Analytical and Bioanalytical Chemistry

, Volume 403, Issue 6, pp 1619–1628 | Cite as

Detection of recombinant human EPO administered to horses using MAIIA lateral flow isoform test

  • Maria Lönnberg
  • Ulf Bondesson
  • Florence Cormant
  • Patrice Garcia
  • Yves Bonnaire
  • Jan Carlsson
  • Marie-Agnes Popot
  • Niclas Rollborn
  • Kristina Råsbo
  • Ludovic Bailly-Chouriberry
Original Paper

Abstract

Doping of horses with recombinant human erythropoietin (rHuEPO) to illegally enhance their endurance capacity in horseracing has been reported during the last years. This leads to increased blood viscosity which can result in sudden death and is of concern for the horse welfare. Additionally, the horse can start production of rHuEPO antibodies, which cross-reacts with endogenous equine EPO and can lead to severe anaemia and even death. In this study, a novel micro-chromatographic method, EPO WGA MAIIA, has been tested for the capability in plasma and urine samples to detect administration of erythropoiesis-stimulating agents, like the rHuEPO glycoprotein varieties Eprex and Aranesp, to horses. After administration of 40 IU Eprex kg−1 day−1 to seven horses during 6 days, the presence of Eprex in horse plasma was detected up to 2–5 days after last injection. In urine samples collected from two horses, Eprex was detected up to 3 days. A single injection of Aranesp (0.39 μg/kg) was detected up to 9 days in plasma and up to 8 days, the last day of testing, in the urine sample. The LC-FAIMS-MS/MS system, with 1 day reporting time, confirmed the presence of Eprex up to 1 day after last injection for six out of seven horses and the presence of Aranesp up to 5 days after last injection in plasma samples. The MAIIA system showed to be a promising tool with high sensitivity and extremely short reporting time (1 h).

Fig

The interior of the MAIIA membrane. EPO protein molecules (blue balls) flow through the WGA lectin ligands immobilised in the network structure. Some types of EPO have oligosaccharide structures with higher interaction strength and will be delayed, while other low reacting types will rapidly reach the anti-EPO antibodies immobilised downstream, during the 5 min. reaction time.

Keywords

Aranesp EPO doping control Eprex Equine EPO Micro-chromatography WGA 

Introduction

The human 30-kDa glycoprotein erythropoietin (HuEPO), produced by human adults mainly in the renal cortex, is essential for red blood cell production. For healthy individuals, a decrease in haemoglobin concentration directly increases the production rate of erythropoietin (EPO), as recently reviewed [1]. The human recombinant EPO (rHuEPO) has successfully been synthesised in mammal cells since the late 1980s [2] and has been administered to millions of anaemic patients with chronic kidney disease, AIDS and cancer.

It has been speculated for horses, due to the large red blood cell storage capacity in the spleen [3], that rHuEPO doping will not enhance their exercise performance. This was tested by administration in healthy horses of rHuEPO, with 50 IU/kg injected three times a week during 2 weeks, which increased the number of red blood cells and improved the oxygenation of main muscles compared with the control group [4]. A substantial 19 % increase in maximal aerobic capacity was shown 1 week after treatment. A higher number of red blood cells leads to increased blood viscosity, which may result in cerebral thromboembolism, as for humans [5]. Moreover, horses treated with rHuEPO have developed immune response, with production of anti-human EPO antibodies cross-reacting with equine EPO (eEPO), and obtained severe anaemia [6]. The use of rHuEPO and its analogues is prohibited in the majority of human and animal sports.

In humans and horses, only minute blood concentrations of rHuEPO, in the nanogrammes per litre range, is required to increase erythropoiesis. The clearance from blood is rapid and shows large variations between individuals. For epoetin alpha, one type of rHuEPO, the biological half-lives were 19.4 ± 10.7 h after subcutaneous (s.c.) and 6.8 ± 2.7 h after intravenous (i.v.) injections to humans [7]. Moreover, the varieties of protein-based erythropoiesis stimulating agents (ESAs) [8,9] are increasing rapidly, with up to 80 biosimilar epoetin products available in 2009 [2], which makes the doping control for humans even more difficult [10]. The EPO variants on the market are differing in biological activity and structure, e.g. glycosylation, due to cell line expression and details in the manufacturing process, like culture conditions and purification methods. EPO-resembling recombinant protein drugs with longer biological half-lives, like Aranesp with a molecular weight of 37 kDa and a biological half-life of 26 h (i.v.) [11], and Mircera, a pegylated rHuEPO, with a molecular weight of 60 kDa and a biological half-life of 134 h (i.v.) [12], are efficient ESAs. They can be used with less frequent injection regimes, which facilitates for the patients.

