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Simple high-performance liquid chromatography method for the simultaneous determination of serum caffeine and paraxanthine following rapid sample preparation
Abstract
A simple reversed-phase high-performance liquid chromatography (HPLC) method for the simultaneous determination of caffeine and paraxanthine in human serum is described. Serum proteins are precipitated with perchloric acid and the resulting supernatant neutralized for direct injection onto an HPLC column. The method uses a phosphate–methanol mobile phase (85:15, v/v) at pH 4.9 with a flow-rate of 1.75 ml/min and quantitation is by UV absorbance at 274 nm. Elution times are approximately 18 min for caffeine and 8 min for paraxanthine. Theobromine and theophylline have elution times of 5.4 and 9.4 min and do not interfere in the assay. The intra-assay and between-assay means for precision and accuracy for both drugs are: 4.5% C.V. and 3.3% deviation. The sensitivity of the method is 50 ng/ml for each drug.
... Despite quantitating metabolites of caffeine (namely paraxanthine), few studies have published the precision, accuracy and recovery data for the paraxanthine assay. Of the methods which simultaneously determine both paraxanthine and caffeine, the only matrix used has been serum (Holland et al., 1998; Koch et al., 1999 ). Furthermore , only a few studies have determined the stability of caffeine while none have reported the stability of paraxanthine and caffeine at room temperature in saliva (Holland et al., 1998; Klebanoff et al., 1998; Koch et al., 1999; Tang and Kalow, 1996). ...
... Of the methods which simultaneously determine both paraxanthine and caffeine, the only matrix used has been serum (Holland et al., 1998; Koch et al., 1999 ). Furthermore , only a few studies have determined the stability of caffeine while none have reported the stability of paraxanthine and caffeine at room temperature in saliva (Holland et al., 1998; Klebanoff et al., 1998; Koch et al., 1999; Tang and Kalow, 1996). In comparison to other matricies, saliva has the advantage of being easily obtainable, economical and non-invasive, making it an ideal matrix to use in some specific populations such as children or frail older people. ...
... The applicability of this method was established in a study of two healthy male volunteers which demonstrated that: (1) the limit of quantification was appropriate to measure concentrations of caffeine and paraxanthine over a 24 h period; (2) saliva and plasma concentration ranges over 24 h fell within the validated range. The limit of quantification in this assay in plasma (0.025 mg/mL) and in saliva (0.05 mg/mL) is lower than that reported in previous studies and therefore would be more suitable for the 100 mg caffeine dose chosen, especially when measuring lower concentrations of caffeine and paraxanthine present at 24 h (Alkaysi et al., 1988; Blanchard and Sawers, 1982; Haughey et al., 1982; Holland et al., 1998; Koch et al., 1999; O'Connell and Zurzola, 1984). As shown inTable 1, a variety of internal standards and columns have been used in caffeine assays. ...
Caffeine has been extensively used as a probe to measure CYP1A2 activity in humans with caffeine clearance or the paraxanthine (major metabolite of caffeine) to caffeine concentration ratio being regarded as the preferred metric. A simple reverse-phased C(18) HPLC assay using ethyl acetate liquid-liquid extraction was developed to quantitate caffeine and paraxanthine concentrations in saliva and plasma. The mobile phase consisted of acetonitrile-acetic acid-H(2)O (100:1:899) and analytes were quantitated with UV detection at 280 nm. The extraction recovery for paraxanthine and caffeine was approximately 70% in both saliva and plasma. The assay was linear over the concentration ranges 0.05-2.50 and 0.05-5.00 µg/mL, for paraxanthine and caffeine, respectively, in saliva. In plasma the assay was linear over the ranges 0.025-2.50 and 0.025-5.00 µg/mL for paraxanthine and caffeine, respectively. Intra- and inter-assay precision and accuracy were less than 15%. Detection limits were 0.015 µg/mL for paraxanthine and caffeine in saliva, while it was 0.005 µg/mL for paraxanthine and caffeine in plasma. Utility was established in samples collected from two healthy volunteers who abstained from caffeine for 24 h and received a single 100 mg oral dose of caffeine. The assay developed is a robust, simple and precise technique to measure caffeine and paraxanthine in saliva and plasma of healthy volunteers after a single oral dose of caffeine.
... Plasma samples were aliquoted into Eppendorfs and stored at j80-C until analysis. Plasma [CAF] was analyzed using high-performance liquid chromatography (HPLC) as described by Holland et al. (16) with the following minor modifications: before injection onto the HPLC column, each sample was individually filtered (Mini-UniPrep syringeless filters; Fisher Scientific, UK) and no guard column was used. The method produced a coefficient of variation (CV) of 1.06% (range, 0.24%-1.45%). ...
... Although the main effect for differences in TAUC-FFA was not significant (P = 0.072), the ES ranged from small (AB vs PARA, ES = 0.47) to large (AB vs TETRA, ES = 0.85; PARA vs TETRA, ES = 1. 16). No significant difference in iAUC-FFA was observed (P = 0.357). ...
Purpose:
To investigate the absorption curve and acute effects of caffeine at rest in individuals with no spinal cord injury (SCI), paraplegia (PARA) and tetraplegia (TETRA).
Methods:
Twenty-four healthy males (8 able-bodied (AB), 8 PARA and 8 TETRA) consumed 3 mg[BULLET OPERATOR]kg caffeine anhydrous (CAF) in a fasted state. Plasma caffeine [CAF], glucose, lactate, free-fatty acid [FFA] and catecholamine concentrations were measured during a 150 min rest period.
Results:
Peak [CAF] was greater in TETRA (21.5 ?M) compared to AB (12.2 ?M) and PARA (15.1 ?M), and mean peak [CAF] occurred at 70, 80 and 80 min, respectively. Moderate and large ES were revealed for TETRA compared to PARA and AB (-0.55 and -1.14, respectively) for the total area under the [CAF] versus time curve. Large inter-individual responses were apparent in SCI groups. The change in plasma catecholamine concentrations following CAF did not reach significance (p>0.05) however both adrenaline and noradrenaline concentrations were lowest in TETRA. Significant increases in [FFA] were seen over time (p<0.0005) but there was no significant influence of SCI level. Blood lactate concentration reduced over time (p=0.022) whereas blood glucose concentration decreased modestly (p=0.695), and no difference between groups was seen (p>0.05).
Conclusion:
Level of SCI influenced the caffeine absorption curve and there was large inter-individual variation within and between groups. Individual curves should be considered when using caffeine as an ergogenic aid in athletes with an SCI. The results indicate TETRA should trial low doses in training and PARA may consider consuming caffeine greater than 60 min prior to exercise performance. The study also supports caffeine's direct effect on adipose tissue, which is not secondary to catecholamine release.
... A high-performance liquid chromatography (HPLC) method [16] was used for determination of plasma caffeine, paraxanthine, theophylline and theobromine concentrations with modifications as described. Plasma samples were thawed immediately before the assay, and deproteinized by adding 350 µL of plasma to 350 µL of 0.8 M perchloric acid. ...
... The column was flushed and re-equilibrated with 18 mL 0.1 % TFA between runs. Caffeine and its metabolites were read at 274 nm and concentrations were calculated from peak area with the following retention times (min): theobromine, 15.20; theophylline, 16 ...
In a randomised, double-blind, placebo-controlled crossover design, 10 females taking monophasic oral contraceptives completed 90 min intermittent treadmill-running 45 min after ingestion of 6 mg∙kg(-1) body mass anhydrous caffeine or artificial sweetener (placebo). Water (3 mL∙kg(-1)) was provided every 15 min during exercise. Venous blood samples were taken before, during and after exercise, as well as after sleep (~15 h post-ingestion), and levels of caffeine, paraxanthine, theobromine and theophylline were measured using high-performance liquid chromatography. Sleep quality was assessed using the Leeds Sleep Evaluation Questionnaire. Plasma caffeine concentration peaked 100 min after ingestion. Caffeine clearance was 0.95±0.14 mL·min(-1)·kg(-1) while the elimination half-life of caffeine was 17.63±8.06 h. Paraxanthine and theophylline levels were significantly elevated at 15 h with no significant change in theobromine. Sleep latency and subsequent quality of sleep was impaired following caffeine supplementation (P<0.05); there were no differences between trials for how participants were feeling upon awakening. This is the first controlled study to examine caffeine supplementation on sleep quality in female athletes taking a low-dose monophasic oral contraceptive steroid following an intermittent-exercise running protocol. The data shows that female athletes using monophasic oral contraceptive steroids will have impaired sleep quality following evening caffeine ingestion.
© Georg Thieme Verlag KG Stuttgart · New York.
... These non-invasive phenotyping methods are based on the HPLC determination of the paraxanthine/caffeine ratio in saliva and urine after oral ingestion of a cup of coffee containing approximately 200 mg caffeine. The major pathway of the biotransformation of caffeine is the metabolism into paraxanthine by CYP1A2 (73 -80%), theobromine (10%) and theophylline (10%) (Holland, Godfredsen, Page and Connor, 1998). As described in the literature, caffeine is a suitable marker substance for phenotyping CYP1A2 (Bendriss, Markoglou and Wainer, 2000;Carillo et al., 2000;Holland, Godfredsen, Page and Connor, 1998;Koch et al., 1999;Nyéki, Biollaz, Kesselring and Décosterd, 2001). ...
... The major pathway of the biotransformation of caffeine is the metabolism into paraxanthine by CYP1A2 (73 -80%), theobromine (10%) and theophylline (10%) (Holland, Godfredsen, Page and Connor, 1998). As described in the literature, caffeine is a suitable marker substance for phenotyping CYP1A2 (Bendriss, Markoglou and Wainer, 2000;Carillo et al., 2000;Holland, Godfredsen, Page and Connor, 1998;Koch et al., 1999;Nyéki, Biollaz, Kesselring and Décosterd, 2001). The substance is well-tolerated and shows a fast and complete gastrointestinal absorption, as well as a short half-life time. ...
The purpose of this research is to impart basic knowledge about the cytochrome P450 metabolism of xenobiotics and its meaning for pharmaceutical and medical problems by means of experiment-led learning. The teaching approach includes an experimental part and bibliographic investigations as well as a discussion of the obtained results. The experimental work describes two simple methods for phenotyping cytochrome P450 1A2 in humans by HPLC-UV measurement of caffeine and its main metabolite paraxanthine in urine and saliva. These simple and inexpensive methods are applicable to demonstrate the importance of cytochrome P450 enzymes concerning drug inefficacy, drug-drug interactions and adverse drug reactions. Two simple and fast HPLC-UV methods for phenotyping cytochrome P450 1A2 in humans are presented. Using these methods, probands can be classified into poor and fast metabolisers by the determination of the paraxanthine/caffeine ratio in saliva and urine. Since our HPLC methods are inexpensive and simple to carry out, the experiments are particularly suitable for pharmaceutical and medical student laboratories.
... Blood plasma was then recovered and immediately frozen in liquid nitrogen and was subsequently stored at −80°C. Plasma caffeine and free carnitine concentrations were assayed at a later date via high-performance liquid chromatography (Holland et al., 1998) and a radioenzymatic assay (Cederblad et al., 1982), respectively. The pHOx Ultra analyzer did not work for one of the participants and so blood Na + and K + concentration data are presented as n = 5. ...
Increasing skeletal muscle carnitine content can manipulate fuel metabolism and improve exercise performance. Intravenous insulin infusion during hypercarnitinemia increases plasma carnitine clearance and Na+‐dependent muscle carnitine accretion, likely via stimulating Na+/K+ ATPase pump activity. We hypothesized that the ingestion of high‐dose caffeine, also known to stimulate Na+/K+ ATPase activity, would stimulate plasma carnitine clearance during hypercarnitinemia in humans. In a randomized placebo‐controlled study, six healthy young adults (aged 24 ± 5 years, height 175 ± 8 cm, and weight 70 ± 13 kg) underwent three 5‐h laboratory visits involving the primed continuous intravenous infusion of l‐carnitine (CARN and CARN + CAFF) or saline (CAFF) in parallel with ingestion of caffeine (CARN + CAFF and CAFF) or placebo (CARN) at 0, 2, 3, and 4 h. Regular blood samples were collected to determine concentrations of blood Na+ and K+, and plasma carnitine and caffeine, concentrations. Caffeine ingestion (i.e., CAFF and CARN + CAFF conditions) and l‐carnitine infusion (i.e., CARN and CARN + CAFF) elevated steady‐state plasma caffeine (to ~7 μg·mL−1) and carnitine (to ~400 μmol·L−1) concentrations, respectively, throughout the 5 h infusions. Plasma carnitine concentration was ~15% lower in CARN + CAFF compared with CARN during the final 90 min of the infusion (at 210 min, 356 ± 96 vs. 412 ± 94 μmol·L−1; p = 0.0080: at 240 min, 350 ± 91 vs. 406 ± 102 μmol·L−1; p = 0.0079: and at 300 min, 357 ± 91 vs. 413 ± 110 μmol·L−1; p = 0.0073, respectively). Blood Na+ concentrations were greater in CAFF and CARN + CAFF compared with CARN. Ingestion of high‐dose caffeine stimulates plasma carnitine clearance during hypercarnitinemia, likely via increased Na+/K+ ATPase activity. Carnitine co‐ingestion with caffeine may represent a novel muscle carnitine loading strategy in humans, and therefore manipulate skeletal muscle fuel metabolism and improve exercise performance.
