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1.
Article in Chinese | WPRIM | ID: wpr-920655

ABSTRACT

@#An innovative approach to quantitatively analyze the histamine and its precursor histidine simultaneously in biological matrices was established for the first time based on double adsorption combined with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).The internal standard was 2-dihydroxybenzoic acid (DHB).The plasma and brain tissue homogenate was protein precipitated with 3-fold acetonitrile, and the supernatant was then sampled for injection analysis.The chromatographic separation of the target components was achieved on an amino chromatography column (ODS-SPXBridge? Amide).Gradient elution was carried out with the mobile phase consisting of solvent A (0.1% formic acid and 1mmol/L ammonium formate in water) and solvent B (acetonitrile).Mass spectrometry was employed for quantitative analysis with ESI ion source in multiple reaction monitoring (MRM) mode.In order to improve the specificity and accuracy, activated carbon and calcite were used for the double adsorption of biological matrices for the first time.The adsorbed matrix was then used for methodology validation.The results showed that histamine and histidine were linear in the quantitative range (correlation coefficient r ≥ 0.999).Accuracy, precision, extraction recovery, matrix effect and stability all met the requirements of biological sample analysis.All results suggested that the present method could not only be efficiently and reliably used for simultaneous quantitative analysis of histamine and histidine in biological samples, but also provide reference for the detection of other endogenous substances.

2.
Article in Chinese | WPRIM | ID: wpr-935782

ABSTRACT

Objective: To establish an ultrahigh performance liquid chromatography tandem mass spectrometry method for the determination of creatinine (Cre) and 2-thiothiazolidine-4-carboxylic acid (TTCA) in urine. Methods: In October 2020, the end-of-shift urine samples of the monitored subjects were taken, and the filtrate was prepared by centrifugation. After separated by ultra high performance liquid chromatography C18 column, acetonitrile and 0.2% acetic acid aqueous solution were used as mobile phases for gradient elution, the three quadrupole tandem mass spectrometry adopted an electrospray ion source (ESI) , the ion source temperature was 500 ℃ , and the air curtain gas flow rate was 31.4 L/min, qualitative and quantitative analysis of Cre and TTCA were carried out under the multiple reaction monitoring mode. Results: The linear range of Cre was 1.0-1 000.0 μg/L, the linear equation was y=947.3x-1605.6, and the correlation coefficient was 0.9994. The detection limit and the limit of quantitation were 0.3, 1.0 μg/L. When the addition concentrations were 50.0, 150.0 and 450.0 μg/L, the recovery rates were 92.8%-94.6% , the intra assay precisions were 3.6%-5.7% , and the inter assay precisions were 3.4%-5.4%. The linear range of TTCA was 0.1-200.0 μg/L, the linear equation was y=1164.7x-2243.9, and the correlation coefficient was 0.9991. The detection limit and the limit of quantitation were 0.03, 0.1 μg/L. When the addition concentrations were 10.0, 40.0 and 160.0 μg/L, the recovery rates were 90.8%-93.6%, the intra assay precisions were 4.6%-7.4%, and the inter assay precisions were 4.4%-6.9%. Conclusion: The sample pretreatment process of the ultra high performance liquid chromatography tandem mass spectrometry method for the determination of Cre and TTCA in urine is simple, and the continuous determination of Cre and TTCA in urine can be realized only by switching mass spectrometry parameters under the same chromatographic conditions, which is accurate and efficient, and each performance index of the method meets the determination requirements.


Subject(s)
Chromatography, High Pressure Liquid , Creatinine , Humans , Tandem Mass Spectrometry , Thiazolidines
3.
Article in Chinese | WPRIM | ID: wpr-935780

ABSTRACT

Objective: To establish a method for rapid determination of bongkrekic acid (BA) in plasma by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Methods: In November 2020, plasma samples were extracted by methanol and acetonitrile (1∶1) and purified directly. The samples were separated by C18 column. Gradient elution was carried out with 5 mmol/L ammonium acetate water acetonitrile solution as mobile phase. Under the optimized instrument conditions, the electrospray ionization multiple reaction monitoring (MRM) mode was used, and the external standard method was used for quantitative analysis. Results: The linear relationship of BA in plasma was good in the concentration range of 2-100 μg/L, the correlation coefficient was 0.9998, the average recovery was 83.7%-112.0%, the relative standard deviation within and between batches was less than 10%, the detection limit of the method was 0.7 μg/L and the lower limit of quantification was 2.0 μg/L. Conclusion: The method is simple, rapid, accurate and sensitive, and can meet the requirements for the determination of BA in blood samples of poisoning patients.


