Interpretation of Pain Management Testing Results Using Case Examples.

BACKGROUND: Because of the increasing volume of opiate-related overdoses, clinical testing of urine for drugs and related compounds in pain management clinics has become increasingly important. Interpreting findings of drugs present in urine specimens requires knowledge of pharmacokinetics, metabolism, drug purity, and cutoff concentrations used to report a positive result. CONTENT: This case-based mini-review provides examples of how to interpret immunoassay and quantitative confirmatory urine drug-testing results. Particular emphasis is placed on interpretation of opiate and benzodiazepine results, as these drugs have complicated metabolic profiles. SUMMARY: Both determining patient medication compliance and identifying the presence of additional drugs provides important information to the treating physician involved in managing pain. Mass spectrometry-based methods are required to identify specific drugs present and can provide important quantitative data for interpreting opiate medication compliance.

A sensitive assay for urinary cocaine metabolite benzoylecgonine shows more positive results and longer half-lives than those using traditional cut-offs.

Cocaine is a common drug of abuse. To detect its use, a screening detection concentration for the cocaine metabolite benzoylecgonine is commonly set at 150 ng/mL and its confirmatory cut-off is set at 100 ng/mL. Studies have suggested that these cut-offs may be set too high, allowing some patients with this substance abuse problem to be missed or improperly monitored. With the advent of liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology it is possible to reliably detect and quantify lower concentrations of its metabolite benzoylecgonine as part of a larger drug panel. One purpose of the study was to establish if there was a significant increase in detection of cocaine use with a ten-fold more sensitive cut-off. A very sensitive dilute and shoot assay for benzoylecgonine was developed with a lower limit of quantitation of 5 ng/mL. Validation of the 5 ng/mL cut-off was achieved by plotting all the positive cocaine observations as a frequency distribution on a logarithmic scale. The number of positive results with measurable concentrations below the typical industry 100 ng/mL cut-off level but above the high sensitivity 5 ng/mL cut-off level was observed to be 51.9% of the observed positives. The lower cut-off also allowed a re-evaluation of the window of detection after cessation of use. It was observed to be between 17 and 22 days. © 2016 Precision Diagnostics, LLC. Drug Testing and Analysis published by John Wiley & Sons, Ltd.

Evaluation of high-resolution mass spectrometry for urine toxicology screening in a pain management setting.

To evaluate liquid chromatography-high-resolution mass spectrometry (LC-HR-MS) for urine toxicology screening, 29 analytes were quantitated in 152 urine specimens from patients with chronic pain using two unique mass spectrometry platforms. De-identified specimens were quantitated in April of 2011 by liquid chromatography-triple quadrupole mass spectrometry (LC-MS-MS) and by full-scan LC-HR-MS at Millennium Laboratories. Considering LC-MS-MS as the reference method, false positive results were identified in 19 specimens measured by LC-HR-MS. Application of relative retention times using deuterium labeled internal standards improved the rate of false positive detection to only five specimens, with four occurring for the same analyte. Ultra-high-resolution mass spectrometry (R = 100,000 at m/z 200) showed no improvement over high-resolution mass spectrometry (R = 10,000 at m/z 200) in the number of false positives detected. Quantitative results measured by LC-MS-MS and LC-HR-MS showed good agreement over four orders of dynamic range. This study demonstrates that LC-HR-MS is a suitable platform for toxicology screening for a pain management population and that quantitative accuracy and sensitivity are comparable to that achieved with LC-MS-MS. The specificity of LC-HR-MS is improved by the addition of deuterium labeled internal standards and the implementation of relative retention time matching.

Observations on the urine metabolic ratio of oxymorphone to oxycodone in pain patients.

The object of this study was to evaluate the metabolism of oxycodone to oxymorphone in a pain patient population using a quantitative liquid chromatography-tandem mass spectrometry analysis of 32,656 urine specimens obtained from pain patients between March 2008 and Feb 2010. The observed excretion was modeled using logarithmic transformation and approximated a Gaussian distribution. Oxycodone excretion into urine had a geometric mean of 1.93 mg/g of creatinine and oxymorphone had a value of 0.41 mg/g of creatinine. Increasing concentrations of oxycodone correlated with a smaller proportion of oxymorphone excretion suggesting saturation of oxycodone metabolism. Urine samples containing oxycodone without oxymorphone allowed an estimation of the proportion of poor metabolizers (2.4 ± 2.1%) in the population. A similar analysis of samples containing oxymorphone without oxycodone gave an estimate of the proportion of ultra-rapid metabolizers (1.8 ± 1.1%) in the population. Samples with concentrations of oxycodone above 10 mg/g of creatinine showed a sub-population of subjects with metabolic ratios roughly 100-fold less than the linear predictive model in this study. This study describes typical ranges for oxycodone and oxymorphone in urine, and showed that it is possible to identify fast or slow metabolizers who may be at risk for adverse events.

