Determination of synthetic cathinone α-pyrrolidinovalero-phenone and its metabolite in urine using solid-phase extraction and gas chromatography-mass spectrometry.

RATIONALE: The presence of α-pyrrolidinovalerophenone (α-PVP) and its metabolites in urine is evidence of the administration of α-PVP. A toxicological challenge is that the metabolites of α-PVP exhibit amphoteric properties, which make them unsuitable for detection using gas chromatography-mass spectrometry (GC/MS). In the study reported, proper derivatization and sample extraction were essential for improving the sensitivity for GC/MS analysis. METHODS: An automated solid-phase extraction (SPE) method has been developed and optimized. The derivatization efficiency was tested using longer reaction time and the addition of polar pyridine into a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane. Method validation, including linearity, limit of detection, precision, accuracy, and recovery, was evaluated using automatic SPE and GC/MS. RESULTS: The results suggested that adding pyridine to BSTFA (1:1, v/v) significantly improved derivatization efficiency and precision. After optimization, the linear range was from 25 to 1000 ng mL-1 with R2 > 0.9950. The limit of detection was 5 ng mL-1 for α-PVP and 25 ng mL-1 for OH-α-PVP. The recovery for SPE was over 88%. The inter-day and intra-day precisions were less than 15%. A forensic sample has been found containing α-PVP (67.3 ng mL-1 ) and OH-α-PVP (560.2 ng mL-1 ). CONCLUSIONS: This study is the first to validate an auto-SPE-GC/MS method for the quantification and qualification of α-PVP and OH-α-PVP in urine. We have successfully improved the derivatization efficiency and developed a sensitive and semi-automatic approach. This approach is desirable for the detection of synthetic cathinone at trace levels in biological samples.

Human in vivo metabolism study of LGD-4033.

Selective androgen receptor modulators (SARMs) are an emerging class of therapeutics targeted to cachexia, sarcopenia, and hypogonadism treatment. LGD-4033 is a SARM which has been included on the Prohibited List annually released by the World Anti-Doping Agency (WADA). The aim of the present work was the investigation of the metabolism of LGD-4033 in a human excretion study after administration of an LGD-4033 supplement, the determination of the metabolites' excretion profiles with special interest in the determination of its long-term metabolites, and the comparison of the excretion time of the phase I and phase II metabolites. The results were also compared to those derived from previous LGD-4033 studies concerning both in vitro and in vivo experiments. Supplement containing LGD-4033 was administered to one human male volunteer and urine samples were collected up to almost 21 days. Analysis of the hydrolyzed (with ß-glucuronidase) as well as of the non-hydrolyzed samples was performed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) in negative ionization mode and revealed that, in both cases, the two isomers of the dihydroxylated metabolite (M5) were preferred target metabolites. The gluco-conjugated parent LGD-4033 and its gluco-conjugated metabolites M1 and M2 can be also considered as useful target analytes in non-hydrolyzed samples. The study also presents two trihydroxylated metabolites (M6) identified for the first time in human urine; one of them was recently reported in an LGD-4033 metabolism study in horse urine and plasma.

Determination of Synthetic Cathinones in Urine Using Gas Chromatography-Mass Spectrometry Techniques.

In recent years, the abuse of synthetic cathinones has increased considerably. This study proposes a method, based on gas chromatography/mass spectrometry (GC-MS), to analyze and quantify six synthetic cathinones in urine samples: mephedrone (4-MMC), methylone (bk-MDMA), butylone, ethylone, pentylone and methylenedioxypyrovalerone (MDPV). In our procedure, the urine samples undergo solid-phase extraction (SPE) and derivatization prior to injection into the GC-MS device. Separation is performed using a HP-5MS capillary column. The use of selective ion monitoring (SIM mode) makes it is good sensitivity in this method, and the entire analysis process is within 18 min. In addition, the proposed method maintains linearity in the calibration curve from 50 to 2,000 ng/mL (r(2) > 0.995). The limit of detection of this method is 5 ng/mL, with the exception of MDPV (20 ng/mL); the limit of quantification is 20 ng/mL, with the exception of MDPV (50 ng/mL). In testing, the extraction performance of SPE was between 82.34 and 104.46%. Precision and accuracy results were satisfactory <15%. The proposed method was applied to six real urine samples, one of which was found to contain 4-MMC and bk-MDMA. Our results demonstrate the efficacy of the proposed method in the identification of synthetic cathinones in urine, with regard to the limits of detection and quantification. This method is highly repeatable and accurate.

