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

By Virendra Vora*, Mukesh Kumar Raikwar and Vikas Bhardvaj
June 2013

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

To develop and validate method for tetracycline antibiotics and their epimers in marine products as per EU commission (2007/657/EC). Now a day it becomes necessary to detect tetracycline as well as their epimers at EUMRL level with high confidence so that effort was made to separate and detect tetracyclines (tetracycline, oxytetracycline, doxytetracyclinea and chlortetracycline) and their epimers (epitetracycline, epichlortetracycline and epioxytetracycline). Here isomers have the same molecular weight and somewhat chemical nature too so that long run time was used to get separate peaks. 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 seven tetracyclines 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 to determine ruggedness and it was within limit.


Antibiotics are used worldwide to control bacterial infection and promote healthy marine production. Because tetracyclines are broad spectrum antibiotics so that they 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 tetracyclines including oxytetracycline, doxytetracycline and chlortetracycline are set to provide for an acceptable daily intake (ADI) and for setting a tolerance for residues in marine. 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 a tolerance 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 humans and 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 marine products to be consumed safely by humans. The existing LCMS method and microbial method for tetracyclines measurements in marine products are not satisfactory (does not include separation of epimers by retention time). Plus, the microbial method can only measure the total tetracyclines present, not being able to distinguish among different tetracyclines. The need for a new procedure with a consistent recovery rate, better specificity, selectivity and higher precisions has been pushed 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

Table 1 · HPLC gradient for sepration of isomers of tetracyclines
Time (min) B%
0.01 12
5 12
17 30
20 30
20.1 12
22 12
Table 2 · Following Precursor and product ions with fragmentor and collision energy were set
Compound Name Precursor Ion Product Ion Frag.
Tetracycline 445 410 100 17 +ve
445 427 100 7 +ve
Oxytetracycline 461 443 100 7 +ve
461 426 100 17 +ve
Doxytetracycline 445 428 100 15 +ve
445 154 100 30 +ve
Chlortetracycline 479 444 100 20 +ve
445 462 100 10 +ve
Epi tetracycline 445 410 100 17 +ve
445 427 100 15 +ve
Epi oxy tetracycline 461 443 100 7 +ve
461 426 100 43 +ve
Epichlortetracycline 479 462 100 13 +ve
479 444 100 28 +ve

All reagents and solvents were HPLC or analytical grade. Tetracycline and their epimer standards were purchased from Erhenstopher. Stock solution (1 mg/mL) was prepared in methanol and kept in the deep freezer (-20 °C). Working solutions were prepared using the stock solution diluted with a methanol. The working solutions were prepared daily. The SPE cartridges were Agilent SampliQ OPT 3 mL, 60 mg. Mcllavaine buffer was prepared by 222 ml of Disodium hydrogen phosphate and 278 ml 0.1 M Citric acid monohydrate in 500ml measuring cylinder. 18.6 gram Na2EDTA was added to dissolv in 100 ml mixture of Mcllvaine solution 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 5 μm, 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 3500 and 500 V.

Sample Preparation

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

SPE Purification: The procedure used for the SPE extraction is as follow. Agilent SampliQ OPT cartridges were preconditioned with 5 mL water and then 5 mL of methanol. A 10 mL extract was passed through the SampliQ OPT cartridge at a speed of 1 mL/min. After the sample effused completely, the cartridge was washed with 3 mL of water. The entire effluent was discarded. The cartridge was dried 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 collected and 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 was analyzed on LCMS.

Results and Discussion

Figure 1 · Chromatogram of Tetracycline and their Epimers
Figure 1

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

Selectivity: To qualify this parameter peaks were separated at different retention time as shown in fig. 1 and further it was assured by using three product ions one known as a quantifier 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 any potentially interfering substance in matrix or any other part of method. Recovery, Trueness, Repeatability, Decision limit (CCα), Detection capability (CCβ) and Ruggedness were performed and calculated as per the EC guidelines (Tab 3). Following results were obtained and which were within the limits of EU commission acceptable criteria.

Table 3 · Validation results for Linearity, Recovery, Trueness, Repeatability, CCα and CCβ
Antibiotics Range in
(r²) %
Tetracycline 10 - 300 >0.99 104.03
104.03 105.78
Oxytetracycline 10 - 300 >0.99 100.23
103.78 105.91
Doxytetracycline 10 - 300 >0.99 103.43
104.49 108.26
Chlortetracycline 10 - 300 >0.99 93.77
102.93 105.94
Epitetracycline 10 - 300 >0.99 104.02
103.83 106.82
Epioxytetracycline 10 - 300 >0.99 98.33
103.76 107.38
Epichlortetracycline 10 - 300 >0.99 103.39
104.23 106.50

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

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

As per EU commission CCα and CCβ individually should not exceed 5% of MRL and CCα respectively. Calibration curve was plotted between the upper and lower range and r² was found greater than 0.99 for all compounds (Fig 2). Coefficient variation of method was less than 2.82 % with batter recovery ranging from 77 % to 89 %. Trueness for each compound was greater than 93 % and lesser than 104 % for which acceptance criteria is 90 % to 110 %. Youden approach was used to determine the ruggedness of method, in which obtained results in different condition were calculated statistically. As per this approach statistically obtained Standard deviation should not be higher than the intraday SD (Tab 4).

Table 4 · Ruggedness calculation for tetracycline as per youden approach
Parameters 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 c
Flow 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
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)  
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
F 98.9271 f 98.8051 0.1220 0.014
G 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.


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 using the 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 in marine products.


  1. Rail Khismatoullin, Rafavel Kuzyaev, Yaroslav Lyapunov, Elena Elovikova. Apiacta,2003, 38, 246-248.
  2. Alippi A.D, Albo G.N, Leniz D, Rivera I, Zanelli M L. Roca A E, Journal of Apicultural Research, 1999, 38, 149-158.
  3. James D.Macneil, Valerie K. Martz, J. AOAC, 1996, 79, 405-417.
  4. Pang Guofang, Cao Yanzhong, Fan Chunlin, Zhang Jinjie, Li Xuemin, Li Zengyin, Jia
  5. Guangqun, Apiculture of China, 2003, 54, 25-29.


Copyright © 2013, ECO Services International