Exploring a green and precise approach based on graphene oxide-cobalt ferrite nanocomposite for detection and quantification of vitamin B9 and antibacterial assessment

Abstract

In this study, magnetic cobalt ferrite/graphene oxide (CoFe₂O₄/GO) sorbent was synthesized and used in magnetic dispersive micro-solid phase extraction (MD-µ-SPE) combined to spectrofluorometric as a green, precise, and effective method to separate and detect of vitamin B9 in food and biological samples. The structure, morphology and magnetic properties of CoFe₂O₄/GO nanocomposite was characterized by Fourier transform infrared (FT-IR) spectroscopy, X-Ray diffraction (XRD), field emission-scanning electron microscopy (FE-SEM), and vibrating sample magnetometer (VSM). Four parameters of pH, sorbent dose, temperature, and sonication time were selected as effective variables on the process and optimized by central composite design (CCD). Under optimized conditions, the linear range, limit of detection, intra-day (repeatability) and inter-day (intermediate) precision were obtained 15–750 ng mL⁻¹, 3.96 ng mL⁻¹, 3.3–3.8%, and 4.2–4.9%, respectively. The material’s antimicrobial potency also evaluated, showcasing notable inhibitory action against both Gram-negative Escherichia coli bacteria, with a zone of inhibition measuring 17.5 mm at the highest concentration, and Gram-positive Staphylococcus aureus bacteria, which displayed a 14.5 mm ZIO at similar concentrations. To the best of our knowledge, this is the first report on using CoFe₂O₄/GO for vitamin B9 extraction and detection. This work introduces a novel, eco-friendly, and effective analytical platform that combines the magnetic properties of CoFe₂O₄ with the high surface area and functional groups of GO for targeted vitamin analysis. These results demonstrate that the synthesized nanocomposite possesses both excellent adsorption capability and antibacterial activity.

Introduction

Vitamins are organic compounds that are essential for the human body as micronutrients. These compounds are not sufficiently synthesized by the body and therefore they are often supplied through the diet¹. Some vitamins, such as B vitamins and vitamin C, are water-soluble, while others, such as vitamins A, D, E, and K, are hydrophobic. As coenzymes, these vitamins play significant role in main metabolic reactions occur in the body, including the metabolism of carbohydrates, lipids, and proteins. They also affect the health of skin, eyes, hair, muscles, and liver². Vitamin B9 (folic acid) is one of the B vitamins that is naturally present in many food sources. The presence of vitamin B9 and its derivatives is necessary in important biochemical processes such as amino acid metabolism, nucleic acid synthesis and cell division. Also, this vitamin can be very vital for women to produce blood during pregnancy. Vitamin B9 can be very effective in preventing diseases such as various types of cancer, stroke and vascular disorders^3,4. Using enough level of this vitamin in the diet can reduce risk of heart diseases⁵. Whereas the deficiency of vitamin B9 may cause several diseases including anemia, cancer, hypertension, diabetes, psychiatric disorders, etc⁶. Therefore, it is considered essential to examine and determine vitamin B9, especially in food and biological samples.

So far, many analytical methods have been reported for determination of vitamin B9, like high performance liquid chromatography (HPLC)⁷, spectrophotometry⁸, chemiluminescence⁹, fluorescence¹⁰, and electrochemistry¹¹. Some of these methods require expensive equipment, materials and solvents, trained personnel, long analysis time, etc., which has seriously limited their application. Among these methods, fluorescence has high speed, sensitivity and selectivity. Furthermore, this technique is simple and accessible in terms of operator^{12, 13–14}. Generally, before detection with instrumental techniques, a sample preparation step is inevitable so as to eliminate the interfering effects of the sample matrix, extraction and separation of the target analytes and pre-concentrate of the compounds are present in the samples in trace amounts¹⁵. But since the sample preparation stage is the most time-consuming and laborious stage of an analytical process, nowadays various sample preparation methods are implemented with the aim of saving time, materials and equipment. In recent years, the development of green analytical procedures has received considerable attention due to growing environmental concerns. Analytical methods based on miniaturized sample preparation, reduced solvent consumption, recyclable materials, and low-energy synthesis approaches are in accordance with the fundamental principles of green chemistry. Therefore, our proposed magnetic dispersive micro-solid phase extraction (MD-µ-SPE) method, in combination with a recyclable CoFe₂O₄/GO nanocomposite and environmentally friendly solvents, represents a promising green alternative for trace analysis of biomolecules¹⁶. MD-µ-SPE method is a cost-effective method compared to other solid phase extraction methods due to perfect adaptability and selectivity, less solvent, time consumption and easier separation of two phases^17,18.

Graphene oxide (GO) is a special kind of carbon materials with abundant oxygen functional groups on the surface, such as hydroxyl, carbonyl, carboxylic and epoxide groups¹⁹. Owing to the large specific surface area, high colloidal stability and low cost, GO has been widely used as the potential adsorbent in water treatment²⁰. In spite of the various advantage of GO, using GO alone as a sorbent in the extraction process, due to its tendency to self-aggregate in the separation step, can cause problems. As a matter of fact, its separation from the liquid phase after the extraction process is an important challenge. Therefore, researches have progressed towards modifying the surface and functionalizing GO with the aim of optimally using it as a sorbent in various separation processes with maximum efficiency. Recently, in this regard, extensive research has been conducted on the bonding of magnetic nanoparticles to graphene oxide, the result of which was the easy separation of GO from the liquid phase after the adsorption process and with the help of a magnetic field^21,22. Combining GO with other nanomaterials, such as metal oxides, can lead to the formation of several nanocomposites with many applications. CoFe₂O₄ is currently used in the synthesis of new sorbents. High physical and chemical stability and high saturation magnetism are unique properties of CoFe₂O₄. Furthermore, studies have shown the higher structural stability of CoFe₂O₄ nanoparticles compared to magnetite (Fe₃O₄) nanoparticles²³. These examinations demonstrated functionalizing GO with CoFe₂O₄ magnetic nanoparticles is an excellent alternative to improve the sorption performance of these materials²⁴. Pathogens such as Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are known as an important source of serious foodborne diseases, and they can intensify the decreasing effectiveness of traditional antibiotics^25,26. Metal oxide nanocomposites have proven powerful antibacterial efficacy owing to their extensive surface area and ability to generate reactive radicals. These features put nanocomposites at the head of the expansion of inorganic antibacterial agents and it indicates them as a beam of hope in the fight against microbial resistance^{27, 28–29}.

