Journal of Analytical Toxicology, Vol. 33, March 2009

High-Performance Liquid Chromatographic Determination of Chlorhexidine in Whole Blood by Solid-Phase Extraction and Kinetics Following an Intravenous Infusion in Rats* Yuying Xue1,2,†, Meng Tang1, Yoko Hieda2, Junko Fujihara2, Koji Takayama2, Hisakazu Takatsuka2, and Haruo Takeshita2 1Department 2Department

of Toxicology, School of Public Health, Southeast University, No.87 Dingjiaqiao, Nanjing 210009, China and of Legal Medicine, Shimane University Faculty of Medicine, 89-1 Enya, Izumo, Shimane 693-8501, Japan

Abstract This paper presents the extraction and analysis of chlorhexidine (CHX) from whole blood using solid-phase extraction (SPE) together with high-performance liquid chromatography (HPLC). Blood samples, spiked with chlorpromazine used as an internal standard, were fortified with sodium acetate buffer and purified with Bakerbond C18 SPE columns. The columns were washed, dried, and eluted with experimental optimized solvent systems. The HPLC was performed using a Capcell Pak C18 MG column (4.6 × 250-mm) and monitored at 260 nm, using a UV detector. A mobile phase consisting of acetonitrile/water (40:60 v/v), containing 0.05% trifluoroacetic acid, 0.05% heptafluorobutyric acid, and 0.1% triethylamine, was employed. The assay was linear over the range of 0.05 to 2.0 µg/g and the limit of detection was 0.01 µg/g for CHX in whole blood. At the concentration range of 0.05 to 2.0 µg/g, the recoveries ranged from 72% to 85%, and the intra- and interday precision, expressed as coefficient of variation, were less than 11% and 13%, respectively. Kinetic characteristics following an intravenous infusion of a CHX product, Maskin® solution, at a dose of 15 mg/kg in rats were evaluated using the present method. The kinetic profiles of CHX conformed to a twocompartment model with an alpha half-life (of distribution) at 0.05 h and a beta half-life (of elimination) at 0.55 h in rats. The method is simple and reliable for the determination of CHX in blood samples and could be expected to apply to forensic and clinical specimens.

connected by a central hexamethylene chain (Figure 1). The presence of two symmetrically positioned basic chlorophenyl guanide groups attached to a lipophilic hexamethylene chain aids in rapid absorption through the outer bacterial cell wall, causing irreversible bacterial membrane injury, cytoplasmic leakage, and enzyme inhibition (1). Due to its wide spectrum of bactericidal and antiviral activity, CHX is used as a common ingredient in various formulations, ranging from skin disinfectants in healthcare products to antiplaque or anticariogenic agents, both in human and veterinary medicine (2,3). In human use, CHX is applied to prevent and treat the redness, swelling, and bleeding gums associated with gingivitis. In veterinary medicine, CHX is used as a generalpurpose disinfectant for cleansing wounds, skin, instruments, and equipment. Because of its antiseptic properties and low potential for systemic or dermal toxicity, CHX has been incorporated into shampoos, ointments, skin and wound cleansers, teat dips, surgical scrubs, etc. (4). Many cases of poisoning have occurred in Japan recently which involve in disinfectants and antiseptics because they are easily accessible. Accidental ingestion by children or the elderly and suicidal ingestion of these compounds has occasionally occurred (5–11). Two cases of accidental intravenous injection to patients (7,8), a fatal case of suicidal injection (9), a survival case of suicidal ingestion (10), and a fatal case of accidental ingestion (11) have been reported.

Introduction Chlorhexidine (CHX) is a symmetrical cationic molecule containing two 4-chlorophenyl rings and two biguanide groups * Presented in part as a poster at the 26th Annual Meeting of Japanese Association of Forensic Toxicology, June 2007, Miyazaki, Japan. † Author to whom correspondence should be addressed. Yuying Xue, Ph.D., Department of Toxicology, School of Public Health, Southeast University, No. 87 Dingjiaqiao, Nanjing 210009, China. E-mail: [email protected].

Figure 1. Structure of CHX.

