In-house validation for the determination of honey adulteration with plant sugars (C4) by Isotope Ratio Mass Spectrometry (IR-MS)

The objective of any analytical measurement is to obtain consistent, reliable, and accurate data. Validated analytical methods play a major role in achieving this goal. Although there have been many studies reporting about the isotopic compositions of honeys, little has been documented regarding the validation of these methods. In this study, an Isotope Ratio Mass Spectrometry (IR-MS) method for the determination of honey adulteration was validated in-house in terms of selectivity, stability, linearity, accuracy, repeatability, sensitivity, and recovery. This study was the first attempt to describe some important method validation parameters, such as the limit of detection (LOD), limit of quantification (LOQ), and recovery for the IR-MS studies. The LOD of the method was 0.11%, and the LOQ was 0.38% based on the percent adulteration ratio. The recovery value for spiked blank honey sample with the in-house standard was 98.57%. To evaluate the usefulness of the method, 13 different brands of honey samples were collected from markets in Turkey and analyzed. The ranges of δ¹³C values of analyzed honey samples and their protein fractions were from −12.87 ± 0.01 to −25.56 ± 0.08‰ and from −23.77 ± 0.09 to −25.98 ± 0.06‰, respectively. Adulteration was found in one honey sample.     

Geographical origin of polished rice based on multiple element and stable isotope analyses

We determined carbon and nitrogen contents (C and N contents) and stable carbon, nitrogen, and oxygen isotopic compositions (δ¹³C, δ¹⁵N, and δ¹⁸O) of polished rice in order to develop a simple method to discriminate its geographical origin. As a first attempt, we examined a single cultivar, Koshihikari rice, from 14 different cultivation areas including Australia (n = 1), Japan (n = 12), and USA (n = 1). For all rice samples, C and N contents and the isotopic compositions are consistent with those of general plant materials, being 37.2–40.0% (C content), 0.8–1.4% (N content), −27.1 to −25.4% (δ¹³C), +0.4 to +9.0% (δ¹⁵N), and +18.8 to +22.9% (δ¹⁸O). However, its cultivated area is clearly distinguished by a pentagonal radar plot based on the elemental and isotopic compositions. Thus, the comparison of C and N contents and δ¹³C, δ¹⁵N, and δ¹⁸O values would potentially be useful for rapid and routine discrimination of geographical origin of the polished rice.     

Determination of Chinese honey adulterated with high fructose corn syrup by near infrared spectroscopy

The use of fibre optic diffuse reflectance near infrared spectroscopy (NIR) in combination with chemometric techniques has been investigated to discriminate authenticity of honey. NIR spectra of unadulterated honey and adulterated honey samples with high fructose corn syrup were registered within 10,000–4000 cm⁻¹ spectral region. Discriminant partial least squares (DPLS) models were constructed to distinguish between unadulterated honey and adulterated honey samples and main bands responsible for the discrimination of samples are in the range of 6000–10,000 cm⁻¹. For these models, the correct classification rate for calibration samples were above 90%. Hundred percentage of unadulterated honey and 95% of adulterated honey samples from test set were correctly classified after appropriate preprocessing of first derivative, 13 smoothing points, followed by mean centering pre-treatment and eight model factors, respectively. Our results showed that NIR spectroscopy data with chemometrics techniques can be applied to rapid detecting honey adulteration with high fructose corn syrup.     

Online determination of <Superscript>2</Superscript>H/<Superscript>1</Superscript>H and <Superscript>13</Superscript>C/<Superscript>12</Superscript>C isotope ratios of cinnamaldehyde from different sources using gas chromatography isotope ratio mass spectrometry

The combination of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) and gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-P-IRMS) is applied to the authenticity assessment of cinnamaldehyde from various sources. For that reason, cinnamon oils were self-prepared by steam distillation from three different varieties of cinnamon bark on the market, C. ceylanicum (ceylon), C. cassia (cassia) and C. burmanii (cassia vera). Furthermore, the so-called wood cinnamon was investigated, which is produced from the outer bark of older branches of cinnamon of minor quality. Self-prepared oils were analysed from commercial cinnamon powder. In addition several commercial samples of cinnamon oil and cinnamaldehyde, some of them declared to be natural, were investigated. ²_V-SMOW and ¹³C_V-PDB values of cinnamaldehyde were determined and characteristic authenticity ranges were deduced, allowing the differentiation between synthetic and natural samples. By correlation of both the ²_V-SMOW and ¹³C_V-PDB values, characteristic authenticity ranges were defined for ceylon, cassia and wood cinnamon. The ²_V-SMOW and ¹³C_V-PDB values of cassia vera samples are in the range of cassia. By comparing the ²_V-SMOW values of different self-prepared samples (ground bark, distillate) of cinnamon determined by TC/EA-IRMS with the corresponding GC-IRMS values, online GC-IRMS methods are proved to be essential in the authentication of complex natural products.     

