Chemical Papers

, Volume 72, Issue 10, pp 2561–2574 | Cite as

Validated and rapid measurement of the ferric reducing antioxidant power in plasma samples

  • Maria L. Gonzalez-Rivera
  • Flavio Martinez-Morales
  • Angel J. Alonso-Castro
  • Juan F. Lopez-Rodriguez
  • Juan R. Zapata-Morales
  • Saray Aranda Romo
  • Othoniel H. Aragon-MartinezEmail author
Original Paper


The ferric reducing antioxidant power assay was developed using rat plasma samples and validated according to the Food and Drug Administration guidelines as well as strategies for the lack of endogenous compounds-free samples of matrix. For validation procedures, the phosphate buffered saline solution was used as the artificial matrix because this led to a direct interpolation in calibration curves. The absorbance responses and antioxidant activities in curves showed a second order polynomial relationship (R2 value = 0.9982). The precision, accuracy, and stability of the method ranged from 1.7 to 7.2%, 89.8 to 100.0%, and 82.7 to 111.6%, respectively. This assay had a short time of analysis (96 samples per min) and absence of interferences during the spectrophotometric monitoring. For the application of the method, the plasma antioxidant capacity, blood distribution of levofloxacin, and biometry hematic were evaluated in samples obtained from rats under different experimental conditions. The in vitro condition applied to blood samples increased the plasma antioxidant capacity and volume of erythrocytes, whereas diminished the levofloxacin concentration in these cells. The high antioxidant activity was produced by a high amount of inosine, which in turn was caused by high oxidative stress leading to an impaired blood distribution of levofloxacin and erythrocyte swelling. This assay is a validated and rapid biomarker for the evaluation of the total antioxidant capacity in plasma samples.

Graphical abstract


Ferric reducing antioxidant power Bioanalytical validation Surrogate matrix Erythrocyte dysfunction Fluoroquinolone 



Acesulfame potassium


Body weight


Food and Drug Administration


Ferric reducing antioxidant power (µmol Fe(II) L−1)


Liquid chromatography


Lower limit of quantification (µmol Fe(II) L−1)




Mean corpuscular hemoglobin concentration (g dL−1)


Mean corpuscular volume (fL)


