Determination of -blockers in animal samples by different extraction methods followed by HPLC-UV and LC-MS analysis.

Organic Chemistry Department Lund University

1

Determination of -blockers in animal samples by different extraction methods followed by HPLC-UV and LC-MS analysis.

Virginia Daz Cerdn

Master Project

D e pa r tm e nt o f Or g a ni c C h e mi s tr y J a n - ke J ns s o n g r o u p S w ed e n

Lund University

2010

Determination of -blockers in animal samples by different extraction methods followed by HPLC-UV and LC-MS analysis.
Virginia Daz Cerdn. Master student at Lund University, Department of Analytical Chemistry, Getingevgen 60, 221 00 Lund, Sweden

ABSTRACT In this work, different analytical extraction methods (Membrane Assisted Solid Extraction MASE, Membrane Assisted Solid Extraction using Molecularly Imprinted Polymers MASE-MIP, Molecularly Imprinted Solid Phase Extraction MISPE) have been compared for the extraction of four -blockers: atenolol, carazolol, metoprolol and propranolol in bovine meat and swine kidney. The procedure involves extraction with ethyl acetate. Extraction parameters such as the type of organic acceptor solvent, extraction time, addition of salt in the aqueous sample and amount of MIP were optimized. All determination was performed applying high-performance liquid chromatography with UV detection at 260 nm. The chromatographic separation was performed in an Agilent Eclipse XDB-C18 column (150 mm x 4.6 mm, 5 m) using a gradient mixture of (A) 10 mM ammonium formate in water (pH 3.9 adjusted with formic acid) and (B) methanol as mobile phase at a flow rate of 0.2 mL/min. LC-MS analysis was used to check the obtained results. The electrospray interface was operated in the positive ion mode. Resulting chromatograms were free of interfering peaks. The results are well below the current maximum residue limit for the carazolol compound. Atenolol is not a good compound to extract with these techniques because it is too hydrophilic (log P=0.10), so in further analysis it was not studied. In general, faster analysis and better extraction of -blockers from animal tissues were achieved with MISPE. It is concluded that LCMS is the more sensitive method than HPLC for this work.

1.

Introduction

Nervous system regulation is done by substances called catecholamines (Fig. 1): norepinephrine and epinephrine (adrenaline). [1]. To exert their action, these chemicals bind to a receptor on the cell surface (adrenergic receptors). When this receptor is stimulated by the arrival of catecholamine; heart rate, blood pressure and cardiac contractility increase.

Beta blocker Atenolol Carazolol Metoprolol Propranolol

Class 1 selective Non-selective 1 selective Non-selective

Table 1. Classification of -blockers.

-Adrenergic receptor blocking agents are used in animals, given by intramuscular injection, to calm them and to prevent death or even the loss of meat quality during transport to the slaughterhouse. Most tranquillizers are rapidly metabolised in the animals body. Any residues are concentrated in the kidney and/or liver. However, if there is a short period of time between treatment and slaughtering, it can lead to considerable residue concentrations in meat, so these organs should be discarded. Eating food containing high levels of these substances can be harmful to consumer health, so the control of -blockers residues is required. Such meat is less marketable and the farmer may be financially penalised. In the EU, maximum residue limits (MRLs) have been elaborated for carazolol (Table 2). [2-3].

Fig.1. Chemical structures of related catecholamines.

-Blockers, also known as -adrenergic blocking agents, are drugs that block these substances from binding to -receptors on nerves and prevent catecholamine stimulation. The final effect is a reduction in heart rate and blood pressure, and contractility (strength) of the heart. -Receptors have been subdivided into (these control the frequency and strength of the heartbeat) and receptors (these control the smooth muscles function) and due to that, there are non-selective (1/) -blockers (Table 1), which block both receptors, and selective -blockers. [1].

Master Project Report. Analytical Department. 2010. Lund University Sweden.