Dielectric properties of honey adulterated with sucrose syrup

Sucrose syrup is a common additive in honey adulteration. To provide information for developing a cheap, simple, convenient and rapid sucrose-adulterated honey detector or sucrose content sensor, the permittivities of pure jujube, yellow-locust and milk-vetch flower honey, pure sucrose syrup and honey-sucrose syrup mixtures with sucrose content from 0% (pure honey) to 80% (pure sucrose syrup) were studied from 10 to 4500 MHz with open-ended coaxial-line technology and a network analyzer at room temperature. The correlations between permittivities and sucrose contents were regressed. The results showed that the dielectric constants of all samples decreased with increasing frequency, while the pure honey had higher dielectric constant than pure sucrose syrup. Dielectric relaxation existed in all samples. The maximum loss factor decreased with increasing sucrose content. The relaxation frequency changed very little with sucrose content. Strong negative linear correlation, R² > 0.98, was found between loss factor around the relaxation frequency and sucrose content.     

C NMR and electrospray ionization mass spectrometric study of sucrose aqueous solutions at high pH: NMR measurement of sucrose dissociation constant

The ¹³C NMR technique is used for the measurement of the first dissociation constant of sucrose (HL) in highly alkaline solutions. In 1.0 M NaCl/NaOH medium and for 25 °C, the concentration dissociation constant (pK₁) was 13.1 ± 0.3; and, for 60 °C, pK₁ = 12.30 ± 0.05. The β-d-fructofuranosyl ring was found to be responsible for dissociation. The NMR data reveal no clear evidence of the second dissociation step below pH 14, either at 25 °C or at 60 °C. In the solutions with 4–10 mol dm⁻³ NaOH content the ¹³C NMR technique indicated the chemical shift changes, treated as the second dissociation step of sucrose and a sodium complex formation. A very rough estimation, for variable ionic strength, gives the value: pK₂ ∼ 15.8 ± 0.8. The anionic species L⁻ and NaH₋₁L⁻ have been registered by electrospray ionization time-of-flight mass spectrometry (ESI-ToF MS) for 0.01 M sucrose solutions with initial pH 13.     

Investigating the impact of botanical origin and harvesting period on carbon stable isotope ratio values (13C/12C) and different parameter analysis of Greek unifloral honeys: A chemometric approach for correct botanical discrimination

The aim of this study was to investigate the impact of botanical origin and harvesting period on carbon stable isotope ratio (¹³C/¹²C), colour intensity (CI), radical scavenging activity (%RSA), P and Sn content of Greek unifloral honeys. Thus, twenty‐four honey samples were collected during harvesting periods 2011–2012 and 2012–2013, from four different regions in Greece. ¹³C/¹²C ratios and minerals were determined using isotope ratio mass spectrometry (IRMS) and inductively coupled plasma optical–emission spectroscopy (ICP‐OES), respectively. CI and %RSA were measured using spectrophotometric assays. Results showed that only ¹³C/¹²C values and %RSA were affected by both botanical origin and harvesting period (P < 0.05). Applying then chemometric analyses to the collected data set, honeys were correctly classified according to honey type (correct classification rate 87.5% and 79.2% using the original and cross‐validation method, respectively). The use of different origin parameters has the potential to aid in honey authentication.     

Contribution to the characterisation of honey-based Sardinian product abbamele: Volatile aroma composition,honey marker compounds and antioxidant activity

Sardinian abbamele is a typical product obtained from the honey recuperation from combs (traditional procedure) or by concentration of the honey diluted in water (industrial procedure). Seven abbamele samples were obtained to study the volatiles’ composition, the presence of honey marker compounds and their relationship with the production procedures. The long thermal treatment applied in abbamele production caused very high (1007.0–4405.8 mg/kg) HMF content (HPLC-DAD), while glucose and fructose amounts were quite similar to the honey ones (HPLC-RI). Total antioxidant activity (FRAP assay) of the samples ranged between 13.3 and 71.2 mmol Fe²⁺/kg, while antiradical activity (DPPH assay) ranged between 3.8 and 23.3 mmol TEAC/kg. Such high antioxidant values were linearly correlated with total phenol amount (1297.8–4469.5 mg GAE/kg) determined by Folin–Ciocalteau method. Thermally derived furan derivatives and terpenes were abundant among the headspace volatiles (HS-SPME), particularly limonene (0.5–76.0%) that probably originated from citrus rinds’ addition during abbamele production. GC and GC–MS analyses of USE isolates revealed HMF predominance as well as the honey marker compounds (if/when existing) such as methyl syringate (up to 49.2%), marker of Asphodelus microcarpus honey. High isophorone percentage (up to 30.9%) determined by HS-SPME followed by minor percentage of 4-ketoisophorone and norisoprenoids in one sample indicated Arbutus unedo L. honey use in the production. HPLC-DAD analysis confirmed the presence of specific honey markers: two samples showed high methyl syringate concentrations (150.4–120.1 mg/kg) while homogentisic acid and other specific markers of A. unedo honey were found in one sample. The compared GC–MS and HPLC-DAD data proved to be useful to obtain information about the use of specific honey in the production and to verify citrus addition.     

