Simultaneous quantification of caffeine and its main metabolites by gas chromatography mass spectrometry in horse urine.

Caffeine is a naturally occurring alkaloid and it is metabolized to paraxanthine, theophylline and theobromine. Analysis of caffeine and its metabolites is challenging since the metabolites theophylline and paraxanthine generate similar product and precursor ions. In this study, a new method was developed for the simultaneous analysis of caffeine, paraxanthine, theobromine and theophylline in horse urine using gas chromatography-mass spectrometry (GC-MS). Urine samples were treated using solid-phase extraction followed by the elution with dichloromethane-isopropanol (90:10) after the pH was adjusted to 6, and then derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide-1% trimethylchlorosilane before analysis with GC-MS. Sample preparation and derivatization steps were optimized and the method permitted elution all of these analytes within 13 min. The method was fully validated according to Commission Decision, 2002/657/EC guidelines. The calibration curves were linear with a correlation coefficient of >0.99. Precision and accuracy were well within the 15% acceptance range and the method was robust. The validation results demonstrated that the method is highly reproducible, easily applicable and selective. The method was applied to urine samples collected from racehorses to demonstrate its applicability.

Investigating the Relations Between Caffeine-Derived Metabolites and Plasma Lipids in 2 Population-Based Studies.

OBJECTIVE: To investigate the relations between caffeine-derived metabolites (methylxanthines) and plasma lipids by use of population-based data from 2 European countries. METHODS: Families were randomly selected from the general population of northern Belgium (FLEMENGHO), from August 12, 1985, until November 22, 1990, and 3 Swiss cities (SKIPOGH), from November 25, 2009, through April 4, 2013. We measured plasma concentrations (FLEMENGHO, SKIPOGH) and 24-hour urinary excretions (SKIPOGH) of 4 methylxanthines-caffeine, paraxanthine, theobromine, and theophylline-using ultra-high-performance liquid chromatography-tandem mass spectrometry. We used enzymatic methods to estimate total cholesterol, high-density lipoprotein cholesterol, and triglyceride levels and the Friedewald equation for low-density lipoprotein cholesterol levels in plasma. We applied sex-specific mixed models to investigate associations between methylxanthines and plasma lipids, adjusting for major confounders. RESULTS: In both FLEMENGHO (N=1987; 1055 [53%] female participants) and SKIPOGH (N=990; 523 [53%] female participants), total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels increased across quartiles of plasma caffeine, paraxanthine, and theophylline (total cholesterol levels by caffeine quartiles in FLEMENGHO, male participants: 5.01±0.06 mmol/L, 5.05±0.06 mmol/L, 5.27±0.06 mmol/L, 5.62±0.06 mmol/L; female participants: 5.24±0.06 mmol/L, 5.15±0.05 mmol/L, 5.25±0.05 mmol/L, 5.42±0.05 mmol/L). Similar results were observed using urinary methylxanthines in SKIPOGH (total cholesterol levels by caffeine quartiles, male participants: 4.54±0.08 mmol/L, 4.94±0.08 mmol/L, 4.87±0.08 mmol/L, 5.27±0.09 mmol/L; female participants: 5.12±0.07 mmol/L, 5.21±0.07 mmol/L, 5.28±0.05 mmol/L, 5.28±0.07 mmol/L). Furthermore, urinary caffeine and theophylline were positively associated with high-density lipoprotein cholesterol in SKIPOGH male participants. CONCLUSION: Plasma and urinary caffeine, paraxanthine, and theophylline were positively associated with plasma lipids, whereas the associations involving theobromine were less clear. We postulate that the positive association between caffeine intake and plasma lipids may be related to the sympathomimetic function of methylxanthines, mitigating the overall health-beneficial effect of caffeine intake.

Comparison of Two Dietary Supplements for Treatment of Uric Acid Renal Lithiasis: Citrate vs. Citrate + Theobromine.

