Determination of melamine in milk powder using gas chromatography-high-resolution isotope dilution mass spectrometry

A GC-high-resolution isotope dilution MS (IDMS) method for the quantification of melamine in milk powder is described. The developed technique is compared to the LC-IDMS/MS technique, typically used for the determination of melamine in various matrices. The accuracy of the GC-high-resolution IDMS method was demonstrated when a small degree of equivalence was obtained in a regional comparative study involving the determination of melamine in milk powder.     

Development of melamine certified reference material in milk using two different isotope dilution mass spectrometry techniques

A new certified reference material (CRM) of melamine in milk GLHK-11-02 was developed aiming to address the great demand from the testing community after the melamine crises. The material was prepared by adding an appropriate quantity of melamine into the skimmed milk samples and the final product was in the form of fine lyophilized powder. Characterization of the material relied on two newly developed gravimetric isotope dilution mass spectrometry (IDMS) methods, one using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and another gas chromatography-mass spectrometry (GC-MS). Experimental parameters with crucial effects on the performance of the two IDMS methods were thoroughly investigated. These included purity of standard used, equilibration time of isotopes, efficiency of extraction methods as well as possible interferences from the matrix and melamine analogues. Precision was found to be excellent with a coefficient of variation of 2.5% for the LC-IDMS/MS (n=46) and 1.9% for the GC-IDMS (n=30) respectively. Using one-tail Student's t-test at 95% confidence interval, analytical data sets generated from the two methods were found to exhibit no significant difference. Measurement accuracy of the methods was further verified through an Asia Pacific Metrology Program (APMP) pilot study. Analytical results of the present LC-IDMS/MS for the two milk test samples at the concentration level of about 0.45 and 3.5 mg kg(-1) were proven to be very good. There were excellent overlaps between our results and the assigned reference values, and the absolute deviation was less than 3.2%. Both the LC-IDMS/MS and GC-IDMS methods were shown to be sufficiently reliable and accurate for certification of the melamine CRM. Certified value of melamine in dry mass fraction in GLHK-11-02 was 1.14 mg kg(-1). Expanded uncertainty due to sample inhomogeneity, long term and short term stability and variability in the characterization procedure was at 7.1% or 0.08 mg kg(-1). The CRM is primarily used to provide a complete method validation for and to improve the technical competence of melamine analysis to food and chemical testing laboratories.     

Rapid simultaneous determination of dexamethasone and betamethasone in milk by liquid chromatography tandem mass spectrometry with isotope dilution

A simple, sensitive and reliable analytical method for the rapid simultaneous determination of dexamethasone and betamethasone in milk by high performance liquid chromatography–negative electrospray ionization tandem mass spectrometry (HPLC–NESI-MS/MS) with isotope dilution was developed. Samples were directly purified through C₁₈ cartridge. Then the eluate was dried under nitrogen and residues were dissolved in mobile phase. Samples were analyzed by HPLC–MS/MS on a Hypercarb graphite column with a mixture of acetonitrile–water–formic acid as mobile phase. The samples were quantified using dexamethasone-D₄ as an internal standard. The procedure was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), trueness, precision, linearity and stability. The method is demonstrated to be suitable for the determination of dexamethasone and betamethasone in milk. The total time required for the analysis of one sample was about 35 min.     

Simultaneous determination of clenbuterol, salbutamol and ractopamine in milk by reversed-phase liquid chromatography tandem mass spectrometry with isotope dilution

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of clenbuterol (CLB), salbutamol (SAL) and ractopamine (RAC) in milk by ultra high performance liquid chromatography–positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS) with isotope dilution. Samples were directly purified through HLB cartridge. Then the eluate was dried under nitrogen and residues were redissolved in mobile phase. Samples were analyzed by LC–MS/MS on an Acquity UPLC^® BEH C₁₈ column with gradient elution. The samples were quantified using clenbuterol-D₉, salbutamol-D₃ and ractopamine-D₆ as internal standards. The proposed method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity, robustness and stability. CCα values were 0.054, 0.006 and 0.008 μg/kg for CLB, SAL and RAC, respectively. CCβ values were 0.058, 0.007 and 0.009 μg/kg for CLB, SAL and RAC, respectively. The mean recoveries, repeatability (expressed as coefficient of variation, CV_r), and reproducibility (CV_R) varied from 95.8 to 106.2%, from 3.60 to 6.44% (CVr), and from 4.77 to 7.53% (CV_R), respectively. The method is demonstrated to be suitable for the determination of clenbuterol, salbutamol and ractopamine in milk. The total time required for the analysis of one sample, including sample preparation, was about 45 min.     

