Determination of macrolide antibiotics in meat and fish by liquid chromatography–electrospray mass spectrometry

Analytica Chimica Acta 492 (2003) 187–197

Determination of macrolide antibiotics in meat and fish by liquid chromatography–electrospray mass spectrometry Masakazu Horie a,∗ , Harumi Takegami a , Kazuo Toya a , Hiroyuki Nakazawa b a

Department of Food Chemistry, Saitama Prefectural Institute of Public Health, 639-1, Kamiokubo, Urawa, Saitama 338-0824, Japan b Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41, Ebara, Shinagawa-ku, Tokyo 142-0063, Japan Accepted 1 July 2003

Abstract A simple and reliable method using liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) has been developed for the determination of macrolide antibiotics, erythromycin (EM), oleandomycin (OM), kitasamycin (KT), josamycin (JM), mirosamicin (MRM), spiramycin (SPM), tilmicosin (TLM) and tylosin (TS) in meat. The LC separation was performed on a TSKgel Super ODS column (100 mm × 2 mm i.d.) with a gradient system of 0.2% acetic acid–acetonitrile (containing 0.2% acetic acid) as the mobile phase at the flow rate of 0.2 ml/min. The positive ionization produced the molecular related ions, (M + 2H)2+ , at m/z 422 and 435 for SPM and TLM, and (M + H)+ , at 734, 688, 772, 828, 728 and 916 for EM, OM, KT, JM, MRM and TS, respectively. The calibration graphs for each drug were rectilinear from 0.05 to 25 ng with selected ion monitoring (SIM). The drugs were extracted with 0.2% metaphosphoric acid–methanol (6:4), and the extracts were cleaned up on an Oasis HLB cartridge (60 mg). The recoveries of the drugs from meat and fish fortified at the 0.2 ␮g/g level was 70.4–93.2% with high precision. The limits of quantification of the drugs in meat and fish were 0.01 ␮g/g. © 2003 Elsevier B.V. All rights reserved. Keywords: Macrolide antibiotics; Erythromycin; Oleandomycin; Kitasamycin; Josamycin; Mirosamicin; Spiramycin; Tilmicosin; Tylosin; Meat; Fish; LC; Mass spectrometry; LC–MS

1. Introduction Macrolide antibiotics are a very important class of antibacterial compounds widely used in medical and veterinary practices. Erythromycin (EM) and oleandomycin (OM) are 14-membered ring macrolide antibiotics. Josamycin (JM), kitasamycin (KT), mirosamicin (MRM), spiramycin (SPM), tylosin (TS) and tilmicosin (TLM) belong to the class of 16membered macrolide antibiotics. Macrolide antibiotics are considered to be medium-spectrum antibiotics. ∗ Corresponding author. Tel.: +81-48-853-5742; fax: +81-48-840-1041. E-mail address: [email protected] (M. Horie).

They are highly active against a wide range of Grampositive bacteria such as Mycoplasma and Chlamydia. The macrolides are the most effective medicine against diseases produced by Mycoplasmas [1]. These drugs are the well absorbed after oral administration and are distributed extensively in tissues, especially lungs, liver and kidneys, with high tissue/plasma ratios [2]. Therefore, these macrolides have been widely used in the rearing of food-producing animals to prevent and treat diseases. Concerns about food hygiene have arisen regarding the presence of drug residues in livestock products [3]. Antibiotic residues may have direct toxic effects on consumers, e.g., allergic reactions in hypersensitive individuals, or may indirectly cause problems through

