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Background: Conventional home blood glucose measurements require a sample of blood that is obtained by puncturing the skin at the fingertip. To avoid the pain associated with this procedure, there is high demand for medical products that allow glucose monitoring without blood sampling. In this review article, all such products are presented. Methods: In order to identify such products, four different sources were used: (1) PubMed, (2) Google Patents, (3) Diabetes Technology Meeting Startup Showcase participants, and (4) experts in the field of glucose monitoring. The information obtained were filtered by using two inclusion criteria: (1) regulatory clearance, and/or (2) significant coverage in Google News starting in the year 2016, unless the article indicated that the product had been discontinued. The identified bloodless monitoring products were classified into three categories: (1) noninvasive optical, (2) noninvasive fluid sampling, and (3) minimally invasive devices. Results: In total, 28 noninvasive optical, 6 noninvasive fluid sampling, and 31 minimally invasive glucose monitoring products were identified. Subsequently, these products were characterized according to their regulatory, technological, and consumer features. Products with regulatory clearance are described in greater detail according to their advantages and disadvantages, and with design images. Conclusions: Based on favorable technological features, consumer features, and other advantages, several bloodless products are commercially available and promise to enhance diabetes management. Paths for future products are discussed with an emphasis on understanding existing barriers related to both technical and non-technical issues.

To compare the Kadish and the modified Dulguerov staging of individual participants to determine the impact of stage and other prognostic factors on disease-free (DFS) and overall survival (OS).Systematic review of EMBASE, MEDLINE, Cochrane Library, and CINAHL databases.The Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) was followed for this study. Articles including patients with olfactory neuroblastoma (ONB) staged with both Kadish and Dulguerov staging systems were reviewed. The raw data from eligible studies were requested to perform an individual participant data (IPD) meta-analysis.Pooled data from 21 studies representing 399 patients with ONB undergoing treatment with curative intent showed that increasing age, treatment with chemotherapy, and positive or unreported margin status portended worse DFS (This study represents the first IPD meta-analysis of ONB directly comparing the outcomes of Kadish and Dulguerov staging systems in patients treated with primary surgery. Both systems correlated with DFS and OS, with superior performance in the Dulguerov system. Furthermore, the Kadish C group represented a heterogeneous group with regard to outcomes after stratification by the Dulguerov system. Dulguerov T4 patients had the worst outcome, with most being approached with open resection.

True pituitary surgical emergencies are rare. These events can occur throughout the perioperative period and are broadly categorized by the timing of occurrence. Acute indications for emergent pituitary surgery include pituitary apoplexy, vision loss, and severe Cushing presentation. Emergencies may also occur intraoperatively, secondary to bleeding. Postoperative emergencies include epistaxis, pneumocephalus, and intracranial bleeding. Cerebrospinal fluid (CSF) leak occurs in about 37.4% of transsphenoidal sellar surgery, yet postoperative CSF leaks are less frequent at approximately 2.6%. As they occur often during pituitary surgery, CSF leaks alone are generally not considered a true surgical emergency unless associated with symptomatic tension pneumocephalus.

The COVID-19 pandemic has strained the biological matrix supply chain. An upsurge in demand driven by numerous COVID-19 therapeutic and vaccine development programs to combat the pandemic, along with logistical challenges sourcing and transporting matrix, has led to increased lead times for multiple matrices. Biological matrix shortages can potentially cause significant delays in drug development programs across the pharmaceutical and biotechnology industry. Given the current circumstances, discussion is warranted around what will likely be increased use of surrogate matrices in support of pharmacokinetic (PK), immunogenicity, and biomarker assays for regulatory filings. Regulatory authorities permit the use of surrogate matrix in bioanalytical methods in instances where matrix is rare or difficult to obtain, as long as the surrogate is appropriately selected and scientifically justified. Herein, the scientific justification and possible regulatory implications of employing surrogate matrix in PK, immunogenicity, and biomarker assays are discussed. In addition, the unique challenges that cell and gene therapy (CGT) and other innovative therapeutic modalities place on matrix supply chains are outlined. Matrix suppliers and contract research organizations (CROs) are actively implementing mitigation strategies to alleviate the current strain on the matrix supply chain and better prepare the industry for any future unexpected strains. To maintain ethical standards, these mitigation strategies include projecting matrix needs with suppliers at least 6 months in advance and writing or updating study protocols to allow for additional matrix draws from study subjects and/or re-purposing of subject matrix from one drug development program to another.

The next-generation artificial pancreas is under development with the goal to enhance tight glycemic control for people with type 1 diabetes. Such technology requires the integration of a chemical sensing unit combined with an insulin infusion device controlled by an algorithm capable of autonomous operation. The potential of near-infrared spectroscopic sensing to serve as the chemical sensing unit is explored by demonstrating the ability to quantify multiple metabolic biomarkers from a single near-infrared spectrum. Independent measurements of β-hydroxy-butyrate, glucose, and urea are presented based on analysis of near-infrared spectra collected over the combination spectral range of 5000-4000 cm-1 for a set of 50 ternary aqueous standard solutions. Spectra are characterized by a 1 μAU root-mean-square (RMS) noise for 100% lines with a resolution of 4 cm-1 and an optical path length of 1 mm. Calibration models created by the net analyte signal (NAS) and the partial least squares (PLS) methods provide selective measurements for each analyte with standard errors of prediction in the upper micromolar concentration range. The NAS method is used to determine both the selectivity and sensitivity for each analyte and their values are consistent with these standard errors of prediction. The NAS method is also used to characterize the background spectral variance associated with instrumental and environmental variations associated with buffer spectra collected over a multiday period.

Purpose of review Evidence-based medicine underpins clinical practice. Ideally, our clinical decision-making stems from systematic reviews of randomized controlled trials. However, in practice, this is not often the case, and we must instead rely on the best available evidence. Recent findings We review the history of evidence-based research, the development of the levels of evidence, and the relationship of evidence and bias present in craniomaxillofacial surgery. We also discuss the recent trends in CMF publications and identify areas for improvement. Summary Because of inherent challenges, the quality of evidence in craniomaxillofacial surgery lags behind other surgical and medical specialties. However, over recent years this has improved significantly, with better reporting of data and a higher rate of randomized controlled trials.

