biomolecules Article

ArticleAnalysis of Amygdalin in Various Matrices Using AnalysisElectrospray of Amygdalin Ionization inand Various Flowing Matrices Atmospheric- Using ElectrosprayPressure Afterglow Ionization Mass and Spectrometry Flowing Atmospheric-Pressure Afterglow Mass Spectrometry Maria Guć *, Sandra Rutecka and Grzegorz Schroeder Maria Gu´c* , Sandra Rutecka and Grzegorz Schroeder Faculty of Chemistry, Adam Mickiewicz University in Poznan, Uniwersytetu Poznańskiego 8, 61-614 Poznań, FacultyPoland; [email protected] Chemistry, Adam (S.R.); Mickiewicz [email protected] University in(G.S.) Poznan, Uniwersytetu Pozna´nskiego 8, 61-614* Correspondence: Pozna´n,Poland; [email protected] [email protected] (S.R.); [email protected] (G.S.) * Correspondence: [email protected] Received: 4 October 2020; Accepted: 13 October 2020; Published: 19 October 2020


Received: 4 October 2020; Accepted: 13 October 2020; Published: 19 October 2020
 Abstract: Amygdalin is a natural cyanogenic compound that plants produce in the fight against Abstract:insects andAmygdalin herbivores. is Excessive a natural amounts cyanogenic of amygdalin compound by that animals plants produceand humans in the can fight potentially against insectslead to and fatal herbivores. intoxication. Excessive However, amounts studies of amygdalin confirm that by animals amygdalin and humanshas antitumor can potentially properties, lead toincluding fatal intoxication. the ability However, to inhibit studies the proliferation confirm that of amygdalin cancer cells has and antitumor to induce properties, their apoptosis. including The the abilityanalysis to of inhibit amygdalin the proliferation in various matrices of cancer is an cells important and to analytical induce their problem apoptosis. today. The publication analysis of amygdalinpresents the in methodology various matrices of direct is an determination important analytical of amygdalin problem in today. water, The sewage, publication and biological presents thematerials methodology using electrospray of direct determination ionization mass of amygdalin spectrometry in water,(ESI-MS) sewage, and a and new biological analytical materials method using electrosprayflowing atmospheric-pressure ionization mass spectrometryafterglow mass (ESI-MS) spectrometry and a new(FAPA-MS). analytical The method methods using of flowinganalyte pre-concentration atmospheric-pressure using afterglow a magnetic, mass molecularly spectrometry imprinted (FAPA-MS). polymer The methods(mag-MIP) of and analyte the pre-concentrationinfluence of interferents using aon magnetic, the recorded molecularly spectra imprinted were discussed. polymer Analytical (mag-MIP) parameters and the influence in ESI-MS of −1 −1 interferentsand FAPA-MS on themethods recorded were spectra established. were discussed. The linearity Analytical range was parameters 4.5 µg L in ESI-MS–45 mg andL in FAPA-MS positive mode ESI-MS and FAPA-MS. The limit of detection (LOD) for1 ESI-MS was1 0.101 ± 0.003 µg L−1 and methods were established. The linearity range was 4.5 µg L− –45 mg L− in positive mode ESI-MS the limit of quantification (LOQ) was 0.303 ± 0.009 µg L−1. In FAPA-MS, the LOD 1was 0.050 ± 0.002 and FAPA-MS. The limit of detection (LOD) for ESI-MS was 0.101 0.003 µg L− and the limit of −1 −1 ± quantificationµg L and the (LOQ) LOQ was 0.3030.150 ± 0.0090.006 µµgg LL1.. InThe FAPA-MS, content of the amygdalin LOD was 0.050in various0.002 matricesµg L 1 wasand ± − ± − thedetermined. LOQ was 0.150 0.006 µg L 1. The content of amygdalin in various matrices was determined. ± − Keywords: amygdalin; mag-MIP; ESI-MS; FAPA-MS

1. Introduction Amygdalin isis a naturala natural chemical chemical compound compound of plant of origin,plant belongingorigin, belonging to the group to the of cyanogenic group of glycosides.cyanogenic Naturalglycosides. amygdalin Natural has amygdalin the (R)-configuration has the (R)-configuration at the chiral phenyl at the center. chiral Under phenyl mild center. basic conditions,Under mild this basic stereogenic conditions, center this isomerizes; stereogenic the ( S)-epimercenter isomerizes; is called neoamygdalin. the (S)-epimer Although is called the synthesizedneoamygdalin. version Although of amygdalin the synthesi is thezed (R )-epimer,version of the amygdalin stereogenic is centerthe (R attached)-epimer, to the the stereogenic nitrile and phenylcenter attached groups easily to the epimerizesnitrile and (Figurephenyl1 gr)[oups1–3]. easily epimerizes (Figure 1) [1–3].

*

Figure 1. Amygdalin structural formula. Biomolecules 2020, 10, x; doi: FOR PEER REVIEW www.mdpi.com/journal/biomolecules

Biomolecules 2020, 10, 1459; doi:10.3390/biom10101459 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 1459 2 of 25

Amygdalin is found in plants belonging to the Rosaceae and the Passifloraceae families. Its harmfulness is associated with hydrogen cyanide released in the metabolic process. This provides a natural defense system. Amygdalin taken orally is highly toxic. Hydrogen cyanide released as a result of enzymatic hydrolysis of amygdalin causes nausea, headaches and dizziness, convulsions, and even coma and death. Its toxicity is due to the high affinity for iron ions (Fe3+). As a consequence, intracellular respiration is blocked and lactic acid is excessively secreted. The metabolism of amygdalin in the body is conditioned by many factors, such as digestive enzymes, intestine microbiome, supplementation, and medications [4–7]. However, amygdalin also has healing properties. The antitumor activity of amygdalin is linked with two hydrolytic enzymes (β-glucosidase and rhodanase). Excessive β-glucosidase and rhodanase deficiency would lead to cell death due to the release and accumulation of toxic hydrogen cyanide. Others, however, mistakenly treated amygdalin as vitamin B17, the deficiency of which was supposed to be the cause of cancer. Finally, in vitro studies conducted at the end of the 20th century confirmed the antitumor activity of amygdalin and its beneficial effects on the circulatory, respiratory, and digestive systems. Amygdalin was found to inhibit the proliferation of cancer cells and induce their apoptosis. In addition, it was noted that, at higher concentrations and longer exposure periods, amygdalin was increasingly effective against cervical, prostate, or liver cancer cells. Currently, the use of amygdalin in cancer therapy has its opponents and supporters [8–11]. The analysis of amygdalin in various matrices (plant material, water, sewage, and biological materials) is being studied by many scientists [12–16]. Xu et al. [12] performed the analysis of amygdalin, neoamygdalin, and amygdalin amide with HPLC-ESI-MS/MS and HPLC-DAD. Juan et al. [17] postulated solid-to-liquid extraction and HPLC/UV determination of amygdalin in apple seeds. Moreover, Feng et al. [18] analyzed amygdalin content in cherry seeds by capillary electrophoresis and Bolarinwa et al. [19] determined its concentration in apple seeds, fresh apples, and apple juices by HPLC. The application of amygdalin as vitamin B17 is the focus of a controversial debate. On the one hand, this compound is attributed highly therapeutic effects and is used as an anticancer drug; on the other hand, amygdalin is cataloged as a dangerous substance capable of producing highly toxic effects, due to the release of highly toxic HCN from the molecule in biological processes. Due to the properties of amygdalin, the analysis of this compound is focused on examining its content in two different samples: in food and in materials of animal origin (rat) after consuming products containing amygdalin. Various techniques have been used to determine the amygdalin contents of different foods and biological materials (Table1).

Table 1. Analytical methods used in the analysis of amygdalin in a deferent matrix.

The Range of Materials Analytical Methods LOD Measured Amygdalin References 1 Concentration (mg g− ) Different tissues of Extraction and loquat fruit - 2.44–16.70 [20] HPLC/UV cv. “Dahongpao” Solid-to-liquid extraction Seeds of apples 0.0505 mg g 1 -[17] and HPLC/UV − Seeds, kernels, Extraction 0.1 µg mL 1 0.01–17.50 [1] and food products and HPLC/UV − Extraction Apricot kernel - 0.217–0.284 [21] and HPLC/UV Biomolecules 2020, 10, 1459 3 of 25

Table 1. Cont.

The Range of Materials Analytical Methods LOD Measured Amygdalin References 1 Concentration (mg g− ) Bitter almond HPLC-ESI-MS2 and products: raw, 2 µg mL 1 -[12] HPLC-DAD − stir-fried, and scalded The enzyme Food extracts 2.7 10 7 M -[22] immunoassay test × − Solid-phase extraction Rat plasma 1.25 ng mL 1 -[23] and LC-MS2 − Rat plasma Extraction and UPLC - - [24] 1 Rat plasma HPLC 4.8 mg L− -[25] Rat urine LC-MS2 --[26]

