INTRODUCTION:

One very important parameter to consider during pharmaceutical development of new drugs as well as new formulations is the stability studies¹. It determines the shelf-life prediction of pharmaceutical preparations^{2, 3, 4}. In fact, stability studies constitute an integral part of pharmaceutical development^5,6,7. According to Tembhare et al.⁸ stability of drug is defined as the capability of a particular formulation in a specific container/closed system, to remain within its physical, chemical, microbiological, therapeutic, and toxicological specifications throughout its shelf life. Iqbal and Muhammed⁹ referred to stability as the period of time under specific storage conditions and in a specific container-closure system that a product will retain within predefined limits all of its original characteristics. The United States Pharmacopeia (USP)¹⁰ defines stability as the extent to which a product retains, within specified limits, and throughout its period of storage and use (i.e. its shelf-life), the same properties and characteristics that it possessed at the time of its manufacture. As opined by NagaRaju et al.², stability studies of pharmaceutical products entails the time during which the pharmaceutical products retain its physical, chemical, microbiological, pharmacokinetic properties and characteristics throughout the shelf life from the time of manufacture. Stability studies which can either be real time (long term), intermediate or accelerated studies play a significant role in the quality, safety and efficacy of finished pharmaceutical products (FPPs) throughout its shelf life and it is required to be conducted in a planned way following the guidelines issued by the International Conference on Harmonization (ICH), World Health Organization (WHO) or other regulatory agencies^2,11. Bajaj et al.¹² in their studies opined that stability testing of pharmaceutical products involve a complex set of procedures which require considerable time, scientific expertise with its attended cost in order to build quality, efficacy and safety in FPPs. Oral liquid and external pharmaceutical preparations which are examples of FPPs are subjected to real time studies in order to guarantee their potencies throughout their shelf life. Forced degradation is also a useful in predicting of the stability of drug substance or product^13,14. According to Panda et al.⁵; Carstensen¹⁵and WHO¹⁶, the stability of FPPs depends on the followings: environmental factors (such as light, heat, moisture conditions) during shipment, storage and handling; physical and chemical properties of the active pharmaceutical ingredients (APIs) and the excipients; the composition of the FPPs; the dosage form; the manufacturing processes followed; the nature/attributes of the container-closure system; properties of the packaging materials. However, the degradation reactions such as hydrolysis, oxidation, reduction which play a pivotal role in the stability of FPPs also depend on conditions such as the pH, radiation, concentration of the reactants, catalysts, the raw materials used during the manufacturing processes, the duration between the usage of FPPs and when they were manufactured^2,15,16.

In addition, the stability of FPPs may also be affected due to the followings: consistency, pH, clarity, content uniformity, change in appearance, moisture contents, particle size and shape and package integrity. As reported by Carstensen¹⁵, physical changes that occur during the storage of FPPs may be because of impact, vibration, abrasion and temperature fluctuations such as freezing, thawing or shearing. The chemical reactions like solvolysis, oxidation, reduction, racemization that occur in the pharmaceutical products may lead to the formation of degradation product, loss of potency of active pharmaceutical ingredient (API), loss of excipient activity like antimicrobial preservative action and antioxidants. Stability of a pharmaceutical product can also be affected because of microbiological changes like growth of microorganisms in non sterile products and changes in preservative efficacy as reported by Matthews¹⁷.

Stability tests are carried out so that recommended storage conditions and shelf life can be included on the label to ensure that the medicine is safe and effective throughout its shelf life.

MATERIALS AND METHODS:

Study Area:

The study covered selected pharmaceutical industries located within North Central, Nigeria.

Collection of Data:

The data for real time stability studies of the following selected oral and external pharmaceutical preparations such as: Vitamin C Syrup, Cough Syrup Expectorant, Cough Syrup (for children), Paracetamol Syrup, Magnesium Trisilicate Suspension, Flaggyl Suspension, Co-trimoxazole Suspension, Hydrogen peroxide (6%), Iodine Tincture, Calamine Lotion, Eusol lotion, Benzyl Benzoate and Methylated Spirit from 2018-2022 were obtained from selected pharmaceutical industries located within North Central, Nigeria. The data obtained covered both physico-chemical and microbiological parameters.

