Bioanalysis

Aim: A novel, sensitive and reproducible method for quantification of dolutegravir (DTG) in dried breast milk spots (DBMS) was developed and validated for use in clinical studies. Its application enabled measurement of DTG pharmacokinetics in breastfeeding mothers and their infants. Results/methodology: Sample extraction was by liquid–liquid extraction using tert-butyl methy-ether, with DTG–d5 as an internal standard. DTG was eluted on a reverse phase C18 Waters XBridge (3.5 μm: 2.1 × 50 mm) column using a gradient mobile phase consisting of 0.1% formic acid in deionised water or methanol. The assay was validated over a calibration range of 10–4000 ng/ml. Conclusion: Stability, inter and intra-assay variability were acceptable according to FDA and EMA bioanalytical method guidelines. The assay is robust, accurate, precise and can be reliably applied for analysis of clinical samples in trials from low resource settings.

An easy, quick, precise, active and reproducible reverse method high performance liquid chromatography (RP-HPLC), as well as Ultra pressure liquid chromatography (UPLC) unique technique, was developed for the bioanalytical method of Etoposide and Cisplatin with Oxaplatin as an internal standard. Chromatographic analysis was performed using HPLC was Waters Alliance e-2695, and UPLC was Waters Acquity UPLC by using x-bridge phenyl 150x4.6mm, 3.5µ column and therefore the mobile phase containing 0.1% triethylamine and acetonitrile at a ratio out of 60:40 v/v. The flow rate was 1 ml/min, and analytes were detected at 283 nm employing a photodiode array detector at ambient temperature in both liquid chromatographic systems. The proposed biological method was proved in both HPLC and UPLC with USFDA guidelines. USP tailing is 1.1, 1.02 for etoposide and 1.16, 1.05 for Cisplatin in HPLC and UPLC. USP plate count is 4794, 3884 for Etoposide and 9289, 14487 for Cisplatin. The calibration curv...

Hamilton Microlab Star liquid handlers have unique and flexible functionalities such as independent liquid aspiration/dispense in each channel, and untethered access to and from any given location on the machine deck layout. However, the STAR control program (Venus) can be difficult to learn and operate. To simplify STAR programming for analysts, a user friendly interface software called HSPA (Hamilton STAR Program Assistant) was generated. HSPA communicates with Venus during the liquid handling process through a text output file. It leaves the instrument action commands to Venus, and allows users to focus on detailed method information and sample location information.

A fast and selective method based on high-performance liquid chromatography with diode array and atmospheric pressure chemical ionization-mass spectrometry detection (HPLC-DAD-APCI-MS) was developed and optimized to assess specific food samples content in carotenoids. Two compounds were chosen as significant carotenoids with respect to nutritional value, beta-carotene, and lycopene. The optimum conditions suitable for samples analysis were: a mobile phase of methanol:water (95:5) (A) and acetonitrile:methylene chloride (90:10) (B), with a gradient elution: 0-4 min 0% B, 4-7 min 20% B, 7-10 min 50% B, 0-10 min flow 0.1 mL/min, 10-30 min 90% B, 10-30 min flow 0.2 mL/min. Analytes were separated within 30 min on a C18 column and a diode array detection (450 nm for beta-carotene and 470 nm for lycopene).The linear dependence between peak area and concentration ranged from 5x10-7 molL-1 to 5x10-5 molL-1 (r=0.9998, n=9) for beta-carotene and respectively 7.5x10-7 molL-1 to 7.5x10-5 molL-1...

Eclipta alba (Bhringraj) in ayurveda has been widely used as a traditional medicine for its multi-therapeutic properties for ages. Luteolin (LTL), wedelolactone (WDL) and apigenin (APG) are the three main bioactive phytochemicals present in Eclipta alba extract. However there was a lack of sensitive bioanalytical method for the pharmacokinetics of these free compounds in plasma which majorly contributes for their activities after oral administration of Eclipta alba. The present study aims to develop a sensitive, rapid and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous estimation of mice plasma concentrations of LTL, WDL and APG using quercetin as an internal standard for the pharmacokinetic analysis. Analytes were separated on Phenomenex Luna C18 (150 x 4.6 mm, 3.0 µm) column with mobile phase containing methanol: acetonitrile (90: 10, v/v) and 0.1% formic acid in 10 mM ammonium formate buffer in the ratio of 70: 30 (v/v) in isocratic mode. Liquid-liquid extraction was optimized using Hansen solubility parameters and diethyl ether finalized as an extraction solvent for the recovery ranging from 61-76% for all analytes in mice plasma. The validated method has an accuracy and precision over the linearity range of 0.1 – 200 ng/mL with a correlation coefficient (r2) of ≥0.997. The intra and inter-day assay accuracy was between 98.17-107% and 95.83-107.89% respectively and the intra and inter day assay precision ranged from 0.37-6.05% and 1.85-10.76%, respectively for all the analytes. This validated method can be used for future clinical investigation studies of Eclipta alba extracts.

