Using Liquid Chromatography-Tandem Mass Spectrometry in Detecting Plasma Lyso-GL3 Levels in Patients with Fabry Disease and the Association Analysis of Phenotype-Genotype of the Disease / 罕见病研究

ObjectiveUsing the liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the plasma level of Lyso-GL3 in patients with Fabry disease and to analyze the clinical application of the method.MethodsThirty-nine patients with a genetic diagnosis of Fabry disease were included, and plasma levels of Lyso-GL3 were measured by LC-MS/MS analysis, and detailed clinical information of the patients was obtained including: α-galactosidase A activity, genetic variants, quantification of urine protein, mean arterial pressure, and estimation of glomerular filtration rate, and the differences in the levels of Lyso-GL3 in different clinical phenotypes and genotypes were statistically analyzed, as well as the association with clinical indicators.ResultsLyso-GL3 showed good linearity within 0.7856-400 ng/mL(r=0.9992).Further analysis of 39 Fabry disease patients diagnosed in Ruijin Hospital, Shanghai Jiao Tong University School of Medicine showed a median Lyso-GL3 concentration of 23.6 ng/mL(4.3-92.9 ng/mL); Lyso-GL3 levels were significantly higher in patients with both the frameshift and the splicing mutations, as well as in patients with the nonsense mutations, than in patients with the missense mutations (median value 119.7 ng/mL vs. 11.9 ng/mL, P=0.006, and median value 97.0 ng/mL vs. 11.9 ng/mL, P=0.015, respectively). Whereas, association analysis revealed that Lyso-GL3 was not significantly associated with urinary protein, mean arterial pressure and estimated glomerular filtration rate.ConclusionsThe using of LC-MS/MS to quantify plasma Lyso-GL showed significant differences in Lyso-GL3 concentrations between classical and atypical phenotypes, suggesting that plasma Lyso-GL3 may help with clinical phenotypes. However, Lyso-GL3 levels is found to be overlapped between genotypes. No significant linear correlation was found between Lyso-GL3 and renal clinical indicators, suggesting the urgent need in finding a more accurate tool to assess renal involvement and prognosis in patients with Fabry disease.

Determination of 22 phospholipids in serum by ultra performance liquid chromatography-tandem mass spectrometry / 中华劳动卫生职业病杂志

Objective: To establish ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of 22 phospholipids in serum. Methods: In September 2022, Using synthetic non endogenous phospholipids as internal standard, phospholipids in serum were extracted by methanol-dichloromethane (2∶1, V/V) protein precipitation method. Chromatographic separation was achieved on an ACQUITY UPLC BEH shield RP18 column, and the mobile phase was methanol/water (5∶95, V/V) containing 10 mM ammonium formate and methanol. Detection was performed in multiple reaction monitoring mode with ion mode switching. And the method was applied by analyzing phospholipids in the serum of coal workers' pneumoconiosis patients. Results: The 22 phospholipids showed good linear relationships in their respective concentration ranges and the correlation coefficients were higher than 0.990. The spiked recoveries of the 22 phospholipids were 81.03%-121.63% at the three spiked levels. The intra-assay were less than 14.52%, and the inter-assay were less than 15.00%. Conclusion: The method with the advantages of simplicity, stability and high sensitivity, and it can be used for the analysis of phospholipids in serum.

UPLC-MS/MS Method for Detection of Etomidate and Its Metabolite Etomidate Acid Quantity in Blood / 法医学杂志

OBJECTIVES@#To establish a method for the simultaneous quantitative analysis of etomidate and its metabolite etomidate acid in blood, and to discuss its application value in actual cases.@*METHODS@#Acetonitrile precipitate protein method was used, and C18 column was selected. Gradient elution was performed with acetonitrile and 5 mmol/L ammonium acetate within 6 min. Electrospray ionization source in positive ion mode was used. The internal standard etomidate acid-d5 was obtained by etomidate-d5 alkaline hydrolysis reaction. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for quantitative analysis. The methodological verification was conducted.@*RESULTS@#Etomidate and etomidate acid in blood showed good linear relationship in the quantitative linear range (r>0.999), with the lower limit of quantification was 2.5 ng/mL and 7.5 ng/mL, respectively. The accuracy, precision, recovery rate, and matrix effect of the method met the professional verification standards. The practical application results showed that etomidate and etomidate acid could be detected in the blood of the abusers, and their mass concentrations ranged from 17.24 to 379.93 ng/mL.@*CONCLUSIONS@#The method established in this study can simultaneously quantify etomidate and etomidate acid in blood, which is simple and convenient to operate with accuracy. It can meet the detection needs of actual cases and provide technical support for law enforcement to crack down on etomidate abuse.

