Neomycin sulfate and triamcinolone acetonide suspended ointment designed for transdermal delivery: formulation and in vitro evaluation

Jiong Wu; Qian Tang; Xufei Zhao; Yan Shen; Ruixi Liao; Hongxiu Zhang; XiuJuan Feng; Aiping Shi

doi:10.1007/s44446-025-00027-1

. 2025 Aug 4;33(4):28. doi: 10.1007/s44446-025-00027-1

Neomycin sulfate and triamcinolone acetonide suspended ointment designed for transdermal delivery: formulation and in vitro evaluation

Jiong Wu ^1,^#, Qian Tang ^1,^#, Xufei Zhao ¹, Yan Shen ¹, Ruixi Liao ¹, Hongxiu Zhang ^2,^✉, XiuJuan Feng ^3,^✉, Aiping Shi ^4,^✉

PMCID: PMC12321736 PMID: 40760257

Abstract

Neurodermatitis and chronic eczema are characterized by severe itching and can lead to complications such as skin infections and folliculitis. Current treatment primarily involves glucocorticoid analogs which may be associated with side effects and lack of effective delivery strategies. The suspended ointment developed in this study aims to address these issues, offering improved therapeutic outcomes. The present study aimed to investigate the relationship between formulation/process variables versus the content uniformity and viscosity of neomycin sulfate and triamcinolone acetonide ointments and to explore the feasibility of using an in vitro approach to assess product sameness. Monofactor analysis was used to evaluate the impact of formulation and process variables. The new formulation was evaluated in terms of the prescription process, quality, stability, in vitro drug release behavior, and distribution of skin. The optimal prescription composition was 0.54% neomycin sulfate, 0.10% triamcinolone acetonide, 5.08% substrate A and 94.28% liquid paraffin. Quality and stability assessments confirmed that the formulation met the required standards for appearance and composition. Mathematical modeling of the drug release profile indicated that the release kinetics evaluated using the vertical diffusion cell method, closely aligned with first-order kinetics. Raman spectroscopy confirmed the successful penetration of triamcinolone acetonide into the skin, triamcinolone acetonide works primarily in the skin. Furthermore, skin irritation tests demonstrated that the formulation caused no detectable irritation. These results demonstrate that it can be a promising drug in treating neurodermatitis and chronic eczema.

Graphic Abstract

Keywords: Transdermal delivery, Suspended ointment, Neomycin sulfate, Triamcinolone acetonide, In vitro release profile, Raman spectroscopy

Introduction

In dermatology, an increasing number of semi-solid formulations for topical application are being marketed for the treatment of certain inflammatory skin diseases(Herbig et al. 2023). Neurodermatitis and chronic eczema are prevalent, long-term inflammatory skin diseases characterized by severe itching(Shahin et al. 2023), recurrent eczematous lesions, and often thickened(Silverberg 2017), rough skin with pigmentation changes(Sm et al. 2020). Both conditions exhibit high prevalence globally, substantially impacting patients'quality of life and presenting significant healthcare challenges(Tran et al. 2023; Walling and Swick 2010). Without effective treatment or management, these diseases may lead to complications such as skin infections and folliculitis(Borg et al. 2024; Zeng et al. 2023), further compromising patients'physical and mental well-being(Yew et al. 2024).

