Determination of 13 C/ 12 C ratios of...

Research Article

Received: 22 January 2014 Revised: 4 March 2014 Accepted: 5 March 2014 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2014, 28, 1194–1202

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Determination of 13C/12C ratios of endogenous urinary 5-amino-imidazole-4-carboxamide 1β-D-ribofuranoside (AICAR)

Thomas Piper1*, Andreas Thomas1, Norbert Baume2, Timothy Sobolevsky3,Martial Saugy2, Grigory Rodchenkov3, Wilhelm Schänzer1 and Mario Thevis11German Sport University Cologne, Center for Preventive Doping Research – Institute of Biochemistry, Am SportparkMüngersdorf 6, 50933 Köln, Germany2Swiss Laboratory for Doping Analysis, University Center of Legal Medicine, Geneva and Lausanne, Centre HospitalierUniversitaire Vaudois and Université Lausanne, Ch. des Croisettes 22, CH-1066 Epalinges, Switzerland3Antidoping Centre Moscow, Elizavetinsky pereulok 10, bldg.1, RU 105005 Moscow, Russia

RATIONALE: AICAR (5-aminoimidazole-4-carboxamide 1β-D-ribofuranoside) is prohibited in sport according to rulesestablished by the World Anti-Doping Agency. Doping control laboratories identify samples where AICAR abuse issuspected by measuring its urinary concentration and comparing the observed level with naturally occurringconcentrations. As the inter-individual variance of urinary AICAR concentrations is large, this approach requires acomplementary method to unambiguously prove the exogenous origin of AICAR. Therefore, a method for thedetermination of carbon isotope ratios (CIRs) of urinary AICAR has been developed and validated.METHODS: Concentrated urine samples were fractionated by means of liquid chromatography for analyte cleanup.Derivatization of AICAR yielding the trimethylsilylated analog was necessary to enable CIR determinations by gaschromatography/combustion/isotope ratio mass spectrometry. The method was tested for its repeatability and stabilityover time and a linear mixing model was applied to test for possible isotopic discrimination. A reference population ofn = 63 males and females was investigated to calculate appropriate reference limits to differentiate endogenous fromexogenous urinary AICAR. These limits were tested by an AICAR elimination study.RESULTS: The developed method fulfills all the requirements for adequate sports drug testing and was found to be fit forpurpose. The investigated reference population showed a larger variability in the CIR of AICAR than of the endogenoussteroids. Nevertheless, the calculated thresholds for differences between AICAR and endogenous steroids can be appliedstraightforwardly to evaluate suspicious doping control samples with the same statistical confidence as established e.g.for testosterone misuse. These thresholds enabled the detection of a single oral AICAR administration for more than 40 h.CONCLUSIONS: Determination of thee CIRs is the method of choice to distinguish between an endogenous and anexogenous source of urinary AICAR. The developed method will enable investigations into doping control samples withelevated urinary concentrations of AICAR and clearly differentiate between naturally produced/elevated and illicitlyadministered AICAR. Copyright © 2014 John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.6891

The World Anti-Doping Agency (WADA) has prohibitedthe use of AICAR (5-aminoimidazole-4-carboxamide 1β-D-ribofuranoside) since 2009. Several studies have demonstratedthe biochemical properties of AICAR acting as an activatorfor adenosine monophosphate activated protein kinase inrat and humans.[1–3] This modulation influences insulinaction and glucose uptake.[4–6] Under these circumstancesit was further shown that AICAR can act as an exercisemimetic.[7,8] These facts together with the reported goodtolerance of this drug candidate might explain whyathletes could be tempted to illegally administer AICAR.[9]

Moreover, recently reported confiscations of illicitly

* Correspondence to: T. Piper, German Sport UniversityCologne, Center for Preventive Doping Research –Institute of Biochemistry, Am Sportpark Müngersdorf6, 50933 Köln, Germany.E-mail: [email protected]

Rapid Commun. Mass Spectrom. 2014, 28, 1194–1202

distributed AICAR preparations have required dopingcontrol laboratories to develop methods for the detectionof this banned substance.[10,11]

AICAR emerges as an intermediate during the de novo purinebiosynthesis in all human beings. As AICAR-5´monophosphate,it is further metabolized to purine and from thereon it can bedegraded to the final urinary end product uric acid. Renalclearance of AICAR itself has also been described.[12]

The first step in developing a method for the detection ofillicit AICAR administration was therefore to investigatenatural urinary concentrations of AICAR in athletes.[13] Basedon these results it was possible to investigate factors influencingurinary AICAR concentrations despite a large inter-individualvariability being found. Significant differences could be shownbetween the genders, in- and out-of-competition sampling anddifferent types of sports.[13] Most recently, the same strategywas applied to red blood cells as AICAR is stored withinthese cells as AICAR-5´monophosphate.[14]

Copyright © 2014 John Wiley & Sons, Ltd.

