Tutorial review on validation of liquid chromatography–mass spectrometry methods: Part II

This is the part II of a tutorial review intending to give an overview of the state of the art of method validation in liquid chromatography mass spectrometry (LC–MS) and discuss specific issues that arise with MS (and MS–MS) detection in LC (as opposed to the “conventional” detectors). The Part II starts with briefly introducing the main quantitation methods and then addresses the performance related to quantification: linearity of signal, sensitivity, precision, trueness, accuracy, stability and measurement uncertainty. The last section is devoted to practical considerations in validation. With every performance characteristic its essence and terminology are addressed, the current status of treating it is reviewed and recommendations are given, how to handle it, specifically in the case of LC–MS methods.     

Tutorial review on validation of liquid chromatography–mass spectrometry methods: Part I

This is the part I of a tutorial review intending to give an overview of the state of the art of method validation in liquid chromatography mass spectrometry (LC–MS) and discuss specific issues that arise with MS (and MS/MS) detection in LC (as opposed to the “conventional” detectors). The Part I briefly introduces the principles of operation of LC–MS (emphasizing the aspects important from the validation point of view, in particular the ionization process and ionization suppression/enhancement); reviews the main validation guideline documents and discusses in detail the following performance parameters: selectivity/specificity/identity, ruggedness/robustness, limit of detection, limit of quantification, decision limit and detection capability. With every method performance characteristic its essence and terminology are addressed, the current status of treating it is reviewed and recommendations are given, how to determine it, specifically in the case of LC–MS methods.     

Simultaneous determination of fluoroquinolones,sulfonamides and tetracyclines in sewage sludge by pressurized liquid extraction and liquid chromatography electrospray ionization-mass spectrometry

A new scheme for the quantitative determination of traces of fluoroquinolones (FQs), tetracyclines (TCs) and sulfonamides (SAs) in sewage sludge was developed. The compounds were simultaneously extracted from sewage sludge by pressurized liquid extraction (PLE). A novel and effective method for PLE was developed. Solid-phase extraction was used for cleaning up the extracts. Identification and quantification of the compounds was done using high-performance liquid chromatography with electrospray ionization mass spectrometry in selected reaction monitoring mode. The best recovery of FQs and TCs was obtained by using hydrophilic–lipophilic balance cartridges, recoveries ranged 59% for norfloxacin to 82% for ofloxacin and 95% for doxycycline; for SAs strong cation-exchange cartridges were more efficient, recoveries were 96% for sulfamethoxazole and 43% for sulfadimethoxine. Limit of quantification ranged from 0.1 ng/g for SAs to 160 ng/g for tetracycline. Method precision for TCs was 5.06% and 1.12%, and for SAs 0.43% and 2.01%. FQs precision ranged from 0.77% to 1.89%.     

Optimization of electrospray interface and quadrupole ion trap mass spectrometer parameters in pesticide liquid chromatography/electrospray ionization mass spectrometry analysis

Optimization of both the ionization process and ion transportation in the mass spectrometer is of crucial importance in order to achieve high sensitivity and low detection limits and acceptable accuracy in liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) analysis. In this paper four optimization procedures of electrospray interface and quadrupole ion‐trap mass spectrometer parameters (ESI‐MS) (nebulizer gas and drying gas flow rate, end plate voltage, capillary voltage, skimmer voltage, octopoles direct current and radio frequency, trap drive and lens voltages) were studied on three pesticides – thiabendazole, aldicarb and imazalil. The results demonstrate that the methodology of optimization strongly influences the effectiveness of finding true optima of the operating parameters. Both eluent flow rate and composition during optimization have to mimic the situation during real analysis as closely as possible in order to achieve parameters giving the highest sensitivity. Therefore, post‐column addition of analyte to the mobile phase identical in composition to the one in which analyte elutes during real analysis combined with software‐based optimization was found to be the most effective and fastest method for achieving intensity maxima. The parameters most strongly affecting ion formation and transportation, hence sensitivity, were capillary voltage, direct current of the first octopole, trap drive and the second lens for all pesticides under study. In addition to sensitivity and detection limit matrix effect was considered in the optimization process. It was found that the matrix effect can be reduced but not eliminated by adjusting the ESI and MS parameters. The optimal parameters from the point of view of the matrix effect can only be found with factorial design. Parameters giving higher sensitivity tended to be more affected by matrix effect causing higher ionization suppression by co‐eluting compounds. Copyright © 2010 John Wiley & Sons, Ltd.     

