiochemistry precursor was produced by converting the hydroxyl moiety into a tosylate leaving group (9) for subsequent radiochemistry development. All compounds were characterised by 1H-NMR, 13C-NMR, 19F-NMR and mass spectrometry  was synthesised in a three-step radiosynthesis and is fully described in the Methods section and ESI. Compound [18F]5 was produced in a non-decay corrected radiochemical yield (RCY n.d.c) of 5.1 ± 0.9% (n = 4) in 170 min with a molar activity of 7.6 GBq/μmol. The LogD7.5 of [18F]5 was determined experimentally using the shake-flask method and found to be 1.03 ± 0.37 . Cells showed a concentration-dependent (with respect to compound 5) accumulation of fluorescence  in the cell lines with lowest uptake, and even higher for 786-O, MCF7, T47D and HCT116, where uptake was 1.4–2.3 fold higher . It was expected that [18F]5 uptake would correlate with total glycogen level however, despite stark differences in uptake in some cell lines, no correlation (R2 = 0.03) with total glycogen was observed  , biodistribution and in vivo metabolism of ing Fig. a. [18F]5i.) Fig. b, c; alt[18F]5 was determined in liver, plasma and urine by analytical radio-HPLC at 60 min p.i. A single polar radioactive metabolite (> 99%) was observed in all measured samples except for liver where < 3% of parent [18F]5 remained Target recognition - maintaining the pharmacophore of CDg4 to ensure a fluorine-18 radiolabelled derivative would have similar biological properties.ii)Fluorescence - retention of the push-pull electron system to maintain similar fluorescence properties to CDg4.iii)PET radionuclide - incorporation of a fluorine atom into a synthetically accessible location to allow for simple fluorine-18 radiochemistry.To develop a dual fluorescence/PET imaging probe based on the structure of CDg4, three important properties were considered:To satisfy all three properties, the terminal hydroxyl group of CDg4 was selected as a suitable location to incorporate a fluorine atom. This modification avoided change to the chalcone core structure and was hypothesised to retain similar glycogen binding properties as CDg4. Lee et al.  had prev5, a fluorine-containing derivative of CDg4, was synthesised in four steps and was used as a fluorescent probe for visualising glycogen by fluorescence microscopy and as a HPLC reference standard to confirm the identity of the fluorine-18 radiolabelled analogue ([18F]5). Compound 5 and CDg4 exhibited similarity in photochemical properties, determined by UV-Vis and fluorescence spectroscopy, which inferred that replacing the hydroxyl group with fluorine had minimal impact on the electron push-pull pairs of the molecule ; therefore, we also expect the quantum yield to be similar to CDg4 (Φ = 0.2). The λex and λem wavelengths were used for the biological evaluation of 5 by fluorescence microscopy.Compound [18F]5, a radiochemistry precursor was synthesised. The terminal hydroxyl moiety of compound 8 was converted into a good leaving group by tosylation, producing a boc-protected precursor (9) for simple SN2 displacement with [18F]fluoride. Producing [18F]5 in a single-step from an appropriate radiochemistry precursor was avoided as the acryloyl group was identified as potentially sensitive to the basic conditions and high temperatures required for the fluorine-18 radiochemistry. The boc-protected precursor (9) allowed effective radiolabelling and installation of the acryloyl moiety post-fluorination in a three-step radiosynthesis. In brief, [18F]10 was synthesised from precursor 9 via nucleophilic substitution (SN2) by displacement of the tosylate leaving group with anhydrous [18F]fluoride. Intermediate [18F]10 was purified from unreacted [18F] fluoride using a tC18 solid-phase extraction (SPE) cartridge and eluted in acetonitrile for further reactions. To reduce radiation exposure, the drying of [18F] fluoride and the first radiolabelling step was automated using the GE FASTlab™ platform  was > 98%, produced in an acceptable RCY (n.d.c) of 5.1 ± 0.9% and molar activity (Am = 7.6 GBq/μmol).To access the fluorine-18 radiolabelled compound 5 and [18F]5 was evaluated in vitro by fluorescence microscopy and PET to determine their ability to bind glycogen in cell-based studies. The in vivo biodistribution and metabolic stability of [18F]5 was evaluated in mice and was conducted with the intention of identifying a promising molecule for preclinical evaluation, and further, clinical translation into humans.The cellular uptake of compound 5 to bind glycogen determined by fluorescence microscopy; three of which  exhibited higher glycogen storage compared to HCT116 which was used as a negative control. As our interests were predominantly in developing a glycogen targeted PET probe, only [18F]5 was evaluated in all 11 cancer cell lines. Compound 5 accumulated in cells in a concentration-dependent manner, and the fluorescent signal was diminished when the cells were treated with α-amylase to digest carbohydrate polymers, suggesting the accumulation of 5 was specific to glycogen. The lowest fluorescence intensity was observed in HCT116 cells which was consistent with the measured glycogen levels. This data supports the observations made by Lee et al.  and that the retention of [18F]5 in cells, and the large difference in uptake between cell lines (1.4–2.3 fold difference), may be due to non-specific interactions; perhaps facilitated by the formation of covalent bonds between [18F]5 and cellular components  through the acrylamide moiety, a potent Michael acceptor. This functional group has been used in the design of covalent irreversible receptor inhibitors.The in vitro uptake of radiolabelled els Fig. . The ave5 in cells was specific to a carbohydrate polymer, the evaluation of [18F]5 did not yield comparable results. The difference in the in vitro performance of 5 and [18F]5 may have resulted from the so-called “mass effect”; the quantity of fluorescent probe 5 (lowest at 4 μM) was 40-fold larger in comparison to PET probe [18F]5 present at ca 100 nM (determined from Am), which may have been sufficient to overcome non-specific reactivity towards cellular components. The use of [18F]5 at lower Am  to replicate the mass of compound used in fluorescent experiments was not performed as the translation of low molar activity tracers into clinical imaging is not routine. A similar phenomenon has been exemplified for covalent epidermal growth factor receptor (EGFR) inhibitors which, despite many being permeability glycoprotein (PGP) substrates, are still effective drugs when high doses are administered , suggesting that retention within cells was based on a non-specific mechanism.Despite promising fluorescence microscopy data, which suggested that the accumulation of [18F]5 showed no correlation to cellular glycogen, the high uptake warranted further investigation into the PK, biodistribution and metabolic fate of this molecule. This understanding may inform the design of new [18F]5 analogues that effectively bind glycogen and are suitable candidates for pre-clinical and clinical translation. Compound [18F]5 rapidly accumulated in the liver within 10 min p.i. followed by the intestines, gallbladder and eventually the bladder after 30 min p.i. suggesting hepato-biliary-intestinal clearance. Rapid clearance is advantageous for tumour imaging by improving the signal-to-background ratio, provided the tumour is not located in the abdominal region.Although [18F]5 in the plasma, liver and urine showed almost complete in vivo metabolism within 60 min into a more polar species. Although the structure of the main metabolite was not elucidated, it is possible that metabolic liability of [18F]5 arose from the highly reactive acrylamide moiety either through a putative glutathione conjugation, or more generally, the formation of covalent bonds with cellular components (i.e. biomolecules)  and investigate its properties as a dual-modality fluorescence/PET imaging probe for glycogen in a panel of cancer cell lines. The fluorine-18 radiolabelled [18F]5 was synthesised in an acceptable RCY n.d.c. (5.1 ± 0.9%), molar activity (Am: 7.6 GBq/μmol) and excellent RCP > 98%. Compound 5 accumulated in glycogen-containing cells visualised by fluorescence microscopy, and the signal was diminished by enzymatic digestion of carbohydrates using α-amylase; however, no correlation between the in vitro uptake of [18F]5 and total glycogen level in a panel of cancer cell lines was observed. Evaluation of [18F]5 in vivo showed the accumulation of radioactivity in the gallbladder and intestines and rapid metabolism in 60 min. The investigation of suitable radiolabelled molecules for binding glycogen, without the inclusion of a potent Michael acceptor moiety is ongoing.We report the development of fluorine-containing analogue of CDg4 (254 320–400 mesh). Thin Layer Chromatography (TLC) was performed on Merck aluminium-backed plates pre-coated with silica  which were visualised by quenching of ultraviolet fluorescence (λ = 254 and 365 nm). 1H-NMR, 13C-NMR and 19F-NMR was obtained using a Bruker AV-400 spectrometer at a frequency of 400, 101 and 376 MHz, respectively. Chemical shifts (δ) are given in parts per million (ppm) and referenced to the appropriate residual solvent peaks. Signals are assigned as s, d, t, dt, m and br for singlet, doublet, triplet, double triplet, multiplet and broad respectively. Mass spectrometry was performed by the Mass Spectrometry Facility of the Chemistry Department of Imperial College London. Excitation (λex) and emission (λem) spectra were obtained using an Infinite 200 PRO plate reader .Anhydrous solvents and reagents were purchased from Sigma Aldrich  and were used without additional purification. N-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde was purchased from abcr GmbH . Flash column chromatography purification was performed on silica gel (Merck Kieselgel 60 FN-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde  and TEA  in dry DCM (10 mL) was added dropwise 4-toluenesulfonyl chloride  in DCM (5 mL). The reaction mixture was stirred under N2 at room temperature overnight, after which time it was concentrated in vacuo. The resulting material was purified by silica gel column chromatography  to provide the product  as a pale yellow solid. 1H NMR  δ 9.75 , 7.94–7.54 , 7.34–7.05 , 6.59 , 4.21 , 3.72 , 3.01 , 2.39 . 13C NMR  δ 190.36, 152.76, 145.26, 132.62, 132.10, 129.99, 127.92, 126.12, 111.25, 66.57, 51.07, 39.45, 21.79. HRMS (ESI) = 334.1120 (M + H)+. Calc. for C17H20NO4S: 334.1113.To a solution of the 2 at 100 °C for 2 h, followed by cooling to room temperature and concentration in vacuo. The residue was taken up in DCM (20 mL), washed with NH4Cl (30 mL) and brine (30 mL), dried over Na2SO4. The organic layer was filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (100% DCM) to afford the product  as a yellow solid. 1H NMR  δ 9.64 , 7.63 , 6.63 , 4.53 , 3.65 , 3.02 . 13C NMR  δ 190.25, 153.29, 132.03, 125.63, 111.15, 81.49 , 52.16 , 39.17. 19F NMR  δ − 222.55 (m). HRMS (ESI) = 182.0988 (M + H)+. Calc. for C10H13NO4F: 182.0981.To a solution of 2-((4-formylphenyl)(methyl)amino)ethyl-4-methylbenzenesulfonate  in dry THF (15 mL) was added dropwise TBAF . The reaction mixture was stirred under N1H NMR  δ 7.92 , 7.75 , 7.53 , 7.36 , 6.77–6.64 , 4.61 , 3.70 , 3.08 . 