Analysis of vitamins by liquid chromatography

CHAPTER

Analysis of vitamins by liquid chromatography

19

Alessandra Gentili, Fulvia Caretti Sapienza University, Rome, Italy

­C HAPTER OUTLINE 19.1 Introduction..................................................................................................... 571 19.2  Liquid Chromatographic Determination of Water-Soluble Vitamins...................... 572 19.2.1  Vitamin B1................................................................................. 574 19.2.2  Vitamin B2................................................................................. 576 19.2.3  Vitamin B3................................................................................. 577 19.2.4  Vitamin B5................................................................................. 578 19.2.5  Vitamin B6................................................................................. 578 19.2.6  Vitamin B8................................................................................. 579 19.2.7  Vitamin B9................................................................................. 580 19.2.8  Vitamin B12................................................................................ 581 19.2.9  Vitamin C................................................................................... 582 19.3  Liquid Chromatographic Determination of Fat-Soluble Vitamins........................... 583 19.3.1  Vitamin A................................................................................... 585 19.3.2  Vitamin D................................................................................... 586 19.3.3  Vitamin E................................................................................... 589 19.3.4  Vitamin K................................................................................... 590 19.4  Multivitamin Methods....................................................................................... 591 References............................................................................................................... 603

19.1 ­INTRODUCTION Vitamins are essential micronutrients, varying widely in chemical structure, ­biological activity, and physicochemical properties [1]. Owing to this heterogeneity, the classification as water-soluble (B-complex, C) and fat-soluble (A, D, E, K) is based on their solubility characteristics. On the whole, 13 groups are recognized in human nutrition (the B-complex groups consisting of vitamins B1, B2, B3, B5, B6, B8, B9, and B12) and each of them is composed of several biologically active forms, known as vitamers, which differ in structure, biopotency, and stability. Liquid Chromatography. http://dx.doi.org/10.1016/B978-0-12-805392-8.00019-0 © 2017 Elsevier Inc. All rights reserved.

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CHAPTER 19  Analysis of vitamins by liquid chromatography

Due to their relevance in the human physiology, the requirement of accurate data on forms and concentrations of vitamins naturally occurring in foods has become more stringent in recent years. Likewise, the determination of the forms added to fortified foods and supplements needs reliable analytical procedures. Although liquid chromatography (LC) is the ideal technique for vitamin quantitative analysis, many of the current international methods are still based on microbiological assays [1–3]; moreover, some of them are outdated, time consuming, expensive, and characterized by a high measurement uncertainty. On the contrary, the scientific literature has continuously presented new LC-based procedures suitable for individual and simultaneous vitamin analysis. At the present moment, the European Committee for Standardization (CEN) and the Association of Official Analytical Chemists (AOAC) International consider LC as the first choice to determine vitamins B1, B2, B6, C [1,2] and the fat-soluble vitamins [1,3]. For the other watersoluble vitamins, the published LC methods have to be tested collaboratively before they can be applied as official methods. Before performing an LC analysis, it is advisable to adopt some preventive measures to restrain losses due to instability problems. The most important factors that cause inactivation are light, air, temperature, pH, trace metals, and ionic strength. Because the majority of the vitamins are photosensitive, the use of low actinic amber glassware and subdued light is recommended for the whole duration of the analysis. Another precaution that cannot be disregarded is the addition of a proper antioxidant to the solvents employed for the preparation of standard solutions and for the extraction. Furthermore, when vitamins have to be dosed in food and biological matrices, there are additional problems to be tackled: (a) (inter- and intragroup) chemical heterogeneity; (b) different and/or low concentrations in the real samples; (c) matrix complexity; and (d) interactions with other constituents, such as polysaccharides (food), proteins, and lipids (food and biological samples). To date, the most used approach consists in analyzing each vitamin individually, by hydrolyzing all bound forms (acidic, alkaline, or enzymatic digestion) and/or by converting the several vitamers into the most stable form (see B9 and B12 determination as examples). In the latter case, it is possible to increase the concentration of the single-target homolog in the final extract, to simplify the analysis and reduce expenditure for the purchase of standards.

19.2 ­LIQUID CHROMATOGRAPHIC DETERMINATION OF WATER-SOLUBLE VITAMINS The choice of the LC mode for the analysis of water-soluble vitamins depends on the extraction procedure employed and the vitamin form to be quantified (Fig.  19.1). The most popular LC modes are normal-phase (NP), reversedphase (RP), ion-pair RP, ion-suppression RP, and ion-exchange chromatography. Recently, hydrophilic interaction liquid chromatography (HILIC) has been evaluated as an alternative to RPLC [4]. In practice, it can be considered a variation of NPLC with a water-rich stationary phase and a semiaqueous mobile phase:

19.2 ­ Liquid chromatographic determination of water-soluble vitamins

typically, 40%–97% acetonitrile in water. The water fraction (~3%–60%) forms a liquid layer on the stationary phase that facilitates partitioning of analytes from the high organic mobile phase to the hydrophilic stationary phase. Advantages include improved peak shape and increased retention for polar compounds, such as water-soluble vitamins. Water-soluble vitamins Vitamin B1 NH2

CH3

⊕ N

N

S

N

H3C

R = -H thiamin R = -PO(OH)2 thiamin monophosphate R = -PO(OH)-O-PO(OH)2 thiamin dishosphate R = -PO(OH)-O-PO(OH)-O-PO(OH)2 thiamin triphosphate

OR

Vitamin B2 O H3C

N

H3C

N

Riboflavin

R = -H NH

N

R = -PO(OH)2 Riboflavin-5¢-phosphate (FMN, flavin mononucleotide)

O

CH2

HO

NH2 N

HO

OH

R

OR

O O P O P O CH2 N OH OH O

OH OH

N N Riboflavin-5¢-adensoyldiphosphate (FAD, flavin adenine dinucleotide)

Vitamin B3 O

O OH

NH2

N

N Nicotinamide

Nicotinic acid Vitamin B5 OH

CH3

HOOCJCH2:CH2:NHJCOJ CHJCJCH2:OH Pantothenic acid

CH3 NH2 CH3

O

N

O

HSJCH2JCH2JNH:CO:CH2:CH2JNH:COJ CHJCJCH2:OJ PJOJPJOJ CH2 Coenzyme A (CoA)

OH CH3

OH

OH

O

N

N N

O OH O P OH OH CH3

O

HSJCH2JCH2JNH:CO:CH2:CH2JNH:COJ CHJCJCH2:OJ PJOJserineJprotein

(A)

Acyl carrier protein (ACP)

OH CH3

OH

FIG. 19.1 Names and structures of the water-soluble vitamins naturally occurring in foods. (A) Vitamins B1, B2, B3, and B5; (Continued)

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CHAPTER 19  Analysis of vitamins by liquid chromatography

Water-soluble vitamins Vitamin B6 CHO

CH2OH HO

CH2OH

HO

H3C

N

H3C

Pyridoxine

CH2NH2 CH2OR

CH2OR

HO

N

H3C

N

R = -H, pyrideoxal

R = -H, pyridoxamine

R = -PO(OH)2, pyridoxal 5¢-phoshate

R = -PO(OH)2, pyridoxamine 5¢-phoshate

Vitamin B8 O NH

HN

D-biotin

CH2(CH2)3COOH

S

Vitamin B9

OH 4

N

N3 H2N

5

1

2

8

N

N

7

C

NH

NH CH CH2

H

5

8

N H

7

NHR H H

7,8-Dihydrofolic acid (DHF)

N 5

CHO 6

8

N H

n Polyglutamates (n = 1–7 glutamate residues)

COOR

H 6

7

H NHR H H

5,6,7,8-Tertrahydrofolic acid (THF)

COOH NH CH CH2 CH2 COOH

COOH

CH2

R = -H folic acid (pteroylmonoglutamic acid)

N

R=

COOH

O 6

CO NH CH CH2 CH2

N 5

6

8

N H

7

CH3 H NHR H H

5-Formyl-THF

N 5

6

8

N H

7

H NHR H H

5-Methyl-THF

(B) FIG. 19.1, CONT'D (B) vitamins B6, B8, and B9;

19.2.1 ­ VITAMIN B1 Vitamin B1 [1] exists in nature both in free (thiamin) and esterified form (thiamin monophosphate, diphosphate, and triphosphate), while thiamin hydrochloride is used as a supplement [5]. To evaluate the total content of vitamin B1 in food, extraction with acid hydrolysis (0.1 M HCl in a water bath at 100°C or in an autoclave at 121°C) followed by enzymatic digestion (diastases possessing a phosphatase activity) is usually required [1,2,6,7]. The acid treatment frees protein-bound forms and converts starch into soluble sugars. The enzymatic treatment may require several hours of incubation (on average 3 h) for complete dephosphorylation of the thiamin esters. A recent procedure, based on dispersive liquid–liquid

19.2 ­ Liquid chromatographic determination of water-soluble vitamins

Water-soluble vitamins Vitamin B12 CH2OH O H

O

H

H

H

O

OH

P O O

H 3C

N

CH3

N

CH3

R group Cyanocobalamin

-CN

Hydroxocobalamin

-OH

Methylcobalamin

-CH3

CH CH2 NH

Coenzyme B12

CO

-H2C

OH OH

O

N

CH2 CH2

CH3

H3C H2NOCJH2C

N

CH2CH2CONH2

N NH2

CH3 N

N

CH3

N Co

H 3C H3C CH2

N

N CH2CH2CONH2 CH3

CONH2

CH3 CH2CONH2 R CH2CH2CONH2 Vitamin C

CH2OH

CH2OH

H

H C OH O HO

(C)

C OH O

O OH

L-ascorbic

acid

O

O

O

L-dehydroascorbic

acid

FIG. 19.1, CONT'D (C) B12, C.

microextraction (DLLME), has been proposed for the extraction of thiamin from different liquid and solid foods [8]. DLLME is a miniaturized liquid-liquid extraction (LLE) technique, which makes use of a ternary solvent system: sample aqueous phase, disperser solvent, and extraction solvent. Typically for the extraction of thiamin the mixture of organic solvents (acetonitrile as dispersing solvent and tetrachloroethane as extraction solvent) is rapidly injected into the aqueous sample (beer, fermented milk, etc.), with formation of minute droplets dispersed throughout the aqueous phase. After centrifugation, the settled solvent droplets are recovered and the extract is analyzed by LC with fluorescence detection. For solid foods, acid hydrolysis with trichloroacetic acid was required before extraction by DLLME [9].

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CHAPTER 19  Analysis of vitamins by liquid chromatography

RP and ion-pair RP chromatography are the most common forms of LC used for the determination of free thiamin [1,6,7,10–13]. Highly deactivated columns restrain peak broadening and tailing caused by interaction of vitamin-B1 basic sites with silanol groups on the stationary phase. Poor retention on RP columns results from the low molecular weight and polarity of vitamin B1; mobile phases containing a high percentage of water and a suitable ion-pair agent (for example alkyl sulfonates) are useful to improve peak shape and to increase retention. HILIC is also effective and, in addition, affords an improved separation of thiamin from polar interferences coextracted from the matrix [14]. Owing to its low molar absorptivity, the use of UV detection is mainly indicated for the analysis of fortified foods with high concentrations of thiamin [1,10]. Low content of endogenous vitamin and high quantities of interfering substances in an extract require a more sensitive and selective detector [11–13]. Fluorescence detection can be used provided the thiamin is oxidized to thiochrome by pre- or postcolumn reaction with alkaline hexacyanoferrate(III). Precolumn derivatization is more widely used but postcolumn methods are more convenient for routine analysis and to eliminate the problems of reducing sugars, produced during acid hydrolysis and competing with thiamin for the oxidizing agent [13]. LC methods for thiamin determination in foodstuffs and other matrices have been reviewed by Lynch and Young [15].

