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Journal of Chromatography A, 1261 (2012) 179 188
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A
jou rn al h om epage: www.elsev ier .com
Sequen lubvegeta
J. Santos . Ha REQUIMTE/De rbo Feb Institute of Fo Madr
a r t i c l
Article history:Available onlin
Keywords:Fresh-cut vegeFat-soluble vitWater-solubleLCMS/MSGreen leafy vegetables
waterlved.is pre
quane devamincress
red attained for the analyzed vitamins in less than 100 min, including extraction and vitamin analysis using2 optimized procedures; good repeatability and linearity was achieved for all vitamins studied, whilerecoveries ranged from 83% to 105%. The most abundant free vitamins found in leafy vegetable productswere vitamin C, provitamin A and vitamin E. The richest sample on vitamin C and provitamin A was pealeaves (154 mg/g fresh weight and 14.4 mg/100 g fresh weight, respectively), whereas lambs lettuce was
1. Introdu
Vitaminessential mfunctions inare classie(FSV) (A, E, vitamins anchemical cotance in theare involvebiological ftion (vitamiE) and blooSeveral vitacatabolism
WSV anlabeled in (e.g. lettuce
CorresponE-mail add
0021-9673/$ http://dx.doi.othe vegetable with the highest content on vitamin E (3.1 mg/100 g fresh weight). Generally, some lossesof vitamins were detected after storage, although the behavior of each vitamin varied strongly amongsamples.
2012 Elsevier B.V. All rights reserved.
ction
s are biologically active organic compounds that areicronutrients involved in metabolic and physiological
the human body. There are 13 vitamins identied thatd according to their solubility into fat-soluble vitaminsD, and K) and water-soluble vitamins (WSV) (B-groupd vitamin C) [1]. These compounds greatly differ in theirmposition, physiological action and nutritional impor-
human diet, even within the same group [2]. The FSVd in complex metabolic reactions related to importantunctions, such as vision (vitamin A), calcium absorp-n D), antioxidant protection of cell membranes (vitamind coagulation (vitamin K), among other functions [3].mins of the B-group act mainly as coenzymes in theof foodstuffs to produce energy [1].d FSV are one of the micronutrients that are usuallyfoods. In this sense, minimally processed vegetables, wild rocket, watercress, spinach) are not an exception.
ding author. Tel.: +34 91 0017956; fax: +34 91 0017905.ress: [email protected] (E. Ibnez).
These products are basically ready-to-eat foods composed by rawvegetables that retain as much of the naturally occurring vitamincontent. However, there are several factors that can lead to vita-min losses in these products, such as temperature, the presence ofoxygen, light, moisture content, water activity, pH, enzymatic mod-ications and metal trace elements, particularly iron and copper[1].
The degree of degradation will vary according to the vitamin andcould also be affected by the processing and storage time to whichthe vegetable is submitted. It is known that WSV are more sus-ceptible to leaching losses during washing, while vitamin C is veryprone to chemical oxidation during processing and storage stages[1]. Vitamins A and E could be destroyed under the presence of oxy-gen, light, heat, trace metal ions and storage time [1]. Therefore,monitoring the vitamin content during processing and storage isof great importance to food technologists and consumers to assurethe nutritive value of foods, and also for quality assurance purposesand regulatory compliance. This requirement creates the need formore rapid and specic methods for vitamin determination [46].
The development of a single method for the multiple and simul-taneous monitoring of WSV and FSV is very challenging due todifferent reasons. The level of vitamins in food may be as low asfew micrograms per 100 g, usually very unstable and accompanied
see front matter 2012 Elsevier B.V. All rights reserved.rg/10.1016/j.chroma.2012.04.067tial determination of fat- and water-sobles during storagea, J.A. Mendiolab, M.B.P.P. Oliveiraa, E. Ibnezb,, Mp. Cincias Qumicas, Faculdade de Farmcia, Universidade do Porto, Rua de Jorge Viteod Science Research, CIAL (CSIC-UAM), Nicols Cabrera 9, Campus Cantoblanco, 28049
e i n f o
e 3 May 2012
tablesamins
vitamins
a b s t r a c t
The simultaneous analysis of fat- and wide range of chemical structures invoand analysis of both types of vitamins to LCMS/MS and LCDAD in order toand after a 10-days storage period. ThC, B1, B2, B3, B5, B6, B9, E and provitgreen lettuce, ruby red lettuce, waterpea leaves, mizuna, garden cress and/ locat e/chroma
le vitamins in green leafy
errerob
rreira, 228, 4050-313 Porto, Portugalid, Spain
-soluble vitamins from foods is a difcult task considering the In this work, a new procedure based on a sequential extractionsented. The procedure couples several simple extraction stepstify the free vitamins contents in fresh-cut vegetables beforeeloped method allows the correct quantication of vitamins
A in ready-to-eat green leafy vegetable products including, swiss chard, lambs lettuce, spearmint, spinach, wild rocket,mustard. Using this optimized methodology, low LOQs were
-
180 J. Santos et al. / J. Chromatogr. A 1261 (2012) 179 188
by an excess of compounds with similar chemical behavior. Tradi-tionally, methods for vitamin determination require the analysis ofeach vitamin individually by using different physical, chemical andbiological methods. Microbiological assays are still the methods ofreference asome vitamtive but alsocertain prec(HPLC) metFSV. The chsitivity requsample madetection isbut showedspecicity ispectrometthe determsimultaneoThe majoriterature focuto analysis [5,1013]. Ourally occubest of our vitamins inout.
Consequand validateple and seqof WSV (vitaand vitaminas well as ththe market
2. Materia
2.1. Chemic
All chemmins standahydrochlorpantothena(E) and -cAldrich (Matrans--Aplated hydroSpain). Am(Barcelona,Methanol (acetate wePoland). Di(Millipore,
Individusolution weand kept inAscorbic acnicotinamidriboavin amethod devdilution of ammoniumof the valuestudy (C: 3.B5: 0.8 g/mcarotene an
xtrac
ml) ae fat soluble vitamins was also prepared before injection at/ml with ethyl acetate.
mples
elve samples of green leafy vegetables from seven differents (Asteraceae, Brassicaceae, Chenopodiaceae, Valerianaceae,ae, Amaranthaceae, and Fabaceae) were obtained from aer of minimally processed vegetables (Odemira, Portugal).mples used were fresh-cut leafs of red ruby lettuce andlettuce (Lactuca sativa var. crispa), watercress (Nasturtiumle), swiss chard (Beta vulgaris), lambs lettuce (Valerianella), spearmint (Mentha spicata), spinach (Spinacia oleracea),cket (Diplotaxis muralis), pea (Pisum sativum), mizuna (Bras-pa var. japonica), garden cress (Lepidium sativum) and redrd (Brassica juncea). The samples were freeze-dried (Telstars-80, Terrassa, Barcelona) upon arrival and after 10 days ofrated storage (3 1 C). The freeze-dried leafs were reducede powder in a knife mill (GM 200, RETSCH, Haan, Germany)red protected from light, oxygen and heat until analysis. Thedried samples were spiked with vitamins standards in ordertify and quantify these vitamins forms in the real samples.
