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@#Objective - To optimize the extraction and quantification methods for the determination of S phenylmercapturic acid - Methods (SPMA) in urine with performance liquid chromatography mass spectrometry. The urine was hydrolyzed with 50.0% sulfuric acid. The hydrolysate was purified by solid phase extraction column. Purified samples were separated by C18 chromatographic column and detected by tandem mass spectrometry. The isotope labeled SPMA was used as the internal Results - standard. The internal standard curve was used for quantification. The linear range of SPMA was 0.50 50.00 μg/L with the correlation coefficient of 0.999 8. The detection limit and the lower limit of quantification were 0.05 and 0.17 μg/L, - - - - respectively. The recovery rate was 97.0% 102.0%. The within run and between run relative standard deviation were 0.6% 1.0% - and 1.7% 6.5%, respectively. The mass concentration of urinary SPMA in the occupational benzene exposure group was - vs P higher than the non occupational benzene exposure group by this method (median: 2.81 0.28 μg/g creatinine, <0.05). Conclusion Compared to the national standard method, this optimized method of solid phase extraction and internal standard for quantification eliminates the matrix effect. This method is accurate and precise, and is suitable for the determination of SPMA acid in urine.
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Cordycepin,which has great immunomodulatory activities such as anticancer,antifungal,antivirus,antileukemia and lipid-lowering ones,is the secondary metabolite of Cordyceps militaris (C.militaris).Liquid submerged fermentation is the common cultivation process to produce cordycepin.To optimize the fermentation process and improve production,monitoring the cordycepin secretion in the fermen-tation is essential.The measurement based on chromatography-mass spectrometry methods is generally involved in the complex sample pretreatments and time-consuming separation,so more rapid and convenient methods are required.Matrix-assisted laser desorption ionization mass spectrometry(MALDI-MS) is more attractive for faster and direct detection.Therefore,MALDI-MS detection combined with isotope-labeled internal standard was applied to the measurement of cordycepin content in the fermentation broth and mycelium.This method made accurate quantification of cordycepin in the range of 5-400 μg/mL with a relative standard deviation of 5.6％.The recovery rates of fermentation samples after the 1,13,and 25 days were 90.15％,94.27％,and 95.06％,respectively.The contents of cordycepin in the mycelium and fermentation broth were 136 mg/g and 148.39 mg/mL on the 20th culture day,respectively.The cordycepin secretion curve of the liquid fermentation of C.militaris was real-time traced over 25 days.
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OBJECTIVE： To establish a method for measuring the paclobutrazol residue in Ophiopogon japonicus from Sichuan and detect the quality of O. japonicus from Sichuan from different sources. METHODS： Totally 50 batches of samples were collected from different origin places， commercial markets and manufacturers. The sample pretreatment method was QuEChERS method， .ie the sample was extracted by aqueous acetonitrile， salted out by QuEChERS extract package （containing anhydrous magnesium sulfate and anhydrous sodium acetate）， the extract solution was purified by QuEChERS purification package （containing anhydrous magnesium sulfate， N-propyl ethylenediamine， octadecylsilane chemically bonded silica， silica gel， graphitized carbon black） and then added into internal standard triphenyl phosphate. The paclobutrazol residue in O. japonicus from Sichuan was determined by GC-MS/MS. The determination was performed on DB-5MS column. The temperature programming was adopted， and the detector was triple quadrupole MS detector. The initial flow rate of carrier gas was 1.3 mL/min； acquisition mode was MRM. Injection method was splitless injection. RESULTS： The linear range of paclobutrazol was 1.01-505 ng/mL （r＝ 0.999 7）. RSDs of precision， stability （24 h） and repeatability tests were 3.94％， 13.62％， 7.54％ （n＝6）， respectively. Average method recovery was 111.26％ （RSD＝5.43％， n＝9）. The paclobutrazol residue in 50 batches of sample were 0.02-2.72 mg/kg. CONCLUSIONS： Established method is simple， accurate， sensitive and reproducible. It also can be used for the determination of paclobutrazol residue in O. japonicus from Sichuan. The contents of paclobutrazol residue in O. japonicus from Sichuan from different sources are different greatly.
