Simultaneous quantification of 2-aminothiazoline-4-carboxylic acid and 2-aminothiazoline-4-oxoaminoethanoic acid utilizing chemical derivatization followed by liquid chromatography-tandem mass spectrometry.

Detecting cyanide compounds in postmortem blood samples is an important matter in forensic science because cyanide is often used as a poison for murder or suicide. However, the direct analysis of cyanide itself has practical limitations because of cyanide's volatility and short half-life at ambient temperature. Here, we focused on the relatively stable cyanide metabolites 2-aminothiazoline-4-carboxylic acid (ATCA) and 2-aminothiazoline-4-oxoaminoethanoic acid (ATOEA) as potential markers of cyanide exposure. We developed an analytical method that uses chemical derivatization of the target compounds with 4-bromoethyl-7-methoxycoumarin followed by liquid chromatography coupled with electrospray ionization-tandem mass spectrometry. The recovery rates for pretreatment and calibration curve linearities were good in the concentration range of 20-1000 ng/mL. Using our approach, we were able to detect and quantify both ATCA and ATOEA concentrations in postmortem blood samples, and in our samples the ratio of ATCA and ATOEA was in the range of 4.5-19.1. To our knowledge, this is the first time ATOEA has been successfully detected in human blood samples. In addition, we found that ATCA and ATOEA concentrations were both significantly higher in the blood of fire victims than in the blood of individuals with a non-fire-related cause of death. Also, we found that there was a significant positive correlation between ATCA concentrations and ATOEA concentrations. Together, our present data suggested that ATCA and ATOEA are both potential markers of cyanide exposure.

Simultaneous determination of fenthion and its metabolites in a case of fenthion self-poisoning.

Fenthion (MPP) is a popular organophosphorus pesticide that acts via inhibition of the enzyme cholinesterase. It is well known that fenthion is metabolized by plants, animals and soil microorganisms to sulfone and sulfoxide by oxidation of thioether and is further metabolized by conversion of P = S to P = O (oxon). Although human fenthion poisonings sometimes occur, details of the distribution of fenthion and its metabolites within the bodies of victims are unclear. In this study, we developed and validated an approach that uses liquid chromatography coupled with electrospray ionization-tandem mass spectrometry to quantify the concentrations of fenthion and its five metabolites (MPP-sulfoxide, MPP-sulfone, MPP-oxon, MPP-oxon sulfoxide and MPP-oxon sulfone) in the fluids [blood, cerebral spinal fluid (CSF) and urine] of a human cadaver. The calibration curves were linear in the concentration range 5-200 ng/mL. Our method allowed for repeatable and accurate quantification with intra- and inter-assay coefficients of variation smaller than 8.6% and 11.0%, respectively, for each target compound. We used the developed method to measure the fenthion concentration in the blood of a dead victim of fenthion poisoning and found the concentration to be in the comatose-fatal range. In addition, we detected for the first time fenthion and all five fenthion metabolites in the cadaveric blood and CSF. The concentrations of the oxidized forms of fenthion, including MPP-sulfone and MPP-sulfoxide, were higher in CSF than in the blood.

Quantification of nine psychotropic drugs in postmortem dried blood spot samples by liquid chromatography-tandem mass spectrometry for simple toxicological analysis.

Dried blood spot (DBS) sampling has evolved to become the method of choice for collecting samples for newborn screening and therapeutic drug monitoring worldwide. The major advantage of this approach is that it requires only a small amount of blood. In addition, the collection of DBSs on filter paper is simple, sample storage costs are small, and the process deactivates microorganisms and viruses. However, despite these advantages, DBS sampling is seldom used in forensic toxicological analyses. Here, we developed and validated an approach that uses liquid chromatography coupled with electrospray ionization-tandem mass spectrometry for quantifying nine psychotropic drugs (citalopram, duloxetine, mirtazapine, olanzapine, paroxetine, quetiapine, sertraline, zolpidem and zopiclone) in cadaveric DBS samples. Most of them are frequently used by self-harm but are not already targeted by an existing drug screening kit. Our method use only one 3-mm disk excised from each DBS and does not require the troublesome purification process. The linearities of the calibration curves were good in the concentration range of 0.05-1.0 µg/mL. Our method allows for repeatable and accurate quantification with intra- and inter-assay coefficients of variation of below 11.9% and below 12.5%, respectively, for each of the target drugs. In addition, the target drug concentrations in the DBSs remained stable for at least one month when stored at - 80 °C. Compared with our institute's routine method for cadaveric blood sampling, the QuEChERS method, quantifiable concentrations showed a good positive correlation for each of the target drugs. In addition, the concentrations of almost all the target drugs obtained with DBS sampling method were comparable with those obtained with the QuEChERS sampling method. Thus, the present findings extend the possible uses of DBS sampling to the quantification of multiple psychotropic drugs in the field of forensic toxicological testing.

