Bioanalytical Method Development and Validation for Quantification of an Anti-Neoplastic Agent - Glasdegib by using LC–MS/MS (ESI) in Human Plasma

Bioanalytical method development and validation of a liquid chromatography-tandem mass spectrometry method for determination of β-lapachone in human plasma

Bioanalytical methods are used for the quantitative analysis of drugs and their metabolites in the biological media like saliva, urine, plasma, serum. Development and validation of bioanalytical method is important to understand the pharmacokinetics of any drug and/or its metabolites. Bioanalytical method development consists of three essential interrelated parts sample preparation, chromatographic separation and detection by using proper analytical method. Validation of a Bioanalytical method is the process by which it is established that the performance characteristics of the method meet the requirements for the intended Bioanalytical application. The validation is further divided into 3 segments full validation, partial validation, and cross validation each of which has its own purpose. This review describes mainly the various aspects for development of bioanalytical method and for the validation of bioanalytical methods. Also the Bioanalytical method transfer is also described.

The present study Estimation of drugs in biological media is increasingly important nowadays, which reveals information like bioavailability, bioequivalence, drug abuse, and pharmacokinetics and drug research. HPLC is the most suitable technique for the analysis of biological fluids owing to its well-developed characteristics and ruggedness. It is an extremely sensitive, precise, accurate, and rapid, separation technique. Clotrimazole is a broad spectrum antifungal agent that is used for the treatment of infections caused by various species of pathogenic dermatophytes, yeasts, and Malassezia furfur. The primary action of Clotrimazole is against dividing and growing organisms. Clotrimazole interacts with yeast 14-α demethylase, a cytochrome P-450 enzyme that converts lanosterol to ergosterol, an essential component of the membrane. In this way, Clotrimazole inhibits ergosterol synthesis, resulting in increased cellular permeability. Few analytical methods were reported for the quantitative determination of Clotrimazole and its combination with other drugs by HPLC. Only one method was reported for Clotrimazole in mice plasma by capillary electrophoresis. But there is no RP-HPLC method reported for the bio-analytical method development in human plasma using protein precipitation method under different chromatographic conditions. The present study is planned to develop newer analytical method for the determination of Clotrimazole by bio-analytical method development in human plasma using protein precipitation by RP-HPLC under different chromatographic conditions. CONCLUSION: A bioanalytical method was developed for the estimation of Clotrimazole by HPLC method. The method was validated for its transferability to other user or other laboratory. The HPLC method was developed by using 0.5%TEA in water (pH 3 adjusted with Orthophosphoric acid) and Acetonitrile in meticulous ratio. The peaks obtained for the drugs of interest by the present method are well resolved from each other without any interference and from the plasma endogenous proteins. The peaks are symmetrical with acceptable tailing factor. The retention time of Clotrimazole was within the limit. The results of linearity, intraday and interday precision study and capability of the extraction method were within the limits of bioanalytical method development. The method was linear with a correlation coefficient of acceptable agreement, which is suitable for the estimation of Clotrimazole in human plasma and other biological fluids. The method demonstrated relative recoveries with acceptable relative standard deviation. The limit of quantification (LOQ) and limit of detection (LOD) for Clotrimazole was found to be nanograms lesser than unity. Hence the developed method is sensitive for the estimation of Clotrimazole in trace amounts. Peak purity studies, with peak purity index values closer to unity reveals that the method developed was specific for the estimation of Clotrimazole in blood and other biological fluids. It can be concluded that the developed RP-HPLC method in human plasma was found to be very simple, reliable and selective for providing satisfactory accuracy and precision. The methods are suitable for routine quantitative analysis in pharmaceutical dosage forms. Hence this developed method can be used further in Bioequivalence and bioavailability studies of clotrimazole. Pharmacokinetic and bio-equivalence study centers

