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Determination of doxycycline in chicken fat by liquid chromatography with UV detection and liquid chromatography–tandem mass spectrometry
Journal of Chromatography B, 928 (2013) 113–120
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of doxycycline in chicken fat by liquid chromatography with UV detection and liquid chromatography–tandem mass spectrometry Anna Gajda a,∗ , Andrzej Posyniak a , Jan Zmudzki a , Grzegorz Tomczyk b a b
Department of Pharmacology and Toxicology, National Veterinary Research Institute, Partyzantów 57, 24-100 Pulawy, Poland Department of Poultry Diseases, National Veterinary Research Institute, Partyzantów 57, 24-100 Pulawy, Poland
a r t i c l e
i n f o
Article history: Received 4 January 2013 Accepted 11 March 2013 Available online 21 March 2013 Keywords: Doxycycline Fat Validation LC–UV LC–MS/MS
a b s t r a c t A sensitive analytical method for determination of doxycycline (DC) residues in chicken fat/fat and skin was developed. The extraction, in the presence of the internal standard (IS) minocycline (MINO), was carried out using solution of oxalic acid (pH 4.0) and ethyl acetate. The samples were cleaned up by solid phase extraction (SPE) procedure using, at first carboxylic acid and then polymeric Strata X cartridges. Chromatographic separation of DC by LC–UV was achieved on a Luna C8 analytical column and for LC–MS/MS analysis Luna C18 column was used. The presented procedures were evaluated according to the Commission Decision 2002/657/EC. Specificity, decision limit (CC␣), detection capacity (CC), recovery (absolute and relative), precision (repeatability and reproducibility) were determined during validation process. The limit of detection (LOD) was 10 g/kg for LC–UV and 1 g/kg for LC–MS/MS method. The limit of quantitation (LOQ) was 15 and 2 g/kg for LC–UV and LC–MS/MS, respectively. The absolute recovery for the LC–UV and relative recovery for the LC–MS/MS method at 300 g/kg concentration level were 79%; 101% for fat and 82%; 99% for fat and skin, respectively. The developed liquid chromatography with ultraviolet detection (LC–UV) and liquid chromatography tandem mass spectrometry (LC–MS/MS) methods have been applied to quantitative determination of doxycycline (DC) in samples of chicken fat tissue obtained from animals treated with DC. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Doxycycline is a member of the tetracycline antibiotics group, that is widely used in the treatment of respiratory and urinary tract infections in various species. It is bacteriostatic compound, which acts by inhibiting protein biosynthesis after binding to the 30 S ribosomal subparticle in bacterial cell. Improper use of doxycycline in food producing animals can cause its residues in animal tissues, which can be toxic and dangerous for human health and may generate the resistance of many microorganisms to antibiotics. Doxycycline is more lipids soluble than other tetracyclines and penetrates to body tissues and fluids in a greater extent after the application. Stronger lipophilic character may result in long persistence of doxycycline in animal body, especially fat, what can cause unacceptable concentration in animal tissues [1,2]. The maximum residue limits (MRLs) established by the European Union for
∗ Corresponding author at: National Veterinary Research Institute, Department of Pharmacology and Toxicology, al. Partyzantow 57, 24-100 Pulawy, Poland. Tel.: +48 81 889 31 27; fax: +48 81 8862595. E-mail address: [email protected] (A. Gajda). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.03.011
doxycycline in pigs, poultry and bovine are as follows: muscle – 100 g/kg, kidney – 600 g/kg, liver – 300 g/kg. The MRL for fat and skin – 300 g/kg has been established only for pigs and poultry, excluding bovine. According to Commission Regulation (EU) 37/2010 [3], quantification of doxycyline in animal tissues requires a determination only a parent compound as the marker residue, without its 4-epimer, contrary to other tetracyclines, where the MRLs are defined as a sum of parent drugs and their 4-epimers. In reference to the international trade and international regulations, there is a need to control the food of animal origin, including fat. For the export purposes, it was required to develop a suitable method for analysis and quantification of doxycycline in fat. Additionally, there is no data on the DC residues and its depletion in chicken fat. According to the EMEA summary report [4] the highest DC residues are found in kidney, followed by liver, skin, muscle, however in fat, doxycycline is not detectable to any great extent. Therefore, it was decided to investigate the DC concentration and its stability in chicken fat after administration of Doksy RW® . The aim of conducted experiment was to prove, that there are also residual concentration of this compound in fat. Many analytical procedures have been developed for the determination of doxycycline in animal tissues [5–7], eggs [7,8], and plasma [9]. Only a few methods have been described for the
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determination of DC in pork fat [10,11]. It has been presented a paper of Cherlet for a quantitative analysis of tetracyclines in pig tissues (muscle, kidney, and liver, including skin + fat) by LC–MS/MS. To our knowledge, no analytical method has been proposed for determining DC residues in chicken fat so far. The determination of DC residue is generally performed by HPLC suitable detectors, including UV [9,12–14], fluorescence [6,7] diode-array [5,8,15] or mass spectrometry [10,16–19] detection. The most important part of analytical procedure are sample preparation and clean-up step. Most LC analysis for determination of TCs used a solid phase extraction (SPE) for clean-up purposes with different kinds of sorbents. Some researchers have successfully adopted matrix solid-phase dispersion (MSPD) technique [16]. It has been also reported a metal chelate affinity chromatography (MCAC) to establish a clean-up method [20]. A sample preparation requires an extraction step with a suitable solvent system. Most extraction techniques use mild acidic buffer pH 3–5 [6]. There have been also reported methods with organic solvents, such as ethyl acetate [7,21,22] for extraction. Due to the increasing use of tetracycline antibiotics, especially most commonly applied in poultry doxycycline, it was necessary to develop a sensitive and accurate method for the determination of doxycycline in chicken fat with a simultaneous UV and MS/MS detection, after one sample preparation step. The developed methods were verified and used to measure the DC residues after the experimental application of this antibiotic to chicken. Additionally, the presented methods have been also successfully applied to fat and skin samples, in natural form. The validation data for fat matrix and for fat with skin are presented in this study. 2. Experimental 2.1. Reagents Doxycycline (DC) standard (purity > 97%) was obtained from Sigma–Aldrich Chemical Company (USA). Minocycline (internal standard, purity > 97%) was also from Sigma Aldrich. All organic solvents were HPLC grade and all chemicals were analytical grade. Acetonitrile, methanol, and Bakerbond SPE cartridges (carboxylic acid 500 mg/3 ml) were from J.T. Baker. Strata X (33 m, 100 mg, 6 ml) polymeric solid phase extraction (SPE) columns were obtained from Phenomenex (USA). Oxalic acid dihydrate (ACS), trichloroacetic acid (TCA), ethyl acetate and sodium sulphate anhydrous were from POCh Gliwice (Poland). Heptafluorobutyric acid (HFBA) was from Sigma–Aldrich. Water was purified using Milli-Q system.
chicken were killed and fat was collected. The birds were sacrificed under the animal welfare rules of the European Council Directive 93/119/CE [23]. The experiment was approved by Ethical Local Committee No 2 in Lublin (Poland) with approval number 22/2011. Samples were homogenized and stored at −20 ◦ C until the analysis. 2.3.2. Incurred chicken fat The experiment was conducted with twenty-four week old chickens (16 animals), treated individually with DC (Doksy RW® , 10 mg/kg BW) administered orally after dilution with drinking water using syringe, once daily, for a period of five consecutive days. Food and water were available ad libitum. The treated chickens were killed at 1st day, 2nd day, 7th day and 8th day (4 animals per every day) after the final administration of drug and fat was collected from thigh muscle. All samples were kept separately at the temperature of −20 ◦ C until the analysis. 2.4. Sample preparation Before the extraction step, fat/fat and skin samples were grinded. Three grams of chicken fat/fat and skin were weighted and internal standard was added. Samples were mixed with 3 ml of 0.02 M oxalic acid buffer, pH 4.0 and then vortex mixing for 15 s. After addition of 15 ml of ethyl acetate, the mixture was vortexed again for 30 s. To the solution, 0.5 ml of 20% trichloracetic acid was added and all was stirred and centrifuged for 10 min at 2500 × g. The supernatant was transferred to a new tube. The extraction was repeated twice with another 15 ml of ethyl acetate and the extract solutions were combined. On the top of carboxylic acid SPE columns, which were earlier preconditioned with 5 ml of ethyl acetate, 2.0 g of sodium sulphate on funnels was placed. The whole supernatant was filtered through a sodium sulphate and poured on SPE columns. After percolation of the whole solution, the columns were washed with 2.5 ml of ethyl acetate and dried (under vacuum) for 10 min. The doxycycline was eluted with 2.5 ml of solution consisting of acetonitrile–methanol–0.02 M oxalic acid pH 2.0 (20:15:65, v/v/v). To an eluate solution, 8 ml of 0.02 M oxalic acid buffer, pH 4.0 was added and combined supernatants was poured onto SPE polymeric columns firstly preconditioned with 2 ml of methanol, 2 ml of water and 3 ml of 0.02 M oxalic acid buffer, pH 4.0. After the percolation of whole solution columns were washed with 3 ml of 0.02 M oxalic acid buffer (pH 4.0) and 2 ml of water, and dried (under vacuum) for 10 min. The DC was eluted with 5 ml of methanol and then the eluate was dried under a gentle nitrogen stream at 40 ◦ C. The dry residue was dissolved with 1 ml of water and after vortex mixing transfer to vials and ready to LC–UV, as well as LC–MS/MS analyze.
