Article pubs.acs.org/JAFC
Simultaneous Determination of 25 Ergot Alkaloids in Cereal Samples by Ultraperformance Liquid Chromatography−Tandem Mass Spectrometry Qiaozhen Guo,†,‡ Bing Shao,† Zhenxia Du,‡ and Jing Zhang*,† †
Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Center for Disease Control & Prevention, Beijing 100013, China ‡ College of Science, Beijing University of Chemical Technology, Beijing 100029, China S Supporting Information *
ABSTRACT: A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of 25 ergot alkaloids in cereal samples. The analytes included both -ine and -inine ergot alkaloids and were extracted using an acetonitrile and ammonium carbonate solution, followed by purification with C-18 sorbent. After full separation on a C18 column, the 25 ergot alkaloids were detected by LC-MS/MS using multiple reaction monitoring (MRM) in the positive ion mode. The linear range was 0.05−5.0 μg/kg for the 25 ergot alkaloids. The mean recoveries at three spiked concentrations varied from 76.5 to 120% with RSD < 15%. This method was validated using a FAPAS proficiency test sample of ergot alkaloids in rye flour and was finally applied to analyze real samples, including rye flours, wheat flours, whole wheat flours, bread, and noodles. KEYWORDS: ergot alkaloids, cereal sample, ultraperformance liquid chromatography−tandem mass spectrometry
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INTRODUCTION Ergot alkaloids are mycotoxins produced by Claviceps fungal species that grow on the ears of rye and related cereal or forage plants. This fungus can parasitize forage plants in the rainy season, especially during the flowering period, and replace the developing grain or seed with a wintering body, known as ergot or sclerotium, that contains alkaloids. The sclerotia are harvested together with the uninfected grain. Humans or animals that ingest the contaminated cereal products might experience poisoning or even abortion.1,2 In 2001, there was an outbreak of ergot poisoning in Arsi and Ethiopia due to the consumption of contaminated barley.3 The common structural feature of ergot alkaloids is the ergoline ring, which is methylated on the N-6 nitrogen atom, is substituted at C-8, and possesses a C89 or C910 double bond (Figure 1). 4 The main ergot alkaloids include pharmacologically active lysergic acid derivatives, derivatives of isolysergic acid, and clavine alkaloids.5 According to international classification, the left-hand rotation isomers of ergot alkaloids (C-8-(R) configuration) (Figure 1) are termed ergopeptines (e.g., ergotamine), and the right-hand rotation diastereomers (C-8-(S) configuration) (Figure 1) are termed ergopeptinines (e.g., ergotaminine). C-8-(R) isomers (-ines) are biologically active, whereas the C-8-(S) isomers (-inines) are inactive.6 The conversion of -ines to -inines is rapid, especially in aqueous acidic or alkaline solutions, and -inines can also convert back into the -ine form in methanol, aqueous organic solvent, and acids.7 Although up to 82% of ergot sclerotia can be effectively removed during grain cleaning and processing,8 ergot alkaloids still occur in food and feed commodities, sometimes at relatively high levels.9 The European Union (EU)Scientific © 2016 American Chemical Society
Panel on Contaminants in the Food Chain of the European Food Safety Authority (EFSA) has recommended that the major ergopeptines and their corresponding epimers be targeted for determination in food and feed to provide data to enable consumer exposure calculations.10 To safeguard the health of animals and human beings, EFSA established a tolerable daily intake (TDI) for total ergot alkaloids of 0.6 μg/ kg of body weight/day. A maximum allowable value of 500 mg of ergot bodies/kg grain (0.05% w/w) was set in the EU.11 The maximum permissible level in the United States and Canada is 300 mg of ergot bodies/kg grain,9 and the limit in China is 0.01% of the total ergot alkaloids content in grain.12 Many methods can be used to extract ergot alkaloids from cereal samples, including liquid−liquid extraction,13,14 solidphase extraction,15−17 and QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe).18 These sample preparation methods can effectively extract the targeted ergot alkaloids from a complicated matrix under optimized conditions. The analytical methods used to determine ergot alkaloids include liquid chromatography coupled to mass spectrometry (LC-MS/ MS)8,13,19−24 with different modes of ionization including electrospray ionization (ESI) and atmospheric pressure photoionization;25 LC coupled to high-resolution mass spectrometry;16,26 and LC coupled to ultraviolet, photodiode array,16,27,28 or fluorescence detection.29−32 However, for rapid screening diagnosis, some authors have suggested that thin layer chromatography27,28,33 or immunoassays34,35 are preferred Received: Revised: Accepted: Published: 7033
June 2, 2016 August 25, 2016 September 1, 2016 September 1, 2016 DOI: 10.1021/acs.jafc.6b02484 J. Agric. Food Chem. 2016, 64, 7033−7039
Article
Journal of Agricultural and Food Chemistry
Figure 1. Structures of the 25 ergot alkaloids.
