Dr Shane Needham received his BS degree in chemistry from Washington State University and his PhD in chemistry from the University of Rhode Island. Dr Needham is Co-Founder and Chief Scientific Officer of Alturas Analytics, Inc. Dr Needham manages all scientific aspects of the HPLC/MS/MS bioanalytical contract laboratory at Alturas Analytics, Inc. Currently, Dr Needham's work is focused on the development and validation of assays for the determination of therapeutic agents and biomarkers from biological matrices. His laboratory leads in the area of dried blood spot analysis, microflow HPLC-MS/MS and LC-MS/MS oflarge molecules (Antibody Drug Conjugates, Biomarkers, New Biological Entities etc.) to support Drug Metabolism and Pharmacokineticsresearch.

Aim: Develop a universal extraction and liquid chromatography-mass spectrometer method to simultaneously analyze cystine-dense peptide (CDP) miniproteins from rat and human plasma. The results of the analysis will be used to assist selection of therapeutic drug candidates from the vast CDP library. Methods & results: A micro-elution solid-phase extraction method was developed for the sample preparation of the CDP peptides in rat and human plasma followed by analysis by microflow liquid chromatography MS/MS. The methods developed for drug discovery were found to be accurate (±≤15.2% from nominal concentrations) and precise (≤13.4% CV), with a dynamic range of 1.00-500 ng/ml and extraction recoveries of 47.2-99.0%. Conclusion: This bioanalytical method can be utilized to screen CDP proteins and other miniproteins for drug discovery, candidate selection and further drug development.

Electrospray ionization mass spectrometry (ESI-MS), coupled to nanobore liquid chromatography (nLC) has revolutionized the identification and analysis of proteins and peptides present in cells, tissues, and biological fluids. Nanofibre LC for protein identification has rapidly evolved into the key platform for the discovery of protein and peptide biomarkers. Nanobore LC-ESI-MS platforms for qualitative proteomics and biomarker discovery are essentially identical. LC-MS has experienced significant technical evolution having established trends toward decreasing column diameter, lower flow rates, and smaller column packing particle sizes. The combination of micro- or nano-LC combined with nanospray ionization would appear to yield a nearly ideal platform for high-sensitivity bioanalysis by LC-MS/MS. Traditional nanospray methods that feature the use of packed-column emitters offer an exceptionally high degree of performance. For optimal performance, nanospray sources are typically equipped with precision translation stages for optimal MS inlet capture, specialized means for the application of high-voltage precolumn, and specialized zero dead volume connections.