Currently, clinical treatment for this type of disease primarily involves glucocorticoid drugs(Islam et al. 2025; Prado et al. 2025; J. Zhang et al. 2025a, b; Zheng et al. 2025). However, prolonged use may lead to adverse effects, including skin atrophy and hyperpigmentation, especially in sensitive areas of the face and body(Holstroma et al. 2025; Nawan et al. 2025; Park et al. 2025; Tanaka 2025). In cases of severe atopic dermatitis, systemic immunosuppressant therapies are often used, yet long-term use can impair liver and kidney function and increase infection risk(Bautista Sanchez et al. 2025; Bhatt et al. 2025; Dabour et al. 2025). Neomycin has been widely recognized internationally in recent years, and it has been shown to have bacteriostatic activity against some drug-resistant strains at a certain minimum inhibitory concentration(Al-Jebouri 2024; Obszynski et al. 2022). Neomycin sulfate is a highly polar, hydrophilic aminoglycoside antibiotic with a molecular weight of approximately 712.72 g/mol. It is readily soluble in water (100 mg/mL) but poorly absorbed through the intact skin, which necessitates optimized formulation strategies to enhance local retention and effectiveness. Therefore, neomycin sulfate is still one of the most important antibiotics in clinical treatment(Pagkalis et al. 2011; Ye et al. 2018). Triamcinolone acetonide, a synthetic glucocorticoid, exhibits both anti-inflammatory and immunomodulatory properties(Formica et al. 2020). It is a moderately lipophilic compound with a molecular weight of 434.50 g/mol and a LogP value of approximately 2.42, favoring dermal absorption. Its crystalline nature and limited aqueous solubility (21 mg/L) necessitate its dispersion in appropriate semisolid bases for effective topical delivery. Its anti-inflammatory effects include reducing capillary permeability to alleviate congestion, inhibiting complement activation in inflammatory responses, stabilizing lysosomal membranes by suppressing phagocyte activity and blocking the release of inflammatory mediators such as kinins and histamine(Duguid et al. 1992; Heidenkummer and Kampik 1995). In addition to these anti-inflammatory actions, corticosteroids also induce vasoconstriction(Sommer et al. 1998). Clinically, triamcinolone acetonide is well-tolerated, with a low incidence of adverse effects, most of which manifest as mild local irritations that are resolved upon discontinuation(Balazic et al. 2025; Maity et al. 2025; Mardhiah et al. 2025; Yuan et al. 2025). No serious adverse events have been reported, underscoring its favorable safety profile(Serpieri et al. 2024; Shi et al. 2024). The combination of neomycin sulfate and triamcinolone acetonide can effectively prevent the factors that trigger and exacerbate the disease, and it can also compulsorily control the scratching behavior that causes lesion hyperplasia and hypertrophy(Liang et al. 2023a). The synergistic application of neomycin sulfate and triamcinolone acetonide is of high value and has some therapeutic effects as shown by the combination of existing products(Liang et al. 2023b). It is a promising undertaking to develop a semi-solid topical combined formulation of neomycin sulfate and triamcinolone acetonide for better quality.

A notable limitation of current treatment methods is the lack of effective drug delivery strategies. Following oral administration, drugs must be absorbed into the bloodstream after passing through the gastrointestinal tract, often undergoing extensive metabolism in the liver during the first-pass effect(Huang et al. 2025; Luo et al. 2025; Martínez et al. 2025; Yang et al. 2025; L. Zhang et al. 2025a, b). This process significantly reduces drug concentration, compromising efficacy. For drugs intended to target localized tissues (e.g., skin or specific lesions), the oral route may result in poor delivery efficiency. While injections can achieve rapid systemic circulation, they generally lack sustained release or precise targeting, leading to widespread distribution and potential side effects. In the case of localized lesions, such as skin lesions, injectable administration may result in suboptimal drug concentration at the target site, further limiting therapeutic efficacy(Dent et al. 2025; Feng et al. 2025; Hong et al. 2025; Prokopov et al. 2025). Neurodermatitis and chronic eczema are both inflammatory diseases with exudation, and most of them are now treated with topical preparations(Davis et al. 2024; Riva et al. 2024). Compared with other routes of administration, transdermal administration of ointments has the following advantages: (1) avoidance of first-pass effect and drug degradation in the gastrointestinal tract; (2) reduction of side effects due to systemic toxicity; (3) large absorption area; (4) slow-release or controlled-release; (5) improvement of the patient's compliance with the medication; (6) better stability of the medication; (7) better uniformity of the content of the preparation. A suspended ointment is a semisolid dosage form in which insoluble drug particles are dispersed within an ointment base. The preparation of such formulations presents several challenges, including the need for precise control of particle size to ensure uniform dispersion and consistent drug release. Stability concerns, such as particle sedimentation and aggregation, must be addressed through the incorporation of appropriate suspending agents or viscosity modifiers(Eichenauer et al. 2023). Additionally, ensuring compatibility between the drug and the ointment base is crucial to prevent drug degradation or precipitation. Optimizing the manufacturing process, particularly mixing and homogenization techniques, is essential for maintaining formulation uniformity and therapeutic efficacy. However, for semi-solid topical formulations, whether the main drug can pass through the skin smoothly and at what rate is directly related to the quality of the preparation(García-Arieta et al. 2023). It was found that the amorphous neomycin sulfate active pharmaceutical ingredient undergoes a crystalline morphology shift, partially from amorphous to crystalline(Masoud et al. 2018). This happens during the production or storage and placement of the formulation, which affects the content uniformity of the ointment(Chow et al. 2023). Therefore, it is important to determine the content uniformity of the ointment.

