Characterizing the Stability of Angiotensin II in 0.9% Sodium Chloride Using High Performance Liquid Chromatography and Liquid Chromatography Tandem Mass Spectrometry

Abstract

Purpose: The purpose of this study was to evaluate the stability of angiotensin II in 0.9% sodium chloride for up to 5 days. Methods: We prepared angiotensin II dilutions, by aseptically diluting 2.5 mg (1 mL) in 249 mL 0.9% sodium chloride creating a solution of 10 000 ng/mL. Admixtures were stored under refrigeration (5 ± 3°C). Stability of the dilution was assessed by: preservation of clarity, consistency of pH, and retention of concentration. Solutions were sampled at times 0, 24, 48, 72, 96, 120 hours. Solutions were analyzed via High-Performance Liquid Chromatography (HPLC-UV) and Liquid Chromatography Mass Spectrometry (LC-MS/MS). Retention of concentration was set a priori at > 90% of initial concentration. Results: Clarity, color, and pH at all sample time points remained constant. Both methods of analysis confirmed similar results. When stored under refrigeration, the concentration of angiotensin II solution remained above 90% of initial concentration throughout the entire sampling period. Conclusions: Angiotensin II in 0.9% sodium chloride stored in infusion bags under refrigeration (5 ± 3°C) maintained at least 90% of their original concentrations for up to 5 days. Stability was also demonstrated based on turbidity, color, and pH assessment.

Keypoints

• Refrigerated solutions of angiotensin II in 0.9% sodium chloride retained >90% concentration out to 5 days.

• Angiotensin II can have a post-dilution refrigeration shelf life of up to 5-days.

Introduction

Angiotensin II (ANGII) is a parenteral vasopressor indicated for the treatment of hypotension in adults with septic or other distributive shock. ¹ Its distinctive mechanism of action targets endogenous angiotensin II receptors resulting in vasoconstriction and increased aldosterone release, which raises blood pressure. Several studies and case reports have investigated the efficacy and safety of ANGII and have shown several advantages over conventional catecholamine vasopressors including rapid correction of hypotension, reduction in excessive catecholamine use, and favorable safety profile.^{2 -5} In addition, in patients receiving kidney transplantation, ANGII has been shown to reverse perioperative hypotension with less arrhythmias compared to catecholamine vasopressors with theoretical benefits on glomerular filtration and renal vasoconstriction potentially decreasing the risk of ischemia and acute kidney injury (AKI) post-transplant.^6,7 Despite these findings, ANGII has an average manufacturer price of $1800/mL (2.5 mg), making it about 17 times more expensive than norepinephrine and 10 times that of epinephrine which could result in underutilization by institutions who cannot justify its cost.^{8 -13}

Extended stability data exists for existing first and second line catecholamine (ie, norepinephrine and epinephrine) allowing for increased flexibility of use, margin, and workflow optimization in the drug solution preparation process, serving as a benefit for institutions.^{8 -12} According to prescribing information, ANGII dilutions should be discarded after 24 hours stored at room temperature or under refrigeration. ¹ The European Medicines Agency Assessment Report suggests use of ANGII may be feasible up to 36 hours after preparation, based on its review of internal data. ¹⁴ However, without additional extended use stability data, the risk of preparation (without use) creates a lack of margin which remains costly. This prospective in vitro study was designed to evaluate the extended use stability of diluted ANGII for a period up to 5 days after preparation.

Methods

Sample Preparation

Dilution of ANGII (angiotensin II, 2.5 mg/mL, 1 mL vial, lot 2457-113-A; La Jolla Pharmaceutical Company, San Diego, California) to a nominal concentration of 10 000 ng/mL was performed under aseptic conditions in an International Standards Organization class 7 environment^1,15 by adding 2.5 mg of angiotensin II to 249 mL of 0.9% sodium chloride for injection (0.9% sodium chloride for injection, 250 mL in polyvinyl chloride bag, lot Y392648; Baxter International, Deerfield, Illinois). Accurate volumes were achieved by injecting exact volumes of 0.9% sodium chloride into an empty polyvinyl chloride bag utilizing a graduated syringe. The preparation was done in duplicate, as well as completed a second time for confirmation analysis. The prepared solutions were then stored in the refrigerator (5 ± 3°C) under normal florescent lighting. ANGII samples (0.5 mL aliquots) were obtained from each of the stock bags kept under the specified storage conditions utilizing sterile technique, placed into cryovials, labeled, and stored at −80°C until analysis. This was done at time zero and repeated every 24 hours for 5 days. Sterility of samples was not assessed.