Relationships between cause of death and concentrations of seven steroids obtained from the serum and cerebrospinal fluid of cadavers.

In this study, we assessed 80 autopsy samples to investigate the relationships between cause of death and the concentrations of multiple steroids in serum and cerebrospinal fluid (CSF). First, we developed and validated analytical methods to quantify seven steroids (cortisol, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycortiocosterone, progesterone, and testosterone) by using liquid chromatography coupled with electrospray ionization-tandem mass spectrometry. Next, we statistically evaluated the levels of each steroid for six causes of death: hypothermia, traumatic injury, fire fatality, asphyxia, intoxication, and internal disease. We observed that cortisol concentrations in serum and CSF obtained from cadavers who died from hypothermia were significantly higher than those in samples obtained from cadavers who died from the remaining causes of death (P < 0.05). Similarly, corticosterone concentrations obtained from cadavers who died from hypothermia were significantly higher than those in samples from several other causes of death. However, concentrations of the remaining steroids analyzed did not differ significantly among the causes of death. We further elucidated the correlations between steroid concentrations in serum and CSF. Except for 11-deoxycorticosterone and progesterone, steroid concentrations were significantly positively correlated in serum and CSF. Although data on cadaveric steroid concentrations are limited-especially in CSF-values obtained were in the approximate range of the living human data reported to date.

Quantification of cyanide metabolite 2-aminothiazoline-4-carboxylic acid in postmortem dried blood spot samples by liquid chromatography-tandem mass spectrometry.

2-Aminothiazoline-4-carboxylic acid (ATCA), which is produced by the reaction of cyanide with endogenous cystine, is a promising biomarker of cyanide exposure because of its physicochemical stability. Analysis of more stable metabolite than the toxic gas itself is sometimes useful for postmortem diagnosis of gas poisoning. Here, we developed and validated an approach that uses liquid chromatography coupled with electrospray ionization-tandem mass spectrometry for quantifying ATCA in dried blood spot (DBS) samples. The linearity of the calibration curve was good in the concentration range of 20-1500 ng/mL. Our method allows for repeatable and the accurate quantification of ATCA, with intra- and inter assay coefficients of variation of below 7.8 % and below 9.3 %, respectively. In addition, the concentration of ATCA in DBSs remained stable for at least one month when stored at -20°C. Our results indicated that our analytical approach can be used to determine past exposure to higher doses of cyanide. In a comparison of ATCA concentrations in DBSs obtained from cadavers with various causes of death, significantly higher ATCA concentrations were observed in fire victims than in non-fire victims, confirming that fire victims inhale large amounts of cyanide gas. Thus, here we extended the possible uses of DBS for quantification of ATCA to forensic toxicological testing for cyanide poisoning.

Assessment of blood 2-aminothiazoline-4-carboxylic acid concentrations: Age and sex differences, and correlation with carboxyhemoglobin in fire victims.

Recently, 2-aminothiazoline-4-carboxylic acid (ATCA), a cyanide (CN) metabolite, has been proposed as a stable diagnostic marker of CN poisoning. In this study, liquid chromatography coupled with electrospray ionization - tandem mass spectrometry was used to quantify ATCA concentrations in human postmortem blood samples, and differences in ATCA concentrations according to age and sex were determined. Both age and sex had significant effects on blood ATCA concentrations. Although ATCA concentrations exhibited an inverted U shape with increasing age in men, in women ATCA concentrations plateaued at around 40-59 years of age. There were significant differences between the sexes in ATCA concentrations for the 20-39 and 40-59 year age groups (P < 0.05 and P < 0.01, respectively). Correlations between ATCA concentrations and carboxyhemoglobin (CO-Hb) saturation were also examined in fire victims. ATCA concentrations increased significantly with increasing CO-Hb saturation (r = 0.382, P < 0.01). In addition, ATCA concentrations were also correlated to CN concentrations (r = 0.309, P < 0.05). The results of our study may provide novel information about the contribution of CN poisoning to the cause of death at fire scenes.

