Simultaneous detection of residues of 34 beta-lactam antibiotics in livestock and fish samples through liquid chromatography-tandem mass spectrometry

Abstract

β-Lactam is one of the widely used veterinary drugs, but simultaneous analytical methods for β-lactam on various animal foods have not been established. In this study, we aimed to detect 34 β-lactam antibiotics simultaneously in livestock samples (beef, pork, chicken, egg, and milk) by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Samples were extracted using phosphate buffer/acetonitrile or water/acetonitrile and then cleaned with 150 mg of C₁₈ and 900 mg of MgSO₄. The method showed acceptable recovery and repeatability of 66.1–119% and 1.5–26%, respectively. The method was employed to monitor 127 real samples from the domestic market to confirm its applicability, and no β-lactam residues were detected. It was also applied to other matrices (eel, flat fish, and shrimp) and showed acceptable recovery (62.1–120%) and repeatability (1.0–28%). The method is expected to improve the efficiency of monitoring veterinary drug residues in domestic livestock products and fishery foods.

Introduction

Antibiotics are widely used veterinary drugs for preventing or controlling clinical diseases in food animals. β-Lactams, which include penicillins, cephalosporins, and carbapenems, are a class of antibiotics primarily used to treat mastitis or listeriosis affecting ruminants (Bush and Bradford, 2014). The World Health Organization (WHO) reported that antibiotic resistance poses a serious threat to human health (WHO, 2014). Therefore, the usage of antibiotics such as β-lactams must be regulated and monitored to prevent the abuse or misuse of antibiotics for human health. In Korea, some penicillins (nafcillin, dicloxacillin, amoxicillin, ampicillin, cloxacillin, and penicillin V) and cephalosporins (cefadroxil, cefacetrile, cefazolin, cephapirin, cefalexin, cefalonium, cefoperazone, cefuroxime, cefquinome, and ceftiofur) are regulated below the maximum residue limits (MRLs). The MRLs of β-lactams range from 0.004 to 1.0 mg/kg in livestock foods, such as beef, pork, chicken, egg, and milk, in Korea (MFDS, 2022). In China, β-lactam residues in chicken muscle include 5–35 μg/kg ampicillin, 3–15 μg/kg penicillin V, and 36–307 μg/kg ceftiofur (Chen et al., 2017). In Brazil, 7.9 µg/L penicillin and 107 μg/L cloxacillin have been detected in milk (Jank et al., 2012, 2015). In 2021, 13,183 kg of cephalosporins (ceftiofur > cefazolin > cephalexin > cephalonium > cefuroxime > cephapirin) and 304,442 kg of penicillins (amoxicillin > ampicillin > penicillin > dicloxacillin > cloxacillin) were sold in Korea (MAFRA, 2022).

Among β-lactams, ceftiofur and cephapirin are monitored together with their metabolites desfuroyl ceftiofur and desacetyl cephapirin, respectively (Hornish et al., 2003; Prescott, 2013). Ceftiofur is rapidly converted to desfuroyl ceftiofur as the primary metabolite, which is further converted to other forms, such as desfuroyl ceftiofur glutathione disulfide and desfuroyl ceftiofur cysteine disulfide. Meanwhile, cephapirin is converted to its desacetyl derivative and rapidly excreted via urine.

Penicillins and cephalosporins have a core four-membered β-lactam ring that is connected to a five-membered thiazolidine ring and a six-membered dihydrothiazine ring, respectively (Chaudhry et al., 2019). Most studies have been conducted to analyze residual β-lactams in beef (Feng et al., 2014; Jung et al., 2022; Rezende et al., 2012; Rocco et al., 2017) or milk (Holstege et al., 2002; Jank et al., 2015). Antibiotics can be qualitatively and quantitatively analyzed using liquid chromatography-mass spectrometry (LC–MS) or UV-diode array detection (DAD) (Cámara et al., 2013; Heller et al., 2006; Karageorgou et al., 2018). Samples are prepared using dispersive solid phase extraction (d-SPE) and solid phase extraction (SPE). d-SPE using QuEChERS (quick, easy, cheap, effective, rugged, and safe) is widely employed in food analysis. A previous study reported that β-lactam compounds show poor accuracy when cleaned with hexane and concentrated to dryness. The recovery of nafcillin is too high when hexane is used (Rocco et al., 2017), and this compound could be lost due to drying (Bessaire et al., 2018; Rezende et al., 2012). Previous analyses on β-lactams focused on bovines and obtained a detection limit of 0.01–371.7 μg/kg (Rezende et al., 2012; Rocco et al, 2017). However, β-lactams are also used in other food animals and regulated in various livestock products. Thus, a method that can be used to detect β-lactam antibiotics simultaneously in livestock products needs to be developed.

In this study, we aimed to detect 34 β-lactam compounds simultaneously in five livestock samples (beef, pork, chicken, egg, and milk) by using a method combining d-SPE and liquid chromatography-tandem mass spectrometry (LC–MS/MS). The method was validated in accordance with CODEX guidelines, and its applicability to real domestic livestock products and fishery foods, such as eel, flat fish, and shrimp, was assessed. This study was conducted to improve the efficiency of monitoring veterinary drug residues in domestic livestock products.

Materials and methods

Materials

All veterinary standards (penicillin and cephalosporin) were obtained from Sigma-Aldrich (St. Louis, MO, USA), Dr. Ehrenstorfer (Augsburg, Germany), and TRC (Windsor, CT, USA) and stored at − 80 °C. HPLC-grade acetonitrile and water were obtained from Merck (Rahway, NJ, USA). Monobasic potassium phosphate and dibasic potassium phosphate were obtained from Sigma-Aldrich. QuEChERS kits (C₁₈ 500 mg, C₁₈ 150 mg + MgSO₄ 900 mg, and 150 mg C₁₈ + 900 mg MgSO₄ + 300 mg PSA) were obtained from Chromatific (Heidenrod, Germany). Nylon and polytetrafluoroethylene (PTFE) syringe filters were obtained from Teknokroma (Barcelona, Spain).

All standard solutions were individually prepared at 1000 mg/L and stored at − 80 °C. Nafcillin, dicloxacillin, cefadroxil, cefazolin, cephapirin, cefalexin, cefotaxime, cefoperazone, cefuroxime, amoxicillin, oxacillin, cloxacillin, penicillin G, and penicillin V were diluted in 25% acetonitrile. Mecillinam, cefamandole, cefalonium, cephaloridine, cefalotin, cefetamet, cefepime, cefpodoxime, cefoxitin, cefsulodin, ceftiofur, desfuroyl ceftiofur, cefpirome, cefixime, ampicillin, piperacillin, and hetacillin were diluted in 50% acetonitrile. Aspoxicillin was diluted in 80% acetonitrile. Mezlocillin was diluted in 50% methanol. Cefacetrile, desacetyl cephapirin, and cefquinome were diluted in dimethyl sulfoxide. For sample extraction, 1% phosphate buffer solution was prepared by dissolving 8.0×g of potassium phosphate monobasic and 2.0 g of potassium phosphate dibasic in 1 L of water and then storing it at room temperature for 1 week.

Instrumental conditions

The analysis was conducted on a Shimadzu LCMS-8060 (Osaka, Japan) equipped with an Imtakt Cadenza CD-C18 column (150 mm × 2.0 mm i.d., 3.0 μm). To enhance sensitivity, peak shape, and resolution, 1% formic acid in water (A) and 1% formic acid in acetonitrile (B) were used. The LC gradient conditions were as follows: 2% B (0 min), increased up to 60% B (4.50 min), increased up to 95% B (5.50 min), held 95% B (8.00 min), rapidly decreased B to 2% (8.10 min), and held for stabilization (12.00 min). The flow rate was 0.3 mL/min, and the injection volume and temperature were 5 μL and 35 °C, respectively. The analytes were protonated or deprotonated in the electrospray ionization (ESI) mode. The capillary temperature was 200 °C, the capillary voltage was 3.6 kV in the positive mode and 2.8 kV in the negative mode, and the collision gas was argon. The specific multiple-reaction monitoring conditions are listed in Table 1.

Table 1.

Multiple reaction monitoring conditions of 34 β-lactams

Compounds	Ionization	Retention time (min)	Exact mass	Precursor ion	Product ion (Underlined ion for quantification)	Collision energy (eV)
Nafcillin	Positive	6.08	414.12	414.95	199.05, 171.00, 115.05	15, 35, 55
Dicloxacillin	Positive	6.24	469.03	469.75	160.10, 310.90, 114.10	15, 15, 40
Mecillinam	Positive	3.93	325.15	326.05	167.10, 139.05, 55.05	22, 31, 50
Cefadroxil	Positive	3.17	363.09	363.95	114.05, 208.05, 86.00	21, 10, 49
Cefamandole	Positive	4.62	462.08	462.95	347.10, 157.95, 140.00	10, 19, 25
Cefacetrile	Positive	3.84	339.05	357.00	280.05, 156.05, 251.90	10, 14, 15
Cefazolin	Positive	4.01	454.03	454.85	322.95, 155.95, 294.90	15, 16, 17
Cephapirin	Positive	3.25	423.06	423.90	292.00, 152.00, 124.05	16, 24, 46
Desacetyl cephapirina	Positive	2.76	381.05	381.90	152.00, 112.10, 291.95	14, 26, 25
Cefalexin	Positive	3.61	347.10	347.95	158.00, 106.05, 174.10	10, 28, 15
Cefalonium	Positive	3.68	458.07	458.85	336.95, 152.00, 123.10	11, 21, 12
Cephaloridine	Positive	3.77	415.07	415.90	152.00, 336.95, 80.05	20, 11, 11
Cefalotin	Positive	5.03	396.05	418.85	359.00, 314.95, 204.00	13, 14, 18
Cefetamet	Positive	3.77	397.05	397.90	241.05, 126.00	16, 28
Cefepime	Positive	2.92	480.13	480.95	395.85, 124.85, 86.00	12, 49, 14
Cefpodoxime	Positive	3.64	427.06	427.85	241.10, 124.95	21, 48
Cefotaxime	Positive	3.79	455.06	455.85	396.10, 323.95, 124.95	11, 14, 52
Cefoperazone	Positive	4.33	645.14	645.85	143.10, 530.10, 290.05	37, 12, 22
Cefoxitin	Positive	4.43	427.05	449.85	389.00, 344.90	11, 15
Cefuroxime	Negative	4.28	424.07	422.85	206.95, 317.95, 362.00	14, 9, 9
Cefsulodin	Positive	3.03	532.07	532.90	123.05, 330.90, 228.00	15, 15, 38
Cefquinome	Positive	3.41	528.13	528.95	134.05, 395.90, 125.00	15, 14, 55
Ceftiofur	Positive	4.72	523.03	523.85	240.95, 125.00	17, 52
Desfuroyl ceftiofura	Positive	4.00	429.10	429.85	125.15, 258.80, 284.65	50, 31, 16
Cefpirome	Positive	3.23	514.11	514.85	120.10, 323.95, 395.95	21, 18, 13
Cefixime	Positive	3.78	453.04	453.85	285.00, 301.05, 126.00	23, 15, 30
Amoxicillin	Positive	3.07	365.10	365.95	349.00, 114.05, 208.00	9, 21, 13
Ampicillin	Positive	3.58	349.11	350.00	106.10, 192.00, 160.05	23, 16, 13
Oxacillin	Positive	5.76	401.10	401.90	243.00, 160.00, 114.00	14, 15, 37
Cloxacillin	Positive	5.99	435.27	435.70	276.95, 159.95, 114.00	15, 14, 39
Penicillin G	Positive	5.30	334.10	334.95	160.00, 176.10, 114.05	12, 13, 33
Penicillin V	Positive	5.58	350.09	350.95	160.00, 114.10, 86.95	12, 31, 46
Piperacillin	Positive	4.96	517.16	517.95	160.00, 115.00, 143.05	21, 55, 12
Hetacillin	Positive	3.57	389.14	390.50	350.05, 106.05	9, 22
Aspoxicillin	Positive	3.22	493.16	493.95	250.00, 101.00, 348.95	19, 30, 18
Mezlocillin	Positive	5.27	539.11	539.85	295.95, 252.95, 131.95	21, 32, 46

^aDesacetyl cephapirin and desfuroyl ceftiofur are metabolites of cephapirin and ceftiofur, respectively

Sample collection

All samples were purchased from a domestic market in Korea, and five types of samples were obtained (beef, pork, chicken, egg, and milk). Except for milk, all other samples were homogenized using a blender and then placed in zipper bags. Milk samples were poured into a 50 mL centrifuge tube. All samples were stored at − 20 °C before extraction. Blank samples confirmed negative for all the target analytes for each sample type were used to check for contamination during sample preparation and instrument analysis.

Sample preparation

Homogenized samples (2 g) were prepared in 50 mL centrifuge tubes. Two extraction solutions as solvents were used for comparison: (1) 3 mL of 1% phosphate buffer and 7 mL of acetonitrile and (2) water:acetonitrile (1:4, v/v). The extraction solvent was then added to the samples. Each mixture was shaken for 10 min and centrifuged at 4700×g for 10 min at 4 °C. For sample clean-up, each extract was decanted into 50 mL centrifuge tubes containing each of the three QuEChERS kits for comparison. Each mixture was shaken for 10 min and then centrifuged at 4700×g for 10 min at 4 °C. The supernatant (5 mL) was transferred into a 15 mL centrifuge tube and then concentrated to less than 1 mL (approximately 0.5 mL) under gentle nitrogen gas at 40 °C. The concentrate was diluted to 1 mL with water, and then the mixture was analyzed using LC–MS/MS after filtering with a syringe filter.

Method validation

In accordance with the CODEX guideline (CAC/GL 71-2009, 2009), the linearity, accuracy, precision, and detection limits of the method were validated. Linearity was represented as the coefficient of determination (r²), and the five points of the calibration curve were ¼ MRL, ½ MRL, MRL, 2 MRL, and 4 MRL. Accuracy and precision were confirmed by repeating five times at three levels (1/2 MRL, MRL, and 2 MRL) and represented as recovery and coefficient of variation (CV), respectively. The limit of detection (LOD) and limit of quantification (LOQ) were calculated as 3.3 and 10 times the standard deviation divided by the slope, respectively. Method validation was performed in three different laboratories to confirm inter-lab reproducibility.