International Journal of PharmacokineticsVol. 2, No. 1 EditorialMeeting the rigorous demands of biosimilar pharmacokineticsSaara Mansouri, Jennifer Zemo & Krystal J AlligoodSaara Mansouri BioAgilytix Labs. 2300 Englert Drive, Durham, NC 27713, USASearch for more papers by this author, Jennifer Zemo BioAgilytix Labs. 2300 Englert Drive, Durham, NC 27713, USASearch for more papers by this author & Krystal J Alligood*Author for correspondence: E-mail Address: Krystal.Alligood@bioagilytix.com BioAgilytix Labs. 2300 Englert Drive, Durham, NC 27713, USASearch for more papers by this authorPublished Online:19 Jan 2017https://doi.org/10.4155/ipk-2016-0024AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: bioanalytical method validationbiosimilarligand-binding assayoriginatorpharmacokineticsReferences1 Cai XY, Gouty D, Baughman S, Ramakrishnan MS, Cullen C. Recommendations and requirements for the design of bioanalytical testing used in comparability studies for biosimilar drug development. Bioanalysis 3(5), 535–540 (2011).Link, CAS, Google Scholar2 DeSilva B, Smith W, Weiner R et al. Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm. Res. 20(11), 1885–1900 (2003).Crossref, CAS, Google Scholar3 Marini JC, Anderson M, Cai XY et al. Systematic verification of bioanalytical similarity between a biosimilar and a reference biotherapeutic: committee recommendations for the development and validation of a single ligand-binding assay to support pharmacokinetic assessments. AAPS J. 16(6), 1149–1158 (2014).Crossref, CAS, Google Scholar4 Colbert A, Umble-Romero A, Prokop S et al. Bioanalytical strategy used in development of pharmacokinetic (PK) methods that support biosimilar programs. MAbs 6(5), 1178–1189 (2014).Crossref, Google Scholar5 Xiao YQ, Halford A, Hayes R. Bioanalytical method development and validation of biosimilars: lessons learned. MOJ Immunol. 1(1), 1–4 (2014).Crossref, Google Scholar6 European Medicines Agency. EMEA/CHMP/BWP/49348/2005. Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: quality issues (2006). www.federalregister.gov/documents/2010/10/05/2010-24853/approval-pathway-for-biosimilar-and-interchangeable-biological-products-public-hearing-request-for.Google Scholar7 US Department of Health and Human Services. Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Center for Biologics Evaluation and Research (CBER). Quality considerations in demonstrating biosimilarity to a reference protein product (2012) (Draft). www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2014/10/WC500176768.pdf.Google Scholar8 US Department of Health and Human Services. Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Center for Biologics Evaluation and Research (CBER). Scientific considerations in demonstrating biosimilarity to a reference product (2012) (Draft). www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2015/01/WC500180219.pdf.Google Scholar9 European Medicines Agency. EMEA/CHMP/EWP/192217/2009. Guideline on bioanalytical method validation (2012). www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf.Google Scholar10 US Department of Health and Human Services. Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Center for Drug Evaluation and Research (CDER). Bioanalytical Method Validation (2013) (Draft). www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm368107.Google Scholar11 US Department of Health and Human Services. Food and Drug Administration. Docket No. FDA-2010-N-0477. Approval pathway for biosimilar and interchangeable biological Products. Public Hearing (2010). www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003953.pdf.Google Scholar12 European Medicines Agency. CHMP/437/04. Guideline on similar biological medicinal products (2015). www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2014/10/WC500176768.pdf.Google Scholar13 European Medicines Agency. EMEA/CHMP/BMWP/42832/2005. Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: non-clinical and clinical issues (2015). www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2015/01/WC500180219.pdf.Google ScholarFiguresReferencesRelatedDetails Vol. 2, No. 1 Follow us on social media for the latest updates Metrics Downloaded 69 times History Published online 19 January 2017 Published in print January 2017 Information© Future Science LtdKeywordsbioanalytical method validationbiosimilarligand-binding assayoriginatorpharmacokineticsFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

Bioanalytical method Validation employed for quantitative determination of drug and their metabolites in biological fluids. Comprises all criteria determining data quality, such as selectivity, accuracy, precision, recovery and senstivity. The main purpose of method validation is to demonstrate that a specific Bioanalytical method can reliably determine the concentration of drug in study sample with high degree of confidence. Validation does not means that method is perfect, but validation means method has met a set of criteria to ensure that it is reliable and consistent. Tizanidine is a central alpha 2 adrenergic agonist –inhibits release of excitatory amino acid in the spinal interneurones. It may facilitate the inhibitory transmitter glycine as well. It inhibits polysyneptic reflexes reduce muscle tone and frequency of muscle spasms without reducing muscle strength. Following oral administration, tizanidine is essentially completely absorbed .The absolute oral bioavailability of tizanidine is approximately 40%,due to extensive first-pass hepatic metabolism. Tizanidine is extensively distributed throughout the body with a mean steady state volume of distribution of 2.4 L/kg following intravenous administration in healthy adult volunteers tizanidine is approximately 30% bound to plasma proteins.