Determination of 13C/12C ratios of endogenous urinary AICAR

Both methods suffer from the large biological variabilityin absolute concentrations which in some cases might notallow for an unambiguous differentiation between naturallyelevated AICAR concentrations and a doping offence,especially as medical treatment or certain diseases can alsoalter the urinary AICAR concentration.[12,15]

GC/C/IRMS (gas chromatography/combustion/isotoperatio mass spectrometry) has been applied to sports drugtesting for more than 10 years as it allows for distinguishingbetween the endogenous and exogenous sources of acompound.[16,17] Mainly steroids have been investigated sofar by comparison of the 13C/12C ratios of an endogenousreference compound (ERC) to that of a target compound(TC). 13C/12C ratios are expressed as δ13C values against theinternational standard Vienna Pee Dee Belemnite (VPDB)based on the equation:[18]

δ13CVPDB ¼

�13C

=

12C

�SAMPLE�̄

13C =

12C

�VPDB

– 1

As absolute δ13C values can show a large variability, indoping control usually deviations of the TC from an ERC

(1)

are investigated. These differences are expressed as Δ valuesbased on the equation:

Δ ‰½ � ¼ δ13CTC � δ13CERC (2)

In this circumstance, AICAR was the TC and four differentendogenous steroids served as the ERCs: androsterone (A, 3α-hydroxy-5α-androstane-17-one), etiocholanolone (E, 3α-hydroxy-5β-androstane-17-one), pregnanediol (PD, 5β-pregnane-3α,20α-diol) and 11-ketoetiocholanolone (K, 3α-hydroxy-5β-androstane-11,17-dione). These steroids have been chosen asthey were readily implemented into the sample preparationmethod and commonly yield sufficient urinary concentrations.The complete method has been validated in accordance

with formerly established tests for IRMS measurements inthe context of sport drug testing.[19]

A reference population encompassing n=63 male and femalesport students was investigated regarding their CIRs to establishreference limits for all Δ values. This population was composedof an arbitrarily chosen subset of a larger reference population(n=499males and females) investigated for their urinaryAICARconcentrations. By means of this it was possible to calculate aconcentration-based decision limit that triggers subsequentIRMS analysis in routine doping controls.In order to test the established reference limits for both

CIRs and urinary concentrations, an excretion studyencompassing a single oral administration of 10 g AICARwas performed. Post-administration samples were clearlyinfluenced beyond the established thresholds for allparameters proving the usefulness of the novel approach todetect AICAR misuse in doping control samples.

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EXPERIMENTAL

Reagents and chemicals

tert-Butyl methyl ether (TBME, distilled before use) and ethylacetate were from KMF Laborchemie (St.Augustin, Germany),acetonitrile (ACN) from VWR (Darmstadt, Germany),

Copyright © 2014Rapid Commun. Mass Spectrom. 2014, 28, 1194–1202

β-glucuronidase from Escherichia coli from Roche DiagnosticsGmbH (Mannheim, Germany), and steroid reference materialsA, E, PD, androsterone-glucuronide (A-Gluc) and 5α-androstan-3β-ol (RSTD) were supplied by Sigma (Steinheim,Germany) together with AICAR and MSTFA (N-methyl-N-(trimethylsilyl)trifluoroacetamide). The AICAR used in theelimination study and 13C2,

15N-labeled AICAR were fromToronto Research Chemicals (Toronto, Ontario, Canada). Kand K-glucuronide (K-Gluc) were purchased from Steraloids(Newport, RI, USA). All solvents and reagents were ofanalytical grade.

Reference population

Urinary concentrations

The reference population included 499 students and employeesof the German Sport University Cologne (n= 152 females andn=347 males) aged between 19 and 59 years. All participantswere sportive and exercised up to 30 h per week. Single spoturine samples were collected in the forenoon and stored at–20 °C until analysis. No participant declared the use ofprohormones or dietary supplements, and the volunteers wereasked to list recently administered medications. The study wasapproved by the local ethical committee, and written consentwas given by all participants.