Partial intracapsular tonsillectomy (PIT) is a well-established technique for reducing post-operative morbidity in pediatric patients with sleep disordered breathing (SDB). Although tonsillar re-growth rates are reported as low, risks of symptom recurrence or need for completion tonsillectomy are clear disadvantages when compared to traditional tonsillectomy (TT). We aim to identify cohort differences to better guide clinical decision making and identify patient-specific factors that may influence this decision. A secondary aim was to evaluate potential risk factors for tonsillar regrowth.Retrospective chart review of pediatric patients who underwent TT or PIT for SDB between 2015 and 2019 at a tertiary care academic medical center. Records were reviewed for age, gender, race, body mass index, comorbidities, diagnosis, apnea-hypopnea index, pre-operative Brodsky tonsil size, length of stay, post-operative hemorrhage, tonsillar regrowth, symptom recurrence, and need for completion tonsillectomy.315 patients were included: 174 underwent TT and 141 underwent PIT. Patients undergoing TT were more likely to have a sleep study showing OSA (OR 3.01, p 0.0001), asthma (OR 4.28, p = 0.000124), and other comorbidities (OR 4.06, p = 0.0258). The overall complication rate was 4.44% (14/315). Tonsillar regrowth was exclusive to the PIT group, occurring in 7/141 patients (4.96%). Age ≤4 years was significantly associated with increased risk of tonsillar regrowth (≤4 years: 7.69%,4 years: 0%; p = 0.049). Race and pre-operative tonsil size were not associated with regrowth.Our study supports the low incidence of tonsillar regrowth in PIT and suggests an association with younger age. Moreover, we found that patients undergoing TT are more likely to be older, have OSA, asthma, and other comorbidities.

Importance Venous thromboembolism (VTE) is associated with substantial morbidity and is the most common factor associated with preventable death among hospitalized patients. Data from otolaryngologic studies suggest that the risk of VTE may be underestimated among high-risk patients, particularly among those undergoing oncologic procedures. The incorporation of prolonged-duration chemoprophylaxis (PDC) into preventive therapy has been associated with substantial decreases in VTE incidence among patients undergoing oncologic surgery. However, bleeding remains a major concern among otolaryngologists, and substantial variation exists in the use of thromboprophylaxis. Objective To assess the association between PDC and VTE in high-risk patients with head and neck cancer undergoing oncologic procedures. Design, setting, and participants This retrospective cohort study identified 750 patients with biopsy-confirmed head and neck cancer and a Caprini risk score of 8 or higher who underwent inpatient oncologic surgery at a tertiary care referral center between January 1, 2014, and February 1, 2020. After exclusions, 247 patients were included in the study; patients were divided into 2 cohorts, traditional and PDC, based on the duration of prophylaxis. Univariate and multivariate analyses were performed to examine the development of VTE and bleeding-associated complications during the 30-day postoperative period. Data were analyzed from April 1 to April 30, 2020. Exposures PDC, defined as 7 or more postoperative days of chemoprophylaxis. Main outcomes and measures VTE and bleeding events during the 30-day postoperative period. Results Among 247 patients (mean [SD] age, 63.1 [11.1] years; 180 men [72.9%]) included in the study, 106 patients (42.9%) received traditional prophylaxis, and 141 patients (57.1%) received PDC. The incidence of VTE was 5 of 106 patients (4.7%) in the traditional cohort and 1 of 141 patients (0.7%) in the PDC cohort (odds ratio [OR], 0.15; 95% CI, 0.003-1.33). In the multivariate logistic regression analysis, PDC was independently associated with reductions in the risk of VTE (OR, 0.04; 95% CI, 0.001-0.46). The incidence of bleeding events was 1 of 106 patients (0.9%) in the traditional cohort and 6 of 141 patients (4.3%) in the PDC cohort (OR, 4.64; 95% CI, 0.55-217.00). Conclusions and relevance The use of chemoprophylaxis for high-risk patients with head and neck cancer remains a high-priority topic. The results of this study suggest that PDC may be associated with reductions in VTE among this patient population. However, the associated increase in nonfatal bleeding events warrants careful consideration and further highlights the need to determine an optimal duration for chemoprophylaxis among this distinct cohort.

Aim: A UHPLC-MS/MS assay was developed to quantify urinary dehydroepiandrosterone (DHEA), 7β-hydroxy-DHEA, cortisone and 6β-hydroxycortisone as potential biomarkers to predict CYP3A activity. Results: A sensitive assay at LLOQ of 0.500 ng/ml with good accuracy and precision was developed for the four analytes in human urine. This UHPLC-MS/MS assay was optimized by eliminating nonspecific loss of the analytes in urine, ensuring complete hydrolysis of the conjugates to unconjugated forms and use of the product ions of [M+H-H2O]+ for multiple reaction monitoring detection of DHEA and 7β-hydroxy-DHEA. Conclusion: This assay was successfully applied to a pilot clinical study. It is also suitable for future drug–drug interaction studies to continue evaluating the potential of these steroids as biomarkers for CYP3A inhibition and induction.

BMS-823778 is a potent 11-β-hydroxysteroid-dehydrogenase 1 (11βHSD-1) inhibitor and a potential therapeutic agent for type 2 diabetes mellitus (T2DM). A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated to enable reliable separation and quantification of both enantiomers of a chiral hydroxy metabolite (BMT-094817) in human plasma. Following liquid-liquid extraction in a 96-well plate format, chromatographic separation of the metabolite enantiomers was achieved by isocratic elution on a Chiralpak IA-3 column. Chromatographic conditions were optimized to ensure separation of both metabolite enantiomers. Metabolite enantiomers and stable isotope-labeled (SIL) internal standards were detected by positive ion electrospray tandem mass spectrometry. The LC-MS/MS assay was validated over a concentration range of 0.200-200ng/mL. Intra- and inter-assay precision values for replicate quality control samples were less than 9.9% for both enantiomers during the assay validation. Mean quality control accuracy values were within ±7.3%. Assay recoveries were high (>75%) and consistent across the assay range. The metabolite enantiomers were stable in human blood for 2h on ice. The analytes were also stable in human plasma for 25h at room temperature, 34days at -20°C and -70°C, and following five freeze-thaw cycles. No interconversion of the metabolite enantiomers was detected under any bioanalytical stress conditions, from blood collection/processing through extracted sample storage. The validated assay was successfully applied to the quantification of both metabolite enantiomers in human plasma in support of a human pharmacokinetic study.

