Correction of creatine-creatinine conversion during serum creatinine quantification by two-dimensional liquid chromatography and double-spike isotope dilution tandem mass spectrometry.

BACKGROUND AND AIMS: Development of a candidate reference method based on bidimensional liquid chromatography coupled to ESI-MS/MS and double spike isotope dilution for serum creatinine quantification capable of correcting for creatinine-creatine interconversion during sample pretreatment. Study of the impact of the creatine-creatinine interconversion during the analysis of human serum samples. MATERIALS AND METHODS: 13C1-creatinine and 13C2-creatine are added to the serum sample. Separation carried out by bidimensional liquid chromatography combining reversed phase and a strong cation exchange chromatography. The heart cut, containing creatine and creatinine, is automatically transferred to the second dimension. Quantification carried out by double spike isotope dilution tandem MS/MS. RESULTS: Minimization of spectral interferences and ion suppression due to matrix effects while increasing sample throughput compared to the direct coupling of cation exchange chromatography to the ESI source. Trueness of the method studied with the satisfactory analysis of two certified reference materials. Satisfactory intra- and inter-day precisions obtained analysing a serum pool and control sera. Analysis of 93 serum samples revealed negligible interconversions with no correlation with creatine levels. CONCLUSIONS: The method provides adequate analytical figures of merit for serum creatinine determination according to CSLI guidelines. Negligible creatine-creatinine interconversion is promoted with the applied sample preparation procedure.

Cadmium in the hyperaccumulating mushroom Thelephora penicillata: Intracellular speciation and isotopic composition.

Thelephora penicillata is an ectomycorrhizal mushroom that can accumulate extraordinarily high concentrations of Cd, As, Cu, and Zn in its fruit-bodies. To better understand its element accumulation ability, we compared the element concentrations in T. penicillata with 10 distinct ectomycorrhizal mushroom species growing at the same site (Karlina Pila, Czech Republic). On average, T. penicillata accumulated 330, 2130, 26, and 4 times more Cd, As, Cu, and Zn, respectively, than other mushrooms. Size-exclusion chromatography and an electrophoretic analysis of T. penicillata cell extracts indicate that intracellular Cd may be present mainly in >1 kDa, presumably compartmentalized, Cd species, and partially binding with 6-kDa cysteinyl-containing peptide(s) resembling metallothioneins. The cadmium isotopic composition of mushroom fruit-bodies, soil digests, and soil extracts was investigated by thermal ionization mass spectrometry (TIMS) with double spike correction. The isotopic composition (δ114/110Cd) of ectomycorrhizal mushrooms from Karlina Pila varied in a wide range of -0.37 to +0.14 ‰. However, remarkably low δ114/110Cd values were observed in the majority of the investigated mushrooms when compared to the relatively homogeneous Cd isotopic composition of bulk soil (δ114/110Cd = +0.09 ‰) and the comparatively heavy isotopic composition of soil extracts (mean δ114/110Cd values of +0.11 ± 0.01 ‰ and +0.22 ± 0.01 ‰, depending on the extraction method). The isotopic composition of Cd hyperaccumulated in T. penicillata essentially matched the mycoavailable soil Cd fraction. However, most isotopic data indicates isotopic fractionation at the soil/fruit-body interface, which could be of environmental significance.

Tibial graft fixation methods and bone tunnel enlargement: A comparison between the TensionLoc implant system and the double-spike plate.

