Development and Validation of an RP-HPLC Method for the Quantitative Analysis of Triclosan in Human Urine

as an antimicrobial agent. TCS has sparked widespread awareness because of its toxicity and possible negative efect on public health in recent years. In this study, a highly sensitive, fast, and cost-efective isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method coupled with solid-phase extraction for analysis of triclosan in human urine samples was developed. Te method utilized methanol and water in a ratio of 90:10 as the mobile phase on a Phenomenex Luna 3 µ m C18(2) 100 ˚A, 150 × 4.60mm stationary phase, with a runtime of 5minutes. Te method showed good resolution of triclosan in the presence of the sample matrix. Validation of the method was performed according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). Linearity was tested over a range of 0.00625 µ g/mL to 6.4 µ g/mL, as accuracy recorded a recovery of 89.25%, 91.0%, and 92.75%. Limits of detection (LOD) and quantifcation (LOQ) were obtained to be 0.0173 µ g/mL and 0.0525 µ g/ mL, respectively. Te method proved to be robust over a temperature range of 26 ° C, 30 ° C, and 35 ° C and a fow rate of 0.5ml, 1.0ml, and 1.5ml. Te developed method was employed to detect and quantify triclosan in 153 urine samples, comprising 60 samples from Ibadan, Nigeria, and 93 samples from Kumasi, Ghana. Triclosan was detected in a total of 52 samples with an average content of 0.054588 µ g/ml. Tis method can therefore be used for the routine analysis of triclosan in urine samples.


Introduction
Triclosan (TCS), 5-chloro-2-(2,4-dichlorophenoxy) phenol (Figure 1), is a synthesized chlorinated phenolic substance discovered in the 1960s.It is an aromatic ether which possesses two functional groups, that is, the phenol and ether functional group.It has served as a preservative and antimicrobial agent over the years.TCS is found in most consumable products such as hand sanitizers, soaps, toothpaste, and mouth rinses, due to its broad antimicrobial spectrum [1].Triclosan has a molecular formula of C 12 H 7 Cl 3 O 2 , the molar mass of 289.54 g/mol, melting and boiling point of 55-57 °C and 120 °C, respectively, and a density of 1.45 g/ml.Te standard permissible limit of triclosan in consumable products is 0.01 mg/L to 0.1 mg/L [2].
Human exposure to TCS may be through the mouth or through the skin.TCS is readily absorbed through the skin, mucous membrane of the mouth and gastrointestinal lining due to its lipophilic properties [3].Mammals as the subject of concern metabolize TCS predominantly through conjugation processes prior to hydroxylation, which result in glucuronide and sulphate conjugates that are eliminated in the faeces, urine, and breast milk [4][5][6].At the ecologically relevant doses of 1 to 5 micromolar, triclosan sulphate, and glucuronide are produced in the liver at almost comparable rates, according to pharmacokinetic studies.Sulfonation is likely to be the main metabolic pathway for triclosan removal when concentrations are less than 1 micromolar [2].
An average of 50% of free TCS is excreted in 24 hours with urine being the principal route of excretion [5].In 2003 and 2004, a detection of 75% of TCS in urine sample was recorded according to Weatherly, among the United States of America's population with a concentration range of 7.9 nM to 13.1 µM [6].
Triclosan, in spite of its broad-spectrum antimicrobial property may impose an adverse efect on human health.Although triclosan is for external use, it still fnds its way into human body through the skin.Tis makes triclosan a public health concern as there could be a potential bioaccumulation of this life-threatening compound.Triclosan can alter hormone regulation, serving as an endocrine disruptor, and contributing to antibiotic resistance [7,8].Research by Savage et al. [9] also shows that triclosan is capable of precipitating asthmatic attack in kids.Studies have shown triclosan worsened fatty liver disease in mice.Elevated levels of triclosan in urine have been linked to immunological malfunction, allergic responses, and the development of asthma [10].TCS has been demonstrated to bind to human serum albumin as well, causing the protein structure to be altered [11].Toxins bonding to serum albumin can obstruct endogenous chemical transport and create structural changes in the protein complex, which can impair activity or modify its physiological function [12,13].
In view of the permissible limit of (0.01 mg/L to 0.1 mg/L) of triclosan [2] and its negative efects on humans, many governmental agencies and organizations have raised concerns on the role of various foods and drugs regulatory authorities in their country.Various analytical methods for the analysis of triclosan in environmental, consumable products, and human samples have been developed over the years including enzyme-linked immunosorbent assay (ELISA) [14,15], cuvette-less microvolume Ultraviolet/visible spectroscopy (UV/Vis) [16], gas chromatography coupled with mass spectrometry (GC-MS) [17,18], and high-performance liquid chromatography (HPLC) [19][20][21].However, most of these methods target TCS in environmental samples or consumable products and not human urine samples.
Te current study, therefore, seeks to develop and validate a reliable, cost-efective reversed-phase highperformance liquid chromatographic method coupled with solid-phase extraction (SPE) to analyze triclosan in urine samples.Te urine samples were obtained from children aged between 6 and 14 years attending Asthma Clinics at University College Hospital, Ibadan, Nigeria, and Komfo Anokye Teaching Hospital (KATH), Kumasi, Ghana.