mples extraction
heme of the extraction procedure developed in the presento simultaneously extract WSV and FSV is shown in Fig. 1. Dur-
extraction process, samples were always protected fromexposition to light and kept on ice to minimize vitaminsation.s Ofcial Methods of Analysis of AOAC International forins (vitamin B5, B6, B9 and B12); these are highly sensi-
laborious to achieve an estimation of mean value with aision [1,6,7]. High performance liquid chromatographichods are often used for the determination of WSV andoice of the method depends on the accuracy and sen-ired, as well on the interferences encountered in the
trix. HPLC, with UV absorbance and/or uorescence well established for both FSV and WSV measurements,
some limitations for certain analytes and also lacksn complex matrices [8]. Liquid chromatographymassry (LCMS) shows more sensitivity and specicity forination of vitamins in these matrices, and permits theus analysis of multiple vitamins in a single analysis [8,9].y of the HPLC multivitamin methods found in the lit-sed only either FSV or WSV and were mainly applied
of pharmaceutical preparations or supplemented foodsnly some of them attempted the determination of nat-rring vitamins on food [8,9,1418]. Moreover, to theknowledge, the determination of a wide group of free
green-leafy fresh-cut vegetables has not been carried
ently, the objective of the present work is to develop a HPLCDADMS/MS-based method that allows a sim-uential extraction and monitoring of several free formsmins C, B1, B2, B3, B5, B6 and B9) and FSV (pro-vitamin A
E) in raw green leafy vegetables to study their contentseir evolution along a typical storage period emulatingconditions.
ls and methods
als and standard solutions
icals used were of analytical reagent grade. Vita-rds (purity > 99.0%), namely, ascorbic acid (C), thiamine
ide (B1), riboavin (B2), nicotinamide (B3), D-calciumte (B5), pyridoxine (B6), folic acid (B9), -tocopherolarotene (provitamin A), were purchased from Sigmadrid, Spain). The internal standards, hippuric acid ando-8-carotenal as well as triethylamine (TEA) and buty-xytoluene (BHT) were also from Sigma Aldrich (Madrid,monium acetate and acetic acid were from Panreac
Spain) and Scharlau (Sentmenat, Spain), respectively.MeOH), methyl tert-butyl ether (MTBE) and ethylre HPLC-grade from Lab-Scan (Gliwice, Sowinskiego,stilled water was deionized by using a Milli-Q systemBedford, MA, USA).al WSVs standard solutions and hippuric acid (1 mg/ml)re prepared in 10 mM ammonium acetate (pH 4.5),
the dark under refrigeration at 4 C until analysis.id was prepared at 5 mg/ml, thiamine hydrochloride,e, D-calcium pantothenate and pyridoxine at 1 mg/ml,t 0.05 mg/ml and folic acid at 0.01 mg/ml. Duringelopment, a mixture of WSVs were prepared daily bythe individual vitamins stock solutions with 10 mM
acetate solution with concentrations within the ranges reported by nutritional tables for the samples under3 g/ml; B1: 0.6 g/ml; B2: 0.75 g/ml; B3: 1.75 g/ml;
l; B6: 0.67 g/ml; B9: 0.33 g/ml). -tocopherol, -d trans--Apo-8-carotenal were dissolved in MeOH
Fig. 1. Eacetate.
(1 mg/of thes0.1 mg
2.2. Sa
TwfamilieAlliaceproducThe sagreen ofcinalocustawild rosica ramustaCryodorefrigeto a nand stofreeze to iden
2.3. Sa
A scwork ting thedirect degradtion scheme for WSV and FSV. US, ultrasounds; AcNH4, ammonium
nd stored at 20 C, protected from light. A mixture
-
J. Santos et al. / J. Chromatogr. A 1261 (2012) 179 188 181
Table 1Gradient elution used on water soluble vitamins analysis.
Time (min) A (%) B (%) C (%)
0 347
1017 20 30
Briey, 010 mM ammBHT. Standa1 mg/ml trapuric acid cfor trans-good sampplaced in aalways contnot rise abo15 min anda 0.45 m trated into ainjected inttent. The sotwice with also in the (14,000 g,through a 0under a N2 sand injecte(-carotene
2.4. Analysi
HPLCMliquid chromwith a diodQuantum trmatograph interface. Xlyze and st(100 2.1 mTechnologie
The metforms of WS[19] with ssolution (pHas mobile phase C. Thwas 0.2 ml/recorded thcompartmeidentify andated rst incapillary teThese condiA second semonitor thevoltage andrespectivelysures of 402 units andscan time w
system was operated in selected reaction monitoring (SRM). SRMparameters were optimized by direct injection of standards. Twoion transitions were monitored for identication but only the mostintense product ion for each precursor ion was used for quantica-
he van en.
alys
FSVatogr
dion (5 ny). ed band yed at us, 100in ared
noid
ethod
ecovepeart sim, the
s waed aion.
and dy wampe opeterson waminand tion o
one g/metecl-to-
Accutage
atisti
SPS and
ne-wncesnces90 10 090 10 050 0 5050 0 500 100 00 100 0
90 10 090 10 0
.250 g of each sample was rst extracted with 16 ml ofonium acetate/methanol 50:50 (v/v) containing 0.1%
rd solutions (40 l of 1 mg/ml hippuric acid and 40 l ofns--Apo-8-carotenal) were added at this stage. Hip-oncentration added was of 2.5 g/ml and of 3.3 g/ml-Apo-8-carotenal. After 15 min of shaking to achievele dispersion in the extraction liquid, samples weren ultrasound bath for 15 min. Bath temperature wasrolled with ice to guarantee that water temperature didve 25 C. The samples were centrifuged at 14,000 g for
the supernatant was withdrawn and ltered throughnylon lter. One ml of the supernatant was concen-
nitrogen stream to evaporate the methanol and it waso a HPLCMS/MS system to determine the WSV con-lid residue from the rst extraction was re-extractedethyl acetate containing 0.1% BHT (6 + 6 ml) for 15 min,ultrasonic bath. Finally, the samples were centrifuged
15 min) and the supernatants combined and ltered.45 m nylon lter. The extract was taken to drynesstream. The residue was dissolved in 3 ml of ethyl acetated in a HPLCDAD system to monitor the pro-vitamin A) and vitamin E (-tocopherol) contents.
