BioanalysisVol. 14, No. 16 EditorialOpen AccessEfficient interactions with bioanalytical contract research organizations: inside scoopAimin Tan, Constantine Fanaras & John C FanarasAimin Tan *Author for correspondence: E-mail Address: tan@nucro-technics.comhttps://orcid.org/0000-0001-5313-8650Nucro-Technics, Scarborough, ON, Canada, Constantine FanarasNucro-Technics, Scarborough, ON, Canada & John C FanarasNucro-Technics, Scarborough, ON, CanadaPublished Online:4 Oct 2022https://doi.org/10.4155/bio-2022-0129AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Keywords: bioanalysiscontract research organizationCROdrug developmentLC–MSoutsourcingBioanalytical outsourcing has been in steady increase over the past a few decades and this trend will continue in the foreseeable future. The main reason behind this is that pharma and biotech companies continue to seek higher efficiency and lower costs for their drug development programs [1–2]. Together with the increase in outsourcing, in-house bioanalytical capabilities in pharma and biotech companies have greatly been reduced or the departments have been shut down. This further reinforces outsourcing due to insufficient internal resources and/or lack of advanced expertise. To meet the increasing demand for outsourcing, bioanalytical contract research organizations (CROs) have been growing and constantly evolving [3]. As working with bioanalytical CROs is inevitable for most drug development programs and placing studies with them is not without risk, efficient interactions with bioanalytical CROs becomes critical. Drawn from our decades of successful experiences in working with worldwide clients/sponsors at the CRO side, we want to share our perspectives on efficient interactions with bioanalytical CROs to achieve win–win results for both sides.Find the right bioanalytical CRODespite the many bioanalytical CROs available, sponsors often find/comment that it is really difficult to find a good one for the job at hand. This is not without reason.First, there are not many truly good bioanalytical CROs in the market. A good bioanalytical CRO should have top-notch scientific expertise, an excellent track record of compliance, fast response, flexibility in timeline and workload, low staff turnover rate (at least for project management and key scientific staff) and preferably the ability to 'baby-sit' the sponsor's studies. Often, a bioanalytical CRO is strong in some of these aspects and weak in others. For bioanalysis, no issue is small. Even a seemingly tiny one, such as contamination, nonspecific binding or carryover, if not identified and addressed in a timely manner, can become a large issue, leading to the failure of a batch/study or even the closure of a laboratory (e.g., closure of MDS Quebec Lab in 2006) [4–8]. On the other hand, most contract bioanalysis work is highly regulated. Relevant staff must be familiar with various regulatory guidelines. It is critical to have scientifically strong staff who are also well versed in both regulations and the overall drug development program at management positions to perform timely evaluation and to make the best decisions. In reality, very few bioanalytical CROs are like this. As a result, many subtle yet critical factors are overlooked or lost/delayed during communications [4–8].Second, bioanalytical CROs are constantly evolving – for example, mergers, closures, reopenings or new openings. Accordingly, staff turnover is usually very high, which leads to a migration of expertise. When a sponsor finds or gets used to a bioanalytical CRO, the personnel or the lab itself might not be there anymore when the sponsor goes back for a new study.Third, some sponsors may not know what they really require or how their needs or stage of drug development impacts the selection of bioanalytical CROs. Prior to approaching bioanalytical CROs, sponsors should examine their needs and evaluate the various aspects of potential CROs. Typically, for bioanalysis during the early stages of drug development, the focus should be on the scientific capability of CROs. As little is known about the analytes (stability and concentration ranges are typically unknown), tough scientific decisions must be made frequently and in a timely manner. On the other hand, bioanalytical needs at a later stage of drug development will differ. In particular, for bioequivalence studies, the focus should be on a good track record of compliance, capacity to take on the work and the availability of validated methods, particularly those successfully used in previous studies. In short, sponsors need to do their homework well and to approach bioanalytical CROs with specific requirements, instead of randomly shopping around.Provide the relevant information & materials on timeEach bioanalytical method corresponds to a specific matrix type, analyte(s) and the associated concentration range(s). Without these specifics, it would be meaningless or difficult to evaluate the feasibility/costs of method development. The challenges and costs associated with the development of a method for quantifying an analyte at μg/ml level in saline are not the same as a method to quantify an unstable endogenous analyte at 1 pg/ml level in whole blood. Additionally, the costs of key materials (e.g., stable-isotope labeled internal standards [9]) can vary significantly from one analyte to the other, such as from less than a dollar to over $10,000 (USD) per 10 mg.Bioanalytical methods developed for previous studies or by a different bioanalytical CRO are usually not as useful/valuable as many sponsors would think. Just as a study in rats cannot save the study in dogs, a bioanalytical method developed for one species may not work for another species due to the potential differences in matrix effects, metabolism, enzyme activities, sensitivity requirements etc. Still, just like an earlier non-Good Laboratory Practices (GLP) study cannot replace a GLP study, a generic bioanalytical method developed for early-stage drug development usually does not meet the standards for GLP method validation. Furthermore, as different bioanalytical CROs may not have the same standard operating procedure systems, regulatory reputation, instrument setup, bioanalytical expertise, strategies and philosophy, a method developed or validated by one bioanalytical CRO is rarely readily transferable to another (the exception might be the multisites of a large company, which have the same standard operating procedures and instrument setup). In most instances, trying to transfer a bioanalytical method from another lab is more trouble than it is actually worth. The usual result is that one ends up spending more time and money to adapt something that may not be optimal rather than starting from scratch.For the aforementioned reasons, sponsors should strive to provide specifics with their bioanalytical request: 1.Sample matrix2.Analyte(s) to be quantified3.Expected concentration range(s)4.Type and degree of compliance5.Number of samples6.TimelineAdditional information, although not essential, such as solubility and stability of the analyte(s) in solution and/or matrix, is helpful. For chiral separation, any information about the chiral separation mode, LC column and mobile phase is beneficial.When the essential information is not readily available, efforts should be made to obtain the missing information, such as conducting a small pilot study for this purpose rather than repeatedly asking the bioanalytical CRO if the method is ready. Even though some initial method development activities, such as compound mass optimization on mass spectrometers and LC optimization, can be initiated, the method development cannot be finalized until the essential information is provided and tested. There is usually no need to involve bioanalytical staff in meetings or discussions regarding study designs. This is typically a waste of their time and outside the scope of their capabilities. What a bioanalytical lab needs are the same specifics outlined previously regardless of the types of studies.If the test article or the analyte is not commercially available, the sponsor needs to send some reference material to initiate the method development as soon as possible. For shipment of reference material, sponsors should provide the certificate of analysis or at a minimum storage condition in advance to avoid unnecessary back-and-forth communications and potential compromise of the received compound (if not properly stored).Leave the professionals to do their job & respect their workSponsors should do their due diligence prior to placing a study with a bioanalytical CRO. But once the bioanalytical CRO is found and timelines and outcomes are established, sponsors should then leave the professionals in the lab to do their job without much interference. It is often useless or even counterproductive to ask for detailed weekly or even daily progress reports, unless the sponsor representative is truly a bioanalytical expert and has time and energy to troubleshoot an unexpected issue (which is rarely the case) [4–8].Sponsors should review documents and results on time, especially study plans and validation protocols. The focus should be on scientific soundness, adherence to guidance and integrity of data, not excessively on p's and q's for what is usually a several hundred-page document. As each bioanalytical CRO may have different ways of doing things (e.g., calibration schemes [10,11]) or different report formats, sponsors should abstain from insisting on one particular way unless it is a mistake or against regulatory guidance. After all, most bioanalytical CROs, especially the veterans, have worked for many different sponsors and their approaches have been examined multiple times by both their clientele and regulatory agencies. Sometimes, as a service provider, a bioanalytical CRO may just follow the sponsor's recommendations merely for the sake of keeping good relations. In this case, the sponsor should not be lured into thinking that the way he/she insisted is the only correct way.Aim for long-term win–win relationshipIt takes time and cost to find a good bioanalytical CRO because truly good ones are rare in the market. There are always risks and potential losses when outsourcing a study or drug development program. Therefore, once a good bioanalytical CRO is found, sponsors should keep working with the CRO to forge a long-term win–win relationship. After all, shopping around may bring uncertainties and the methods already established may not be readily transferable. The new CRO has to start from scratch to accumulate the relevant experience. Furthermore, the new CRO needs to purchase reagents and materials that the existing CRO may already have. All these costs will be ultimately billed to the sponsor one way or the other. Therefore, forging a long-term relationship is greatly beneficial to both sides.Financial & competing interests disclosureThe authors are employed in a contract research organization. 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.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/References1. Zimmer D. Bioanalytical outsourcing: the two sides of the medal. Bioanalysis 5(17), 2095–2099 (2013).Link, CAS, Google Scholar2. Dong K, Tang D. Bioanalytical outsourcing strategy in China. Bioanalysis 9(15), 1157–1159 (2017).Link, CAS, Google Scholar3. Premkumar N, Lowes S, Jersey J et al. Formation of a global contract research organization council for bioanalysis. Bioanalysi. 2(11), 1797–1800 (2010).Link, CAS, Google Scholar4. Tan A, Hussain S, Musuku A, Massé R. Internal standard response variations during incurred sample analysis by LC–MS/MS: case by case trouble-shooting. J. Chromatogr. B. 877, 3201–3209 (2009).Crossref, Medline, CAS, Google Scholar5. Tan A, Gagnon-Carignan S, Lachance S et al. Beyond successful incurred sample reanalysis (ISR): case by case investigations for unmatched reassay results when ISR passed. Bioanalysis 3(9), 1031–1038 (2011).Link, CAS, Google Scholar6. Tan A, Wu Y, Gu G, Fanaras JC. Self-initiated and concentration-dependent degradation of tetracaine in neat standard solutions: a trouble-shooting story. J. Chromatogr. B. 1033, 112–116 (2016).Crossref, Medline, Google Scholar7. Tan A, Fanaras JC. Nonspecific binding in LC–MS bioanalysis. In: Targeted Biomarker Quantitation by LC–MS. Weng NJian W (Eds).John Wiley & Sons, NJ, USA,137–147 (2017).Crossref, Google Scholar8. Tan A, Fanaras JC. How much separation for LC–MS/MS quantitative bioanalysis of drugs and metabolites. J. Chromatogr. B. 1084, 23–35 (2018).Crossref, Medline, CAS, Google Scholar9. Tan A, Awaiye K. Use of internal standards in LC–MS bioanalysis. In: Handbook of LC–MS Bioanalysis: Best Practices, Experimental Protocols, and Regulations. Li WZhang JTse FLS (Eds). John Wiley & Sons, NJ, USA, 217–227 (2013).Crossref, Google Scholar10. Tan A, Awaiye K, Jose B et al. Comparison of different linear calibration approaches for LC–MS bioanalysis. J. Chromatogr. B. 911, 192–202 (2012).Crossref, Medline, CAS, Google Scholar11. Tan A, Awaiye K, Trabelsi F. Impact of calibrator concentrations and their distribution on accuracy of quadratic regression for liquid chromatography–mass spectrometry bioanalysis. Anal. Chim. Acta 815, 33–41 (2014).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetails Vol. 14, No. 16 Follow us on social media for the latest updates Metrics History Received 30 June 2022 Accepted 24 August 2022 Published online 4 October 2022 Published in print August 2022 Information© 2022 Aimin Tan, Constantine Fanaras & John C FanarasKeywordsbioanalysiscontract research organizationCROdrug developmentLC–MSoutsourcingFinancial & competing interests disclosureThe authors are employed in a contract research organization. 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.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. 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The purpose of this study was to evaluate the ocular pharmacokinetics, bio-distribution and local tolerability of γ-cyclodextrin (γCD) based irbesartan 1.5% eye drops and candesartan 0.15% eye drops after single and multiple topical administration in rabbit eyes. In this randomized, controlled study, a total number of 59 New Zealand White albino rabbits were consecutively assigned to two study groups. Group 1 (n = 31) received irbesartan 1.5% and group 2 (n = 28) candesartan 0.15% eye drops. In both groups, single dose and multiple administration pharmacokinetic studies were performed. Rabbits were euthanized at five predefined time points after single-dose administration, whereas multiple-dose animals were dosed for 5 days twice-daily and then euthanized 1 h after the last dose administration. Drug concentration was measured by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the retinal tissue, vitreous humor, aqueous humor, corneal tissue and in venous blood samples. Pharmacokinetic parameters including maximal drug concentration (Cmax), time of maximal drug concentration (Tmax), half-life and AUC were calculated. To assess local tolerability, six additional rabbits received 1.5% irbesartan eye drops twice daily in one eye for 28 days. Tolerability was assessed using a modified Draize test and corneal sensibility by Cochet Bonnet esthesiometry. Both γCD based eye drops were rapidly absorbed and distributed in the anterior and posterior ocular tissues. Within 0.5 h after single administration, the Cmax of irbesartan and candesartan in retinal tissue was 251 ± 142 ng/g and 63 ± 39 ng/g, respectively. In the vitreous humor, a Cmax of 14 ± 16 ng/g for irbesartan was reached 0.5 h after instillation while Cmax was below 2 ng/g for candesartan. For multiple dosing, the observed Cmean in retinal tissue was 338 ± 124 ng/g for irbesartan and 36 ± 10 ng/g for candesartan, whereas mean vitreous humor concentrations were 13 ± 5 ng/g and <2 ng/g, respectively. The highest plasma concentrations of both irbesartan (Cmax 5.64 ± 4.08 ng/mL) and candesartan (Cmax 4.32 ± 1.04 ng/mL) were reached 0.5 h (Tmax) after single administration. Local tolerability was favorable with no remarkable differences between the treated and the control eyes. These results indicate that irbesartan and candesartan in γCD based nanoparticle eye drops can be delivered to the retinal tissue of the rabbit’s eye in pharmacologically relevant concentrations. Moreover, safety and tolerability profiles appear to be favorable in the rabbit animal model.

