Application of paper spray-MS in PK studies using sunitinib and benzethonium as model compounds.

AIM: Current bioanalytical methods applied in nonclinical PK studies for screening drug candidates demand significant amount of time and resources, hence, the need to develop alternative methods. RESULTS: A proof-of-concept paper spray-MS method for the detection and quantitation of small molecules in plasma has been developed and validated using sunitinib and benzethonium as model compounds. The method includes single oral or intravenous administration of sunitinib to mice and serial micro-volume (20 µl) blood collection at different time intervals. The method is rapid with overall analysis time of 1 min and a full PK profile of sunitinib was obtained from a single mouse. CONCLUSION: The paper spray-MS approach is simple, sensitive and can potentially enable significant reduction of animal use and cost.

Direct quantitative bioanalysis of drugs in dried blood spot samples using a thin-layer chromatography mass spectrometer interface.

The CAMAG thin-layer chromatography mass spectrometer (TLC-MS) interface has been assessed as a tool for the direct quantitative bioanalysis of drugs from dried blood spot (DBS) samples, using an MS detector, with or without high-performance liquid chromatography (HPLC) separation. The approach gave acceptable sensitivity, linearity, accuracy, and precision data for bioanalytical validations with and without the inclusion of HPLC separation. In addition, the direct elution technique was shown to increase assay sensitivity for a range of analytes representing a wide "chemical space" for pharmaceutical-type molecules over that obtained by conventional manual extraction of samples (punching of DBS and elution with solvent prior to HPLC-MS analysis). Investigations were performed to optimize extraction time, minimize sample-to-sample carry-over, and compare chromatographic performance. On the basis of this preliminary assessment, it has been demonstrated that the TLC-MS interface has the potential to be an effective tool for the direct analysis of drugs in DBS samples at physiologically relevant concentrations, an approach that could provide significant time and cost savings and greatly simplify bioanalytical procedures compared to current manual practices. Further, the increased sensitivity compared to that of manual extraction may enable the analysis of analytes not currently amenable to DBS sampling due to limitations in assay sensitivity.

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.

[Survey of synthetic disinfectants in grapefruit seed extract and its compounded products].

Grapefruit seed extract (GSE), derived from the seeds of grapefruit (Citrus paradisi MCAF.), is listed as a natural food additive in Japan. Products containing GSE are used as disinfectants made from only natural sources, especially after Japanese researchers found that GSE prevents the growth of norovirus. On the other hand, recent overseas studies indicated that synthetic disinfectants, such as benzalkonium and benzethonium chlorides, were present in some commercial GSE products. To confirm the quality of commercial GSE products available in Japanese markets, we carried out comprehensive research to identify the major constituents of commercial GSE products which are used as food additives (13 products from 6 manufacturers), dietary supplements (5 products from 4 manufacturers), cosmetic materials (16 products from 10 manufacturers) and disinfectant or deodorant sprays (7 products from 7 manufacturers). By means of NMR and LC/MS analysis, synthetic disinfectants such as benzethonium or benzalkonium salts were detected in most of the commercial GSE products.

Simultaneous identification and quantification by liquid chromatography of benzethonium chloride, methyl paraben and triclosan in commercial products labeled as grapefruit seed extract.

A HPLC method has been developed which permits the quantification of methyl paraben, benzethonium chloride and triclosan in various samples of grapefruit seed extract (GSE). The best results were obtained with a Phenomenex Gemini C18 column using gradient mobile phase of water (0.1% acetic acid) and acetonitrile (0.1% acetic acid) with a flow rate of 1.0 mL per minute. The detection wavelength was 254 nm for methyl paraben, and 275 nm for benzethonium chloride and triclosan. The main synthetic antimicrobial agent identified in commercial GSE samples was benzethonium chloride in concentrations from 0.29-21.84%. Positive ion electrospray MS of a commercial GSE sample showed a molecular ion at m/z 412 [M+], which matched that of a standard of benzethonium chloride. Triclosan was detected in two samples at 0.009 and 1.13%concentrations; while methyl paraben was not detected in the samples analyzed.

Determination of benzethonium chloride in anthrax vaccine adsorbed by HPLC.

A novel and sensitive HPLC method for the determination of benzethonium chloride (BZC) in anthrax vaccine was developed. Adjuvant Alhydrogel was removed by syringe filter after a simple sample pretreatment - acidification prior to injection. Chromatography was performed by isocratic reverse phase separation with methanol/262 mM ammonium acetate (80/20, v/v) on an endcapped C18 column with diode array detector (DAD). The method showed excellent recovery (100+/-1.5%). The results indicated that this method could accurately determine BZC at the limit of detection (LOD) of 0.5 ppm and the limit of quantitation (LOQ) of 1.5 ppm with dynamic range up to 100 ppm. The comparison of analysis between new HPLC and old titrimetric methods is also reported. The HPLC method is proven to be more accurate and precise with much less vaccine sample and human labor required.

Studies on the formation of electrostatic complexes between benzethonium chloride and anionic polymers.

The chemistry of a mixture of benzethonium chloride and a copolymer of methoxyethylene-maleic anhydride was investigated. This mixture was of interest because it was effective in reducing dental plaque, calculus, and gingival inflammation in vivo. Evidence from dialysis, pH measurements, and stoichiometry demonstrated that the benzethonium cation and the anion of the hydrolyzed copolymer formed an electrostatic complex. An emulsion was produced when a stoichiometric excess of either component was present, but this mixture coacervated at stoichiometric quantities. The stability of the complex was pH dependent, and it did not form in 50% acetone. The complex was decomposed by simulated saliva, mainly due to calcium and magnesium ions, but was unaffected by salivary proteins. Other anionic polymers also formed this type of complex.

Direct spectorphotometric assay of quaternary ammonium compounds using bromthymol blue.

Benzalkonium chloride, benzethonium chloride, and chlorhexidine gluconate were assayed quantitatively by a direct spectrophotometric method with bromthymol blue buffered at pH 7.5. The method shows good results at concentrations of 0--300 microgram/ml and in the presence of epinephrine bitartrate, phenylephrine hydrochloride, pilocarpine hydrochloride, and polyvinyl alcohol.

Development of an agar-diffusion method for the assay of quaternary ammonium germicides.

Five quaternary ammonium germicides (QAGs) were tested for their adsorption by agar. This was found to be in the following ascending order: alkylbenzylmethyl-ammonium chloride, alkyltrimethylammonium bromide, cetyltrimethylammonium bromide, cetylpyrimidinium chloride and cetylbenzyldimethylammonium chloride. An inverse relationship was established between the extent of agar binding of the QAGs and their inhibition zones. In an attempt to develop a sensitive cup-plate assaying technique suitable for QAGs, important factors affecting the agar-diffusion of QAGs were investigated. These included the influence of various polysorbates, buffer ions, agars and test organisms. Furthermore, the effect of the pH and/or the concentration of the selected polysorbate and the buffer were studied. The best medium developed for the sensitive agar-diffusion assay of QAGs was nutrient agar-Tris (0.05 M, pH 8) provided that distilled water and polysorbate 20 (0.5%) were used as diluents for the mixed alkyl and the pure cetyl QAGs, respectively.