Subject(s)
Bongkrekic Acid , Chromatography, High Pressure Liquid , Humans , Solid Phase Extraction , Tandem Mass Spectrometry
4.
Article in Chinese | WPRIM | ID: wpr-935746

ABSTRACT

Objective: To establish a method for the determination of methyl isobutyl ketone (MIBK) in urine samples by headspace-gas chromatography-mass spectrometry. Methods: Automatic headspace sampling technique was adopted to optimize the headspace conditions (headspace bottle heating temperature and equilibration time) and gas chromatographic conditions. A total of 5 ml samples were taken and added with 3.0 g ammonium sulfate into a 20 ml headspace bottle. After heated at 60 ℃ for 30 mins, gas from the upper part of headspace bottle was injected into gas chromatography with an injection volume of 100 μl. The target was separated by HP-5MS UI (30 m×0.25 mm×0.25 μm) capillary column and then detected by mass spectrometry detector. The retention time and external standard method were used for qualitative and quantitative analysis of MIBK in samples, respectively. Results: The standard curve of MIBK showed significant linearity between 20.0-1 000.0 μg/L. The standard curve was y=62.9x-652.5, and the correlation coefficient r=0.9998. The detection limit of MIBK was 5.0 μg/L and the quantification limit of MIBK was 16.0 μg/L. The average recovery rate was 95.3%~100.2% at three spiked concentrations of low (50.0 μg/L) , medium (200.0 μg/L) and high (500.0 μg/L) . The intra-day and inter-day precisions were 1.7%~3.8% (n=6) and 1.2%~4.0% (n=6) respectively. This method was stable for the determination of MIBK, and the urine could be kept 14 d at -20 ℃ without significantly loss. Conclusion: This method is proved to be simple, practical and highly sensitive. It can satisfy the request for the determination of urine samples of workers exposed to MIBK.


Subject(s)
Gas Chromatography-Mass Spectrometry , Humans , Methyl n-Butyl Ketone
5.
Article in Chinese | WPRIM | ID: wpr-934398

ABSTRACT

Objectives:To establish a candidate reference measurement procedure based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for cyclosporin A, tacrolimus, sirolimus, and everolimus measurements in human whole blood.Methods:The isotope labeled cyclosporine A, tacrolimus, sirolimus, and everolimus were selected as the internal standards. Samples were accurately weighed while protein precipitation and solid phase extraction were selected for the sample preparation. The standard curve method was applied for quantification. The ultra-high liquid chromatography coupled with triple quadrupole mass spectrometer was used for analysis. The specificity, matrix effect, detection limit, quantification limit, precision, accuracy, and uncertainty of the method were evaluated.Results:The method showed good selectivity and specificity. No apparent interferences or matrix effects were found in the target analyte measurements. The detection limits and quantification limits of cyclosporin A, tacrolimus, sirolimus and everolimus met clinical requirements. Intra-batch coefficients of variation ( CV) were from 1.4% to 1.8% for CSA, from 1.7% to 2.8% for TAC, from 1.3% to 3.7% for SRL and from 2.3% to 3.2% for EVR, and total CVs were from 1.8% to 2.9% for CSA, from 1.7% to 3.8% for TAC, from 2.6% to 4.7% for SRL and from 3.5% to 4.6% for EVR. The relative recoveries were from 97.9% to 100.3% for CSA, from 98.4% to 103.1% for TAC, from 99.4% to 102.0% for SRL and from 98.3% to 99.4% for EVR, and the relative expanded uncertainties at four concentrations were from 4.2% to 4.4% for CSA, from 1.5% to 2.4% for TAC, from 4.4% to 4.9% for SRL and from 2.2% to 2.7% for EVR. Conclusion:A candidate reference measurement procedure for the cyclosporine A, tacrolimus, sirolimus, and everolimus in human whole blood was established by ID-LC-MS/MS.

