Analysis of Antibiotics in Honey by an Integrated On-Line Extraction UHPLC-MS/MS System

Zicheng Yang and Louis Maljers, Bruker Daltonics Inc.
Poster presented as part of NACRW 2015 Conference, St. Petersburg, FL., 19-22July 2015.

Introduction

The most critical aspects of reliable food contamination analysis are the reduction of interferences from the sample matrix and analyte recovery. Traditionally, SPE, SLE, Liquid-Liquid, syringe filtration, and centrifugation have been used to reduce matrix interference prior to LC/MS analysis. However, these techniques are time consuming, adversely impact recovery, require expensive consumables, and use large amounts of solvent. Improved sample prep methods were developed using eXtreme|FV for contaminant analysis of antibiotics honey.

Equipment

UHLC Conditions

  • Trap Column: YMC-Pack ODS-AQ, 10 µm, 10 mm x 3.0 mm I.D.
  • Mobile Phase C: 0.1% Formic Acid in water
  • Equilibration flow: 1000 µL (4.0 min)
  • Loading Flow: 500 µL
  • Analytical Column: YMC- UltraHT Pto C18 , 2 µm, 100 mm × 2.0 mm I.D.
  • Column Temperature: 40 ˚C
  • Injection Volume: 10 µL (100 µL Loop)
  • Gradient
    • Mobile Phase A: 0.1% FA in water
    • Mobile Phase B: 100% MeOH
    • LC Gradient
      Time minMobile Phase A(%)Mobile Phase B(%)Flow Rate µL/min
      0.08020200
      0.28020200
      4.00100200
      6.00100200
      6.18020200
      8.08020200
  • EVOQ Conditions
    • Source parameters
      Source:HESI
      Spray Voltage (+)4000 V
      Cone Gas Flow20
      Cone Temperature350˚ C
      Heated Probe Gas Flow45
      Heated Probe Temperature400˚ C
      Nebulizer Gas Flow55
      Exhaust GasOn

Sample Preparation

  • Weigh about 50mg of honey in the Thomson eXtreme|FV (p/n 85531).
  • Add MeOH/Water, 50/50, v/v make 100 mg/mL solution.
  • Mix by pipet and depress the filter vial plunger, 0.2 µm PVDF completely to filter.
  • Solution is ready for injection.

Results

Store bought honey samples analyzed by UHPLC-MS/MS ciprofloxacin, tetracycline, enrofloxacin and erythromycin were analyzed utilizing nine point calibration curves for the individual antibiotics, see Table 3. Simple sample prep was achieved using the Thomson eXtreme Filter Vial, 0.2um PVDF. Excellent linearity was achieved from 0.05ng/mL to 20ng/mL. The LOQ was determined to be < 0.5ng/g. Chromatograms at 0.05ng/mL of spiked honey show over lapping peaks that are resolved by mass for the ciprofloxacin, tetracycline, and enrofloxacin. While the erythromycin is nicely resolved by both LC and MS. In the chromatogram s in fig.? 0.5ng antibiotics were spiked into 1.0g honey to yield a concentration of 0.05ng/mL.

Store bought honey from the US (3 different brands), Canada, China and India were analyzed for ciprofloxacin, tetracycline, enrofloxacin and erythromycin. Calculation is base on matrix calibration curve (=100/(detected amount/spiked amount).The recovery for iprofloxacin and erythromycin looks consistent across all levels. The enrofloxacin signal enhanced in matrix and tetracycline signal enhanced at low concentration. Results with an ND are < 0.05ng/mL.

Table.1Antibiotics analyzed in this method
Compound NameRetention TimeQ1 First MassQ3 First MassCollision EnergyQuantifier IonsQual Mass
Ciprofloxacin3.168332.2314-18314
230.9-34230.90
245-18
Enrofloxacin3.201360.3342-18342.00
286-29286
316-15316
Tetracycline3.169445.2410-15410.0
154-24154
427.1-9427.1
Erythromycin4.370734158.1-27158.1
576.3-16576.3

Structures, Formula & LOQ for the Antibiotics Used

honey antibiotics structures 1

Structure of Erythromycin, Formula C37H67NO13, Tolerance Limit 5 (ug/kg, ppb)

honey antibiotics structures 2

Structure of Ciprofloxacin, Formula C17H18FN3O3, Tolerance Limit 5 (ug/kg, ppb)

honey antibiotics structures 3

Structure of Enrofloxacin, Formula C19H22FN3O3, Tolerance Limit 5 (ug/kg, ppb)

honey antibiotics structures 4

Structure of Tetracycline, Formula C22H24N2O8, Tolerance Limit 5 (ug/kg, ppb)

Table.3Calibration curve levels for antibiotics in honey
Calibration Levelng/gng/mL
10.50.05
210.1
320.2
450.5
5101
6202
7505
810010
920020
6

fig.19 point calibration curves.

honey antibiotic chromatograms

fig.2Chromatograms of antibiotics spiked into honey, USA-1.

Table.4Store bought honey from the US (3 different brands), Canada, China and India were analyzed for ciprofloxacin, tetracycline, enrofloxacin and erythromycin. ND is <0.5ng/g. test result based on calibration curve of antibiotics in honey. * Honey from China contains approximately 0.2ng/g Ciproloxacin
AntibioticsCiprofloxacinEnrofloxacinErythromycinTetracycline
Honey SourceConcentration in ng/g
USA-1NDNDNDND
USA-2NDNDNDND
USA-3NDNDNDND
CanadaNDNDNDND
ChinaNDNDNDND
IndiaNDNDND3.8

Recovery

Table.5Recovery of antibiotics spiked into store bought honey, USA-1.
Standard (ng/g)CiprofloxacinEnrofloxacinErythromycinTetracycline
0.5109.6-85.5191
1121.1-85.7278
2114.26666.788.2233
5158.9511.883.8335
1093.2207.486.1116
20111.3202.888.9169
50103.3180.793.4132
100109.8179.196.2127
200133.4190.599.3120

Conclusion

Bruker UHPLC combined with the EVOQ Elite Triple Quadrupole MS was used for identification and quantification of ciprofloxacin, tetracycline, enrofloxacin and erythromycin in store-bought honey utilizing the Thomson eXtreme|FV, 0.2um PVDF. Simple sample prep consisting of diluting the sample, filtering and injecting onto the UHPLC-MS/MS achieved LOQ of < 0.05ng/mL and LOD of 0.02ng/mL.

Title

Analysis of Antibiotics in Honey by an Integrated On-Line Extraction UHPLC-MS/MS System

Description

The most critical aspects of reliable food contamination analysis are the reduction of interferences from the sample matrix and analyte recovery. ... Improved sample prep methods were developed using eXtreme|FV for contaminant analysis of antibiotics honey.

PDF Application

Product Used in Application

eXtreme|FV® - PVDF 0.2µm

Pre-Split Septum, Red Cap

pn#85531-200

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