Simultaneous analysis of carcinogenic N-nitrosamine impurities in metformin tablets using on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry.

Contamination of active pharmaceutical ingredients (APIs) and pharmaceutical preparations with carcinogenic N-nitrosamines has led to recalls of these products and supply shortages to patients. The present study describes the development of a highly sensitive method for simultaneous analysis of seven N-nitrosamines using on-line in-tube solid-phase microextraction (IT-SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine their actual contamination in metformin tablets. Using a Carboxen 1006 PLOT capillary as the extraction device for IT-SPME, these compounds were efficiently extracted and concentrated 6‒24-fold by subjecting 40 µL of sample to 25 repeated draw/eject cycles at a rate of 0.2 mL/min. The seven N-nitrosamines were separated within 11 min by gradient elution with 0.1 % formic acid solution and acetonitrile as the mobile phase using a CAPCELL PAK C18 MGII column and detected by multiple reaction monitoring in positive ion mode. The calibration curve showed linearity in the range 0.2‒50 ng/mL and detection limits (S/N = 3) in the range 3‒112 pg/mL. The intra-day and inter-day precisions were less than 5.5 % and 7.0 % (n = 6), respectively, with accuracies ranging from 93‒117 %. Following ultrasonic extraction with water, centrifugation and filtration of the supernatant liquid through a membrane filter, the N-nitrosamine impurities in metformin tablets could be analyzed by IT-SPME/LC‒MS/MS. Their limits of quantification (S/N = 10) were 0.1‒5.1 pg/mg API and recoveries ranged from 87‒102 %. Analysis of eight metformin tablets from eight manufacturers showed that 5.8‒7.5 pg/mg N-nitrosodimethylamine were present in three tablets, with no other N-nitrosamines detected in any of the eight tablets. This method may be useful in testing for N-nitrosamine impurities in pharmaceutical preparations.

Solid-Phase Microextraction and Related Techniques in Bioanalysis.

Living organisms, such as microorganisms, plants and animals, are composed of complex constituents, which may include bioactive components that maintain their functions [...].

Online In-Tube Solid-Phase Microextraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry for Automated Analysis of Four Sulfated Steroid Metabolites in Saliva Samples.

Accurate measurement of sulfated steroid metabolite concentrations can not only enable the elucidation of the mechanisms regulating steroid metabolism, but also lead to the diagnosis of various related diseases. The present study describes a simple and sensitive method for the simultaneous determination of four sulfated steroid metabolites in saliva, pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), cortisol sulfate (CRTS), and 17ß-estradiol-3-sulfate (E2S), by online coupling of in-tube solid-phase microextraction (IT-SPME) and stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). These compounds were extracted and concentrated on Supel-Q PLOT capillary tubes by IT-SPME and separated and detected within 6 min by LC-MS/MS using an InertSustain swift C18 column and negative ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. Calibration curves using their stable isotope-labeled internal standards showed good linearity in the range of 0.01-2 ng mL-1 for PREGS, DHEAS, and CRTS and of 0.05-10 ng mL-1 for E2S. The limits of detection (S/N = 3) of PREGS, DHEAS, CRTS, and E2S were 0.59, 0.30, 0.80, and 3.20 pg mL-1, respectively. Moreover, intraday and interday variations were lower than 11.1% (n = 5). The recoveries of these compounds from saliva samples were in the range of 86.6-112.9%. The developed method is highly sensitive and specific and can easily measure sulfated steroid metabolite concentrations in 50 µL saliva samples.

Risk Assessment of Passive Smoking Based on Analysis of Hair Nicotine and Cotinine as Exposure Biomarkers by In-Tube Solid-Phase Microextraction Coupled On-Line to LC-MS/MS.

