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Rapid determination of minority organic acids in honey by high-performance liquid chromatography

A rapid high-performance liquid chromatographic method for the determination of organic acids in honey is reported. Malic, maleic, citric, succinic and fumaric acids were identified and quantified in 15 min. First time repeatibility, reproducibility and recoveries were determined out for these acids in honey samples. Maleic acid was also quantified for first time by a chromatographic method. The organic acids were removed from honey by using a solid-phase extraction procedure with anion-exchange cartridges. Previously, the solution of honey was adjusted to pH 10.50 with 0.1 M NaOH and stirred for 15 min at room temperature. Then, this solution was adjusted to pH 5.00 with 0.1 M H2SO4. This procedure was carried out to avoid interferences in the baseline. The chromatographic separation was achieved with only one Spherisorb ODS-2 S5 column thermostated at 25 degrees C. Metaphosphoric acid (pH 2.20) was used as mobile phase at a flow-rate of 0.7 ml/min. Organic acids were detected with a UV-vis detector (215 nm). The precision results showed that the relative standard deviations of the repeatability and reproducibility were < or =3.20% and < or =4.86%, respectively. The recoveries of the organic acids ranged from 62.9 to 99.4%. Under optimum conditions the detection limits ranged from 0.0064 to 7.57 mg/kg and the quantification limits ranged from 0.025 to 10.93 mg/kg.

Significance of nonaromatic organic acids in honey.

Although organic acids represent < 0.5% of honeys constituents, they make important contributions to the organoleptic, physical, and chemical properties of honey. To date, approximately 30 nonaromatic organic acids have been identified in honey, but relatively little attention has been paid to these components. This article reviews the current literature related to the significance of nonaromatic organic acids in honey; it was written with a goal of attracting researchers to study these interesting honey components. Previous research contributions on nonaromatic organic acids in honey may be classified into five main areas: (i) the antibacterial activities of these acids, (ii) the antioxidant activities of these acids, (iii) the use of these acids as possible indicators of incipient fermentation, (iv) the use of these acids for treatment of Varroa infestation, and (v) the use of these acids as factors for the characterization of both botanical and geographical origins of honeys. We conclude that nonaromatic organic acids are of interest for diverse reasons and that there is a particular need for studies regarding their possible antibacterial and antioxidant activities.

Analytical Methods for the Determination of Organic Acids in Honey

Although organic acids represent less than 0.5% of honeys constituents, they make important contributions to organoleptic, physical, and chemical properties of honey. They could be used as fermentation indicators, for the treatment of Varroa infestation, and to discriminate among honeys according to their botanical and/or geographical origins. This article reviews the current literature related to the analytical methods (enzymatic, chromatographic and electrophoretic) that have been applied recently to the determination of honeys organic acids. The advantages and disadvantages of all the procedures described are also discussed. This review has been written to make the study of these interesting honey component easier.

Solid-phase extraction procedure to remove organic acids from honey.

A solid-phase extraction procedure was applied to remove organic acids from honey. Malic, maleic, citric, succinic and fumaric acids were isolated with an anion-exchange cartridge. The different parameters which affected the extraction procedure were studied and optimised to establish the optimal conditions for maximum recovery of organic acids and minimum extraction of interferences. The optimised procedure used a cartridge which was activated with 10 ml of 0.1 M sodium hydroxide solution (percolation rate 3 ml/min). A 10 ml volume of honey solution was passed at a flow-rate of 0.5 ml/min. The cartridge was washed with 10 ml of water (3 ml/min) and organic acids were eluted with 4 ml of 0.1 M sulfuric acid (0.5 ml/min). This solution was injected directly into the chromatograph. When this procedure was carried out on standard solutions of organic acids, recoveries between 99.2 and 103.4% were found. If this procedure was applied to honey samples these recoveries were also satisfactory and ranged from 62.9 to 99.4%.

Enzymatic determination of L-malic acid in honey

Abstract l -Malic acid determination has been carried out in honey using a direct enzymatic method. The sample solution was prepared from 2.5 g honey in 100 ml Milli-Q water. The enzymatic determination was measured spectrophoto metrically at 340 nm, using glutamate-oxaloacetate transaminase and l -malate dehydrogenase. The direct method combines precision (CV was 3.5%, at worst), good recovery (100 ± 3.5%), zero interference, simplicity, and low cost (cost was reduced by 50% using a microtest). This direct enzymatic method was applied to 20 floral honeys of Galicia (northwestern Spain) and the results ranged between 94 and 596 mg kg −1 (mean 246 mg kg −1 ) of l -malic acid, which is in keeping with value ranges obtained by other authors. Different clarifications [as polyvinyl-polypyrrolidone (PVPP), Carrez, Carrez with NaOH, Carrez with KOH, Carrez together with PVPP and activated charcoal] and a pair of controls have also been used but the precision and the recovery of direct enzymatic method of l -malic acid in honey did not improve.

