Effect of ethoxylated sorbitan ester surfactants on the chromatographic efficiency of poly(ethylene glycol)-based monoliths.

The effect of adding ethoxylated sorbitan ester surfactants (Tweens®) to poly(ethylene glycol) diacrylate-based monolithic recipes was investigated. Five different Tweens® have been evaluated to investigate the exact role of non-ionic surfactants in poly(ethylene glycol) diacrylate-based monolith preparations. These monoliths were characterized by scanning electron microscopy, infrared spectroscopy, and nitrogen physisorption analysis. Different morphological features, and surface areas were observed when different types of Tween® were included in the recipe; Tween® 20 and 85 showed small globules, while Tween® 40, 60 and 80 exhibited larger globular structures with different sizes and degrees of coalescence. The different Tween®-based monoliths were investigated for the chromatographic separation of mixtures consisting of hydroxybenzoic acids and alkylbenzenes. These columns were mechanically stable, except for Tween® 80. The highest methylene selectivity and the best overall performance were achieved by Tween® 60. The efficiency was increased by increasing the concentration of the Tween® 60 and the amount of poly(ethylene glycol) diacrylate Mn 700 in the recipes up to 30 wt%, each. Further increases in either Tween® 60 or poly(ethylene glycol) diacrylate Mn 700 led to formation of non-permeable columns. The optimized column was successfully used for separation of mixtures of nonsteroidal anti-inflammatory and sulfa drugs, with a maximum efficiency of 60,000 plates/m.

Development and Validation of Chromatographic Method for the Standardization of Homeopathic Formulation of Syzygium Cumini.

BACKGROUND: Syzygium cumini (Lam.), family Myrtaceae, has a long history of use in folk and traditional systems of indigenous medicine. Many homeopathic formulations of Jamun seeds are available in the market for their crucial usage as an anti-diabetic. Despite the popularity of homeopathic products, a lack of standard quality is a significant impediment in their acceptance. The present study aimed to develop and validate a chromatographic method for the standardization of the homeopathic formulation of Syzygium cumini. METHODS: The seeds of Syzygium cumini were studied for physicochemical evaluation and preliminary phytochemical screening. Also, the in-house standard and marketed homeopathic formulations of Syzigium cumini were standardized for pH, total fatty content, total phenolic and flavonoid content, with quantitative high-performance liquid chromatography- photodiode array detector (HPLC-PDA) analysis by using ellagic acid as a marker. RESULTS: The physicochemical characteristics of crude material were found to be within pharmacopeial limits. The phytochemical screening showed the presence of various secondary metabolites. The total phenolic and flavonoid content was higher in the in-house standard than in marketed formulations. A validated quantitative HPLC-PDA analysis showed variations of ellagic acid content in different homeopathic formulations. CONCLUSION: Physicochemical analysis and the HPLC method for quantitative estimation of ellagic acid can be used to standardize a homeopathic formulation of Syzygium cumini.

Scale-Up of Plasmid DNA Downstream Process Based on Chromatographic Monoliths.

Purification of high-quality plasmid DNA in large quantities is a crucial step in its production for therapeutic use and is usually conducted by different chromatographic techniques. Large-scale preparations require the optimization of yield and homogeneity, while maximizing removal of contaminants and preserving molecular integrity. The advantages of Convective Interaction Media® (CIM®) monolith stationary phases, including low backpressure, fast separation of macromolecules, and flow-rate-independent resolution qualified them to be used effectively in separation of plasmid DNA on laboratory as well as on large scale. A development and scale-up of plasmid DNA downstream process based on chromatographic monoliths is described and discussed below. Special emphasis is put on the introduction of process analytical technology principles and tools for optimization and control of a downstream process.

Perspectives on updates, clarifications and controversies in chromatographic assay guidance for bioanalytical method validation from major regulatory agencies and organizations.

Bioanalysis, a key supporting function for generating data for pre-clinical and clinical studies in drug development, is under the regulation of local agencies as well as global organizations to ensure the data integrity and quality in submission. As major regulatory agencies and organizations, the US Food and Drug Administration, the European Medicines Agency and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use have been updating their industry guidance for bioanalytical method validation, to keep up with the development new modalities, technologies and regulations. This article summarizes the recent updates and any clarifications and controversies triggered by those updates. Perspectives and recommendations are given based on our own experience as well as commonly accepted practice in the bioanalytical community.