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0003-2670(03)00891-2

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the induction of resistant strains of bacteria. Therefore, simple and reliable analytical methods are required to monitor these drug residues in the edible tissues of livestock animals. Generally, the determination of antibiotics, including macrolide antibiotics, is mainly carried out by microbiological assays [4,5]. These assays excel as a qualitative means by which samples may be screened for residual amounts of antibacterial substances. However, the assays tended to lack specificity, and their use involves difficulty in confirming what kinds of drugs remain in the animal tissues and fish. In order to overcome these problems, chemical analyses such as high-performance liquid chromatographic (HPLC) techniques have been used for the determination of macrolide antibiotics [6–8]. For the HPLC of macrolide antibiotics, the most popular detector is a UV spectrophotometer. Some macrolide antibiotics, such as TS, TLM, SPM and JM have relatively strong UV absorptions, so that UV detection is sufficiently sensitive for the determination of these antibiotics [9]. However, EM and OM do not have a specific UV chromophore. Liquid chromatography–mass spectrometry (LC– MS) is the most promising technique for separation and has recently been used in the determination of antibiotics [10–13]. This paper describes a simple, rapid and reliable LC–MS method for the simultaneous determination of eight macrolide antibiotics at 0.01 ␮g/g in meat and fish. 2. Experimental 2.1. Materials and reagents EM, OM and TLM were obtained from Dainippon Pharmaceuticals (Tokyo, Japan), Taito-Pfizer Pharmaceuticals (Tokyo, Japan) and Eli Lilly Japan (Tokyo, Japan), respectively. JM, KT, MRM, SPM and TS were kindly supplied by Yamanouchi Pharmaceuticals (Tokyo, Japan), Asahi Chemical Industry (Tokyo, Japan), Asahi Chemical Industry, Kyowa Hakko Kogyo (Tokyo, Japan) and Takeda Pharmaceuticals (Osaka, Japan), respectively. Neospiramycin (NSPM) was obtained from Hayashi Pure Chemical Industries (Osaka, Japan). Oasis HLB (60 mg) cartridges were purchased from Waters (Milford, MA, USA). The cartridges were con-

ditioned by washing with 5 ml of methanol and then 10 ml of water before use. Hyflo Super-Cel was obtained from Johns-Manville (Denver, CO, USA). All other chemicals were of analytical-reagent or HPLC grade. Water was purified using a Milli-Q water system (Millipore, Bedford, MA, USA) prior to use. The mobile phase A was 0.2% acetic acid, while the mobile phase B was acetonitrile containing 0.2% acetic acid. 2.2. Preparation of standard solutions Standard stock solutions of the nine antibiotics were prepared by dissolving 50 mg of each compound in 50 ml of methanol. The standard solutions were kept at 5 ◦ C in amber glass vessels and were stable for up to 6 months. The standard working solutions were prepared by diluting the stock solution with 30% methanol. The working standard solutions were stored in a refrigerator and were stable for up to 3 days. 2.3. Apparatus The experiments were carried out using a Agilent (Palo Alto, CA, USA) Model 1100 Series LC/MSD system with an electrospray ionization (ESI) interface. The separation was performed on a TSKgel Super ODS column (2 ␮m, 100 mm ×2 mm i.d., Tosoh) with a gradient system of 0.2% acetic acid–acetonitrile containing 0.2% acetic acid as the mobile phase at a flow rate of 0.2 ml/min. The gradient was initiated with 15% eluent B followed by a linear increase to 50% eluent B over 20 min. The LC–MS operating conditions are summarized in Table 1. The other instruments used were a Model N-1 rotary evaporator (Tokyo Rikakikai, Tokyo, Japan) and a Model NS-50 Physcotron homogenizer (Niti-on, Chiba, Japan). 2.4. Calibration graphs Standards at concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 2.0 and 5.0 ␮g/ml of the nine macrolide antibiotics were prepared from the standard stock solutions. A 5 ␮l volume of these solutions was injected into the column. Calibration graphs were obtained by measurement of the peak areas on the (M+H)+ or (M+2H)2+ selected ion monitoring (SIM) chromatograms for the amount of the antibiotics.

M. Horie et al. / Analytica Chimica Acta 492 (2003) 187–197 Table 1 Operating conditions of LC–MS for macrolide antibiotics MS conditionsa Ionization Fragmentor Nebulizer Drying gas V-cap

ESI, positive Time program N2 (40 psi) N2 (10 l/min, 350 ◦ C) 4500 V

HPLC conditionsb Column Eluent Flow rate Oven temperature Injection size SIM ion

TSK-gel Super ODS (100 mm × 2 mm) Gradient 0.2 ml/min 40 ◦ C 5 ␮l m/z (M + H)+ , (M + 2H)2+

a For time of 5.0, 11.5 and 15.5 min, fragmentor voltage was 60, 100 and 150 V, respectively. b For time of 0 min, 85% of 0.2% acetic acid (A) and 15% of acetonitrile (containing 0.2% acetic acid, B) were used. For time of 20 and 25 min, 50% A and 50% B were used.