To determine outcomes of patients with displaced nasal bone fractures after closed nasal reduction (CNR).Retrospective patient review.Review of all patients presenting to the emergency department of a tertiary-care, level 1 trauma hospital with a nasal bone fracture over a 2-year period, followed by telephone survey after CNR.Six hundred seven patients presented to the emergency department in 2015 and 2016 with a diagnosis of nasal bone fracture. Of these, 134 patients met inclusion criteria and underwent CNR without septal reduction. Those with sports-related injuries and those with a septal fracture identified on computed tomography imaging were significantly more likely to undergo CNR. Ninety-one patients completed the post-CNR telephone survey. Over 90% of patients were satisfied with the procedure. However, patients with septal fractures reported worse outcomes, as 53.6% versus 24.1% (P = .0025) disagreed that CNR improved nasal breathing. Of all patients, 11 (2%) eventually underwent septorhinoplasty, with the presence of septal fracture on imaging a significant risk factor.Nasal bone fractures are a common injury, often managed initially with CNR. Patients with septal fractures should be counseled on the high risk of posttraumatic nasal deformity and obstruction despite CNR. In addition, addressing a septal fracture found on imaging may be warranted with either closed septal reduction or early aggressive management given the poorer outcomes seen in the present study. Although these patients are more likely to have definitive treatment, many forego later intervention despite persistent symptoms, emphasizing the need for early intervention or close follow-up.3 Laryngoscope, 129:1784-1790, 2019.

The 11th Global CRO Council Closed Forum was held in Universal City, CA, USA on 3 April 2017. Representatives from international CRO members offering bioanalytical services were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The second CRO–Pharma Scientific Interchange Meeting was held on 7 April 2017, which included Pharma representatives’ sharing perspectives on the topics discussed earlier in the week with the CRO members. The issues discussed at the meetings included cumulative stability evaluations, matrix stability evaluations, the 2016 US FDA Immunogenicity Guidance and recent and unexpected FDA Form 483s on immunogenicity assays, the bioanalytical laboratory's role in writing PK sample collection instructions, biosimilars, CRO perspectives on the use of chiral versus achiral methods, hybrid LBA/LCMS assays, applications of fit-for-purpose validation and, at the Global CRO Council Closed Forum only, the status and trend of current regulated bioanalytical practice in China under CFDA's new BMV policy. Conclusions from discussions of these topics at both meetings are included in this report.

Background: The bioanalytical strategy for antibody–drug conjugates (ADC) includes numerous measurements integrally designed to provide comprehensive characterization of PK, PD and immunogenicity. This manuscript describes the utilization of reagents specifically tailored to an ADC with a microtubule polymerization inhibitor payload and cathepsin B cleavable linker. Methods: The PK strategy includes the evaluation of physiological levels of total antibody, active ADC, total ADC, antibody-conjugated payload and unconjugated payload. These data are evaluated in the context of target and antidrug antibody levels to elucidate bioactive ADC. Results & conclusion: Herein, we discuss how this strategy has been applied and present our preliminary observations. Continuously evolving to meet pipeline demands, the integrated bioanalytical data will provide critical insights into the exposure–response relationship.

Herein we report a rapid, accurate and robust UHPLC–MS/MS assay for the quantitation of BMS-911453, a Janus kinase 2 inhibitor under clinical development for the treatment of myeloproliferative disorders, in human plasma. A systematic method development approach was used to optimize the mass spectrometry, chromatography, and sample extraction conditions, and to minimize potential bioanalytical risks. The validated method utilizes stable-isotope labeled 13 C 4 -BMS-911543 as the internal standard. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved within 2 min on a Zorbax Extend-C18 column with an isocratic elution. BMS-911543 and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The assay range was from 1 to 500 ng/mL, and the standard curve was fitted with 1/ x 2 weighted linear regression. The intra-assay precision was within 5.0% CV and the inter-assay precision was within 2.6% CV. The inter-assay mean accuracy, expressed as percents of theoretical, was between 99.8% and 102.3%. The assay has high recovery (∼80%) and minimal matrix effect (0.95–1.00). BMS-911543 was stable in human plasma for at least 24 h at room temperature, 90 days at −20 °C, and following three freeze–thaw cycles. The validated method was successfully applied to sample analysis in clinical studies.

Aims: A cross-laboratory experiment has been performed on a novel dried blood sampler in order to investigate whether it overcomes issues associated with blood volume and hematocrit (HCT) that are observed when taking a subpunch from dried blood spot samples. Materials & methods: An average blood volume of 10.6 μl was absorbed by the samplers across the different HCTs investigated (20–65%). Results: No notable change of volume absorbed was noted across the HCT range. Furthermore, the variation in blood sample volumes across six different laboratories was within acceptable limits. Conclusion: The novel volumetric absorptive microsampling device has the potential to deliver the advantages of dried blood spot sampling while overcoming some of the issues associated with the technology.

Apixaban (Eliquis™) was developed by Bristol-Myers Squibb (BMS) and Pfizer to use as an antithrombotic/anticoagulant agent and has been recently approved for the prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. A clinical study of apixaban, sponsored by BMS and Pfizer, included a pilot exploratory portion to evaluate the potential for future drug concentration monitoring using dried blood spot (DBS) sample collection. For DBS sample collection, a fixed blood volume was dispensed onto a DBS card by either regular volumetric pipette (venous blood collection) or capillary dispenser (finger prick blood collection). A 96-well semi-automated liquid-liquid extraction sample preparation procedure was developed to provide clean extracts for UHPLC-MS/MS quantitation. Assays using both partial-spot center punch and whole spot punch were developed and validated. The linear dynamic ranges for all the analyses were from 0.5 to 500 ng/mL. The coefficient of determination (r(2)) values was >0.9944 for all the validation runs. For the center punch approach, the intra-assay precision (%CV) was within 4.4% and inter-assay precision was within 2.6%. The assay accuracy, expressed as %Dev., was within ± 5.4% of the nominal concentrations. One accuracy and precision run was performed using the whole spot approach, the intra-assay precision (%CV) was within 7.1% and the accuracy was within ± 8.0% of the nominal concentrations. In contrast to the center punch approach, the whole spot approach eliminated the effect of hematocrit and high lipids on the analysis of apixaban in human DBS when an accurate sample blood volume was collected on DBS cards.

Dual or triple combination regimens of novel hepatitis C direct-acting antivirals (DAA, daclatasvir, asunaprevir, or beclabuvir) provide high sustained virological response rates and reduced frequency of resistance compared to clinical monotherapy. To support pharmacokinetic (PK) assessments in clinical studies, a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitation of daclatasvir, asunaprevir, beclabuvir (BMS-791325) and its active metabolite (BMS-794712) in human plasma was developed and validated. Human plasma samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis, which was conducted in a multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) were 1 ng/mL for daclatasvir, asunaprevir, and BMS-794712, and 2 ng/mL for beclabuvir. Intra-run precision (≤4.5% CV), inter-run precision (≤2.9% CV), and accuracy (±5.3% deviation) based on different concentration levels (low, geometric mean, mid and high) of the quality control samples (QCs) provided evidence of the methods accuracy and precision. Selectivity and matrix effect on LC-MS/MS detection, stability in plasma, and potential interference of coadministered drugs (ribavirin and interferon) were all evaluated and the results were acceptable. Method reproducibility was demonstrated by the reanalysis of a portion of study samples. The cross-validation results for QCs demonstrated the equivalency between this method and two single-analyte methods which were previously validated for quantitation of daclatasvir in human plasma. This approach of using a multiplexed LC-MS/MS method for the simultaneous quantitation of three DAAs is time- and cost-effective, and can maintain good data quality in sample analysis.