Results and discussion

Liquid chromatographic (LC) method

To obtain an optical instrumental method, LC methods were compared for their capacities to separate 34 β-lactam compounds simultaneously. The following three mobile phases were compared: (1) 0.1% formic acid in water and 0.1% formic acid in acetonitrile, (2) 0.1% formic acid in water and 0.1% formic acid in acetonitrile:methanol (50:50, v/v), and (3) 0.05 mM ammonium formate in water and 0.1% formic acid in methanol (Bessaire et al., 2018; FSIS, 2011). The first mobile phase showed better sensitivity, resolution, and peak shape than the other mobile phases. Cefacetrile and desfuroyl ceftiofur were unsuitable for the second and third mobile phases because of their poor resolution and peak shape. In the first mobile phase, cefacetrile showed a higher sensitivity at the oven temperature of 35 °C than 40 °C. Therefore, the 34 β-lactam compounds were isolated using 0.1% formic acid in water and 0.1% formic acid in acetonitrile at 35 °C.

Optimization of clean-up and filtration without livestock samples

QuEChERS was performed for clean-up and filtration without any sample to obtain sufficient quantities of the 34 β-lactam compounds. The three QuEChERS kits were used, and the filters were nylon and PTFE. The comparison was performed without food samples, and the analytes (20 ng) were spiked into 10 mL of water/acetonitrile (1:4, v/v) and repeated five times. As a result, most analytes were sufficient when PTFE was used after clean-up with C₁₈. By contrast, the nylon filter showed low recoveries for some compounds, such as cefsulodin, cefixime, ceftiofur, and dicloxacillin. Various types of syringe filters are widely used for sample preparation in analytical laboratories. However, incorrect filter selection can result in analyte loss owing to surface absorption. This loss has been related to the chemical properties, such as acidity, of the analyte (Carlson and Thompson, 2000). In the present study, PTFE was more suitable for β-lactams than nylon. PSA showed low recovery for cefsulodin and cefixime while excessively high recovery for cefuroxime. PSA is a sorbent that retains fatty acids and other organic acids, and it is also reportedly unsuitable for β-lactams because of its carboxylic group (Rocco et al., 2017). Therefore, the method was optimized with a PTFE filter, and sample clean-up was compared without PSA.

Some penicillins are unsuitable for hexane washing (Rocco et al., 2017), and dry concentrations may cause the loss of target compounds. Thus, hexane-based clean-up and dry concentration methods were not used in the present study. Analysis of the effects of concentration showed that when the sample was concentrated to less than 1 mL (approximately 0.5 mL), the intensity was equal to or 1.1–1.3 times higher than that of the dry concentration. In particular, desfuroyl ceftiofur was the most sensitive to concentration, showing 6.3 times higher intensity than that of the dry concentration.

Optimization of clean-up method and extraction solvent

Two QuEChERS kits (C₁₈ 500 mg and C₁₈ 150 mg + MgSO₄ 900 mg) were compared to identify an appropriate clean-up method for the five types of samples. As shown in Fig. 1, all of the livestock samples, except for pork, were sufficient when the QuEChERS kit C₁₈ 150 mg + MgSO₄ 900 mg was used. C₁₈ helps obtain clean extracts by removing hydrophobic matter, such as animal origin matrix, from samples. In addition, salts such as MgSO₄, NaCl, and NaSO₄ are effective in extracting analytes in the aqueous layer into the organic layer. As a result, sample clean-up was more efficient in the presence than in the absence of a salt. Therefore, C₁₈ 150 mg + MgSO₄ 900 mg was used in the present study. However, some targets, such as desacetyl cephapirin, cefuroxime, cefquinome, and desfuroyl ceftiofur, showed low recovery and repeatability.

Fig. 1.

Results of three methods to compare two types of clean-up kits and extraction solvents for five livestock samples (n = 5). *In methods 1 and 2, samples were purified with 500 mg C₁₈ and 150 mg C₁₈ + 900 mg MgSO₄, respectively, and then extracted with water/ACN (1:4, v/v). In method 3, samples were purified with 150 mg C₁₈ + 900 mg MgSO₄ and then extracted with 1% phosphate buffer/ACN (3:7, v/v). **Y-axis represents the number of β-lactams that satisfy recovery (%) and CV (%) in accordance with the CODEX guidelines. This number includes 34 β-lactams and two metabolites (desacetyl cephapirin and desfuroyl ceftiofur). ACN acetonitrile

In previous studies, β-lactams were extracted from food samples with water/acetonitrile, acetic acid, phosphate buffer, etc. Acetonitrile and water/acetonitrile are commonly used solvents for antibiotic extraction. The extraction solvents were compared to obtain satisfactory results for all targets (Fig. 1). All targets showed sufficient recovery and repeatability when 1% phosphate buffer and acetonitrile were used, but the recovery and repeatability of some targets, such as cefacetrile, cefazolin, cefotaxime, desfuroyl ceftiofur, and cefixime, were insufficient to be quantified in chicken. Only chicken samples showed satisfactory results when water/acetonitrile (1:4, v/v) was used. Additionally, 1% acetic acid in acetonitrile was tested on pork, only 27 targets satisfied the CODEX guidelines. Therefore, 1% phosphate buffer with acetonitrile was used for the beef, pork, egg, and milk samples, whereas water/acetonitrile (1:4, v/v) was used for the chicken samples.

Considering that different extraction solvents must be used depending on the type of sample, some components are presumed to be affected by the matrix. Therefore, future studies may require the development of methods that can improve these limitations and improve the efficiency of food analysis.

Method validation

Method validation was conducted for six types of samples (beef, pork, fat, chicken, egg, and milk) and evaluated for linearity, accuracy, precision, and detection limit. Linearity was represented as the coefficient of determination (r²), which was greater than 0.98 in all targets. The accuracy and precision of the method are represented as recovery and CV (Tables 2 and 3). The recovery of the method was 70.4–117% for beef, 66.1–117% for pork, 73.7–118% for fat (pork), 72.1–105% for chicken, 75.8–117% for egg, and 76.2–119% for milk. The CV of the method was 1.8–23% for beef, 1.5–25% for pork, 2.4–23% for fat (pork), 1.7–26% for chicken, 2.1–25% for egg, and 1.9–23% for milk. Overall, the recovery and CV of the method were 66.1–119% and 1.5–26%, respectively. Inter-lab results were expressed as inter-average and inter-CV, which were 76.1–111% and 2.8–26%, respectively, for the six types of samples. All results were within the range of values ​​required for each spiking concentration in accordance with the CODEX guidelines.

Table 2.

Accuracy and precision of the method for detecting 34 β-lactams in beef, pork, and fat (pork) samples from three laboratories (n = 5)

