Introduction

Vitamin D is the summarizing term for bioactive lipid compounds (vitamin D2–D7) which are essential micronutrients in human nutrition [1]. These secosteroids vary in the number of carbons (C27–C29) and double bonds (∆3–4), as well as the substitution pattern and stereochemistry in the side chain (SC) attached to C-17 (Fig. 1, SC2–SC7) [2, 3]. Due to their comparably higher concentrations in foods, cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2) play the predominant role in human nutrition. Both molecules are formed from distinct precursor sterols (i.e., cholesterol in animals and ergosterol in mushrooms) which are eventually transferred into the corresponding vitamins under ultraviolet (UV) radiation [4,5,6]. Hence, vitamin D is also known as the ‘sunshine’ vitamin, and levels in foods and humans naturally vary in dependence on UV exposure and lifestyle [5, 7, 8]. Notably, increasing numbers of vitamin D deficiencies in humans have been reported in the Western world [9]. Specifically, only a few dietary sources of animal origin (e.g., fatty fish, cod liver oil, and beef liver) contain noteworthy amounts of vitamin D3 (1 − 250 µg/100 g fresh weight (fw)) [10, 11], while mushrooms are the only relevant source of vitamin D2 of non-animal origin. However, typical vitamin D2 contents of edible mushrooms (< 10 µg/100 g fw) are comparably low but they are rich in provitamin ergosterol [12,13,14,15,16].

Fig. 1
figure 1

Structure and IUPAC numbering of the carbon atoms of the vitamin D backbone and different side chains (precursor sterols in parenthesis) with SC2 = vitamin D2 or ergocalciferol (ergosterol), SC3 = vitamin D3 or cholecalciferol (7-dehydrocholesterol), SC4 = vitamin D4 (ergosta-5,7-dienol or 22,23-dehydroergosterol), SC5 = vitamin D5 (poriferasta-5,7-dienol or 7-dehydrositosterol), SC6 = vitamin D6 (poriferasta-5,7,22-trienol or 7-dehydrostigmasterol), SC7 = vitamin D7 (7-dehydrocampesterol or campesta-5,7-dienol)

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In the European Union, UV-treated button mushrooms and mushroom powders with increased vitamin D2 levels (5–20 µg/100 g in fresh mushrooms and 100–130 mg/100 g in powders) were recently approved as Novel Food (EU 2020/1163 based on EU 2015/2283) [17]. By UV treatment of mushrooms, provitamin ergosterol is converted via C–C bond dissociation into previtamin D2 and subsequently into vitamin D2 [18]. Next to vitamin D2 formation, various isomeric by-products [18,19,20] can be formed which are difficult to characterize due to their complexity and comparably low amounts. Several co-elutions are known to exist in high-performance liquid chromatography which cannot even be resolved by LC/MSMS which currently represents the ‘gold standard method’ in vitamin D analysis [21]. Contrary to vitamin D3 which is also analyzed for metabolites in blood serum [13, 16, 22,23,24], knowledge on liable and comprehensive analytical methods for isomeric photoproducts of vitamin D2 is only scattered found and difficult to combine [25,26,27,28].

To overcome these difficulties, UV irradiation experiments were performed with ergosterol and vitamin D2. A rather long irradiation time was chosen in order to generate a high variety of isomeric photoproducts in sufficient amounts. Reaction products were investigated by high-performance liquid chromatography (HPLC) with diode array detector (DAD) and gas chromatography with mass spectrometry (GC/MS). In addition, HPLC with circular dichroism (CD) detection was used for the first time in this research area. Selected compounds were isolated by repeated HPLC fractionation and analyzed by 1H NMR.

