- Email: [email protected]
Highly sensitive method for aldehydes detection: Application to furfurals analysis in raisin and bovine milk powder
Accepted Manuscript Highly sensitive method for aldehydes detection: Application to furfurals analysis in raisin and bovine milk powder Yao Sun, Zhaobing Guan, Hongwei Cai, Yiyong Huang, Yawei Lin, Xiaosong Hu PII:
S0003-2670(17)30969-8
DOI:
10.1016/j.aca.2017.08.032
Reference:
ACA 235405
To appear in:
Analytica Chimica Acta
Received Date: 19 June 2017 Revised Date:
13 August 2017
Accepted Date: 17 August 2017
Please cite this article as: Y. Sun, Z. Guan, H. Cai, Y. Huang, Y. Lin, X. Hu, Highly sensitive method for aldehydes detection: Application to furfurals analysis in raisin and bovine milk powder, Analytica Chimica Acta (2017), doi: 10.1016/j.aca.2017.08.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highly sensitive method for aldehydes detection: application to furfurals analysis
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in raisin and bovine milk powder
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Yao Sun#, Zhaobing Guan#, Hongwei Cai, Yiyong Huang, Yawei Lin* and Xiaosong
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Hu*
Department of Chemistry, School of Chemistry, Chemical Engineering and Life
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Sciences, Wuhan University of Technology, 430070 Wuhan, China
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* Corresponding author
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Dr. Yawei Lin, Department of Chemistry, School of Chemistry, Chemical
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Engineering and Life Sciences, Wuhan University of Technology, 430070 Wuhan,
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China; Email: [email protected], Tel: +86-18062409780.
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Dr. Xiaosong Hu, Department of Chemistry, School of Chemistry, Chemical
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Engineering and Life Sciences, Wuhan University of Technology, 430070 Wuhan,
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China; Email: [email protected], Tel: +86-18502707636.
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Yao Sun and Zhaobing Guan contribute equally to this work.
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Abstract A pre-column fluorescent derivatization method based on nitrone formation has
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been applied to determine furfurals (e.g. furfural (F), 5-methyfurfural (5-MF) and
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5-hydroxymethylfurfural (5-HMF)) in food samples for the first time. An
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N-substituted hydroxylamine reagent 4-((hydroxyamino)butyl)-7-hydroxycoumarin
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(HAHC) was used to react with the aldehyde group of furfurals to form stable nitrone
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derivatives with high fluorescence intensities. The reactions proceeded under mild
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conditions in 30 min with high derivatization yields (> 93%). A baseline-separation of
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three furfurals derivatives was subsequently achieved within 25 min on a
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reversed-phase column. The detection limits were at the low femtomol level (S/N = 3,
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20 µL per injection). The linear range of the calibration curve was 0.4-4000 nM with
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good correlation coefficients (R2 ≥ 0.9991). The proposed method was further applied
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for food sample analysis, such as bovine milk powder and raisin. Satisfactory
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recoveries were obtained in the range of 94.7%-103.5%. Above all, this pre-column
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derivatization method is simple, fast and highly sensitive, providing an effective and
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promising way for future studies of aldehydes in different matrices.
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Keywords:
4-((hydroxyamino)butyl)-7-hydroxycoumarin;
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Furfurals; High performance liquid chromatography; Fluorescence detection.
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2
Nitrone
formation;
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1. Introduction Furfurals,
including
furfural
(F),
5-methylfurfural
(5-MF)
and
5-hydroxymethylfurfural (5-HMF) as a group of important furanic aldehydes, can be
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found in a various of food products, such as milk [1, 2], raisin [3, 4], bread [5],
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concentrated juice [6], honey [7], vinegar [8] and wines [9, 10], among others. They
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are generated by the degradation of saccharides [11] as a result of thermal processing
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or long storage time [3, 9]. Furfurals appearance can lead to color, texture and flavor
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changes in these foods products [12, 13]. Moreover, several studies have pointed out
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that F, 5-MF and 5-HMF might have mutagenic and genotoxic effects [14, 15]. These
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compounds have negative impacts on human central nervous system, liver, kidney,
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heart and other organs when their concentrations exceed a certain limit of the body
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absorption [16]. Therefore, it is of vital importance to develop a simple, fast and
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sensitive method to determine the presence of furfurals in different food products.
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A number of methods have been developed, including spectrophotometric [17-19]
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and chromatographic (e.g. gas chromatography (GC) [10, 20-22] and high
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performance liquid chromatography (HPLC)) methods [23-26]. On the one hand,
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spectrophotometric method are simpler and of lower cost, however, they are often
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hindered by matrix effects and insufficient selectivity. On the other hand, liquid
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chromatography with UltraViolet detection (LC-UV) has been applied for furfurals
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determination in food products, yet its detection limit is often 2-3 orders of
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magnitudes higher than that of GC coupled with mass spectrometric method (GC-MS).
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Unfortunately, GC-MS often requires several analytical steps, including extraction
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and derivatization before analysis [21, 27]. HPLC methods couple with MS or fluorescence detection (FLD) are of high
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sensitivity and selectivity and they are often applied for efficient analysis in a given
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matrix. Widely used to enhance the hydrophobicity and the spectroscopic response of
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the analytes, fluorescent derivatization has also been used in the determination of
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furfurals by HPLC-FLD. For example, Donnarumma et al. [25] reported the
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determination of 5-HMF in human plasma using a fluorescent dansylhydrazine
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(DNSH) labeling and achieved a low limit of detection (LOD) at picomole level.
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lab
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synthesized
an
aldehyde-reactive
agent,
4-((hydroxyamino)butyl)-7-hydroxycoumarin (HAHC) [28]. This reagent has shown
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good fluorescence properties with a high quantum yield of 0.61. In HAHC, coumarin
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serves as the fluorophore core and the N-monosubstituted hydroxylamine group as the
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reactive end (Fig. 1). A furanic aldehyde thus can be labeled through condensation
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reaction with HAHC to afford a nitrone derivative without isomeric forms (Fig. 2).
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Moreover, the long aliphatic carbon chain is also expected to increase the
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hydrophobicity of the derivative and improve its chromatographic behavior [29].
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The aim of this work is to achieve a nitrone formation method using HAHC as the
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derivatization reagent that allows the qualitative and quantitative analysis of furfurals
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in foods using HPLC-FLD. Several reaction parameters were optimized to achieve
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high derivatization yields under mild conditions. We also investigated the signal
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enhancements of the labeled furfurals with HAHC in both fluorescence and mass
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spectrometric detection. Finally, the method was validated by analyzing furfurals in
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with N-substituted hydroxylamines reagents has a great potential in sensitive analysis
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of aldehydes in various sources.
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2. Experimental
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2.1. Materials and Chemicals
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4-((hydroxyamino)butyl)-7-hydroxycoumarin (HAHC) was synthesized in our
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laboratory. 5-hydroxymethyl furfural, furfural, 5-methyfurfural and dansylhydrazine
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were purchased from J&K Scientific (Beijing, China). Aliphatic aldehydes C1-C6,
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including formaldehyde (C1), ethanal (C2), propanal (C3), butanal (C4), pentanal
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(C5), hexanal (C6), were purchased from Aladdin (Shanghai, China). Acetic acid,
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sodium acetate, ammonium acetate, phosphoric acid, potassium dihydrogen phosphate,
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trifluoroacetic acid and aniline were purchased from Sinopharm Chemical Reagent Co.
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Ltd (Shanghai, China). Raisin was purchased from Tenwow Food Co. Ltd (Anhui,
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China). Bovine milk powder was purchased from Mengniu Dairy Co. Ltd
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(Neimenggu, China). All reagents used in the current study were of analytical reagent
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grade, unless otherwise noted. Filter (N66, 13 mm × 0.22 µm) was purchased from
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Jinteng (Tianjin, China). Ultracentrifuge tube (regenerated cellulose, 5K MWCO) was
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purchased from MilliporeAmicon (MA, USA).
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2.2. Equipment and reagents
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Chromatographic analyses were performed on a Shimadzu (Kyoto, Japan)
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LC-20A system with an injector with a 20 µL sample loop, a Shimadzu RF-20 5
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were performed on a reversed phase HPLC column (Agilent Eclipse XDB-C18, 5 µm,
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4.6 × 150 mm). Liquid chromatography grade acetonitrile (ACN) was purchased from
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Fisher Scientific (Pittsburgh, PA, USA). Water was Milli-Q grade. Fluorescence
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spectra were recorded on a RF5301 fluorescence spectrometer (Shimadzu, Tokyo,
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Japan) and UV/Vis spectra were recorded on a Lambda 10 UV/Vis spectrometer
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(Perkin-Elmer) with a 1×1 cm quartz cell. A pH meter (PB-10, Sartorius, USA) was
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used for the determination of pH values.
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2.3. NanoLC-ESI-MS analysis
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Electrospray-ionization mass spectra (ESI-MS) were measured on a Triple-TOF
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5600 System (AB SCIEX, USA) with a nanospray source. The elute mode for
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samples separation was done on a NanoLC Ultra System (Eksigent, USA) with a C18
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(5 µm, 0.15 × 150 mm) column. The column temperature was kept at 25 ºC. Elution
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was carried out with solvent A and B, which were acetonitrile-water (0.5/9.5, v/v) and
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acetonitrile-water (9.5/0.5, v/v) containing 0.1% formic acid, respectively. 25 µL of
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solvent A and 25 µL of methanol solution were used to dissolve the samples. A 2.4 µL
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aliquot of the solution was loaded into trap column at a flow rate of 2.0 µL min-1 for
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10 min. Gradient elution with a flow rate of 300 nL min-1 was used to conduct
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analytical separation and data acquisition was obtained using an ion source gas of
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3 psi, a curtain gas of 35 psi, an ion spray voltage of 2.3 kV, and an interface heater
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temperature of 150 ºC. ESI-MS was performed in positive ion mode with the mass
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range of 20-2000 m/z. IDA (information-dependent acquisition) mode was applied to
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1.2 software (AB SCIEX, USA) was used to process the resulting data.