19.2.2 ­VITAMIN B2 Vitamin B2 [1] is a generic term for a group of compounds characterized by equal biological activity: riboflavin, riboflavin-5′-phosphate (FMN, flavin mononucleotide), and riboflavin-5′-adenosyldisphosphate (FAD, flavin adenine dinucleotide). In animal tissues, FMN and FAD are coenzymes bound tightly but not covalently to the corresponding apoenzymes. Forms for food fortification are FMN and riboflavin hydrochloride [5]. An extraction protocol, analogous to that described for vitamin B1, permits all flavins (endogenous and supplemented) to be determined [1,7,11,13]: acidic hydrolysis promotes the release of the protein-bound forms and converts FAD to FMN; the succeeding enzymatic digestion (with takadiastase, amylase, acid phosphatase, claradiastase) is used to dephosphorylate FMN and to hydrolyze starch. Recently, solid-phase extraction (SPE) with a molecularly imprinted polymer (MIP) as sorbent was used to extract riboflavin from milk and infant formula [16]. MIPs are materials obtained for polymerization of functional and cross-linking monomers around a template molecule; subsequently, the template is removed, leaving a cavity complementary to the target compound [17]. A MIP for specific sorption of riboflavin was developed using 2,6-bis(acrylamide)pyridine as functional monomer (hydrogen-bond interactions with the riboflavin imide group), pentaerythritol triacrylate as cross-linking agent, and riboflavin tetraacetate as template. After loading 1 mL of milk onto the MIP cartridge, matrix interferences were removed by rinsing with 2 mL of ultra-pure water and riboflavin eluted with 3 mL of 1% (v/v) acetic acid in methanol [16].

19.2 ­ Liquid chromatographic determination of water-soluble vitamins

RP and ion-pair RP chromatography on C18 stationary phases with UV-vis [18,19] or fluorescence detection [7,11,13] are the LC methods most frequently employed; occasionally separations on C8 [20] and amide-C16 columns [21] have been used. Free flavins in aqueous solution exhibit an intense yellowish-green fluorescence (450 nm excitation/522 nm emission) due to their 3-imino group, whereas protein-bound forms do not fluoresce, since their interaction is established through this functional group [1]. The UV-vis spectrum of riboflavin shows four bands centered at 223, 266, 373, and 445 nm [1]. The use of a 254-nm fixed-wavelength absorbance detector is common [18], but the detection at 446 nm is less susceptible to interferences [19].

19.2.3 ­ VITAMIN B3 Two forms of vitamin B3, also known as niacin, are found in food [1,2]: nicotinic acid and nicotinamide. In living tissues, nicotinamide is a component of the coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP); in meat, it is found in the free form because of postmortem hydrolysis of NAD [1]. Nicotinamide is also used for food fortification [5]. Nicotinic acid is the prevalent vitamer in mature cereal grains; nevertheless, it is unavailable due to conjugation with polysaccharides (niacytin) and polypeptides (niacinogen) [1]. The determination of “total” (free plus bound) or “free” (bioavailable) niacin is based on well-established extraction procedures [1,2,22]: total niacin is released from the food matrix by autoclaving at 121°C with base [23] or 1–2.5 N mineral acid [24]; free niacin is isolated by boiling with 0.1 N mineral acid for 1 h [25]; or incubating with NAD glycohydrolase (NADase) at 37°C for 18 h [26]. SPE can be used for the clean-up of a digest from a complex matrix [27,28]. For example, MI-SPE was used for the isolation of nicotinamide from an alkaline digest of pork liver. The MIP was synthesized from methacrylic acid monomer and ethylene glycol dimethacrylate cross-linker with nicotinamide as a template. This MIP allowed a recovery of 87% [27]. Most studies have used absorbance detection at 254–264 nm for both B3 vitamers [23–25,29]. Their absorptivity is affected by pH: in an acidic solution, it is higher with a λmax almost unchanged at 261 nm [1]. The UV detection is convenient but neither sensitive nor selective and clean-up is generally required to remove interfering compounds. To improve detection limits and selectivity without recourse to purification of hydrolysates, some researchers [26,30,31] proposed a LC method based on the UV irradiation of the column eluent in the presence of H2O2 and Cu2 + to obtain fluorescent compounds (280 nm excitation/380 nm emission). When the extraction procedure is applied to the determination of total niacin, nicotinic acid is the only B3 vitamer to be monitored by LC because all nicotinamide is converted into the acid form. Anion exchange chromatography [24] was applied less frequently than RP chromatography using ion-suppression and ion-pairing conditions [23,25], while a two-dimensional chromatography system, composed by C18 and anion-exchange columns, was assembled to increase the selectivity of the UV

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CHAPTER 19  Analysis of vitamins by liquid chromatography

detection at 254 nm [32]. By using electrospray (ES) ionization, interference problems similar to those found with other LC determinations of niacin persisted in the selected ion recording mode [28], while they were solved by the additional selectivity of the Multiple Reaction Monitoring (MRM) acquisition mode [33].

19.2.4 ­VITAMIN B5 Vitamin B5 occurs in three biologically active forms in foods [1]: pantothenic acid, coenzyme A (CoA), and acyl carrier protein (ACP). Calcium or sodium pantothenate is the form generally used as supplements in infant formula [5]. The total quantification of vitamin B5 requires the release of pantothenic acid from CoA and ACP. Since it consists of pantoic acid linked through an amide linkage to β-alanine, chemical hydrolysis cannot be used. The only alternative to free pantothenic acid from CoA is digestion with a number of enzymes (pepsin, alkaline phosphatase, pantetheinase); nevertheless, this treatment is unable to release the vitamin from ACP [34,35]. For the extraction of free pantothenic acid from milk and calcium pantothenate from infant formula, an acid deproteination is often used, followed by centrifugation and filtration [36,37]. Ion-suppression RP (trifluoroacetic acid, formic acid, phosphate buffer) on C18 [34–36] and C8 columns [37] is the commonly used chromatographic mode. The poor selectivity and sensitivity of the UV detector (a very weak absorbance at 204 nm due to the carbonyl group) makes LC-UV unsuitable for the determination of low vitamin B5 concentrations in nonformulated foods. Some researchers have overcome these problems using multiwavelength UV detection (at 200, 205, and 240 nm) [37], fluorimetric detection (postcolumn derivatization of β-alanine with o-­phthaldialdehyde in the presence of 2-mercaptoethanol) [34], and mass spectrometry (MS) with ES ionization [35,38,39]. The latter method provides a limit of quantitation (LOQ) adequate for the determination of pantothenic acid at greater than 0.024 mg/100 mg, such as those occurring in starch-containing foods [35]. Also, fluorometric detection is suitable for the determination of both free and total pantothenic acid in foodstuff, but the approach is probably too complex for routine analyses [34].

19.2.5 ­VITAMIN B6 Six vitamers of vitamin B6, with equivalent biopotency, are found in nature [1,22]: pyridoxine or pyridoxol, pyridoxal, pyridoxamine, and their 5′-phosphate esters; acid pyridoxic and pyridoxine-glucoside are inactive forms occurring in plant tissues. Regulation 1925/2006/EC cites pyridoxine hydrochloride, pyridoxine 5­ ′-phosphate, and pyridoxine dipalmitate as the forms used to enrich foods [5]. The preferred technique for vitamin B6 assay is LC since its selectivity permits the simultaneous separation and quantitation of the homologs of vitamin B6. Different extraction protocols can be applied before LC analysis to estimate the ­total or bioavailable vitamin B6 [1,2,22]. For routine analysis, hydrolysis of the conjugated forms is used; in this way, the chromatography is limited to pyridoxine, pyridoxamine, and

19.2 ­ Liquid chromatographic determination of water-soluble vitamins

pyridoxal, and the quantitation results are comparable to those obtained by microbiological assay. Traditional methods using acid and high temperature (0.1 N HCl, 121°C) denature proteins, disintegrate the food matrix, and hydrolyze phosphorylated and glycosilated forms completely [22,40]; the inconvenience may be an overestimation of bioavailable vitamin. A selective extraction at room temperature with a deproteinizing agent (sulfosalicylic acid, trichloroacetic acid, metaphosphoric acid, and perchloric acid) frees pyridoxal by hydrolysis of Schiff bases formed with food proteins, preserves all vitamers (free, phosphorylated, and glycosylated), and allows their individual quantification; in this case, the chromatographic separation of all homologs is more complex [41–43]. The two most common approaches are based on a combination of chemical hydrolysis [44,45] or deproteinization [46,47] with enzymatic digestion. The latter is usually performed with acid phosphatase [46] or takadiastase [44]; β-glucosidase is indispensable to determine the bioavailable vitamin B6 in plant foods [45]. Ndaw et al. [40] tested a mixture of enzymes (α-amylase, papain, acid phosphatase) to release, in a single step, the bound forms of vitamins B1, B2, and B6; the acid hydrolysis proved to be superfluous, due to the presence of a protease. Recently, exciting results were obtained using a MIP specific for pyridoxine. One of the main challenges in the MIP technology is the design of sorbents for watersoluble compounds, that is for compounds that are insoluble in nonpolar solvents used for the MIP synthesis. An ion-pair complex between the pyridoxine ion and the dodecyl sulfate ion as template molecule was the solution: this ion-pair is soluble in chloroform, in which the polymerization is carried out (acrylic acid is the monomer). The MIP obtained has a high affinity for vitamin B6 and is a promising material for selective clean-up of complex matrices [48]. Usually, the individual and simultaneous separation of free [40,44,46,47] and conjugated B6 [41–43] vitamers is carried out by means of RP chromatography on C18 columns with acidic mobile phases. These methods use the native fluorescence of B6 vitamers [1,40–47], increasing the weak intensity of phosphate esters at low pH by means of postcolumn derivatization with sodium hydrogensulfite.

19.2.6 ­ VITAMIN B8 The only biologically active form of vitamin B8 is d-(+)-biotin, a unique stereoisomer found in nature among the eight theoretically possible isomers [1,49]. In animal and plant tissues, most biotin is covalently bound to a lysine residue, occurring as free amino acid (d-biocytin) or belonging to biotin-dependent enzymes through an amide attachment. Biotin is also the form used for food enrichment [5]. Extraction of total biotin is obtained by acidic hydrolysis (2–3 M HCl at 100°C or 1–3 M H2SO4 by autoclaving at 121°C), which breaks protein bonds and totally converts d-biocytin into d-biotin [50,51]. Possible losses of vitamin depend on both the acid concentration and the duration of autoclaving. Enzymatic digestion with papain for 18 h leaves biocytin intact and allows the determination of available biotin (biotin plus biocytin); takadiastase is added for starchy foods, such as cereals [51].

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CHAPTER 19  Analysis of vitamins by liquid chromatography

Owing to the low natural concentration and the absence of a strong chromophore, the RPLC determination of vitamin B8 in nonsupplemented foods is difficult [1,2]. Most methods [50,51] use avidin (the egg-white protein) labeled with a fluorescent marker (fluorescein 5-isothiocyanate) as a postcolumn derivatizing agent; its fluorescence is enhanced on binding to biotin and biocytin, its specific ligands. Avidin, after a brief digestion with trypsin, was also used to prepare an affinity column for the determination of biotin in several foods (natto, beans, nuts, eggs, etc.). After acid hydrolysis, biotin was derivatized with 9-anthryldiazomethane forming a fluorescent ester, which affording a detection limit of 70 fmol [52]. An LC-tandem MS method [51], using an ESI source in the positive ion mode and biotin-d6 as internal standard, was able to determine biotin in foods at low concentrations after hydrolysis with sulfuric acid and enzymatic digestion with papain. The whole procedure was reliable and faster than the microbiological assay.