ACE2 immunohistochemistry in salivary and tracheal glands related to age.

OBJECTIVE: SARS-CoV-2 is the cause of COVID-19, the rapidly spreading pandemic. When SARS-CoV-2 enters the target cells in the respiratory system, the spike glycoprotein binds to a cellular receptor angiotensin converting enzyme 2 (ACE2). The susceptibility to infection in individuals under 20 years of age is approximately half that of adults aged over 20 years. In this study, we investigated the immunohistochemical protein expressions of ACE2 in mandibular salivary glands and tracheal glands from forensic autopsy specimens covering adults and children. RESULTS: The ACE2 immunohistochemistry of autopsy specimens was performed, and the percentages of the immuno-positive areas in the cell layers of the glands were calculated. Our results demonstrate that the ACE2 positivity in mandibular salivary gland and tracheal glands showed the statistically significant decrease with the increase of age, which indicates that the susceptibility of aged individuals to SARS-CoV-2 may be due to various factors including but not limited to ACE2 protein expressions.

Simultaneous quantification by LC/ESI-MS/MS of chlorinated tyrosine derivatives in the autopsy sample of a victim of chlorine exposure.

Direct detection and accurate quantification of chlorine in autopsy samples are difficult because of the volatility and rapid metabolism of chlorine. Here, we developed and validated a method for quantitative analysis of 3-chloro-l-tyrosine (Cl-Tyr) and 3,5-dichloro-l-tyrosine (DiCl-Tyr) as stable markers of chlorine exposure. Chemical derivatization followed by liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) enabled us to simultaneously analyze both Cl-Tyr and DiCl-Tyr in an autopsy sample from the victim of chlorine exposure. Cl-Tyr was detected in the heart blood (53.6 ng/mL), urine (9.5 ng/mL), and lung tissue (211.1 ng/g); however, DiCl-Tyr was detected only in the lung tissue (10.3 ng/g). In contrast, in autopsy samples obtained from cases without exposure to chlorine, DiCl-Tyr was not detected in any matrixes. Our result suggested that the simultaneous detection of Cl-Tyr and DiCl-Ty may provide a better appreciation of chlorine exposure. To our knowledge, this is the first time Cl-Tyr and DiCl-Tyr have been determined simultaneously in a real human autopsy sample from a victim of chlorine exposure.

Quantification of 2-aminothiazoline-4-carboxylic acid as a reliable marker of cyanide exposure using chemical derivatization followed by liquid chromatography-tandem mass spectrometry.

In this research, we have developed a novel and simple liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for quantification of 2-aminothiazoline-4-carboxylic acid (ATCA), which is produced by the direct reaction of cyanide (CN) with endogenous cystine. In forensic science, detection of CN is important because CN is a poison that is often used for murder or suicide, in addition to being produced by the thermal decomposition of natural or synthetic materials. However, because CN disappears rapidly from body tissue, ATCA is thought to be a more reliable indicator of CN exposure. For the method reported herein, human blood samples (20 µL) were subjected to protein precipitation followed by derivatization with 4-bromoethyl-7-methoxycoumarin. Blood spiked with ATCA at concentrations ranging from 50 to 1500 ng/mL was used to prepare a calibration curve (lower limit of quantification; 50 ng/mL, lower limit of detection; 25 ng/mL). Our method uses chemical derivatization, so unlike previously reported methods, it does not require tedious pretreatment procedures, hydrophilic interaction liquid chromatography columns, or specialized equipment. In addition, our method allows for repeatable and accurate quantification of ATCA, with intra- and inter-assay coefficients of variation of below 5.0% and below 6.0%, respectively. We used the method to analyze ATCA in postmortem human blood samples, including samples from people who had intentionally ingested CN or were fire victims. Blood ATCA concentrations were higher among people who had ingested CN or were fire victims than among people in a control group (P < 0.0001). The data reported herein demonstrate that our LC/ESI-MS/MS method can be used to detect and quantify ATCA in postmortem blood samples and that CN exposure strongly affects ATCA concentration, providing a useful tool for detection of CN poisoning.

A fatal poisoning case of acetone cyanohydrin and citalopram.