Dapagliflozin (Farxiga), alone, or in the fixed dose combination with metformin (Xigduo), is an orally active, highly selective, reversible inhibitor of sodium-glucose cotransporter type 2 (SGLT2) that is marketed in United States, Europe, and many other countries for the treatment of type 2 diabetes mellitus. Here we report a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical assay of dapagliflozin in human plasma. A lower limit of quantitation (LLOQ) at 0.2 ng/mL with 50 μL of plasma was obtained, which reflects a 5-fold improvement of the overall assay sensitivity in comparison to the previous most sensitive assay using the same mass spectrometry instrumentation. In this new assay, acetate adduct ions in negative electrospray ionization mode were used as the precursor ions for selective reaction monitoring (SRM) detection. Sample preparation procedures and LC conditions were further developed to enhance the column life span and achieve the separation of dapagliflozin from potential interferences, especially its epimers. The assay also quantifies dapagliflozin's major systemic circulating glucuronide metabolite, BMS-801576, concentrations in human plasma. The assay was successfully transferred to contract research organizations (CROs), validated, and implemented for the sample analysis of pediatric and other critical clinical studies. This assay can be widely used for bioanalytical support of future clinical studies for the newly approved drug Farxiga or any combination therapy containing dapagliflozin.

Biological therapeutics can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAbs). Functional (usually cell-based) NAb assays are preferred to determine NAb presence in patient serum, but are often subject to interferences from numerous serum factors, such as growth factors and disease-related cytokines. Many functional cell-based NAb assays are essentially drug concentration assays that imply the presence of NAbs by the detection of small changes in functional drug concentration. Any drug contained in the test sample will increase the total amount of drug in the assay, thus reducing the sensitivity of NAb detection. Biotin-drug Extraction with Acid Dissociation (BEAD) has been successfully applied to extract ADA, thereby removing drug and other interfering factors from human serum samples. However, to date there has been no report to estimate the residual drug level after BEAD treatment when the drug itself is a human monoclonal antibody; mainly due to the limitation of traditional ligand-binding assays. Here we describe a universal BEAD optimization procedure for human monoclonal antibody (mAb) drugs by using a LC-MS/MS method to simultaneously measure drug (a mutant human IgG4), NAb positive control (a mouse IgG), and endogenous human IgGs as an indicator of nonspecific carry-over in the BEAD eluate. This is the first report demonstrating that residual human mAb drug level in clinical sample can be measured after BEAD pre-treatment, which is critical for further BEAD procedure optimization and downstream immunogenicity testing.

In September 2013, the FDA released a draft revision of the Bioanalytical Method Validation (BMV) Guidance, which included a number of changes to the expectations for bioanalysis, most notably the inclusion of biomarker assays and data. To provide a forum for an open, inclusive discussion of the revised draft BMV Guidance, the AAPS and FDA once again collaborated to convene a two-and-a-half day workshop during early December 2013 in Baltimore, MD, USA. The resulting format embodied extensive open discussion and each thematic session included only brief, concise descriptions by Agency and industry representatives prior to opening the floor discussion. The Workshop was built around four thematic sessions (Common Topics, Chromatographic, Ligand-Binding Assays, and Biomarkers) and a final session with international regulators, concluding with a review of the outcomes and recommendations from the thematic sessions. This Workshop report summarizes the outcomes and includes topics of agreement, those where the FDA will consider the Industry's perspective, and those where the workshop provided a first open dialogue. This article will be available to the bioanalytical community at http://www.aaps.org/BMV13 .

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years’ editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies’ Input. Part 1 (Small molecules bioanalysis using LCMS) was published in the Bioanalysis issue 6(22) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the Bioanalysis issue 6(24).

Aim: To support drug development of a PEGylated anti-CD28 domain antibody, a sensitive and robust LC–MS/MS assay was developed for the first in-human multiple ascending dose study. Materials & methods: The procedure consists of a protein precipitation with acidified acetonitrile, followed by trypsin digestion of the supernatant. A surrogate peptide from the complementarity determining region was quantified with an LC–MS/MS assay using a stable isotope-labeled internal standard with flanking amino acids. An acid dissociation step was found to be essential to achieve full analyte recovery in the presence of antidrug antibodies and soluble target CD28. Results & conclusion: The fully validated LC–MS/MS assay demonstrates good accuracy (% deviation ≤6.3) and precision (%CV ≤5.2) with an lower limit of quantitation of 10 ng/ml.

The last 10 years have witnessed robust debate within the bioanalytical community and regulatory authorities on the topic of metabolite monitoring and safety assessment. Of particular interest to regulated bioanalytical laboratories was the acceptance by the US FDA and other major regulatory bodies of a tiered approach to bioanalytical assay validation. The tiered approach defines a sliding scale of regulatory rigor for the evaluation of significant human metabolites that encompasses a range of assessments from semi-quantitative assays to fully validated assays, all of which can be used in support of regulatory submissions. This article describes the utilization of a tiered approach at Bristol-Myers Squibb and the decision trees guiding the selection of the appropriate level of assay qualification. Case studies illustrate how decisions are made, how different scientific situations influence the assay choice, and what criteria may be set to continue or discontinue metabolite monitoring in later drug development.

Background: Increasing assay sensitivity is critical for promoting the application of LC–MS/MS quantitative bioanalysis of therapeutic proteins. A sample processing method that can selectively remove the abundant background peptides in the serum tryptic digests and retain the target peptides can greatly improve the assay sensitivity. Results: Mixed-mode strong-cation exchange SPE was systematically investigated as an orthogonal sample separation technique to reversed-phase UHPLC for the analysis of a test monoclonal antibody, BMS-986012, in monkey serum after pellet digestion. Strong cation exchange SPE efficiently removed most of the background peptides and reduced the matrix effect and background level in the monitored mass transition channels. As a result, improved sensitivity was observed for the surrogate peptides VVSV and SLIY. Conclusion: This orthogonal approach provides a simple and easy-to-develop sample preparation method that can selectively remove most background peptides and extract the target peptides, therefore, improving the LC–MS/MS assay sensitivity.