Beta blocker Carazolol

Animal species Bovine

Carazolol

Porcine

MRLs 5 g/kg 25 g/kg 25 g/kg 25 g/kg 5 g/kg

Tissue Muscle+fat Liver Kidney Liver, kidney Muscle, skin+fat

Table 2. MRLs for beta blockers.

In this context, the present work focused on the detection and quantification of four -blockers presenting a wide range of lipophilicity (log P ranging from 0.10 to 3.59), similar molecular weights (300 g/mol) and pKa values (9.2) as Table 3 shows. -blocker Atenolol Carazolol Metoprolol Propranolol Molar mass (g/mol) 266.34 298.34 267.37 259.34 logP 0.100.28 3.590.52 1.320.26 3.100.25 pKa 9.16 9.20 9.17 9.14

Fig. 2. Chemical structures of studied -blockers.

Solvents and reagents Standard stock solutions of each compound were prepared (1000 ppm) into 10 mL volumetric flasks and made up to the mark with the ammonium formate buffer, used as mobile phase A for HPLC; reference solutions at concentrations of 10, 3.33, 1.6, 1, 0.33, and 0.01 ppm were also prepared. The standard stock solutions were stored in the refrigerator. -Receptor molecularly imprinted polymers (LOTE MH05-1189) were kindly supplied by MIP Technologies. Gradient grade methanol and 10 mM ammonium formate buffer in deionized water adjusted to pH 3.9 with formic acid (which was stored in a dark place) were employed. Cyclohexane, toluene, n-hexane, dichloromethane, ethyl acetate, tert-buthylmethylether, sodium chloride, formic acid, methanol, Millipore water, ammonium dihydrogen phosphate, orthophosphoric acid, 0.1 M HCl and acetonitrile were also used.

Table 3. Physico-chemical characteristics of studied -blockers.

The aim of this study was the selective extraction of -blockers in bovine meat samples and the comparison of two different preparation methods (MASE and SPE) and the survey of the selectivity of MIPs in these techniques. For this purpose, three different extraction techniques were developed (MASE, MASEMIP and MISPE) followed by HPLC analysis. Further, LC-MS method was used to check the obtained results.

2.

Materials and apparatus

Samples Minced bovine meat (Organic Natural Pasture Meat) and swine kidney (Scan) were bought from a local supermarket in Lund (ICA Clemenstorget, Lund, Sweden).

-blockers The target analytes (Fig. 2): atenolol, metoprolol, carazolol and propranolol, were obtained from MIP Technologies (Lund, Sweden).

Instruments and laboratory apparatus The instruments used included an HPLC system with autosampler (Agilent 1100), an LC-MS system (see below), microprocessor pH meter pH211 (Hanna Instrument), vortex machine, analytical balance (Sartorius basic), balance SG-5001 (Fisher Scientific), multi-position magnetic stirrer RO 10 power (IKA), SPE cartridges (supplied by MIP Technologies), solid phase extraction unit (VacMaster), test tube shaker-vibrating REAX 2000 (Heidolph), paper filter N 3 (Munktell), 0.45 m membrane filter (Whatman), centrifuge machine, homogenizer machine T25

Master Project Report. Analytical Department. 2010. Lund University Sweden.

digital ULTRA-TURRAX (IKA), rotary evaporator Laborota 4000 WB (Heidolph). The device used for membrane-assisted solvent extraction produced by Gerstel is described in several publications [4-5-6-7] and is shown in Figure 3. The extraction cell consisted of a conventional 20 mL headspace vial with a membrane insert. The material of the membrane bag is dense propylene (4 cm long with a wall thickness of 0.03 mm and an internal diameter of 6 mm). This synthetic polymer is resistant to most organic solvents and stays stable during agitation.

Mobile phase A was 10 mM ammonium formate buffer in deionized water adjusted to pH 3.9 with formic acid. Mobile phase B was gradient grade methanol for HPLC. All the compounds were separated and identified by their retention time: metoprolol (11.3 min), carazolol (12.4 min) and propranolol (13.9 min).

