550 Turnipseed et al.: Journal of AOAC International Vol. 98, No. 3, 2015 SPECIAL GUEST EDITOR SECTION
Review: Application of High Resolution Mass Spectrometry to Monitor Veterinary Drug Residues in Aquacultured Products Sherri B. Turnipseed and Jack J. Lohne
U.S. Food and Drug Administration, Animal Drugs Research Center, Denver Federal Center, PO Box 25087, Denver, CO 80225
Joe O. Boison
Centre for Veterinary Drug Residues (CVDR) Laboratory, Canadian Food Inspection Agency, 116 Veterinary Rd, Saskatoon SK, S7N 2R3, Canada
High resolution MS (HRMS) instruments provide accurate mass measurements. With HRMS, virtually an unlimited number of compounds can be analyzed simultaneously because full-scan data are collected, rather than preselected ion transitions corresponding to specific compounds. This enables the development of methods that can monitor for a wide scope of residues and contaminants in aquacultured fish and shellfish including antibiotics, metabolites, and emerging contaminants. Applications of HRMS to the analysis of veterinary drug residues in aquacultured products are summarized in this review including methods for screening, quantifying, and identifying drug residues in these matrixes. The use of targeted, semi-targeted, and nontargeted analysis of HRMS data and the implications to the global aquaculture industry are also reviewed.
A
quaculture is a growing, global industry. It is projected that by 2030 approximately 100 million tons or over 60% of the fish destined for human consumption will be supplied by aquaculture. Much of the farmed fish and shellfish will originate from China, Southeast Asia, and Latin America (1, 2). Veterinary drugs (e.g., antibiotics, parasiticides, and antifungal agents) are commonly administered to fish in an aquacultured environment to treat disease or proactively prevent infection. The degree to which different countries regulate and report the use of chemotherapeutics in aquaculture varies (2–4). Several reports have been published documenting chemical residues found in aquacultured fish and shellfish (4, 5). A primary concern with the use of antibiotics in aquaculture, as with the animal food industry in general, is the potential for increased antibiotic resistance (6). Several examples of antibiotic resistance have been correlated to the use of chemotherapeutics in aquaculture (7, 8). Other chemicals, such as chloramphenicol or the triphenylmethane dyes used as antifungal agents, also have potential adverse human health effects. Therefore, it is important to have effective analytical methods to monitor for residues of these drugs in aquacultured products. Analytical methodology to monitor for residues and chemical Guest edited as a special report on “Methods of Analysis for Residues and Chemical Contaminants in Aquaculture” by Joe Boison and Sherri Turnipseed. Corresponding author’s e-mail: [email protected] DOI: 10.5740/jaoacint.14-265
contaminants in aquacultured species has changed significantly in recent years. Originally methods were developed specifically for one residue, or for several analytes from a specific class of drugs (e.g., sulfonamides or tetracyclines), in a single species of fish or shellfish. A thorough review of these types of class-specific methods in fish was published in 2009 (9). With the increased use of MS, there has been a trend towards multiclass, multiresidue methods to monitor for dozens of analytes in a single method (10). These procedures utilize LC tandem MS (LC-MS/MS with either ion trap (11, 12) or triple quadrupole (13, 14) mass analyzers. This approach greatly increases the monitoring capacity by analyzing for many residues in a single sample. A disadvantage of these methods, however, is that they are still limited to preselected target analytes because the data acquisition program consists of specific MSn product ion scans or selected reaction monitoring (SRM) transitions. If other analytes of interest are present in the sample, they will not be detected. High resolution (HR) MS (HRMS) instruments provide accurate mass measurements, defined as the ability to determine the mass of a compound within a few parts-per-million (ppm) of its theoretical mass. With these instruments, virtually an unlimited number of compounds can be simultaneously analyzed