Study of the Microbiological Stability of a Pediatric Oral Solution of Paracetamol, after Introduction of the Dosing Pipette for 5 days of use

 

Nihal Bhirich^1,2*, Hicham Elhorr^3,4, Ghita Salime Meknassi^1,2, Ali Cherif Chefchaouni^1,2, Mohamed Yafout^3,4, Amal Ait haj Said^3,4, Younes Rahali^1,2

¹Department of Galenic Pharmacy and Pharmacotechnics. Faculty of Medicine and Pharmacy,

Mohammed V University, Rabat, Morocco.

²Ibn Sina University Hospital Center, Rabat, Morocco.

³Laboratory of Drugs Sciences, Biomedical Research and Biotechnology. Faculty of Medicine and Pharmacy, Hassan II University of Casablanca, Morocco.

⁴Ibn Rochd University Hospital Center, Casablanca, Morocco.

 

ABSTRACT:

Background: In Morocco as in other countries around the world, the galenic form most used for the administration of paracetamol in children is the syrup form, most often accompanied by an administration device "dosing pipette" also called "dose pipette -weight” which is presented in the form of a graduated syringe, in units corresponding to the weight of the child in kilograms (Kg). The primary objective: In the present study, we are interested in the effect of the use of dose-weight pipette dosing devices on the microbiological stability of the oral solution after opening. This concerns in particular the effect of opening and frequent introduction of this pipette into the medicinal product during its use during treatment. Methods: For this study we used an oral pediatric paracetamol solution, we simulated the use of this solution, by taking 4 doses every 6 hours, for 5 days (average duration of a febrile state), using the dosing pipette provided. The microbiological contamination of the solution was systematically tested immediately after opening (control) over a period of five days after daily introduction of the pipette (every 6 hours). At the same time, we carry out the same simulation, with another bottle of paracetamol of the same brand. The bottle will be kept in a refrigerator at a temperature between 2° and 8°, after each introduction of the dosing pipette, unlike the first simulation. In accordance with the specifications described in European Pharmacopoeia monograph 2.6.12. A 1:10 dilution of the product was prepared using a buffered sodium chloride-peptone solution at pH 7.0. More precisely, for each sample, 2.5grams were weighed and diluted according to the protocol prescribed in European Pharmacopoeia. Results: Based on the results of the study, the health risk associated with the use of the pipette was identified mainly in terms of microbial contamination by staphylococci. Thus, in accordance with the specifications of the European Pharmacopoeia, the microbiological quality is judged to be non-compliant for the solution used for a simulation of use for 5 days. Conclusion: This study therefore underlines the importance of taking into consideration during stability studies after opening, of these pharmaceutical forms, in general, the impact of the frequent introduction of "dosing pipette" type devices as well as compliance with the rules for using this type of device in particular, cleaning and storage instructions.

 

 

INTRODUCTION: 

Fever in children is an extremely common reason for consultation; according to numerous studies carried out in general medicine and pediatrics, it is most often the subject of symptomatic treatment¹. Thus, 29.7% of pediatric consultations are solely motivated by the presence of a fever, which places fever in second place among reasons for consultation behind cough (46.5%)². The average duration of fever according to these studies is 4.9 days³.

 

Currently, antimicrobials are the most widely used category of drugs in the world⁴, but among the prescribed molecules, we find PARACETAMOL, N- acetyl -p- aminophenol, which is slowly decomposed under acidic or alkaline conditions into acetic acid and p-aminophenol^5,6,7. It is a synthetic non-opiate derivative of para- aminophenol and an active metabolite of phenacetin⁸. It was first introduced by von Mering in 1893 but did not come into wide use until the 1950s. It has become the most widely used analgesic-antipyretic in the world today, due to its excellent risk/benefit ratio and its low potential for harmful drug interactions at standard doses⁹.

 

Oral route is the most preferred route for better patient compliance and easy administration for children. In Paediatrics patient there are more compliance with ease administration and more palatable and attractive dosage forms has significantly importance in the design of novel drug delivery system¹⁰.

