The correlation between serum total bile acid and alanine aminotransferase of pregnant women and the disorders of neonatal hyperbilirubinemia-related amino acid metabolism.

BACKGROUND: To explore the association between liver metabolism-related indicators in maternal serum and neonatal hyperbilirubinemia (NHB), and further investigate the predictive value of these indicators in NHB-related amino acid metabolism disorders. METHODS: 51 NHB and 182 No-NHB newborns and their mothers who treated in the Fourth Hospital of Shijiazhuang from 2018 to 2022 were participated in the study. The differences in clinical data were compared by the Mann-Whitney U test and Chi-square test. Multivariate logistic regression was used to analyze the relationship between maternal serum indicators and the occurrence of NHB. The correlation analysis and risk factor assessment of maternal serum indicators with NHB-related amino acid metabolic disorders were performed using Spearman correlation analysis and multivariate logistic regression. RESULTS: Compared to the non NHB group, the NHB group had higher maternal serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), ALT/AST, and total bile acid (TBA), while lower levels of serum albumin (ALB), total cholesterol (TC) and high-density lipoprotein (HDL). The levels of alanine (ALA), valine (VAL), ornithine (ORN), and proline (PRO) in the newborns were reduced in NHB group, while arginine (ARG) showed a tendency to be elevated. Multiple logistic regression analysis showed that maternal ALT, AST, ALT/AST, and TBA levels were all at higher risk with the development of NHB, whereas ALB, TC, and HDL levels were negatively associated with NHB development. Increasing maternal TBA level was associated with lower ALA (r=-0.167, p = 0.011), VAL (r=-0.214, p = 0.001), ORN (r=-0.196, p = 0.003), and PRO in the newborns (r=-0.131, p = 0.045). Maternal ALT level was negatively associated with ALA (r=-0.135, p = 0.039), VAL (r=-0.177, p = 0.007), ORN (r=-0.257, p < 0.001), while ALT/AST was positively correlated with ARG (r = 0.133, p = 0.013). After adjustment for confounding factors, maternal serum TBA and ALT were the independent risk factor for neonatal ORN metabolic disorders [(adjusted odds ratio (AOR) = 0.379, 95%CI = 0.188-0.762, p = 0.006), (AOR = 0.441, 95%CI = 0.211-0.922, p = 0.030)]. Maternal ALT level was an independent risk factor for neonatal VAL metabolic disorders (AOR = 0.454, 95%CI = 0.218-0.949, p = 0.036). CONCLUSIONS: The levels of high TBA, ALT, AST, and low HDL, TC of maternal were associated with the risk of NHB. Maternal TBA and ALT levels were independent risk factors for NHB-related amino acid disturbances which have value as predictive makers.

The association between maternal iron, zinc, copper levels in serum and pregnancy outcomes.

AIMS: To investigate the associations of serum trace elements (iron, zinc, and copper) between women with different pregnancy outcomes. METHODS: About 774 pregnant women who came to The Fourth Hospital of Shijiazhuang for prenatal examination were investigated. The concentrations of trace elements in the serum of pregnant women in the third trimester were collected. Multivariate logistic regression was used to analyze the relationship between serum trace element levels and the different pregnancy outcomes. Multiple linear regression was used to analyze the relationship between serum trace elements levels and hypersensitive-C-reactive-protein. RESULTS: Results of the multiple logistic regression showed that zinc, copper and copper/zinc ratio were found to be associated with the risk of gestational diabetes mellitus, and zinc was a protective factor (p = 0.002) while copper and copper/zinc ratio as risk factors (p = 0.030 and p = 0.001, respectively) after adjusting for major confounders. It was found that iron and zinc were negatively associated with the risk of moderate or severe anemia (p = 0.022 and p = 0.001, respectively). In contrast, the copper/zinc ratio was positively related to the risk of moderate or severe anemia (p = 0.021). The adjusted relationships between copper and copper/zinc ratio with premature rupture of membranes were statistically significant (p = 0.007 and p = 0.037). Iron and zinc were negatively associated with the risk of chorioamnionitis, while copper and copper/zinc ratio were positively associated with the risk of chorioamnionitis (all p < 0.05). CONCLUSIONS: Serum iron, zinc and copper levels are closely related to pregnancy outcomes.

The association between vitamin D levels in the second trimester of pregnancy and gestational diabetes mellitus.