LC-MS technique To check the achieved results in the sample by HPLC analysis, a LC system composed by an L-6000 (Merck Hitachi) pump, a vacuum degasser, an autosampler Triathlon, an ODS-2 hypersil (5m, 100 x 2.1mm (Thermo, Kungens kurva, Sweden) column, a C18 precolumn (Phenomenex, Torrance, USA)) and a single quadrupole mass spectrometer (Waters Micromass ZMD Benchtop, Yvelines Cedez, France) was used. All the compounds were separated, enabling each molecule to be identified by their retention time.

Fig. 3. Experimental arrangement.

3.

Experimental

Selective ion monitoring (SIM) was used to detect ions with m/z of 260, 268 and 299, which correspond to the pseudo-molecular ions of the studied molecules: Analyte Metoprolol Carazolol Propranolol Rt (min) 2.5 3.5 8.4 Molar mass (g/mol) 267 298 259 Ion selected for quantification (SIM) 268 299 260

High-performance liquid chromatography Chromatographic separation was performed on an Agilent Eclipse XDB-C18 column (5 m particle size; 150 mm x 4.6 mm inner diameter; 90% acetonitrile, 10% water). The separation was performed at room temperature with a flow rate of 0.2 mL/min. The injection volume was 100 L and a linear gradient was used: the methanol content was increased linearly from 20 to 40% (v/v) in 6 min, then up to 100% in 9 min, and decreased to 17% in 3 min. It was adjusted back to 20% (v/v) in 0.5 min and held for 1.5 min (Fig. 4). UV detection was performed at 260 nm.

Membrane-assisted extraction (MASE) MASE is an alternative to LLE: It is based on small-scale LLE with a low density polyethylene (LDPE) membrane separating the aqueous sample from the organic solvent. MASE can be performed fully automated. Extracts can be directly injected into the HPLC system. The membranes have to be pre-conditioned in order to exclude interfering compounds from the membrane material. For this, the membranes together overnight, with the tubular metal jackets, are cleaned in an ultrasonic bath with 40 mL of the solvent, which is used for extraction. The efficiency of cleaning may be enhanced by one exchange of the solvent, so after a few hours, the solvent was replaced with a fresh one. The extraction cell caps were preconditionated too by the same way. Once this preconditioning, the vial of the extraction cell was filled with 15 mL of aqueous sample. The membrane bag was attached

100

Methanol Buffer

80

60

Content (%)

40

20

0 0 5 10 15 20

Time (min)

Fig. 4. Elution gradient used.

Master Project Report. Analytical Department. 2010. Lund University Sweden.

to the metal funnel and fixed with a PTFE ring, and the funnel was suspended in the opening of the vial. Subsequently, the membrane bag was filled with 1000 L of organic acceptor solution. Vials were closed with metallic crimp caps. The extraction step took place (shaking of the vials in an agitator). At least three parallel extractions were performed simultaneously. After the preset extraction time, the organic extract was automatically withdrawn and it was transferred to a 2 mL vial. The bag was rinsed with another fresh organic solvent and mixed with the original in the 2 mL vial. The 2 mL combined organic solvent was reduced to almost dryness with a gentle stream of nitrogen. Later, 0.5 mL of ammonium formate buffer was added and the dissolved sample was injected into the HPLC (100 L). To clean the membrane bags, the outside was rinsed with deionized water to remove all the remaining salts. Then the membrane bags still attached to the metal funnels and the PTFE rings were soaked into about 40 mL of acetonitrile. They were dried and soaked in about 40 mL of cyclohexane over night (refreshing the organic solvent at least one time).

The application of these synthetic polymers as sorbents allows not only pre-concentration and cleaning of the sample but also selective extraction of the target analyte, which is important, particularly when the sample is complex and impurities can interfere with quantification.