Compounds	Beef					Pork					Fat (pork)
	Spiking level (mg/kg)	Recovery (%)			Inter-lab.	Spiking level (mg/kg)	Recovery (%)			Inter-lab.	Spiking level (mg/kg)	Recovery (%)			Inter-lab.
		Lab. A	Lab. B	Lab. C			Lab. A	Lab. B	Lab. C			Lab. A	Lab. B	Lab. C
Nafcillin	0.15	94.4 ± 6.7	90.1 ± 5.1	105 ± 4.8	96.0 ± 8.0	0.005	95.9 ± 5.3	92.5 ± 6.2	103 ± 13	96.7 ± 8.4	0.005	102 ± 9.0	83.7 ± 2.8	107 ± 2.5	98.2 ± 12
	0.3	97.0 ± 4.7	97.5 ± 4.1	106 ± 11	99.5 ± 7.5	0.01	93.5 ± 11	97.1 ± 7.2	93.8 ± 6.4	94.6 ± 8.4	0.01	87.0 ± 12	86.9 ± 2.2	96.0 ± 3.1	89.4 ± 8.9
	0.6	102 ± 6.3	95.3 ± 3.0	107 ± 11	102 ± 7.8	0.02	79.8 ± 9.8	103 ± 2.9	95.5 ± 6.5	90.4 ± 13	0.02	85.7 ± 11	92.0 ± 4.4	101 ± 4.0	91.7 ± 10
Dicloxacillin	0.15	93.6 ± 9.1	100 ± 6.3	99.2 ± 7.3	97.0 ± 7.8	0.15	102 ± 4.9	95.1 ± 4.9	103 ± 1.0	101 ± 5.2	0.15	109 ± 14	97.1 ± 4.0	98.9 ± 2.1	103 ± 11
	0.3	99.3 ± 2.4	104 ± 3.8	97.5 ± 10	100 ± 5.6	0.3	103 ± 12	96.0 ± 6.1	99.0 ± 5.7	100 ± 9.1	0.3	83.4 ± 10	98.4 ± 7.8	108 ± 2.9	96.5 ± 13
	0.6	110 ± 8.9	100 ± 4.6	90.6 ± 8.5	101 ± 11	0.6	93.8 ± 11	103 ± 1.5	102 ± 4.4	98.7 ± 8.3	0.6	104 ± 12	99.1 ± 5.5	103 ± 7.2	103 ± 8.9
Mecillinam	0.005	98.0 ± 2.7	97.1 ± 4.4	98.8 ± 4.6	98.0 ± 3.4	0.005	95.4 ± 3.0	95.3 ± 9.8	87.0 ± 5.7	93.1 ± 6.9	0.005	90.2 ± 3.7	102 ± 6.0	103 ± 6.9	96.8 ± 8.1
	0.01	94.1 ± 3.4	99.4 ± 5.1	94.2 ± 3.1	95.6 ± 4.3	0.01	91.9 ± 4.4	94.0 ± 8.7	108 ± 4.9	96.8 ± 9.0	0.01	88.1 ± 7.2	99.2 ± 2.9	94.5 ± 5.1	92.9 ± 7.3
	0.02	87.3 ± 4.6	105 ± 0.5	98.9 ± 2.2	95.2 ± 8.8	0.02	90.9 ± 3.7	97.0 ± 2.9	102 ± 4.9	95.7 ± 6.3	0.02	95.0 ± 4.6	101 ± 3.5	94.6 ± 3.2	96.6 ± 4.7
Cefadroxil	0.005	104 ± 4.9	112 ± 8.1	103 ± 5.0	106 ± 6.5	0.005	107 ± 9.6	96.3 ± 6.3	85.4 ± 7.5	98.3 ± 13	0.005	101 ± 9.0	90.8 ± 14	104 ± 4.1	99.0 ± 10
	0.01	102 ± 4.0	101 ± 17	116 ± 6.7	106 ± 10	0.01	101 ± 7.0	103 ± 2.8	104 ± 3.1	102 ± 5.0	0.01	103 ± 9.7	93.6 ± 7.9	104 ± 7.8	101 ± 9.1
	0.02	95.2 ± 6.0	95.4 ± 13	108 ± 3.7	98.6 ± 9.1	0.02	103 ± 4.0	106 ± 5.7	100 ± 4.1	103 ± 4.6	0.02	117 ± 9.4	104 ± 9.4	102 ± 3.1	109 ± 10
Cefamandole	0.005	103 ± 5.4	99.0 ± 10	105 ± 8.7	102 ± 7.2	0.005	101 ± 3.6	85.6 ± 6.5	98.0 ± 3.6	95.8 ± 7.9	0.005	102 ± 8.8	94.1 ± 7.9	92.4 ± 11	97.2 ± 9.5
	0.01	111 ± 6.0	102 ± 2.3	106 ± 1.0	107 ± 5.4	0.01	106 ± 4.3	93.0 ± 7.2	105 ± 4.3	102 ± 7.3	0.01	107 ± 11	95.4 ± 4.7	90.1 ± 6.3	99.4 ± 12
	0.02	107 ± 3.6	99.5 ± 4.9	102 ± 4.1	104 ± 4.8	0.02	102 ± 5.9	98.2 ± 2.2	100 ± 3.5	100 ± 4.4	0.02	116 ± 9.7	102 ± 2.0	94.1 ± 2.5	106 ± 12
Cefacetrile	0.015	112 ± 9.2	103 ± 6.8	99.6 ± 13	106 ± 10	0.005	117 ± 15	105 ± 4.8	87.5 ± 11	106 ± 17	0.005	101 ± 13	99.9 ± 8.4	95.6 ± 4.9	99.1 ± 9.5
	0.03	113 ± 5.4	105 ± 11	93.5 ± 1.6	105 ± 10	0.01	110 ± 9.6	103 ± 4.2	90.1 ± 5.4	103 ± 11	0.01	99.3 ± 6.8	95.5 ± 2.5	95.1 ± 2.9	97.1 ± 5.2
	0.06	113 ± 5.1	98.5 ± 2.5	100 ± 10	106 ± 8.9	0.02	97.7 ± 4.8	84.3 ± 5.1	97.3 ± 5.6	93.9 ± 8.0	0.02	103 ± 6.5	101 ± 5.9	104 ± 4.6	102 ± 5.4
Cefazolin	0.025	104 ± 5.0	101 ± 7.0	93.2 ± 11	101 ± 8.2	0.025	107 ± 2.8	96.6 ± 13	95.8 ± 2.6	101 ± 7.9	0.025	93.9 ± 4.0	96.9 ± 7.3	98.5 ± 4.2	95.9 ± 5.0
	0.05	106 ± 5.0	101 ± 2.6	88.6 ± 8.0	99.8 ± 8.9	0.05	98.4 ± 5.1	96.7 ± 8.0	99.9 ± 3.5	98.4 ± 5.2	0.05	91.6 ± 7.1	95.8 ± 0.8	111 ± 9.6	97.9 ± 11
	0.1	99.4 ± 8.4	101 ± 6.7	98.1 ± 4.6	99.4 ± 6.5	0.1	97.9 ± 5.4	95.2 ± 2.8	102 ± 3.8	98.3 ± 4.9	0.1	99.4 ± 3.3	103 ± 3.6	92.7 ± 6.7	98.6 ± 5.7
Cephapirin	0.025	97.8 ± 2.9	108 ± 3.5	95.3 ± 5.6	99.9 ± 6.4	0.005	93.1 ± 5.5	98.9 ± 8.8	94.9 ± 0.4	95.2 ± 5.9	0.005	88.6 ± 4.9	99.4 ± 13	97.3 ± 3.4	93.9 ± 8.8
	0.05	96.0 ± 2.6	105 ± 4.6	104 ± 1.4	101 ± 5.2	0.01	95.3 ± 3.9	99.2 ± 8.8	102 ± 0.8	98.1 ± 5.4	0.01	87.1 ± 5.8	99.6 ± 1.8	95.8 ± 2.7	92.9 ± 7.2
	0.1	92.7 ± 4.4	95.9 ± 9.3	104 ± 1.2	96.8 ± 7.2	0.02	98.9 ± 4.7	99.3 ± 2.0	96.1 ± 1.4	98.3 ± 3.5	0.02	94.3 ± 5.2	99.2 ± 9.0	97.8 ± 4.0	96.6 ± 6.0
Desacetyl cephapirin	0.025	102 ± 5.8	104 ± 11	100 ± 14	102 ± 8.8	0.005	102 ± 3.7	94.1 ± 7.9	93.8 ± 4.7	97.4 ± 6.2	0.005	95.4 ± 6.4	84.7 ± 16	102 ± 1.4	94.4 ± 11
	0.05	101 ± 4.1	90.1 ± 15	99.0 ± 7.4	97.4 ± 8.9	0.01	101 ± 4.3	104 ± 9.5	97.7 ± 4.5	101 ± 6.0	0.01	81.7 ± 5.0	98.5 ± 9.2	99.6 ± 6.2	91.2 ± 12
	0.1	96.1 ± 5.7	88.0 ± 21	104 ± 2.9	95.9 ± 11	0.02	102 ± 6.9	103 ± 11	88.0 ± 6.8	98.7 ± 10	0.02	94.7 ± 4.3	101 ± 17	96.2 ± 4.2	96.9 ± 9.0
Cefalexin	0.1	99.5 ± 2.9	99.7 ± 5.3	96.6 ± 2.6	98.7 ± 3.5	0.1	99.6 ± 5.2	102 ± 5.8	98.1 ± 5.0	100 ± 5.0	0.1	105 ± 8.5	102 ± 9.9	106 ± 2.2	104 ± 7.2
	0.2	99.8 ± 2.0	104 ± 5.8	101 ± 1.6	102 ± 3.6	0.2	95.3 ± 1.8	98.6 ± 10	106 ± 3.9	99.0 ± 6.8	0.2	108 ± 3.9	97.0 ± 3.8	97.9 ± 3.9	102 ± 6.3
	0.4	96.7 ± 5.4	91.7 ± 4.5	104 ± 2.4	97.2 ± 6.3	0.4	95.1 ± 4.4	99.3 ± 3.7	104 ± 2.0	98.6 ± 5.0	0.4	115 ± 3.8	100 ± 4.5	97.5 ± 1.0	106 ± 8.8
Cefalonium	0.005	98.0 ± 5.6	104 ± 4.4	93.5 ± 6.5	98.3 ± 6.4	0.005	93.5 ± 7.5	99.6 ± 5.8	91.7 ± 11	94.6 ± 7.9	0.005	97.0 ± 4.5	102 ± 4.9	101 ± 1.9	99.6 ± 4.4
	0.01	98.0 ± 5.3	100 ± 3.5	93.5 ± 5.9	97.4 ± 5.3	0.01	96.7 ± 1.9	95.6 ± 12	96.8 ± 11	96.4 ± 7.4	0.01	93.4 ± 9.0	95.3 ± 2.0	100 ± 4.1	95.8 ± 6.7
	0.02	94.1 ± 8.6	97.9 ± 6.2	93.8 ± 2.4	95.1 ± 6.5	0.02	98.5 ± 5.8	91.1 ± 3.1	94.3 ± 4.7	95.4 ± 5.7	0.02	96.4 ± 5.0	98.0 ± 2.9	95.6 ± 14	96.6 ± 7.1
Cephaloridine	0.005	101 ± 3.6	99.5 ± 8.7	100 ± 12	100 ± 7.1	0.005	97.6 ± 3.1	98.3 ± 9.1	99.1 ± 3.0	98.2 ± 4.8	0.005	95.7 ± 1.0	106 ± 6.2	103 ± 7.0	100 ± 6.3
	0.01	100 ± 5.9	93.4 ± 2.7	98.5 ± 1.0	97.9 ± 5.1	0.01	98.4 ± 1.5	93.0 ± 7.0	95.0 ± 5.2	96.0 ± 4.7	0.01	94.4 ± 8.7	104 ± 4.2	101 ± 16	98.8 ± 10
	0.02	94.9 ± 6.2	94.7 ± 6.5	103 ± 4.7	97.0 ± 6.5	0.02	95.7 ± 5.8	91.0 ± 2.1	100 ± 1.9	95.6 ± 5.4	0.02	102 ± 4.2	101 ± 1.3	95.6 ± 1.4	99.7 ± 3.9
Cefalotin	0.005	99.8 ± 5.8	89.6 ± 7.1	90.5 ± 6.4	94.5 ± 7.8	0.005	98.2 ± 2.9	92.9 ± 9.3	105 ± 4.0	98.6 ± 6.6	0.005	113 ± 8.7	107 ± 12	95.9 ± 7.8	107 ± 11
	0.01	98.3 ± 2.8	97.5 ± 4.3	101 ± 4.4	98.8 ± 3.6	0.01	98.2 ± 5.7	95.8 ± 5.8	99.7 ± 5.6	97.9 ± 5.3	0.01	112 ± 8.1	99.6 ± 7.9	91.0 ± 9.0	103 ± 12
	0.02	99.4 ± 5.7	97.9 ± 3.7	99.5 ± 3.7	99.0 ± 4.4	0.02	93.1 ± 7.3	98.1 ± 4.5	98.3 ± 7.8	95.9 ± 6.7	0.02	117 ± 5.7	101 ± 4.1	93.2 ± 6.2	106 ± 11
Cefetamet	0.005	104 ± 7.9	106 ± 3.0	99.8 ± 2.9	104 ± 5.9	0.005	104 ± 4.1	102 ± 9.7	98.7 ± 3.3	102 ± 5.7	0.005	97.2 ± 4.5	103 ± 5.5	95.9 ± 0.8	98.4 ± 4.9
	0.01	106 ± 5.8	102 ± 1.8	98.8 ± 3.7	103 ± 5.2	0.01	102 ± 3.4	94.1 ± 11	97.3 ± 1.6	98.4 ± 6.2	0.01	98.0 ± 4.7	100 ± 1.5	100 ± 2.0	99.1 ± 3.3
	0.02	96.5 ± 6.6	97.2 ± 9.8	107 ± 7.8	99.6 ± 8.5	0.02	97.5 ± 5.0	84.5 ± 2.8	101 ± 2.8	95.0 ± 8.1	0.02	101 ± 5.7	102 ± 2.8	94.4 ± 2.0	99.5 ± 5.1
Cefepime	0.005	109 ± 8.1	106 ± 5.4	109 ± 6.5	108 ± 6.5	0.005	71.8 ± 20	101 ± 22	98.5 ± 6.5	87.0 ± 23	0.005	93.9 ± 12	100 ± 12	106 ± 0.9	99.4 ± 10
	0.01	97.5 ± 2.5	108 ± 10	103 ± 9.9	102 ± 8.1	0.01	89.8 ± 12	98.2 ± 13	105 ± 4.5	96.1 ± 12	0.01	84.1 ± 23	98.7 ± 4.9	108 ± 5.7	95.7 ± 17
	0.02	92.5 ± 8.0	92.6 ± 3.9	95.8 ± 8.3	93.4 ± 6.7	0.02	91.4 ± 16	108 ± 5.1	97.0 ± 7.6	97.5 ± 13	0.02	81.1 ± 13	96.0 ± 5.5	111 ± 3.7	93.4 ± 16
Cefpodoxime	0.005	94.5 ± 6.1	100 ± 12	94.8 ± 6.6	96.1 ± 7.9	0.005	97.6 ± 16	106 ± 9.4	92.4 ± 5.1	98.5 ± 12	0.005	83.1 ± 7.2	107 ± 2.9	106 ± 3.1	95.9 ± 14
	0.01	92.2 ± 4.6	95.4 ± 6.0	99.5 ± 3.4	95.1 ± 5.3	0.01	94.1 ± 2.8	93.1 ± 7.8	97.5 ± 3.7	94.8 ± 4.6	0.01	87.6 ± 3.3	99.4 ± 1.9	94.2 ± 5.3	92.6 ± 6.4
	0.02	89.9 ± 3.5	97.3 ± 4.5	103 ± 8.7	95.6 ± 8.0	0.02	93.6 ± 6.1	84.9 ± 2.4	98.9 ± 0.5	92.7 ± 7.2	0.02	91.3 ± 5.1	99.5 ± 1.2	99.2 ± 10	95.7 ± 7.2
Cefotaxime	0.005	98.6 ± 4.1	98.7 ± 8.1	98.1 ± 10	98.5 ± 6.3	0.005	104 ± 5.4	98.6 ± 13	97.2 ± 1.1	101 ± 7.4	0.005	94.4 ± 5.6	94.9 ± 6.3	103 ± 1.8	97.0 ± 6.1
	0.01	96.5 ± 5.2	106 ± 5.1	95.5 ± 6.5	98.7 ± 6.7	0.01	99.1 ± 5.8	94.5 ± 12	104 ± 2.9	99.1 ± 7.3	0.01	87.8 ± 7.6	98.4 ± 4.1	95.6 ± 3.1	92.8 ± 7.4
	0.02	93.6 ± 8.0	108 ± 6.6	105 ± 2.7	101 ± 9.0	0.02	93.6 ± 6.5	80.4 ± 1.9	103 ± 13	92.6 ± 12	0.02	96.5 ± 2.5	106 ± 2.3	98.4 ± 4.0	99.5 ± 4.8
Cefoperazone	0.005	100 ± 4.1	105 ± 3.6	94.9 ± 1.1	100 ± 4.9	0.005	90.2 ± 4.8	94.6 ± 7.0	93.3 ± 6.0	92.2 ± 5.6	0.005	102 ± 3.3	99.4 ± 3.5	91.4 ± 14	98.5 ± 7.9
	0.01	98.4 ± 2.9	103 ± 4.3	104 ± 2.9	101 ± 4.0	0.01	103 ± 3.6	96.1 ± 4.9	96.8 ± 2.5	99.3 ± 4.7	0.01	96.5 ± 12	96.0 ± 3.4	90.1 ± 3.6	94.6 ± 8.4