In this study, concerning the commercially available neomycin sulfate ointment, a new compound preparation was prepared by adding triamcinolone acetonide first. Additionally, a more comprehensive preparation quality evaluation criteria was established, and a comprehensive study was carried out on the prescription process and in vitro release of the drug in the preparation and other properties. Furthermore, the stability and content uniformity of the preparation proved to be better compared to the existing reference products.

Material and methods

Materials

The materials and reagents used in this study were as follows: Neomycin sulfate was obtained from Yichang Sanxia Pharmaceutical Co., Ltd., China, and triamcinolone acetonide was purchased from Jiangsu Ruidian Pharmaceutical Co., Ltd., China. Liquid paraffin was supplied by Xi’an Jinxiang Pharmaceutical Excipients Co., Ltd., China. Reference standards for HPLC analysis were provided by the National Institutes for Food and Drug Control, China. Healthy male ICR mice (20 ± 2 g) were supplied by Nanjing Anokang Biological Technology Co., Ltd., China. All animal care and experimental protocols strictly adhered to established ethical guidelines and received prior approval from the China Pharmaceutical University Animal Ethical Experimentation Committee.

Design of experiments and preparation of compound neomycin sulfate ointment

This study referenced commercially available formulations combining neomycin sulfate and triamcinolone acetonide, with modifications made to the original prescription. The final formulation included a single dose of 35,000 units of neomycin sulfate and 10 mg of triamcinolone acetonide. The batch size was set at 50 g, and the detailed composition included neomycin sulfate, triamcinolone acetonide, liquid paraffin, and substrate A. Substrate A, a polyethylene composite, was incorporated as an additive or modifier to enhance the ointment’s rheological properties and bioadhesion(Nikseresht et al. 2024). The optimal concentration of Substrate A was determined through screening experiments.

To prepare the formulation, the prescribed amount of liquid paraffin and Substrate A was weighed and heated until completely melted. The mixture was maintained at a constant temperature for further use. Triamcinolone acetonide was then added to the molten substrate and stirred thoroughly, maintaining the temperature throughout. Separately, the prescribed amount of neomycin sulfate was weighed and mixed with 60% liquid paraffin, first by shearing the solution at room temperature, followed by heating. The neomycin sulfate solution was combined with the triamcinolone acetonide solution, after which heating was stopped, and the mixture was cooled using circulating tap water. As the viscosity increased, the preparation was processed using a high-speed homogenizer. After homogenization, the mixture was stirred for a specified period and left to stand for 2–3 h, yielding the final product. The flowchart is shown in Fig. 1.

Fig. 1 — Preparation process of compound neomycin sulfate ointment

The formulation and process parameters were optimized through mono-factor analysis. For the formulation, key variables such as the particle size of the active pharmaceutical ingredients (APIs) and the proportion of oily substrate were systematically varied. For the process, mixing temperature, mixing time, and cooling temperature were individually adjusted. At each condition, the resulting preparation was evaluated based on its appearance and content uniformity, which served as the primary criteria for optimization. The optimal formulation was selected as the one exhibiting the best combination of uniform drug distribution and desirable physical characteristics. These evaluation criteria were chosen because content uniformity directly reflects dispersion efficiency, while appearance indicates formulation stability and patient acceptability. Specific operations are as follows. 1) Particle Size Investigation of Raw Materials: The original API was processed into different particle sizes using air-flow pulverization, and ultra-micronization. API particle size was determined three times in succession using a laser particle sizer. These APIs with varying particle sizes were incorporated into ointments to examine the impact of particle size on the ointment's appearance, viscosity, and content uniformity. 2) Optimization of Oily Substrate Quantity: To determine the optimal amount of the oily matrix, three formulation gradients were designed. 4.0616%, 5.0770%, and 6.0924% for substrate A, respectively. 3) Mixing Temperature Investigation: With other variables unchanged, temperatures of 80 °C, 90 °C, and 100 °C were tested to determine their impact on the formulation’s appearance, viscosity, and content uniformity. 4) Mixing Time Investigation: Emulsification and mixing times of 1, 2, and 3 h were tested to evaluate their impact on the appearance, viscosity, and content uniformity of the final product. 5) Cooling Temperature Investigation: Cooling temperatures of 0 °C, −10 °C, and −20 °C were examined to determine their effects on appearance, viscosity, and content uniformity.

