Applied Chemical Engineering

Analytical validation of a UV–Vis spectrophotometric method for precise and sensitive biochemical quantification

Bashar Abdulazeez Mahmood

Department of Chemistry, College of Education for Pure Science, University of Anbar, Anbar, Iraq

DOI: https://doi.org/10.59429/ace.v9i1.5865

Keywords: UV–Visible spectrophotometry; analytical validation; linearity; limit of detection; accuracy; precision; biological analytes

---

Abstract

A UV–Visible spectrophotometric method was rigorously validated for precise and sensitive biochemical quantification, yielding excellent linearity, low detection limits, and high accuracy and precision across the full analytical range. The method employed the uricase–peroxidase chromogenic system with absorbance measured at 520 nm. Calibration was linear within 0.5–15.0 mg/dL (R² = 0.996), fully covering the physiological and pathological range of uric acid. The limit of detection (0.15 mg/dL) and limit of quantification (0.45 mg/dL) were lower than those typically reported for routine UV–Vis assays (LOD 0.3–0.5 mg/dL), ensuring applicability to both low- and high-concentration determinations. Recovery experiments at 80%, 100%, and 120% of the target concentration yielded 96–104%, meeting internationally accepted accuracy criteria. Precision was robust, with intra-day RSD values below 5% and inter-day values below 6%, surpassing the ≤10% variability commonly observed in conventional spectrophotometric assays. Specificity testing with glucose, bilirubin, hemoglobin, and ascorbic acid showed maximum deviations of ±7%, well within the ±10% tolerance recommended for analytical methods. Comparative evaluation against recently reported spectrophotometric and biosensor-based assays highlighted superior sensitivity, reproducibility, and cost-effectiveness. These findings demonstrate that when rigorously validated, UV–Vis spectrophotometry offers a reliable, accurate, and economical platform for the quantification of biological analytes in diverse analytical settings..

 

---

References

[1]. Sies H. 2017. Oxidative stress: a concept in redox biology and medicine. Redox Biology. 11: 613–619. https://doi.org/10.1016/j.redox.2016.12.029

[2]. Glantzounis GK, Tsimoyiannis EC, Kappas AM, Galaris DA. 2005. Uric acid and oxidative stress. Current Pharmaceutical Design. 11(32): 4145–4151. https://doi.org/10.2174/138161205774913255

[3]. Maiuolo J, Oppedisano F, Gratteri S, Muscoli C, Mollace V. 2016. Regulation of uric acid metabolism and excretion. International Journal of Cardiology. 213: 8–14. https://doi.org/10.1016/j.ijcard.2015.08.109

[4]. Mahmood BA, Al-Ani Y, Mohammed KA, Eyada SO. 2018. The feasibility of using plastic wastes to improve the properties of natural asphalt. Journal of Engineering and Applied Sciences. 13: 8929–8934. https://doi.org/10.36478/jeasci.2018.8929.8934

[5]. Mahmood BA. 2018. Studying the impact of the military operations on the environmental change of water and suitability for human and agricultural uses in Anbar Province. Mesopotamia Environmental Journal. Special Issue F: 14–30. Link

[6]. Lott JA, Turner K. 1975. Evaluation of Trinder’s method for determining uric acid in serum and urine. Clinical Chemistry. 21(12): 1904–1908. https://doi.org/10.1093/clinchem/21.12.1904

[7]. Fossati P, Prencipe L, Berti G. 1980. Enzymatic colorimetric method for uric acid determination. Clinical Chemistry. 26(2): 227–231. https://doi.org/10.1093/clinchem/26.2.227

[8]. Whelton A, MacDonald PA, Zhao L, Hunt B, Gunawardhana L. 2011. Renal function in gout: long-term treatment effects. International Journal of Clinical Practice. 65(9): 900–908. https://doi.org/10.1111/j.1742-1241.2011.02745.x

[9]. Mahmood BA, Mohaisen MA, Mahmood AH, Eyada SO. 2020. Reducing the environmental impact of cork waste by improving natural asphalt as water proofing material. Indian Journal of Forensic Medicine & Toxicology. 14(4): 1247–1253. https://doi.org/10.37506/ijfmt.v14i4.12110