Determination of trace elements in Croatian floral honey originating from different regions

The concentrations of As, Cd, Cu, Hg and Pb were determined in 54 multi-floral honey samples collected from five regions of Croatia during 2009 and 2010. Element contents decreased in the following order: Cu > Pb > As > Hg > Cd. Significant differences in lead and copper levels were observed between regions. Mean levels of elements (μg kg⁻¹) in all honey samples measured were: 19.7 for As, 1.51 for Cd, 1074 for Cu, 2.72 for Hg and 65.2 for Pb. Copper and lead were the most abundant elements in the Centre region, with range and mean contents of 108–41,271 and 3232 μg kg⁻¹ and 22.0–440 and 131 μg kg⁻¹, respectively. The highest element contents were: As 23.8 μg kg⁻¹ in the South region, Cd 2.11 μg kg⁻¹ in the Southwest region and Hg 2.63 μg kg⁻¹ in the Northeast region. The finding that lead contents in Croatian honey were higher than most reported lead levels in honey from other European countries is of particular concern. These indicate that attention must be focused on setting positions for honey production hives in areas distant from highways and railways.     

Determination of important biochemical properties of honey to discriminate pure and adulterated honey with sucrose (Saccharum officinarum L.) syrup

The aims of the present study were to determine biochemical properties of honey samples and to discriminate pure and adulterated honey produced by the standard bee feeding method (control honey), the shaking method (pure blossom honey), and overfeeding (100 kg/colony syrup) with sucrose syrup (adulterated honey). The biochemical properties evaluated were moisture, ash, acidity, hydroxymethylfurfural (HMF), specific sugars (i.e. fructose, glucose, fructose–glucose, sucrose, and maltose), diastase activity, δ¹³C value (honey), δ¹³C value (protein), electrical conductivity, potassium, vitamin C, and proline. Fifteen honey samples were analyzed by discriminant analysis stepwise method. Proline, electrical conductivity and sucrose were found as discriminative characters of samples. Based on these three properties 100% of original group cases (samples) correctly classified in their real group. We found that the honey produced by feeding with 100 kg sucrose syrup per colony contained the sucrose as low as pure blossom honey. Therefore, the sugar (sucrose, fructose and glucose) content of honey cannot be used to distinguish between adulterated (sucrose syrup) and pure blossom honey.     

Antioxidant profile of strawberry tree honey and its marker homogentisic acid in several models of oxidative stress

The antioxidant activity of several honeys was evaluated considering the different contribution of entire samples. The strawberry tree honey emerged as the richest in total phenols and the most active honey in the DPPH and FRAP tests, and could protect cholesterol against oxidative degradation (140 °C). Homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA), the main phenolic compound from strawberry tree honey, showed interesting antioxidant and antiradical activities, and protective effect against thermal-cholesterol degradation, comparable to those of well known antioxidants. Moreover, the pre-treatment with HGA significantly preserved liposomes and LDL from Cu²⁺-induced oxidative damage at 37 °C for 2 h, inhibiting the reduction of polyunsaturated fatty acids and cholesterol and the increase of their oxidative products. This phenol had no toxic effect in human intestinal epithelial Caco-2 cells within the concentration range tested (5–1000 μM). HGA was able to pass through the Caco-2 monolayers, the apparent permeability coefficients (P_app) in the apical-to-basolateral and basolateral-to-apical direction were 3.48 ± 1.22 × 10⁻⁶ and 2.18 ± 0.34 × 10⁻⁶ cm/s, respectively, suggesting a passive diffusion pathway as the dominating process. The results of the work qualify HGA as natural antioxidant, able to exert a significant in vitro protective effect and to contribute to the strawberry tree honey antioxidant activity.     