13C NMR  δ 188.43, 150.82, 150.43, 143.88, 130.91, 130.32, 129.29, 123.76, 117.38, 114.02, 111.98, 81.77 , 52.44 , 39.23. 19F NMR  δ − 222.25 (m). HRMS (ESI) = 299.1571 (M + H)+. Calc. for C18H20N2OF: 299.1560.To a stirred solution of N-Methyl-N-(2-fluoroethyl)-4-aminobenzaldehyde  and 4′-aminoacetophenone  in EtOH (20 mL) was added 4 M NaOH (0.5 mL) at RT. The reaction mixture was stirred at 50 °C for 16 h. The solution was then cooled and neutralised with cold 1 M HCl, followed by in vacuo removal of bulk solvent. The resulting material was purified by silica gel column chromatography  to provide the product  as an orange solid. 4  and TEA  in acetonitrile (2 mL) was added the acryloyl chloride  at RT and stirred for 2 h. After concentration in vacuo, the residue was purified by silica gel chromatography (1% MeOH in DCM) to provide the product  as a yellow solid. 1H NMR  δ 8.02 , 7.78 , 7.73 , 7.55 , 7.35 , 6.70 , 6.48 , 6.30 , 5.82 , 4.63 , 3.72 , 3.10 . 13C NMR  δ 189.35, 163.80, 150.81, 145.56, 141.64, 134.90, 130.99, 130.69, 129.88, 128.82, 123.37, 119.36, 117.07, 112.02, 81.76 , 52.46 , 39.30. 19F NMR  δ − 222.31 (m). HRMS (ESI) = 353.1659 (M + H)+. Calc. for C21H22N2O2F: 353.1665.To a mixture of compound 2 at RT for 16 h, after which time it was concentrated in vacuo. The resulting material was purified by silica gel column chromatography  to provide the product  as a pale white solid. 1H NMR  δ 7.91 , 7.45 , 6.70 , 2.56 , 1.53 . 13C NMR  δ 197.00, 152.27, 143.02, 132.02, 129.99, 117.54, 81.46, 28.42, 26.52. HRMS (ESI) = 236.1287 (M + H)+. Calc. for C13H18NO3: 236.1287.To a solution of the 4′-aminoacetophenone  and TEA  in dry DCM (20 mL) was added di-tert-butyl dicarbonate  in DCM (5 mL). The reaction mixture was stirred under NN-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde  in EtOH (5 mL) at RT. The reaction mixture was stirred at 50 °C for 16 h, followed by in vacuo removal of bulk solvent. The resulting material was purified by silica gel column chromatography  to provide the product  as a pale yellow solid. 1H NMR  δ 7.97 , 7.74 , 7.49 , 7.31 , 6.88 , 6.73 , 3.84 , 3.55 , 3.04 , 1.53 . 13C NMR  δ 189.25, 152.43, 151.47, 145.29, 142.47, 133.48, 130.57, 129.94, 123.23, 117.66, 116.88, 112.14, 81.28, 60.24, 54.70, 39.08, 28.41. HRMS (ESI) = 397.2113 (M + H)+. Calc. for C23H29N2O4: 397.2127.To a stirred solution of compound 7  was added NaOH (10 mg) and 8  was dissolved in anhydrous pyridine (8 mL) under N2. The solution was stirred at 0 °C and 4-toluenesulfonyl chloride  in pyridine (1 mL) was added slowly. Reaction was stirred for 4 h at 0 °C and then at RT for 16 h. After completion of the reaction, the mixture was partitioned between EtOAc (15 mL) and H2O (15 mL). The organic layer was washed with H2O (2 × 10 mL), 1 M HCl solution (2 × 10 mL), brine (2 × 10 mL) and dried over Na2SO4. The solvent was removed in vacuo. The resulting residue was purified by silica gel column chromatography  to provide the product  as a pale yellow solid. 1H NMR  δ 7.98 , 7.73 , 7.66 , 7.51 , 7.44 , 7.33 , 7.25–7.17 , 7.13 , 6.52 , 4.16 , 3.63 , 2.91 , 2.35 , 1.50 . 13C NMR  δ 188.95, 152.44, 149.97, 145.07, 144.82, 142.66, 133.22, 132.51, 130.35, 129.86, 129.83, 127.79, 123.46, 117.61, 117.14, 111.79, 81.05, 66.80, 50.91, 39.09, 28.33, 21.67. HRMS (ESI) = 551.2218 (M + H)+. Calc. for C30H35N2O6S: 551.2210.Compound 5 was freshly prepared in DMSO at the indicated concentrations of 0.01, 0.1, 1, 10 and 50 μM. Each concentration was assayed in triplicate in a 96-well dark plate using a plate reader . Absorption spectra were first determined, where the absorbance was plotted against the measured wavelength ranging from 380 nm to 460 nm. The wavelength of maximum absorption was used as the excitation wavelength to obtain emission (fluorescence) spectra of compound 5. All spectra shown in ESI.Compound [18F]5 was partly automated using the GE FASTLab™ platform. An automated sequence was developed  of K222 (12 mg/mL) and KHCO3 (18 mg/mL) respectively. The [18F] fluoride was dried by evaporation at 120 °C under vacuum and a flow of nitrogen for 8 min then 70 °C for 5 min. To the dry fluoride was added precursor 9 (1.6 mg) in MeCN (1.2 mL) and the reactor sealed and heated to 90 °C for 20 min, after which the reactor was cooled. The crude reaction mixture containing intermediate [18F]10 was expelled into an external dilution vial containing H2O (25 mL) and loaded through a preconditioned tC18 SPE cartridge [conditioned with 5 mL MeCN and 10 mL H2O prior to the experiment]. The cartridge was subsequently washed with H2O (10 mL), dried under nitrogen for 60 s, and the radioactive product eluted into a Wheaton vial with MeCN (1 mL) and analysed by analytical radio-HPLC  and heated to 80 °C for 15 min. Hydrolysis of the boc-protecting group to yield [18F]4 was confirmed by analytical radio-HPLC analysis  and trapped on a preconditioned tC18 SPE cartridge [conditioned with 5 mL MeCN and 10 mL H2O prior to the experiment]. The cartridge was washed with H2O (10 mL) and dried under the vigorous flow of nitrogen for 5 mins. Compound [18F]4 was eluted into a dry Wheaton vial using anhydrous MeCN (1 mL) followed by the addition of acryloyl chloride (50 μL), the vessel was heated to 50 °C for 15 min and reaction progress monitored by analytical radio-HPLC  and purified by semi-preparative HPLC (mobile phase: 60% MeCN/ 40% H2O / 0.1% TFA) with the desired product eluting at tR = 11 min. The desired product was cut into H2O (30 mL) and trapped on a preconditioned tC18 SPE [conditioned with 5 mL EtOH and 10 mL H2O prior to the experiment]. The final product ([18F]5) was eluted in EtOH (0.5–1 mL) into a clean vial for biological evaluation. Identity of [18F]5 was confirmed by radio-HPLC analysis and co-elution with the authentic reference compound 5  was used in conjunction with an Agilent Zorbax XDB C18 column . The retention time of parent compound [18F]5 was determined by injecting a radioactive sample onto the metabolite radio-HPLC system. The liver was excised, and a portion homogenised in ice cold MeCN (1 mL) using a Precellys tissue homogeniser fitted with the Cryolys cooling module . Solid tissues and protein were pelleted by centrifugation  and the supernatant was removed and filtered (0.22 μm syringe filter) before being diluted in H2O + 0.1% TFA for radio-HPLC analysis. Urine was diluted in H2O + 0.1% TFA and filtered prior to radio-HPLC analysis. Plasma was obtained from whole blood by centrifugation  to separate the blood cells from the plasma; the plasma was removed and precipitated in ice cold MeCN (1 mL) and centrifuged  to pellet the proteins. The supernatant was filtered (0.22 μm syringe filter) and diluted in H2O + 0.1% TFA for radio-HPLC analysis. The HPLC injection loop (1 mL) was washed with mobile phase between each injection. The efficiency of extracting radioactivity from each plasma and liver sample was determined by counting the activity  in a small aliquot (20 μL) of the supernatant of a known volume and the whole protein pellet, in a λ-counter . The extraction efficiency of radioactivity from plasma and liver was 73.7 ± 2.5 and 45.2 ± 4.3% of total activity, respectively. Radio-HPLC chromatograms were integrated using Laura 6 software .Liver, urine and blood plasma were analysed for radioactive metabolites by radio-HPLC (Agilent 1100 system) fitted with an in-line posiRAM metabolite detector . An isocratic mobile phase , MCF-7 , T47D , MDA-MB-231 , HCT116 , 786-O , RCC4 plus vector alone , RCC4 plus VHL , U87 shCtrl and U87 shPYGL  were cultured in DMEM media (Sigma-Aldrich) supplemented with 10% foetal calf serum (FCS), 1% L-glutamine and 2% penicillin-streptomycin. IGROV-1  was maintained in RPMI 1640 media (Sigma-Aldrich) with the same supplements as described above. All cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO2SO4 and absolute ethanol, was kept in fridge overnight to induce glycogen precipitation. On the next day, the mixture was centrifuged and re-incubated with 70% ethanol for 30 min to purify the precipitation. After the last centrifuge, glycogen precipitates were dissolved in deionised water and ready for glycogen assay. Amyloglucosidase was firstly added to break down glycogen into glucose. Then the resulted glucose was measured using a Glucose (GO) assay kit (Sigma) according to manufacturer’s instructions. Assay reagent was added to samples and incubated at 37 °C for 45 min. 12 N sulfuric acid (H2SO4) was added to terminate the reaction and absorbance was measured at 540 nm using a standard microplate reader. Glycogen levels in unknown samples were determined based on the glycogen standard curve and normalised to total intracellular protein as measured by BCA.Cells were each seeded at an appropriate density and allowed to attach overnight. After 24 h, cells were trypsinised, centrifuged and washed to obtain pellets. Glycogen was extracted by boiling cell pellets in 30% KOH for 15 min. The mixture, in the presence of 2% Na5 was added to cells and incubated at 37 °C for 1 h in dark. Hoechst 33342 (2 μg/mL) was used as nuclear staining. After 1 h incubation, cells were gently washed twice with PBS and images were obtained using a standard fluorescence microscope (Olympus BX51). For glycogen digestion, fresh media containing 0.5 mg/mL of α-amylase was added to cells after the 1 h-staining. Control cells were incubated in normal media for 15 min, in the absence of α-amylase.At 24 h after seeding, fresh media containing 4 μM or 8 μM of compound [18F]5 for 1 h at 37 °C in a humidified condition of 5% CO2. Cells were then washed three times with ice-cold PBS and lysed in 1 mL of RIPA buffer for 10 min on ice. The radioactivity of 800 μL lysate from each sample was counted on a WIZARD2 Automatic Gamma Counter. Data were expressed as a percentage of radioactivity incorporated into cells, normalised to total cellular protein as measured by BCA assay.Cells were seeded at each appropriate density and allowed to attach for 24 h. On the day of uptake, cells were washed three times with pre-warmed PBS and incubated with 1 mL of fresh DMEM media containing approximately 0.74 MBq [18F]5 via a lateral tail vein cannula. Imaging was performed under 2% isoflurane/O2 anaesthesia. After tracer injection, emission scans were acquired in list-mode format (over 0–60 min - dynamic scans) to give decay-corrected values of radioactivity accumulation in tissues. The collected data were reconstructed with a 3-dimensional maximum likelihood estimation method 3D ML-EM (Sofie Biosciences). Cumulative images of the data were used for visualization of radiotracer uptake and to define tissue volumes of interest (VOIs) using Siemens Inveon Research Workplace software . Tissue radioactivity uptake values were normalized to injected dose and mouse weight.For PET imaging, BALB/c mice  were anesthetized and scanned on a dedicated small animal PET scanner  following a bolus injection of 1.48 MBq of Data were expressed as mean values ± standard deviation (SD). Correlation analysis was determined using GraphPad Prism v.6.Additional file 1 Materials and methods; 1H, 13C and 19F NMR data; UV-Vis spectra; detailed radiochemistry and automation methods for the synthesis of [18F]5; UV-HPLC and radio-HPLC chromatograms.


