Immunoassay

MONITORING CAFFEINE IN HUMAN SALIVA USING ANEWLY DEVELOPED ELISA

Jose Joao Carvalho,1 Michael G. Weller,1 Ulrich Panne,1,2 andRudolf J. Schneider11BAM Federal Institute for Materials Research and Testing, Berlin, Germany2Chemistry Department, Humboldt-Universitat zu Berlin, Berlin, Germany

Caffeine is a useful indicator to quickly assess liver function. High-throughput tests are

needed for single-point caffeine measurements, with low cross-reactivity toward its major

metabolite, paraxanthine. A newly developed ELISA was compared with an LC-MS/MS

reference method, using 60 saliva samples from 10 individuals, before and after caffeine

intake. Bland-Altman plot, Student t-test and F-test were used to compare the two methods.

Proteins were precipitated using organic solvent and the caffeine recoveries compared with

those obtained using sample microfiltration. The antibody, with a low cross-reactivity

toward paraxanthine (0.08%), allows quantification of caffeine in saliva samples from

2.5lg/L to 125lg/L with high precision and the ELISA shows comparable results to those

obtained by LC-MS/MS. A one-step protein precipitation using an organic solvent provides

comparable results to a more costly and time-consuming microfiltration pre-treatment of

samples. The new ELISA is a fit-for-purpose method to accurately and precisely determine

caffeine in saliva samples.

Keywords: Caffeine; Cytochrome P450 CYP1A2; Enzyme-linked immunosorbent assay; LC-MS=MS;

Liver function tests; Saliva

INTRODUCTION

Few molecules have such widespread use and so many applications as caffeine.Narrowing the spotlight to the clinical field, caffeine is used for treating apnea inpremature infants and neonates (Lee et al. 1996; Pesce, Rashkin, and Kotagal1998; de Wildt et al. 2001), as a psychostimulant substance (Brice and Smith 2001;Smith 2002; Haskell et al. 2005; Hewlett and Smith 2007), a physical performanceenhancer (Chambaz, Meirim, and Decombaz 2001; Hogervorst et al. 2008), and as

Received 8 April 2012; accepted 17 May 2012.

J. J. Carvalho thanks BAM Federal Institute for Materials Research and Testing for a grant within

its doctoral program. The contribution of K. Petsch, BAM, is acknowledged for repeating all the measure-

ments, confirming the results. Special thanks to all colleagues who provided the samples.

Address correspondence to Rudolf J. Schneider, BAM Federal Institute for Materials Research and

Testing, Richard-Willstaetter-Str. 11, D-12489 Berlin, Germany. E-mail: rudolf.schneider@bam.de

Analytical Letters, 45: 2549–2561, 2012

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032719.2012.696226

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a diagnostic tool for hepatic function and renal clearance studies in humans andanimals (Golden et al. 1994; Carrillo et al. 2000; Tarantino et al. 2006).

Serum has been the classical testing matrix to monitor caffeine in humans, butsaliva has become a trend in the 1990s (Teeuwen, Elbers, and Vanrossum 1991; Leeet al. 1996; Biederbick et al. 1997)—an understandable improvement as salivarysampling is less invasive than blood sampling, particularly important for weakpremature neonates, providing comparable results and similar precision (Lee et al.1996; de Wildt et al. 2001).

Hepatic microsomal function in adults can be assessed by a simple caffeineclearance test (Jost et al. 1987; Wahllander, Mohr, and Paumgartner 1990). In fact,caffeine clearance was suggested to be a more sensitive indicator of structural liverdisease than conventional liver function tests, especially for alcoholic liver disease;caffeine clearance measurement is 100% sensitive for alcohol-related liver diseasesand 89% sensitive for all liver diseases when compared to sensitivities of conven-tional liver function tests with 96% and 83%, respectively (McDonagh et al. 1991).Additionally, caffeine clearance after ingestion of a standardized oral dose discrimi-nated best between patients with cirrhosis, patients with non-cirrhotic liver disease,and healthy individuals (Wang et al. 1985).

