Time (min) B%

0.01 12

5 12

17 30

20 30

20.1 1222 12

Table 1 · HPLC gradient forsepration of isomers of tetracyclines

Compound Name Precursor Ion Product Ion Frag.energy

CollisionEnergy Polarity

Tetracycline445 410 100 17 +ve

445 427 100 7 +ve

Oxytetracycline461 443 100 7 +ve

461 426 100 17 +ve

Doxytetracycline445 428 100 15 +ve

445 154 100 30 +ve

Chlortetracycline479 444 100 20 +ve

445 462 100 10 +ve

Epi tetracycline445 410 100 17 +ve

445 427 100 15 +ve

Epi oxy tetracycline461 443 100 7 +ve

461 426 100 43 +ve

Epichlortetracycline479 462 100 13 +ve

479 444 100 28 +ve

Table 2 · Following Precursor and product ions with fragmentor and collision energy were set

Method Development and Validation of Tetracycline Antibiotics and their Epimers in Marine Products asper the EU Guidelines

By Virendra Vora*, Mukesh Kumar Raikwar and Vikas BhardvajJune 2013

The authors are Specialists at the Department of Pesticide Residue, Reliable Analytical Laboratories Pvt Ltd, in Bhiwandi, Thane, India*Corresponding Author → See also:

«Study of Contamination Level of Pesticide Residues in Grapes (Vitis vinifera) in Maharashtra»«Assessment and Method Validation of Chlormequat Chloride (CCC) in Food (Fruit, Vegetable & Grain) by Liquid Chromatography MassSpectrometry»

AbstractTo develop and validate method for tetracycline antibiotics and their epimers in marine products as per EU commission (2007/657/EC). Now a day itbecomes necessary to detect tetracycline as well as their epimers at EUMRL level with high confidence so that effort was made to separate anddetect tetracyclines (tetracycline, oxytetracycline, doxytetracyclinea and chlortetracycline) and their epimers (epitetracycline, epichlortetracycline andepioxytetracycline). Here isomers have the same molecular weight and somewhat chemical nature too so that long run time was used to get separatepeaks. For sample extraction Mcvalline buffer was used and then extract was purified by passing through a C18 solid phase extraction (SPE) column.The prepared samples were analyzed on LC/MSMS. Tetracycline antibiotic residues in marine products were analyzed. The recovery rates of seventetracyclines in sample preparation ranged from 77.03 % to 89.95 % in fortified blanks; the coefficient variation were between 0.35 % and 2.89 %.The Decision limits (CCα) were 102.93 to 104.23 ng/g and Detection Capability (CCβ) was 105.78 to 108.26 ng/g. Youden approach was used todetermine ruggedness and it was within limit.

Introduction

Antibiotics are used worldwide to control bacterial infection and promote healthy marine production. Because tetracyclines are broad spectrum antibiotics so thatthey are widely used. However, it is very undesirable to have them in the marine products (like fish, shrimp and crap) supply. FDA’s regulations for tetracyclinesincluding oxytetracycline, doxytetracycline and chlortetracycline are set to provide for an acceptable daily intake (ADI) and for setting a tolerance for residues inmarine. The ADI for total residues of these compounds as per EU countries is 100 micrograms per kilogram of body weight per day. With the establishment of atolerance of 100 ppb for the sum of residues of tetracycline, a tolerance of 100 ppb for each of the seven tetracyclines is also accepted.

Tetracyclines are commonly used in aquaculture to prevent certain diseases in marine products. However, residue of these antibiotics can be carried over to humansand get enriched to a high level that could harm human health. The common requirements are that the total residue of tetracyclines is < 100 ng/g for marineproducts to be consumed safely by humans. The existing LCMS method and microbial method for tetracyclines measurements in marine products are notsatisfactory (does not include separation of epimers by retention time). Plus, the microbial method can only measure the total tetracyclines present, not being able todistinguish among different tetracyclines. The need for a new procedure with a consistent recovery rate, better specificity, selectivity and higher precisions has beenpushed further by EU commission in marine products that means-importing countries will not accept any products that fail to meet the above requirements (1, 2, 3 &

4).

