Isotope Dilution Gas Chromatography-mass Spectrometry Research Articles

Trans Fatty Acids in Plasma, Serum, and Red Blood Cells

Trans‐fatty acids (TFA) intake was associated with increased risk of cardiovascular disease. However, little is known about TFA concentrations in human blood. Studies suggested that individuals with the same fatty acid (FA) intake may have different concentrations of regular FA in red blood cells (RBC), plasma, and serum due to a number of factors including genetics and lifestyle. This created the need to assess TFA concentrations in different blood specimens from people to obtain information about TFA exposure and metabolism. We investigated the TFA distribution in RBC, plasma, and serum using convenience samples from 66 adults. Samples were analyzed using an isotope dilution‐gas chromatography‐mass spectrometry method validated for the analysis of 28 FA, including the 4 major TFA: palmitelaidic acid, elaidic acid, vaccenic acid, and linoelaidic acid. The TFAs were detected in all samples, with median total TFA concentrations of 21.12 μM in RBC, 17.86 μM in plasma, and 20.88 μM in serum. The median percent of total TFA was similar for elaidic and vaccenic acids in all three matrices. The median percent of total TFA was 1/3 lower in the RBC than in the plasma and serum for palmitelaidic acid and half as high for linoelaidic acid in RBC as in plasma and serum. The differences in TFA distributions suggest differences in metabolism of individual TFA.

Simultaneous determination of creatinine and creatine in human serum by double-spike isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS).

This work describes the first multiple spiking isotope dilution procedure for organic compounds using (13)C labeling. A double-spiking isotope dilution method capable of correcting and quantifying the creatine-creatinine interconversion occurring during the analytical determination of both compounds in human serum is presented. The determination of serum creatinine may be affected by the interconversion between creatine and creatinine during sample preparation or by inefficient chemical separation of those compounds by solid phase extraction (SPE). The methodology is based on the use differently labeled (13)C analogues ((13)C1-creatinine and (13)C2-creatine), the measurement of the isotopic distribution of creatine and creatinine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the application of multiple linear regression. Five different lyophilized serum-based controls and two certified human serum reference materials (ERM-DA252a and ERM-DA253a) were analyzed to evaluate the accuracy and precision of the proposed double-spike LC-MS/MS method. The methodology was applied to study the creatine-creatinine interconversion during LC-MS/MS and gas chromatography-mass spectrometry (GC-MS) analyses and the separation efficiency of the SPE step required in the traditional gas chromatography-isotope dilution mass spectrometry (GC-IDMS) reference methods employed for the determination of serum creatinine. The analysis of real serum samples by GC-MS showed that creatine-creatinine separation by SPE can be a nonquantitative step that may induce creatinine overestimations up to 28% in samples containing high amounts of creatine. Also, a detectable conversion of creatine into creatinine was observed during sample preparation for LC-MS/MS. The developed double-spike LC-MS/MS improves the current state of the art for the determination of creatinine in human serum by isotope dilution mass spectrometry (IDMS), because corrections are made for all the possible errors derived from the sample preparation step.

Validation of an isotope dilution gas chromatography–mass spectrometry method for combined analysis of oxysterols and oxyphytosterols in serum samples