2.2. Preparation of standard solutions 2.5. Liquid chromatography–UV analysis 2.2.1. Standard solutions Stock standard solution (1 mg/ml) prepared by weighing out 10.0 ± 0.1 mg of standard substances and dissolving in 10 ml of methanol, was stable for six months when stored at the temperature below −20 ◦ C in amber glass. Working standard solutions (100 g/ml, 10 g/ml) prepared in acetonitrile by diluting suitable aliquot of stock standard were stable for three months, stored at 2–8 ◦ C in amber glass. Working standard solutions in mobile phase were prepared on the day of the analysis.
The instrumental analysis was performed using Varian Prostar HPLC system, equipped with quaternary pump, autosampler, column oven, and UV/Vis detector ( = 355 nm), controlled by Galaxie Workstation software. Chromatographic analyses were performed on Luna (Phenomenex) C8 column (5 m, 250 mm × 4.6 mm) with mobile phase consisting of acetonitrile–0.02 M oxalic acid, pH 2.0 in gradient mode at 1.0 ml min−1 flow rate (Table 1). The injection volume for LC–UV was 100 l. The column oven temperature was controlled at 30 ◦ C.
2.3. Sample collection 2.6. Liquid chromatography–mass spectrometry 2.3.1. Blank control chicken fat Blank fat samples were taken from 4 chicken never treated with doxycycline, used as control, bought from Chicken Farms in Poland. Birds were kept in groups, in separate rooms, at the temperature of 20 ◦ C. The average body weight was 2.6 kg. After 5 days
Analyses were performed on an Agillent 1200 LC system consisting of a quaternary pomp, an autosampler, a column heater (kept at 25 ◦ C), switching valve and an automatic degasser (Agilent Technologies, Palo Alto, CA, USA) and an API 4000 triple quadropule
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Table 1 Gradient elution for LC–UV and LC–MS/MS methods. LC–UV
LC–MS/MS
Time (min)
ACN (%)
0.02 M oxalic acid (%)
Time (min)
ACN (%)
0.025% HFBA (%)
0.00 1.00 5.00 7.00 10.00 12.00 15.00
5 5 20 30 20 5 5
95 95 80 70 80 95 95
0.00 12.00 12.01 15.00 15.01 23.00
5 60 90 90 5 5
95 40 10 10 95 95
mass analyser with a TurboIonSpray source (Applied Biosystems, Toronto, ON, Canada). The MS detector was configured as ESI (electrospray ionization). The ESI was operated in the positive ion mode and MS data acquisition was performed in the MRM mode, selecting one precursor ion to two products ion transitions for DC. The Analyst 1.5 software controlled the LC–MS/MS system processed the data. Nitrogen was used as nebulizer gas, curtain gas and collision gas. TurboIonSpray source was operated at 300 ◦ C with the capillary voltage set at 5500 V. Collision energy (CE) was optimized to maximize the relative abundance for ion transition. The chromatographic separation was performed on a Luna C18 (150 × 2.0 mm, 3 ) analytical column (Phenomenex) coupled with octadecyl guard column (2 mm × 4 mm) (Phenomenex). The mobile phase was consisted of solvent A: acetonitrile and solvent B: 0.025% of HFBA in water (v/v). The elution was performed in a gradient mode with injection volume of 30 l (Table 1). The column was operated at 35 ◦ C with a flow rate of 0.25 ml min−1 , and time of analyze – 23 min.
the repeatability and analysing on two different days with the same instrument and the different operators. The overall CVs were calculated. The accuracy was obtained as a relative recovery for LC–UV and for LC–MS/MS. The recovery was evaluated in the same experiment as repeatability. The relative recovery was determined by comparing peak area ratios (DC/internal standard) from fortified matrix samples with peak area ratios (DC/internal standard) from direct injections of equivalent quantities of standards. Additionally, for LC–UV the absolute recovery was calculated. The CC␣ and CC were determined by the matrix calibration curve procedure. CC␣ was calculated with a statistical certainty of 1 − ˛ (˛ − 0.05), and CC was calculated with a statistical certainty of 1 − ˇ (ˇ − 0.05).
2.7. Validation study
Signal suppression (%) =
The validation of the methods was performed according to the recommendations of the Commission Decision 2002/657/EC [24]. Parameters such as linearity, specificity, precision, accuracy, detection limit and limit of quantification, decision limit and detection capability were established. The method was validated at the 150, 300 and 450 g/kg. Calibration curves for the DC standards and extracted samples were constructed. Linearity was tested by preparing matrix calibration curve in a range of 50–600 g/kg. Blank chicken fat/fat and skin samples were fortified with working standard solution of DC at five concentration levels. Specificity was verified by analyzing 20 blank fat/fat and skin chicken samples of different origin, in order to verify the absence of potential interfering compounds at DC expected retention time. The LOD and LOQ values were considered as the concentration giving a signal to noise ratio of 3 and 10, respectively. Five different concentration levels from 10 to 30 g/kg were prepared by duplicate in order to establish the LOD and LOQ in LC–UV, whereas five levels from 0.1 to 6 g/kg were prepared in order to obtain LOD and LOQ using LC–MS/MS. For the evaluation of precision (repeatability and withinlaboratory reproducibility), as well as recovery, blank fat/fat and skin samples were spiked with the DC working standard solution to levels corresponding to 150, 300 and 450 g/kg, respectively. The experiments were carried out on three consecutive days. The repeatability was determined by fortifying six blank samples at each of three concentration levels 150, 300 and 450 g/kg with DC compound. The samples were analyzed on the same day with the same instrument and the same operators, and the coefficient of variations (CV) was calculated. The within-laboratory reproducibility was determined by fortifying other two sets of blank samples at the same concentration levels of the analyzed compound as for
where IF is the DC peak area in fat extract spiked after the extraction at 300 g/kg and IW is DC peak area in high-quality pure water extract spiked after extraction at 300 g/kg.
2.8. Matrix effect study for LC–MS/MS method In this study the interference caused by fat matrix during extraction was evaluated. To calculate the signal suppression for DC, the following equation was used [25]:
1−
IF IW
× 100
2.9. Stability The stability of DC in acetonitrile was evaluated in standard solutions stored at −20 ◦ C and +4 ◦ C at concentration of 10 g/ml at different time intervals: 1 day, 10 and 20 days, 1, 2, 3, 4, 5 and 6 months. Stability was also investigated in fat samples spiked with DC at the level of 100 g/kg. The spiked sample were analyzed on the day of the preparation. Samples were prepared in duplicate. Then, the spiked samples were stored at −20 and +4 ◦ C, and analyzed every week for 4 weeks. A short-term stability was evaluated on extracts stored at room temperature for 72 h into automatic injector. Sample were analyzed at 0, 24, 48,and 72 h. Samples were prepared at concentration of 100 g/kg. 3. Results and discussion 3.1. Sample preparation The effective isolation of tetracycline residues from biological matrices is difficult because of their binding with sample proteins and chelating with metal ions. In numerous studies for the determination of doxycycline, it has been presented an extraction by use of EDTA and oxalic acid buffer, citrate buffer to overcome such undesirable properties [5,14,15,18,26,27]. The difficulty in isolating some antibiotics from fat is connected with its polarity. According to Cherlet et al. [10] it seems to be a decreasing recovery with decreasing polarity of the component (from oxytetracycline, one
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Table 2 Multiple reaction monitoring (MRM) mode for the detection of doxycycline. Analyte
RT (min)
Precursor (m/z)
Product (m/z)
DP (V)
CE (V)
CXP (V)
Peak area ratio ± tolerance (%)
Doxycycline
12.52 10.97
428 154 441
55 55 72
25 40 25
15 15 12
0.09 ± 50
Minocycline (IS)
445 445 458
RT: retention time; DP: declustering potential; CE: collision energy; CXP: cell exit potential.