Phosphate buffered saline


Quality control


Coefficient of determination





This work was supported by Scientific Research Funding obtained from the Autonomous University of San Luis Potosi (Grant Number C16-FAI-09-32.32). Maria L. Gonzalez-Rivera is a CONACYT fellow (Grant Number: 584981).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aragon-Martinez OH, Galicia O, Isiordia-Espinoza MA, Martinez-Morales F (2014) A novel method for measuring the ATP-related compounds in human erythrocytes. Tohoku J Exp Med 233:205–214. CrossRefPubMedGoogle Scholar
  2. Aragon-Martinez OH, Isiordia-Espinoza MA, Galicia O, Aranda Romo S, Gómez Gómez A, Romano-Moreno S, Martinez-Morales F (2017a) Measurement of levofloxacin in human plasma samples for a reliable and accessible drug monitoring. Clin Biochem 50:73–79. CrossRefPubMedGoogle Scholar
  3. Aragon-Martinez OH, Martinez-Morales F, Isiordia-Espinoza MA, Luque Contreras D, Zapata Morales JR, Gonzalez-Rivera ML (2017b) Bacterial resistance and failure of clinical cure could be produced by oxidative stress in patients with diabetes or cardiovascular diseases during fluoroquinolone therapy. Med Hypotheses 103:32–34. CrossRefPubMedGoogle Scholar
  4. Armbruster DA, Pry T (2008) Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev 29:S49–S52PubMedPubMedCentralGoogle Scholar
  5. Benzie IF, Strain JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem 239:70–76. CrossRefPubMedGoogle Scholar
  6. Benzie IF, Strain JJ (1999) Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol 299:15–27. CrossRefPubMedGoogle Scholar
  7. Bolanos de la Torre AA, Henderson T, Nigam PS, Owusu-Apenten RK (2015) A universally calibrated microplate ferric reducing antioxidant power (FRAP) assay for foods and applications to Manuka honey. Food Chem 174:119–123. CrossRefPubMedGoogle Scholar
  8. Candioti LV, De Zan MM, Cámara MS, Goicoechea HC (2014) Experimental design and multiple response optimization. Using the desirability function in analytical methods development. Talanta 124:123–138. CrossRefPubMedGoogle Scholar
  9. Colino CI, García Turiño A, Sanchez Navarro A, Lanao JM (1998) A comparative study of ofloxacin and ciprofloxacin erythrocyte distribution. Biopharm Drug Dispos 19:71–77.CrossRefPubMedGoogle Scholar
  10. Cong WN, Wang R, Cai H, Daimon CM, Scheibye-Knudsen M, Bohr VA, Turkin R, Wood WH 3rd, Becker KG, Moaddel R, Maudsley S, Martin B (2013) Long-term artificial sweetener acesulfame potassium treatment alters neurometabolic functions in C57BL/6 J mice. PLoS ONE 8:e70257. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Crotty GF, Ascherio A, Schwarzschild MA (2017) Targeting urate to reduce oxidative stress in Parkinson disease. Exp Neurol. CrossRefPubMedGoogle Scholar
  12. deGraft-Johnson J, Kolodziejczyk K, Krol M, Nowak P, Krol B, Nowak D (2007) Ferric-reducing ability power of selected plant polyphenols and their metabolites: implications for clinical studies on the antioxidant effects of fruits and vegetable consumption. Basic Clin Pharmacol Toxicol 100:345–352. CrossRefPubMedGoogle Scholar
  13. Dudzinska W (2014) Purine nucleotides and their metabolites in patients with type 1 and 2 diabetes mellitus. J Biomed Sci Eng 7:38–44. CrossRefGoogle Scholar
  14. Duplancic D, Kukoc-Modun L, Modun D, Radic N (2011) Simple and rapid method for the determination of uric acid-independent antioxidant capacity. Molecules 16:7058–7068. CrossRefPubMedGoogle Scholar
  15. El-Sharif HF, Phan QT, Reddy SM (2014) Enhanced selectivity of hydrogel-based molecularly imprinted polymers (HydroMIPs) following buffer conditioning. Anal Chim Acta 809:155–161. CrossRefPubMedGoogle Scholar
  16. Festing MF, Altman DG (2002) Guidelines for the design and statistical analysis of experiments using laboratory animals. ILAR J 43:244–258CrossRefPubMedGoogle Scholar
  17. Firuzi O, Mladenka P, Riccieri V, Spadaro A, Petrucci R, Marrosu G, Saso L (2006) Parameters of oxidative stress status in healthy subjects: their correlations and stability after sample collection. J Clin Lab Anal 20:139–148. CrossRefPubMedGoogle Scholar
  18. Franco-Martínez L, Romero D, Rubio CP, Tecles F, Martínez-Subiela S, Teles M, Tvarijonaviciute A (2018) New potential biomarkers of oxidative stress in Mytilus galloprovincialis: analytical validation and overlap performance. Comp Biochem Physiol B: Biochem Mol Biol 221–222:44–49. CrossRefGoogle Scholar
