Absolute Peak Areas Research Articles

Logistic regression in approximation of the results of gas chromatographic analysis of thermally unstable compounds

Regularities and peculiarities of gas chromatographic analysis of thermally unstable compounds were considered on the example of mixture of the reaction products of isopropylbenzene (cumene) free-radical chlorination. The principal constituent in this mixture is (1-chloro-1-methylethyl)benzene, which has the lowest thermal stability, and is partially converted to a-methylstyrene, the only product of its thermal destruction at the chromatograph injector temperatures up to 300 °С. Nevertheless, the results of the study confirms that the gas chromatographic analysis of chloroalkylarenes is possible without their decomposition with the injector temperatures up to 200 °С, even if the analytes contain chlorine atoms at the tertiary carbon atoms and in the “benzylic” positions relative to the aromatic fragment. Similar control of thermal stability of analytes can be recommended for other samples contained potentially unstable constituents. It is shown that thermal decomposition of thermally unstable constituents of samples cannot be revealed from the results of gas chromatographic analysis with capillary columns using variations of their absolute peak areas. Such task can be solved only by using relative peak areas calculated in respect to thermally stable compounds. The dependencies of relative peak areas of unstable constituents vs. temperature (descending), as well as those of their decomposition products (ascending) are characterized by presence of two limits. Low temperature limits correspond to the real content of unstable constituents or their decomposition products is the samples, while the upper limits – to the composition of such samples at their hypothetically complete destruction. Such dependencies can be approximated by logistic regression equation if sampling into capillary columns is carried out at relatively high split ratios (approx. not less than 10 : 1). At lower split ratios the temperature dependencies of peak areas of unstable constituents and products of their transformation are strongly distorted by so-called sample’s composition discrimination effects that make impossible data approximation using logistic regression.

Croatian Native Grapevine Varieties' VOCs Responses upon Plasmopara viticola Inoculation.

The Plasmopara viticola pathogen causes one of the most severe grapevine diseases, namely downy mildew. The response to P. viticola involves both visible symptoms and intricate metabolomic alterations, particularly in relation to volatile organic compounds, and depends on the degree of resistance of a particular variety. There are numerous native grapevine varieties in Croatia, and they vary in susceptibility to this oomycete. As previously reported, in vitro leaf disc bioassay and polyphenolic compound analysis are complementary methods that can be used to separate native varieties into various resistance classes. This research used the Solid Phase Microextraction-Arrow Gas Chromatography-Mass Spectrometry method to identify the early alterations in the VOCs in the leaves after P. viticola inoculation. Based on the absolute peak area of sesquiterpenes, some discrepancies between the sampling terms were noticed. The presence of certain chemical compounds such as humulene, ylangene, and α-farnesene helped distinguish the non-inoculated and inoculated samples. Although specific VOC responses to P. viticola infection of native varieties from various resistance classes could not be identified, the response of less susceptible native varieties and resistant controls was associated with an increase in the absolute peak area of several compounds, including geranylacetone, ß-ocimene, and (E)-2-hexen-1-ol.

Isotope Ratio Outlier Analysis (IROA) for HPLC-TOFMS-Based Metabolomics of Human Urine.

Metabolic fingerprinting by mass spectrometry aims at the comprehensive, semiquantitative analysis of metabolites. Isotope dilution, if successfully implemented, may provide a more reliable, relative quantification. Therefore, the 13C labeled yeast extract of the IROA TruQuant kit was added as an internal standard (IS) to human urine samples measured in full-scan mode on a high-performance liquid chromatography-time-of-flight mass spectrometer (HPLC–TOFMS) system. The isotope ratio approach enabled the analysis of 112 metabolites. The correlation with reference data did not improve significantly using 12C/13C ratios compared to absolute 12C peak areas. Moreover, using an intricate 13C-labeled standard increased the complexity of the mass spectra, which made correct signal annotation more challenging. On the positive side, the ratio approach helps to reduce batch effects, but it does not perform better than computational methods such as the “removebatcheffect” function in the R package Limma.