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Although CHX, one of the most popular disinfectants, is associated with many gingival and dermal allergic problems (12–15), its relative lack of acute toxicity has not drawn much serious attention from forensic toxicologists. The toxicokinetics of CHX following intravenous administration have not been previously reported. The ability to detect and identify disinfectants is inherently useful for suspected disinfectant poisoning events. A procedure for extracting CHX from biological samples and determining the concentration in blood is thus required in the forensic field. The kinetic characteristics of CHX administered intravenously are imperative to signify the effects of CHX in the body, even though the specific physicochemical properties of the compound make the determination an intricate task. High-performance liquid chromatography (HPLC) has been widely used for the determination of CHX in various formulations (16–18). A number of reports have dealt with determination of CHX by LC using ion-pair reversed-phase HPLC (19,20). An isocratic reversed-phase LC method has been developed for determination of five active substances, including CHX, in an ointment (21). The hydrolytic pathway of CHX has been investigated (22). HPLC methods with gradient mode have been used for the assay of CHX and its known degradation products (22–24). CHX has been determined together with other antimicrobial agents used in cosmetics using ion interaction reversed-phase HPLC (25). A new method for determination of CHX release from preparations on artificial fissures was recently described. CHX determination was conducted in a microplate reader and the reduced intensity of fluorescence of the microplates was used for CHX quantification (26). However, none of these techniques are related to biological specimens. There are several reports about the determination of CHX in biological fluids using HPLC with a UV detector (8,27–33). The samples determined involved saliva, urine, raw dairy milk, serum, and plasma. There is no simple, sensitive, and reproducible analytical method available to quantify CHX in whole blood by HPLC. Other methods reported in the literature used LC–electrospray ionization (ESI)-mass spectrometry (MS) for the determination of CHX in saliva (34) and in hemolyzed blood (9). However, these approaches do need extensive sample preparation and expensive devices which are not available in all departments. In our laboratory, great endeavors have been made for the determination of disinfectants or surfactants in biological samples and evaluation of toxicological characteristics of these compounds for a forensic purpose. We have previously reported a sensitive HPLC method to determine benzalkonium chloride, a cationic surfactant used as a disinfectant, in blood and tissue samples (35), and also a specific determination of linear alkylbenzenesulfonates, anionic surfactants used widely in household products, in whole blood using an HPLC method (36). The kinetic characteristics and toxic effects of benzalkonium chloride following various routes of administration in rats has also been investigated (37,38). Because whole blood is a common matrix used in forensic investigation, the aim of this study was to develop a sensitive and simple method for the assay of CHX, a widely used

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disinfectant, in blood using the HPLC method. The blood sample was purified and CHX was extracted by solid-phase extraction (SPE) method. The kinetic characteristics of CHX in rats were investigated following an intravenous infusion of a CHX product, Maskin solution, using the present method.

Experimental Reagents and materials

Standard chlorhexidine dihydrochloride (CHX hydrochloride) and chlorhexidine digluconate (CHX gluconate) (20% solution in water) were purchased from Sigma-Aldrich (St. Louis. MO) and stored at 4°C in the dark. A product of CHX, Maskin solution (20 w/v% CHX gluconate in water), which is one of the most common disinfectants in Japan, was purchased from Maruishi Pharmaceutical (Osaka, Japan). Chlorpromazine hydrochloride, used as an internal standard (IS) was purchased from ICN Biomedicals (Solon, OH), which was imported by Wako Pure Chemical Industries (Osaka, Japan). Trifluoroacetic acid, heptafluorobutyric acid, and triethylamine were purchased from Wako Pure Chemical Industries too. Acetonitrile for mobile phase was of HPLC grade, and all other reagents were of analytical grade. An SPE cartridge (Bakerbond SPE Octadecyl C18, 3 mL, 500 mg) was purchased from J.T. Baker (Phillipsburg, NJ). Stock solutions of CHX hydrochloride and CHX gluconate, as well as the solution of IS, were prepared in methanol to give the concentration of 1.0 mg/mL as free chlorhexidine or free Chlorpromazine, respectively. These solutions were stored in a refrigerator at 4°C and were stable at least a half year. Each solution was further diluted to the required concentrations before use. Instrumentation

