ObjectiveTo investigate the effects and potential mechanisms of Babaodan （BBD） against acetaminophen （APAP）-induced acute liver injury （ALI） based on transcriptomics. MethodsA total of 36 male C57BL/6 mice were randomly divided into 6 groups （n=6 per group）： normal group， model group， N-acetylcysteine group （NAC， 120 mg·kg^-1）， and BBD low-， medium-， and high-dose groups （BBD-L， BBD-M， BBD-H groups， 75， 150， 300 mg·kg^-1， respectively）. Except for the normal group， all other groups were subjected to APAP-induced ALI. The serum levels of aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， total cholesterol （TC）， triglyceride （TG）， and low-density lipoprotein cholesterol （LDL-C） were measured in each group. Hepatic levels or activities of malondialdehyde （MDA） and glutathione peroxidase （GSH-Px） were detected using commercial kits. Hematoxylin-eosin （HE） staining was performed to evaluate the degree of liver histopathological damage. Transcriptomic analysis was employed to screen differentially expressed genes （DEGs）， and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis was performed to identify differential pathways involved in BBD intervention against ALI. Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot were applied to validate the expression of differential genes and related pathway proteins. Additionally， glucose （GLU） consumption， as well as lactate （LD） and adenosine triphosphate （ATP） content were assessed across all mouse groups. ResultsPharmacodynamic evaluation showed that， compared with the normal group， the model group exhibited significantly elevated serum levels of ALT， AST， TC， TG， and LDL-C （P<0.01）， significantly increased MDA level （P<0.01）， and significantly decreased GSH-Px level （P<0.05）. Compared with the model group， BBD intervention at different doses significantly reduced the serum levels of ALT， AST， TC， TG， and LDL-C （P<0.05， P<0.01）， increased GSH-Px level （P<0.05， P<0.01）， significantly decreased MDA level （P<0.01）， and ameliorated hepatic histopathological injury. Liver transcriptomic analysis revealed that， following high-dose BBD intervention， the core genes were mainly enriched in pathways related to inflammatory responses and energy metabolic reprogramming， including the interleukin-17 （IL-17） signaling pathway， the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway， fructose and mannose metabolism， and glycolysis. Real-time PCR validation demonstrated that， compared with the normal group， the mRNA expression levels of glycolysis-related genes ［hexokinase 1 （HK1）， hexokinase 2 （HK2）， 6-phosphofructo-2-kinase/fructose-2，6-bisphosphatase 3 （PFKFB3）， 6-phosphofructo-2-kinase/fructose-2，6-bisphosphatase 4 （PFKFB4）， pyruvate kinase M （PKM）， and lactate dehydrogenase A （LDHA）］， as well as inflammatory cytokines ［tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-1β （IL-1β）］ were significantly upregulated in the model group （P<0.05， P<0.01）. Compared with the model group， the BBD-H group showed significantly decreased mRNA expression of HK1， HK2， PFKFB3， PFKFB4， PKM， LDHA， TNF-α， IL-6， and IL-1β （P<0.05， P<0.01）. Western blot results indicated that， compared with the normal group， the model group had significantly increased expression of glycolysis-related proteins ［glucose transporter 1 （GLUT1）， HK1， PFKFB3， and PKM］， inflammatory proteins ［interleukin-18 （IL-18）， TNF-α， and IL-1β］， and phosphorylated （p）-PI3K and p-Akt proteins （P<0.05， P<0.01）. Compared with the model group， the BBD-H group exhibited significantly decreased expression of GLUT1， HK1， PFKFB3， PKM， IL-18， TNF-α， IL-1β， p-PI3K， and p-Akt （P<0.05， P<0.01）. Metabolic indicator measurements showed that， compared with the model group， the BBD-H group showed significantly reduced GLU consumption， LD and ATP content （P<0.05， P<0.01）. ConclusionBBD may alleviate APAP-induced ALI through dual regulation of metabolic reprogramming and inflammatory responses， potentially via inhibition of the PI3K/Akt signaling pathway.

