The introduction of imatinib, an oral tyrosine kinase inhibitor, as first-line standard therapy in patients with unresectable, metastatic, or recurrent gastro-intestinal stromal tumor (GIST), ...strongly improved their treatment outcomes. However, therapeutic drug monitoring (TDM) is recommended for this drug due to the large inter-individual variability in plasma concentration when standard dose is administered. A Cmin higher than 760 ng/mL was associated with a longer progression free survival. Thus, a LC-MS/MS method has been developed and fully validated to quantify imatinib and its active metabolite, norimatinib, in finger-prick dried blood spot (DBS). The influence of hematocrit, sample homogeneity, and spot size and the correlation between finger-prick and venous DBS measurements were also assessed. The method showed a good linearity (R2 > 0,996) between 50-7500 ng/mL for imatinib and 10-1500 ng/mL for norimatinib. Analytes were extracted from DBS samples by simply adding to 3 mm-discs 150 μL of acidified methanol containing IMA-D8. The collected extract was then injected on a LC Nexera system in-house configured for the on-line cleanup, coupled with an API-4000 QT. The chromatographic separation was conducted on a Synergi Fusion-RP column (4 μm, 2x50 mm) while the trapping column was a POROS R1/20 (20 μm, 2x30 mm). The total run time was 8.5 min. DBSs stored at room temperature in plastic envelopes containing a silica-gel drying bag were stable up to 16 months. The proposed method was applied to 67 clinical samples, showing a good correlation between patients' finger-prick DBS and plasma concentrations, measured by the reference LC-MS/MS method, internally validated. Imatinib and norimatinib concentrations found in finger-prick DBS were adjusted by hematocrit or by an experimental correction factor to estimate the corresponding plasma measurements. At the best of our knowledge, the proposed LC-MS/MS method is the first analytical assay to measure imatinib and norimatinib in DBS samples.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In this study immunosuppressants, i.e. cyclosporin A (CyA), tacrolimus (TRL), sirolimus (SRL) and everolimus (RAD) were quantified in whole blood samples from immunosuppressant treated transplant ...recipients by an integrated on-line solid phase extraction–high performance liquid chromatography–tandem mass spectrometry (SPE–HPLC–MS/MS) system. This method has been developed to improve the following characteristics: speed, robust analysis, simultaneous determination and low cost. This can be achieved by the use of a perfusion column as an extraction cartridge in combination with a short HPLC column and highly selective and sensitive atmospheric pressure ionisation tandem mass spectrometry (API–MS/MS) in the multiple reaction monitoring (MRM) detection mode. This high throughput technique is perfectly appropriate for routine therapeutic drug monitoring (TDM) of organ transplanted patients.
Congenital disorders of N-glycosylation (CDG) are a large group of rare metabolic disorders caused by defects in the most common post-translational modification of proteins. CDGs are often difficult ...to diagnose as they are manifested with non-specific symptoms and signs. Analysis of serum transferrin (TRF) isoforms, as the classical procedure used to identify a CDG patient, enables to predict pathological steps in the N-linked glycosylation process.
We devised a new strategy based on liquid chromatography-mass spectrometry (LC-MS) for the analysis of TRF isoforms by combining a simple and fast sample preparation with a specific chromatographic cleanup/separation step followed by mass-spectrometric measurement. Single TRF isoform masses were obtained through reconstruction of multiply charged electrospray data collected by quadrupole-MS technology. Hereby, we report the first analyzed serum samples obtained from 20 CDG patients and 100 controls.
The ratio of desialylated isoforms to total TRF was calculated for patients and controls. CDG-Type I patients showed higher amounts of bi-sialo isoform (range: 6.7-29.6%) compared to controls (<5.5%, mean percentage 3.9%). CDG-Type II pattern showed an increased peak of tri-sialo isoforms. The mean percentage of tri-sialo-TRF was 9.3% (range: 2.9-12.9%) in controls, which was lower than that obtained from two patients with COG5-CDG and MAN1B1-CDG (18.5 and 24.5%). Intraday and between-day imprecisions were less than 9 and 16%, respectively, for bi-sialo- and less than 3 and 6% for tri-sialo-TRF.
