Improving species-specific IDMS: the advantages of triple IDMS

Triple isotope dilution mass spectrometry (triple IDMS) has been applied for the first time on protein quantification, especially on transferrin. Transferrin as an acute phase protein is a marker for several inflammation processes in the human body. Therefore, in Germany, the accurate and precise measurement of this important analyte is required. In this work, a new approach to triple IDMS is described and compared to double IDMS. Also, complete uncertainty budgets for both methods were set up to demonstrate the ability of this method to be used as a reference procedure. The relative expanded uncertainty (k?=?2) for triple IDMS (3.6 %) is smaller than the one for double IDMS (4.0 %). The content of transferrin found in the human serum reference material ERM-DA470k/IFCC ((2.41?±?0.08) g/kg) with both methods was in good agreement with each other and with the certificate. For triple IDMS ((2.426?±?0.086) g/kg) and for double IDMS ((2.317?±?0.092) g/kg), transferrin was determined. Although triple IDMS is a little more time consuming compared to double IDMS, there is the advantage that the isotopic composition of the spike material does not have to be determined. This is very useful especially in case of a marginal isotopic enrichment in the spike or problems with the accurate measurement of the spike isotope ratio.

Construction of a Standard Reference Plasmid for Detecting GM Cottonseed Meal

A plasmid was constructed for quantification of genetically modified (GM) cottonseed meal in the gene-specific level. The Cry1Ab/c gene was connected with the Sad1 gene by fusion PCR. The fusion gene was cloned into the pMD®19-T Simple Vector. The plasmid DNA was then digested with a restriction endonuclease SmaI to reduce the characteristic differences between the plasmid DNA and genomic DNA. For a rough quantitative analysis of GM cotton meal contents, a rapid method for measurement of the copy numbers of the transgenic Cry and cotton endogenous Sad1 gene using a real-time PCR system with the plasmid DNA as a calibrator was established. The inter-run and intra-run coefficients of variation were less than 1.48% and 2.36%, respectively. The limits of detection and quantitation of the Cry and Sad1 genes were 9 and 91 copies of pMDCS, respectively. These results prove that the standard plasmid represents a valuable alternative to genomic DNA as a certified reference material for the quantification of GM cotton and is a useful tool to establish a feasible identification management for GM cottonseed meal content in the feed industry.     

Comparison of GC-ICP-MS and HPLC-ICP-MS for species-specific isotope dilution analysis of tributyltin in sediment after accelerated solvent extraction

This study describes a direct comparison of GC and HPLC hyphenated to ICP–MS determination of tributyltin (TBT) in sediment by species-specific isotope dilution analysis (SS-IDMS). The certified reference sediment PACS-2 (NRC, Canada) and a candidate reference sediment (P-18/HIPA-1) were extracted using an accelerated solvent extraction (ASE) procedure. For comparison of GC and LC methods an older bottle of PACS-2 was used, whilst a fresh bottle was taken for demonstration of the accuracy of the methods. The data obtained show good agreement between both methods for both the PACS-2 sediment (LC–ICP–IDMS 828±87 ng g^–1 TBT as Sn, GC–ICP–IDMS 848±39 ng g^–1 TBT as Sn) and the P-18/ HIPA-1 sediment (LC–ICP–IDMS 78.0±9.7 ng g^–1 TBT as Sn, GC–ICP–IDMS 79.2±3.8 ng g^–1 TBT as Sn). The analysis by GC–ICP–IDMS offers a greater signal-to-noise ratio and hence a superior detection limit of 0.03 pg TBT as Sn, in the sediment extracts compared to HPLC–ICP–IDMS (3 pg TBT as Sn). A comparison of the uncertainties associated with both methods indicates superior precision of the GC approach. This is related to the better reproducibility of the peak integration, which affects the isotope ratio measurements used for IDMS. The accuracy of the ASE method combined with HPLC–ICP–IDMS was demonstrated during the international interlaboratory comparison P-18 organised by the Comité Consultatif pour la Quantité de Matière (CCQM). The results obtained by GC–ICP–IDMS for a newly opened bottle of PACS-2 were 1087±77 ng g^–1 Sn for DBT and 876±51 ng g^–1 Sn for TBT (expanded uncertainties with a coverage factor of 2), which are in good agreement with the certified values of 1090±150 ng g^–1 Sn and 980±130 ng g^–1 Sn, respectively.     

