Comparative Study in Preservation Efficiency of Oral Calcium/Vitamin D 3 /Vitamin B 12 Syrup

Columbia International Publishing American Journal of Pharmaceutical Sciences and Nanotechnology doi:10.7726/ajpsn.2017.1001 Short Communication Comparative Study in Preservation Efficiency of Oral Calcium/Vitamin D 3 /Vitamin B 12 Syrup Mostafa Eissa 1* Received: 15 December 2016; Accepted: 12 March 2017; Published online: 26 August 2017 Columbia International Publishing 2017.Published at www.uscip.us Abstract Preservation of pharmaceutical multidose products with water activities greater than 0.60 is very important aspect in ensuring the microbiological safety of medicinal products. In the current study five different formulae (three were chemically preserved and two were not preserved) for the commercial drug products for the same medicinal use in the market were tested for comparative study of preservative efficacy test (PET) against pharmacopoeial five standard strains viz. Staphylococcus aureus, Escherichia coli,, Candida albicans, Aspergillus brasiliensis and Burkholderia cepacia as a frequently isolated waterborne isolate. The test was preceded by verification of the validity of microbial recovery and neutralization technique using microbiological membrane filtration method. All formulae showed strong antibacterial effect, moderate activity against yeast and static activity against mold spores mainly. Interestingly, two formulae were not chemically preserved, yet showed comparable antimicrobial activity with those preserved with Potassium Sorbate, Sodium benzoate and Methyl/Propyl Paraben. The five formulae passed USP antimicrobial efficacy test. Keywords: Multidose products; Water activity; Preservative efficacy test; Neutralization technique; Membrane filtration; Potassium Sorbate; Sodium Benzoate; Methyl/Propyl Paraben 1. Introduction Microbial contamination of non-sterile oral products is considered a critical issue that constitute health hazard. Orth (1993) included updated list of objectionable microorganisms that included spp., Burkholderia spp., Escherichia coli, Staphylococcus aureus, and pathogenic yeasts (Candida albicans). Most products at high risk are those with water activity that allows for microbial proliferation. This could be demonstrated in the United states Pharmacopeia Chapter <1112>, Application of Water Activity Determination to Non-sterile Pharmaceutical Products (USP, 2015). *Corresponding e-mail: mostafaessameissa@yahoo.com 1 Quality Unit, HIKMA Pharma for Pharmaceutical Industry, Egypt 1

Products that are prone to microbial spoilage include syrup, simple aqueous solution, suspension, emulsion, creams, lotions, ointment, oil and shampoo. Manifestations of microbial spoilage are variable and affected by several factors. They embrace toxicity due to irritant substances, metabolic products and toxin release, in addition to affecting product activity, olfactory and physically visible changes in the products (Smart and Spooner, 1972). Selection of appropriate antimicrobial preservatives for pharmaceutical formulae is challenging task. Regulatory agencies have approved few number of preservative compounds that can be included into the topical and oral multidose pharmaceutical compounds (Elder and Crowley, 2012). The limitation in the choice of preservatives can be observed when reviewing the pharmacopial guidelines (JP, 2006; EP, 2010a; USP, 2010a). The current work aimed to investigate five non-sterile pharmaceutical oral liquid Calcium/D 3/B 12 supplement products from the market were they have very close composition and to determine the preservative efficacy test (PET) of them after applying an effective method of neutralization and the recovery of low level bioburden. 2. Materials and Methods Five non-sterile liquid oral Calcium/D 3/B 12 products were collected based on the fact that they were very close in composition to facilitate the comparison in the antimicrobial effect as seen in Table (1). Pharmaceutical products were initially tested for microbiological cleanliness. Microbial tests of pharmaceutical materials were done using culture media that passed growth promotion tests according to compendial guideline (USP 38-NF 33, 2015). Bacterial visualization was facilitated using colorless Triphenyltetrazolium Chloride (TTC) dye which turns red by viable cells. Negative control samples were included concurrently with the test. Selection of the preliminary verification method of neutralization was done as demonstrated by Eissa and Mahmoud (2015). Acceptance criteria for the recovery method, microbial limit test and preservative efficacy test were done according to Pharmacopeial method (USP 38-NF 33, 2015). Identification of the water-borne bacteria and the verification of standard strains culture purity was done according to Ashour et al., 2011. Environmental monitoring (EM) samples from surfaces and air were taken as described by Eissa (2014) with every campaign test performed in biological safety cabinet. All experimental testing were done in Biological safety Cabinet (BSC) (Jouan MSC 9 Class II A2 BioSafety Cabinet, Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, California 95134). All the nutrient media and chemicals were purchased from OXOID (Basingstoke, Hampshire) and Sigma-Alrich (St. Louis, MO 63103), respectively. All organisms were stored at -80 C in a validated - 86C Ultra low temperature freezer (-86 Degree ULT Freezers, Qingdao Shandong, China) in a validated cryogenic environment, and revived prior to conducting the study. All culture media were sterilized by autoclaving in steam sterilizer (FEDEGARI FOB4L-TS, Fedegari Autoclavi SpA, SS 235 km 8, 27010 Albuzzano (PV), Italy). Microbial test suspensions were used once the results of serial dilutions could be enumerated using digital colony counter (Digital Colony Counter Model: 361, Laxman Mahtre Rd. Navagaon, Dahisar West, Mumbai). Identifications kits BBL Crystal Identification System purchased from BD (Becton Dickinson Microbiology Systems, Cockeysville, Md.) were used to identify critical water-borne isolate. Plastic 9mm sterile plates were purchased from Sterilin Limited (solaar house, 19 mercers row, cambridge, UK). All statistical procedures and 2