BACKGROUND AND AIMS: 25-hydroxyvitamin D (25(OH)D) affects glucose metabolism by increasing insulin secretion and insulin receptor expression. However, whether 25(OH)D deficiency will increase the risk of gestational diabetes mellitus (GDM) has not been clearly reported. The purpose of this study is to assess the relationship between vitamin D levels in the second trimester of pregnancy and the risk of GDM. METHODS: According to the inclusion and exclusion criteria, 247 pregnant women came to the fourth hospital of Shijiazhuang (The affiliated obstetrics and gynecology hospital of Hebei Medical University) for obstetrics were investigated during the period of January 1, 2019 to December 31, 2020. The levels of 25(OH)D in the second trimester (16-20 weeks) and oral 75 g glucose tolerance test (OGTT) at 24-28 weeks of pregnancy were reviewed. The sociodemographic data were collected from questionnaire. Multivariate logistic regression was used to analyze the relationship between vitamin D levels and GDM. RESULTS: The incidence of GDM in the observation group (25(OH)D ≤ 26 ng/ml) was higher than that in the control group (25(OH)D > 26 ng/ml) (p = 0.039). Compared with control group, the observation group had significantly higher level of fasting plasma glucose (FPG) (4.7 [4.5-5.0] mmol/L vs. 4.6 [4.4-4.8] mmol/L, p = 0.012). In the whole study, the level of 25(OH)D was negatively correlated with FPG (r = - 0.164，p = 0.010). After adjusting for age, pre-pregnancy BMI, parity and adverse pregnancy history, compared with the observation group (25 (OH) D ≤ 26 ng/ml), the risk of developing GDM decreased by 50.9% in control group (25(OH)D > 26 ng/ml) (odds ratio [OR] = 0.491, 95% confidence interval [CI] = 0.243-0.989, p = 0.047). CONCLUSION: Adequate vitamin D levels during the second trimester of pregnancy may reduce the risk of GDM.

Association between Maternal Serum 25(OH)-Vitamin D3 Levels in Late Pregnancy and Profiles of Newborn Amino Acid Concentrations.

BACKGROUND: Vitamin D deficiency is common during pregnancy. 25(OH)-Vitamin D3 is the major vitamin D circulating form in human organism. However, the effects of 25(OH)-vitamin D3 deficiency in late pregnancy on the infant's amino acid metabolism has still not been studied. The aim of this study was to evaluate the relationship between maternal serum 25(OH)-vitamin D3 levels in late pregnancy and profiles of newborn amino acid concen-trations. METHODS: A total of 539 women in late pregnancy and their newborns enrolled in this study. The concentrations of 25(OH)-vitamin D3 in maternal serum were measured by ABI 4500 high performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS). For newborns, their amino acids levels were measured by ABI 3200 LC/MS/MS. T-test and Spearman's correlation analyses were used in the study as statistical analysis methods. RESULTS: The concentrations of arginine (Arg) and glycine (Gly) in newborn blood spots were significantly different in each maternal serum 25(OH)-vitamin D3 status group. There was a significant correlation between maternal serum 25(OH)-vitamin D3 status and Arg concentration in their offspring (p = 0.03). CONCLUSIONS: Maternal serum 25(OH)-vitamin D3 concentration in late pregnancy may affect their newborn's amino acid metabolism, but the precise mechanisms underlying the relationship need further investigation.

Performance of LC/MS/MS in Analyzing Multiple Amino Acids and Acylcarnitines in Dried Blood Spot of Very Low Birth Weight, Low Birth Weight, and Normal Weight Neonates.

BACKGROUND: Amino acid (AA) and acylcarnitine (AC) are important biomarkers of protein and fatty acid metabolism. Examining their levels in newborns may reveal multiple inherited metabolic diseases. However, they have rarely been assessed in very low birth weight (VLBW) neonates, low birth weight (LBW) neonates and rarely been compared with normal weight (NW) neonates. The aim of the study was to identify the AA and AC profiles in dried blood spot (DBS) specimens of LBW and VLBW neonates, then compare with NW neonates, and make a contribution to the determination of cutoff values of VLBW and LBW neonates. METHODS: Liquid Chromatography tandem mass spectrometry (LC/MS/MS) is an excellent tool for quantitatively detecting AA and AC profiles. This article verified the precision, accuracy, and linearity of the LC/MS/MS method in AA and AC detection, then analyzed AA and AC profiles in DBS of VLBW, LBW and NW neonates, and compared the difference of AA and AC in the three groups. RESULTS: The results showed that the LC/MS/MS method had wide linear range, satisfied precision and reproducibility in detecting AA and AC in DBS specimens; most AA and AC concentrations significantly correlated with birth weight in DBS samples (p < 0.05). CONCLUSIONS: The results suggested that VLBW and LBW neonates have different metabolic or nutritional status with NW neonates and different AA and AC cutoffs should be defined for them to reduce the risk of false-positive cases.