Several authors showed the correlation between salivary and plasma concen-tration of caffeine in hepatic function assessment (Cook et al. 1976; Newton et al.1981; Wahllander et al. 1990; Carrillo et al. 2000), with salivary sampling alone start-ing to be accepted (Haskell et al. 2005). Salivary caffeine is becoming a useful stan-dard to evaluate cytochrome P450 1A2 (CYP1A2) activity (Carrillo et al. 2000) andit was also proposed to phenotype the activities of several other (iso)enzymes—NAT2, XO and CYP2A6—in epidemiologic and drug-drug interaction studies(Hakooz 2009). Some authors even suggest the use of a single-point measurementto evaluate cirrhotic dysfunction (Jost et al. 1987; Tarantino et al. 2006). A singlecaffeine determination in saliva—total overnight salivary caffeine assessments(TOSCA)—was proposed as a useful diagnostic tool for cirrhotic patients andeventually also for discriminating between the cirrhosis types (Tarantino et al. 2006).

Gas Chromatography (GC) (Newton et al. 1981; Wang et al. 1985; Teeuwenet al. 1991), High-Performance Liquid Chromatography (HPLC) (Miceli, Aravind,and Ferrell 1983; Ou, Frawley, and Ellis 1984; Zysset, Wahllander, and Preisig1984; Wahllander et al. 1990; Lee et al. 1996; Biederbick et al. 1997; Ptolemy et al.2010) and Enzyme-Multiplied Immunoassay Technique (EMIT) (Golden et al. 1994;Chambaz et al. 2001; Haskell et al. 2005; Tarantino et al. 2006; D. R. Mendu et al.2007a) are the most frequently used methods for caffeine measurements in biologicalmatrices. The EMIT assay appears especially popular within clinical laboratoriesowed to its simplicity, relatively low analysis costs per sample, and comparableresults to the ones obtained by HPLC (Miceli et al. 1983; Ou et al. 1984; Zyssetet al. 1984; Wahllander et al. 1990; de Wildt et al. 2001); even though, the lack oflow-end precision (D. Mendu et al. 2007a) and the high cross-reactivity (28%)toward caffeine’s major metabolite, paraxanthine, are of concern (Zysset et al.1984; Jost et al. 1987; Perera, Gross, and McLachlan 2010). Attempts to improvethe precision were recently successfully made (Mendu et al. 2007b) and a polyclonalserum from sheep showed low cross-reactivity for paraxanthine (0.05%), even if 16%cross-reactivity with theophylline was observed (Fickling et al. 1990).

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We developed an ELISA, based on a monoclonal antibody, for measuringcaffeine in several non-biological matrices with a low detection limit and relativelylow cross-reactivities (Carvalho et al. 2010). Herein, we describe the application ofthe assay to saliva samples as a useful tool for single-point caffeine measurements(Tarantino et al. 2006), doping control (Chambaz et al. 2001; Hogervorst et al.2008), veterinary medicine (Golden et al. 1994), and cognitive and behavioral studies(de Leon et al. 2003; Rogers et al. 2005; Hewlett and Smith 2007).

MATERIALS AND METHODS

Reagents

The anti-caffeine monoclonal antibody (mouse IgG 2b, catalogue numberC0110-06, clone 1.BB.877, lot L0251502M, 1.37mg=mL) was purchased fromUSBiological Inc. (Swampscott, MA, USA). The secondary (capture) antibody, apolyclonal anti-mouse IgG ‘‘whole molecule’’ (R12569, Lot 20243, 2.2mg=mL), fromsheep, was obtained from Acris Antibodies GmbH (Herford, Germany). Theenzymatic tracer (horseradish peroxidase conjugate) was synthesized in-house basedon a novel coupling hapten (Carvalho, Emmerling, and Schneider 2007) as previouslydescribed (Carvalho et al. 2010). 3,30,5,50-Tetramethylbenzidine (TMB), researchgrade, and Tween 20, pure, were purchased from Serva (Heidelberg, Germany).Buffer salts (sodium phosphate dibasic dihydrate, sodium phosphate monobasicdihydrate, potassium phosphate monobasic, potassium dihydrogen citrate, sodiumchloride, sodium carbonate, sodium bicarbonate) were of Fluka ‘‘ultra’’ quality(Sigma-Aldrich, Taufkirchen, Germany). Tris(hydroxymethyl)aminomethane (p.a.‘‘for buffer’’) was purchased fromMerck (Darmstadt, Germany). Hydrogen peroxide30% Trace select1 and ammonium acetate (puriss., LC-MS additive) were fromFluka. Sulfuric acid 95–97%, and hydrochloric acid 32%, were of ‘‘Baker analyzedgrade’’, methanol and acetonitrile were HPLC gradient grade (Mallinckrodt Baker,Griesheim, Germany). Ultrapure reagent water (hereafter referred to as Milli-Qwater) was obtained by running demineralized water (by ion exchange) through aMilli-Q water purification system (Millipore Synthesis A 10, Schwalbach, Germany).Caffeine (puriss.) was purchased fromFluka and 13C-trimethyl-caffeine (99 atom.% 13C)was supplied by Sigma-Aldrich (Taufkirchen, Germany).