We describe the validation of a method for the analysis of oxysterols, i.e. oxycholesterols and oxyphytosterols, in human serum using gas chromatography–mass spectrometry selected ion monitoring (GC–MS-SIM). Concentrations of 7α- and 7β-hydroxy-, and 7oxo-cholesterol, -campesterol, and -sitosterol as well as 4β-hydroxycholesterol and side-chain oxygenated 24S-, 25-, and 27-hydroxycholesterol were determined by isotope dilution methodology.After saponification at room temperature the oxysterols were extracted, separated from their substrates, cholesterol, campesterol, and sitosterol, by solid phase extraction, and subsequently derivatised to their corresponding trimethylsilyl-ethers prior to GC–MS-SIM. In order to prevent artificial autoxidation butylated hydroxytoluene and ethylenediaminetetraacetic acid were added. The validation of the method was performed according to the International Conference on Harmonisation guidance, including limits of detection and quantification, ranges, recovery and precision. Due to improved instrumental settings and work-up procedure, limits of detection and quantification ranged between 8.0–202.0pg/mL and 28.0–674pg/mL, respectively. Recovery data in five calibration points varied between 91.9% and 116.8% and in serum samples between 93.1% and 118.1%. The mean coefficient of variation (CV) for the recovery of all compounds was <10%. Well satisfying CVs for within-day precision (2.1–10.8%) and for between-day precision (2.3–12.1%) were obtained. More than 20 samples could be processed in a single routine day and test series of about 300 samples can be realised without impairment of the validation parameters during a sequence. Comparison of oxysterol and oxyphytosterol content in serum and plasma revealed no difference.A fully validated isotope dilution methodology for the quantification of oxycholesterols and oxyphytosterols from human serum or plasma is presented.

S1 certification of alpha-endosulfan, beta-endosulfan, and endosulfan sulfate in a candidate certified reference material (organochlorine pesticides in tea) by isotope dilution gas chromatography-mass spectrometry.

This paper presents the certification of alpha-endosulfan, beta-endosulfan, and endosulfan sulfate in a candidate tea certified reference material (code: GLHK-11-03) according to the requirements of the ISO Guide 30 series. Certification of GLHK-11-03 was based on an analytical method purposely developed for the accurate measurement of the mass fraction of the target analytes in the material. An isotope dilution mass spectrometry (IDMS) method involving determination by (i) gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS) and (ii) gas chromatography-electron ionization-high-resolution mass spectrometry (GC-EI-HRMS) techniques was employed. The performance of the described method was demonstrated through participation in the key comparison CCQM-K95 "Mid-Polarity Analytes in Food Matrix: Mid-Polarity Pesticides in Tea" organized by the Consultative Committee for Amount of Substance-Metrology in Chemistry in 2012, where the study material was the same as the certified reference material (CRM). The values reported by using the developed method were in good agreement with the key comparison reference value (KCRV) assigned for beta-endosulfan (727 ± 14μgkg(-1)) and endosulfan sulfate (505 ± 11μgkg(-1)), where the degree of equivalence (DoE) values were 0.41 and 0.40, respectively. The certified values of alpha-endosulfan, beta-endosulfan, and endosulfan sulfate in dry mass fraction in GLHK-11-03 were 350, 730, and 502μgkg(-1), respectively, and the respective expanded uncertainties, due to sample inhomogeneity, long-term and short-term stability, and variability in the characterization procedure, were 27μgkg(-1) (7.8%), 48μgkg(-1) (6.6%), and 33μgkg(-1) (6.6%).

Rapid analysis of heterocyclic acetals in wine by stable isotope dilution gas chromatography–mass spectrometry

Heterocyclic acetals from acetaldehyde and glycerol have been proposed as markers of age and oxidation in fortified wines. Here the analysis of these acetals (cis- and trans-5-hydroxy-2-methyl-1,3-dioxane and cis- and trans-4-hydroxymethyl-2-methyl-1,3-dioxolane) by liquid–liquid extraction and gas chromatography–mass spectrometry was optimized for rapid monitoring of these compounds in aged and oxidized table wines. The method provides a wide range of linearity and reproducibility below 3.0% RSD, while the detection limits, 2.85–13.5 μg/L, were significantly lower than typical levels measured in wines. Stable isotope dilution improved the accuracy of the method. Oxidation of red wines was shown to correlate with increased levels of the acetals. The accuracy and speed of the new method will allow for quantifying oxidation in large experiments such as wine aging or micro-oxygenation trials, and to assess the potential value of these substances as markers of wine oxidation.