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2.4. Fluorescence analysis
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HAHC was dissolved in acetonitrile-water (1.0/9.0, v/v）to a concentration of
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2 µM. For the emission spectrum, the excitation wavelength was maintained at
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322 nm and the emission wavelength was scanned from 350 to 600 nm. For the
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excitation spectrum, the emission wavelength was maintained at 447 nm and the
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excitation wavelength was from 200 to 400 nm.
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2.5. Derivatization procedure
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In the standard derivatization procedure, 20 µL of HAHC solution (1 mM),
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10 µL of the standard furfurals solution, 10 µL of aniline solution (20 mM) and 10 µL
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of sodium acetate buffer (0.1 M, pH 3.5) were mixed in a plastic centrifuge tube. The
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mixture was kept at room temperature (20 ºC) for 30 min followed by high
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performance liquid chromatography with fluorescence detection (HPLC-FLD)
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analysis. Reagent blanks were carried through the same procedure except for the
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addition of standard furfurals.
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2.6. Chromatographic separation
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Samples were analyzed by a HPLC system using a C18 column at a flow rate of
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1 mL min-1 with fluorescence detection at excitation and fluorescence emission
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wavelengths of 322 and 447 nm, respectively. Before the analysis, the C18 column was
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solution was injected. The column temperature was kept at 30 ºC. The elution
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followed a binary gradient separation. Eluent A was acetonitrile-water (0.5/9.5, v/v)
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and eluent B was acetonitrile. Gradient: 0-12 min, 13% B; 12-30 min, 20% B.
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2.7 The comparison of chromatograms after HAHC and DNSH labeling
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DNSH labeling: The derivatization procedure of three furfurals with DNSH was
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adopted according to the reference [25]. The injection concentration of three furfurals
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was 0.02 mM each. The chromatographic separation was conducted as described by
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the reference [25]. Excitation and fluorescence emission wavelengths of the
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fluorescence detector were set at 350 and 525 nm, respectively. The separation
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followed a binary gradient mode. Eluent A was phosphate buffer (20 mM, pH 2.5) and
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eluent B was acetonitrile. Gradient: 0-4 min, 30% B; 4-9 min, 30% B up to 60% B.
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The flow rate was kept at 1 mL min-1 and the column temperature was kept at 30 ºC.
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HAHC labeling: The derivatization procedure of three furfurals was described in
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section 2.5. The injection concentration of three furfurals was 0.02 mM each. The
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chromatographic separation was described in section 2.6.
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2.8 Method validation
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The linearity was calculated by analyzing standard mixtures of three furfurals
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with different concentrations starting from LOQ (signal-to-noise ratio = 10) values to
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4 µM.
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Intra-day precision was measured by six replicates of standard furfurals samples 8
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on the same day and inter-day precision was determined by using the same method on
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three different days. Recoveries were determined by the analysis of spiked samples at medium and
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high concentrations (0.4 µM and 1 µM) before the sample preparation procedure. The
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calculated and expected concentrations (C) of the spiked sample were compared to
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determine the recovery values using following equation: Recovery, % = [Cspiked
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sample/Cexpected] × 100
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2.9 Sample preparation
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Raisin: The sample preparation of dried raisin was conducted according to the
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reference [4] with slight modifications. Briefly, 5.0 g sample was accurately weighed
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in a 50 mL centrifuge tube. Then 5 mL of methanol and 20 mL of 0.02 M ammonium
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acetate solution (pre-adjusted to pH 4.5 with acetic acid) were added to this centrifuge
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tube and mixed ultrasonically for 30 min. The supernatant of the mixture was filtered
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through a 0.22 µm filter (Nylon 66) and transferred to a new plastic centrifuge tube
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before derivatization. The samples were diluted 10 times before derivatization.
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Milk powder: The sample pretreatment followed the procedure described
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previously [30] with slight changes. Briefly, 5.0 g sample was accurately weighed and
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dissolved with warm water to 25 mL. The solution was well mixed ultrasonically for
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10 min. Then 1 mL acetic acid-water (2.5/7.5, v/v) was added to the solution followed
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by the addition of methanol to the volume of 50 mL. The mixture was well mixed and
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then centrifuged at 6000 rpm for 2 h in an ultrafiltration tube (regenerated cellulose
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membrane) with a molecular weight cut-off of 5000 Da. The filtrate was then filtered 9
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were diluted 2.5 times before derivatization.
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3. Results and discussion
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3.1. Derivatization with HAHC
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HAHC is a new fluorescent reagent synthesized by the authors. The synthetic
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procedure and the characterization of HAHC have been described in our previous
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work [28]. The excitation and fluorescence emission wavelengths of HAHC were
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determined to be 322 and 447 nm, respectively (Fig. 3). In our initial attempt, we
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chose F as the model furanic aldehyde to react with HAHC for 1 h at 50 °C followed
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by HPLC-FLD analysis. Compared with the control, one new peak (tR: 9 min) was
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observed in the HPLC spectrum and identified as the F-HAHC derivative (see Fig.
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4A). The eluate corresponding to that peak was then collected and subjected to
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fluorescence and ESI-MS detection. The resulting fluorescence spectrum (Fig. S1)
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shows that F-HAHC derivative and HAHC have identical λex and λem, indicating that
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the fluorescence of the F-HAHC derivative is determined by the HAHC moiety.
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Ion of m/z 328.12 was detected by ESI-MS (Fig. 4B), corresponding to the
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F-HAHC derivative with a theoretical m/z value of 328.1182. Furthermore, a close
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look of the ESI-MS/MS spectrum discloses that the F-HAHC derivative ion could
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fragment on the carbon-nitrogen bond and the carbon-nitrogen double bond adjacent
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to the furan (Fig. 5). The major fragment ion (m/z 217.09) belongs to the HAHC part
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and the less prominent ion (m/z 81.03) belongs to the furfural part.
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producing a single F-HAHC nitrone derivative. This observation is consistent with the
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findings of previous studies [31,32], where the authors showed that the condensations
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of aldehyde with N-monosubstituted hydroxylamines gave out single nitrone product
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without
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2,4-dinitrophenylhydrazine
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O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) [27] give two
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peaks for each furfural, because their derivatives can exist in both E- and Z- isomeric
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forms. On the contrary, our method of derivatization only produces one unambiguous
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isomer, which simplifies the chromatograms and facilitates the analysis of furfurals
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mixtures.
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3.2 Optimization of derivatization conditions
forms.
Please
noted
(DNPH)
that [26],
labeling
reagents
DNSH
[25]
such
as
and
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We next used single factor analysis to investigate various parameters that could
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affect the derivatization efficiency, including reaction time, reaction temperature, pH
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and derivatization reagent amount. The results are presented in Fig. 6. We use the area
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of the product peak in the HPLC spectrum to estimate the derivatization efficiency.
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The bigger the peak area, the higher the derivatization efficiency.
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The effects of reaction time were studied by monitoring the reaction performed
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in a water bath at 50 °C. As shown in Fig. 6A, the peak area of the furfural derivative
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plateaued at the reaction time of 4 h. Therefore, 4 h was determined as the optimal
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reaction time.
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shown in Fig. 6B, the peak area of the derivative was the greatest at 50 °C. When the
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reaction temperature was over 50 °C, the peak areas of derivative decreased, which is
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presumably due to the instability of the nitrone derivative at higher temperatures.
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Thus, 50 °C was determined as the optimized reaction temperature.
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Since the condensation of aldehyde and hydroxylamine is favored in acidic
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condition [33], further optimization of the derivatization reaction was performed in
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the pH range of 3.0-5.0 using sodium acetate buffer (Fig. 6C). The peak area
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maximized at pH 3.5 and this value was determined as the optimal pH value.
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Generally, in fluorescent derivatization, excessive amounts of the labeling
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reagents are required to ensure the derivatization efficiency and reproducibility.
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However, excess reagents may cause the overloading of LC column and
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chromatographic peak tailing, therefore an appropriate amount of reagent needs to be
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selected. As shown in Fig. 6D, increasing the HAHC concentration initially led to an
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increase of the peak area. However, when HAHC concentration reached 0.4 mM and
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above, the peak area almost remained constant. Therefore, 0.4 mM was selected as the
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optimized HAHC in the derivatization.
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These results show that the derivatization reaction conducted at 50 °C for 4 hours
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would offer the highest derivatization efficiency. However, 5-HMF can be formed in
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seconds in juices [34] when heated at 85 °C, indicating that high temperature should
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be avoided during real sample analysis, especially in sugar-rich foods. Therefore, to
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compensate good derivatization efficiency at a relatively low temperature, an
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appropriate catalyst needs to be chosen. Aniline has been reported as a nucleophilic catalyst for imine formation reactions
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(e.g. oxime formation and reductive amination [35]) according to the imine exchange
259
mechanism [36]. In this study, we found, for the first time, aniline also has
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considerable catalytical effects for the nitrone formation reaction. As shown in Fig.
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7A, when no aniline was added, the peak areas of the derivative maximized at 4 h.
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When aniline was introduced, the peak areas maximized in much shorter reaction
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times (e.g. 15 to 30 min). The enlarged graph (Fig. 7B) shows that the catalytical
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effects of 4 mM aniline was the greatest. However, sharp decreases of the
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derivatization yields were also observed with the increasing of reaction time, which is
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probably due to the competitive amination reaction between aniline and furfurals at
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50 °C [37]. When the reaction temperature was lowered to 20 °C (Fig. 7C), the peak
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areas reached maximum and remained constant, suggesting that the competitive
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reaction of aniline was minimized at room temperature. Also, among different aniline
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concentrations (0.4-4 mM), the greatest derivatization yield was achieved in 15 min
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when 4 mM of aniline was introduced. Therefore, 4 mM was chosen as the optimal
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aniline concentration and 30 min was chosen as the reaction time for high accuracy.
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Using the optimized derivatization method, two other furfurals (5-MF and 5-HMF)
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were
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NanoLC-ESI-MS (Fig. S2-S3, supporting information).
