Application Note #1824322Quantitative LC-MS/MS analysis of Sulfonamides in Honey using Advance UHPLC-EVOQ™ Elite LC-MS/MS system

AbstractA simple, robust and effective LC-MS/MS method operated in multi-

ple reaction monitoring (MRM) mode is developed for simultaneous

estimation of “Sulfonamide Drugs” in food grade honey with easy

and minimal sample preparation. In this experiment, two MRM tran-

sitions were tracked for each targeted analyte, one for quantifi cation

and the other for qualifi cation. The limit of quantifi cation achieved for

all Sulfonamides was 0.1 ppb with consistent ion ratio of quantifi er to

qualifi er ions. The method was found to be linear and repeatable with

regression (R)2 of ≥ 0.996 and relative standard deviation of <12% for

all nine Sulfonamides under study. Use of EVOQTM Elite Triple Quad

LC-MS/MS system offers high sensitivity, specifi city and fl exibility

of the method and allows high sample throughput for screening and

quantifi cation of Sulfonamides in honey.

Introduction Sulfonamide drugs are commonly used for the prevention & treat-

ment of bacterial growth mainly because of their high and broad

spectrum activity. In addition, they are also used to fi ght disease in

honey bees. Residues of these Sulfonamide drugs are quite often

found in honey samples and are a matter of great concern to con-

sumers around the world due to their toxic and/or allergic reactions

they induce in hypersensitive humans and potential transfer of

drug-resistant bacteria’s from honey to humans.

Because of these harmful effects, different countries have estab-

lished MRLs (maximum residues limits) for these antibiotics in food

of animal origin products. The European Union has set a MRPL (min-

imum required performance level) for Sulfonamide’s at a level of

10μg/kg in honey1. There are several techniques available for deter-

mination and quantifi cation of these Sulfonamides; however, due to

insuffi cient sensitivity and poor selectivity posed by very complex

matrix, the targeted MRPL’s cannot be measured using those tech-

niques. The use of LC-MS/MS has shown good selectivity and sen-

sitivity to quantify the sulphonamides at the required MRPL levels in

complex food matrices.

This application note describes a quick, simple, sensitive & rugged

method for simultaneous determination of Sulfadimethoxin (SMM),

Sulfadiazin (SD), Sulfadoxin (SDM), Sulfamethazine (SM2), Sulfadim-

idine (SDN), Sulfamerazine (SM1), Sulfathiazole (ST), Sulfamethoxy-

pyridazin (SMP) and Sulfamethizole (SMZ) residues in honey using

Bruker’s Advance UHPLC system hyphenated with EVOQTM Elite Tri-

ple Quad MS/MS system. The method was validated as per the cri-

teria specifi ed in European Union Commission Decision 2002/657/

EC1.

The method repeatability was studied on three different days by spik-

ing blank honey matrix at levels of 0.1, 1 and 10 ppb of Sulfonamides.

Experimental

Chemicals and reagentsLC-MS/MS grade Acetonitrile (ACN), Formic Acid and Water were

purchased from Sigma-Aldrich. Triple distilled water was used for re-

agent preparation. The hydrochlorides of all nine Sulfonamides under

study [SMM, SD, SDM, SM2, SDN, SM1, ST, SMP and SMZ] were

purchased from Sigma-Aldrich.

Calibration & Quality Control Solution preparationStock solution of 1 mg/mL of each standard was prepared in meth-

anol and stored below 00C. Mixed standards solution (containing

all nine Sulfonamides standards) at concentration level of 1000 ng/

mL, was prepared by diluting individual stock solutions using diluent

(ACN: Water (20:80)). The calibration solutions of 0.1 ppb, 0.25 ppb,

0.5 ppb, 1 ppb, 2.5 ppb, 5 ppb & 10 ppb were prepared by spiking an

appropriate volume of the mixed standards solution in blank honey

matrix. All standard solutions were stored in a refrigerator at 40C.

Sample Preparation 2.5 g of honey (purchased from the open market) was weighed in

polypropylene tube of 50 mL and acidifi ed with 2 mL of 1% formic

acid in water and vortexed for 2 minutes, followed by addition of 8

mL of ACN and 10 minutes of ultrasonic extraction. 1gm powder

from BR73763 Kit (6gm MgSO4, 1.5gm NaOAc) was added to the

tube and shaken for 30 seconds. This mixture was vortexed for 15

min to achieve complete solubilisation followed by centrifugation for

3 min. at 3000 rpm.