For doping control of rHuEPO administered to humans, the only difference between exogenous and endogenous EPO is the composition of the three N-linked and one O-linked oligosaccharide complex, constituting 40 % of the protein molecular weight. The main methodologies, as recently reviewed [13], combine electrophoretic or chromatographic separation of EPO glycoforms, with sensitive anti-EPO antibody based detection methods. The methods often require an additional purification and/or concentration step before urine and plasma samples analysis [14,15]. Endogenous and recombinant HuEPO glycosylation can be distinguished by differences in charge [16,17], isoelectric point [18, 19, 20], molecular mass [21, 22, 23] or interaction with lectins, e.g. wheat germ agglutinin (WGA) [24,25] for the glycoproteins. The methods differ in how well they distinguish a certain type of glycosylation and how much EPO is required for analysis.

For EPO doping control in horses, a rapid screening method for rHuEPO using a human EPO immunoassays with low reactivity for eEPO compared with HuEPO has been used [26] to select samples to be confirmed with the more resource demanding methods. Exogenous rHuEPOs and analogues present in equine samples have been detected by isoelectric focusing (IEF) with double-immunoblotting [26], due to their differences in oligosaccharide composition. Equine and human EPO have 84 % identity in the amino acid structure [27] which has been utilised for LC-MS/MS analyses of peptide sequences specific for HuEPO appearing after trypsin digestion of EPO [28, 29, 30, 31, 32]. However, if recombinant eEPO will appear at the market MS based methods cannot be used for its detection, but methods showing differences in glycosylation can still be used.

MAIIA is a novel analytical micro-chromatographic system [33] for separation and detection of low abundant protein isoforms with good resolution [34] and high sensitivity [35]. The lateral flow based micro-column has been equipped with ligands suitable for ion-exchange chromatography [36,37] and with lectin ligands, like WGA [25], for affinity chromatography. The MAIIA (EPO WGA MAIIA) method, using WGA as chromatographic ligand and EPO detection was recently found to distinguish ten different rHuEPOs and analogues from human endogenous EPO [25]. It was also shown, when testing samples from a major sport competition, that the use of the MAIIA test revealed 6 % of the athletes as positive for EPO doping while the accredited IEF method for human athletes found no positive samples.

In this study, the MAIIA method is evaluated to detect the daily s.c. administration of 40 IU/kg of Eprex during six consecutive days and the single administration of 0.39 μg/kg of Aranesp to horses.

Materials and methods

Sample collection from horses administered with rHuEPO (Eprex) and analogues (Aranesp)

The details for the administration series performed at Laboratoire des Courses Hippiques (L.C.H.; Verrières le Buisson, France) have recently been presented [30]. Briefly, eight thoroughbred horses were administered subcutaneously in the neck. Seven horses (H579 to H585) received 40 IU kg bodyweight−1 day−1 of Eprex (epoetin alpha) during 6 days, and horse H626 received a single dose of 0.39 μg/kg of Aranesp (darbepoetin alpha). Heparin plasma samples were obtained before, during and after the end of administrations as indicated in Fig. 1. During the 6 days with Eprex injections, samples were collected 24 h after previous injection. Urine samples were also collected from three horses (H579, H580 and H626). All samples were stored at −20 °C until their analysis. The study was led in agreement with animal welfare rules at the administration and sampling Centre of the Fédération Nationale des Courses Françaises (FNCF).
Fig. 1

The MAIIA test gives both the total EPO concentration and the PMI value as shown for the plasma samples collected after Eprex (left panel) and Aranesp (right panel) administration. Low PMI value indicates presence of Eprex or Aranesp. For the Eprex administration series, the samples collected up to day 4 after final injection showed significantly lower PMI values compared with the results for eEPO in the pre-administered samples. The samples collected up to 9 days after injection of Aranesp were all positive. The longer detection period for Aranesp, as seen when comparing the left and right panels, is mainly due to its slower release from the circulation. The EPO concentration was back to base-line values 7 days after last injection for Aranesp while it is interpolated that base-line was reached at day 3 after last Eprex injection, after administration of comparable amounts of the recombinant proteins

Reference urine and plasma samples used for EPO WGA MAIIA

As reference plasma samples, one of the pre-administration samples, collected 2 days prior to the administration, was selected from the eight thoroughbred horses (eight samples) and ten samples were leftovers from routine analysis (post-racing). For the urine analysis, six reference samples were used, three samples were collected before injection and three samples were leftovers from routine analysis.

