PII: S0003-2670(02)00199-X - CBD · 2004. 7. 30. · Title: PII: S0003-2670(02)00199-X Created Date: 5/18/2002 2:16:51 PM

Analytica Chimica Acta 460 (2002) 289–307

Strategies for internal quality control in antidoping analyses

Cintia Jiméneza,b, Rosa Venturaa,b,∗, Xavier de la Torrea,b, Jordi Seguraa,ba Unitat de Farmacologia, Institut Municipal d’Investigació Mèdica (IMIM),

Doctor Aiguader 80, 8003 Barcelona, Spainb Universitat Pompeu Fabra, Barcelona, Spain

Received 28 May 2001; received in revised form 26 November 2001; accepted 12 March 2002

Abstract

Internal quality control (IQC) is one of the most important elements contributing to quality assurance in the laboratory.In this study, the strategy for the implementation of an IQC program to monitor performance of the analytical proceduresused in an antidoping control laboratory is presented. Different IQC parameters have been defined according to the aim ofthe method (qualitative or quantitative, screening or confirmation). They are based on the analysis of control and calibrationsamples in each analytical batch and on the use of an internal standard in chromatographic methods. IQC parameters forchromatographic and immunological methods and the acceptance criteria used to check the quality control data obtained aredescribed and discussed. These IQC procedures have been applied during routine antidoping analyses of more than 5000samples per year in a laboratory accredited by the International Olympic Committee (IOC) and meeting the requirements ofthe quality standard ISO 17025. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Internal quality control; Antidoping analysis

1. Introduction

Results obtained in antidoping control laboratoriesare used to take decisions that can severely affectthe athlete’s career and it is, therefore, of paramountimportance to ensure a high level of quality in theanalytical results. In agreement with National andInternational Sports Federations’ requirements, lab-oratories have to be accredited according to interna-tional quality standards. Laboratories have to pass anannual re-accreditation by the International OlympicCommittee (IOC) and, in addition, ISO accreditationas a testing laboratory is a pre-requisite for the IOCaccreditation beyond the year 2000[1].

∗ Corresponding author. Tel.:+34-93-221-10-09;fax: +34-93-221-32-37.E-mail address: [email protected] (R. Ventura).

Internal quality control (IQC) is one of the mostimportant steps to ensure the quality and reliability ofthe analytical results. The first step is the use of meth-ods validated to guarantee that the requirements for aspecific intended use are fulfilled. Furthermore, cal-ibration of equipment, quality of reference materialsand reagents, training of personnel and environmentalconditions have to be adequate to achieve the objec-tives of quality proposed. The second level of qualityassurance is the implementation of an IQC program tomonitor performance of the procedures when they areused during routine work. Finally, the highest level ofquality assurance is the participation in external qual-ity assessment programs[2–5].

IQC has been defined as the set of procedures under-taken by laboratory staff for the continuous monitor-ing of the operation and the results of measurements inorder to decide whether the results are reliable enough

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S0003-2670(02)00199-X

290 C. Jimenez et al. / Analytica Chimica Acta 460 (2002) 289–307

to be released[6]. Abundant literature can be foundon quality control in the clinical laboratory[7–12],but since most of the analytical methods applied inthese laboratories are quantitative, recommendationsare commonly addressed to these type of assays. Theuse of Shewhart’s control charts is the most extendedprocedure to establish an IQC system for quantitativemethods[7–9]. The most important control rules usedfor the interpretation of these control charts were for-mulated by Westgard. They are based on the use ofcontrol limits. In general, the control limits set at±2S.D. (S.D. = standard deviation) from the mean valueare indicative of unacceptable trends in assay perfor-mance and those set at±3 S.D. are indicative of sig-nificant assay problems. Recommendations for IQChave been also proposed in different areas of toxico-logical analysis[13–19].

In antidoping control, mainly qualitative methodsare used for screening and confirmation purposes.Contrary to quantitative methods, few studies areavailable on IQC procedures for qualitative assaysand the existing recommendations have a limitedscope and are mainly addressed to the composition ofthe analytical batch[10,12].

In this study, the implementation of IQC proce-dures in qualitative and quantitative methods usedin the Barcelona antidoping control laboratory isdescribed. Quality control parameters to identify dif-ferent sources of errors for each type of analyticalprocedures have been studied.

2. Antidoping control analytical procedures

The list of prohibited classes of substances, prohi-bited methods and classes of drugs subjected to cer-tain restrictions according to the IOC is presented inTable 1 [20]. Stimulants, narcotics, anabolic agents,diuretics and peptide hormones, mimetics and ana-logues are included. For each one of the classes thereis a list of representative examples, but other com-pounds with similar chemical structure or pharmaco-logical activity are also prohibited. For some specificcompounds, for which it is difficult to distinguish be-tween the social or therapeutic use and the misuse, orto differentiate between physiological concentrationsand exogenous administration, cut-off concentrationshave been established (Table 1).

