> Boosting the performance of LC/MS/MS analysis of mycotoxins and their transformation products by use of innovative separation techniques Florian übner
> overview analytes of interest separation techniques first example T-2 and T-2 in human cells second example multi mycotoxin analysis third example determination of TA and TA metabolites summary 2
> analytes of interest mycotoxins deoxynivalenol (DN) 3-acetyldeoxynivalenol (3-Ac-DN) zearalenone (ZN) N 2 N 2 fumonisin B 1 (FB 1 ) fumonisin B 2 (FB 2 ) T-2 toxin (T-2) T-2 toxin (T-2) ochratoxin A (TA) N Cl 3
> analytes of interest discussed metabolites of TA 22 22 14 N (S) 16 14 N (S) 16 12 7 12 7 Cl 9 1 (R) 3 10 C 3 11 4R/4S - hydroxy - ochratoxin A 9 1 (R) 3 10 C 3 11 ochratoxin A - hydrochinone 22 14 N (S) 16 12 7 Cl 9 1 (R) 3 10 C 3 11 ochratoxin A 22 22 14 N (S) 16 14 N (S) 16 12 7 12 7 Cl 9 1 10 (R) 10 (R) ochratoxin B 9 1 C 3 11 open-lactone-ochratoxin A C 3 11 12 7 Cl 9 1 (R) 3 10 C 3 11 ochratoxin α 12 7 Cl 9 1 (R) 3 10 C 3 11 ochratoxin α glucuronide 22 16 (S) 12 14 N 7 Cl 9 1 10 (R) 3 C 3 11 ochratoxin A glucuronide 22 14 N (S) 16 12 7 Cl 9 1 (S) 3 11 10 C 2 11-hydroxy-ochratoxin A 4
> separation techniques ion mobility (AB SCIEX S SelexIN device) analyte and matrix ESI towards MS separation voltage (SV) compensation voltage (CV) 5
> separation techniques MRM³ Q1 collision cell Q3 ion-trap linear ion-trap product ion spectrum of m + + + + m 1 m 2 m 3 m 2a + m 2b + m 2c + 6
> separation techniques ekspert microlc 200 system micro-lc: flow-rate: 15-50 µl/min injection volume: 0.5-5 µl pressure: up to 600 bar column i.d.: 0.5-1.0 mm improved performance: - better ionization during ESI - more focused peaks - faster separation 7
> 1 st example T-2 and T-2 on LC-MS 4000 QTRAP (AB SCIEX) 75 70 60 50 40 30 20 10 0 430 400 300 200 100 1 ng/ml area: 152 T-2 442 215 4.32 5.16 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 5.21 10 ng/ml area: 2084 4.28 T-2 484 215 0.5 ng/ml area: 836 5 ng/ml area: 4707 quantitation of T-2 and T-2 in cell culture studies column: Synergi Fusion RP18, 150 mm x 2 mm i.d.; 4 µm mobile phase: A: 2 + 5 mm N 4 Ac B: ACN + 5 mm N 4 Ac fragments of [M+N 4 ] + 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Time, 8.0 min 9.0 10.0 11.0 12.0 13.0 14.0 8
> 1 st example T-2 and T-2 on LC-MS QTRAP 5500 (AB SCIEX) 1184 7.57 5.64 1000 750 500 250 0 12000 10000 7500 5000 2500 0 1 ng/ml area: 1443 4.88 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 10 ng/ml area: 19780 4.74 5.38 0.5 ng/ml area: 10560 5 ng/ml area: 106800 7.37 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 quantitation of T-2 and T-2 in cell culture studies column: Synergi Fusion RP18, 150 mm x 2 mm i.d.; 4 µm mobile phase: A: 2 + 5 mm N 4 Ac B: ACN + 5 mm N 4 Ac fragments of [M+N 4 ] + 9
> 1 st example T-2 and T-2 on microlc-ms QTRAP 5500 (AB SCIEX) 250 0 10000 1184 1000 750 500 7500 5000 2500 T-2 447 345 1 ng/ml area: 1835 1.77 1.75 2.00 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 10 ng/ml area: 17290 1.99 T-2 489 387 0.5 ng/ml area: 3226 5 ng/ml area: 24530 quantitation of T-2 and T-2 in cell culture studies column: AL RP18, 100 mm x 0.5 mm i.d.; 2.7 µm mobile phase: A: 2 + 0.1 % FA B: ACN + 0.1 % FA fragments of [M+Na] + 0 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 10
