Quality assessment of fluoxetine and fluvoxamine pharmaceutical formulations purchased in different countries or via the Internet by 19F and 2D DOSY 1H NMR

A simple and selective (19)F NMR method has been validated for the quantitation of fluoxetine (FLX) and fluvoxamine (FLV) in methanol solutions and in human plasma and urine. The regression equations for FLX and FLV showed a good linearity in the range of 1.4-620 microg mL(-1) (3.3 x 10(-6)-1.8 x 10(-3) mol L(-1)) with a limit of detection of approximately 0.5 microg mL(-1) (1.3 x 10(-6) mol L(-1)) and a limit of quantification of approximately 2 microg mL(-1) (4.6 x 10(-6) mol L(-1)). The precision of the assay depends on the concentrations and is comprised between 1.5 and 9.5% for a range of concentrations between 1.2 x 10(-3) and 3.2 x 10(-6) mol L(-1). The accuracy evaluated through recovery studies ranged from approximately 96 to 103% in methanol solutions and in urine, and from approximately 93 to 104% in plasma, with standard deviations <7.5%. The low sensitivity of the method precludes its use for the assay of these antidepressants in biofluids at least at therapeutic doses as the ranges of FLX and FLV plasma levels are 0.15-0.5 microg mL(-1) and 0.15-0.25 microg mL(-1), respectively. The method was applied successfully to the determination of FLX and FLV contents in pharmaceutical samples (brand-named and generic) purchased in several countries or via the Internet. All the commercial formulations contain the active ingredient in the range 94-103% of stated concentration. A "signature" of the formulations (solid and liquid) was obtained with 2D Diffusion-Ordered SpectroscopY (DOSY) (1)H NMR which allowed the characterisation of the active ingredient and excipients present in the formulations studied. Finally, the DOSY separation of FLX and FLV whose molecular weights are very close was obtained by using beta-cyclodextrin as host-guest complexing agent.     

Generic ciprofloxacin tablets contain the stated amount of drug and different impurity profiles: A 19F, 1H and DOSY NMR analysis

The objective of this study was to control the purity of 16 commercial formulations of ciprofloxacin tablets purchased in different countries or via the Internet using 19F and 1H nuclear magnetic resonance (NMR). Twelve out of the sixteen commercial formulations of ciprofloxacin measured by 19F NMR contain the active ingredient within 100+/-5% of stated concentration. Three formulations have a lower ciprofloxacin content between 90 and 95% and one shows a higher concentration superior to 105%. The impurity profile was characterised using 19F and 1H NMR, and is characteristic of the manufacturer. Four to twelve fluorinated impurities among them fluoride ion and two already known compounds were detected and quantified in the sixteen formulations analysed by 19F NMR. Two other non-fluorinated impurities were observed in the seven formulations analysed with 1H NMR. The total content of impurities as well as their individual levels are in agreement with those reported previously in the few studies devoted to ciprofloxacin purity. However, all the formulations do not comply with the limits for impurities given in the ciprofloxacin monograph of the European Pharmacopeia. Finally, a "signature" of the formulations was obtained with Diffusion-Ordered SpectroscopY (DOSY) 1H NMR which allowed the characterisation of some excipients present in the formulations studied.     

Quantifying trifluoroacetic acid as a counterion in drug discovery by 19F NMR and capillary electrophoresis

Drug discovery compounds are often isolated as salts of trifluoroacetate from preparative high performance liquid chromatography, which are then used for biological assays in order to assess their efficacy against the biochemical target of interest. It is, therefore, imperative to determine the TFA content in order to ascertain the correct formula weight and when required, to ensure that the TFA has been completely exchanged for another counterion in order to have superior pharmacokinetic properties and to avoid potential toxicity effects. In this paper, we present capillary electrophoresis and (19)F nuclear magnetic resonance methods for determining the TFA content of drug discovery compounds. Furthermore, these methods have been successfully applied in a high-throughput fashion, which is a key feature for general applicability in a pharmaceutical setting.     

