Hospital Pharmacy - June 2020 - 190

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of this study is to determine the physicochemical stability of
lorazepam 167 µg/mL in polypropylene syringes at 5 ± 3°C
and to compare results with stability data of lorazepam in
syringes stored at room temperature, in glass bottles at 5 ±
3°C, and in glass bottles at room temperature.

Method
Solutions Preparation
Eight polypropylene syringes (Becton Dickinson, lot
1709211, Madrid, Spain) and 6 glass bottles were prepared
under aseptic conditions. Each was prepared by diluting 1
mL of lorazepam solution 4 mg/mL (Temesta®; Pfizer, lot
4070, Brussels, Belgium) in 23 mL of sodium chloride solution (NaCl 0.9%; Baxter, lot 18A14G60, Lessines, Belgium)
to obtain a final lorazepam concentration of 167 µg/mL.

Storage
Five syringes and 3 bottles were stored at 5 ± 3°C. Three
syringes and 3 bottles were stored at room temperature.
Immediately after the preparation (day 0) and after 1, 2, 3, 4,
and 7 days, 1 mL of each solution was withdrawn from each
syringe and bottle to perform the physical stability tests
while 20 µL intended for chemical stability evaluation were
frozen at 80°C.

Physical Stability
At each time point, samples were visually inspected with the
unaided eye in front of a black and a white background to check
the absence of particle, haze, precipitation, and color change.
The samples were also centrifuged at 2150 g for 5 minutes
(Heraeus multifuge 1S; Thermo Scientific, Waltham,
Massachusetts) before observing the pellet under an optical
microscope 12.5× (Jenamed; Carl Zeiss, Germany) to search
crystals.
The optical densities (ODs) were measured with a spectrophotometer (Genesys 10 UV; Spectronic Unicam, Thermo
Scientific, Waltham, Massachusetts) at 350, 410, and 550 nm to
detect subvisible particles and assess turbidity of solution.11
The pH of each sample was measured with a glass
-electrode pH meter (InoLab; WTW GmbH, Germany).

Chemical Stability
Chromatographic conditions
A high-performance liquid chromatography (HPLC) system
connected to an ultraviolet (UV) detector (Alliance 2695,
detector 2489; Waters, Milford, Connecticut) and a processing software (Empower 3 software; Waters) was used.
The chromatographic separation was performed on
a reverse phase column (Synergi 4µm Hydro-RP 80A

Hospital Pharmacy 55(3)
150 × 4.6 mm; Phenomenex, Torrance, California) with a
mobile phase containing 65% of KH2PO4 (Merck, lot
A0360673202, Darmstadt, Germany) 10 mM pH 2.4
(adjusted with phosphoric acid 85 wt. % in water, SigmaAldrich, lot MKCB7708, St. Louis, Missouri) and 35% of
acetonitrile (Biosolve, lot 1228451, Dieuze, France). The
flow rate was set at 2.5 mL/min, the column temperature at
30°C, and the detection wavelength at 220 nm.
Five standard solutions (26, 50, 99, 160, 200 µg/mL of
lorazepam) and 3 control levels (26, 99, 200 µg/mL) were
prepared before each analysis. Fifty microliters of 1/10
diluted calibrators, controls, and therapeutic solutions were
analyzed during a run time of 7 minutes. The elution time of
lorazepam was 4 minutes.

Validation of the HPLC Method
The method was validated according to precision, linearity,
limit of detection (LOD), limit of quantification (LOQ), and
stability indication, as recommended in the ICH Q2(R1)
guidelines.12
Precision.  The within- and between-day variations were calculated based on 3 levels of lorazepam concentration
(approximately 26, 99, and 200 µg/mL) with 10 repetitions
for each level.
Linearity.  Linearity was evaluated by the analysis of 14 solutions obtained from the 2-fold serial dilution in water of a
200 µg/mL lorazepam solution.
LOD and LOQ.  Ten blank (mobile phase) measurements were
used to determine LOD and LOQ. They were calculated as
follows:
LOD = mean + 3SD.
LOQ = mean +10SD.
Stability Indication. The stability-indicating capability of
the chromatographic method was assessed using partially
decomposed solutions of lorazepam. The solution was analyzed in natural (pH 5.8), acidic (adjusted at pH 1.7 with
HCl 5M), and basic (adjusted at pH 12.3 with NaOH 5M)
conditions before and after heating at 100°C for 1 hour.

Chemical Stability Study
All frozen samples withdrawn from a same container were
injected the same day in HPLC to reduce results variability
due to experiment conditions.
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