Occurrences of benzalkonium chloride in streams near a pharmaceutical manufacturing complex in Korea and associated ecological risk.

Benzalkonium chloride (BKC) is a commonly used preservative in personal care products and pharmaceutical preparations. However, its ecological risks are not well understood because of lack of monitoring data and ecotoxicological information. In the present study, occurrence of BKC was investigated in the waters near a pharmaceutical manufacturing complex of South Korea and its acute and chronic ecotoxicities were evaluated using Daphnia magna and Japanese medaka (Oryzias latipes). Associated ecological risks were estimated by calculating hazard quotients (HQs). In addition, endocrine disruption potency of BKC was compared with those of other frequently used preservatives using human adrenal (H295R) and rat pituitary (GH3) cells. High concentration of BKC was detected at locations near the pharmaceutical manufacturing plants, i.e., 35.8 µg/L for dodecyl benzyl dimethyl ammonium chloride (BKC-C12), and 21.6 µg/L tetradecyl benzyl dimethyl ammonium chloride (BKC-C14). In Daphnia, 48 h immobilization EC50 and 21 d reproduction NOEC were determined at 41.1 µg/L and ≥10.8 µg/L, respectively. For O. latipes, 96 h LC50 was determined at 246 µg/L while the growth inhibition NOEC was ≥113.4 µg/L following early life stage exposure. BKC significantly up-regulated vitellogenin gene of juvenile fish, indicating its endocrine disrupting potential in fish. Exposure to BKC increased steroid hormone level in H295R cells, and induced cytotoxicity in GH3 cells. HQ values of BKC were determined at greater than one in the ambient water near pharmaceutical manufacturing facilities. Considering high ecological risk and endocrine disrupting potential, long-term consequences of BKC contamination in aquatic ecosystem need to be examined.

Benzalkonium chloride alters phenotypic and genotypic antibiotic resistance profiles in a source water used for drinking water treatment.

Antibiotic resistance is a major public health concern. Triclosan is an antimicrobial compound with direct links to antibiotic resistance that was widely used in soaps in the U.S. until its ban by the U.S. Food and Drug Administration. Benzalkonium chloride (BAC), a quaternary ammonium compound, has widely replaced triclosan in soaps marketed as an antibacterial. BAC has been detected in surface waters and its presence will likely increase following increased use in soap products. The objective of this study was to determine the effect of BAC on relative abundance of antibiotic resistance in a bacterial community from a surface water used as a source for drinking water treatment. Bench-scale microcosm experiments were conducted with microbial communities amended with BAC at concentrations ranging from 0.1 µg L-1 to 500 µg L-1. Phenotypic antibiotic resistance was quantified by culturing bacteria in the presence of different antibiotics, and genotypic resistance was determined using qPCR to quantify antibiotic resistance genes (ARGs). BAC at concentrations ranging from 0.1 µg L-1 to 500 µg L-1 was found to positively select for bacteria resistant to ciprofloxacin and sulfamethoxazole, and negatively select against bacteria with resistance to six other antibiotics. Exposure to BAC for 14 days increased the relative abundance of sul1 and blaTEM. This study re-highlights the importance of employing both culture and non-culture-based techniques to identify selection for antibiotic resistance. The widespread use of BAC will likely impact antibiotic resistance profiles of bacteria in the environment, including in source waters used for drinking water, wastewater treatment plants, and natural waterways.

Application of an algal growth inhibition assay to determine distribution coefficients of benzalkonium ions between kaolinite and water.

Benzalkonium compounds are widely used and found in environmental samples. Due to their amphiphilic nature, it is important to know sorption coefficients to account their bioavailability. However, currently available models describing their partitioning were developed using low molecular weight homologues and it cannot be ascertained whether they are applicable to their higher molecular weight homologues. Reasons for the scarcity of data on highly sorptive compounds include the lack of reliable quantification techniques for analyzing these chemicals at environmentally relevant levels. This study, therefore, reports on an algal growth inhibition assay-based method for the determination of kaolinite/water distribution coefficients for benzalkonium compounds at their environmentally relevant concentration range. Sorption to clay was computed using the difference between median effective concentration determined in a culture with kaolinite and that derived from a culture grown in standard medium. A kinetic model was used to account for uptake into algal cells and to calculate free concentrations. Due to the sensitivity of the algal species, Pseudokirchneriella subcapitata, it was possible to determine distribution coefficients below micromole per liter concentrations. The computed distribution coefficients showed a linear increase with number of carbon atoms in the alkyl chain up to 14. The proposed bioassay-based method should be applicable to determine distribution coefficients for highly hydrophobic chemicals and ionic liquids at a concentration range lower than typical analytical limits.

