Article pubs.acs.org/JAFC
Folate Profiling in Potato (Solanum tuberosum) Tubers by UltrahighPerformance Liquid Chromatography−Tandem Mass Spectrometry Jeroen Van Daele,† Dieter Blancquaert,‡ Filip Kiekens,† Dominique Van Der Straeten,‡ Willy E. Lambert,† and Christophe P. Stove*,† †
Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, Gent 9000, Belgium Laboratory of Functional Plant Biology, Department of Physiology, Ghent University, K.L. Ledeganckstraat 35, Gent 9000, Belgium
‡
S Supporting Information *
ABSTRACT: An ultrahigh-performance liquid chromatography−tandem mass spectrometry method was developed and validated for the profiling of six folate species in potatoes. The calibration curves cover a wide, linear range (the lower and upper limits of quantitation range between 0.22−0.24 and 216.07−242.28 μg/100 g of fresh weight), allowing sensitive determination in small amounts of potato flesh. With a single exception, the acceptance criteria for intra- and interday precision and accuracy were met: for all quality controls, the percent relative standard deviation and the percent bias were lower than 15% (or 20% at the lower limit of quantitation). Application of the method on tubers at different stages of maturation demonstrated the large variability within a single variety: the folate content and polyglutamylation rate varied between 10.35 and 24.01 μg/100 g of fresh weight and between 4.96% and 60.49%, respectively. Additionally, the two-dimensional folate profiling of mature tubers demonstrated an increase in folate from center to peel, combined with a stable species distribution and polyglutamylation rate. KEYWORDS: folate, folate profiling, potato, Solanum tuberosum, UHPLC-MS/MS, tandem mass spectrometry, variability
■
INTRODUCTION Folate (vitamin B9, Figure S1, Supporting Information) is the generic name for a group of water-soluble vitamins with similar structural and physiological properties. They consist of three components: a pterin residue, a para-aminobenzoic acid (pABA) moiety, and one or more glutamate moieties. Naturally occurring folates differ in the oxidation state of the pterin ring, the substitution of one-carbon units on N5 or N10, and the number of glutamate residues.1 Multiple methods for folate determination in potato have been described in the literature2−19 (Table S1, Supporting Information, gives an overview of relevant publications, ranked by technique and year), and the reported folate concentrations cover a wide range: for fresh samples, values varied from 3.0 to 76.0 μg/100 g, with an exceptionally high reported value of 125.1 μg/100 g of fresh weight (FW) by McKillop et al.;6 for lyophilized samples, the values varied from 20.7 to 303.1 μg/100 g of dry weight (DW). Three main causes may underlie these divergent values. First of all, there is a considerable biological variation, both between different varieties and within a single variety. Moreover, depending on the harvest or storage time, there will also be a substantial difference in the folate content of tubers from the same variety.8,9,12 Second, there is a lack of consensus on sample treatment prior to folate analysis: homogenization was either performed by mixing or by grinding in liquid nitrogen,12,19 and lyophilization was performed or not to express folate content in DW or FW. Remarkably, while most authors use amylase and protease (in addition to conjugase) treatments, Konings et al. stated that this was not necessary, because none of the extracted folate was matrix bound.14 The third cause may lie in the technique that was applied to determine folate content. Microbiological assays, historically © 2014 American Chemical Society
the most used technique for folate determination in foods, are associated with several problems, among which the different response of the utilized bacteria to different folate forms and the intrinsic inability to distinguish different folate forms.14 The latter is only possible when using chromatography-based methods, which are capable of separating the different vitamers. In particular, folate determination has been performed using high-performance liquid chromatography (HPLC) with ultraviolet, fluorescence, diode array, electrochemical, or mass spectrometric detection. Most of the published methods only determined folic acid, tetrahydrofolate, and/or 5-methyltetrahydrofolate.11,15,18,19 While three methods are capable of determining more than three folates, these require sample preparation via affinity-based purification (using folate-binding protein)14,20 or solid-phase extraction,12,13 which strongly complicates the procedure. Moreover, the majority of hitherto reported methods starts from relatively large quantities of fresh potato flesh. This may pose sensitivity problems when only small amounts of starting material are available, as is the case when very small tubers are to be analyzed or when the folate profile of an individual potato tuber has to be determined. Two authors already reported that 5-methyltetrahydrofolate and total folate concentrations are higher in the peel than in the flesh.5,19 However, a complete folate profile from center to peel (as has already been described for minerals21 and ascorbic acid22) has not been described, yet. As such, it could be useful to compose a folate two-dimensional profile of potato tubers. Received: Revised: Accepted: Published: 3092
October 6, 2013 March 19, 2014 March 22, 2014 March 22, 2014 dx.doi.org/10.1021/jf500753v | J. Agric. Food Chem. 2014, 62, 3092−3100
Journal of Agricultural and Food Chemistry
Article
