Scholarly article on topic 'Comparison of Tandem Quadrupole Mass Spectrometry and Orbitrap High Resolution Mass Spectrometry for Analysis of Pharmaceutical Residues in Biota Samples'

Comparison of Tandem Quadrupole Mass Spectrometry and Orbitrap High Resolution Mass Spectrometry for Analysis of Pharmaceutical Residues in Biota Samples Academic research paper on "Chemical sciences"

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Academic research paper on topic "Comparison of Tandem Quadrupole Mass Spectrometry and Orbitrap High Resolution Mass Spectrometry for Analysis of Pharmaceutical Residues in Biota Samples"

Materials Science and Applied Chemistry

doi: 10.1515/msac-2016-0001


Comparison of Tandem Quadrupole Mass Spectrometry and Orbitrap High Resolution Mass Spectrometry for Analysis of Pharmaceutical Residues in Biota Samples

Ingars Reinholds1, Iveta Pugajeva2, Vadims Bartkevics3

l-3Faculty of Chemistry, University of Latvia, Latvia

Abstract - This research demonstrates the development of reliable multi-component methods for the detection of antibiotic residues in environmental biota. The efficiency of analytical performance of ultra high performance liquid chromatography (UHPLC) techniques with triple quadrupole (QqQ-MS/MS) and Orbitrap high-resolution mass spectrometry (Orbitrap-HRMS) detectors is discussed. Antibiotic residues in biota samples collected in Latvia were analysed. The contamination status was determined as rather low within acceptable levels set by the European Union Regulations.

Keywords - Antibiotic residues, Orbitrap high resolution mass spectrometry, tandem mass spectrometry, ultra-high performance liquid chromatography.

I. Introduction

Human and veterinary drugs have been continually released into the environment mainly as a result of manufacturing processes, improper disposal or metabolic excretion [1]. Antibiotics have received a special attention among pharmaceuticals for their increased application in human therapy, aquaculture and livestock agriculture [2], [3]. The maximum acceptable residue levels (MRLs) for antibiotics in foodstuff are determined in the European Union by the Commission Regulation No. 10/370 [4]. The variety of multi-class compounds distributed in aquatic systems has tendency to increase causing issues of bacterial resistance [5].

Several techniques have been proposed for the analysis of antibiotics that include microbial assays [6], capillary electrophoresis [7], liquid chromatography with fluorescence [8], ultraviolet or diode array [9] and mass spectrometric (MS) [10]—[12] detection. The technique based on the tandem triple quadrupole mass spectrometry (QqQ-MS/MS) is the most used technique for multi-component analysis of contaminants in foodstuff and environmental samples where fish play a major role as biomarkers of biota pollution [13]—[15].

High-resolution mass spectrometry (HRMS) based on Orbitrap technology has been shown lately to be a promising approach for routine screening and confirmation of antibiotics residues in products of animal origin; it has exhibited a good performance in multi-contaminant analysis in environmental samples [16]—[18].

The present study is dedicated to the development of a sensitive Orbitrap high-resolution mass spectrometric (UHPLC-Orbitrap-HRMS) method for assessment of antibiotic residues in environmental biota. Samples of fish were used as indicators of contamination. The detection performance of Orbitrap-HRMS and optimized tandem quadrupole mass spectrometry QqQ-MS/MS technique were evaluated within validation studies of accuracy, repeatability and reproducibility of both methods at 50 % of MRLs (maximum residue limits) set in the European Union.

II. Materials and Methods A. Reagents and Chemicals

HPLC grade acetonitrile and methanol were obtained from Merck-Millipore (Darmstadt, Germany). ACS grade formic acid (> 96.0 %) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was generated by a MilliQ™ system (Billerica, MA, USA). Disposable Ultrafree®-MC centrifugal filters (0.22 ^m) were obtained from Merck Millipore. Phree™ phospholipid removal tubes (1 mL), were purchased from Phenomenex (Torrance, CA, USA).

Standards of 23 compounds from several antibiotic groups such as macrolides (erythromycin, josamycide, lincomycin, spiramycine, tylosin), penicillins (ampicillin, cloxacillin, dicloxacillin, oxacillin), sulfonamides (sulfachloropyridazine, sulfadimethoxine, sulfadimidine, sulfamethizole, sulfathiazole), tetracyclines (doxycycline, oxytetracycline, tetracycline), and quinolones (ciprofloxacin, difloxacin, enrofloxacin, flumequine and nalidixic acid) were purchased from Sigma-Aldrich (St. Louis, MO, USA), but danofloxacin, marbofloxacin and chlortetracycline were obtained from Dr. Ehrenstorfer (Augsburg, Germany).

