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Sensitive and Fast Measurement of in Milk, Meat or Eggs by HILIC-MS/MS and Identi cation using MRM Spectrum Mode

ASMS 2017 TP-184

Mikaël LEVI1, Hisashi KATO2, David BAKER3, Ichiro HIRANO1 1 SHIMADZU Corporation, MS Business Unit, Kyoto, Japan; 2 BIOTAGE Japan, Tokyo, Japan; 3 SHIMADZU MS Business Unit Overseas, Manchester, UK. Sensitive and Fast Measurement of Aminoglycoside Antibiotics in Milk, Meat or Eggs by HILIC-MS/MS and Identi cation using MRM Spectrum Mode

Introduction Aminoglycoside are an family widely used for the commodities (e.g. in eggs) or have low MRL. treatment of bacterial in , sheep, pigs and So in the case of a positive sample, a strict identi cation of poultry. Due to their high af nity for tissues, the the compound is necessary to con dently report potential consumption of meat, milk or eggs containing fraud. (AGs) can be potentially hazardous for AGs are very polar compounds poorly retained by human health. Regulatory agencies have set maximum reversed-phase liquid chromatography and ion-pairing residue limits (MRL) for these compounds with veterinary reagents are not desirable when users share several use. Depending on the countries, the animal species, the methods on a single system. commodity or the AG, these MRL are different. For food Here we present a method using hydrophilic interaction safety laboratories testing large numbers of samples, a liquid chromatography (HILiC) with high sensitivity mass method capable to cover as many compounds, matrices spectrometer to reach limits of quanti cation at and regulated range as possible would be of great help. femtogram level on column, combined with MRM In addition, some AGs are strictly banned for some Spectrum Mode for formal identi cation.

Methods and Materials Sample Preparation Frozen meat samples were homogenized using a knife Further puri cation was then perfomed by Solid Phase mill (Grindomix GM200, Retsch). 5 g of homogenized Extraction (SPE) using mixed-mode sorbent (WCX Express sample or mixed eggs or 10 mL of milk were placed in a 96-well plate 30 mg, Biotage) and Extrahera automate polypropylene tube. After addition of 20 mL of extraction (Biotage). 1 mL of extract was loaded without prior solution (10 mM NH4OAc, 0.4mM EDTA, 0.5% NaCl, 2% conditioning. Then the sample was washed with 1 mL of trichloroacetic acid in water), sample were vortex mixed ammonium acetate buffer 50mM pH7. After sorbent and shaked for 10 min. After centrifugation for 10 min at drying, target compounds were eluted with 250 µL of 4000 rpm, the supernatant was transferred to a clean PP aqueous formic acid 10% (v/v). Puri ed extracts were tube. Extraction was repeated and supernatant combined. then diluted 25 times with aqueous formic acid 1% (v/v) Extract pH was then adjusted to pH 6.5 +/- 0.25. prior to transfer into a polypropylene vial and analysis.

Analytical Conditions Two methods were used. First method for fast column but with a longer gradient and 15 MRM per quantitative screening is using a fast gradient with two compound. Parameters are described parameters in Table MRM by compound. The second method for positive 1 and 2. sample con rmation, used same mobile phases and

2 Sensitive and Fast Measurement of Aminoglycoside Antibiotics in Milk, Meat or Eggs by HILIC-MS/MS and Identi cation using MRM Spectrum Mode

Table 1 HILiC conditions

System : Nexera X2 Column : GL Sciences Inertsil Amide 3µm 100x2.1mm Temperature : 50°C Mobile Phases : A: Water + 250 mM ammonium formate + 1% formic acid B: Acetonitrile Flow Rate : 800 µL/min Injection Volume : 5 µL (Quant screening) Gradient : 80 % B (0.1min) to 30%B in 1 min. 30%B (0.5 min). 30%B to 80%B in 0.1 min Total Run Time : 3.5 min (ID Con rmation) Gradient : 70 % B (0.1min) to 60%B in 6 min. 60%B to 50 (3min). 50%B to 70%B in 0.1 min Total Run Time : 11 min

Table 2 MS/MS conditions for fast quantitative screening

System : LCMS-8060 Ionization : Heated ESI Probe Voltage : +1.5 kV (positive ionization) Temperature : Interface: 400°C Desolvation Line: 150°C Heater Block: 300°C Gas Flow : Nebulizing Gas: 3 L/min Heating Gas: 20 L/Min Drying Gas: 3 L/min Dwell Time / Pause time : 6 ms / 1.5 ms MRM : Compound MRM Quant MRM Qual Spectinomycin 351.1 > 207.0 351.1 > 98.2 540.3 > 217.1 540.3 > 378.3 584.3 > 263.2 584.3 > 246.0 C1a 450.2 > 322.2 450.2 > 163.0 Gentamicin C1 478.3 > 322.3 478.3 > 157.1 Gentamicin C2 464.3 > 322.1 464.3 > 160.0 528.3 > 177.1 528.3 > 352.1 Kanamycin 485.3 > 163.0 485.3 > 324.2 B 615.3 > 161.1 615.3 > 163.1 582.3 > 263.2 582.3 > 246.12 586.3 > 425.2 586.3 > 163.3 476.3 > 299.2 476.3 > 191.2 616.3 > 163.1 616.3 > 293.2 Sisomycin 448.3 > 254.1 448.3 > 271.2 468.3 > 324.0 468.3 > 163.0