Preparation of Stock and Working Solutions. All folate stock solutions were prepared in a 50 mM phosphate buffer, pH 7.5 or 3 (for 5,10-CH+THF, as this compound is more stable at acidic pH) containing 1% ascorbic acid and 0.5% DTT/methanol (50/50, v/v). Both standard and IS stock solutions were stored at −80 °C. Working solutions were prepared daily by appropriate dilution with the extraction buffer, which was a 50 mM phosphate buffer at pH 7.5, containing 1% ascorbic acid, 0.5% DTT, and the IS (with a concentration of 20 ng/mL for [13C5]-THF and 40 ng/mL for the other ISs). Lyophilization. Dry matter content determination and freezedrying of frozen homogeneous samples were performed by an Amsco FINN-AQUA GT4 freeze-dryer (GEA, Köln, Germany). The cycle comprised primary drying at −20 °C overnight, followed by a secondary drying step at 0 °C for three hours. The pressure for both cycles was held at 100 μbar. Chromatography. Chromatography was performed on a Waters Acquity UHPLC system including a binary pump, controlled by analyst 1.5.2 software (AB Sciex, Toronto, Canada). The column oven temperature was maintained at 60 °C, and the autosampler was at 4 °C. An Acquity HSS T3 column (C18, 150 mm × 2.1 mm; 1.8 μm particle size, from Waters) with a VanGuard precolumn (C18, 5 mm × 2.1 mm; 1.8 μm particle size, from Waters) was used. The injection volume was 10 μL. For gradient elution, the mobile phase consisted of eluent A (0.1% formic acid in water) and eluent B (0.1% formic acid in acetonitrile) and was pumped at a flow rate of 0.6 mL/min. Initially, the same gradient was applied as described by De Brouwer et al.26 However, we adapted this gradient to eliminate an interference coeluting with FA. The starting condition (100% A) was kept for 1 min. Subsequently, there was a nonlinear, convex increase to 95% B in 3 min, which was kept for 1 min. The mobile phase was then immediately adjusted to its initial composition and held for 3 min for re-equilibration, resulting in a total time of 8 min. MS Instrumentation and Settings. The detection was performed by positive electrospray ionization (ESI) utilizing heated auxiliary gas (Turbospray), in the scheduled MRM mode on an Applied Biosystems API 4000 tandem mass spectrometer (Ontario, Canada). On the basis of the largest peak width at the base (5MTHF), an MRM detection window of 30 s was chosen, together with a 0.5 s target scan time, generating at least 27 data points for 5-MTHF. The source conditions were optimized and were set as follows: source temperature, 500 °C; ion spray voltage, 3.5 kV; Q1 and Q3, unit mass resolution. The interface heater was on (100 °C); nitrogen was used for the nebulizer (gas 1), heater (gas 2), curtain, and collisionally activated dissociation (CAD) gas. Gasses 1 and 2 and the curtain gas had respective pressure settings at 70, 90, and 25 psi. The vacuum setting for CAD was set at 8, a configuration-dependent arbitrary value of the instrument. Compound parameters, together with retention times are given in Table 1. Sample Preparation. The general sample preparation protocol included boiling, homogenization, extraction with trienzyme treatment (α-amylase, protease, and conjugase), and additional heat treatments to stop enzyme activities. The resulting solutions were ultrafiltrated at 12 851g for 15 min prior to UHPLC-MS/MS analysis. Before all manipulations, 1.5 mL of the extraction buffer was added to ±200 mg of fresh potato flesh, taken with a kitchen knife from the center of a single tuber. When samples were prepared in bulk (for optimization and validation purposes), a corresponding volume of extraction buffer was added to central sections of potato tubers (8−10 g, collected from 2−3 mature tubers). This quantity was afterward divided into aliquots corresponding to 200 mg of fresh potato flesh. Several variables in distinct steps of this general protocol were evaluated to obtain the best folate recovery. These included a comparison of homogenization by mixing or grinding with liquid nitrogen, boiling or not, freeze-drying or not, and varying the volumes of the three enzymes: 0, 20, 30, or 40 μL of α-amylase (0, 470, 705, or 940 units, respectively); 0 or 150 μL of the charcoal-treated protease solution (0 or 2.1 units, respectively); 100 or 150 μL of the charcoal-treated rat serum conjugase solution. Since starch is a main component of potato flesh, we opted to test three increasing volumes of α-amylase. The evaluation of boiling and
This way, information about the difference in folate distribution within or between individual potatoes of the same variety, age, and size can be used to gain further insight into the divergence of reported results. Here, we report on the development, the validation, and the application of an ultrahigh-performance liquid chromatography−tandem mass spectrometry (UHPLC-MS/MS) method, capable of providing a folate profile (encompassing six folate monoglutamates), starting from only 200 mg (FW) of potato flesh. To expand the use of our method, we opted for a single calibration curve, covering a wide dynamic range. This way, potatoes with higher folate content (obtained, for example, through biofortification via breeding2 or genetic engineering23−25) can also be analyzed. The method was used to investigate the differences in folate distribution in tubers of the potato variety Désirée at different stages of maturation. Moreover, this method was used to compose, for the first time, a two-dimensional folate profile throughout potato tubers in order to visualize the biological variability both within and between potato tubers of the potato variety Désirée.
