Food Control 40 (2014) 19e25

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Food Control

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Species determination of nuts in commercial samples causing pine nut syndrome

Aase Æ. Mikkelsen a,1, Flemming Jessen b, Nicolai Z. Ballin c,*,1 a Department of Food Chemistry, Danish Veterinary and Food Administration (DVFA), Sønderskovvej 5, DK-8520 Lystrup, Denmark b National Food Institute, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark c Department of Food Chemistry, Danish Veterinary and Food Administration (DVFA), Søndervang 4, DK-4100 Ringsted, Denmark article info abstract

Article history: Consumption of pine nuts from the species of Pinus armandii has been reported to cause dysgeusia, Received 15 July 2013 commonly known as pine mouth, or pine nut syndrome (PNS). However, the number of reports on pine Received in revised form nut consumptions of the different species and PNS is limited. This open the possibility that other 18 November 2013 pine species than P. armandii could be involved in PNS as well. This study investigated 18 samples Accepted 19 November 2013 involved in PNS and received at the Danish Veterinary and Food Administration in 2011 through 2012. Samples were subjected to gas chromatographic analysis of fatty acids. The content of 11 individual fatty Keywords: acids was used together with the diagnostic index and the sum of D5-fatty acids as diagnostic param- Authentication Dysgeusia eters. Diagnostic parameters from samples were then compared to reference material and literature data Fatty acid profile to determine the species. In a limited number of samples, the diagnostic parameters matched neither our PCA reference materials nor literature data. However, the morphology, the fatty acid analysis, and externally Pine nut syndrome obtained DNA sequencing data suggest a P. armandii subspecies or a variety. With these possible Species determination P. armandii subspecies, P. armandii was identified in all analyzed samples. The application of principal component analysis (PCA) to the data set showed a satisfactory separation of the majority of the 13 pine species included in the study. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction agent is still unknown and symptoms have, so far, been limited to ingestion of pine nuts from the species of Pinus armandii (Destaillats, Pine nut related dysgeusia, now referred to as pine mouth or pine Cruz-Hernandez, Giuffrida, & Dionisi, 2010; Destaillats et al., 2011; nut syndrome (PNS), was first described in 2001 (Mostin, 2001). In Köbler et al., 2011; Zonneveld, 2011). 2010 reports showed up in the literature again, one reporting around P. armandii is one out of more than 100 different Pinus species 3000 cases in France in 2009 (Flesch et al., 2011). Complaints of pine that exist worldwide. According to Food and Agriculture Organi- nut related dysgeusia increased from 2008, peaked in 2009 (Flesch zation of the United Nations (FAO) http://www.fao.org/docrep/ et al., 2011) and in 2011 (Kwegyir-Afful et al., 2013), and then X0453E/X0453e12.htm, 29 of these species produce edible nuts; decreased in 2012. The symptoms of PNS are characterized by a not all are used commercially. The most common edible pine nuts constant bitter or metallic taste, often intensified by the ingestion of belong to the species of Pinus koraiensis, Pinus sibirica, and to a certain foods (Ballin, 2012; Flesch et al., 2011; Kwegyir-Afful et al., lesser extent Pinus gerardiana, Pinus pinea, Pinus pumila, Pinus 2013). These symptoms appear 1e2 days after ingestion, resolve tabuliformis, Pinus wallichiana (formerly known as Pinus griffithii), within 14 days, and show no spontaneous relapse or other side ef- and Pinus yunnanensis. Common inedible pine nuts belong to the fects within a year (Ballin, 2012). A large investigation based on a species of P. armandii and Pinus massoniana. The genetic variation of questionnaire showed that the bitter or metallic taste lasted for P. armandii within a limited geographic Chinese area (Chen & Chen, 9.3 4.8 days (n ¼ 57) (Kwegyir-Afful et al., 2013). The causative 2011) suggests that different subspecies or varieties might end up as a commodity for international trade. According to The Interna- tional Names Index (IPNI, 2012), the recognized 8 subspecies Abbreviations: DI, diagnostic index; FA, fatty acid(s); GC, gas chromatography; and varieties of P. armandii include Pinus armandii var. amamiana, NMR, nuclear magnetic resonance; PCA, principal component analysis; PNS, pine P. armandii Franch (Pinus armandii var. armandii), Pinus armandii nut syndrome; RM, reference material; S, sample. var. dabeshanensis, Pinus armandii var. farjonii, Pinus armandii var. * Corresponding author. Tel.: þ45 72276083; fax: þ45 72276000. E-mail addresses: [email protected], [email protected] (N.Z. Ballin). fenzeliana, Pinus armandii var. mastersiana, Pinus armandii subsp. 1 Contributed equally to this work. mastersiana, and Pinus armandii subsp. yuana. Unfortunately, most

