Hydrochloric acid (hcl)

Analytical grade reagents were required in the experimental procedures: n-Hexane (96%), methanol (MeOH, ≥99%), and ethanol absolute (EtOH, ≥99.8) for HPLC gradient quality; acetonitrile (ACN) and formic acid (FA) of MS quality. Hydrochloric acid (HCl, 37%) and sulfuric acid (H₂SO₄, 95–97%) were supplied by Scharlab (Barcelona, Spain). Dimethyl sulfoxide (DMSO, ≥99.9%), 2N Folin–Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH, ≥99.9%), sodium methoxide (95%), trichloroacetic acid (TCA, 99%), thiobarbituric acid (≥98%), sanitary ethanol (96%), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), curcumin (>65%), and LB broth with agar and Tryptic Soy Broth (TSB) were all supplied by Sigma-Aldrich (St. Louis, MO, USA). Curcumin was stored at −20 °C prior to analysis. Amyloid β protein fragment 1–42 (Aβ_1–42) (A9810, CAS: 107761-42-2, MW 4514.04 g mol⁻¹) was also obtained from Sigma-Aldrich (St. Louis, MO, USA) for incubation assays and stored at −80 °C until analysis. The standard Trolox (≥97%) was provided by Sigma-Aldrich (Burghasen, Germany). 2-Propanol for HPLC gradient (C₃H₈O, PrOH), Aluminum chloride 6-hydrate (99%), sodium carbonate anhydrous, sodium hydroxide (NaOH, 98%), p-iodonitrotetrazolium chloride (INT, 98%), ferrous-sulfate heptahydrate (FeSO₄·7H₂O, 98%), and sodium nitrite (≥98%) were obtained from Panreac (Barcelona, Spain). Silica was purchased from Fisher Scientific (Pittsburgh, PA, USA).
Fatty acid standards FAME 37 component SUPELCO Ref CRM47885, tridecanoic acid (C13:0, ≥99%), and ethyl nonanoate (˃98%) were purchased from Sigma-Aldrich (Barcelona, Spain).
α-tocopherol (≥96%), γ-tocopherol (≥96%), and δ-tocopherol (≥90%) standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). β-tocopherol stock solution was supplied by Sigma-Aldrich (St. Louis, MO, USA) and was already dissolved in methanol, and the remaining stock solutions of 1000 mg·L⁻¹ were prepared, dissolving the adequate quantity of analyte in methanol as well. Those solutions were later further diluted for the purpose of a calibration curve into solutions of 0.3, 0.5, 2.0, 5.0, 10.0, 20.0, 50.0, 75.0, and 100 mg·L⁻¹. Stock solution stability was also considered, and it was established in three months, at the very least, via conservation in darkness in the freezer at −20 °C.
Phenolic standards gallic acid monohydrate (≥98.0%), chlorogenic acid (≥95.0%), dihydroxybenzoic acid (≥97.0%), caffeic acid (≥98.0%), catechin (≥98.0%), p-coumaric acid (≥98.0%), epicatechin (≥98.0%), rutin trihydrate (≥95.0%), trans-ferulic acid (98%), myricetin (≥96.0%), resveratrol (≥99.0%), quercetin (≥95.0%), kaempferol (≥97.0%), and naringin (≥95.0%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Hesperidin (≥98.0%) was provided by European Pharmacopoeia. Phenolic stock solutions (200 mg·L⁻¹) were prepared in MeOH, an ethanol–water mixture 80:20 (v/v) (quercetin), or a 5% (v/v) DMSO aqueous solution (hesperidin). They were stored in the dark at 4 °C or at −80 °C (hesperidin, trans-ferulic, myricetin, and caffeic acid) for up to one month. Fresh working standard solutions were prepared daily by diluting stock solutions as required. Nylon membrane filters with 0.22 μm pore size (Teknokroma, Barcelona, Spain) were used for mobile phase filtration before chromatographic analysis.
The antimicrobial activity of the extracts was evaluated against Gram-positive bacteria Staphylococcus aureus (ATCC 29213) and Gram-negative bacteria Escherichia coli (ATCC 25922). Streptomycin sulfate salt was used as a positive standard and was provided by Sigma-Aldrich (St. Louis, MO, USA).