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also
successfully
derivatized
and
identified
by
HPLC-FLD
and
The derivatization yields for F, 5-MF and 5-HMF were also measured as 93.41%,
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95.41% and 96.59%, respectively (see supporting information), demonstrating that the
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highly efficient derivatization were achieved under mild conditions in the presence of
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aniline.
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3.3
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3.3.1 Chromatographic separation
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A reversed-phase mode is usually used in HPLC for the separation of the
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derivatives of furfurals in food sample analysis [1]. The separation of three derivatives
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of furfurals was then investigated on a C18 column and the flow rate was set at
285
1.0 mL min-1 at room temperature. Initially, isocratic elution of the three analytes was
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investigated and a long separation time was needed. Hence, the gradient elution
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program, described in the experimental section 2.6, was adopted, using the
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conventional acetonitrile-water system as the mobile phase. As shown in Fig. 8, a
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baseline separation of these three derivatives was achieved within 25 min (Fig. 8B).
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The derivatives of F, 5-MF and 5-HMF were all identified by ESI-MS and
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ESI-MS/MS (Fig. S4-S5, supporting information).
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To investigate the stability of the nitrone products, the reaction mixture was kept
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at room temperature for 1 h, 24 h and 36 h before it was subjected to HPLC-FLD
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analysis at each time interval. The chromatograms obtained showed no obvious
295
changes and the peak areas of the derivatives remained constant (Fig. S6), indicating
296
the excellent stability of the nitrone derivatives.
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3.3.2 Matrix interference 14
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aliphatic aldehydes or sugars) may interfere or even hinder the analysis of furfurals.
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To evaluate the potential interference, we investigated the furfurals samples spiked
301
with carbohydrates (e.g. glucose and maltoheptaose) and aliphatic aldehydes (C1-C6).
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In case of the carbohydrates analysis, no obvious product peaks were observed. A
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possible explanation for this is that sugars are less reactive toward HAHC under room
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temperature, because they are relatively stable and exist as hemiacetals in cyclic forms.
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In contrast, aliphatic aldehydes (C1-C6) react with HAHC readily, but their
306
derivatives are well separated from furanic aldehydes using the same elution program
307
as mentioned above (Fig. 9). These results highlight the selective coupling of HAHC
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with free aldehyde groups to afford stable derivatives with different chromatographic
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properties, facilitating the reversed-phase HPLC analysis. It also implies that the
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nitrone formation method could be further adapted to the analysis of various
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aldehydes.
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3.3.3 Enhancement of the detection sensitivity by HAHC derivatization
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To further evaluate the new derivatization method, we compared HAHC with a
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conventional fluorescent reagent DNSH. Furfurals with the same starting
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concentration were derivatized with HAHC and DNSH individually, followed by
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HPLC-FLD analysis. From the resulting chromatograms (Fig. S7-S9), HAHC labeling
317
produced one chromatographic peak for each analyte, while DNSH labeling produced
318
two. Moreover, the peak areas of each HAHC derivatives was considerably larger
319
than the corresponding DNSH derivatives. The peak area enhancement folds over 15
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DNSH derivatives were measured. For all the furfurals, the peak areas were more than
321
ten times as large as those of DNSH labeling. We also found that HAHC labeling can significantly improve the MS responses
323
of furfurals. The extracted ion chromatograms (EIC) from LC-MS for furfurals before
324
and after derivatization were obtained (Fig. S10, supporting information). The
325
magnitudes of signal enhancement, estimated by the peak area ratio of the derivatized
326
furfurals over un-derivatized one over was measured (Fig. S11). Impressively, all
327
three furfurals showed signal improvements over two orders of magnitudes, with the
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HAHC-labeled F in particular achieving an improvement of > 104-fold over its
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underivatized counterpart. The significant enhancement of MS response can be
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attributed to the hydrophobic fluorophore tag and the long alkyl chain [29] introduced
331
by HAHC labeling. Additionally, from the MS/MS spectra of all three furfurals
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derivatives (Fig. 5 and Fig. S4-S5 in supporting information), we observed the same
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characteristic fragmentation ion (m/z 217.09) from the HAHC part. These results
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demonstrate the great potential of HAHC as a mass tag, which not only enhances the
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mass spectrometric response but also provides characteristic fragment ions that may
336
facilitate the profiling of aldehydes by mass spectrometric analysis.
337
3.4 Method validation
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For method validation, standard mixtures of three furfurals with different
339
concentrations were analyzed with the new derivatization method, a more detailed
340
description was provided in section 2.8. The linear calibration ranges, regression
341
equations, correlation coefficients and detection limits of furfurals were calculated 16
ACCEPTED MANUSCRIPT and summarized in Table 1. The calibration curves showed good linearity in wide
343
dynamic ranges (0.4-4000 nM) between concentration and peak areas with correlation
344
coefficients (R2) from 0.9991 to 0.9998; We also observed low detection limits
345
ranging from 0.10 nM to 0.80 nM (signal to noise = 3, injection volume 20 µL). The
346
reproducibility of the proposed method, expressed by intra-day and inter-day relative
347
standard deviation (RSD), was also satisfactory, ranging from 0.74% to 1.46% and
348
from 4.59% to 5.06%, respectively. These results show that the new derivatization
349
method can provide sensitive and selective analysis of furfurals with high precision.
350
3.5 Real sample analysis
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Furfurals are not present in fresh food, however they can be found in a wide
352
variety of heat processed foods, such as dried fruits and dairy products. To
353
demonstrate the practical application of this new derivatization method, we attempted
354
to determination the amount of F, 5-MF and 5-HMF in raisin and milk powder
355
samples. After simple sample preparation procedures (described in section 2.9), the
356
samples were derivatized with HAHC, prior to being sent directly for HPLC-FLD
357
analysis.
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The HPLC results of raisins unspiked and spiked with standard furfurals mixture
359
solutions were shown in Fig. 10. All three furfurals were well separated and detected
360
without any interference. The detailed data were listed in Table 2. The recovery
361
percentages, in the ranged of 94.7%-103.5%, were achieved, compensating the
362
accuracy of the measurement. In the analysis of bovine milk powder (Fig. 11), F and
363
5-HMF were identified with good recoveries (96.7%-98.8%) (see Table 3). 17
ACCEPTED MANUSCRIPT These results show that, F and 5-HMF are present in thermally-processed
365
carbohydrate-containing foods, including raisin and milk powder. 5-HMF level is
366
relatively high, indicating 5-HMF need to be carefully monitored in these products.
367
Additionally, no 5-MF was observed in milk powder sample, which corroborates with
368
other studies on milk powder analysis [1, 2]. This is possibly the consequence of
369
different formation mechanisms of furfurals in different food sources.
370
3.6. Comparison with other methods
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As shown in Table 4, the LODs of other methods using pre-column derivatization,
372
such as GC-MS, HPLC-UV and HPLC-FLD, are all higher than 7.9 nM. In contrast,
373
the LOD of our method is much lower, in the range between 0.1-0.8 nM. Furthermore,
374
other chromatographic methods typically require extraction procedures such as solid
375
phase extraction or solid phase micro-extraction prior to the analysis, adding more
376
costs and time. Our method, on the other hand, does not require any prior purification
377
or extraction, and the derivatized sample was directly subjected to LC-FLD analysis.
378
Moreover, our derivatization method with HAHC can be conducted at room
379
temperature in the presence of aniline within a short period of reaction time, avoiding
380
the artificial formation of 5-HMF at high temperature. Therefore, our method has
381
advantages of high detection sensitivity and good reproducibility. With all these
382
features, we believe our method would be a better choice to detect and analyze these
383
furfurals than existing methods.
384
4. Conclusions
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18
ACCEPTED MANUSCRIPT In summary, we have detailed a new pre-column derivatization method based on
386
nitrone formation, which has been applied to determine F, 5-MF and 5-HMF for the
387
first time. Furfurals can be labeled with HAHC with high derivatization yields under
388
mild conditions, producing stable derivatives that have significant signal responses for
389
both fluorescence and MS detection. Additionally, combined with LC separation, the
390
method is selective and of low interference, allowing simultaneous analysis of three
391
furfurals in food samples without complicated purification or extraction procedures.
392
We obtained sub-nM level detection of furfurals, which is much more sensitive when
393
compared with other methods. The results showed good reproducibility and high
394
accuracy. We expect that this nitrone formation method can find wide applications in
395
the labeling of various aldehydes; in the meantime, we are actively pursuing the
396
development of new N-substituted hydroxylamine reagents.
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Acknowledgements
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We gratefully acknowledge the National Natural Science Foundation of China
400
(21405117 to Y. Lin and 21472144 to X. Hu), the Fundamental Research Funds for
401
the Central Universities (WUT: 2016-IB-005, 2016-IB-007, 2017-IB-007) for
402
financial support.
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Conflict of interest The authors declare that they have no conflicts of interest. 19
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bases, Rev. Chim. 66(2015) 1965-1967.
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Figure Captions
529 530
Fig. 1. The structure of the N-monosubstituted hydroxylamine reagent HAHC.
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Fig. 2. Schematic representation of nitrone formation reaction by the condensation of
533
N-monosubstituted hydroxylamine and furanic aldehyde to afford nitrone derivative.
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532
534
Fig. 3. The fluorescence excitation and emission spectrum of HAHC (2.0 µM).
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536
Fig. 4. The derivatization of F (20 µM) with HAHC. (A) Typical chromatogram of
538
F-HAHC derivative; The elution followed an isocratic mode and the mobile phase
539
was acetonitrile-water (3.0/7.0, v/v); Flow rate: 1.0 mL min-1; (B) ESI mass spectrum
540
of F-HAHC derivative.
542
543
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Fig. 5. On-line MS/MS spectrum of F-HAHC derivative.
544
Fig. 6. Effects of derivatization conditions on the peak areas of F-HAHC derivative,
545
including (A) reaction time, (B) reaction temperature, (C) buffer pH and (D) the
546
concentration of HAHC. The data represent error bars of triplicate measurements.
