Oxylipins were extracted using polymer-based reversed-phase Strata-X cartridges (33 μm, 200 mg/6 mL; Phenomenex, PA). All SPE steps were performed via gravity flow. The cartridge was preconditioned with 6 mL methanol and 6 mL water. Just before loading each sample, the supernatant from plasma sample obtained after centrifugation or standards in the final solution was transferred with glass Pasteur pipette to pre-cooled Kimble glass centrifuge tubes containing 6 mL of cold water in an ice bath and were vortexed briefly. The diluted supernatant was immediately decanted into a SPE cartridge and passed through the cartridge. The cartridge was then washed with 6 mL of 10% methanol in water and air-dried for approximately 2 min. The remaining washing solution in the cartridge tip was removed. Afterwards, the metabolites were eluted with 6 mL methanol containing 0.0004% w/v BHT into a glass tube containing 10 μL of 30% glycerol in methanol. The eluted solutions were evaporated under a stream of nitrogen to dryness at 30 °C. The residues were reconstituted with 40 μL of methanol, transferred to amber autosampler vials, and an aliquot (10 μL) was injected into the LC-MS/MS system.
Strata x cartridge
Manufactured by Phenomenex
Sourced in United States, Spain
Strata-X cartridges are solid-phase extraction (SPE) products designed for sample preparation and analyte concentration. They feature a polymeric sorbent material that can be used for a variety of applications, including the extraction of analytes from complex matrices. The core function of Strata-X cartridges is to facilitate the isolation and concentration of target compounds prior to instrumental analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ultracel ym 10, by Merck Group (2 mentions)
8050 triple quadrupole mass spectrometer, by Shimadzu (1 mentions)
Oasis mcx cartridge, by Waters Corporation (1 mentions)
2h3 cotinine, by Merck Group (1 mentions)
β glucuronidase, by Merck Group (1 mentions)
Mc lr, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Mc rr, by Merck Group (1 mentions)
Mc yr, by Merck Group (1 mentions)
Mc lw, by Merck Group (1 mentions)
Ammonium hydroxide solution nh4oh, by Merck Group (1 mentions)
Saliva collection system, by Greiner (1 mentions)
Oasis hlb, by Waters Corporation (1 mentions)
Strata sdb l, by Phenomenex (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
19 protocols using strata x cartridge
1
Solid-Phase Extraction of Oxylipins
2
Quantitative Lipidomics Protocol for SARS-CoV-2
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Concentrated samples (500 µL) were denatured with 500 µL of LC‐MS–grade MeOH containing the internal standards then warmed at 60º Celsius for 30 minutes to inactivate SARS‐CoV‐2, denatured overnight (−20° Celsius), and then centrifuged (10,000 g) to remove the denaturated proteins. Under these conditions, no significant degradation of internal standards and selected lipids and lipid mediators was observed (Figure S1). Supernatants were diluted with water containing 0.01% acetic acid and lipids were extracted by solid phase extraction using Strata‐X cartridges (Polymeric Reversed Phase, Phenomenex, USA) as described before.12 Lipids were separated using the same column and LC program as in,12 quantification was done using a Shimadzu 8050 triple quadrupole mass spectrometer and analyses were performed using multiple reaction monitoring for the specific mass transitions of each lipid as well as the deuterated internal standard used (Table S2). Due to the large number of analytes being quantitated and/or commercial availability, some lipid mediators were analyzed with a surrogate, deuterated internal standard.
3
Quantitative Analysis of Kava Constituents
4
Microcystin Quantification in Water
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All chemicals were reagent grade or higher in quality. Formic acid (FA) and ammonium hydroxide solution (NH4OH) were purchased from Sigma-Aldrich (Milan, Italy). HPLC gradient grade methanol (MeOH), acetonitrile (ACN) and ultrapure water (H2O) were supplied by Carlo Erba (Cornaredo, Italy). Strata™-X cartridges (200 mg, 3 mL) were purchased from Phenomenex (Castel Maggiore, Italy). DA (≥90%), OA sodium salt (MQ 100) and analytical grade standard MC-RR, MC-LR, MC-YR and MC-LW solutions were supplied by Sigma-Aldrich (Milan, Italy). Individual stock standard solutions of all analytes (1 μg mL−1) were prepared in MeOH, stored in the dark (−20 °C) and renewed weekly.
