FACULTY OF CHEMISTRY UNIVERSITY OF LATVIA
Vadims Bartkevičs
DETERMINATION OF VETERINARY DRUGS RESIDUES
USING MASSPECTROMETRIC METHODS
Abstract of dissertation
Riga, 2004
Introduction
Veterinary drugs are widely used nowadays for enhancement of efficiency of
agricultural production, i.e. for treatment of different animal diseases, for increase of weight
gain of animals etc. Due to application of veterinary drugs in agricultural production, the
residues of medicines could be transferred to the food of animal origins. Drug residues may
cause negative impact to human health. During the last years the attention of society to the
issues of food safety has been increased. Therefore the reliable control of drugs residues in
animal origin products should be established.
One of the most important conditions of efficiency of veterinary drugs residue control is
the application of accurate, precise and fast methods of analysis. In addition to the residue
control the reliable methods of analysis are necessary for the investigations of
pharmacokinetic parameters, for determination of metabolic pathways and determination of
purity of veterinary drugs. During the last years the fast development of masspectrometric
methods of analysis are observed. This tendency is caused by possibilities to increase the
sensitivity of methods, gain additional information about structure of analyte and confirm
presence of analyte in the sample with high level of certainty. Taking into account high
selectivity of masspectrometric methods it is possible to simplify the sample preparation
procedures. One additional advantage of masspectrometric methods is the possibility to apply it
to the wide range of chemical substances. Therefore it is possible to elaborate methods, which
simultaneously allow analysing of number of different compounds.
Necessity for the implementation of veterinary drugs control is determined in European
Council directive 96/23/EEC (29.04.1996.) and regulation of Minister Cabinet No.277
,,Residue control and order for it financing". Food and Veterinary Service performs the
execution of residue control in Latvia.
Taking into account the importance of residue control the different researches are
performed in order to elaborate methods with following features:
• the lowest limits of detection;
• simple sample preparation method;
• possibility to apply method for determination of the wide range of compounds;
• possibility to confirm the presence of veterinary drug residues according to the
official requirements.
Due to the low concentration of veterinary drug residues and necessity to confirm the
presence of medicines the number of researches involving gas chromatography with
masspectrometric detection (GC-MS) and liquid chromatography with tandem
masspectrometric detection (LC-MS-MS) is growing.
The aims of the work
1) Elaboration of method for determination of levamisol residues using gas chromatography
- positive chemical ionisation masspectrometry and liquid chromatography - tandem
masspectrometry.
2) Elaboration of GC-MS method for simultaneous analysis of residues of steroid compounds
in animal origin products. Method should be able to detect stilbenes (diethy 1 stilbestrol,
hexestrol, dienostrol), steroids (17a-methylltestosterone, boldenone, nandrolone, estradiol,
testosterone, epitestosterone, progesterone, megestrole acetate, medroxyprogesterone acetate,
estriol, estrone, cis-androsterone, hydroxiprogesterone) and lactones of resorcylic acid
(zeranol, taleranol) using one common sample preparation procedure.
3) Elaboration of GC-MS method for simultaneous determination of 5 non-steroidal antiinflammatory drugs (phenylbutazone, ketoprofen, naproxen, fenoprofen and diclofenac)
residues in meat.
4) Elaboration of LC-MS-MS
method for simultaneous determination of trimetoprim,
sulphanilamides and nitroimidazoles residues in animal origin products.
5) Application of schemes developed for the analysis of veterinary drugs residues for
investigations of pharmacokinetic parameters of fentanyl.
6) Evaluation of the possibility to apply a gel filtration chromatography for unified sample
preparation for the analysis of levamisole, phenylbutazone, steroid and sulphanilamides.
7) Collection of information concerning presence of veterinary drug residues in animal origin
products and quality of Latvian food products.
Results of the work
1. Method for determination of residues of levamisol
In order to apply maspectrometric methods for qualitative and quantitative analysis and to
choose optimal detection parameters the mass spectra of levamisol was measured in different
ionisation modes and fragmentation pattern was proposed. A mass spectrum of levamizol in
positive chemical ionization (PCI) mode exhibits less fragmentation in comparison with
electron impact ionization (El) mode. Base peak of PCI spectra (M+H)+ is formed by proton
transfer process. Following fragmentation pattern for PCI mode has been proposed:
Fig. 1. Fragmentation of levamisole in PCI mode
The formation of ion 178 Da is influenced by instability of chemical bonds C-N and C-S.