1H and 19F NMR relaxation studies of fleroxacin with Micrococcus luteus

In order to investigate and characterize interaction processes between the fluoroquinolone fleroxacin and bacterial cells we used non-selective (all resonances are excited), selective (observed resonance is excited) spin-lattice relaxation rates and spin-spin relaxation measurements. The signals of three hydrogens at different moieties of the fleroxacin molecule were considered to get an insight in the complexation behavior. The enhancement of selective relaxation rates was observed with increasing fleroxacin concentrations and keeping the bacterial mass constant. The obtained relaxation rates of the affected hydrogens were analyzed via a Lineweaver-Burk-plot to determine the KD values. Furthermore, 19F NMR spectra were recorded and spin-spin relaxation rates (R2) were determined by a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence. Because of the dependency of the line width of NMR peaks on transversal relaxation time T2, we compared the line width at half-height at different fleroxacin concentrations in order to investigate the involvement of fluorine atoms in different positions in the complexation. All findings point to core quinolone moiety to be involved in the interaction with bacterial cells.     

In vivo 19F nuclear magnetic resonance spectroscopy: a potential monitor of 5-fluorouracil pharmacokinetics and metabolism

Summary In vivo ¹⁹F nuclear magnetic resonance (NMR) spectroscopy has the potential to non-invasively measure the concentration of 5-fluorouracil (FUra) and some of its metabolites in humans. Such a measure could be useful in predicting and optimizing the response of individual patients treated with FUra. The ability of ¹⁹F NMR to monitor FUra metabolism in situ in rodent tumors and liver and in human liver has been demonstrated. However, the potential impact of this technique as a predictor of FUra response in individual patients is limited by both the sensitivity (i.e., limit of detection) and the resolution (i.e., ability to distinguish among magnetically similar metabolites) of NMR. To date, the ability of in vivo ¹⁹F NMR spectroscopy to provide information that can distinguish FUra-sensitive from FUra-insensitive tumors has not been established. This crucial point should be addressed in the immediate future in studies using the best of experimental conditions (i.e., optimum sensitivity and resolution in well-defined rodent tumor models with NMR methodology appropriate for measurement of absolute metabolite concentrations). The information gained from such studies and any new technical developments to enhance in vivo NMR sensitivity should be directly applicable to any future application of ¹⁹F NMR spectroscopy in clinical FUra therapy.     

采用核磁共振氟谱定性与定量分析盐酸氟西汀

采用核磁共振氟谱检测盐酸氟西汀,在δ14.15出现信号峰,随着浓度减小(2.00～0.05 mmol.L1),化学位移略往高场移动(δ14.158到14.145),定性分析的进一步确定可以采用盯梢重叠实验。氟谱灵敏度可以达到17 mg.L1,符合药典要求,但需要扫描500次与使用合适的参数。定量分析上制作出浓度氟谱积分的标准工作曲线,藉由内插或公式法进行快速定量分析。氟标准曲线的制作中,本文提出使用毛细管内标法,利用毛细管中的三氟乙酸作为积分的标定参考。此法操作简便并获得极佳的线性拟合度;使用三氟乙醇直接添加法制作的标准曲线的线性拟合度不如预期。     

Modification of the radioreceptor assay technique for estimation of serum neuroleptic drug levels leads to improved precision and sensitivity

This report describes a series of modifications in the radioreceptor assay procedure for monitoring of serum neuroleptic levels which resulted in a substantial increase in precision and in a higher sensitivity than is the case with existing assays. The use of a dopamine receptor preparation from pig striatum yielded a lower limit of sensitivity for haloperidol of 0.08 ng/ml, when 0.5 ml serum was used in the assay; the intra- and inter-assay coefficients of variation were 3 and 9%, respectively. The relative antipsychotic potency of various neuroleptics correlated with their optimal daily dosage. Anticonvulsants (carbamazepine, phenytoin, barbiturates), anxiolytics (diazepam, bromazepam), as well as other drugs or hormones such as lithium, chloral hydrate, l-tryptophan or l-thyroxine did not interfere with the assay. Simultaneous determination of haloperidol by the radioreceptor assay and radioimmunoassay in serum showed good correlation (r=0.994). Good correlation was also noted between the average optimal clinical daily dose of the neuroleptics and their affinity to the dopamine receptor of the porcine striatum (r=0.891). Cross-laboratory assessment indicated good correlation between the estimation of haloperidol by a dopamine receptor preparation from bovine caudata and porcine striata (r=0.830). The sensitivity of the present assay was improved about 30-fold compared to those previously reported.     

Human urinary excretion of doxifluridine and metabolites during a 5-day chemotherapeutic schedule using fluorine-19 nuclear magnetic resonance spectrometry

The urinary excretion of doxifluridine (5 dFUrd) and its metabolites was determined during five days of chemotherapy using fluorine-19 nuclear magnetic resonance spectrometry. The daily urinary excretion of 5 dFUrd and its metabolites was high (-100% of the 5 dFUrd administered) and nearly constant through out the treatment. By far the major excreted compounds were unchanged 5 dFUrd and -fluoro--alanine which made up respectively -40% and -50% of the total. Neither accumulation of 5 dFUrd nor significant modifications in its metabolism were observed during the treatment.     