Table 1IOC list of classes of prohibited substances and methods of dopingand summary of urinary concentrations above which IOC-accre-dited laboratories must report findings for specific substances[20]

(I) Prohibited classes of substances(A) Stimulants(B) Narcotics(C) Anabolic agents

(1) Anabolic androgenic steroids(2) �2-Agonists

(D) Diuretics(E) Peptide hormones, mimetics and

analogues

(II) Prohibited methods(A) Blood doping(B) Pharmacological, chemical and

physical manipulation

(III) Classes of drugs subject to certainrestrictions

(A) Alcohol(B) Cannabinoids(C) Local anesthetics(D) Corticosteroids(E) �-Blockers

Caffeine >12�g ml−1

Carboxy-THC >15 ng ml−1

Cathine >5�g ml−1

Ephedrine >10�g ml−1

Epitestosterone >200 ng ml−1

Methylephedrine >10�g ml−1

Morphine >1�g ml−1

19-Norandrosterone >2 ng ml−1 in males>5 ng ml−1 in females

Phenylpropanolamine >25�g ml−1

Pseudoephedrine >25�g ml−1

SalbutamolAs stimulant >100 ng ml−1

As anabolic agent >1000 ng ml−1

T/E ratio >6

Antidoping control is performed using screeningand confirmation methods. Several screening meth-ods, addressed to the detection of a wide number ofcompounds and/or metabolites with similar physic-ochemical properties, are applied. If the presenceof a compound is suspected, a confirmatory test,specific for the compound detected, is applied. Theconfirmatory test should be at least as sensitive andpreferably more specific than the screening test. Massspectrometry (MS) is mandatory for confirmation pur-poses, whenever possible. Criteria to demonstrate the

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Table 2Summary of screening procedures used in human doping control in Barcelona

Procedure Detected substance Number ofanalytes

Sample preparation Instrumentalanalysis

A Stimulants, narcotics,local anesthetics

73 Alkaline extraction (t-butyl-methylether) and salting-out effect

GC/NPD, GC/MSD

B Anabolic steroids (free+ conjugated),�2-agonists,narcotics (morphine, buprenorphine),stimulants (ethamivan, pemoline),diuretics (triamterene), THCmetabolite, probenecid

73 Sample clean-up GC/MSDEnzymatic hydrolysisAlkaline extraction witht-butyl methyl etherDerivatization withMSTFA/NH4I/2-mercaptoethanol

C Anabolic steroids (free in urine),stimulants (mesocarb), diuretics(amiloride)

12 Alkaline extraction (ethylacetate) and salting-out effect

GC/MSD

Derivatization withMSHFB/TMSIm and MBHFB

D Anabolic steroids (free+ conjugated),�2-agonists

11 Same as procedure B GC/ITD (MS/MS)

E Diuretics, probenecid 22 Alkaline extraction (ethylacetate) and salting-out effect

GC/MSD

Derivatization with methyl iodide

F Clenbuterol,�-adrenergic agonistsand antagonists

– Incubation with theanalyte–enzyme conjugate

ELISA

Washing with bufferIncubation with enzyme substrate

G Cocaine metabolite, cannabinoids, opiates – No sample preparation is required FPIA

H Hormones (�-Hcg, LH) – No sample preparation is required MEIA

presence of prohibited substances using chromatogra-phy coupled to MS have been proposed by differentorganizations[19,21].

The analytical methodology used in antidopinganalyses has been extensively described[22–29].The techniques used for screening and confirmationpurposes depend on each specific group of com-pounds. Physicochemical properties of the analytesand the sensitivity and specificity required are themost important factors to take into account. In gen-eral, capillary gas chromatography (GC) coupled tonitrogen–phosphorus detector (NPD) or to mass se-lective detector (MSD) is the most used technique.High performance liquid chromatography (HPLC)with variable wavelength ultraviolet (UV), diode ar-ray detector (DAD) or MSD is also used in somelaboratories for specific compounds. Due to the highrequirements in sensitivity and specificity, GC cou-pled to high resolution MS (HRMS) or to tandem

MS (MS/MS) has also been used for anabolic agents[30–33]. A selection of the screening methods usedin our laboratory for antidoping control in athletesand a summary of the sample preparation procedureis presented inTable 2. For screening purposes, im-munological methods (enzyme-linked immunosorbentassay (ELISA), fluorescence polarization immunoas-say (FPIA) and microparticle enzyme immunoassay(MEIA)) are also applied (Table 2) [26–28,34].

3. Internal quality control in Barcelonaantidoping laboratory

In addition to instrumental analysis, analytical pro-cedures involve many different steps during samplepreparation (hydrolysis, liquid–liquid or solid phaseextraction, derivatization, etc.) (Table 2), thus increas-ing the number of possible sources of error. As an


Table 3List of factors affecting the quality of the analytical results

Variable IQC parameters

Sample preparation

Aliquoting of samples Pipetting precision/accuracy Concentration values of PCU (inquantitative methods)

ISTD Pipetting precision/accuracy Detector signal (area/height) of ISTDConcentration accuracy

Hydrolysis pH adjustment accuracy Hydrolysis rate of a conjugated standard in PCUEnzyme stability and efficacyIncubation time/temperature precision/accuracy