26
ACCEPTED MANUSCRIPT 547
Fig. 7. (A) Effects of the aniline concentration on the derivatization efficiency at
549
50 ºC, (■) Caniline = 0 M, (●) Caniline = 0.8 mM, (▲) Caniline = 4.0 mM, (▼) Caniline =
550
8.0 mM; (B) Enlarged view of partial Fig. 7A; (C) Effects of the aniline concentration
551
on the derivatization efficiency at 20 ºC, (■) Caniline = 0.4 mM, (●) Caniline = 2.0 mM
552
(▲) Caniline = 4.0 mM.
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Fig. 8. (A) Typical chromatogram of reagent blank carried through the same
555
derivatization procedure except for the addition of furfurals; (B) Typical
556
chromatogram of standard mixture of three furfurals (2.0 µM each). Shown are the
557
derivatives of 5-HMF (tR: 9.5 min), F (tR: 19.8 min) and 5-MF (tR: 25.2 min). Mobile
558
phase: A was acetonitrile-water (0.5:9.5, v/v); B was acetonitrile; Gradient: 0-12 min,
559
13% B; 12-30 min, 20% B; Flow rate: 1.0 mL min-1.
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Fig. 9. Representative chromatogram of a standard mixture of three furfurals (2.0 µM
562
each) spiked with six aliphatic aldehydes (C1-C6, 2.0 µM each) subjected to
563
derivatization. Named are the derivatives peaks for C1-C2 (tR: 2.2 min), C3 (tR:
564
7.6 min), 5-HMF (tR: 9.5 min), C4 (tR: 14.7 min), F (tR: 19.7 min), C5 (tR: 21.6 min),
565
5-MF (tR: 25.0 min), C6 (tR: 31.6 min). The same elution program as described in Fig.
566
8 was adopted.
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ACCEPTED MANUSCRIPT 567
568
Fig. 10. Typical chromatograms obtained from (A) raisin sample and (B) raisin
569
sample spiked with F, 5-MF and 5-HMF (1.0 µM each). Chromatographic conditions
570
were as described in Fig. 8. * = F derivative, ● = 5-MF derivative,
571
derivative.
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= 5-HMF
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572
◆
Fig. 11. The chromatograms obtained from (A) bovine milk powder sample and (B)
574
the same powder sample spiked with F and 5-HMF (1.0 µM each). Chromatographic
575
conditions were as described in Fig. 8. * = F derivative,
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◆
= 5-HMF derivative.
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Figures
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578
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580 581 582
Fig. 1. The structure of the N-monosubstituted hydroxylamine reagent HAHC.
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ACCEPTED MANUSCRIPT 584
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586 587
Fig. 2. Schematic representation of nitrone formation reaction by the condensation of
589
N-monosubstituted hydroxylamine and furanic aldehyde to afford a nitrone derivative.
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ACCEPTED MANUSCRIPT 591
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Fig. 3. The fluorescence excitation and emission spectrum of HAHC (2.0 µM).
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ACCEPTED MANUSCRIPT 596
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Fig. 4. The derivatization of F (20 µM) with HAHC. (A) Typical chromatogram of
600
F-HAHC derivative; The elution followed an isocratic mode and the mobile phase
601
was acetonitrile-water (3.0/7.0, v/v); Flow rate: 1.0 mL min-1; (B) ESI mass spectrum
602
of F-HAHC derivative.
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ACCEPTED MANUSCRIPT 604
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Fig. 5.On-line MS/MS spectrum of the F-HAHC derivative.
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ACCEPTED MANUSCRIPT 609
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Fig. 6. Effects of derivatization conditions on peak areas of the F-HAHC derivative,
613
including (A) reaction time, (B) reaction temperature, (C) buffer pH and (D) the
614
concentration of HAHC. The data represent error bars of triplicate measurements.
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ACCEPTED MANUSCRIPT 616
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Fig. 7. (A) Effects of the aniline concentration on the derivatization efficiency at
620
50 ºC, (■) Caniline = 0 M, (●) Caniline = 0.8 mM, (▲) Caniline = 4.0 mM, (▼) Caniline =
621
8.0 mM; (B) Enlarged view of partial Fig. 7A; (C) Effects of the aniline concentration
622
on the derivatization efficiency at 20 ºC, (■) Caniline = 0.4 mM, (●) Caniline = 2.0 mM
623
(▲) Caniline = 4.0 mM.
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ACCEPTED MANUSCRIPT 625
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627
Fig. 8. (A) Typical chromatogram of reagent blank carried through the same
629
derivatization procedure except for the addition of furfurals; (B) Typical
630
chromatogram of standard mixture of three furfurals (2.0 µM each). Shown are the
631
derivatives of 5-HMF (tR: 9.5 min), F (tR: 19.8 min) and 5-MF (tR: 25.2 min). Mobile
632
phase: A was acetonitrile-water (0.5:9.5, v/v); B was acetonitrile; Gradient: 0-12 min,
633
13% B; 12-30 min, 20% B; Flow rate: 1.0 mL min-1.
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ACCEPTED MANUSCRIPT 635
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Fig. 9. Representative chromatogram of a standard mixture of three furfurals (2.0 µM
639
each) spiked with six aliphatic aldehydes (C1-C6, 2.0 µM each) subjected to
640
derivatization. Named are the derivatives peaks for C1-C2 (tR: 2.2 min), C3 (tR:
641
7.6 min), 5-HMF (tR: 9.5 min), C4 (tR: 14.7 min), F (tR: 19.7 min), C5 (tR: 21.6 min),
642
5-MF (tR: 25.0 min), C6 (tR: 31.6 min). The same elution program as described in Fig.
643
8 was adopted.
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Fig. 10. Typical chromatograms obtained from (A) raisin sample and (B) raisin
649
sample spiked with F, 5-MF and 5-HMF (1.0 µM each). Chromatographic conditions
650
were as described in Fig. 8; * = F derivative, ● = 5-MF derivative,
651
derivative.
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ACCEPTED MANUSCRIPT 653
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Fig. 11. The chromatograms obtained from (A) bovine milk powder sample and (B)
657
the same powder sample spiked with F and 5-HMF (1.0 µM each). Chromatographic
658
conditions were as described in Fig. 8. * = F derivative,
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Tables
660 661
Table 1
663
Linear calibration ranges, regression equations, detection limits, correlation
664
coefficient and reproducibility of the proposed method.
5-HMF F 5-MF
Regression Equationa Y = AX + B Y = 1.99X – 1.93 × 104 Y = 1.78X + 3.45 × 104 Y = 1.58X + 1.31 × 105
Calibration Range (nM) 2-4000 0.8-4000 0.4-4000
R2 0.9991 0.9998 0.9995
a
X = sample concentration (pM), Y = peak area (mV·min).
666
b
Signal-to-noise ratio = 3.
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667
40
RSD (%)
Intra-day
0.74 1.32 1.46
SC
Furfurals
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Inter-day
4.93 5.06 4.59
LODb (nM) 0.8 0.2 0.1
ACCEPTED MANUSCRIPT 668 669 670
Table 2
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Results of furfurals determination in raisin samples.
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Samples
Raisin RSD (%, n = 6) Recoverya Intra-day Inter-day / 2.74 6.24 94.7 ± 2.2 2.32 5.48 96.5 ± 1.7 1.76 4.52
Found (µM) 1.85 2.13 2.75
F
0.0 0.4 1.0
0.14 0.52 1.18
/ 96.3 ± 2.6 103.5 ± 1.3
2.98 2.70 1.26
5.26 4.73 4.13
1.68 ± 0.05
5-MF
0.0 0.4 1.0
0.17 0.56 1.16
/ 98.2 ± 2.1 99.1 ± 3.5
4.03 2.14 3.53
6.50 4.76 3.65
2.31 ± 0.07
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Recovery are given as mean ± SD (n = 6, %).
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b
Value are given as mean ± SD (n = 6, mg/Kg).
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Added (µM) 0.0 0.4 1.0
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Contentb
29.16 ± 0.80
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Table 3
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Results of furfurals determination in milk powder samples.
F
Found (µM) 1.44 1.79 2.36
0.0 0.4 1.0
0.14 0.53 1.12
/ 98.1 ± 2.4 98.8 ± 3.1
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a
Recovery are given as mean ± SD (n = 6, %).
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b
Value are given as mean ± SD (n = 6, mg/Kg).
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5-HMF
Added (µM) 0.0 0.4 1.0
Bovine milk powder RSD (%, n = 6) Recoverya Intra-day Inter-day / 2.73 4.34 97.3 ± 2.0 2.06 5.32 96.7 ± 2.7 2.79 5.16 3.79 2.45 3.14
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6.03 3.92 4.52
Contentb
11.35 ± 0.31
0.84 ± 0.03
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684
Table 4
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Comparison with other methods. Reaction time/temp.
LOD (nM) 33-2545
Sample extraction /
/
/
400-430
/
/
/
7.9
PMME
DNPH
1 h/50 °C
16.6
SPME
PFBHA
GC-MSe
48.7-82.5
HS-SPME PFBHA
HPLC-FLD
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SPE
HPLC-FLD
0.1-0.8
/
HPLC-PAD c
HPLC-UV GC-MS
d
Reference [2]
[24]
[26]
DNSH
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b
Reagent
20 min/50 °C
[25]
HAHC
30 min/20 °C
This work
30 min/30 °C
[27]
27 min/45 °C
[21]
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Analytical method HPLC-DADa
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686
a
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detection (PAD) using a modified electrode with nickel nanoparticles; c HPLC-UV
688
detection with polymer monolith microextraction (PMME) using DNPH as the
689
derivatization reagent;
690
chromatography and mass spectrometry detection;
691
microextraction (HS-SPME) combined with gas chromatography and mass
692
spectrometry detection.
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HPLC with pulsed amperometric
Solid-phase microextraction (SPME) combined with gas
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HPLC with diode array detection (DAD);
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e
Headspace solid-phase
ACCEPTED MANUSCRIPT
Highlights First paper using nitrone formation as a pre-column derivatization method to determine furfurals in raisin and bovine milk powder.
responses were achieved by the derivatized furfurals.
