Characterizing the critical role of metabolism in osteosarcoma based on establishing novel molecular subtypes.

OBJECTIVE: Osteosarcoma is the third most frequently diagnosed cancer among adolescents. Immunotherapy is an effective curative treatment for metastatic osteosarcoma patients. This study aimed to further reveal the significance of metabolism in tumor progression, and to categorize molecular subtypes for guiding personalized therapy. MATERIALS AND METHODS: Univariate Cox regression analysis was performed to screen metabolism-related genes associated with osteosarcoma prognosis. A molecular subtyping system was developed by unsupervised consensus clustering. Survival analysis and functional analysis were used to evaluate the performance of subtyping and characterize the TME of subtypes. Stepwise Akaike information criterion (stepAIC) was employed to optimize the prognostic model. RESULTS: C1 and C2 subtypes showed distinct prognosis, with more favorable survival in C2 subtype. C2 subtype presented a higher immune infiltration and active anti-tumor response. Notably, C2 subtype was predicted to have better immune response to immune checkpoint blockade. In addition, a 5-gene prognostic signature with robust ability to classify patients into high-risk and low-risk groups was developed. CONCLUSIONS: The study revealed the critical role of metabolism in tumorigenesis by comparing the features between the two subtypes. Oncogenic pathways including epithelial mesenchymal transition (EMT), glycolysis and hypoxia may be closely involved in the correlation with metabolism. Importantly, we developed a novel subtyping system and a 5-gene signature with high potential to be applied in clinical practice.

Identification of amygdalin and its major metabolites in rat urine by LC-MS/MS.

Amygdalin and its metabolites in rat urine were identified using liquid chromatography-electrospray ionization (ESI) tandem ion-trap mass spectrometry. The purified rat urine sample was separated using a reversed-phase C18 column with 10 mM sodium phosphate buffer (pH 3.1) containing 30% methanol as the mobile phase, amygdalin and its metabolites were detected by on-line mass detector in selected ion monitoring (SIM) mode. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. At least seven metabolites and the parent drug were found in rat urine after i.v. injection of 100 mg/kg doses of amygdalin. Among them, six metabolites were reported for the first time.

Identification of dauricine and its metabolites in rat urine by liquid chromatography-tandem mass spectrometry.

A rapid, sensitive and specific liquid chromatographic-electrospray ionization (ESI) tandem ion trap mass spectrometric method has been developed for identification of dauricine and its metabolites in rat urine. Six healthy rats were administrated a single dose (100 mg/kg) of dauricine by oral gavage. The urine were sampled from 0 to 24 h and purified by using a C18 solid-phase extraction (SPE) cartridge, then the purified urine samples were separated on a reversed-phase C18 column using methanol/2 mmol/L ammonium acetate (70:30, v/v, adjusted to pH 3.5 with formic acid) as mobile phase and detected by an on-line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular mass (Deltam) and full scan MS(n) spectra with those of the parent drug. At least eight metabolites (such as N-demethyl, dehydrogenate, demethoxyl, hydroxyl, glucuronide conjugated and sulfate conjugated metabolites) and the parent drug were found in rat urine.

[Separation and determination of baicalin in prescriptions containing Scutellaria baicalensis Georgi by HPCE].

Qingkailing Oral Tonic and Shuanghuanglian Oral Tonic are two kinds of complex formlae of Chinese traditional patent medicine, and both contain Scutellaria baicalensis Georgi. Baicalin is referred to the main active component with functions of dispelling wind-heat to reduce fever and detoxicating, and takes effect on uppering respiratory tract infection and tonsillities. In this work a high performance capillary electrophoresis (HPCE) method was established for separation and quantitative analysis of baicalin in prescriptions containing Scutellaria baicalensis Georgi, based on the mode of capillary zone electrophoresis. Electrophoretic conditions are as follows: uncoated fused silica capillary (34.8/39.5 cm x 50 microns i.d.), 40 mmol/L borax buffer(pH 9.0), applied voltage 17 kV, polarity anode (inlet) to cathode (outlet), temperature 25 degrees C, detection wavelength 285 nm, injection 68.95 kPa x s. Under the optimum conditions, baicalin was separated successfully from other components within 15 minutes. The relative peak area of baicalin increased linearly with the increase of its concentration in the range of 10-640 mg/L. The recoveries were (100.31 +/- 1.98)% for Qingkailing Oral Tonic and (100.60 +/- 2.36)% for Shuanghuanglian Oral Tonic. The analytical results demonstrate the method is simple, rapid and well reproducible, and can be used as a reliable tool for the quality control of Chinese traditional medicines containing Scutellaria baicalensis Georgi.

