Dietmar Stöckl

Full-scan mass spectral evidence for 3-epi-25-hydroxyvitamin D3 in serum of infants and adults

Abstract Background: Since the introduction of liquid chromatography-mass spectrometry (LC/MS) for assessing vitamin D status, it has been shown that the C-3 epimer can account for a significant proportion of circulating 25-hydroxyvitamin D3 (25OHD3) concentrations in infants. However, some question whether monitoring a single MS transition at a chromatographic retention time typical for 3-epi-25OHD3 sufficiently warrants conclusions about the identity of the substance generating the signal. Therefore, we aimed to substantiate the evidence for 3-epi-25OHD3 in infants by collision induced dissociation (CID)-MS/MS product ion scans. A second objective was mass spectrometric investigation of the presence and prevalence of the 3-epi metabolite in serum from adults. Methods: Serum samples from six infants and 32 adults were studied using an ultra performance LC/tandem MS (UPLC/MS/MS) method designed to separate the 3-epi-25OHD3 from 25OHD3. Samples were submitted to liquid/liquid extraction and Sephadex LH-20 fractionation, prior to column-switching UPLC with MS/MS recording of CID product ion spectra of the [M+H]+ precursor ion. The respective standards were analyzed under identical UPLC/MS/MS conditions for comparison. Results: In the chromatograms of all samples, two peaks eluted with retention characteristics and spectra closely matching those observed for the 25OHD3 and the 3-epi standards. The percentage of the 3-epi metabolite relative to 25OHD3 in infants ranged from 15% to 41%, and in adults from 2.5% to 17%. Conclusions: This preliminary finding suggests that the prevalence of 3-epi-25OHD3 in serum of infants is considerable, and that even in adults the concentrations of this form should not be neglected.

Specifications for trueness and precision of a reference measurement system for serum/plasma 25-hydroxyvitamin D analysis

BACKGROUND The divergence in analytical quality of serum/plasma 25-hydroxy-vitamin D analysis calls for defining specifications for a reference measurement system. METHODS Fundamentally, in a reference measurement system, there should be a relationship between the analytical specifications for higher- (reference) and lower-order (routine) measurements. Therefore, when setting specifications, we started with limits for routine imprecision (CV(rou)) and bias (B(rou)) using 4 models: (1) the misclassifications in diagnosis, (2) biological variation data (reference interval (RI) and monitoring), (3) expert recommendations, and (4) state-of-the-art performance. Then, we used the derived goals to tailor those for reference measurements and certified reference materials (CRMs) for calibration by setting the limits for CV(ref) at 0.5 CV(rou), B(ref) at 0.33 B(rou)(,) max. uncertainty (U(max)) at 0.33 B(ref). RESULTS The established specifications ranged between CV(rou)<or=22%, B(rou)<or=10%, CV(ref)<or=11%, B(ref)<or=3.3%, U(max) 1.1% (model 3) and CV(rou)<or=4%, B(rou)<or=2.6%, CV(ref)<or=2%, B(ref)<or=0.9%, U(max) 0.3% (model 2, monitoring). CONCLUSIONS Model 2 (monitoring) gave the most stringent goals, model 3, the most liberal ones. Accounting for state-of-the-art performance and certification capabilities, we used model 2 (RI) to recommend achievable goals: for routine testing, CV(rou)<or=10%, B(rou)<or=5%, for reference measurements, CV(ref)<or=5%, B(ref)<or=1.7%, and for CRMs, U(max) 0.6%.

Isotope dilution-liquid chromatography electrospray ionization-tandem mass spectrometry for the determination of serum thyroxine as a potential reference method.

A new candidate reference method is presented for the determination of thyroxine in serum. The method is based on isotope dilution-liquid chromatography/tandem mass spectrometry using electrospray for ionization. The internal standard used was 13C6-thyroxine, sample pretreatment consisted of protein precipitation and a two-step liquid/liquid extraction procedure, HPLC was performed on a C-18 column with an eluent containing methanol/water/formic acid (60:40:0.1, by volume), and finally thyroxine and its isotopically labeled analogue were measured in the selected reaction monitoring mode for the transitions m/z 777.7--> 731.7 and m/z 783.7--> 737.7, respectively. The detection limit for thyroxine was 6 pg, the within-run coefficient of variation was 1.1%. The samples were measured in six-fold: in duplicate on three independent days. The mean overall coefficient of variation of the method was 1.6%. The new method was evaluated by measuring nine control sera previously determined by an existing ID-GC/MS method. The differences between the results of the two methods ranged from-1.6% to +3.3%, with a mean of +0.2%.

Method validation across the disciplines--critical investigation of major validation criteria and associated experimental protocols.