BACKGROUND: Uric acid (UA) renal lithiasis has a high rate of recurrence and a prevalence ranging from 10% and 15%, depending on the population. The most important etiological factor is persistence of urinary pH below 5.5 and one of the most common treatments is alkalization with citrate. Recent studies demonstrated that theobromine, which is abundant in chocolate and cocoa, is a potent inhibitor of UA crystallization. AIM: The aim was to compare the efficacy of citrate versus citrate + theobromine as treatment for UA lithiasis. METHODS: This randomized cross-over trial investigated the efficacy of two treatments in 47 patients with UA renal lithiasis. Urine volume, pH, UA excretion, theobromine excretion, and risk of UA crystallization (RUAC) at baseline and at the end of each intervention period were measured. RESULTS: Each treatment significantly reduced the risk of UA crystallization compared to basal values. The RUAC after citrate + theobromine was lower than the RUAC after citrate, although this difference was not statistically significant. CONCLUSION: The combined consumption of citrate and theobromine may be a promising strategy for the prevention of UA kidney stones.

Evaluating Novel Markers for Specimen Validity Testing.

CONTEXT.­: Synthetic urine products are commercially marketed for the purpose of specimen substitution for urine drug screens. These products are widely popular because they yield negative drug screen results, meet criteria for specimen validity testing, and are easily accessible and affordable. Current specimen validity criteria are ineffective for detecting these synthetic products, and new markers of specimen validity are required. OBJECTIVE.­: To develop and evaluate a multicomponent liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for urine specimen validity testing. DESIGN.­: A quantitative LC-MS/MS assay was developed for caffeine, cotinine, theobromine, and urobilin in urine. The assay was applied to known synthetic urine products (n = 10) as well as human specimens received for pre-employment testing (n = 500), for-cause workplace testing (n = 100), and medical pain management monitoring (n = 200). Specimens devoid of all 4 validity markers were subjected to follow-up testing that involved microscopic urinalysis and comprehensive gas chromatography mass spectrometry for drugs, pharmaceuticals, hormones, and lipids. RESULTS.­: Of the experimental groups, 10 of 10 synthetic urine products (100%), 12 of 500 pre-employment specimens (2.4%), and 4 of 200 pain management specimens (2.0%) failed the experimental LC-MS/MS assay. Follow-up testing indicated that each of the failed specimens was nonphysiologic in nature. CONCLUSIONS.­: Simultaneous application of the 4 experimental validity markers appeared to be a robust method for detecting nonphysiologic specimens. New markers of specimen validity must be developed in order to identify commercially available synthetic urine products.

On-line in-tube solid phase microextraction coupled to capillary liquid chromatography-diode array detection for the analysis of caffeine and its metabolites in small amounts of biological samples.

In-tube solid phase microextraction (IT-SPME) coupled on-line to capillary liquid chromatography with diode array detection provides a simple and fast analytical methodology for the simultaneous quantitation of caffeine and its three primary metabolites (theobromine, paraxanthine and theophylline) in micro samples of serum, saliva and urine matrices. The sample amount required for one analysis was only 2.5 µL of saliva, 6.25 µL of serum or 40 µL of urine, a requirement for its implementation in a hospital laboratory for preterm newborns, where sample availability is a major problem. In standard conditions, 25 µL of diluted saliva or serum (or 100 µL of urine) were processed by IT-SPME in 30 cm of commercially available capillary GC column coated with ZB-FFAP (100% nitroterephthalic modified polyethylene glycol). The retained compounds were desorbed from the IT-SPME capillary by the mobile phase (a gradient mixture of water and methanol) and the separation was carried out in a C18 column (150 mm × 0.5 mm i.d., 5 µm particle size). Analytes eluted before 14 min, at a flow rate of 15 µL min-1, and were detected by absorbance at 275 nm. The calibration graphs presented good linearity (R2 > 0.99), without the presence of matrix effect, and recoveries between 84 and 112% were obtained. Limits of detection (S/N = 3) were 0.1 µg·mL-1 in serum and 0.5 µg·mL-1 in saliva and urine samples, for all compounds, and the intra- and inter-day variation coefficients (n = 3) were between 3 and 17%. Analytical figures of merit were similar to those proposed by other methodologies, but using lower sample volume and a faster and simpler sample treatment and analysis. Paired samples of serum and saliva from preterm newborns treated with caffeine at the pediatric intensive care unit were analyzed by the method, with statistically equivalent results for caffeine concentrations.