s of water soluble vitamins (HPLCDADMS/MS)
S/MS analyses of WSV were performed using an Accelaatograph (Thermo Scientic, San Jose, CA) equippede array detector (DAD), an autosampler and a TSQiple quadrupole analyzer (Thermo Scientic). The chro-was coupled to a MS analyzer via an electrospray (ESI)calibur software (Thermo Scientic) was used to ana-ore the data. The column used was an ACE-100 C18m i.d., 3 m particle size) (Advanced Chromatographics, Aberdeen, UK).hod developed to simultaneously separate the sevenV in a single run was based on the work of Vazquez et al.ome modications, using 10 mM ammonium acetate
4.5) as mobile phase A, MeOH with 0.1% acetic acidphase B and MeOH with 0.3% acetic acid as mobilee gradient used is described in Table 1. The ow ratemin whereas the injection volume was 10 L. The DADe spectra from 200 to 680 nm. Column and autosamplernts were thermostated at 20 and 5 C, respectively. To
quantify the WSV, the mass spectrometer was oper- the negative ESI mode, for 1.7 min. Spray voltage andmperature were set at 3000 V and 250 C, respectively.
tion. TcollisioTable 2
2.5. An
Thechrompler, acolumGermapublishv/v/v) emploried ou43 min1 ml/mmonitocarote
2.6. M
A rusing Sried ouof thempoundextractextractbeforeery stusame s
Oncparamprecisiied vit(n = 5) centraof eachB3: 0.7its of da signang/ml.percen
2.7. St
IBMoration95%. OdiffereDifferetions were the most suitable for ascorbic acid detection.gment of 10.3 min followed, using positive ESI mode to
presence of the other WSV. In this segment the spray capillary temperature were set at 5000 V and 250 C,. Nitrogen was used as sheath and auxiliary gas at pres-
and 19 a.u., respectively. Ion sweep gas pressure was collision gas (Ar) pressure, 1.3 mTorr. Scan width andere xed at 0.020 (m/z) and 0.1 s, respectively, and the
3. Results
Two difffree forms oone deals wanalysis anmethods foforms. Althlues corresponding to the tube lens offset voltage andergy for each selected ion transitions are indicated in
is of fat soluble vitamins (HPLCDAD)
determination was performed in an Agilent 1100 HPLCaph (Agilent, Palo Alto, CA) equipped with an autosam-de array detector (DAD) and an YMC C30 analyticalm particle size, 250 4.6 mm i.d.) (YMC, Schermbeck,
The method used was based on the method previouslyy Jaime et al. [20]. Methanol/water/TEA (90:10:0.1,MTBE/methanol/water/TEA (90:6:4:0.1, v/v/v/v) weres mobile phases A and B, respectively. Elution was car-ing the following gradient: 0 min, 6.5%B; 8 min, 6.5%B;%B; 46 min, 6.5%B; 55 min, 6.5%B. The ow rate was
nd the injection volume 10 L. Chromatograms wereat 295 nm for -tocopherol content and at 450 nm forcompounds.
validation
ry study for each vitamin was performed as followsmint as model sample: 2 sets of extractions were car-ultaneously following the optimized procedure. In one
sample spiked with a known amount of the target com-s extracted whereas in the other, the same sample wasnd spiked with the same concentration of vitamins afterPeak areas of vitamins from the same sample spikedafter the extraction process were compared. The recov-as performed in three different days, always with thele in order to check its repeatability.timum separation conditions and MS/MS detection
were optimized, instrumental intraday and interdayas assessed. For this purpose, the mixture of the stud-s was consecutively injected ve times in the same dayalso in three different days (n = 15). The vitamins con-f this mixture was in the middle range of the linearity
(vitamin C: 1.32 g/ml; B1: 0.24 g/ml; B2: 0.3 g/ml;l; B5: 0.33 g/ml; B6: 0.27 g/ml; B9: 0.13 g/ml). Lim-
tion (LOD) and quantication (LOQ) were calculated asnoise ratio of 3 and 10, respectively, and expressed inracy of the method was assessed by using the recoverys of spiked samples.
cal analysis
S Statistics software v.19 was employed for data elab- statistical analysis using a level of signicance set atay analysis of variance (ANOVA) was employed to assess
among vitamin contents at the different storage times. were considered statistically signicant if p < 0.05.
and discussion
erent approaches can be followed to determine severalf water- and fat-soluble vitamins in real samples. Firstith the separation of all the vitamin forms in one singled the second with the optimization of two individualr a more accurate measurement of the different vitaminough several works have been published dealing with
-
182 J. Santos et al. / J. Chromatogr. A 1261 (2012) 179 188
Table 2Retention times (min) and MS/MS detection parameters for the water soluble vitamins analyzed.
Peak Vitamin tR (min) Precursor ion (m/z) SRM transitions Tube lens offset (V)
Quantier ion (m/z)ion en
Qualier ion (m/z)
1 (14) 2 (10)3 (11) 4 16) 5 (12) 6 (10) 7 (13) 8 (23)
the simultagle chromatnone of thebut insteadand parentemain goal omethod for leafy vegetcold storagaccurate quMain problsingle run uthe differenorganic acidamounts), wconditions, for both groefforts weremethods ththat could carried out HPLCDAD
3.1. HPLCM
During tseven WSVphase colum1.9 m part3 m partica better septhat were wacid (vitamanalysis timWSV studiethe separatachieved. AHILIC separto Goldschmnicotinamidthis type ofstudied herdevelopme
Althouglished methmethanol aalso tested.formic acid)aqueous mowas also tesdeterminatin this appli
nol wto sucorbi, B6,
promtic ay, dif
wast elufcieg th
of ble 1and gs, as e thearamrd sotudit ionst abt iont ionplo
2 shound
empic aciizatndarred ate (
paraname5000re (0luesed ine theetersecisih the(collis
Ascorbic acid (C) 1.41 174.9 [MH] 115.2Thiamine (B1) 1.81 265.1 [M+H]+ 122.1Pyridoxine (B6) 2.30 169.9 [M+H]+ 152.1Nicotinamide (B3) 3.26 123.0 [M+H]+ 80.3 (Pantothenic acid (B5) 3.72 220.0 [M+H]+ 202.1Hippuric acid (IS) 5.90 180.1 [M+H]+ 105.2Folic acid (B9) 6.24 442.0 [M+H]+ 294.9Riboavin (B2) 6.82 377.1 [M+H]+ 243.0
neous determination of several WSV and FSV in a sin-ographic run with DAD and MS detectors [15,19,21,22],m refer to the analysis in real complex food samples
analyze pharmaceutical preparations, functional drinksral nutrition admixtures. As mentioned previously, thef the present work was to develop a useful and rapidthe determination of both types of vitamins in real greenables and to use it to follow vitamin evolution duringe; therefore, the method should be able to provide anantication of all the free vitamin forms and its changes.ems arisen in the determination of WSV- and FSV- in asing real complex samples deal with coelutions amongt vitamins and with other major components such ass and polyphenols (that obviously are found in higherith the difculty of selecting the best MS detection
including the ionization technique, which are differentups of vitamins; etc. Considering all these aspects, our
focused in the development of two different sequentialat could provide good quantication capabilities andbe employed in parallel. Therefore, optimization wasfor the separation of WSV by HPLCMS/MS and FSV by; and for the extraction protocol to recover both.
