Quick screening of priority beta-agonists in urine using ...

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Quick screening of priority beta-agonists in urine usingautomated TurboFlow™ - LC/Exactive mass

spectrometryThorsten Bernsmann, Peter Fuerst, Michal Godula

To cite this version:Thorsten Bernsmann, Peter Fuerst, Michal Godula. Quick screening of priority beta-agonists in urineusing automated TurboFlow™ - LC/Exactive mass spectrometry. Food Additives and Contaminants,2011, 28 (10), pp.1352-1363. �10.1080/19440049.2011.619504�. �hal-00743048�

https://hal.archives-ouvertes.fr/hal-00743048

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This paper describes a method for the determination of priority β-agonists in urine based on a fully automated sample preparation procedure using the online TurboFlow™ chromatography clean-up step and determination on the Orbitrap™ mass analyzer technology. The principle of the method

after enzymatic hydrolysis over night

on a small column packed with a special stationary phase (TurboFlow™) while flushing away sample matrix and interfering compounds. Thereafter the analytes are transferred onto an analytical column and detected by

id chromatography/high resolution mass spectrometry in full scan mode at a resolution of R=50,000 FWHM (full width at half maximum) and in HCD (Higher Energy Collisional Dissociation) scan mode at a resolving power of 10,000 FWHM. The optimization of each step of the method, such as selection of the TurboFlowTM and analytical column as well as sample loading and elution parameters were performed using a standard solution containing salbutamol, clenbuterol and mabuterol at a

veloped automated sample preparation significantly improved the throughput and efficiency of the previous used

screening method and resulted in a considerable reduction in analysis time. Validation experiments including 24 β-agonists in urine gave

0.35 ug/L. The repeatability of analyses for urine samples spiked at 0.5 ug/L was within the range of 5-26% and recoveries for all compounds were found to be within 89-107%.

http://mc.manuscriptcentral.com/tfac Email: [email protected]

Food Additives and Contaminants

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* Corresponding author. Email: [email protected]

Quick screening of priority beta-agonists in urine using automated

TurboFlowTM

- LC/Exactive mass spectrometry

Thorsten Bernsmanna*

, Peter Fürsta and Michal Godula

b

aChemical and Veterinary Analytical Institute Münsterland-Emscher-Lippe, Joseph-König-

Straße 40, 48147 Münster, Germany

bThermo Fisher Scientific, Slunečná 27, 10000 Praha 10, Czech Republic

Abstract

This paper describes a method for the determination of priority β-agonists in urine based on a

fully automated sample preparation procedure using an online TurboFlowTM

chromatography

clean-up step and determination with OrbitrapTM

mass analyzer technology. The principle of

the method was the enrichment of the β-agonists after enzymatic hydrolysis overnight on a

small column packed with a special stationary phase (TurboFlowTM

) while flushing away

sample matrix and interfering compounds. Thereafter the analytes were transferred onto an

analytical column and detected by liquid chromatography/high resolution mass spectrometry

in full scan mode at a resolution of R=50,000 FWHM (full width at half maximum) and in

HCD (Higher Energy Collisional Dissociation) scan mode at a resolving power of 10,000

FWHM. The optimization of each step of the method, such as selection of the TurboFlowTM

and analytical column as well as sample loading and elution parameters were performed using

a standard solution containing salbutamol, clenbuterol and mabuterol at a concentration of 100

µg/L. The developed automated sample preparation significantly improved the throughput and

efficiency of the previous used screening method and resulted in a considerable reduction in

analysis time. Validation experiments including 24 β-agonists in urine gave decision limits

(CCα) between 0.05-0.35 µg/L. The repeatability of analyses for urine samples spiked at 0.5

µg/L was within the range of 5-26% and recoveries for all compounds were found to be

within 89-107%.

Key Words: Beta-agonists, urine, TurboFlowTM

, OrbitrapTM

mass analyzer technology,

Exactive MS

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Introduction

Beta-agonists are synthetically produced compounds that, in addition to their bronchodilatory

and tocolytic effects, can promote live weight gain in food producing animals. There have

been documented cases when consumption of liver and meat from animals illegally treated

with clenbuterol has resulted in serious human intoxication (Botsoglou et al. 2001). Due to

their adverse effects, the use of clenbuterol and its analogues from the β-agonist group has

been banned by the European Union (EU 1996) and other regulatory agencies worldwide.

