The concentrations of NIRB and DOX in samples were quantified using both HPLC–UV and MS analysis. Briefly, NIRB and DOX were extracted from liposome samples by tenfold dilution with methanol. The resultant samples were then centrifuged at 3000 RPM, 4 °C for 15 min in glass centrifuge tubes to separate extracted drug from lipid. The supernatant was then assayed directly for HPLC–UV or diluted accordingly for MS detection.
HPLC–UV analysis of samples was performed using an Agilent 1260 infinity series LC (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was achieved using an Agilent Eclipse XDB-C18 column (4.6 × 150 mm, 5.0 μm) at 25 °C with a mobile phase composed of acetonitrile and methanol 50:50 (A) and 50 mM ammonium acetate, pH 4 (B). The initial mobile phase was 40% A with a flow rate of 1.0 mL/min, which was gradually increased to 55% A over 4 min. Following a 4-min equilibration, the composition was changed back to 40% A over a duration of 30 s. The mobile phase was then maintained for another 1.5 min until run completion. Detection of both drugs was achieved using an Agilent 1260 Infinity II Diode Array Detector with detection of NIRB and DOX at 310 nm and 480 nm wavelengths, respectively.
HPLC–MS analysis was performed using an Agilent 1260 infinity series LC equipped with an Agilent EC-C18 column (2.1 × 50 mm, 1.9 μM) heated to 40 °C. A gradient elution was applied using methanol (A) and water (B) both with 0.1% formic acid (v/v). The initial mobile phase was 75% A with a flow rate of 0.3 ml/min which was gradually decreased to 0% A over 4 min. This composition was maintained for another 6 min before it was rapidly changed back to 75% A where it was maintained for the remaining 3 min of the run.
Mass spectrometry detection of NIRB and DOX was achieved using a ThermoFisher Scientific TSQ Endura Triple Quadrupole Mass Spectrometer (Mississauga, ON, Canada). Analysis was performed on positive ion mode with optimal ion source settings as follows: spray voltage of 3500 V, sheath gas of 5 a.u., auxiliary gas of 2 a.u., and ion transfer tube temperature of 275 °C. Selected precursor ions for NIRB were m/z 321.2 → 180.0, 205.0, 207.0, 232.0, 304.083 while precursor ions for DOX were m/z 544.2 → 320.9, 345.9, 361.0, 378.9, 396.9. Collision energies ranged from 19.0 V to 42.0 V for NIRB and 11.2 V to 41.2 V for DOX.
The encapsulation efficiency of drug in liposomes was calculated using the following equation:
HPLC–UV analysis of samples was performed using an Agilent 1260 infinity series LC (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was achieved using an Agilent Eclipse XDB-C18 column (4.6 × 150 mm, 5.0 μm) at 25 °C with a mobile phase composed of acetonitrile and methanol 50:50 (A) and 50 mM ammonium acetate, pH 4 (B). The initial mobile phase was 40% A with a flow rate of 1.0 mL/min, which was gradually increased to 55% A over 4 min. Following a 4-min equilibration, the composition was changed back to 40% A over a duration of 30 s. The mobile phase was then maintained for another 1.5 min until run completion. Detection of both drugs was achieved using an Agilent 1260 Infinity II Diode Array Detector with detection of NIRB and DOX at 310 nm and 480 nm wavelengths, respectively.
HPLC–MS analysis was performed using an Agilent 1260 infinity series LC equipped with an Agilent EC-C18 column (2.1 × 50 mm, 1.9 μM) heated to 40 °C. A gradient elution was applied using methanol (A) and water (B) both with 0.1% formic acid (v/v). The initial mobile phase was 75% A with a flow rate of 0.3 ml/min which was gradually decreased to 0% A over 4 min. This composition was maintained for another 6 min before it was rapidly changed back to 75% A where it was maintained for the remaining 3 min of the run.
Mass spectrometry detection of NIRB and DOX was achieved using a ThermoFisher Scientific TSQ Endura Triple Quadrupole Mass Spectrometer (Mississauga, ON, Canada). Analysis was performed on positive ion mode with optimal ion source settings as follows: spray voltage of 3500 V, sheath gas of 5 a.u., auxiliary gas of 2 a.u., and ion transfer tube temperature of 275 °C. Selected precursor ions for NIRB were m/z 321.2 → 180.0, 205.0, 207.0, 232.0, 304.083 while precursor ions for DOX were m/z 544.2 → 320.9, 345.9, 361.0, 378.9, 396.9. Collision energies ranged from 19.0 V to 42.0 V for NIRB and 11.2 V to 41.2 V for DOX.
The encapsulation efficiency of drug in liposomes was calculated using the following equation:
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