Iron (II) Determination in Lipstick Samples using Spectrophotometric and Microfluidic Paper-based Analytical Device (µPADs) Platform via Complexation Reaction with Iron Chelator 1, 10-phenanthroline: A Comparative Study

: This study was undertaken to introduce a fast, accurate, selective, simple and environment-friendly colorimetric method to determine iron (II) concentration in different lipstick brands imported or manufactured locally in Baghdad, Iraq. The samples were collected from 500-Iraqi dinars stores to establish routine tests using the spectrophotometric method and compared with a new microfluidic paper-based analytical device (µPAD) platform as an alternative to cost-effective conventional instrumentation such as Atomic Absorption Spectroscopy (AAS). This method depends on the reaction between iron (II) with iron(II) selective chelator 1, 10-phenanthroline(phen) in the presence of reducing agent hydroxylamine (HOA) and sodium acetate (NaOAc) buffer to yield a reddish/orange colour change proportional to the iron(II) concentration measured at λ max = 510 nm. Under optimum conditions, the calibration curve was linear in the range between (0.5-150) mg L -1 with a limit of detection of 0.09 mg L -1 . Compared to a spectrophotometric detection method, µPAD measured colour intensity using captured images using Samsung mobile phone and image J program to give proof of concept that µPAD platform fulfils the purpose of accuracy and at the same time remaining cost-effective and simplistic to be used in both developing and developed countries gave same linear calibration curve with a limit of detection 0.12 mg L -1 . ANOVA test was used to compare the proposed method results with conventional method results showing the method was accepted. The antimicrobial activity showed no significant effect from lipstick samples on tested microbes.

health awareness and consumers wellbeing. Women ingest lipstick due to eating and/or drinking, kissing, and licking their lips. Therefore it is said, "women without intention eat about 4 lb of lipsticks in a lifetime" 9 . Oral ingestion of lipstick is considered as the significant route for the bioaccumulation of heavy metals particularly hazardous elements such as iron in the human body as lipstick products are directly applied to the mouth in addition to hand-to-mouth contact and systemically transported to vital internal organs exhibiting their toxicities 10 . The mechanism of toxic metal in the human body is attributed to the absorption and formation of complexes between them and proteins amines (-NH 2 ), carboxylic acids (-COOH), and thiols (-SH) 11,12 , results in malfunctioning, damage or death of the cell and consequently leads to a variety of diseases 13 .
Iron is an essential micronutrient utilized in almost every aspect of a living organism particularly crucial for the conservation of energy. Nevertheless, iron concentration in body tissue must be monitor and regulated as iron forms free radicals which in excessive amounts can cause tissue damage 14 . Also, a broad spectrum of clinical manifestations are associated with iron overwhelms in cells that lead to the increase of iron toxicity including neurodegenerative diseases, anaemia, diabetes and cardiovascular disease 15,16 . Extensive screening of literature was carried out on the analysis of iron levels in different lipstick and a variety of analytical techniques have been published including Atomic Absorption Spectrometry (AAS) [17][18][19][20][21][22] , and Inductivity Coupled Plasma-Optical Emission Spectrophotometer(ICP-OES) 9,23 .
However, inherited problems related to cost-effective, the need for trained people, and laborious procedures have driven researchers to develop simple, portable, and easy to use detection methods and colorimetric detection is one of the most reliable and promising approaches that can be a good alternative to conventional instrumentation [24][25][26][27][28] .
The increasing interest in cosmetics and the lack of a simple and sensitive method for the determination of hazard concentration of iron (II) in cheaply priced lipsticks that are sold in the open market and imported from countries or manufacture locally where there is a lack of regulatory conditions necessitated in this study. In this context, the method emphasizes that iron (II) reaction with phen to form Fe-Phen complex that can be compatible with the colorimetric technique including spectrophotometric and µPADs platform methods considered as a good alternative to conventional techniques such as atomic absorption spectroscopy permitting independent evaluation of toxic metal (iron) in cosmetic (lipstick), these techniques allow fast and accurate method which is in agreement with green chemistry.