6.
Article in Chinese | WPRIM | ID: wpr-934397

ABSTRACT

Objective:To analyze the serum and urinary amino acid (AA) profiles of urolithiasis patients to explore the potential biomarkers for clinical screening and early diagnosis.Methods:Case-control study. Serum and urine samples were collected from 74 urolithiasis patients (aged 20-82 years, 41 men, 33 female) in the department of urology of the First Affiliated Hospital of Fujian Medical University and 35 healthy controls (HC, aged 22-80 years old, 20 men, 15 female) from the health examination center from February 2015 to October 2017. Serum and urinary AA levels of patients and HC were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) based metabolomic strategy. The multivariate statistical analysis methods of principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA) were employed for modeling. The variable importance projection (VIP) value of OPLS-DA model>1 and P<0.05 of t test were selected to screen the differential amino acid metabolites. The diagnostic capabilities of potential markers were evaluated by receiver operating characteristic (ROC) curve and binary logistic regression analysis. Results:Five AA metabolites including serine, glutamate, aspartic acid, isoleucine and glycine were found, which had statistically significant differences between the patient group and the control group ( P<0.05) and were associated with seven metabolic pathways. Serum serine, glutamate, aspartic acid, isoleucine and urine glycine and aspartic acid were combined into an integrated marker panel whose AUC value was 0.890, the sensitivity was 78.0%, and the specificity was 96.4%. Conclusion:Five amino acids in serum and urine could be used as an integrated biomarker panel for the clinical screening and early diagnosis of urolithiasis, which could provide some experimental basis for molecular urolithiasis research.

7.
Article in Chinese | WPRIM | ID: wpr-934396

ABSTRACT

Objective:To establish a candidate reference method for serum progesterone using isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) in our laboratory, validate the analytic performance of five clinical routine detection systems to explore the comparability of serum progesterone detection by different detection systems.Methods:A candidate reference method for serum progesterone using ID-LC/MS/MS method was established. The sample was pretreated by liquid-liquid extraction method, and the reversed phase liquid phase separation in positive ion mass spectrometry mode was used to detect progesterone in human serum, and the detection time of a single sample was controlled within 5 minutes by gradient elution. In order to improve the accuracy of the method, the bracketing calibration method (BCM) was used to establish the standard curve. The sensitivity, accuracy, precision and specificity of BCM and classical calibration curve method were evaluated according to CLSI C62-A, EP15-A2, EP6-A2 and EP9-A3, and the analytical performance and comparability of five clinical routine progesterone detection systems were evaluated,compared with ID-LC/MS/MS method, the bias at medical decision level 2 and 25 ng/ml was evaluated to see if they were <1/2TEa (12.5%).Results:The limit of detection (LOD) of ID-LC/MS/MS was 0.005 ng/ml. The recoveries of BCM method and classical calibration curve method are 97.95%-101.58% and 96.88%-110.70%, respectively. The measurement results of BCM method for certified reference materials are within its declared uncertainty range. The intra-and inter-assay coefficient of variation ( CV) of BCM method was less than 3.0%, which was better than that of classical calibration curve method ( CV: 2.48%-9.33%). The precision and linear range of the five clinical routine detection systems can meet the detection requirements. The measurement bias of detection system 1, 3 and 5 at 25 ng/ml of medical decision level was less than 1/2TEa, and the measurement bias at 2 ng/ml of medical decision level was more than 1/2TEa. The measurement bias of detection system 2 and 4 at two medical decision levels was less than 1/2TEa. Conclusion:The candidate reference method for serum progesterone ID-LC/MS/MS established in our laboratory meets the requirements of the reference method. BCM has better detection performance than classical calibration curve method. The precision and linearity of the five progesterone clinical detection systems are satisfactory. The five clinical detection systems could meet the clinical requirements at the medical determination level of 25 ng/ml, however, only two of the five clinical detection systems meet the clinical requirements at the medical determination level of 2 ng/ml.

8.
Article in Chinese | WPRIM | ID: wpr-934395

ABSTRACT

Objective:To determine the analytical performance of a candidate reference measurement procedure for 17α-hydroxyprogesterone in human serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS).Methods:The serum spiked with a deuterium-labeled internal standard was extracted from serum from individual undergoing physical examination by liquid-liquid extraction with n-hexane/ethyl acetate (3∶2, v/v), separated by C18 reversed-phase chromatography and detected by positive electrospray ionization mass spectrometry. According to the Clinical and Laboratory Standards Institute (CLSI) C62-A documents, the analytical performance including linearity, limit of detection,limit of quantitation,relative matrix effect,precision and trueness,carry-over and specificity was evaluated.Results:The linear range of 17α-hydroxyprogesterone by LC-MS/MS was 0.21-119.67 μg/L. The limit of detection and limit of quantitation were 5.218 ng/L and 0.116 μg/L. The relative matrix effects were -0.02%, -0.40% and -0.90% for sera and solution mixtures in 3 different ratios (50∶50, 80:20 and 20∶80). The coefficients of variation ( CVs) of intra-assay were 1.73%-2.11%, 0.98%-1.71%, 0.47%-0.87% at 0.164 μg/L, 14.81 μg/L, 81.63 μg/L and the CVs of inter-assay were 1.82%, 1.03%, 0.80% at above three concentrations. The average recovery rates of 3 levels (0.5, 20 and 100 μg/L) were 100.4%, 101.7%, 102.8%, respectively. The measured values of GBW09829 of National Institute of Metrology were within the specified uncertainty range. Conclusion:The candidate reference measurement procedure for 17α-hydroxyprogesterone in human by LC-MS/MS is established with good accuracy and precision, which can be clinically used for measurement traceability.