Individual 1 mgmL-1 stock solutions of each analyte in methanol were prepared and stored at -18 °C. The mix of working standard solutions of all antibiotics in methanol at 10 ^gmL-1 concentration was prepared and stored at -18 °C up to one month. Further diluted working standard solutions were prepared in methanol at concentration ratios according to the MRLs in fish muscle [4]. The working solutions were used for spiking of blank samples and stored at +4 °C for a month.

©2016 Ingars Reinholds, Iveta Pugajeva, Vadims Bartkevics. This is an open access article licensed under the Creative Commons Attribution License (, in the manner agreed with De Gruyter Open.

B. Sample Preparation

Each homogenized sample (2 g) was weighed into a 15 mL polypropylene centrifuge tube. Calibration and quality control samples were fortified with the appropriate volumes of the standard solution corresponding to 50 % of EU MRLs related to muscles of biota samples. Then acetonitrile (3 mL) was added to each sample. The samples were vigorously shaken for 20 min using an automatic shaker (Multi RS-60, BioSan, Latvia). The sample tubes were centrifuged for 15 min at 4500 rpm to separate phases using the Heraeus Multifuge 3L-R centrifuge (Thermo Fisher Scientific, USA) and the supernatant was collected and loaded for purification onto Phree™ phospholipid removal tubes (1 mL) after preconditioning with 0.5 mL of acetonitrile. The obtained extracts (2 mL) were collected into clean sample tubes and the SPE tube was washed with additional 0.3 mL of acetonitrile. The obtained combined acetonitrile extracts were evaporated to dryness under nitrogen stream at 55 °C, using a TurboVap LV concentration evaporator (Biotage, Sweden). The retained residues were eluted at a low flow-rate using 1 mL of 0.1 % formic acid solution in 10/90 % (v/v) methanol/water. The samples were filtered through 0.22 ^m centrifuge filters at 3000 rpm and transferred to autosampler vials for analysis. An aliquot of 10 ^l was injected into the chromatographic system.

C. UHPLC-q-Orbitrap Instrument and Conditions

An Accela 1250 UHPLC (Thermo Fisher Scientific, San Jose, CA, USA) was used for chromatographic separation of antibiotics performed on a 50 x 2.1 mm i.d., 2.6 ^m Kinetex analytical column (Phenomenex, Torrance, CA, USA). The mobile phase consisting of 0.1 % formic acid in water (phase A) and 0.1 % formic acid solution in methanol (phase B) was set as follows: the initial mobile phase of 80 % A and 20 % B was held constant for 0.5 min. From 0.5 min to 5.0 min the percentage of phase B was linearly raised from 20 % to 100 % and held constant until 6.5 min. Then the percentage of phase B was decreased to 20 % by 6.6 min and was kept at this level until 10.0 min. The flow rate of the mobile phase was 0.4 mLmin-1 and the injection volume was equal to 10 ^L. The column and autosampler were maintained at temperatures of 40 °C and 10 °C.

The described UHPLC system was coupled to Q Exactive™ Orbitrap-HRMS (Thermo Scientific™, USA) equipped with a heated electrospray ionisation probe (H-ESI II) operating in the positive detection mode. The following ionization parameters were applied: 3.20 kV electrospray voltage, auxiliary gas heater temperature equal to 450 °C, capillary temperature of the ESI interface equal to 270 °C, sheath gas (N2) and auxiliary (N2) gas flow rates were 50 and 10 arbitrary units (a.u.), respectively, the tube lens voltage was adjusted to 90 V, and the S-lens radio frequency level was at 50 %. The mass spectrometer was operated in the full MS - data dependent MS2 (dd-MS2) mode. This type of scanning comprises a full MS scan (the selected range was from 200 m/z to 1000 m/z and the resolution was 70,000 full width

half maximum (FWHM)) with an isolation window applied, followed by a data dependent scan. External calibration of the Q Exactive mass spectrometer was performed daily over the mass range of 50 to 2000 m/z according to the guidelines provided by the instrument supplier. The principal scheme and mass equation used in the Orbitrap analyser are shown in Fig. 1.