Materials and Methods: Sample Collection and treatment
All different brands of lipsticks were purchased from different 500 ID cosmetic stores in Baghdad the capital of Iraq with unknown origins which sold cheap items suitable for poor and middle-class Iraqi society. The samples were coded as Lip1 (orange), Lip 2 (brown), Lip 3 (coral), Lip 4 (green), Lip 5 (maroon), Lip 6 (golden yellow), Lip 7 (lilac), and Lip 8 (beige). The samples were acid digested for elemental determination in lipstick following the Nnorom et al procedure 18 . A weight of 0.2 g of cosmetic sample (lipstick) was placed into a porcelain crucible and allowed to be heated on a hotplate (Ijlassco, India) near dryness in a fume hood until no fumes were observed(an indication of digestion process end). The content was left to cool to room temperature then 1 mL of concentrated Nitric acid (purity 69.5%, BDH, England) was added. After digestion, the digested sample was filtered through Whatman No. 40 filter paper to remove undigested materials such as glitters, wax, etc. After mineralization, the clear residue was transferred quantitatively into a 10 mL volumetric flask, diluted with deionized water and stored for further experiments. mL volumetric flask, diluted to mark with deionized water and mixed thoroughly, this solution was prepared daily.

Spectrophotometric Calibration Curve
To construct the calibration graph; 1 mL of HOA(reducing agent) 0.2 M was added to an increasing volume of iron (III) standard solution 400 mg L -1 into a series of 10 mL volumetric flask covering the range between (0.5-150) mg L -1 . Afterwards, 1 mL of phen (ligand) 0.04 M and 0.2 mL of 0.1 M NaOAc (buffer) were added and completed to the mark with deionized water. The resulted reddish/orange chromogenic product was measurable at 510 nm versus reagent blank (prepared under the same conditions without iron metal addition) after 10 minutes. The calibration curve was constructed from plotting standards concentrations versus absorbance reported as triplicate measurements to ensure precision and accuracy. The accuracy of the procedure was determined by measuring the recovery of metal added to lipstick matrix owing to the unavailability of certified material 29 . Uv-vis spectrophotometer (Shimadzu, Kyoto-Japan) digital double-beam was used equipped with a quartz cell of 1 cm for λmax determination and all absorbance measurements.

Microfluidic paper-based analytical device (µPAD) Calibration Curve
The chromogenic product calibration curve was formed on a μPADs device fabricated according to Peter et al method 30 . Initially, by dropping 7 µL of HOA 0.2 M, and 7 µL of iron solution series working solution prepared from standard iron solution (400 mg L -1 ) into the detection zone using Eppendorf micropipette, allow drying at room temperature (~ 25° C). Afterwards, 7 μL of phen 0.04 M and 3 μL of 0.1 M NaOAc buffer were pipetted into each detection zone (eight separated spots on each chip) producing a coloured product. For additional characterization, images were taken using a Samsung note 9 camera and analysed using Image J freeware (National Institute of Health, USA).

Standard Addition Calibration Graph for Lipsticks Samples
The standard addition method was chosen to determine iron(II) concentration in both spectrophotometric and μPADs methods as in this method the influence of the interference can be eliminated 31 . This method was done by the addition of 1 mL of HOA 0.2M, increment concentration (0, 10, 60, and 100) mg L -1 of iron standard solution to 1mL of the solution of prepared samples. Then 1 mL of phen 0.04 M and 0.2 mL of 0.1 M NaOAc buffer were transferred into four volumetric flasks containing the above concentration of iron and sample. The mixture was allowed to react for 10 minutes and measured using spectrophotometric. For μPADs method, a similar method was undertaken using optimum conditions 7 μL of the phen 0.04 M, and 3 μL of 0.1 M NaOAc buffer which was added to detection zone containing 7 µL of HOA 0.2 M, increment concentration (0, 10, 60, 100) mg L -1 of iron(ІІІ) standard solution and 5 µL of lipstick sample.