Background/objective: TensionLoc (Arthrex, Naples, Florida, USA), a tibial graft fixation system for anterior cruciate ligament (ACL) reconstruction, is expected to apply the preoperatively determined level of graft tension and allow setting of lower initial tension. Considering its mechanism, we hypothesised that TensionLoc would prevent postoperative bone tunnel enlargement (TE) through fixation with lower initial tension. Therefore, the present study aimed to compare TE between ACL reconstructions using the double-spike plate (DSP; Smith and Nephew, Andover, Massachusetts) and TensionLoc implant system. Methods: A total of 40 patients who underwent anatomical single-bundle ACL reconstruction with a hamstring tendon graft were retrospectively analysed. In the group in which DSP and screw were used, the initial graft tension was set to 40 N at 20° of knee flexion (group D). In the other group in which TensionLoc was used, the initial graft tension was set to 30 N at 20° of knee flexion (group T). Both groups included 20 patients each. Tunnel areas were measured using computed tomography images at one week and three months after surgery, and the TE ratio was calculated according to the following equation: TE ratio (%) = (tunnel area at three months after surgery - tunnel area at one week after surgery)/tunnel area at one week after surgery × 100. Results: The femoral TE ratios were significantly higher in group T (80.5% ± 28.8%) than in group D (45.5% ± 34.6%) (p = 0.001). However, the tibial TE ratios did not significantly differ between the two groups. Conclusion: Compared with ACL reconstruction using DSP and screw, ACL reconstruction using TensionLoc fixed the graft with lower initial tension but showed greater femoral TE and restricted knee extension in the early postoperative period.

A double-spike MC TIMS measurement procedure for low-amount Ca isotopic analysis of limited biological tissue samples.

The application of Ca isotopic analysis in biomedical studies has great potential to identify changes in Ca metabolism and bone metabolism. Reliable measurement of Ca isotope-amount ratios is challenging considering limited Ca amounts and significant procedural blank levels. In this study, Ca purification was performed using the DGA Resin, optimized for low procedural blanks and separation of Ca from matrix elements and isobaric interferences (Na, Mg, K, Ti, Fe, Ba), while maintaining quasi-quantitative recoveries which are sufficient since a 42Ca-48Ca double-spike (DS) was applied. Ca isotopic analysis was performed using multicollector thermal ionization mass spectrometry (MC TIMS). The obtained procedural Ca blank of ≤10 ng enables processing of limited Ca amounts down to 670 ng. Data reduction of the measured Ca isotope-amount ratios was performed using an in-house developed software solving the DS algorithm. Data quality was improved by extension of equilibration time of the sample-DS mixture and implementation of a normalization strategy for raw isotopic data. The reported δ(44Ca/40Ca)NIST SRM 915a of NIST SRM 915a processed as a sample was found to be 0.01 ‰ ± 0.08 ‰ (2 SD, n = 15). Ca isotope-amount ratios of the reference material NIST SRM 1400 (bone ash), NIST SRM 1486 (bone meal), GBW07601 (human hair), and IAPSO (seawater) were in good agreement within uncertainty with literature data. Novel data on additional reference materials for biological tissues (hair) is presented, which might indicate a potential fractionation of Ca incorporated into hair tissue when compared to the blood pool.

Novel nickel isolation procedure for a wide range of sample matrices without using dimethylglyoxime for isotope measurements using MC-ICP-MS.

Nickel isotope ratios have traditionally been used as an important tracer in cosmochemistry, and recently, it has gained attention in geochemistry, biochemistry, and environmental sciences with the development of MC-ICP-MS. Purification of Ni before isotope measurement is mandatory for obtaining precise data, which has been commonly achieved with ion-exchange chromatography, employing dimethylglyoxime (DMG) as a chelating agent for Ni. However, it has been pointed out that the use of DMG can adversely affect the isotope measurement due to insufficient Ni recovery and mass bias during measurement caused by the remaining DMG. Ni isolation procedures without the usage of DMG were innovated, but they have disadvantages such as the necessity of complex separation methods, high Ni blank, and matrix-dependent Ni recovery. Here, we present a simple Ni isolation procedure without using DMG but with the aid of oxalic acid along with common inorganic acids, achieving near-complete recovery of Ni with low blanks [0.7 ± 0.3 ng (2SD, n = 4)] only using three ion exchange column steps. To validate our method and strengthen the Ni isotope database of reference materials, 60Ni/58Ni of 20 geological reference materials, covering wide matrix compositions, were measured by MC-ICP-MS using the double-spike method. The results have shown that high recovery of Ni, independent of the sample matrix elements was achieved (98 ± 4%) and the 60Ni/58Ni was measured with a 2SD of 0.006-0.084‰ from samples containing 100-200 ng Ni.

