Antioxidant content of and migration from commercial polyethylene, polypropylene, and polyvinyl chloride packages

Antioxidants commonly used in polyolefins were studied in commercial food packages made of low- and high-density polyethylene (LDPE and HDPE), polypropylene (PP), polyvinylchloride (PVC), and polyethylene terephtalate (PET) and in a LDPE film extruded at the laboratory. The phenolic antioxidants BHA, BHT, AO 2246, AO 425, Ethanox 330, Irganox 1010, and Irganox 1076 were studied together with the phosphite Irgafos 168 and their two degradation products, phosphate and DBP. Antioxidants were extracted from polyolefins using microwave energy and analyzed using high-performance liquid chromatography (HPLC) to determine the antioxidant content in the diverse commercial films. Irganox 1010 and Irganox 1076 were found in the majority of the samples generally together with the phosphite Irgafos 168 and its oxidized product (phosphate). Specific migration levels of each antioxidant were determined by HPLC after pretreatment with solid-phase extraction (SPE) in aqueous food simulants and after their dilution with tetrahydrofuran (THF) in fatty food simulant. These levels were much lower than limits allowed by legislation.     

Liquid chromatographic determination of seven antioxidants in dry food

The liquid chromatographic determinative step of the official method for propyl gallate, trihydroxybutyrophenone, tert-butylhydroquinone, nordihydroguaiaretic acid, butylated hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxymethylphenol, and butylated hydroxytoluene (BHT) in fats and oils has been applied to their determination in a number of dry foods. A representative sample (10 g) is homogenized first with hexane (25 mL), then with 5 mL added water, and finally with 75 mL added acetonitrile. The hexane and acetonitrile are decanted, filtered, and separated; the hexane and rehydrated food are reextracted with 2 additional portions of acetonitrile, and the combined acetonitrile extracts are concentrated and diluted to 10 mL. An aliquot is analyzed as described in the official method, using a 150 x 4.6 mm 5 microns C-18 column. The need for rehydration to maximize the recovery of BHA and other antioxidants from marketplace dry food samples such as potato flakes, dry coffee whiteners, and dessert topping mixes was demonstrated. Rehydration was not required for cheese snacks, breakfast cereals, cake mixes, and some other foods. The need for rehydration should be determined by analyzing other foods with and without the addition of water. Potato and corn chips, popcorn and cheese snacks, breakfast cereals, dry beverage mixes, rice, potato flakes, french fried potatoes, and cake mixes were spiked with the above antioxidants at 10-50 ppm. Overall recoveries ranged from 64.3 to 105.6% and repeatabilities ranged from 0.7 to 10.8%. A total of 109 samples of the above foods were analyzed, and 64% contained detectable (greater than 1-2 ppm) antioxidants, mainly BHA and BHT.     

Determination of potential migrants in polycarbonate containers used for microwave ovens by high-performance liquid chromatography with ultraviolet and fluorescence detection

The determination of several compounds present in a commercial polycarbonate container intended to be used in microwave ovens which could be considered as potential migrants has been carried out by reversed-phase high-performance liquid chromatography (HPLC) with both ultraviolet (UV) and fluorescence detectors. Total dissolution with dichloromethane and polymer reprecipitation with methanol have been used to evaluate 100% potential migration as the worst case. The extract consisted of a complex mixture containing monomers, oligomers, UV stabilizers, antioxidants, degradation products, and other additives. Phenol, Bisphenol A, 2,4-di-tert-butylphenol, Cyasorb UV5411, bis(2-ethylhexylphthalate), Irganox 1076, and Irgafos 168 were identified by both retention times and fluorescence-to-UV ratios. Additional confirmation was achieved by HPLC with diode array detection and gas chromatography-mass spectrometry. Recovery percentages were in the range of 73.8-94.4%, the lowest one being for the antioxidant Irgafos 168 due to its transformation into the phosphate form and 2,4-di-tert-butylphenol. The concentrations of the studied analytes present in the polycarbonate container ranged between 0.9 and 240 microg.g(-)(1). The total dissolution conditions that may affect the final concentration of analytes, mainly Bisphenol A, are discussed.     