ObjectiveTo investigate the effects and potential mechanisms of Babaodan （BBD） against acetaminophen （APAP）-induced acute liver injury （ALI） based on transcriptomics. MethodsA total of 36 male C57BL/6 mice were randomly divided into 6 groups （n=6 per group）： normal group， model group， N-acetylcysteine group （NAC， 120 mg·kg^-1）， and BBD low-， medium-， and high-dose groups （BBD-L， BBD-M， BBD-H groups， 75， 150， 300 mg·kg^-1， respectively）. Except for the normal group， all other groups were subjected to APAP-induced ALI. The serum levels of aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， total cholesterol （TC）， triglyceride （TG）， and low-density lipoprotein cholesterol （LDL-C） were measured in each group. Hepatic levels or activities of malondialdehyde （MDA） and glutathione peroxidase （GSH-Px） were detected using commercial kits. Hematoxylin-eosin （HE） staining was performed to evaluate the degree of liver histopathological damage. Transcriptomic analysis was employed to screen differentially expressed genes （DEGs）， and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis was performed to identify differential pathways involved in BBD intervention against ALI. Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot were applied to validate the expression of differential genes and related pathway proteins. Additionally， glucose （GLU） consumption， as well as lactate （LD） and adenosine triphosphate （ATP） content were assessed across all mouse groups. ResultsPharmacodynamic evaluation showed that， compared with the normal group， the model group exhibited significantly elevated serum levels of ALT， AST， TC， TG， and LDL-C （P<0.01）， significantly increased MDA level （P<0.01）， and significantly decreased GSH-Px level （P<0.05）. Compared with the model group， BBD intervention at different doses significantly reduced the serum levels of ALT， AST， TC， TG， and LDL-C （P<0.05， P<0.01）， increased GSH-Px level （P<0.05， P<0.01）， significantly decreased MDA level （P<0.01）， and ameliorated hepatic histopathological injury. Liver transcriptomic analysis revealed that， following high-dose BBD intervention， the core genes were mainly enriched in pathways related to inflammatory responses and energy metabolic reprogramming， including the interleukin-17 （IL-17） signaling pathway， the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway， fructose and mannose metabolism， and glycolysis. Real-time PCR validation demonstrated that， compared with the normal group， the mRNA expression levels of glycolysis-related genes ［hexokinase 1 （HK1）， hexokinase 2 （HK2）， 6-phosphofructo-2-kinase/fructose-2，6-bisphosphatase 3 （PFKFB3）， 6-phosphofructo-2-kinase/fructose-2，6-bisphosphatase 4 （PFKFB4）， pyruvate kinase M （PKM）， and lactate dehydrogenase A （LDHA）］， as well as inflammatory cytokines ［tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-1β （IL-1β）］ were significantly upregulated in the model group （P<0.05， P<0.01）. Compared with the model group， the BBD-H group showed significantly decreased mRNA expression of HK1， HK2， PFKFB3， PFKFB4， PKM， LDHA， TNF-α， IL-6， and IL-1β （P<0.05， P<0.01）. Western blot results indicated that， compared with the normal group， the model group had significantly increased expression of glycolysis-related proteins ［glucose transporter 1 （GLUT1）， HK1， PFKFB3， and PKM］， inflammatory proteins ［interleukin-18 （IL-18）， TNF-α， and IL-1β］， and phosphorylated （p）-PI3K and p-Akt proteins （P<0.05， P<0.01）. Compared with the model group， the BBD-H group exhibited significantly decreased expression of GLUT1， HK1， PFKFB3， PKM， IL-18， TNF-α， IL-1β， p-PI3K， and p-Akt （P<0.05， P<0.01）. Metabolic indicator measurements showed that， compared with the model group， the BBD-H group showed significantly reduced GLU consumption， LD and ATP content （P<0.05， P<0.01）. ConclusionBBD may alleviate APAP-induced ALI through dual regulation of metabolic reprogramming and inflammatory responses， potentially via inhibition of the PI3K/Akt signaling pathway.