2.5. Sample preparation The sample preparation was done as follows based on previous studies [14]. A 5 g sample was homogenized at high speed for 2 min with 100 ml of 0.2% metaphosphoric acid–methanol (6:4, v/v) used as the deproteinizing extractant. The homogenate was filtered through ca. 2 mm of Hyflo Super-Cel coated on a suction funnel. For a liver sample, several grams of Hyflo Super-Cel were added to the homogenized solution before filtration. After a slight mixing, the obtained mixture was filtered. The filtrate was evaporated under reduced pressure at 40 ◦ C to approximately 25 ml volume. The flask contents were passed through an Oasis HLB cartridge. After washing with 10 ml of water, the cartridge was then eluted with 5 ml of methanol. The eluate was evaporated to dryness under reduced pressure at 40 ◦ C, and the residue was dissolved in 1 ml of 30% methanol. A 5 ␮l sample of the solution was then injected into the LC–MS system.

3. Results and discussion 3.1. LC–MS conditions 3.1.1. MS conditions For the interface, ESI was selected, which is excellent for the manipulation and suitable for ionizing

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polar and non-polar compounds. Macrolide antibiotics are built from the macrocyclic lactone ring attached to the amino sugar(s) as the basic structure. Therefore, the positive mode was found suitable for the eight macrolide antibiotics. ESI is a mild ionization method that produces some molecular related ions. These ions are typically protonated molecular ions (M + H)+ . Information on the molecular weight of these compounds can be easily obtained, but little structural information is obtained by ESI. Therefore, the common method consists of changing the collision condition with nitrogen molecules in the ion source by changing the voltage at the capillary exit (fragmentor voltage) to obtain the fragment ions. By changing the voltage of the fragmentor, various degrees of fragmentation may be achieved. With a low voltage, there is a little fragmentation, but at higher voltages, a molecular related ion is fragmented to a greater degree. As shown in Fig. 1, the strength of the generated ion species changed as the fragmentor voltage changed. To detect with sufficient sensitivity these antibiotics was difficult at the same fixed fragmentor voltage. Therefore, the fragmentor voltage was set time programmed (Table 1). In addition, the best measurement conditions for the nebulizer pressure, dry gas pressure, temperature, capillary voltage, etc., were examined, and these conditions are shown in Table 1. We used molecular related ions for the quantitative determination and monitored the fragment ions at the same time to obtain more accurate results (Table 2). The detectability limit of the antibiotics, which had been obtained under these conditions, was ca. 0.005 ␮g/ml (25 pg as the absolute

Table 2 Typical ions detected for macrolide antibiotics using LC–ESI-MS Compound EM OM KT JM MRM SPM NSPM TLM TS

Mw 733.9 688.9 771.9 828.0 727.9 843.1 698.8 869.2 916.1

Base peak ions 734.5 688.4 772.5 828.5 728.4 422.3 350.2 435.3 916.5

+ H)+

(M (M + H)+ (M + H)+ (M + H)+ (M + H)+ (M + 2H)2+ (M + 2H)2+ (M + 2H)2+ (M + 2H)2+

Main other ions 716.4, 670.4, 702.5, 860.4, 554.3 843.5, 721.5, 869.5, 742.3,

576.3 544.3 558.3 786.4 699.5, 540.3 699.5, 540.3 695.5 582.3

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Relative intensity (%)

100

m/z 350.3(NSPM) m/z 721.5(NSPM) m/z 422.4(SPM) m/z 843.5(SPM)

50

0 20

60

100

140

180

Fragmentor voltage (V)

Relative intensity (%)

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m/z 435.3(TLM) m/z 869.5(TLM) m/z 688.4(OM) m/z 544.3(OM) m/z 728.4(MRM) m/z 554.3(MRM)

50

m/z 734.4(EM) m/z 576.3(EM)

0 20

60

100

140

180

Fragmentor voltage (V)

Relative intensity (%)

100

m/z 916.5(TS) m/z 742.4(TS) m/z 772.5(KT) m/z 558.3(KT) m/z 828.4(JM)

50

0 20

60

100

140

180

Fragmentor voltage (V) Fig. 1. Effect of fragmentor voltage on the relative intensity of molecular related ions and major fragment ions of macrolide antibiotics.