of the most polar tetracyclines to doxycycline) for muscle, liver and kidney tissues. Surprisingly, the recovery for DC in fat was relatively good. In Cherlet et al. study, the extraction of tetracyclines was performed using sodium succinate solution. In this method doxycycline was extracted by oxalic acid to dechelate the doxycycline-metal complex. Different pH values of solvent extraction were checked (pH 2.0, pH 4.0 and pH 6.0). The best results were obtained when the pH 4.0 of oxalic acid buffer was used. The addition of 20% trichloracetic acid to the extraction step coagulated and removed the protein, that may occur in matrix. The first sample preparation step in presented study was an improvement of the previously described analytical method for the determination of tetracyclines in animal tissue and eggs [7]. Fats consist of a wide group of compounds that are generally soluble in organic solvents and generally insoluble in water. When the acetonitrile (as more polar solvent) was used to extraction, the recovery was much lower. It was confirmed that ethyl acetate (a mid-polar solvent) is suitable for a quantitative isolation of doxycycline from biological matrices, also from fat/fat and skin and the use of triple extraction gives better and more reliable results. For the determination of tetracyclines, including doxycycline, in animal tissues several types of cartridges for clean-up purposes were investigated such as: C18 [5,13,15], polymeric (Strata X) [26], carboxylic acid (COOH) [7], SDB1 (styrene-divinylbenzene) [12] or Bond Elut ENV [18]. It has been also described on-line MCAC cleanup [14], or Oasis (HLB) cartridges after pressurized liquid extraction (PLE) [19]. However, the most suitable for clean-up step in fat tissues were carboxylic acid columns and then polymeric cartridges. The usage of Strata X columns allowed concentrating an extract and obtaining more sensitive method, with a very low DC concentration level detection. Additionally, after two step clean-up, the final extract was clean and pure. An application of methanol to the DC elution from Strata X columns, gave the possibility to obtain a suitable extract for LC–MS/MS analysis. The presented methods were verified and checked for DC extraction from incurred chicken fat, as well as they were performed well for DC isolation from spiked fat and skin samples. Their usefulness for fat of other species has been proved. Many analytical methods for the analysis of DC in animal tissues involve demeclocycline, as an internal standard [10,18,19,22]. Because of a better repeatability and stability for minocycline, (inhouse data), as well as consistent retention time, minocycline, as a member of tetracyclines group not used in food producing animals, was chosen in presented study.
3.2. Liquid chromatography In other studies, the usage of polymeric [6,10,12,15,27], phenyl [13] or C18 analytical columns to analyze tetracyclines in animal tissues [15,16], has been presented. For the DC quantification by LC–UV, C8-bonded silica column was used based on the results of Neils and De Leenheer [28]. The use of this column eliminated the need to use ion-pairing agents in the mobile phase that are generally required to reduce tetracycline tailing with conventional silanol LC columns. At C8 column, DC was observed at 11 min with stable retention time. To avoid DC adsorption on RP column and
forming chelate complexes various acids – citric, oxalic, phosphoric acid [6,12,18,22], were tested. It has been also reported an ion exchange column with hydrochloric acid:acetonitrile eluent for the analysis of tetracyclines [27]. In the presented method, RP column was chosen. The best results for this type of column were obtained when solution of oxalic acid was used. The asymmetries of DC peak depend on the pH value of the aqueous oxalic acid. The optimum pH was 2.0. LC mobile phase consisting of acetonitrile–0.02 M oxalic acid in gradient mode proved to be suitable for symmetrical and sharp peak. Application of gradient elution was necessary to obtain good separation for DC and MINO (IS). The oxalic acid appears to strip or mask any metal ions that may affect doxycycline analyses, effectively. Furthermore, at = 355 nm no interference was observed with endogenous substances of the blank tissues. Hence, the wavelength of = 355 nm was chosen for quantitation of doxycycline. Fig. 1 shows the chromatograms obtained with LC–UV corresponding to the blank fat sample, DC spiked sample and the samples from the treated animals. In the blank sample, the only substance observed corresponds to the internal standard (Fig. 1a). In chromatogram B, DC was added at the concentration of 150 g/kg (Fig. 1b). For the LC–MS/MS analysis, good results for DC were achieved using C18 analytical column. Oxalic buffer of the mobile phase was replaced by 0.025% of HFBA in water (v/v). The use of ion-paring agent in the mobile phase reduced DC tailing. For the DC to be positively confirmed, the retention time had to match that of a standard to within 2.5%. MS data acquisition was performed in the multiple reaction monitoring (MRM) mode, selecting one precursor ion to two products ion transitions for analyte. The most abundant ion, as precursor ion m/z 445.0 was chosen. Declustering potential (DP), collision cell exit potential (CXP) and collision energy (CE) were established. The optimal conditions with transition parameters for detection of DC, LC–MS/MS retention time of analyte and ion ratio are reported in Table 2. The presented LC–MS/MS method fulfills all the EU requirements for confirmatory method with consistent retention time and can be used for DC presence confirmation in fat. Fig. 2 shows the typical MRM chromatograms of DC and internal standard in the spiked fat sample at the concentration of 150 g/kg. 3.3. Method validation 3.3.1. Linearity The linearity was evaluated by five point’s calibration curve with triple analysis. Correlation coefficient (r = 0.998) was obtained for calibration curve using standard solutions. Matrix-calibration curve for DC fat/fat and skin spiked samples had correlation coefficient above 0.99 for LC–UV, as well as for LC–MS/MS, what showed a good linearity between DC concentration and peak area (Table 3). 3.3.2. Specificity Specificity is the ability of a method to distinguish between the analyte of interest and other substances (impurities or matrix components) that may be present in a test sample. In the evaluation of the specificity blank fat/fat and skin samples were analyzed by both UV and MS detectors. The results obtained with blank samples have been compared with DC spiked samples and it is observed that no
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Fig. 1. Chromatograms of (a) fat blank sample; (b) fat blank sample with internal standard; (c) fat DC spiked sample at 150 g/kg; (d) incurred fat sample at 1 day after last treatment.
Fig. 2. LC–MS/MS chromatograms in MRM mode of DC and internal standard at the level of 150 g/kg.
Table 3 Linearity, working range, limit of detection (LOD), limit of quantification, decision limit and detection capability of suggested methods. Parameters
Correlation coefficient (r) Linearity (working range) (g/kg) Limit of detection (g/kg) Limit of quantification (g/kg) Decision limit (g/kg) Detection capability (g/kg)
LC–UV
LC–MS/MS
Fat
Fat + skin
Fat
Fat + skin
0.991 50–600 10 15 324 356
0.992 50–600 10 15 336 361
0.995 50–600 1 2 317 344
0.994 50–600 1 2 322 350
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Table 4 Precision and recovery of the presented method for doxycycline in chicken fat and fat with skin samples (n = 18). Parameters
Spiked level (g/kg) 150
LC–UV Recovery (absolute, %) Recovery (relative, %) Repeatability, CV (%) Reproducibility, CV (%) LC–MS/MS Recovery (relative, %) Repeatability, CV (%) Reproducibility, CV (%)
300
Fat
Fat + skin
77 95 9.5 13.3
79 93 9.7 14.0
99 8.6 11.9
96 9.0 13.2
interfering peaks with LC–UV were found in the retention time of the target analyte (Fig. 1). Fig. 3a shows the results obtained with LC–MS/MS of fat spiked sample at 150 g/kg. 3.3.3. Precision and recovery In the present work, recoveries and precision assays of the proposed method were evaluated using spiked samples including the MRL concentration level (Commission Regulation (EU) 37/2010) [1]. Thus, chicken fat/fat and skin samples were spiked at 150, 300 and 450 g/kg in 18 replicates, respectively. The relative DC recovery for LC–UV method ranged between 92–95% for fat samples and 93–96% for fat and skin, respectively. For LC–MS/MS relative recovery were established at a range of 97–101% for fat and 96–99% for fat with skin. Additionally an absolute obtained recovery for LC–UV was 77–81% for fat and 79–82% for fat and skin. All the results are presented in Table 4. The repeatability and within-laboratory reproducibility for DC were lower than 10 and 14%, respectively at all spiking levels for fat and fat with skin. The satisfactory results of precision expressed as coefficient of variation (CV) showed that the presented methods can be used as validated methods and can be useful for confirmation of DC residues in fat/fat and skin. At each level, the accuracy and precision fell within the required ranges for that specific concentration.
3.3.4. Limits of detection and quantification The procedure was satisfactorily sensitive; the limit of detection was established at the level of 10 g/kg and 1 g/kg for LC–UV and LC–MS/MS for fat/fat and skin, respectively. The limit of quantification was 15 g/kg and 2 g/kg for LC–UV and LC–MS/MS for fat/fat and skin. The obtained results indicate LC–MS/MS being a more sensitive method. Values obtained with LC–MS/MS, as well as LC–UV technique were much lower than MRL established by the European Union. All values of LOD and LOQ are shown in Table 3.
