Relative Response Factor Values Research Articles

Evaluation of the variation in relative response factors of GC-MS analysis with the internal standard methods: Application for the alcoholic products quality control

This paper focuses on the evaluation of variation of relative response factors (RRFs) obtained by two internal standard (IS) methods that are used to control the quality of alcoholic products. A standard IS method using 1-pentanol was compared with an “Ethanol as IS” method. The variation of RRF values for both methods was determined using standard solutions based on 20, 40 and 96% ethanol-water matrices. For this purpose, solutions of the ten most abundant volatile compounds were analysed at four different concentrations (250, 500, 1000 and 5000 mg L−1 absolute alcohol, AA) within these matrices. Each solution was measured four times by gas chromatography-mass spectrometry (GC-MS) in single ion monitoring (SIM) mode under repeatability conditions. Our results showed that for the 40% and 96% matrices, the ethanol and standard IS methods showed similar relative standard deviations (RSDs) variation of no more than 2% within the 250–1000 mg L−1 AA volatile compounds concentration range. For the 20% matrix as well as for the 250–5000 mg L−1 AA concentration range the resulting variation in calibration factors reaches 10% for both methods. As for the whole range of 20–96% alcohol by volume (ABV) and 250–5000 mg L−1 AA volatile concentration range, the resultant RRFs for the standard IS method (8% RSD) were more stable than those for the ethanol IS method (almost 40% RSD). Nonlinearity of the signal-to-amount dependence was also assessed with respect to the injection and detection processes.

Quality control of cannabis inflorescence and oil products: Response factors for the cost-efficient determination of ten cannabinoids by HPLC

The quality control of medicinal cannabis should include quantification of as many cannabinoids as practicable in a routine analytical lab, to accurately reflect the quality of the product. However, the cost and availability of some cannabinoid standards is an impediment to their routine use. This work seeks to overcome this obstacle by analysing samples using relative retention times (RRT) and relative response factors (RRF), relative to CBD and CBDA reference standards which are readily available. A high-performance liquid chromatography-photodiode array method was developed to quantify ten cannabinoids (Δ9-THC, Δ8-THC, THCA-A, CBN, CBD, CDBA, CBC, CBDV, CBG, and CBGA) in dried cannabis inflorescence and cannabis oil. This method was validated according to ICH guidelines. The proposed method has detection limits ranging from 20 to 78 µg/g, which provided sufficient sensitivity for the panel of cannabinoids. Non-cannabinoid surrogate matrices were used for spike recovery studies to determine method accuracy – analyte recoveries for the inflorescence and oil ranged from 90.1 to 109.3% (inflorescence mean, 100.9%; oil mean, 99.6%). The RRT and RRF values determined independently by three analysts were comparable, indicating the method is robust. The validity of analysis using RRT and RRF was further confirmed by testing six inflorescence samples, as it was found that concentrations above the order of magnitude of the LoQ agreed satisfactorily (range, 95.0 to 111.9%; mean, 100.0%) with the concentrations obtained through the conventional approach of multipoint calibration using pure standards. The proposed method is therefore suitable for the rapid and simple determination of a panel of ten cannabinoids without having to repeatedly purchase every expensive pure standard. Accordingly, analysts in the medicinal cannabis field may explore the use of RRF and RRT for their methods and instruments.

Use of qNMR to determine HPLC relative response factors for botanical reference standards used in pharmacopeial monographs