Monitoring programs have shown that β-agonists are still illegally used by food producers

(Fiori et al. 2002, Mazzanti et al. 2003). Moreover, newly developed analogues with modified

structures are obviously being continuously introduced in routine practice. Many papers have

been published in the past describing the analysis of β-agonists in various matrices using GC-

MS. Typically are those methods based on the determination of compounds after

derivatization by GC-MS (Montrade et al. 1993, Damasceno et al. 2000, Henze et al. 2001) or

GC-MS/MS (Biancotto et al. 1999, Amendola et al. 2002, Bocca et al. 2003). In most cases

the sample preparation steps include time-consuming evaporation of water based re-extracts

or solid phase extraction (SPE) clean-up steps.

In order to avoid derivatization steps the recently published methods use liquid

chromatography coupled to tandem mass spectrometry (MS/MS) instruments. In the case of

liver and kidney samples, the β-agonists are typically extracted from the tissue after enzymatic

digestion and the extracts are cleaned up using liquid-liquid extraction and SPE (Fesser et al.

2005). Beta-agonists from urine are generally alkaline extracted after enzymatic hydrolysis

and acidic re-extraction is then performed. After the evaporation of the acidic re-extract and

reconstitution in the mobile phase the analytes are determined using electrospray ionization

(ESI) LC-MS/MS (Thevis et al. 2003). Alternatively, the clean-up of the sample can be

improved using SPE and as an alternative to ESI LC-MS/MS atmospheric pressure chemical

ionization (APCI) is also used for LC-MS/MS determination (Dickson et al. 2005). The very

efficient alternative to the commonly used SPE approach is the use of molecularly imprinted

polymer (MIP) columns. Several papers have been published using MIP columns to

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effectively remove sample matrix and to allow sub ng/mL determination of β-agonists in

various matrices (Widestrand et al. 2004, Fiori et al. 2005, Kootstra et al. 2005). Although

most of the above cited approaches make it possible to reach the required detection limits their

main limitation is the time-consuming and expensive column clean-up step.

Based on the annual national production figures, the EU Commission stipulates sampling

levels and frequencies for β-agonists and other veterinary drugs in animal products and

matrices in order to check for illegally administered substances and in the case of authorized

compounds for compliance with prescribed withdrawal periods. Compared to time and effort

to fulfill these requirements, the number of positive findings is relatively low. This indicates

that there is a clear need for quick and simple screening methods to routinely and accurately

control levels of β-agonists in samples of animal origin, such as urine, plasma, and tissues.

The principle of TurboFlowTM

chromatography is the separation of analytes from the matrix

using specific columns packed with large particles. In combination with high linear velocity of

the mobile phase the conditions are induced on the column that allow the retention of smaller

molecules (i.e. veterinary drugs) while the large molecules (such as proteins and lipids) are

passing through the column unretained (Quinn and Takarewski 1997). The application of

TurboFlowTM

chromatography has been already described on different examples of analyzing

specific groups of analytes in a range of matrices (milk, honey or pig tissues) (Mottier et al,

2008, Krebber et al 2009, Stolker et al. 2010).

The use of mass spectrometers based on high resolution and high mass accuracy

measurements offers certain benefits over traditional tandem mass spectrometry. As

documented already (Kaufman et al 2011), the selectivity and detection limits obtained by

high resolution mass spectrometer is comparable or better than those obtained by triple

quadrupole mass spectrometers. The advantage of the high resolution MS approach is the

simple operation with no specific compound setup and the fact that the data acquisition is

done most often in full scan mode. This allows not only to detect targeted analytes but also to

potentially search for non targeted analytes (metabolites e.g.). The importance of the various

factors affecting the quality of data produced by high resolution mass spectrometers,

especially the resolving power and mass accuracy achieved has been also discussed

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(Kaufmann et al 2010, Kellman et al 2009).To our knowledge, till today no work has been

documented analyzing a complete set of β-agonists in urine using the combination of the

automated TurboFlowTM

sample preparation and detection based on the use of high resolution

Orbitrap mass spectrometer. The newly developed approach results in a simplification of

current methodologies and a considerable reduction in analysis time.