3 Sensitive and Fast Measurement of Aminoglycoside Antibiotics in Milk, Meat or Eggs by HILIC-MS/MS and Identi cation using MRM Spectrum Mode

Results Calibration As the method should t any type of sample, calibration de ne the highest calibration level. When no MRL was standards were prepared in aqueous 1% formic acid. The de ned, the Japan rule using 10 ng/g as default MRL was calibration range was set up by combining all MRL from employed for the highest level. Finally, taking into Europe, Japan and USA for all target compounds and all account the sample preparation protocol, volumetric commodities. For each compound, the lowest MRL concentration were established. The table 3 summarize divided by 10, or the practical achievable concentration the calibration ranges used. Some typical chromatograms with S/N > 10 was considered as the target limit of at LOQ are provided in gure 1. quanti cation. The highest MRL +50% was used to

Table 3 Calibration ranges

Calibration range (pg/mL) Compound lowest (Low MRL/10) highest (high MRL + 50%) Amikacin 20 150 Apramycin 30 30000 Dihydrostreptomycin 5 30000 Gentamicin(*) 80 75000 Hygromycin 10 150 Kanamycin 20 37500 Neomycin (+) 100 150000 Netilmicin 10 150 Paromomycin 40 22500 Sisomycin 10 150 Spectinomycin 10 75000 Streptomycin 5 30000 Tobramycin 10 150

(*) sum of C1, C1a and C2 congeners (+) using Neomycin B as marker

4 Sensitive and Fast Measurement of Aminoglycoside Antibiotics in Milk, Meat or Eggs by HILIC-MS/MS and Identi cation using MRM Spectrum Mode

SPC (10) APR (30) DTP (5) NEO (100) STP (5) SIS (10) TOB (10)

Q 351.10>207.00 6.89e3Q 540.30>217.10 2.08e3Q 584.30>263.15 1.75e4Q 615.30>161.10 1.05e4Q 582.30>263.15 2.02e4Q 448.30>254.05 2.41e3Q 468.30>324.00 1.93e3

2.0e4 R1 134.55% 2.0e3 R1 60.72% R1 31.01% 1.0e4 R1 69.67% R1 33.83% R1 66.94% R1 107.39% 2.3e3 2.0e3 9.0e3 1.6e4 1.8e4 1.8e3 9.0e3 1.8e3 8.0e3 2.0e3 1.4e4 1.6e4 1.6e3 8.0e3 1.6e3 7.0e3 1.8e3 1.2e4 1.4e4 1.4e3 7.0e3 1.4e3 1.5e3 6.0e3 1.2e4 1.2e3 1.0e4 6.0e3 1.2e3 5.0e3 1.3e3 1.0e3 5.0e3 1.0e4 8.0e3 1.0e3 1.0e3 4.0e3 8.0e3 8.0e2 4.0e3 8.0e2 6.0e3 3.0e3 6.0e3 7.5e2 6.0e2 3.0e3 6.0e2 4.0e3 5.0e2 2.0e3 4.0e2 2.0e3 4.0e3 4.0e2

2.0e3 1.0e3 2.0e2 1.0e3 2.0e3 2.5e2 2.0e2

0.0e0 0.0e0 0.0e0 0.0e0 0.0e0 0.0e0 0.0e0

1.0 1.2 1.4 1.2 1.4 1.6 1.2 1.4 1.6 1.2 1.4 1.6 1.2 1.4 1.6 1.2 1.4 1.6 1.2 1.4 1.6

AMI (20) NET (10) PAR (40) GENC1a GENC1 (80) GENC2 HYG (10) KAN (20)

Q 586.30>425.20 1.67e3Q 476.30>299.15 1.15e4Q 616.30>163.10 7.84e3Q 450.20>322.20 1.08e4Q 478.30>322.25 1.12e5Q 464.30>322.10 2.89e4Q 528.30>177.05 1.66e3Q 485.30>163.00 2.41e3

1.8e3 R1 107.96% R1 51.80% R1 47.35% R1 38.56% 1.1e5 R1 104.13% R1 71.82% R1 79.53% R1 18.99% 1.1e4 2.8e4 2.3e3 1.0e4 2.3e3 7.0e3 1.6e3 1.0e4 1.0e5 2.5e4 2.0e3 9.0e3 2.0e3 9.0e3 9.0e4 1.4e3 6.0e3 2.3e4 8.0e3 1.8e3 1.8e3 8.0e3 8.0e4 2.0e4 1.2e3 5.0e3 7.0e3 1.5e3 7.0e3 7.0e4 1.8e4 1.5e3 1.0e3 6.0e3 6.0e4 1.3e3 6.0e3 4.0e3 1.5e4 1.3e3 5.0e3 8.0e2 5.0e3 5.0e4 1.3e4 1.0e3 1.0e3 3.0e3 4.0e3 4.0e3 4.0e4 1.0e4 6.0e2 7.5e2 7.5e2 3.0e3 3.0e3 2.0e3 3.0e4 7.5e3 4.0e2 5.0e2 5.0e2 2.0e3 2.0e3 2.0e4 5.0e3 1.0e3 2.0e2 2.5e2 2.5e2 1.0e3 1.0e3 1.0e4 2.5e3