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amount), when the monitor ions were presumed to be molecular related ions in the SIM mode.

3.2. Determination method and analytical specificity of SIM

3.1.2. LC conditions A disadvantage of the HPLC of basic substances on silica-based reversed-phases is peak tailing due to an interaction with the residual silanols on the silica-gel. Similar to a number of other macrolide antibiotics, EM, OM, JM, KT, MRM, SPM, TLM and TS, which are basic molecules containing amino sugar(s) in their structure, are strongly affected by the silanol groups remaining in the column packing material [15]. Therefore, the HPLC column used was a TSKgel Super ODS, an end-capped C18 column based on pure silica gel. Next, the effect of the acetic acid concentration of the mobile phase on peak shape and retention times of the drugs was studied. The simple aqueous acetonitrile mobile phase has a pH of around 7, which, it is thought, would dissociate any residual silanols into weakly cationic species that could strongly retain the basic macrolides. To prevent strong broad tailing of the peaks, the mobile phase pH was adjusted to acidic. The asymmetry and retention time of the peaks decreased with the increasing acetic acid concentration in the mobile phase, but as the concentration increased, the detection sensitivity decreased. As a result of the above investigations, eluent A, 0.2% acetic acid, and eluent B, acetonitrile (containing 0.2% acetic acid), were chosen as the mobile phase. Generally, macrolide antibiotics, especially EM, are unstable in acidic solutions. However, investigations of the stabilities of 2.0 ␮g/ml of the macrolide antibiotics in the mobile phase, 0.2% acetic acid–acetonitrile (80:20), at 40 ◦ C for 30 min demonstrated that they were relatively stable under these conditions. Consequently, the use of an acidic HPLC mobile phase did not present a problem. The capacity factor (k ) significantly decreased for the nine macrolides as the acetonitrile concentration rose. However, there were significant differences in the affinity for the column among the macrolides, so that analysis by the isocratic elution system was difficult. The gradient elution method was then adopted in this experiment. Fig. 2 shows the typical chromatograms of the standard mixture of the macrolide antibiotics obtained under these conditions.

Sixteen-membered macrolides are mostly produced as complex mixtures of related components [1]. SPM consists of three components, spiramycin I (SPM-I), II and III; SPM-I is the major component. TS is composed of four components, tylosin A, B, C and D; tylosin A (TS-A) is the main and the most important component. Similarly, KT consists of several components, leucomycin A1, A3-9 and A13, with leucomycin A5 (LM-A5) as the major component. TLM is composed of cis and trans isomers in a ratio of about 85:15%. It is difficult to monitor all the components of these macrolide antibiotics in animal tissues. When the residual level is high enough to detect each component, it is preferable to monitor each component. However, the residual drug level in animal tissues is actually too low. Therefore, it is thought to be a practical measure to assume the main component of these drugs to be an indicator to evaluate the residual level. Accordingly, SPM-I, TS-A, LM-A5 and cis-TLM were indicators for SPM, TS, KT and TLM residues, respectively. It is known that SPM is metabolized to NSPM in animals [16,17]. The primary metabolite NSPM has an estimated antimicrobial activity of ca. 90% compared to SPM [17]. Therefore, the primary metabolite neospiramycin I (NSPM-I) was also simultaneously analyzed. Next, the compositions of the EM, OM, JM and MRM preparations, defined as a single component, were determined [9]. As a result, each preparation included 90% or more of EM, OM, JM and MRM. The calibration curves of nine macrolide antibiotics were drawn based on SIM, which detected molecular related ions. The calibration curves of each ion showed excellent linearity within the range of 0.05–25 ng. The detection and quantitative determination were fully available up to 0.05 ␮g/ml (=0.01 ␮g/g of tissues). Moreover, the five replicate measurements taken for each injection rate (0.1 and 1 ng) to obtain the relative standard deviations (R.S.D.) in the peak area were satisfactory found within 2% for each injection rate. 3.3. Sample preparation The extract from a tissue sample contains many diverse compounds in addition to the possible traces of

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Fig. 2. Typical LC–ESI-MS–SIM chromatograms of standard mixture (0.05 ␮g/ml).

