Evaluation of Rubber Closure Compliance and Container-Closure Characteristics in Syrian Ceftriaxone Sodium Products

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Peer Review Article | Open Access | Published 29th September 2026 | Submitted 12th July 2026

Evaluation of Rubber Closure Compliance and Container-Closure Characteristics in Syrian Ceftriaxone Sodium Products

Authors: Abeer Srour^a*, Basima Arous^a, and Mhd Amer Al-Mardini^a- Damascus University, Syria | EJPPS | 313, (2026) | Cite this article |

Summary

Objectives: Elevated concentrations of butylated hydroxytoluene (BHT), a known rubber-closure additive, have previously been reported in Syrian ceftriaxone sodium products. The present study aimed to evaluate whether the rubber closures used in these products comply with United States Pharmacopeia (USP) physicochemical requirements and to characterize variations in container geometry that may be associated with previously reported differences in BHT concentrations.

Methods: Four hundred rubber closures used in ceftriaxone sodium products manufactured by eight Syrian pharmaceutical companies were evaluated using USP turbidity and reduced substances tests. Vial dimensions of products from the same manufacturers were measured, and inner diameters were calculated from outer diameter and wall thickness. Selected samples were additionally analysed for qualitative BHT identification using GC-MS with reference standard and spiking confirmation.

Results: All rubber closures complied with USP requirements for turbidity and reduced substances. Vial geometry varied among manufacturers, with inner vial diameters ranging from 16.28 to 20.95 mm. Qualitative GC–MS analysis confirmed the presence of BHT in selected ceftriaxone sodium samples.

Conclusions: Compliance with USP physicochemical requirements did not preclude the presence of BHT in ceftriaxone sodium products. Variations in container geometry, together with previously reported differences in BHT concentrations, suggest that container–closure characteristics may influence leachable exposure. These findings underscore the importance of dedicated extractables and leachables assessments in addition to routine pharmacopoeial testing during the evaluation of injectable drug products.

Keywords: rubber closures; ceftriaxone sodium; butylated hydroxytoluene (BHT); turbidity test; reduced substances test; vial geometry.

Introduction

Pharmaceutical packaging plays a crucial role in maintaining drug quality, stability, and safety.¹ Rubber closures, commonly used to seal vials, are preferred for their sealing properties, elasticity, and penetrability.^2,3 However, these closures contain various additives, such as vulcanizing agents, antioxidants, and plasticizers, which may migrate into drug formulations.^2-4 This migration raises concerns regarding extractables and leachables, as these substances can compromise drug efficacy, introduce toxicity risks, and interfere with analytical testing.^1-4

One notable leachable is butylated hydroxytoluene (BHT),^1-6 an antioxidant incorporated into rubber closures at concentrations of 0.5–1% to enhance colour stability.⁷ BHT has been shown to leach from rubber closures into parenteral drug formulations, such as ceftriaxone sodium,^2,5,6 a widely used antibiotic supplied as a powder for injection.^2,4,5,8,9 This migration occurs during storage, with BHT adsorbing to the C3-side chain of ceftriaxone sodium.^2,5 Given BHT's poor water solubility, this interaction leads to the formation of turbidity in reconstituted solutions.^2,5,7 This issue is particularly concerning given previous findings, where BHT levels in some ceftriaxone sodium samples exceeded the toxicological threshold by up to 20-fold.⁶

Beyond its impact on drug clarity, BHT poses potential toxicological risks, as it accumulates in body fat and may enhance IgE-dependent allergic responses, increasing histamine release. Studies have shown that BHT exhibits hepatocarcinogenic effects in animal models, with DNA damage and tumour-promoting activity.⁶ Given its potential health risks and presence as a leachable in injectable drugs, its safety warrants careful evaluation.