Materials

Transparent microtiter plates with 96 flat-bottom wells with high proteinbinding capacity (MaxiSorp) were purchased from Nunc (Thermo Scientific). Micro-filtration filters with a cut-off of 3 kDa, Vivaspin 500, were purchased from Sartorius(Gottingen, Germany). Polypropylene tubes were purchased from Eppendorf(Hamburg, Germany); HPLC vials were from Agilent (Waldbronn, Germany).

Saliva Collection

Sixty samples from ten individuals were collected every hour for five hours, afterthe intake of 200mg caffeine. The saliva samples were collected directly, after

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stimulation using Parafilm, into 2-mL Eppendorf tubes. After vortexing, the tubes werecentrifuged (15�C, 15,000 g, 5min) and placed overnight at 4�C until pretreatment.

Participants and Given Instructions

The group of ten individuals was composed by regular caffeine consumers: fivewomen and five men, three smokers and seven non-smokers, five were betweentwenty and thirty years old, three between thirty and forty, one between forty andfifty and one over fifty. The participants were advised on saliva sampling proceduresby the following written instructions:

- collect the first sample before brushing/flossing the teeth, eating, drinking or apply-ing makeup; refraining from brushing, flossing the teeth or eating within one hourof the subsequent saliva collections.

- avoid caffeine, alcohol and nicotine during the 12 hours before the first saliva’scollection (T¼ 0h).

- do not drink=eat coffee, tea, coke, cacao or chocolate during the day of thesampling;

- rinse the mouth at least twice with cool water before chewing the provided piece ofParafilm for 1–2 minutes (instructing also: ‘‘If you do not want to chew the Paraf-ilm, saliva collection is still possible but may take longer’’).

- remove the cap of the Eppendorf tube and collect the saliva directly into the tube,avoiding touching the tube aperture with the hands.

- fill the tube at least 3=4 full and close it, evenly and securely.

Sample Pretreatment

Two methods were tested to remove proteins and cells from the samples:

1. Microfiltration using filters with a cut-off of 3 kDa (Vivaspin 500);2. Protein precipitation using an organic solvent: acetonitrile (Hoetelmans et al.

1998; Chi et al. 2002).

Twelve saliva samples from different individuals were spiked with 10mg=L of13C3-trimethyl-caffeine (1.5mL saliva þ 15mL of 1000mg=L of 13C3-trimethyl-caffeine) and the proteins removed using both methods. From each spiked sample, 2aliquots of 200mL were passed through a Vivaspin 500 filter and centrifuged for 20minutes, 15,000g at 6�C; 2 other aliquots with the same volume were placed intoEppendorf tubes and 1000mL acetonitrile was added prior to centrifugation, underthe same conditions. All four aliquots were centrifuged simultaneously. The filtrateswere diluted 100-fold in Milli-Q water prior to LC-MS=MS analysis and theacetonitrile extracts diluted 20-fold in Milli-Q water. For calculation purposes, thedilution factor for the acetonitrile extracts was obtained considering the thermo-dynamic properties of the mixtures waterþacetonitrile at 20.0�C (293.15K) and atmos-pheric pressure (del Carmen Grande et al. 2006; Zarei, Lavasani, and Iloukhani 2008).

After showing the equivalence between the two extraction methods, samples wereextracted using the acetonitrile method and analyzed by ELISA and LC-MS=MS in the

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same day, using the same calibration standards. The extracts of the samples as well asthe control samples were evaporated until dryness under a nitrogen stream and recon-stituted in an equivalent volume of Milli-Q water. Thereafter, the extracts were dilutedappropriately to fall within the calibration range.

Immunoassay Procedure

The direct competitive ELISA was performed at room temperature. The samebuffer was used for all the steps (TRIS-NaCl buffer, 10mM Tris(hydroxymethyl)-aminomethane, 150mM NaCl, pH adjusted to 8.50) with the exception of thesubstrate, which was prepared in substrate buffer (200mM citric acid containing3mM hydrogen peroxide and 0.01% sorbic acid potassium salt, pH 4.0). Thewashing buffer was prepared by adding 0.05% (v=v) Tween 20 to a PBS buffer(1mM sodium phosphate, 0.015M NaCl, pH 7.6).