This LC-MS-based approach provides a simple, sensitive and fast analytical tool for characterizing CDG disorders in a routine clinical biochemistry while improving diagnostic accuracy and speeding clinical decision-making.
Plasma lipoproteins, such as high‐density lipoprotein (HDL), can serve as carriers for a wide range of proteins that are involved in processes such as lipid metabolism, thrombosis, inflammation and ...atherosclerosis. The identification of HDL‐associated proteins is essential with regards to understanding these processes at the molecular level. In this study, a combination of proteomic approaches including 1‐DE and 2‐DE MALDI‐TOF, isotope‐coded affinity tag and Western blot analysis were employed to identify proteins associated with human HDL. To minimize potential losses of HDL‐associated proteins during isolation, a one‐step ultracentrifugation technique was applied and the quality of purified HDL was confirmed by nephelometry, high‐performance gel chromatography, and Western blot analysis. MS analysis revealed the presence of 56 HDL‐associated proteins including all known apolipoproteins and lipid transport proteins. Furthermore, proteins involved in hemostasis and thrombosis, the immune and complement system were found. In addition, growth factors, receptors, hormone‐associated proteins and many other proteins were found to be associated with HDL. Our approach thus resulted in the identification of a large number of proteins associated with HDL. The combination of proteomic technologies proved to be a powerful and comprehensive tool for the identification of proteins on HDL.
The aim of this study was to set up a robust method suitable for large-scale studies (screening) with a minimized preparation process and with reduced running costs, for measuring five enzyme ...activities on dried blood spots by a new and simplified tandem mass spectrometry-based method. After incubation, all 5 reaction mixtures, carried out separately, were stopped, combined together, and centrifuged. The cleaning-up of the injected mixture was performed through a fast online trapping step preceding a liquid chromatography/tandem mass-spectrometry measurement. This method takes only 4 min as analysis run time and without any purification following the enzymatic reaction. We assessed the effectiveness of this approach in assaying the enzymatic activities on dried blood spots from 10 patients affected by “Pompe”, 6 by “Gaucher”, 12 by “Fabry”, 3 by “Niemann-Pick” A/B, and 2 by “Krabbe” diseases. Reference values were established on 5000 healthy newborns and 300 healthy adults. All affected patients showed enzymatic activities below the normal range. In heterozygous carriers (18 for Fabry, 10 for Pompe, and 4 for Gaucher disease) the activities were slightly lower than in control subjects. The results show that the method set out in its simplicity, low costs, and low processes preparations can be fully applicable to a mass screening.
Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry ...(MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile.
Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method.
Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified.
The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.
Newborn screening for congenital adrenal hyperplasia (CAH) involves measurement of 17alpha-hydroxyprogesterone (17-OHP), usually by immunoassay. Because this testing has been characterized by high ...false-positive rates, we developed a steroid profiling method that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure 17-OHP, androstenedione, and cortisol simultaneously in blood spots.
Whole blood was eluted from a 4.8-mm (3/16-inch) dried-blood spot by an aqueous solution containing the deuterium-labeled internal standard d(8)-17-OHP. 17-OHP, androstenedione, and cortisol were extracted into diethyl ether, which was subsequently evaporated and the residue dissolved in LC mobile phase. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray. The steroids were quantified in the selected-reaction monitoring mode by use of peak areas in reference to the stable-isotope-labeled internal standard. We analyzed 857 newborn blood spots, including 14 blood spots of confirmed CAH cases and 101 of false-positive cases by conventional screening.
