Applicability of bioanalysis of multiple analytes in drug discovery and development: review of select case studies including assay development considerations

The development of sound bioanalytical method(s) is of paramount importance during the process of drug discovery and development culminating in a marketing approval. Although the bioanalytical procedure(s) originally developed during the discovery stage may not necessarily be fit to support the drug development scenario, they may be suitably modified and validated, as deemed necessary. Several reviews have appeared over the years describing analytical approaches including various techniques, detection systems, automation tools that are available for an effective separation, enhanced selectivity and sensitivity for quantitation of many analytes. The intention of this review is to cover various key areas where analytical method development becomes necessary during different stages of drug discovery research and development process. The key areas covered in this article with relevant case studies include: (a) simultaneous assay for parent compound and metabolites that are purported to display pharmacological activity; (b) bioanalytical procedures for determination of multiple drugs in combating a disease; (c) analytical measurement of chirality aspects in the pharmacokinetics, metabolism and biotransformation investigations; (d) drug monitoring for therapeutic benefits and/or occupational hazard; (e) analysis of drugs from complex and/or less frequently used matrices; (f) analytical determination during in vitro experiments (metabolism and permeability related) and in situ intestinal perfusion experiments; (g) determination of a major metabolite as a surrogate for the parent molecule; (h) analytical approaches for universal determination of CYP450 probe substrates and metabolites; (i) analytical applicability to prodrug evaluations-simultaneous determination of prodrug, parent and metabolites; (j) quantitative determination of parent compound and/or phase II metabolite(s) via direct or indirect approaches; (k) applicability in analysis of multiple compounds in select disease areas and/or in clinically important drug-drug interaction studies. A tabular representation of select examples of analysis is provided covering areas of separation conditions, validation aspects and applicable conclusion. A limited discussion is provided on relevant aspects of the need for developing bioanalytical procedures for speedy drug discovery and development. Additionally, some key elements such as internal standard selection, likely issues of mass detection, matrix effect, chiral aspects etc. are provided for consideration during method development.     

An automated blood sampling system to measure lovastatin level in plasma and faeces

The aim of this study was to develop an automated sampling method to measure lovastatin in a conscious and freely moving rat. The blood samples were collected by means of the automated blood sampling system DR-II and the faecal samples were collected using a metabolic cage. The concentration of lovastatin was determined by a reversed-phase liquid chromatographic system with a UV absorbance detector. The mobile phase contained acetonitrile and 10 mm NaH2PO4 in the proportions 60:40 (v/v) with a flow-rate of 1 mL/min. The calibration curve was linear in concentration ranges of 0.05-100 and 0.1-100 microg/mL for lovastatin in blood and faecal samples, respectively. Following pharmacokinetic analysis, we identified that the maximum plasma concentration was around 1.18 +/- 0.08 microg/mL at concentration peak time 120 min and almost 78% of loading dose was accumulated in the faeces within 48 h after lovastatin administration (500 mg/kg, p.o.).     

A validated high-performance liquid chromatographic assay for determination of lumiracoxib in human plasma

Lumiracoxib {2-[(2-fluoro-6-chlorophenyl)amino]-5-methyl-benzeneacetic acid} is a highly selective and potent cyclooxygenase-2 (COX-2) inhibitor, which is chemically distinct from other coxibs in that it contains a carboxylic group and is weakly acidic. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of lumiracoxib in human plasma. The analyte was separated on a reversed-phase column with acetonitrile and 0.05% trichloracetic acid in water (35:65, v/v) as mobile phase at a flow rate of 1 mL/min, and UV detection at 270 nm. The retention times for lumiracoxib and niflumic acid (internal standard) were 16.9 and 10.4 min, respectively. The validated quantitation range for lumiracoxib was 10-10,000 ng/mL. The developed procedure was applied to assess the pharmacokinetics of lumiracoxib following administration of a single oral 200 mg dose to a healthy male volunteer.     

