“The ability of a drug substance or drug product to remain within specifications established to ensure its identity, strength, quality and purity throughout the retest or expiration dating period” is the definition of stability in the context of pharmaceutical products.¹ Drug instability may result in an undesirable change in performance that results in the failure of the product. Every pharmaceutical product must have an expiration date as it represents a critical quality parameter. Ideally, alongside the expiration date, there should also be guidance on the recommended storage conditions.²

Stability studies are one of the most crucial steps in the creation of a pharmaceutical since they ensure the identity, potency and purity of both the raw materials and the finished goods.³ After it has been produced, a pharmaceutical product’s stability is influenced by a number of environmental factors, such as the ambient temperature, humidity and light levels, as well as factors unique to the product, such as the chemical and physical properties of the active ingredients and pharmaceutical excipients, the composition of the dosage form, the manufacturing process, the type of container closure system and the characteristics of the packing material.^4–6 Stability studies are an integral part of the drug development process, which consists of six distinct phases namely Stage 1, Stage 2, Stage 3, Stage 4, Stage 5, Stage 6.⁷ The objective of performing stability testing is to establish shelf-life and processing specifications for new products, to investigate whether the breakdown of active medications may result in the formation of toxic compounds, to ensure that the brand is fit for usage for the duration that it is on the market and has all functionally acceptable features to preserve the manufacturer’s good name.^8–14 The stability studies are also done to check if any deviation have been made during the manufacturing process or formulation process which might lead to detrimental effects on product stability. Stability studies provide valuable insights in selecting excipients, formulations and container-closing techniques. More importantly, stability studies are the only method to definitively determine whether or not a pharmaceutical drug fulfills the criteria for approval. According to various studies and investigations there are many factors which will affect drug stability. The drug stability is influenced by many such factors are temperature^15–17 moisture, pH, excipients, Oxygen, light, etc. Apart from the mentioned factors, there are also factors such as physical stability,¹⁸ chemical stability, microbiological stability and therapeutic stability of the drug substances.

Stability testing is a customary approach applied throughout the diverse phases of pharmaceutical substance and product development. In the initial stages, accelerated stability tests are employed to evaluate the type of degradation observed after prolonged storage. The primary objectives of pharmaceutical stability testing are to ensure that products remain suitable for consumption until the final pharmaceutical unit is used and that they maintain their acceptable quality throughout their market presence.¹⁹ There are four categories of stability testing methodologies widely employed namely real-time stability testing, accelerated stability testing, retained sample stability testing and cyclic temperature stress testing. The Real-time stability test Utilizes trend analysis, relevant data is collected at an appropriate frequency to distinguish instability from everyday fluctuations throughout the testing process.²⁰ Accelerated stability testing exposes a product to higher temperatures and various stress factors to determine heat-induced failure. Stressors like humidity, heat, turbulence, weight, pH and packaging are applied and samples are stretched, cooled and then analysed quickly to reduce measurement instability compared to real-time testing. For thermo-labile and protein components, reliable stability predictions require avoiding denaturing stress temperatures. Four different stress levels are recommended for statistical validity.^21,22 The Arrhenius equation is used to forecast stability based on degradation rates at high temperatures and it can predict degradation under “stress” at lower temperatures.²³ According to Retained sample stability testing, it is customary to test samples at predefined intervals for stability if a product has a shelf life of five years, therefore stability samples are examined at 3, 6, 9, 12, 18, 24, 36, 48 and 60 months. The constant interval procedures are the accepted method for gathering stability data on samples that have been stored.²⁴ Cyclic stress testing primarily emphasises aspects such as stability, potency, purity and formulation, as opposed to physical stressors such as temperature cycling.

Stability chambers are environmental specialist chambers that can stimulate storage conditions and real-time stability, expedited stability and protocol for the long term. They are used for stability testing and allow for the evaluation of product stability. There are 2 different kinds of light sources employed in the photostability chamber such as cool white and near UV fluorescent tubes are combined and daylight artificial lamps (e.g., metal halide or xenon). A maximum exposure of 1.2 million lux hours is required. A lux metre is used to determine the amount of visible light necessary and the number of hours of exposure.²⁵

Stability Climate zone testing was done using regional average annual temperature and relative humidity.²⁵ The structured approach within the drug development process is stability testing. Stability data for the pharmaceutical compound play a crucial role in determining suitable storage conditions and packaging for large quantities of the substance.²⁶ The goal of conducting stability studies on the drug product is to determine its shelf life or expiration date.^27–29

Connect the pH meter to a power source and allow it to warm up for duration of 5 to 10 min. Switch it to pH mode when you’re ready to record the pH values.