This review illustrates the need to use nuclear magnetic resonance (NMR) spectroscopy for the quantitative analysis of small molecules in their crude forms and in mixtures.

We provide the basic concepts of quantitative NMR (qNMR), a brief description of important acquisition and processing parameters responsible for obtaining high-quality, reproducible NMR spectra in order to maximize accuracy, and the latest referencing techniques used for quantitative analysis.

We also describe methods that are used for quantitative analysis including calibration-curve and standard-addition methods. Further, we briefly address validation of qNMR spectroscopy and its major applications in various scientific disciplines

A new method is described for simultaneous determination of 3-methylindole (3MI) and indole in porcine adipose tissue. Sample preparation included liquid–liquid extraction with n-hexane and 75% aqueous acetonitrile. The acetonitrile extracts were analysed by liquid chromatography–mass spectrometry (LC–MS) using atmospheric pressure chemical ionization (APCI) with selective ion monitoring of protonated ions [M + H]+. This method showed excellent linearity over the concentration range tested (from 2 to 500 ng mL−1 for 3MI and from 1 to 500 ng mL−1 for indole) and good accuracy (recovery of 92 ± 10% for 3MI and 91 ± 10% for indole). This new LC–MS method was compared with traditional colorimetric method for 3MI equivalent. Additionally, the correlation between 3MI concentrations in adipose tissue and plasma was studied. The described LC–MS method can be used to quantify 3MI and indole in porcine adipose tissue in various endocrinological or meat science studies.

The misuse of medicines for performance enhancement in sport (doping) is not approved by regulatory agencies, and is illegal in many countries. In addition to the 'traditional' doping agents such as steroids, β-blockers and blood transfusions, the list of agents and techniques used in doping is increasing and now includes newer medicines such as erythropoiesis-stimulating agents and growth hormones. Innovative new medicines are of particular interest as would-be dopers may believe them to be undetectable by current methods. Close collaboration between the biopharmaceutical industry and anti-doping agencies such as the World Anti-Doping Agency is critical to a successful anti-doping strategy. Industry is ideally placed to identify the doping potential of new medicines at early stages and to support early development of detection assays. A strong, united front between the biopharmaceutical industry and anti-doping agencies is essential to counter the misuse of medicines for pe...

In this review article, a brief discussion of Bioanalytical method development and Validation. The development process to ensure quality and reliability of method development of bio-analytical sample by using Various Validation parameters like accuracy, precision, reproducibility and stability are performed it's limits sets by the FDA (Food and drug Administration).The bioanalytical method includes such as solid phase extraction, Chromatography method etc.

An ultrasensitive nano UHPLC-ESI-MS/MS method is developed to simultaneously monitor three low-concentration neuromedin-like peptides in microdialysates. Peptide preconcentration and sample desalting is performed online on a trap column. A shallow gradient slope at 300 nl/min on the analytical column maintained at 35°C, followed by two saw-tooth column wash cycles, results in the highest sensitivity and the lowest carryover. The validated method allows the accurate and precise quantification of 0.5 pM neurotensin and neuromedin N (2.5 amol on column), and of 3.0 pM neuromedin B (15.0 amol on column) in in vivo microdialysates without the use of internal standards. The assay is an important tool for elucidating the role of these neuromedin-like peptides in the pathophysiology of neurological disorders.

Aim: Several neuropsychopharmacological properties have been attributed to the 3α-reduced pregnane steroids, allopregnanolone and pregnanolone, as well as to dehydroepiandrosterone sulfate because of their ability to modulate γ-aminobutyric acid (GABA A) receptors in the CNS. In order to understand better their role in several mechanisms in CNS, a new methodology is proposed to monitor these compounds in human plasma. Methodology & results: The analytes were first derivatized with 2-hydrazinopyridine and extracted from plasma using SPE. Then, the compounds were separated and detected by LC–MS/MS. A mobile phase of formic acid (0.1%) in water and methanol through a gradient of composition and a flow rate of 0.3 ml min-1 resulted in good separations of the analytes. Linear responses in wide range of concentrations and LOQs ranging from 10 (dehydroepiandrosterone 3-sulfate) to 40 pg ml-1 (dehydroepiandrosterone) were obtained in <9 min. The method proposed has been validated and then applied to monitor these neurosteroids in plasma samples from ten volunteers. Conclusion: For the first time, a straightforward and reliable method for the chromatographic separation of allopregnanolone, epiallopregnanolone and pregnanolone, as well as of dehydroepiandrosterone and dehydroepiandrosterone 3-sulfate was carried out, with optimal accuracy, sensitivity and specificity.