In Morocco as in other countries around the world, the galenic form most used for the administration of paracetamol in children is the oral solution form, most often accompanied by an administration device "dosing pipette" also called "pipette dose-weight” which is presented in the form of a graduated syringe, in units corresponding to the weight of the child in kilograms.

 

We also find in other products more classic dosing devices such as the measuring spoon which remains widely used in France in particular and in other European countries¹¹.

 

In the present study, we are interested in the effect of the use of dose-weight pipette type dosing devices on the microbiological stability of the oral solution after opening. This concerns in particular the effect of opening and frequent introduction of this pipette into the medicinal product during its use during treatment.

 

MATERIALS AND METHODS:

Material:

For this study we used an oral pediatric paracetamol solution, marketed in Morocco and purchased in a pharmacy in a 90ml bottle. Its cost is two Dollars, and its expiry date was: 03/2026, packaged in tinted glass, the validity period after opening mentioned on the bottle is 3 months. According to the instructions, the composition of this medicine is as follows:

·       Paracétamol

·       Macrogol 6000

·       Sucrose solution

·       Sorbic acid (E 200)

·       Sodium methyl parahydroxybenzoate E 219

·       Sodium propyl parahydroxybenzoate E 217

·       Purified water qs. 100 ml of oral solution

 

This specialty is supplied with a dose-weight pipette.

 

Duration and conditions of the study:

We simulated the use of paracetamol solution for pediatric oral use, by taking 4 doses every 6 hours, for 5 days (average duration of a febrile state3), using the dosing pipette provided. The doses taken are directly eliminated without any contact with the handler and the child. The pipette is then rinsed each time according to the recommendations in the instructions:

 

“After use, close the bottle of the oral solution, rinse the oral dosing pipette well with water and dry the oral dosing pipette. Then immediately store the dosing pipette for oral administration in its box in a place inaccessible to children. Never separate the dosing pipette for oral administration from the other medication packaging elements (bottle, box, instructions).”

 

The study took place during a cold period of the year, the vial is stored in a place at room temperature for the duration of the study. The first sample is taken after the first opening on March 20, 2024, and the second sample after 5 days on March 25, 2024.

 

At the same time, we carry out the same simulation, with another bottle of paracetamol of the same brand. The bottle will be kept in a refrigerator at a temperature between 2° and 8°, after each introduction of the dosing pipette, unlike the first simulation.

 

Evaluation of microbiological contamination:

The microbiological contamination of the solution was systematically tested immediately after opening (control) then over a period of five days after daily introduction of the pipette (every 6hours). This assessment adheres to established microbiological quality standards referenced in the European Pharmacopoeia, encompassing rigorous testing protocols for total aerobic microbial counts, identification of specific pathogens and verification of the absence of specified microorganisms. This methodical approach aims to evaluate and compare the microbiological integrity of the solution with the introduction of the pipette for five days compared to the control solution, thus providing valuable information on the potential microbial risks associated (Table I) with the use of the dosing pipette in the administration of liquid forms.

 

Table 1: Acceptance criteria for the microbiological quality of aqueous solutions¹²

Route of administration	TAMC* (CFU/g or CFU/ mL)	TYMC* (CFU/g or CFU/ mL)	Specified microorganismes
Aqueous preparations for oral use	10 2	10 1	Absence of Escherichia coli (1 g or 1 mL)

 

TAMC: Total aerobic microbial count

TYMC: Total yeast and mold count

CFU: Colony -forming unit

In accordance with the specifications described in European Pharmacopoeia Monograph 2.6.12, a dilution procedure was undertaken. A 1:10 dilution of the product was prepared using a buffered sodium chloride-peptone solution at pH 7.0. More precisely, for each sample, 2.5grams were weighed and diluted according to the protocol prescribed in Table II.