Evaluation of an enzyme immunoassay for the detection of methadone metabolite EDDP [2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine] in urine.

BACKGROUND: We evaluated a new EDDP [2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine] enzyme immunoassay (EDDPI; Lin-Zhi International, Inc., Sunnyvale, CA) for the detection of this primary methadone urinary metabolite. METHODS: All specimens were tested with two different cutoff calibrators at 150 and 300 ng/ml EDDP on an ADVIA 1200 Chemistry System auto-analyzer. Controls containing 0, -25% (negative control), and +25% (positive control) of the cutoff calibrators (Lin-Zhi) were analyzed with each batch. All urine specimens were then analyzed by high-pressure liquid chromatography/ mass spectrometry/mass spectrometry (HPLC-MS/MS) for EDDP. RESULTS: Approximately, 42% (151) of the 362 specimens yielded positive results by the EDDP assay at 150 and/or 300 ng/ml cutoff values. Of these specimens, HPLC-MS/MS confirmed the presence of EDDP > 25 ng/ml in all 151 specimens. No specimen yielding negative EDDPI results contained EDDP by HPLC/MS/MS. At 150 ng/ml cutoff, the EDDPI demonstrated a sensitivity of 1.00, a specificity of 0.986, and an overall agreement of HPLC/MS/MS of >99%. At 300 ng/ml cutoff, the EDDPI demonstrated a sensitivity of 1.00, a specificity of 0.959, and an overall agreement of HPLC/MS/MS results of 97.5%. CONCLUSION: The Lin-Zhi EDDPI provides a precise, reliable method for the routine detection of methadone metabolite in urine specimens, particularly in pain management compliance testing.

Formation of 1,2:3,4-diepoxybutane-specific hemoglobin adducts in 1,3-butadiene exposed workers.

1,3-Butadiene (BD) is an important industrial chemical that is classified as a human carcinogen. BD carcinogenicity has been attributed to its metabolism to several reactive epoxide metabolites and formation of the highly mutagenic 1,2:3,4-diepoxybutane (DEB) has been hypothesized to drive mutagenesis and carcinogenesis at exposures experienced in humans. We report herein the formation of DEB-specific N,N-(2,3-dihydroxy-1,4-butadiyl)valine (pyr-Val) in BD-exposed workers as a biomarker of DEB formation. pyr-Val was determined in BD monomer and polymer plant workers that had been previously analyzed for several other biomarkers of exposure and effect. pyr-Val was detected in 68 of 81 (84%) samples ranging from 0.08 to 0.86 pmol/g globin. Surprisingly, pyr-Val was observed in 19 of 23 administrative control subjects not known to be exposed to BD, suggesting exposure from environmental sources of BD. The mean ± SD amounts of pyr-Val were 0.11 ± 0.07, 0.16 ± 0.12, and 0.29 ± 0.20 pmol/g globin in the controls, monomer, and polymer workers, respectively, clearly demonstrating formation of DEB in humans. The amounts of pyr-Val found in this study suggest that humans are much less efficient in the formation of DEB than mice or rats at similar exposures. Formation of pyr-Val was more than 50-fold lower than has been associated with increased mutagenesis in rodents. The results further suggest that formation of DEB relative to other epoxides is significantly different in the highest exposed polymer workers compared with controls and BD monomer workers. Whether this is due to saturation of metabolic formation or increased GST-mediated detoxification could not be determined.

The metabolism of [14C]-zibotentan (ZD4054) in rat, dog and human, the loss of the radiolabel and the identification of an anomalous peak, derived from the animal feed.