LC/UV/ESI-MS analysis of isoflavones in Edamame and Tofu soybeans

High-performance liquid chromatography coupled with ultraviolet and electrospray ionization mass spectrometry (HPLC/UV/ESI-MSD) was applied to the study of isoflavones in both Edamame and Tofu soy varieties, from which the immature fresh soybeans or the mature soybean seeds are consumed, respectively. Positive atmospheric pressure interface (API) MS and MS/MS were used to provide molecular mass information and led to the identification of a total 16 isoflavones, including three aglycones, three glycosides, two glycoside acetates, and eight glycoside malonates. The major isoflavones in soybean seeds were daidzein and genistein glycoside and their malonate conjugates. Trace levels of daidzein and genistein acetyl glycosides were found only in the mature dry soybean seeds. To facilitate quantitative analysis, acid hydrolysis during extraction of soy samples was selected to convert the various phytoestrogen conjugates into their respective isoflavone aglycones, allowing accurate quantitation of total phytoestrogens as aglycones. On the basis of HPLC combined with UV and MS detection, all three targeted soy isoflavone aglycones, daidzein, genistein and glycitein in hydrolyzed extracts were successfully quantified within 25 min with formononetin used as the internal standard. The standard curves of UV detection were fitted in the range of 14.16-29000 ng/mL for daidzein, 15.38-31500 ng/mL for genistein, and 11.72-24000 ng/mL for glycitein. For MS detection, the standard curves were established in the range of 3.54-1812.5 ng/mL for daidzein, 3.85-1968.75 ng/mL for genistein, and 2.93-1500 ng/mL for glycitein. Good linearities (r(2) > 0.999 for UV and r(2) > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and precision (RSD) were within 10% for UV detection and 15% for MS detection (n = 10). Using this method, the phytoestrogen levels of total isoflavone aglycones from 30 soybean seed varieties were then evaluated for confirmation of the technique. Total isoflavones ranged across the varieties from 0.02 to 0.12% in the Edamame varieties, which are harvested while the seeds are still immature, and from 0.16 to 0.25% in Tofu varieties, harvested when the seeds are physiologically mature. While the literature has focused on the isoflavone content of soy products and processing soy, this report provides a reliable analytical technique for screening of authenticated fresh immature Edamame soybeans and Tofu soybeans.     

Competitive enzyme-linked immunoassay for sialoglycoprotein of edible bird's nest in food and cosmetics

The proliferation of fake and inferior edible bird's nest (EBN) products has recently become an increasingly serious concern. To identify and classify EBN products, a competitive enzyme-linked immunoassay (ELISA) was developed to quantitate sialoglycoprotein in EBN used in food and cosmetic applications. The characteristic sialoglycoprotein in EBN was found, extracted, purified, and analyzed. Sialoglycoprotein, considered the main carrier of sialic acid in EBN, consisted of 106 and 128 kDa proteins. A monoclonal antibody that could recognize both proteins was prepared. The heat-treated process did not change the affinity of sialoglycoprotein with the antibody. An optimized ELISA method was established with a cross-reactivity of less than 0.1% and an IC(50) of 3.3 μg/mL. On the basis of different food and cosmetic samples, the limits of detection (LOD) were 10-18 μg/g. Recoveries of fortified samples at levels of 20 and 80 μg/g ranged from 81.5 to 96.5%, respectively. The coefficients of variation were less than 8.0%.     

Microwave heating causes rapid degradation of antioxidants in polypropylene packaging, leading to greatly increased specific migration to food simulants as shown by ESI-MS and GC-MS

Microwave heating of commercial microwavable polypropylene packaging in contact with fatty food simulants caused significant antioxidant degradation and increased specific migration as shown by electrospray ionization-mass spectrometry (ESI-MS) and gas chromatography-mass spectrometry (GC-MS). Degradation of the antioxidants Irgafos 168 and Irganox 1010 was not detected during conventional heating of polypropylene packaging at the same temperature. The migration into aqueous food simulants was primarily restricted by the water solubility of the migrants. Using isooctane as fatty food simulant caused significant swelling and greatly enhanced overall migration values compared to the other fatty food simulant, 99.9% ethanol, or the aqueous food simulants 10% ethanol, 3% acetic acid, or water. ESI-MS spectra clearly reflected the overall migration values, and the number and amount of compounds detected decreased as the hydrophilicity of the food simulant increased. ESI-MS was shown to be an excellent tool for the analysis of semivolatile migrants and a good complement to GC-MS analysis of volatile migrants.     