9.
Article in Chinese | WPRIM | ID: wpr-934375

ABSTRACT

Changes in protein glycosylation modification have been found to be closely related to cancer and other diseases. Profiling glycosylation change has become an effective way to explore the diagnosis and treatment markers. The accuracy of quantitative technology is the key to reveal the mystery of glycosylation, and the screening and validation of glycosylation disease markers is dependant on the study of large clinical samples. Therefore, glycomic technology are expected to be simple, rapid, high-throughput, accurate and practical. In this paper, the characteristics of some commonly used glycomic analysis techniques and their applications in glycan markers are briefly discussed, and the characteristics, progress and applications of high-throughput precision glycome quantitative methods based on MALDI MS are emphasized.

10.
Article in Chinese | WPRIM | ID: wpr-934368

ABSTRACT

Objective:This study explored the consistency of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay for the detection of steroid hormones. The diagnostic value of multiple steroid hormones in 21-hydroxylase deficiency (21-OHD) was investigated and the follow-up indicators were screened.Methods:This experimental group included 109 patients with typical 21-OHD who received standard treatment, and the control group included 94 normal children. 17-hydroxyprogesterone (17-OHP), androstenedione (Δ4-A), testosterone and cortisol were detected by immunoassay and LC-MS/MS method. At the same time, 16 other adrenal steroids were detected by LC-MS/MS method. The experimental group was divided into: (1) overtreatment group: 17OHP<4 ng/ml; (2) well controlled group: 4 ng/ml≤17-OHP<12 ng/ml; (3) poorly controlled group: 17-OHP≥12 ng/ml. The following studies were carried out. (1) The consistency of immunoassay and LC-MS/MS detection results was analyzed; (2) The serum concentrations of various steroid hormones in patients with 21-OHD and the control group were compared to explore the diagnostic value of multiple steroid hormones detection; (3) The concentration differences of 20 kinds of steroid hormones in 21-OHD patients with different therapeutic effects were compared to screen more valuable follow-up indicators.Results:(1) among the four indicators detected by LC-MS/MS and immunoassay, the consistency of T and 17-OHP was high. The concentrations of cortisol and Δ4-A determined by immunoassay were higher than those determined by LC-MS/MS. (2) Among the 20 kinds of steroid hormones secreted by adrenal gland detected by LC-MS/MS, 6 kinds of hormones were significantly higher and 6 kinds of hormones were significantly lower in 21-OHD patients compared with the control group, ,and 8 kinds of steroids showed no statistical difference. (3) 17-OHP decreased and 11-deoxycortisol increased in over-inhibition group, while 17-OHP, pregnenolone, progesterone, 17-hydroxypregnenolone, 21-deoxycortisol, Δ4-A and estrone increased in the poorly controlled group.Conclusions:LC-MS/MS can detect many kinds of steroid hormones at one time with better evaluate dimensions. During the follow-up, only 8 of the 20 hormones were closely related to the control status of patients, suggesting that unnecessary testing work could be reduced.

11.
Article in Chinese | WPRIM | ID: wpr-934359

ABSTRACT

Objective:To screen out the differentially regulated metabolites by the analysis of serum metabolic fingerprints, and to provide potential biomarkers for diagnosis of lung cancer.Methods:A total of 228 subjects were enrolled in Changhai Hospital from January 27, 2021 to June 4, 2021, including 97 newly diagnosed lung cancer patients and 131 healthy individuals. Serum samples were collected from the enrolled cohort according to a standard procedure, and the enrolled cohort was divided into a training set and a completely independent validation set by stratified random sampling. The metabolic fingerprints of serum samples were collected by previously developed nano-assisted laser desorption/ionization mass spectrometry (nano-LDI MS). After age and gender matching of the training set, a diagnostic model based on serum metabolic fingerprints was established by machine learning algorithm, and the classification performance of the model was evaluated by receiver operating characteristic (ROC) curve.Results:Serum metabolic fingerprint for each sample was obtained in 1 minute using a novel nano-LDI MS, with consumption of only 1 μl original serum sample. For the training set, the area under ROC curve (AUC) of the constructed classifier for diagnosis of lung cancer was 0.92 (95% CI 0.87-0.97), with a sensitivity of 89% and specificity of 89%. For the independent validation set, the AUC reached 0.96 (95% CI 0.90-1.00) with a sensitivity of 91% and specificity of 94%, which showed no significant decrease compared to training set. We also identified a biomarker panel of 5 metabolites, demonstrating a unique metabolic fingerprint of lung cancer patients. Conclusion:Serum metabolic fingerprints and machine learning were combined to establish a diagnostic model, which can be used to distinguish between lung cancer patients and healthy controls. This work sheds lights on the rapid metabolic analysis for clinical application towards in vitro diagnosis.