One-step optimization strategy in the simulated moving bed process with asynchronous movement of ports: A VariCol case study.

The VariCol process is a variant of the conventional simulated moving bed (SMB) process, distinguished by the asynchronous shifting of the inlet and outlet ports of the chromatographic column train. This feature allows for a more flexible operation in column utilization and can also achieve higher separation performances. However, to take full benefit out of it, the operating parameters, such as the strategy for port switching, must be optimal. in this paper, a novel methodology for optimizing those parameters, based on a single NLP (non-linear programming), is proposed. The main advantage of this approach is that it significantly reduces the complexity of the original MINLP (mixed-integer non-linear programming) formulation currently discussed in the literature. The proposed optimization problem is built, considering that the average column configuration of three zones provides the necessary and sufficient information to describe the VariCol process. Several optimization scenarios for the enantioseparation of 1,1´-bi-2-naphthol and aminoglutethimide were considered to evaluate the proposed methodology and to compare the performance of VariCol and SMB processes. The results have shown that with the single NLP approach, it is possible to explore the optimal solution in all the VariCol process domains with less computational effort than other optimization strategies reported in the literature. That is a great advantage, especially in the context of real-time applications.

[Recommendations for aminoacids chromatography analysis]. / Recommandations concernant l'analyse de la chromatographie des acides aminés.

Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Amino acid chromatography allows the identification and quantification of more than forty compounds. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from SFEIM recommends an approach to accredit amino acid chromatography. Validation parameters and recommendations are discussed in this specific framework.

Cross-validation of equations to predict whole-body sweat sodium concentration from regional measures during exercise.

We have previously published equations to estimate whole-body (WB) sweat sodium concentration ([Na+ ]) from regional (REG) measures; however, a cross-validation is needed to corroborate the applicability of these prediction equations between studies. The purpose of this study was to determine the validity of published equations in predicting WB sweat [Na+ ] from REG measures when applied to a new data set. Forty-nine participants (34 men, 15 women; 75 ± 12 kg) cycled for 90 min while WB sweat [Na+ ] was measured using the washdown technique. REG sweat [Na+ ] was measured from seven regions using absorbent patches (3M Tegaderm + Pad). Published equations were applied to REG sweat [Na+ ] to determine predicted WB sweat [Na+ ]. Bland-Altman analysis of mean bias (raw and predicted minus measured) and 95% limits of agreement (LOA) were used to compare raw (uncorrected) REG sweat [Na+ ] and predicted WB sweat [Na+ ] to measured WB sweat [Na+ ]. Mean bias (±95% LOA) between raw REG sweat [Na+ ] and measured WB sweat [Na+ ] was 10(±20), 0(±19), 9(±20), 22(±25), 23(±24), 0(±15), -4(±18) mmol/L for the dorsal forearm, ventral forearm, upper arm, chest, upper back, thigh, and calf, respectively. The mean bias (±95% LOA) between predicted WB sweat [Na+ ] and measured WB sweat [Na+ ] was 3(±14), 4(±12), 0(±14), 2(±17), -2(±16), 5(±13), 4(±15) mmol/L for the dorsal forearm, ventral forearm, upper arm, chest, upper back, thigh, and calf, respectively. Prediction equations improve the accuracy of estimating WB sweat [Na+ ] from REG and are therefore recommended for use when determining individualized sweat electrolyte losses.

Correcting the Analytical Evaluation Threshold (AET) and Reported Extractable's Concentrations for Analytical Response Factor Uncertainty Associated with Chromatographic Screening for Extractables/Leachables.

It is generally acknowledged that quantitation in extractables and leachables (E&L) can be variably reproducible and accurate, depending on the quantitation approach taken. This is especially true for "simple" quantitation, which is the practice of estimating an analyte's concentration based on its response relative to that of an internal standard that has been added to the sample in a known amount. Simple quantitation is prone to error and variation as it is based on the largely false premise that the response factors for all extractables, leachables, and internal standard candidates are the same. It has been proposed that this uncertainty (inaccuracy and variation) be accounted for by adjusting two key parameters in E&L assessment, the reported concentrations themselves and the analytical evaluation threshold (AET) via an uncertainty factor (UF). This paper examines quantitation variation and discusses the means of establishing and utilizing the UF to adjust the AET to lower values and to adjust reported concentrations to higher values, enabling an impact assessment performed with this data to be more protective of patient safety. Although adjustment of the AET lower with the UF is supported, flaws in the concept of using the UF to adjust reported concentrations upward are considered, and it is recommended that the UF not be used in this manner. Rather, E&L quantitation should be based on compound-specific relative response factors, collected and collated in an E&L database.