Proteomic Difference Analysis of Whole Blood and Bloodstains / 法医学杂志

OBJECTIVES@#To study the changes of protein levels in peripheral blood after it dried.@*METHODS@#The proteins from whole blood and bloodstains were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and normalized by the label-free quantification (LFQ) method. The differential proteins were analyzed by using R 4.2.1 software, limma and edgeR package. The analysis of biological function, signaling pathway and subcellular localization for the differential proteins was then performed.@*RESULTS@#A total of 623 and 596 proteins were detected in whole blood and bloodstains, respectively, of which 31 were statistically significant in the quantitative results, including 10 up-regulated and 21 down-regulated proteins in bloodstains.@*CONCLUSIONS@#The protein abundances in whole blood and bloodstains are highly correlated, and the variation of protein abundances may be related to the changes of endogenous and structural proteins in cells. The application of proteomics technology can assist the screening and identification of protein biomarkers, thereby introducing new biomarkers for forensic research.

Detection and Analysis of Blood Dexmedetomidine in Drug-Facilitated Cases / 法医学杂志

OBJECTIVES@#To establish a simple and rapid qualitative and quantitative detection method of dexmedetomidine in blood.@*METHODS@#Blood was separated on the Allure PFP Propyl liquid chromatography column with isocratic elution after it was precipitated by acetonitrile and filtered. Qualitative and quantitative analysis of dexmedetomidine was performed using positive ion scan mode and multi-reaction monitoring mode.@*RESULTS@#The limit of detection of dexmedetomidine in blood was 0.2 ng/mL and the limit of quantification was 0.5 ng/mL. The linearity of the method was good in the range of 0.5-1 000 ng/mL, and the correlation coefficient was greater than 0.99. The accuracy of the method was 90.34%-112.67% and the extraction recovery was 50.05%-91.08%, with no significant matrix effect.@*CONCLUSIONS@#This method is simple, selective and suitable for the qualitative and quantitative analysis of dexmedetomidine in blood, which can provide a reference for drug-facilitated cases involving dexmedetomidine.

Rapid Determination of Bucinnazine in Blood by UPLC-MS/MS / 法医学杂志

OBJECTIVES@#To establish a rapid method for the analysis of bucinnazine in blood by UPLC-MS/MS and to apply the method to the practical case.@*METHODS@#After the internal standard was added to blood, the protein was precipitated with 900 μL mixed solution (Vacetonitrile∶Vwater=8∶2). After vortex and centrifugation, the protein was measured through 0.22 μm filter membrane. The separation was performed on C18 chromatography column, with acetonitrile and 5 mmol/L ammonium acetate containing 0.1% formic acid aqueous as mobile phase gradient elution at the flow rate of 0.4 mL/min. Multiple reaction monitoring scan was performed in electrospray positive ion mode, quantitative measurement was performed by internal standard method, and methodological verification was carried out.@*RESULTS@#The linear relationship of bucinnazine in blood was good in the range of 0.5-200 μg/L, the correlation coefficient (r) was 0.999 7, the limit of detection was 0.1 μg/L, the limit of quantitation was 0.5 μg/L, and the recovery was 78.3%-83.8% at 1, 10 and 100 μg/L mass concentration levels. The matrix effect was 69.4%-73.8%, the intra-day precision was 1.9%-2.8%, and the inter-day precision was 2.8%-3.2%, the accuracy was 3.1%-3.5%. The stability test results of 1 and 100 μg/L mass concentrations at -25 ℃ showed that the accuracy (bias) of 10 d was less than 4.5%.@*CONCLUSIONS@#This method has the advantages of simple pre-treatment process, fast sample processing speed, high sensitivity of instrument analysis, good stability of content determination and reliable identification results, and can meet the needs of case identification.