box plot figure were performed using GraphPad Prism v6.01 for Windows. Any figure interpretation and complex calculation were performed using Microsoft Excel 2007. Table 1 Chemical compositions of the five pharmaceutical liquid oral Calcium/D 3/B 12 supplement products Category of contents Active Pharmaceutical Ingredients (API) Preservatives NP Inactive Ingredients Syrup Formula F1 F2 F3 F4 F5 Calcium Levulinate Calcium glubionate Vitamin D 3 Vitamin D 3 Vitamin B 12 Vitamin B 12 Potassium Sodium Methyl/Propyl Sorbate 6.5 benzoate NP Paraben 2/0.5 mg/ 5 ml 10 mg/5 ml mg/5 ml Butylhydroxy anisole, Butylhydroxy anisole, Butyl Hydroxytoluene, Cremophor RH 40, Sorbitol, Glycerin, Acesulfame Potassium, Fragaria x ananassa flavor, EDTA Disodium, HCl, Carmoisine, Propylene Glycol Butylhydroxy anisole, Citric Acid, Cremophor RH 40, Sorbitol, Glycerin, Fragaria x ananassa flavor, EDTA Disodium, Carmoisine, Propylene Glycol Butylhydroxy toluene, Citric Acid, Cremophor RH 40, Sorbitol, Glycerin, Acesulfame Potassium, Fragaria x ananassa flavor, EDTA Disodium, Carmoisine, Propylene Glycol ph range 4.5 6.5 = All formulae were prepared using pharmaceutical grade purified water USP. = Strawberry. = APIs concentrations were the same for F1, F2 and F3 group but F4 and F5group have the same concentrations but different the previous triplicate group. = Not present i.e. the formula did not contain any specific preservative compounds. = Macrogol- Glycerolhydroxystearate Ph. Eur and Polyoxyl 40 Hydrogenated Castor Oil USP/NF. 3. Results and Discussion Preservative- free pharmaceuticals and cosmetics are considered superior over their chemically preserved counterparts (Elder and Crowley, 2012). Thus, F1 and F4 formulae have added advantage of the remaining chemically preserved products. All EM samples results complied during testing activity. All tested products were clean microbiologically and all negative control plates were free from any microbial growth. The identified microorganism was Gram-negative rod with identification profile of B. cepacia and all microbial cultures were pure before performing the experiments. EP (2010b) and USP (2010b) have recommended incorporation of Escherichia coli in PET as well as any other microorganisms that are associated with manufacturing environment and may cause problems if they get into the medicinal products such as B. cepacia which is considered opportunistic pathogen. The neutralization procedure is a critical step which was done initially to ensure complete accuracy of data derived from PET test without carryover of the residual biocide (Sutton et al., 2002). All products passed the test using membrane filtration technique with recovery >70% from positive control as demonstrated in Table (2). There are no outlier results as 3