Analysis of Total Thiols in the Urine of a Cystathionine ß-Synthase-Deficient Mouse Model of Homocystinuria Using Hydrophilic Interaction Chromatography.

Homocysteine and related thiols (cysteine, cysteinylglycine, and glutathione) in the urine of a cystathionine ß-synthase (CBS)-deficient mouse model were quantified using hydrophilic interaction chromatography with fluorescence detection. Urine samples were incubated with tris(2-carboxyethyl) phosphine to reduce disulfide bonds into thiols. After deproteinization, thiols were fluorescently derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Homocysteine, cysteine, cysteinylglycine, and glutathione in mouse urine were analyzed using an amide-type column with a mobile phase of acetonitrile/120 mM ammonium formate buffer (pH 3.0) (81:19). The developed method was well-validated. Thiol concentrations in the urine of CBS-wild type (-WT), -heterozygous (-Hetero), and -knockout (-KO) mice were quantified using the developed method. As expected, total homocysteine concentration in CBS-KO mice was significantly higher than that in CBS-WT and CBS-Hetero mice. The developed method shows promise for diagnoses in preclinical and clinical studies.

Purification of ADCs by Hydrophobic Interaction Chromatography.

Antibody-drug conjugate (ADC) in vitro potency has been shown to be dependent on drug load, with higher drug load providing lower IC50 values. However, in vivo potency is affected by intrinsic biological effects as well, such as plasma clearance, dose-limiting toxicity, etc. Developing a preparative HIC process for ADC purification to isolate species with a specific drug loading involves several steps including conjugation optimization, resin selection, solubility studies gradient screening, and step gradient development (buffer selection). In this chapter, the rationale and general considerations for developing a preparative hydrophobic interaction chromatography (HIC) method are described for isolation of an example ADC with specific drug load, e.g., two monomethyl auristatin E (MMAE) payloads (E2).

False positives from impurities result in incorrect functional characterization of receptors in chemosensory studies.

The discovery of chemoreceptors and technological advances have greatly increased our understanding of chemosensory mechanisms. However, some of this rapid progress may have been severely compromised by insufficient attention given to the possible effects of impurities in the chemical standards used in identifying ligands for target receptors. Here, we show that even trace amounts of impurities in test stimuli can completely obscure true ligand-receptor relationships. Responses to impurities may go unrecognized because of two main factors. First, the sensitivity of receptors to ligands may be greater than that of the instruments used to check sample purity. Second, the concentrations of impurities actually reaching the chemoreceptor during experiments may be orders of magnitude higher than that of the putative stimulus, due to large differences in vapour pressure between the impurities and the putative stimulus. Errors caused by impurities are not limited to receptor-ligand studies, but can also affect related areas of chemosensory research, such as neural processing, downstream behaviours, and "in-silico" bioinformatics predictions of response profiles. The purity of standards is always implied but must be checked rigorously to prevent skewed or invalid results or conclusions, such as we exemplify here for Drosophila melanogaster and its olfactory receptor DmOr7a.

Isotopic Ratio Outlier Analysis (IROA) for Quantitative Analysis.

There is always a tension within the omics sciences between trying to measure biological molecules rapidly and measuring accurately. Metabolomics as an omics science tries to measure the small biochemicals rapidly, in a single pass, but the current state of the art cannot provide the reproducibility or accuracy needed for clinical use or even daily reproducibility for larger experiments. The IROA TruQuant measurement system uses a daily "Long-Term Reference Standard (LTRS)" and a chemically identical Internal Standard (IS) to provide validated chemical identity, daily QA/QC on instrument and sample preparation, and accurate reproducible quantitation that is comparable across days, instruments, and even, for most compounds, chromatographic methods. The LTRS is, as the name implies, a Long-Term Reference Standard that is always the same and should therefore provide very similar results on a large but finite collection of compounds. All of the compounds in the LTRS are isotopically signed with formula indicating IROA patterns so they cannot be mistaken for one another. Because of the precise IROA patterns, a software-driven analysis of the compounds seen daily can determine the performance of the instrument in terms of sensitivity, in-source fragmentation, and chromatographic and injection stability and provide completely reproducible quantitation.