because full-scan data, rather than preselected ion transitions corresponding to specific compounds, are collected. In addition, exact mass measurement combined with the ability to generate product ions facilitates the detection and identification of unknown compounds. This can lead to the development of methods that can monitor for a wide scope of residues and contaminants including those that are not on any target list, allowing agencies to be more proactive in discovering possible adulteration of the food supply including aquacultured products. HRMS Instrumentation Mass resolution is the ability to discriminate between ions with very similar m/z values. For modern nonsector instruments this is defined as R = m/Δm where Δm is the full width of an ion peak at half maximum (FWHM). Using this definition, the resolution will automatically be dependent on the mass of the ion used to calculate the value. Quadrupole mass analyzers typically have a mass resolution of 3000–5000 at m/z 200–500. HR instruments have a mass resolution of 10 000 to 100 000 or higher at m/z 200. The higher the mass resolution, the better the instrument is able to fully differentiate an analyte from isobaric interferences. Without sufficient mass resolution, coeluting
Turnipseed et al.: Journal of AOAC International Vol. 98, No. 3, 2015 551 peaks with the same nominal mass (from the matrix or other analytes) can cause inaccurate mass measurements by shifting the centroid mass. Mass accuracy, or exact mass measurement error, is the difference between the measured and theoretical exact mass. Because this difference is very small with HRMS instruments, it is often expressed in milliDalton (mDa) or as a ppm difference, where ppm = [(measured mass – theoretical mass)/theoretical mass] * 106. Overviews of mass analyzers used in veterinary drug analyses, including HRMS instruments, have recently been published (15, 16). The simplest HR mass analyzer is the time-of-flight (ToF) instrument. With a ToF mass analyzer, ions that have the same kinetic energy but different m/z values are separated in a field-free flight tube and reach the detector at different times. The smallest ions are fastest and will be detected first, while the larger, slower, ions will reach the detector at a later time. The flight path (L) is fixed and the potential (V) at which ions are accelerated is kept constant. Therefore, the time it takes an ion to reach the detector (t) is proportional to its m/z value according to the following equation: m/z = 2eVt2/L2. Most ToF instruments also employ a reflectron, or electrostatic mirror, reversing the direction of the ions in the middle of the flight tube. This increases the mass resolution because the electrostatic reflectron corrects for small differences in initial (prepulse) velocities of ions with the same m/z. It also effectively doubles the length of the flight tube without requiring additional laboratory space. ToF mass spectrometers offer medium to high resolution (up to 40 000 FWHM), accurate mass measurement (
Review: Application of High Resolution Mass Spectrometry to Monitor Veterinary Drug Residues in Aquacultured Products Sherri B. Turnipseed and Jack J. Lohne
U.S. Food and Drug Administration, Animal Drugs Research Center, Denver Federal Center, PO Box 25087, Denver, CO 80225
Joe O. Boison
Centre for Veterinary Drug Residues (CVDR) Laboratory, Canadian Food Inspection Agency, 116 Veterinary Rd, Saskatoon SK, S7N 2R3, Canada
High resolution MS (HRMS) instruments provide accurate mass measurements. With HRMS, virtually an unlimited number of compounds can be analyzed simultaneously because full-scan data are collected, rather than preselected ion transitions corresponding to specific compounds. This enables the development of methods that can monitor for a wide scope of residues and contaminants in aquacultured fish and shellfish including antibiotics, metabolites, and emerging contaminants. Applications of HRMS to the analysis of veterinary drug residues in aquacultured products are summarized in this review including methods for screening, quantifying, and identifying drug residues in these matrixes. The use of targeted, semi-targeted, and nontargeted analysis of HRMS data and the implications to the global aquaculture industry are also reviewed.