Carbon isotope ratios

The subpopulation investigated for this purpose included 63subjects (n = 27 females and n= 36 males). All specimens havebeen drawn following a protocol of simple random samplefrom the complete population and all selected samples havebeen included in the calculations of the reference limits. Thisapproach was in accordance with the guidelines of theInternational Federation of Clinical Chemistry (IFCC).[20]

The study was approved by the local ethical committee, andwritten consent was given by all participants.

Excretion study

One healthy male volunteer was orally administered 10 g ofAICAR dissolved in 750 mL of water. Single spot morningurine samples were collected 3 days and directly before theapplication. Post-administration, all urine specimens weresampled for 48 h followed by single spot morning urinecollection for another 3 days. All specimens were stored at–20 °C until analysis. The excretion studywas conducted underthe ethical approval of the Ethical Commission of the Ministryof Sport of Russian Federation (Ref. No. 302-1).

Urinary AICAR concentrations

All samples were investigated following an establishedprotocol for the determination of urinary AICARconcentrations.[13] Elevated urinary concentrations were cor-rected for specific gravity.[21]

Sample preparation for GC/C/IRMS

For valid CIR determinations the analytes have to beefficiently isolated and purified in order to avoid co-elutionof compounds and to maintain the ability to measuredifferently concentrated analytes in comparable amounts.

wileyonlinelibrary.com/journal/rcmJohn Wiley & Sons, Ltd.

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Figure 1. Structural formula of 5-aminoimidazole-4-carboxamide 1β-D-ribofuranoside (AICAR).

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Here, direct injection of urine on a high-performance liquidchromatography (HPLC) system together with subsequentfraction collection was employed.Depending on the prior determined urinary AICAR

concentration, up to 3 mL of urine were concentrated in avacuum centrifuge at 60 °C for 2.5 h, operating at 1500 rpmand a pressure below 20 mbar. The urine concentrate wastransferred into autosampler vials and, if necessary, dilutedwith water to a final volume of 180 μL. These aliquots wereforwarded to HPLC cleanup.

HPLC cleanup

In order to remove all interfering or co-eluting compounds,two consecutive HPLC fractionation steps were necessaryfor AICAR purification. Both were performed on an Agilent1100 HPLC system (Waldbronn, Germany) utilizing twodifferent columns.The first separation was performed on a BETASIL Phenyl-

Hexyl column (250 × 4.5 mm, 3 μm particle size) from ThermoFisher (Bremen, Germany) protected with a BETASIL Phenyl-Hexyl guard-column (10 × 4 mm). The gradient startedwith 100% water, held for 1.5 min, then decreased to90% water/10% ACN in 5 min, changed to 95% ACN in12 min, held for 3 min with subsequent re-equilibrationfor 8 min at 100% water. The flow rate was constant at1 mL/min and the injection volume was 60 μL.Before each batch of samples, a standard mixture

containing 4 μg of AICAR and 10 μg of A-Gluc and K-Gluceach dissolved in 120 μL of 1% aqueous urea was injectedtwice to check the system stability and to determine theretention times of the target analytes for accurate fractioncollection. Standards were detected at 270 and 195 nm.Fractions were collected with an automated fraction collectorFOXY R1 (Axel Semrau, Sprockhövel, Germany) from 5.8 to6.8 min and from 9.0 to 12.0 min containing AICAR and thesteroid glucuronides, respectively. Each sample was injectedthree times and the collected fractions were combined andevaporated at 50 °C under a stream of nitrogen.The fraction containing steroids was reconstituted in 1 mL

of phosphate buffer (pH 7), 50 μL of β-glucuronidase wasadded and the sample was incubated for 1 h at 50 °C. Aftercooling to room temperature 0.5 mL of sodium bicarbonatebuffer (pH 9.6) was added together with 5 mL of TBME toperform a liquid-liquid extraction. After shaking and centri-fugation the organic layer was transferred into a new test-tubeand evaporated to dryness under a stream of nitrogen. Thedried residues were forwarded to derivatization.The fraction containing AICAR underwent a second HPLC