Efficient UPLC and CE methods for the simultaneous determination of azelastine hydrochloride and its genotoxic impurity.

A novel stability-indicating UPLC and CE method was established and validated for the determination of azelastine hydrochloride (AZL) and its genotoxic impurity, benzohydrazide, in the presence of benzalkonium chloride. The developed UPLC method was based on chromatographic separation using a C18 column as a stationary phase and acetonitrile-(0.1% w/v) aqueous sodium lauryl sulfate (55:45, v/v, pH 5 with phosphoric acid) as a mobile phase with a flow rate of 1.2 mL/min and UV detection at 215 nm. The chromatographic run time was ~2 min. The developed CE method depended on using a stationary phase of Standard Bare Fused Silica Capillaries (75 µm i.d. × 59 cm and 50 cm detection length) and the applied voltage was 30 kV using 40 mm phosphate buffer (pH 2 with aqueous H3 PO4 ); the detection wavelength was 225 nm. The analysis time was about 6 min. The suggested methods were successfully applied for the analysis of AZL in a pharmaceutical preparation. The validity of the developed methods was assessed by applying the standard addition technique and no interference from excipients was observed. The results obtained by the proposed methods were statistically analyzed and compared with the manufacturer's method and no significant difference was found between the compared methods.

Liquid chromatography-tandem mass spectrometry multiresidue method for the analysis of quaternary ammonium compounds in cheese and milk products: Development and validation using the total error approach.

Quaternary ammonium compounds (QACs) are both cationic surfactants and biocidal substances widely used as disinfectants in the food industry. A sensitive and reliable method for the analysis of benzalkonium chlorides (BACs) and dialkyldimethylammonium chlorides (DDACs) has been developed that enables the simultaneous quantitative determination of ten quaternary ammonium residues in dairy products below the provisional maximum residue level (MRL), set at 0.1mgkg-1. To the best of our knowledge, this method could be the one applicable to milk and to three major processed milk products selected, namely processed or hard pressed cheeses, and whole milk powder. The method comprises solvent extraction using a mixture of acetonitrile and ethyl acetate, without any further clean-up. Analyses were performed by liquid chromatography coupled with electrospray tandem mass spectrometry detection (LC-ESI-MS/MS) operating in positive mode. A C18 analytical column was used for chromatographic separation, with a mobile phase composed of acetonitrile and water both containing 0.3% formic acid; and methanol in the gradient mode. Five deuterated internal standards were added to obtain the most accurate quantification. Extraction recoveries were satisfactory and no matrix effects were observed. The method was validated using the total error approach in accordance with the NF V03-110 standard in order to characterize the trueness, repeatability, intermediate precision and analytical limits within the range of 5-150µgkg-1 for all matrices. These performance criteria, calculated by e.noval® 3.0 software, were satisfactory and in full accordance with the proposed provisional MRL and with the recommendations in the European Union SANTE/11945/2015 regulatory guidelines. The limit of detection (LOD) was low (<1.9µgkg-1) and the limit of quantification (LOQ) ranged from 5µgkg-1 to 35µgkg-1 for all matrices depending on the analytes. The validation results proved that the method is suitable for quantifying quaternary ammoniums in foodstuffs from dairy industries at residue levels, and could be used for biocide residues monitoring plans and to measure the exposition consumer to biocides products.

Tolerance development in Listeria monocytogenes-Escherichia coli dual-species biofilms after sublethal exposures to pronase-benzalkonium chloride combined treatments.

This study was designed to assess the effects that sublethal exposures to pronase (PRN) and benzalkonium chloride (BAC) combined treatments have on Listeria monocytogenes-Escherichia coli dual-species biofilms grown on stainless steel in terms of tolerance development (TD) to these compounds. Additionally, fluorescence microscopy was used to observe the changes of the biofilm structure. PRN-BAC exposure was carried out using three different approaches and TD was evaluated treating biofilms with a final 100 µg/ml PRN followed by 50 µg/ml BAC combined treatment. Results showed that exposure to PRN-BAC significantly decreased the number of adhered L. monocytogenes (P < 0.05), while E. coli counts remained generally unaltered. It was also demonstrated that the incorporation of recovery periods during sublethal exposures increased the tolerance of both species of the mixed biofilm to the final PRN-BAC treatment. Moreover, control biofilms became more resistant to PRN-BAC if longer incubation periods were used. Regardless of the treatment used, log reduction values were generally lower in L. monocytogenes compared to E. coli. Additionally, microscopy images showed an altered morphology produced by sublethal PRN-BAC in exposed L. monocytogenes-E. coli dual-species biofilms compared to control samples. Results also demonstrated that L. monocytogenes-E. coli dual-species biofilms are able to develop tolerance to PRN-BAC combined treatments depending on way they have been previously exposed. Moreover, they suggest that the generation of bacterial tolerance should be included as a parameter for sanitation procedures design.