Comparison of LC–UV, LC–ELSD and LC–MS Methods for the Determination of Sesquiterpenoids in Various Species of Artemisia

Simple and specific analytical methods for the quantitative determination of sesquiterpenoids from various species of Artemisia plant samples were developed. By LC–UV, LC–ELSD, the separation was achieved by reversed-phase chromatography on a C18 column with water and acetonitrile both containing 0.025% trifluoroacetic acid as the mobile phase. In the LC–MS system, trifluoroacetic acid was replaced by 0.1% formic acid. The wavelength used for quantification of sesquiterpenoids with a diode array detector was 205 nm. The limits of detection by LC–MS was found to be 5, 10, 25, 50, 50 ng mL^?1. The limits of detection by LC–UV and LC–ELSD were found to be 5.0, 3.0, 100, 100, 7.5 μg mL^?1, by LC–UV and 50, 25, 30, 100 and 75 μg mL^?1 by LC–ELSD. LC–mass spectrometry coupled with electrospray ionization (ESI) interface is described for the identification and quantification of sesquiterpenoids in various plant samples. This method involved the use of the [M + H]⁺ ions of sesquiterpenoids in the positive ion mode with extractive ion monitoring.     

Development of a certified reference material for the determination of perfluorooctanoic acid

A certified reference material (CRM) for the determination of perfluorooctanoic acid (PFOA) has been issued as NMIJ CRM 4056-a by the National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (NMIJ/AIST). Purity (kg kg^?1) based on a titration method was determined by subtracting the mass fractions of impurities measured using liquid chromatography/mass spectrometry (LC/MS) from those of acids expressed as PFOA measured by a neutralization potentiometric titration. To validate an obtained result, purity based on a mass balance method was determined by subtracting the mass fractions of impurities, measured using LC/MS, Karl-Fischer titration (KFT), and vacuum evaporation, from 1 kg kg^?1. Results from both titration and mass balance methods were in agreement within the accepted limits of uncertainty. The certified purity of NMIJ CRM 4056-a was determined to be 0.959 kg kg^?1, calculated as the mean of the results obtained with the two methods. The standard uncertainty of the certified purity was evaluated from purity evaluations as well as from sample homogeneity and stability obtained from LC/MS and KFT analyses. Consequently, the expanded uncertainty was estimated to be 0.005 kg kg^?1 with a coverage factor of k = 2.     

Bias reduction in the quantitative analysis of a target analyte present in a limited quantity in human plasma using dual-mode heart-cutting two-dimensional liquid chromatography coupled with isotope dilution mass spectrometry

Dual-mode heart-cutting two-dimensional liquid chromatography (DMHC 2D-LC) was applied to isotope dilution mass spectrometry (IDMS) to reduce the bias in the quantitative analysis of a target analyte present in a limited quantity in human plasma. Based on a Waters I-Class LC system, the DMHC 2D-LC system was operated in one- and two-dimensional modes to facilitate the determination of heart-cutting time and the efficient trapping of the target LC eluate. Experiments to determine the feasibility of coupling with IDMS were performed with triple quadrupole mass spectrometry using folic acid standards and/or ¹³C₅-folic acid. To validate the performance of the DMHC 2D-LC/IDMS system on a complex sample, human plasma was analyzed for folic acid and the result was compared with that obtained using conventional single-column LC. The total run time of the DMHC 2D-LC system was 20 min, the same as that of the single-column LC system. The peak profile of the spiked ¹³C₅-folic acid obtained with single-column LC/MS was affected by matrix effects, but resolved with DMHC 2D-LC/MS, thus improving the accuracy of the analysis. The DMHC 2D-LC/IDMS system showed reliable performance in analyzing the target analyte in human plasma, eliminating matrix effects and saving analysis time.     