	0.02	98.4 ± 3.2	101 ± 1.3	112 ± 5.4	103 ± 6.7	0.02	108 ± 2.9	96.1 ± 1.1	92.0 ± 9.2	100 ± 8.4	0.02	104 ± 6.2	101 ± 4.1	82.1 ± 6.8	97.1 ± 11
Cefoxitin	0.005	102 ± 11	100 ± 11	94.8 ± 4.9	99.4 ± 9.1	0.005	83.3 ± 11	84.3 ± 12	98.4 ± 4.3	87.7 ± 12	0.005	118 ± 14	95.8 ± 16	91.9 ± 2.5	105 ± 17
	0.01	95.9 ± 7.1	106 ± 9.9	90.0 ± 7.9	97.1 ± 9.8	0.01	90.8 ± 3.9	97.7 ± 20	114 ± 1.1	98.9 ± 14	0.01	101 ± 8.9	104 ± 4.9	89.2 ± 5.7	98.5 ± 9.1
	0.02	94.2 ± 3.6	110 ± 7.1	90.5 ± 5.0	97.5 ± 9.6	0.02	94.0 ± 3.1	95.3 ± 6.0	102 ± 12	96.5 ± 7.3	0.02	105 ± 6.1	105 ± 4.1	95.9 ± 17	103 ± 9.2
Cefuroxime	0.01	117 ± 23	104 ± 7.1	98.0 ± 2.7	108 ± 18	0.005	103 ± 23	86.2 ± 15	69.4 ± 12	89.3 ± 25	0.005	104 ± 5.8	85.4 ± 14	88.1 ± 15	94.4 ± 13
	0.02	101 ± 11	103 ± 4.9	91.6 ± 16	99.2 ± 11	0.01	91.2 ± 25	91.7 ± 9.0	74.3 ± 13	85.7 ± 18	0.01	106 ± 7.7	96.2 ± 5.0	101 ± 18	102 ± 11
	0.04	113 ± 6.6	101 ± 4.3	103 ± 2.7	107 ± 7.5	0.02	107 ± 11	91.3 ± 8.6	89.9 ± 9.9	98.2 ± 13	0.02	106 ± 8.4	106 ± 6.9	104 ± 11	106 ± 7.9
Cefsulodin	0.005	95.8 ± 7.1	106 ± 9.9	93.8 ± 7.6	98.1 ± 9.1	0.005	93.4 ± 10	100 ± 14	91.6 ± 6.8	94.7 ± 11	0.005	94.5 ± 8.6	88.6 ± 16	106 ± 7.7	96.0 ± 12
	0.01	99.3 ± 5.9	98.8 ± 19	95.9 ± 8.6	98.2 ± 10	0.01	90.5 ± 8.1	105 ± 9.5	111 ± 2.3	100 ± 12	0.01	82.5 ± 4.8	95.5 ± 3.3	107 ± 2.3	92.6 ± 12
	0.02	95.6 ± 6.5	94.8 ± 14	102 ± 10	97.1 ± 9.4	0.02	98.3 ± 5.8	106 ± 5.0	95.3 ± 6.0	99.6 ± 6.7	0.02	98.3 ± 6.1	101 ± 15	98.2 ± 7.1	98.9 ± 8.6
Cefquinome	0.025	97.6 ± 5.4	104 ± 8.1	97.6 ± 8.9	99.4 ± 7.1	0.025	101 ± 1.8	93.6 ± 6.6	99.1 ± 3.5	98.6 ± 4.8	0.025	76.6 ± 16	96.2 ± 7.0	106 ± 3.2	90.0 ± 18
	0.05	98.5 ± 6.4	97.8 ± 14	97.6 ± 1.6	98.1 ± 7.6	0.05	101 ± 5.1	95.3 ± 13	89.7 ± 15	96.2 ± 10	0.05	95.6 ± 13	96.7 ± 5.9	93.2 ± 5.7	95.2 ± 9.4
	0.1	95.9 ± 7.0	93.9 ± 13	105 ± 9.0	97.7 ± 9.3	0.1	99.5 ± 4.0	94.5 ± 2.4	95.0 ± 1.9	96.9 ± 3.9	0.1	108 ± 10	101 ± 5.7	93.6 ± 4.2	102 ± 9.7
Ceftiofur	0.5	99.6 ± 2.1	93.8 ± 5.8	97.1 ± 3.3	97.4 ± 4.1	0.5	103 ± 7.0	91.7 ± 12	94.8 ± 1.8	97.6 ± 8.8	1	106 ± 6.8	94.8 ± 10	97.0 ± 3.8	101 ± 8.3
	1	92.6 ± 5.0	94.6 ± 4.6	99.3 ± 0.8	95.0 ± 4.8	1	108 ± 8.3	92.9 ± 6.0	100 ± 1.5	101 ± 8.8	2	94.2 ± 7.2	98.0 ± 3.9	87.2 ± 3.4	93.2 ± 6.8
	2	101 ± 6.2	97.8 ± 6.6	100 ± 2.0	99.7 ± 5.1	2	108 ± 4.2	92.5 ± 2.3	98.7 ± 2.0	101 ± 7.6	4	98.5 ± 5.9	103 ± 1.6	87.7 ± 0.5	96.5 ± 7.4
Desfuroyl ceftiofur	0.5	97.2 ± 5.5	103 ± 5.8	98.2 ± 4.2	99.0 ± 5.4	0.5	110 ± 11	100 ± 3.6	102 ± 11	105 ± 9.5	1	106 ± 4.7	76.8 ± 8.2	105 ± 5.0	97.7 ± 15
	1	84.3 ± 6.2	94.2 ± 9.8	102 ± 6.9	91.7 ± 11	1	103 ± 8.7	92.9 ± 6.5	108 ± 1.7	102 ± 8.6	2	96.9 ± 6.9	95.3 ± 4.7	87.2 ± 2.2	93.8 ± 6.9
	2	72.5 ± 11	95.0 ± 15	105 ± 2.4	87.5 ± 19	2	95.5 ± 6.2	92.6 ± 8.9	108 ± 4.1	98.0 ± 8.6	4	102 ± 3.8	99.2 ± 4.6	94.7 ± 2.7	99.1 ± 4.6
Cefpirome	0.005	97.3 ± 12	107 ± 6.6	90.8 ± 21	98.1 ± 14	0.005	66.1 ± 12	87.1 ± 17	108 ± 1.4	83.3 ± 24	0.005	90.4 ± 17	101 ± 23	87.2 ± 11	92.3 ± 17
	0.01	94.4 ± 8.3	101 ± 5.8	105 ± 11	99.2 ± 9.2	0.01	79.4 ± 8.5	91.4 ± 10	92.4 ± 10	86.2 ± 11	0.01	91.6 ± 13	111 ± 1.6	89.8 ± 6.7	96.5 ± 13
	0.02	94.6 ± 9.9	95.5 ± 4.6	108 ± 2.1	98.6 ± 9.0	0.02	78.5 ± 4.4	106 ± 5.0	91.3 ± 5.9	89.6 ± 14	0.02	109 ± 16	94.8 ± 3.2	85.3 ± 2.7	98.4 ± 15
Cefixime	0.005	113 ± 8.9	94.4 ± 7.2	96.1 ± 17	104 ± 13	0.005	84.6 ± 12	92.6 ± 13	94.9 ± 0.4	89.6 ± 11	0.005	89.7 ± 7.4	98.1 ± 5.5	97.0 ± 3.9	94.0 ± 7.0
	0.01	102 ± 5.4	97.2 ± 1.7	95.6 ± 9.4	99.0 ± 6.2	0.01	91.3 ± 2.2	83.0 ± 18	85.8 ± 4.9	87.5 ± 9.2	0.01	84.6 ± 7.4	99.7 ± 10	105 ± 4.2	94.2 ± 12
	0.02	73.6 ± 14	93.3 ± 5.5	103 ± 7.1	87.1 ± 18	0.02	92.6 ± 6.6	81.9 ± 12	101 ± 7.8	92.0 ± 11	0.02	94.8 ± 5.5	101 ± 17	99.7 ± 11	97.8 ± 10
Amoxicillin	0.025	99.3 ± 2.9	107 ± 4.4	103 ± 5.2	102 ± 4.7	0.025	98.9 ± 7.1	104 ± 11	97.4 ± 2.9	99.9 ± 7.4	0.025	101 ± 5.0	91.4 ± 9.3	102 ± 2.3	98.7 ± 7.0
	0.05	96.6 ± 3.3	99.7 ± 5.3	102 ± 2.7	98.8 ± 4.1	0.05	94.7 ± 5.2	101 ± 11	94.9 ± 1.3	96.5 ± 6.8	0.05	92.6 ± 6.7	93.8 ± 2.1	100 ± 2.8	95.0 ± 5.7
	0.1	89.8 ± 5.6	87.8 ± 3.4	103 ± 3.9	92.9 ± 8.3	0.1	93.1 ± 6.4	103 ± 0.9	93.8 ± 2.5	95.9 ± 6.1	0.1	99.0 ± 2.8	92.5 ± 7.2	95.8 ± 3.3	96.3 ± 4.9
Ampicillin	0.025	99.1 ± 2.9	104 ± 3.7	95.9 ± 1.3	99.7 ± 4.2	0.025	97.0 ± 3.6	98.7 ± 9.3	101 ± 0.2	98.5 ± 5.0	0.025	94.2 ± 5.6	97.2 ± 4.4	101 ± 2.9	96.8 ± 5.1
	0.05	94.2 ± 2.8	101 ± 3.9	98.9 ± 2.2	97.3 ± 4.1	0.05	92.5 ± 4.7	98.4 ± 12	103 ± 2.4	96.9 ± 7.8	0.05	91.9 ± 5.9	97.6 ± 3.4	98.0 ± 5.1	95.1 ± 5.6
	0.1	93.7 ± 5.3	94.4 ± 1.6	95.8 ± 2.7	94.5 ± 3.7	0.1	89.4 ± 4.4	99.2 ± 2.8	104 ± 1.6	96.1 ± 7.6	0.1	98.1 ± 3.1	97.7 ± 1.9	94.6 ± 1.8	97.0 ± 2.8
Oxacillin	0.005	102 ± 7.1	101 ± 4.2	101 ± 6.7	101 ± 5.7	0.005	110 ± 4.4	92.6 ± 9.4	93.7 ± 3.0	101 ± 10	0.005	109 ± 9.4	97.1 ± 5.8	95.7 ± 13	102 ± 11
	0.01	99.8 ± 1.8	100 ± 4.3	102 ± 3.6	101 ± 2.9	0.01	103 ± 11	91.9 ± 7.0	111 ± 2.2	102 ± 10	0.01	97.6 ± 12	93.9 ± 4.7	93.2 ± 5.4	95.4 ± 8.8
	0.02	105 ± 6.5	94.8 ± 2.0	99.7 ± 5.7	101 ± 6.6	0.02	91.9 ± 8.6	98.4 ± 0.8	113 ± 5.0	99.3 ± 11	0.02	101 ± 12	101 ± 5.3	93.4 ± 1.7	98.9 ± 8.8
Cloxacillin	0.15	96.5 ± 6.0	104 ± 6.3	110 ± 3.1	102 ± 7.5	0.15	107 ± 7.4	92.5 ± 8.6	104 ± 5.1	102 ± 8.9	0.15	104 ± 6.8	92.3 ± 4.4	106 ± 1.1	101 ± 7.5
	0.3	98.7 ± 4.2	104 ± 3.2	102 ± 8.2	101 ± 5.4	0.3	117 ± 10	92.6 ± 5.3	110 ± 7.1	108 ± 12	0.3	98.4 ± 15	95.8 ± 5.3	93.7 ± 6.8	96.4 ± 11
	0.6	105 ± 5.8	100 ± 0.5	95.6 ± 7.9	101 ± 6.4	0.6	108 ± 10	98.9 ± 1.4	97.7 ± 5.1	103 ± 8.5	0.6	100 ± 12	102 ± 2.8	97.3 ± 7.3	99.8 ± 8.6
Penicillin G	0.025	88.6 ± 8.7	85.0 ± 7.7	109 ± 8.3	86.2 ± 7.7	0.025	97.9 ± 18	84.9 ± 8.3	103 ± 11	95.9 ± 15	0.005	89.9 ± 12	78.0 ± 4.8	84.7 ± 18	85.3 ± 13
	0.05	87.6 ± 11	91.6 ± 7.9	101 ± 3.7	78.8 ± 26	0.05	105 ± 13	98.5 ± 7.9	72.7 ± 13	93.4 ± 19	0.01	74.4 ± 20	79.3 ± 9.1	-	76.9 ± 4.5
	0.1	70.4 ± 12	87.5 ± 7.1	101 ± 3.6	76.1 ± 15	0.1	82.9 ± 13	98.7 ± 7.3	77.5 ± 16	86.0 ± 15	0.02	73.7 ± 20	96.1 ± 9.7	75.9 ± 15	80.4 ± 19
Penicillin V	0.005	102 ± 8.3	106 ± 7.7	94.4 ± 3.4	101 ± 7.9	0.005	109 ± 4.0	98.1 ± 1.1	92.4 ± 6.0	102 ± 8.3	0.005	113 ± 8.0	96.9 ± 2.7	93.9 ± 2.4	103 ± 11
	0.01	112 ± 3.7	102 ± 3.9	102 ± 2.9	106 ± 5.7	0.01	105 ± 5.7	93.3 ± 5.0	101 ± 3.9	101 ± 7.0	0.01	110 ± 7.8	96.8 ± 4.5	100 ± 6.7	104 ± 8.7
	0.02	114 ± 8.0	102 ± 3.4	99.7 ± 2.0	107 ± 8.6	0.02	95.8 ± 8.6	97.9 ± 4.5	96.9 ± 4.6	96.7 ± 6.2	0.02	113 ± 10	99.7 ± 3.3	101 ± 5.8	106 ± 9.5
Piperacillin	0.005	104 ± 4.2	105 ± 5.3	98.2 ± 2.3	102 ± 4.6	0.005	96.3 ± 4.1	98.8 ± 1.2	97.5 ± 3.3	97.3 ± 3.2	0.005	98.8 ± 7.7	99.1 ± 3.2	99.1 ± 7.4	99.0 ± 6.0
	0.01	99.1 ± 4.7	98.6 ± 4.7	102 ± 1.3	99.7 ± 3.9	0.01	106 ± 4.4	96.7 ± 5.6	104 ± 2.3	103 ± 5.6	0.01	91.0 ± 13	94.9 ± 6.4	89.1 ± 1.2	91.5 ± 9.3
	0.02	102 ± 6.6	93.0 ± 5.7	102 ± 3.6	99.5 ± 6.7	0.02	105 ± 4.8	97.5 ± 7.7	103 ± 0.7	102 ± 5.5	0.02	97.4 ± 8.5	99.5 ± 0.9	89.7 ± 2.6	95.9 ± 7.0
Hetacillin	0.005	92.5 ± 8.3	110 ± 4.4	97.6 ± 4.7	98.5 ± 9.5	0.005	99.2 ± 8.4	105 ± 9.3	100 ± 3.9	101 ± 7.4	0.005	79.0 ± 7.8	99.5 ± 1.6	93.9 ± 3.4	88.7 ± 12
	0.01	99.2 ± 8.7	98.1 ± 6.5	98.0 ± 11	98.6 ± 7.9	0.01	103 ± 6.2	94.7 ± 11	97.5 ± 2.4	99.4 ± 7.4	0.01	78.1 ± 5.8	101 ± 5.2	98.2 ± 3.8	89.9 ± 13
	0.02	94.3 ± 6.0	91.8 ± 5.1	94.9 ± 3.8	93.8 ± 4.9	0.02	108 ± 2.5	91.9 ± 10	91.7 ± 7.6	99.2 ± 10	0.02	89.4 ± 2.4	96.8 ± 1.1	100 ± 2.8	94.4 ± 5.6
Aspoxicillin	0.005	104 ± 5.4	108 ± 4.3	99.8 ± 7.5	104 ± 5.9	0.005	92.6 ± 6.2	102 ± 8.6	99.7 ± 7.2	97.1 ± 7.9	0.005	95.5 ± 14	92.8 ± 2.9	94.1 ± 13	94.4 ± 11
	0.01	98.0 ± 5.0	99.1 ± 8.8	103 ± 6.0	99.7 ± 6.1	0.01	93.9 ± 3.2	100 ± 9.8	111 ± 4.9	100 ± 9.2	0.01	93.6 ± 8.7	92.8 ± 3.8	99.5 ± 3.8	95.0 ± 6.7
	0.02	91.2 ± 9.5	91.7 ± 3.9	98.5 ± 2.7	93.4 ± 7.2	0.02	90.8 ± 5.8	105 ± 2.0	101 ± 7.2	97.5 ± 8.4	0.02	99.8 ± 4.6	99.9 ± 4.1	97.3 ± 7.4	99.1 ± 4.9
Mezlocillin	0.005	103 ± 5.6	102 ± 4.4	101 ± 1.3	102 ± 4.2	0.005	111 ± 4.1	91.9 ± 7.8	95.8 ± 0.9	101 ± 9.7	0.005	111 ± 9.8	101 ± 4.0	98.2 ± 3.1	104 ± 8.9
	0.01	102 ± 3.0	99.2 ± 3.3	98.4 ± 2.5	100 ± 3.3	0.01	104 ± 9.7	92.0 ± 6.2	102 ± 2.0	100 ± 8.8	0.01	99.7 ± 12	97.5 ± 3.6	95.6 ± 0.4	98.0 ± 8.1
	0.02	110 ± 5.6	98.7 ± 3.2	100 ± 3.3	104 ± 6.7	0.02	95.1 ± 9.4	98.3 ± 5.8	102 ± 1.2	97.9 ± 7.1	0.02	103 ± 12	97.8 ± 0.9	95.3 ± 1.2	99.3 ± 8.7