In this study, the evaluation of CoFe₂O₄/GO nanocomposite as a solid-phase sorbent with the characteristics such as good selectivity, high sorption capacity and eco-friendly characteristics dedicated to isolate and quantify of vitamin B9 in food and biological samples prior to their quantification by fluorescence spectroscopy. A multivariate optimization approach has been used to optimize the influential variables on the extraction recovery of vitamin B9. While previous research, such as the work by Chang et al. (2020)³⁰ focused on the application of CoFe₂O₄/GO composites for the adsorption of organic pollutants from environmental samples, the present study extends the use of this nanocomposite to the analytical determination of a biologically relevant compound. Furthermore, the antimicrobial potential of the synthesized nanocomposite was assessed through antibacterial assays against E. coli and S. aureus, demonstrating its multifunctionality and potential applicability in food safety and biomedical monitoring.

Experimental

Reagents and solutions

Vitamin B9 was purchased from Aldrich (Chemical Co., Milwaukee, WI, USA). Sulfuric acid (H₂SO₄) 98%, graphite powder, potassium permanganate (KMnO₄), hydrogen peroxide (H₂O₂), acetone, iron (III) nitrate nonahydrate (Fe (NO₃)₃⋅9H₂O), cobalt chloride hexahydrate (CoCl₂⋅6H₂O), sodium borohydride (NaBH₄), acetonitrile as desorption solvent and 0.1 M NaOH and 0.1 M HCl solutions (to adjust the pH) were provided from Merck (Darmstadt, Germany,) and utilized without any additional purification. A standard solution of vitamin B9 with a concentration of 100 mg L⁻¹ prepared in deionized water. Then, working solutions with desired concentrations obtained by diluting the standard solution with deionized water. In all experiments utilized deionized water and for washing the dishes used HNO₃ (10%) and deionized water.

Apparatus and software

To record the fluorescence spectra used the Spectrofluorometer model Shimadzu RF-5301PC: (Shimadzu, Japan). For pH adjustment, the pH meter model 2211 HI of the American company HANNA applied. The ultrasonic bath (James Ultrasonic system, 37 kHz & 160 W, England) and magnetic stirrer (Heidolph, model D-91126, Schwabach) device utilized to synthesis and the proposed extraction processes.

Design-Expert Software Version 12.0.3.0 (Stat-Ease Inc., Minneapolis, MN, USA) applied to optimize the parameters affecting process.

Synthesis of graphene oxide (GO)

The modified Hummer method used for the synthesis of GO³¹. First, 83.33 mmol of graphite powder (1.0 g) and 23 mL of 98% sulfuric acid transferred to a 250 mL round bottom flask and stirred in an ice bath (20 °C). 18.98 mmol potassium permanganate (KMnO₄) (3.0 g) added to the mixture under the same temperature. Next, the resulting mixture stirred at 30 °C for 30 min until a light brown precipitate was obtained. Then, 46 mL of deionized water slowly added to the precipitate and the mixture stirred at 98 °C for 15 min. 140 mL of deionized water and 30 mL of 30% H₂O₂ added to the mixture, and after transferring to centrifuge tubes, it was centrifuged at 5000 rpm for 10 min. In the following, precipitate was washed 3 times with 5% HCl (to remove sulfate ions), 5 times with deionized water (to release chloride ions and obtain a neutral pH) and finally, 3 times with acetone (to remove the remaining moisture), filtered and dried for 24 h in a vacuum oven at 65 °C.

Synthesis of Cobalt ferrite nanoparticles (CoFe₂O₄)

CoFe₂O₄ nanoparticles synthesized by hydrothermal method³⁰. First, 4.45 mmol of Fe (NO₃)₃·9H₂O (1.8 g) and 2.06 mmol of Co (NO₃)₂·6H₂O (0.6 g) dissolved in 50 mL of deionized water. The molar ratio of Fe³⁺ to Co²⁺ precursors was maintained at approximately 2.16:1, close to the theoretical stoichiometric ratio required for CoFe₂O₄ formation, to ensure phase purity and homogeneous particle distribution. The solution placed in an ultrasonic bath for 30 min to achieve uniform dispersion and minimize agglomeration. Then, 37.01 mmol of sodium borohydride (NaBH₄) (1.4 g) dissolved in 20 mL of deionized water and used as a reducing agent. Next, the prepared NaBH₄ solution added dropwise to the prepared metal ions solution over 1 h at room temperature to control the nucleation rate and promote uniform particle growth. Finally, the solution transferred to a 100 mL hydrothermal reactor (autoclave with Teflon coating) and placed in an oven at 200 °C for 2 h. These reaction conditions provide a highly pressurized and thermodynamically stable environment for controlled crystal growth. After cooling to room temperature, the product was centrifuged 3–4 times with deionized water (5 mL) to remove residual ions, and finally dried in an oven at 60 °C for 12 h.

