Torbjörn Arvidsson

Survey and qualification of internal standards for quantification by 1H NMR spectroscopy

In quantitative NMR (qNMR) selection of an appropriate internal standard proves to be crucial. In this study, 25 candidate compounds considered to be potent internal standards were investigated with respect to the ability of providing unique signal chemical shifts, purity, solubility, and ease of use. The (1)H chemical shift (delta) values, assignments, multiplicities and number of protons (for each signal), appropriateness (as to be used as internal standards) in four different deuterated solvents (D(2)O, DMSO-d(6), CD(3)OD, CDCl(3)) were studied. Taking into account the properties of these 25 internal standards, the most versatile eight compounds (2,4,6-triiodophenol, 1,3,5-trichloro-2-nitrobenzene, 3,4,5-trichloropyridine, dimethyl terephthalate, 1,4-dinitrobenzene, 2,3,5-triiodobenzoic acid, maleic acid and fumaric acid) were qualified using both differential scanning calorimetry (DSC) and NMR spectroscopy employing highly pure acetanilide as the reference standard. The data from these two methods were compared as well as utilized in the quality assessment of the compounds as internal standards. Finally, the selected internal standards were tested and evaluated in a real case of quantitative NMR analysis of a paracetamol pharmaceutical product.

Procedures for direct injections of untreated blood plasma into liquid chromatographic columns with emphasis on a pre-column venting technique.

The stability of reversed-phase liquid chromatographic systems when untreated blood plasma samples are applied was studied with respect to retention, peak efficiency and column back-pressure. The influence of the content of organic solvents in the eluent, the flow-rate and the particle diameter of the support was investigated. Many of the column-deteriorating effects seem to be due to denaturation of the plasma proteins and the kinetics of this process. The stability is increased by decreasing the content of organic modifier in the eluent. The flow-rate should be moderate, as both high and low flow-rates give decreased stability. The stability increases with increasing particle size of the solid phase. A small pre-column is used (1) as a guard column and (2) to effect a pre-separation of the solute from plasma matrix components by a pre-column venting technique. The pre-column venting technique considerably increases the lifetime of the system and especially the separation column. Under optimal conditions hundreds of injections (10 microliters of plasma) can be performed without loss of stability.

A general analytical platform and strategy in search for illegal drugs.

An effective screening procedure to identify and quantify active pharmaceutical substances in suspected illegal medicinal products is described. The analytical platform, consisting of accurate mass determination with liquid chromatography time-of-flight mass spectrometry (LC-QTOF-MS) in combination with nuclear magnetic resonance (NMR) spectroscopy provides an excellent analytical tool to screen for unknowns in medicinal products, food supplements and herbal formulations. This analytical approach has been successfully applied to analyze thousands of samples. The general screening method usually starts with a methanol extraction of tablets/capsules followed by liquid chromatographic separation on a Halo Phenyl-Hexyl column (2.7μm; 100mm×2.1mm) using an acetonitrile/0.1% formic acid gradient as eluent. The accurate mass of peaks of interest was recorded and a search made against an in-house database containing approximately 4200 substances, mostly pharmaceutical compounds. The search could be general or tailored against different classes of compounds. Hits were confirmed by analyzing a reference substance and/or by NMR. Quantification was normally performed with quantitative NMR (qNMR) spectroscopy. Applications for weight-loss substances like sibutramine and orlistat, sexual potency enhancement (PDE-5 inhibitors), and analgesic drugs are presented in this study. We have also identified prostaglandin analogues in eyelash growth serum, exemplified by isopropyl cloprostenate and bimatoprost. For creams and ointments, matrix solid-phase dispersion (MSPD) was found to give a clean extracts with high recovery prior to LC-MS analyses. The structural elucidation of cetilistat, a new weight-loss substance recently found in illegal medicines purchased over the Internet, is also presented.

Chromatographic resolution of enantiomers using albumin as complexing agent in the mobile phase

Enantiomers of carboxylic acids have been separated with albumin as a chiral complexing agent in the mobile phase. Stereoselective separation has been obtained for different types of acids, in some cases with very high separation factor, as shown for di-p-toluoyltartaric acid (alpha(s) = 5.8). The stereoselectivity and retention properties depend on pH and on the concentration of albumin in the mobile phase. Retention can also be regulated by modifying the nature of the solid phase as well as by the use of additives in the mobile phase. Acids with low molar absorptivity, or with absorbance in the same wavelength range as albumin, can be detected by an indirect technique based on the use of a cationic mobile phase additive, such as 1-ethylquinolinium, which has high UV-absorptivity at a wavelength remote from that of albumin.

Elimination of peak deformation in the liquid chromatographic separation of a strongly protein-bound drug from directly injected blood plasma samples.

Direct injection of blood plasma samples into reversed-phase columns resulted in skewed chromatographic peaks for the drug naproxen. The skew is shown to be due to strong binding of naproxen to albumin present in the blood plasma. Methods to eliminate the peak skew have been investigated. They include changes of the composition of the eluent and of the sample solution in order to decrease the degree of binding of the drug to albumin. The methods studied were dilution, addition of displacers, change of pH and change of methanol concentration. Calculations based on known binding constants indicate that the degree of peak skew was directly influenced by the degree of protein binding of the drug in the sample solution.