Table 3.

Accuracy and precision of the method for detecting 34 β-lactams in chicken, egg, and milk samples from three laboratories (n = 5)

Compounds	Chicken					Egg					Milk
	Spiking level (mg/kg)	Recovery (%)			Inter-lab.	Spiking level (mg/kg)	Recovery (%)			Inter-lab.	Spiking level (mg/kg)	Recovery (%)			Inter-lab.
		Lab. A	Lab. B	Lab. C			Lab. A	Lab. B	Lab. C			Lab. A	Lab. B	Lab. C
Nafcillin	0.005	86.3 ± 8.0	101 ± 4.0	107 ± 3.6	96.1 ± 11	0.005	102 ± 4.6	101 ± 10	100 ± 1.7	101 ± 5.5	0.015	113 ± 2.2	93.1 ± 10	98.9 ± 3.6	104 ± 10
	0.01	87.9 ± 6.3	91.7 ± 2.1	94.3 ± 3.8	90.7 ± 5.4	0.01	99.9 ± 5.7	108 ± 6.6	104 ± 2.0	103 ± 5.8	0.03	94.3 ± 7.9	92.4 ± 12	91.5 ± 2.8	93.0 ± 7.4
	0.02	83.9 ± 4.4	91.6 ± 0.7	96.7 ± 6.6	89.5 ± 7.6	0.02	97.1 ± 3.8	96.9 ± 5.2	105 ± 2.2	99.1 ± 5.0	0.06	86.3 ± 13	90.5 ± 8.4	95.3 ± 2.5	89.9 ± 9.8
Dicloxacillin	0.15	88.9 ± 14	102 ± 3.7	98.3 ± 0.8	94.9 ± 11	0.005	108 ± 6.0	95.4 ± 15	98.0 ± 5.9	102 ± 9.9	0.015	115 ± 5.8	108 ± 7.4	101 ± 2.1	109 ± 7.4
	0.3	99.9 ± 11	101 ± 3.9	99.8 ± 1.3	100 ± 7.4	0.01	102 ± 14	108 ± 2.4	106 ± 3.2	105 ± 9.0	0.03	95.9 ± 11	94.4 ± 10	100 ± 0.6	96.7 ± 8.6
	0.6	94.9 ± 11	96.0 ± 3.1	103 ± 1.2	97.4 ± 7.7	0.02	94.0 ± 5.1	97.3 ± 11	107 ± 7.1	98.4 ± 8.7	0.06	88.0 ± 18	85.7 ± 11	102 ± 2.9	91.2 ± 14
Mecillinam	0.005	89.3 ± 4.9	99.2 ± 4.4	101 ± 0.7	95.3 ± 7.1	0.005	105 ± 3.4	87.9 ± 18	96.4 ± 8.5	98.0 ± 11	0.005	101 ± 7.9	98.0 ± 7.8	102 ± 2.2	100 ± 6.4
	0.01	95.2 ± 1.0	95.7 ± 3.5	101 ± 2.1	97.0 ± 3.3	0.01	102 ± 3.4	105 ± 12	98.0 ± 12	102 ± 8.3	0.01	99.1 ± 4.7	99.7 ± 6.6	101 ± 0.7	99.8 ± 4.2
	0.02	93.2 ± 4.4	93.4 ± 2.8	95.4 ± 1.8	93.9 ± 3.3	0.02	101 ± 5.4	106 ± 8.2	104 ± 6.1	103 ± 6.1	0.02	100 ± 6.6	96.3 ± 11	96.4 ± 2.5	98.0 ± 6.8
Cefadroxil	0.005	74.1 ± 16	103 ± 14	101 ± 1.5	89.2 ± 20	0.005	91.5 ± 16	96.3 ± 2.9	106 ± 5.3	96.8 ± 12	0.005	101 ± 4.2	94.1 ± 2.8	118 ± 4.6	104 ± 10
	0.01	93.6 ± 17	90.0 ± 8.0	101 ± 0.6	94.6 ± 12	0.01	97.0 ± 6.9	108 ± 2.9	111 ± 3.6	104 ± 7.8	0.01	98.0 ± 7.7	97.5 ± 5.6	101 ± 2.2	98.8 ± 5.8
	0.02	89.0 ± 14	87.0 ± 2.5	103 ± 3.3	92.3 ± 12	0.02	107 ± 9.8	115 ± 3.6	109 ± 8.9	110 ± 8.0	0.02	96.7 ± 6.0	89.9 ± 2.0	102 ± 2.4	96.2 ± 6.3
Cefamandole	0.005	91.5 ± 13	102 ± 1.9	103 ± 3.1	97.4 ± 9.9	0.005	106 ± 8.3	101 ± 5.7	92.3 ± 4.4	101 ± 8.6	0.005	112 ± 10	95.3 ± 6.9	107 ± 4.0	106 ± 10
	0.01	100 ± 9.3	94.1 ± 0.9	98.5 ± 4.9	98.2 ± 7.0	0.01	115 ± 3.5	108 ± 5.2	104 ± 11	110 ± 7.2	0.01	107 ± 3.8	94.5 ± 4.7	105 ± 2.1	103 ± 6.3
	0.02	103 ± 10	87.4 ± 4.2	98.6 ± 0.9	97.3 ± 9.9	0.02	112 ± 5.2	104 ± 7.6	107 ± 1.2	108 ± 5.7	0.02	91.1 ± 15	97.1 ± 9.4	89.1 ± 2.3	92.2 ± 11
Cefacetrile	0.005	76.8 ± 17	104 ± 15	97.8 ± 11	89.9 ± 19	0.005	101 ± 18	102 ± 12	101 ± 5.6	101 ± 13	0.025	110 ± 11	94.6 ± 4.2	102 ± 5.3	104 ± 10
	0.01	80.3 ± 12	106 ± 9.0	96.3 ± 11	92.8 ± 16	0.01	99.8 ± 12	97.3 ± 10	101 ± 7.0	99.6 ± 9.6	0.05	119 ± 3.7	98.9 ± 6.9	98.0 ± 1.5	108 ± 11
	0.02	90.7 ± 18	106 ± 5.8	101 ± 2.4	99.3 ± 11	0.02	87.5 ± 12	104 ± 7.2	105 ± 7.7	96.7 ± 13	0.1	116 ± 4.9	101 ± 7.2	101 ± 3.7	108 ± 8.9
Cefazolin	0.005	97.0 ± 13	104 ± 6.5	103 ± 1.5	101 ± 7.7	0.005	107 ± 10	91.2 ± 10	102 ± 14	101 ± 12	0.025	107 ± 5.8	94.2 ± 4.7	99.0 ± 2.0	101 ± 7.3
	0.01	92.2 ± 26	94.0 ± 2.0	100 ± 2.2	95.1 ± 15	0.01	102 ± 6.2	108 ± 4.5	94.9 ± 9.9	102 ± 7.9	0.05	100 ± 7.2	103 ± 6.6	101 ± 0.8	106 ± 7.1
	0.02	100 ± 17	92.8 ± 2.3	99.7 ± 2.0	97.6 ± 9.6	0.02	99.7 ± 5.2	103 ± 1.4	105 ± 4.5	102 ± 4.6	0.1	107 ± 4.8	105 ± 3.8	104 ± 1.4	106 ± 3.8
Cephapirin	0.005	86.7 ± 9.6	106 ± 7.0	100 ± 1.2	95.5 ± 11	0.005	100 ± 5.4	95.7 ± 5.3	102 ± 7.3	99.4 ± 5.9	0.015	99.8 ± 7.8	91.2 ± 7.7	103 ± 2.5	98.3 ± 7.8
	0.01	90.0 ± 3.9	93.2 ± 1.8	105 ± 2.9	95.0 ± 7.7	0.01	99.4 ± 4.2	113 ± 3.1	114 ± 2.4	107 ± 7.6	0.03	99.4 ± 4.7	99.4 ± 2.3	101 ± 1.4	99.7 ± 3.3
	0.02	89.6 ± 4.3	85.4 ± 3.7	105 ± 2.0	92.5 ± 9.1	0.02	97.3 ± 7.4	109 ± 5.7	112 ± 11	105 ± 9.8	0.06	97.5 ± 7.1	93.7 ± 15	103 ± 1.1	98.0 ± 8.6
Desacetyl cephapirin	0.005	102 ± 9.2	106 ± 1.3	99.8 ± 7.6	103 ± 7.1	0.005	102 ± 15	101 ± 11	100 ± 12	102 ± 12	0.015	97.7 ± 7.9	84.1 ± 6.0	109 ± 3.2	97.1 ± 12
	0.01	94.8 ± 5.1	89.9 ± 5.0	100 ± 0.6	95.0 ± 5.7	0.01	105 ± 12	104 ± 8.3	98.4 ± 5.1	103 ± 9.2	0.03	98.0 ± 3.2	84.2 ± 4.8	107 ± 2.8	96.6 ± 9.6
	0.02	91.6 ± 9.3	90.7 ± 4.8	97.4 ± 1.3	92.9 ± 6.9	0.02	99.5 ± 6.6	101 ± 5.7	102 ± 6.1	101 ± 5.7	0.06	94.0 ± 8.0	96.7 ± 12	101 ± 2.8	96.5 ± 7.8
Cefalexin	0.1	97.7 ± 13	97.9 ± 1.8	102 ± 1.4	98.9 ± 8.2	0.005	99.9 ± 10	91.5 ± 2.5	94.6 ± 8.3	96.2 ± 8.6	0.05	103 ± 4.3	93.8 ± 4.6	106 ± 3.5	101 ± 6.1
	0.2	99.8 ± 8.0	96.8 ± 1.4	104 ± 1.3	100 ± 5.8	0.01	103 ± 4.9	109 ± 2.1	96.5 ± 7.4	103 ± 6.6	0.1	101 ± 3.4	104 ± 9.3	107 ± 0.9	104 ± 5.3
	0.4	93.2 ± 6.9	93.7 ± 4.3	103 ± 1.0	96.0 ± 6.6	0.02	104 ± 5.4	113 ± 3.4	98.1 ± 8.0	105 ± 7.6	0.2	98.2 ± 4.4	92.3 ± 3.5	105 ± 2.5	98.4 ± 6.0
Cefalonium	0.005	81.6 ± 5.9	90.6 ± 2.0	107 ± 2.8	90.9 ± 13	0.005	97.1 ± 4.9	86.3 ± 13	94.9 ± 3.6	93.5 ± 8.2	0.005	100 ± 11	100 ± 6.1	104 ± 4.8	101 ± 7.7
	0.01	86.6 ± 7.5	101 ± 3.9	104 ± 6.8	95.3 ± 11	0.01	92.1 ± 4.6	103 ± 3.6	99.6 ± 11	97.2 ± 7.9	0.01	102 ± 5.5	106 ± 4.6	94.8 ± 1.6	101 ± 5.9
	0.02	85.4 ± 6.6	104 ± 2.2	101 ± 3.8	94.9 ± 11	0.02	100 ± 7.7	103 ± 3.1	94.1 ± 3.2	99.3 ± 6.4	0.02	100 ± 6.7	93.9 ± 11	98.1 ± 2.0	97.9 ± 6.9
Cephaloridine	0.005	83.1 ± 7.0	103 ± 5.3	99.6 ± 2.1	93.1 ± 12	0.005	99.6 ± 3.8	91.3 ± 2.0	101 ± 5.4	97.7 ± 5.6	0.005	97.9 ± 9.1	101 ± 8.1	99.5 ± 3.2	99.2 ± 7.1
	0.01	90.0 ± 5.3	93.0 ± 1.6	103 ± 2.7	94.4 ± 7.0	0.01	99.5 ± 2.8	107 ± 6.6	98.0 ± 4.8	101 ± 5.6	0.01	98.2 ± 3.4	99.7 ± 1.9	92.9 ± 2.5	97.2 ± 3.9
	0.02	89.9 ± 4.5	93.0 ± 3.0	103 ± 0.5	94.3 ± 6.7	0.02	102 ± 5.1	105 ± 4.4	101 ± 4.6	102 ± 4.6	0.02	97.7 ± 6.4	95.2 ± 13	97.8 ± 1.6	97.0 ± 7.2
Cefalotin	0.005	93.5 ± 4.1	105 ± 7.7	99.7 ± 7.8	98.4 ± 7.7	0.005	91.1 ± 5.5	85.6 ± 12	92.8 ± 6.9	90.1 ± 7.6	0.005	107 ± 7.8	94.4 ± 13	94.6 ± 2.9	100 ± 10
	0.01	99.7 ± 4.9	105 ± 6.5	105 ± 0.9	103 ± 5.0	0.01	102 ± 6.3	104 ± 2.7	103 ± 2.8	103 ± 4.4	0.01	101 ± 6.5	99.1 ± 13	95.9 ± 3.3	98.9 ± 7.6
	0.02	96.1 ± 1.7	96.0 ± 3.9	104 ± 1.6	98.2 ± 4.3	0.02	107 ± 5.5	103 ± 2.9	104 ± 13	105 ± 7.2	0.02	96.4 ± 6.0	98.1 ± 6.4	105 ± 0.9	99.2 ± 6.1
Cefetamet	0.005	86.4 ± 6.5	108 ± 2.7	101 ± 1.5	96.3 ± 11	0.005	101 ± 4.3	97.9 ± 4.6	101 ± 4.8	100 ± 4.3	0.005	108 ± 7.8	96.3 ± 11	104 ± 3.0	104 ± 8.3
	0.01	92.7 ± 3.0	95.3 ± 0.7	103 ± 1.0	96.2 ± 5.0	0.01	102 ± 3.4	105 ± 6.1	101 ± 4.7	102 ± 4.4	0.01	105 ± 5.1	96.5 ± 3.5	101 ± 3.4	101 ± 5.3
	0.02	94.6 ± 7.5	88.4 ± 2.0	101 ± 2.9	94.7 ± 7.3	0.02	105 ± 4.9	104 ± 4.2	104 ± 0.3	104 ± 3.6	0.02	101 ± 4.1	95.2 ± 8.8	103 ± 1.2	100 ± 5.6
Cefepime	0.005	105 ± 12	85.5 ± 8.2	103 ± 8.5	98.9 ± 13	0.005	79.8 ± 25	86.9 ± 28	103 ± 4.5	88.0 ± 22	0.005	119 ± 18	90.1 ± 8.9	103 ± 12	107 ± 19
	0.01	101 ± 10	94.7 ± 1.4	107 ± 0.6	101 ± 8.1	0.01	83.9 ± 18	98.5 ± 12	102 ± 7.7	92.8 ± 15	0.01	104 ± 11	88.1 ± 4.4	107 ± 4.2	101 ± 11
	0.02	91.9 ± 5.1	97.4 ± 2.0	97.5 ± 0.3	94.9 ± 4.5	0.02	98.3 ± 2.1	101 ± 7.5	98.1 ± 5.6	99.1 ± 4.7	0.02	102 ± 14	89.9 ± 13	103 ± 3.3	99.0 ± 12
Cefpodoxime	0.005	76.0 ± 8.1	92.5 ± 6.3	102 ± 3.0	87.6 ± 15	0.005	107 ± 12	92.6 ± 8.7	99.5 ± 2.7	101 ± 11	0.005	107 ± 9.1	94.4 ± 4.6	107 ± 1.7	103 ± 8.4
	0.01	83.9 ± 10	98.7 ± 1.2	101 ± 2.9	92.6 ± 11	0.01	101 ± 3.3	107 ± 4.9	96.3 ± 7.1	101 ± 6.0	0.01	107 ± 4.1	101 ± 2.7	103 ± 0.7	104 ± 4.0
	0.02	85.4 ± 8.6	100 ± 3.1	101 ± 3.3	93.5 ± 10	0.02	101 ± 6.0	105 ± 1.3	94.6 ± 1.2	100 ± 5.5	0.02	102 ± 8.2	91.8 ± 6.0	104 ± 2.0	99.8 ± 7.9
Cefotaxime	0.005	85.1 ± 3.2	102 ± 5.1	93.4 ± 0.7	93.6 ± 8.6	0.005	99.7 ± 6.4	90.4 ± 3.5	103 ± 3.9	98.0 ± 7.0	0.005	96.3 ± 7.9	98.5 ± 7.1	103 ± 3.6	98.8 ± 6.8
	0.01	82.5 ± 9.4	91.0 ± 3.2	96.3 ± 4.1	88.6 ± 9.2	0.01	99.1 ± 4.5	103 ± 6.9	109 ± 2.4	103 ± 5.9	0.01	102 ± 5.2	99.4 ± 5.4	96.1 ± 0.4	99.8 ± 4.9
	0.02	85.9 ± 11	96.3 ± 2.5	101 ± 2.4	92.8 ± 9.9	0.02	102 ± 7.5	101 ± 3.0	108 ± 2.3	103 ± 5.7	0.02	102 ± 6.9	99.1 ± 11	101 ± 2.6	101 ± 6.8
Cefoperazone	0.005	89.6 ± 5.5	97.3 ± 4.6	99.5 ± 4.2	94.4 ± 6.6	0.005	100 ± 5.5	97.0 ± 5.0	93.3 ± 8.2	97.3 ± 6.3	0.015	119 ± 7.4	93.2 ± 8.9	100 ± 2.1	107 ± 13
	0.01	95.6 ± 3.3	99.3 ± 1.5	98.6 ± 1.3	97.4 ± 2.9	0.01	98.4 ± 5.0	111 ± 6.9	104 ± 5.8	103 ± 7.3	0.03	111 ± 7.6	92.1 ± 19	101 ± 2.0	103 ± 12