electrophoresis36 or gas chromatography,28,37 which are usually accompanied by MS. In China, a unified analysis standard
because they are quick and cost-saving. Moreover, other instrumental procedures have been used, such as capillary zone 7034
DOI: 10.1021/acs.jafc.6b02484 J. Agric. Food Chem. 2016, 64, 7033−7039
Article
Journal of Agricultural and Food Chemistry
obtained from Waters (Milford, MA, USA). The -ine and -inine type ergot alkaloid standards were dissolved in chloroform, sealed, and stored in the dark at −80 °C individually. Working solutions were prepared by evaporating the chloroform and reconstituting in acetonitrile. The other standards were dissolved in acetonitrile. The 25 mixed standards were also prepared in acetonitrile. Sample Preparation. All of the cereal samples (rye flour, wheat flour, bread, and noodles) were purchased from local supermarkets and Taobao, the largest internet-based retailer in China. The FAPAS proficiency test sample of ergot alkaloids in rye flour was obtained from the Food and Environment Research Agency (Sand Hutton, York, UK). The noodles, pasta, and bread were ground first. Approximately 1.0 g of sample was weighed in a 50 mL polypropylene centrifuge tube, and 10 mL of acetonitrile and ammonium carbonate (85:15, v/v) was added. After 30 s of shaking, 30 s of vortexing, and 5 m of centrifuging at 9000 rpm below 4 °C, 5 mL of the supernatants was transferred to another tube that contained 150 mg of C18 sorbent for purification. The mixture was vortexed for 30 s and centrifuged for 5 min at 9000 rpm below 4 °C, and then the upper layer was withdrawn for the UPLC-MS/MS analysis. LC-MS/MS Analyses. The analyses were performed on an UPLC system (Waters) coupled to a Xevo-TQS tandem mass spectrometer (Waters). The LC separation column used was a 100 mm × 2.1 mm i.d., 1.7 μm, BEH C18, with a 5 mm × 2.1 mm i.d. guard column of the same material (Waters) at 30 °C. The flow rate was 0.2 mL/min, and the injection volume was 5 μL. The initial composition of the mobile phase was 25% acetonitrile (A) and 75% aqueous ammonium carbonate (3.0 mmol/L) (B). Phase A was increased to 40% in 2.0 min, to 60% in 6.0 min, to 78% in 9.0 min, and then to 90% in 11 min. Finally, the mobile phase was returned to the initial composition in 1.0 min and equilibrated for 2 min before the next injection. For mass spectrometric acquisition, multiple-reaction monitoring (MRM) was conducted in positive ESI mode. The source temperature was set to 150 °C and the desolvation gas temperature to 500 °C. The desolvation gas flow was held at 700 L/h, and the collision pressure was 3.1 × 10−3 mbar. The capillary voltage and cone voltage were 2.5 kV and 30 V, respectively, and the other parameters are listed in Table 2. Method Validation. After separation and detection by UPLCMS/MS, the calibration curves were generated in MassLynx 4.1 software using the equation y = ax + b, where y is the analyte peak area abundance and x is the analyte concentration. The method was validated by determining the matrix effect (ME), limit of detection (LOD), limit of quantitation (LOQ), linearity of the calibration, recoveries, and repeatability (% RSD).
method for grain crops contaminated by ergots was based on the physical counting of the ergots in the grain sample.12 Agroclavine is a potent inhibitor of the early stage of pregnancy in the rat and mouse with potent toxicity.38 However, the studies to date on the analysis of ergot alkaloids mainly focused on the six major ergots and their epimers, and few studies concerned the determination of the other ergot alkaloids, which might result in an underestimation of the actual occurrence. Therefore, an objective systematic evaluation of the levels of ergot alkaloids in cereals with an established comprehensive analytical method is needed. The objective of this work was to develop a rapid and simple sample preparation and an accurate analytical method for the simultaneous determination of 25 ergot alkaloids in cereal samples.