BioanalysisVol. 9, No. 24 EditorialFree AccessMicrospray and microflow liquid chromatography: the way forward for LC–MS bioanalysisShane R NeedhamShane R Needham*Author for correspondence: E-mail Address: sneedham@alturasanalytics.com Alturas Analytics, Inc. 1324 Alturas Drive Moscow, ID 83843, USASearch for more papers by this authorPublished Online:5 Dec 2017https://doi.org/10.4155/bio-2017-0219AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Keywords: bioanalysisLC–MSLC–MS/MSmicroflowmicrospraynanosprayIt is well known that the efficiency of ionization improves as the liquid flow rate to the source decreases for ESI. The improved ionization efficiency leads to better MS signal [1]. Microflow liquid chromatography (MFLC) has also been shown to be advantageous for over 40 years [2]. Conventional LC–MS flow rates are in the range of 250–600 μl/min with column internal diameters of 2.1 mm. Nanospray, a technique highly developed by Gary Valaskovic utilizes flow rates in the order of sub μl/min with column internal diameters of <100 μm [3]. Microspray (also known as ‘high flow nanospray’) is a technique that typically consists of flow rates of 5–100 μl/min with column diameters from 0.25–1.0 mm. Microspray and MFLC combined, are a great means to achieve the benefits of improved MS signal from nanospray and optimal analysis times shown by conventional LC–MS flow rates [4,5].The ‘sampling advantage’ of MFLC–MSThe benefits of MFLC–MS include improved signal, low solvent consumption, reduced matrix effects and reduced source contamination. However the biggest benefit in using MFLC–MS/MS in bioanalysis is what we like to call the effective ‘sampling advantage’ [5]. The improved signal afforded by MFLC–MS compared with conventional LC–MS allows the bioanalyst to inject less sample and achieve the same signal as conventional LC–MS. As more assays transition to microsampling and micro volumes, the bioanalyst relies on a smaller sample size for a more conveniently collected and/or more concentrated sample [6]. As an example, if the bioanalyst has 100 μl of final sample volume and uses conventional LC (2.1 mm ID column), the injection volume could likely be 40 μl to achieve the desired MS signal. In comparison with MFLC, the bioanalyst could use an injection volume of 1–5 μl and achieve similar MS signal from the original final sample volume. In our laboratory, our bioanalysts call this a ‘peace of mind’, since many (>10) injections could be made from the same sample with minimal concern of running out of sample. In the bioanalytical world, equipment failures, misinjections, stopped runs and other issues are not desired but expected to happen. Thus, the ability for many injections from the same sample gives the bioanalyst a ‘peace of mind’ to think that ‘if all else fails, I can re-inject my sample(s)’. For any scientist that has analyzed samples over-night only to arrive in the morning to see there was a misinjection, this convenience (ability to inject numerous samples) is invaluable and is the true high performance of MFLC–MS.The reason to implement MFLC are as compelling as the motivation to switch from 4.6 mm ID columns (common in LC–MS in the early 1990s) to 2.1 mm ID columns. With the effective ‘sampling advantage’, it is difficult to imagine bioanalytical laboratories returning to 4.6 mm ID columns at 1–2 ml/min flowrate [7].System volume challengesAs column volumes are miniaturized, attention to the overall plumbing and connection scheme, starting at the outlet of the HPLC pump is required. An MFLC column of dimensions, 0.3 mm ID × 50 mm in length has an approximate total column volume of 3 μl. Thus, total system volume, the injection volume, as well as pre- and postcolumn volume is critical for the efficiency of the chromatographic peaks and optimal run times. To minimize the overall run time and the gradient delay from the LC pump, the precolumn volume (tubing connections with pump, volume through autosampler and connections to the column) should be reduced as much as feasible. In order to maintain efficient and symmetrical peaks, the postcolumn connections to the ESI source should be minimized as much as possible. Integration of the LC column into the source of the ESI emitter provides such a benefit. In fact, a packed emitter column called PicoFrit™ has been used in proteomics for the past 20 years [8]. Recently, integrated columns have shown utility in bioanalysis [9,10]. Integrated columns in bioanalysis yielded improved chromatographic peak shape and improved signal to noise compared with conventional LC columns.Barriers to adoption of MFLC–MSA discussion of the challenges of the wide spread implementation of MFLC–MS is only fair with respect to science. As with any technology, the widespread adoption typically lies with the end user (bioanalyst) not being familiar with or comfortable with the technology. I think there are two reasons that have slowed the wide spread implementation of MFLC–MS. Reason number one is mostly user based hesitation and the other is the apparent challenge coupling a miniaturized system (minimal system dwell volumes required) with the MS. Both of these challenges lead to an inconvenient experience by the user and both challenges can be solved with a good collaboration of users and instrument manufacturers.The first reason/or end user dilemma is that current liquid flow pumps are only capable of one mode (MFLC or conventional HPLC) of flow rate operation. Thus, the bioanalyst is required to change from one pump to the other when conventional and MFLC methods are developed on the same mass spectrometer, which is common practice in the bioanalytical laboratory. Even if the change-over process is a few minutes, this is still a loss of productivity and the process is difficult to perform on an unattended run overnight. The second reason for widespread adoption of the technology is the inefficient coupling of MFLC with the MS. Where MFLC columns can have total column volumes of 2–10 μl, the proximity of the MFLC column to the autosampler, HPLC pumps and MS requires excessive tubing and connections to the MS interface. Current set-up of MFLC–MS instruments may have extra column dwell volumes greater than the column volume. This leads to poor chromatographic peak shapes and suboptimal run times. Both of these challenges are not insurmountable and with patience and collaboration (instrument manufacturers and users) new technology will be developed for a liquid flow pump to deliver sub μl/min to ml/min flow rates. Manufacturers will also optimize instruments where the LC column is integrated into the source. The integrated MFLC–MS of the future with the autosampler, LC and MS could even look like one encompassed unit with no external connections. These changes will minimize system dwell volumes; will expand the user base and acceptance of MFLC–MS as routine.ConclusionMFLC–MS is convenient (it works) and this truly is the high performance of MFLC–MS. The acceptance of MFLC–MS will continue to grow. Liquid flow pumps will be developed that handle flow rates for MFLC and HPLC in the same unit. Integrated technologies will be developed to interface autosamplers, MFLC and MS to minimize connections and dwell volumes. Especially as more large molecules are developed as therapeutics, MFLC–MS will be the perfect fit. The way forward for bioanalytical MS is MFLC–MS, yet bioanalysts today may not even recognize the look of the fully ‘integrated’ instrument 20 years from now.AcknowledgementsA special thanks to G Valaskovic of New Objective and MS Lee of Milestone Development Services for insightful discussions. The author also like to thank C. Christianson and A Hoffman of Alturas Analytics, Inc. for their vision for the future of MFLC-MS.Financial & competing interests disclosureThe author has 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.References1 Kebarle P, Tang L. From ions in solution to ions in the gas phase: the mechanism of electrospray ionization mass spectrometry. Anal. Chem. 65, A972–A986 (1993).Crossref, CAS, Google Scholar2 Ishii D, Asai K, Hibi K, Jonokuchi T, Nagaya M. A study of micro-high-performance liquid chromatography. J. Chromatogr. A 144(2), 157–168 (1977).Crossref, CAS, Google Scholar3 Valaskovic GA, Utley L, Lee MS, Wu JT. Ultra-low flow nanospray for the normalization of conventional liquid chromatography/mass spectrometry through equimolar response: standard-free quantitative estimation of metabolite levels in drug discovery. Rapid Comm. Mass Spec. 20(7), 1087–1096 (2006).Crossref, Medline, CAS, Google Scholar4 Johnson CJL, Christianson CD, Needham SR. The advantages of microflow LC–MS/MS compared with conventional HPLC–MS/MS for the analysis of methotrexate from human plasma. Bioanalysis 5(11), 1387–1396 (2013).Link, Google Scholar5 Needham SR, Valaskovic G. Peptide and protein bioanalysis using integrated column-to-source technology for high-flow nanospray. Chapter 5. In: Protein Analysis Using Mass Spectrometry: Accelerating Protein Biotherapeutics from Lab to Patient. Lee MS, Li QC (Eds). John Wiley and Sons, Inc. (2017).Crossref, Google Scholar6 Stove C, Spooner N. DBS and beyond. Bioanalysis 7(16), 1961–1962 (2015).Link, CAS, Google Scholar7 Covey TE, Henion J. High-speed liquid chromatography/tandem mass spectrometry for the determination of drugs in biological samples. Anal Chem. 58, 2453–2460 (1986).Crossref, Medline, CAS, Google Scholar8 Emmett M, Caprioli R. Microspray-electrospray mass spectrometry: ultra-high-sensitivity analysis of peptides and proteins. J. Am. Soc. Mass Spectrom. 5(7), 605–613 (1994).Crossref, Medline, CAS, Google Scholar9 Johnson CC, DeChenne CS, Needham S, Valaskovic G. Validation of an in-source micro flow LC–MS/MS method for bioanalysis. Presented at: The 62nd Conference on American Society for Mass Spectrometry. Baltimore, MD, 15–19 June 2014.Google Scholar10 Kleinnijenhuis A, Ingola M, Toersche J, van Holthoon F, van Dongen W. Quantitative bottom up analysis of infliximab in serum using protein A purification and integrated LC-electrospray chip IonKey MS/MS technology. Bioanalysis 8(9), 891–904 (2016).Link, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByDetermination of drug-to-antibody ratio of antibody–drug conjugate in biological samples using microflow-liquid chromatography/high-resolution mass spectrometryKazuko Inoue, Toshiki Mochizuki, Nana Kasamori & Takafumi Komori24 February 2023 | Bioanalysis, Vol. 14, No. 24On the potential of micro-flow LC-MS/MS in proteomics18 October 2022 | Expert Review of Proteomics, Vol. 19, No. 3Molecular dynamics simulations of amino acid adsorption and transport at the acetonitrile–water–silica interface: the role of side chains1 January 2021 | RSC Advances, Vol. 11, No. 35Collective Solvation and Transport at Tetrahydrofuran–Silica Interfaces for Separation of Aromatic Compounds: Insight from Molecular Dynamics Simulations3 February 2021 | Langmuir, Vol. 37, No. 6Evaluation of OptiFlow™-MS/MS for bioanalysis of pharmaceutical drugs and metabolitesJason Barricklow, Joseph Tweed, Christopher L Holliman & Ragu Ramanathan18 December 2019 | Bioanalysis, Vol. 12, No. 1Current strategies for quantification of estrogens in clinical researchThe Journal of Steroid Biochemistry and Molecular Biology, Vol. 192Optimization of microflow LC–MS/MS and its utility in quantitative discovery bioanalysisJun Zhang, Wilson Shou, Tairo Ogura, Shu Li & Harold Weller28 June 2019 | Bioanalysis, Vol. 11, No. 11Instrumental and technical evolution over the past decade in bioanalysisRobert MacNeill4 April 2019 | Bioanalysis, Vol. 11, No. 7An overview of the Brazilian contributions to Green Analytical Chemistry1 January 2019 | Anais da Academia Brasileira de Ciências, Vol. 91, No. suppl 1 Vol. 9, No. 24 Follow us on social media for the latest updates Metrics History Received 29 September 2017 Accepted 5 October 2017 Published online 5 December 2017 Published in print December 2017 Information© 2017 Future Science LtdKeywordsbioanalysisLC–MSLC–MS/MSmicroflowmicrospraynanosprayAcknowledgementsA special thanks to G Valaskovic of New Objective and MS Lee of Milestone Development Services for insightful discussions. The author also like to thank C. Christianson and A Hoffman of Alturas Analytics, Inc. for their vision for the future of MFLC-MS.Financial & competing interests disclosureThe author has 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

High performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) is the go-to technique for high-throughput analysis of small-molecule therapeutics, metabolites, and biomarkers. Through technological advancements in the last decade, developing quality methods for a novel analyte in the contract research environment has become easier and faster than ever. Increasingly shorter run times, higher sensitivity, and greater separation have all become possible in a standard method. This is, in part, because of column technologies that have enabled the standardization of the method development process. Method efficiency and productivity are also improving because of emerging column technologies such as sub-2-mu m particles coupled with ultrahigh-pressure liquid chromatography (UHPLC)-MS-MS, superficially porous particle columns, and microflow HPLC-MS-MS. Increasing efficiency and productivity in high-throughput bioanalysis is becoming more important as the applications for HPLC-MS-MS expand to large molecules such as peptides, proteins, and oligonucleotides.

BioanalysisVol. 7, No. 9 EditorialFree AccessMicrospray and microflow LC–MS/MS: the perfect fit for bioanalysisShane R Needham & Gary A ValaskovicShane R NeedhamAuthor for correspondence: E-mail Address: sneedham@alturasanalytics.comAlturas Analytics, Inc., 1324 Alturas Drive, Moscow, ID 83843, USA & Gary A ValaskovicNew Objective, Inc., 2 Constitution Way, Woburn, MA 01801, USAPublished Online:3 Jun 2015https://doi.org/10.4155/bio.15.42AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Keywords: bioanalysisLC–MS/MSmicroflowmicrospraynanosprayFigure 1. A PicoFuzeTM Column Integrated into an AB SCIEX ESI Probe.Figure 2. Overlayed Chromatograms from the PicoFuzeTM, MFLC-MS/MS, and HPLC-MS/MS Analysis of MAOB Digest from Plasma.The advantages of microflow LC (MFLC) and nanoflow liquid chromatography coupled with a mass spectrometer are well known and include improved signal, low solvent consumption, reduced matrix effects and reduced source contamination [1]. In the past, microflow and nanoflow MS/MS have mainly been used in academic and fundamental research labs requiring high sensitivity analysis with limited sample volumes for qualitative or comparative analysis. The analytical advantages of low flow methods have led the technological development of specialized LC pumps that accurately deliver ≤100 µl/min of solvent, for use with columns with an inside diameter of ≤1 mm. With the use of these specialized LC pumps and rugged API–MS ionization sources, the realm of using MFLC–MS/MS for quantitative bioanalysis is a possibility.Researchers have used nanoflow or MFLC–MS/MS for analysis of proteomic samples, food samples, the petroleum products and other applications [2,3]. Nanoflow LC–MS/MS publications as early as 1994 include applications in discovery bioanalysis [4]. However, application of nano or MFLC-MS/MS methods in regulated bioanalysis including application in pharmaceutical and clinical space has not been extensively studied. The adoption of MFLC–MS/MS in regulated bioanalytical laboratories has been limited by the perception that the technique lacks sufficient robustness, and the technical hurdles of implementation. The high volume requirement of bioanalytical quantitation typically demands operation on a continuous 24-7 basis. Downtime is not acceptable. Fortunately, development on the instrumentation side from vendors, and the application side of leading bioanalytical labs, have fostered many developments in the design and coupling of sources for nanospray ESI-MS to improve the ruggedness and ease of use of MFLC-MS/MS. More recently a design successful in coupling the HPLC column within the conventional ESI has been shown to provide better signal and better chromatography compared with traditional segregated techniques [5].Integration of miniaturized LC with nanospray ESI–MS is a key for successLC–MS has experienced significant technical evolution having established trends toward decreasing column diameter, lower flow rates and smaller column packing particle sizes. State of the art 'conventional' LC has evolved from 4.6 mm inside diameter columns (ID), operating at ca. 1 ml/min, to 1–2 mm ID columns, operating at less than 200 µl/min. Even smaller micro- (0.2–0.3 mm ID) and nanoscale (< 0.2 mm ID) column formats, operating at 10 and 0.3 µl/min, respectively, have strong application specific roles, particularly when high sensitivity is required and/or sample volumes are strictly limited.The driving factors in the trend to smaller diameter columns are many. Primary benefits in the switch to smaller columns include reduced solvent consumption, improved cleanliness of the mass spectrometer source/inlet/vacuum system, and perhaps most importantly, reduced sample injection volume. A 0.3 mm ID column, on average, will consume one twentieth and one one-hundredth of the solvent required for 2 and 4.6 mm ID columns, respectively [6]. Less solvent consumption reduces both purchase and chemical waste disposal costs. The low flow rates associated with small ID columns achieve the same front-end cleanliness goals as the typical postcolumn LC divert valve approach. Less mobile phase flowing through the ESI source, and presented to the front-end inlet of the mass spectrometer, translates directly into a cleaner MS, reducing maintenance expense and increasing MS acquisition time without the need for a switching valve.The reduced injection volume required by small ID columns, and the resulting relative concentration advantage when using reverse-phase gradient elution HPLC, results in clear experimental advantages. As shown previously, the concentration of analyte on the head of the column, for injections of a fixed volume, increases as column diameter decreases [7]. A 0.3 mm ID column has a 40-fold concentration advantage compared with a 2.1 mm ID column, while a 75 µm ID column has a nearly 800-fold advantage [7]. This concentration benefit is often described in the literature as an 'increase in (column) sensitivity,' it is, and however, perhaps more correctly viewed as an effective sampling advantage. Micro- and nanobore columns enable the handling and analysis of much smaller absolute sample sizes and/or concentration of trace components present in larger sample volumes. Reduced sampling volume has an under-appreciated workflow benefit, unrelated to sensitivity, giving incredible sample security or a 'peace of mind' to the analyst for unattended analyses. A precious 100 µl sample is sufficient for at least 50 injections of 1.0 µl with micro-scale LC, compared with only two 40 µl injections using conventional LC. The reasons to implement 0.3 mm ID (and smaller) columns are as compelling as the initial push to switch to 2.1 mm ID columns. Certainly, it is hard to see those labs ever moving back to the routine use of 4.6 mm columns.Along with the compelling advantages provided by miniaturized sampling and separation, detection by ESI–MS must also fit the paradigm of miniaturization. The driving factor to trend to lower flow rates for ESI-MS is an improvement in ionization efficiency. As shown by Kebarle et al. [8], early works on ESI response demonstrated an increase in proportional MS signal-to-noise ratio, as the mobile phase flow rate of ESI is reduced. Thus a system that provides optimal chromatographic benefits and optimal flow rates to the MS is an ideal situation for high-throughput, high-sensitivity LC–MS bioanalysis. Nanobore chromatography, using LC columns having a typical ID of 300 µm or less, is conveniently coupled to MS via high flow nanospray ('microspray') ionization, a low-flow variant of ESI. Microspray provides a highly efficient means to transport liquid ions to gas phase ions with little, or no added thermal energy. The low-flow rates associated with nanospray (10–500 nl/min) result in the generation of submicrometer droplets, generating a maximal surface area-to-volume ratio of column effluent. Such high surface area translates directly to high ionization efficiency [9]. The lower thermal requirement for microspray compared with traditional ESI is an added benefit as it is directly compatible with high molecular weight, thermally sensitive, peptides, proteins and small molecules. Additional advantages at low-flow rates (<100 µl/min) include reduced ion suppression [10], a trend toward equimolar response [11], linear ionization response [12] and improved signal-to-noise ratio [13].In addition to the smaller droplets afforded by low-flow rates, the overall physical size of the ESI plume also scales with flow rate. Conventional flow (ml/min) ESI–MS ESI generates an aerosol plume centimeters in diameter. Compared with the (sub) millimeter MS inlet, only a small fraction of the plume is sampled. Under conventional conditions, the MS inlet is an atmospheric pressure flow splitter, with greater than 99% of the generated plume flowing to waste. High-flow nanospray and microspray, with a (sub) µl/min flow rate, has a plume that is on the same dimensional scale as the MS inlet [14]. This gain in apparent sensitivity is best thought of in terms of utilization and sampling efficiency. Near zero postcolumn waste directly translates into the ability to handle micro-scale samples with complete efficiency.Micro- & nano-LC are well suited for quantitative bioanalysisIt would seem that the rational combination of micro- or nano-LC combined with nanospray ionization, would yield a nearly ideal platform for high sensitivity bioanalysis by LC–MS/MS. In practiced hands, that is often the case, but experimental challenges are present. Assembling a high-performance system is at present time the domain of the expert user. Challenges in pre- and postcolumn plumbing, sample preparation of complex matrices, and mass spectrometer tuning requires training and persistence in method development.A common misconception is that miniaturized LC is 'too slow' to use in applications requiring short run times and high throughput. The casual observation of the mobile phase flow rate (0.3–10 µl/min) would suggest this is the case. However, it is important to realize that the linear velocity of analyte through a micro- or nanobore column is identical to a conventional column. This misperception appears to have two different origins: many minutes (ten or more) were required to move mobile phase from the piston of the pump through the autosampler and onto the column. Second, much of the application literature for miniaturized LC is qualitative peptide analysis using injection-to-injection cycle times measured in hours or days. The reason for these long cycle times is not the speed of the sample flow through the column. The long cycle time is a necessity based on the mixture complexity faced in qualitative discovery experiments. Shallow gradients and long LC (≥25 cm) columns must be employed to achieve sufficient chromatographic peak capacity for the mass spectrometer to effectively sample the mixture.Attention to the overall plumbing and connection scheme, starting at the outlet of the HPLC pump is required critical factors including total system volume, the injection volume, as well as pre- and postcolumn volume. Precolumn volume is clearly important as the gradient delay time from pump, through the auto-sampler, and onto the column, places the ultimate limit on the minimum injection cycle time. Controlling and reducing postcolumn volume is perhaps the most critical parameter to maintaining the quality of LC peak shape and overall cycle times. Integration of the LC column into body the ionization emitter provides just such a benefit that will minimize connections and thus precolumn and postcolumn volumes. Indeed, the technology of the so-called 'packed-emitter' column (PicoFrit™ column), has been the de facto standard in qualitative proteomics for the past 20 years [15]. More recently, integrated columns for use in microspray have shown the ruggedness and ease of use to have great utility in quantitative bioanalysis [5,16]. An LC column integrated into the ESI–MS source is shown in Figure 1 with a corresponding chromatogram shown in Figure 2. This integrated system is known as the PicoFuze™. As presented, the minimization of extra pre and postcolumn connection volumes yields improved chromatographic peak shape and signal to noise compared with nonintegrated LC columns. Additionally, unlike operation at conventional flow rates, no switching valve or external column heater is needed for these high performance experiments.ConclusionLooking to future, organizations will be looking for high-performance solutions that improve productivity and efficiency while maintaining workflows. Microflow-LC–MS, especially integrated column LC–MS are solutions that meet this need to support therapeutic advancements and clinical applications. For integrated systems, the reduction of solvent use, minimized source contamination, lower injection volumes, no need for switching valves or external column heaters, improves productivity and lower costs. The end result is an easy to use system that is also high performance. As in many technology-driven industries, the miniaturization of process, components and workflow provides optimal results – we see this to be no different for the future of LC–MS.AcknowledgementsThe authors wish to thank M Lee of Milestone Development Services for insightful discussions during the preparation of this manuscript.Financial & 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.References1 Gibson GT, Mugo SM, Oleschuk RD. Nanoelectrospray emitters: trends and perspective. Mass Spectrom. Rev. 28(6), 918–936 (2009).Crossref, Medline, CAS, Google Scholar2 Wood TD, Moy MA, Dolan AR et al. Miniaturization of electrospray ionization mass spectrometry. Appl. Spec. Rev. 38(2), 187–244 (2003).Crossref, CAS, Google Scholar3 Wickremsinhe ER, Singh G, Ackermann BL, Gillespie TA, Chaudhary AK. A review of nanoelectrospray ionization applications for drug metabolism and pharmacokinetics. Curr. Drug Metab. 7(8), 913–928 (2006).Crossref, Medline, CAS, Google Scholar4 Skor H, Visswanathan R. Quantitative bioanalysis by microflow LC–MS to support discovery-based pharmacokinetic studies. In: Eliminating Bottlenecks for Efficient Bioanalysis: Practices and Applications in Drug Discovery and Development. Shou WZ, Weng N (Eds). Future Science Ltd, London, UK 66–84 (2014).Crossref, Google Scholar5 Johnson C, Christianson C, DeChenne S, Needham S, Valaskovic G. Validation of an in-source micro flow LC-MS/MS method for bioanalysis. Proceedings from the 62nd Conference on American Society for Mass Spectrometry. Baltimore, MD (2014). www.asms.org.Google Scholar6 Chapter 4 HPLC Columns for Pharmaceutical Analysis. In: Handbook of Pharmaceutical Analysis by HPLC. Ahuha S, Dong M (Eds). Elsevier Hobooken, NJ 91 (2005).Google Scholar7 Tomer KB, Moseley MA, Deterding LJ, Parker CE. Capillary liquid chromatography/mass spectrometry. Mass Spectrom. Rev. 13, 431–457 (1994).Crossref, CAS, Google Scholar8 Kebarle P, Tang L. From ions in solution to ions in the gas phase: the mechanism of electrospray ionization mass spectrometry. Anal. Chem. 65, 972A–986A (1993).CAS, Google Scholar9 Juraschek R, Dulcks T, Karas M. Nanoelectrospray–more than just a minimized-flow electrospray ionization source. J. Am. Soc. Mass Spectrom. 