  19. Fujieda Y, Yamaoka K, Ito T, Nakagawa T (1996) Local absorption kinetics of levofloxacin from intestinal tract into portal vein in conscious rat using portal-venous concentration difference. Pharm Res 13:1201–1204. CrossRefPubMedGoogle Scholar
  20. Gawlik K, Naskalski JW, Fedak D, Pawlica-Gosiewska D, Grudzień U, Dumnicka P, Małecki MT, Solnica B (2016) Markers of antioxidant defense in patients with type 2 diabetes. Oxid Med Cell Longev 2016:2352361. CrossRefPubMedGoogle Scholar
  21. Gómez Ruiz B, Roux S, Courtois F, Bonazzi C (2016) Spectrophotometric method for fast quantification of ascorbic acid and dehydroascorbic acid in simple matrix for kinetics measurements. Food Chem 211:583–589. CrossRefPubMedGoogle Scholar
  22. González JA, Callejón Mochón M, de la Rosa FJ (2000) Spectrofluorimetric determination of levofloxacin in tablets, human urine and serum. Talanta 52:1149–1156. CrossRefPubMedGoogle Scholar
  23. Gudkov SV, Shtarkman IN, Smirnova VS, Chernikov AV, Bruskov VI (2006) Guanosine and inosine display antioxidant activity, protect DNA in vitro from oxidative damage induced by reactive oxygen species, and serve as radioprotectors in mice. Radiat Res 165:538–545. CrossRefPubMedGoogle Scholar
  24. Güngör N, Ozyürek M, Güçlü K, Cekiç SD, Apak R (2011) Comparative evaluation of antioxidant capacities of thiol-based antioxidants measured by different in vitro methods. Talanta 83:1650–1658. CrossRefPubMedGoogle Scholar
  25. Gupta A, Kant S, Gupta SK, Prakash S, Kalaivani M, Pandav CS, Rai SK, Misra P (2016) Serum FRAP levels and pre-eclampsia among pregnant women in a rural community of northern India. J Clin Diagn Res 10:LC12–LC15. CrossRefPubMedPubMedCentralGoogle Scholar
  26. International Conference on Harmonization (ICH) (2005) In: International conference on harmonization of technical requirements for the registration of pharmaceuticals for human use, validation of analytical procedures: text and methodology Q2(R1). Geneva, SwitzerlandGoogle Scholar
  27. Kaplan IV, Attaelmannan M, Levinson SS (2001) Fibrinogen is an antioxidant that protects beta-lipoproteins at physiological concentrations in a cell free system. Atherosclerosis 158:455–463. CrossRefPubMedGoogle Scholar
  28. Kolarzyk E, Pietrzycka A, Zając J, Morawiecka-Baranek J (2017) Relationship between dietary antioxidant index (DAI) and antioxidants level in plasma of Kraków inhabitants. Adv Clin Exp Med 26:393–399. CrossRefPubMedGoogle Scholar
  29. Krebs HA (1950) Chemical composition of blood plasma and serum. Annu Rev Biochem 19:409–430CrossRefPubMedGoogle Scholar
  30. Kumar P, Chand S, Maurya PK (2016) Quercetin-modulated erythrocyte membrane sodium-hydrogen exchanger during human aging: correlation with ATPase’s. Arch Physiol Biochem 122:141–147. CrossRefPubMedGoogle Scholar
  31. Lee SG, Wang T, Vance TM, Hubert P, Kim DO, Koo SI, Chun OK (2017) Validation of analytical methods for plasma total antioxidant capacity by comparing with urinary 8-isoprostane level. J Microbiol Biotechnol 27:388–394. CrossRefPubMedGoogle Scholar
  32. Lu LL, Li YH, Lu XY (2009) Kinetic study of the complexation of gallic acid with Fe(II). Spectrochim Acta A Mol Biomol Spectrosc 74:829–834. CrossRefPubMedGoogle Scholar
  33. Marrocco I, Altieri F, Peluso I (2017) Measurement and clinical significance of biomarkers of oxidative stress in humans. Oxid Med Cell Longev 2017:6501046. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Mexican Official Norm NOM-007-SSA3-2011 (2012) For the operation and organization of clinic laboratories. Official Journal of the Federation, Mexico CityGoogle Scholar
  35. Mexican Official Norm NOM-062-ZOO-1999 (2001) Technical specifications for production, care, and use of laboratory animals. Official Journal of the Federation, Mexico CityGoogle Scholar
  36. Mexican Official Norm NOM-177-SSA1-2013 (2013) Tests and procedures to prove that a medication is interchangeable. Official Journal of the Federation, Mexico CityGoogle Scholar
  37. Modun D, Music I, Vukovic J, Brizic I, Katalinic V, Obad A, Palada I, Dujic Z, Boban M (2008) The increase in human plasma antioxidant capacity after red wine consumption is due to both plasma urate and wine polyphenols. Atherosclerosis 197:250–256. CrossRefPubMedGoogle Scholar
