Specific examples of recent methodology

In the next section, a few specific important examples are examined in more detail. These include methods for the analysis of sulfonamides in milk and dairy products, chloramphenicol in shellfish, nitrofurans in various foods and triphenylmethane dyes in farmed fish and shrimp.

4.3.1 Sulfonamides in dairy products

Sulfonamide residues in dairy products have been a regulatory issue for a long time. These drugs effectively treat bovine mastitis and can then be transferred into the cow's milk. While some of these drugs are approved for such use, appropriate withdrawal times need to be observed to prevent the occurrence of residues in the milk and other dairy products. Several reviews for the analysis of sulfonamide residues in food have been published [3,73].

Many innovations in analytical chemistry have been applied to sulfonamide residue methods. The detection of these drugs is fairly straightforward since the compounds have a strong chromophore of around 270 nm. The primary amine group on these compounds can also be derivatized with fluorescent groups to provide even greater sensitivity and selectivity for LC detection. There have been several examples utilizing unique extraction and isolation techniques for sulfonamide residues in food. Supercritical fluid (CO2) along with in-line adsorption on alumina has been used for the extraction of sulfonamides from chicken tissue [184]. A hot-water matrix solid-phase dispersion method has also been used to extract these drugs from milk and eggs [66]. Roybal et al. illustrated the use of size-exclusion chromatography for the isolation of sulfonamides from shrimp [68]. Van Rhijn et al. isolated these drugs from small quantities of milk after a simple clean-up using only ultrafiltration with molecular weight cutoff

Table 1 Selected veterinary drug residue methods

Drug class Function (used to Examples of Typical matrices treat) compounds

Sulfonamides Pneumonia, Sulfamethazine, Milk, bovine, other respiratory sulfadimethoxine, swine and diseases, mastitis, sulfamerazine, poultry tissue, diphtheria, diarrhea sulfathiazole fish, eggs, shrimp, honey



Colibacillosis, bacterial enteritis, salmonellosis, mastitis

Enteritis, pneumonia, anaplasmosis, growth efficiency

Penicillin, ampicillin, amoxicillin, cloxacillin, cephapirin, ceftioflur

Tetracycline, Oxytetracycline, Chlortetracycline, doxycycline

Milk, bovine, swine and fish tissue

Milk, bovine, poultry and swine tissue, shrimp, fish, eggs, honey


Macro lides

Broad-spectrum antibiotics, growth efficiency

For gram-positive organisms and strains of Listeria and Mycoplasma, growth efficiency

Streptomycin, gentamicin, neomycin

Erythromycin, tylosin, oleandomycin, spiramycin, tilmicosin

Milk, bovine, poultry and swine tissue (kidney)

Milk, bovine, swine and fish tissue, eggs, honey


Comments References

LC with UV Multi-residue [21,60-72]

(270 nm), liquid methods Review: [73]

chromatography- available fluorimetric detector (LC-FLD) after derivatization, LC-MS

LC-UV penicillins: 210-230 nm, cephalsosporins: 260-295 nm, LC-MS

May use tungstic [74-83]

or trichloro- Review: [4] acetic acid in extraction

LC or ion chromatography with UV at 270/ 350 nm; LC-FLD of metal complexes, LC-MS

LC-FLD w/ derivatization; GC-ECD, LC-MS

Chelating agents used in extraction and LC mobile phase

Poor chromo-phore, may need ion pair reagents for LC

after derivatization, chromophores LC-MS



Broad spectrum effective against gram positive, fluoroquinolones are effective for gram-negative species

Infections, bovine respiratory disease

Oxolinic acid, nalidixic acid, sarafloxacin, enrofloxacin, ciprofloxacin, flumequine

Chloramphenicol, florfenicol, thiamphenicol

Milk, bovine, poultry, swine and fish tissue, eggs

Milk, bovine, poultry, swine and fish tissue, shrimp, eggs

Ionophores and other coccidiostats

Parasites, growth efficiency

Monensin, lasalocid, salinomycin, halofuginone,

Bovine and poultry tissue, eggs



Broad spectrum antibiotics

Growth efficiency

Furazolidone, furaltadone, nitrofurazone, nitrofurantion

Bacitracin, avoparcin, virginiamycin, Colistin

Poultry and swine tissue, shrimp, honey

Bovine, swine and poultry tissue, milk,

LC-FLD and/or UV Concerns [110-120]

detection, LC-MS regarding Reviews:

antibiotic [8,100] resistance to these drugs

LC-UV, GC-ECD or GC-MS after derivatization, negative ion LC-MS

LC with visible or fluorescence detection after derivatization, LC-MS

LC-MS after derivatization with nitrobenzaldehyde

LC-MS, LC-UV, LC with electrochemical detection

No tolerance or maximum residue limit (MRL) for chloramphenicol residues

Ionophores form sodium adducts that are detected by LC-MS

Need to hydrolyze bound metabolites, no tolerance

Concerns regarding antibiotic resistance to these drugs

Drug class

Function (used to treat)