Extraction procedure: MIP particles were placed inside of the membrane bag, followed by 1000 L of toluene. The membrane bag was compressed in order to mix the organic solvent with the MIP particles. The membrane bag should be wiped with a tissue (to avoid any residual organic solvent on the outside of the bag) before putting it into the vial with 15 mL of saturated aqueous sample with sodium chloride and a magnetic stirrer. The extraction cell was closed with the cell caps and with Parafilm, then it was placed onto the multi-position magnetic stirrer and extraction proceeded for 180 min. At least three parallel extractions were performed simultaneously. After extraction, the organic acceptor inside the membrane bag was transferred into an empty cartridge (provided with a filter at the bottom), which is placed onto the solid phase extraction unit. Each membrane bag was rinsed again with 1000 L of toluene, for quantitative transfer of the MIP particles to the cartridge. Toluene was separated from MIP particles by opening the SPE valve and allowing it to flow out by gravity at about 0.5 mL/min. Later, the membrane bag was rinsed with 1000 L of dichloromethane which was also passed through the cartridge with the MIP particles. A full vacuum was applied during 2 min. Thereafter, the trapped analytes were eluted with three portions of 1000 L (3 X 1000 L) of methanol, which were allowed to pass one after another completely though the cartridge. The first two fractions were used firstly to rinse the membrane bag as well for any remaining of MIP particles. The extracts were reduced with nitrogen blowing to almost dryness. Afterwards, 0.5 mL of ammonium formate buffer was added and the dissolved sample was injected into the HPLC (100 L). The same procedure to clean the membrane bags as with MASE technique was followed in this one.

Membrane-assisted extraction using molecularly imprinted polymers (MASE-MIP technique) Molecularly imprinted polymers (MIPs) are synthetic materials having a predefined selectivity for the compound for which they were designed. Selectivity is introduced during MIP synthesis (Fig. 5) in which a template molecule, designed to mimic the analyte, guides the formation of specific cavities or imprints that are sterically and chemically complementary to the target analyte(s). [8-9-10].

Solid phase extraction using molecularly imprinted polymers (MISPE technique) To overcome some of the disadvantages of liquid-liquid extraction (e.g., its tediousness), solid phase extraction (SPE) was developed. This technique saves time, it is rapid, and can provide more reproducible results. [11].

Fig. 5. Schematic pictures of the imprinting process.

Master Project Report. Analytical Department. 2010. Lund University Sweden.

However, conventional SPE (Fig.6) has well-known limitations because of the lack of selectivity. Specific SPE sorbents based on molecularly imprinted polymers can avoid this problem by providing selective extraction. The cartridge with a filter at the bottom was filled with MIP particles. Another filter was placed on top of that to hold it place with help of a glass rod. MIP particles were conditioned with 1000 L of methanol followed by 1000 L of millipore water and 1000 L of 25 mM ammonium phosphate (NH4H2PO4) buffer at pH 3, adjusted with ortho-phosphoric acid. NOTE: Conditioning was done without allowing the cartridges to dry. Then, 18 mL of aqueous sample (1 ppm) were passed at about 0.5 mL/min. The washing step was performed passing 1000 L of 0.1 M HCl followed by 1000 L of millipore water. NOTE: Gentle vacuum was applied between each step followed by full vacuum during 20 min to remove any residual moisture from the cartridge. 1500 L of dichloromethane were then applied for a second washing step. And after that, a full vacuum was applied for 2 min to remove any residual solvent. The elution step was performed with 3X1000 L of methanol at about 0.2 mL/min, applying a gentle vacuum between each fraction and full vacuum at the end. The extract was reduced by nitrogen blowing to almost dryness. Afterwards, 0.5 mL of ammonium formate buffer was added and it was injected into the HPLC (100 L). [12]

Sample pre-treatment 5 g minced beef meat were homogenized with 20 mL of ethyl acetate and vortexed for 2 min. Then it was centrifuged for 6 min at 2000 rpm. The supernatant was evaporated to dryness and the residue was reconstituted in 10 mL of Millipore water. Thereafter, the aqueous sample was filtered twice (first once with a Munktell filter paper N 3 and followed by a 0.45 m Whatman membrane filter). 5 mL in total were used in all the cleaning process and due to that the final aqueous sample was 15 mL. pH of the extracts was about 4.5, and it was used as the aqueous sample and extracted by the above techniques.