A
quaculture is a growing, global industry. It is projected that by 2030 approximately 100 million tons or over 60% of the fish destined for human consumption will be supplied by aquaculture. Much of the farmed fish and shellfish will originate from China, Southeast Asia, and Latin America (1, 2). Veterinary drugs (e.g., antibiotics, parasiticides, and antifungal agents) are commonly administered to fish in an aquacultured environment to treat disease or proactively prevent infection. The degree to which different countries regulate and report the use of chemotherapeutics in aquaculture varies (2–4). Several reports have been published documenting chemical residues found in aquacultured fish and shellfish (4, 5). A primary concern with the use of antibiotics in aquaculture, as with the animal food industry in general, is the potential for increased antibiotic resistance (6). Several examples of antibiotic resistance have been correlated to the use of chemotherapeutics in aquaculture (7, 8). Other chemicals, such as chloramphenicol or the triphenylmethane dyes used as antifungal agents, also have potential adverse human health effects. Therefore, it is important to have effective analytical methods to monitor for residues of these drugs in aquacultured products. Analytical methodology to monitor for residues and chemical Guest edited as a special report on “Methods of Analysis for Residues and Chemical Contaminants in Aquaculture” by Joe Boison and Sherri Turnipseed. Corresponding author’s e-mail: [email protected] DOI: 10.5740/jaoacint.14-265
contaminants in aquacultured species has changed significantly in recent years. Originally methods were developed specifically for one residue, or for several analytes from a specific class of drugs (e.g., sulfonamides or tetracyclines), in a single species of fish or shellfish. A thorough review of these types of class-specific methods in fish was published in 2009 (9). With the increased use of MS, there has been a trend towards multiclass, multiresidue methods to monitor for dozens of analytes in a single method (10). These procedures utilize LC tandem MS (LC-MS/MS with either ion trap (11, 12) or triple quadrupole (13, 14) mass analyzers. This approach greatly increases the monitoring capacity by analyzing for many residues in a single sample. A disadvantage of these methods, however, is that they are still limited to preselected target analytes because the data acquisition program consists of specific MSn product ion scans or selected reaction monitoring (SRM) transitions. If other analytes of interest are present in the sample, they will not be detected. High resolution (HR) MS (HRMS) instruments provide accurate mass measurements, defined as the ability to determine the mass of a compound within a few parts-per-million (ppm) of its theoretical mass. With these instruments, virtually an unlimited number of compounds can be simultaneously analyzed because full-scan data, rather than preselected ion transitions corresponding to specific compounds, are collected. In addition, exact mass measurement combined with the ability to generate product ions facilitates the detection and identification of unknown compounds. This can lead to the development of methods that can monitor for a wide scope of residues and contaminants including those that are not on any target list, allowing agencies to be more proactive in discovering possible adulteration of the food supply including aquacultured products. HRMS Instrumentation Mass resolution is the ability to discriminate between ions with very similar m/z values. For modern nonsector instruments this is defined as R = m/Δm where Δm is the full width of an ion peak at half maximum (FWHM). Using this definition, the resolution will automatically be dependent on the mass of the ion used to calculate the value. Quadrupole mass analyzers typically have a mass resolution of 3000–5000 at m/z 200–500. HR instruments have a mass resolution of 10 000 to 100 000 or higher at m/z 200. The higher the mass resolution, the better the instrument is able to fully differentiate an analyte from isobaric interferences. Without sufficient mass resolution, coeluting
Turnipseed et al.: Journal of AOAC International Vol. 98, No. 3, 2015 551 peaks with the same nominal mass (from the matrix or other analytes) can cause inaccurate mass measurements by shifting the centroid mass. Mass accuracy, or exact mass measurement error, is the difference between the measured and theoretical exact mass. Because this difference is very small with HRMS instruments, it is often expressed in milliDalton (mDa) or as a ppm difference, where ppm = [(measured mass – theoretical mass)/theoretical mass] * 106. Overviews of mass analyzers used in veterinary drug analyses, including HRMS instruments, have recently been published (15, 16). The simplest HR mass analyzer is the time-of-flight (ToF) instrument. With a ToF mass analyzer, ions that have the same kinetic energy but different m/z values are separated in a field-free flight tube and reach the detector at different times. The smallest ions are fastest and will be detected first, while the larger, slower, ions will reach the detector at a later time. The flight path (L) is fixed and the potential (V) at which ions are accelerated is kept constant. Therefore, the time it takes an ion to reach the detector (t) is proportional to its m/z value according to the following equation: m/z = 2eVt2/L2. Most ToF instruments also employ a reflectron, or electrostatic mirror, reversing the direction of the ions in the middle of the flight tube. This increases the mass resolution because the electrostatic reflectron corrects for small differences in initial (prepulse) velocities of ions with the same m/z. It also effectively doubles the length of the flight tube without requiring additional laboratory space. ToF mass spectrometers offer medium to high resolution (up to 40 000 FWHM), accurate mass measurement (
