Analyses were performed on a Shimadzu Prominence HPLC system, consisting of one LC-20AP solvent delivery module, a DGU 20As online degasser, an automatic sample injector SIL-10AF, a RID-10A differential refractive index detector, a CTO-20A column oven and a CBM-20A system controller. Isocratic separations were conducted at 35 °C using an EC 250/4 Nucleodur 100–5 NH2 RP column (250 × 4.6 mm, Macherey-Nagel)—a multimode column protected by a pre-column (30 mm × 4.6 mm), with a flow rate of 1 mL. Min−1 acetonitrile in water (80:20 v/v%) as the mobile phase—an optimized Macherey-Nagel procedure [71 ]; the injection volume was 10 μL. Under these conditions, baseline separations of the target carbohydrates were accomplished in less than 10 min. The compounds’ identification was based both on comparison of the retention times with those of standards and on co-elution after spiking with authentic standards. Quantification was performed by the external standard method. Calibration curves were established using a mixture of fructose, glucose and sucrose standards, at five concentrations (Table 4 ). Recoveries were calculated by analyzing spiked samples, revealing percentages in the range of 95.27% for fructose and 98.83% for sucrose.
Sil 10af
Manufactured by Shimadzu
Sourced in Japan, Germany
The SIL-10AF is an autosampler module designed for use in high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) systems. It is capable of automatically injecting samples into the chromatographic system.
Automatically generated - may contain errors
Lab products found in correlation
Lc 10at vp, by Shimadzu (4 mentions)
Lc 20ad, by Shimadzu (3 mentions)
Spd m20a, by Shimadzu (3 mentions)
Spd m10a, by Shimadzu (2 mentions)
Lc 10ad, by Shimadzu (2 mentions)
Scl 10at vp, by Shimadzu (2 mentions)
Hplc system, by Shimadzu (2 mentions)
Dgu 20a, by Shimadzu (2 mentions)
Labsolutions version 5, by Shimadzu (2 mentions)
Nucleosil 120 5 c18, by Macherey-Nagel (2 mentions)
Cto 20a, by Macherey-Nagel (2 mentions)
Lc 20ap, by Shimadzu (1 mentions)
Prominence hplc system, by Shimadzu (1 mentions)
Raffinose pentahydrate, by Merck Group (1 mentions)
Anhydrous glucose, by Merck Group (1 mentions)
Dgu 20a5r, by Shimadzu (1 mentions)
Zorbax nh2, by Agilent Technologies (1 mentions)
Prominence lc 20ad, by Shimadzu (1 mentions)
Labsolution system, by Shimadzu (1 mentions)
Lc 20at pump, by Shimadzu (1 mentions)
16 protocols using sil 10af
1
HPLC Quantification of Carbohydrates
2
Quantification of Flavonoid Aglycones in Fruit Extracts
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Flavonoids were quantified according to the method applied by De Carvalho et al. (2018) (link). Briefly, extracts were obtained by mixing 5 g of pulp and 10 mL of ethanol: water (1:1) solution. One-mL of the extract was mixed to 0.2 mL of methanol 50% with HCl (1.2 N) and tert-butylhydroquinone (TBHQ, 0.4 g/L) to get flavonoid aglycones, sonicated and filtered through a HV 0.45 mm millex filter. a Shimadzu® HPLC system, equipped with a high-pressure pump model LC-10AT VP, an autosampler model SIL-10AF and UV visible diode array detector model SPD-M10A (Shimadzu®, Kyoto, Japan) was used to separation, identification and quantification. Chromatograms were obtained by using 270, 340 and 380 nm wavelengths. Mobile phases were (A) Water: Tetrahydrofuran: Trifluoroacetic Acid (99.79:0.2:0.01) and (B) Acetonitrile. For vitexine, isovitexine, orientin and isoorientine, running was carried out with 80% A 20% B, with a flow rate of 0.5 mL/minute for 25 min. For hesperitin, the flow of 0.7 mL/min for 25 min was used with 70% A, 30% B.
