Determination of flavonoids in a Citrus fruit extract by LC–DAD and LC–MS

Flavonoids are a group of polyphenolic compounds with health-related properties. Citrus fruits are rich in flavonoids and their extracts are being used as functional ingredients for several industrial products. A new high performance liquid chromatography technique with an UV photodiode-array detector was used to analyze flavonoids of an extract of Citrus species. To our knowledge this is the first study that reports isoquercitrin presence at a level of 77.3 mg/100 g in a sample made of Citrus fruits; four other flavonoids were quantified as rutin (326.59 mg/100 g), naringin (338.36 mg/100 g), quercetin (96.35 mg/100 g) and naringenin (2.35 mg/100 g). Identification was confirmed by a liquid chromatography mass spectrometer system. Method validation was achieved, providing an analytical technique that can be used to detect trace amounts of these compounds in Citrus extracts with an extremely rapid sample preparation.     

Determination of catechins in green tea infusions by reduced flow micellar electrokinetic chromatography

A sulfated-β-cyclodextrin (s-β-CD) modified reduced flow micellar electrokinetic chromatography (RF-MEKC) method was developed and validated for the determination of catechins in green tea. The optimal electrolyte consisted of 0.2% triethylamine, 50 mmol/L SDS and 0.8% s-β-CD (pH = 2.9), allowing baseline separation of five catechins in 4 min. The samples and standards were injected at 0.6 psi for 5 s under constant voltage of −30 kV. Sample preparation simply involved extraction of 2 g of tea with 200 mL water at 95 °C under constant stirring for 5 min. The method demonstrated excellent performance, with limits of detection (LOD) and quantification (LOQ) of 0.02–0.1 and 0.1–0.5 μg/mL, respectively, and recovery percentages of 94–101%. The method was applied to six samples of Brazilian green tea infusions. Epigallocatechin gallate (23.4–112.4 μg/mL) was the major component, followed by epigallocatechin (18.4–78.9 μg/mL), epicatechin gallate (5.6–29.6 μg/mL), epicatechin (4.6–14.5 μg/mL) and catechin (3.2–8.2 μg/mL).     

GC–MS quantification and sensory thresholds of guaiacol in orange juice and its correlation with Alicyclobacillus spp.

Guaiacol is a profoundly negative taint/off-flavour produced by the increasingly common microbial contamination of fruit juices with Alicyclobacillus spp. The objectives of this study included: determining sensory thresholds for guaiacol in orange juice (OJ), developing an analytical method whose detection limits were equivalent to sensory thresholds and determining levels of Alicyclobacillus spp. that would produce detectable levels of guaiacol. A 12 member trained panel was used to establish guaiacol detection and recognition thresholds. Guaiacol ortho and retro nasal detection thresholds in OJ were 0.70 and 0.53 μg/l respectively. Odour recognition threshold was 2.0 μg/l. A SPME GC–MS Selected Ion Monitoring (SIM) procedure was optimised to achieve analytical detection limits of 0.5 μg/l. Optimum guaiacol detection limit was achieved using only responses from m/z 109 and 124. Ion ratios (m/z 109/124) and linear retention index value matching were used to confirm the identification of guaiacol. Quantification was achieved using peak areas from standard guaiacol additions in orange juice between 0.5 and 100 μg/l. Alicyclobacillus growth of 2.2 log CFU/ml in OJ produced just detectable levels (0.7 μg/l) of guaiacol.     

Liquid chromatography–tandem mass spectrometry for the determination of chrysoidine in yellow-fin tuna

A high performance liquid chromatography (HPLC) coupled to electrospray ionisation tandem mass spectrometry (MS/MS) method was described for the residue detection of chrysoidine in yellow-fin tuna in the present study. Samples were cleaned up with solid phase extraction (SPE) cartridge, and then injected into HPLC for separation. Multiple-reaction monitoring (MRM) was applied for quantitative determination. Results showed that the low limit of detection (LOD) of the method was 1.25 × 10⁻¹² g, and the low limit of quantification (LOQ) was 0.42 μg/L. The standard calibration curve was y = 2333.9x −845 (r² > 0.99) with the linear range of 0.63–100 μg/L. The average recoveries of chrysoidine ranged from 86.0% to 108.0% when the spiked concentration was from 0.5 μg/kg to 20 μg/kg. And the developed method also showed the good test precisions (RSD%: 4.38–14.27%).     