Reagents contamination Absence of interfering peaks in negativecontrol samples (NCU, REBL)

Extraction pH accuracy Detector signal (area/height) of ISTD andtarget compounds in PCU

Reagents contamination Absence of interfering peaks in negativecontrol samples (NCU, REBL)

Evaporation Water bath N2 pressure/temperatureprecision/accuracy

Detector signal (area/height) of ISTD and targetcompounds in PCU

Derivatization Reagents stability/volume precision/accuracy Detector signal (area/height) of ISTD and targetcompounds in PCUIncubation time/temperature precision/accuracy

Reagents contamination Absence of interfering peaks in negative controlsamples (NCU, REBL, DEBL)

Instrumental conditions

GC

Injector Injection volume precision/accuracy Detector signal (area/height) of ISTD and targetcompounds in PCUInjector suitability (reproducibility, linearity)

Injector temperature Peak shapes of ISTD and target compounds in PCU

Carry-over Injection orderAbsence of interfering peaks in negative controlsamples (NCU, REBL, DEBL, SOBL)

Column Column performance (capacity,resolution, retention)

RTs of ISTD and target compounds in PCUPeak shape/resolution of ISTD and targetcompounds in PCU

Carrier gas Flow rate accuracy RTs of ISTD and target compounds in PCU

Split Split rate accuracy Detector signal (area/height) of ISTD and targetcompounds in PCU

Temperature program System heating/thermostattingperformance (accuracy, precision,stability, ramping characteristics)

RTs of ISTD and target compounds in PCU

Detector Detector performance (output,response, background noise, drift,selectivity, linearity)

Detector signal (area/height) of ISTD and targetcompounds in PCUS/N ratio of ISTD and target compounds in PCU


Table 3 (Continued )

Variable IQC parameters

Instrumental conditions

HPLC

Injector Injection volume precision/accuracy Detector signal (area/height) of ISTD and targetcompounds in PCUInjector suitability (reproducibility, linearity)

Carry-over Injection orderAbsence of interfering peaks in negative controlsamples (NCU, REBL, DEBL, SOBL)

Column Column performance (capacity,resolution, retention)

RTs of ISTD and target compounds in PCUPeak shape/resolution of ISTD and targetcompounds in PCU

Mobile phase Composition suitability RTs of ISTD and target compounds in PCUSolvents stability Peak shape/resolution of ISTD and target

compounds in PCUMobile phase delivery system(precision, accuracy, pulse-free)Flow rate precision/accuracyGradient

Detector Detector performance (output, response,background noise, drift, selectivity, linearity)

Detector signal (area/height) of ISTD and targetcompounds in PCU

Wavelength accuracy (DAD) S/N ratio of ISTD and target compounds in PCU

example, a list of possible sources of error in differ-ent steps of chromatographic procedures is given inTable 3. Due to the large number of variables involved,it is difficult to identify all specific causes for analyti-cal errors. An IQC program based on a detailed check-ing of individual error sources could be difficult toimplement and can become quite inefficient and verytime-consuming. For this reason, a general IQC pro-cedure with few parameters to control a wide numberof variables has been chosen.

3.1. General strategy for internal quality control

IQC procedures implemented in the laboratory arebased on the use of control and calibration sampleswhich are included in the analytical batch and treatedin the same way as the test samples. In chromato-graphic methods, they are also based on the use of in-ternal standards (ISTDs). The verification of differentparameters in the control and calibration samples andthe assessment of the ISTD peak in each sample ofthe analytical batch allow to control the most relevantpossible sources of error described inTable 3.

The composition of the analytical batch dependson the aim of the analytical procedure (qualitative orquantitative, screening or confirmation) and the type

of target analytes. Control and calibration samplesincluded in some screening and confirmation proce-dures used in our laboratory are listed inTable 4. Forqualitative procedures, only control samples (posi-tive or negative) are used. For quantitative methods,calibration samples (CAL) are also included in eachanalytical batch and control samples are used toverify the calibration. In screening procedures, testsamples are processed in batches of 20–25 samplesand the analytical batch includes a minimum of onepositive and one negative control sample, normally anegative control urine (NCU) and a positive controlurine (PCU) (Table 4). For confirmatory analyses, ifonly identification is required (confirmation of ben-zoylecgonine and ecgonine methyl ester inTable 4)the batch consists of the sample of interest, a reagentsblank (REBL), a NCU sample and a PCU sample.

PCU samples are prepared using urine as samplematrix and the analytes are added at a concentrationcompatible with the expected range after therapeu-tic doses or with the clients or official requirements.To ensure that adequate sensitivity is achieved inqualitative procedures, positive control samples withconcentrations of the analytes slightly higher than therecommended limits of detection[35] are used. Alist of the composition of CAL and PCU samples for

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chromatographic screening procedures used in our lab-oratory is presented inTable 5. Furthermore, in someprocedures, PCU samples at different concentra-tion levels of some analytes are used (i.e. PCU1-B,PCU2-B and PCU3-B in procedure B;Table 5). Cal-ibration samples are also prepared using urine asa matrix except for the calibration of endogenouscompounds, which is performed using a calibrationsample prepared using water as a matrix (see CAL1-Bfor calibration of endogenous steroids in procedureB; Table 5).