Top 4 mL of organic layer was transferred to another centrifuge

tube and evaporated to dryness at 450C under Nitrogen. The extract

was reconstituted with 0.5 mL of initial mobile phase (Water:MeOH

(80:20)) and fi ltered through a 0.22 μm nylon fi lter (Millipore, India) di-

rectly into an HPLC vial and injected.

Instrumentation

Results and DiscussionFigure 1 shows representative chromatograms of all nine Sulfon-

amides investigated at LOQ concentration level of 0.1 ppb in hon-

ey with peak-to peak (PP) S/N ratio. The S/N ratio for all nine Sulfon-

amides in honey matrix at 0.1ppb is well above 10. Figure 2 shows

the overlaid chromatograms of all nine Sulfonamides at LOQ level

and the blank honey matrix. Figure 2 clearly shows that blank honey

matrix is very clean and does not contain any of the targeted Sulfon-

amides.

In this study European Union decision 2002/657/EC has been fol-

lowed which specifi es several parameters in order to confi rm de-

tection of the targeted compounds. As Sulfonamides are forbidden

Group A substances, minimum of 4 Identifi cation Points (IP) are re-

quired to confi rm detection of compound of interest. This is satis-

fi ed by acquiring 2 MRM Transitions where 1.0 IP is assigned for the

“precursor ion” and 1.5 IP’s are assigned for each “product ions”. The

ion ratio of pair of MRM transitions acquired for each target analyte

must match to those of the certifi ed reference material within speci-

fi ed tolerance and were found consistent for all the analyte taken for

this study. Figure 3 shows a comparison of ion ratios of MRM tran-

sitions for a representative analyte acquired experimentally vis-a-vis

the standard reference ion ratio for same analyte. Calibration curves

(seven levels from 0.1 to 10 ppb) for all nine Sulfonamides showed

linear response with R2 > 0.996 as shown in Figure 4. The method

was found repeatable (intra-day and inter-day) with relative standard

deviations (RSD’s) below 12% (Table 2). Results obtained for all nine

Sulfonamides under study at LOQ (0.1ng/g) level concentration were

well within the acceptable range of accuracy and precision. The LOQ

of 0.1ng/g achieved for all nine Sulfonamides under study were much

lower than the MRL’s specifi ed by different country’s regulations.

Figure 1: Chromatograms of all nine Sulfonamides investigated at LOQ con-centration level of 0.1 ppb in honey with peak-to peak (PP) S/N ratio.

Table 1: MRM Transitions acquired

aMRM transition used as Quantifi cation ion

Figure 2: Overlaid chromatograms of all nine Sulfonamides at LOQ level and the honey matrix blank.

Figure 3: Experimental vs. certifi ed reference material ion ratio comparison for Sulfamethazine.

Bru

ker

Dal

toni

cs is

con

tinua

lly im

prov

ing

its p

rodu

cts

and

rese

rves

the

rig

ht

to c

hang

e sp

ecifi

catio

ns w

ithou

t no

tice.

© B

ruke

r D

alto

nics

10-

2013

, #18

2432

2

For research use only. Not for use in diagnostic procedures.

Bruker Daltonik GmbH Bruker Daltonics Inc. Bruker Daltonics Inc.

Bremen · GermanyPhone +49 (0)421-2205-0Fax +49 (0)421-2205-103sales@bdal.de

www.bruker.com/ms

Billerica, MA · USAPhone +1 (978) 663-3660Fax +1 (978) 667-5993ms-sales@bdal.com

Fremont, CA · USAPhone +1 (510) 683-4300Fax +1 (510) 490-6586ms-sales@bdal.com

Conclusions Bruker’s Advance UHPLC coupled with the EVOQTM Elite Triple Quad

LC-MS/MS system demonstrated excellent detection limits and re-

peatability for all nine Sulfonamides under study in the complex hon-

ey matrix. Additionally, the use of Advance UHPLC reduced the total

chromatographic run time. The validation results revealed that the

method can be used for the screening and quantifi cation of all nine

Sulfonamides under study in honey as per the requirements of Euro-

pean Union and other nations.

AuthorsGouri Satpathy

ReferencesCommission Decision 657/2002 of August 2002 on implementing

Council Directive 96/23/EC concerning performance of analytical

methods and the interpretation of results. Offi cial Journal of the Eu-

ropean Union, L221, 8-36, 2002.

AcknowledgementsSima Labs Pvt Ltd., New Delhi for active collaboration and providing

experimental resources and Standards.

Table 2: Sulfonamides in honey performance summary

Figure 4: Calibration curves for all nine Sulfonamides (seven levels from 0.1 to 10 ppb)