3.3.5. Decision limit and detection capability According to Council Directive 2002/657/EC an interpretation of the results requires decision limit (CC␣) and detection capability (CC) to be determined. CC␣ is defined as the concentration above which it can be decided with a statistical certainty of (1 − ˛) that the identified analyte content is truly above MRL. CC␣ value is established with an error of 5% (probability of false non compliant ≤5%) and CC is determined with an error ˇ = 5% (probability of false compliant samples ≤5%). Table 3 summarizes the obtained CC␣ and CC for chicken fat/fat and skin at the level of 300 g/kg. The results showed that method can be useful for detection and quantification DC residues in fat, as well as in fat and skin samples.
Fat
450 Fat + skin
Fat
Fat + skin
79 94 9.0 12.8
82 96 9.4 13.3
81 92 9.1 12.1
80 96 8.9 12.9
101 7.9 10.8
99 8.8 12.9
97 7.6 11.2
97 7.3 12.4
3.4. Stability The stability of standard solutions was examined. The DC standard solution stored at −20 ◦ C was stable for 6 months, while the stability of DC solutions dissolved in acetonitrile at the concentration of 10 g/kg stored at +4 ◦ C was 3 months. Fat samples spiked at the level of 100 g/kg were examined every 1 week. The stability of drug was determined in two replicates each week using the formula, where the value of concentration after some period was divided by value of concentration in freshly prepared matrix × 100%. The stability of the analyzed sample freezed at −20 ◦ C has been estimated for at least 4 weeks. Spiked samples stored at +4 ◦ C were stable for 1 week. To avoid an decreasing concentration of DC is better to freeze extracts, when the analysis of samples is impossible at the same day of preparation. For the evaluation of the short-term stability, three spiked samples at the concentration of 100 g/kg were prepared and kept in automatic injector at room temperature. For DC compound, the slope of the linear regression did not deviate significantly from zero and there are no significant variations in concentration with samples prepared and analyzed immediately. That indicates the analyte to be stable over the time of 72 h. 3.5. Matrix effect Mass spectrometry with electrospray ionization (ESI-MS) can cause some difficulties in the quantitative analysis by so-called matrix effects in which the matrix coextracted with the analytes can change the signal response and signal suppression or enhancement of the analyte signal might take place. As a result, decrease of method sensitivity and poor accuracy or linearity can be noticed. Due to that, an evaluation of matrix effects should become an integrated part of quantitative LC-ESI-MS method development and validation. To compensate for a matrix signal suppression, different methods are applied: the use of internal standards, the application of standard dilution method and the dilution of the extracts before the instrumental determination. A stronger signal suppression was observed for DC, when the dry residue was dissolved in 500 l of mobile phase (36%). When the volume increased to 1 ml, the signal enhancement got lower (19%). The results obtained by Kasprzak-Horden B., indicated that among many pharmaceuticals, doxycycline is very susceptible to matrix components [25]. Therefore, the internal standard was used and matrix calibration curve was prepared to quantification of DC in fat tissue to take into account matrix influence. Additionally, solid-phase two step clean-up with combination of polymeric and carboxylic acid cartridges, allowed to eliminate matrix interferences and reduce matrix effect.
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Table 5 Residual contents of DC in positive samples of fat from chicken receiving DC over 5 consecutive days via the drinking water in a dose of 10 mg/kg BW/day through oral consumption. Day of slaughter
1 2 7 8
DC concentration (g/kg) LC–MS/MS (n = 4)
DC concentration (g/kg) LC–UV (n = 4) Range
Mean value
Range
Mean value
410–998 45–78 18–22 ≤15
636.5 64.2 19.5 –
460–880 56–83 17–26 8–18
624.5 72.5 21.5 12.5
Fig. 3. Ion reconstituted chromatograms obtained for the analysis of DC spiked fat sample at 150 g/kg (a) and medicated fat sample with DC concentration at 460 g/kg by LC–MS/MS (b). Peaks: (1) MINO (IS); (2) DC.
4. Application to treated chicken samples The proposed methods have been applied to determinate DC in chicken fat sample from animals treated with DC. The highest concentration of DC was found on the first day after treatment. The DC stayed at the concentrations above CC␣ and CC only for one day. The residual concentration of DC was sharply decreased on the day 2 and the residues of DC were found to be below the MRL values. Withdrawal time for applicated product contained DC was 7 days, so animals were slaughtered also after 7 and 8 days form the final administration. The DC concentrations at 7 and 8 day were close to LOD, LOQ values of the presented method. Fig. 1c shows chromatogram corresponding to the animal slaughter after 1 day of taking away of medication. Fig. 3 b shows the results obtained with LC–MS/MS of incurred fat sample with DC concentration at 460 g/kg. The quantitation was achieved using a matrix-calibration curve. The concentrations of DC residues present in incurred chicken fat were determined and the results are expressed in Table 5. Only with LC–MS/MS, it was possible to quantify DC in sample from one chicken slaughtered 8 days after the final administration of drug. The obtained results are similar both LC–UV and LC–MS/MS technique. 5. Conclusions The presented LC–UV and LC–MS/MS methods provided a satisfactory determination of the DC in fat/fat and skin. It was also checked the usefulness of presented method to analysis and isolation of DC from pig fat with the satisfactory results. However, due to the experiment on chickens, this study showed isolation and validation for chicken fat only. The method validation results indicate that the developed methods could successfully extract DC from fortified fat/fat with skin samples, as well as described method was applied to incurred chicken fat in order to determine its reliability in performing residue analysis of DC in such difficult matrix.
The results of the conducted experiment shows, that DC accumulate in fat and there are found residual concentrations of this compound in fat. In the conducted experiment DC concentration sharply decreasing at 2 day after a final administration of medicinal product containing DC. There are not many studies, describing successful extraction of DC from fat. Additionally, proposed method is able to simultaneous screening and confirmation the presence of DC after using one sample preparation step, which is less time consuming, because both analyses are completed in a one single day. The presented method is selective, sensitive, reliable and accurate, which was confirmed by the method validation results. References [1] M.D.F. Santos, H. Vermeersch, J.P. Remon, M. Schelkens, P. De Backer, H.J.J. Van Bree, R. Ducatelles, F. Haesebrouck, J. Vet. Pharmacol. Therap. 19 (1996) 274. [2] F. Yang, H.W. Liu, M. Li, H.Z. Ding, X.H. Huang, Food Addit. Contam. Part A 29 (1) (2012) 73. [3] Commission Regulation (EU) No. 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. [4] The European Agency for the Evaluation of Medicinal Products: Committee for veterinary medicinal products - Doxycycline- Summary Report (2), EMEA/MRL/97-FINAL. [5] A.R. Shalaby, N.A. Salama, S.H. Abou-Raya, W.H. Emam, F.M. Mehaya, Food Chem. 124 (2011) 1660. [6] S. Croubels, H. Vermeersch, P. De Backer, M.D.F. Santos, J.P. Remon, C. Van Peteghem, J. Chromatogr. B 708 (1998) 145. [7] N. Haagsma, P. Scherpenisse, Euroresidue II, Veldhoven, The Netherlands, 1993. [8] N. Furusawa, Chromatographia 53 (2001) 47. [9] M.D.F. Santos, H. Vermeersch, J.P. Remon, M. Schelkens, P. De Backer, R. Ducatelle, F. Haesebrouck, J. Chromatogr. B 682 (1996) 301. [10] M. Cherlet, M. Schelkens, S. Croubels, P. De Backer, Anal. Chim. Acta 492 (2003) 199. [11] M. Crivineanu, V. Trifan, G.H. Paraschiv, V. Nicorescu, Lucrari Stinifice Medicina Vetrinaria, Timisoara XLI (2008) 114. ˙ S. Semeniuk, J. Niedzielska, R. Ellis, Biomed. Chro[12] A. Posyniak, J. Zmudzki, matogr. 12 (1989) 294. [13] E.J. Mulders, D. Van De Lagemaat, J. Pharm. Biomed. Anal. 7 (1989) 1829.
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[14] A.D. Cooper, G.W.F. Stubbings, M. Kelly, J.A. Tarbin, W.H.H. Farrington, G. Shearer, J. Chromatogr. A 812 (1998) 321. [15] M. Castellari, J.A. Garcia-Regueiro, Chromatographia 58 (2003) 789. [16] S. Bogialli, R. Curini, A. Di Corcia, A. Lagana, G. Rizzuti, J. Agric. Food Chem. 54 (2006) 1564. [17] H. Oka, Y. Ito, Y. Ikai, T. Kagami, K. Harada, J. Chromatogr. A 812 (1998) 309. [18] H. Nakazawa, S. Ino, K. Kato, T. Watanabe, Y. Ito, H. Oka, J. Chromatogr. B 732 (1999) 55. [19] C. Blasco, A. Di Corcia, Y. Pico, Food Chem. 116 (2009) 1005. [20] S.M. Croubels, K.E.I. Vanoosthuyze, C.H. Van Pethegem, J. Chromatogr. B 690 (1997) 173. [21] H.J.E.M. Reeuwijk, U.R. Tjaden, J. Chromatogr. A 353 (1986) 339.
