NON-SPECIFIC METHODS FOR DETECTING RESIDUES OF CLEANING AGENTS DURING CLEANING VALIDATION

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1 Available on line at Association of the Chemical Engineers AChE Chemical Industry & Chemical Engineering Quarterly 17 (1) (2011) CI&CEQ DRAGAN M. MILENOVIĆ 1 DRAGAN S. PEŠIĆ 1 SNEŽANA S. MITIĆ 2 1 Zdravlje-Actavis Company, Leskovac, Serbia 2 Faculty of Sciences and Mathematics, University of Niš, Niš, Serbia SCIENTIFIC PAPER UDC DOI /CICEQ M NON-SPECIFIC METHODS FOR DETECTING RESIDUES OF CLEANING AGENTS DURING CLEANING VALIDATION Cleaning validation procedures are carried out in order to assure that residues of cleaning agents are within acceptable limits after the cleaning process. Cleaning agents often consist of a mixture of various surfactants which are in a highly diluted state after the water rinsing procedure has been completed. This makes it difficult to find appropriate analytical methods that are sensitive enough to detect the cleaning agents. In addition, it is advantageous for the analytical methods to be simple to perform and to give results quickly. In this study, three different non-specific analytical methods are compared: visual detection of foam, ph and conductivity measurements. The analyses were performed on different dilutions of the cleaning agents Bactericidal Hydroclean and Tickopur R33. The results demonstrated that the most appropriate method for these detergents are conductivity measurements, by which it is possible to detect concentrations of cleaning agents down to 10 µg/ml. In this case, ph is an inadequate method (non-linear) and visual detection of foam is a semi- -quantitative method. All these methods are easy to perform, gives a quick results, and requires no expensive instrumentation. Key words: detergents; cleaning validation; residues; swab analysis. The main objective of cleaning validation procedures is to provide documented evidence that the equipment is safe for the manufacture of the next product. For this purpose, cleaning agents are used for removal of the previously manufactured product and also for the removal microorganisms. Because cleaning agents often consist of a mixture of various surfactants, often very diluted, it may be difficult to find appropriate analytical methods that are sensitive enough for detection of the compounds. The first step in cleaning equipment is the selection of an appropriate cleaning product for the type of residue to be removed. Once this is done, an analytical strategy must be devised to determine the amount of cleaning agent residue left on surface that has been cleaned. This determination is important because if a cleaning agent effectively removes the drug- -product residue but leaves behind its own residue, then one type of contamination has been exchanged for another, and the equipment has not been cleaned effectively. Detergent selection is a critical step in the Correspondening author: D. Milenović, Zdravlje-Actavis Company, 199 Vlajkova St., Leskovac, Serbia. Paper received: 24 May, 2010 Paper revised: 10 August, 2010 Paper accepted: 01 September, 2010 development a cleaning validation. Detergents are not part of the manufacturing process and are only added to facilitate cleaning during the cleaning process. Thus, they should be easily removable. Otherwise, different detergents should be selected [1]. When developing an analytical strategy for cleaning agents residue determination, the first issue that must be addressed is whether to center the analysis on a specific assay for a single component of the cleaning agent or on a "whole-product" analysis. The latter involves the use of a single assay to determine the presence of cleaning agent components in the residue without identifying which particular component or components make up the residue. In this work we will focus on a "whole-product" analysis without trying to determine which particular component is present. If the cleaning agent is effective, then it should not only remove the target substance but should also rinse freely. This would support the applicability of a non- -specific method to determine whether cleaning agent residue of any kind was left behind [2,3]. The swabbing experiment involves mechanically removing cleaning agent residue from a surface with a swab, extracting the cleaning agent from the swab, and analyzing the resultant solution. This experiment will demonstrate that any cleaning agent residue that 39