 

Table 2: Microbiological control plan for enumeration test of a solution^{12,13,14,15,16}

90 ml of buffered sodium chloride-peptone solution pH 7.0
+ 10 ml of the product to be examined (Test or control)
Bacteria	Mushrooms	Escherichia coli
2*1 ml in casein soybean agar (TSA) digestion medium	2*1 ml of Sabouraud-dextrose agar medium (SAB)	10 ml in 100 ml of soy casein Bean digestion broth
05 days at 30°C-35°C	07 days at 20°C-25°C	18-24 hours at 30°C-35°C
		MacConky broth medium
		24-48 hours at 42-44°C
		Conky Agar Media
		18-72 hours at 30-35°C

 

In total, three tests were performed for each bottle, including: two test samples after 5 days of pipette use and one control sample after immediate opening, for each category, giving a total of six tests.

 

In the case of existing colonies, we will use the Analytical Profile Index or API, a standardized system designed for genera identification, which uses miniaturized biochemical tests and a specially adapted database. The full list of bacteria that can be identified using the API is available in the corresponding manual¹⁷.

 

RESULTS:

1/Results of the 1^st sample and 2^nd sample after first opening:

The culture of the three categories (bacteria, fungi and E coli) proved to be sterile: absence of bacteria, including Escherichia coli, after subculture on MacConkey agar medium. As well as the absence of fungi in a Sabouraud-dextrose agar (SAB) medium, for both bottles.

 

2/ Result of the 3^rd sample after 5 days stored at room temperature:

The culture was negative for: SAB medium indicating the absence of fungi and also on MacConkey agar medium after subculturing, which means the absence of Escherichia coli.

 

While twenty colonies were recorded on TSA medium, nine on the first Petri dish and eleven on the second. Following Gram staining, the presence of Gram-positive cocci was identified in Figure 1 and Figure 2.

 

Figure 1. Petri dish No. 1 with 9 colonies on TSA medium

 

Figure 2. Petri dish No. 2 with 11 colonies.

 

Positive and coagulase negative tests were then carried out, followed by exploration on an electron microscope to confirm that it could be a Staphylococcus Figure 3.

 

Figure 3. Exploration on an electron microscope.

 

When subculturing using the API® Staph Analytical Profile Index, it was determined to be Staphylococcus hominis with an obtained score of 6210013 and Staphylococcus warneri with a probability of 79.3%, Staphylococcus hominis 16.3%, Staphylococcus auricularis 3.1% and Staphylococcus saprophyticus 1.0% figures 4,5 and 6.

Figure 4. Transplanting into the API T0 gallery.

 

Figure 5. Reaction of transplanting in the API gallery.

 

Figure 6. score obtained after revealing the reactions in the API gallery.

 

2/Result of the fourth sample after 5 days stored in a refrigerator:

The culture of the three categories (bacteria, fungi and E coli) proved to be sterile: absence of bacteria, including Escherichia coli, after subculture on MacConkey agar medium. As well as the absence of fungi in a Sabouraud-dextrose agar (SAB) medium.

 

3/Interpretation of the results:

Taking into account the 20 colonies obtained, the average number of colonies in the two Petri dishes is 10 and taking into account a dilution factor of 10 for 1ml of the suspension of diluted concentrate, i.e. the result of the formation: 100 colonies. The units per gram are clearly at the limit of 10² CFU/g. Thus, in accordance with the specifications of the European Pharmacopoeia, the microbiological quality is judged to be non-compliant for the solution used for simulation for 5 days.

 

DISCUSSION:

We could not find any similar studies in literature to compare our study. Based on our results, the health risk associated with the use of the pipette was identified mainly in terms of microbial contamination: Coagulase negative staphylococci (CNS) include many species. Several species are commensal to the skin and mucous membranes of children. They can be responsible for endogenous (SCN originating from the patient's microbiota) and exogenous (SCN transmitted by the environment) infections, particularly in premature newborns or immunocompromised children¹⁸. The presence of colonies identified as S. warnari and S. hominis indicates the possibility of microbial contamination within the solution. These bacteria can present health risks, particularly for pediatric patients whose immune systems are weakened, given the infections they can cause. S. warneri is a catalase-positive, oxidase-negative, and coagulase-negative organism that is a commensal of skin flora. It constitutes <1% of the Staphylococcus skin flora¹⁹. It is a rare cause of sepsis and an immunocompromised state is a predisposing factor for such infections^20,21.

