Characterization of Five Oxidative Degradation Impurities and One Process Impurity of Suvorexant Drug Substance by LC-MS/MS, HR-MS and 1D, 2D NMR: Validation of Suvorexant Drug Substance and Process Impurities by HPLC and UPLC.

During the oxidative (10% H2O2) degradation of suvorexant drug substance, around 1.0% of one impurity and less than 1.0% four impurities were found by a new high-performance liquid chromatography (HPLC) assay and related substance method. The mass numbers of 1.0% impurity was 469 [M + H]+, remaining four impurities were 172 [M + H]+, 467 [M + H]+, 483 [M + H]+ and 485 [M + H]+. The 469 [M + H]+, 485[M + H] and 172 [M + H]+ impurities were characterized by using the LC-MS/MS, HR-MS and 1D, 2D NMR spectroscopic data. The 172 [M + H]+ impurity was prepared synthetically and co-injected in HPLC. The retention time of synthesized 172 [M + H]+ impurity was matching with the unknown degradation impurity in HPLC. The developed mass compatible HPLC and ultra performance liquid chromatography methods were validated for drug substance and process impurities by following ICH Q2 (R1) guidelines.

Study of degradation behavior of besifloxacin, characterization of its degradation products by LC-ESI-QTOF-MS and their in silico toxicity prediction.

Knowledge and understanding of the stability profile of a drug is important as it affects its safety and efficacy. In the present work, besifloxacin, a new, fourth-generation fluoroquinolone antibiotic, was subjected to different forced-degradation conditions as per International Conference on Harmonization (ICH) guidelines such as hydrolysis (acid, base and neutral), oxidation, thermal and photolysis. The drug degraded under acidic, basic, oxidative and photolytic conditions while it was found to be stable under dry heat and neutral hydrolytic conditions. In total, five degradation products (DPs) were formed under different conditions-DP1 and DP2 (photolysis), DP3 (oxidation), DP4 (acidic), DP3 and DP5 (basic). The chromatographic separation of besifloxacin and its degradation products was achieved on a Sunfire C18 (250 mm × 4.6 mm, 5 µm) column with 0.1% aqueous formic acid-acetonitrile as a mobile phase. The gradient RP-HPLC method was developed and validated as per ICH guidelines. The degradation products were characterized with the help of LC-ESI-QTOF mass spectrometric studies and the most likely degradation pathway of the drug was proposed. In silico toxicity assessment of the drug and its degradation products was carried out, which indicated that DP3 and DP4 carry a mutagenicity alert.

Besifloxacin: A Critical Review of Its Characteristics, Properties, and Analytical Methods.

Bacterial conjunctivitis has high impact on the health of the population, since it represents more than a third of ocular pathologies reported by health services worldwide. There is a high incidence of bacterial resistance to the antimicrobials most commonly used for the treatment of conjunctivitis. In this context, besifloxacin stands out, since it is a fluoroquinolone developed exclusively for topical ophthalmic use, presenting a low risk of developing resistance due to its reduced systemic exposure. Bausch & Lomb markets it as ophthalmic suspension, under the trade name Besivance™. Literature review on besifloxacin is presented, covering its pharmaceutical and clinical characteristics, and the analytical methods used to measure the drug in pharmaceutical products and biological samples. High performance liquid chromatography is the most used method for this purpose. A discussion on Green Chemistry is also presented, focusing the importance of the development of green analytical methods for the analysis of drugs.

Tissue Distribution of Suvorexant in Three Forensic Autopsy Cases.

Suvorexant (Belsomra®) is a relatively new insomnia medication that has been available in USA and Japan since 2014. It is a dual orexin receptor antagonist that promotes sleep by inhibiting the binding of orexin neurons to the OX1R and OX2R receptors. In this report, we describe the detection and quantitation of suvorexant from the postmortem specimens of three separate autopsy cases handled by our department. Suvorexant was identified by fast gas chromatography/mass spectrometry during routine screening, and quantitated by a fully validated liquid chromatography-tandem mass spectroscopy method. Quantitation was achieved by positive electrospray ionization in the selected reaction monitoring mode. Monitored transitions were m/z 451 > 186 for quantitation and m/z 451 > 104 for qualification. To our knowledge, this is the first instance of suvorexant being quantitated from actual autopsy cases. It is likely that this compound will be encountered more often by the forensic toxicology community going forward.