Materials and methods

Chemicals and standards

Pyridine (> 99% puriss.), ergosterol (≥ 95%), and vitamin D2 (CRM ≥ 99%) were from Sigma-Aldrich (Darmstadt, Germany). Previtamin D2, lumisterol2 (97%), and 5,6-trans vitamin D2 (95%)—the subscript number “2” refers to the same side chain substitution as in vitamin D2—were from Santa Cruz Biotechnology (Dallas, TX, USA). Tachysterol2 (> 90%) was from Toronto Research Chemicals (North York, Canada). 5α-cholestane (98%) was from Acros Organics (Geel, Belgium). Methanol, ethanol, and n-hexane (all HPLC grade) were from Th. Geyer (Renningen, Germany). Acetonitrile (> 99.5%) was from Bernd Kraft (Duisburg, Germany). The silylation reagent Silyl 991 (N,O-bis(trimethylsilyl)-trifluoroacetamide and trimethylchlorosilane (TMCS), 99:1 (v/v)) was from Macherey–Nagel (Karlsruhe, Germany). Double distilled water was from the in-house system of the University of Hohenheim (Stuttgart, Germany).

Irradiation of standard solutions ergosterol and vitamin D2

UV lamps (n = 2) with a main emission line of λmax = 310 nm were from Waldmann (Villingen-Schwenningen, Germany). The distance between the two UV lamps and samples was 30 cm, respectively. Standard solutions of ergosterol and vitamin D2 were prepared in ethanol (1 mg/mL, respectively) and placed in UV transmitting quartz glass vessels. Initial experiments with different exposure times showed that a long irradiation time of 60 min was best suited to generate relatively high amounts and a high variety of photoproducts. Therefore, standard solutions were irradiated for 60 min except in the case of preparative HPLC fractionation and subsequent 1H NMR analysis (90 min irradiation time).

HPLC–UV and HPLC-CD analysis, and preparative HPLC fractionation

Before and after UV irradiation, standard solutions (in ethanol) were analyzed on a UFLC system (injection volume 10 µL) equipped in series with an SPD-M20A (diode array) UV–vis detector (Shimadzu, Kyoto, Japan) and a CD-2095 circular dichroism (chiral) detector (CD; Jasco, Pfungstadt, Germany). The simple CD-2095 detector does not record full CD spectra but displays peaks at a given HPLC retention time and excitation wavelength as a positive or negative signal. Noteworthy, the direction of the signal (±) in the CD must not necessarily reflect the one of the optical rotation (α[D]20 value).

Due to the serial connection, HPLC retention times in the CD detector were ~ 30 s higher than in the UV detector. An analytical ET 250/8/4 Nucleosil 7 C18 column (250 mm × 4 mm i.d. × 7 µm particle size, Macherey–Nagel, Düren, Germany) was used in combination with a binary gradient system (A: acetonitrile, B: methanol) according to Wittig et al. [25]. The solvent flow was set to 1 mL/min with 100% A at the beginning for 10 min. Then, over 10 min to 95% A and 5% B, which was held for a further 10 min. Finally, again to 100% A within 5 min and isocratic (100% A) for 8 min to equilibrate the system. Data were collected in the full spectral range of 190–800 nm (DAD) and at an excitation wavelength of 278 nm (CD). The HPLC fractionation of irradiated standard solutions (ergosterol or vitamin D2) for subsequent GC/MS analysis was based on positive and negative signals in the HPLC-CD detector. At these points, the effluent was manually collected in different brown glass flasks (1 mL and 25 mL depending on fraction size).

Silylation and GC/MS analysis

Sample solutions were evaporated to dryness by a gentle N2 stream and derivatized (60 min at 55 °C) with trimethylsilylation reagent (50 µL Silyl 991 and 25 µL pyridine) as described previously in detail [29, 30]. Silylated residues were then re-dissolved in 25 µL internal standard solution (1.2 µg 5α-cholestane/mL n-hexane). Silylated ergosterol, vitamin D2, further standards, and HPLC fractions of photoproducts were analyzed on an Agilent 6890/5973 N GC/MS system connected with a GC PAL autosampler (CTC Analytics PAL system, Zwingen, Switzerland) as described previously [30, 31]. The GC was equipped with a deactivated guard column (1 m × 0.25 mm, Zebron Phenomenex, Aschaffenburg, Germany) hyphenated to an Optima 5-HT chromatographic column (30 m × 0.25 mm i.d. × 0.25 µm film thickness, Macherey–Nagel, Düren, Germany). Chromatographic separation was achieved as described previously [30, 31]. Data were acquired in full scan mode (m/z 50–650).

