Article pubs.acs.org/ac
Identification of α-Cobratoxin in Equine Plasma by LC-MS/MS for Doping Control Ludovic Bailly-Chouriberry,*,† Florence Cormant,† Patrice Garcia,† Albert Kind,‡ Marie-Agnès Popot,† and Yves Bonnaire† †
Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, 91370 Verrières le Buisson, France CYCADS Laboratory, Iowa State College of Veterinary Medicine, 1600 S 16th Street, Ames, Iowa 50011, United States
‡
ABSTRACT: Cobra venom (Naja kaouthia) contains a toxin called αcobratoxin (α-Cbtx). This toxin is a natural protein containing 71 amino acids (MW 7821 Da) with a reported analgesic potency greater than morphine. In 2007, in USA, this substance was found in the barns of a thoroughbred trainer and since then till date, the lack of a detection of this molecule has remained a recurring problem for the horseracing industry worldwide. To solve this problem, the first method for the detection of α-cobratoxin in equine plasma has now been developed. Plasma sample (3 mL) was treated with ammonium sulfate at the isoelectric point of α-Cbtx, and the pellet was dissolved in a phosphate buffer and mixed with methanol for precipitation. The supernatant was then concentrated prior to its extraction on WCX SPE cartridges. The eluate was concentrated with two consecutive filtration steps before the trypsin digestion. The samples were analyzed using a LC-MS/MS Q Exactive instrument at 70,000 resolution on the product ions of the doubly charged precursor of the target peptide (24TWCDAFCSIR33). The method was validated (n = 18) at 5 μg/L (640 pmol/L) according to the Association of Official Racing Chemists (AORC) requirements. The lower limit of detection was 1 μg/L (130 pmol/L). The present method has made it possible for us to confirm the presence of α-Cbtx in a horse plasma sample 24 h post the administration of α-Cbtx. Thus, the present method provides the first sensitive, specific, and reliable analytical method to confirm the presence of α-Cbtx in equine plasma. the reported method is specific to α-cobratoxin. The αcobratoxin (α-Cbtx) or alpha-elapitoxin-Nk2a (recommended name) is a protein-based drug toxin produced by Naja kaouthia (monocled cobra). α-Cbtx is a member of the second group and contains 71 amino acids (MW 7821 Da) with 5 disulfide bridges identified by X-ray crystallography7−11 and NMR12 studies. The monomeric form of α-Cbtx binds with high affinity to muscular and neuronal α-7 nicotinic acetylcholine receptors (nAChR) and causes paralysis by preventing acetylcholine binding to the nAChR13−16 without modification of the acetylcholine release.17 The homodimeric form binds with low affinity to α-7 nAChRs, whereas it acquires the capacity to block α-3/β-2 nAChRs unlike its monomeric form.14 Despite numerous investigations conducted by several laboratories, and unlike growth hormone18 or erythropoietin19−23 detection, there is no method available to confirm the presence of α-cobratoxin for doping control analysis. Thus, this new method provides the first sensitive, specific, reliable, and validated analytical strategy to confirm the presence of α-Cbtx in equine plasma sample at low concentrations (1−5 μg/L or
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eptide toxins are usually found in animal venom which are from a wide variety of species such as cone snails, spiders, scorpions, anemones, frogs, wasps, and snakes.1 Surprisingly, in 2007, vials of α-cobratoxin (α-Cbtx) were found in the barns of a thoroughbred trainer in USA.2 This molecule was therefore strongly suspected to be used as an undetectable analgesic substance3−5 to relieve pain due to injuries in the horse in the horseracing industry. Moreover, this molecule was also suspected to be used for the same reason on barefoot race horses in Europe. Cobra venoms contain a complex mixture of molecules that consist of several toxic proteins with diverse biological activities such as neurotoxin which acts on nicotinic acetylcholine receptors (nAChR) and cardiotoxin which acts on cell membranes as well as phospholipase and D-amino acid oxidase.6 The alignment of different sequences of snake venom neurotoxins with their phylogenetically related proteins was described in 1973 by Evert Karlsson.7 The author has concluded that neurotoxins are low molecular weight basic proteins tightly cross-linked by disulfide bridges which can be split into two groups. The first group consists of smaller toxins (MW 7000 Da) with 60 to 62 amino acid residues in a peptide chain cross-linked by 4 disulfide bridges and a second group with larger proteins (MW 8000 Da) containing 71 to 74 amino acids and 5 disulfide bridges. Besides these homologies, there are significant differences of toxin among different species, and © 2013 American Chemical Society
Received: February 28, 2013 Accepted: April 12, 2013 Published: April 12, 2013 5219
dx.doi.org/10.1021/ac4006342 | Anal. Chem. 2013, 85, 5219−5225
Analytical Chemistry
Article
130−640 pmol/L). Furthermore, the detection of α-Cbtx in a postadministration plasma sample is presented.