19.2.7 ­VITAMIN B9 Folate and folacin [1] are generic terms referring to a group of compounds with vitamin B9 activity, including folic acid (pteroylglutamic acid), dihydrofolic acid, tetrahydrofolic acid (H4 folic acid), 5-formyl-H4 folic acid, and 5-methyl-H4 folic acid. Folic acid is not considered a natural physiological form and is the chemical form used as a food supplement [5]. All folates exist in nature at low levels and predominantly as polyglutamates containing no more than seven glutamate residues with a γ-peptide linkage. Even though the theoretical number of folates approaches 150, so far only 50 of these have been observed in nature. Multiplicity, instability, and low concentrations of folates in animal and plant tissues have constituted an obstacle to the development of LC methods for their determination [1,2,53]. For the same reasons, sample preparation and purification are key steps carried out according to a well-established protocol [2,54]. During extraction, a number of precautions should be taken to avoid the pH-dependent interconversion of some species, oxidative losses (adding antioxidants, such as ascorbate and 2-mercaptoethanol), and thermal degradation [55]. Folates are released from the food matrix by autoclaving the food sample in a buffered aqueous medium: particles are broken up, starch is gelatinized, and folate-binding proteins are denatured as well as enzymes catalyzing degradation or interconversion of folates. The autoclaved sample is submitted to a tri-enzyme treatment [54]. The first digestion is performed with protease to free the protein-bound folates definitively. The second uses α-amilase to release the starch-bound folates. Finally, the third treatment, carried out with the folic acid conjugase, deconjugates the polyglutamates to the corresponding monoglutamates. A clean-up step on SPE cartridges is often employed to remove interfering compounds co-extracted with folates from the real matrix [56,57]. The most used stationary phases include strong anion-exchange (SAX) materials, phenyl-bonded silica, and affinity chromatographic sorbents with immobilized folate-binding protein. Both provide high recoveries, but only the affinity columns purify and concentrate the extracts efficiently.

19.2 ­ Liquid chromatographic determination of water-soluble vitamins

LC with UV detection (at 280 or 290 nm) is used for the analysis of foods with low concentrations of folates [58,59], after tri-enzyme digestion and clean-up on affinity sorbents to concentrate the extracts about 10-fold [58]. Nevertheless, fluorimetric detection is more efficient for determining naturally occurring folates [60] as well as ESI-MS [57,61] that, combined with isotope dilution, allows accurate quantification [62–65]. Ion-suppression RP chromatography is the most suitable mode for coupling with both detectors [60]. In fact, folates show native fluorescence (288 nm ­excitation/353–356 nm emission) that is enhanced in an acidic medium (mobile phase at pH 2.3 using phosphate buffer) for the reduced forms but not for folic acid; the solution adopted for the latter is to produce a fluorescent pterin fragment through postcolumn oxidative cleavage [66]. Acidic mobile phases support ES ionization of folates, but the phosphate buffer has to be substituted by the more volatile formic acid [62,67–69]. Ion-pair RP chromatography is carried out at neutral or basic pH, but it requires acidification of the column eluent to make fluorescence detection possible [70].

19.2.8 ­ VITAMIN B12 Vitamin B12 [1] is a term that generically describes a group of cobalt-containing organic compounds with antipernicious anemia activity, known as cobalamins. The major forms occurring in foods of animal origin include 5­ ′-deoxyadenosylcobalamin (coenzyme B12) and methylcobalamin, two coenzymes covalently bound to their apoenzymes; hydroxocobalamin is their photooxidation product. The general extractive protocol [71], applied for determining total vitamin B12, provides for the protein-bound vitamers to be freed and converted into a single and stable form, for example cyanocobalamin. To this end, a food sample is dissolved in a buffered solution (pH 4) containing sodium cyanide (at 100°C for 35 min) and, then, is digested with pepsin (at 37°C for 3 h) [72]. The determination of vitamin B12 by LC-UV is difficult to perform in nonsupplemented foodstuffs because of the very low concentrations of the vitamin and the poor sensitivity and selectivity of the detection system. Despite this, the concentration of the digest, obtained with the conventional protocol, on an immunoaffinity column with detection at 361 nm allowed the determination of total vitamin B12 in nonfortified milk-based products [73,74]. Liebiedzińska et al. [10] proposed α-amilase and papain for digesting salmon samples after autoclaving at 121°C in the presence of cyanide; cyanocobalamin was then monitored by an electrochemical detector. Fluorescence detection [75] offers high sensitivity and can be used after chemical or enzymatic hydrolysis to detect α-ribazole, a characteristic fluorescent fragment of vitamin B12. Nevertheless, the latter may occur in foods as a vitamin B12 metabolite; therefore, it is essential to extensively purify extracts before hydrolysis. LC-MS using ES ionization was successfully applied to the analysis of vitamin B12 in some fortified foods [76] and cultivated mushrooms [77]. This technique is also valid for cobalamin speciation in food and biological samples since the high detection sensitivity and selectivity permit omitting the derivatization step [78–80]. Some of the most recent methods dealing with this issue have proposed extraction procedures able to preserve all natural forms of vitamin B12. These protocols are simple and rapid so as to limit cobalamin photodegradation and to avoid artifacts.

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CHAPTER 19  Analysis of vitamins by liquid chromatography

Szterk et al. [78] developed an extraction procedure from beef meat and liver, based on the thermal protein denaturation in a weakly acidic medium, followed by LLE and subsequent SPE clean-up on OASIS HLB cartridges. Major forms of B12 found in meat were 5′-deoxyadenosylcobalamin and, in smaller amounts, hydroxycobalamin. Pérez-Fernández et al. [80] proposed a fast extraction based on (i) dilution/protein precipitation of the milk sample with 50 mM sodium acetate buffer (pH 4.0), (ii) centrifugation, (iii) filtration of the supernatant; (iv) SPE clean-up combining two types of sorbents in the same cartridge: two disks of buckypaper (BP) separated by a Teflon frit from OASIS HLB (500 mg). BP is a felt composed of entangled unoriented oxidized multiwalled carbon nanotubes (MW-CNTs); the good sorbent properties, the high porosity and surface area are characteristics that make BP particularly attractive as a sorbent for SPE. The LC-MRM analysis of the final extract determined methylcobalamin as the major B12 form in cow's milk, hydroxocobalamin as a minor form and cyanocobalamin as an occasional form. Viñas et al. [81] analyzed the juice of homogenized seafood after dilution, filtration, and clean-up on a mini-column filled with carbon nanotubes; probably due to the poor sensitivity of the UV detector, only cyanocobalamin was found in the samples.

19.2.9 ­VITAMIN C l-Ascorbic acid (AA) and l-dehydroascorbic acid (DHAA) are the two main homologs of vitamin C occurring in nature [1]. In food analysis, the measurement of the vitamin C total content should account for both forms, since DHAA is readily reduced to AA in the animal body. d-Isoascorbic acid (d-IAA), also known as erythorbic acid or d-araboascorbic acid, has analogous reductive properties but only 5% of the antiscorbutic activity of l-AA; this epimer is a by-product of vitamin C, and is approved within the European Community as an antioxidant additive [82]. The capability of LC to distinguish the two ascorbic acid isomers and their primary oxidation products is very useful for analyzing processed foods. Forms used for supplementation are AA, sodium-, calcium-, or potassium-l-ascorbate and l-ascorbyl 6-palmitate [5]. AA is susceptible to both chemical and enzymatic oxidation [1]. Chemical oxidation is catalyzed by minerals, such as Cu(II) and Fe(III), in the presence of oxygen at a pH-dependent rate, while enzymatic oxidation is catalyzed by the ascorbate oxidase occurring in plant tissues. Light and heat are other factors that promote its degradation. For these reasons, the extraction procedure should be designed to stabilize the vitamin; for example, metaphosphoric acid [83–87] denatures proteins, inactivates enzymes, provides a medium below pH 4 (degradation rate is minimal at pH 2), and inhibits metal catalysis, whereas EDTA [88,89] chelates metals efficiently. Recently, microextraction by packed sorbent (MEPS) was used for isolating AA from beverages [90]. MEPS is a miniaturized version of classical SPE; the sorbent material (1–2 mg) is usually inserted into the barrel of a syringe (100–250 μL). In a typical experiment, 300 μL of sample (ice-tea or fruit juice) is passed through the silica sorbent and the entrapped AA quantitatively recovered by eluting with 60 μL

19.3 ­ Liquid chromatographic determination of fat-soluble vitamins

of methanol-water (10%, v/v) solution. Compared to the classical procedures, MEPS reduces sample preparation time and organic solvent consumption. Several LC methods have been proposed for vitamin C analysis [1,82–97]. The good selectivity of aminopropylsiloxane-bonded silica-based columns in separating vitamin C vitamers is probably due to the hydrogen bonding between hydroxyl protons of vitamin C and the neutral amino group of the stationary phase rather than to differences in the pKa values of the vitamers [84,91]. A disadvantage is the short lifetime of the stationary phase due to possible reaction of the amine group with the carbonyl group of reducing sugars, or of other compounds, to form Schiff bases. Other separation modes include ion exclusion (poorly selective) [92], RP with and without ion suppression [83,85,93], and ion-pair RP chromatography [94,95]. The problem with RP packings is that AA is only weakly retained and, therefore, requires the addition of a cationic ion-pair agent to the mobile phase. To this end, several primary, secondary, and tertiary amines have been used as hydrophobic modifying reagents to obtain sharp and well-defined peaks; tetrabutylammonium has been the most used [1]. Recently, HILIC was successfully applied for the determination of AA [96] and DHAA [89,97] using either diol or zwitterionic stationary phases. Analyte retention is adequately increased, but a disadvantage is the exclusive use of stabilizers soluble in the organic phase (acetonitrile), which excludes the most effective stabilizer of vitamin C, that is metaphosphoric acid. The UV (254 nm) [85,86,98] and amperometric detection (a potential of +0.7 V using either a platinum or a glassy carbon electrode) [92,93] is used for the direct monitoring of AA, while fluorimetric detection requires chemical derivatization [84]. DHAA has a weak molar absorptivity and is electrochemically inactive. A precolumn reduction to AA using cystein or dithiotreitol makes possible absorbance detection but not electrochemical (high noise) detection [99]. LC-MS with ES ionization in the negative ion mode [100–102] was applied for the analysis of vitamin C in several food commodities; the main advantage was the simultaneous determination of AA and DHAA with no need for oxidation-reduction or derivatization steps [101]. LC methods for vitamin C determination in foods have recently been reviewed by Spínola et al. [103].