 

Among SCNs S. hominis, normally found on human skin, is the second of the four most frequently identified isolates in the blood of neonates and immunocompromised patients and has been associated as a causative agent of bacteremia, septicemia and endocarditis²². S. saprophyticus is a frequent colonizer of the human gastrointestinal tract, cervix, urethra, vagina, perineum and rectum²³. Possible complications such as acute pyelonephritis, urethritis, and endocarditis²⁴, particularly in immunocompromised individuals, have been documented. If these bacteria are transferred into medications and then ingested, this could increase the risk of infection, particularly in the pediatric population with compromised health.

 

Knowing that the study did not include contact with the patient's oral flora which may contain other microbiological species, the risk would be greater in reality and therefore the contamination would be greater with agents that could be pathogenic in the case. serious illnesses (tuberculosis, angina, pneumonia, etc.).

 

We have also noticed the effectiveness of storage at a temperature between 2° and 8°, on bacterial proliferation. This could reduce the risk of contamination of the entire solution. without forgetting the factor of the microbiological quality of the water which depends on the urban and rural environment, this could further contaminate the dosing pipette at the time of rinsing²⁵.

 

It is therefore recommended that pharmaceutical companies further research the stability of liquid forms intended for pediatric use, particularly those that involve the use of dose-weight pipettes for drug administration. This is all the more crucial in light of the results of studies concerning the microbiological stability of the product. It is also recommended to re-evaluate the administration devices and move to dispensing using devices which do not require simultaneous introduction into the pharmaceutical product and into the patient's mouth, such as the spoon. For that, many processes should be applied for controlling of pathogens to preserve infant lives. Microbes are present everywhere, and hence, it is very likely and very easy for organisms to contaminate our drugs²⁶.

 

The composition of excipients, especially preservatives, in our case sodium methyl parahydroxybenzoate and sodium propyl parahydroxybenzoate, must also be checked, especially in the case of this study which proves the non-effectiveness of the latter after 5 days, this requires updating, adjustment of doses of preservatives and/or reformulation to obtain the best protection of the product in question, thus minimizing the risk to the user's health^27,28,29,30.

 

LIMITATION OF THE STUDY:

This study, carried out on a limited number of samples, aimed to test, in a one-off manner, the microbiological stability of pediatric pharmaceutical forms used with a dosing pipette.

 

The results show that, however, it is necessary to carry out additional studies including a greater number of specialties and a greater number of samples in order to identify this research topic.

 

CONCLUSION:

In conclusion, our study highlights the dual nature of dosing pipettes: effective in taking and administering a precise dose in relation to the child's weight, but also posing risks of microbial contamination. A balance between these factors is crucial to ensure the safety of pediatric patients who rely on this drug delivery device. Vigilance in monitoring their impact is essential to optimize their benefits and minimize the associated risks in the pediatric population.

 

This study therefore underlines the importance of taking into consideration during stability studies after opening of these pharmaceutical forms, in general, the impact of the frequent introduction of "dosing pipette" type devices as well as compliance with the rules for using this type of device in particular, cleaning and storage instructions. It would be interesting to conduct similar studies on a larger number of samples of similar shape based on other active ingredients.

 

CONFLICT OF INTEREST:

Authors declare that there is no conflict of interest

 

REFERENCES:

1.      Charkaluk M.L. Kalach N. El Kohen R. Kremp O. Utilisation familiale de l’ibuprofène chez l’enfant fébrile: une etude prospective aux urgences d’un hôpital lillois. Archives de pédiatrie 2005; 12(8): 1209–1214: https://doi.org/10.1016/j.arcped.2005.02.024.

2.      Boivin J.-M. Weber F. Fay R. Monin P. Prise en charge de la fièvre de l’enfant: les connaissances et pratiques des parents sont-elles satisfaisantes ? Archives de pédiatrie 2007; 14(4): 322–329: https://doi.org/10.1016/j.arcped.2006.12.018.

