Simultaneous determination of tylosin and josamycin residues in muscles, liver, eggs and milk by MLC with a monolithic column and time-programmed UV detection: application to baby food and formulae
© Nasr et al.; licensee Chemistry Central Ltd. 2014
Received: 18 April 2014
Accepted: 13 June 2014
Published: 20 June 2014
Tylosin and Josamycin are macrolide antibiotics. They are used in the treatment of pneumonia, arthritis and mastitis in cattle, and mycoplasma infections in poultry. The incorrect use of antibiotics has lead to the presence of antibiotic residues in foods. The residues cause toxic effects on consumers.
A simple and sensitive method was optimized and validated for the analysis of tylosin and josamycin residues in food samples. Analytical separation was performed in less than 10 min using a RP C18 monolithic column with time-programmed UV detection at 287 nm and 232 nm and a micellar solution of 0.17 M sodium dodecyl sulphate, 14% methanol and 0.3% triethylamine in 0.02 M phosphoric acid buffered at pH 4 as the mobile phase. The method was fully validated in accordance with ICH guidelines. The micellar method was successfully applied to quantitatively determine tylosin and josamycin residues in spiked chicken muscles, chicken liver, bovine muscles, liver, milk and eggs. It was also extended to the determination of tylosin and josamycin residues in chicken-based baby food and baby formulae. The compounds were separated by a monolithic column which, on account of its particular structure, could bear higher flow rates than usually found for this kind of analysis. High extraction efficiency for tylosin and josamycin was obtained without matrix interference in the extraction process and in the subsequent chromatographic determination. No organic solvent was used during the pretreatment step. Hence, it is considered an interesting technique for “green” chemistry.
The proposed method was validated and successfully applied for the determination of tylosin and josamycin residues in spiked chicken muscles, chicken liver, bovine muscles, liver, milk and eggs. It was also extended to the determination of tylosin and josamycin residues in chicken-based baby food and baby formulae.
KeywordsMicellar liquid chromatography Tylosin Josamycin Monolithic column Chicken muscles Eggs
Macrolide antibiotics are a very important class of antibacterial compounds widely used in human and veterinary practices for both therapeutic and prophylactic treatments . They are active agents against Gram-positive and some Gram-negative bacteria . Macrolides are also employed as growth promoters in stock farming in food producing animals .
There is no current legislation which establishes limits of veterinary drug residues in meat-based baby food and baby formulae. As a result of this lack of regulations for veterinary drugs, a zero-tolerance policy is applied for veterinary drug residues in baby food and formulae  which means that the presence of these compounds is illegal at any level. Therefore, the application of the “zero tolerance” concept requires the development of sensitive analytical methods to determine the presence of these compounds at very low concentration levels .
Literature revealed several HPLC methods for the simultaneous determination of TS and JM residues in muscle tissues. TS and JM residues were determined together with other macrolide antibiotics in meat , bovine, porcine and poultry muscles  using UV detection. They were also determined in sheep’s milk using UV-diode array detector . Liquid chromatography coupled with electrospray ionization tandem mass spectrometry was used for the determination of TS and JM residues among other veterinary drugs in bovine, porcine and chicken muscles  and with other macrolide antibiotics in meat and fish . HPLC [17, 18] and UPLC  coupled with tandem mass spectrometry were also used.
Micellar liquid chromatography (MLC) allows complex matrices to be analyzed without the aid of extraction and with direct injection of samples . Micelles tend to bind proteins competitively, thereby releasing protein-bound drugs and proteins, rather than precipitating into the column. Proteins are solubilized and washed harmlessly away, eluting with the solvent front. This means that costs and analysis times are cut considerably . Micellar mobile phases usually need less quantity of organic modifier and generate less amount of toxic waste in comparison to aqueous-organic solvents, so that they are less toxic, non-flammable, biodegradable and relatively inexpensive . Because of these advantages, MLC is considered an interesting technique for “green” chemistry that copes with current concern about the environment . MLC has proved to be a useful technique in the determination of diverse groups of compounds in several matrices [23–25], including food samples [26, 27].
Nowadays, the most challenging trend in liquid chromatography is the development of new sorbents, which are able to separate complicated substances efficiently. One of these novel types of sorbents is monolithic silica. They have a different structure compared to conventional silica . Monolithic columns contain a special silica (or another material), which is not formed by particles. They are made by sol–gel technology, which enables formation of highly porous material, containing macropores and mesopores in its structure . Due to these facts, higher flow rates can be used while the resolution of the silica rod column is much less affected in comparison to particulate materials after increasing the flow-rate and column back-pressure is still low. Another practical advantage is a short time needed for column equilibration when a mobile phase gradient is used . There are a few works that deal with the practical application of monolithic columns in LC [30, 31].
The present study describes, for the first time, a rapid, simple, sensitive and selective MLC–monolithic method with UV detection for the simultaneous determination of TS and JM residues in chicken muscles, chicken liver, bovine meat, liver, eggs and milk. The proposed procedure benefits from the two main advantages associated to the use of micellar mobile phases, namely, the use of environment-friendly eluents and fast and easy sample preparation. The procedure makes use also of the main advantage of monolithic columns which is the use of high flow rate without increasing the column back pressure. The procedure could also be extended to the analysis of these residues in chicken or meat-based baby food in addition to baby formulae.