Bioanalysis is an essential part in drug discovery and development. Bioanalysis is related to the analysis of analytes (drugs, metabolites, biomarkers) in biological samples and it involves several steps from sample collection to sample analysis and data reporting. The first step is sample collection from clinical or preclinical studies; then sending the samples to laboratory for analysis. Second step is sample preparation and it is very important step in bioanalysis. In order to reach reliable results, a robust and stable sample preparation method should be applied. The role of sample preparation is to remove interferences from sample matrix and improve analytical system performance. Sample preparation is often labour intensive and time consuming. This guideline defines key elements necessary for the validation of bioanalytical methods. The guideline focuses on the validation of the bioanalytical methods generating quantitative concentration data used for pharmacokinetic and toxicokinetic parameter determinations. Guidance and criteria are given on the application of these validated methods in the routine analysis of study samples from animal and human studies. Measurement of drug concentrations in biological matrices (such as serum, plasma, blood, urine, and saliva) is an important aspect of medicinal product development. It is therefore paramount that the applied bioanalytical methods used are well characterised, fully validated and documented to a satisfactory standard in order to yield reliable results. This review provides an overview of bioanalytical method development and validation and main principles of method validation stages discussed.
 Keywords: Bioanalysis, Sample Preparation, Bioanalytical Method Development and Validation

Bioanalytical method development and validation are essential for reliable quantification of drugs in biological matrices. This research focuses on developing and validating a UPLC-MS/MS method for the simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma, adhering to established regulatory guidelines for bioanalytical method validation. The development and validation focus on creating a robust and sensitive assay suitable for bioequivalence studies and routine therapeutic drug monitoring. This method utilizes bisoprolol D5 and hydrochlorothiazide C13 D2 as internal standards to enhance accuracy and precision. Chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm particle size) with an isocratic mobile phase consisting of 10 mM ammonium formate buffer, methanol, and 0.1% ammonia solution (10:90, v/v). The flow rate was set at 0.3 mL/min, with a retention time of 2.2 min. Multiple reaction monitoring (MRM) was used with positive ESI for bisoprolol (m/z 326.36 → 116.13, internal standard m/z 331.36 → 121.13) and negative ESI for hydrochlorothiazide (m/z 296.11 → 269.00, internal standard m/z 299.11 → 270.00). The analytes and their respective internal standards were co-extracted using a liquid-liquid extraction method with tert-butyl methyl ether as the extraction solvent. Linearity for bisoprolol and hydrochlorothiazide was maintained over a concentration range of 1-100 ng/mL for bisoprolol and 1.0-300 ng/mL for hydrochlorothiazide, respectively, using a weighted least squares linear regression model (1/x). This method achieved a lower limit of quantification (LLOQ) of 1.0 ng/mL, making it highly sensitive for the detection of these analytes. Moreover, the method demonstrated high accuracy, precision, selectivity, and reduced overall analysis time, making it well suited for routine analysis and bioequivalence studies of 10 mg bisoprolol and 25 mg hydrochlorothiazide tablets.

The process of developing a method that will allow a compound of interest to be located and measured in a biological matrix is known as bioanalytical method development. A substance can frequently be measured using a variety of techniques, and selecting an analytical technique requires careful thought. Different extraction techniques, such as liquid-liquid extraction, solid phase extraction (SPE), and protein precipitation, are used to analyse drugs and their metabolites in a biological matrix. Samples from these extraction methods are spiked with calibration (reference) standards and quality control (QC) samples. The process of establishing if a quantitative analytical method is suitable for biomedical applications is known as bioanalytical method. includes all processes that will be presented measuring analytes quantitatively in particular biological samples such blood plasma, serum, or urine. To evaluate a drug's effectiveness and safety, clinical and non-clinical toxicokinetic and pharmacokinetic studies are used. Therefore, in order to produce accurate results, it is crucial that the applied bioanalytical procedures utilised are thoroughly defined, verified, and documented to a suitable quality. An overview of the development and validation of bioanalytical methods is given in this article, along with key considerations for each stage of method validation. The created procedure is then verified. Evaluation and interpretation of bioavailability, bioequivalence, pharmacokinetic, and pharmacodynamic investigations heavily rely on bioanalytical validations. where various parameters are carried out, including accuracy, precision, selectivity, sensitivity, reproducibility, and stability.