Validation and pharmacokinetic application of a method for determination of doxazosin in human plasma by high-performance liquid chromatography

A simple high-performance liquid chromatographic method for the determination of doxazosin in human plasma was developed and validated. Prazosin was used as internal standard. After extraction twice with ethyl acetate, chromatographic separation of doxazosin in human plasma was carried out using a reversed-phase Apollo C18 column (250 x 4.6 mm, 5 microm) with mobile phase of methanol-acetonitrile-0.04 m disodium hydrogen orthophosphate (22:22:56, v/v/v) adjusted to pH 4.9 with 0.9 m phosphoric acid and quantified by fluorescence detection operated with an excitation wavelength of 246 nm and an emission wavelength of 389 nm. The lower limit of quantification (LLOQ) of this assay was 1 ng/mL using 500 microL human plasma. Linearity was established over the range 1-25 ng/mL (r2 > 0.9994). The intra- and inter-day accuracy ranged from 90.5 to 104.4% and the coefficient of variation were not more than 8.6% for both intra- and inter-day precision, over the range of the calibration curve. The absolute recoveries of doxazosin and prazosin from human plasma were more than 91%. Doxazosin demonstrated acceptable short-term, long-term and freeze-thaw stability in human plasma. The assay has been successfully applied to plasma sample ana-lysis for pharmacokinetic study.     

Simple determination of pirfenidone in rat plasma via high-performance liquid chromatography

A simple, rapid and reliable high-performance liquid chromatographic method was developed and validated for the determination of pirfenidone and its major metabolites in rat plasma. Plasma proteins were precipitated with perchloric acid (10%, v/v) and the supernatant after centrifugation was determined using high-performance liquid chromatography. The analysis was carried out on a Lichrospher C(18) column (250 x 4.6 mm i.d., 5 microm). The mobile phase consisted of acetonitrile-water containing 0.2% acetic acid (23:77, v/v) at a flow-rate of 1 mL/min. The eluant was detected at 310 nm. The calibration curves were linear over a concentration range from 0.15 to 76.67 microg/mL. The accuracy (relative error) of the assay ranged from -2.6 to 7.9% and the precision (coefficient of variation) was less than 4.5%. The established method has been successfully applied to a pharmacokinetic study of pirfenidone following a single oral dose to rats.     

Species Differences in Metabolism of Soluble Epoxide Hydrolase Inhibitor,EC1728, Highlight the Importance of Clinically Relevant Screening Mechanisms in Drug Development

There are few novel therapeutic options available for companion animals, and medications rely heavily on repurposed drugs developed for other species. Considering the diversity of species and breeds in companion animal medicine, comprehensive PK exposures in the companion animal patient is often lacking. The purpose of this paper was to assess the pharmacokinetics after oral and intravenous dosing in domesticated animal species (dogs, cats, and horses) of a novel soluble epoxide hydrolase inhibitor, EC1728, being developed for the treatment of pain in animals. Results: Intravenous and oral administration revealed that bioavailability was similar for dogs, and horses (42 and 50% F) but lower in mice and cats (34 and 8%, respectively). Additionally, clearance was similar between cats and mice, but >2× faster in cats vs. dogs and horses. Efficacy with EC1728 has been demonstrated in mice, dogs, and horses, and despite the rapid clearance of EC1728 in cats, analgesic efficacy was demonstrated in an acute pain model after intravenous but not oral dosing. Conclusion: These results demonstrate that exposures across species can vary, and investigation of therapeutic exposures in target species is needed to provide adequate care that addresses efficacy and avoids toxicity.     

A liquid chromatography–tandem mass spectrometry method for the quantitation of actarit in rabbit plasma: application to pharmacokinetics and metabolic stability

Actarit (ATR), 4‐acetylaminophenylacetic acid is an orally effective disease‐modifying anti‐rheumatic drug widely prescribed for the treatment of rheumatoid arthritis. The present study demonstrates the first report on a selective and sensitive liquid chromatography–tandem mass spectrometry method for the quantification of ATR in rabbit plasma using p‐coumaric acid as an internal standard (IS). Following liquid–liquid extraction, chromatographic separation of the reconstituted samples was achieved isocratically on a Syncronis‐C18 column with a mobile phase consisting of aqueous ammonium acetate (10 mM, pH 4)‐ methanol and acetonitrile mixture (8 : 92, v/v) at a flow rate of 0.6 ml/min. ATR and IS were detected using electrospray ionization operated in negative multiple reaction monitoring mode. The calibration curve was linear (r² ≥ 0.990) over the concentration range of 1–4000 ng/ml with a lower limit of quantitation of 1 ng/ml. The mean extraction recovery of ATR and IS from rabbit plasma was greater than 85%. The method complied well with US Food and Drug Administration guidelines for selectivity, sensitivity, accuracy, precision, matrix effect, dilution integrity, carry‐over effect and stability. The method was successfully applied to in vitro metabolic stability (using rabbit liver microsomes) and in vivo pharmacokinetic study after oral administration of ATR at a dose of 10 mg/kg in New Zealand rabbits. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparative pharmacokinetics of prim-O-glucosylcimifugin and cimifugin by liquid chromatography-mass spectrometry after oral administration of Radix Saposhnikoviae extract, cimifugin monomer solution and prim-O-glucosylcimifugin monomer solution to rats

