1. Introduction
β-agonists such as clenbuterol, cimaterol, salbutamol, ractopamine, and
zilpaterol have been used to improve feed efficiency and increase
muscle mass in animals. However, presence of β-agonists in animal
derived products such as meat, and dairy products have been linked
with hepatotoxicity in humans. Analysis of β-agonists using liquid
chromatography-tandem mass spectrometry (LC-MS/MS) in meat
matrices have been reported in literature but less emphasis has been
placed on dairy products.
Milk based infant formula is a complex matrix due to its high fat, protein
and minerals. Additionally, selection of optimum ratio between infant
formula powder and reconstitution solvent, and ratio between infant
formula powder and extraction solvent can also affect the extraction of
analytes. Prior to LC-MS/MS detection, a cleanup step in usually
introduced in order to minimize matrix interference. In this work, we
have utilized QuEChERS to remove matrix interference prior to LC-
MS/MS detection of β-agonists in milk based infant formula.
Analytes
Sample Preparation
Instrument Method
Table 3. MS/MS Transitions
Results and Discussion
Stock solutions (1 mg/mL) were prepared in methanol, calibration
standards were prepared in water and linearity was investigated as
shown in figure 2.
Extraction was performed by using acetonitrile:isopropanol (4:1).
Quechers kit contained sodium chloride (1.2 gm), sodium sulfate (4 gm)
and magnesium sulfate (0.5 gm). Infant formula powder samples were
fortified at 1 µg/kg. recoveries of all β-agonists ranged from 60-130 %
and RSD values were below 8% as shown in table 4
Table 4: Fortification recoveries of β-agonists in infant formula.
Conclusions
A quick and simple method was developed for quantitative analysis of β-
agonists in infant formula. The cleanup and quantification was
performed by QuEChERS and LC-MS/MS respectively. Presented
method can serve as an efficient tool to quantitatively determine β-
agonists at 1 µg/kg in milk based infant formula.
Determining the optimum ratio between infant formula powder,
reconstiution solvent and extraction solvent remains the critical factor
which affect the extraction of analytes.
Future studies are aimed at performing method validation to determine
selectivity/specificity and precision (repeatability and between day
reproducibility.
References
CLG-AGON1.07, USDA Food Safety and Inspection Service,Office of
Public Science, “Screening, Determination and Confirmation of β-
agonists by HPLC/MS/MS”. January 11, 2018.
Utilizing QuEChERS-based Methodology and LC-MS/MS
Detection for the Quantitative Analysis of β-agonists in
Animal-derived Foods
Ujwal S. Patil, Sarah Ruiz, Cheryl Stephenson and John Reuther
Eurofins Central Analytical Laboratories, 2219 Lakeshore Drive, New Orleans, LA 70122
Figure 2. Calibration curves for
β-agonists
Table 1. LC-MS/MS conditions.
Table 2. LC Gradient Program.
Contact information: UjwalPatil@eurofinsUS.com
Clenbuterol Cimaterol
Zilpaterol
Ractopamine Salbutamol
Figure 1: Total ion chromatogram of β-agonists. The extracted ion
chromatograms of A) Cimaterol, B) Salbutamol, C) Zilpaterol, D)
Clenbuterol and E) Ractopamine are shown in the inset.
A B C
D E
Time (min) Flow rate
(mL/min)
Mobile phase
B (%)
0.1 0.4 5
1.3 0.4 5
7.3 0.4 65
7.36 0.4 5
12 0.4 5
Analyte Precursor Ion Quantifier Ion
Clenbuterol 277.2 203,132
Cimaterol 220.2 143,160
Ractopamine 302.2 164, 121
Salbutamol 240.1 148,121
Zilpaterol 262.2 185,157
Clenbuterol
R2: 0.9982
Salbutamol
R2: 0.9995
Cimaterol
R2: 0.9985
Zilpaterol
R2: 0.9992
Ractopamine
R2: 0.9968
Analytes
Fortification
Levels (µg/kg)
Fortification
Recovery (%)
RSD (%)
Clenbuterol 1 128 7
Cimaterol 1 111 4
Ractopamine 1 102 2
Salbutamol 1 124 1
Zilpaterol 1 60 8
Instrumental
Details
LC Instrument Shimadzu Nexera 2
Column: Phenomenex Kinetex-C18 column, 100 x 2.1 mm, 2.6 µm
Injection Volume: 5 µL
Temperature 35°C
Flow Rate 0.4 mL/min
Mobile Phase A 0.1 % Formic acid in water
Mobile Phase B Acetonitrile
MS Instrument AB Sciex 5500 Triple Quad
Curtain Gas 20
Ion Spray Voltage 4000
Temperature 350
Ion Spray Gas 1 60
Ion Spray Gas 2 75
Analyte conc./IS conc.
Analytearea/ISarea