Fig. 1. Scheme of the Orbitrap mass analyser.

The accurate masses for the precursor and transition ions, collision energies and the retention times for the analysed antibiotics are shown in Table I. The control of the instrument performance and data processing was performed by using

Thermo Scientific Xcalibur™ and TraceFinder™ software (USA).

D. UHPLC-QqQ-MS/MS Instrument and Conditions

An Ultimate 3000 UHPLC system coupled to TSQ Quantiva™ triple quadrupole QqQ-MS/MS tandem mass spectrometer (Thermo Scientific, USA) equipped with an electrospray source was used for comparative reasons on analytical performance assessment. The separation was performed on a Phenomenex Kinetex column. The separation was achieved on the same conditions of elution system and the injection volume (10 ^L). The gradient profile used for solvent B was as follows: the initial mobile phase of 90 % A and 10 % B was held constant for 4 min. From 4.0 min to 5.0 min the percentage of phase B was raised from 10 % to 30 % and from 5.0 min to 8.0 min it was raised from 30 % to 95 % and was held constant until 10.5 min. Then the percentage of phase B was decreased back to 10 % by 11.0 min and was kept at this level until 15.0 min. The flow rate of the mobile phase was 0.3 mLmin-1. The column and autosampler were maintained at temperatures of 30 °C and 10 °C.

The analysis of target compounds was performed by multiple reactions monitoring (MRM) in the positive detection mode. ESI-QqQ-MS/MS analysis was performed with the following parameters: vaporiser temperature - 280 °C, source temperature (i.e., ion transfer tube temperature) - 320 °C, ion spray voltage - 5.0 kV, curtain gas nebuliser - 35 psi, aux gas - 15 psi and sweep gas 2 psi, respectively. The control of the instrument conditions and the data processing were performed using the TraceFinder™ software (Thermo Scientific, USA). The information about the abundant molecular ions, fragment ions and their collision energies are summarised in the Table I.


Chromatographic Detection Parameters for All Compounds: Retention times, Data for Orbitrap-HRMS (Accurate Masses for Precursor and Transition Ions, Collision Energy - CE) and for QqQ-MS/MS (Ion Transitions - MRM1, MRM2 and Their Collision Energies: CE1 and CE2)


Antibiotic Acronym Retention time

(min) Precursor ion Transition ion CE MRM1 CE1 MRM2 CE2

(m/z) (m/z) (eV) (m/z) (eV) (m/z) (eV)

