Ultra-high pressure LC for astaxanthin determination in shrimp by-products and active food packaging.

Nowadays, there is increasing interest in natural antioxidants from food by-products. Astaxanthin is a potent antioxidant and one of the major carotenoids in crustaceans and salmonids. An ultra-high pressure liquid chromatographic method was developed and validated for the determination of astaxanthin in shrimp by-products, and its migration from new packaging materials to food simulants was also studied. The method uses an UPLC® BEH guard-column (2.1 × 5 mm, 1.7 µm particle size) and an UPLC® BEH analytical column (2.1 × 50 mm, 1.7 µm particle size). Chromatographic separation was achieved using a programmed gradient mobile phase consisting of (A) acetonitrile-methanol (containing 0.05 m ammonium acetate)-dichloromethane (75:20:5, v/v/v) and (B) ultrapure water. This method was evaluated with respect to validation parameters such as linearity, precision, limit of detection, limit of quantification and recovery. Low-density polyethylene films were prepared with different amounts of the lipid fraction of fermented shrimp waste by extrusion, and migration was evaluated into food simulants (isooctane and ethanol 95%, v/v). Migration was not detected under the tested conditions.

HPLC method validation for measurement of sulforaphane level in broccoli by-products.

A simple and specific analytical method was developed and tested for the determination of sulforaphane in broccoli by-products. The method includes the optimization of the conversion of glucoraphanin to sulforaphane, followed by purification of extracts using solid-phase extraction and high-performance liquid chromatography (HPLC) analysis. The response surface methodology was used to find optimum conditions for the preparation and purification procedure. Chromatographic conditions for reversed-phase HPLC with UV photodiode array detection were as follows: column, Exil ODS C(18), 25 x 0.46 cm, 5 microm; column temperature, 36 degrees C; mobile phase, a 30 : 70 (v/v) mixture of acetonitrile:water; flow rate, 0.6 mL/min. The detection wavelength was UV 202 nm. Under these conditions, excellent linearity was obtained (r(2) = 1), and the overall recovery was 97.5 and 98.1% for fresh florets and lyophilized florets, respectively. The precision results showed that the relative standard deviation of the repeatability for florets fresh and lyophilized was 3.0 and 4.0%, respectively. Sulforaphane contents were determined in the edible portion of fresh broccoli, and broccoli crop remains.

High-performance liquid chromatography with fluorescence detection for quantitation of tryptophan and tyrosine in a shrimp waste protein concentrate.

A new, simple, and reproducible isocratic high-performance liquid chromatography (HPLC) method has been developed for the determination of free and total tyrosine and tryptophan in a protein concentrate. To determine total amino acids, the method involves alkaline hydrolysis of the proteins with sodium hydroxide at 120 degrees C for 4h in the absence of air. Best results were achieved with a SS Exil ODS column 5microm (25cmx0.46cm i.d.), with an eluent of methanol: 40mM sodium acetate buffer (adjusted to pH 4.5 with acetic acid; 20:80, v/v), a flow rate of 0.80mL/min at 26 degrees C, and with programmable fluorescence detection. Under optimum conditions excellent linearity was obtained, and the overall recovery was 90.5, and 95.9% for total tryptophan and tyrosine, respectively. The precision results showed that the relative standard deviation of the repeatability and reproducibility were < or =4.78 and < or =4.65, respectively. This method was used to quantify the cited analytes in the protein concentrate obtained during the lactic acid fermentation of shrimp waste.

Quantitation of glucosamine from shrimp waste using HPLC.

This work presents a high-performance liquid chromatography (HPLC) method for the quantitation of glucosamine in chitin. The method includes an acid hydrolysis of chitin. The chromatographic separation is achieved using a Hypersil ODS 5-microm column (250 x 4.6 mm) at 38 degrees C, with precolumn derivatization with 9-fluorenylmethyl-chloroformate and UV detection (lambda = 264 nm). The mobile phase is a mixture of mobile phase A [30 mM ammonium phosphate (pH 6.5) in 15:85 methanol-water (v/v)], mobile phase B [15:85 methanol-water (v/v)], and mobile phase C [90:10 acetonitrile-water (v/v)], with a flow rate of 1.2 mL/min. The HPLC method proposed showed adequate repeatability (relative standard deviation, 5.8%), accuracy (92.7% recovery), and sensitivity, with a detection limit of 2 microg/mL. The method is successfully applied to the quantitation of glucosamine for the determination of the purity of chitin from shrimp waste.

Quantification of astaxanthin in shrimp waste hydrolysate by HPLC.