Nitrile IR intensities directly measure electric fields in protic and non-protic environments

Nitriles are useful vibrational probes; the interpretation of their frequencies, however, is complicated by hydrogen bonding in protic environments. Here we observed a vibrational Stark effect (VSE) which correlates electric field to transition dipole moment (and, by extension, the nitrile peak area) instead of to frequency. This VSE governs the transition dipole moment for the CO stretch in acetophenone and the CN stretch in benzonitrile, with studies performed in bulk-phase solvents. While modest for the acetophenone carbonyl CO, the VSE for the benzonitrile nitrile CN is dramatic, and a >300% increase in absolute peak area from hexanes to water.

Decreasing the uncertainty of gas chromatographic quantification using the solvent’s signal in the method of external standard

Uncertainties of the results of quantitative determinations in gas chromatography using the methods based on the absolute peak areas (including the external standard method) are rather “sensitive” to the reproducibility of injections. The effective way to compensate for such errors is to introduce the additional standards into the samples, followed by replacing the absolute peak areas by their ratios to peak areas of the standards. It is important to underline that any constituents of the samples can be used as additional standards, including the solvents. Solvents can be used for these purposes even if the heights of their peaks are restric­ted when the analytical signals exceed the amplifier limits. Using the relative peak areas does not require any extra sample processing besides the registration of peak areas for solvents. Comparing the commonly known and modified methods of external stan­dard demonstrates that using the relative peak areas instead of the absolute ones does not influence the overall precision of determinations (according to the criterion “intro­duced-determined”) but improve the reproducibility by 2-3 times. The problem of increasing the reliability of such statistical evaluations of results is discussed and to solve it, it is proposed to change the “design” of the experiments. Instead of series of successive analyses of similar origin samples, the use of parallel determinations is preferable. This can be realized, for example, during the fulfillment of student’s practical works.

New Analytical Method for Determination of Phthalates in Wastewater by on Line LC-GC-MS Using the TOTAD Interface and Fraction Collector

There is an increasing demand for automatic, reliable and sensitive analytical methods for determining trace levels of phthalic acid esters (PAEs) in environmental samples. While on line coupled liquid chromatography-gas chromatography (LC-GC) has been proof to be a powerful tool for trace-level analyses in complex matrices, the present work presents a new totally automated on line LC-GC method, using the Through Oven Transfer Adsorption Desorption (TOTAD) interface, for the analysis of four of the main phthalates, dibuthyl phthalate (DBP), diethyl phthalate (DEP), dimethyl phthalate (DMP) and diethylhexyl phthalate (DEHP), in a matrix as complex as leachate. The sample is directly injected into the LC injector valve with no sample pretreatment other than simple filtration. The LC step separates the target analytes from matrix interference. Two different LC fractions are collected in a purposely designed fraction collector and then transferred to the TOTAD interface, which concentrates the analytes, totally eliminates the solvent and transfers the analytes to the GC-MS system, where the analysis is carried out. The LOD of the method varied from 0.1 µg/L (DEHP) to 1.4 µg/L (DMP), RSD for retention time below 0.14% and for absolute peak areas below 12% and linearity from 1 µg/L to 1000 µg/L (R2 > 0.99), except in the case of DEHP (linearity from 1 to 250 µg/L, R2 = 0.94). The method was applied to the analysis of the target analytes in samples collected from a municipal solid waste (MSW) landfill in Rosario (Argentina).

Study on preparation extraction process of Linggui Zhugan Decoction

To verify the appropriate preparation process of extracts for the solid substance benchmark of Linggui Zhugan Decoction. The extracts were prepared by different preparation processes, namely the traditional process(process 1), the extract combined with volatile oil separated from traditional process extract liquid(process 2), the modern secondary reflux extraction process(process 3) and the process that volatile oil was extracted first, then prepared according to the traditional process, and combined with extract(process 4); based on the characteristic spectrum, index components of cinnamaldehyde, glycyrrhizin, ammonium glycyrrhizinate, cinnamic acid, and the dry extract rate of process 1, the differences and similarities of four extracts were compared. The results showed that the similarity of the characteristic spectrum of process 2, process 4 and process 1 were all greater than 0.97, while there was no significant difference for the content of 4 quality control components and dry extract rate; the similarity of the characteristic spectrum of process 3 and process 1 was 0.91, the absolute peak area of 13 out of 21 peaks and the relative peak area of 7 peaks increased significantly, and the content of 3 out of 4 quality control components and dry extract rate also significantly increased. In conclusion, the material standards of extracts by the process 2 and 4 are consistent with that of the traditional process, so the two processes are suitable.