Simultaneous analysis of 10 polycyclic aromatic hydrocarbons in roasted coffee by isotope dilution gas chromatography-mass spectrometry: Optimization, in-house method validation and application to an exploratory study

The presence of polycyclic aromatic hydrocarbons (PAHs) in food implies a potential risk to human health. Although the coffee has been appointed as an important matrix to be monitored for contamination by PAHs, there is still a gap in the literature regarding to the methods of analysis appropriately validated for this scope and also related to the occurrence of contamination in Brazilian commercial samples. In this paper, a method for the determination of ten priority PAHs in roasted coffee by isotope dilution gas chromatography coupled to mass spectrometry was optimized, validated and applied to an exploratory study with 24 commercial samples. The samples were extracted with simultaneous clean-up using pressurized liquid extraction (PLE), followed by liquid–liquid extraction (LLE) and clean-up using silica columns. Linearity was obtained in the concentration range from 0.25 to 4.00 μg kg−1. Matrix effects were not significant. The precision was demonstrated with relative standard deviations varying from 3.3 to 24 % under repeatability and from 3.3 to 33 % under intermediate precision conditions. The average recovery values ranged between 87 and 111%. These values, as well as the limits of detection, limits of quantification and uncertainties of measurements, met the performance criteria established by European Union regulations. The contamination of the commercial samples ranged from 1.00 ± 0.35 to 11.29 ± 2.33 μg kg−1, indicating the need of further occurrence studies and establishment of monitoring programs in Brazil.

Influence of chemical straightening on the stability of drugs of abuse in hair.

Chemical straightening, also known as a relaxer, is ubiquitously used among African American women to obtain straighter hair compared with their natural tresses. This study focused on the stability of drugs of abuse in hair after a single application of the relaxer. Commercially available 'Lye' or 'No-Lye' chemical straightening products (Silk Elements™) were applied in vitro to drug-fortified hair (standard reference materials (SRM) 2379 and 2380) and hairs clipped from established drug users. Target analytes (cocaine (COC), benzoylecgonine (BZE), cocaethylene (CE), phencyclidine and tetrahydrocannabinol) were isolated using solid-phase extraction and then analyzed with isotope dilution gas chromatography-mass spectrometry with selective ion monitoring. After either treatment, drug concentrations were significantly (P < 0.05) decreased in both the SRM sample and the hair from authentic abusers. In the SRM groups, 6-67% of the original concentration remained after a single chemical treatment. Similarly, only 5-30% of the original concentration remained in authentic drug hairs that had formerly tested positive for COC, BZE and CE.

Monitoring urinary metabolites resulting from sulfur mustard exposure in rabbits, using highly sensitive isotope-dilution gas chromatography-mass spectrometry.

A highly sensitive method for the determination of sulfur mustard (SM) metabolites thiodiglycol (TDG) and thiodiglycol sulfoxide (TDGO) in urine was established and validated using isotope-dilution negative-ion chemical ionization (NICI) gas chromatography-mass spectrometry (GC-MS). TDGO in the samples was reduced with TiCl3, and then determined together with TDG as a single analyte. The sample preparation procedures, including two solid-phase-extraction (SPE) clean-up steps, were optimized to improve the sensitivity of the method. The limits of detection (LOD) for both TDG and TDG plus TDGO (TDG + TDGO) were 0.1 ng mL(-1), and the limits of quantitation (LOQ) for both were 0.3 ng mL(-1). The method was used in a rabbit cutaneous SM exposure model. Domestic rabbits were exposed to neat liquid SM at three dosage levels (0.02, 0.05, and 0.15 LD50), and the urinary excretion of four species of hydrolysis metabolites, namely free TDG, free plus conjugated TDG (total TDG), free TDG + TDGO, and free plus conjugated TDG + TDGO (total TDG + TDGO), was evaluated to investigate the metabolic processes. The total urinary excretion profiles of the metabolites, including the peak time, time window, and dose-response and time-response relationships, were clarified. The results revealed that the concentrations of TDG and TDG + TDGO in the urine increased quickly and then decreased rapidly in the first two days after SM exposure. The cumulative amount of total TDG + TDGO excreted in urine during the first five days accounted for 0.5-1% of the applied dose of SM. It is also concluded that TDG and TDGO in urine existed mainly in free form, the levels of glucuronide and of sulfate conjugates of TDG or TDGO were very low, and most hydrolysis metabolites were present in the oxidized form (TDGO). The study indicates that the abnormal increase of TDG and TDGO excretion levels can be used as a diagnostic indicator and establishes a reference time-window for retrospective analysis and sampling after SM exposure.

