One-Pot Synthesis of Fluorescent Nitrogen and Sulfur-Carbon Quantum Dots as a Sensitive Nanosensor for Trimetazidine Determination

Fathalla Belal1, Mokhtar Mabrouk2, Sherin Hammad2, Aya Barseem3, * and Hytham Ahmed3
1Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt

2Department of pharmaceutical analytical Chemistry, Faculty of Pharmacy, Tanta University, Tanta, Egypt

3Pharmaceutical Analysis Department, Faculty of Pharmacy, Menoufia University, Egypt

*Correspondence to [[email protected]]

Abstract
A water-soluble and highly stable N,S-doped CQDs (N,S-CQDs) have been synthesized by a low-cost strategy using citric acid (CA) and thiosemicarbazide (TSC) in one-step as a fluorescent nanosensor. The achieved N, S-CQDs have strong emission at 446 nm upon excitation at 370 nm and a high quantum yield of 58.5%. The quenching effect on the prepared N,S-CQDs has been utilized for determination of Trimetazidine (TMZ) spectrofluorimetrically in a wide linear range of 0.04-0.5µM (0.0106 – 0.133 µg mL-1) and low limit of detection of 0.01μM (0.002 µg mL-1). Furtherer, CDs was used to determine TMZ in its pharmaceutical formulations as well as in human plasma as a simple and rapid fluorescent probe. The method was tested in compliance with the ICH guidelines. The results obtained were statistically compared to those given by a reported method showing no significant variations regards accuracy and precision.

Keywords
Heteroatom-doped carbon quantum dots; trimetazidine; fluorescent nanosensor; plasma.

1. Introduction
Trimetazidine dihydrochloride (TMZ) is 1-[(2,3,4-trimethoxyphenyl) methyl]-piperazine dihydrochloride (Fig. 1). It is indicated for the treatment of angina pectoris. TMZ enhances the use of myocardial glucose by several actions such as reactive oxygen species level reduction, modification of the composition of cellular lipids and oxidation inhibition of mitochondrial fatty acids [1]. Several analytical methods were documented for the determination of TMZ in human plasma and dosage forms, viz HPLC [2-10], LC-MS [11-15], GC-MS[16,17], HPTLC [18-20], spectrophotometry [21-29], spectrofluorimetry[29], potentiometry [30,31], voltammetry [32], and chemiluminescence[33].
The use of nanomaterials in analytical research became a rising field of interest. The fluorescent nanoparticles have the advantages of high sensitivity and reproducibility, and low-cost instrumentation [34,35]. The fluorescence sensor is an alternative technique for measuring extremely low concentrations of analytes. Fluorescence probe technique could be used to evaluate a number of analytes, through quenching of fluorophore fluorescence, such as metal ions [36,37], pollution molecules[38] and biomolecules[39]. Nevertheless, fluorescent probes approaches have been seldom reported in TMZ determination, consequently, seeking for an easily available and efficient TMZ fluorescent sensor is essential and significant.
Carbon quantum dots as a new group of fluorescent nanoparticles are inexpensive, less toxic with high photostability, reliable photoluminescence and excellent biocompatibility. In the areas of bioimaging, biosensors and pharmaceutical delivery, CDs were widely used[40-42]. Moreover, heteroatom CDs are currently being developed to improve the optical and electrical properties of these nanoparticles. For example, N, S- doped CQDs express a high degree of photostability and quantum yield [43-47]. Their fluorescence intensity can be reduced by interesting analytes and thus provide a basis for expanded applications of analysis.

In this work, nitrogen and sulfur doped carbon quantum dots (N,S-CQDs) with high luminescence and water solubility were prepared successfully. Citric acid and thiosemicarbazide were used as a carbon source and as N and S source, respectively in a reflux hydrothermal treatment. TMZ was found to be a quencher of the fluorescent N,S-CQDs; leading to a simple method for TMZ determination (Scheme 1). Finally, these N,S-CQDs were successfully utilized for the determination of TMZ in its tablets and human plasma samples.