12.
Article in Chinese | WPRIM | ID: wpr-934333

ABSTRACT

Objective:To screen the potential biomarkers for the diagnosis and differential diagnosis of immune-mediated demyelinating diseases by tandem mass tags (TMT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology.Methods:Twenty patients with demyelinating diseases (demyelinating group) and 10 patients with noninflammatory neurological diseases (NND group) from Beijing Tiantan Hospital affiliated to Capital Medical University from January 2020 to January 2021 were enrolled in this study. The demyelinating group included 10 patients with Guillain-Barre syndrome (GBS subgroup) and 10 patients with multiple sclerosis (MS subgroup). TMT proteomics was used to screen out the different protein expression patterns between the demyelinating group and the NND group and between the GBS subgroup and the MS subgroup (difference>2 or<0.5 and with statistical significance), and String database was used to perform gene ontology (GO) analysis and Kyoto encyclopedia of gene and genomes (KEGG) analysis on the pathways involved in the differently expressed proteins between the groups. In addition, 80 demyelinating patients (demyelinating diseases validation group) and 40 healthy subjects (healthy control group) were selected for retrospective analysis of general lipid indexes. The demyelinating diseases validation group included 40 GBS patients (GBS validation group) and 40 MS patients (MS validation group). Receiver operating characteristic (ROC) curve was obtained to evaluate the value of general lipid indexes for the diagnosis of demyelinating diseases and the differential diagnosis between GBS and MS groups.Results:A total of 362 proteins were detected by TMT proteomics. There were 101 differentially expressed proteins between the demyelinating group and the NND group, and 45 differentially expressed proteins between the GBS group and the MS group. Compared with the NND group, GO enrichment analysis showed that the top five enrichment pathways in the demyelinating group were macrophage colony stimulating factor and receptor complex, negative regulation of cholesterol input, negative regulation of very low density lipoprotein particle clearance, triglyceride-rich lipoprotein particle remodeling, and cholesterol reverse transport. Compared with MS group, the top five enriched pathways in GBS group were high-density lipoprotein particle receptor binding, negative regulation of very low density lipoprotein particle remodeling, negative regulation of cholesterol input, negative regulation of very low density lipoprotein particle clearance, and medium density lipoprotein particle. KEGG enrichment analysis results showed that differentially expressed proteins in the demyelinating group and the NND group were enriched in 8 pathways, including phosphatidylinositide 3-kinases-protein kinase B signaling pathway, complement and coagulation cascade reaction, extracellular matrix and its receptor interaction, Staphylococcus aureus infection, cholesterol metabolism, RAS signaling pathway, phagosome, and mitogen-activated protein kinase signaling pathway. Differentially expressed proteins in GBS group and MS group were enriched in 9 pathways: cholesterol metabolism, complement and coagulation cascade, platelet activation, peroxisome proliferators-activated receptors signaling pathway, vitamin digestion and absorption, novel coronavirus infection, fat digestion and absorption, axon guidance, and neutrophil extracellular trap formation pathway. The levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and apolipoprotein B (apoB) were significantly higher, while high density lipoprotein cholesterol (HDL-C) and apolipoprotein A1 (apoA1) levels were significantly lower in the demyelinating disease validation group than in the healthy control group (all P<0.05 or 0.01). Area under the curve (AUC) of TG, TC, HDL-C, LDL-C, apoA1 and apoB alone or in combination for the diagnosis of immune-mediated demyelinating diseases was 0.746, 0.643, 0.798, 0.703, 0.806, 0.708 and 0.868, respectively. The AUC of HDL-C, apoA1, LDL-C and apoB for differential diagnosis between GBS and MS was 0.692, 0.653, 0.632, 0.695 and 0.718, respectively. Conclusions:There are differences in cerebrospinal fluid proteomics between patients with immune-mediated demyelinating disease and patients with NND, GBS and MS, and the differentially expressed protein patterns mainly exist in the pathways related to lipid metabolism. Lipid related indicators may be used as biomarkers for the diagnosis and differential diagnosis of immune-mediated demyelinating disease.