Application of LC-NMR and HR-NMR to the characterization of biphenyl impurities in the synthetic route development for vestipitant,a novel NK1 antagonist

Vestipitant (1) is a novel NK1 antagonist currently under investigation for the treatment of CNS disorders and emesis. The first synthetic step comprised a Grignard synthesis. An impurity was identified and initially expected to be a symmetric biphenyl. This paper reports the work to synthesise the supposed structure and the spectroscopic analyses (LC-NMR and HR-NMR) to correctly identify the real structure and understand the chemical pathway of the impurity.     

Formation of pyridinium species of haloperidol in human liver and brain

Recent interest in the neurotoxicity of haloperidol is based on its oxidation in rodents to the pyridinium derivative, HPP⁺, a structural analog of the neurotoxin, 1-methyl-4-phenylpyridinium (MPP⁺). Recently, we reported that HPP⁺ and a newly identified reduced pyridinium, RHPP⁺, were present in blood and urine of haloperidol-treated schizophrenics and that the concentrations of RHPP⁺ exceeded those of HPP⁺. In this study, we examined pathways for formation of RHPP⁺ in subcellular fractions of human liver (n=5) and brain (basal ganglia;n=5). The major pathway was reduction of HPP⁺ (20 µM) to RHPP⁺ in cytosol (0.17–0.39 and 0.03–0.07 µM RHPP⁺/g cytosolic protein per h in liver and brain, respectively). The reactions were inhibited significantly by menadione and in brain also by daunorubicin. The inhibition profile, cytosolic location and strict NADPH dependence suggest that the enzymes involved are ketone reductases. A second pathway was oxidation of reduced haloperidol (50 µM), a major metabolite of haloperidol in blood and brain, to RHPP⁺. In liver microsomes, 0.17–0.63 µmol RHPP⁺ was formed /g microsomal protein per h. A potent inhibitor of the pathway was ketoconazole (IC₅₀, 0.8 µM), which suggests that P-450 3A isozymes could be involved. In brain mitochondria but not microsomes, reduced haloperidol (120 µM) was oxidised to RHPP⁺ at a small but significant rate (0.005–0.020 µmol RHPP⁺/g mitochondrial protein per h) which was not attenuated by SKF 525A, quinidine, ketoconazole, or monoamine oxidase inhibitors. Further studies are warranted to establish the biological importance of these metabolites in vivo.     

Measurement of the Diffusion of 2,2,2-trifluoroacetamide Within Thermoresponsive Hydrogels Using NMR Imaging

Pharmaceutical Research -     

19F NMR as a Probe of Ligand Interactions with the iNOS Binding site of SPRY Domain‐Containing SOCS Box Protein 2

SPRY domain‐containing SOCS box protein 2 (SPSB2) regulates inducible nitric oxide synthase (iNOS) by targeting it for proteasomal degradation. Inhibiting this interaction prolongs the intracellular lifetime of iNOS, leading in turn to enhanced killing of infectious pathogens such as bacteria and parasites. SPSB2 recognizes a linear motif (DINNN) in the disordered N‐terminus of iNOS, and ligands that target the DINNN binding site on SPSB2 are potentially novel anti‐infective agents. We have explored ¹⁹F NMR as a means of probing ligand binding to SPSB2. All six Trp residues in SPSB2 were replaced with 5‐fluorotryptophan (5‐F‐Trp) by utilizing a Trp auxotroph strain of Escherichia coli. The labeled protein was well folded and bound a DINNN‐containing peptide with similar affinity to native SPSB2. Six well‐resolved 5‐F‐Trp resonances were observed in the ¹⁹F NMR spectrum and were assigned using site‐directed mutagenesis. The ¹⁹F resonance of W207 was significantly perturbed upon binding to DINNN‐containing peptides. Other resonances were perturbed to a lesser extent although in a way that was sensitive to the composition of the peptide. Analogues of compounds identified in a fragment screen also perturbed the W207 resonance, confirming their binding to the iNOS peptide‐binding site on SPSB2. ¹⁹F NMR promises to be a valuable approach in developing inhibitors that bind to the DINNN binding site.     