In modern botanical pharmacopeial monographs, one measurement of content is the quantitation of relevant constituents as marker compounds. The use of suitable reference standards (RSs) to quantify multiple compounds by HPLC is recommended in the U.S. Pharmacopeial (USP) botanical monographs. However, these substances may be expensive and difficult to develop into an RS. Surrogate RSs could be used instead of the actual constituents, provided that the relative response factors (RRFs) of each analyte to the selected surrogate RS are known. USP monographs of both Sichuan Lovage Rhizome and Dong Quai Root recognize Z-ligustilide as a major characteristic marker compound, making quantitation of Z-ligustilide and its analog(s) relevant for quality control. However, because Z-ligustilide is unstable, it is difficult to develop it into a quantitative RS. Instead, oxybenzone was selected as a surrogate external quantitative RS because of its similar chromatographic behavior to Z-ligustilide, its stability, and affordable cost. The RRF determination of Z-ligustilide to oxybenzone by the conventional HPLC procedure is challenging due to both the instability of the purified Z-ligustilide at ambient temperature and the difficulty of determining its purity. Therefore, a qNMR method was used to overcome these challenges as it enables to directly measure the mass ratio of Z-ligustilide to oxybenzone in the stock solution without the need for weighing and purity information. In the present study, RRF values of 1.01, 0.46, and 0.89 for Z-ligustilide, senkyunolide A, and ferulic acid relative to oxybenzone, respectively, were determined using the qNMR-based methodology.

Investigating the Ionization of Dissolved Organic Matter by Electrospray.

Electrospray ionization (ESI) operating in the negative mode coupled to high-resolution mass spectrometry is the most popular technique for the characterization of dissolved organic matter (DOM). The vast molecular heterogeneity and the functional group diversity of this complex mixture prevents the efficient ionization of the organic material by a single ionization source, so the presence of uncharacterized material is unavoidable. The extent of this poorly ionizable pool of carbon is unknown, is presumably variable between samples, and can only be assessed by the combination of analysis with a uniform detection method. Charged aerosol detection (CAD), whose response is proportional to the amount of nonvolatile material and is independent from the physicochemical properties of the analytes, is a suitable candidate. In this study, a fulvic acid mixture was fractionated and analyzed by high-pressure liquid chromatography–mass spectrometry in order to investigate the polarity and size distributions of highly and poorly ionizable material in the sample. Additionally, DOM samples of terrestrial and marine origins were analyzed to evaluate the variability of these pools across the land–sea aquatic continuum. The relative response factor values indicated that highly ionizable components of aquatic DOM mixtures are more hydrophilic and have lower molecular weight than poorly ionizable components. Additionally, a discrepancy between the samples of terrestrial and marine origins was found, indicating that marine samples are better represented by ESI than terrestrial samples, which have an abundant portion of hydrophobic poorly ionizable material.

Study of relative response factors and mass balance in forced degradation studies with liquid chromatography/photo-diode array detector/evaporative light scattering detector/mass spectrometry system

A case study was performed using photodiode array detection (PDA) in combination with evaporative light scattering (ELS) detection and mass spectrometry (MS) to assess both mass balance and the relative response factors (RRFs) in the forced degradation studies of the drug substance, glimepiride. The RRF value, which is the ratio of the response factor of the impurity to that of the API, was first determined using calibration curves of standards. This conventional technique was compared to a second, multi-detection technique, which used the PDA and ELS detectors to determine both the ultraviolet (UV) peak area and the concentration (based on measurement by ELS) in a single analysis. The resulting RRF values were then applied to forced degradation studies (acidic hydrolysis and oxidation) of glimepiride drug substance. This analysis was used to assess mass balance as well as impurity quantification. The impact of applying impurity RRF values was evaluated and found to have a significant impact on the percent of impurity quantified and on mass balance calculations at higher degradation levels. In addition, MS was used to quantify a non-chromophoric by-product and assess its impact on mass balance. Analysis of the forced degradation studies by MS detection also provided confirmation of the degradation pathway with new insights. Specifically, MS revealed differences in the stereoselectivity of the degradation processes: the acidic hydrolysis degradation was found to be stereoselective with the formation of the trans by-product while the oxidation degradation produced both the cis/trans isomers in a non-stereoselective reaction.

Determination of Relative Response Factors of Cefazolin Impurities by Quantitative NMR.