3.      Chouchane S. Chouchane C.H. Ben Meriem C.H. Seket B. Hammami S. Nouri S. Monastiri K. Guediche M.N. Les fièvres prolongées de l’enfant. Étude rétrospective de 67 cas. Archives de pédiatrie 2004; 11: 1319–1325 : https://doi.org/10.1016/j.arcped.2004.07.018

4.      Wal Pranay, Wal Ankita, Srivastava Rishabh, Rastogi Prateek, Rai Awani K. Antibiotic Therapy in Pediatric Patients. Research J. Pharm. and Tech. 2010; 3(1): 118-120.

5.      Mamatha T. Raheem Md. Imam Pasha S. Zubair Md. Comparative In-Vitro Evaluation of Marketed Paracetamol Tablets. Research J. Pharma. Dosage Forms and Tech. 2009; 1(2): 100-102. DOI: 10.5958/0975-4377.2017.00002.7

6.      Abhishek S. Pujari, Nitin A. Gaikwad, Indrajeet V. Mane, Ganesh B. Vambhurkar, Pravin P. Honmane. In-Vitro Evaluation of Different Marketed Brands of Paracetamol Tablets Using Quality Control Tests. Asian J. Pharm. Tech. 2018; 8(3): 119-122. doi: 10.5958/2231-5713.2018.00019.3

7.      Ganesh B. Vambhurkar, Asha M. Jagtap, Akshata S. Gavade, Manish D. Rajput, Hrutuja V. Kambale, Manohar D. Kengar. Evaluation of five Different Marketed Brands of Paracetamol Tablets using Quality Control Tests. Asian J. Pharm. Tech. 2018; 8(4): 227-230. doi: 10.5958/2231-5713.2018.00035.1

8.      Arana A. Morton NS. Hansen TG. Treatment with paracetamol in infants. Acta Anaesthesiol Scand. 2001; 45(1): 20-29. doi:10.1034/j.1399-6576.2001. 450104.x

9.      Bannwarth B. Péhourcq F. Pharmacological Rationale for the Clinical Use of Paracetamol: Pharmacokinetic and Pharmacodynamic Issues.  Drugs. 2003; 63(Special Issue 2): 5-13 DOI: 10.2165/00003495-200363992-00003

10.   Sonali T. Gade A. Review article on Oral Jellies for Pediatrics. Asian J. Pharm. Tech. 2020; 10(3): 207-212. doi: 10.5958/2231-5713.2020.00034.3

11.   Walsh J. Math M.C. Breitkreutz J. Zerback T. Wachtel H. Devices for oral and respiratory paediatric medicines: What do healthcare professionals think?, Int J Pharmaceut 2015; 492(1-2): 304-315. http://dx.doi.org/10.1016/j.ijpharm.2015.05.041

12.   Ph. Eur. 11.0, 50104 (01/2021). Qualite microbiologique des préparations pharmaceutiques non stériles et des substances a usage pharmaceutique.

13.   Ph. Eur. 11.0, 20613 (01/2021). Examen microbiologique des produits non stériles : test de micro-organismes specifies.

14.   Ph. Eur. 11.0, 20612 (01/2021). Examen microbiologique des produits non stériles : tests de dénumération microbienne.

15.   Nagarnaik M. Sarjoshi A. LingeP.  Bhore S. PandyaG. A Microbial Study of Some Cosmetics and Raw Materials Used in Personal Care Products in Urban Area. Research J. Topical and Cosmetic Sci. 2015; 6(1): 48-51. doi: 10.5958/2321-5844.2015.00008.4

16.   Kumar Jain S. Kumar Jain R. Evolution of GMP in Pharmaceutical Industry. Research J. Pharm. and Tech. 2017; 10(2): 601-606. doi: 10.5958/0974-360X.2017.00118.4

17.   18_api-ref-guide_v7. [Cited 19 décembre 2023]. Disponible sur : https://www.biomerieux-usa.com/sites/subsidiary_us/files/18_api-ref-guide_v7.pdf

18.   Tran T.-A. Filleron A. Infections à staphylocoques de l'enfant : aspects physiopathologiques, bactériologiques et cliniques. Pédiatrie - Maladies Infectieuses. 2020; 4: 283 Doi: 10.1016/S1637-5017(20)65459-X