Quantification of highly selective sigma-1 receptor antagonist CM304 using liquid chromatography tandem mass spectrometry and its application to a pre-clinical pharmacokinetic study.

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for quantification of CM304, a novel and highly selective sigma-1 receptor antagonist that has recently entered into human clinical trials. A structural analogue of CM304, SN56, was used as the internal standard (IS). Chromatographic separation was achieved on an Acquity UPLC™ BEH C18 (1.7 µm, 2.1 mm × 50 mm) column using a mobile phase [water:methanol (0.1%v/v formic acid; 50:50, %v/v)] at a flow rate of 0.2 mL/min. Mass spectrometric detection was performed in the positive ionization mode with multiple reaction monitoring (MRM) using m/z transitions of 337 > 238 for CM304 and 319 > 220 for the IS. The method was found to be linear and reproducible with a regression coefficient consistently >0.99 for the calibration range of 3 to 3000 ng/mL. The extraction recovery ranged from 91.5 to 98.4% from spiked (7.5, 300 and 2526 ng/mL) plasma quality control samples. The precision (%RSD; 1.1 to 2.9%) and accuracy (%RE; -1.9 to 1.8%) were within acceptable limit. The validated method was successfully applied to a single dose oral and intravenous (I.V.) pharmacokinetic study of CM304 in rats. Following I.V. administration, the compound exhibited adequate exposure along with high extravascular distribution and insignificant amount of extra hepatic metabolism. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous quantification of gatifloxacin, moxifloxacin, and besifloxacin concentrations in cornea and aqueous humor by LC-QTOF/MS after topical ocular dosing.

The fourth-generation fluoroquinolones are widely used as ophthalmic antimicrobials. This study aimed to validate a new analytical technique for simultaneous quantification of gatifloxacin, moxifloxacin, and besifloxacin concentrations in the cornea and aqueous humor by liquid chromatography (LC) coupled to quadrupole time-of-flight mass spectrometry (QTOF/MS) at 10min and 1h after instillation of topical ophthalmic antimicrobial suspensions. It was used twenty-two male dogs without ocular lesions verified by ophthalmic and histologic examinations. Methanol:water (4:1) was used for the extraction procedure for cornea and acetonitrile:water (4:1) was used for aqueous humor. The chromatographic separations were carried out on a C18 column with a linear gradient of water and methanol, both containing 0.1% formic acid. The total chromatographic run time was 4min. Mass spectrometry analyses were performed on a Xevo™ G2-S QTof tandem mass spectrometer, operated in a positive ion electrospray ionization (ESI+) mode. The retention times were approximately 1.42min for gatifloxacin, 1.87min for moxifloxacin, and 3.01min for besifloxacin. No interference peak was detected for the three tested antimicrobials in samples obtained from both cornea and aqueous humor, ensuring that the peak response was exclusive to the analyte of interest. The limit of detection for the three antimicrobials was 0.11µg/mL and the limit of quantification was 0.42µg/mL for both cornea and aqueous humor samples. At both time points post instillation of the three antimicrobials, moxifloxacin had the highest corneal concentration and besifloxacin demonstrated the highest concentration in the aqueous humor.

Simple Isocratic HPLC Method for Determination of Enantiomeric Impurity in Besifloxacin Hydrochloride.