Determination of drug and content uniformity testing

Within one week after ointment preparation and packaging, individual units of packed aluminum tubes were selected for determination and content uniformity(Dong et al. 2018; Thakkar et al. 2020). For each unit, the bottom seal was cut off and a vertical cut was made from the bottom to the top of the tube to expose the ointment drug product. The product was inspected for any changes in its physical appearance. Divide the contents into 3 equal parts (upper, middle, and lower), accurately weigh the appropriate amount, add petroleum ether, ultrasonic shake, make it completely dissolved, put it in the separation funnel, extract with pure water, and measure the content of API in each section of the ointment by HPLC.

High-performance liquid chromatography (HPLC)

The HPLC system consisted of an Agilent 1260 Series equipped with a degasser, binary solvent pump, autosampler, temperature-controlled column compartment, and a diode array detector. The current method of neomycin sulfate employed a chromatographic column with octadecylsilane-bonded silica gel as the filler. The column temperature was maintained at 25 °C. The mobile phase consisting of a 2% trifluoroacetic acid solution (0.01% pentafluoropropionic acid + 0.6% sodium hydroxide) was pumped at a flow rate of 0.8 mL/min. A sample volume of 25 μL was injected into the column. Detection was carried out by an integrating pulse amperometric electrochemical detector with a gold electrode (3 mm diameter recommended) as the detection electrode, an Ag/AgCl composite electrode as the reference electrode, a titanium alloy counter electrode, and post-column alkaline solution; 50% NaOH solution with a recommended flow rate of alkaline solution of 0.3 mL/min. The current method of triamcinolone acetonide employed chromatographic columns packed with octadecylsilane-bonded silica gel (Agilent Eclipse Plus C18, 4.6 × 150 mm, 5 μm). The column temperature was maintained at 30 °C. The mobile phase consisting of methanol: water (60:40, v/v) was pumped at a flow rate of 1 mL/min. A sample volume of 20 μL was injected into the column and the triamcinolone acetonide was detected at 240 nm.

Stability of compound neomycin sulfate ointment

Influencing Factor Test: Light and moisture significantly affect the quality of the preparation over time, especially because each preparation is exposed to these factors after being opened(Ubhe et al. 2024). To address this, samples were filled into high-density aluminum tubes to simulate practical conditions. The samples were stored under high-temperature conditions (40 °C), high-humidity conditions (75% RH), and light exposure (4500 lx ± 500 lx) for 30 days to evaluate the potential impacts on product quality.

Accelerated Stability Test: Three batches of the preparation were sealed in aluminum tubes and stored in a constant temperature and humidity chamber at 30 °C ± 2 °C/65% RH ± 5% RH. Samples were taken after one, two, and three months(Lacassia et al. 2024). Key properties, including appearance, viscosity, and content were analyzed to assess stability over time.

Long-term Stability Test: The same three batches were also sealed in aluminum tubes and stored under long-term conditions at 25 °C ± 2 °C/60% RH ± 5% RH. Samples were collected in March to evaluate appearance and content, ensuring the formulation's stability under typical storage conditions(Padamwar and Pokharkar 2006).

Rheological characterization

Rheological behaviors of ointments were evaluated using a hybrid rheometer (Discovery HR-10) equipped with a Peltier stage (25 °C) and a fixture (40 mm). For each test, approximately 2 mL of ointment sample was placed on the lower plate before slowly lowering the upper plate to the preset trimming gap. After trimming excessive material, the following procedures were performed in sequence on each sample to characterize ointment rheological behavior: 1) The stress and viscosity change with shear rate (0.1–1000 s⁻¹) was recorded at 25 °C using a 40 mm fixture. 2) The energy storage modulus (G') and loss modulus (G") are evaluated at a fixed frequency of 10 rad/s and a strain range of 0.1% to 1000%. 3) The shear strain was fixed at 2.0%, the angular frequency range was set to 0.1 rad/S-100 rad/s, and the curve of energy storage modulus and loss modulus with frequency was recorded. 4) The sample was scanned between 25 °C and 45 °C at a fixed strain of 2% and angular frequency of 10 rad/s. Changes in the energy storage modulus (G') and loss modulus (G'') were monitored to determine the fluid's temperature sensitivity(Andjic et al. 2024).

