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A Simple and Rapid Spectrophotometric Method for Nitrite Detection in Small Sample Volumes

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A Simple and Rapid Spectrophotometric Method for Nitrite Detection in Small Sample Volumes chemosensors Article A Simple and Rapid Spectrophotometric Method for Nitrite Detection in Small Sample Volumes Yudtapum Thipwimonmas 1,2, Janjira Jaidam 1, Kritsada Samoson 1,3,4, Vacharachai Khunseeraksa 1,3,4, Apichai Phonchai 1,2, Adul Thiangchanya 1, Kah Haw Chang 5, Ahmad Fahmi Lim Abdullah 5 and Warakorn Limbut 1,2,3,4,* 1 Division of Health and Applied Sciences, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand; [email protected] (Y.T.); [email protected] (J.J.); [email protected] (K.S.); [email protected] (V.K.); [email protected] (A.P.); [email protected] (A.T.) 2 Forensic Innovation Center, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand 3 Center of Excellence for Trace Analysis and Biosensors (TAB-CoE), Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand 4 Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand 5 Forensic Science Programme, School of Health Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia; [email protected] (K.H.C.); [email protected] (A.F.L.A.) * Correspondence: [email protected]; Tel.: +66-74-288-563 Abstract: A simple, rapid, and environmentally-friendly spectrophotometric method for nitrite detection was developed. Detection was based on a redox reaction with iodide ions in an acidic Citation: Thipwimonmas, Y.; Jaidam, condition. The reaction was evaluated by detecting the increase in absorbance of the colored product J.; Samoson, K.; Khunseeraksa, V.; of iodine at 362 nm wavelength. To obtain a good spectrophotometric performance, the iodide Phonchai, A.; Thiangchanya, A.; Chang, K.H.; Abdullah, A.F.L.; ions concentration, hydrochloric acid concentration, and reaction time were optimized. In the Limbut, W. A Simple and Rapid optimal condition, the developed spectrophotometric method provided a linear range of 0.0625 to −1 −1 Spectrophotometric Method for 4.00 mg L (r = 0.9985), reaction time for 10 min, a limit of detection of 25 µg L , and a limit of −1 Nitrite Detection in Small Sample quantitation of 85 µg L . This method showed good repeatability (RSD < 9.21%), high sample − Volumes. Chemosensors 2021, 9, 161. throughput (9 samples min 1), and good accuracy (recovery = 88 ± 2 to 99.5 ± 0.4%). The method https://doi.org/10.3390/ has the potential to be used in crime scene investigations as a rapid screening test for gunshot residue chemosensors9070161 detection via nitrite detection. Academic Editor: Yoav Broza Keywords: nitrite; spectrophotometric method; 96 well microplate Received: 18 May 2021 Accepted: 22 June 2021 Published: 25 June 2021 1. Introduction Publisher’s Note: MDPI stays neutral The gunshot residue (GSR) is the product of primer detonation, gunpowder combus- with regard to jurisdictional claims in tion, and emitted particles of cartridge, projectile, and gun barrel. Deposition of GSR on published maps and institutional affil- surfaces in the vicinity of a shooting [1–3] made it useful forensic evidence in investiga- iations. tions of criminal use of firearms, aiding suspect identification, predicting firing distance, differentiating between suicide and homicide, and also indicating a bullet hole [4,5]. In routine forensic analysis, GSR is identified through morphological and elemental analysis using scanning electron microscopy coupled with energy dispersive X-ray analysis [6]. Additionally, inductively coupled plasma-mass spectrometry [7] and atomic absorption Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. spectroscopy [8] were also reported in analyzing GSR. These techniques had demonstrated This article is an open access article good accuracy and high sensitivity; however, these techniques could also be limited by distributed under the terms and their respective cost, time, availability, and accessibility of instrumentation, as well as the conditions of the Creative Commons need for well-trained operators [9,10]. Therefore, a fast, inexpensive, and simple screening Attribution (CC BY) license (https:// technique for determining GSR could benefit the forensic science community prior to the creativecommons.org/licenses/by/ application of specific confirmatory techniques. 