Péter Jankovics

Determination of paracetamol and its main impurity 4-aminophenol in analgesic preparations by micellar electrokinetic chromatography

A micellar electrokinetic chromatographic (MEKC) method was developed for the quality control of paracetamol containing pharmaceutical preparations. The influence of several factors (surfactant concentration, buffer concentration, pH and applied voltage) was studied during development and optimisation of the method. Phosphate buffer (pH 9.0) containing sodium dodecyl sulphate (75 mM) was found as the ideal running buffer for the separation; the applied voltage was 25 kV and the analysis time was 10 min. The limit of quantitation for 4-aminophenol was 6 microgml(-1); the linearity of the method was studied in the concentration ranges 20-260 microgml(-1) for paracetamol and 20-150 microgml(-1) for 4-aminophenol. The method was successfully applied for the quality control of paracetamol containing products.

Qualitative and quantitative analysis of PDE-5 inhibitors in counterfeit medicines and dietary supplements by HPLC–UV using sildenafil as a sole reference

Due to their popularity, medicinal products containing the phophodiesterase type 5 enzyme (PDE-5) inhibitors sildenafil, vardenafil and tadalafil are often subject to counterfeiting. In addition, illicit herbal dietary supplements adulterated with these substances or their analogs have appeared on the market offering an easy and anonymous sale. This paper describes an analytical method for qualitative and quantitative screening of sildenafil, vardenafil, tadalafil and 11 of their designer analogs in illegal erectile dysfunction products by high-performance liquid chromatography with UV detection (HPLC-UV). Sildenafil served as a single external standard for both identification and quantification of all analytes. Relative retentions and reference UV spectra were used for qualitative, and correction factors for quantitative analyses, respectively. The separation was performed on a Kinetex C18 reverse-phased column at 25°C using gradient elution. Mobile phase A consisted of 200mM ammonium acetate solution while mobile phase B was a 1:1 (v/v) mixture of methanol and acetonitrile with a flow rate of 0.5ml/min and injection volume of 5μl. Detection wavelength was set to 290nm. The method was validated in accordance with the appropriate guideline of the International Conference on Harmonization (ICH) in terms of specificity, selectivity, precision, linearity, limit of quantitation, limit of detection, accuracy, robustness and stability, and was successfully applied to the analysis of natural dietary supplements and herbal remedies with an indication for enhanced male sexual potency. The proposed method offers a cheap and simple alternative to LC-MS screening used by control laboratories for routine analysis of suspicious products.

Detection and structure elucidation of hydroxythiovardenafil as an adulterant in a herbal dietary supplement

A new derivative of vardenafil was detected in an alleged herbal dietary supplement and identified as 2-(2-ethoxy-5-(4-(2-hydroxyethyl)piperazin-1-ylsulfonyl)phenyl)-5-methyl-7-propyl-imidazo[1,5-f][1,2,4]triazin-4(3H)-thione. Structure elucidation was carried out by LC-UV-MS/MS and NMR. Results obtained with high resolution MS and IR spectroscopy confirmed the proposed chemical structure. The compound was distinguished from hydroxyvardenafil, a second active substance identified in the same product, by the conversion of the oxo group to a thio group on the imidazo-triazin moiety. Hydroxythiovardenafil was therefore suggested as a proprietary name for the new molecule. This is the first paper to describe a thio-analog of vardenafil in a commercially available product.

Qualitative and quantitative analysis of N-acetyllactosamine and lacto-N-biose, the two major building blocks of human milk oligosaccharides in human milk samples by high-performance liquid chromatography-tandem mass spectrometry using a porous graphitic carbon column

This study presents a validated, porous graphitic carbon stationary phase-based LC-MS/MS method for the identification and quantification of lacto-N-biose (LNB) and N-acetyllactosamine (LacNAc). These compounds are the major building blocks of human milk oligosaccharides, however the presence of their unbound form in human milk has not been examined so far. The separation of these highly related structures in their alditol form was accomplished by a gradient LC method and multiple reaction monitoring (MRM) analysis after appropriate sample preparation including size-exclusion chromatography and solid-phase extraction. Baseline separation of the components provides the selectivity for the method. Validation was performed according to the European Medicines Agency (EMA) Guidelines and the method was found to be precise and accurate. Using our developed and validated method we were able to identify and quantify both saccharides in human milk for the first time. Based on our results the LacNAc concentration is in the range of 6.7-31μg/mL while LNB concentration decreased from 26μg/mL below the detection limit during the first week of lactation. The presence of LNB and LacNAc in human milk also implies new biological functions which can lead us closer to the understanding of the various functions of this complex biofluid.

Exploring the possibilities of capacitively coupled contactless conductivity detection in combination with liquid chromatography for the analysis of polar compounds using aminoglycosides as test case

The analysis of highly polar (often charged) compounds which lack a strong UV absorbing chromophore is really challenging. Despite the numerous analytical methods published, the demand for a simple, robust and cheap technique for their analysis still persists. Here, reversed phase (RP) liquid chromatography (LC) with capacitively coupled contactless conductivity detection (C(4)D) was explored for the first time as a possible method for separation and detection of various aminoglycoside (AMG) antibiotics which were taken as typical test compounds: tobramycin (TOB), spectinomycin, streptomycin, amikacin, kanamycin A and kanamycin B. C(4)D was performed using a commercially available as well as a laboratory made cell. As ion-pairing reagents (IPR) four perfluorinated carboxylic acids were used: pentafluoropropionic acid, heptafluorobutyric acid, nonafluoropentanoic acid (NFPA) and pentadecafluorooctanoic acid (PDFOA). 0.125 mM NFPA-acetonitrile (ACN) (90:10) or 0.125 mM PDFOA-ACN (70:30) as mobile phases were suitable to detect TOB with reasonable retention times. However, NFPA was preferred for practical reasons. Its applicable concentration range in the mobile phase was strongly restricted by loss of chromatographic performance at lower levels and excessive background conductivity at higher levels. Overall repeatability and robustness of the method were rather poor which was explained by the relatively low IPR levels. Selectivity between the tested AMGs was mainly influenced by the number of protonated amino groups per molecule making it impossible to separate compounds of equal net charges. Problems encountered with gradient elution, hydrophilic interaction liquid chromatography (HILIC) and separation at high pH without IPRs are also discussed.