10, 300–308 (1999).Crossref, Medline, CAS, Google Scholar10 Hatsis P, Valaskovic G, Wu JT. Online nanoelectrospray/high-field asymmetric waveform ion mobility spectrometry as a potential tool for discovery pharmaceutical bioanalysis. Rapid Commun. Mass Spectrom. 23(23), 3736–3742 (2009).Crossref, Medline, CAS, Google Scholar11 Valaskovic GA, Utley L, Lee MS, Wu JT. Ultra-low flow nanospray for the normalization of conventional liquid chromatography/mass spectrometry through equimolar response: standard-free quantitative estimation of metabolite levels in drug discovery. Rapid Commun. Mass Spectrom. 20(7), 1087–1096 (2006).Crossref, Medline, CAS, Google Scholar12 Wickremsinhe ER, Singh G, Ackermann BL, Gillespie TA, Chaudhary AK. A review of nanoelectrospray ionization applications for drug metabolism and pharmacokinetics. Curr. Drug Metab. 7(8), 913–928 (2006).Crossref, Medline, CAS, Google Scholar13 Zhou F, Lu Y, Ficarro SB, Webber JT, Marto JA. Nanoflow low pressure high peak capacity single dimension LC-MS/MS platform for high-throughput, in-depth analysis of mammalian proteomes. Anal. Chem. 84(11), 5133–5139 (2012).Crossref, Medline, CAS, Google Scholar14 Schneider BB, Javaheri H, Covey TR. Ion sampling effects under conditions of total solvent consumption. Rapid Commun. Mass Spectrom. 20(10), 1538–1544 (2006).Crossref, Medline, CAS, Google Scholar15 Emmett MR, Caprioli RM. Micro-electrospray mass spectrometry: ultra-high-sensitivity analysis of peptides and proteins. J. Am. Soc. Mass Spectrom. 5(7), 605–613 (1994).Crossref, Medline, CAS, Google Scholar16 Gallagher R, Dillon L, Grimsley A, Murphy J, Samuelsson K, Douce D. The application of a new microfluidic device for the simultaneous identification and quantitation of midazolam metabolites obtained from a single micro-litre of chimeric mice blood. Rapid Commun. Mass Spectrom. 28(11), 1293–1302 (2014).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByMicroflow Liquid Chromatography – Multi-Emitter Nanoelectrospray Mass Spectrometry of OligonucleotidesJournal of Chromatography A, Vol. 65Improving the LC-MS/MS analysis of neuromedin U-8 and neuromedin S by minimizing their adsorption behavior and optimizing UHPLC and MS parametersJournal of Pharmaceutical and Biomedical Analysis, Vol. 2On-line solid-phase extraction to enhance sensitivity in peptide biomarker analysis by microseparation techniques coupled to mass spectrometry: Capillary liquid chromatography versus capillary electrophoresisMicrochemical Journal, Vol. 183Microflow LC–MS/MS to improve sensitivity for antisense oligonucleotides bioanalysis: critical role of sample cleannessDi Jiang & Long Yuan10 January 2023 | Bioanalysis, Vol. 14, No. 21Bioanalytical LC–MS/MS method for simultaneous estimation of atorvastatin, its major active metabolites and ezetimibeEhab F Elkady, Bushra M Jaadan, Elsayed Ramadan & Ayman Abo Elmaaty9 January 2023 | Bioanalysis, Vol. 14, No. 21On the potential of micro-flow LC-MS/MS in proteomics18 October 2022 | Expert Review of Proteomics, Vol. 19, No. 3TinMiniaturization in Separation Techniques1 July 2022Miniaturization in Separation Techniques18 December 2021Micro‐flow hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry detects modified nucleosides in the transfer RNA pool of cyanobacteria11 July 2021 | Journal of Separation Science, Vol. 44, No. 17Intact Protein Mass Spectrometry for Therapeutic Protein Quantitation, Pharmacokinetics, and Biotransformation in Preclinical and Clinical Studies: An Industry Perspective1 September 2020 | Journal of the American Society for Mass Spectrometry, Vol. 32, No. 8How much separation sciences fit in the green chemistry canoe?Current Opinion in Green and Sustainable Chemistry, Vol. 30An integrated Qual/Quan strategy for ganglioside lipidomics using high‐resolution mass spectrometry and Skyline software2 February 2021 | Rapid Communications in Mass Spectrometry, Vol. 35, No. 7Robust Microflow LC-MS/MS for Proteome Analysis: 38 000 Runs and Counting17 February 2021 | Analytical Chemistry, Vol. 93, No. 8Miniaturized LC in Molecular Omics12 August 2020 | Analytical Chemistry, Vol. 92, No. 17Miniaturized liquid chromatography focusing on analytical columns and mass spectrometry: A reviewAnalytica Chimica Acta, Vol. 1103Evaluation of OptiFlow™-MS/MS for bioanalysis of pharmaceutical drugs and metabolitesJason Barricklow, Joseph Tweed, Christopher L Holliman & Ragu Ramanathan18 December 2019 | Bioanalysis, Vol. 12, No. 1More sensitivity is always better: Measuring sub-clinical levels of serum thyroglobulin on a µLC–MS/MS systemClinical Mass Spectrometry, Vol. 15Analysis of endocannabinoids in plasma samples by biocompatible solid-phase microextraction devices coupled to mass spectrometryAnalytica Chimica Acta, Vol. 1091Evaluation of meter-long monolithic columns for selected reaction monitoring mass spectrometryJournal of Bioscience and Bioengineering, Vol. 128, No. 3Optimization of microflow LC–MS/MS and its utility in quantitative discovery bioanalysisJun Zhang, Wilson Shou, Tairo Ogura, Shu Li & Harold Weller28 June 2019 | Bioanalysis, Vol. 11, No. 11Quantification below the LLOQ in regulated LC–MS/MS assays: a review of bioanalytical considerations and cautionsJeffrey X Duggan17 April 2019 | Bioanalysis, Vol. 11, No. 8Simultaneous extraction and analysis of multiple cystine-dense peptides by μSPE and microflow-MS/MS from plasmaMi-Youn Brusniak, Chad Christianson, Emily Witthuhn, Shane Needham & James M Olson20 March 2019 | Bioanalysis, Vol. 11, No. 6Solid Phase Microextraction-mass spectrometry: MetanoiaTrAC Trends in Analytical Chemistry, Vol. 112Profiling of polyunsaturated fatty acids in human serum using off-line and on-line solid phase extraction-nano-liquid chromatography-quadrupole-time-of-flight mass spectrometryJournal of Chromatography A, Vol. 1537Open Port Probe Sampling Interface for the Direct Coupling of Biocompatible Solid-Phase Microextraction to Atmospheric Pressure Ionization Mass Spectrometry13 February 2017 | Analytical Chemistry, Vol. 89, No. 7Characterization of peak capacity of microbore liquid chromatography columns using gradient kinetic plotsJournal of Chromatography A, Vol. 1485Micro-liquid chromatography mass spectrometry for the analysis of antineoplastic drugs from wipe samples21 September 2016 | Analytical and Bioanalytical Chemistry, Vol. 408, No. 28Challenges for the in vivo quantification of brain neuropeptides using microdialysis sampling and LC–MSYannick Van Wanseele, An De Prins, Dimitri De Bundel, Ilse Smolders & Ann Van Eeckhaut24 August 2016 | Bioanalysis, Vol. 8, No. 18Advances in LC: bioanalytical method transferPatricia Wright & Adrian Wright5 August 2016 | Bioanalysis, Vol. 8, No. 17Biocompatible Solid-Phase Microextraction Nanoelectrospray Ionization: An Unexploited Tool in Bioanalysis29 December 2015 | Analytical Chemistry, Vol. 88, No. 2Advances in quantitative bioanalysis of oligonucleotide biomarkers and therapeuticsLaixin Wang & Chengjie Ji10 December 2015 | Bioanalysis, Vol. 8, No. 2Top-Down Mass Spectrometry: Proteomics to Proteoforms15 December 2016 Vol. 7, No. 9 Follow us on social media for the latest updates Metrics History Published online 3 June 2015 Published in print May 2015 Information© Future Science LtdKeywordsbioanalysisLC–MS/MSmicroflowmicrospraynanosprayAcknowledgementsThe authors wish to thank M Lee of Milestone Development Services for insightful discussions during the preparation of this manuscript.Financial & 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