Transparent high-binding microtiter plates were coated with polyclonalanti-mouse IgG (H&L,‘‘whole molecule’’) serum (1mg=L in TRIS-NaCl buffer,200 mL per well), covered with Parafilm and incubated overnight (� 18 hours) on aTitrama 101 plate shaker (Heidolph, Schwabach, Germany) at 750 rpm. The plateswere then washed three times with washing buffer using an automatic 96-channelplate washer (ELx405 Select, BioTek Instruments, Bad Friedrichshall, Germany).

The anti-caffeine monoclonal antibody was diluted in TRIS-NaCl buffer(0.137 mg=mL, 200 mL per well), added to the pre-incubated plate and shaken for90min. After washing the plate three times, the caffeine standards and samples wereincubated (100 mL per well) together with the tracer (0.008 mg=mL in TRIS-NaClbuffer, 100 mL per well) for 40 minutes with permanent shaking. The calibrationcurve was established using 9 calibrators, from 2.5 mg=L to 125 mg=L in Milli-Qwater. After washing away excess standards=samples and unbound tracer, 200 mLof substrate solution was added into the wells and incubated for 30 minutes whileshaking. The substrate solution was prepared according to the procedure describedby Frey et al. (2000). The reaction was stopped using 1M sulfuric acid, 100 mL perwell. The optical density (OD) was read on a microplate spectrophotometer Spectra-Max Plus384 (Molecular Devices) controlled by SoftMax Pro software (v 5.2,Molecular Devices). The OD was measured at 450 nm using 650 nm as reference.For quantitation purposes the calibrators were fitted with a logistic model curve(sigmoidal) using Origin 8.0 (OriginLab, Northampton, USA). Both calibratorsand samples were determined in triplicate on each plate.

Cross-Reactivity Determination

The immunoassay selectivity for caffeine with respect to related xanthines wastested by assaying an aqueous dilution series of theophylline, theobromine, para-xanthine, xanthine, caffeine-trimethyl-13C3, and caffeine itself. The cross-reactivitywas calculated as the ratio of mass concentrations at the inflection points (midpoints,parameter C in the 4-parameters sigmoidal curve� half maximum inhibitoryconcentration, IC50) of the corresponding calibration curves and expressed inpercent relative to the caffeine midpoint, as previously described (Bahlmann et al.2009; Carvalho et al. 2010).

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LC-MS/MS Reference Method for Caffeine Determination

The LC-MS=MS experiments were carried out using an Agilent 1100 LiquidChromatography system (Agilent, Waldbronn, Germany) and an API 4000 triple-quadrupole mass spectrometer from Applied Biosystems. A C18 reversed-phasecolumn, 250mm� 2.1mm, 5mm (Phen, UltraSep ES, SepServ, Berlin, Germany) wasused and the ionization performed in electrospray positive ion mode (ESIþ). Thechromatographic method was previously described in detail (Carvalho et al. 2010).

Spiking Experiments

An aliquot from a saliva sample (at t¼ 180min) of each individual (n¼ 10) wasspiked with caffeine and a second one with 13C3-trimethyl-caffeine (13C3-CAF), atapproximately half the caffeine concentration found in the original sample.

Quality Control Samples

The saliva samples were divided in batches of 10, one batch per each samplecollection time. The following control samples were included in each batch: Milli-Qwater spiked with caffeine to obtain a final concentration of 75 mg=L (around themidpoint of the calibration curve) and passed through the process; a Milli-Q watersample was also extracted to serve as a blank control; a control standard of 75 mg=L(prepared independently from the one used for calibration) was analyzed togetherwith each batch to evaluate the fitting of the calibration curves. The antibody doesnot distinguish well between caffeine and 13C3-trimethyl-caffeine (13C3-CAF) andtherefore the sample could not be spiked with the latter. The matrix effects wereevaluated using the spiking experiments described. The calibrators were includedin each ELISA plate and per each twenty samples with the LC-MS=MS method.