S/MS determination of water-soluble vitamins
he optimization procedure for the separation of the studied, the performance of two different reversedns was tested. A short UPLC C18 column (50 2.1 mm,
icle diameter) and a longer C18 column (100 2.1 mm,le size) were compared. The longer column providedaration among the tested compounds, including thoseeakly retained in the C18 stationary phase like ascorbic
in C) and thiamine (vitamin B1), while maintaining shortes (7 min). In Fig. 2, the SRM chromatograms of the 7d and hippuric acid are shown. As it can be observed,ion of all the studied compounds was appropriatelylthough other type of columns adequate to performations had been used to separate WSV [5], accordingidt and Wolf [5], thiamine (B1), pyridoxine (B6) ande (B3) were the only vitamins that could benet from
separations. Thus, considering the wider group of WSVe, the applicability of the HILIC approach in the present
methauseful like asmins Calso to[8]. Acequentl0.3%. Itthe lassome eallowinportion(see Tapeaks pound
Onction pstandawere sproducthe moproducproducwas emTable compovaluesascorbESI ionnal stamonitotothenrest ofmins, (3000pressuThe vadescrib
Oncparamday prthrougnt would be of limited value.h the method was developed based on a previously pub-od [19] using 10 mM ammonium acetate (pH 4.5) ands mobile phases, other alternative mobile phases were
The use of acidied water (using 1% of either acetic or did not improve the obtained results using the bufferedbile phase. Regarding the organic modier, acetonitrileted. This solvent has been used in several multivitaminion methods present in the literature [5,11,23], althoughcation the separation was not improved. Thus, the use of
WSV standfor three diThe obtainefor the sambetween 0.days were sretention tiareas and 2also testedity (R2 > 0.9ergy, V) (collision energy, V)
87.3 (18) 76144.1 (16) 48134.1 (19) 5578.3 (24) 67184.1 (12) 6077.4 (10) 59176.0 (34) 69147.1 (37) 93
as maintained. The acidication of the solvent could beppress the dissociation of vitamins with an acidic group,c acid, pyridoxine, pantothenic acid and folic acid (vita-
B5 and B9, respectively), improving peak shapes, andote a better ionization of the basic sites of all vitamins
cid provided better peak shapes than formic acid. Conse-ferent levels of acetic acid were tested, namely 0.1% and
observed that the higher proportion of acid improvedting peaks, whereas the less retained compounds lostncy. Thus, it was decided to employ a ternary system,e introduction of more acidied methanol as the pro-the organic modier increased during the separation). Under these conditions, sufcient resolution betweenood peak shapes were obtained for all the studied com-it can be observed in Fig. 2.
separation was optimized, the different MS/MS detec-eters were studied. By using the direct infusion oflutions, both positive and negative ESI ionization modesed for the production of characteristic precursor ands of each compound. Precursor ions were selected asundant mass-to-charge (m/z) values. Subsequently, twos for each precursor were chosen. The most intense
was used for the quantication whereas the otheryed to conrm the identity of the studied compounds.ws the precursor and product ions selected for eachas well as the collision energies and tube lens offsetloyed for their detection. As it can be observed, onlyd (vitamin C) showed more intense ions in the negativeion mode. The rest of vitamins as well as the inter-d were detected as [M+H]+. Thiamine (vitamin B1) wasas the loss of associated chloride, whereas calcium pan-vitamin B5) was determined as pantothenic acid. Themeters involved in the ESI detection of the studied vita-ly, capillary temperature (250350 C), spray voltage
V), sheath gas pressure (2060 a.u.) and auxiliary gas40 a.u.), were optimized using a univariate method.
that provided the best response for all vitamins are Section 2.4.
optimum separation conditions and MS/MS detection were selected, the instrumental intra-day and inter-on was evaluated. Intra-day precision was assessed
consecutive injection (n = 5) of a mixture of the seven
ards consecutively in the same day, that was repeatedfferent days for inter-day precision evaluation (n = 15).d results are summarized in Table 3. The RSD values,e day, ranged between 0.9% and 7.2% for peak areas and1 and 2.0% for retention times. The RSD values betweenlightly higher (3.69.1% for peak areas and 0.31.5% formes), although they were always below 10% for peak% for retention times (see Table 3). The linear range was
using a least square t, which showed a good linear-9) for all WSV, within the range selected. Instrumental
-
J. Santos et al. / J. Chromatogr. A 1261 (2012) 179 188 183
LODs (0.18noise (S/N)WSV as 10 tdata veriefor a rapid a
3.2. HPLCD
The meti.e., provitawas based
Table 3Method evalua
Vitamin
Ascorbic acidThiamine (BRiboavin (BNicotinamidPantothenicPyridoxine (Folic acid (BHippuric aci-tocophero-carotene (-Apo-8-ca
tR, retention tia Mean of Fig. 2. HPLCMS/MS chromatogram of the seven WSV standards and the IS (hippu
42.28 ng/ml) calculated as three times the signal-to- ratio and LOQs (0.56128.13 ng/ml) obtained for eachimes the S/N ratio, are also presented in Table 3. Thesed the suitability of the optimized HPLCMS/MS methodnd sensitive detection of the 7 WSV in a single run.
