Abstract
Ultratrail running is a sport with growing number of adherents. To complete ultratrail despite physical issues such as joint and muscle pain, many runners use nonsteroidal anti‐inflammatory drugs (NSAIDs) and acetaminophen. Studies asking participants about their consumption of drugs during ultratrail revealed a prevalence of NSAIDs and acetaminophen up to 70% and 25%, respectively. The aims of the present study were to determine the prevalence of NSAIDs and acetaminophen for 81 runners during the 2021 Ultratrail du Mont Blanc® (UTMB®) using direct analysis of dried blood spots (DBS) and oral fluid (OF) and to compare results with the declaration of consumption by runners; this is to identify the most relevant method to study the prevalence of drugs. Our results show a prevalence of NSAIDs of 46.6% using DBS, 18.5% using OF, and 13.8% based on a questionnaire. Prevalence of acetaminophen were 30.1%, 30.9%, and 22.5% using DBS, OF, and questionnaire, respectively. From this study, we conclude that the analysis of drugs directly in DBS is the most relevant tool to determine the prevalence in ultratrail events.
Keywords: acetaminophen, dried blood spot, nonsteroidal anti‐inflammatory drugs, oral fluid, ultratrail
In the present work, the prevalence of the use of NSAIDs and acetaminophen in ultratrail was studied using three tools: direct analysis of dried blood spots (DBS), direct analysis of oral fluid (OF), and a questionnaire filled by runners. Comparative results showed that the analysis of drugs in DBS is the most relevant tool to determine the prevalence in ultratrail events.
1. INTRODUCTION
Running is a sport with a continually growing number of adherents. Moreover, distances longer than marathon, that is, long distance, ultratrail, trail running, become more and more popular, 1 and many events in the world propose race distances over 100 km. To complete ultratrails, runners frequently exceed their physical and mental limits. Physical issues mostly reported by ultratrail runners are pain, nausea, and/or vomiting. Several studies indicated that to prevent muscle and osteo‐articular pains, many ultratrail runners use NSAIDs and acetaminophen before, during, and/or after the race. 2 , 3 Indeed, in many ultratrail events, prevalence of the use of NSAIDs, estimated using a questionnaire completed by the runners, were higher than 50%. 3 , 4 , 5 , 6 Ibuprofen is the most common NSAID used and in most cases without medical prescription. 3 , 4 , 7 However, NSAIDs are not free from adverse effects (gastrointestinal, cardiovascular, hepatic, renal, cerebral, and pulmonary complications) and only 30%–40% of athletes have sufficient knowledge about them. 7 The International Trail Running Association (ITRA) aims to carry out measures to protect runner's health and to contribute to a cleaner sport. 8 To achieve this goal, ITRA promotes the development of the Quartz event program and its implementation in trail running races around the world. According to this program, runners cannot participate to the event in case of using NSAIDs within 24 h before the start or during the race. Most of the data concerning the prevalence come from the declarations of athletes. The runner can, however, omit to declare that he/she has taken an NSAID, have forgotten that he/she has taken one or has taken another drug (e.g., acetaminophen) and declare that he has taken an NSAID due to pharmacological ignorance. Thus, it is relevant to perform a detection of NSAIDs and acetaminophen in biological matrix from ultratrail runners in order to obtain direct and objective data on the prevalence. Ultratrail races bring together many participants, take place often in outlying places with sometimes extreme temperature conditions, and often partially or totally during the night. Therefore, the samples must be based on a simple device, a low or no invasive procedure and must be rapidly executed. In this context, a method for the quantification of 19 NSAIDs in oral fluid (OF) was previously validated. 9 However, the transport and the storage of OF samples may present some difficulties when it is abroad. An alternative is the use of dried blood spot (DBS). DBS is a micro blood sampling procedure already implemented for drug detection in therapeutic drug monitoring, in sports drug testing and in doping. 10 , 11 , 12 The use of DBS in the context of doping has experienced significant development in recent years. The World Anti‐Doping Agency (WADA) has recently edited a technical document concerning DBS 13 and the Union Cycliste International (UCI) started a pilot study for the analysis of tramadol in DBS. 14
In order to evaluate the relevance of the declaration of consumption of NSAIDs and acetaminophen by runners and to identify the most relevant tool to study prevalence, we performed this comparative study with direct research in DBS and OF.
