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Farmacia Hospitalaria

On-line version ISSN 2171-8695Print version ISSN 1130-6343

Farm Hosp. vol.40 n.5 Toledo Sep./Oct. 2016 



Microbiological quality of pediatric oral liquid formulations

Calidad microbiológica de las formulaciones orales líquidas pediátricas



M.a José Cabañas Poy1, Carme Cañete Ramírez1, Sabina X González di Lauro2, Virginia Rodríguez Garrido2, Gloria Roig Carbajosa2, Aurora Fernández-Polo1 and Susana Clemente Bautista1

1Servei de Farmacia, Àrea Maternoinfantil.
2Servei de Microbiologia i Parasitologia Clíniques. Hospital Universitari Vali d'Hebron, Barcelona. Spain.





The oral administration of drugs to the pediatric population involves the extemporaneous preparation of liquid formulations. These formulations have studies on their physicochemical stability, but they often lack microbiological studies. The objective of this study is to check the microbiological quality of five oral liquid formulations prepared with different excipients, which represent five major combinations, in two conditions: kept unopened until the day of the test, and in a multi-dose vial opened daily. The formulations were prepared according to standard operating procedures. Half of each batch was packaged in vials that remained closed until the day of testing, and the other half in a single container which was opened daily. Both the vials and the containers had been previously sterilized. Microbiological tests were performed weekly during the first month of the study, and then every two weeks, until the expiration date. The microbiological quality of oral liquid formulations is determined by the Royal Spanish Pharmacopoeia. The conclusion was that none of the formulations prepared that were packaged in sterilized containers became contaminated, either in unopened vials or in multi-dose containers when they were opened daily.

Key words: Stability; Contamination; Sterility; Formulation; Paediatrics.


La administración oral de fármacos a la población pediátrica implica la preparación de fórmulas líquidas extemporáneas. Estas fórmulas tienen estudios de estabilidad fisicoquímica pero en muchas ocasiones carecen de estudios microbiológicos. El objetivo del estudio es comprobar la calidad microbiológica de cinco fórmulas orales líquidas, preparadas con diferentes excipientes, que representan mayoritariamente cinco combinaciones, en dos condiciones: conservadas sin abrir hasta el día del análisis y abriendo diariamente el envase multidosis. Se prepararon las fórmulas según los procedimientos normalizados de trabajo. La mitad del lote de cada fórmula se envasó en viales que estuvieron cerrados hasta el día del análisis y la otra mitad en un solo frasco que se abría diariamente. Tanto los viales como los frascos para el envasado estaban esterilizados previamente. El análisis microbiológico se realizó cada semana durante el primer mes de estudio y después cada dos semanas hasta llegar al periodo de caducidad. La calidad microbiológica de las fórmulas orales líquidas viene marcada por la Real Farmacopea Española. Se concluye que ninguna de las fórmulas elaboradas envasadas en contenedores esterilizados se contaminó ni en los viales cerrados ni en los frascos multidosis cuando se abrieron diariamente.

Palabras clave: Estabilidad; Contaminación; Esterilidad; Formulación; Pediatría.


Contribution to Scientific Literature

This is the first study that addresses exclusively the microbiological stability of five types of paediatric oral liquid formulations. There are publications about the physicochemical stability of liquid formulations, but microbiological stability has only been addressed as a secondary aspect.

The outcomes of this study and our own experience can encourage other Hospital Pharmacy Units to conduct research on the microbiological quality of the formulations they prepare. We believe that it is important to publish these articles, in order to inform about some work procedures that can be extrapolated to Pharmacotechnology areas, with the aim to improve the microbiological quality of oral liquid formulations.



The paediatric population requires modifications or adaptations of solid pharmaceutical products, tablets or capsules, in order to achieve dose individualization and facilitate oral administration. The preparation by the Pharmacist of liquid formulations allows an accurate dose measurement, which must be frequently modified according to weight.