An LC-MS/MS assay was developed and fully validated for the simultaneous quantitation of two coadministered human monoclonal antibodies (mAbs), mAb-A and mAb-B of IgG4 subclass, in monkey serum. The total serum proteins were digested with trypsin at 50 °C for 30 min after methanol denaturation and precipitation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated with a C18 column (2.1 × 100 mm, 1.7 μm) with mobile phases of 0.1% formic acid in water and acetonitrile. Four peptides, a unique peptide for each mAb and two confirmatory peptides from different antibody domains, were simultaneously quantified by LC-MS/MS in the multiple reaction-monitoring mode. Stable isotopically labeled peptides with flanking amino acids on C- and N-terminals were used as internal standards to minimize the variability during sample processing and detection. The LC-MS/MS assay showed lower limit of quantitation (LLOQ) at 5 μg/mL for mAb-A and 25 μg/mL for mAb-B. The intra- and interassay precision (%CV) was within 10.0% and 8.1%, respectively, and the accuracy (%Dev) was within ±5.4% for all the peptides. Other validation parameters, including sensitivity, selectivity, dilution linearity, processing recovery and matrix effect, autosampler carryover, run size, stability, and data reproducibility, were all evaluated. The confirmatory peptides played a critical role in confirming quantitation accuracy and the integrity of the drugs in the study samples. The robustness of the LC-MS/MS assay and the data agreement with the ligand binding data demonstrated that LC-MS/MS is a reliable and complementary approach for the quantitation of coadministered antibody drugs.

Automated sample extraction for regulated bioanalysis by liquid chromatography/tandem mass spectrometry (LC-MS/MS) still presents significant challenges. A new sample preparation methodology with a simplified and completely automated workflow was developed to overcome these challenges using cap piercing for direct biofluid transfer and evaporation-free solid phase extraction (SPE). Using pierceable cap sample tubes, a robotic liquid handler was able to sample without uncapping or recapping during sample preparation. Evaporation for SPE was eliminated by using a mobile phase-compatible elution solvent followed by sample dilution prior to LC-MS/MS analysis. Presented here are three LC-MS/MS assays validated using this methodology to support three CNS drug development programs: (1) BMS-763534 and its metabolite, BMS-790318, in dog plasma; (2) BMS-694153 in monkey plasma; and (3) Pexacerfont (BMS-562086) and two metabolites, BMS-749241 and DPH-123554, in human plasma. These assays were linear from 1.00 to 1000 or 2.00 to 2000ng/mL for each analyte with excellent assay accuracy, precision and reproducibility. These assays met acceptance criteria for regulated bioanalysis and have been successfully applied to drug development study samples. The methodology described here successfully eliminated all manual intervention steps achieving fully automated sample preparation without compromising assay performance. Importantly, this methodology eliminates the potential exposure of the bioanalyst to any infectious biofluids during sample preparation.

The paradigm shift of globalized research and conducting clinical studies at different geographic locations worldwide to access broader patient populations has resulted in increased need of correlating bioanalytical results generated in multiple laboratories, often across national borders. Cross-validations of bioanalytical methods are often implemented to assure the equivalency of the bioanalytical results is demonstrated. Regulatory agencies, such as the US FDA and European Medicines Agency, have included the requirement of cross-validations in their respective bioanalytical validation guidance and guidelines. While those documents provide high-level expectations, the detailed implementation is at the discretion of each individual organization. At Bristol-Myers Squibb, we practice a fit-for-purpose approach for conducting cross-validations for small-molecule bioanalytical methods using LC–MS/MS. A step-by-step proposal on the overall strategy, procedures and technical details for conducting a successful cross-validation is presented herein. A case study utilizing the proposed cross-validation approach to rule out method variability as the potential cause for high variance observed in PK studies is also presented.

Background: Creatinine is an endogenous compound generated from creatine by normal muscular metabolism. It is an important indicator of renal function and the serum level is routinely monitored in clinical labs. Results & methodology: Surrogate analyte (d3-creatinine) was used for calibration standard and quality control preparation and the relative instrument response ratio between creatinine and d3-creatinine was used to calculate the endogenous creatinine concentrations. Conclusion: A fit-for-purpose strategy of using a surrogate analyte and authentic matrix was adopted for this validation. The assay was the first human plasma assay using such strategy and was successfully applied to a clinical study to confirm a transient elevation of creatinine observed using an existing clinical assay.

Dried saliva spot (DSS) sampling is a non-invasive sample collection technique for bioanalysis that can be potentially implemented at the patient's home. A UHPLC-MS/MS assay was developed using detergent-assisted sample extraction to quantify BMS-927711, a drug candidate in development for the treatment of migraines, in human DSS. By implementing DSS sampling at the patients' home, the bioanalytical sample collection for pharmacokinetic evaluation can be done at the time of the acute migraine attack without the need for clinical visits. DSS samples were prepared by spotting 15 μL of liquid saliva onto regular Whatman FTA™ DMPK-C cards and verified with a UV lamp (at λ 254 nm or 365 nm) during DSS punching. The 4-mm DSS punches in a 96-well plate were sonicated with 200 μL of [ 13 C 2 , D 4 ]-BMS-927711 internal standard (IS) solution in 20/80 MeOH/water for 10 min, followed by sonication with 50 μL of 100 mM NH 4 OAc with 1.0% Triton-X-100 (as detergent) prior to liquid-liquid extraction with 600 μL EtOAc/Hexane (90:10). UHPLC-MS/MS was performed with an Aquity ® UPLC BEH C18 Column (2.1 × 50 mm, 1.7 μm) on a Triple Quad™ 5500 mass spectrometer. The assay was linear with a concentration range from 2.00 to 1000 ng mL −1 for BMS-927711 in human saliva. The intra- and inter-assay precision was within 8.8% CV, and the accuracy was within ±6.7% Dev of the nominal concentration values. This UHPLC–MS/MS assay has been successfully applied to determine the drug's pharmacokinetics within a clinical study. For the first time, we observed BMS-927711 exposure in human DSS, confirming the suitability of this sampling technique for migraine patients to use at home. Detergent-assisted extraction with Triton-X-100 could be very useful in DSS or other dried matrix spot (DMS) assays to overcome low or inconsistent analyte recovery issues.