In vitro release testing (IVRT)

The vertical diffusion cell (VDC) used in this study had a volume of 8 mL and a release area of 2.2 cm², and the formulation was uniformly applied to the artificial membrane using a spatula. The temperature was maintained at (32.0 ± 0.5) °C with 600 rpm magnetic stirring during the experiment, and 2 mL of samples were taken at 1, 2, 4, 6, 10, 12, 24, 36, and 48 h. 2 mL of the blank acceptor medium was added. The sample solution was filtered through a 0.45 μm organic filter membrane, and 1 mL of the filtrate was discarded as the test solution. Control solution: dry neomycin sulfate/triamcinolone acetonide 105℃ for 3 h, take about 7 mg, weigh precisely, put in a 50 mL volumetric flask, add the appropriate amount of acceptance medium to dissolve, then dilute to the scale, shake well as a control solution. According to Liquid chromatographic conditions used before, inject the control solution and test solution into HPLC, calculate the concentration of the drug by peak area (A) according to the external standard method, and calculate the cumulative release of the drug. The amount of drug released per unit area can also be calculated according to the following formula(Siepmann and Peppas 2011).

Q_{n} = C_{n} \frac{V_{c}}{A_{c}} + \frac{V_{s}}{A_{c}} \sum_{i = 1}^{n} C_{i - 1}

Qn (µg·cm⁻²) is the cumulative amount of drug released per unit area at time (n); Cn (µg·cm⁻³) is the concentration of drug in the receiving medium at different sampling times (n); V_s is the sample volume (cm³); V_c is the receiving chamber volume; and A_c is the effective diffusion area (cm²).

The amount of drug released per unit area was plotted against the square root of time, and the slope of the straight line obtained by linear fitting was the release rate.

When neomycin sulfate was released in vitro by the vertical diffusion cell method, a phosphate buffer of pH 5.8 was chosen as the acceptor medium, and 3.0 μm polyethersulfone membranes were selected for subsequent in vitro release studies.

When triamcinolone acetonide was released in vitro by vertical diffusion cell method, ethanol–water (30:70) was chosen as the acceptor medium, and 3 μm polyethersulfone membranes were selected for subsequent in vitro release assay studies.

Distribution of skin

Raman spectroscopic determination of APIs and blank excipients was carried out to determine the assay conditions, followed by Raman spectroscopic studies on skin (mouse skin).

Neomycin Sulfate Raman Spectroscopy: An appropriate amount of Neomycin Sulfate API was placed on a slide and evenly spread. The experimental conditions were as follows: laser wavelength of 532 nm, exposure time of 0.1 s (10 Hz), 200 cumulative scans, laser power of 8.0 mv, 50X objective lens, step size of 3.0 μm, and depth of 12 μm. The Raman spectrum of Neomycin Sulfate API was measured six times(Lu et al. 2024).

Triamcinolone Acetonide Raman Spectroscopy: A suitable amount of Triamcinolone Acetonide API was placed on a slide and evenly spread. The experimental parameters included a laser wavelength of 532 nm, exposure time of 0.1 s (10 Hz), 200 cumulative scans, laser power of 8.0 mv, 50X objective lens, step size of 3.0 μm, and depth of 12 μm. Each sample was measured six times to obtain the Raman spectrum of Triamcinolone Acetonide API.

Raman Spectroscopy of Blank Excipients: An appropriate amount of the blank excipient mixture was placed on a slide and spread evenly. The instrumental conditions were identical to the API scans: laser wavelength of 532 nm, exposure time of 0.1 s (10 Hz), 200 cumulative scans, laser power of 8.0 mv, 50X objective lens, step size of 3.0 μm, and depth of 12 μm. Six measurements were taken for each sample.

Raman Spectroscopic Study of Mouse Skin: A confocal Raman imaging microscope (Thermo DXR3xi, U.S.A.) was used for skin measurements. Experimental parameters included a laser wavelength of 532 nm, exposure time of 0.1 s, 100 cumulative scans, laser power of 8.0 mv, and 50X magnification. Two-dimensional scanning was performed along the depth of the XZ plane with a step size of 12 μm, while the scanning depth along the XY plane was 90 μm(Ohashi et al. 2022).