Affinity purification of EPO from biological samples as a pre-step for EPO WGA MAIIA analysis

EPO Purification Kit, Art. No. 0250, was obtained from MAIIA Diagnostics (Uppsala, Sweden). EPO from 0.5 mL equine plasma samples (for 20 % of the samples 0.47 to 0.06 mL was used) or 20-mL equine urine samples was purified according to the instructions from the producer, using the recommended addition of detergent and bovine serum albumin to the reagents. Purified EPO was finally obtained in a volume of 220 μL for equine urine. EPO from the plasma samples was obtained in an eluate volume of 55 μL, except for the high EPO concentration samples collected during 1 to 3 days after first injection of Eprex, for which 220-μL eluate was collected. EPO Quantification Kit Art. no. 0100 (MAIIA Diagnostics), was used for the determination of EPO concentration in the eluate. The EPO affinity purification recovery was measured and found to be in accordance with recent evaluations [14,15] showing 75 and 55 % recovery for NeoRecormon® (Roche GmBH, Mannheim, Germany) added to buffer and equine plasma, respectively, and 39 % for Aranesp (Thousands Oak, CA, USA) added to equine plasma.

EPO WGA MAIIA isoform analysis

A MAIIA kit (EPO WGA MAIIA prototype kit, MAIIA Diagnostics), containing MAIIA lateral flow strips and reagents for performing WGA lectin chromatography and EPO immunoassay, was used with EPO affinity purified samples in accordance with the instructions from the supplier. The procedure and scanner equipment has been described recently [25] together with the standardisation regime. The MAIIA lateral flow strip contains both a WGA zone (lectin affinity) and downstream this, an anti-EPO zone (capturing antibody for immunoassay). Briefly, after immersing the strip in a well containing the sample, all EPO is bound in the first tenth of the 8-mm long WGA zone. Then the strip is moved to a second well for desorption of EPO, from its WGA interaction, by a WGA competing sugar derivate, N-acetylglucosamine (GlcNAc). At least two strips are used to obtain total (using high concentration of GlcNAc, 100 mM) and retarded (using low concentration of GlcNAc) desorption of EPO for each sample. The membrane lot and the selected GlcNAc concentrations were: (a) results in Table 1, L100112—3 mM GlcNAC, (b) plasma runs, L091118—2 mM GlcNAc, and (c) urine runs, L090108—10 mM GlcNAc. Desorption from the WGA binding starts the migration of EPO and the most rapidly migrating ones can pass the WGA zone and be captured in the subsequent anti-EPO zone on the strip. Desorption is interrupted after 5 min by removing the WGA zone by cutting. EPO bound to the anti-EPO zone is reacted with anti-EPO bound to carbon black nano-string, and the obtained grey to black signal intensity is quantified with an image scanner.
Table 1

Different types of EPOs and their MAIIA PMI values

 

Buffer prep

Plasma

Urine

Plasma runs (2 mM GlcNAc)

Urine runs (10 mM GlcNAc)

n

PMI

n

PMI

n

PMI

n

PMI

n

PMI

Comparison of PMI values (mean ± SD) for a variety of EPOs (3 mM GlcNAc)a

 rHuEPO

 NeoRecormon

4

8.0 (±0.7)

        

 rHuEPO analogues

 Aranesp

2

3.0 (±0.7)

        

 Mircera

3

57.6 (±6.9)

        

 Endogenous EPO

 Human EPO

  

2

25.7 (±3.6)

3

14.1 (±2.8)

    

 Equine EPO

  

3

87.9 (±6.9)

3

57.1 (±3.2)

    

Optimised WGA interaction for the studyb

 Equine EPO

      

26

90.0 (±7.3)

6

77.0 (±2.5)

 NeoRecormon in buffer

      

11

13.4 (±2.0)