... Serum caffeine and paraxanthine concentrations were measured by high-performance liquid chromatography (HPLC) using a method modified from Holland et al. (1998). In Fig. 1 Schematic of experimental procedures. ...
PurposeTo determine the influence of two commonly occurring genetic polymorphisms on exercise, cognitive performance, and caffeine metabolism, after caffeine ingestion.Methods
Eighteen adults received caffeine or placebo (3 mg kg−1) in a randomised crossover study, with measures of endurance exercise (15-min cycling time trial; 70-min post-supplementation) and cognitive performance (psychomotor vigilance test; PVT; pre, 50 and 95-min post-supplementation). Serum caffeine and paraxanthine were measured (pre, 30 and 120-min post-supplementation), and polymorphisms in ADORA2A (rs5751876) and CYP1A2 (rs762551) genes analysed.ResultsCaffeine enhanced exercise performance (P < 0.001), but effects were not different between participants with ADORA2A ‘high’ (n = 11) vs. ‘low’ (n = 7) sensitivity genotype (+ 6.4 ± 5.8 vs. + 8.2 ± 6.8%), or CYP1A2 ‘fast’ (n = 10) vs. ‘slow’ (n = 8) metabolism genotype (+ 7.2 ± 5.9 vs. + 7.0 ± 6.7%, P > 0.05). Caffeine enhanced PVT performance (P < 0.01). The effect of caffeine was greater for CYP1A2 ‘fast’ vs. ‘slow’ metabolisers for reaction time during exercise (− 18 ± 9 vs. − 1.0 ± 11 ms); fastest 10% reaction time at rest (− 18 ± 11 vs. − 3 ± 15 ms) and lapses at rest (− 3.8 ± 2.7 vs. + 0.4 ± 0.9) (P < 0.05). There were no PVT differences between ADORA2A genotypes (P > 0.05). Serum caffeine and paraxanthine responses were not different between genotypes (P > 0.05).Conclusion
Caffeine enhanced CYP1A2 ‘fast’ metabolisers’ cognitive performance more than ‘slow’ metabolisers. No other between-genotype differences emerged for the effect of caffeine on exercise or cognitive performance, or metabolism.
... Samples were frozen immediately at − 20°C, before being transferred to a − 80°C freezer. Plasma caffeine, paraxanthine, theobromine and theophylline concentrations were quantified via high-performance liquid chromatography [18]. The ratio of paraxanthine to caffeine (PX:CA) at the end of the PRE trial, i.e. when the concentration of both metabolites was at a steady state, was calculated as an index of CYP1A2 activity. ...
Background:
Pre-exercise supplements containing low doses of caffeine improve endurance exercise performance, but the most efficacious time for consumption before intense endurance exercise remains unclear, as does the contribution of caffeine metabolism.
Methods:
This study assessed the timing of a commercially available supplement containing 200 mg of caffeine, 1600 mg of β-alanine and 1000 mg of quercetin [Beachbody Performance Energize, Beachbody LLC, USA] on exercise performance, perception of effort and plasma caffeine metabolites. Thirteen cyclists (V̇O2max 64.5 ± 1.4 ml kg- 1 min- 1 (± SEM)) completed four experimental visits consisting of 30 min of steady-state exercise on a cycle ergometer at 83 ± 1% V̇O2max followed by a 15-min time trial, with perceived exertion measured regularly. On three of the visits, participants consumed caffeine either 35 min before steady-state exercise (PRE), at the onset of steady-state (ONS) or immediately before the time trial (DUR) phases, with a placebo consumed at the other two time points (i.e. three drinks per visit). The other visit (PLA) consisted of consuming the placebo supplement at all three time points. The placebo was taste-, colour- and calorie-matched.
Results:
Total work performed during the time trial in PRE was 5% greater than PLA (3.53 ± 0.14 vs. 3.36 ± 0.13 kJ kg- 1 body mass; P = 0.0025), but not ONS (3.44 ± 0.13 kJ kg- 1; P = 0.3619) or DUR (3.39 ± 0.13 kJ kg- 1; P = 0.925), which were similar to PLA. Perceived exertion was lowest during steady-state exercise in the PRE condition (P < 0.05), which coincided with elevated plasma paraxanthine in PRE only (P < 0.05).
Conclusion:
In summary, ingestion of a pre-exercise supplement containing 200 mg caffeine 35 min before exercise appeared optimal for improved performance in a subsequent fatiguing time trial, possibly by reducing the perception of effort. Whether this was due to increased circulating paraxanthine requires further investigation.
Trial registration:
ClinicalTrials.Gov, NCT02985606 ; 10/26/2016.
... 16 The remaining 5 mL was dispensed into tubes containing clotting activator and left for approximately 1 h prior to centrifugation at 1750 × g for 10 min at 4 • C. The resulting serum was stored at −21 • C for the subsequent determination of cortisol and prolactin with ELISA (DRG diagnostic, Germany) and caffeine with reverse-phase HPLC. 17 All data were analysed using IBM SPSS statistics version 22.0. Normality of distribution was determined using the Shapiro-Wilk test. ...
Objectives:
This study investigated the influence of a moderate caffeine dose on endurance cycle performance and thermoregulation during prolonged exercise in high ambient temperature.
Design:
Double-blind cross-over study.
Methods:
Eight healthy, recreationally active males (mean±SD; age: 22±1 years; body mass: 71.1±8.5kg; VO2peak: 55.9±5.8mLkg(-1)min(-1); Wmax: 318±37W) completed one VO2peak test, one familiarisation trial and two experimental trials. After an overnight fast, participants ingested a placebo or a 6mgkg(-1) caffeine dose 60min before exercise. The exercise protocol consisted of 60min of cycle exercise at 55% Wmax, followed by a 30min performance task (total kJ produced) in 30°C and 50% RH.
Results:
Performance was enhanced (Cohen's d effect size=0.22) in the caffeine trial (363.8±47.6kJ) compared with placebo (353.0±49.0kJ; p=0.004). Caffeine did not influence core (p=0.188) or skin temperature (p=0.577) during exercise. Circulating prolactin (p=0.572), cortisol (p=0.842) and the estimated rates of fat (p=0.722) and carbohydrate oxidation (p=0.454) were also similar between trial conditions. Caffeine attenuated perceived exertion during the initial 60min of exercise (p=0.033), with no difference in thermal stress across trials (p=0.911).
Conclusions:
Supplementation with 6mgkg(-1) caffeine improved endurance cycle performance in a warm environment, without differentially influencing thermoregulation during prolonged exercise at a fixed work-rate versus placebo. Therefore, moderate caffeine doses which typically enhance performance in temperate environmental conditions also appear to benefit endurance performance in the heat.
... The remaining blood (5 mL) was dispended into tubes containing clotting activator and left at room temperature for at least 60 min before centrifugation at 1750 g for 10 min at 4°C. The supernatant was stored at −21°C for the determination of serum prolactin and cortisol in duplicate via ELISA (DRG diagnostics, Germany) and serum caffeine in duplicate with reverse-phase HPLC as previously described (Holland, Godfredsen, Page, & Connor, 1998). The intra-assay coefficient of variation (CV) for serum prolactin, cortisol and caffeine was 4.9%, 5.3% and 2.9%, respectively. ...
This study examined effects of 4 weeks of caffeine supplementation on endurance performance. Eighteen low-habitual caffeine consumers (<75 mg · day⁻¹) were randomly assigned to ingest caffeine (1.5–3.0 mg · kg⁻¹day⁻¹; titrated) or placebo for 28 days. Groups were matched for age, body mass, V̇O2peak and Wmax (P > 0.05). Before supplementation, all participants completed one V̇O2peak test, one practice trial and 2 experimental trials (acute 3 mg · kg⁻¹ caffeine [precaf] and placebo [testpla]). During the supplementation period a second V̇O2peak test was completed on day 21 before a final, acute 3 mg · kg⁻¹ caffeine trial (postcaf) on day 29. Trials consisted of 60 min cycle exercise at 60% V̇O2peak followed by a 30 min performance task. All participants produced more external work during the precaf trial than testpla, with increases in the caffeine (383.3 ± 75 kJ vs. 344.9 ± 80.3 kJ; Cohen’s d effect size [ES] = 0.49; P = 0.001) and placebo (354.5 ± 55.2 kJ vs. 333.1 ± 56.4 kJ; ES = 0.38; P = 0.004) supplementation group, respectively. This performance benefit was no longer apparent after 4 weeks of caffeine supplementation (precaf: 383.3 ± 75.0 kJ vs. postcaf: 358.0 ± 89.8 kJ; ES = 0.31; P = 0.025), but was retained in the placebo group (precaf: 354.5 ± 55.2 kJ vs. postcaf: 351.8 ± 49.4 kJ; ES = 0.05; P > 0.05). Circulating caffeine, hormonal concentrations and substrate oxidation did not differ between groups (all P > 0.05). Chronic ingestion of a low dose of caffeine develops tolerance in low-caffeine consumers. Therefore, individuals with low-habitual intakes should refrain from chronic caffeine supplementation to maximise performance benefits from acute caffeine ingestion.
... Plasma theobromine and caffeine levels were measured by high-performance liquid chromatography to confirm chocolate consumption. 17 Platelet function was assessed using a Platelet Function Analyzer (PFA-100 TM ; Dade Behring, West Sacramento, CA, USA), which measures shear stress-induced platelet aggregation using both collagen-epinephrine and collagen-adenosine diphosphate (ADP) cartridges. Results are reported as closure times indicating the speed for blood to clot up to a maximum time of 300 s. 18 ...
Aims
Poor prognosis in chronic heart failure (HF) is linked to endothelial dysfunction for which there is no specific treatment currently available. Previous studies have shown reproducible improvements in endothelial function with cocoa flavanols, but the clinical benefit of this effect in chronic HF has yet to be determined. Therefore, the aim of this study was to assess the potential therapeutic value of a high dose of cocoa flavanols in patients with chronic HF, by using reductions in N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) as an index of improved cardiac function.
Methods and results
Thirty‐two patients with chronic HF, stable on guideline‐directed medical therapy, were randomized to consume 50 g/day of high‐flavanol dark chocolate (HFDC; 1064 mg of flavanols/day) or low‐flavanol dark chocolate (LFDC; 88 mg of flavanols/day) for 4 weeks and then crossed over to consume the alternative dark chocolate for a further 4 weeks. Twenty‐four patients completed the study. After 4 weeks of HFDC, NT‐proBNP (mean decrease % ± standard deviation) was significantly reduced compared with baseline (−44 ± 69%), LFDC (−33 ± 72%), and follow‐up (−41 ± 77%) values. HFDC also reduced diastolic blood pressure compared with values after LFDC (−6.7 ± 10.1 mmHg).
Conclusions
Reductions in blood pressure and NT‐proBNP after HFDC indicate decreased vascular resistance resulting in reduced left ventricular afterload. These effects warrant further investigation in patients with chronic HF.
... mg/L [22], 0.05-4.94 mg/L [23] of plasma caffeine levels after coffee (1-2 cups) or caffeine (200-350 mg) intake. A typical plasma chromatogram is shown on Figure 2. Peaks are numbered from 1-4, caffeine represented by peak 4 with a retention time of 5.5 minutes on average. ...
Austin Journal of Pharmacology and Therapeutics
Open Access
Research Article
Does CYP1A2 Genotype Influence Coffee Consumption?