Methanol Precipitation. Methanol (7 mL, room temperature) was added to the dissolved pellet and left under stirring for 60 min prior centrifugation (3500 g, 5 min). The supernatant was collected and evaporated at 48 °C to about 500 μL in a TurboVap LV Evaporator (Zymark, Hopkinton, MA, USA) under a steady stream of nitrogen. The resulting sample was stored at 4 °C until next step. Solid-Phase Extraction. Weak Cation eXchange (WCX) SPE cartridges (60 mg, 3 mL, 80 Å), obtained from Waters (Milford, MA, USA), were sequentially conditioned with 2 mL each of methanol and water, using a VacElut vacuum manifold (Sigma-Aldrich, St. Louis, MO, USA). The 500 μL sample from methanol precipitation procedure above was mixed with 1 mL of high purity water and 1 mL of phosphoric acid (0.6 M) prior to loading onto the column and allowed to pass through the column bed. The SPE column was then washed with 3 mL of ammonium hydroxide (0.4 M) followed with 3 mL of 80/20 water/acetonitrile (v/v). Analytes were eluted with 3 mL of 25/ 75 water/acetonitrile (v/v) containing 0.1% TFA. The eluate was evaporated at 48 °C to 500 μL under a gentle stream of nitrogen as described above. Trypsin Digestion. The above sample was filtered (18 000 g, 15 min) on a 30 kDa MWCO (Millipore, Bedford, MA, USA). The filtrate was then mixed with BSA (10 μL, 3.75 μM) and concentrated (18 000 g, 15 min) two times on the same 3 kDa MWCO filter with two washes with ammonium bicarbonate (150 μL, 50 mM). The retentate (20 μL) was transferred into a 200 μL tube and mixed with trypsin (6 μg). The digestion occurred in a Thermomixer Comfort (Eppendorf, Le Pecq, France) for 18 h at 37 °C with alternating rotation set at 800 rpm. The peptides mixture was carefully transferred into a glass HPLC insert and completed with 5 μL acetonitrile and the peptide external standard solution (5 μL, 80 pM). Samples must be analyzed within two days. Drug Administration and Sample Collection. Drug administration protocol was established in agreement with animal welfare rules and performed at the administration and sampling Center of Fédération Nationale des Courses Françaises (FNCF). A 22 year-old, 526 kg thoroughbred stallion, identified as H719, was subcutaneously administered 2 mg of cobratoxin prepared in physiological saline solution (3.8 μg/kg bw) in two points at the pastern (internal and external). Whole blood was collected from the jugular vein into lithium heparinized tubes (Greiner Bio-One SAS, Courtaboeuf, France). Samples were collected at 36, 24, and 12 h before drug administration and at 0.5, 1, 2, 4, 6, 8, 24, 48 h, Day+3, Day+4, and Day+7 after drug administration. Cells were separated from plasma by centrifugation (10 min, 1500 g). All plasma samples were stored at −80 °C until analysis. Samples (6 each of mares, stallions, and geldings) were randomly selected from L.C.H. (French horse doping control laboratory) following routine analysis and were declared to be negative for which reason they were used for method validation in this study. LC-MS/MS Analysis. Peptide Standard. Analysis of the αcobratoxin target peptide (24TWCDAFCSIR33) was prepared at 10 ng/μL in methanol/water (50:50, v/v) containing 0.2% formic acid and was directly introduced into the linear ion trap (LTQ XL mass spectrometer; ThermoFisher Scientific, San Jose, CA, USA) with an electrospray ionization (ESI) source operated in the positive mode. The flow was set at 5 μL/min via Flow Injection Analysis (FIA). Parameters of the linear ion trap mass spectrometer were 275 °C and 4.8 kV for ion transfer
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MATERIALS AND METHODS Chemicals and Reagents. α-Cobratoxin was purchased from Latoxan (Valence, France). Synthetic peptides with amino acid sequences 24TWCDAFCSIR33 used as an oxidized target peptide and MGGPGAGSGPGAGGSGA was used as an external standard were purchased from Millegen (Labège, France). Trypsin (sequencing grade) was purchased from Promega (Charbonières-les-Bains, France). HPLC-grade methanol, acetonitrile, and ammonium hydroxide were purchased from Carlo Erba (Val de Reuil, France). Formic acid (FA), trifluoroacetic acid (TFA), ammonium bicarbonate, phosphoric