Utility of dry load injection for an efficient natural products isolation at the semi-preparative chromatographic scale.

Semi-preparative HPLC is one of the main techniques used for the purification of natural products (NPs). Generally, the sample has to be solubilized in organic solvent and injected on column through a loop valve. Since the solubility of crude natural extracts is often limited, a high solvent volume is needed for injection. This significantly compromises the resolution and increases the risk of overpressure in the system. To overcome this problem, a dry load injection procedure was developed to ensure optimum resolution even at high sample loading. The approach was first validated with a representative mixture of NPs standards, and successfully applied to two representative crude plant extracts: the dichloromethane extract of Annacardium occidentale and the methanolic extract of Hypericum perforatum. In all cases, the dry loading injection setup enabled an efficient introduction of the samples in the semi-preparative HPLC system. Different overload conditions of the columns were tested and the results demonstrated the robustness of the method and the possibility of applying it with a limited loss of resolution compared to liquid injection and without increasing pressure. The chromatographic resolutions were close to those obtained at the analytical level and separation were of much better quality when compared to liquid injection. This approach is especially relevant when purifying compounds isolated with high resolution from extracts that are poorly soluble in low volume of injection solvent due to the presence of lipophilic compounds and are thus not compatible for loop injection in typical reversed phase conditions. In addition, the dry load setup was also found to be useful when relatively polar components have to be separated in reversed phase conditions. In this case, loop injection with methanol generates strong peak distortion and broadening, while the dry load injection affords symmetrical peaks.

Detailed efficiency analysis of columns with a different packing quality and confirmation via total pore blocking.

We report on a systematic study involving columns with a clearly different efficiency (4 distinct quality groups) obtained by packing the columns that were C18 bonded and endcapped with a different carbon loading. Using B-term analysis (via peak parking) and theoretical models to estimate the magnitude of the Cm- and Cs-term contributions, it could be concluded that the difference in efficiency among the groups was entirely due to a difference in eddy dispersion. As such, the columns provided an ideal testing ground to verify how well the total pore blocking (TPB)-method can be used to probe differences in packing heterogeneity. In agreement with earlier literature observations, it turns out the TPB-method is much more sensitive to packing heterogeneities than the eddy dispersion (Heddy)-contribution measured under open-pore conditions via B- and C- term subtraction. Typically, differences in Heddy on the order of 0.1-0.5µm translate into a difference on the order of 0.5-2µm in the TPB mode. This confirms the TPB as a powerful technique to make very sensitive measurements of the homogeneity of packed beds.

Lessons from a comparison of immuno-chromatographic and chemiluminescent micro-particle immunoassay in the diagnosis of syphilis.

OBJECTIVE: To synthesize lessons from comparison of results obtained from the immuno-chromatographic SD Bioline testing method and the chemiluminescent micro-particle immunoassay Architect in the diagnosis of syphilis at Livingstone Central hospital laboratory. RESULTS: The specificity and sensitivity of SD Bioline syphilis 3.0 against the chemiluminescent immunoassay using the Architect syphilis Treponema pallidum (TP) was 85.3% and 91.3% respectively with substantial agreement between the two test methods (88%, ĸ  = 0.76; p < 0.0005). We recommend further comprehensive study with a larger sample size and clinical details to ascertain the validity of our findings. We also recommend using a non-treponemal test with the current treponemal tests being used to aid diagnosis.

International Council for Standardization in Haematology (ICSH) Recommendations for Laboratory Measurement of Direct Oral Anticoagulants.

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH) for providing haemostasis-related guidance documents for clinical laboratories. This inaugural coagulation ICSH document was developed by an ad hoc committee, comprised of international clinical and laboratory direct acting oral anticoagulant (DOAC) experts. The committee developed consensus recommendations for laboratory measurement of DOACs (dabigatran, rivaroxaban, apixaban and edoxaban), which would be germane for laboratories assessing DOAC anticoagulation. This guidance document addresses all phases of laboratory DOAC measurements, including pre-analytical (e.g. preferred time sample collection, preferred sample type, sample stability), analytical (gold standard method, screening and quantifying methods) and post analytical (e.g. reporting units, quality assurance). The committee addressed the use and limitations of screening tests such as prothrombin time, activated partial thromboplastin time as well as viscoelastic measurements of clotting blood and point of care methods. Additionally, the committee provided recommendations for the proper validation or verification of performance of laboratory assays prior to implementation for clinical use, and external quality assurance to provide continuous assessment of testing and reporting method.