cleanup. The dried residue was dissolved in 200 μL ofmethanol, transferred to an autosampler vial, evaporated todryness in a desiccator and reconstituted in 100 μL of ACNcontaining 10% (v/v) of water. Then separation wasperformed on a NUCLEODUR 100-5 NH2-RP column(250× 4.6, 5 μm particle size; Macherey&Nagel, Düren,Germany). The gradient started with 95% ACN/5% waterto 59% ACN in 12 min, to 10% ACN in 0.1 min held constantfor 6 min and then the column was re-equilibrated for 5 minat 95% ACN. The flow was 1 mL/min and the injectionvolume was 2 × 50 μL, i.e. each sample was divided intotwo separate injections. Before each batch of samples, theretention time was fixed with two injections of a standard

wileyonlinelibrary.com/journal/rcm Copyright © 2014 John Wile

containing 4 μg AICAR in 100 μL ACN/water (90/10, v/v)and the fraction containing AICAR was collected from 8.4to 9.3 min. Again the fractions were combined, evaporatedto dryness under a stream of nitrogen and forwarded toderivatization.

Derivatization of AICAR

In order to minimize the number of carbon atoms added tothe target analyte during derivatization, different strategieswere assessed. Selective derivatization of the hydroxylgroups andmaintaining the nitrogen atoms unaffected (Fig. 1)were desirable and thesewere accomplished by adding 100 μLof a solution containing ethyl acetate/MSTFA (80/20, v/v)and incubating the samples for 30 min at 70 °C. Afterevaporating the incubation mixture under a stream ofnitrogen, the residues were reconstituted in 200 μL of ethylacetate, transferred into autosampler vials and evaporated ina desiccator. Before injection onto the IRMS system theresidues were reconstituted in an appropriate amount of ethylacetate containing 20 μg/mL RSTD also as trimethylsilyl(TMS) derivative.

This protocol resulted in mono-TMS derivatives for A, Eand K, bis-TMS derivatives of PD, and tris-TMS derivativesof AICAR.

GC/C/IRMS measurements

The carbon isotope ratios were measured on a Delta V Plusisotope ratio mass spectrometer (Thermo Fisher) coupled toa Trace 1310 gas chromatograph (Thermo Fisher) equippedwith a TriPlus RSH autosampler. The systems were connectedvia a GC IsoLink CNH operated at 950 °C and a ConFlo IVinterface (both Thermo Fisher). Injections were performed insplitless mode at 280 °C with 2.5 μL of ethyl acetate. TheGC column was a J&W Scientific DB-17MS (length 30 m, i.d.0.25 mm, film thickness 0.25 μm) from Agilent (Waldbronn,Germany). The initial oven temperature was 60 °C,maintained for 2 min, increased at 40 °C/min to 265 °C, thenat 2 °C/min to 285 °C, and finally at 40 °C/min to 310 °C, andheld for 2 min before cool-down to the initial conditions. Thecarrier gas was helium (purity grade 5.0) with a constant flowrate of 1.5 mL/min. Isodat 3.0 software (Thermo Fisher) wasused for data acquisition and evaluation.

GC/TOFMS measurements for specificity

During method development each aliquot was re-injectedinto an Agilent 7200 Accurate-Mass quadrupole time-of-flight(Q-TOF) system coupled to an Agilent 7890A gas chroma-tograph (Santa Clara, CA, USA) to ensure peak purity andto enable identification of possible co-eluting compounds.Injections were performed in splitless mode at 280 °C withan injection volume of 0.5 μL. The same GC column and an

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identical temperature program to that mentioned above wereused with a reduced flow rate of 1.2 mL/min helium (puritygrade 4.6). Data was acquired in TOFmode over a range fromm/z 50 to 800 and evaluated with Mass Hunter software(version B.06, Agilent).

Correction for the trimethylsilyl moieties

During derivatization carbons with a different CIR areincorporated into the analyte. In order to compare themono-, bis- and tris-TMS derivatives, it was necessary tocorrect the obtained δ13C values for the TMS moieties. Herethe simple mass balance formula (Eqn. (3)) should besufficient as no kinetic isotope effects are expected for TMSderivatization.[22,23]

ncdδ13Ccd ¼ ncδ13Cc þ ndδ13Cd (3)

with n=number of moles of carbon, c = compound of interest,d =derivative group, and cd=derivatized compound.For steroidal compounds this derivatization technique

proved to be suitable.[24,25] Nevertheless, in addition topossible isotopic fractionation occurring during the formationof derivatized compounds, their combustion to CO2 shouldalso be considered as a source of biased results. Siliconentering the combustion tube will not only form siliconoxides, but also silicon carbide, retaining carbon atomsmainly derived from the TMS group.[26] Taking this intoaccount, Eqn. (3) should be modified as:[27]

ncdδ13Ccd ¼ ncδ13Cc þ ndδ13Cdcorr (4)