This paper presents an overview of a cross-species investigation of the metabolic fate of [(14)C]-zibotentan (ZD4054), with particular focus on the main analytical challenges encountered during the study. A combination of detection methods were used including HPLC coupled to UV, RAD and/or MS(MS), and (1)H NMR spectroscopy. The objective was to characterise and identify the major metabolites found in the circulation and excreta of rat and dog for comparison with those produced in human. Initial investigations in rat, using [(14)C]-labelled zibotentan positioned on the oxadiazole ring and HPLC-UV-RAD analysis, revealed seven labelled resolved metabolite peaks. Parallel analysis by HPLC-UV-MS (with in-source fragmentation) uncovered two additional metabolites, indicating loss of the radiolabel during biotransformation. Hence, in subsequent studies in rat, dog and human, dual-radiolabelled zibotentan was employed with the (14)C-label positioned on the pyridine ring, which was shown to be less prone to metabolism. A total of 12 metabolites were found in the excreta and plasma in all species. One of these metabolites was found in the circulation in humans, which warranted further investigations. Characterisation of the isolated human circulating metabolite by (1)H NMR was complicated by the co-extraction of a matrix component with a similar UV-chromophore to zibotentan, which was identified as daidzein, an isoflavone derived from the animal feed.

Urine analysis of 3,4-methylenedioxypyrovalerone in opioid-dependent patients by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry (GCMS) procedure was developed for the quantitative analysis of the new designer drug methylenedioxypyrovalerone (MDPV) in urine together with the common stimulants amphetamine, methamphetamine, and methylenedioxymethamphetamine (MDMA). The procedure involved electron ionization (EI) GCMS in the selected ion monitoring (SIM) mode after liquid-liquid extraction with toluene and derivatization with heptafluorobutyric acid anhydride. All MDPV findings were confirmed by positive chemical ionization GCMS in SIM mode. Positive chemical ionization-GCMS allowed the protonated molecule M+H+ m/z 276 to be used as a target ion with 3 abundant fragments as qualifier ions. By electron ionization-GCMS, the limit of quantification (LOQ) for MDPV was 0.02 mg/L; and for amphetamine, methamphetamine, and MDMA, the LOQ was 0.05 mg/L. The method was applied to monitoring urine samples from opioid-dependent patients undergoing opioid substitution treatment. Nine of the 34 urine samples (26%) analyzed were MDPV positive by the GCMS procedure. The positive samples were obtained from 2 female and 7 male patients with a mean age of 31 years. The median (range) MDPV concentration was 0.16 mg/L (0.04-3.9 mg/L) based on the 7 samples for which a numeric value was obtained, whereas the concentration was below the LOQ but above the limit of detection in 2 samples. The method revealed amphetamine in approximately 40% of the cases, and there was no statistical difference between the MDPV-positive and MDPV-negative groups. Urine amphetamine concentrations were on average 10 times higher than those of MDPV. The opioid-dependent patients used MDPV mainly as a substitute for amphetamine, judging from the laboratory findings of this study and the information from our patients.

Toxicological determination and in vitro metabolism of the designer drug methylenedioxypyrovalerone (MDPV) by gas chromatography/mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometry.

A method for the toxicological screening of the new designer drug methylenedioxypyrovalerone (MDPV) is described; with an emphasis on its application for anti-doping analysis. The metabolism of MDPV was evaluated in vitro using human liver microsomes and S9 cellular fractions for CYP450 phase I and uridine 5'-diphosphoglucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II metabolism studies. The resulting metabolites were subsequently liquid/liquid extracted and analyzed using gas chromatography/mass spectrometry (GC/MS) as trimethylsilyl (TMS) derivatives. The structures of the metabolites were further confirmed by accurate mass measurement using a liquid chromatography/quadrupole time-of-flight (LC/QTOF) mass spectrometer. The studies demonstrated that the main metabolites of MDPV are catechol and methyl catechol pyrovalerone, which are in turn sulfated and glucuronated. The method for the determination of MDPV in urine has been fully validated by assessing the limits of detection and quantification, linearity, repeatability, and accuracy. This validation demonstrates the suitability for screening of this stimulant substance for anti-doping and forensic toxicology purposes.

Gas chromatography-mass spectrometry method for the determination of methadone and 2-ethylidene-1,5-dimethyl-3, 3-diphenylpyrrolidine (EDDP).