The relationship between the physicochemical properties of antioxidants and their ability to inhibit lipid oxidation in bulk oil and oil-in-water emulsions

Chain-breaking antioxidants differ in their effectiveness at inhibiting lipid oxidation because of their chemical properties and physical location within a food. Our objective was how the physicochemical properties of four structurally related lipid-soluble antioxidants were related to their antioxidant activity. Antioxidants differed in the number of methyl (alpha-tocopherol and delta-tocopherol) or hydroxyl (butylated hydroxytoluene (BHT) and 4-hydroxymethyl-2,6-ditertiarybutylphenol) groups. Surface activity of the antioxidants was in the order of delta-tocopherol > alpha-tocopherol approximately 4-hydroxymethyl-2,6-ditertiarybutylphenol > BHT. Free-radical scavenging activity was similar between alpha-tocopherol and delta-tocopherol as well as BHT and 4-hydroxymethyl-2,6-ditertiarybutylphenol. In bulk menhaden oil, BHT was a more effective antioxidant than 4-hydroxymethyl-2,6-ditertiarybutylphenol while alpha-tocopherol was more effective than delta-tocopherol. In menhaden oil-in-water emulsions, BHT was a more effective antioxidant than 4-hydroxymethyl-2,6-ditertiarybutylphenol while delta-tocopherol was more effective than alpha-tocopherol. These results indicate that the surface activity and polarity of lipid-soluble antioxidants were not the only determinants of their antioxidant effectiveness in food lipids.     

Antioxidant activity and diffusion of catechin and epicatechin from antioxidant active films made of poly(L-lactic acid)

Active membranes and food packaging containing antioxidants like catechin and epicatechin, combined with the use of materials made of biopolymers obtained from renewable sources, could create a novel alternative to reduce oxidation in food, pharmaceutical, and cosmetic products. Poly(94% L-lactic acid) films containing 1.28% catechin and 1.50% epicatechin were extruded in a pilot plant-scale extrusion machine. The diffusion kinetics of catechin and epicatechin into 95% ethanol at 20, 30, 40, and 50 °C and 50% ethanol at 40 °C displayed Fickian release behavior and diffusion coefficients between 0.5 and 50 × 10(-11) cm(2)/s. According to the Arrhenius equation, the energy of activation for the diffusion of catechin and epicatechin in the films was 110.43 and 98.92 kJ/mol, respectively. The antioxidant activity of the films was measured in methanol extracts containing 46.42 μg/mL of catechin and 57.52 μg/mL of epicatechin as 32.90 and 36.68% of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical, respectively.     

基质固相分散萃取-高效液相色谱串联质谱法测定土壤中残留的磺酰脲类除草剂

建立了基质固相分散萃取-高效液相色谱串联质谱法(MSPD-HPLC-MS/MS)测定土壤中3种磺酰脲类除草剂(氯磺隆、甲磺隆、苯磺隆)残留的分析方法。对基于球磨的基质固相分散萃取条件进行了详细优化,最终确定最佳条件为:0.2 g土壤样品、0.8 g HC-C18粉末状分散剂与直径为8 mm的小钢珠一起球磨10 min后,转移至空的玻璃萃取小柱,用10 m L乙腈洗脱,氮气吹干后用甲醇定容至0.6 m L,再经0.22μm的滤膜抽滤后装入自动进样瓶中。用Syncronis C18反相色谱柱分离,以甲醇(A)~1‰甲酸溶液(B)为流动相进行梯度洗脱,选择反应监测(SRM)模式下进行检测。氯磺隆在20~200μg·kg~(-1),甲磺隆和苯磺隆在10~200μg·kg~(-1)范围内线性良好,相关系数r在0.997 9~0.999 5。土壤样品的平均加标回收率在84.7%~104.6%,相对标准偏差在4.5%~7.9%(n=5)。方法的检出限(S/N=3)0.32~0.68μg·kg~(-1)。该方法简单、效率高、干扰少、回收率高,满足土壤中除草剂的残留分析要求。     