2. MATERIAL AND METHODS
2.1. Chemicals and reagents
Standard solutions or powder of flurbiprofen, indomethacin, ketoprofen, mefenamic acid, para‐aminosalicylic acid, piroxicam, celecoxib, diclofenac, ibuprofen, naproxen, parecoxib, tenoxicam, tiaprofenic acid, mefenamic acid 13C6, and ketoprofen D3 came from Sigma‐Aldrich™ (Saint Louis, USA). Celecoxib 13C3, diclofenac D4, ibuprofen 13C6, sulfasalazine D4, piroxicam D3, and sulindac D6 were supplied by Toronto Research Chemicals (Toronto, Canada). Aceclofenac, etodolac, etoricoxib, meloxicam, niflumic acid, salicylic acid, and sulindac were purchased from LoGiCal® Standards (Luckenwalde, Germany). Acetaminophen came from B. Braun (Saint Cloud, France). Solvents, all HPLC‐grade, were obtained from Biosolve Chimie (Dieuze, France) and Carlo Erba (Val de Reuil, France). Deionized water was provided with an Elga Purelab (Flex system™, High Wycombe, UK). DBS collection device HemaXis DB 10 and OF device Quantisal® were supplied by DBS system SA (Gland, Switzerland) and Immunalysis (Pomona, California, USA), respectively.
2.2. Sample collection and preparation
A volume of 10 μl of capillary whole blood was collected using HemaXis DB 10 device. Before analysis, the entire spot was cut and transferred into 1 ml of ACN/water (60/40; v/v). After the addition of 10 ng of internal standards, samples were vortexed and sonicated for 15 min. Then after removal of the spot, tubes were centrifugated at 13,000 g for 5 min. After acidification of the supernatant with 50 μl of acetic acid, a liquid/liquid extraction (LLE) was carried out by mixing with 500 μl of chloroform. Chloroform was then collected and evaporated under nitrogen. The dry residue was resuspended in 100‐μl mobile phase, and 10 μl were injected into the liquid chromatography–mass spectrometry/high‐resolution mass spectrometer (LC–MS/HRMS) device. OF samples were prepared as previously described. 9
2.3. LC–MS/HRMS analysis
Analyses were carried out on an UltiMate 3000 system coupled with a Q‐Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific™, Bremen, Germany). LC–MS/HRMS conditions were as previously described. 9 Quantification was performed using internal standard method (Table S1).
In DBS, a quantitative method was validated according to EMA guideline for aceclofenac, diclofenac, etodolac, etoricoxib, ibuprofen, indomethacin, ketoprofen, meloxicam, naproxen, niflumic acid, para‐aminosalicylic acid, parecoxib, piroxicam, sulindac, tenoxicam, and tiaprofenic acid. Thus, the concentrations of all of these NSAIDs were determined. Salicylic acid, celecoxib, flurbiprofen, mefenamic acid, and acetaminophen were validated only for qualitative analysis because at least one of the EMA validation criteria was out of range. Consequently, only the presence or the absence of these five drugs was monitored.
2.4. Application
OF and DBS were collected by a nurse from ultratrail runners who participated to the UTMB® in August 2021. Sampling was performed just after the finish of the race. For each participant, a short questionnaire concerning the consumption of NSAIDs before and during the race was proposed (Table S2). The study was approved by the French ethical committee (Id number: 2021‐A01751‐40, CPP Sud Méditerranée I 13/07/2021).
3. RESULTS
3.1. Inclusion
Eighty‐one ultratrailers (61 men and 20 women), presenting an average age of 38.8 years (±8.8), were enrolled in the present study. All participants accepted OF sampling, 80 participants filled the questionnaire, and 73 accepted DBS sampling. The HemaXis device is based on microfluidics to collect an accurate volume of 10 μl of capillary whole blood. Although the volume collected at the fingertip is very low, the puncture remains an invasive procedure. Thus, some ultratrailers (9.9%) did not accept blood collection with the DBS device due to the fear of the needle or of a vagal discomfort.