The sequence of analysis is also defined for eachscreening and confirmation procedure (Table 4). Inscreening procedures, IQC samples are always in-jected at the beginning of the sequence before testsamples. For confirmatory analyses, in identificationprocedures the sample to be confirmed is alwaysinjected after negative control samples and beforeany positive control sample to prevent contamination

Table 5Composition of control and calibration samples used in screening procedures in human antidoping control

[21]. In quantitative confirmation procedures withseveral calibration samples, aliquots of the sampleto be confirmed are also injected after a negativecontrol sample, except for quantitation of the testos-terone/epitestosterone (T/E) ratio (Table 4). In thiscase, due to the fact that measurement of T/E ratioin urine may be influenced by several instrumentalfactors, the sequence of injection of calibration andtest samples recommended by previous authors toavoid differences due to instrumental parameters ispreferred[36].

3.2. Internal quality control parameters forchromatographic methods

IQC parameters and their acceptance criteria, de-fined for some of the screening and confirmation chro-matographic procedures used in our laboratory arelisted inTable 4.


Table 5 (Continued )

3.2.1. Internal standardIn chromatographic methods, an ISTD is added to

the sample at the beginning of the analytical proce-dure to compensate for sample losses occurring duringextraction, clean-up, derivatization and final chro-matographic analysis. Ideally the ISTD should be asclose as possible to the analytes. However, due to thehigh number of compounds analyzed in most proce-dures and the wide differences in chemical structures

between some of them, a compromise had to beachieved in the selection of the ISTDs (Table 4).Deuterated ISTDs are preferably used in GC/MSD,especially for quantitative purposes (screening proce-dure B and confirmation of T/E ratio;Table 4).

The evaluation of the ISTD (peak shape, peak re-tention time (RT) and chromatographic signal (peakarea/height)), provides a control of each test sample.Based on experience and also depending on different


factors, such as the instrumental technique (HPLCor capillary GC), the oven temperature program inGC analyses and absolute RT values, among others,acceptance ranges from±0.1 to 0.3 min have beendefined for the ISTD RT in each procedure. Further-more, a minimum peak area/height for the ISTD sig-nal has been established to ensure that adequate limitsof detection are attained (Table 4). Some of the vari-ables affecting preparation and instrumental analysisof samples controlled through the ISTD are listed inTable 3.

Resemblance between the ISTD injections made atthe start and at the end of the chromatographic batchis also checked. Differences in peak shapes betweensamples are indicative of a change in the chromato-graphic column performance during the analysis.

3.2.2. Negative control samplesNegative control samples are samples free from

the substances of interest, which are used to checkfor potential sources of contamination and to ver-ify the selectivity/specificity of the analytical proce-dure. Different negative control samples used are listedbelow.

• Solvent blank (SOBL): is the same solvent as usedto clean the injection syringe or to reconstitute thedry extract before chromatographic analysis, whenno derivatization step is used (e.g. methanol oracetone).

• Derivatization blank (DEBL): is the derivatizationreagent(s).

• REBL: is a sample of water processed as a testsample.

• Blank urine (NCU): is a urine free from the an-alyte(s) of interest, which is processed as a testsample.

The different negative control samples are used tocontrol the absence of contamination or interferencesin those parts of the procedure where they are involved(chromatographic system, derivatization reagents, ex-traction reagents or sample matrix). In all cases, theabsence of interfering peaks at the RT of the analyte(s)is evaluated (Table 4). The type of negative controlsamples included in the analytical batch depends onthe type of analytical procedure. Although for rou-tine screening procedures only a NCU sample is reallyneeded, a DEBL or a SOBL is additionally used in our

laboratory. For confirmation methods and for methodsnot frequently applied, a REBL is also included.

As indicated before, the position of the negativecontrol samples in the injection sequence is alsoimportant and a negative control sample is alwaysinjected before test samples to control a possiblecarry-over effect (Table 4).

3.2.3. Positive control samplesPositive control samples are prepared in the labo-

ratory by spiking blank samples with the analytesof interest, following the instructions described inthe corresponding standard operating procedures(SOPs). Control samples are prepared at large vol-umes, aliquoted and stored at−20◦C until requiredfor analysis. The control of preparation of a new batchis performed by analysis in parallel of aliquots of thenew and the old batch. For qualitative analytes, thesignal-to-noise (S/N) ratio is verified; additionally,for quantitative analytes the mean value of concentra-tion is calculated after analysis of several replicates(N = 20 in different analytical batches). This value isalso used to define the acceptance range of the controlsample.

In some cases, where reference materials (parentcompounds or metabolites present in urine) are notcommercially available, quality control samples areobtained from excretion studies after the administra-tion of the drug to healthy volunteers. The excretionstudies are performed using a clinical protocol ap-proved by the corresponding Ethical Committee. SOPsdescribing how to obtain these samples and the qualitycontrol procedures used to check their adequacy arealso available. These samples are verified by the anal-ysis with the suitable procedure and comparing theresults obtained with those described in the literature.