AD determination of fat-soluble vitamins
hod chosen to determine the free fat-soluble vitamins,min A (-carotene) and vitamin E (-tocopherol),on a previously published method [20,24] with some
modicatiowas employextract. Thiseparate dtocopherolsC18 stationselectivity and E [28]. the gradientions betwereal sample
tion parameters: LOD and LOQ values, repeatability, linearity and mean recovery percen
Detection LOD (ng/ml) LOQ (ng/ml) Repeatability (RSD %)
Intra-day Inter-day
Area tR Area tR
(C) MS/MS 42.28 128.13 5.6 2.0 6.3 2.0 1) MS/MS 0.79 2.41 3.1 0.7 3.6 0.7 2) MS/MS 0.07 0.20 2.5 0.2 4.3 0.2 e (B3) MS/MS 4.35 13.17 2.3 0.5 9.8 0.5
acid (B5) MS/MS 7.67 23.25 0.9 0.6 9.1 0.6 B6) MS/MS 0.18 0.56 1.7 0.6 9.1 0.6 9) MS/MS 0.63 1.90 7.2 0.1 8.4 0.0 d (IS) MS/MS 4.93 14.9 1.5 0.3 5.7 0.5 l (E) UV 170 520 0.7 0.1 2.1 0.5 pro-A) UV 70 200 0.4 0.1 0.3 0.2 rotenal (IS) UV 17 51 0.5 0.2 1.8 0.4
me.ve extractions.ric acid) under the optimum analysis conditions.
ns. A reversed phase column with C30 stationary phaseed to separate the compounds present on the lipophilics type of column has demonstrated to be capable toifferent geometric isomers of both carotenoids and
in different matrices [14,2527]. In fact, compared toary phases, the less polar C30 phases exhibit superiorfor isomer separation of carotenoids, and vitamins AStarting from the conditions already published [20,24],t elution program was modied in order to avoid coelu-en -tocopherol and some carotenoids present on thes. An isocratic period was included at the beginning
tages.
Linearity Mean recovery
Range tested (ng/ml) R2 Added (ng/ml) %a RSD
705280 0.993 3300 102.8 10.61.5960 0.993 600 87.3 8.50.91500 0.996 750 93.9 7.4
42800 0.995 1750 85.4 3.6201670 0.998 830 88.2 12.60.8530 0.997 670 82.8 8.90.4530 0.995 330 104.8 16.63.15000 0.998 2500 100.5 4.6
6250100,000 0.999 50,000 87.5 4.96250250,000 0.999 50,000 105.3 12.56250100,000 0.999 3300 88.7 2.5
-
184 J. Santos et al. / J. Chromatogr. A 1261 (2012) 179 188
Fig. 3. (A) Rec l vs 8after the one (
of the elutiparticular c
Besides,analyzed, wsamples (seof the seleccorrespondmitted the extract to spectra simvalues wereboth paramretention ticurves show
3.3. Optimi
A sequenof WSV andThe methodrial using ufollowing aextraction ototal time. volume of v/v) contaivitamins. Thsolution another hand,vitamins likfact that BHtect fat solusample fromthis was nobeen descrimight potenmetaphospto cause iobined use ostudied vitapromote thinto the sotemperaturprocedurescentrifugati
a N2 usineter
actiot wi
was tion ate ayclesgh th
sin proccetas obtof sohe pwas (6 + 6to jution ct prome. . Thiion, dcern
is og th
Howthe deratoveries of water-soluble vitamins (%) obtained after 1 vs 2 extraction cycles (16 m12 ml) and two (6 + 6 ml) extraction cycles.
on, which signicantly improved the separation. Theonditions employed are described in Section 2.4.
the DAD detector was sensitive enough for both FSVithin the concentration range expected in the studiede Table 3). Although identication and quanticationted compounds was achieved by comparison with theiring commercial standards, the DAD detector also per-spectral analysis of other compounds present in theidentify other tocopherols and carotenoids based onilarity. The intra-day (n = 5) and inter-day (n = 15, 3 days)
also assessed. Low RSD (%) values were obtained foreters being always below 2.1 and 0.6% for peak areas andmes, respectively (see Table 3). The linear calibrationed a high coefcient of determination (R2 > 0.999).
zation of the extraction procedure
tial and simple method for the extraction of free forms FSV from different vegetables is proposed in this work.
is based on the sequential extraction of the plant mate-ltrasounds. The method optimization was carried out
univariate procedure in order to achieve the maximumf the free forms of the studied vitamins in the shortestBasically, the freeze-dried material was mixed with a10 mM ammonium acetate (pH 4.5)/methanol (50:50,ning 0.1% BHT in order to obtain the water-solublee use of methanol in the mixture allowed the better dis-
d extraction of the less polar studied WSV [8,21]. On the BHT was included to prevent the oxidation of the some
under 15 min
To dof extrried ou16 ml extrac(triplicutive calthouThus, awholeethyl aResult12 ml WSV, tcycles cycles pared extracdid notion tistreamdetect
Concedureallowinesters.due to the gene vitamin C or -carotene and -tocopherol. Despite theT is not soluble in water and is normally used to pro-ble compounds, the inclusion in this stage protected the
oxidation since the beginning of extraction. Althought the best stabilizer for vitamin C, its presence hasbed to partly inhibit its oxidation [8]. Other chemicalstially have a higher protective effect in this process, like
horic acid or EDTA, but their presence is also referredn suppression in MS detection [8,29]. Thus, the com-f BHT and methanol, provided a compromise for all themins. A 15 min ultrasonic extraction was performed toe dissolution of the vitamins from the vegetal matrixlvent. Due to the labile nature of most vitamins, thee of the ultrasound bath was always controlled, and all
were performed without direct exposure to light. Afteron of the extracts, the supernatant was evaporated
determinatleafy vegetof FSV esteied vegetabfrom the ta
3.4. Extract
To assesstudy was out simultaSection 2.3.with a knowhereas inwith the sacates of the 8 ml) for WSV. (B) Recoveries (%) of fat-soluble vitamins obtained
stream. The solid residue was then re-extracted forg ethyl acetate as extracting solvent to recover FSVs.mine the optimum volume of solvents and the numbern cycles suitable for all vitamins, optimization was car-th spiked samples. Concerning the extraction of WSV,xed as solvent volume and the possibility of using an
step or two extraction steps (8 ml each) was studiedssays). As it can be observed in Fig. 3A, using two consec-, the amount of extracted vitamins was slightly higher,e differences were not statistically signicant (p > 0.05).gle extraction step was selected in order to make theedure faster. On the other hand, different volumes ofte (8, 12 and 15 ml) were tested for the FSV extraction.ained showed an improvement in the nal results whenlvent were used compared to 8 and 15 ml. Similarly toossibility of dividing the extraction volume in differentstudied. It was observed that the use of 2 extraction
ml) signicantly improved the results obtained com-st one extraction cycle (see Fig. 3B). The use of a thirdycle was also explored but discarded considering that
duce signicant gains while increasing the total extrac-The extracts obtained were concentrated under a N2s step was especially necessary for the -tocopherolue to its low signal in the DAD detector.ing the determination of FSV, a hot saponication pro-ften used to remove chlorophylls and lipids whilee release of the free vitamin forms that are found asever, in our approach, this procedure was not suitableegradation of carotenoids by thermal isomerization andion of some artifacts that can interfere in the vitamins
ion. Moreover, this step is not recommended for greenables, tomatoes and carrots, due to their low contentsrs [14,30]. Instead, the chlorophylls found in the stud-les were, in our optimized method, properly separatedrget compounds during the chromatographic analysis.