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Significant signal enhancements in fluorescence and mass spectrometric
The detection limit was as low as 0.1 × 10 9 M using high performance liquid −
chromatography with fluorescence detection.
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This method provides a tool for the highly sensitive detection of furfurals in food
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samples.
S0003-2670(17)30969-8
DOI:
10.1016/j.aca.2017.08.032
Reference:
ACA 235405
To appear in:
Analytica Chimica Acta
Received Date: 19 June 2017 Revised Date:
13 August 2017
Accepted Date: 17 August 2017
Please cite this article as: Y. Sun, Z. Guan, H. Cai, Y. Huang, Y. Lin, X. Hu, Highly sensitive method for aldehydes detection: Application to furfurals analysis in raisin and bovine milk powder, Analytica Chimica Acta (2017), doi: 10.1016/j.aca.2017.08.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highly sensitive method for aldehydes detection: application to furfurals analysis
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in raisin and bovine milk powder
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Yao Sun#, Zhaobing Guan#, Hongwei Cai, Yiyong Huang, Yawei Lin* and Xiaosong
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Hu*
Department of Chemistry, School of Chemistry, Chemical Engineering and Life
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Sciences, Wuhan University of Technology, 430070 Wuhan, China
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* Corresponding author
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Dr. Yawei Lin, Department of Chemistry, School of Chemistry, Chemical
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Engineering and Life Sciences, Wuhan University of Technology, 430070 Wuhan,
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China; Email: [email protected], Tel: +86-18062409780.
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Dr. Xiaosong Hu, Department of Chemistry, School of Chemistry, Chemical
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Engineering and Life Sciences, Wuhan University of Technology, 430070 Wuhan,
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China; Email: [email protected], Tel: +86-18502707636.
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#
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Yao Sun and Zhaobing Guan contribute equally to this work.
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Abstract A pre-column fluorescent derivatization method based on nitrone formation has
24
been applied to determine furfurals (e.g. furfural (F), 5-methyfurfural (5-MF) and
25
5-hydroxymethylfurfural (5-HMF)) in food samples for the first time. An
26
N-substituted hydroxylamine reagent 4-((hydroxyamino)butyl)-7-hydroxycoumarin
27
(HAHC) was used to react with the aldehyde group of furfurals to form stable nitrone
28
derivatives with high fluorescence intensities. The reactions proceeded under mild
29
conditions in 30 min with high derivatization yields (> 93%). A baseline-separation of
30
three furfurals derivatives was subsequently achieved within 25 min on a
31
reversed-phase column. The detection limits were at the low femtomol level (S/N = 3,
32
20 µL per injection). The linear range of the calibration curve was 0.4-4000 nM with
33
good correlation coefficients (R2 ≥ 0.9991). The proposed method was further applied
34
for food sample analysis, such as bovine milk powder and raisin. Satisfactory
35
recoveries were obtained in the range of 94.7%-103.5%. Above all, this pre-column
36
derivatization method is simple, fast and highly sensitive, providing an effective and
37
promising way for future studies of aldehydes in different matrices.
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Keywords:
4-((hydroxyamino)butyl)-7-hydroxycoumarin;
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Furfurals; High performance liquid chromatography; Fluorescence detection.
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2
Nitrone
formation;
ACCEPTED MANUSCRIPT 42
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1. Introduction Furfurals,
including
furfural
(F),
5-methylfurfural
(5-MF)
and
5-hydroxymethylfurfural (5-HMF) as a group of important furanic aldehydes, can be
45
found in a various of food products, such as milk [1, 2], raisin [3, 4], bread [5],
46
concentrated juice [6], honey [7], vinegar [8] and wines [9, 10], among others. They
47
are generated by the degradation of saccharides [11] as a result of thermal processing
48
or long storage time [3, 9]. Furfurals appearance can lead to color, texture and flavor
49
changes in these foods products [12, 13]. Moreover, several studies have pointed out
50
that F, 5-MF and 5-HMF might have mutagenic and genotoxic effects [14, 15]. These
51
compounds have negative impacts on human central nervous system, liver, kidney,
52
heart and other organs when their concentrations exceed a certain limit of the body
53
absorption [16]. Therefore, it is of vital importance to develop a simple, fast and
54
sensitive method to determine the presence of furfurals in different food products.
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A number of methods have been developed, including spectrophotometric [17-19]
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and chromatographic (e.g. gas chromatography (GC) [10, 20-22] and high
57
performance liquid chromatography (HPLC)) methods [23-26]. On the one hand,
58
spectrophotometric method are simpler and of lower cost, however, they are often
59
hindered by matrix effects and insufficient selectivity. On the other hand, liquid
60
chromatography with UltraViolet detection (LC-UV) has been applied for furfurals
61
determination in food products, yet its detection limit is often 2-3 orders of
62
magnitudes higher than that of GC coupled with mass spectrometric method (GC-MS).
63
Unfortunately, GC-MS often requires several analytical steps, including extraction
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and derivatization before analysis [21, 27]. HPLC methods couple with MS or fluorescence detection (FLD) are of high
66
sensitivity and selectivity and they are often applied for efficient analysis in a given
67
matrix. Widely used to enhance the hydrophobicity and the spectroscopic response of
68
the analytes, fluorescent derivatization has also been used in the determination of
69
furfurals by HPLC-FLD. For example, Donnarumma et al. [25] reported the
70
determination of 5-HMF in human plasma using a fluorescent dansylhydrazine
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(DNSH) labeling and achieved a low limit of detection (LOD) at picomole level.
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Our
lab
recently
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synthesized
an
aldehyde-reactive
agent,
4-((hydroxyamino)butyl)-7-hydroxycoumarin (HAHC) [28]. This reagent has shown
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good fluorescence properties with a high quantum yield of 0.61. In HAHC, coumarin
75
serves as the fluorophore core and the N-monosubstituted hydroxylamine group as the
76
reactive end (Fig. 1). A furanic aldehyde thus can be labeled through condensation
77
reaction with HAHC to afford a nitrone derivative without isomeric forms (Fig. 2).
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Moreover, the long aliphatic carbon chain is also expected to increase the
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hydrophobicity of the derivative and improve its chromatographic behavior [29].
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The aim of this work is to achieve a nitrone formation method using HAHC as the
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derivatization reagent that allows the qualitative and quantitative analysis of furfurals
82
in foods using HPLC-FLD. Several reaction parameters were optimized to achieve
83
high derivatization yields under mild conditions. We also investigated the signal
84
enhancements of the labeled furfurals with HAHC in both fluorescence and mass
85
spectrometric detection. Finally, the method was validated by analyzing furfurals in
4
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with N-substituted hydroxylamines reagents has a great potential in sensitive analysis
88
of aldehydes in various sources.
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2. Experimental
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2.1. Materials and Chemicals
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4-((hydroxyamino)butyl)-7-hydroxycoumarin (HAHC) was synthesized in our
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laboratory. 5-hydroxymethyl furfural, furfural, 5-methyfurfural and dansylhydrazine
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were purchased from J&K Scientific (Beijing, China). Aliphatic aldehydes C1-C6,
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including formaldehyde (C1), ethanal (C2), propanal (C3), butanal (C4), pentanal
95
(C5), hexanal (C6), were purchased from Aladdin (Shanghai, China). Acetic acid,
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sodium acetate, ammonium acetate, phosphoric acid, potassium dihydrogen phosphate,
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trifluoroacetic acid and aniline were purchased from Sinopharm Chemical Reagent Co.
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Ltd (Shanghai, China). Raisin was purchased from Tenwow Food Co. Ltd (Anhui,
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China). Bovine milk powder was purchased from Mengniu Dairy Co. Ltd
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(Neimenggu, China). All reagents used in the current study were of analytical reagent
101
grade, unless otherwise noted. Filter (N66, 13 mm × 0.22 µm) was purchased from
102
Jinteng (Tianjin, China). Ultracentrifuge tube (regenerated cellulose, 5K MWCO) was
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purchased from MilliporeAmicon (MA, USA).
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2.2. Equipment and reagents
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Chromatographic analyses were performed on a Shimadzu (Kyoto, Japan)
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LC-20A system with an injector with a 20 µL sample loop, a Shimadzu RF-20 5
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were performed on a reversed phase HPLC column (Agilent Eclipse XDB-C18, 5 µm,
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4.6 × 150 mm). Liquid chromatography grade acetonitrile (ACN) was purchased from
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Fisher Scientific (Pittsburgh, PA, USA). Water was Milli-Q grade. Fluorescence
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spectra were recorded on a RF5301 fluorescence spectrometer (Shimadzu, Tokyo,
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Japan) and UV/Vis spectra were recorded on a Lambda 10 UV/Vis spectrometer
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(Perkin-Elmer) with a 1×1 cm quartz cell. A pH meter (PB-10, Sartorius, USA) was
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used for the determination of pH values.
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2.3. NanoLC-ESI-MS analysis
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Electrospray-ionization mass spectra (ESI-MS) were measured on a Triple-TOF
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5600 System (AB SCIEX, USA) with a nanospray source. The elute mode for
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samples separation was done on a NanoLC Ultra System (Eksigent, USA) with a C18
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(5 µm, 0.15 × 150 mm) column. The column temperature was kept at 25 ºC. Elution
120
was carried out with solvent A and B, which were acetonitrile-water (0.5/9.5, v/v) and
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acetonitrile-water (9.5/0.5, v/v) containing 0.1% formic acid, respectively. 25 µL of
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solvent A and 25 µL of methanol solution were used to dissolve the samples. A 2.4 µL
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aliquot of the solution was loaded into trap column at a flow rate of 2.0 µL min-1 for
124
10 min. Gradient elution with a flow rate of 300 nL min-1 was used to conduct
125
analytical separation and data acquisition was obtained using an ion source gas of
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3 psi, a curtain gas of 35 psi, an ion spray voltage of 2.3 kV, and an interface heater
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temperature of 150 ºC. ESI-MS was performed in positive ion mode with the mass
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range of 20-2000 m/z. IDA (information-dependent acquisition) mode was applied to
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1.2 software (AB SCIEX, USA) was used to process the resulting data.