0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.11.030 20 A.Æ. Mikkelsen et al. / Food Control 40 (2014) 19e25 commercial pine nuts are not labeled with its subspecies or variety, Pinus densata (RM3), P. gerardiana (RM4), P. massoniana (RM6), and it is therefore difficult to obtain reference material for sub- P. pumila (RM8), P. wallichiana (RM11) (formerly known as species comparison. P. griffithii), and P. yunnanensis (RM12A) were purchased from The inedible pine nuts from P. armandii are commercialized for Prime , Denmark. P. koraiensis (RM5B) was kindly gifted from animal feed production and industrial purposes. In and the the University of Copenhagen, Arboretum Hørsholm, Denmark. P. US, P. armandii batches have within recent years been sold labeled pinea (RM7) was collected in a store and authenticated through a as P. sibirica or as other edible species. Efficient control of pine nut morphological evaluation and a comparison of the FA content with species is, therefore, a focal point in preventing future problems literature data (Wolff et al., 2000). P. tabuliformis (RM10) was pur- with mislabeling and potential cases of PNS. chased from Le Semences du Puy, France. Pinus bungeana (RM2), DNA sequencing (Handy et al., 2011) and nuclear magnetic P. yunnanensis (RM12B), and Pinus cembra (RM13) were purchased resonance (NMR) has been applied to pine speciation with suc- from OMC Seeds, Lithuania. cess. NMR has been coupled to principal component analysis (PCA), which is an effective tool in handling large and compli- 2.2. Chemicals and standards cated data sets (Köbler et al., 2011). Köbler et al. (2011) presented a fast screening method that was able to discriminating groups of The FAs myristic acid 14:0, palmitic acid 16:0, palmitoleic acid species and exclude nuts involved in PNS (Köbler et al., 2011). 16:1n-7, margaric acid 17:0, heptadecanoic acid 17:1n7, stearic acid Gas chromatographic (GC) analysis of FAs (FA) (Wolff, Pedrono, 18:0, oleic acid 18:1n9, vaccenic acid 18:1n7, linoleic acid 18:2n6, Pasquier, & Marpeau, 2000) is another speciation strategy, arachidic acid 20:0, eicosenoic acid 20:1n9, linolenic acid 18:3n3, especially in less equipped laboratories. A review of the FA docosadienoic acid 20:2n-6, behenic acid 22:0, and lignoceric acid content of 144 Pinus species (Wolff et al., 2000) has shown 24:0 were included in the GLC 87, 606, 546B, 17AA0, 68A standard species specific FA differences. Seven of these species specificFAs mixes purchased from Nu Chek Prep, INC., Elysian, MN, USA. made it possible to calculate a diagnostic index (DI) value Methanol, heptane, and n-heptane (all HPLC grades) were pur- capable of differentiating between different Pinus species chased from Rathburn Chemicals Ltd, Walkerburn Scotland. 3N (Destaillats et al., 2010). However, further exploration of the DI hydrogen chloride in methanol (for GC derivatization) was pur- showed problems in the analysis of samples with mixed species chased from SigmaeAldrich, Denmark. (Fardin-Kia,Handy,&Rader,2012). In this study, the homoge- neity of pine nut samples involved in PNS was evaluated based 2.3. Samples on the different species morphology. In cases of mixtures, pine nuts were carefully separated into groups of similar morphology Eighteen samples (S1eS18) were either analyzed as part of a prior to analysis. This study shows that the use of the DI in national regulatory control project or on a commercial basis. species determination should be confirmed by additional diag- Commercial samples were analyzed for Danish importers and for nostic parameters. In addition to the DI value, we extended the the Ministry for Health, the Elderly and Community Care (MHEC), diagnostic parameters to include the sum of the D5-FAs and Environmental Health Directorate, Malta. All samples (S1eS18) analysis of eleven individual FAs (denoted diagnostic FAs). Prin- originated from batches involved in PNS consumer complaints, and cipal component analysis (PCA) was used to visualize how the FA samples were received and analyzed from February 2011 through content enabled pine species discrimination. 2012.