■
MATERIALS AND METHODS
All manipulations involving folates were done under subdued light. The air in the headspace was not displaced by use of an inert gas, but the solutions used for preparation of the stock solutions, the working solutions, and the extraction buffer were flushed with nitrogen. Tetrahydrofolate (THF), 5-methyltetrahydrofolate (5-MTHF), 5,10-methenyltetrahydrofolate (5,10-CH+THF), 10-formyl folic acid (10-CHOFA), 5-formyl tetrahydrofolate (5-CHOTHF), and folic acid (FA) were purchased from Schirck’s Laboratories (Jona, Switzerland). [13C5]-THF, [13C5]-5-MTHF, [13C5]-5,10-CH+THF, and [13C5]-FA were obtained from Merck Eprova (Schaffhausen, Switzerland) and were used as internal standards (ISs, labeling yield for all >98%) for their respective analogues. [13C5]-FA was also used as an IS for 5CHOTHF and 10-CHOFA, based on its chromatographic retention characteristics. Ultrapure water was obtained from a Synergy ultrapure water purification system (Millipore, Brussels, Belgium). Formic acid, sodium phosphate, ascorbic acid, dithiotreitol (DTT), and activated charcoal were purchased from Sigma (Diegem, Belgium). Methanol and acetonitrile of LC-MS grade were purchased from Biosolve (Valkenswaard, The Netherlands). BCR-121 wholemeal flour was obtained from the European Commission, Institute for Reference Materials and Measurement (Brussels, Belgium). α-Amylase (type I-A, from porcine pancreas, 23.5 units/μL, EC no. 232-565-6) and protease (type XIV, from Streptomyces griseus, ≥3.5 units/mg, EC no. 232-909-5) were purchased from Sigma. Male rat serum was acquired from Harlan (Horst, The Netherlands). Protease was dissolved in distilled water (4 mg/mL). To remove most of the endogenous folates, protease and rat serum were mixed with a 1/10th volume of activated charcoal and stirred for 1 h on ice, centrifuged, filtered, and divided into 500 μL aliquots. Both enzymes were stored at −80 °C. α-Amylase was used without pretreatment and stored at 4 °C. Homogenization was either performed by manually grinding single samples in liquid nitrogen with mortar and pestle or with an IKA-18 basic Ultra-Turrax (Labo service, Kontich, Belgium). Larger volumes were mixed and subsequently divided into representative aliquots of actual samples. For ultrafiltration, 3 mL (Amicon, Millipore) 3 kDa molecular weight cutoff membrane filters were used. Ultrafiltration was done in an Eppendorf centrifuge 5804 R at 4 °C (Hamburg, Germany). The potato variety Nicola was bought in a local supermarket (for optimization of sample preparation) or provided by a local distributor (for profiling). The potato variety Désirée, which we used for genetic engineering purposes,25 was provided by the Laboratory of Functional Plant Biology (for determination at different stages of maturation) or bought in a local supermarket (for validation experiments). 3093
dx.doi.org/10.1021/jf500753v | J. Agric. Food Chem. 2014, 62, 3092−3100
Journal of Agricultural and Food Chemistry
Article
nominal concentration and multiplied by 100), in accordance with the protocol described by Almeida et al.27 Linearity of the most appropriate model for each folate was then tested by performing analysis of variance (ANOVA) lack of fit (LOF) tests. Calibration curves with seven calibrators were constructed by spiking a mix of the six standards to a 1:20 dilution of potato matrix in extraction buffer. Four quality controls (QCs), including the lower and upper limits of quantitation (ULOQ), were prepared similarly but in a 1:2 diluted potato matrix, except the LLOQ QC, which was prepared in a 10-fold diluted matrix, so the endogenous folate content would not exceed the spiked concentration. The concentrations (in μg/100 g of FW) spiked to the calibrators and QCs are depicted in Table S2 (Supporting Information). After completing the sample preparation protocol, extracts of these samples were injected onto the UHPLC-MS/MS, not randomly but in increasing order of calibrator concentration. To make sure this did not contribute to heteroscedasticity, the carry-over effect was investigated by injecting a blank water sample after injection of the calibrator with the highest concentration. Lack of a significant carry-over requires that the signal obtained at the retention times of the respective folates should at all times be less than 20% or less than 5% of the LLOQ of the folate standards and ISs, respectively. Accuracy, Precision and Measurement Uncertainty. Experiments for accuracy and precision were performed at four QC levels on three nonconsecutive days, in duplicate (n = 3 × 2). Every day, fresh calibrators and QCs were prepared and analyzed as described above. Zero samples (i.e., samples with only IS spiked) were also included to subtract the endogenous folate content of the matrix. Intra- and interday precision (% relative standard deviation or %RSD), together with accuracy (%bias) were calculated as reported by Wille et al.28 and should be less than 