5
DNA Hydrolysis and Purification Protocol
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The DNA samples
were dissolved in 0.6 mL of 10 mM sodium succinate (pH 7.4) buffer
containing 5 mM CaCl2 and 5 fmol [13C1015N2]TpMeT as internal standard, followed by
the addition of deoxyribonuclease I (0.75 units), phosphodiesterase
I (0.005 units), and alkaline phosphatase (0.4 units). The solution
was then incubated overnight at 37 °C. On the next day, 20 μL
of hydrolysate was collected for dGuo analysis by HPLC-UV and DNA
quantitation.8 (link) The remaining hydrolysate
was filtered through 10 K centrifugal filters (Ultracel YM-10, Millipore),
and the filtrates were loaded on 30 mg Strata X cartridges (Phenomenex)
activated with 2 mL of MeOH and 2 mL of H2O. The cartridges
were washed with 2 mL of H2O and 1 mL of 10% MeOH sequentially
and finally eluted with 2 mL of 50% MeOH. The 50% MeOH fraction containing
analytes was concentrated to dryness in a centrifugal evaporator.
The residue was redissolved in 15 μL of deionized H2O prior to analysis by LC-NSI-HRMS/MS.
were dissolved in 0.6 mL of 10 mM sodium succinate (pH 7.4) buffer
containing 5 mM CaCl2 and 5 fmol [13C1015N2]TpMeT as internal standard, followed by
the addition of deoxyribonuclease I (0.75 units), phosphodiesterase
I (0.005 units), and alkaline phosphatase (0.4 units). The solution
was then incubated overnight at 37 °C. On the next day, 20 μL
of hydrolysate was collected for dGuo analysis by HPLC-UV and DNA
quantitation.8 (link) The remaining hydrolysate
was filtered through 10 K centrifugal filters (Ultracel YM-10, Millipore),
and the filtrates were loaded on 30 mg Strata X cartridges (Phenomenex)
activated with 2 mL of MeOH and 2 mL of H2O. The cartridges
were washed with 2 mL of H2O and 1 mL of 10% MeOH sequentially
and finally eluted with 2 mL of 50% MeOH. The 50% MeOH fraction containing
analytes was concentrated to dryness in a centrifugal evaporator.
The residue was redissolved in 15 μL of deionized H2O prior to analysis by LC-NSI-HRMS/MS.
6
DNA Hydrolysates Purification Protocol
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DNA hydrolysates were partially purified by solid-phase extraction using Strata-X cartridges (30 μm, Phenomenex, Torrance, CA) that were activated with 3 mL of MeOH and preconditioned with 1 mL of H2O. The hydrolysates were loaded on the cartridges and washed sequentially with 1 mL of H2O and 1 mL of 5% MeOH in H2O, and the analytes were eluted with 1 mL of 100% MeOH and 1 mL of MeOH containing 2% formic acid, and both fractions were evaporated to dryness and reconstituted in 2% MeOH in H2O (LC-MS grade, Fluka) to a final volume of 20 μL prior to MS analysis.