Intra- and interassay CVs for 17-OHP were 7.2-20% and 3.9-18%, respectively, at concentrations of 2, 30, and 50 microg/L. At a cutoff for 17-OHP of 12.5 microg/L and a cutoff of 3.75 for the sum of peak areas for 17-OHP and androstenedione divided by the peak area for cortisol, 86 of the 101 false-positive samples were within reference values by LC-MS/MS, whereas the 742 normal and 14 true-positive results obtained by conventional screening were correctly classified.
Steroid profiling in blood spots can identify false-positive results obtained by conventional newborn screening for CAH.
Bile acids play a pivotal role in the metabolism of cholesterol and lipids. Their blood concentrations are important prognostic and diagnostic indicators of hepatobiliary and intestinal dysfunction. ...This class of molecules comprises a heterogeneous group of compounds with a common cholesterol scaffold. Recently, the introduction of liquid chromatography coupled to tandem mass spectrometry methods has revealed an innovative path in the quantisation of specific bile acids in biological specimens. A robust and sensitive method has been developed based on high performance liquid chromatography separation coupled to an electrospray triple-quadrupole mass spectrometer. Human plasma samples were analysed on a C18 reverse-phase column. The elution profiles were monitored in multiple reaction-monitoring mode, quantifying and identifying each analyte by its own unique precursor to product patterns. A linear correlation over a broad range of bile acid concentrations (0.1–100 μM) was observed. The average recovery period for all of the analysed bile acids was 98±3%. Intra-day and inter-day precision averages were 2% and 5.4%, respectively. The determination was achieved within a single chromatographic run for all unconjugated, glycine-and taurine-conjugated isomeric forms of bile acids. As a proof of principle this method has been validated on a small subset of cholestatic patients (n = 7) and compared to appropriate clinical controls (n = 10). Based upon our encouraging experimental results, the described HPLC separation coupled to tandem mass spectrometry method for the analysis of bile acids in biological samples is deemed a robust and accurate procedure. Consequently, we propose this technique as a suitable candidate method for the identification and quantitation of bile acids in routine analysis.
We describe a new fully validated enantioselective LC–MS/MS method for stereospecific quantification of both the racemic forms of Warfarin (WF), Phenprocoumon and Acenocoumarol in human plasma. ...Measurement specificity was assessed by using different blank donor plasma samples, where no interfering reagent peak appeared at the retention time (RT) of the targeted analytes. Response was linear for all analytes. Typical linear regression coefficients have >0.99. The recoveries ranged from 98% to 118%. Determinations in 10 normal healthy individuals revealed a high reproducibility of RTs. These findings confer to the method suitability for large population studies.
Total homocysteine (tHcy) has emerged as an important independent risk factor for cardiovascular disease. Analytical methods are needed to accommodate the high testing volumes for tHcy and provide ...rapid turnaround.
We developed liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) method based on the analysis of 100 microL of either plasma or urine with homocystine-d(8) (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the multiple reaction monitoring mode in which tHcy and Hcy-d(4) were detected through the transition from the precursor to the product ion (m/z 136 to m/z 90 and m/z 140 to m/z 94, respectively). The retention time of tHcy and Hcy-d(4) was 1.5 min in a 2.5-min analysis.
Daily calibrations between 2.5 and 60 micromol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 micromol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for the comparison between our previous HPLC method (y) and the LC-MS/MS method (x) was y = 1.097x - 1.377 (r = 0.975; S(y|x) =1.595 micromol/L; n = 367), and for comparison between a fluorescence polarization immunoassay (Abbott IMx; y) and LC-MS/MS (x) was y = 1.039x + 0.025 (r = 0.969; S(y|x) =1.146 micromol/L; n = 367). Inter- and intraassay CVs were 2.9-5.9% and 3.6-5.3%, respectively, at mean concentrations of 3.9, 22.7, and 52.8 micromol/L. Mean recovery of tHcy was 94.2% (20 micromol/L) and 97.8% (50 micromol/L).
The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy. Reagents are inexpensive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation.
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