Establishment of chromatographic fingerprint of Squama Manis and its applications in animal source identification and quality grade discrimination

Squama Manis, or "Chuanshanjia" in Chinese, is a traditional Chinese medicine (TCM) for promoting blood circulation and reducing swelling and discharge; the only animal source used in TCM is the scales of Manis pentadactyla. However, in today's pharmaceutical market, there are many scales from other species of the same genus that are difficult to distinguish from Squama Manis. High-quality and low-quality scales are also severely confused. To solve the above problems, various analytical methods have been developed, such as thin-layer chromatography, mass spectrometry and DNA detection. Owing to their low resolving ability, high equipment cost, and inconvenient operation, none of these methods are appropriate for routine identification of Squama Manis. A chromatographic fingerprint can comprehensively reflect the synergic action of multiple chemical compositions in TCM and has been widely used for the quality control of TCM. In the present study, we established a fingerprint of Squama Manis and explored its feasibility in identifying the origin and quality grade of scales. First, Squama Manis powder was hydrolyzed by hydrochloric acid (1 mol/L). Next, the extract was analyzed on a Symmetry 300 C18 column by linear gradient elution, using 0.1% trifluoroacetic acid (v/v) in water and 0.1% trifluoroacetic acid (v/v) in acetonitrile as the mobile phase and 280 nm as the detection wavelength. The established method was systematically validated, demonstrating good precision, repeatability and sample stability (relative standard deviation (RSD)<5%). Subsequently, samples of different sources and quality grades were distinguished by similarity evaluation and discrimination analysis based on the fingerprint data. In the similarity evaluation, the reference fingerprint was defined as the average fingerprint of twelve first-class samples, and seventeen chromatographic peaks were identified as common peaks. Similarities between the reference fingerprint and fingerprints with different base sources and quality grades were calculated using the absolute area of common peaks as original data. The similarities between Squama Manis and scales from other animals were all less than 0.776, while the similarities between Squama Manis of different grades overlapped significantly, varying from 0.988 to 0.996 for first-class samples and 0.950 to 0.995 for general samples. The results reflected the feasibility of similarity evaluation for discriminating base source and its limitation in the distinguishing between quality grades. Nonetheless, first-class scales showed higher average similarity and lower RSD than general scales, which indicates some level of revelation between fingerprint similarity and quality grade. Thus, a better algorithm or discriminant model is required to distinguish between quality grades. Therefore, a supervised chemometric technique, kernel-based support vector machine (SVM), was applied to construct predictive models. The SVM is a common discriminant model that classifies samples by constructing a separate hyperplane in n-dimensional space, maximizing the margin between classes. Combination with a kernel function can effectively avoid "dimension disaster" when dealing with nonlinear data. In the model, the quality grade was defined as a sample label, and the absolute peak areas constituted the data matrix. Verified by 10-fold cross-validation, the unbiased prediction accuracy was up to 95.83%. The predicted results were highly consistent with the actual classifications. The results indicate the high feasibility of the established model for determining quality grade, as it performed significantly better than the similarity evaluation. Samples from batches A and B were completely discriminated and only two samples from batch S were incorrectly classified. Given the batch bias, we believe that model error may have been caused by man-made tag errors rather than the model itself. In conclusion, we established a chromatographic fingerprint for Squama Manis quality analysis and demonstrated its feasibility in animal source identification and quality determination by combining different data analysis methods. The established strategy may provide a new method for improving the the validity and accuracy of Squama Manis in clinical use.

The Perfect Timing For Sodium Bicarbonate Supplementation: Greater Possibilities With Probabilities

