Liquid chromatography coupled to isotope ratio mass spectrometry: a new perspective on honey adulteration detection

A new procedure to determine individual sugar (sucrose, glucose, and fructose) 13C isotope ratios, using liquid chromatography-isotope ratio mass spectrometry (HPLC-IRMS), has been developed to improve isotopic methods devoted to the study of honey authenticity. For this purpose 79 commercial honey samples from various origins were analyzed. Values of delta13Choney ranged from -14.2 to -27.2", and delta13Cprotein ranged from -23.6 to -26.9". A very strong correlation is observed between the individual sugar 13C ratios, which are altered in the event of sugar addition, even at low levels. The use of Deltadelta13C [fruct-glu], Deltadelta13C [fruct-suc], and Deltadelta13C [gluc-suc] systematic differences as an authenticity criterion permits the sugar addition [C3, beet sugar; or C4, cane sugar, cane syrup, isoglucose syrup, and high-fructose corn syrup (HFCS)] to be reliably detected (DL = 1-10%). The new procedure has advantages over existing methods in terms of analysis time and sensitivity. In addition, it is the first isotopic method developed that allows beet sugar addition detection.     

Application of Fourier transform midinfrared spectroscopy to the discrimination between Irish artisanal honey and such honey adulterated with various sugar syrups

A collection of authentic artisanal Irish honeys (n = 580) and certain of these honeys adulterated by fully inverted beet syrup (n = 280), high-fructose corn syrup (n = 160), partial invert cane syrup (n = 120), dextrose syrup (n = 160), and beet sucrose (n = 120) was assembled. All samples were adjusted to 70 degrees Bx and scanned in the midinfrared region (800-4000 cm(-1)) by attenuated total reflectance sample accessory. By use of soft independent modeling of class analogy (SIMCA) and partial least-squares (PLS) classification, authentic honey and honey adulterated by beet sucrose, dextrose syrups, and partial invert corn syrup could be identified with correct classification rates of 96.2%, 97.5%, 95.8%, and 91.7%, respectively. This combination of spectroscopic technique and chemometric methods was not able to unambiguously detect adulteration by high-fructose corn syrup or fully inverted beet syrup.     

Stable carbon isotopic composition of the wine and CO2 bubbles of sparkling wines: detecting C4 sugar additions

Sparkling wines have become a popular beverage in recent years, and the production of these wines is subject to adulteration during fermentation. This study investigated the stable carbon isotopic composition (expressed as delta(13)C) of the wine and of the CO(2) bubbles produced during the second fermentation for a number of sparkling wines produced in different countries around the world. Carbon isotope ratio analyses were used to estimate the addition of sugar obtained from C(4) plants (sugar cane or corn). The average delta(13)C values of the Brazilian brut, demi-sec, and doux sparkling wines were -20.5 +/- 1.2 per thousand (n = 18), -18.1 +/- 1.3 per thousand (n = 9), and -15.8 per thousand (n = 1), respectively. These values were statistically heavier (more positive carbon isotope ratio values) than the average delta(13)C of sparkling wines produced in other parts of South America (Argentina and Chile, -26.1 +/- 1.6 per thousand, n = 5) and Europe (France, Germany, Italy, Portugal, and Spain, -25.5 +/- 1.2 per thousand, n = 12), but not statistically different from sparkling wines produced in the United States or Australia. The most likely explanation for differences in the carbon isotope ratios of wines from these different regions is the addition of C(4) sugar during the production of some sparkling wines from Australia, Brazil, and the United States. The isotopic composition of the CO(2) bubbles (delta(13)C-CO(2)) followed similar trends. The average delta(13)C-CO(2) of most of the Brazilian and Argentine sparkling wines was -10.8 +/- 1.2 per thousand (n = 23), indicating that the likely source of carbon for the second fermentation was sugar cane. Conversely, the average delta(13)C-CO(2) of most of the sparkling wines produced in Chile and Europe was -22.0 +/- 1.2 per thousand (n = 13), suggesting that a different sugar (most likely sugar beet) was most used in the second fermentation. It was concluded that in many cases, the carbon isotope ratios of sparkling wine and CO(2) bubbles can provide valuable information about the sugar sources.     