Chemical characterization of a traditional honey-based Sardinian product: Abbamele

The first chemical characterization of abbamele, a traditional honey decoction from Sardinia (Italy) is hereby reported. Water content (from 17.7% to 27.7%), electrical conductivity (from 0.19 to 0.81 mS cm⁻¹), pH (from 3.21 to 3.92), free acidity (from 26.1 to 87.6 meq kg⁻¹), invertase activity (from 0 to 1.02 U kg⁻¹), 5-(hydroxymethyl)-2-furaldehyde, HMF (from 881 to 4776 mg kg⁻¹), total polyphenols (from 188 to 984 mg kg⁻¹) and free amino acid contents of thirteen abbamele samples, from industrial and traditional producers, were obtained in an attempt to compare this traditional product with honey and to study the relationship between its main features and the production procedures. The long thermal treatment involved in the production of abbamele has been identified as the main cause of very low (or absent) invertase activity and free amino acid content as well as the very high content of HMF.     

Influence of technological processing on apple aroma analysed by high resolution gas chromatography–mass spectrometry and on-line gas chromatography-combustion/pyrolysis-isotope ratio mass spectrometry

Extracts obtained by simultaneous distillation extraction (SDE) from industrial raw materials, namely single strength apple juices, and concentrates and aromas made thereof (each n = 31, from one production line; origin Poland, Germany, Turkey, Romania and China), as well as commercially available juices (n = 27), were analysed by standard controlled capillary gas chromatography–mass spectrometry (HRGC–MS). During the technological processing from juice to the aroma, no qualitative changes in the apple aroma profile were observed. Major constituents of the juices and aromas under study were found to be 1-hexanol (juice, 0.06–5.9 mg/l; aroma, 47–685 mg/l), 1-butanol (juice, 0.1–4.7 mg/l; aroma, 17–370 mg/l); E-2-hexenol (juice, 0.01–3.4 mg/l; aroma, 12–300 mg/l); E-2-hexenal (juice, 0–3.0 mg/l; aroma 0–470 mg/l), and butyl acetate (juice, 0–1.7 mg/l; aroma, 0–165 mg/l). By far the major component of the apple juice concentrates under study was furfural (2.4–56 mg/kg). The observed occurrence of 3-methyl-1-butanol (juice, 0.01–2.1 mg/l; aroma, 1.5–134 mg/l) and, in part, its acetate (juice, 0–0.3 mg/l; aroma, 0–3.3 mg/l), both known not to be genuine apple constituents, was obviously caused by fermentative effects in the course of industrial juice production. In addition, on-line capillary gas chromatography–isotope ratio mass spectrometry was used in the combustion (C) and the pyrolysis (P) modes (HRGC–C/P–IRMS) for the determination of δ¹³C_V-PDB and δ²H_V-SMOW values of selected apple flavour constituents to check potential isotope discrimination during distillative aroma production. As shown by means of the representative examples of E-2-hexenal, 1-hexanol and E-2-hexenol, their δ²H_V-SMOW values were slightly depleted. However, authenticity assessment by stable IRMS will not be influenced by this effect.     

Tissue turnover in ovine muscles and lipids as recorded by multiple (H,C, O,S) stable isotope ratios

Multiple stable isotope ratios (δ²H, δ¹³C, δ¹⁸O and δ³⁴S) were measured in muscle, muscle lipids and lipid fractions collected from 28 lambs, subjected to a diet-switch and raised on two energy allowances (EAs), to determine tissue turnover and diet-tissue fractionation. The diet-muscle fractionations prior to the diet-switch were estimated to be −44.0‰, +1.9‰ and 0‰ for H, C and S, respectively, while the drinking water was demonstrated to be the main source of muscle O and thus δ¹⁸O variation. The diet-intra-muscular lipid fractionations prior to the diet-switch were estimated to be −172.7‰, −1.3‰ and −11.5‰ for H, C and O, respectively. The C half-lives of muscle were determined to be 75.7 and 91.6 days for animals receiving the high and low EA, respectively. Extracting temporally resolved pre-slaughter dietary information from meat by analysing bulk muscle, muscle lipids and muscle lipid fractions appeared to be not practicable due to possible incomplete turnover of lipids.     