Synthesis of graphene oxide-cobalt ferrite nanocomposite (CoFe₂O₄/GO)

0.073 mmol of GO (0.15 g) and 0.32 mmol of CoFe₂O₄ (0.075 g) dissolved in 50 mL of ethanol by stirring for 1 h at room temperature. Then the solution transferred to a 100 mL autoclave and placed in an oven for 4 h at 100 °C. Eventually, the obtained product centrifuged 3–4 times with deionized water (5 mL) and put it for 24 h in an oven at 60 °C to dry³¹. This synthesis was performed under mild and environmentally friendly conditions without the use of hazardous reagents or high-temperature calcination. The overall procedure is consistent with the principles of green chemistry, as it involves low energy consumption and the use of non-toxic, recyclable solvents.

Extraction procedure based CoFe₂O₄/GO nanocomposite

To perform the extraction process, a 50 mL test tube for transferring a certain amount of vitamin B9 with a concentration of 300 ng mL⁻¹ used. The pH of the solution adjusted to 5.5 with 0.1 M acetate buffer solution. Next, 18.0 mg of CoFe₂O₄/GO nanocomposite added to the solution and diluted with deionized water to 50 mL. Solution sonicated for 16 min at 35 °C in an ultrasonic bath. After the adsorption process, in order to separate the phases, the sorbent containing the analyte kept at the bottom of the test tube using an external magnet. The supernatant solution decanted and 0.7 mL of acetonitrile desorption solvent added. Then, using an external magnet, the sorbent completely separated from the solution containing the desired analyte. Finally, the fluorescence spectrum recorded at 352 nm with an excitation wavelength of 285 nm.

Preparation of real samples

To verify the potential use of the prepared nanocomposite, real samples containing mint, broccoli, lettuce, and urine were chosen. In this study, a collection of real samples achieved from a local supermarket in Birjand city (Iran).

To prepare of the mint sample, 5.0 g of powdered mint added to a 250 mL Erlenmeyer flask containing 100 mL of deionized water. The solution heated to 100 °C for 10 min until the mint contents extracted. The final solution filtered and stored at 4 °C³².

Broccoli sample was washed with water and dried in an oven for 24 h. After that, 1.0 g of grounded sample added to 5 mL of HNO₃ and 5 mL of H₂O₂ and heated until 2 mL of the solution remained. The obtained solution filtered and diluted to a volume of 200 mL with deionized water³³.

Lettuce leaves were washed with water, and after being cut into pieces, dried in an oven at 80 °C for 10 h. Next, 1.0 g of grounded lettuce sample transferred to a 250 mL beaker. Then, 10 mL of concentrated HNO₃ and 5 mL of concentrated H₂SO₄ added and the solution gently heated at the same time. Finally, the solution transferred to a 200 mL flask and made up to volume with deionized water.

A urine sample was collected from a healthy adult human volunteer who had not taken any medication prior to sample collection. Written informed consent was obtained before sampling. All procedures involving human participants were conducted in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments. All experimental protocols were approved by the Ethics Committee of the University of Birjand. However, no formal approval number was provided, as is common practice at this institution. A 10 mL aliquot of the sample was centrifuged at 4000 rpm for 15 min to remove suspended solids. The resulting clear supernatant was then subjected to the proposed MD-µ-SPE procedure using the synthesized sorbent under optimized extraction conditions.

Results and discussion

Structure and morphology of the as-synthesized samples

The FT-IR spectra of synthesized compounds is shown in Fig. 1a. The peaks reported at 3374.33 cm⁻¹ for GO, 3426.40 cm⁻¹ for CoFe₂O₄ and 3354.09 cm⁻¹ for CoFe₂O₄/GO are all related to the stretching vibration of OH bonds. In the spectrum of GO, the absorption peaks located at 1718.59 cm⁻¹ and 1618. 63 cm⁻¹ are devoted to the stretching vibration of the C = O bond. Moreover, in this spectrum, absorption bands assigned to C-O epoxy and C-O alkoxy groups can be seen at 1223.49 cm⁻¹ and 1052.27 cm⁻¹, respectively³⁴. Regarding CoFe₂O₄ and CoFe₂O₄/GO nanocomposite, the observed peaks at 590.22 cm⁻¹ and 588.95 cm⁻¹ indicate the vibration of the Fe-O bond in the tetrahedral structure. The peaks around 454.51 cm⁻¹ and 451.36 cm⁻¹ are related to the vibration of the Co-O bond in the octahedral structure³⁵. The weak peak observed at 1634.86 cm⁻¹ for CoFe₂O₄ can also be attributed to the bending vibrations of the OH bond in the water molecule. Thus, the FT-IR spectra further affirm the successful synthesis of GO, CoFe₂O₄, and the CoFe2O4/GO composites.

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Fig. 1

(a) FT-IR spectra, (b) XRD pattern, (c) VSM analysis, (d) EDX analysis and (e) Mapping analysis images related to synthesized materials of GO, CoFe₂O₄ and CoFe₂O₄/GO.

XRD patterns of as-synthesized CoFe₂O₄ and CoFe₂O₄/GO are presented in Fig. 1b. In the case of CoFe₂O₄, the peaks at 18.48˚, 30.22˚, 35.58˚, 43.22˚, 53.57˚, 57.12˚, 62.67˚and 74.12˚ can be assigned to the plates (111), (220), (311), (400), (422), (511), (440) and (533) indexed. Furthermore, in the XRD pattern of CoFe₂O₄/GO nanocomposite, all the peaks related to CoFe₂O₄, the GO peak is located at 11.92˚, which is due to the crystal plane (002). Indeed, this spectrum confirms the existence of GO phase, CoFe₂O₄ spinel and the absence of other phases or impurity diffraction peaks shows the high purity of the synthesized compounds³¹.