Another type of positive control sample, the deriva-tization standard (DEST) is obtained by direct deriva-tization of the analytes. DEST is important speciallywhen no other more direct parameters to controlthe derivatization process are available, for example,the use of a suitable ISTD (screening procedure E;Table 4).

For qualitative screening procedures, the generalevaluation criterion for positive control samples isthe verification of the presence of all the substancescontained. Furthermore, RRTs of the analytes withinthe established acceptance range are also verified


(Table 4). Peak shapes of some critical analyteshighly sensitive to the chromatographic conditionsare specially evaluated. A list of variables controlledusing control samples is given inTable 3. In Fig. 1,results obtained after analysis of the control samplePCU1-B in procedure B are presented. In the upperpart, chromatograms of the ions used to estimate theconcentrations of different endogenous compoundsare presented; in the lower part, chromatograms ofthe three diagnostic ions used for the identification ofsome compounds detected in procedure B are shown.

PCU samples are used in different procedures tocontrol some critical steps that can severely affect thequality of the analytical results. This is the case ofthe evaporation step in procedure A, used to detectvolatile compounds (stimulants such as amphetaminesand other phenylalkylamine derivatives). These ana-lytes are included in PCU1-A and CAL1-A samples(Table 5) to have a close control of possible lossesduring the evaporation process.

Control samples are also used to verify the quantita-tion of analytes performed in some of the procedures,i.e. ephedrines and caffeine in screening procedureA and testosterone and epitestosterone in screeningprocedure B (Table 4). In screening procedures Aand B, calibration is performed using the responsefactor obtained with a single calibration sample(Tables 4 and 5) and the quantitation is verified witha control sample using a Shewhart’s chart. In thesecases, the acceptance range of the concentration ofthe control sample is based on the previous analysisof the sample (N = 20) and is set at±3 S.D. from themean of the concentration values obtained. The ac-ceptance range is calculated for each batch of controlsample. As an example, a Shewhart’s control chart ofephedrine in PCU1-A sample is presented inFig. 2.

In procedure B, concentrations of testosterone andepitestosterone, and the T/E areas ratio are evaluated.As testosterone and epitestosterone are endogenouscompounds, a blank urine (NCU1-B) is used as acontrol sample for these two analytes. The T/E ratioof the CAL1-B is also evaluated. Shewhart’s controlcharts for T/E ratios of the calibration sample CAL1-Bin two different GC/MSD systems are presented inFig. 3. According to data obtained, the IQC parame-ters can vary depending on the instrument used for theanalysis, even with instruments of the same model.Statistical differences (P < 0.05) have been found in

T/E ratios when analyzing control samples of the samebatch in two different GC/MSD systems (HP 5970MSD). Thus, different control charts must be preparedto evaluate control data obtained in each instrument,even using the same batch of calibration sample.

For quantitative confirmation procedures, the accep-tance range of concentrations of the analytes in PCUsamples has been established at±20% of the assignedconcentration (Table 4). Concentration values of caf-feine in the PCU sample of different caffeine quanti-tation analytical batches are presented inFig. 4.

3.2.4. Calibration samplesCalibration samples are prepared by spiking blank

samples (with or without matrix) with known con-centrations of the analytes. Calibration samples usedin screening procedures are also prepared at high vol-umes, aliquoted and stored at−20◦C. The control ofpreparation is performed by analysis of several repli-cates of the sample. A deviation of the concentrationof ±20% with respect to the nominal value is ac-cepted. The acceptance range of the IQC parameter,when needed, is defined by analysis of 20 replicatesin different batches.

For confirmatory quantitative procedures, calibra-tion samples are prepared the day of the analysis.Calibration samples are independently prepared fromdifferent weightings or initial dilutions of the refer-ence material or obtained from other sources thancontrol samples to prevent a possible undetectablebias into the analytical results.

The type of calibration samples depends on thepurpose of the procedure. In screening procedures(procedures A and B;Table 4), the estimation of theconcentration of the analytes of interest is performedusing the response factor obtained with a single cal-ibration sample. For confirmation purposes, to eval-uate if the concentration of analyte in the sampleis higher than the cut-off concentration, a statisticalcomparison is performed between the relative signalof the analyte to the ISTD in the test sample and therelative signal in a calibration sample at the cut-offconcentration (i.e. CA21, CA22 and CA23 for con-firmation of caffeine; CA11, CA12 and CA13 forconfirmation of T/E ratio). In addition, a calibrationgraph at three concentration levels: lower than thecut-off (CA11 and CA12), at the cut-off concentration(CA21, CA22 and CA23) and higher than the cut-off



Fig. 2. Shewhart control chart of ephedrine concentrations in PCU1-A used in procedure A.

(CA31 and CA32) is used to calculate the concentra-tion of the analyte in the test sample (confirmationof caffeine; Table 4). In the procedure for confir-mation of T/E ratio (Table 4), the estimation of theT/E ratio is performed using a calibration point witha T/E ratio at the cut-off level (T/E= 6) with anepitestosterone concentration similar to that esti-mated in the screening step, as described by otherauthors[36].