Chromatographic analyses were carried out using a Shimadzu Prominence HPLC system (Shimadzu Corporation, Japan) with a LC-20 AD pump, DGU-20 A5 degasser, CBM-20A interface, and SPD-20A UV–VIS detector with 20 μL injection loop. The columns used were reversed-phase Chromolith® Performance (RP-18e, 100 mm × 4.6 mm i.d.) column obtained from Merck (Darmstadt, Germany) and Nucleodur MN-C18 column (150 mm × 4.6 mm i.d., 5 μm particle size), Macherey-Nagel, Düren, Germany. Centrifugation was carried out using TDL-60B Centrifuge (Anke, Taiwan). BHA-180 T Sonicator (Abbotta Corporation, USA) was used. Tissue homogenization was made using Tissue Master-125 Homogenizer (Omni International, Georgia, USA).
Reagents and materials
All reagents and solvents used were of HPLC grade. Tylosin tartrate and Josamycin analytical standard was of 100% purity from Sigma-Aldrich (Seelze, Germany). Methanol, 1-propanol, acetonitrile and sodium dodecyl sulphate (SDS) were from Sigma-Aldrich (Seelze, Germany). Triethylamine and phosphoric acid were from Riedel-deHaën (Seelze, Germany). Regenerated cellulose membrane filters and syringe filters (Minisart RC25) with pore size 0.45 μm were from Sartorius-Stedim (Goettingen, Germany). Chicken muscles, chicken liver, bovine meat and liver samples and eggs were purchased from the local market. Milk was from Juhayna® (Cairo, Egypt) and Infant Formula Milk was from Hero® (Spain). Chicken noodles baby food, precooked, was from Gerber® (USA).
Preparation of solutions
Stock solutions of 0.2 mg mL−1 of each TS and JM were prepared by dissolving in methanol. Working solutions were prepared by diluting the stock solutions with the mobile phase. Stock solutions were found to be stable for one week if kept in the refrigerator protected from light.
Preparation of calibration curves
Working solutions containing 1 – 200 μg mL−1 of TS and 5 – 500 μg mL−1 of JM were prepared by serial dilutions of aliquots of the stock solutions. 20 μL aliquots were injected (triplicate) and eluted with the mobile phase under the reported chromatographic conditions. The average peak areas of each drug were plotted versus the concentrations of drug in μg mL−1. Alternatively, the corresponding regression equations were derived.
Analysis of bulk substance
The method mentioned under the previous section was applied to the determination of the purity of TS and JM raw materials. The percentage recoveries were calculated by referring to the calibration graphs previously prepared or by applying the regression equations.
2.5 g of each of samples were accurately weighed and 5 mL of milk were measured and spiked with aliquots of TS and JM solutions. The spiked samples were mixed with 25 mL of 0.17 M SDS solution of pH 4. The solid samples were then homogenized at 5000 rpm for 5 min, then, the homogenate was sonicated for 15 min, and then centrifuged at 3000 rpm for 5 min. The egg and milk samples were not homogenized, but they were only sonicated for 2 min without centrifugation. The supernatant of all samples was filtered through 0.45 μm membrane filters using vacuum pump and diluted with the mobile phase. Aliquots of 20 μL were injected (triplicate) and eluted with the mobile phase under the reported chromatographic conditions.
Results and discussion
Selection and optimization of chromatographic conditions
Optimization of experimental factors affecting the chromatographic performance of the proposed method
Number of theoretical plates (N)
Organic modifier nature
Methanol concentration (%)
SDS concentration (M)
Choice of column
Two different columns were tested for performance investigations, including: reversed-phase Chromolith® Performance C18 column, and Nucleodur MN-C18 column. The experimental studies revealed that the first column was more suitable, since it produced well-resolved peaks with a very high sensitivity within a reasonable analytical run time.
Choice of appropriate detection wavelength and time program
The UV detector responses of TS and JM were carefully studied and the best wavelengths were found to be 287 nm and 232 nm for TS and JM, respectively showing the highest sensitivity. Programmable UV detection was employed to allow sensitive determination of both TS and JM simultaneously. TS was detected at 287 nm within 0–5 min, while, JM was detected at 232 nm after 5 min.
Choice of a suitable flow rate
As the peak of JM was very late at flow rate 1 mL min−1, a flow rate of 2 mL min−1 was chosen in order to have a reasonable analytical run time (10 min) for the assay. The use of this considerably high flow rate was possible due to the use of a monolithic column which has the advantage of using high flow rates without affecting the column back pressure.