AIM:To investigate the expression profile and biological significance of long noncoding RNA(lncRNA)zinc finger antisense 1(ZFAS1)in the brains of mice with diabetic encephalopathy(DE).METHODS:Ten db/m mice served as the normal control group,while twenty 22-week-old db/db mice were used to establish the DE model and randomly divided into two subgroups:ten as the db/db model control and the remaining ten receiving ZFAS1 gene knockdown(db/db+sh-ZFAS1)via lentiviral transfection.Weekly measurements of body weight and blood glucose levels were performed.Brain tissues were collected for Nissl staining to evaluate neuronal damage,TUNEL assay to detect apop-tosis,and immunofluorescence staining to examine neural biomarker expression.Serum levels of tumor necrosis factor-α(TNF-α)and oxidative stress markers,including reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px),were determined.Western blot was conducted to quantify the protein expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),p38 mitogen-activated protein kinase and phosphorylated p38(p-p38)in brain tissues.The expression levels of ZFAS1 and caspase-3 mRNA were determined by RT-qPCR.RESULTS:Knockdown of ZFAS1 in db/db mice significantly improved cognitive function,alleviated hippocampal neuronal damage,and reduced body weight and blood glucose levels(P<0.01).More-over,oxidative stress was mitigated,as evidenced by decreased MDA and ROS levels(P<0.01)and increased activity of antioxidant enzymes,GSH-Px,SOD and CAT(P<0.01 or P<0.05).Meanwhile,ZFAS1 silencing down-regulated Bax and p-p38/p38 protein expression(P<0.01 or P<0.05)while up-regulating Bcl-2(P<0.01).Consistently,RT-qPCR confirmed significant down-regulation of ZFAS1 and caspase-3 mRNA levels(P<0.01).CONCLUSION:lncRNA ZFAS1 is highly expressed in the hippocampus of DE mice.Down-regulation of ZFAS1 expression enhances cognitive func-tion,suppresses oxidative stress,and inhibits neuronal apoptosis,thereby attenuating neural damage in DE.

AIM:To investigate the expression profile and biological significance of long noncoding RNA(lncRNA)zinc finger antisense 1(ZFAS1)in the brains of mice with diabetic encephalopathy(DE).METHODS:Ten db/m mice served as the normal control group,while twenty 22-week-old db/db mice were used to establish the DE model and randomly divided into two subgroups:ten as the db/db model control and the remaining ten receiving ZFAS1 gene knockdown(db/db+sh-ZFAS1)via lentiviral transfection.Weekly measurements of body weight and blood glucose levels were performed.Brain tissues were collected for Nissl staining to evaluate neuronal damage,TUNEL assay to detect apop-tosis,and immunofluorescence staining to examine neural biomarker expression.Serum levels of tumor necrosis factor-α(TNF-α)and oxidative stress markers,including reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px),were determined.Western blot was conducted to quantify the protein expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),p38 mitogen-activated protein kinase and phosphorylated p38(p-p38)in brain tissues.The expression levels of ZFAS1 and caspase-3 mRNA were determined by RT-qPCR.RESULTS:Knockdown of ZFAS1 in db/db mice significantly improved cognitive function,alleviated hippocampal neuronal damage,and reduced body weight and blood glucose levels(P<0.01).More-over,oxidative stress was mitigated,as evidenced by decreased MDA and ROS levels(P<0.01)and increased activity of antioxidant enzymes,GSH-Px,SOD and CAT(P<0.01 or P<0.05).Meanwhile,ZFAS1 silencing down-regulated Bax and p-p38/p38 protein expression(P<0.01 or P<0.05)while up-regulating Bcl-2(P<0.01).Consistently,RT-qPCR confirmed significant down-regulation of ZFAS1 and caspase-3 mRNA levels(P<0.01).CONCLUSION:lncRNA ZFAS1 is highly expressed in the hippocampus of DE mice.Down-regulation of ZFAS1 expression enhances cognitive func-tion,suppresses oxidative stress,and inhibits neuronal apoptosis,thereby attenuating neural damage in DE.

OBJECTIVE To establish an UHPLC-MS method for simultaneous determination of 9 bile acids components in Shedan Chuanbei liquid. To analyze the differences of bile acids components from different manufacturers and different batches of product. METHODS Thermo Fisher Scientfic Bremen HYPERSIL GOLD(2.1 mm×100 mm, 1.9 µm) was selected as the chromatographic column. The mobile phase was 0.1% formic acid-water and methanol with gradient elution at a flow rate of 0.2 mL·min−1, and the column temperature was 40 ℃. HESI negative ion mode was used for mass spectrometry, and PRM scanning mode was used for mass spectrometry data. RESULTS The calibration curves of 9 biles acids were linear in their own range(r≥0.999 2). The average recoveries ranged from (95.5±1.52)% to (103.1±3.81)%. The RSD ranged from 1.1% to 5.5%. Nine bile acids components including taurocholic acid, tauroursodesoxycholic acid, 7-ketone-3α,12-α-hydroxyl cholanic acid, taurochenodeoxy-cholic acid, sodium taurodeoxycholate, cholic acid, sodium taurocholate, chenodeoxycholic acid and deoxycholic acid were tentatively identified from 19 batches of test samples. Taurocholic acid, taurochenodeoxycholic acid and taurodeoxycholate sodium were detected in all the test batches. CONCLUSION This method is simple and stable, and has certain reference value for improving the quality control and evaluation of Shedan Chuanbei liquid.