1

31

aAn example of WGA interaction optimised (3 mM GlcNAc) to measure both the EPO variety with the strongest (Aranesp, only 3.0 % passed the WGA zone) and with the weakest binding (purified eEPO from plasma, 87.9 % passed the WGA zone)

bThe WGA interaction optimised for this study to allow almost all equine EPO to migrate through the WGA zone. Urine eEPO migrated slower through the WGA zone than plasma eEPO when compared in Table 1a, and a reduced WGA reactivity was required to obtain higher relative passage of eEPO in urine. The use of 2- and 10-mM GlcNAc for plasma runs and urine runs, respectively, is indicated by the increased migration of the control NeoRecormon in urine runs to 31 % migrating compared with 13.4 % for the plasma runs

In this study, 25 μL of affinity-purified EPO sample was applied to each strip. For each sample, four strips were utilised since duplicates were used for determination of the amount of EPO that had passed the WGA zone during the 5 min of total and retarded desorption. By using the appropriate low GlcNAc concentration for retarded desorption, a minor part of rHuEPO (about 13–31 %), and most of endogenous eEPO (>75 %), were allowed to migrate into the anti-EPO zone. The amount of EPO reaching the anti-EPO zone was calculated using a standard curve of NeoRecormon (3 to 1,000 ng/L). Stored signal values for the standard curve were used for runs performed with the same batch of reagents. The MAIIA value characterises the WGA interaction with the EPO glycosylation by the unit percentage of migrated isoforms (PMI), [EPO amount released during retarded conditions using low GlcNAc concentration]/[total amount of EPO released using 100 mM GlcNAc] × 100. In each of the 11 plasma runs, two control preparations were included, an affinity purified equine plasma and a buffer preparation with NeoRecormon. The test procedure took about 30 min for processing 56 strips.

Concentration determination using the EPO WGA MAIIA

EPO concentration in the eluates from the affinity purification was determined from the MAIIA strips used with 100 mM GlcNAc in the desorption step. The concentration of EPO in the plasma samples was estimated by adjusting for applied sample volume in the affinity purification step, and correcting for the 55 and 39 % recovery obtained for the controls with NeoRecormon and Aranesp applied to equine plasma. The variation in recovery was recently estimated to 15 % in coefficient of variation for the affinity purification of NeoRecormon added to buffer [15]. The concentration of the EPO analogue Aranesp was underestimated with 18 % when using NeoRecormon as standard, as reported earlier for the antibodies used in the sandwich immunoassay part of the test [35]. The EPO concentration of samples containing eEPO might also be underestimated as the used anti-EPO antibodies were obtained by injection with HuEPO, and no purified eEPO preparation was available for standardisation.

Enzyme immunoassay of EPO in horse plasma samples

An ELISA for HuEPO, Quantikine EPO, was purchased from R&D Systems (Minneapolis, MN, USA) and used at L.C.H. for selecting horse plasma samples above 170 ng/L (5 pM) for further LC-MS/MS analysis. The conversion factor used to obtain units to mass for the rHuEPO standard was 1 IU = 8.4 ng.

Mass spectrometry analysis, LC-FAIMS-MS/MS

The method [30], developed and performed at L.C.H., combines a rapid pre-analytical purification and concentration device with detection using LC-high-field asymmetric waveform ion mobility spectrometry (FAIMS)-MS/MS. EPO was purified and concentrated from 4-mL plasma samples or 10-mL urine samples with the EPO Purification kit (MAIIA Diagnostics) further elaborated [30] to be used prior to MS analysis. The target peptides for human EPO were T6 and T17 as previously described [28] with a retention time of 9.9 and 9.0 min, respectively. Each peptide showed four SRM transitions and a signal-to-noise ratio above 3. Performances were validated with blank equine plasma and urine samples, and the same samples spiked with Aranesp. The validated limit of confirmation was defined at 250 ng Aranesp/L and the lower limit of detection was found at 100 ng/L [30]. In the validation, Aranesp was selected as reference as this molecule was less efficiently captured than NeoRecormon in the affinity purification process. In the present study, a sample was identified as positive when Association of Official Racing Chemists (AORCs) minimum criteria for MS identification of small molecules were fulfilled.

Statistics

Values are means ± 1 standard deviation (SD). Differences between results for samples collected before and after injection were examined by paired t test (SigmaPlot 12, Systat Software, San Jose, CA, USA) and statistical significance was accepted at p < 0.05. For the MAIIA test, the one-tailed 99.9 % confidence limit (CL) were calculated from the mean result for the reference samples and results outside CL were termed positive.