Different IQC parameters evaluated in calibrationsamples of quantitative procedures are listed inTable 4(confirmations of caffeine and T/E ratio). When a cal-ibration graph is used, it is accepted when the cor-relation coefficient of the linear function is >0.990.An acceptance criterion for rejection of duplicates ofcontrol and calibration samples has been also defined.

�

Fig. 1. Extract of GC/MSD results obtained after analysis of the positive control urine in procedure B (PCU1-B). Top: extract ofan endogenous steroids report. Chromatograms ofm/z 432 (testosterone bis-O-TMS (T) and epitestosterone bis-O-TMS (E)), m/z 446(methyltestosterone bis-O-TMS (MT) ISTD), m/z 435 (testosterone-d3 bis-O-TMS (T-d3) ISTD), m/z 434 (androsterone bis-O-TMS (A)and etiocholanolone bis-O-TMS (Et)), comparison ofm/z 272 and 434 (characteristic ions of androsterone mono-O-TMS and androsteronebis-O-TMS). Bottom: extract of an exogenous steroids report. Chromatograms of three characteristic ions to ascertain the presence ofthe TMS derivatives of some exogenous steroids:m/z 405, 420 (norandrosterone bis-O-TMS (nandrolone met1)) andm/z 422 (VitaminE derivative co-eluting with norandrosterone bis-O-TMS), m/z 405, 420, 315 (noretiocholanolone bis-O-TMS (nandrolone met2)),m/z143, 435, 345 (17�-methyl-5�-androstane-3�,17�-diol bis-O-TMS, 17�-methyl-5�-androstane-3�,17�-diol bis-O-TMS (MT met1,2)),m/z433, 448, 343 (1�-methyl-5�-androstan-3�-ol-17-one bis-O-TMS (mesterolone met1)),m/z 195, 208, 446 (methenolone bis-O-TMS(methenolone)),m/z 431, 446, 432 (1-methylen-5�-androstan-3�-ol-17-one bis-O-TMS/DHEA bis-O-TMS (methenolone met/DHEA)),m/z451, 466, 468 (4-chloroandrost-4-en-3�-ol-17-one bis-O-TMS (clostebol met1)),m/z 453, 468, 470 (4-chloro-5�-androstan-3�-ol-17-onebis-O-TMS (clostebol met2)).

This criterion involves the rejection of those replicatesthat differ from the mean in a value higher than thevalue of uncertainty of the method.

3.2.5. Derivatization parametersCorrect derivatization is assessed by using a suit-

able ISTD, a DEST or PCU samples, or by mon-itoring the derivatization of endogenous compounds(Table 4). In procedure B, derivatization of hydroxyand keto groups of anabolic androgenic steroids is ac-complished by using a mixture of MSTFA, ammo-nium iodide and a reducing agent[37]. To control thecomplete derivatization of keto groups, the ratio ofthe peak areas of two derivatives of some endogenouscompounds, such as mono-TMS derivative (indicativeof uncomplete derivatization) and bis-TMS derivative

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Fig. 4. Concentration of caffeine obtained after analysis of PCU1, PCU2 and PCU3 by the confirmation of caffeine procedure. Resultsobtained in different analytical batches are compared.

(indicative of complete derivatization) of androsteroneand etiocholanolone, is evaluated (Fig. 1). This ratiomust be lower than 5% to ensure the nearly completederivatization of keto groups needed to achieve ade-quate limits of detection for endogenous and exoge-nous anabolic steroids.

3.3. Internal quality control parameters forimmunological methods

NCU and PCU samples are also used in im-munological methods (procedure F;Table 4). Formany immunological methods, control samples arecommercially available or directly supplied by themanufacturers with the immunoassay kits. However,in-house prepared NCU and PCU samples are alsoused. Immunological methods are applied for screen-ing purposes but, while FPIA and MEIA providequantitative results of the analytes in the test samples,ELISAs are used as qualitative tests. According tothis, different IQC parameters have been establishedfor both types of immunological methods, but theShewhart’s control chart procedure has been adoptedto monitor assay performance in all three cases.

For qualitative ELISA tests, different ways of dataexpression were evaluated in order to select the mostappropriate IQC parameter: concentration of the tar-get analyte in the PCU sample, optical density (OD),

control activity (CA% = [ODPCU/ODNCU] × 100)and relative control activity (RCA%= [(ODNCU −ODPCU)/ODNCU] × 100), where ODNCU is the opti-cal density of the NCU sample and ODPCU the opticaldensity of the PCU sample[38]. RCA% was chosen asthe most suitable IQC parameter for its higher stability,which was reflected in the low coefficients of variationobtained for the control samples in the ELISA tests in-vestigated. The behavior of RCA% as IQC parameterwas studied by analysis of variance and showed thatthe inherent variability of ELISA tests remains ratherhigh, being the ELISA reagents manufacturer’s batchthe major source of variance (P < 0.05). Accordingto these results, acceptance limits should be calculatedfor each reagent batch and for each quality controlsample batch but, in practice, this would be difficultdue to the early expiry date of the reagents. It was alsoobserved that the variability was not constant amongdifferent reagent batches of the same ELISA test. Forthese reasons, the use of generic limits, independentof the reagent batch and a wide acceptance criterion(mean±4 S.D.) has been adopted for the IQC variableselected[38].