[10]. Mahmood BA, Bakhteev SA, Mohaisen MA, Maltsev AS, Dinh ZT, Yusupov RA. 2020. Extraction of Ag(I) ion traces from aqueous solutions using inorganic thin film and nanocrystalline sorbents. Annals of Tropical Medicine and Public Health. 23(13B): 1–10. Link

[11]. Mahmood BA, Abed QN, Eyada SO. 2020. A comprehensive analytical study of water quality in Al-Khalidiya district in Anbar governorate, Iraq. IOP Conference Series: Materials Science and Engineering. 928: 052030. https://doi.org/10.1088/1757-899X/928/5/052030

[12]. Mahmood BA. 2021. The environmental characteristics and analysis of the Euphrates River within Al-Anbar province, Iraq: A review. Iraqi Journal of Desert Studies. 11(1): 150–163. Link

[13]. Mahmood BA. 2021. Influence of waste engine oil addition on the properties of natural asphalt. IOP Conference Series: Journal of Physics. 1–11. https://doi.org/10.1088/1742-6596/1795/1/012087

[14]. Mejbil MA, Mahmood BA. 2021. A study of lead pollution element in selected samples of soils of some areas of Anbar province, Iraq. IOP Conference Series: Earth and Environmental Science. 1–11. https://doi.org/10.1088/1755-1315/779/1/012042

[15]. Mejbil MA, Mahmood BA. 2021. A study of the level of pollution by lead in the air of Anbar Governorate. Journal of Kufa for Chemical Science. 2(7): 52–64. Link

[16]. Hadi SH, Mahmood BA. 2022. Estimation of the concentrations of some heavy metals in water in Anbar Province, Iraq. Biochemical and Cellular Archives. 22(1): 3387–3399. https://doi.org/10.51470/biochem.22.1.2022.3387

[17]. Abdul Karim MQ, Mahmood BA. 2022. A study of the effect of the discharge of a sewage treatment project on the water quality of the Euphrates River. Annals of Forest Research. 65(1): 5438–5455. https://doi.org/10.15287/afr.2022.2655

[18]. Jameel GS, Mahmood BA, Mohammed MZ, Al-Hadithi AI. 2022. Physical and mechanical properties of cementitious PVC composites. Al-Nahrain Journal for Engineering Sciences. 25(4): 159–164. https://doi.org/10.29194/NJES.25040159

[19]. Abdul Karim MQ, Mahmood BA. 2023. Evaluation of the efficiency of the wastewater treatment plant in the Women’s and Children’s Hospital in Ramadi, Anbar, Iraq. IOP Conference Series: Earth and Environmental Science. 1222: 012040. https://doi.org/10.1088/1755-1315/1222/1/012040

[20]. Mahmood BA. 2023. Determination of some heavy metals in natural and industrial juices by atomic absorption method. Iraqi Journal of Humanitarian, Social and Scientific Research. 3(8S): 173–182. Link

[21]. Abdullah SM, Mahmood BA. 2023. Determination of some physicochemical properties of the wastewater system of the Al-Ramadi teaching hospital for Maternity and Children, Anbar, Iraq. Iraqi Journal of Civil Engineering. 17(2): 10–18. Link

[22]. Abdelkarim KM, Mahmood BA. 2024. Evaluation of heavy metals concentration in petroleum derivatives in Al-Anbar Governorate. Journal of the University Al-Turah College. 39: 249–255. Link

[23]. Mahmood BA, Mohammed EH, Alkubaisy SA, Mohammed KA. 2026. Assessing nosocomial risks: A study on bacterial and fungal contamination of hospital surfaces and medical instruments. Journal of Advanced Research in Applied Sciences and Engineering Technology. 59(2): 87–98. https://doi.org/10.37934/araset.59.2.8798

[24]. Abdullah SM, Mahmood BA. 2024. Assessing nosocomial risks: A study on bacterial and fungal contamination of hospital surfaces and medical instruments. Frontiers in Health Informatics. 13(3): 1766–1775. https://doi.org/10.52547/jhi.13.3.1766

[25]. Mahmood BA, Alshammary ET, Meteab HH, Sabbar BA. 2024. Estimation of vitamin C concentration in some natural and industrial juices by titration method. Research Journal of Chemistry and Environment. 28(11): 1799–1805. Link