[22] A.P. De Leenheer, H.J. Nelis, J. Pharm. Sci. 68 (8) (1979) 999. [23] Council Directive 93/119/EC of 22 December 1993 on the protection of animals at the time of slaughter or killing. [24] Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. OJ 2002, L 221, pp. 8–36. [25] B. Kasprzyk-Hordern, R.M. Dinsdale, A.J. Guwy, J. Chromatogr. A 1161 (2007) 132. [26] A. Gajda, A. Posyniak, Bull. Vet. Inst. Pulawy 53 (2009) 263. [27] X. Ding, S. Mou, J. Chromatogr. A 897 (2000) 205. [28] H.J. Neils, A.P. De Leenheer, J. Chromatogr. A 195 (1980) 35.
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of doxycycline in chicken fat by liquid chromatography with UV detection and liquid chromatography–tandem mass spectrometry Anna Gajda a,∗ , Andrzej Posyniak a , Jan Zmudzki a , Grzegorz Tomczyk b a b
Department of Pharmacology and Toxicology, National Veterinary Research Institute, Partyzantów 57, 24-100 Pulawy, Poland Department of Poultry Diseases, National Veterinary Research Institute, Partyzantów 57, 24-100 Pulawy, Poland
a r t i c l e
i n f o
Article history: Received 4 January 2013 Accepted 11 March 2013 Available online 21 March 2013 Keywords: Doxycycline Fat Validation LC–UV LC–MS/MS
a b s t r a c t A sensitive analytical method for determination of doxycycline (DC) residues in chicken fat/fat and skin was developed. The extraction, in the presence of the internal standard (IS) minocycline (MINO), was carried out using solution of oxalic acid (pH 4.0) and ethyl acetate. The samples were cleaned up by solid phase extraction (SPE) procedure using, at first carboxylic acid and then polymeric Strata X cartridges. Chromatographic separation of DC by LC–UV was achieved on a Luna C8 analytical column and for LC–MS/MS analysis Luna C18 column was used. The presented procedures were evaluated according to the Commission Decision 2002/657/EC. Specificity, decision limit (CC␣), detection capacity (CC), recovery (absolute and relative), precision (repeatability and reproducibility) were determined during validation process. The limit of detection (LOD) was 10 g/kg for LC–UV and 1 g/kg for LC–MS/MS method. The limit of quantitation (LOQ) was 15 and 2 g/kg for LC–UV and LC–MS/MS, respectively. The absolute recovery for the LC–UV and relative recovery for the LC–MS/MS method at 300 g/kg concentration level were 79%; 101% for fat and 82%; 99% for fat and skin, respectively. The developed liquid chromatography with ultraviolet detection (LC–UV) and liquid chromatography tandem mass spectrometry (LC–MS/MS) methods have been applied to quantitative determination of doxycycline (DC) in samples of chicken fat tissue obtained from animals treated with DC. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Doxycycline is a member of the tetracycline antibiotics group, that is widely used in the treatment of respiratory and urinary tract infections in various species. It is bacteriostatic compound, which acts by inhibiting protein biosynthesis after binding to the 30 S ribosomal subparticle in bacterial cell. Improper use of doxycycline in food producing animals can cause its residues in animal tissues, which can be toxic and dangerous for human health and may generate the resistance of many microorganisms to antibiotics. Doxycycline is more lipids soluble than other tetracyclines and penetrates to body tissues and fluids in a greater extent after the application. Stronger lipophilic character may result in long persistence of doxycycline in animal body, especially fat, what can cause unacceptable concentration in animal tissues [1,2]. The maximum residue limits (MRLs) established by the European Union for
∗ Corresponding author at: National Veterinary Research Institute, Department of Pharmacology and Toxicology, al. Partyzantow 57, 24-100 Pulawy, Poland. Tel.: +48 81 889 31 27; fax: +48 81 8862595. E-mail address: [email protected] (A. Gajda). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.03.011
doxycycline in pigs, poultry and bovine are as follows: muscle – 100 g/kg, kidney – 600 g/kg, liver – 300 g/kg. The MRL for fat and skin – 300 g/kg has been established only for pigs and poultry, excluding bovine. According to Commission Regulation (EU) 37/2010 [3], quantification of doxycyline in animal tissues requires a determination only a parent compound as the marker residue, without its 4-epimer, contrary to other tetracyclines, where the MRLs are defined as a sum of parent drugs and their 4-epimers. In reference to the international trade and international regulations, there is a need to control the food of animal origin, including fat. For the export purposes, it was required to develop a suitable method for analysis and quantification of doxycycline in fat. Additionally, there is no data on the DC residues and its depletion in chicken fat. According to the EMEA summary report [4] the highest DC residues are found in kidney, followed by liver, skin, muscle, however in fat, doxycycline is not detectable to any great extent. Therefore, it was decided to investigate the DC concentration and its stability in chicken fat after administration of Doksy RW® . The aim of conducted experiment was to prove, that there are also residual concentration of this compound in fat. Many analytical procedures have been developed for the determination of doxycycline in animal tissues [5–7], eggs [7,8], and plasma [9]. Only a few methods have been described for the
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determination of DC in pork fat [10,11]. It has been presented a paper of Cherlet for a quantitative analysis of tetracyclines in pig tissues (muscle, kidney, and liver, including skin + fat) by LC–MS/MS. To our knowledge, no analytical method has been proposed for determining DC residues in chicken fat so far. The determination of DC residue is generally performed by HPLC suitable detectors, including UV [9,12–14], fluorescence [6,7] diode-array [5,8,15] or mass spectrometry [10,16–19] detection. The most important part of analytical procedure are sample preparation and clean-up step. Most LC analysis for determination of TCs used a solid phase extraction (SPE) for clean-up purposes with different kinds of sorbents. Some researchers have successfully adopted matrix solid-phase dispersion (MSPD) technique [16]. It has been also reported a metal chelate affinity chromatography (MCAC) to establish a clean-up method [20]. A sample preparation requires an extraction step with a suitable solvent system. Most extraction techniques use mild acidic buffer pH 3–5 [6]. There have been also reported methods with organic solvents, such as ethyl acetate [7,21,22] for extraction. Due to the increasing use of tetracycline antibiotics, especially most commonly applied in poultry doxycycline, it was necessary to develop a sensitive and accurate method for the determination of doxycycline in chicken fat with a simultaneous UV and MS/MS detection, after one sample preparation step. The developed methods were verified and used to measure the DC residues after the experimental application of this antibiotic to chicken. Additionally, the presented methods have been also successfully applied to fat and skin samples, in natural form. The validation data for fat matrix and for fat with skin are presented in this study. 2. Experimental 2.1. Reagents Doxycycline (DC) standard (purity > 97%) was obtained from Sigma–Aldrich Chemical Company (USA). Minocycline (internal standard, purity > 97%) was also from Sigma Aldrich. All organic solvents were HPLC grade and all chemicals were analytical grade. Acetonitrile, methanol, and Bakerbond SPE cartridges (carboxylic acid 500 mg/3 ml) were from J.T. Baker. Strata X (33 m, 100 mg, 6 ml) polymeric solid phase extraction (SPE) columns were obtained from Phenomenex (USA). Oxalic acid dihydrate (ACS), trichloroacetic acid (TCA), ethyl acetate and sodium sulphate anhydrous were from POCh Gliwice (Poland). Heptafluorobutyric acid (HFBA) was from Sigma–Aldrich. Water was purified using Milli-Q system.