15%, except at the LLOQ, where they should be less than 20%. The measurement uncertainty is represented by a 95% confidence interval (CI). The interval was constructed by the combination of %bias and the %RSD (interday) of the measured value. This way, the interval becomes [measured value ± (|%bias| + 2 × %RSD)]. Matrix Effect and Recovery. To evaluate the matrix effect (ME, where percentage values 100% represent ion enhancement) and recovery (REC), three separate batches of samples (n = 3) were prepared as suggested by Matuszewski et al.29 Batch A contained the “neat” standards and consisted of extraction buffer, spiked at three QC levels (LOW, MED, and HIGH). Batches B and C contained 1:2 diluted potato matrix spiked after and before sample preparation, respectively. For the latter two batches, zero samples were again included. The ME and REC were then calculated as B/A*100 and C/B*100, respectively, where the zero sample signals were subtracted from B and C. Short-Term Stability (STS) and Freeze−Thaw Stability (FTS). Aliquots of sample extracts (n = 3), spiked at the four QC levels, were immediately injected onto the UHPLC-MS/ MS (and stayed in the autosampler at 4 °C for reanalysis after 24 h) or were frozen at −20 °C (FTS). Three freeze−thaw cycles were also performed in one day and after each cycle analysis was performed. Analysis of Reference Material BCR-121 (Wholemeal Flour). Samples of 200 mg (n = 5) were treated the same way
Table 1. Compound Parameters for Six Folates and Their Internal Standardsa precursor ion (m/z)
product ion (m/z)
tr (min)
DP (V)
EP (V)
CE (V)
CXP (V)
THF
446.2
3.19
5-MTHF
460.3
5,10-CH +THF
456.2
10-CHOFA
470.1
5-CHOTHF
474.2
FA
442.1
[13C5]-THF [13C5]-5MTHF [13C5]-5,10CH+THF [13C5]-FA
451.1 465.3
299.3 166.4 313.2 194.2 412.1 282.1 295.4 176.3 327.2 299.3 295.3 176.1 299.3 313.2
3.19 3.34
70 70 70 70 75 75 65 65 61 61 61 61 70 70
10 10 10 10 10 10 10 10 10 10 10 10 10 10
30 59 30 53 40 65 35 59 29 45 30 57 30 30
20 12 18 14 10 16 16 14 8 16 18 14 20 18
461.0
416.2
3.56
75
10
40
10
447.2
295.3
4.09
65
10
20
16
3.34 3.56 3.91 3.96 4.09
a
tr, retention time; DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, collision cell exit potential; V, volt. The first transition was used as quantifier, the second was used as qualifier. lyophilization prior to storage was done in a joint experiment: skinless fresh potato was boiled or kept on ice and immediately homogenized by mixing. Subsequently, representative aliquots were weighed individually before and after lyophilization. These samples were kept at −80 °C until analysis.
■
VALIDATION Selectivity. Selectivity for the folate monoglutamates was investigated by injecting aliquots of neat buffer solutions, spiked with the six monoglutamates and the four ISs (individually and together). Sensitivity. The limit of detection (LOD) and lower limit of quantitation (LLOQ) are defined as the concentrations with a signal-to-noise ratio equal to or larger than 3 and 10, respectively. In this case, the LLOQ was arbitrarily set as the lowest point of the calibration curve because of the lack of a blank matrix. For determination of the LOD, potato matrix was diluted 1:20 in ultrapure water and boiled in the presence of light to destroy most endogenous folates. As this still yielded a measurable signal for 5-MTHF, the matrix was further diluted 1:20 and ultrafiltrated to approach a blank matrix. The matrix was prepared this way because the LOD is a qualitative descriptor of sensitivity, in contrast to the LLOQ, where possible matrix effects can affect calibration. It was then used to serially dilute the six folate standards. The average short-term noise of five independent injections of these serially diluted standards was used to correlate the peak heights with the corresponding concentrations. Linearity and Calibration. Prior to testing the linearity of a calibration model, homoscedasticity of the calibrators was investigated. Therefore, residual plots were generated and Fisher’s tests (p < 0.01) were performed. To obtain a linear model, the most common weighting factors (1/√x, 1/x, 1/x2, 1/√y, 1/y, and 1/y2) and transformations (logarithmic or square rooted) were tested by comparing the sums of percent relative error (%RE, calculated as the difference between the obtained and the nominal concentration, divided by the 3094
dx.doi.org/10.1021/jf500753v | J. Agric. Food Chem. 2014, 62, 3092−3100
Journal of Agricultural and Food Chemistry
Article
Figure 1. (a) MRM chromatogram of the lowest calibrator, i.e., spiked at the LLOQ and (b) MRM chromatogram representing the folate profile of potato variety Désirée (scan windows are depicted for each folate and its internal standard below the chromatogram). (1) THF; (2) 5-MTHF; (3) 5,10-CH+THF; (4) 10-CHOFA; (5) 5-CHOTHF; (6) FA; (7) [13C5]-THF; (8) [13C5]-5-MTHF; (9) [13C5]-5,10-CH+THF; (10) [13C5]-FA; (i) signal at first 5-CHOTHF MRM transition; (ii) signal at first FA MRM transition.