PURPOSE:Orally administered angiotensin II receptor blockers (ARBs) decrease intraocular pressure (IOP). Topical administration may reduce systemic side effects and result in a useful glaucoma drug. The aim of this study is to test the ocular delivery and pharmacologic effect of nanoparticle eye drops containing ARBs (e.g. irbesartan and candesartan).METHODS:1.5% irbesartan and 0.15% candesartan eye drops were applied to rabbits. The pharmacokinetics in cornea and aqueous humour after single eye drop application were studied in 49 rabbits. The effect of the eye drops on IOP was studied in 10 rabbits using an iCare (® TonoVet Plus, iCare, Finland) tonometer and compared with 0.5% timolol eye drops.RESULTS:Candesartan lowered IOP from 24.6 ± 5.1 mmHg at baseline to 19.0 ± 2.9 mmHg (mean ± SD, p = 0.030, n = 10) 4 hr after application. Irbesartan lowered IOP from 24.2 ± 1.7 mmHg to 20.2 ± 0.9 mmHg (p = 0.14, n = 10). Timolol decreased the IOP from 24.9 ± 4.2 mmHg to 20.4 ± 4.8 mmHg (mean ± SD, p = 0.036, n = 10). The pharmacokinetics data show that both formulations deliver effective amounts of drug into the intraocular tissues, with irbesartan and candesartan reaching concentrations of 121 ± 69 and 30.43 ± 13.93 ng/g (mean ± SD), respectively, in the aqueous humour 3 hr after a single-dose administration.CONCLUSIONS:Topical application of irbesartan and candesartan eye drops delivers effective drug concentrations to the anterior segment of the eye in rabbits, achieving drug concentrations 100 times above the IC50 for angiotensin II receptor and showing an IOP-lowering effect. Angiotensin receptor blocker (ARB) eye drops have potential as a new class of glaucoma drugs.

