Galenic Stability of Propofol Emulsions After Expiry: Assessment After 3 Years

Meryem Chennaq^1,2, Zineb Aliat^1,2, Safae Elmedkouri^1,2, Ali Cherif Chefchaouni^1,2,

Nadia ou-kheda^1,2,4, Aicha Fahry^1,2,3, Abdelkader Laatiris^1,2,3, Nawal Cherkaoui^1,2,3,

Karim souly^1,2,4, Yassir Alaoui^1,2,3, Younes Rahali^1,2,3

¹Faculty of Medicine and Pharmacy, Mohammed V University in Rabat, 10 170 Rabat, Morocco.

²Ibn Sina University Hospital Center, 10 170 Rabat, Morocco.

³Team of Formulation and Quality Control of Health Products, Faculty of Medicine and Pharmacy,

Mohammed V University- Rabat, 10 170 Rabat, Morocco.

⁴Central Bacteriology Laboratory, CHU Ibn Sina, Rabat.

*Corresponding Author E-mail: meryemchennaq12@gmail.com

ABSTRACT:

Introduction: Propofol (2,6-diisopropylphenol) is a potent intravenous hypnotic widely used for the induction and maintenance of general anesthesia and sedation in various medical settings. Despite its widespread use, propofol emulsions, formulated as oil-in-water (O/W) due to its low solubility in water, pose formulation-related challenges. Emulsion stability is critical to prevent phase separation and droplet aggregation, which can lead to embolisms. This study builds upon a previous investigation assessing the stability of propofol emulsions up to six months post-expiration, extending the evaluation to three years after expiry by examining visual appearance, pH, droplet diameter, sterility and zeta potential. Methods: Fifteen ampoules of Propofol (Propofol Fresenius, 10 mg/ml) from the same batch, expired for three years, were used. Stability measurements included visual observation, pH, droplet diameter, polydispersity index, sterility, and zeta potential. The study was conducted under ICH Q1A storage conditions (30°C ± 2°C). Samples were prepared and analyzed using a Zeta nanosizer v6.12 (Malvern Instrument) for droplet size and zeta potential, and an Eutech pH meter 510 for pH measurements. Sterility testing, performed according to USP <71> guidelines, while statistical analyses using Jamovi software (version 2.6.2). Results were compared to the previous study conducted six months after expiration to identify trends in galenic stability. Results: No phase separation or visible particles were observed in any of the six tubes during visual examination. The mean droplet diameter ranged from 94.42 nm to 106.37 nm, significantly lower than the 109.52–125.15 nm reported in the six-month post-expiration study, reflecting enhanced galenic stability over time. The polydispersity index ranged from 0.14 to 0.16, also lower compared to the previous study, indicating improved droplet uniformity. The pH measured between 6.85 and 7.07, and the zeta potential ranged from -42.7 mV to 68.9 mV, with trends consistent with the earlier findings. All samples maintained unimodal droplet size distribution, aligning with USP guidelines for injectable lipid emulsions. Additionally, sterility testing confirmed the absence of microbial contamination in all samples, further supporting their safety and stability over time. Conclusion: The propofol emulsion demonstrated galenic stability up to three years post-expiration, showing consistent improvements in droplet diameter and uniformity compared to the six-month study. These results suggest the potential for post-expiry use of propofol emulsions in emergency situations, particularly during shortages. Further comprehensive stability studies, including bacterial endotoxin detection, and an assay of the active substance to detect any potential degradation are recommended to ensure the safety and efficacy of post-expiry use.

KEYWORDS: Propofol, Galenic stability, Lipid emulsion, Post-expiration, Intravenous anesthesia.

INTRODUCTION:

Propofol (2,6-diisopropylphenol) is a potent intravenous hypnotic, first introduced in Europe in 1986 and in the USA in 1989¹. It rapidly became popular thanks to its favorable pharmacokinetic and pharmacodynamic profile, making it the intravenous anesthetic of choice for many surgical procedures over the last three decades.^2-4 Commonly used for the induction and maintenance of general anesthesia, as well as for sedation in a variety of medical settings, propofol is particularly suited to outpatient procedures thanks to its rapid recovery and reduced side effects such as nausea and vomiting.^5-8

