Screening Natural Content of Water-Soluble B Vitamins in Fish: Enzymatic Extraction, HILIC Separation, and Tandem Mass Spectrometric Determination

Abstract

Despite the potential of LC with tandem MS (MS/MS) in improving sensitivity and selectivity, analytical methods are scarce for the determination of protein-bound and phosphorylated forms of B vitamins in food. This prompted us to develop a method for LC-MS/MS determination of naturally occurring nicotinamide, nicotinic acid, thiamine, pyridoxine, riboflavin, pantothenic acid, biotin, folic acid, and cyanocobalamin in fish. Baseline separation of the vitamins was achieved in a hydrophilic interaction LC condition. An ultrasonication-assisted enzymatic extraction protocol for sample preparation was optimized and validated. The time required for extraction was significantly reduced (to 4 h), while maintaining good extraction efficiency. Acetonitrile content (80%, v/v) in the prepared sample was found to be optimum for excellent peak shape and sensitivity. The dynamic linear range of the vitamins ranged from 2.5 to 500 ng/g, and the regression coefficient values were greater than 0.99. LOQ values ranged from 0.4 to 50 ng/g for the different vitamins. The spike recovery values at 50 and 100 ng/g ranged from 87.5 to 97.5%. The intra- and interday precision values were satisfactory. Accuracy of the developed method was determined by analysis of a Certified Reference Material. The method could also be used for unambiguous determination of the natural content of the target vitamins in fish.

Validated B Vitamin Quantification from Lentils by Selected Reaction Monitoring Mass Spectrometry

“…This mainly due to the gradual release of B3 (nicotinic acid and nicotinamide) within the first four hours incubation (increased 17% to 54% comparing to those from 1 h incubation), and its significant loss during overnight incubation (reduced 69% to 98% comparing to those from 4 h incubation, Figure 1). Similar observation was reported by Chatterjee et al (2017), with a 4 h extraction approach resulting in a higher level of B3 (nicotinic acid and nicotinamide) in fish comparing to 18 h incubation. Note that since biotin (B8) or cyanocobalamin (B12) were not detected in lentil seeds, for samples containing these two B vitamins, the extraction condition may need to be optimized accordingly.…”

Development of a LC-MS/MS method using stable isotope dilution for the quantification of individual B6 vitamers in fruits, vegetables, and cereals

“…Recoveries described in various foods [ 24 , 26 ], e.g., carrot [ 32 ], anchovy [ 32 ], garlic [ 32 ], chinoa [ 32 ], kiwi [ 43 ], tomato pulp [ 43 ], fish [ 66 ], cooked sausages [ 28 ], cow milk [ 23 , 55 , 68 ], Italian pasta [ 44 ], wheat [ 29 ], maize flour [ 43 ], and whole wheat flour [ 27 ], ranged between 15 and 118% depending on the matrix, extraction method, detection apparatus, and vitamer. Important factors when assessing the recoveries of B 6 molecules refer to the extraction procedure and sample workup, since incomplete extraction or dephosphorylation/deglycosylation by enzymes has a crucial impact on the method.…”

“…Inter- and intra-day precisions for foodstuff reported in the literature vary between 2 and 12 and between 1 and 5% depending on the matrix, vitamer, and method, respectively [ 23 , 24 , 26 , 28 , 43 , 58 , 66 , 68 ]. Hereby, Gentili et al reported inter-day precisions < 12% for B 6 vitamers in maize flour, while Kall et al noted an internal reproducibility of 5.6% for PN in Graham flour [ 26 ].…”

“…LODs and LOQs of B 6 were reported for various food samples [26], e.g., Italian pasta [44], corn steep liquor [65], sea food [24,66], cow milk [23], maize flour [43], green or golden kiwis [43], tomato pulp [43], cooked sausage [28], and vary between 0.000016 and 0.043 mg/kg (LOD) and between 0.000033 and 0.129 mg/kg (LOQ) depending on the vitamer and matrix. Data specifically regarding PNG are scarce in the literature because the vitamer is commonly determined as PN after enzymatic treatment.…”

Determination of selected water-soluble vitamins (thiamine, riboflavin, nicotinamide and pyridoxine) from a food matrix using hydrophilic interaction liquid chromatography coupled with mass spectroscopy

“…However, as highlighted by Fatima et al (2019) [36], a very limited number of validated methods utilising HILIC separation coupled to MS detection have been developed to analyse water soluble vitamins in complex food matrices. A study by Chatterjee et al (2017) [37] developed a LC-MS/MS method targeting nine water-soluble vitamins in fish using HILIC separation for increased sensitivity. The authors optimised an enzymatic hydrolysis method to subsequently extract B vitamins; including nicotinamide, nicotinic acid, pyridoxine, pantothenic acid, biotin, thiamine, riboflavin and cyanocobalamin from fish flesh.…”