3
Quantitative Analysis of Bioactive Compounds in Subcritical Extracts
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Subcritical extracts were centrifuged at 2500 rpm for 10 min prior to HPLC. The centrifuged extracts were added with 5 μL of 10 g/L o-cresol (99% purity, Fujifilm Wako Pure Chemical Industries Co., Ltd., Osaka, Japan) as an intermediate marker to 1 mL of supernatant. Ten microliters of the obtained solution was sampled using an HPLC autosampler (SIL-10 AF, Shimadzu Corporation, Kyoto, Japan) and injected into the HPLC apparatus. The flow rate was 1.0 mL/min, and the wavelength was set to 280 nm for PB2 [45 (link),46 (link)], 5CQA [47 (link),48 ], and epicatechin [49 (link),50 (link)].
Because it is difficult to resolve all individual peaks, a gradient method with two mobile phases [51 (link)] was used. Mobile phase A consisted of 2.5 vol.% aqueous acetic acid, and mobile phase B was acetonitrile. The gradient conditions were set as follows: 0 min A–B (97:3), 5 min A–B (91:9), 15 min A–B (84:16), 33 min A–B (64:36), 38 min A–B (0:100), 48 min A–B (97:3), and 60 min A–B (97:3).
HPLC pumps A (LC10-AD, Shimadzu Corporation, Kyoto, Japan) and B (LC20-AD, Shimadzu Corporation, Kyoto, Japan) were used. The separation column STR ODS-II (4.6 mm × 250 mm; Shinwa Chemical Industry, Kyoto, Japan) was used at a temperature of 40 °C. The HPLC detector was an SDP-M10A diode array detector (Shimadzu Corp., Kyoto, Japan).
Because it is difficult to resolve all individual peaks, a gradient method with two mobile phases [51 (link)] was used. Mobile phase A consisted of 2.5 vol.% aqueous acetic acid, and mobile phase B was acetonitrile. The gradient conditions were set as follows: 0 min A–B (97:3), 5 min A–B (91:9), 15 min A–B (84:16), 33 min A–B (64:36), 38 min A–B (0:100), 48 min A–B (97:3), and 60 min A–B (97:3).
HPLC pumps A (LC10-AD, Shimadzu Corporation, Kyoto, Japan) and B (LC20-AD, Shimadzu Corporation, Kyoto, Japan) were used. The separation column STR ODS-II (4.6 mm × 250 mm; Shinwa Chemical Industry, Kyoto, Japan) was used at a temperature of 40 °C. The HPLC detector was an SDP-M10A diode array detector (Shimadzu Corp., Kyoto, Japan).
4
HPLC Analysis of Carbohydrates
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Carbohydrate analysis was carried out on a Shimadzu (Kyoto, Japan) HPLC system which consisted of an LC-20AD prominence solvent delivery module, a DGU-20A5R degassing unit, an SIL-10 AF automatic sample injector, and an RID-10A refractive index detector. The instrument was coupled with a computer equipped with LabSolutions Lite Version 5.52 software. The analytical column used for carbohydrate (fructose, glucose, sucrose, maltose, melezitose, raffinose and xylose) separation was an Agilent Technologies ZORBAX NH2 (Santa Clara, CA, USA) (4.6 × 250 mm, 5 μm particle size). The mobile phase consisted of HPLC-grade acetonitrile (J. T. Baker, Devnter, The Netherlands) and ultrapure water (70/30, v/v), while the operating conditions were: an injection volume of 10 µL, a mobile phase flow of 1 ml/min and a temperature of 30 °C. Carbohydrates were identified according to their retention times, and quantification was performed via external calibration carried out with carbohydrate standards suitable for HPLC analysis. Fructose, anhydrous glucose, sucrose, raffinose pentahydrate and melezitose hydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA), while xylose and maltose monohydrate were purchased from Kemika (Zagreb, Croatia) [21 ].