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2.4. Fluorescence analysis
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HAHC was dissolved in acetonitrile-water (1.0/9.0, v/v）to a concentration of
133
2 µM. For the emission spectrum, the excitation wavelength was maintained at
134
322 nm and the emission wavelength was scanned from 350 to 600 nm. For the
135
excitation spectrum, the emission wavelength was maintained at 447 nm and the
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excitation wavelength was from 200 to 400 nm.
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2.5. Derivatization procedure
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In the standard derivatization procedure, 20 µL of HAHC solution (1 mM),
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10 µL of the standard furfurals solution, 10 µL of aniline solution (20 mM) and 10 µL
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of sodium acetate buffer (0.1 M, pH 3.5) were mixed in a plastic centrifuge tube. The
141
mixture was kept at room temperature (20 ºC) for 30 min followed by high
142
performance liquid chromatography with fluorescence detection (HPLC-FLD)
143
analysis. Reagent blanks were carried through the same procedure except for the
144
addition of standard furfurals.
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2.6. Chromatographic separation
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Samples were analyzed by a HPLC system using a C18 column at a flow rate of
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1 mL min-1 with fluorescence detection at excitation and fluorescence emission
148
wavelengths of 322 and 447 nm, respectively. Before the analysis, the C18 column was
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150
solution was injected. The column temperature was kept at 30 ºC. The elution
151
followed a binary gradient separation. Eluent A was acetonitrile-water (0.5/9.5, v/v)
152
and eluent B was acetonitrile. Gradient: 0-12 min, 13% B; 12-30 min, 20% B.
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2.7 The comparison of chromatograms after HAHC and DNSH labeling
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DNSH labeling: The derivatization procedure of three furfurals with DNSH was
155
adopted according to the reference [25]. The injection concentration of three furfurals
156
was 0.02 mM each. The chromatographic separation was conducted as described by
157
the reference [25]. Excitation and fluorescence emission wavelengths of the
158
fluorescence detector were set at 350 and 525 nm, respectively. The separation
159
followed a binary gradient mode. Eluent A was phosphate buffer (20 mM, pH 2.5) and
160
eluent B was acetonitrile. Gradient: 0-4 min, 30% B; 4-9 min, 30% B up to 60% B.
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The flow rate was kept at 1 mL min-1 and the column temperature was kept at 30 ºC.
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HAHC labeling: The derivatization procedure of three furfurals was described in
163
section 2.5. The injection concentration of three furfurals was 0.02 mM each. The
164
chromatographic separation was described in section 2.6.
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2.8 Method validation
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The linearity was calculated by analyzing standard mixtures of three furfurals
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with different concentrations starting from LOQ (signal-to-noise ratio = 10) values to
168
4 µM.
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Intra-day precision was measured by six replicates of standard furfurals samples 8
ACCEPTED MANUSCRIPT 170
on the same day and inter-day precision was determined by using the same method on
171
three different days. Recoveries were determined by the analysis of spiked samples at medium and
173
high concentrations (0.4 µM and 1 µM) before the sample preparation procedure. The
174
calculated and expected concentrations (C) of the spiked sample were compared to
175
determine the recovery values using following equation: Recovery, % = [Cspiked
176
sample/Cexpected] × 100
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2.9 Sample preparation
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Raisin: The sample preparation of dried raisin was conducted according to the
179
reference [4] with slight modifications. Briefly, 5.0 g sample was accurately weighed
180
in a 50 mL centrifuge tube. Then 5 mL of methanol and 20 mL of 0.02 M ammonium
181
acetate solution (pre-adjusted to pH 4.5 with acetic acid) were added to this centrifuge
182
tube and mixed ultrasonically for 30 min. The supernatant of the mixture was filtered
183
through a 0.22 µm filter (Nylon 66) and transferred to a new plastic centrifuge tube
184
before derivatization. The samples were diluted 10 times before derivatization.
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Milk powder: The sample pretreatment followed the procedure described
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previously [30] with slight changes. Briefly, 5.0 g sample was accurately weighed and
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dissolved with warm water to 25 mL. The solution was well mixed ultrasonically for
188
10 min. Then 1 mL acetic acid-water (2.5/7.5, v/v) was added to the solution followed
189
by the addition of methanol to the volume of 50 mL. The mixture was well mixed and
190
then centrifuged at 6000 rpm for 2 h in an ultrafiltration tube (regenerated cellulose
191
membrane) with a molecular weight cut-off of 5000 Da. The filtrate was then filtered 9
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193
were diluted 2.5 times before derivatization.
194
3. Results and discussion
195
3.1. Derivatization with HAHC
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HAHC is a new fluorescent reagent synthesized by the authors. The synthetic
197
procedure and the characterization of HAHC have been described in our previous
198
work [28]. The excitation and fluorescence emission wavelengths of HAHC were
199
determined to be 322 and 447 nm, respectively (Fig. 3). In our initial attempt, we
200
chose F as the model furanic aldehyde to react with HAHC for 1 h at 50 °C followed
201
by HPLC-FLD analysis. Compared with the control, one new peak (tR: 9 min) was
202
observed in the HPLC spectrum and identified as the F-HAHC derivative (see Fig.
203
4A). The eluate corresponding to that peak was then collected and subjected to
204
fluorescence and ESI-MS detection. The resulting fluorescence spectrum (Fig. S1)
205
shows that F-HAHC derivative and HAHC have identical λex and λem, indicating that
206
the fluorescence of the F-HAHC derivative is determined by the HAHC moiety.
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Ion of m/z 328.12 was detected by ESI-MS (Fig. 4B), corresponding to the
208
F-HAHC derivative with a theoretical m/z value of 328.1182. Furthermore, a close
209
look of the ESI-MS/MS spectrum discloses that the F-HAHC derivative ion could
210
fragment on the carbon-nitrogen bond and the carbon-nitrogen double bond adjacent
211
to the furan (Fig. 5). The major fragment ion (m/z 217.09) belongs to the HAHC part
212
and the less prominent ion (m/z 81.03) belongs to the furfural part.
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214
producing a single F-HAHC nitrone derivative. This observation is consistent with the
215
findings of previous studies [31,32], where the authors showed that the condensations
216
of aldehyde with N-monosubstituted hydroxylamines gave out single nitrone product
217
without
218
2,4-dinitrophenylhydrazine
219
O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) [27] give two
220
peaks for each furfural, because their derivatives can exist in both E- and Z- isomeric
221
forms. On the contrary, our method of derivatization only produces one unambiguous
222
isomer, which simplifies the chromatograms and facilitates the analysis of furfurals
223
mixtures.
224
3.2 Optimization of derivatization conditions
forms.
Please
noted
(DNPH)
that [26],
labeling
reagents
DNSH
[25]
such
as
and
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isomeric
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We next used single factor analysis to investigate various parameters that could
226
affect the derivatization efficiency, including reaction time, reaction temperature, pH
227
and derivatization reagent amount. The results are presented in Fig. 6. We use the area
228
of the product peak in the HPLC spectrum to estimate the derivatization efficiency.
229
The bigger the peak area, the higher the derivatization efficiency.
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The effects of reaction time were studied by monitoring the reaction performed
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in a water bath at 50 °C. As shown in Fig. 6A, the peak area of the furfural derivative
232
plateaued at the reaction time of 4 h. Therefore, 4 h was determined as the optimal
233
reaction time.
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235
shown in Fig. 6B, the peak area of the derivative was the greatest at 50 °C. When the
236
reaction temperature was over 50 °C, the peak areas of derivative decreased, which is
237
presumably due to the instability of the nitrone derivative at higher temperatures.
238
Thus, 50 °C was determined as the optimized reaction temperature.
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Since the condensation of aldehyde and hydroxylamine is favored in acidic
240
condition [33], further optimization of the derivatization reaction was performed in
241
the pH range of 3.0-5.0 using sodium acetate buffer (Fig. 6C). The peak area
242
maximized at pH 3.5 and this value was determined as the optimal pH value.
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Generally, in fluorescent derivatization, excessive amounts of the labeling
244
reagents are required to ensure the derivatization efficiency and reproducibility.
245
However, excess reagents may cause the overloading of LC column and
246
chromatographic peak tailing, therefore an appropriate amount of reagent needs to be
247
selected. As shown in Fig. 6D, increasing the HAHC concentration initially led to an
248
increase of the peak area. However, when HAHC concentration reached 0.4 mM and
249
above, the peak area almost remained constant. Therefore, 0.4 mM was selected as the
250
optimized HAHC in the derivatization.
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These results show that the derivatization reaction conducted at 50 °C for 4 hours
252
would offer the highest derivatization efficiency. However, 5-HMF can be formed in
253
seconds in juices [34] when heated at 85 °C, indicating that high temperature should
254
be avoided during real sample analysis, especially in sugar-rich foods. Therefore, to
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compensate good derivatization efficiency at a relatively low temperature, an
256
appropriate catalyst needs to be chosen. Aniline has been reported as a nucleophilic catalyst for imine formation reactions
258
(e.g. oxime formation and reductive amination [35]) according to the imine exchange
259
mechanism [36]. In this study, we found, for the first time, aniline also has
260
considerable catalytical effects for the nitrone formation reaction. As shown in Fig.
261
7A, when no aniline was added, the peak areas of the derivative maximized at 4 h.
262
When aniline was introduced, the peak areas maximized in much shorter reaction
263
times (e.g. 15 to 30 min). The enlarged graph (Fig. 7B) shows that the catalytical
264
effects of 4 mM aniline was the greatest. However, sharp decreases of the
265
derivatization yields were also observed with the increasing of reaction time, which is
266
probably due to the competitive amination reaction between aniline and furfurals at
267
50 °C [37]. When the reaction temperature was lowered to 20 °C (Fig. 7C), the peak
268
areas reached maximum and remained constant, suggesting that the competitive
269
reaction of aniline was minimized at room temperature. Also, among different aniline
270
concentrations (0.4-4 mM), the greatest derivatization yield was achieved in 15 min
271
when 4 mM of aniline was introduced. Therefore, 4 mM was chosen as the optimal
272
aniline concentration and 30 min was chosen as the reaction time for high accuracy.