Chemicals and Reagents.
Triclosan reference standard was purchased from Sigma-Aldrich with lot number of BCBW3642.HPLC grade methanol was obtained from VWR International (Chromanorm), and trifuoroacetic acid was obtained from Wagtech Projects Ltd. (Table 1).Distilled water was obtained from KNUST Central Laboratory.

Equipment.
Equipment used in this study included the Stuart SMP10 Melting Point Apparatus for the determination of melting point.Analytical balance (Sartorius TE214S) for accurate weighing of chemicals.UV/Vis Spectrometer and Infrared Spectrometer (Bruker) were used for the identifcation test of reference standard used.An HPLC Chromatographic instrument with the specifcation of (Perkin Elmer 785A Flexar PDA detector) having a Phenomenex Luna 3 µm C18(2) 100 Å, 150 × 4.60 mm column was used.Te HPLC possesses a binary mixing Flexar LC pump and Flexar LC Autosampler, making use of Chromera software and Flexar LC Solvent Manager.

Identifcation Test for Triclosan Standard.
Te following identifcation tests were performed to ascertain the integrity of the reference standard obtained (triclosan): infrared spectroscopy, UV-Vis spectrophotometry, and melting point determination.

RP-HPLC Method Development.
Based on the preliminary studies conducted, the isocratic RP-HPLC chromatographic mode was used for eluting triclosan.Te mobile phase selected (90 : 10, methanol: water) was pumped through the Phenomenex Luna 3 µm C18(2) 100 Å, 150 × 4.60 mm column at a fow rate of 1.0 ml/min with the 2 Journal of Chemistry column kept at an ambient temperature.Equilibration of the column was performed by running the mobile phase through it for 30 minutes preceding injection of triclosan.Te detection of triclosan by photodiode array (PDA) was monitored at 280 nm.Te working solution of 50 μg/ml was injected at a fow rate of 1 ml/min, and triclosan was eluted at a mean retention time of 2.863 minutes.Te chromatogram recorded showed good resolution and a sharp peak.Te procedure was repeated three consecutive times and the results were found to be reproducible.

ICH Guided Method Validation
(1) Linearity.To ascertain the relative relationship of the obtained response against analyte concentration, the linearity of the method was tested over a range of calibration points using the calibration point solution prepared.Te solutions, whose concentration ranged from (0.00625-6.4)µg/ml, were analyzed in triplicate per concentration with blank analysis between them using the developed method.Te results from the analysis were recorded, and a calibration curve of response versus concentration was plotted using a GraphPad prism.
(2) Limit of Detection and Limit of Quantifcation.Using the statistically approved formulae, LOD and LOQ values were calculated using the following equations: where σ � the standard deviation of the y-intercept of the calibration curve and S means slope of the calibration line [22,23]. (

3) Intraday Precision (Repeatability).
A nominal concentration of 1.6 µg/ml of triclosan was injected 9 times at a 15minute interval.Te peak area and relative standard deviation were recorded to ascertain intraday precision.
(4) Interday Precision (Intermediate Precision).In four consecutive days, using a nominal concentration of 1.6 µg/ml of triclosan, the interday precision was evaluated by analyzing the solution in triplicate.Te peak areas were recorded, and relative standard deviation (RSD) values were calculated.
(5) Accuracy.Te accuracy of the developed method was evaluated by a spiking blank urine sample with three different concentration triclosan reference standards corresponding to 80%, 100%, and 120% of the target concentration of 0.32 μg/mL, 0.40 μg/mL, and 0.48 μg/mL.
Te obtained solutions were analyzed in triplicate, and the percentage recoveries and relative standard deviation of their responses were calculated according to ICH guidelines.
(6) Suitability.Te parameters such as the limit of detection, the limit of quantifcation, correlation coefcient, and the concentration range were all determined to ascertain that the chosen method is specifc and selective.
(7) Selectivity/Specifcity. Te specifcity of the developed method was evaluated by comparison of chromatograms of triclosan standard and triclosan in the urine sample.It was noticed that there was no interference and that there was a good correlation existing between the retention times of the standard and those of the sample.Using a concentration 0.8 µg/mL, the fow rate of the mobile phase solution was varied between 0.5 min, 1.0 min, and 1.5 min.At each of the stated fow rates, the sample was analyzed in triplicate to assess its robustness.Te area under the peaks was recorded and the relative standard deviation was calculated.

