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.

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.

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.

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.

Phenotyping of flavin-containing monooxygenase using caffeine metabolism and genotyping of FMO3 gene in a Korean population.

Flavin-containing monooxygenase (FMO) activity was determined in 82 Korean volunteers by taking molar concentration ratio of theobromine and caffeine present in the 1 h urine (between 4 and 5 h) samples collected after administration of a cup of coffee containing 110 mg of caffeine. Among 82 volunteers, there were 19 women and 63 men (30 smokers and 52 non-smokers). Volunteers were divided into two groups comprising low (0.53-2.99) and high (3.18-11.95) FMO activities separated by an antimode of 3.18. Peripheral bloods were sampled from these volunteers and their genomic DNAs were amplified by polymerase chain reaction with oligonucleotides designed from intronic sequences of human FMO3 gene. Comparing nucleotide sequences of the amplified FMO3 gene originating from randomly selected individuals with low and high FMO activities, nine point mutations were identified in the open reading frame sequences. Among these nine mutations, three FMO3 mutant types (FMO3/Stop148, Lys158 and Gly308) were selected and correlated with FMO activities observed in our Korean population. A rare FMO3/Stop148 mutant allele originating from FMO3/Gly148 occurred by substitution of G442T in exon 4 and yielded a premature TGA stop codon. The stop codon was detected in one individual having the second lowest FMO activity and he had the mutation in heterozygous state. In a pedigree study, he was found to have inherited the mutation from his mother who also had a heterozygous stop codon and equally low FMO activity. In our volunteers, two other common mutations were detected in exons 4 and 7. The one in exon 4 resulted from a G472A change eliminating a HinfI restriction site and produced an amino acid substitution from Glu158 to Lys. The other mutation in exon 7 resulted from an A923G change generating a DraII restriction site and produced a non-conservative replacement of Glu308 to Gly. Based on the secondary structure maps of FMO3 enzyme proteins for these two mutant types, FMO3/Gly308 mutation transformed the helix structure into a sheet shape and indicated that dysfunctional FMO3 may be produced. FMO3/Lys158 mutation did not alter the secondary structure. Approximately 80% of volunteers with homozygous and/or heterozygous mutations on either one or two of these mutations had low FMO activities. Thus, individuals with these FMO3 gene mutations may have defective metabolic activity for many clinically used drugs and dietary plant alkaloids which are oxidized primarily by hepatic FMO3.

High levels of methylxanthines in chocolate do not alter theobromine disposition.

Theobromine disposition was measured twice in 12 normal men, once after 14 days of abstention from all methylxanthines and once after 1 week of theobromine (6 mg/kg/day) in the form of dark chocolate. Mean theobromine t 1/2, apparent volume of distribution, and clearance after abstinence from all methylxanthines were 10.0 hours, 0.76 L/kg, and 0.88 ml/min/kg. High daily doses of chocolate for 1 week did not change these values. After subjects abstained from methylxanthines, urinary radioactivity over 72 hours after a single, oral dose of [8-14C]theobromine consisted of 42% 7-methylxanthine, 20% 3-methylxanthine, 18% theobromine, 10% 7-methyluric acid, and 10% 6-amino-5[N-methylformylamino]-1-methyluracil. A week of daily theobromine consumption in the form of dark chocolate also did not alter this urinary profile of theobromine and its metabolites. Although these results might appear to differ from other reports of inhibition of theobromine elimination after five consecutive daily doses of theobromine in aqueous suspensions, both the rate and extent of absorption of theobromine in chocolate were less than that of theobromine in solution. Relative bioavailability of theobromine in chocolate was 80% that of theobromine in solution. This reinforces the fundamental principle that both the metabolic and the therapeutic consequences of a particular chemical can differ when that chemical is given in the pure compared with the dietary form.

Urinary excretion of an uracilic metabolite from caffeine by rat, monkey and man.