For the swine kidney sample, the pre-treatment was the same. However, for these, pH of the extracts was about 3.8.

4.

Results and discussion

External calibration curve Parameter Slope Intercept R Range (mg/L) Metoprolol 18 -1 0.99973 0.01-10 -blocker Carazolol 230 1 0.99999 0.01-1

Propranolol 63 -6 0.9981 0.01-10

Table 4. Summary of external calibration curve used by HPLC.

Optimized parameters (MASE) Various parameters were optimized such as the type of organic acceptor solution, the extraction time, and the addition of salt in the aqueous sample.

pH value: The aqueous sample should be between 9 and 13.8 to ensure the neutrality of the analytes (since the extraction is possible only if the molecule is uncharged, which depends on the pKa values of the blockers ).

Due to that, all further experiments were carried out with pH12.4.
NOTE: I read them out from a pH electrode. Due to these values are above 11, these are wrong values because of the alkali error. However, I still used the values that I could read.

Fig. 6. Step by step in MISPE.

Master Project Report. Analytical Department. 2010. Lund University Sweden.

Organic acceptor solution (Figs.7-8): A main challenge in MASE was to find a solvent, which is suitable for extraction of the analytes and compatible with the HPLC system as well. In this context a large variety of organic solvents were tested: toluene, cyclohexane, nhexane, dichloromethane: ethyl acetate (1:1), dichloromethane (DCM), and tert-buthylmethylether.

750 rpm and room temperature since it provides good extraction yields with MASE for all our compounds (an increase in the extraction time resulted in higher enrichment).

3,0

2,5

Metoprolol Carazolol Propranolol

Concentration (ppm)

2,0

20

1,5

Concentration (mg/L)

15

Metoprolol Carazolol Propranolol

1,0

0,5
10

0,0 30min
5

105min

150min

180min

Extraction time (min)

0 Ciclohexane DCM DCM:ethyl ac MTBE n-Hexane Toluene

Organic acceptor solution

Fig. 9. Optimization of extraction time (n=3, 0.25ppm each compound, extraction: 15 mL of aqueous sample, pH 12.5. Extraction at room temperature, extraction solvent: 1000 L of toluene, 100 L injection volume).

Fig.7. Optimization of organic acceptor solution in MASE (n=3, 1 ppm each compound, extraction: 15 mL of aqueous sample, pH 12.5, 120 min of extraction time, at room temperature, extraction solvent: 1000 L, 100 L injection volume).
Ciclohexane Toluene nHexane DCM:ethyl acetate (1:1) DCM MTBE

The reason why the time required is too extended could be because we tried to extract analytes with a high molecular weight ( 300 g/mol).

20

15

Concentration (mg/L)

10

Extraction overnight was checked as well, but higher recoveries were not achieved.

0 metoprolol carazolol propranolol

-blocker

Fig. 8. Optimization of organic acceptor solution in MASE (n=3, 1 ppm each compound, extraction: 15 mL of aqueous sample, pH 12.5, 120 min of extraction time, at room temperature, extraction solvent: 1000 L, 100 L injection volume).

Salt addition (Fig. 10): The influence of NaCl addition to the aqueous samples on the extraction of our analytes was also studied. To investigate the impact of salt, 4.5 g of sodium chloride were added to 15 mL of the aqueous sample (saturated solution, 300 g/L).

Toluene was the best organic acceptor solution for the proposed MASE extraction because the best extraction yield of -blocker was achieved using this solvent.