As the δ13Cd value is not known, the δ13Cdcorr value isestimated empirically by consecutive measurements of boththe native and the derivatized compound. Subsequently, thevirtual δ13C value for the TMS moiety can be determinedand utilized for correction. While steroids can be measuredboth underivatized and as TMS derivatives on the GC/C/IRMS system this was not possible for AICAR. Therefore,the CIR of native AICAR was determined by an elementalanalyzer/IRMS system: an EA 1110 elemental analyzer(CE Instruments, Milan, Italy) coupled to a MAT 253isotope ratio mass spectrometer (Thermo Fisher) via aConFlo IV interface.In order to estimate the influence of incomplete combustion

on δ13C-AICAR values, a standard addition approach using13C2-labeled AICAR was used.[28] By comparing the measuredδ13C values for the TMS derivatives with the calculated CIRsfor AICARmixedwith isotopically labelled AICAR at differentlevels (here ranging from –5 to +16‰) a statistical value for theCO2molecules derived from carbon atoms from the TMS groupduring combustion can be calculated.

Linear mixing models

In addition to the careful investigation of possible isotopicfractionation occurring during derivatization or combustion,the complete method including all steps of sample preparationwas also investigated for its influence on the measured CIR.The linear mixing model approach utilized here was, inaccordance with investigations on urinary steroids, a variationof a Keeling plot.[29,30] By means of a two-pool mixing modeltogether with the above-mentioned mass balance formula it is


possible to detect and estimate isotopic fractionationsassociated with sample preparation. The detailed procedurewas published earlier.[19,31]

In brief, two batches of a blank urine containing 220 ng/mLof endogenous AICAR were fortified with 0, 200, 400, 600,1000, and 1500 ng of AICAR, processed and measured.

As the sample preparation and the measurements forthe two batches were performed on different days, it waspossible to test for both repeatability and reproducibility.If isotopic fractionation were to occur during samplepreparation, it would be observable because the line ofbest fit would not intercept the y-axis by a numericalvalue representing the δ13C value of the added standard.By calculating the least-squares fit, it is possible toevaluate standard deviations (SDs) for the describedmethod, if it is assumed that errors for the concentrationsare negligible.

Blank urine

A pooled urine sample was collected over a period of 24 h(total amount of 1 L) from a healthy male volunteer whodeclared that he did not use any prohibited substances orany nutritional supplements. The urine was aliquoted to50 mL portions and stored at –20 °C until analysis. Duringthe sample preparation for reference values, the blank wasprocessed with every batch resulting in a total of eightindependent preparations and measurements providing dataabout the long-term repeatability and stability of the method.In addition, the urine was used to conduct the experimentsconcerning the linear mixing model.

Statistical analyses

All the δ13C values, Δ values and urinary concentrationsobtained for the reference population were tested forGaussian distribution using the Shapiro-Wilk test at asignificance level of p≤0.05. As a log-normal distributionwas expected for urinary concentrations, the values wereconverted accordingly. Reference limits were calculatedparametrically by adding the 3-fold standard deviation tothe mean value of each distribution. This treatment is inaccordance with the requirements of the InternationalFederation of Clinical Chemistry (IFCC).[20,32]

The lines of best fit were determined with the Gaussianmethod of least squares; generalized linear models wereused to test for significant correlations within the refe-rence population.

RESULTS AND DISCUSSION

Method

While CIR determinations of urinary steroids are wellestablished in sports drug testing, the investigations intoAICAR were new territory in doping controls. Therefore,possible factors influencing the reliable measurement ofAICAR have been investigated in more detail than thoseof steroids.


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Sample preparation

As all structural information is lost during combustion ofanalytes in IRMS measurements, the clean-up proceduremust be highly effective to ensure peak purity and to avoidco-elutions from the GC column. Chromatograms of thestandards and urinary extracts are shown in Fig. 2, todemonstrate the efficiency of the developed method. Whilethe employed second HPLC cleanup for AICAR wasnecessary to achieve the well-separated TC, this effort wasnot spent on the steroids used as ERCs. Subsequently, thechromatograms here are not as pure; however, this had noinfluence on the obtained δ13C values as revealed bycomparison of the data with formerly obtained values onreference populations (vide infra).

