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High Throughput Determination of Multiple Toxic Alkaloids in Food By Application Note Food Testing & Agriculture High-Throughput Determination of Multiple Toxic Alkaloids in Food by UHPLC/MS/MS Authors Abstract Guoyin Lai, Lijian Wu, Zhongda Lin, Liyi Lin, Dunming Xu, This Application Note presents a simple sample preparation procedure followed Zhigang Zhang with an Agilent 1290 Infinity II LC combined with an Agilent 6470A triple quadrupole Technigue Center, LC/MS for simultaneous, high-throughput determination of multiple alkaloids in food Xiamen Customs, matrices. The matrix-matched calibration shows very good linear relationships for Xiamen, China the 18 alkaloids in the concentration range of 0.5 to 50 µg/L in the diluted, extracted matrices of bread, milk, wine, and rice powder. This corresponds to 5.0 to 500 µg/kg Meiling Lu in the matrices when considering the dilution factor of 10. The linear regression Agilent Technologies (China) coefficients for all the analytes are higher than 0.99. The signal-to-noise ratios Co., Ltd. (S/Ns) for all analytes are higher than 10 at the lowest calibration level of 0.5 µg/L, corresponding to 5.0 µg/kg in the samples. The recoveries for the analytes in four different matrices at 5, 50, and 250 µg/kg levels ranged from 90 to 110%, and the corresponding relative standard deviations were within 2.3 to 7.9%. The results demonstrated that the developed method has the advantages of high sensitivity, accuracy, and precision. The simple sample preparation combined with rapid LC/MS/MS detection allows high-throughput screening of alkaloid residues in food matrices. Introduction Experimental (pH 3). The tube was vortexed thoroughly for two minutes, then centrifuged Alkaloids are naturally occurring alkaline Standards and sample at 7,500 rpm for five minutes. The substances with nitrogen-containing preparation procedure supernatant was collected and filtered heterocyclic structures. They often through a 2.0 µm membrane and All five classes 18 alkaloid compounds exhibit high bio-activity, and are analyzed by LC/MS. Liquid samples such were purchased from Anpu Tech. commonly the primary components as milk and wine followed the same (Shanghai, China), which were prepared in traditional herb medicine. Alkaloids extraction procedure except without the at 1.0 g/L in methanol for stock at are found in some plants, and initial hydration step. –20 °C. Calibration standard solutions may cause adverse effects when were then diluted from the stock ingested by accident.1 According LC system solutions using the corresponding to Commission Regulation (EU) Agilent 1290 Infinity II LC with the matrix extract. 2016/239, the maximum residue levels following module configuration: (MRLs) of tropane alkaloids including Solid samples (2.0 g) were added • Agilent 1290 Infinity II binary pump atropine and scopolamine in certain to 50 mL centrifugation tubes, and with degasser cereal-based foods for infants and 5 mL of water was transferred to the • Agilent 1290 Infinity II autosampler young children are as low as 1 µg/kg.2 tube for hydration. Acetonitrile (10 mL) with needle seat backflush function Colchicine is in the prohibited list in was then added to the tube, followed 3 • Agilent 1290 Infinity II multiple Regulation (EU) No. 37/2010. China by 5 mL of Na2EDTA-Mcllvaine buffer allows atropine to be used in all food column compartments matrices of animal origin without MRL, LC conditions and no other regulations restrict the amount of alkaloids in food matrices Parameter Value in China.4 However, the accidental Column Agilent ZORBAX Eclipse Plus C18 (3.0 × 50 mm, 1.8 µm) mixing of alkaloid-containing weeds Mobile Phases A: 0.1% formic acid in water; B: methanol into crops or the accidental ingestion Flow Rate 0.50 mL/min of alkaloid-containing weeds by farm Column Temperature 40 °C animals may increase the risk of Injection Volume 2.0 µL exposure to the public. It is essential to Time (min) %A %B 0 90 10 develop sensitive and reliable methods 1 80 20 Gradient 7 20 80 for routine monitoring of alkaloids in food 9 90 10 matrices. In reality, alkaloids vary greatly 10 90 10 in physicochemical properties, and they are also involved in many complicated MS conditions matrices. It is challenging to develop a Parameter Value reliable method for routine, simultaneous MS Agilent 6470A triple quadrupole LC/MS monitoring of multiclass alkaloids. Ion Source Agilent Jet Stream ESI Available reports are mainly focused on Ionization Mode Positive the detection of alkaloids with similar Capillary Voltage 3,500 V 5,6 chemical properties. This Application Nozzle Voltage 500 V Note presents a simple sample Nebulizer Gas Pressure N2 (35 psi) preparation procedure followed by rapid Drying Gas (N ) Temperature 300 °C LC/MS/MS analysis for simultaneous, 2 Drying Gas Flow Rate 8 L/min high-throughput determination of Sheath Gas (N ) Temperature 400 °C five classes of alkaloids in four types 2 Sheath Gas Flow Rate 12 L/min of food matrices, including milk, wine, Acquisition Mode MRM bread, and rice powder. 