Flavonoids with potent antioxidant activity found in young green barley leaves

Saponarin, a flavonoid found in young green barley leaves, possesses potent antioxidant activities, which are determined by its inhibition of malonaldehyde (MA) formation from various lipids oxidized by UV light or Fenton's reagent. Lipids used were squalene, ethyl linoleate, ethyl linolenate, ethyl arachidonate, octadecatetraenoic acid (ODTA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), cod liver oil, lecithin I, lecithin II, and blood plasma. The addition of saponarin inhibited the formation of MA from squalene upon UV irradiation at the level of 2 μmol/mL by almost 100%, whereas BHT inhibited its formation by 75% at the same level. Saponarin showed potent antioxidant activity toward fatty acid ethyl esters at levels >100 μg/mL. Saponarin inhibited MA formation in ODTA by 60%, in EPA by 50%, and in DHA by 43% at the level of 15 μmol/mL. Saponarin exhibited strong antioxidant activities with dose-response levels toward cod liver oil and lipoproteins (lecithins I and II), higher than those of α-tocopherol. A mixture of saponarin/lutonarin (4.5:1, w/w) inhibited MA formation appreciably from all lipids tested with dose response. This mixture exhibited highest effect toward cod liver oil (86%), followed by DHA, lecithin II, blood plasma, EPA, and lecithin I. Supplementation of young green barley leaves containing saponarin should be beneficial to health and may prevent diseases caused by oxidative damage such as various cancers, inflammations, and cardiovascular diseases.     

Novel application of square-wave adsorptive-stripping voltammetry for the determination of xanthohumol in spent hops

This paper reports the development of a novel electrochemical assay for xanthohumol (XN) by square-wave adsorptive-stripping voltammetry (SWAdSV) with a hanging mercury drop electrode. The method showed good repeatability (CV < 2%) and linearity (between 10 and 250 μg L(-1)), as well as suitable limits of detection (2.6 μg L(-1)) and quantification (8.8 μg L(-1)). The method was applied for the quantification of this compound in spent hops, and the results obtained were compared with the HPLC-UV method. XN contents determined by the SWAdSV method were 16 ± 1 and 100 ± 4 μg L(-1) for aqueous and methanolic extracts, respectively. The developed new methodology considerably reduces the analysis time, approximately from 25 min (HPLC-UV method) to 7 min, enabling a high sample throughput. In addition, the detection and quantification limits were approximately 5-fold lower than those obtained with the chromatographic method.     

High-performance capillary electrophoresis with indirect UV detection for determination of alpha-galactosides in Leguminosae and Brassicaceae

A rapid, easy, and reproducible capillary electrophoresis method for determination of raffinose family oligosaccharides (alpha-galactosides) was developed. Sucrose, raffinose, stachyose, verbascose, and ajugose were determined with indirect UV detection at moderate alkaline pH 9.2, using pyridine-2,6-dicarboxylic acid as background electrolyte in a sodium tetraborate buffer with added cetyltrimethylammonium bromide. The separation efficiency measured by the number of theoretical plates (N) ranged from 1.4 x 10(5) to 2.3 x 10(5). The precision of the method, measured by the relative standard deviation (RSD), was less than 0.53% for the migration times and better than 3.4% for normalized areas (NA), considering all sugars except verbascose (RSD(NA) = 11.8%). Detection limits were about 110 microg/mL, corresponding to 150-320 microM. Relative response factors (RRF) were calculated on the basis of linearity studies and used for quantification of alpha-galactosides in a lupine sample (Lupinus angustifolius).     

Widespread occurrence of low levels of alternariol in apple and tomato products, as determined by comparative immunochemical assessment using monoclonal and polyclonal antibodies

This study investigated the production of polyclonal (pAB) antibodies and the first time production of monoclonal (mAB) antibodies against the mycotoxin alternariol, and their implementation in enzyme immunoassay (EIA) for the rapid determination of alternariol in foods. Both EIAs were highly sensitive, with detection limits (IC??) of 35 ± 6.9 pg/mL (mAb EIA) and 59 ± 16 pg/mL (pAb EIA). Food products (n = 109; apple and tomato products, white wine) from German retail shops were analyzed. At a detection limit of 1-2 μg/kg, alternariol at 1-13 μg/kg was found with high frequency in apple (67%) and tomato (93%) products. Tomatoes with visible signs of Alternaria infection, stored at room temperature for up to 4 weeks, contained alternariol at levels up to 50 mg/kg, as determined by EIA and HPLC-FLD. It is concluded that the alternariol immunoassays present a versatile screening tool which could facilitate food control for Alternaria toxins.     