  38. Moslemnezhad A, Mahjoub S, Moghadasi M (2016) Altered plasma marker of oxidative DNA damage and total antioxidant capacity in patients with Alzheimer’s disease. Casp J Intern Med 7:88–92Google Scholar
  39. National Research Council (US) (2011) Committee for the update of the guide for the care and use of laboratory animals, guide for the care and use of laboratory animals, 8th edn. National Academies Press (US), Washington, DCGoogle Scholar
  40. Parham H, Rahbar N (2009) Solid phase extraction-spectrophotometric determination of salicylic acid using magnetic iron oxide nanoparticles as extractor. J Pharm Biomed Anal 50:58–63. CrossRefPubMedGoogle Scholar
  41. Pizzino G, Irrera N, Cucinotta M, Pallio G, Mannino F, Arcoraci V, Squadrito F, Altavilla D, Bitto A (2017) Oxidative stress: harms and benefits for human health. Oxid Med Cell Longev 2017:8416763. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Pohanka M, Bandouchova H, Sobotka J, Sedlackova J, Soukupova I, Pikula J (2009) Ferric reducing antioxidant power and square wave voltammetry for assay of low molecular weight antioxidants in blood plasma: performance and comparison of methods. Sensors 9:9094–9103. CrossRefPubMedGoogle Scholar
  43. Quattrocchi OA, de Andrizzi SA, Laba RF (1992) Introducción a la HPLC, Aplicación y Práctica, 1st edn. Artes Gráficas Farro SA, Buenos AiresGoogle Scholar
  44. Raudonis R, Raudone L, Jakstas V, Janulis V (2012) Comparative evaluation of post-column free radical scavenging and ferric reducing antioxidant power assays for screening of antioxidants in strawberries. J Chromatogr A 1233:8–15. CrossRefPubMedGoogle Scholar
  45. Rivero-Pérez MD, Muñiz P, Gonzalez-Sanjosé ML (2007) Antioxidant profile of red wines evaluated by total antioxidant capacity, scavenger activity, and biomarkers of oxidative stress methodologies. J Agric Food Chem 55:5476–5483CrossRefPubMedGoogle Scholar
  46. Rubio CP, Martinez-Subiela S, Hernández-Ruiz J, Tvarijonaviciute A, Ceron JJ (2017) Analytical validation of an automated assay for ferric-reducing ability of plasma in dog serum. J Vet Diagn Investig 1040638717693883:1–5. CrossRefGoogle Scholar
  47. Sies H (2015) Oxidative stress: a concept in redox biology and medicine. Redox Biol 4:180–183. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Tavazzi B, Di Pierro D, Amorini AM, Fazzina G, Tuttobene M, Giardina B, Lazzarino G (2000) Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress. Eur J Biochem 267:684–689. CrossRefPubMedGoogle Scholar
  49. Thakare R, Chhonker YS, Gautam N, Alamoudi JA, Alnouti Y (2016) Quantitative analysis of endogenous compounds. J Pharm Biomed Anal 128:426–437. CrossRefPubMedGoogle Scholar
  50. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine (USDHHS, FDA, CDER & CVM) (2001) Guidance for industry: bioanalytical method validation. United Stated of America, Maryland.…/Guidances/ucm070107.pdf. Accessed 2 Oct 2017
  51. van de Merbel NC (2008) Quantitative determination of endogenous compounds in biological samples using chromatographic techniques. Trends Anal Chem 27:924–933. CrossRefGoogle Scholar
  52. van Wijk R, van Solinge WW (2005) The energy-less red blood cell is lost: erythrocyte enzyme abnormalities of glycolysis. Blood 106:4034–4042. CrossRefPubMedGoogle Scholar
  53. Yokotani K, Umegaki K (2017) Evaluation of plasma antioxidant activity in rats given excess EGCg with reference to endogenous antioxidants concentrations and assay methods. Free Radic Res 51:193–199. CrossRefPubMedGoogle Scholar

Copyright information

© Institute of Chemistry, Slovak Academy of Sciences 2018

Authors and Affiliations

  • Maria L. Gonzalez-Rivera
    • 1
  • Flavio Martinez-Morales
    • 1
  • Angel J. Alonso-Castro
    • 2
  • Juan F. Lopez-Rodriguez
    • 3
  • Juan R. Zapata-Morales
    • 2
  • Saray Aranda Romo
    • 4
  • Othoniel H. Aragon-Martinez
    • 1
    • 5
    Email author
  1. 1.Department of Pharmacology, School of MedicineAutonomous University of San Luis PotosiSan Luis PotosiMexico
  2. 2.Division of Natural and Exact Sciences, Department of PharmacyUniversity of GuanajuatoGuanajuatoMexico
  3. 3.Animal Laboratory, School of MedicineAutonomous University of San Luis PotosiSan Luis PotosiMexico
  4. 4.Diagnostic Clinic, Dentistry SchoolAutonomous University of San Luis PotosíSan Luis PotosiMexico
  5. 5.Laboratory of Natural Compounds (LABCON)San Luis PotosiMexico

Personalised recommendations