Materials and Methods

Samples

The urine samples from cattle and pigs were routinely taken within the frame of the national

residue control plan. Until analysis the samples were stored at -18 °C.

Chemicals and Reagents:

Acetonitrile, methanol, isopropanol (all picograde) were purchased from Promochem (Wesel,

Germany). Ultrapure water was produced by reverse osmoses. Acetic acid glacial (purity AR-

A) and ammonium acetate (purity AR-U) were obtained from Biosolve (Valkenswaard, The

Netherlands) and an aqueous solution of ß-glucuronidase Type H-2 (from Helix pomatia) with

an activity of 130,200 units/mL came from Sigma Aldrich (St. Louis, USA).

Reference standards

Beta agonist standards: clenbuterol, fenoterol, isoxsuprine, ritodrin, salbutamol, salmeterol,

terbutalin were purchased from Sigma Aldrich (St. Louis, USA). Brombuterol,

chlorbrombuterol, cimbuterol, clencyclohexerol, clenhexerol, clenpenterol, clenproperol,

hydroxymethylclenbuterol, mabuterol, mapenterol, tulobuterol were purchased from Witega

(Berlin, Germany). Ractopamin-hydrochlorid was purchased from Dr. Ehrensdorfer

(Augsburg, Germany). Carbuterol, metoprolol, pirbuterol and zilpaterol were obtained from

the former Federal Institute for Consumer Health Protection and Veterinary Medicine (now

Federal Office of Consumer Protection and Food Safety, Berlin, Germany). Cimaterol was

purchased from Tocris bioscience (Bristol, UK).

Sample Preparation:

Urine samples were centrifuged at 3500 rpm for 10 min. One mL of the supernatant was

diluted with 580 µL water, 400 µL 50 mM ammonium acetate buffer (pH 5.2) and 20 µL ß-

glucoronidase solution. The mixture was kept at 37 °C over night in a shaker. After the

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hydrolysis step the mixture was centrifuged again at 3500 rpm. The supernatant was filtrated

through a 0.45 µm filter into an autosampler vial.

TLX-Exactive analysis

The online clean-up of urine samples was carried out using a TurboFlowTM

system TLX

Aria-1 (Thermo Fisher Scientific, San Jose, CA, USA) consisting of loading and eluting

pumps, two valve switching modules and automatic liquid sampler. TurboFlowTM

online

sample preparation was carried out using different columns: C18-P XL 0.5 mm I.D x 50mm

length; C18-XL 0.5x50mm; C8 0.5x50mm, Cyclone 0.5x50mm, Cyclone-P 0.5x50mm;

Cyclone-MAX 0.5x50mm (all Thermo Fisher Scientific, Franklin, MA, USA). Separation of

the compounds was carried out on a reversed-phase phenyl-hexyl analytical column (150 mm

x 4.6 mm x 3.5µm particle size (Eclipse Plus Phenyl-Hexyl, Agilent Technologies, Santa

Clara, CA, USA). The injected sample volume was 50 to 100 µL.

The online TurboFlowTM

system Aria-1 (schematics shown in Figure 1) was connected to a

single stage mass spectrometer LC-MS ExactiveTM

with an HCD cell, equipped with the

heated electrospray interface HESI-II (Thermo Fisher Scientific, San Jose, CA, USA). The

Exactive was operated in positive ionization mode using the following MS parameters:

electrospray, voltage: 4.0 kV; sheath gas: 65 arbitrary units; auxiliary gas: 15 arbitrary units;

capillary temperature 250 °C, heater temperature 300 °C, capillary voltage 60 V, tube lens

voltage 120 V. These settings are a compromise between the different source parameters for

each analyte and gave the best sensitivity over the whole group. The other instrument

parameters were automatically tuned for maximum ion signal. The system was operated in full

scan mode at a resolving power of 50,000 FWHM and in HCD scan mode at a resolving

power of 10,000 FWHM with an HCD voltage of 30 V. No specific lock mass was used for

internal mass axis correction. Mobile phases used in the Aria-1 system were as follows:

Loading pump:

A: 10 mM ammonium acetate in water, adjusted to pH 8 with ammonium hydroxide

B: methanol

C: isopropanol:acetonitrile:cyclohexane (30:30:40, v/v/v)

D: 0.1% acetic acid in water

Eluting pump:

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A: 0.1% acetic acid in water

B: methanol

The typical conditions used for the online sample clean-up on a TurboFlowTM

column and the

chromatographic separation on the analytical column are shown in Table 1.