Zinc isotopic variation of water and surface sediments from the German Elbe River.

Recent studies suggested the use of the isotopic composition of Zn as a possible tracer for anthropogenic Zn emissions. Nevertheless, studies mainly focused on sampling areas of a few km2 with well-characterized anthropogenic Zn emissions. In contrast, this study focused on analyzing a large sample set of water and sediment samples taken throughout the course of the Elbe River, a large, anthropogenically impacted river system located in Central Europe. The primary objective was to evaluate the use of the isotopic composition of Zn to trace anthropogenic Zn emission on a large regional scale. In total 18 water and 26 surface sediment samples were investigated, covering the complete course of over 700 km of the German Elbe between the German/Czech border and the German North Sea, including six tributaries. Stable isotope abundance ratios of Zn were assessed by multi-collector inductively coupled plasma mass spectrometry (MC ICP-MS) in water filtrates (<0.45 µm) and total digests of the sieved surface sediment fraction (<63 µm) after analyte/matrix separation using Bio-Rad AG MP-1 resin via a micro-column approach and application of a 64Zn/67Zn double spike. Measured isotopic compositions of δ66Zn/64ZnIRMM-3702 ranged from -0.10 ‰ to 0.32 ‰ for sediment samples, and from -0.51 ‰ to 0.45 ‰ for water samples. In comparison to historical data some tributaries still feature high mass fractions of anthropogenic Zn (e.g. Mulde, Triebisch) combined with δ66Zn/64ZnIRMM-3702 values higher than the lithogenic background. The dissolved δ66Zn/64ZnIRMM-3702 values showed a potential correlation with pH. Our results indicate that biogeochemical processes like absorption may play a key role in natural Zn isotopic fractionation making it difficult to distinguish between natural and anthropogenic processes.

Determination of Lead Elemental Concentration and Isotopic Ratios in Coal Ash and Coal Fly Ash Reference Materials Using Isotope Dilution Thermal Ionization Mass Spectrometry.

The rapid expansion of coal-fired power plants around the world has produced a huge volume of toxic elements associated with combustion residues such as coal fly ash (CFA) and coal ash (CA), which pose great threats to the global environment. It is therefore crucial for environmental science to monitor the migration and emission pathway of toxic elements such as CFA and CA. Lead isotopes have proved to be powerful tracers capable of dealing with this issue. Unfortunately, up to now, few high precision lead isotope data of CFA and CA certified reference materials (CRMs) determined by using the double spike technique have been reported. Hence, to facilitate the application of lead isotopes in environmental science, it is indispensable and urgent to determine a suite of high precision Pb isotope ratios and Pb elemental contents for CFA and CA CRMs. Here, we measured lead isotope ratios from four CFA and CA CRMs using thermal ionization mass spectrometry (TIMS) combined with the 204Pb-207Pb double spike method. Lead isotope ratios values of CRMs (GBW11124, GBW08401, GBW11125d, and JCFA-1) covered wide variation ranges from 17.993 to 19.228 for 206Pb/204Pb, from 15.513 to 15.675 for 207Pb/204Pb, and from 38.184 to 39.067 for 208Pb/204Pb. Lead isotope ratios of these CRMs, except for GBW11124, show good external reproducibility (2 RSD, n = 8), which is better than 0.05% for 206Pb/204Pb and 207Pb/204Pb, 0.07% for 208Pb/204Pb, 0.04% for 206Pb/207Pb, and 0.05% for 208Pb/206Pb. The Pb concentrations of these CRMs were determined using 207Pb single spike method. The reproducibility (1 RSD, n = 4) of Pb elemental content was <0.60%. This indicates the distribution of Pb elements in these CRMs is homogeneous. With the exception of GBW11124, the suite of CRMs can be used for determining CFA and CA matrix composition for quality control of Pb isotope analyses.