Rapid analysis of cyclamate in foods and beverages by gas chromatography-electron capture detector (GC-ECD)

A rapid method for determination of sodium cyclamate in foods and beverages was developed. Sodium cyclamate was converted to N,N-dichloridecyclohexylamine by reaction with sodium hypochlorite under acid condition. N,N-dichloridecyclohexylamine was subsequently extracted by n-hexane and determined by gas chromatography. Conditions such as derivatization time, the concentration of sodium hypochlorite and sulphuric acid were optimised. Amino acids, aliphatic amines, and food additives such as preservatives, dyes and sweeteners showed no interference for quantification of cyclamate. The correlation coefficient of calibration curve was 0.9993 in the range of 5.0–250 mg/L. The limits of detection (LOD) and limits of quantification (LOQ) were calculated as three or ten times the signal-to-noise ratio (S/N), respectively. The LOD and LOQ for yellow wine and fruit juice were 0.05 and 0.2 mg/L, respectively. The LOD and LOQ for cake and preserved fruit were 0.25 and 0.8 mg/kg, respectively. The intra-day and inter-day RSD were 0.28% and 1.1% (n = 5), respectively. The method was successfully applied for determination of cyclamate in yellow wine, cake, fruit juice and preserved fruit. This method was simple, fast, and sensitive. It was suitable for the determination of cyclamate in foods and beverages for safety and quality control inspections.     

Simultaneous quantitative determination of Sudan dyes using liquid chromatography–atmospheric pressure photoionization–tandem mass spectrometry

Atmospheric pressure photoionization–tandem mass spectrometry (APPI–MS/MS) method has been developed for quantitative determination of Sudan I to IV dyes. This study demonstrates the applicability of a simple isocratic normal phase HPLC method using isopropanol (0.3%) in n-hexane as the mobile phase for the separation of these dyes. A simple extraction procedure using n-hexane has been applied for the extraction of these dyes from spiked samples of chilli powder and tomato sauce. The quantitative determination of Sudan I to IV is obtained from the spiked tomato sauce and chilli powder samples by external standard method under single reaction monitoring (SRM) mode. The study includes a detailed investigation on LOD, LOQ, linearity and recovery of Sudan I to IV dyes. The LOD ranged from 5–18 μg/l and LOQ ranged from 10–24 μg/l. The present method can be a powerful analytical tool for the simultaneous quantitative determination of Sudan dyes present in food products.     

Distribution of fungi and aflatoxins in a stored peanut variety

The objective of the present study was to evaluate the mycoflora and occurrence of aflatoxins in stored peanut samples (hulls and kernels) from Tupã, State of São Paulo, Brazil. The samples were analyzed monthly over a period of one year. The results showed a predominance of Fusarium spp. (67.7% in hulls and 25.8% in kernels) and Aspergillus spp. (10.3% in hulls and 21.8% in kernels), and the presence of five other genera. The growth of Aspergillus flavus was mainly influenced by temperature and relative humidity. Analysis of hulls showed that 6.7% of the samples were contaminated with AFB₁ (mean levels = 15–23.9 μg/kg) and AFB₂ (mean levels = 3.3–5.6 μg/kg); in kernels, 33.3% of the samples were contaminated with AFB₁ (mean levels = 7.0–116 μg/kg) and 28.3% were contaminated with AFB₂ (mean levels = 3.3–45.5 μg/kg). Analysis of the toxigenic potential revealed that 93.8% of the A. flavus strains isolated were producers of AFB₁ and AFB₂.     

Fluorometric determination of beta-hydroxybutyrate in milk and blood plasma

Determination of β-hydroxybutyrate (BHBA) in blood and milk samples is an important tool in the diagnosis of ketosis in dairy cattle. Apart from semiquantitative cow-side tests, well-established laboratory methods exist for measurements in blood serum or plasma. These spectrophotometric methods are, however, neither convenient nor reliable when transferred to analyses of milk. Due to its nontransparent nature, milk needs extensive pretreatment if traditional analyses are to be used. This paper describes a fluorometric determination of BHBA that is useful without pretreatment in opaque matrices such as milk and in blood plasma. The method is easy to automate, saves labor expenses, and is inexpensive. The analytical accuracy and precision are reliable for intensive as well as large-scale analysis; for example, in-line sampling from automatic milking systems. Analysis of 2500 random milk samples showed a BHBA content ranging from 10 to 631 μM (mean 49 μM). Furthermore, selected samples (n = 295) from diagnosed ketotic animals taken on d −35 to +35 from peak level ranged from 10 to 684 μM (median 79 μM, mean 141 μM). Using the same 1240 blood plasma samples, the fluorometric method was closely correlated with a traditional spectrophotometric method (r = 0.987). Hemolysis of samples does not appear to affect the fluorometric determination of BHBA.     