Mobile phase composition
Several modifications in the micellar mobile phase composition were performed in order to study the possibilities of changing the selectivity of the chromatographic system. These modifications included the change of the concentration and type of organic modifier, the surfactant concentration, and the pH. The mobile phase was prepared using 0.3% triethylamine and 0.02 M phosphoric acid. The effect of changing the type of organic modifier on the selectivity and retention times of TS and JM was investigated using mobile phases containing 10% methanol, 1-propanol or acetonitrile. Methanol was the best, giving well-resolved peaks and the highest number of theoretical plates. The effect of changing the concentration of organic modifier on the selectivity and retention times of TS and JM was investigated using mobile phases containing concentrations of 10–16% methanol and containing 0.15 M SDS and buffered at pH 4. 14% of methanol was the best, giving well-resolved peaks and the highest number of theoretical plates. Hence, a small amount of methanol is added to accelerate and control the elution of the drug. The effect of changing the concentration of surfactant on the selectivity and retention times of TS and JM was investigated using mobile phases containing SDS concentrations in the range 0.1–0.2 M and containing 14% methanol and buffered at pH 4. 0.17 M SDS was the best, giving well-resolved peaks and highest number of theoretical plates. The effect of changing the pH of the mobile phase on the selectivity and retention time of TS and JM was investigated using mobile phases of pH ranging from 2.5–5.0 with 0.17 M SDS concentration and 14% methanol. pH of 4.0 was most appropriate, giving well-resolved peaks and the highest number of theoretical plates. Values of pH higher than 5.0 resulted in very low number of theoretical plates.
After these experimental investigations, the assay was carried out using a Chromolith® Performance C18 column with mobile phase consisting of 0.17 M sodium dodecyl sulphate–14% methanol–0.3% triethylamine–0.02 M phosphoric acid at pH 4.0 and programmable UV detection with a flow rate of 2.0 mL min−1.
System suitability test parameters
To ascertain the reproducibility of the MLC method, system suitability tests were performed using the working standard solutions of TS and JM. Resolution (Rs), theoretical plates number (N) and tailing factor (T) were measured as the criteria for system suitability testing. These results are satisfactory compared to the minimum values necessary for an acceptable method.
The validity of the proposed MLC method was tested in terms of linearity, ranges, limits of detection, limits of quantification, accuracy and precision.
Linearity and range
Where P is the peak area and C is the concentration of drug in μg mL−1 and r is the regression coefficient.
The high values of the correlation coefficients (r-values >0.999) indicate good linearity of the calibration graphs. Statistical analysis of the data gave small values of the % relative error, (% Er) 1.21% and 0.56% for both TS and JM, respectively .
Limit of quantitation (LOQ) and limit of detection (LOD)
The limit of quantitation (LOQ) was determined by establishing the lowest concentration that can be measured according to ICH Q2B recommendations  below which the calibration graph is non linear and was found to be 3.6 μg g−1 and 9.9 μg g−1 for TS and JM, respectively. The limit of detection (LOD) was determined by establishing the minimum level at which the analyte can be reliably detected (S/N = 3); it was found to be 1.1 μg g−1 (1.0 × 10−7 M) and 3.0 μg g−1 (3.6 × 10−7 M) for TS and JM, respectively.
Statistical analysis of the results obtained by the proposed and reference methods for pure samples of tylosin and josamycin
Variance ratio F-valuea
Accuracy and precision data for tylosin and josamycin using the proposed method
Concentration (μg mL−1)
Recovery (mean ± S.D.)
Recovery (mean ± S.D.)
99.8 ± 0.2
99.1 ± 0.6
99.5 ± 0.6
100.2 ± 0.4
99.6 ± 0.6
99.7 ± 0.5
100.8 ± 0.3
100.3 ± 0.3
99.6 ± 0.5
100.9 ± 0.4
99.7 ± 0.5
99.6 ± 0.6
Assay of tylosin and josamycin in food samples using the proposed and reference methods
Mean recovery a
Variance ratio F-valueb
Mean recovery a
Variance ratio F-valueb
Mean recovery a
Variance ratio F-valueb
Mean recovery a
Variance ratio F-valueb
The proposed procedure is useful for food quality testing and control areas to determine the content of TS and JM in chicken muscles, liver, bovine muscles, liver, milk and egg samples. Moreover, it allows the detection of traces of TS and JM residues in meat or chicken-based baby food and baby formula milk with high sensitivity. One advantage of this procedure is the possibility of injecting the samples directly into the chromatographic system with no previous treatment other than homogenization, dilution and filtration, thus avoiding tedious extractions from matrices. Validation according to ICH regulation provides satisfactory results in terms of sensitivity, linearity, accuracy and recoveries and at the ppm level. It is noteworthy that the use of micellar mobile phases endows the procedure advantages such as non flammability, biodegradability and low cost. Current concern about the environment also reveals MLC as an interesting technique for “green” chemistry because it uses mobile phases containing low amounts of organic solvents. These micellar mobile phases have a low toxicity and are not producing hazardous wastes. It can be also concluded that, under our conditions, the monolithic column could operate at a higher flow rate than a conventional RP column with a reduced pressure and shorter washing and re-equilibration times.
Micellar liquid chromatography
Limit of detection
Limit of quantification.
This work was supported by L’Oréal-UNESCO Pan Arab Fellowship ‘For Women in Science’ 2010, in partnership with the Arab Science and Technology Foundation (ASTF).
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