SPE was carried out using a 12-position Sep-Pak® Waters vacuum manifold supplied by Millipore (Billerica, MA). HPLC was performed on a Shimadzu (Kyoto, Japan) SCL-10 system comprised of a SCL-10Avp system controller, LC-10ADvp pump, LC-10AD pump, SIL-10ADvp autoinjector, and CTO-2A column thermostat. UV signals were monitored by a Waters (Milford, MA) 2487 detector and recorded by a Hitachi (Tokyo, Japan) D-7500 integrator. The HPLC conditions were performed based on the method of Kudo et al. (8) with minor modifications. Briefly, separation was carried out on a Capcell Pak C18 MG column (4.6 × 250 mm, S-5 µm) supplied by Shiseido (Tokyo, Japan). The mobile phase was operated isocratically using a mixture of acetonitrile/water (40:60) containing 0.05% trifluoroacetic acid, 0.05% heptafluorobutyric acid, and 0.1% triethylamine. A flow rate of 1.0 mL/min was maintained throughout the analysis. An injection volume of 10 µL was used. The temperature of the column was maintained at 40°C. The detector was set to record at 260 nm. Extraction procedure for biological samples

SPE conditioning. The Bakerbond SPE columns were

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conditioned with 1 × 3 mL methanol, followed by 1 × 3 mL distilled water and 1 × 3 mL sodium acetate buffer (0.1 M, pH 5.0). These were allowed to flow through the column under gravity. The height of the aqueous liquid was halted just above the column bed to prevent drying out of the columns. Sample pretreatment. Rat whole blood (0.05–1.0 g) was added with 2–4 mL water and 1 mL hydrochloric acid (1 N) (this was used to release CHX from the CHX-protein complex in blood samples, because CHX combined protein easily). The mixture was shaken for 15 min, then centrifuged at 3000 rpm for 20 min at 4°C. The supernatant was added with 9 mL of sodium acetate buffer (0.1 M, pH 5.0), and spiked by 100 µL of internal standard solution (10 µg/mL), and then centrifuged at 18,000 rpm for 60 min at 4°C. Washing. The supernatant liquids were loaded onto the conditioned sorbent and allowed to pass through with the aid of gravity. After the samples had percolated through the columns, the sorbent was washed with 3 × 3 mL sodium acetate buffer (0.1 M, pH 5.0), followed by 3 × 3 mL of methanol. The columns were dried under a full vacuum for 5 min (if this is not done, the recovery will be very low at approximately 30%). The columns were further washed with 3 × 3 mL ethyl acetate, and were dried for 5 min under a full vacuum. Elution. CHX were eluted from the columns using 1 × 3 mL of methanol/ethyl acetate (1:1, v/v) containing 0.01% ammonium chloride. The eluants were collected in a clean polypropylene test tube at a rate of approximately 1 mL/min. The eluants were dried under a stream of nitrogen in a ventilator at room temperature. The residue was reconstituted in 150 µL of acetonitrile/water (1:1, v/v) and filtered through a 0.2-µm syringe filter (Millex®-GN). Ten microliters of the reconstituted sample was injected into the HPLC. Animal experiments

The experimental protocols were approved by Shimane University Faculty of Medicine Animal Experimental Committee. A total of 14 male Sprague-Dawley rats (BW 350410 g, Charles River Breeding Labs, Yokohama, Japan) were used in this study. Rats were under anesthesia throughout the experimental procedures. A mix of droperidol 1.25 mg/mL and fentanyl 0.025 mg/mL was used as anesthetic. A dose of 2 mL/kg (droperidol 2.5 mg/kg and fentanyl 0.05 mg/kg) was initially administered intramuscularly and was given at 0.3–0.6 mL/kg thereafter as needed. Eight rats were decapitated under anesthesia, and blank blood was collected for the development of assay. All the samples were stored at –20°C until analysis.

collected from the femoral vein at 2, 5, 10, 30, 60, 120, and 180 min, respectively, and the same volume of saline was substituted each time for the blood loss. The volume of blood removed increased from 0.1 to 1.5 mL to allow quantification of lower blood CHX levels at later time points. Approximately 15–17% of the total volume of blood was taken (40). Rats were decapitated to collect trunk blood at 240 min. All the blood samples were stored at –20°C until analysis.