Santos RM1*, Cotta K1, Jiang S2 and Lima DRA3
1Department of Pharmaceutical Sciences, South University School of Pharmacy, USA 1Department of
Pharmaceutical Sciences, South University School of Pharmacy, USA
2Department of Biomedical Science, Mercer University School of Medicine, USA
3Instituto of Neurology, Federal University of Rio de Janeiro, Brazil
*Corresponding author: Santos RM, Department of Pharmaceutical Sciences, South University School of
Pharmacy, 709 Mall Boulevard, Savannah, GA 31406, USA, Tel: 1-912-201-8131; Fax: 1-912-201-8153;
Email:
rsantos@southuniversity.edu
Received: November 26, 2014; Accepted: February 05,
2015; Published: February 09, 2015
Abstract
Coffee is the major source of caffeine in the American diet. Caffeine is 95%
metabolized by CYP 1A2, which has a polymorphic genetic binomial distribution within the
population. The homozygous wild type confers a fast metabolizer phenotype and the
homozygous variant allele confers a slow metabolism of caffeine, the latter being the
least predominant in the normal population. Our study objective was to examine whether genetic
variability of caffeine metabolism (CYP 1A2) is associated with plasma caffeine levels and can
influence the coffee consumption in young healthy volunteers. We found an inverse relationship
between caffeine metabolization by CYP 1A2 and caffeine plasma levels at 45-60 minutes after intake
of one cup of standardized brewed coffee (150 mg of caffeine). Our sample size was small (15
volunteers) but all possible genotypes were represented in accordance with the normal distribution
in the population. We did not find a correlation between CYP 1A2 genotype and frequency
or type of coffee selection (caffeinated or decaffeinated). We concluded that a bigger
sample size is needed in order to identify a probable influence of caffeine metabolism phenotype on
coffee consumption.
Keywords: Coffee consumption; Caffeine; Plasma levels; CYP1A2
genotype
... Briefly, Holland et al. assessed paraxanthine and caffeine in serum samples after simple sample preparation using an RP analytical column and UV detection, a single run was approx. 10 min and LOQ was 50 µg/L [16]. Krul and Hageman reported a HPLC -UV method for the determination of caffeine and its five metabolites in urine, but the analysis length was 30 min, which is unacceptable for the batch analysis of large sets of samples [17]. ...
Abstract:
Objective: The aim of this work was to develop a new, simple, rapid, sensitive and reproducible RP-HPLC method for the determination of caffeine, 1,7 dimethylxanthine, 1,7-dimethyluric acid, 5-acetylamino-6-formylamino-3-methyluracil, 5-acetylamino-6-amino-3-methyluracil, 1-methylxanthine, and 1-methyluric acid in human urine and saliva as a method of determining the metabolic activity of the human enzyme CYP1A2.
Methods: A Luna C18(2) (150mm×4.6mm i.d.) analytical column was used for the separation. The mobile phase consisted of sodium acetate trihydrate (pH 5.0) and methanol 85:15 (v/v). The flow rate was maintained at 0.8 mL/min. The absorbance of the eluent was monitored at 263 nm (5-acetylamino-6-amino-3-methyluracil), 285 nm (5-acetylamino-6-formylamino-3-methyluracil, 1,7-dimethyluric acid, 1-methyluric acid), 272 nm (caffeine, 1,7 dimethylxanthine) and 268 nm (1-methylxanthine). Acetaminophen as an internal standard was used to ensure the precision and accuracy of this method, and it was monitored at 245 nm.
Results: All compounds, including the internal standard, were eluted within 18 min. Analytes were extracted by liquid-liquid extraction. Limits of quantitation varied from 7.3 to 74.2 μg/L for individual analytes in saliva and from 8.4 to 82.4 μg/L in urine.
Conclusion: This method may become a useful alternative to urine caffeine metabolic ratio measurement with respect to CYP1A2 metabolic activity assessment in clinical practice.
... CF-derived metabolites determined in plasma corresponded to the structurally related (2)-epicatechin metabolites (SREMs) and 5-(3#,4#dihydroxyphenyl)-g-valerolactone metabolites (g-VLMs) and were measured by using validated methods as previously described (22,25). Methylxanthines, including theobromine, caffeine, paraxanthine, and theophylline, were quantified to assess compliance as previously described (26). ...
Background:
Evidence from dietary intervention studies shows that the intake of flavanols and procyanidins can be beneficial for cardiovascular health. Nevertheless, there is a clear need for advancing our understanding with regard to safe amounts of intake for these bioactives.
Objective:
The aim was to investigate in healthy adults the effects of cocoa flavanol (CF) intake amount and intake duration on blood pressure, platelet function, metabolic variables, and potential adverse events (AEs).
Design:
This investigation consisted of 2 parts. Part 1 was an open-label, intake-amount escalation study, in which 34 healthy adults (aged 35-55 y) consumed escalating amounts of CFs, ranging from 1000 to 2000 mg/d over 6 wk. Primary outcomes were blood pressure and platelet function, select metabolic variables, and the occurrence and severity of AEs. Secondary outcomes included plasma concentrations of CF-derived metabolites and methylxanthines. On the basis of the outcomes of study part 1, and assessing the same outcome measures, part 2 of this investigation was a controlled, randomized, double-masked, 2-parallel-arm dietary intervention study in which healthy participants (aged 35-55 y) were asked to consume for 12 consecutive weeks up to 2000 mg CFs/d (n = 46) or a CF-free control (n = 28).
Results:
Daily intake of up to 2000 mg CFs/d for 12 wk was not associated with significant changes in blood pressure or platelet function compared with CF-free controls in normotensive, healthy individuals who exhibited a very low risk of cardiovascular disease. There were no clinically relevant changes in the metabolic variables assessed in either of the groups. AEs reported were classified as mild in severity and did not significantly differ between study arms.
Conclusion:
The consumption of CFs in amounts up to 2000 mg/d for 12 wk was well tolerated in healthy men and women. This trial was registered at clinicaltrials.gov as NCT02447770 (part 1) and NCT02447783 (part 2).
... Twenty-one subjects had ABPM recordings at both 6 and 12 weeks (reasons for missing recordings: failure to attend the clinic on the required day, or BP monitor recording failure at either 6 or 12 weeks). Caffeine and theobromine were measured by HPLC following published methodology [27]. Plasma lipids (total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides) were measured by the Pathology Laboratories at Barts Health NHS trust. ...
... mg/L [22], 0.05-4.94 mg/L [23] of plasma caffeine levels after coffee (1-2 cups) or caffeine (200-350 mg) intake. A typical plasma chromatogram is shown on Figure 2. Peaks are numbered from 1-4, caffeine represented by peak 4 with a retention time of 5.5 minutes on average. ...
Coffee is the major source of caffeine in American diet. Caffeine is 95% metabolized by CYP 1A2, which has a polymorphic genetic binomial distribution within the population. The homozygous wild type confers a fast metabolizer phenotype and the homozygous variant allele confers a slow metabolism of caffeine. The latter being the least predominant in the normal population. Our study objective was to examine whether genetic variability of caffeine metabolism (CYP 1A2) is associated with plasma caffeine levels and can influence the coffee consumption in young healthy volunteers. We found an inverse relationship between caffeine metabolization by CYP 1A2 and caffeine plasma levels at 45-60 minutes after intake of one cup of standardized brewed coffee (150 mg of caffeine). Our sample size was small (15 volunteers) but all possible genotypes were represented according with the normal distribution in the population. We did not find a correlation between CYP 1A2 genotype and frequency or type of coffee selection (caffeinated or decaffeinated). We concluded that a bigger sample size is needed in order to identify a probable influence of caffeine metabolism phenotype on coffee consumption.
... Caffeine was analysed by high-performance liquid chromatography (HPLC) as described previously (Holland et al. 1998). Briefly, 150 µl of 0.8 M perchloric acid was added to 150 µl of serum from each sample. ...
Purpose:
In athletes, caffeine use is common although its effects on sleep have not been widely studied. This randomised, double-blind, placebo-controlled crossover trial investigated the effects of late-afternoon caffeine and carbohydrate-electrolyte (CEB) co-ingestion on cycling performance and nocturnal sleep.
Methods:
Six male cyclists/triathletes (age 27.5 ± 6.9 years) completed an afternoon training session (TS; cycling 80 min; 65% VO₂max) followed by a 5 kJ kg(-1) cycling time trial (TT). Caffeine (split dose 2 × 3 mg kg(-1)) or placebo was administered 1 h prior and 40 min into the TS. A 7.4% CEB (3 ml kg(-1) every 15 min) was administered during the TS, followed 30 min after by a standardised evening meal. Participants retired at their usual bedtime and indices of sleep duration and quality were monitored via polysomnography.
Data:
mean ± SD.
Results:
All participants performed better in the caffeine TT (caffeine 19.7 ± 3.3; placebo 20.5 ± 3.5 min; p = 0.006), while ratings of perceived exertion (caffeine 12.0 ± 0.6; placebo 12.9 ± 0.7; p = 0.004) and heart rate (caffeine 175 ± 6; placebo 167 ± 11 bpm; p = 0.085) were lower in the caffeine TS. Caffeine intake induced significant disruptions to a number of sleep indices including increased sleep onset latency (caffeine 51.1 ± 34.7; placebo 10.2 ± 4.2 min; p = 0.028) and decreased sleep efficiency (caffeine 76.1 ± 19.6; placebo 91.5 ± 4.2%; p = 0.028), rapid eye movement sleep (caffeine 62.1 ± 19.6; placebo 85.8 ± 24.7 min; p = 0.028) and total sleep time (caffeine 391 ± 97; placebo 464 ± 49 min; p = 0.028).
Conclusions:
This study supports a performance-enhancing effect of caffeine, although athletes (especially those using caffeine for late-afternoon/evening training and competition) should consider its deleterious effects on sleep.
... Extraction methods using organic solvents need lengthy sample processing time. Protein precipitation has been achieved in nonextraction methods by addition of acetonitrile 15 for Glyburide analysis, perchloric acid 16 for analysis of caffeine and paraxanthine, methanol 17 for the measurement of 10,11-dihydro-10-hydroxycarbamazepine and hydrochloric acid/ammonium sulphate 18 in paracetamol analysis. Direct injection after filtering the sample through 0.45 μm disposable filters 19 was also reported in Dyphylline and Doxofylline in serum by HPLC. ...
Paracetamol is one of the most commonly used over-the-counter pain relievers and fever-reducers. However, overdosing of paracetamol causes liver damage which may lead to patients' death upon delayed treatment. To provide accurate diagnosis and fast treatment of paracetamol poisoning, rapid analysis of paracetamol in patient tissues is necessary. In this study a rapid and simple high performance liquid chromatographic method was developed to analyse plasma/serum and urinary paracetamol using 100 μL of specimen. Paracetamol is chromatographically resolved, with an isocratic mobile phase followed by UV detection after protein precipitation. β-Hydroxyethyltheophylline was used as the internal standard. The elution of paracetamol and the internal standard was achieved within 8 minutes. The calibration curve was linear over 0.25-200 mg/L. This method produced excellent accuracy and precision in all matrices tested. Intra and inter day variability was less than 5%. The limits of detection were 0.13 mg/L for plasma and 0.43 mg/L for urine, whereas the limits of quantification were 0.68 mg/L for plasma; and 2.25 mg/L for urine. No matrix effects were observed with endogenous substances. This method is applied to analyze paracetamol levels in serum of accidental or self poisioning patients.
... by Holland et al. 48 The limits of quantitation for both serum caffeine and serum paraxanthine were set at 0.10 μg/ml, with accuracy and precision assessments <10%. ...
Four popular ephedra-free dietary supplements were evaluated for their effects on heart rate (HR), blood pressure (BP), and electrocardiographic (ECG) parameters. Twelve healthy men participated in a study randomized for product sequence, with a 21-day washout period between supplement-administration phases. Throughout the study, Holter monitors were used to assess ECG and HR activity. BP was assessed automatically on multiple occasions. The supplements were ingested three times daily for 3 days. Caffeine content, microbial load, and serum caffeine concentrations were determined. Mean systolic (SBP) and diastolic BP (DBP) readings showed significant increases relative to baseline (10.8 ± 2.5 and 5.3 ± 3.1 mm Hg, respectively; P < 0.05). All supplements significantly increased HR and decreased bradycardia runs; abnormal atrial/ventricular events were frequently noted. Gastrointestinal and sympathomimetic symptoms were also common. Two supplements were heavily contaminated with Bacillus species. In light of these findings, the use of ephedra-free dietary supplements should be discouraged in individuals with hypertension, diabetes, or other cardiovascular diseases.Clinical Pharmacology & Therapeutics (2013); advance online publication 30 January 2013. doi:10.1038/clpt.2012.241.