Aqueous solutions and suspensions are more unstable from a physicochemical and microbiological point of view than solid formulations, and require stability tests after their preparation in order to determine their time of validity. Even though physicochemical validity is an aspect widely studied within magistral formulations, this is not the same for the microbiological aspect1. The contamination of non-sterile formulations can reduce or inactivate the therapeutic activity of the drug, or even lead to drug toxicity that can affect the patient's health. On the other hand, the ingestion of pathogenic microorganisms could originate particularly severe infections in newborns and immunodepressed patients.

In order to prepare a liquid formulation, it is required to have an active ingredient, which comes from a pharmaceutical product or raw materials, as well as excipients providing physicochemical and microbiological stability, and offering acceptable organoleptic characteristics. In order to avoid the proliferation of microorganisms, preservatives are frequently added on, particularly in the case of multi-dose preparations.

It is very important to determine the microbiological validity of multi-dose formulations that will be opened and closed very frequently during their period of physicochemical validity. Thus the importance of meeting the quality assurance rules by the Royal Spanish Pharmacopoeia, which sets some limitations. The criteria for acceptance of the microbiological quality of aqueous oral non-sterile formulations are: 102 CFUs in the total count of aerobic microorganisms (maximum number acceptable: 200) 101 CFUs in the total count of yeasts and molds (maximum number acceptable: 20), and lack of Escherichia coli in 1 g or 1 ml2-4.

Previous Study

For selecting the formulations to be tested, the standard liquid formulations in the Pharmacotechnology Area of the Hospital Vall d'Hebron Pharmacy Unit were classified according to type of excipients. Five formulations were selected, which represent the potential combinations between them. Some contain preservatives (Ora Sweet®, Ora Plus®, National Formulary (NF) syrup and plain syrup), while others do not (methylcellulose 1%).

Table 1 shows the composition of the formulations representing each type of excipients, and their storage requirements and expiration dates.

Once the formulations had been selected, and taking into account the period of validity of each, a work schedule was designed. A weekly microbiological test would be conducted during the first month of validity. From then on, tests were conducted every two weeks, until the date of expiration for each formulation. Throughout the period studied, the formulations were stored under the conditions recommended for the validity period. The work schedule appears in table 2.

The objective was to assess the microbiological stability during the period of physicochemical validity of five multi-dose liquid formulations that represent five types of vehicles prepared under controlled conditions and packaged in sterile vials. The study determined if there was any microbiological contamination of the formulation when not handled during the period of validity, and also when the vial was opened and closed every day until its expiration date, in a simulation of their conditions of use. This was intended to confirm if the type of excipient leads to the contamination of the formulation when prepared and packaged in the conditions previously described.



This study was conducted in the Maternity and Child Area of the Pharmacy Unit and in the Microbiology and Parasitology Lab of the Hospital Universitario Vall d'Hebron between June and September, 2015.

Formulation Preparation

The preparation of solutions and suspensions was conducted according to the rules for adequate preparation of magistral formulations5.

A batch of each formulation was prepared following the relevant Standard Operating Procedure (SOP). Thus, 250 ml of ursodeoxycholic acid suspension and caffeine solution were prepared, as well as 125 ml of spironolactone suspension and atenolol, and 120 ml of propanolol suspension. Half of the volume prepared (sample) was packaged in a 125 ml topaz glass multi-dose vial, with a shutter and a stopper with first-opening indicator. The other half of the volume prepared (control) was packaged in 10 ml topaz vials, with rubber lids and plate caps, that were kept closed until testing day. All vials were stored in the fridge.

Every day, the sample vials were removed from the fridge, left to warm up, the suspensions were shaken for homogenization, and they were left opened for at least 5 minutes and maximum 30 minutes.

On testing day, approximately 10 ml of preparation was extracted from the sample vial, packaged in a topaz vial, and closed with a rubber lid and an aluminium cap. The sample vial and one of the control vials were then microbiologically tested.