In Vitro Antimicrobial Activity Assay
To investigate the antimicrobial of lipsticks from microbial contamination, a range of different gram-positive and gram-negative bacteria was selected in addition to fungi. The isolates included (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, E.coli, Klebsiella sp., Pseudomonas aeruginosa and Candida albicans) isolate. The selection of S. aureus and S. epidermidis bacteria depended on the basis that these bacteria are naturally a skin habitat. Additionally, they also can cause skin infections with Strep. pyogenes, and P. aeruginosa. The microbial activity of 8 different lipsticks was determined by the agar well diffusion method described by Parekh and Chanda 32 . In this method, a pure isolate of 24hrs growth was cultured in Muller-Hinton Agar plate (HiMedia, Mumbai, India) by using a sterile swab to achieve a confluent growth. The plates were allowed to dry and a sterile cork borer of diameter 5.0 mm was used to bore one well in each agar plate. Each well was filled with lipstick suspension (volume 100 μl) from each sample by micropipette into Muller-Hinton Agar plate. The plates were allowed to stand for 1hr or more to allow pre-diffusion to take place and then incubated at 37ºC for 24hrs. The zone of inhibition was recorded in millimetres. Each experiment was performed in a duplicate. Results and Discussion:

Method A (Determination of Iron via Conventional Spectrophotometer Technique)
The absorption spectra of the coloured complex formed by complexion reaction between iron metal 80 mg L -1 and phen solution 0.04 M in the presence of HOA 0.2 M and 0.2mL of 0.1 M NaOAc buffer were recorded via spectrophotometer, as the complex turned from colourless to reddish/orange complex after 10 minutes with maximum absorbance at 510 nm. The absorbance was proportional to the concentration of iron that exists in the lipstick sample as can be seen in Fig. 1.

Figure 1. (I) represent the absorption spectra of (a) phen versus deionized water, (b) iron (III) versus deionized water and (c) 80 mg L -1 iron (II) complicated with phen against reagent blank. (II) a photograph of iron chelator phen complex and blank coloured reaction.
Based on literature; the reaction between ferrous ion and phen is highly selective and sensitive to ferrous to form a reddish/orange complex 33 . The mechanism is based on the reduction of ferric to ferrous carried out by a reducing agent such as hydroxylamine, followed by metal complexion with phen which is considered as a selective ligand to iron metal through the implication of three nitrogen groups with iron (II) 34 . The proposed mechanism is shown in scheme 1. Scheme 1. Illustrate the mechanism of iron (II) metal with 1,10-phenanthroline reaction to form the coloured complex 35 .
The present study was undertaken to determine the iron concentration in lipstick samples by conducting batch experiments at the variation that influence the absorption intensity of the coloured product including reagent phen volume, HOA volume, pH, order of addition, and reaction time. All experiments were measured in triplicate and the obtained results were corresponding to the average value. Optimization was carried out by changing one parameter and keeping the other fixed and measured at 510 nm versus reagent blank and so on. The Effect of 1,10-phenanthroline Volume: The influence of phen volume on the absorption of the reddish/orange product was studied using different volumes ranged from (0.5 -2.5) mL. The results presented in Fig. 2a reveal that the absorbance increased with the increase of phen 0.04M volume up to 1 mL, then there was a decrease in absorbance. Therefore, 1 mL of the phen ligand was used in all consequent experiments. The Effect of HOA Volume: A series of HOA 0.2M volumes range of (0.5 -2.5) mL was examined. It was observed in Fig. 2b that 1 mL of HOA was enough to obtain a maximum colour intensity absorbance as there was a drop in absorbance with increasing volume. As a result, it was used in all further experiments. The Effect of pH: The pH solution plays a substantial role in the formation of the metal complex with phen and to avoid error in the analysis, NaOAc buffer solution (pH =5) was added to control the solution pH since the complex formed is very sensitive to the change in pH. Thus, the effect of pH was studied in the range of 3 to 8 using 359 different pH NaOAc buffer solutions. The result is shown in Fig. 2c reveals that the absorbance increased with pH increasing and reached the maximum at pH 5.0 for iron complexes this can be attributed to low hydrogen-ion activity, which avoids the nitrogen atoms protonation leading to strong bond formation. Afterwards, the absorbance gradually decreased because a higher pH deters iron ions into iron hydroxyl species that alter the complexion between Iron metal and phen 36,37 . The Effect of Order Addition: The development of complex colour is highly affected by the order of reagents addition. Diverse types of addition were carried out (Metal (M), phen (L), HOA (B), NaOAc buffer (AB)) as shown in Fig. 2d. Results show the addition of base, metal, reagent and acetate buffer was recommended as the absorbance reaches 1.00, which can be attributed to the fact that the base reduces ferric to ferrous and later ferrous reacts with the ligand to form a reddish /orange complex product and thus was followed in the subsequent experiments since it resulted in obtaining maximum absorbance. The Effect of Reaction Time: The optimum reaction time is a fundamental parameter for the iron determination procedure. Therefore, it is essential to study the influence of time over the time interval from (0-35) minutes. Figure 2e shows that there was an initial increase in absorbance with time up to 10 minutes. As a result, in subsequent experiments, 10 minutes was considered more than sufficient for complex colour development at room temperature and the complex was stable at least for one week in solution.