Results

MAIIA test optimisation of the percentage of EPO migrating through the WGA zone

The interaction strength with WGA for different types of EPO related molecules has recently been shown to be considerable different [25]. The resolution between two types of EPO populations can be increased by optimising the concentration of GlcNAc used for competitive desorption of EPO [25]. However, as the binding strength of the WGA ligand bound to the membrane also can vary, the selected concentration of GlcNAc has to be optimised for each membrane lot. In Table 1, the target for the optimisation was to compare the WGA interaction from the EPO-like molecule with the strongest interaction (Aranesp) to the EPO population with the weakest interaction (equine EPO in plasma). Aranesp has increased WGA interaction due to the two additional carbohydrate structures; only 3 % (=3.0 PMI) was passing the WGA zone. Equine EPO bound with considerable less strength, having values of 57.1 and 87.9 PMI in urine and plasma, respectively, compared with NeoRecormon with 8.0 PMI. Human endogenous EPO in urine and plasma showed values of 14.1 and 25.7 PMI, respectively. Both for humans and horses, the oligosaccharides on EPO in urine showed more structures interacting with WGA, with lower PMI values for urine compared with plasma EPOs.

In the present administration study, a slightly different WGA interaction strength was selected, specific for plasma and for urine runs, to allow almost all eEPO in each run to migrate through the WGA zone. For the plasma runs, 90 % (90.0 PMI) of eEPO were allowed to pass the WGA zone, which was comparable to the interaction obtained for the results presented in Table 1. For the urine run, 77 % of eEPO was allowed to pass the zone. Compared with the results found in Table 1, 57.1 %, the WGA interaction was reduced for the urine run to obtain better resolution from recombinant EPO and Aranesp. The results for NeoRecormon, with 13.4 and 31 PMI for the plasma and the urine runs, respectively, makes it possible to compare that different interaction strengths has been used.

For the administration series, the rHuEPO type Eprex was injected to the horses. This EPO variety has recently been compared with NeoRecormon and showed comparable WGA interaction with values of 25.1 and 30.0 PMI, respectively [25].

MAIIA results for the Eprex and Aranesp administration series

The PMI values obtained by MAIIA analysis of equine plasma during the administration of Eprex and Aranesp are shown in Fig. 1. Included in the figure is also the total EPO concentration in plasma, although both eEPO and Aranesp are likely to be underestimated when using rHuEPO for standardisation. The low PMI values for EPO in the samples during the administration of Eprex indicate strongly the presence of exogenous EPO glycoforms in the equine plasma. The results for horse plasma samples collected 1 day after injection (n = 47) showed a highly significant (p < 0.001) reduction in PMI values to 21.8 ± 4.8 PMI after Eprex injections, compared with 88.7 ± 7.2 PMI for eEPO present in the eight samples collected 2 days prior to the initial injection. Samples collected 2 (p < 0.001) and 4 days (p = 0.019) after last injection of Eprex from seven horses were also significantly different from the samples collected before injection. No plasma sample was collected 3 days after injection. The samples collected 5 days after last injection and later on could not be differentiated from eEPO. The samples from horse 581, collected during the administration period after the last three injections, showed considerable lower EPO concentrations compared with samples from other horses, which was also confirmed with the EPO ELISA immunoassay. The PMI values were not as low as for the other horses, although still clearly aberrant from eEPO.

For the diagnostics application, to settle if a single sample is aberrant, 18 plasma reference samples were tested and a mean value of 88.0 ± 7.1 PMI was obtained. There was no significant difference between the ten left-over samples (87.2 ± 7.7 PMI) and the eight pre-administration samples (89.0 ± 6.7 PMI) used as reference samples. The one-tailed 99.9 % CL was 65.9 PMI. Samples with PMI values outside 99.9 % CL were regarded as positive and the results are shown in Table 2. All plasma samples (n = 7) collected 2 days after injection were positive. The samples from horse H579, collected 4 and 5 days after injection, were also positive, hence 14.3 % of the horses were positive on days 4 and 5. For the Aranesp injection of one horse, see Table 3, the PMI values were positive up to 9 days after the single injection.
Table 2

MAIIA analysis of samples collected during and after Eprex administration

 

Days after last injection

Eprex horse 579

Eprex horse 580

Eprex horse 581

Eprex horse 582

Eprex horse 583

Eprex horse 584

Eprex horse 585

Plasma

1

POS

POS

POS

POS

POS

POS

POS

2

POS

POS

POS

POS

POS

POS

POS

3

       