Characterization strategy and quality control via 1H NMR analysis

Photoproducts characterized either with available reference standards (i.e. previtamin D2, vitamin D2, tachysterol2, lumisterol2, and 5,6-trans vitamin D2) or 1H NMR were assigned to level A (structure verified; il-A, not labeled in the following). All other isomers in UV irradiated solutions were specified by HPLC (λmax in UV spectra, retention times, and positive/negative signals in the CD detector) and GC/MS data (full scan mass spectra and relative retention times). In case of a good match with literature values, they were labeled identification level B (il-B) compared to il-C for tentative assignments.

Solutions of vitamin D2, UV irradiated for 90 min (signals in HPLC-CD detector were deemed too weak after 60 min), were fractionated by HPLC using the column above along with a serially connected second HPLC column (ET 250/8/4 NUCLEOSIL 7 C18, Macherey–Nagel, Düren, Germany) and isocratic elution (5% methanol, 95% acetonitrile). Six or seven HPLC runs were fractionated between HPLC-CD retention time (tR) 14.9–15.9 min (suprasterol2 II, il-B) and 18.6–20.1 min (suprasterol2 I, il-B), were pooled, respectively, evaporated to dryness, and taken up in CDCl3 for 1H NMR analysis (500 MHz).

Results and discussion

Next to vitamin D2 (3), several other isomers including lumisterol2 (4), tachysterol2 (5), and suprasterol2 isomers (8, 9), are formed during UV irradiation of ergosterol (1). These isomers can be of tetracyclic (ergosterol and three further isomers), tricyclic (vitamin D2 and four other isomers), and pentacyclic (two isomers, Fig. 2) structure [2, 18, 20, 32]. For peak assignment, both ergosterol (1) and vitamin D2 (3) were irradiated for 60 min (if not denoted otherwise).

Fig. 2
figure 2

Trivial names and structures including stereochemistry of the isomers ergosterol (1) and vitamin D2 (3), the tricyclic photoisomers previtamin D (2) and (5, 6, 7), the tetracyclic photoisomer lumisterol2 (4) and the thermal isomerization products pyro- (10) and isopyro vitamin D2 (11) and the pentacyclic photoproducts suprasterol2 I (8) and II (9). Asterisk According to IUPAC nomenclature, the letter ‘Z’ specifies the orientation of the ∆5,6 double bond to the ring system which bears the 3-OH group. Letters before and after ‘Z’ refer to cis- (c) or trans- (t) orientation of the other double bonds (at ∆10,19 and ∆7,8, respectively) relative to the one at ∆5,6. Subscript letters 2 refer to the side chain as found in vitamin D2. Blue bullet Isomers marked with a blue dot (6, 7) are isomerized to pyro- and isopyro products of unknown structure at GC injection

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HPLC–UV and HPLC-CD analysis of ergosterol and vitamin D2 standards before and after UV irradiation