2. Materials and methods 2.4. GC instrumentation and chromatographic conditions

2.1. Reference material (RM) Two different gas chromatographic (GC) systems were used. The GC-flame ionization detector (FID) system was used for routine Reference materials (RM) were named RM1eRM13. Species, RM determination of pine species, and the GC-mass spectrometer (MS) names, origin, and year of crop are presented in Table 1. P. armandii system was used to identify FAs that were not included in the FA (RM1), P. koraiensis (RM5A), and P. sibirica (RM9) were kindly gifted standard mixes commercially available. from the International Nut and Dried Fruit Council, Spain (INC). The GC-FID system: A polar SP2560 (Supelco, SigmaeAldrich, Denmark) or a CPSil88 (Agilent Technologies, Denmark) L i.d. m fi Table 1 100 m 0.25 mm column with a 0.2 m lm was used on a 6890A Identity of reference samples, their origin and year of harvest. GC system equipped with an FID (Agilent Technologies, Denmark). The injector temperature was 250 C. Oven temperature program: Species Name Origin Year of harvest Initial oven temperature at 100 C, raised to 140 Cat10C/min and P. armandii RM1 2010 held for 3 min, to 180 C min at 5 C/min, to 200 Cat1C/min, to P. bungeana RM2 China 2010 fi P. densata RM3 Benxi, China 2009 215 Cat5 C/min, held for 10 min, and nally raised to 230 Cat P. gerardiana RM4 Kashmir 2009 5 C/min and held for 10 min. A post run for 3 min at 100 Cwas P. koraiensis RM5A China 2010 included. Inlet pressure of helium carrier gas was 29 Psi, split 60:1, P. koraiensis RM5B Copenhagen, Denmark 2011 with a 4 mm ID split liner. Injection volume was 0.5 mL with a sy- P. massoniana RM6 Lishui, Zhejiang 2009 ringe size of 5 mL. Constant column helium flow was 0.8 ml/min. P. pinea RM7 Unknowna Unknowna fl P. pumila RM8 Kamchatka, Russia 2010 Detector temperature was 275 C. Hydrogen ow was 40.0 ml/min, P. sibirica RM9 China 2010 the air flow was 450.0 ml/min, and the helium makeup flow was P. tabuliformis RM10 China 2009 45.0 ml/min. P. wallichiana RM11 Kashmir 2010 The GCeMS system: The CPSil88 column (dimensions described P. yunnanensis RM12A Yuannan province of China 2009 e P. yunnanensis RM12B China 2011 above) was used on a Saturn GC MS system (Varian Inc.). Chro- P. cembra RM13 Germany 2010/2011b matographic conditions were similar as for the GC-FID. The GCeMS operated in the electric ionization (EI) mode and was used to obtain a Pine nuts were obtained from a local supermarket with no information on the geographic origin and year of harvest. a total ion chromatogram (TIC). Scans were performed from 40 to b A mixture of two different crops. 500 m/z, with a multiplier offset at 200 V, and a scan time of 0.740 s. A.Æ. Mikkelsen et al. / Food Control 40 (2014) 19e25 21

2.5. Separation of species and fatty acid analysis 1.7%; 5,11,14e20:3, 1.9%. The relatively low RSDs (%) were considered satisfactory. All pine nuts were either purchased unshelled or had their shell removed with a mortar and pestle or with a nutcracker prior to the 2.7. Diagnostic parameters in species determination morphological separation. The morphological pine nut character- istics such as size, color, and shape (eFloras, 2008) were used to The diagnostic parameters included the DI value, the sum of D5- separate pine nuts into subsamples of presumably distinct species. FAs, and the eleven diagnostic FAs (Supporting information, However, close morphological characteristics of pine nuts from Tables 1 and 2). The DI values (Destaillats et al., 2010) were auto- different species (eFloras, 2008) challenged the separation into matically calculated in the GC Chemstation software (Rev.B.03.02) distinct species. Only pine nuts with the most distinct morpho- according to Equation (1).