Caffeine (C) metabolism has been studied in rat, monkey (Macaca cynomolgus) and man after oral administration of the compound. Eleven metabolites were quantified in urine; particular attention has been drawn to 4-amino [5-formyl methylamino]1,3-dimethyl uracil (ADMU) because of its structural analogies with 5-fluorouracil. The rat has been found to produce a much larger fraction of ADMU (about 30%) in respect to monkey an man (1-2%). The potential toxicological implications of this findings are discussed.

Determination of methylxanthine stimulants in urine of racing greyhounds by high-performance liquid chromatography. Resolution of a contested drug administration case.

Results of urine drug analyses for three racing greyhounds that tested positive for caffeine in this laboratory were contested by the animal trainer, who asserted that positives were achieved from deliberate chocolate feeding by a rival kennel. The metabolism and excretion of methylxanthines was examined by high-performance liquid chromatography of extracts of the urine of racing greyhounds force-fed either caffeine (No Doz) or chocolate (Hersheys chocolate drops). Samples from untreated animals served as controls. Study results showed that test animals fed chocolate could be very easily distinguished from those fed caffeine. While the former animals exhibited a prominent theobromine peak with trace amounts of caffeine and theophylline, the caffeine-treated animals gave a prominent caffeine peak with moderate theophylline levels and almost nondetectable theobromine amounts. When these results were compared with the results from the positive authentic racing animals, chocolate feeding was clearly ruled out as an alternative to caffeine administration.

Detection and determination of theobromine and caffeine in urine after administration of chocolate-coated peanuts to horses.

The objective of this study was to determine the urinary excretion of methylxanthines in horses following ingestion of chocolate over eight days. The study was performed in response to gas chromatography-mass spectrometry (GC-MS) confirmation of the presence of caffeine in a positive urine test in a racehorse. The trainer of the horse alleged that he often administered chocolate-coated peanuts as treats to his horses, and he believed that the ingestion of chocolate was responsible for the positive urine test. The urinary excretion of theobromine and caffeine after the ingestion of chocolate-coated peanuts was investigated in three horses. Enzyme-linked immunoassay (ELISA), high-performance liquid chromatography (HPLC), and GC-MS assays were performed on all urine specimens. Theobromine (HPLC) was detected for 72 h and caffeine (GC-MS) for 48 h after chronic ingestion of chocolate-coated peanuts. Methylxanthines were detected by ELISA for 120 h after administration of chocolate.

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.

The excretion of theobromine in Thoroughbred racehorses after feeding compounded cubes containing cocoa husk--establishment of a threshold value in horse urine.

Thoroughbred geldings were fed racehorse cubes containing a predetermined concentration of theobromine in the form of cocoa husk. They were offered 7 kg of cubes per day, divided between morning and evening feed, and food consumption was monitored. Urinary concentrations of theobromine were determined following the consumption of cubes containing 11.5, 6.6, 2.0 and 1.2 mg per kg of theobromine, to verify whether or not such concentrations would produce positive urine tests. Pre-dose urine samples were collected to verify the absence of theobromine before each experiment. It became apparent from the results of the first three administrations that the limit of detection of theobromine, using such procedures, would be reached at a feed level of about 1 mg per kg theobromine. Therefore the final administration, using cubes containing 1.2 mg per kg theobromine, was singled out for additional analytical work and quantitative procedures were developed to measure urinary concentrations of theobromine. It was anticipated that the results would form a basis for discussions relating to the establishment of a threshold value for theobromine in horse urine. The Stewards of the Jockey Club subsequently gave notice that they had established a threshold level for theobromine in urine of 2 micrograms/ml.

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.

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.

Urinary excretion of theobromine in horses given contaminated pelleted food.

A high pressure liquid chromatographic (HPLC) method for measuring the theobromine content in cocoa husks, pelleted food and horse urine is described. Starting with 2 ml of urine, concentrations of 500 ng/ml could easily be detected. When feed containing 38.4 mg of theobromine was given twice daily to horses for 2 1/2 days, two days were needed after the last intake before the theobromine concentrations fell below the threshold value of 2 micrograms/ml. The time at which the peak excretion rate occurred varied from 2 to 12 h after the last administration, while the excretion rate seemed to be dependent on the urinary flow. Theobromine could not be detected in plasma after administration in this way.