Method B (Determination of iron via μPADs Method)
Investigations were carried out using the μPADs method platform once the spectrophotometric method was established and accurate results were obtained. The initial concept is to prove that the same reproducible results can be achieved using both methods via complex colour change using iron (II) via phen reaction. Preliminary experiments showed that the addition of HOA 0.2 M, standard iron solution, phen solution 0.04 M and 0.1 M NaOAc buffer on paper device lead to colour development as the paper natural matrix would permit the reagents to wick in the detection zone and easily visualize by the human eye (Fig. 3). Sequentially, iron concentrations related to varied complex absorbance were measurable via the image J programme.

Figure 3. Picture of μPAD platform for iron detection viewing the detection zone containing iron complex with phen in the presence of HOA and NaOAc buffer (first and second vertical spots to the left) and its blank (third and fourth vertical spots).
Parameters that affect the colour development including volume of phen and HOA and reaction time were examined to give the best absorbance for rapid and sensitive measurements. All experiments were done using 80 mg L -1 of iron solution. The Effect of 1,10-phenanthroline Volume: Experiments on the influence of spotted reagent volume were done as the insufficient volume may lead to absorbance reduction at the same time the excess of volume may cause to solution spread outside the detection zone 36 . Phen 0.04 M volume was examined in the range (1-9) µL. As can be seen from Fig. 4a the best volume that gave the higher absorbance was 7 µL, therefore, it was chosen for further experiments. The Effect of Hydroxylamine Hydrochloride Volume: The HOA 0.2 M different volumes of influence were tested to determine the optimum volume that gives the highest intensity. Figure 4b shows there was a slight increase in intensity through the addition of 1, 3, and 7 µL of the base. Then a decrease was observed after 7µL, these results indicate that the optimum base volume that gives the optimum intensity was 7µL. The Effect of NaOAc Volume: NAOAc solution volume effect on maximum colour intensity was tested and the results showed that 3 µL of NaOAc 0.1 M gave the highest colour intensity, therefore it was selected for further experiments (Fig. 4c). The Effect of Reaction Time: One of the most fundamental parameters influencing complex formation is reaction time and to ensure maximal colour intensity, reaction time was optimized over time intervals from (0-35) minutes. The colour development initially increased with time up to 10 minutes which was deemed adequate to obtain maximum colour intensity and used for subsequent experiments (Fig. 4d).