For FPIA and MEIA analyses, commercially avail-able positive quality control materials are used. TheShewhart’s chart method is used and the acceptancerange has been established at mean± 3 S.D. At least20 individual values in different analytical batches are


needed to calculate this acceptance range. The concen-trations assigned by the manufacturers with an accep-tance range of±20% is used provisionally as referencevalues for IQC purposes until determined in-house.

NCU samples prepared in the laboratory are alsoanalyzed by FPIA. A cut-off concentration has beenadopted for evaluation of FPIA results, according tointernational recommendations[39]. In these cases,a concentration of the target analyte lower than thecut-off value was defined as acceptance criterion forthe NCU sample.

4. Discussion

As other areas of toxicological analysis (i.e. drugsof abuse testing), antidoping control involves twosteps: screening, to detect samples not containingthe analytes of interest and not requiring additionalanalyses; and confirmation of the presence of specificanalytes in the presumptive positive samples detectedin the screening step. However, due to the large num-ber of analytes to be detected, their chemical nature,their metabolic and urinary excretion characteristicsand the limits of detection to be achieved, most ofthe screening procedures applied in antidoping con-trol are based in chromatographic techniques (mainlyGC/MSD) [22–29]. In addition, immunological testsare also used for some specific drugs[26–28,34]. An-other important difference with respect to other areasof toxicological analysis is the need of a qualitativedetection of most of the analytes; cut-off concen-trations are only defined for a few compounds[20].Taking into consideration all these factors, screen-ing analysis in antidoping control is an expensiveand time-consuming step. Procedures with the abilityof keeping the rate of both false negative and falsepositive results to a minimum have to be applied; areduced rate of false positive results is also requiredduring the screening step to avoid an extra work inthe confirmation step, increasing the time and the costof the analyses. In addition of having procedures withsuch characteristics, a continuous and close monitor-ing of the quality of results during routine work isalso needed to ensure the fulfillment of the quality re-quirements. For this reason, IQC procedures are of ex-treme importance for both screening and confirmationprocedures.

The strategy proposed in this paper for IQC inantidoping analyses is based on the use of controland calibration samples that are treated in the sameway as the test samples, as described in other areasof toxicological analysis[12–19]. Additionally, forchromatographic methods, the use of an ISTD pro-vides also the basis of some IQC procedures that areapplied to all samples in the analytical batch. Dif-ferent IQC parameters have been defined accordingto the aim of the method (qualitative or quantitative,screening or confirmation) (Table 4). These param-eters allow to control the performance of the wholeanalytical procedure including the sample preparationstep and the instrumental analysis (Table 3).

Contrary to other fields of clinical chemistry, insports drug testing, control and calibration samplesare not generally commercially available and theyhave to be prepared in the laboratory. Quality controlsamples are representative of the test samples withregard to matrix composition and type and concen-tration range of the analytes[10]. All control andcalibration samples are prepared using urine as sam-ple matrix, except when calibration of endogenouscompounds has to be done. Although the effect ofthe urine matrix has been shown to be important forT/E ratio determination[36,40], a calibration samplewith water as matrix (procedure B;Table 4) or withstandards (confirmation of T/E ratio;Table 5) is useddue to the difficulties in obtaining urines with sup-pressed endogenous levels as blank matrix at the highvolumes needed for routine purposes.

Owing to the large number of analytes to be deter-mined, PCU samples with a large number of analytescompared to other fields of toxicological analysis, areused (Table 5). The most critical analytes are alwaysincluded in the control samples (e.g. oxandrolone,3′-hydroxystanozolol and triamterene in procedureB; volatile analytes in procedure A). In order toguarantee adequate sensitivity during routine work,control samples spiked with the analytes near therecommended limits of detection (LODs) are used,especially for critical analytes. In the confirmationstep, the concentration of the analytes in the PCUsample in identification procedures is similar to thatof the sample to be confirmed and a concentration notdifferent than a factor of five is used[21].

The analytical batch includes at least one controlfor every 10 test samples, with a minimum of one


positive and one negative control sample in agree-ment with international recommendations[13,17,18].For analytes with cut-off concentrations, a calibrationsample is always included in the screening step. Forconfirmatory analyses, the composition of the ana-lytical batch recommended in the field of antidopingcontrol is used[19,21].

The order of the sequence of injection is also im-portant to control all components of error during therun. Random position of the control and calibrationsamples is recommended to have a better estimationof the analytical imprecision; analysis of the controlsample just before the calibration sample can falselyreduce the estimation of the analytical imprecision[10]. However, in screening methods in antidopinganalyses, all control and calibration samples are in-jected at the beginning of the sequence to check thatthe entire system is working correctly. A SOBL orDEBL is always the first sample injected to check forcontamination due to the chromatographic system.Additionally, a blank sample is always injected beforethe test samples to ensure that no carry-over exists.For confirmatory analysis, the recommended order ofinjection into the analytical instrument is as follows:REBL (if pertinent), a NCU sample, the sample beingconfirmed, a NCU sample and a PCU sample; thus,negative control samples are always injected beforethe sample to be confirmed to eliminate any possibil-ity of cross-contamination between samples[19,21].