Histamine dihydrochloride (HIS), benzoyl chloride (BC), tetraethyl orthosilicate (TEOS), and zinc nitrate hexahydrate solution (Zn(NO₃)₂·6H₂O) were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Ammonia solutions with a concentration of 25% (w/v), high-purity sodium silicate, acetonitrile, methanol, 2-propanol, acetone, and ethanol (of HPLC grade) were obtained from Merck (Darmstadt, Germany). Sodium hydroxide (NaOH), sodium chloride (NaCl), and hydrochloric acid (HCl) were purchased from Scharlau (Barcelona, Spain). Ultrapure water (DI) was used as the primary water source in all experiments. The Accurel Q3/2 polypropylene hollow fiber was purchased from Membrana GmbH (Wuppertal, Germany).

Mitoxantrone (98%) was purchased from Bosche Scientific (New Brunswick, NJ, USA). Tetraethyl orthosilicate (TEOS, 98%), hexadecyltrimethylammonium bromide (CTAB ≥ 98%), dichloromethane anhydrous (DCM, ≥ 99.8%), iron (II) chloride tetrahydrate (FeCl₂·4H₂O, 98%), iron (III) chloride hexahydrate (FeCl₃·6H₂O, 97%), titanium (IV) oxysulfate solution (TiOSO₄, 1.9 − 2.1%), hydrogen peroxide (H₂O₂, 32%), glacial acetic acid (≥ 99%), ammonium hydroxide solution (NH₄OH, 30–33%), 3-(triethoxysilyl)propyl isocyanate (95%), human serum (HS), dimethyl sulfoxide (DMSO), Dulbecco’s Modified Eagle’s Medium (DMEM) (with 4,5 g L^− 1 of glucose, stable glutamine and sodium bicarbonate), trypsin (1X), penicillin-streptomycin, L-glutamine, non-essential aminoacids, phosphate buffered saline (PBS) and Thiazolyl Blue Tetrazolium Bromide (MTT) were obtained from Sigma-Aldrich (Madrid, Spain). Absolute ethanol (EtOH), petroleum ether, sodium hydroxide and hydrochloric acid (35% w/w) were bought from Scharlab (Barcelona, Spain). Triton X-100 was purchased from PanReac AppliChem (Barcelona, Spain). DAPI (4′,6-diamidino-2-phenylindole) and Alexa Fluor 647 Conjugated Wheat Germ Agglutinin (WGA) were obtained from Fisher Scientific (Madrid, Spain).

Prednisolone sodium phosphate (PSP) and prednisolone base (PB), both of USP grade, were obtained from Medisca (Mascot, NSW, Australia). Excipients in the CDS tablets were polyethylene glycol (PCCA, Matraville, NSW, Australia), hydrogenated polyoxyl 40 castor oil (Cremophor^® RH40, BP grade, Ingredient Plus, Rydalmere, NSW, Australia), xanthan gum (PharmAust, Malaga, WA, Australia), steviol (100% pure organic stevia extract powder, Hemmant, QLD, Australia), and Jamaica dark chocolate (Springer Foods, Myaree, WA, Australia). Chemicals used in the study include methanol (Macron Fine Chemicals, Center Valley, PA, USA), hydrochloric acid (Scharlau, Barcelona, Spain), hydrogen peroxide (Ajax Finechem, Sydney, NSW, Australia), acetonitrile, and sodium hydroxide pellets (AR grade, Merck, Darmstadt, Germany). Double deionised water (PSI Water Filters, South Launceston, TAS, Australia) and HPLC-grade organic solvents were used throughout.