Specificity

The specificity of the method was ensured by GC/TOFMSmeasurements. The mass spectrum of a tris-TMS AICARstandard is depicted in Fig. 3. During method development,urinary AICAR extracts were forwarded for GC/TOFMSmeasurements after CIR determination to check peak purityand to exclude possibly underlying co-elutions. The referencepopulation samples were only re-injected if co-elutions couldnot be excluded by the IRMS chromatogram.

Figure 2. GC/C/IRMS chromatogramsAICAR_TMS and RSTD_TMS and a purmixture containing RSTD_TMS, A_TMS,purified urine sample.


Correction for the TMS moieties

As mentioned above, the derivatization itself might notnecessarily cause issues when using MSTFA. It is more likelythat the incomplete combustion of TMS derivatives wouldresult in retained carbon from the TMS moiety within thecombustion furnace. The used standard addition approachdemonstrated that statistically 8.7 carbon atoms of the 9added carbon atoms were combusted to CO2 as the resultingline of best fit had a slope of 0.5203 instead of 0.5. Taking intoaccount the calculated measurement uncertainty of ±0.01775for the slope, the determined value of 0.5203 is not statisticallydifferent from the intended value of 0.5 (t-test, p≤0.05).Consequently, systematic errors introduced by incompletecombustion of AICAR-TMS can be excluded. A possibleinfluence on the CIR values, if any, was highlyreproducible as demonstrated hereafter by a linear mixingmodel and incorporated in the threshold calculations bymeansof the reference population.

Linear mixing model

The approach of the linear mixing model had proved to besuitable for testing the validity of an IRMSmethod several timespreviously and it was thus also applied to AICAR in the presentstudy. The key figures obtained were –18.4± 0.55 ‰ for theslope of the line of best fit and –4.6± 0.33‰ for the y-intercept.

of (a) a standard mixture containingified urine sample and (b) a standardE_TMS, PD_TMS and K_TMS and a


Figure 3. High-resolution EI mass spectrum of tris-trimethylsilylated AICAR (M+=m/z 474). The annotatedchemical compositions reflect possible fragments with a masserror <5 ppm.

Table 1. Results obtained for the blank urine duringreference population measurements (n = 8). All values inδ13CVPDB [‰]

AICAR A E PD K

Mean –21.5 –22.5 –23.5 –22.7 –23.7SD 0.77 0.36 0.31 0.44 0.47


The slight deviation of the intercept from the assigned value forthe AICAR standard (–5.2±0.2) might be due to the above-mentioned carbon retention during the combustion process.Overall, however, themixingmodel revealed no systematic biaspotentially introduced by the sample preparation, as illustratedin Fig. 4.

Repeatability of the blank urine test results

One blank urine sample was prepared with every batch ofsamples belonging to the reference population determi-nations, resulting in a total of eight preparations over a timeperiod of 4 months. The results are listed in Table 1 and allanalytes could be determined with excellent SDs (<0.5 ‰).The slightly larger SD found for AICAR is attributed to thenecessary correction for the TMS moiety. The TMS derivativeitself could be measured with a SD of ±0.38 ‰.

Reference population

AICAR concentrations

To date only one study has reported on endogenous urinaryconcentrations of AICAR in healthy and active volunteers.[13]

Regular doping control samples were analyzed for theirAICAR content after the absence of any other doping agenthad been ascertained. This procedure always entails risks as

Figure 4. Linear mixing model for AICAR; the determinedδ13C values (CIR) are plotted against the ratio of endogenousAICAR concentration divided by the concentration of themixture (Ce/Cm). The open squares represent the firstsample preparation, the grey diamonds the second one andthe open circle the value determined for the standard. Thesolid line represents the best fit.


no further information on the subpopulation is availableand as it cannot entirely be excluded that drugs such asAICAR have been misused by some of the athletes.

The reference population investigated herein showedsystematically lower urinary AICAR concentrations as formerlyreported with a mean value of 632±362ng/mL. In agreementwith earlier data, significantly different concentrations (t-test,p<0.001) were found for men (682±367ng/mL) and women(519±321ng/mL). Further investigations also showeddifferences between different kinds of sports but all these wereinsignificant after taking into account the gender difference.Interestingly, a small but significant (generalized linear model,p<0.01) increase in AICAR concentrations with increasingquantities of hours exercised per week was detected. Thisrelationship cannot explain the difference found betweenthe two populations (sport students vs athletes) but mightgive a hint for further studies on AICAR excretion and itssurrounding metabolism.