After elimination of CH 2=CH2 group the ion join 2 protons due to the high probability of
proton transfer processes in PCI mode. Under specified conditions splitting of C-S has higher
probability in comparison with cleavage of ring. This condition causes formation of ion 145
Da. The formation of 127 Da ion is caused by cleavage of chemical bond between two rings
similarly to the EI ionisation mode. Following fragmentation of ion 127 Da proceeds with
elimination of CH2=CH2 group and splitting of two C-N bonds.
Fragmentation patterns of levamisol are strongly influenced by ionisation type and
ionisation
energy.
Mostly
fragmentation
takes
place
in
2,3,5,6 tetrahydroimidazo[2,lb]thiazole cycle. The most intensive fragmentation is characteristic to
the spectra obtained in El mode, spectra obtained in PCI and negative chemical ionization
(NCI) modes exhibit strong molecular ion peak.
In order to compare the sensitivity of GC-MS method in different ionisation modes the
mass spectra of levamisol standard solution (1 mg/ml) has been measured in El, PCI and NCI
mode. The intensity of base peak shows that El and PCI modes are more appropriate for
analysis in comparison with NCI mode. The highest sensitivity could be obtained in PCI
mode:
Fig.2. Intensity of base line of mass spectra of levamisole in different ionisation modes
Results could be explained by low energy level needed for the protonation of levamisol and
thus high yield of protonated molecules in process of positive ion chemical ionisation.
Checking of methods parameters.
Linearity of GC-MS detection in El mode was checked by measuring of
chromatograms of levamisol standard solutions with concentrations 1, 2, 5 and 10 g/ml.
Linear calibration curve was obtained (R 2=0,991). Calibration curve for GC-MS analysis in
PCI mode in concentration interval 0,5-5,0 g/ml is described by 2nd order polinomial
equation (R2=0,993). Calibration curve for LC-MS-MS methods was constructed using
standard solutions with concentrations 0,1, 0,5, 1,0, 5,0, 10,0, 20,0, 50,0 ng/ml. Calibration
curve is linear and determination coefficient is R2=0,998.
For the determination of limit of determination of levamisol in standard solution ratio
signal / noise = 3 has been used. Following results are obtained: GC-MS in El mode -140
ng/ml, GC-MS in El mode - 8,9 ng/ml, LC-MS-MS - 0,19 ng/ml.
For the validation of method the matrix (liver) was spiked by levamisol at three
different concentrations (0,5, 1 and 1,5 times of maximum permitted concentration) and
analysed using GC-MS PCI method in three different days. For each concentration level six
parallel analyses have been performed (in total - 54 analysis). Results are calculated using
internal standard of levamisol:
Table 1
Recovery and repeatability of GC-MS method for determination of levamisol
Average recoveries of levamisol (n=18) for concentration level 50 g/kg (93%), 100
g/kg (97%) and 150 g/kg (96%) comply with requirements to the analytical methods. These
criteria require the recovery for concentrations levels > 10 g/kg to be better than 80%.
Elaboration of sample preparation method. Determination of residues of fentanyl.
Two general sample preparation methods (gelfiltration chromatography and solid
phase extraction) have been checked in order to elaborate sample preparation method
applicable for analysis of different veterinary drug residues.
Optimisation of conditions of gelfiltration chromatography was done using mixture of
polichlorobiphenyls (PCB) (interval of MW 200-400 Da). Recoveries of polichlorobiphenyls
using fraction volume 24-49 ml were 90-95%. Application of gelfiltration chromatography for
analysis of levamisoi showed that average recovery is 62% and repeatability - 6,9% (n=5).
Taking into account that gelfiltration chromatography is general method for separation of
substances with low molecular weight from fats and proteins this method could be applied for
analysis of other drug residues (as demonstrated for analysis of sulphanilamides further in this
work).
Possibilities to apply Oasis MCX solid phase extraction columns have been checked.
Absolute recovery of levamisol on Oasis MCX column is 71% and repeatability is 10% (n=3).
The consumption of organic solvents is significantly lower in comparison with liquid-liquid
extraction procedures. Besides the solid phase extraction requires less time necessary for the
sample preparation before analysis of levamisol. Due to the high selectivity of sorbent the
columns effectively eliminates interfering compounds.