Besifloxacin is a unique chiral broad-spectrum flouroquinolone used in the treatment of bacterial conjunctivitis. R-form of besifloxacin hydrochloride shows higher antibacterial activity as compared to the S-isomer. Therefore, it is necessary to establish chiral purity. To establish chiral purity a high-performance liquid chromatography (HPLC) method for determination of R-besifloxacin and S-besifloxacin (BES impurity A) was developed and validated for in-process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD), and lower limit of quantification (LOQ) were determined according to International Council for Harmonization ICH Q2(R1) guidelines. HPLC separation was achieved on Chiralpak AD-H (250 x 4.6 mm, 5 µm) column using n-heptane: ethanol: ethylenediamine: acetic acid (800:200:0.5:0.5) (v/v/v/v) as the mobile phase in an isocratic elution. The eluents were monitored by UV/Visible detector at 290 nm. The resolution between S-isomer and besifloxacin hydrochloride was more than 2.0. Based on a signal-to-noise ratio of 3 and 10 the LOD of besifloxacin was 0.30 µg/mL, while the LOQ was 0.90 µg/mL. The calibration curves were linear in the range of 0.9-7.5 µg/mL. Precision of the method was established within the acceptable range. The method was suitable for the quality control enantiomeric impurity in besifloxacin hydrochloride. Chirality 28:628-632, 2016. © 2016 Wiley Periodicals, Inc.

Production of microparticles of molinate degrading biocatalysts using the spray drying technique.

Previous studies demonstrated the capability of mixed culture DC1 to mineralize the thiocarbamate herbicide molinate through the activity of molinate hydrolase (MolA). Because liquid suspensions are not compatible with long-term storage and are not easy to handle when bioremediation strategies are envisaged, in this study spray drying was evaluated as a cost-effective method to store and transport these molinate biocatalysts. Microparticles of mixed culture DC1 (DC1) and of cell free crude extracts containing MolA (MA) were obtained without any carrier polymer, and with calcium alginate (CA) or modified chitosan (MCt) as immobilizing agents. All the DC1 microparticles showed high molinate degrading activity upon storage for 6 months, or after 9 additions of ∼0.4 mM molinate over 1 month. The DC1-MCt microparticles were those with the highest survival rate and lowest heterogeneity. For MA microparticles, only MA-MCt degraded molinate. However, its Vmax was only 1.4% of that of the fresh cell free extract (non spray dried). The feasibility of using the DC1-MCt and MA-MCt microparticles in bioaugmentation processes was assessed in river water microcosms, using mass (g):volume (L) ratios of 1:13 and 1:0.25, respectively. Both type of microparticles removed ∼65-75% of the initial 1.5 mg L(-1) molinate, after 7 days of incubation. However, only DC1-MCt microparticles were able to degrade this environmental concentration of molinate without disturbing the native bacterial community. These results suggest that spray drying can be successfully used to produce DC1-MCt microparticles to remediate molinate polluted sites through a bioaugmentation strategy.

Assessment of combined electro-nanoremediation of molinate contaminated soil.

Molinate is a pesticide widely used, both in space and time, for weed control in rice paddies. Due to its water solubility and affinity to organic matter, it is a contaminant of concern in ground and surface waters, soils and sediments. Previous works have showed that molinate can be removed from soils through electrokinetic (EK) remediation. In this work, molinate degradation by zero valent iron nanoparticles (nZVI) was tested in soils for the first time. Soil is a highly complex matrix, and pollutant partitioning between soil and water and its degradation rates in different matrices is quite challenging. A system combining nZVI and EK was also set up in order to study the nanoparticles and molinate transport, as well as molinate degradation. Results showed that molinate could be degraded by nZVI in soils, even though the process is more time demanding and degradation percentages are lower than in an aqueous solution. This shows the importance of testing contaminant degradation, not only in aqueous solutions, but also in the soil-sorbed fraction. It was also found that soil type was the most significant factor influencing iron and molinate transport. The main advantage of the simultaneous use of both methods is the molinate degradation instead of its accumulation in the catholyte.

Shiga-like toxin B subunit of Escherichia coli as scaffold for high-avidity display of anti-immunocomplex peptides.