Calibration Graphs and Figure of Merits
The linear calibration graph was constructed for methods A and B using a series of iron concentration solutions via suitable dilution of stock solution. Figure 5a and b show a calibration graph for the spectrophotometric method under optimum experimental variables measured at λmax 510 nm. Figure 5c and d show the calibration graph for a μPADs method using optimum conditions. Table 1 summarizes the regression equation, correlation coefficient, molar absorptivity, Sandell's sensitivity, the limit of the quantification (LOQ) and limit of detection (LOD) for both iron methods.   The precision and accuracy for iron (II) proposed methods were studied by calculating the values of relative standard deviation (RSD %) and percentage of relative error (Er %). The results in Table 2 showed that RSD values were low between (0.39 -1.85), relative error less than 3%, and acceptable recoveries values between (98. 18 -101.33) were obtained indicating to good precision and accuracy of the proposed methods.

Analytical Application and Comparison Method
The ability of the proposed method to determine iron (II) in commercial lipstick collected from 500 I.D. stores in Baghdad were investigated using the standard addition method to eliminate interferences influence by comparing the proposed method results with standard analytical method results that are usually used for iron analysis; therefore atomic absorption spectrometry (AAS) was adopted. The results presented in Fig. 6 showed that the concentration of iron (II) from Spectrophotometric and µPAD were not significantly different from those of conventional AAS. Table 3 presents the analysis of variance including eight samples indicating F tab = 3.4668 is much higher than F Stat = 0.2526 Therefore, it can be concluded that there was an insignificant difference between the three methods. Also, comparing the results from spectrophotometric, paper-based device and atomic absorption spectrophotometric methods showed a good correlation between results via the three methods i.e. (r = 0.9987), (r = 0.9969) and (r = 0.9981), respectively.

Study of Biological Activity
The antimicrobial activity of local lipsticks against clinical and normal flora microorganisms was examined using agar well diffusion method. The antimicrobial efficacy testing revealed that all the lipsticks samples showed no effect on  Fig. 7a. For gram-negative bacterial isolates, all isolates were sensitive to all lipsticks samples and sample 7 showed the most antimicrobial effects on Pseudomonas aeruginosa isolate Fig. 7b. While the lipsticks samples showed no antimicrobial activity against Candida albicans isolate Fig. 7c.  The lack of expiry date and an ingredients label on lipsticks with unknown brands pose a potential high risk to consumer health. The possible contamination of lipsticks by microorganisms before and after use by the consumer is a major problem for the cosmetic industry. Staphylococcus aureus, E. coli, and Pseudomonas aeruginosa and Candida albicans are the most common tests strains that are recommended to test antimicrobial efficacy in addition to normal flora such as Staphylococcus epidermidis 38 . Using pyrosequencing analysis, a study detected 105 bacterial genera in 20 lipstick samples and Streptococcus were one of four genera predominant in 92% of the 19,863 total sequence reads while Staphylococcus, Pseudomonas, Escherichia a potentially pathogenic bacteria represented 27.6% of the total 105 genera. According to a recent study by Siya 2019, S. epidermidis represented 40% while S. aureus represented 32% from identified microorganisms 39 . S. epidermidis is responsible for biofilm formation in the prosthetic devices and implants 40 and S. aureus is involved in causing bacteremia in patients suffering from endocarditis and metastatic infections 41 .

Conclusion:
Two new, simple, reliable and economical colorimetric assays including spectrophotometric and paper-based analytical microfluidic devices (µPADs) were used for the development of in situ system for measuring the iron (II) concentration in cheap lipsticks samples collected from the local market of Baghdad, Iraq as this metal could cause toxic metal poisoning to lipstick consumers due to daily use. This paper used the employment of 1,10phenanthroline as a mostly used ligand in coordination chemistry for iron (II) detection to form a measurable complex and its resulted absorption due to colour change is proportional to the concentration of iron(II) that exist in lipstick. This gives easily interpretable results without sophisticated equipment such as atomic absorption spectroscopy, additional advantage relies upon a short time frame to complete the colourimetric assay. The important findings of this study, iron (II) levels were found to be lower than the permissible limit in lipstick samples, antimicrobial activity testing revealed that the tested microbes showed varied sensitivity all lipsticks samples with exception of Staphylococcus aureus and Candida albicans that were not affected by lipsticks samples and sample 7 showed the highest effect, and optimization of experimental results showed that these two methods give similar accuracy.