HPLC–UV chromatograms (reversed phase mode) of ergosterol and vitamin D2 standards verified the much faster elution of the more polar tricyclic vitamin D2 (3) (ΔtR ~ 20 min, Fig. 3a,c) [25], and both isomers produced negative peaks in the HPLC-CD chromatogram (Fig. 3b,d). The split peak in the HPLC-CD chromatogram of the vitamin D2 standard (Fig. 3b) indicated the presence of two co-eluting compounds which could not be distinguished in the UV detector (Fig. 3a). Noteworthy, another vitamin D2 standard from a different supplier did not show this split peak (Fig. S1, supporting information). Interestingly, in the case of vitamin D3 (cholecalciferol), HPLC co-elution of 3α- and 3β-hydroxy epimers of 25-hydroxy-vitamin D3 was recently described by Al-Zohily et al. [33]. The details of the C3-epimerization process are not fully understood but can reportedly occur at all major vitamin D metabolites [33, 34]. The presence of the corresponding and hitherto unreported 3-hydroxy epimers of vitamin D2 could thus explain our measurements, and this observation directly underlined the benefits the HPLC-CD detector added to the present study.

Fig. 3
figure 3

HPLC chromatograms with (a, c, e, g) UV detection (brown lines) and (b, d, f, h) CD detection (black lines) of standard solutions of vitamin D2 and ergosterol in ethanol (a–d) before and (e–h) after UV irradiation (60 min) with 5,6-trans vitamin D2 (peak #1), previtamin D2 (peak #2), and the non-isomeric ergosta-3,5,7,9,22-pentaene (peak #4). Peak #3 consisted of five isomers which were eventually assigned to vitamin D2 (peak #3a; (c−)Zt form), (trans-vitamin D2 (peak #3b; (c+)Zt-vitamin D2), lumisterol2 (peak #3c), tachysterol2 (peak #3d), and suprasterol2 (peak #3e). Subscript letters 2 refer to the side chain as found in vitamin D2

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HPLC–UV chromatograms of UV irradiated ergosterol and vitamin D2 together featured four additional signals, which were eluted before vitamin D2 (peak #1 and peak #2), co-eluted with vitamin D2 (peak cluster #3), or eluted between vitamin D2 and ergosterol (peak #4) (Fig. 3e,g). HPLC analysis of additional standards (Sect. 2.1) verified that peaks #1 and #2 originated from 5,6-trans vitamin D2 (7) and previtamin D2 (2), respectively, due to identical HPLC retention times and UV spectra (λmax are given in Table 1 and UV spectra in Fig. S2, supporting information). The broad and fronting peak at the retention time of vitamin D2 (3) in both HPLC–UV chromatograms indicated that several compounds including c(+)Zt trans-vitamin D2 (peak #3b) as well as lumisterol2 (peaks #3c) and tachysterol2 (peak #3d)—both verified by standards—co-eluted with vitamin D2 (peak #3a) (Fig. 3e, g). This could be substantiated by five co-eluting compounds detected in the HPLC-CD chromatograms (Fig. 3f, h). While vitamin D2 (peak #3a) and ergosterol (peak #5) standards generated negative signals in the CD (Fig. 3b, d), most newly generated peaks in the irradiated solutions including peaks #3b-d generated positive signals in the HPLC-CD detector (Fig. 3f, h). In agreement with that, the UV peak purity test and UV full spectrum analysis at HPLC-CD retention times of peak #3a,c (generated by UV irradiation of ergosterol, Fig. 3h) showed that vitamin D2 (peak #3a, λmax at 265 nm) co-eluted with lumisterol2 (peak #3c, λmax at 280 nm and 271 nm) (Figs. S1 and S2, supporting information). According to the CD signal, vitamin D2 was only present at trace levels in both UV irradiated solutions, although the HPLC–UV chromatogram, which did not allow to distinguish both forms, showed an abundant peak at the corresponding retention time (Fig. 3e, g). At this point, it became obvious that HPLC–UV data based on single wavelengths were not sufficient for peak characterization. Subsequent fractionation of peak #3 according to the HPLC-CD signal or recording of the UV full range spectrum (190–800 nm) followed by a peak purity test allowed to elaborate further details. Accordingly, peak #3b in the UV irradiated vitamin D2 solution showed λmax at 265 nm which is in full agreement with vitamin D2 (Figs. S1 and S2, supporting information). However, the positive signal in the CD detector (Fig. 3f) unequivocally showed that the bulk of peak #3b could not originate from vitamin D2 [which produced a negative signal in the CD, (#3a, Fig. 3b)] but indicated the presence of a vitamin D2 conformer without differences in the conjugated system. In line with that, previous investigations on the photochemistry of vitamin D (not specified in which form) reported the existence of two stable vitamin D conformers, namely c(−)Zt-vitamin D (3, Fig. 2) and the less twisted c(+)Zt trans-vitamin D (6) [18, 32]. These two compounds were tentatively assigned to peaks #3a and #3b in Fig. 3f. The vitamin D conformers (3, 6, Fig. 2) differ in the orientation of the cisoid diene moiety [18, 32] which could be the reason for the reversal of the CD signal while λmax (265 nm) remained the same (Fig. S3, supporting information). Accordingly, at this long irradiation time of 60 min, vitamin D2 (3) was barely detected at all and predominantly transformed into c(+)Zt trans-vitamin D (6, Fig. 2). This indicated the strong dependency of vitamin D2 formation and degradation on the reaction conditions and, again, the value added by implementing the CD detector. Further details were elaborated by supplementary measurements with GC/MS (Sect. 3.2).