Diabetic retinopathy is a major cause of vision loss in adults. Novel eye-drop formulations of candesartan and irbesartan are being developed for its cure or treatment. To support a preclinical trial in rabbits, it was critical to develop and validate a new LC-MS/MS method for simultaneous quantification of candesartan and irbesartan in rabbit eye tissues (cornea, aqueous humor, vitreous body and retina/choroid). Eye tissue samples were first homogenized in H2 O-diluted rabbit plasma. The candesartan and irbesartan in the supernatants together with their respective internal standards (candesartan-d4 and irbesartan-d4 ) were extracted by solid-phase extraction. The extracted samples were injected onto a C18 column for gradient separation. The MS detection was in the positive electrospray ionization mode using the multiple reaction monitoring transitions of m/z 441 → 263, 445 → 267, 429 → 207, and 433 → 211 for candesartan, candesartan-d4 , irbesartan and irbesartan-d4 , respectively. For the validated concentration ranges (2-2000 and 5-5000 ng/g for candesartan and irbesartan, respectively), the within-run and between-run accuracies (% bias) were within the range of -8.0-10.0. The percentage CV ranged from 0.6 to 7.3. There was no significant matrix interference nor matrix effect from different eye tissues and different rabbits. The validated method was successfully used in the Good Laboratory Practice (GLP) study of rabbits.

