A facile nano-iron oxide sensor for the electrochemical detection of the anti-diabetic drug linagliptin in the presence of glucose and metformin
- 128 Downloads
A highly sensitive sensor for the electrochemical determination of an antidiabetic drug Linagliptin (LG) was constructed using carbon paste electrode (CPE) modified with iron oxide nanoparticles (Fe2O3NPs). The electrochemical performance of LG was examined analytically, and some dynamics were considered for the first time.
This work indicates that the oxidation reaction of LG on CPE/Fe2O3NPs is a one electron and one proton process, which is controlled by both diffusion and adsorption. The simultaneous determination of LG with glucose and metformin (MET) was also considered by square wave voltammetry in universal buffer pH 7.4. Experimental results specify a linear relation between LG peak current and its concentration in the range of 0.03 to 86 μg/ml, leading to a detection limit of 8.0 ng/ml.
This novel sensor was successfully used to determine LG in commercialized tablets and in spiked urine samples.
KeywordsDiabetes Iron oxide nanoparticles Linagliptin Carbon paste electrode
Finding new methods for the problems in the medical field is the center of researchers everywhere. Diabetes mellitus is a worldwide public health problem. People who have diabetes can develop serious or life-threatening complications, so taking medication(s) may help to manage diabetes and improve health (Association AD 2014). Insulin deficiency and hyperglycemia cause the concentration of blood glucose to increase or decrease than the normal range of 80–120 mg/dL (4.4–6.6 mM), which leads to death (Waldhäusl et al. 1979). The complications of fighting diabetes, include heart disease, kidney failure, or blindness, which can be reduced through personal control of blood glucose (Group UPDS 1998). Linagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor developed by Boehringer Ingelheim for treatment of type II diabetes in patients who cannot control blood sugar levels alone. Linagliptin works by increasing the amount of insulin released and decreasing the amount of sugar made by the body by slowing the breakdown of insulinotropic hormone glucagon-like peptide (GLP)-1 for better glycemic control.
Combination therapy has a pivotal role in type II diabetes mellitus management in patients unable to maintain normal glycemic level using metformin alone. Addition of linagliptin, dipeptidyl peptidase-IV inhibitor, to metformin improves glycemic control. Various analytical techniques are applied for simultaneous determination of binary diabetes drugs mixture such as LG and MET mixture (Haak 2015) including HPLC with UV detector (Vemula et al. 2015), LC-MS/MS (Abbas Moussa et al. 2019), and UV-Vis diffuse reflectance spectroscopy (El-Bagary et al. 2013). No electrochemical reported work applied for the determination of either the LG or our studied mixture, only voltammetric determination for MET (Brocks et al. 2010).
Magnetic nanoparticles (NPs) are in the focus of much interest especially iron oxide nanoparticles due to their enormous and unique physical and chemical properties. The most considered materials with promising properties are those with magnetic properties due to their enormous and unique physical and chemical properties and applications such as drug delivery, biomedical uses, and tumor labeling (Jalilian et al. 2009; Polyak and Friedman 2009). These applications require that the nanoparticles have high magnetization values, a size smaller than 100 nm, and a narrow particle size distribution. The nano-size of iron oxide nanoparticles and its biocompatibility make them perfect for surface engineering and functionalization (Laurent et al. 2011; Prodan et al. 2013). Also, Fe2O3 NPs provide a path to a new generation of chemical and electrochemical sensors for glucose biosensor applications with some materials such as Pt nanoparticles and multi-walled carbon nanotubes (Li et al. 2010; Rong et al. 2007), Ni oxide nanoparticles (Salimi et al. 2007), and silicon dioxide (Baby and Ramaprabhu 2010). The goal of the present work is to carry out a study on the effect of different parameters such as pH, scan rate, and the effect of changing the percentage of Fe2O3Nps added, on the determination of LG raw material, on pharmaceutical formulation, and in human urine samples.
Linagliptin (LG) was supplied from the European Egyptian Pharmaceutical Industries, Egypt, iron nanoparticles. Graphite powder and paraffin oil were purchased from sigma Aldrich. Universal buffer (B–R buffer) 0.04 M of pH 2–11 (CH3COOH + H3BO3 + H3PO4), was used as the supporting electrolyte, then 0.2 M NaOH was used to gain the desired pH value.
All voltammetric measurements were performed by stationary electrode using 797VA Computrace software (1.0) from Metrohm, Switzerland, electrochemical analyzer. A three-electrode cell system incorporating the carbon paste electrode as a working electrode and the impedance measurements were done by the three-electrode electrochemical workstation EC-Lab SP-150 potentiostat. A platinum wire as an auxiliary electrode was used with respect to calomel reference electrode. Energy-dispersive X-ray measurements were performed by EDX spectroscopy unit of Model Quanta 250 FEG (Field Emission Gun) with accelerating voltage 30 KV.