Table 1 HPLC/UV-CD and GC/MS data of the photoproducts detected after UV irradiation and characterized after HPLC fractionation of irradiated standard solutions (ergosterol and vitamin D2)
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GC/MS analysis of silylated standards

In hot GC/MS injector ports, trimethylsilylated vitamin D2 (3, Table 1 and Fig. 2) was converted into two characteristic tetracyclic isomers, i.e. pyro vitamin D2 with 9S,10S-configuration (10, Table 1) and the later eluting isopyro vitamin D2 with 9R,10R-configuration (11, Table 1) (Fig. 4a) [22]. Note that this problem cannot be overcome by using an on-column injector because this leads to uncontrolled isomerization on the column during heating. Both isomers were not formed during UV irradiation but they are structurally closely related to ergosterol (1) with 9S,10R-configuration, and lumisterol2 (4) with 9R,10S-configuration (Fig. 2). The four tetracyclic isomers showed very similar GC/MS spectra but could be distinguished due to different GC retention times (Table 1). Similarly, 5,6-trans vitamin D2 (7)—which was available as standard—was degraded in the GC injector port to give pyro-5,6-trans vitamin D2 (RRT 1.32) and isopyro-5,6-trans vitamin D2 (RRT 1.37) (Table 1). Accordingly, isopyro and pyro forms of vitamin D2 and 5,6-trans vitamin D2 could be GC separated [28, 35]. Most remarkably, however, the (pure) tricyclic previtamin D2 (2) standard was partly converted into pyro- (10) and isopyro vitamin D2 (11) in the GC injector. Hence, the simultaneous presence of previtamin D2 (2) in samples will lead to an over-determination of vitamin D2 (3) when GC/MS is used for quantification. In GC/MS, previtamin D2 (2) was also partly co-eluted with lumisterol2 (4) (Table 1) while tachysterol2 (5) eluted slightly before ergosterol from the GC/MS column (Fig. 4a). In the following, we aimed to minimize these limitations of HPLC and GC by a shared evaluation of UV irradiated samples with both methods.

Fig. 4
figure 4

GC/MS full scan chromatograms of (a) trimethylsilylated standard solutions of vitamin D2 (quantitatively isomerized to give in pyro- and isopyro vitamin D2), tachysterol, and ergosterol and the resulting solutions after UV irradiation (1 h in ethanol) of (b) vitamin D2 and (c) ergosterol. * and ** refer to coelutions as shown above the peaks. #A-#D are four minor isomers that could not be fully characterized

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HPLC and GC/MS evaluation of photoproducts derived from the UV irradiation of vitamin D2 (over-irradiation products)