4

POS

NEG

NEG

NEG

NEG

NEG

NEG

5

POS

NEG

NEG

NEG

NEG

NEG

NEG

6

NEG

NEG

NEG

NEG

NEG

NEG

NEG

7

NEG

NEG

NEG

NEG

NEG

NEG

NEG

8

       

9

NEG

NEG

NEG

NEG

NEG

NEG

NEG

Urine

1

POS

POS

     

2

POS

POS

     

3

POS

POS

     

The diagnostic application of the MAIIA test, using a cut-off at 99.9 % CL, showed that all horses were positive up to 2 days after last injection with Eprex when analysing plasma samples, while all urine samples were positive up to 3 days, the last day of testing. Plasma samples collected 3 days after injection were not available. One horse was positive up to 5 days after injection

Table 3

MAIIA analysis of samples collected during and after Aranesp administration

Days after last injection

Plasma

Urine

1

POS

 

2

POS

POS

3

POS

 

4

POS

 

5

POS

 

6

POS

POS

7

POS

 

8

POS

POS

9

POS

 

10

NEG

 

After Aranesp injection (H626) the MAIIA test showed positive results in plasma up to 9 days after injection and in urine sample up to 8 days, the last day of testing

For urine samples, the six reference samples showed a mean value of 77.0 ± 2.5 PMI, and the 99.9 % CL was 69.0 PMI. The urine samples collected up to 3 days after last injection with Eprex (n = 2), and up to 8 days after the single injection with Aranesp (n = 1), were clearly positive. No urine samples were tested after these collections days.

EPO concentration in plasma sample determined with EPO WGA MAIIA

The estimated EPO concentration in equine plasma shown in Fig. 1 for seven horses injected with 40 IU (0.34 μg) Eprex/kg and one horse injected with 0.39 μg Aranesp/kg bodyweight reveal the quite different elimination pattern for Eprex and the more glycosylated Aranesp.

For samples collected 1 day after injection with Eprex, the EPO concentration was 891 ± 179 ng/L (n = 21) after the first three injections, and 522 ± 208 ng/L (n = 21) after the three following injections. The samples collected 1 day after the last injection was 375 ± 165 ng/L (n = 7) and after 2 days 52 ± 26 ng/L (n = 7), a decline to 10 % when adjusted for the mean baseline values at 13 ng/L for these horses. For the horse injected with a single dose of Aranesp, the sample collected 1 day after injection showed a value at 3.400 ng/L, which declined to 2.800 ng/L (84%) after further 1 day. Between days 3 (1562 ng/L) and 4 (487 ng/L), the concentration of Aranesp declined to 33 %, when correcting for the baseline value at 10 ng/L for this horse. The concentration was back to baseline 3–4 and 7–8 days after injection with Eprex and Aranesp, respectively, while still the PMI values were aberrant.

It was also found that the estimated concentration of eEPO was much lower in the samples collected after injection than before. The eEPO concentration was reduced to 52 % (p = 0.026) for samples collected at days 10–14 after the initial injection (5 to 9 days after last injection) compared with the pre-administration concentration.

Imprecision for EPO WGA MAIIA

The immunoassay measurement of the affinity purified plasma samples included in the injection study tested in desorption mode with low and high GlcNAc concentration showed a median coefficient of variation (CV) of 5.7 (n = 163; mean, 27 ng/L) and 3.9 % (n = 163; mean, 97 ng/L), respectively, between the duplicates. The mean inter-assay CVs for the PMI values were 10.5 and 15.2 % for the controls at 74.4 ± 7.9 PMI (an affinity-purified plasma eEPO) and 13.4 ± 2.0 PMI for NeoRecormon in buffer, when measured in duplicate at 11 different plasma runs.

LC-FAIMS-MS/MS results

In order to confirm that the results of the administration study was in accordance with other studies analysed with mass spectrometry, liquid chromatography-ion-mobility MS/MS [30] was performed on some selected samples. Samples having a plasma concentration above 170 ng/L, as tested with ELISA, were analysed. This limit was a realistic target concentration in regards of the validated (n = 10) limit of identification obtained at 250 ng/L and the lower limit of detection (LLOD) at 100 ng/L (detectable but was not able to validate at n = 10) [30] when using 4-mL plasma or 10-mL urine sample. In addition, one of the two pre-administration plasma samples was tested for all seven horses and found to be negative. Two pre-administered urine samples for horse H579 and H626 were also negative with the method.