Methadone is a synthetic opioid used to relieve pain, treat opioid withdrawal, and wean heroin addicts. Measurement of methadone and its major metabolite EDDP in urine is useful for assessing compliance with addiction rehabilitation and pain management programs. This method quantitatively measures methadone and its metabolite EDDP in urine. Methadone and EDDP are recovered from urine by solid phase extraction at pH 6.0. Analysis is performed by gas chromatography with mass spectrometry detection, using selective ion monitoring.

New designer drug alpha-pyrrolidinovalerophenone (PVP): studies on its metabolism and toxicological detection in rat urine using gas chromatographic/mass spectrometric techniques.

The aim of the present study was to identify the metabolites of the new designer drug alpha-pyrrolidinovalerophenone (PVP) in rat urine using GC/MS techniques. Eleven metabolites of PVP could be identified suggesting the following metabolic steps: hydroxylation of the side chain followed by dehydrogenation to the corresponding ketone; hydroxylation of the 2''-position of the pyrrolidine ring followed by dehydrogenation to the corresponding lactam or followed by ring opening to the respective aliphatic aldehyde and further oxidation to the respective carboxylic acid; degradation of the pyrrolidine ring to the corresponding primary amine; and hydroxylation of the phenyl ring, most probably in the 4'-position. The authors' screening procedure for pyrrolidinophenones allowed the detection of PVP metabolites after application of a dose corresponding to a presumed user's dose. In addition, the involvement of nine different human cytochrome P450 (CYP) isoenzymes in the side chain hydroxylation of PVP was investigated and CYP 2B6, 2C19, 2D6, and 3A4 were found to catalyze this reaction.

Metabolism, distribution, and excretion of a next generation selective estrogen receptor modulator, lasofoxifene, in rats and monkeys.

Disposition of lasofoxifene (LAS; 6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalen-2-ol. tartrate) was investigated in rats and monkeys after oral administration of a single oral dose of [(14)C]LAS. Total mean recoveries of the radiocarbon were 96.7 and 94.3% from rats and monkeys, respectively. The major route of excretion in both species was the feces, and based on a separate study in the bile duct-cannulated rat, this likely reflects excretion in bile rather than incomplete absorption. Whole-body autoradioluminography suggested that [(14)C]LAS radioequivalents distributed rapidly in the rat with most tissues achieving maximal concentrations at 1 h. Half-life of radioactivity was longest in the uvea (124 h) and shortest in the spleen ( approximately 3 h). LAS was extensively metabolized in both rats and monkeys because no unchanged drug was detected in urine and/or bile. Based on area under the curve((0-24)) values, >78% of the circulating radioactivity was due to the metabolites. A total of 22 metabolites were tentatively identified by liquid chromatography-tandem mass spectrometry. Based on the structures of the metabolites, six metabolic pathways of LAS were identified: hydroxylation at the tetraline ring, hydroxylation at the aromatic ring attached to tetraline, methylation of the catechol intermediates by catechol-O-methyl transferase, oxidation at the pyrrolidine ring, and direct conjugation with glucuronic acid and sulfuric acid. LAS and its glucuronide conjugate (M7) were the major circulating drug-related moieties in both rats and monkeys. However, there were notable species-related qualitative and quantitative differences in the metabolic profiles. The catechol (M21) and its sulfate conjugate (M10) were observed only in monkeys, whereas the glucuronide conjugate of the methylated catechol (M8) and hydroxy-LAS (M9) were detected only in rats.

Simultaneous determination of ketamine, tramadol, methadone, and their metabolites in urine by gas chromatography-mass spectrometry.

An automated solid-phase extraction procedure combined with gas chromatography-mass spectrometry methodology, without derivatization, has been developed for the identification and quantitation of ketamine, norketamine, tramadol, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, and 2-ethyl-5-methyl-3,3-diphenylpyrroline in urine. The analytical approach is simple and rapid, yet reliable. Good linearity (r(2) > 0.995 over the concentration range of 30 to 600 ng/mL), sensitivity (limits of quantitation 15-30 ng/mL), accuracy (81.0-109.9%), precision (RSD < 13.8%), and recovery (> 79.6% in average) were achieved for all analytes. Ninety-one urine specimens from suspected drug users and 21 clinical urine specimens from methadone substitution therapy patients were analyzed to validate the method compatibility and stability. Results have demonstrated that this GC-MS method is a good confirmation and quantitation test scheme for the six target compounds in urine.

