Drug Testing and Analysis

HPLC-diode array detector method was developed and validated for quantification of the antidote 2-PAM in porcine urine allowing the evaluation of its elimination in vivo in a pesticide poisoning scenario.

Various non-column and column procedures applied for 2-PAM analysis are reviewed demonstrating that ion pair chromatography is the most popular method well established for biological samples.

Harald John, John Mikler, Franz Worek, Horst Thiermann ^“Application of an enantioselective LC-ESI MS/MS procedure to determine R- and S-hyoscyamine following intravenous atropine administration in swine”.

For the first time R- and S-hyoscyamine enantiomers were quantified in swine plasma by a LC-ESI MS/MS procedure that allowed to monitor concentration-time profiles in an in vivo atropine study.

Developmental lots of HI-6 DMS provided by different manufacturers were examined with respect to their purity with a view to their future use as an active pharmaceutical ingredient (API). An unknown impurity in HI-6 could be identified as an isomer. These findings supply data regulatory required for the description of the pharmaceutical quality in accordance with module 3 of a Common Technical Document (CTD) and thus contribute to marketing authorization of HI-6 as an API for autoinjectors and infusions.

Tested under conditions of ICH Q1B HI-6 showed a marked degradation after exposure to day-light; the mechanism of degradation could be detected as photoisomerism. Based on quantitative results of the isomerisation rate a standardised in-vitro test showed a decreased ability of light exposed HI-6 to reactivate sarin- and paraoxon-inhibited human acetylcholinesterase. These results have an impact on the further development of antidotes containing HI-6 as light protection will probably be necessary during handling, packaging, storage and application of HI-6.

A sensitive and fast UPLC-ESI-MS/MS method for measurements of N-butylscopolamine in plasma was developed and validated using protein precipitation for the clean up of the plasma and N-methylhomatropine with internal standard (IS). The acquisition was performed in the multiple reaction monitoring mode monitoring the transitions: m/z 360.0 → 194.0 for N-butylscopolamine and m/z 290.3 → 138.0 for SI with positive ion electrospray ionization using a mobile phase of acetonitrile: 5mM ammonium acetate + 0.1% formic acid (90:10, v/v). The method was linear in 0.03 (lower limit of quantitation; LLOQ) - 10.00 ng/mL and demonstrated to be selectivity, linearity, precision, accuracy, robustness and was successfully applied to a pharmacokinetic study of healthy human volunteers; the results showed that the two scopolamine butylbromide formulations tested are not bioequivalent in rate and extent of absorption.

Rapid LC methods allow substantial improvements in quality monitoring programmes of stockpiled medicines. The suggested RRLC method is suitable for atropine containing products that are used in case of organophosphate poisoning.

Atropine is used in the daily clinical practice for the treatment of poisonings caused by anticholinesterase pesticides. In order to investigate the cause of the adverse effects that appear during atropine administration a simple, rapid, sensitive, specific and fully validated GC/MS method for the determination of atropine in serum samples is needed. The developed method proved to be useful for the investigation of adverse effects that appear during atropine treatment of patients poisoned by anticholinesterase pesticides.

A total number of 554 spectra covering 291 compounds were extracted from 109 publications. These spectra were matched to the MSforID library. Sensitivity of library search was found to exceed 95%, which clearly proves that the MSforID library can successfully handle data from a huge variety of mass spectrometric instruments to allow accurate compound identification.

The utility of state-of-the-art mass spectrometers and recent instrumental developments such as new and/or improved hybrid analyzer are discussed and selected applications presented, focusing particularly on high resolution/high accuracy mass spectrometry.

Detection methods for organophosphorus and carbamate toxicants are reviewed, compared and evaluated.

Conjugates of DFPase and linear PEGs are characterizedusing ESI-ToF-MS and their in vitro and in vivo efficacy against toxic organophosphorus compounds is determined. A PEGylated variant of DFPase is shown to be able to protect rats from a 3x LD₅₀ challenge with soman.

A review of the development of organophosphorus hydrolase (OPH) for use as in vivo catalytic bioscavengers is presented. As improved candidate enzymes are continually developed, our understanding of the contributions of the catalytic parameters (kcat, K_M and catalytic efficiency) to efficacy expands, and the role immunogenicity, absorption, distribution and elimination contribute to the level of protection afforded by the protein is appreciated.

Review of the potential and limitations of the Ellman assay for kinetic interactions of AChE, OP and oximes. Application of the assay requires the consideration of potential matrix effects, side reactions and other determinants. The Ellman assay allows the precise and reproducible determination of kinetic constants as a basis for the understanding of toxic OP effects and for the development of improved therapies against poisoning by OP.

A set of bispyridinium compounds structurally related to SAD-128 were tested in competition binding experiments with recombinant human M₅ muscarinic acetylcholine receptors. Five of the six investigated bispyridinium compounds interacted with the orthosteric binding site, with affinities in the the low micromolar range. These data indicate that interaction of bispyridinium compounds with muscarinic receptors may contribute to their therapeutic efficacy.

Fourier Tarnsform Infrared Spectroscopy (ATR-FTIR) is discussed with respect to new applications like Process Analytical Chemistry (PAC), the quantitation of drugs in complex matrix formulations, the analysis of protein binding and function and in combination with IR microscopy to the emergence of IR imaging technologies.

Creating mass doublets by mixing a natural isotopic abundance drug with a ¹³C-labelled isotopomer and applying stable isotope filtering to high resolution mass spectrometry allows one to rapidly identify drug metabolites in complex samples. Applying this approach to drug metabolism requires the synthesis of stable isotope labelled drugs. We have developed efficient strategies for stable isotope labelling of complex molecules based on the rich chemistry of [¹³C]methyl phenyl sulfide, where the phenylthio is a carrier for the valuable ¹³C-label.

A rapid mass spectrometric based screening technique for the detection of organophosphorous chemical warfare agent metabolites in unprepared urine.