Examples of compounds

Typical matrices




Benz imidazoles



Bovine and

LC-UV (290 nm),

May form sulfone




swine tissue,



Review: [11]








Milk, bovine,


Form sodium




swine and fish

after derivatization,

adducts that

Review: [12]





are detected


by LC-MS


Fungus and

Malachite green,

Fish, shrimp

LC-visible, LC-MS

Convert leuco


methane dyes


crystal violet,

metabolites to

Review: [30]


brilliant green

chromic form

for detection,

no tolerance


Reduce stress and











transport aid pre





zine, xylazine,





Milk, bovine


5-Hydroxy flunixin




pheny lbu tazone,

tissue (kidney)

is flunixin



filters [64]. In-tube solid-phase microextraction (SPME) was used to isolate five sulfonamides from milk; low levels (ng/mL) were detected by LC-UV after chromatographic separation on a capillary monolithic LC column [72]. Because maximum or safe levels for these drugs have been established (10 ng/mL in the U.S. and 100 ng/mL according to EU regulations), it has not been necessary to develop methods with lower detection limits. Instead, many of these innovative methods have been able to reduce the sample size needed, down from a 10-20 mL portion to 1mL or less and still successfully detect and measure the drugs at the required concentrations.

The emergence of MS has had an influence in this area of analysis with early methods utilizing GC-MS [185] and many more recent methods taking advantage of the capability of LC-MS [64,66,67,186]. For example, a detection and confirmation LC-MS-MS method for sulfonamides in milk was developed that can detect 14 residues at levels below 10 ng/mL [67]. Another paper describes the analysis of these drugs in processed dairy products such as condensed milk and soft cheeses using LC-MS-MS [21]. An example of the analysis of three sulfonamides in condensed milk using this method is shown in Figure 4.

The development of rapid screening tests for sulfonamides has also been of interest for many years. These drugs do not respond very well to traditional microbial inhibition testing, so effective alternative screening methods are important. Early chemical methods for the screening of milk for sulfonamide residues included thin layer chromatography methods [187]. There have also been rapid test kits available for quite some time [43]. The detection of

100 ii



Sulfadimethoxine iXjjiljIia^jlii i


SRM ms2 2510-25


SRM ms2 2560-25


SRM ms2 2500-25


SRM ms2 2650-25


SRM ms2 2790-25


SRM ms2 2850-25


SRM ms 2 3110-25



SRM ms2 3010-25

Time (min)

Figure 4 LC-MS-MS combined ion chromatograms for sulfathiazole, sulfamethazine and sulfadimethoxine in control condensed milk fortified at 5 ng/mL. Reprinted with permission from Ref. [21]. Copyright 2005 by AOAC International.

sulfonamides in milk was one of the earliest applications of the use of biosensors in animal drug residue methodology [188] and is still in use today [189].

4.3.2 Chloramphenicol Residues in Seafood and Other Products

As mentioned earlier, chloramphenicol residues are of concern because of the drug's unique capability to produce aplastic anemia in susceptible individuals. A review of the analytical methodology for chloramphenicol is also a good illustration of how the field of veterinary drug residue methodology has changed over the last several years. Initially, methods to detect residues of chloramphenicol or analogous compounds in food used either LC-UV, or GC with (ECD) or MS after derivatization. For example, one of the first methods for chloramphenicol in shrimp involved GC-ECD analysis of a silyl derivative after liquid-liquid extraction and isolation [190]. In an extension of that method, chloramphenicol, florfenicol, florfenicol amine and thiamphenicol were silylated and analysed by GC-ECD after liquid-liquid extraction and isolation from shrimp by a series of SPE columns; the quantification limit for this method was 5 mg/kg [121].

In 2001 and 2002, chloramphenicol residues in food became an urgent problem, specifically in seafood and honey imported to EU countries, Canada and the U.S. from China. More sensitive methods were needed to adequately monitor for these residues. With the advent of widely available LC-MS instrumentation, the detection limits for chloramphenicol were decreased significantly to below 0.5 mg/kg. For this reason, there were many methods published in 2003-2004 describing the analysis of chloramphenicol in these food products using LC-MS [115,122-127,132]. Most of these methods utilized negative ion ESI to monitor the deprotonated molecular ion of chloramphenicol at m/z 321. A triple quadrupole LC-MS-MS instrument was most commonly utilized and the methods were capable of both quantification and confirmation of the residue. A few methods described the use of a single quadrupole or ion trap instrumentation. Regardless of the MS instrumentation used, the chloramphe-nicol molecule breaks apart into consistent fragment or product ions that can be used for confirmation. In some methods, the chlorine isotope ratio was also used for identification purposes. Internal standards were often used for quantification including mefa-chloramphenicol, deuterated chloramphenicol or an analogous compound such as thiamphenicol. The extraction procedure for many of these methods was still fairly extensive with liquid-liquid extraction and SPE isolations.