19.3 ­ LIQUID CHROMATOGRAPHIC DETERMINATION OF FAT-SOLUBLE VITAMINS Fat-soluble vitamins occur in the lipid fraction of foods [1], composed mainly of triglycerides and partly of sterols, phospholipids, and other lipid constituents. These substances, having solubility analogous to that of the fat-soluble vitamins, complicate the vitamin isolation and constitute a source of interference during the following analysis [1,3]. Hot saponification [1,3,104–106] is the most effective tool for removing the majority of fatty material, hydrolyzing ester linkages of glycerides, phospholipids, sterols, and carotenols. Moreover, alkaline hydrolysis frees bound forms of vitamins (for instance, the esterified and protein-bound forms) and degrades chlorophylls in

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CHAPTER 19  Analysis of vitamins by liquid chromatography

water-soluble products. Also, gelatin of the vitamin premix, added to supplemented foods, is dissolved. Starch-containing products, such as breakfast cereals, are first digested with takadiastase and then saponified to avoid the formation of undissolved particles [105]. Normally, saponification is carried out with a mixture of ethanol and 50% (w/v) aqueous KOH solution in the presence of an antioxidant (pyrogallol, ascorbic acid, butylated hydroxytoluene) for 30 min at about 80°C [1,3]. The unsaponifiable fraction (fat-soluble vitamins, carotenoids, sterols, etc.) is extracted from the alkaline digest by LLE using a water-immiscible organic solvent (hexane, petroleum ether, or petroleum ether: diethyl ether 1:1, v/v). Hexane has the advantage of providing extracts that do not contain soaps and that are nearly neutral; nevertheless, it is advisable to maintain the ethanol percentage below 40% (v/v) to avoid losses of slightly polar vitamins, such as retinols and tocopherols. Saponification can be used for vitamins A, D, and E, but it is unsuitable for the K vitamers, which are rapidly decomposed in alkaline media at high temperature [1,3]. Alcoholysis [1] is milder (ambient temperature) and faster (2 min) than saponification. Methanol reacts with KOH to form potassium methoxide, which, in turn, converts glycerides to methyl esters and soaps. It was used to determine vitamin A palmitate in defatted milk and vitamin D in whole milk [107]. Enzymatic hydrolysis [108,109] with lipase (from Candida rugosa or from porcine pancrease) is an alternative procedure to remove glycerides in vitamin K determinations. Addition of papain (from Carica papaya) aids the digestion of meat and other foods of animal origin. The hydrolysate is first alkalinized (potassium carbonate in ethanol) to precipitate fatty acids as soaps and then extracted with a water ­immiscible organic solvent (hexane or pentane). It has also been used in combination with supercritical fluid extraction (SFE) [110]. Also, direct solvent extraction [111] is applied for the isolation of vitamers susceptible to degradation in alkaline media (retinyl esters, vitamers K), but it is ineffective for removing interfering fatty substances. In some cases, ultrasonication has been employed to break up the lipoproteic complex encapsulating fat-soluble vitamins [112]. Several LC modes have been used for the separation of fat-soluble vitamins. The choice depends on vitamin forms to be determined, nature of the food matrix, and sample treatment. Adsorption chromatography has two main advantages: (a) geometric and positional isomers are generally resolved on silica stationary phases [113,114]; and (b) relatively high loads of lipid material can be tolerated by this type of column. The latter feature allows the direct injection of extracts obtained by means of direct liquid extraction [115] or sample dissolution in hexane (e.g., vitamin E from oils) [116], where recourse to saponification is not essential for isolation of analytes. In these cases, fluorescence detection is preferred to absorption detection, which may reveal intrusive peaks of lipid origin. The majority of LC separations of fat-soluble vitamins is based on RP chromatography on C18 columns, but the use of a triacontyl stationary phase (C30) has become more common due to its superior shape selectivity [117,118]. Shape discrimination with C30 columns at subambient temperature improves the resolution of

19.3 ­ Liquid chromatographic determination of fat-soluble vitamins

geometric and positional isomers partly. Nevertheless, C30 columns are less efficient and peaks are broader than those obtained with C18 columns. Nonaqueous reversed-phase (NARP) chromatography [119] was employed for the separation of fat-soluble vitamins [120] and carotenoids [121]. This technique uses either C18 columns with a high carbon loading (20%) or C30 columns and low polarity mobile phases. A typical NARP mobile phase consists of a polar solvent (e.g., acetonitrile), and a solvent of lower polarity (e.g., dichloromethane) to solubilize analytes and to adjust the mobile phase strength. Good selectivity results from the small difference in polarity between the mobile phase and the stationary phase. Supercritical fluid chromatography (SFC) is an attractive technique for separating low-polarity compounds [122]. A recently developed technology, known as ultra-performance convergence chromatography (UPCC or UPC2), combines the advantages of SFC with those of ultra-performance liquid chromatography (UPLC) providing an increase in selectivity [123]. Convergence chromatography (CC) has features in common with NPLC, and orthogonal to RPLC, and is used to obtain fast, clean, and low cost separations. In CC, the separation is achieved by varying the density and composition of a SF-based mobile phase. The primary mobile phase is CO2 in either a supercritical or liquefied state. Pure CO2 has limited solvating power, so it is often mixed with co-solvents (typically methanol) and additives (diethylamine, formic acid, ammonium formate, or small amounts of water). CC is compatible with solvents used in the preliminary extraction process, and with elution gradients enabling utilization of detection techniques such as UV, diode array (DAD), and MS detection. Since all compounds with log P values between −2 and 9 are suitable for CC, fat-soluble vitamins are ideal candidates. Most studies so far deal with the analysis of vitamin E in particular [124–130].

19.3.1 ­ VITAMIN A Vitamin A-active compounds [1] are present in foods of animal origin as retinoids (retinol, retinyl esters, retinal, retinoic acid) and in those of plant origin as carotenoids (only carotenoids with one unsubstituted β-ionone ring and with an 11-carbon polyene chain at least are provitamins A). Retinyl palmitate is the main form used as a food supplement [5]. Food sample pretreatment usually consists of either (a) saponification to quantify the free forms (retinol or xanthophylls can occur free or esterified in foods) [131,132] or (b) direct extraction to determine the unaltered vitamin A vitamers [111,115]. Alkaline hydrolysis is also used to simplify vitamin A analysis, since retinol is the only form to be quantified; nevertheless, the use of an antioxidant (ascorbic acid, hydroquinone, or pyrogallol) is crucial to prevent photo-oxidation. A drawback of hot saponification is the generation of artifacts, that is geometric isomers of retinol and carotenoids [133]. DLLME, combined with a preliminary alkaline digestion, was used to extract retinol from fruit juice, achieving a high enrichment factor [134]. Retinol and its esters can be monitored by UV (325 nm) [115,132] and fluorescence (324–328 nm excitation/470 nm emission) detection [135]; the latter has the

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CHAPTER 19  Analysis of vitamins by liquid chromatography

advantage that β-carotene does not interfere with the vitamin A determination even if there is coelution, while the disadvantage is a limited linear dynamic range. RP chromatography on a C18 column with semiaqueous mobile phases is most often used for vitamin A analysis [3,136,137], but adsorption chromatography on silica is the more efficient approach for separating geometric isomers of retinol [113,132]. Carotenoids show a characteristic three peak spectrum in the UV-vis region [138]. The absorption maxima of all-trans β-carotene, the main provitamin A, occur at 428, 453, and 478 nm (in hexane or ethanol); its cis isomers may be identified according to the following indications: (a) a small hypsochromic shift of λmax (usually 2–6 nm for mono-cis, 10 nm for di-cis, and 50 nm for poli-cis isomers); (b) an ipochromic effect; (c) a reduction in the fine structure; and (d) the appearance of a cis-peak in the nearUV range, between 330 and 350 nm. RP chromatography is preferred to NP for the determination of provitamins A, because many carotenoids can be monitored within the same separation: the xanthophylls are eluted early, while the carotenes require strong mobile phases (little or no water) for their displacement [139]. Carotenes and their cis isomers are poorly resolved on monomeric C18 phases, while their separation on polymeric C18 or C30 phases depends on the organic modifier selected [140]. In addition, the C30 sorbent provides the highest selectivity at low temperature (19°C); only under these conditions, are lutein and zeaxanthin (structural isomers) adequately separated [117,118]. Lately, LC-MS analysis of carotenoids and retinoids has been reported for food matrices such as fish eggs [141], milk [142], infant formula [143,144], and tomato fruits [145]. Atmospheric pressure chemical ionization (APCI) is the method of choice for their detection in the positive ion mode: retinol gives an intense ­dehydrated pseudomolecular ion, [MH–H2O]+, at m/z 269; retinyl esters are fragmented in the ion source producing [MH–fatty acid–H2O]+ ions (i.e., retinol dehydrated at m/z 269); carotenoids generate several ions such as [M+H]+, [M]+•, and [M−H]+. The ES interface is better for the ionization of xanthophylls when semiaqueous mobile phases are used for their separation. HPLC-DAD-MS/MS hyphenation was used recently to elucidate the retinoid composition of cow, buffalo, ewe, and goat's milk [142]. Since APCI detection is not completely selective, a reliable identification of retinyl esters was accomplished by fully separating the analytes on a tandem C18/C30 column system by nonaqueous and reversed-phase chromatography. Retinyl palmitate was found to be the most abundant vitamin A vitamer, with retinyl oleate the prevalent form in caprine milk; moreover, buffalo and ewe's milk were characterized for the first time. The relative abundance of the MRM transitions (two for each analyte) was used as an extra tool for the distinction of structural isomers and the related families of geometrical isomers. When applied to tomatoes, up to 44 carotenoids were identified [145].

19.3.2 ­VITAMIN D Vitamin D is the name given to a series of compounds with antirachitic activity [1]: cholecalciferol (vitamin D3) is present in foods of animal origin, whereas ergocalciferol (vitamin D2) is produced by plants, fungi, and yeast (Fig.  19.2). In animal

19.3 ­ Liquid chromatographic determination of fat-soluble vitamins

Fat-soluble vitamins Vitamin A X

X = -CH2OH Retinol

X = -COH

X = -COOH Retinoic acid X = -OCOR

Retinal Retinyl esters

All-trans-b-carotene

Vitamin D

HO

HO

(A)

Cholecalciferol

Ergocalciferol

Fat-soluble vitamins Vitamin E R1 HO R2

O R3

a-Tocopherol b-Tocopherol g-Tocopherol δ-Tocopherol

R1,R2,R3 = - CH3 R2 = -H R1, R3 = -CH3 R1 = -H R2, R3 = -CH3 R1,R2 = -H R3 = -CH3 R1 HO O

R2 R3

R1,R2,R3 = - CH3 R2 = -H R1, R3 = -CH3 R1 = -H R2, R3 = -CH3 R1,R2 = -H R3 = -CH3

a-Tocopherol b-Tocopherol g-Tocopherol δ-Tocopherol

Vitamin K O

O

CH3

trans-Phylloquinone

O n CH3

(B)

O

Menaquinones-n

FIG. 19.2 Names and structures of the fat-soluble vitamins and pro-vitamins A carotenoid naturally occurring in foods. (A) Vitamins A and D; (B) vitamins E and K.