Physical Evaluation

Quantitative and qualitative physical stability was assessed every 24 hours and included assessments of pH, color, and turbidity for the 5 consecutive days of the study period. The pH meter (Seven2Go, Mettler Toledo, Columbus, OH) was calibrated with commercially available standards at the beginning of each testing session. Visual examinations were used for color changes against and white and black background and for the formation of precipitate to assess turbidity.

Sample Analysis

ANGII concentrations were determined by 2 validated methods to cross-validate and confirm results. We utilized high-performance liquid chromatography (HPLC-UV) for set #1 and Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) for set #2.

High-performance liquid chromatography methodology

ANGII concentrations were determined using HPLC with ultraviolet light detection. The HPLC system consisted of an autosampler (Shimadzu SiL-20AC), a delivery pump (Shimadzu LC-20AD), a C8 biUK-C8 column, 3 μm, 50 mm × 2 mm (part no. UK822; Shimadzu, Columbia, Maryland), a variable wavelength ultraviolet light detector set to 254 nm (Shimadzu SPD-20A), a controller (Sciex Analyst 1.6.3), and a solvent waste container. Mobile phase A consisted of 0.1% Trifluoroacetic Acid (v/v) in water, and mobile phase B consisted of 100% acetonitrile. The mobile phase was run in a gradient elution program. For the first 1.0 minute solution B was 10%, at 5.0 minutes solution B was increased to 80% and maintained at the same level until 6.1 minutes when solution B was changed to initial condition to equilibrate the column until 7 minutes. The flow rate was set at 0.5 mL/min. Angiotensin II (Giapreza, LaJolla Pharmaceutical Company, San Diego, CA, Lot: 2457-113-A) was used for validation and quality control of the HPLC assay. Given the control was from a Food and Drug Administration (FDA) regulated product, the purity falls within the acceptable allowance per FDA regulations. Angiotensin II samples (20 µL) were then injected into the HPLC system for analysis.

Liquid chromatography tandem mass spectrometry methodology

Additional ANGII samples were analyzed for retention of concentration using liquid chromatography mass spectrometry (LC-MS/MS). The LC-MS/MS system consisted of XBridge Peptide BEH C₁₈ Column, 130 Å, 5 μm, 2.1 mm × 50 mm (part no. 186003574; Waters Corporation, Milford, MA) with an attached guard column (SecutrityGuard EVO-C₁₈ 2 × 3 mm part no. AJ0-9297; Phenomonex, USA). Mobile phase used for chromatography was water containing 0.1% (v/v) formic acid (solution A), and methanol containing 0.1% formic acid (solution B). The mobile phase was run in a gradient elution program. For the first 1.0 minute solution B was 5%, at 3.0 minutes B was increased to 85% and maintained at the same level for next 2 minutes (until 5 minutes), at 5.5 minutes solution B changed to the initial condition to equilibrate the column until 8 minutes. The mobile phase flow was 0.3 mL/min. Mass spectrometry conditions were as follows: H-ESI in positive mode, spray voltage 4000 V; sheath gas 40 Arb (arbitrary units), auxiliary gas 20 Arb.

Analytical grade angiotensin II (Ang-13C&15N, ANASPEC; California) was used for validation and quality control. Water (Optima LC-MS grade; w6-4) Acetonitrile (Optima LC-MS grade; A955-212), Methanol (Optima LC-MS grade; A456-4), Formic acid (Optima LC-MS grade; A117) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Solutions were diluted with water and methanol (50:50, v/v) at a concentration ranging from 100 µg/mL to 0.1 µg/mL. The calibration curve was in the range of 10 ng/mL to 10 000 mg/mL with total of 10 points. The working concentration of internal standard was 200 ng/mL. Working solution of quality control (QC) samples were prepared by dilution from stock in methanol and water (50:50, v/v) at the concentration of 8 μg/mL, 1.6 μg/mL and 0.32 μg/mL. The final concentration of QC samples were 9000 ng/mL, 1800 ng/mL and 360 ng/mL. All the stock and working solutions were stored in −20°C before and after use. Saline water was spiked with appropriate volume of each working calibration standards and quality controls to get calibration samples.