Clinical and Pharmaceutical Solutions through Analysis, São Paulo, Brazil, 3-5 August 2015 The 2nd Annual Symposium on Clinical and Pharmaceutical Solutions through Analysis was held on 3-5 August 2015 at Club Transatlântico, São Paulo, Brazil. This annual meeting began in 2014 and was the first industry-led event in Brazil to focus on the specific needs of industry researchers while bringing together technology and regulators. The goal of CPSA is to provide an in-depth review of innovative technology and industry practices through open discussion of industry-related issues and needs. Education and specialized training are the foundation of all CPSA events. As the industry has evolved so has CPSA. The CPSA annual meeting thrived with high quality scientific content, open interaction from industry opinion leaders and a collegial environment.

The 17th Annual Symposium on Clinical and Pharmaceutical Solutions through Analysis (CPSA) 29 September-2 October 2014, was held at the Sheraton Bucks County Hotel, Langhorne, PA, USA. The CPSA USA 2014 brought the various analytical fields defining the challenges of the modern analytical laboratory. Ongoing discussions focused on the future application of bioanalysis and other disciplines to support investigational new drugs (INDs) and new drug application (NDA) submissions, clinical diagnostics and pathology laboratory personnel that support patient sample analysis, and the clinical researchers that provide insights into new biomarkers within the context of the modern laboratory and personalized medicine.