Despite these observed differences, one threshold (ratherthan various different ones) for urinary AICAR is suggestedfor reasons of simplification. In accordance with the CIR data,a 99.7% reference limit at 3500 ng/mL is proposed anddoping control samples above this value should be subjectedto the established IRMS approach.

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Carbon isotope ratios of AICAR

The reference limits established by means of referencepopulation investigations have the advantage that calculatedmean values and their SDs include all potentially influentialfactors. These factors can originate, amongst others, fromsample preparation and measurement, and include a possiblebias resulting from the TMS derivatization. The computedthresholds described herein are fit-for-purpose and allow forthe differentiation between endo- and exogenous AICAR.

The absolute δ13C values of both the TC AICAR and thesteroids serving as the ERC are listed in Table 2. While thesteroidal analytes show the expected range for an Europeanpopulation,[33] the CIRs of AICAR cover an unexpectedlylarge range. The widespread distribution cannot be attributedonly to the larger SD found in AICAR measurements asdemonstrated for the repeated preparation of the sameurine sample within the reproducibility test (Table 1).As the steroid analyses yielded the expected values thatfit perfectly into distributions of reference populationsinvestigated with different methods,[19,33–37] the derivatizationusing MSTFA can also not be responsible for the largerscattering. Hence, it can be hypothesized that AICAR itselfhas a larger inter-individual variance, potentially due to itsposition within the high turnover purine biosynthesis pathway.Since the carbon atomswithin theAICARbackbone are derived


9

Table 2. δ13CVPDB values of the investigated referencepopulation (n = 63). Listed are the mean values, thestandard deviations (SD) and the ranges

AICAR A E PD K

Mean –20.9 –22.9 –23.9 –22.9 –23.1SD 1.96 0.72 0.67 0.73 0.73Min –25.8 -24.8 –25.2 –24.5 –24.7Max –16.2 –21.5 –22.4 –21.2 –21.8

Table 4. Δ values of the investigated reference population(n = 63) and resulting reference limits (mean+ 3SD) for thesteroids among themselves

A-PD A-K E-PD E-K

Mean 0.04 0.36 �0.97 �0.64SD 0.71 0.68 0.71 0.58RefLim 2.2 2.4 1.2 1.1

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from ribose, carbon dioxide, glycine and folic acid,[38] differentsources with possibly different CIRs can contribute to theoverall δ13C values measured for AICAR. Moreover, otherfactors that might have an impact on AICAR biosynthesis andexcretion, such as the observed tendency towards a correlationbetween urinary AICAR concentrations and the extent ofphysical activity, might also affect AICAR CIRs. Here furtherstudies seem appropriate and advisable.No significant gender-specific difference was found for the

CIRs of AICAR and the measured steroidal ERCs.

Reference limits

As all the calculated Δ values were found to be Gaussian-distributed, the 99.7% reference limits could be calculatedby adding the threefold standard deviation to the meanvalue.[20] The results are listed in Table 3. Due to theaugmented standard deviation of the AICAR values theresulting thresholds were also elevated. Taking into accountthe CIR of exogenous/synthetic AICAR measured so far,these comparably high limits should serve the intendedpurpose. Two different standards from Sigma had measuredvalues of –5.2 ‰ and –3.3 ‰, one from TRC (used for theadministration study) had a measured value of –3.4 ‰, andtwo black market products had values of –3.5 ‰ and –3.7 ‰(n=6, SD±0.3 ‰), i.e., all these products were more than 10 ‰enriched in 13C compared with the endogenous values forAICAR found within the reference population.Considering only data for the steroids (Table 4) revealed

again that the method using TMS derivatives cannot beresponsible for the increased SD. Here the Δ values are inperfect agreement with already published data.[34–36]

The applicability of the calculated thresholds waseventually demonstrated in the course of an AICARadministration study.

Table 3. Δ values of the investigated reference population(n= 63) and resulting reference limits (mean+ 3SD) forAICAR vs all ERCs

AICAR -A

AICAR -E

AICAR -PD

AICAR -K

Mean 1.94 2.94 2.06 2.40SD 2.11 2.04 1.90 1.98RefLim 8.3 9.1 7.8 8.4


AICAR administration study

Due to its low bioavailability after oral administration, lowdoses of AICAR would most probably not result in increasedserum concentrations.[9] Hence, the dosage was adjustedaccordingly within this excretion study and the results arepresented in Fig. 5. As expected, the urinary concentrationsrise rapidly after ingestion of 10 g of AICAR and reach themaximum of approximately 9800 ng/mL after 8 h. Fluctuationsin AICAR concentrations post-administration were attributedto a high diuresis causing comparably dilute urine samples.Nevertheless, suspicious levels (>3500 ng/mL) were foundfor approx. 20 h after administration followed by a slow declineback to basal values ranging from 400 to 900 ng/mL.