Characterization of a new candidate isotopic reference material for natural Pb using primary measurement method.

A lead isotopic standard solution with natural abundance has been developed by applying a mixture of a solution of enriched 208Pb and a solution of enriched 204Pb (208Pb-204Pb double spike solution) as bracketing method. The amount-of-substance ratio of 208Pb:204Pb in this solution is accurately measured by applying EDTA titrimetry, which is one of the primary measurement methods, to each enriched Pb isotope solution. Also metal impurities affecting EDTA titration and minor lead isotopes contained in each enriched Pb isotope solution are quantified by ICP-SF-MS. The amount-of-substance ratio of 208Pb:204Pb in the 208Pb-204Pb double spike solution is 0.961959 ± 0.000056 (combined standard uncertainty; k = 1). Both the measurement of lead isotope ratios in a candidate isotopic standard solution and the correction of mass discrimination in MC-ICP-MS are carried out by coupling of a bracketing method with the 208Pb-204Pb double spike solution and a thallium internal addition method, where thallium solution is added to the standard and the sample. The measured lead isotope ratios and their expanded uncertainties (k = 2) in the candidate isotopic standard solution are 18.0900 ± 0.0046 for 206Pb:204Pb, 15.6278 ± 0.0036 for 207Pb:204Pb, 38.0626 ± 0.0089 for 208Pb:204Pb, 2.104406 ± 0.00013 for 208Pb:206Pb, and 0.863888 ± 0.000036 for 207Pb:206Pb. The expanded uncertainties are about one half of the stated uncertainty for NIST SRM 981, for 208Pb:204Pb, 207Pb:204Pb and 206Pb:204Pb, or one eighth, for 208Pb:206Pb and 207Pb:206Pb, The combined uncertainty consists of the uncertainties due to lead isotope ratio measurements and the remaining time-drift effect of mass discrimination in MC-ICP-MS, which is not removed by the coupled correction method. In the measurement of 208Pb:204Pb, 207Pb:204Pb and 206Pb:204Pb, the latter contribution is two or three times larger than the former. When the coupling of a bracketing method with the 208Pb-204Pb double spike solution and a thallium internal addition method is applied to the analysis of NIST SRM 981, the measured lead isotope ratios are in good agreement with its certified values. This proves that the developed method is not only consistent with the conventional one by NIST SRM 981 but also enables measurement of the lead isotope ratios with higher precision.

High Precision Zinc Stable Isotope Measurement of Certified Biological Reference Materials Using the Double Spike Technique and Multiple Collector-ICP-MS.

Biological reference materials with well-characterised stable isotope compositions are lacking in the field of 'isotope biochemistry', which seeks to understand bodily processes that rely on essential metals by determining metal stable isotope ratios. Here, we present Zn stable isotope data for six biological reference materials with certified trace metal concentrations: fish muscle, bovine muscle, pig kidney, human hair, human blood serum and human urine. Replicate analyses of multiple aliquots of each material achieved reproducibilities (2sd) of 0.04-0.13‰ for δ66/64Zn (which denotes the deviation of the 66Zn/64Zn ratio of a sample from a pure Zn reference material in parts per 1000). This implies only very minor isotopic heterogeneities within the samples, rendering them suitable as quality control materials for Zn isotope analyses. This endorsement is reinforced by (i) the close agreement of our Zn isotope data for two of the samples (bovine muscle and human blood serum) to previously published results for different batches of the same material and (ii) the similarity of the isotopic data for the samples (δ66/64Zn ≈ -0.8 to 0.0‰) to previously published Zn isotope results for similar biological materials. Further tests revealed that the applied Zn separation procedure is sufficiently effective to enable accurate data acquisition even at low mass resolving power (M/ΔM ≈ 400), as measurements and analyses conducted at much higher mass resolution (M/ΔM ≈ 8500) delivered essentially identical results.