Evaluating the relationship between carisoprodol concentrations and meprobamate formation and inter-subject and intra-subject variability in urinary excretion data of pain patients.

Using urinary carisoprodol data from pain patients, our objectives were to determine the relationship between carisoprodol concentration and its conversion to meprobamate, and quantify the intra-subject and inter-subject variability in carisoprodol metabolism. Liquid chromatography-tandem mass spectrometry was used to quantitate carisoprodol and meprobamate concentrations in urine specimens. The log creatinine-corrected carisoprodol versus log creatinine-corrected meprobamate showed a marginal positive relationship (R(2) = 0.395), with a 29.1-fold variance between subjects at the mean carisoprodol concentration. The geometric mean carisoprodol and meprobamate urine concentrations were 0.519 ± 3.38 mg and 28.2 ± 2.34 mg analyte per gram creatinine, respectively. The log metabolic ratio (MR) versus log creatinine-corrected carisoprodol displayed a marginal positive correlation. A subpopulation of outliers with higher carisoprodol and lower meprobamate levels were considered poor metabolizers and represented 0.483% (n = 21) of the study population. Using a curve-fit mathematical model, we estimated 0.318% (n = 10) to be ultra-rapid metabolizers. The inter-subject population geometric standard deviation (SD) of the MR was 3.64. The intra-subject geometric median and mean SD of the MR were 1.60 (interquartile range: 1.28, 2.07) and 1.72 ± 1.60, respectively. Inter-subject variability was 2.27 times greater than the median intra-subject variability. With a better understanding of urine carisoprodol and meprobamate concentrations and variability, urine drug testing provides a useful monitoring reference for clinicians.

Evaluating the relationship of methadone concentrations and EDDP formation in chronic pain patients.

Methadone is used to treat moderate to severe pain in patients not responsive to non-narcotic analgesics and for maintenance treatment of opioid addiction. Methadone is primarily metabolized by N-demethylation to an inactive metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidene (EDDP) by CYP3A4 and CYP2B6. Establishing expected concentrations for metabolism of methadone to EDDP using urine excretion data may be useful for monitoring "medications" and toxicity. Urine specimens from chronic pain patients were collected during routine clinic visits. Methadone and EDDP were quantified by liquid chromatography-tandem mass spectrometry. Approximately 8,000 subjects who reported taking methadone had creatinine concentrations ≥20 mg/dL, and excreted concentrations of methadone and EDDP above ≥100 ng/mL were selected. The median methadone urine concentration was 3.03 mg/g cr. Ninety-five percent of the population had concentrations between 0.175 and 20.9 mg/g cr. EDDP was, on average, twice the methadone concentration. The wide variance in relationship of methadone to its metabolite was not concentration-dependent. Variability between subjects was larger than variability within subjects. As the urinary pH increased, the proportion of excreted EDDP increased, implying a preferred excretion of EDDP.

Observations on the metabolism of morphine to hydromorphone in pain patients.

Morphine is one of several opioids used to treat chronic pain. Because of its high abuse potential, urine drug tests can confirm "consistency with prescribed medications." Hydromorphone is a recently described minor metabolite of morphine, but few data exist on the characteristics of this metabolic pathway or the relationship of morphine and hydromorphone between and within subjects. Part I of this retrospective study shows that formation of hydromorphone from morphine is concentration-dependent and possibly saturated at high concentrations of morphine. In addition, the percentage of ultra-rapid metabolizers and poor metabolizers can be determined using the lower asymptote of a sigmoidal mathematical fit and are estimated to be 0.63 and 4.0%, respectively. Expected limits of morphine and hydromorphone (as a result of morphine metabolism) concentrations in the urine were established. Part II of this study used the metabolic ratio (hydromorphone-morphine) to determine the inter-patient and intra-patient variability in morphine metabolism to hydromorphone. Metabolic ratio values varied over a large range; 25-fold and 7-fold, respectively. The expected limits established in this study can assist in assessing the cause for possible variances in metabolism, such as drug interactions. The wide variability between and within subjects may explain unpredictable, adverse effects.