The M − H plot of the synthesized CoFe₂O₄ and CoFe₂O₄/GO composites recorded at room temperature. According to Fig. 1c, the residual loops show that the saturation magnetization (Ms) of CoFe₂O₄ is about 65.29 emu g⁻¹, which confirms the paramagnetic behavior of cobalt ferrite nanoparticles. It is obvious that the saturation magnetization of CoFe₂O₄/GO nanocomposite should be lower than CoFe₂O₄ (21.39 emug⁻¹), as the placement of CoFe₂O₄ nanoparticles on the sheets of the GO organic compound leads to a decline in the magnetic properties of this compound. Thus, because of superior magnetic performance, the CoFe₂O₄/GO nanocomposites can be easily separated from the aqueous solution by an external magnetic field.

To study the elemental composition of CoFe₂O₄/GO nanocomposite X-ray energy diffraction (EDX) spectroscopy used. The results obtained from this analysis in Fig. 1d illustrate the elemental distribution as Co = 12.9%, O = 16.4%, Fe = 23.7% and C = 47%, which is almost similar to the atomic ratios in the CoFe₂O₄/GO nanocomposite. The presence of C in the EDX analysis is due to the GO present in CoFe₂O₄/GO, and the rest of the elements such as Fe and Co refer to the spinel phase of CoFe₂O₄³¹. The surface composition of CoFe₂O₄/GO nanocomposite was also confirmed by the EDX-mapping analysis (Fig. 1e). Mapping analysis shows the frequency distribution of the elements of a composition. A brighter area in the mapping indicates a higher concentration of the corresponding element in that area. As is widely known, the elements are almost homogeneously distributed on the surface of CoFe₂O₄/GO nanocomposite.

FE-SEM analysis performed in different magnifications to further study the morphology of the synthesized CoFe₂O₄/GO nanocomposite. As can be seen in Fig. 2a, the CoFe₂O₄ particles with a spherical structure are homogeneously placed on the GO sheets. Some aggregation in the CoFe₂O₄/GO nanocomposite structure is probably due to the magnetic property exists between the CoFe₂O₄ nanoparticles and the large surface of GO. Based on the FE-SEM analysis, the average diameter of about 34 nm achieved for the synthesized CoFe₂O₄/GO nanocomposite as sorbent.

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Fig. 2

(a) FE-SEM images, and (b) Zeta potential diagram of CoFe₂O₄/GO nanocomposite.

To determine the point of zero charge (pH_pzc), 25 mL of 0.01 M potassium nitrate (KNO₃) solutions transferred to several Erlenmeyer flasks and the pH of the solutions adjusted to certain values. Then, 0.005 g of CoFe₂O₄/GO added to each flask and the solutions stirred for 24 h and the final pH of each solution was measured. In Fig. 2b, the pH_pzc of CoFe₂O₄/GO is 6.05.

Model fitting, statistical analysis, and interpretation of results

Based on preliminary tests, the variables affecting the MD-µ-SPE method for enrichment and extraction of vitamin B9, including pH (A), sorbent dose (B), temperature (C) and sonication time (D) were selected and their effect range reported in Table 1. Hence, a five-level central composite design (CCD) was considered to peruse the variables affecting the process and their interaction effects simultaneously³⁶.

Table 1. Effective variables, their levels, and ANOVA results in the CCD for the determination of vitamin B9.

Variable	Symbol	Levels of variable
		-α	–1	0	+ 1	+α
pH	A	4	4.5	5	5.5	6
Sorbent dose (mg)	B	12	14	16	18	20
Temp (°C)	C	25	30	35	40	45
Ult. time (min)	D	10	12	14	16	18

Source	Sum of Squares	DFa	Mean Square	F Value	p-value Prob > F
Model	2821.56	14	201.54	29.77	< 0.0001	significant
A	51.28	1	51.28	7.57	0.0165
B	214.85	1	214.85	31.73	< 0.0001
C	126.04	1	126.04	18.62	0.0008
D	55.33	1	55.33	8.17	0.0134
AB	0.5873	1	0.5873	0.0867	0.7730
AC	379.51	1	379.51	56.05	< 0.0001
AD	355.57	1	355.57	52.52	< 0.0001
BC	205.07	1	205.07	30.29	0.0001
BD	409.71	1	409.71	60.51	< 0.0001
CD	23.14	1	23.14	3.42	0.0874
A2	49.68	1	49.68	7.34	0.0179
B2	71.95	1	71.95	10.63	0.0062
C2	868.56	1	868.56	128.28	< 0.0001
D2	26.33	1	26.33	3.89	0.0702
Residual	88.02	13	6.77
Lack of Fit	70.32	10	7.03	1.19	0.4971	not significant
Pure Error	17.70	3	5.90
Cor. Total b	3457.80	29

^a Degrees of freedom. ^bTotals of all information corrected for the mean.