Lietal.[23]analyzedthepharmacokineticsofamygdalininrats. Studies of metabolic pharmacokinetics of amygdalin in rats after consumption of feed containing amygdalin are particularly important in order to establish the threshold of pro-health and toxic concentrations. However, a direct analysis of amygdalin from biological materials is difficult due to the interference of other compounds. In addition, there are many ions in this material that form stable complexes with the amygdalin molecule. Therefore, the quantitative determination of amygdalin by electrospray ionization mass spectrometry (ESI-MS) is not possible. Due to analytical difficulties associated with the analysis of amygdalin, molecularly imprinted polymers (MIPs) were used. MIPs, based on the creation of specific recognition sites in strict polymer networks that have complementary shape, size, and functional groups toward the imprinted molecule, have attracted increasing interest as selective adsorbents for the isolation and enrichment of small organic molecules, metal ions, biomacromolecules, etc. [27,28]. Molecular imprinting of polymers is currently the most generic, versatile, scalable, and cost-effective approach to create synthetic molecular receptors. A number of advantageous properties contribute to the growing interest in MIPs: their high affinity and selectivity, enhanced stability (which is superior to that exhibited by natural biomolecules), and simplicity of their preparation. Recently, special attention has been paid to magnetic MIPs (mag-MIPs). In the new core–shell synthesis of mag-MIPs, a molecularly imprinted polymer surface (shell) covers magnetic iron oxide nanoparticles (core). After the synthesis, the material retains its magnetic properties and thus can be easily manipulated using a neodymium magnet, while providing selectively adsorptive capabilities. Additionally, the decreased size of the mag-MIP particles increases the surface area, enhancing the activity per unit mass of the polymer. Moreover, the recognition sites are located on the surface of the material, facilitating the access of the analyte to the selective cavities and its easy removal. In comparison to the MIPs obtained by traditional synthesis methods, mag-MIPs present a wide set of advantageous properties. Due to the large number of recognition sites located on the surface of MIPs in each mag-MIP particle, the quantification of analytes using these materials is expected to be more sensitive and selective. Mag-MIPs have been successfully used in the analysis of amygdalin by flowing atmospheric-pressure afterglow mass spectrometry (FAPA-MS) [27–30]. FAPA-MS techniques involve the generation of a direct current or radiofrequency electrical discharge between a pair of electrodes in contact with a flowing inert gas, creating a stream of ionized molecules, radicals, excited state neutral atoms, and electrons. The plasma species are directed toward the sample, resulting in desorption and ionization of the analyte. Ambient plasma MS techniques have many advantages including simple instrumentation, rugged construction of the measuring system, no solvent requirement, and generation of singly charged analyte species that are more easily identifiable than multiple charged ions and various adducts produced by spray-based techniques. Biomolecules 2020, 10, 1459 4 of 25

In this paper, we present the results of amygdalin analysis in various matrices (water, sewage, extract, skin, and artificial blood). In addition, we examined the effect of the presence of different ions in the sample on the analysis. We compared the quantitative analysis of amygdalin determination directly and with the use of mag-MIP for pre-concentration of the analyte. The results were obtained by ESI-MS and FAPA-MS.

2. Materials and Methods

2.1. Chemicals All reagents used were commercial products. FeCl 4H O, FeCl 6H O, amygdalin, 2· 2 3· 2 3-vinyltriethoxysilane (3-VTES), 4-vinylpyridine (4-VP), ethylene glycol dimethacrylate (EGDMA), 2,2-azobisisobutyronitrile (AIBN), ammonia solution, acetonitrile, dimethyl sulfoxide (DMSO), acetone, and methanol were purchased from Merck (Darmstadt, Germany). Formic acid, acetic acid, hydrochloric acid, sodium chloride, potassium chloride, sodium hydroxide, calcium perchlorate, lead perchlorate, copper (II) nitrate (V), and cadmium nitrate (V) tetrahydrate were obtained from POCH (Gliwice, Poland). Buffer solutions—pH 2 (hydrochloric acid, sodium chloride, and aminoacetic acid), pH 7 (disodium hydrogen phosphate, and citric acid), and pH 10 (boric acid, potassium chloride, and sodium hydroxide)—were from Eurochem BGD (Tarnów, Poland). Transparent tape 3M (19 mm 65.8 m) from Scotch (Minneapolis, MN, USA) was used for skin testing. Certified organic × 1 sweet apricot kernels containing 30 mg of amygdalin per 4.8 g material (6.25 mg g− ) were from Sunfood (El Cajon, CA, USA). Bitter almonds were from Uzbekistan, purchased from Skworcu (Siemianowice Sl´ ˛askie,Poland). The apples and apricots came from the local market (Pozna´n,Poland). Human serum from male AB plasma USA origin, sterile-filtered were purchased from Merck (Darmstadt, Germany) was used as the biological material.

2.2. Synthesis

2.2.1. Fe3O4

Magnetite nanoparticles (Fe3O4) were synthesized by using FeCl2 4H2O and FeCl3 6H2O as precursors. FeCl2 4H2O (2.0 g) and FeCl3 6H2O (5.2 g) were dissolved in 25 mL deoxygenated water, and then 0.85 mL of concentrated HCl was added. The resulting solution was added dropwise into 250 mL of 1.5 M NaOH solution upon vigorous stirring and N2 protection at 80 ◦C. The synthesized magnetic nanoparticles (MNPs) were separated from the solution by a powerful magnet and washed with 200 mL deionized water three times. Fe3O4 was used for the preparation 1 of a stable aqueous suspension. A total of 10 mL of an aqueous solution of citric acid (0.5 g mL− ) was added to the vigorously stirred suspension of washed nanoparticles. The pH value was set to 5.2 with a concentrated ammonia solution and heated to 80 ◦C. After 90 min, the pH value of the solution was elevated to 10. Lastly, the suspension was centrifuged for 5 min at 4000 rpm to remove any agglomerated nanoparticles. The obtained MNPs were dried at 60 ◦C.

2.2.2. Fe3O4@SiO2@VIN

Fe3O4 (2.4 g) and 125 mL DMSO were added to a round bottom flask and placed in an ultrasonic bath for 1 h. 3-VTES (12 mmol) was slowly added to the suspension. The mixture was stirred using a magnetic stirrer for 24 h. Then, the precipitate was isolated with a magnet and washed with distilled water and acetone. The resulting product Fe3O4@SiO2@VIN (Fe3O4 as a magnetic core covered with a thin layer of silica and vinyl (VIN) groups) was dried.

2.2.3. Fe3O4@SiO2@VIN@MIP Amygdalin (1 mmol) as a template molecule and 4-VP (4 mmol) as a functional monomer were dissolved in 50 mL acetonitrile and 35 mL methanol. Subsequently, 20 mmol of EGDMA Biomolecules 2020, 10, 1459 5 of 25

(previously purified with aluminum oxide, activated, basic, Brockman I) as a cross-linking agent and 1 g of Fe3O4@SiO2@VIN were added. The ratio of the template:monomer:cross-linking agent was 1:4:20. The pre-polymerization solution was sonicated and purged with nitrogen for

30Biomolecules min. Afterward, 2020, 10, x FOR 1 mLPEERAIBN REVIEW as initiator was added. The tube was sonicated and purged5 of 26 with nitrogen for an additional 5 h at 70 ◦C, then sealed and placed in an oven for 24 h at 50 ◦C. Afterwas polymerization,extracted with Feacetonitrile3O4@SiO2@VIN@MIP-amygdalin via Soxhlet extraction was for dried 24 underh. Then, reduced the pressurefinal product and grounded.Fe3O4@SiO Amygdalin2@VIN@MIP was was extracted dried at with60 °C acetonitrile under vacuum via Soxhlet and grounded. extraction for 24 h. Then, the final product Fe3O4@SiO2@VIN@MIP was dried at 60 ◦C under vacuum and grounded. 2.3. Instruments 2.3. Instruments ESI-MS and ESI-MSn spectra were recorded using an amaZon SL ion trap (Bruker, Bremen, n Germany)ESI-MS equipped and ESI-MS withspectra an electrospray were recorded ion source using in an infusion amaZon mode. SL ion The trap sample (Bruker, solution Bremen, was Germany)introduced equipped into the ionization with an electrospray source at a flow ion sourcerate of 5 in µL infusion min−1 using mode. a syringe The sample pump. solutionThe apparatus was 1 introducedwas operated into theusing ionization the so-called source “enhanced at a flow rate resolution of 5 µL min mode”− using (mass a syringe range: pump.50–2200 The m/ apparatusz, scanning wasrate: operated 8100 m using/z perthe second). so-called The “enhanced capillary voltage resolution was mode” set at (mass −4.5 kV range: and 50–2200 the endm plate/z, scanning offset was rate: at 8100 m/z per second). The capillary voltage was set at 4.5 kV and the end plate offset was at 500 V. −500 V. The source temperature was 80 °C and the desolvation− temperature was 250 °C. Helium− was Theused source as the temperature cone gas and was desolvating 80 ◦C and the gas desolvation (nitrogen) at temperature flow rates of was 50 250 L h◦−1C. and Helium 800 L wash−1, respectively. used as the 1 1 coneThe gas mass and spectrometer desolvating gas was (nitrogen) operated at in flow the rates ESI ofpositive 50 L h− andand negative 800 L h− ionization, respectively. mode. The In mass MSn n spectrometerexperiments, was the operatedwidth of the in theselection ESI positive window and was negative set at 2 Da ionization and the mode.amplification In MS ofexperiments, the excitation thewas width set according of the selection to the experiment window was (from set 0.2 at to 2 Da1.5 andV). Mass the amplification spectrometers of were the equipped excitation optionally was set accordingin a V-FAPA to the (Figure experiment 2) NOVA011 (from 0.2 ambient to 1.5 V).plasma Mass source spectrometers (ERTEC, Wroclaw, were equipped Poland). optionally in a V-FAPA (Figure2) NOVA011 ambient plasma source (ERTEC, Wroclaw, Poland).

Figure 2. Scheme of V-FAPA (flowing atmospheric-pressure afterglow) ambient plasma source. Figure 2. Scheme of V-FAPA (flowing atmospheric-pressure afterglow) ambient plasma source. V-FAPA was used to generate plasma, allowing the ionization of analyte particles released V-FAPA was used to generate plasma, allowing the ionization of analyte particles released thermally from a heated crucible with temperature regulation from 20 to 400 ◦C, at a temperature thermally from a heated1 crucible with temperature regulation from 20 to 400 °C, at a temperature increase rate of 3 ◦C s− . The temperature of maximum desorption of the analyte was 340 10 ◦C. −1 ± Theincrease mini crucible rate of 3 allowing °C s . The the temperature temperature-controlled of maximum desorption desorption was of the placed analyte approx. was 340 10 mm± 10 below°C. The themini ion crucible stream. Theallowing distance the betweentemperature-controlled the inlet to the massdesorption spectrometer was placed and theapprox. FAPA 10 ion mm source below was the aboution stream. 10 cm (FigureThe distance3). The between experimental the inlet details to the of mass the FAPA spectrometer method and were the presented FAPA ion in previoussource was publicationsabout 10 cm [29 (Figure,31]. 3). The experimental details of the FAPA method were presented in previous publicationsThe amygdalin [29,31]. content of the solution was determined using a chromatography system (Alliance, type 2690, Waters, Milford, MA, USA) coupled to a UV photodiode array and MS detectors. Quantification was performed using a C18 column (Atlantis T3 column, silica-based, reversed-phase, 100 Å, 3 µm, 3 mm 100 mm, 1/pk from Waters (Warsaw, Poland)) at 25 C. The wavelength × ◦ range in the UV spectrum was 200–500 nm and the range of recorded masses was 100–1000 m/z. Chromatography was performed using a flow rate of 0.5 mL per minute and an isocratic eluent program, and a mixture of 25% volume of methanol and 75% volume of water was used as the eluent. The injector volume was 10 µL and the total elution time was 20 min. The X-ray fluorescence (XRF) spectra were produced on a PANalytical MiniPal2 XRF spectrometer (Malvern, UK) equipped with a rhodium X-ray vacuum tube under the given conditions: time of the analysis, 200 s and X-ray tube voltage, 15 kV. The current generated in the electron beam varied between 20 and 53 µA.

