Available online at www.sciencedirect.com International Journal of Pharmaceutics 353 (2008) 170–175 Sterilization by pure oxygen plasma and by oxygen–hydrogen peroxide plasma: An efficacy study M.R. Boscariol a,∗ , A.J. Moreira b,2 , R.D. Mansano b,2 , I.S. Kikuchi a,1 , T.J.A. Pinto a,1 a Faculty of Pharmaceutical Sciences, USP (University of S˜ ao Paulo), Av. Prof. Lineu Prestes 580, 05508-000, bloco 13, S˜ ao Paulo, Brazil b Integrated Systems Laboratory, PSI, Polytechnic School of the University of S˜ ao Paulo, Av. Prof. Luciano Gualberto trav 3, 158, 05508-900 S ˜ ao Paulo, Brazil Received 6 July 2006; received in revised form 10 November 2007; accepted 15 November 2007 Available online 18 January 2008 Abstract Plasma is an innovative sterilization method characterized by a low toxicity to operators and patients, and also by its operation at temperatures close to room temperatures. The use of different parameters for this method of sterilization and the corresponding results were analyzed in this study. A low-pressure inductive discharge was used to study the plasma sterilization processes. Oxygen and a mixture of oxygen and hydrogen peroxide were used as plasma source gases. The efficacy of the processes using different combinations of parameters such as plasma-generation method, type of gas, pressure, gas flow rate, temperature, power, and exposure time was evaluated. Two phases were developed for the processes, one using pure oxygen and the other a mixture of gases. Bacillus subtilis var. niger ATCC 9372 (Bacillus atrophaeus) spores inoculated on glass coverslips were used as biological indicators to evaluate the efficacy of the processes. All cycles were carried out in triplicate for different sublethal exposure times to calculate the D value by the enumeration method. The pour-plate technique was used to quantify the spores. D values of between 8 and 3 min were obtained. Best results were achieved at high power levels (350 and 400 W) using pure oxygen, showing that plasma sterilization is a promising alternative to other sterilization methods. © 2007 Elsevier B.V. All rights reserved. Keywords: Gas plasma; Inductively coupled plasma; Bacillus subtilis (Bacillus atrophaeus); Oxygen; Hydrogen peroxide 1. Introduction The term sterilization implies complete inactivation of microorganisms. Spores of certain bacterial species that are highly resistant to sterilization are used as biological indica- tors to evaluate the extent to which effective sterilization is achieved by different sterilization processes (Philips, 1975). Sterilizing irradiation is a process that can be used with heat- sensitive materials. However, this process is very costly, not only because of the equipment required but also because of the strict safety precautions that must be observed. Further- more, although irradiation is carried out at low temperatures and stabilizers are incorporated into the process, the formation ∗ Corresponding author. Tel.: +55 11 3091 3302/3649/3674; fax: +55 11 3091 3626. E-mail address: micabosca@yahoo.com.br (M.R. Boscariol). 1 Tel.: +55 11 3091 3302/3674. 2 Tel.: +55 11 3091 9729. of free radicals, cross-linking reactions, and double bonds cause mechanical and visual alterations that very often prevent the viable application of this process (Pinto et al., 2003). Another widely used sterilization method uses ethylene oxide, which also works at low temperatures, is commonly used for dental, medical and hospital equipment. Its mutagenic properties, how- ever, make strict control of the process essential (Alfa et al., 1997). Plasma is an innovative alternative method, and appears to have advantages over other sterilization methods currently in use. Advantages of plasma include the fact that the technique is effective in inactivating the required microbial load (safety assurance level), can be performed at room temperature, and do not use toxic gases (Moore, 2004). It has the characteris- tics of an ideal sterilant, namely, a high degree of efficacy, fast action, penetrability, lack of toxicity, compatibility with different materials, and cost-effectiveness. In the present case, plasma is sustained by applying a radio-frequency electric field to precur- sor gases, which in turn produce highly reactive species that lead 0378-5173/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2007.11.059