Efficacy of benzalkonium chloride against bioluminescent P. aeruginosa ATCC9027 constructs.

Whole cell biosensors have been seldom used in the pharmaceutical and cosmetics industries for preservative efficacy testing (PET). According to several pharmacopoeias, preservatives should be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. The aim of the study was to assess the applicability of Pseudomonas aeruginosa ATCC9027 and its validated bioluminescent strains for preservative efficacy tests using benzalkonium chloride (BKC). Applicability of five constitutively-expressed bioluminescent strains was evaluated for preservative efficacy tests (PET) using bacterial replication, bioluminescence and fluorescence in a three-way study. PET using BKC showed no significant difference between bioluminescence and enumeration. Good correlations between bioluminescence, colony-forming units (CFU) count and fluorescence were obtained for BKC concentrations (R>0.9) between 0.0003-0.0025% against strains containing the constructs lys-pMElux, lpp-pMElux and tat-pMElux. Furthermore, two-way ANOVA analysis showed that the bioluminescent method and traditional plate counting method were equivalent for concentrations of BKC (0.0003-0.01%) during preservative efficacy tests. PET testing with BKC showed that tat-pMElux (R>0.9) had consistently high correlation coefficients between CFU and relative bioluminescence; P. aeruginosa ATCC9027 tatH5-pMElux is the best construct for testing various antimicrobial agents.

Analytical Method for the Detection of Residual Active Ingredients Found in Neutralized Suspensions of Antimicrobial Products.

An analytical method for determining the presence and levels of residual active ingredients found in neutralized suspensions of phenolic and quaternary ammonium salt-based antimicrobial products was developed using solid-phase extraction in combination with LC-tandem MS. A single-laboratory validation of the method was performed at three concentration levels for the quaternary ammonium compounds (also referred to as benzalkonium chlorides or BACs) and the phenols in the presence of letheen broth neutralizer at 2.5 and 2.75 µg/mL, respectively, as well as at dilutions of 1:10 and 1:100 in those concentrations. The method's lowest LODs were 0.005 µg/g for BACs and 0.006 µg/g for phenols. The average recovery of the fortified samples for both active ingredients ranged between 80 and 124%, and RSDs were generally <20%. In a related study, the effectiveness of letheen broth with and without sodium thiosulfate was evaluated as a neutralizer for sodium hypochlorite. The results showed that letheen broth without sodium thiosulfate neutralizes chlorine concentrations up to 60 ppm, and that 200 µg sodium thiosulfate are required to neutralize a 72 ppm concentrated chlorine solution in letheen broth.

Determination of benzalkonium chloride in wet wipes by using a validated capillary electrophoresis method.

Benzalkonium chloride (BAC), which is a mixture of C8 and C18 alkylbenzyldimethylammonium chlorides, has an important biocide character and is used in many cosmetics, especially wet wipes, as a preservative and/or antibacterial agent. The concentration range of BAC is 0.005­0.5 % and it is an irritant substance when used at high concentrations. Thus, the concentration of BAC should be carefully monitored in commercial products intended for skin use. In this study, a capillary electrophoresis (CE) analysis method for BAC quantification was developed. The quantitative analysis was carried out by the external standard method. The electrophoretic separation was performed by using 75 mM (pH 6.0) phosphate buffer solution containing 30% acetonitrile as the electrolyte. The separation voltage was 10 kV and the temperature was held at 18°C. Samples were introduced into the capillary column hydrodynamically using 50.0-mbar pressure over a 3-s period. The developed method was validated and applied on samples prepared by wringing out antibacterial wet wipes containing BAC without any further extraction. The linearity of the method was controlled by applying the Mandel test. The limit of detection (LOD) values for the developed method were 0.313 and 0.309 µg/ml and the limit of quantification (LOQ) values were 1.042 and 1.029 µg/ml for C10 and C12 derivatives, respectively.