Preparation of CPE/Fe2O3NP sensor
Carbon paste electrode (CPE) was prepared by mixing graphite powder (0.5 g) with drops of nujol oil in a glass mortar. CPE/Fe2O3NP-modified electrode was prepared by mixing ratios of 5, 10, 12, 15, and 20% modified paste containing 95, 90, 88, 85, and 80 mg of graphite powder and 5, 10, 12, 15, and 20 mg of Fe2O3NP modifier mixed, respectively, with suitable amount of paraffin oil. The components were stirred for 10 min in a glass mortar at room temperature to achieve homogenous paste that was used for voltammetric measurements without preconditioning.
Validation in pharmaceutical samples
Five tablets of the commercial pharmaceutical Trajenta (5 mg LG/Tablet) were powdered and dissolved in deionized water. After supersonic treatment for 30 min, it was filtered and the concentration of LG in the working range was achieved, and then, SWV were recorded using the standard addition method.
Analysis of spiked urine samples
Urine was supplied from healthy volunteers; the spiked urine sample was centrifuged for 10 min at 13,000 rpm to get rid of protein residues, and then, the supernatant was taken carefully. An aliquot (1 mL) of the clear solution was added to a mixture of 9 mL B–R buffer to achieve a final concentration of 1.0 mM, at an optimum pH value to perform a calibration curve.
Effect of pH: The effect of solution pH on the electrochemical response of LG at CPE/Fe2O3NPs was recorded in B–R buffers and was found to be discerning over the pH range of 2–11. The peak potentials decrease gradually in solutions as pH is raised, manifesting that protons have taken part in the electrode reaction processes. The results show that pH 7 and 9 gave the maximum anodic peak current response, because the surface of iron oxide atoms act as Lewis acids and coordinate with water, which dissociates giving amphoteric hydroxyl group. These hydroxyl groups carry negative charges (Lefebure et al. 1998). And since the pKa value of LG is 9.86, therefore, an electrostatic attraction force was induced between the positively charged LG at alkaline pH values and the negatively charged iron oxide nanoparticles surface which give a high current response. So, the rest of the work will be performed at pH 7.4 (physiological pH of the body).
The surface areas were evaluated to be 0.072 cm2 and 0.143, for CPE and CPE/Fe2O3NPs, respectively, which indicates that the addition of iron oxide nanoparticles doubled the surface area of the sensor and therefore increases the electroactive sites that enhanced the current response of LG.
The electron transfer coefficient was found to be 0.53, and then, n was calculated to be 1.2 ≈ 1 which indicated that only one electron was involved in the oxidation of LG. The value of ks can be calculated from the intercept of the Laviron equation and was found to be 1.06 × 102 s−1.
Comparison of the proposed method with other methods
Linear range (μg/ml)
(Shehata et al. 2016)
(Shen et al. 2009)
(Vassilyev et al. 1985)
Methanol and phosphate buffer
(Vemula et al. 2015)
Also, the reduced LG donates electrons to Fe2O3NP surface. Therefore, resistance is decreased, or conductance is increased.
Influence of the square wave frequency (f)
The square wave frequency determines the intensity of the signal and the sensitivity of the technique. The relation between the anodic current and the square wave frequency shows a linear relation up to 140 s−1(Fig. 6, inset B). For larger values of frequency, the effect is almost negligible (O’Dea et al. 1993). This behavior corresponds to a totally irreversible process controlled by adsorption of the analyte on the electrode surface. Also, the peak potential should vary linearly with the logarithm of the frequency following the relationship:
A value of 0.66 was determined for αn, and as the electron transfer coefficient was found to be 0.53, therefore, n = 1.2, indicating the transference of 1 electron per LG molecule, confirming the data obtained in the scan rate section. Thus, the suggested mechanism for LG is presented in Scheme 1.
Specificity of the method is monitored by measuring the current response of an analyte in the presence of other interferences. Glucose sensors account for approximately 85% of the biosensor industry. Non-enzymatic glucose electrodes give higher oxidation current response, higher stability, lower cost, and greater sensitivity. Electrocatalytic processes are important for glucose oxidation, since it is a kinetically very slow process with negligible faradaic current, showing a non-diffusion-controlled process (Vassilyev et al. 1985). The electrocatalysis occurs via the adsorption of analyte on the electrode surface, which involves the d-electrons to form bonds with the analyte (Pletcher 1984).