	0.02	95.2 ± 4.2	93.7 ± 6.4	97.2 ± 1.1	95.4 ± 4.1	0.02	97.0 ± 8.8	102 ± 2.8	106 ± 4.5	101 ± 7.1	0.06	89.9 ± 11	85.3 ± 3.7	103 ± 2.7	92.2 ± 10
Cefoxitin	0.005	90.4 ± 11	103 ± 7.9	88.2 ± 6.4	93.3 ± 11	0.005	98.8 ± 10	90.7 ± 18	93.7 ± 7.1	95.2 ± 11	0.005	99.5 ± 10	100 ± 7.1	93.8 ± 3.8	98.1 ± 7.9
	0.01	93.7 ± 8.5	107 ± 1.9	91.2 ± 11	96.7 ± 9.9	0.01	93.2 ± 8.6	100 ± 15	94.8 ± 4.0	95.5 ± 9.3	0.01	104 ± 4.7	98.9 ± 14	102 ± 9.1	102 ± 8.1
	0.02	91.6 ± 4.7	113 ± 4.1	103 ± 2.8	101 ± 10	0.02	103 ± 3.9	97.3 ± 20	98.9 ± 10	101 ± 10	0.02	87.9 ± 11	85.5 ± 4.3	96.4 ± 3.2	89.6 ± 8.7
Cefuroxime	0.005	101 ± 7.8	89.5 ± 6.4	94.6 ± 14	95.0 ± 10	0.005	108 ± 16	100 ± 11	77.8 ± 25	97.5 ± 20	0.01	109 ± 23	103 ± 4.2	111 ± 2.9	108 ± 15
	0.01	91.9 ± 18	104 ± 1.7	107 ± 5.8	99.2 ± 13	0.01	101 ± 9.1	97.6 ± 8.4	95.4 ± 8.2	98.4 ± 8.1	0.02	83.5 ± 11	86.3 ± 15	108 ± 6.6	90.8 ± 15
	0.02	98.6 ± 17	111 ± 4.5	96.5 ± 4.1	102 ± 11	0.02	91.6 ± 6.3	99.3 ± 5.7	115 ± 4.9	100 ± 12	0.04	76.2 ± 5.8	72.4 ± 31	109 ± 4.7	84.2 ± 23
Cefsulodin	0.005	97.4 ± 2.8	109 ± 6.9	107 ± 1.6	103 ± 6.7	0.005	98.8 ± 9.8	101 ± 15	92.9 ± 5.4	97.7 ± 10	0.005	93.6 ± 8.8	-	106 ± 5.8	99.8 ± 8.8
	0.01	86.3 ± 5.5	104 ± 4.8	103 ± 3.7	95.5 ± 10	0.01	95.2 ± 9.2	99.1 ± 12	105 ± 2.7	98.8 ± 8.8	0.01	101 ± 6.8	90.3 ± 3.2	101 ± 4.9	97.8 ± 7.2
	0.02	80.1 ± 9.5	107 ± 9.8	99.5 ± 9.3	92.7 ± 16	0.02	102 ± 5.7	104 ± 9.6	104 ± 7.6	103 ± 6.6	0.02	101 ± 9.1	93.8 ± 9.4	102 ± 9.1	99.2 ± 8.9
Cefquinome	0.005	88.0 ± 9.4	97.6 ± 7.7	109 ± 1.5	96.2 ± 11	0.005	117 ± 3.6	102 ± 5.4	107 ± 2.5	110 ± 7.0	0.01	105 ± 15	89.1 ± 12	99.5 ± 4.6	99.0 ± 13
	0.01	91.6 ± 6.6	92.5 ± 4.6	111 ± 4.7	97.2 ± 11	0.01	110 ± 8.9	108 ± 9.8	103 ± 3.2	108 ± 7.8	0.02	104 ± 6.6	99.2 ± 16	92.5 ± 1.3	99.6 ± 9.7
	0.02	94.0 ± 6.5	99.9 ± 0.5	110 ± 3.5	100 ± 8.3	0.02	103 ± 7.4	114 ± 1.8	104 ± 8.4	106 ± 7.5	0.04	104 ± 9.5	81.3 ± 7.8	95.7 ± 4.4	95.6 ± 13
Ceftiofur	0.005	89.1 ± 7.0	102 ± 7.4	100 ± 2.7	95.5 ± 8.5	0.005	103 ± 4.9	98.6 ± 7.4	98.6 ± 2.8	101 ± 5.2	0.05	112 ± 4.2	92.7 ± 18	106 ± 1.7	105 ± 11
	0.01	93.6 ± 4.3	94.8 ± 1.3	97.4 ± 0.4	95.0 ± 3.2	0.01	103 ± 7.4	109 ± 5.4	103 ± 5.7	105 ± 6.5	0.1	100 ± 5.2	85.2 ± 0.4	109 ± 1.0	98.6 ± 10
	0.02	92.7 ± 4.9	91.1 ± 1.4	98.1 ± 1.1	93.7 ± 4.4	0.02	102 ± 3.9	104 ± 4.1	104 ± 1.6	103 ± 3.3	0.2	91.6 ± 7.4	87.1 ± 1.9	110 ± 2.4	95.3 ± 11
Desfuroyl ceftiofur	0.005	90.0 ± 16	111 ± 4.5	88.6 ± 8.3	95.9 ± 15	0.005	88.3 ± 12	98.8 ± 19	99.1 ± 9.2	94.1 ± 13	0.05	102 ± 5.0	96.7 ± 4.2	73.6 ± 6.9	92.8 ± 14
	0.01	97.3 ± 16	116 ± 2.7	96.0 ± 11	102 ± 14	0.01	80.9 ± 7.8	75.3 ± 20	110 ± 7.0	87.3 ± 20	0.1	94.6 ± 8.7	91.9 ± 10	72.3 ± 6.3	87.8 ± 14
	0.02	101 ± 11	90.0 ± 11.1	97.5 ± 6.6	97.1 ± 11	0.02	81.8 ± 15	75.7 ± 4.2	101 ± 11	85.4 ± 16	0.2	86.2 ± 4.5	96.8 ± 12	82.8 ± 5.9	88.2 ± 9.5
Cefpirome	0.005	82.6 ± 14	96.9 ± 3.2	104 ± 2.9	92.3 ± 13	0.005	101 ± 5.8	98.5 ± 19	95.9 ± 3.3	98.8 ± 9.5	0.005	106 ± 16	99.5 ± 5.6	92.8 ± 15	101 ± 14
	0.01	95.7 ± 9.3	92.8 ± 8.6	95.6 ± 5.3	94.9 ± 7.5	0.01	93.6 ± 5.0	108 ± 6.1	118 ± 3.6	104 ± 11	0.01	101 ± 6.2	99.5 ± 6.8	89.2 ± 4.3	97.1 ± 7.5
	0.02	94.6 ± 5.8	87.4 ± 7.2	97.0 ± 1.0	93.3 ± 6.4	0.02	88.6 ± 11	109 ± 5.3	109 ± 12	99.7 ± 14	0.02	101 ± 15	88.0 ± 12	96.5 ± 5.4	96.3 ± 13
Cefixime	0.005	86.9 ± 15	98.4 ± 17	92.7 ± 11	92.1 ± 14	0.005	93.2 ± 9.0	77.8 ± 5.9	89.0 ± 4.5	87.8 ± 10	0.005	101 ± 7.5	94.2 ± 3.3	98.2 ± 9.3	98.5 ± 7.2
	0.01	88.9 ± 17	105 ± 4.2	88.8 ± 8.0	93.1 ± 14	0.01	98.8 ± 10	108 ± 3.6	95.1 ± 8.1	100 ± 9.0	0.01	104 ± 6.2	102 ± 8.4	94.8 ± 2.8	101 ± 7.0
	0.02	89.5 ± 11	99.8 ± 6.0	99.2 ± 6.6	95.0 ± 9.6	0.02	95.5 ± 7.5	111 ± 11	102 ± 4.5	101 ± 9.7	0.02	97.7 ± 12	92.0 ± 12	101 ± 1.9	97.0 ± 9.8
Amoxicillin	0.025	96.8 ± 11	93.5 ± 4.8	104 ± 1.1	97.8 ± 8.3	0.005	93.4 ± 14	97.0 ± 1.6	94.9 ± 22	94.8 ± 13	0.002	100 ± 6.9	92.7 ± 8.5	105 ± 4.1	99.6 ± 7.8
	0.05	105 ± 3.5	93.3 ± 2.0	105 ± 1.5	102 ± 5.9	0.01	97.4 ± 9.4	114 ± 3.9	108 ± 6.3	105 ± 9.6	0.004	103 ± 7.0	97.3 ± 14	105 ± 0.3	102 ± 7.9
	0.1	101 ± 6.2	94.3 ± 4.6	98.2 ± 1.5	98.5 ± 5.5	0.02	98.0 ± 6.8	108 ± 3.4	93.4 ± 6.9	99.4 ± 7.8	0.008	96.2 ± 3.3	90.5 ± 12	106 ± 4.1	97.2 ± 8.3
Ampicillin	0.025	92.8 ± 7.5	104 ± 2.2	101 ± 3.3	98.0 ± 7.1	0.005	103 ± 4.4	97.3 ± 14	107 ± 4.0	103 ± 7.7	0.002	101 ± 6.5	92.0 ± 8.0	104 ± 2.7	99.3 ± 7.3
	0.05	93.8 ± 2.3	97.2 ± 1.4	102 ± 0.7	97.0 ± 4.0	0.01	103 ± 3.9	110 ± 2.8	105 ± 10	105 ± 6.1	0.004	100 ± 5.3	106 ± 1.1	102 ± 1.0	102 ± 4.0
	0.1	92.0 ± 5.0	91.7 ± 3.7	106 ± 0.4	95.7 ± 7.6	0.02	103 ± 5.4	106 ± 2.4	103 ± 2.4	104 ± 3.9	0.008	97.8 ± 6.7	89.2 ± 9.2	106 ± 2.9	97.7 ± 8.9
Oxacillin	0.005	91.9 ± 2.1	90.2 ± 6.1	100 ± 1.4	93.7 ± 5.4	0.005	110 ± 6.9	95.6 ± 11	108 ± 2.7	105 ± 8.8	0.005	109 ± 5.4	95.9 ± 5.2	100 ± 1.9	103 ± 7.1
	0.01	94.0 ± 3.1	97.9 ± 2.5	93.2 ± 1.9	94.8 ± 3.2	0.01	103 ± 5.8	104 ± 4.5	102 ± 4.6	103 ± 4.7	0.01	95.9 ± 6.7	97.8 ± 4.8	99.4 ± 1.9	97.4 ± 5.0
	0.02	90.0 ± 2.7	103 ± 1.6	98.9 ± 2.1	95.9 ± 6.4	0.02	105 ± 5.8	96.4 ± 1.4	103 ± 5.8	102 ± 5.8	0.02	84.0 ± 8.1	103 ± 3.7	102 ± 4.3	93.9 ± 12
Cloxacillin	0.15	87.5 ± 4.1	90.6 ± 7.9	102 ± 2.5	92.4 ± 8.3	0.005	107 ± 7.0	98.3 ± 12	110 ± 5.7	105 ± 8.4	0.015	114 ± 5.9	101 ± 3.9	116 ± 6.7	111 ± 7.9
	0.3	97.3 ± 6.1	99.7 ± 2.7	102 ± 2.2	99.3 ± 4.7	0.01	107 ± 11	98.4 ± 9.1	112 ± 9.2	106 ± 11	0.03	96.6 ± 8.4	95.4 ± 8.2	109 ± 9.5	99.6 ± 9.7
	0.6	88.6 ± 4.7	101 ± 2.4	102 ± 3.1	95.7 ± 7.8	0.02	92.4 ± 5.3	95.8 ± 5.2	113 ± 7.9	98.9 ± 11	0.06	89.7 ± 11	99.4 ± 3.4	105 ± 1.5	96.4 ± 9.6
Penicillin G	0.025	81.8 ± 15	97.0 ± 2.0	78.8 ± 6.3	85.5 ± 13	0.002	80.9 ± 12	91.4 ± 2.7	85.5 ± 4.3	85.0 ± 9.1	0.002	118 ± 3.8	93.5 ± 5.5	87.2 ± 3.8	103 ± 15
	0.05	79.6 ± 13	84.4 ± 3.1	70.0 ± 5.8	78.3 ± 12	0.004	91.6 ± 21	110 ± 7.7	93.2 ± 3.6	97.0 ± 16	0.004	94.4 ± 11	83.1 ± 4.5	85.5 ± 8.2	88.9 ± 10
	0.1	72.1 ± 13	89.0 ± 4.1	86.7 ± 8.8	80.7 ± 13	0.008	89.8 ± 10	108 ± 4.0	91.6 ± 3.3	95.2 ± 11	0.004	76.2 ± 7.0	84.8 ± 4.1	88.6 ± 4.9	81.9 ± 8.6
Penicillin V	0.005	82.4 ± 6.7	92.2 ± 3.8	105 ± 5.9	91.2 ± 12	0.005	103 ± 4.5	97.8 ± 13	110 ± 6.2	103 ± 8.2	0.005	116 ± 2.8	90.9 ± 2.9	101 ± 2.8	106 ± 11
	0.01	91.5 ± 6.0	98.9 ± 2.4	102 ± 2.1	96.6 ± 6.6	0.01	104 ± 4.0	108 ± 4.1	109 ± 4.8	106 ± 4.4	0.01	110 ± 6.9	97.8 ± 2.4	100 ± 1.8	104 ± 7.2
	0.02	90.9 ± 4.8	101 ± 1.5	100 ± 3.5	96.1 ± 6.2	0.02	101 ± 4.8	97.7 ± 3.9	102 ± 2.1	100 ± 4.0	0.02	98.3 ± 9.4	99.5 ± 8.6	100 ± 5.2	99.1 ± 7.5
Piperacillin	0.005	93.8 ± 8.7	95.9 ± 6.2	102 ± 1.1	96.6 ± 7.1	0.005	103 ± 5.4	93.1 ± 8.0	95.3 ± 4.7	98.2 ± 7.1	0.005	114 ± 9.8	85.9 ± 7.7	104 ± 2.8	104 ± 14
	0.01	103 ± 4.5	96.0 ± 1.0	99.4 ± 1.4	100 ± 4.2	0.01	95.5 ± 4.6	107 ± 9.7	101 ± 3.6	100 ± 7.4	0.01	105 ± 7.1	95.0 ± 2.9	99.6 ± 1.3	101 ± 6.4
	0.02	100 ± 6.8	96.3 ± 0.8	102 ± 1.8	99.5 ± 5.0	0.02	91.5 ± 7.6	103 ± 1.8	98.9 ± 3.8	96.7 ± 7.3	0.02	93.9 ± 1.9	102 ± 5.7	103 ± 1.1	98.4 ± 5.3
Hetacillin	0.005	76.9 ± 2.7	93.9 ± 5.4	98.2 ± 9.3	89.7 ± 12	0.005	75.8 ± 12	109 ± 4.6	108 ± 2.6	93.7 ± 19	0.005	92.1 ± 22	88.0 ± 15	98.6 ± 9.3	92.8 ± 16
	0.01	75.2 ± 11	98.1 ± 2.5	103 ± 14	88.9 ± 18	0.01	92.2 ± 5.2	103 ± 14	109 ± 7.7	99.5 ± 11	0.01	93.4 ± 15	73.2 ± 1.4	102 ± 7.5	90.1 ± 17
	0.02	80.3 ± 6.1	106 ± 0.8	100 ± 9.2	92.8 ± 14	0.02	107 ± 8.3	97.0 ± 4.3	101 ± 0.5	102 ± 7.1	0.02	106 ± 5.6	76.2 ± 3.3	99.2 ± 6.8	96.1 ± 15
Aspoxicillin	0.005	85.6 ± 6.0	97.2 ± 3.7	104 ± 7.7	93.7 ± 10	0.005	90.9 ± 7.3	104 ± 9.4	111 ± 5.5	100 ± 11	0.005	103 ± 8.6	82.3 ± 6.6	110 ± 11	99.2 ± 14
	0.01	90.6 ± 7.9	103 ± 4.4	107 ± 5.4	98.6 ± 9.7	0.01	95.0 ± 5.1	111 ± 5.2	114 ± 12	105 ± 11	0.01	99.7 ± 6.9	107 ± 3.0	107 ± 5.0	103 ± 6.0
	0.02	89.1 ± 6.2	99.7 ± 3.8	101 ± 4.8	95.3 ± 7.8	0.02	103 ± 7.0	111 ± 5.6	117 ± 17	109 ± 11	0.02	97.8 ± 7.3	91.5 ± 11	103 ± 0.7	97.5 ± 8.0
Mezlocillin	0.005	94.8 ± 4.8	95.3 ± 4.3	97.5 ± 3.3	95.7 ± 4.1	0.005	104 ± 6.7	96.3 ± 4.1	94.2 ± 0.7	99.0 ± 6.6	0.005	112 ± 2.5	104 ± 2.0	103 ± 3.5	107 ± 4.7
	0.01	98.4 ± 6.8	99.4 ± 1.5	97.9 ± 1.9	98.5 ± 4.5	0.01	97.2 ± 4.1	105 ± 3.7	102 ± 3.4	100 ± 4.7	0.01	98.0 ± 5.1	106 ± 0.6	98.9 ± 0.6	100 ± 4.6
	0.02	99.7 ± 6.3	97.2 ± 1.4	96.1 ± 1.0	98.0 ± 4.5	0.02	90.1 ± 2.5	103 ± 2.2	98.8 ± 3.1	96.0 ± 6.5	0.02	94.1 ± 7.5	95.7 ± 4.5	99.3 ± 2.1	95.9 ± 5.7