Analysis of Data:

The analysis of variance (ANOVA) of the data obtained for the real time stability studies was carried out using IBM SPSS Statistics Version 23. All data were expressed as mean ± standard error of the mean. The values with different superscripts along the same column were significantly different (P < 0.05).

RESULTS:

The results of real time stability studies for Vitamin C Syrup, Cough Syrup Expectorant, Cough Syrup (for children), Paracetamol Syrup, Magnesium Trisilicate Suspension, Flaggyl Suspension, Co-trimoxazole Suspension, Hydrogen peroxide (6%), Iodine Tincture, Calamine Lotion, Eusol lotion, Benzyl Benzoate and Methylated Spirit conducted between 2018 to 2022 are presented in Tables 1-13.

Table 1 shows the real time stability studies of Vitamin C Syrup from 2018-2020. The results obtained for filling volume range from 99.52-100.00ml. There was a reduction in pH values from 2.08 to 2.03; viscosity reduces from 2440.5 mpa.s to 1509.93 mpa.s; ascorbic acid reduces from 101.11% to 95.57% but Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, total viable aerobic mesophilic bacteria plate count (TVAMBPC) and fungi recorded 0.00 cfu/ml respectively.

Table 2 shows the real time stability studies of Cough Syrup Expectorant from 2018-2021. The results obtained for filling volume range from 100.08-100.28ml. The pH values range from 4.39 to 4.45; viscosity reduces from 1731.73 to 1530.93 mpa.s; diphenhydramine hydrochloride reduces from 103.54 to 96.50%; sodium citrate reduces from 101.22 to 95.76%; ammonium chloride reduces from 102.84 to 99.47% while TVAMBPC recorded 66.57cfu/ml in 2020 and 92.67 cfu/ml in 2021. On the other hand, 0.00cfu/ml was obtained for E. coli, S. aureus, S. typhimurium, P. aeruginosa and fungi respectively.

Table 3 shows the real time stability studies of Cough Syrup (for Children) from 2018-2021. The results obtained for filling volume range from 99.66-100.05ml. The pH values range from 4.03 to 4.37; viscosity reduces from 1629.95 to 1535.70 mpa.s; diphenhydramine hydrochloride reduces from 102.68 to 97.33%; sodium citrate reduces from 100.65 to 95.42%; TVAMBPC of 23.33cfu/ml and 30.00cfu/ml were obtained for in 2020 and 2021 while 3.33cfu/ml was recorded for fungi in 2020. On the other hand, E. coli, S. aureus, S. typhimurium and P. aeruginosa had 0.00cfu/ml each.

Table 4 shows the real time stability studies of Paracetamol Syrup from 2018-2021. The filling volume range from 60.05-60.18ml; pH values range from 6.44 to 6.56; viscosity reduces from 2137.43 to 1557.13 mpa.s and acetaminophen reduces from 102.24 to 95.62%. Meanwhile, E. coli, S. aureus, S. typhimurium, P. aeruginosa, TVAMBPC and fungi had 0.00 cfu/ml each.

The results for real time stability studies of Magnesium Trisilicate Suspension from 2018-2021 are presented in Table 5. The filling volume range from 197.08-200.28 ml; 8.05-8.83 values were obtained for pH; viscosity reduces from 14078.77 to 12436.80mpa.s; magnesium content reduces from 102.35 to 92.81%; sodium bicarbonate drops from 100.88 to 94.00; E. coli, S. aureus, S. typhimurium, P. aeruginosa had 0.00 cfu/ml each while TVAMBPC were 23.33cfu/ml in 2019, 40.00 cfu/ml in 2020, 36.67cfu/ml in 2021 but fungi were 10.00cfu/ml in 2019, 3.33cfu/ml in 2020 and 6.67cfu/ml in 2021.

Furthermore, the results for real time stability studies of Flaggyl Suspension from 2018-2021 are presented in Table 6. The filling volume range from 59.93-60.27ml; pH values range from 5.21-6.56; reduction in viscosity from 2137.43 to 1996.13 mpa.s; metronidazole benzoate reduces from 101.75 to 95.37% while E. coli, S. aureus, S. typhimurium, P. aeruginosa; TVAMBPC and fungi recorded 0.00 cfu/ml each.