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OBJECTIVE： To establish a method for determination of fluconazole concentration in human plasma. METHODS： UPLC-MS/MS method was adopted to determine plasma after precipitated with acetonitrile. Using isotope fluconazole-d4 as internal standard， the determination was performed on ACQUITY UPLC BEH C18 column with mobile phase consisted of 0.1％ formic acid-acetonitrile （gradient elution） at the flow rate of 0.3 mL/min. The column temperature was 40 ℃， and the sample size was 3 μL. ESI was used for positive ion scanning by multiple reaction monitoring mode. The ion pairs for quantitative analysis were m/z 307.1→220.0 （fluconazole） and m/z 311.1→223.0 （internal standard）. RESULTS： The linear range of fluconazole was 10-5 000 ng/mL （r＝0.998 1）. The limits of quantitation was 10 ng/mL. RSDs of intra-day and inter-day were less than 8％； accuracy ranged 95.8％-106.7％. The extraction recovery ranged 97.3％-107.3％ （RSD＜5.0％， n＝6）， and matrix effect， dilution effect and residual effect didn’t influence quantitative analysis of the substance to be measured. CONCLUSIONS： The method is simple， rapid， specific and accurate， which can be used for therapeutic drug monitoring and pharmacokinetic study of fluconazole.
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Objective@#To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the detection of serum oleic acid (OA), and preliminarily evaluate the role of OA in insulin resistance (IR) of type 2 diabetes (T2DM). @*Methods@#OA-［ 13 C 5 ］ was used as isotope-labeled internal standard, and the ion pairs of OA and OA-［ 13 C 5 ］ were 281.3/281.3 and 286.3/286.3, respectively. The ultrapure water was used as mobile phase A and methanol: acetonitrile (1∶1, v/v) as mobile phase B in a ZORBAX SB-Aq C18 reversed phase column. Meanwhile, the gradient elution system with a flow rate of 0.3 mL/min was used. According to the CLSI guidelines (EP15-A3), the reliability of the established method was evaluated by detecting the performance indicators such as precision, trueness, linear range, stability and carrying contamination rate. Serum OA levels were detected by the established HPLC-MS/MS method in 109 patients with clinically diagnosed T2DM and 100 healthy controls. The insulin resistance index (HOMA-IR) was calculated to evaluate IR, and the relationship between OA and IR was further analyzed. @*Results@#The established HPLC-MS/MS method for the detection of serum OA had good specificity and linearity in the range of 10-1 000 μmol/L (y=0.007 55x+0.004 83,r=0.997 7), and the low limit of quantification (LLOQ) was 10 μmol/L. It also had good precision, and the within-run coefficient of variation (CV) and total CV were not more than 1.62% and 1.73%, respectively, indicating that the method was suitable for the detection of serum OA. The serum OA levels in T2DM patients ［(425.58 ± 220.17) μmol/L］ were significantly higher than that in the healthy controls ［(113.20±58.00) μmol/L］, and serum OA levels were significantly correlated with HOMA-IR in T2DM patients and healthy controls. The area under the receiver operating characteristic (ROC) curve (AUC) of OA for the diagnosis of IR was 0.689. When the cut-off value identified by Youden index was 235.8 μmol/L, the sensitivity and specificity were 70.4% and 63%, respectively. When OA combined with fasting blood glucose (FBG) to diagnose IR, the AUC increased to 0.806, which was significantly higher than that of OA (P＜0.05). @*Conclusion@#A scientific and efficient HPLC-MS/MS method for the quantitative detection of serum OA is established successfully, which provides a reliable method for the dynamic monitoring of the changes of OA levels in the patients with metabolic diseases.
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To reveal the extraction regularity of volatile oil from galangal by GC-MS analysis. The volatile oil in galangal was extracted by steam distillation. The extract was collected every 30 min, the oil part and the water part were separated. GC-MS was used to analyze the extraction liquid collected at different time periods. A total of 140 volatile components were obtained by GC-MS analysis. Among them, the main components were eucalyptus oil alcohol, alpha-pine oil alcohol and 4-terpene alcohol; 22 special components were dissolved in water, 77 special components were dissolved in oil and 41 components were dissolved in both oil and water. With the increase of specific components in water, the content of Eucalyptus in water increased in a linear manner. The increase of eucalyptus oil further promoted the dissolution or dispersion of alpha PN in water, and the change of specific components in oil was positively correlated with the content of Eucalyptus and alpha-terpilenol in oil. The results of principal component analysis show that the physical and chemical properties of the compounds were important factors affecting the distribution of components. PC1 (molecular weight, melting point, boiling point positive correlation), PC2 (negative correlation of refractive index) and PC3 (positive correlation of water solubility) were the main components that lead to the differences in composition distribution. The process of extracting volatile oil from galangal through steam distillation was affected by the physical and chemical properties of volatile components. Some components were specifically distributed in the fragrance and volatile oil system. The endemic components of aromatic water increased the content of the main components in the water system, which may lead to the "emulsification", reduction of the yield and low quality of the volatile oil.