An Effective High-Performance Liquid Chromatographic–Mass Spectrometric Assay for Catecholamines, as the N(O,S)-Ethoxycarbonyl Ethyl Esters, in Human Urine

The purpose of this study was to develop a simple and accurate analytical method for determination of norepinephrine, epinephrine, and dopamine in urine. The method involves liquid–liquid extraction then liquid chromatography–mass spectrometry (LC–MS). Alkyl chloroformate derivatives were prepared, as the N(O,S)-alkoxycarbonyl alkyl esters of the analytes, in the aqueous samples. The optimum derivatizing reagent for preparation of the N(O,S)-alkoxycarbonyl alkyl esters was chosen by comparing the efficiency of LC of the derivatized analytes after liquid–liquid extraction. The optimum conditions for liquid–liquid extraction from the aqueous matrix were pH 3.0, no salt, and diethyl ether as extraction solvent. Limits of detection (LOD) were 0.5 ng mL^?1 for dopamine and epinephrine and 0.1 ng mL^?1 for norepinephrine. Limits of quantification (LOQ) for urine samples were 1.0 ng mL^?1 for all three compounds. The precision of intra- and inter-day assays was 1.65–581 and 7.17–9.73% (relative standard deviation, RSD), respectively. The range of inaccuracy for intra- and inter-day assays was ?6.47 to 11.9% and ?7.5 to 7.76% (bias) at concentrations of 5 and 50 ng mL^?1, respectively.     

Novel concept for the mass spectrometric determination of absolute isotopic abundances with improved measurement uncertainty: Part 1 – theoretical derivation and feasibility study

The development of a new method for the experimental determination of absolute isotopic abundances using a modified isotope dilution mass spectrometry (IDMS) technique is described. The intention and thus main application will be the quantification of molar masses M of highly enriched materials with improved measurement uncertainty (U_rel(M) ≈ 10^?8 with k = 2). In part 1 of the current work, the theoretical foundation of the new method and its mathematical derivation is shown in detail, while part 2 will cover the experiments based on the new method described. Its core idea is the introduction of a virtual element (VE) consisting of all isotopes but the one having the largest or smallest abundance. IDMS is used to determine the mass fraction of this VE in its matrix, namely the element itself. A new set of equations serve to calculate all isotopic abundances (even the large one omitted with the introduction of the VE) merely from the mass fraction of the VE. A comprehensive uncertainty budget according to the Guide to the Expression of Uncertainty in Measurement (GUM) was set up in order to discuss and validate the novel concept. The hypothetical input data of the uncertainty budget were estimated to resemble a silicon material highly enriched with respect to ²⁸Si used in the context of the international Avogadro Project. Considering the calculated results, the experimental determination of the molar mass of the above mentioned silicon seems very promising. As far as the authors know, this will be the first time IDMS was applied to determine a molar mass.     

Determination of Shikimic Acid in Fruits of Illicium Species and Various Other Plant Samples by LC–UV and LC–ESI–MS

A simple and specific analytical method for the quantitative determination of shikimic acid from the methanol extract of the fruits of Illicium species and from various plant samples was developed. The LC–UV separation was achieved by reversed-phase chromatography on a C18 column with potassium dihydrogen phosphate and methanol as the mobile phase. In the LC–MS method, the separation was achieved by a C12 column using water and acetonitrile, both containing 0.1% acetic acid as the mobile phase. The methods were successfully used to study the percentage compositions of shikimic acid present in nine species of Illicium and various other plant samples. The detector response was linear with concentrations of shikimic acid in the range from 1.0–500.0 μg mL^?1 by LC–UV and 100–1000 ng mL^?1 by LC–MS. Mass spectrometry coupled with electrospray ionization interface is described for the identification of shikimic acid in various plant samples. This method involved the use of the [M-H]^? ions of shikimic acid at m/z 173.0455 (calculated mass) in the negative ion mode with extractive ion monitoring.     