A capillary liquid chromatography method for benzalkonium chloride determination as a component or contaminant in mixtures of biocides.

A method for quantifying benzalkonium chloride (BAK), an alkyl dimethyl benzyl ammonium compound, in several biocides formulations is proposed. A tertiary amine like N-(3-aminopropyl)-N-dodecyl-1,3-propanediamine (TA) and a straight-chain alkyl ammonium compound like trimethyl-tetradecyl ammonium chloride (TMTDAC), have been employed as trade surfactants besides BAK. Two capillary analytical columns with different polarities are tested: inertsil CN-3 capillary column (150mm×0.5mm i.d., 3µm particle diameter) and a non endcapped Zorbax C18 capillary column (35mm×0.5mm i.d., 5µm particle diameter). This latter column provided the best separation of the BAK homologues in less than 12min using acetonitrile:acetate buffer (50mM, pH 5) 85:15 at 20µLmin(-1). The proposed method combines on-line in-tube solid-phase microextraction (IT-SPME) coupled to capillary liquid chromatography (CapLC) and UV diode array detection. Matrix effect was present when TA were in excess to BAK. If TMTDAC is the co-biocide, matrix effect is always present. A decreasing of analytical response mainly for C12-BAK homologue was found using both chromatographic columns. The charged amount of mixture in the system was the most important parameter for obtaining reliable results. 1mL was the on line processed sample volume optimum for concentrations lower than 35µgmL(-1) of total surfactants. LODs were 0.03µgmL(-1) and 0.006µgmL(-1) for C12-BAK and C14-BAK, respectively. This method is also of use to evaluate the unwanted presence of BAK in biocide formulations due to industrial processes.

Development and validation of a rapid ultra-high performance liquid chromatography method for the assay of benzalkonium chloride using a quality-by-design approach.

A rapid robust reversed-phase UHPLC method has been developed for the analysis of total benzalkonium chloride in preserved drug formulation. A systematic Quality-by-Design (QbD) method development approach using commercial, off the shelf software (Fusion AE(®)) has been used to optimize the column, mobile phases, gradient time, and other HPLC conditions. Total benzalkonium chloride analysis involves simple sample preparation. The method uses gradient elution from an ACE Excel 2 C18-AR column (50mm×2.1mm, 2.0µm particle size), ammonium phosphate buffer (pH 3.3; 10mM) as aqueous mobile phase and methanol/acetonitrile (85/15, v/v) as the organic mobile phase with UV detection at 214nm. Using these conditions, major homologs of the benzalkonium chloride (C12 and C14) have been separated in less than 2.0min. The validation results confirmed that the method is precise, accurate and linear at concentrations ranging from 0.025mg/mL to 0.075mg/mL for total benzalkonium chloride. The recoveries ranged from 99% to 103% at concentrations from 0.025mg/mL to 0.075mg/mL for total benzalkonium chloride. The validation results also confirmed the robustness of the method as predicted by Fusion AE(®).

Benzalkonium runoff from roofs treated with biocide products - In situ pilot-scale study.

Roof maintenance practices often involve the application of biocide products to fight against moss, lichens and algae. The main component of these products is benzalkonium chloride, a mixture of alkyl benzyl dimethyl ammonium chlorides with mainly C12 and C14 alkyl chain lengths, which is toxic for the aquatic environment. This paper describes, on the basis of an in-situ pilot scale study, the evolution of roof runoff contamination over a one year period following the biocide treatment of roof frames. Results show a major contamination of roof runoff immediately after treatment (from 5 to 30 mg/L), followed by an exponential decrease. 175-375 mm of cumulated rainfall is needed before the runoff concentrations become less than EC50 values for fish (280 µg/l). The residual concentration in the runoff water remains above 4 µg/L even after 640 mm of rainfall. The level of benzalkonium ions leaching depends on the roofing material, with lower concentrations and total mass leached from ceramic tiles than from concrete tiles, and on the state of the tile (new or worn out). Mass balance calculations indicate that a large part of the mass of benzalkonium compounds applied to the tiles is lost, probably due to biodegradation processes.

Quality by design based optimization of a high performance liquid chromatography method for assay determination of low concentration preservatives in complex nasal formulations.