> 1 st example summary 5500 QTRAP about 10 times more sensitive than QTRAP 4000 LC-MS system T-2 1 ng/ml T-2 10 ng/ml T-2 0.5 ng/ml T-2 5 ng/ml 4000 QTRAP QTRAP 5500 QTRAP 5500 + micro LC 152 2084 836 4707 1443 19780 10560 106800 1835 17290 3226 24530 micro-lc system allows for 3 times faster runs while maintaining similar signal to noise levels 11
> 2 nd example basic goal for many mycotoxins maximum levels are decreed by European Law, e.g.: deoxynivalenol: 200 1750 µg/kg fumonisins (sum of FB 1 and FB 2 ): 200 4000 µg/kg ochratoxin A: 0.5 15 (80) µg/kg zearalenone: 20 400 µg/kg T-2 & T-2 toxin:? dilute and shoot -method preferred e.g. 10 g of sample + 20 ml of solvent (70% organic) supernatant diluted 1:5 (15% organic), PLC starting conditions filtrated if necessary and directly injected therefore the method should be suited for minimum levels of: 10 ng/ml for DN, 3-Ac-DN, FB 1 and FB 2 1 ng/ml for ZN 0.01 ng/ml for TA 12
> 2 nd example separation of eight mycotoxins on QTRAP 5500 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 2.01 DN 3.04 3-Ac-DN 4.18 FB 1 basic method mycotoxin standards: DN & 3-Ac-DN: 25 ng/ml FB 1, FB 2,T-2 & T-2: 5 ng/ml ZN: 2 ng/ml TA: 0.1 ng/ml 1.0e4 4.77 FB 2 0.0 6864 6000 5000 4000 3000 2000 1000 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 4.53 T-2 4.94 T-2 ZN measured in negative ionization mode 5.52 TA 5.59 ZN 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 column: Synergi Fusion RP18, 150 mm x 2 mm i.d.; 4 µm mobile phase: A: 2 + 0.1 % FA B: Me + 0.1 % FA 13
> 2 nd example separation of eight mycotoxins on QTRAP 5500 9.8e4 1.26 3-Ac-DN 8.0e4 6.0e4 4.0e4 1.44 FB 1 1.58 FB 2 method directly transferred to a micro-lc system mycotoxin standards: DN & 3-Ac-DN: 25 ng/ml FB 1, FB 2,T-2 & T-2: 5 ng/ml ZN: 2 ng/ml TA: 0.1 ng/ml 2.0e4 0.0 1.8e4 1.5e4 1.0e4 5000 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 0.64 DN 1.64 T-2 1.89 T-2 1.97 TA 1.99 ZN column: AL RP18, 100 mm x 0.5 mm i.d.; 2.7 µm mobile phase: A: 2 + 0.1 % FA B: ACN + 0.1 % FA 0.0 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 14
> 2 nd example separation of eight mycotoxins on QTRAP 5500 1.6e5 1.2e5 8.0e4 4.0e4 1.40 3-Ac-DN 1.68 FB 2 1.55 FB 1 optimized micro-lc method mycotoxin standards: DN & 3-Ac-DN: 10 ng/ml FB 1 & FB 2 : 5 ng/ml T-2, T-2 & ZN: 1 ng/ml TA: 0.01 ng/ml 0.0 6500 5000 4000 3000 2000 1000 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 1.08 DN 1.77 T-2 2.01 T-2 2.08 TA 2.09 ZN column: AL RP18, 100 mm x 0.5 mm i.d.; 2.7 µm mobile phase: A: 2 + 0.1 % FA B: ACN + 0.1 % FA 0 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 15
> 2 nd example summary very sensitive equipment allows for dilute and shoot methods even at very low concentrations needed for the determination of TA most important mycotoxin contaminants in grains and grain products can be analyzed in a 4 min PLC run 16