Relationship between the concentration of hydrocodone and its conversion to hydromorphone in chronic pain patients using urinary excretion data.

Hydrocodone in combination with acetaminophen is commonly used to control moderate pain and is metabolized by cytochrome P4502D6 to form the active metabolite, hydromorphone. The purpose of this study was to determine the metabolic relationship and variability between hydrocodone and its conversion to hydromorphone using urinary excretion data from chronic pain patients. Liquid chromatography-tandem mass spectrometry was used to quantitate hydrocodone and hydromorphone concentrations in urine specimens. The first visits of 25,200 subjects who took hydrocodone and not hydromorphone and had measurable concentrations were included in this study. The geometric mean (95% confidence index) of hydrocodone and hydromorphone urine concentrations were 1.39 (1.37-1.41) mg per gram of creatinine and 0.224 (0.221-0.227) mg per gram of creatinine, respectively. The log of creatinine-corrected hydromorphone versus the log of creatinine-corrected hydrocodone showed a positive relationship (R(2) = 0.20), with 60-fold variability between subjects. The plot of the log of the metabolic ratio ([hydromorphone] divided by [hydrocodone]) versus the log of creatinine-corrected hydrocodone had a coefficient of determination of R(2) = 0.42, with 125-fold variability between subjects. Ultra-rapid metabolizers represented 0.6% of the population, whereas 4% were poor metabolizers. Within-subject variability for the excretion of hydrocodone in urine was 23-fold, whereas between-subject variability was 134-fold. Hydrocodone and hydromorphone urine concentrations showed great variability within and between subjects.

Therapeutic monitoring of benzodiazepines in the management of pain: current limitations of point of care immunoassays suggest testing by mass spectrometry to assure accuracy and improve patient safety.

BACKGROUND: Concomitant use of opioids and benzodiazepines can result in significant untoward effects. Point of care (POC) urine testing devices are commonly used tools to monitor patient use of medications. These useful devices are relatively inexpensive and yield immediate results that can be acted upon at the time of the appointment, although numerous limitations have been identified for specific medications or medication classes. We established the diagnostic accuracy of a commonly used POC testing method for benzodiazepines. METHODS: One thousand patients, from a single interventional pain practice receiving opioid therapy provided urine specimens as part of the usual practice of monitoring consistency with prescribed medications. These de-identified urine specimens were tested using LC-MS/MS and the results were compared using the standard calculations for sensitivity, specificity, and predicted value. Five specimens were excluded from the study because the prescribed flurazepam could not be confirmed by LC-MS/MS (the LC-MS/MS instrumentation was not set to identify flurazepam), resulting in 995 specimens. RESULTS: Point of care assays yielded false negative results for patients prescribed benzodiazepines nearly 20% of the time (98 out of 498 patients). The point of care cup often failed to produce positive results for persons who were shown by LC-MS/MS to be taking lorazepam or clonazepam. Although only 26 out of 498 patients (5%) were prescribed ≥2 benzodiazepines, 73 out of 498 patients (15%) were found to be positive for that drug class. CONCLUSIONS: POC immunoassay for benzodiazepines could fail to provide accurate information regarding patient specific medication use. The false positive and false negative rates of the immunoassay were particularly high for clonazepam and lorazepam. Further testing of patient specimens using more accurate methods such as LC-MS/MS is necessary to provide definitive data that can assist in clinical decision making, and potentially protect these patients from untoward effects, morbidity and mortality.

Dilute and shoot: analysis of drugs of abuse using selected reaction monitoring for quantification and full scan product ion spectra for identification.

Our objective was to develop a "dilute and shoot" liquid chromatography-tandem mass spectrometry confirmatory procedure that uses full scan product ion spectra to identify drugs that are present above cutoff values as determined by isotope dilution relative to a deuterium-labeled internal standard. Deuterium-labeled internal standards are added to urine which is then diluted prior to analysis. Full scan product ion spectra were obtained in the data-dependent mode using a linear ion trap (ABI 4000 Qtrap). Identification was based on a purity fit of greater than 70. Ninety-seven urine specimens were analyzed by the method described, and results were compared to values obtained from a reference laboratory using selected reaction monitoring (SRM). The ion trap provided about 30-fold increase in signal-to-noise ratio as compared with the same instrument operated in a traditional full scan product ion mode. The assays were linear to at least 10 times the cutoff. Selecting appropriate triggers for obtaining full scan product ion spectra minimized space charging for specimens that contained high concentrations of drugs. There was 100% concordance between the full scan identification and the SRM results for identification of amphetamine, methamphetamine, benzoylecgonine, morphine, codeine, hydrocodone, and hydromorphone. The ability to "dilute and shoot" reduces the turnaround time for results. The data acquired with SRM and full scan product ion spectra provide accurate quantification and a high degree of specificity.