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.     

Identification and quantification of S-allyl-l-cysteine in heated garlic juice by HPLC with ultraviolet and mass spectrometry detection

This paper presents a new high performance liquid chromatography (HPLC) method for the identification and quantification of S-allyl-l-cysteine (SAC), which is the most important bioactive compound present in heated garlic juice (HGJ). This method uses LC combined with an ultraviolet (LC–UV) and mass spectrometry detection (LC–MS/MS) method and is based on the dansyl chloride derivatization step. The method validation included selectivity, linearity, precision, accuracy, and sensitivity. The linear correlation coefficients (r) were always greater than 0.998. Intra- and inter-day precision for SAC at three concentrations demonstrated a relative standard deviation (RSD) of less than 15.3% for both UV and MS methods. The limit of quantification (LOQ) was 0.71 μg/g and 0.07 μg/g for UV and MS methods, respectively. The results were not significantly different between the SAC contents determined by the UV and MS detection in real HGJ samples. Both methods are proposed as useful quality control tools for accurate determination of SAC content of garlic preparations.     

Exploration of Vanilla pompona from the Peruvian Amazon as a potential source of vanilla essence: Quantification of phenolics by HPLC-DAD

This study provides the first chemical investigation of wild-harvested fruits of Vanilla pompona ssp. grandiflora (Lindl.) Soto-Arenas developed in their natural habitat in the Peruvian Amazon. Flowers were hand-pollinated and the resulting fruits were analysed at different developmental stages using an HPLC-DAD method validated for the quantification of glucovanillin and seven other compounds. The method showed satisfactory linearity (r² > 0.9969), precision (coefficient of variation <2%), recoveries (70–100%), limit of detection (0.008–0.212 μg/ml), and limit of quantification (0.027–0.707 μg/ml). The evaluation of crude and enzyme-hydrolyzed Soxhlet-extracted samples confirmed the leading role of glucosides in fruit development. LC–ESI-MS studies corroborated the identities of four glucosides and seven aglycones, among them vanillin (5.7/100 g), 4-hydroxybenzyl alcohol (3.6/100 g), and anisyl alcohol (7.1/100 g) were found in high concentrations. The attractive flavor/aroma profile exhibited by wild V. pompona fruits supports studies focused on the development of this species as a specialty crop.     

Determination of steroidal saponins in different organs of yam (Dioscorea pseudojaponica Yamamoto)

Yams (Dioscorea spp.) are perennial trailing rhizome plants. Steroidal saponins, furostanol and spirostanol glycosides are the marked functional compounds in yams. In this investigation, a C18 solid phase extraction method was developed for yam saponins purification. The contents of saponins in various organs of yam (Dioscorea pseudojaponica Yamamoto) were also determined. Results showed that the recoveries of yam saponins extracted by the developed method were about 99.48–100.08% when the saponins (each saponin weighed 0.20, 0.50 and 1.00 mg) passing through the C18 cartridge. The extractive method could efficiently reduce the interferences from impurities in yam saponin extracts prior to HPLC analysis. The recoveries of added saponins in different yam organs were 98.34–99.92% for tuber flesh, 95.98–98.89% for tuber cortex, 97.89–99.44% for rhizophor, 93.82–98.01% for leaf and 93.87–97.65% for vine, respectively. The yam tuber cortex had the highest amount of saponins (582.53 μg/g dw), which was higher than that existed in the tuber flesh (227.86 μg/g dw) about 2.55 times. The contents of saponins in the rhizophor, leaf and vine of yam were 29.39, 24.41 and 23.96 μg/g dw, respectively.     