Ampicillin AMP 1.13 350.1169 106.0655 25 350/106 20 350/160 15

Chlortetracycline CTC 1.92 479.1216 444.0843 20 479/444 35 479/154 45

Ciprofloxacin CPX 1.18 332.1405 288.1499 40 332/288 22 332/314 15

Cloxacycline CLO 3.68 436.0729 160.0424 15 468/160 25 468/436 20

Danofloxacin DNX 1.41 358.1562 255.0566 70 358/283 24 358/340 20

Dicloxacillin DCX 3.80 470.0339 160.0429 60 470/160 25 470/311 20

Difloxacin DFX 1.62 400.1467 356.1560 30 400/356 23 400/382 23

Doxycycline DOX 2.55 445.1605 428.1339 20 445/321 45 445/428 30

Enrofloxacin ERX 1.36 360.1718 316.1812 25 360/245 24 360/316 24

Erythromycin ERM 3.31 734.4685 158.1175 35 735/158 35 735/577 25

Flumequne FLU 3.20 262.0874 238.0506 70 262/202 10 262/244 20

Josamycine JSM 3.61 828.4740 109.0646 45 828/174 40 861/174 40

Lincomycin LCM 0.58 407.2210 126.1278 35 407/126 25 407/359 16

Marbofloxacin MAR 0.76 363.1463 277.0619 30 363/345 35 363/205 40

Nalidixic acid NX 3.10 233.0921 205.0604 50 233/187 20 233/215 15

Oxacillin OXA 3.57 402.1118 160.0430 15 402/160 20 402/243 30

Oxytetracycline OTC 0.94 461.1555 426.1180 15 461/426 30 461/443 20

Spiramycine SPM 1.39 843.5213 174.1126 45 844/142 30 844/174 45

Sulfachloropyrid 285/156 285/92

SMZ 1.26 285.0208 156.0115 35 16 33

Sulfadimethoxine SDM 2.45 311.0809 156.0764 35 311/156 25 311/92 35

Sulfadimidine SDD 1.00 279.0910 204.0437 35 279/124 23 279/156 20

Sulfamethizole SMZ 0.97 271.0318 156.0112 30 271/156 14 271/92 28

Sulfathiazole STZ 0.59 256.0209 156.0112 35 256/92 30 256/156 15

Tetracycline TTC 0.89 445.1605 410.1232 20 445/427 20 445/410 25

Trimethoprim TMP 0.69 291.1452 123.0667 45 291/110 30 291/123 30

Tylosin TYL 3.37 916.52643 174.1127 35 917/174 45 948/174 45

E. Method Validation

A full validation study was performed according to the EU guidelines for the validation of screening methods [19]. For estimation of accuracy, blank and determined biota (i.e. fish muscle) samples were fortified with a mixture of antibiotics corresponding to 50 % of the MRLs set in the European Union for muscle samples representing animal origin products [4]. Ten replicate test portions were analysed on three different days in case of the elaborated UHPLC-Orbitrap-HRMS method. For estimation of method precision repeatability and reproducibility within the laboratory were calculated. The UHPLC-QqQ-MS/MS method was fully validated using alternative approach with InterVal software for in-house method validation [20]. According to the results of validation, both methods were compared mainly to confirm the reliability of the rather novel Orbitrap-HRMS based detection method developed in the research. The data of validation parameters is summarised in Table II.

III. Results and Discussion A. Performance Studies of Detection

It was necessary to adjust the procedure for sample preparation during the initial stage of our study. The developed method for the analysis of pharmaceutical residues in biota samples included an extraction by acetonitrile at the first stage followed by purification procedure on Phree™ phospholipid removal columns. The overall procedure was rapid, rather cheap and highly effective regarding the removal of complex biological matrix compounds from samples. Thus, the application of rather novel Phree™ columns demonstrated an advantage on sample purification by affecting the reduction of the analytical background level during the detection (e.g., decreasing of matrix influence on the selectivity) in most cases. The chromatograms of determined compounds in biota samples plotted in Fig. 2 confirm good selectivity of target compounds determined in the matrix samples of obtained chromatograms.

100-1 500100-1

RT: 0.91

RT: 1.00


NL: 6.60E6

m/z: 445.1583-445.1628 ESI Full ms Fish SP1


NL: 2.15E6

m/z: 271.0304-271.0331 ESI Full ms Fish SP1

0.0 0.5 1.0

1.5 2.0

t (min)

2.5 3.0

Fig. 2. The chromatograms of spiked antibiotics (tetracycline, and sulfamethiazole) in biota samples.

No disrupting matrix signals or background drift could be observed, thus confirming effectiveness of the selected purification procedure and high selectivity of detection technique.

Further optimisation of method was dedicated to evaluation of elution system. Methanol and acetonitrile were both

assessed for their efficiency and the concentration of additional formic acid was evaluated at the volume content range from 0.1 % to 0.5 %. The results of solvent evaluation indicated that methanol provides a superior separation and sensitivity to the antibiotics compared to that of acetonitrile or a 50/50 (v/v) % mixture of both solvents. Thus, methanol was adjusted as the preferable mobile phase B of gradient profile, where the lowest evaluated concentration at 0.1 % level of formic acid was adjusted for addition to mobile phases to enhance the ionisation efficiency (e.g., minimise the ion suppression).

Two different HPLC columns (Thermo Hypersil Gold C18 and Phenomenex Kinetex C18) were compared and used during the initial elaboration of the method. The Phenomenex Kinetex column indicated a better chromatographic separation of the analytes and gave the best stability with the chosen content of the mobile phase as the column was used for analysis of real samples using the elaborated method based on Orbitrap-HRMS detection technique, which showed advantage on analysis compared to the conventional QqQ-MS/MS method on target assessment.


Validation Parameters of UHPLC-Q-Orbitrap-HRMS and UHPLC-QqQ-MS/MS Methods for Analysis of Antibiotics in Biota Samples.