The relative response factors (RRFs) of ten cefazolin impurities were determined by quantitative nuclear magnetic resonance (qNMR) and high-performance liquid chromatography (HPLC) equipped with an ultraviolet (UV) detector. The purities of these ten cefazolin impurities were successfully measured by qNMR for the purpose of RRFs determination by HPLC. The RRF values and their uncertainties determined by the two approaches are comparable. While the qNMR approach is effective and makes it easier to determine the RRFs for impurities, it also has the advantage of allowing the universal detection of protons without the limitations of common mass detectors. The use of qNMR provides a reliable and universal method for the RRF determination of impurities.

DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING RP-HPLC METHOD FOR THE ESTIMATION OF DIACEREIN IMPURITIES IN API AND PHARMACEUTICAL FORMULATION

A sensitive, stability-indicating gradient RP-HPLC method has been developed for the quantitative determination of impurities of Diacerein in API and pharmaceutical formulation. Efficient chromatographic separation was achieved on a Hypersil BDS C18, 250 × 4.6 mm, 5 µm column with mobile phase containing a gradient mixture of solvents A and B. The flow rate of the mobile phase was 1.0 mL min−1 with column temperature of 40°C and detection wavelength at 254 nm. The relative response factor values of Rhein, Aloe Emodin, Monoacetyl Rhein I, and Monoacetyl Rhein II were 0.86, 0.80, 0.81, and 0.89, respectively. Diacerein formulation sample was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Diacerein was found to degrade significantly in acid, base, and peroxide stress conditions. The degradation products were well-resolved from Diacerein and its impurities. The peak purity test results confirmed that the Diacerein peak was homogenous and pure in all stress samples and the mass balance was found to be more than 98%, thus proving the stability-indicating power of the method. The developed method was validated according to ICH guidelines with respect to specificity, linearity, limits of detection and quantification, accuracy, precision, and robustness.

Relative response factor determination of β-artemether degradants by a dry heat stress approach

During the stability evaluation of β-artemether containing finished drug products, a consistent and disproportional increase in the UV-peak areas of β-artemether degradation products, when compared to the peak area decline of β-artemether itself, was observed. This suggested that the response factors of the formed β-artemether degradants were significantly higher than β-artemether. Dry heat stressing of β-artemether powder, as a single compound, using different temperatures (125-150 °C), times (10-90 min) and environmental conditions (neutral, KMnO(4) and zinc), resulted in the formation of 17 degradants. The vast majority of degradants seen during the long-term and accelerated ICH stability study of the drug product, were also observed here. The obtained stress results allowed the calculation of the overall average relative response factor (RRF) of β-artemether degradants, i.e. 21.2, whereas the individual RRF values of the 9 most prominent selected degradants ranged from 4.9 to 42.4. Finally, Ames tests were performed on β-artemether as well as a representative stressed sample mixture, experimentally assessing their mutagenic properties. Both were found to be negative, suggesting no mutagenicity problems of the degradants at high concentrations. Our general approach and specific results solve the developmental quality issue of mass balance during stability studies and the related genotoxicity concerns of the key antimalarial drug β-artemether and its degradants.

Evaluation of Relative Response Factor Methodology for Demonstrating Attainment of Ozone in Houston, Texas

In 2007, the U.S. Environmental Protection Agency (EPA) released guidance on demonstrating attainment of the federal ozone (O3) standard. This guidance recommended a change in the use of air quality model (AQM) predictions from an absolute to a relative way. This was accomplished by using a ratio, and not the absolute difference of AQM O3 predictions from a historical year to an attainment year. This ratio of O3 concentrations, labeled the relative response factor (RRF), is multiplied by an average of observed concentrations at every monitor. In this analysis, whether the methodology used to calculate RRFs is severing the source-receptor relationship for a given monitor was investigated. Model predictions were generated with a regulatory AQM system used to support the 2004 Houston-Galveston-Brazoria State Implementation Plan. Following the procedures in the EPA guidance, an attainment demonstration was completed using regulatory AQM predictions and measurements from the Houston ground-monitoring network. Results show that the model predictions used for the RRF calculation were often based on model conditions that were geographically remote from observations and counter to wind flow. Many of the monitors used the same model predictions for an RRF, even if that O3 plume did not impact it. The RRF methodology resulted in severing the true source-receptor relationship for a monitor. This analysis also showed that model performance could influence RRF values, and values at monitoring sites appear to be sensitive to model bias. Results indicate an inverse linear correlation of RRFs with model bias at each monitor (R2 = 0.47), resulting in a change in future O3 design values up to 5 parts per billion (ppb). These results suggest that the application of RRF methodology in Houston, TX, should be changed from using all model predictions above 85 ppb to a method that removes any predictions that are not relevant to the observed source-receptor relationship.

