• Application Note

Rapid, High Sensitivity Detection of Recombinant Bovine Somatotropin in Cattle

Rapid, High Sensitivity Detection of Recombinant Bovine Somatotropin in Cattle

  • Gaud Pinel
  • Sandrine Roulet-Rochereau
  • Marie-Hélène Le Breton
  • Sylvain Chéreau
  • Fabrice Monteau
  • Paul Silcock
  • Bruno Le Bizec
  • Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), ONIRIS
  • Nestlé Research Center, Nestec Ltd.
  • Waters Corporation
A herd of beef cattle grazing on farm land

Abstract

This application note describes the use of Waters Xevo TQ MS for the specific detection of rbST in plasma samples collected from an animal treated with the recombinant hormone.

Benefits

The results presented are amongst the very first allowing unambiguous detection of the administration of recombinant bovine somatotropin some days after injection.

  • The high sensitivity offered by the Xevo TQ MS allows the unambiguous detection of rbST in biological matrices according to 2002/657/EC requirements.
  • Sensitivity of the detection is perfectly adapted to the detection of the circulating hormone at trace level in blood over several days after the administration to the animal.
  • The robustness of the Xevo TQ MS shows good repeatability of results, with very low matrix effect, which is an important aspect for routine analysis in the framework of monitoring control plans

Introduction

Controlling safety with respect to food products of animal origin is a priority for governments, international regulatory bodies and organizations that process and handle products prior to consumption. Food safety issues arising from commodity products often become globally reported and have the potential to impact consumer confidence and trade at international levels.

Recombinant bovine somatotropin (rbST), also called growth hormone, is used in some countries as a general growth promoter in pigs and cattle but also in lactating cows to increase milk production1,2. This practice is strictly regulated, especially in the European Union, with a complete ban of this substance3,4. However, this regulation faces a lack of analytical tools able to detect the (mis)use of this hormone. The difficulty of analysis is due to the protein nature of the hormone, its low level of concentration in biological fluids, and the complexity of the matrices of interest. Up to now, methods have been limited to immunoassays with the problem that native and recombinant forms were not differentiated. A few attempts have been published but they were all unable to specifically detect rbST at physiological levels5,6.

Only recently, a successful strategy has been developed for the direct detection of rbST in biological matrices7,8. The analysis is based on the detection of the tryptic N-terminal peptide, specific of the recombinant form of the hormone. Indeed, the Alanine N-terminal amino acid present in the natural form is replaced by a methionine in the case of the recombinant hormone as present in formulations for injection.

This application note describes the use of Waters Xevo TQ MS for the specific detection of rbST in plasma samples collected from an animal treated with the recombinant hormone. The results presented are amongst the very first allowing unambiguous detection of the administration of recombinant bovine somatotropin some days after injection.

Experimental

Standards of recombinant hormones

Protein standards of rbST and recombinant equine somatotropin reST were obtained from the Harbor-UCLA Medical Center, National Hormone and Pituitary Program (Torrance, USA) and Bresagen Limited (Thebarton, Australia), respectively.

Sample preparation

Sample extraction and purification procedure for recombinant bovine somatotropin in plasma has already been described7,8.

LC conditions

LC system:

ACQUITY UPLC System

Runtime:

8.00 min

Column:

ACQUITY BEH C18, 1.7 μm, 2.1 x 100 mm

Porosity:

130 A

Mobile phase A:

0.1% formic acid dissolved in water

Mobile phase B:

CH3CN + 0.1% formic acid

Flow rate:

0.6 mL/min

Injection volume:

8.0 μL

Mobile phase gradient is detailed in Table 1.

ACQUITY UPLC mobile phase gradient. Table 1. ACQUITY UPLC mobile phase gradient.

MS conditions

MS system:

XEVO TQ MS

Ionization mode:

ESI positive

Capillary voltage:

3 kV

Source temp:

150 °C

Desolvation temp:

550 °C

Desolvation gas:

800 L/hr

Collision gas flow:

0.15 mL/min

MRM method parameters

MRM transitions were first generated using Waters’ IntelliStart9. The various parameters were then optimized individually for each diagnostic signal. These can be found in Table 2.

Xevo TQ MS MRM mode acquisition parameters. Table 2. Xevo TQ MS MRM mode acquisition parameters.

Results and Discussion

Optimization of the LC-MS/MS conditions

The ACQUITY UPLC System allowed a good retention and separation of the tryptic N-terminal peptides with a total run time of 8 min, and retention times of 2.74 and 2.79 min for the N-terminal peptides rbST and reST, respectively (Figure 1). 

UPLC (ESI+)-MS/MS MRM diagnostic signals. Figure 1. UPLC (ESI+)-MS/MS MRM diagnostic signals of N-terminal rbST (913 > 774 and 913 > 1047) and N-terminal reST (623 > 795 and 623 > 530)obtained from the injection of a standard solution at 10 ng mL-1.