GC/MS chromatograms of UV irradiated standards indicated isomeric photoproducts other than those mentioned above produced only one signal. Specifically, the GC/MS chromatogram of irradiated vitamin D2 solution featured four additional peaks, three of which will be characterized in this section. Two prominent peaks of suprasterol2 I (8) and suprasterol2 II (9) in the GC/MS chromatogram (Fig. 4b) gave no signal in the HPLC–UV chromatograms which indicated the lack of conjugated double bonds. However, analysis of HPLC fractions by GC/MS allowed us to determine their HPLC retention times (i.e. within the peak #3 cluster) and subsequent isolation by preparative HPLC (Sect. 2.2) and 1H NMR analysis confirmed their structures (Fig. S4, supporting information). Namely, the chemical shifts verified the pentacyclic backbones as reported by Kobayashi et al. [28]. Moreover, the peak abundance ratio of suprasterol2 II (9) to suprasterol2 I (8) in the present GC/MS chromatograms of ~ 3: 1 (Fig. 4b) was in accordance with the one reported by Okamura et al. (2001) [27]. A small negative CD signal indicated the presence of the suprasterol2 isomers (8, 9) in the irradiated vitamin D2 solution (peak #3e, Fig. 3f) but underrated their high relevance according to GC/MS. Last but not least, the identical GC/MS spectra of the suprasterol2 isomers (M+, trimethyl silylated, m/z 468) featured the diagnostic base peak at m/z 118 [M-TMSiO-H-259]+ with the more abundant suprasterol2 II (9) eluting first [28, 35,36,37]. A further diagnostic GC/MS fragment ion of silylated suprasterols2 was detected at m/z 209 which still included the TMSiO group [M-259]+ (Table 1). In HPLC, suprasterol2 II (peak #3e) eluted slightly after and partly coeluted with c( +)Zt trans-vitamin D (6) which produced the prominent positive peak #3b (Fig. 3f).

The third newly formed peak formed during the UV irradiation of vitamin D2 originated from 5,6-trans vitamin D2 (7) (verified by a standard). In GC/MS, 5,6-trans vitamin D2 (7) and c(+)Zt trans vitamin D2 (6) were converted into pyro- and isopyro-vitamin D2 (i.e. the same compounds, Fig. 4b). The pyro form of the trans-isomers left the column slightly earlier than isopyro vitamin D2 (Fig. 4b). In HPLC, the isomers 5,6-trans vitamin D2 (peak #1; 7) and c(+)Zt trans-vitamin D (peak #3b; 6) were resolved by ∆tR ≈ 3 min (Fig. 3f). Furthermore, 5,6-trans vitamin D2 (7) and c(+)Zt trans-vitamin D2 (6) with three conjugated double bonds each, differ in the number of alkyl substituents (Fig. 2). The higher number of five alkyl substituents on the conjugated triene system in the case of 5,6-trans-vitamin D2 (7) versus four in c(+)Zt trans-vitamin D2 (6) could be the reason for the shift of λmax from 265 nm (6) to 272 nm (7) (Fig. 2, Table 1) [38].

Again, λmax at 265 nm in combination with the positive signal detected at this retention time in the CD detector (Fig. 3f) unequivocally verified that the bulk of peak #3b originated from c(+)Zt trans-vitamin D and not from vitamin D2. In this context, it is important to note that vitamin D2 was more abundant at shorter irradiation times (data not shown). Given the fact that all compounds of the peak #3 cluster in HPLC (virtually the same tR) were isomers, a differentiation could not be achieved by LC/MS (Fig. S5, supporting information). Additional HPLC measurements with a second connected HPLC column and UHPLC/HRMS measurements (Fig. S5, supporting information) showed that the separation of some (i.e. tachysterol2 and lumisterol2) but not all isomers (i.e. c(+)Zt and c(−)Zt isomers) can be improved using advanced chromatographic systems (e.g. UHPLC or 2D-LC). However, problems in the identification of the isobaric substances remained due to the identical mass spectra which were dominated by a prominent [M + H]+ ion at m/z 397.3461 (Fig. S5, supporting information). This example again illustrates the high value added by implementing the CD detector.

