Urinary concentration of 8OHdG was analyzed using HPLC with coularray electrochemical detection as previously published [19 (link)]. Briefly, 8OHdG was extracted from 1 ml urine with the Oasis-HLB 3 cc (60 mg) cartridge (Waters Corporation) following the manufacturer’s instructions. The eluents were dried under ultra-pure N2 stream and reconstituted in buffer (10 mM ammonium acetate in 2% MeOH, pH 4.3) for analysis with the same HPLC-ECD system.
Oasis hlb 3cc 60 mg cartridge
Manufactured by Waters Corporation
Sourced in United States
The Oasis HLB 3cc 60 mg cartridge is a solid-phase extraction (SPE) product used for sample preparation. It is designed to extract and retain analytes from liquid samples prior to analysis. The cartridge contains a hydrophilic-lipophilic balanced (HLB) sorbent material that can retain a wide range of polar and non-polar compounds.
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4 protocols using oasis hlb 3cc 60 mg cartridge
1
Urinary 8OHdG Quantification by HPLC-ECD
2
Oasis HLB Extraction Protocol
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Oasis HLB 3cc 60 mg cartridge (hydrophilic-lipophilic balanced sorbent, Waters, USA) was conditioned with 3 ml methanol, equilibrated with 3 ml water and then 3 ml cultivation broth (pH adjusted to 9.0 with ammonium hydroxide) was loaded. Subsequently, the cartridge was washed with 3 ml water and absorbed substances were eluted with 1.5 ml methanol. The eluent was evaporated to dryness, reconstituted in 150 μl methanol and centrifuged at 13000 rpm for 5 min. The extract was then diluted 10× with methanol:water (1:1 v/v) and 2 μl were injected into LC–MS.
3
Solid-Phase Extraction Protocol for Metabolite Analysis
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The solid-phase extraction (SPE) procedure was performed as described elsewhere [136 (link)]. Briefly, each strain’s supernatant was isolated by centrifugation at 10,000× g at 4 °C for 10 min, and the pH was adjusted to 3–4 using formic acid (Merck, Darmstadt, Germany). An Oasis HLB 3cc 60 mg cartridge (hydrophilic-lipophilic balanced sorbent, Waters, Milford, MA, USA) was conditioned with 3 mL methanol, equilibrated with 3 mL Milli-Q water (Sigma-Aldrich Co. St. Louis, MO, USA), and subsequently, 3 mL of culture supernatant was loaded. The cartridge was then washed with 3 mL of water, and the absorbed substances were eluted with 1.5 mL of methanol.
4
Extraction and Purification of Secondary Metabolites
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Secondary metabolites were taken from the culture supernatants extracted using ethyl acetate (Rajan and Kannabiran, 2014 (link)), QuEChERS (Schenck and Hobbs, 2004 (link)), or solid phase extraction (Kamenik et al., 2010 (link)), which was found to be the most suitable and was carried out as follows. An Oasis HLB 3cc 60 mg cartridge (hydrophilic-lipophilic balanced sorbent, Waters, USA) was conditioned with 3 mL methanol (LC-MS grade, Biosolve, Netherlands), equilibrated with 3 mL water (prepared using Milli-Q water purifier, Millipore, USA) and then 3 mL culture supernatant (pH adjusted to 3 with formic acid, 98–100%, Merck, Germany) was loaded. Subsequently, the cartridge was washed with 3 mL water and absorbed substances were eluted with 1.5 mL methanol. The eluent was evaporated to dryness (Concentrator Plus, 2013 model, Eppendorf), reconstituted in 200 μL 50% methanol and centrifuged at 12,000 × g for 5 min.
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