Uv 2075plus intelligent uv vis detector

Reversed-phase liquid chromatography was performed using a Jasco system containing an UV-2075Plus Intelligent UV/Vis Detector, an AS-2050Plus Intelligent Sampler, an LG-2080-02 Ternary Gradient Unit, a PU-2080Plus Intelligent HPLC Pump, a DG-2080-53 3-Line Degasser, and an LC-NetII/ADC (Jasco, Gross-Umstadt, Germany). The mobile phase acetonitrile/water 10:90 was degassed prior to use and the flow rate was 1 mL min⁻¹ with the following gradient: a linear gradient for 2 min from 89% solvent A (water containing 10% acetonitrile) to 33% solvent B (100% acetonitrile) holding it for 4 min, a linear gradient for 4 min to 100% solvent B holding it for 2 min, a linear gradient for 0.1 min to 89% solvent A holding it for 3 min. A reversed-phase ec MN Nucleodor C18 (5 µm, 4.0 × 125 mm²) column (Macherey-Nagel, Düren, Germany) was adjusted to 40 °C by a column thermostat (W.O. Electronics, Austria). For each measurement, 10 µL of a sample was injected and the absorbance was monitored at 240 nm. The relative product level was evaluated using the relative peak area of the product compared to the combined peak areas of educt and product. The values of the three samples were averaged out (mean average), and standard deviations were calculated.

To observe changes in the appearance and pH and for high-performance liquid
chromatography (HPLC), we first slowly added 0.4 mL of each sample dropwise to
8.4 mL of PPN solution, and we observed the visual changes (color tone/turbidity)
immediately after the dropwise addition. After recording changes in appearance, we stirred the
mixture for 30 sec using a vortex mixer and measured the pH and midazolam concentration
of each sample. The pH was measured using a pH meter (F-72 pH/ION Meter, HORIBA, Ltd.). The
midazolam content was analyzed through HPLC with reference to Kobo’s method,⁸ under the following conditions: column, TS Kgel ODS
- 100 V (5 μm, 4.6 mm×15 cm, Tosoh Corp.); column temperature, room
temperature, flow rate, 0.8 mL/min; detector, UV - 2075 Plus intelligent UV/Vis detector
(JASCO Corp.); detection wavelength, 254 nm, mobile phase, 1 mM citrate-phosphate
buffer (pH 5.0): methanol: acetonitrile=1:1:1 (V/V). The measurement was performed using
an absolute calibration curve method using a midazolam injection solution as a standard
solution. The calibration curve showed linearity in the range 3.1–50.0 μg/mL. We
determined the relationship between the midazolam content as measured through HPLC and the
midazolam concentration of the mixture by fitting the approximate curve. We performed a
Pearson’s regression analysis to determine the correlation.