2 might be left on the surface can be accurately removed and quantified through a swabbing procedure [4,5]. Several different analytical methods have been used in the analyses of surfactants, including reversed-phase chromatography with UV detection [6], ion chromatographic method with conductivity detection [7], total organic carbon analysis, ph, visual detection [8-15]. These analytical methods, along with others, are often used in the validation of cleaning validation procedures according to the demands of the Food and Drug Administration (FDA) [16]. Our analytical approach is to take advantage of the fact that many cleaning agents contain high concentrations of surfactants, i.e., are able to generate visually detectable foam, and also contain strong alkali/acids or have a high ionic content. The goal of this study is to compare the sensitivity of these three potential analytical methods: visual detection of foam, ph and conductivity measurement in the analysis of the cleaning agents Bactericidal Hydroclean and Tickopur R33, and validate the most appropriate of them in support of cleaning validation. The second aim is to evaluate the effectiveness of using swabbing to recover detergent from stainless steel surfaces. EXPERIMENTAL Materials Bactericidal Hydroclean and Tickopur R33 were purchased from Arrow Chemicals Ltd., England. Purified water was obtained with Arium Laboratory Equipment (RO, UV) by Sartorius AG Gottingen (Germany). The extraction-recovery sampling was done with Alpha Swab polyester on polypropylene handle - TX714A (ITW Texwipe, Mahwah, NJ, USA). Equipment Conductivity and ph were measured with a Metrohm 712 conductometer and a Metrohm 827 ph lab, respectively. Standard solutions preparation Stock linearity solution (30 mg/ml) was prepared. From this solution appropriate dilutions by purified water were prepared and ph and conductivity were measured. The stock linearity solution concentrations of the Tickopur R33 and Bactericidal hydroclean were based on total mass of the concentrate. Sample preparation The tip of the swab was placed in diluent purified water. It was than shaken gently twice to remove excess liquid. After that the surfaces were sampled with swabs. The selected stainless steel surfaces (5 cm 5 cm), previously cleaned and dried, were sprayed with 500 µl of a standard solution of detergents at the three concentration levels. Six untreated swabs and plates were spiked at the 15, 7.5 and 1.5 mg per swab level, allowed to dry in air and then extracted with 15 ml of purified water. Each sample tube was shaken vigorously by hand before sonicating for 1 min. After drying, the selected surface was wiped with the first swab soaked with purified water, passing it in various ways, and an another dry cotton swab was used to wipe the wet surfaces of the plate (according to our experience, we used the wet swab first and the dry swab second) [17-19]. The background control sample was prepared from the purified water. The negative swab control was prepared in the same way as the samples, using swabs which have not been in contact with the test surface. Regarding production sample, a more practical solution, where possible, is to perform a swab sampling, place the swab in 15 ml of water, mix it, and check the presence of foam, measure conductivity and ph and in that way prove the presence of detergent residues on the equipment. Samples/standards for all methods were analyzed in triplicate. Properties of the Cleaning agents used in this study 1) Bactericidal Hydroclean. Bactericidal Hydroclean is an alkaline detergent which is a safe and effective product for the removal of soils and grease from hard surfaces, with an added broad spectrum biocide which is active against bacteria, fungi, yeasts, algae and spores. It is odourless in use, non-flammable, non-caustic and biodegradable. The density of Bactericidal is 1.07 g/ml at 25 C, and ph value of concentrated solution is ) Tickopur R33. Tickopur R33 is a universal cleaner for the ultrasonic bath. It is a concentrate, mildly alkaline, with corrosion protection, which cleans and degreases machines and their components, tools, equipment and parts in industry, handicraft and laboratories. Tickopur R33 has been especially designed for cleaning in ultrasonic cleaning units, as well as for cleaning by immersion, wiping or high pressure techniques. Properties: liquid, highly concentrated, for ultrasonic application, ph 9.9 at 1%, with corrosion protection, emulsifying, biodegradable. Analytical methods Visual Detection of Foam. 15 ml of cleaning agent solution of appropriate concentration was added to a 25 ml glass test tube. The test tubes were shaken 1 min, and were then visually inspected within 40

3 1 min in order to detect the presence of foam on top of the solution. Visual detection of foam is affirmative only in cases that all observers affirm observation explicitly. A purified water blank was used as a negative reference. ph and conductivity measurement. Measurements involving the ph meter and conductometer were performed according to British Pharmacopoeia [20]. RESULTS AND DISCUSSION Based on the most toxicological component and 10 ppm criteria (the detergent does not have therapeutical impact) for the detergent, we determined that the acceptable cleaning limit is 4.5 mg/25 cm 2. Both of the cleaning agents that were used in this study have similar properties, which caused a small variation in detection limits of each method. Table 1 shows the results from the visual detection of foam method obtained in the introductory study. conductivity giving a coefficient of determination >0.99 and a conductivity increase by >5 compared to water blank. In the introductory study, which included only the visual detection of foam, the analysis of Bactericidal Hydroclean and Tickopur R33 produced consistent values for the detection limits (n = 3), as shown in Table 1. Residues of both of detergents could be visually detected by foam generation at a concentration of which were given in Table 1, probably because both detergents are formulated with higher level of foaming surfactant. The results obtained from the ph method indicated that ph measurement is not a viable method for detecting residues of both of detergents; ph measurements gave a detection limits of above 200 µg/ml. When conductivity measurements were carried out, the cleaning agents Bactericidal Hydroclean and Tickopur R33 were detected in solutions containing 10 µg/ml. Table 1. Visual detection of foam in solutions of Bactericidal Hydroclean and Tickopur R33 Visual detection of foam Solution number (with three different observers) Concentration, μg/ml Bactericidal Hydroclean Tickopur R No No 2 25 No Yes 3 50 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes These results, together with the extended study, including also determination of ph and conductivity were used in order to determine the detection limit for cleaning agents, as presented in Table 2. Definitions of each detection limit for the respective analytical methods are [5]: 1) The detection limit for visual detection of foam is defined by an obvious foam regeneration, after shaking, compared to a water blank. 2) The detection limit for ph is defined by a ph deviation from water blank by >1 ph units for the ph meter. 3) The detection limit for conductivity is defined by a linear relationship between concentration and Linearity A linear relationship between the conductometar response (triplicate determination) and the concentration of detergent (Bactericidal Hydroclean) in the range from 10 to 1000 µg/ml was demonstrated with a coefficient of determination of and by Eq. (1): y = x , 2 r = (1) In the case of Tickopur R33, a linear relationship between the conductometar response (triplicate determination) and the concentration of detergent in the range from 10 to 1000 µg/ml was demonstrated with a coefficient of determination of and by Eq. (2): Table 2. Detection limits (μg/ml) of the analyzed cleaning agents for each respective analytical method Detergent Visual detection of foam Detection by ph Detection by conductivity Bactericidal Hydroclean (µg/ml) Tickopur R33 (µg/ml)