... Caffeine taken into the body from many different foods such as coffee and tea. The average cup of coffee contains approximately 100 mg of caffeine (Holland, Godfredsen, Page, & Corner, 1998). It is believed that drinking tea is advantageous to human health, because it contains a lot of polyphenols, which are known to have high antioxidant activity, but tea also contains caffeine, which stimulates the central nervous system and causes certain diseases (Horie, Nesumi, Ujihara, & Kohata, 2002). ...
In order to determine the amount of caffeine and theobromine, spectrophotometry was used as a simple, rapid and economical method. Because of severe overlapping between these components, artificial neural network was used. The 230–300 nm spectral window with 1 nm interval was used for data acquisition. An artificial neural network (5-5-3) with linear transfer function between input-hidden and hidden-output layers was trained and applied for prediction of concentration of these methylxanthines in four Iranian tea samples. The model was compared with PLS modeling method. HPLC technique was used as a standard method.
... Analytic methods. Serum concentrations of caffeine and paraxanthine were quantified by HPLC with ultraviolet absorbance detection according to the method of Holland et al. 25 Chlorzoxazone and 6-hydroxychlor- zoxazone serum concentrations were measured by HPLC by use of ultraviolet absorbance detection as previously described by Frye and Stiff. 26 The HPLC method with fluorescence detection described by Frye and Branch 27 was used for the quantitation of debrisoquin and 4-hydroxydebrisoquin in urine. ...
Phytochemical-mediated modulation of cytochrome P450 (CYP) activity may underlie many herb-drug interactions. Single-time point phenotypic metabolic ratios were used to determine whether long-term supplementation of Citrus aurantium , Echinacea purpurea , milk thistle (Silybum marianum), or saw palmetto (Serenoa repens) extracts affected CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity.
Twelve healthy volunteers (6 women, 6 men) were randomly assigned to receive C aurantium , E purpurea , milk thistle, or saw palmetto for 28 days. For each subject, a 30-day washout period was interposed between each supplementation phase. Probe drug cocktails of midazolam and caffeine, followed 24 hours later by chlorzoxazone and debrisoquin (INN, debrisoquine), were administered before (baseline) and at the end of supplementation. Presupplementation and postsupplementation phenotypic trait measurements were determined for CYP3A4, CYP1A2, CYP2E1, and CYP2D6 by use of 1-hydroxymidazolam/midazolam serum ratios (1-hour sample), paraxanthine/caffeine serum ratios (6-hour sample), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour sample), and debrisoquin urinary recovery ratios (8-hour collection), respectively. The content of purported "active" phytochemicals was determined for each supplement.
Comparisons of presupplementation and postsupplementation phenotypic ratios suggested that these particular supplements had no significant effect on CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity. Phytochemical profiles indicated that C aurantium was devoid of the CYP3A4 inhibitor 6',7'-dihydroxybergamottin. Quantities of fatty acids, flavonolignans, and cichoric acid were consistent with label claims for saw palmetto, milk thistle, and E purpurea , respectively.
Botanical supplements containing C aurantium , milk thistle, or saw palmetto extracts appear to pose a minimal risk for CYP-mediated herb-drug interactions in humans. Although the effects of E purpurea on CYP activity were minor, further study into the interaction potential of this botanical is merited.
... erum concentrations of caffeine and paraxanthine were determined by high-performance liquid chromatography (HPLC) (Pickard et al. 1986, Holland et al. 1998). The analysis was performed with an automated HP 1100 liquid chromatograph on a Waters Symmetry C8 column paraxanthine was 20 g/L. ...
... In Studies II to IV, plasma caffeine and paraxanthine concentrations were determined by HPLC, with -OH-ethyltheophylline as the internal standard (Pickard et al. 1986, Holland et al. 1998). The day-to-day CV of caffeine and paraxanthine was less than 6% at relevant concentrations. ...
... After rinsing their mouths with water, participants chewed on the cotton swab for 45 sec. Samples were centrifuged and frozen at −70°C until assayed using high-performance liquid chromatography (Deroche et al. 1990;Holland et al. 1998; Global Lifescience Solutions, LLC, Ann Arbor, MI, USA). The minimum detection threshold was 0.02 μg/ml and concentrations below this threshold were recorded as zero. ...
Caffeine typically produces positive effects on mood and performance. However, tolerance may develop following habitual use, and abrupt cessation can result in withdrawal symptoms, such as fatigue. This study investigated whether caffeine has a greater stimulant effect in a withdrawn state compared to a normal caffeinated state, among moderate daily caffeine consumers.
Using a within-subjects design, 17 caffeine consumers (mean +/- sd = 375 +/- 101 mg/day) ingested placebo or caffeine (250 mg) following 30-h of caffeine abstention or normal dietary caffeine use on four separate days. Self-reported mood and performance on choice reaction time, selective attention, and memory tasks were measured.
Caffeine had a greater effect on mood and choice reaction time in the abstained state than in the normal caffeinated state, but caffeine improved selective attention and memory in both states.
Although improvements in mood and reaction time may best explained as relief from withdrawal symptoms, other performance measures showed no evidence of withdrawal and were equally sensitive to an acute dose of caffeine in the normal caffeinated state.
... Plasma concentrations of caffeine and paraxanthine were quantified by HPLC-UV according to the method of Holland et al. [10]. Omeprazole and 5-hydroxyomeprazole plasma concentrations were measured by LC-MS/MS with reference to the method of Gonzalez et al. [11]. ...
Three kinds of herbal medicines, commonly used in Korea, Angelicae tenuissima radix, Angelicae dahuricae radix and Scutellariae radix were studied to evaluate their effect on cytochrome P450 (CYP) activities in healthy volunteers. A total of 24 healthy male volunteers were assigned to one of three parallel herbal treatment groups, each consisting of eight volunteers. A cocktail of probe drugs for CYP enzymes was orally administered before and after multiple administrations of herbal medicines, three times a day for 13 days. Probe drugs used to measure CYP activities were caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1) and midazolam (CYP3A4). The probe drugs and their metabolites were quantified in plasma or urine using HPLC or LC-MS/MS. Changes in each CYP activity was evaluated by metabolic ratio of the probe drug (concentration ratio of metabolite to parent form at reference time point) following the herbal medication period, compared to the baseline values. A. dahuricae radix significantly decreased CYP1A2 activity to 10% of baseline activity (95% CI: 0.05-0.21). S. radix also showed significant changes in CYP2C9 and CYP2E1 activities. Compared to baseline values, the metabolic activities of losartan were decreased to 71% (0.54-0.94). In addition, S. radix showed a 1.42-fold (1.03-1.97) increase in chlorzoxazone metabolic activity. However, CYP activities were not meaningfully influenced by A. tenuissima radix. Changes in certain CYP activities were observed after the administration of S. radix and A. dahuricae radix in healthy volunteers. Therefore, herbal medicines containing S. radix or A. dahuricae radix are candidates for further evaluation of clinically significant CYP-mediated herb-drug interactions in human beings.
... After rinsing out their mouths with water, a saliva sample was obtained by chewing on a Salivette® cotton swab for 45 s (Sarstedt, Newton, NC, USA). Samples were frozen at −70°C until caffeine concentrations ([CAF]) were assayed using high-performance liquid chromatography (Deroche et al. 1990;Holland et al. 1998; Global Lifescience Solutions, LLC, Ann Arbor, MI, USA). The minimum detection threshold was 0.02 μg/ml. ...
The field of research regarding the effects of habitual caffeine use is immense and frequently utilizes self-report measures of caffeine use. However, various self-report measures have different methodologies, and the accuracy of these different methods has not been compared.
Self-reported caffeine use was estimated from two methods (a retrospective interview of weekly caffeine use and a 7-day prospective diary; n = 79). These estimates were then tested against salivary caffeine concentrations in a subset of participants (n = 55).
The estimates of caffeine use (mg/day) from the interview- and diary-based methods correlated with one another (r = 0.77) and with salivary caffeine concentrations (r = 0.61 and 0.68, respectively). However, almost half of the subjects who reported more than 600 mg/day in the interview reported significantly less caffeine use in the diary.
Self-report measures of caffeine use are a valid method of predicting actual caffeine levels. Estimates of high caffeine use levels may need to be corroborated by more than one method.
... Theobromine was determined by a method based on that described by Holland et al (1998) with some modi®cations. Plasma (100 ml) was mixed with 100 ml metaphosphoric acid (10%) and centrifuged at 3000 g for 10 min at 4 C. ...
To evaluate the plasma kinetics in man of epicatechin from black chocolate.
An intervention study with 8 volunteers. Each served as his own control. Theobromine was used as control marker of the chocolate intake.
Metabolic Unit, Nestle Research Center, Vers-chez-les-Blanc, Switzerland.
Eight healthy male volunteers (4 smokers and 4 non-smokers) were enrolled in this study. They abstained from foods rich in polyphenols (coffee, tea, wine, fruit juice, cocoa products) for 24 h prior to the test until its completion.
Volunteers ate 40 g and 80 g of black chocolate (Nestle Noir) together with bread with a one-week interval. Blood samples were drawn every hour during the first 4h and a last one at 8 h after chocolate consumption. Plasma samples were analysed for epicatechin and theobromine content by HPLC.
Plasma concentrations of epicatechin and theobromine increased markedly after chocolate consumption (P = 0.002 and P= 0.001, respectively), reaching a maximum between 2 and 3 h. The maximal concentration and area under the curve of plasma kinetics of both substrates correlated very well with the dose of chocolate.
Epicatechin is absorbed from chocolate and is rapidly eliminated from plasma. Attainable plasma values are 0.7 micromol/l from 80g of black chocolate.
... Serum concentrations of caffeine and paraxanthine were quantified by HPLC with ultraviolet absorbance detection according to the method of Holland et al. 25 Chlorzoxazone and 6-hydroxychlorzoxazone serum concentrations were measured by HPLC with ultraviolet absorbance detection as previously described by Frye and Stiff. 26 The HPLC method with fluorescence detection that was described by Frye and Branch 27 was used for the quantitation of debrisoquin and 4-hydroxydebrisoquin in urine. ...
Phytochemical-mediated modulation of cytochrome P450 (CYP) activity may underlie many herb-drug interactions. Single-time point phenotypic metabolic ratios were used to determine whether long-term supplementation of St John's wort, garlic oil, Panax ginseng, and Ginkgo biloba affected CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity.
Twelve healthy volunteers (6 females) were randomly assigned to receive either St John's wort, garlic oil, P ginseng, or G biloba for 28 days. For each subject, a 30-day washout period was interposed between each supplementation phase. Probe-drug cocktails of midazolam, caffeine, chlorzoxazone, and debrisoquin (INN, debrisoquine) were administered before supplementation (baseline) and at the end of supplementation. Presupplementation and postsupplementation phenotypic trait measurements were determined for CYP3A4, CYP1A2, CYP2E1, and CYP2D6 with the use of 1-hydroxymidazolam/midazolam serum ratios (1-hour sample), paraxanthine/caffeine serum ratios (6-hour sample), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour sample), and debrisoquin urinary recovery ratios (8-hour collection), respectively.
Comparisons of presupplementation and postsupplementation ratios indicated that St John's wort significantly induced the activity of CYP2E1 and CYP3A4 (P <.0001). Among female subjects, St John's wort produced significantly greater increases in CYP3A4 phenotypic ratios that appeared to be unrelated to body mass index. This finding is suggestive of a sexual dimorphism in CYP3A4 inducibility. Garlic oil reduced CYP2E1 activity by 39% (P =.030), whereas no significant effect on CYP activity was observed for P ginseng and G biloba.
Single-time point phenotypic metabolic ratios may provide a practical means of predicting CYP-mediated herb-drug interactions in humans.