The effects of seven different chromatographic parameters and five sample preparation parameters in a high performance liquid chromatography (HPLC) method for assay determination of benzalkonium chloride (BKC) in a nasal formulation were evaluated using two fractional factorial experimental designs. The design space of the analytical method was modeled using Umetrics Modde software and the optimal method conditions were predicted. The optimum HPLC chromatographic conditions were obtained using a Luna CN column (150 x 4.6 mm, 3 µm). The results show that mobile phase pH, amount of acetonitrile in the mobile phase and column temperature are the most important factors in obtaining good separation of BKC homologs from an interfering peak. In the sample preparation step, the use of an aqueous solution for dissolving the samples was the most important factor since it eliminated the interfering effect of the active compound. The optimal method was validated for linearity, accuracy and precision. The use of experimental designs enables obtaining the maximum amount of information with the least possible number of experiments. Such designs are an economical manner in evaluating a variety of different factors and their interactions.

Comparison of the ocular surface changes following the use of two different prostaglandin F2α analogues containing benzalkonium chloride or polyquad in rabbit eyes.

OBJECTIVE: The aim of this study is to compare the effect of prostaglandin analogues preserved with either 0.015% or 0.001% benzalkoium chloride (BAK); or 0.001% polyquad (PQ) on the ocular surface of rabbit eyes. METHODS: Forty white rabbits were randomized to receive four-times daily instillation of either 0.0015% tafluprost (TF) preserved with 0.001% BAK (TF-BAK); 0.004% travoprost (TR) with 0.015% BAK (TR-BAK) or 0.001% PQ (TR-PQ); or preservative-free artificial tears in one eye for a 4-week period. Tear samples collected from the 40 rabbits were analyzed by enzyme-linked immunosorbent assays (ELISA) to identify the presence of inflammatory cytokines: interleukin (IL)-1ß and IL-6 on day 14. Subsequently, harvested cornea and bulbar conjunctiva were evaluated using light and transmission electron microscopy (TEM). RESULTS: IL-6 was significantly increased in TF-BAK and TR-BAK groups compared to controls and TR-PQ group (p = 0.005); however, IL-1ß level was not significantly different among four groups (p = 0.360). Rabbits treated with TR-BAK showed decreased goblet cell density of bulbar conjunctiva and increased pyknotic change and vacuolization of corneal epithelial cells on light microscopy; similar change occurred but was less severe in TF-BAK group. The TR-PQ group showed similar results as the controls. The destruction of the microvillar architecture of bulbar conjunctiva and cornea was most prominent in the TR-BAK group. CONCLUSIONS: Preservatives included in the anti-glaucoma eye-drops showed different ocular surface changes according to the concentration and type in the rabbits. Prostaglandin analogues preserved with higher level of BAK may cause more harmful effects on the ocular surface than PQ-preserved medications.

Environmental risk assessment of selected pharmaceuticals in Turkey.

In this study, environmental risks of selected pharmaceuticals were investigated to assess potential hazards. Ciprofloxacin, Clarithromycin, Cefuroxime axetil, antibiotics, Benzalkoniuman antiseptic, Paracetamol, an analgesic, and Naproxen, an anti-inflammatory, were selected due to their high rate of usage in Turkey. Ciprofloxacin was found to have the highest risk due to its high PEC/PNEC ratio (28.636). Benzalkonium, Paracetamol and Clarithromycin have a potential to cause environmental hazards. The biodegradation and biological concentration factors (BCF) of the drugs were also determined using EPA/STWIN and EPA/BCFWIN programs. The results illustrated that these pharmaceuticals are nonbiodegradable in wastewater treatment plants. The BCFs of Benzalkonium and Clarithromycin were found to be very high, 70.790 L/kg and 56.490 L/kg, respectively. It was suggested that alternative treatment methods other than biological ones should be investigated for these pharmaceuticals because of their low biodegradability. Also, unnecessary use of antibiotics is supposed to be discouraged to reduce environmental hazards.

A comparative study of novel spectrophotometric methods based on isosbestic points; application on a pharmaceutical ternary mixture.

This work represents the application of the isosbestic points present in different absorption spectra. Three novel spectrophotometric methods were developed, the first method is the absorption subtraction method (AS) utilizing the isosbestic point in zero-order absorption spectra; the second method is the amplitude modulation method (AM) utilizing the isosbestic point in ratio spectra; and third method is the amplitude summation method (A-Sum) utilizing the isosbestic point in derivative spectra. The three methods were applied for the analysis of the ternary mixture of chloramphenicol (CHL), dexamethasone sodium phosphate (DXM) and tetryzoline hydrochloride (TZH) in eye drops in the presence of benzalkonium chloride as a preservative. The components at the isosbestic point were determined using the corresponding unified regression equation at this point with no need for a complementary method. The obtained results were statistically compared to each other and to that of the developed PLS model. The specificity of the developed methods was investigated by analyzing laboratory prepared mixtures and the combined dosage form. The methods were validated as per ICH guidelines where accuracy, repeatability, inter-day precision and robustness were found to be within the acceptable limits. The results obtained from the proposed methods were statistically compared with official ones where no significant difference was observed.