Quantification of human growth hormone by amino acid composition analysis using isotope dilution liquid-chromatography tandem mass spectrometry

We describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-HPLC-mass spectrometry (ID-HPLC-MS) method. Sample purity was confirmed using capillary zone electrophoresis, HPLC and MS. The analyte protein, human growth hormone (hGH), was effectively hydrolyzed by incubation with 8 M hydrochloric acid at 130 °C for 48 h, where at least 1 μM of hGH was treated to avoid possible degradation of released amino acids during hydrolysis. Using a reversed-phase column, the analytes (isoleucine, phenylalanine, proline and valine) were separated within 5 min using an isocratic eluent comprising 10% acetonitrile containing 0.1% trifluoroacetic acid. The detection limit (signal to noise ratio of 3) of amino acids was 5.5-6.2 fmol per injection. The quantification precision (RSD) of amino acids for intra- and inter-day assays was less than 0.98% and 0.39%, respectively. Comparison with other biochemical and instrumental methods revealed substantially higher accuracy and reproducibility of the ID-HPLC-MS/MS method as expected. The optimized hydrolysis and analytical conditions in our study were suitable for accurate quantification of hGH.     

Evaluation of matrix effect in isotope dilution mass spectrometry based on quantitative analysis of chloramphenicol residues in milk powder

In the present study, we developed a comprehensive strategy to evaluate matrix effect (ME) and its impact on the results of isotope dilution mass spectrometry (IDMS) in analysis of chloramphenicol (CAP) residues in milk powder. Stable isotope-labeled internal standards do not always compensate ME, which brings the variation of the ratio (the peak area of analyte/the peak area of isotope). In our investigation, impact factors of this variation were studied in the extraction solution of milk powder using three mass spectrometers coupled with different ion source designs, and deuterium-labeled chloramphenicol (D5-CAP) was used as the internal standard. ME from mobile phases, sample solvents, pre-treatment methods, sample origins and instruments was evaluated, and its impact on the results of IDMS was assessed using the IDMS correction factor (θ). Our data showed that the impact of ME of mobile phase on the correction factor was significantly greater than that of sample solvent. Significant ion suppression and enhancement effects were observed in different pre-treated sample solutions. The IDMS correction factor in liquid–liquid extraction (LLE) and molecular imprinted polymer (MIP) extract with different instruments was greater or less 1.0, and the IDMS correction factor in hydrophilic lipophilic balance (HLB) and mix-mode cation exchange (MCX) extract with different instruments was all close to 1.0. To the instrument coupled with different ion source design, the impact of ME on IDMS quantitative results was significantly different, exhibiting a large deviation of 11.5%. Taken together, appropriate chromatographic conditions, pre-treatment methods and instruments were crucial to overcome ME and obtain reliable results, when IDMS methods were used in the quantitative analysis of trace target in complex sample matrix.     

Calibration graphs in isotope dilution mass spectrometry

Isotope-based quantitation is routinely employed in chemical measurements. Whereas most analysts seek for methods with linear theoretical response functions, a unique feature that distinguishes isotope dilution from many other analytical methods is the inherent possibility for a nonlinear theoretical response curve. Most implementations of isotope dilution calibration today either eliminate the nonlinearity by employing internal standards with markedly different molecular weight or they employ empirical polynomial fits. Here we show that the exact curvature of any isotope dilution curve can be obtained from three-parameter rational function, y = f(q) = (a₀ + a₁q)/(1 + a₂q), known as the Padé[1,1] approximant. The use of this function allows eliminating an unnecessary source of error in isotope dilution analysis when faced with nonlinear calibration curves. In addition, fitting with Padé model can be done using linear least squares.     

