LC-MS / MS Method , Development and Validation to Determine Three Tetracyclines and Their Epimers in Shrimp Samples

A modest liquid chromatography/tandem mass spectrometric method was developed and validated to determine three tetracycline antibiotics and their epimers tetracycline, oxytetracycline, chlortetracycline, 4-epi tetracycline, 4-epi oxytetracycline, 4-epi chlortetracycline in shrimp samples respectively. The procedure was involved the extraction of tetracyclines and its epimers from homogenized sample with EDTA-McIlvaine buffer followed by solid phase extraction clean up with Oasis HLB cartridge. The analysis was carried out using Inertsil ODS-3V, 5 micron, 150×4.6 mm column and mobile phase consists of 5 mM Oxalic acid in water (A) and 0.1% formic acid in methanol (B) in gradient mode at a flow rate of 0.8 mL/minutes. The final extracts were analyzed by the sensitive and selective LC/ESI/MS/MS operating in positive ion multiple reaction monitoring (MRM) mode.


INTRODUCTION
Tetracyclines (TCs) are a family of broadspectrum antibiotics that are highly effective against a number of gram-positive and gram-negative bacteria, these antibiotics used worldwide to control bacterial infection and promote aquatic production [1][2][3][4] .Therefore, the easy availability and efficiency for the treatment of bacterial infected disease, they were widely used in animal husbandry and aquaculture [5][6][7] .As with other veterinary antibiotics, when tetracycline is used in food animals, it has the potential to generate drug residues in the animals and animal products which can lead to increases in microbial resistance.Due to the misuse, the antibiotic residues in products of aquatic animal origin (Fish, Shrimp etc.) brought a concern to consumers.The residue of this kind of drugs can be directly toxic or else cause allergic reactions in some hypersensitive individuals 8 .Thus in order to protect human health, the European Union and other regulatory authorities worldwide have established maximum residue limits (MRL) for antibiotic residues in animal products entering the human food chain.European Union Commission Regulation 37/2010/EC establishes the MRLs for tetracyclines are 100 µg/kg (ppb) in muscle and 300 µg/kg (ppb) in liver for all food producing species 9 .
In this respect, it is of great interest to develop analytical procedures capable of determining with good accuracy and sensitivity, the animal tissue concentrations of TCs and to evaluate their presence in edible animal products to protect human health.Accurate monitoring of chemical residue levels in food and agriculture products is essential to assure the safety of the food supply and manage global health risks.The analysis of chemical residues requires techniques sensitive enough to detect and quantify contaminants at or below the maximum residue limit (MRL) of the compound in a given sample matrix.In addition, because of increased food safety regulations and the growing numbers of samples to be analyzed, it is critical that the analytical techniques provide high sample throughput.Several analytical methods have been successfully proposed for the determination of TCs in various matrixes.In particular liquid chromatography in reverse phase mode (RP-HPLC) coupled with different detection systems such as UV [10][11][12][13][14][15] , PDA [16][17][18] , fluorescence is widely used in routine analysis of TCs in different matrixes 19,20 .On the other hand, during last decade LC-MS/MS methods have been developed and implemented effectively for the determination of TCs mainly due to the known advantages of MS detection over conventional detection methods [21][22][23][24][25][26][27][28] .However based on the author's knowledge no LC-MS/MS method with full validation has been reported for the determination of tetracycline antibiotics along with their epimers in shrimp samples.Therefore the aim of this study is to develop rapid, simple and reliable LC-MS/MS method for the determination of three tetracyclines (chlortetracycline, oxytetracycline and tetracycline) along with their epimers (4-epi chlortetracycline, 4-epi oxytetracycline and 4-epi tetracycline) in shrimp samples.

Chemicals and materials
Methanol, acetonitrile and ammonium acetate were purchased from Sigma-Aldrich.Ethylacetate was purchased from Merck & Co.All the other inorganic chemicals and organic solvents were of analytical reagent grade or higher.Standards of tetracycline, chlortetracycline, oxytetracycline, 4-epi chlortetracycline, 4-epi oxytetracycline, 4-epi tetracycline were acquired from Sigma-Aldrich.

Preparation of stock solution (standard solution)
The standard stock solutions were prepared by accurately weighed 10 mg of each standard and is transferred into 10m l volumetric flask and made up to the mark with HPLC grade methanol and stored at -20°C.After proper dilution these stock solutions were used to prepare calibration curve and to fortify the blank shrimp samples to perform the recovery study.

Shrimp samples
Shrimp samples were collected from local market and stored at 20 o C before the analysis.

Equipment Liquid chromatography
The extracts were separated on Inertsil ODS-3V 5 micron, 150×4.6 mm HPLC column using Agilent 1290 HPLC system with a Binary pump, an auto sampler with temperature control, and a column oven kept at 30 °C.Sample aliquots of 5 µL were injected to the HPLC column, mobile phase consists of 5 mM Oxalic acid in Water (A) and 0.1% Formic acid in Methanol (B) in gradient mode with a flow rate of 0.8 ml/minutes.The gradient programme was given in the Table 1

Tandem MS analysis
The flow from the LC column was transferred to a triple quadrupole mass spectrometer (Agilent 6470) equipped with an ESI source.The analysis was performed in positive polarity mode for all compounds.Instrument control, data acquisition and evaluation were done with Mass Hunter software, Version B.06.00.Determination was performed by two MRM (multiple reaction monitoring) transitions (one for quantitation and second one for conformation) mode, the MRM dynamics were given in the table 2. The typical source parameters were: Gas Temperature: 350 o C, Gas flow: 10 l/min.Nebulizer: 45 psig, Sheath Gas Temperature: 400 o C, Sheath Gas flow: 11 L/min.V Cap: 3500 V, Nozzle Voltage: 0V.