2 Table 1. Optimized MRM parameters for sensitive detection. Results and discussion Retention Precursor Product Fragmentor Collision Alkaloids can easily be protonated Analyte Time (min) Ion (m/z) Ion (m/z) Voltage (V) Energy (V) under acidic conditions. Therefore, Physostigmine 2.250 275.8 219.2, 162.2* 110 7, 22* MRM in positive ESI ionization mode Scopolamine 1.898 303.8 137.9, 156.1* 120 23, 15* was selected. Different mobile phase Colchicine 4.771 399.9 358.1, 309.9* 135 22, 32* combinations were examined, including Strychnine 2.368 334.9 156.1, 184.1* 135 55, 44* water/methanol, water/acetonitrile, Cortisone 5.307 360.9 162.9, 121.2* 135 25, 36* 0.1% formic acid/methanol, and Pseudoephedrine 1.719 166 148.1, 133.0* 80 10, 20* 0.1% formic acid/acetonitrile. Most Camptothecin 5.262 348.8 305.0, 249.0* 135 25, 33* alkaloids exhibited a better response in 1-(1-Naphthyl)thiourea 3.912 202.8 185.8, 144.1* 135 20, 20* methanol than in acetonitrile. Compared Atropine 2.554 290 124.1, 93.0* 135 26, 35* to the narrower and more symmetrical Mesasonitine 4.906 632.1 571.8, 105.0* 135 35, 60* peaks with higher intensity in the acidic Hypaaconitine 5.059 616.2 556.6, 105.3* 135 50, 50* water, tailing peaks with low intensity 10,11-Dimethoxystrychnine 2.594 394.9 243.6, 323.9* 135 46, 46* were observed if water without acid was Nuciferin 4.122 295.8 265.1, 249.9* 135 15, 17* used. Hence, acidified water/methanol Podophyllotoxin 5.532 414.8 246.6, 397.1* 135 10, 5* was selected as the binary mobile 10-Hydroxycamptothecin 4.773 364.8 320.5, 235.7* 135 30, 33* phase in the final conditions. MRM Aconitine 5.119 646.2 586.6, 105.3* 135 45, 50* parameters were then optimized under Gelsemine 2.171 322.8 70.1, 235.8* 135 32, 37* the selected mobile phase. Table 1 lists Solanine 4.965 868.5 98.3, 398.1* 135 92, 92* the resultant parameters. * Quantitative ion For those compounds not separated by chromatography, the characteristic ×105 MRMs enabled accurate compound 8 quantitation. Figure 1 shows the typical MRM overlays of 18 alkaloids from ) 6 different classes. 4 2 Response counts (cps 0 1 2 3 4 5 6 Retention time (min) Figure 1. MRM overlay of 18 alkaloids from different classes at a level of 50 µg/L for each. 3 5 mL 10 mL 15 mL Sample preparation optimization and 140 method performance 120 ) Methanol and acetonitrile were 100 investigated as extraction solvents. It 80 60 was found that acetonitrile can provide Recovery (% 40 better recovery. The volume of extraction 20 solvent required was also examined. As 0 shown in Figure 2, with the extraction e e n e ison Atropine Nuciferin Aconitine Solanine volume increasing from 5 to 10 mL, the ColchicineStrychnineCort Gelsemin Scopolamine Mesasonitine Physostigmine Samptothecin Hypaaconitin recoveries for all alkaloids increased, Podophyllotoxi Pseudoephedrine with the majority of values higher Dimethoxystrychnine 1-(1-Naphthyl)thiourea Hydroxycamptothecin than 60%. When the volume increased from 10 to 15 mL, recoveries for two Figure 2. Effect of extraction solvent volume on recovery. compounds decreased, though others continued to increase. Considering pH 3 pH 4 pH 5 140 the sensitivity in the real samples with 120 ) extraction-induced dilution, 10 mL of 100 extraction solvent was selected for target 80 60 compounds extraction. Recovery (% 40 Alkaloids are easily dissolved in water 20 under acidic conditions, and present as 0 cations. The Na EDTA-Mcllvaine buffer e e n e 2 ison was added to the extraction solvent to Atropine Nuciferin Aconitine Solanine ColchicineStrychnineCort Gelsemin Scopolamine Mesasonitine Physostigmine Samptothecin Hypaaconitin achieve acidic pH. As shown in Figure 3, Podophyllotoxi Pseudoephedrine higher recovery values can be achieved 1-(1-Naphthyl)thiourea Hydroxycamptothecin for most compounds at pH 3 and 4 than 10,11-Dimethoxystrychnine at pH 5. Therefore, pH 3 was selected for Figure 3. The effect of pH for the extraction solvent on the recovery of each analyte. the buffer during extraction. Matrix effect was evaluated using the t Wine Rice powder Milk Bread linear response slopes obtained in the solven 140 e matrix and in the pure solvent. Matrices 120 100 affected cortisone and podophyllotoxin to slop 80 more severely, particularly bread matrix e 60 (Figure 4). 40 20 0 e e n e Ratio of slop ison Atropine Nuciferin Aconitine Solanine ColchicineStrychnineCort Gelsemin Scopolamine Mesasonitine Physostigmine Samptothecin Hypaaconitin Podophyllotoxi Pseudoephedrine Dimethoxystrychnine 1-(1-Naphthyl)thiourea Hydroxycamptothecin Figure 4. Matrix effect of alkaloid detection.
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