... Serum concentrations of clozapine, N-desmethylclozapine, clozapine-N-oxide, caffeine and paraxanthine were determined by high-performance liquid chromatography (HPLC) (Pickard et al. 1986;Weigmann & Hiemke 1992;Schulz et Mean∫S.D. concentrations of clozapine, N-desmethylclozapine, clozapine-N-oxide, caffeine, paraxanthine and C-reactive protein, and Ndesmethylclozapine/clozapine and clozapine-N-oxide/clozapine concentration ratios during study phases. 1995;Holland et al. 1998). The quantitation limit for clozapine, N-desmethylclozapine and clozapine-N-oxide was 30 nmol l ª1 . ...
Clozapine and caffeine are metabolised mainly by the cytochrome P4501A2 (CYP1A2) enzyme. Studies suggest that caffeine in coffee inhibits clozapine metabolism and increases serum clozapine concentrations. Our objective was to study whether coffee in the amounts usually consumed has an effect on steady-state serum clozapine concentrations. A randomised placebo-controlled cross-over design with two phases was used. Twelve hospitalised clozapine-using patients volunteered in the study where, after one-week run-in period, either caffeine-containing or decaffeinated instant coffee was available ad libitum for seven days. Serum concentrations of clozapine, N-desmethylclozapine, clozapine-N-oxide, caffeine, paraxanthine and C-reactive protein were measured after run-in period and on days 4 and 8 of the following study phases. Two patients were excluded from the statistical analysis because of non-compliance based on serum caffeine and paraxanthine determinations. In six fully compliant patients caffeine-containing coffee increased the mean serum trough concentration of clozapine by 26% (non-significant (NS), 95% CI -3% to +54%, P=0.07), N-desmethylclozapine by 6% (95% CI 1% to 12%, P=0.03), and clozapine-N-oxide by 7% (NS, 95% CI -6% to +20%, P=0.22). The ratio of N-desmethylclozapine/clozapine decreased by 13% (NS, 95% CI -1% to +27%, P=0.06) and that of clozapine-N-oxide/clozapine by 7% (NS, 95% CI -5% to +17%, P=0.19). In the analysis of combined data (including day 4 data of the four patients compliant up to that point) serum trough concentration of clozapine was 20% (95% CI 3% to 37% to P=0.03) higher, and that of N-desmethylclozapine 7% (95% CI 2% to 13%, P=0.02) higher during the caffeine phase than during the decaffeinated phase. We conclude that the effect of instant coffee drinking on serum clozapine concentrations is of minor clinical relevance in most of the patients, but some individuals may be more sensitive to this interaction due e.g. to genetic factors. The increase in serum clozapine concentration was most likely due to the inhibition of the CYP1A2 enzyme by caffeine.
... Volunteers abstained from all caffeine-containing food and beverages for a period of at least 15 h prior to the study. To confirm that there was no residual caffeine present in plasma at this time, plasma levels of caffeine and paraxanthine were measured by HPLC using a method derived from those of Schreiber-Deturmeny and Bruguerolle [15] and Holland et al. [16]. Caffeine was undetect-able at this time, whilst paraxanthine levels were very low-5 F 1.5 AM. ...
This study investigated the effect of in vitro exposure to caffeine, and its major metabolite paraxanthine, at concentrations relevant to typical caffeine consumption in humans, on lipopolysaccharide (LPS)-stimulated cytokine production in human whole blood. In addition, a role for the cyclic AMP/protein kinase A (PKA) pathway in the immunomodulatory effect of caffeine was investigated. Diluted whole blood (taken following >/=15 h abstinence from caffeine-containing food and beverages) was preincubated with caffeine or paraxanthine (10-100 microM) and stimulated with LPS (1 proportional, variant g/ml) for 24 h. The proinflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-12, and the antiinflammatory cytokine IL-10 were measured in cell-free supernatants. Whilst caffeine and paraxanthine had little or no effect on IL-10, IL-1beta, or IL-12 production, TNF-alpha production was suppressed in all individuals studied. The effect was statistically significant at 100 microM and consistent across seven experiments performed. Although not statistically significant, a similar effect was observed with paraxanthine. Caffeine (100 microM) also increased intracellular cyclic AMP concentrations in LPS-stimulated monocytes isolated from whole blood. Moreover, the effect of caffeine on TNF-alpha production was abolished by pretreatment with the protein kinase A inhibitor Rp-8-Br-cAMPS (10(-4) and 10(-5)M). To conclude, this study demonstrates that concentrations of caffeine that are relevant to human consumption consistently suppress production of the proinflammatory cytokine TNF-alpha in human blood and that this effect is mediated by the cyclic AMP/protein kinase A pathway.
Background and Aims: Caffeine is a xanthine alkaloid found naturally in plants. Caffeine has cardiotonic and stimulant effects in humans and animals. For this reason, caffeine is on the monitoring list for human sports and is listed as a feed contaminant in horse racing. The aim of this study was to develop a rapid, practical, and specific method for the determination of caffeine in horse urine.
Methods: In the new method, the pH of the sample was adjusted by the addition of phosphate buffer, and after solid phase extraction, it was dissolved in methanol before being analysed by gas chromatography mass spectrometry without derivatization. The method was validated according to the European Commission’s 2002/657/EC criteria.
Results: The effects of different cartridge brands, pH, and elution solution were determined. Intraday and interday CV% values are 2.8 and 5.2 for the International Residue Limit (IRL), respectively. Five levels (blank, 0.5xIRL, IRL, 1.5xIRL, and 2xIRL) were used in constructing the curve, and the R2 value was greater than 0.99. The analysis run was 11.8 min. The decision limit (CCα) was determined to be 56.7 ng/mL due to IRL. The detection limit of the method was calculated to be 3.3 ng/mL. The method was determined to be robust according to changes in extraction pH, phosphate buffer concentration, centrifugation time, hexane volume in the wash step, different grades of methanol, inlet temperature, and operator.
Conclusion: The applicability of the method was demonstrated by analysing positive and negative horse urine samples. Validation parameters showed the method to be selective, specific, and easy to apply.
Anahtar Kelimeler: Caffeine, Gas chromatography-mass spectrometry, Urine
The Cytochrome P450 CYP1A2 is a central enzyme in the metabolism of drugs and xenobiotics. The overall activity of this enzyme is influenced by a complex array of biochemical, dietary, and genetic factors. One of the simplest ways to probe the overall output of CYP1A2 is to measure the ratio between the concentration of a precursor and a product of its activity. With the growing interest in the Paraxanthine/Caffeine ratio, the need arises to develop improved analytical methods specifically optimized for the rapid and sensitive determination of paraxanthine and caffeine in biological samples. We report a new optimized method for the determination of caffeine and paraxanthine in various human matrices. The method involved direct determination following protein precipitation based on ultra high performance liquid chromatographic separation with tandem mass spectrometric detection (UHPLC-ESIMS/MS). The method offers an improvement in the detection limit over previously published methods by at least 10-fold (0.1 pg), rapid chromatographic separation (ca. 5 min), the utilization of a green chromatographic solvent (5% v/v ethanol), direct determination with little sample preparation, and the employment of isotopically labeled internal standards and qualifier ions to ensure accuracy. Method validation in urine, saliva, and plasma was performed by spiking at various concentration levels where the recovery and repeatability were within ±15% and ±10%, respectively. The method was applied to investigate the levels of caffeine and paraxanthine in volunteers following controlled caffeine administration and to investigate the inter- and intra-individual variability in the paraxanthine/caffeine ratio in volunteers following an unrestricted caffeine diet. In conclusion, the developed UHPLC-ESIMS/MS method is optimized specifically for the simultaneous determination of the paraxanthine/caffeine ratio in multiple biological matrices, offers several advantages over the current methods, and is well suitable for application in large clinical studies.
Caffeine is a common xanthine alkaloid found in tea leaves, coffee beans, and other natural plants, and is the most widely used psychotropic substance in the world. Accumulating evidence suggests that low plasma levels of caffeine and its metabolites may serve as reliable diagnostic markers for early Parkinson's disease patients. In this study, we demonstrated a new MEKC method for determining caffeine and its three main downstream metabolites, paraxanthine, theobromine and theophylline, in human plasma. Plasma samples were collected and analyzed after solid‐phase extraction, by using MEKC. The running buffer was composed of 35 mM phosphate, pH of 10.5, and 25 mM SDS. The separation voltage was 15 kV and the detection wavelength was at 210 nm. Under the optimum conditions, four distinct analytes were completely separated and detected in less than 12 min. Method limits of detection were as low as 7.5 ng/mL for caffeine, 5.0 ng/mL for theobromine and 4.0 ng/mL for both paraxanthine and theophylline. The recoveries were between 88.0%–105.9%. This method was successfully applied to 27 human plasma samples. The results indicate that the plasma concentrations of the four analytes are significantly lower in patients with early Parkinson's disease than in control subjects (p <0.05). The area under curve was improved to 0.839 when caffeine and its three main metabolites were included, suggesting that MEKC testing of caffeine, theophylline, theobromine, and paraxanthine may serve as a potential method for early diagnosis of Parkinson's disease.
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This study systematically examined the influence of carbohydrate (sucrose), sodium and caffeine on the fluid retention potential of beverages under euhydrated conditions, using the beverage hydration index (BHI) method. Three cohorts, each of 12 young, healthy, active men, ingested 1L of beverages containing four different concentrations of a single component (sucrose, sodium or caffeine) in a double blind, crossover manner. Urine output was collected for the subsequent 4-h. Cumulative urine output was lower and net fluid balance were higher after 10% and 20% sucrose beverages than 0% and 5% sucrose beverages (P<0.05), and after 27mmol/L and 52mmol/L sodium beverages than 7mmol/L and 15mmol/L sodium beverages (P<0.05). No difference in urine output or net fluid balance was apparent following ingestion of caffeine at concentrations of 0 - 400 mg/l (P=0.83). Consequently, the calculated BHI was greater in beverages with higher sucrose or sodium content, but caffeine had no effect. No difference was observed in arginine vasopressin or aldosterone between any trials. These data highlight that the key drivers promoting differences in the fluid retention potential of beverages when euhydrated are energy density, likely through slowed fluid delivery to the circulation (carbohydrate content effect), or electrolyte content through improved fluid retention (sodium content effect). These data demonstrate that beverage carbohydrate and sodium content influence fluid delivery and retention in the 4-h after ingestion, but caffeine up to 400mg/L does not. Athletes and others can use this information to guide their daily hydration practices.
A new reversed-phase ultraperformance liquid chromatography method with a photodiode array detector was developed for the quantification of ascorbic acid (AA) and caffeine (CAF) in 11 different commercial drinks consisting of one energy drink and 10 ice tea drinks. Separation of the analyzed AA and CAF with an internal standard, caffeic acid, was performed on a Waters BEH C18 column (100 mm × 2.1 mm, 1.7 μm i.d.), using a mobile phase consisting of acetonitrile and 0.2M H3PO4 (11:89, v/v) with a flow rate of 0.25 mL/min and an injection volume of 1.0 μL. Calibration graphs for AA and CAF were computed from the peak area ratio of AA/internal standard and CAF/internal standard detected at 244.0 nm and 273.6 nm, respectively. The developed reversed-phase ultraperformance liquid chromatography method was validated by analyzing standard addition samples. The proposed reversed-phase ultraperformance liquid chromatography method gave us successful results for the quantitative analysis of commercial drinks containing AA and CAF substances.
Caffeine (trimehylxantkine) is an alkaloid present in many beverages and rood sources. It is widely consumed and it is known for its stimulating properties and potential performance-enhancing effects. World Anti-Doping Agency (WADA) has placed caffeine on the 2006 monitoring program for stimulants in-competition only substance if its presence in the urine exceeding 12.0 μg/ml. Turkish coffee is the most commonly used type of coffee in the Middle-East region. However, athletes who arc Turkish coffee consumers may not easily appreciate how much caffeme they can ingest before being accused of breaching the new WADA regulation of caffeine monitoring program for stimulants in-competition. Such problem could be avoided by establishing caffeine content in each cup of Turkish coffee, based on the understanding of the cultural practice in drinking and preparing Turkish coffee, and estimating the dose of caffeine present m each cup of Turkish coffee. Twenty-four volunteer participated in (lie study as group A and B and consumed Turkish coffee cups (40ml each) with caffeine mean dose of S.56 (4 cups) and 12.84 (6 cups) mg-'kg respectively over a two-hour interval. Volunteers of Group A who had 4 cups showed minary maximum caffeine concentration of 8.86±2.3d μg/ml three horns post ingestion. However, Group B who consumed 6 cups of coffee under the same experiment conditions showed a maximum urinary concentration of 12.36±2.02 μg/ml. such caffeine concentration is above the permitted level of 12.0 μg/ml. In Conclusion, athletes who arc Turkish coffee drinkers are advised to lower their intake of Turkish caffeine to less than two cup of regular coffee during or before sport competition especially if the athlete ingests soft or hot dunks containing caffeine or taking permitted drugs containing caffeine. The small volume of Turkish coffee cup of 40ml could be misleading to athletes, and a cross over with the new WADA regulations of caffeine misuse will be reported if the athlete consumed 6 regular cups of Turkish coffee only.