13.
Article in Chinese | WPRIM | ID: wpr-933379

ABSTRACT

Objective:To analyze clinical characteristics of 17α-hydroxylase deficiency, and to facilitate the understanding and management of the disease.Methods:A retrospective analysis of the clinical characteristics and biochemical results of 5 cases with 17α-hydroxylase deficiency diagnosed and treated from 2018 to 2020.Results:All 5 patients were female as social gender, and reached adulthood upon first clinic visit to our department and got diagnosed. All 5 cases had hypertension, hypokalemia, bilateral adrenal hyperplasia or adenoma, osteoporosis, and typical hormone changes related to steroid synthesis.Conclusion:Steroid hormone tests with liquid chromatography tandem mass spectrometry(LC-MS/MS) enable early diagnosis of 17α-hydroxylase deficiency, assessment of the type and degree of enzyme deficiency, and choice of treatment. For such patients, it is necessary to give appropriate anti-osteoporosis therapy.

14.
Article in Chinese | WPRIM | ID: wpr-931257

ABSTRACT

A rapid and sensitive method for analyzing trace β-blockers in complex biological samples,which involved magnetic solid-phase extraction(MSPE)coupled with Fourier transform ion cyclotron reso-nance mass spectrometry(FTICR-MS),was developed.Novel nanosilver-functionalized magnetic nano-particles with an interlayer of poly(3,4-dihydroxyphenylalanine)(polyDOPA@Ag-MNPs)were synthesized and used as MSPE adsorbents to extract trace β-blockers from biological samples.After extraction,the analytes loaded on the polyDOPA@Ag-MNPs were desorbed using an organic solvent and analyzed by FTICR-MS.The method was rapid and sensitive,with a total detection procedure of less than 10 min as well as limits of detection and quantification in the ranges of 3.5-6.8 pg/mL and 11.7-22.8 pg/mL,respectively.The accuracy of the method was also desirable,with recoveries ranging from 80.9%to 91.0%following the detection of analytes in human blood samples.All the experimental results demonstrated that the developed MSPE-FTICR-MS method was suitable for the rapid and sensitive analysis of trace β-blockers in complex biological samples.

15.
Article in Chinese | WPRIM | ID: wpr-931256

ABSTRACT

Docosanol is the only US Food and Drug Administration(FDA)approved over-the-counter topical product for treating recurrent oral-facial herpes simplex labialis.Validated analytical methods for docosanol are required to demonstrate the bioequivalence of docosanol topical products.A gas chromatography/selected ion monitoring mode mass spectrometry(GC/SIM-MS)method was developed and validated for docosanol determination in biological samples.Docosanol and isopropyl palmitate(internal standard)were separated on a high-polarity GC capillary column with(88%cyanopropy)aryl-polysiloxane employed as the stationary phase.The ions of m/z 83 and 256 were selected to monitor docosanol and isopropyl palmitate,respectively;the total run time was 20 min.The GC/SIM-MS method was validated in accordance with US FDA guidelines,and the results met the US FDA acceptance criteria.The docosanol calibration standards were linear in the 100-10000 ng/mL concentration range(R2>0.994).The recoveries for docosanol from the receptor fluid and skin homogenates were>93.2%and>95.8%,respectively.The validated method was successfully applied to analyze ex vivo human cadaver skin permeation samples.On applying Abreva?cream tube and Abreva?cream pump,the amount of doco-sanol that penetrated human cadaver skin at 48 h was 21.5±7.01 and 24.0±6.95 ng/mg,respectively.Accordingly,we concluded that the validated GC/SIM-MS was sensitive,specific,and suitable for quantifying docosanol as a quality control tool.This method can be used for routine analysis as a cost-effective alternative to other techniques.