The aim of the present study was to compare two methods, C0 (through level) and AUC(0–12) (area under the concentration time curve), for assessing cyclosporine and tacrolimus concentrations. The study included 340 kidney recipients. The AUC(0–12) was estimated using a Bayesian estimator and a three-point limited sampling strategy. Therapeutic drug monitoring of tacrolimus performed by using AUC(0–12) and C0 showed that tacrolimus in most cases is overdosed when considering C0, while determination of the AUC(0–12) showed that tacrolimus is effectively dosed for 27.8–40.0% of patients receiving only tacrolimus and for 25.0–31.9% of patients receiving tacrolimus with MMF (mycophenolate mofetil). In the 1–5 years post-transplantation group, 10% higher CsA (cyclosporine) dose was observed, which was proportionate with a 10% higher AUC(0–12) exposure value. This indicates good compatibility of the dosage and the AUC(0–12) method. The Bland–Altman plot demonstrated that C0 and AUC(0–12) might be interchangeable methods, while the ROC (receiver operating characteristic) curve analysis of the C0/AUC(0–12) ratio in the tacrolimus-receiving patient group demonstrated reliable performance to predict IFTA  after kidney transplantation, with an ROC curve of 0.660 : 0.576–0.736), p < 0.01. Moreover, AUC(0–12) and C0 of tacrolimus depend on concomitant medication and adjustment of the therapeutic range for AUC(0–12) might influence the results.The AUC (area under the concentration time curve) is considered the pharmacokinetic exposure parameter best associated with clinical effects. Unfortunately, no prospective studies of clinical outcomes have been conducted in adult transplant recipients to investigate properly the potential benefits of AUC There were significant differences in C0, C2, and the calculated AUC after shifting to single daily dosing of cyclosporine [Due to the narrow immunosuppressants’ therapeutic range, highly variable pharmacokinetics, inter-individual differences, and a tendency of interactions mainly occurring during the metabolism, which are cytochrome P450 3A4 (CYP3A4)-mediated therapeutic window and fairly common adverse effects ,2, prescosporine , which losporine .(0–12) monitoring compared to the C0-guided therapy. In the present study, two pharmacokinetic methods, the C0 and the AUC(0–12), for assessing cyclosporine and tacrolimus concentrations were analyzed, as well as these methods’ ability to contribute to determining the risk of kidney alteration in long-term treated patients.The AUC is considered the pharmacokinetic exposure parameter best associated with clinical effects. Unfortunately, no prospective studies of clinical outcomes have been conducted in adult and pediatric transplant recipients to investigate properly the potential benefits of AUCAnonymized medical records of 817 patients receiving immunosuppressant therapy after renal transplantation hospitalized at the Limoges University Hospital (France) during the study period from 2011 to 2012 were reviewed. Brief patient screening is available in Renal transplant recipients aged from 19 to 83 years who underwent immunosuppressant (cyclosporine or tacrolimus) monitoring at the university hospital of Limoges during a 1-year period were included in the study.The inclusion criteria were age of more than 18 years, stable kidney transplant, and immunosuppression with either cyclosporine or tacrolimus. Patients were excluded if they received immunosuppression with other medicaments and/or underwent transplantation of other organs. All patients received prednisolone orally according to the standard hospital practice. Protocol biopsies were taken to identify interstitial fibrosis and tubular atrophy (IFTA) .The work described was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).For PK sampling, patients were admitted to hospital. After a first blood sampling for trough concentration, approximately 12 h after the evening dose, the fasting patients were given cyclosporine/tacrolimus and mycophenolate mofetil (if not monotherapy) doses at a precise time by the nurse. Then, blood samples were collected at 60 min ± 15 min and 180 min ± 30 min after administration into EDTA-containing vacutainers.0) and drug blood concentration 1 (C1) and 3 (C3) hours after the use of a CsA dose. CsA whole blood concentrations were measured using a validated turbulent flow chromatography–tandem mass spectrometry technique [−1 using Cyclone P®, a 50 μm particle size  column , in alkaline conditions. Chromatographic separation was performed in acidic conditions  using Propel MS C18, a 5 μm  column (Thermo Fisher) kept at 60 °C with a constant flow rate of 300 μL/min−1. Detection was performed using a TSQ Quantum Discovery tandem mass spectrometer equipped with an orthogonal electrospray ionization source and controlled by the XCalibur software (Thermo Fisher). Tandem mass spectrometry detection was performed in the positive ion multiple reaction monitoring (MRM) mode following 3 transitions for cyclosporine (m/z 1220.0→1203.0 for quantification and m/z 1220.0→1185.0 and m/z 1220.0→425.0 for confirmation) and 2 transitions (m/z 1234.0→1217.0 for quantification and m/z 1234.0→119.0 for confirmation) for its analog cyclosporine D used as an internal standard (IS). Methanol/aqueous zinc sulphate  containing the internal standard at 25 μg/L−1 was added to the whole blood (100 μL). The mixture was vortex-mixed for 45 s, centrifuged at 13,000 rpm, and the supernatant was introduced into a 200 μL vial for injection. Calibration standards at 0, 10, 20, 50, 100, 200, 500, 1000, and 2000 μg/L−1 of CsA were prepared by spiking blank blood. The limits of detection (LOD) and quantification (LOQ) were 10 μg/L−1 and 20/μg L−1, and the calibration curves obtained using quadratic regression from the LOQ to 2000 μg/L−1 yielded r2 > 0.99.The blood samples were collected in EDTA (ethylenediaminetetraacetic acid) tubes to measure the CsA trough level  column  in alkaline conditions. Chromatographic separation was performed in acidic conditions using Propel MS C18, a 5 mm  column  heated to 60 °C, with a constant flow rate of 300 mL/min−1. Detection was performed using a TSQ Quantum Discovery MS/MS system  equipped with an orthogonal electrospray ionization source and controlled by the XCalibur software (Thermo Fisher). MS/MS detection was performed in the positive ion, multiple reaction monitoring mode following two transitions for tacrolimus  and two for the internal standard ascomycin .Tacrolimus was determined using a previously reported validated turbulent flow chromatography–tandem mass spectrometry (TFC–MS/MS) method . Briefly−1 and the calibration curves obtained from the lower limit of quantification up to 100 mg/L−1 yielded r2 > 0.998. The method was found to be accurate and precise with the bias from −4.4% to 0.6% and a low coefficient of variation from −3.8% to 6.4% [This method was fully validated for tacrolimus determination in whole blood. The calibration curves using a 1/x weighted quadratic regression were used to obtain the best fit across the calibration range, based on the standard error of the fit and minimization of the calibrator’s bias. The lower limit of quantification was 1 mg/L to 6.4% .® version VI  nonlinear mixed-effects population pharmacokinetic model and the Bayesian estimator of a three-point limited sampling strategy developed at the Limoges University Hospital were used to determine cyclosporine [(0–12)). Briefly, concentration data obtained in de novo or stable renal transplant patients were fitted using a one-compartment open model with first-order elimination combined with a gamma model of absorption with two parallel absorption routes; or a two-compartment open model with first-order elimination and an Erlang absorption model. Based on these PK models, several maximum a posteriori Bayesian estimators allowing the determination of individual PK parameters and AUC estimation were developed using the iterative two-stage Bayesian approach and/or nonlinear mixed-effects modeling. For each of them, either external or internal validation  was performed before any clinical use.The NONMEMosporine ,13 and tosporine  area undThe G∗Power 3.1.9.4 version was used to calculate the sample size. Statistical test, MANOVA , with effect size of f2(V) = 0.07 was used. The total calculated sample size was 136 patients with an actual power of 0.95 for each group: cyclosporine-receiving patients and tacrolimus-receiving patients.