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MSD1 TIC, MS File (MLS\MLS_1023.D) API-ES Positive 20000 15000 10000 5000 0

TIC 0 5 10 MSD1 350, EIC=349.9:350.9 (MLS\MLS_1023.D) API-ES Positive

20000 15000 10000 5000 0

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m/z 350.2 0 5 10 MSD1 422, EIC=422:423 (MLS\MLS_1023.D) API-ES Positive

20000 15000 10000 5000 0

m/z 422.3 0 5 10 MSD1 435, EIC=435:436 (MLS\MLS_1023.D) API-ES Positive

20000 15000 10000 5000 0

m/z 435.3 0 5 10 MSD1 689, EIC=688.1:689.1 (MLS\MLS_1023.D) API-ES Positive

20000 15000 10000 5000 0

m/z 688.4 0 5 10 MSD1 729, EIC=728.1:729.1 (MLS\MLS_1023.D) API-ES Positive

20000 15000 10000 5000 0

m/z 728.4 0 5 10 MSD1 735, EIC=734.2:735.2 (MLS\MLS_1023.D) API-ES Positive

20000 15000 10000 5000 0

m/z 734.5 0 5 10 MSD1 917, EIC=916.2:917.2 (MLS\MLS_1023.D) API-ES Positive

20000 15000 10000 5000 0

m/z 916.5 0 5 10 MSD1 773, EIC=772.2:773.2 (MLS\MLS_1023.D) API-ES Positive

20000 15000 10000 5000 0

m/z 772.5 0 5 10 MSD1 829, EIC=828.2:829.2 (MLS\MLS_1023.D) API-ES Positive

20000 15000 10000 5000 0

m/z 828.5 0

5

10

Fig. 3. Typical LC–ESI-MS–SIM chromatograms of chicken muscle extract.

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M. Horie et al. / Analytica Chimica Acta 492 (2003) 187–197 MSD1 TIC, MS File (MLS\MLS_1020.D) API-ES Positive 20000 10000

TIC

0

0 5 10 MSD1 350, EIC=349.9:350.9 (MLS\MLS_1020.D) API-ES Positive

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20000 10000

m/z 350.2

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0 5 10 MSD1 422, EIC=422:423 (MLS\MLS_1020.D) API-ES Positive 20000 10000

m/z 422.3

0

0 5 10 MSD1 435, EIC=435:436 (MLS\MLS_1020.D) API-ES Positive 20000 10000

m/z 435.3

0

0 5 10 MSD1 689, EIC=688.1:689.1 (MLS\MLS_1020.D) API-ES Positive 20000 10000

m/z 688.4

0

0 5 10 MSD1 729, EIC=728.1:729.1 (MLS\MLS_1020.D) API-ES Positive 20000 10000

m/z 728.4

0

0 5 10 MSD1 735, EIC=734.2:735.2 (MLS\MLS_1020.D) API-ES Positive 20000 10000

m/z 734.5

0

0 5 10 MSD1 917, EIC=916.2:917.2 (MLS\MLS_1020.D) API-ES Positive 20000 10000

m/z 916.5

0

0 5 10 MSD1 773, EIC=772.2:773.2 (MLS\MLS_1020.D) API-ES Positive 20000 10000

m/z 772.5

0

0 5 10 MSD1 829, EIC=828.2:829.2 (MLS\MLS_1020.D) API-ES Positive 20000 10000

m/z 828.5

0 0

5

10

Fig. 4. Typical LC–ESI-MS–SIM chromatograms of liver extract of cattle.