Passive smoking due to environmental tobacco smoke is a serious public health concern because it increases the risk of lung cancer and cardiovascular disease. However, the current status and effect of passive smoking in various lifestyles are not fully understood. In this study, we measured hair nicotine and cotinine levels as exposure biomarkers in non-smokers and assessed the risk from the actual situation of passive smoking in different lifestyle environments. Nicotine and cotinine contents in hair samples of 110 non-smoker subjects were measured by in-tube solid-phase microextraction with on-line coupling to liquid chromatography-tandem mass spectrometry, and self-reported lifestyle questionnaires were completed by the subjects. Nicotine and cotinine were detected at concentrations of 1.38 ng mg-1 and 12.8 pg mg-1 respectively in the hair of non-smokers, with levels significantly higher in subjects who reported being sensitive to tobacco smoke exposure. These levels were also affected by type of food intake and cooking method. Nicotine and cotinine in hair are useful biomarkers for assessing the effects of passive smoking on long-term exposure to environmental tobacco smoke, and our analytical methods can measure these exposure levels in people who are unaware of passive smoking. The results of this study suggest that the environment and places of tobacco smoke exposure and the lifestyle behaviors therein are important for the health effects of passive smoking.

Headspace Solid-Phase Microextraction/Gas Chromatography-Mass Spectrometry for the Determination of 2-Nonenal and Its Application to Body Odor Analysis.

The odors and emanations released from the human body can provide important information about the health status of individuals and the presence or absence of diseases. Since these components often emanate from the body surface in very small quantities, a simple sampling and sensitive analytical method is required. In this study, we developed a non-invasive analytical method for the measurement of the body odor component 2-nonenal by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry by selective ion monitoring. Using a StableFlex PDMS/DVB fiber, 2-nonenal was efficiently extracted and enriched by fiber exposition at 50 °C for 45 min and was separated within 10 min using a DB-1 capillary column. Body odor sample was easily collected by gauze wiping. The limit of detection of 2-nonenal collected in gauze was 22 pg (S/N = 3), and the linearity was obtained in the range of 1-50 ng with a correlation coefficient of 0.991. The method successfully analyzed 2-nonenal in skin emissions and secretions and was applied to the analysis of body odor changes in various lifestyles, including the use of cosmetics, food intake, cigarette smoking, and stress load.

Online In-Tube Solid-Phase Microextraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry for the Determination of Tobacco-Specific Nitrosamines in Hair Samples.

Active and passive smoking are serious public health concerns Assessment of tobacco smoke exposure using effective biomarkers is needed. In this study, we developed a simultaneous determination method of five tobacco-specific nitrosamines (TSNAs) in hair by online in-tube solid-phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS). TSNAs were extracted and concentrated on Supel-Q PLOT capillary by in-tube SPME and separated and detected within 5 min by LC-MS/MS using Capcell Pak C18 MGIII column and positive ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. The calibration curves of TSNAs showed good linearity in the range of 0.5-1000 pg mL-1 using their stable isotope-labeled internal standards. Moreover, the limits of detection (S/N = 3) of TSNAs were in the range of 0.02-1.14 pg mL-1, and intra-day and inter-day precisions were below 7.3% and 9.2% (n = 5), respectively. The developed method is highly sensitive and specific and can easily measure TSNA levels using 5 mg hair samples. This method was used to assess long-term exposure levels to tobacco smoke in smokers and non-smokers.

Proposal and validation of polyconvex strain-energy function for biological soft tissues.

BACKGROUND: Mechanical simulations for biological tissues are effective technology for development of medical equipment, because it can be used to evaluate mechanical influences on the tissues. For such simulations, mechanical properties of biological tissues are required. For most biological soft tissues, stress tends to increase monotonically as strain increases. OBJECTIVE: Proposal of a strain-energy function that can guarantee monotonically increasing trend of biological soft tissue stress-strain relationships and applicability confirmation of the proposed function for biological soft tissues. METHOD: Based on convexity of invariants, a polyconvex strain-energy function that can reproduce monotonically increasing trend was derived. In addition, to confirm its applicability, curve-fitting of the function to stress-strain relationships of several biological soft tissues was performed. RESULTS: A function depending on the first invariant alone was derived. The derived function does not provide such inappropriate negative stress in the tensile region provided by several conventional strain-energy functions. CONCLUSIONS: The derived function can reproduce the monotonically increasing trend and is proposed as an appropriate function for biological soft tissues. In addition, as is well-known for functions depending the first invariant alone, uniaxial-compression and equibiaxial-tension of several biological soft tissues can be approximated by curve-fitting to uniaxial-tension alone using the proposed function.