Material and Method

All reagents and solvents were HPLC or analytical grade. Tetracycline and their epimer standards were purchased fromErhenstopher. Stock solution (1 mg/mL) was prepared in methanol and kept in the deep freezer (-20 °C). Working solutions wereprepared using the stock solution diluted with a methanol. The working solutions were prepared daily. The SPE cartridges wereAgilent SampliQ OPT 3 mL, 60 mg. Mcllavaine buffer was prepared by 222 ml of Disodium hydrogen phosphate and 278 ml 0.1 MCitric acid monohydrate in 500ml measuring cylinder. 18.6 gram Na2EDTA was added to dissolv in 100 ml mixture of Mcllvainesolution and volume was makeup to the marks. The analysis was performed on an RRLC (rapid resolution liquid chromatography)Agilent 1200 HPLC with mass spectrometer (Agilent triple quadrapole 6460). The analytical column was an Agilent Eclipse - C18 5μm, 150 mm × 4.6 mm id. Flow rate was set at 0.5 mL/min, Column temperature was 30 °C and Injection volume was 10 μL.Simple mobile phase comprised of A: 0.1 % formic acid in water and B: Acetonitrile was used.

Following MS parameteters were set for tetracycline analysis nitrogen gas temperature was set at 330, gas flow was 6 lit./min,Nebulizer pressure was 40 psi, capillary and Nozzele voltage was set at 3500and 500 V.

Sample Preparation

A 200 g sample of shrimp was homogenized with a tissue disintegrator, placed ina clean, sealed container, and stored in a freezer below -20 °C. A 1 ghomogeneous sample (accurate to 0.01 g) was placed into a 50 mLpolypropylene centrifuge tube with 20 mL 0.1 M Na2EDTA-Mcllvaine buffersolution and vortex mixed for 1 min. The sample was then centrifuged at a rotatespeed of 5000 rpm for 5 minutes (below 15 °C). The supernatant was removedand saved in a clean tube. The extraction was repeated twice with 20 mL and 10mL successively. The combined supernatant fluid was brought to 50 mL withbuffer, mixed well, centrifuged at a rotate speed of 7000 rpm for 10 min (below15 °C), and filtered with fast filter paper.

SPE Purification: The procedure used for the SPE extraction is as follow. AgilentSampliQ OPT cartridges were preconditioned with 5 mL water and then 5 mL ofmethanol. A 10 mL extract was passed through the SampliQ OPT cartridge at aspeed of 1 mL/min. After the sample effused completely, the cartridge waswashed with 3 mL of water. The entire effluent was discarded. The cartridge wasdried under negative pressure below 2.0 kPa for 3 minutes. Finally, the cartridge was eluted with 10 mL of 10 mM oxalic acid in methanol. The eluent was collectedand dried under nitrogen below 40 °C. The resulting residue was dissolved and made to a constant volume of 0.5 mL using the methanol. Then the residue wasanalyzed on LCMS.

Results and Discussion

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Figure 1 · Chromatogram of Tetracycline and their Epimers

Figure 2 · Linearity of Epitetracyline and Tetracycline rangine from 25 to 250 µg/kg

Validation was performed as per the commission decision dated on 12th August 2002 byimplementing Council Directive 96/23/EC who are concerning with the performance ofanalytical methods and the interpretation of results. It is necessary to ensure the qualityand comparability of the analytical results generated by laboratories approved for officialresidue control. This should be achieved by using quality assurance systems andspecifically by applying of methods validated according to common procedures andperformance criteria and by ensuring traceability to common standards or standardscommonly agreed upon.As per EU commission following parameters should be includedin validation procedure.

Selectivity: To qualify this parameter peaks were separated at different retention time asshown in fig. 1 and further it was assured by using three product ions one known as aquantifier ion and other two as a qualifier ions (Tab. 2).

Specificity of method was checked by analyzing twentyrelevant blank (matrix) samples and straight base line was observed. This says that this method has not anypotentially interfering substance in matrix or any other part of method. Recovery, Trueness, Repeatability, Decision limit (CCα), Detection capability (CCβ) andRuggedness were performed and calculated as per the EC guidelines (Tab 3). Following results were obtained and which were within the limits of EU commissionacceptable criteria.