The saturation of the aqueous samples with salt increased the recovery of all our compounds. When salt is added to the aqueous sample, the water molecules solvate the electrolyte ions and therefore the water solubility of polar compounds is decreased (salting out effect).

Extraction time (Fig. 9): The extraction-time profiles of the analytes were studied from 30 min to 180 min. We decided to select for all subsequent experiments 3 h of extraction at

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Master Project Report. Analytical Department. 2010. Lund University Sweden.

The best extraction yield was achieved with 50 mg of MIP.

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 Metoprolol Carazolol

deionised water salt addition

Optimized parameters (MISPE) Amount of MIP (Fig.12): A range between 25 to 75 mg was tested.
Metoprolol Carazolol Propranolol

Extraction efficiency (%)

18 16
Propranolol

-blockers

Concentration (ppm)

14 12 10 8 6 4 2 0

Fig. 10. Impact of NaCl on the extraction efficiency of MASE (n=3, 0.25 ppm each compound, extraction: 15 mL of aqueous sample, pH 12.5, 180 min. Extraction time at room temperature, extraction solvent: 1000 L of toluene, 100 L injection volume).

Therefore, all further experiments were carried out with saltsatured solutions. Extraction efficiency (E) was calculated as:

20

30

40

50

60

70

80

MIP (mg)

Fig. 12. Varying the amount of MIP in the cartridges.

100 = 15 0.5

The best extraction yield for MASE-MIP was achieved with 50 mg of MIP.

Optimization of the liquid chromatographic mass spectrometric parameters The mass spectrometer settings were optimized with direct injection of standard solution. The electrospray interface was operated in the positive ion mode + ([M+H] ions) with a capillary voltage of 3.25 kV and a cone voltage of 30 V. Source temperature was 150 C, desolvation temperature 350 C. Desolvation gas flow (N2) was set to 540 L/h, extractor: 4 V, resolution: 12 (to produce mass resolution). Chromatographic separation of the analytes was achieved which an isocratic elution of 63% ammonium formate buffer (adjusted a pH 3.9 with formic acid) and 37% methanol at a flow rate of 0.3 mL/min. The injection volume was 5 L.

Optimized parameters (MASE-MIP) Amount of MIP (Fig. 11): A range between 25 to 100 mg was tested.

4,4 4,2 4,0 3,8 3,6

Metoprolol Carazolol Propranolol

Concentration (ppm)

3,4 3,2 3,0 2,8 2,6 2,4 2,2 2,0 1,8 1,6 20 30 40 50 60 70 80 90 100 110

Fig. 11. Varying the amount of MIP in the acceptor phase (n=3, 0.25 ppm each compound, extraction: 15 mL of aqueous sample, pH 12.5, 180 min. Extraction time at room temperature, extraction solvent: 1000 L of toluene, 100 L injection volume).

MIP (mg)

Analysis of real samples with all these techniques. After the pre-treatment of the sample (beef meat or swine kidney), the filtrate was used as aqueous sample for the different techniques.

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Master Project Report. Analytical Department. 2010. Lund University Sweden.

Samples for extraction with MASE and MASE-MIP were adjusted to pH12.4 (NOTE: alkali error) and were saturated with sodium chloride (4.5 g of NaCl), while those for MISPE were extracted without any further processing.

LC-MS analysis Parameter Slope Intercept R Range (mg/L) Metoprolol 4E7 -1E5 0.998992 0.01-0.2 -blocker Carazolol 4E7 -2E5 0.998952 0.01-0.2

Propranolol 3E7 -4E4 0.999189 0.01-0.2

Minced beef sample:

Table 6. Summary of external calibration curve used.

HPLC analysis The obtained chromatogram (Fig. 13) showed that -blockers seemed to be extracted from meat in small concentrations (about 20.9 g/kg metoprolol, 3.4 g/kg carazolol and 15.9 g/kg propranolol). (Table 5).

Fig 14. Chromatogram in full scan mode: MASE extract of minced beef sample.