Isotopically-labeled ISTDs are preferably usedin GC/MSD for quantitative purposes[36,41,42].Following this recommendation, deuterated ISTDs(testosterone-d3 and epitestosterone-d3) are used forendogenous steroids quantitation in screening proce-dure B and confirmation of T/E ratio (Table 4). Thevariation in response factors for endogenous steroids,calculated from the quotient between the concentra-tion and the areas ratio of the target analyte and theISTD in the calibration sample, is reduced when usingthe respective deuterated steroid as ISTD, resulting inan increase in precision[41].

The hydrolysis efficiency is indirectly controlled bymonitoring the analytes that are present in the urinematrix as conjugates (i.e. testosterone and epitestos-terone concentrations in NCU in screening procedure4B). A closer control could be achieved by spikingthe urine matrix with the analyte in the glucuronideform (when commercially available) and monitoring

the concentration of the free analytes. However, themost direct way to control the hydrolysis efficiencyis the use of conjugates of the deuterated analyte asISTD in all the samples, as described for the screeningof anabolic steroids by using deuterated androsteroneglucuronide[43].

An acceptance range of the ISTD signal based onmean± 2 S.D. or mean± 3 S.D. of absolute val-ues of peak adequate areas or peak heights was notfeasible for any procedure. Since the absolute valuecan largely vary depending on different factors (e.g.instrumental tuning), too wide acceptance ranges ofareas or heights are obtained, which would provide alow diagnostic value for IQC purposes. For this rea-son, a minimum peak area/height was established toguarantee that adequate sensitivity was achieved.

Reference values of RT and RRT of each analytehave been defined for each screening and confirma-tion procedure. As indicated before, the acceptanceranges of RT and RRT for both the ISTD and theanalytes in the PCU and CAL samples have beenestablished using different criteria. In most cases, adeviation of±2% with respect to reference values isaccepted (Table 4). In some cases, larger deviationsare accepted for RT, especially in confirmation meth-ods not often applied. However, a more restrictivecriterion is used to evaluate the positivity of a testsample: differences in RRT not higher than 1% areaccepted between the test sample and the PCU sampleanalyzed in the same batch[21].

Some IQC parameters in screening proceduresare controlled by using Shewhart’s control charts(Table 4). In these cases, an acceptance range ofmean± 3 S.D. has been defined. This acceptancerange is wider than normally used according to theWesgard rules (mean± 2 S.D.) [7–9], but it is ac-cepted since the results obtained with these screeningmethods must be further confirmed using more ac-curate confirmation procedures. For ELISA tests, awider acceptance range (mean± 4 S.D.) has beendefined for the IQC parameter due to the intrinsicvariability of the ELISA reagents used[38].

For quantitative confirmation procedures, the ac-ceptance range for the concentration of the analyte inPCU samples has been defined as the assigned valueof concentration±20% (Fig. 4), in agreement withthe criterion of accepted accuracy during method val-idation [44,45]. In these cases, a percentage range is


preferred since these quantitative confirmation meth-ods are not frequently applied and a large number ofdeterminations is needed to set control limits at mul-tiple S.D. from the mean according to the Shewhart’schart procedure.

The IQC procedures presented in this paper havebeen applied during routine antidoping control workfor more than two years with a large number ofsamples (>5000 samples per year). These IQC proce-dures met the requirements of the ISO 17025 qualitystandard and they have been shown to provide theinformation needed to detect and prevent possiblesources of errors, requiring a short time of evaluation.

5. Summary

As part of the quality assurance system, IQC isone of the most important elements contributing toachieve the high degree of reliability in the analyti-cal results needed by the analytical laboratory. Strate-gies for the implementation of an IQC program in anantidoping control laboratory have been presented inthis paper, taking into account recommendations andrequirements described by official organizations andquality standards.

Since in general quantitative methods are the mostused, guidelines and recommendations provided bythe literature are usually related to IQC in this type ofanalytical procedures. However, in antidoping controlqualitative methods are more often applied for screen-ing and confirmation purposes as in other fields of clin-ical and analytical chemistry. For this reason, specialattention has been paid in this work to IQC proceduresfor qualitative methods, with the aim of covering thelack of guidance existing in this area.

Thus, IQC parameters and acceptance criteria toassess the quality control data obtained in qualitativeand quantitative chromatographic and immunologicalprocedures have been presented. These criteria can bealso applied to other fields of clinical and analyticalchemistry.

Acknowledgements

The co-operation and positive attitude of all ourcolleagues involved in routine antidoping control is

gratefully acknowledged. The authors would like tothank Dr. Rafael de la Torre and Dr. José AntonioPascual for helpful discussions on IQC issues. Thegrant for Grup de Recerca Altament Qualificat (CIRIT1999SGR00242) of Generalitat de Catalunya is ac-knowledged.

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