4.0/). Chemosensors 2021, 9, 161. https://doi.org/10.3390/chemosensors9070161 https://www.mdpi.com/journal/chemosensors Chemosensors 2021, 9, 161 2 of 11 Nitrite ion is one of the most common inorganic compounds upon primer detonation and gunpowder combustion [11,12]. Detection of nitrite ions can provide valuable forensic information in the preliminary analysis of GSR on any related evidence, including the shooter, cartridge case, firearm, and surfaces nearby a shooting activity. The detection and measurement of nitrite could be employed as a screening method for the presence of GSR. Literatures reported various techniques for determination of nitrite, including electrochemical methods [13,14], ion chromatography [15,16], electrophoresis [17,18], and spectrophotometry [19,20]. Among these techniques, the spectrophotometry technique received increasing attention due to its simplicity, convenience, low cost, and good detection limit [21–23]. During the forensic investigation of firearms-related cases, suspects are frequently approached, and suspected GSR samples are collected, commonly from the hands, face, or clothing. In such cases, the forensic evidence is not limited to one sample, but several samples recovered from various sites of collection. Apart from that, the numbers of suspected GSR samples would be greatly increased if more suspects are identified. Given this, a fast screening procedure to determine the presence of GSR via the detection of nitrite would be needed, prior to the application of confirmatory SEM-EDX analysis. Spectrophotometric analysis in 96-well microplate read mode could be utilized, in which it enabled multi-sample analyses and low consumption of chemical reagent. Moreover, the interpretation of the absorbance values captured by the instrumentation through a microplate reader and directly stored in the computer would also provide quick and accurate data [20]. Most spectrophotometric techniques for the detection of nitrite were based on dye formation via a diazo-coupling reaction to produce the end product allowable for deter- mination, and typically an aromatic amine was used. Aromatic amines employed for this purpose included p-rosaniline [24], 3-nitroaniline [25], p-nitroaniline [26], 1-amino- 4-naphtalenesulfonic acid [27], p-aminobenzoic acid [28], 4-aminobenzotrifluoride [29], 4-aminophenylmercaptoacetic acid [30], safranin [31], and sulfanilamide [32]. However, these substances often possess some drawbacks, including the diazotization temperature, coupling time, and pH dependence. Furthermore, these techniques frequently employ large volumes of amines, and sometimes they are carcinogenic and toxic [22,33]. This present work reports a simple, inexpensive, and environmentally-friendly spec- trophotometric method for the determination of nitrite based on a redox reaction with iodide ions in an acidic condition. The method was optimized utilizing a UV/Vis 96- well microplate spectrophotometer and its analytical performance was evaluated in terms of its linearity, limit of detection (LOD), the limit of quantification (LOQ), repeatability, accuracy, and interference-effect. Finally, as a proof-of-concept for the application, we investigated the performance of the developed spectrophotometric method by applying sodium nitrite powder to various substrate surfaces, such as hands, clothes, tables, phones, notebook computers, and tiled floors. It is hoped that the developed method can be used to screen large numbers of suspected GSR samples, serving as a useful determination method complementing the SEM-EDX analysis in forensic laboratories. 2. Materials and Methods 2.1. Reagents and Apparatus Sodium nitrite (NaNO2) was purchased from Ajax Chemical Finechem Pty Ltd. (New South Wales, Australia). Potassium iodide (KI) and hydrochloric acid (HCl, 37%) were obtained from Merck KGaA (Darmstadt, Germany). Deionized water (18.2 MW cm) (Barn- steadTM Easy PureTM II water purification system, Thermo ScientificTM, Waltham, MA, USA) was employed for the preparation of all solutions. Stock nitrite standard solutions were prepared at a concentration level of 1000 mg L−1. Working solutions were prepared through dilution of a pre-determined volume of the stock nitrite standard solution with distilled water. A UV/Vis microplate spectrophotometer (Thermo Fisher Scientific, Multi- skan TM, GO, USA) was utilized in this study and the analytical procedure was controlled by Skanlt software 3.2. Chemosensors 2021, 9, 161 3 of 11 2.2. Analytical Procedure A few milliliters of nitrite ions solution were transferred into a microtube which contained 0.020 mol L−1 of hydrochloric acid and 0.050 mol L−1 of iodide ion solution. When a color change was observed by the naked eye, the mixture solution was dropped into wells of a 96-well microplate and mixed for 10 min. The absorbances detected by UV/Vis microplate spectrophotometer at a wavelength of 362 nm against a blank solution were recorded for each analysis. Note that the blank solution was prepared in a similar procedure but without nitrite ions. Figure1 illustrates the scheme for the detection of nitrate, comprising the preparation, incubation, and measurement steps. Figure 1. The scheme for the detection of nitrite. 2.3. Optimization In order
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