2. Experimental
2.1. Materials and Reagents:
Analytical-grade chemicals and reagents were purchased from Merck Company (Germany). Chemicals were: Sodium hydroxide, Sodium chloride, hydrochloric acid, citric acid, disodium phosphate and boric acid. Highly pure distilled water was used during the analysis. TMZ was kindly provided by the Egyptian Drug Authority (EDA)-Egypt. Vastarel® Tablets, 20 mg TMZ per tablet (product of Servier, Philippines) were obtained from commercial sources in the local market. Human plasma samples were kindly provided by Menoufia University Hospital, Menoufia, Egypt and kept at -80 °C until use after gentle thawing.

Standard TMZ stock solution (1.0 mM) was obtained by weighing 0.026 gm of TMZ powder and dissolving distilled water to 100 mL then diluting as appropriate by the same solvent to give different concentrations. Teorell and Stenhangn Buffer (Citrate-phosphate-borate/HCl buffer) stock solution was obtained by dissolving a total of 8.803 g disodium phosphate, 7.0 g citric acid, 3.54 g boric acid in water, adding 0.243 L of 1M Sodium hydroxide in a 1.0 Volumetric flask , completing to the mark by a freshly boiled and cooled distilled water. Different pH buffer solutions (2.0 – 12.0) were formed by adding definite volumes of 1M hydrochloric acid solution to 20 mL of the stock buffer solution.

2.2 Instrumentation:
• The particle size of S, N-CDs was characterized by using the TEM-2100 Transmittance Electron Microscope (Korea) with resolution as high as 0.19 nm (in UHR configuration and paired AMT Camera.
• IRAffinity-1S FTIR Spectrophotometer from Shimadzu (Columbia, united states) was used for FT-IR spectrum measurement, With wavelength 7,800 to 350 cm- and KBr disc system.
• A Shimadzu Scanning Spectrophotometer, UV-1601PC model, with wavelength range from 190-1100 nm.
• Fluorescence spectra were recorded by Jasko Fp-6300 Spectrofluorimeter with high sensitivity, the signal-to-noise ratio is in excess of 550:1, and provides wavelength range from 220 to 750 nm.
• Reax Top Vortex mixer (541-10000-00), Heidolph, rotation speed range (0 – 2,500 rpm)- Germany.
• Bench top centrifuge Hunan, China.
• Adwa pH Meter of AD1030, pH ranges from 2.0 to 14.0, pH resolution (0.01 pH / 0.001 pH).

2.3 Preparation of N,S-CQDs

N,S-CQDs were synthesized by hydrothermal treatment of CA and TSC as precursors. Briefly,
2.5 mMole of citric acid was mixed and dissolved with 7.5 mMole of thiosemicarbazide in 20 mL of distilled water. The solution was heated under reflux at 160 °C overnight till the appearance of an orange color. The solution was cooled down to room temperature, filtered by a membrane filter (0.2 μm). Then it was stored in refrigerator and turned into room temperature before use and characterization. The solution showed high stability without precipitation for 3 weeks.

2.4. Quantum yield measurements
The efficiency of photon emission, the N,S-CQDs quantum yield, was estimated using the following equation (47,48):
Φx=Φst×(Fx/Fst) ×(ηx/ηst)2×(Ast/Ax)
Where:X subscript is the unknown sample and st subscript is the reference sample. Φ is the yield of quantum.
F refers to integrated measured emission intensity. η refers to refractive index of solvent.
A is absorbance.The reference was quinine sulfate in H2SO4 (0.1 M), QY was 0.54 at 350 nm. ηx/ηst is equivalent to 1 in aqueous solutions.