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CHAPTER 19  Analysis of vitamins by liquid chromatography

organisms, vitamin D is metabolized to its biologically active forms, 25-hydroxy- and 1α,25-dihydroxycholcalciferol. The extraction of vitamin D from fatty foods requires alkaline hydrolysis [1,3,112]. Thermal isomerization of vitamin D to previtamin D during hot saponification entails losses of 10%–20% making quantification difficult. Overnight cold saponification (prolonged digestion at room temperature) is not affected by this problem and, moreover, demands less operator attention and yields higher recovery than hot saponification. Clean-up or fractionation procedures include sterol precipitation in a digitonin solution stored at −20°C overnight, SPE, gel permeation chromatography, and LC on a semipreparative scale [1,22,112,146–148]. These steps are essential: (a) to remove the excessive amounts of sterols, which might alter the retention of the vitamin and interfere with its UV detection (265 nm in ethanol or hexane); (b) to achieve an adequate enrichment factor of the final extract as vitamin D occurs at low concentrations in nature. SPE is more advantageous than the other methods; the most used sorbent is C18, but polar sorbents, such as silica, aminopropyl silica, and Florisil, are also efficient in removing sterols, carotenoids, vitamin E, and other interfering compounds. Magnetic-SPE, a recent modification of the classical SPE on cartridge, has been used to extract vitamin D from milk samples. Polypyrrolecoated magnetite nanoparticles can capture vitamin D2 and vitamin D3 without a preliminary saponification or protein precipitation step with recoveries exceeding 72% [149]. An alternative procedure for the reliable quantification of total vitamin D is the conversion of both D vitamers to isotachisterol [150], a compound more stable to heat and light; moreover, the absorption maximum at 301 nm and the high extinction coefficient contribute to improved selectivity and sensitivity. Ultraviolet and electrochemical detectors are the most widely used for the quantification of vitamin D. The UV detector has the advantage that all vitaminD-active compounds absorb within the range of 250–265 nm [1,144,146]; 280 nm has also been used to reduce interference problems [151]. The great sensitivity, selectivity, and linear dynamic range of the electrochemical detector [148] allow the analysis of samples containing low concentrations of vitamin D and the simplification of sample treatment. LC-APCI-MS is advantageous for the same reasons and for the reliable identification low concentrations of cholecalciferol [105,152–156] and its metabolites [157] in complex matrices such as milk, meat, and fish products. Although, APCI has a pronounced sensitivity, the low endogenous concentrations of vitamin D homologs are often difficult to detect and quantify because of low ionization efficiencies. However, the conjugated diene group of vitamin D makes it a specific target for Diels-Alder derivatization. In fact, some highly reactive 4-substituted 1,2,4-triazoline-3,5-diones (TADs or Cooksontype reagents) have been used to increase the ESI and APCI detection sensitivity of vitamin D and its metabolites by 100–1000-fold [158,159]. By treating foods with a high lipid content such as milk and its processed products, interferences from coeluting unsaponified material isobaric to D3 or its analogs, are often observed. A solution is either to improve the chromatographic separation or to use derivatization [159].

19.3 ­ Liquid chromatographic determination of fat-soluble vitamins

NP chromatography [1,112] has the advantage of tolerating relatively heavy loads of fatty material and of separating vitamin D from its hydroxylated metabolites; nevertheless, it cannot resolve vitamins D2 and D3. Hexane containing a small percentage (less than 5%, v/v) of a polar solvent (isopropanol, dichloromethane, or ethyl acetate) is the most useful mobile phase [112]. RP columns with a high carbon load [146–148] can differentiate vitamin D2 from D3, making possible the use of one homolog as an internal standard for the other [146], as well as their hydroxylated metabolites [147].

19.3.3 ­VITAMIN E Vitamin E is of plant origin and has eight biologically active forms [1]: four tocopherols (with a saturated isoprenoid side chain) and four tocotrienols (with an unsaturated isoprenoid side chain), designed as α-, β-, γ-, and δ- according to the number and position of methyl groups on the chromanol ring. Of these, α-tocopherol is the most active and widespread form in nature. During sample treatment, vitamin E must be protected from light and oxygen (flushing with nitrogen and adding an antioxidant) to quantify its actual content. Its concentration in oils can be performed by means of direct injection of a sample diluted with hexane [116] onto a NP column, but hot saponification is almost always required for other foods, especially if characterized by a high fat content [104–106]. However, alkaline conditions (volumes of alkali and ethanol, time, and temperature) must be carefully balanced to avoid losses [104]; moreover, the highest recovery is obtained when the digestion is completed under reflux conditions [1]. Recently, pressurized liquid extraction (PLE) was used to accelerate the extraction of tocol from plant foods [160–162]. PLE is an emerging green technique based on the use of solvents at elevated temperature and pressure; methanol or a methanol:isopropanol (1:1, v/v) solution at 50°C and 1600 psi were the applied conditions for vitamin E extraction. PLE was also combined with DLLME: depending on the food concentrations of tocols, 1/250, or 1/25 of the PLE extract was further purified by means of the miniaturized technique [161]. DLLME was also used alone, after a small-scale saponification step to isolate tocols from rice, barley, oat, wheat, corn, and millet [163]. NP chromatography [114,164] completely resolves the eight homologs, which elute in order of increasing polarity: α-tocopherol, α-tocotrienol, β-tocopherol, γtocopherol, β-tocotrienol, γ-tocotrienol, δ-tocopherol, and δ-tocotrienol. The decreasing number of methyl groups and the unsaturation in the side chain make these compounds more polar and therefore more retentive. Separation of β- and γ- positional isomers is due to the diverse interactions that the methyl groups establish with the silanol groups of the silica stationary phase. RP chromatography [164] is less effective for the separation of vitamin E vitamers; in fact, β- and γ- positional isomers coelute on C18 columns, but a separation can be achieved on C30 columns [165,166]. Pentafluorophenylsiloxane-bonded silica stationary phases (PFP), based on Fused-Core technology, are more efficient than NP for separating all eight tocols under RP conditions. The PFP-phase retains hydrophobic compounds less than C18,

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CHAPTER 19  Analysis of vitamins by liquid chromatography

but separates closely related compounds such as β- and γ-tocopherols and tocotrienols [161,167]. Similar selectivity was obtained by CC [124], which resolved the eight vitamers in less than 6 min [125,126]. Tocopherols and tocotrienols show a low intensity of absorption between 292 and 298 nm in ethanol [1,124], so fluorescence detection (290–296 nm excitation/330 nm emission) is preferred to analyze vitamin E in complex food matrices [116,167–169]. Polar solvents, such as diethyl ether and alcohols, provide a strong fluorescence response, and their inclusion in the hexane mobile phase improves the detection limits in NP chromatography [1]. The first description of LC-MS analysis dates back to 1998 [170]: tocopherols and carotenoids were detected by ESI as silver ion adducts, after postcolumn treatment with acetone containing silver perchlorate. Despite the low detection limits, the poor solubility of this salt in organic solvents causes contamination problems and this ionization technique is unsuitable for routine analysis. ESI in the negative ion mode was used for the quantification of tocopherols and tocotrienols after PLE with methanol from cereal samples [162]. Lanina et al. [171] compared the performances of ESI and APCI sources in both ionization modes for the detection of vitamin E. They observed that the positive ion mode is less efficient than the negative ion mode because of signal dispersion between [M+H]+ and [M]•+. They chose APCI in the negative ion mode for the detection of tocopherols in sunflower oil and milk due to the larger dynamic range and lower detection limits. Positive ion APCI was the preferred ionization mode used by other researchers [172,173].

19.3.4 ­VITAMIN K The homologs of vitamin K [1] are a family of 2-methyl-1,4-naphthoquinones possessing cofactor activity for γ-glutamylcarboxylase, which differ in the side chain attached at C3. Phylloquinone (vitamin K1) has a phytyl side chain and occurs in green plants. Vitamin K2 includes a group of compounds synthesized by bacteria and characterized by a polyisoprene side chain; each of which is designated menaquinone-n (MK-n, with n from 4 to 13) according to the number of isoprenyl units. Menadione (vitamin K3), menadiol (vitamin K4), and vitamin K1(25) are synthetic forms [1,5]. Owing to their instability to alkali, hot saponification is unworkable, but milder conditions (cold saponification and a minimal amount of alkali) can be applied to extract vitamin K vitamers from fatty foods with a low degree of degradation [174]. Enzymatic hydrolysis [108,109,175,176] and direct solvent extraction [175–179] are the most-common techniques employed for extraction. Liquid-phase reductive extraction with zinc chloride in an acid medium was used to extract K vitamers from dairy products (foods with high lipid content) as their hydroquinone forms are soluble in acetonitrile, used as selective extractant in place of hexane [176,177]. Acid hydrolysis with a short digestion time (10 min) [176] was used to isolate long chain menaquinones from cheese. Semipreparative LC [176,178,180] and SPE [177] have sometimes been employed as a clean-up and concentration step after solvent extraction. Matrix solid-phase dispersion (MSPD) followed by PLE (with ethyl acetate at 50°C and 1500 psi [181] or with n-heptane:ethyl acetate (4:1, v/v) at 75°C and

19.4 ­ Multivitamin methods

1800 psi [182]) and SFE (using carbon dioxide at 8000 psi and 60°C [183]) are fast, alternative procedures for extracting phylloquinone. Phylloquinone and menaquinones exhibit a UV spectrum characteristic of the naphthoquinone ring (five absorption maxima at 242, 248, 260, 269, and 325 nm), but UV detection is seldom used [184] due to its poor sensitivity and selectivity. Most methods employ fluorescence detection (238–244 nm excitation/418–430 nm emission) after a postcolumn reduction with a methanolic solution containing zinc chloride, sodium acetate, and acetic acid in a reactor packed with zinc metal powder [108,109,175–177]; this LC method is sufficiently sensitive for the determination of the low concentrations of menaquinones in animal products [108,176]. Electrochemical detection is an alternative for phylloquinone in foods of animal and plant origin [178,180]. In the last few years, APCI-MS detection has become competitive with fluorescence detection and the number of applications continues to grow [174,182,185,186]. The homologs of vitamin K are separated on C18 columns [108,176–178,180], but their geometric isomers can be resolved only on C30 columns [109,175] and silica columns [184]. Due to the lipophilic nature of the long chain menaquinones, NARP chromatography is best suited to their separation and determination [174].

19.4 ­MULTIVITAMIN METHODS Chromatographic techniques are suitable for quantitative multianalyte determinations. In particular, LC is the technique of choice for the direct analysis of polar, nonvolatile, and heat-sensitive compounds, such as water-soluble vitamins (see Fig. 19.3 for an example); moreover, having no molecular weight limitations, it can be used for the separation of cobalamins, polyglutamates, FAD, and CoA. LC is also the most common technique used for the concurrent analysis of fat-soluble vitamins and provitamin A carotenoids (see Fig. 19.4 for an example). The recently introduced UPLC technology has been employed for both the individual and simultaneous analysis of vitamins (see Table  19.1). The use of columns packed with sub-2 μm particles produces both enhanced resolution, useful to separate vitamin homologs efficiently, and higher detectability, useful for the determination of low endogenous concentrations of some vitamin forms. Another alternative, which has been used to improve efficiency and for faster separations, is the Fused-Core or Core-Shell technology [161,167,202,203]. These columns use superficially porous particles, which offer analogous performances to sub-3 μm and sub-2 μm particles but with a larger particle size and a lower column backpressure compatible with conventional HPLC instrumentation. The shorter diffusion path allows for faster mass transfer of the analytes (the C term in the van Deemter plot decreases). So far core shell column technology has been explored only partially for vitamin analysis [161,167,202,203]. The key step of a multivitamin method is the development of a simultaneous and quantitative extraction procedure. The intra- and intergroup heterogeneity of watersoluble vitamins makes it difficult to realize this goal. The application of an acid

591

3.0e4

0.0

2.88 Pyridoxamine m/z 169/152

2

4

6

8

10

Max. 3.0e4 cps.

12

14

16

Intensity, cps

Intensity, cps

CHAPTER 19  Analysis of vitamins by liquid chromatography

0

Nicotinamide m/z 123/80 Max. 4490.0 cps.

5.87

4490

2

4

6

9160

2.95 Pyridoxamine 5P m/z 249/232

Max. 9160.0 cps.