LC Method for Quantification of Lutein in Rat Plasma: Validation, and Application to a Pharmacokinetic Study

A reversed-phase LC method has been developed for quantitative analysis of lutein in rat plasma and applied to a study of the pharmacokinetics of lutein in rats. From a variety of compounds and solvents tested, astaxanthin was selected as the internal standard. n-Hexane was found to be the best solvent for extracting lutein from plasma. LC analysis of the extracts was performed on a C₁₈ column equipped with a guard pre-column. Linearity was good (r > 0.99) over the range 10–100 ng mL^?1. Recovery from plasma was 82.7–92.9% the intra-day and inter-day precision were always better than 3%. The limits of detection (LOD) and quantification (LOQ) were 2.5 and 8.3 ng mL^?1, respectively. The LC method was used to quantify lutein and zeaxanthin in rat plasma in a 36-h pharmacokinetic study in which experimental rats received a single oral dose of lutein (20 mg kg^?1). The results are presented.     

LC–MS–MS Determination of Troxerutin in Plasma and Its Application to a Pharmacokinetic Study

A highly sensitive liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the determination of troxerutin in human plasma using tramadol as internal standard (IS) has been developed and validated. Sample preparation involved liquid–liquid extraction with ethyl acetate–isopropanol (95:5, v/v). The analyte and IS were separated by RP–LC with gradient elution using 10 mM ammonium acetate containing 0.1% formic acid and methanol at a flow rate of 0.9 mL min^?1. LC–MS–MS in the positive ion mode employed multiple reaction monitoring of the transitions at m/z 743.2→435.3 and m/z 264.1→58.0 for troxerutin and IS, respectively. The assay was linear in the concentration range 0.01–10 ng mL^?1 with precision and accuracy within assay variability limits as per FDA guidelines. The assay was successfully applied to a pharmacokinetic study involving oral administration of 300 mg troxerutin to eight healthy Chinese volunteers.     

Development and Validation of Atorvastatin by LC–ESI–MS and Application in Bioequivalence Research in Healthy Chinese Volunteers

The aim of this research was to develop a sensitive liquid chromatographic–electrospray ionization–mass spectrometric (LC–MS) method for direct measurement of the concentration of Atorvastatin in human plasma. Plasma samples (1 mL) were extracted with 3 mL ethyl acetate, and by a simple reversed-phase chromatography. Pitavastatin was used as internal standard (IS). The LOQ was 0.25 ng mL^?1 (RSD 4.24%). The assay was linear from 0.25–20 ng mL^?1. And the correlation coefficient for the calibration regression line was 0.9996 or better. Intra-day and inter-day accuracy were better than 15%. The method has been successfully used for a pharmacokinetic study with human subjects. A two-period crossover designed bioequivalence research was also progressed in healthy Chinese volunteers. Among the pharmacokinetic data obtained, T _max was 1.36 ± 0.68 h for reference formulation and 0.81 ± 0.54 h for test formulation. C _max was 8.54 ± 5.06 ng mL^?1 for reference formulation and 9.54 ± 3.68 ng mL^?1 for test formulation. t _1/2 was 8.50 ± 2.74 h for reference formulation and 9.24 ± 3.17 h for test formulation. AUC _0?48h was 54.77 ± 21.82 h ng mL^?1 for reference formulation and 55.66 ± 20.91 h ng mL^?1 for test formulation. The method was successfully applied to the study of pharmacokinetics of Atorvastatin in healthy Chinese volunteers.     