Analysis of illegally manufactured formulations of tadalafil (Cialis) by 1H NMR, 2D DOSY 1H NMR and Raman spectroscopy

Counterfeit and/or imitation medicines are becoming a major health problem not only in developing countries but also in wealthier countries. The need of new and easy analytical methods for quality control of drugs is essential. We describe the use of Raman spectroscopy, 1H nuclear magnetic resonance (NMR) and 2D diffusion-ordered spectroscopy (DOSY) NMR to analyse genuine Cialis and seven illegally manufactured formulations of this drug purchased via the internet. Seven out of the eight commercial formulations of tadalafil contain the active ingredient, measured by high performance liquid chromatography (HPLC), within 100+/-5% of stated concentration. Vardenafil and homosildenafil instead of tadalafil were found in the Chinese imitation. 2D DOSY NMR spectra clearly showed similarities and differences in the composition of the pharmaceutical formulations of tadalafil, thus giving a precise and global "signature" of the manufacturer. Our data show that the quality of the Cialis imitations manufactured in India and Syria is correct, whereas the Chinese formulation is adulterated with active pharmaceutical ingredients.     

Characterization of a novel impurity in bulk drug eprosartan by ESI/MS and NMR

A simple and sensitive liquid chromatography tandem multi-stage mass spectrometry (HPLC/MSⁿ) method suitable for eprosartan analysis was developed, by which an unknown impurity in bulk drug eprosartan was detected. The fragmentation behavior of eprosartan and the impurity in negative mode was investigated. Two molecules of CO₂ lost from eprosartan precursor ion were observed, while four molecules of CO₂ were extruded from the deprotonated molecular ion to the MS³ product ions of the impurity. Furthermore, a characteristic fragmentation ion at m/z 335 was observed in both eprosartan and the impurity indicated that the impurity might have two eprosartan units. The unknown impurity was initially proposed to be eprosartan dimer connected via methylene unit at the thiophene moiety on the basis of the multi-stage mass spectrometric and exact mass evidences, and it was finally elucidated as 4,4′-(5,5′-(1E,1′E)-3,3′-(4,4′-methylenebis(thiophene-4,2-diyl))bis(2-carboxyprop-1-ene-3,1-diyl)bis(2-butyl-1H-imidazole-5,1-diyl))bis(methylene) dibenzoic acid by NMR experiments including 1D (¹H NMR, ¹³C NMR, DEPT135⁰) and 2D (¹H-¹HCOSY, HMQC and HMBC) data.     

Fluorine-19 or phosphorus-31 NMR spectroscopy: a suitable analytical technique for quantitative in vitro metabolic studies of fluorinated or phosphorylated drugs

Fluorine-19 or phosphorus-31 NMR (¹⁹F NMR or ³¹P NMR) spectroscopy provides a highly specific tool for identification of fluorine- or phosphorus-containing drugs and their metabolites in biological media as well as a suitable analytical technique for their absolute quantification. This article focuses on the application of in vitro ¹⁹F or ³¹P NMR to the quantitative metabolic studies of some fluoropyrimidine or oxazaphosphorine drugs in clinical use. The first part presents an overview of the advantages (non-destructive and non-selective direct quantitative study of the biological matrices) and limitations (expensive cost of the spectrometers, limited mass or concentration sensitivity) of NMR spectroscopy. The second part deals with the criteria to be considered for successful quantification by NMR (uniform excitation over the entire spectral width of the spectrum, resonance signals properly characterised by taking into account T₁ values and avoiding NOE enhancements, optimisation of the data processing, choice of a suitable standard reference). The third and fourth parts report some examples of quantification of 5-fluorouracil, its prodrug capecitabine, 5-fluorocytosine and their metabolites in bulk solutions (biofluids, tissue extracts, perfusates and culture media) and heterogeneous media (excised tissues and packed intact cells) as well as cyclophosphamide and ifosfamide in biofluids. These two parts emphasise the high potential of in vitro ¹⁹F or ³¹P NMR for absolute quantification, in a single run, of all the fluorine- or phosphorus-containing species in the matrices analysed. The limit of quantification in bulk solutions is 1–3 μM for ¹⁹F NMR and approximately 10 μM for ³¹P NMR. In heterogeneous media analysed with ¹⁹F NMR, it is 2–5 nmol in excised tissues and cell pellets.     

The application of absolute quantitative 1H NMR spectroscopy in drug discovery and development

ABSTRACT

Introduction: The identification of a drug candidate and its structural determination is the most important step in the process of the drug discovery and for this, nuclear magnetic resonance (NMR) is one of the most selective analytical techniques.