as fresh potato flesh, apart from the homogenization step, and underwent the full trienzyme treatment. Application to the Potato Variety Désirée. The developed procedure was applied to the potato variety Désirée at three stages of maturation. Potato samples (200 mg, taken from the center of the tubers) were treated or not with conjugase to gain information about the polyglutamylation status. Immature tubers were grown at the Laboratory of Functional Plant Biology, harvested 17 weeks after plantation, and kept frozen at −20 °C until analysis. Store-bought mature tubers were kept at room temperature and analyzed that same day or after two months (when tubers were sprouting). Folate Profiling of Potato Variety Nicola. Three potatoes with the same level of maturation and of uniform size were chosen. From each of these, a central slice was cut with a kitchen knife and divided into segments, to form a twodimensional representation of the tuber. From the basal to the horizontal opposite apical end, 13 segments were taken, including 1 central and 2 marginal segments containing peel. From the upper surface to below, seven segments were taken, again including one central and two marginal segments. Each of these segments was further subdivided into two subsections. The first subsection was used for determination of dry matter content, the second subsection was further divided into two 200 mg sections to create genuine replicates.
we switched the linear increase of the gradient to a convex type, which resulted in a more swift increase of mobile phase B. This modification caused a change in retention of the matrix-specific interference, now allowing a selective detection of FA with its first transition (442.1 → 295.3). Application of this gradient also improved the peak shape of 5-CHOTHF, which now eluted in a shorter time interval. Representative chromatograms, obtained with the optimized gradient, are depicted in Figure 1. Figure 1a shows a chromatogram of diluted potato extracts, spiked at the lowest calibrator level, that is, at LLOQ, while Figure 1b depicts the folate profile of potato variety Désirée. In the former, the respective scan windows of the scheduled MRM algorithm are indicated for each folate and IS at the bottom of the chromatogram. It should be noted that the fact that 10-CHOFA and 5-CHOTHF are not baseline separated does not pose a problem, since MS can distinguish these compounds. We deliberately chose not to detect or distinguish between 5,10-methylenetetrahydrofolate (5,10CH2THF) and 10-formyl tetrahydrofolate (10-CHOTHF), since these are highly susceptible to interconversion and oxidation at neutral (extraction protocol) and acidic (chromatographic run) pH, even with the use of antioxidants.31,32 Sample Preparation. Experiments involving the trienzyme treatment were performed twice. The influence of the treatment was initially investigated using a previously validated HPLC-MS/MS method.30 α-Amylase and protease were added in increasing amounts to evaluate folate recovery, and conjugase was added in two volumes to evaluate the deconjugation efficiency (Figure S2a, Supporting Information). A one-way ANOVA and complementary t tests showed no significant impact (p < 0.05) on folate recovery when using αamylase or protease, supporting the findings by Konings et al.14 There was also no significant difference (p < 0.05) between the addition of 100 and 150 μL of conjugase. These results were confirmed using the newly optimized UHPLC-MS/MS
■
RESULTS AND DISCUSSION Eliminating Coeluting Interferences in Potato Matrix. The experiments involving sample preparation were conducted on the variety Nicola with a HPLC-MS/MS procedure as described by De Brouwer et al.30 The initially determined total and free monoglutamate contents of this variety were respectively 21.78 ± 1.42 and 13.62 ± 0.35 μg/100 g, indicating a polyglutamylation status of 37.5%. Because of a coeluting interference, FA had to be quantified using its second MRM transition (442.1 → 176.2). To eliminate this problem, 3095
dx.doi.org/10.1021/jf500753v | J. Agric. Food Chem. 2014, 62, 3092−3100
Journal of Agricultural and Food Chemistry
Article
Table 2. Sensitivity and Calibration Data THF 5-MTHF 5,10-CH+THF 10-CHOFA 5-CHOTHF FA a
LODa
linear rangea
0.0429 0.0057 0.0574 0.0474 0.0233 0.0135
0.2349−234.8611 0.2161−216.0722 0.2242−224.1688 0.2423−242.2778 0.2252−225.1700 0.2423−242.2778
slope [95% CI] 31.9469 33.5891 33.8356 38.2897 49.3652 25.9820
[29.4908−34.4030] [29.1018−38.0763] [30.7014−36.9699] [32.7726−43.8076] [44.9580−53.7723] [24.9862−26.9778]
intercept [95% CI] −0.0024 −0.0017 −0.0005 −0.0014 −0.0001 −0.0005
[−0.0077 to 0.0028] [−0.0037 to 0.0003] [−0.0030 to 0.0021] [−0.0021 to −0.0008] [− 0.0009 to 0.0007] [−0.0027 to 0.0017]
R2 0.9970 0.9909 0.9941 0.9859 0.9945 0.9989
Values in μg/100 g.