PURPOSE: To describe the reliability of blood bicarbonate pharmacokinetics to sodium bicarbonate (SB) supplementation and, based on those data, to estimate probabilities of SB ingestion timing before exercise using currently accepted thresholds. METHODS: Thirteen males (age 27±5 y; body mass (BM) 77.4±10.5 kg; height 1.75±0.06 m) ingested 0.3 g·kg-1BM SB in capsules on 3 occasions (SB1, SB2 and SB3). Blood was obtained at baseline and every 10 min following SB ingestion for 3h, then every 20 min for a further hour to determine bicarbonate concentration. Time-to-peak (Tmax), absolute peak (Cmax), absolute peak change (ΔCmax) and area under the curve (AUC) were determined and analysed using mixed models, as was the intraclass correlation coefficient (ICC), coefficient of variation (CV) and typical error (TE). Individual variation in pharmacokinetic responses was assessed using a Bayesian simulation approach using multilevel models with random intercepts. RESULTS: No significant differences between sessions were shown for blood bicarbonate regarding Cmax, ΔCmax or AUC (all p>0.05), although Tmax occurred significantly earlier in SB2 (127±36 min) than in SB1 (169±54 min, p=0.0088) and SB3 (159±42 min, p=0.05). ICC, CV and TE showed moderate to poor reliability for these variables. Bayesian modelling estimated that over 80% of individuals from the population experience elevated blood bicarbonate levels greater than +5 mmol·L-1 between 75-240 min after ingestion, and between 90-225 min for elevations greater than +6 mmol·L-1 (Table 1). CONCLUSIONS: Assessing SB supplementation using discrete values showed only moderate reliability at the group level, and poor reliability at the individual level, while Tmax was not reproducible. However, when analysed as modelled curves, a 0.3 g·kg-1BM dose was shown to create a long-lasting window of ergogenic potential, which has practical utility for athletes and suggests that individually tailored timings are not required.

HPLC methods of fexofenadine quantitative analysis in rabbits’ liver

The investigation of pharmacokinetics of marker substrates of carrier protein P-glycoprotein (Pgp, ABCB1-protein) including fexofenadine, is one of the methods of its functional activity evaluation.The aim of the study was to work out the HPLC methods of the quantitative determination of fexofenadine in rabbits’ liver.Materials and methods. The quantitative determination of fexofenadine was performed using Stayer chromatographic system (Akvilon, Russia) with UVV 104 ultraviolet detector. Reverse-phased chromatographic column Luna C18 100Å (250*4.6) was used with 5 µm granulation at 45°С. The сoncentration of fexofenadine was determined by methods of absolute peak area calibration.Results. The work was conducted in the isocratic mode. The composition of the mobile phase consisted of deionized water, acetonitrile and glacial acetic acid at the ratio of 267.4:120:4.33 brought to pH=6.7 with triethylamine. The sample processing was in the form of homogenization of 500 mg of ground liver in 500 µl of purified water with the subsequent centrifugation (1750 g) and selection of the supernatant. The proteins were precipitated by acetonitrile (2.5 ml) acidified with 375 µl of hydrochloric acid by shaking at 500 rev/min. The supernatant was transported into a separate test tube, where methylene chloride, diethyl ether and ethyl acetate were added (2 ml each). Then the solution was again shaken for 10 minutes (500 rev/min). After that, the solution was centrifuged (1750 g) and the supernatant was evaporated on a rotor-vacuum evaporator at 50°С. 300 µl of the mobile phase was added to the dry residue, and 100 µl was injected into the chromatograph. The method was validated in the linear range from 3 to 60 µg/g of fexofenadine with the acceptable intra- and intercycle accuracy, precision and stability. The method was tested on rabbits after the intravenous administration of fexofenadine at the dose of 11 mg/kg.Conclusion. The HPLC methods of fexofenadine quantitative determination in the hepatic tissue of rabbits has been worked out. It can be used for the evaluation of the functional activity of Pgp in preclinical studies.Abbreviations: Pgp – P-glycoprotein, HPLC – high performance liquid chromatography, rev/min – revolutions per minute

Specific Features of the Gas Chromatographic Analysis of Samples in High-Boiling Solvents Using Capillary Columns with a Low Split Ratio of the Carrier Gas Flow

Gas chromatographic analysis of samples in high-boiling polar organic solvents is characterized by several features. First, the resolution of the components depends on the injector temperature (Tinj), which should not be lower than the boiling point (Tbp) of the solvent. Second, the retention indices (RIs) of the components eluted before the polar solvent zone are somewhat overestimated compared to the retention indices of the components eluted after this zone. And, third, the so-called “discrimination effects ” are observed for these samples, first of all, the dependence of the absolute peak areas of the components from Tinj. It is confirmed that such dependence does not affect the results of quantitative analysis by the method of internal normalization. Nevertheless, the possibility of using application of this and other methods is recommended, and the condition of Tinj ≥ Tbp should be satisfied to verify the thermal stability and mutual compatibility of the components of the test samples. These features are set in the analysis of both a solution of model analytes in 1,3-butanediol (Tbp = 207°C) and a sample of the food flavoring, which is a solution of a complex mixture of components in dipropylene glycol (Tbp 222–236°C). The reason for these effects is a slowdown in the process of sample evaporation and dynamic modification of the stationary phase of the chromatographic column by high-boiling polar solvents.