Ethyl glucuronide, ethyl sulfate, and ethanol in urine after intensive exposure to high ethanol content mouthwash.

To determine the degree of ethanol absorption and the resultant formation and urinary excretion of its conjugated metabolites following intensive use of high ethanol content mouthwash, 10 subjects gargled with Listerine(®) antiseptic 4 times daily for 3» days. First morning void urine specimens were collected on each of the four study days and post-gargle specimens were collected at 2, 4, and 6 h after the final gargle of the study. Urine ethanol, ethyl glucuronide (EtG), ethyl sulfate (EtS), and creatinine were measured. Ethanol was below the positive threshold of 20 mg/dL in all of the urine specimens. EtG was undetectable in all pre-study urine specimens, but two pre-study specimens had detectable EtS (6 and 82 ng/mL; 16 and 83 µg/g creatinine). Only one specimen contained detectable EtG (173 ng/mL; 117 µg/g creatinine). EtS was detected in the urine of seven study subjects, but was not detected in the single specimen that had detectable EtG. The maximum EtS concentrations were 104 ng/mL and 112 µg/g creatinine (in different subjects). Three subjects produced a total of eight (non-baseline) urinary EtS concentrations above 50 ng/mL or 50 µg/g creatinine and three EtS concentrations exceeding 100 ng/mL or 100 µg/g creatinine. In patients being monitored for ethanol use by urinary EtG and EtS concentrations, currently accepted EtG and EtS cutoffs of 500 ng/mL are adequate to distinguish between ethanol consumption and four times daily use of high ethanol content mouthwash.

Determination of illicit drug cutoff values in a pain patient population.

BACKGROUND: When properly selected, cutoff levels minimize the reporting of false negative and false positive test results and allow the laboratory to accurately determine the prevalence of marijuana, cocaine and methamphetamine use. Selecting the ideal cutoff requires the collection of drug excretion data for a large patient population to determine the expected range of drug concentrations. The cutoff can then be set to capture a high percentage of positives at a concentration within the dynamic range of the method. We used quantitative urine drug excretion data to calculate cutoffs needed to best determine the presence of these illicit drugs in urine. METHODS: This study used liquid chromatography tandem mass spectrometry (LC-MS/MS) as the analytical method. The study group was the pain patient population which is well-known to have a significant incidence of use of these illicit drugs. Frequency distributions were plotted for the creatinine normalized and raw data for all positive specimens with values greater than or equal to the method limit of quantitation. A non-parametric 2.5% estimator was applied to each data set to establish the cutoff for each drug. RESULTS: The urinary excretion data for the three drugs studied suggest cutoffs of approximately 30 ng/ml (benzoylecgonine), 10 ng/ml (carboxy-THC), and 50 ng/ml (methamphetamine) to identify 97.5% of the users of these drugs in this population. CONCLUSIONS: Evaluation of urinary excretion data provides an objective method to validate the selection of cutoffs. These data provide additional support for the revised SAMHSA cutoffs which could increase the positivity rates for both benzoylecgonine and methamphetamine by 7%.

Ethyl glucuronide, ethyl sulfate, and ethanol in urine after sustained exposure to an ethanol-based hand sanitizer.

To assess the degree of ethanol absorption and subsequent formation of urinary ethyl glucuronide (EtG) and ethyl sulfate (EtS) following sustained application of hand sanitizer, 11 volunteers cleansed their hands with Purell(™) hand sanitizer (62% ethanol) every 5 min for 10 h on three consecutive days. Urine specimens were obtained at the beginning and end of each day of the study, and on the morning of the fourth day. Urinary creatinine, ethanol, EtG, and EtS concentrations were measured. EtG was undetectable in all pre-study urine specimens, but two pre-study specimens had detectable EtS (73 and 37 ng/mL). None of the pre-study specimens had detectable ethanol. The maximum EtG and EtS concentrations over the course of the study were 2001 and 84 ng/mL, respectively, and nearly all EtG- and EtS-positive urine specimens were collected at the conclusion of the individual study days. Only two specimens had detectable EtG at the beginning of any study day (96 and 139 ng/mL), and only one specimen had detectable EtS at the beginning of a study day (64 ng/mL), in addition to the two with detectable EtS prior to the study. Creatinine-adjusted maximum EtG and EtS concentrations were 1998 and 94 µg/g creatinine, respectively. In patients being monitored for ethanol use by urinary EtG concentrations, currently accepted EtG cutoffs do not distinguish between ethanol consumption and incidental exposures, particularly when urine specimens are obtained shortly after sustained use of ethanolcontaining hand sanitizer. Our data suggest that EtS may be an important complementary biomarker in distinguishing ethanol consumption from dermal exposure.