Rapid determination of melamine and cyanuric acid in milk powder using direct analysis in real time-time-of-flight mass spectrometry

The use of fast semi-automated method employing direct analysis in real time (DART) ion source coupled to time-of-flight mass spectrometry (TOFMS) for analysis of melamine (MEL) and cyanuric acid (CYA) in milk powder and milk based products has been demonstrated in this study. Simple sample extraction procedure employing methanol–5% aqueous formic acid mixture, which enabled disruption of melamine–cyanurate complex, was followed by direct, high-throughput (30 s per run) examination of sample extracts spread on a glass rod by mass spectrometry under ambient conditions, without any prior chromatographic separation. After optimization of instrument parameter settings, limits of detection (LODs) 170 and 450 μg kg⁻¹ were achieved for MEL and CYA, respectively. In the final phase of study, the possibility of minimizing spectral interference, thus improving method performance characteristics through the use of ultrahigh resolving power offered by Orbitrap based mass analyzer is demonstrated.     

Determination of phthalate esters in Chinese spirits using isotope dilution gas chromatography with tandem mass spectrometry

Phthalate esters are additives used in polyvinylchloride and are found as contaminants in many food products. An isotope dilution mass spectrometry technique has been developed for accurate analysis of 16 phthalate esters in Chinese spirits by adopting the 16 corresponding isotope‐labeled phthalate esters. The ethanol in the spirit sample was first removed by heating with a water bath at 100°C with a stream of nitrogen, after which the residue was extracted with n‐hexane twice. The phthalates collected were identified and quantified by gas chromatography with tandem mass spectrometry in multiple reaction monitoring mode. The spiking recoveries of 16 analytes ranged from 94.3 to 105.3% with relative standard deviation values of <6.5%. The detection limits for 16 analytes were <10.0 ng/g. The expanded relative uncertainties were from 3.0 to 14%. A survey was performed on Chinese spirits from the market. Six of the nine analyzed samples were contaminated by phthalates. Di‐n‐butyl phthalate and di‐2‐ethylhexyl phthalate showed higher detection frequency and concentrations. This isotope dilution gas chromatography with tandem mass spectrometry method is simple, rapid, accurate, and highly sensitive, which qualifies as a candidate reference method for the determination of phthalates in spirits.     

Analysis of urinary biomarkers for exposure to alkyl benzenes by isotope dilution gas chromatography-mass spectrometry

A validated GC-MS method for the analysis of urinary metabolites of alkyl benzenes is reported. Metabolites for exposure to toluene, xylene and ethylbenzene were analyzed simultaneously using stable isotope substituted internal standards. The method entailed acidic deconjugation of urine samples followed by extractive alkylation with pentafluorobenzyl bromide as alkylating agent. The resulting pentafluorobenzyl derivatives of ortho-, meta-, para-cresol, mandelic acid (MA), hippuric acid (HA) and ortho-, meta-, para-methylhippuric acid (MHA) were then quantified by SIM. Optimized reaction conditions for the extractive alkylation step are reported. The derivatives were found to be sufficiently stable for overnight batch analysis. The LODs were below 0.1 micromol/L for the cresols and below 1 micromol/L for MA and the HAs. Within-batch precision for o-MHA was 7%, for m-MHA 5%, for p-MHA 5.2% and below 5% for the rest of the analytes.     

Paradigms in isotope dilution mass spectrometry for elemental speciation analysis

Isotope dilution mass spectrometry currently stands out as the method providing results with unchallenged precision and accuracy in elemental speciation. However, recent history of isotope dilution mass spectrometry has shown that the extent to which this primary ratio measurement method can deliver accurate results is still subject of active research. In this review, we will summarize the fundamental prerequisites behind isotope dilution mass spectrometry and discuss their practical limits of validity and effects on the accuracy of the obtained results. This review is not to be viewed as a critique of isotope dilution; rather its purpose is to highlight the lesser studied aspects that will ensure and elevate current supremacy of the results obtained from this method.     