Objective: To investigate the prevalence of frailty and its influencing factors among the elderly, so as to provide the evidence for prevention and control of frailty. Methods: The elderly population at ages of 65 years and older were selected from 14 administrative villages or communities in Wuzhong District, Suzhou City, Jiangsu Province, using the random cluster sample method from July to November, 2022. Demographic information, smoking and alcohol consumption were collected through questionnaire surveys. Physical activity, sleep quality and frailty were evaluated using the International Physical Activity Questionnaire-Short, Pittsburgh Sleep Quality Index and the FRAIL Scale, respectively. Factors affecting frailty among the elderly were evaluated using a multinomial logistic regression model. Results: A total of 8 782 elderly peolple were surveyed, including 4 259 males (48.50%) and 4 523 females (51.50%). The median age was 71.00 (interquartile range, 8.00) years. There were 2 145 cases with pre-frailty (24.42%) and 189 cases with frailty (2.15%). Multinomial logistic regression analysis showed that age (75-<85 years, OR=1.330, 95%CI: 1.186-1.492; ≥85 years, OR=2.452, 95%CI: 1.882-3.195), smoking (current smoking, OR=0.838, 95%CI: 0.714-0.983), physical activity level (low, OR=1.161, 95%CI: 1.010-1.333) and sleep quality (poor, OR=2.248, 95%CI: 1.822-2.774) were associated with pre-frailty; age (75-<85 years,OR=2.629, 95%CI: 1.921-3.596; ≥85 years, OR=3.067, 95%CI: 1.621-5.801), educational level (junior high school and above, OR=0.488, 95%CI: 0.298-0.798), body mass index (underweight, OR=1.848, 95%CI: 1.023-3.337; obesity, OR=1.798, 95%CI: 1.180-2.740), smoking (quit smoking, OR=1.787, 95%CI: 1.007-3.171; current smoking, OR=0.448, 95%CI: 0.242-0.830), alcohol consumption (yes, OR=0.532, 95%CI: 0.291-0.972), physical activity level (low, OR=2.757, 95%CI: 1.646-4.616) and sleep quality (poor, OR=3.911, 95%CI: 2.438-6.273) were associated with frailty. Conclusion Older, low physical activity level, poor sleep quality, underweight and obesity are associated with frailty of the elderly.

OBJECTIVE To establish a method for simultaneous determination of 15 bile acids in Tongren niuhuang qingxin pills， and to determine the contents of 15 batches of samples. METHODS Using dehydrocholic acid as internal standard， the determination was performed by ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method. The determination was performed on Hypersil GOLD C18 column with methanol-0.1% formic acid solution as the mobile phase by gradient elution at the flow rate of 0.2 mL/min. The column temperature was 40 ℃ ， and the sample size was 2 µL. Using heated electrospray ion source， parallel reaction monitoring mode scanning was performed in negative ion mode. SPSS 24.0 software was used for chemical pattern recognition analysis of content determination results. RESULTS The 15 bile acid components had a good linear relationship with peak area （all R2≥0.998 9）； their precision， repeatability and stability were all good （all RSD≤5.49%）； the average recoveries were 93.8%-105.7% （RSD was 0.5%-5.8%）. The average contents of taurocholic acid， 7-oxodeoxycholic acid， 12-dehydrocholic acid， glycocholic acid， 3-oxo-7α， 12α-hydroxy-5β-cholanoic acid， taurochenodeoxycholic acid， 3α-hydroxy- 7-oxo-5β -cholanic acid， hyocholic acid， taurodeoxycholic acid sodium salt hydrate， hyodeoxycholic acid， cholic acid， glycochenodeoxycholic acid， glycodeoxycholic acid， chenodeoxycholic acid and deoxycholic acid were 670.56， 25.97， 10.54， 280.12， 4.04， 29.81， 182.98， 813.55， 120.95， 220.31， 797.37， 18.37， 68.59， 30.13， 59.82 μg/g， respectively. Both cluster analysis and principal component analysis divided 15 batches of Tongren niuhuang qingxin pills into 2 categories， S1-S12 as one category and S13-S15 as the other category. CONCLUSIONS The established method is accurate， sensitive and specific， and can determine many types of bile acids. It also can quickly achieve the quantitative analysis of 15 bile acids in Tongren niuhuang qingxin pills， which is suitable for the quality control of this drug.