Figure 3. The measuring system: plasma torch, heated sample holder, and amaZon SL mass spectrometer. Biomolecules 2020, 10, x FOR PEER REVIEW 5 of 26 was extracted with acetonitrile via Soxhlet extraction for 24 h. Then, the final product Fe3O4@SiO2@VIN@MIP was dried at 60 °C under vacuum and grounded.

2.3. Instruments

ESI-MS and ESI-MSn spectra were recorded using an amaZon SL ion trap (Bruker, Bremen, Germany) equipped with an electrospray ion source in infusion mode. The sample solution was introduced into the ionization source at a flow rate of 5 µL min−1 using a syringe pump. The apparatus was operated using the so-called “enhanced resolution mode” (mass range: 50–2200 m/z, scanning rate: 8100 m/z per second). The capillary voltage was set at −4.5 kV and the end plate offset was at −500 V. The source temperature was 80 °C and the desolvation temperature was 250 °C. Helium was used as the cone gas and desolvating gas (nitrogen) at flow rates of 50 L h−1 and 800 L h−1, respectively. The mass spectrometer was operated in the ESI positive and negative ionization mode. In MSn experiments, the width of the selection window was set at 2 Da and the amplification of the excitation was set according to the experiment (from 0.2 to 1.5 V). Mass spectrometers were equipped optionally in a V-FAPA (Figure 2) NOVA011 ambient plasma source (ERTEC, Wroclaw, Poland).

Figure 2. Scheme of V-FAPA (flowing atmospheric-pressure afterglow) ambient plasma source.

V-FAPA was used to generate plasma, allowing the ionization of analyte particles released thermally from a heated crucible with temperature regulation from 20 to 400 °C, at a temperature increase rate of 3 °C s−1. The temperature of maximum desorption of the analyte was 340 ± 10 °C. The mini crucible allowing the temperature-controlled desorption was placed approx. 10 mm below the ion stream. The distance between the inlet to the mass spectrometer and the FAPA ion source was aboutBiomolecules 10 cm2020 (Figure, 10, 1459 3). The experimental details of the FAPA method were presented in previous6 of 25 publications [29,31].

FigureFigure 3. TheThe measuring measuring system: system: plasma plasma torch, torch, heated heat sampleed sample holder, andholder, amaZon and SL amaZon mass spectrometer. SL mass spectrometer. X-ray photoelectron spectroscopy (XPS) was utilized to evaluate the chemical composition of the investigated MIPs. The measurements were carried out on an Escalab 250Xi spectroscope (Thermo Fisher Scientific, Waltham, MA, USA). The XR 50 source was used, and 60 eV pass energy and 90 eV bias voltage were utilized. The peak deconvolution was carried out using the Avantage software provided by the spectroscope manufacturer. All measurements were made three times. The presented results are average values.

2.4. Analysis

2.4.1. Analysis of Amygdalin in Aqueous Solutions by ESI-MS

1 1 The amygdalin solutions were prepared at concentrations ranging from 0.045 µg L− to 45 mg L− . ESI-MS measurements were performed in buffer solutions with pH 2, 7, and 10. The linear range of the method, limit of detection (LOD), and limit of quantification (LOQ) were determined. To study the effect of ions present in the solution on the determination of amygdalin by ESI-MS, 1 2+ 2+ the following ions were added separately to 10 mL of 4.5 mg L− amygdalin solutions: Cu , Pb , Ca2+, Na+, and K+ in a molar ratio of 1:1 relative to amygdalin.

2.4.2. Analysis of Amygdalin in Aqueous Solutions by FAPA-MS

1 1 The amygdalin solutions were prepared at concentrations ranging from 0.045 µg L− to 45 mg L− . FAPA-MS measurements were performed in a buffer solution with pH 7. The linear range of the method, LOD, and LOQ were determined.

2.4.3. Binding and Release of Amygdalin from Fe3O4@SiO2@VIN@MIP/Fe3O4@SiO2@VIN@MIP-Amygdalin in Aqueous Solutions

In order to investigate the binding of amygdalin by Fe3O4@SiO2@VIN@MIP in aqueous solutions, 1 1 1 three amygdalin concentrations of 0.0045 mg L− , 0.45 mg L− , and 45 mg L− were prepared. Then, 20 mg of Fe3O4@SiO2@VIN@MIP was added to 5 mL of each solution and stirred for 1 h. Subsequently, Fe3O4@SiO2@VIN@MIP-amygdalin was removed from the solution. To determine the amount of non-adsorbed amygdalin by Fe3O4@SiO2@VIN@MIP, the remaining amygdalin in solutions was measured by ESI-MS. The amount of adsorbed amygdalin in Fe3O4@SiO2@VIN@MIP-amygdalin was measured by FAPA-MS. Biomolecules 2020, 10, 1459 7 of 25

To test the degree of amygdalin release from Fe3O4@SiO2@VIN@MIP-amygdalin, 100 mg of empty Fe3O4@SiO2@VIN@MIP was placed in aqueous solutions containing 2 mg, 5 mg, and 7 mg of amygdalin. After all the amygdalin was bound to the polymer structure, Fe3O4@SiO2@VIN@MIP-amygdalin was isolated from the solution, washed, and dried. The three Fe3O4@SiO2@VIN@MIP-amygdalin polymers prepared in this way containing respectively 2, 5, and 7 mg of amygdalin per 100 mg of polymer were placed in 10 mL of an aqueous solution, acidified with formic acid to pH 2, and intensively stirred for 1 h to completely release amygdalin. After this time, both the solution (ESI-MS method) and Fe3O4@SiO2@VIN@MIP (FAPA-MS method) were analyzed for amygdalin content.

2.4.4. Extraction of Amygdalin from Fruit Seeds

Water Extraction at 40 ◦C Bitter almond kernels were grounded in a blender and 2 g was weighed into a conical flask (200 mL). Water (50 mL) was added and the flask was placed in a shaker with temperature control. Amygdalin extractions were carried out for 120 min. The extracts were filtered and analyzed with two methods. In the first method, the analysis of the amygdalin water extract with the ESI-MS method was performed. In the second method, 10 mg of Fe3O4@SiO2@VIN@MIP was added to the solution to pre-concentrate the analyte (amygdalin), followed by a direct analysis of amygdalin from the polymer structure using FAPA-MS. The amygdalin determination process was repeated three times. Apple and apricot extracts were prepared and analyzed identically.

Ethanol Extraction at 40 ◦C The ethanol extraction was performed identically as described above, with ethanol used as a solvent, instead of water.

2.4.5. Analysis of Amygdalin Skin Penetration In order to analyze amygdalin found on the skin, hands were accidentally stained with amygdalin solution during work, and the tape-stripping technique was used [32–34]. The 3M adhesive tape was used to collect the skin sample. The tape (1 cm2) was glued to the palm and after 1 min it was torn off. Then, the tape containing the test substance was immersed in ethanol (5 mL) and left for 1 h. After the set time had elapsed, 200 µL of solution for ESI-MS analysis was taken. The tape was glued four more times in the same place. The protocol was approved by the Ethics Committee (Komisja Bioetyczna przy Uniwersytecie Medycznym im. Karola Marcinkowskiego w Poznaniu, 469/16, 16 June 2016) and informed consent was obtained. All measurements were taken three times.

2.4.6. Analysis of Amygdalin in Human Serum To determine amygdalin in human serum from human male AB plasma, 5 mg of amygdalin was added to 1 mL human serum. The ESI-MS spectrum was recorded.

3. Results and Discussion

3.1. Analysis of Amygdalin in Aqueous Solutions by ESI-MS The analysis of amygdalin in aqueous solutions by ESI-MS showed signals with different m/z values, depending on the pH value of the solution. In the positive mode ESI-MS spectrum for amygdalin in a buffer solution of pH 7.0, we observed m/z 480 [M + Na]+ and m/z 496 [M + K]+ signals and the m/z 427 signal from the fragmentation ion. The fragment ion structure is shown in the spectrum in Figure4a. In the negative mode ESI-MS spectrum, we observed signals m/z 456 [M H] , m/z 492 [M + Cl] , and m/z 554 [M + H PO ] . − − − 2 4 − This was a consequence of measurements in the buffer in which the component was disodium hydrogen phosphate (Figure4b). Biomolecules 2020, 10, 1459 8 of 25 Biomolecules 2020, 10, x FOR PEER REVIEW 8 of 26

480 [M+Na]+

+ H

427 496 [M+K]+

(a)

456 [M-H]-

554 - [M+H2PO4]

492 [M+Cl]-

(b)

Figure 4. TheThe electrospray electrospray ionization ionization mass mass spectrometry spectrometry (ESI-MS) (ESI-MS) spectrum of amygdalin performed inin a a buffer buffer solution solution of of pH pH 7.0 7.0 (disodium (disodium hydrogen hydrogen phosphate phosphate and and citric citric acid): acid): (a) positive (a) positive mode, mode, (b) negative(b) negative mode. mode.