Background/Aim: Curcumin is being widely investigated for its anticancer properties and several studies in the literature suggest that curcumin is distributed to a higher degree in cancer cells compared to normal cells. The goal of this study was to investigate the disposition of curcumin in the form of Lipocurc™ in multiple myeloma (MM)-causing plasma cell lines and B-lymphocytes from healthy individuals and compare the uptake to previously published data for red blood cells (RBCs), peripheral blood mononuclear cells (PBMCs) from healthy individuals and PBMCs from patients with chronic lymphocytic leukemia (CLL-cells). Materials and Methods: Two MM-producing cell lines were studied: RPMI-8266, an IgG lambda cell line, and NCL-H929, an IgA kappa line. The distribution of liposomal curcumin and its metabolism to the major stable metabolite tetrahydrocurcumin (THC) were measured in vitro in the cell lines and B-lymphocytes. The cells were incubated in plasma protein-supplemented media with liposomal curcumin (Lipocurc™) for 15 min at 37°C and the levels of curcumin and THC in cells and medium were determined by liquid chromatography tandem mass spectrometry. Results: Extremely intense uptake was seen in both MM lines compared to that in B-lymphocytes and previously published data in RBCs, PBMCs and CLL cells. The levels of curcumin in RPMI-8266 and NCI-H929 cells were 14,225±847 and 12,723±500 pg/106 cells compared to 19±5,587±86 and 3,122±166 pg/106 cells in RBCs, PBMCs and CLL cells, respectively. Conversion of curcumin to THC was greatest in PBMCs, considerably less in CLL cells and minimal or absent in B-lymphocytes and MM cell lines. Conclusion: The extremely intense uptake of curcumin (as Lipocurc™) in both MM lines further suggests that Lipocurc™ should be investigated in the treatment of patients with this disease.