Solid Phase Extraction (SPE).
To obtain better results after preliminary studies, modifcation of the SPE protocol on the SPE cartridge purchased (strata X 33 µm polymeric sorbent) was used.Te SPE catridges were conditioned with 3 ml of HPLC grade methanol; the cartridge was then equilibrated with 3 ml of distilled water to wet the SPE surface.Two millilitres of the sample were then loaded onto the SPE cartridge, which was then washed with 6 ml of 50 : 50 distilled water and methanol in a series of 3 ml.Te cartridges were then dried for 10 minutes following elution with 4 ml of HPLC grade methanol in a series of 2 ml.Te eluate was collected and evaporated over nitrogen gas till the fnal volume reached 2 ml.Te obtained eluate was then analyzed using the developed method.
(1) SPE Recovery.To ascertain the performance of the developed SPE method, SPE recovery was performed by frst analyzing 0.2 µg/ml of standard TCS in triplicate using the developed RP-HPLC method.Te area under the peaks was recorded.Two millilitres of three sets of the same urine Journal of Chemistry sample was prespiked with 0.2 µg/ml TCS each.Te samples were then run following the SPE method developed.Te eluates were then analyzed with the RP-HPLC method and the area under the peaks was recorded, the mean area was then computed.Te percentage recovery was then calculated using the following formula:

Sample Preparation and Analysis.
Te urine samples were placed in an ultrasonic bath and sonicated for 15 min at a frequency of 35 kHz with the temperature function turned of.Te samples were fltered using a 0.45 µm nylon membrane flter into a 5 ml falcon tube.Te fltered samples were then run through the developed solid-phase extraction method for purifcation and to concentrate the analyte of interest.Te pH of the samples was adjusted to pH 4.00 using trifuoroacetic acid (TFA).Te HPLC method developed was used to quantitatively determine amount of triclosan present in the human urine sample in triplicate.Te response of each sample was recorded and GraphPad Prism, Excel, and Minitab, statistical software were used to analyze the data.

Identifcation Test for Triclosan Standard.
To ascertain the authenticity and integrity of the obtained triclosan reference standard, infrared absorption spectroscopic and ultraviolet-visible spectrophotometric analyses of triclosan were performed according to United Stated Pharmacopoeia 2017 (197E and 197U, respectively) specifcation, as well as melting point determination.Te wavelength of maximum absorbance was obtained from the UV spectrum to be λ max � 281 nm in methanol, which falls within the USPspecifed wavelength range of 280-281 nm [24].Melting point was determined in the range of 55-57 °C [25].Te results of the identifcation tests are as shown in Table 2, Figure 2, and S1-S2.3 and 4).Comparing this chromatographic condition to [28], who recorded a retention time of 5.35 minutes for HPLC analysis of triclosan in cream and spray.Tohidi and Cai [17] had a retention time of 17.22 min for GC-MS analysis of triclosan.Aminu et al. [29] also obtained a retention time of 12.47 min for HPLC analysis of triclosan in stimulated saliva.Te retention times, as well as the total run time for these studies, are relatively longer in comparison to this study, making this method relatively faster.

HPLC Method Validation.
To evaluate the overall performance of a developed method, the ICH method validation protocol plays an important role.Te developed method was validated to test its integrity and applicability (Table 3).Te linearity of the method was tested within a calibration range of 0.00625 µg/mL (Figure S3) to 6.4 µg/ mL for triclosan.An r 2 value of 0.9999 was obtained which indicated good linearity (Figure 5).Table S4 shows the Te precision of the developed method was evaluated by both intraday and interday precision.Te intraday precision gave a percentage relative standard deviation (%RSD) of 1.723, while the interday precision was 0.106, 1.489, 0.071, and 1.326 for four diferent days.Based on the results obtained, it can be concluded that the proposed method is precise as all the results fall within the ICH acceptable limit of ≤ ±2.0%.
Te accuracy of the developed method was evaluated by spiking a blank urine sample with three diferent concentrations of triclosan reference standard, corresponding to 80%, 100%, and 120% of the target concentration of 0.32 μg/mL, 0.40 μg/mL, and 0.48 μg/mL.A recovery of 91.00% was obtained for 0.32 µg/mL, 92.75% was obtained for 0.40 µg/mL, and 89.25% was obtained for 0.48 µg/mL.Tey all fall within the ICH acceptable recovery values of 80-120%, indicating the method was accurate.Specifcity or selectivity of the method was justifed as there was no signifcant interference from the constituent of the urine sample in any peak region, demonstrating that the method is selective.