Engineering high-performance hairpin stacking circuits for logic gate operation and highly sensitive biosensing assay of microRNA.

Recently, hairpin stacking circuits (HSC) based on toehold-mediated strand displacement have been engineered to detect nucleic acids and proteins. However, the three metastable hairpins in a HSC system can potentially react non-specifically in the absence of a catalyst, limiting its practical application. Here, we developed a unique hairpin design guideline to eliminate circuit leakage of HSC, and the high-performance HSC was successfully implemented on logic gate building and biosensing. We began by analyzing the sources of circuit leakage and optimizing the toehold lengths of hairpins in the HSC system based on the surface plasmon resonance (SPR) technique. Next, a novel strategy of substituting two nucleotides in a specific domain, termed 'loop-domain substitution', was introduced to eliminate leakages. We also systematically altered the positions and numbers of the introduced substitutions to probe their potential contribution to circuit leakage suppression. Through these efforts, the circuit leakage of HSC was significantly reduced. Finally, by designing different DNA input strands, the logic gates could be activated to achieve the output signal. Using miRNA as a model analyte, this strategy could detect miRNA down to pM levels with minimized circuit leakage. We believe these work indicate significant progress in the DNA circuitry.

Evaluation of alanine aminotransferase/aspartate aminotransferase ratio and high-density lipoprotein for predicting neonatal adverse outcomes associated with intrahepatic cholestasis of pregnancy.

Background: To determine the association between lipid metabolism and intrahepatic cholestasis of pregnancy (ICP), and explore the value of maternal alanine aminotransferase/aspartate aminotransferase (ALT/AST) and high-density lipoprotein (HDL) in predicting adverse neonatal outcomes in women with ICP. Methods: A total of 147 pregnant women with ICP admitted to The Fourth Hospital of Shijiazhuang and 120 normal pregnant women in the same period were selected in this study. The Mann-Whitney U test and Chi-square tests were used to compare the differences in clinical data. Multivariate logistic regression was used to analyze the relationship between ALT/AST and the occurrence of adverse pregnancy outcomes in patients with ICP. The combined predictive value of ALT/AST and HDL was determined by receiver operating characteristic (ROC) curve analysis. Results: Among 147 women with ICP, 122 women had total bile acid (TBA) levels of 10-39.9 µmol/L, and 25 had TBA ≥ 40 µmol/L. There was significantly lower gestational age in patients with severe ICP than in those with mild and control groups (all p < 0.05), and the weight of newborns in the maternal ICP group was significantly lower than in the control group (p < 0.05). Increasing TBA levels was associated with higher AST, ALT, ALT/AST, and lower HDL level (all p < 0.05). Meanwhile, higher levels of ALT/AST was positively associated with neonatal hyperbilirubinemia [adjusted odds ratio (AOR) = 4.019, 95% CI [1.757-9.194, p = 0.001] and cardiac injury [AOR = 3.500, 95% CI [1.535-7.987], p = 0.003]. HDL was a significant protective factor for neonatal hyperbilirubinemia and cardiac injury [AOR = 0.315, 95% CI [0.126-0.788], p = 0.014; AOR = 0.134 (0.039-0.461), p = 0.001]. The area under the ROC curve (AUC) for prediction of neonatal hyperbilirubinemia by ALT/AST combined with HDL was 0.668 [95% CI [56.3-77.3%], p = 0.002], and the sensitivity and specificity were 47.1% and 84.0%, respectively. To predict neonatal cardiac injury, the AUC value was 0.668 [95% CI [56.4-77.1%], p = 0.002], with sensitivity and specificity were 41.2% and 87.1%, respectively. Conclusions: The levels of higher ALT/AST and lower HDL were significantly associated with the risk of ICP-related adverse neonatal outcomes. Moreover, ALT/AST combined with HDL has moderate clinical value in predicting the adverse outcomes of neonatal hyperbilirubinemia and cardiac injury.