Cyclodextrin nanoparticles form water-soluble complexes with lipophilic and poorly water-soluble drugs and thus can greatly improve drug transport from the ocular surface to the posterior eye segment. The aim of the current study was to evaluate the ocular pharmacokinetics and biodistribution of two newly developed □-cyclodextrin based angiotensin receptor antagonist formulations for topical administration: cyclodextrin-Irbesartan 1.5% and cyclodextrin-Candesartan 0.15%. 59 rabbits were included in the study to receive one of the two study drugs. For each drug group, single and multiple dose pharmacokinetics were performed in a randomized fashion: Single dose rabbits were euthanized 0.5, 1.5, 3 or 6 hours post eye drop administration, whereas multiple dose animals were dosed for 5 days twice daily. Pharmacokinetic parameters including maximal drug concentration (C max ) and time of maximal drug concentrations (T max ) for single dosing and mean concentrations for multiple dosing were calculated for aqueous humor (AH) and retina/choroid (RT). Analysis was done using LC-MS/MS. Topical administration of the study drugs was well tolerated. Single dose Irbesartan eye drop administration led to a RT C max of 251 ng/g ± 143 ng/g at 0.5 hours whereas in the AH a C max of 121 ng/g ± 69 ng/g was reached at 3 hours post instillation. For single dose Candesartan eye drops, RT C max was 63 ng/g ± 39 ng/g at 0.5 hours after application. AH reached a C max of 30 ng/g ± 14 ng/g at the 3 hours time point. For multiple dosing mean RT concentration reached 338 ng/g ± 124 ng/g for Irbesartan and 36 ng/g ± 10 ng/g for Candesartan, whereas mean AH concentrations were 231 ng/g ± 68 ng/g and 70 ng/g ± 22 ng/g, respectively. The present data confirm that cyclodextrin based Candesartan and Irbesartan eye drops deliver drugs to the posterior pole of the eye in biologically relevant concentrations.