During the whole process, the rules for correct preparation were followed, as currently used in the Pharmacotechnology Area of the Pharmacy Unit. The equipment used for formulation preparation was washed according to the SOP, and the final rinse was made with highly purified (deionized) water. All packaging materials were sterilized (the vial, shutter, and lid with first-opening-indicator, and the vials, rubber stoppers and aluminium caps), as an extra procedure in order to minimize the biological burden.

Microbiological Assay

In order to determine whether the formulations met the criteria for acceptance of microbiological quality, the procedures indicated by the Royal Spanish Pharmacopoeia were followed, regarding microbiological control of non-sterile products (count tests of microbes and specified microorganisms)2,3. Fertility and sterility tests were conducted (both with and without the product to be tested). Each test was conducted in duplicate. The microorganisms researched were mesophyll bacteria and fungi.

For the fertility test without the product, ATCC strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas Aeruginosa were used. Starting with a known concentration of 1.5 x102 CFU/ml in TSB (Tryptic Soy Broth), the pouring plate method was used in a PCA (Plate Count Agar) medium for S. aureus. In order to spread the McConKey agar medium (E. coli y P. aeruginosa), the spread-plate technique was used with Digralsky loops.

The sterility test was conducted with 1 ml of TSB spread in PCA, McConkey broth and Sabouraud agar.

The fertility test with product was conducted starting with a 1/10 syrup dilution, adding the ATCC strain in a dilution of 1.5 x 104 CFU/ml, and spread in the media previously described for the fertility test.

Finally, the test for product sterility was conducted by preparing a 1/10 dilution of the product to be tested in TSB broth. 1 ml of this dilution was spread in PCA agar, Sabouraud agar, and McConkey broth.

For each test, the McConkey broths and plates and the PCA plates were incubated at between 35 and 37oC during 48 hours; while the Sabouraud agar plates were incubated at between 20 and 25oC during 5 days. The former were observed every day, while the latter were examined at the third and fifth days, looking for any growth.


Outcomes and Discussion

Our outcomes show that there was no microbiological growth in any of the cultures during the period of the study. None of the samples of the formulations extracted from the multi-dose vials or any of the control vials were contaminated during the period of validity. The controls used were adequate for assessing the technique.

The type of excipient did not alter the contamination pattern of oral liquid formulations prepared under controlled conditions and packaged in sterile vials.

The spironolactone 5 mg/ml suspension used Ora Sweet® and Ora Plus® (1:1) in its formulation, and contained parabens as preservatives. The propanolol 1 mg/ml suspension was prepared exclusively with plain syrup with parabens. In these two situations, the vehicle osmolarity and the presence of preservatives favoured the lack of contamination.

The oral 20 mg/ml caffeine citrate solution was prepared with a fifty-fifty proportion of plain syrup without preservatives and sterile water, and the concentration of sugar and preservatives was reduced by half in the final preparation. The case of the 20 mg/ml ursodeoxycholic acid suspension was very similar to the previous one, because the 1% methylcellulose suspension does not contain any preservatives.

The plain syrup preservatives used in our study were nipagin and nipasol bases, at a 0.02% and 0.01% concentration (P/V) respectively; and for NF syrup, sodium nipagin and nipasol at a 0.06% and 0.03% concentration (P/V)(in bases form) respectively. The dilution by half of NF syrup offered paraben concentrations still protecting against microbiological contamination, taking into account the recommendations by Rowe RC6, which indicate nipagin base concentrations between 0.015 and 0.2%, and nipasol base concentrations between 0.01 and 0.02%. This could be the reason for the sustained microbiological stability. On the contrary, the concentration of parabens in the plain syrup diluted by half lost its protection action. At the time of designing the plain syrup concentration, we chose one that contained the minimum level of parabens, in order to reduce the use of these preservatives in the paediatric population; and therefore, when diluting it by half, the preservative concentration was insufficient. Even though it has not become contaminated throughout the period of the study, for daily practice we recommend using the vial within 14 days after its opening, following the guidelines by the Pharmacotechnology Work Team1.