16.
Article in Chinese | WPRIM | ID: wpr-931244

ABSTRACT

Diagnosing Alzheimer's disease(AD)in the early stage is challenging.Informative biomarkers can be of great value for population-based screening.Metabolomics studies have been used to find potential biomarkers,but commonly used tissue sources can be difficult to obtain.The objective of this study was to determine the potential utility of erythrocyte metabolite profiles in screening for AD.Unlike some commonly-used sources such as cerebrospinal fluid and brain tissue,erythrocytes are plentiful and easily accessed.Moreover,erythrocytes are metabolically active,a feature that distinguishes this sample source from other bodily fluids like plasma and urine.In this preliminary pilot study,the erythrocyte metab-olomes of 10 histopathologically confirmed AD patients and 10 patients without AD(control(CTRL))were compared.Whole blood was collected post-mortem and erythrocytes were analyzed using ultra-performance liquid chromatography tandem mass spectrometry.Over 750 metabolites were identified in AD and CTRL erythrocytes.Seven were increased in AD while 24 were decreased(P<0.05).The ma-jority of the metabolites increased in AD were associated with amino acid metabolism and all of the decreased metabolites were associated with lipid metabolism.Prominent among the potential bio-markers were 10 sphingolipid or sphingolipid-related species that were consistently decreased in AD patients.Sphingolipids have been previously implicated in AD and other neurological conditions.Furthermore,previous studies have shown that erythrocyte sphingolipid concentrations vary widely in normal,healthy adults.Together,these observations suggest that certain erythrocyte lipid phenotypes could be markers of risk for development of AD.

17.
Article in Chinese | WPRIM | ID: wpr-931242

ABSTRACT

Posttranslational modifications of antibody products affect their stability,charge distribution,and drug activity and are thus a critical quality attribute.The comprehensive mapping of antibody modifications and different charge isomers(CIs)is of utmost importance,but is challenging.We intended to quanti-tatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity.The CIs of antibodies were fraction-ated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection,followed by stepwise structural characterization at three levels.First,the differences between CIs were explored at the intact protein level using a top-down mass spectrometry approach;this showed differences in glycoforms and deamidation status.Second,at the peptide level,common modifications of oxidation,deamidation,and glycosylation were identified.Peptide mapping showed nonuniform deamidation and glycoform distribution among CIs.In total,10 N-glycoforms were detected by peptide mapping.Finally,an in-depth analysis of glycan variants of CIs was performed through the detection of enriched glycopeptides.Qualitative and quantitative analyses demonstrated the dynamics of 24 N-glycoforms.The results revealed that sialic acid modification is a critical factor ac-counting for charge heterogeneity,which is otherwise missed in peptide mapping and intact molecular weight analyses.This study demonstrated the importance of the comprehensive analyses of antibody CIs and provides a reference method for the quality control of biopharmaceutical analysis.

18.
Article in Chinese | WPRIM | ID: wpr-931233

ABSTRACT

Endogenous ribonucleotides(RNs)and deoxyribonucleotides(dRNs)are important metabolites related to the pathogenesis of many diseases.In light of their physiological and pathological significances,a novel and sensitive pre-column derivatization method with N-(t-butyldimethylsilyl)-N-methyltri-fluoroacetamide(MTBSTFA)was developed to determine RNs and dRNs in human cells using high-performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS).A one-step extraction of cells with 85%methanol followed by a simple derivatization reaction within 5 min at room temper-ature contributed to shortened analysis time.The derivatives of 22 nucleoside mono-,di-and tri-phosphates were retained on the typical Cig column and eluted by ammonium acetate and acetonitrile in 9 min.Under these optimal conditions,good linearity was achieved in the tested calibration ranges.The lower limit of quantitation(LLOQ)was determined to be 0.1-0.4 μM for the tested RNs and 0.001-0.1 μM for dRNs.In addition,the precision(CV)was<15%and the RSD of stability was lower than 10.4%.Furthermore,this method was applied to quantify the endogenous nucleotides in human colorectal carcinoma cell lines HCT116 exposed to 10-hydroxycamptothecin.In conclusion,our method has proven to be simple,rapid,sensitive,and reliable.It may be used for specific expanded studies on intracellular pharmacology in vitro.