Detection of sugar adulterants in apple juice using fourier transform infrared spectroscopy and chemometrics

Fourier transform infrared spectroscopy and attenuated total reflection sampling have been used to detect adulteration of single strength apple juice samples. The sample set comprised 224 authentic apple juices and 480 adulterated samples. Adulterants used included partially inverted cane syrup (PICS), beet sucrose (BS), high fructose corn syrup (HFCS), and a synthetic solution of fructose, glucose, and sucrose (FGS). Adulteration was carried out on individual apple juice samples at levels of 10, 20, 30, and 40% w/w. Spectral data were compressed by principal component analysis and analyzed using k-nearest neighbors and partial least squares regression techniques. Prediction results for the best classification models achieved an overall (authentic plus adulterated) correct classification rate of 96.5, 93.9, 92.2, and 82.4% for PICS, BS, HFCS, and FGS adulterants, respectively. This method shows promise as a rapid screening technique for the detection of a broad range of potential adulterants in apple juice.     

Specific natural isotope profile studied by isotope ratio mass spectrometry (SNIP-IRMS): (13)C/(12)C ratios of fructose, glucose, and sucrose for improved detection of sugar addition to pineapple juices and concentrates

The delta(13)C values of fructose, glucose, and sucrose have been determined in authentic pineapple juices. The sugar fraction is separated from the organic acids by an anionic exchange process. Then the individual components (fructose, glucose, and sucrose) are isolated on a preparative HPLC device using a NH(2)-type column. It is demonstrated that no significant isotope fractionation occurs when close to 100% of material is recovered and when the hydrolysis of sucrose is avoided. The control of the recovery rates and of the sucrose hydrolysis rate after purification is recommended for a reliable interpretation of the results. Correlations between the delta(13)C values of fructose (delta(13)Cf), glucose (delta(13)Cg), and sucrose (delta(13)Csu) can be characterized by systematic differences between these values. For the set of measurements on authentic pineapple juices and concentrates, the mean and the standard deviation of the differences are delta(13)Cf - delta(13)Cg = -0.6 +/- 0.6 per thousand, delta(13)Cf - delta(13)Csu = -1.3 +/- 0. 6 per thousand, and delta(13)Cf - delta(13)Csu = -0.7 +/- 0.5 per thousand. The determinations of the (13)C content of fructose, glucose, and sucrose enable a refinement of the detection of added sugars in fruit juices, re-enforcing the SNIP-IRMS method.     

Detection of sugar syrups in apple juice by delta(2)H per thousand and delta(13)C per thousand analysis of hexamethylenetetramine prepared from fructose

An improved procedure for determining (13)C and (2)H isotope ratios, using gas chromatography-isotope ratio mass spectrometry (GC-IRMS), has been developed for identifying the addition of low cost commercial sugar syrups to apple juices and related products. Isotopic techniques are commonly used to identify the addition of low cost sugars to fruit juices and are difficult to circumvent as it is not economically viable to change the isotopic ratios of the sugars. The procedure utilizes the derivative hexamethylenetetramine, which is produced through chemical transformation of a sugar degradation product and provides position-specific (13)C and (2)H ratios that relate to the parent sugar molecule. The new procedure has advantages over methods using nitro-sugar derivatives in terms of analysis time and sensitivity. The differences between the delta(2)H per thousand and delta(13)C per thousand values of the 100 authentic apple juices and beet and cane commercial sugar syrups permit their addition to be reliably detected.     

Honey protein as internal standard for stable carbon isotope ratio detection of adulteration of honey

Using the difference in stable carbon isotope ratio between a honey and its protein fraction permits objective evaluation of possible adulteration of honey with small amounts (7-20%) as well as larger amounts of corn or cane sugar. The present uncertainty in interpretation of results from pure honey with delta 13C values outside the generally accepted limits for pure honey of -27.5% to -23.5% is eliminated; likewise TLC testing to resolve questionable samples with delta 13C values between -23.5 and -21.5% is not needed. Fifty certified samples of pure honey were used to establish criteria for purity, and 38 other samples with delta 13C values in the "questionable" or "adulterated" range for the AOAC official method were tested. A difference of 1.0% or more between honey and protein fractions is proposed to indicate adulteration.     

Detection of exogenous citric acid in fruit juices by stable isotope ratio analysis

A new method has been developed for measuring the D/H ratio of the nonexchangeable sites of citric acid by isotope ratio mass spectrometry (IRMS). Pure citric acid is transformed into its calcium salt and subsequently analyzed by pyrolysis-IRMS. The citric acid isolated from authentic fruit juices (citrus, pineapple, and red fruits) systematically shows higher D/H values than its nonfruit counterpart produced by fermentation of various sugar sources. The discrimination obtained with this simplified method is similar to that obtained previously by applying site specific isotopic fractionation-nuclear magnetic resonance (SNIF-NMR) to an ester derivative of citric acid. The combination of carbon 13 and deuterium measurements of extracted citric acid is proposed as a routine method for an optimum detection of exogenous citric acid in all kinds of fruit juices.     