illustrated by box plot in Fig. 1 but there was significant difference in microbial recovery between formulae as demonstrated in Table (3) which is the finding that requires further analysis. However, the source of variation was found to be due significant difference between F3 and F4 in over all microbial recovery by Two-Way ANOVA using Turkey s Multiple Comparison Test at α=0.05. Hence, factor of experimental variability could not be excluded. So, further investigation is required. Formulae F1 and F3 were almost equal in the overall preservative activity while F4 and F5 formulae are higher with F4 slightly lower than F5 in total antimicrobial activity. F2 is slightly more active than F5 in the performance when compared after 28 days. However, after 14 days and the overall activity of F2 was lesser than F4. These findings were concluded from Table (4, 5, 6, 7 and Table 2 Neutralization efficacy test results using filtration technique showing microbial recovery of low-level inoculums for each microorganism in each formula against the control group Control Test groups recovery percent (%) group (CFU/Plate) F1 F2 F3 F4 F5 Staphylococcus aureus 54-78 89.7 92.3 88.5 91.4 111.1 Escherichia coli 39-80 79.4 87.3 77.8 148.7 97.5 29-85 94.1 97.6 89.4 113.8 93.3 Burkholderia cepacia 22-35 85.7 77.1 82.9 125.7 113.6 Candida albicans 26-99 74.4 79.5 76.9 100.0 99.0 Aspergillus brasiliensis 16-21 90.5 76.2 71.4 88.2 81.3 8). On the other hand, C. albicans could be viewed as a moderately affected organism by the preservation system in the formulae. However, each product showed different pattern of kinetics of count reduction for C. albicans and this is illustrated in Fig. 2. F5 showed the highest rate at the beginning then almost constant from 14 to 28 days. F2 showed relatively slower but steady rate which eventually turned from lower LR after 14 days than F5 to higher one after 28 days. On the other hand F4 followed by F3 where lower in the rate and the final LR than F2. Moreover, further investigation is required to determine the impact of water activity on the antimicrobial activity of the formula of each product, notably those formulae which are self-preserved. 4

M ic r o b ia l R e c o v e r y % Mostafa Eissa / American Journal of Pharmaceutical Sciences and Nanotechnology 2 0 0 1 5 0 1 0 0 F 1 F 2 F 3 F 4 F 5 5 0 0 F 1 F 2 F 3 F 4 F 5 T y p e o f F o r m u la Fig. 1. Box-and-whisker diagram showing the difference in total microbial recovery efficacy from each product formula with no outliers detected (no recovery exceptionally high or low) Table 3 Two-Way ANOVA using Tukey's multiple comparisons test of neutralization efficacy Table Analyzed Neutralization Efficacy Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significance Factor 15 0.2032 ns No Formula Factor 47 0.0021 ** Yes ANOVA table SS DF MS F (DFn, DFd) P value Factor 1231 5 246 F (5,20) = 1.6 P = 0.2032 Formula Factor 3791 4 948 F (4,20) = 6.2 P = 0.0021 Residual 3060 20 153 5