2.5. Recommended Procedure:

Into a series of measuring flasks (10 mL), 100 μL aliquots of N, S-CQDs stock solution (1 g L- 1) were mixed with working standard TMZ solutions (10μM) having different concentrations. Then, 3 mL aliquots of Teorell and Stenhagen buffer of pH 3.0 were added to each flask; the solutions were shaken well and completed to the mark with distilled water to reach the desired range of 0.04 – 0.5 μM. The fluorescence quenching values (after excitation at 370 nm) were measured at 446 nm at 298, 310 and 318 K in a Quartz cuvette of 3cm. The fluorescence
quenching values (ΔF) was plotted against final drug concentrations (µM) to give the standard calibration curve. The calculations of linear data regression were also conducted.

2.6. Determination of TMZ in tablets:
Ten Vastarel ®Tablets were carefully pulverized and weighed. An accurately weighed amount of the powder equivalent to 2.6 mg of TMZ was transferred into a small flask in a quantitative manner. The powder was then extracted with 5 mL of distilled water using ultrasonic vibration for 5 min. Then the solution was transferred quantitively into 10 mL volumetric flask. The volume was finalized with the same solvent then filtered to get a solution (1.0 mM). This solution was diluted by transfering100 μL into 10 mL volumetric flask, then diluting with water up to the mark to provide a (10 μM) solution. More serial dilutions were made, and the process was completed as above to meet the concentration range (section 2.5). The percentage recoveries were calculated adopting the regression equation.

2.7. Determination of TMZ in spiked human plasma
Drug-free human plasma (1 mL) was spiked with 100 μL of standard TMZ stock solution into 10 mL centrifuge tube, vortex mixed for 1 min, 3 mL of acetonitrile were added, then the solution was centrifuged for 20 min at 5000 rpm. In order to provide the working range, various volumes of the clear supernatant were withdrawn and diluted with water as appropriate. As in the Recommended Procedure (section 2.5), the same steps were followed. The % recovery was calculated using the corresponding regression equation.

3. Results and Discussion:
TMZ was tested in a simple, sensitive and cost-efficiency way by the proposed study. N, S- CQDs were successfully prepared in a single step. CA and TSC have been used in a hydrothermal reflux treatment respectively as a carbon source and a N and S source (Scheme 1). Our method has greater sensitivity in comparison with other reported spectroscopic methods (Table 1).

3.1. Characterization of N, S-CQDs

The prepared N, S-CQDs was confirmed by different techniques presented in (Fig. 2) as following: ultraviolet–visible spectroscopy was presented in (Fig. 2A), an obvious UV-Vis absorption peak for N,S-CQDs was observed at 378 nm. This peak is due to n-π* transition of
the carbonyl/amine functional groups as the surface states confine the excited-state energy producing strong emission[48,49]. Excitation and emission fluorescence spectrum was shown in (Fig. 2B), the orange aqueous solution of S,N-CDs showed high blue fluorescence at λem = 446 nm upon excitation at 370 nm. The prepared S,N-CDs showed a high quantum yield (58.5%) using quinine sulfate as a reference. The FT-IR analysis of N-S-CQDs functional groups (Fig. 2C) was also performed. It shows vibrations of 1212 cm-1 and 1100 cm-1 that represent the -C-C- and C = S, moieties stretching modes, respectively. A vibration of 1770 cm-1 due to (-C=O) carbonyl groups absorption. A broad band of the NH / OH groups in the region between 3600-3100 cm-1[50]. To indicate the exact size and shape of N,S-CQDs, the synthesized N,S-CQDs were characterized by TEM and shown in(Fig. 2D), the TEM micrograph displayed the well separated spherical N,S-CQDs with a range of ( 2.7 – 7) nm in diameter.