In-tube solid-phase microextraction: Current trends and future perspectives.

In-tube solid-phase microextraction (IT-SPME) was developed about 24 years ago as an effective sample preparation technique using an open tubular capillary column as an extraction device. IT-SPME is useful for micro-concentration, automated sample cleanup, and rapid online analysis, and can be used to determine the analytes in complex matrices simple sample processing methods such as direct sample injection or filtration. IT-SPME is usually performed in combination with high-performance liquid chromatography using an online column switching technology, in which the entire process from sample preparation to separation to data analysis is automated using the autosampler. Furthermore, IT-SPME minimizes the use of harmful organic solvents and is simple and labor-saving, making it a sustainable and environmentally friendly green analytical technique. Various operating systems and new sorbent materials have been developed to improve its extraction efficiency by, for example, enhancing its sorption capacity and selectivity. In addition, IT-SPME methods have been widely applied in environmental analysis, food analysis and bioanalysis. This review describes the present state of IT-SPME technology and summarizes its current trends and future perspectives, including method development and strategies to improve extraction efficiency.

A sensitive method for the determination of tobacco-specific nitrosamines in mainstream and sidestream smokes of combustion cigarettes and heated tobacco products by online in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry.

A simple and sensitive method, using automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), was developed for the determination of four tobacco-specific nitrosamines (TSNAs) in mainstream and sidestream smoke of combustion cigarettes and heated tobacco products. These TSNAs were separated within 4 min on a Capcell Pak C18 MGⅢ column and detected in the positive ion mode by multiple reaction monitoring. The optimum in-tube SPME conditions were 30 draw/eject cycles of 40 µL of sample at a flow rate of 200 µL min-1 using a Supel-Q PLOT capillary column as an extraction device. The extracted TSNAs were easily desorbed from the column by passage of the mobile phase, with no carryover observed. This in-tube SPME LC-MS/MS method showed good linearity for four TSNAs in the 0.5-100 pg mL-1 ranges, with correlation coefficients above 0.9998 (n = 24), using stable isotope-labeled TSNAs as internal standards. The validation assays for limit of detection, limit of quantification, specificity, precision and accuracy of the analytes were consistent with the requirements recommended by the ICH guidelines. The validated method was utilized successfully to analyze four TSNAs in mainstream and sidestream smoke samples without any interference peaks. Overall recoveries of TSNAs spiked into smoke sample solutions were 97.3-104.6%. The developed method can automate the extraction, concentration and analysis of samples, and is sensitive and selective for TSNAs. This method was used to analyze TSNAs in mainstream and sidestream smoke samples of several commercially available combustion cigarettes and heated tobacco products.

Analysis of nicotine and cotinine in hair by on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry as biomarkers of exposure to tobacco smoke.

Smoking not only increases the risk of lung cancer but is strongly related to the onset of cardiovascular disease. Particularly, passive smoking due to sidestream smoke is a critical public health problem. To assess active and passive exposure to tobacco smoke, we developed a simple and sensitive method, consisting of on-line in-tube solid phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), to determine nicotine and its metabolite cotinine in hair samples. These compounds were separated within 5 min using a Polar-RP80A column and detected in the positive ion mode by multiple reaction monitoring. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 µL of sample at a flow rate of 200 µL/min using a Carboxen 1006 capillary column as an extraction device. The extracted compounds in the stationary phase on the inner wall of the capillary could be dissolved easily into the mobile phase and transferred to an LC column. Using the in-tube SPME LC-MS/MS method, the calibration curves were linear in the 5-1000 pg/mL ranges for nicotine and cotinine, and the detection limits (signal to noise ratio of 3) were 0.45 and 0.13 pg/mL, respectively. The intra-day and inter-day precisions were below 3.4% and 6.0% (n = 5), respectively. This method was utilized successfully to analyze pg/mg levels of nicotine and cotinine in 1 mg of hairs without interference peaks, and good recoveries were obtained. The concentration of cotinine in hair was two orders of magnitude lower than that of nicotine, but a good positive correlation was found between the concentrations of these compounds. This method can automate the extraction, concentration and analysis of samples, and is useful for the assessment of long-term exposure to tobacco smoke.