Antibiotic MRL Fortification UPLC-Q-Orbitrap-HRMS UPLC-QqQ-MS/MS

acronym (Mg-kg-1) level i T -1\ Recovery Repeatability Reproducibility Recovery Repeatability Reproducibility

(ng mL ') (%) (%) (%) (%) (%) (%)

AMP 50 25 95 12 13 102 9 10

CTC 100 50 98 12 13 98 10 11

CPX 100 50 102 12 15 104 6 7

CLO 300 150 85 11 10 95 7 10

DNX 200 100 91 11 14 104 8 10

DCX 200 100 98 9 11 98 5 6

DFX 400 200 102 12 13 116 7 9

DOX 100 50 102 10 15 88 8 12

ERX 100 50 95 10 13 98 5 8

ERM 200 100 100 10 15 102 5 7

FLU 200 100 75 9 12 80 6 8

JSM 200 100 95 8 14 101 7 11

LCM 100 50 78 11 15 85 7 12

MAR 150 75 101 13 13 96 5 9

NX 40 20 104 9 14 100 6 8

OXA 300 150 102 8 13 103 6 15

OTC 100 50 108 11 12 117 6 14

SPM 200 100 95 13 14 100 9 10

SMZ 100 10 101 12 15 98 11 11

SDM 100 10 103 13 13 104 6 8

SDD 100 10 85 11 13 101 5 7

SMZ 100 10 103 9 12 100 5 8

STZ 100 10 98 8 14 103 6 9

TTC 100 50 102 10 13 117 6 16

TMP 50 25 103 11 12 102 5 7

TYL 100 50 98 8 9 102 7 7

As known, the European Commission Decision 2002/657/EC defines the following criteria for confirmation of veterinary drug residues in samples of animal origin: 4 identification points must be obtained, therefore at least two ions are required to be included in the high resolution mass spectrometric analysis. In case of QqQ-MS/MS the multi reaction mechanism of two target ions gives the confirmation of the compound. Thus, the elaborated method using dd-MS2 scanning mode in case of Orbitrap-HRMS resolves this issue by obtaining daughter ion spectra for all compounds included in the method. This quantification mode provides an unambiguous confirmation of the antibiotics in full compliance with the requirements of the EC Decision 2002/657/EC.

B. Validation Studies of Orbitrap-HRMS and QqQ-MS/MS

The developed UHPLC-QqQ-MS/MS method was fully assessed using InterVal software for the in-house method validation. The UHPLC-q-Orbitrap method was validated over three days at level of 50 % of maximum residue limit set in the European Union [4]. The results of validation studies for both methods are summarised in Table II.

The experiments on the spiked samples of biota showed that the average recoveries of the UHPLC-q-Orbitrap method ranged from 85 % to 110 %, which is in a good agreement with requirements of EU guidelines [4]. In case of UHPLC-QqQ-MS/MS the values are in the range from 80 % to 117 %, similarly to those obtained by the UHPLC-q-Orbitrap-HRMS method with exception of difloxacine, tetracycline and oxytetracycline with the mean values of recovery reaching 116 % to 117 %, which are out of the profile, but at a sufficient level.

The precision of elaborated methods was evaluated in relation to inter-day repeatability and intra-day reproducibility over the three-day period. The coefficients of variation of repeatability are in the range from 8.9 % to 16 %. In case of UHPLC-QqQ-MS/MS, it demonstrated better precision (< 15 %) and accuracy; however, this technique has a limitation related to the maximum number of compounds which could be included into the scope of analysis.

Thus, the elaborated UHPLC-Orbitrap-HRMS method was chosen as more advantageous for the target assessment and was applied for the analysis of biota samples.

C. Concentration Levels of Determined Antibiotic Residues

The elaborated Orbitrap-HRMS method was applied to the analysis of 10 fish samples that originated from the Latvian market. The concentration levels of all analysed compounds were assessed in accordance to the validation parameters of elaborated methods. No traces of a major part of target compounds (25 antibiotics) were found in analysed samples. Meanwhile, the analytical signals of 2 compounds (tetracycline and sulfamethiazole) were found at rather low levels in one sample of farmed fish. However, the calculated concentrations of target compounds were significantly below the validated level and, therefore, the presence of these pharmaceutical compounds could not be regarded as confirmed in accordance to strict requirements of the European Union. Taking into account the wide application of tetracycline and sulphanilamide class compounds in veterinary and human therapy and their wide occurrence in biological

samples and environmental objects, further development of reliable analytical techniques has an utmost importance in control of pharmaceutical residues in water ecosystems by using biota samples [21], [22]. While real concentration levels of residues are low, their presence should be taken into account and monitoring research programs should be implemented.