The CIR of AICAR (Fig. 5, bottom) showed a more distinctpattern of influence directly after the oral administration ofthe therapeutic agent. Already after 15 min the δ13C values

Figure 5. Effects of 10 g of AICAR administered orally att = 0. Top: Urinary AICAR concentrations. Bottom: δ13Cvalues (CIR) of AICAR (grey triangles) and androsterone(black circles). The horizontal dashed lines represent therespective proposed thresholds.


Table 5. δ13CVPDB values of samples with increasedurinary concentrations of AICAR

sample AICAR A AICAR-A

1 –20.4 –21.4 1.032 –20.1 –22.2 2.113 –22.5 –22.4 –0.034 –22.4 –21.7 –0.665 –23.0 –23.1 0.106 –21.1 –22.2 1.117 –21.3 –22.4 1.088 –22.2 –22.3 0.079 –20.3 –22.5 2.2310 –19.8 –22.9 3.0411 –20.3 –22.5 2.22


of urinary AICAR (–4.0 ‰) were close to the CIR of theadministered substance (–3.4 ‰). Obviously, and in accor-dance with theory, the low specific gravity of the urine hadno influence on the measured CIR. The maximum valueof –3.1 ‰ was reached 2 h post-administration. Withinthe measurement uncertainty of the method, this resultreflects the value of the administered AICAR. Interestingly,after 24 h, an additional small peak of 13C-enriched AICARwas visible, which was not reflected by an increasedurinary concentration. The reason for this is currentlyunknown as further factors influencing the CIR of AICARhave not yet been studied. Fluctuation caused by samplepreparation or the like can be excluded as all samples wereprocessed and analyzed twice yielding similar results.Considering the suggested thresholds, Δ values beyond the

reference limit were found for more than 40 h. Similar todoping controls concerning natural/endogenous steroids,IRMS analyses can provide a prolonged detection windowfor exogenous AICAR in comparison with relying solely onurinary concentrations alone.These results, even if only obtained as an exemplarily

conducted excretion study with only one volunteer, clearlydemonstrate the potential of CIR to detect AICAR misuseand to confirm its exogenous source.

Doping control samples

Until now, 11 samples with elevated urinary AICAR concen-trations (between 2000 and 3300 ng/mL) have been investigatedwith the developed method. All these samples yielded clearlyendogenous δ13C signatures for both TC and ERC (Table 5).The Δ values fit perfectly to those from the reference populationwith a mean of 1.1±1.2 ‰. In contrast to older studies,[13] nosamples were found beyond the concentration limit of3500 ng/mL within the investigated period.[39]

120

CONCLUSIONS

As for all other endogenously produced substances relevantto doping controls, it is of utmost importance to stipulaterelevant decision limits that provide both high sensitivityand high specificity. The considerable inter-individualvariability found especially for urinary concentrations often


impede this approach. Consequently, in accordance with themanagement of testosterone in sports drug testing, it wasnecessary to develop a confirmation procedure for AICARthat can unambiguously distinguish between endogenousand exogenous sources of the analyte.

The developed method presented herein allows for simul-taneous determinations of CIR of AICAR and four differentsteroids serving as ERCs. The method was fully validatedand the calculated reference limits can serve as decision limitsto distinguish between endogenous and exogenous sources ofurinary AICAR. The threshold calculated for urinary con-centrations is intended to be used as a decision limit to screendoping control samples. Those found with elevated con-centrations should be forwarded to IRMS.

Further studies will be necessary to investigate factorsinfluencing both AICAR concentrations and isotope ratios toelucidate the sources of the higher variability found in theCIRs. In addition, more available reference material and blackmarket products should be investigated to verify the uniformityin CIRs found so far for all products.

AcknowledgementsThis project was partly funded by the Federal Ministry of theInterior of the Federal Republic of Germany, the World Anti-Doping Agency (Grant #12A2MT), and the Irish SportsCouncil (Dublin, Ireland).

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