Table 4 Preservative efficacy of the formula (F1) from Calcium/D 3/B 12 supplement syrup showing Staphylococcus aureus 3.1x10 5 >4.5 >4.5 Escherichia coli 1.6x10 6 1.5 3.5 4.5x10 5 3.0 >4.7 Burkholderia cepacia 1.3x10 6 3.8 >5.1 Candida albicans 5.8x10 5 1.2 0.5 Aspergillus brasiliensis 1.0x10 5 0.0 0.0 Table 5 Preservative efficacy of the formula (F2) from Calcium/D 3/B 12 supplement syrup showing Staphylococcus aureus 2.4x10 5 3.5 >4.4 Escherichia coli 1.5x10 6 4.1 >5.2 1.0x10 5 >4.0 >4.0 Burkholderia cepacia 1.2x10 6 5.1 >5.8 Candida albicans 7.5x10 5 2.4 4.6 Aspergillus brasiliensis 1.2x10 5 0.0 0.6 Interestingly, F1 showed initial reduction followed by increase in the yeast count. This finding requires further extensive study to find the relation between formula composition and the effect on yeast count reduction. Although all bacteria were inhibited effectively by the five formulae, A. brasiliensis spores showed the greatest resistance to the all formulae, where the products showed almost no significant LR with the exception of F2 which showed slight decrease in fungal count but after 28 days. Water activity (a w) is another aspect that should be investigated thoroughly for pharmaceutical product with water activity above 0.60 a w since some researchers showed that the vast majority of pathogenic bacteria are inhibited by 0.85 a w (most Gram-negative bacteria are inhibited below 0.93 a w) and spoilage molds at 0.75 a w. So, one way to achieve natural preservation is to reduce the water activity in a product (Clontz, 2009). In some formulations, it is possible to use these factors, or hurdles to reduce or eliminate the use of preservatives (Kabara and Orth 1997; Kabara and Orth 1998). Hence, it is not surprising that two out of five formulae which are not chemically preserved showed good preservation effect. In addition, other non-preservative components may modify the preservation system such as acids, colors, fragrances, flavors, humectants (propylene glycol, glycerol and sorbitol), chelating agents (tetrasodium ethylenediaminetetraacetic acid and citric acid) and phenolic antioxidants (t-butylhydroxytoluene (BHT)) (Orth and Milstein 1989). Both BHT and t-butylhydroxyanisole (BHA) possess antimicrobial activity with the later being more effective than the former (Lim et al., 1987). 6

Table 6 Preservative efficacy of the formula (F3) from Calcium/D 3/B 12 supplement syrup showing Staphylococcus aureus 3.6x10 5 1.4 >4.6 Escherichia coli 1.7x10 6 2.2 5.2 3.5x10 5 3.0 >4.5 Burkholderia cepacia 1.3x10 6 4.6 >5.1 Candida albicans 8.7x10 5 0.8 1.2 Aspergillus brasiliensis 1.0x10 5 0.1 0.0 Table 7 Preservative efficacy of the formula (F4) from Calcium/D 3/B 12 supplement syrup showing Staphylococcus aureus 4.3x10 5 >4.6 >4.6 Escherichia coli 1.4x10 6 >5.1 >5.1 9.8x10 5 >4.9 >4.9 Burkholderia cepacia 1.2x10 5 >4.1 >4.1 Candida albicans 3.4x10 5 1.7 2.8 Aspergillus brasiliensis 1.4x10 5 0.0 0.0 Table 8 Preservative efficacy of the formula (F5) from Calcium/D 3/B 12 supplement syrup showing Staphylococcus aureus 2.4x10 6 >5.4 >5.4 Escherichia coli 1.9x10 6 >5.3 >5.3 1.6x10 6 >5.2 >5.2 Burkholderia cepacia 2.0x10 6 >5.3 >5.3 Candida albicans 7.0x10 5 3.2 3.3 Aspergillus brasiliensis 1.4x10 5 0.1 0.2 7

Log Reduction (LR) Mostafa Eissa / American Journal of Pharmaceutical Sciences and Nanotechnology 5 4 3 2 1 0 0 5 10 15 20 25 30 Time (Days) F1 F2 F3 F4 F5 Fig. 2. Kinetics of the preservation capabilities against yeast for the five tested pharmaceutical formulae on Candida albicans over the course of 28 days of testing 4. Conclusion The currently tested pharmaceutical liquid oral Calcium/D 3/B 12 supplement syrup products either not directly preserved (i.e. antimicrobial effect is due inactive non-preservatives ingredients) or preserved using potassium sorbate, sodium benzoate and methyl/propyl parabens mixture were effective in preserving the medicinal products from microorganisms with Aspergillus brasiliensis being the most resistant while Burkholderia cepacia are the most sensitive. Further work will be required to correlate antimicrobial activity of the products (especially self-preserved formulae) with water activity. Furthermore, It is recommended to include water activity measurement in the future as part of quality control monitoring for multidose medicinal drugs with more than 0.60 a w as a part of safety measure to ensure protection against dedicated group of risk pathogens or objectionable microorganisms. Acknowledgements This work was supported partially financially by HIKMA Pharma pharmaceutical company 2 nd Industrial zone - 6 th of October city. Thanks to Research and development department team of HIKMA Pharma for providing data and information about the composition of the products. The practical part of all experiments was performed in the microbiology laboratory in the quality control department by Ahmed Saber Nouby. Data gathering and issuing was performed by HIKMA microbiology laboratory team. Reference and writing style review was performed by Dr. Engy Refaat Rashed. 8

