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A method was developed for the quantification of human growth hormone ( hGH ) by protein purification and isotope dilution-high performance liquid chromatography mass spectrometry ( HPLC-IDMS ) . The hGH was purified and fractionated by fast protein liquid chromatography ( FPLC ) , then hGH molecular weight was accurately determined by Fourier transform ion cyclotron resonance mass spectrometer ( FTICR-MS). The purified hGH was hydrolyzed and the separation was performed on an KINETEX C18 column (150 mmí2 mm I. D. , 2. 6 μm) with water ( containing 0. 1% TFA) and acetonitrile isocratic solution as the mobile phase at a flow rate of 0 . 2 mL/min and 40℃. The electrospray source was operated in the positive ion mode, and monitored in the multiple reaction monitoring ( MRM) mode. The measured hGH molecular weight by FTICR-MS was only 0. 31 Da difference from theoretical value. Three amino ( proline, valine and phenylalanine) were clearly separated by isocratic elution within 5 min. Under the optimized conditions, the content of hGH was 186 . 80 μg/g with a RSD of 0 . 5%. The detection results of hGH in international comparison by this method were consistent with the reference value, which validated the feasibility of the established method. The developed method is simple, practical, accurate, reliable and reproducible, and can be used for the hGH quantitation of pure hGH CRM to provide reference for the routine detection of hGH.
ABSTRACT
Objective To establish a method for measuring serum cotinine by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) and provide an assay that can be applied to theevaluation of the level of smoke exposure and to the risk analysis of smoking related diseases.Methods Blood samples were collected from 94 apparently healthy subjects from October to December in 2010 and centrifuged,and the sera were separated.Serum samples were mixed with [ D3 ] -cotinine ( as the internal standard) and treated with acetonitriles to precipitate protein.After centrifugation,the supernatants were transferred and evaporated under a stream of nitrogen until dryness and reconstituted with mobile phase.Then the residuals were analyzed by LC/MS/MS system with multiple reaction monitor model; the concentration of cotinine were quantified by the isotope internal standard method and the stand curve was employed with a series of calibration.To estimate the precision of the method,five frozen serum pools were repeatedly analyzed in five runs,and every pool was analyzed in triplicate.In addition,the recovery rates were analyzed with the serum sample added with different levels of standard.The stability of cotinine in serum preserved at room temperature,4 ℃ and - 80 ℃,respectively.Finally,the levels of cotinine of 94 healthy subjects were measured to evaluate the distribution of cotinine with different smoke statuses.Results Serum cotinine measured by ID-LC/MS/MS was separated well with few interferences.The correlation coefficients between the peak area ratios and cotinine concentrations were higher than 0.9993.The values of within-run coefficients of variation (CV) of five frozen serum pools (0.68,48.42,94.34,250.95 and 287.04 μg/L) were 2.19％,0.78％,0.75％,0.65％ and 0.67％,respectively.The values of total CV were 4.71％,1.40％,1.98％,1.10％ and 1.03％,respectively.The limit of detection (LOD) and limit of quantitation ( LOQ ) were 0.013 and 0.050 μg/L,respectively.The analytical recoveries ranged from 99.22％ to 102.67％.The samples could maintain stability within 2 d at room temperature,7 d at 4 ℃ and 3 months at -80 ℃ resulting the accuracy of measurements from 99.28％ to 100.87％ and the CV＜5％.The levels of cotinine of 94 healthy subjects were measured and shown skewed and leptokurtic distribution.The concentrations of twenty smokers,fourteen former smokers and sixty non-smokers were 116.40 (63.17 -241.12),0.67 (0.15 - 0.95 ) and 0.22 (0.15 - 0.42 ) μg/L,respectively.Furthermore,the level of cotinine of former smokers (Z =-2.12,P ＜0.05) and smokers (Z =-6.67,P ＜0.001) were statistically higher than non-smokers.Conclusions An ID-LC/MS/MS method for serum cotinine detection has been established.It is hoped that the method will be applied to the assessment of smoke exposure and its association with the risks of smoking related diseases since it is simple,specific,precise,sensitive and accurate.
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Characteristics and measurement principles of reference methods in clinical biochemistry were described.Implementation of reference systems is one of the most effective approaches to improve the accuracy and comparability of clinical laboratory test results.Reference methods are the key components of reference systems.Reference methods should have measurement uncertainties that meet the requirements of the intended use,and thus should be based on reliable measurement principles.For the well-defined biochemistry analytes,reference methods have been almost all based on instrumental analysis.Isotope dilution mass spectrometry (ID/MS) is considered most reliable and has been the major analytical principle of the reference methods.ID/MS analysis is accurate but expensive.Use of other validated instrumental analyses as reference measurement principles would be justified.
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Objective To develop a method for the determination of total cholesterol in serum by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS).Methods Serum samples were supplemented by addition of [3,4-~(13)C_2]-cholesterol,hydrolyzed with alcoholic sodium hydroxide and oxidized into cholest-4-ene-3,6-dione by chromic acid.The oxidation products were analyzed by LC/MS/MS using atmospheric pressure chemical ionization (APCI) source and detection modes of multiple reaction monitoring (MRM) and single ion recording (SIR).Signals (peak areas) of the internal standard were corrected for the contributions of cholesterol and the signal ratios of cholesterol to internal standard for the calibrations were linearly regressed against cholesterol concentrations.The resulted regression equation was used for the calculation of serum cholesterol concentrations.Results The correlation coefficients between the peak area ratios and cholesterol concentrations were 0.999 9 and higher.Under MRM mode,the average within-run CV of the results obtained on 3 serum samples was 0.95% (ranged from 0.92% to 0.99%) and the total CVwas 0.86% (0.82% to 0.89%),and under SIR mode,the within-run CV was 0.64% (from 0.54% to 0.77%) and the total CVwas O.69% (0.62% to 0.81%),respectively. Results on certified reference materials (SRM 1951 a Level Ⅰ and Level Ⅱ;GBW 09145 and GBW 09147) showed an average bias of 0.23% (0.14% to 1.00%) under MRM mode,and 0.24% (0.07% to 1.27%) under SIR mode.Conclusions An ID-LC/MS/MS method for serum cholesterol has been developed.It is specific and precise and may be used as a candidate reference method.
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Objective To develop a candidate reference method for the measurement of progesterone in human serum.Methods The serum sample is mixed with the internal standard [3,4-~(13)C_2] progesterone.After extraction with n-hexane and purified by a aqueous solution of 2-Hydroxypropyl-?- cyclodextrin (HP-?-CD),the serum progesterone and labeled progesterone are converted to the 3-enol heptafluorobutyrate and analyzed by gas chromatography mass spectrometry (GC/MS) with selected ion monitoring.The concentration of serum progesterone is calculated by bracketing method.Results The results gave coefficients of variation (CVs) of 0.69% to 2.12%.The analytical recoveries ranged from 98.3% to 100.1%.The results of measuring certified reference materials of serum progesterone are agree with the target value.Conclusion The procedure for measuring progesterone in serum is a highly accurate and precise method and may be used as a candidate reference method for serum progesterone assays.