5000 0

2

4

6

8

10

12

14

16

Intensity, cps

Intensity, cps

Time, min

2

4

6

8

10

12

14

16

Intensity, cps

Intensity, cps

Max. 1.5e4 cps.

3.39 0.0

2

4

6

5.10 Ascorbic acid m/z 177/141

2

4

6

8

10

7.00

1.00e5

12

Max. 4.9e6 cps.

14

16

6

8 10 12 Time, min

Pyridoxal m/z 168/150 5.83 2

4

4

6

6

16

Max. 2.7e4 cps.

8 10 12 Time, min

5.84

2

14

14

16

Max. 5760.0 cps. Pyridoxine m/z 170/152

8 10 12 Time, min

14

16

Intensity, cps

4

Intensity, cps

5760 4000 2000 0

0.0

2

Intensity, cps

Intensity, cps Intensity, cps

2.7e4 2.0e4

1.0e4 0.0

12

14

16

Pantothenic acid m/z 220/202 Max. 1.0e5 cps.

2

4

6

8

10

12

14

16

1.00e4 0.00

Folic acid m/z 442/295

Max. 1.2e4 cps.

7.80 2

4

6

8

10

12

14

16

Time, min

Pyridoxal 5P m/z 248/150 Max. 1.7e4 cps.

5.84

10

0.00

Time, min 1.7e4

8

Time, min Intensity, cps

Intensity, cps

0.0

16

Max. 3810.0 cps. 0

Time, min 4.9e6

14

Time, min

Thiamin m/z 265/125

1.5e4 1.0e4

8 10 12 Time, min

5.88 Nicotinic acid m/z 124/80

3810

Time, min

Intensity, cps

592

780 500 0

1.00e4

Cyanocobalamin m/z 678/147 7.89

2

4

6

Riboflavin m/z 377/243

8 10 12 Time, min 8.58

Max. 780.0 cps.

14

16

Max. 1.0e4 cps.

0.00 2

4

6

8 10 12 Time, min

D-Biotin m/z 245/227

3.0e4

14

16

Max. 3.0e4 cps.

9.13 0.0

2

4

6

8 10 12 Time, min

14

16

FIG. 19.3 Extracted ion current profiles of the water-soluble vitamins detected in a green kiwi extract (see Ref. [187] for the details). The LC-MS/MS chromatogram was acquired by a highflow ESI source (TurboIonSpray source). Each analyte was identified on the basis of its retention, the two selected MRM transitions and their relative abundance. Only the most intense MRM ion current is reported in the figure.

treatment to hydrolyze the bound forms can be used for the simultaneous extraction of vitamins B1, B2, B3, B6, B8, B12, and C [188,204], but it is not appropriate for B5 and B9, which are sensitive to low pH [188]. Since the heterogeneity of the fat-­soluble vitamins is less marked, it is simpler to develop a common extraction

Intensity, cps Intensity, cps

7.6e5 5.0e5

Intensity, cps

5000

Intensity, cps

8000 5000

3992

Intensity, cps

Intensity, cps

4700

Intensity, cps

19.4 ­ Multivitamin methods

γ-Tocopherol m/z 417/151

8.12

Max. 4700.0 cps.

8.78 0

0.0

0

0

0

2

4

α-Tocopherol m/z 431/165

2

4

6

8

2

4

6

10

4

6

8

18

20

22

24

26

28

10

12

14 16 Time, min

18

20

22

24

26

28

8.80 Max. 5033.3 cps

8

10

8

12

14 16 Time, min

18

20

22

24

Menaquinone-4 m/z 445/187

10

8.80

12

14 16 Time, min

26

28

Max. 8000.0 cps

18

20

22

24

Zeaxanthin m/z 569/135

26

28

Max. 3991.7 cps

9.19 2

4

6

8

10

12

14 16 Time, min

18

20

22

24

Phylloquinone 10.80 m/z 451/187

2

4

6

8

10

12

5000.00

14 16 Time, min

15.22 2

4

6

8

10

26

28

Max. 5.2e4 cps

18

20

22

24

All-trans β-carotone 15.83 m/z 537/177

1.00e4 0.00

14 16 Time, min

Max. 7.6e5 cps

8.98

2

12

8.75

Lutein m/z 551/459

5.0e4

0.0

6

12

14 16 Time, min

26

28

Max. 1.2e4 cps 16.29 18

20

22

24

26

28

FIG. 19.4 Extracted ion current profiles of the fat-soluble vitamins detected in a green kiwi extract. This is an example of NARP chromatography (see Table 19.1, Ref. [199], for details): a nonaqueous mobile phase was chosen as the best compromise between chromatographic resolution and support for the positive APCI ionization of analytes.

protocol. Hot saponification is the most common procedure [105,146,196–198], especially for foods with a high fat content. It is indicated for the majority of fat-soluble vitamins and carotenoids but causes rapid decomposition of K vitamers; nevertheless, if a stoichiometric amount of alkalis is used to hydrolyze the food triglycerides at room temperature, the saponification can be applied as a collective procedure with acceptable yields for the vitamin K homologs [200]. MSPD is an effective technique

593

594

CHAPTER 19  Analysis of vitamins by liquid chromatography

Table 19.1  LC Methods for the Simultaneous Determination of Vitamins Analytes

Matrix

Analytical Technique

Separative Conditions

B1 (thiamin), B2 (riboflavin), B3 (nicotinamide, nicotinic acid), B5 (pantothenic acid), B6 (pyridoxine, pyridoxal, pyridoxamine), B9 (folic acid)

Italian pasta and fortified pasta

(a) HPLC-UV B3 λ= 260 nm; folic acid λ= 280 nm (b) HPLC-FL derivatized B1 λex/λem = 366/435 nm; B2 λex/λem = 422/522 nm (c) HPLC-ESI(+)-QqQ and HPLC-APCI(+)-QqQ; 1 MRM transition. Separated acquisition for B9 and B5

B1 (thiamin), B2 (riboflavin), B3 (nicotinamide), B5 (pantothenic acid), B6 (pyridoxine), B9 (folic acid), B12 (cyanocobalamin), C (ascorbic acid)

Infant formulas, infant milk, vitamin-enriched milk

B5 (pantothenic acid), B8 (biotin), B9 (folic acid), B12 (cyanocobalamin)

Fortified milk and rice powders

HPLC-DAD-FL FL detection: B2 λex/λem = 400/520 nm; B6 λex/λem = 290/410 nm. UV detection: B1 λ = 245 nm; B3 λ = 261 nm; B5 λ = 195 nm; B9 and C λ = 282 nm; B12 λ = 370 nm UPLC-ESI(+)-QqQ; 2 MRM transitions. IS: methotrexate

For (a) and (b): Supelcosil C18 (250 mm × 4.6 mm; 5 μm) Isocratic elution with: 60:40 (v/v) methanol-sodium acetate buffer (pH 4.5) for B1 and B2; A mobile phase containing sodium acetate buffer, acetic acid, and sodium 1-heptanesulfonate monohydrate for B3; 640 mL of a sodium acetate and sodium sulfate (pH 5.3) mixture + 360 mL of acetonitrile for B9. For (c): Discovery RP-Amide C16 column (150 × 4.6 mm; 5 μm). Gradient elution with aqueous ammonium formate (pH 3.75) and methanol. Flow-rate of 0.75 mL/min Spherisorb ODS-2 C18 column (250 mm × 4.6 mm; 3 μm) kept at 40°C. Gradient elution with an aqueous phosphate buffer (pH 2.95) and methanol, at a flow rate of 1 mL/min

B1 (thiamin), B2 (riboflavin), B3 (nicotinic acid, nicotinamide), B5 (pantothenic acid), B6 (pyridoxine, pyridoxal, pyridoxamine, pyridoxal 5′-phosphate, pyridoxamine 5′-phosphate), B8 (biotin), B9 (folic acid), B12 (cyanocobalamin), C (ascorbic acid)

Tomato pulp, green and golden kiwi, maize flour

Water-soluble vitamins

HPLC-ESI(+)-QqQ; 2 MRM transitions

BEH C18 column (100 mm × 2.1 mm; 1.7 μm) kept at 35°C. Gradient elution with 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile, at a flow rate of 0.2 mL/min Alltima C18 column (250 mm × 4.6 mm; 5 μm). Gradient elution with 5 mM aqueous formic acid and 5 mM formic acid in acetonitrile at a flow-rate of 1 mL/min

19.4 ­ Multivitamin methods

595

Method Performance Extraction Procedure

Recovery (%)

Limits

Ref.

For B1, B2, B3, and B6, a ground sample (1 g) was autoclaved with HCl (120°C, 30 min), cooled, diluted to known volume of aqueous ammonium acetate, vortexed, and centrifuged. The filtered supernatant was injected onto the LC column. For B5, a ground sample (4 g), was treated with acetate buffer (pH 5.6) and autoclaved (121°C, 15 min), cooled, diluted with aqueous ammonium formate, vortexed, and centrifuged. The filtered supernatant was injected onto the LC column. For B9, a ground sample (2.5 g) was mixed with a sodium phosphate-sodium citrate/ascorbate buffer (pH 8), heated, cooled, and incubated with papain and di-α-amylase (40°C, 2 h). The solution was diluted with aqueous ammonium formate, vortexed, and centrifuged. The supernatant was filtered and injected into the LC-MS system. For LC-UV analysis, the extract was loaded onto a preconditioned SAX cartridge. The cartridge was washed with a sodium sulfate and sodium chloride solution and adjusted to pH 5.3 with acetic acid. Elution with 1 mL of the washing solution (adjusted pH to 2.5 with TFA) To a reconstituted, homogenized solid sample (0.5 g) or liquid sample (5.0 g), a precipitation solution (containing zinc acetate, phosphotungstic polyhydrated, and glacial acetic acid in water) was added. The mixture was vortexed, centrifuged, filtered, and injected into the LC column

Higher than 90%

LODs for LC-ESI-MS/ MS in the range of 0.5–5 μg/L;LODs for LC-APCI-MS/MS in the range 0.5–2.7 μg/L

[188]

Only for B5 (95%–98%) and B12 (95%–104%)

CCαs in the range of 0.003–0.580 mg/kg.CCβs in the range of 0.005–0.950 mg/kg

[189]

To a 1-g sample, spiked with the IS, an aqueous ammonium acetate was added. After magnetic agitation and ultrasonic extraction, 10 mL of chloroform were added. The solution was shaken again, centrifuged, filtered, and finally injected for UPLC-MS/MS analysis A homogenized sample (2 g), was mixed with BHT as antioxidant and, only for tomato and kiwifruits, also with diatomaceous earth (1 g). The extraction cartridge was prepared by packing a layer of first of C18 (0.5 g) and then the sample layer. Teflon frits were placed above and below the sorbent-food matrix bed. Elution with 14 mL of ethanol-water (50:50, v/v) solution; 100 μL was injected into the LC-MS/MS system

In the range of 85%–105%

Instrumental LODs in the range of 0.005–0.03 μg/L

[190]

Maize flour: higher than 70%, with the exception of vitamin C (19%), pyridoxal5′-phosphate (40%), and B9 (40%). Tomato pulp: higher than 64%, except vitamin C (41%). Kiwi higher than 73%, except nicotinamide (30%)

LODs in the range of 0.68–239 ng/g; LOD of vitamin C was 30.22 μg/g/

[187]

Continued

596

CHAPTER 19  Analysis of vitamins by liquid chromatography

Table 19.1  LC Methods for the Simultaneous Determination of Vitamins—cont'd Analytes