RESULTS

Sample Pretreatment-Microfiltration vs. Organic SolventPrecipitation

Protein precipitation using acetonitrile shows slightly better recovery of 13C-CAF than microfiltration, both in saliva samples and in Milli-Q water (Figure 1).A major drawback however seems to be the relatively higher dispersion andskewness of the recoveries as illustrated by the box plots in Figure 1. The lowestvalue for acetonitrile precipitation in the distribution is clearly an outlier (84.5%)but it was not removed as the plot-shape does not change significantly by doingso. Moreover, the differences between the sample pretreatment methods are notsignificant regarding the absolute recovery value in either method: 84.5–99.8% foracetonitrile precipitation and 87.6–95.3% for the microfiltration.

Caffeine concentrations were also compared and the same outcome observed:slightly higher values when acetonitrile is used, as illustrated by the slope of thecorrelation curve between the methods when the acetonitrile extraction is plottedon the ordinate and filtration as abscissa (y¼ 1.055.xþ 0.048; R2¼ 0.9985).The standard deviations (n¼ 3) for each sample follow the same trend as for

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13C3-CAF (data not shown), with the acetonitrile extraction producing slightly lowerstandard deviations than the filtration method.

Assay Validation

Cross-reactivities of the ELISA. The cross-reactivities of the assay withrelated compounds were the following: caffeine (100%), caffeine-trimethyl-13C3

(93%), theophylline 12%, theobromine (0.13%), paraxanthine (0.08%), and xanthine(<0.01%).

Comparison with LC-MS/MS reference method. A typical calibrationcurve for this ELISA is shown in Figure 2. The calibration standards were selectedto provide almost equidistant concentrations when a linear curve transformation isused, as illustrated by the insert in Figure 2. The linear transformation was obtainedby plotting – log [OD] on the ordinate. The displayed precision profile, calculatedusing the model proposed by Ekins (1981), shows low errors overall (<8%, rightordinate), with the lowest ones being found for the two lower standards (<2%),and the highest ones for the upper calibrators (around 7%).

Bland-Altman plot, Student’s t-test and F-test for comparing the twomethods. The results from both methods (LC-MS=MS and ELISA), after dilutionfactor corrections, were plotted using a Bland-Altman plot (Bland and Altman 1986)and are shown in Figure 3. The entire batch—with the exception of two samples—lies within the 95% confidence interval, thus providing evidence of good agreementbetween the methods. The plot also shows a rather equal distribution above andbelow the 0% line.

Figure 1. Box plot representing the 13C3-CAF recovery in 12 saliva samples using microfiltration and

acetonitrile precipitation, respectively, to remove proteins=enzymes and cells. The black circles represent

caffeine recovery in Milli-Q water to serve as a reference point.

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A Student’s t-test (one-tailed) shows no evidence of a significant differencebetween the methods for p¼ 0.05 and 58 degrees of freedom [t(critical)¼ 0.42;t(table)¼ 2.00]. The F-test (p¼ 0.05, n� 1¼ 59) also shows no evidence of significantdifferences between the methods’ variances [F(critical)¼ 1.03=F(table)¼ 1.54],proving good trueness of the ELISA.

Figure 3. Bland-Altman plot for the saliva samples measured using both methods. The x-coordinate

represents the average concentration of the values obtained using both methods and the ordinate the

respective differences, in percentages.

Figure 2. ELISA calibration curve used to quantify the saliva samples. The right ordinate represents the

relative error of concentration calculated from the precision profile (Ekins 1981); the points obtained are

represented by open triangles and connected by a dashed line. The transformation of the calibration curve

towards a linear model is represented by the inset.

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Recoveries. Caffeine recoveries from spiked Milli-Q water were between 96%and 104% and the control standard of 75 mg=L caffeine showed variations between�7.1% and þ1.4%. Caffeine was never detected in blank samples.

Recoveries above 77% were observed when saliva samples were spiked with13C3-CAF and above 87% for caffeine itself. Table 1 summarizes the results andthe data provides confidence regarding the suitability of the acetonitrile-basedextraction procedure. The apparently lower recoveries observed for some samplesspiked with 13C3-CAF (when compared with caffeine itself) are probably due to thelower final concentration after the spiking – the lowest recoveries with 13C3-CAF areobserved in the 3 samples spiked with the lowest amounts (500 mg=L and 1000 mg=L).Moreover, the difference in average recoveries for caffeine (93� 3%) and 13C3-CAF(84� 3%) become insignificant once the cross-reactivity of the antibody toward 13C3-CAF (93� 2%) is considered.