Development of a new high-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry method for the determination of low molecular mass organic acids in plant tissue extracts

A liquid chromatography-electrospray ionization time-of-flight mass spectrometry method has been developed for the direct and simultaneous determination of 10 low molecular mass organic acids in different plant tissue extracts. The method does not include a derivatization step. Quantification was accomplished using (13)C-labeled malic and succinic acids as internal standards. Good limits of detection (0.05-1.27 pmol) were obtained for malic, 2-oxoglutaric, succinic, quinic, shikimic, cis-aconitic, and citric acids, whereas for oxalic, ascorbic, and fumaric acids limits of detection were 255, 25, and 11 pmol, respectively. Repeatability values for the retention time and peak area were <5%, with the exception of ascorbic acid. Analyte recovery was between 92% and 110% in most cases, with the exception of oxalic (39-108%), 2-oxoglutaric (44-69%), and ascorbic (22-86%) acids, which may require specific extraction procedures and use of the corresponding (13)C-labeled organic acid as internal standards to improve accuracy. The method was applied to three types of plant materials: sugar beet leaf extracts, tomato xylem sap, and commercial orange juice.     

A new methodology based on GC-MS to detect honey adulteration with commercial syrups

Honey adulterations can be carried out by addition of inexpensive sugar syrups, such as high fructose corn syrup (HFCS) and inverted syrup (IS). Carbohydrate composition of 20 honey samples (16 nectar and 4 honeydew honeys) and 6 syrups has been studied by GC and GC-MS in order to detect differences between both sample groups. The presence of difructose anhydrides (DFAs) in these syrups is described for the first time in this paper; their proportions were dependent on the syrup type considered. As these compounds were not detected in any of the 20 honey samples analyzed, their presence in honey is proposed as a marker of adulteration. Detection of honey adulteration with HFCS and IS requires a previous enrichment step to remove major sugars (monosaccharides) and to preconcentrate DFAs. A new methodology based on yeast (Saccharomyces cerevisiae) treatment has been developed to allow the detection of DFAs in adulterated honeys in concentrations as low as 5% (w/w).     

Use of differential scanning calorimetry (DSC) as a new technique for detection of adulteration in honeys. 1. Study of adulteration effect on honey thermal behavior.

Differential scanning calorimetry (DSC) was used to study the thermal behavior of authentic honeys (Lavandula, Robinia, and Fir honeys) and industrial sugar syrups. Thermal or thermochemical parameters such as the glass transition temperature (Tg), enthalpies of fusion (DeltaH(fus)), and heat capacity variation (DeltaC(p)) were measured. The syrups and honeys showed significant differences in thermal phenomena, as well as in their amplitude and position on the temperature scale. Results showed good reproducibility of the method for all samples studied. The effect of adulteration of honey with different amounts of syrup (5, 10, 20, 40, and 60%) was investigated. A linear relationship was found between the percentage of added syrup and the glass transition temperature. A similar relationship was obtained from the enthalpy of fusion results in the temperature range of 40-90 degrees C. Under applied conditions, the effects of adulteration of honeys by industrial syrups appeared to be detectable from a level as low as 5%.     

Improved characterization of the botanical origin of sugar by carbon-13 SNIF-NMR applied to ethanol

Until now, no analytical method, not even isotopic ones, had been able to differentiate between sugars coming from C4-metabolism plants (cane, maize, etc.) and some crassulacean acid metabolism plants (e.g., pineapple, agave) because in both cases the isotope distributions of the overall carbon-13/carbon-12 and site-specific deuterium/hydrogen isotope ratios are very similar. Following recent advances in the field of quantitative isotopic carbon-13 NMR measurements, a procedure for the analysis of the positional carbon-13/carbon-12 isotope ratios of ethanol derived from the sugars of pineapples and agave using the site-specific natural isotopic fractionation-nuclear magnetic resonance (SNIF-NMR) method is presented. It is shown that reproducible results can be obtained when appropriate analytical conditions are used. When applied to pineapple juice, this new method demonstrates a unique ability to detect cane and maize sugar, which are major potential adulterants, with a detection limit in the order of 15% of the total sugars, which provides an efficient mean of controlling the authenticity of juices made from this specific fruit. When applied to tequila products, this new method demonstrates a unique ability to unambiguously differentiate authentic 100% agave tequila, as well as misto tequila (made from at least 51% agave), from products made from a larger proportion of cane or maize sugar and therefore not complying with the legal definition of tequila.     