273
Using the optimized derivatization method, two other furfurals (5-MF and 5-HMF)
274
were
275
NanoLC-ESI-MS (Fig. S2-S3, supporting information).
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also
successfully
derivatized
and
identified
by
HPLC-FLD
and
The derivatization yields for F, 5-MF and 5-HMF were also measured as 93.41%,
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95.41% and 96.59%, respectively (see supporting information), demonstrating that the
278
highly efficient derivatization were achieved under mild conditions in the presence of
279
aniline.
280
3.3
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3.3.1 Chromatographic separation
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A reversed-phase mode is usually used in HPLC for the separation of the
283
derivatives of furfurals in food sample analysis [1]. The separation of three derivatives
284
of furfurals was then investigated on a C18 column and the flow rate was set at
285
1.0 mL min-1 at room temperature. Initially, isocratic elution of the three analytes was
286
investigated and a long separation time was needed. Hence, the gradient elution
287
program, described in the experimental section 2.6, was adopted, using the
288
conventional acetonitrile-water system as the mobile phase. As shown in Fig. 8, a
289
baseline separation of these three derivatives was achieved within 25 min (Fig. 8B).
290
The derivatives of F, 5-MF and 5-HMF were all identified by ESI-MS and
291
ESI-MS/MS (Fig. S4-S5, supporting information).
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To investigate the stability of the nitrone products, the reaction mixture was kept
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at room temperature for 1 h, 24 h and 36 h before it was subjected to HPLC-FLD
294
analysis at each time interval. The chromatograms obtained showed no obvious
295
changes and the peak areas of the derivatives remained constant (Fig. S6), indicating
296
the excellent stability of the nitrone derivatives.
297
3.3.2 Matrix interference 14
ACCEPTED MANUSCRIPT In sugar-rich food products, other compounds with aldehyde groups (e.g.
299
aliphatic aldehydes or sugars) may interfere or even hinder the analysis of furfurals.
300
To evaluate the potential interference, we investigated the furfurals samples spiked
301
with carbohydrates (e.g. glucose and maltoheptaose) and aliphatic aldehydes (C1-C6).
302
In case of the carbohydrates analysis, no obvious product peaks were observed. A
303
possible explanation for this is that sugars are less reactive toward HAHC under room
304
temperature, because they are relatively stable and exist as hemiacetals in cyclic forms.
305
In contrast, aliphatic aldehydes (C1-C6) react with HAHC readily, but their
306
derivatives are well separated from furanic aldehydes using the same elution program
307
as mentioned above (Fig. 9). These results highlight the selective coupling of HAHC
308
with free aldehyde groups to afford stable derivatives with different chromatographic
309
properties, facilitating the reversed-phase HPLC analysis. It also implies that the
310
nitrone formation method could be further adapted to the analysis of various
311
aldehydes.
312
3.3.3 Enhancement of the detection sensitivity by HAHC derivatization
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To further evaluate the new derivatization method, we compared HAHC with a
314
conventional fluorescent reagent DNSH. Furfurals with the same starting
315
concentration were derivatized with HAHC and DNSH individually, followed by
316
HPLC-FLD analysis. From the resulting chromatograms (Fig. S7-S9), HAHC labeling
317
produced one chromatographic peak for each analyte, while DNSH labeling produced
318
two. Moreover, the peak areas of each HAHC derivatives was considerably larger
319
than the corresponding DNSH derivatives. The peak area enhancement folds over 15
ACCEPTED MANUSCRIPT 320
DNSH derivatives were measured. For all the furfurals, the peak areas were more than
321
ten times as large as those of DNSH labeling. We also found that HAHC labeling can significantly improve the MS responses
323
of furfurals. The extracted ion chromatograms (EIC) from LC-MS for furfurals before
324
and after derivatization were obtained (Fig. S10, supporting information). The
325
magnitudes of signal enhancement, estimated by the peak area ratio of the derivatized
326
furfurals over un-derivatized one over was measured (Fig. S11). Impressively, all
327
three furfurals showed signal improvements over two orders of magnitudes, with the
328
HAHC-labeled F in particular achieving an improvement of > 104-fold over its
329
underivatized counterpart. The significant enhancement of MS response can be
330
attributed to the hydrophobic fluorophore tag and the long alkyl chain [29] introduced
331
by HAHC labeling. Additionally, from the MS/MS spectra of all three furfurals
332
derivatives (Fig. 5 and Fig. S4-S5 in supporting information), we observed the same
333
characteristic fragmentation ion (m/z 217.09) from the HAHC part. These results
334
demonstrate the great potential of HAHC as a mass tag, which not only enhances the
335
mass spectrometric response but also provides characteristic fragment ions that may
336
facilitate the profiling of aldehydes by mass spectrometric analysis.
337
3.4 Method validation
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For method validation, standard mixtures of three furfurals with different
339
concentrations were analyzed with the new derivatization method, a more detailed
340
description was provided in section 2.8. The linear calibration ranges, regression
341
equations, correlation coefficients and detection limits of furfurals were calculated 16
ACCEPTED MANUSCRIPT and summarized in Table 1. The calibration curves showed good linearity in wide
343
dynamic ranges (0.4-4000 nM) between concentration and peak areas with correlation
344
coefficients (R2) from 0.9991 to 0.9998; We also observed low detection limits
345
ranging from 0.10 nM to 0.80 nM (signal to noise = 3, injection volume 20 µL). The
346
reproducibility of the proposed method, expressed by intra-day and inter-day relative
347
standard deviation (RSD), was also satisfactory, ranging from 0.74% to 1.46% and
348
from 4.59% to 5.06%, respectively. These results show that the new derivatization
349
method can provide sensitive and selective analysis of furfurals with high precision.
350
3.5 Real sample analysis
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Furfurals are not present in fresh food, however they can be found in a wide
352
variety of heat processed foods, such as dried fruits and dairy products. To
353
demonstrate the practical application of this new derivatization method, we attempted
354
to determination the amount of F, 5-MF and 5-HMF in raisin and milk powder
355
samples. After simple sample preparation procedures (described in section 2.9), the
356
samples were derivatized with HAHC, prior to being sent directly for HPLC-FLD
357
analysis.
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The HPLC results of raisins unspiked and spiked with standard furfurals mixture
359
solutions were shown in Fig. 10. All three furfurals were well separated and detected
360
without any interference. The detailed data were listed in Table 2. The recovery
361
percentages, in the ranged of 94.7%-103.5%, were achieved, compensating the
362
accuracy of the measurement. In the analysis of bovine milk powder (Fig. 11), F and
363
5-HMF were identified with good recoveries (96.7%-98.8%) (see Table 3). 17
ACCEPTED MANUSCRIPT These results show that, F and 5-HMF are present in thermally-processed
365
carbohydrate-containing foods, including raisin and milk powder. 5-HMF level is
366
relatively high, indicating 5-HMF need to be carefully monitored in these products.
367
Additionally, no 5-MF was observed in milk powder sample, which corroborates with
368
other studies on milk powder analysis [1, 2]. This is possibly the consequence of
369
different formation mechanisms of furfurals in different food sources.
370
3.6. Comparison with other methods
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As shown in Table 4, the LODs of other methods using pre-column derivatization,
372
such as GC-MS, HPLC-UV and HPLC-FLD, are all higher than 7.9 nM. In contrast,
373
the LOD of our method is much lower, in the range between 0.1-0.8 nM. Furthermore,
374
other chromatographic methods typically require extraction procedures such as solid
375
phase extraction or solid phase micro-extraction prior to the analysis, adding more
376
costs and time. Our method, on the other hand, does not require any prior purification
377
or extraction, and the derivatized sample was directly subjected to LC-FLD analysis.
378
Moreover, our derivatization method with HAHC can be conducted at room
379
temperature in the presence of aniline within a short period of reaction time, avoiding
380
the artificial formation of 5-HMF at high temperature. Therefore, our method has
381
advantages of high detection sensitivity and good reproducibility. With all these
382
features, we believe our method would be a better choice to detect and analyze these
383
furfurals than existing methods.
384
4. Conclusions
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18
ACCEPTED MANUSCRIPT In summary, we have detailed a new pre-column derivatization method based on
386
nitrone formation, which has been applied to determine F, 5-MF and 5-HMF for the
387
first time. Furfurals can be labeled with HAHC with high derivatization yields under
388
mild conditions, producing stable derivatives that have significant signal responses for
389
both fluorescence and MS detection. Additionally, combined with LC separation, the
390
method is selective and of low interference, allowing simultaneous analysis of three
391
furfurals in food samples without complicated purification or extraction procedures.
392
We obtained sub-nM level detection of furfurals, which is much more sensitive when
393
compared with other methods. The results showed good reproducibility and high
394
accuracy. We expect that this nitrone formation method can find wide applications in
395
the labeling of various aldehydes; in the meantime, we are actively pursuing the
396
development of new N-substituted hydroxylamine reagents.
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Acknowledgements
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We gratefully acknowledge the National Natural Science Foundation of China
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(21405117 to Y. Lin and 21472144 to X. Hu), the Fundamental Research Funds for
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the Central Universities (WUT: 2016-IB-005, 2016-IB-007, 2017-IB-007) for
402
financial support.
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Conflict of interest The authors declare that they have no conflicts of interest. 19
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Figure Captions
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Fig. 1. The structure of the N-monosubstituted hydroxylamine reagent HAHC.
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Fig. 2. Schematic representation of nitrone formation reaction by the condensation of
533
N-monosubstituted hydroxylamine and furanic aldehyde to afford nitrone derivative.
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Fig. 3. The fluorescence excitation and emission spectrum of HAHC (2.0 µM).
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Fig. 4. The derivatization of F (20 µM) with HAHC. (A) Typical chromatogram of
538
F-HAHC derivative; The elution followed an isocratic mode and the mobile phase
539
was acetonitrile-water (3.0/7.0, v/v); Flow rate: 1.0 mL min-1; (B) ESI mass spectrum
540
of F-HAHC derivative.