Validation of a quantitative NMR method for suspected counterfeit products exemplified on determination of benzethonium chloride in grapefruit seed extracts

A 1H-nuclear magnetic resonance (NMR) spectroscopy method for quantitative determination of benzethonium chloride (BTC) as a constituent of grapefruit seed extract was developed. The method was validated, assessing its specificity, linearity, range, and precision, as well as accuracy, limit of quantification and robustness. The method includes quantification using an internal reference standard, 1,3,5-trimethoxybenzene, and regarded as simple, rapid, and easy to implement. A commercial grapefruit seed extract was studied and the experiments were performed on spectrometers operating at two different fields, 300 and 600 MHz for proton frequencies, the former with a broad band (BB) probe and the latter equipped with both a BB probe and a CryoProbe. The concentration average for the product sample was 78.0, 77.8 and 78.4 mg/ml using the 300 BB probe, the 600MHz BB probe and CryoProbe, respectively. The standard deviation and relative standard deviation (R.S.D., in parenthesis) for the average concentrations was 0.2 (0.3%), 0.3 (0.4%) and 0.3mg/ml (0.4%), respectively.

The cyanobacterial amino acid β-N-methylamino-l-alanine perturbs the intermediary metabolism in neonatal rats

The neurotoxic amino acid β-N-methylamino-l-alanine (BMAA) is produced by most cyanobacteria. BMAA is considered as a potential health threat because of its putative role in neurodegenerative diseases. We have previously observed cognitive disturbances and morphological brain changes in adult rodents exposed to BMAA during the development. The aim of this study was to characterize changes of major intermediary metabolites in serum following neonatal exposure to BMAA using a non-targeted metabolomic approach. NMR spectroscopy was used to obtain serum metabolic profiles from neonatal rats exposed to BMAA (40, 150, 460mg/kg) or vehicle on postnatal days 9-10. Multivariate data analysis of binned NMR data indicated metabolic pattern differences between the different treatment groups. In particular five metabolites, d-glucose, lactate, 3-hydroxybutyrate, creatine and acetate, were changed in serum of BMAA-treated neonatal rats. These metabolites are associated with changes in energy metabolism and amino acid metabolism. Further statistical analysis disclosed that all the identified serum metabolites in the lowest dose group were significantly (p<0.05) decreased. The neonatal rat model used in this study is so far the only animal model that displays significant biochemical and behavioral effects after a low short-term dose of BMAA. The demonstrated perturbation of intermediary metabolism may contribute to BMAA-induced developmental changes that result in long-term effects on adult brain function.

Use and qualification of primary and secondary standards employed in quantitative 1H NMR spectroscopy of pharmaceuticals

Standards are required in quantitative NMR (qNMR) to obtain accurate and precise results. In this study acetanilide was established and used as a primary standard. Six other chemicals were selected as secondary standards: 3,4,5-trichloropyridine, dimethylterephthalate, maleic acid, 3-sulfolene, 1,4-bis(trimethylsilyl)benzene, and 1,3,5-trimethoxybenzene. The secondary standards were quantified using the primary standard acetanilide. A protocol for qualification and periodic checks of these secondary standards was developed, and used for evaluation of the stability of the compounds. Periodic monitoring of purity was performed for several years. The purity was higher than 99% for all secondary standards. All standards maintained the initial purity during the time period of monitoring, with very small variations in purity (0.3-0.4%). The selected secondary standards were shown to be suitable qNMR standards and that periodic requalification of the standards by qNMR ensures reliable analytical results. These standards have been used in our laboratory for compliance testing of pharmaceutical active substances and approved medicinal products as well as for analysis of suspected illegal medicines. In total more than 1000 samples have been tested using both internal and external standardization and examples are given.

Quantitative determination of salbutamol in tablets by multiple-injection capillary zone electrophoresis

A multiple-injection capillary zone electrophoresis (MICZE) method has been developed for the assay of salbutamol in Ventoline Depot tablets (GlaxoSmithKline). In the developed method, seven sample sets, each consisting of three samples, were sequentially injected into the capillary and analyzed within a single run. This enabled a total of twenty-one sequential injections, i.e., six standards and fifteen samples, containing salbutamol and the injection marker oxprenolol. The injected sample plugs were separated by plugs of background electrolyte, through application of a short-term voltage (30kV) over the capillary for different time periods, i.e., t(PE1) and t(PE2). The samples in each set were isolated from each other by partial electrophoresis for 2.35min (t(PE1)), while the sample sets were separated for 10.50min (t(PE2)). After the final injection, all the applied samples were subjected to electrophoresis for a time period corresponding to that in conventional single-injection CZE. The method was validated regarding linearity, accuracy, precision and robustness before it was applied to the determination of salbutamol in 15 tablets of Ventoline Depot with a labeled content of 8mg salbutamol. The average salbutamol content was determined to 7.8mg (+/-0.3mg) from simultaneous analyses of the 15 different tablets.

precolumn-venting plug technique for direct injection of untreated blood plasma samples into reversed-phase liquid chromatography systems

A simple column switching technique for the direct injection of untreated blood plasma samples is presented for the determination of drugs and related compounds. The system consists of injector valves, a precolumn, a three port valve and a separation column. The precolumn is used for trace enrichment and sample clean-up. Aqueous plugs are introduced on both sides of the plasma sample before it enters the precolumn. This results in a stable system, because contact between plasma proteins and the organic solvent usually present in the mobile phase is prevented. The proteins are eluted to waste with the aqueous plug fluid. The stability of the chromatographic system was studied with respect to efficiency and column back-pressure. The influence of the concentration of organic solvent in the eluent, type and particle size of the packing material, precolumn filters, pH, and ionic strength was investigated for large sample volumes. Optimal stability was obtained at low concentrations of organic solvent in the eluent, with plugs of phosphate buffer (mu = 0.1) and with a spreader at the inlet and a screen (2 microns) at the outlet of the precolumn. The precolumn was packed with 10-microns particles for trace enrichment. Under favourable conditions 40-50 large-volume (0.5-ml) plasma injections can be made into a single precolumn.