Results Chromatography

Standard chromatograms were recorded for both CHX hydrochloride and CHX gluconate, and no differences were observed for these two compounds. Because of that finding and the same component as the commercial product of CHX, CHX gluconate was used in the study later on as a standard compound. Figure 2 shows the typical chromatograms of standard CHX (A), extracts obtained from the Maskin solution (B), the blank rat blood (C), and rat blood spiked with standard CHX and IS solutions (D). CHX and IS were detected at 10 min and 20 min, respectively. No interfering peaks derived from biological materials appeared around the corresponding peaks in whole blood samples. Extraction

Liquid–liquid extraction (LLE) was initiated with blank rat whole blood based on the methods described by Kudo and Soskolne et al. (8,31). The interfering peaks derived from endogenous components were too large to record the CHX peak correctly. To avoid using a large volume of organic solvents, the SPE method was developed empirically depending on our previous experiences on cationic (35) and anionic surfactants (36). CHX is a dicationic base and should be expected to exist in an ionic state over a wide pH range (Figure

Intravenous infusion

Six rats were used for the investigation of kinetic properties of CHX. Each rat was implanted in the left femoral vein with a catheter for blood sampling. A dose of 15 mg/kg CHX (1.5 mL/kg as 20-fold diluted Maskin solution) (1% of CHX in the solution) was infused over 1 min via caudal vein. This dose (15 mg/kg) was based on the LD50 (21 mg/kg i.v. in rats) reported by Case (39) and a minimum lethal dose (data not shown). The blood samples of 0.1, 0.3, 0.5, 0.5, 0.8, 1.0, and 1.5 mL were

Figure 2. Typical chromatograms obtained from standard CHX and IS (A), extract of Maskin solution (B), extract of rat blank whole blood (C), and extract of rat blood spiked with standard CHX and IS (D). The retention times of CHX and IS were at 10 min and 20 min, respectively. The concentration of CHX added in A, B, or D was 0.5 µg/g.

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1). As such, a complex set of equilibria can govern its physiochemical behavior and affect matrix extraction efficiency. To increase the retention capacity of CHX on the C18 column, samples were fortified with sodium acetate buffer (0.1 M, pH 5.0) and this buffer was also selected for SPE conditioning instead of distilled water. The buffer solution could make residual silanol ionized to retain CHX on the SPE column. Chloroform, ethyl acetate, and a mixture of methanol/ethyl acetate in different ratios were tried as elution solvents. Good recoveries were obtained by using methanol/ethyl acetate (1:1, v/v) containing 0.01% ammonium chloride, which is evaporated easily as well. The recoveries of CHX in whole blood ranged from 72 to 85% calculated when the standard spiked in water was 100% (Table I). Six concentrations at the level of 0.05, 0.1, 0.5, 1.0, 1.5, and 2.0 µg/g, with six-time repeated tests, were determined. The precision for intra- and interday assays was determined at the concentrations of 0.05–2.0 µg/g in blood samples. The coefficients of variation for intra- and interday precision were less than 11% (n = 6) and 13% (n = 5), respectively (Table II). The linearity of CHX in whole blood was determined by plotting the peak-area ratio of CHX to IS on rat blood spiked with the standard solution (Figure 3). Good linearity was obtained at the concentration range of 0.05–2.0 µg/g in blood. The detection limit was determined empirically by running a series of standard samples extracted from spiked blank blood samples. The lowest level at which CHX could be detected was found at the concentration of 0.01 µg/g in blood when the signal-to-noise ratio was set at 3.

Kinetics following intravenous infusion

The time-course profile in the blood concentrations of CHX following a caudal vein infusion was shown in Figure 4. The blood concentrations of CHX were observed to fall rapidly after a dose of 15 mg/kg CHX within 30 min, from mean ± SD being 99.35 ± 26.17 µg/g at 2 min to 5.84 ± 2.51 µg/g at 30 min. The concentrations of CHX then declined much slower after 30 min, and Mean ± SD was 0.71 ± 0.21 µg/g at 240 min. After a single intravenous dose of 15 mg/kg, the kinetic profiles of CHX conformed to a two-compartment model. The kinetic parameters were calculated for each rat using Drug and Statistical Software (Version 2.0, Mathematical Pharmacology Professional Committee of China, Shanghai, China). The alpha half-life (of distribution) (t½α) and the beta half-life (of elimination) (t ½ β) evaluated from the curve of blood concentrations of CHX until 240 min were 0.05 h and 0.55 h, respectively, in caudal vein infusion in rats (Table III).