Anthocyanin-based natural colorants: a new source of antiradical activity for foodstuff

The antiradical capacity (radical scavenger capacity, RSC) of anthocyanin-based fruit extracts prepared in the laboratory (black chokeberry, black-thorn, and strawberry) was studied by using the 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH(*)). To determine their RSC, the second-order rate constant (k(2)) for the oxidation of these extracts by DPPH(*) was calculated. The value of k(2) was compared to that used in the food industry as natural (alpha-tocopherol) or synthetic (butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA)) antioxidants, as well as for a commercial elderberry concentrate and a synthetic colorant (Ponceau 4R). The k(2) values ((mg/mL)(-)(1) s(-)(1)), in methanol at 25 degrees C, were 1.87, 0.7, 0.42, 0.2, 0.05, 0.03, and 0.008 for alpha-tocopherol, black chokeberry, BHA, black-thorn, BHT, strawberry, and elderberry, respectively. Ponceau 4R lacked RSC. Therefore, these natural colorants proved to be a combined source of color and RSC for food material.     

On-line HPLC-DPPH screening method for evaluation of radical scavenging phenols extracted from apples (Malus domestica L.)

An on-line HPLC-DPPH screening method for phenolic antioxidants in apple methanol/water (80:20, v/v) extracts was applied. The determination of antioxidants was based on a decrease in absorbance at 515 nm after postcolumn reaction of HPLC-separated antioxidants with the 2,2'-diphenyl-1-picrylhydrazyl radicals (DPPH*). Each of the antioxidants separated by the HPLC column was observed as a negative peak corresponding to its antioxidative activity. The on-line method was applied for quantitative analysis of the antioxidants. A linear dependence of negative peak area on concentration of the reference antioxidants was observed. For validation of the on-line method the limit of detection, LOD (microg/mL), and the limit of quantification, LOQ (microg/mL), of the phenolic compounds were determined. Comparison of the UV and DPPH radical quenching chromatograms with authentic compounds identified catechin, chlorogenic acid, caffeic acid, epicatechin, and phloridzin in the apple cultivars (Lobo, Golden Delicious, and Boskoop), and the distribution of total antioxidant activity was calculated.     

Simultaneous determination of ethidimuron, methabenzthiazuron, and their two major degradation products in soil

An analytical method has been developed for the quantification of two herbicides (ethidimuron and methabenzthiazuron) and their two main soil derivatives. This method involves fluidized-bed extraction (FBE) prior to cleanup and analysis by reverse-phase liquid chromatography with UV detection at 282 nm. FBE conditions were established to provide efficient extraction without degradation of the four analytes. (14)C-labeled compounds were used for the optimization of extraction and purification steps and for the determination of related efficiencies. Extraction was optimal using a fexIKA extractor operating at 110 degrees C for three cycles (total time = 95 min) with 75 g of soil and 150 mL of a 60:40 v/v acetone/water mixture. Extracts were further purified on a 500 mg silica SPE cartridge. Separation was performed on a C18 Purosphere column (250 mm x 4 mm i.d.), at 0.8 mL min(-1) and 30 degrees C with an elution gradient made up of phosphoric acid aqueous solution (pH 2.2) and acetonitrile. Calibration curves were found to be linear in the 0.5-50 mg L(-1) concentration range. Besides freshly spiked soil samples, method validation included the analysis of samples with aged residues. Recovery values, determined from spiked samples, were close to 100%. Limits of detection ranged between 2 and 3 microg kg(-1) of dry soil and limits of quantification between 8 and 10 microg kg(-1) of dry soil. An attempt to improve these performances by using fluorescence detection following postcolumn derivatization by orthophthalaldehyde-mercaptoethanol reagent was unsuccessful.     