Table 7 shows the results for real time stability studies for Co-Trimoxazole Suspension from 2018-2021. The filling volume range from 50.02-50.17 ml; pH values range from 5.44-5.71; reduction in viscosity from 1693.13 to 1584.67 mpa.s; sulphamethoxazole reduces from 101.18 to 95.52%; Trimethoprim reduces from 103.04 to 95.26% while E. coli, S. aureus, S. typhimurium, P. aeruginosa; TVAMBPC and fungi recorded 0.00 cfu/ml each.

Table 8 shows the results for real time stability studies of Hydrogen Peroxide (6%) from 2018-2020. The filling volume range from 100.01-100.02ml; pH values range from 2.53-2.75; reduction in hydrogen peroxide (6%) from 6.49-5.75% while E. coli, S. aureus, S. typhimurium, P. aeruginosa; TVAMBPC and fungi recorded 0.00 cfu/ml each.

In addition, Table 9 shows the results for real time stability studies of Iodine Tincture from 2018-2020. The filling volume range from 15.01-15.02 ml; reduction in iodine from 2.33-2.28%; reduction in potassium iodide from 2.37-2.31% while E. coli, S. aureus, S. typhimurium, P. aeruginosa; TVAMBPC and fungi recorded 0.00 cfu/ml each.

Table 10 shows the results for real time stability studies of Calamine Lotion from 2018-2020. The filling volume range from 100.01-100.03 ml and the value of residue on ignition reduces from 19.95-19.12%. E. coli, S. aureus, S. typhimurium, P. aeruginosa; TVAMBPC and fungi had 0.00cfu/ml each.

The results for real time stability studies of Eusol Lotion from 2018-2020 are shown in Table 11. The filling volume was 100.01 ml and the value of available chlorine reduces from 0.38-0.25%. E. coli, S. aureus, S. typhimurium, P. aeruginosa; TVAMBPC and fungi had 0.00cfu/ml each.

The results for real time stability studies of Benzyl Benzoate from 2018-2020 are shown in Table 12. The filling volume range from 100.02-100.07ml and the value of benzyl benzoate reduces from 25.08-24.80% while E. coli, S. aureus, S. typhimurium, P. aeruginosa; TVAMBPC and fungi had 0.00cfu/ml each.

Table 13 shows the results for real time stability studies of Methylated Spirit from 2018-2022. The filling volume range from 100.00-100.03ml; alkalinity ranges from 0.80-0.86ml; acidity ranges from 0.10-0.20ml; apparent density increases from 805.04-817.09 Kgm^-3; the value of non-volatile matter ranges from 0.00-0.01 %w/v; aldehydes values range from 10.08 to 10.15 ppm; the highest sensitivity values recorded by S. aureus and C. albicans were 21.00mm and the lowest values were 18.00mm respectively.

The level of significance difference for all the values obtained was determined at p<0.05. The values with the same superscript in the same column are not significantly different (Tables 1-13).

Table 1: Real Time Stability Studies of Vitamin C Syrup from 2018-2020

Parameters	2018	2019	2020	Expected/Limits
Filling Volume (ml)	100.00±0.06^a	99.52±0.51^b	99.96±0.04^b	95-105 ml**
pH	2.08±0.04^a	2.05±0.01^a	2.03±0.01^a	2.0-6.0**
Viscosity (mpa.s)	2440.50±118.07^b	1542.54±32.12^c	1509.93±55.52^c	1500-2000 mpa.s**
Assay: Ascorbic Acid (%)	101.11±1.29^a	96.30±0.96^b	95.57±0.86^b	95.0-107.5%*
Escherichia coli (cfu/ml)	0.00±0.00^a	0.00±0.00^a	0.00±0.00^a	0 cfu/ml*
Staphylococcus aureus (cfu/ml)	0.00±0.00^a	0.00±0.00^a	0.00±0.00^a	0 cfu/ml*
Salmonella typhimurium (cfu/ml)	0.00±0.00^a	0.00±0.00^a	0.00±0.00^a	0 cfu/ml*
Pseudomonas aeruginosa (cfu/ml)	0.00±0.00^a	0.00±0.00^a	0.00±0.00^a	0 cfu/ml*
TVAMBPC (cfu/ml)	0.00±0.00^a	0.00±0.00^a	0.00±0.00^a	≤1 x 10³*
Fungi (cfu/ml)	0.00±0.00^a	0.00±0.00^a	0.00±0.00^a	≤1 x 10²*