Rapid capillary zone electrophoresis method for the determination of metal cations in beverages.

A rapid and reliable capillary zone electrophoresis method for the determination of inorganic cations was developed. The complete separation of K(+), Ba(2+), Ca(2+), Na(+), Mg(2+), Mn(2+), Ni(2+), Cd(2+), Li(+) and Cu(2+) can be achieved in 4min with a simple electrolyte composed by 10mM imidazole as the carrier buffer and background absorbance provider and acetic acid as the complexing agent (pH 3.60). Injection was performed hydrostatically by elevating the sample at 10cm for 30s. The running voltage was +25kV at room temperature. Indirect UV-absorption detection was achieved at 185nm. The detection limit was in the range between 0.06mg/l (Mg(2+)) and 0.57mg/l (K(+)) and the quantification limits ranged from 0.10mg/l (Ni(2+)) to 0.80mg/l (Cu(2+)). The calibration graphs were linear in the concentration range from the quantification limit till at least 1g/l in K(+), 10mg/l in Ba(2+), Ca(2+), Mg(2+), Mn(2+), Ni(2+) and Cd(2+), 40mg/l in Na(+) and 12mg/l in Li(+) and Cu(2+). The repeatability, intraday and interday analysis were </=1.55% and </=3.64% for migration time and </=3.38% and </=3.63% for peak area. The method developed has been applied to several beverage samples with only a simple dilution and filtration treatment of the sample. The proposed method is simple, fast, cheap and it is achieved with common products in either laboratory. For these reasons, it is a very useful method for routine analysis.

Variation of apparent ethanol content of unspoiled northwestern Spanish honeys during storage

Abstract This paper describes, for the first time, the variation of apparent ethanol content during storage of 33 honey samples. Moisture content was determined by measuring refractive index at 20°C. Apparent ethanol content was determined in a double cuvette, according to a modification of the Boehringer–Mannheim enzymatic method. Four different types of apparent ethanol evolution were observed: constant increment, increment followed by decrease, values oscillation and constant decrease.

Different forms of maleic and fumaric acids (cis and trans of 2-butenedioic acid) in honey

Abstract The total contents of maleic and fumaric acids (cis and trans-2-butenedioic acids, respectively) were quantified by a high performance liquid chromatography method, in 50 floral honeys of Galicia (north-western Spain). Honey pH, activity coefficients, dissociation constants of the acids (K1 and K2), and the molar concentrations of the forms of maleic and fumaric acids naturally present in honey ([AH2], [AH−] and [A2−]) have been calculated for the first time. The contents of maleic and fumaric acids can be determined either as total maleic and fumaric acids ([AH2]) or as total maleate and fumarate ([A2−]), but there are other forms of these acids in honey. Therefore the calculation of the forms of the maleic and fumaric acids present would illuminate their origin at honey pH. The predominant acid form depends on honey pH value. Maleic acid was quantifiable in 44 honeys. The [AH−] form was found as a major component in all samples. Fumaric acid was quantifiable in 49 honeys. The [A2−] form was found as a major component in most honeys (28 samples) and the [AH−] was predominant in 21 samples. No honey analysed had a [AH2] form as predominant. Although maleic and fumaric acids have the same molecular weight and they are both dicarboxylic acids, their pH relationships differ. The relationships between maleic and fumaric acids and honey pH and between the total content of maleic and fumaric acids and their forms in honey have been calculated.

Nonaromatic Organic Acids of Honeys

Pot honeys have delicate, sweet and sour flavors, and are highly appreciated in tropical areas. Their acidity is usually high, and therefore, free acid values could contribute to characterize stingless bee honeys. Organic acids contribute to several honey properties and have been considered potentially useful to determine the origin of honeys. Among other components, organic acids have been studied as possible contributors to honeys’ antioxidant and antibacterial activities. Some honey nonaromatic organic acids have been used as treatments against varroasis and small hive beetles. High acetic acid contents could indicate honey fermentation. The most important procedures to determine honey nonaromatic organic acids are enzymatic assays, chromatographic techniques, and capillary electrophoresis procedures. At the end of this chapter the advantages and disadvantages of each of them are summarized.