Sample preparation
Collected blank shrimp samples were cleaned thoroughly, cut in to small pieces and homogenized.5 g of homogenized sample was weighed and taken into 50 mL centrifuge tube, to this 15  SPE Clean UP: The aqueous layer was applied to on preactivated with methanol (6ml), water 93 ml) and Mcvallin buffer (6 ml) HLB 500 mg/ 6cc spe cartridge previously activated with methanol and milli-Q water.After sample loading, the cartridge was given washings with Milli-Q water (6 ml) and dried the cartridge with the help of vacuum.TC antibiotics were eluted with methanol (6 ml) and the eluent was evaporated under a stream of nitrogen at 35 o C , and the residue was dissolved in 2 mL 0.1% Formic acid: Methanol (9:1) and analyse on LC-MS/MS.

Method validation
The proposed method was validated for system suitability, specificity, selectivity, sensitivity (limits of detection and limit of quantification), recovery (accuracy), ruggedness, decision limit (CC α) and detection capability (CC β) according to 2002/657/EC decision.

System suitability
System suitability was performed by injecting six replicates of known concentration of standard solution.The results were within the acceptance criteria i.e. % RSD of area was less than 5%.The results have been given in Table 3.

Specificity
Specificity was performed by injecting 20 representative blank samples and sample spiked at MRL, observed that there was no interferences at the retention of analyte.The results have been given in Table 4.

Calibration Curve (Linearity)
Linearity was checked for each compound using six standard solutions with concentrations ranging in concordance with the level of the maximum residue limit (MRL).Three sets Linearity was established using matrix-matched calibration curves.Calibration curves were prepared at levels of matrix blank, 10 ppb, 50 ppb, 100 ppb, 150 ppb and 200 ppb in Shrimp and analyzed with each batch.The results of coefficient of determination greater than 0.99 and the results have been shown in Table 5

Limit of Detection and Limit of Quantification
Calculated the Limit of Detection (LOD) and Limit of Quantification (LOQ) by using standard deviation (STEYX) of the response and the slope of the linearity and the obtained LOD and LOQ values are given in the Table 6.

Repeatability and Within Laboratory Reproducibility
Repeatability was performed by injecting seven spiked samples at 50 ppb, 100 ppb and 150 ppb and calculated the mean, standard deviation and % RSD.The results were within the acceptance criteria i.e. % RSD of area ≤20.For within Laboratory reproducibility the above procedure was performed and calculated the mean, standard deviation and %RSD for all the analyses at 50 ppb (0.5 MRL), 100 ppb (MRL) and 150 ppb (1.5 MRL).

Recovery/Accuracy (Trueness)
Recovery was performed by injecting seven spiked samples at 50 ppb, 100 ppb and 150 ppb and calculated the mean, the standard deviation and the coefficient of variance for these  7.

Ruggedness
Ruggedness was performed by injecting seven spiked samples with two different analysts and two different lots of extraction solvents at permitted level.Calculated the Mean, standard deviation and % RSD.The results were within the acceptance criteria i.e. % RSD of area ≤7 and the results were given in Table-8.

Decision Limit (CC α)
Decision limit was performed by injecting seven spiked samples at 50 ppb, 100 ppb and 150 ppb in three batches and calculated the y-intercept and slope.The decision limit (at á = 5 %) was calculated at permitted limit for all the compounds, the concentration at permitted limit (MRL) plus 1.64 times the standard deviation of the within-laboratory reproducibility.The results were given in table 9. Detection capability (β = 5 %) was calculated, the concentration at decision limit plus 1.64 times the standard deviation of the 20 samples fortified at decision limit.The results were shown in table 9 and the recoveries of the 20 samples are meeting the criteria.

CONCLUSIONS
A simple positive ion LC-MS/MS method was developed and validated for the determination of tetracyline antibiotic residues in raw shrimp meat.Based on the results obtained, acceptance criteria for all validation parameters such as system suitability, specificity, calibration curve (Linearity), repeatability, within laboratory reproducibility, recovery, decision limit (CC α) and detection capability (CC β) have been met for the method used to estimate the tetracylines in shrimp samples as per protocol and as well as EU guideline council Directive 2002/657/EC.The method requires only a small sample volume, needs minimal manual sample preparation and reasonable run time.For this reason, the method could be useful for determination of tetracylines in shrimp samples.Henceforth considered the method is validated and can be used for further intended purpose.
. The resultant chromatograms are shown in Figure.1.
mL of Disodium EDTA McIlvaine buffer is added, vortexed for 5 min.separated the aqueous layer by centrifuging the sample with 4500 rpm at 4 o C. The supernatant was transferred into a clean tube and repeated the extraction by adding 15 ml of McIlvaine buffer and combined the aliquots.