chicken were killed and fat was collected. The birds were sacrificed under the animal welfare rules of the European Council Directive 93/119/CE [23]. The experiment was approved by Ethical Local Committee No 2 in Lublin (Poland) with approval number 22/2011. Samples were homogenized and stored at −20 ◦ C until the analysis. 2.3.2. Incurred chicken fat The experiment was conducted with twenty-four week old chickens (16 animals), treated individually with DC (Doksy RW® , 10 mg/kg BW) administered orally after dilution with drinking water using syringe, once daily, for a period of five consecutive days. Food and water were available ad libitum. The treated chickens were killed at 1st day, 2nd day, 7th day and 8th day (4 animals per every day) after the final administration of drug and fat was collected from thigh muscle. All samples were kept separately at the temperature of −20 ◦ C until the analysis. 2.4. Sample preparation Before the extraction step, fat/fat and skin samples were grinded. Three grams of chicken fat/fat and skin were weighted and internal standard was added. Samples were mixed with 3 ml of 0.02 M oxalic acid buffer, pH 4.0 and then vortex mixing for 15 s. After addition of 15 ml of ethyl acetate, the mixture was vortexed again for 30 s. To the solution, 0.5 ml of 20% trichloracetic acid was added and all was stirred and centrifuged for 10 min at 2500 × g. The supernatant was transferred to a new tube. The extraction was repeated twice with another 15 ml of ethyl acetate and the extract solutions were combined. On the top of carboxylic acid SPE columns, which were earlier preconditioned with 5 ml of ethyl acetate, 2.0 g of sodium sulphate on funnels was placed. The whole supernatant was filtered through a sodium sulphate and poured on SPE columns. After percolation of the whole solution, the columns were washed with 2.5 ml of ethyl acetate and dried (under vacuum) for 10 min. The doxycycline was eluted with 2.5 ml of solution consisting of acetonitrile–methanol–0.02 M oxalic acid pH 2.0 (20:15:65, v/v/v). To an eluate solution, 8 ml of 0.02 M oxalic acid buffer, pH 4.0 was added and combined supernatants was poured onto SPE polymeric columns firstly preconditioned with 2 ml of methanol, 2 ml of water and 3 ml of 0.02 M oxalic acid buffer, pH 4.0. After the percolation of whole solution columns were washed with 3 ml of 0.02 M oxalic acid buffer (pH 4.0) and 2 ml of water, and dried (under vacuum) for 10 min. The DC was eluted with 5 ml of methanol and then the eluate was dried under a gentle nitrogen stream at 40 ◦ C. The dry residue was dissolved with 1 ml of water and after vortex mixing transfer to vials and ready to LC–UV, as well as LC–MS/MS analyze.
2.2. Preparation of standard solutions 2.5. Liquid chromatography–UV analysis 2.2.1. Standard solutions Stock standard solution (1 mg/ml) prepared by weighing out 10.0 ± 0.1 mg of standard substances and dissolving in 10 ml of methanol, was stable for six months when stored at the temperature below −20 ◦ C in amber glass. Working standard solutions (100 g/ml, 10 g/ml) prepared in acetonitrile by diluting suitable aliquot of stock standard were stable for three months, stored at 2–8 ◦ C in amber glass. Working standard solutions in mobile phase were prepared on the day of the analysis.
The instrumental analysis was performed using Varian Prostar HPLC system, equipped with quaternary pump, autosampler, column oven, and UV/Vis detector ( = 355 nm), controlled by Galaxie Workstation software. Chromatographic analyses were performed on Luna (Phenomenex) C8 column (5 m, 250 mm × 4.6 mm) with mobile phase consisting of acetonitrile–0.02 M oxalic acid, pH 2.0 in gradient mode at 1.0 ml min−1 flow rate (Table 1). The injection volume for LC–UV was 100 l. The column oven temperature was controlled at 30 ◦ C.
2.3. Sample collection 2.6. Liquid chromatography–mass spectrometry 2.3.1. Blank control chicken fat Blank fat samples were taken from 4 chicken never treated with doxycycline, used as control, bought from Chicken Farms in Poland. Birds were kept in groups, in separate rooms, at the temperature of 20 ◦ C. The average body weight was 2.6 kg. After 5 days
Analyses were performed on an Agillent 1200 LC system consisting of a quaternary pomp, an autosampler, a column heater (kept at 25 ◦ C), switching valve and an automatic degasser (Agilent Technologies, Palo Alto, CA, USA) and an API 4000 triple quadropule
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Table 1 Gradient elution for LC–UV and LC–MS/MS methods. LC–UV
LC–MS/MS
Time (min)
ACN (%)
0.02 M oxalic acid (%)
Time (min)
ACN (%)
0.025% HFBA (%)
0.00 1.00 5.00 7.00 10.00 12.00 15.00
5 5 20 30 20 5 5
95 95 80 70 80 95 95
0.00 12.00 12.01 15.00 15.01 23.00
5 60 90 90 5 5
95 40 10 10 95 95
mass analyser with a TurboIonSpray source (Applied Biosystems, Toronto, ON, Canada). The MS detector was configured as ESI (electrospray ionization). The ESI was operated in the positive ion mode and MS data acquisition was performed in the MRM mode, selecting one precursor ion to two products ion transitions for DC. The Analyst 1.5 software controlled the LC–MS/MS system processed the data. Nitrogen was used as nebulizer gas, curtain gas and collision gas. TurboIonSpray source was operated at 300 ◦ C with the capillary voltage set at 5500 V. Collision energy (CE) was optimized to maximize the relative abundance for ion transition. The chromatographic separation was performed on a Luna C18 (150 × 2.0 mm, 3 ) analytical column (Phenomenex) coupled with octadecyl guard column (2 mm × 4 mm) (Phenomenex). The mobile phase was consisted of solvent A: acetonitrile and solvent B: 0.025% of HFBA in water (v/v). The elution was performed in a gradient mode with injection volume of 30 l (Table 1). The column was operated at 35 ◦ C with a flow rate of 0.25 ml min−1 , and time of analyze – 23 min.
the repeatability and analysing on two different days with the same instrument and the different operators. The overall CVs were calculated. The accuracy was obtained as a relative recovery for LC–UV and for LC–MS/MS. The recovery was evaluated in the same experiment as repeatability. The relative recovery was determined by comparing peak area ratios (DC/internal standard) from fortified matrix samples with peak area ratios (DC/internal standard) from direct injections of equivalent quantities of standards. Additionally, for LC–UV the absolute recovery was calculated. The CC␣ and CC were determined by the matrix calibration curve procedure. CC␣ was calculated with a statistical certainty of 1 − ˛ (˛ − 0.05), and CC was calculated with a statistical certainty of 1 − ˇ (ˇ − 0.05).
2.7. Validation study
Signal suppression (%) =
The validation of the methods was performed according to the recommendations of the Commission Decision 2002/657/EC [24]. Parameters such as linearity, specificity, precision, accuracy, detection limit and limit of quantification, decision limit and detection capability were established. The method was validated at the 150, 300 and 450 g/kg. Calibration curves for the DC standards and extracted samples were constructed. Linearity was tested by preparing matrix calibration curve in a range of 50–600 g/kg. Blank chicken fat/fat and skin samples were fortified with working standard solution of DC at five concentration levels. Specificity was verified by analyzing 20 blank fat/fat and skin chicken samples of different origin, in order to verify the absence of potential interfering compounds at DC expected retention time. The LOD and LOQ values were considered as the concentration giving a signal to noise ratio of 3 and 10, respectively. Five different concentration levels from 10 to 30 g/kg were prepared by duplicate in order to establish the LOD and LOQ in LC–UV, whereas five levels from 0.1 to 6 g/kg were prepared in order to obtain LOD and LOQ using LC–MS/MS. For the evaluation of precision (repeatability and withinlaboratory reproducibility), as well as recovery, blank fat/fat and skin samples were spiked with the DC working standard solution to levels corresponding to 150, 300 and 450 g/kg, respectively. The experiments were carried out on three consecutive days. The repeatability was determined by fortifying six blank samples at each of three concentration levels 150, 300 and 450 g/kg with DC compound. The samples were analyzed on the same day with the same instrument and the same operators, and the coefficient of variations (CV) was calculated. The within-laboratory reproducibility was determined by fortifying other two sets of blank samples at the same concentration levels of the analyzed compound as for
where IF is the DC peak area in fat extract spiked after the extraction at 300 g/kg and IW is DC peak area in high-quality pure water extract spiked after extraction at 300 g/kg.
2.8. Matrix effect study for LC–MS/MS method In this study the interference caused by fat matrix during extraction was evaluated. To calculate the signal suppression for DC, the following equation was used [25]:
1−
IF IW
× 100
2.9. Stability The stability of DC in acetonitrile was evaluated in standard solutions stored at −20 ◦ C and +4 ◦ C at concentration of 10 g/ml at different time intervals: 1 day, 10 and 20 days, 1, 2, 3, 4, 5 and 6 months. Stability was also investigated in fat samples spiked with DC at the level of 100 g/kg. The spiked sample were analyzed on the day of the preparation. Samples were prepared in duplicate. Then, the spiked samples were stored at −20 and +4 ◦ C, and analyzed every week for 4 weeks. A short-term stability was evaluated on extracts stored at room temperature for 72 h into automatic injector. Sample were analyzed at 0, 24, 48,and 72 h. Samples were prepared at concentration of 100 g/kg. 3. Results and discussion 3.1. Sample preparation The effective isolation of tetracycline residues from biological matrices is difficult because of their binding with sample proteins and chelating with metal ions. In numerous studies for the determination of doxycycline, it has been presented an extraction by use of EDTA and oxalic acid buffer, citrate buffer to overcome such undesirable properties [5,14,15,18,26,27]. The difficulty in isolating some antibiotics from fat is connected with its polarity. According to Cherlet et al. [10] it seems to be a decreasing recovery with decreasing polarity of the component (from oxytetracycline, one
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Table 2 Multiple reaction monitoring (MRM) mode for the detection of doxycycline. Analyte
RT (min)
Precursor (m/z)
Product (m/z)
DP (V)
CE (V)
CXP (V)
Peak area ratio ± tolerance (%)
Doxycycline
12.52 10.97
428 154 441
55 55 72
25 40 25
15 15 12
0.09 ± 50
Minocycline (IS)
445 445 458
RT: retention time; DP: declustering potential; CE: collision energy; CXP: cell exit potential.