Determination of γ-glutamyl-valyl-glycine in raw scallop and processed scallop products using high pressure liquid chromatography–tandem mass spectrometry

The determination of the kokumi peptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) in raw scallop and processed scallop products was carried out using high pressure liquid chromatography–tandem mass spectrometry (LC/MS/MS). The detection of γ-Glu-Val-Gly was achieved using a multiple reaction monitoring (MRM) method. The optimised condition enabled the precise determination of γ-Glu-Val-Gly. Raw scallop contained 0.08 μg/g γ-Glu-Val-Gly, and the γ-Glu-Val-Gly levels in processed scallop products, such as dried-scallop and scallop extract, were measured to be 0.64 and 0.77 μg/g, respectively. This is the first report to confirm the existence of γ-Glu-Val-Gly in foodstuff.     

Development of a simple capillary electrophoretic determination of glucosamine in nutritional supplements using in-capillary derivatisation with o-phthalaldehyde

A simple capillary electrophoretic method was developed for the determination of glucosamine using in-capillary derivatisation. Glucosamine in commercial products was extracted with purified water. The CE separation was achieved on an uncoated fused-silica capillary using a 20 mM borate buffer (pH 9.2) containing 5 mM o-phthalaldehyde (OPA) and 5 mM 3-mercaptopropionic acid (MPA) at 25 kV, followed by UV detection at 340 nm. The detector response was linear (r² > 0.999) in the concentration range 10–1000 μg/mL. The limit of detection (LOD) was 1.3 mg/g. Spiked glucosamine recoveries at 50 and 100 mg/g level were 95.1% and 104.3%, respectively. The method was applied to 16 commercial products. The concentrations of glucosamine were 109–705 mg/g, and the ratios of detected glucosamine content to the labelled value were 88.8–124%. No significant bias was observed (r² = 0.989, p < 0.01), between results obtained by the proposed CE method and an official colorimetric method (Japanese Health Food & Nutrition Food Association).     

Monitoring and risk assessment of pesticide residues in yuza fruits (Citrus junos Sieb. ex Tanaka) and yuza tea samples produced in Korea

The objective of this study was to establish an analytical method to measure pesticides used to cultivate yuza (Citrus junos Sieb. ex Tanaka) and to analyze pesticide residue levels of yuza and yuza tea samples. Risk assessments were also performed by calculating estimated daily intake (EDI) and acceptable daily intake (ADI). An excellent linear correlation was achieved with coefficient correlation values of 0.9750–0.9999. Percent recoveries were 80.4–109.9% for most pesticides with a <6.9% relative standard deviation (RSD). The limits of quantification for the method were 0.10–0.67 μg/ml. The RSD of intra-day and inter-day variability was <15.3%. Seven pesticides in yuza (n = 80) and yuza tea (n = 75) were analyzed with the optimized analytical method. Acequinocyl, spirodiclofen and carbendazim were detected in yuza samples in the concentration range of 0.07–0.15 μg/g, 0.11–1.89 μg/g, and 0.03–5.15 μg /g, respectively, whereas chlorpyrifos, prothiofos, phosalone, and deltamethrin were not detected in yuza or yuza tea. The concentrations of acequinocyl, spirodiclofen and carbendazim ranged from 0.18–1.05 μg/g, 0.13–0.29 μg/g, and 0.17–2.36 μg/g, respectively, in yuza tea samples. The percent ratios of EDI to ADI for acequinocyl, spirodiclofen, and carbendazim were 24.6%, 22.7%, and 58.5%, respectively.     

New vitamin E isomers (gamma-tocomonoenol and alpha-tocomonoenol) in seeds,roasted seeds and roasted seed oil from the Slovenian pumpkin variety ‘Slovenska golica’

The Štajerska region in north-eastern Slovenia and the Styria region in southern Austria have a long tradition of growing pumpkins (Cucurbita pepo L.) as an oil crop. GC-MS determination of the free and esterified minor compounds in oil of roasted pumpkin seeds from the Slovenian C. pepo L. variety ‘Slovenska golica’ revealed the presence of two previously unreported compounds: alpha-tocomonoenol and gamma-tocomonoenol. Using the GC-MS data, reference samples (Crude Palm Oil) and tocopherol and tocotrienol standards it was possible to assign and quantify alpha-tocomonoenol (17.6 ± 0.6 μg/g) and gamma-tocomonoenol (118.7 ± 1.0 μg/g) compounds in roasted ‘S. golica’ seed oil using HPLC. The concentrations of alpha-tocopherol and gamma-tocopherol were 77.9 ± 1.9 μg/g and 586.0 ± 4.6 μg/g, respectively. Surprisingly the gamma-tocotrienol concentration found was only 6.9 ± 0.2 μg/g. Analysis of the seeds from which the oil was pressed showed the initial gamma-tocotrienol amount was even lower (1.6 ± 0.1 and 2.2 ± 0.1 μg/g in the ground and roasted seeds, respectively) than in the roasted seed oil.     