Stable isotope dilution gas chromatography-mass spectrometry for quantification of thymoquinone in black cumin seed oil.

Black cumin seed (Nigella sativa L.) is a widely used spice and herb, where thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone) is the major bioactive compound. Here, a stable isotope dilution (SID) gas chromatography-mass spectrometry (GC-MS) technique was developed for the quantification of thymoquinone. A doubly deuterated thymoquinone ([(2)H2]-thymoquinone) was synthesized for the first time with more than 93% deuteration degree shown by mass spectrometry and proton nuclear magnetic resonance ((1)H NMR). This compound was used as an internal standard for the quantification of thymoquinone using a SID GC-MS method. The validation experiment showed a recovery rate of 99.1 ± 1.1% relative standard deviation (RSD). Standard addition and external calibration methods have also been used to quantify thymoquinone, which cross-validated the developed stable isotope dilution assay (SIDA). In comparison to external calibration and standard addition methods, the SIDA method is robust and accurate. The concentration of thymoquinone in five marketed black cumin seed oils ranged between 3.34 and 10.8 mg/mL by use of SID GC-MS.

The brominated flame retardants, PBDEs and HBCD, in Canadian human milk samples collected from 1992 to 2005; concentrations and trends

Human milk samples were collected from individuals residing in various regions across Canada mostly in the years 1992 to 2005. These included five large cities in southern Canada as well as samples from Nunavik in northern Quebec. Comparative samples were also collected from residents of Austin, Texas, USA in 2002 and 2004. More than 300 milk samples were analysed for the brominated flame retardants (BFRs), PBDEs and HBCD, by extraction, purification and quantification using either isotope dilution gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-MS. The Canadian total PBDE values in the years 2002–2005 show median levels of about 20μg/kg on a lipid basis; a value significantly higher than in the 1980s and 1990s. Milk samples from Inuit donors in the northern region of Nunavik were slightly lower in PBDE concentrations than those from populated regions in the south of Quebec. Milk samples from Ontario contained slightly lower amounts of PBDEs in two time periods than those from Texas. HBCD levels in most milk samples were usually less than 1ppb milk lipid and dominated by the α-isomer. This large data set of BFRs in Canadian human milk demonstrates an increase in the last few decades in human exposure to BFRs which now appears to have stabilized.

Alkylpyrazine contents of coffee beverages using stable isotope dilution gas chromatography–mass spectrometry

In the presented study the effects of key brewing parameters (temperature, holding time and storage conditions of coffee powder) on 12 alkylpyrazines in coffee beverages were investigated. The results show that the alkylpyrazine profiles of coffee beverages are similar to those of the coffee used. In total, 70–82% of the alkylpyrazines (113.3 ± 3.5 to 132.6 ± 0.6 mg/kg), depending on the brewing method, passed into beverages during the brewing process. At temperatures higher than 50 °C the alkylpyrazine contents of coffee beverages were almost independent of the brewing temperature. During a 30 min holding time at room temperature the alkylpyrazine contents of a freshly brewed cup of coffee decreased by about 10%. Furthermore, storing coffee in closed systems for up to 29 days at +4 °C, −20 °C or room temperature (+22 °C) had minor effects on the alkylpyrazine contents of beverages produced from the coffee. The results show that under customary conditions more than 70% of the alkylpyrazines present in ground coffee are transferred to beverages during the brewing process. Estimated total amounts of alkylpyrazines ingested by the consumer per cup of coffee beverage are in the milligram range, sufficient to have significant potential health effects.