Effects of the Discrimination of Sample Composition with the Use of Split Injection into Gas Chromatographic Capillary Columns

The manifestation of noticeable effects of the discrimination of sample composition upon split injection into short capillary columns with large diameters at small splitting ratios is discussed. They consist in the anomalously strong dependences of the peak areas of even volatile components on the injector temperature, the solvents used and, to a lesser extent, on the amounts of injected samples. It is proposed to consider the following criteria for assessing the degree of manifestation of these factors: (1) the dependence of the absolute peak areas of different analytes on the injector temperature and (2) the analogous dependence of the relative peak areas of the same components in different solvents. In this case, the relative peak areas of various components in the same solvents remained almost constant regardless of the injector temperature. The above effects complicate quantitative determinations based on the measurement of absolute peak areas (the method of internal normalization and the determination of distribution coefficients in partition chromatography). For these purposes, it is preferable to use standard capillary columns with split sample injection at a sufficiently high splitting ratio.

Odor characteristics of white croaker and small yellow croaker fish during refrigerated storage.

White croaker and small yellow croaker both belong to the fish family Sciaenidae, but their economic value and odor characteristics are quite different. In this study, electronic nose and gas chromatography-mass spectrometry were utilized to explore the odor characteristics of the two stored for different refrigeration periods. The results showed that their odor profiles could be clearly distinguished by principal component analysis. Compounds associated with fresh white croaker were found to be more complex than smaller yellow croaker through the load graph, while the result was opposite in later cold storage. The absolute peak areas of compounds like trimethylamine and 3-methyl-butanol were 6.42 and 1.42, respectively, in the white croaker, which were higher than in the small yellow croaker at the first day of refrigeration. And compound such as indole was first produced in white croaker during late cold storage. However, there were more compounds related to spoilage in the small yellow croaker; compounds like phenylethyl alcohol and benzeneacetaldehyde were not detected in the white croaker. PRACTICAL APPLICATIONS: White croaker and small yellow croaker are almost indistinguishable in appearance, especially after being cooked. But there are vast differences in their meat quality and odor characteristics, which affect their commercial values. As a result, a lot of white croakers are dyed and sold as small yellow croakers, although this does not change their eating or odor qualities. Principal component analysis of the odor characteristics of the two species of fish stored for different periods of refrigeration might provide some scientific basis for exploring the causes of their economic value differences.

Capillary Isoelectric Focusing-Mass Spectrometry Method for the Separation and Online Characterization of Intact Monoclonal Antibody Charge Variants.

We report a new online capillary isoelectric focusing-mass spectrometry (CIEF-MS) method for monoclonal antibody (mAb) charge variant analysis using an electrokinetically pumped sheath-flow nanospray ion source and a time-of-flight MS with pressure-assisted chemical mobilization. To develop a successful, reliable CIEF-MS method for mAb, we have selected and optimized many critical, interrelating reagents and parameters that include (1) MS-friendly anolyte and catholyte; (2) a glycerol enhanced sample mixture that reduced non-CIEF electrophoretic mobility and band broadening; (3) ampholyte selected for balancing resolution and MS sensitivity; (4) sheath liquid composition optimized for efficient focusing, mobilization, and electrospray ionization; (5) judiciously selected CIEF running parameters including injection amount, field strength, and applied pressure. The fundamental premise of CIEF was well maintained as verified by the linear correlation (R2 = 0.99) between pI values and migration time using a mixture of pI markers. In addition, the charge variant profiles of trastuzumab, bevacizumab, infliximab, and cetuximab, obtained using this CIEF-MS method, were corroborated by imaged CIEF-UV (iCIEF-UV) analyses. The relative standard deviations (RSD) of absolute migration time of pI markers were all less than 5% (n = 4). Triplicate analyses of bevacizumab showed RSD less than 1% for relative migration time to an internal standard and RSD of 7% for absolute MS peak area. Moreover, the antibody charge variants were characterized using the online intact MS data. To the best of our knowledge, this is the first time that direct online MS detection and characterization were achieved for mAb charge variants resolved by CIEF as indicated by a well-established linear pH gradient and correlated CIEF-UV charge variant profiles.