ion recovery study
s the suitability of the complete method, a recoveryperformed. Namely, 2 sets of extractions were carriedneously following the optimized procedure described in
In one of them, a vegetable sample (spearmint) spikedwn amount of the target compounds was extracted
the other, the same sample was extracted and spikedme concentration of vitamins after extraction. Tripli-
extraction protocols were performed and the extracts
-
J. Santos et al. / J. Chromatogr. A 1261 (2012) 179 188 185
Fig. 4. Mean amount of WS
were analyzvalues obtaeffect was oranged betwered as appcompounds
3.5. Green l
Once theand FSV wadifferent fremine how conditions different prrocket, swispea leaves, were no prand after a 1determinatmin per 100found in hiacid and viples variedpea leaves tpea leaves,ples on thisvitamin C sstrong decrOther sampment thesesignicant ia signicanshown thatwork, ascorto dehydroain the planterating tocothe decreasvegetables
B-groupied vegetabvitamins arfoods and c
generally the richest in all the studied samples. In fact, in all cases,the amount of free vitamin B5 detected in the samples after the10-days storage period was always signicantly higher than the
ts found before. These increments were in the range of a 2-creas
(e.gservom cy a chsphaaminole ving m
afte freshceptioces, shrs of post-c aci, at
spinworkm aer hples100 han feren
the reneriboost ans in(see
n Tansid
sambles
in soial ged po
theatogrns inomperol content of water-soluble vitamins of sample spiked with the sameV standards before and after extraction.
ed using the two methods by duplicate. Comparing theined, it was possible to deduce that no signicant matrixbserved during the extraction (Fig. 4). The recoverieseen 83% and 105% (see Table 3) which was consid-
ropriate for a method that simultaneously determines with different structures and chemical properties.
eafy vegetables analysis
complete method of extraction and analysis of WSVs optimized, the procedure was applied to the study ofsh-cut green leafy vegetables. The aim was to deter-the vitamin contents evolved during storage undersimilar to those found in the market. To do that, 12oducts (namely, green lettuce, ruby red lettuce, wilds chard, watercress, spinach, lambs lettuce, spearmint,mizuna, red mustard and garden cress), for which thereevious published results, were analyzed upon arrival0-days storage at 3 C. The results obtained from these
ions are summarized in Table 4, expressed as g of vita- g fresh weight (fw). As it can be observed, the vitaminsghest amounts in general were provitamin A, ascorbictamin E. The amounts of vitamin C found in the sam-
in great extent, from more than 150 mg/100 g fw ino undetectable amounts in swiss chard. Together with
garden cress and wild rocket were the richest sam-
contenfold inhigherthis obacid frated bor phothe vitthe whproducetablesload ofbe susfact, prcuttingsourceof the
Folilettuceacid inlished the forthe othied sam1.5 g/lower tthe dif
Forples. Gthan rin almvitamimin B1cress ihigh costudiedvegetamentsmicrobobservexplainchromvitamiother ctocoph component. For most of the samples, the contents ofignicantly decreased after the 10-days storage. Veryeases were observed for red mustard and garden cress.les, such as wild rocket or watercress did not experi-
great losses. Interestingly, a slight but no statisticallyncrease (p > 0.05) was observed for pea leaves, whereast increment was found for lambs lettuce. It has been
over a storage period similar to that employed in thisbic acid present in fresh-cut vegetables can be oxidizedscorbic acid as a result of the function of this compound
as protector against oxygen species [31], even regen-pherols [32]. Vitamin C could be also responsible fore in enzymatic browning observed in some fresh-cutduring storage [33].
vitamins were found in lower amount in all the stud-les, as it could be expected considering that most of Be mainly contained in higher extent in animal-derivedereals. Among them, pantothenic acid (vitamin B5) was
high resolvdeterminat
The leveby far high(-carotenemaximum poorest soulower thanthe levels o10-days stotuce and spwere obserpresented aprovitamin
The amobles after s3663 g/10(p < 0.05) oe, although for some vegetables, the increase was even., Mizuna). There are several possible explanations toation; on one hand, a catabolic release of pantothenicoenzyme A (CoA) and acyl carrier protein (ACP) medi-ain of enzymatic steps including hydrolases (for ACP)
tases and pyrophosphatases (for CoA) [34] thus freeing B5 from CoA; and on the other hand an increase onitamin B5 content due to a higher vitamin B5-bacterialicrobiological load of the green leafy ready-to-eat veg-
r storage. A recent study showed an increase in bacterial-cut salads commercialized in Portugal [35] that might
ble of synthesizing higher amounts of this vitamin. Inses associated to fresh-cut products, such as washing,edding and slicing, have been identied as potentialmicrobial contamination [36]. An increase as a resultharvest metabolism should not be also discarded [37].d (vitamin B9) was only found in spinach and lambslevels lower than 11.5 g/100 g; the contents on folicach were by far lower than those reported in other pub-s [38], although it should be kept in mind that only
s folic acid was determined in the present work. Onand, pyridoxine (vitamin B6) was found in all the stud-, although maintaining very low levels, even as low asg. In general, the amount of vitamin B6 after storage wasbefore, with some exceptions. Even in those latter cases,ces were small.est of B-group vitamins, the levels varied among sam-ally, nicotinamide (vitamin B3) was more abundantavin and thiamin (vitamins B2 and B1, respectively)ll samples. For some samples, the contents on thesecreased after the 10-days storage time, mainly for vita-
wild rocket, spinach, spearmint, mizuna and gardenble 4). Nevertheless, these increments were not tooering the amounts of these free vitamins found in theples. Similar behaviors have been observed for other
at the end of prolonged storage periods [39]. The incre-me of them, such as vitamin B2, might be related torowth in the samples [39]. Besides, other authors havest-harvest synthesis of these vitamins, which could also
increments found [37]. Fig. 5 shows the correspondingams obtained during the quantication of the studied
garden cress. As it can be observed in Fig. 5B, someounds could be tentatively assigned to carotenoid orfamily, thanks to the use of a DAD together with theing power of the C30 stationary phase employed in thision.ls of fat-soluble vitamins found in the samples wereer than those of the B-group vitamins. Provitamin A) was the most abundant FSV in all samples reachingvalues for pea leaves, garden cress and spinach. Therce of provitamin A was ruby red lettuce, with values
3.5 mg/100 g fw. For 4 out of the 12 samples studied,f provitamin A did not varied signicantly after therage period, namely, mizuna, pea leaves, lambs let-inach. For other samples, small to moderate decreasesved, being green lettuce the fresh-cut vegetable that
higher percentage of loss of its initial content on this (ca. 50%).unt of vitamin E (-tocopherol) in the studied vegeta-torage ranged from 321 g/100 g fw for watercress to0 g fw for pea leaves. A statistically signicant increasen the concentration of vitamin E after storage was
-
186J.