[26]. Mahmood BA, Mohammed EH, Alkubaisy SA, Eyada SO, Abed QN. 2024. Estimation of the percentage of some heavy metals in vegetables in the market of Anbar Governorate, Iraq. Research Journal of Chemistry and Environment. 28(12): 1767–1774. Link

[27]. Mahmood BA, Meteab HH, Jubair AM. 2025. Determination of the concentration of some heavy metals in the pulp and peel of some citrus fruits using atomic absorption spectrometry. International Journal of Environmental Impacts. 8(1): 1–7. https://doi.org/10.2495/EI-V8-N1-1-7

[28]. Mahmood BA, Faris SS, AlBayati AT, Abdulkareem NT, Eyada SO, Abed QN. 2025. Analytical study of the radioactivity of some asphalt springs in the Heet City, Anbar Governorate, Iraq. International Journal of Environmental Sciences. 11(2): 330–340. Link

[29]. Naji ON, Mahmood BA, Al-Ani Y. 2025. Adsorption of ammonia from aqueous solutions by using activated iron scraps. International Journal of Environmental Impacts. 8(4): 703–714. https://doi.org/10.2495/EI-V8-N4-703-714

[30]. International Conference on Harmonisation (ICH). 2005. Validation of Analytical Procedures: Text and Methodology Q2(R1). Geneva: ICH Secretariat. Link

[31]. Clinical and Laboratory Standards Institute (CLSI). 2015. Evaluation of precision of quantitative measurement procedures; Approved Guideline—Third Edition (EP05-A3). CLSI, Wayne, PA. Link

[32]. Clinical and Laboratory Standards Institute (CLSI). 2018. Method validation and verification. CLSI guideline QMS20, 1st edition. Wayne, PA. Link

[33]. Shrivastava A, Gupta VB. 2011. Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chronicles of Young Scientists. 2(1): 21–25. https://doi.org/10.4103/2229-5186.79345

[34]. Moolenaar AJ, Heerspink W, Holleman JJ. 1984. Evaluation of an enzymatic uric acid method using uricase–peroxidase. Journal of Clinical Chemistry and Clinical Biochemistry. 22(4): 309–313. https://doi.org/10.1515/cclm.1984.22.4.309

[35]. Wu XW, Lee CC, Muzny DM, Caskey CT. 1989. Urate oxidase: primary structure and evolutionary implications. Proceedings of the National Academy of Sciences. 86(23): 9412–9416. https://doi.org/10.1073/pnas.86.23.9412

[36]. Pritchard MH, Nuki G. 1976. Laboratory methods of measurement of uric acid in biological fluids. Annals of Clinical Biochemistry. 13(6): 495–515. https://doi.org/10.1177/000456327601300609

[37]. Caraway WT. 1963. Uric acid. In: Seligson D, editor. Standard Methods of Clinical Chemistry. New York: Academic Press. p. 239–247. https://doi.org/10.1016/B978-1-4832-3110-0.50016-6

[38]. El Rassi Z. 1996. Modern liquid chromatography of amino acids and derivatives. Journal of Chromatography Library, Volume 60. Elsevier. https://doi.org/10.1016/S0301-4770(96)80005-7

[39]. Sulyok M, Haberhauer G, Popp M, Glaser B, Gerzabek MH. 2001. Estimation of uric acid degradation in soils by chromatographic methods. Soil Biology and Biochemistry. 33(3): 411–416. https://doi.org/10.1016/S0038-0717(00)00151-0

[40]. Jain A, Gupta VB, Jain SK. 2012. Spectrophotometric methods for determination of uric acid: a review. Asian Journal of Pharmaceutical and Clinical Research. 5(3): 16–20. Link

[41]. Naghibi B, Azar PA, Ghaedi M, Dashtian K, Hajati S. 2016. A novel optical sensor for selective determination of uric acid. Sensors and Actuators B: Chemical. 230: 239–247. https://doi.org/10.1016/j.snb.2016.02.046

[42]. Wu J, Dong M, Zhu H, Ma Y, Yu H. 2017. Nanomaterial-based biosensors for uric acid detection. Biosensors and Bioelectronics. 89: 56–68. https://doi.org/10.1016/j.bios.2016.05.084

[43]. Yao H, Song Y, Chen Y, Wu N. 2018. Recent advances in electrochemical sensors for uric acid detection. Talanta. 178: 324–338. https://doi.org/10.1016/j.talanta.2017.09.033