Anomalous observations of hydrocodone in patients on oxycodone.

BACKGROUND: Urine drug monitoring is used by physicians treating chronic pain patients with opioid therapy. Patients are tested in part to insure that they are not taking other drugs. Therefore, the finding of hydrocodone in a patient who is only prescribed oxycodone has clinical implications. Oxycodone preparations are known to have small amounts of hydrocodone as an impurity estimated to be < 0.1%. We established the concentration of unexpected hydrocodone in patients taking oxycodone. METHODS: Urine drug testing specimens from a population of 30,000 pain patients prescribed oxycodone in various formulations were quantitatively measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The frequency and concentration of hydrocodone as a function of oxycodone concentration were determined. RESULTS: There were 187 specimens with > 100,000 ng/ml of oxycodone. Of these, 72% were positive for hydrocodone. Of the 311 specimens with oxycodone concentrations > 50,000-100,000 ng/ml, 33% were positive for hydrocodone. Of the 1067 specimens with oxycodone > 20,000-50,000 ng/ml, 16% were positive for hydrocodone. Of the 8508 specimens with oxycodone > 1000-20,000 ng/ml, 16% were positive for hydrocodone. CONCLUSIONS: The high frequency of hydrocodone in samples containing high concentrations of oxycodone was ascribed to the manufacturing process of the oxycodone medications. However, a significant number of patients also took hydrocodone that was not listed on their prescribed medications. When oxycodone is > 100,000 ng/ml, hydrocodone should be <1500 ng/ml. When oxycodone is < 100,000 ng/ml then hydrocodone should be <500 ng/ml. Values greater than these indicate non-prescribed hydrocodone use. Clinicians and laboratories testing urine for drugs should be aware of the possibility of low concentrations of hydrocodone in the urine of patients taking high doses of oxycodone.

Competitive dopamine receptor antagonists increase the equiactive cocaine concentration during self-administration.

Competitive dopamine receptor antagonists increase the rate of cocaine self-administration. As the rate of self-administration at a particular unit dose is determined by the satiety threshold and the elimination half-life (t(½)) of cocaine, we investigated whether dopamine receptor antagonists altered these parameters in rats. The plasma cocaine concentration at the time of each self-administration was constant during a session demonstrating that this satiety threshold concentration represents an equiactive cocaine concentration. The plasma cocaine concentration at the time of self-administration was increased by SCH23390, consistent with pharmacological theory. In rats trained to reliably self-administer cocaine, SCH23390 had no effect on the plasma steady-state cocaine concentration produced by constant infusions of cocaine. Therefore, this antagonist had no effect on cocaine t(½) at a dose that accelerated cocaine self-administration. A constant cocaine infusion at a rate that maintained steady state concentrations above the satiety threshold stopped self-administration. SCH23390, or the D2 dopamine receptor antagonist (-)eticlopride, reinstated self-administration in the presence of the constant cocaine infusion. This is consistent with SCH23390 and eticlopride raising the satiety threshold above the steady state level produced by the constant cocaine infusion. It is concluded that the antagonist-induced acceleration of cocaine self-administration is the result of a pharmacokinetic/pharmacodynamic interaction whereby the rate of cocaine elimination is faster at the higher concentrations, as dictated by first-order kinetics, so that cocaine levels decline more rapidly to the elevated satiety threshold. This results in the decreased interinjection intervals.

Variability of transdermal fentanyl metabolism and excretion in pain patients.