[Separation and determination of glibenclamide in xiaotangling tablets by micellar electrokinetic capillary chromatography].

A high performance capillary electrophoresis method has been developed for separation and quantitative analysis of glibenclamide in Xiaotangling tablets. Electrophoretic conditions were as follows: micellar electrokinetic capillary chromatography (MECC) mode, trimethoprimum as internal standard, uncoated fused silica capillary (34.8 cm/39.5 cm(effective/total length), 50 microns i.d.), 25 mmol/L borax-30 mmol/L SDS(pH 9.0), applied voltage 17 kV, (+)-->(-), temperature 28 degrees C, detection wavelength 228 nm, pressure injection 68.95 kPa.s. Glibenclamide was separated successfully from other components within 14 minutes under the optimum conditions, and the relative peak area of glibenclamide increased linearly with the increase of its concentration within the range of 25 mg/L-275 mg/L. The recovery was (100.6 +/- 1.4)%. The method is simple, rapid and well reproducible, and can be used as a reliable tool for the quality control of Chinese traditional medicine containing glibenclamide.

[Determination of baicalin and chlorogenic acid in yinhuang granule by capillary electrophoresis].

Yinhuang Granule is a kind of complex formulae made up of extract of Flos lonicerae and that of Radix scutellariae, of the fuctions of dispelling wind-heat to reduce fever, detoxicating, relieving inflammation, promoting urination and tranquilizimg the mind and can be applied to upper respiratory tract infection and tonsillitis. Baicalin and chlorogenic acid are referred to main effective components of it. In this paper, the separation and determination of baicalin and chlorogenic acid in Yinhuang Granule by capillary electrophoresis is described. The experimental conditions are stated as follow: separation column: bare fused-sillica capillary (50 microns i.d., 370 microns o.d., total length 47 cm, 40 cm from the inlet to UV detector); electrolyte: 25 mmol/L borax, pH 8.5; detection wavelength: 310 nm; sampling: 17 kPa.s; running voltage: 25 kV, polarity from anode (inlet) to cathode (outlelt); temperature: 25 degrees C; internal standard: p-nitrobenzoic acid. The linear ranges of determination for baicalin and chlorogenic acid were 160-960 mg/L(r = 0.9993, RSD = 1.76%-2.33%) and 80-960 mg/L(r = 0.9989, RSD = 1.07%-2.51%), respectively. The recoveries of baicalin and chlorogenic acid were (102.09 + 1.74)% (RSD = 1.71%, n = 6) and (99.81 + 3.11)% (RSD = 3.12%, n = 6). The method is simple, fast and accurate. It can be used for the quality control of Yinhuang Granule and its related Chinese medicines.

Separation of five antipyretic analgesics by micellar electrokinetic capillary chromatography.

The derivatives of antipyrine and phenylbutazone are important antipyretic analgesics commonly used in clinical medicine. Although high performance liquid chromatography has been the conventional method used for the analysis of these drugs, in recent years capillary electrophoresis was validated to be a useful method in the analysis of antipyretic analgesics. However, there has been no report on the separation of antipyrine (AP), 4-aminoantipyrine (4-AAP), aminopyrine (APY), dipyrone (DIP) and phenylbutazone (PHE) in the literature. In this paper, a micellar electrokinetic capillary chromatographic (MECC) separation method was described for the five antipyretic analgesics.