The samples from six horses collected 1 day after the last injection, with an Eprex concentration of 218–353 ng/L (ELISA), were all positive. H579, having only one target peptide positive when analysed in plasma, was signed as positive in regards of the minimum AORCs requirements while the urine sample, collected the same day, was clearly positive. For the remaining horse injected with Eprex, H581, the sample collected 1 day after last injection was not analysed as the EPO concentration was only 97 ng/L. The urine sample from H580, 1 day after injection, was not tested although the plasma concentration showed that it was measurable.

For the horse injected with Aranesp, H626, the samples collected 4 and 5 days after injection were positive (176–294 ng/L) while the urine sample collected 6 days after injection was negative as expected due to its low plasma EPO concentration (75.6 ng/L) that day.

Discussion

Detecting administration of rHuEPO

For MAIIA, all seven horses were positive both 1 and 2 days after last s.c. injection of Eprex, 40 IU/kg, and one of the horses were positive at 4 and 5 days after last injection when analysing the plasma samples. Urine samples from horse 579 and 580, collected 3 days after last injection, were all positive while blood samples were not collected that day. It is likely that the MAIIA test can detect the presence of Eprex up to 3 days after last injection for all horses. LC-FAIMS-MS/MS identified the presence of Eprex for six of the seven horses in the plasma samples collected 1 day after last injection.

Both MAIIA and LC-FAIMS-MS/MS used the same type of EPO purification test prior to the analysis, although further elaborated for use prior to mass spectrometry. For the MAIIA test, 0.5-mL plasma was sufficient for identification of Eprex even though, 2–5 days after injection, some of the samples were as low as 20 ng/L. LC-FAIMS-MS/MS required 4 mL of plasma, and only samples above 170 ng/L was selected for analysis due to the confirmation limit validated at 250 ng/L with LLOD at 100 ng/L.

Other laboratories have presented studies involving fewer horses and different administration regimes (differing in amount of ESA, s.c. or i.v. administration, single or several doses), which make comparisons difficult. The accredited EPO doping method for human athletes based on isoelectric focusing IEF, showed that s.c. administration of 36 IU Eprex/kg bodyweight was not detectable in equine urine from the three horses after 48 h from last injection [26]. By LC-MS/MS (limit of identification at 200 ng/L), the single i.v. injection of 8.4 and 34 IU/kg of epoetin alpha, to one horse, could be identified in plasma up to 24- and 48-h post-administration, respectively [28]. The results for the MAIIA test, with detection up to 2–5 days after s.c. administration of 40 IU/kg of Eprex, seem very competitive to previously presented methods for doping control of horses, although enhanced exercise performance have been found 1 week after injection [4]. Additional out-of-competition testing will hence be valuable for anti-doping purpose.

Detecting administration of Aranesp

After the single s.c. administration 0.39 μg Aranesp/kg, to one horse, positive plasma samples were found up to 9 days after last injection for MAIIA. In the urine samples, Aranesp was detectable up to 8 days after last injection, the last day of testing, with MAIIA. The LC-FAIMS-MS/MS method confirmed the presence of Aranesp in plasma up to 5 days after last injection.

Other studies using the IEF method showed that a single s.c. administration of 0.37 μg/kg of Aranesp to one horse was detectable in equine urine up to 5 days post-administration [26]. In a study using LC-MS/MS, an identification limit of 100 ng/L was obtained for Aranesp and an i.v. dose of 0.37 μg/kg was identified 168 h (7 days) post-administration for one horse [29]. Another study using LC-MS/MS detected the injection of 100 μg Aranesp to one horse up to 4 days [38] while the long-reacting pegylated epoetin beta (Mircera) was found up to 120 h (5 days) after s.c. injection of 100 μg into three horses. The MAIIA method seems to be very competitive also for Aranesp detection with detection up to 9 days post-drug administration.

Suspected anti-HuEPO antibody production

The estimated concentration of eEPO with the MAIIA test was significantly lower in samples collected about 1 week after ceasing administration (13.7 ng/L) compared with pre-administration samples (26.5 ng/L). This might depend on production of equine anti-HuEPO antibodies, which can interfere by shielding the EPO epitope for the capturing antibody in the affinity purification system, or disturb the interaction with one or two of the antibodies in the immunoassay, resulting in lower amount of EPO captured or detected. Formation of anti-HuEPO antibodies is also likely to be the reason why the estimation of the Eprex concentration in samples collected 1 day after injection was lower after the last three injections (522 ng/L) compared with after the first three injections (891 ng/L).