InIn the the positive positive mode mode ESI-MS ESI-MS spectrum spectrum for for amygdalin amygdalin in a a buffer buffer solution of pH 2.0, we we observed observed thethe main signal m/z 480 [M + Na]+ andand a a signal signal of of lower lower intensity intensity mm//zz 533.533. The The mm//zz 533533 signal signal resulted resulted from the attachment of aminoacetic acid acid present in the buffer buffer to the amygdalin molecule. We We also observed signals mm//z 427 and m/z 404 from the fragmentation ions (Figure5 5a).a). TheThe structurestructure ofof thethe fragmentation ion ion m//z 404 is presented in the spectrum in Figure5 5a.a. InIn thethe negativenegative modemode ESI-MSESI-MS spectrum, we observed only one signal m/z 492 [M + Cl]Cl]− (Figure(Figure 5b).5b). BiomoleculesBiomolecules 20202020,, 1010,, x 1459 FOR PEER REVIEW 99 of of 26 25

480 [M+Na]+

+ Na

404

427 533 + [M+C2H5NO2+H]

(a)

492 [M+Cl]-

(b)

FigureFigure 5. 5. TheThe ESI-MS ESI-MS spectrum spectrum of of amygdalin amygdalin performed performed in in a a buffer buffer solution solution of of pH pH 2.0 2.0 (hydrochloric (hydrochloric acid,acid, sodium sodium chloride, chloride, and and aminoacetic aminoacetic acid): acid): ( (aa)) positive positive mode, mode, ( (bb)) negative negative mode. mode.

InIn the positivepositive mode mode ESI-MS ESI-MS spectrum spectrum for amygdalinfor amygdalin in a buinff aer buffer solution solution of pH 10.0,of pH we 10.0, observed we + + observedtwo low-intensity two low-intensity signals msignals/z 480 m [M/z 480+ Na] [M + Na]and+ andm/z m496/z 496 [M [M+ K] + K]and+ and two two signalssignals from the the fragmentationfragmentation ions ions ( (mm//zz 427427 and and mm//zz 443).443). The The structure structure of of the the fragmentation fragmentation ion ion mm//zz 443443 is is shown shown in in thethe spectrum spectrum in in Figure Figure 66a.a. In the negative mode ESI-MS spectrum, we observed only one signal mm//zz 492492 [M [M ++ Cl]Cl]− −(Figure(Figure 6b).6b). Biomolecules 20202020,, 10,, x 1459 FOR PEER REVIEW 1010 of of 26 25

˙ 443

+ H O 427 2

496 + 480 [M+K] [M+Na]+

(a)

492 [M+Cl]-

(b)

FigureFigure 6. TheThe ESI-MS ESI-MS spectrum spectrum of of amygdalin amygdalin performed performed in in a a buffer buffer solution solution of of pH pH 10.0 10.0 (boric (boric acid, acid, potassium chloride, and sodium hydroxide): ( aa)) positive positive mode, mode, ( (b) negative mode.

BasedBased on on the obtained results, further tests were carried out in neutral solutions. The presence ofof other components in in the sample composition also also affects affects the determination of amygdalin in environmentalenvironmental samples. samples. Amygdalin Amygdalin easily easily complexe complexess ions ions found found in in solution, solution, which which we we took took into 2+ 2+ 2+ accountaccount in in the the conducted conducted research. research. Solutions Solutions with with the the addition addition of ofvarious various ions ions (Cu (Cu2+, Pb,2+ Pb, Ca2+,, CaNa+,, + + andNa K, and+) were K )analyzed were analyzed (Figure (Figure7a–e). The7a–e). results The are results shown are in shown the spectra. in the In spectra. the sample In the with sample both amygdalinwith both amygdalinand copper and ions, copper a plurality ions, a of plurality complexes of complexes were formed. were The formed. ions fo Thermed ions as formed a result as of a attachingresult of attachingubiquitous ubiquitous sodium in sodium the environment in the environment were still were visible. still visible.They were They also were present also present in the samplein the sample with added with addedlead ions. lead Furthermore, ions. Furthermore, amygdalin amygdalin with lead with ions lead also ions formed also formed complexes. complexes. In the remainingIn the remaining samples, samples, amygdalin amygdalin also formed also complexe formed complexess with calcium, with calcium,sodium, or sodium, potassium or potassium ions. The testedions. The ions tested are very ions common are very in common the environment. in the environment. They occur They naturally occur in naturally various biological in various materials. biological Thematerials. presence The of presence these ofions these in ionsthe inanalyzed the analyzed samp samplele practically practically prevented prevented the the correct correct analysis analysis of of amygdalinamygdalin usingusing thethe ESI-MS ESI-MS method. method. The The ESI-MS ESI-MS spectra spectra reflected reflected the constituents the constituents present present in solution; in solution;however, however, this process thismay process be limited may be for limited several for reasons.several reasons. The changing The changing solution solution conditions conditions during duringdroplet droplet evaporation evaporation in a spectrometer, in a spectrometer, such as changes such as in changes pH, ionic in strength, pH, ionic unequal strength, evaporation unequal evaporation of constituents, or occurrence of charge reduction, may alter the metal complex or the Biomolecules 2020, 10, 1459 11 of 25

Biomolecules 2020, 10, x FOR PEER REVIEW 11 of 26 of constituents, or occurrence of charge reduction, may alter the metal complex or the equilibrium conditions.equilibrium Moreover, conditions. species Moreover, which species are stable which in are solution stable mayin solution not be may stable not in be the stable gas phasein the duegas to thephase absence due ofto the a solvent absence [35 of– 37a solvent]. In the [35–37]. case of In metal–amygdalin the case of metal–amygdalin complexes complexes where the where structure the is determinedstructure is by determined electrostatic by interactions,electrostatic interactions the ESI-MS, spectrathe ESI-MS reflect spectra the composition reflect the composition of the solution of the [26 ]. Electrostaticsolution [26]. interactions Electrostatic are interactions greatly strengthened are greatly strengthened in a solvent environment.in a solvent environment. As a consequence, As a consequence, metal–ligand complexes with strong electrostatic interactions are more stable in the gas metal–ligand complexes with strong electrostatic interactions are more stable in the gas phase [38]. phase [38]. In addition, as a consequence of the dynamic character of liquid-based ionization in the In addition, as a consequence of the dynamic character of liquid-based ionization in the ESI process, it ESI process, it is possible in this process to monitor liquid-phase reactions of metal ions in solution is possible in this process to monitor liquid-phase reactions of metal ions in solution [39]. [39].

582 2+ + [M+Cu NO3]

519 489 [M+Cu+]+ [2M+Cu++H]2+

480 [M+Na]+

(a)

664 [M+Pb]+

480 [M+Na]+

(b)

Figure 7. Cont. BiomoleculesBiomolecules2020 2020, 10, 10, 1459, x FOR PEER REVIEW 12 of12 26 of 25

477 [2M+Ca]2+ 596 + [M+Ca(ClO4)]

(c)

480 [M+Na]+

(d)

496 [M+K]+

(e)

Figure 7. The ESI-MS spectrum of amygdalin performed in solutions with the addition of various ions: (a) Cu2+,(b) Pb2+,(c) Ca2+,(d) Na+,(e)K+. Biomolecules 2020, 10, x FOR PEER REVIEW 13 of 26

Biomolecules 2020, 10, 1459 13 of 25 Figure 7. The ESI-MS spectrum of amygdalin performed in solutions with the addition of various ions: (a) Cu2+, (b) Pb2+, (c) Ca2+, (d) Na+, (e) K+. A linear relationship between signal intensity and amygdalin concentration was established by selectingA linearm/z relationship458 [M + H]+ between, m/z 480 signal [M + Na] intensity+, and mand/z 496 amygdalin [M + K]+ concentrationas analytical signals.was established The linearity by selecting m/z 458 [M + H]+, m/z 480 [M1 + Na]+, and1 m/z 496 [M + K]+ as analytical signals. The linearity range of the method was 4.5 µg L− –45 mg L− in positive mode ESI-MS. The LOD for ESI-MS was range of the method1 was 4.5 µg L−1–45 mg L−1 in positive 1mode ESI-MS. The LOD for ESI-MS was 0.101 0.003 µg L− and the LOQ was 0.303 0.009 µg L− . 0.101 ±± 0.003 µg L−1 and the LOQ was 0.303 ± ±0.009 µg L−1. 3.2. Analysis of Amygdalin in Aqueous Solutions by FAPA-MS

3.2. Analysis of Amygdalin in Aqueous Solutions by FAPA-MS 1 1 Amygdalin was determined in aqueous solutions in the range from 0.045 µg L− to 45 mg L− . In theAmygdalin FAPA-MS was method, determined tests were in performedaqueous so inlutions negative in the and range positive from ion 0.045 modes. µg L After−1 to 45 a preliminary mg L−1. In theanalysis FAPA-MS of the method, results, thetests rest were of the performed measurements in negati wereve performedand positive in ion the modes. FAPA-MS After positive a preliminary ion mode. analysisIn FAPA-MS of the mass results, spectra, the mrest/z 371 of the and measurementsm/z 476 signals were observed.performed These in the signals FAPA-MS were derivedpositive fromion mode.the reaction In FAPA-MS products mass of amygdalin spectra, m with/z 371 water and inm/z the 476 plasma signals stream were andobserved. the products These signals of hydrolysis were derivedand fragmentation. from the reaction As aproducts result of of the amygdalin hydrolysis with of water nitriles, in the amides plasma were stream formed and (Equation the products (1)). ofAmides hydrolysis can be and hydrolyzed fragmentation. to carboxylic As a result acids. of Moreover, the hydrolysis the molecule of nitriles, can fragment amides (Equationwere formed (2)). (EquationStructural (1)). formulas Amides of compoundscan be hydrolyzed are presented to carboxylic in the massacids. spectrum Moreover, in the Figure molecule8. can fragment (Equation (2)). Structural formulas of compounds are presented in the mass spectrum in Figure 8. hydrolisis R1 CN R1 CONH2 (1) −R1-CN →⎯⎯⎯⎯⎯ R−1-CONH2 (1) hydrolisis fragmentation R1 CONH R1 COOH R2 COOH (2) R−1-CONH ⎯⎯→⎯⎯⎯ R1−-COOH ⎯⎯⎯⎯⎯→⎯⎯⎯⎯ R2-COOH− (2)

371

+ H + H + H

476 355

329

-1 FigureFigure 8. 8. TheThe positive positive mode mode FAPA-MS FAPA-MS spectrum spectrum of of amygdalin amygdalin in in 45 45 mg mg L L-1 aqueousaqueous solution. solution.

m z µ 1 1 TheThe m//z 371371 signal was selectedselected as as the the analytical analytical signal. signal. The The linearity linearity range range was was4.5 4.5g Lµg− –45L−1–45 mg mg L− for FAPA-MS. The LOD was 0.050 0.002 µg L 1 and the LOQ was 0.150 0.006 µg L 1. L−1 for FAPA-MS. The LOD was 0.050± ± 0.002 −µg L−1 and the LOQ was 0.150± ± 0.006− µg L−1. 3.3. Comparison of Analytical Parameters of the ESI-MS and FAPA-MS Methods 3.3. Comparison of Analytical Parameters of the ESI-MS and FAPA-MS Methods To compare the methods of determining amygdalin in aqueous solutions, ESI-MS and FAPA-MS To compare the methods of determining amygdalin in aqueous solutions, ESI-MS and FAPA- techniques were analyzed and the results obtained are presented in Table2. MS techniques were analyzed and the results obtained are presented in Table 2.