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MATERIALS AND METHODS
Materials. The standards of 25 ergot alkaloids (ergometrinine, ergometrine hydrogenmaleate, ergosine, ergosinine, ergotamine tartrate, ergotaminine, ergocornine, ergocorninine, ergocristine, ergocristinine, α-ergocryptine, α-ergocryptinine, β-ergocryptine (purity > 73.5%), erginine (purity > 98%), agroclavine, chanoclavine-I, elymoclavine, festuclavine, lysergol, dihydroegocristine, dihydroergocryptine, dihydroergotamine, dihydroergocornine, dihydroergine, and dihydrolysergol) were purchased from Alfarma (Prague, Czech Republic). The chemical structures of these 25 compounds are seen in Figure 1 and Table 1. HPLC grade acetonitrile was obtained from J.
Table 1. Information of 25 Ergot Alkaloids no.
compound
formula
mass
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
agroclavine festuclavine elymoclavine chanoclavine erginine lysergol dihydrolysergol dihydroergine dihydroergocornine dihydroergocryptine dihydroergotamine dihydroergocristine ergometrinine ergometrine ergosinine ergosine ergocorninine ergocornine α-ergocryptine α-ergocryptinine β-ergocryptine ergotaminine ergotamine ergocristinine ergocristine
C16H18N2 C16H20N2 C16H18N2O C16H20N2O C16H17N3O C16H18N2O C16H20N2O C16H19N3O C31H41N5O5 C32H43N5O5 C33H37N5O5 C35H41N5O5 C19H23N3O2 C19H23N3O2 C30H37N5O5 C30H37N5O5 C31H39N5O5 C31H39N5O5 C32H41N5O5 C32H41N5O5 C32H41N5O5 C33H35N5O5 C33H35N5O5 C35H39N5O5 C35H39N5O5
238.15 240.16 254.14 256.15 267.14 254.14 256.16 269.15 563.31 577.33 583.28 611.31 325.18 325.18 547.28 547.28 561.30 561.30 575.31 575.31 575.31 581.26 581.26 609.30 609.30
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RESULTS AND DISCUSSION Optimization of the LC Conditions. The mobile phase composition for the chromatographic separation of the 25 ergot alkaloids was based on previous papers8,18,20 with minor modifications. Acetonitrile and ammonium carbonate constituted the mobile phase for the BEH C18 column. The flow rate and column temperature were evaluated to obtain good separation and high resolution for the 25 ergot alkaloids. The resolution was highest when the column was maintained at 30 °C, and the flow rate was 0.2 mL/min. All 25 ergot alkaloids were well separated except β-ergocryptine and ergocristine. Although these two compounds could not achieve baseline separation, their resolution was acceptable for LC-MS/MS quantitation. After the mobile phase optimization, a single run of 25 ergot alkaloids was able to finish in 9.5 min (Figure 2). Optimization of the Sample Preparation. Extraction Solvent. Due to the particularity of the target compounds, an aprotic solvent was used in the experiment to avoid transformations of the epimers. Different proportions of the acetonitrile and ammonium carbonate mobile phase were
T. Baker (Deventer, The Netherlands). Chloroform and formic acid (99% purity) were from Acros Organics (Morris Plains, NJ, USA). Ammonium carbonate is from Sigma-Aldrich (St. Louis, MO, USA), dissolved in water at 288 mg/L (3.0 mmol/L, pH 8.9 ± 0.3). Ultrapure water was obtained from a Milli-Q water purification system (Millipore, Milford, MA, USA). The C18 and primary−secondary amine (PSA) sorbent were from Supelco (Bellefonte, PA, USA), and the MycoSep 150 Ergot SPE cartridge was supplied by Romer Laboratories (Union, MO, USA). The Oasis MCX SPE cartridge was 7035
DOI: 10.1021/acs.jafc.6b02484 J. Agric. Food Chem. 2016, 64, 7033−7039
Article
Journal of Agricultural and Food Chemistry Table 2. Retention Times and MS Analysis Parameters for 25 Ergot Alkaloids no.