More recently published methods for chloramphenicol residue analysis generally utilize simplified generic or novel extraction methods and have expanded the analysis to additional matrices such as crab, honey, milk and eggs [26,128,131]. LC-MS-MS analysis of chloramphenicol in honey by electrospray with negative ion detection and quantification with meffl-chloramphenicol as an internal standard is illustrated in Figure 5 [26]. Biosensor-based assay test kits have expanded the possibility of rapidly screening food matrices for chloram-phenicol residues at or below a concentration of 0.07mg/kg [191].

1500 -1000 -500 0

2000 1000 0

1500 z 1000 i 500 0

200 100 0

RT: 7.41

RT: 5.95

RT: 5.61

200 100 0

iTr 11 ri' nrrrfr)

Figure 5 LC-MS-MS ion chromatograms for chloramphenicol of (a) 0.5 ng/g standard, (b) control honey with internal standard meta-chloramphenicol and (c) a chloramphenicol-positive honey sample. Ions monitored are (from top to bottom) m/z 152, 176, 194, 257 and 207. Reprinted with permission from Ref. [26]. Copyright 2006 by AOAC International.

iTr 11 ri' nrrrfr)

5 500

2000 1500 ^ 1000 i 500 0


Figure 5 LC-MS-MS ion chromatograms for chloramphenicol of (a) 0.5 ng/g standard, (b) control honey with internal standard meta-chloramphenicol and (c) a chloramphenicol-positive honey sample. Ions monitored are (from top to bottom) m/z 152, 176, 194, 257 and 207. Reprinted with permission from Ref. [26]. Copyright 2006 by AOAC International.


321 >176


4.3.3 Nitrofuran residues in seafood, poultry and honey

Nitrofuran antibiotics are broad-spectrum agents effective against infections common to poultry, swine and aquacultured shrimp. Nitrofurans may also be used to treat honey bees. The most common nitrofurans associated with food production are furazolidone, furaltadone, nitrofurazone and nitrofurantion. The drugs metabolize rapidly so the parent compounds are not detected as residues. The metabolites of these drugs, which correspond to a side chain of the original molecule bound to proteins, do persist in tissue for a significant amount of time. The metabolites generated are 3-amino-2-oxazolidinone, 3-amino-5-morpholinomethyl-2-oxazolidinone, semicarbazide and 1-aminohy-dantion from furazolidone, furaltadone, nitrofurazone and nitrofurantion, respectively. There are concerns regarding the carcinogenicity and mutagenicity of these metabolites, making monitoring for these compounds both important and a significant analytical challenge [32]. Because of the human health concerns, there are no tolerances or maximum residue limits set for nitrofuran metabolite residues. The EU has established a minimum residue performance level of 1 mg/kg.

Methods have been developed for nitrofuran metabolite residues in tissue [139,147], shrimp [140,141], eggs [139] and honey [145]. In general, these methods have several attributes in common. A hydrolysis step, usually incubation with dilute hydrochloric acid, is needed to release the protein-bound metabolites. The molecules are then derivatized with nitrobenzaldehyde. While LC detection by UV is possible at this point, LC-MS is usually utilized to obtain the necessary detection limits as well as residue confirmation. There are some variations in this basic protocol. The clean-up and isolation of the residues after hydrolysis, but before derivatization, can be accomplished with liquid-liquid extraction or with a SPE cartridge, or by using a combination of both. In some methods, the matrix is first washed to remove any unbound residue, so that only compounds covalently attached to proteins are measured in the final extract; other procedures do not incorporate this step. One particular concern is for the residues of semicarbazide. While the metabolism of nitrofurazone is one source of semicarbazide in food, there are other possible explanations for the presence of this residue. Axodicarbonamide is an industrial chemical used as a blowing agent for rubber gaskets on jars (such as baby food) and in some cases as a food additive in flour. This chemical can also thermally degrade to form semicarba-zide. It is also possible that semicarbazide could originate from the treatment of nitrogen-rich food with hypochlorite, a procedure that occurs when seaweed products are processed into gelling agents. It was originally thought that any protein-bound semicarbazide would necessarily originate from nitrofurazone exposure and that washing unbound residue prior to hydrolysis could reduce the amount of semicarbazide residues in the matrix from these other sources. However, a more extensive study of this issue is still needed [32].