Small compounds cannot bind simultaneously to two antibodies, and thus, their immunodetection is limited to competitive formats in which the analyte is indirectly quantitated by measuring the unoccupied antibody binding sites using a competing reporter. This limitation can be circumvented by using phage-borne peptides selected for their ability to specifically react with the analyte-antibody immunocomplex, which allows the detection of these small molecules in a noncompetitive format (PHAIA) with increased sensitivity and a positive readout. In an effort to find substitutes for the phage particles in PHAIA, we explore the use of the B subunit of the Shiga-like toxin of Escherichia coli, also known as verotoxin (VTX), as a scaffold for multivalent display of anti-immunocomplex peptides. Using the herbicides molinate and clomazone as model compounds, we built peptide-VTX recombinant chimeras that were produced in the periplasmic space of E. coli as soluble pentamers, as confirmed by multiangle light scattering analysis. These multivalent constructs, which we termed nanopeptamers, were conjugated to a tracer enzyme and used to detect the herbicide-antibody complex in an ELISA format. The VTX-nanopeptamer assays performed with over a 10-fold increased sensitivity and excellent recovery from spiked surface and mineral water samples. The carbon black-labeled peptide-VTX nanopeptamers showed great potential for the development of a lateral-flow test for small molecules with a visual positive readout that allowed the detection of up to 2.5 ng/mL of clomazone.

Quantitative evaluation of besifloxacin ophthalmic suspension by HPLC, application to bioassay method and cytotoxicity studies.

Besifloxacin (BSF) is a synthetic chiral fluoroquinolone developed for the topical treatment of ophthalmic infections. The present study reports the development and validation of a microbiological assay, applying the cylinder-plate method, for determination of BSF in ophthalmic suspension. To assess this methodology, the development and validation of the method was performed for the quantification of BSF by high performance liquid chromatography (HPLC). The HPLC method showed specificity, linearity in the range of 20-80 µg mL(-1) (r=0.9998), precision, accuracy and robustness. The microbiological method is based on the inhibitory effect of BSF upon the strain of Staphylococcus epidermidis ATCC 12228 used as a test microorganism. The bioassay validation method yielded excellent results and included linearity, precision, accuracy, robustness and selectivity. The assay results were treated statistically by analysis of variance (ANOVA) and were found to be linear (r=0.9974) in the range of 0.5-2.0 µg mL(-1), precise (inter-assay: RSD=0.84), accurate (101.4%), specific and robust. The bioassay and the previously validated high performance liquid chromatographic (HPLC) method were compared using Student's t test, which indicated that there was no statistically significant difference between these two methods. These results confirm that the proposed microbiological method can be used as routine analysis for the quantitative determination of BSF in an ophthalmic suspension. A preliminary stability study during the HPLC validation was performed and demonstrated that BSF is unstable under UV conditions. The photodegradation kinetics of BSF in water showed a first-order reaction for the drug product (ophthalmic suspension) and a second-order reaction for the reference standard (RS) under UVA light. UVA degraded samples of BSF were also studied in order to determine the preliminary in vitro cytotoxicity against mononuclear cells. The results indicated that BSF does not alter the cell membrane and has been considered non-toxic to human mononuclear cells in the experimental conditions tested.

Liquid chromatographic determination of CPZEN-45, a novel anti-tubercular drug, in biological samples.

CPZEN-45 is a new drug candidate being considered for the treatment of tuberculosis (TB). The aim of this study was to develop and validate a reverse-phase high-performance liquid chromatographic (HPLC) method suitable to determine CPZEN-45 concentrations in biological samples. CPZEN-45 was extracted from biological fluids and tissues (plasma, lung and spleen from guinea pig) by sequential extraction with acetonitrile and quantified by a Waters HPLC Alliance System coupled with a ZORBAX Bonus-RP column, guard column and UV detection at 263nm. The mobile phase was 20:80 acetonitrile:ultrapure-water with 0.05% TFA. The CPZEN-45 peak was eluted at 5.1min with no interference from the inherent peaks of plasma, bronchoalveolar lavage (BAL), lung or spleen tissues. Recovery of CPZEN-45 from biological samples was >96% of the spiked amount. The limit of detection was 0.05µg/ml and the limit of quantitation was 0.29µg/ml which was more than 5 and 21 times lower than the reported minimal inhibitory concentration of CPZEN-45 (MIC=1.56µg/ml for Mycobacterium tuberculosis and 6.25µg/ml for MDR-TB, respectively). Thus, HPLC method was deemed reliable, sensitive, reproducible and accurate for the determination of CPZEN-45 concentrations in plasma, BAL, lung and spleen tissues. Therefore, this method was used in subsequent studies in the guinea pig model to determine the disposition of CPZEN-45 after administration of solutions by the IV and SC routes.