Funding Source None Mostafa Eissa / American Journal of Pharmaceutical Sciences and Nanotechnology Conflict of Interest None References Ashour, M. S., Mansy, M. S., & Eissa, M. E. (2011). Microbiological environmental monitoring in pharmaceutical facility. Egypt Acad J biolog Sci, 3(1), 63-74. Clontz, L. (2009). Microbial limit and bioburden tests (1st ed.). Boca Raton: CRC Taylor & Francis. Eissa, M. (2014). Studies Of Microbial Resistance Against Some Disinfectants: Microbial Distribution & Biocidal Resistance in Pharmaceutical Manufacturing Facility. Saarbrücken: LAP LAMBERT Academic Publishing. Eissa, M.E. & Mahmoud A.M. (2015). Establishment Of Methods For Microbial Recovery: Miscellaneous Non- Sterile Pharmaceutical Dosage Forms (Study I). European Journal of Biomedical and Pharmaceutical Sciences,2(3),1272-1281. Elder, D. P. & Crowley, P. (2012). Antimicrobial Preservatives Part One: Choosing a Preservative System. Americanpharmaceuticalreview.com. Retrieved 13 March 2017, from http://www.americanpharmaceuticalreview.com/featured-articles/38886-antimicrobial- Preservatives-Part-One-Choosing-a-Preservative-System/ European Pharmacopoeia (EP). (2010a). EP 6.4. European Directorate for Quality of Medicines, Strasbourg, France. Japanese Pharmacopeia (JP). (). 15th Edition. Society of Japanese Pharmacopeia, Tokyo, Japan. Kabara, J. & Orth, D. (1997). Preservative-free and self-preserving cosmetics and drugs: Principles and Practice. (1st ed., pp. 1-14). New York: Marcel Dekker. Lim, C. M., Kyung, K. H., & Yoo, Y. J. (1987). Antimicrobial effects of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Korean Journal of Food Science and Technology, 19(1), 54-60. Orth, D. (1993). Handbook of cosmetic microbiology (1st ed.). New York: M. Dekker. Orth, D. S., & Kabara, J. J. (1998). Preservative-free and self-preserving cosmetics and drugs application of hurdle technology. Cosmetics and toiletries, 113(4), 51-58. Orth, D. S., & Milstein, S. R. (1989). Rational development of preservative systems for cosmetic products. Cosmetics and toiletries, 104(10), 91-103. Smart, R., & Spooner, D. F. (1972). Microbiological spoilage in pharmaceuticals and cosmetics. J Soc Cosmet Chem, 23, 721-737. Sutton, S. V., Proud, D. W., Rachui, S., & Brannan, D. K. (2002). Validation of microbial recovery from disinfectants. PDA Journal of Pharmaceutical Science and Technology, 56(5), 255-266. United States Pharmacopeia (USP). (2010a). USP 34-NF29, US Pharmacopeia. Rockville, Maryland, USA. United States Pharmacopeia (USP). (2015). USP 38-NF 33. Baltimore, MD, USA: USPC Official. United States Pharmacopeia (USP). (2010b). General Chapter <51> Antimicrobial Effectiveness Testing. USP 34-NF29, US Pharmacopeia, Rockville, Maryland. European Pharmacopoeia (EP). (2010b). EP 5.1.3 Efficacy of Antimicrobial Preservation. EP 6.4, European Directorate for Quality of Medicines, Strasbourg, France. 9