was heteroscedastic. There was no carry-over: signals at the respective retention times of each folate or its IS, obtained after injection of a water sample, were all less than 5% of the LLOQ. Evaluation of several weighting factors and transformations demonstrated that applying a weighting factor of 1/x2 yielded the lowest sum of %RE. Subsequent performing of ANOVA LOF tests on this model showed a linear relationship for the chosen calibration ranges of all six folates. Intra- and interday precision (%RSD) and accuracy (%bias) were determined on three nonconsecutive days, in duplicate, by analyzing QCs that covered the whole calibration range (including the LLOQ and ULOQ). The data on precision and accuracy are summarized in Table 3, together with the 95% confidence limits of measurement uncertainty. Requirements for both intra- and interday precision were met for the six folates: all QCs had a % RSD lower than 15%, even at the LLOQ level. The accuracy also met acceptance criteria, except for 5-CHOTHF at its LLOQ, where accuracy was 22.69%. The IS-corrected matrix effect exceeded the 85−115% range (Table 4, together with the recovery), though not excessive. While normalization using the IS did not compensate for matrix effects, it did compensate for the losses during sample preparation. Recoveries of more than 60% were observed for all folates at the three tested concentration levels. The (relatively) lower recovery of THF can be explained by its labile nature, while finding the actual cause for the recovery obtained for 5,10-CH+THF is more difficult: interconversion into 5-CHOTHF and the very unstable 10-CHOTHF at neutral pH, in the presence of antioxidants, has been described.32 Subsequent oxidation of 10-CHOTHF to 10CHOFA and other, nonfolate degradation products could result in a low recovery of 5,10-CH+THF. Alternatively, 10CHOTHF and 5-CHOTHF might interconvert to 5,10-CH +THF at high temperature in acidic environment (as is the case during the chromatographic run). Since the recovery of 5,10CH+THF was reproducible (%RSDs of 6.42, 13.67, and 13.58 for LOW, MED, and HIGH, respectively) and since other validation parameters yielded acceptable results, we consider quantitative determination of 5,10-CH+THF sufficiently reliable. In general, our newly developed UHPLC-MS/MS shows improved sensitivity and precision at LLOQ, in comparison with a previously described method in rice.26 In addition, changing to a scheduled MRM mode lowered the LODs. Although the matrix effects exceeded 15% at some instance, IScorrected ME did not exceed the 75−125% range and given the acceptable results obtained for accuracy and precision can be regarded as “under control”. Stability. Two postextraction stability experiments were set up to find out whether or not the processed samples could be preserved over a short period of time for subsequent reanalysis. The resulting extracts could easily be kept in the autosampler
method. Yet, although trienzyme treatment did not significantly affect folate recovery from the center of the potato matrix, we still opted to include this treatment: the resulting extracts were less gummy and easier to manipulate after consecutive heating and cooling steps. In addition, implementation of trienzyme treatment may overcome potential variability, introduced by the somewhat different composition of different sections of a tuber; for example, the dry matter content is typically lowest in the center.33 Evaluation of the homogenization step revealed that there was no significant difference (p < 0.05) in folate recovery between mixing and grinding with liquid nitrogen. We therefore used mixing, which is less laborious, for further experiments and validation of the method. Since boiling prior to homogenization may aid in degrading the matrix and reducing the enzymatic and oxidative degradation of folates,1,34−36 the impact of this manipulation on recovery was also investigated (Figure S2b, Supporting Information). In the same experiment, samples were also lyophilized or not after homogenization. The purpose of this manipulation was to find out if freeze-drying compensates for the increased variability caused by differences in moisture content, rather than preparing them for storage. Samples that were only boiled had a significantly higher folate content (13.32 ± 0.85 μg/100 g of FW, p < 0.05) than samples that were left untreated or that were lyophilized (with or without prior boiling). The advantage that lyophilization may offer, that is, compensating for the variable moisture content