Establishment and application of detection methods of dicamba in drinking water / 环境与职业医学

Background Dicamba is widely used in agricultural production in China, but it is extremely soluble in water and can be harmful to human health when it enters the body via water drinking. It is necessary to establish an accurate, sensitive, and rapid detection method to determine the residues of dicamba in domestic drinking water. Objective To establish two methods for the determination of dicamba residues in drinking water by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) respectively. Methods The conditions of the proposed method using HPLC-MS/MS included CAPCELL PAK ST chromatographic column, ammonium formate water solution and methanol as the mobile phase, and isocratic elution. The system was operated under multiple reaction monitoring mode and electrospray negative ionization mode. Trimethylsilylated diazomethane was used as a derivatizing agent for GC-MS/MS, and an external standard curve was used to evaluate the system. The residues of dicamba in seven water samples of tap water or secondary water supply from six regions in Chengdu were detected by the established systems to evaluate their applicability and to understand the status quo of dicamba residues in drinking water. Results For the HPLC-MS/MS, the linear range of dicamba was 1.00-100 μg·L−1, the regression equation was \begin{document}$\hat Y $\end{document}=1250.9X+2681.5, the correlation coefficient was 0.9988, the relative standard deviations were 1.23%-26.3%, the limit of detection was 0.95 μg·L−1, and the spiked recoveries were 91.8%-111%. For the GC-MS/MS, the linear range of dicamba was 0.200-10.0 μg·L−1, the regression equation was \begin{document}$\hat Y $\end{document}=190597X+40911, the correlation coefficient was 0.9993, the relative standard deviations were 0.64%-3.90%, the limit of detection was 0.18 μg·L−1, and the spiked recoveries were 97.3%-105%. No dicamba residue was identified in the seven water samples of tap water or secondary water supply from six regions in Chengdu by the proposed methods. Conclusion The two detection methods established in this study are sensitive and rapid, meet the requirements from the detection of dicamba residues in drinking water, and provide an experimental basis for subsequent research on the detection of dicamba residues. In the future, it is necessary to continue to pay attention to the pollution of dicamba in drinking water in Chengdu.

Determination of bongkrekic acid in corn flour by direct extraction ultra-high performance liquid chromatography tandem mass spectrometry / 公共卫生与预防医学

Objective To establish a direct extraction ultra-high performance liquid chromatography tandem mass spectrometry method for the determination of bongkrekic acid in corn flour. Methods Bongkrekic acid was directly extracted with 80% methanol from corn flour samples, and the supernatant after vortex and centrifugation was determined after passing through membrane filtration. At the same time, the corn flour samples were extracted by solid phase extraction. The determination results of the two methods were compared. Results The linearity of standard series was good within the range of2-20 μg/L, and the linearity coefficient was＞0.999. The determination result of the positive sample by direct extraction method was 193.40 mg/kg (n=6). Adding the standard to the blank sample at the levels of 2, 6, and 10 μg/L, the calculated recovery rate was 75.82% - 99.33%, and the relative standard deviation was 3.54 % - 8.45%. The detection limit of the method reached 6 μg/kg. After extraction by solid phase extraction, the determination result of the positive sample was 196.84 mg/kg (n=6). The recovery rate was 77.12% -100.83%, with a relative standard deviation of 8.32% - 9.54%. Conclusion Compared with the solid phase extraction, the direct extraction method for the extraction of bongkrekic acid from corn flour has the advantages of rapidity, simplicity, and cost savings.

Simultaneous determination of 15 bile acids in Tongren niuhuang qingxin pills by UPLC-MS/MS / 中国药房

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.

Study on Clinical Application and Supervision of LC-MS in in Vitro Diagnosis Test / 中国医疗器械杂志

From the perspective of technical evaluation, this study reviewed the current situation of application and clinical application of medical device products were detected by liquid chromatography-tandem mass spectrometry in the market in recent years. The regulatory requirements of these products in China, USA, EU and Japan were compared and analyzed, and the monitoring situation of adverse events after listing, the standards for reference and the domestic and foreign regulatory documents were combined, the clinical application and regulatory risks of the product were analyzed. The problems such as pre-treatment, system matching, adequacy of performance index requirements, inter-room consistency, reference interval and registration unit were discussed and suggestions for supervision were given, with a view to the field of product R&D and production, review and approval of supervision to provide technical reference.