Although widely used, propofol emulsions present a number of formulation-related challenges. Due to its low solubility in water⁹, propofol is formulated as an oil-in-water (O/W) emulsion, giving it a milky-white appearance.¹⁰ However, these emulsions are thermodynamically unstable, which can lead to galenic stability problems such as phase separation and droplet aggregation¹¹. Emulsion stability is important, as large droplets can cause embolisms when they enter pulmonary capillaries. Research has shown that zeta potential, pH, and droplet size are key factors influencing the stability of propofol emulsions.^12,13

In a previous study¹⁴, we evaluated the galenic stability of a batch of propofol over a six-month period following its expiration date, and concluded that the emulsion maintained good stability for up to three months after expiry¹⁵. This earlier work emphasized the potential for extending the use of expired propofol in specific situations, particularly during shortages. However, the study only covered a short post-expiration period.¹⁴

To build on these findings, the present study aims to assess the stability of ampoules from the same lot three years after their expiration date¹⁶. By comparing the new results with those obtained in the previous study, we seek to better understand the long-term behavior of propofol emulsions post-expiry and explore whether they remain suitable for use over extended periods. Such insights could have significant implications for economic efficiency and clinical practice, especially in resource-limited settings. ^14,17-19

MATERIALS AND METHODS:

In total, we had 50 ampoules of Propofol (Propofol Fresenius) (10 mg/mL, batch number: 10LD9330) received from the pharmacy of the National Institute of Oncology in Rabat, with a common use-by date of May 2021. Although the ampoules used in this study were different from those utilized in the previous study¹⁴, they were obtained from the same batch of Propofol. They were presented in colorless glass containers; each 20 mL ampoule contained 200 mg of Propofol. The active substance is propofol, and the other components of this preparation include refined soybean oil, purified egg lecithin, glycerol, oleic acid, sodium hydroxide, and water for injection.

Galenic stability measurements were initiated three years after the expiration date and conducted over a period of six months. These measurements, similar to those in the previous study¹⁴, included visual examination, pH determination, mean droplet diameter, polydispersity index, and zeta potential assessment. This allowed for a comparative evaluation of the long-term post-expiry stability against the results of the earlier study conducted up to six months after expiry.

For this purpose, the ampoules were stored under the conditions dictated by ICH Q1A (30°C ± 2°C) for intermediate stability studies.

Dry tubes from the same hospital were used to take samples from the propofol ampoules.

1) Preparation of the samples:

First, 36 sample tubes to be used for the six-month study were prepared under a laminar flow hood with sterile syringes and needles. Then, 20 µL of each emulsion sample was diluted with 1 mL of distilled water in a clean Malvern sample bottle adapted to the Zeta nanosizer v6.12 (Malvern Instrument) for measurements.

2) Visual examination:

During each measurement, the prepared sample was visually examined for phase separation, color changes, or macroscopic precipitation. The reversibility of creaming was tested by three successive inversions. Samples were observed against black and white backgrounds for 5 seconds to detect visible particles, as in the previous study¹⁴.

3) Droplet size and Zeta potential:

The average droplet diameter (DD), polydispersity index (PDI), and zeta potential (ZP) of the emulsion at each monthly interval were measured using a Zeta nanosizer v6.12 (Malvern Instrument) at 25°C²⁰.

· Dynamic light scattering was used to measure droplet size distribution, capable of detecting droplets ranging in size from 1 nm to 1 µm.

· Electrophoretic light scattering measured the electrophoretic mobility of the emulsion droplets, converted into the zeta potential.

A dip cell (zen1002, Malvern Instruments) with a pair of parallel Pd electrodes was used to provide an electrical trigger on the charged particles. In line with the previous study, measurements were assessed against the American Pharmacopoeia recommendations, which require lipid globule sizes to remain under 5 µm, with less than 0.05% of globules exceeding this threshold. ^21,22

The zeta potential represents the potential difference between the tightly bound ion layer at the particle surface and the electroneutral region in the surrounding solution. A relatively high zeta potential (absolute value of 25 mV or more) indicates significant stability, whereas values below 25 mV suggest lower stability.

Each month, six dry tubes of propofol were prepared, with measurements for each tube performed in triplicate to calculate the average (Table 1).

4) pH:

An Eutech pH meter 510 was used to measure pH (calibrated against pH 9 and pH 4 buffers). The pH meter was regularly checked and recalibrated to avoid contamination errors. As in the prior work, pH was measured immediately after dilution to minimize exposure to atmospheric changes.