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Distillation , Gas Chromatography-Mass Spectrometry , Kinetics , Oils, Volatile , Plant Oils , Steam , Zingiberaceae , ChemistryABSTRACT
Objective To reveal the dissolution rule of volatile oil in the extraction processfrom Foeniculum vulgare by GC-MS analysis. Methods The volatile oil of F. vulgare was extracted by steam distillation; The volatile oil/perfume system was collected at fixed time interval (30 min); The volatile oil part and perfume part were separated. Using N-docosane and methyl myrisate as double internal standard, GC-MS was selected for analysis and quantification. The main components were extracted by thermography, and the distribution regularity of components was definited. The principal components with the impact on the composition distribution were investigated according to the physical and chemical properties of the molecular weight, melting point, boiling point, and density of the compounds. Results The GC-MS analysis results concluded 123 volatile components. The main components were estragole, anethole, and (R)-(+)-limonene. There were 60 and 27 endemic components in aromatic aqueous solution and volatile oil, respectively, in which 27were common composition. The content of anethole in water was positively related to the endemic components in aromatic water, and even had a positive correlation with anethole artemisia content and (R)-(+)-limonene content. The specific components in the oil were positively related to the content of the main components (R)-(+)-limonene in the volatile oil. The principal component analysis showed that the physical and chemical properties of the compounds were important factors affecting the distribution of components; PC1 (molecular weight and positive correlation of melting point), PC2 (refractive index positive correlation), and PC3 (water-soluble negative correlation) were the principal components that lead to differences in component distribution. Conclusion In the process of extracting volatile oil from F. vulgare, steam distillation is affected by the physical and chemical properties of volatile components. Some components are specifically distributed in aromatic perfume and volatile oil system. The endemic components of aromatic water increase the content of the main components in the water system, which may lead to theproduction of "emulsification" during the extraction process of volatile oil and reduce the yield and quality of volatile oil.
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Vorapaxar,a novel antagonist of the protease-activated receptor 1 (PAR-1 ),can inhibit the clotting process. Deuterium-labeled vorapaxar was required for the analysis of clinical sample as an internal stand-ard. Starting for unlabeled vorapaxar,four-step reactions including hydrolysis,condensation,transesterification and hydrogen-deuterium exchange were carried out to synthesize [D8]vorapaxar effectively for the first time. All intermediates and final products were confirmed by NMR and high resolution mass spectrometry (HRMS).Impor-tantly,the prepared [D8]vorapaxar could meet the requirements of sample analysis as the internal standard.
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Objective To provide a proper concentration of Internal Standard Solution by comparing five different degrees to the simultaneous analysis of 10 metals including V,Cr,Mn,Fe,Ni,Cu,As,Cd,Pb and Hg in Cardeniajasminoides.Method An Inductively Coupled Plasma-Mass Spectrometry method with Microwave-Assisted Digestion was developed with10,50,200,500,1 000 μg/L of Ge,In,Bi as internal standard.Result The degree of 500 μg/L concentration of internal stand solution can both correct the matrix effect and alleviate the contamination as the 1 000 μg/L.Conclusion With excellent detection limits,high sensitivity and wide linear range,the 500 μg/L internal standard may serve as reference in the determination of elements in other traditional Chinese medicine (TCM).
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Objective To establish a kind of simple,rapid,accurate and reliable method to analyze the concentration of alcohol in blood by headspace gas chromatography (HS-GC) with dual-column and dual-detector.Methods The samples were pre-treated by headspace sampler,which was the basis on the extraction principle of the gas extracting volatile substances.Next,these samples were analyzed by HS-GC that the tertiary butyl alcohol was acted as the internal standard substance.The HS-GC was equipped with two chromatographic column (the DB-ALC2 chromatographic column of 001 channel;the DB-ALC1 chromatographic column of 002 channel).At the same time,the HS-GC was also equipped with two hydrogen flame ionization detector (FID1 detector;FID2 detector).The retention time of the peak was finally performed as qualitative parameter and the standard curves method of internal standard were acted as quantitative basis.Results The liner range of the method was 0.2-2.0 mg/mL.The linear regression equation of 001 channel was Y=1.057 7X+0.048 2 and the correlation coefficient was R2=0.999 05.Besides,the linear regression equation of 002 channel was Y=1.039 5X+0.046 5 and the correlation coefficient was R2=0.999 25.In short,the average recovery rate of the method was 99.70%.Relative standard deviation(RSD) was less than 4% between the analysis results of 001 channel and 002 channel for the determination of the plan sample.Conclusion The method shown satisfactorily that it could not only be applied to determine the alcohol of blood of forensic toxicological analysis,but also be applied to determine the plan sample of ability test and verify of laboratory ability accreditation.