These results indicate that these substances did not interfere with LG, which confirms the specificity of the proposed method, since the tolerance limit was less than 2%.
Application of linagliptin
In pharmaceutical formulation: For the purpose of the practical applicability of the proposed method and the modified sensor, the SWV method was applied for the investigation of LG in five Trajenta (5 mg/tablet) tablets without any sample extraction to obtain 1.0 mM LG solution. The validity of the proposed method was evaluated by the standard addition technique, and the accuracy has been determined giving satisfactory recoveries from 99.98 to 101.9%, and relative standard deviation from 0.04 to 0.63% reveals that the excipient presented in the tablet does not interfere with the active ingredient. The results suggested that CPE/Fe2O3NPs has high reproducibility and would be useful sensor for quantitative analysis of LG in pharmaceutical formulations.
In spiked urine sample: The validation of CPE/Fe2O3NPs for the quantitative assay of LG in urine was examined in B–R buffer pH 7.4. Six different concentrations on the calibration curve are chosen to be repeated for five times to evaluate the accuracy and precision of the proposed method, which shows a recovery values from 99.9–100.1%. Also, the statistical analysis of the results was obtained by applying the proposed and the reported methods, and the mean ± S.D. of LG in urine sample was found to be 100.05 ± 0.25 for the proposed method with t (2.776) value of 0.575 and F (19) value of 3.760 (n = 4), while the mean ± S.D. for the reported method was found to be 99.95 ± 0.13 when the values between parentheses was 85%.
This work describes a new and very simple CPE sensor for the electrochemical determination of linagliptin in pharmaceutical formulations, in urine samples, and in the presence of glucose and the co-administered drug MET using magnetic iron oxide nanoparticles. Some dynamic parameters are considered for the first time. The results showed that the method was sensitive, precise, and selective with no significant effects of the excipients background. A linear relation between LG peak current and its concentration in the range 0.03 to 86 μg/ml leads to a detection limit of 0.008 μg/ml.
The authors acknowledge with thanks NODCAR for the technical support.
This work has no funds.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
All authors contributed in this work and in writing the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The manuscript does not contain studies involving human participation.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- Abbas Moussa B, Mahrouse MA, Fawzy MG (2019) A validated LC-MS/MS method for simultaneous determination of linagliptin and metformin in spiked human plasma coupled with solid phase extraction: application to a pharmacokinetic study in healthy volunteers. J Pharm Biomed Anal 30(163):153–161CrossRefGoogle Scholar
- Bard AJ, Faulkner LR, Leddy J, Zoski CG (1980) Electrochemical methods: fundamentals and applicationsEditor. Wiley, New York, p 2Google Scholar
- Compton R G, Banks C E (2007) Understanding Voltammetry, World ScientificGoogle Scholar
- El-Bagary RI, Elkady EF, Ayoub BM (2013) Spectrophotometric methods for the determination of linagliptin in binary mixture with metformin hydrochloride and simultaneous determination of linagliptin and metformin hydrochloride using high performance liquid chromatography. Int J Biomed Sci 9:41PubMedPubMedCentralGoogle Scholar
- IHT Guideline (1997) Fed Regist:62Google Scholar
- Jerez LB, García-Pérez U, Zambrano-Robledo P, Hernández-Moreira J (2014) Carbon paste electrode modified with BiVO4 to sense metformin. Int J Electrochem Sci 9:4643–4652Google Scholar
- Mohamed MA, Fekry AM, El-Shal MA, Banks CE (2017) Incorporation of tetrazolium blue (TB)/gold nanoparticles (GNPs) into carbon paste electrode: application as an electrochemical sensor for the sensitive and selective determination of sotalol in micellar medium. Electroanalysis 29:2551CrossRefGoogle Scholar
- Padmaja N, Veerabhadram G (2015) Development and validation of analytical method forsimultaneous estimation of empagliflozin and linaglptin in bulk drugs and combined dosage forms using UV-visiblespectroscopy. Pharm Lett 7:306–312Google Scholar
- Prodan AM, Iconaru SL, Chifiriuc CM, Bleotu C, Ciobanu CS, Motelica-Heino M, Sizaret S, Predoi D (2013) Magnetic properties and biological activity evaluation of iron oxide nanoparticles. J Nanomater 2013:7Google Scholar
- Vemula P, Dodda D, Balekari U, Panga S, Veeresham C (2015) Simultaneous determination of linagliptin and metformin by reverse phase-high performance liquid chromatography method: an application in quantitative analysis of pharmaceutical dosage forms. J Adv Pharm Technol Res 6(1):25–28CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.