Simultaneous analysis of multiple urinary biomarkers for the evaluation of oxidative stress by automated online in-tube solid-phase microextraction coupled with negative/positive ion-switching mode liquid chromatography-tandem mass spectrometry.

This study described an automated online method for the simultaneous determination of 8-isoprostane, 8-hydroxy-2'-deoxyguanosine, and 3-nitro-l-tyrosine in human urine. The method involves in-tube solid-phase microextraction using a Carboxen 1006 PLOT capillary column as an extraction device, followed by liquid chromatography with tandem mass spectrometry using a CX column and detection in the negative/positive switching ion-mode by multiple reaction monitoring. Using their stable isotope-labeled internal standards, each of these oxidative stress biomarkers showed good linearity from 0.02 to 2.0 ng/mL. Their detection limits (S/N = 3) were 3.4-21.5 pg/mL, and their intra- and inter-day precisions (relative standard deviations) were >3.9 and 6.5% (n = 5), respectively. This method was applied successfully to the analysis of urine samples, without any other pretreatment and interference peaks.

Spatial correlativity of atmospheric particulate components simultaneously collected in Japan.

The simultaneous sampling of total suspended particles was performed at 14 sites in Japan during July 2008-June 2009. The spatial correlativity of each particulate composition toward Osaka was obtained for nine selected sites to overview the chemical composition and geographical distribution of particulate components across a wide range of areas nationwide. The spatial correlatives of atmospheric particulate components were extended to an even wider range of areas up to 950 km distance (meso-alpha scale region, >200 km) for a far-reaching distance analysis unique in the literature. Overall, the spatial correlations of ionic species and both organic and elemental carbons were significant, suggesting their shared advections, including their long-range transport from East Asia. Although sulfate ions are widely dispersed across Japan, such is not necessarily correlated with organic and elemental carbon, possibly indicating that the sulfate emission source, including long-range transport, differs from that of carbonaceous particulates. By contrast, the characteristics of spatial correlatives of metallic constituents vary; for example, particulate Pb and Cd show a significantly wide range of spatial correlatives to Osaka, while Mn-though limited to cities neighboring Osaka-shows significant spatial correlations. Other metallic constituents showed no significant spatial correlatives, indicating the effects of local pollutants. Moreover, the extent of the spatial dispersion of the particulate components and the relationships among chemical components were analyzed via factor analysis to highlight the effects of long-range inflow and local original emissions. In this treatment, 13 particulate components among the 19 measured were implicated in long-range transport.

Determination of the oxidative stress biomarker urinary 8-hydroxy-2'-deoxyguanosine by automated on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry.

A simple and sensitive method for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage in human urine, was developed using automated on-line in-tube solid-phase microextraction (SPME) coupled with stable isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Creatinine was also analyzed simultaneously to normalize urine volume by the in-tube SPME LC-MS/MS method, and 8-OHdG and creatinine were separated within 3 min using a Zorbax Eclipse XDB-C8 column. Electrospray MS/MS for these compounds was performed on an API 4000 triple quadruple mass spectrometer in the positive ion mode by multiple reaction monitoring. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 µL of sample at a flow rate of 200 µL/min using a Carboxen 1006 PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. The calibration curve for 8-OHdG using its stable isotope-labeled internal standard was linear in the range of 0.05-10 ng/mL, and the detection limit was 8.3 pg/mL. The intra-day and inter-day precision (relative standard deviations) were below 3.1% and 9.6% (n=5), respectively. This method was applied successfully to the analysis of urine samples without any other pretreatment and interference peaks, with good recovery rates above 91% in spiked urine samples. The limits of quantification of 8-OHdG and creatinine in 0.1 mL urine samples were about 0.32 and 0.69 ng/mL (S/N=10), respectively. This method was utilized to assess the effects of smoking, green tea drinking and alcohol drinking on the urinary excretion of 8-OHdG.