Formation and characteristics of polystyrene nanoplastic-plant protein corona / 生物工程学报

To investigate the formation of polystyrene nanoplastic-plant protein corona and its potential impact on plants, three differently modified polystyrene nanoplastics with an average particle size of 200 nm were taken to interact with the leaf proteins of Impatiens hawkeri for 2 h, 4 h, 8 h, 16 h, 24 h, and 36 h, respectively. The morphological changes were observed by scanning electron microscopy (SEM), the surface roughness was determined by atomic force microscopy (AFM), the hydrated particle size and zeta potential were determined by nanoparticle size and zeta potential analyzer, and the protein composition of the protein corona was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proteins were classified in terms of biological processes, cellular components, and molecular functions to study the adsorption selection of nanoplastics to proteins, investigate the formation and characteristics of polystyrene nanoplastic-plant protein corona and predict the potential impact of protein corona on plants. The results showed that the morphological changes of the nanoplastics became clearer as the reaction time extends, as evidenced by the increase in size and roughness and the enhancement of stability, thus demonstrating the formation of protein corona. In addition, the transformation rate from soft to hard protein corona was basically the same for the three polystyrene nanoplastics in the formation of protein corona with leaf proteins under the same protein concentration conditions. Moreover, in the reaction with leaf proteins, the selective adsorption of the three nanoplastics to proteins with different isoelectric points and molecular weights differed, and the particle size and stability of the final formed protein corona also differed. Since a large portion of the protein fraction in protein corona is involved in photosynthesis, it is hypothesized that the formation of the protein corona may affect photosynthesis in I. hawkeri.

Serum Metabonomics Study of Difference in Lymph Node Metastasis of Pancreatic Ductal Adenocarcinoma Based on LC-MS / 肿瘤防治研究

Objective To investigate the differential metabolites of lymph node metastasis in pancreatic ductal carcinoma (PDAC) and provide new ideas for the pathogenesis, early diagnosis and treatment of metastatic pancreatic cancer. Methods Forty serum specimens of patients with pancreatic ductal carcinoma were collected and divided into lymph node metastasis group (18 cases) and non-metastasis group (22 cases). Thirty-one serum specimens were also collected from the healthy control group. Liquid chromatographytandem mass spectrometry was used to analyze the differential metabolites and metabolic pathways between patients with PDAC and healthy controls as well as between lymph node metastasis and non-metastasis groups. Results Principal component analysis and partial least squares-discriminant analysis revealed statistically significant differences in metabolites and metabolic pathways between patients with PDAC and the healthy controls and between lymph node metastasis and non-metastasis groups. The differences in profiles were also statistically significant. Seventy-six different metabolites and 11 metabolic pathways were screened between patients with PDAC and the healthy controls, among which phenylalanine metabolism and histidine metabolism were the two most influential metabolic pathways. Four different metabolites were screened between lymph node metastasis and non-metastasis groups, and the expression of ethopropazine and phenylalanine were upregulated but the expression of tetrahydrodeoxycorticosterone and oxprenolol were downregulated. Conclusion Metabolites are significantly altered in the lymph node metastasis group of patients with PDAC compared with the non-metastasis group. Ethopropazine, phenylalanine, tetrahydrodeoxy corticosterone, and oxprenolol are potential biomarkers of lymph node metastasis in patients with PDAC.

The trend of changes in serum vitamin A, D, and E levels during pregnancy / 中华检验医学杂志