7
Saliva Collection and Extraction Protocol
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The principle steps of the sample preparation workflow are summarized in Fig. 1 . An equivalent volume of 500 μl neat oral fluid was submitted to SPE after the addition of stable isotope-labeled internal standards (10 μl, buprenorphine-D4 and norbuprenorphine-D3 with 1000 ng/ml all other with 500 ng/ml) and centrifugation (2200×g, 10 min). SPE was accomplished on Strata-X cartridges (33 μm, 200 mg/3 ml, Phenomenex, Torrance, USA). SPE columns were washed with 2 ml MeOH and equilibrated with 2 ml water. Next, the sample was rinsed through the column. After washing with 3 ml water and 2 ml 30% MeOH in water (v/v), columns were dried under vacuum for 10 min to enable elution with two times 750 μl of 2% FA in ACN (v/v). The eluate was evaporated to dryness at 25 °C under a gentle stream of nitrogen. Finally, the dry residue was reconstituted in 50 μl aqueous 0.5% HOAc solution (v/v).![]()
Overview on the principle steps of the sample preparation workflow employed for processing saliva samples collected with the Greiner Bio-One (GBO) saliva collection system
8
DNA Hydrolysis and Purification for HPLC-MS
9
Solid-phase extraction for diverse analytes
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Strata-X cartridges (500 mg, 6 mL) (Phenomenex, Le Pecq, France) are ionic-based polymeric sorbents, chosen to retain compounds according to their pKa: Strata X-AW (for pKa < 5), Strata X-CW (for pKa > 8), Strata X-A (for pKa < 2), Strata X-C (for pKa > 10.5). Oasis HLB (200 mg, 6 mL) (Waters, Milford, MA, USA) is a reversed-phase cartridge used to select a wide range of substances, including neutral, basic, acidic, and polar ones. Silica-based cartridges Strata C18 (200 mg, 6 mL) and Strata SDBL (500 mg, 6 mL) (Phenomenex, Le Pecq, France) are employed to retain neutral hydrophobic and non-polar molecules. ENV+ (500 mg, 6 mL) (Biotage, Glamorgen, UK) is a polymeric phase appropriate for the retention of polar analytes. C18/ENV+ (400 mg, 6 mL) (Biotage, Glamorgen, UK) is a layered cartridge composed of the previous ENV+ phase as the bottom layer and a C18 phase as the top layer to improve the range of analytes possibly extractable. Finally, a homemade Multilayer cartridge made of Oasis HLB (200 mg), Isolute ENV+ (150 mg), Strata X-AW (100 mg), and Strata X-CW (100 mg) was prepared according to the method developed by Kern et al. [27 (link)].
10
Extraction and Purification of Antibiotics
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Whole or skim milk samples (10 g ± 0.1 g) were added to 50-mL centrifuge tubes. Cream or casein samples (1 ± 0.01 g) and ultra-pure water (9 mL) were added to centrifuge tubes (50 mL).
Amoxicillin was extracted from whole and skim milk, casein, and cream using 1 M HCl (10 mL, Carlo Erba, Barcelona, Spain) and the supernatants underwent solid-phase extraction using Strata X cartridges (Phenomenex). The Strata X cartridges were activated with 3 mL of methanol and 3 mL of distilled water. After samples were passed through the system, the cartridge was cleaned with 3 mL of distilled water to decrease matrix interference. After 1 min of drying, the analytes were eluted with 1 mL of 0.1% trifluoroacetic acid and acetonitrile (92:8, vol/vol).
Tylosin was extracted from whole and skim milk, casein, and cream using 30 mL of acetonitrile, and the supernatants underwent solid-phase extraction using Strata X cartridges. The Strata X cartridges were activated with 10 mL of methanol and 10 mL of distilled water. After samples were passed through the system, the cartridge was cleaned with 10 mL of distilled water followed by 10 mL of 25% acetonitrile to decrease matrix interference. After 1 min of drying, the analytes were eluted with 1 mL of 0.1M ammonium acetate (Merck).
Amoxicillin was extracted from whole and skim milk, casein, and cream using 1 M HCl (10 mL, Carlo Erba, Barcelona, Spain) and the supernatants underwent solid-phase extraction using Strata X cartridges (Phenomenex). The Strata X cartridges were activated with 3 mL of methanol and 3 mL of distilled water. After samples were passed through the system, the cartridge was cleaned with 3 mL of distilled water to decrease matrix interference. After 1 min of drying, the analytes were eluted with 1 mL of 0.1% trifluoroacetic acid and acetonitrile (92:8, vol/vol).
Tylosin was extracted from whole and skim milk, casein, and cream using 30 mL of acetonitrile, and the supernatants underwent solid-phase extraction using Strata X cartridges. The Strata X cartridges were activated with 10 mL of methanol and 10 mL of distilled water. After samples were passed through the system, the cartridge was cleaned with 10 mL of distilled water followed by 10 mL of 25% acetonitrile to decrease matrix interference. After 1 min of drying, the analytes were eluted with 1 mL of 0.1M ammonium acetate (Merck).
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