LC-MS/MS method development for investigation of DHA lipid peroxidation products and polyphenol profiling of U.S. honey of various geographical origins
Authors:   , Nyarko Kate 
Published: November 2024

Polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and arachidonic acid (ARA) are highly abundant in the brain cell membranes and have neuroprotective properties. These properties have been associated with the peroxidation products, 4-hydroxyhexenal (4-HHE) and 4-hydroxynonenal (4-HNE), produced during the lipid peroxidation process of DHA and ARA, respectively. However, not much is known whether DHA supplementation alters the levels of 4-HHE and 4-HNE in the brain and studies to examine whether dietary intakes of DHA are positively correlated to the blood or tissue levels of DHA in the brain remains unknown. In this study, we present a sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to analyze the levels of 4-HHE and 4-HNE in brain tissue of offspring mice after administration of DHA in the maternal diet. We hypothesized that increased maternal DHA supplementation may increase production of 4-HHE in the offspring, which may enhance protective and antioxidant effects. To test this hypothesis, pregnant mice were fed with either a control or a 1 percent DHA-enriched diet during gestation and lactation followed by subsequent measurements of 4-HHE and 4-HNE in brain tissues. Our results with the DHA-enriched diet resulted in significant increases in 4-HHE (4763 [plus or minus] 4.3 ng/g tissue), a peroxidative product of DHA, with reduced levels of 4-HNE (3098 [plus or minus] 5.1 ng/g tissue) in the offspring brain tissues. Although the 4-HNE levels were present in appreciable amounts, this peroxidation product was not altered bymaternal supplementation of DHA. The rationale for detecting 4-HNE was due to the physiological phenomenon associated with the endogenous 4-HNE levels in the brain. Our method provides a reliable and accurate measure of the targeted 4-HHE and 4-HNE metabolites for assessing the neurophysiological functions of DHA in the brain. The presence of phenolic compounds in honey can serve as potential authenticity markers for honey's botanical or geographical origins. The composition and properties of honey can vary greatly depending on the floral and geographical origins. This study focuses on identifying the specific markers that can distinguish honey based on their geographical areas in the United States. The main approach presented in this study to identify the geographic origins of honey involves chemometric methods combined with phenolic compound fingerprinting. Sample clean-up and phenolic compound extraction was carried out using solid phase extraction (SPE). Reversed phase liquid chromatography in combination with tandem mass spectrometry were utilized for the separation of the compounds. The honey physicochemical qualities were predominantly determined via spectrophotometric methods. Multivariate statistical tools such as principal component analysis (PCA), analysis of variance (ANOVA), and partial-least squares discriminant analysis (PLS-DA) were employed as both classification and feature selection tools. Overall, the present study was able to identify the presence of 12 potential markers to differentiate the honey's geographical origins. The total phenolic content ranged from 81.6 to 105.7 mg GAE/100 g corresponding to honey from Colorado and Washington, respectively (GAE: gallic acid equivalents). The regression analysis shows a tendency for the total phenolic content of honey to increase as the color of honey increases. The most important result obtained in this study is the demonstration that the geographical origin of honey plays a critical role in predicting the physical properties and phenolic composition of honey. [alpha]-Dicarbonyls are significant degradation products resulting from the Maillard reaction during food processing. Their presence in foods can indicate the extent of heat exposure, processing treatments, and storage conditions. Despite their importance, the occurrence of [alpha]-dicarbonyls in honey produced in the United States has not been extensively studied. This study aims to assess the concentrations of [alpha]-dicarbonyls in honey samples from different regions across the United States. The identification and quantification of [alpha]-dicarbonyls were conducted using reverse-phase liquid chromatography after derivatization with o-phenylenediamine (OPD), which was detected using ultraviolet (UV) and mass spectrometry methods. The study investigated the effects of pH, color and derivatization reagent on the presence of [alpha]-dicarbonyls in honey. Three major OPD-derivatized [alpha]-dicarbonyls, including methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG), were quantified in all the honey samples. 3-Deoxyglucosone (3-DG) was identified as the predominant [alpha]-dicarbonyl in all the U.S. honey samples, with concentrations ranging from 10.80 to 50.24 mg/kg. The total [alpha]-dicarbonyl content ranged from 16.81 to 55.74 mg/kg, with the highest concentration measured for Southern California honeys. The method validation results yielded excellent recovery rates for 3-DG (82.5 percent), MGO (75.8 percent), and GO (67.0 percent). The method demonstrated high linearity with a limit of detection and limit of quantitation ranging from 0.0015 to 0.002 mg/kg and 0.005 to 0.008 mg/kg, respectively. Our results provide insights on the occurrence and concentrations of [alpha]-dicarbonyl compounds in U.S. honey varieties, offering valuable information on their quality and susceptibility to thermal processing effects.