Objective:High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to quantify the levels of vitamins A, D and E in pregnant women during the second trimester, and to investigate the change trends of serum vitamins A, D and E levels during pregnancy.Method:A total of 720 pregnant women with an average age of (29.7±4.4) years and 12-22 weeks of gestation were included from October 1, 2021 to October 30, 2022 in the obstetrics department of the People′s Hospital of Wuhan University. The concentrations of vitamins A, D and E were determined by HPLC-MS/MS. The concentration levels of each group were statistically analyzed and the deficiency rate were calculated.Results:The distribution range of vitamin A, D and E (95% CI) was 0.74-2.74 μmol/L, 2.88-25.37 ng/ml and 6.18-35.08 μmol/L, with the deficiency rates were 9.30%, 93.76% and 35.83%, respectively. Vitamin A, D and E levels in the twin group were (1.67±0.51) μmol/L, (13.18±7.44) ng/ml and 11.97 (8.85, 14.60) μmol/L, respectively. They were significantly higher than those in the singlet group (1.45±0.36) μmol/L, (10.87±5.26) ng/ml and 10.46 (6.99, 14.11) μmol/L, with statistical significance by independent sample t-test ( P<0.001). The concentration of vitamin D in the lower BMI group (<22 kg/m 2) was (12.54±5.74) ng/ml, significantly higher than that in the fat group (≥22 kg/m 2) (10.46±4.90) ng/ml, and the rank-sum test was statistically significant ( P<0.001). Conclusion:In this study, the levels of three vitamins were monitored in mid-pregnancy using HPLC-MS/MS, and the changes of serum vitamin A, D, and E levels during pregnancy were analyzed.

Development of a LC-MS/MS method for the determination of vancomycin in human serum and validation of its clinical value / 中华检验医学杂志

Objective:The aim of our study is to develop an LC-MS/MS method using isotope internal standard for the determination of vancomycin in human blood serum and to validate its clinical value.Method:We conducted a methodological evaluation study using serum samples from 221 hospitalized patients (142 males and 79 females; mean age (59.31±15.32) years) who received treatments of vancomycin at the Sir Run Run Shaw Hospital of Zhejiang University between March 2021 and June 2022. In addition, thirty clinical residual serum samples from healthy individuals (15 males and 15 females; mean age (35.65±9.86) years) undergoing physical examination were used for methodological evaluation. The method was established using AB Sciex Triple Quad 4500 MD liquid chromatography-tandem mass spectrometer and chromatographic separation was carried out using a Phenyl-Hexyl column with gradient elution. The mobile phase was composed of 0.1% formic acid in water and methanol; the column temperature was 40℃; Vancomycin-[d12] TFA salt was used as the internal standard (IS). The sensitivity, specificity, linearity, accuracy, imprecision, matrix effect, and carry-over of the method were evaluated.Results:The detection limit of vancomycin was 0.2 mg/L and the lowest limit of quantification was 0.5 mg/L. It showed good linearity ( R2=0.998 4) in the 1 to 50 mg/L concentration range. Accuracy (recovery rate 87.45%-112.69%), intra-day and inter-day imprecision ( CV 4.91%-7.69%), internal standard standardized matrix factor (90.22%-104.29%). Carryover pollution was negligible. Of the 221 patients, the mean trough concentrations of vancomycin in serum was (13.15±8.56) mg/L. Conclusion:The LC-MS/MS method for the detection of serum vancomycin established in our laboratory meets the requirements of the reference method, and can be used for the monitoring of clinical therapeutic drugs.

Simultaneous determination of 18 common disinfection by-product residues in drinking water by high performance liquid chromatography-tandem mass spectrometry / 上海预防医学

ObjectiveA method was developed for the rapid determination of 18 common disinfection by-products including halogenated oxides and haloacetic acid （HAAs） in drinking water by high performance liquid chromatography tandem mass spectrometry （HPLC-MS/MS）. MethodThe water sample was filtered by 0.22 μm hydrophilic membrane then the analytes were separated on a PFP （2.1 mm× 100 mm， 2.7 μm） pentafluorophenyl column with 0.1% acetic acid and acetonitrile as mobile phase gradient elution. Ionization in anionic electrospray mode was detected by multi-reaction monitoring （MRM） mode. The external standard method was used for quantitation. ResultsThe correlation coefficients of 18 disinfection by-products were above 0.999 in the corresponding linear range. The average spiked recoveries of 1， 10 and20 times of LOQ of each analyte were 91.6%‒101.8%， and the relative standard deviation （RSD） was 1.2%‒6.4%. The LOD and LOQ were 0.020‒2 μg·L-1 and 0.050‒5 μg·L-1， respectively. ConclusionThis method is simple， sensitive and accurate， and could be used for the routine analysis of 18 common disinfection by-products in drinking water.