Results and Discussion

The simplification and increased throughput of current analytical methods is of major concern

in many labs performing routine analyses for residues of veterinary drugs in samples of

animal origin. The automated online sample preparation technique based on the high

turbulent-flow liquid chromatography (HTLC) has the potential to offer significant advantage

over manually performed procedures. The principle of the approach is the enrichment of

analytes on a small column packed with special stationary phase (TurboFlowTM

) while

flushing away sample matrix and interfering compounds. Thereafter, the analytes are

transferred onto an analytical column and detected by mass spectrometry as shown in Figure

1. However, before successful application in routine analysis, it is necessary to optimize the

performance of the whole system.

The most important steps in the optimization procedure are:

(I) TurboFlowTM

column selection – evaluation of different types of columns with respect

to the retention of analytes

(II) Sample loading parameters – optimization of the pH of the loading mobile phase to

retain the analytes on the TurboFlowTM

column

(III) Sample elution parameters – optimization of the elution mobile phase pH, flow rate

and time during the elution step from the TurboFlowTM

column

(IV) Analytical column focusing parameters – optimization of initial gradient composition

to effectively trap and focus all compounds of interest during the chromatographic

separation step.

The full optimization of each step of the method was performed using a standard solution

containing salbutamol, clenbuterol and mabuterol each at a level of 100 µg/L. The three

compounds were selected from all conceivable β-agonists due to their physico-chemical

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properties and strong dependency on the pH of the mobile phase which substantially

influences their behavior in the chromatographic system.

The selection of the TurboFlowTM

column with a suitable stationary phase was the first

optimization step. The following TurboFlowTM

columns were tested with respect to their

retention capabilities:

� C18-P XL 0.5x50mm

� C18 XL 0.5x50mm

� C8 0.5x50mm

� Cyclone 0.5x50mm

� Cyclone-P 0.5x50mm

� Cyclone-MAX 0.5x50mm

The standard solution containing the three compounds was injected on the different

TurboFlowTM

columns using water with 10 mM of ammonium acetate as loading solvent at a

flow rate of 1.5 mL/min. Elution from the column was performed with a 200 µL loop filled

with a solution of acetonitrile:water 80/20 v/v with 0.1% formic acid added. The extracted ion

chromatograms (EIC) of the [M+H]+-ions for the three test compounds salbutamol,

clenbuterol and mabuterol shown in Figure 2 document how the column selectivity influences

the retention of the tested compounds. From all tested columns, the Cyclone-P column was

identified as the most promising one due to the fact that only a small portion of salbutamol

was not retained. The other compounds (mabuterol and clenbuterol) were fully retained. On

all other columns salbutamol was not retained at all due to its amphoteric properties leading to

charging of the compound under the applied conditions.

The next step to optimize was the composition of the loading mobile phase. As already

mentioned, due to the amphoteric nature of some β-agonists, it was necessary to select the

optimal pH of the mobile phase used during this step.

The following mobile phase compositions were tested at a flow rate of 1.5 mL/min:

� water + 0.1% formic acid, pH 3

� water + 10 mM ammonium acetate, adjusted to pH 6

� water + 10 mM ammonium acetate, adjusted to pH 8

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The chromatograms shown in Figure 3 demonstrate how the pH influences the retention of

compounds on the Cyclone-P column. Clenbuterol and mabuterol are fully retained at all pH

conditions tested. However, at lower pH values (3-6) salbutamol is not fully retained on the

column. The efficiency of salbutamol retention is only about 60%. At pH 8 more than 85% of

salbutamol stays on the column. For this reason, a loading phase with pH 8 was found optimal

for all tested analytes.