Simultaneous analysis of phthalates, adipate and polycyclic aromatic hydrocarbons in edible oils using isotope dilution-gas chromatography–mass spectrometry

A method for simultaneous determination of 12 priority phthalates, adipate and polycyclic aromatic hydrocarbons (PAHs) in edible oils by isotope dilution-gas chromatography–mass spectrometry (ID-GC–MS) was developed for fast, accurate and trace analysis. The extraction and clean-up procedures were optimised, and using stable isotope-labelled internal standards for each analyte, relative standard deviations (RSDs) of 0.92–10.6% and spiked sample recoveries of 80.6–97.8% were obtained. Limits of detection for PAHs were in the range of 0.15–0.77 µg/kg and those for phthalates were in the range of 4.6–10.0 µg/kg. The calibration curves exhibited good linearities with regression coefficients of R2 ≥ 0.99. Twelve edible oils were examined to evaluate the efficiency of this method. Among the 12 analytes, dibutyl phthalates (DBP), diethylhexyl phthalates (DEHP), diethylhexyl adipate (DEHA), benzo[a]anthracene (B[a]A), chrysene (Chry) and benzo[b]fluoranthene (B[b]F) were detected in the range of 1.17–806 µg/kg.

Pharmacologic Stimulation of Cytochrome P450 46A1 and Cerebral Cholesterol Turnover in Mice

Cytochrome P450 46A1 (CYP46A1) is a brain-specific cholesterol 24-hydroxylase responsible for the majority of cholesterol elimination from the brain. Genetically increased CYP46A1 expression in mice leads to improved cognition and decreases manifestations of Alzheimer disease. We found that four pharmaceuticals (efavirenz (EFV), acetaminophen, mirtazapine, and galantamine) prescribed for indications unrelated to cholesterol maintenance increased CYP46A1 activity in vitro. We then evaluated the anti-HIV medication EFV for the mode of interaction with CYP46A1 and the effect on mice. We propose a model for CYP46A1 activation by EFV and show that EFV enhanced CYP46A1 activity and cerebral cholesterol turnover in animals with no effect on the levels of brain cholesterol. The doses of EFV administered to mice and required for the stimulation of their cerebral cholesterol turnover are a hundred times lower than those prescribed to HIV patients. At such small doses, EFV may be devoid of adverse effects elicited by high drug concentrations. CYP46A1 could be a novel therapeutic target and a tool to further investigate the physiological and medical significance of cerebral cholesterol turnover.

Quantitative analysis and stability of the rodenticide TETS (tetramine) in finished tap water

The determination of the rodenticide tetramethylenedisulfotetramine (TETS) in drinking water is reportable through the use of automated sample preparation via solid phase extraction and detection using isotope dilution gas chromatography-mass spectrometry. The method was characterized over twenty-two analytical batches with quality control samples. Accuracies for low and high concentration quality control pools were 100 and 101%, respectively. The minimum reporting level (MRL) for TETS in this method is 0.50 μg L−1. Five drinking waters representing a range of water quality parameters and disinfection practices were fortified with TETS at ten times the MRL and analyzed over a 28 day period to determine the stability of TETS in these waters. The amount of TETS measured in these samples averaged 100 ± 6% of the amount fortified suggesting that tap water samples may be held for up to 28 days prior to analysis.

Determination of endogenous agmatine in rat plasma by isotope dilution-gas chromatography-mass spectrometry

A method for the determination of endogenous agmatine in rat plasma was developed by isotope dilution-gas chromatography-negative chemical ionization mass spectrometry (GC-NCI/MS). The plasma samples were analyzed after protein precipitation, evaporation, derivatization by hexafluoroacetone (HFAA), and clean-up on a Florisil SPE column. The GC-MS analysis utilized stable isotope d8-agmatine as internal standard. The samples after treatme were tested by negative chemical ionization with selected ion monitoring (SIM) which was set at m/z 492 (molecular ion of agmatine) and m/z 500 (molecular ion of internal standard). The limit of detection (LOD) of agmatine standard solution was 0.005 7 ng/mL. The calibration curve of the agmatine spiked in rat plasma showed a good linear relationship at the range of 1.14-57.0 ng/mL (r = 0.997). The recoveries of agmatine spiked in rat plasma ranged from 92.3% to 109.8%. Inter-day and intra-day precisions were less than 15%. The average concentration level of agmatine in rat plasma was (22 +/- 9) ng/mL, and there was no significant difference between male and female SD rats (p > 0.05). The method is high sensitive and specific, and can be used for the determination of endogenous agmatine in plasma. It provides a strong support for the subsequent research of agmatine.

