Standard additions: myth and reality

Ellison, Stephen L. R.; Thompson, Michael

doi:10.1039/b717660k

Cited by 145 publications

(122 citation statements)

References 4 publications

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“…One-point standard addition, in which a single known amount of CRP standard is added to the serum sample being measured, is effective in eliminating potential matrix effect bias. 35 As the CRP standard used in this study was purified from human serum, the structural and molecular differences between CRP in the standard and samples will be minimized and offer the greatest affinity equivalency possible. If there is molecular and structural identity between the intrinsic serum CRP and the CRP added, any bias in the affinity chromatographic step should equally affect the intrinsic protein and the protein added for standard addition and thus be eliminated.…”

Section: Discussionmentioning

confidence: 99%

Reference Measurement Procedure Development for C-Reactive Protein in Human Serum

Kilpatrick

Bunk

2009

Anal. Chem.

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This paper describes the development of a reference measurement procedure to quantify human C-reactive protein (CRP) in serum using affinity techniques prior to tryptic digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the certification of reference materials in clinically relevant ranges. The absence of a suitable internal standard for the CRP measurement, necessary to eliminate potential measurement bias in both the affinity purification and trypsin digestion steps, was addressed using the method of standard addition. The standard addition quantification approach was combined with affinity purification, using an anti-CRP monoclonal antibody conjugated to polystyrene beads, trypsin digestion of the purified protein, and LC-MS/MS analysis of CRP tryptic peptides. The effectiveness of intact protein affinity purification was evaluated through the measurement of CRP in several serum-based CRP control materials, yielding levels that were comparable to their expected mean concentration values. Quantitative results were confirmed with an external calibration approach. This study demonstrates the feasibility of affinity purification with LC-MS/MS for the reference measurement procedure development of low abundance serum protein analytes.Reference measurement procedures are used for a variety of purposes including (1) the assessment of the measurement quality of routine measurement procedures, (2) the value assignment of high-order calibration solutions (the so-called "master calibrators") for routine assays, and (3) the value assignment of certified reference materials. 1,2 In these roles, reference measurement procedures are considered "higher-order" measurement procedures, as they aim to achieve a higher level of accuracy and precision than the routine assay for the analyte being measured, producing a value with a low uncertainty of defined magnitude. In order for a reference measurement procedure to be fit for this purpose, it must be thoroughly evaluated to identify sources of bias and assess their magnitude. [3][4][5] Minimizing or eliminating bias is necessary to measure the most accurate, true concentration of the analyte. Because of the need for high levels of accuracy and precision, reference measurement procedures are typically low-throughput, labor intensive, and often costly to perform, in sharp contrast to what is desirable for routine measurements.Reference measurement procedures are key elements in the establishment of measurement standardization and metrological traceability in clinical chemistry. For more than 30 years, isotope dilution mass spectrometry (IDMS) has been one of the analytical techniques used frequently in reference measurement procedures, particularly for small molecule and inorganic analytes of clinical importance. 6 Despite this, few reference measurement procedures exist for clinically relevant proteins. This is one reason why so many clinical protein analytes are measured in arbitrary International Units (IUs); without a reference measurement procedure to...

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Section: Discussionmentioning

confidence: 99%

Reference Measurement Procedure Development for C-Reactive Protein in Human Serum

Kilpatrick

Bunk

2009

Anal. Chem.

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“…A concentração do analito na matriz biológica é determinada pela extrapolação da reta, definida pelas demais concentrações analisadas (Figura 2c). 31,35 Considerando que, em geral, os métodos bioanalíticos utilizam etapas de tratamento com significativa manipulação da amostra (por exemplo, extração, diluição, centrifugação, concentração, etc), na maioria das vezes utiliza-se a padronização interna para a elaboração da curva de calibração.…”

Section: Figura 1 Sistema De Infusão Pós-colunaunclassified

Validação em métodos cromatográficos para análises de pequenas moléculas em matrizes biológicas

Cassiano¹,

Barreiro²,

Martins³

et al. 2009

Quím. Nova

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“…However, for the rest of the products, the margin of error was well below the 10 % threshold set for mass fraction of ≥1,000 μg kg −1 in accordance with CD2002/657/EC [21], although the direct application of this standard in a standard additions technique does not seem feasible given the complexity of the sample, nor has it been reported previously. The observed degradation of precision is expected in standard additions procedures as described by Ellison and Thompson [22], although such variations are also expected to arise from the detailed extraction procedures required for this type of formulation [12,23]. Table 4 summarises the equations of the lines obtained for the standard addition curves prepared for each of the samples analysed.…”

Section: Assay Precisionmentioning

confidence: 98%

An LCMS method for the assay of melittin in cosmetic formulations containing bee venom

et al. 2015

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There is a growing interest in the potential of bee venom in cosmetics as a rejuvenating agent. Products currently on the market do not specify exactly their content of bee venom (BV). Therefore, we developed a method for the detection and quantification of melittin, as a marker of bee venom content, in selected commercial creams which contained BV according to their marketing claims, in order to gauge the relative quality of such formulations. A quantitative method was achieved following a rigorous extraction procedure involving sonication, liquid-liquid extraction and solid phase extraction since carryover of excipients was found to cause a rapid deterioration in the chromatographic performance. The method employed a standard additions approach using, as spiking standard, purified melittin isolated from bee venom and standardised by quantitative NMR. The aqueous extracts of the spiked creams were analysed by reversed phase LCMS on an LTQ Orbitrap mass spectrometer. The purity of the melittin spiking standard was determined to be 96.0%. The lowest measured mean melittin content in the creams was 3.19 ppm (±1.58 ppm 95% CI) while the highest was 37.21 ppm (±2.01 ppm 95% CI). The method showed adequate linearity (R (2) ≥ 0.98) and a recovery of 87.7-102.2% from a spiked blank cream. An assay precision of <20% RSD was achieved for all but one sample where the RSD value was 27.5%. The method was sensitive enough for use in routine assay of BV-containing cosmetic creams. Differences in the melittin content of the commercial products assayed were nearly tenfold.

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Standard additions: myth and reality

Cited by 145 publications

References 4 publications

Reference Measurement Procedure Development for C-Reactive Protein in Human Serum

Reference Measurement Procedure Development for C-Reactive Protein in Human Serum

Validação em métodos cromatográficos para análises de pequenas moléculas em matrizes biológicas

An LCMS method for the assay of melittin in cosmetic formulations containing bee venom

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