(0–12) and C0) of CsA and Tacro were assessed (compliance within the therapeutic ranges) and compared between the patients’ groups. The unpaired t-test was used to compare the study groups . Probability values of less than 0.05 were considered significant. GraphPad was used for scatter plots and MedCalc—for ROC analyses. ROC analyses were performed looking for cut-off values.Statistical analysis was performed using IBM SPSS 20.0. Pharmacokinetic parameters  exposure or C0 values outside the determined therapeutic range were evaluated as non-compliance in this study. Therapeutic range of the AUC(0–12) exposure was 120–150 µg/h/L for tacrolimus and 3.05–3.75 mg/h/L and 2.70–2.98 mg h/L for cyclosporine post-transplantation, respectively. The therapeutic range of C0 was 4–6 µg/L and 3–5 µg/L for tacrolimus 1–5 years and >5 years post-transplantation, respectively; and 75–150 µg/L for cyclosporine. Therapeutic ranges of AUC(0–12) and C0 are summarized in Tacrolimus or cyclosporine AUCIn total, 340 kidney recipients were enrolled: 196 kidney recipients received cyclosporine (CsA) and 144 kidney recipients received tacrolimus (Tacro), post-transplantation time > 1 year, 2 BID (twice (two times) a day) regimens.Tacrolimus-receiving patients’ group consisted of 144 patients: 116 patients were tacrolimus- and MMF-treated, and 28 patients were tacrolimus-treated only. Patient age varied from 19 to 75 years, mean: 53.31 ± 12.31 (SD) years. All patients were stable kidney recipients with post-transplantation time > 1 year  years). The Tacro dose varied from 1.0 to 13.0 mg/day, mean: 4.99 ± 2.40 (SD) mg/day. These patients were divided into four following groups depending on the post-transplantation time (1–5 years after transplantation and >5 years after transplantation) and the medication they received (Tacro + MMF or Tacro monotherapy). For the detailed information, see (0–12) exposure and C0 values were assessed in these groups. The gauge was compliance within the therapeutic range. The summarized data of the AUC(0–12) exposure compliance within the therapeutic range in the patients receiving tacrolimus is provided in 0 compliance within the therapeutic range in the patients receiving tacrolimus is provided in 0 demonstrated that Tacro C0 values were not in compliance within the established therapeutic ranges and Tacro was prescribed in high doses or overdosed. Compliance within the therapeutic range rates (60.0%) was only observed in the patients on tacrolimus monotherapy (post-transplantation time: 1–5 years).AUC(0–12) assessment (120–150 µg h/L) in all groups of patients receiving Tacro, while there are several therapeutic ranges for the assessment of C0. Therapeutic ranges of C0 depend on the post-transplantation time. The initial treatment doses were higher and the therapeutic width was in a higher range for early post-transplant stage patients. The dose of the immunosuppressive drug was reduced and the therapeutic width was adjusted within switching to maintenance treatment. Therapeutic ranges for both AUC(0–12) and C0 assessments are given in To note, there is only one therapeutic range for the AUCThe cyclosporine-treated patients’ group consisted of 196 subjects: 147 patients were cyclosporine- and MMF-treated, and 49 patients were cyclosporine-treated only. Patient age varied from 22 to 83 years, mean: 58.26 ± 13.85 (SD) years. All patients were stable kidney recipients with post-transplantation time > 1 year  years). The CsA dose varied from 60 to 400 mg/day, mean: 185.00 ± 52.53 (SD) mg/day. These patients were divided into four following groups depending on the post-transplantation time (1–5 years after transplantation and >5 years after transplantation) and the medication they received (CsA + MMF or CsA monotherapy). The baseline data of each cyclosporine-treated group are provided in (0–12) and C0 within the therapeutic ranges of AUC(0–12) and/or C0. To assess CsA concentrations in blood and the calculated AUC(0–12), the previously determined therapeutic latitudes for the AUC(0–12) exposure were used [(0–12) targeting to 3.8 mg/L.The evaluation included cyclosporine dosing, compliance and non-compliance rates of AUCere used  instead (0–12) demonstrated higher compliance within therapeutic range rates in the patients receiving only tacrolimus (27.8–40.0%) versus the patients receiving tacrolimus together with MMF, where compliance rates were 25.0–31.9%. Low dosing was more often observed in the patients receiving only tacrolimus (44.4–60.0%), while higher rates of overdosing were observed in the patients receiving tacrolimus with MMF (29.5–36.1%)  .(0–12) exposure below the range, which was possibly related to low tacrolimus dosing in the maintenance state and higher transplanted organ rejection risk. This risk remained increased regardless of whether patients received Tacro monotherapy or Tacro with MMF (44.4% vs. 45.5%). The patients receiving only Tacro with grafts aged 1–5 years were at an even higher organ rejection risk (60.0%). The results are presented in Low tacrolimus dosing is often associated with transplanted organ rejection. This study’s data showed that around 45% of the patients with grafts older than 5 years had AUC0 compliance within the therapeutic range. The most accurate dosing was observed in the patients with grafts aged 1–5 years receiving Tacro monotherapy (60.0%). Cases of tacrolimus overdosing or cases when tacrolimus C0 values were above the therapeutic range were observed in the patients with grafts aged 1–5 years receiving Tacro with MMF (81.9%); in patients with grafts aged > 5 years receiving Tacro + MMF or only Tacro—93.2% and 88.9%, respectively. The results are presented in Slightly different results were obtained while analyzing the C0 assessment presented more cases of Tacro overdosing than of accurate dosing, while the AUC(0–12) assessment presented more cases of Tacro overdosing or low dosing. The results are presented in The data showed that the C(0–12) exposure values in different groups was 146.97 ± 53.04 µg/h/L in patients receiving Tacro with MMF (post-transplantation time: 1–5 years) and 113.90 ± 17.95 µg/h/L in patients receiving Tacro monotherapy (post-transplantation time: 1–5 years); 129.73 ± 36.70 µg/h/L in patients receiving Tacro with MMF (post-transplantation time > 5 years) and 129.72 ± 38.09 µg h/L in patients receiving Tacro monotherapy (post-transplantation time > 5 years) ((0–12) values’ distribution from the median in Tacro-receiving study groups (median ± SE): 134.00 ± 6.251 µg/h/L  versus 110.50 ± 5.675 µg/h/L ; 128.50 ± 5.532 µg/h/L  versus 124.50 ± 8.978 µg/h/L . An independent sample t-test showed that the data for the groups receiving tacrolimus with MMF or only tacrolimus statistically significantly differ in patients with the post-transplantation time of 1–5 years. These results suggested that Tacro AUC(0–12) statistically significantly depends on the concomitant medication used with Tacro, either MMF or no MMF .The distribution of mean AUC5 years) . Data di5 years)  displayi0 values were as follows: 8.23 ± 2.81 µg/L  versus 6.12 ± 1.47 µg/L ; 7.23 ± 2.14 µg/L  versus 7.54 ± 2.67 µg/L  : 7.62 ± 0.33 µg/L  versus 5.77 ± 0.46 µg/L ; 6.88 ± 0.32 µg/L  versus 7.16 ± 0.63 µg/L .Mean Tacro Ctherapy) . Data ditherapy)  displayit-test results of C0 were compared in two different post-transplantation time groups: post-transplantation time of 1–5 years and post-transplantation time > 5 years. Patients were receiving either Tacro with MMF or Tacro monotherapy. Results of the independent sample t-test showed a statistically significant difference between mean C0 values in the post-transplantation time of 1–5 years groups, p = 0.023 (p < 0.5). These results confirm that the C0 value might depend on the concomitant medication used with Tacro, while no statistically significant difference between mean C0 values in the post-transplantation time > 5 years groups was not noticed, p = 0.635 (p > 0.5).Independent sample 0 statistically significantly differed between the groups, p = 0.022 (p < 0.05)  .Patients using high medication doses  are at higher risk of nephropathy, especially when the medication is nephrotoxic, like tacrolimus. Non-compliance was associated with the increased risk of interstitial fibrosis and tubular atrophy. The results are presented in 0 enabled to identify a larger number of subjects with interstitial fibrosis and tubular atrophy (IFTA) than the assessment of AUC(0–12). IFTA was obtained in 17 out of 72 (23.6%) patients receiving Tacro + MMF (post-transplantation time: 1–5 years). Non-compliance was obtained in 13 out of 17 (76.5%) patients with C0 > 6 µg/L. The assessment of AUC(0–12) enabled to identify 4 out of 17 patients (23.5%) with AUC(0–12) > 150 µg/h/L. Similar results were obtained in other tacrolimus-receiving patients’ groups (The assessment of C’ groups .IFTA was observed in 34 (23.6%) patients out of 144 patients. ROC curve analyses were performed to identify the cut-off values of increased risk of Tacro-induced IFTA.(0–12) sensitivity and specificity for IFTA. According to this cut-off, 18 (12.50%) patients’ AUC(0–12) values were below this threshold, and in 11 (61.10%) of these cases, IFTA was observed, while among the 126 patients with the AUC(0–12) value > 95.00 µg/h/L, 23 (18.30%) corresponded to IFTA. This cut-off corresponds to a sensitivity of 32.35% and a specificity of 93.64%.A cut-off of 95.00 µg/h/L was the optimal one to maximize AUC0 sensitivity and specificity for IFTA. According to this cut-off, 43 (29.86%) patients’ C0 values were below this threshold, and in 14 (32.60%) of these cases, IFTA was observed, while among the 101 patients with the C0 value > 6.21 µg/L, 20 (19.80%) corresponded to IFTA. This cut-off corresponds to a sensitivity of 41.18% and a specificity of 73.64%.A cut-off of 6.21 µg/L was the optimal one to maximize C0/AUC(0–12) ratio’s sensitivity and specificity for IFTA. According to this cut-off, 84 (58.33%) patients’ C0/AUC(0–12) ratios were below this threshold, and in 9 (10.70%) of these cases, IFTA was observed, while among the 60 patients with the C0/AUC(0–12) ratio > 0.059, 25 (41.67%) corresponded to IFTA. This cut-off corresponds to a sensitivity of 73.53 % and a specificity of 70.00%.A cut-off of 0.059 was the optimal one to maximize the C(0–12), C0, and the C0/AUC(0–12) ratio represented by the area under the receiver operator curve (AUROC) in kidney recipients with IFTA. Discrimination of the C0/AUC(0–12) ratio showed reliable performance to predict IFTA after kidney transplantation, with an ROC curve of 0.660 : 0.576–0.736), p < 0.01. However, ROC curves of AUC(0–12) and C0 were not sufficiently informative for IFTA diagnosis (p > 0.05).