4 y = x , 2 r = (2) Linearity data are presented in Table 3. Table 4 shows the influence of purified water, swabs and plates on ph and conductivity measurements. Table 3. Linearity data for Bactericidal hydroclean and Tickopur R33 Detergent Property Concentration, µg/ml Bactericidal Hydroclean Mean conductivity, µs/cm ph (mean value) Tickopur R33 Mean conductivity, µs/cm ph (mean value) Accuracy and precision Recovery from swabs. The accuracy and precision of detergent extraction from swabs and plates were determined by conductometric measurements. Six untreated swabs and plates were spiked at the 1.5, 7.5 and 15 mg per swab level. The average measured recovery, the 95% confidence interval and the relative standard deviation (%RSD) were calculated. Table 5 presents the data obtained from the typical recovery experiments for the detergent extraction from the swabs into the diluent-purified water. Since the determined accuracy, precision and recovery are in the expected limits the proposed method is excellent for determination the traces of the detergents Bactericidal hydroclean and Tickopur R33 in cleaning validation. Recovery from stainless steel plates. To determine the accuracy and precision of the method, six stainless steel plates, each 5 cm 5 cm were cleaned thoroughly, rinse with purified water and allowed to dry before treating each plate with 500 μl of the detergent standard solution for positive swab control at the 1.5, 7.5 and 15 mg per swab level described in the method. The plates were allowed to dry before swabbing them and analysing the swabs according to the description of the method. The average measured recovery, the 95% confidence interval and the relative standard deviation (%RSD) were calculated (Table 6). This shows that the method is acceptable with respect to accuracy and precision for the extraction of detergent from plates and swabs into the diluentpurified water. CONCLUSION By comparing the methods that were used, we conclude that the ph analysis is less sensitive; while Table 4. Influence of purified water, swabs and plates on ph and conductivity measurements Detergents Sample Conductivity, µs/cm ph Bactericidal Hydroclean Background sample Negative swab control Negative swab control from plates St 100 µg/ml Tickopur R33 Background sample Negative swab control Negative swab control from plates St 100 µg/ml Table 5. Accuracy and precision; recovery from swabs Detergent Swabs, mg per swab (n = 6) 95% Confidence interval Mean recovery, % RSD / % Bactericidal hydroclean Tickopur R