Dapagliflozin is used for controlling blood glucose levels in patients with type 2 diabetes. It is a sodium-glucose cotransporter 2 inhibitor, which enhances the elimination of blood glucose through the urine by inhibiting the protein involved in the transport mechanism of SGLT2. Dapagliflozin requires a selective and sensitive bioanalytical RP-HPLC method. Reverse phase - high performance liquid chromatography technique was used to develop and validate a bioanalytical method for the quantification of dapagliflozin (DAPA) in human plasma. The internal standard (IS) used was azilsartan medoxomil. In isocratic mode, the mobile phase consisted of 50:50 v/v acetonitrile and 0.1% orthophosphoric acid in water at a flow rate of 1.0 mL/min. The chromatogram was recorded at 224 nm. For the chromatographic separation, a Kromasil C18 column (250 mm × 4.6 mm; 5μ) was used. The drug was extracted from plasma samples by the protein precipitation method. The chromatographic run time was 15 min. Dapagliflozin and IS eluted at 4.6 and 5.7 min, respectively. The method was selective and sensitive, with a limit of quantification of 1.50 µg/mL. The developed method was found to be linear in the range of 1.50-60 µg/mL (r2 = 0.9994). The accuracy and precision obtained from six sets of quality control (QC) samples ranged from 96.23% to 108.67% and 1.35% to 3.19%, respectively. The extraction recovery of dapagliflozin in three QC samples ranged from 87.39% to 90.78%. The bench-top stability, stock solution stability, stability of processed extracted samples at room temperature, and freeze-thaw stability evaluations showed no evidence of degradation of dapagliflozin. The stability, selectivity, sensitivity, and reproducibility of the developed method make it suitable for the determination of dapagliflozin in human plasma.

The concepts, importance and application of bioanalytical method development have been discussed for a long time and validation of bioanalytical methods is widely accepted as pivotal before they are taken into routine use. Now it is widely accepted that bioanalysis is an integral part of the clinical diagnosis, biomarker discovery, pharmacokinetic/ pharmacodynamic characterization of a novel chemical entity from the time of its discovery and during various stages of drug development, leading to its market authorization. Bioanalytical methods, based on a variety of physico-chemical and biological techniques such as chromatography, immunoassay and mass spectrometry, must be validated prior to and during use to give confidence in the results generated. This study describes the development of an innovative, rapid, precise, selective and sensitive reverse phase high-performance liquid chromatography method for the quantitative determination of Saxagliptin (SAXA) in human plasma and pharmaceutical dosage form. Extraction of drug from plasma was done by employing optimized liquid-liquid extraction procedure. The sample was analyzed using methanol: acetonitrile in the ratio of 50:50v/v as mobile phase. Chromatographic separation was achieved on Thermo C18 analytical column (250mm×4.6mm i.d., 5.0μm) as stationary phase using isocratic elution (at a flow rate of 1 ml/min). The peak was detected using UV-PDA detector set at 230 nm and the total time for a chromatographic separation was 20 min. The calibration curve obtained was linear (r2 = 0.999) over the concentration range of 5-25μg/ml for SAXA. Method was validated for precision, robustness and recovery.

BackgroundSHetA2 is an oral anticancer agent being investigated for cancertreatment and prevention. The aim of this study was to develop and validatea simple, cost-effective, and sensitive HPLC-UV method for thequantification of SHetA2 in biological samples and to apply the method topharmacokinetic studies of the drug.MethodsSample preparation for mouse and human plasmas involved liquid-liquidprecipitation and extraction using chilled acetonitrile with 2,3-Diphenylquinoxaline as an internal standard. The separation of SHetA2 andinternal standard was achieved via Waters XBridge™ BEH 130 C18 (3.5μm, 2.1×150 mm) column coupled with a WatersXBridge™ C-18 (3.5 μm, 2.1×10 mm) guard column using65% v/v acetonitrile: distilled water as a mobile phase in anisocratic mode with a flow rate of 0.18 ml/min. The analytes were eluted ata detection wavelength of 341 nm at a column temperature of25°C.ResultsThe method was validated across a range of 5-1000 ng/ml for SHetA2 inplasma, with a lower limit of quantification of 5 ng/ml. The method showedhigh recovery in human (79.9-81.8%) and mouse (95.4-109.2%)plasma with no matrix effect. The intra- and inter-day accuracy andprecision studies demonstrated that the method was specific, sensitive, andreliable. Stability studies showed that SHetA2 is stable for 20 hpostoperatively in the auto sampler, and for six weeks at -80°C inplasma. Repetitive freezing and thawing may be avoided by preparing thealiquots and storing them at -80°C. The developed method wassuccessfully applied to study the plasma pharmacokinetics of SHetA2 intumor-bearing nude mice after intravenous and oral administration.ConclusionA novel method for quantifying SHetA2 in mouse and human plasmas hasbeen validated and is being applied for pharmacokinetic evaluation of SHetA2in tumor-bearing mice. The developed method will be utilized for thequantification of SHetA2 in clinical studies.