The United States Pharmacopeia (USP) sets strict guidelines for elastomeric closures used in parenteral formulations and classifies them into two types: Type I closures, which are intended for aqueous formulations, and Type II closures, designed for non-aqueous formulations. Since ceftriaxone sodium is reconstituted with water, it falls under the category of aqueous formulations, and therefore, Type I closures must be used to ensure compatibility with the drug and compliance with safety requirements. These closures must comply with specific biological, physicochemical, and functional requirements during shipping and in their final ready-to-use state. Among the key physicochemical tests, the reduced substances test, conducted through volumetric assay, assesses oxidizable substances. Additionally, the turbidity test detects substances affecting solution clarity.¹⁰

Although prior research has investigated BHT migration, no study has examined whether USP compliance of rubber closures in Syrian ceftriaxone sodium products correlates with reduced BHT leachability, nor the impact of vial geometry on leachable levels. This study aims to fill this gap by evaluating closure compliance via reduced substances and turbidity testing and analyzing how vial dimensions affect BHT concentration, using data from previous work.⁶ The findings provide critical insights into the safety and compatibility of rubber closures in injectable drugs.

Materials and Methods:

Materials:

Chemicals and Reagents:

Sodium thiosulphate, potassium iodide (BDH Laboratory), hexamethylenetetramine (Merck), hydrazine sulfate (E. Merck AG Darmstadt), potassium permanganate. A BHT reference standard (Sigma-Aldrich) was used for GC–MS identification.

Rubber Closures:

A total of 400 rubber closures representing those used in ceftriaxone sodium products from eight Syrian pharmaceutical companies were collected. The closures from each company were designated as a separate sample (Samples 1–8), corresponding to Companies A–H, respectively. Companies A, B, E, and F used closures from Source I, while Companies C and G used closures from Source II. Companies D and H used closures from Sources III and IV, respectively.

Ceftriaxone Products:

Forty ceftriaxone sodium products from the same eight pharmaceutical companies (A–H), representing different production batches, were evaluated for container geometry assessment. Additionally, one representative sample from each manufacturer (n = 8) was analyzed for qualitative BHT identification by GC–MS, with each sample analyzed in triplicate (three replicates).

Methods:

Turbidity and Reduced Substances Tests:

The turbidity and reduced substances tests were performed according to the USP guidelines for elastomeric closures used in parenteral formulations.¹⁰

Preparation of Solution S:

For each manufacturer, closures were selected and used uncut, corresponding to a surface area of 100 ± 10 cm², as per United States Pharmacopeia (USP) guidelines. Water for injection (WFI) was initially added, and the closures were then boiled for 5 minutes and rinsed four times with WFI. After rinsing, 200 mL of WFI was added. The samples were weighed, then autoclaved at 121 °C for 30 minutes. After autoclaving, they were weighed again, and any reduction in weight was compensated by adding an equivalent volume of water. This final solution constituted Solution S for subsequent testing.

Turbidity Test:

The closures were tested for turbidity according to USP guidelines for elastomeric closures used in parenteral formulations. Turbidity was assessed visually using USP Procedure A. The opalescence of Solution S was compared with USP reference suspensions (A, B, C, D) under standardized conditions. Closures pass the test if the turbidity of Solution S is not greater than that for reference suspension B.

Reduced Substances Test:

Within 4 hours of preparing Solution S, a volumetric assay was conducted using potassium permanganate and sodium thiosulphate titration, as outlined in the USP. The difference between titration volumes for the test solution and the blank was calculated. The closures passed the test if the difference did not exceed 3.0 mL for Type I closures.

The methodology is summarized in Figure 1.

Vial Geometry:

To explore the potential association between container geometry and previously reported leachable exposure,⁶ vial dimensions were measured using a precision micrometer (USSR). The outer diameter and wall thickness of each vial were recorded, and the inner diameter was calculated using the following equation:

Inner Diameter = Outer Diameter – (2 × Wall Thickness)

This value was used to estimate the surface area exposed to potential headspace leachables during storage.