Determination of thermodynamic and transport parameters of naphthenic acids and organic process chemicals in oil sand tailings pond water.

Oil sand tailings pond water contains naphthenic acids and process chemicals (e.g., alkyl sulphates, quaternary ammonium compounds, and alkylphenol ethoxylates). These chemicals are toxic and can seep through the foundation of the tailings pond to the subsurface, potentially affecting the quality of groundwater. As a result, it is important to measure the thermodynamic and transport parameters of these chemicals in order to study the transport behavior of contaminants through the foundation as well as underground. In this study, batch adsorption studies and column experiments were performed. It was found that the transport parameters of these chemicals are related to their molecular structures and other properties. The computer program (CXTFIT) was used to further evaluate the transport process in the column experiments. The results from this study show that the transport of naphthenic acids in a glass column is an equilibrium process while the transport of process chemicals seems to be a non-equilibrium process. At the end of this paper we present a real-world case study in which the transport of the contaminants through the foundation of an external tailings pond is calculated using the lab-measured data. The results show that long-term groundwater monitoring of contaminant transport at the oil sand mining site may be necessary to avoid chemicals from reaching any nearby receptors.

Combining poly(dimethyldiphenylsiloxane) and nitrile phases for improving the separation and quantitation of benzalkonium chloride homologues: In-tube solid phase microextraction-capillary liquid chromatography-diode array detection-mass spectrometry for analyzing industrial samples.

The retention and separation of four homologues of benzalkonium chloride (alkyl (C12, C14, C16, C18) dimethylbenzylammonium chloride) have been studied in poly(dimethyldiphenylsiloxane) (TRB) and nitrile capillary phases, respectively. Under the optimized conditions (50% acetonitrile in processed samples, 35% of diphenyl content of the TRB, capillary length 43cm and water:methanol 60:40 as replacing solvent), the extraction efficiency was similar for all the homologues with satisfactory reproducibility and independently of the amount and proportion of homologues. Industrial samples with high viscosity or with complex composition and washes waters have been analyzed without previous treatment. The coupling of IT-SPME-CapLC-DAD to MS detection allowed the determination of the minority homologues (C16 and C18) in the industrial samples and washes waters. No matrix effect was found.

Localisation and quantification of benzalkonium chloride in eye tissue by TOF-SIMS imaging and liquid chromatography mass spectrometry.

Benzalkonium (BAK) chloride is the most commonly used preservative in eye drops. It is generally composed of benzyldimethyldodecylammonium C12 and benzyldimethyltetradecylammonium C14 and is supposed to increase penetration of active compounds. However, numerous studies have reported its toxic effect to ocular surface especially in long-term treatments like against glaucoma, a sight-threatening disease. Albino rabbits were treated with a hyperosmolar solution and a high concentration of BAK solution for 1 month. Enucleated eyes were cryo-sectioned and analysed by mass spectrometry. Mass spectrometry imaging using time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used to characterize the spatial distribution and to determine the relative quantity of BAK at the surface of rabbit eye sections. Liquid chromatography coupled with mass spectrometry (LC-MS) using a hybrid linear ion trap-Orbitrap® mass spectrometer was used to obtain relative quantification of BAK at the sample surface. TOF-SIMS images of BAK ions indicated a distribution at the ocular surface and in deeper structures. Didecyldimethylammonium (DDMAC), which is used in hospitals as a substitute for BAK, was also detected and showed an accumulation around the eyes. After extraction with acetonitrile and chromatographic separation using a Gemini C18 column and an original elution gradient, the relative quantities of BAK and DDMAC present in the whole eye section surface were determined. This LC-MS method was validated in terms of limits of quantification, linearity, repeatability and reproducibility and its feasibility was evaluated in surgically obtained human samples. Specimens of iris, lens capsule or trabecular meshwork were found with significant levels of BAK and DDMAC, thus confirming the penetration of BAK in deep ocular structures, with potential deleterious effects induced by this cytotoxic compound. The analytical method developed here could therefore be of primary interest in the field of pharmaco-toxicology in order to localise, identify and quantify drugs or xenobiotic compounds present at biological sample surfaces.