Development and Validation of a Stability-Indicating LC Method for Simultaneous Analysis of Aceclofenac and Paracetamol in Conventional Tablets and in Microsphere Formulations

A stability-indicating reversed-phase LC method for analysis of aceclofenac and paracetamol in tablets and in microsphere formulations has been developed and validated. The mobile phase was 80:20 (v/v) methanol–phosphate buffer (10 mM at pH 2.5 ± 0.02). UV detection was at 276 nm. The method was linear over the concentration ranges 16–24 and 80–120 μg mL^?1 for aceclofenac and paracetamol, respectively, with recovery in the range 100.9–102.22%. The limits of detection and quantitation for ACF were 0.0369 and 0.1120 μg mL^?1, respectively; those for PCM were 0.0631 and 0.1911 μg mL^?1, respectively.     

A new liquid chromatography–fluorescence method for determination of perfluorooctanesulphonyl fluoride upon derivatisation with 1-naphthol

Perfluorooctanesulphonyl fluoride (PFOSF), as a main precursor of perfluorooctanesulphonate (PFOS) that is ubiquitous in the environment, has been released to the environment with substantial quantity. Determination of PFOSF presents significant analytical challenges for using liquid chromatography with UV (LC–UV) and fluorescence detection (LC–FLD) due to the lack of chromophore in the molecular structure. In this study, a new method was developed by derivatising PFOSF with 1-naphthol to form 1-naphthylperfluorooctanesulphonate (NPFOS), which allowed rapid qualitative and quantitative analysis using LC–UV and LC–FLD. The derivatising product was confirmed from the analyses by proton nuclear magnetic resonance and quadrupole–time of flight mass spectrometry. The LC–FLD method demonstrated good linearity in the NPFOS concentration range from 20 pg µL^?1 to 20 ng µL^?1 with a correlation coefficient better than 0.999, with the instrument detection limit of 1.5 pg µL^?1.     

Nickel tetrathiooxalate as a cathode material for potassium batteries

We report a nickel tetrathiooxalate (NiTTO) coordination polymer as a cathode material for potassium batteries. In a potential range of 1.3–3.6 V vs. K⁺/K, the specific capacity of the material is 209 mA h g^?1 at a current density of 0.1 A g^?1, which roughly corresponds to the two-electron reduction of polymer repeating units. The charge–discharge mechanisms of NiTTO in potassium cells were examined using operando Raman spectroscopy.     

Simple Determination of 4-Methylimidazole in Soft Drinks by Headspace SPME GC–MS

A simple method to detect 4-methylimidazole in soft drinks is described. This method is based on headspace solid-phase micro-extraction and gas chromatography–mass spectrometry (HS-SPME GC–MS). The HS-SPME parameters (selection of fiber, extraction temperature, heating time, and pH) were optimized and selected. Under the established condition, the detection and the quantification limit were 1.9 and 6.0 μg L^?1 using 4 mL of the liquid sample, respectively. The relative standard deviation for five independent determinations at 100.0 and 500.0 μg L^?1 was less than 8 %. The calibration curve was y = 0.6027x–0.0033 with a linearity of r ² = 0.997. Using the proposed method, the levels of 4-MEI were detected in a range from 94.0 to 324.8 μg L^?1. The comparison of liquid chromatography tandem mass spectrometry (LC–MS/MS) with the proposed method was performed and the agreement with LC–MS/MS for all samples was acceptable.     

Determination of reference interval values for inorganic elements in whole blood samples of humans and laboratory animals by X-ray fluorescence spectrometry