The discrepancy distribution of macrophage subsets in preeclampsia placenta with or without fetal growth restriction from a small cohort.

OBJECTIVES: To identify the effect of distribution characteristic of macrophages on placental function and angiogenesis in pregnancies with preeclampsia (PE) in presence of fetal growth restriction (FGR) or preeclampsia without FGR. MATERIAL AND METHODS: The study tested the hypothesis that there was association between distribution characteristic of macrophage subsets (marked by CD68, CD163, respectively) and placental capillary development, leading to placental dysfunction in PE pregnancies with FGR (n = 36). Changes in placental parameters related with efficiency and angiogenesis and macrophage phenotypes (CD68 and CD163) were evaluated by immunohistochemistry. Pearson correlation analysis was performed to analysis the association between macrophage phenotype and placental function as well the CD34 staining, respectively. Additionally, the localization of CD68 and CD163 was assessed by using immunoflurorescence staining. RESULTS: Pearson correlation analysis had shown the positive association between CD68 expression and microvessel formation and the reverse linear relationship between CD163 staining and placental sufficiency in PE + FGR placenta. The co-localization of CD163 and CD34 may pointed to the compensatory role of CD163 distribution involved in prompting neovascularization. CONCLUSIONS: The association between disturbed distribution of macrophages and placental efficiency and angiogenesis were only found in PE with FGR not in PE pregnancies without FGR, underlying the discrepancy role of macrophage subsets depending on the clinical phenotype of PE pregnancies.

Correlation between high serum ferritin levels and adverse pregnancy outcomes in women with gestational diabetes mellitus.

Aims: To explore the association between higher serum ferritin (SF) levels in mid-pregnancy and adverse pregnancy outcomes in gestational diabetes mellitus (GDM) pregnancies, then develop a predictive cut-off value that might effectively predict the risk of adverse pregnancy outcomes in future clinical. Methods: The study involved 201 pregnant women with GDM. 201 gestational age and parity matched normoglycemic pregnant women were taken as control group. The differences in clinical data were compared by the Mann-Whitney U test and Chi-square tests. Multivariate logistic regression was used to determine the relationship between SF and GDM-relate adverse pregnancy outcomes. The predicted value of SF level was determined through receiver operating characteristic (ROC) curve analysis. Results: SF level was significantly higher in women with GDM [16.10 (27.30-9.50) (ng/mL) vs. 12.04 (18.11-7.06) (ng/mL), (p < 0.001)]. Meanwhile, higher levels of SF were also discovered in GDM women with preeclampsia and neonatal hypoglycemia and respiratory distress (all p < 0.05). In the adjusted model, a positive association was shown between SF and preeclampsia [adjusted odds ratio (AOR) = 1.032, 95%CI = 1.004-1.060, p = 0.024], neonatal hypoglycemia [adjusted odds ratio (AOR) = 1.047, 95%CI = 1.022-1.072, p < 0.001] and respiratory distress outcomes (AOR = 1.034, 95%CI = 1.011-1.058, p = 0.004) respectively. The area under ROC curve (AUC) for prediction of preeclampsia by SF combined with serum calcium, age, pre-pregnancy BMI and gestational weight gain (GWG) was 0.658 (95% CI = 50.8-80.8%, p = 0.028) with the cut-off value of 24.45 ng/mL, and the sensitivity and specificity were 58.8.0% and 64.3%, respectively. To predict neonatal hypoglycemia, the clinical point value of SF was 27.43 ng/mL with AUC was 0.800, sensitivity and specificity was 90.5% and 68.0% respectively. Predicting neonatal respiratory distress, the AUC value of the SF level was 0.730, with a cut-off value of 27.37 ng/mL and the sensitivity and specificity were 52.0% and 86.5%, respectively. Conclusions: Higher level of SF in mid-pregnancy was significantly associated with the risk of GDM and GDM-relate adverse pregnancy outcomes. Moreover, SF levels have moderate clinical value in predicting the adverse outcomes of maternal preeclampsia, neonatal hypoglycemia and respiratory distress.

A colorimetric biosensor for ultrasensitive detection of the SURF1 gene based on a dual DNA-induced cascade hybridization reaction.