Specific screening for color precursors and colorants in beet and cane sugar liquors in relation to model colorants using spectrofluorometry evaluated by HPLC and multiway data analysis

A comparison was made of the fluorophores in beet thick juice and cane final evaporator syrup, which are comparable in the production of cane and beet sugar; that is, both represent the final stage of syrup concentration prior to crystallization of sugar. To further elucidate the nature of the color components in cane and beet syrup, a series of model colorants was also prepared, consisting of mildly alkaline-degraded fructose and glucose and two Maillard type colorants, glucose--glycine and glucose--lysine. Fluorescence excitation--emission landscapes resolved into individual fluorescent components with PARAFAC modeling were used as a screening method for colorants, and the method was validated with size exclusion chromatography using a diode array UV--vis detector. Fluorophores from the model colorants were mainly located at visible wavelengths. An overall similarity in chromatograms and absorption spectra of the four model colorant samples indicated that the formation of darker color was the distinguishing characteristic, rather than different reaction products. The fluorophores obtained from the beet and cane syrups consisted of color precursor amino acids in the UV wavelength region. Tryptophan was found in both beet and cane syrups. Tyrosine as a fluorophore was resolved in only beet syrup, reflecting the higher levels of amino acids in beet processing. In the visible wavelength region, cane syrup colorant fluorophores were situated at higher wavelengths than those of beet syrup, indicating formation of darker colorants. A higher level of invert sugar in cane processing compared to beet processing was suggested as a possible explanation for the darker colorants.     

Gas chromatographic determination of formaldehyde in maple syrup as 2,4-dinitrophenylhydrazone derivative

A procedure is presented for the quantitative extraction and determination of formaldehyde in maple sap and syrup. The method is based on the reaction between formaldehyde and 2,4-dinitrophenylhydrazine and determination of the derivative by gas chromatography. The procedure was used to evaluate formaldehyde in saps and syrups of paraformaldehyde implanted trees. Average recoveries were 101.5 +/- 5.7%. The detection limit was 0.078 mg/kg.     

Fluorometric and spectrophotometric determination of pirbuterol hydrochloride in authentic and dosage forms

Pirbuterol hydrochloride has been assayed in alkaline medium by using a fluorometric method to measure fluorescence intensity at 372 nm with excitation at 310 nm and by the delta A method at 242 nm. The linearity ranges are 0.5-4 micrograms/mL and 10-50 micrograms/mL, respectively. An authentic pirbuterol HCl sample was analyzed by nonaqueous potentiometric titration using 0.1N perchloric acid, and the results were compared with those for fluorometric and delta A methods. The mean percent recoveries for the authentic sample were 98.72 +/- 1.13 and 99.24 +/- 0.85, respectively. When applied to commercial capsules containing 10 mg and 15 mg each, the fluorometric method gave mean percent recoveries of 101.11 +/- 1.05 and 98.12 +/- 0.93; the delta A method gave mean percent recoveries of 100.57 +/- 0.83 and 97.80 +/- 0.75, respectively.     

Stable isotopic carbon composition of apples and their subfractions--juice, seeds, sugars, and nonvolatile acids

The 13C:12C ratios of 8 authentic apple juice samples and their subfractions were determined by mass spectrometry. Apples from Argentina, Mexico, New Zealand, and the United States were processed into juice; pulp was collected from the milled fruit and seeds were collected from the press-cake. Sugars, nonvolatile acids, and phenolics were isolated from the juice by treatment with ion-exchange resins and polyvinylpyrrolidone (PVPP). The mean value for all juice samples was -24.2% which is close to the values reported by other investigators. Juice from apples grown in Argentina, Mexico, and New Zealand did not differ from U.S. samples. The isotopic composition of the subfractions ranged from -22.0 to -31.0%. The values for the pulp were essentially the same as for juice. The sugar fraction was slightly less negative than the juice; the nonvolatile acid and phenolic fractions were more negative. The levels of nonvolatile acids and phenolics in apple juice are low, however, so these compounds contribute little to overall delta 13C values in juice.     

Determination of authenticity, regional origin, and vintage of Slovenian wines using a combination of IRMS and SNIF-NMR analyses

The authenticity and geographical origin of wines produced in Slovenia were investigated by a combination of IRMS and SNIF-NMR methods. A total of 102 grape samples of selected wines were carefully collected in three different wine-growing regions of Slovenia in 1996, 1997, and 1998. The stable isotope data were evaluated using principal component analysis (PCA) and linear discriminant analysis (LDA). The isotopic ratios to discriminate between coastal and continental regions are the deuterium/hydrogen isotopic ratio of the methylene site in the ethanol molecule (D/H)(II) and delta(13)C values; including also delta(18)O values in the PCA and LDA made possible separation between the two continental regions Drava and Sava. It was found that delta(18)O values are modified by the meteorological events during grape ripening and harvest. The usefulness of isotopic parameters for detecting adulteration or watering and to assess the geographical origin of wines is improved only when they are used concurrently.     