Confirmatory BHT Identification:

To support the interpretation of previously reported BHT leaching findings, selected ceftriaxone sodium samples (n = 8) were analysed for the presence of BHT using a previously published GC-MS method.³ Briefly, chromatographic separation was performed using the conditions described in the original method, with the oven temperature initially maintained at 50 °C for 3 min, followed by a temperature increase to 290 °C at a rate of 220 °C/min, and maintained at 290 °C for 10 min. The inlet temperature was set at 280 °C. BHT identification was performed by comparison of the chromatographic response with a BHT reference standard and confirmed by analysis of spiked samples.

Results:

All rubber closure samples met the USP acceptance criteria: the volume difference in sodium thiosulphate titration between the blank and Solution S remained below the USP limit of 3.0 mL, and turbidity levels did not exceed the specified threshold. The results are presented in Table 1.

Measurements of vial geometry revealed differences among products from the eight manufacturers. Inner vial diameters, calculated from outer diameter and wall thickness measurements, ranged from 16.28 mm to 20.95 mm. The measured dimensions and closure-source classifications are presented in Table 2.

Although closures from the same source were identical in type and size, differences in vial dimensions were observed among manufacturers. Figure 2 illustrates the vial designs used by the eight manufacturers.

BHT was qualitatively identified in the analysed ceftriaxone sodium samples (n = 8). Identification was confirmed by comparison of chromatographic responses with a BHT reference standard and by analysis of spiked samples, which demonstrated corresponding chromatographic behaviour. Representative chromatograms of the BHT standard and spiked samples are presented in Figure 3, while the qualitative identification results are summarized in Table 3.

Discussion:

All tested closures complied with the USP turbidity and reduced substances tests, indicating acceptable performance with respect to the pharmacopoeial criteria assessed.

However, when the pharmacopoeial tests results obtained in the present study were compared with previously reported BHT concentrations in the corresponding ceftriaxone sodium products,⁶ an important observation emerged. Despite compliance with USP requirements, BHT had previously been detected in all examined products, and 47.62% of samples contained concentrations exceeding the Threshold of Toxicological Concern (TTC).⁶ These findings indicate that satisfactory performance in routine pharmacopoeial tests does not necessarily preclude the presence of relevant leachables.

The USP emphasizes that compliance with biological, physicochemical, and functional requirements alone is insufficient for the selection and qualification of packaging components for injectable drug products.¹⁰ Accordingly, the USP recommends the application of dedicated extractables and leachables assessments and encourages the development of analytical procedures capable of detecting and quantifying compounds that may migrate from packaging materials into pharmaceutical products.¹¹

The inability of the turbidity and reduced substances tests to identify closures associated with elevated BHT levels may be explained by the physicochemical properties of BHT itself. The preparation of Solution S relies on aqueous extraction followed by evaluation of turbidity and oxidizable substances. Because BHT exhibits very low water solubility,⁷ extraction into the aqueous medium used for these tests is likely limited. Consequently, closures containing appreciable quantities of BHT may still satisfy USP acceptance criteria. This observation highlights the importance of supplementing routine pharmacopoeial testing with targeted analytical methods when specific leachables are known or suspected to be present.

In the present study, additional confirmatory analysis of selected ceftriaxone sodium samples demonstrated the presence of BHT. The identity of the detected compound was confirmed by comparison with a BHT reference standard and by analysis of spiked samples, providing further evidence that closure-derived BHT may persist despite compliance with routine USP physicochemical requirements. Although this analysis was performed on a limited number of samples and was not intended as a comprehensive quantification study, these findings are consistent with previous reports of BHT migration into ceftriaxone sodium products,⁶ and further emphasize the need for targeted extractables and leachables assessments when specific compounds are of concern.