In this work, a simple and ultrasensitive colorimetric biosensor for detection of SURF1 gene fragments (Leigh syndrome) has been developed based on a dual DNA-induced cascade hybridization reaction. Firstly, a biotin labeled capture probe was immobilized on a streptavidin labeled 96-well transparent plate surface. Then the target SURF1 fragment and auxiliary probe S1 were added into the reaction system to form a "Y" structure with the capture probe. Furthermore, to achieve a highly efficient signal amplification strategy, digoxin labeled P1, P2, P3 and P4 probes were used to cause a dual DNA-induced cascade hybridization reaction on the "Y" structure of the 96-well plate surface. As a detection probe, the HRP anti-digoxin antibody was combined on the surface to produce a colorimetric response to the SURF1 fragment in the presence of TMB. Under the optimal conditions, the established method exhibited a wide linear range from 1.0 × 10-13 M to 1.0 × 10-8 M and a detection limit to SURF1 as low as 1.73 × 10-14 M. In addition, the strategy has been successfully applied to the detection of SURF1 in spiked human serum samples. Therefore, the established biosensor has potential application prospects in gene fragment analysis and early diagnosis of clinical diseases.

Aptamer based high throughput colorimetric biosensor for detection of staphylococcus aureus.

To develop a high throughput colorimetric biosensor for detection of Staphylococcus aureus (SA) based on specific aptamer and catalysis of dsDNA-SYBR Green I (SG I) complex. SA specific aptamer was immobilized on a 96-well plate by hybridization with the capture probe anchored on the plate surface through streptavidin-biotin binding. In presence of SA, the aptamer was dissociated from the capture probe-aptamer duplex due to the stronger interaction between the aptamer and SA. The consequent single-strand capture probe could be hybridized with a three-way junction (TWJ) probe. With the presence of SG I, the dsDNA-SG I complex catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) under photo-irradiation, producing sensitive photo-catalyzed colorimetric response to SA. Under the optimal conditions, the proposed method could directly detect SA with the limit of detection (LOD) at 81 CFU mL-1 in PBS buffer in 5.5 hours, which demonstrated the sensitive and fast quantification of target pathogenic bacteria. The method showed weak colorimetric signal to Escherichia coli and Pseudomonas aeruginosa, indicating the high specificity for SA. In addition, the method can simultaneously detect 96 samples which can be used for high throughput analysis. The designed method may become a powerful tool for pathogenic microorganisms screening in clinical diagnostics, food safety and environmental monitoring.

1H-NMR based metabolomic profiling of cord blood in gestational hypothyroidism.

BACKGROUND: Gestational hypothyroidism (GHT) is a common pregnancy-related thyroid disfunction. The adverse outcomes by GHT has been increasingly recognized, leading to more public awareness of the disease. However, comprehensive understanding of the prognosis of GHT has not yet achieved. Metabolomics is a powerful tool in evaluation of disease outcomes, and cord blood represents an excellent candidate for the investigation of gestational outcomes. METHODS: In the present study, we performed 1H-NMR based metabolomics on cord blood of 18 pregnant women with GHT and 18 non hypothyroidism (NHT) control. RESULTS: The metabolomic profile of GHT was separated with the NHT control. A total of 8 metabolites with altered abundances were observed, among which Creatinine and O-Phosphocholine were elevated and the others were downregulated in GHT. Spearman rank correlation suggested that the eight differential metabolites were correlated with the GHT related thyroid hormones. Pathway analysis of the differential metabolites indicated that two metabolic pathways were significantly altered in GHT (adjusted P<0.05), including tyrosine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis. Enrichment analysis of the differential metabolites against disease-associated metabolite sets suggested that GHT was associated with disease risks of non-insulin dependent diabetes mellitus, isovaleric acidemia, and methylmalonic aciduria. CONCLUSIONS: The results of this study revealed GHT associated metabolic changes in cord blood, providing insights into the metabolic intermediates between GHT and its related disease risks.

A simple electrochemical aptasensor for ultrasensitive protein detection using cyclic target-induced primer extension.