5) Microbiological test:

Sterility testing was performed according to the USP <71> guidelines to ensure the absence of viable microorganisms in the propofol emulsion samples. For this purpose, a total of 6 different ampoules were tested²³. Each sample was aseptically transferred to sterile culture media under a laminar flow hood to prevent contamination. The samples were incubated for 14 days.

The cultures were visually examined daily for evidence of microbial growth. Positive controls containing known microorganisms were used to confirm the efficacy of the culture media.

6) Statistical Analysis:

Statistical analyses were performed using the Jamovi software (version 2.6.2, Jamovi Project). Normality of the data was assessed using the Shapiro-Wilk test, and paired comparisons between measurements at 6 months and 3 years post-expiration were conducted using the paired t-test. The significance threshold was set at p < 0.05. The analyses were conducted to evaluate changes in key parameters, including mean droplet diameter, polydispersity index, zeta potential, and pH, over the extended storage period.

RESULTS AND DISCUSSION:

1) Visual examination:

In all prepared tubes containing propofol, neither phase separation nor particles visible to the naked eye were observed. An emulsion would be considered thermodynamically unstable if the dispersed and continuous phases transformed into separate oil and water phases, either by melting or by droplet coalescence²⁰. The European Pharmacopoeia guidelines state that an injectable lipid emulsion should not exhibit signs of phase separation, a criterion that was consistently met in this study.²⁴

No changes in color or signs of precipitation were detected during the visual inspections. Furthermore, after centrifugation (6000 rpm for 30 minutes), no phase separation was observed in any of the six tubes from each month, even three years post-expiration. These results align with the findings of the previous study¹⁴, which also reported no phase separation during the six months following expiration. The consistency in these observations reinforces the robustness of the galenic stability of propofol emulsions under proper storage conditions.

2) Mean droplet diameter and polydispersity index:

The study of post-expiration measurements revealed that the mean droplet diameter of the propofol emulsion ranged from 94 ± 1.66 nm to 106 ± 21.87 nm (Figure 1a), with a maximum of 152.27 nm recorded in the sixth tubes (Table 2). These values are significantly lower compared to the previous study, where the mean droplet diameter ranged from 109.52 ± 25,92 nm to 125.15 ± 30.46 nm, with a maximum of 169.5 nm.

The Shapiro-Wilk test confirmed that the data for the mean droplet diameter (DD) followed a normal distribution (p-value = 0.460 > α = 0.05) (Table 3), validating the assumption of normality required for conducting parametric tests such as the paired t-test. Consequently, the paired t-test was applied and revealed a statistically significant reduction in mean droplet diameter between the two studies (t = 5.44, p = 0.003 < α = 0.05) (Table 4). This reduction highlights a structural change in the emulsion, indicating enhanced stability over the extended storage period.

Table 1: Monthly Measurement of Propofol Emulsion Parameters After 3 Years Post-Expiration.

Month

Tube

Visuel Aspect

Measure 1

Measure 2

Measure 3

IPD

Table 2: Stability Parameters (Mean Droplet Diameter, Polydispersity Index, Zeta Potential, and pH) of Propofol Emulsions Monitored Over Six Months, Three Years Post-Expiration.

Month	Mean droplet diameter (nm)				Polydispersity index
Month	Mean	Sd	Min	Max	Mean	Sd	Min	Max
1	99.36	5.92	93.63	115.2	0.14	0.06	0.09	0.24
2	99.80	5.27	93.61	109.06	0.16	0.05	0.08	0.22
3	94.42	1.37	92.84	98.37	0.10	0.02	0.08	0.14
4	98.33	3.10	93.39	104.7	0.14	0.05	0.09	0.20
5	99.26	5.23	94.45	113.5	0.14	0.04	0.08	0.22
6	106.37	16.16	93.23	152.27	0.11	0.06	0.06	0.25

Table 2: cont……

Month	Zeta potential (mV)				pH
Month	Mean	Sd	Min	Max	Mean	Sd	Min	Max
1	-27	1.35	-28.2	-25.9	6.93	0.08	6.86	7.04
2	-42.1	1.27	-43.2	-41	6.91	0.02	6.89	6.92
3	40.5	0.49	40.1	40.9	6.85	0.02	6.83	6.87
4	45.5	0.26	45.3	45.7	7.07	0.04	7.03	7.11
5	68.9	0.21	68.7	69.1	6.96	0.005	6.95	6.96
6	-42.7	0.52	-43.2	-42.1	6.91	0.02	6.89	6.93

Sd: standard deviation

Min: minimum

Max: maximum

Table 3: Shapiro-Wilk Normality Test Results for Stability Parameters of Propofol Emulsions (6-Month Post-Expiry vs. 3-Year Post-Expiry).