Fig. 3. GC-MS chromatogram of liver sample spiked -with levamisol at 10 g/kg after
purification on Oasis MCX column
Solid phase extraction using Oasis MCX columns and LC-MS-MS determination method was
apply also for determination of fentanyl residues. Elaborated method of analysis was used for
determination of pharmacokinetic parameter of fentanyl in chickens. Method is selective,
sensitive (limit of determination is 0,05 ng/ml) and linear in concentration interval 0,2 - 150
ng / ml of plasma (R2 = 0,999).
2. Method for determination of residues of steroid compounds
Derivatization of steroid compounds with heptafluorobutyric acid (HFBA) and analysis
of reaction products by GC-MS in El, NCI and PCI mode have been selected for elaboration of
method of analysis. Results indicated that all compounds could be divided into two groups substances reacting with one or two molecules of HFBA. Estradiol (I) and
estrone (II) could be mentioned as example.
Fig. 4. Derivatization scheme of estriol and estrone
Table 2 shows the difference between two groups performing analysis using
masspectrometric methods
Table 2
Different sensitivities of steroids derivatization products in PCI and NCI mode
It can be inferred from the results that NCI mode is not so sensitive for compounds
containing only one HFB group. In this case the use of PCI or EI modes is advisable. For
compounds containing two HFB groups use of NCI is recommendable because of highest
selectivity and sensitivity of analysis.
During elaboration of analytical method the derivatisation with pentafluoropropionic
acid anhydride (PFPA) was checked as well. Results show that derivatization with PFPA
gives products with lower GC-MS response in comparison with HFBA. Besides HFBA
derivatisation yields products with increased retention times and diagnostic ion masses (50 Da
for one HFB group). As a consequence the better selectivity of chromatographic detection is
achieved.
Method parameters
Linearity of detection system was checked using standard solutions with five different
concentrations. Results indicate good correlation between GC-MS signal and concentration of
steroid compounds (R 2 ranging from 0,993 for diethylstilbestrol to 1,000 for megestrol
acetate).
Nine compounds representing three different groups (stilbene, steroids, lactones of
resorcylic acid) with different chemical properties were selected for the validation of
analytical method. Each compound was added to the matrix at three different levels and
analysed using GC-MS in three different days. Six parallel analyses have been performed for
each concentration level (54 analyses in total). Results are calculated using internal standards of
steroid compounds:
Table 3
Reproducibility and recovery of stilbene determination method
Table 4
Reproducibility and recovery of zeranol and taleranol determination method
Table 5
Reproducibility and recovery of steroid determination method
3. Method for determination of sulphanilamides and nitroimidazoles
After optimization of gel filtration chromatography performed during the elaboration of
levamisol residues determination method the same parameters were applied for sample
preparation method intended for use for analysis of sulphanilamides:
Table 6
Recovery of sulphanilamides after clean up of samples using gel filtration
Results show that average absolute recovery of sulphanilamides during the gel filtration
chromatography is 79%. That indicates that gel filtration chromatography is applicable for
clean-up of biological samples prior to analysis with masspectrometric methods. Therefore
extract of sample obtained after clean-up with gel filtration method could be used for
analysis of both levamisol and sulphanilamides residues.
Parameters of method
In order to perform quantitative analysis of sulphanilamides the linearity of LC-MS-MS
calibration curve has been checked:
Table 7
Linearity of detection of sulphanilamides
It could be inferred from the results that LC-MS-MS detection method has acceptable
linearity with correlation coefficients ranging from 0,987 to 0,999.
Calibration on matrix was performed using standard addition of sulphanilamides at
concentration levels 20, 50, 80 and 100 g/kg. For these calibrations the linear curves are
obtained with following determination coefficients: R 2=0,996 (sulphadimetoxine), R2=0,995
(trimetoprim), R2=0,997 (sulphachloropyridazine), R2=0,994 (sulphadimidine), R2=0,999
(sulphamethizole), R 2=0,996 (sulphathiazole), R 2 = 0,999 (ronidazol) and R2 = 0,994
(metronidazol). Therefore an LC-MS-MS method exhibits good correlation between analytical
signal and content of sulphanilamides or nitroimidazoles in biological samples.
Limits of detection of method
According to the legislation of Latvia the maximum permitted concentration of
sulphanilamides in animal origin products is 100 ug/kg. The limit of detection of LC-MS-MS
method is calculated for the standard solution (c=0,5 ng/l) and spiked sample with
concentration of sulphanilamides 100 ug/kg and concentration of nitroimidazoles 50 g/kg.