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543
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Fig. 5. On-line MS/MS spectrum of F-HAHC derivative.
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Fig. 6. Effects of derivatization conditions on the peak areas of F-HAHC derivative,
545
including (A) reaction time, (B) reaction temperature, (C) buffer pH and (D) the
546
concentration of HAHC. The data represent error bars of triplicate measurements.
26
ACCEPTED MANUSCRIPT 547
Fig. 7. (A) Effects of the aniline concentration on the derivatization efficiency at
549
50 ºC, (■) Caniline = 0 M, (●) Caniline = 0.8 mM, (▲) Caniline = 4.0 mM, (▼) Caniline =
550
8.0 mM; (B) Enlarged view of partial Fig. 7A; (C) Effects of the aniline concentration
551
on the derivatization efficiency at 20 ºC, (■) Caniline = 0.4 mM, (●) Caniline = 2.0 mM
552
(▲) Caniline = 4.0 mM.
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Fig. 8. (A) Typical chromatogram of reagent blank carried through the same
555
derivatization procedure except for the addition of furfurals; (B) Typical
556
chromatogram of standard mixture of three furfurals (2.0 µM each). Shown are the
557
derivatives of 5-HMF (tR: 9.5 min), F (tR: 19.8 min) and 5-MF (tR: 25.2 min). Mobile
558
phase: A was acetonitrile-water (0.5:9.5, v/v); B was acetonitrile; Gradient: 0-12 min,
559
13% B; 12-30 min, 20% B; Flow rate: 1.0 mL min-1.
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Fig. 9. Representative chromatogram of a standard mixture of three furfurals (2.0 µM
562
each) spiked with six aliphatic aldehydes (C1-C6, 2.0 µM each) subjected to
563
derivatization. Named are the derivatives peaks for C1-C2 (tR: 2.2 min), C3 (tR:
564
7.6 min), 5-HMF (tR: 9.5 min), C4 (tR: 14.7 min), F (tR: 19.7 min), C5 (tR: 21.6 min),
565
5-MF (tR: 25.0 min), C6 (tR: 31.6 min). The same elution program as described in Fig.
566
8 was adopted.
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Fig. 10. Typical chromatograms obtained from (A) raisin sample and (B) raisin
569
sample spiked with F, 5-MF and 5-HMF (1.0 µM each). Chromatographic conditions
570
were as described in Fig. 8. * = F derivative, ● = 5-MF derivative,
571
derivative.
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Fig. 11. The chromatograms obtained from (A) bovine milk powder sample and (B)
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the same powder sample spiked with F and 5-HMF (1.0 µM each). Chromatographic
575
conditions were as described in Fig. 8. * = F derivative,
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Fig. 1. The structure of the N-monosubstituted hydroxylamine reagent HAHC.
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Fig. 2. Schematic representation of nitrone formation reaction by the condensation of
589
N-monosubstituted hydroxylamine and furanic aldehyde to afford a nitrone derivative.
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Fig. 3. The fluorescence excitation and emission spectrum of HAHC (2.0 µM).
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Fig. 4. The derivatization of F (20 µM) with HAHC. (A) Typical chromatogram of
600
F-HAHC derivative; The elution followed an isocratic mode and the mobile phase
601
was acetonitrile-water (3.0/7.0, v/v); Flow rate: 1.0 mL min-1; (B) ESI mass spectrum
602
of F-HAHC derivative.
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Fig. 5.On-line MS/MS spectrum of the F-HAHC derivative.
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Fig. 6. Effects of derivatization conditions on peak areas of the F-HAHC derivative,
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including (A) reaction time, (B) reaction temperature, (C) buffer pH and (D) the
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concentration of HAHC. The data represent error bars of triplicate measurements.
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Fig. 7. (A) Effects of the aniline concentration on the derivatization efficiency at
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50 ºC, (■) Caniline = 0 M, (●) Caniline = 0.8 mM, (▲) Caniline = 4.0 mM, (▼) Caniline =
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8.0 mM; (B) Enlarged view of partial Fig. 7A; (C) Effects of the aniline concentration
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on the derivatization efficiency at 20 ºC, (■) Caniline = 0.4 mM, (●) Caniline = 2.0 mM
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(▲) Caniline = 4.0 mM.
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Fig. 8. (A) Typical chromatogram of reagent blank carried through the same
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derivatization procedure except for the addition of furfurals; (B) Typical
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chromatogram of standard mixture of three furfurals (2.0 µM each). Shown are the
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derivatives of 5-HMF (tR: 9.5 min), F (tR: 19.8 min) and 5-MF (tR: 25.2 min). Mobile
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phase: A was acetonitrile-water (0.5:9.5, v/v); B was acetonitrile; Gradient: 0-12 min,
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13% B; 12-30 min, 20% B; Flow rate: 1.0 mL min-1.
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Fig. 9. Representative chromatogram of a standard mixture of three furfurals (2.0 µM
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each) spiked with six aliphatic aldehydes (C1-C6, 2.0 µM each) subjected to
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derivatization. Named are the derivatives peaks for C1-C2 (tR: 2.2 min), C3 (tR:
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7.6 min), 5-HMF (tR: 9.5 min), C4 (tR: 14.7 min), F (tR: 19.7 min), C5 (tR: 21.6 min),
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5-MF (tR: 25.0 min), C6 (tR: 31.6 min). The same elution program as described in Fig.
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8 was adopted.
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Fig. 10. Typical chromatograms obtained from (A) raisin sample and (B) raisin
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sample spiked with F, 5-MF and 5-HMF (1.0 µM each). Chromatographic conditions
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were as described in Fig. 8; * = F derivative, ● = 5-MF derivative,
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derivative.
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◆
= 5-HMF
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Fig. 11. The chromatograms obtained from (A) bovine milk powder sample and (B)
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the same powder sample spiked with F and 5-HMF (1.0 µM each). Chromatographic
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conditions were as described in Fig. 8. * = F derivative,
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◆
= 5-HMF derivative.
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Tables
660 661
Table 1
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Linear calibration ranges, regression equations, detection limits, correlation
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coefficient and reproducibility of the proposed method.
5-HMF F 5-MF
Regression Equationa Y = AX + B Y = 1.99X – 1.93 × 104 Y = 1.78X + 3.45 × 104 Y = 1.58X + 1.31 × 105
Calibration Range (nM) 2-4000 0.8-4000 0.4-4000
R2 0.9991 0.9998 0.9995
a
X = sample concentration (pM), Y = peak area (mV·min).
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b
Signal-to-noise ratio = 3.
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RSD (%)
Intra-day
0.74 1.32 1.46
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Furfurals
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Inter-day
4.93 5.06 4.59
LODb (nM) 0.8 0.2 0.1
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Table 2
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Results of furfurals determination in raisin samples.
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Samples
Raisin RSD (%, n = 6) Recoverya Intra-day Inter-day / 2.74 6.24 94.7 ± 2.2 2.32 5.48 96.5 ± 1.7 1.76 4.52
Found (µM) 1.85 2.13 2.75
F
0.0 0.4 1.0
0.14 0.52 1.18
/ 96.3 ± 2.6 103.5 ± 1.3
2.98 2.70 1.26
5.26 4.73 4.13
1.68 ± 0.05
5-MF
0.0 0.4 1.0
0.17 0.56 1.16
/ 98.2 ± 2.1 99.1 ± 3.5
4.03 2.14 3.53
6.50 4.76 3.65
2.31 ± 0.07
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5-HMF
a
Recovery are given as mean ± SD (n = 6, %).
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b
Value are given as mean ± SD (n = 6, mg/Kg).
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Added (µM) 0.0 0.4 1.0
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Contentb
29.16 ± 0.80
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Table 3
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Results of furfurals determination in milk powder samples.
F
Found (µM) 1.44 1.79 2.36
0.0 0.4 1.0
0.14 0.53 1.12
/ 98.1 ± 2.4 98.8 ± 3.1
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a
Recovery are given as mean ± SD (n = 6, %).
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b
Value are given as mean ± SD (n = 6, mg/Kg).
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5-HMF
Added (µM) 0.0 0.4 1.0
Bovine milk powder RSD (%, n = 6) Recoverya Intra-day Inter-day / 2.73 4.34 97.3 ± 2.0 2.06 5.32 96.7 ± 2.7 2.79 5.16 3.79 2.45 3.14
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Samples
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6.03 3.92 4.52
Contentb
11.35 ± 0.31
0.84 ± 0.03
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Table 4
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Comparison with other methods. Reaction time/temp.
LOD (nM) 33-2545
Sample extraction /
/
/
400-430
/
/
/
7.9
PMME
DNPH
1 h/50 °C
16.6
SPME
PFBHA
GC-MSe
48.7-82.5
HS-SPME PFBHA
HPLC-FLD
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SPE
HPLC-FLD
0.1-0.8
/
HPLC-PAD c
HPLC-UV GC-MS
d
Reference [2]
[24]
[26]
DNSH
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b
Reagent
20 min/50 °C
[25]
HAHC
30 min/20 °C
This work
30 min/30 °C
[27]
27 min/45 °C
[21]
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Analytical method HPLC-DADa
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a
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detection (PAD) using a modified electrode with nickel nanoparticles; c HPLC-UV
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detection with polymer monolith microextraction (PMME) using DNPH as the
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derivatization reagent;
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chromatography and mass spectrometry detection;
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microextraction (HS-SPME) combined with gas chromatography and mass
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spectrometry detection.
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HPLC with pulsed amperometric
Solid-phase microextraction (SPME) combined with gas
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d
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HPLC with diode array detection (DAD);
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e
Headspace solid-phase
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Highlights First paper using nitrone formation as a pre-column derivatization method to determine furfurals in raisin and bovine milk powder.
responses were achieved by the derivatized furfurals.
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Significant signal enhancements in fluorescence and mass spectrometric
The detection limit was as low as 0.1 × 10 9 M using high performance liquid −
chromatography with fluorescence detection.
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This method provides a tool for the highly sensitive detection of furfurals in food
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samples.