ObjectiveTo investigate the principle and scientific connotation of Euodiae Fructus（EF） processed with Glycyrrhizae Radix et Rhizoma（Gly） by comparing the effects of unprocessed products of EF（UEF） and processed products of EF with the different proportions of Gly（GEFs） at toxic doses on oxidative stress and autophagy in the liver of mice. MethodSeventy mice were randomly divided into 7 groups， namely the control group， the UEF group， the group of the processed products of EF without Gly（PEF） and 4 groups of GEFs（the mass ratios of EF to Gly were 100∶3， 100∶6， 100∶12 and 100∶24， respectively， hereinafter referred to as the processed products of EF with the mass ratios of 100∶3， 100∶6， 100∶12 and 100∶24 of Gly）. The mice were given purified water， the decoction of UEF， PEF and GEFs by gavage at a dose of 30 g·kg^-1. PEF and GEFs were prepared according to the method under EF in the 2020 edition of Chinese Pharmacopoeia. Levels of alanine aminotransferase（ALT） and aspartate aminotransferase（AST） were determined by ultraviolet-visible spectrophotometry， hematoxylin-eosin（HE） staining was used to evaluate the pathological changes of liver tissue， the level of reactive oxygen species（ROS） was detected by fluorescence method， the mRNA expression of heme oxygenase-1（HO-1）， quinone oxidoreductase-1（NQO1）， glutathione-S-transferase 1（GSTA1）， Kelch-like epichlorohydrin-associated protein 1（Keap1） and p62 were measured by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， western blot was used to detect the protein expression of phosphorylated mammal target of rapamycin（p-mTOR）， phosphorylated ribosomal p70 S6 protein kinase（p-p70S6K）， p62， microtubule-associated protein 1 light chain 3Ⅰ（LC3Ⅰ） and LC3Ⅱ. ResultCompared with the control group， after 7 d of administration， the increase in body mass of mice in the UEF group began to slow down and the difference gradually increased， and the liver body index significantly increased（P<0.01）， pathomorphological observation showed that the structure of hepatic lobules was disordered， and local hepatic sinuses were narrowed or disappeared， and there were inflammatory infiltration and local bleeding， the levels of ALT and AST in serum and ROS in liver tissue were significantly increased（P<0.01）， and the expressions of Keap1， HO-1， NQO1， GSTA1， p62 mRNA and p-mTOR， p-p70S6K， p62 protein in liver tissue were significantly decreased（P<0.01）， and LC3Ⅱ/LC3Ⅰ was significantly increased（P<0.01）. Compared with the UEF group， the body mass of mice increased， and the liver body index， the levels of ALT and AST in serum， and the level of ROS in liver tissue all decreased in the groups of PEF and GEFs. Among these groups， only the liver lobules in GEF（100∶6） group were intact， and the size of liver sinuses was close to that in the control group. The mRNA expressions of Keap1， HO-1， NQO1， GSTA1 and p62 in liver tissue showed an overall upward trend in the groups of PEF and GEFs. Among these groups， only the ones of the above mRNA in the GEF（100∶6） group had a significant increase（P<0.05， P<0.01）. The protein expressions of p-mTOR， p-p70S6K， p62 and LC3Ⅱ/LC3Ⅰ had a callback in the groups of PEF and GEFs， of which the protein expressions of p-mTOR， p-p70S6K and LC3Ⅱ/LC3Ⅰ in the GEF（100∶6） group and the expression of p62 protein in the GEF（100∶24） group had the largest callback. Except for p-mTOR protein， other protein expressions were statistically significant（P<0.05， P<0.01）. ConclusionThe hepatotoxicity of EF is closely related to its ability to induce oxidative stress， which leads to pathological autophagy and hepatocyte damage. This ability can be reduced by the processing with different proportions of Gly， especially the ratio of 100∶6.