The 16th Annual Symposium on Clinical and Pharmaceutical Solutions through Analysis (CPSA) 7-10 October 2013, Sheraton Bucks County Hotel, Langhorne, PA, USA. The 2013 CPSA brought together the various US FDA regulated analytical fields affecting a 'patient' for the first time - bioanalysts supporting IND and NDAs, clinical diagnostic and pathology laboratory personnel, and clinical researchers that provide insights into new biomarkers. Although the regulatory requirements are different for each of the above disciplines, the unique analytical perspectives that affect the patient were shared - and the goal of the 2013 CPSA - 'Connecting Patients and Subject Numbers Through Analysis' was achieved.

Chapter 21 Beyond Dried Blood Spots—Application of Dried Matrix Spots Shane R. Needham, Shane R. NeedhamSearch for more papers by this author Shane R. Needham, Shane R. NeedhamSearch for more papers by this author Book Editor(s):Wenkui Li, Wenkui Li Novartis Institutes for BioMedical Research, East Hanover, NJ, USASearch for more papers by this authorMike S. Lee, Mike S. Lee Milestone Development Service, Newtown, PA, USASearch for more papers by this author First published: 13 June 2014 https://doi.org/10.1002/9781118890837.ch21Citations: 2 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Summary Dried blood spot (DBS) techniques have been shown to play a beneficial role in drug development for the bioanalysis of drugs and metabolites from blood. In this discussion, we describe the technique of dried matrix spotting (DMS) to indicate the DBS technique can be applied to non-blood matrices. The DMS method employs a color-indicating process that enhances the ability to analyze transparent fluids spotted onto collection paper by allowing the analyst to visually verify the location of the dried sample spot. Matrices to date that have been analyzed using DMS include cerebral spinal fluid (CSF), synovial fluid, saliva, tears, urine and plasma. The addition of color-indicating dye has been shown to have additional benefits including improved method accuracy and precision by allowing addition of the internal standard with the color-indicating dye. Citing Literature Dried Blood Spots: Applications and Techniques RelatedInformation

BioanalysisVol. 5, No. 11 EditorialMicro-LC–MS/MS: the future of bioanalysisDon W Arnold & Shane R NeedhamDon W Arnold* Author for correspondenceEksigent, part of AB Sciex, 1201 Radio Road, Redwood Shores, CA 94065, USA. Search for more papers by this authorEmail the corresponding author at dwarnold@eksigent.com & Shane R NeedhamAlturas Analytics, Inc. 1324 Alturas Dr. Moscow, ID 83843, USASearch for more papers by this authorPublished Online:6 Jun 2013https://doi.org/10.4155/bio.13.31AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleKeywords: delay volumemicro-LCmicro-LC–MS/MSUHPLCReferences1 Knox JH. Theoretical aspects of LC with packed and open small-bore columns. J. Chromatogr. Sci.18(9),453–461 (1980).Crossref, CAS, Google Scholar2 Reese CE, Scott RPW. Microbore columns – design, construction, and operation. J. Chromatogr. 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Bioanalysis5(11),1387–1396 (2013).Link, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByComparison of pharmacokinetic study profiles of insulin in rat plasma through conventional sampling and microsampling by micro-LC–MS/MSGaudry Bruno Troché, Tue Søeborg, Thóra Brynja Bödvarsdottir, Mads Bjelke & Nikoline Juul Nielsen14 April 2023 | Bioanalysis, Vol. 15, No. 5Bioanalytical LC–MS/MS method for simultaneous estimation of atorvastatin, its major active metabolites and ezetimibeEhab F Elkady, Bushra M Jaadan, Elsayed Ramadan & Ayman Abo Elmaaty9 January 2023 | Bioanalysis, Vol. 14, No. 21Research Progress on Quantification Methods of Drug Concentration of Monoclonal AntibodiesCurrent Pharmaceutical Analysis, Vol. 18, No. 7On the potential of micro-flow LC-MS/MS in proteomics18 October 2022 | Expert Review of Proteomics, Vol. 19, No. 3Omics in medicinal plantsPharmacokinetic and metabolic analysis of an Alzheimer's disease therapeutic in rat serum via microfluidic CZE–MS4 October 2021 | Biomedical Chromatography, Vol. 36, No. 1Evaluation of OptiFlow™-MS/MS for bioanalysis of pharmaceutical drugs and metabolitesJason Barricklow, Joseph Tweed, Christopher L Holliman & Ragu Ramanathan18 December 2019 | Bioanalysis, Vol. 12, No. 1Metaproteomics of the human gut microbiota: Challenges and contributions to other OMICSClinical Mass Spectrometry, Vol. 14Microflow UPLC and high-resolution MS as a sensitive and robust platform for quantitation of intact peptide hormonesZhuo Chen, Yun W Alelyunas, Mark D Wrona, Jonathan R Kehler, Matthew E Szapacs & Christopher A Evans12 July 2019 | Bioanalysis, Vol. 11, No. 13Instrumental and technical evolution over the past decade in bioanalysisRobert MacNeill4 April 2019 | Bioanalysis, Vol. 11, No. 7Qualitative and quantitative characterization of protein biotherapeutics with liquid chromatography mass spectrometry20 April 2016 | Mass Spectrometry Reviews, Vol. 36, No. 6Peptide and Protein Bioanalysis Using Integrated Column‐to‐Source Technology for High‐Flow Nanospray2 June 2017SWATH-MS as a tool for biomarker discovery: From basic research to clinical applications16 February 2017 | PROTEOMICS, Vol. 17, No. 3-4Micro-liquid chromatography mass spectrometry for the analysis of antineoplastic drugs from wipe samples21 September 2016 | Analytical and Bioanalytical Chemistry, Vol. 408, No. 28Advances in LC: bioanalytical method transferPatricia Wright & Adrian Wright5 August 2016 | Bioanalysis, Vol. 8, No. 17Key Mass Spectrometry Techniques Used in Clinical Biomarker Research23 August 2016Method development strategies in bioanalysis28 October 2015Sense and nonsense of miniaturized LC–MS/MS for bioanalysisMartijn Hilhorst, Chad Briscoe & Nico van de Merbel23 December 2014 | Bioanalysis, Vol. 6, No. 24Quantitative bioanalysis by microflow LC–MS to support discovery-based pharmacokinetic studies24 October 2014Applications of low-flow LC–SRM for the analysis of large molecules in pharmaceutical R&DMichael E Lassman & Carmen Fernandez-Metzler26 August 2014 | Bioanalysis, Vol. 6, No. 13Quantitation of human peptides and proteins via MS: review of analytically validated assaysDerek L Chappell, Michael E Lassman, Thomas McAvoy, Mingxiang Lin, Daniel S Spellman, Omar F Laterza26 August 2014 | Bioanalysis, Vol. 6, No. 13 Vol. 5, No. 11 STAY CONNECTED Metrics Downloaded 546 times History Published online 6 June 2013 Published in print June 2013 Information© Future Science LtdKeywordsdelay volumemicro-LCmicro-LC–MS/MSUHPLCFinancial & competing interests disclosureDW Arnold is a Vice President and Principal Scientist at Eksigent, part of AB SCIEX, a manufacturer of micro-LC–MS/MS systems. The authors have no other 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 apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