The atenolol 2 mg/ml suspension was the formulation more sensitive to contamination, because 1% methyl-cellulose solution was exclusively used, and it contained no preservatives. Even though its expiration period is determined at 28 days, once the vial is opened it should be used within 14 days maximum, as indicated by the Pharmacotechnology Work Group1.

The 1% methylcellulose solution is prepared with the raw material and with sterile distilled water. After preparation, it has a 14-day expiration period if not sterilized; on the other hand, after sterilization in autoclave, its validity period is increased up to 6 months. We always use sterilized methylcellulose for preparation of liquid formulations.

Oral liquid formulations don't need to be sterile, but it is very necessary to apply all strategies in order to minimize the bacterial burden and meet the Pharmacopoeia rules. One of them is the use of sterile water when required in the formulation. In our Pharmacy Unit, all liquid formulations are prepared with sterile water.

The Good Practice Guidelines for preparation of medications in Hospital Pharmacy Units7 recommend using purified water for the final rinse of lab and packaging materials. In our case, we use highly purified water for the final rinse, with a better microbiological quality.

Another aspect to highlight is the fact of using sterilized packaging materials as an additional quality measure for liquid formulations, because it can favour a reduction in the initial bacterial burden of the preparation, which could act as a contamination source. The fact of sterilizing the packaging materials allows to reduce the amount of preservatives to the higher extent possible.

Finally, another source of contamination for the final formulation must be considered: the raw materials or the initial solid formulations containing the active ingredient.

One limitation of the study is that the daily dynamics of opening the multi-dose vials has been reproduced, but the formulation has not been handled by extracting a sample volume, which would be closer to a real situation. On the other hand, some formulations are not for single daily administration, but must be administered several times per day; in these cases, the frequency of opening the vials is higher, and it is unknown if this factor can affect microbiological stability.



Our study has demonstrated that the microbiological stability of oral liquid formulations prepared under controlled conditions and packaged in multi-dose sterile vials will be sustained during the period of physicochemical validity, both when the vial is kept closed and when it is opened every day, regardless of whether the vehicle contains preservatives or not.

Even so, it is always advisable to adjust the volume of the vial to the volume required by a patient during the period until the expiration date, taking into account that vials should not be kept open for over 14 days, if they contain no preservatives.


Conflict of interests

No conflict of interests.



1. Período de validez y caducidad de formas farmacéuticas no estériles orales líquidas. Farmacotecnia, boletín informativo SEFH BOLETIN_1_2015.pdf. (citado 9 de abril de 2016). Disponible:         [ Links ]

2. 2.6.12. Control microbiológico de productos no estériles: ensayos de recuento microbiano. Real Farmacopea Española en internet 5a ed. (citado 9 de abril de 2016) Disponible:         [ Links ]

3. 2.6.13. Control microbiológico de productos no estériles: ensayo de microorganismos especificados. Real Farmacopea Española en internet 5a ed. (citado 9 de abril de 2016). Disponible:         [ Links ]

4. 5.1.4. Calidad microbiológica de las preparaciones farmacéuticas y de las sustancias para uso farmacéutico no estériles. Real Farmacopea Española en internet 5a ed (citado 9 de abril de 2016). Disponible:         [ Links ]

5. Real Decreto 175/2001, de 23 de febrero, por el que se aprueban las normas de correcta elaboración y control de calidad de fórmulas magistrales y preparados oficinales. Boletín Oficial del Estado, no 65, (16 de marzo de 2001).         [ Links ]

6. Rowe RC, Sheskey PJ, Quinn ME, editors . Handbook of Pharmaceutical Excipients. 6a ed. London: Pharmaceutical Press; 2009.         [ Links ]

7. Guía de Buenas Prácticas de Preparación de Medicamentos en Servicios de Farmacia Hospitalaria - Ministerio de Sanidad, Servicios Sociales e Igualdad 2014.GuiaBPP3.pdf (citado 9 de abril de 2016). Disponible:         [ Links ]



Correo electrónico:
(M.a José Cabañas Poy).

Recibido el 12 de abril de 2016;
aceptado el 13 de junio de 2016.

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