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Objective: To establish a GC determination method for the content and dissolution of magnesium valproate tablets. Methods:Magnesium valproate tablets were detected by a GC internal standard method. The samples were dissolved in 0. 1 mol·L-1 hydrochloric acid solution, and then extracted by dichloromethane. Eicosane was used as the internal standard. The dissolution was de-termined by the first method described in ChP 2015 edition. The dissolution medium was 0. 1 mol·L-1 hydrochloric acid solution and the rotation speed was 100 r·min-1 with the sampling time at 45 min. The samples were extracted by dichiormethane, and eicosane was used as the internal standard as well. Results: The dissolution of magnesium valproate tablets showed good linearity within the range of 0. 005-1. 000 mg·ml-1(r=0. 9999). The recovery was 99. 2% (RSD=0. 5%, n=9). The dissolution curve showed that magnesium valproate released above 80% in 45 minutes. Conclusion:The method has good specificity and high accuracy, and can be used for the content determination and dissolution detection of magnesium valproate tablets.
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Objective To establish a method for the determination of 8 heavy metal elements of Pb,Cd,As,Hg,Co,V,Se,Mo in iron dextran by inductively coupled plasma optical emission spectrometry (ICP-OES).Method Through selection of detection wavelengths,optimization of instrument parameters,verification of matrix effect interference and investigation of internal standard for correction of matrix effect interference,the 8 heavy metal elements were analyzed by ICP-OES.Results The recovery rate of the detected elements were increased from 83％-90％ to 94％-100％,after correction of matrix effect interference by internal standard method.The accuracy of the method were good.The spiked recoveries of the detected elements were from 94.3％ to 101.5％.The precision of the method were good (RSD ＜ 3.5％,n =6).The linearities of the detected elements were good,and the correlation coefficients were all greater than 0.999.The detection limits were from 0.22 to 8.54 ng/mL.The quantization limits were from 0.68 to 25.73 ng/mL.Conclusion The established method was accurate,sensitivity,rapid and reliable,which can be applied to the determination of contents of 8 heavy metal elements in iron dextran.
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Objective To establish a method for the determination of 8 heavy metal elements of Pb,Cd,As,Hg,Co,V,Se,Mo in iron dextran by inductively coupled plasma optical emission spectrometry (ICP-OES).Method Through selection of detection wavelengths,optimization of instrument parameters,verification of matrix effect interference and investigation of internal standard for correction of matrix effect interference,the 8 heavy metal elements were analyzed by ICP-OES.Results The recovery rate of the detected elements were increased from 83％-90％ to 94％-100％,after correction of matrix effect interference by internal standard method.The accuracy of the method were good.The spiked recoveries of the detected elements were from 94.3％ to 101.5％.The precision of the method were good (RSD ＜ 3.5％,n =6).The linearities of the detected elements were good,and the correlation coefficients were all greater than 0.999.The detection limits were from 0.22 to 8.54 ng/mL.The quantization limits were from 0.68 to 25.73 ng/mL.Conclusion The established method was accurate,sensitivity,rapid and reliable,which can be applied to the determination of contents of 8 heavy metal elements in iron dextran.
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BACKGROUND: Circulating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application. METHODS: We designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients. RESULTS: Compared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R²=0.916, P<0.0001). CONCLUSIONS: This duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.
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Adult , Female , Humans , Male , Middle Aged , DNA/blood , Healthy Volunteers , Real-Time Polymerase Chain Reaction/methods , Reference Values , Wounds and Injuries/bloodABSTRACT
A suitable ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of 11 mycotoxins with isotope internal standard in malt. The mycotoxins in malt were extracted and purified by one-step ultrasonic extraction procedure using acetonitrile/water/acetic acid (80:19:1), and then detected and confirmed by UPLC-MS/MS, and quantified by isotope labeled AFB1 ([13C17]-AFB1) and ZEN ([13C18]-ZEN) internal standards. Rapid separation of the 11 mycotoxins was successfully achieved on a Phenomenex Kinetex C18 column (100 mm × 2.1 mm, 2.6 μm) with gradient elution using the mobile phase of methanol containing 0.1% formic acid and 2 mmol·L-1 ammonium acetate in water. Simultaneous acquisition was performed in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI) source operated in both positive and negative ionization modes. The established method provided a good linearity for the 11 mycotoxins within their respective linear ranges with correlation coefficients all higher than 0.999 1. The average recoveries ranged from 75.0% to 117.0% with relative standard deviations (RSDs) below 5.1%. The limits of detection (LODs) and quantitation (LOQs) ranged from 0.05 to 30 μg·kg-1 and 0.15 to 87.5 μg·kg-1, respectively, which were below the maximum residue levels (MRLs) set by the European Union. Twenty malt samples were analyzed and nine samples were detected with mycotoxins, which were confirmed according to the same fragment ions found in positive samples and the standards at the same retention time. This study has demonstrated that the one-step extraction procedure of mycotoxins from complex matrices coupled to UPLC-MS/MS method is simple, quick, accurate and sensitive for quantitative and qualitative analysis of multiple mycotoxins in malt.