In order to statistically check the parameters and validate the model, analysis of variance (ANOVA) was used as stated by the F test and at a confidence level of 95%. High F values and low P values are desirable. P-value less than 0.05 for each parameter indicates its significance. By a high F-value (F model = 29.77) and a very low p-value (P model < 0.0001), the high significance of the model was certified. In addition to the model, the effect of parameters A, B, C, D, AC, AD, BC, BD, A²B²and C² is similarly significant (Table 1). The result of the best correlation between the response (extraction recovery, ER %) and different parameters was achieved as a coded quadratic polynomial equation (Eq. 1):

1

The p-value for the lack of fit is 0.4971, approves model fit to a set of experimental data. The closeness of R² value (R² = 0.9697) to 1 indicates a good correlation between data. A good matching of adjusted R² (0.9372) and predicted R² (0.8535) demonstrate the validity of the proposed model. Adequate precision (signal-to-noise ratio) for values ≥ 4 are acceptable, the obtained value of 27.59 is sufficient for experimental design suitability.

The simultaneous effect of pH and dosage of sorbent on the extraction recovery (ER %) of vitamin B9 is shown in Fig. 3a. Due to increasing sorption sites and active groups at higher sorbent dosages, the ER% of vitamin B9 is increased with adding amounts of more than 16.0 mg of CoFe₂O₄/GO nanocomposite. In amounts < 14.0 mg, analyte molecules are not adsorbed well on the sorbent, and the ER% decrease. Additionally, the highest ER % of vitamin B9 obtained at pH = 5.0 −5.5, because of the increasing interaction between vitamin B9 and CoFe₂O₄/GO. Indeed, the large specific surface area, layer structure and the π-π interaction between vitamin B9 molecules and π-conjugation regions of GO are also beneficial to the adsorption properties of CoFe₂O₄/GO nanocomposite.

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Fig. 3

3-D response surface, counter, and diagnostic plots for determination of vitamin B9. (a) pH and sorbent dose, (b) Sorbent dose and temperature, (c) Temperature and ultrasonic time, (d) Normal probability plot, and (e) The predicted values vs. the actual values.

Regarding Fig. 3b, by increasing the temperature of the solution up to 35 °C, as a result of the rise in the kinetic energy of the molecules, the diffusion and mass transfer of vitamin B9 and finally the %ER increases. At temperatures higher than 37 °C, caused by the self-quenching effect, the intensity of the response and %ER reduces. As the adsorption time (ultrasonic time) increases, the extraction efficiency increases (Fig. 3c). Generally, the sorbent is sufficiently dispersed in the sample solution, which creates a large contact surface. Thus, more effective interactions and increases in %ER occur.

The residuals can be analyzed using the normal probability plot (NPP) (Fig. 3d). If the distribution of residuals is normal, the resulting graph appears as a straight line. According to the plot, the distribution of the residues in the process of vitamin B9 extraction is normal and the data are centered on the straight line, it displays that the existing error is random and there is no systematic error.

The predictive power of the model is checked by plotting the predicted response values in terms of the actual (experimental) responses. In this study, as can be seen in Fig. 3, the predicted and the actual values have a good correlation that indicates the high predictive power of the proposed model³⁷.

Based on the desirability function in Design-Expert software, the optimal points obtained. The optimal points of variables affecting the process of extraction and detection of vitamin B9 with the desirability value of 0.95, obtained as follows: pH = 5.5, sorbent dose = 18.0 mg, temperature = 35 °C and ultrasonic time = 16 min. The extraction process repeated three times under optimal conditions, and the average obtained responses (92.45 ± 3.50) were in good agreement with the predicted ones (95.36 ± 2.60).

Desorption conditions

The most important step in the MD-µ-SPE method is desorption of the analyte from the sorbent surface. As well, in order to the final detection with the desired analytical instrument, it should not interfere with the analyte response. In this research, with the aim of achieving the highest desorption rate of vitamin B9 from the CoFe₂O₄/GO sorbent, various desorption solutions such as HCl 1.0 M, methanol, ethanol, acetone, and acetonitrile used. Regarding to the reported results in Fig. 4a, the highest %ER of vitamin B9 was obtained using acetonitrile solvent. It is possible that acetonitrile, in addition to hydrogen bonding with vitamin B9, can also make π-π interaction with the aromatic rings of this compound and easily desorb it from the sorbent. With a view to complete desorption of the analyte from the sorbent and using the minimum volume of solvent, different volumes of acetonitrile from 0.5 to 1.5 mL were considered. Based on the results in Fig. 4b, with the volume of 0.7 mL of acetonitrile, the highest %ER of vitamin B9 was gained. In volumes less than 0.7 mL, the amount of solvent probably was not enough to desorb completely the analyte from the sorbent. In subsequent studies, 0.7 mL of acetonitrile used as the desorption solvent because increasing the volume of the eluent (more than 0.7 mL) resulted in a dilution effect and a decrease in the response.

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Fig. 4

(a) The effect of desorption solvent type on the determination of vitamin B9, (b) The effect of desorption solvent volume on the determination of vitamin B9.

Analytical method validation

After optimization, the introduced method for vitamin B9 determination was validated following ICH Q2 (R1) guidelines.

Linearity

At the optimized conditions, the analytical specification of the recommended method inspected. A wide linear dynamic range in the concentration range of 15–750 ng mL⁻¹ (R² = 0.98) attained. The limit of detection (LOD) and limit of quantification (LOQ) of the introduced method as the signal-to-noise ratio of 3 and 10, were 3.96 and 13.07 ng mL⁻¹, respectively. The enrichment factor value was 64.80. This amount was based on the ratio of vitamin B9 concentration in the final extract to that in the initial water sample.