Rapid determination of four herbicides in urine with ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry / 预防医学

Objective@#To establish a ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry method for rapid simultaneous determination of quinclorac, acetochlor, butachlor and metolachlor in urine.@*Methods@#Urine samples were diluted 10 times, prepared into the mixed standard solution, and subjected to gradient elution on the ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) with 0.1% formic acid and acetonitrile as the mobile phase. The quinclorac, acetochlor, metolachlor and butachlor levels were determined using electrospray ionization-positive ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry with the multiple reaction monitoring mode.@*Results@#Four herbicides were effectively separated on the ACQUITY UPLC BEH C18 column (100 mm× 2.1 mm, 1.7 μm), and good linear relationships were observed for quinclorac, acetochlor and butachlor at 1 to 25 μg/L and for metolachlor at 0.2 to 25 μg/L, with all linear correlation coefficients of >0.999. The detection limts of quinclorac, acetochlor, butachlor and metolachlor were 0.10, 0.10, 0.20 and 0.01 μg/L, respectively. The recovery rates of quinclorac, acetochlor and butachlor were 107.42%, 93.94% and 90.27% from urine samples at a spiked level of 5 µg/L, with relative standard deviations of 4.82%, 3.84% and 6.76%, and the recovery rate of metolachlor was 89.51% at a spiked level of 0.5 µg/L, with a relative standard deviation of 8.98%.@*Conclusion@#The chromatography and mass spectrometry conditions are optimized in this ultra-high performance liquid chromatography-tandem mass spectrometry, which is effective for rapid simultaneous determination of quinclorac, acetochlor, metolachlor and butachlor in urine samples.

Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique / 法医学杂志

OBJECTIVES@#To establish an LC-MS/MS method based on single hair micro-segmental technique, and verify the detection of 42 psychoactive substances in 0.4 mm hair segments.@*METHODS@#Each piece of single hair was cut into 0.4 mm segments and extracted by sonication and the segments were immersed in dithiothreitol-containing extraction medium. Mobile phase A was the aqueous solution containing 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. Mobile phase B was acetonitrile. An electrospray ionization source in positive ion mode was used for data acquisition in multiple reaction monitoring (MRM) mode.@*RESULTS@#The 42 psychoactive substances in hair had a good linear relationship within their respective linear ranges (r>0.99), the limits of detection were 0.2-10 pg/mm, the limits of quantification were 0.5-20 pg/mm, the intra-day and inter-day precisions were 1.5%-12.7%, the intra-day and inter-day accuracies were 86.5%-109.2%, the recovery rates were 68.1%-98.2%, and the matrix effects were 71.3%-111.7%. The method was applied to hair samples collected from one volunteer at 28 d after a single dose of zolpidem, with zolpidem detected in 5 hairs was 1.08-1.60 cm near the root tip, and the concentration range was 0.62-20.5 pg/mm.@*CONCLUSIONS@#The micro-segmental technique of single hair analysis can be applied to the investigation of drug-facilitated sexual assault cases.

Rapid screening of 28 alkaloids in food poisoning samples by liquid chromatography-tandem mass spectrometry / 中国热带医学