of potatoes,2 was clearly lost, so we decided not to include a lyophilization step but to immediately freeze the samples after boiling and homogenization. Freeze-drying did provide information about the water content of the center of boiled and raw potatoes: 84.34% and 79.41%, respectively, in accordance with literature findings.15 Therefore, freeze-drying can still be used to determine the dry matter content of frozen potato segments that do not undergo folate analysis. In conclusion, sample treatment comprised an initial boiling step followed by mixing. Subsequently, the samples were subjected to a trienzyme treatment: α-amylase (30 μL, 20 min at 37 °C) followed by an incubation with protease (150 μL, 1 h at 37 °C) and with deconjugase (100 μL rat serum, 2 h at 37 °C). The protease and conjugase incubations were each followed by a boiling step (10 min at 100 °C) to stop the respective enzyme activities. After each boiling step, samples were cooled down on ice. Samples intended for direct analysis of free monoglutamate content were spiked with water (instead of conjugase) and kept on ice (Figure S3, Supporting Information). Validation. No signals from other folates or the IS were seen at the respective retention times of the injected standards, demonstrating the selectivity of the method. The calculated LODs of the six folates can be found in Table 2, together with the linear ranges, slopes, intercepts, and R2 values of the final calibration curves. Visual evaluation of residual plots and performing Fisher’s tests showed that the calibrator distribution 3096
dx.doi.org/10.1021/jf500753v | J. Agric. Food Chem. 2014, 62, 3092−3100
for 24 h after injection (STS, 24 h at 4 °C): the decrease was below 10% for all of the folates at the four concentration levels (Figure S4a, Supporting Information). This is sufficient to allow the samples to be reanalyzed the next morning, in case of a technical failure. The same was observed for vials with extracts that were submitted to three freeze−thaw cycles (FTS, three cycles at −20 °C) on the day of analysis. Again, the decrease did not exceed 10% (Figure S4b, Supporting Information). After performing these experiments, we concluded that reanalysis of treated samples can be performed after storage for up to 24 h at 4 °C or after freezing samples at −20 °C, followed by thawing up to three times. Analysis of Reference Material BCR-121. Analysis of the BCR-121 wholemeal flour reference material confirmed the validity of our method: the total folate content (53.26 ± 4.93 μg/100 g of DW) as measured by UHPLC-MS/MS agreed well with the certified value (50 ± 7 μg/100 g), determined with a microbiological assay. Furthermore, the folate species concentration and distribution (1.01 ± 0.12, 5.34 ± 0.24, 5.34 ± 0.80, 10.31 ± 1.59, 29.04 ± 4.99, and 2.26 ± 0.17 μg/100 g for THF, 5-MTHF, 5,10-CH+THF, 10-CHOFA, 5-CHOTHF, and FA, respectively) were also in accordance with the literature.18,37 Application to Potato Tubers at Different Stages of Maturation (Variety Désirée). The folate content and distribution of individual folates (Figure 2a, b) in immature and mature Désirée tubers was comparable to those in Nicola tubers (determined with a previous method). The total folate content at these growth stages was 23.58 ± 1.51 and 24.01 ± 0.23 μg/100 g of FW, respectively, with polyglutamylation rates of only 5.4% and 5%. Hence, a comparable folate content and distribution was observed in lab-grown immature and storebought mature potatoes. The polyglutamate content of sprouting potatoes was remarkably higher (60.49%), although their folate content had decreased (10.35 ± 0.95 μg/100 g of FW). This significantly lower folate content (p < 0.05) contrasts with Goyer and Navarre,8,9 who reported a higher folate content in stored potatoes near sprouting. This discrepancy may be explained by the fact that our determination was performed on central potato flesh. Several authors4,8,16 reported on the fact that the folate concentration is higher in potato peels, and Goyer and Navarre hypothesized that folate levels may increase when a tuber is sprouting (occurring at the peel).8 The higher polyglutamylation level in sprouting potatoes approached the one reported by Konings et al. (90%),14 but the distribution was different: 5-MTHF was still the predominant species, but it only accounted for 41% instead of 95% (Konings et al.)14 of the total folate content. Composing a Two-Dimensional Folate Profile of Mature Potato Tubers (Variety Nicola). To further investigate the biological