Repeatability and intermediate precision

The precision of the developed MD-µ-SPE method was evaluated in terms of repeatability and intermediate precision, in accordance with standard validation guidelines. Repeatability was assessed by analyzing replicate samples of vitamin B9 under identical conditions within a single day by the same analyst. The relative standard deviation (%RSD) values obtained from six consecutive measurements at three different concentrations of vitamin B9 (low, medium, and high levels) were 3.8%, 3.5%, and 3.3% for concentrations of 100.0 ng mL⁻¹, 300.0 ng mL⁻¹, and 600.0 ng mL⁻¹, respectively, demonstrating excellent short-term consistency of the method. Intermediate precision was evaluated by performing the same procedure over three non-consecutive days, also by the same analyst. The %RSD values across different days remained below 5.0%, 4.9%, 4.5%, and 4.2% for concentrations of 100.0 ng mL⁻¹, 300.0 ng mL⁻¹, and 600.0 ng mL⁻¹, respectively, confirming the method’s stability and reliability under routine laboratory conditions.

Accuracy and application

To validate the applicability and reliability of the suggested method, the MD-µ-SPE procedure based on the CoFe₂O₄/GO nanocomposite sorbent was applied for the pre-concentration and quantification of vitamin B9 in real samples, including mint, broccoli, lettuce, and urine. The accuracy of the method was evaluated using the standard addition technique, and recovery percentages (%Recovery) were calculated to determine the agreement between the measured concentrations and the known added amounts of analyte. As shown in Table 2, the relative recoveries (%RR) ranged from 84.79 to 97.65%, with RSD values below 5.0% (n = 3), indicating satisfactory accuracy and precision. Furthermore, statistical comparison with the standard HPLC-UV method confirmed that there were no significant differences, thereby validating the proposed method for practical applications.

Specificity and selectivity

The specificity and selectivity of the proposed method for the determination of vitamin B9 (300 ng mL⁻¹) were assessed by examining potential interferences from co-existing compounds. A deviation of ± 5% in fluorescence intensity was considered the acceptable threshold to indicate no significant interference. As shown in Table 3, most of the tested substances, within their typical concentration ranges, had negligible effects on both the extraction efficiency and the fluorescence response of vitamin B9. These results confirm the high selectivity of the developed method, indicating its ability to distinguish vitamin B9 even in the presence of structurally or chemically related compounds. Therefore, the method exhibits excellent specificity and is suitable for reliable quantification of vitamin B9 in complex matrices, such as fortified food products and biological samples.

Table 2. Determination of vitamin B9 in food and biological samples with the introduced method (n = 3).

Matrix	LDR (ng mL−1)	R2	Spiked (ng mL−1)	Found (ng mL−1)	Recovery (%)	HPLC-UV (ng mL−1)
Mint			0	47.43 ± 4.30a	-	49.74 ± 3.87
	25–600	0.9674	40	82.72 ± 4.00	88.21	83.59 ± 3.50
			100	139.52 ± 3.70	92.08	131.14 ± 4.54
Broccoli			0	52.21 ± 5.00	-	54.13 ± 6.06
	30–650	0.9723	40	90.07 ± 4.40	94.66	95.28 ± 5.12
			100	149.85 ± 3.90	97.65	165.62 ± 4.70
Lettuce			0	65.21 ± 3.98	-	68.14 ± 5.09
	40–700	0.9765	40	99.13 ± 4.45	84.79	105.74 ± 4.42
			100	158.96 ± 3.42	93.76	164.23 ± 3.80
Urine			0	N.Db	-	N.D
	35–630	0.9826	40	35.93 ± 3.12	89.82	37.93 ± 4.32
			100	94.30 ± 3.64	94.30	97.10 ± 4.16

^a Mean ± RSD (%). ^b Not detected.

Robustness analysis

The robustness of the recommended MDµ-SPE procedure was evaluated by introducing deliberate small variations in critical experimental parameters to assess their influence on the extraction efficiency of vitamin B9 (300 ng mL⁻¹). The tested variables included pH (± 0.2 units around the optimized value of 5.5), sorbent amount (± 1.0 mg from the optimized 18.0 mg), sonication time (± 2 min around 16 min), and sonication temperature (± 2 °C from 35 °C). In each case, the extraction and fluorescence detection were performed under slightly altered conditions while keeping all other parameters constant. As shown in Table 4, none of these minor variations caused significant changes in the extraction efficiency of vitamin B9 (deviation ≤ ± 5%), confirming the method’s robustness. Therefore, the proposed MDµ-SPE procedure can be considered reliable and reproducible for the routine determination of vitamin B9, even in the presence of slight fluctuations in experimental conditions.

Table 3. The effect of different substances on the extraction recovery of vitamin B9 (300 Ng mL− 1).

Interferences	Tolerance ratio (ng mL−1/ng mL−1)
Ca2+, Mg2+, Na+, K+, SO42−, Br−, F−, NO3−, Cl−	850 (3.5)a
Zn2+, Cu2+, Fe2+, Mn2+	600 (2.8)
Glucose	500 (3.4)
Urea	350 (4.1)
Uric acid	200 (3.7)
Vitamin C (ascorbic acid)	100 (4.0)
Vitamin B1 (riboflavin)	80 (3.8)

^aThe relative standard deviation (n = 3).

Table 4. Robustness evaluation of the developed method for vitamin B9 determination (n = 3).

Parameter	Tested Value	Recovery (%)	RSD (%)	ΔRecovery (%)
pH of solution	5.5 (optimal)	92.45	3.5	-
	5.3	92.16	3.2	0.29
	5.7	92.60	2.8	0.15
Sorbent amount (mg)	18.0 (optimal)	92.45	3.5	-
	17.0	91.40	3.6	1.05
	19.0	93.10	3.1	0.65
Sonication time (min)	16 (optimal)	92.45	3.5	-
	14	91.25	2.5	1.20
	18	93.56	3.8	1.11
Temperature (°C)	35 (optimal)	92.45	3.5	-
	33	91.20	4.0	1.25
	37	92.75	3.3	0.30

All variations led to deviations within the ± 5% limit, indicating good robustness of the method under slightly altered conditions.