Adulteration of apple with pear juice: emphasis on major carbohydrates, proline, and arbutin

Detection of juice-to-juice adulteration based on chemical composition studies is a common method used by government regulatory agencies and food companies. This study investigated the use of major carbohydrate (fructose, glucose and sucrose), polyol (sorbitol), proline, and phenolic profiles as indicators of pear adulteration of apple juice (PAAJ). For this work, a total of 105 authentic apple juice samples from 13 countries and 27 authentic pear juice samples from 5 countries were analyzed. Because the major carbohydrate ranges for these juices showed significant overlap their use as markers for PAAJ detection would be very limited. It was found that sorbitol and proline means for apple and pear were significantly different; however, their broad natural ranges would afford PAAJ at levels up to 30% without detection. In addition, careful selection of the pear juice used as the adulterant would further limit the usefulness of these markers for PAAJ detection. Arbutin was conclusively identified as a marker for pear juice on the basis of its presence in all 27 authentic pear samples and its absence (<0.5 microg/mL) in all 105 apple juice samples analyzed in this study. The application of the developed HPLC-PDA method for arbutin analysis to detect PAAJ at levels as low as 2% (v/v) was demonstrated. A confirmation method for the presence of arbutin in pure pear juice and apple adulterated with pear juice was introduced on the basis of the hydrolysis of arbutin to hydroquinone employing beta-glucosidase, with reactant and product monitoring by HPLC-PDA.     

Detecting Corn Syrup in Barley Malt Extracts

Methods for detecting corn syrup in barley (Hordeum vulgare L.) malt extract were evaluated. Twelve samples representative of commercially available 2‐rowed and 6‐rowed malting barleys were malted. Extracts prepared from the finely ground malts were analyzed for ¹³C/¹²C ratios, expressed as δ¹³C, and concentrations of protein and sugars. The ¹³C/¹²C ratios were sufficiently different to distinguish corn syrup from malt extract. By calculating the mean values for the barleys, it was determined that a δ¹³C > ‐24.3‰ indicated that the malt extract had been adulterated with corn syrup (99% confidence). Protein concentrations <4.5% (2‐rowed malt) or <5.0% (6‐rowed malt) of the extracts also indicated probable adulteration with corn syrup, which is devoid of protein. Because of differences in sugar concentrations between the malt extracts and corn syrup, carbohydrate analysis also indicated probable mixtures. These findings were confirmed by analysis of extracts from composite 2‐rowed and 6‐rowed barley malts that had been mixed with known quantities of corn syrup. The regressions for δ¹³C, protein concentration, and most sugar concentrations against percent dilution with corn syrup in the mixtures were significant.     

气候变化情景下新疆棉花和甜菜需水量的变化趋势

利用改进的非参数统计检验Mann-Kendall(MK)方法进行序列趋势检验时,因考虑了时间序列自相关性的影响而使检验结果更趋严格。本文采用MK法及改进的MK法对新疆地区41站1961-2010及2015-2099年主要经济作物棉花和甜菜需水量ETc和相关气候要素的变化趋势进行了检验,探讨了序列自相关性对ETc趋势检验结果的影响,并给出了各站及全疆ETc的变化趋势。结果表明:1)当自相关阶数大于0时,应采用改进MK法判断ETc的变化趋势;2)1961-2010棉花和甜菜的ETc具有一定的相依结构,2015-2099年棉花的ETc具有非常明显的长程相关性,但2015-2099年甜菜的ETc均具有时间独立性;3)无论对于棉花还是甜菜ETc,各站点Sen斜率和线性斜率值很接近,满足极好的线性关系,表明两者在反映趋势幅度时具有高度一致性;4)ETc序列是否存在相依性对于趋势显著性有重要影响,最终的趋势检验需用MK法和改进的MK法综合判断。1961-2010和2015-2099年棉花和甜菜的ETc在多数站点呈下降趋势,但其显著性各异。ETc序列的下降说明未来新疆棉花和甜菜用水需求降低,遭受干旱的风险降低。棉花及甜菜ETc的趋势特征对于气候变化背景下干旱-半干旱区作物灌溉制度的制定和防灾抗旱有重要参考价值。