3.2. Mechanism of fluorescence response of N,S-CQDs to TMZ
The quenching mechanism is static as evident from the UV–vis absorption spectra (fig.2A). The absorbance peak of TMZ was at 269 nm and the N,S-CQDs-TMZ system also showed the absorbance peak at 269 nm. The absorbance of TMZ doesn’t overlap with excitation or emission of N,S-CDs. The spectrum of UV absorption of N,S-CQDs-TMZ system and that of sum absorption between N,S-CQDs and TMZ were not overlaid within the experimental error, which indicated the complex ground state formation between N,S-CQDs and TMZ. The absorption spectrum of N,S-CQDs -TMZ system showed a minute decrease in absorption at 269 nm compared to the sum absorption spectrum of N,S-CQDs and TMZ which implies the hypochromic effect following the formation of a N,S-CQDs-TMZ complex. These findings showed the static quenching mechanism of N,S-CQDs -TMZ system exclude dynamic types[51]. Furthermore, Stern-volmer equation was applied to prove the static quenching mechanism. The fluorescence quenching mechanism of N,S-CQDs-TMZ system has been evaluated by Stern-Volmer’s equation. The F0/F was plotted versus TMZ level (Fig. 3) using the Stern-Volmer Equation:
(F0/F=1 + KSV [Q] = 1 + KqƮ0 [Q])Where: F0 and F are fluorescence intensity of N,S-CQDs, without and with addition of the studied drug, respectively. KSV is a constant (the curve slope), [Q] is studied drug concentration, Kq refers to a constant of the fluorescence quenching, and Ʈ0 is lifetime of fluorescence in quencher absence.

The average lifetime is 2.89 x 10-9 s for N,S-CQDs. The quenching efficiency was studied at three different temperatures. The calibration graph shows KSV values at 298, 310 and 318 K as: 0.5723×106, 0.4352×106 and 0.3937×106 L mol-1, respectively. Applying the quenching results to the previous equation resulted in Kq values of 0.198 x1015, 0.150 x1015 and 0.136 x1015 at 298, 310 and 318 K, respectively. There is a confirmed largest Kq (1–2) x 1010 Lmol- 1S-1 collision quenching [52]. From these data we concluded that, the quenching process is a static one, as KSV decreased with increasing the temperature while Kq values exceeded (1–2) x 1010 Lmol-1S-1.

3.3. Optimization of Experimental Conditions:
3.3.1. pH
In order to investigate the pH influence on the fluorescence of N,S-CQDs, a wide range of pH was used between 2.0 and 12.0 using Teorell and Stenhangen buffer. At λem = 446 nm, the pH that gave the highest ΔF value was pH 3.0 (Fig. 4A).

3.3.2. Buffer Volume
Through testing various volumes of Teorell and Stenhangen buffer of pH 3.0, the maximum quenching effect was achieved upon adding 3.0 mL of the buffer (Fig. 4B).

3.3.3. Incubation time
After adding TMZ to N,S-CQDs solution, the incubation period is noticed. Time intervals between 0.5 to 30 min were tested for quenching N,S-CQDs fluorescence intensity. All experiments were found to be completed in 1 minute and showed stable fluorescence signals for over at least 30 minutes (Fig. 4C).

3.3.4. Ionic Strength
The ionic strength was studied by the addition of 0.1 M to 2M sodium chloride solution to the buffer solution. However, N,S-CQDs fluorescence intensities were found to be constant in all cases pointing out to the stability of N,S-CQDs towards the ionic activity (Fig. 4D).

3.4. Validation:
Testing the validity of the proposed method has been carried out in compliance with ICH guidelines [53].

3.4.1. Linearity and range
It was observed that by increasing the concentration of TMZ, the fluorescence of the prepared CQDs decreased in a quantitative manner (Fig. 5). The fluorescence emission intensities of N,S-CQDs with TMZ were measured after optimization of conditions and the curve of calibration was designed by relating the ∆F against the drug concentration (Fig. 5). A wide linear correlation is determined between fluorescence intensity of N,S-CQDs and TMZ concentrations in the range of 0.04 – 0.5 μM. Table 2 displayed the output data and statistical parameters which show high regression coefficients, with low Sa, Sb and Sy /x values that refer to standard intercept and slope deviation and residual standard deviations, respectively.

3.4.2. LOD and LOQ
The values of limit of detection (LOD) and limit of quantitation (LOQ) were based on guidelines of ICH Q2 (R1) [53], LOD was 0.01 μM and LOQ was 0.032 μM that point out the method sensitivity.