Uncertainty in liquid chromatographic analysis of pharmaceutical product: influence of various uncertainty sources

An ISO GUM measurement uncertainty estimation procedure was developed for a liquid-chromatographic drug quality control method-assay of simvastatin in drug formulation. In quantification of uncertainty components several practical approaches for including difficult-to-estimate uncertainty sources (such as uncertainty due to peak integration, uncertainty due to nonlinearity of the calibration curve, etc.) have been presented. Detailed analysis of contributions of the various uncertainty sources was carried out. The results were calculated based on different definitions of the measurand and it was demonstrated that unequivocal definition of the measurand is essential in order to get rigorous uncertainty estimate. Two different calibration methods - single-point (1P) and five-point (5P) - were used and the obtained uncertainties and uncertainty budgets were compared. Results calculated using 1P and 5P calibrations agree very well. The uncertainty estimate for 1P is only slightly larger than with 5P calibration.     

Analysis of selenomethylselenocysteine and selenomethionine by LC-ESI-MS/MS with diethyl ethoxymethylenemalonate derivatization

A simple method to identify and determine Selenomethionine (SeMet) and Selenomethylselenocysteine (Se-MeSeCys) with diethyl ethoxymethylenemalonate (DEEMM) derivatization and LC-ESI-MS/MS determination was developed. Separation of SeMet and Se-MeSeCys was achieved in 15.3 minutes. The calibration graph was linear in the range of 0.32 pmol to 49 pmol for SeMet and 0.34 pmol to 40 pmol for Se-MeSeCys. To prevent oxidation of SeMet, 2-mercaptoethanol was introduced to the calibration solutions. Detection limits were 0.1 pmol, which are comparable to LC-ICP-MS analysis. The developed method therefore offers an alternative to LC-ICP-MS offering similar sensitivity and additionally allows identification. The method was used to determine Se-MeSeCys and SeMet in onion samples.     

A review of analytical techniques for determination of Sudan I–IV dyes in food matrixes

Sudan dyes are a family of lipophilic azo dyes, extensively used in industrial and scientific applications but banned for use as food colorants due to their carcinogenicity. Due to the continuing illicit use of Sudan dyes as food colorants their determination in different food matrices – especially in different chilli and tomato sauces and related products – has during the recent years received increasing attention all over the world. This paper critically reviews the published determination methods of Sudan I–IV dyes. LC–UV–vis and LC–MS are the dominating methods for analysis of Sudan I–IV dyes. Sudan dyes are usually found in food at mg kg⁻¹ levels at which it may be necessary to use a preconcentration step in order to attain the desired detection limits. Liquid–solid extraction is the dominating sample preparation procedure. In recent years it has been supplemented by ultrasonic-assisted extraction and pressurized liquid extraction. Various solid phase extraction types have been used for sample cleanup. The large majority of works use conventional C18 columns and conventional LC eluents. Traditionally the UV–vis absorbance detection has been the most frequently used. In the recent years MS detection is applied more and more often as it offers more reliable identification possibilities.     

Control of Two-photon Fluorescence of Common Dyes and Conjugated Dyes

We present a comprehensive study of the selective excitation of two-photon fluorescence from various pairs of dyes and dyes in different conjugation states with tailored pulse shapes found with a genetic algorithm (GA). We investigate a number of biologically important dyes, and include dyes conjugated to trastuzumab (Herceptin®) and to a poly(amidoamine) dendrimer. We consider in detail the ability of tailored pulse shaping to discriminate dyes with significant spectral overlap. Our procedure for adaptive pulse shaping includes power-law and chirp-scaling checks to prevent trivial convergences. The GA uses a multiplicative fitness parameter in a graded search method that converges on pulse shapes that not only differentiate two-photon processes, but do so in a high signal regime. We consider the results in terms of not only the absolute maximum ratio of discrimination achieved, but also present the evolutionary course of the GA and compare the improvement to a quantitative measure of the noise level. We also implement a time-domain acousto-optic measurement of two-photon excitation cross-section spectra. The results show that the ability to discriminate dyes is determined almost entirely by their differences in two-photon excitation cross section.     

Influence of boric acid on electrospray ionization efficiency

Liquid chromatography electrospray ionization mass-spectrometry (LC-ESI-MS) was used to analyze 9-fluorenylmethylmethoxycarbonyl chloride (Fmoc-Cl) and diethyl ethoxymethylenemalonate (Deemm) derivatives of three amino acids and five other compounds. Influence of boric acid on their ionization was investigated and dramatic impact on the signal was observed. The strongest signal suppression (6% of signal remains) was observed for the Deemm derivative of beta-Alanine (with ammonium acetate in eluent). With only formic acid as the eluent pH modifier, signal enhancement was observed, being largest for Fmoc-Cl derivative of Phenylalanine, 267%. Investigation of the influence of boric acid shows that it is a possible signal enhancer for LC-ESI-MS analysis.     

Cataract diagnosis by measurement of backscattered light

We present a portable optical cataract assessment technology which measures with a circular photodetector the fraction of light scattered backwards by the human eye lens when illuminated by a laser diode. As our signal arises directly from the fundamental pathology-increased scattering in the lens-it directly assesses cataract extent and progression. Initial clinical results in undilated human eyes show device reading correlations in agreement with clinical examination and Scheimpflug photography.