Molinate quantification in environmental water by a glutathione-S-transferase based biosensor.

A glutathione-S-transferase (GST) based biosensor was developed to quantify the thiocarbamate herbicide molinate in environmental water. The biosensor construction was based on GST immobilization onto a glassy carbon electrode via aminosilane-glutaraldehyde covalent attachment. The principle supporting the use of this biosensor consists of the GST inhibition process promoted by molinate. Differential pulse voltammetry was used to obtain a calibration curve for molinate concentration, ranging from 0.19 to 7.9 mg L(-1) and presenting a detection limit of 0.064 mg L(-1). The developed biosensor is stable, and reusable during 15 days. The GST-based biosensor was successfully applied to quantify molinate in rice paddy field floodwater samples. The results achieved with the developed biosensor were in accordance with those obtained by high performance liquid chromatography. The proposed device is suitable for screening environmental water analysis and, since no sample preparation is required, it can be used in situ and in real-time measurements.

Simultaneous quantification of stemocurtisine, stemocurtisinol and stemofoline in Stemona curtisii (Stemonaceae) by TLC-densitometric method.

Stemona curtisii Hook. F. (Family Stemonaceae), a prominent species distributed in the south and southwest of Thailand, has widely been used as a natural pesticide and as treatment for head lice and skin diseases. This study developed a thin-layer chromatography (TLC)-densitometric method for the simultaneous quantification of major components--stemocurtisine, stemocurtisinol and stemofoline--in the extracts from the roots of S. curtisii collected from 10 locations in Thailand. Components were found in the ranges of 0.0353-0.1949, <0.0121-0.0859 and 0.0733-0.1689 percent dry weight, respectively. The method was validated for linearity, precision, accuracy, robustness, limit of detection and limit of quantitation. The linearity was found over the range of 40-320 ng/spot with a good correlation coefficient (r > 0.9866). Intra-day and inter-day precision showed a relative standard deviation of less than 6%. The accuracy of the method was determined by a recovery study, and the average recoveries were 100.4, 100.2 and 100.3% for stemocurtisine, stemocurtisinol and stemofoline, respectively. The proposed TLC-densitometric method was found to be simple, precise, specific and inexpensive, and can be used simultaneously for the routine quality control of raw materials of S. curtisii roots, extracts and their products, and also other products containing these markers.

Nanopeptamers for the development of small-analyte lateral flow tests with a positive readout.

There is a great demand for rapid tests that can be used on-site for the detection of small analytes, such as pesticides, persistent organic pollutants, explosives, toxins, medicinal and abused drugs, hormones, etc. Dipsticks and lateral flow devices, which are simple and provide a visual readout, may be the answer, but the available technology for these compounds requires a competitive format that loses sensitivity and produces readings inversely proportional to the analyte concentration, which is counterintuitive and may lead to potential misinterpretation of the result. In this work, protein-multipeptide constructs composed of anti-immunocomplex peptides selected from phage libraries and streptavidin/avidin as core protein were used for direct detection of small compounds in a noncompetitive two-site immunoassay format that performs with increased sensitivity and positive readout. These constructs that we termed "nanopeptamers" allow the development of rapid point-of-use tests with a positive visual end point of easy interpretation. As proof of concept, lateral flow assays for the herbicides molinate and clomazone were developed and their performance was characterized with field samples.

Determination of enantiomeric impurity in besifloxacin hydrochloride by chiral high-performance liquid chromatography with precolumn derivatization.