Table I. Percent Recoveries of CHX in Whole Blood (n = 6) Relative to Water Using the Same Extraction Concentration of CHX (µg/g) 0.05 0.1 0.5 1.0 1.5 2.0

Mean ± SD (%)

Figure 3. A linear relationship of CHX in blood with triplicate tests. The peak-area ratio of CHX to IS was plotted at the concentration (µg/g) of rat blood spiked with the standard as y- and x-axis, independently.

85.14 ± 6.58 83.82 ± 1.87 73.45 ± 0.42 74.19 ± 4.11 76.48 ± 2.58 71.68 ± 2.85

Table II. Intra- and Interday Precision: Mean ± Standard Deviation (Coefficient of Variation in Percent) for CHX Determination in Blood Concentration of CHX (µg/g)

Intraday (n = 6)

Interday (n = 5)

0.05 0.1 0.5 1.0 1.5 2.0

0.0503 ± 0.0057 (11.31) 0.0868 ± 0.0078 (8.97) 0.4909 ± 0.0141 (2.87) 1.0526 ± 0.0407 (3.85) 1.5026 ± 0.0053 (0.35) 1.9606 ± 0.0736 (3.75)

0.0508 ± 0.0065 (12.79) 0.0902 ± 0.0086 (9.53) 0.5014 ± 0.0179 (3.57) 1.0273 ± 0.0042 (4.09) 1.5102 ± 0.0250 (1.66) 2.0211 ± 0.0281 (1.39)

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Figure 4. The time-course changes in blood CHX concentrations following caudal vein administration at the dose of 15 mg/kg. Data express mean ± SD (n = 6). The kinetic profiles of CHX conformed to a two-compartment model with an alpha half-life (of distribution) at 0.05 h and the beta half-life (of elimination) at 0.55 h in rats.

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Discussion CHX is a cationic biguanide compound that has been in use as an antiseptic since the early 1950s. It is widely available for disinfection of skin, mucous membranes, and medical instruments. Maskin is a popular brand of disinfectant containing CHX and is in common use in Japan. It has been ubiquitous in hospitals and the community for over four decades. Accidental ingestion or suicidal injection seems to have occurred more frequently than previously reported (5–11). Although CHX has been considered ordinarily to have low toxicity because of its poor absorption via oral or dermal administration, its high toxic effects when given parenterally are not well characterized. The bioavailability of CHX via oral or dermal administration can be estimated to be quite low because of its ionic properties. However a high risk of CHX injection should not be overlooked because its high bioavailability can be reached by the direct injection. A marked difference of CHX in the LD50 between oral (> 3000 mg/kg in rat) and intravenous (21 mg/kg in rat) administration (39) also implies a potential risk of CHX injection. A fatal case caused by accidental ingestion of approximately 200 mL of Maskin (5% CHX) was reported (11). The patient developed hypotension and rapid deterioration of consciousness, and died of acute respiratory distress syndrome (ARDS). The CHX was suspected to absorb through the pulmonary alveoli following aspiration, not from the gastrointestinal tract. Another case also developed ARDS by unintentional injection of CHX (7). The determination of CHX in whole blood samples by HPLC and the systemic kinetic characteristics of CHX have not been reported to our knowledge. The aim of this study was to establish a simple and sensitive assay to determine CHX in blood samples and to investigate the kinetic properties of CHX in rats following intravenous administration. We first tried to extract CHX in rat whole blood using the method reported by Kudo et al. (8). The result showed that the interfering peaks were very large and totally covered the CHX peak. The method was modified according to other publications (31,33), the peaks of CHX and IS were observed, but were still partly covered by interferences. Because the LLE method had disadvantages such as time consumption, poor recovery, and use of large quantities of flammable and toxic solvents, the SPE method was employed in this study. CHX is a strong basic Table III. Kinetic Parameters in the Rats That Were Administered 15 mg/kg of CHX Via Caudal Vein* Parameter