SPME techniques for biomedical analysis.

Biomedical analyses of drugs and their metabolites are important in new drug development, therapeutic drug monitoring and forensic toxicology. In these analyses, sample preparation is very important to isolate target compounds from complex biological matrices and markedly influences the reliability and accuracy of determination. SPME is a simple and convenient sample preparation technique that has enabled automation, miniaturization and high-throughput performance. This article focuses on current developments, their biomedical applications and future trends with emphasis on new extraction devices using selective polymer coating materials in novel SPME techniques, including fiber SPME, in-tube SPME and related techniques.

Biomonitoring method for the determination of polycyclic aromatic hydrocarbons in hair by online in-tube solid-phase microextraction coupled with high performance liquid chromatography and fluorescence detection.

Polycyclic aromatic hydrocarbons (PAHs) are formed from the incomplete combustion or pyrolysis of organic matter during industrial processing and various human activities, but human exposure to PAHs has not yet been elucidated in detail. To assess long-term exposure to PAHs, we developed a simple and sensitive method for measuring PAHs in hair by online in-tube solid-phase microextraction using a CP-Sil 19CB capillary column as an extraction device, followed by high-performance liquid chromatography using a Zorbax Eclipse PAH column and fluorescence detection. Seventeen PAHs could be analyzed simultaneously, with good linearity from 20 to 1000pg/mL each as determined using stable isotope-labeled PAH internal standards. The detection limits of PAHs were 0.5-20.4pg/mL. PAHs in human hair samples were extracted by ultrasonication in 50mM NaOH in methanol, and successfully analyzed without any interference peaks, with good recovery rates above 70% in spiked hair samples. Using this method, we evaluated the suitability of using hair PAHs as biomarkers for long-term exposure.

Analysis of urinary 8-isoprostane as an oxidative stress biomarker by stable isotope dilution using automated online in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry.

We have developed a simple and sensitive method for the determination of the oxidative stress biomarker 8-isoprostane (8-IP) in human urine by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Zorbax Eclipse XDB-8 column and 0.1% formic acid/methanol (25/75, v/v) as a mobile phase. Electrospray MS/MS for 8-IP was performed on an API 4000 triple quadruple mass spectrometer in negative ion mode. The optimum in-tube SPME conditions were 20 draw/eject cycles with a sample size of 40 µL using a Carboxen 1006 PLOT capillary column for the extraction. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Total analysis time of this method including online extraction and analysis was about 30 min for each sample. The in-tube SPME LC-MS/MS method showed good linearity in the concentration range of 20-1000 pg/mL with a correlation coefficient r = 0.9999 for 8-IP using a stable isotope-labeled internal standard, 8-IP-d4. The detection limit of 8-IP was 3.3 pg/mL and the proposed method showed 42-fold higher sensitivity than the direct injection method. The intra-day and inter-day precisions (relative standard deviations) were below 5.0% and 8.5% (n = 5), respectively. This method was applied successfully to the analysis of urine samples without pretreatment or interference peaks. The recovery rates of 8-IP spiked into urine samples were above 92%. This method is useful for assessing the effects of oxidative stress and antioxidant intake.

Development of exposure assessment method based on the analysis of urinary heterocyclic amines as biomarkers by on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry.