Biomolecules 2020, 10, x FOR PEER REVIEW 14 of 26

Biomolecules 2020, 10, 1459 14 of 25 Table 2. Results of amygdalin determination in aqueous solutions by ESI-MS and FAPA-MS methods.

Analysis of Amygdalin in Aqueous Analysis of Amygdalin in Aqueous TableAmygdalin 2. Results of amygdalin determination in aqueous solutions by ESI-MS and FAPA-MS methods. Solutions by ESI-MS Solutions by FAPA-MS Concentration cmin cmax cmean ± SD cmin cmax cmean ± SD (mg L−1) Analysis of Amygdalin in Aqueous Solutions Analysis of Amygdalin in Aqueous Solutions Amygdalin Concentration (mg L−1) (mg byL−1 ESI-MS) (mg L−1) (mg L−1) (mg byL−1 FAPA-MS) (mg L−1) 1 (mg L− ) cmin cmax cmean SD cmin cmax cmean SD 45.0 42.0 1 50.0 1 45.7 ± 1.4± 1 38.0 1 52.0 1 44.7 ± 2.2 ± 1 (mg L− ) (mg L− ) (mg L− ) (mg L− ) (mg L− ) (mg L− ) 0.45 0.40 0.51 0.45 ± 0.02 0.39 0.52 0.46 ± 0.04 45.0 42.0 50.0 45.7 1.4 38.0 52.0 44.7 2.2 ± ± 0.00450.45 0.00380.40 0.0054 0.51 0.0047 0.45± 0.00020.02 0.0036 0.39 0.0053 0.52 0.0045 ± 0.46 0.00020.04 ± ± 0.0000450.0045 nd0.0038 nd 0.0054 0.0047 nd 0.0002 nd 0.0036 nd 0.0053 0.0045 nd 0.0002 ± ± 0.000045 ndnd, not detected; nd SD, standard nd deviation. nd nd nd nd, not detected; SD, standard deviation. Both the methods were characterized by similar values of analytical parameters (Figure 9): the scatter of results and the average value of the measurement in relation to the tested systems (Table Both the methods were characterized by similar values of analytical parameters (Figure9): the 3). scatter of results and the average value of the measurement in relation to the tested systems (Table3).

Figure 9. Dependence of signal intensity vs. amygdalin concentration in the sample. Figure 9. Dependence of signal intensity vs. amygdalin concentration in the sample. Table 3. Summary of analytical parameters. Table 3. Summary of analytical parameters. Analytical Method Analytical Parameter Analytical Method Analytical Parameter ESI-MS FAPA-MS ESI-MS FAPA-MS Linear range 4.5 µg L 1–45 mg L 1 4.5 µg L 1–45 mg L 1 Linear range 4.5 µg L−1–45 mg L−1− 4.5 µg L−1–45 mg− L−1 − LOD 0.101 0.003 µg L 1 0.050 0.002 µg L 1 −−1 −1 − LOD 0.101 ±± 0.003 µg L 1 0.050 ± 0.002 µg± L 1 LOQ 0.303 0.009 µg L− 0.150 0.006 µg L− LOQ 0.303 ±± 0.009 µg L−1 0.150 ± 0.006 µg± L−1

The FAPA-MS methodmethod hadhad lowerlower LOD,LOD, but the linearity range for FAPA-MS andand ESI-MSESI-MS was the same.same. The attractiveness of of the the FAPA-MS FAPA-MS method in relation to ESI-MSESI-MS lies in thethe possibilitypossibility of analyzing amygdalinamygdalin directlydirectly fromfrom FeFe33OO44@SiO2@VIN@MIP-amygdalin. This This eliminates eliminates the influence influence of interferants, concentrates the sample, and facilitates the analysis of real samples with a low concentration of thethe analyteanalyte inin thethe presencepresence ofof otherother compoundscompounds inin thethe sample.sample.

3.4. Comparison of the HPLC-UV/MS HPLC-UV/MS and FAPA-MS MethodsMethodswith withCertified Certified Reference Reference Materials Materials of of Apricot Apricot Kernels Containing Amygdalin Kernels Containing Amygdalin For the quantification of amygdalin content in solution, the calibration curve for HPLC-UV/MS was For the quantification of amygdalin content in solution, the calibration curve for HPLC-UV/MS constructed using five different solutions of amygdalin in methanol (Figures 10–13). The chromatogram was constructed using five different solutions of amygdalin in methanol (Figures 10–13). The of amygdalin solution (0.5 mg mL 1) is presented in Figure 10. chromatogram of amygdalin solution− (0.5 mg mL−1) is presented in Figure 10. Biomolecules 2020, 10, x FOR PEER REVIEW 15 of 26 Biomolecules 2020, 10, x FOR PEER REVIEW 15 of 26 BiomoleculesBiomolecules 20202020, ,1010, ,x 1459 FOR PEER REVIEW 1515 of of 26 25

−1 Figure 10. HPLC-diode array chromatogram of amygdalin solution (0.5 mg mL ). Figure 10. HPLC-diode array chromatogram of amygdalin solution (0.5 mg mL−11). FigureFigure 10. 10. HPLC-diodeHPLC-diode array array chromatogram chromatogram of of amygdalin amygdalin solution solution (0.5 (0.5 mg mg mL mL−−1).).

Figure 11. UV spectrum of amygdalin, HPLC retention time tR = 6.33 min. Figure 11. UV spectrum of amygdalin, HPLC retention time tR = 6.33 min. Figure 11. UV spectrum of amygdalin, HPLC retention time tR = 6.33 min. Figure 11. UV spectrum of amygdalin, HPLC retention time tR = 6.33 min.

Figure 12. Full scan spectrum of amygdalin, HPLC retention time t = 6.33 min, m/z 456 [M − H] , Figure 12. Full scan spectrum of amygdalin, HPLC retention time tR = R6.33 min, m/z 456 [M − H] , m/z− + + − + 950mFigure/z [2M950 12.+ [2M Cl] Full−+, mCl] scan/z− 492, m spectrum/ [Mz 492 + Cl] [M of−,+ m amygdalin,Cl]/z −475, m [M/z 475 + HPLCNa] [M+,+ m retentionNa]/z 496, m [M/z time 496+ K] [Mt+R, and=+ 6.33K] m /min,,z and938 m m[2M//zz 456938 + Na] [M [2M +−. +H]Na]−, m/z. Figure 12. Full scan spectrum of amygdalin, HPLC retention time tR = 6.33 min, m/z 456 [M − H]−, m/z 950 [2M + Cl]−, m/z 492 [M + Cl]−, m/z 475 [M + Na]+, m/z 496 [M + K]+, and m/z 938 [2M + Na]+. 950 [2M + Cl]−, m/z 492 [M + Cl]−, m/z 475 [M + Na]+, m/z 496 [M + K]+, and m/z 938 [2M + Na]+. Biomolecules 2020, 10, x FOR PEER REVIEW 16 of 26

A good regression equation (R2 = 0.9973) for HPLC-UV/MS determination of amygdalin was obtained, where the signal intensity of amygdalin was denoted in the y-axis and amygdalin −1 Biomoleculesconcentration2020, 10 (mg, 1459 mL ) was denoted in the x-axis (Figure 13). 16 of 25