Fig 13. Chromatogram of MASE extract of minced beef sample followed by HPLC.

Technique

-blocker Metoprolol Carazolol Propranolol

MASE

Concentration (mg/L) 0.2 0.03 0.2

Concentration (g/kg) 20.9 3.4 15.9

Table 5. Extracted results.

Because the peaks seemed to be of similar height as noise, all these achieved results were checked by LC-MS,
Fig 15. Chromatogram in SIM mode: metoprolol (268ES+), carazolol (299ES+), propranolol (260ES+). MASE extract of minced beef meat sample.

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Master Project Report. Analytical Department. 2010. Lund University Sweden.

Technique

Beta-blocker Metoprolol Carazolol Propranolol

MASE

Concentration (mg/L) 0.007 0.007 0.004

Concentration (g/kg) 0.7 0.7 0.4

Table 7. Extracted results.

As Figs. 14-15 and Table 7 show, the signals were quite low although they were not signals for LOD (noise), those were the target analytes. However, due to the fact that the sample was organic meat, in which it is forbidden to use any chemical, it should not contain -blockers. As a conclusion, this might be a result of dirtiness in the material used (membrane bags, vials).

Swine kidney sample:

Fig 17. Chromatrogram of MASE-MIP extract of swine kidney sample followed by HPLC.

HPLC analysis

Fig 16. Chromatogram of MASE extract of swine kidney sample followed by HPLC.

Fig 18. Chromatrogram of MISPE extract of swine kidney sample followed by HPLC.

The obtained chromatograms (Figs. 16-18) showed that blockers seemed to be extracted better with MISPE technique than with the two others techniques. (Table 8).

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Master Project Report. Analytical Department. 2010. Lund University Sweden.

Technique

-blocker Metoprolol Carazolol Propranolol Metoprolol Carazolol Propranolol Metoprolol Carazolol Propranolol

MASE MASEMIP MISPE

Concentration (mg/L) 0,1 0,2 0,4 0,3 0,1 0,8 0,2 0,3

Concentration (g/kg) 7,4 0,1 16,3 39,2 2,7 13,9 85,3 20,5 31,1

For all the samples, the levels of carazolol determined by the three extraction procedures do not exceed the maximum limit (MRL) stipulated by EU for pig kidney samples, which is 25 g/kg.

LC-MS analysis The pump was changed, and due to that another standard calibration was done. Parameter Slope Intercept R Range (mg/L) Metoprolol 4E7 -2E5 0,998892 0.01-0.2 -blocker Carazolol 2E7 -3E5 0,999633 0.0825-0.8

Table 8. Extraction results by MASE, MASE-MIP and MISPE techniques and analysis by HPLC.

90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 Metoprolol Carazolol Propranolol

MASE MASE-MIP MISPE

Propranolol 2E7 -2E6 0,996459 0.0825-3.33

Table 9. Summary of external calibration curve used.

Concentration of -blocker (ug/kg)

Beta-blocker

Fig. 19. Extracted concentration of swine kidney extracts (MASE, MASEMIP and MISPE).

Fig. 21. Chromatogram in total ion current mode. MASE extract swine kidney sample.

Of the total compounds studied, three of them were found in the sample, in the range of 0.1 - 16.3 g/kg (RSD 7.4 %) by MASE, 2.7 - 39.2 g/kg (RSD 20.9 %) by MASE-MIP and 20.5 85.3 g/kg (RSD 12.5%) by MISPE (Fig.19).

MASE MASE-MIP MISPE

20

15

% RSD

10

0 Metoprolol Carazolol Propranolol

Beta-blocker

Fig. 20. Comparison of % RSD values (n=3) in the extraction of swine kidney extracts (MASE, MASE-MIP and MISPE). Fig. 22. Chromatogram in SIM mode: metoprolol (268ES+), carazolol (299ES+), propranolol (260ES+). MASE extract of swine kidney sample.