M. Horie et al. / Analytica Chimica Acta 492 (2003) 187–197

the target analytes. To exclude these interfering substances, a number of cleanup methods have been developed for the analysis of macrolide antibiotics [6–8]. In a previous paper [9], we reported the simultaneous determination of five macrolide antibiotics (SPM, MRM, TS, KT and JM) in animal tissues in which the samples were extracted with 0.3% metaphosphoric acid–methanol (7:3), the cleanup procedure used a prepacked cation exchange resin cartridge (Bond Elut SCX), and the antibiotics were detected at 232 or 287 nm. This method is quite excellent for the cleanup effect, but the manipulation is rather complicated. The most widely used solid-stationary phase for extraction and cleanup is octadecyl silane bonded to silica gel, called C18 or ODS. As the LC–MS measurement technique has an excellent selectivity, we then decided to use a reverse-phase system cartridge, which is also excellent for preparing samples. Several reverse-phase cartridges were examined and all of them showed sufficient recovery rates. The polymer type Oasis HLB was adopted this time. Next, the extracting solvents were examined. As mentioned above, we have previously reported that samples were extracted with 0.3% metaphosphoric acid–methanol (7:3). However, when this extracting solvent were used, the pH of the extracts condensed to about 25 ml was 3.9–4.5. Generally, macrolide antibiotics, especially EM, are unstable in acidic so-

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lutions, with almost all macrolide antibiotics being converted by acid to degradation products at pH < 4.5 [6]. Therefore, we used the 0.2% metaphosphoric acid–methanol (6:4) for the sample extraction. When this extracting solvent were used, the pH of the extracts condensed to about 25 ml was 4.5–5.0. In addition, the extraction with 0.1% metaphosphoric acid–methanol (6:4) was not effective enough with regard to deproteinization. Figs. 3 and 4 show the chromatograms of chicken muscle extracts and cattle liver extracts obtained under the established conditions. There were no interfering peaks on the LC–MS SIM chromatograms in each sample.

4. Recovery Linear calibration graphs (r 2 > 0.995) were obtained from 0.05 to 25 ng (equivalent to 0.01–1.0 ␮g/g) for the antibiotics. Table 3 summarizes the recoveries of the drugs from samples of meat and fish fortified with 0.2 ␮g/g. Greater than 70% overall mean recoveries and within ca. 10% R.S.D. were obtained with each sample. The detection limits of the method were 0.01 ␮g/g for the antibiotics (signal-to-noise ratio >5) in each sample. Fig. 5 shows the typical LC–ESI-MS–SIM chromatograms of yellowtail extract fortified at 0.2 ␮g/g of the macrolide antibiotics.

Table 3 Recoveries of macrolide antibiotics from meat and fisha Sample

Recovery (mean ± S.D., n = 5) (%) SPMb

OM

TLM

MRM

EM

TS

KT

JM

Intra-assay Cattle muscle Cattle liver Swine muscle Swine liver Chicken muscle Chicken liver Yellowtail Red sea bream