19.
Article in Chinese | WPRIM | ID: wpr-930996

ABSTRACT

Objective:To study the incidence, clinical features and genetic mutation profiles of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) using screening strategy.Methods:From September 2015 to September 2020, neonates in Xuzhou area were prospectively screened for genetic metabolic diseases using tandem mass spectrometry. Suspected infants were further confirmed using urinary organic acid test and SLC25A13 gene mutation analysis. The clinical manifestations, biochemical and gene mutation results, treatment and prognosis of the confirmed cases were analyzed.Results:A total of 468,494 live-birth newborns were screened with 112 cases suspected and 95 cases received urinary organic acid test and SLC25A13 gene mutation analysis. 13 cases of NICCD were diagnosed with a prevalence of 1/36,038. Most confirmed cases presented with delayed disappearance of neonatal jaundice, feeding difficulties and poor weight gain. Biochemical changes included increased bile acid, abnormal liver enzymes, increased alpha-fetoprotein, hypoglycemia, decreased hemoglobin, abnormal coagulation function and increased blood ammonia. Tandem mass spectrometry showed increased citrulline, methionine, arginine, tyrosine and phenylalanine, and in some cases with slightly increased acylcarnitine. Urine organic acid analysis mainly showed increased 4-hydroxyphenyllactic acid and 4-hydroxyphenylpyruvate. All confirmed cases received genetic mutation tests and a total of 13 mutation loci were detected, including c.852_855delTATG, c.511dupG, c.1638_1660dup, IVS16ins3kb, c.1078C>T, c. 615+5G>A, c.742G>A, c.44G>A, c.1311+1G>A, c.1399C>T, c.889G>T, c.1177+1G>A, c.1841+3_1841+4del, among which, c.852_855delTATG was the most common one. A total of 5 novel mutation loci were discovered in this study with c.1841+3_1841+4del, c.511dupG and c.889G>T predicted as pathogenic variants. Special formula of lactose-free and fortified medium-chain triglyceride (MCT) were used in confirmed cases and most of the symptoms were relieved within 1 year and abnormal indicators significantly improved.Conclusions:The prevalence of NICCD in Xuzhou was 1/36,038. c.852_855delTATG mutation is the most frequent one. Five novel mutation loci are discovered, expanding the SLC25A13 gene mutation spectrum. Most infants with NICCD have a good prognosis, requiring early diagnosis, treatment and life-long follow-up.

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Article in Chinese | WPRIM | ID: wpr-930436

ABSTRACT

Objective:To detective the cut-off values of amino acid levels in premature infants in Sichuan.Methods:Data of newborns screening for inherited metabolic diseases (IMD) by tandem mass spectrometry in Sichuan Province from January 2018 to December 2019 were retrospectively analyzed.They were divided into premature infant group ( n=2 264, 1 312 males and 952 females) and full-term infant group ( n=53 275, 28 269 males and 25 006 females). The cut-off values of amino acids in dry blood spots were expressed as percentage ( P0.5 - P99.5), and rank sum test was used for comparison between preterm and full-term infants. Results:(1) The distribution of 11 amino acids [alanine (ALA), arginine (ARG), citrulline (CIT), glycine(GLY), leucine (LEU), methionine (MET), ornithine (ORN), phenylalanine (PHE), proline (PRO), tyrosine (TYR) and valine (VAL)] in premature infants were abnormal.(2) The cut-off values of amino acids in premature infants were as follows: ALA: 135.20-552.33 μmol/L, ARG: 1.34-47.04 μmol/L, CIT: 5.66-32.02 μmol/L, GLY: 181.48-909.93 μmol/L, LEU : 71.10-283.29 μmol/L, MET: 4.21-34.51 μmol/L, ORN: 40.58-293.76 μmol/L, PHE: 23.60-106.30 μmol/L, PRO: 77.76-358.24 μmol/L, TYR: 27.52-352.91 μmol/L, VAL: 53.74-228.37 μmol/L.(3) The cut-off values of amino acid in full-term infants were as follows: ALA: 135.20-552.33 μmol/L, ARG: 1.30-42.73 μmol/L, CIT: 5.92-30.35 μmol/L, GLY: 208.17-980.09 μmol/L, LEU: 72.91-287.49 μmol/L, MET: 4.27-33.90 μmol/L, ORN: 48.40-305.59 μmol/L, PHE: 27.63-92.27 μmol/L, PRO: 97.38-372.75 μmol/L, TYR: 40.19-276.54 μmol/L, VAL: 65.75-237.92 μmol/L.(4) Except for PHE ( Z=-0.58, P>0.05), the other indicators were significantly different between 2 groups [ALA ( Z=-15.32, P<0.05), ARG ( Z=-5.62, P<0.05), CIT ( Z=-5.86, P<0.05), GLY ( Z=-14.52, P<0.05), LEU ( Z=-5.62, P<0.05), MET ( Z=-5.22, P<0.05), ORN ( Z=-13.01, P<0.05), PRO ( Z=-22.09, P<0.05), TRY ( Z=-2.09, P<0.05), VAL ( Z=-17.82, P<0.05)]. Conclusions:The establishment of the cut-off values of amino acids in premature infants in Sichuan provides a theoretical basis for laboratory diagnosis of IMD screening, which enhances the accuracy of diagnosis and avoids excessive medical treatment.

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