acid, trizma base, and sodium hydroxide were from SigmaAldrich (St. Louis, MO). Ammonium sulfate, chloridric acid, sodium dihydrogenophosphate, and disodium hydrogenophosphate were purchased from VWR International (Leuven, Belgium). Bovine Serum Albumin (BSA) was from Jackson ImmunoResearch (Newmarket, England). Preparation of Standard Solutions. Standard solution of α-Cbtx was prepared by adding 1 mL of high purity water in the vial resulting in 1 mg/mL and stored at 4 °C. Standard solutions of synthetic peptides were prepared in water/ acetonitrile (90/10 v/v, 0.2% FA) at 1 mg/mL and stored at 4 °C. Working standard solutions were prepared by consecutive 1/10 dilutions and used up to 3 months with storage at 4 °C. Standard powder was stored at −20 °C pending analysis. Preparation of Reagents. All buffers and solutions were exclusively prepared with high purity water with conductivity of >18 MΩ·cm and filtered online through a 0.22 μm membrane (Millipore, Bedford, MA, USA). Tris/HCl pH 8.6. A solution of HCl was prepared by adding 1.67 mL of HCl 37% in a final volume of 100 mL of high purity water. The Tris/HCl pH 8.6 buffer was prepared by mixing 9.692 g of Trizma base with 97.6 mL of HCl in 400 mL of high purity water. Saturated Solution of Ammonium Sulfate pH 8.6. To 250 mL of high purity water was added 90 g of ammonium sulfate under stirring. After waiting for complete dissolution, 60 g was added and then 50 g. After 1 h under stirring, the saturated solution is adjusted at pH 8.6 with sodium hydroxide 32%. Phosphate Buffer pH 6. Fourteen g of NaH2PO4, 2 H2O and 2.2 g of NaHPO4, 2 H2O were dissolved in a final volume of 1 L of high purity water. Phosphoric Acid pH 1.4. Four mL of H3PO4 85% was added to a final volume of 100 mL of high purity water. Ammonium Hydroxide pH 11.6. Five mL of NH4OH was added to a final volume of 100 mL of high purity water. Ammonium Bicarbonate 50 mM. 0.040 g of ammonium bicarbonate was added to a final volume of 10 mL of high purity water. Sample Preparation. Ammonium Sulfate Precipitation. To 3 mL of plasma, 15 mL of tris/HCl buffer (0.2 M, pH 8.6) was added and mixed. Saturated solution of ammonium sulfate (pH 8.6, 12 mL) was then added in a dropwise manner with gentle stirring until 40% saturation was achieved. Stirring continued for 90 min at room temperature after which the mixture was stored overnight at 4 °C to induce protein precipitation. Following centrifugation (3500 g, 20 min) at room temperature, the supernatant was discarded, and the protein pellet was dissolved in 3 mL of phosphate buffer (0.1 M, pH 6.0). 5220
dx.doi.org/10.1021/ac4006342 | Anal. Chem. 2013, 85, 5219−5225
Analytical Chemistry
Article
improve the detection of α-Cbtx in terms of sensitivity and specificity, we decided to focus on a specific peptide. The oxidized peptide candidate 24TWCDAFCSIR33 was synthesized and directly introduced into the linear ion trap mass analyzer in order to obtain its characteristic MS2 and MS3 product ions (Figure 1). The peptide was characterized by its doubly charged state ([M+2H]2+, m/z 600.8), as is common with peptides resulting from trypsin digestion with minor detection of the singly charged state and no detection of a triply charged state. Figure 1 shows product ion spectra in MS2 and MS3 mode of the doubly charged state target peptide of α-Cbtx ([M+2H]2+, m/z 600.8). Identification of the three most abundant fragment ions, m/z 260.2, m/z 288.2, and m/z 912.4 corresponding to a2, b2, and y8, respectively, was achieved in MS2. A minor ion was also observed at m/z 375.3 corresponding to y3. These results are in accordance with the structure of the peptide for which the disulfide bridge between cysteine 26 and cysteine 30 do not allow fragmentation. Indeed, the resulting major product ions are present on each side of the disulfide bridge. In order to achieve good sensitivity with a high degree of reliability, the analyses of plasma samples were carried out on a Q Exactive (ThermoFisher Scientific, San Jose, CA, USA) mass spectrometer. The resolution fixed at 70,000 (m/z 200) allowed sufficient scan rate with a high level confidence on the identity product ions of α-Cbtx as the target peptide (