The positive electrospray ionization leads to a doubly-charged form for N-terminal peptide rbST: [M+2H]2+=913 which was selected as precursor ion. According to the Commission Decision 2002/657/EC10. Two diagnostic signals were selected in the MRM method for identification purposes. Recombinant equine somatotropin (reST) was used as internal standard with a fortification at 100 ng mL-1 in samples. The ionization of its N-terminal peptide lead to a main triply-charged form [M+3H]3+= 623, which was selected as precursor ion.

Detection of rbST in samples collected from a treated animal

This method has been applied to samples collected from a treated animal. A lactating cow was treated once subcutaneously with a dose of 500 mg of Lactatropin.

The chromatograms corresponding to plasma samples collected before treatment(D0) and 2 days (D2) after treatment are shown in Figure 2.

UPLC-ESI+-MS/MS MRM diagnostic signals. Figure 2. UPLC-ESI+-MS/MS MRM diagnostic signals for rbST obtained from a plasma sample collected before treatment (D0) and 2 days after treatment (D2).

The results shown in Figure 2 show that it’s possible to detect rbST in plasma two days after its administration to the animal. Analysis of the signals acquired leads to unambiguous identification of the protein according to 2002/657/EC requirements10. This confirms the suitability of the detection method’s sensitivity in accordance with expected levels of the protein in animal plasma. Little evidence of matrix effects was observed and very good linearity in matrix allowed further relative quantification of rbST up to around 50 ppb in plasma two days after the administration of the hormone, which is in accordance with previous results7,11. The robustness of Xevo TQ MS allowed good repeatability at low levels in complex samples as shown in Table 3.

Repeatability data for a 10-ppb fortified plasma sample. Table 3. Repeatability data for a 10-ppb fortified plasma sample.

Conclusion

  • The high sensitivity offered by the Xevo TQ MS allows the unambiguous detection of rbST in biological matrices according to 2002/657/EC requirements.
  • Sensitivity of the detection is perfectly adapted to the detection of the circulating hormone at trace level in blood over several days after the administration to the animal.
  • The robustness of the Xevo TQ MS shows good repeatability of results, with very low matrix effect, which is an important aspect for routine analysis in the framework of monitoring control plans.

References

  1. Burton JL, McBride BW, Block E, Glimm DR and Kennelly JJ. A review of bovine growth hormone. Canadian Journal of Animal Science. 1994; 74: 167-201.
  2. Bauman DE and Vernon RG. Effects of exogenous bovine somatotropin on lactation. Annual review of Nutrition. 1993, 13: 437-461.
  3. Council Decision of 20 December 1994 amending Decision 90/218/EEC concerning the placing on the market and administration of bovine somatotropin (BST). Official Journal of the European Communities. 1994/936/EC
  4. Council Decision of 17 December 1999 concerning the placing on the market and administration of bovine somatotropin (BST) and repealing Decision 90/218/EEC. Official Journal of the European Communities. 1999, 1999/879/EC.
  5. Blokland MH, Sterk SS, Van Ginkel LA, Stephany RW, and Heck AJR. Analysis for endogenous and recombinant porcine somatotropine in serum. Analytica Chimica Acta 2003, 438, 201-206.
  6. Pinel G, Buon R, Aviat F, Larre C, André-Fontaine G, André F, and Le Bizec B. Recombinant bovine somatotropin misuse in cattle. Evaluation of western blotting and 2D electrophoresis methods on biological samples for the demonstration of its administration. Analytica Chimica Acta 2005, 529, 41-46.
  7. Le Breton M H, Rochereau-Roulet S, Pinel G, Bailly-Chouriberry L, Rychen G, Jurjanz S, Goldmann T, and Le Bizec B. Direct determination of recombinant bovine somatotropin in plasma from a treated goat. Rapid Communication in Mass Spectrometry. 2008; 22: 3130-3136.
  8. Le Breton M H, Rochereau-Roulet S, Pinel G, Cesbron N and Le Bizec B. Elimination kinetic of recombinant somatotropin in bovine. Analytica Chimica Acta. 2009; 637: 121-127.
  9. Monteau F, Antignac JP, Pinel G, Silcock P, Hancock P, Le Bizec B. Xevo TQ MS:Adressing new challenges in the field of growth promoters in biological samples. Waters Application Note 2009.
  10. Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Official Journal of the European Communities. 2002, 2002/657/EC.
  11. Le Breton M H, Rochereau-Roulet S, Chereau S, Pinel G, Delatour T and Le Bizec. Identification of cows treated with recombinant bovine somatotropine. Journal of Agricultural and Food Chemistry, DOI:10.1021/jf903032q.

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