0.0e0 0.0e0 0.0e0 0.0e0 0.0e0 0.0e0 0.0e0 0.0e0

1.2 1.4 1.6 1.2 1.4 1.6 1.2 1.4 1.6 1.2 1.4 1.6 1.2 1.4 1.6 1.2 1.4 1.6 1.2 1.4 1.6 1.2 1.4 1.6

Figure 1 Chromatograms at the LOQ (concentrations in brackets, in pg/mL)

Recovery Several meat samples, eggs and milk samples were concentration. Results are presented in table 4. purchased from the local supermarket. All samples were The mean recoveries for each compound were superior to processed as described in 2.1. Blank samples and samples 80% and moreover, were homogenous within the type of spiked at 100 ng/g before extraction were analysed. No samples tested. This illustrates the good extraction compound were found in blank samples. Peak areas were recoveries and low matrix effect obtained. compared to an aqueous standard at the same

Identi cation using MRM Spectrum Mode In MRM Spectrum Mode, 15 MRM transitions were Thanks to ultrafast MRM features of the mass acquired per compound. Signals were merged by the spectrometer used, there is no signi cant difference in software to create a spectrum with optimized sensitivity sensitivity when acquiring 2 or 15 MRM per compound. for each fragment. By comparing this spectrum to a Figure 2 illustrates this approach. prede ned library, identi cation becomes unambiguous.

5 Sensitive and Fast Measurement of Aminoglycoside Antibiotics in Milk, Meat or Eggs by HILIC-MS/MS and Identi cation using MRM Spectrum Mode

Table 4 Total recovery in real samples

SPC APR DTP GEN C1a GEN C1 GEN C2 HYG KAN Eggs 93% 83% 86% 83% 86% 96% 89% 85% Fat Milk 94% 81% 109% 88% 86% 88% 84% 82% Low Fat Milk 88% 90% 106% 85% 89% 82% 88% 78% Low Fat Beef 99% 88% 99% 78% 90% 87% 89% 101% Fat Beef 93% 82% 102% 82% 80% 89% 76% 73% Chicken Leg 94% 100% 97% 109% 93% 106% 77% 103% Chicken Liver 83% 86% 90% 90% 81% 87% 88% 89% Chicken Breast 93% 83% 96% 89% 90% 81% 84% 81% Pork Cutlet Muscle 86% 87% 102% 84% 83% 88% 86% 86% Pork Cutlet Fat 94% 83% 92% 82% 91% 91% 88% 90% Pork Bacon 92% 79% 98% 96% 93% 96% 89% 96% Mean 92% 86% 98% 88% 87% 90% 85% 88% %RSD 4.9% 6.9% 6.9% 9.7% 5.2% 7.9% 5.6% 10.6%

NEO STP AMI NET PAR SIS TOB Eggs 79% 89% 84% 84% 85% 89% 81% Fat Milk 80% 101% 83% 83% 90% 73% 87% Low Fat Milk 81% 105% 75% 85% 73% 85% 87% Low Fat Beef 80% 100% 98% 85% 82% 84% 87% Fat Beef 89% 103% 83% 83% 73% 83% 59% Chicken Leg 98% 99% 108% 108% 106% 105% 109% Chicken Liver 82% 95% 84% 85% 87% 86% 95% Chicken Breast 89% 93% 78% 90% 91% 87% 90% Pork Cutlet Muscle 79% 103% 82% 74% 88% 92% 94% Pork Cutlet Fat 75% 92% 88% 88% 84% 86% 86% Pork Bacon 89% 93% 102% 99% 97% 96% 85% Mean 84% 98% 88% 88% 87% 88% 87% %RSD 8.0% 5.5% 11.8% 10.3% 11.0% 9.3% 13.6%

6 Sensitive and Fast Measurement of Aminoglycoside Antibiotics in Milk, Meat or Eggs by HILIC-MS/MS and Identi cation using MRM Spectrum Mode

15 MRM Dihydrostreptomycin

Generate optimized sensitivity spectrum for library search

2 MRM No match Same sensitivity

Streptomycin

Figure 2 MRM Spectrum Mode ID con rmation

Conclusions A very sensitive HILiC-MS/MS method was developed to been optimized to providehigh-throughput. The good detect a large panel of aminoglycoside antibiotics without recoveries obtained across the tested matrices eliminate ion-pairing. the use of matrix-matched calibration standards. One method can be used for all kind of animal species or In addition to the fast quantitative screening method, a ID commodities, covering major food safety regulations. con rmation method using MRM Spectrum Mode can be The complete workow, including sample preparation has performed with same mobile phases and column.

First Edition: June, 2017

© Shimadzu Corporation, 2017