3.4.2. Accuracy
Calculation of the average percent recovery of pure TMZ samples across the given concentration range for both the developed and the reference methods tested the accuracy. The comparison method [23] relied on measuring the absorbance of the formed chromogen due to the reaction between TMZ and chloranil acetaldehyde reagent at 627 nm. The Student’s t-test and the variance ratio F-test were statistically utilized and the findings of both the proposed and comparison methods revealed that no substantial difference, regarding accuracy and precision occurred (Table 3).

3.4.3. Intra-day and inter-day precision:
By measuring three different concentrations within the range of the bulk form of TMZ and then performing on the same day, three times replicate analysis, the intra-day precision was acheived. The small value of % RSD was tested for intraday precision.The results are shown in Table 4. Repeated study of selected levels of TMZ in pure form for three consecutive days tested the intermediate precision (inter-day).The low-percent RSD (Table 4) Verified the high precision of the method developed.

3.4.4. Robustness
Robustness was evaluated when slight variations were made to the experimental conditions. The pH was modified by ± 0.2 unit, the volume of buffer was changed by ± 0.2 mL and the incubation period has been modified by ± 0.5 min. The small variations during daily work did not have any impact on the results.

3.4.4. Selectivity
Selectivity of the method was shown by quantifying TMZ in the presence of excipients of tablet dosage form such as (mannitol, maize starch, talc, …) and in the presence of plasma matrix. The method selectivity was determined by % recovery estimation. If a relative error of less than ± 5% in the fluorescence intensity of CDs caused by the interfering substances, no interference in TMZ determination is considered. The mean % recovery of Vastarel® 20 mg tablet was found to be 100.91±1.09% (Table 5). Also, TMZ was successfully determined in spiked human plasma with mean % recovery 96.71 ± 1.03 (Table 6). The good % recovery indicate the reliability and selectivity of the sensing system for TMZ.

3.5. Applications:
3.5.1. Analysis of TMZ in dosage form:
The frequently applied t-test and F-test have been used for this method to evaluate TMZ in its tablets (Table 5). The satisfactory % recoveries obtained indicate that the excipients often found do not interfere with the assay. This shows the suitability of the proposed approach for the assessment of the drugs tested in their dosage forms. The measured and theoretical values at 95% of confidence were in good agreement with the reference method.

3.5.2. Determination of TMZ in human plasma:
The developed procedure was adopted to assess the drug in plasma by spiking into human plasma samples. As stated earlier, samples have been prepared and analyzed. Following oral dosage of 20 mg of TMZ, the mean plasma peak concentration was 53.6 μg L-1 (0.2 µM)[54].The proposed method could achieve the reported TMZ plasma concentration, as its levels in plasma fall within its linearity range as referred to in Table 6. The results obtained show that the mean absolute %recovery and the SD in plasma samples is 96.71 ± 1.03.

4. Conclusion
The development of the eco-friendly optical sensor (N,S-CQDs) for determination of TMZ in spiked human plasma samples and commercial tablets was successfully achieved. The synthesized N,S-CQDs by CA and TSC has a small size of 4.7 nm and high strength of fluorescence and photostability. The sensing probe has many advantages like low cost, simplicity, high selectivity and sensitivity. The studies show that N,S-CQDs and TMZ react rapidly and can effectively be used in the field of pharmaceutical and bioequivalence research and in pharmaceutical enterprises.

Author Declarations

Conflict of Interest: The authors have no conflicts of interest to declare that are relevant to the content of this article.

Funding: No funds, grants, or other support was received.

Ethics approval: Not applicable.

Consent to participate: Not applicable.

Consent for publication: Not applicable.

Data availability: All data generated or analyzed during this study are included in this published article.

Code availability: Not applicable.

Authors’ contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Aya Barseem]. The first draft of the manuscript was written by [Aya Barseem] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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