Figure 13. Dependence of signal intensity vs. amygdalin concentration in the sample. Figure 13. Dependence of signal intensity vs. amygdalin concentration in the sample. A good regression equation (R2 = 0.9973) for HPLC-UV/MS determination of amygdalin was obtained,In order whereto validate thesignal the method intensity of determinat of amygdalinion wasof amygdalin denoted inin theplanty-axis materials, and amygdalin Sunfood’s 1 −1 concentrationcertified organic (mg sweet mL− )apricot was denoted kernels in containing the x-axis 30 (Figure mg of 13 amygdalin). per 4.8 g material (6.25 mg g ) wereIn used. order The to validate apricot the kernels method (4.8 of g) determination were extracte ofd amygdalinwith 50 mL in of plant water materials, or ethanol, Sunfood’s and amygdalin certified 1 organicconcentration sweet apricot in the kernels extracts containing was 30determin mg of amygdalined by HPLC-UV/MS per 4.8 g material and (6.25 FAPA-MS mg g− ) wereusing used. the TheFe3 apricotO4@SiO kernels2@VIN@MIP (4.8 g) material were extracted for the withselectiv 50 mLe concentration of water or ethanol, of the analyte. and amygdalin concentration in theA extracts comparison was determined of the results by of HPLC-UV HPLC-UV/MS/MS and and FAPA-MS FAPA-MS using methods the Fe for3O 4the@SiO determination2@VIN@MIP of materialamygdalin for thein apricot selective kernels concentration is presented of the in analyte.Table 4. A comparison of the results of HPLC-UV/MS and FAPA-MS methods for the determination of amygdalinTable in4. apricotResults kernelsof amygdalin is presented determination in Table in4 apricot. kernels (6.25 mg g−1) by HPLC-UV/MS and FAPA-MS methods. Table 4. Results of amygdalin determination in apricot kernels (6.25 mg g 1) by HPLC-UV/MS and −FAPA-MS Used FAPA-MSAmygdalin methods. HPLC-UV/MS Fe3O4@SiO2@VIN@MIP Concentration cmin cmax cmean ± SD cmin cmax cmean ± SD -1 HPLC-UV/MS FAPA-MS Used Fe3O4@SiO2@VIN@MIP Amygdalin(6.25 Concentration mg g ) −1 −1 -1 −1 −1 −1 1 (mgcmin g ) (mgcmax g ) cmean(mg gSD) (mgcmin g ) (mgcmax g ) cmean(mg gSD) (6.25 mg g− ) 1 1 ± 1 1 1 ± 1 Water extract (mg5.11 g− ) (mg 6.31 g− ) 5.68(mg g±− 0.21) (mg 6.22 g− ) (mg 6.67 g− ) 6.46(mg g±− 0.12) Water extract 5.11 6.31 5.68 0.21 6.22 6.67 6.46 0.12 Ethanol extract 5.82 6.36 6.10± ± 0.17 6.45 6.81 6.63± ± 0.14 Ethanol extract 5.82 6.36 6.10 0.17 6.45 6.81 6.63 0.14 ± ± The applied methods showed that, in the case of extraction of amygdalin with water or ethanol, followedThe applied by determination methods showed with HPLC-UV/MS, that, in the case the of extraction results were of amygdalin lower than with those water declared or ethanol, in the followedcertified bymaterial. determination On the other with HPLC-UVhand, in the/MS, case the of results extraction were with lower water than or those ethanol, declared followed in the by certifiedselective material. adsorption On by the Fe other3O4@SiO hand,2@VIN@MIP in the case and of FAPA-MS extraction determination, with water or the ethanol, results followed were higher by selectivethan those adsorption declared by in Fe the3O certified4@SiO2@VIN@MIP material. and FAPA-MS determination, the results were higher than those declared in the certified material. 3.5. Characteristic of Amygdalin Binding and Release from 3.5. Characteristic of Amygdalin Binding and Release from Fe3O4@SiO2@VIN@MIP/Fe3O4@SiO2@VIN@MIP-Amygdalin in Aqueous Solutions Fe3O4@SiO2@VIN@MIP/Fe3O4@SiO2@VIN@MIP-Amygdalin in Aqueous Solutions As a result of multistage synthesis, the hybrid material Fe3O4@SiO2@VIN@MIP was obtained As a result of multistage synthesis, the hybrid material Fe O @SiO @VIN@MIP was obtained (Figure 14). 3 4 2 (Figure 14). After removing the template, the resulting system was capable of binding analyte–amygdalin according to the equilibrium shown in Figure 15. XRF analysis confirmed that a significant part of the Fe3O4@SiO2@VIN@MIP mass consisted of a magnetic core (Fe3O4) covered with a thin silica coating. The mass ratio of Fe:Si was approximately 100:1 (Figure 16). Biomolecules 2020, 10, x FOR PEER REVIEW 17 of 26

Biomolecules 2020, 10, 1459 17 of 25 Biomolecules 2020, 10, x FOR PEER REVIEW 17 of 26

Figure 14. Scheme of Fe3O4@SiO2@VIN@MIP synthesis.

After removing the template, the resulting system was capable of binding analyte–amygdalin Figure 14. Scheme of Fe3O4@SiO2@VIN@MIP synthesis. according to the equilibriumFigure shown 14. Schemein Figure of Fe15.3O 4@SiO2@VIN@MIP synthesis.

After removing the template, the resulting system was capable of binding analyte–amygdalin according to the equilibrium shown in Figure 15.

Figure 15. Release and binding of amygdalin in Fe3O4@SiO2@VIN@MIP-amygdalin/Fe3O4@SiO2@VIN@MIP. BiomoleculesFigure 2020 , 15.10, x FORRelease PEER REVIEWand binding of amygdalin in Fe3O4@SiO2@VIN@MIP-18 of 26 amygdalin/Fe3O4@SiO2@VIN@MIP. Fe

3 4 2 XRFFigure analysis 15. confirmedRelease thatand a significant binding part of of theamygdalin Fe O @SiO @VIN@MIPin Fe3O4@SiO mass2@VIN@MIP- consisted of a magneticamygdalin/Fe core (Fe33OO44)@SiO covered2@VIN@MIP. with a thin silica coating. The mass ratio of Fe:Si was approximately 100:1 (Figure 16). XRF analysis confirmed that a significant part of the Fe3O4@SiO2@VIN@MIP mass consisted of a magnetic core (Fe3O4) covered with a thin silica coating. The mass ratio of Fe:Si was approximately 100:1 (Figure 16).

Signal intensity (-) (-) Signal intensity Fe Si

Energy (eV)

Figure 16. X-ray fluorescence (XRF) spectrum of Fe3O4@SiO2@VIN@MIP. Figure 16. X-ray fluorescence (XRF) spectrum of Fe3O4@SiO2@VIN@MIP.

Moreover, we performed XPS analyses for Fe3O4@SiO2@VIN@MIP and Moreover, we performed XPS analyses for Fe3O4@SiO2@VIN@MIP and Fe3O4@SiO2@VIN@MIP- Fe3O4@SiO2@VIN@MIP-amygdalin to confirm the structure of the formed core–shell particles. amygdalin to confirm the structure of the formed core–shell particles. The results of XPS analyses are collectively presented in Figures S1 and S2. The XPS analyses were surface tests and the surface of the obtained materials was a polymer coating. The low-intensity signal from the magnetic core formed by Fe3O4 confirmed that it was covered with a thin layer of silica and then a polymer layer. The observed signals came mainly from carbon and oxygen, i.e., elements building the polymer structure. Furthermore, we observed a slight difference in oxygen and nitrogen content between empty Fe3O4@SiO2@VIN@MIP and the polymer with amygdalin. It was an additional confirmation that during the synthesis, the polymer bound amygdalin molecules in its structure, and during extraction, the molecules were successfully washed away. In the first method, ESI-MS was used to examine the decrease in amygdalin concentration in aqueous solutions after the addition of Fe3O4@SiO2@VIN@MIP. The binding of amygdalin by the polymeric material was performed for three different initial concentrations of amygdalin solutions. The decrease in amygdalin concentration in these solutions over time is shown in Figure 17.

(a) (b) (c)

Figure 17. The concentration of amygdalin over time in solution after addition of

Fe3O4@SiO2@VIN@MIP. C0 = 45 mg L−1 (a), 0.45 mg L−1 (b), and 4.5 μg L−1 (c).

In solutions with different concentrations of amygdalin, it was most effectively bound in the polymer structure at different time points. However, for all samples, the total binding time of the analyte did not exceed 1 h; therefore, 1 h was considered as the optimal binding time of the analyte from the real samples. All experiment times with the use of mag-MIP were assumed to be 1 h. The second FAPA-MS method examined the content of amygdalin in Fe3O4@SiO2@VIN@MIP-amygdalin isolated from complementary aqueous solutions of amygdalin. The parallel determination of the analyte by the FAPA-MS method was carried out directly from the polymer structure. Due to the magnetic properties of the material, it was easily isolated from solutions using a small neodymium magnet. Then, the polymer was washed three times with water. In the next step, a magnet was placed in the FAPA-MS measuring system and a direct analysis of amygdalin from the polymer structure Biomolecules 2020, 10, x FOR PEER REVIEW 18 of 26

Fe

Signal intensity (-) (-) Signal intensity Fe Si

Energy (eV)

Figure 16. X-ray fluorescence (XRF) spectrum of Fe3O4@SiO2@VIN@MIP.

BiomoleculesMoreover,2020, 10 we, 1459 performed XPS analyses for Fe3O4@SiO2@VIN@MIP and Fe3O4@SiO2@VIN@MIP-18 of 25 amygdalin to confirm the structure of the formed core–shell particles. The results of XPS analyses are collectively presented in Figures S1 and S2. The XPS analyses were surface tests and the surface of The results of XPS analyses are collectively presented in Figures S1 and S2. The XPS analyses were the obtained materials was a polymer coating. The low-intensity signal from the magnetic core surface tests and the surface of the obtained materials was a polymer coating. The low-intensity formed by Fe3O4 confirmed that it was covered with a thin layer of silica and then a polymer layer. signal from the magnetic core formed by Fe3O4 confirmed that it was covered with a thin layer of The observed signals came mainly from carbon and oxygen, i.e., elements building the polymer silica and then a polymer layer. The observed signals came mainly from carbon and oxygen, i.e., structure. Furthermore, we observed a slight difference in oxygen and nitrogen content between elements building the polymer structure. Furthermore, we observed a slight difference in oxygen and empty Fe3O4@SiO2@VIN@MIP and the polymer with amygdalin. It was an additional confirmation nitrogen content between empty Fe3O4@SiO2@VIN@MIP and the polymer with amygdalin. It was an that during the synthesis, the polymer bound amygdalin molecules in its structure, and during additional confirmation that during the synthesis, the polymer bound amygdalin molecules in its extraction, the molecules were successfully washed away. structure, and during extraction, the molecules were successfully washed away. In the first method, ESI-MS was used to examine the decrease in amygdalin concentration in In the first method, ESI-MS was used to examine the decrease in amygdalin concentration in aqueous solutions after the addition of Fe3O4@SiO2@VIN@MIP. The binding of amygdalin by the aqueous solutions after the addition of Fe3O4@SiO2@VIN@MIP. The binding of amygdalin by the polymeric material was performed for three different initial concentrations of amygdalin solutions. polymeric material was performed for three different initial concentrations of amygdalin solutions. The decrease in amygdalin concentration in these solutions over time is shown in Figure 17. The decrease in amygdalin concentration in these solutions over time is shown in Figure 17.

(a) (b) (c)

FigureFigure 17. TheThe concentration concentration of ofamygdalin amygdalin over over time time in in solution solution after after addition addition of −1 1 −1 1 −1 1 FeFe33OO44@SiO@SiO2@[email protected]@VIN@MIP. C C0 =0 =4545 mg mg L L −(a),( a0.45), 0.45 mg mg L L(−b),(andb), and4.5 µg 4.5 Lµg ( Lc−). (c).