A methodology for the accurate calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays and its application in supporting microdose absolute bioavailability studies are reported for the first time. For simplicity, this calculation methodology and the strategy to minimize the isotopic interference are demonstrated using a simple molecule entity, then applied to actual development drugs. The exact isotopic interferences calculated with this methodology were often much less than the traditionally used, overestimated isotopic interferences simply based on the molecular isotope abundance. One application of the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC-MS/MS bioanalytical assay. The second application is the selection of an SIL analogue for use in intravenous (i.v.) microdosing for the determination of absolute bioavailability. In the case of microdosing, the traditional approach of calculating isotopic interferences can result in selecting a labeling scheme that overlabels the i.v.-dosed drug or leads to incorrect conclusions on the feasibility of using an SIL drug and analysis by LC-MS/MS. The methodology presented here can guide the synthesis by accurately calculating the isotopic interferences when labeling at different positions, using different selective reaction monitoring (SRM) transitions or adding more labeling positions. This methodology has been successfully applied to the selection of the labeled i.v.-dosed drugs for use in two microdose absolute bioavailability studies, before initiating the chemical synthesis. With this methodology, significant time and cost saving can be achieved in supporting microdose absolute bioavailability studies with stable labeled drugs.

Biological sample dilution is a rate-limiting step in bioanalytical sample preparation when the concentrations of samples are beyond standard curve ranges, especially when multiple dilution factors are needed in an analytical run. We have developed and validated a Microsoft Excel-based robotic sample preparation program (RSPP) that automatically transforms Watson worklist sample information (identification, sequence and dilution factor) to comma-separated value (CSV) files. The Freedom EVO liquid handler software imports and transforms the CSV files to executable worklists (.gwl files), allowing the robot to perform sample dilutions at variable dilution factors. The dynamic dilution range is 1- to 1000-fold and divided into three dilution steps: 1- to 10-, 11- to 100-, and 101- to 1000-fold. The whole process, including pipetting samples, diluting samples, and adding internal standard(s), is accomplished within 1 h for two racks of samples (96 samples/rack). This platform also supports online sample extraction (liquid-liquid extraction, solid-phase extraction, protein precipitation, etc.) using 96 multichannel arms. This fully automated and validated sample dilution and preparation process has been applied to several drug development programs. The results demonstrate that application of the RSPP for fully automated sample processing is efficient and rugged. The RSPP not only saved more than 50% of the time in sample pipetting and dilution but also reduced human errors. The generated bioanalytical data are accurate and precise; therefore, this application can be used in regulated bioanalysis.

A liquid chromatography and tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine the concentrations of saxagliptin (Onglyza™, BMS-477118) and its major active metabolite, 5-hydroxy saxagliptin to support pharmacokinetic analyses in clinical studies. The dynamic range of the assay was 0.1-50 ng/mL for saxagliptin and 0.2-100 ng/mL for 5-hydroxy saxagliptin. Protein precipitation (PPT) with acetonitrile was used to extract the analytes from plasma matrix before injecting on an Atlantis(®) dC18 column (50 mm × 2.1 mm, 5 μm) for LC-MS/MS analysis. The sample pre-treatment process was carefully controlled to disrupt DPP4-specific binding and non-specific binding observed at lower concentrations. The recoveries for both analytes were >90%. The assay was selective, rugged and reproducible; storage stability of at least 401 days at -20°C was demonstrated. Under these chromatographic conditions, the isomers of saxagliptin and 5-hydroxy saxagliptin were chromatographically separated from saxagliptin and 5-hydroxy saxagliptin. The assay has been used to support multiple clinical studies and regulatory approvals.

Currently, mass spectrometry-based protein bioanalysis is primarily achieved through monitoring the representative peptide(s) resulting from analyte protein digestion. However, this approach is often incapable of differentiating the measurement of protein analyte from its post-translational modifications (PTMs) and/or potential biotransformation (BTX) products. This disadvantage can be overcome by direct measurement of the intact protein analytes. Selected reaction monitoring (SRM) on triple quadrupole mass spectrometers has been used for the direct measurement of intact protein. However, the fragmentation efficiency though the SRM process could be limited in many cases, especially for high molecular weight proteins. In this study, we present a new strategy of intact protein bioanalysis by high-resolution (HR) full scan mass spectrometry using human lysozyme as a model protein. An HR linear ion-trap/Orbitrap mass spectrometer was used for detection. A composite of isotopic peaks from one or multiple charge states can be isolated from the background and used to improve the signal-to-noise ratio. The acquired data were processed by summing extracted ion chromatograms (EIC) of the 10 most intense isotopic ions of octuply protonated lysozyme. Quantitation of the plasma lysozyme was conducted by utilizing high resolving power and an EIC window fitting to the protein molecular weight. An assay with a linear dynamic range from 0.5 to 500 μg/mL was developed with good accuracy and precision. The assay was successfully employed for monitoring the level of endogenous lysozyme and a potential PTM in human plasma. The current instrumentation limitations and potential advantages of this approach for the bioanalysis of large proteins are discussed.

BACKGROUND Multiple laboratory tests are used to diagnose and manage patients with diabetes mellitus. The quality of the scientific evidence supporting the use of these tests varies substantially. APPROACH An expert committee compiled evidence-based recommendations for the use of laboratory testing for patients with diabetes. A new system was developed to grade the overall quality of the evidence and the strength of the recommendations. Draft guidelines were posted on the Internet and presented at the 2007 Arnold O. Beckman Conference. The document was modified in response to oral and written comments, and a revised draft was posted in 2010 and again modified in response to written comments. The National Academy of Clinical Biochemistry and the Evidence-Based Laboratory Medicine Committee of the American Association for Clinical Chemistry jointly reviewed the guidelines, which were accepted after revisions by the Professional Practice Committee and subsequently approved by the Executive Committee of the American Diabetes Association. CONTENT In addition to long-standing criteria based on measurement of plasma glucose, diabetes can be diagnosed by demonstrating increased blood hemoglobin A1c (HbA1c) concentrations. Monitoring of glycemic control is performed by self-monitoring of plasma or blood glucose with meters and by laboratory analysis of HbA1c. The potential roles of noninvasive glucose monitoring, genetic testing, and measurement of autoantibodies, urine albumin, insulin, proinsulin, C-peptide, and other analytes are addressed. SUMMARY The guidelines provide specific recommendations that are based on published data or derived from expert consensus. Several analytes have minimal clinical value at present, and their measurement is not recommended.

To compete globally, pharmaceutical companies desire to use bioanalytical data and reports as a single version for all filings; not revising for specific countries or regions. Historically, this meant following the US FDA and International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidance/guidelines; finding them sufficient to achieve global acceptance. However, a growing challenge of the past decade has been additional country-specific and regional regulations that have been released. The differences between the bioanalytical regulations among countries have been recognized as a challenge to the pharmaceutical industry and its CRO partners. Harmonization of the regulations at a global level has been the subject of a number of recent articles and editorials, and the topic has been vigorously discussed at several conferences over the past year. Since all have been in agreement about the need to harmonize regulations, this article will not focus on harmonization but rather it will provide a comparison of the USA/Canadian regulations versus those of South America, in particular Brazil, noting the additional work needed to achieve compliance with country-specific regulations. All countries discussed have specific guidance or regulations on clinical bioequivalence studies, and due to the higher standards for these studies, the regulations for bioequivalence studies will be used as the basis for comparison in the article.

