HPLC analysis was performed using a C18 Onyx Monolithic column 100 mm × 4.6 mm (Phenomenex, Torrance, CA, USA) coupled in sequence to a C18 Chromolith Performance RP-18e column 100 mm × 4.6 mm (Merck, Darmstadt, Germany) in a chromatographic system consisting of a System Gold Programmable Solvent Module 126 Pump (Beckman, San Ramon, CA, USA) equipped with an HT 800 L autosampler (HTA, Brescia, Italy) fitted with a 20 µl loop. The FLD detection was obtained by means of a 821 FP fluorescence detector (Jasco, Tokyo, Japan) set at the excitation wavelength of 340 nm and emission wavelength of 460 nm. The system was computer-controlled by a Beckman Coulter 32 Karat Software (Beckman, San Ramon, CA, USA). OTA separation was archived using isocratic elution and at room temperature, under conditions similar to those adopted by Altafini et al. [21 (link)]. The mobile phase consisted of 70% of a mixture of water, isopropyl alcohol-acetonitrile and acetic acid 1% (79:7:7:7 v/v) and 30% of acetonitrile. The flow rate was set at 1 mL/min, while the injection volume was 20 µL.
821 fp fluorescence detector
Manufactured by Jasco
Sourced in Japan
The 821 FP fluorescence detector is a laboratory instrument designed to measure fluorescence intensity. It is capable of detecting and quantifying fluorescent compounds within a sample. The device operates by exciting the sample with a light source and measuring the resulting fluorescent emission.
Automatically generated - may contain errors
6 protocols using 821 fp fluorescence detector
1
HPLC Analysis of Ochratoxin A
2
Quantifying 5-FC and 5-FU Concentrations
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The solution strengths of 5-FC and 5-FU were quantified. Prior to HPLC, molecules were transformed into a lactone form by acidification. Acidified methanol, comprising 5 µL 1 N HCI/mL methanol, was used to dilute the samples. These underwent centrifugation (14,000× g, 2 min). The resulting supernatant was passed at a flow rate of 1 mL/min through a 4-p,m Nova-Pak C,8 column, of dimensions 300 × 3.9 mm, for which 75 mM ammonium acetate, 25% acetonitrile (pH 4.0) had been used for equilibration. Elution of 5-FC occurred under these parameters after 4.3 min, and 5-FU, after 6.3 min. A Jasco 82 1-FP fluorescence detector was utilized for product identification, using excitation and emission wavelengths of 375 nm and 550 nm, respectively. System Gold software (32 Karat Software, version 3.0) was employed for data analysis. Detection limits were 20 pg/pA for 5-FC and 2 pg/pA for 5-FU.
3
Pulmonary Hydroxyl Radical Assessment
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The level of MG formation in the BALF has been used as an index for pulmonary hydroxyl radical formation [74 (link)]. The lavage supernatant was diluted 1:100, and MG was measured using a spectrofluorimetric detector (Jasco 821-FP Fluorescence Detector, Tampa, FL, USA) with a fluorescent excitation maximum at 395 nm and emission maximum at 500 nm. The assay was calibrated by a standard curve of authentic MG (Sigma M0377, St. Louis, MO, USA), generated by various concentrations of MG (0, 50, 100, 150, and 200 mg/mL). The coefficient of variance in percentage was 3.8% with a detection limit of 1 mg/mL.
4
Aflatoxin M1 Analysis by HPLC-FLD
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AFM1 analysis was performed by HPLC-FLD on a chromatographic apparatus consisting of a System Gold Programmable Solvent Module 126 Pump (Beckman, San Ramon, CA, USA) equipped with an HT 800 L autosampler (HTA, Brescia, Italy) fitted with a 100 µL loop. Chromatographic separation was achieved in isocratic elution mode and at room temperature using an analytical column Zorbax Eclipse Plus C18 150 × 4.6 mm 5 µm (Agilent, Santa Clara, CA, USA). The mobile phase consisted of 61% of deionized water, and 39% of acetonitrile. The flow rate was set at 0.7 mL/min, and the injection volume was 100 µL. The FLD detection was obtained by means of a 821 FP fluorescence detector (Jasco, Tokyo, Japan) set at the excitation wavelength of 360 nm and emission wavelength of 440 nm. The system was computer-controlled by a Beckman Coulter 32 Karat Software.
5
HPLC Quantification of Analytes
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For OTA analysis and quantification, a HPLC system consisting of a System Gold Programmable Solvent Module 126 pump (Beckman Coulter, Brea, CA, USA) equipped with an HT 800 L autosampler (HTA, Brescia, Italy) fitted with a 20 l loop and an 821 FP fluorescence detector (Jasco, Tokyo, Japan) was used. Fluorescence excitation and emission wavelengths were 340 and 460 nm respectively. The system was computer-controlled by a Beckman Coulter 32 Karat Software. The analytical conditions were those developed by Armorini et al. (2016) , and optimized for use in this study.
Chromatographic separation was achieved in gradient elution mode using two monolithic columns coupled in sequence: a C18 Onyx Monolithic 100 mm x 4.6 mm (Phenomenex, Torrance, CA, USA) and a C18 Chromolith Performance RP-18e 100 mm x 4.6 mm (Merck, Darmstad, Germany). The mobile phase consisted of eluent A containing water/acetonitrile/isopropyl alcohol/acetic acid 1% (79:7:7:7, v/v) and eluent B consisting of acetonitrile. The gradient program started with 58% A and 42% B, reaching 49% B in 3.5 min with linear increase. The flow rate was 1.1 ml/min, while the sample injection volume was 20 µl.
Chromatographic separation was achieved in gradient elution mode using two monolithic columns coupled in sequence: a C18 Onyx Monolithic 100 mm x 4.6 mm (Phenomenex, Torrance, CA, USA) and a C18 Chromolith Performance RP-18e 100 mm x 4.6 mm (Merck, Darmstad, Germany). The mobile phase consisted of eluent A containing water/acetonitrile/isopropyl alcohol/acetic acid 1% (79:7:7:7, v/v) and eluent B consisting of acetonitrile. The gradient program started with 58% A and 42% B, reaching 49% B in 3.5 min with linear increase. The flow rate was 1.1 ml/min, while the sample injection volume was 20 µl.
6
HPLC Analysis of Fluorescent Compounds
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The HPLC system consisted of a System Gold Programmable Solvent Module 126 pump (Beckman Coulter, Brea, CA, USA) equipped with an HT 800 L autosampler (HTA, Brescia, Italy) fitted with a 20 μl loop and a 821 FP fluorescence detector (Jasco, Tokyo, Japan); fluorescence excitation and emission wavelengths were 340 and 460 nm respectively. The system was computer-controlled by a Beckman Coulter 32 Karat Software. The HPLC column was a C18 Onyx Monolithic column 100 mm x 4.6 mm (Phenomenex, Torrance, CA, USA) coupled in sequence to a C18 Chromolith Performance RP-18e column 100 mm x 4.6 mm (Merck, Darmstad, Germany). Chromatographic separation was achieved in gradient conditions and at room temperature. The mobile phase consisted of water-acetonitrile-isopropyl alcohol-acetic acid 1% mixtures in various ratios. Mobile phase A: water/acetonitrile/isopropyl alcohol/acetic acid 1% (79:7:7:7 v/v) and mobile phase B: acetonitrile. Gradient: from 80% A and 20% B to 58% A and 42% B in 11 min with linear increase. Flow rate: 1 ml/min. The injection volume was 20 µl.
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