Santos et
al. /
J. Chrom
atogr. A
1261 (2012) 179 188
Table 4WSV and FSV contents (free forms) found in the fresh-cut green leafy vegetables at the indicated storage times. Values shown as mean sd relative to fresh weight (fw). Asterisks indicate values not statistically different (p > 0.05)between day 1 and day 10.
Samples Ascorbic acid (C) Thiamine (B1) Riboavin (B2) Nicotinamide (B3) Pantothenic acid (B5) Pyridoxine (B6) Folic acid (B9) -tocopherol (E) -carotene (provit A)mg/100 g fw sd g/100 g fw sd g/100 g fw sd g/100 g fw sd g/100 g fw sd g/100 g fw sd g/100 g fw sd mg/100 g fw sd mg/100 g fw sd
Green lettuceDay 1 0.1 0.0* 79 27* 28 4* 130 20 147 24 2 0.2 n.d. n.d. 5.5 0.3Day 10 0.1 0.0* 80 3* 30 5* 80 6 215 22 3 0.3 n.d. n.d. 2.6 0.2
Ruby red lettuceDay 1 0.1 0.0* 68 15* 28 4 195 13 77 19 15 2 n.d. 1.0 0.1 3.4 0.0Day 10 0.1 0.0* 55 6* 18 3 55 3 127 16 6 0 n.d. 0.6 0.0 2.0 0.0
Wild rocketDay 1 85.1 5.7 11 2 43 13* 143 19 309 75 10 1 n.d. 1.6 0.1* 9.3 0.9Day 10 3.6 0.7 24 0.8 54 4* 85 6 790 15 28 2 n.d. 1.4 0.0* 6.8 0.6
Swiss chardDay 1 n.d. 14 2 120 6 201 12 247 7 22 1 n.d. 1.2 0.1* 7.2 0.2Day 10 n.d. 11 2 111 4 139 19 490 42 46 3 n.d. 1.0 0.1* 6.3 0.2
WatercressDay 1 59.6 2.3 70 21* 143 7 111 10 263 3 13 0.1* n.d. 2.5 0.2 8.3 0.0Day 10 48.2 0.8 99 11* 163 9 77 5 560 63 16 2* n.d. 0.3 0.1 9.3 0.1
SpinachDay 1 14.4 2.8 194 14 257 21 167 12* 345 20 8 0.2 1 0.1* 2.9 0.1 11.2 1.2*Day 10 1.7 0.0 244 24 223 17 179 15* 526 59 13 0.6 1 0.1* 3.6 0.3 13.4 1.5*
Lambs lettuceDay 1 59.1 10.0 131 6* 169 32 241 46* 585 38 20 5 11 0.6 3.1 0.2* 9.2 0.1*Day 10 13.7 8.3 130 13* 111 8 194 5* 698 45 9 1 6 1 3.2 0.4* 9.4 0.1*
SpearmintDay 1 0.5 0.0 121 10 169 25* 282 7 557 31 8 0.9 n.d. 1.4 0.0* 10.6 1.3Day 10 0.3 0.0 216 13 144 14* 239 13 1622 118 5 0.1 n.d. 1.5 0.2* 8.8 0.0
Pea leavesDay 1 154.0 2.8* 178 15* 112 14* 131 2 637 30 20 1 n.d. 2.7 0.1 14.4 0.1*Day 10 174.0 19.3* 189 6* 128 11* 104 5 1186 33 48 1 n.d. 3.7 0.1 13.6 0.6*
MizunaDay 1 33.0 1.6 6 0.5 53 5* 58 8 125 11 8 0.5 n.d. 1.1 0.0 7.8 0.2*Day 10 14.7 1.2 25 4 56 5* 35 3 693 47 21 3 n.d. 2.7 0.0 9.4 0.8*
Red mustardDay 1 43.9 1.1 12 2 64 4 108 13 113 7 5 1* n.d. 2.1 0.2* 8.2 0.1Day 10 0.1 0.0 18 2 28 1 67 6 278 23 4 0.7* n.d. 1.9 0.0* 6.3 0.0
Garden cressDay 1 10.1 1.2 36 3 125 10* 191 21 587 18 4 0.2 n.d. 1.9 0.2 12.7 0.2Day 10 0.2 0.0 102 5 122 3* 159 15 1222 100 17 1 n.d. 2.5 0.1 10.8 0.1
-
J. Santos et al. / J. Chromatogr. A 1261 (2012) 179 188 187
garden
detected insamples webeen alread
4. Conclus
In this stwater solubsoluble vitaLCDAD, reused to quables before (3 C). The has revealedeterminatvitamins, wples. The prin less thanand determsuitable LOprecisions.
Acknowled
This wo(Programa S2009/AGRgrateful to FPOPH-QRENthank MICINalso thank Istudy.
nces
.F. Baa Rato. FingHerreg andFig. 5. Chromatograms of WSV (A) and FSV (B) from an extracted sample (
spinach, pea leaves, mizuna and garden cress. Thesere also among the richest on vitamin C, which, as it hasy mentioned, is able to regenerate vitamin E [32].
ions
Refere
[1] G.MBoc
[2] P.M[3] M.
plinudy, a methodology to extract and quantify several freele vitamins (vitamins C, B1, B2, B3, B5, B6 and B9) and fatmins (vitamin E and provitamin A) by LCMS/MS andspectively, has been optimized. The method has beenntify the vitamins level in 12 different fresh-cut vegeta-and after a 10-days storage period under refrigerationoptimized sequential extraction-analysis procedured as an appropriate methodology for the sequentialion of a whole range of free water and fat-solubleith different chemical structures, in real complex sam-ocedure allows a complete analysis of all the vitamins
100 min of total analysis time, including extractionination, with recoveries ranging from 83 to 105%,D and LOQ and appropriate intra-day and inter-day
gments
rk has been nanced by CSD2007-00063 FUN-CFOODCONSOLIDER-INGENIO 2010) and Project ALIBIRD
-1469 (Community of Madrid) projects. J. Santos isCT for a PhD grant (SFRH/BD/66476/2009) nanced by
and subsidized by ESF and MCTES. M.H. would like toN for a Ramn y Cajal research contract. The authors
berian Salads S.A for the vegetables samples used in this
[4] K.W. PhinAnal. Che
[5] R. Goldsc[6] P. Chen, R[7] C. Blake, [8] A. Gentili
Rapid Co[9] A. Lepora
Chim. Ac[10] S. Albal-
J. Chroma[11] O. Heudi,[12] B. Lu, Y.
225.[13] M. Huang[14] A. Gentili[15] J.A. Mend
Ibnez, J.[16] C.-H. Che
Inst. Chem[17] M. Ciulu,
Talanta 8[18] I. Koncza[19] R. Vazqu
EJHP Sci. [20] L. Jaime,
F.J. Senor[21] B. Klejdu
Kubn, A[22] H. Li, F. C[23] P. Chen, W[24] D.E. Breit[25] H.M. Akh[26] L.Y. Kong
(2011) 86[27] W.J. Lessi[28] A. Klaus, cress). Peak id: t: tocopherols; c: carotenoids.