The detection limits are represented as LOD and LOQ (Table 4). The respective LODs and LOQs of the method were 0.0003–0.1 and 0.001–0.4 mg/kg for beef, 0.0005–0.05 and 0.002–0.2 mg/kg for pork, 0.0005–0.04 and 0.002–0.1 mg/kg for fat (pork), 0.0002–0.04 and 0.0006–0.1 mg/kg for chicken, 0.0002–0.07 and 0.0006–0.2 mg/kg for egg, and 0.0002–0.02 and 0.0008–0.05 mg/kg for milk. The LOQs of all targets were below half of the MRLs. Therefore, the method is sufficient for the quantitative analysis of β-lactam compounds in livestock products.

Table 4.

Limits of detection (LODs) and limits of quantification (LOQs) of the method in six livestock samples (mg/kg)

Compounds	Beef		Pork		Fat (pork)		Chicken		Egg		Milk
	LOD	LOQ	LOD	LOQ	LOD	LOQ	LOD	LOQ	LOD	LOQ	LOD	LOQ
Nafcillin	0.03	0.09	0.001	0.004	0.0006	0.002	0.001	0.002	0.002	0.001	0.004	0.01
Dicloxacillin	0.03	0.09	0.03	0.1	0.03	0.09	0.04	0.1	0.002	0.001	0.003	0.01
Mecillinam	0.0003	0.001	0.0007	0.002	0.0008	0.003	0.0004	0.002	0.0008	0.003	0.001	0.004
Cefadroxil	0.0008	0.003	0.002	0.007	0.001	0.004	0.0009	0.003	0.001	0.003	0.001	0.004
Cefamandole	0.0006	0.002	0.001	0.004	0.001	0.004	0.002	0.006	0.001	0.004	0.0007	0.002
Cefacetrile	0.001	0.004	0.001	0.004	0.001	0.003	0.003	0.01	0.001	0.004	0.005	0.02
Cefazolin	0.002	0.006	0.004	0.01	0.005	0.02	0.0006	0.002	0.001	0.003	0.005	0.02
Cephapirin	0.004	0.01	0.0008	0.003	0.001	0.004	0.0006	0.002	0.0009	0.003	0.003	0.009
Desacetyl Cephapirin	0.005	0.02	0.001	0.003	0.0005	0.002	0.001	0.003	0.001	0.004	0.002	0.008
Cefalexin	0.02	0.06	0.01	0.04	0.02	0.05	0.006	0.02	0.001	0.004	0.007	0.02
Cefalonium	0.0005	0.002	0.0009	0.003	0.0009	0.003	0.0008	0.003	0.0007	0.003	0.0009	0.003
Cephaloridine	0.0005	0.002	0.0006	0.002	0.001	0.004	0.0007	0.002	0.001	0.004	0.001	0.003
Cefalotin	0.0006	0.002	0.0008	0.003	0.0008	0.003	0.0004	0.001	0.0008	0.003	0.0009	0.003
Cefetamet	0.0007	0.002	0.0006	0.002	0.0008	0.003	0.0004	0.001	0.0008	0.003	0.001	0.004
Cefepime	0.001	0.003	0.001	0.004	0.004	0.01	0.001	0.004	0.0009	0.003	0.004	0.01
Cefpodoxime	0.0005	0.002	0.0008	0.003	0.001	0.003	0.0007	0.002	0.0006	0.002	0.0006	0.002
Cefotaxime	0.0006	0.002	0.0007	0.002	0.001	0.004	0.0009	0.003	0.001	0.004	0.0009	0.003
Cefoperazone	0.0007	0.002	0.0009	0.003	0.0007	0.002	0.0007	0.002	0.001	0.004	0.003	0.01
Cefoxitin	0.0009	0.003	0.001	0.004	0.001	0.004	0.0009	0.003	0.002	0.008	0.0006	0.002
Cefuroxime	0.0007	0.002	0.0007	0.002	0.001	0.004	0.0009	0.003	0.001	0.004	0.001	0.003
Cefsulodin	0.0008	0.002	0.001	0.004	0.001	0.004	0.001	0.004	0.0009	0.003	0.003	0.009
Cefquinome	0.004	0.01	0.005	0.08	0.006	0.02	0.0003	0.001	0.0005	0.002	0.002	0.006
Ceftiofur	0.05	0.2	0.05	0.2	0.02	0.07	0.0006	0.002	0.001	0.004	0.009	0.03
Desfuroyl ceftiofur	0.1	0.4	0.03	0.09	0.04	0.1	0.002	0.007	0.07	0.2	0.2	0.05
Cefpirome	0.001	0.004	0.001	0.004	0.001	0.003	0.0009	0.003	0.0008	0.003	0.001	0.004
Cefixime	0.001	0.004	0.001	0.004	0.0005	0.002	0.005	0.02	0.001	0.005	0.001	0.004
Amoxicillin	0.003	0.01	0.003	0.009	0.005	0.02	0.003	0.01	0.001	0.004	0.0004	0.002
Ampicillin	0.003	0.009	0.001	0.004	0.005	0.02	0.002	0.006	0.0007	0.003	0.0002	0.0008
Oxacillin	0.0008	0.003	0.0005	0.002	0.0008	0.003	0.0004	0.002	0.0002	0.0006	0.0008	0.003
Cloxacillin	0.02	0.08	0.03	0.1	0.02	0.07	0.02	0.07	0.0005	0.002	0.003	0.01
Penicillin G	0.0003	0.001	0.004	0.01	0.002	0.006	0.005	0.02	0.0005	0.002	0.0004	0.001
Penicillin V	0.0009	0.003	0.001	0.004	0.001	0.004	0.0005	0.002	0.001	0.004	0.0005	0.002
Piperacillin	0.0005	0.002	0.001	0.003	0.001	0.004	0.0003	0.0009	0.0006	0.002	0.0008	0.003
Hetacillin	0.0007	0.002	0.0005	0.002	0.0007	0.002	0.0005	0.002	0.001	0.004	0.0009	0.003
Aspoxicillin	0.0007	0.002	0.0008	0.003	0.001	0.004	0.0006	0.002	0.001	0.003	0.0009	0.003
Mezlocillin	0.0005	0.002	0.0007	0.002	0.001	0.003	0.0002	0.0006	0.0003	0.001	0.0008	0.003

This method was also tested in different laboratories to confirm its reproducibility. The recovery of the method was 88.6–116% for beef, 69.4–114% for pork, 70.0–111% for chicken, 77.8–118% for egg, and 72.3–118% for milk. The CV of the method was 0.78–21% for beef, 0.16–16% for pork, 0.35–14% for chicken, 0.31–25% for egg, and 0.33–14% for milk. Overall, the recovery and CV of the method were 69.4–118% and 0.16–25%, respectively, and all results were within the range of values ​​required for each spiking concentration in accordance with the CODEX guideline.

Application to real samples

In this study, a method for the simultaneous quantitative analysis of 34 β-lactams was developed. The method showed sufficient linearity, accuracy, precision, and detection limits for five types of livestock samples (beef, pork, chicken, egg, and milk). To confirm the applicability of the method to livestock products, 25 beef, 52 pork, 25 chicken, 13 egg, and 12 milk samples were purchased. A total of 127 samples were obtained from the domestic market and analyzed following the method described in this study. All the samples in the results were below the LODs of the 34 β-lactams.

This method is expected to help improve the efficiency of domestic food analysis for some β-lactam substances (cefacetrile, cephapirin, desacetyl cephapirin, cefalonium, cefuroxime, cefquinome, and desfuroyl ceftiofur) that could not be quantified by the domestic simultaneous test method. The method was verified for meat, poultry, egg, and milk, but it is expected that prior evaluation may be required when applied to other livestock products in consideration of the matrix effect.

Analysis of β-lactams in fishery foods

The method was also used to analyze β-lactams in fishery foods, including eels, flat fish, and shrimp. It was conducted following the method with two extraction solvents: (1) 3 mL of 1% phosphate buffer and 7 mL of acetonitrile and (2) water:acetonitrile (1:4, v/v). Comparison results showed that the solvent containing phosphate buffer was more suitable than the one containing water. Extraction using water:acetonitrile (1:4, v/v) yielded unacceptable results for cefacetrile, cefalexin, cefixime, and hetacillin. Cefalexin is an important compound because it has an MRL of 0.2 mg/kg for flat fish and shrimp in Korea. Therefore, the method using phosphate buffer was validated by the CODEX guidelines, but some compounds have yet to be quantified (cefacetrile for flat fish, cefixime for eel and flat fish, and desfuroyl ceftiofur for shrimp).

Except for cefixime and desfuroyl ceftiofur, all analytes showed a linearity of > 0.98. The accuracy, precision, and detection limits of the method are presented in Table 5. The recovery of the method was 75.2–120% for eel, 62.1–118% for flat fish, and 82.2–119% for shrimp. The CV of the method was 1.5–26% for eel, 1.5–26% for flat fish, and 1.0–28% for shrimp, and all results were within the range of values ​​required for each spiking concentration in accordance with the CODEX guidelines. The respective LODs and LOQs of the method were 0.0008–0.2 and 0.003–0.8 mg/kg for eel, 0.0006–0.2 and 0.002–0.5 mg/kg for flat fish, 0.0008–0.01 and 0.003–0.04 mg/kg for shrimp.

Table 5.