5
Quantification of 3-Deoxyanthocyanins in Samples
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The 3-deoxyanthocyanins: luteolinidin (LUT), apigeninidin (API), 5-methoxyluteolinidin (5-MeO LUT) and 7-methoxyapigeninidin (7-MeO API) contents were determined according to a method proposed by Yang, Allred, Geera, Allred, and Awika (2012) , modified by Cardoso et al. (2014) . The compounds were extracted from 1 g of sample with 10 mL of 1% HCl in methanol. Analyzes were performed in an HPLC system (Shimadzu, SCL 10AT VP, Japan) equipped with diode array detector (Shimadzu, SPD-M10A, Japan), high pressure pump (Shimadzu, LC-10AT VP, Japan), autosampler with loop of 500 mL (Shimadzu, SIL-10AF, Japan), and helium degassing system using the chromatographic conditions described by Cardoso et al. (2014) .
Identification was performed by correlating the retention time and the absorption spectrum of peaks of the standards and samples, analyzed under the same conditions. The quantification of each compound was performed by comparison of peak areas with those of standard curves constructed through injection, in duplicate, of six different standard concentrations (R 2 ranged from 0.9939 to 0.9992). The 5-MeO-LUT and 7-MeO-API contents were quantified using standards of luteolinidin and apigeninidin, respectively, as well as with the appropriate molecular weight correction factor (Dykes, Seitz, Rooney, & Rooney, 2009) . Results were expressed in lg/g dry matter.
Identification was performed by correlating the retention time and the absorption spectrum of peaks of the standards and samples, analyzed under the same conditions. The quantification of each compound was performed by comparison of peak areas with those of standard curves constructed through injection, in duplicate, of six different standard concentrations (R 2 ranged from 0.9939 to 0.9992). The 5-MeO-LUT and 7-MeO-API contents were quantified using standards of luteolinidin and apigeninidin, respectively, as well as with the appropriate molecular weight correction factor (Dykes, Seitz, Rooney, & Rooney, 2009) . Results were expressed in lg/g dry matter.
6
HPLC Analysis with Shimadzu System
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HPLC was performed using an HPLC system from Shimadzu USA Manufacturing Inc. (Canby, OR, USA) consisting of a low-pressure gradient flow LC-20AT pump, a DGU-20A online solvent degasser, an SPD-M20A photodiode array detector, an SIL-10AF sample injector, and an FRC-10A fraction collector. Data were monitored using a Shimadzu LabSolution system.
7
HPLC Quantification of Aflatoxin B1 Adsorption
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AFB1 quantification in the buffer solutions was achieved by direct
injection into a Shimadzu HPLC (Tokyo, Japan) system consisting of a
fluorescence detector (RF-10A XL) and an autosampler (SIL-10AF). An ODS column 5
μm 4.6 × 150 mm (Phenomenex, Torrance, CA, USA) was used. The system was
stabilized for one hour at a flow rate of 1 mL/min at room temperature. The
mobile phase was a solution of water, acetonitrile and methanol (60:20:20) at a
flow rate of 1 mL/min. The excitation detection was performed at a wavelength of
360 nm, and emission was monitored at 440 nm. Under the above conditions, the
detection limit for AFB1 was 0.01 ng/mL, and the retention time was
approximately 10.5 min with a retention window of ± 10%.
The quantification of the percentage of AFB1 adsorbed was performed
usingEq. 1 , where A represents
the percentage of AFB1 adsorbed by the sample, B the area of positive
control chromatographic peak (AFB1 in buffer solution), C the area of
sample chromatographic peak (AFB1 in buffer solution + sample) and D
the area of negative control chromatographic peak (buffer solution +
sample).
injection into a Shimadzu HPLC (Tokyo, Japan) system consisting of a
fluorescence detector (RF-10A XL) and an autosampler (SIL-10AF). An ODS column 5
μm 4.6 × 150 mm (Phenomenex, Torrance, CA, USA) was used. The system was
stabilized for one hour at a flow rate of 1 mL/min at room temperature. The
mobile phase was a solution of water, acetonitrile and methanol (60:20:20) at a
flow rate of 1 mL/min. The excitation detection was performed at a wavelength of
360 nm, and emission was monitored at 440 nm. Under the above conditions, the
detection limit for AFB1 was 0.01 ng/mL, and the retention time was
approximately 10.5 min with a retention window of ± 10%.