High-pH or basic/alkaline mobile phases are not commonly used in LC-MS or LC-MS/MS bioanalysis because of the deeply rooted concern with column instability and reduced detection sensitivity for basic compounds in high-pH mobile phases owing to charge neutralization. With the advancement of LC column technology and the wide recognition of the "wrong-way-round" phenomena, high-pH mobile phases are more and more used in LC-MS or LC-MS/MS bioanalysis to improve chromatographic peak shape, retention, selectivity, resolution, and detection sensitivity, not only for basic compounds, but also for many other compounds. In this article, the benefits, practical considerations, application examples and cautions for using high-pH mobile phases in LC-MS or LC-MS/MS bioanalysis are reviewed, with a focus on quantification. Furthermore, the future trends in this field are also envisaged. A total of 84 references are cited in this review.

Background/Aim: Curcumin is being widely investigated for its anticancer properties and studies in the literature suggest that curcumin distributes to a higher degree in tumor versus non-tumor cells. In the current study, we report on investigation of the distribution of curcumin and metabolism to THC in PBMC from healthy individuals and chronic lymphocytic leukemia (CLL) patients following exposure to Lipocurc™ (liposomal curcumin). Materials and Methods: The time and temperature-dependent distribution of liposomal curcumin and metabolism to tetrahydrocurcumin (THC) were measured in vitro in human peripheral blood mononuclear cells (PBMC) obtained from healthy individuals, PBMCHI(cryopreserved and freshly isolated PBMC) and CLL patients (cryopreserved PBMC) with lymphocyte counts ranging from 17-58×106 cells/ml (PBMCCLL,Grp 1) and >150×106 cells/ml (PBMCCLL,Grp 2). PBMC were incubated in plasma protein supplemented media with Lipocurc™ for 2-16 min at 37°C and 4°C and the cell and medium levels of curcumin determined by LC-MS/MS. Results: PBMC from CLL patients displayed a 2.2-2.6-fold higher distribution of curcumin compared to PBMCHICurcumin distribution into PBMCCLL, Grp 1/Grp 2 ranged from 384.75 - 574.50 ng/g w.w. of cell pellet and was greater compared to PBMCHIthat ranged from 122.27-220.59 ng/g w.w. of cell pellet following incubation for up to 15-16 min at 37°C. The distribution of curcumin into PBMCCLL,Grp 2 was time-dependent in comparison to PBMCHIwhich did not display a time-dependence and there was no temperature-dependence for curcumin distribution in either cell type. Curcumin was metabolized to THC in PBMC. The metabolism of curcumin to THC was not markedly different between PBMCHI(range=23.94-42.04 ng/g w.w. cell pellet) and PBMCCLL,Grp 1/Grp 2 (range=23.08-48.22 ng/g. w.w. cell pellet). However, a significantly greater time and temperature-dependence was noted for THC in PBMCCLL,Grp 2 compared to PBMCHIConclusion: Curcumin distribution into PBMC from CLL patients was higher compared to PBMC from healthy individuals, while metabolism to THC was similar. The potential for a greater distribution of curcumin into PBMC from CLL patients may be of therapeutic benefit.

LC-MS/MS has been the dominant analytical technology for quantitative bioanalysis of drugs and metabolites for more than two decades. Despite this, a very fundamental question like how much separation is required for LC-MS/MS quantitative bioanalysis of drugs and metabolites has not been adequately addressed. Some think that no or only very limited separation is necessary thanks to the unparalleled selectivity offered by tandem mass spectrometry. Others think that the more separation, the better, because of the potential detrimental impact of matrix effect (ion suppression or enhancement). Still others just use a rule-of-thumb approach by keeping the adjusted retention/capacity factor always between 2 and 5. The purpose of this article is to address this fundamental question through rational thinking together with various real case examples drawn from regulated bioanalytical laboratories.

A statistical strategy for full analytical validation has been applied to ensure the reliability of a bioanalytical method and to control the risk associated with future use. To this end, the applicability of this approach, called uncertainty profile, has been demonstrated by evaluating the liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) bioanalysis, for the determination of Doxycycline in human plasma. This innovative procedure combines two main concepts, namely analytical validation and measurement uncertainty; its aim is to guarantee that a known amount of future results obtained with the bioanalytical method will be within the acceptance limits set beforehand. In addition, this approach allows to calculate the measurement uncertainty of the method without any additional effort, by using data coming from the analytical validation when we respect as best as possible the intermediate precision conditions at each concentration level. Thus, the LC-MS/MS method for the determination of Doxycycline in human plasma was found to be valid in the studied concentrations range since the tolerance intervals of type β-content, γ-confidence fell into the acceptable limits of ±15%, and the relative expanded uncertainty did not exceed 11% of the values of β-proportion and α-risk equal to 90% and 5% respectively.