Weight per milliliter (weight per mL) of a liquid refers to the weight in grams of 1 mL of the liquid at 20°C unless specified otherwise. This method is employed in tests for oral solutions and suspensions. To calculate the weight per milliliter (g per mL) for content determination, a known quantity of the solution or suspension is weighed and its weight per mL is obtained by dividing the weight of the liquid filling a specific pycnometer at the specified temperature by the pycnometer’s capacity in milliliters at that temperature. The pycnometer’s capacity is determined by measuring the weight of water required to fill it at that temperature.

The following products are sourced from Topley House at 52 Wash Lane, Bury, Lancashire, UK, BL96AS: Nutrient broth, nutrient agar, Mac Conkey agar, Mannitol salt agar, Cetrimide agar, Mueller-Hinton agar, Salmonella-Shigella agar and Saboraud Dextrose agar.

Two tablets from each blister-packed paracetamol, brand were placed in 10 mL of sterile normal saline, mixed for 5 min using a vortex mixer to dislodge microbes and the liquid above settled solid particles (supernatant) was used for further analysis.

1 mL from each brand’s dispersion was plated on duplicate nutrient agar plates. This process was repeated with MacConkey agar, Mannitol salt agar, cetrimide agar, Salmonella-Shigella agar and Saboraud Dextrose agar. Saboraud Dextrose agar plates were incubated at 25°C for 72-96 hr, while the others were incubated at 37°C for 48 hr. After incubation, plates were inspected for microbial growth.

Multivitamin syrup was diluted in sterile peptone water at ratios of 1:10, 1:100 and 1:1000. Each dilution (0.1 mL) was inoculated onto duplicate Petri dishes with various agar media. These plates were incubated at 37°C for 24-48 hr, except for Saboraud Dextrose agar, which was incubated at 25°C for 72-96 hr. After incubation, plates were checked for microbial growth. Bacterial isolates from growth-positive plates underwent standard microbiological identification tests to confirm their identity and purity, including colony morphology assessment and conventional biochemical tests.

The antibiotic susceptibility testing, both Gram-negative and Gram-positive isolates were assessed using the disc diffusion method, following Clinical and Laboratory Standards Institute (CLSI) guidelines. Gram-negative bacteria were tested against eight antibiotics, including augmentin (30 μg), ofloxacin (5 μg), gentamycin (10 μg), nalidixic acid (30 μg), nitrofurantoin (200 μg), cotrimoxazole (25 μg), amoxycillin (25 μg) and tetracycline (25 μg). Gram-positive bacteria were tested against three antibiotics, which were oxacillin (1 μg), cefuroxime (30 μg) and vancomycin (30 μg).

To perform the susceptibility tests, colonies from a nutrient agar plate were suspended in sterile distilled water and adjusted to a turbidity equivalent to a 0.5 McFarland standard. This suspension was spread onto Mueller Hinton agar plates and allowed to acclimatize and grow at 37°C for 20 min. Antibiotic discs were placed on the agar surface and refrigerated for 30 min for proper diffusion. A control strain, E. coli ATCC 25922, was used as a reference. After 18 hr of incubation at 37°C, the diameters of inhibition zones were measured in millimeters and interpreted according to CLSI guidelines.

A 1000 mL volumetric flask was filled with 4.4 g of dibasic potassium phosphate and 500 mg of sodium 1-octanesulfonate and the mixture was then diluted with water to volume. A pH of 8.20 0.05 was achieved by adding phosphoric acid to the mixture. Then, the 45:40:15 mixtures of acetonitrile, octane sulfonate buffer and methanol was filtered and degassed to create the mobile phase.

Initially, 1.7 g of monobasic ammonium phosphate were placed into a 1 L volumetric flask and subsequently filled with water to reach the desired volume. The pH was then meticulously adjusted to 10.00±0.05 using ammonium hydroxide. Subsequently, a diluent labeled as “A” was formulated by combining ammonium phosphate buffer (pH 10), methanol and acetonitrile in a precise ratio of 35:35:30.

Initially, a standard azithromycin solution was created by placing 20 mg of USP azithromycin RS into a 50 mL volumetric flask. Diluent A was then introduced to reach the flask’s calibration mark, followed by sonication to achieve a solution with a concentration of 0.4 mg of azithromycin per milliliter.

The multivitamin syrup was taken. This was mixed with 75 mL of Diluent A, sonicated for 15 min and diluted to the flask’s mark. From this solution, 6.0 mL were transferred to a 50 mL flask, further diluted with Diluent A to reach a 0.4 mg/mL syrup concentration and filtered through a 0.45 μm filter. The chromatographic system used a liquid chromatograph with a 210-nm detector, a 4.6 mm × 25 cm column with 5-mm L1 packing and operated at a flow rate of 1.5 mL/min for a total run time of 30 min. The column was consistently maintained at 50°C and HPLC was executed, verifying column equilibrium with a standard solution (50 μL) until the relative standard deviation stayed below 2% for six consecutive injections.