The GC/MS spectra of 5,6-trans-vitamin D2 (7) and trans-vitamin D2 (6) showed the base beak at m/z 119 (Fig. 5a). Most likely, m/z 119 represents the dimethyl-substituted tropylium cation [C9H11]+, which could be formed from M+ by loss of TMSiOH, and carbons C-8 to C-28 (ring C, ring D, and the entire side chain) (Fig. 5a). This was further substantiated by fragment ions m/z 93 and m/z 79 in the GC/MS which resulted from the further loss of C2H2 (m/z 119—26 u) and C3H4 (m/z 119—40 u), respectively (Fig. 5a). Accordingly, the distinct prominence of the (dimethyl-substituted) tropylium cation at m/z 119 allowed to distinguish 5,7-dienic trans-vitamin D forms (6, 7) from both 6- monoenic D-vitamin (2, 5) isomers and tetracyclic sterols (1, 4) including pyro- and isopyro-vitamin D2 (10,11) by GC/MS (Table 2). Namely, the tetracyclic isomers (1, 4, 10, 11) produced the diagnostic fragment ion at m/z 131 of ∆5,7-sterols [30, 39]. Likewise, the base peak of the tricyclic trans forms at m/z 119 is 1 u higher than the base peak of the suprasterol2 isomers (m/z 118, see above) which also allows to demarcate these two substance classes by GC/MS (Table 2).

Fig. 5
figure 5

Structures and GC/MS full scan spectra of trimethylsilylated (a) 5,6-trans vitamin D2 with inserted postulated formation of m/z 119 and (b) tachysterol2

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Table 2 Diagnostic GC/MS fragment ions of tri-, tetra-, and pentacyclic isomers for the differentiation of sterols, D-vitamins, trans-D-vitamins, and suprasterols. * base peak
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HPLC and GC/MS evaluation of photoproducts derived from the UV irradiation of ergosterol

HPLC–UV chromatograms of UV-irradiated ergosterol also featured previtamin D2 (2) (peak #2, λmax 261 nm, Table 1, Fig. 2g,h) [25, 40, 41], tachysterol2 (peak #3d, λmax 280 nm, Table 1, Fig. 2g,h) and lumisterol2 (peak #3c, λmax 271 nm, Table 1, Fig. 2g,h). Contrary to very similar GC/MS spectra, ergosterol and lumisterol2 (4) can be distinguished via HPLC–UV and GC/MS retention time. For instance, λmax (271 nm) was unique for lumisterol2 (Fig. 4c) and these details agreed well with literature data [25, 38]. Also, the CD spectra of both isomers are different [25, 38], and in agreement with that, ergosterol (1, peak #5) generated a negative and lumisterol2 (4, peak #3c) a positive signal in the HPLC-CD chromatogram (Fig. 3b,h) (Sect. 3.1). The shorter GC and HPLC retention times of lumisterol2 (4) compared with ergosterol (1) or other sterols (Table 1) were likely due to the 10(S) configuration and resulting distinctly different geometry caused by the trans-configuration of ring system A-B (1, 9(R),10(S) versus (2), 9(S),10(R), respectively) which results in a ‘boat’ versus ‘chair’ form and also differs from the open-chained and more planar vitamin D2 structures.