Effect of Consumption of Cocoa-Derived Products on Uric Acid Crystallization in Urine of Healthy Volunteers.

The purpose of this study was to determine the effects of consumption of different cocoa-derived products on uric acid crystallization in urine of 20 healthy volunteers. Participants were requested to select the specific diet that they wished to follow during the 12 h prior to collection of urine. The only restriction was that the diet could not include any product with cocoa, coffee, or caffeine. On the first day, each volunteer followed their selected diet, and an overnight 12 h urine sample was collected as the baseline urine. After seven days on an unrestricted diet, each volunteer repeated the same diet with 20 g of milk chocolate, chocolate powder, or dark chocolate during breakfast and another 20 g during dinner. Overnight 12 h urine samples were then collected. Urine volume, pH, oxalate, creatinine, uric acid, theobromine, and a uric acid crystallization test were determined for each sample. The results for all 20 patients show that uric acid crystallization was significantly lower following the consumption of chocolate powder or dark chocolate relative to baseline or following the consumption of milk chocolate. The results indicated that increased concentrations of urinary theobromine reduced the risk of uric acid crystallization.

Correction.

In the article by Guessous et al (Guessous I, Pruijm M, Ponte B, Ackermann D, Ehret G, Ansermot N, Vuistiner P, Staessen J, Gu Y, Paccaud F, Mohaupt M, Vogt B, Pechère-Bertschi A, Martin PY, Burnier M, Eap CB, Bochud M. Associations of ambulatory blood pressure with urinary caffeine and caffeine metabolite excretions. Hypertension. 2015;65:691­696. doi: 10.1161/HYPERTENSIONAHA.114.04512), which published online ahead of print December 8, 2014, and appeared in the March 2015 issue of the journal, a correction was needed.One of the author surnames was misspelled. Antoinette Pechère-Berstchi has been corrected to read Antoinette Pechère-Bertschi.The authors apologize for this error.

HPLC method for urinary theobromine determination: Effect of consumption of cocoa products on theobromine urinary excretion in children.

OBJECTIVE: To validate a simple method of urinary theobromine determination, to assess urinary theobromine levels in 80 healthy children and to relate these levels to consumption of cocoa products. DESIGN AND METHODS: Urine samples were diluted, directly injected into an HPLC system, separated by gradient elution on a C18 column, and detected by UV spectrometry. The method was validated for linearity, limits of detection and quantification, imprecision, accuracy, recovery and interferences. The proposed method was used to assess 12-h day and 12-h night urinary theobromine excretion by 80 healthy children, divided into four groups based on consumption of cocoa products. In addition, urinary excretion of magnesium and oxalate, also present in cocoa, was measured in these four groups. RESULTS: The method was linear to a theobromine concentration of 278µmol/L (50mg/L). LOD and LOQ for urine samples, diluted 1:5 (vol/vol) with water, were 1.1 and 3.6µmol/L respectively. Within-run and between-run imprecisions (CV) were each <2%. Average recovery was 99%, and analysis of a certified reference sample showed an error <2.5%. Theobromine excretion levels were significantly higher in healthy children with higher consumption of cocoa products (p<0.001), but oxalate (p=0.098) and magnesium (p=0.068) excretion levels did not differ significantly. CONCLUSION: This validated method resulted in urinary theobromine determination with 100% recovery, without sample pretreatment. Urinary theobromine levels in healthy children were directly related to their consumption of cocoa products.

Associations of ambulatory blood pressure with urinary caffeine and caffeine metabolite excretions.