As the graphs in show, the % RSD obtained by MASE is lower than that achieved by the others techniques.

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Master Project Report. Analytical Department. 2010. Lund University Sweden.

Fig. 23. Chromatogram in total ion current mode MASE-MIP extract swine kidney sample.

Fig. 26. Chromatogram in SIM mode: metoprolol (268ES+), carazolol (299ES+), propranolol (260ES+). MISPE extract of swine kidney sample.

The obtained chromatograms (Figs.21-26) showed that blockers seemed to be extracted better with MISPE technique than with the others two techniques (Table 10).

Technique
Fig. 24. Chromatogram in SIM mode: metoprolol (268ES+), carazolol (299ES+), propranolol (260ES+). MASE-MIP extract of swine kidney sample.

Beta-blocker Metoprolol Carazolol Propranolol Metoprolol Carazolol Propranolol Metoprolol Carazolol Propranolol

MASE MASEMIP MISPE

Concentration (mg/L) 0.004 0.01 0.09 0.02 0.03 0.1 0.01 0.05 0.1

Concentration (g/kg) 0.4 1.4 9.1 1.9 2.7 11.6 1.1 5.2 11.9

Table 10. Extraction results by MASE, MASE-MIP and MISPE technique and analysis by LC-MS.

Fig. 25. Chromatogram in total ion current mode. MISPE extract swine kidney sample.

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Master Project Report. Analytical Department. 2010. Lund University Sweden.

used only once and SPE consists of more manual steps (sample loading, washing, drying, elution of analytes and so on).
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Concentration of -blocker (ug/kg)

10

MASE MASE-MIP MISPE

The advantages of molecularly imprinted polymers (MIPs) as selective sorbents have been demonstrated for the determination of -blockers in animal tissues. Solvent evaporation seems to be a critical step in all these methods, as analyte losses may occur during the evaporation process. This problem should be solved by increasing the sample volume.

0 Metoprolol Carazolol Propranolol

Beta-blocker

Fig. 27. Extracted concentration of swine kidney extracts (MASE, MASE-MIP and MISPE).

In general, MISPE technique provided better recoveries, being faster and simpler. Furthermore, MISPE extraction is a powerful method for the clean-up and the direct selective extraction of trace levels of various compounds from complex matrices. The results of -blockers by both techniques were compared, indicating that extraction of -blockers from animal tissues by MASE technique is less effective than MASE-MIP, and this is less than MISPE technique.

The results (Fig. 27) show a level below the MRL, as HPLC showed as well.

However, when HPLC and LC-MS results for the same samples are compared, the variation of the analyte concentrations obtained is quite different. LC-MS is, in general, more sensitive than HPLC. The more trustable results are the LC-MS values since the machine was calibrated the same day as the analysis day, whereas the HPLC instrument was used the last days by another user so it only could be used during a short period of time. Due to that, the samples were measured but the machine was not calibrated again.

As a conclusion, this work shows that MISPE can be conveniently combined with HPLC and LC-MS.

6.

Acknowledgements

MIP Technologies is gratefully acknowledged for providing MIP cartridges and MIP particles, as well as the -blockers studied in this work. 5. Conclusions

The analytical procedures described provide a simple comparison of the analysis of three -blockers by different techniques: MASE, MASE-MIP and MISPE followed by HPLC and LC-MS, and they were successfully applied for the determination of -blockers in animal tissues. SPE is a commonly used and very well known method, whereas MASE is relatively new. Moreover, the MASE extraction procedure is relatively simple, and due to the re-use of the membranes MASE is a low cost extraction method. Although, the extraction ability of MASE techniques towards more polar compounds (logP < 1.3) will probably be limited due to the nonpolar polypropylene material. MASE with other (more polar) materials is not yet commercially available. On the other hand, SPE cartridges are purchased by many different suppliers. Nevertheless, each SPE cartridge is usually

7. 1. 2. 3. 4.

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