Use of capillary electrophoresis and poly(ethylene oxide) as the coating agent for the determination of substances related to heroin addiction and treatment.

The purpose of this study was to identify and quantify morphine, codeine, methadone, and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine using capillary electrophoresis in urine specimens. Adequate peak separation was achieved using nearly neutral pH phosphate buffer and poly(ethylene oxide) as the coating agent. This dynamic coating of the inner surface of the capillary was obtained by rinsing with a solution containing this compound. The electroosmotic flow and the interactions between analytes and the capillary wall surface were reduced, while resolution and reproducibility were thus improved. Detection limits were appropriate for usual analytical requirements in forensic laboratories.

Studies on the metabolism and toxicological detection of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) in rat urine using gas chromatography-mass spectrometry.

The aim of the presented study was to identify the metabolites of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) in rat urine using GC-MS techniques. After enzymatic hydrolysis, extraction and various derivatizations, seven metabolites of MPBP could be identified suggesting the following metabolic steps: oxidation of the 4'-methyl group to the corresponding alcohol and further oxidation to the respective carboxy compound, hydroxylation of the pyrrolidine ring followed by dehydrogenation to the corresponding lactam or reduction of the keto group to the 1-dihydro compound. A previously published GC-MS-based screening procedure for pyrrolidinophenones involving enzymatic hydrolysis and mixed-mode solid-phase extraction of urine samples allowed detection of MPBP metabolites. Assuming similar metabolism and dosages in humans, an intake of MPBP should be detectable via its metabolites in urine.

Metabolism of the new designer drug alpha-pyrrolidinopropiophenone (PPP) and the toxicological detection of PPP and 4'-methyl-alpha-pyrrolidinopropiophenone (MPPP) studied in rat urine using gas chromatography-mass spectrometry.

R,S-alpha-pyrrolidinopropiophenone (PPP) is a new designer drug with assumed amphetamine-like effects which has appeared on the illicit drug market. The aim of this study was to identify the PPP metabolites using solid-phase extraction, ethylation or acetylation as well as to develop a toxicological detection procedure in urine using solid-phase extraction, trimethylsilylation and gas chromatography-mass spectrometry (GC-MS). Analysis of urine samples of rats treated with PPP revealed that PPP was extensively metabolized by hydroxylation of the pyrrolidine ring with subsequent dehydrogenation to the corresponding lactam, hydroxylation of the aromatic ring in position 4' or double dealkylation of the pyrrolidine ring to the corresponding primary amine (cathinone) partly followed by reduction of the keto group to the corresponding secondary alcohol (norephedrines). As cathinone and the norephedrine diastereomers are also formed after intake of other drugs of abuse or medicaments, special attention must be paid to the detection of the unequivocal metabolite 2"-oxo-PPP as an unambiguous proof for the intake of PPP. The hydroxy groups were found to be partly conjugated. Based on these data, PPP could be detected in urine via its metabolites by full-scan GC-MS using mass chromatography for screening and library search for identification by comparison of the spectra with reference spectra. The same toxicological detection procedure can be applied to other designer drugs of the pyrrolidinophenone type, like MOPPP, MDPPP, MPHP, and MPPP. The detection of the latter will also be presented here.

Methadone and metabolite urine concentrations in patients maintained on methadone.

As regulatory control over methadone maintenance relaxes, the need for methods of monitoring compliance will increase. In community clinics, monitoring would most likely involve immunoassays of outpatients' trough urine specimens. There are no published norms for such data. Therefore, we determined concentrations of methadone in 1093 urine specimens collected thrice weekly in 27 outpatients during up to 17 weeks of observed methadone ingestion (35 to 80 mg/day) using a semiquantitative homogeneous enzyme immunoassay (CEDIA). We used a separate CEDIA assay to measure methadone's main metabolite, 2-ethylidene-3,3-diphenylpyrrolidine (EDDP), which may help detect compliance in fast metabolizers or patients who adulterate samples to simulate compliance. Methadone concentrations were more variable than those of EDDP. Concentrations of methadone were < 100 ng/mL in one specimen, between 100 and 300 ng/mL in 27, and >or= 300 ng/mL in all others. EDPP concentrations were >or= 100 ng/mL in all specimens, suggesting that EDDP should be detectable in urine from compliant patients. Methadone and EDDP concentrations significantly increased with methadone dose and (in one participant with poor clinic attendance) significantly decreased following missed methadone doses. Nevertheless, variability was too great to permit estimation of methadone dose (or detect a single missed administration) from any single specimen.