Journal of Chemistry
A purposeful change in temperature of the column at 26 °C, 30 °C, and 35 °C, as well as fow rate, did not have any signifcant efect on the result.Giving an indication that the method is robust.For sensitivity evaluation of the analytical method, the limit of detection (LOD) and limit of quantifcation (LOQ) as validation parameters are key.Te LOD and LOQ of this proposed method were calculated to be µg/mL and 0.0525 µg/mL, respectively.

SPE Method Development and Validation.
A solid-phase extraction method was developed to concentrate the analyte of interest and for purifcation.Te method made use of strata X 33 µm polymeric sorbent SPE cartridge which is a surface-modifed styrene divinylbenzene.Due to its balance between lipophilic and hydrophilic retention characteristics, it enhances the retention of neutral, acidic, or basic aromatic compounds.Moreover, urine samples usually contain a lot of highly polar metabolites, and these can easily be washed of on such columns.Triclosan molecules, which pose lipophilic and hydrophilic moieties, and are therefore classifed as moderately polar, can therefore be selectively retained whiles getting rid of the several highly polar metabolites in the urine matrix.A double elution with concentrated methanol as mobile phase was employed as it gave better recovery.Although acetonitrile, n-hexane, MeOH-DCM, or acetone could have been used, as used by [17,30], keeping the method simple, less costly, and less timeconsuming was what informed the choice.Te SPE recovery process was undertaken and a recovery of 91.02% was obtained, which represents a good recovery (Table 4).(3)

Determination of Triclosan in Urine
Samples.Te developed and validated method was used to assay 153 urine samples (Figure 6), among which triclosan was detected and quantifed in 52 samples, whereas the remaining 101 were below the limit of detection.Te least detected concentration of free triclosan was 0.0246 µg/mL, while the maximum was 0.1595 µg/mL, giving a range of (0.0246-0.1595) µg/ml.Te mean concentration of TCS among the detected samples was obtained to be 0.054588 µg/ml (Table 5).Tis data suggests a signifcant number of people in the study area are exposed to triclosan.

Conclusion
A highly sensitive, fast, cost-efective, and accurate isocratic reversed phase high-performance liquid chromatography (RP-HPLC) and solid-phase extraction (SPE) methods were developed for the analysis of triclosan in human urine samples.Te HPLC method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines.Te RP-HPLC method was shown to be linear, precise, accurate, and robust.Te developed method was used to assay 153 urine samples, among which triclosan was detected and quantifed in 52 samples with 101 were below the limit of detection.Te concentration of detected samples was within a range of 0.0246 µg/mL-0.1595µg/mL with a mean concentration of 0.054588 µg/ml.

( 8 )
Robustness (a) Temperature variationTo validate robustness, the temperature of the column was varied between temperatures of 26, 30, and 35 °C for the assay of triclosan in triplicate per sample.Te areas of the peaks were recorded, and relative standard deviations were calculated.(b) Flow rate variation

Figure 6 :
Figure 6: Chromatogram of one of the urine samples from the patient (with code 02GO25), showing triclosan at a concentration of 0.03 µg/ mL eluting at a retention time of 2.81 min.
) 2.3.5.Sample Collection.Urine samples were obtained following parental consent and subject assent from 153 children aged 6-14 years attending Asthma Clinics in Ghana (Komfo Anokye Teaching Hospital, Kumasi, n � 93) and Nigeria (University College Hospital, Ibadan, Nigeria, n � 60) as part of a study to investigate the possible efect of triclosan exposure on asthma symptoms.Ethical clearance was obtained from the Committee on Human Research, Publication, and Ethics, School of Medicine and Dentistry (KNUST, CHRPE/AP/070/20), and the University of Ibadan/University College Hospital, Ibadan Joint Ethics Committee, respectively.Te children had no other comorbidities apart from asthma.Urine collection was done under supervision of hospital staf.Each sample was collected into a tight-sealing, amber-coloured glass container, labelled, and stored in a refrigerator at −20 °C until they were used.Samples from Nigeria were shipped by air securely in a frozen state from Ibadan to Kumasi.

Table 5 :
RP-HPLC representative Results for TCS determination in urine samples.