High-precision measurements of seawater Pb isotope compositions by double spike thermal ionization mass spectrometry.

A new method for the determination of seawater Pb isotope compositions and concentrations was developed, which combines and optimizes previously published protocols for the separation and isotopic analysis of this element. For isotopic analysis, the procedure involves initial separation of Pb from 1 to 2L of seawater by co-precipitation with Mg hydroxide and further purification by a two stage anion exchange procedure. The Pb isotope measurements are subsequently carried out by thermal ionization mass spectrometry using a (207)Pb-(204)Pb double spike for correction of instrumental mass fractionation. These methods are associated with a total procedural Pb blank of 28±21 pg (1sd) and typical Pb recoveries of 40-60%. The Pb concentrations are determined by isotope dilution (ID) on 50 mL of seawater, using a simplified version of above methods. Analyses of multiple aliquots of six seawater samples yield a reproducibility of about ±1 to ±10% (1sd) for Pb concentrations of between 7 and 50 pmol/kg, where precision was primarily limited by the uncertainty of the blank correction (12±4 pg; 1sd). For the Pb isotope analyses, typical reproducibilities (±2sd) of 700-1500 ppm and 1000-2000 ppm were achieved for (207)Pb/(206)Pb, (208)Pb/(206)Pb and (206)Pb/(204)Pb, (207)Pb/(204)Pb, (208)Pb/(204)Pb, respectively. These results are superior to literature data that were obtained using plasma source mass spectrometry and they are at least a factor of five more precise for ratios involving the minor (204)Pb isotope. Both Pb concentration and isotope data, furthermore, show good agreement with published results for two seawater intercomparison samples of the GEOTRACES program. Finally, the new methods were applied to a seawater depth profile from the eastern South Atlantic. Both Pb contents and isotope compositions display a smooth evolution with depth, and no obvious outliers. Compared to previous Pb isotope data for seawater, the (206)Pb/(204)Pb ratios are well correlated with (207)Pb/(206)Pb, underlining the significant improvement achieved in the measurement of the minor (204)Pb isotope.

Platinum stable isotope analysis of geological standard reference materials by double-spike MC-ICPMS.

We report a method for the chemical purification of Pt from geological materials by ion-exchange chromatography for subsequent Pt stable isotope analysis by multiple-collector inductively coupled plasma mass spectrometry (MC-ICPMS) using a 196Pt-198Pt double-spike to correct for instrumental mass bias. Double-spiking of samples was carried out prior to digestion and chemical separation to correct for any mass-dependent fractionation that may occur due to incomplete recovery of Pt. Samples were digested using a NiS fire assay method, which pre-concentrates Pt into a metallic bead that is readily dissolved in acid in preparation for anion-exchange chemistry. Pt was recovered from anion-exchange resin in concentrated HNO3 acid after elution of matrix elements, including the other platinum group elements (PGE), in dilute HCl and HNO3 acids. The separation method has been calibrated using a precious metal standard solution doped with a range of synthetic matrices and results in Pt yields of ≥90% with purity of ≥95%. Using this chemical separation technique, we have separated Pt from 11 international geological standard reference materials comprising of PGE ores, mantle rocks, igneous rocks and one sample from the Cretaceous-Paleogene boundary layer. Pt concentrations in these samples range from ca. 5 ng g-1 to 4 µg g-1. This analytical method has been shown to have an external reproducibility on δ198Pt (permil difference in the 198Pt/194Pt ratio from the IRMM-010 standard) of ±0.040 (2 sd) on Pt solution standards (Creech et al., 2013, J. Anal. At. Spectrom. 28, 853-865). The reproducibility in natural samples is evaluated by processing multiple replicates of four standard reference materials, and is conservatively taken to be ca. ±0.088 (2 sd). Pt stable isotope data for the full set of reference materials have a range of δ198Pt values with offsets of up to 0.4‰ from the IRMM-010 standard, which are readily resolved with this technique. These results demonstrate the potential of the Pt isotope system as a tracer in geochemical systems.