3.2. Prevalence of NSAIDs and acetaminophen
In 15 OF (18.5%) and 34 DBS (46.6%), one or more NSAIDs were identified. Based on the questionnaire, 11 runners (13.8%) reported taking an NSAID 24 h before and/or during the race. For two positive OF samples, no DBS were collected. Overall, NSAIDs were identified in 36 samples from 81 runners (44.4%). In 21 cases, NSAIDs were found in DBS, and not in OF (Figure 1). For four of them, a metabolite of NSAID (hydroxyibuprofen) was found in OF, without the parent drug. Only one participant declared the consumption of ibuprofen during the race, but neither ibuprofen nor its metabolites were detected in DBS and OF (Figure 1).
FIGURE 1.
Distribution of positive samples for NSAIDs between DBS and/or OF and/or questionnaire. Data from 73 runners for whom OF, DBS and questionnaire were available. DBS, dried blood spot; NSAIDs, nonsteroidal anti‐inflammatory drugs; OF, oral fluid.
Six different NSAIDs were identified in DBS: ibuprofen (n = 23), diclofenac (n = 6), ketoprofen (n = 3), salicylic acid (n = 3), naproxen (n = 2), and niflumic acid (n = 2). In most cases (80.6% of positive cases), one NSAID was observed. However, two NSAIDs were found for six runners and three NSAIDs for one runner. Concentrations of NSAIDs in DBS and saliva from runners are presented in Table 1, except for salicylic acid that was analyzed only qualitatively. For the majority of samples, the concentrations found were lower than maximum concentrations after therapeutic dose: 20–30 μg/ml for ibuprofen, 0.9–3 μg/ml for diclofenac, 7–10 μg/ml for ketoprofen, 60–80 μg/ml for naproxen, and 20 μg/ml for niflumic acid. 15 , 16 , 17 , 18 , 19 For five runners, concentrations of ibuprofen in DBS were lower than the lower limit of quantitation (LLOQ) (0.01 μg/ml) but higher than the limit of detection (LOD) (0.004 μg/ml). It was the same for four runners for diclofenac (LLOQ = 0.05 μg/ml and LOD = 0.001 μg/ml), for one runner for ketoprofen (LLOQ = 0.05 μg/ml and LOD = 5.10−4 μg/ml), for one runner for naproxen (LLOQ = 0.1 μg/ml and LOD = 0.006 μg/ml), and for two runners for niflumic acid (LLOQ = 0.05 μg/ml and LOD = 4.10−4 μg/ml).
TABLE 1.
DBS and saliva concentrations of NSAIDs identified in runner's samples.
| NSAIDs | Runner | Concentrations in DBS (μg/ml) | Concentrations in saliva (ng/ml) |
|---|---|---|---|
| Ibuprofen | 1 | 31.5 | 5.2 |
| 2 | 21.1 | 7.3 | |
| 3 | 20.1 | 8.3 | |
| 4 | 19.1 | 23.2 | |
| 5 | 15.6 | <2 | |
| 6 | 8.7 | <2 | |
| 7 | 3.3 | <2 | |
| 8 | 1.32 | NF | |
| 9 | 0.67 | NF | |
| 10 | 0.52 | NF | |
| 11 | 0.45 | NF | |
| 12 | 0.29 | NF | |
| 13 | 0.22 | NF | |
| 14 | 0.22 | NF | |
| 15 | 0.20 | NF | |
| 16 | 0.16 | NF | |
| 17 | 0.14 | NF | |
| 18 | 0.11 | NF | |
| 19 | <0.1 | NF | |
| 20 | <0.1 | NF | |
| 21 | <0.1 | NF | |
| 22 | <0.1 | NF | |
| 23 | <0.1 | NF | |
| Diclofenac | 24 | 0.4 | <10 |
| 25 | 0.06 | NF | |
| 6 | <0.05 | NF | |
| 18 | <0.05 | NF | |
| 26 | <0.05 | NF | |
| 27 | <0.05 | NF | |
| Ketoprofen | 18 | 0.14 | <2 |
| 15 | 0.07 | NF | |
| 28 | <0.05 | <2 | |
| Naproxen | 29 | 40.9 | <50 |
| 1 | <0.1 | NF | |
| Niflumic acid | 30 | <0.05 | NF |
| 31 | <0.05 | NF |
Abbreviations: DBS, dried blood spots; NF, not found; NSAIDs, nonsteroidal anti‐inflammatory drugs.
