Simultaneous quantification of riboflavin, nicotinamide and nicotinic acid in anchovies (Engraulis enrasicolus) by HPLC-MS/MS triple quadrupole Giovanni Caprioli1, Sauro Vittori1, Gianni Sagratini1, Elisabetta Torregiani1 1School of Pharmacy, University of Camerino, Camerino, Italy Summary: A new analytical method that uses high performance liquid chromatography–triple quadrupole mass spectrometry (HPLC-MS/MS) was developed for the simultaneous analysis of vitamins B2 (riboflavin) and B3 (nicotinamide and nicotinic acid) in anchovies. Keywords: riboflavin, niacin, anchovies 1 Introduction Vitamins are crucial for maintaining good health in humans; lack of a sufficient amount of them can cause serious disease [1]. Essential for the calculation of dietary intake of nutrients from food is the use of reliable, accurate and precise analytical methods for nutrients in the foods. The water-soluble vitamins act mainly as coenzymes, while the fat-soluble ones act in different and more complex ways [2]. In foodstuffs, the vitamins B2 (riboflavin) and B3 (niacin) may be present in free (riboflavin, nicotinamide and nicotinic acid) and binding forms (essentially riboflavin-5’-phosphate (FMN), riboflavin- 5’-adenosyldiphosphate (FAD), nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP)). Furthermore, they may be bound tightly but non-covalently to proteins and polysaccharides [3]. Anchovies are a good source of those two vitamins, based on the various nutrition tables available. Thus, the aim of this paper was to evaluate the content of vitamin B2 (riboflavin) and B3 (niacin, i.e. nicotinic acid plus nicotinamide) in anchovies samples provided by a company called “Dimar Sapore di Mare”. 2 Experimental Anchovies canned in olive or sunflower oil were purchased from the local supermarket, while normal anchovy samples were kindly supplied by D.I.MAR. Srl - Sapore di Mare (Corridonia, Macerata, Italy). Different extraction procedures were tested and the best one was chosen for the accurate quantification of riboflavin, nicotinic acid and nicotinamide in different types of anchovy samples. Extraction Method n°1 consisted in acidic hydrolysis: ground anchovies were hydrolysed with 30 mL of 0.1 N HC1 at 100 °C for 30 min under magnetic stirring. Method n°2 consisted in the addition of TCA 50% for protein precipitation after acidic hydrolysis. Method n°3 consisted in acidic hydrolysis plus enzymatic hydrolysis with different enzymes or mixture of them (claradiastase, papain, takadiastase and amylase). Method n°4 consisted only in the enzymatic digestion. UHPLC–MS/MS studies were performed using an Agilent 1290 Infinity series and a Triple Quadrupole 6420 from Agilent Technology (Santa Clara, CA) equipped with an ESI source operating in positive ionization mode. The separation of riboflavin, nicotinic acid and nicotinamide was achieved using a Kinetex Hilic analytical column (100 mm × 4.6 mm i.d., particle size 2.6 µm) from Phenomenex (Torrance, CA, USA). Detection was performed by electrospray ionization (ESI)-MS in the “multiple reaction monitoring” (MRM) mode. The MRM peaks areas were integrated for quantification; the most abundant product ion was used for quantification, and the rest of the products ions were used for qualification. 3 Results The analytical procedure developed is fast (2.5 minutes of chromatography run time), the method is sensitive (LOQ for all compounds is in the range 1-5 µg kg-1), linear (R2 for all compounds was higher than 0.9996), accurate and robust as it is possible to apply the method to both normal and under-oil/canned anchovies. The results obtained indicated that method 1 was the best of the four optimized extraction procedures in terms of recoveries (range 96.2 - 105.3%, with RSDs lower than 9.2%), quantitative results obtained, and rapidity, and thus it was chosen for the analysis of vitamins B2 and B3 in 23 anchovy samples (both normal and canned ones). The application to real samples indicates that the highest niacin levels were found in normal anchovies (101.21-150.92 mg kg-1), while the highest level of riboflavin was in anchovies canned in olive oil (ranging from 2.90 to 3.35 mg kg-1). 4 Conclusions A new analytical method that uses HPLC-MS/MS triple quadrupole was developed for the first time for the analysis of riboflavin, nicotinamide and nicotinic acid in anchovies. Samples were extracted using different extraction procedures, and acidic hydrolysis (method 1) was found to be the best in terms of recoveries, quantitative results and rapidity. In order to understand the dietary intake of vitamins from different types of anchovies, the method was applied to 23 samples including eleven normal anchovy samples and twelve canned ones. References 1. A. Zafra-Gomez, A. Garballo, J.C. Morales, L.E. Garcia-Ayuso. Journal of Agricultural and Food Chemistry, 54 (2006), pp. 4531-4536. 2. A. Gentili, F. Caretti, G. D’Ascenzo, S. Marchese, D. Perret, D. Di Corcia, L.M. Rocca. Rapid Communications in Mass Spectrometry, 22 (2008), pp. 2029-2043. 3. S. Ndaw, M. Bergaentzlè, D. Aoudè-Werner, C. Hasselmann. Enzymatic extraction procedure for the liquid chromatographic determination of niacin in foodstuffs. Food Chemistry, 78 (2002), pp. 129-134.
