Rapid Determination of Chloramphenicol in Tilapia by ultra-high performance liquid chromatography-mass spectrometry

A rapid method for the determination of residues of chloramphenicol in tilapia by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established. The samples were extracted with acetonitrile, and separated on a C18 column using methanol-water solution as mobile phase, and then detected under ESI- multiple reaction monitoring mode. The method showed a good linearity for the analysts over the range of 0.1-100μg/L. The detection limits were 0.10μg/kg. The recoveries ranged from 88.6% to 108% at spiked concentrations with the relative standard deviations lower than 5%. The results shows that this method has the advantages of easy to operate, fast to perform, with high sensitivity and accuracy, and it is suitable for detection of residues of chloramphenicol in tilapia.


INTRODUCTION
Chloramphenicol (CAP) is an effective broad-spectrum antibiotic active against the main species of pathogenic gram-positive and gram-negative bacteria, as well as against other groups of micro-organisms [1] . CAP had been used globally to cure infectious diseases in both humans and animals. The Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives (JECFA) concluded that it was not appropriate to establish an acceptable daily intake (ADI) for CAP because CAP intake may induce aplastic anemia and it exhibits genotoxic effects [2] . According to JECFA's recommendation, the use of CAP is restricted in many countries and it is totally banned for use in food-producing animals in China, the EU. Nevertheless, because of its low price, high effectiveness and excellent pharmacokinetic properties, residual CAP has been detected in a variety of imported foods such as fish in china.
Numerous analytical approaches for the determination of residues of CAP have been published. Include gas chromatography [3] , gas chromatography-mass spectrometry [4] , liquid chromatography-mass spectrometry [5] , liquid chromatography-tandem mass spectrometry [6,7] , enzyme-linked immunosorbent assay [8] . The ability to detect CAP at regulated levels has been dramatically developed by the application of mass spectrometry. With the development of technology, the ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS/MS) has become an important determination and confirmation method [9][10][11][12][13] . The aim of this study was to develop a quick UPLC-MS/MS method with high selectivity, sensitivity, easy to operate and fast to perform to detect CAP residues in tilapia samples.

REAGENTS, INSTRUMENTS AND MATERIALS
Acetonitrile and methanol were chromatographic grade obtained from Spectrum and DIMA respectively. CAP, CAP-D5 were of chromatographic grade (purity≥98%) and purchased from Dr. Ehrenstorfer company. While, CAP-D5 was employed as the internal standard (IS) for the quantification of CAP. Their stock solutions were prepared individually at a concentration of 100mg/L in acetonitrile, and stored in low-actinic glassware protected from light at −20℃(stable for at least 3 months). All working solutions were prepared by serial dilution of the stock solutions with acetonitrile and stored at 4℃.
Tilapia was purchased from aquatic market of Zhanjiang. Once in the laboratory, all of the samples were accuracy weighed, and their head, bone and fat were removed. Fresh Tilapia were separated, homogenised and stored at -18℃. The samples with no detectable residues of the analytes confirmed were used as negative samples.

SAMPLE PREPARATION
Tilapia muscles sample 5.00g, D5-CAP, acetonitrile (20mL) was added into a 50mL centrifuge tube in turn. The mixture was homogenized for 2min at 5000r/min and shaken periodically for 3min. Then, the mixture was extraction by ultrasonic for 1min followed by centrifuging at 6000r/min for 8min. The supernatant was decanted into a 50mL comparison tube.
All solution was collected and heating in a water bath set at 50℃, the contents were rotoevaporated just to dryness under a reduced pressure.The obtained contents was dissolved with 1.00mL methanol-water solution followed by centrifuging at 15000 r/min for 8 min. The solution was passed through a 0.2μm membrane which was finally subjected to UPLC-MS/MS determination and confirmation.

INSTRUMENT CONDITIONS
A gradient UPLC system using methanol and water at a flow rate of 0.30 mL/min, was used to separate CAP on a Waters ACQUITY UPLC BEH C18 column (50×2.1mm,1.7μm) in Table 1. The column temperature was 40℃. The injection volume was 10.0μL.  The analysis was performed using negative ion electrospray interface (ESI-) with multiple reaction monitoring mode. Interface conditions were as follows: capillary voltage was 3.0kV; source temperature was 110℃; desolvation temperature was 600℃; the flow rates of cone and desolvation gas (nitrogen) were 150L/h and 600L/h, respectively; collision gas was argon; MS/MS parameters were shown in Table 2.

Optimisation of mobile phase
Because the kinds of mobile phase have a important roles in UPLC analysis effect, the use of different mobile phase(acetonitrile-water, methanol-water, acetonitrile-5mmol/L ammonium acetate solution) were tested. The results shows that when methanol-water used as mobile phase to seperater CAP in ACQUITY UPLC BEH C18 can get a better separation effect, CAP have a higer signal/noise and a better chromatographic peak shape (Fig.1).

Linearity
The calibration curves for CAP were constructed by plotting the peak area (y) versus concentration (x) of each analyte which were expressed by the equation given as: y=0.8152x+0.0084 with a correlation coefficient (r 2 ) of 0.9994. The calibration curves were generated daily from the peak area responses of standards with concentrations ranging from 0.1 to 100μg/L.

Limit of detection
The negative samples were spiked with the standard solution, then pretreated and analyzed following the method described above. The LOD for CAP were obtained from the analysis of the negative samples spiking with mixed standard solution. The LOD based on three times the signal to noise ratio were 0.10μg/kg.

Accuracy of methods
For estimation of accuracy, negative samples were added with CAP standard solution. Six replicate tests, at each of the three fortification levels, were analysed. The recovery of the method was determined using tilapia samples fortified at 0.1、0.5、2.0、5.0μg/kg. Mean recovery (n=6) of the analytes, determined in three separate assays shown in Table 3 was between 88.6% to 108% and the relative standard deviations lower than 5%.

CONCLUSIONS
In this study, a rapid method was developed for determination of CAP in tilapia. The results shows that this method has the advantages of easy to operate, fast to perform, with high sensitivity and accuracy, and it is suitable for detection of residues of chloramphenicol in tilapia.