Previously reported LC-MS methods for quantifying 8-alpha-hydroxy-mutilin (a marker residue of tiamulin) in tissues all used a pseudo MRM transition (from protonated molecular ion to protonated molecular ion, m/z 337 -> 337) due to difficulties in finding a product ion, leading to suboptimal selectivity and sensitivity for detection. By using electrospray negative ionization in a basic medium, we, for the first time, found a highly selective and sensitive true MRM transition for 8-a-hydroxy-mutilin, m/z 335 -> 179. With this newly found MRM transition and the use of pleuromutilin as the internal standard, a very sensitive, selective, and robust LC-MS/MS method has been developed and validated for quantifying 8-a-hydroxy-mutilin in rabbit tissues (muscle, liver, kidney, and fat). In comparison with the previously published methods, the selectivity and sensitivity were significantly improved. For the concentration range validated (0.2-10 ppm or 0.2-10 mu g/g), the within-run and between-run accuracies (% bias) ranged from -5.0 to 3.1 and -4.9 to 3.0, respectively. The% CV ranged from 2.2 to 6.6 and 4.7 to 8.3 for within-run and between-run precisions, respectively. The validated method was successfully used to support two GLP tissue residue depletion studies in rabbits.

Nonspecific binding (NSB) is an undesirable yet very common phenomenon in LC-MS bioanalysis. This chapter explores how people would even know NSB exists; ways to evaluate its severity; why is NSB particularly problematic for biomarker quantitation; and how it can be differentiated from a seemingly similar stability issue. The questions are addressed using representative application examples from the LC-MS bioanalysis of both small and large molecules. To confirm NSB or evaluate the severity, many different approaches can be taken, such as multiple sequential transfers, the deliberate preparation of small and large volumes, and frequent comparisons with fresh spiking. The key behind these various approaches is to amplify the impact of NSB by exposing the compounds of interest to as large of a surface area as possible and by exposing as many times as possible, so that it will not go unnoticed.

BACKGROUND/AIM:The aim of this study was to investigate the distribution of curcumin (in the form of Lipocurc™) and its major metabolite tetrahydrocurcumin (THC) in Beagle dog and human red blood cells, peripheral blood mononuclear cells (PBMC) and hepatocytes.MATERIALS AND METHODS:Lipocurc™ was used as the source of curcumin for the cell distribution assays. In vitro findings with red blood cells were also compared to in vivo pharmacokinetic data available from preclinical studies in dogs and phase I clinical studies in humans.RESULTS:High levels of curcumin were measured in PBMCs (625.5 ng/g w.w. cell pellet or 7,297 pg/106 cells in dog and 353.7 ng/g w.w. cell pellet or 6,809 pg/106 cells in human) and in hepatocytes (414.5 ng/g w.w. cell pellet or 14,005 pg/106 cells in dog and 813.5 ng/g w.w. cell pellet or 13,780 pg/106 cells in human). Lower curcumin levels were measured in red blood cells (dog: 78.4 ng/g w.w. cell pellet or 7.2 pg/106 cells, human: 201.5 ng/g w.w. cell pellet or 18.6 pg/106 cells). A decrease in the medium concentration of curcumin was observed in red blood cells and hepatocytes, but not in PBMCs. Red blood cell levels of THC were ~5-fold higher in dog compared to human and similar between dog and human for hepatocytes and PBMCs. The ratio of THC to curcumin found in the red blood cell medium following incubation was 6.3 for dog compared to 0.006 for human, while for PBMCs and hepatocytes the ratio of THC to curcumin in the medium did not display such marked species differences.CONCLUSION:There was an excellent correlation between the in vitro disposition of curcumin and THC following incubation with red blood cells and in vivo plasma levels of curcumin and THC in dog and human following intravenous infusion. The disposition of curcumin in blood cells is, therefore, species-dependent and of pharmacokinetic relevance.

