Zorbax extend c18 column

In order to analyze the loading efficiency of EPI, the unloaded drug concentration of EPI-CAO-AuNPs was determined by means of HPLC (Agilent Technologies 1260, USA) at wavelength of 233 nm. A Zorbax-Extend-C₁₈ column (4.6 mm × 150 mm, 5 μm, Agilent Technologies, USA) was used for determination of EPI with acetonitrile and water as a mobile phase. The volume ratio of acetonitrile/water was 30/70 with 0.1% trifluoroacetic acid (TFA), the column temperature was remained at 25 °C, and the flow rate was 1.0 mL/min. Methanol (10 mL) was added into 500 μL of EPI-CAO-AuNPs dispersion to make it disintegrate for releasing the loaded EPI drug. Then the mixture was oscillated for 1 min, sonicated for 5 min, and centrifuged at 10,000 rpm for 10 min. The process was repeated as above steps one time. The supernatant solution was collected and measured to calculate the residual content of EPI. The entrapment efficiency (EE) and drug loading efficiency (LE) percentages were calculated according to the following equations: EE (%, w/w) = (Mass of drug in nanoparticles/Mass of initial added drug) × 100; LE (%, w/w) = (Mass of drug in nanoparticles/Mass of nanoparticles) × 100.

Tea infusions were subjected to HPLC coupled with a PDAD (Waters, Milford, MA, USA) and an Agilent Zorbax Extend-C18 column (4.6 × 250 mm, 5 µm) for determination of phenolic compounds and caffeine. The HPLC method referred to previous literature with minor modification [23 (link)]. In brief, the mobile phase A and B were methanol and 0.1% formic (v/v), respectively, and the temperature was 35 °C with the flow rate of 1.0 mL/min. The elution gradient was set as follows: 5% A (0 min), 20% A (10 min), 22% A (15 min), 25% A (20 min), 40% A (40 min), 42% A (50 min), 50% A (60 min), 95% A (70 min), 5% A (70.1 min) and 5% A (75 min). Phenolic compounds and caffeine in tea infusions were identified via comparing their retention time and ultraviolet-visible (UV-vis) spectra with those of standard compounds. Quantitative analysis was performed according to the peak area under the maximal absorbance wavelength, and the contents were expressed as mg/g DW. The limit of detection (LOD) and limit of quantity (LOQ) of this method were 2 and 5 µg/mL, respectively.

The HPLC analysis of DH herbal pair extract with different proportions (20 mg/mL) was performed on the Agilent 1290 liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA). The Agilent Zorbax Extend C-18 column (150 mm × 3.0 mm, 3.5 μm) was used for separation at a flow rate of 0.4 mL/min. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). The detailed elution program was as follows: 5–8% B at 0–6 min, 8–10% B at 6–14 min, 10–15% B at 14–23 min, 15–17% B at 23–38 min, 17–22% B at 38–48 min, 22–26% B at 48–58 min, 26–40% B at 58–68 min, 40–45% B at 68–73 min, 45–60% B at 73–83 min, 60–90% B at 83–93 min, 90–100% B at 93–95 min, and 100% B at 95–98 min. The injection volume was 5 μL, the column temperature was 35 °C, and the UV detection wavelength was 280 nm.

An Agilent 1100 HPLC system was used for reversed-phase liquid chromatography
(RPLC) separation with an Agilent Zorbax Extend-C18 column (5 μm,
150 nm × 2.1 mm) and UV detection at 210 nm and 280 nm. Mobile
phase A and mobile phase B were set to ACN-H₂O (2:98, v/v)
and ACN-H₂O (90:10, v/v), respectively, and the flow rate
was set to 300 μL/min. The gradient elution conditions were
as follows: 0–8 min, 98% A; 8.00–8.01 min, 98–95%
A; 8.01–38 min, 95–75% A; 38–50 min, 75–60%
A; 50–50.01 min, 60–10% A; 50.01–60 min, 10%
A; 60–60.01 min, 10–98% A; and 60.01–65 min,
98% A. The samples were collected between 8 and 50 min; the eluate
buffer was collected every minute into centrifuge tubes numbered 1–15
and cycled in this order until the end of the gradient. After collection,
frozen samples were prepared for mass spectrometry.

Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were quantified using modified ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) by the Lam method [39 (link)]. Octadeuterated endocannabinoids AEA-d₈ and 2-AG-d₈ were added as internal standards to the cell lysates, and all cannabinoids were isolated using solid phase extraction (SPE). UPLC–MS/MS analysis was performed using an Agilent 1290 UPLC system with a Zorbax Extend C18 column (2.1 × 150, 1.8 mm, Agilent, Santa Clara, CA, USA) and interfaced with an Agilent 6460 triple quadrupole mass spectrometer with an electrospray ionization source (ESI). The samples were analyzed in positive-ion mode using multiple reaction monitoring (MRM). Transition of the precursor to the product ion was as follows: m/z 348.3→62.1 for AEA; m/z 379.3→287.2 for 2-AG. The LODs were as follows: 2 pg/mL for AEA and 40 pg/mL for 2-AG. Analyses were performed in three independent experiments. Obtained results were normalized for milligrams of protein. Endocannabinoids concentrations are expressed as a percentage of the concentrations found in control cells (15.9 ± 0.7 and 238 ± 16 fmol/mg protein for AEA and 2-AG, respectively).