Acetone cyanohydrin (ACH) is a readily available source of cyanide and is widely used in basic and applied sciences. In toxicology, ACH is classified as extremely hazardous as it readily decomposes on contact with water, with the potential rapid release of highly toxic hydrogen cyanide (HCN). We report the case of a young woman found dead from the intentional ingestion of ACH and citalopram, an antidepressant of the selective serotonin reuptake inhibitor class. The autopsy findings included bright reddish-purple hypostasis and mild pulmonary edema. As ACH can decompose to acetone and HCN, we quantified the concentration of each compound and thiocyanate separately in various body fluids and organs and determined their whole-body distributions by using gas chromatography-mass spectrometry (GC-MS). We observed high concentrations of both acetone and cyanide in the blood (0.63 mg/mL and 17.99 mM, respectively) and gastric contents (9.76 mg/mL and 472.44 mM). The whole-body distributions of acetone and cyanide were similar (i.e., the concentration of each compound was the highest in the lung, followed by the heart, and then the liver). Our results suggest that not only the route of administration but also the dose taken could greatly affect the body distributions of cyanide in humans. In addition, as toxicological screening detected citalopram, which was not prescribed to the deceased, we performed a chiral analysis by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We determined that only (S)-citalopram was ingested antemortem; its concentration was 0.36 µg/mL, which is in the toxic range.

Development of an LC-MS/MS method for quantification of 3-chloro-L-tyrosine as a candidate marker of chlorine poisoning.

A simple and sensitive liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for the determination of 3-chloro-L-tyrosine (Cl-Tyr) was developed and validated. For sample preparation, 50 µL of the body fluids or tissue extracts were processed by protein precipitation followed by the derivatization with dansyl chloride. The calibration curve was linear over the concentration range of 2.0-200 ng/mL blood or 4.0-400 ng/g tissue. Our method allowed the reproducible and accurate quantification. That is, the intra- and inter-assay coefficients of variation were below 7.73 and 6.94%, respectively in both the blood and lung. We applied the developed method to the analysis of Cl-Tyr in the human autopsy samples, which were suspected of chlorine poisoning, and detected 55.2 ng/mL and 206.6 ng/g Cl-Tyr in left heart blood and lung, respectively. Furthermore, in more than 20 autopsy samples, which were obtained from other causes of death including burn, drowning, hanging, internal disease, trauma and drug poisoning, Cl-Tyr was almost not detected in their both body fluids and organ tissues. In conclusion, the data here reported demonstrate that the LC/ESI-MS/MS method allows the Cl-Tyr in the autopsy samples and that chlorine exposure strongly affects its level, providing a basis for novel identification tool of chlorine poisoning.

Hyperketonemia as the diagnostic basis for hypothermia: An experimental study in diabetic and control mice.

Hypothermia is an important cause of death in forensic pathology. For the forensic diagnosis of hypothermia, some reports point out the possibility that hypothermia without diabetes may cause ketoacidosis. In this study, we evaluated the diagnostic value of ketoacidosis in a murine model of hypothermia, using the cold stress at 4 °C for 3 or 5 hrs in genetically diabetic (BKS.Cg-+Leprdb/+Leprdb/J) mice, compared with control (BKS.Cg- Dock7m+/Dock7m+/J) mice. The core temperature decrease was larger in diabetic mice than in control mice. We observed a novel finding that ketoacidosis assessed by elevated serum 3-hydroxybutyrate (3HB) occurs in hypothermia both in diabetic and control mice. Diabetic mice showed a prominent elevation of serum 3HB under cold stress. The protein expressions of monocarboxylate cotransporter 1 (MCT1), the channel protein used for the uptake of 3HB in skeletal muscles, showed a statistically significant decrease under cold stress for 3 hrs in control mice, indicating that the serum 3HB increase may be partially due to the decrease in the cellular uptake through the channel protein. Our results suggest the usefulness of hyperketonemia for the diagnosis of hypothermia not only in diabetic but also in non-diabetic cases.

Determination of 3-chloro-l-tyrosine as a novel indicator of chlorine poisoning utilizing gas chromatography-mass spectrometric analysis.