Mean ± SD

AUC0~4 h (µg/mL.h) t½α (h) t½β (h) Clt (mL/h/kg) Vc (mL/kg)

22.9 ± 8.4 0.05 ± 0.02 0.55 ± 0.58 588.6 ± 153.7 103.2 ± 30.6

* The kinetic parameters were estimated by a two-compartment model analysis. AUC0~4 h: area under blood CHX concentrations during initial 4 h; t½α: alpha half-life or half-life of distribution; t½β: beta half-life or half-life of elimination; Clt: total body clearance; and Vc: volume of central compartment.

dicationic compound with pKa values of 10.3 and 2.2 (8); we initially chose strong cation exchange over the LLE procedures. Unfortunately, the complex ionic behavior of CHX made it difficult to select an appropriate elution solvent for consistent and quantitative extraction from the ion-exchange resin bed. By diluting the blood sample and conditioning the C18 SPE column with an acetate buffer at pH 5.0, CHX could be retained on a C 18 sorbent and the sample could be cleaned up sufficiently. A series of elution solvents including chloroform, ethyl acetate, and a mixture of methanol/ethyl acetate in different ratios were tested and the methanol/ethyl acetate (1:1, v/v) containing 0.01% ammonium chloride was found to be a suitable solvent for CHX eluted from C18 SPE column. HPLC separation was thus achieved by using a polymer-coated silica-based column under the conditions based on the method reported by Kudo et al. (8). Good recoveries and linearity were obtained in the concentrations of CHX in whole blood range from 0.05 to 2.0 µg/g. The method was simple, sensitive, and accurate for the determination of CHX in blood samples. There has been no report concerning the toxicokinetics of CHX following intravenous infusion. In this study, the timeconcentration profile of CHX in blood following a caudal vein infusion until 240 min was investigated using the method described here. The kinetic profiles of CHX conformed to a twocompartment model with an alpha half-life (of distribution) at 0.05 h and the beta half-life (of elimination) at 0.55 h in rats administered intravenously. At the toxic level of 15 mg/kg, the rats stopped breathing for 20–60 s immediately after the infusion, but recovered soon. However, five rats died of dyspnea during or soon after the infusion via jugular vein in our preliminary experiment. Two of six rats developed hematuria during the kinetic experiment. Ascites was observed in autopsy at the end time point of 240 min. Blood concentrations after CHX poisoning were reported by at least two groups of investigators (8,9). An accidental injection of the 20% CHX (10 mL) into a patient had a concentration of CHX at 39.5 µg/mL in serum. A suicidal injection of unknown amount of CHX had a high concentration in hemolyzed blood at 352 µg/mL which was determined by the LC–ESI-MS method. Blood concentrations in rats were very high immediately after infusion and declined quickly with the lapse of time. These results are corresponding to our previous studies on the cationic disinfectant (37,38). However, the systemic toxic effects and tissue distribution were not addressed in this study. A further investigation is needed to evaluate the toxicity and distribution of CHX parenteral administration.

Conclusions A simple method for the quantification of CHX in whole blood has been developed using SPE with HPLC separation. The sample was fortified with an acetate buffer and purified by SPE cartridges. The determination was not disturbed by endogenous substances from the whole blood. The toxicokinetic characteristics of CHX following an intravenous infusion were investigated using the present method. The

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determination is sensitive and reliable for the measurement of CHX in blood samples with a lower detection limit of 0.01 µg/g and could be expected to apply to the forensic and clinical specimens.

Acknowledgements The author Yuying Xue received a Postdoctoral Fellowship for Foreign Researchers (ID No. P 05226) awarded by Japan Society for the Promotion of Science (JSPS). This work was supported by JSPS under Grant-in-Aid for Scientific Research (No. 17·05226) and also supported by National Basic Research Program of China (No. 2006CB705602), National Natural Science Foundation of China (No. 30671782).

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Manuscript received June 27, 2008; revision received August 5, 2008.

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