Matrix ion suppression/enhancement is a well-observed and discussed phenomenon in electrospray ionization mass spectrometry. Nonuniform matrix ion suppression/enhancement across different types of samples in an analytical run is widely believed to be well compensated for by using a stable isotope-labeled internal standard (SIL-IS) in bioanalysis using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Therefore, the risk of nonuniform matrix ion suppression/enhancement is usually deemed low when an SIL-IS is used. Here, we have identified, evaluated, and proposed solutions to control bioanalytical risks from nonuniform matrix ion suppression/enhancement even with an SIL-IS through a case study using omeprazole. Two lots of human blank urine were tested, and ion enhancement of about 500% for omeprazole was observed in one lot but not in the other. When a quadratic regression model had to be used, the assay failed the industry acceptance criteria due to unacceptable positive bias for the middle and high quality control (QC) samples. The failure was attributed to different extents of matrix ion enhancement between the standards (STDs) and QCs, which resulted in the misaligned results from the regression model. It was concluded that, for the same amount of drug, nonuniform ion enhancement for different types of samples (STD or QC) resulted in different ion intensities, therefore leading to different response behaviors (linear or nonlinear) at the mass spectrometer detector. A simplified mathematical model was used to evaluate the risk when unmatched response models occurred for different types of samples. A diagnostic factor Q (Q = X(ULOQ)(-A/B)) was proposed to monitor the risks, where X(ULOQ) is the upper limit of quantitation of the assay, A is the quadratic slope of the curve, and B is the linear slope of the curve. The potential maximum errors were estimated on the basis of the mathematical model for different scenarios, and Q values were given to control the risks under these conditions for bioanalysis using LC-MS/MS.

A sensitive method was developed and validated for the measurement of ixabepilone (BMS-247550, Ixempra) using a demethylated analogue of ixabepilone (BMS-212188) as an internal standard. A 0.050 mL portion of each plasma sample was extracted with 0.450 mL of acetonitrile containing the internal standard via protein precipitation. The supernatant was analyzed on a LC-MS/MS system. Chromatography was carried out on a 2.0 mm x 100 mm YMC ODS-AQ 3 microm column using an isocractic mobile phase consisting of acetonitrile:10 mM ammonium acetate, pH 5.0 (70:30, v/v) at a flow rate of 0.30 mL/min. The mass spectrometer was fitted with a TurboIonSpray source and operated in negative ionization mode. Detection of ixabepilone and BMS-212188 were accomplished using multiple reaction monitoring (MRM) of precursor>product ion pairs of m/z 505.2>405.2, and 492.1>392.1, respectively. The assay range was 2.00-500 ng/mL and was fitted to a 1/x(2) weighted quadratic regression model. Replicate sample analysis indicated that intra- and inter-day accuracy and precision are within +/-15.0%. The recovery of ixabepilone from 0.050 mL of plasma containing 5.00 and 400 ng/mL was greater than 94%. The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies. This paper also discussed approaches used for resolving a curve splitting issue observed during quantitative analysis of ixabepilone in biological matrices. Finally, to adapt the methodology to pharmacokinetics of ixabepilone after oral administration, the potential interference of chemical degradants on the determination of ixabepilone was evaluated.

The ex vivo removal of urea during hemodialysis treatments is monitored in real time with a noninvasive near-infrared spectrometer. The spectrometer uses a temperature-controlled acousto optical tunable filter (AOFT) in conjunction with a thermoelectrically cooled extended wavelength InGaAs detector to provide spectra with a 20 cm−1 resolution over the combination region (4000–5000 cm−1) of the near-infrared spectrum. Spectra are signal averaged over 15 seconds to provide root mean square noise levels of 24 micro-absorbance units for 100% lines generated over the 4600–4500 cm−1 spectral range. Combination spectra of the spent dialysate stream are collected in real-time as a portion of this stream passes through a sample holder constructed from a 1.1 mm inner diameter tube of Teflon. Real-time spectra are collected during 17 individual dialysis sessions over a period of 10 days. Reference samples were extracted periodically during each session to generate 87 unique samples with corresponding reference concentrations for urea, glucose, lactate, and creatinine. A series of calibration models are generated for urea by using the partial least squares (PLS) algorithm and each model is optimized in terms of number of factors and spectral range. The best calibration model gives a standard error of prediction (SEP) of 0.30 mM based on a random splitting of spectra generated from all 87 reference samples collected across the 17 dialysis sessions. PLS models were also developed by using spectra collected in early sessions to predict urea concentrations from spectra collected in subsequent sessions. SEP values for these prospective models range from 0.37 mM to 0.52 mM. Although higher than when spectra are pooled from all 17 sessions, these prospective SEP values are acceptable for monitoring the hemodialysis process. Selectivity for urea is demonstrated and the selectivity properties of the PLS calibration models are characterized with a pure component selectivity analysis.

BMS-378806 is a human immunodeficiency virus (HIV) entry inhibitor that is being developed for the oral treatment of HIV infection. Human plasma and urine LC/MS/ MS methods have been developed and validated for the quantitation of BMS-378806. For human plasma method, methyl t-butyl ether was used to extract BMS-378806 from plasma in a 96-well format, and the organic layers were dried down and then reconstituted for the injection, while a dilute-and-shoot approach was used for human urine method in a 96-well format. Chromatographic separation was achieved isocratically on a Phenomenex C18 (2) Luna column (2 x 50 mm 2 , 5 μm). The mobile phase contained 60:40 v/v of 0.1% formic acid in water and ACN. Detection was by positive ion electrospray MS/MS. The standard curves ranged from 1.25 to 1000 ng/mL for the plasma assay and from 10 to 5000 ng/mL for the urine assay. The curves were fitted to a 1/x 2 weighted quadratic regression model for both methods. The validation results demonstrated that both methods had satisfactory precision and accuracy across the calibration ranges. The methods were applied to the analysis of human plasma and urine samples from a single ascending dose clinical study to assess the pharmacokinetics of the drug. The pharmacokinetic analysis results indicated the absorption and disposition of the drug was rapid. The systemic exposure of BMS-378806 was generally dose proportional among the doses from 100 to 1200 mg, but not dose proportional to 1600 mg. There were modest increases in the systemic exposure when the drug was given with food or given as a solution formulation. Renal excretion was not a substantial elimination pathway of the drug. BMS-378806 was safe and well tolerated over a dose range of 100 - 1600 mg administered as a single oral dose.