Chlorine gas exposure occurs in chemical warfare, industrial and household accidents. In forensic science, the generation of chlorine gas by mixing sodium hypochlorite detergent and strong acid detergent cannot be overlooked because of the possibility of suicide method (NaClO + 2HCl â†’ NaCl + H2O + Cl2). Though typical autopsy findings are obtained in chlorine exposure, such as pulmonary edema, useful biomarkers don't exist. In this research, we developed an analytical method of 3-chloro-l-tyrosine (Cl-Tyr) in blood as a novel marker of chlorine poisoning utilizing gas chromatography-mass spectrometry (GC-MS). Cl-Tyr was purified using protein precipitation and cation-exchange solid phase extraction, derivatized by the silylation agent and subjected to GC-MS. The quantification range was 10-200 ng/mL and good reproducibility was obtained. We applied the developed method to analyze Cl-Tyr in autopsy sample, which is suspected of chlorine poisoning, and detected 59.7 ng/mL Cl-Tyr in left heart blood. To our knowledge, this is the first report of determination of the chlorinated biomolecule in the human autopsy sample from chlorine poisoning.

Immunohistochemistry of advanced glycation end product Nε-(carboxymethyl)lysine in coronary arteries in relation to cardiac fibrosis and serum N-terminal-pro basic natriuretic peptide in forensic autopsy cases.

OBJECTIVE: Advanced glycation end products (AGEs) are known to play important roles in the development of diabetes mellitus and atherosclerosis.　Nε-(carboxymethyl)lysine (CML) is the major AGE, and is found in the arterial walls in the heart. The CML involvement in myocardial ischemia has been reported. We studied the immunohistochemical localization of CML in the hearts from forensic autopsies in relation to the age, serum N-terminal-pro basic natriuretic peptide (NT-proBNP), heart weights, and the degree of peri-myocardial fibrous tissues reflecting coronary microvascular infarction and myocardial remodeling. RESULTS: The CML immunoreactivity in the endothelial cells and intima of arterial walls in the interstitium of ventricular muscles was significantly stronger in the aged group, compatible with the progression of atherosclerosis. The blood level of NT-proBNP, a known useful marker for heart failure, had the positive correlation with the CML immunoreactivity. The degree of fibrosis, heart weights and the histories of hypertension and hyperlipidemia showed positive correlations with the CML immunoreactivity. Our results show the novel positive correlation between the CML immunohistochemistry in the heart vessels and heart conditions, and its future usefulness in the cardiovascular evaluation in histopathology.

Ror2 signaling regulates Golgi structure and transport through IFT20 for tumor invasiveness.

Signaling through the Ror2 receptor tyrosine kinase promotes invadopodia formation for tumor invasion. Here, we identify intraflagellar transport 20 (IFT20) as a new target of this signaling in tumors that lack primary cilia, and find that IFT20 mediates the ability of Ror2 signaling to induce the invasiveness of these tumors. We also find that IFT20 regulates the nucleation of Golgi-derived microtubules by affecting the GM130-AKAP450 complex, which promotes Golgi ribbon formation in achieving polarized secretion for cell migration and invasion. Furthermore, IFT20 promotes the efficiency of transport through the Golgi complex. These findings shed new insights into how Ror2 signaling promotes tumor invasiveness, and also advance the understanding of how Golgi structure and transport can be regulated.

Effect of polymer containing a naphthyl-alanine derivative on the separation selectivity for aromatic compounds in temperature-responsive chromatography.

A novel polymer-grafted stationary phase of high-performance liquid chromatography (HPLC) was developed, utilizing a temperature-responsive polymer containing an aromatic moiety. Firstly, we synthesized novel functional polymer poly(N-isopropylacrylamide-co-N-acryloyl-3-(2-naphthyl)-L-alanine methyl ester) [poly(NIPAAm-co-Nap)], which has temperature-responsiveness and selective retention of aromatic compounds by an intermolecular π-π interaction. The polymer exhibited a significant reversible phase transition from hydrophilic to hydrophobic in the vicinity of its lower critical solution temperature. Employing the developed polymer-grafted silica column, temperature-responsive chromatography was conducted using water as a sole mobile phase. A comparison with a conventional ODS column or a homogeneous PNIPAAm-grafted silica column showed that the retention of aromatic compounds was dramatically increased on the poly(NIPAAm-co-Nap)-grafted stationary phase. Introducing the naphthyl-alanine derivative caused a significant effect on the retention selectivity for aromatic compounds.

Reaction monitoring of tocopherols with active nitrogen oxides by ultra high-speed liquid chromatography.

