RESUMEN

Protoporphyrins are organic compounds with cyclic structure that are synthesised by a wide variety of organisms. In humans, these compounds are detected in blood and urine, with significantly higher levels in blood. Their potential as biomarkers of anemia and other diseases is currently being investigated, as their levels change according to the biochemical processes associated with the disease. The most widely used biomarker of anemia is serum ferritin, but it is unreliable in patients with inflammatory bowel disease (IBD) because its levels can be altered by acute inflammation and/or infections. There is therefore a need to look for new markers to help diagnose anemia in IBD patients. This work develops and validates a method for the determination of three protoporphyrins in human urine: protoporphyrin IX (PPIX), protoporphyrin IX complex with Zn (ZnPPIX) and protoporphyrin IX complex with Fe (II) (FePPIX), the latter also known as heme. The aim is to evaluate their potential as biomarkers of anemic disease in patients diagnosed with IBD. The proposed analytical method is based on high performance liquid chromatography (HPLC) with dual detection based on photodiode array (PDA) and fluorescence (FD). Quantification of the analytes at very low concentrations is possible due to the efficient preconcentration provided by dispersive liquid-liquid microextraction (DLLME) and the sensitivity of the detection systems. The method was validated by evaluating linearity (25-1000 ng mL-1), matrix effect, sensitivity (limits of quantification were between 5 and 11 ng mL-1), selectivity, accuracy, carry-over, dilution integrity, stability and precision (< 12.1 %). Finally, statistical analyses applied to the sample quantification results showed these three markers, together with five clinical markers, were significantly different between anemic and non-anemic IBD patients.

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RESUMEN

The nitrosyl-heme complex is considered the pigment responsible for the development of reddish colour in cooked hams. However, the same reddish colour was observed in a nitrite-free product elaborated with polyphenols, suggesting the presence of other red pigments that can contribute to generate this colour. In this study, the protoporphyrins composition of the pigment solution obtained from nitrite and nitrite-free cooked hams was analysed using 80% (v/v) acetone/water solution for extraction. Chromatographic analysis using a combination of diode array and fluorescence detectors revealed the presence of protoporphyrin IX and Zn-protoporphyrin IX in this solution, and these protoporphyrins were subsequently identified with complete certainty by mass spectrometry. These results show how the colour of cooked hams can be developed by a mixture of different protoporphyrins and also demonstrate the absence of selectivity of acetone/water extraction for measuring the content of nitrosyl-heme in cooked hams.

RESUMEN

Nitrosamines (NAs), which are catalogued as carcinogenic compounds, may be present in meat products due to the conversion of nitrites and as result of migration from elastic rubber nettings used. A method based on ultrasonic assisted extraction coupled with dispersive liquid-liquid microextraction as sample treatment and gas chromatography-mass spectrometry as separation and detection technique was proposed for the determination of twelve NAs in cooked ham samples. The method was validated by evaluating linearity (0.5-1000 ng g-1), matrix effect, sensitivity (detection limits were between 0.15 and 1.4 ng g-1) and precision, which was below 12%. Five NAs were found in the samples with levels ranging from not quantifiable to 40 ng g-1. The effect of the elastic rubber nettings on the nitrosamine content of meat was evaluated by comparing the levels found in products made with several plastics or thread in the presence of additives.

RESUMEN

Ranitidine drug products were recently recalled because they contained carcinogenic nitrosamines (NAs), such as N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA). This episode emphasises the importance of developing analytical methods to determine NAs in this type of product. This study describes the development and validation of an analytical method for the determination of nine NAs (NDMA, N-nitrosomethylethylamine (NEMA), NDEA, N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR) N-nitrosodi-n-propylamine (NDPA) N-nitrosopiperidine (NPIP), N-nitrosodi-n-butylamine (NDBA) and N-nitrosodiphenylamine (NDPhA)) in ranitidine drug samples using a combination of microextraction and gas chromatography-mass spectrometry. The procedure involved the dissolution of 1 g of sample in 10 mL of water. For the dispersive liquid-liquid microextraction, 0.5 g of NaCl was added to this aqueous solution, followed by a mixture containing 0.5 mL methanol as dispersant and 150 µL chloroform as extractant solvent. The recovered organic phase was injected into the GC-MS system and a 20 min oven programme was applied. Quantification limits were in the 0.21-21 ng g-1 range, corresponding the lower limit to NDPhA and the higher to NDMA. Relative standard deviations lower than 12% were achieved in all cases, which indicates the adequate precision of the method. Seven pharmaceutical products containing two different amounts of ranitidine (150 and 300 mg) were analysed. None of the samples contained NEMA, NDEA, NPYR, NMOR, NDPA or NPIP, while NDMA, NDBA and NDPhA were found in three products.

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An analytical procedure is proposed for determining three cyanotoxins (microcystin RR, microcystin LR, and nodularin) and two phycotoxins (domoic and okadaic acids) in seawater and algae-based food supplements. The toxins were first isolated by a salting out liquid extraction procedure. Since the concentration expected in the samples was very low, a dispersive liquid-liquid microextraction procedure was included for preconcentration. The ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate (80 mg) was used as green extractant solvent and acetonitrile as disperser solvent (0.5 mL) for a 10 mL sample volume at pH 1.5, following the principles of green analytical chemistry. Liquid chromatography with electrospray ionization and quadrupole time of flight-mass spectrometry (LC-Q-TOF-MS) was used. The selectivity of the detection system, based on accurate mass measurements, allowed the toxins to be unequivocally identified. Mass spectra for quadrupole time of flight-mass spectrometry (Q-TOF-MS) and Q-TOF-MS/MS were recorded in the positive ion mode and quantification was based on the protonated molecule. Retention times ranged between 6.2 and 17.9 min using a mobile phase composed by a mixture of methanol and formic acid (0.1%). None of the target toxins were detected in any of the seawater samples analyzed, above their corresponding detection limits. However, microcystin LR was detected in the blue green alga sample.

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