InIn solutions solutions with with different different concentrations of of am amygdalin,ygdalin, it it was was most most effectively effectively bound bound in in the polymer structure at different different time points. However, However, for for all all samples, samples, the the total total binding binding time time of of the analyteanalyte did notnot exceedexceed 1 1 h; h; therefore, therefore, 1 h1 wash was considered considered as theas the optimal optimal binding binding time time of the of analyte the analyte from fromthe real the samples. real samples. All experiment All experiment times times with the with use the of use mag-MIP of mag-MIP were assumed were assumed to be 1 to h. be The 1 secondh. The secondFAPA-MS FAPA-MS method method examined examined the content the ofcontent amygdalin of amygdalin in Fe3O4 @SiOin Fe32O@VIN@MIP-amygdalin4@SiO2@VIN@MIP-amygdalin isolated isolatedfrom complementary from complementary aqueous aqueous solutions solutions of amygdalin. of amygdalin. The parallel The determination parallel determination of the analyte of the by analytethe FAPA-MS by the methodFAPA-MS was method carried was out carried directly out from directly the polymer from the structure. polymer structure. Due to the Due magnetic to the magneticproperties properties of the material, of the material, it was easily it was isolated easily fromisolated solutions from solutions using a smallusing neodymiuma small neodymium magnet. magnet.Then, the Then, polymer the polymer was washed was washed three timesthree withtimes water.with water. In the Innext the next step, step, a magnet a magnet was was placed placed in inthe the FAPA-MS FAPA-MS measuring measuring system system and and a a direct direct analysis analysis of of amygdalinamygdalin fromfrom thethe polymer structure structure was performed. The complementary results obtained with both methods are presented in Table5. The Fe3O4@SiO2@VIN@MIP polymer bound amygdalin from the solution very efficiently, and the amygdalin content determined by the FAPA-MS method directly from the polymer structure corresponded to the amount of the analyte in the test sample.

Table 5. Amygdalin adsorption capacity by Fe3O4@SiO2@VIN@MIP.

The Amount of Amygdalin Remaining in the Aqueous Amount of Adsorbed Amygdalin in The Amount of Solution after Adding Fe3O4@SiO2@VIN@MIP (20 mg), Fe3O4@SiO2@VIN@MIP-Amygdalin (20 mg), Determined Amygdalin in 5 Determined by ESI-MS by FAPA-MS mL Sample (µg) amount amount (µg) amount (µg) amount (µg) amount (µg) amount (µg) mean min max mean min max SD (µg) ± 225.0 nd nd nd 206.0 257.1 230.3 7.2 ± 2.25 nd nd nd 1.97 2.41 2.27 0.11 ± 0.0225 nd nd nd 0.0179 0.0299 0.0231 0.0009 ± 0.00022 nd nd nd 0.00015 0.00031 0.00024 0.00002 ± nd, not detected; SD, standard deviation. Biomolecules 2020, 10, x FOR PEER REVIEW 19 of 26 was performed. The complementary results obtained with both methods are presented in Table 5. The Fe3O4@SiO2@VIN@MIP polymer bound amygdalin from the solution very efficiently, and the amygdalin content determined by the FAPA-MS method directly from the polymer structure corresponded to the amount of the analyte in the test sample.

Table 5. Amygdalin adsorption capacity by Fe3O4@SiO2@VIN@MIP.

The Amount of Amygdalin Remaining in Amount of Adsorbed Amygdalin in The Amount the Aqueous Solution after Adding Fe3O4@SiO2@VIN@MIP-Amygdalin (20 of Amygdalin Fe3O4@SiO2@VIN@MIP (20 mg), mg), Determined by FAPA-MS in 5 mL Determined by ESI-MS Sample (μg) amountmin amountmax amountmean amountmin amountmax amountmean ± (μg) (μg) (μg) (μg) (μg) SD (μg) 225.0 nd nd nd 206.0 257.1 230.3 ± 7.2 2.25 nd nd nd 1.97 2.41 2.27 ± 0.11 0.0231 ± 0.0225 nd nd nd 0.0179 0.0299 0.0009 0.00024 ± 0.00022 nd nd nd 0.00015 0.00031 0.00002 nd, not detected; SD, standard deviation. Biomolecules 2020, 10, 1459 19 of 25 The resulting Fe3O4@SiO2@VIN@MIP showed high amygdalin-binding capacity in aqueous solutions of 11.5 mg amygdalin/g material. The resulting Fe3O4@SiO2@VIN@MIP showed high amygdalin-binding capacity in aqueous The process of amygdalin release from Fe3O4@SiO2@VIN@MIP-amygdalin in an aqueous solutions of 11.5 mg amygdalin/g material. solution was also analyzed. The release of the analyte into the solution was performed from three The process of amygdalin release from Fe O @SiO @VIN@MIP-amygdalin in an aqueous solution polymer matrices containing different amounts3 4of amygdalin.2 The analysis was carried out for 1 h, was also analyzed. The release of the analyte into the solution was performed from three polymer after which time the amygdalin content in the solution did not increase. The optimal time for the matrices containing different amounts of amygdalin. The analysis was carried out for 1 h, after which complete release of amygdalin from the material was 1 h. In the first method, the increase in time the amygdalin content in the solution did not increase. The optimal time for the complete release amygdalin concentration in the solution was examined by ESI-MS. The results are shown in Figure of amygdalin from the material was 1 h. In the first method, the increase in amygdalin concentration 18. in the solution was examined by ESI-MS. The results are shown in Figure 18.

Figure 18. The amount of amygdalin released over time in solution after addition

FigureFe3O4@SiO 18.2 @[email protected] amount of amygdalin released over time in solution after addition Fe3O4@SiO2@VIN@MIP-amygdalin. After 1 h, Fe3O4@SiO2@VIN@MIP was isolated from the solution, washed with water, and the remainingAfter 1 amygdalin h, Fe3O4@SiO with2@VIN@MIP the polymer was material isolated was from analyzed the solu by FAPA-MS.tion, washed The with results water, are shown and the in remainingTable6. amygdalin with the polymer material was analyzed by FAPA-MS. The results are shown in Table 6. Table 6. Release of amygdalin from Fe O @SiO @VIN@MIP-amygdalin in aqueous solution. 3 4 2 The Amount of Amygdalin in 10 mL Solution after Remaining Amygdalin in 100 mg Release from 100 mg Fe O @SiO @VIN@MIP after the Release Process, Fe O @SiO @VIN@MIP-Amygdalin, 3 4 2 The Amount of Amygdalin in 100 mg 3 4 2 Determined by FAPA-MS Determined by ESI-MS Fe3O4@SiO2@VIN@MIP-Amygdalin (mg) amount amount amount (mg) amount (mg) mean amount (mg) amount (mg) mean min max SD (mg) ± min max SD (mg) ± 7.0 6.74 7.50 6.9 0.4 0.01 0.05 0.02 0.02 ± ± 5.0 4.85 5.23 5.04 014 0.001 0.008 0.005 0.003 ± ± 2.0 1.92 2.14 2.02 0.09 nd nd nd ± nd, not detected; SD, standard deviation.

The amygdalin bound in the polymer structure in the solution was released practically completely within 1 h. The amount of unreleased amygdalin did not exceed 1% of the initial value; thus, it did not significantly affect the obtained results.

3.6. The Use of Hybrid Material Fe3O4@SiO2@VIN@MIP in the Analysis of Amygdalin in Samples In order to check the usefulness of the proposed analytical method, the content of amygdalin in aqueous and ethanolic extracts of bitter almonds by ESI-MS was determined. The material Fe3O4@SiO2@VIN@MIP was used as a molecular scavenger to pre-concentrate amygdalin from aqueous solutions. In the next step, the obtained magnetic scavenger was used to pre-concentrate amygdalin in the water and alcohol extracts, and a direct analysis on the polymer material by the FAPA-MS method was performed. The obtained results are presented in Table7. Biomolecules 2020, 10, 1459 20 of 25

Table 7. The content of amygdalin in bitter almond kernels.

Amygdalin Content in Bitter Amygdalin Content in Bitter Almond Kernels (mg/g), Almond Kernels (mg/g), Determined by FAPA-MS from Materials Determined by ESI-MS from Fe3O4@SiO2@VIN@MIP-Amygdalin the Extract after Binding of the Analyte from the Extract min. max. mean SD min. max. mean SD ± ± Water extract 19.9 26.1 22.1 0.7 21.9 30.1 27.7 1.2 ± ± Ethanol extract 34.8 44.5 37.9 1.7 40.7 61.2 48.4 1.8 ± ± SD, standard deviation.

The concentration of amygdalin in bitter almonds was 22.1 mg/g in the aqueous extract and 37.9 mg/g in the ethanol extract, as determined by ESI-MS. When using the FAPA-MS method in combination with Fe3O4@SiO2@VIN@MIP,the content was about 26% higher. The analysis of amygdalin in bitter almonds allowed to design an analytical method. Then, in the same way, extracts of apple and apricot seeds were prepared. Using the FAPA-MS method combined with Fe3O4@SiO2@VIN@MIP, it was determined that the amygdalin content in apple seeds and apricot seeds ranged from 0.87 to 1.43 mg/g and from 1.59 to 2.98 mg/g, respectively. The obtained results were compared with the results obtained using the HPLC-UV method by Bolarinwa et. al. [1], which showed that the presented method gave complementary results to the results obtained by other researchers.

3.7. Analysis of Amygdalin Skin Penetration Using the tape-stripping technique, the amount of amygdalin that penetrates the skin when poured in an aqueous solution was determined (Table8).

Table 8. Analysis of amygdalin skin penetration.

Amount of Amygdalin per 1 cm2 of Skin after Sample Number Release from Tape, Determined by ESI-MS (mg) min. max. mean SD ± 1 26.119 31.211 28.783 0.962 ± 2 0.055 0.081 0.067 0.004 ± 3 0.004 0.005 0.0040 0.0002 ± 4 nd nd nd nd, not detected; SD, standard deviation.

Based on the measurements, it was found that amygdalin from aqueous solutions very poorly penetrated subsequent layers of the skin. As much as 99.7% of amygdalin was found in the epidermis layer.