(0–12) demonstrated higher compliance within therapeutic range rates in the patients receiving only CsA (23.9–66.7%) versus the patients receiving CsA together with MMF, where compliance rates were 20.8–29.4%. Low dosing was more often observed in the patients receiving CsA together with MMF (35.4–51.0%), while higher rates of overdosing were observed in the patients receiving only CsA (47.8%)  .(0–12) exposure values were below the range possibly had a higher transplanted organ rejection risk. This risk was increased in the patients who received either CsA monotherapy or CsA with MMF (35.4% vs. 51.0%). The results are presented in Low CsA dosing, as well as low tacrolimus dosing is often associated with transplanted organ rejection. Patients whose AUC0 compliance within the therapeutic range. Compliance rates between the groups were quite similar and ranged from 63.0% to 71.9%. The most accurate dosing was observed in patients with grafts aged 1–5 years receiving CsA together with MMF (71.9%). Cases of CsA overdosing or cases where CsA C0 values were above the therapeutic range were observed in patients with grafts aged 1–5 years receiving CsA with MMF (19.6%) and in patients with grafts aged > 5 years, with an expected overdose of CsA of 5.2% (CsA + MMF) and 19.6% (CsA), respectively. The results are presented in Slightly different results were obtained while analyzing the C(0–12) assessment was more related with CsA overdosing or low dosing than with accurate dosing, while the C0 assessment was related with accurate CsA dosing with compliance rates of 63.0–71.9%. The results are presented in The data showed that the AUC(0–12) exposure values in different groups was 3.31 ± 0.94 mg/h/L in patients receiving CsA with MMF (post-transplantation time: 1–5 years) and 2.96 ± 0.79 mg/h/L in patients receiving CsA monotherapy (post-transplantation time: 1–5 years); 3.01 ± 0.96 mg/h/L in patients receiving CsA with MMF (post-transplantation time > 5 years) and 2.98 ± 0.70 mg/h/L in patients receiving CsA monotherapy (post-transplantation time > 5 years) ((0–12) values’ distribution from the median in the CsA-receiving study groups (median ± SE): 3.02 ±0.13 mg/h/L  versus 3.56 ± 0.55 mg/h/L ; 2.84 ± 0.80 mg/h/L  versus 2.91 ± 0.10 mg/h/L .The distribution of mean AUC5 years) . Data di5 years)  displayit-test showed that the data statistically significantly differed between the groups receiving CsA with MMF. The patients having grafts aged 1–5 years received 10% higher CsA doses versus the patients with grafts aged > 5 years (204.41 ± 53.82 mg vs. 183.18 ± 50.14 mg) . Accordingly, the obtained mean C0 and AUC(0–12) values statistically significantly differed. The mean AUC(0–12) exposure value was 10% higher in the patients with grafts aged 1–5 years than in the patients with grafts aged > 5 years (3.31 ± 0.94 mg/h/L vs. 2.97 ± 0.79 mg/h/L) , while the mean C0 value was 23% higher in the patients with grafts aged 1–5 years versus the patients with grafts aged > 5 years (128.55 ± 51.84 µg/L vs. 97.90 ± 33.23 µg/L) (p < 0.05).The independent sample t-test results also showed that the CsA dose statistically significantly differed between patients’ groups: patients receiving CsA + MMF (post-transplantation time > 5 years) had 10% higher CsA doses than patients receiving CsA monotherapy (post-transplantation time > 5 years) (183.18 ± 50.14 mg vs. 164.13 ± 48.55 mg) ; patients receiving CsA monotherapy with post-transplantation time of 1–5 years had 30% higher CsA doses than patients receiving CsA monotherapy with post-transplantation time > 5 years (233.33 ± 28.87 mg vs. 164.13 ± 48.55 mg)  (The independent sample  < 0.05) . No othe0 values’ distribution from the median in CsA-receiving study groups (median ± SE): 120.00 ± 7.26 µg/L  versus 132.00 ± 24.23 µg/L ; 94.00 ± 3.39 µg/L  versus 101.50 ± 5.51 µg/L .Data distribution is presented in scatter plots  displayi0 and the CsA dose statistically significantly differed between the groups (p < 0.05)  .Non-compliance within the therapeutic range was associated with the appearance of IFTA. The results are presented in (0–12) enabled to identify a larger number of subjects with IFTA than the assessment of C0. IFTA was obtained in 20 out of 96 patients receiving CsA + MMF (post-transplantation time > 5 years). Non-compliance was obtained in 8 out of 20 (40.0%) patients with AUC(0–12) > 2.99 mg/h/L. At the same time, the assessment of C0 enabled to identify 2 out of 20 patients (10.0%) with C0 > 150 µg/h/L. Similar results were obtained in other CsA-receiving patients’ groups (The assessment of AUC’ groups .IFTA was observed in 39 (19.9%) patients out of 196 patients. ROC curve analyses were performed to identify the cut-off values of the increased risk of CsA-induced IFTA.(0–12) sensitivity and specificity for IFTA. According to this cut-off, 34 (17.35%) patients’ AUC(0–12) values were below this threshold, and in 11 (32.40%) of these cases, IFTA was observed, while among the 162 patients with the AUC(0–12) value > 2.34 mg/h/L, 28 (17.30%) corresponded to IFTA. This cut-off corresponds to a sensitivity of 28.21% and a specificity of 85.35%.A cut-off of 2.34 mg/h/L was the optimal one to maximize AUC0 sensitivity and specificity for IFTA. According to this cut-off, 136 (69.39%) patients’ C0 values were below this threshold, and in 24 (17.60%) of these cases, IFTA was observed, while among the 60 patients with the C0 value > 120 µg/L, 15 (20.00%) corresponded to IFTA. This cut-off corresponds to a sensitivity of 38.46% and a specificity of 71.34%.A cut-off of 120 µg/L was the optimal one to maximize C0/AUC(0–12) ratio’s sensitivity and specificity for IFTA. According to this cut-off, 122 (62.24%) patients’ C0/AUC(0–12) ratios were below this threshold, and in 20 (16.40%) of these cases, IFTA was observed, while among the 74 patients with the C0/AUC(0–12) ratio > 0.059, 19 (25.70%) corresponded to IFTA. This cut-off corresponds to a sensitivity of 48.72 % and a specificity of 64.97%.A cut-off of 36.755 was the optimal one to maximize the C(0–12), C0, and the C0/AUC(0–12) ratio represented by the area under the receiver operator curve (AUROC) in kidney recipients with IFTA. However, ROC curves of AUC(0–12), C0, and the C0/AUC(0–12) ratio were not sufficiently informative for IFTA diagnosis (p > 0.05).0 and AUC(0–12) difference correlated with either tacrolimus or cyclosporine exposure was evaluated. The data of Bland–Altman plot analyses are provided in p < 0.01) and interchangeable.T-test showed that the CMany transplantation centers around the world use therapeutic drug monitoring (TDM) in clinical practice as a tool to achieve a predetermined target—drug concentration and to balance between efficacy and toxicity . However(0–12) of CsA and Tacro, and to correlate these deviations with the treatment outcomes. In the study conducted by K.A. Barraclough et al., the relationship between tacrolimus exposure AUC(0–12) or C0 values and acute rejection was not observed. Although tacrolimus C0 values indicated in this study are sufficient and high, a large part of the subjects (38% on day 4 and 42% at 1 month) had the AUC(0–12) exposure below the lower limit of the target range (150–250 µg/h/L). K.A. Barraclough et al. associated these results with a poor or average correlation between AUC(0–12) and C0 or, consequently, with the inappropriate AUC(0–12) target range [It is important to assess the non-compliance within the therapeutic ranges of AUCet range . The res(0–12) exposure values was made using the standard therapeutic range. The differentiation of the therapeutic range of AUC(0–12) exposure values depending on the time after transplantation and the co-administered drug could possibly improve the results or lead to similar or better results than those obtained by assessing C0 compliance within therapeutic ranges.The results of this study also showed that attention should be drawn to the fact that the assessment of AUCThe potential interaction between tacrolimus and MMF was considered based on the results of scarce in vitro or clinical studies ,22,23. TIn MMF-treated organ transplant recipients, lower mycophenolic acid (MPA) plasma concentrations have been found in cyclosporine- compared with tacrolimus-based immunosuppressive regimens. CsA-mediated inhibition of the biliary excretion of MPAG (mycophenolic acid glucuronide) by the Mrp2 transporter is the mechanism responsible for the interaction between CsA and MMF .New research also shows that proposed tacrolimus AUC target levels need to be redefined due to the circadian variation and flat real-life non-fasting PK-profiles. The association between high-peak concentrations and side effects of tacrolimus may be overestimated given the flat real-life non-fasting PK profiles. The effect of real-life dosing of tacrolimus may very well be present for other drugs and should be investigated for drugs where TDM is indicated . K.A. BaThe research shows that low tacrolimus dosing that does not correspond to the therapeutic range is associated with an increased risk of organ rejection  As the cM.A. Halim et al. concluded that the CsA dose should be individualized in renal transplant recipients, especially if they have viral hepatitis and a single daily dosing of cyclosporine has the advantage of decreasing dosage and CsA-related adverse effects while maintaining optimal graft function .8), contrary to C0 [(0–12) calculated by the Bayesian method is a strong pharmacokinetic parameter [0/AUC ratio is proposed to identify those patients who are good candidates to analyze AUC-Tacro instead of C0 [Thus, the research carried out and the results of this study show that TDM is undoubtedly significant in reducing the risk of organ rejection or overdose. Recent studies besides the AUC calculated by the Bayesian method offer tacrolimus sampling in the elimination phase  and C0 showed that tacrolimus is in most cases overdosed when considering C0, while the determination of AUC(0–12) showed that tacrolimus is effectively dosed for 27.8–40.0% of the patients receiving only tacrolimus and for 25.0–31.9% of the patients receiving tacrolimus with MMF. In the 1–5 years post-transplantation group, a 10% higher CsA dose was observed, which was proportionate with a 10% higher AUC(0–12) exposure value. This indicates good compatibility of the dosage and the AUC(0–12) method.Precise dosage was more often observed in patients with grafts aged 1–5 years receiving either cyclosporine or tacrolimus monotherapy than in patients with grafts aged > 5 years and receiving several medicaments. Therapeutic drug monitoring of tacrolimus performed by using AUC0 and AUC(0–12) might be interchangeable methods, while the ROC curve analysis of the C0/AUC(0–12) ratio in the tacrolimus-receiving patient group demonstrated reliable performance to predict IFTA after kidney transplantation, with the ROC curve of 0.660 : 0.576–0.736), p < 0.01. Moreover, AUC(0–12) and C0 of tacrolimus depend on the concomitant medication and adjustment of the therapeutic range for AUC(0–12) might influence the results.The Bland–Altman plot demonstrated that C