Ultra high-speed liquid chromatography (LC) has become increasingly popular in analytical research fields. This analytical system provides fast and efficient chromatographic separation over a wide range of flow rate and pressure. In this study, we applied an ultra high-speed LC system to monitor the reaction of α-, γ-, and δ-tocopherols with active nitrogen species. By using an ultra high-speed LC system equipped with a photo-diode array detector, short time analysis and detection of a wide range of reaction products were accomplished efficiently. The analysis time of tocopherol and its major oxidation products were greatly shortened compared to conventional HPLC methods (more than 10 times). The ultra reversed-phase LC was demonstrated to be as a powerful tool for monitoring rapid oxidation reactions of tocopherols with active nitrogen species.

Separation of phosphorylated peptides utilizing dual pH- and temperature-responsive chromatography.

The phosphorylation of a peptide is considered to be one of the most important post-translational modification reactions that can alter protein function in mammalian cells. To separate and purify, we developed a dual temperature- and pH-responsive chromatography based on terpolymer composed of N-isopropylacrylamide, N,N'-dimethylaminopropylacrylamide and butylmethacrylate. The property of the surface of the terpolymer-grafted stationary phase altered from hydrophilic to hydrophobic, and from changed to non-charged by changes in the temperature and the pH, respectively. In addition, it was possible to appear and hide ion-exchange groups on the polymer chain surface by temperature changes. These phenomena resulted from changes in the charge and the hydrophobicity of the pH- and temperature-responsive polymer on the stationary surface by controlling the temperature. In the developed environmental-responsive chromatographic system, the ionizable dimethylamino group of N,N'-dimethylaminopropylacrylamide in terpolymer played a key role for the separation. We applied the developed chromatographic system to the separation of phosphorylated compounds, such as phospho-tyrosine, phosphopeptide and oligonucleotides. At a low column temperature, the electrostatic interaction plays a predominant role for retain anionic phosphorylated compounds, because of the strong interaction between the cationic dimethylamino group in the stationary phase and the anionic phosphoric group in the analyte. On the contrary, the hydrophobic interaction became predominant upon increasing the temperature. The results showed that both the electrostatic and the hydrophobic interactions became controllable with a temperature change during the chromatographic process. Dual pH- and temperature-responsive chromatography would be very useful for biomacromolecules separation and purification.

Dual temperature- and pH-responsive fluorescence molecular probe for cellular imaging utilizing a PNIPAAm-fluorescein copolymer.

The thermally controlling fluorescence intensity of a molecular probe for cellular imaging has been investigated. A reversible temperature-induced phase transition of N-isopropylacrylamide/fluorescein copolymers [poly(NIPAAm-co-FL)] was used as a molecular switch to control the fluorescence intensity of the imaging probe. The copolymer displayed environmentally sensitive fluorescence properties, in which the fluorescence intensity changed with the response to both the temperature and the pH. Utilizing these features, we monitored the thermal-aggregation process of BSA by the fluorescence resonance energy transfer (FRET) method. Additionally, the cellular uptake in RAW264.7 cells of poly(NIPAAm-co-FL) conjugated with lipid was studied using a confocal laser scanning microscope.

Aqueous chromatographic system for the quantification of propofol in biological fluids using a temperature-responsive polymer modified stationary phase.

A new method for the quantitative analysis of monkey serum propofol, which is widely used as an anaesthetic agent, was developed by utilizing a temperature-responsive polymer of N-isopropylacrylamide (NIPAAm) and butyl methacrylate (BMA) as the stationary phase of HPLC-fluorescence detection. This poly(NIPAAm-co-BMA) copolymer undergoes a reversible phase transition from a hydrophilic to a hydrophobic microstructure when triggered by change in the temperature. Also this chromatographic system is possible to separate the analytes by using only water as a mobile phase. A pretreatment of the serum (80 microL) was only solid-phase extraction, and the recovery rate of propofol and internal standard was more than 77%, respectively. This method covered the calibration range from 0.5 microg/mL to 10 microg/mL and allowed a reproducible quantification of the serum propofol in administrated monkey serum. The intra- and inter-assay relative standard deviations were less than 14.1%. In addition, there was good relationship of the quantification values between the developed method and the widely used reversed-phase HPLC method. Our developed method has proven to be useful for a simple analysis of propofol in clinical practice, because the avoidance of complicated mobile phase preparation was possible, and only temperature changing could regulate the retention time of the analyte. In addition, by using water instead of fossil fuel, it is the ideal analytical method according to green chemistry.