Besifloxacin hydrochloride is a novel chiral broad-spectrum fluoroquinolone developed for the treatment of bacterial conjunctivitis. R-besifloxacin hydrochloride is used in clinics as a consequence of its higher antibacterial activity. To establish an enantiomeric impurity determination method, some chiral stationary phases (CSPs) were screened. Besifloxacin enantiomers can be separated to a certain extent on Chiral CD-Ph (Shiseido Co., Ltd., Japan), Chiral AGP, and Crownpak CR (+) (Daicel Chemical IND., Ltd., Japan). However, the selectivity and sensitivity were both unsatisfactory on these three CSPs. Therefore, Chiral AGP, Chiral CD-Ph, and Crownpak CR (+) were not used in the enantiomeric impurity determination of besifloxacin hydrochloride. The separation of enantiomers of besifloxacin was further performed using a precolumn derivatization chiral high-performance liquid chromatography method. 2,3,4,6-Tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate was used as the derivatization reagent. Besifloxacin enantiomer derivates were well separated on a C(18) column (250 × 4.6 mm, 5 µm) with a mobile phase that consisted of methanol-KH(2)PO(4) buffer solution (20 mM; pH 3.0) (50:50, v/v). Selectivity, sensitivity, linearity, accuracy, precision, stability, and robustness of this method were all satisfied with the method validation requirement. The method was suitable for the quality control of enantiomeric impurity in besifloxacin hydrochloride.

Ultra-sensitive electrochemical immunosensor using analyte peptidomimetics selected from phage display peptide libraries.

Immunosensors for small analytes have been a great addition to the analytical toolbox due to their high sensitivity and extended analytical range. In these systems the analyte is detected when it competes for binding to the detecting antibody with a tracer compound. In this work we introduce the use of phage particles bearing peptides that mimic the target analyte as surrogates for conventional tracers. As a proof of concept, we developed a magneto-electrochemical immunosensor (EI) for the herbicide molinate and compare its performance with conventional formats. Using the same anti-molinate antibody and phage particles bearing a molinate peptidomimetic, the EI performed with an IC(50) of 0.15 ngmL(-1) (linear range from 4.4 × 10(-3) to 10 ngmL(-1)). Compared to the conventional ELISA, the EI was faster (minutes), performed with a much wider linear range, and the detection limit that was 2500-fold lower. The EI produced consistent measurements and could be successfully used to assay river water samples with excellent recoveries. By using the same EI with a conventional tracer, we found that an important contribution to the gain in sensitivity is due to the filamentous structure of the phage (9 × 1000 nm) which works as a multienzymatic tracer, amplifying the competitive reaction. Since phage-borne peptidomimetics can be selected from phage display libraries in a straightforward systematic manner and their production is simple and inexpensive, they can contribute to facilitate the development of ultrasensitive biosensors.

High-throughput polymer monolith in-tip SPME fiber preparation and application in drug analysis.

BACKGROUND: A simple, low-cost and reproducible automated procedure has been developed to prepare in-tip solid-phase microextraction (SPME) fibers coated with polymer monoliths using a photopolymerization technique. Up to 96 fibers were prepared at one time using a polymerization mixture consisting of ethylene glycol dimethacrylate, dimethoxy-α-phenylacetophenone and 1-decanol. RESULTS: The optimization procedures that affected polymer morphology, such as compositions of the crosslinkers and porogens, polymerization time and fiber thickness as well as extraction efficiency of the immobilized Oasis hydrophilic-lipophilic-balanced extraction sorbent were investigated. Also, the reproducibility of automated in-tip SPME fiber preparation, as well as sample process parameters, such as sample extraction and desorption volumes, are discussed. CONCLUSION: The performance of the polymer monoliths in-tip SPME assessed with a model drug compound from clinical studies and a head-to-head comparison using in-tip SPME and conventional SPE clearly demonstrated that the SPME is a feasible approach for routine drug analysis in the pharmaceutical industry.

Development and validation of a liquid chromatography method with electrospray ionization tandem mass spectrometry for the determination of brotizolam residues in beef and commercial whole milk.

In this work, a liquid chromatography-tandem mass spectrometric detection technique was developed and validated for the determination of brotizolam residues in beef muscle and commercial whole milk. This procedure involves the extraction of the analyte from the samples via liquid-solid extraction, and caffeine was used as an internal standard. The analyte was successfully separated on an XTerra-C(18) column, with a mobile phase composed of 0.01% formic acid in acetonitrile and 1 mm ammonium formate-0.01% formic acid in water. The one-step extraction method evidenced good selectivity, precision (RSD = 9.87-26.47%), and the recovery of the extractable analyte was 92.61-115.98% in the matrices. The limits of quantification ranged between 0.4 and 0.5 µg/kg. The developed method is simple since it requires no additional cleanup procedures.