ð5; 9e18 : 22 þ 5; 9; 12e18 : 3 þ 5; 11; 14e20 : 3Þ Diagnostic index ðDIÞ¼ 10 (1) ð18 : ln9 þ 18 : 1n7 þ 18 : 2n6 þ 20 : 2n6Þ

logical characteristics were therefore chosen for further analysis. The sum of D5-FAs was calculated according to Equation (2) (the From the groups of separated pine nuts, 0.5e10 g were homoge- 5,9,12,15e18:4 FA appeared only in trace amount (see Supporting nized in a mortar. 0.1 g of homogenized pine nuts was used for information) and was excluded). preparation of FA methyl esters (FAME’s) as described elsewhere (Destaillats et al., 2010). In brief, 100 mg of the homogenized nuts The sum of D5 FAs ¼ 5; 9e18 : 2 þ 5; 9; 12e18 : 3 were esterified with 2.0 ml of methanol, 2.0 ml of 3 N hydrogen þ 5; 11e20 : 2 þ 5; 11; 14e20 : 3 chloride in methanol, and 2.0 ml of n-heptane in a 30 ml closed (2) tube placed for 1 h at 100 C in a Turbovap (Zymark Turbovap, Sotax, Switzerland). The esterification mixture was then cooled to The eleven diagnostic FAs included 16:0; 18:0; 18:1n9; 18:2n6; room temperature before 2.0 ml of water were added to stop the 5,9e18:2; 18:3n3; 5,9,12e18:3; 20:1n9; 5,11e20:2; 5,11,14e20:3; reaction. The mixture was whirl mixed for 30 s and centrifuged at and 7,11,14e20:3. 3500 RCF for 5 min (Heraeus Megafuge 3.0R, Thermo Fisher Sci- For simplicity, the content of the unidentified peaks and the entific, Inc.). Finally, one part from the heptane layer was diluted minor FAs 14:0; 17:1n7; 5,9,12,15e18:4; and 24:0 (Fig. 1)were with four parts of heptane and applied to GC vials. Each RM, sam- included in a sum of minor FAs. The minor FAs were not used as ple, or subsample was analyzed in duplicate unless otherwise diagnostic parameters in the species determination. stated. Each analysis consisted of a single injection into the GC The FA content obtained from RM in this study was used system. Fatty acids in the pine nuts were identified through com- together with literature data (Fardin-Kia et al., 2012; Wolff et al., parison to the following FA standard mixes: GLC 87, 606, 546B, 2000)(Supporting information, Table 1) to calculate intervals for 0 17AA , and 68A. Commercial standards that contain the cis-D5 all three diagnostic parameters. These intervals were denoted unsaturated polymethylene interrupted FAs (UPIFA) and bisho- reference intervals. The reference interval resembles a confidence mopinolenic acid 7,11,14e20:3 were not commercially available. A interval and calculated according to Equation (3). comparison of the obtained mass spectra with the mass spectra pffiffiffi present in the lipid library database, www.lipidlibrary.aocs.org,was Reference interval ¼ x t s= n ; (3) therefore used to confirm the identity of the cis D5-UPIFA and the 7,11,14e20:3 FA. The cis D5-UPIFA included taxoleic acid 5,9e18:2; where x is the mean and s the standard deviation. The 97.5% fractile pinolenic acid 5,9,12e18:3; coniferonic acid 5,9,12,15e18:4; kete- in the t-distribution with n 1 degrees of freedom was used for the leeronic acid 5,11e20:2; and sciadonic acid 5,11,14e20:3. The total reference interval. The unknown samples were assigned to the FAs chromatographic peak areas represent 100%. Quantification of species where most of the diagnostic parameters were within the the individual FA contents was calculated as peak area % compared reference intervals of the RM. Samples with more than three results to the 100% peak area. outside the reference interval were considered mixtures of species or species not included in our collection of RM. These samples were generally categorized as unknowns. 2.6. Quality assurance In two cases, additional analyses were provided by Sara Handy (US Food and Drug Administration) using the DNA sequencing Each analytical series consisted of a blank (reagents only), approach described in Handy et al. (2011). relevant RM, and samples. Reference material was analyzed twice on different days, except for P. armandii (RM1), P. koraiensis (RM5A), and P. sibirica (RM9) that were analyzed four times on different 2.8. Principal component analysis (PCA) days, and P. densata (RM3) that was analyzed once. Samples were For the visualization of the overall variation in the FA data, PCA analyzed in double determination. Detection limits of all FAs were Ò determined from 3 the noise, which correspond to 0.03e0.04% of was performed using Unscrambler (Version 9.1; CAMO, Trond- the total content of FAs (data not shown). The method was heim, Norway). accredited by The Danish Accreditation and Metrology Fund (DANAK) in August 2011. 3. Results and discussion Relative standard deviation (RSD) for duplicate analysis (n ¼ 77) was calculated for the DI index and four individual FA’s. Species determination through a comparison of diagnostic pa- The RSD (%) was: DI, 0.79%; 16:0, 1.5%; 18:1n9, 0.83%; 5,9e18:2, rameters in samples with diagnostic parameters in RM requires 22 A.Æ. Mikkelsen et al. / Food Control 40 (2014) 19e25