ObjectiveTo explore the effect of Babaodan （BBD） on the NOD-like receptor pyrin domain containing 3/cysteine aspartate-specific protease-3 （NLRP3/Caspase-1） pathway proteins in mice with acetaminophen （APAP）-induced acute liver injury. MethodC57BL/6 mice were randomly grouped， and BBD （75， 150， 300 mg·kg^-1， ig） was administered twice a day for three days. After 2 hours of the last administration， the mice were treated with APAP （400 mg·kg^-1， ip）， and the eyeballs were removed to collect blood after 14 hours. Then they were sacrificed by cervical dislocation for sample collection. Hematoxylin-eosin （HE） staining was used to observe the morphological changes of liver tissue cells， and biochemical methods were used to detect the activities of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， superoxide dismutase （SOD）， malondialdehyde （MDA） and myeloperoxidase （MPO） in serum of mice in each group. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was performed to determine the mRNA expression of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β） and IL-6， and Western blot was performed to determine the protein expression of cyclooxygenase-2 （COX-2）， inducible nitric oxide synthase （iNOS）， NLRP3， Caspase-1 and IL-18 in the liver of mice. ResultCompared with the conditions in normal group， the hepatic lobule structure of mice in the model group was partially destroyed， and the hepatic sinusoids were dilated. And the expression levels of ALT and AST in serum， the protein levels of NLRP3， Caspase-1， iNOS， IL-18 and COX-2 and the mRNA levels of IL-1β， IL-6 and TNF-α were increased （P<0.05， P<0.01）. Compared with the model group， the administration groups had improvement in liver cell rupture and hepatic sinusoidal compression， and a dose-dependent decrease in the levels of ALT and AST in serum as well as the protein levels of NLRP3， Caspase-1， iNOS， IL-18 and COX-2 and the the mRNA levels of IL-1β， IL-6 and TNF-α in liver tissue （P<0.05， P<0.01）. ConclusionBBD can reduce APAP-induced acute liver injury in mice. The mechanism may be related to anti-oxidative stress， inhibition of NLRP3/Caspase-1 pathway， and decreased expression levels of IL-1β， IL-18， TNF-α and IL-6.

OBJECTIVE To establish a method for concentration determination of caffeine and its three metabolites， theophylline， paraxanthine and theobromine in urine， and apply it in clinical practice. METHODS Using caffeine-13C3-d3 as internal standard （IS）， and the urine samples were protein precipitated with acetonitrile； HPLC-MS/MS method was adopted to determine the concentrations of caffeine and its three metabolites. The determination was performed on Waters ACQUITY UPLC® BEH HILIC column with mobile phase consisting of 60 mmol/L ammonium acetate （A）-acetonitrile （B） （gradient elution） at the flow rate of 0.5 mL/min. The column temperature was set at 38 ℃ ， and the sample size was 2 μL. The electrospray ionization detection was operated in a positive mode by multiple reaction monitoring. The detection ions for quantitative analysis were m/z 195.1→110.0 for caffeine， m/z 181.1→124.0 for theophylline， m/z 181.1→124.0 for paraxanthine， m/z 181.1→138.0 for theobromine， and m/z 198.1→ 140.1 for IS. The above method was used to determine the concentrations of caffeine and its three metabolites in the urine of 19 infants with apnea of prematurity （AOP）. RESULTS The linear ranges of mass concentration of caffeine， theophylline， paraxanthin and theobromine were 0.200-200， 0.050-50.0，0.050 0-50.0， and 0.100-100 μg/mL， respectively. The lower limits of quantification were 0.200， 0.050， 0.050 and 0.100 μg/mL （r＞0.990）， respectively. RSDs of intra-day and intra- day precision were not above 10.37%， and matrix factors were 85.68%-109.90%； extraction recoveries were 93.53%-109.40% （RSD≤15%）， and RSDs of stability tests were all lower than 15%. The concentrations of caffeine and its three metabolites in the urine of 19 cases were （27.346±7.951）， （0.351±0.223）， （0.428±0.395） and （0.472±0.374） μg/mL， respectively. CONCLUSIONS The established HPLC-MS/MS method is simple， sensitive and can be used for the determination of caffeine and its three metabolites in urine samples of AOP.