Determination of 19-norandrosterone in CCQM-P68. NIST results using an isotope dilution liquid chromatography/tandem mass spectrometry method

The US National Institute of Standards and Technology (NIST) participated in an international interlaboratory study under the auspices of the Consultative Committee for Amount of Substance (CCQM) for the determination of 19-norandrosterone (19-NA) in urine, the principal metabolite of nandrolone and certain other synthetic testosterone substitutes banned for use by the World Antidoping Agency (WADA). Prior to this study, NIST developed a candidate reference measurement procedure based upon isotope dilution liquid chromatography/tandem mass spectrometry. This method was applied to a urine sample distributed to the participants in the study by the Australian National Measurement Institute, Pymble, Australia (NMIA). The NIST results were in very good agreement with those from the other participants, all of whom used methods based upon gas chromatography/mass spectrometry. All known significant sources of uncertainty were evaluated, resulting in a relative expanded uncertainty of less than 5% (coverage factor k = 2).     

Determination of uranium in marine sediment pore waters by isotope dilution inductively coupled plasma mass spectrometry

Inductively coupled plasma mass spectrometry (ICP-MS) was used in an isotope dilution mode to assay small-volume (0.25 ml) sediment pore waters for their uranium contents, using ²³⁶U as the spike. The only pretreatment required was a simple dilution by a factor of 20, which gave sufficient volume for three replicate analyses per sample. Rapid and accurate results were obtained for a variety of samples and standards, ranging in concentration from 0.05 to 10 ng U ml^?1. A suite of 30 samples can be analysed in less than 6 h by this method. The relative standard deviation was better than 1.9%, with a detection limit, based on 3σ background, of 2 pg U ml^?1 in solution (40 pg ml^?1 in samples). Sea water is a difficult matrix for ICP-MS and thus the method is generally suitable for uranium determinations in many other sample solutions.     

Microwave-assisted extraction and determination of cyanuric acid residue in pet food samples by liquid chromatography-tandem mass spectrometry

Cyanuric acid (CYA) is attracting more attention due to its potential toxicity. In the present work, microwave-assisted extraction method in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was proposed for the determination of CYA in pet food samples. Among different solvents, diethylamine-acetonitrile-water mixture (1:5:4, v/v) was found to be the best one as the extractant due to the strong polarity of CYA in the pet food. An internal standard, (13) C(3) -labeled CYA, was used in the extractions. The separation was performed on a MERCK ZIC HILIC column (150 mm × 2.1 mm id, 5 μm) with gradient elution of 20 mM ammonium acetate solution-acetonitrile. CYA was well retained (Rt = 5.10 min) and eluted with good peak shape. The method could respond linearly with CYA at concentrations from 1.0 to 50 ng/mL with a quantification limit of 0.25 mg/kg. The intra- and inter-day precision was less than 4.0% and the recovery of the assay was in the range of 90.4-108.1%. In the analysis of practical spiked pet food samples, the new method yielded satisfactory results. Due to its simplicity and accuracy the straightforward method is particularly suitable for routine CYA detection.     

Determination of trace sulfur in biodiesel and diesel standard reference materials by isotope dilution sector field inductively coupled plasma mass spectrometry

A method is described for quantification of sulfur at low concentrations on the order of mg kg⁻¹ in biodiesel and diesel fuels using isotope dilution and sector field inductively coupled plasma mass spectrometry (ID-SF-ICP-MS). Closed vessel microwave-assisted digestion was employed using a diluted nitric acid and hydrogen peroxide decomposition medium to reduce sample dilution volumes. Medium resolution mode was employed to eliminate isobaric interferences at ³²S and ³⁴S related to polyatomic phosphorus and oxygen species, and sulfur hydride species. The method outlined yielded respective limits of detection (LOD) and limits of quantification (LOQ) of 0.7 mg kg⁻¹ S and 2.5 mg kg⁻¹ S (in the sample). The LOD was constrained by instrument background counts at ³²S but was sufficient to facilitate value assignment of total S mass fraction in NIST SRM 2723b Sulfur in Diesel Fuel Oil at 9.06 ± 0.13 mg kg⁻¹. No statistically significant difference at a 95% confidence level was observed between the measured and certified values for certified reference materials NIST SRM 2773 B100 Biodiesel (Animal-Based), CENAM DRM 272b and NIST SRM 2723a Sulfur in Diesel Fuel Oil, validating method accuracy.     