Fig. 1. A routine chromatogram that shows the identified fatty acids and the 5 unknown compounds in P. armandii. Identification of peaks: 1, unidentified; 2, 14:0; 3, 16:0; 4, unidentified; 5, 16:1n7; 6, 17:0; 7, 17:1n7; 8, 18:0; 9, 18:1n9; 10, 18:1n7; 11, 5,9e18:2; 12, unidentified; 13, 18:2n6; 14, 20:0; 15, unidentified; 16, 5,9,12e18:3; 17, unidentified; 18, 20:1n9; 19, 18:3n3; 20,5,11e20:2; 21, 5,9,12,15e18:4; 22, 20:2n6; 23, 22:0; 24,5,11,14e20:3; 25,7,11,14e20:3; 26, 24:0. that the RM encompasses the natural variation of the FAs. The RM samples contained a subspecies or a variety of P. armandii. To gather should, therefore, include year to year climatic changes and more evidence for this indication, samples S2 and S4 were DNA geographical differences. The variation of our RM was limited to sequenced at an external laboratory (see 3.2 Subspecies and one or two RMs within each species. Literature data (Fardin-Kia varieties). Sample S3 was equivalent to sample S2 and S4. et al., 2012; Wolff et al., 2000) on the FAs present in different The samples S1 and S5eS8 were morphologically homogenous pine species was, therefore, used to introduce variation in the and showed a content of the diagnostic FAs within most of the reference interval for the different RM (Supporting information, reference intervals for P. armandii. Table 1). Samples S9eS14 were separated into two morphologically In the evaluation of the literature data, one should be aware that different subsamples. Subsamples S9aeS14a showed a content of the different diagnostic parameters are calculated and reported diagnostic FAs close to the reference intervals of P. armandii. Sub- differently. This difference hampers direct comparison and should sample S9beS14b showed a content of diagnostic FAs close to the be accounted for. For example, Fardin-Kia et al. (2012) calculates the reference intervals of P. sibirica, except for subsample S14b that was DI value excluding the 18:1n7 FA. In the present and other work closest to the reference intervals of P. koraiensis. (Destaillats et al., 2010), the 18:1n7 FA was included in the DI. To Sample S15 was also separated into two morphologically enable comparison of the DI values, we recalculated the DI values different subsamples S15a and S15b. Subsample S15a showed a from Fardin-Kia et al. (2012) to include the 18:1n7 FA (Supporting content of diagnostic FAs close to the reference intervals of information, Table 1). P. armandii. Subsample S15b showed more than three diagnostic FAs outside the reference interval, and was therefore not species 3.1. Samples determined. Samples S16eS18 were separated into three morphologically The FA results obtained from analysis of samples (Fig. 1, and different subsamples S16aeS16c. Subsample S16a showed a con- Supporting information, Table 2), were compared to the reference tent of diagnostic FAs that match the reference intervals of intervals of RM in Supporting information, Table 1. The samples P. armandii. Subsample S16b showed a content of diagnostic FAs were assigned to the species where most of the diagnostic pa- closest to the reference intervals of P. sibirica. Subsample S16c rameters (the DI value, the sum of D-FAs, and the diagnostic FA) showed a content of diagnostic FAs close to the reference intervals were within the reference intervals (Supporting information, of P. koraiensis. Table 2). Samples with more than three results outside the refer- Sample S17 was separated into three morphologically different ence interval were considered mixtures of species or species not subsamples S17aeS17c. Subsample S17a showed a content of included in our collection of RM. These samples were therefore diagnostic FAs close to the reference intervals of P. armandii. Sub- categorized as unknowns. sample S17b showed a content of diagnostic FAs close to the Three homogenous samples S2eS4 (morphologically resem- reference intervals of P. cembra. Subsample S17c showed a content bling P. armandii) showed a content of diagnostic FAs that did not of diagnostic FAs close to the reference intervals of P. koraiensis. match any of the RMs (Supporting information, Table 2). Results Sample S18 was also separated into three morphologically were, nevertheless, closest to the reference interval of P. armandii. different subsamples S18aeS18c. Subsample S18a showed more These unmatched FA results and the fact that the samples had been than three diagnostic FAs outside the reference interval, and was involved in PNS consumer complaints could indicate that the therefore not species determined. Subsample S18b showed a A.Æ. Mikkelsen et al. / Food Control 40 (2014) 19e25 23 content of diagnostic FAs close to the reference intervals of P. koraiensis. Subsample S18c showed a content of diagnostic FAs close to the reference intervals of P. armandii. Several subsamples from the binary mixtures contain FAs that do not match the specific reference intervals perfectly but were moved toward the other subsample in the binary mixture (Supporting information, Tables 1 and 2). This was evident in subsamples S9b, S11beS13b characterized as P. sibirica. Subsamples S9b, S11beS13b matched the reference intervals of P. sibirica excluding a maximum of three diagnostic FAs that were moved toward the reference intervals of P. armandii, which was the other subsample present in samples. This shift in diagnostic FAs was also evident in subsample S14a categorized as P. armandii. Sample S14a matched the reference intervals of P. armandii excluding two diagnostic FAs that were moved toward the reference intervals of P. koraiensis, which was the other subsample in sample S14. Sample S14b