Matrix

Analytical Technique

Separative Conditions

B1 (thiamin), B2 (riboflavin), B3 (nicotinamide), B5 (pantothenic acid), B6 (pyridoxine), B8 (biotin), B9 (folic acid)

NIST SRM 1849 infantadult nutritional formula

HPLC-ESI(+)-QqQ; 1 MRM transition. IDMS. Isotopically labeled vitamins used as ISs: 4,5,4-methyl[13C3]-thiamine chloride; 4,5-bis(hydroxymethyl)-[13C4]pyridoxine hydrochloride; 2,4,5,6-[2H4]-nicotinamide; calcium pantothenate[13C615N2]; biotin-2H2; [13C415N2]-riboflavin;folic acid-[13C5]

B2 (riboflavin), B3 (nicotinic acid), B5 (pantothenic acid), B9 (folic acid), C (ascorbic acid)

Honey

HPLC-UV. B3 and C λ = 254 nm; B2, B5 and B9 λ = 210 nm

B1 (thiamin), B2 (riboflavin), B3 (nicotinamide), B5 (panthothenic acid), B6 (pyridoxine)

NIST SRM 3280 multivitaminmultielement tablets and SRM 1849 infantadult nutritional formula

B1(thiamin), B2 (riboflavin and FAD), B3 (nicotinamide), B6 (pyridoxal)

Human milk NIST SRM 1849

B1 (thiamin), B2 (riboflavin), B3 (nicotinic acid), B3 (nicotinamide), B6 (pyridoxal), B9 (folic acid)

Cornflakes

HPLC-ESI(+)-MS; acquisition in SIM mode. IDMS. Isotopically labeled vitamins used as ISs: nicotinamide-[2H4]; thiamine chloride-[13C3]; calcium pantothenate monohydrate[13C3,15N]; pyridoxine hydrochloride-[13C4] UPLC-ESI(+)-QqQ; 2 MRM transition. IDMS. Isotopically labeled vitamins used as ISs: thiamin(4-methyl-13C-thiazol-5yl-13C3) hydrochloride, riboflavin-dioxo-pyrimidine13 C4,15N2, and pyridoxalmethyl-d3 hydrochloride HPLC-UV and HPLC-FL. (a) UV detection: 268 nm for B1, 260 nm for both B3 species and 284 nm for B9. (b) FL detection: λex/λem = 268/513 nm for B2; λex/ λem = 284/317 nm for B6

RP LC:Synergi HydroRP column (250 mm × 2 mm; 4 μm). Gradient elution with: (a) 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile or (b) aqueous formic acid-ammonium formate buffer (pH 0.7) and 0.1% formic acid in acetonitrile. HILIC:ZIC-HILIC column(150 mm × 2 mm; 3.5 μm). Gradient elution with aqueous formic acid-ammonium formate buffer (pH 3.7) and 0.025% formic acid in acetonitrile Alltima C18 column (250 mm × 4.6 mm; 5 μm). Gradient elution with 0.025% aqueous trifluoroacetic acid and acetonitrile, at a flow rate of 1 mL/min Cadenza CD-C18 column (250 mm × 4.6 mm; 3 μm), kept at 22°C. Gradient elution with aqueous ammonium formate (pH 4) and methanol, at a flow rate of 0.8 mL/min

ACQUITY UPLC HSS T3 column (2.1 mm × 50 mm, 1.8 μm) kept at 40°C. Gradient elution with ammonium formate (10 mM) and acetonitrile at a flow rate of 0.3 mL/min

ZORBAX HILIC Plus silica column (100 mm × 4.6 mm; 3.5 μm), kept at 30°C. Mobile phase: (A) water:acetonitrile (95:5,v:v); (B) acetonitrile:water (95:5, v:v); both of them 10 mM ammonium acetate (pH 5.0) Gradient elution from 100% to 60% solvent B at a flow rate of 0.8 mL/min

19.4 ­ Multivitamin methods

597

Method Performance Extraction Procedure

Recovery (%)

Limits

Ref.

A sample (0.2–0.5 g) was spiked with the labeled ISs and extracted with a 0.1 M phosphate buffer (at pH 2). Centrifugation, filtration and injection into the LC system

–

–

[191]

A reconstituted, homogenized sample (10 g) was treated with NaOH and phosphate buffer (pH 5.5). The solution was diluted with water, filtered and injected into the LC-UV system

In the range of 98%-104%

[192]

A sample (2 g) was treated with the extraction solution (1% aqueous acetic acid), placed on vortex and then in an ultrasonic bath. After addition of acetonitrile, the sample was placed at −20°C overnight. The extract was centrifuged, filtered, and injected into the LC-MS system

–

LODs B2, B3 = 0.25 mg/kg; B5 = 0.58 mg/kg; B9 = 0.15 mg/kg; C = 0.1 mg/kg –

A sample (20–50 μL) was diluted to 100 μL and spiked with the ISs. After protein precipitation with methanol, it was mixed and centrifuged. 500 μL of the supernatant was evaporated and reconstituted in 120 μL of water containing 13C-caffeine. The nonpolar constituents were extracted with diethyl ether followed by incubation at 4°C to favor precipitation. After centrifugation, the supernatant was filtered and analyzed

In the range of 73%–100%

LODs B1 = 0.01 μg/L; B2 = (riboflavin) 0.1 μg/L, (FAD) 0.1 μg/L; B3 = 0.5 μg/L; B6 = 4 μg/L

[194]

Ground cornflakes (1 g) were hydrolyzed with 0.1 M HCl, incubated at 100°C for 30 min and cooled. The pH was adjusted at 4.5 with sodium acetate buffer. The sample was digested with papain and claradiastase at 37°C for 16 h, heated at 100°C for 4 min to deactivate the enzymes. The digest was centrifuged, treated with acetonitrile, centrifuged a second time, and the ensuing supernatant was filtered and injected directly into HPLC

–

LODs B1 (UV) = 83.9 ng/mL; B2 (FL) = 7.7 ng/mL; nicotinic acid (UV) = 47.8 ng/mL; nicotinamide (UV) = 18.1 ng/mL; B6 (FL) = 97.2 ng/mL; B9 (UV) = 31.5 ng/mL

[4]

[193]

Continued

598

CHAPTER 19  Analysis of vitamins by liquid chromatography

Table 19.1  LC Methods for the Simultaneous Determination of Vitamins—cont'd Analytes

Matrix

Analytical Technique

Separative Conditions

B1 (thiamin, dibenzoyl thiamine and bisbentiamine), B2 (riboflavin and riboflavin tetrabutylate), B3 (nicotinic acid and nicotinamide), B5 (pantothenate), B6 (pyridoxine), B8 (biotin), B9 (folic acid), B12 (cyanocobalamin), C (ascorbic acid, dehydroascorbic acid and ascorbic acid 2-glucoside)

SRM 3280 multivitamin/ multielement tablets, energy drinks, and dietary supplements.

UPLC-ESI(±)-QqQ; 2 MRM transitions. Chromatographic analysis utilized a multimode C18 column, which did not require adding ion pair reagents to the mobile phase and which provided reverse-phase, anion- and cation-exchange capacities, favoring retention of both acid and basic compounds

Scherzo SM-C18 column (150 mm × 2.0 mm, 3 μm) kept at 40°C. Gradient elution with: (A) 5 mM ammonium formate aqueous solution with formic acid 0.05% (v/v) and (B) acetonitrile with formic acid 0.3% (v/v). Flow rate of 0.2 mL/min

A (retinol, retinyl acetate, retinyl palmitate, β-carotene, α-carotene), E (α-tocopherol, γ-tocopherol, tocopheryl acetate), Carotenoids (lutein, lycopene)

Natural and fortified milk products

HPLC-DAD λ = 326 nm for retinoids; λ = 294 nm for tocopherols; λ = 450 nm for carotenoids. IS: β-cryptoxanthin

Spheri-5 ODS column(220 mm × 4.6 mm; 5 μm). Isocratic elution with acetonitrilemethylene chloride-methanol (70:20:10, v/v/v) at a flow rate of 1.3 mL/min. Gradient elution to confirm lutein and zeaxanthin, not resolved under isocratic conditions

A (retinyl acetate), E (tocopheryl acetate), D (cholecalciferol), K (phylloquinone)

Fortified milk, powdered milk

HPLC-DAD λ = 230 nm for K, D, and E; λ = 280 nm for A, E, D, and K; λ = 300 nm for K, D, and E

A (retinol, β-carotene, β-cryptoxanthin, 13Z-βcarotene, 9Z-β-carotene), E (α-tocopherol, γ-tocopherol), Carotenoids (lutein zeaxanthin, violaxanthin, neoxanthin, 5,6 epoxide, antheraxanthin)

Milk (forage, plasma)

UPLC-DAD λ = 325 nm for vitamin A; λ = 292 nm for vitamin E; λ = 450 nm for carotenoids. ISs in milk: echinenone and δ-tocopherol

Microsorb C18 column (250 × 4.6 mm; 5 μm) kept at 30°C. Isocratic elution with a 3% (w/v) sodium dodecylsulphate aqueous solution pH 7 (phosphate buffer) with the presence of 15% (v/v) butyl alcohol. Flow rate of 2 mL/min Acquity UPLC HSS T3 column (150 mm × 2.1 mm; 1.8 μm), kept at 35°C. Gradient elution with acetonitrile/dichlorometane-methanol (75:10:15, v/v/v) and aqueous 0.05 M ammonium acetate, at a flow rate of 0.4 mL/min. Nucleosil C18 column (150 mm × 4.6 mm; 3 μm). Isocratic elution with acetonitriledichlorometane-0.05 M ammonium acetate in methanol-water (70:10:15:5, v/v/v/v) at a flow rate of 2 mL/min

Fat-soluble vitamins

19.4 ­ Multivitamin methods

599

Method Performance Extraction Procedure

Recovery (%)

Limits

Ref.