Quantification of Riboflavin, Nicotinamide and Nicotinic Acid in Anchovies (Engraulis Enrasicolus) by HPLC-MS/MS Triple Quadrupole
Giovanni Caprioli;Sauro Vittori;Gianni Sagratini;
2017-01-01
Abstract
Simultaneous quantification of riboflavin, nicotinamide and nicotinic acid in anchovies (Engraulis enrasicolus) by HPLC-MS/MS triple quadrupole Giovanni Caprioli1, Sauro Vittori1, Gianni Sagratini1, Elisabetta Torregiani1 1School of Pharmacy, University of Camerino, Camerino, Italy Summary: A new analytical method that uses high performance liquid chromatography–triple quadrupole mass spectrometry (HPLC-MS/MS) was developed for the simultaneous analysis of vitamins B2 (riboflavin) and B3 (nicotinamide and nicotinic acid) in anchovies. Keywords: riboflavin, niacin, anchovies 1 Introduction Vitamins are crucial for maintaining good health in humans; lack of a sufficient amount of them can cause serious disease [1]. Essential for the calculation of dietary intake of nutrients from food is the use of reliable, accurate and precise analytical methods for nutrients in the foods. The water-soluble vitamins act mainly as coenzymes, while the fat-soluble ones act in different and more complex ways [2]. In foodstuffs, the vitamins B2 (riboflavin) and B3 (niacin) may be present in free (riboflavin, nicotinamide and nicotinic acid) and binding forms (essentially riboflavin-5’-phosphate (FMN), riboflavin- 5’-adenosyldiphosphate (FAD), nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP)). Furthermore, they may be bound tightly but non-covalently to proteins and polysaccharides [3]. Anchovies are a good source of those two vitamins, based on the various nutrition tables available. Thus, the aim of this paper was to evaluate the content of vitamin B2 (riboflavin) and B3 (niacin, i.e. nicotinic acid plus nicotinamide) in anchovies samples provided by a company called “Dimar Sapore di Mare”. 2 Experimental Anchovies canned in olive or sunflower oil were purchased from the local supermarket, while normal anchovy samples were kindly supplied by D.I.MAR. Srl - Sapore di Mare (Corridonia, Macerata, Italy). Different extraction procedures were tested and the best one was chosen for the accurate quantification of riboflavin, nicotinic acid and nicotinamide in different types of anchovy samples. Extraction Method n°1 consisted in acidic hydrolysis: ground anchovies were hydrolysed with 30 mL of 0.1 N HC1 at 100 °C for 30 min under magnetic stirring. Method n°2 consisted in the addition of TCA 50% for protein precipitation after acidic hydrolysis. Method n°3 consisted in acidic hydrolysis plus enzymatic hydrolysis with different enzymes or mixture of them (claradiastase, papain, takadiastase and amylase). Method n°4 consisted only in the enzymatic digestion. UHPLC–MS/MS studies were performed using an Agilent 1290 Infinity series and a Triple Quadrupole 6420 from Agilent Technology (Santa Clara, CA) equipped with an ESI source operating in positive ionization mode. The separation of riboflavin, nicotinic acid and nicotinamide was achieved using a Kinetex Hilic analytical column (100 mm × 4.6 mm i.d., particle size 2.6 µm) from Phenomenex (Torrance, CA, USA). Detection was performed by electrospray ionization (ESI)-MS in the “multiple reaction monitoring” (MRM) mode. The MRM peaks areas were integrated for quantification; the most abundant product ion was used for quantification, and the rest of the products ions were used for qualification. 3 Results The analytical procedure developed is fast (2.5 minutes of chromatography run time), the method is sensitive (LOQ for all compounds is in the range 1-5 µg kg-1), linear (R2 for all compounds was higher than 0.9996), accurate and robust as it is possible to apply the method to both normal and under-oil/canned anchovies. The results obtained indicated that method 1 was the best of the four optimized extraction procedures in terms of recoveries (range 96.2 - 105.3%, with RSDs lower than 9.2%), quantitative results obtained, and rapidity, and thus it was chosen for the analysis of vitamins B2 and B3 in 23 anchovy samples (both normal and canned ones). The application to real samples indicates that the highest niacin levels were found in normal anchovies (101.21-150.92 mg kg-1), while the highest level of riboflavin was in anchovies canned in olive oil (ranging from 2.90 to 3.35 mg kg-1). 4 Conclusions A new analytical method that uses HPLC-MS/MS triple quadrupole was developed for the first time for the analysis of riboflavin, nicotinamide and nicotinic acid in anchovies. Samples were extracted using different extraction procedures, and acidic hydrolysis (method 1) was found to be the best in terms of recoveries, quantitative results and rapidity. In order to understand the dietary intake of vitamins from different types of anchovies, the method was applied to 23 samples including eleven normal anchovy samples and twelve canned ones. References 1. A. Zafra-Gomez, A. Garballo, J.C. Morales, L.E. Garcia-Ayuso. Journal of Agricultural and Food Chemistry, 54 (2006), pp. 4531-4536. 2. A. Gentili, F. Caretti, G. D’Ascenzo, S. Marchese, D. Perret, D. Di Corcia, L.M. Rocca. Rapid Communications in Mass Spectrometry, 22 (2008), pp. 2029-2043. 3. S. Ndaw, M. Bergaentzlè, D. Aoudè-Werner, C. Hasselmann. Enzymatic extraction procedure for the liquid chromatographic determination of niacin in foodstuffs. Food Chemistry, 78 (2002), pp. 129-134.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