@#Abstract: Objective　To investigate a poisoning incident caused by eating eight treasure congee, and establish liquid chromatography (LC)-mass spectrometry (MS)/MS screening method of 28 alkaloids to provide references for disposal of similar poisoning incidents. Methods　LC-MS/MS was used for screening 28 alkaloids in the urine, eight treasure congee and food raw material, and the detected alkaloids were quantified. Samples were extracted with 0.4% formic acid aqueous solution and separated by a Acquity UPLC BEH C18 column (1.7 μm, 100 × 2.1 mm). Acetonitrile-0.2% formic acid aqueous solution was used as the mobile phase and gradient elution was adopted. The ionization mode was electrospray positive ionization mode, and the detection method was multi-reaction monitoring (MRM). Analytes were quantified with the external standard method. Results　In the concentration range of 0-100 ng/mL, the linear correlation coefficient r were greater than 0.999 for 28 alkaloids. The recovery of 28 alkaloids in urine sample ranged from 63.0% to 105.0%, and the relative standard deviations (RSDs) were between 5.8% and 8.6%. The recovery of 28 alkaloids in eight treasure congee sample ranged from 72.0% to 109.0%, and the RSDs were between 6.3% and 9.7%. The recovery of 28 alkaloids in semen sesami nigrum sample ranged from 60.0% to 95.0%, and the RSDs were between 4.8% and 8.2%. Hyoscyamine (2 380.0 ng/mL), scopliamine (3.6 ng/mL) and rac-anisodamine (4.7 ng/mL) were detected in the patient's urine. Hyoscyamine (63.3 μg/g), scopliamine (5.7 μg/g) and rac-anisodamine (2.1 μg/g) were detected in eight treasure congee. Hyoscyamine (901.0 μg/g), scopliamine (80.0 μg/g) and rac-anisodamine (30.1 μg/g) were detected in the seed of Datura stramonium L. The ratio of scopliamine and hyoscyamine in the seed of D. stramonium was 1∶11, which complies with the characteristics of D. stramonium L. In urine sample, the proportion of scopliamine and rac-anisodamine was 0.15% and 0.20%, and hyoscyamine accounted for 99.65%. Conclusion　Seed morphology, the content range and proportion of three alkaloids are all in accord with the characteristics of D. stramonium. Combined with the clinical symptoms of atropine poisoning, it can be deduced that this incident is a family food poisoning caused by accidental consumption of seed of D. stramonium L. The method can provide technical support for the clinical diagnosis and treatment of alkaloid poisoning patients, and also provide a basis for emergency detection and disposal of alkaloid poisoning events.

The PK bioanalysis method study of c-Met antibody-drug conjugate (RC108) in cynomolgus monkey / 药学学报

Antibody-drug conjugate (ADC) has the characteristics of low toxicity and high efficiency, and plays an important role in cancer treatment. However, due to the complexity of its structure, it brings difficulties in pharmacokinetic (PK) bioanalysis. This study established an analytical method for the detection of ADC (RC108) in cynomolgus monkey plasma by ligand-binding assay (LBA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which was used to analyze and quantify the total antibody, bound antibody and free drug in cynomolgus monkey plasma. Based on the LBA method, rabbit anti-RC108 Fab and mouse anti-MMAE (monomethyl auristatin E) mAb were pre-coated in 96-well plates as the total antibody and antibody binding reagents, respectively. The samples to be tested were added, and then the detection reagents were added in turn. Goat anti-human IgG (H+L)-HRP, chromogenic solution tetramethylbenzidine (TMB), H2SO4 terminate the reaction, read data at 450 nm/630 nm wavelength of microplate reader; LC-MS/MS analysis method quantifies MMAE concentration, and refer to relevant regulations for methodological validation. The analytical method for quantifying total antibody, bound antibody and free drug of RC108 drug obtained good accuracy and precision, and the selectivity, dilution linearity, hook effect, parallelism and stability were verified. Meet the requirements of biological analysis. Finally, a bioanalytical method for the determination of the concentration of the test substance RC108 (total antibody, conjugated antibody, free MMAE) in cynomolgus monkey plasma with high sensitivity and high throughput was established by LBA and LC-MS/MS method. Subsequent non-clinical research on PK research in cynomolgus monkeys will provide technical support.

Simultaneously quantitative analysis of 35 components in gualoupi injection using hydrophilic interaction liquid chromatography tandem mass spectrometry / 药学学报

A hydrophilic interaction chromatography tandem mass spectrometry method was developed for simultaneous quantification of 35 components in gualoupi injection. The analytes were separated with an ACQUITY XBridge Amide column using 20 mmol·L-1 ammonium formate aqueous solution (pH 3.0) as mobile phase A and 20 mmol·L-1 ammonium formate (pH 3.0)∶acetonitrile (1∶9) as mobile phase B for gradient elution. Mass spectrometry with dynamic multiple reaction monitoring and external standard method were used for quantitative analysis. A total of 35 components were determined in 10 batches of gualoupi injection. The results showed that the 35 compounds had a good linear relationship within their respective concentration ranges with the correlation coefficients (R2 > 0.998 0), the recoveries ranged from 76.6% to 118.5%. The results showed that γ-aminobutyric acid, trigonelline, alanine, threonine, homoserine, citrulline, and leucine were abundant in gualoupi injection, while nicotinamide, methylsuccinic acid, cytosine and choline account for a low percentege. The present study provides an important reference for elucidation of the effective material basis and the improvement of quality standard of gualoupi injection.