BACKGROUND: The rising popularity of the fentanyl transdermal patch and the striking number of deaths attributed to its prescribed use have brought attention to the large variability of fentanyl metabolism and the need for predictive models to prevent toxicity. OBJECTIVE: The purpose of this study was to determine the amount of both intrasubject and intersubject variability in fentanyl metabolism and excretion, using urinary excretion data from patients with chronic pain prescribed the fentanyl transdermal patch. METHODS: Liquid chromatography tandem mass spectrometry analytical technique was used to quantitate fentanyl and norfentanyl concentrations in spot urine specimens, after incubation with glucuronidase. Descriptive statistics and graphical analysis were conducted using Microsoft Excel 2007. Analysis was conducted on 206 subjects with > or = 2 visits listing transdermal fentanyl as current medication. Outliers and subjects with no detectable levels of drug were excluded, resulting in subject populations of 200 (all subjects analyzed) and 166 (subjects with drug concentrations above the instrument detection limit for all visits). RESULTS: The geometric mean metabolic ratio (MR) of norfentanyl to fentanyl was 6.2 x division by 2.4. A wide distribution was observed in total fentanyl load (1,000-fold) and MR (200-fold). The intersubject geometric standard deviation in MR was 2.4 (95% confidence interval [CI] for MR: 1-37) and the intrasubject geometric standard deviation was 1.8 (95% CI for MR: 2-20). CONCLUSION: The level of intrasubject variability over time in the pharmacokinetics of the fentanyl patch is much greater than previously observed and may be due to variability in absorption, interference of metabolism by concomitant medications, and variable metabolism due to genetic polymorphisms. The variation in the MR between subjects and within subjects may explain the unpredictable adverse effects observed with use of transdermal fentanyl.

A chimeric human/murine anticocaine monoclonal antibody inhibits the distribution of cocaine to the brain in mice.

The predominantly human sequence, high-affinity anticocaine monoclonal antibody (mAb) 2E2 was cleared slowly from mouse blood by a first-order process with an elimination t(1/2) of 8.1 days. Infused 2E2 also produced a dramatic dose-dependent increase in plasma cocaine concentrations and a concomitant decrease in the brain cocaine concentrations produced by an i.v. injection of cocaine HCl (0.56 mg/kg). At the highest dose of 2E2 tested (3:1, mAb/drug), cocaine was not detectable in the brain. Pharmacokinetic studies showed that the normal disappearance of cocaine from plasma was described by a two-compartment pharmacokinetic model with distribution t(1/2alpha) and terminal elimination t(1/2beta) values of 1.9 and 26.1 min, respectively. In the presence of an equimolar dose of mAb 2E2, there was a 26-fold increase in the area under the plasma cocaine concentration-time curve (AUC) relative to the AUC in the absence of 2E2. Consequently, 2E2 decreased the volume of distribution of cocaine from 6.0 to 0.20 l/kg, which approximated that of 2E2 (0.28 l/kg). However, cocaine was still rapidly cleared from plasma, and its elimination was now described by a single-compartment model with an elimination t(1/2) of 17 min. Importantly, 2E2 also produced a 4.5-fold (78%) decrease in the cocaine AUC in the brain. Therefore, the effect of 2E2 on plasma and brain cocaine concentrations was predominantly caused by a change in the distribution of cocaine with negligible effects on its rate of clearance. These data support the concept of immunotherapy for drug abuse.

Age-related changes in plasma coenzyme Q10 concentrations and redox state in apparently healthy children and adults.

BACKGROUND: Coenzyme Q10 (CoQ) is an endogenous enzyme cofactor, which may provide protective benefits as an antioxidant. Because age-related CoQ changes and deficiency states have been described, there is a need to establish normal ranges in healthy children. The objectives of this study are to determine if age-related differences in reduced CoQ (ubiquinol), oxidized CoQ (ubiquinone), and CoQ redox state exist in childhood, and to establish reference intervals for these analytes in healthy children. METHODS: Apparently healthy children (n=68) were selected from individuals with no history of current acute illness, medically diagnosed disease, or current medication treatment. Self-reported healthy adults (n=106) were selected from the ongoing Princeton Follow-up Study in greater Cincinnati. Participants were assessed for lipid profiles, ubiquinol concentration, ubiquinone concentration, total CoQ concentration, and CoQ redox ratio. RESULTS: Mean total CoQ and ubiquinol concentrations are similar in younger children (0.2-7.6 years) and adults (29-78 years); however, lipid-adjusted total CoQ concentrations are significantly increased in younger children. Also CoQ redox ratio is significantly increased in younger and older children compared with adults. CONCLUSIONS: Elevated CoQ and redox ratios in children may be an indication of oxidative stress effects, which are associated with early development of coronary heart disease.