Concentrations of Cocaine and Benzoylecgonine in Femoral Blood from Cocaine-Related Deaths Compared with Venous Blood from Impaired Drivers

The concentrations of cocaine and its major metabolite benzoylecgonine (BZE) were determined in femoral blood from 132 cocaine-related deaths and compared with venous blood from 988 apprehended drivers. Cocaine and BZE were determined by solid-phase extraction and isotope dilution gas chromatography-mass spectrometry with limits of quantitation of 0.02 mg/L for both substances. Significantly more men (95-98%) than women (2-5%) abused cocaine, although their mean age was about the same (29-30 years). Mean age (±SD) of cocaine-related deaths was 29 ± 7 years, which was not significantly different from 30 ± 8 years in traffic cases (P > 0.05). The median concentration of cocaine in blood in 61 fatalities was 0.10 mg/L compared with 0.06 mg/L in traffic cases (P < 0.001). In drug intoxication deaths, the median concentration of cocaine was 0.13 mg/L (N = 25), which was not significantly different from 0.09 mg/L (N = 36) in other causes of death. Cocaine-related deaths mostly involved mixed drug intoxications including co-ingestion of heroin, cannabis, amphetamines as well as legal drugs, such as benzodiazepines and/or ethanol. The concentrations of cocaine in blood from living and deceased persons overlapped, which makes it infeasible to predict toxicity from the analytical toxicology results alone.

Preparation of serum total cholesterol, total glycerol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol reference materials

Objective To prepare serum reference materials for total cholesterol (TC),total glycerol (TTG),high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Methods Individual blood samples were collected from 28 healthy donors (14 males and 14 females) aged from 20 to 50 years old in November 2008 in Beijing Hospital,and the sera were separated and mixed into 3 serum pools according to cholesterol levels.The materials were tested for homogeneity and stability using routine methods.The methods of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) and high performance liquid chromatography (HPLC) were used to determine the concentration of TC.HPLC,ID-LC/MS/MS and isotope dilution gas chromatography mass spectrometry (ID-GC/MS) methods were used for TTG.The values of HDL-C and LDL-C were assigned by the designated comparison method and ultracentrifugation/HPLC method,respectively. Results The materials were homogeneous and were tested to be stable for at least 1 year at -70℃ using the isochronous stability study.The certified values (reference value±expanded uncertainty) of the 3 materials were: TC, 6.798±0.075, 4.470±0.050, 3.221±0.042; TTG, 1.327±0.024, 0.824±0.017, 0.659±0.014; HDL-C, 1.640±0.028, 1.652±0.027, 1.184±0.022; LDL-C, 4.577±0.097, 2.492±0.072, 1.788±0.041. Conclusion Certified reference materials for serum TC, TTG, HDL-C and LDL-C have been prepared.(Chin J Lab Med,2013,36:1013-1017) Key words: Cholesterol; Triglycerides; Cholesterol; HDL; Cholesterol; LDL; Reference standards

An improved reference measurement procedure for triglycerides and total glycerides in human serum by isotope dilution gas chromatography–mass spectrometry