Studied parameters			W	p
D (6 months expiry)	-	D (3 years expiry)	0.914	0.460
IPD (6 months expiry)	-	IPD (3 years expiry)	0.891	0.322
PZ (6 months expiry)	-	PZ (3 years expiry)	0.875	0.247
pH (6 months expiry)	-	pH (3 years expiry)	0.876	0.253

Table 4: Paired t-Test Results Comparing Stability Parameters of Propofol Emulsions (6 Months vs. 3 Years Post-Expiry).

Studied parameters			Statistique	ddl	p
D (6 months expiry)	D (3 years expiry)	t de Student	5.44	5.00	0.003
IPD (6 months expiry)	IPD (3 years expiry)	t de Student	4.42	5.00	0.007
PZ (6 months expiry)	PZ (3 years expiry)	t de Student	-2.52	5.00	0.053
pH (6 months expiry)	pH (3 years expiry)	t de Student	-1.16	5.00	0.298
Note. Hₐ μ_Mesure₁_-_Mesure₂ ≠ 0

3) pH and Zeta potential:

The study of the samples 3 years after expiration shows that the average pH measured ranged from 6.85 to 7.07 (Figure 3). For optimal stability, the pH of this emulsion is ideally maintained between 7 and 8.5^14,26. In another study evaluating the stability of two propofol formulations under normal conditions of use, the pH ranged from 7 to 8.5 for the formulation containing edetate disodium and from 4.5 to 6.4 for the emulsion containing sodium metabisulfite. Additionally, it is well known that oil-in-water (o/w) emulsions tend to develop better stability in a basic pH environment. The effect of pH reduction on the stability of phospholipid-stabilized emulsions has already been well-documented, with lower pH levels leading to reduced electrostatic repulsion and increased risk of flocculation or coalescence. ^26-28

Statistical analyses further revealed that the differences in pH mean between 6 months and 3 years after expiration were not statistically significant (t = -1.16, p = 0.298) (Table III). These findings suggest that the pH of the emulsion remained relatively stable over time, within the acceptable range for maintaining emulsion integrity.

Regarding the zeta potential, values obtained included both positive and negative measurements, ranging from -42.7 mV to 68.9 mV across the six post-expiration tubes (Figure 4). Emulsion droplets with zeta potentials ranging between -40 mV and -50 mV are generally considered to have sufficient surface charge to maintain stability. Conversely, values below 30 mV (absolute) are indicative of increased risks of aggregation, instability, flocculation, or coagulation. ^29,30

The paired t-test analysis of zeta potential measurements revealed no significant difference between the two periods (t = -2.52, p = 0.053) (table II) . However, the observed trend indicates a decrease in zeta potential over the 3-year period, aligning with the slight pH reduction noted. These findings are consistent with previously reported results for parenteral emulsions stabilized by phospholipids, which typically exhibit zeta potentials around -50 mV at pH 8 and show a progressive decrease as pH declines.

While the p-value for zeta potential differences does not meet the strict threshold for statistical significance, it is important to note that in pharmaceutical and medical studies, statistical significance alone does not fully explain the practical or clinical relevance of observed differences. The trend observed in zeta potential values suggests a potential decrease in electrostatic stabilization over time, which may have implications for the long-term stability of emulsions. The interplay between pH and zeta potential highlights the critical role of electrostatic forces in maintaining the stability of oil-in-water emulsions over extended storage periods.

4) Sterility Testing:

Sterility testing, conducted in accordance with USP <71>, confirmed that all post-expiry samples remained free of microbial contamination throughout the study period. This finding is of paramount importance in assessing the safety of using expired propofol emulsions, as microbial contamination poses a significant risk, particularly in intravenous formulations. The sterility of the emulsions indicates that the manufacturing process, packaging integrity, and storage conditions were sufficient to prevent microbial ingress, even three years after the labeled expiration date.

These results strongly support the argument for extending the shelf-life of propofol emulsions in emergencies, particularly in resource-limited settings or during drug shortages. While the study demonstrates microbiological safety, it is essential to emphasize that sterility testing should always accompany a comprehensive stability study, including endotoxin detection and active ingredient assay, to ensure both the safety and efficacy of the product beyond its expiration date. These additional evaluations would provide a more robust framework for regulatory consideration of post-expiry use.