Table 8
Detection limits of compounds
Results shows that elaborated LC-MS-MS method for analyses of sulphanilamides,
trimetoprim and nitroimidazole ensures appropriate level of sensitivity required by legislation.
Additionally the accuracy of method was successfully proven in proficiency testing organised
by EU reference laboratory (AFSSA).
4. Method for determination of non-steroidal anti-inflammatory drugs
Mass spectra of non-steroidal anti-inflammatory drugs (NSAID) in different ionisation
modes were obtained during initial steps of elaboration of method. Measurements in positive
ion chemical ionisation mode showed that only insignificant fragmentation of molecules
occurs and mostly adducts ions [M+C2H5]+ and [M+C3H5]+ are observed. More intensive
fragmentation is characteristic to the spectrum acquired in electron impact chemical ionization
mode. Following ions were chosen for analysis in PCI and EI mode of GC-MS:
Table 9
Choice of ions for determination of NSAID compounds using GC-MS method
Derivatization studies were performed in order to elaborate a multi-residue method for
simultaneous determination of phenylbutazone with other compounds from NSAID group.
These acidic compounds should undergo derivatization prior to the gas chromatographic
analysis. Methytation with methanol - sulphuric acid solution and trimethylsilyldiazomethane,
propylation with BF3 / C3H7OH and butylation with BF3 / C4H9OH were chosen for the
experiments. Possible pathways of derivation reactions are shown below:
Fig.5. Derivatization pattern of phenylbutazone using different reagents
The experiments showed that no reaction occurs using the silylating reagent (BSTFA). Gas
chromatographic analysis of methylation reaction mixture reveals the presence of methylated
and unchanged phenylbutazone with chromatographic peak area ratio Me-PBZ / PBZ = 1.7.
The peak area ratio for propyl-PBZ and unchanged PBZ is 0.38 and butyl-PBZ to unchanged
phenylbutazone is 0.27. The results show that derivatization reaction proceeds less efficiently
with increasing mass of alkyl chain. The steric reasons determine absence of analytical use of
propylation and butylation reactions. After comparison of reaction yield the methylation using
trimethylsilyldiazomethane has been applied for elaboration of multi-residue method for
detection of NSAID group compounds.
The property of NSAID molecules to distribute differently between organic and water
phases at different pH levels was used during the elaboration of method. Hexane, ethyl acetate
and dichloromethane were checked for these procedures. Hexane was found to be the most
appropriate solvent for extraction of interfering compounds (mostly fat) from alkalified water
phase and dichloromethane - for extraction of phenylbutazone from acidified water phase.
Series of liquid - liquid extraction using different pH levels ensure effective separation of
analytes from interfering compounds.
Selectivity of NSAID determination method was checked by analysis of different
blank meat and milk samples. Chromatograms of matrixes contained no signals interfering
with peaks of NSAID compounds. Selectivity of phenylbutazone determination method is
shown below (chromatogram of blank sample is overlaid on chromatogram of sample spiked
with phenylbutazone at concentration 50 ug/kg):
Fig. 6. Chromatogram of blank meat sample overlaid on chromatogram of spiked meat sample
(phenylbutazone concentration 50 Hg/kg)
Linearity and sensitivity of method
Comparison of information obtained from measurements of standard solution showed that
GC-MS in El mode is the most sensitive technique (LOD for standard solution of
phenylbutazone is 10 ng/ml). GC-MS in PCI mode exhibits five times lower sensitivity than
in EI mode.
The GC-MS analysis of samples spiked in concentration range 10 - 100 g/kg reveals
good correlation between the peak height and added amount of NSAID compound (R2 = 0,998
(diclofenac), R2 =0,998 (fenoprofen), R2 = 0,999 (naproxen) R2 - 0,989 (ketoprofen) R2 =
0,998 (phenylbutazone)).
The recovery and repeatability of the analytical method were measured by the analysis of
six blank meat samples fortified at each of three concentrations (5, 10 and 25 g/kg) on three
separate occasions.
Table 10
Reproducibity and recovery of GC-MS method for determination of NSAID
Results of validation show that recovery of method complies with requirements to the
analytical methods. These requirements determines that recovery should be >70% for
concentration interval 1 -10 g/kg and >80% for concentration interval above 10 g/kg.