Intake of caffeinated beverages might be associated with reduced cardiovascular mortality possibly via the lowering of blood pressure. We estimated the association of ambulatory blood pressure with urinary caffeine and caffeine metabolites in a population-based sample. Families were randomly selected from the general population of Swiss cities. Ambulatory blood pressure monitoring was conducted using validated devices. Urinary caffeine, paraxanthine, theophylline, and theobromine excretions were measured in 24 hours urine using ultrahigh performance liquid chromatography tandem mass spectrometry. We used mixed models to explore the associations of urinary excretions with blood pressure although adjusting for major confounders. The 836 participants (48.9% men) included in this analysis had mean age of 47.8 and mean 24-hour systolic and diastolic blood pressure of 120.1 and 78.0 mm Hg. For each doubling of caffeine excretion, 24-hour and night-time systolic blood pressure decreased by 0.642 and 1.107 mm Hg (both P values <0.040). Similar inverse associations were observed for paraxanthine and theophylline. Adjusted night-time systolic blood pressure in the first (lowest), second, third, and fourth (highest) quartile of paraxanthine urinary excretions were 110.3, 107.3, 107.3, and 105.1 mm Hg, respectively (P trend <0.05). No associations of urinary excretions with diastolic blood pressure were generally found, and theobromine excretion was not associated with blood pressure. Anti-hypertensive therapy, diabetes mellitus, and alcohol consumption modify the association of caffeine urinary excretion with systolic blood pressure. Ambulatory systolic blood pressure was inversely associated with urinary excretions of caffeine and other caffeine metabolites. Our results are compatible with a potential protective effect of caffeine on blood pressure.

Quantification of theobromine and caffeine in saliva, plasma and urine via liquid chromatography-tandem mass spectrometry: a single analytical protocol applicable to cocoa intervention studies.

Targeted analyses of clinically relevant metabolites in human biofluids often require extensive sample preparation (e.g., desalting, protein removal and/or preconcentration) prior to quantitation. In this report, a single ultra-centrifugation based sample pretreatment combined with a designed liquid chromatography-tandem mass spectrometry (LC-MS/MS) protocol provides selective quantification of 3,7-dimethylxanthine (theobromine) and 1,3,7-trimethylxanthine (caffeine) in human saliva, plasma and urine samples. The optimized chromatography permitted elution of both analytes within 1.3 min of the applied gradient. Positive-mode electrospray ionization and a triple quadruple MS/MS instrument operated in multiple reaction mode were used for detection. (13)C(3) isotopically labeled caffeine was included as an internal standard to improve accuracy and precision. Implementing a 20-fold dilution of the isolated low MW biofluid fraction prior to injection effectively minimized the deleterious contributions of all three matrices to quantitation. The assay was linear over a 160-fold concentration range from 2.5 to 400 micromol L(-1) for both theobromine (average R(2) 0.9968) and caffeine (average R(2) 0.9997) respectively. Analyte peak area variations for 2.5 micromol L(-1) caffeine and theobromine in saliva, plasma and urine ranged from 5 and 10% (intra-day, N=10) to 9 and 13% (inter-day, N=25) respectively. The intra- and inter-day precision of theobromine and caffeine elution times were 3 and <1% for all biofluids and concentrations tested. Recoveries for caffeine and theobromine ranged from 114 to 118% and 99 to 105% at concentration levels of 10 and 300 micromol L(-1). This validated protocol also permitted the relative saliva, plasma and urine distribution of both theobromine and caffeine to be quantified following a cocoa intervention.

Direct injection liquid chromatography/electrospray ionization mass spectrometric horse urine analysis for the quantification and confirmation of threshold substances for doping control. II. Determination of theobromine.

In equine sport, theobromine is prohibited with a threshold level of 2 microg mL(-1) in urine, hence doping control laboratories have to establish quantitative and qualitative methods for its determination. Two simple liquid chromatography/mass spectrometry (LC/MS) methods for the identification and quantification of theobromine were developed and validated using the same sample preparation procedure but different mass spectrometric systems: ion trap mass spectrometry (ITMS) and time-of-flight mass spectrometry (TOFMS). Particle-free diluted urine samples were directly injected into the LC/MS systems, avoiding the time-consuming extraction step. 3-Propylxanthine was used as the internal standard. The tested linear range was 0.75-15 microg mL(-1). Matrix effects were evaluated analyzing calibration curves in water and different fortified horse urine samples. A great variation in the signal of theobromine and the internal standard was observed in different matrices. To overcome matrix effects, a standard additions calibration method was applied. The relative standard deviations of intra- and inter-day analysis were lower than 8.6 and 7.2%, respectively, for the LC/ITMS method and lower than 5.7 and 5.8%, respectively, for the LC/TOFMS method. The bias was less than 8.7% for both methods. The methods were applied to two case samples, demonstrating simplicity, accuracy and selectivity.