19.   Kloos WE. Natural populations of the genus Staphylococcus. Annu Rev Microbiol. 1980; 34: 559-592. doi:10.1146/annurev.mi.34.100180.003015

20.   Kloos WE. Schleifer KH. Isolement et caractérisation des staphylocoques de la peau humaine II. Descriptions de quatre nouvelles espèces : Staphylococcus warneri, Staphylococcus capitis, Staphylococcus hominis et Staphylococcus simulans. Int J Syst Bactériol. 1975; 25: 62-79. https://doi.org/10.1099/00207713-25-1-62

21.   Kanuparthy A, Challa T, Meegada S, Siddamreddy S, Muppidi V. Staphylococcus warneri: Skin Commensal and a Rare Cause of Urinary Tract Infection. Cureus. 2020 May 28; 12(5): e8337. doi: 10.7759/cureus.8337. PMID: 32494545; PMCID: PMC7263002.

22.   Lawal OU. Barata M. Fraqueza MJ. et al. Staphylococcus saprophyticus From Clinical and Environmental Origins Have Distinct Biofilm Composition. Front Microbiol. 2021;12: 663768. Published 2021 Jun 7. doi:10.3389/fmicb.2021.663768

23.   TRS 902 - Annex 9: Guidelines on packaging for pharmaceutical products. WHO Technical Report Series, No. 902, 2002 https://www.who.int/publications/m/item/annex-9-trs-902

24.   Sangeetha N. Guidelines for Antibiotic Therapy of Urinary Tract Infection in Acute Pyelonephritis, Bacteriuria in Adolescents. Asian J. Pharm. Res. 2018; 8(2): 83-86. doi: 10.5958/2231-5691.2018.00014.X

25.   Lakshmi Ganapati P. Soujanya K. Machiraju P.V.S. Bacteriological Quality of the Drinking Water and Health Survey in Koyyalagudem mandal of West Godavari District, Andhra Pradesh, India. Asian J. Research Chem. 2019; 12(1): 31-36. doi: 10.5958/0974-4150.2019.00008.7

26.   El-Waseif M. A. Heba A. El-Ghareeb.Walied B. Ahmed M. Motaz M. Chemical, Microbial and Sensory Stability Study of Infant Formula during Consumption. Research Journal of Pharmacy and Technology. 2022; 15(9): 4173-1. doi: 10.52711/0974-360X.2022.00700

27.   Doron S. Friedman M. Falach M. Sadovnic E. Zvia H. Antibacterial effect of parabens against planktonic and biofilm Streptococcus sobrinus. Int J Antimicrob Agents. 2001; 18(6): 575-578. doi:10.1016/s0924-8579(01)00436-8

28.   Sathish Kumar Konidala, Adinarayana Penumala, Vinod Kumar Mugada, Govinda Rao Kamala. Development and validation of RP-HPLC method for simultaneous estimation of Paracetamol and Flupirtine Maleate. Asian J. Pharm. Ana. 2015; 5(2): 105-111. doi: 10.5958/2231-5675.2015.00017.4

29.   Vaishali N. Sonawane Chaitali A. Yeola Vijayraj N. Sonawane, Khemchand R. Surana1, Dhananjay M. Patil, Deepak D. Sonawane. Estimation of Paracetamol in various brands of Paracetamol Tablets and their Comparative Study. Asian Journal of Pharmaceutical Analysis. 2023; 13(3): 155-1. doi: 10.52711/2231-5675.2023.00025

30.   Mali A. Hake A. Patil P. Bathe R. Tamboli A. Simultaneous UV Spectrophotometric Methods for Estimation of Paracetamol and Domperidone in Bulk and Tablet Dosage Form. Asian J. Res. Pharm. Sci. 2016; 6(1): 21-26. doi: 10.5958/2231-5659.2016.00003.5

 

 

 

Received on 29.10.2024      Revised on 27.01.2025 Accepted on 22.04.2025      Published on 12.06.2025 Available online from June 14, 2025 Research J. Pharmacy and Technology. 2025;18(6):2834-2839. DOI: 10.52711/0974-360X.2025.00406 © RJPT All right reserved
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