Abstract Introduction Reference materials based on human cerebrospinal fluid were certified for the mass concentration of amyloid beta (Aβ)1‐42 (Aβ 42 ). They are intended to be used to calibrate diagnostic assays for Aβ 42 . Methods The three certified reference materials (CRMs), ERM‐DA480/IFCC, ERM‐DA481/IFCC and ERM‐DA482/IFCC, were prepared at three concentration levels and characterized using isotope dilution mass spectrometry methods. Roche, EUROIMMUN, and Fujirebio used the three CRMs to re‐calibrate their immunoassays. Results The certified Aβ 42 mass concentrations in ERM‐DA480/IFCC, ERM‐DA481/IFCC, and ERM‐DA482/IFCC are 0.45, 0.72, and 1.22 μg/L, respectively, with expanded uncertainties ( k = 2) of 0.07, 0.11, and 0.18 μg/L, respectively. Before re‐calibration, a good correlation (Pearson's r > 0.97), yet large biases, were observed between results from different commercial assays. After re‐calibration the between‐assay bias was reduced to < 5%. Discussion The Aβ 42 CRMs can ensure the equivalence of results between methods and across platforms for the measurement of Aβ 42 .
This work provides a multidimensional method for the simultaneous, direct quantification of intact human insulin and five insulin analogs in human plasma. This investigation solves both the selectivity and sensitivity problems encountered for accurate quantification of insulins in plasma since the former is not possible with conventional assays and the latter with conventional LC-MS/MS. The method uses a mixed-mode SPE and a multidimensional LC method including a solid-core particle column containing an anion exchange stationary phase. Matrix factors for all analogs were calculated in 6 sources of human plasma and CVs of the matrix factors were <15% in all cases supporting the selectivity of the method, while achieving LLOQs of 50-200 pg/mL (1.4-5.6 μIU/mL) for each insulin from 250 μL of human plasma. The average accuracy for the standard curve points in extracted human plasma was 99-100%. Average inter- and intraday accuracies for QC samples were 98% and 94%, respectively. Average inter- and intraday precisions for QC samples were 7.5 and 5.3%, respectively. Patient samples were analyzed in a blind study and results concurred with their diabetes multidosing regimes. The study also demonstrated that the presence of high levels of human insulin and bovine insulin does not interfere with quantification of any of the analyzed analogs. We propose this method for the accurate pharmacokinetic monitoring of diabetic patients, for sport antidoping and forensic toxicology analysis.
Ethinylestradiol (EE) is the active component in most birth control products. It is especially difficult to analyze due to the presence of many closely related endogenous steroids. Endogenous components can coelute with EE making selective extraction and chromatographic separation challenging. Current MS systems are more sensitive to background, contamination and the overall cleanliness of samples and solvents, placing additional emphasis on sample preparation methodology.UPLC was combined with a sensitive triple quadrupole MS and a three-step sample preparation method to highlight and resolve method development challenges.EE was adequately resolved using an unendcapped high-strength silica C(18) column. The average matrix factor in six sources of plasma was 1.14 with a %CV of 4.48. Standard curves were linear with 1/x weighting and r(2) value of 0.999 over three orders of magnitude. Average accuracy for standard curves and quality control samples was 96%. LOD of 0.001 ng/ml was achieved.
INT RODUCT ION A previous application note (720003682en) described in detail the development of a fast, flexible SPE/LC/MS/MS platform for the quantification of multiple amyloid beta (aβ) peptides from human or monkey CSF for use in a biomarker or preclinical discovery setting. In this work, the mass spectrometry platform has been updated from the Xevo® TQ MS to the Xevo TQ-S mass spectrometry system. This change facilitated both a 4X reduction in required sample size and a 4-5X increase in assay sensitivity.
Increased pressure to obtain more, higher sensitivity data from less sample is especially critical for large peptides, whose already optimized LC-MS methods are heavily challenged by traditional ligand-binding assays.Critical bioanalytical assays were adapted to integrated microscale LC to reduce sample volumes while increasing sensitivity. Assays for teriparatide, glucagon and human insulin and five analogs were transferred from 2.1 mm analytical scale LC to a 150 µm scale system. This resulted in a 15-30 fold overall improvement in sensitivity derived from increased signal to noise, three to six fold reduction in injection volumes, and a two to five fold reduction in sample consumption.Integrated microscale LC reduces sample consumption while enabling single picomolar quantification for therapeutic and endogenous peptides.
Objective: Insulin glargine is a long-acting insulin analogue with biotransformation, at the injection site, to a primary active metabolite (glargine-M1). Combined therapy with rapid-acting insulin aspart is common, but pharmacokinetics with supra-therapeutic doses have not been reported. We present clinical and pharmacokinetic findings for 2 episodes of overdose with glargine and aspart in a 43-year-old male with type 1 diabetes.
Extraction recoveries ranged from 73 to 105% with an average of 92% and matrix effects were less than 20% for all compounds with only 3 greater than 15%. Calibration curves were linear from 2-500 ng/mL, with accurate and precise results from quality control samples. The analysis of several different classes of these drugs should render this method applicable to newly developed related compounds with little, if any, modification necessary.
Measurement of the 42 amino acid form of amyloid β (Aβ42) in cerebrospinal fluid (CSF) is a well-established biomarker for Alzheimer's disease (AD) and is routinely measured using different immunoassays. However, broad-scale use of these antibody based techniques in clinical routine is hampered by high variability. Thus, there is a need for harmonization of CSF AD biomarker assays with high analytical precision, selectivity, stability and with low variability across centers. Mass spectrometry (MS) has been used for quantification of small molecules in the clinic for many years. A key difference of MS quantification compared to immunoassays is the ability to use stable isotope-labeled internal standards (IS). Thereby, MS has the potential to provide unbiased quantification unaffected by matrix effects, thus decreasing inter-laboratory variation. In the current study we test this hypothesis for CSF Aβ42 measurement in a round robin study involving 4 laboratories involved in the Global Consortium for Biomarker Standardization (GCBS) of the Alzheimer's Association. Aliquots from twelve pools of human CSF were distributed to the participating laboratories. Samples were spiked with 15N uniformly labeled Aβ42 (15N-Aβ42) as an IS and external calibration was performed either in human or artificial CSF. Spiked samples were denatured with guanidine hydrochloride, extracted using solid phase extraction (SPE) and injected on a reversed phase column prior to MS analysis in the selected reaction monitoring mode. The inter-laboratory variation showed excellent correlation (R2 > 0.98). The average intra-laboratory CVs was 4.7% while the average inter-laboratory CV was 12.2%. Since no reference material exists for CSF Aβ42, we evaluated the potential benefit of such a material by designating one of the samples as reference. Following this correction, the average inter-laboratory CV was 8.3%. Using SPE under denaturing conditions in combination with stable isotope-labeled IS overcomes the immune-affinity based center-to-center variations possibly due to elimination of matrix-effects and lot-to-lot variations between kits. Our study shows that MS Aβ42 assays established in four laboratories using different instrumentation, calibration methods, and different calibrant preparations, produce similar results. The use of a common reference sample further decreases inter-laboratory variation, highlighting the importance of developing a standard reference material.
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