Ibuprofen was the most consumed one because it has been detected in 23 samples (16 from DBS and 7 from DBS and OF). Ibuprofen was identified in OF only when the concentration in DBS was greater than 3 μg/ml (Figure 2). The median of OF/DBS ibuprofen concentration ratio was estimated as 0.0003 [0.0001–0.0012].
FIGURE 2.
Concentration of ibuprofen in DBS. Light bars represent cases for which ibuprofen was identified in DBS and OF. Dark bars represent cases for which ibuprofen was identified only in DBS. DBS, dried blood spot; OF, oral fluid.
Acetaminophen was detected in 25 cases (30.9%) in OF; in 22 cases (30.1%) in DBS and 18 runners (22.5%) declared having taken it. Based on DBS analysis, 12 runners (16.4%) consumed both ibuprofen and acetaminophen.
4. DISCUSSION
In the present work, the prevalence of NSAIDs and acetaminophen use by ultratrailers during the 2021 UTMB® was determined. First, prevalence was higher for NSAIDs than acetaminophen. This is in agreement with previously reported data for similar distances of ultratrail. 2 , 4 Second, the prevalence observed for NSAIDs (46.6%) is rather high but still lower than those reported in literature for trails over 100 km: 60.5% in the 2009 edition of a 161 km race in the USA 4 and 60.3% in a 112 km of Ultra Mallorca Serra de Tramuntana in Spain. 3 The lower prevalence of NSAIDs in the present study may be explained by the implementation of the Quartz event rules in the regulations of UTMB®. We can also wonder if information campaigns on the risks of NSAIDs may have change mentalities. A third hypothesis is a shift towards other painkillers. Acetaminophen may be excluded regarding the results of the present study because the prevalence was similar to those reported in the literature. 7 However, other painkillers such as tramadol or codeine were not monitored in the present work. Thus, it will be relevant to include these compounds in further studies.
In previous studies, the prevalence of NSAIDs and acetaminophen consumption by ultratrailers were determined using a questionnaire only. 2 , 3 , 4 , 5 In the present work, prevalence was studied using three approaches: one based on a questionnaire filled by runners and two based on a direct research of NSAIDs in biological samples (OF and whole blood). For NSAIDs, prevalence was very different depending on the three tools. Value from the questionnaire was lower than those from the direct research in biological samples. This difference may be explained by a lack of knowledge of trailers about the name of the drugs belonging to NSAIDs. It could also be due to runners lying because they would be afraid to devaluate their performances. Nonetheless, the difference between questionnaire and direct research in DBS or OF is lesser for acetaminophen, which is not in favor of the second hypothesis. Anyway, the present data suggest that the study of the prevalence using a questionnaire may lead to an underestimation of the consumption.