> 3 rd example TA exposition adults Mean consumption analyzed TA levels (g/person/day) (µg/kg) nonalcoholic beer 21.3 <LD 0.08 beer 192.3 <LD 0.29 wine and grape juice 29.51 <LD 7.00 coffee 18.03 <LD 6.32 bread 146.99 <LD 5.54 pasta 14.02 <LD 29.77 chocolates and sweets 51.82 <LD 3.60 breakfast cereals 67.55 <LD 31.80 sausages 46.86 <LD 4.56 Source: Directorate-general ealth and Consumer protection, European Commission, SCP, Task 3.2.7 Assessment of the dietary intake of ochratoxin A by the population of the EU member states, 2002 17
> 3 rd example analysis of urine samples on QTRAP 5500 intensity, [cps] 9.0e4 7.5e4 5.0e4 2.5e4 TA 1 ng/ml in urine (spiked sample) m/z 404,1 239 17.67 - N Cl C 3 5000.0 0.00 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 9500 5000 15.00 open lactone TA-glucuronide 2 ng/ml in urine (spiked sample) m/z 596.0 420.0 - N Cl C 3 1000 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 18
> 3 rd example tuning of DMS (SelexIN) 9000 Tα TB parameters: - separation voltage - modifier - gas flow - temperature 6000 3000 P-Tαglucuronide P-TAglucuronide TA 4-TA P-TA 500-4.5-3.5-2.5-1.5-0.5 0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 compensation voltage CV [V] separation voltage: 2700 V; DMS temperature: 300 C (high); modifier: methanol; modifier composition: 132 µl/min (low); offset: 4.4 19
> 3 rd example reduction of background signals using DMS Intensity, cps 9.0e4 7.5e4 5.0e4 2.5e4 TA 1 ng/ml in urine (spiked sample) m/z 404,1 239 17.67 Max. 9.0e4 cps. 5000.0 0.00 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time, min Intensity, cps 1.8e4 1.5e4 1.0e4 using DMS signal intensity lowered by a factor of 5 background signal lowered by a factor of 7.5 17.64 Max. 1.8e4 cps. 5000.0 1000.0 0.00 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time, min 20
> 3 rd example fragmentation of open lactone TA glucuronide - cleavage of glucuronic acid in the collision cell N Cl fragmentation in the linear ion-trap (Q3) C 3 21
> 3 rd example improving selectivity by using MRM³ 9500 5000 open lactone TA glucuronide 2 ng/ml in urine (spiked sample) m/z 596.0 420.0 P-TA-glucuronide MRM 1000 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 3.1e6 2.0e6 1.0e6 open lactone TA glucuronide 2 ng/ml in Urin (dotiert) m/z 596.0 420.0 332.0 P-TA-glucuronide MRM 3 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 22
> summary while dilute and shoot methods are often convenient, there is a lack of selectivity caused by co-elution of matrix compensation of lacking selectivity can be achieved by innovative separation techniques for example MRM³ and ion mobility can be used for a more selective isolation of analytes use of a micro-lc system is not only faster and due to much less consumption of solvents more friendly to the environment, but can also boost the performance of methods by more focused peaks thus improving signal to noise ratios 23
> acknowledgments Prof. Dr. ans-ulrich umpf and Dr. Benedikt Cramer Maria Weidner and Katharina von Bargen (PhD students) Julian Dopstadt and Irina Schimanowski (MSc) and AB SCIEX for support 24