matched the reference intervals of P. koraiensis excluding two diagnostic FAs that were moved toward the reference intervals of P. armandii, which was the other subsample in sample S14. These Fig. 2. Principal component 2 versus 1 scores from a PCA of the fatty acids content (% results showed that a morphological separation of pine nuts into of total fatty acids) of the reference materials (RM) from Supporting information, distinct species is difficult as pine nuts can be fractured, have their Table 1. The labels denote the species of the RM (score): A (P. armandii), B tip moved, be dried, or heat treated. (P. bungeana),C(P. densata), D (P. densata), G (P. gerardiana), K (P. koraiensis), M (P. massoniana), PI (P. pinea), P (P. pumila), S (P. sibirica), T (P. tabuliformis), W (P. wallichiana) and Y (P. yunnanensis). Circles illustrate grouping of RM from the same 3.2. Subspecies and varieties species. The first two PCs accounted for 93% and 4% of the total variation between RM, respectively. An attempt was made to investigate further, the identity of samples S2 and S4. Sample S2 and S4 were subjected to DNA sequencing analogously to the method described by Handy et al. because the best fit(P. armandii) showed 4 diagnostic fatty acids (2011). The DNA sequence was close to the vouchers of outside the reference intervals. The PCA plot showed, nevertheless, P. armandii but far from the other species included in the study that sample S18a was placed within the P. armandii group. This could (Handy et al., 2011). The fact that both the FA profiles and the DNA indicate that the species determination criterion that only three results (data not shown) from samples S2 and S4 were different but diagnostic fatty acids can be outside the reference intervals might be closest to the results obtained from P. armandii RM support the too narrow. It is, therefore, possible that the sample S18a is in fact notion that different subspecies of P. armandii are involved in PNS. P. armandii, despite its morphological difference from sample S18c The identification of possible subspecies was also visualized in characterized as P. armandii. The morphological differences between the PCA. sample S18a and S18c could originate from e.g. a natural batch variation or a mixture of pine nuts from different year of crop. 3.3. Principal component analysis (PCA) 4. Conclusion Principal component analysis (PCA) was used to gain insight into the large data set obtained from the FA analysis (Supporting P. armandii seems to be present in all the samples involved in information). Fig. 2 shows principal component (PC) 2 versus 1 PNS. However, in samples S2eS4 the species determination was scores from the PCA of the FAs content of the reference materials. It ambiguous, but the FA results were closer to P. armandii than to any is evident that samples from the same species grouped together in of the other species included in the RM. In addition, the the plot. The main variation (along PC1, accounting for 93% of the morphology of samples S2eS4 resembled P. armandii and the DNA total variation) was due to P. gerardiana (G) and P. pinea (PI) sequencing data close to P. armandii, suggested a subspecies or a differing substantially from all the other species. Also P. bungeana variety of P. armandii, which is different from the P. armandii sub- (B) differed considerably from the other species, mainly due to jected to analysis. This indicates that different subspecies or vari- variation along PC2. The PCA had 4 components and in Fig. 3 these eties might be involved in PNS. dimensions are illustrated. Species that were not separated by PC1 Subsamples S9a, S11aeS14a showed results on two or three and PC2 could be differentiated by PC3 (e.g. P. massoniana (M) and diagnostic FAs outside the reference interval but shifted toward the P. densata (D) as seen in Fig. 3, panel B) or PC4 (e.g. P. massoniana species present in the corresponding subsample and vice versa. (M) and P. wallichiana (W) as seen in Fig. 3, panel D). However, the This indicates an incomplete morphological separation of pine nuts species P. densata (D) and P. sibirica (S) could not be separated into distinct species, suggesting that much effort should be put into clearly by any of the 4 PCs. the separation process prior to species determination. To demonstrate the discriminating power of the PCA of the FAs The reference interval for the DI showed overlapping values content of the reference materials the samples from the present between species (Supporting information, Table 1). As a conse- study was used as a test set. In Fig. 4 it can be seen that the present quence, species determination based exclusively on the DI is not samples (S1eS18) grouped according to species nearly coincident possible. Other diagnostic parameters were therefore added and with the samples from the reference material. Thus it was shown included the sum of the D5-FAs and the diagnostic FAs (eleven that the species specific variation in these two data sets was quite individual FAs). The use of data from RM obtained in this study in similar. The PCA did also succeed in placing the samples S2eS4, away combination with literature data, ensured reference intervals that from other groups of RM and samples (although close to the encompass the natural variation of the FA content within species. P. armandii group), which supports the notion that they are sub- However, the data used to obtain the reference intervals were species. Sample S18a was characterized as an unknown species limited, and more studies could improve the range of the reference 24 A.Æ. Mikkelsen et al. / Food Control 40 (2014) 19e25