This study aimed to investigate how prolonged storage of adult retinal pigment epithelial (ARPE-19) cell sheets affects cell metabolism, morphology, viability, and phenotype. ARPE-19 cell sheets were stored at three temperatures  for three weeks. Metabolic status and morphology of the cells were monitored by sampling medium and examining cells by phase-contrast microscopy, respectively, throughout the storage period. Cell viability was analyzed by flow cytometry, and phenotype was determined by epifluorescence microscopy after the storage. Lactate production and glucose consumption increased heavily, while pH dropped considerably, through storage at 37 °C compared to 4 °C and 16 °C. During storage, morphology started to deteriorate first at 4 °C, then at 37 °C, and was maintained the longest at 16 °C. Viability of the cells after three weeks of storage was best preserved at 16 °C, while cells stored at 4 °C and 37 °C had reduced viability. Dedifferentiation indicated by reduced expression of retinal pigment epithelium-specific protein 65 (RPE65), zonula occludens protein 1 (ZO-1), and occludin after three weeks of storage was noticed in all experimental groups compared to control. We conclude that storage temperature affects the metabolic status of ARPE-19 cells and that 16 °C reduces metabolic activity while protecting viability and morphology. The retinal pigment epithelium (RPE) is a monolayer of pigmented cells located between the neurosensory retina and the choroid ,2. Loss The cells can be delivered to the subretinal space by means of the cell suspension or as an RPE patch ,29. HoweThrough several studies ,34,35,362, and pCO2), the storage medium was sampled every alternate day. Lactate concentration was dramatically increased after storage at 37 °C (0.5–8.6 mmol/L), while it was only slightly increased after storage at 4 °C and 16 °C   A.2 was maintained in all temperature groups throughout the storage period   , while only a slight reduction was noted in cultures stored at 4 °C and 16 °C  B. The pHt 37 °C) D. The pCt 37 °C) E. The paTo study the morphological effects of storage duration at the three different storage temperatures, phase contrast photomicrographs were captured every alternate day. Prior to storage, cells were generally well apposed and showed typical ARPE morphology A,M,Y.At the end of the three-week storage period, cells stored at 16 °C showed a morphology most similar to the control. Signs of apoptosis (marked with a black arrow) and intercellular spacing  and 37 °C , but not at 16 °C , compared to the control (94% ± 1%) (p < 0.001), but not at 4 °C  or 16 °C , compared to the control (5% ± 1%) (p < 0.001), but not at 4 °C  or 16 °C , compared to the control (0.2% ± 0.2%) (Cell viability after three weeks of storage (defined as the percentage of cells that were annexin V- and PI-negative) was significantly reduced at 4 °C  B. Necrot5% ± 1%) C. Simila ± 0.2%) D. The vip = 0.011; 16 °C: 29% ± 7%, p < 0.001; 37 °C: 19% ± 8%, p < 0.001)  A,B.p = 0.035; 35% ± 10%, p = 0.0155 and 17% ± 8%, p = 0.0510 for 4, 16 °C and 37 °C, respectively). Occludin, another tight junction marker, was also significantly reduced following storage at all storage temperatures  compared to the non-stored control  compared to the control (p = 0.070) at 37 °C  after three weeks , Belgium. Minimum essential medium (MEM) was purchased from Invitrogen . Pipettes, 25 cm2 flasks, 15 mL and 50 mL centrifugation tubes, 1 L glass bottles, and pipette tips were supplied by VWR International . Vacuum filtration rapid filter mix was supplied by BioNordika . Mouse anti-RPE65, rabbit anti-occludin, FITC-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG antibodies were obtained from Abcam . Mouse anti-ZO-1 and Alexa Fluor 568 phalloidin were purchased from Life Technologies .ARPE-19 cells were obtained from the American Type Culture Collection (ATCC) . Dulbecco’s Modified Eagle’s Medium: nutrient mixture F12 (hereafter named DMEM:F12), fetal bovine serum (FBS), bovine serum albumin (BSA), trypsin-EDTA, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium bicarbonate, gentamycin, phosphate-buffered saline (PBS), penicillin, streptomycin, 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), Tween-20 and PAP pen were purchased from Sigma-Aldrich . Fluorescein isothiocyanate (FITC)-labeled annexin V (to bind PS), annexin V-binding buffer containing 10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2 at 37 °C in DMEM:F12 containing 10% FBS, 50 units/mL penicillin and 50 µg/mL streptomycin. The cells at passage 6 were seeded  in 25 cm2 culture flasks with filter closer. The culture medium was changed every other day, and confluent cultures were obtained on the sixth day. Control cultures, which were not subjected to subsequent storage, were immediately prepared for the various analyses.Human ARPE-19 cells were routinely cultured in 95% air and 5% CO2 supply. The storage containers have been described previously [After the six-days culture period, the T25 flasks were removed from the incubator, and the culture medium was replaced by a storage medium consisting of 9.53 g MEM, 25 mM HEPES, 600 mg/L sodium bicarbonate and 50 µg/mL gentamycin in 1 L distilled water. The filter cap of the flasks was changed to a solid cap to avoid evaporation during storage. Thereafter, the cultures were randomized for storage at three temperatures  for three weeks in storage containers without COeviously . The sta2) and partial pressure of carbon dioxide (pCO2). The experiment was repeated eight times (n = 8).Samples of the storage medium (2 mL) were taken every alternate day from day 1 to day 21 and were analyzed using a Radiometer ABL 700 blood gas analyzer  at room temperature. The following parameters were studied: pH, glucose, lactate, partial pressure of oxygen (pOn = 4). Photomicrographs were captured at 400× magnification using a Leica DM IL LED microscope and a Canon EOS 5D Mark II camera.Morphology of the stored ARPE-19 cell cultures was assessed by light microscopy every alternate day during the storage period. The experiment was repeated four times  using FITC-conjugated annexin V and PI. Annexin V binds selectively to phosphatidylserine , which is anchored at the cytosolic face of the plasma membrane in viable cells. During the early phases of apoptosis, phosphatidylserine is re-localized to the outer surface of the plasma membrane, where it can be detected with fluorescently labeled annexin V . Apoptotn = 4). Briefly, RPE cells were trypsinized and centrifuged at room temperature. The supernatant was aspirated from the cell pellet, which was resuspended in 200 µL of annexin V-binding buffer containing annexin V-FITC (1 µL/mL) and incubated for 25 min at room temperature. PI dye (10 µg/mL) was added for further 5-min incubation at room temperature before the suspension was analyzed using a flow cytometer.The analysis was performed according to the protocol provided by the supplier and repeated four times , anti-ZO-1 (1:50) and anti-occludin (1:50) antibodies were diluted in blocking solution (PBS with 1% BSA). In the negative controls, primary antibodies were substituted with PBS. Samples were incubated for one hour at room temperature. FITC-conjugated goat anti-mouse secondary antibodies (diluted 1:200 in blocking solution) and FITC-conjugated goat anti-rabbit secondary antibodies (1:250) were added for one hour at room temperature. Specimens were washed three times in PBS, and 1 µg/mL DAPI was added during the last wash to stain the nuclear DNA. To visualize the actin cytoskeleton, samples were fixed, blocked, and permeabilized as described above and stained with 100 units/mL, which is equivalent to approximately 20 µM Alexa Fluor 568 phalloidin. After incubating for 1 h at room temperature, specimens were washed in PBS and stained with DAPI.n = 4).The samples were studied using a Nikon Eclipse Ti fluorescence microscope and photographed at ×200 magnification with a DS-Qi1 black-and-white camera. Identical exposure length and gain were maintained for all compared samples, and the image brightness was within the dynamic range of the camera. The experiments were repeated four times  as described previously , with soP values below 0.05 were considered significant.A one-way analysis of variance with Tukey’s post hoc comparisons (SPSS ver. 19.0 or GraphPad prism 8.2.1) was used for statistical evaluation of the results.