In the present study, a simple and rapid reversed-phase HPLC method for the determination of astaxanthin in shrimp waste hydrolysate has been developed and validated. The analytical procedure involves the direct extraction of astaxanthin from the lipid fraction with methanol. The analytical column, SS Exil ODS, was operated at 25C. The mobile phase consisted of a mixture of water:methanol:dichloromethane:acetonitrile (4.5:28:22:45.5 v/v/v/v) at a flow rate of 1.0 mL/min. Detection and identification were performed using a photodiode array detector (lambda(detection) = 476 nm). The proposed HPLC method showed adequate linearity, repeatability and accuracy.

Analysis of free amino acids in fermented shrimp waste by high-performance liquid chromatography.

This work presents an HPLC method for the quantification of free amino acids in lyophilized protein fraction from shrimp waste hydrolysate which is obtained by acid lactic fermentation and analyzed using pre-column derivatization with 9-fluorenylmethyl-chloroformate. The amino acids were separated in a Hypersil ODS 5 microm column (250 mm x 4.6 mm) at 38 degrees C. The mobile phase was a mixture of phase A: 30 mM ammonium phosphate (pH 6.5) in 15:85 (v/v) methanol/water; phase B: 15:85 (v/v) methanol/water; and phase C: 90:10 (v/v) acetonitrile/water, with flow rate 1.2 ml/min. Fluorescence detection was used at an excitation wavelength of 270 nm and an emission wavelength of 316 nm. Method precisions for the different amino acids were between 4.4 and 7.1% (relative standard deviation, RSD); detection limits were between 23 and 72 ng/ml; and the recoveries were between 89.0 and 95.0%. The amino acid present at the highest concentration was tyrosine.

High-performance liquid chromatography method to measure alpha- and gamma-tocopherol in leaves, flowers and fresh beans from Moringa oleifera.

A high-performance liquid chromatography method for the microscale determination of alpha- and gamma-tocopherol in leaves, flowers and fresh beans from Moringa oleifera is reported. The method includes microscale saponification and extraction with n-hexane. Optimized conditions for reversed-phase HPLC with UV detection were as follows: column, 25 cm x 0.46 cm, Exil ODS 5-microm; column temperature, 25 degrees C; mobile phase, a 20:80 (v/v) mixture of methanol:acetonitrile; flow rate, 1.0 ml/min. With these conditions, method precision (relative standard deviation) was 5.6% for alpha-tocopherol and 4.9% for gamma-tocopherol. We used this method to measure alpha- and gamma-tocopherol in samples from M. oleifera as part of nutritional studies in edible plants cultivated in the Northwest México.

High-performance liquid chromatography method for the simultaneous quantification of retinol, alpha-tocopherol, and cholesterol in shrimp waste hydrolysate.

This study presents an HPLC method for the simultaneous quantification of retinol, alpha-tocopherol, and cholesterol in shrimp waste hydrolysate lipid fraction. The method includes microscale saponification and extraction with n-hexane. Liposoluble vitamins and cholesterol were quantified by HPLC with UV detection (HPLC-UV), on a 25 cm x 0.46 cm SS Exil ODS 5 microm column, mobile phase 68:28:4 (v/v/v) methanol:acetonitrile:water; flow rate 1.4 ml/min; column temperature 36 degrees C. The detection was operated using two channels of a diode-array spectrophotometer, 325 nm for retinol and 208 nm for alpha-tocopherol and cholesterol. With these conditions, the overall recovery was 95.7, 100.8, and 98.0% for retinol, alpha-tocopherol, and cholesterol, respectively. The method precision (relative standard deviation) was 1.83% for retinol, 2.32% for alpha-tocopherol, and 1.98% for cholesterol. This method was used to quantify the cited analytes in the hydrolysate obtained during lactic acid fermentation of shrimp waste. This hydrolysate may be a valuable supplement of nutrients in fish production.

An HPLC method for the quantification of sterols in edible seaweeds.

This study presents an HPLC method for the quantification of sterols in edible seaweeds. Sterols were identified by HPLC/mass spectrometry (HPLC-MS) in positive APCI mode. The samples were saponified by refluxing with 1 m ethanolic KOH, and the non-saponifiable fraction was extracted with hexane. Sterols were quantified by HPLC with UV detection (HPLC-UV), on a 15 x 0.4 cm Kromasil 100 C(18) 5 micro m column (mobile phase 30:70 v/v methanol:acetonitrile; fl ow rate 1.2 mL/min; column temperature 30 degrees C; detection wavelength 205 nm). Method repeatability for fucosterol was good (coefficient of variation 2.4%). Sterol contents were determined in canned or dried brown seaweeds (Himanthalia elongata, Undaria pinnatifida, Laminaria ochroleuca) and red seaweeds (Palmaria sp., Porphyra sp.). The predominant sterol was fucosterol in brown seaweeds (83-97% of total sterol content; 662-2320 micro g/g dry weight), and desmosterol in red seaweeds (87-93% of total sterol content; 187-337 micro g/g dry weight).