Validation of the method for fishery food (eel, flat fish, and shrimp)

Compounds	Eel				Flat fish				Shrimp
	Spiking level (mg/kg)	Recovery (%, n = 5)	LOD (mg/kg)	LOQ (mg/kg)	Spiking level (mg/kg)	Recovery (%, n = 5)	LOD (mg/kg)	LOQ (mg/kg)	Spiking level (mg/kg)	Recovery (%, n = 5)	LOD (mg/kg)	LOQ (mg/kg)
Nafcillin	0.005	90.2 ± 22	0.008	0.03	0.005	87.1 ± 6.3	0.003	0.01	0.005	103 ± 7.7	0.003	0.009
	0.01	119 ± 15			0.01	84.2 ± 7.2			0.01	100 ± 3.0
	0.02	100 ± 18			0.02	93.1 ± 11			0.02	101 ± 2.9
Dicloxacillin	0.005	94.8 ± 22	0.005	0.02	0.005	92.6 ± 7.0	0.003	0.01	0.005	107 ± 8.2	0.004	0.01
	0.01	118 ± 4.8			0.01	89.7 ± 6.6			0.01	104 ± 4.8
	0.02	96.6 ± 18			0.02	94.2 ± 8.6			0.02	100 ± 3.4
Mecillinam	0.005	94.4 ± 3.9	0.003	0.01	0.005	92.7 ± 9.9	0.0006	0.002	0.005	99.9 ± 3.3	0.004	0.01
	0.01	99.5 ± 5.2			0.01	92.7 ± 4.0			0.01	100 ± 1.6
	0.02	99.0 ± 5.7			0.02	93.0 ± 7.5			0.02	98.7 ± 4.1
Cefadroxil	0.005	101 ± 4.8	0.002	0.006	0.005	118 ± 21	0.005	0.02	0.005	101 ± 8.8	0.003	0.009
	0.01	105 ± 2.8			0.01	93.9 ± 13			0.01	104 ± 4.9
	0.02	101 ± 7.4			0.02	99.8 ± 6.2			0.02	103 ± 14
Cefamandole	0.005	108 ± 13	0.002	0.008	0.005	94.3 ± 9.1	0.003	0.009	0.005	106 ± 8.9	0.003	0.01
	0.01	110 ± 9.1			0.01	97.7 ± 8.5			0.01	103 ± 7.0
	0.02	97.8 ± 11			0.02	103 ± 9.9			0.02	100 ± 3.3
Cefacetrile	0.005	104 ± 12	0.003	0.01	0.005	–	–	–	0.005	93.2 ± 17	0.007	0.02
	0.01	100 ± 8.9			0.01	–			0.01	111 ± 7.6
	0.02	97.2 ± 18			0.02	–			0.02	102 ± 6.0
Cefazolin	0.005	102 ± 12	0.001	0.005	0.005	83.9 ± 17	0.005	0.02	0.005	103 ± 6.2	0.002	0.006
	0.01	106 ± 7.2			0.01	97.0 ± 5.8			0.01	104 ± 1.7
	0.02	101 ± 7.0			0.02	104 ± 15			0.02	100 ± 3.7
Cephapirin	0.005	86.7 ± 1.4	0.005	0.02	0.005	84.8 ± 11	0.002	0.005	0.005	100 ± 10	0.003	0.009
	0.01	99.5 ± 8.6			0.01	92.7 ± 4.4			0.01	99.2 ± 1.0
	0.02	100 ± 8.0			0.02	89.1 ± 5.3			0.02	100 ± 5.1
Desacetyl cephapirin	0.005	90.0 ± 16	0.006	0.02	0.005	80.7 ± 26	0.003	0.01	0.005	97.2 ± 8.7	0.005	0.02
	0.01	111 ± 14			0.01	88.2 ± 5.4			0.01	102 ± 6.0
	0.02	103 ± 9.8			0.02	92.6 ± 4.9			0.02	95.2 ± 5.4
Cefalexin	0.1	100 ± 5.1	0.07	0.2	0.1	87.1 ± 15	0.06	0.2	0.005	108 ± 14	0.001	0.005
	0.2	110 ± 5.1			0.2	95.4 ± 9.0			0.01	118 ± 5.7
	0.4	106 ± 7.4			0.4	105 ± 10			0.02	119 ± 6.9
Cefalonium	0.005	97.5 ± 4.6	0.004	0.01	0.005	79.3 ± 20	0.003	0.008	0.005	94.6 ± 6.8	0.003	0.009
	0.01	108 ± 5.6			0.01	84.3 ± 4.7			0.01	95.4 ± 3.2
	0.02	106 ± 6.5			0.02	96.7 ± 5.0			0.02	96.5 ± 7.1
Cephaloridine	0.005	94.1 ± 5.3	0.004	0.01	0.005	92.3 ± 9.4	0.0009	0.003	0.005	100 ± 9.3	0.002	0.006
	0.01	103 ± 6.6			0.01	90.7 ± 1.9			0.01	101 ± 2.6
	0.02	106 ± 5.2			0.02	95.5 ± 8.8			0.02	98.6 ± 4.5
Cefalotin	0.005	99.6 ± 8.9	0.003	0.01	0.005	86.9 ± 12	0.005	0.02	0.005	98.6 ± 12	0.001	0.004
	0.01	112 ± 11			0.01	92.6 ± 6.6			0.01	100 ± 6.1
	0.02	102 ± 13			0.02	97.7 ± 9.6			0.02	102 ± 5.6
Cefetamet	0.005	102 ± 4.6	0.001	0.005	0.005	90.1 ± 10	0.002	0.005	0.005	95.1 ± 3.6	0.004	0.01
	0.01	102 ± 4.7			0.01	92.0 ± 7.4			0.01	102 ± 2.2
	0.02	101 ± 5.1			0.02	98.6 ± 12			0.02	101 ± 4.7
Cefepime	0.005	88.3 ± 14	0.006	0.02	0.005	108 ± 5.6	0.005	0.02	0.005	100 ± 21	0.002	0.006
	0.01	114 ± 25			0.01	96.9 ± 8.5			0.01	94.6 ± 8.4
	0.02	101 ± 8.2			0.02	83.4 ± 7.9			0.02	91.2 ± 7.8
Cefpodoxime	0.005	98.3 ± 7.3	0.002	0.007	0.005	83.2 ± 18	0.003	0.01	0.005	88.2 ± 8.9	0.003	0.009
	0.01	106 ± 6.8			0.01	91.6 ± 11			0.01	92.1 ± 4.3
	0.02	99.2 ± 3.6			0.02	95.5 ± 9.8			0.02	92.0 ± 4.5
Cefotaxime	0.005	99.4 ± 5.3	0.002	0.005	0.005	80.0 ± 26	0.003	0.01	0.005	98.6 ± 9.6	0.003	0.01
	0.01	104 ± 4.4			0.01	82.5 ± 10			0.01	99.0 ± 2.6
	0.02	101 ± 5.5			0.02	99.3 ± 8.1			0.02	98.1 ± 4.6
Cefoperazone	0.005	97.9 ± 8.4	0.002	0.005	0.005	95.3 ± 3.0	0.003	0.01	0.005	101 ± 11	0.002	0.006
	0.01	108 ± 11			0.01	91.6 ± 4.0			0.01	98.9 ± 1.1
	0.02	97.8 ± 9.6			0.02	99.3 ± 10			0.02	97.1 ± 3.8
Cefoxitin	0.005	111 ± 4.6	0.001	0.005	0.005	98.5 ± 10	0.002	0.008	0.005	113 ± 25	0.002	0.007
	0.01	105 ± 5.2			0.01	97.8 ± 7.9			0.01	105 ± 9.7
	0.02	98.6 ± 6.8			0.02	92.5 ± 7.1			0.02	91.2 ± 4.5
Cefuroxime	0.005	119 ± 17	0.003	0.01	0.005	62.1 ± 11	0.002	0.006	0.005	94.4 ± 14	0.003	0.01
	0.01	117 ± 14			0.01	84.5 ± 25			0.01	100 ± 7.4
	0.02	107 ± 11			0.02	105 ± 21			0.02	108 ± 2.7
Cefsulodin	0.005	94.4 ± 6.5	0.007	0.02	0.005	102 ± 18	0.002	0.006	0.005	82.1 ± 28	0.003	0.009
	0.01	98.7 ± 10			0.01	95.0 ± 12			0.01	86.6 ± 12
	0.02	104 ± 14			0.02	83.4 ± 3.3			0.02	92.8 ± 12
Cefquinome	0.005	85.3 ± 18	0.006	0.02	0.005	99.5 ± 15	0.001	0.004	0.005	105 ± 12	0.003	0.009
	0.01	105 ± 20			0.01	101 ± 3.1			0.01	102 ± 4.1
	0.02	102 ± 13			0.02	94.2 ± 6.6			0.02	96.1 ± 4.4
Ceftiofur	0.2	106 ± 8.6	0.2	0.6	0.2	95.1 ± 7.4	0.1	0.3	0.005	102 ± 7.4	0.0008	0.003
	0.4	119 ± 7.6			0.4	94.1 ± 8.3			0.01	98.1 ± 3.5
	0.8	107 ± 11			0.8	96.6 ± 11			0.02	96.4 ± 1.7
Desfuroyl ceftiofur	0.2	82.8 ± 15	0.2	0.8	0.2	85.0 ± 9.7	0.2	0.5	0.005	–	–	–
	0.4	92.7 ± 10			0.4	87.0 ± 6.6			0.01	–
	0.8	75.1 ± 9.2			0.8	99.4 ± 7.0			0.02	–
Cefpirome	0.005	99.2 ± 9.8	0.004	0.01	0.005	95.1 ± 15	0.003	0.01	0.005	97.3 ± 19	0.005	0.02
	0.01	105 ± 12			0.01	94.0 ± 5.7			0.01	101 ± 7.7
	0.02	98.0 ± 9.6			0.02	86.3 ± 5.0			0.02	93.8 ± 6.3
Cefixime	0.005	–	–	–	0.005	–	–	–	0.005	105 ± 19	0.003	0.01
	0.01	–			0.01	–			0.01	82.6 ± 16
	0.02	–			0.02	–			0.02	84.0 ± 15
Amoxicillin	0.025	100 ± 5.5	0.02	0.07	0.025	89.0 ± 14	0.004	0.01	0.025	97.4 ± 9.2	0.01	0.04
	0.05	108 ± 4.6			0.05	95.3 ± 4.2			0.05	96.4 ± 4.6
	0.1	103 ± 10			0.1	87.2 ± 4.9			0.1	92.9 ± 10
Ampicillin	0.025	93.5 ± 5.6	0.03	0.09	0.025	88.6 ± 14	0.01	0.07	0.025	102 ± 4.4	0.01	0.04
	0.05	102 ± 5.1			0.05	94.1 ± 5.1			0.05	99.9 ± 1.4
	0.1	99.8 ± 7.4			0.1	95.0 ± 8.2			0.1	94.1 ± 3.5
Oxacillin	0.005	114 ± 11	0.001	0.005	0.005	95.7 ± 1.4	0.002	0.007	0.005	102 ± 7.9	0.002	0.007
	0.01	117 ± 3.8			0.01	88.8 ± 8.2			0.01	100 ± 4.8
	0.02	105 ± 12			0.02	90.2 ± 8.8			0.02	100 ± 4.5
Cloxacillin	0.005	114 ± 14	0.003	0.009	0.005	93.8 ± 3.7	0.002	0.008	0.005	104 ± 4.7	0.003	0.009
	0.01	115 ± 5.3			0.01	89.5 ± 5.6			0.01	99.4 ± 3.5
	0.02	99.7 ± 15			0.02	93.7 ± 9.2			0.02	96.0 ± 3.1
Penicillin G	0.005	80.9 ± 16	0.0008	0.003	0.005	76.9 ± 14	0.004	0.01	0.005	101 ± 8.4	0.002	0.005
	0.01	100 ± 25			0.01	82.4 ± 10			0.01	95.5 ± 3.7
	0.02	86.9 ± 14			0.02	91.9 ± 9.4			0.02	94.0 ± 2.9
Penicillin V	0.005	112 ± 10	0.002	0.005	0.005	94.5 ± 5.7	0.001	0.004	0.005	102 ± 6.7	0.003	0.009
	0.01	114 ± 6.4			0.01	94.6 ± 7.1			0.01	104 ± 1.9
	0.02	102 ± 13			0.02	100 ± 9.6			0.02	105 ± 1.5
Piperacillin	0.005	111 ± 5.1	0.002	0.006	0.005	89.2 ± 4.5	0.002	0.007	0.005	104 ± 7.1	0.002	0.007
	0.01	112 ± 7.8			0.01	90.2 ± 7.3			0.01	103 ± 1.3
	0.02	99.8 ± 9.8			0.02	95.6 ± 9.8			0.02	99.5 ± 3.0
Hetacillin	0.005	94.2 ± 4.8	0.006	0.02	0.005	115 ± 24	0.004	0.01	0.005	102 ± 4.4	0.002	0.006
	0.01	101 ± 2.7			0.01	110 ± 11			0.01	98.3 ± 4.3
	0.02	98.0 ± 5.6			0.02	109 ± 9.3			0.02	95.9 ± 5.9
Aspoxicillin	0.005	98.6 ± 7.2	0.004	0.01	0.005	94.8 ± 16	0.002	0.006	0.005	106 ± 6.5	0.004	0.01
	0.01	110 ± 6.3			0.01	98.0 ± 6.0			0.01	100 ± 6.4
	0.02	103 ± 9.1			0.02	81.5 ± 6.5			0.02	93.4 ± 8.5
Mezlocillin	0.005	114 ± 7.8	0.003	0.008	0.005	95.4 ± 2.8	0.001	0.004	0.005	102 ± 8.3	0.002	0.008
	0.01	113 ± 7.6			0.01	89.5 ± 5.0			0.01	98.5 ± 3.7
	0.02	96.0 ± 12			0.02	91.6 ± 7.9			0.02	97.8 ± 2.7

LOD limit of detection, LOQ limit of quantification

In Korea, the use of β-lactams such as cefadroxil, cefalexin, ceftiofur, desfuroyl ceftiofur, amoxicillin, and ampicillin in fish and amoxicillin and ampicillin in shellfish is regulated (MFDS, 2022). And other compounds, including penicillin G, cloxacillin, dicloxacillin, and oxacillin, are also regulated by the European Union (Cañada-Cañada et al., 2009; European Commission, 2008). Methods for some penicillins and cephalosporins have already been prepared in previous studies (Guidi et al, 2017; Turnipseed et al., 2017). However, more substances may be included in this method and it showed good accuracy and precision in three types of fishery food and can be extended to other matrices in future studies.

Considering the introduction of PLS ​​in Korea and the regulation of some β-lactams in fish products, it is necessary to develop an analytical method for this. Therefore, validation of fish products in this study is meaningful, and further studies and applicability evaluation may be required to include some unquantified substances (cefacetrile, cefixime, and desfuroyl ceftiofur).

Declarations

Conflict of interest

All authors declares that they have no conflict of interest to disclose.