A simple electrochemical aptasensor was developed for ultrasensitive protein detection by combining a novel strategy of cyclic target-induced primer extension (CTIPE) with an aptamer-hairpin probe and enzyme-amplified electrochemical readout. In the presence of protein target, the immobilized aptamer-hairpin probe recognized the protein to trigger primer extension reaction by target-induced conformational transition, which released the protein from replicated DNA duplex. The released target could cyclically bind with other aptamer-hairpin probes and trigger new primer extension, leading to formation of numerous biotin-tagged DNA duplex, which significantly amplified the protein recognition event and facilitated the subsequent enzymatic signal enhancement, leading to an ultrasensitive electrochemical aptasensor. Using human vascular endothelial growth factor as a model protein, the designed aptasensor could detect protein down to 0.82 pg mL(-1) with a linear range from 1 pg mL(-1) to 1 ng mL(-1). The proposed aptasensor was amenable to quantification of protein in complex biological matrixes, and would become a simple and powerful tool for bioanalysis and clinic diagnostic application.

Label-free and high-sensitive detection of Salmonella using a surface plasmon resonance DNA-based biosensor.

A method based on surface plasmon resonance (SPR) DNA biosensor has been developed for label-free and high-sensitive detection of Salmonella. A biotinylated single-stranded oligonucleotide probe was designed to target a specific sequence in the invA gene of Salmonella and then immobilized onto a streptavidin coated dextran sensor surface. The invA gene was isolated from bacterial cultures and amplified using a modified semi-nested asymmetric polymerase chain reaction (PCR) technique. In order to investigate the hybridization detection, experiments with different concentration of synthetic target DNA sequences have been performed. The calibration curve of synthetic target DNA had good linearity from 5 nM to 1000 nM with a detection limit of 0.5 nM. The proposed method was applied successfully to the detection of single-stranded invA amplicons from three serovars of Salmonella, i.e., Typhimurium, Enterica and Derby, and the responses to PCR products were related to different S. typhimurium concentrations in the range from 10(2) to 10(10) CFU mL(-1). While with this system to detect E. coli and S. aureus, no significant signal was observed, demonstrating good selectivity of the method. In addition, the hybridization can be completed within 15 min, and the excellent sensor surface regeneration allows at least 300 assay cycles without obvious loss of performance.

Electrochemical immunosensor for competitive detection of neuron specific enolase using functional carbon nanotubes and gold nanoprobe.

An electrochemical immunosensor for detection of neuron specific enolase (NSE) was designed by immobilizing NSE covalently functionalized single-walled carbon nanotubes (NSE-SWNTs) on a glassy carbon electrode. The NSE-SWNTs not only enhanced electrochemical signal but also presented abundant antigen domains for competitive immunological recognition to anti-NSE primary antibody and then gold nanoprobes labeled with alkaline phosphatase conjugated secondary antibody (AP-anti-IgG/AuNPs). The AP-anti-IgG/AuNPs exhibited highly catalytic activity toward enzyme substrate and significantly amplified the amperometric signal for target molecule detection. Based on the dual signal amplification of SWNTs and gold nanoprobe, the immunosensor could response down to 0.033 ng mL(-1) NSE with a linear range from 0.1 ng mL(-1) to 2 µg mL(-1), and showed acceptable precision and reproducibility. The designed immunosensor was amenable to direct quantification of target protein with a wide range of concentration in complex clinical serum specimens. The assay results were in a good agreement with the reference values. The proposed electrochemical immunosensor provided a pragmatic platform for convenient detection of tumor markers in clinical diagnosis.

[Determination of ethyl glucuronide in human urine by solid phase extraction-gas chromatography-mass spectrometry].

A solid phase extraction (SPE) and gas chromatography (GC) with mass spectrometry (MS) method for determination of ethyl glucuronide (EtG) in human urine was established. One mL urine sample was deproteinated by 100 microL 3 mol/L hydrochloric acid and cleaned up through a solid phase extraction column. The target analytes were eluted from an NH2-column with 4% ammonia solution and then treated with bis (trimethylsilyl) trifluoroacetamide (BSTFA) + trimethylchlorosilane (TMCS) (99:1) for derivatization. The derivatized samples were analyzed by GC-MS. Data were acquired in the selected ion monitoring (SIM) mode and the quantitation of EtG was done through internal standard method. Good linearity was obtained at the mass concentration range of 0.1 - 3.2 mg/L with a correlation coefficient (r) of 0.9921. The limit of detection (LOD) was 28.4 microg/L. The range of recoveries was 92.5% - 108.7%, and the relative standard deviations (RSDs) of intra-day and inter-day were all less than 5%. This method is sensitive, specific, accurate and can be applied to the determination of EtG for medicolegal identification and clinical laboratory.