Composition,antimicrobial, antiradical and spasmolytic activity of Ferula heuffelii Griseb. ex Heuffel (Apiaceae) essential oil

The essential oil from underground parts of Ferula heuffelii from N.E. Serbia, was analysed using GC and GC–MS. The main compounds of the essential oil were elemicin (35.4%) and myristicin (20.6%). The essential oil exhibited the best antimicrobial activity against two strains of Candida albicans (MIC = 7.0 and 13.7 μg/ml), as well as against Micrococcus luteus (MIC = 13.7 μg/ml), Staphylococcus epidermidis (MIC = 17.6 μg/ml), Bacillus subtilis (MIC = 21.1 μg/ml) and Micrococcus flavus (MIC = 28.2 μg/ml). In the DPPH radical scavenging assay, essential oil showed substantial activity with SC₅₀ = 22.43 μl/ml. The essential oil was also tested for antispasmodic activity. It inhibited spontaneous contraction of isolated rat ileum dose-dependently, and at the concentration of 86.64 μg/ml exhibited 50% of the maximum effect of atropine. After incubation with 75.00 μg/ml of essential oil, acetylcholine did not induce contractions of ileum, and at 250.00 μg/ml, the essential oil almost completely abolished the spasmodic effect of potassium chloride (80 mM).     

Determination of six lignans in Schisandra chinensis (Turcz.) Baill. Fruits and related Chinese multiherb remedies by HPLC

A simple, rapid and specific HPLC method was established for simultaneous determination of six major lignans in Schisandra chinensis and related Chinese multiherb remedies (CMRs) containing this herb. The six lignans were successfully separated on a C₁₈ column by gradient elution using acetonitrile and water as the mobile phase, and detection wavelength was set at 225 nm. The method was validated through the following performance criteria: linearity, precision, repeatability, stability, accuracy, limit of detection (LOD) and quantification (LOQ). This assay was successfully used for determination of six lignans in 10 raw herbs collected from different regions in China and five Chinese multiherb remedies. Significant variations were demonstrated in the contents of six compounds in these samples. This HPLC method established is suitable for routine quantitative analysis of S. chinensis and multiherb remedies containing this herb.     

Speciation of organic and inorganic selenium in selenium-enriched rice by graphite furnace atomic absorption spectrometry after cloud point extraction

A new method was developed for the determination of organic and inorganic selenium in selenium-enriched rice by graphite furnace atomic absorption spectrometry detection after cloud point extraction. Effective separation of organic and inorganic selenium in selenium-enriched rice was achieved by sequentially extracting with water and cyclohexane. Under the optimised conditions, the limit of detection (LOD) was 0.08 μg L⁻¹, the relative standard deviation (RSD) was 2.1% (c = 10.0 μg L⁻¹, n = 11), and the enrichment factor for selenium was 82. Recoveries of inorganic selenium in the selenium-enriched rice samples were between 90.3% and 106.0%. The proposed method was successfully applied for the determination of organic and inorganic selenium as well as total selenium in selenium-enriched rice.     

Determination of hyperoside and 2″-acetylhyperoside from Ligularia fischeri by high performance liquid chromatography

Ligularia fischeri and its main flavonoids, hyperoside and 2″-acetylhyperoside, posses antioxidant properties. This study was carried out to investigate the contents of hyperoside and 2″-acetylhyperoside in L. fischeri by using high performance liquid chromatography (HPLC). An HPLC–photodiode array (PDA) detection method was established for the simultaneous determination of hyperoside and 2″-acetylhyperoside in L. fischeri. Two flavonoids were successfully separated in less than 20 min using an YMC RP 18 column. The mobile phase was composed of water (A) and acetonitrile (B) with isocratic elution system (23% B) at a flow rate of 1 mL/min. Their calibration curves showed good linear regression (r > 0.9992) within the test ranges. The method was validated for specificity, accuracy, precision, and limits of detection. The determined two compounds were well separated with a linear range of 18–180 μg/mL. The contents of hyperoside and 2″-acetylhyperoside were 0.387 ± 0.002 and 0.526 ± 0.006 mg/g in L. fischeri, respectively.