Background
Herb-drug interactions may be of particular concern in the elderly. Single time-point, phenotypic metabolic ratios were used to determine whether supplementation of Ginkgo biloba, Panax ginseng, garlic oil, or St. John's wort extracts affected CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity in elderly subjects.Methods
Twelve healthy volunteers between the ages of 60 and 76 (mean= 66 years) were randomly assigned to receive each supplement for 28 days followed by a 30-day washout period. Probe drug cocktails of midazolam, caffeine, chlorzoxazone, and debrisoquine were administered before and at the end of supplementation. Pre- and post-supplementation phenotypic ratios were determined for CYP3A4, CYP1A2, CYP2E1, and CYP2D6 using 1-hydroxymidazolam/midazolam serum ratios (1-hr), paraxanthine/caffeine serum ratios (6-hr), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hr), and debrisoquine urinary recovery ratios (8-hr), respectively.ResultsComparisons of pre- and post-St. John's wort phenotypic ratios revealed significant induction of CYP3A4 (~140%) and CYP2E1 activity (~28%). Garlic oil inhibited CYP2E1 (~22%). Ginseng inhibited CYP2D6, but the magnitude (~7%) did not appear clinically relevant. CYP1A2 activity in the elderly was unaffected by herb supplementation.Conclusions
Elderly subjects, like their younger counterparts, are susceptible to herb-mediated changes in CYP activity, especially those involving St. John's wort.Clinical Pharmacology & Therapeutics (2005) 77, P16-P16; doi: 10.1016/j.clpt.2004.11.063
A rapid, selective and convenient liquid chromatography–mass spectrometric method for the simultaneous determination of paracetamol
and caffeine in human plasma was developed and validated. Analytes and theophylline [internal standard (I.S.)] were extracted
from plasma samples with diethyl ether-dichloromethane (3:2, v/v) and separated on a C18 column (150×4.6mmID, 5μm particle size, 100Å pore size). The mobile phase consisted of 0.2% formic acid–methanol (60:40,
v/v). The assay was linear in the concentration range between 0.05 and 25μgmL−1 for paracetamol and 10–5,000ngmL−1 for caffeine, with the lower limit of quantification of 0.05μgmL−1 and 10ngmL−1, respectively. The intra- and inter-day precision for both drugs was less than 8.1%, and the accuracy was within ±6.5%. The
single chromatographic analysis of plasma samples was achieved within 4.5min. This validated method was successfully applied
to study the pharmacokinetics of paracetamol and caffeine in human plasma.
Capillary electrophoresis (CE) offers the possibility of fast, cheap and reproducible separations for pharmaceutical preparations. Alkylxanthines make up a family of compounds that are used in the treatment and prevention of bronchi asthma and chronic pulmonary disease. The group of analysed compounds include caffeine, dyphylline, theophylline, theobromine and enprofylline. This paper shows a simple capillary zone electrophoretic (CZE) method for separation of this group of xanthines. Using 20 mM borate buffer at pH 9.4 as running buffer at 55 °C it was possible to complete a total separation of a sample in 2 min. Limits of detection in the range 1.9–2.5 mg l−1 were achieved with %R.S.D. of 0.06–0.22% (n = 5). The technique is applied to a range of samples containing the analytes, including tablets and chocolate. Reproducibility (%R.S.D.) of the chocolate analysis technique by CZE was less than 4.5%.
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.
Outside pregnancy, acute caffeine consumption is associated with insulin resistance. We investigated if during pregnancy plasma concentrations of caffeine and its metabolite, paraxanthine, were associated with insulin resistance. Caffeine, paraxanthine, glucose, and insulin were measured and insulin resistance estimated by homeostasis model assessment (HOMA) in banked samples from 251 fasting subjects at mean gestational age of 20.3 ± 2.0 weeks. Analysis of covariance and adjusted logistic regression were performed. Most (96.4%) women had caffeine and/or paraxanthine present. Caffeine concentrations in the upper two quartiles (>266 ng/mL) were associated with threefold higher odds of having higher insulin resistance estimated by log HOMA ≥75th percentile (third quartile odds ratio [OR], 3.02; 95% confidence interval [CI]: 1.21 to 7.54 and fourth quartile OR, 2.95; 95% CI: 1.19 to 7.31). Paraxanthine concentrations in the upper quartile (>392 ng/mL) were also associated with threefold higher odds of having higher insulin resistance (OR, 3.04; 95% CI: 1.28 to 7.25). Adjusted mean HOMA in the first caffeine-to-paraxanthine ratio quartile was 1.5 ± 2.2 versus 1.3 ± 2.3 in the fourth quartile ( P < 0.01). Both high caffeine and paraxanthine concentrations were associated with insulin resistance, but slow versus fast metabolism did not make an important difference.
The cytochrome P450 1A2 (CYP1A2) is one of the major metabolizing enzymes. The muscle relaxant tizanidine is a selective substrate of CYP1A2, and the non-steroidal anti-inflammatory drug (NSAID) rofecoxib was thought to modestly in-hibit it. Cases suggesting an interaction between tizanidine and rofecoxib had been reported, but the mechanism was unknown. Also other NSAIDs are often used in combination with muscle relaxants. The aims of this study were to investigate the effect of rofecoxib, several other NSAIDs and female sex steroids on CYP1A2 ac-tivity in vitro and in vivo, and to evaluate the predictability of in vivo inhibition based on in vitro data. In vitro, the effect of several NSAIDs, female sex steroids and model inhibitors on CYP1A2 activity was studied in human liver microsomes, without and with preincubation. In placebo controlled, cross-over studies healthy volunteers ingested a single dose of tizanidine after a pretreament with the inhibitor (rofecoxib, tolfenamic acid or celecoxib) or placebo. Plasma (and urine) concentrations of tizanidine and its metabolites were measured, and the pharmacodynamic effects were recorded. A caffeine test was also performed. In vitro, fluvoxamine, tolfenamic acid, mefenamic acid and rofecoxib potently in-hibited CYP1A2. Ethinylestradiol, celecoxib, desogestrel and zolmitriptan were moderate, and etodolac, ciprofloxacin, etoricoxib and gestodene were weak inhibi-tors of CYP1A2. At 100 µM, other tested NSAIDs and steroids inhibited CYP1A2 less than 35%. Rofecoxib was found to be a mechanism-based inhibitor of CYP1A2. In vivo, rofecoxib greatly increased the plasma concentrations (over ten-fold) and the pharmacodynamic effects of tizanidine. Also the metabolism of caf-feine was impaired by rofecoxib. Despite the relatively strong in vitro CYP1A2 inhibitory effects, tolfenamic acid and celecoxib did not have a significant effect on tizanidine and caffeine concentrations in humans. Competitive inhibition model and the free plasma concentration of the inhibitor predicted well the effect of fluvoxam-ine and the lack of effect of tolfenamic acid and celecoxib on tizanidine concentra-tions in humans, and mechanism-based inhibition model explained the effects of rofecoxib. However, the effects of ciprofloxacin and oral contraceptives were un-derestimated from the in vitro data. Rofecoxib is a potent mechanism-based inhibitor of CYP1A2 in vitro and in vivo. This mechanism may be involved in the adverse cardiovascular effects of rofecoxib. Tolfenamic acid and celecoxib seem to be safe in combination with tizanidine, but mefenamic acid might have some effect on tizanidine concentrations in vivo. Con-sidering the mechanism of inhibition, and using the free plasma concentration of the inhibitor, many but not all CYP1A2 interactions can be predicted from in vitro data. Sytokromi P450 1A2 (CYP1A2) on yksi tärkeimmistä lääkeaineita metaboloivista entsyymeistä. Lihasrelaksantti titsanidiini metaboloituu pääosin sen välityksellä maksassa. Titsanidiinin ja tulehduskipulääke rofekoksibin yhteiskäytön on raportoitu johtavan lisääntyneisiin haittavaikutuksiin, mutta yhteisvaikutuksen mekanismi on ollut tuntematon. Muitakin tulehduskipulääkkeitä käytetään monesti yhdessä lihasrelaksanttien kanssa. Tutkimuksen tarkoituksena oli tutkia yleisesti käytettyjen kipulääkkeiden ja hormonien vaikutusta CYP1A2:n toimintaan sekä koeputkiolosuhteissa (in vitro) että terveillä koehenkilöillä (in vivo), ja vertailla soveltuvin osin tuloksia toisiinsa. Kipulääkkeiden ja hormonien vaikutusta CYP1A2:n aktiivisuuteen tutkittiin in vitro maksan mikrosomeilla. Myös metaboliasta riippuvaa estoa tutkittiin. Vaihtovuoroisissa tutkimuksissa terveille koehenkilöille annettiin esilääkkeenä rofekoksibia, tolfenaamihappoa tai selekoksibia, ja vertailuvaiheessa lumetta muutaman päivän ajan. Sitten annettiin kerta-annos titsanidiinia. Titsanidiinin ja metaboliittien pitoisuudet mitattiin plasmasta (ja virtsasta). Lisäksi mm. verenpainetta ja väsymystä seurattiin. Myös toinen CYP1A2:n aktiivisuutta mittaava testi, kofeiinitesti, suoritettiin. Fluvoksamiini, tolfenaamihappo, mefenaamihappo ja rofekoksibi estivät CYP1A2:ta voimakkaasti in vitro. Etinyylestradioli, selekoksibi, desogestreeli ja tsolmitriptaani olivat kohtalaisia, ja etodolaakki, siprofloksasiini, etorikoksibi ja gestodeeni heikkoja CYP1A2:n estäjiä. Muut tutkitut aineet (100 µM) estivät CYP1A2:ta alle 35%. Rofekoksibi osoittautui irreversiibeliksi CYP1A2-estäjäksi in vitro, ja koehenkilöillä se nosti voimakkaasti titsanidiinin (yli kymmenkertaisiksi) ja kofeiinin pitoisuuksia ja voimisti titsanidiinin aiheuttamaa verenpaineen laskua huomattavasti. Tolfenaamihapolla ja selekoksibilla ei ollut vaikutusta titsanidiinin ja kofeiinin pitoisuuksiin, vaikka ne estivät CYP1A2:ta kohtalaisesti in vitro. Kompetitiivinen estomalli ja vapaa CYP1A2-estäjän plasmapitoisuus ennustivat hyvin fluvoksamiinin, tolfenaamihapon ja selekoksin vaikutukset titsanidiinin pitoisuuksiin. Irreversiibeli estomalli pystyi ennustamaan rofekoksibin interaktiot. Toisaalta siprofloksasiinin ja ehkäisyvalmisteiden vaikutuksia ei pystytty ennustamaan. Rofekoksibi on voimakas CYP1A2:n irreversiibeli (mechanism-based) estäjä in vitro ja in vivo. Tämä mekanismi saattaa selittää jopa osan rofekoksibin kardiovaskulaarihaittavaikutuksista, jotka johtivat sen käytöstä poistamiseen. Tolfenaamihappo ja selekoksibi näyttävät sopivan yhdessä titsanidiinin kanssa käytettäviksi, mutta mefenaamihapolla saattaa olla lievää vaikutusta titsanidiinin pitoisuuksiin. Kun metabolianeston mekanismi otetaan huomioon ja käytetään metabolianestäjän vapaata plasmapitoisuutta, monet, muttei kaikki, CYP1A2-välitteiset interaktiot voidaan ennustaa in vitro tulosten perusteella.