Figure 3^24,25: pH Values (± SD) of Propofol Emulsions Monitored Over Six Months, Three Years Post-Expiration.

Figure 4: Zeta Potential (± SD) of Propofol Emulsions Measured Over Six Months, Three Years Post-Expiration.

Figure 5:Droplet Size Distribution by Intensity and Mass.

4) GENERAL DISCUSSION:

The expiration date guarantees the stability and efficacy of a drug until that date in its original container. However, this date primarily reflects the manufacturer's assurance that the labeled activity will last at least until that point, rather than an absolute stability limit³¹. FDA regulations do not mandate determining the duration for which drugs remain stable beyond the expiration date, allowing manufacturers to set these dates without fully evaluating long-term stability. Few studies have explored the stability of injectable lipid emulsions over extended periods, making this investigation particularly relevant.³²

The stability of injectable lipid emulsions is a critical factor in parenteral administration, as their physical characteristics directly impact their safety and efficacy. Among these, droplet size and distribution are the most important parameters. An increase in droplet size is often the first sign of formulation instability, and droplets larger than 5 μm pose a significant risk of pulmonary embolism, as they can become trapped in the lungs.³³ Unlike the United States Pharmacopeia (USP), which defines strict limits for droplet size, the European Pharmacopoeia does not provide specific guidelines for injectable lipid emulsions, further emphasizing the need for detailed stability studies.³⁴

In terms of zeta potential, high absolute values (whether negative or positive) indicate electrical stabilization of the emulsion, while low absolute values increase the likelihood of coagulation or flocculation, resulting in poor galenic stability. When the zeta potential is high, the repulsive forces between droplets exceed the attractive forces, leading to a relatively stable emulsion system.³⁵

The results of this study suggest that the propofol emulsion maintains galenic stability up to 3 years post-expiration under controlled storage conditions. This finding highlights the potential for extending the use of propofol emulsions beyond their expiration date in specific situations, particularly during shortages or emergencies. However, a more comprehensive stability study is required to confirm these findings, including sterility testing, bacterial endotoxin detection, and active ingredient assay to monitor potential degradation of the active substance over time³⁶.

While these results are promising, the inherently thermodynamically unstable nature of lipid emulsions warrants careful consideration. Special monitoring protocols should be established if post-expiry use of the emulsion is contemplated³⁷. Such protocols would ensure that the emulsion remains safe and effective, mitigating risks associated with long-term storage.

In the context of long-term storage and post-expiry use, identifying potential degradation by-products remains a critical component of safety evaluation. Degradation of propofol emulsions can lead to the formation of impurities, which, if not detected and quantified, may compromise safety and efficacy₃₈. According to USP guidelines, testing for specific impurities, such as free fatty acids and propofol-related compounds (e.g., compound A and B), provides a framework for assessing the chemical stability of injectable emulsions³⁹. Analytical techniques, including high-performance liquid chromatography (HPLC) and gas chromatography (GC), as outlined in USP <621>, could complement the current study⁴⁰.

Future investigations should focus on testing the impurity profile of propofol emulsions, ensuring compliance with pharmacopeial standards such as limits for organic impurities and bacterial endotoxins (NMT 0.33 EU/mg of propofol). Incorporating these assessments would provide a robust validation of post-expiry safety, reinforcing the clinical applicability of extended shelf-life recommendations.

CONCLUSION:

The study of the propofol emulsion, based on key parameters such as pH, droplet diameter, sterility and zeta potential, demonstrated a galenic and microbiological stability extending up to 3 years beyond the expiration date. These findings suggest that, under appropriate storage conditions, the emulsion may retain its galenic integrity for an extended period, offering potential advantages for its use in emergency situations, particularly during shortages.

However, to fully validate the post-expiry use of this emulsion, a comprehensive stability study must be conducted. This study should include, bacterial endotoxin detection, and an assay of the active substance to detect any potential degradation. Establishing such evidence-based guidelines could pave the way for safe and effective post-expiry use of propofol emulsions, leading to significant economic benefits for hospital structures and better resource allocation.

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Received on 25.03.2025 Revised on 14.07.2025

Accepted on 21.10.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):215-222.

DOI: 10.52711/0974-360X.2026.00031

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