Table 3 · Validation results for Linearity, Recovery, Trueness, Repeatability, CCα and CCβ

Antibiotics Range inµg/kg (r²) %

Trueness%CV

%Recovery

CCα(µg/kg)

CCβ(µg/kg)

Tetracycline 10 - 300 >0.99104.0395.95

103.14

2.511.771.05

82.5589.5387.05

104.03 105.78

Oxytetracycline 10 - 300 >0.99100.2395.9096.13

1.911.272.98

89.9188.0490.00

103.78 105.91

Doxytetracycline 10 - 300 >0.99103.4394.69

102.77

2.660.961.16

78.2280.0984.92

104.49 108.26

Chlortetracycline 10 - 300 >0.9993.7793.83

102.88

2.822.232.02

80.7888.5681.35

102.93 105.94

Epitetracycline 10 - 300 >0.99104.0298.07

100.23

1.740.741.29

77.0389.4584.19

103.83 106.82

Epioxytetracycline 10 - 300 >0.9998.3399.5696.40

0.930.351.06

77.9481.4283.94

103.76 107.38

Epichlortetracycline 10 - 300 >0.99103.3993.3999.51

2.460.571.06

79.1287.7688.50

104.23 106.50

Following equation was used for the determination of CC α and CCβ at MRL value.

CCβ: CCα+1.64*SD of 20 fortified blanks at CCαCCα: MRL+1.64*SD of 20 fortified blanks at MRL

As per EU commission CCα and CCβ individually should not exceed5% of MRL and CCα respectively. Calibration curve was plottedbetween the upper and lower range and r² was found greater than0.99 for all compounds (Fig 2). Coefficient variation of method wasless than 2.82 % with batter recovery ranging from 77 % to 89 %.Trueness for each compound was greater than 93 % and lesser than104 % for which acceptance criteria is 90 % to 110 %. Youdenapproach was used to determine the ruggedness of method, in whichobtained results in different condition were calculated statistically. Asper this approach statistically obtained Standard deviation should notbe higher than the intraday SD (Tab 4).

Table 4 · Ruggedness calculation for tetracycline as per youden approachParameters Fac 1 Fac 2 Fac 3 Fac 4 Fac 5 Fac 6 Fac 7 Fac 8

pH of mobile phase A A A A a a a a

Gradient B B b b B B b b

Column Temp C c C c C c C cFlow Rate D D d d d d D D

Room Temp E e E e e E e E

Sheath Gas Temp F f f F F f f F

Light G g g G g G G g

Results

A1 (ppb) 98.39 97.02 96.52 100.73 100.25 96.02 99.86 99.46

A2 (ppb) 96.17 98.85 99.09 96.93 99.54 101.67 101.40 99.95

Avg. (ppb) 97.28 97.93 97.80 98.82 99.89 98.84 100.63 99.70+ (X) Σ/4 - (Y) Σ/4 D ( X – Y) D²

A 97.9623 a 99.7699 -1.8076 3.267

B 98.4895 b 99.2427 -0.7532 0.567

C 98.9035 c 98.8287 0.0749 0.005

D 98.8891 d 98.8431 0.0460 0.002

E 98.4093 e 99.3228 -0.9135 0.834

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F 98.9271 f 98.8051 0.1220 0.014G 98.8965 g 98.8357 0.0608 0.003

Intraday SD 1.69 SD 1.1934

Capital alphabet shows that parameter was modified and lower alphabets shows that parameter was not changed.

Conclusions

Tetracyclines have their epimers which are similar in chemical nature. This method shows a simple way to detect and monitor the isomers with high confidence usingthe highly selective LC/MS/MS technology and improved chromatography making the results more reliable to the EU Commission.

As this method is fully validated and all parameters comply with the requirement of EU commission, the method can be used for the analysis of EIC samples inmarine products.

References

Rail Khismatoullin, Rafavel Kuzyaev, Yaroslav Lyapunov, Elena Elovikova. Apiacta,2003, 38, 246-248.1.Alippi A.D, Albo G.N, Leniz D, Rivera I, Zanelli M L. Roca A E, Journal of Apicultural Research, 1999, 38, 149-158.2.James D.Macneil, Valerie K. Martz, J. AOAC, 1996, 79, 405-417.3.Pang Guofang, Cao Yanzhong, Fan Chunlin, Zhang Jinjie, Li Xuemin, Li Zengyin, Jia4.Guangqun, Apiculture of China, 2003, 54, 25-29.5.

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