retention time (min)
MRM transitiona
collision energy (eV)
no.
retention time (min)
MRM transitiona
collision energy (eV)
1
5.53
239.1 → 208.1 239.1 → 183.1
17 17
14
2.44
326.2 → 223.1 326.2 → 208.1
23 28
2
5.91
241.1 → 168.1 241.1 → 154.0
28 32
15
7.39
548.2 → 268.1 548.2 > 223.1
23 31
3
2.81
255.1 → 224.0 255.1 → 196.1
15 19
16
5.16
548.2 → 268.1 548.2 → 223.1
23 31
4
2.59
257.1 → 226.1 257.1 → 168.1
10 21
17
8.30
562.3 → 268.1 562.3 → 223.1
25 37
5
3.26
268.1 → 223.1 268.1 → 208.1
20 24
18
6.13
562.3 → 268.1 562.3 → 223.1
25 37
6
2.92
255.1 → 240.1 255.1 → 197.1
20 22
19
6.64
576.3 → 268.1 576.3 → 223.1
25 38
7
2.85
257.2 → 208.1 257.2 → 182.1
23 27
20
8.86
576.3 → 268.1 576.3 → 223.1
25 38
8
2.28
270.1 → 210.1 270.1 → 168.1
22 13
21
6.80
576.3 → 268.1 576.3 → 223.1
25 38
9
5.76
564.3 → 270.1 564.3 → 168.10
30 53
22
7.84
582.2 → 223.1 582.2 → 208.1
34 44
10
6.28
578.30 → 270.1 578.30 → 253.1
31 31
23
5.46
582.2 → 223.1 582.2 → 208.1
34 44
11
5.45
584.4 → 270.4 584.4 → 253.1
29 33
24
9.10
610.2 → 268.1 610.2 → 223.1
26 35
12
6.48
612.3 → 350.2 612.3 → 270.1
25 32
25
6.83
610.2 → 268.1 610.2 → 223.1
26 35
a
13
3.50
326.2 → 223.1 326.2 → 208.1
Underlined ions are the quantitation ions. The compound number is same as that in Table 1.
23 28
used as the extraction solvent. This extraction solvent was a minor modification of that used by Kokkonen and Jestoi.20 Under this condition, the recoveries of the 25 ergot alkaloids were >85%. Selection of Sorbent for Sample Cleanup. To remove the fat, protein, and carbohydrates in the cereal matrix for purification, the sorbents PSA and C18 were optimized. C18 cleaned the cereal matrix slightly better than the PSA sorbent (Figure 3). Similar recovery results were obtained for the MycoSep 150 Ergot column, a one-step cleanup column for this group of mycotoxins, and the C18 sorbent (Figure 3). To shorten the cleanup process and reduce the cost of sample preparation, we used the C18 sorbent as an alternative. Optimization of the Purified Sorbent Dosage. The quantity of the C18 sorbent was investigated in this experiment. Above a concentration of 30 mg/mL of the C18 sorbent, the recovery was not significantly different for the 25 ergot alkaloids. Therefore, we selected 150 mg of C18 as the purified sorbent for 5 mL of extraction liquid.
Figure 2. Chromatographic separation of the 25 ergot alkaloids.
optimized. When the proportion of acetonitrile in the extraction solvent was low, the effectiveness of the extraction for nonpolar substances significantly decreased. To balance the extraction of polar and nonpolar compounds, acetonitrile and 3.0 mmol/L ammonium carbonate, equal to 85:15 (v/v), was 7036
DOI: 10.1021/acs.jafc.6b02484 J. Agric. Food Chem. 2016, 64, 7033−7039
Article
Journal of Agricultural and Food Chemistry
Figure 3. Recoveries of 25 ergot alkaloids in rye by prepurification with three different purification procedures.
