C18 reverse phase column

PB standard concentrations from 0.04 to 0.8 mg/mL were made in acetonitrile: water (98:2% v/v) ratio. The flow rate was kept at 1.5 mL/min with an auto-sampler injection volume of 40 µL. The HPLC setup was a low-pressure gradient Shimandzu Prominence HPLC system (Pump model: LC-20AT, UV detector: SPD-20A and injector model: SIL-20AC, Shimandzu Corporation, Kyoto, Japan) at a wavelength of 242 nm. The separation was performed using a Phenomenex C-18 reverse phase column with an internal diameter of 4.5 mm and a length of 25 cm attached with the guard column.
PB extraction from blood and other tissues was performed by mixing serum or tissues with ice-cold acetonitrile in a 1:1 ratio. After centrifugation, 40 µL of purified serum (supernatant) was mixed with 160 µL mobile phase acetonitrile, gently vortexed for 20 s, and then centrifuged at 15,000 rpm for 20 min. After this, 20 µL of supernatant was transferred for analysis to an auto-sampler HPLC vial.

For the analysis of the samples, an Agilent Technologies 1200 high-resolution liquid chromatograph (HPLC) consisting of a quaternary array of pumps (Agilent Technologies G1311A, Santa Clara, CA, USA) connected to an automatic injector (Agilent Technologies G1329A, Santa Clara, CA, USA) was used. A total 20 μL of the tissue extract was injected and subjected to chromatography with an isocratic elution system with a flow rate of 0.6 mL min⁻¹ in a C₁₈ reverse-phase column (Phenomenex, Torrance, CA, USA) of 250 mm × 4.6 mm. The samples were analyzed with a fluorescence detector (Agilent Technologies G1321A, Santa Clara, CA, USA) at an emission length of 280 nm and an excitation length of 340 nm. The presence of compounds in the analyzed samples was determined by the retention times of IAA and of IAA-Ala, IAA-Leu, IAA-Glu, and IAA-Asp conjugates (Supplementary Figures S1–S3), for which co-injections of the standards and the samples were analyzed, to determine whether they co-eluted. The calibration curves were made with free IAA and the conjugate standards using the area under each curve for each compound.

Chromatographic analysis was performed on a reverse-phase Shimadzu HPLC system (Shimadzu Corp., Kyoto, Japan) with a Shimadzu LC-20AR solvent pump, coupled to a SPD-20A UV/VIS detector. Separation was performed on a Phenomenex C₁₈ reverse-phase column (4.6 × 150 mm, 5 μm) using a gradient solvent system comprising acetonitrile (A) and water (B), with a composition by volume of 10% A at 0 min and 50% A at 40 min. The flow rate was 2 mL/min; the reaction was monitored spectrophotometrically at 254 nm.

D-cyphenothrin was quantified with Waters 2690 HPLC system equipped with a Phenomenex C₁₈ reverse phase column (250 nm × 4.60 mm, 5 μm) and UV detector. Mobile phase was composed of acetonitrile and deionized water (75:25) at a flow rate of 1.0 mL·min⁻¹. Injection volume and detection wavelength were 10 μL and 253 nm, respectively. Retention times of D-cyphenothrin, permethrin, bifenthrin, tetramethrin, allethrin, and chlorempenthrin remained as 8.97, 14.04, 20.99, 6.78, 7.22, and 10.78 min, respectively.
D-cyphenothrin metabolites were identified in Agilent 6890N/5975 GC/MS system equipped with auto-sampler, an on-column, split/splitless capillary injection system, and HP-5MS capillary column (30.0 m × 250 μm × 0.25 μm) with array detector. Helium was used as carrier gas at a flow rate of 1.5 mL·min⁻¹. Analytical mode was scanned from 30–500 nm. The column temperature was first held at 90 °C for 2 min, raised at 6 °C·min⁻¹ to 150 °C for 1 min, 10 °C·min⁻¹ to 180 °C for 4 min, and finally 20 °C·min⁻¹ to keep at 260 °C for 10 min. Temperatures corresponding to transfer line and ion source were 280 and 230 °C, respectively. The column outlet was directly inserted into electron ionization source block at 70 eV. The injection volume was 1.0 μL with splitless sampling at 250 °C [37 (link)].

The amount of RG7388 and Entinostat within NPs
was assessed using
high performance liquid chromatography (HPLC) and absorbance spectrometry,
respectively. The NP pellet was lysed using a mixture of 1:1 acetonitrile
and dimethyl sulfoxide (DMSO) to release any entrapped drug. RG7388
entrapment was detected by HPLC using a C18 reverse phase column (Phenomenex,
150 × 4.6 mm, 5 μM). The flow rate was set to be constant
at 1 mL/min at 25 °C. 30 μL of 1 mg/mL of sample was injected
per run, and the absorbance was detected at 273 nm and compared to
a series of standards prepared by spiking known amounts of free RG7388
into blank NPs (BNPs) in 1:1 acetonitrile:DMSO. Entinostat entrapment
was quantified by measurement of absorbance at 330 nm using a plate
reader (Biotek) and again compared to a series of standards prepared
by spiking known amounts of free Entinostat into BNPs in 1:1 acetonitrile:DMSO
(Supplementary Figure 1). Drug loading
was calculated using the formula below.