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.

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.

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.

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.

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.

Immunochromatographic strip assay for the rapid and sensitive detection of Salmonella Typhimurium in artificially contaminated tomato samples.

This study was designed to confirm the applicability of a liposome-based immunochromatographic assay for the rapid detection of Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella Typhimurium) in artificially contaminated tomato samples. To determine the detection limit and pre-enrichment incubation time (10, 12, and 18 h pre-enrichment in 1% buffered peptone water), the tests were performed with different cell numbers of Salmonella Typhimurium (3 × 10(0), 3 × 10(1), 3 × 10(2), and 3 × 10(3) CFU·mL(-1)) inoculated into 25 g of crushed tomato samples. The assay was able to detect as few as 30 Salmonella Typhimurium cells per 25 g of tomato samples (1.2 cells·g(-1)) after 12 h pre-enrichment incubation. Moreover, when the developed assay was compared with traditional morphological and biochemical culture-based methods as well as colloidal gold nanoparticle-based commercial test strips, the developed assay yielded positive results for the detection of Salmonella Typhimurium within a shorter period time. These findings confirm that the developed assay may have practical application for the sensitive detection of Salmonella Typhimurium in various food samples, including raw vegetables, with a relatively low detection limit and shorter analysis time.

External quality assessment of HbA1c and its effect on comparison between Swedish pediatric diabetes clinics. Experiences from the Swedish pediatric diabetes quality register (Swediabkids) and Equalis.

BACKGROUND: To explore to what extent measurement error can explain the variation of mean patient HbA(1c) between clinics. METHODS: For each year 2005-2010 data from 5380-6985 children, age <18 years, in 35-43 Swedish pediatric clinics was analyzed. Each year 13,000-19,000 HbA(1c) analyses were evaluated. Year mean HbA(1c) for each patient was calculated for HbA(1c) values when insulin dose was ≥0.5 U/kg. In Sweden HbA(1c) values were during the study period standardized to the Mono S level, HbA(1c)(Mono S)%, but are given also in the international unit HbA(1c)(IFCC), mmol/mol. Performance of locally measured HbA(1c) is monitored by Equalis through monthly external quality assessment (EQA) schemes. RESULTS: The yearly mean bias term for each clinic varied from -0.54 to 0.41 HbA(1c)(Mono S)%. The bias between clinic HbA(1c) and target value improved during the 6 years and the mean bias was for 79%-88% of clinics within the recommended level ±0.14 HbA(1c)% the last 2 years. Inter-clinic mean HbA(1c) had a wide interquartile range, 0.30-0.43 HbA(1c)(Mono S)% [3.2-4.5 HbA(1c)(IFCC)mmol/mol]. CONCLUSIONS: Regular participation in EQA schemes is necessary when comparing HbA(1c) values. The measurement error decreased during the 6-year period and explained from 28% to <10% of the inter-clinic variation in year mean clinic HbA(1c).

Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column.

A stationary phase composed of silica-bonded cyclofructan 6 (FRULIC-N) was evaluated for the separation of four cyclic nucleotides, six nucleoside monophosphates, four nucleoside diphosphates, and five nucleoside triphosphates via hydrophilic interaction chromatography (HILIC) in both isocratic and gradient conditions. The gradient conditions gave significantly better separations by narrowing peak widths. Sixteen out of nineteen nucleotides were baseline separated on the FRULIC-N column in one run. Unlike other known HILIC stationary phases, there can be dual-retention mechanisms unique to this media. Traditional hydrogen bonding/dipolar interactions can be supplemented by dynamic ion interaction effects for anionic analytes. This occurs because the FRULIC-N stationary phase is able to bind certain buffer cations. The extent of the ion interaction is tunable, in comparison to stationary phases with embedded charged groups, where the inherent ionic properties are fixed. The best mobile phase conditions were determined by varying the organic modifier (acetonitrile) content, as well as salt type/concentration and electrolyte pH. The thermodynamic characteristic of the FRULIC-N column was investigated by evaluating the column temperature effect on retention and utilizing van't Hoff plots. This study shows that there is a greater entropic contribution for the retention of nucleotide di and triphosphates, whereas there is a greater enthalphic contribution for the cyclic nucleotides with the FRULIC-N column.

A fast and inexpensive procedure for the isolation of synthetic cannabinoids from 'Spice' products using a flash chromatography system.

In the age of the Internet, the variety of drugs offered online is constantly increasing, and new drugs emerge every month. One group of drugs showing such an enormous increase is that of synthetic cannabinoids. Since their first identification in 'herbal mixtures', new structural modifications continue to appear on the market. In order to keep up with this process, toxicological screening methods need to be up to date. This can become extremely difficult if no reference material is available. In this article, a fast and effective way to extract and purify synthetic cannabinoids from 'herbal mixtures' is presented. This method opens a new opportunity for a timely reaction by obtaining reference material straight out of the 'herbal mixtures' ordered via the Internet. Isolation was carried out on a flash chromatography system with gradient elution on a C18 column using methanol and 0.55 % formic acid as mobile phases. The obtained purity of all compounds exceeded 99 %. In addition to the isolation of single compounds, the method proved to be suitable for the separation of various synthetic cannabinoids in one mixture, including the diastereomers cis- and trans-CP-47,497-C8. This approach for obtaining pure standards of new drugs proved to be effective, inexpensive and much quicker than waiting for the substances to be commercially available as reference material.