After successful enrichment of the analytes it is important to provide their efficient transfer

onto an analytical column. The transfer from the TurboFlowTM

column to an analytical

column is achieved by the elution with a solvent located in the fixed loop with a typical

volume of 100-200 µL (see Figure 1). The solvent filled in the loop typically consists of the

organic phase (methanol or acetonitrile) with a certain water percentage. The aim of this

optimization step is to use the mixture with the lowest possible organic phase percentage and

to accomplish the elution with the lowest possible flow rate. Those two conditions are

required to allow efficient focusing of the analytes at the head of the analytical column. To

optimize the elution composition and flow rate a mobile phase containing methanol:water +

0.1 % formic acid at different ratios was used. Methanol was chosen due to its higher polarity

compared to acetonitrile, expected better solubility for compounds and better compatibility

with LC-MS solvents.

The solvent elution ratios used were from 100% to 50% of methanol in 10% increments. The

results of the optimization are illustrated in Figure 4. When the percentage of methanol in the

elution solvent dropped down to 70%, clenbuterol was no longer completely eluted from the

TurboFlowTM

column. At 50% of methanol about 25% of clenbuterol was retained on the

column. Consequently, the flow rate of the mobile phase during elution was tested and it was

found that the elution of all compounds from the column took about 1 min when a flow rate of

100 µL/min was applied. At 75 µL/min this process took already 2 min. Therefore, for the

next experiments, a flow rate of 100 µL/min was selected as optimal condition and 70% of

methanol in the eluting solvent was used.

The last step in the method optimization was the evaluation of the combinations of flow rates

and mobile phase compositions to efficiently focus analytes in the front part of the analytical

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column. The initial conditions tested were:

Loading pump: methanol : water + 0.1 % formic acid (70:30, v/v), flow rate 100 µL/min

Eluting pump: methanol : water with 0.1% formic acid (2:98, v/v), flow rate 700 µL/min.

Both solvent flows were combined in the T-piece on the valve 2. Using these conditions, a

peak broadening of salbutamol was observed on the analytical column, indicating improper

focusing. Further optimization was then performed to improve the focusing step to obtain

good peak shapes for all compounds. Different mobile phases, pH, flow ratios from eluting

and analytical pump and percentages of the organic phase in the HPLC mobile phase were

tested. However, at all settings employed, the peak shape of salbutamol was still not

satisfactory. Consequently, the modification of the mobile phase with acetic acid instead of

formic acid was tested. Probably due to its better interactions with the stationary phase of the

phenyl based column, acetic acid at the concentration levels of 0.1% in the water phases

significantly improved the focusing step and provided excellent peak shapes for all analyzed

agonists (see Figure 5). The final conditions that were optimized for the online TurboFlowTM

sample preparation are summarized in Table 2.

Method validation

It is necessary to ensure the quality and comparability of analytical results generated by a

newly developed method before its use in official residue control. This should be achieved by

using quality assurance systems and especially by performing a validation according to

internationally recognized and harmonized procedures and performance criteria. Thus,

following the optimization of the TurboFlowTM

and HPLC column conditions, the method

was thoroughly validated by following validation criteria laid down in Decision 2002/657/EC.

Where the Decision has set no criteria, such as for using a high resolution mass spectrometer

coupled with liquid chromatography, respective criteria were taken from the Document

SANCO/10684/2009 which lays down the method validation and quality control procedures

for pesticide residues analysis in food and feed. For the identification of the different analytes

the retention time and the [M+H]+-ion in full scan mode (based on Decision 2002/657/EC) at

a resolution of 50,000 FWHM were used. For confirmation purposes other ions, such as [M-

H2O +H]+ or other adduct ions like sodium or ammonium in full scan mode or a product ion in

HCD mode were utilized. The most sensitive ions with their exact masses used for

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identification (mass accuracy < 5 ppm; one fragment ion), unequivocal confirmation and

quantification are listed in Table 3. Extracted ion chromatograms of the recorded ions of a

standard solution (0.5 µg/L) and a spiked urine sample (0.1 µg/L) are shown in Figures 6 & 7.