The quantification of the percentage of AFB1 adsorbed was performed
using
the percentage of AFB1 adsorbed by the sample, B the area of positive
control chromatographic peak (AFB1 in buffer solution), C the area of
sample chromatographic peak (AFB1 in buffer solution + sample) and D
the area of negative control chromatographic peak (buffer solution +
sample).
8
HPLC Analysis of Pinostrobin Compound
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The Shimadzu HPLC system (Kyoto, Japan) consisting of LC-20AB prominence liquid chromatograph, SIL-10AF auto-injector, SPD-10AV UV-VIS detector, and SCL-10A system controller was used for analysis. Separation was achieved using a C18 Phenomenex Kinetex column (5 μm, 250 x 4.6 mm) with UV detection at 275 nm. The mobile phase was prepared by mixing acetonitrile:water:methanol (75:20:5 v/v), filtered through a 0.2 um nylon filter and degassed using reduced pressure prior to use. Separation was carried out isocratically at ambient temperature (22±1°C) with a flow rate of 0.6 mL/min. Pinostrobin was used as the internal standard. Shimadzu EZStart (Version 7.4) software was used for data acquisition and integration. Samples were freshly prepared on day of analysis and injected into the HPLC system.
9
Quantification of Methylglyoxal via HPLC-DAD
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MGO was analyzed as its corresponding quinoxaline derivative and quantified relative to the unheated samples (t = 0 min) by HPLC-DAD [41 (link)]. For derivatization, aliquots of the model systems were incubated with ortho-phenylendiamine (OPD): 0.1 mL of OPD solution (50 mmol/L in water/methanol 1:1, v/v) was added to the sample (0.1 mL). Subsequently, samples were stored in darkness at room temperature for 24 h. After derivatization, samples were diluted (1:50) and analyzed by HPLC-DAD. The following setup was used: degasser, Shimadzu DGU-20As; pump, Shimadzu LC-20AD; autosampler, Shimadzu SIL-10AF; column oven, Shimadzu CTO-20A; column, Nucleosil® 120-5 C18 (Macherey-Nagel GmbH & Co. KG, Düren, Germany); detector Shimadzu SPD-M20A; software, Shimadzu LabSolutions Version 5.90. The following settings were used: column temperature, 35 °C; flow rate, 0.5 mL/min; eluent A, 0.075% acetic acid in water (v/v); eluent B, methanol; eluent gradient, 0 min, 40% B; 10 min, 40% B; 15 min, 60% B; 25 min, 60% B; 26 min, 90% B; 34 min, 90% B; 35 min, 40% B; wavelength for quantitation, 318 nm.
10
HPLC-DAD Quantification of Phytochemical Conversions
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The conversion of FF, NF, MAL, and PA was quantified relative to the unheated samples (t = 0 min) by HPLC-DAD. Samples were diluted (1:20) and analyzed with the following setup: degasser, Shimadzu DGU-20As; pump, Shimadzu LC-20AD; autosampler, Shimadzu SIL-10AF; column oven, Shimadzu CTO-20A; column, Nucleosil® 120-5 C18 (Macherey-Nagel GmbH & Co. KG, Düren, Germany); detector Shimadzu SPD-M20A; software, Shimadzu LabSolutions Version 5.90. The following settings were used: column temperature, 45 °C; flow rate, 1.0 mL/min; eluent A, 0.075% acetic acid in water (v/v); eluent B, methanol; eluent gradient, 0 min, 5% B; 10 min, 20% B; 15 min, 90% B; 20 min, 90% B; 21 min, 5% B; wavelength for quantitation, 285 nm.
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