Area covered: The present review illustrates the various perspectives of absolute quantitative ¹H NMR spectroscopy in drug discovery and development. It deals with the fundamentals of quantitative NMR (qNMR), the physiochemical properties affecting qNMR, and the latest referencing techniques used for quantification. The precise application of qNMR during various stages of drug discovery and development, namely natural product research, drug quantitation in dosage forms, drug metabolism studies, impurity profiling and solubility measurements is elaborated. To achieve this, the authors explore the literature of NMR in drug discovery and development between 1963 and 2015. It also takes into account several other reviews on the subject.

Expert opinion: qNMR experiments are used for drug discovery and development processes as it is a non-destructive, versatile and robust technique with high intra and interpersonal variability. However, there are several limitations also. qNMR of complex biological samples is incorporated with peak overlap and a low limit of quantification and this can be overcome by using hyphenated chromatographic techniques in addition to NMR.     

The inhibitory effect of the antipsychotic drug haloperidol on HERG potassium channels expressed in Xenopus oocytes

1. The antipsychotic drug haloperidol can induce a marked QT prolongation and polymorphic ventricular arrhythmias. In this study, we expressed several cloned cardiac K⁺ channels, including the human ether-a-go-go related gene (HERG) channels, in Xenopus oocytes and tested them for their haloperidol sensitivity.
2. Haloperidol had only little effects on the delayed rectifier channels Kv1.1, Kv1.2, Kv1.5 and I_sK, the A-type channel Kv1.4 and the inward rectifier channel Kir2.1 (inhibition <6% at 3 μM haloperidol).
3. In contrast, haloperidol blocked HERG channels potently with an IC₅₀ value of approximately 1 μM. Reduced haloperidol, the primary metabolite of haloperidol, produced a block with an IC₅₀ value of 2.6 μM.
4. Haloperidol block was use- and voltage-dependent, suggesting that it binds preferentially to either open or inactivated HERG channels. As haloperidol increased the degree and rate of HERG inactivation, binding to inactivated HERG channels is suggested.
5. The channel mutant HERG S631A has been shown to exhibit greatly reduced C-type inactivation which occurs only at potentials greater than 0 mV. Haloperidol block of HERG S631A at 0 mV was four fold weaker than for HERG wild-type channels. Haloperidol affinity for HERG S631A was increased four fold at +40 mV compared to 0 mV.
6. In summary, the data suggest that HERG channel blockade is involved in the arrhythmogenic side effects of haloperidol. The mechanism of haloperidol block involves binding to inactivated HERG channels.

     

Solid-state NMR and IR for the analysis of pharmaceutical solids: Polymorphs of fosinopril sodium

The two polymorphic modifications of fosinopril sodium have been characterized as to their differences in melting behaviour, powder X-ray diffraction patterns, Fourier transform infrared spectra (FTIR), and solid-state³¹P- and¹³C-NMR spectra. The polymorphs were found to be enantiotropically related based upon melting point, heat of fusion, and solution mediated transformation data. Analysis of the solid-state FTIR and¹³C-NMR data indicated that the environment of the acetal side chain of fosinopril sodium differed in two polymorphs, and that there might becis-trans isomerization about the C₆—N peptide bond. These conformational differences are postulated as the origin of the observed polymorphism.     

Syntheses of [3H2]T0901317 and a labeled structural isomer,and characterization of the dispersed labeled compounds via 19F NMR

The synthesis and characterization of [³H₂]T0901317 and a structural isomer are described. The structural assignments of the closely related labeled compounds were primarily accomplished via ¹⁹F NMR analyses of the corresponding ethanolic compound dispersions.     

氟脲嘧啶和呋氟脲嘧啶的β-环糊精包合物的组成和稳定常数的核磁共振研究

利用氟脲嘧啶(5-Fu)和呋氟脲嘧啶(FT-207)与β-环糊精(β-CD)形成主—客包合物后¹⁹F化学位移的变化研究了这些包合物的组成和稳定常数。5-Fu和FT-207均与β-CD形成1:1包合物,包合物的稳定常数在40℃分别为17.0±1.7mol^-1·L和9.0±0.1mol^-1·L。研究结果还表明,与β-CD形成包合物后,5-Fu和FT-207的¹⁹F化学位移的变化有较明显的差异,提示了它们分子中的氟脲嘧啶环在β-CD疏水性内腔中的位置是不同的。