Development of in-tube solid-phase microextraction coupled to pressure-assisted CEC and its application to the analysis of propranolol enantiomers in human urine.

A successful hyphenation of in-tube solid-phase microextraction (SPME) and pressure-assisted CEC (pCEC) was developed by installing a poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolithic capillary to the six-port valve in a CEC system. The device designed was appropriate for on-line in-tube SPME coupled to pCEC or muHPLC. The evaluation of this hyphenation was first carried out for in-tube SPME-muHPLC with analytical capillaries packed with 3 microm octadecyl silica (ODS). Theobromine (TB), theophylline (TP), and caffeine (CA) were chosen as model drugs for an easy comparison with the results obtained by in tube SPME-HPLC. The detection limits of these three analytes were improved more than 100 times when compared with the direct analysis by muHPLC. Then in-tube SPME-pCEC with CEC capillaries packed with perphenylcarbamoylated beta-CD-bonded silica particles was applied to the determination and analysis of propranolol enantiomers in human urine. Under optimal extraction and separation conditions, the experimental LODs were 4 and 7 ng/mL for (S)-propranolol and (R)-propranolol, respectively. The calibration curves showed good linearity for both (S)-propranolol (R(2) = 0.9997) and (R)-propranolol (R(2) = 0.9996) over the concentration range from 20 to 5000 ng/mL. Reproducibility of the method was also investigated with intra- and interday precisions lower than 10% for both enantiomers at different concentration levels.

Urinary excretion of dietary contaminants in horses.

REASONS FOR PERFORMING STUDY: Presence of drugs is completely prohibited in post racing urine samples by most of racing and competition authorities, even if environmental contamination might occur. OBJECTIVES: To assess the daily dose of several contaminants absorbed through the diet that would result in detectable concentrations in urine. METHODS: Caffeine, theobromine, theophylline, atropine, scopolamine, bufotenine, DMT or morphine were administered orally to 6 horses, in different dosages, for 3 days before their urine was sampled for regular anti-doping tests. RESULTS: Theobromine, theophylline, bufotenine and morphine daily intake >10 mg, 2 mg, 10 mg and 200 microg, respectively, by a performance horse, were found to result in detectable urinary concentrations. At the 2 tested doses, atropine (5 and 15 mg) and dimethyltryptamine (3 and 10 mg) were not detected in urine. For caffeine and scopolamine, even the lowest dosage tested (5 mg/horse/day and 2 mg/horse/day respectively) induced detectable concentrations of the molecule in urine. CONCLUSIONS: Horses fed dietary contaminants, even at level much below the effective dosage, may be positive to antidoping urine analysis. Further research is needed to gain more confident results on a daily safe intake for caffeine and scopolamine. POTENTIAL RELEVANCE: Selection of feed materials appears to be of great importance to prevent non voluntary positive result to anti-doping tests.

Urinary methylxanthine and autistic disorder: absence of previously reported correlation.

We were unable to reveal significant difference in the levels of xanthine and methylxanthines in the urine samples from 59 patients diagnosed with autistic symptoms and 64 age- and sex-matched normal volunteers. Our data suggest that abnormalities in xanthine and methylxanthine excretion (US Patent 20020019406 A1, Feb. 12, 2002) represent distincly uncommon symptoms in autism.

Separation, pre-concentration, and HPLC analysis of methylxanthines in urine samples.