BACKGROUND:In support of bioanalysis, there has always been a desire to improve detection limits and reduce scale. Microflow LC (MFLC) coupled with MS accomplishes both of these goals.RESULTS:As such, MFLC coupled with an MS system was used to generate bioanalytical validation data that met US FDA criteria. The MFLC-MS/MS data was compared with the same method with the use of conventional HPLC-MS/MS and a more than 14× S/N improvement was found with the MFLC-MS/MS method. Methotrexate was used as a model molecule to demonstrate the validation of the method from human plasma. The MFLC-MS/MS method was demonstrated to be accurate (±7%) and precise (12.9% at the LLOQ and a maximum of 11.6% at all other concentrations) across the dynamic range of the assay (1-1000 ng/ml) and compared well with the HPLC-MS/MS method. The MFLC bioanalytical validation was performed at a flow rate of 35 µl/min on a 0.5-mm inner diameter (I.D.) column, whereas, for the same linear velocities on the 2.0-mm I.D. column, the conventional HPLC bioanalytical validation was performed at 700 µl/min. Since the flow rate of the MFLC system is 20-times less than the HPLC system, the consumable solvent and disposal cost to perform the MFLC validation was significantly less.CONCLUSION:MFLC-MS/MS can be used to perform bioanalytical method validations with increased MS signal, reduced source contamination and reduced solvent consumption.

DBS techniques for the bioanalysis of drugs and metabolites from whole blood have been demonstrated to be a useful tool in drug development. The term dried matrix spot (DMS) has been used to indicate that the DBS technique has been applied to nonblood matrices. DMS methods often employ a color-indicating process that enhances the ability to analyze these mostly transparent fluids when spotted onto collection paper. The color-indicating dye allows the analyst to visually confirm the location of the dried sample spot. Other benefits of using a color-indicating dye include improved method accuracy and precision, because the process of adding the dye allows for the concurrent addition of the IS prior to sample addition and extraction. To date, matrices that have been analyzed using DMS include cerebrospinal fluid, synovial fluid, saliva, tears, urine and plasma.

This annual meeting began in 1998 and was the first industry-led event to focus on the specific needs of industry researchers. The goal of Clinical and Pharmaceutical Solutions Through Analysis (CPSA) is to provide an in-depth review of innovative technology and industry practices through open discussion of industry-related issues and needs. Education and specialized training are the foundation of all CPSA events. As the industry has evolved so has CPSA. Thus, the year the name was changed from Chemical and Pharmaceutical Structural Analysis to CPSA was to reflect the growing focus on clinical applications and the emergence of personalized medicine. Most importantly, the CPSA annual meeting has retained the same high-quality scientific content, open interaction from industry opinion leaders and a collegial environment.

The Global CRO Council for Bioanalysis (GCC) was formed in September 2010. Since then, the representatives of the member companies come together periodically to openly discuss bioanalysis and the regulatory challenges unique to the outsourcing industry. The 4th GCC Closed Forum brought together experts from bioanalytical CROs to share and discuss recent issues in regulated bioanalysis, such as the impact of coadministered drugs on stability, some differences between European Medicines Agency and US FDA bioanalytical guidance documents and lessons learned following recent Untitled Letters. Recent 483s and agency findings, as well as issues on method carryover, were also part of the topics discussed.

This annual meeting began in 1998 and was the first industry-led event to focus on the specific needs of industry researchers. The goal of Chemical and Pharmaceutical Structure Analysis is to provide an in-depth review of innovative technology and industry practices through open discussion of industry-related issues and needs. Education and specialized training are the foundation of all Chemical and Pharmaceutical Structure Analysis events - where technology and solutions meet.

) Guideline on Bioanalytical Method Validation (BMV), during the 4th GCC (23 October 2011, Washington DC, USA) and 5th GCC (14 November 2011, Barcelona, Spain) Closed Forums. These North American and European events provided a unique opportunity for CRO leaders to openly share opinions and perspectives and to agree on unified bioanalytical recommendations specifically in relation with the new EMA guideline.The Global CRO Council for Bioanalysis (GCC)