A further observation arising from the present study was the variability in vial dimensions among products utilizing closures from the same source. Comparison of the vial geometry measurements obtained in this work with previously reported BHT concentrations,⁶ suggested a potential association between inner vial diameter and BHT levels. In comparisons where closure source, closure type, and storage conditions were similar, products with larger inner vial diameters tended to exhibit higher BHT concentrations. Although the mechanism underlying this relationship remains unclear, differences in the geometry of the container-closure system may influence the extent of exposure of the drug product to leachables during storage.

Nevertheless, this finding should be interpreted with caution. The comparison was based on a limited number of products and relied on BHT concentrations reported in a previous study.⁶Consequently, the observed association should be regarded as exploratory and hypothesis-generating rather than conclusive.

Conclusions:

Compliance with USP physicochemical requirements remains an important indicator of rubber-closure quality; however, it does not necessarily exclude the presence of leachables. The findings of this study, considered together with previously reported BHT data, support the need for dedicated extractables and leachables assessments in addition to routine pharmacopoeial testing. For ceftriaxone sodium products, where closure-derived BHT has been detected, the implementation of sensitive analytical methods for monitoring relevant leachables may provide an additional safeguard for product quality, stability, and patient safety. Further studies are warranted to clarify the potential contribution of container–closure geometry to leachable migration and accumulation.

Acknowledgements:

We would like to express our sincere gratitude to the Syrian Ministry of Health for providing the rubber closure and ceftriaxone sodium product samples essential to this study. Special thanks are extended to Dr. Linda Hsien for her valuable support and contributions throughout the course of this work.

Conflict of Interest:

The authors declare that there is no conflict of interest.

Funding Sources:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References

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07. Rowe RC, Sheskey PJ, Quinn ME, eds. Butylated hydroxytoluene. In: Handbook of Pharmaceutical Excipients. 6th ed. London, Pharmaceutical Press and American Pharmacists Association, 2009; 75–76.

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Figures

Figure 1. Method Overview for Turbidity and Reduced Substances Tests. — **Figure 1.** Method Overview for Turbidity and Reduced Substances Tests.

An illustrated schematic showing the tested rubber closures, addition of potassium permanganate and potassium iodide, and visual representation of turbidity formation in samples. Arrows indicate the sequence of steps

Figure 2. Vial Designs of Ceftriaxone Sodium Products from Eight Syrian Manufacturers. — **Figure 2.** Vial Designs of Ceftriaxone Sodium Products from Eight Syrian Manufacturers.

Visual comparison of vial designs used by eight Syrian manufacturers (labeled A–H) for generic ceftriaxone sodium preparations. Each vial corresponds to a distinct company. A-1 represents Company A’s vial design prior to 2024, while A-2 shows the updated design adopted in 2024 and thereafter.

Figure 3. GC–MS Chromatograms of BHT Reference Standard and Spiked Ceftriaxone Sodium Samples. — **Figure 3.** GC–MS Chromatograms of BHT Reference Standard and Spiked Ceftriaxone Sodium Samples.

(A) BHT reference standard.

(B) Representative ceftriaxone sodium sample spiked with BHT reference standard.

Tables

Table 1: Compliance of Rubber Closures from 8 Generic Ceftriaxone Sodium Manufacturers with USP Standards

Sample No	User Company (A-J)	Closure Type	Closure Size	Turbidity Test	Reduced Substances Test	USP compliance
1	A	Bromo-Butyl	20-A	< Sus B	< 3 mL	Complies
2	B	Bromo-Butyl	20-A	< Sus B	< 3 mL	Complies
3	C	Bromo-Butyl	20-A	< Sus B	< 3 mL	Complies
4	D	Chloro-Butyl	20-A	< Sus B	< 3 mL	Complies
5	E	Bromo-Butyl	20-A	< Sus B	< 3 mL	Complies
6	F	Not available	Not available	< Sus B	< 3 mL	Complies
7	G	Bromo-Butyl	20-A	< Sus B	< 3 mL	Complies
8	H	Not available	Not available	< Sus B	< 3 mL	Complies