of the most polar tetracyclines to doxycycline) for muscle, liver and kidney tissues. Surprisingly, the recovery for DC in fat was relatively good. In Cherlet et al. study, the extraction of tetracyclines was performed using sodium succinate solution. In this method doxycycline was extracted by oxalic acid to dechelate the doxycycline-metal complex. Different pH values of solvent extraction were checked (pH 2.0, pH 4.0 and pH 6.0). The best results were obtained when the pH 4.0 of oxalic acid buffer was used. The addition of 20% trichloracetic acid to the extraction step coagulated and removed the protein, that may occur in matrix. The first sample preparation step in presented study was an improvement of the previously described analytical method for the determination of tetracyclines in animal tissue and eggs [7]. Fats consist of a wide group of compounds that are generally soluble in organic solvents and generally insoluble in water. When the acetonitrile (as more polar solvent) was used to extraction, the recovery was much lower. It was confirmed that ethyl acetate (a mid-polar solvent) is suitable for a quantitative isolation of doxycycline from biological matrices, also from fat/fat and skin and the use of triple extraction gives better and more reliable results. For the determination of tetracyclines, including doxycycline, in animal tissues several types of cartridges for clean-up purposes were investigated such as: C18 [5,13,15], polymeric (Strata X) [26], carboxylic acid (COOH) [7], SDB1 (styrene-divinylbenzene) [12] or Bond Elut ENV [18]. It has been also described on-line MCAC cleanup [14], or Oasis (HLB) cartridges after pressurized liquid extraction (PLE) [19]. However, the most suitable for clean-up step in fat tissues were carboxylic acid columns and then polymeric cartridges. The usage of Strata X columns allowed concentrating an extract and obtaining more sensitive method, with a very low DC concentration level detection. Additionally, after two step clean-up, the final extract was clean and pure. An application of methanol to the DC elution from Strata X columns, gave the possibility to obtain a suitable extract for LC–MS/MS analysis. The presented methods were verified and checked for DC extraction from incurred chicken fat, as well as they were performed well for DC isolation from spiked fat and skin samples. Their usefulness for fat of other species has been proved. Many analytical methods for the analysis of DC in animal tissues involve demeclocycline, as an internal standard [10,18,19,22]. Because of a better repeatability and stability for minocycline, (inhouse data), as well as consistent retention time, minocycline, as a member of tetracyclines group not used in food producing animals, was chosen in presented study.
3.2. Liquid chromatography In other studies, the usage of polymeric [6,10,12,15,27], phenyl [13] or C18 analytical columns to analyze tetracyclines in animal tissues [15,16], has been presented. For the DC quantification by LC–UV, C8-bonded silica column was used based on the results of Neils and De Leenheer [28]. The use of this column eliminated the need to use ion-pairing agents in the mobile phase that are generally required to reduce tetracycline tailing with conventional silanol LC columns. At C8 column, DC was observed at 11 min with stable retention time. To avoid DC adsorption on RP column and
forming chelate complexes various acids – citric, oxalic, phosphoric acid [6,12,18,22], were tested. It has been also reported an ion exchange column with hydrochloric acid:acetonitrile eluent for the analysis of tetracyclines [27]. In the presented method, RP column was chosen. The best results for this type of column were obtained when solution of oxalic acid was used. The asymmetries of DC peak depend on the pH value of the aqueous oxalic acid. The optimum pH was 2.0. LC mobile phase consisting of acetonitrile–0.02 M oxalic acid in gradient mode proved to be suitable for symmetrical and sharp peak. Application of gradient elution was necessary to obtain good separation for DC and MINO (IS). The oxalic acid appears to strip or mask any metal ions that may affect doxycycline analyses, effectively. Furthermore, at = 355 nm no interference was observed with endogenous substances of the blank tissues. Hence, the wavelength of = 355 nm was chosen for quantitation of doxycycline. Fig. 1 shows the chromatograms obtained with LC–UV corresponding to the blank fat sample, DC spiked sample and the samples from the treated animals. In the blank sample, the only substance observed corresponds to the internal standard (Fig. 1a). In chromatogram B, DC was added at the concentration of 150 g/kg (Fig. 1b). For the LC–MS/MS analysis, good results for DC were achieved using C18 analytical column. Oxalic buffer of the mobile phase was replaced by 0.025% of HFBA in water (v/v). The use of ion-paring agent in the mobile phase reduced DC tailing. For the DC to be positively confirmed, the retention time had to match that of a standard to within 2.5%. MS data acquisition was performed in the multiple reaction monitoring (MRM) mode, selecting one precursor ion to two products ion transitions for analyte. The most abundant ion, as precursor ion m/z 445.0 was chosen. Declustering potential (DP), collision cell exit potential (CXP) and collision energy (CE) were established. The optimal conditions with transition parameters for detection of DC, LC–MS/MS retention time of analyte and ion ratio are reported in Table 2. The presented LC–MS/MS method fulfills all the EU requirements for confirmatory method with consistent retention time and can be used for DC presence confirmation in fat. Fig. 2 shows the typical MRM chromatograms of DC and internal standard in the spiked fat sample at the concentration of 150 g/kg. 3.3. Method validation 3.3.1. Linearity The linearity was evaluated by five point’s calibration curve with triple analysis. Correlation coefficient (r = 0.998) was obtained for calibration curve using standard solutions. Matrix-calibration curve for DC fat/fat and skin spiked samples had correlation coefficient above 0.99 for LC–UV, as well as for LC–MS/MS, what showed a good linearity between DC concentration and peak area (Table 3). 3.3.2. Specificity Specificity is the ability of a method to distinguish between the analyte of interest and other substances (impurities or matrix components) that may be present in a test sample. In the evaluation of the specificity blank fat/fat and skin samples were analyzed by both UV and MS detectors. The results obtained with blank samples have been compared with DC spiked samples and it is observed that no
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Fig. 1. Chromatograms of (a) fat blank sample; (b) fat blank sample with internal standard; (c) fat DC spiked sample at 150 g/kg; (d) incurred fat sample at 1 day after last treatment.
Fig. 2. LC–MS/MS chromatograms in MRM mode of DC and internal standard at the level of 150 g/kg.
Table 3 Linearity, working range, limit of detection (LOD), limit of quantification, decision limit and detection capability of suggested methods. Parameters
Correlation coefficient (r) Linearity (working range) (g/kg) Limit of detection (g/kg) Limit of quantification (g/kg) Decision limit (g/kg) Detection capability (g/kg)
LC–UV
LC–MS/MS
Fat
Fat + skin
Fat
Fat + skin
0.991 50–600 10 15 324 356
0.992 50–600 10 15 336 361
0.995 50–600 1 2 317 344
0.994 50–600 1 2 322 350
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Table 4 Precision and recovery of the presented method for doxycycline in chicken fat and fat with skin samples (n = 18). Parameters
Spiked level (g/kg) 150
LC–UV Recovery (absolute, %) Recovery (relative, %) Repeatability, CV (%) Reproducibility, CV (%) LC–MS/MS Recovery (relative, %) Repeatability, CV (%) Reproducibility, CV (%)
300
Fat
Fat + skin
77 95 9.5 13.3
79 93 9.7 14.0
99 8.6 11.9
96 9.0 13.2
interfering peaks with LC–UV were found in the retention time of the target analyte (Fig. 1). Fig. 3a shows the results obtained with LC–MS/MS of fat spiked sample at 150 g/kg. 3.3.3. Precision and recovery In the present work, recoveries and precision assays of the proposed method were evaluated using spiked samples including the MRL concentration level (Commission Regulation (EU) 37/2010) [1]. Thus, chicken fat/fat and skin samples were spiked at 150, 300 and 450 g/kg in 18 replicates, respectively. The relative DC recovery for LC–UV method ranged between 92–95% for fat samples and 93–96% for fat and skin, respectively. For LC–MS/MS relative recovery were established at a range of 97–101% for fat and 96–99% for fat with skin. Additionally an absolute obtained recovery for LC–UV was 77–81% for fat and 79–82% for fat and skin. All the results are presented in Table 4. The repeatability and within-laboratory reproducibility for DC were lower than 10 and 14%, respectively at all spiking levels for fat and fat with skin. The satisfactory results of precision expressed as coefficient of variation (CV) showed that the presented methods can be used as validated methods and can be useful for confirmation of DC residues in fat/fat and skin. At each level, the accuracy and precision fell within the required ranges for that specific concentration.