variability, we first tried to exclude as many confounding (maturation stage, growth, harvest, and storage facilities) factors as possible: a batch of Nicola tubers at the same stage of maturation were obtained from a local distributor. From that batch, three tubers with the same dimensions were chosen for profiling of their cross sections. Comparison of genuine replicates within a tuber and corresponding segments of different potatoes were used for evaluating the variability within a certain potato variety. Analysis of all segments resulted in a horizontal and vertical (x and y) profile, as shown in Figure 3a. In these tubers, the folate concentration was consistently higher in segments containing peel, than in central segments. The average total folate concentration of all the different segments was 17.95 ±
Five QCs were used because one outlier confirmed with the Grubbs test (p < 0.01) was excluded. bValues in μg/100 g (calculated as [measured value ± (|%bias| + 2 × %RSD)]). a
HIGH
Article
[221.70−295.21] [188.84−292.44] [213.11−300.00] [18.42−32.92] [20.55−29.41] [20.59−31.36] [1.84−3.17] [2.02−2.97] [2.07−3.11] [0.20−0.37] [0.15−0.40] [0.19−0.35] 3.77 7.33 5.52 11.15 3.40 6.77 11.52 4.10 6.65 5.88 12.00 9.24 2.80 3.94 4.83 1.88 2.87 3.30 6.26 3.38 5.48 4.70 7.34 6.18 6.68 6.87 5.89 3.53 10.70 6.89 18.28 22.69 10.46
5.94 10.94 7.20
[17.77−26.12] [17.88−25.06] [14.77−28.12]
MED LOW
[2.01−3.16] [1.73−2.79] [1.87−2.55] [0.21−0.32] [0.19−0.29] [0.20−0.30]
LLOQ HIGH
8.18 5.80 2.52 6.07 8.03 13.39
MED LOW
3.78 9.58 7.08a 2.41 4.66 5.41
LLOQ HIGH
1.42 4.53 2.48 6.07 6.76 5.46
MED LOW
3.70 2.49 4.92a 1.62 3.20 4.74 −1.82 1.50 3.77
LLOQ HIGH MED
−6.56 −0.65 −4.33
LOW
10.16 4.58 −1.23a
LLOQ
13.93 12.07 10.49
THF 5-MTHF 5,10-CH +THF 10-CHOFA 5-CHOTHF FA
uncertainty [95% CI]b interday precision (%RSD) intraday precision (%RSD) accuracy (% bias)
Table 3. Accuracy, Intra- and Interday Precision, and Measurement Uncertainty (n = 3 × 2)
[188.67−272.51] [190.57−248.04] [212.15−253.10]
Journal of Agricultural and Food Chemistry
3097
dx.doi.org/10.1021/jf500753v | J. Agric. Food Chem. 2014, 62, 3092−3100
Journal of Agricultural and Food Chemistry
Article
Table 4. Matrix Effects and Recovery (n = 3) for LOW, MED, and HIGH QCs matrix effects without IS (%) THF 5-MTHF 5,10-CH+THF 10-CHOFA 5-CHOTHF FA
matrix effects with IS (%)
recovery (%)
LOW
MED
HIGH
LOW
MED
HIGH
LOW
MED
HIGH
80 91 75 97 109 71
90 91 80 94 98 84
92 92 71 92 86 110
121 110 116 122 124 75
110 105 119 102 110 90
109 118 96 95 89 108
63 86 64 98 76 79
64 97 68 88 94 97
74 99 63 74 97 81
Figure 2. (a) Total and free folate monoglutamate content (error bars indicate the standard deviation) and (b) relative monoglutamate and polyglutamate distribution of immature, mature, and sprouting Désirée tubers.
3.17 μg/100 g of FW, but the average profiles of the horizontal and vertical sections (Figure 3b, c) show that this was not representative for a homogeneous distribution in the tuber: the average folate concentration in central segments was 14.08 ± 2.16 μg/100 g, increasing up to 86% toward segments containing peel (26.26 ± 11.24, 21.54 ± 5.61, 23.52 ± 3.92, and 20.31 ± 4.83 μg/100 g of FW for the peels at the basal, apical, upper, and lower ends, respectively). Interestingly, despite the increase of total folate concentration toward the peel, folate species distribution and polyglutamylation rates remained the same throughout the entire sections: 5-MTHF was still the predominant folate, representing an average 69% of the total folate. From this total folate, an average 78% was polyglutamylated. The comparable profiles of the three individual tubers also confirmed that 5-MTHF was the predominant folate in mature potato tubers variety Nicola.
The median variability (%RSD, Figure 3d) of genuine replicates within a single potato was 12%, increasing to 30% when considering corresponding segments of different potatoes. To make sure these results were not biased by the differences in dry matter content, the folate concentrations were also converted to their DW equivalents. This resulted in a moderate decrease in median variability (to 11% and 26% within and between the individual potatoes, respectively), demonstrating that folate concentrations in potato tubers may be expressed as FW as well as their DW equivalent. The distribution of total folate, folate species, and glutamylation was similar throughout the cross sections of the three individual tubers. In addition, the “inter-tuber” variability of corresponding segments exceeded that of genuine replicates (