The quantification of these metabolites can be complicated. In most cases, selective reaction monitoring using a triple quadrupole LC-MS-MS system is used to quantify the residues. Additional ion transitions can be monitored for confirmatory purposes. The fragmentation patterns of these derivatized metabolites have been established [192]. Ion trap and single quadrupole instruments can be used as well, but the results may not be as reliable for quantitative purposes as those obtained with a triple quadrupole procedure [140]. Matrix effects, i.e., ion suppression, can be an issue in obtaining good quantitative LC-MS methods [193]. For these methods, matrix effects can be overcome with matrix-based standards; but more often deuterated internal standards of one or more of the analytes are added to compensate for any ion suppression as well as for lack of recovery through the extraction procedure. The results of a proficiency study for nitrofuran metabolites in shrimp highlight some of the issues with these methods [140].

4.3.4 Triphenylmethane dyes in fish

Triphenylmethane dyes are used as inexpensive and effective water bath treatments for fungal and parasite infections in fish. Malachite green is the most commonly used treatment within this class of compounds. The suspected carcinogenicity and teratogenicity of malachite green has rendered its use inappropriate for fish raised for human consumption [33]. Crystal violet (also known as gentian violet) and brilliant green are structurally similar dyes with similar antifungal and toxicity properties [33,194,195]. Regardless of the international restrictions on the use of malachite green, illegal residues are regularly found in fish tissue. Reports of crystal violet abuse have also been noted [196]. The use of these therapeutic dyes in fish has been reviewed [30,195,197].

Methods to detect triphenylmethane dyes and their reduced leuco metabolites in fish tissue often include extraction by mixing the ground fish tissue with an acidic buffer and acetonitrile. This is followed by defatting and sample clean-up and residue isolation with liquid-liquid partition and/or SPE. The cationic triphenylmethane dyes have strong chromophores in the visible region of the spectrum, yet their reduced leuco metabolites are colorless. Since it is the leuco moiety that is found in greatest concentration in tissues, methods must emphasize the detection of this metabolite. Many traditional malachite green analytical methods have taken advantage of the sensitivity offered by LC with detection in the visible region by including a lead oxide, post-LC column reactor to oxidize the leuco residues to their chromic analogs [198-203]. The lead oxide reactor, however, is often difficult to prepare and can lead to rapid depletion and peak broadening resulting in a decrease in method sensitivity for regulatory use.

Because all of the triphenylmethane dyes are banned, regulatory action can be taken in many countries if any level of residue is found in fish tissue. Method detection limits therefore drive regulatory action and should be as sensitive as possible. Recent methodology for the detection of malachite green in fish tissues has included several different approaches to meet minimum performance limits (2.0 mg/kg and lower) of international agencies [204]. In the first approach, leucomalachite green residues were oxidized to malachite green using 2,2,7-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) prior to chromatographic analysis, enabling the measurement of the sum of malachite green and leucomalachite green residues by LC with visible detection at 1.0 mg/kg [163,168]. In another approach, residues at the 0.5 mg/kg level were determined using LC separation followed by the visible detection of malachite green at 620 nm and florescence detection of leucomalachite green at 360 nm (265 nm excitation) [171]. Both of these methods are advantageous for screening numerous samples with less expensive instrumentation.

Mass spectrometric analysis of malachite green and leucomalachite green residues are divided by those that use an oxidation procedure to detect leucomalachite green as malachite green and those that detect leucomalachite green directly. Malachite green is cationic and more sensitively detected than leucomalachite green by LC-MS. In the former case, DDQ has been used prior to sample analysis with no-discharge LC-MSn to detect the sum of malachite green and leucomalachite green at 0.25 mg/kg [165,168]. This method was used to detect and confirm malachite green residues in store-bought basa fish (Figure 6). The lead oxide reactor has also been used to detect leucomalachite green as low as 0.1 mg/kg [164,166,172]. Several other methods rely on detection of malachite green and leucomalachite green individually using triple quadrupole LC-MS-MS, which is more sensitive for the quantification of leucomalachite green [162,167,170,205]. These methods offer excellent sensitivity with limits of detection


Area: 256,730,573 SN: 16,141


Area: 187,769,285 SN: 15,042 I


cd QC


Area: 73,898,029



Area: 132,887,237

285.2 284.21


_1.16E7 m/z= 313.0-315.0 ms2 329.000 50.00 [150.00-350.00]




313.3 314.2

Figure 6 LC-MSn extracted ion chromatograms and product ion spectrum from the malachite green (m/z 329) product ion trace in an extract from retail basa (diluted 1:5). Extracted ion ranges (from top to bottom) are m/z 329, 313-315, 284-286, 251 and 208. Reprinted with permission from Ref. [168]. Copyright 2006 American Chemical Society.

below 0.5 mg/kg. Some of these newer methods have also been applied to the concurrent determination of crystal violet and brilliant green residues [170,206]. Finally, an ELISA test kit based on DDQ oxidation for the detection of malachite green at 0.5 mg/kg in fish tissue has recently been commercialized [207].

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