We developed a simple assay method for the determination of serum and urine norfloxacin and enoxacin using reversed-phase high-performance liquid chromatography and perchloric acid precipitation for sample pre-treatment. Optimized conditions can permit detection of norfloxacin and enoxacin in the same chromatogram, so either compound can be used as an internal standard for another determinant. Supernatants of the precipitated samples were analyzed by the octadecylsilyl silica-gel column under ambient temperature and an ultraviolet wavelength of 272 nm. A mobile phase solvent consisting of 20 mm sodium dihydrogenphosphate (pH 3.0) and acetonitrile (85:15, v/v) was pumped at a flow rate of 1.0 mL/min. The calibration curves for norfloxacin and enoxacin at a concentration of 62.5-1000 ng/mL for serum and 250-4000 ng/mL for urine were linear (r > 0.9997). The recoveries of norfloxacin and enoxacin from serum and urine were >94% with the coefficient of variations (CV) <5%. The CVs for intra- and inter-day assay of norfloxacin and enoxacin were <4.2 and <5.5%, respectively. This method can be applied to the pharmacokinetic study of norfloxacin and enoxacin after repeated administration to assess changes in CYP1A2 activity in healthy subjects.
The anodic oxidn. of 4-chlorophenoxyacetic acid (CPA) on synthetic B-doped diamond thin film electrodes in acid media was studied, using cyclic voltammetry and bulk electrolysis. The results showed that in the potential region where the supporting electrolyte is stable, reactions occur, resulting in the loss of activity due to electrode fouling. Electrolysis at high anodic potentials in the region of electrolyte decompn. causes complex oxidn. reactions that lead to the complete incineration of CPA. There is no indication of electrode fouling under these conditions. The exptl. were compared with a theor. model. This model is based on the assumption that the electrochem. oxidn. of CPA by electrogenerated hydroxyl radicals is a fast reaction and that the process is diffusion-controlled. [on SciFinder (R)]
This paper describes design of a new cartridge for selective solid phase extraction (SPE) using molecularly imprinted polymers (MIPs). The apparatus which is termed solvent extraction-MISPE (SE-MISPE) cartridge, consisted of a modified conventional micro test tube and has been developed to perform simultaneous forward-extraction of analyte from aqueous sample solution to an organic phase and back-extraction to MIP solid phase. In order to evaluate the performance of the proposed method, extraction of theophylline (THP) from human serum sample was investigated. An appropriate amount of THP-imprinted polymer was placed in the bottom of the micro tube and an organic solvent pipetted onto it and left to swell the polymer completely. A polyethylene frit to secure MIP particles was positioned by two Teflon rings such that it was fixed below the level of the organic layer. Then, aqueous sample solution containing THP was layered over the organic phase and the lid was closed. After completion of extraction, the organic and aqueous phases were removed and the adsorbed analyte was desorbed using a polar organic solvent. In order to reach the highest recovery, the experimental parameters such as the type of organic solvent, pH and ionic strength of aqueous phase, organic to aqueous volume ratio, time of extraction, type and amount of desorbent solvent were optimized. Under the experimental conditions, a plot of HPLC peak areas vs. initial concentrations of THP in the concentration interval of 0.5-30 microg ml(-1) showed a good linearity (r=0.9974). The limit of detection (LOD) and limit of quantification (LOQ) based on three and ten times of the noise of HPLC profile were 0.09 and 0.3 microg ml(-1), respectively. The relative standard deviation (RSD) of the proposed method for the extraction and determination of 5 microg THP from 200 microl standard sample solution for 3 replicate measurements was 3.5%. The results showed that by means of the proposed cartridge, THP could significantly separate from the other structurally related compounds such as theobromine (THB) and caffeine (CAF). The added THP could be quantitatively recovered (79-83%) from the serum samples by the proposed procedure, being thus a guarantee of the accuracy of the SE-MISPE procedure. In addition, the loss of capability of the SE-MISPE cartridge was not considerably observed after 10 times loading and elution cycles.
A HPLC assay and solid-phase extraction technique from human plasma has been developed and validated for the novel anticancer agent CT2584, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, which has recently completed a phase I trial at the Christie Hospital, Manchester under the auspices of the CRC phase I/II committee. Following addition of CT2576, 1-(11-octylamino-10-hydroxylundecyl)-3,7-dimethylxanthine, as internal standard, a solid-phase extraction cartridge (100 mg cyanopropyl) was used to isolate the drug CT2584 from human plasma. Analysis was performed by reversed-phase chromatography. CT2576 was used as internal standard at a concentration of 4 microg ml(-1) for the quantification of CT2584 from plasma for the duration of this work. The lower limit of quantification for the drug CT2584 in buffer using this assay was found to be 0.0122 microM (0.008 microg ml(-1)) and 0.048 microM (0.027 microg ml(-1)) when extracted from human plasma.
In this study the validation of a reversed-phase high-performance liquid chromatography (HPLC) method, with UV-detection, for both caffeine and paraxanthine in human serum is described. This method is feasible for cytochrome P450 1A2 (CYP1A2) phenotyping, according to the results of a pilot study. With this HPLC method caffeine and paraxanthine can be determined selectively and specifically. In the expected concentration range, caffeine recoveries were 98-108% (within-run variation 4.0-6.4%, between-run variation 6.4-8.8%), paraxanthine recoveries were 96.6-97.5% (within-run variation 5.0-7.2%, between-run variation 7.2-10.8%). The limits of detection for caffeine and paraxanthine using this HPLC system were 0.3 and 0.1 mg/L, respectively. Linear calibration curves for both caffeine and paraxanthine were obtained in the concentration range 0.5-30 mg/L (r > 0.9999. Serum samples were stable for a week, when stored at -20 and +4 degrees C.
An improved extraction procedure and a new RP-HPLC method were developed for selective and rapid analysis of caffeine and 14 of its metabolites in urine. Analytes were isolated by solid-phase extraction and separated on an Eclipse XDB-C18 column. Recoveries ranged between 83 and 99%. Precision, linearity and accuracy of the chromatographic method were found to be within required limits. Using this procedure, caffeine metabolic ratios were determined in 20 subjects with characteristic CYP1A2 activities, relatively to smoking habit and contraceptives intake. The method might be useful to point out induction and inhibition of CYP1A2 activity.
Our objective was to study the effect of fluvoxamine on the pharmacokinetics and pharmacodynamics of tizanidine, a centrally acting skeletal muscle relaxant.
In a double-blind, randomized, 2-phase crossover study, 10 healthy volunteers took 100 mg fluvoxamine or placebo orally once daily for 4 days. On day 4, each ingested a single 4-mg dose of tizanidine. Plasma concentrations of tizanidine and fluvoxamine and pharmacodynamic variables were measured. A caffeine test was performed on day 3 to examine the role of cytochrome P450 (CYP) 1A2 in tizanidine pharmacokinetics.
On average, fluvoxamine increased the total area under the concentration-time curve [AUC(0- infinity )] of tizanidine 33-fold (range, 14-fold to 103-fold; P =.000002) and the peak plasma concentration 12-fold (range, 5-fold to 32-fold; P =.000001). The mean elimination half-life of tizanidine was prolonged from 1.5 to 4.3 hours (P =.00004) by fluvoxamine. The AUC(0- infinity ) of tizanidine and its increase by fluvoxamine correlated with the caffeine/paraxanthine ratio and its increase, respectively (P <.03). All pharmacodynamic variables revealed a significant difference between the fluvoxamine and placebo phases, eg, in the maximal effects on systolic blood pressure (-35 mm Hg, P =.000009), diastolic blood pressure (-20 mm Hg, P =.00002), heart rate (-4 beats/min, P =.007), Digit Symbol Substitution Test (P =.0003), subjective drug effect (P =.0000001), and drowsiness (P =.0002). In particular, the decrease in systolic blood pressure, to the level of 80 mm Hg or even less, was an alarming finding.
Fluvoxamine seriously affects the pharmacokinetics of tizanidine and increases the intensity and duration of its effects. Inhibition of tizanidine-metabolizing enzyme(s), mainly CYP1A2, by fluvoxamine seems to explain the observed interaction. Because of the potentially hazardous consequences, the concomitant use of tizanidine with fluvoxamine, or other potent inhibitors of CYP1A2, should be avoided.
A reversed-phase liquid chromatographic column switching system was described for the determination of caffeine (CF), theophylline (TH) and theobromine (TB) in human plasma with a direct injection procedure. A short protein-coated mu Bondapak CN silica pre-column (20 x 3 mm, i.d.) was used for enrichment of the drugs and clean up from weakly retained plasma components using phosphate buffer saline pH 7.4. After washing step, the retained drugs were flushed into a reversed-phase column (5 microm TSK gel ODS-80 TM, 150 x 4.6 mm i.d.) with a mobile phase of methanol-0.01 M phosphate buffer, pH 3.5 (30:70, v/v) for the final separation. The eluent was monitored with a UV detector at 275 nm. The resulting chromatograms showed no interference from endogenous plasma components. A linear relationship between the concentration of drug and peak height was confirmed in the range of 0.5-20 microg/mL for all drugs. High extraction recoveries from plasma ranging from 96.12 to 100.32% were achieved. Validation of the method was examined performing intra- and inter-day accuracy and precision and was found to be satisfactory. The coefficients of variation of the three drugs were less than 3% for intra-day and less than 4% for inter-day run assays.
This is a summary report of the conference on “Analytical Methods Validation: Bioavailability, Bioequivalence and Pharmacokinetic Studies.” The conference was held from December 3 to 5, 1990, in the Washington, D.C., area and was sponsored by the American Association of Pharmaceutical Scientists, the U.S. Food and Drug Administration, Federation International Pharmaceutique, Health Protection Branch (Canada), and the Association of Official Analytical Chemists. The report presents our assessment of the major agreements and issues discussed at the conference. The report is also intended to provide guiding principles for validation of analytical methods used in bioavailability, bioequivaience, and pharmacokinetics studies in humans and animais. The objectives of the conference were as follows: (1) to reach a consensus on what should be required in analytical methods validation and the procedures to establish validation; (2) to determine processes of application of the validation procedures in bioavailability, bioequivalence, and pharmacokinetics studies; and (3) to develop a report on analytical methods validation that may be referred to in developing future formal guidelines. Acceptable standards for documenting and validating analytical methods with regard to processes, eters, or data treatments are discussed because of their importance in assessing pharmacokinetic, bioavailability, and bioequivalence studies. Other topics that were considered essential in the conduct of pharmacokinetic studies or in establishing bioequivalency criteria, including measurement of drug metabolites and stereoselective determinations, are also discussed.
A fast, simple, and cost-effective HPLC method for the quantitation of the antiviral drug ganciclovir is described. The serum samples are extracted with perchloric acid and neutralized with potassium phosphate buffer, and urine samples are diluted with distilled water. A reversed-phase column with isocratic elution by 15 mM potassium phosphate buffer (pH 2.5) containing 0.25% acetonitrile is used to separate ganciclovir; quantitation is by UV absorbance at 254 nm. Total turnaround time is 22 min; more than 3000 samples can be run on a single column without loss of peak quality. The limit of quantitation is 0.05 micrograms/ml. Recoveries varied from 91 to 107% with coefficients of variation ranging from 0.387 to 7.95%.
An automated reverse phase high performance liquid chromatography (HPLC)—photodiode array method using a multi linear gradient elution is described for the simultaneous analysis of nine xanthines: xanthine, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, isocaffeine, theobromine, paraxanthine, theophylline and caffeine. The separation method development was based on mobile-phase optimisation and off-line solid-phase extraction (SPE) from human biological fluids: blood serum and urine. Eluent consisted of 0.05 M CH3COONH4 and methanol (90:10 v/v) changing to (70:30 v/v) over a period of 20 min. Identification of xanthines was achieved by photodiode—array detector and quantitation was performed at 270 nm. Isocaffeine was used as internal standard at a concentration of 3.06 ng/μL. High extraction recoveries were achieved from Merck RP-18 cartridges using 1% hydrochloric acid as eluent, requiring small volumes, 40 μL of blood serum and 100 μL of urine.The separation of xanthines was achieved on octylsilica, using a Silasorb C8, 10μm, 250 × 4.6 mm i.d. analytical column thermostated at 32 °C and proved to be highly selective, sensitive, reproducible, accurate and rapid regarding the nine compounds. Detection limits ranged from 2 to 3 ng tor 20 μL injected volume while linearity holds up to 20 ng/μL for each compound.