Table 3. Matrix Effect (ME) of 25 Ergot Alkaloids in Rye with Different Sample Preparation Procedures
no.a
extracts of acetonitrile and ammonium carbonate (%)
PSA sorbent purified (%)
C18 sorbent purified (%)
MCX cartridge purified (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
88.0 112 110 122 104 101 100 109 107 106 109 114 106 99 108 108 104 98.0 101 68.0 105 101 108 113 106
99.0 117 112 133 106 96.0 103 114 102 112 111 118 115 107 113 107 114 98.0 93 77.0 94.0 112 107 122 110
78.0 115 105 122 103 98.0 97.0 104 99.0 112 115 117 112 97 113 103 108 97.0 90 87.0 81.0 104 108 118 107
95.0 118 101 108 107 92.0 95.0 100 138 143 127 148 89 94 169 128 136 118 128 59.0 132 161 118 183 146
a
Table 4. Occurrence of Ergot Alkaloids in the FAPAS Proficiency Test Sample
a
no.a
level (μg/kg)
FAPAS value
Z score
13 14 15 16 17 18 19 + 21 20 22 23 24 25
26.7 6.20 4.90 24.8 2.00 5.80 13.6 3.60 5.80 34.2 6.20 30.4
not stated 6.38 10.2 not stated 1.93 6.10 11.5 4.00 not stated 25.2 9.40 33.2
not applicable −0.15 −2.38 not applicable 0.22 −0.24 0.83 −0.45 not applicable 1.62 −1.53 −0.39
The compound number is same as that in Table 1.
blank rye flour was kindly provided by China Agriculture University; and the other blank samples were purchased from local markets and analyzed according to the method reported by Kokkonen and Jestoi20 as well as this newly developed method. Compared to the neat standard solution, slight ion enhancement or ion suppression was observed for the 25 target compounds in matrix-matched solution of rye flour (Table 3). Subsequently, several cereal-related samples, for example, wheat, bread, noodle, and pasta, were evaluated for the ME, which presented results similar to that of rye. To compensate for the ME, matrix-matched standard curves were used for the calibration. Linearity, LOQ and LOD. The calibration was evaluated by spiking the ergot alkaloid standards into cleaned up extracts of uninfected samples at seven concentrations. The linearity range was 0.05−5.0 μg/kg for the 25 ergot alkaloids. Good linearity of the compounds was obtained, and the correlation coefficients (r) were >0.99. The LOD and LOQ were determined as the analyte concentration corresponding to the mean of 3 and 10 standard deviations (SDs), respectively, of the response measured in an uninfected sample matrix. Recovery and Repeatability. The recoveries of the analytes were measured by spiking uninfected blank samples (wheat, rye, bread, noodle, and pasta) with the 25 ergot alkaloids at three different concentrations. Samples were extracted according to the method described above. After UPLC-MS/ MS analysis, the recoveries were calculated and varied from 76.5 to 120% with RSD < 15%. Application of the Method to the Analysis of Cereal Samples. To certify the accuracy of our method, we purchased
The compound number is the same as that in Table 1.
Although the extraction solvent was slightly different from that used previously,20 our work used little solvent compared with liquid−liquid extraction13 and was simple, time-saving, and inexpensive compared to SPE-PSA14 and SPE-C18,19 making it suitable for commercialization. LC-MS/MS Analyses. ME. ME is quantitated by the use of the following formula: ME % = 100 × slope in spiked extract/ slope in pure solvent. A value of 100% indicates that there is no ME. The signal is enhanced if the value is >100%, and the signal is suppressed if the value is +3 or < −3) is considered unsatisfactory. In our analysis, only the ergosinine result was questionable, and the others were satisfactory. Therefore, the developed analytical method was used to test 123 cereal samples purchased from Taobao (15) and local supermarkets (108), including rye flours (9), wheat flours (52), wheat flour noodles (43), and breads (19). Fortunately, no ergot alkaloid was detected in the flour samples and related food products purchased from supermarkets. Two rye and three whole wheat flour samples obtained from the internet were found to contain 13 -ine and -inine ergot alkaloids at a concentration range of 1.01−593 μg/ kg (Table 5). The analysis of each positive sample was conducted in six replicates, the resulting RSDs being