Stability Analysis

The change in percentage from initial concentration of ANGII at each time point was determined. Samples were considered stable if there was <10% change in initial concentration. Additional stability data was defined as consistency of pH, maintenance of color, and clarity.

Statistical Analysis

The pH, visual inspection results, and ANGII concentrations were determined for each study time point. Additionally, the percentage of initial ANGII concentration remaining (ie, relative concentration) was calculated for each sample. This was calculated by the [ANGII]_{Day X} ÷ [ANGII]_{Day zero} × 100. The samples were aggregated, and the pH and concentrations were described with the average ± standard deviation (SD).

Results

ANGII diluted with 0.9% sodium chloride kept under refrigeration was physically stable throughout the study with all samples remaining clear with no precipitation observed. The average ± SD pH of the solutions was 5.05 ± 0.17. The results of the HPLC-UV and LC-MS/MS analyses are shown in Tables 1 and 2 respectively demonstrating the retention of ≥90% initial concentration throughout the 5-day study period with both analysis methodologies.

Table 1.

HPLC-UV Refrigeration^* Stability Results.

	Day	% Initial concentration remaining	pH	Visual results
0.9% NaCl + ANG II	0	100	5.38	Clear
0.9% NaCl + ANG II	1	102.5195768	5.0	Clear
0.9% NaCl + ANG II	2	105.3073091	5.02	Clear
0.9% NaCl + ANG II	3	96.83531008	5.0	Clear
0.9% NaCl + ANG II	4	97.75182388	5.0	Clear
0.9% NaCl + ANG II	5	93.70563605	4.9	Clear

^*Refrigeration conditions were 5 ± 3°C.

Table 2.

LC-MS/MS Refrigeration^* Stability Results.

	Day	% Initial concentration remaining	pH	Visual results
0.9% NaCl + ANG II	0	100	5.28	Clear
0.9% NaCl + ANG II	1	96.01917229	5.16	Clear
0.9% NaCl + ANG II	2	93.27897908	5.21	Clear
0.9% NaCl + ANG II	3	92.8759758	5.23	Clear
0.9% NaCl + ANG II	4	96.62106405	5.25	Clear
0.9% NaCl + ANG II	5	94.12302642	5.15	Clear

^*Refrigeration conditions were 5 ± 3°C.

Discussion

To the authors’ knowledge, there is no available data published regarding ANGII extended stability in 0.9% sodium chloride. The refrigerated solutions of ANGII in 0.9% sodium chloride retained >90% initial ANGII concentration out to 5 days. These results were observed in 2 separate acceptable methods for validation. As each experiment utilized a different method to confirm concentration retention among samples, the results appear reliable. This data suggests that ANGII can have a post-dilution refrigeration shelf life of up to 5-days which would allow for increased flexibility, workflow optimization, potential reduction in waste, and ultimately cost-avoidance.

As with any experiment there are limitations. As the study design called for samples to be frozen at −80°C until the time of analysis, it was assumed that ANGII samples would not degrade at this low temperature and that no volume losses would occur during storage. The refrigerated samples would support this assumption, but it is possible there was some pre-analysis loss. Given that concentrations were retained throughout the study period, this loss is likely negligible. Additionally, in vitro determination of stability does not automatically guarantee the pharmacodynamic activity of the drug will remain unchanged in vivo. Ideally, follow-up pharmacokinetic and pharmacodynamic studies would be performed in vivo to confirm these results. Lastly, though we tested for physical stability, we did not test for sterility which could affect extended usage. However, the infusion bags used were sterile and the solutions were prepared in a sterile environment using sterile technique which would limit the likelihood of sterility loss.

Conclusions

According to qualitative and quantitative HPLC-UV and LC-MS/MS analyses of samples obtained over a 5-day period, ANGII in 0.9% sodium chloride at a concentration of 10 000 ng/mL stored in infusion bags under refrigeration were stable, maintaining at least 90% of their original concentrations, for up to 5 days. The results of this study provide information on potentially extending the expiration date on ANGII solutions.