Combined use of HMF and furosine to assess fresh honey quality

BACKGROUND: The quality of honey can be affected by practices such as adulteration, inadequate storage or the application of severe heat treatments. Because hydroxymehylfurfural (HMF) is an indicator of honey freshness and furosine (ε‐2‐furoylmethyl lysine) has proved to be a useful chemical indicator of the progress of the Maillard reaction in foods, the aim of this work was to assess their usefulness as indicators of fresh honey quality. The effect of heat treatment, storage and adulteration with different types of syrups on HMF and furosine content has been studied. RESULTS: In fresh honey, HMF and furosine values ranged from 0.9 to 14.6 mg kg^?1 of product and from 3.06 to 12.06 g kg^?1 of protein, respectively. Heating of honey samples with different pH (3.76 and 5.14) produced slight increases in HMF content and negligible changes were detected in furosine values. The storage of fresh honey for 2 years caused a high increase in the HMF level, reaching values above EU limits. However, furosine showed a different behavior depending on the type of honey sample. Adulteration assays using different syrups produced an increase in HMF and a decrease of furosine values by dilution effect. HMF content of adulterated honey samples with syrup of known origin did not exceed EU limits. CONCLUSION: These results show the influence of long periods of storage or adulteration, using different percentages of corn or invert sugar syrups, on HMF and furosine content of fresh honey. This seems to indicate that the combination of HMF and furosine may be useful for evaluating the quality of fresh honey. Copyright © 2009 Society of Chemical Industry     

Study on distribution pattern of stable carbon isotope ratio of Chinese honeys by isotope ratio mass spectrometry

The δ ¹³C values of 26 varieties of Chinese pure single‐flower honeys, 323 census samples of six varieties of single‐flower honeys and one multi‐flower honey as well as 20 888 commercial honey samples from 135 honey‐related enterprises in 25 provinces of China were analysed by stable carbon isotope ratio mass spectrometry between 1998 and 2004. It was found that the δ ¹³C values of different Chinese honeys fell within the ranges of values proposed by JW White. This shows that White's theory of the stable carbon isotope ratio of honeys is applicable to Chinese honeys and further demonstrates that the theory is universal to honeys from all over the world. The study also confirmed that the δ ¹³C values of honeys do not bear much relation to the environment in which the honey plants are grown, e.g. geographical area, water and soil, climate, etc., but do vary slightly with the honey plant species. Copyright © 2005 Society of Chemical Industry     

Curing and diffusion coefficient study in pastırma,a Turkish traditional meat product

Changes in water activity (a_w), moisture and salt contents and salt effective diffusion coefficients (D_eff) of past?rma samples during the curing process were determined. At the end of the curing stage, a_w values decreased to 0.942. The average initial moisture content of the samples decreased from 74.56% to 66.64%, depending on the curing time and the average salt content increased to 15.65 g NaCl/100 g dry matter at the end of the 48-hour curing process. Past?rma samples were assumed the geometry of endless slices, and the analytical solution of Fick's second equation was used for determination of salt D_eff values. Salt D_eff values were found to vary between 1.49 × 10^− 9–4.08 × 10^− 9 m²/s.     

Flavonoid pattern of sage (Salvia officinalis L.) unifloral honey

The aim of the present paper was to determine the flavonoids in monofloral sage (Salvia officinalis L.) honey which is characteristic and specific for the area of Croatian coast and islands. For that purpose 38 sage honey samples from two production seasons were analysed. After specific pollen content determination, and analyses of selected physicochemical parameters which confirmed that samples are in compliance with national and international regulations and can be regarded as unifloral sage honeys, flavonoid fraction was isolated and analysed using RP-HPLC/DAD method. The HPLC analysis showed that all examined sage honey samples contain quercetin (3,3′,4′,5,7-pentahydroxyflavone), luteolin (3′,4′,5,7-tetrahydroxyflavone), kaempferol (3,4′,5,7-tetrahydroxyflavone), apigenin (4′,5,7-trihydroxyflavone), chrysin (5,7-dihydroxyflavone) and galangin (3,5,7-trihydroxyflavone), as well as p-coumaric (trans-4-hydroxycinnamic acid) and caffeic acid (3,4-dihydroxycinnamic acid). Total amount of identified flavonoids varied from 109.4 μg/100 g of honey to 589.9 μg/100 g of honey, with the average of 288.5 μg/100 g of honey. All analysed honey samples showed common and specific flavonoid profile which could be the basis for differentiating sage from other monofloral honeys.     

Effects of prolonged heating on antioxidant activity and colour of honey

The kinetics of changes in total antioxidant activity as assessed by DPPH radical and brown pigment formation (BPF) in honey heated at different temperatures (50, 60 and 70 °C) for up to 12 days were studied. Antioxidant activity and BPF increased with treatment temperature and time. BPF increased following zero-order kinetics with the activation energy value of 122 kJ/mol⁻¹ at 50–70 °C. However, antioxidant activity variation showed different trends according to heating temperatures following second-order, first-order and zero-order kinetics at 50, 60 and 70 °C, respectively. Heating of honey at 70 °C was found to be more effective than 50 and 60 °C for both two parameters. The results demonstrated that antioxidant activity was correlated with increased browning of the samples.