High Throughput Determination of Multi-Class Toxic Alkaloids In

Poster Reprint

ASMS 2019 MP194

High Throughput Determination of Multiple Toxic Alkaloids in Food by High Performance Liquid Chromatography and Tandem Mass Spectrometry

Guoyin Lai1, Lijian Wu1, Zhongda Lin1, Liyi Lin1, Dunming Xu1, Zhigang Zhang1, Meiling Lu 2 1 Xiamen Customs Technique Center 2 Agilent Technologies (China) Co., Ltd. Introduction Experimental

Alkaloids are natural-occurring alkaline substances LC Conditions with nitrogen heterocyclic structure in variety of food matrices, which may lead to adverse effects when Agilent 1290 Infinity II UHPLC system: ingested by accident1. Hence, it is essential to develop 1290 Infinity II binary pump with degasser reliable analytical methods to detect toxic alkaloids in 1290 Infinity II autosampler with needle seat some alkaloids-relevant food matrices. Current backflush function available reports are mainly focused on the detection 1290 Infinity II multiple column compartment of alkaloids with similar chemical properties. Here, we Column：Agilent ZORBAX Eclipse Plus C18 (3.0×50 report a simple sample preparation procedure mm, 1.8 µm); followed by rapid LC-MS/MS analysis for Mobile phases: A. 0.1% formic acid in water simultaneous determination of 5 classes of alkaloids in order to screen for alkaloids in food in a high- B. Methanol throughput fashion. Flow rate：0.50 mL/min Column temperature：40 ℃ Injection volume：2.0 µL Method

Gradient Condition: Sample Preparation Procedure Time /min A (%) B (%)

0 90 10

1 80 20

7 20 80

Add samples (2.0 g) in 50 mL of 9 90 10 centrifugation tube 10 90 10 Add 5 mL of water to the tube ( ignore for liquid samples) MS Conditions Add 10 mL of acetonitrile to the tube MS: Agilent 6470 QQQ Ion source: Agilent JetStream ESI Add 5 mL of Na2EDTA-Mcllvaine buffer Ionization mode: Positive Vortex the tube thoroughly for 2 min Capillary voltage 3500 V Nozzle voltage: 500 V Centrifugation @7500 rpm for 5 min Nebulizer gas press.: N2 (35 psi)

Drying gas (N2) temp.: 300 ºC Collect the supernatant and filter through 2.0 m membrane Drying gas flow rate: 8 L/min

Sheath gas (N2) temp.: 400 ºC LC/MS analysis Sheath gas flow rate:12 L/min Acquisition mode: MRM Results and Discussion

Table 1: Optimized MRM Parameters for Sensitive Detection Retention Precursor Ion Product Ion Fragmentor Vol. Collision Energy Analyte Time (min) （m/z）（m/z）（V） (V） Physostigmine 2.250 275.8 219.2, 162.2* 110 7, 22* Scopolamine 1.898 303.8 137.9, 156.1* 120 23, 15* Colchicine 4.771 399.9 358.1, 309.9* 135 22, 32* Strychnine 2.368 334.9 156.1, 184.1* 135 55, 44* Cortisone 5.307 360.9 162.9, 121.2* 135 25, 36* Pseudoephedrine 1.719 166 148.1, 133.0* 80 10, 20* Camptothecin 5.262 348.8 305.0, 249.0* 135 25, 33* 1-(1-naphthyl)thiourea 3.912 202.8 185.8, 144.1* 135 20, 20* Atropine 2.554 290 124.1, 93.0* 135 26, 35* Mesasonitine 4.906 632.1 571.8, 105.0* 135 35, 60* Hypaaconitine 5.059 616.2 556.6, 105.3* 135 50, 50* 10,11-Dimethoxystrychnine 2.594 394.9 243.6, 323.9* 135 46, 46* Nuciferin 4.122 295.8 265.1, 249.9* 135 15, 17* Podophyllotoxin 5.532 414.8 246.6, 397.1* 135 10, 5* 10-Hydroxycamptothecin 4.773 364.8 320.5, 235.7* 135 30, 33* Aconitine 5.119 646.2 586.6, 105.3* 135 45, 50* Gelsemine 2.171 322.8 70.1, 235.8* 135 32, 37* Solanine 4.965 868.5 98.3, 398.1* 135 92, 92* Alkaloids can be easily protonated under acidic water without acid was used. Acidified water/methanol conditions. Therefore, MRM under positive ESI ionization was thus used as the binary mobile phases in the final mode was selected. Different mobile phases conditions. MRM parameters were then optimized under combinations were examined, including water/methanol, the selected mobile phases, and the resultant water/acetonitrile, 0.1% formic acid/methanol, and 0.1% parameters were listed in Table 1. For those compounds formic acid/acetonitrile. It was observed that most not separated by chromatography, the characteristic alkaloids exhibited better response in methanol than in MRMs allow to quantitate the compounds accurately. acetonitrile. Compared to the narrower and more The typical MRMs overlays of 18 alkaloids from different symmetric peaks with higher intensity in the acidic classes are shown in Figure 1.