CNB (99.79% purity) and CNB-H impurity were provided from Metrochem API Pvt Ltd, India. Xcopri® tablets were supplied from SK life science, Inc. Paramus, NJ07652. The tablets are labeled to contain 100.0 mg CNB. Both acetonitrile and methanol were HPLC grade and obtained from Scharlau, Spain. Diluent is prepared by mixing acetonitrile and purified water in the ratio of 40:60, % v/v. Excipients used in preparation of spiked placebo include microcrystalline cellulose (FMC, USA), colloidal silicon dioxide, (Evonik, Germany), lactose monohydrate (DFE Pharma, Germany), sodium starch glycolate (JRS Pharma, USA), magnesium stearate (Italmatch, Italy), and film coating agents: FD&C Blue# 2/indigo carmine aluminum lake, iron oxide red, iron oxide yellow (Karma, Egypt), polyethylene glycol 3350 (IMCD, Egypt), polyvinyl alcohol-part hydrolyzed (East Hony, China), Talc (Elgomohorya, Egypt), and titanium dioxide (Kronos, USA). Hydrogen peroxide (Merck, Germany, 30%), hydrochloric acid (Scharlau, Spain, 36.5–38%) and sodium hydroxide pellets (Scharlau, Spain, 98.5%) were utilized in the study of degradation conditions.

Primary polyclonal antibodies, such as rabbit anti-E. coli, were used (GTX13626, GeneTex, Irvine, CA, USA).
Rabbit anti-E. coli serotype O/K (PA1-7213, ThermoFisher, Waltham, MA, USA), rabbit anti-E. coli serotype O/K (ab31499, Abcam, Cambridge, UK), and polyclonal (chicken) anti-B-Galactosidase (AB3403-I, Sigma-Merck, San Luis, MO, USA) were used.
As primary monoclonal antibodies, mouse anti-E. coli LPS (ab35654, Abcam) and mouse anti-E. coli (6911,35-660, Prosci-Inc., Poway, CA, USA) were used.
The specific antigens were different concentrations of LPS from Escherichia coli eBioscience™ (Thermo Fisher Scientific, Waltham, MA, USA).
Indirect ELISA reagents included H₂SO₄ solution, 0.1 M hydrochloric acid (Scharlab, Barcelona, Spain), and commercial TMB substrate (Thermo Scientific, Waltham, MA, USA).
The secondary antibodies used comprise GARPO polyclonal anti-rabbit IgG peroxidase (Abcam, Cambridge, UK) and GAMPO polyclonal anti-mouse IgG peroxidase (Abcam, Cambridge, UK).
Samples were inoculated in a pH 9.6 0.05 M carbonate buffer. This buffer was chosen to maintain a stable pH environment for antigen–antibody interaction. Each sample was inoculated in duplicate to ensure reproducibility and reliability of the results.
The carbonate buffer was sourced from Merck (Darmstadt, Germany). The negative control was PBS (phosphate-buffered saline) solution. A conventional solution of lipopolysaccharides (LPSs) from Escherichia coli was used as the positive control. LPS is a component of the outer membrane of Gram-negative bacteria like E. coli and serves as a reliable positive control to verify the sensor’s functionality and sensitivity.
Seven serial dilutions were created from 1 mL of E. coli antigens from strains 101, 425, 418, and 4558 (Spanish Type Culture Collection, CECT, Valencia, Spain). These dilutions were prepared in 0.05 M carbonate buffer pH 9.6, resulting in 10 to 10⁵ CFU/mL concentrations.
A stock solution of LPS E. coli antigen from Escherichia coli eBioscience™ was prepared, and the diluent used was 0.05 M carbonate buffer at pH 9.6. Seven serial dilutions of LPS E. coli antigen from Escherichia coli eBioscience™ (Termo Fischer Scientific, Waltham, MA, USA) were prepared at concentrations from 0.06 ng/mL to 4 ng/mL in 0.05 M carbonate buffer pH 9.6.
Absorbance measurements were conducted at two wavelengths, 450 nm (OD450) and 650 nm (OD650), with the use of the Varioskan Flash multimode scanning microplate reader (Multilabel Victor 1420 Counter). The criteria for positive control designation (P/N 2.1) were based on the OD450 values being 2.1 times higher than the negative controls.