Inorganic elements are responsible for essential bodily functions, such as osmotic regulation, cardiac frequency and contractibility, blood clotting and neuromuscular excitability. The determination of inorganic elements in corporeal fluids such as blood, serum, plasma and urine is used as a monitor for a part or the whole organism; their values, then, are compared with reference interval values. In this study, the energy dispersive X-ray fluorescence spectrometry (EDXRF), applying the Fundamental Parameters method, for the determination of inorganic elements in whole blood samples from humans and laboratory animals, was used. Peripheral blood samples were collected and, before coagulation, 100 μL of sample were deposited onto Whatman No. 41 filter paper and dried, using infrared spotlight. The reference interval values for healthy Brazilian population of Na were found to be 1,788–1,826 μg g^?1, of Mg 63–75 μg g^?1, of P 602–676 μg g^?1, of S 1,519–1,718 μg g^?1, of Cl 2,743–2,867 μg g^?1, of K 1,508–1,630 μg g^?1, of Ca 214–228 μg g^?1, of Fe 170–184 μg g^?1, of Cu 4–6 μg g^?1 and of Zn 1–3 μg g^?1. The reference interval values for golden hamster (Mesocricetus auratus) of Na were found to be 1,714–1,819 μg g^?1, Mg 51–79 μg g^?1, P 970–1,080 μg g^?1, S 1,231–1,739 μg g^?1, Cl 2,775–2,865 μg g^?1, of K 1,968–2,248 μg g^?1, of Ca 209–257 μg g^?1, of Fe 145–267 μg g^?1, of Cu 4–6 μg g^?1 and of Zn 3–5 μg g^?1. A comparative study between EDXRF and instrumental neutron activation analysis data was carried out and the results for both techniques are statistically equal (α = 0.05). The results contribute for the establishment of reference interval values for Na, Mg, P, S, Cl, K, Ca, Cu and Zn in the healthy Brazilian population and the referred laboratory animal species.     

Ion-Pair LC Method for Simultaneous Determination of Aliskiren Hemifumarate,Amlodipine Besylate and Hydrochlorothiazide in Pharmaceuticals

A rapid and precise LC method was developed for the simultaneous determination of aliskiren hemifumarate (ALS), amlodipine besylate (AML) and hydrochlorothiazide (HCZ) using acetonitrile:25 mM octane sulfonic acid sodium salt monohydrate in water (60:40 v/v) as the mobile phase. The flow rate was maintained at 1.2 mL min^?1 on a stationary phase composed of Supelco, Discovery^® HS (C18) column (25 cm × 4.6 mm, 5 μm). Isocratic elution was applied throughout the analysis. Detection was carried out at λ _max (232 nm) at ambient temperature. The method was validated according to ICH guidelines. Linearity, accuracy and precision were satisfactory over the concentration ranges of 32–320, 2–44 and 4–64 μg mL^?1 for ALS, AML and HCZ, respectively. LOD and LOQ were estimated and found to be 0.855 and 2.951 μg mL^?1, respectively, for ALS, 0.061 and 0.202 μg mL^?1, respectively, for AML as well as 0.052 and 0.174 μg mL^?1, respectively, for HCZ. The method was successfully applied for the determination of the three drugs in their co-formulated tablets. The results were compared statistically with reference methods and no significant difference was found. The developed method is specific and accurate for the quality control and routine analysis of the cited drugs in pharmaceutical preparations.     

LC–MS–MS Analysis of Mirtazapine in Plasma, and Determination of Pharmacokinetic Data for Rats

A sensitive LC–MS–MS method with electrospray ionization has been developed for analysis of mirtazapine in rat plasma. After addition of diazepam as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 × 4.6 mm, 5 μm particle, ODS column with 84:16 (v/v) methanol–water containing 0.1% ammonium acetate and 0.01% glacial acetic acid as mobile phase. LC–MS–MS was performed in selected-ion-monitoring (SIM) mode using target fragment ions m/z 195.09 for mirtazapine and m/z 192.80 for the IS. Calibration plots were linear over the range of 0.516–618.8 ng mL^?1. The lower limit of quantification was 0.516 ng mL^?1. Intra-day and inter-day precision were better than 12.6 and 8.8%, respectively. Mean recovery of mirtazapine from plasma was in the range 87.41–90.06%; average recovery was 88.40% (RSD 3.95%). Significant gender differences between mirtazapine pharmacokinetic data were observed in this study.