Then, concerning the two studied biological matrices, the number of positive cases for NSAIDs was much higher with DBS than with OF. This can be explained by the fact that blood concentrations of NSAIDs are higher than salivary concentrations. As NSAIDs present percentage of binding to plasmatic proteins about 95%–99%, only a very small free fraction (unbound to plasmatic proteins) can cross from blood into OF because drugs have to be free to diffuse. This has been illustrated in this study by an OF/DBS concentration ratio of about 0.0003 for ibuprofen and 0.005 for ketoprofen. It was not possible to calculate the ratio for the other NSAIDs because they were under LLOQ or not found in saliva. In the literature, only few works reported this ratio for NSAID: 0.003 for piroxicam and 0.03 for salicylic acid. 20 , 21
The ability to detect a compound in the blood depends on several parameters such as the maximum blood concentration (Cmax), which is related to the dose absorbed, the elimination half‐life, and the LOD of the analytical method used. Elimination half‐life (t ½) is the time required to produce a 50% reduction in blood concentration. Based on these parameters, an estimation of the maximum time interval (MTI) during which the NSAIDs could be detected in blood was proposed according to the LOD obtained with the present assay. The MTI were (the values of Cmax and t 1/2 used to make the estimate was indicated in bracket) 26 h for ibuprofen (Cmax = 30 μg/ml, t ½ = 2 h), 22 h for diclofenac (Cmax = 3 μg/ml, t ½ = 2 h), 28 h for ketoprofen (Cmax = 10 μg/ml, t ½ = 2 h), 60 h for niflumic acid (Cmax = 20 μg/ml, t ½ = 4 h), and 150 h for naproxen (Cmax = 80 μg/ml, t ½ = 12 h). Thus, for NSAIDs that present t 1/2 around 2 h (e.g., ibuprofen, diclofenac, and ketoprofen), only the consumption during the trail can be detected because the runners take between 24 and 48 h to complete the 171‐km course. The same approach could not be applied for OF because no data concerning the kinetics of these NSAIDs in OF were available in literature. Thus, an estimation of the MTI for OF was proposed only for ibuprofen and ketoprofen based on the three previous parameters and the OF/DBS concentrations ratio. The MTI were (the value of the ratio used was indicated in bracket) 8 h for ibuprofen (ratio = 0.0003) and 18 h for ketoprofen (ratio = 0.005). Therefore, the detection window of NSAIDs in OF was about two‐ to three‐fold shorter than in blood. Table 2 summarizes advantages and limits of the three compared approaches to study prevalence.
TABLE 2.
Advantages and limits of questionnaire and direct research in dried blood spots (DBS) and oral fluid (OF) for prevalence determination of drug use during ultratrails
| Advantages | Limits | |
|---|---|---|
| Questionnaire | Inexpensive, non‐invasive | Indirect determination (leading to underestimation?), based on trust and knowledge |
| DBS | Direct determination, transport, storage | Detection interval ~24 h (depending on NSAIDs), cost (sampling and analysis), slightly invasive |
| OF | Direct determination, non‐invasive | Short detection interval, cost (sampling and analysis), transport, storage |
DBS and saliva were selected in this work thanks to their low intrusiveness, low invasiveness, and easy sampling. Urine could also be a relevant matrix because it is non‐invasive and drugs could be detected with a longer detection interval after intake than in blood and saliva. However, some limits can also be identified, such as difficulties to collect: dehydration of runners, practical organization for taking samples, intrusiveness of sampling. 22 Issues concerning transport and storage have to be considered if urine spot is not selected as a sample. Considering these limits, urinary samples were not included in this work.
5. CONCLUSION
The present work is the first study to determine the prevalence of NSAIDs and acetaminophen consumption by ultratrail runners during the race using a direct analysis in biological matrices. The comparison of results obtained by a questionnaire and the research in DBS and OF showed clearly that the DBS samples are the most relevant method to obtain the prevalence. The prevalence of NSAIDs determined from the results of DBS is close to 50%, which is high in view of the criteria of the Quartz event program (no consumption within 24 h before the start and during the race). It is therefore necessary to enhance the information concerning the consumption of NSAIDs in trail running, both from the point of view of the ethics of the sport and on the aspects of health and risks for the runners.
CONFLICT OF INTEREST
No potential conflict of interest was reported by the authors.
Supporting information
Table S1. NSAIDs studied and their corresponding internal standard
Table S2. Questionnaire filled by runners.
ACKNOWLEDGEMENTS
We thank Dr Lars Petter Jordheim for editing the manuscript and Marie Sammons for her help on UTMB®.
Mashal MS, Bevalot F, Citterio‐Quentin A, et al. Comparative study between direct analysis in whole blood, oral fluid, and declaration of consumption for the prevalence of nonsteroidal anti‐inflammatory drugs and acetaminophen in ultratrail runners. Drug Test Anal. 2023;15(1):97‐103. doi: 10.1002/dta.3374
Funding information Mohammad Shafiq Mashal was supported by a French Government Bourse from the Foreign Ministry of France.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1. NSAIDs studied and their corresponding internal standard
Table S2. Questionnaire filled by runners.