New and highly efficient column chromatographic extraction and simple purification of camptothecin from Camptotheca acuminata and Nothapodytes pittosporoides.

INTRODUCTION: Camptothecin, a widely used natural anti-cancer drug, is difficult to extract and purify effectively from plants. OBJECTIVE: To develop new and highly efficient extraction and purification methods for analysis and production of camptothecin from leaves and fruits of Camptotheca acuminata and Nothapodytes pittosporoides roots. METHODS: Dried materials were loaded in empty columns with fivefold 60% ethanol for leaves or 70% ethanol for fruits of C. acumnata, and sixfold 70% ethanol for N. pittosporoides roots. The columns were eluted with the same solvents at room temperature. Eluent was collected as extraction solution. Extraction solution from leaves and fruits of C. acuminata was vacuum-evaporated to remove ethanol, precipitated at pH 8.0 to remove alkaline insolubles and fractionated with chloroform at pH 3.0, which yields a crude product with 70% purity. Extraction solution from N. pittosporoides roots was concentrated to 1/10 volume and precipitated at pH 3.0, which yields a crude product with 60% purity. All crude products were purified by crystallisation. All steps were monitored by HPLC. RESULTS: Camptothecin was extracted from the three plant materials at a 98% rate with 15- or 18-fold solvent for content analysis, or at a 97% rate with five- or sixfold solvent for production. All crude products were purified to 98%. The overall recovery rates of camptothecin from plant materials to purified products reached 92% or higher. CONCLUSION: The new procedures are simple and highly efficient, and have multiple advantages for quantitative analysis and large production of camptothecin from plants.

Chromatographic methods for simultaneous determination of diiodohydroxyquinoline and metronidazole in their binary mixture.

Two chromatographic methods were developed for analysis ofdiiodohydroxyquinoline (DIHQ) and metronidazole (MTN). In the first method, diiodohydroxyquinoline and metronidazole were separated on TLC silica gel 60F254 plate using chloroform: acetone: glacial acetic acid (7.5: 2.5: 0.1, by volume) as mobile phase. The obtained bands were then scanned at 254 nm. The second method is a RP-HPLC method in which diiodohydroxyquinoline and metronidazole were separated on a reversed-phase C18 column using water : methanol (60 :40, V/V, PH=3.6 )as mobile phase at a flow rate of 0.7 mL.min-1 and UV detection at 220 nm. The mentioned methods were successfully used for determination of diiodohydroxyquinoline and metronidazole in pure form and in their pharmaceutical formulation.

Evaluation of rapid immunochromatographic NS1 test, anti-dengue IgM test, semi-nested PCR and IgM ELISA for detection of dengue virus.

Dengue virus (DENV) causes various clinical symptoms of differing severity based on time of infections. The existing laboratory methods, semi-nested PCR and Dengue IgM ELISA, still have limitations for diagnosis. A commercially available rapid immunochromatographic dengue NS1 antigen and IgM antibody tests in comparison with semi-nested PCR and IgM ELISA for confirmation of DENV infection were evaluated. In total, 237 single acute serum specimens and 50 paired sera of dengue patients were examined using the rapid dengue NS1 antigen test, IgM antibody test, semi-nested PCR and Dengue IgM ELISA. The NS1 and IgM rapid tests showed sensitivity of 70.6%, and 75.6%, respectively, and specificity of 73.4% and 97.1%, respectively. The combination of NS1 and IgM tests enhanced diagnosis. Thus rapid dengue NS1 antigen and IgM antibody tests are highly appropriate for diagnosis of dengue infection as it is rapid, easily applicable, sensitive and highly specific.

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.

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.

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.

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.

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).

[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.

An effective method for hemoglobin E detection: DEAE sepharose microcolumn.

Hemoglobin E (Hb E) is a variant hemoglobin that can lead to considerable morbidity when it occurs in a compound heterozygous state with beta-thalassemia. Therefore, its detection is important because it permits antenatal counseling. Hb E is prevalent in economically weaker regions of the world. Thus, its recognition is often hindered by the high costs of sophisticated Hb E-detection assays. We developed a simple visual test based on identifying Hb E in blood samples with DEAE Sepharose microcolumn chromatography. The diagnostic accuracy of our new Hb E microcolumn assay was 100% for sensitivity, specificity, and positive and negative predictive values. Furthermore, the Hb E microcolumn assay is rapid, reproducible, and economical; hence, it offers affordable screening for Hb E in less well-equipped laboratories.