Limits of quantitation (LOQ). The lower limit of quantitation (LLOQ) fallsbelow the first standard of the curve (2.5 mg=L) (data not shown. By contrast, theupper limit of quantitation (ULOQ) had to be established: 125 mg=L. A higherstandard (150 mg=L) was also measured and thereafter excluded, as the precisionwas not satisfactory in all the plates.

Caffeine profiles in saliva. Figure 4 shows caffeine metabolic profiles ofsamples from individuals obtained using the newly developed ELISA. A secondLC-MS=MS injection was performed seven days later to show the variation withintwo LC-MS=MS determinations (the extracts were frozen at �20�C during this timeinterval).

The variations between the concentrations obtained by ELISA and the firstLC-MS=MS injection, performed in the same day as the ELISA, are also representedand show values between �13% and þ10%. Moreover, similar quantitative resultswere found for all the 10 individuals.

Table 1. Recoveries of caffeine and 13C3-trimethyl-caffeine in ten samples from different individuals (A–J)

collected at t¼ 180min after caffeine intake

Caffeine 13C3-Caffeine

Sample

concentration,

mg=L

Spiking

concentration,

mg=L

Concentration

determined,

mg=LRecovery,

%

Spiking

concentration,

mg=L

Concentration

determined,

mg=LRecovery,

%

Sample

A 5002 2500 7033 93.8 2500 2218 88.7

B 3465 1250 4346 92.2 1250 1129 90.3

C 4391 1250 5408 95.9 1250 1103 88.3

D 3592 1250 4575 94.5 1250 1084 86.8

E 2539 1250 3283 86.6 1250 1031 82.5

F 3255 1250 4221 93.7 1250 1120 89.6

G 1150 500 1497 90.7 500 395 79.1

H 2127 1000 2872 91.8 1000 779 77.9

I 4244 1250 5304 96.5 1250 1054 84.3

J 2270 1000 3130 95.7 1000 765 76.5

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DISCUSSION

Immunoassays often use wide calibration ranges (more than 3 orders of mag-nitude) while LC-MS=MSmethods employ calibration ranges of less than 2 orders ofmagnitude. Because the calibration range is of paramount importance for the com-parison of two methods, the same range as well as the same standards should be usedto establish the calibration curves in both methods. The same sample extracts, ordilutions of them, were analyzed by ELISA and LC-MS=MS in parallel. By doingso, calibration-derived deviations are minimized as well as sample preparationerrors, which could cause a misinterpretation of the data. The concentrations of caf-feine obtained using the ELISA were comparable to those obtained by LC-MS=MS.

The monoclonal antibody used exhibits low cross-reactivity toward caffeine’smajor metabolite, paraxanthine (0.08%), allowing the ELISA to be used as a toolfor single-point caffeine measurements in clinical chemistry, doping control, veterin-ary medicine and cognitive studies. The assay’s potential can be further explored byclinical laboratories for the determination of caffeine in other biological samples, likesera and urine (Perera et al. 2010). Theophylline presented the highest cross-reactivity (12%) but advantageously is a minor metabolite of caffeine and was notdetected in any of the saliva extracts dilutions measured by LC-MS=MS. The assayshould however be carefully applied when measuring saliva samples from individualsunder theophylline treatment, as high concentrations of theophylline could lead toan overestimation of caffeine concentrations.

The assay was adequate for the samples (saliva from healthy individuals) andconditions described. Further validation studies, in a clinical routine context, will berequired to evaluate reproducibility, unknown cross-reactants from patients underdifferent pharmacologic treatments, and other interferences that could arise fromthe intake or use of juices, toothpastes, and mouthwashes.

Figure 4. Caffeine salivary concentration over time using ELISA (filled stars), LC-MS=MS (filled

squares), and a 2nd LC-MS=MS, later, analysis (filled triangles). Lines represent the caffeine decay with

time: ELISA (bold), LC-MS=MS dotted 1st (black) and 2nd (grey) analysis. Open symbols represent %

deviation of concentrations obtained by ELISA (open stars) and the 2nd LC-MS=MS analysis (open

triangles) using the 1st LC-MS=MS results as reference.

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A batch of 62 samples was analyzed by ELISA within 3 hours, while 45 hourswere needed for the same number of samples by LC-MS=MS. The high throughputtogether with the low cross-reactivity towards paraxanthine is the major advantageof this newly proposed ELISA.

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