A sensitive and reliable liquid chromatography–mass spectrometry method has been developed and validated for simultaneous determination of cimifugin and prim‐O‐glucosylcimifugin in rat plasma after oral administration of Radix Saposhnikoviae (RS) extract, prim‐O‐glucosylcimifugin monomer solution and cimifugin monomer solution. Plasma samples were pretreated by protein precipitation with acetonitrile containing the internal standards puerarin and daidzein. LC separation was achieved on a Zorbax SB‐C₁₈ column (150 × 4.6 mm i.d., 5 µm) with 0.1% formic acid in water and methanol by isocratic elution. The detection was carried out in select‐ion‐monitoring mode with a positive electrospray ionization interface. The fully validated method was successfully applied to the pharmacokinetic study of the analytes in rats. A bimodal phenomenon appeared in the concentration–time curve of prim‐O‐glucosylcimifugin and cimifugin after oral administration of RS extract. Prim‐O‐glucosylcimifugin mainly transformed to cimifugin when it was absorbed into blood. Both absorption and elimination of cimifugin after oral administration of RS were longer than after administration of single cimifugin. The pharmacokinetic parameters (AUC_0–t, AUC_0–∞ and t_1/2) of prim‐O‐glucosylcimifugin and cimifugin by giving cimifugin monomer solution, prim‐O‐glucosylcimifugin monomer solution and RS extract had significant differences (P < 0.05). Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitation of glucosamine sulfate in plasma by HPLC-MS/MS after administration of powder for oral solution formulation

A rapid method for the quantification of glucosamine in human plasma using high‐performance liquid chromatography coupled to tandem mass spectrometry was developed and validated. The sample preparation includes a simple deproteinization step, using d ‐[1‐¹³C] glucosamine hydrochloride as an internal standard. Chromatographic separation was performed on an ACE Ciano column using isocratic elution with acetonitrile and aqueous 2 mm ammonium acetate containing 0.025% formic acid (80:20). Selected reaction monitoring was performed using the transitions m/z 180.1 → m/z 72.1 and m/z 181.0 → m/z 74.6 to quantify glucosamine and internal standard, respectively. The method was validated and proved to be linear, accurate and precise over the range 50–5000 ng/mL of glucosamine. Recovery rates higher than 90% were obtained for both glucosamine and internal standard. No matrix effect was detected in the samples. The validated method was successfully applied to a pharmacokinetic study after oral administration of a powder for oral solution formulation containing glucosamine sulfate. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of atractylenolide II and atractylenolide III by liquid chromatography-tandem mass spectrometry in rat plasma and its application in a pharmacokinetic study after oral administration of Atractylodes Macrocephala Rhizoma extract

Atractylenolide II (AII) and atractylenolide III (AIII) are the major active components in Atractylodes Macrocephala Rhizoma (AMR). In this study, a sensitive, rapid and selective liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous determination of AII and AIII in rat plasma using loliolide as internal standard (IS). After protein precipitation with ethyl acetate, the analytes were injected into an LC‐MS/MS system for quantification. Chromatography was performed using a C₁₈ column, eluting with water and acetonitrile (45:55, v/v) at 0.2 mL/min. All analytes including IS were monitored under positive ionization conditions by multiple reaction monitoring with an electrospray ionization source. The validated method was successfully applied to the pharmacokinetic study of AII and AIII in rat plasma after oral administration of AMR extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine. Copyright © 2012 John Wiley & Sons, Ltd.