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ObjectiveTo evaluate the uncertainty for the determination of ethanol in human blood by auto-headspace gas chromatography (HS-GC) with internal standard curve method.Methods Each source of uncertainty, arising from the procedure of testing, was analyzed and conifrmed according to the guidelines of the uncertainty in measurement . After each uncertainty component was quantized, the combined standard uncertainty and the expanded uncertainty of the result were calculated.Results The relative uncertainty brought from the measurement repeatability, standard solution of the ethanol, the sample of blood, internal standard solution of the tert-butyl alcohol, the calibration cure, gas chromatography were 3.4%,0.71%,0.61%,0.41%,1.1% and 1.3% respectively; the relative expanded uncertainty of ethanol in blood was 3.9%.Conclusion The measurement uncertainty of the concentration of ethanol was came primarily from the measurement repeatability of sample, HS-GC and standard curve of ethanol.
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Objective:To evaluate the measurement uncertainty in the determination of sodium valproate tablets by GC with an in -ternal standard method , and determine the main sources of uncertainty .Methods:A GC internal standard method was selected to sys-tematically analyze the uncertainty in the determination of sodium valproate tablets , including the sample quantity , dilution ratio, purity and area repeatability of chromatographic peaks .Results: The expanded uncertainty of sodium valproate tablets was 2.7%, and the determination range of sodium valproate tablets was (96.3 ±2.7)%(k=2).Conclusion:The established GC internal standard meth-od for the uncertainty evaluation is reliable , which is helpful to improving the quality evaluation and control of sodium valproate tablets .
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OBJECTIVE: To establish a1 H-NMR internal standard method for measurement of glucosamine hydrochloride in glucosamine hydrochloride capsules. METHODS: For the HPLC-UV method, a China national drug tentative standard (No. YBH11442006) was employed. For the H-NMR analysis, the optimal determination conditions were selected as follows; the pulse sequence was zgpr, hydrogen spectral width was 6 009 Hz, pulse width was 11. 95μs, delay time was 6.50μs, the temperature was 27℃ and the number of scans was 32. RESULTS: The peak of δ 3.05 ppm of the glucosamine hydrochloride and the peak of δ 0 ppm of TSP were selected as quantitative peak and internal standard peak, respectively, and the concentration of the internal standard TSP was 1.02 mmol·L-1. A good linear relationship was obtained with a regression equation as Y = 2.717 IX +0.149 9(r =0.9998) in the concentration range of 2.0-12.0 mg·mL-1. The average recovery rate and the RSD of glucosamine hydrochloride capsules were 103.59% and 1.09% (n = 9), respectively. CONCLUSION: With simple pretreatment, high sensitivity, good repeatability and short analysis time, the NMR-based method could be applied to the measurement of glucosamine hydrochloride in glucosamine hydrochloride capsules.
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OBJECTIVE:To establish a method for the uncertainty measurement evaluation of the content determination of pred-nisolone API. METHODS:HPLC internal standard method was adopted to determine the content of prednisolone API,establish mathematical model for uncertainty evaluation,analyze influential factors and evaluate the factors. RESULTS:HPLC internal stan-dard method showed the contont was 98.8%,and expanded uncertainty of prednisolone API was 1.6%,and the determination result was(98.8±1.6)%,k=2. CONCLUSIONS:The method is suitable for the uncertainty measurement evaluation of the content deter-mination of prednisolone API by HPLC internal standard method.
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Objective] To discuss the applicability of internal standard in determination of cycla-mate in wine by HPLC-tandem mass spectrometry . [ Methods] After dilution wine sample was deter-mined by HPLC-tandem mass spectrometry and quantified by external and internal standard methods respec-tively. [Results] The results by the 2 quantitation methods differed 11.2%in average, and the maxi-mum relative deviation was 16.2%.And the result by external standard quantitation method proved to be consistent with that by the international standard GB/T 5009.97-2003. [ Conclusion] The difference in results is thought to originate from matrix effect in HPLC-tandem mass spectrometry and the improper in-ternal standard compound applied .