Enhanced Analysis of Steroids by Gas Chromatography/Mass Spectrometry using Microwave-Accelerated Derivatization

Derivatization of steroids is typically required before analysis by gas chromatography/mass spectrometry (GC/MS); nevertheless, the derivatization process can often be time-consuming and irreproducible. Although several strategies have been employed to enhance this process, few have the potential of microwave-accelerated derivatization (MAD) to be more efficient than traditional thermal derivatization methods. MAD using a synthesis microwave system was evaluated and compared to traditional thermal derivatization methods in terms of yield, reproducibility, and overall analysis time. Parameters affecting MAD, including reaction temperature, time, and power, were systematically optimized for several silyl reagents (BSTFA with TMCS, MSTFA, and BSA) and other derivatization procedures (MOX reagent and MTBSTFA). MSTFA was found to derivatize best with the microwave, as demonstrated by the enhanced relative response factors (RRFs). BSTFA with TMCS, on the other hand, did not couple as well, but RRF values improved significantly upon addition of polar solvents. The rapid (1 min) derivatization reactions associated with MAD had comparable RRFs for all reagents with those obtained with thermal heating (>30 min). This study highlights the best methods for analyzing a comprehensive variety of steroids and also provides ideal strategies for MAD of steroids on an individual or class level.

Determination of relative response factors of impurities in paclitaxel with high performance liquid chromatography equipped with ultraviolet and charged aerosol detectors

A case study was conducted to determine the relative response factors (RRFs) of paclitaxel-related impurities by high performance liquid chromatography (HPLC) equipped with an ultraviolet (UV) detector and charged aerosol detector (CAD) in tandem. The peak response using CAD was independent of analyte structure in an isocratic analysis for this application. After a sample containing known and unknown impurities was analyzed with HPLC–UV–CAD, an empirical approach was developed to calculate the RRFs for all impurities. The RRFs of known impurities were also determined by linear calibration curves. For known impurities, the RRFs values determined with two approaches are comparable. The new approach is effective yet simpler to determine the RRFs for unknown impurities or degradation products since the need for obtaining authentic pure materials was eliminated.

Separation of Δ5- and Δ7-Phytosterols by Adsorption Chromatography and Semipreparative Reversed Phase High-Performance Liquid Chromatography for Quantitative Analysis of Phytosterols in Foods

A method for the separation, isolation, and identification of phytosterols was developed. A commercial phytosterols mixture, Generol 95S, was fractionated first by adsorption silica gel column chromatography and then separated by means of a semipreparative reverse phase high-performance liquid chromatography fitted with a Polaris C8-A column (250 mm x 10 mm i.d., 5 microm) using isocratic acetonitrile:2-propanol:water (2:1:1, v/v/v) as the mobile phase. Milligram scales of six individual phytosterols, including citrostadienol, campesterol, beta-sitosterol, Delta7-avenasterol, Delta7-campesterol, and Delta7-sitosterol, were obtained. Purities of these isolated sterols were 85-98%. Relative response factors (RRF) of these phytosterols were calculated against cholestanol as an authentic commercial standard. These RRF values were used to quantify by gas chromatography-mass spectrometry (GC-MS) the phytosterols content in a reference material, oils, and chocolates.

Stable Isotope Tagging of Epitopes

Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.