Equine anti-HuEPO is of analytical interest as the antibodies remain in the circulation far longer than rHuEPO. It is also of methodological interest; if available, it will be advantageous to make sure that the mouse monoclonal anti-HuEPO antibodies on the affinity matrix, or in the immunoassay, do not cross-react with the same EPO epitope as the dominating equine polyclonal anti-HuEPO antibodies.

Doping test

Doping control is traditionally performed by mass spectrometry which is the best technology for molecular characterisation of small drugs through specific and characteristic fragmentation pathways. However, for large molecules such as proteins (insulin, growth hormone, EPO and IGF-1), for which the amino acid sequence of the recombinant and endogenous forms are identical, other or additional analytical tools would be needed to reveal differences. The requirement of unique MS/MS mass spectral data for equine forensic drug testing [29] limits the detectability for proteins used for doping, like rHuEPO, for which the MAIIA test can show aberrant EPO forms in samples containing several times lower EPO concentration. Presence of rhEPO was found in samples with an EPO concentration at 20 ng/L in this study when using 0.5-mL plasma samples. Recent studies have shown that the presence of rhEPO was detected in human samples at such low EPO concentration as 0.2 ng/L when using 20–30 mL of urine [25]. Moreover, with the advent of equine recombinant EPO, both the presently used screening method and the MS/MS methods will fail to distinguish recombinant eEPO forms from endogenous eEPO.

Besides high sensitivity and specificity for the doping substance, the potential to set up high-throughput analysis (including sample pre-treatment) is of high importance. This will reduce the analysis cost for the final customer and enable more samples to be analysed. More samples analysed with a highly sensitive doping test will increase the risk for identification of doping and thus hampering it. Even though LC-MS/MS is a rapid detection technique, the pre-analytical handling takes considerable resources using traditionally affinity purification methods, denaturation and trypsin digestion steps, followed by liquid chromatography separation. The replacement of the conventional immunoaffinity process required for biological samples based on, e.g. beads [28,29], by the easy-to-use kit with the anti-EPO monolith [30], reduce both the hands-on-time and total time considerably. Besides, using the disposable anti-EPO monolith instead of cleaning and re-using the anti-EPO matrix will omit the risk for carry-over between doping samples.

A doping test performed with the MAIIA test, including rapid affinity purification using the disposable anti-EPO monolith, takes about one hour totally to proceed when using 0.5 mL of equine plasma. Further development of both the affinity purification and the MAIIA test seems to be possible to enable the use of suitable equipment for large-scale analysis.

Conclusions

The MAIIA test is a promising tool exhibiting high sensitivity and short reporting time (1 h) for equine doping analysis of ESAs in urine or plasma samples. Introduction of such easy-to-use test will most likely increase the analysis frequency, which will be valuable for horse welfare.

Notes

Acknowledgements

The authors thank Maria Andrén, Malin Drevin, Mikael Lönnberg and Trikien Quach for technical assistance, and the Swedish Foundation for Equine Research, Stockholm, Sweden, and MAIIA Diagnostics, Uppsala, Sweden, for support. The authors are indebted to Dr. Jean-Jacques Garin, veterinary surgeon at FNCF, to the horse farm manager in Coye la Forêt and to the staff who participated in drug administration, sampling and horse care.

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Maria Lönnberg
    • 1
  • Ulf Bondesson
    • 2
  • Florence Cormant
    • 3
  • Patrice Garcia
    • 3
  • Yves Bonnaire
    • 3
  • Jan Carlsson
    • 4
  • Marie-Agnes Popot
    • 3
  • Niclas Rollborn
    • 4
  • Kristina Råsbo
    • 1
  • Ludovic Bailly-Chouriberry
    • 3
  1. 1.Department of Chemistry-Biomedical CenterUppsala UniversityUppsalaSweden
  2. 2.Department of Chemistry, Environment and Feed Hygiene, The National Veterinary Institute (SVA), Uppsala, and Division of Analytical Pharmaceutical Chemistry, Biomedical CenterUppsala UniversityUppsalaSweden
  3. 3.L.C.H., Laboratoire des Courses HippiquesVerrières le BuissonFrance
  4. 4.MAIIA DiagnosticsUppsalaSweden

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