High accuracy analysis by isotope dilution mass spectrometry using an iterative exact matching technique

The selection of an appropriate isotope dilution mass spectrometry technique for the practical application of this potentially primary method of analysis is highly important. The NARL approach for the application of the exact matching double isotope dilution mass spectrometry technique developed by Henrion is presented. NARL's approach utilises exact matching to minimise the effect of measurement biases within the method but also includes the thorough examination of all other biasing factors. The approach has been successfully tested in international CCQM intercomparisons with other national metrology institutes.     

New mathematical models with associated equations for isotope dilution mass spectrometry (IDMS)

Vector models which progressively lead to a general model for isotope dilution mass spectrometry (IDMS) are presented for the case of two 'monitor isotopes' and one blend involved. They enable one to find the boundary conditions for performing IDMS, and cover the cases of highly enriched isotopes, radioactive isotopes and ratios that are given with different denominator. The models identify the key measurements in their simplest form as well as the conditions which minimise the measurement effort and in some cases the propagated measurement uncertainties. The equations are discussed and compared with other published IDMS equations. Combined with discussion on fundamental aspects of IDMS, this results in an even more 'general' but also more complex IDMS equation.     

Direct quantification of creatinine in human urine by using isotope dilution extractive electrospray ionization tandem mass spectrometry

Urinary creatinine (CRE) is an important biomarker of renal function. Fast and accurate quantification of CRE in human urine is required by clinical research. By using isotope dilution extractive electrospray ionization tandem mass spectrometry (EESI–MS/MS) a high throughput method for direct and accurate quantification of urinary CRE was developed in this study. Under optimized conditions, the method detection limit was lower than 50 μg L⁻¹. Over the concentration range investigated (0.05–10 mg L⁻¹), the calibration curve was obtained with satisfactory linearity (R² = 0.9861), and the relative standard deviation (RSD) values for CRE and isotope-labeled CRE (CRE-d3) were 7.1–11.8% (n = 6) and 4.1–11.3% (n = 6), respectively. The isotope dilution EESI–MS/MS method was validated by analyzing six human urine samples, and the results were comparable with the conventional spectrophotometric method (based on the Jaffe reaction). Recoveries for individual urine samples were 85–111% and less than 0.3 min was taken for each measurement, indicating that the present isotope dilution EESI–MS/MS method is a promising strategy for the fast and accurate quantification of urinary CRE in clinical laboratories.     

液相色谱-同位素稀释串联质谱法测定动物源性食品中硝基呋喃类代谢物残留

加入同位素标记物的动物源性样品(龙虾、肠衣),在酸性条件下用2-硝基苯甲醛,使样品中呋喃唑酮、呋喃西林、呋喃它酮、呋喃妥因实现衍生。经乙酸乙酯提取浓缩、用甲醇水溶液溶解过滤后,滤液用液相色谱-串联质谱定量。在0.5,1.0和2.0μg/kg3个浓度水平上进行添加回收试验,回收率为80.1％～92.2％,检出限(LOQ)...     

Determination of B in body fluids by isotope dilution inductively coupled mass spectrometry with direct injection nebulization

The determination of B in small volumes of undigested blood plasma and urine by isotope dilution and high efficiency direct injection nebulization (DIN) inductively coupled plasma mass spectrometry (ICP-MS) is proposed. The C interference over ¹¹B was removed by precipitating the samples proteins. Samples aliquots of 1 ml were spiked with an enriched ¹⁰B solution and shaken during 1 h to attain the isotopic equilibrium. Thereafter, the sample proteins were denaturated with nitric acid and the supernatant was analyzed. This procedure was effective to reduce C concentrations in approximately 94%. Sample volumes of 50 μl were introduced into the ICP by the direct injection nebulizer producing transient signals lasting 30 s for B isotopes measurements. Precision of ¹⁰B/¹¹B measurements was characterized by relative standard deviation (RSD) lower than 1%. The instrumental mass discrimination factor was lower than 5%. Total B concentrations from 100 to 135 μg L⁻¹ in plasma and 0.499 to 3.021 mg L⁻¹ in urine samples were found. Reproducibility of triplicate samples was characterized by RSD<2.0% for plasma and lower than 1.3% for urine samples. Limit of detection (3σ) of 0.6 ng ml⁻¹ was calculated from synthetic blood plasma blank. Results of the denatured supernatant and digested plasma and urine samples were comparable at the 95% confidence level.