BackgroundTriglycerides are widely tested in clinical laboratories using enzymatic methods for lipid profiling. As enzymatic methods can be affected by interferences from biological samples, this together with the non-specific nature of triglycerides measurement makes it necessary to verify the accuracy of the test results with a reference measurement procedure. Several such measurement procedures had been published. These procedures generally involved lengthy and laborious sample preparation steps. In this paper, an improved reference measurement procedure for triglycerides and total glycerides was reported which simplifies the sample preparation steps and greatly shortens the time taken. MethodsThe procedure was based on isotope dilution gas chromatography–mass spectrometry (ID GC–MS) with tripalmitin as the calibration standard. Serum samples were first spiked with isotope-labeled tripalmitin. For the measurement of triglycerides, the serum samples were subjected to lipid extraction followed by separation of triglycerides from diglycerides and monoglycerides. Triglycerides were then hydrolyzed to glycerol, derivatized and injected into the GC–MS for quantification. For the measurement of total glycerides, the serum samples were hydrolyzed directly and derivatized before injection into the GC–MS for quantification. ResultsAll measurement results showed good precision with CV <1%. A certified reference material (CRM) of lipids in frozen human serum was used to verify the accuracy of the measurement. The obtained values for both triglycerides and total glycerides were well within the certified ranges of the CRM, with deviation <0.4% from the certified values. The relative expanded uncertainties were also comparable with the uncertainties associated with the certified values of the CRM. The validated procedure was used in an External Quality Assessment (EQA) Program organized by our laboratory to establish the assigned values for triglycerides and total glycerides.

Presence of appreciable amounts of ethylene glycol, propylene glycol, and diethylene glycol in human urine of healthy subjects

In a previous study, we found that appreciable amounts of ethylene glycol (EG), propylene glycol (PG), and diethylene glycol (DEG) were present in the blood of nonoccupational healthy humans. In this study, we measured the three glycols in the urine of healthy subjects by the same isotope dilution gas chromatography–mass spectrometry method, and found the concentrations to be much higher than those in blood. The concentrations of EG, PG, and DEG in urine samples (mean ± standard deviation) obtained from 23 subjects at random were 604 ± 360, 5,450 ± 9,290, and 59.0 ± 49.3 ng/ml, respectively. These values were 9.44, 30.1, and 5.31 times higher than those in whole blood samples, respectively. The much higher concentrations of the three glycols found in urine samples of nonoccupational healthy humans suggest that the three glycols, once incorporated into the human body, are rapidly excreted into urine. To our knowledge, this is the first demonstration of the presence of relatively high concentrations of EG, PG, and DEG in urine of healthy human subjects.

Simultaneous analysis of urinary phthalate metabolites of residents in Korea using isotope dilution gas chromatography–mass spectrometry

Phthalates are used in industry products, household items, and medical tools as plasticizers. Human exposure to phthalates has raised concern about its toxicity. In the present study, optimization was conducted for the simultaneous analysis of eight kinds of phthalate metabolites using gas chromatography–mass spectrometry (GC-MS): MEP, MiBP, MnBP, MBzP, MiNP, MEHP, MEOHP, and MEHHP. In order to minimize the matrix effect and to do quantitative analysis, isotope dilution and LLE-GC-MS methods were performed. Urine samples were enzymatically hydrolyzed, extracted with a mixture of n-hexane and ethyl ether (8:2; v:v), and subsequently derivatized with trimethylsilylation. All eight kinds of analytes showed clear resolution and high reproducibility in GC-MS results. The method detection limit ranged from 0.05ng/mL to 0.2ng/mL. Calibration curves were found to be linear from 0.2 to 100ng/mL with –2>0.992. The relative standard deviation of the intraday precision using water and urine ranged from 2.1% to 16.3%. The analysis was performed with urine samples that were collected from adults residing in the Republic of Korea. The analyzed concentration results were compared according to gender and region. As a result, DEHP metabolites showed the highest detected concentration (75.92μg/g creatinine, 100%), and MiNP, a metabolite of DiNP, showed the lowest detected concentration (0.42μg/g creatinine, 22.5%). On average, female urine (200.76μg/g creatinine) had a higher detected concentration of ∑8 phthalate metabolites than male urine. Samples from rural regions (211.96μg/g creatinine) had higher levels than samples from urban regions.