Stability and reusability of CoFe₂O₄/GO sorbent

The recycling and reusability of a sorbent are effective in reducing the time and cost of the extraction process. Under the optimized conditions, the reusability of the prepared CoFe₂O₄/GO nanocomposite was evaluated by repeating the sorption/desorption process eight times. As illustrated in Fig. 5a, the %ER gradually decreased from 98% in the first cycle to 74% in the eighth cycle. This decline remains within an acceptable range, indicating good operational stability. Moreover, FT-IR analysis confirmed that the structural integrity of the sorbent remained unchanged (Fig. 5b). These results demonstrate that the CoFe₂O₄/GO nanocomposite possesses high stability and excellent reusability when applied in the MD-µ-SPE method. The minimal loss in performance highlights its potential as a sustainable, cost-effective, and environmentally friendly sorbent, further emphasizing the green aspects of the developed extraction method.

[See PDF for image]

Fig. 5

a) Evaluation of stability and reusability of CoFe₂O₄/GO nanocomposite, (b) FT-IR analysis of (i) fresh CoFe₂O₄/GO, and (ii) CoFe₂O₄/GO after eight cycles.

Comparative study

The proposed MD-µ-SPE method based on the CoFe₂O₄/GO for extraction/preconcentration and detection of vitamin B9 was compared with some reported methods. Compared to the most of the other reported methods, the proposed method has a lower RSD, better LOD, and comparable linear range, considering the results in Table 5. A combination of separation methods, such as HPLC and liquid chromatography-mass spectrometry (LC-MS) with sample pretreatment techniques, suffers from some disadvantages like the using of toxic solvents and expensive equipment, the necessity for tedious sample pre-treatments for complex matrices, and the resultant waste products. While in this study, an economical and fast analytical instrument was used to detect vitamin B9 that it was easy process, and sensitive sample preparation method prior to the inherently high selectivity and sensitivity characteristics of fluorescence. Additionally, consumption of a low volume of extraction solvent, short processing time, and high extraction efficiency are the main advantages of the MD-µ-SPE method.

Anti-bacterial activity of synthesized compounds

The antibacterial activity of synthesized GO, CoFe₂O₄ and CoFe₂O₄/GO compounds within the range of 0.06 to 0.48 mg mL⁻¹ against E. coli (Gram-negative) and S.aureus (Gram-positive) microorganisms were investigated using the well diffusion method. As demonstrated in Fig. 6, by measuring the diameter of the growth inhibition zone around the agar wells containing a certain amount of synthesized compounds, the results showed that all three compounds have a better antimicrobial activity on E. coli (Fig. 6a) bacteria compared to S. aureus (Fig. 6b). Notably, the maximum antimicrobial impact was observed at 0.48 mg/mL concentration against E. coli, which it was 17.5 mm. The zones of inhibition for under studied microorganisms are itemized in Table 6. There are some factors play a crucial role in the toxicological effects of the nanocomposite such as particle size and surface anomalies. A greater zone of inhibition was shown for the CoFe₂O₄/GO nanocomposite compared to other compounds for both S. aureus and E. coli. Although the observed antimicrobial activity-particularly against E. coli-was not directly incorporated into the current analytical procedure, it was investigated to highlight the added value and broader applicability of the nanocomposite. This dual functionality suggests promising opportunities for future applications in areas where both selective analyte extraction and microbial inhibition are advantageous. For instance, such multifunctional materials could be further developed for smart food packaging systems or incorporated into biosensors and safety monitoring tools in pharmaceutical and biomedical fields^{43, 44–45}.

[See PDF for image]

Fig. 6

Inhibition zones of E. coli(a) and S. aureus(b) microorganisms based on four concentrations of 0.06, 0.12, 0.24 and 0.48 mg mL⁻¹ of GO, CoFe₂O₄ and CoFe₂O₄/GO materials.

Table 5. Comparison of the proposed method with reported methods in the determination of vitamin B9.

Method	Instrumental analysis	Matrix	LDR (ng mL −1)	LOD (ng mL −1)	LOQ (ng mL −1)	RSD%	References
PFSPEa	HPLC-UV	Urine	50-10000	12	41	3.20–5.20	38
MIP-DSPEb	LC-MS	Food	10-1000	3	-	5.3	39
MIP-MSPE	HPLC-UV	Food	40-1400	1.68	5.54	3.94	40
Electrochemistry	2FTNECMNPPEc	Food and biological	44-221000	15	49.5	1.70–3.40	41
Fluorescence	CQDs sensors	Biological	500-21000	168	-	≤10.9.	12
Fluorescence	QDY-MIPs sensors	Blood plasma	221–8800	14.12	46.6	4.2	42
MD-µ-SPE	Spectrofluorometer	Food	15–750	3.96	13.07	3.35	This work

a: Packed-fiber solid-phase extraction. b: Molecularly imprinted polymer-dispersive solid phase extraction. c: 2FTNE-modified CMNP paste electrodes.

Table 6. E. coli and S. aureus growth inhibition zone (mm) based on the concentration of synthesized compounds (mg mL⁻¹).

		Zone of inhibition (mm)
Bacteria	Concentration (mg mL −1)	GO	CoFe2O4	CoFe2O4/GO
E. coli	0.06	7	9.5	10
	0.12	7	10	10.5
	0.24	9	11.5	13.5
	0.48	10	14	17.5
	0.06	0*	7	8.5
S. aureus	0.12	7	7.5	9.5
	0.24	8.5	8.5	11.5
	0.48	10	11.5	14.5

*No growth inhibition zone was observed.