3.8. Analysis of Amygdalin in Human Serum Studies show that amygdalin in mammals undergoes enzymatic hydrolysis and is converted to two glucose molecules (glucose and prunasin) as well as mandelonitrile, which due to its unstable nature, is spontaneously converted to HCN and benzaldehyde [40–42]. We used artificial blood human serum, from human male AB plasma, sterile-filtered and fortified with amygdalin to establish the level of detection of this analyte in this biological matrix. This human AB serum is used in tissue engineering, transplantation, and cell therapy applications. The fortification was performed at the level of 5 mg of amygdalin per 1 mL of human serum. The toxicity of amygdalin through oral administration is higher than intravenous route. After oral administration, amygdalin produces Biomolecules 2020, 10, 1459 21 of 25

more toxic hydrocyanic acid in the organism. In rats, the fatal dose (LD50) of oral administration of 1 amygdalin is 880 mg kg− body weight [43]. The human lethal dose for intravenous injection is 5 g. Humans can present systemic toxicity after oral administration of amygdalin, 4 g per day for half a month or intravenous injection for a month. Moreover, the digestive system toxicity response is more common, with changes in atrial premature beats and electrocardiogram T wave. The toxicity response above can disappear after drug withdrawal. If the dose is reduced to daily oral doses of 0.6–1 g, toxicity can be avoided [44]. In the FAPA-MS spectra of human serum fortified with amygdalin, the signals m/z 371 (at 200 ◦C) and m/z 313 (at 340 ◦C) were observed. The signal intensity at m/z 371 strongly depended on the disposable equipment used. When using plastic equipment, the intensity of this signal increased sharply. The m/z 371 signal is characteristic of bis(2-ethylhexyl) adipate, + plasticizer (DEHA), or polyethylene glycol (PEG) [A8B + H] ((C2H4O)nH2O). However, no signals characteristic of amygdalin were observed. The MS2 fragmentation spectrum of m/z 371 signal showed two signals from the fragmentation ions, m/z 352 [371-19 (H3O)+]+ and m/z 265 [371-106 + (CH2CH2)2H2O] . Low-intensity m/z 371 signal was observed when only glass equipment was used in the sample preparation process. No signals characteristic of amygdalin were observed. In serum solution, amygdalin undergoes a very rapid hydrolysis reaction to acid, which gave the low-intensity 2 m/z 371 signal. This compound was converted to m/z 313 at a higher temperature (at 340 ◦C). The MS fragmentation spectrum of m/z 313 showed signals from the fragmentation ions m/z 296, m/z 285, m/z 235, m/z 180, and m/z 145 characteristic for the structure distribution with mass m/z 313 (Figure 19). Biomolecules 2020, 10, x FOR PEER REVIEW 22 of 26

313

180

145 296

235 285

2 FigureFigure 19. 19. FAPA-MSFAPA-MS2 fragmentationfragmentation spectrum spectrum of of the the mm/z/z 313313 ion ion observed observed for for amygdalin. amygdalin.

TheThe structures structures of of both both compounds compounds are are presente presentedd in inFigures Figures 20 and20 and 21. 21The. Thesignal signal compound compound m/z 313m/z is313 well-suited is well-suited for foridentifying identifying serum serum amygdali amygdalinn concentration. concentration. Unfortunately, Unfortunately, the the detection detection thresholdthreshold was was relatively relatively high high and and amounted amounted to to 0.1 0.1 mg mg of of amygdalin amygdalin in in 1 1 mL mL of of human human serum serum in in the the FAPA-MSFAPA-MS method. method.

371

+ H

Figure 20. FAPA-MS spectrum of amygdalin in human serum at 200 °C. Biomolecules 2020, 10, x FOR PEER REVIEW 22 of 26

313

180

145 296

235 285

Figure 19. FAPA-MS2 fragmentation spectrum of the m/z 313 ion observed for amygdalin.

The structures of both compounds are presented in Figures 20 and 21. The signal compound m/z 313 is well-suited for identifying serum amygdalin concentration. Unfortunately, the detection thresholdBiomolecules was2020, 10relatively, 1459 high and amounted to 0.1 mg of amygdalin in 1 mL of human serum 22in ofthe 25 FAPA-MS method.

371

+ H

Biomolecules 2020, 10, x FOR PEER REVIEW 23 of 26 FigureFigure 20. 20. FAPA-MSFAPA-MS spectrum spectrum of of amygdalin amygdalin in human serum at 200 °C.◦C.

313

+ H

FigureFigure 21. 21. FAPA-MSFAPA-MS spectrum spectrum of of amygdalin amygdalin in in human human serum serum at at 340 340 °C.◦C.

Due to the rapid transformation of amygdalin in the human serum, it was impossible Due to the rapid transformation of amygdalin in the human serum, it was impossible to pre- to pre-concentrate and separate amygdalin from the sample using Fe3O4@SiO2@VIN@MIP. concentrate and separate amygdalin from the sample using Fe3O4@SiO2@VIN@MIP. The synthesized The synthesized material showed selectivity toward amygdalin molecules, and the binding time material showed selectivity toward amygdalin molecules, and the binding time of the analyte in the of the analyte in the structure was up to 1 h. Therefore, it was impossible to bind amygdalin to structure was up to 1 h. Therefore, it was impossible to bind amygdalin to Fe3O4@SiO2@VIN@MIP in theFe3 Osample4@SiO 2in@VIN@MIP a time shorter in the than sample the intime a time duri shorterng which than the the timetransformation during which of amygdalin the transformation in the biologicalof amygdalin environment in the biological takes place. environment It was not takespossible place. to lower It was the not LOD possible for amygdalin to lower in the FAPA-MS LOD for byamygdalin combining in FAPA-MS this bymethod combining with this methodthe an withalyte the analytepre-concentration pre-concentration technique technique using using Fe3O4@SiO2@VIN@MIP. Fe3O4@SiO2@VIN@MIP. 4. Conclusions 4. Conclusions This paper presented the methodology of direct determination of amygdalin in water, extracts, This paper presented the methodology of direct determination of amygdalin in water, extracts, and biological materials using the ESI-MS and FAPA-MS methods. Both methods had a similar range and biological materials using the ESI-MS and FAPA-MS methods. Both methods had a similar range of linearity, LOD, and LOQ. However, the FAPA-MS method was less sensitive to the presence of of linearity, LOD, and LOQ. However, the FAPA-MS method was less sensitive to the presence of interferents in the analyzed sample. During the analysis of aqueous solutions of amygdalin with the interferents in the analyzed sample. During the analysis of aqueous solutions of amygdalin with the addition of various metal cations, many of the complexes formed were observed in the ESI-MS spectrum. addition of various metal cations, many of the complexes formed were observed in the ESI-MS This was not recorded in the FAPA-MS method. Moreover, the FAPA-MS method was compared spectrum. This was not recorded in the FAPA-MS method. Moreover, the FAPA-MS method was with the HPLC-UV/MS technique using a certified reference material. In order to pre-concentrate compared with the HPLC-UV/MS technique using a certified reference material. In order to pre- concentrate and isolate amygdalin from biological samples, the synthesis of Fe3O4@SiO2@MIP as molecular scavengers was proposed. They were used successfully to pre-concentrate amygdalin in aqueous and ethanol extracts. Moreover, the process of binding and releasing amygdalin from this material Fe3O4@SiO2@MIP was characterized. The aim of the research was the synthesis of amygdalin- selective Fe3O4@SiO2@MIP, which will enable the isolation and pre-concentration of the analyte from real samples. As a result, the analysis was to be characterized by better analytical parameters, and the material was to be used for the analysis of amygdalin using the FAPA-MS technique. The obtained sorption material could not be used for the pre-concentration and isolation of amygdalin from human serum. The mechanism of changes of amygdalin taking place in the human serum was proposed, as well as the structure of the resulting compounds, and analytical signals enabling the determination of amygdalin in this medium were selected. The described methods allow for effective determination of amygdalin in aqueous solutions, environmental samples, and biological materials.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: XPS spectra obtained from Fe3O4@SiO2@VIN@MIP-amygdalin, Figure S2: XPS spectra obtained from Fe3O4@SiO2@VIN@MIP- amygdalin.

Author Contributions: Conceptualization, M.G., S.R. and G.S.; methodology, S.R.; software, M.G.; validation, M.G.; formal analysis, S.R.; investigation, M.G.; resources, S.R.; data curation, G.S.; writing—original draft Biomolecules 2020, 10, 1459 23 of 25

and isolate amygdalin from biological samples, the synthesis of Fe3O4@SiO2@MIP as molecular scavengers was proposed. They were used successfully to pre-concentrate amygdalin in aqueous and ethanol extracts. Moreover, the process of binding and releasing amygdalin from this material Fe3O4@SiO2@MIP was haracterized. The aim of the research was the synthesis of amygdalin-selective Fe3O4@SiO2@MIP, which will enable the isolation and pre-concentration of the analyte from real samples. As a result, the analysis was to be characterized by better analytical parameters, and the material was to be used for the analysis of amygdalin using the FAPA-MS technique. The obtained sorption material could not be used for the pre-concentration and isolation of amygdalin from human serum. The mechanism of changes of amygdalin taking place in the human serum was proposed, as well as the structure of the resulting compounds, and analytical signals enabling the determination of amygdalin in this medium were selected. The described methods allow for effective determination of amygdalin in aqueous solutions, environmental samples, and biological materials.

Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/10/1459/s1, Figure S1: XPS spectra obtained from Fe3O4@SiO2@VIN@MIP-amygdalin, Figure S2: XPS spectra obtained from Fe3O4@SiO2@VIN@MIP-amygdalin. Author Contributions: Conceptualization, M.G., S.R. and G.S.; methodology, S.R.; software, M.G.; validation, M.G.; formal analysis, S.R.; investigation, M.G.; resources, S.R.; data curation, G.S.; writing—original draft preparation, M.G.; writing—review and editing, G.S.; visualization, M.G. and G.S.; supervision, G.S.; project administration, G.S.; funding acquisition, G.S. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the National Science Centre, Poland, under Grant Number 2016/21/B/ST4/02082 and Project No. POWR.03.02.00-00-I023/17 co-financed by the European Union through the European Social Fund under the Operational Program Knowledge Education Development. Conflicts of Interest: The authors declare no conflict of interest.

References

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