IV. Conclusion

A sensitive, rapid and reliable multi-class method based on UHPLC-q-Orbitrap-HRMS technique was elaborated for the identification and quantification of 26 compounds from different groups of antibiotics (sulfonamides, macrolides, tetracyclines, aminoglycosides, penicillins and quinolones) in biota samples for assessment of pharmaceutical residue contamination levels in water ecosystems.

The adjusted novel sample purification procedure was applied to the biota sample using PhreeTM phospholipid removal columns - that showed an excellent performance in achievement of the method selectivity. Validation experiments on spiked samples showed that the average recoveries of elaborated UHPLC-q-Orbitrap-HRMS method were at acceptable level and ranged from 80 % to 112 %, and coefficients of variation were in the range from 8 % to 15 %. While UHPLC-QqQ-MS/MS demonstrated better precision (< 15 %), this technique has a limitation related to selectivity of detection and the maximum number of compounds, which could be included into the scope of analysis. The developed UHPLC-q-Orbitrap-HRMS methodology was applied to determine antibiotics residues in 10 fish samples from Latvia. It demonstrated very low occurrence of pharmaceutical compounds - levels of all antibiotics were under the validated levels of detection.


The presented work and obtained results in this publication have received funding from Norwegian Financial Mechanism 2009-2014 co-financed project "Establishing of the scientific capacity for the management of pharmaceutical products residues in the environment of Latvia and Norway", Contract No NFI/R/2014/010. 1.


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Ingars Reinholds gained his Dr. chem. degree at the University of Latvia in 2014. Since 2014 he is a senior expert and a principal researcher at the Institute of Food Safety, Animal Health and Environment "BIOR" and also a researcher at the Department of Chemistry, University of Latvia. He specializes in fields of analysis of environmental contaminants, materials science of composite materials and properties of their components. E-mail:, ORCID: 0000-0002-6910-3054.

Iveta Pugajeva is a PhD student of analytical chemistry at the University of Latvia (UL); she gained her Master's degree at UL in the field of analytical chemistry in 2007. Since 2004 she is a senior expert and a principal researcher. Since 2015 she also is the Head of the Division of Liquid Chromatography at the Institute of Food Safety, Animal Health and Environment "BIOR". She is experienced in method elaboration and optimisation of liquid chromatography coupled to Orbitrap-high resolution MS and triple quadrupole MS methods for analysis of contaminants. E-mail:

Vadims Bartkevics was awarded his Doctoral degree in analytical chemistry (Dr. chem.) at University of Latvia (UL) in 2005. He is an Associate Professor at the Faculty of Chemistry, UL since 2013. Since 2005 he is the Head of Food and Environment Investigation Laboratory at the Institute of Food Safety, Animal Health and Environment "BIOR". He has an extensive experience in training of laboratory personnel for modern methods in area of instrumental analysis of food quality and safety.

E-mail:, ORCID: 0000-0002-6193-7409.

Ингарс Реинхолдс, Ивета Пугаева, Вадиме Барткевицс. Сравнение методов тандемной масспектрометрии и Орбитрап-масспектрометрии высокого разрешения для определения остаточных количеств фармацевтических соединений в образцах биоты. В данном исследовании предложен чувствительный и надёжный метод, который основан на применении колонок твердофазной экстракции, отделяющих фосфолипиды, и двух методов детектирования - жидкостной хроматографии, соединённой с тандемной квадрупольной масспектрометрией (ВЭЖХ-МС/МС) и гибридной квадрупольной -Орбитрап-масспектрометрией (ВЭЖХ-Орбитрап-МС). Разработанная процедура применена для определения 26 антибиотиков из различных классов лекарственных соединений. Предложенная методика подготовки образцов селективна для анализа всех включённых в метод антибиотиков. Применение метода ВЭЖХ-Орбитрап-МС для практического определения остаточных количеств антибиотиков в десяти образцах рыбы свидетельствует о низком уровне распространения антибиотиков в Латвии.