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Instrumentation for Assessing mTBI Events
Published in Mark A. Mentzer, Mild Traumatic Brain Injury, 2020
Once the deformation is read from the kinetics spectrum, several quantities may be extracted, which can be used to further characterize different candidate materials. These parameters are illustrated in Figure 3.7. The principle used is the Beer–Lambert law, which is normally used to determine the concentration dependence C of a solute in a solvent from absorbance data A: A = εlC, where l is path length and ε is the extinction coefficient or molar absorptivity. In our case all material parameters may be assumed fixed, with the path length l being replaced by mass m due to delamination. Thus, the transmittance T is proportional to the variation ion path length or, equivalently, the mass change. Therefore a priori measurement of T(t) versus known m can be used to compute the change of mass: T(t) = εmρ where ρ is the density and t is the observation time.
Determination of oxygen status in human blood
Published in John Edward Boland, David W. M. Muller, Interventional Cardiology and Cardiac Catheterisation, 2019
A = log (I0/Is), where I0 = measured reference light intensity with a clear cuvette sample (i.e. water), IS = measured intensity with the blood cuvette sample. Subsequent calculations of saturations are based on the Lambert-Beers Law: Ayλ = E yλ × Cyλ × L, where E = proportionality constant (termed molar absorptivity, or the molar extinction coefficient of the particular derivative compound), A = absorbance, L = light path (thickness of cuvette), y = compound derivative, λ = wavelength, C = molar concentration of the compound derivative.17,18 Thus, the calculation involves measuring the absorbance of both an unknown sample containing haemoglobin and a sample containing a known amount of haemoglobin, and with algebraic rearrangement, calculating the amount of unknown haemoglobin.19
Spectroscopy and Fluorimetry
Published in Joseph Chamberlain, The Analysis of Drugs in Biological Fluids, 2018
The molar absorptivity of the solute in spectrophotometry is determined by the chemical structure. A functional group on a molecule which is responsible for absorption is termed a chromophore. Table 4.2 lists the absorption characteristics of a number of functional groups found in drugs.
Evaluation of analytical similarity between trastuzumab biosimilar CT-P6 and reference product using statistical analyses
Published in mAbs, 2018
Jihun Lee, Hyun Ah Kang, Jin Soo Bae, Kyu Dae Kim, Kyoung Hoon Lee, Ki Jung Lim, Min Joo Choo, Shin Jae Chang
Molar absorptivity was determined according to Beer-Lambert Law. First, measurements of OD280 and OD320 were performed using a Beckman DU730 spectrophotometer. Then, protein concentration was obtained from the amino acid analysis using the concentration of ‘robust’ amino acids (where % deviation between observed and expected results was ≤ 5%) such as aspartic acid, glycine, arginine, alanine, proline, valine and leucine. Molar extinction coefficient was calculated with UV absorbance at 280 nm, concentration of protein and molecular weight of CT-P6 and Herceptin®.
An optimised spectrophotometric assay for convenient and accurate quantitation of intracellular iron from iron oxide nanoparticles
Published in International Journal of Hyperthermia, 2018
Mohammad Hedayati, Bedri Abubaker-Sharif, Mohamed Khattab, Allen Razavi, Isa Mohammed, Arsalan Nejad, Michele Wabler, Haoming Zhou, Jana Mihalic, Cordula Gruettner, Theodore DeWeese, Robert Ivkov
To develop our assay, the chromogenic compound ferene-s was chosen because its higher molar absorptivity compared to ferrozine (Figure 1, Supplementary Table S1). The molar absorptivity was calculated from the Beer–Lambert law: A=ɛlc; where A is the absorbance, ɛ is the molar absorptivity, l is the path length and c is the concentration of the sample. When using a standard UV/Vis cuvette (usually with a fixed path length of 1 cm) the light passes through the sample horizontally and is therefore independent of the sample volume. The ferene-s assay protocol reported here uses a 96-well plate format for absorbance readings to facilitate high throughput processing. It is important to note however that microplate readers use a vertical light path and the distance for light to travel through the sample depends on the sample volume. To compare the absorbance readings between standard 1-ml UV/Vis cuvettes and 96-well plates we performed four independent experiments with iron standards ranging in concentration from 0 to 4.0 μg Fe/ml. In each experiment the same sample was used for both readings. The results are shown in Supplementary Tables S3 and S4. Absorbance readings were about 20% lower when measured with 96-well plates. This is expected because the approximate height of the 300 μl solution in each well is ∼0.88 cm. When adjusted to a height of 1 cm the readings remained about 8–9% lower than when measured with 1-ml cuvettes. The plate reader used for our absorbance measurements incorporates a path-check option in its software which automatically adjusts the absorbance readings to match the readings from a 1-ml cuvette. When using this option the absorbance readings from the 96-well plates were similar to those obtained using a 1-ml cuvette (compare Supplementary Tables S3 and S5).
Analytical similarity assessment of rituximab biosimilar CT-P10 to reference medicinal product
Published in mAbs, 2018
Kyoung Hoon Lee, Jihun Lee, Jin Soo Bae, Yeon Jung Kim, Hyun Ah Kang, Sung Hwan Kim, So Jung Lee, Ki Jung Lim, Jung Woo Lee, Soon Kwan Jung, Shin Jae Chang
The techniques used to compare the primary structure of CT-P10, EU- and US-Rituximab include amino acid analysis, molar absorptivity, peptide mapping (HPLC and LC-MS), N/C-terminal sequencing and determination of intact mass. Amino acid analysis using HCl hydrolysis followed by RP-HPLC showed the molar ratios for the most robust amino acids (including glycine, valine and alanine) were similar to the expected ratio for all 3 product (data not shown). Molar absorptivity determined by measuring the optical density at a wavelength of 280 nm and protein molarity derived from the amino acid analysis also showed similar results for the 3 products (Table 2). The UV-based tryptic peptide map revealed a highly similar peak profile among all the samples without missing or additional new peaks with comparative retention time of each peak, suggesting identical primary sequence of the 3 products. The representative tryptic peptide maps for each product are shown in Fig. 1. The amino acid sequence was further confirmed by LC-MS peptide mapping. The detected peptides by trypsin or Asp-N of the 3 products matched the expected peptides from the amino acid sequence. In addition, the MS/MS data (data not shown) confirmed that the amino acid sequences of CT-P10, EU- and US-Rituximab matched and sequence coverage by MS/MS was 100%. The N- and C-terminal sequences of the heavy and light chain were also confirmed by peptide mapping in combination with MS/MS. The detected N- and C-terminal sequences of the light and heavy chains matched the expected sequences of rituximab for all 3 products. Peptide mapping by LC-MS was also used to identify the post-translational modifications. The results show highly similar post-translational modifications of CT-P10 compared to EU- or US-Rituximab (Table 2). The levels of asparagine deamidation and methionine oxidation were similarly very low in all 3 products. With regards to the N/C-terminal variants, the first residue was detected predominantly as pyro-glutamate in both the light and heavy chains. The majority of C-terminal sequence in the heavy chain was detected as a lysine-clipped form. Although the relative content of N/C-terminal variants was generally conserved among the 3 products, CT-P10 contains slightly lower levels (about 2%) of N-terminal pyro-glutamate in the light chain compared to EU- and US-Rituximab. Intact mass analysis yielded 4 possible masses corresponding to G0F-G0F, G0F-G1F, G1F-G1F or G0F-G2F and G2F-G2F. In all samples, the observed mass closely matches the expected mass.