An SPE method, using RP18 phases, for the simultaneous extraction of caffeine, theobromine, theophylline, paraxanthine, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, 1-methyluric acid, 1,3-dimethyluric acid, 1,7-dimethyluric acid, and 1,3,7-trimethyluric acid from urine has been developed. Besides a gradient HPLC system for the analysis of the compounds of interest on a LiChrosorb RP-18 (7 microm) column with mobile phase containing 0.05% aq. solution of trifluoroacetic acid and acetonitrile has been elaborated. The procedure has been successfully applied to the analysis of methylxanthines and methyluric acids in urine of patients with chronic asthma treated with theophylline and in urine of healthy subjects.

Electrospray ionization mass spectrometric characterization and quantitation of xanthine derivatives using isotopically labelled analogues: an application for equine doping control analysis.

Isotope-dilution mass spectrometry has been employed successfully in numerous fields of analytical chemistry enabling the establishment of fast and reliable procedures. In equine sports, xanthine derivatives such as caffeine and theobromine are prohibited, and doping control laboratories analyze horse urine specimens regarding these illicit performance-enhancing drugs. Theobromine has to exceed a threshold level of 2 microg/mL, hence a robust and reliable quantitation is required. Stably deuterated theobromine and caffeine were synthesized by the reaction of xanthine or theobromine with iodomethane-d3 in the presence of N-methyl-N-trimethylsilyltrifluoroacetamide or potassium carbonate in acetonitrile, respectively. Both compounds were characterized by nuclear magnetic resonance spectroscopy and electrospray ionization tandem mass spectrometry, and a robust and fast assay for the qualitative and quantitative analysis of theobromine in equine urine samples was validated. Urine specimens were extracted by means of solid-phase extraction cartridges, and concentrated extracts were analyzed by liquid chromatography interfaced to a triple-quadrupole mass spectrometer. In addition, the dissociation behavior of deuterated analogues to caffeine and theobromine allowed proposals for fragmentation routes of xanthine derivatives after atmospheric pressure ionization and collisionally activated dissociation.

Simultaneous determination of caffeine, theobromine, and theophylline by high-performance liquid chromatography.

This work relates the development of an analytical methodology to simultaneously determine three methylxanthines (caffeine, theobromine, and theophylline) in beverages and urine samples based on reversed-phase high-performance liquid chromatography. Separation is made with a Bondesil C18 column using methanol-water-acetic acid or ethanol-water-acetic acid (20:75:5, v/v/v) as the mobile phase at 0.7 mL/min. Identification is made by absorbance detection at 273 nm. Under optimized conditions, the detection limit of the HPLC method is 0.1 pg/mL for all three methylxanthines. This method is applied to urine and to 25 different beverage samples, which included coffee, tea, chocolate, and coconut water. The concentration ranges determined in the beverages and urine are: < 0.1 pg/mL to 350 microg/mL and 3.21 microg/mL to 71.2 microg/mL for caffeine; < 0.1 pg/mL to 32 microg mL and < 0.1 pg/mL to 13.2 microg/mL for theobromine; < 0.1 pg/mL to 47 microg/mL and < 0.1 pg/mL to 66.3 microg/mL for theophylline. The method proposed in this study is rapid and suitable for the simultaneous quantitation of methylxanthines in beverages and human urine samples and requires no extraction step or derivatization.

The pharmacokinetics, metabolism and urinary detection time of caffeine in camels.

The pharmacokinetics of caffeine were determined in 10 camels after an intravenous dose of 2.35 mg kg(-1). The data obtained (median and range) were as follows. The elimination half-life (t(1/2)) was 31.4 (21.2 to 58.9) hours, the steady state volume of distribution (V(SS)) was 0.62 (0.51 to 0.74) litre kg(-1)and the total body clearance (Cl(T)) was 14.7 (8.70 to 19.7) ml kg(-1)per hour. Renal clearance estimated in two camels was 0.62 and 0.34 ml kg(-1)per hour. In vitro plasma protein binding (mean +/-SEM, n = 10) to a concentration of 2 and 8 microg ml(-1)was 36.0 +/- 0.24 and 39.2 +/- 0.36 per cent respectively. Theophylline and theobromine were identified as caffeine metabolites in serum and urine. The terminal elimination half-life of the former, estimated in two camels, was 70. 4 and 124.4 hours. Caffeine could be detected in the urine for 14 days.