Beverages were ultrasonically degassed, diluted, filtered and injected. Ground dietary supplements were treated with water/acetonitrile (95:5 v/v) with acetic acid 1% (v/v) at 65°C for 10 min and then centrifuged. The supernatant was diluted, filtered and injected

In the range of 93.2%–106.9%

–

[195]

Individual forms: a sample aliquot (200 mL) was heated (85°C) and mixed; IS was added to 1-mL sample before deproteinization with ethanol. Two extractions with a 0.01% BHT hexane-methylene chloride (5:1, v/v) solution (ultrasonic bath, 5 min) followed by centrifugation. Pooled organic phases were evaporated, reconstituted, filtered and injected into the LC system. Total content of vitamins A and E: 1-mL sample was spiked with the IS and pyrogallic acid, and submitted to alkaline hydrolysis with methanolic KOH; the sample was vortexed and ultrasonicated at 45°C for 15 min. The cooled sample was extracted twice with aqueous 5% NaCl, followed by isopropanol and hexane-methylene (5:1, v/v). Organic phases were pooled, washed with water until pH < 7, evaporated, reconstituted, filtered, and injected into the LC system Cold overnight saponification (30-g sample) with ethanolic KOH and ascorbic acid. LLE with hexane (three times). Organic phases were pooled, washed two times with water, and evaporated to dryness (40°C). Residue was dissolved in 5 mL of methanol, filtered, and injected into the LC system

LLE: recoveries of retinyl palmitate and α-tocopherol were >85%. Alkaline hydrolysis: recoveries of retinyl palmitate and αtocopheryl acetate were 95%

LOQs < 0.03 μmol/L for retinol, retinyl acetate, and retinyl palmitate; LOQs = 0.02– 0.04 μmol/L for lycopene, α- and β-carotene, βcryptoxanthin, lutein, and zeaxanthin. LOQs <0.23 μmol/L for α- and γ-tocopherol

[196]

–

Instrumental LODs in the range of 0.81–1.12 mg/L

[197]

Milk sample (2 mL) was deproteinized with ethanol containing ISs, vortexed, and extracted twice with a hexane/ethyl acetate (9:1, v/v) solution; it was again vortexed, and, finally, centrifuged. Xanthophylls and vitamin A extraction: pooled organic phases were added of ethanol-water (9:1, v/v), vortexed and centrifuged; the step was repeated and the two ethanolic phases were collected and evaporated. Carotene and vitamin purification: the upper hexanic phase was saponified with ethanolic KOH (60°C, 1 h), followed by two extractions with a hexane-ethyl acetate (9:1, v/v) solution. Hexanic and ethanolic phases were pooled, evaporated to dryness. Residue was dissolved in THF + acetonitriledichlorometane-methanol (75:10:15, v/v/v) and injected into the UPLC or the HPLC system

Up to 70% for carotenoids and vitamins

LODs for HPLC system: between 1.3 and 10 ng injected. LODs for UPLC system between 0.8 and 8 ng injected

[198]

Continued

600

CHAPTER 19  Analysis of vitamins by liquid chromatography

Table 19.1  LC Methods for the Simultaneous Determination of Vitamins—cont'd Analytes

Matrix

Analytical Technique

Separative Conditions

Provitamins A (β-carotene, β-cryptoxanthin), D (ergocalciferol), E (α-tocopherol, γtocopherol, δ-tocopherol), K (phylloquinone, menaquinone-4), Carotenoids (lutein, zeaxanthin) A (retinyl acetate, retinyl palmitate), D (ergocalciferol, cholecalciferol), E (tocopheryl acetate), K (phylloquinone)

Maize flour, green and golden kiwi

HPLC-DAD-APCI(+)-QqQ; 2 MRM transitions. DAD detection (λ = 450 nm). ISs: trans-β-apo-8′-carotenal, α-tocopheryl acetate, 1α-hydroxyvitamin D3, phylloquinone-[2H7]

ProntoSIL C30 column (250 mm × 4.6 mm; 3 μm). Gradient elution with methanol and isopropanol-hexane (50:50, v/v) solution, at a flow rate of 1 mL/min

NIST SRM 3280 multivitaminmultielement tablets and SRM 1849 infant/ adult nutritional formula

ACE C18 column (250 mm × 4.6 mm; 5 μm), kept at 25°C. Isocratic elution with a 5 mM ammonium acetate in acetonitrilemethanol (60:40, v/v) mobile phase at a flow rate of 1 mL/min

A (all-trans-β-cryptoxanthin, all-trans-β-carotene, alltrans-retinol), Carotenoids nonprovitamins A (alltrans-lutein, all-transzeaxanthin), E (α-tocopherol, γ-tocopherol, δ-tocopherol), D (ergocalciferol, cholecalciferol), K (phylloquinone, and menaquinone-4)

Cow's, buffalo's, goat's, ewe's, and donkey's milk

HPLC-APCI(+)-MS; acquisition in SIM mode. IDMS. Isotopically labeled vitamins used as ISs: ergocalciferol-[2H3], cholecalciferol-[2H3], phytonadione-[2H4], retinyl acetate-[2H6], retinyl palmitate-[2H4] HPLC-DAD-PCI(+)-QqQ; 2 MRM transitions for MS detection. DAD detection (λ = 450 nm; range from 250 to 750 nm). ISs: α-tocopherol-d6 [α-tocopherol-(ring5,7-dimethyl-d6)], cholecalciferol-d3 [cholecalciferol (6,19,19-d3)], phylloquinone-d7 (5,6,7,8d4, 2-methyl-d3), and trans-β-apo-8′-carotenal

D (ergocalciferol and cholecalciferol), K (phylloquinone, menaquinone-4 and menadione)

Infant foods, certified reference material infant/ adult nutritional formula SRM 1849, green vegetables

HPLC-DAD-APCI(±)-MS; acquisition in SIM mode

Separation of fat soluble vitamins: two C18 columns, a Supelcosil C18 (4.6 mm × 50 mm, 5 μm) and an Alltima C18 (4.6 mm × 250 mm, 5 μm), were connected in series to increase efficiency in separating analytes from interfering compounds. Separation of carotenoids: it was carried out on a ProntoSIL C30 column (250 mm × 4.6 mm; 3 μm) kept at 19°C. In both cases, gradient elution with methanol and isopropanol-hexane (50:50, v/v) solution was performed at a flow rate of 1 mL/min Zorbax Eclipse ODS nonendcapped (25 cm × 0.46 cm; 5 μm). The mobile phase consisted of acetonitrile, isopropanol and water, operating under gradient elution conditions at 1 mL/min of flow rate

Acronyms: APCI, atmospheric pressure chemical ionization; BHT, butylated hydroxytoluene; DAD, diode array detector/ detection; ESI, electrospray ionization; FL, fluorescence detection; HILIC, hydrophilic-interaction LC; HPLC, high performance liquid chromatography; IDMS, isotope-dilution mass spectrometry; IS, internal standard; LLE, liquid-liquid extraction; LOD, limit of detection; LOQ, limit of quantitation; MRM, multiple reaction monitoring; MSPD, matrix solid-phase dispersion; QqQ, triple quadrupole; RP, reversed phase; SIM, selected Ion monitoring; SRM, standard reference material; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; UPLC, ultraperformance liquid chromatography.

19.4 ­ Multivitamin methods

601

Method Performance Extraction Procedure

Recovery (%)

Limits

Ref.

MSPD: a 2-g sample was blended with BHT (antioxidant) and C18 sorbent (maize flour) or diatomaceous earth and Na2SO4 (kiwi fruits). A syringe-like glass tube was filled with the dispersion (Teflon frits above and below the sorbent-food matrix bed). Elution in sequence with methanol, isopropanol, and hexane. The pooled extracts were concentrated to 500 μL, diluted to 1 mL with isopropanol:exane (50:50, v/v), centrifuged, and injected into the LC-MS/MS system

Recoveries for maize flour were higher than 78%; recoveries for kiwifruits were higher than 60%

LODs. Maize flour: in the range of 0.0004–0.075 μg/g. Kiwi fruits: in the range of 0.001–0.221 μg/g

[199]

A sample (1.5–2.5 g) was treated with ethyl acetate, ultrasonicated, and rotated-mixed overnight. Ethyl acetate was collected after centrifugation, and a new solvent aliquot was added; the procedure was repeated for a total of five times. The pooled ethyl acetate solution was evaporated, centrifuged, and injected

–

–

[193]

A milk sample (6 mL) was spiked with the ISs and, after equilibration, submitted to overnight cold saponification (18 mL of ethanol containing 0.1% (w/v) BHT, and x mL of 50% (w/v) aqueous KOH; x = 1 mL for donkey's and cow's milk; x = 3 mL for buffalo's, goat's, and ewe's milk). At the end of incubation, the digest was diluted with Milli-Q water and the analytes were extracted with several 12-mL aliquots of hexane with 0.1% (w/v) BHT. After centrifugation, the combined hexane layers were washed with Milli-Q water to remove alkalis. The extract was then evaporated to 100 μL at 30°C, diluted to 200 μL and, eventually, injected into the HPLC-DAD-MS/MS system

Phylloquinone = 67%; menaquinone-4 = 54 %; the recoveries of all other analytes ranged between 80% and 100%

LODs for cow's milk as an example. Retinol = 15.6 μg/L; β-carotene = 7.41 μg/L; for all other analytes LODs ranged between 0.90 to 3.70 μg/L

[200]

Each sample was treated with acetonitrile (3 mL) and centrifuged. The supernatant was recovered and used as dispersive solvent. Carbon tetrachloride (150 μL) was used as extractant solvent. The mixture was rapidly injected into 6 mL of water using a micropipette, and gently shaken manually for several seconds. After centrifugation, the extraction solvent was settled at the bottom of the conical tube. The sediment was collected and evaporated to dryness. The residue was reconstituted with 50 μL of acetonitrile and a volume of 20 μL was injected into the LC

In the range of 88%–105%

LODs by HPLC-DAD between 0.2 and 0.6 ng/mL. LODs by HPLC-MS between 0.2 and 4.0 ng/mL

[201]

602

CHAPTER 19  Analysis of vitamins by liquid chromatography

for the simultaneous isolation of both fat-soluble [199] and water-soluble vitamins [187] from plant foods. Recently, DLLME was used for the extraction of vitamin D and vitamin K homologs from vegetables [201], vitamin A vitamers from fruit juices [205], and vitamin A and vitamin E from oil samples [206]. The UV/DAD [192,196–198,204,207] and fluorescence detection [189,208] have been used to develop multivitamin LC methods, which, however, remain limited to a few analytes with adequate response to the same detection system and extracted by the same procedure. Moreover, UV detection is limited by the absence of strong chromophores for some vitamins (pantothenic acid and biotin), which absorb with modest intensity in the low UV region only, where selectivity is scarce (absorption of interfering compounds). An effective detector for the development of multivitamin LC methods is the mass spectrometer, since it can overcome problems due to poor ­chromatographic resolution by extracting the characteristic ion currents associated with target analytes. The ESI interface is the most efficient for the ionization of polar substances such as water-soluble vitamins, which, endowed with acidic or basic groups, can be deprotonated or protonated. Moreover, ESI can ionize high-molecular-mass compounds and produce multicharged pseudomolecular ions, such as [M + nH]n+ or [M − nH]n−. This feature is useful for the detection of cobalamins that give intense mono-, double- and triple-charged pseudomolecular ions [80,187]. Since all water-soluble vitamins respond in the positive ion mode, this has been chosen for the development of multivitamin methods [187,190,191,193]. Similarly, the APCI interface operating in the positive ion mode represents the best compromise for the simultaneous detection of fat-soluble vitamins and carotenoids [105,145,193,199,200]. The use of a mass spectrometer as a chromatographic detector offers another great advantage in vitamin analysis: the possibility of simplifying the extraction procedure. The selectivity of the LC-MS technique reduces problems due to intrusive peaks from matrix components, while its sensitivity (ng or pg injected for real samples) can sometimes permit the direct injection of an extract, eliminating the concentration step and the exposure to heat (most water-­ soluble vitamins possess low thermal stability). Sample preparation time is reduced as well as the duration of exposition to air and light (most vitamins and carotenoids are susceptible to these factors). Recently, convergent chromatography was shown to be able to separate 17 vitamins (retinyl acetate, retinyl palmitate, β-carotene, ergocalciferol, α-tocopherol, phylloquinone, menaquinone-4, thiamin, riboflavin, nicotinic acid, nicotinamide, pantothenic acid, pyridoxine, biotin, cyanocobalamin, and ascorbic acid) covering a wide polarity range (log P from −2.11 for thiamin up to 10.12 for retinyl palmitate) in a single separation. This method used a gradient with the mobile phase changing from pure carbon dioxide to pure methanol in 4 min. This novel approach presents a great opportunity to analyze very different compounds by means of a single chromatographic system [130]. In the last few years, the number of LC methods proposed for multivitamin determination in dietary supplements and food products has been growing. The most emblematic multivitamin methods, published in the last decade, are summarized in Table 19.1.

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