82.2 73.2 81.7 75.1 83.1 75.1 85.1 87.2

± ± ± ± ± ± ± ±

4.6 9.4 4.7 8.7 4.6 7.8 3.7 3.6

90.1 89.7 92.5 85.1 90.0 88.7 91.1 93.2

± ± ± ± ± ± ± ±

3.6 4.6 4.3 3.1 2.6 3.7 2.8 1.8

86.6 83.0 85.1 81.7 85.1 81.5 87.1 89.2

± ± ± ± ± ± ± ±

3.2 5.6 2.7 4.6 4.2 4.3 3.3 3.1

88.6 83.9 87.3 83.7 87.1 85.9 87.4 88.4

± ± ± ± ± ± ± ±

3.4 2.8 3.8 3.5 3.3 4.6 3.3 4.2

82.1 71.2 81.4 70.4 82.5 75.1 82.1 83.0

± ± ± ± ± ± ± ±

5.9 11.2 4.9 10.8 5.9 8.6 4.3 4.5

87.3 80.3 86.7 78.1 86.1 80.1 90.1 92.0

± ± ± ± ± ± ± ±

4.6 7.6 5.3 6.2 3.9 6.4 4.3 5.0

87.5 78.3 81.1 76.7 83.1 79.8 86.4 88.7

± ± ± ± ± ± ± ±

4.9 10.2 3.9 9.1 5.2 6.2 4.7 3.9

83.5 79.3 81.5 77.1 84.2 78.5 90.1 91.5

± ± ± ± ± ± ± ±

3.3 7.5 2.8 5.8 3.6 6.4 5.3 4.1

Inter-assay Cattle muscle Cattle liver Swine muscle Swine liver

84.4 74.3 83.0 76.6

± ± ± ±

4.2 10.5 5.7 6.7

87.5 90.1 90.4 84.3

± ± ± ±

2.9 3.8 5.3 5.1

87.8 80.4 86.9 80.8

± ± ± ±

5.8 6.3 3.5 6.3

87.1 85.9 88.1 82.7

± ± ± ±

3.8 5.7 4.1 4.2

80.1 67.2 85.4 73.1

± ± ± ±

5.7 9.2 4.2 8.3

80.2 75.4 80.0 73.7

± ± ± ±

3.5 6.7 4.3 6.0

85.2 79.0 88.4 76.8

± ± ± ±

2.7 7.7 4.0 7.5

82.2 78.2 85.7 77.3

± ± ± ±

5.1 4.8 4.7 5.7

a b

Samples were spiked with 0.2 ␮g/g of each drug. Average value of the recovery of spiramycin I and NSPM I.

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M. Horie et al. / Analytica Chimica Acta 492 (2003) 187–197 MSD1 TIC, MS File (MLS\MLS_1030.D) API-ES Positive 300000 200000 100000

TIC

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Neospiramycin

60000 40000 20000

m/z 350.2

0

0 5 10 MSD1 422, EIC=422:423 (MLS\MLS_1030.D) API-ES Positive

Spiramycin

100000 75000 50000 25000 0

m/z 422.3 0 5 10 MSD1 435, EIC=435:436 (MLS\MLS_1030.D) API-ES Positive

Tilmicosin 100000 50000

m/z 435.3

0

0 5 10 MSD1 689, EIC=688.1:689.1 (MLS\MLS_1030.D) API-ES Positive

15

Oleandomycin

100000 50000

m/z 688.4

0

0 5 10 MSD1 729, EIC=728.1:729.1 (MLS\MLS_1030.D) API-ES Positive

15

Mirosamicin 200000 100000

m/z 728.4

0

0 5 10 MSD1 735, EIC=734.2:735.2 (MLS\MLS_1030.D) API-ES Positive

15

Erythromycin

80000 60000 40000 20000 0

m/z 734.5 0 5 10 MSD1 917, EIC=916.2:917.2 (MLS\MLS_1030.D) API-ES Positive

15

Tylosin

100000 50000

m/z 916.5

0

0 5 10 MSD1 773, EIC=772.2:773.2 (MLS\MLS_1030.D) API-ES Positive

15

Kitasamycin

150000 100000 50000

m/z 772.5

0

0 5 10 MSD1 829, EIC=828.2:829.2 (MLS\MLS_1030.D) API-ES Positive

15

20

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Josamycin

200000 150000 100000 50000 0

m/z 828. 0

5

10

15

20

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Fig. 5. Typical LC–ESI-MS–SIM chromatograms of yellowtail extract fortified at 0.2 ␮g/g of each drug.

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5. Conclusions The method described for the determination of eight macrolide antibiotics in meat and fish samples yielded good recoveries and precision. In addition, the detection limits of this method were 0.01 ␮g/g for these antibiotics in samples, and the time required for the analysis of one sample was less than 1.5 h. HPLC-UV is generally used for the residual analysis of veterinary drugs, but it is necessary to use an extremely complicated pretreatment. However, as LC–MS is quite excellent in selectivity and detection sensitivity, it became possible to use a simple pretreatment method. Therefore, we recommend this proposed method for the routine analysis of the residual eight macrolide antibiotics in livestock products and fish samples. It is now expected that LC–MS will be applied as a qualitative and quantitative analysis method for toxic chemical substances in foodstuffs, including pharmaceutical drugs for animals. References [1] S. Omura (Ed.), Macrolide Antibiotics: Chemistry, Biology and Practice, Academic Press, Orlando, 1984, p. 26. [2] K. Ninomiya, Antibiotics for Animals, Youkendo Press, Tokyo, 1987, p. 307.

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