3.3.4. Limits of detection and quantification The procedure was satisfactorily sensitive; the limit of detection was established at the level of 10 g/kg and 1 g/kg for LC–UV and LC–MS/MS for fat/fat and skin, respectively. The limit of quantification was 15 g/kg and 2 g/kg for LC–UV and LC–MS/MS for fat/fat and skin. The obtained results indicate LC–MS/MS being a more sensitive method. Values obtained with LC–MS/MS, as well as LC–UV technique were much lower than MRL established by the European Union. All values of LOD and LOQ are shown in Table 3.
3.3.5. Decision limit and detection capability According to Council Directive 2002/657/EC an interpretation of the results requires decision limit (CC␣) and detection capability (CC) to be determined. CC␣ is defined as the concentration above which it can be decided with a statistical certainty of (1 − ˛) that the identified analyte content is truly above MRL. CC␣ value is established with an error of 5% (probability of false non compliant ≤5%) and CC is determined with an error ˇ = 5% (probability of false compliant samples ≤5%). Table 3 summarizes the obtained CC␣ and CC for chicken fat/fat and skin at the level of 300 g/kg. The results showed that method can be useful for detection and quantification DC residues in fat, as well as in fat and skin samples.
Fat
450 Fat + skin
Fat
Fat + skin
79 94 9.0 12.8
82 96 9.4 13.3
81 92 9.1 12.1
80 96 8.9 12.9
101 7.9 10.8
99 8.8 12.9
97 7.6 11.2
97 7.3 12.4
3.4. Stability The stability of standard solutions was examined. The DC standard solution stored at −20 ◦ C was stable for 6 months, while the stability of DC solutions dissolved in acetonitrile at the concentration of 10 g/kg stored at +4 ◦ C was 3 months. Fat samples spiked at the level of 100 g/kg were examined every 1 week. The stability of drug was determined in two replicates each week using the formula, where the value of concentration after some period was divided by value of concentration in freshly prepared matrix × 100%. The stability of the analyzed sample freezed at −20 ◦ C has been estimated for at least 4 weeks. Spiked samples stored at +4 ◦ C were stable for 1 week. To avoid an decreasing concentration of DC is better to freeze extracts, when the analysis of samples is impossible at the same day of preparation. For the evaluation of the short-term stability, three spiked samples at the concentration of 100 g/kg were prepared and kept in automatic injector at room temperature. For DC compound, the slope of the linear regression did not deviate significantly from zero and there are no significant variations in concentration with samples prepared and analyzed immediately. That indicates the analyte to be stable over the time of 72 h. 3.5. Matrix effect Mass spectrometry with electrospray ionization (ESI-MS) can cause some difficulties in the quantitative analysis by so-called matrix effects in which the matrix coextracted with the analytes can change the signal response and signal suppression or enhancement of the analyte signal might take place. As a result, decrease of method sensitivity and poor accuracy or linearity can be noticed. Due to that, an evaluation of matrix effects should become an integrated part of quantitative LC-ESI-MS method development and validation. To compensate for a matrix signal suppression, different methods are applied: the use of internal standards, the application of standard dilution method and the dilution of the extracts before the instrumental determination. A stronger signal suppression was observed for DC, when the dry residue was dissolved in 500 l of mobile phase (36%). When the volume increased to 1 ml, the signal enhancement got lower (19%). The results obtained by Kasprzak-Horden B., indicated that among many pharmaceuticals, doxycycline is very susceptible to matrix components [25]. Therefore, the internal standard was used and matrix calibration curve was prepared to quantification of DC in fat tissue to take into account matrix influence. Additionally, solid-phase two step clean-up with combination of polymeric and carboxylic acid cartridges, allowed to eliminate matrix interferences and reduce matrix effect.
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Table 5 Residual contents of DC in positive samples of fat from chicken receiving DC over 5 consecutive days via the drinking water in a dose of 10 mg/kg BW/day through oral consumption. Day of slaughter
1 2 7 8
DC concentration (g/kg) LC–MS/MS (n = 4)
DC concentration (g/kg) LC–UV (n = 4) Range
Mean value
Range
Mean value
410–998 45–78 18–22 ≤15
636.5 64.2 19.5 –
460–880 56–83 17–26 8–18
624.5 72.5 21.5 12.5
Fig. 3. Ion reconstituted chromatograms obtained for the analysis of DC spiked fat sample at 150 g/kg (a) and medicated fat sample with DC concentration at 460 g/kg by LC–MS/MS (b). Peaks: (1) MINO (IS); (2) DC.
4. Application to treated chicken samples The proposed methods have been applied to determinate DC in chicken fat sample from animals treated with DC. The highest concentration of DC was found on the first day after treatment. The DC stayed at the concentrations above CC␣ and CC only for one day. The residual concentration of DC was sharply decreased on the day 2 and the residues of DC were found to be below the MRL values. Withdrawal time for applicated product contained DC was 7 days, so animals were slaughtered also after 7 and 8 days form the final administration. The DC concentrations at 7 and 8 day were close to LOD, LOQ values of the presented method. Fig. 1c shows chromatogram corresponding to the animal slaughter after 1 day of taking away of medication. Fig. 3 b shows the results obtained with LC–MS/MS of incurred fat sample with DC concentration at 460 g/kg. The quantitation was achieved using a matrix-calibration curve. The concentrations of DC residues present in incurred chicken fat were determined and the results are expressed in Table 5. Only with LC–MS/MS, it was possible to quantify DC in sample from one chicken slaughtered 8 days after the final administration of drug. The obtained results are similar both LC–UV and LC–MS/MS technique. 5. Conclusions The presented LC–UV and LC–MS/MS methods provided a satisfactory determination of the DC in fat/fat and skin. It was also checked the usefulness of presented method to analysis and isolation of DC from pig fat with the satisfactory results. However, due to the experiment on chickens, this study showed isolation and validation for chicken fat only. The method validation results indicate that the developed methods could successfully extract DC from fortified fat/fat with skin samples, as well as described method was applied to incurred chicken fat in order to determine its reliability in performing residue analysis of DC in such difficult matrix.
The results of the conducted experiment shows, that DC accumulate in fat and there are found residual concentrations of this compound in fat. In the conducted experiment DC concentration sharply decreasing at 2 day after a final administration of medicinal product containing DC. There are not many studies, describing successful extraction of DC from fat. Additionally, proposed method is able to simultaneous screening and confirmation the presence of DC after using one sample preparation step, which is less time consuming, because both analyses are completed in a one single day. The presented method is selective, sensitive, reliable and accurate, which was confirmed by the method validation results. References [1] M.D.F. Santos, H. Vermeersch, J.P. Remon, M. Schelkens, P. De Backer, H.J.J. Van Bree, R. Ducatelles, F. Haesebrouck, J. Vet. Pharmacol. Therap. 19 (1996) 274. [2] F. Yang, H.W. Liu, M. Li, H.Z. Ding, X.H. Huang, Food Addit. Contam. Part A 29 (1) (2012) 73. [3] Commission Regulation (EU) No. 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. [4] The European Agency for the Evaluation of Medicinal Products: Committee for veterinary medicinal products - Doxycycline- Summary Report (2), EMEA/MRL/97-FINAL. [5] A.R. Shalaby, N.A. Salama, S.H. Abou-Raya, W.H. Emam, F.M. Mehaya, Food Chem. 124 (2011) 1660. [6] S. Croubels, H. Vermeersch, P. De Backer, M.D.F. Santos, J.P. Remon, C. Van Peteghem, J. Chromatogr. B 708 (1998) 145. [7] N. Haagsma, P. Scherpenisse, Euroresidue II, Veldhoven, The Netherlands, 1993. [8] N. Furusawa, Chromatographia 53 (2001) 47. [9] M.D.F. Santos, H. Vermeersch, J.P. Remon, M. Schelkens, P. De Backer, R. Ducatelle, F. Haesebrouck, J. Chromatogr. B 682 (1996) 301. [10] M. Cherlet, M. Schelkens, S. Croubels, P. De Backer, Anal. Chim. Acta 492 (2003) 199. [11] M. Crivineanu, V. Trifan, G.H. Paraschiv, V. Nicorescu, Lucrari Stinifice Medicina Vetrinaria, Timisoara XLI (2008) 114. ˙ S. Semeniuk, J. Niedzielska, R. Ellis, Biomed. Chro[12] A. Posyniak, J. Zmudzki, matogr. 12 (1989) 294. [13] E.J. Mulders, D. Van De Lagemaat, J. Pharm. Biomed. Anal. 7 (1989) 1829.
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