5 Table 6. Accuracy and precision; recovery from plates Detergent Plates, mg per swab (n = 6) 95% Confidence interval Mean recovery, % RSD / % Bactericidal Hydroclean Tickopur R the visual detection of foam is more sensitive, it is only a semi-quantitative method. In addition, visual detection of foam is easy to perform, gives a quick result, and requires no expensive instrumentation. Conductivity measurements also provide for simple analysis, with the same sensitivity as visual detection of foam, and has the advantage of being a quantitative analysis. Conductivity measurements were found to be the most sensitive method for detection of both of detergents in this study. When choosing a method for detecting the residues of cleaning agents, it is extremely crucial to unerstand the properties of the residual cleaning agent; ph is a good method to use for strongly alkaline or acidic agents. Residues of ionic cleaning agents can be detected by conductivity, and visual detection of foam works well if the cleaning agent contains high amounts of surfactants. Since cleaning agents often contain a mixture of surfactants and other compounds, it is advisable to use more than one method for detecting the residues of cleaning agents. In summary, all of the methods that were tested can be used to detect small amounts of cleaning agents. In the cleaning agents that were tested, visual detection of foam and conductivity were able to detect levels down to 25 and 10 µg/ml, respectively. ph-measurement may be suitable for detecting significantly acidic or alkaline cleaning agents, but is generally a less sensitive method than the others in the study. REFERENCES [1] B. Wallace, R. Stevens, M. Purcell, Pharm. Technol. Aseptic Process. (2004) [2] K.M. Jenkins, A.J. Vanderwielen, Pharm. Technol. 18 (1994) [3] J.M. Smith, Pharm. Technol. 17 (1993) [4] P. Yang, K. Burson, D. Feder, F. Macdonald, Pharm. Technol. 29 (2005) [5] M.J. Shifflet, M. Shapiro, Am. Pharm. Rev. 4 (2002) [6] J. Zayas, H. Colón, O. Garced, L.M. Ramos, J. Pharm. Biomed. Anal. 41 (2006) [7] W. Resto, D. Hernandez, R. Rey, H. Colón, J. Zayas, J. Pharm. Biomed. Anal. 44 (2007) [8] K.M.Jenkins, A.J. Vanderwielen, J.A. Armstrong, L.M. Leonard, G.P. Murphy, N.A. Piros, PDA J. Pharm. Sci. Technol. 50 (1996) [9] L. Westman, G. Karlsson, PDA J. Pharm. Sci. Technol. 54 (2000) [10] R.J. Forsyth, J. Roberts, T. Lukievics, V.V. Nostrand, Pharm. Technol. 30 (2006) [11] R.J. Forsyth, J.L. Hartman, V.V. Nostrand, Pharm. Technol. 30 (2006) [12] R.J. Forsyth, V.V. Nostrand, Pharm. Technol. 29 (2005) [13] R.J. Forsyth, V.V. Nostrand, G.P. Martin, Pharm. Technol. 28 (2004) [14] G.L. Fourman, M.V. Mullen, Pharm. Technol. 17 (1993) [15] D.A. LeBlanc, Pharm. Technol. 22 (1998) [16] Guide to Inspections Validation of Cleaning Processes, U.S. Food and Drug Administration, Washington, available at: /ucm htm (accessed 25 February, 2010) [17] D.M. Milenović, Z.B. Todorović, Acta Chromatogr. 21 (2009) [18] Z.B.Todorović, M.L.Lazić, V.B.Veljković, D.M.Milenović, J.Serb.Chem.Soc. 74 (2009) [19] D.M.Milenović, M.L.Lazić, V.B.Veljković, Z.B.Todorović, Acta Chromatogr. 20 (2008) [20] British Pharmacopoeia, Volume IV, Appendix V,

6 DRAGAN M. MILENOVIĆ 1 DRAGAN S. PEŠIĆ 1 SNEŽANA S. MITIĆ 2 1 Kompanija Zdravlje-Actavis, Leskovac, Srbija 2 Prirodno matematički fakultet, Univerzitet u Nišu, Niš, Srbija NAUČNI RAD NESPECIFIČNE METODE ZA ODREĐIVANJE TRAGOVA DETERDŽENATA TOKOM VALIDACIJE ČIŠĆENJA Procedura validacije čišćenja se izvodi kako bi se obezbedilo prisustvo tragova deterdženata unutar prihvatljivih granica nakon procesa čišćenja. Deterdženti se sastoje od smeše različitih površinski aktivnih supstanci koje se u visoko razblaženom stanju nakon završetka procedure ispiranja opreme vodom. Iz tih razloga veoma je teško pronaći adekvatnu analitičku metodu koja je dovoljno osetljiva za detekciju tragova detedženata. Pored toga, prednost je za analitičku metodu ukoliko je ona jednostavna za izvođenje i daje rezultate veoma brzo. U ovim radu, poređene su tri različite nespecifične metode: vizuelna detekcija pene, merenje ph i merenje provodljivosti. Analize su izvođene pri različitim razblaženjima deterdženata Bactericidal Hydroclean i Tickopur R33. Rezultati su pokazali da je najpogodnija metoda za ove deterdžente konduktometrijska metoda, pomoću koje je moguće detektovati koncentracije deterdženata i do 10 µg/ml. U ovom slučaju, merenje ph je neadekvatna metoda (nelinearna), a vizuelna detekcija pene je semi-kvantitativna. Sve ove metode su lake za izvođenje, brzo daju rezultate i ne zahtevaju skupu opremu. Ključne reči: deterdženti; validacija čišćenja; ostaci u tragovima; analiza brisa. 44

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