In equal quantities, approximately 50 μL of the multivitamin syrup,³⁰ were independently introduced into the chromatograph. The peak area responses (n=3) were recorded for all peaks. Subsequently, the percentage content of azithromycin was determined using the following formula:

Survey was conducted based on the parameters which are given in Table 4 following results.

This is performed to assess and address instances where a product or sample does not meet established specifications or quality standards. This test is done to check if any deviating from the defined parameters of a product and if any findings suggest any deviation it is labeled as an “OOS” result, OOS causes can be categorized as Assignable (when the cause of the error is identified) and Non-assignable (When the cause of the error remains unidentified).These determinations are communicated by the Quality Control (QC) team to designated personnel responsible for OOS classification, distinguishing between assignable and non-assignable causes.

An analytical result that whilst being in the specification, falls outside of the expected normal or pre-defined for that test parameter. This shall be identified during analysis by comparing against trend and results obtained at the extreme of specification. In general, Out of Trend can be described as a result of sequence of results that are within specification limits but are unexpected and not in line with the routine.

It refers to testing that is carried out to either substantiate or rule out potential root causes based on initial assumptions. This involves conducting additional tests to investigate what could have occurred, which can encompass various aspects such as further testing related to sample filtration, sonication/extraction, or potential equipment malfunctions, among others. This process allows for the exploration of multiple hypotheses.

The test was conducted on the stability of multivitamin syrup under storage conditions at a temperature of 300°C±20°C/ relative humidity 65%±5% with a test duration of 6 months according to ICH guidelines. The production batch size of the product is 1000 L. The test started on 05/10/2022 and ended on 04/09/2024, as shown in Table 1.

According to the specifications, the syrup was found to be yellow-colored syrup contained in a duly graded amber-colored PET bottle with a yellow metallic cap. In the 1st month, 3rd month and 6th month, the targets specified in Table 1 are met.

The specification of the drug product pH is 3.0-4.5. 1^st month it was found to be 4.02. In 3^rd month pH was found to be 4.01 and 6^th month, the pH was found to be 4.0. The pH of the product compiles the specifications in Table 1.

The product specification of the weight for mL is 1.00 to 1.15 g/ mL. In the 1st month, the result was found to be 1.1020 g/mL. In the 3^rd month, the result was found to be 1.1018 g/mL and in the 6^th month, the result was found to be 1.1019 g/mL. The weight per mL of the product compiles the specification. The date of analysis of the above tests is as per specifications shown in Table 1.

The specification of Total yeast and mold count is not more than 100 cfu/mL and not less than 10 cfu/mL. The first month it was found to be 25 cfu/mL and less than 10 cfu/mL. The 3^rd month it was found to be 25 cfu/mL and less than 10 cfu/mL and 6^th month it was found to be 30 cfu/mL and less than 10 cfu/mL as shown in Table 2.

The specification of Escherichia coli should be absent. In the 1^st, 3^rd and 6^th months, there were no Escherichia coli was present.

The specification of Salmonella should be absent. In the 1^st, 3^rd and 6^th months, there were no Salmonella present.

The specification of Pseudomonas aeruginosa should be absent. In the 1^st, 3^rd and 6^th months there were no Pseudomonas aeruginosa was present.

The specification of Staphylococcus aureus should be absent. In the 1^st, 3^rd and 6^th months, there were no Staphylococcus aureus was present.

The results of microbial limit studies of the product were found as per ICH specification. This means the product is stable.

We conducted purity and quantity tests on syrup, following ICH specifications (not less than 90% purity). Here are the results for each component shown in Table 3.

Thiamine Hydrochloride BP: 1^st month:3.2012 mg (160.06%), 3^rd month: 3.1982 mg (159.91%).

6^th month: 3.1892 mg (159.46%).

Riboflavin Sodium Phosphate BP: 1^st month: 3.9984 mg (157.42%), 3^rd month: 3.9910 mg (157.13%), 6^th month: 3.9674 mg (156.20%).

Pyridoxine Hydrochloride BP: 1^st month: 3.2984 mg (164.92%), 3r^d month: 3.2965 mg (164.83%), 6^th month: 3.2940 mg (164.70%).

Niacinamide BP: 1^st month: 21.3300 mg (106.65%), 3^rd month: 21.3285 mg (106.64%), 6^th month: 21.3225 mg (106.61%).

D-Panthenol BP: 1^st month: 6.5855 mg (109.76%), 3^rd month: 6.5827mg (109.71%),

6^th month: 6.5778 mg (109.63%).