AB

CD

Fig. 3. Scores from a PCA of the fatty acids content (% of total fatty acids) of the reference materials (RM). Panel A: PC2 versus PC1; Panel B: PC3 versus PC1; Panel C: PC3 versus PC2 and Panel D: PC4 versus PC3. The labels denote the species of RM as given in legend to Fig. 2.

intervals to better reflect the natural variation. Nevertheless, PCA showed that samples grouped according to species nearly coinci- dent with the samples from the reference material. The success of PCA to discriminate the different pine species shows its potential in future speciation projects to avoid P. armandii. Nuclear magnetic resonance, DNA sequencing and FA profiling present a variety of methodologies, which are important to meet the specific equipment present in different laboratories. Further- more, in cases of ambiguous speciation results, different methods can act as complementary tools.

Acknowledgments

We gratefully acknowledge Sara M. Handy at the Food and Drug Administration, US, for performing the DNA sequencing analyses. Kirsten Halkjær Lund is acknowledged for critical comments on the manuscript. The Ministry for Health, the Elderlyand Community Care (MHEC), Environmental Health Directorate, Malta, is acknowledged for allowing inclusion of results obtained from their samples.

Appendix A. Supplementary data

Fig. 4. The position of samples from this study (Supporting information, Table 2) in the Supplementary data related to this article can be found at http:// scores plot from the PCA of the fatty acids content (% of total fatty acids) of the dx.doi.org/10.1016/j.foodcont.2013.11.030. reference materials. The labels of the reference material samples (pale gray) denote the species of the sample as given in legend to Fig. 2. The labels of the samples from the present study denote the species of the samples: a (P. armandii), c (P. cembra), k References (P. koraiensis), s (P. sibirica) and u (unknown species). The circles illustrate grouping of reference material sample from the same species. S2eS4 refer to samples assumed to Ballin, N. Z. (2012). A trial investigating the symptoms related to pine nut syn- originate from a P. armandii subspecies. drome. Journal of Medical Toxicology, 8,278e280. A.Æ. Mikkelsen et al. / Food Control 40 (2014) 19e25 25

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