A matrix-considering validation protocol was used which takes into account the uncertainty

due to a potential matrix influence. (Jülicher et al. 1998; 1998 a; Gowik et. al. 1998). To build

up the experimental design and to calculate all relevant validation parameters the software

program “Interval” from the company “quo data” was used. The factors that may influence the

measurements were determined based on practical considerations. Two factors were chosen

which could have an impact on the analytical results.

As can be seen from Table 4, the validation experiment considered species (urine from

different cows and pigs collected in the frame of the National Residue Control Plan) as a

matrix-related factor and two analysts as leading factors. For this design a factorial plan was

calculated by the programme, which required a randomised performance of the experiment.

The experimental plan was replicated for each concentration level. The concentration levels,

which were chosen for each compound (in matrix), were 0.00 (blank sample), 0.10, 0.25, 0.50,

1.0, 1.5 and 2.0 µg/L. For this purpose, pretested blank urine samples from cows and pigs

were spiked with the different concentrations of the β-agonists. After spiking, the urine

samples were prepared in the same way as described above. In total, the validation study was

based on 112 different analyses for each substance. The model allows the estimation of

critical concentrations for given α-errors and the calculation of the corresponding power

function which are important to evaluate the performance of an analytical method. The

relevant parameters are:

• Decision limit (CCα): This means the limit at and above which it can be concluded

with an error probability of α that a sample is non-compliant (Decision 2002/657/EC)

• Detection capability (CCβ): This describes the smallest content of the substance that

may be detected, identified and/or quantified in a sample with an error probability of β.

In the case of substances for which no permitted limits have been established, the

detection capability is the lowest concentration at which a method is able to detect

truly contaminated samples with a statistical certainty of 1 – β (Decision 2002/657/EC)

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• In-house validation (Decision 2002/657/EC)

Table 5 summarizes the data obtained from the method validation study. The decision limits

and detection capabilities calculated range from 0.05-0.35 µg /L and from 0.13-0.70 µg /L

urine, respectively. The repeatability of sample analyses spiked at 0.5 µg /L is within the

range of 5-26% whilst within-laboratory reproducibility (precision obtained in the same

laboratory under stipulated conditions concerning e.g. method, test materials, operators,

environment over long time intervals) range between 11-31%. Recoveries for all compounds

were between 89 and 107%. The TLX system showed less than 1 % cross-contamination. The

main source for the cross-contamination was the auto-sampler where an intensive washing

procedure with an acetic acid/methanol solution after each injection is necessary, which can

be done during the chromatographic run.

The validation has proven that the developed method is fit-for-purpose. It allows the

determination of 24 β-agonists in urine at the concentration of interest whilst considerably

reducing the manual work load for the laboratory technicians. Moreover, due to the substantial

shorter analysis time as a result of the unattended automatic sample preparation process, the

laboratory’s sample throughput can be significantly increased. According to our calculations,

the overall sample preparation time excluding the enzymatic hydrolysis which is necessary to

release bound forms of β-agonists is approximately 30 min. This is more than 3x less time

than what is needed for a typical method based on simple sample preparation (Thevis et al

2003). It is worthy of mention that the flexibility of the overall system makes it possible to

perform also traditional HPLC or UHPLC separations without any hardware modifications.

As already mentioned in the introduction, the possibility of acquiring the MS data in full scan

mode offers the future extension of the method to screen for other groups of analytes as well

as search for metabolites or degradation products.

Acknowledgements

The authors would like to acknowledge Dr. Francois Espourteille from Thermo Fisher

Scientific, Franklin, MA (USA) for his help with the initial TurboFlowTM

method

optimization and column selection

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tandem mass spectrometry for the multi-residue analysis of beta agonists in calves urine.

Analytica Chimica Acta, 529(1-2): 207-210

Gowik P, Jülicher B, Uhlig B. Multi-residue method for non-steroidal anti-inflammatory

drugs in plasma using high-performance liquid chromatography–photodiode-array detection:

Method description and comprehensive in-house validation. Journal of Chromatography B

716 (1998): 221-232

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agonists and confirmation of fenoterol, orciprenaline, reproterol and terbutaline with gas

chromatography-mass spectrometry as tetrahydroisoquinoline derivatives. Journal of

Chromatography B 751: 93-105

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means of a statistically based in-house validation concept. Analyst 123: 173-179

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the investigation of the measurement uncertainty using fractional factorial experiments.