Caffeine is metabolized extensively (on average 80%) to paraxanthine. With regular caffeine consumption, average serum levels of paraxanthine are two thirds those of caffeine. Both caffeine and paraxanthine competitively and nonselectively inhibit adenosine receptors in vitro. To examine the contribution of paraxanthine to the pharmacologic activity of caffeine, we administered to 12 subjects in a crossover design oral caffeine (2 or 4 mg/kg) versus placebo or oral paraxanthine (same dose as caffeine) versus placebo, each after 3 days of methylxanthine abstinence. Both caffeine and paraxanthine significantly increased diastolic blood pressure, plasma epinephrine levels, and free fatty acids. Caffeine and paraxanthine produced a similar magnitude of response at 4 mg/kg; however, caffeine appeared to produce greater responses than paraxanthine at 2 mg/kg. Caffeine and paraxanthine have similar sympathomimetic actions. The activity of paraxanthine needs to be considered in understanding the clinical pharmacology of caffeine, particularly with chronic, repetitive caffeine consumption.
The plasma protein binding of caffeine in young and elderly males was evaluated using an ultrafiltration technique. In spite of a significantly lower plasma albumin concentration in the elderly subjects the observed percent bound (∼35%) was essentially identical in both subject groups. The binding of caffeine to human plasma albumin (4.5% w/v) in vitro was also examined using ultracentrifugation and it was observed to be bound to the extent of 37.8%. In both the plasma and albumin binding studies the free fraction remained constant over the range of concentrations examined. Although there was no apparent correlation between the percent bound and the albumin concentration in the plasma of either subject group the close agreement between the degree of binding of caffeine to albumin and human plasma indicates that albumin is likely the major plasma binding protein for caffeine.
Caffeine was analyzed in human plasma and saliva by a simple, rapid, and sensitive radioimmunoassay procedure. Immunization of rabbits with an antigen prepared by coupling 7-(5-carboxypentyl)-1,3-dimethylxanthine to bovine serum albumin resulted in the formation of antibodies selective for caffeine as opposed to various mono- and dimethylxanthines, mono-, di-, and trimethyluric acids and a variety of common drugs. The radioligand used for competitive binding studies was 7-(2,3-3H2-propyl)-1,3-dimethylxanthine. The procedure permits direct analysis of caffeine in plasma or saliva without extraction. Comparison with a high pressure liquid chromatography method for the analysis of caffeine gave satisfactory results and showed no evidence for interference by metabolites. A caffeine half-life of 4.0 hours determined by the radioimmunoassay was in agreement with previous work. Comparison of human plasma and saliva levels by the radioimmunoassay procedure indicated approximately equal concentrations in the two fluids.
The purpose of this study was to assess the pharmacokinetics of caffeine and its metabolites in the lactating rabbit and suckling pup. The ability of a diffusional model to predict milk-to-serum drug concentration ratios (M/S) observed in vivo from in vitro experiments was established. The distribution into milk of caffeine, paraxanthine, theobromine, and theophylline was measured in lactating New Zealand White rabbits following an iv bolus dose of caffeine (5 mg/kg). M/S ratios were determined in vivo (M/Sobs; caffeine = 0.875 +/- 0.052; paraxanthine = 0.358 +/- 0.019; theobromine = 0.829 +/- 0.038; and theophylline = 0.412 +/- 0.054) under single dose conditions using area under the milk and serum concentration-time profiles. Predicted M/S values (M/Spred; caffeine = 0.797 +/- 0.040; paraxanthine = 0.316 +/- 0.029; theobromine = 0.692 +/- 0.062; and theophylline = 0.385 +/- 0.039) were calculated from in vitro measurements of the unbound fractions of drug in skim milk and serum (fm and fs, respectively), the skim-to-whole milk drug concentration ratio (S/W), milk and serum pH, and the pKa of the model compound. The pharmacokinetic profile of caffeine in the suckling pup following iv bolus administration (5 mg/kg) was more prolonged compared with adult rabbits. The mean systemic clearance of total caffeine (CIs) in the adults and the pups was 3.83 +/- 1.94 and 1.14 +/- 0.80 ml/min/kg, respectively. The mean unbound systemic clearance (CIs,u) for caffeine was 5.09 +/- 2.60 ml/min/kg in the adults and 1.41 +/- 0.71 ml/min/kg in the pups.(ABSTRACT TRUNCATED AT 250 WORDS)
An improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its three primary metabolites (theophylline, theobromine and paraxanthine) in human plasma is described. The four substances were separated on a reversed-phase column (5 microns TSK gel ODS-80TM, 150 mm x 4.6 mm I.D.) by use of the mobile phase methanol-0.1 M NaH2PO4 (30:70, v/v) with a flow-rate of 0.8 ml/min. Absorbance was monitored at 274 nm. The detection limit was 5 ng/ml for theobromine and caffeine and 10 ng/ml for paraxanthine and theophylline. The linearity and reproducibility were sufficient for drug monitoring of caffeine and its primary methylxanthines.
Some recent epidemiologic studies have reported a nonlinear dose-response in the relationship between coffee consumption and health risks, such that the risks increase disproportionately to the increase in dose. Assuming caffeine contributes to the adverse health effects of coffee, a possible explanation for the nonlinear dose-response relationship is dose-dependent metabolism of caffeine. We examined the hypothesis that under chronic dosing conditions the metabolism of caffeine is dose-dependent. Nine healthy subjects were given, in randomized 5-day treatment blocks, placebo, 4.2 (low) and 12 (high) mg/kg/day caffeine in decaffeinated coffee, in six divided doses spaced throughout the day. On the third day of each dosing period, 25 mg of stable-isotope labeled caffeine (2-13C, 1,3-15N2) was given intravenously. Clearance of labeled caffeine fell from 0.118 (placebo treatment) to 0.069 (low dose; p less than 0.005) and to 0.54 (high dose; p less than 0.001) L/hr/kg. The formation and metabolite clearances of paraxanthine, the major primary metabolite of caffeine, also decreased comparing the low and high doses (p less than 0.05). We conclude that caffeine metabolism is dose-dependent, resulting in nonlinear accumulation of methylxanthines in the body. Dose-dependent metabolism of caffeine may explain in part why people who drink large amounts of coffee are at greater risk for cardiovascular disease.
The absolute bioavailability of orally administered caffeine was investigated in 10 healthy adult male volunteers, aged 18.8 to 30.0 years. The subjects were administered a 5 mg/kg dose of caffeine as either an aqueous oral solution or an intravenous infusion, on separate occasions about 1 week apart, in a randomized crossover fashion. Plasma samples were collected over the 24-h period following each dose and assayed for their caffeine content using a high-performance liquid chromatographic technique. The oral absorption was very rapid, reaching a peak (Tp) plasma concentration after 29.8 +/- 8.1 min (mean +/- SEM). In addition, the variation in the maximum plasma concentration (Cmax) was low, 10.0 +/- 1.0 micrograms/ml. The absolute bioavailability was assessed by comparing the areas under the plasma concentration vs. time curves for the intravenous and oral doses of caffeine. The rapid absorption resulted in essentially complete bioavailability of the oral caffeine, F(%) = 108.3 +/- 3.6%. The caffeine plasma half-lives varied from 2.7 to 9.9 h, indicating substantial inter-subject variability in its elimination.
Elimination kinetics of theophylline and its major metabolites were investigated in 14 healthy adults in single-dose studies and in a multiple-plateau study. The plasma concentrations of theophylline and the metabolites 3-methylxanthine (3-MX), 1-methyluric acid (1 MU), and 1,3-dimethyluric acid (13-MU) were monitored to about 0.020 mg/l and became convex descending at concentrations below 1 mg/l after single theophylline doses. Renal clearance values of 3-MX, 1-MU, and 13-MU were 12.0 +/- 1.3 l/hr, 22.5 +/-1.5 l/hr, and 22.6 +/- 1.6 l/hr. Metabolite formation of the three metabolites followed Michaelis-Menten kinetics and became capacity limited within the therapeutic range of theophylline. The apparent Michaelis-Menten parameters for each metabolite formation step were obtained by computer fitting. For the formation of 3-MX. 1-MU, and 13-MU, the approximate mean maximal rate of formation of metabolite (Vmax) values were 5 mg/hr, 13 mg/hr, and 34 mg/hr and the apparent concentration of theophylline at which metabolite formation rate is half of Vmax values were 2.7 mg/l, 9.3 mg/l, and 14.2 mg/l. The elimination of each of the metabolites was rate limited by the elimination of theophylline. Concomitant measurement of theophylline urinary excretion rate showed the renal clearance of the drug to be highly dependent on urine flow. The initial renal clearance, elevated due to diuresis, and the distribution phase tended to counterbalance the saturable metabolic formation clearance after a single therapeutic dose. Therefore, plasma theophylline concentration decayed roughly in a log-linear fashion and the convex-descending curve, characterized by capacity-limited elimination kinetics, was observed only at lower concentrations.
This paper describes the disposition of caffeine and its metabolites, theophylline, theobromine and paraxanthine in the 20-day fetal and adult brains following a single maternal dose of 5 or 25 mg/kg caffeine. Brains and plasma were collected 5 and 30 min, and 1, 3, 8 and 24 h after dosing. It was found that fetal and adult caffeine AUC (area under the concentration-time curve) values did not differ between the brain and plasma at either dose. Caffeine's primary metabolites theophylline, theobromine and paraxanthine did, however, accumulate in the fetal brain at both doses resulting in a 3-fold increase in brain metabolite exposure compared to fetal circulatory levels. In contrast to the fetus, metabolite AUC values after a dose of 25 mg/kg were found to be lower in the brains of adults compared with plasma. This suggests that caffeine's primary metabolites might be selectively excluded from the adult brain. In conclusion we have shown that, unlike the adult, the fetal rat brain accumulates theophylline, theobromine and paraxanthine when exposed to caffeine doses comparable to those attainable by normal human consumption. Since many aspects of caffeine metabolism are similar in the rat and human, we suggested that particular attention should be paid to the consumption of caffeine during pregnancy.
Sample preparation using protein precipitation is
- J Blanchard
J. Blanchard, J. Chromatogr. 226 (1981) 455. Sample preparation using protein precipitation is
575 (1992) 311. caffeine and paraxanthine in human serum has been
- E Tanaka
- J Chromatogr
E. Tanaka, J. Chromatogr. 575 (1992) 311. caffeine and paraxanthine in human serum has been
14 to a wide range of storage and handling conditions (1983) 93. likely to be experienced by serum samples obtained
- J Blanchard
- S J A Sawers
- Eur
J. Blanchard, S.J.A. Sawers, Eur. J. Clin. Pharmacol. 14 to a wide range of storage and handling conditions (1983) 93. likely to be experienced by serum samples obtained
Skelly, routinely used in our laboratory [13]. Samples as
- V P Shar
- K K Midha
- S Dighe
- I J Mcgilveray
V.P. Shar, K.K. Midha, S. Dighe, I.J. McGilveray, J.P. Skelly, routinely used in our laboratory [13]. Samples as A. Yacobi, T. Layloff, C.T. Viswanathan, C.E. Cook, R.D.
) 1415. phylline. Both CA and PX were shown to be stable
- J Blanchard
J. Blanchard, J. Pharm. Sci. 71 (1982) 1415. phylline. Both CA and PX were shown to be stable
drugs and specific, with no interferences from two Chromatogr
- I N Papadoyannis
- V F Samanidou
- K A Georga
- J Liq
I.N. Papadoyannis, V.F. Samanidou, K.A. Georga, J. Liq. drugs and specific, with no interferences from two Chromatogr. Rel. Technol. 19 (1996) 2559. other caffeine metabolites, theobromine and
simple, cheap and fast and does not require the Benowitz
- C P Denero
- C R Brown
- M Wilson
- P Jacob Iii
C.P. Denero, C.R. Brown, M. Wilson, P. Jacob III, N.L. simple, cheap and fast and does not require the Benowitz, Clin. Pharmacol. Ther. 48 (1990) 277. addition of an internal standard. The method is
9 (1992) small as 100 ml can be used. Quantitation is by use 588. of external standards and the sensitivity is around 50 ng / ml for each drug. The method has been applied
- K A Mcdowell
- S Pittman
- Spector
McDowell, K.A. Pittman, S. Spector, Pharm. Res. 9 (1992) small as 100 ml can be used. Quantitation is by use 588. of external standards and the sensitivity is around 50 ng / ml for each drug. The method has been applied
A simple reversed-phase HPLC method, with UV Ther
- .-S Tang-Liu
- R L Williams
- S Riegelman
D.D.-S. Tang-Liu, R.L. Williams, S. Riegelman, Clin. Pharm. A simple reversed-phase HPLC method, with UV Ther. 31 (1982) 358. detection, for the simultaneous determination of