Concentrations of 5-HT and 5-HIAA in the cell-free supernatant and placental perfusate were determined using a Shimadzu LC20 Performance HPLC chromatograph (Shimadzu, JP) as described previously [3 (link)]. A Kinetex EVO C18 100 A 150 × 3 mm, particle size 5 μm column (Phenomenex, USA) with a guard column was used. Analytes were eluted with a biphasic mobile phase consisting of A − 3:97 (v/v) methanol:acetic acid (0.1 M, pH 4.5, adjusted with NaOH) and B - methanol. The proportion of B was 0% for 0–8.4 min, then linearly increased to 20% at minute 9.6, held at 20% until minute 22.6, linearly decreased to 0% after minute 23.2 then held at 0% until minute 30. Excitation and emission wavelengths of the fluorescence detector were set for individual compounds: 280/334 nm for 5-HT from 0 to 6.5 min and 276/333 nm for 5-HIAA from 6.5 to 18 min.
Lc20 performance hplc chromatograph
Manufactured by Shimadzu
Sourced in Japan
The LC20 Performance HPLC chromatograph is a high-performance liquid chromatography system designed for analytical and preparative separations. It features a dual-plunger pump, gradient mixer, and a variety of detection options to meet the diverse needs of researchers and analysts.
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4 protocols using lc20 performance hplc chromatograph
1
HPLC Analysis of Placental 5-HT and 5-HIAA
2
HPLC Analysis of Neurotransmitters
3
HPLC Determination of Indoleamines
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The HPLC analyses were performed using Shimadzu LC20 Performance HPLC chromatograph (Shimadzu, Kyoto, Japan) equipped with UV and fluorescence detector. For simultaneous chromatographic separation of all tested compounds, Phenomenex Kinetex 5 μm EVO C18 100 A 150 × 3 mm with a guard column was used. An isocratic elution, at a flow rate of 0.5 mL/min, was performed with mobile phase consisting of 0.1 M acetic acid, pH 4.5 (adjusted with NaOH), and methanol 97 + 3. All analytes were eluted within 8.5 min.
Excitation and emission wavelengths of fluorescence detector were set for individual compounds: 275/333 nm for 5-OH-TRP from 0 to 3.1 min and 280/334 nm for 5-HT and TRP from 3.1 min. KYN was detected by UV detection with wavelength set to 369 nm. Additionally, in cases of TRP concentrations higher than the range of fluorescence detection, UV detection was used with wavelength set to 300 nm.
Excitation and emission wavelengths of fluorescence detector were set for individual compounds: 275/333 nm for 5-OH-TRP from 0 to 3.1 min and 280/334 nm for 5-HT and TRP from 3.1 min. KYN was detected by UV detection with wavelength set to 369 nm. Additionally, in cases of TRP concentrations higher than the range of fluorescence detection, UV detection was used with wavelength set to 300 nm.
4
Quantification of Neurotransmitters and Metabolites via HPLC
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Concentrations of TRP, 5-OH-TRP, 5-HT, 5-HIAA, KYN and KYNA in cell-free supernatants were determined using a Shimadzu LC20 Performance HPLC chromatograph (Shimadzu, JP), as described previously [14 ]. A Kinetex EVO C18 100 A 150 × 3 mm, particle size 5 μm column (Phenomenex, USA) with a guard column was used at temperature of 20°C and a flow of 0.5 ml/min. 5-OH-TRP, 5-HT and TRP were eluted with a mobile phase consisting of 3:97 (v/v) methanol:acetic acid (0.1 M, pH 4.5, adjusted with NaOH). The excitation and emission wavelengths of the fluorescence detector were set at 276/333 nm. A mobile phase of 7:93 (v/v) methanol:acetic acid (0.2 M) with the same fluorescence detection wavelengths was used for the analysis of HIAA. Kynurenine was determined using a mobile phase consisting of 2:98 (v/v) methanol:acetic acid (0.1 M, pH 6.8, adjusted with NaOH) with UV detection at 289 nm. A mobile phase of 97:3 (v/v) zinc acetate (0.05 M with 0.025% acetic acid):acetonitrile was used for KYNA with excitation and emission wavelengths of 330/385 nm. Levels of QUIN were determined by a GC-MS method using deuterated quinolinic acid [43 ].
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