Ascorbic Acid BP: 1^st month: 100.2912 mg (133.72%), 3^rd month: 100.2619 mg (133.68%),

6^th month: 100.2022 mg (133.60%).

All components met or exceeded the required 90% purity level.

We surveyed pharmaceutical tablet preparations manufactured by the production department. The syrup was stored at 30°C±20°C/RH 65%±5% for 6 months using blister packaging as the primary packaging. No color or physical changes occurred in the packaging material over this period. The assay showed 90% drug content, with no color or physical changes in the tablets. Microbial studies found no bacteria, fungi, or pyrogens.

The product was found to be in the specification as there is no Assignable (when the cause of the error is identified) and Non-assignable (When the cause of the error remains unidentified) errors observed in the syrup this proves that the syrup meets all the requirements as per specification in the guideline.

As shown in Table 4 the product meets all specifications and is considered stable. The same formulation of syrup with different batches also showed stability under the same storage conditions, with no color or physical changes, compliance in identification and purity tests and no microbial contamination. The OOT Test Results of the syrup No abnormal observations noted during the testing. The formulations meet well within the specification and hence root causes analysis was ruled out in Hypothesis/ Investigative Testing.

The stability studies conducted on the multivitamin syrup showed positive results, indicating that the product is stable under the specified storage conditions. The syrup maintained its yellow color and met the specified targets throughout the 6-month test period. The pH of the product also complied with the specifications, remaining within the range of 3.0 to 4.5.

The weight per mL of the syrup was within the specified range of 1.00 to 1.15 g/mL, demonstrating that the product met the weight specification. Microbial limit tests were conducted to assess the presence of microorganisms in the syrup. The results showed that the total yeast and mold count remained below the maximum limit of 100 cfu/mL and above the minimum limit of 10 cfu/ mL throughout the test period. Additionally, no Escherichia coli, Salmonella, Pseudomonas aeruginosa, or Staphylococcus aureus were detected in any of the monthly tests, indicating that the product was free from these harmful bacteria.

Assay tests were performed to determine the purity and quantity of the components in the syrup. All components, including Thiamine Hydrochloride BP, Riboflavin Sodium Phosphate BP, Pyridoxine Hydrochloride BP, Niacinamide BP, D-Panthenol BP and Ascorbic Acid BP, met or exceeded the required 90% purity level. This indicates that the syrup contained the specified amounts of each component and was of high quality.

A survey was also conducted on multivitamin syrup preparations manufactured by the production department. The syrup showed no color or physical changes in the packaging material. The assay tests on the syrup revealed a 90% drug content and microbial studies found no presence of bacteria, fungi, or pyrogens. This further confirms the stability and quality of the product.

Overall, the stability studies, microbial limit tests and assay tests demonstrate that the multivitamin syrup meets all specifications and is considered stable. The consistent results obtained from different batches of the syrup further support its stability and quality. No out-of-specification errors were observed, indicating that the product meets all requirements as per the guidelines.

The stability studies conducted on the multivitamin syrup showed positive results, indicating that the product is stable under the specified storage conditions. The syrup maintained its yellow color and met the specified targets throughout the 6-month test period. The pH of the product also complied with the specifications, remaining within the range of 3.0 to 4.5.

The weight per mL of the syrup was within the specified range of 1.00 to 1.15 g/mL, demonstrating that the product met the weight specification. Microbial limit tests were conducted to assess the presence of microorganisms in the syrup. The results showed that the total yeast and mould count remained below the maximum limit of 100 cfu/mL and above the minimum limit of 10 cfu/ mL throughout the test period. Additionally, no Escherichia coli, Salmonella, Pseudomonas aeruginosa, or Staphylococcus aureus were detected in any of the monthly tests, indicating that the product was free from these harmful bacteria.

Assay tests were performed to determine the purity and quantity of the components in the syrup. All components met or exceeded the required 90% purity level. This indicates that the syrup contained the specified amounts of each component and was of high quality.

A survey was also conducted on syrup preparations manufactured by the production department. The syrup showed no color or physical changes in the packaging material. The assay tests on the tablets revealed a 90% drug content and microbial studies found no presence of bacteria, fungi, or pyrogens. This further confirms the stability and quality of the product.

Overall, the stability studies, microbial limit tests and assay tests demonstrate that the multivitamin syrup meets all specifications and is considered stable. The consistent results obtained from different batches of the syrup further support its stability and quality. No out-of-specification errors were observed, indicating that the product meets all requirements as per the guidelines.

The authors would like to convey thanks to the NGSM Institute of Pharmaceutical Science for providing the infrastructure for carrying out the present study.