water, tailing peak with low intensity was observed if (cps)

counts Figure 1. MRMs overlay of 18 alkaloids from different classes

at a level of 50 µg/L for each Response

Retention Time (min)

3 Results and Discussion

Sample Preparation Optimization and Method Performance

)

(

Recovery Recovery

Figure 3. Matrix effect of alkaloids detection. Figure 2. Effect of extraction solvent vol. on the recovery. Note: relative slope was used to evaluate the matrix effects. Both methanol and acetonitrile were investigated as continued increase. Considering the sensitivity in the real extraction solvent. It was found that acetonitrile can provide samples with extraction induced dilution, 10 mL of better recovery. Furthermore, the volume for extraction extraction solvent was selected for target compounds solvent was examined. As shown in Figure 2, with the extraction. extraction volume increasing from 5 mL to 10 mL, the recoveries for all alkaloids increased with majority values Matrix effect was evaluated using the linear response higher than 60%. When the volume increased from 10 to 15 slopes obtained in the matrix and that in the pure solvent. mL, recoveries for two compounds decreased though others It was observed that cortisone and podophyllotoxin were affected by matrices more severely, particularly in bread

Bread L3 (Figure 3). Therefore, matrix-matched calibration curve Solanine was applied for quantitation. The result demonstrated Gelsemine Bread L2 that the alkaloids compounds exhibited excellent linearity within 0.5-50 µg/L, with regression coefficients higher Aconitine Bread L1 than 0.998. The S/Ns are all higher than 10 for 18 10-Hydroxycamptothecin Rice analytes at the lowest calibration level of 0.5 µg/L, Powder L3 corresponding to 5.0 µg/kg in the samples when Podophyllotoxin Rice considering the 10-folder dilution factor of the method. Powder L2 Nuciferin Rice The method recovery and precision was evaluated by Powder L1 analyzing the spiking samples at three levels (5, 50, 250 10,11-Dimethoxystrychnine Milk L3 µg/kg) in 4 matrices with 6 replicates each matrix, the Hypaaconitine corresponding concentration for injection solution were Milk L2 0.5, 5.0, and 25 µg/L. As shown in Figure 4, the recoveries Mesasonitine Milk L1 at each level in all matrices range from 82%-119%, with Atropine %RSD within 2.3%-7.9%, suggesting that this method is Wine L3 reliable. 1-(1-naphthyl)thiourea Wine L2 Camptothecin Wine L1 Conclusions Pseudoephedrine • An reliable UHPLC-MS/MS method for simultaneous Cortisone determination of 18 alkaloids in wine, milk, bread, and rice powder is demonstrated. Strychnine • The method has excellent linearity, high sensitivity, Colchicine good accuracy and precision. Scopolamine • The method is easy of operation, can be applied in the Physostigmine routine monitoring of alkaloids in food reliably. 20 40 60 80 100 120 References Figure 4. Recoveries and precisions for the target alkaloids spiking in four matrices at three levels (n=6) 1. Meng P. J. Chem. Bull. 2010, 6, 484-489.