Analytical method development should aim at delivering reliable measurements within a given application. This implies that method validation is integrated in the development process, because it enables to establish a methods performance capabilities, and to demonstrate its fitness-for-purpose. Although analytical chemists mostly are familiar with the validation guidelines within the discipline of their responsibility, we believe that they may take advantage of a better acquaintance with recommendations among disciplines. Therefore, we review the guidance given in 4 disciplines (laboratory medicine, pharmacy, environment, and food), with emphasis on the proposed experimental protocols, acceptance criteria and interpretation strategies by statistical significance testing. Last but not least, we give incentive towards a modernized validation design.

Quantitative analysis of urinary C-peptide by liquid chromatography-tandem mass spectrometry with a stable isotopically labelled internal standard.

We describe the first results of a quantitative LC-tandem mass spectrometry method for urinary C-peptide with the use of [2H14]C-peptide as internal standard. LC was based on gradient elution of a Hypersil PEP C18 column. Mass spectrometry was performed in the negative electrospray ionization mode and by monitoring of the transitions at m/z 1514/1334 ([2H14]C-peptide) and 1507/1320 (C-peptide). For sample preparation, we applied ultrafiltration. The analytical performance of the method in terms of measurement precision gave an RSD of <2% (n=10). The overall imprecision was investigated from independent analysis of two urine samples in six-fold and resulted in an RSD<5%. The limit of detection, expressed as signal-to-noise ratio 3, was approximately 0.15 ng C-peptide injected. Analysis of 10 random urine samples from laboratory volunteers showed interference-free ion chromatograms at a signal-to-noise ratio of approximately 75 on average. The C-peptide concentrations calculated from quantification by the bracketing calibration technique ranged from 32 to 165 ng/ml.

Trueness verification in European external quality assessment schemes: time to care about the quality of the samples

An external quality assessment (EQA) survey on 14 fresh‐frozen, single‐donation sera assigned with reference measurement procedure (RMP) values revealed a mean bias of +5.2% and +3.7% for the cholesterol oxidase and the photometric glucose oxidase procedure groups, respectively. Conversely, on lyophilized sera, the same procedure groups showed almost bias‐free results, the differences from the RMP values being only −0.8% for cholesterol and +0.7% for glucose. These data, which are in fairly good agreement with the literature, suggest the existence of artificial matrix effects in processed materials. Therefore they indicate that, currently, assessment of trueness is hampered in many European EQA schemes, as most of them use lyophilized sera. This approach may give a false impression about the trueness of laboratory results as well as carrying the risk that laboratories calibrated on the RMP values of the survey samples could make errors in patient testing. Consequently, if European EQA is willing to fulfil a post‐market vigilance function of the performance of in vitro diagnostic medical devices, then the time has come to tackle the problem of the quality of the survey samples. EQA organizers urgently need to make an effort to seek out materials that analytically behave like authentic clinical specimens. In the meantime, alternative approaches should be used. Although not ideal, the special survey described in this article is one of the possibilities. Naturally, it implies logistic problems and increased costs for the individual EQA schemes. However, both can be overcome with the cooperation of the predominantly nationally organized schemes.

Models for combining random and systematic errors. assumptions and consequences for different models.

Abstract A series of models for handling and combining systematic and random variations/errors are investigated in order to characterize the different models according to their purpose, their application, and discuss their flaws with regard to their assumptions. The following models are considered 1. linear model, where the random and systematic elements are combined according to a linear concept (TE=|bias|+z·σ), where TE is total error, bias is the systematic error component, σ is the random error component (standard deviation or coefficient of variation) and z is the probability factor; 2. squared model with two sub-models of which one is the classical statistical variance model and the other is the GUM (Guide to Uncertainty in Measurements) model for estimating uncertainty of a measurement; 3. combined model developed for the estimation of analytical quality specifications according to the clinical consequences (clinical outcome) of errors. The consequences of these models are investigated by calculation of the functions of transformation of bias into imprecision according to the assumptions and model calculations. As expected, the functions turn out to be rather different with considerable consequences for these types of transformations. It is concluded that there are at least three models for combining systematic and random variation/errors, each created for its own specific purpose, with its own assumptions and resulting in considerably different results. These models should be used according to their purposes.

Calibration for isotope dilution mass spectrometry : Description of an alternative to the bracketing procedure

A calibration method for isotope dilution mass spectrometry is presented that fully accounts for non-linearity of calibration functions, caused by the interference of the analyte on the m/z used for measurement of the internal standard and vice versa. In this way, it is also possible to use incompletely labelled molecules or analogues with a mass increment of only 1 u for isotope dilution, on condition that certain restrictions are respected. In addition, the proposed method is less time consuming than bracketing. The method works with the computer-stored full calibration curve and a single calibration point that is measured daily. The calibration curve is constructed from the experimentally determined mass spectral overlap between the unlabelled analyte and the labelled internal standard at the m/z values chosen for measurement. Measurement results for samples with unknown analyte concentration are calculated on the basis of a linear relationship between their ion abundance ratio and that of the daily single calibration point, but corrected by a factor derived from the theoretical calibration function. All calculations are performed with commonly available spreadsheet software. An application is presented for determining serum uric acid with a candidate gas chromatographic/ isotope dilution mass spectrometric reference method.