Bioanalytical methods should be reproducible and consistent when applying to clinical sample analysis. Incurred sample reanalyses (confirmatory reanalyses) are performed to support clinical data, and regulatory agencies evaluate the same before approval of bioequivalent products/drugs. A confirmatory reanalysis was demonstrated for metformin after administration of 2/500 mg repaglinide + metformin fixed dose formulation under fasted and fed conditions. The liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of metformin in human plasma using metformin-d6as an internal standard has been developed and validated. The ions transitions recorded in multiple reaction monitoring (MRM) were m/z 130.1→60.0 and 136.2→60.0 for metformin and metformin-d6, respectively. The compounds were isolated by solid phase extraction and separated on a C12 reverse phase (Synergi MAX-RP 80A) column, using isocratic mobile phase flow at a flow rate of 0.8 mL/min. No matrix effect was observed within t...

According to ICH Q3A(R), the impurity in a new drug substance is “any component of a new drug substance that is not the chemical entity defined as a new drug substance”. As Per ICH Q3B(R), the impurity in a new drug product is “any component of the drug product that is not the drug substance and excipients in the drug product. “The forced degradation studies are used to facilitate the development of analytical methodology, to achieve a better understanding of the drug substance and the drug product stability, and to determine degradation pathways and the degradation products. This study will help to get the most stable formulation. The bioanalytical method development and validation is an essential part in the drug discovery and development. There is need to develop and validate bioanalytical methods, as sponsors have to submit clinical pharmacology, bioavailability, bioequivalence, pharmacokinetic evaluation along with non-human pharmacology and toxicology studies and preclinical studies to regulatory authorities. There are number of spectroscopic methods includes Ultraviolet spectroscopy, Mass spectroscopy, Nuclear magnetic resonance spectroscopy and Chromatographic methods includes HPLC,HPTLC,GC,UPLC as well as hyphenated techniques like LC-MS, LC/ESI-MS, LC-NMR-MS used for identification and characterization of impurities in an API and the drug products forced degradation study to obtain stability data and bioanalytical methods.
 The uniqueness of this review is that it describes the detail information and background explaining impurities, forced degradation and bioanalytical method development and validation as well as all literature available regarding development and validation of all said methods for the drugs and the drug products used to treat type 2 diabetes.

Novel bio analytical method development, validation and application for simultaneous determination of nebivolol and S-amlodipine in human plasma using ultra performance liquid chromatography-tandem mass spectrometry

Triclabendazole is an anthelmintic or anti-worm medication. Asimple and sensitive bio-analytical HPLC method with UV detection was developed and validated triclabendazole in human plasma. The analytes were extracted from human plasma samples by liquid-liquid extraction technique. Triclabendazole showed maximum absorbance i.e.; λmax at 305 nm. The developed analytical method was validated as per ICH guidelines. The chromatographic separation was achieved with Hypersil BDS C18 (250 mm x 4.6 mm, Particle size: 5 µm), software Autochrom 3000 using a mobile phase composition of acetonitrile and buffer (60:40 V/V) at a flow rate of 1.2 mL/min with a run time of 7 min. The method showed good linearity in correlation coefficient (r) of >0.9998. The LOD and LOQ were found to be 1.00 µg mL-1 and 3.02 mg mL-1, respectively. Accuracy and precision data were found to be less than 2%, indicating the suitability of method. The developed HPLC method in human plasma using protein precipitation extraction for sample preparation was found to be very simple, reliable, precise, accurate, sensitive and selective analytical method for the estimation of Tricalbendazole. The method is suitable for routine quantitative analysis in pharmaceutical dosage forms. The method developed can be used in therapeutic drug monitoring units, bioequivalence and bioavailability studies, pharmacokinetic and toxicology studies of Tricalbendazole.