Heterocyclic amines (HCAs) formed in cooked meats and fish are mutagens and carcinogens in rodents and nonprimates. Exposure to HCAs may also be a risk factor for human tumors, but the association between dietary intake and human cancer risk has not been determined. To assess recent exposure to HCAs, we developed a simple and sensitive method for measuring HCAs in urine by automated on-line in-tube solid-phase microextraction (SPME) using a Supel-Q PLOT capillary column as an extraction device, in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Thirteen HCAs were separated within 15 min using a ZORBAX Eclipse XDB-C8 column and detected selectively by multiple reaction monitoring using MS/MS. This method can be applied easily to the analysis of small amounts of urine samples without any other pretreatment except for alkaline hydrolysis of bound forms of HCAs. The quantification limits of HCAs in 0.2 mL of urine samples were about 1.7-4.1 pg/mL (S/N = 10). Using this method, we evaluated the exposure to HCAs in persons who consumed well-done pan-fried beef and the suitability of using urinary HCAs as exposure biomarkers. We also analyzed the ability of vegetable consumption to prevent carcinogenic risks from exposure to HCAs by measuring free and bound forms of HCAs in urine.

Noninvasive analysis of volatile biomarkers in human emanations for health and early disease diagnosis.

Early disease diagnosis is crucial for human healthcare and successful therapy. Since any changes in homeostatic balance can alter human emanations, the components of breath exhalations and skin emissions may be diagnostic biomarkers for various diseases and metabolic disorders. Since hundreds of endogenous and exogenous volatile organic compounds (VOCs) are released from the human body, analysis of these VOCs may be a noninvasive, painless, and easy diagnostic tool. Sampling and preconcentration by sorbent tubes/traps and solid-phase microextraction, in combination with GC or GC-MS, are usually used to analyze VOCs. In addition, GC-MS-olfactometry is useful for simultaneous analysis of odorants and odor quality. Direct MS techniques are also useful for the online real-time detection of VOCs. This review focuses on recent developments in sampling and analysis of volatile biomarkers in human odors and/or emanations, and discusses future use of VOC analysis.

Analysis of heterocyclic amines in hair by on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry.

Mutagenic and carcinogenic heterocyclic amines (HCAs) are formed during heating of various proteinaceous foods, but human exposure to HCAs has not yet been elucidated in detail. To assess long-term exposure to HCAs, we developed a simple and sensitive method for measuring HCAs in hair by automated on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using a Zorbax Eclipse XDB-C8 column, 16 HCAs were analyzed within 15 min. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 µL sample at a flow rate of 200 µL min(-1) using a Supel-Q PLOT capillary column as an extraction device. The extracted HCAs were easily desorbed from the column by passage of the mobile phase, with no carryover observed. This in-tube SPME LC-MS/MS method showed good linearity for HCAs in the range of 10-2000 pg mL(-1), with correlation coefficients above 0.9989 (n=18), using stable isotope-labeled HCA internal standards. The detection limits (S/N=3) of 14 HCAs except for MeAαC and Glu-P-1 were 0.10-0.79 pg mL(-1). This method was successfully utilized to analyze 14 HCAs in hair samples without any interference peaks, with quantitative limits (S/N=10) of about 0.17-1.32 pg mg(-1) hair. Using this method, we evaluated the exposure to HCAs in cigarette smoke and the suitability of using hair HCAs as exposure biomarkers.

Simultaneous determination of testosterone, cortisol, and dehydroepiandrosterone in saliva by stable isotope dilution on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry.

We have developed a simple and sensitive method for the simultaneous determination of testosterone (TES), cortisol (CRT), and dehydroepiandrosterone (DHEA) in saliva by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Discovery HS F5 column. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 µL of sample at a flow rate of 200 µL/min using a Supel-Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. The in-tube SPME LC-MS/MS method showed good linearity with correlation coefficients r ≥ 0.9998 for TES, CRT, and DHEA using their respective stable isotope-labeled internal standards. The intra-day and inter-day precisions (relative standard deviations) were below 4.9 and 8.5 % (n = 5), respectively. This method was successfully utilized to analyze TES, CRT, and DHEA in saliva samples without any other pretreatment or interference peaks, and the quantification limits (S/N = 10) of TES, CRT and DHEA were about 0.01, 0.03 and 0.29 ng/mL saliva, respectively. The recoveries of these compounds spiked into saliva samples were each above 94%. This method was applied to analyze changes in salivary TES, CRT, and DHEA levels resulting from stress and fatigue load.