Validation of candidate reference methods based on ion chromatography for determination of total sodium, potassium, calcium and magnesium in serum through comparison with flame atomic emission and absorption spectrometry.

OBJECTIVES We report on the validation of candidate reference methods based on ion chromatography for the determination of total sodium, potassium, calcium, and magnesium in serum. The current study is a continuation of previous investigations in which we were able to show the potential of ion chromatography to serve as a reference method principle. The validation consisted of comparison of the ion chromatographic methods with generally recognized reference methods based on flame atomic emission (for sodium/ potassium) and absorption (for calcium/magnesium) spectrometry. METHODS Sample preparation consisted of acidic dilution and pre-purification with reversed phase cartridges. Ion chromatography with suppressed conductivity was performed with polymeric stationary phases chemically modified with carboxylate and sulfonate functional groups for sodium/potassium and calcium/magnesium, respectively. Single-point calibration was used in combination with individual adaptation of serum sampling and diluting to give similar responses in samples and standards. In the study, more than 25 control sera were analyzed in parallel with the ion chromatographic methods and the traditional reference methods. The measurement protocol consisted of quadruplicate analysis of each sample per run, in 3 different runs. In each measurement series, internal quality control with certified reference materials from the National Institute of Standards and Technology and the Bureau Communautaire de Référence was applied. The performance criteria to fulfill were pre-defined (i.e., the maximum within-day and overall coefficient of variation, the confidence interval, the daily and overall deviation from the certified value and the maximum total analytical error). RESULTS The comparison is presented as difference plot (i.e., % deviation of the results obtained with ion chromatography from those obtained with the respective traditional reference methods). The mean deviations were +0.9% for sodium, +1.0% for potassium, +/- 0.0% for calcium, and +0.1% for magnesium. With the exception of 2 values each for sodium and potassium, all deviations were within the limit of 2 times the allowed total analytical error. From the data obtained for internal quality control during the different measurement campaigns, we further estimated the long-term precision and accuracy of our methods. The overall coefficient of variation (respectively the bias) amounted to 0.7% (+0.1%) for sodium, 0.9% (-0.3%) for potassium, 1.3% (-0.1%) for calcium, and 1.0% (+/- 0.0%) for magnesium. CONCLUSION From the precision and accuracy reached by our ion chromatographic methods, we conclude that they can be proposed as candidate reference methods for the determination of total sodium, potassium, calcium, and magnesium in serum.

Standardization of C-Peptide Measurements in Urine by Method Comparison with Isotope-Dilution Mass Spectrometry

In the past, standardization of measurements of diagnostically important polypeptides and proteins was hampered by the noncommutability of primary standards (1). One way to overcome this problem is to establish a method comparison with a reference measurement procedure. Until now, reference measurement procedures, such as isotope-dilution mass spectrometry (ID-MS), have been scarce. Recent developments in the MS field, however, have made the technique easily applicable to the analysis of polypeptides and proteins (kinetic studies, sequence analysis, and determination of molecular mass and posttranslational modifications). To the best of our knowledge, only two groups have used ID-MS for the quantitative determination of a specific polypeptide/protein. One of these groups described the measurement of apolipoprotein A-I after enzymatic digestion (2), the other described offline ID-liquid chromatography (LC)-MS assays for serum proinsulin, insulin, and C-peptide (3)(4). Neither group, however, examined the potential of ID-MS for standardization of the respective routine test systems (usually, immunoassays). Here we report on the use of an ID-MS measurement procedure for standardization/recalibration of C-peptide measurements in urine by use of a method-comparison study with split-sample measurements. In view of the model character of the study for future applications, we chose urinary C-peptide over the clinically more important serum C-peptide because of the ease of sample collection and MS measurement. The measurement procedure applies online ID-LC-electrospray tandem MS (ID-LC-MS/MS) and is described in detail elsewhere (5). For calibration, it makes use of a commercial C-peptide preparation with a peptide content of 89% and a purity by HPLC of >99% (according to the manufacturer’s information). This purity was taken into account for calculation of the C-peptide content in the calibrators. The C-peptide preparation was delivered in a vial containing 250 μg of freeze-dried material; a calibration solution was prepared by carefully weighing the added volume (∼1 mL) of …