Mouse skin samples were collected from the abdominal area, stored at −20 °C, and thawed in saline for 10 min before use. Skin slices measuring 2 cm × 2 cm were prepared and cleaned to remove dirt and subcutaneous fat. The samples were placed in petri dishes, washed with saline, and subjected to three in vitro transdermal experiments conducted simultaneously(Fujii et al. 2019). Skin samples were removed at 6, 12, and 24 h, respectively, for Raman spectroscopic analysis.

Healthy rats were randomly assigned to three experimental groups: the compound neomycin sulfate ointment group, the blank excipient group, and the blank saline group. Hair on both sides of the spine was removed 24 h before the experiment, ensuring the skin remained intact. For self-control, the depilated area was divided into three sections. In one section, the epidermal layer was intentionally disrupted using a sterilized sharp needle, followed by the application of compound neomycin sulfate ointment. The other two sections received equal amounts of blank excipient and saline, respectively, each covering an area of 3 cm × 3 cm. The applied materials were secured using non-irritating medical adhesive tape. After 72 h, skin reactions were assessed, including erythema, edema, hyperpigmentation, hemorrhage, and skin roughness, along with the time of onset and resolution(Bajgai et al. 2021). At the end of the observation period, skin samples were excised and fixed in 10% neutral buffered formalin. Paraffin sections were then prepared, stained with hematoxylin and eosin (H&E), and examined under a light microscope. The skin irritation response was scored according to Table 1 and Table 2.

Table 1.

Skin irritation scale

Stimulus response	Score
No erythema, no edema	0
Erythema and edema are barely visible	2
Erythema is visible, with an edematous margin higher than the surrounding skin	4
Erythema is moderate to severe, edema is elevated by about 1 mm and well-defined	6
Purple-red spots with crust formation, edema elevated 1 mm or more and expanded	8

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Table 2.

Evaluation Criteria for Skin Irritation Intensity

Evaluation of irritation	Score
Non-irritation	0.00–0.49
Mildly irritation	0.50–2.99
Moderate irritation	3.00–5.99
Intense stimulation	6.00–8.00

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Statistical analysis

The data collected for studies were analyzed using Origin software determining the impact of formulation and process variables. The data were also analyzed for significant differences. One asterisk: p-value less than 0.05, a statistically significant difference. Two asterisks: p-value less than 0.01, a very significant difference. Three asterisks: p-value less than 0.001, the difference is very, very significant.

Results

Determination of drug and content uniformity testing

The results of three API particle size determinations are shown in Table 3. The preparation using the original API exhibited a poor appearance, with the larger particle-sized API (D₅₀ = 31.29 μm) and the oily substrate remaining dispersed independently within the ointment. The content uniformity results indicated that the original API did not adequately combine with the oily substrate to form a uniform suspension. As shown in Fig. 2A, the micronized and airflow-pulverized neomycin sulfate met the requirements for homogeneous suspension, resulting in a more uniform and refined formulation with improved content uniformity. As shown in Fig. 2B, for triamcinolone acetonide, due to its low concentration in the formulation, only the ultra-fine powdered API (D₅₀ = 4.496 μm) ensured content uniformity that met the required standards.

Table 3.

Particle size distribution results for two APIs

Drug	D(μm)	Original API	Air-flow pulverization	Ultra-micronization
Neomycin sulfate	D₁₀	6.530 ± 0.031	5.580 ± 0.025	5.110 ± 0.021
	D₅₀	31.29 ± 0.042	16.04 ± 0.027	11.90 ± 0.028
	D₉₀	84.68 ± 0.025	42.74 ± 0.045	26.38 ± 0.032
Triamcinolone acetonide	D₁₀	5.421 ± 0.024	2.192 ± 0.032	1.269 ± 0.034
	D₅₀	6.342 ± 0.015	5.455 ± 0.036	4.496 ± 0.019
	D₉₀	11.379 ± 0.02	10.590 ± 0.032	7.079 ± 0.026

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The evaluation results were summarized in Fig. 2C and 2D. There was a significant effect on content uniformity when the substrate A content was 4.0616%. Additionally, overly thin formulations failed to maintain uniform API suspension, resulting in particle sedimentation. The quality standard was best met when the content of substrate A was 5.0770%.