Simultaneous determination of thiamine and riboflavin in edible marine seaweeds by high-performance liquid chromatography.

This study presents a high-performance liquid chromatography (HPLC) method for simultaneous determination of thiamine and riboflavin and the results of its application to a number of edible seaweeds that are sampled in dried form (Himanthalia elongata, Laminaria ochroleuca, Undaria pinnatifida, Palmaria sp., and Porphyra sp.) or as canned food (H. elongata and Saccorhiza polyschides). Samples are prepared by acid and enzymatic hydrolysis. Optimized conditions for reversed-phase HPLC with fluorescence detection are as follow: column, Kromasil 100 C18; column temperature, 35 degrees C; mobile phase, a 72:28 (v/v) mixture of 0.005 M ammonium acetate (pH 6.7)-methanol; and flow rate, 1.35 mL/min. With these conditions, recovery is 95.52% for thiamine and 90.08% for riboflavin, and the method precision (relative standard deviation) is 2.66% for thiamine and 2.21% for riboflavin. On a dry weight basis, thiamine contents range from 0.14 microg/g in dried H. elongata to 2.02 microg/g in dried Porphyra and riboflavin contents from 0.31 microg/g in canned H. elongata to 6.15 microg/g in dried Porphyra.

Determination of the uronic acid composition of seaweed dietary fibre by HPLC.

A high-performance liquid chromatographic (HPLC) method is described for determination of the ratio of beta-d-mannuronic acid to alpha-l-guluronic acid (M/G ratio) in dietary fibre of edible seaweeds. Total dietary fibre (TDF) content was determined gravimetrically. The TDF fraction was hydrolysed with 12 m and 1 m H(2)SO(4), then neutralized with AG 4 x 4 resin. The uronic acids were separated in a Tracer Extrasil SAX 5 micro m column (25 cm x 4 mm) at 35 degrees C, with 2 mm KH(2)PO(4) containing 5% methanol as mobile phase at a fl ow rate of 1.5 mL/min. The detection wavelength was UV 210 nm. The chromatographic identifications of beta-d-mannuronic acid and alpha-l-guluronic acid were confirmed by liquid chromatography-mass spectrometry (LC-MS). The method precision was 1.4% for beta-d-mannuronic acid and 3.5% for alpha-l-guluronic acid. The method was used to determine M/G ratio in canned seaweeds (Saccorhiza polyschides and Himanthalia elongata) and in dried seaweeds (H. elongata, Laminaria ochroleuca, Undaria pinnatifida, Palmaria sp. and Porphyra sp.).

Evaluating the migration of ingredients from active packaging and development of dedicated methods: a study of two iron-based oxygen absorbers.

The behaviour of two commercial oxygen-scavenging products with respect to migration of active ingredients into foodstuffs was investigated. Migrants were identified, and by using appropriate analytical methods, migration was determined in a variety of liquid, solid or gelled food simulants and foods. Simulants were chosen to cover a range of water activities and viscosities. Foods and the gelled food simulant agar were packed with and without vacuum, and with the oxygen scavenger in various locations relative to the packed food. The main migrants, as identified by X-ray fluorescence spectrometry, infrared spectroscopy and scanning electron microscopy with energy-dispersive spectrometry were Na(+) and Cl(-) in non-acidic aqueous simulants, and Na(+), Cl(-) and Fe(2+) in 3% acetic acid. Migration into aqueous simulants exceeded the current European Union limit for total migration from plastic materials (assumed to be currently applicable to these systems) and was probably excessive by any reasonable standard. However, neither oxygen scavenger appeared to release significant quantities of migrants into solid foods when the scavenger was properly located in the package and the packing process does not favour the contents becoming wet by water released from the food.

High-performance liquid chromatographic determination of alpha-tocopherol in macroalgae.

A high-performance liquid chromatographic (HPLC) method for the microscale determination of alpha-tocopherol in macroalgae is reported. The method includes microscale saponification and extraction with n-hexane. The presence of alpha-tocopherol in macroalgae samples was confirmed by HPLC-MS. Alpha-tocopherol levels as determined in samples by HPLC with UV and fluorescence detection did not differ significantly; however, fluorescence detection has a higher sensitivity (detection limit 10.4 ng/ml, vs. 104 ng/ml with UV detection), as well as good precision (relative standard deviation 1.81%) and recovery (94.3%). Fluorescence detection is also faster. We used this method to determine the alpha-tocopherol contents of four commercial macroalgae products from northwest Spain as part of nutritional studies in dehydrated Himanthalia elongata and Laminaria ochroleuca, and also in canned Himanthalia elongata and Saccorhiza polychides.