Multivariate approaches for stability control of the olive oil reference materials for sensory analysis - part II: applications.

BACKGROUND: The organoleptic quality of virgin olive oil depends on positive and negative sensory attributes. These attributes are related to volatile organic compounds and phenolic compounds that represent the aroma and taste (flavour) of the virgin olive oil. The flavour is the characteristic that can be measured by a taster panel. However, as for any analytical measuring device, the tasters, individually, and the panel, as a whole, should be harmonized and validated and proper olive oil standards are needed. RESULTS: In the present study, multivariate approaches are put into practice in addition to the rules to build a multivariate control chart from chromatographic volatile fingerprinting and chemometrics. Fingerprinting techniques provide analytical information without identify and quantify the analytes. This methodology is used to monitor the stability of sensory reference materials. CONCLUSION: The similarity indices have been calculated to build multivariate control chart with two olive oils certified reference materials that have been used as examples to monitor their stabilities. This methodology with chromatographic data could be applied in parallel with the 'panel test' sensory method to reduce the work of sensory analysis. © 2018 Society of Chemical Industry.

Considerations regarding the validation of chromatographic mass spectrometric methods for the quantification of endogenous substances in forensics.

The requirement for correct evaluation of forensic toxicological results in daily routine work and scientific studies is reliable analytical data based on validated methods. Validation of a method gives the analyst tools to estimate the efficacy and reliability of the analytical method. Without validation, data might be contested in court and lead to unjustified legal consequences for a defendant. Therefore, new analytical methods to be used in forensic toxicology require careful method development and validation of the final method. Until now, there are no publications on the validation of chromatographic mass spectrometric methods for the detection of endogenous substances although endogenous analytes can be important in Forensic Toxicology (alcohol consumption marker, congener alcohols, gamma hydroxy butyric acid, human insulin and C-peptide, creatinine, postmortal clinical parameters). For these analytes, conventional validation instructions cannot be followed completely. In this paper, important practical considerations in analytical method validation for endogenous substances will be discussed which may be used as guidance for scientists wishing to develop and validate analytical methods for analytes produced naturally in the human body. Especially the validation parameters calibration model, analytical limits, accuracy (bias and precision) and matrix effects and recovery have to be approached differently. Highest attention should be paid to selectivity experiments.

Human rotavirus A detection: Comparison of enzymatic immunoassay and rapid chromatographic test with two quantitative RT-PCR assays.

OBJECTIVE: The aim of this study was to compare results of two commercially available kits used for routine detection of Rotavirus A in human stool samples with results of commercial quantitative reverse-transcription PCR (RT-qPCR) test and in-house RT-qPCR. MATERIAL AND METHODS: In total, 749 stool samples were screen-ed with the use of four different methods. The samples were collected from four diagnostic laboratories from March 2016 to June 2017. Diagnose of gastrointestinal disorders was stated in one third of tested patients, the rest of samples was collected from patients with other primary diagnose. The samples were tested with the enzymatic immunoassay (EIA) (RIDASCREEN® Rotavirus) and with rapid diagnostic immunochromatographic test (RDT) (IMMUNOQUICK® No-Rot-Adeno). As a reference method a commercial RT-qPCR test was used (Primerdesign Genesig® Kit) and it was compared with in-house RT-qPCR test prepared in our laboratory. The samples which in the reference RT-qPCR gave positive signal of reaction in cycle 28 or higher (Ct 28) were assessed as negatives in order to include only samples with some clinical relevance into sensitivity determination. RESULTS: Diagnostic sensitivity was assessed as 84.2% for EIA and 82.5% for RDT. The specificity of those tests was calculated as 97.8% for EIA and 96.4% for RDT. The performance of both diagnostic tests describing their positive predictive value was determined to be 87.3% for EIA and 80.3% for RDT. Negative predictive value was calculated to be 97.2% for EIA and 96.8% for RDT. Proportion of RVA-positive samples determined with the reference RT-qPCR test with our own cut-off level was 15.2% (n=114). Comparisons of the in-house and reference RT-qPCR tests showed very good agreement of results. The sensitivity of the in-house test was 100% and its specificity 99.7%. CONCLUSIONS: RT-qPCR is more sensitive for surveillance of rotavirus gastroenteritis than routinely used EIA or RDT methods. The specificity of both evaluated tests was very high. However, EIA was in all performance parameters assessed better than RDT.