Conclusions
1. Several innovations are proposed for the first time in this work:
• methods for the determination of levamisol residues using gas chromatography masspectrometry and liquid chromatography - tandem masspectrometry were
proposed. These methods exhibits higher sensitivities in comparison with wellknown GC-MS method in EI mode;
• method for determination of steroid compounds allowing detection of stilbenes
(diethylstilbestrol, hexestrol,
dienostrol), steroids
(17-methyltestosterone,
boldenone, nandrolone, estradiol, testosterone, epitestosterone, progesterone,
megestrol acetate, medroxyprogesterone acetate, estriol, estrone, cis-androsterone,
hidroxyprogesterone) and resorcylic acid lactones (zeranol, taleranol) using one
common sample preparation and derivatization step was elaborated;
• method
for
determination
of
non-steroidal
anti-inflammatory
drugs
(phenylbutazone, ketoprofen, naproxen, fenoprofen and diclofenac) in meat using
methylation with trilsilyldiazomethane and gas chromatography - masspectrometry
was elaborated;
• LC-MS-MS
method
for
simultaneous
determination
of
trimetoprim,
sulphanilamides and nitroimidazoles was elaborated;
• schemes elaborated for determination of veterinary drug residues were applied for
investigations of pharmacokinetic parameters of fentanyl;
• applicability of gel filtration chromatography for clean up of samples for the
determination of veterinary drug residues is demonstrated.
2.
In total the new methods for determination of 33 compounds are proposed in this
work. These substances represent different groups of veterinary drugs: antibiotics,
antihelmintic drugs, steroid compounds and non-steroid anti-inflammatory drugs. Quality of
elaborated methods is checked by validation of method checking different parameters
(selectivity, linearity, recovery, repeatability).
3. Elaborated methods have been applied for control of veterinary drug residues in
Latvian animal origin food products. More than 500 analyses assure safety of animal origin
products. Nevertheless the findings of some veterinary drugs (levamisol) and steroids
(progesterone) residues prove the necessity of continuous systematic control applying accurate
and sensitive methods.
The results of this thesis have been presented in following
conferences
1. V.Bartkevičs, G.Kučkovska. Determination of non-steroidal anti-inflammatory drugs in
meat and milk by masspectrometric methods. - Euroresidue V conference,
Noordwijkerhout, The Netherlands, May 10-12, 2004; Book of abstracts, Nr. 1, pp. 291297.
2. V.Bartkevičs. Levamizola atlieku noteikšana pārtikas produktos ar gelfiltrācijas
hromatogrāfiju, GC-MS PCI režīmā un LC-MS-MS. - II Pasaules latviešu zinātnieku
kongress, Rīga, 2001.gada 14.-15.augusts, Tēžu krājums 279 lpp.
Papers
1. V.Bartkevičs, G.Kučkovska, J.Ģībietis. Levamizola atlikumdaudzumu noteikšana
pārtikas produktos ar hromatogrāfijas un masspektrometrijas metodēm. - Latvijas
ķīmijas žurnāls, 2003, Nr.2,159.-170.lpp.
2. V.Barkevičs, J.Ģībietis. Acylation of steroid compounds with fluorinated anhydrides:
reaction pattern, masspectrometric fragmentation and applicability of derivates for
analysis in chemical ionization mode. - RTU zinātniskie raksti, Materiālzinātne un
lietišķā kīmija, 2003, Nr.6, 33.-39.lpp.
3. V.Bartkevičs, J.Ģībietis. Determination of residual phenylbutazone in food products
using gas chromatography - mass spectrometry. - Latvijas ķīmijas žurnāls, 2004, Nr.l,
51.-57.1pp.
4. V.Bartkevičs,
J.Ģībietis.
Nitroimidazolu,
sulfanilamidu
un
trimetoprima
atlikumdaudzumu noteikšana pārtikas produktos ar šķidruma hromatogrāfijas - tandēma
masspektrometrijas metodi. - Latvijas ķīmijas žurnāls, 2004, Nr.3, 263.-269.Ipp. (in
press).
5. N.Berzina, G.Smimova, V.Titajevs, V.Bartkevics, I.Bula. Chickens as an animal model
for the investigation of pharmacokinetics of injection formulation. - Baltic J. Lab. Anim.
Sci, 2003, Nr.l3,pp.l3-18.