ll, Vitamins in Foods. Analysis, Bioavailability, and Stability, CRC Press,n, 2006, p. 3.las, U. Faure, P.J. Wagstaffe, Fresenius, J. Anal. Chem. 345 (1993) 180.ro, A. Cifuentes, E. Ibnez, in: J. Pawliszyn (Ed.), Comprehensive Sam-
Sample Preparation, Elsevier, Amsterdam, 2012, p. 3.
ney, C.A. Rimmer, J.B. Thomas, L.C. Sander, K.E. Sharpless, S.A. Wise,m. 83 (2010) 92.hmidt, W. Wolf, Anal. Bioanal. Chem. 397 (2010) 471.. Atkinson, W.R. Wolf, J. AOAC Int. 92 (2009) 680.
Anal. Bioanal. Chem. 389 (2007) 63., F. Caretti, G. DAscenzo, S. Marchese, D. Perret, D. Di Corcia, L. Rocca,mmun. Mass Spectrom. 22 (2008) 2029.ti, D. Catellani, M. Suman, R. Andreoli, P. Manini, W.M. Niessen, Anal.ta 531 (2005) 87.Hurtado, M.T. Veciana-Nogus, M. Izquierdo-Pulido, A. Marin-Font,togr. A 778 (1997) 247.
T. Kilinc , P. Fontannaz, J. Chromatogr. A 1070 (2005) 49.Ren, B. Huang, W. Liao, Z. Cai, X. Tie, J. Chromatogr. Sci. 46 (2008)
, D. Winters, R. Crowley, J. AOAC Int. 92 (2009) 1728., F. Caretti, J. Chromatogr. A 1218 (2011) 684.iola, F.R. Marin, F.J. Senorns, G. Reglero, P.J. Martn, A. Cifuentes, E.
Chromatogr. A 1188 (2008) 234.n, Y.-H. Yang, C.-T. Shen, S.-M. Lai, C-M.J. Chang, C.-J. Shieh, J. Taiwan. Eng. 42 (2011) 124.
S. Solinas, I. Floris, A. Panzanelli, M.I. Pilo, P.C. Piu, N. Spano, G. Sanna,3 (2011) 924.k, P. Roulle, Food Res. Int. 44 (2011) 2339.ez, M-D.L. Hoang, J. Martin, Y.A. Yahia, H. Graffard, F. Guyon, B. Do,15 (2009) 28.J.A. Mendiola, E. Ibnez, P.J. Martin-lvarez, A. Cifuentes, G. Reglero,ns, J. Agric. Food Chem. 55 (2007) 10585.s, J. Petrlov, D. Potesil, V. Adam, R. Mikelov, J. Vacek, R. Kizek, V.nal. Chim. Acta 520 (2004) 57.hen, Chromatographia 54 (2001) 270.
. Wolf, Anal. Bioanal. Chem. 387 (2007) 2441.haupt, Food Chem. 86 (2004) 449.tar, M. Bryan, Food Chem. 111 (2008) 255., B.G. Su, Z.B. Bao, H.B. Xing, Y.W. Yang, Q.L. Ren, J. Chromatogr. A 121864.n, S.J. Schwartz, J. Agric. Food Chem. 45 (1997) 3728.Trends Anal. Chem. 17 (1998) 648.
-
188 J. Santos et al. / J. Chromatogr. A 1261 (2012) 179 188
[29] K.M. Phillips, M.T. Tarrago-Trani, S.E. Gebhardt, J. Exler, K.Y. Patterson, D.B.Haytowitz, P.R. Pehrsson, J.M. Holden, J. Food Compos. Anal. 23 (2010)253.
[30] A. Rodrguez-Bernaldo de Quirs, H.S. Costa, J. Food Compos. Anal. 19 (2006)97.
[31] A. Martnez-Snchez, M.C. Luna, M.V. Selma, J.A. Tudela, J. Abad, M.I. Gil,Postharvest Biol. Technol. 63 (2012) 1.
[32] A. Conte, G. Conversa, C. Scrocco, I. Brescia, J. Laverse, A. Elia, M. Del Nobile,Postharvest Biol. Technol. 50 (2008) 190.
[33] E. DeglInnocenti, A. Pardossi, F. Tognoni, L. Guidi, Food Chem. 104 (2007)209.
[34] G.F. Combs, The Vitamins: Fundamental Aspects in Nutrition and Health, 3rded., Elsevier Academic Press, Burlington, MA, 2008, p. 608.
[35] M.I. Santos, A. Cavaco, J. Gouveia, M.R. Novais, P.J. Nogueira, L. Pedroso, M.A.S.Ferreira, Food Control 23 (2012) 275.
[36] M. Abadias, J. Usall, M. Anguera, C. Solsona, I. Vinas, Int. J. Food Microbiol. 123(2008) 121.
[37] N. Hounsome, B. Hounsome, D. Tomos, G. Edwards-Jones, Postharvest Biol.Technol. 52 (2009) 173.
[38] N.P. Nangula, A. Oelofse, K.G. Duodu, M.J. Bester, M. Faber, J. Food Compos. Anal.23 (2010) 499.
[39] M.C. Snchez-Mata, M. Cmara, C. Dez-Marqus, Food Chem. 80 (2003) 317.
Sequential determination of fat- and water-soluble vitamins in green leafy vegetables during storage1 Introduction2 Materials and methods2.1 Chemicals and standard solutions2.2 Samples2.3 Samples extraction2.4 Analysis of water soluble vitamins (HPLCDADMS/MS)2.5 Analysis of fat soluble vitamins (HPLCDAD)2.6 Method validation2.7 Statistical analysis
3 Results and discussion3.1 HPLCMS/MS determination of water-soluble vitamins3.2 HPLCDAD determination of fat-soluble vitamins3.3 Optimization of the extraction procedure3.4 Extraction recovery study3.5 Green leafy vegetables analysis
4 ConclusionsAcknowledgmentsReferences