Tetrahydrocurcumin (THC), a major metabolite of curcumin, is often quantified by LC-MS or LC-MS/MS using acidic mobile phases due to the concern of its instability in a basic medium. However, acidic mobile phases often lead to poor chromatography (e.g. split or double peaks) and reduced detection sensitivity in the commonly used negative ionization mode. To overcome these shortcomings, a basic mobile phase was used for the first time in the LC-MS/MS quantification of THC. In comparison with the acidic mobile phases, a single symmetrical chromatographic peak was obtained and the sensitivity increased by 7-fold or more under the equivalent conditions. The new LC-MS/MS method using the basic mobile phase has been successfully validated for the quantification of THC in human EDTA plasma over the concentration range of 5-2500ng/ml. The within-batch accuracy (% nominal concentration) was between 88.7 and 104.9 and the between-batch accuracy ranged from 96.7 to 108.6. The CVs for within- and between-batch precisions were equal to or less than 5.5% and 9.1%, respectively. No significant matrix interference or matrix effect was observed from normal or lipemic and hemolytic plasma matrices. In addition, the common stabilities with adequate durations were established, including up to 5days of post-preparative stability. Furthermore, when the validated method was applied to a clinical study, the passing rate of ISR samples was 83%, indicating the good reproducibility of the method. The success of the unconventional approach presented in this article demonstrates that a mobile phase could be selected based mainly on its merits to facilitate LC separation and/or MS detection. There is no need for excessive concern about the stability of the compound(s) of interest in the selected mobile phase because the run time of modern LC-MS or LC-MS/MS methods is typically only a few minutes.

This paper presents the trouble-shooting for a very unusual stability case. Tetracaine was found unstable in neat solutions only at high concentrations, but not at low concentrations. Moreover, its stable-isotope labeled internal standard did not show similar behavior. A series of trouble-shooting experiments were conducted to uncover the root cause. Some generally applicable precautions/insights can be drawn from this investigation to avoid potential stability issues during bioanalytical method development and validation.

Curcumin possesses multiple attractive pharmacological activities and it is being actively developed to treat several serious diseases, e.g. cancer and neurologic disorders. To support drug development programs, sensitive LC-MS methods for curcumin and its metabolite, tetrahydrocurcumin in biological matrices, are essential. However, due to instability, all existing methods have used acidic mobile phases, which led to low sensitivity in the commonly used negative ionization mode, poor chromatography, and split peaks. This is especially problematic for tetrahydrocurcumin because its concentration in various matrices is quite low and it is most subject to these drawbacks. Contrary to conventional thinking, basic mobile phases were evaluated to significantly increase the detection sensitivity and at the same time improve the chromatography for curcumin and tetrahydrocurcumin.

The current approach in regulated LC-MS bioanalysis, which evaluates the precision and trueness of an assay separately, has long been criticized for inadequate balancing of lab-customer risks. Accordingly, different total error approaches have been proposed. The aims of this research were to evaluate the aforementioned risks in reality and the difference among four common total error approaches (β-expectation, β-content, uncertainty, and risk profile) through retrospective analysis of regulated LC-MS projects. Twenty-eight projects (14 validations and 14 productions) were randomly selected from two GLP bioanalytical laboratories, which represent a wide variety of assays. The results show that the risk of accepting unacceptable batches did exist with the current approach (9% and 4% of the evaluated QC levels failed for validation and production, respectively). The fact that the risk was not wide-spread was only because the precision and bias of modern LC-MS assays are usually much better than the minimum regulatory requirements. Despite minor differences in magnitude, very similar accuracy profiles and/or conclusions were obtained from the four different total error approaches. High correlation was even observed in the width of bias intervals. For example, the mean width of SFSTP's β-expectation is 1.10-fold (CV=7.6%) of that of Saffaj-Ihssane's uncertainty approach, while the latter is 1.13-fold (CV=6.0%) of that of Hoffman-Kringle's β-content approach. To conclude, the risk of accepting unacceptable batches was real with the current approach, suggesting that total error approaches should be used instead. Moreover, any of the four total error approaches may be used because of their overall similarity. Lastly, the difficulties/obstacles associated with the application of total error approaches in routine analysis and their desirable future improvements are discussed.