BMS-068645 is a selective adenosine 2A agonist that contains a methyl ester group which undergoes esterase hydrolysis to its acid metabolite. To permit accurate determinations of circulating BMS-068645 and its acid metabolite, blood samples must be rapidly stabilized at the time of collection. A sensitive, rapid and specific liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantitation of BMS-068645 and its acid metabolite in human plasma has been developed and validated using diisopropyl fluorophosphate (DFP) as the esterase inhibitor to prevent BMS-068645 from converting to its acid metabolite. The D 5 -stable isotope labeled analogs of BMS-068645 and its metabolite were used as the internal standards (IS). Analytes and IS in plasma containing 20 mM DFP were acidified and extracted into methyl tert -butyl ether. The liquid–liquid extraction effectively eliminated the strong matrix effect caused by the esterase inhibitor. The chromatographic separation was achieved on a Waters Atlantis C18 column with a run time of 4 min. Detection was performed on a Sciex API 4000 with positive ion electrospray mode (ESI/MS/MS), monitoring the ion transitions m / z 487 > 314 and 473 > 300 for BMS-068645 and its acid metabolite, respectively. The method was validated over the range from 0.020 to 10.0 ng/mL for BMS-068645 and 0.050 to 10.0 ng/mL for its acid metabolite. Inter- and intra-run precision for the quality control samples during validation were less than 8.7% and 4.0%, respectively, for the two analytes. The assay accuracy was within ±5.4% of the nominal values. The esterase inhibitor effectively stabilized BMS-068645 during blood collection and storage. Blood collection tubes containing DFP were easily prepared and used at the clinical sites and could be stored at −30 °C for 3 months. This method demonstrated adequate sensitivity, specificity, accuracy, precision, stability and ruggedness to support the analysis of human plasma samples in pharmacokinetic studies.

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of over 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. It is once again a 5-day week long event – a full immersion bioanalytical week – specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations that emerged from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to advance scientific excellence, improve quality and deliver better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 1 covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (hybrid LBA/LCMS and regulatory agencies’ inputs) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will also be published in volume 7 of Bioanalysis, issues 23 and 24, respectively.

Asunaprevir (BMS-650032) is a selective hepatitis C virus (HCV) NS3 protease inhibitor with potent activity against HCV genotypes 1, 4, 5 and 6. It has been developed in conjunction with direct-acting antiviral agents, in interferon- and ribavirin-free regimen, to improve existing therapies for HCV infection. To support the pharmacokinetic analyses in asunaprevir clinical studies, we have developed and validated a highly sensitive and robust LC-MS/MS method to quantify asunaprevir in human EDTA plasma with an LLOQ of 0.05ng/mL, which was a 20-fold sensitivity improvement over a previously reported assay for asunaprevir. A deuterated labeled [D9]-asunaprevir was used as the internal standard (IS). The analyte and the IS were extracted using a semi-automated liquid-liquid extraction (LLE) at pH 7 with methyl-t-butyl ether (MTBE) in a 96-well plate containing 10μL of 10% CHAPS as the surfactant to prevent non-specific binding issue. Chromatographic separation was achieved on a Genesis C8 column (2.1×50mm, 4μm) with a gradient elution using 0.1% formic acid in water as mobile phase A and a mixture of methanol: acetone: formic acid (95:5:0.1; v/v/v) as the mobile phase B. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 748→648 for asunaprevir and m/z 757→649 for [D9]-asunaprevir,and a collision energy of 30 electron Volts (eV). The assay was validated over a standard curve range from 0.05 to 50ng/mL for asunaprevir in human plasma. The intra- and inter assay precisions were within 7.1% CV, and the % deviation was within 5.5% of their nominal concentrations. This assay has been successfully applied to multiple clinical studies with excellent assay ruggedness and reproducibility.

The objective of this study was to assess the pharmacokinetics of a newly identified active metabolite of buspirone, 6-hydroxybuspirone (6OHB), over the therapeutic dose range of buspirone. A 26-day, open-label, nonrandomized, single-sequence, dose-escalation study in normal healthy volunteers was conducted (N = 13). Subjects received escalating doses of buspirone with each dose administered for 5 days starting at a dose of 5 mg twice daily and increasing up to 30 mg twice daily. Plasma concentrations of 6OHB were approximately 40-fold greater than those of buspirone. 6OHB was rapidly formed following buspirone administration, and exposure increased proportionally with buspirone dose. Further research regarding the safety and efficacy of 6OHB itself is warranted.

Tight control of blood glucose levels has been shown to dramatically reduce the long-term complications of diabetes. Current invasive technology for monitoring glucose levels is effective but underutilized by people with diabetes because of the pain of repeated finger-sticks and the cost of reagent strips. Optical sensing of glucose could potentially allow more frequent monitoring and tighter glucose control for people with diabetes. The key to a successful optical non-invasive measurement of glucose is the collection of an optical spectrum with a very high signal-to-noise-ratio in a spectral region with significant glucose absorption. Unfortunately, the optical throughput of skin is very small due to absorption and scattering. To overcome these difficulties, we have developed a high-brightness tunable laser system for measurements in the 2.0-2.5 μm wavelength range. The system is based on a 2.3 micron wavelength, strained quantum-well laser diode incorporating GaInAsSb wells and AlGaAsSb barrier and cladding layers. Wavelength control is provided by coupling the laser diode to an external cavity that includes an acousto-optic tunable filter. Tuning ranges of greater than 110 nm have been obtained. Because the tunable filter has no moving parts, scans can be completed very quickly, typically in less than 10 ms. We describe the performance of the laser system and its potential for use in a non-invasive glucose sensor.

The ability to measure glucose noninvasively in human subjects is a major objective for many research groups. Success will revolutionize the treatment of diabetes by providing a means to improve glycemic control, thereby delaying the onset of the medical complications associated with this disease. This article focuses on the current state of the art and attempts to identify the principal areas of research necessary to advance the field. Two fundamentally different approaches are identified for the development of noninvasive glucose sensing technology. The indirect approach attempts to measure glucose on the basis of its effect on a secondary process. The direct approach is based on the unique chemical structure of the glucose molecule. Advances for each approach are limited by issues of selectivity. Several critical parameters are discussed for the direct approach, including issues related to the optical path length, wavelength range, dimensionality of the multivariate calibration model, net analyte signal, spectral variance, and assessment of the chemical basis of measurement selectivity. A set of publication standards is recommended as a means to enhance progress toward a successful noninvasive monitor.