The current practice of using calibration curves with narrow concentration ranges during bioanalysis of new chemical entities has some limitations and is time consuming. In the present study we describe a split calibration curve approach, where sample dilution and repeat analysis can be avoided without compromising the quality and integrity of the data obtained. A split calibration curve approach is employed to determine the drug concentration in plasma samples with accuracy and precision over a wide dynamic range of approximately 0.6 to 15,000 ng/ml for dapsone and approximately 1 to 25,000 ng/ml for cyclophosphamide and glipizide. A wide dynamic range of concentrations for these three compounds was used in the current study to construct split calibration curves and was successfully validated for sample analysis in a single run. Using this method, repeat analysis of samples can be avoided. This is useful for the bioanalysis of toxicokinetic studies with wide dose ranges and studies...

We have applied high-field asymmetric waveform ion mobility spectrometry (FAIMS) to the analysis of the phosphopeptides APLpSFRGSLPKSYVK, APLSFRGpSLPKSYVK, and APLSFRGSLPKpSYVK. The peptides have identical amino acid sequences and differ only in the site of phosphorylation. The results show that FAIMS is capable of at least partially separating these species. Separation was confirmed by coupling FAIMS with high-resolution electron transfer dissociation (ETD) mass spectrometry. Phosphorylation is retained on the ETD peptide fragments thereby allowing assignment of the site of the modification. Co-eluting phosphopeptides which differ only in the site of modification are frequently observed in liquid chromatography/tandem mass spectrometry phosphoproteomics experiments, and therefore these proof-of-principle results have implications for the application of FAIMS in that field. Copyright © 2009 John Wiley & Sons, Ltd.

Introduction Recently, the USFDA issued the new 2018 guidance document for industry on bio-analytical validation. Due to this occasion, it would be worthy to look back in time as well into the (near) future on potential practical impacts the 2018 guidance document might have on bioanalytical method validation and laboratory operations. Bioanalytical method development and validation is the most important part in regulated bioanalysis. Validated bioanalytical methods are used for the quantitative measurement of drugs and their metabolites, endogenous compounds, and biomarkers in biological fluids. Drug concentrations are indispensable for the evaluation and interpretation of pharmacokinetic (PK), toxicokinetic (TK), and bioequivalence (BE) study data. The bioanalytical methods are not only applied for quantification of small molecules (molecular weight ≤ 900 Da) but also for larger molecules such as proteins , antibodies, and peptides. Bioanalysis can be quite challenging due to the complexity of the biological sample matrix. In addition to the sample complexity, data quality obtained from analyzed samples is directly related to the bioanalytical method's performance. Without any doubt, it is of utmost importance that bioanalyt-ical methods used in bioanalysis have to provide reliable data. According to the USFDA is the purpose of bioanalytical method validation: 1) to validate operation conditions, limitations, and 2) to determine the method suitability for its intended purpose and 3) to ensure that the bioanalytical method is optimized for sample analysis. For a long time, method validation procedures and strategies used in bioanalysis, as well as acceptance criteria needed for validation procedures, were a matter of personal prejudice. Many years there existed a lack of guidance uniformity on bioanalytical method development and validation within the bioanalytical community. This suddenly changed when industrial committees and regulatory agencies initiated the development and introduction of guidance documents on bioanalytical method validation for industry [1]. The first USFDA guidance document for industry on bioanalytical method validation was issued as a draft guidance in January 1999. Two years later this draft guidance document was finalized after incorporation of public comments, and the guidance was released as an official guidance document in May 2001. The guidance docu

The European Bioanalysis Forum dried blood spots/microsampling consortium is reporting back from the experiments they performed on further documenting the potential hurdles of the DBS technology. Their experiments focused on the impact of hematocrit changes, IS addition, spot homogeneity, aging of spots and stability of fresh blood and cards. Results from these experiments demonstrate that the issues of DBS in regulated bioanalysis are real and that the technology will need additional improvements to be ready for use as a general tool for regulated bioanalysis. In addition, results on fresh blood and card stability were shared at international meetings and will be reported at a later date.