Analyst 124: 537-545

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confirmative performance of liquid chromatography coupled to high�resolution mass

spectrometry compared to tandem mass spectrometry Rapid Communication in Mass

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improved high resolution mass spectrometry based multi-residue method for veterinary drugs

in various food matrices 2010 Anaytica Chimica Acta, doi:10.1016/j.aca.2010.11.034

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1464–1476

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Figure 1. Schematics of the online TurboFlowTM

clean-up system

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Figure 2. The influence of TurboFlowTM

column chemistry on the retention of the β-agonists

used for optimization (extracted ion chromatograms, EIC). Columns with different stationary

phases were used (C18P, C18, C8, Cyclone, Cyclone P, Cyclone Max). Cyclone P shows the

best retention behavior for all compounds.

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Figure 3. The pH optimization of the mobile phase chosen for the loading of the β-agonists

used for optimization (extracted ion chromatograms, EIC) on the TurboFlowTM

column.

Increasing the pH from 3 to 8 (left to right) improves the retention of salbutamol significantly.

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Figure 4. Loop filling optimization (100% to 50% of methanol in the loop) for the β-agonists

used for optimization (extracted ion chromatograms, EIC). At 70% methanol clenbuterol is

not fully eluted anymore.

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Figure 5. Improper mobile phases composition showing poor refocusing of the analytes

(extracted ion chromatograms, EIC) on the analytical column (left). Peak shapes after

optimization step (right)

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Figure 6. EICs of all 24 analysed β-agonists in a standard solution at a concentration of 0.5

ug/L. (numbering of analyts see Table 3).

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Figure 7. EICs of all 24 analyzed β-agonists in a urine sample spiked at 0.1 ug/L. (numbering

of compounds see Table 3)

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Table 1: TurboFlowTM

loading and eluting conditions

Loading pump Valve

1

Valve

2 Eluting pump

Step Start Sec Flow Grad %A %B %C %D Tee Loop Flow Grad %A %B

1 00:00 30 1.5 Step 100 - - - - Out 0.8 Step 98 2

2 00:30 120 0.1 Step - - - 100 T In 0.9 Step 98 2

3 02:30 30 1.0 Step - 80 20 - In 0.8 Ramp 70 30

4 03:00 120 1.0 Step - - 80 20 - In 0.8 Ramp 55 45

5 05:00 320 1.0 Step - - 80 20 - In 0.8 Ramp 20 80

6 10.20 180 1.0 Step 70 30 In 0.8 Ramp 5 95

7 13.20 120 1.0 Step 70 30 In 0.8 Step 5 95

8 15.20 160 1.5 Step 100 Out 0,8 Step 98 2

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Table 2: Optimized conditions for online TurboFlowTM

sample preparation

TurboFlowTM

Column Cyclone-P, 0.5 x 50 mm

Loading conditions water with10 mM NH4Ac, pH 8, 1.5 mL/min, 30 s

Eluting conditions methanol : 0.1% acetic acid, 70/30, 0.10 mL/min, 120 s

Wash step 1. methanol/0.1% acetic acid, 80:20, 1.5mL/min

2. acetonitrile/acetone/isopropanol; 30/30/40 (v/v/v), 1.5

mL/min, 30 s

Analytical column Eclipse Plus Phenyl-Hexyl, 4.6 mm x 150 mm x 3.5 µm

HPLC mobile phase A: water + 0.1% acetic acid; B: methanol

For gradient see Table 1

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Table 3: List of analysed compounds with molecular formula and exact masses for

identification and quantification

No. Name

Molecular

formula

Quant.

Ion

Qual.