The duration of mixing affects particle dispersion and viscosity. When the mixing time was 0.5 h, large particles and low viscosity were visible indicating incomplete drug dispersion. As shown in Figs. 3A and 3B, the particle size became uniform and content uniformity met the required standards at 2 h. Thus, 2 h was selected as the optimal mixing time.

Fig. 3 — Results of content uniformity: (A) Effect of mixing time on content uniformity of neomycin sulfate; (B) Effect of mixing time on content uniformity of triamcinolone acetonide; (C) Effect of mixing temperature on content uniformity of neomycin sulfate; (D) Effect of mixing temperature on content uniformity of triamcinolone acetonide; (E) Effect of cooling temperature on content uniformity of neomycin sulfate; (F) Effect of cooling temperature on content uniformity of triamcinolone acetonide. Note: * p < 0.05; **p < 0.01; ***p < 0.001

The temperature required to emulsify Substrate A impacts the quality of the ointment. When the emulsification temperature was below 80 °C, the substrate did not fully melt, resulting in an uneven appearance and large agglomerates. At 90 °C, the mixing state improved, yielding better appearance and content uniformity, similar to results at 100 °C. However, considering energy consumption and equipment constraints, a temperature of 90 °C was selected for the emulsification process. Content uniformity results are shown in Fig. 3C and 3D.

Cooling temperature also affects content uniformity. At −20 °C, the viscosity reached 72,500 cP, meeting formulation requirements. The content uniformity results are shown in Figs. 3E and 3F. Therefore, −20 °C was chosen as the optimal cooling temperature.

Under the same preparation process, formulations with smaller API particle sizes exhibited higher viscosity as shown in Table 3. As shown in Fig. 4A, the quantity of the oily matrix significantly influenced the viscosity of the ointment. Excessive viscosity compromised extrudability, while insufficient viscosity increased fluidity, reducing the product’s ability to adhere to the skin(Chen et al. 2019). As shown in Fig. 4D, mixing time has little effect on viscosity. As shown in Fig. 4E, the higher the mixing temperature, the higher the viscosity. The results of viscosity are provided in Fig. 4F. At 0 °C and −10 °C, the viscosity increase was minimal, resulting in high fluidity and poor consistency.

Fig. 4 — Results of viscosity: (A) Effect of neomycin sulfate particle size on viscosity; (B) Effect of triamcinolone acetonide particle size on viscosity; (C) Effect of substrate A content on viscosity; (D) Effect of mixing time content on viscosity; (E) Effect of mixing temperature content on viscosity; (F) Effect of cooling temperature content on viscosity. Note: * p < 0.05; **p < 0.01; ***p < 0.001

After experimental optimization, the final formulation was determined to contain 0.54% neomycin sulfate, 0.10% triamcinolone acetonide, 5.08% substrate A, and 94.28% liquid paraffin.

Stability of compound neomycin sulfate ointment

The results of the influencing factor test, accelerated stability test and long-term stability test are shown in Tables 4, 5, and 6. Under long-term experimental conditions, the preparation was stable for 3 months under the condition of 25℃ ± 2℃/60%RH ± 5%RH.

Table 4.

Results of influencing factor test

Conditions	The content of neomycin sulfate (%)		The content of triamcinolone acetonide (%)
Conditions	10 days	30 days	10 days	30 days
High temperature 40℃	101.75	100.18	98.05	98.18
High humidity 75%RH	101.03	100.30	99.83	99.30
Illumination(4500 lx ± 500 lx)	101.84	101.60	100.54	100.21

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Table 5.

Results of the acceleration test

Sample	Time	The content of neomycin sulfate (%)	The content of triamcinolone acetonide (%)
Sample 1	0 day	102.18	99.58
	1 month	101.58	99.18
	2 months	101.77	98.87
	3 months	105.12	98.52
Sample 2	0 day	102.16	101.16
	1 month	102.03	99.53
	2 months	100.57	100.97
	3 months	100.95	99.95
Sample 3	0 day	104.50	99.50
	1 month	104.07	99.07
	2 months	104.06	98.86
	3 months	103.39	98.79

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Table 6.

Results of long-term stability test

Sample	Time	The content of neomycin sulfate (%)	The content of triamcinolone acetonide (%)
Sample 1	0 day	103.18	99.18
Sample 1	3 months	102.75	98.75
Sample 2	0 day	101.16	100.16
Sample 2	3 months	101.79	99.79
Sample 3	0 day	104.50	101.21
Sample 3	3 months	103.34	101.54

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