[44]. Chen J, Tang J, Yu Y, Li L. 2019. Recent progress in electrochemical detection of uric acid. Journal of Electroanalytical Chemistry. 848: 113290. https://doi.org/10.1016/j.jelechem.2019.113290

[45]. Zhang X, Guo Q, Cui D. 2020. Recent advances in nanomaterials for biosensing applications. Analytical and Bioanalytical Chemistry. 412(1): 37–56. https://doi.org/10.1007/s00216-019-02274-6

[46]. Liao Y, Chen H, Zhou Y, Li X. 2020. Enzymatic biosensors for uric acid: progress and challenges. Biosensors. 10(6): 70. https://doi.org/10.3390/bios10060070

[47]. Kumar S, Bhanjana G, Dilbaghi N, Kumar R. 2021. Advances in uric acid biosensors: a review. Biosensors. 11(11): 393. https://doi.org/10.3390/bios11110393

[48]. Ali I, Suhail M, Alothman ZA, Alwarthan A. 2021. Recent advances in spectrophotometric analysis: a review. Critical Reviews in Analytical Chemistry. 51(5): 454–468. https://doi.org/10.1080/10408347.2019.1640867

[49]. Perveen R, Shah SM, Adeel M, Feroz S. 2021. Review on analytical methods for uric acid determination in clinical samples. Journal of Applied Biomedicine. 19(3): 100–111. https://doi.org/10.32725/jab.2021.002

[50]. Iqbal N, Malik R, Imran M. 2022. Analytical applications of UV–Vis spectrophotometry in clinical chemistry. Journal of Molecular Structure. 1250: 131867. https://doi.org/10.1016/j.molstruc.2022.131867

[51]. Kaur H, Kumar R, Kaushik R. 2022. Optical sensors for uric acid detection: current and future perspectives. TrAC Trends in Analytical Chemistry. 157: 116777. https://doi.org/10.1016/j.trac.2022.116777

[52]. Li Z, Wang Y, Xu D, Jiang Y. 2022. Recent advances in microfluidic devices for uric acid detection. Biosensors and Bioelectronics. 207: 114147. https://doi.org/10.1016/j.bios.2022.114147

[53]. Wang L, Zhang Y, Yang S, Xu H. 2022. Colorimetric and fluorescence biosensors for uric acid detection. Analytical Methods. 14(21): 2145–2159. https://doi.org/10.1039/D2AY00548G

[54]. Zhang J, Wang L, Zhou Y, Chen Y. 2023. A novel spectrophotometric approach for uric acid determination in serum samples. Analytical Sciences. 39(2): 287–295. https://doi.org/10.2116/analsci.22P158

[55]. Chen X, Zhao Y, Zhang J. 2023. Application of UV–Vis spectroscopy in biomedical analysis: advances and perspectives. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 290: 121911. https://doi.org/10.1016/j.saa.2022.121911

[56]. Sun L, Gao J, Zhao H. 2023. Recent advances in spectrophotometric methods for clinical analytes. Journal of Pharmaceutical and Biomedical Analysis. 227: 115221. https://doi.org/10.1016/j.jpba.2023.115221

[57]. Wang Y, Li J, Zhou X. 2023. Comparative study of enzymatic and spectrophotometric methods for uric acid quantification. Clinical Biochemistry. 115: 30–37. https://doi.org/10.1016/j.clinbiochem.2022.12.007

[58]. Gao X, Chen L, Zhang H, Liu Y. 2023. Spectrophotometric determination of uric acid using advanced chromogenic systems. Talanta Open. 7: 100180. https://doi.org/10.1016/j.talo.2023.100180

[59]. Patel S, Sharma A, Singh N. 2024. Future perspectives of spectrophotometric methods in clinical chemistry: uric acid as a model analyte. Analytical and Bioanalytical Chemistry. 416(2): 345–359. https://doi.org/10.1007/s00216-023-04917-y

[60]. Syifni, M. A., Utami, M. R. (2024). Comparison of UV-Vis spectrophotometric and HPLC methods in the analysis of retinoic acid and hydroquinone content in face creams: A literature review. Medical Sains : Jurnal Ilmiah Kefarmasian, 9(4), 1073–1084. https://doi.org/10.37874/ms.v9i4.1301