Ion

HCD

Ion

1 clenproperol C11H16N2OCl2 263.0713 265.0364 245.0607

2 brombuterol C12H18N2OBr2 366.9818 366.9838 350.9729

3 salbutamol C13H21NO3 240.15940 - 148.0757

4 mabuterol C13H18N2OClF3 311.1133 313.1104 237.0400

5 clenbuterol C12H18N2OCl2 277.0869 279.0837 203.0138

6 ractopamin C18H23NO3 302.1751 284.1645 284.1645

7 terbutalin C12H19NO3 226.1438 152.0705 152.0705

8 isoxsuprine C18H23NO3 302.1751 284.1645 284.1645

9 pirbuterol C12H20N2O3 241.15467 - 223.1441

10 fenoterol C17H21NO4 304.1543 326.1363 286.1438

11 salbutamol C12H18NOCl 228.1150 230.1119 154.0415

12 zilpaterol C14H19N3O2 262.1550 244.1440 202.0976

13 cimbuterol C13H19N3O 234.1601 160.0867 160.0867

14 ritodrin C17H21NO3 288.15940 270.1489 270.1489

15 clencyclohexerol C14H20N2O2Cl2 319.0975 321.0942 301.0868

16 clenpenterol C13H20N2OCl2 291.1026 293.0996 203.0682

17 mapenterol C14H20N2OClF3 325.1289 327.0396 217.0337

18 carbuterol C13H21N3O3 268.1656 - 194.0916

19 clenhexerol C14H22N2OCl2 305.1182 307.1154 287.1076

20 chlorbrombuterol C12H18N2OClBr 323.0343 321.0364 248.9608

21 salmeterol C25H37NO4 416.27954 438.2615 398.2690

22 cimaterol C12H17N3O 220.14440 - 143.0602

23 metoprolol C15H25NO3 268.19072 250.1802 250.1802

24 hydroxymethylclenbuterol C12H18Cl2N2O2 293.08240 295.0790 275.0712

Quant. Ion: Quantification ion is the most intensive ion in the full scan experiment. Mostly it

is the [M+H]+-ion.

Qual. Ion: Qualification ion

HCD Ion is the most intensive ion in the HCD experiment.

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Table 4: Details of validation experiment

Analyte(s): brombuterol; carbuterol; chlorbrombuterol; cimaterol;

cimbuterol; clenbuterol; clencyclohexerol; clenhexerol;

clenisopenterol; clenpenterol; clenproperol; fenoterol;

hydroxymethylclenbuterol; isoxsuprin; mabuterol;

mapenterol; metoprolol; pirbuterol; ractopamin; ritodrin;

salbutamol; salmeterol; terbutalin; tulobuterol; zilpaterol

Number of factors: 1 + 1 leading factor

Number of leading factors 2

Number of runs: 16

Number of concentrations 7

Total number of analyses: 112

Runs per day: 8

Leading factor: persons (2)

matrix-related factor: species (urine from different cows and pigs)

Sample preparation

technique: dilution

Analytical technique: TurboFlowTM

HRMS (Exactive)

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Table 5: Results of the method validation

Compound name CCα

(µg/L) CCβ

(µg/L)

Repeatability (%)

(0.5 µg/L)

Within-laboratory

reproducibility (%)

(0.5 µg/L)

Recovery

(%)

brombuterol 0.19 0.46 20 24 97

carbuterol 0.22 0.58 25 27 100

chlorbrombuterol 0.14 0.38 14 26 99

cimaterol 0.13 0.35 15 19 100

cimbuterol 0.08 0.20 9 13 100

clenbuterol 0.14 0.33 13 25 98

clencyclohexerol 0.08 0.21 9 12 100

clenhexerol 0.22 0.49 22 23 98

clenpenterol 0.20 0.49 22 25 99

clenproperol 0.10 0.24 11 15 100

fenoterol 0.11 0.24 11 14 99

hydroxymethylclenbuterol 0.09 0.21 9 14 100

isoxsuprine 0.10 0.25 11 18 100

mabuterol 0.09 0.23 9 17 100

mapenterol 0.10 0.24 10 18 99

metoprolol 0.10 0.30 11 25 100

pirbuterol 0.35 0.70 11 29 89

ractopamin 0.05 0.13 5 13 100

ritodrin 0.05 0.13 6 12 100

salbutamol 0.24 0.60 26 27 103

salmeterol 0.16 0.42 18 21 99

terbutalin 0.20 0.52 22 26 101

tulobuterol 0.10 0.32 11 31 107

zilpaterol 0.08 0.21 9 14 100

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Quick screening of priority beta-agonists in urine using ...

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