Ulva sp., Caulerpa sp. and Codium sp.; Rhodophyta (red), Dasya sp., Grateloupia sp. and Centroceras sp.; Ochrophyta (brown), Ecklonia sp., Sargassum sp.], using liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS). The total phenolic content (TPC), total flavonoid content (TFC) and total tannin content (TTC) were determined. The antioxidant potential of seaweed was assessed using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, a 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radical scavenging assay and a ferric reducing antioxidant power (FRAP) assay. Brown seaweed species showed the highest total polyphenol content, which correlated with the highest antioxidant potential. The LC-ESI-QTOF-MS/MS tentatively identified a total of 54 phenolic compounds present in the eight seaweeds. The largest number of phenolic compounds were present in Centroceras sp. followed by Ecklonia sp. and Caulerpa sp. Using high-performance liquid chromatography-photodiode array (HPLC-PDA) quantification, the most abundant phenolic compound was p-hydroxybenzoic acid, present in Ulva sp. at 846.083 ± 0.02 μg/g fresh weight. The results obtained indicate the importance of seaweed as a promising source of polyphenols with antioxidant properties, consistent with the health potential of seaweed in food, pharmaceutical and nutraceutical applications.Seaweed is an important food widely consumed in Asian countries. Seaweed has a diverse array of bioactive compounds, including dietary fiber, carbohydrate, protein, fatty acid, minerals and polyphenols, which contribute to the health benefits and commercial value of seaweed. Nevertheless, detailed information on polyphenol content in seaweeds is still limited. Therefore, the present work aimed to investigate the phenolic compounds present in eight seaweeds [Chlorophyta (green),  Seaweed has been utilized as a food for humans for centuries, and the current global market is valued at more than USD 6 billion per annum with an annual volume of approximately 12 million tonnes in 2018 . Like plA promising bioactive property of polyphenols relates to their antioxidant activity and redox potential, allowing them to reduce the reactive oxygen species (ROS) that are involved in a range of human disorders . Strong Total phenolic, flavonoid and tannin contents in seaweed can be indirectly measured using assays for total phenolic content (TPC), total flavonoid content (TFC) and total tannin (TTC), respectively. The antioxidant activities of seaweed can be quantified using various assays based on different mechanisms, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays based on free-radical scavenged by antioxidant compounds, and ferric reducing of antioxidant power (FRAP) assay based on the reducing capacity of antioxidants . HoweverThe objectives of the current study were: (1) to extract phenolic compounds from a range of seaweeds; (2) quantify the total phenolic and antioxidant capacities of seaweed extracts using different assays and (3) apply LC-ESI-QTOF-MS/MS and HPLC-PDA to characterize and quantify individual phenolic compounds.Ecklonia sp. showed significantly higher TPC (1044 ± 2.5 μg GAE/gf.w.) and TTC (167 ± 23.2 μg CE/gf.w.) contents than other seaweed (p < 0.05). The presence of higher total phenolics in brown seaweed Ecklonia sp. compared to green seaweed Ulva sp. and red seaweed Porphyra sp. was previously observed by García-Casal, et al. −m/z at 169.0138 was only detected from red seaweed Centroceras sp., and characterized as gallic acid based on the product ion at 125 m/z, corresponding to the loss of CO2 (44 Da) from precursor ion [Himanthalia elongate [p-Hydroxybenzoic acid (Compound 5 with [M − H]− ion at m/z 137.0240) present in Ulva sp., Caulerpa sp. and Centroceras sp. was identified and confirmed by MS2 experiments  from the parent ion − at m/z 397.1144, with product ions at m/z 223 (sinapic acid ion) and m/z 179 (sinapic acid − COO) in Centroceras sp. and Ecklonia sp., which was previously characterized in extracts of arnica flower [8) was also found in Codium sp. and Ulva sp. The presence of cinnamoyl glucose was confirmed by a [M − H]−m/z at 309.0992, which yielded product ions at m/z 147, m/z 131 and m/z 103, indicating the expected loss of hexosyl moiety (162 Da), C6H10O6 (178 Da) and C7H10O7 (206 Da), respectively −m/z at 341.0882, was tentatively characterized as caffeoyl glucose and was present in Ecklonia sp. and Centroceras sp. The MS2 analysis showed the product ions at m/z 179 [M − H − 162] and m/z 161 [M − H − 180], consistent with losses of hexosyl moiety and further loss of H2O [14 was tentatively characterized as caffeoyl tartaric acid found in Grateloupia sp. and Centroceras sp. based on [M − H]−m/z at 311.0403. The identification was further supported by the MS2 spectrum, which exhibited typical product ion at m/z 161, formed by the neutral loss of 150 mass units as a result of tartaric acid fission [O-glucuronide found in Caulerpa sp. (Compound 10 with [M − H]−m/z of 355.0671), MS/MS fragmentation yielded the predominant ion at m/z 179 after the loss of glucuronide moiety (176 Da), indicating the presence of caffeic acid ion − at m/z 179.0350, 258.9929, 273.0086 and 193.0513, respectively. The identification of caffeic acid was confirmed by the product ions at m/z 151 [M − H − 28], m/z 143 [M − H − 36] and m/z 133 [M − H − 46], representing the loss of CO, two H2O units and HCOOH, respectively, from the precursor ion  and m/z 149 [M − H − SO3 − CO2], consistent with the presence of isoferulic acid ion (193 Da) and further loss of CO2 −m/z at 223.0621. In the MS2 spectrum of sinapic acid, the product ions at m/z 205, 179 and 163 were due to the loss of H2O (18 Da), CO2 (44 Da) and two CH2O units (60 Da) from the parent ion, respectively, which was comparable with the fragmentation rules of sinapinic acid [Sinapic acid (Compound nic acid .18 with [M − H]−m/z at 163.0406), yielding a main product ion at m/z 119, which corresponded to loss of CO2 (44 Da), was found in Caulerpa sp. [Coumaric acid  were detected in all seaweeds except Centroceras sp. and Codium sp. Compound (26) showing precursor ion [M − H]− at m/z 305.0668 in negative mode, was the most widely distributed flavanol and was identified as gallocatechin presenting in Caulerpa sp., Ulva sp., Dasya sp., Ecklonia sp. and Sargassum sp. The presence of gallocatechin derivatives in brown seaweed ascophyllum nodosum was reported by Agregán, Munekata, Franco, Dominguez, Carballo and Lorenzo  was due to the loss of CO2 and at m/z 219 [M − H − 86] was caused by the loss of C3O2 and H2O [O-methylcatechin (Compound 27 with [M − H]-m/z of 303.0886) was identified in Grateloupia sp. in the present study, with the product ions at m/z 271  and m/z 163  −m/z of 289.0731. The MS2 spectrum showed the product ions at m/z 245, m/z 205, and m/z 179, indicating the loss of CO2 (44 Da), flavonid A ring (84 Da) and flavonid B ring (110 Da) from the precursor ion, respectively −m/z of 329.0674. The further identification of 3,7-dimethylquercetin was achieved by comparing the previous study, which characterized the same compound from Ipomoea batatas leaves and showed the product ions at m/z 314, m/z 299 and m/z 271, corresponding to the loss of CH3 (15 Da), two CH3 (30 Da) and two CH3 plus CO unit from the precursor ion, respectively [Three flavonols were detected in negative mode in ectively .32 with [M − H]−m/z at 577.1588) was the only flavone identified in Centroceras sp. with the product ions at m/z 413 (M − H − 164) and m/z 269 (M − H − 308), representing the loss of rhamnose moiety and H2O (164 Da) and hexosyl moiety plus rhamnose moiety (308 Da) from the parent ion [Rhoifolin (Compound rent ion . This is33) was only detected in Ecklonia sp. in negative mode with [M − H]−m/z at 299.0918. The identity was confirmed by comparing the previous study which characterized sativanone in Dalbergia odorifera using LC-MS/MS, and the spectrum displayed the product ions at m/z 284  and at m/z 269  and at m/z 225  [37 with [M − H]-m/z at 267.0666 exhibited characteristic fragment ions at m/z 252 [M − H − CH3], m/z 224 [M − H − CH3 − CO] and m/z 180 [M − H − CH3 – CO − CO2] was identified as dalbergin −m/z at 227.0341 was assigned as compound 41, from Grateloupia sp. MS/MS identification by product ions at m/z 198  and 182 m/z  + at m/z 347.1843, was detected in Dasya sp., Ulva sp., Grateloupia sp., Ecklonia sp. and Codium sp. The product ions at m/z 301 and m/z 231 came from the loss of a unit of H2O and CO (46 Da), and cleavage of molecules pentene, water, and carbon monoxide −m/z at 331.1912, was found in Ecklonia sp. Dasya sp., Codium sp. and Sargassum sp. The molecular ion of carnosic acid (m/z 331.1912) produced the major fragment ion at m/z 287 and m/z 269, corresponding to the loss of CO2 and further loss of H2O from the parent ion − ions at m/z 315.1091. In the MS2 spectrum of hydroxytyrosol 4-O-glucoside, the typical loss of hexosyl moiety (162 Da) was observed from precursor, resulting in product ions at m/z 153 [47 with [M − H]−m/z at 319.1200 was only detected from Caulerpa sp., and characterized as 3,4-DHPEA-EDA based on the product ions at m/z 301, m/z 275 and m/z 195, corresponding to loss of H2O (18 Da), CO2 (44 Da) and C5H6(CHO)2 (124 Da) from the precursor ion [Compounds ( m/z 153 . Compounrsor ion . This isrsor ion  in Corni49) with [M − H]− at m/z 125.0242, which was proposed as phloroglucinol appearing in brown seaweed Ecklonia sp. and Sargassum sp. The identity was confirmed by the MS2 spectrum, which produced a major fragment ion at m/z 97, resulting from the loss of CO (28 Da) from the precursor ion −m/z 371.1509. Fragmentation of arctigenin yielded product ions at m/z 356, m/z 312 and m/z 295, corresponding to the loss of CH3 (15 Da), unit of CH3 and CO2 (59 Da), and unit of CH3, CO2 and OH (76 Da), respectively [54 (deoxyschisandrin) displaying the [M + H]+m/z at 417.2286 and was found in Ecklonia sp. and confirmed by the characteristic ions at m/z 402 [M − H − CH3], m/z 347 [M − H − C5H10], m/z 316 [M − H − C5H10 − OCH3] and m/z 301 [M − H − C5H10 − OCH3 − CH3] [Compounds ectively . Compoun3 − CH3] . Lignans3 − CH3] . Previou3 − CH3] ,63. In a3 − CH3] ,65.The screening and characterization of polyphenolic compounds showed that some of the polyphenols presented in these seaweeds have strong antioxidant potential. Hydroxycinnamic acid derivatives, hydroxybenzoic acids and their derivatives, protocatechuic acid, anthocyanins, flavonoids and their derivatives, hydroxybenzaldehydes, hydroxytyrosol, phloroglucinol and quercetin derivatives are regarded as potential compounds showing considerable free radical scavenging capacity ,69,70,71p-hydroxybenzoic acid and coumaric acid) and one flavonoid (catechin).The quantitative analysis of targeted phenolic compounds was performed based on peak area computation using the calibration of corresponding standards and the result are presented as μg/g fresh weight of seaweeds .. In totp-hydroxybenzoic acid (Compound 5), which was present in Ulva sp. with the concentration of 846.0 ± 0.02 μg/gf.w. The p-hydroxybenzoic acid content of eight green and red seaweeds in South Africa was previously reported as ranging from 0.51 ± 0.01 to 13.53 ± 0.03 μg/g dry weight (d.w.) [Ulva sp. in the present study. Gallic acid (Compound 1), chlorogenic acid (Compound 2) and caftaric acid (Compound 4) were detected in Centroceras sp. with the concentration of 138.9 ± 0.02 μg/gf.w., 122.7 ± 0.01 μg/gf.w. and 19.7 ± 0.01 μg/gf.w., respectively. Coumaric acid (Compound 6) was quantified in Ulva sp. with concentrations of 505.4 ± 0.03 μg/gf.w. Caffeic acid (Compound 3) and catechin (Compound 7) were present in Caulerpa sp. with a concentration of 612.9 ± 0.02 μg/gf.w. and 29.5 ± 0.03 μg/gf.w., respectively. Concentrations of gallic acid, chlorogenic acid and caffeic acid in brown seaweed Himanthalia elongate were also previously reported, being measured as 96.3 ± 3.12 μg/gd.w., 38.8 ± 1.94 μg/gd.w. and 44.4 ± 2.72 μg/gd.w., respectively [The most abundant targeted phenolic compound was t (d.w.) , which wectively . About 1ectively . The prep-hydroxybenzoic acid, coumaric acid and catechin, were purchased from Sigma-Aldrich . Water was deionized to reach a resistivity of 18.2 MΩ/cm using a Millipore Milli-Q Gradient Water Purification System  and was filtered through a 0.45 µm type Millipak® Express 20 Filter .Unless otherwise stated, all chemicals used for extraction, characterization and antioxidant assays were analytical grade and purchased from Sigma-Aldrich . Gallic acid, quercetin, catechin, ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), aluminum chloride, iron (III) chloride, vanillin, potassium persulfate and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma-Aldrich . Sulfuric acid 98% was from RCI Labscan  and sodium carbonate anhydrous was from Chem-Supply Pty Ltd. . Analytical-grade methanol, ethanol, hydrochloric acid, anhydrous sodium acetate and hydrated sodium acetate were from Fisher Chemical . Acetic acid solution and acetonitrile, which comprised the mobile phases for HPLC and LC-MS, were from Sigma-Aldrich  and LiChrosolv , respectively. The HPLC reference standards including gallic acid, caftaric acid, chlorogenic acid, caffeic acid, Ulva sp., Caulerpa sp. and Codium sp.), Rhodophyta  and Ochrophyta  were freshly collected from Brighton Beach in March 2019, VIC, Australia. Seaweeds were morphologically identified to the genus level. Classifications for Rhodophyta and Chlorophyta were verified using cytochrome c oxidase subunit I (COI-5P) and Elongation factor Tu 1-Escherichia coli (strain K12) tufA sequence data, respectively, following the protocol of Saunders and Kucera [Eight seaweeds which were identified as Chlorophyta  at 10,000 rpm for 20 s. Then, incubation was carried out in a shaking incubator  at 120 rpm at 4 °C for 16 h. Then, all the samples were centrifuged  at 10,000 rpm for 10 min. The supernatant was collected and stored at −20 °C for further analysis. For HPLC and LC-MS analysis, the extracts were filtered through a 0.45 μm syringe filter .Extracts were prepared by modifying the previous studies ,76, 2 g ® Go microplate photometer .For polyphenol estimation, TPC, TFC and TTC were measured, while for antioxidant potential, three different antioxidant assays, including DPPH, FRAP, and ABTS, were performed using the method of Feng, et al. . The datv/v) folin reagent solution and 200 µL water were added to the wells in a 96-well plate  and incubated at 25 °C for 5 min. Then, 25 µL of 10% (w/w) sodium carbonate was added and further incubated for 1 h at 25°C. The absorbance was measured at 765 nm against a blank using a Multiskan® Go microplate photometer . The calibration curve was plotted using a gallic acid standard ranging from 0 to 200 µg/mL in ethanolic solution and the results were presented as microgram equivalents of gallic acid equivalents (GAE) per gram ± standard error (SE) on the basis of fresh weight (f.w.) .The total phenolic content of seaweed was determined using the Folin-Ciocalteu’s method  with somw/v) aluminum chloride  and 120 µL 50 g/L sodium acetate was added the wells in the plate followed by the incubation at 25 °C for 2.5 h in the dark. The calibration curve was plotted using quercetin standards ranging from 0 to 50 μg/mL and the results are presented as microgram equivalents of quercetin equivalents (QE)/gf.w. ± SE .The total flavonoid content was measured by aluminum chloride colorimetry according to Chan, Matanjun, Yasir and Tan , with sow/v) methanolic vanillin solution and 25 µL 32% (v/v) sulfuric acid (diluted with methanol) were mixed in a 96-well plate and incubated at room temperature for 15 min. The absorbance was measured at 500 nm wavelength against a blank using the microplate reader. The calibration curve was plotted by catechin methanolic solution ranging from 0 to 1000 µg/mL and the results are presented as microgram equivalents of catechin (CE)/gf.w. ± SE .Total tannin content was measured by modifying the method of Rebaya, et al. . Sample/f.w. ± SE . DPPH radical scavenging activities of different extracts were determined based on Chan et al.  with somv/v). 20 µL samples/standards were added into the 96-well plate and mixed with 280 µL FRAP reagent. The mixture was incubated at 37 °C in the plate reader for 10 min before absorbance was measured at 593 nm. A standard curve was generated using ascorbic acid aqueous solution ranging from 0 to 50 µg/mL and the results are expressed as the microgram AAE/gf.w. ± SE .The ferric reducing capabilities of the samples were measured using the FRAP method described by Matanjun, et al. , with sl+ was prepared by mixing 5 mL of 7 mM ABTS solution and 88 µL of 140 mM potassium persulfate solution, and the mixture was placed in the dark for 16 h to allow free radical generation. The stock solution was further diluted with 45 mL analytical-grade ethanol while the absorbance of the dye was fixed at approximately 0.7 at 734 nm. Quantities of 10 µL of sample/standards and 290 µL prepared dye solution were added into a 96-well plate followed by incubation at room temperature for 6 min and the absorbance was measured at 734 nm wavelength. The standard curve was plotted using ascorbic acid aqueous solution ranging from 0 to 200µ/mL and the results are expressed as the microgram AAE/gf.w. ± SE .The antioxidant activities of seaweeds were also measured by an ABTS assay according to Matanjun, Mohamed, Mustapha, Muhammad and Ming , with sov/v; eluent A) and acetonitrile/acetic acid/ water . The gradient profile was described as follows: 10–25% B (0–25 min), 25–35% B (25–35 min), 35–40% B (35–45 min), 40–55% B (45–75 min), 55–80% B (75–79 min), 80–90% B (79–82 min), 90–100% B (82–84 min), 100–10% B (84–87 min), isocratic 10% B (87–90 min). A volume of 6 µL was injected for each standard or sample and the flow rate was set at 0.8 mL/min. Nitrogen gas nebulization was set at 45 psi with a flow rate of 5L/min at 300 °C and the sheath gas was set at 11 L/min at 250 °C. The capillary and nozzle voltage were set at 3.5 kV and 500 V, respectively. A complete mass scan ranging from m/z 50 to 1300 was used, MS/MS analyses were carried out in automatic mode with collision energy  for fragmentation. Peak identification was performed in both positive and negative modes while the instrument control, data acquisition and processing were performed using MassHunter workstation software  .LC-ESI-QTOF-MS/MS analysis was performed with an Agilent 1200 series HPLC  equipped with an Agilent 6520 Accurate-Mass Q-TOF LC-MS  via an electrospray ionization source (ESI). The separation was achieved by a Synergi Hydro-RP 80 Å, LC Column   at room temperature and the sample temperature was set at 10 °C. LC-MS/MS analysis were performed by modifying the method of Chao et al . The mobThe quantitative measurement of individual phenolic compounds present in seaweed samples was performed with an Agilent 1200 HPLC equipped with a photodiode array (PDA) detector by adopting the protocol of Peng et al. . The sam® 19 for windows . A significant difference was considered at the level of p ≤ 0.05 using Tukey’s HSD test.All analyses were performed in triplicates and the results are presented as mean ± standard error (n = 3). Data were analyzed using Tukey’s one-way analysis of variance (ANOVA) by Minitabp-hydroxybenzoic acid being the most abundant. This is the first report that applied different antioxidant assays to estimate the antioxidant potential and applied LC-MS technique to isolate and characterize the polyphenols in some abundant Australian seaweed species. The presence of the various polyphenols with antioxidant potential was identified. Further toxicity, pharmacological and clinical studies should be explored before the application of these Australian seaweeds as ingredients in food, nutraceuticals and pharmaceutical products.Brown seaweed species showed significantly higher polyphenolic content and potential antioxidant capacity than green and red seaweeds. The antioxidant properties varied across different species. Application of LC-ESI-QTOF-MS/MS enabled the isolation and identification of 54 phenolic compounds present in seaweeds. Quantitative analysis of targeted compounds was achieved by calibration of standards using HPLC-PDA. Seven targeted compounds were quantified in seaweeds, with