Tachysterol2 (5, peak #3d, Fig. 2d) showed a similar HPLC retention time as vitamin D2 (peak #3a) but λmax differed remarkably (280 nm compared to 265 nm of vitamin D2, Table 1) [25, 41]. Silylated tachysterol2 (5) eluted slightly earlier than silylated ergosterol (1) from the GC column, but it could not be fully resolved from the much higher abundant ergosterol (Fig. 4a, c). However, the GC/MS spectrum (silylated) of tachysterol2 (5) could be distinguished from ergosterol (1) due to the presence of three equally abundant fragment ions, i.e. m/z 253 [M-TMSOH-SC]+, m/z 157, and m/z 119 (~ 50% of the abundance of the base peak at m/z 69, Fig. 5b) while ergosterol showed the base peak at m/z 363 (Table 1). Noteworthy, tachysterol2 was only present in UV irradiated ergosterol but not in UV irradiated vitamin D2 solution (peak, #3d, Fig. 3f,h).

Next to the compounds discussed so far, four additional isomeric photoproducts (isomers #A-D), trimethylsilylated M+ at m/z 468, Table 1) were detected by GC/MS. The GC/MS spectrum of isomer #D was similar to the one of pyro and isopyro vitamin D2 (10, 11), ergosterol (1), and lumisterol (4) which was surprising because these four tetracyclic isomers cover all structural variants in 9 and 10 positions, respectively (Fig. 2). The GC/MS spectrum of isomer #C was similar to trans-vitamin D2, (6) and included the diagnostic base peak at m/z 119 (dimethyl-substituted tropylium cation) diagnostic for tricyclic, 5,7-dienic trans-vitamin D forms (Table 2) which could be a 3-hydroxy epimer of trans-vitamin D2 due to the similar GC retention time as likewise observed for tentatively identified vitamin D2 3-hydroxy epimer (Sect. 3.1). Isomer #A (present in both UV irradiated solutions, Fig. 4b,c) showed the base peak at m/z 253 [M-SC-TMSOH]+, which is also found in (silylated) tachysterol2 (5) but otherwise, both isomers differed in their fragmentation patterns. Finally, isomer #B showed a unique GC/MS spectrum with the base peak at m/z 143.

In addition, a few smaller peaks originated from non-isomeric compounds or impurities already present in the standard. Exemplarily, two non-isomeric compounds (trimethylsilylated M+ at m/z 466, base peak m/z 376, Table 1, and non-silylated M+ at m/z 376, base peak m/z 251, peak #4 in Fig. 3g,h, Table 1) were detected which were most likely dehydrogenation products of ergosterol.

Conclusion

HPLC and GC/MS methods were used for the characterization of isomers of vitamin D2 formed by the UV-irradiation of ergosterol and the title compound. Both separation methods, HPLC and GC, were found to have drawbacks. For instance, co-elution of vitamin D2 with up to four isomers was observed in HPLC, which may have implications when only LC/MS is used for vitamin D2 determination. These existing problems could be addressed by the serial linking of UV and CD detectors. UV full range spectra enabled the classification of several isomers by λmax. However, suprasterol2 I and II could not be detected due to the lack of conjugated double bonds in these main irradiation products of vitamin D2. Particularly valuable proved to be the CD detector which was used for the first time in this research area. The serial linking of the CD after the UV detector allowed to indicate several co-elutions with vitamin D2, by means of the positive and negative signals. However, one disadvantage of the CD was the weak correlation between peak abundance and the amount of a compound. This problem could be addressed by the application of GC/MS. Despite the isomerization of vitamin D2 and related isomers at mandatorily high temperatures in GC analysis, the good separation was paired with a comparably objective impression of the amounts of the isomers of vitamin D2 and ergosterol. GC/MS allowed indicating the suprasterol2 isomers which could be isolated by HPLC and characterized by 1H NMR. After addressing the benefits and drawbacks of the individual methods, the shared application and evaluation of HPLC–UV, HPLC-CD, and GC/MS enabled the detection of > 10 additional isomers in irradiated ergosterol and vitamin D2 solutions. The methods and data generated in this study are ready for being used in the evaluation of vitamin D2 and isomeric photoproducts in food samples such as (UV-irradiated) mushrooms which are the most important non-animal source of vitamin D in human nutrition.