The antibacterial mechanism of cofe₂o₄/go

The antibacterial activity of the CoFe₂O₄/GO nanocomposite can be attributed to several synergistic mechanisms rooted in its unique physicochemical structure. One key pathway involves the generation of reactive oxygen species (ROS), including superoxide anions (O₂⁻), hydroxyl radicals (•OH), and hydrogen peroxide (H₂O₂), which can form under ambient conditions due to the redox-active nature of CoFe₂O₄ in combination with graphene oxide. These ROS are known to damage bacterial cell walls, membranes, proteins, and DNA, ultimately leading to microbial cell death. In addition, the large surface area and sharp edges of GO nanosheets can physically disrupt bacterial membranes upon direct contact, compromising membrane integrity and causing leakage of intracellular components. The presence of magnetic CoFe₂O₄ nanoparticles further enhances the interaction between the nanocomposite and bacterial cells, promoting localized damage^{46, 47–48}. Collectively, these mechanisms suggest that the CoFe₂O₄/GO nanocomposite exerts its antibacterial effects through a multifaceted mode of action, making it a promising candidate for applications requiring both antimicrobial and analytical functionalities.

Evaluating the greenness and practicality

The greenness and practicality of the method were assessed using the Analytical Greenness (AGREE) tool⁴⁹ and the Blue Applicability Grade Index (BAGI)⁵⁰. The AGREE evaluation considers 12 criteria corresponding to the twelve principles of Green Analytical Chemistry (GAC). Each input variable is normalized on a scale from 0 to 1, and the final score is visualized as a clock-like pictogram. This pictogram uses a color gradient to reflect the performance of each principle, with the central area color ranging from red to green based on the overall score. A score higher than 0.6 indicates a green analytical method. As shown in Fig. 7a, the AGREE pictogram for the proposed method exhibited no red zones. However, four orange zones were noted for the following principles: use of a direct analytical technique (principle 1), positioning of the detection device (principle 3), waste generation (principle 7), and analysis throughput (principle 8). Two prominent yellow zones were associated with automation and miniaturization (principle 5) and the use of sustainable chemicals (principle 10). Green zones were observed for principles indicating strong environmental sustainability, including small sample size (principle 2), reduced number of steps (principle 4), absence of derivatization (principle 6), minimal energy consumption (principle 9), low toxicity (principle 11), and safety (principle 12). The method achieved an overall AGREE score of 0.64, represented by a predominantly green central circle, indicating minimal ecological impact and confirming its classification as a green analytical method.

[See PDF for image]

Fig. 7

Assessment of the greenness and practical applicability of the developed method using (a) the AGREE tool and (b) the BAGI tool.

The practicality of the method was further evaluated using the BAGI tool, which assesses the practical aspects of the analytical process and sample preparation. BAGI evaluates both strengths and limitations using a 10-point scale and presents the results in the form of asteroid pictograms, which visually represent each factor along with its score. As illustrated in Fig. 7b, the BAGI pictogram showed two white zones corresponding to the number of analytes simultaneously identified (only one) and the level of automation, which is typically low in most spectrofluorometric methods. A blue zone was also observed for the implementation of a miniaturized extraction procedure. The developed method received a BAGI score of 65.0, indicating its satisfactory practicality.

Conclusions

In summary, CoFe₂O₄/GO nanocomposite successfully synthesized as sorbents in MD-µ-SPE method with the aim of enrichment/separation and determination of vitamin B9. To assess CoFe₂O₄/GO nanocomposite adsorption capability toward vitamin B9, some of its structural characteristics studied and established by FT-IR, XRD, VSM, FE-SEM, EDX and mapping analyses. The optimum conditions of the MD-µ-SPE process achieved according to the desirability function method in multivariate optimization approach. The selected variables exhibited significant effects on the model according to ANOVA analysis. To analyze the random distribution of factors in the model the residual plots applied. Due to the high surface area, strong π-π stacking interaction between CoFe₂O₄/GO and vitamin B9, and the inherently high sensitivity and selectivity characteristics of fluorescence detection, the recommended method demonstrated good linearity (15–750 ng mL⁻¹), desirable repeatability (RSD < 4.0%), and intermediate precision (RSD < 5.0%). The extraction recoveries of vitamin B9 in mint, broccoli lettuce, and urine samples were between 84.79 and 97.65%. Additionally, the method was validated through comparison with HPLC-UV analysis. Reusability tests indicated that the CoFe₂O₄/GO nanocomposite could be used for at least eight extraction cycles without significant loss in performance. Moreover, the nanocomposite exhibited notable antibacterial activity, especially against E. coli, compared to S. aureus, highlighting its multifunctional potential. Importantly, the proposed MD-µ-SPE method aligns with key green chemistry principles, including the use of low-toxicity solvents in minimal volumes, reusability of the magnetic nanocomposite sorbent, and a low-energy, environmentally friendly synthesis process. These features render the method not only efficient and sensitive, but also sustainable and eco-friendly, with promising potential for future applications, particularly in fields where both selective extraction and antibacterial activity are desirable.

Acknowledgements

The financial support from the University of Birjand, is gratefully acknowledged.

Author contributions

Sampling, sample preparation, and data collection was performed by Zahra Khaleghi. Rouhollah Khani contributed to the funding acquisition, supervision, conception and design of the study, Software, and writing. Conceptualization, Validation, Writing-reviewing and editing by Maryam Moudi. All the authors participated in the writing and revision of the draft manuscript and approved the final version.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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