Pharmacokinetics of alizapride in children receiving chemotherapy for solid tumour.

The purpose of the present study was to determine the pharmacokinetics of alizapride to optimize its use in children aged 1 month to 15 years old who were receiving chemotherapy. Seventeen children were given a single 4 mg/kg alizapride infusion prior to the administration of cytostatic drugs. Blood and urine samples were collected within 10 h after onset of the infusion. Kinetic parameters were calculated and showed a decrease in plasma clearance expressed per unit of body weight with age. The current data suggest that dosage expressed per unit of body weight should be higher in children than in adults and higher in infants than in children.

Rapid determination of methadone and its major metabolite in biological fluids by gas-liquid chromatography with thermionic detection for maintenance treatment of opiate addicts.

A rapid gas-liquid chromatographic assay is developed for the quantification of methadone (Mtd) and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in biological fluids of opiate addicts. After alkaline extraction from samples with lidocaine hydrochloride as internal standard, Mtd and EDDP are separated on SP-2250 column at 220 degrees C and detected with a thermionic detector. The chromatographic time is about 6 min. The relative standard deviations (R.S.D.) of Mtd and EDDP standards are between 1.5 and 5.5%. Most drugs of abuse (morphine, codeine, narcotine, cocaine, benzoylecgonine, cocaethylene, dextropropoxyphene etc) are shown not to interfere with this technique. The method has been applied to study the levels of Mtd and EDDP metabolite in serum, saliva and urine of patients under maintenance treatment for opiate dependence. EDDP levels were found higher than those of Mtd in urine samples from four treated patients, but lower in serum and undetectable in saliva. However, Mtd concentrations were higher in saliva than in serum.

Identification of a pyrovalerone metabolite in the rat by gas chromatography-mass spectrometry and determination of pyrovalerone by gas chromatography-nitrogen-phosphorus detection.

Pyrovalerone and its hydroxylated metabolite have been identified by gas chromatography-mass spectrometry in rat urine and plasma. A sensitive gas chromatographic method for the quantitative analysis of pyrovalerone in rat urine and plasma is described. The method also permits the quantitative monitoring of the urinary excretion of the drug and its metabolite. Pyrovalerone and its hydroxylated metabolite are detected up to 18 h after a single oral administration to the rat at a dose of 20 mg/kg.

Preliminary determination of the association between symptom expression and urinary metabolites in subjects with chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) patients have a urinary metabolite labeled CFSUM1 with increased incidence (P < 0.004) and relative abundance (P < 0.00003). The relative abundances of urinary CFSUM1 and beta-alanine were associated with alterations in metabolite excretion and symptom incidence. In 20 CFS patients and 45 non-CFS subjects, symptom/metabolite associations were investigated by assessing symptom sensitivity and specificity, and symptom indices of total symptom incidence, CFS core symptoms, cognitive, neurological, musculoskeletal, gastrointestinal, infection-related and genitourinary symptom indices, as well as a visual analogue pain scale of average pain intensity. Thirty-three symptoms had significant (P < 0.005) sensitivity and specificity in the CFS patients compared to that in the non-CFS controls. Severe fatigue was the only symptom with 100% sensitivity and specificity and CFSUM1 excretion was the primary metabolite for expression of this symptom. All nine symptom indices had elevated responses in the CFS patients (all P < 0.0000001). Multiple regression analyses indicated that all the symptom indices had significant correlations (R) with changes in the urinary excretion of metabolites (P < 0.0001). CFSUM1 and beta-alanine were the first and second metabolites correlated with the CFS core symptom index and CFSUM1 was primarily associated with infection-related and musculoskeletal indices whereas beta-alanine was primarily associated with gastrointestinal and genitourinary indices. The strong associations of CFSUM1 and beta-alanine with CFS symptom expression provide a molecular basis for developing an objective test for CFS.