Factors influencing benzene formation from the decarboxylation of benzoate in liquid model systems

Benzene may occur in foods due to the oxidative decarboxylation of benzoate in the presence of hydroxyl radicals. This study investigated factors influencing benzene formation in liquid model systems. The type of buffer, other sources of hydroxyl radical formation in food (photo oxidation of riboflavin and lipid oxidation), transition metal ion concentrations, and the inhibitory effect of antioxidants were tested in benzoate containing model systems. Regarding the hydroxyl radical sources tested, the highest benzene formation was observed in light exposed model systems containing ascorbic acid, Cu(2+), and riboflavin in Na-citrate buffer (1250 ± 131 μg kg(-1)). In practice, it seems that the combination ascorbic acid/transition metal ion remains the biggest contributor to benzene formation in food. However, the concentration of Cu(2+) influences significantly benzene formation in such a system with highest benzene yields observed for Cu(2+) 50 μM (1400 μg kg(-1)). The presence of antioxidants with metal chelation or reduction properties could prevent completely benzene formation.     

High Levels of α-Tocopherol in Norwegian Alpine Grazing Plants

Antioxidants prevent oxidation of fatty acids in milk and meat. In the present study, the content of tocopherol antioxidants (vitamin E) in vegetative and reproductive parts of 22 grazing plants was estimated in two alpine areas used for summer farming. The overall mean content of α-tocopherol was 135 ± 34 μg g(-1) DW, and grasses had much lower content (28 ± 11 μg g(-1) DW) than herbs (215 ± 94 μg g(-1) DW), sedges (186 ± 78 μg g(-1) DW), and woody species (178 ± 52 μg g(-1) DW). Highest and lowest species-specific levels were 649 ± 91 and 2 ± 1 μg g(-1) DW, respectively. Plants from light and shady habitats did not differ in their α-tocopherol content, which was idiosyncratic as indicated by significant interactions between species, sampling occasion, site, and tissue type. Our results show that alpine ranges provide fodder with high levels of α-tocopherol.     

LC-MS/MS analysis of neonicotinoid insecticides in honey: methodology and residue findings in Austrian honeys

An analytical method for the simultaneous determination of residues of eight neonicotinoid insecticides and two metabolites in honey using LC-MS/MS was developed and validated. Two approaches of sample preparation were investigated, with the final method involving acetonitrile extraction and subsequent cleanup by dispersive solid-phase extraction (QuEChERS type). Validation was based on quintuplicate analysis at three fortification levels and showed satisfactory recoveries (60-114%) and high precision (RSDs between 2.7 and 12.8%). Low limits of detection and quantification could be achieved for all analytes ranging from 0.6 to 5 μg/kg and from 2 to 10 μg/kg, respectively. Investigations of Austrian honey samples revealed the presence of acetamiprid, thiacloprid, and thiamethoxam residues in honey; however, no sample exceeded the maximum residue limits. On average, flower honey samples contained neonicotinoid residues in higher quantities compared to forest honey samples.     

Determination of chlorophylls in Taraxacum formosanum by high-performance liquid chromatography-diode array detection-mass spectrometry and preparation by column chromatography

Taraxacum formosanum, a well-known Chinese herb shown to be protective against hepatic cancer as well as liver and lung damage, may be attributed to the presence of abundant carotenoids and chlorophylls. However, the variety and content of chlorophylls remain uncertain. The objectives of this study were to develop an high-performance liquid chromatography-diode array detection-mass spectrometry method for determination of chlorophylls in T. formosanum and preparation by column chromatography. An HyPURITY C18 column and a gradient mobile phase of water (A), methanol (B), acetonitrile (C), and acetone (D) could resolve 10 chlorophylls and an internal standard Fast Green FCF within 30 min with a flow rate at 1 mL/min and detection at 660 nm. Both chlorophylls a and a' were present in the largest amount (1389.6 μg/g), followed by chlorophylls b and b' (561.2 μg/g), pheophytins a and a' (31.7 μg/g), hydroxychlorophyll b (26.5 μg/g), hydroxychlorophylls a and a' (9.8 μg/g), and chlorophyllides a and a' (0.35 μg/g). A glass column containing 52 g of magnesium oxide-diatomaceous earth (1:3, w/w) could elute chlorophylls with 800 mL of acetone containing 50% ethanol at a flow rate of 10 mL/min. Some new chlorophyll derivatives including chlorophyllide b, pyropheophorbide b, hydroxypheophytin a, and hydroxypheophytin a' were generated during column chromatography but accompanied by a 63% loss in total chlorophylls. Thus, the possibility of chlorophyll fraction prepared from T. formosanum as a raw material for future production of functional food needs further investigation.