Features of injection and chromatographic separation of samples in high boiling viscous organic solvents using the capillary columns at a low split ratio

The features of injection and gas chromatographic separation of stock analytes in their solutions in high boiling viscous polar organic solvents (on the example of 1,3-butanediol) using a short capillary column of large internal diameter (Megabore type) at low split ratios are considered. The manifestation of so-called discrimination effects is confirmed, with the main of them being the dependence of absolute peak areas vs . injection temperature as well as similar dependence of relative peak areas in different solvents. However, these effects are manifested in a lesser degree than the same effects for the samples dissolved in low boiling solvents. It is demonstrated that the dependencies of peak areas vs . injector temperature ( S = aT + b ) are principally different for solvents with various boiling points. Thus, for the solutions of butyl acetate, cyclohexanone, anisol, butyl butyrate, and acetophenone in low boiling 2-propanol the linear dependencies S ( T ) are revealed for the injector temperature range of 120-180°С only (with correlation coefficients r = 0.986 – 0.999), while at the higher injector temperatures (210-240°С) the significant deviations from linearity are typical. At the same time, the solutions of the same components in higher boiling 1,3-butanediol demonstrate the linear S ( T ) dependencies within the whole temperature range (120-240°С). It is confirmed that the discrimination effects have no significant influence on the results of gas chromatographic analyses of the samples in high boiling solvents if the method of internal normalization is used. The main uncertainties of the results are caused by the column’s overloading with the solvent, which leads to the broadening and asymmetry of its peaks and their overlapping with the peaks of some analytes. DOI: http://dx.doi.org/10.15826/analitika.2018.22.3.005 Keywords : High boiling viscous organic solvents, gas chromatographic analysis, capillary columns, discrimination effects (Russian) Igor G. Zenkevich, Eugene Leleev St. Petersburg State University, Russian Federation, 198504, St. Petersburg, Universitetskii prosp., 26

Freeze-thaw stability and long-term stability of 84 synthetic cannabinoids in serum.

Information about stability of synthetic cannabinoids is important to give recommendations for storage conditions in cases in which use of synthetic cannabinoids is suspected. In this study, freeze-thaw stability (3 cycles at -20 °C at 1.5 ng/mL) and long-term stability at room temperature, 4°C and -20°C (for 150 days) were tested by a validated liquid chromatographic-mass spectrometric method for 80 synthetic cannabinoids. Results demonstrated good freeze-thaw stability for most of the substances. For 5 F-ABICA (72.6% of peak area of time point zero), 5 F- 2 ADB PINACA 2 Isomer (68.7 %), 5Cl-AB-PINACA (84.2 %), AB FUBINACA 2-Isomer (33.5 %), 5 F-PCN (89.7 %), ADB-FUBINACA (78.0 %), EG 018 (88.9 %), and PX-1 (89.4 %) three freeze-thaw cycles led to absolute peak areas < 90% compared to 6 control samples. For 51 of the 84 substances, stability in serum could be demonstrated after 315 days of storage at -20°C. For 82 of the 84 substances, stability in serum could be shown for at least 1 month at -20°C. Long- term stability at 4°C or 20°C showed worse results. Therefore, it is our recommendation to store real serum samples at -20°C until analysis. Copyright © 2016 John Wiley & Sons, Ltd.

Identification and comparative oridonin metabolism in different species liver microsomes by using UPLC-Triple-TOF-MS/MS and PCA

Oridonin (ORI) is an active natural ent-kaurene diterpenoid ingredient with notable anti-cancer and anti-inflammation activities. Currently, a strategy was developed to identify metabolites and to assess the metabolic profiles of ORI in vitro using ultra-high-performance liquid chromatography-Triple/time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). Meanwhile, the metabolism differences of ORI in the liver microsomes of four different species were investigated using a principal component analysis (PCA) based on the metabolite absolute peak area values as the variables. Based on the proposed methods, 27 metabolites were structurally characterized. The results indicate that ORI is universally metabolized in vitro, and the metabolic pathway mainly includes dehydration, hydroxylation, di-hydroxylation, hydrogenation, decarboxylation, and ketone formation. Overall, there are obvious inter-species differences in types and amounts of ORI metabolites in the four species. These results will provide basic data for future pharmacological and toxicological studies of ORI and for other ent-kauranes diterpenoids. Meanwhile, studying the ORI metabolic differences helps to select the proper animal model for further pharmacology and toxicological assessment.

