Relative response factor determination

ID significant peaks by RRT and MW (LC-MS) only, unless more work is necessary for RRF (relative response factor) determination or project needs... 

The water residue level is also determined from the relative responses of the analytes to the internal standards. The sample residue levels are calculated by comparison with an average response factor determined by triplicate analysis of a five-point calibration curve. Samples receive 5ng of each internal standard (0.1 ngmL ) and are concentrated 50-fold by Ci8 SPE before analysis to achieve adequate instrumental sensitivity. The calculations to determine the residue level in water are outlined in Section 7.3.3. 

To allow for this, before the peak areas are normalised we must first correct each area so as to get the area we would have obtained had the detector response been the same for each of the three compounds. We will now use the results from our mixture to determine calibration factors (relative response factors) for the detector, and then use these for the analysis of a commercial tablet. 

H. Wang, G.J. Provan and K. Helliwell, HPLC determination of catechins in tea leaves and tea extracts using relative response factors. Food Chem. 81 (2003) 307-312. 

The truncated peptide analogs were used to demonstrate the specificity of the method and to evaluate the limit of quantitation of potential impurities. Potential impurities were spiked into a solution of IB-367 at 0.05%, 0.1%, 0.2%, 0.5%, and 1% to assay the linearity of potential impurities at low concentrations. The method exhibited acceptable linearity for impurities from 0.05 to 1%. The relative response factors of these analogs were assessed to determine area normalization feasibility. 

Determined by GC on an HP-5 or a Supelco Simplicity 1 fused-silica capillary column. The percentage compositions were obtained from electronic integration measurements, without taking into account relative response factors. 

Relative response factor the ratio of the response factor of individual related substance to that of a drug substance to correct for differences in the response of related substances and that of the drug substance. It can be determined using the following equation ... 

If a linearity curve (Figure 3.1) is constructed for both the related substance and the drug substance by plotting the response versus the concentration, the relative response factor can also be determined by... 

In this approach, one of the substances is considered for internal normalisation. For example, if compound 3 is used to determine the relative response factors K /3 and K2/3 of compounds 1 and 2 with respect to 3, one obtains ... 

To determine the relative response factor /cS/nms of the serotonin (S), with respect to its N-methyl derivative (NMS), the following relation is used ... 

Subunits that react with phloroglucinol are derived from proanthocyanidin extension subunits. Subunits that have not reacted with phloroglucinol are derived from proanthocyanidin terminal subunits or were present as monomeric flavan-3-ols. For quantitation, the sample peak areas of the individual subunits are compared with a(+)-catechin standard, and individual quantities are determined using the relative response factors shown in Table 11.4.1. 

The CLND is limited, of course, to mobile phases that do not contain nitrogen. Acetonitrile and amine modifiers, commonly used in HPLC, are therefore precluded. In addition, the CLND is not readily amenable to non-volatile buffers in the mobile phase. However, it is still possible to determine RRF values for samples run under these non-CLND-compatible HPLC conditions. In such cases, a two-step process is used. First, a CLND-compatible mobile phase (e.g., methanol/water/trifluoroacetic acid) is used to separate the compounds of interest and determine RRF values under those conditions (RRF ). Separately, the UV peak areas obtained using both the CLND-compatible and non-compatible HPLC conditions are compared by analyzing a common sample by both sets of HPLC conditions (apart from the CLND). The peaks of interest must, of course, be tracked to avoid misassignment (e.g., through UV spectra comparison). The relative response factor (RRF ) obtained for the CLND-compatible method can then be used to determine the relative response factor (RRF2)... 

True quantification of pyrazines in the powdered sarples presented seme difficulty. Due to sensitivity problems in sampling reconstituted sairples, the decision was made to purge the powdered NFCM. An internal standard could not be easily added to a dry sample, so quantification was accomplished calculating concentrations relative to response factors determined for external standard solutions. Standardized concentration units were determined using the following relationships ... 

At sufficiently high temperatures, the detector also responds to a variety of nitroaro-matics and to (V-nitrosodimethylamine. NOz yields of 0.70 and 0.90 were obtained for 2,4-DNT and TNT, respectively. The detection system exhibits a linear response for all nitrogen-containing compounds studied, and the detection limit for the determination of organic nitrates was found to be 0.05pmol. The relative response factors for aromatic nitro compounds and nitrosamine for the pyrolyser/luminol detector are presented in Table 3. 

Note The relative response factor for 8-tocopheryl propionate and for 3- plus y-tocopheryl propionates has been determined empirically to be the same as that for a-tocopheryl propionate. 

The relative response factor (RRF) of 1 defines that an impurity and active at identical concentrations have the same analytical response. Generally, no corrections for RRF need to be performed if the RRF of particular impurities are between 0.8 and 1.2. If the relative response factors are outside this region, the impurities can be overestimated or underestimated. For example, if impurity X has an RRF of 0.5 compared to the active, then it would be underestimated and if impurity Y has an RRF of 1.5 compared to the active, then it can be overestimated. In these cases the RRF must be taken into account when determining the percent of related substances during evaluation of the purity of the DS or the DR The following equation could be used. 

The external standard method has a particular advantage in that the reference standard (or standards) that are chosen can be identical to the solute (or solutes) of interest in the sample. This also means that the relative response factors between the reference solute and those of the components of interest are no longer required to be determined. In addition, using the external standard method, the reference mixture can be made to have standard concentrations that are closely similar to the components of the sample. As a consequence, errors that might arise from any slight non-linearity of the detector response are significantly reduced. 

The relative response factor of DneiK and RCMLA to Coomassle Blue R staining was determined by SDS-PAGE analysis of samples of known concentrations of DnaK and RCMLA. Aliquots of RCMLA and DnaK stock solutions of identical concentrations and Tris-HCl buffer (20 mM 19 mM NaCl. pH 7.3) were mixed to afford different [DnaK] [RCMLA] molar ratios (2 1 1 1 1 2 1 3) the concentration of DnaK was kept constant at 4.4 pM. These solutions were treated with SDS sample buffer and analyzed by densitometry as described above. The relative... 

When impurity reference standards are available only in limited quantities, relative response factors (RRFs) to the active ingredient can be used to quantitate impurity concentrations. RRFs can be determined spectrophotometrically by comparing the molar absorptivity of the impurity to that of the active component. However, in our experience, RRFs determined by HPLC by comparing peak area responses of the impurity to those of the active ingredient have been more accurate than those determined by spectrophotometric method. 

Demonstration of Linearity and Range Determination of Relative Response Factor... 

Blanco CG, Canga JS, Dominguez A, et al. 1992. Flame ionization detection relative response factors of some polycyclic aromatic compounds Determination of the main components of the coal tar pitch volatile fraction. J Chromatography 607 295-302. 

The GC-FID analysis is conducted by injection of 1 to 2 fil of FI or F3 into a gas chromatograph equipped with a high resolution capillary column (operated in sphtless injection mode). The injector and detector temperatures are set at 290 and 300°C, respectively. The GC temperature program is selected to achieve near-baseline separation of all of the saturated hydrocarbons. Quantitation of the individual components is performed by the internal standard method. The relative response factor (RRF) for each component is calculated relative to the internal standard. The TPH is also quantified by the internal standard method using the baseline corrected total area of the chromatogram and the average hydrocarbon response factor determined over the entire analytical range. ... 

For quantification 4-(4 -chlorobenzoyl)pyridine as internal standard was added to the extract. Concentrations of WPA were calculated with determined relative response factors. 

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