Quality Assessment of Ijung-tang Preparations Using a HPLC Analysis

ABSTRACT Objectives : Ijung-tang (IJT) is a traditional herbal formula and has been used to treat digestive diseases such as abdominal pain, vomiting, and diarrhea. IJT consists of four herbal medicines, Ginseng radix, Atractylodis rhizoma alba, Zingiberis rhizoma, and Glycyrrhizae radix et rhizoma, containing various bioactive compounds. Quality assesment of IJT preparations was performed by analytical method for determining marker compounds.Methods : Determination of seven marker compounds in IJT preparations was quantitatively conducted by high- performance liquid chromatography equipped with a diode-array detector. The marker compounds were separated on a reversed-phase C18 column and the analytical method was successfully validated. Chemometric analysis was performed to compare IJT water extracts and commercial IJT granules.Results : Limit of detection and limit of quantification values were in the ranges of 0.093–2.649 μg/mL and 0.283–8.027 μg/mL, respectively. Precisions were 0.30–3.87% within a day and 0.23–2.35% over three consecutive days. Recoveries of the marker compounds ranged from 87.35-107.05%, with relative standard deviation (RSD) values < 6.15%. Repeatabilities were < 1.20% and < 1.71% of RSD value for retention time and absolute peak area, respectively. The results from quantitative analysis showed that the quantities of seven marker compounds of IJT samples varied, as were found in principal component analysis and hierarchical clustering analysis.Conclusions : The analytical method developed in the present study was precise and reliable to simultaneously determine marker compounds of IJT. Therefore, it can be used for the quality assessment of IJT preparations.

Decreasing the uncertainty of chromatographic quantitation by using the solvent as an additional standard

The principal sources of results uncertainties of chromatogra­phic quantification methods such as external standard and standard addition are non-controlled losses of the samples injected into the chromatographic column. To suppress such uncertainties we have proposed the modifications of these methods by applying additional standards. It meant replacing the absolute peak areas by their relative values. Further elaboration of this approach showed that solvents contained in the samples can be used as additional standards. This was illustrated by the analyses of model mixtures with the concentrations of target analytes ca . 20-30 mg/mL where the relative standard deviations of peak ratios appeared to be about 1-5 % despite the irre­pro­ducibility of the injections. The total uncertainty of the results decreased by 4-20 times. The approach under the consideration required no additional procedures of sample preparation, as well as no restrictions on the chemical origins of the solv­ents. This was due to the parameters of its peaks being required only for the calculation of rela­ti­ve areas of chromatographic peaks. Keywords: Chromatography, quantification, external standard, standard addition, application of a solvent as an additional standard. (Russian) DOI: http://dx.doi.org/10.15826/analitika.2016.20.2.005 Igor G. Zenkevich*, Denis V. Prokofiev St. Petersburg State University, Universitetskii prosp., 26, St. Petersburg, 198504, Russian Federation

Desirability functions as response in a d‐optimal design for evaluating the extraction and purification steps of six tranquillizers and an anti‐adrenergic by liquid chromatography‐tandem mass spectrometry

AbstractInternal standards can be added at different stages of an analytical procedure. When they are added at the beginning of a multiresidue method and their behavior is not exactly the same as that of the analytes, the intended correction for small variations within the analytical process could not be achieved. Because of this, in the present work, the use of d‐optimal designs together with desirability functions is proposed to state the experimental response under study. The overall desirability function used relates two analytical criteria: to assess a similar chemical behavior of each analyte in relation to its internal standard and to avoid a significant reduction of the absolute peak area of the internal standards. This strategy has been applied to the analysis of the effect of four factors related to the extraction and purification steps of six tranquillizers and a β‐blocker from pig muscle analyzed by liquid chromatography–tandem mass spectrometry. The effect of those factors has been evaluated by means of an ad hoc d‐optimal design consisting of only 11 experiments. The resulting levels of the four factors that enable to achieve the greatest overall desirability have also been compared with those obtained when either the standardized or absolute peak area has been considered as response. Differences in both the significant factors and their optimum levels have been observed. It is noticeable that the experimental effort necessary to study the effect of the factors has been reduced by more than 50% thanks to the d‐optimal design. Copyright © 2015 John Wiley &amp; Sons, Ltd.